Annual Review of Pharmacology and Toxicology 2005 PDF

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Annual Review of Pharmacology and Toxicology

Volume 45, 2005
Annu. Rev. Pharmacol. Toxicol. 2005.45:689-723. Downloaded from arjournals.annualreviews.org

by Universitaet Heidelberg on 10/01/05. For personal use only.
CONTENTS
FRONTISPIECEMinor J. Coon
CYTOCHROME P450: NATURES MOST VERSATILE BIOLOGICAL
CATALYST, Minor J. Coon
CYTOCHROME P450 ACTIVATION OF ARYLAMINES AND
HETEROCYCLIC AMINES, Donghak Kim and F. Peter Guengerich
GLUTATHIONE TRANSFERASES, John D. Hayes, Jack U. Flanagan,
and Ian R. Jowsey
PLEIOTROPIC EFFECTS OF STATINS, James K. Liao and Ulrich Laufs

FAT CELLS: AFFERENT AND EFFERENT MESSAGES DEFINE NEW
APPROACHES TO TREAT OBESITY, Max Lafontan
FORMATION AND TOXICITY OF ANESTHETIC DEGRADATION
PRODUCTS, M.W. Anders
THE ROLE OF METABOLIC ACTIVATION IN DRUG-INDUCED
HEPATOTOXICITY, B. Kevin Park, Neil R. Kitteringham, James L. Maggs,
Munir Pirmohamed, and Dominic P. Williams
xii
1
27
51
89
119
147
177
NATURAL HEALTH PRODUCTS AND DRUG DISPOSITION, Brian C. Foster,

J. Thor Arnason, and Colin J. Briggs
203
BIOMARKERS IN PSYCHOTROPIC DRUG DEVELOPMENT: INTEGRATION

OF DATA ACROSS MULTIPLE DOMAINS, Peter R. Bieck
and William Z. Potter
NEONICOTINOID INSECTICIDE TOXICOLOGY: MECHANISMS OF

SELECTIVE ACTION, Motohiro Tomizawa and John E. Casida
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, APOPTOSIS,
AND NEURODEGENERATIVE DISEASES, De-Maw Chuang,
Christopher Hough, and Vladimir V. Senatorov
NON-MICHAELIS-MENTEN KINETICS IN CYTOCHROME

P450-CATALYZED REACTIONS, William M. Atkins
EPOXIDE HYDROLASES: MECHANISMS, INHIBITOR DESIGNS,
AND BIOLOGICAL ROLES, Christophe Morisseau
and Bruce D. Hammock
227
247
269
291
311
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CONTENTS
NITROXYL (HNO): CHEMISTRY, BIOCHEMISTRY, AND

PHARMACOLOGY, Jon M. Fukuto, Christopher H. Switzer,
Katrina M. Miranda, and David A. Wink
335
TYROSINE KINASE INHIBITORS AND THE DAWN OF MOLECULAR

CANCER THERAPEUTICS, Raoul Tibes, Jonathan Trent,

and Razelle Kurzrock
357
ACTIONS OF ADENOSINE AT ITS RECEPTORS IN THE CNS: INSIGHTS

FROM KNOCKOUTS AND DRUGS, Bertil B. Fredholm, Jiang-Fan Chen,
Susan A. Masino, and Jean-Marie Vaugeois
385
REGULATION AND INHIBITION OF ARACHIDONIC ACID

(OMEGA)-HYDROXYLASES AND 20-HETE FORMATION,
Deanna L. Kroetz and Fengyun Xu
413
CYTOCHROME P450 UBIQUITINATION: BRANDING FOR THE

PROTEOLYTIC SLAUGHTER? Maria Almira Correia, Sheila Sadeghi,
and Eduardo Mundo-Paredes
439
PROTEASOME INHIBITION IN MULTIPLE MYELOMA: THERAPEUTIC

IMPLICATION, Dharminder Chauhan, Teru Hideshima,
and Kenneth C. Anderson
465
CLINICAL AND TOXICOLOGICAL RELEVANCE OF CYP2C9:

DRUG-DRUG INTERACTIONS AND PHARMACOGENETICS,
Allan E. Rettie and Jeffrey P. Jones
477
CLINICAL DEVELOPMENT OF HISTONE DEACETYLASE INHIBITORS,

Daryl C. Drummond, Charles O. Noble, Dmitri B. Kirpotin, Zexiong Guo,
Gary K. Scott, and Christopher C. Benz
495
THE MAGIC BULLETS AND TUBERCULOSIS DRUG TARGETS,

Ying Zhang
529
MOLECULAR MECHANISMS OF RESISTANCE IN ANTIMALARIAL

CHEMOTHERAPY: THE UNMET CHALLENGE, Ravit Arav-Boger
and Theresa A. Shapiro
565
SIGNALING NETWORKS IN LIVING CELLS, Michael A. White

and Richard G.W. Anderson
587
HEPATIC FIBROSIS: MOLECULAR MECHANISMS AND DRUG TARGETS,

Sophie Lotersztajn, Boris Julien, Fatima Teixeira-Clerc, Pascale Grenard,
and Ariane Mallat
ABERRANT DNA METHYLATION AS A CANCER-INDUCING

MECHANISM, Manel Esteller
THE CARDIAC FIBROBLAST: THERAPEUTIC TARGET IN MYOCARDIAL
REMODELING AND FAILURE, R. Dale Brown, S. Kelley Ambler,
M. Darren Mitchell, and Carlin S. Long
605
629
657
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vii
EVALUATION OF DRUG-DRUG INTERACTION IN THE HEPATOBILIARY

AND RENAL TRANSPORT OF DRUGS, Yoshihisa Shitara, Hitoshi Sato,
and Yuichi Sugiyama
689
DUAL SPECIFICITY PROTEIN PHOSPHATASES: THERAPEUTIC TARGETS

FOR CANCER AND ALZHEIMERS DISEASE, Alexander P. Ducruet,
Andreas Vogt, Peter Wipf, and John S. Lazo
725

INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 4145
Cumulative Index of Chapter Titles, Volumes 4145
ERRATA
An online log of corrections to Annual Review of Pharmacology and
Toxicology chapters may be found at
http://pharmtox.annualreviews.org/errata.shtml
751
773
776

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10.1146/annurev.pharmtox.45.120403.100030
Annu. Rev. Pharmacol. Toxicol. 2005. 45:125

doi: 10.1146/annurev.pharmtox.45.120403.100030
c 2005 by Annual Reviews. All rights reserved
Copyright
First published online as a Review in Advance on August 13, 2004
CYTOCHROME P450: Natures Most Versatile

Biological Catalyst
Minor J. Coon
Victor C. Vaughan Distinguished University Professor of Biological Chemistry, Emeritus,
Department of Biological Chemistry, Medical School, The University of Michigan,
Ann Arbor, Michigan 48109; email: mjcoon@umich.edu
Key Words resolution and reconstitution of the microsomal P450 system,

purification and characterization of multiple P450s, coupling of reductase and
P450 reaction cycles, oxygen activation to give multiple functional oxidants
Abstract The author describes studies that led to the resolution and reconstitution
of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence
for multiple isoforms of the oxygenases with distinct chemical and physical properties
and different but somewhat overlapping substrate specificities. Present knowledge of
the individual steps in the P450 and reductase reaction cycles is summarized, including
evidence for the generation of multiple functional oxidants that may contribute to the
exceptional diversity of the reactions catalyzed.
BACKGROUND
To my knowledge, my forebears, who migrated to the state of Colorado in the
United States from Germany (via Russia) and from the Netherlands, had no scientific credentials. However, my father and my paternal grandmother were highly
interested in reading about scientific advances despite having had no formal training, and it was my good fortune that my parents were fully supportive, even pleasantly surprised, at my own scientific bent. I also had the benefit of exposure to
rigorous courses in the Denver public schools. Our teachers frequently told us that
the schools in our city were ranked among the ten best in the country. We did not
ask for documentation of that fact, but the science courses in my high school were
challenging and so interesting that I considered a future career in several such
fields. Geology particularly intrigued me, possibly because from our classroom
windows we could see in the foothills of the Rocky Mountains the formations we
were studying. The chemistry courses, however, opened a new world to me, and I
knew I would continue to pursue some branch of this subject. That turned out to be
the relatively new field of biochemistry, which I first learned about as an undergraduate at the University of Colorado, Boulder. Professor Reuben Gustavson, whose
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training had been in steroid biochemistry at the University of Chicago, taught the
freshman chemistry course, in which he included examples of problems in biology
waiting to be solved by the application of chemistry. His enthusiasm and engrossing stories about the early history of science and the personalities involved made
the subject come alive. I readily accepted his invitation to join his small research
group working on estrogen metabolism, and, at his recommendation, spent the
summer following my junior year at the University of Chicago in the laboratory of
Professor F.C. Koch. From my experience, I am convinced that research in some
field of scholarly endeavor is as crucial to undergraduate education as the usual
didactic studies.
GRADUATE STUDY
Present-day academic institutions advertise widely to attract the best qualified
faculty members, postdoctoral associates, and even medical and graduate students,
and they spend much effort in interviewing and impressing applicants. It was much
simpler in 1943, when I had a brief discussion with Dr. Gustavson about my desire
to enter graduate school. He suggested a few top institutions and we decided on
the University of Illinois. As a result of his correspondence with Professor William
C. Rose, Head of the Biochemistry Division in the Chemistry Department there, I
moved to Urbana in September as a Graduate Research Assistant, and I undertook
a laboratory project a few weeks later.
There were no laboratory rotations in the 1940s, but after considering several
attractive possibilities, I chose Professor Will Rose as my mentor. I paraphrase here
what I have written at greater length elsewhere about his personality and accomplishments (1). Roses research interests included the intermediary metabolism of
amino acids, creatine, uric acid, and related compounds, and he was renowned
for the discovery, isolation, and identification of a new amino acid, -amino-hydroxy-n-butyric acid, which he named threonine. This was the culmination of
experiments in which rats failed to grow on diets containing the 19 previously
known amino acids. When I arrived in Urbana, the identity of the 10 amino acids
essential for growth in rats and the 8 essential for the maintenance of nitrogen
equilibrium in the human (that is, male graduate students) was already known. It
fell to my lot to isolate or synthesize, purify, and analyze amino acids and then
feed them to my fellow students enlisted as human guinea pigs to establish the
quantitative requirements for the essential amino acids and the availability of their
D-isomers or N-acetyl derivatives. In those days, the recruits were grateful for the
free synthetic diets, the dollar a day they were paid, and the prospect of seeing
their initials in Roses widely read publications, but they required my constant encouragement because the rations were unpalatable. I have often remarked that any
skills I developed to persuade my recalcitrant fellow students to continue in these
difficult experiments (and therefore lead to completion of my Ph.D. thesis) became
useful many years later when, as chair of a biochemistry department, I had to deal
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FUNCTIONS OF MULTIPLE P450S

with faculty colleagues having contrary views. Roses students were somewhat in
awe of the professor, probably wondering whether they could meet his exacting
standards or hope to emulate the seeming ease with which he succeeded in all his
professional endeavors. I learned in time that behind his somewhat reserved and
formal manner was genuine warmth and an understanding that young scientists
develop their full potential only by profiting from their mistakes; we became close
friends until his death at age 98.
FACULTY AND SABBATICAL POSITIONS

Following completion of my Ph.D. thesis in 1946, I stayed at the University of
Illinois for an additional year as a postdoctoral fellow, in part because I was
courting another student, Mary Lou Newburn, whom I later married. A year later, I
accepted an attractive faculty position elsewhere. Again, to illustrate the simplicity
of the process at that time, Professor Rose heard of a suitable vacancy at the
University of Pennsylvania and wrote to Professor D. Wright Wilson, Chairman
of the Department of Physiological Chemistry, as it was then called, in the School
of Medicine, on my behalf. Dr. Wilson, a man of few words, took me to a brief
lunch at a national meeting in Chicago, offered me the position, and I accepted
it never having been in Philadelphia or having met other faculty members in
his department. He and I had faith in each other, and no time or expense was
devoted to a more formal interview at that institution. However, when I arrived
in Philadelphia some months later and learned from him, to my dismay, that I
had been hired as a nutritionist (whereas my intent was to pursue intermediary
metabolism as a biochemist), I realized that we had exchanged too few words
previously. Before long, he accepted my career plan, but in my opinion our present
system of thoroughly interviewing numerous applicants is much more likely to
lead to success. Furthermore, it is fairer to potential candidates not having personal
connections.
The Physiological Chemistry faculty at the University of Pennsylvania was
among the first in this country to work with radioactive carbon-14 as a tracer,
and several of the senior members were widely known for their studies: John
Buchanan on purine biosynthesis, Wilson on pyrimidine biosynthesis, and Samuel
Gurin on fatty acid -oxidation. In addition, Otto Meyerhof, the famous biochemist
who had come to the department as a refugee from Germany, was continuing
his investigations on energy relationships in glycolysis. I undertook studies on
amino acid metabolism, beginning with leucine, the oxidation of which is difficult
because classical -oxidation is not possible owing to the branched structure of
the intermediate, isovaleryl-CoA. We discovered that a novel ATP-dependent CO2
fixation was involved that led to intermediates in acetoacetate and cholesterol
synthesis (26), and I have pursued unusual oxidative reactions ever since.
Another advantage of my position at Pennsylvania was the generosity of
Dr. Wilson in allowing me to be absent for a year (although I had not yet earned a
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sabbatical leave) to gain knowledge of enzymology in the laboratory of Professor

Severo Ochoa, then Chairman of Pharmacology at the School of Medicine of New
York University. As mentioned elsewhere (1), Ochoas facilities were crowded and
had limited equipment, but it was an exciting place to pursue research. Ochoas
scientific insight was supplemented by daily discussions with luminaries such as
Otto Loewi, Ephraim Racker, Sarah Ratner, and a legion of visiting postdoctoral
fellows, present and former students, and sabbatical guests from all corners of
the world. The environment was ideal for a visitor to master enzymology as an
essential tool to pursue the complexities of intermediary metabolism.
In 1955, I accepted an offer of a full professorship in Biological Chemistry
at the University of Michigan, and moved with my wife and children Larry and
Susan to Ann Arbor, not without regrets at leaving my friends and colleagues at the
University of Pennsylvania. Michigan has now been a permanent and supportive
home for my scientific career for almost 50 years, during 20 of which I served
as chair of my department. As described below, my research interests gradually
turned from amino acid metabolism, biotin function in CO2 fixation, and pyruvate
kinase properties and function to cytochrome P450 and its role in the metabolism
of drugs and many other foreign compounds, as well as substrates of physiological
importance.
Because of my increasing interest in mechanistic aspects of enzyme catalysis,
I spent a sabbatical leave in 196162 with Professor Vladimir Prelog, Director of
the Organic Chemistry Laboratory of the Eidgenossische Technische Hochschule
(Swiss Federal Institute of Technology) in Zurich. Well known for his investigations on natural products and his outstanding contributions to stereochemistry
(1), he had started to investigate enzyme stereoselectivity, and I began to work
on hydride transfer from decalin ketones by oxidoreductases. The Curvularia enzymes that we employed proved to be difficult to purify, and we eventually found
that a more suitable enzyme for our studies was the 3-oxoacyl-acyl carrier protein
reductase component of a fatty acid synthetase (7). Many of my colleagues hesitate to take sabbatical leaves, believing they cant be spared from the day-to-day
operations of their laboratories and institutions. On the contrary, more important
insights may often be gained at a distance from our usual overloaded schedules. In
my own case, the opportunity to reflect on my future research plans in a different
setting undoubtedly contributed to my striking out in new directions.
My mentors differed in their personal characteristics and research interests, but
all were completely dedicated to science. Severo Ochoa, for example, stated in a
personal essay entitled The Pursuit of a Hobby (8) that in his life biochemistry had
been his only and real hobby. In that connection, I recall being at the University of
Sheffield when Hans Krebs, who had built an excellent department there before his
move to the chair at Oxford, returned to give a seminar. During the question period,
a student asked Sir Hans to what he owed the secret of his success. He modestly
replied, Luck. When the applause died down, he became more serious and said,
I had a certain amount of luck in my life, but then I made a correlationthe
harder I worked, the luckier I got.
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OMEGA OXIDATION: A TRAIL LEADING TO RESOLUTION

AND RECONSTITUTION OF THE LIVER MICROSOMAL
P450-CONTAINING ENZYME SYSTEM
Although most biological oxidations occur according to the predictions of known
chemical principles, those that do not are often found to involve particularly interesting cofactors, such as previously unsuspected metals or organic coenzymes. In
other instances, novel functions of amino acid residues in the enzymes are discovered, thus altering our concepts of biological catalysis. I have long been intrigued
by difficult oxidations of unfunctionalized alkyl groups, as in the conversion of
the side chain of leucine to acetoacetate (as described above); the anabolism and
catabolism of poorly soluble lipids; the degradation of natural products such as
terpenoids; and the transformations of some chemically unreactive foreign substances such as drugs, solvents, and pesticides to products that may be more or
less toxic than their precursors. Even the highly inert alkanes in petroleum have
been known for many years to undergo microbial oxidation.
In the late 1950s, I picked fatty acid -oxidation, in which the attack occurs
at the least reactive position, the terminal methyl group, as a model for such
difficult oxidations. In 1932, Verkade et al. (9) in the Netherlands had discovered
this unexpected conversion when they fed fatty acids of intermediate chain length
to dogs and to human volunteers and isolated the resulting urinary dicarboxylic
acids. Halina Den, a graduate student in my laboratory, was able to show that a
14
C-labeled ,-dimethyl-substituted fatty acid underwent terminal oxidation in
liver tissue (10), but the instability and insolubility of the enzyme system prevented
further progress.
We then turned to the microbial oxidation of hydrocarbons as even more inert
substrates. A postdoctoral associate from Illinois, James Baptist, isolated from
soil samples a strain of Pseudomonas oleovorans, called the gasoline bug by
our colleagues, which grew well on alkanes such as hexane. Cell-free extracts
were soon obtained that required NADH for the aerobic conversion of octane
to octanol (11, 12) and, of particular interest, the -oxidation of fatty acids as
demonstrated by Masamichi Kusunose and his wife Emi, visitors from Japan (13).
After the successful resolution of the enzyme system into three enzyme fractions
by Bill Peterson (14), the components were eventually purified and characterized
as rubredoxin, a red nonheme iron protein (15) previously only known to occur
in anaerobic bacteria; a flavoprotein containing FAD and functioning as NADHrubredoxin reductase that was characterized by Tetsufumi (Ted) Ueda (16, 17); and
the -hydroxylase, an almost colorless protein that aggregated extensively and was
activated by the addition of ferrous ions (18, 19). The properties of the bacterial
hydroxylase made it a difficult candidate for further mechanistic studies, but it
has continued to be investigated by others, who have established that it contains a
nonheme diiron cluster (20).
In the hope that our findings with bacteria would be applicable to mammalian
metabolism, we returned to the liver system we had abandoned approximately
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ten years previously. Fortunately, Anthony Lu joined my research group in 1966

as a postdoctoral associate upon completion of his graduate studies in biochemistry at the University of North Carolina. My immediate impression was that this
extremely capable young scientist deserved to be given a suitably challenging
problem. I doubt that I made it clear just how challenging the hepatic microsomal
system would be, but I advised him to begin with the methods that had succeeded
with the pseudomonad. The lack of success with this approach did not discourage
either of us, nor did the dearth of knowledge at that time about membrane-bound enzymes. After more than two years, Anthonys dedicated efforts eventually resulted
in solubilization of the catalytically active rabbit liver microsomal -hydroxylation
system by the use of various detergents with glycerol and other agents to prevent
enzyme denaturation. As is now well known, ion exchange column chromatography resolved the system into three components, which upon recombination under
controlled conditions, catalyzed the -hydroxylation of 14C-labeled lauric acid
(21, 22). As shown in Figure 1, these included a reddish-brown fraction that we
soon identified, to our surprise and considerable delight, as cytochrome P450 by
the spectral change upon reaction with carbon monoxide after dithionite reduction, and a yellow fraction containing NADPH-cytochrome P450 reductase that
was fully active in electron transfer to P450, unlike preparations isolated by others
after solubilization by protease treatment, with loss of the hydrophobic peptide
at the NH2-terminus. The third fraction contained an active component that was
colorless, heat-stable, and extractable by organic solvents. This was later found
by Henry Strobel, another talented postdoctoral associate from North Carolina,
to contain microsomal phospholipids, of which phosphatidylcholine was the most
effective (23). Thus, we had in our hands the solubilized, reconstituted enzyme
system that would allow us to purify and characterize the enzymes involved. A
variety of drugs, including aminopyrine, benzphetamine, hexobarbital, ethylmorphine, norcodeine, and p-nitroanisole, were also found to be oxidized by the reconstituted system (24), and, of much interest, Robert Kaschnitz (25) and Wilfried
Duppel & Jean-Michel Lebeault (26) found that the same methods used with rabbit
liver were successful with human liver and with Candida tropicalis, respectively.
Our findings were greatly aided by previous knowledge that the microsomal CObinding pigment of unknown function (2729) had been characterized as a b type
cytochrome by Omura & Sato (30). In addition, it was known that this catalyst in
hepatic microsomes is involved in the hydroxylation of several steroids and drugs,
as established in pioneering photochemical action spectroscopic experiments by
Omura et al. in 1965 (31).
In his Bernard Brodie Award Lecture, Anthony Lu (32) has also commented
on our limited knowledge of membrane-bound enzymes in the early days and
the challenge of working on mammalian cytochrome P450. To indicate the many
important questions remaining at that time, a brief summary of the proceedings
of the first Symposium on Microsomes and Drug Oxidations held in Bethesda,
Maryland, in 1968 (33) is in order. The idea came from the Committee on Drug
Safety, Drug Research Board of the National Academy of Sciences. Organized by
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Figure 1 Resolution of -hydroxylation enzyme system into three components by

DEAE-cellulose column chromatography of rabbit liver microsomal extract. Elution
was with a KCl gradient, and 15-ml fractions were collected. For assay of P450 (),
undiluted samples were reduced with dithionite and flushed with carbon monoxide, and
the CO-dependent A450A490 difference was determined in a 1-cm light path. NADPHcytochrome P450 reductase activity () was estimated spectrally by cytochrome c
reduction and expressed as the increase in A550 per minute per 0.4-ml aliquot of column
eluate. The effect of the third component ( e) on laurate hydroxylation in the presence
of the P450 and reductase components was determined and expressed as mmol of
-hydroxylaurate formed per minute per 0.05 ml of column eluate. Taken from Reference 21.
James Gillette, an expert on biochemical pharmacology, and other distinguished

scientists, including Allan Conney, George Cosmides, Ronald Estabrook, James
Fouts, and Gilbert Mannering, the program included 27 lectures by experts from
around the world on microsomal morphology and what was known about drug
metabolism. (Posters had not yet been invented.) The properties of the endoplasmic reticulum were described, and evidence was presented that approximately
20 compounds, encompassing several drugs, steroids, and hydrocarbons, as well as
fatty acids (34), undergo oxidation in liver microsomes from experimental animals.
Hydroxylation, including drug N-demethylation, was the only reaction considered.
Carbon monoxide and SKF-525A were the inhibitors mentioned, and phenobarbital and 3-methylcholanthrene the two chief inducers. Debate ensued on how
many forms of P450 exist, with one camp believing in only a single enzyme.
The interesting proposal was also made on the basis of the effects of inducers on
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the activities and spectra of liver microsomes isolated from the treated animals
for two types of CO-binding pigments, or possibly two interconvertible forms of
a single cytochrome. With respect to the active oxidant produced by P450, oxene,
analogous to compound I of peroxidases, was proposed. All in attendance agreed
that the very intriguing field of drug metabolism was on the threshold of major
progress.
This prefatory chapter is concerned with my research interests that led our laboratory to study the biochemical aspects of drug metabolism, and no attempt is
made to provide a general review of what has become a huge field of endeavor.
However, mention should be made of the outstanding contributions of the Gunsalus laboratory with bacterial P450cam, a nonmembranous cytochrome that is
specific for camphor oxidation (35, 36) and has served as a model for the versatile
but less tractable mammalian P450s. Readers interested in developments in this
field over the years are referred to the proceedings of several series of international
meetings, all with an emphasis on basic science: Symposia on Microsomes and
Drug Oxidations, as already mentioned; Conferences on the Biochemistry and
Biophysics of Cytochrome P450 (37), originated in 1976 by Klaus Ruckpaul, who
was working at Berlin-Buch to overcome the barriers that had divided eastern and
western Europe since the end of World War II, and whose valiant efforts in this
endeavor attracted worldwide support as acknowledged by Sinisi Maricic, the organizer of the first conference (38); meetings on Cytochrome P450 Diversity (37),
with an emphasis on microbial and plant systems, initiated by Hans-Georg Mueller,
a colleague of Ruckpauls at Berlin-Buch; and meetings of the International Society for the Study of Xenobiotics, started by Bruce Migdaloff in discussions with
Fred DiCarlo, John Baer, and Ina Snow at the 1980 Gordon Conference on Drug
Metabolism and launched the following year. Perhaps surprisingly, sufficient new
results are obtained from laboratories around the world to justify all of these and
other related meetings on a regular basis. I had the pleasure of chairing the committees that provided oversight for the Microsomes and Drug Oxidations symposia
and P450 conferences for many years. Without a doubt, the collaborations and
friendships that grew out of such international meetings were a major stimulus to
the rapid development of this broad field, including its application to drug design
and development.
MULTIPLICITY OF P450 CYTOCHROMES

In a recent review of the induction of drug-metabolizing enzymes, Allan Conney
(39) has summarized the extensive evidence from his laboratory and elsewhere
that treatment of animals with different microsomal inducers results in different
profiles of catalytic activity for the metabolism of foreign compounds and steroid
hormones. Such studies in many laboratories strongly suggested the occurrence
of multiple cytochromes P450 with different substrate selectivities and provided a
stimulus for biochemical characterization of the proposed catalysts. Furthermore,
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enhancement by induction of the levels of individual enzymes in particular species,

tissues, and organelles could facilitate purification. It was also widely recognized,
however, that the catalytic changes seen in induced microsomes could be explained
otherwise, by posttranslational modification by proteolysis or by addition of other
chemical groups to a single cytochrome; by alteration of the membrane environment; or even by changes in other components of the system that might alter the
rate-limiting steps, such as NADPH-cytochrome P450 reductase, cytochrome b5,
or phospholipids. In addressing this important question, Nebert et al. (40) showed
that in genetically responsive mice, but not in aromatic hydrocarbon-treated
nonresponsive mice, the inducible hydroxylase activity is localized exclusively
in the P450, or what was then called the P448 fraction. A similar conclusion had
been reached by Lu et al. (41) upon reconstitution of P450 and P448 separately
with the reductase and phospholipid from microsomes of both phenobarbital- and
3-methylcholanthrene-treated rats.
A sophisticated understanding of drug metabolism, including the complexities
of regulation and formation of diverse products that occasionally lead to toxicities, clearly required thorough characterization of the proposed individual P450
enzymes as well as the reductase. Progress toward this goal was made possible by
the availability of the detergent-solubilized P450 system from microsomes, but it
was difficult because these hydrophobic enzymes displayed a high degree of similarity and a tendency to aggregate (42). Indeed, it took over four years for the first
mammalian P450, the phenobarbital-inducible form in rabbit liver microsomes,
to be purified and characterized (4346). The procedures that were developed,
including column chromatography, had to be carried out in the presence of detergents. The resulting isolated cytochrome, now designated P450 2B4 according
to the nomenclature on the basis of divergent evolution as judged by sequence
similarity (47) (originally called LM2, or liver microsomal form 2), differs from
-naphthoflavone-inducible P450 1A2 (form 4) in its physical and chemical properties (45, 46), including electrophoretic behavior; monomeric molecular weight;
immunological reaction with specific antibodies (48, 49); absorption spectra in the
oxidized, reduced, and CO-bound states (46); CD spectra (50); and fluorescence
properties (51). Such individual P450s, which arise from genetically controlled
de novo protein synthesis (52), are called isoforms or isozymes. The latter term
was coined years ago to describe multiple forms of an enzyme identical in function
but differing in some other property such as maximum activity, substrate affinity,
or regulation. A 2B4-like pseudogene was also isolated and characterized; the
alterations supported the view that it would not encode a functional cytochrome
(53). The individual members of what is now called the P450 superfamily often
have numerous functions (sometimes overlapping, as described below) but are
still commonly called isozymes. Further evidence for multiple microsomal P450s
was obtained from the differences in amino acid composition, COOH- and NH2terminal amino acid residues (46, 54), and eventually the complete amino acid
sequences (55, 56). P450 2B4 was the first example of a mature protein found to
have retained its so-called signal peptide. Research in several laboratories turned to
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the question of how many of these unique catalysts occur in different species. The
reductase, which fortunately occurs in only a single form, was purified from both
rat (57) and rabbit liver microsomes (58), the structural features were established
(59), and the crystal structure was subsequently reported (60). The interactions
among the various purified components of the enzyme system have been investigated by several techniques (61, 62), including the puzzling effects of cytochrome
b5, which may be stimulatory or inhibitory (6366).
Among the other rabbit cytochromes identified and then isolated in our laboratory were P450s 2C3 (form 3b) (67), 3A6 (form 3c) (68), and 1A1 (form 6)
(69). In addition, the P450 that catalyzes the 12-hydroxylation of 7-hydroxy4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic
acid, was purified in collaboration with Kyu-Ichiro Okuda and his associates in
Hiroshima (70). Thanks to the painstaking work of Dennis Koop, Edward Morgan,
and George Tarr (71), ethanol-inducible cytochrome P450 was discovered in rabbit
liver microsomes, purified, and characterized as a unique isozyme with unusually
interesting properties. This cytochrome, designated 2E1 (form 3a), displayed the
highest activity of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and was also found to oxidize other alcohols, aniline, and several drugs (72).
The existence of such a microsomal ethanol-oxidizing system, first proposed by
Lieber & DeCarli (73), had previously been the subject of much debate. Although
this may not be a major pathway for alcohol oxidation under most circumstances,
the increased levels of 2E1 resulting from the diabetic state, fasting, and exposure
to ethanol and several other diverse agents, including acetone, imidazole, benzene,
and isoniazid, is a cause for concern because of resulting toxicities (74). In particular, acetaminophen, a widely used antipyretic and analgesic drug, is normally
nontoxic, but in large doses it produces acute hepatic necrosis when converted to a
reactive metabolite. Of a series of P450 isoenzymes examined, 2E1 was one of the
most active in producing this metabolite (75). As summarized elsewhere (76), the
predominant role of alcohol-inducible P450 in oxidative damage involves activation of carcinogenic nitrosamines (77) and the leakiness of this cytochrome in
generating hydrogen peroxide and oxygen radicals (78), as well as alkoxy radicals
in the cleavage of lipid hydroperoxides (79). The rabbit is apparently unique in
having two genes in the alcohol-inducible P450 subfamily, the exon-intron organization of which was determined by restriction mapping and sequence analysis
(80). The genes are not coordinately expressed or regulated, and chemical inducers
act through changes in the rate of synthesis or degradation of the enzyme, rather
than through increased gene transcription (81). The corresponding enzymes are
97% identical in amino acid sequence and have similar substrate selectivity, with
2E2 always somewhat less active. The regulation of 2E1 is particularly complex
and includes effects of insulin and thyroid hormone on mRNA turnover (82) as
well as of cytokines on the transcriptional regulation of both 2E isoforms (83).
Todd Porter, already an expert on NADPH-cytochrome P450 reductase when
he joined the Biological Chemistry Department as a faculty member, contributed
greatly to our research progress with his experience in molecular biology and
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molecular genetics. As he has recently commented elsewhere (84), although the

use of bacteria had become the most common approach to heterologous protein
expression, there was skepticism whether this technique would lead to the retention of activity by membrane-bound enzymes. However, Michael Watermans
laboratory, working with 17- hydroxylase (85), and our laboratory, working with
2E1 (86), were successful with this approach, which often requires modification
of the NH2-terminal signal peptide. Studies by Larson & Porter with 2E1 (86) and
by Steve Pernecky et al. with 2B4 (87) contributed to this highly useful method
for P450 expression and characterization. Of particular importance to those who
crystallized functional truncated microsomal cytochromes, as indicated below,
was our surprising finding that the NH2-terminal segment of these cytochromes is
unnecessary for catalytic activity.
It should also be noted that P450 cytochromes occur in a variety of tissues
other than liver. For example, Xinxin Ding purified several from rabbit olfactory
and respiratory nasal mucosa (88), including 2A10/11 (form NMa) (89) and 2G1
(form NMb) (90), which is active in steroid metabolism and uniquely expressed
in the olfactory mucosa of nasal microsomes in animals. Ding & Kaminsky (91)
have recently reviewed human extrahepatic P450s. The procedures that led to unequivocal evidence for the multiplicity of microsomal cytochrome P450s in the
rabbit were soon applied to other species, including rats and mice, as described in
a comprehensive review by Lu & West (92). Expansion of knowledge about cytochrome P450, aided by rapid progress in molecular genetics, has shown that this
remarkable catalyst occurs throughout nature, including bacteria, fungi, and plants,
as well as animals (93). Multiplicity of isoforms is typical in the various species,
and with the availability of techniques for cloning and heterologous expression,
the purified enzymes are readily available for characterization with respect to substrate specificity and other properties. Of biomedical importance in understanding
the complexities of drug metabolism, the human species is now known to have 59
functional P450 genes (93).
MICROSOMAL P450 CYTOCHROMES CATALYZE

NUMEROUS REACTIONS WITH COUNTLESS SUBSTRATES
When the components of the microsomal oxygenating system had been purified and
characterized, we turned our attention to the individual steps in the hydroxylation
reaction, which has the following overall stoichiometry:
RH + O2 + NADPH + H+ ROH + H2 O + NADP+ ,
where RH represents a drug or some other typical substrate and ROH is the product.
Our findings over many years are summarized in the schemes in Figures 2A,B,
which indicate how reducing equivalents are transferred from NADPH via the
reductase cycle to the P450 cycle, with one atom of molecular oxygen inserted
into the substrate and the other reduced to water. Jan Vermilion and colleagues
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Figure 2 Joint function of P450 and reductase in drug metabolism. The schemes
account for the oxygenase, oxidase, and peroxygenase reactions of cytochrome P450
with electron transfer from NADPH via the reductase. (A) The reductase cycle is
modified from that in Reference 94 with the model for rapid interflavin electron transfer
in Reference 95. (B) The P450 cycle is based on that in Reference 96. Fe represents the
heme iron atom, RH a drug or other substrate, and ROH the corresponding product.
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(94) carried out stopped-flow experiments with reductase preparations in which

the FMN was replaced by artificial flavins that had a range of redox potentials. Her
results supported an electron transfer sequence in the holoreductase, NADPH
FAD FMN P450, and led to the conclusion that the flavoprotein cycles
mainly between the 1e- and 3e-reduced states during turnover (Figure 2A). Electron
donation to P450 occurs via the reaction FMNH2 FMNH, the semiquinone. In
addition, Dan Oprian (95), on the basis of a multiwavelength analysis by stoppedflow spectrophotometry, developed a model that successfully predicted the spectral
course of each phase of the reaction of NADPH with the reductase under anaerobic
conditions. This model was based on the model developed for xanthine oxidase by
Olson et al. (97) at Michigan. Oprians findings corresponded to those predicted for
rapid electron transfer between the two flavins in which the distribution of electrons
was governed at any time by the reduction potentials for the individual flavins. As
indicated by the scheme in Figure 2A, the flavoprotein in its fully oxidized state
is primed for its function by reduction of FAD by NADPH (Reaction 1). This
is followed by electron redistribution (Reaction 2) to give the flavin diradical in
equilibrium with FMNH2-FAD (Reaction 3), which can then donate an electron
to P450 to yield the Le-reduced flavoprotein (Reaction 4). Reduction of FAD by
NADPH (Reaction 5), followed by electron redistribution (Reaction 6), provides
FMNH2 as a potential donor to P450 (Reaction 7). Alternatively, FMNH2-FAD
may be reduced by NADPH to give FMNH2-FADH2 (Reaction 8) as a donor
(Reaction 9). Thus, FMNH2 serves its role in providing reducing equivalents for
oxygen activation by P450, regardless of whether the FAD moiety is in the fully
reduced, semiquinone, or oxidized state.
The scheme in Figure 2B includes the basic reaction cycle for oxygen activation proposed in 1980 by White & Coon (98). The individual steps are based
on experimental work in our own and other laboratories, and they are in accord
with the expected stoichiometry as follows: substrate (RH) binding to ferric P450
(Reaction 1), first electron transfer from FMNH2 (Reaction 2), dioxygen binding
(Reaction 3), second electron transfer from FMNH2 (Reaction 4), uptake of two
protons and heterolytic splitting of the oxygen-oxygen bond with generation of the
putative iron-oxene species (Reaction 5), proposed formation of a substrate radical
as a transient intermediate on the basis of a collaborative investigation with John
Groves on norbornane and 2B4 (Reaction 6) (99), oxygen insertion into substrate
(Reaction 7), and product dissociation with return of P450 to the resting state (Reaction 8). Figure 2B includes additional reactions discovered subsequently (96),
such as the ability of ferrous P450 to donate electrons in a stepwise fashion to bring
about substrate reduction, as in the cleavage of a lipid hydroperoxide [shown as
LCH(OOH)R ] to yield a ketone accompanied by hydrocarbon formation (100).
For example, 13-hydroperoxy-9,11-octadecadienoic acid was found to give rise
to 13-oxo-9,11-tridecadienoic acid and pentane. Lipid peroxidation is generally
looked on as a destructive process in membranes of living cells, with formation
of pentane and other hydrocarbons by various species, including the human, as
a measure of this pathophysiological process. Also shown on the lower left in
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the P450 cycle in Reaction 10 is the oxidative deformylation of an aldehyde with

loss of the aldehyde carbon as formate (101). This reaction, which we believe to
proceed via a peroxyhemiacetal-like adduct as indicated, is a model for the oxidative demethylation that accompanies steroid aromatization (102) and indicates
a possible route for modification of drugs that contain a carbonyl function, as in
aldehydes and ketones. The diverse chemical transformations carried out by this
extremely versatile enzyme system include, in addition to those already mentioned,
N-oxidation; sulfoxidation; epoxidation; oxidative ester and amide cleavage; N-,
S-, and O-dealkylation; peroxidation; ipso-substitution (103); dehalogenation;
desulfuration; and deamination; as well as reduction of epoxides, N-oxides, azo
groups, and nitro groups. Additional chemical reactions attributable to P450 continue to be discovered (104), and it is likely that still more will be found, considering
the major role of this cytochrome in the plant and microbial worlds, perhaps with
counterparts in animals and the human species.
In the early days of P450 research, only a few types of organic compounds
were thought to serve as P450 substrates, but this list continues to grow rapidly.
Most of the following have been employed in our laboratory as well as by many
other investigators: xenobiotics, including drugs, solvents, anesthetics, pesticides,
petroleum products, antioxidants, dyes, and plant products such as flavorants and
odorants, and compounds of physiological importance, such as steroids, fatty acids,
and lipid hydroperoxides, as already mentioned, but also fat-soluble vitamins,
amino acids, eicosanoids, and retinoids. The oxygenation and other alterations of
such a variety of substrates by microsomal P450s may seem indiscriminate, but in
many instances the modification is positionally and even stereochemically specific
(105, 106).
Also shown in the scheme in Figure 2B is the release of products of O2 reduction that are not coupled to substrate oxygenation, such as hydrogen peroxide
(Reaction 11); superoxide (Reaction 12); and, in the 4-electron NADPH oxidase
reaction (Reaction 13), water when the (FeO)3+ species is reduced by electrons
from NADPH (107). Reaction 9 illustrates the well-known peroxide shunt in which
H2O2 (108) or an alkyl hydroperoxide (109), peracid, or iodosobenzene donates
the oxygen atom for substrate hydroxylation with no requirement for molecular
oxygen or for NADPH as an electron donor. Homolytic cleavage of the oxygenoxygen bond occurs as shown, but heterolytic cleavage is also possible with some
hydroperoxides, in which case Fe = O would be formed directly, as observed with
iodosobenzene. A large variety of such donors is known from the work of Bob
Blake (110, 111).
The availability of purified individual microsomal P450s soon made it clear that
they do not conform to the typical textbook definition of an enzyme as a highly
specific biological catalyst. For example, 2B4 and 1A2 were both found as early
as 1975 to catalyze the oxidation of several substrates, including benzphetamine,
ethylmorphine, p-nitroanisole, aniline, biphenyl, and testosterone; furthermore, the
attack on the latter two substrates occurs in more than one position (45). To comment briefly on the total number of substrates for the hepatic microsomal P450s,
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no investigators in the field are surprised by the prediction of a million or more.

The present availability of combinatorial techniques in chemical synthesis further
inflates this prediction. It is widely recognized that almost all drugs, including
those to be produced in future years by the pharmaceutical industry, will serve as
P450 substrates. In most instances the metabolic changes lead to drug inactivation
and excretion of the more polar products, but some compounds that function as
prodrugs become activated and others yield products that inactivate the cytochrome
itself, as in the case of phencyclidine, which was first developed as a short-acting
dissociative anesthetic. As shown by Yoichi Osawa (112), the mechanism-based
inactivation of 2B4 is brought about by this drug and its oxidation product, the
iminium compound.
MULTIPLE OXIDANTS AND MULTIPLE MECHANISMS

IN P450 CATALYSIS
As already indicated, my interest in cytochrome P450 grew out of intense curiosity as to how an enzyme could accomplish with ease in an aqueous environment
at neutral pH and mild temperatures one of the most difficult reactions in nature,
hydroxylation of the unfunctionalized alkyl group in hydrocarbons and fatty acids.
The details of such reactions have intrigued chemists and biochemists for decades.
In studying this problem, my laboratory has been fortunate in having available
microsomal P450s that oxidize virtually any organic compound that might be of
mechanistic interest. We have also benefited greatly by collaboration with organic
chemists, biochemists, and pharmacologists who were attracted to study the enzyme system that my Illinois colleague Steve Sligar calls Natures Blowtorch.
An early example was a study by Groves et al. (99) of deuterated norbornane in
which mass spectral analysis of the exo- and endo-2-norborneol products indicated
a very large isotope effect and significant epimerization in the hydroxylation reaction. The results indicated an initial hydrogen abstraction to give a presumed carbon
radical intermediate in what has been called the hydrogen abstraction-oxygen rebound pathway. In another reaction not involving molecular oxygen, the effect of
a series of meta- and para-substituents on cumene hydroperoxide as the oxygen
donor and toluene as the oxygen acceptor was determined (110). The results supported a homolytic mechanism of oxygen-oxygen bond cleavage but not the heterolytic formation of a common iron-oxo intermediate from the various peroxides.
The surprising range of substrates modified by P450 2B4 is also indicated by the
aromatization of a bicyclic steroid analog, 3-oxodecalin-4-ene-10-carboxaldehyde
(113). The products were formate and 3-hydroxy-6,7,8,9-tetrahydronaphthalene,
thus showing that the artificial substrate is a relevant model for the conversion
of androgens to estrogens. Even the number of P450 inhibitors appears to be almost unlimited. For example, the human placental aromatase P450 binds and is
inhibited by a variety of substituted pyridines and other nonsteroidal compounds
(114).
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More recently, we have gained further insight into the details of oxygen activation by site-directed mutagenesis of mammalian 2B4 and 2E1 in which the highly
conserved I helix threonine residue was replaced by alanine (115, 116). The impetus for our studies was the finding by the Ishimura (117) and Sligar (118) laboratories that the analogous mutation in P450cam apparently caused disruption of proton
delivery, thereby interfering with the conversion of dioxygen to the oxenoid species
and, therefore, the oxidation of the substrate, camphor. Replacement of threonine302 by alanine in 2B4 virtually obliterated benzphetamine demethylation and also
caused decreases in cyclohexane hydroxylation and phenylethanol oxidation. In
sharp contrast, the deformylation of cyclohexane carboxaldehyde was increased
approximately tenfold (115, 119). On the basis of these findings and our previous evidence that P450-dependent aldehyde deformylation is supported by added
H2O2, we concluded that the iron-peroxo species, not oxenoid-iron, is the direct
oxygen donor (115). Furthermore, in a study of olefin epoxidation (with cyclohexene, styrene, and the cis- and trans-isomers of 2-butene as substrates) by the T302A
and T303A mutants of P450s 2B4 and 2E1, respectively, we obtained evidence for
hydroperoxo-iron (as well as oxenoid-iron) as an electrophilic oxidant (116). Thus,
our results support the involvement of three functional species produced during the
reduction of molecular oxygen: peroxo-iron, hydroperoxo-iron (or its protonated
version, iron-complexed hydrogen peroxide), and oxo-iron, as shown in Figure 3.
In the past few years, in collaboration with Martin Newcomb and Paul Hollenberg, we have examined hydroxylations of unactivated C-H bonds in hydrocarbons
Figure 3 Versatility in P450 oxygenating species. The iron-oxygen intermediates

in P450 catalysis and their proposed roles as oxidants. Modified from References
120 and 121.
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and related compounds by use of highly reactive radical clocks. These mechanistic probes, including trans-2-methoxy trans-3-phenylmethylcyclopropane and
methylcubane, were chosen to differentiate between cationic and radical species
because for these two kinds of intermediates different structural rearrangements
occur. When these probes were used with several P450s, cationic products were
found, and the small amounts of radical-derived products indicated that radicallike species were very short lived (subpicosecond) (122). A recent review presents
our knowledge of the very complex P450-catalyzed hydroxylation reaction (121).
In summary, in addition to the commonly accepted iron-oxo species, a second
electrophilic oxidant is believed to exist. This scheme also takes into account
computational work by Shaik et al. (123) and Yoshizawa et al. (124) that suggests the iron-oxo species may function in multiple spin states, possibly one that
would involve oxygen insertion as envisioned in the early days of P450 mechanistic research (125, 126), and the other that would, by hydrogen abstraction,
give a radical intermediate and thus resemble the oxygen-rebound pathway (99).
A related long-standing question is whether the thiolate provided by a cysteine
residue as the proximal heme ligand contributes to the chemical reactivity of these
catalysts. Replacement of the active site cysteine-436 by serine has recently been
shown to convert P450 2B4 into an NADPH oxidase with negligible monooxygenase activity (119). Remaining problems of oxygen activation will continue to
be solved, but it is now clearly evident that the occurrence of multiple oxidizing
species contributes to the remarkable versatility of the P450 family of isozymes
in the modification of drugs and other substrates.
Of major importance, some crystal structures of mammalian P450s are now
known. The first, reported in 2000 by Cosme & Johnson (127) and Williams
et al. (128), was that of isoform 2C5 that had been engineered to delete the single
N-terminal transmembrane domain and to mutate a peripheral membrane-binding
site. More recently, Scott et al. (129) have reported the structure of 2B4, which
revealed a large open cleft that extends from the protein surface directly to the
heme iron. Differences between the two structures suggest that defined regions of
these xenobiotic-metabolizing cytochromes may assume a substantial range of energetically accessible conformations. This flexibility is likely to facilitate substrate
access, metabolic versatility, and product egress. The structural and functional data
available suggest that conformational flexibility may be central to the ability of
family 2 cytochromes to bind such a diverse array of xenobiotics. Thus, both the
structural features of the cytochromes and the generation of multiple oxidants with
different properties (120) may contribute to their exceptional diversity in catalysis.
Sixty years have passed since I decided on the branch of science I would pursue. During that time, technological achievements and increasing overlap among
disciplines have made advances possible in fields that were poorly understood, and
I have been fortunate to share in such progress. In addition to the thrill of research
discoveries, I have enjoyed friendships and interactions with students, postdoctoral
associates, and other collaborators. These have included biochemists, pharmacologists, toxicologists, chemists, biophysicists, molecular biologists, and occasionally
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even clinicians interested in the application of basic science to biomedical problems

related to drug metabolism. Regretfully, not all could be adequately recognized in
this brief presentation, and readers are referred to the ever-increasing literature in
the vast P450 field.

The Annual Review of Pharmacology and Toxicology is online at

http://pharmtox.annualreviews.org
LITERATURE CITED
1. Coon MJ. 2002. Enzyme ingenuity in
biological oxidations: a trail leading to
cytochrome P450. J. Biol. Chem. 277:
2835163
2. Coon MJ, Gurin S. 1949. Studies on the
conversion of radioactive leucine to acetoacetate. J. Biol. Chem. 180:115967
3. Coon MJ. 1950. The metabolic fate of the
isopropyl group of leucine. J. Biol. Chem.
187:7182
4. Bachhawat BK, Robinson WG, Coon MJ.
1954. Carbon dioxide fixation in heart extracts by -hydroxyisovaleryl coenzyme
A. J. Am. Chem. Soc. 76:3098
5. del Campillo-Campbell A, Dekker EE,
Coon MJ. 1959. Carboxylation of methylcrotonyl coenzyme A by a purified
enzyme from chicken liver. Biochim. Biophys. Acta 31:29092
6. Coon MJ, Kupiecki FP, Dekker EE,
Schlesinger MJ, del Campillo A. 1959.
The enzymic synthesis of branched-chain
acids. In Ciba Foundation Symposium on
the Biosynthesis of Terpenes and Sterols,
ed. GEW Wolstenholme, M OConnor,
pp. 6272. London: J & A Churchill
7. Dutler H, Coon MJ, Kull A, Vogel H,
Waldvogel G, Prelog V. 1971. Fatty acid
synthetase from pig liver. 1. Isolation of
the enzyme complex and characterization of the component with oxidoreductase activity for alicyclic ketones. Eur. J.
Biochem. 22:20312
8. Ochoa S. 1980. The pursuit of a hobby.
Annu. Rev. Biochem. 49:130
9. Verkade PE, Elzas M, van der Lee J, de
Wolff HH, Verkade-Sandbergen A, van
10.
11.
12.
13.
14.
15.
16.
17.
der Sande D. 1932. Untersuchen u ber den

fettstoffwechsel. Proc. K. Ned. Akad. Wet.
35:25166
Den H. 1958. Biological omega oxidation.
PhD thesis, Univ. Michigan, Ann Arbor
Baptist JN, Gholson RK, Coon MJ. 1963.
Hydrocarbon oxidation by a bacterial enzyme system. I. Products of octane oxidation. Biochim. Biophys. Acta 69:4047
Gholson RK, Baptist JN, Coon MJ. 1963.
Hydrocarbon oxidation by a bacterial enzyme system. II. Cofactor requirements
for octanol formation from octane. Biochemistry 2:115559
Kusunose M, Kusunose E, Coon MJ.
1964. Enzymatic -oxidation of fatty
acids. II. Substrate specificity and other
properties of the enzyme system. J. Biol.
Chem. 239:213539
Peterson JA, Basu D, Coon MJ. 1966. Enzymatic -oxidation. I. Electron carriers
in fatty acid and hydrocarbon hydroxylation. J. Biol. Chem. 241:516263
Peterson JA, Coon MJ. 1968. Enzymatic
-oxidation. III. Purification and properties of rubredoxin, a component of the
-hydroxylation system of Pseudomonas
oleovorans. J. Biol. Chem. 243:329
34
Ueda T, Coon MJ. 1972. Enzymatic oxidation. VII. Reduced diphosphopyridine nucleotide-rubredoxin reductase:
properties and function as an electron
carrier in -hydroxylation. J. Biol. Chem.
247:501016
Ueda T, Lode ET, Coon MJ. 1972. Enzymatic -oxidation. VI. Isolation of
3 Dec 2004
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18.

19.
20.
21.
22.
23.
24.
25.
26.
homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J.

Biol. Chem. 247:210915
McKenna EJ, Coon MJ. 1970. Enzymatic
-oxidation. IV. Purification and properties of the -hydroxylase of Pseudomonas
oleovorans. J. Biol. Chem. 245:388289
Ruettinger RT, Olson ST, Boyer RF,
Coon MJ. 1974. Identification of the hydroxylase of Pseudomonas oleovorans
as a nonheme iron protein requiring phospholipid for catalytic activity. Biochem.
Biophys. Res. Commun. 57:101117
Shanklin J, Achim C, Schmidt H, Fox
GB, Munck E. 1997. Mossbauer studies of alkane omega-hydroxylases: evidence for a diiron cluster in an integralmembrane enzyme. Proc. Natl. Acad. Sci.
USA 94:298186
Lu AYH, Coon MJ. 1968. Role of hemoprotein P450 in fatty acid -hydroxylation
in a soluble enzyme system from liver microsomes. J. Biol. Chem. 243:133132
Lu AYH, Junk KW, Coon MJ. 1969.
Resolution of the cytochrome P-450containing -hydroxylation system of
liver microsomes into three components.
J. Biol. Chem. 244:371421
Strobel HW, Lu AYH, Heidema J, Coon
MJ. 1970. Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J. Biol.
Chem. 245:485154
Lu AYH, Strobel HW, Coon MJ. 1969.
Hydroxylation of benzphetamine and
other drugs by a solubilized form of cytochrome P-450 from liver microsomes:
lipid requirement for drug demethylation.
Biochem. Biophys. Res. Commun. 36:
54551
Kaschnitz RM, Coon MJ. 1975. Drug
and fatty acid hydroxylation by solubilized human liver microsomal cytochrome P-450phospholipid requirement. Biochem. Pharmacol. 24:295
97
Duppel W, Lebeault JM, Coon MJ. 1973.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
19
Properties of a yeast cytochrome P-450containing enzyme system which catalyzes the hydroxylation of fatty acids,
alkanes, and drugs. Eur. J. Biochem. 36:
58392
Ryan KJ, Engel LL. 1957. Hydroxylation
of steroids at carbon 21. J. Biol. Chem.
225:10314
Klingenberg M. 1958. Pigments of rat
liver microsomes. Arch. Biochem. Biophys. 75:37686
Garfinkel D. 1958. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch. Biochem. Biophys. 77:493
509
Omura T, Sato R. 1962. A new cytochrome in liver microsomes. J. Biol.
Chem. 237:PC137576
Omura T, Sato R, Cooper DY, Rosenthal
O, Estabrook RW. 1965. Function of cytochrome P-450 of microsomes. Federation Proc. 24:118189
Lu AYH. 1998. The Bernard B. Brodie
lecture. A journey in cytochrome P450
and drug metabolism research. Drug
Metab. Dispos. 26:116873
Gillette JR, Conney AH, Cosmides GJ,
Estabrook RW, Fouts JR, Mannering GJ,
eds. 1969. Microsomes and Drug Oxidations. New York: Academic. 547 pp.
Coon MJ, Lu AYH. 1969. Fatty acid oxidation in a soluble microsomal enzyme
system containing P-450. See Ref. 33,
pp. 15166
Gunsalus IC, Sligar SG. 1978. Oxygen
reduction by the P450 monooxygenase
systems. Adv. Enzyme Regul. 47:144
Coon MJ, Sligar SG. 2003. Irwin C.
Gunsalus, talented and creative scientist.
Biochem. Biophys. Res. Commun. 312:1
6
Ruckpaul K. 2003. Early years of
cytochrome P450 research in BerlinBuch: its present state and origin of
the biochemical and biophysical conferences. Biochem. Biophys. Res. Commun.
312:6574
3 Dec 2004
20:34

20
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
P1: GCE
COON
38. Maricic S. 1977. Foreward to proceedings of the Scientific Conference on Cytochrome P-450structural aspects (Primosten, Yugoslavia), ed. MJ Coon, IC
Gunsalus, S Maricic. Croat. Chem. Acta
49:163388
39. Conney AH. 2003. Induction of drugmetabolizing enzymes: a path to the discovery of multiple cytochromes P450.
Annu. Rev. Pharmacol. Toxicol. 43:1
30
40. Nebert DW, Heidema JK, Strobel HW,
Coon MJ. 1973. Genetic expression of
aryl hydrocarbon hydroxylase induction.
Genetic specificity resides in the fraction
containing cytochrome P448 and P450. J.
Biol. Chem. 248:763136
41. Lu AYH, Kuntzman R, West S, Conney AH. 1972. Reconstituted liver microsomal enzyme system that hydroxylates
drugs, other foreign compounds, and endogenous substrates. I. Determination of
substrate specificity by the cytochrome P450 and P-448 fractions. Biochem. Biophys. Res. Commun. 42:12006
42. Autor AP, Kaschnitz RM, Heidema JK,
Coon MJ. 1973. Sedimentation and
other properties of the reconstituted
liver microsomal mixed-function oxidase system containing cytochrome P450,
reduced triphosphopyridine nucleotidecytochrome P450 reductase, and phosphatidylcholine. Mol. Pharmacol. 9:93
104
43. Coon MJ, van der Hoeven TA, Kaschnitz
RM, Strobel HW. 1973. Biochemical
studies on cytochrome P-450 solubilized
from liver microsomes: partial purification and mechanism of catalysis. Ann. NY
Acad. Sci. 212:44957
44. van der Hoeven TA, Coon MJ. 1974.
Preparation and properties of partially
purified cytochrome P-450 and reduced nicotinamide adenine dinucleotide
phosphate-cytochrome P-450 reductase
from rabbit liver microsomes. J. Biol.
Chem. 249:630210
45. Haugen DA, van der Hoeven TA, Coon
46.
47.
48.
49.
50.
51.
52.
53.
54.
MJ. 1975. Purified liver microsomal cytochrome P-450. Separation and characterization of multiple forms. J. Biol.
Chem. 250:356770
Haugen DA, Coon MJ. 1976. Properties of electrophoretically homogeneous phenobarbital-inducible and naphthoflavone-inducible forms of liver
microsomal cytochrome P-450. J. Biol.
Chem. 251:792939
Nebert DW, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, et al. 1987. The
P450 gene superfamily: recommended
nomenclature. DNA 6:111
Dean WL, Coon MJ. 1977. Immunochemical studies on two electrophoretically homogeneous forms of rabbit liver microsomal cytochrome P-450: P-450LM2 and
P-450LM4. J. Biol. Chem. 252:325561
Park SS, Persson AV, Coon MJ, Gelboin
HV. 1980. Monoclonal antibodies to rabbit liver cytochrome P-450LM. FEBS Lett.
116:23135
Chiang YL, Coon MJ. 1979. Comparative study of two highly purified forms
of liver microsomal cytochrome P-450:
circular dichroism and other properties.
Arch. Biochem. Biophys. 195:17887
Inouye K, Coon MJ. 1985. Properties of
the tryptophan residue in rabbit liver microsomal cytochrome P-450 isozyme 2
as determined by fluorescence. Biochem.
Haugen DA, Coon MJ, Nebert DW. 1976.
Induction of multiple forms of mouse
liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with naphthoflavone or phenobarbital. J. Biol.
Chem. 251:181727
Zaphiropoulos PG, Folk WR, Coon MJ.
1986. Isolation and characterization of
a novel cytochrome P-450-like pseudogene. Biochem. Biophys. Res. Commun.
134:499505
Haugen DA, Armes LG, Yasunobu
KT, Coon MJ. 1977. Amino-terminal
sequence of phenobarbital-inducible
3 Dec 2004
20:34
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
P1: GCE

55.
56.
57.
58.
59.
60.
61.
62.
cytochrome P-450 from rabbit liver

microsomes: similarity to hydrophobic
aminoterminal segments of preproteins.
96773
Tarr GE, Black SD, Fujita VS, Coon
MJ. 1983. Complete amino acid sequence
and predicted membrane topology of
phenobarbital-induced cytochrome P-450
(isozyme 2) from rabbit liver microsomes.
Proc. Natl. Acad. Sci. USA 80:6552
56
Fujita VS, Black SD, Tarr GE, Koop DR,
Coon MJ. 1984. On the amino acid sequence of cytochrome P-450 isozyme 4
from rabbit liver microsomes. Proc. Natl.
Acad. Sci. USA 81:426064
Vermilion JL, Coon MJ. 1974. Highly
purified detergent-solubilized NADPHcytochrome P-450 reductase from phenobarbital-induced rat liver microsomes.
131522
French JS, Coon MJ. 1979. Properties of
NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes. Arch.
Biochem. Biophys. 195:56577
Black SD, Coon MJ. 1982. Structural
features of liver microsomal NADPHcytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region. J. Biol. Chem. 257:5929
38
Wang M, Roberts DL, Paschke R, Shea
TM, Masters BSS, Kim J-JP. 1997.
Three-dimensional structure of NADPHcytochrome P450 reductase: prototype
for FMN- and FAD-containing enzymes.
French JS, Guengerich FP, Coon MJ.
1980. Interactions of cytochrome P450, NADPH-cytochrome P-450 reductase, phospholipids, and substrate in the
reconstituted liver microsomal enzyme
system. J. Biol. Chem. 255:411219
Ruckpaul K, Rein H, Ballou DP, Coon MJ.
1980. Analysis of interactions among purified components of the liver microsomal
63.
64.
65.
66.
67.
68.
69.
70.
21
cytochrome P-450-containing monooxygenase system by second derivative spectroscopy. Biochim. Biophys. Acta 626:41
56
Vatsis KP, Theoharides AD, Kupfer D,
Coon MJ. 1982. Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450:
participation of cytochrome b5. J. Biol.
Chem. 257:1122129
Morgan ET, Coon MJ. 1984. Effects
of cytochrome b5 on cytochrome P450-catalyzed reactions. Studies with
manganese-substituted cytochrome b5.
Drug Metab. Dispos. 12:35864
Pompon D, Coon MJ. 1984. On the mechanism of action of cytochrome P-450.
Oxidation and reduction of the ferrous
dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5. J. Biol.
Chem. 259:1537785
Gorsky LD, Coon MJ. 1986. Effects
of conditions for reconstitution with cytochrome b5 on the formation of products
in cytochrome P-450-catalyzed reactions.
Koop DR, Coon MJ. 1979. Purification
and properties of P-450LM3b, a constitutive form of cytochrome P-450, from rabbit liver microsomes. Biochem. Biophys.
Res. Commun. 91:107581
Koop DR, Persson AV, Coon MJ. 1981.
Properties of electrophoretically homogeneous, constitutive forms of liver microsomal cytochrome P-450. J. Biol. Chem.
256:1070411
Koop DR, Coon MJ. 1984. Purification of liver microsomal cytochrome P450 isozymes 3a and 6 from imidazoletreated rabbits. Evidence for the identity
of isozyme 3a with the form obtained
by ethanol treatment. Mol. Pharmacol.
25:494501
Ishida H, Noshiro M, Okuda K, Coon MJ.
1992. Purification and characterization
of 7-hydroxy-4-cholesten-3-one 12hydroxylase. J. Biol. Chem. 267:21319
23
3 Dec 2004
20:34

22
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
P1: GCE
COON
71. Koop DR, Morgan ET, Tarr GE, Coon MJ.

1982. Purification and characterization of
a unique isozyme of cytochrome P-450
from liver microsomes of ethanol-treated
rabbits. J. Biol. Chem. 257:847280
72. Morgan ET, Koop DR, Coon MJ. 1982.
Catalytic activity of cytochrome P-450
isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. J. Biol.
Chem. 257:1395157
73. Lieber CS, DeCarli LM. 1968. Ethanol
oxidation by hepatic microsomes: adaptive increase after ethanol feeding. Science 162:91718
74. Koop DR, Coon MJ. 1986. Ethanol oxidation and toxicity: role of alcohol P450 oxygenase. Alcohol. Clin. Exp. Res.
10:44S49
75. Morgan ET, Koop DR, Coon MJ. 1983.
Comparison of six rabbit liver cytochrome
P-450 isozymes in formation of a reactive
metabolite of acetaminophen. Biochem.
76. Coon MJ, Roberts ES, Vaz ADN. 1991.
Predominant role of alcohol-inducible P450s in oxidative damage. In Oxidative
Damage and Repair: Chemical, Biological and Medical Aspects, ed. KJA Davies,
pp. 72631. New York: Pergamon
77. Yang CS, Tu YY, Koop DR, Coon MJ.
1985. Metabolism of nitrosamines by
purified rabbit liver cytochrome P-450
isozymes. Cancer Res. 45:114045
78. Ingelman-Sundberg M, Johansson I, Pentilla KE, Glaumann H, Lindros KO. 1988.
Centrilobular expression of ethanolinducible cytochrome P-450 (IIE1) in rat
liver. Biochem. Biophys. Res. Commun.
157:5560
79. Vaz ADN, Roberts ES, Coon MJ. 1990.
Reductive -scission of the hydroperoxides of fatty acids and xenobiotics: role
of alcohol-inducible cytochrome P-450.
80. Khani SC, Porter TD, Fujita VS, Coon
MJ. 1988. Organization and differential expression of two highly similar
genes in the rabbit alcohol-inducible cy-
81.
82.
83.
84.
85.
86.
87.
88.
89.
tochrome P-450 subfamily. J. Biol. Chem.

263:717075
Porter TD, Khani SC, Coon MJ. 1989.
Induction and tissue-specific expression
of rabbit cytochrome P450IIE1 and IIE2
genes. Mol. Pharmacol. 36:6165
Peng H-M, Coon MJ. 1998. Regulation
of rabbit cytochrome P450 2E1 expression in HepG2 cells by insulin and thyroid
hormone. Mol. Pharmacol. 54:74047
Peng H-M, Coon MJ. 2000. Promoter
function and the role of cytokines in
the transcriptional regulation of rabbit
CYP2E1 and CYP2E2. Arch. Biochem.
Biophys. 382:12937
Porter TD. 2004. Jud Coon: 35 years of
P450 research. A synopsis of P450 history. Drug Metab. Dispos. 32:16
Barnes HJ, Arlotto MP, Waterman MR.
1991. Expression and enzymatic activity of recombinant cytochrome P450 17
alpha-hydroxylase in Escherichia coli.
Larson JR, Coon MJ, Porter TD. 1991.
Purification and properties of a shortened
form of cytochrome P-450 2E1: deletion
of the NH2-terminal membrane-insertion
signal peptide does not alter the catalytic activites. Proc. Natl. Acad. Sci. USA
88:914145
Pernecky SJ, Olken NM, Bestervelt LL,
Coon MJ. 1995. Subcellular localization,
aggregation state, and catalytic activity of
microsomal P450 cytochromes modified
in the NH2-terminal region and expressed
in Escherichia coli. Arch. Biochem. Biophys. 318:44656
Ding X, Coon MJ. 1988. Purification and
characterization of two unique forms of
cytochrome P-450 from rabbit nasal microsomes. Biochemistry 27:833037
Peng H-M, Ding X, Coon MJ. 1993.
Isolation and heterologous expression of
cloned cDNAs for two rabbit nasal microsomal proteins, CYP 2A10 and CYP
2A11, that are related to nasal cytochrome
P450 form a. J. Biol. Chem. 268:17253
60
3 Dec 2004
20:34
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
P1: GCE


90. Ding X, Porter TD, Peng H-M, Coon
MJ. 1991. cDNA and derived amino acid
sequence of rabbit nasal cytochrome P450NMb (P-450IIG1), a unique isozyme
possibly involved in olfaction. Arch.
91. Ding X, Kaminsky LS. 2003. Human extrahepatic cytochromes P450:
function in xenobiotic metabolism and
tissue-selective chemical toxicity in the
respiratory and gastrointestinal tracts.
73
92. Lu AYH, West SB. 1980. Multiplicity of
mammalian microsomal cytochrome P450s. Pharmacol. Rev. 31:27795
93. Nelson DR. 1999. Cytochrome P450
and the individuality of species. Arch.
94. Vermilion JL, Ballou DP, Massey V, Coon
MJ. 1981. Separate roles for FMN and
FAD in catalysis by liver microsomal
NADPH-cytochrome P-450 reductase. J.
Biol. Chem. 256:26677
95. Oprian DD, Coon MJ. 1982. Oxidationreduction states of FMN and FAD
in NADPH-cytochrome P-450 reductase
during reduction by NADPH. J. Biol.
Chem. 257:893544
96. Coon MJ, Ding X, Pernecky SJ, Vaz ADN.
1992. Cytochrome P450: progress and
predictions. FASEB J. 6:66973
97. Olson JS, Ballou DP, Palmer G, Massey
V. 1974. The mechanism of action of
xanthine oxidase. 1974. J. Biol. Chem.
249:436382
98. White RE, Coon MJ. 1980. Oxygen activation by cytochrome P-450. Annu. Rev.
Biochem. 49:31556
99. Groves JT, McClusky GA, White RE,
Coon MJ. 1978. Aliphatic hydroxylation
by highly purified liver microsomal cytochrome P-450. Evidence for a carbon
radical intermediate. Biochem. Biophys.
100. Vaz ADN, Roberts ES, Coon MJ. 1990.
Reductive -scission of the hydroperoxides of fatty acids and xenobiotics: role
101.
102.
103.
104.
105.
106.
107.
108.
23
of alcohol-inducible cytochrome P-450.

503
Roberts ES, Vaz ADN, Coon MJ. 1991.
Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde
metabolism: deformylation with olefin
formation. Proc. Natl. Acad. Sci. USA
88:896366
Akhtar M, Calder MR, Corina DL, Wright
JN. 1982. Mechanistic studies on C-19
demethylation in oestrogen biosynthesis.
Biochem. J. 201:56980
Vatsis KP, Coon MJ. 2002. Ipsosubstitution by cytochrome P450 with
conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for
hydroperoxo-iron as the active oxygen
species. Arch. Biochem. Biophys. 397:
11929
Guengerich FP. 2001. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.
Chem. Res. Toxicol. 14:61150
Fasco MJ, Vatsis KP, Kaminsky LS, Coon
MJ. 1978. Regioselective and stereoselective hydroxylation of R and S warfarin
by different forms of purified cytochrome
P-450 from rabbit liver. J. Biol. Chem.
253:781320
Deutsch J, Vatsis KP, Coon MJ, Leutz JC,
Gelboin HV. 1979. Catalytic activity and
stereoselectivity of purified forms of rabbit liver microsomal cytochrome P-450
in the oxygenation of the () and (+)
enantiomers of trans-7,8-dihydroxy-7,8dihydrobenzo[a]pyrene. Mol. Pharmacol.
16:101118
Gorsky LD, Koop DR, Coon MJ. 1984.
On the stoichiometry of the oxidase
and monooxygenase reactions catalyzed
by liver microsomal cytochrome P-450.
Products of oxygen reduction. J. Biol.
Chem. 259:681217
Nordblom GD, White RE, Coon MJ.
1976. Studies on hydroperoxidedependent substrate hydroxylation by
purified liver microsomal cytochrome
3 Dec 2004
20:34
24
109.

110.
111.
112.
113.
114.
115.
116.
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
P1: GCE
COON
P-450. Arch. Biochem. Biophys. 175:524
33
Ellin A, Orrenius S. 1975. Hydroperoxide-supported cytochrome P-450linked fatty acid hydroxylation in liver
microsomes. FEBS Lett. 50:37881
Blake RC II, Coon MJ. 1981. On the
mechanism of action of cytochrome P450. Evaluation of homolytic and heterolytic mechanisms of oxygen-oxygen
bond cleavage during substrate hydroxylation by peroxides. J. Biol. Chem.
256:1212733
Blake RC II, Coon MJ. 1989. On the
mechanism of action of cytochrome P450. Spectral intermediates in the reaction
with iodosobenzene and its derivatives. J.
Biol. Chem. 264:3694701
Osawa Y, Coon MJ. 1989. Selective
mechanism-based inactivation of the major phenobarbital-inducible cytochrome
P-450 from rabbit liver by phencyclidine
and its oxidation product, the iminium
compound. Drug Metab. Dispos. 17:713
Vaz ADN, Kessell KJ, Coon MJ. 1994.
Aromatization of a bicyclic steroid
analog, 3-oxodecalin-4-ene-10-carboxaldehyde, by liver microsomal cytochrome P450 2B4. Biochemistry 33:
1365161
Vaz ADN, Coon MJ, Peegel H, Menon
KMJ. 1992. Substituted pyridines: nonsteroidal inhibitors of human placental aromatase cytochrome P450. Drug
Vaz ADN, Pernecky SJ, Raner GM, Coon
MJ. 1996. Peroxo-iron and oxenoid-iron
species as alternative oxygenating agents
in cytochrome P450-catalyzed reactions:
switching by T302A mutagenesis of cytochrome P450 2B4. Proc. Natl. Acad.
Sci. USA 93:464448
Vaz ADN, McGinnity DF, Coon MJ.
1998. Epoxidation of olefins by cytochrome P450: evidence from sitespecific mutagenesis for hydroperoxoiron as an electrophilic oxidant. Proc.
Natl. Acad. Sci. USA 95:355560
117. Imai M, Shimada H, Watanabe Y,

Matsushima-Hibiya Y, Makino R, et al.
1989. Uncoupling of the cytochrome P450cam monooxygenase reaction by a
single mutation, threonine-252 to alanine or valine: a possible role of the
hydroxy amino acid in oxygen activation. Proc. Natl. Acad. Sci. USA 86:7823
27
118. Martinis SA, Atkins WM, Stayton PS,
Sligar SG. 1989. A conserved residue of
cytochrome P-450 is involved in hemeoxygen stability and activation. J. Am.
Chem. Soc. 111:925253
119. Vatsis KP, Peng H-M, Coon MJ. 2002.
Replacement of active-site cysteine-436
by serine converts cytochrome P450 2B4
into an NADPH oxidase with negligible monooxygenase activity. J. Inorg.
Biochem. 91:54243
120. Coon MJ, Vaz ADN, McGinnity DF, Peng
H-M. 1998. Multiple activated oxygen
species in P450 catalysis: contributions
to specificity in drug metabolism. Drug
121. Newcomb M, Hollenberg PF, Coon MJ.
2003. Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations. Arch. Biochem. Biophys. 409:72
79
122. Newcomb M, Shen R, Choi S-Y, Hollenberg PF, Vaz ADN, Coon MJ. 2000. Cytochrome P450-catalyzed hydroxylation
of mechanistic probes that distinguish between radicals and cations. Evidence for
cationic but not for radical intermediates.
J. Am. Chem. Soc. 122:267786
123. Shaik S, Filatov M, Schroder D, Schwarz
H. 1998. Electronic structure makes a
difference: cytochrome P450 mediated
hydroxylations of hydrocarbons as a twostate reactivity paradigm. Chem. Eur. J.
4:19399
124. Yoshizawa K, Kamachi T, Shiota Y.
2001. A theoretical study of the dynamic
behavior of alkane hydroxylation by a
compound I model of cytochrome P450.
J. Am. Chem. Soc. 123:980616
3 Dec 2004
20:34
AR
AR232-PA45-01.tex
AR232-PA45-01.sgm
LaTeX2e(2002/01/18)
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125. Ullrich V, Staudinger H. 1968. Aktiviering von sauerstoff in modellsystemen. In
Nineteenth Colloquium des Gesellschaft
fur Biologische Chemie: Biochemie des
Sauerstoffs, ed. B Hass, H Staudinger,
pp. 22948. Berlin: Springer
126. Hamilton GA. 1974. Chemical models and mechanisms for oxygenases. In
Molecular Mechanisms of Oxygen Activation, ed. O Hayaishi, pp. 40551. New
York: Academic
127. Cosme J, Johnson EF. 2000. Engineering microsomal cytochrome P450 2C5 to
be a soluble, monomeric enzyme. Muta-
25
tions that alter aggregation, phospholipids

dependence of catalysis and membrane
binding. J. Biol. Chem. 275:254553
128. Williams PA, Cosme J, Sridhar V, Johnson
EF, McRee DE. Mammalian microsomal
cytochrome P450 monooxygenase: structural adaptations for membrane binding
and functional diversity. Mol. Cell 5:121
31
129. Scott EE, He YA, Wester MR, White MA,
Chin CC, et al. 2003. An open conformation of mammalian cytochrome P450 2B4
at 1.6 A resolution. Proc. Natl. Acad. Sci.
USA 100:13196201
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Copyright

CYTOCHROME P450 ACTIVATION OF ARYLAMINES

AND HETEROCYCLIC AMINES
Donghak Kim and F. Peter Guengerich
Department of Biochemistry and Center in Molecular Toxicology,
Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146;
email: f.guengerich@vanderbilt.edu
Key Words food pyrolysis, N-hydroxy arylamines, N-acetyl transferase, DNA

adducts, mutations
Abstract Arylamines and heterocyclic arylamines (HAAs) are of particular interest because of demonstrated carcinogenicity in animals and humans and the broad
exposure to many of these compounds. The activation of these, and also some arylamine
drugs, involves N-hydroxylation, usually by cytochrome P450 (P450). P450 1A2 plays
a prominent role in these reactions. However, P450 1A1 and 1B1 and other P450s are
also important in humans as well as experimental animals. Some arylamines (including
drugs) are N-hydroxylated predominantly by P450s other than those in Family 1. Other
oxygenases can also have roles. An important issue is extrapolation between species
in predicting cancer risks, as shown by the low rates of HAA activation by rat P450
1A2 and low levels of P450 1A2 expression in some nonhuman primates.
INTRODUCTION
Many arylamines, e.g., 2-naphthylamine (2-NA), benzidine, and 4-aminobiphenyl
(4-ABP), are of industrial importance because of their use as intermediates in
the synthesis of azo dyes, antioxidants in rubber products, and other commercial
materials (1, 2). Epidemiological observations of the toxicity of arylamines were
first reported in aniline dye factories by Rehn in 1895, with the report that German
and Swiss workers suffered urinary bladder tumors (2, 3). A major toxicological
issue is reaction with DNA and induction of carcinomas, primarily in the urinary
bladder, liver, or other tissues in humans and experimental animals (1, 2, 46).
In 1939, the Swedish chemist Widmark demonstrated that extracts of fried horse
meat induced cancer when applied to mouse skin (7, 8). Sugimura and his associates
investigated the smoke produced by broiling fish and meat; they demonstrated that
the smoke condensate and charred surfaces of broiled fish and meat were highly
mutagenic in Salmonella typhimurium test systems (Table 1) (911). Subsequently,
the heterocyclic arylamine (HAA) products formed as a consequence of pyrolysis
of amino acids or protein-containing foods were isolated, their structures were
0362-1642/05/0210-0027$14.00
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TABLE 1
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Mutagenicity of HAAs in S. typhimurium tester strainsa

Revertants/g HAA
HAA
TA98
2-amino-3-methylimidazo[4,5-f]quinoline (IQ)
433,000
7000
2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ)
661,000
30,000
75,000
1500
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)
145,000
14,000
2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx)
183,000
8000
2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx)
163,000
9900

2-amino-3-methylimidazo[4,5-f]quinoline (IQx)
TA100
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)
1800
120
3-amino-1,4-dimethyl-5H-pyrido[4,3,-b]-indole (Trp-P-1)
39,000
1700
104,000
1800
49,000
3200
1900
1200
41
23
56,800
3-amino-1-methyl-5H-pyrido[4,3,-b]-indole (Trp-P-2)
2-amino-6-methyldipyrido[1,2-a:3 ,2 -d]imidazole (Glu-P-1)
2-aminodipyrido[1,2-a:3 ,2 -d]imidazole (Glu-P-2)

2-amino-5-phenylpyridine (Phe-P-1)
2-amino-9H-pyrido[2,3-b]indole (AC)
a
Reference 11.
determined, and their biological effects were examined, specifically mutagenicity

and carcinogenicity in animals (1216).
The formation of HAAs is the result of a Maillard reaction (8, 1720). This
reaction occurs when amino acids (proteins) and reducing sugars (carbohydrates)
are heated together. More than 20 HAAs have been identified (Figure 1) (8, 14,
2123). HAAs were also identified in cigarette smoke condensate and shown to
be genotoxic (24, 25). Several antimicrobial drugs contain arylamine moieties,
e.g., sulfamethoxazole (SMX) and dapsone. Potential roles of N-hydroxy arylamine metabolites in mediating the idiosyncratic reactions and the importance
of oxidative metabolism in their toxicities have been investigated (26, 27). Arylamines are also formed in commercial hair dyes, and their contributions to an increased risk of bladder, breast, colon, and lymphatic cancer have been investigated
(2830).
CHEMISTRY OF BIOACTIVATION
Arylamines and HAAs require metabolic activation to be mutagenic or carcinogenic (31). The major metabolic process is N-oxidation, which is mediated primarily by cytochrome P450 (P450) enzymes but also by flavin-containing monooxygenases (FMOs) and peroxidases (3139). The resulting N-hydroxylamine
products can be further activated to produce highly reactive ester derivatives that
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P450 AND HETEROCYCLIC AROMATIC AMINES
Figure 1 Some of the HAAs found in food.

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bind covalently to DNA. At least four enzyme systems are known to be involved in
this secondary activation step in mammals: N-acetyltransferase (NAT), sulfotransferase, prolyl tRNA synthetase, and kinases, yielding reactive N-acetoxy, N-sulfonyloxy, N-prolyloxy, and N-phosphatyl esters, respectively (4043). The N-hydroxy HAAs can react directly with DNA (32), but the reaction is facilitated when
reactive ester derivatives undergo heterocyclic cleavage to yield reactive aryl nitrenium ion species, which preferentially react to form DNA adducts (Figure 2).
NAT-catalyzed acetylation of N-hydroxy HAAs and arylamines enhances genotoxic activity and DNA adduct levels through formation of reactive N-acetoxyl
esters (4448). In a similar way, the sulfur esters formed by the action of sulfotransferases are unstable and react (42, 49). The role of the sulfotransferases
has been given less attention than NAT in human epidemiology studies. Even less
information is available about the in vivo roles of the prolyloxy and phosphatyl
esters.
Arylamines and HAAs yield adducts primarily with guanine (50, 51), reacting
at the N2 and C8 atoms. Wild et al. (52) showed that the photoactivated azide
derivatives of IQ, MeIQx, and PhIP bind to DNA to form the same adducts as
the N-acetoxy species, indicating that the nitrenium ion may be a common intermediate for both reactive intermediates. Mechanisms for the reaction at the C8
atom have been less clear (53). A direct reaction is possible (54), and a stepwise
mechanism via an N7-guanyl intermediate has also been proposed (55, 56). The
latter has an advantage of also explaining several accompanying products (e.g.,
8-oxo-7,8-dihydroguanine, depurination, imidazole ring opening) (56).
Several approaches to syntheses of DNA adducts of these amines have been
reported. HAA-DNA adducts (including IQ, MeIQ, MeIQx, 4,8-DiMeIQx, PhIP,
Glu-P-1, and Trp-P-2) have been synthesized and characterized spectroscopically
(5763). Oligonucleotides containing guanyl-C8 2-aminofluorene (2-AF) (and Nacetyl 2-AF) derivatives have been synthesized, and structures have been determined using NMR spectroscopy and mutagenic properties have been examined
in cell-based systems (Figure 3). PhIP has been site-specifically incorporated into
oligonucleotides by a biomimetic approach in which N-acetoxy-PhIP was reacted
with oligonucleotides containing a single guanine (64). In a nonbiomimetic approach, the Rizzo group has reported the synthesis of C8-guanyl adduct of IQ
through palladium-catalyzed N-arylation of a protected 8-bromo-2 -dG derivative
with IQ as the key reaction (65).
SYSTEMS FOR ANALYSIS OF MUTATION AND CANCER

Arylamines have long been known to be mutagenic following metabolic activation (66), and an early study showed the mutagenicity of hair dyes (67). HAAs
have shown strong mutagenicity in S. typhimurium strains since their discovery
(9, 10, 68). HAAs preferentially induce the frameshift mutations in CG repeat of
the hisD+ gene, as opposed to causing base pair mutations (69, 70). This type of
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Figure 2 General pathway for activation of arylamines and HAAs, as shown for IQ.
31
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Figure 3 Structures of some carcinogenic arylamines.
mutation response is also found in other bacterial genes, such as lacZ, lacZ, and
lacI of Escherichia coli (71, 72). An E. coli lacZ reversion mutation assay has
also been applied to study HAA genotoxicity (7376); a tester stain carrying a
(-GC) copy of lacZ gene can regain functional lacZ activity following induction
of frameshifts by HAAs (76, 77). Systems have also been developed that incorporate the heterologous expression of P450s and NADPH-P450 reductase (74).
This system allows the detection of HAA mutagenicity by recombinant human
P450 without a need for rat liver fractions. These bacteria have also been genetically engineered to express S. typhimurium NAT, and the DNA nucleotide excision
repair system has been inactivated (UvrABC) to improve the sensitivity (74, 77,
78). S. typhimurium tester strains have also been used to express human P450s
that activate arylamines and HAAs (73, 79, 80). The E. coli strains overexpressing P450s and NAT have been used to characterize P450 1A2 allelic and random
variants (8184). Another use of this genotoxicity system has been the screening
and characterization of P450 inhibitors. For instance, a P450 1B1based system
was used to characterize the potent inhibition of the enzyme by tetramethoxystilbene (85) and a P450 1A2based system was sensitive to the drug oltipraz (86).
Other bacterial systems have utilized the SOS response in S. typhimurium NM2009
as a measure of DNA damage (47). This strain contains a plasmid-based umuDC
gene linked to a lacZ reporter gene and is activated by induction of the SOS pathway (47). Sensitivity to arylamines and HAAs was also improved by incorporating
plasmids coding for P450 enzymes, NADPH-P450 reductase, and NAT (48).
DNA adducts are considered biomarkers of potential mutagenesis and carcinogenesis (87, 88), and HAAs are thought to induce mutagenesis by producing
mutations in oncogenes and tumor suppressor genes in experimental animals (23,
89). Although base pair mutations are not a major feature of HAAs in bacterial test
systems, one was dominantly induced by IQ in the p53 gene in rat Zymbals gland
tumors and monkey hepatocellular carcinoma (89, 90). Also, a C T transversion
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in rasH was predominantly observed in mouse forestomach and rat Zymbals gland
tumors induced by MeIQ. However, PhIP was reported to induce a number of characteristic one-base deletions in the lacI gene of the colon mucosa of transgenic
mice (91).
The major analytical methods used for the detection and quantification of arylamine and HAA adducts are the 32P-postlabeling assay and mass spectrometry.
32
P-postlabeling assays use polynucleotide kinase to label the adducted or nonadducted 3 -nucleotides with [ -32P]ATP after digestion of DNA to mononucleotides
(92). The radiolabeled 3 , 5 -bisphosphonucleotide adducts can be separated by
two-dimensional thin-layer chromatography (TLC) or high-pressure liquid chromatography (HPLC) (93). A combination of HPLC and electrospray ionizationtandem mass spectrometry can provide structural information on adducts, and the
incorporation of stable isotopically labeled internal standards in assays provides
precise and accurate quantification of the DNA adducts (93).
N-Hydroxylamines and other metabolites of arylamines and HAAs are mainly
identified and measured with combinations of HPLC, using [3H]- or [14C]radiolabeled substrates, and mass spectrometry (36, 37, 94, 95). Care is necessary because of the instability of these oxidized amine species. The in vitro
N-hydroxylation of arylamines and HAAs can be measured colorimetrically by a
modification of a Fe3+-reduction method using 4,7-diphenyl-1,10-phenanthroline
(83, 84, 96). Owing to the inherent instability of aryl N-hydroxylamines and the
large numbers of assays required in enzyme kinetic studies (83), this method can
be used quite sensitively and routinely.
Many cancer studies on arylamines have been done following the initial epidemiological findings in workers (2, 3), beginning with the induction of urothelial
tumors in dogs with 2-NA by Hueper (97).
The carcinogenicity of HAAs has been intensively studied in rodent models.
HAAs induce tumors at multiple organs in rats and mice, including liver, lung,
colon, small and large intestine, forestomach, the hematopoietic system, prostate,
mammary gland, Zymbals gland, clitoral gland, oral cavity, and urinary bladder
(20, 98100).
ACTIVATION BY P450
Studies of arylamine oxidation go back to the 1940s, with early work on azo dyes by
the Millers (101). Several lines of evidence implicated the N-hydroxyl derivatives
of the arylamines as being responsible for carcinogenic activity, as well as for the
methemoglobenemia induced by some drugs (2, 31). N-Hydroxylation was first
demonstrated with the acetamide derivative of 2-AF (102). Subsequently, P450 was
demonstrated to be involved in this reaction (103), and further studies extended
the work to unsubstituted arylamines (31, 96).
In early work on the multiplicity of P450s, several lines of investigation had suggested the role of Ah locus-linked P450 enzymes (i.e., now recognized as Family 1)
in the N-hydroxylation of 2-acetylaminofluorene (AAF) (104, 105). Subsequently,
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these P450s were recognized as being involved in the N-hydroxylation of this substrate and several arylamines in various species (32, 33, 106). This is generally
the case, with many arylamines preferentially N-hydroxylated by rat P450 1A2
and, to a lesser extent, by P450 1A1 (36). However, 4-ABP is N-hydroxylated by
rat P450 proteins in the order 1A2 > 1A1 > 2A6 > 2C11 > 2B1 (36). In the
same study, the order for N-hydroxylation of 4,4 -methylene-bis(2-chloroaniline)
(MOCA) was 2B1 > 2B2 > 1A2 > 2A6 > 1A1.
The human P450 enzymes involved in the metabolism of arylamines, HAAs,
and other chemical carcinogens have long been a subject of interest. Some efforts
had been made at analysis with early preparations of human P450s (107). A key
development was the purification of the human P450 involved in phenacetin Odeethylation, now recognized as P450 1A2 (108). Analysis of the animal models
and subsequent correlations of hepatic expression levels with the N-hydroxylation
of 4-ABP (36) led to the view that this enzyme, then known as P450PA, has a major role in the N-hydroxylation of many arylamines and HAAs. Further evidence
followed, with the demonstration that the same enzyme is involved in caffeine
N3-demethylation (37) and that many HAA activations can be attributed to this enzyme (109). Phenacetin metabolism had been studied in humans in vivo (110), and
the characterization of human P450 1A2 (108) led to insight into the inducibility
of P450 1A2 in humans. However, phenacetin can no longer be used as a human
drug because of concerns about its carcinogenicity, and the demonstration of caffeine N3-demethylation led to the use of caffeine as a noninvasive in vivo probe
(37, 111).
The roles of human P450s in the bioactivation of arylamines and HAAs have
been considerably documented (24, 37, 109, 112116). P450s 1A1 and 1A2 have
been generally recognized to be the major forms involved in the bioactivation of
arylamines and HAAs in human liver and lung microsomes (109, 113, 116). A representative study with HAAs is presented in Table 2. The findings with P450 1A2
have been confirmed in vivo in human studies, at least with PhIP and MeIQx. The
P450 1A2selective inhibitor furafylline blocked most of the in vivo elimination
in studies in which the human volunteers consumed burned meat (117). Another
P450 Family 1 member, P450 1B1, has been also shown to be an important enzyme
involved in the activation of HAAs and has been considered regarding mechanisms
of development of human cancers (Table 2) (118, 119). It should be emphasized
that some of the P450s (other than Family 1) do have measurable activity with some
of the arylamine and HAA substrates. MOCA N-hydroxylation, in contrast with
other arylamines, was shown to be preferentially catalyzed by P450 3A4 in human
liver (114). Kitada & Kamataki (120) have shown that P450 3A7 can activate some
HAAs to mutagens in human fetal liver, where P450 1A2 is not expressed.
Although the early epidemiology linked P450 2D6 with lung cancer incidence
(121), subsequent efforts to provide a basis were not fruitful. We have been unable
to find any carcinogens that are preferentially activated by P450 2D6, including
arylamines, HAAs, and crude cigarette smoke fractions (112, 122).
We have treated P450 1A2 (and other P450s) only in terms of the wild-type
(or more correctly, the predominant) allele thus far. The possibility exists that
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TABLE 2 Activation of HAAs and arylamines by recombinant P450 in an

S. typhimuriumbased genotoxicity systema
Concentration,
Mb
HAA
Glu-P-1

PhIP
1.5
220
Activity (umu, units/min/nmol P450)

1A1
1A2
1B1
2C9
2D6
2E1
3A4
27
91
10
13
24
MeIQx
0.3
16
442
MeIQ
0.05
24
179
IQ
0.5
10
214
Trp-P-1
536
321
232
179
Trp-P-2
578
39
138
555
2-AA
0.1
90
374
50
23
2-AF
12.5
46
676
22
13
25
Reference 48.
Lowest concentration used in assay. See original reference for more results (48).
some individuals will have variants that provide unusual catalytic properties. For
instance, we have utilized screens involving the activation of MeIQ to a genotoxic
N-hydroxylamine to identify P450 1A2 mutants with high activity in laboratorygenerated random libraries (81, 83). Some of these variants have activities (Nhydroxylation) 12-fold higher than the wild type. To our knowledge, none of these
has been reported in the population. We have expressed the known allelic variants
and found catalytic efficiencies (kcat/Km) for N-hydroxylation of several HAAs
within a threefold range, although one P450 1A2 variant failed to incorporate
heme and was inactive (84).
Although the discussion is mainly about the activation of arylamines and HAAs,
we should also emphasize that P450s are involved in detoxication of the same
compounds by other routes (36, 123). This aspect can be important, as shown
in the classic Richardson experiment (124) in which enzyme induction by 3methylcholanthrene lowered the tumorigenicity of an aminoazobenzene compound
to rats.
A study with P450 1A2/ mice indicated that P450 1A2 plays an important
role in DNA adduct formation with PhIP and IQ in vivo (125). Differences owing
to the absence/presence of P450 1A2 were seen in liver, kidney, and colon but
not in mammary glands. However, a neonatal bioassay study with P450 1A2/
mice suggests that an unknown pathway, unrelated to P450 1A2, appears to be
responsible for the carcinogenesis of PhIP (126).
Interspecies differences in metabolism of HAAs by rat and human P450 1A2
were found in the metabolism of MeIQx and PhIP (127, 128). Although rat and
human P450 1A2 have 75% amino acid sequence identity (129), relatively high
levels of P450 1A2 expression in human liver and catalytic activities for HAA
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N-hydroxylation compared to the rat P450 1A2 were observed (127, 128). Important differences between human and rat P450s 1A2 were also found in the C8- and
N-oxidation of MeIQx (130, 131). These suggest that the interspecies differences
in P450 enzyme expression and catalytic activities may be significant and must be
carefully considered when assessing human health risk.
The carcinogenicity of IQ, MeIQ, and PhIP was examined in cynomologous
monkeys, with up to seven years of administration (132134). IQ and PhIP were
potent liver carcinogens. IQ and PhIP formed high levels of DNA adducts in a
number of organs, particularly the liver, kidney, and heart. However, low mutagenic and carcinogenic activation of MeIQx was observed in this species. The
poor activation of MeIQx was explained by the lack of constitutive expression
of P450 1A2 and an inability of other P450s to hydroxylate this quinoxaline
(133).
Arylamines can also be N-hydroxylated by FMO and peroxidases (32, 33, 135,
136). These reports, along with those demonstrating DNA adduct formation by
these routes, suggested that the formation of a common DNA-reactive species
could be generated by alternate pathways and these pathways could be considered
to contribute to the burden of DNA adducts found in extrahepatic tissues. This
appears to be the case because P450 1A2 is not expressed in extrahepatic tissues,
and some N-hydroxy HAAs are too unstable to be transported from the liver to
distant sites (3335, 136, 137), although contributions of P450s such as 1A1 and
1B1 may also be an issue.
OTHER BIOCHEMICAL CONSIDERATIONS

The mechanisms of catalysis by P450 are considered elsewhere, including Nhydroxylation (138). N-Oxygenation is considered an inherent part of the repertoire of P450 chemistry available, even in cases where N-dealkylation is preferred
(39). A mechanism involving one-electron oxidation of the amine and subsequent
homolytic collapse of the intermediated pair is attractive in that there is a basis
with accepted mechanisms for other reactions with amines (e.g., N-dealkylation)
(138, 139). A problem with this hypothesis is a lack of correlation in the Hammett
plots, i.e., limited effect of change in rates owing to the presence of electronwithdrawing groups (39). An alternative is a further one-electron transfer within
the intermediate to yield an [Ar-N+/FeO] species that recombines (31, 33, 39,
138).
The N-hydroxy products of some HAAs have been observed to further oxidize
and produce the nitroso compounds, as judged by autocatalytic NADPH oxidation,
reduction cycling, and direct identification of the species (140) (Figure 4). This reaction cycling with human (and also rat and rabbit) P450 1A2 seems to be selective
among the HAA substrates and may contribute to toxicity, either through covalent
binding to proteins and DNA or possibly oxygen toxicity owing to depletion of
NADPH (140). The nitroso derivatives of HAAs react with DNA and protein (26,
43, 140).
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Figure 4 Cycling of a HAA with the hydroxylamine and nitroso

derivatives (140).
Peroxidases generate nitro compounds from HAAs as well as N-hydroxy products (141, 142). Nitroaromatic hydrocarbons, which are abundant in the environment and can cause cancer (143), undergo nitroreduction to produce N-hydroxy
intermediates in bacteria or in mammalian cells and then follow the same process
for mutagenesis as described for amines (77, 112, 136).
RELEVANCE TO DRUG TOXICITY

Today the inclusion of an arylamine moiety in a new drug would trigger a structural
alert and, at the least, special attention. However, many older drugs on the market
contain the moiety and are in wide use. A number of simple drugs, e.g., phenacetin,
are metabolized to nitroso derivatives that have toxicity (144, 145).
SMX causes a variety of unpredictable idiosyncratic drug reactions, including
fever, lymphadenopathy, skin rashes, hepatitis, nephritis, and blood dyscrasias in
approximately 2%3% of patients (146). SMX is metabolized not only to stable
metabolites, such as the acetamide and glucuronide, but also to a potentially toxic
hydroxylamine, which can undergo further oxidation to a nitroso metabolite (SMXNO) (Figure 5). The N-hydroxylation of SMX is catalyzed primarily by P450 2C9
in humans (147). Several studies proposed that SMX-NO may be responsible for
these idiosyncratic toxicities (26, 148152).
Dihydralazine has been implicated in sporadic incidence of drug-induced hepatitis (Figure 6). The drug is oxidized by human P450 1A2 and yields autoimmune
antibodies (in vivo) that recognize (unmodified) P450 1A2 (153). The relationship
of these events to the etiology of the disease remains to be determined.
Dapsone is a drug of choice for treatment of leprosy. Like the simple arylamines,
it is known to cause hemolytic problems (154), apparently owing to the N-hydroxyl
and probably nitroso derivatives. Several human P450s have been shown to be
capable of dapsone N-hydroxylation, including P450s 3A4 (155157), 2E1 (157),
and 2C9 (157, 158), with P450 2C9 alleged to be most important at low dapsone
concentrations (158).
A general conclusion is that the variability of (human) P450s responsible for
N-hydroxylation of arylamine drugs will be more considerable than for the carcinogenic arylamines and HAAs, based on what is presently known. Thus, a general
hypothesis about a role of P450 Family 1 enzymes with these drugs will probably
not be tenable.
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Metabolism of SMX.
CONCLUSIONS AND FURTHER QUESTIONS

Human cancer risk associated with HAAs depends on the level of dietary exposure
in the population, the biologically effective doses arising from those exposures
within relevant target tissues, and the relationship between these effective doses and
predicted increased cancer risk (159). Industrial exposure to known carcinogenic
Figure 6 Other drugs known to undergo Nhydroxylation.
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arylamines is probably minimal today, at least in developed nations. Exposure to

arylamines does occur via cigarette smoke and hair dyes. Other sources may be an
issue in that some arylamine adducts are detected in both smokers and nonsmokers
(160163).
It is difficult but necessary to estimate the risk of HAAs to humans because of
the general exposure and the differences of polymorphisms in metabolic enzymes.
HAAs have produced tumors in rodent liver, colon, forestomach, Zymbals gland,
and mammary gland (23) and in nonhuman primate liver. HAA-DNA adducts
(PhIP) have been detected in normal human breast and colon (28, 164166). The
exact role of metabolic polymorphisms in the risks of individuals is not yet clear,
and consistently epidemiological studies have shown that exposure levels need to
be included in evaluations (166, 167). HAAs must be considered major candidates
for contributing to human cancer.
ACKNOWLEDGMENTS
Work in this laboratory was supported by USPHS grants R35 CA44353, R01
CA90426, and P30 ES00267. We thank F.F. Kadlubar for comments on a draft of
the manuscript and for collaborations in this area for more than 20 years. We also
particularly thank P.D. Josephy, R.J. Turesky, and T. Shimada for their roles in our
studies with these interesting compounds.
LITERATURE CITED
1. Radomski JL. 1979. The primary aromatic amines: their biological properties
and structure-activity relationships. Annu.
Rev. Pharmacol. 19:12957
2. Garner RC, Martin CN, Clayson DB.
1984. Carcinogenic aromatic amines and
related compounds. In Chemical Carcinogens, ed. CE Searle, 1:175276. Washington, DC: Amer. Chem. Soc. 2nd ed.
3. Rehn L. 1895. Uber

Blasentumoren bei
Fuchsinarbeitern. Archiv. Clin. Chirgurie
50:588600
4. Heflich RH, Neft RE. 1994. Genetic
toxicity of 2-acetylaminofluorene, 2aminofluorene and some of their metabolites and model metabolites. Mutat. Res.
318:73114
5. Verna L, Whysner J, Williams GM. 1996.
6.
7.
8.
9.
2-Acetylaminofluorene mechanistic data

and risk assessment: DNA reactivity, enhanced cell proliferation and tumor initiation. Pharmacol. Ther. 71:83105
Kriek E. 1974. Carcinogenesis by aromatic amines. Biochim. Biophys. Acta
355:177203
Widmark EMP. 1939. Presence of cancerproducing substances in roasted food.
Nature 143:984
Murkovic M. 2004. Formation of heterocyclic amines in model systems. J. Chromatog. B 802:310
Sugimura T, Nagao M, Kawachi T. 1977.
Mutagens-carcinogens in foods, with special reference to highly mutagenic pyrolytic products in broiled foods. In Origins of Human Cancer, ed. HH Hiatt, JD
3 Dec 2004
20:35
40
10.

11.
12.
13.
14.
15.
16.
17.
18.
19.
AR
KIM
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
GUENGERICH
Watson, JA Winsten, pp. 156177. Cold

Spring Habor: Cold Spring Harbor Lab.
Press
Nagao M, Honda M, Seino Y, Yahagi
T, Sugimura T. 1977. Mutagenicities of
smoke condensates and the charred surface of fish and meat. Cancer Lett. 2:221
26
Sugimura T. 1995. History, present and
future, of heterocyclic amines, cooked
food mutagens. In Heterocyclic Amines
and Human Cancer, ed. RH Adamson,
J-A Gustafsson, N Ito, M Nagao, K Wakabayashi, Y Yamazoe, 23:21431. Princeton, NJ: Princeton Sci. Publ.
Nagao M, Sugimura T, Matsushima T.
1978. Environmental mutagens and carcinogens. Annu. Rev. Genet. 12:11759
Sugimura T. 1978. Lets be scientific
about the problem of mutagens in cooked
food. Mutat. Res. 55:14952
Sugimura T. 1986. Studies on environmental chemical carcinogenesis in Japan.
Science 233:31218
Jagerstad M, Grivas S, Olsson K, Laser
Reutersward A, Negishi C, et al. 1986.
Formation of food mutagens via Maillard
reactions. Prog. Clin. Biol. Res. 206:155
67
Hatch FT, Felton JS. 1986. Toxicologic
strategy for mutagens formed in foods
during cooking: status and needs. Prog.
Clin. Biol. Res. 206:10931
Jagerstad M, Reutersward AL, Oeste R,
Dahlqvist A, Grivas S, et al. 1983. Creatinine and Maillard reaction products
as precursors of mutagenic compounds
formed fried beefs. In The Maillard Reaction in Foods and Nutrition, ed. GR
Waller, MS Feather, pp. 50719. Washington, DC: Amer. Chem. Soc.
Grivas S, Nyhammar T, Olsson K,
Jagerstad M. 1985. Formation of a new
mutagenic DiMeIQx compound in a
model system by heating creatinine, alanine and fructose. Mutat. Res. 151:17783
Nyhammar T, Grivas S, Olsson K,
Jagerstad M. 1986. Formation of 4,8-
20.
21.
22.
23.
24.
25.
26.
27.
28.
DiMeIQx from the model system fructose, alanine and creatinine. Comparison
with the isomeric 5,8-DiMeIQx. Mutat.
Res. 174:59
Eisenbrand G, Tang W. 1993. Food-borne
heterocyclic amines. Chemistry, formation, occurrence and biological activities.
A literature review. Toxicology 84:182
Sugimura T. 1986. Past, present, and future of mutagens in cooked foods. Environ. Health Perspect. 67:510
Felton JS, Jagerstad M, Knize MG, Skog
K, Wakabayashi K. 2000. Contents in
foods, beverages and tobacco. In Food
Borne Carcinogens Heterocyclic Amines,
ed. M Nagao, T Sugimura, pp. 3171.
West Sussex, UK: Wiley
Turesky RJ. 2002. Heterocyclic aromatic
amine metabolism, DNA adduct formation, mutagenesis, and carcinogenesis.
Drug Metab. Rev. 34:62550
Shimada T, Guengerich FP. 1991. Activation of amino--carboline, 2-amino-1methyl -6 -phenylimidazo[4,5-b]pyridine,
and a copper phthalocyanine cellulose
extract of cigarette smoke condensate by
cytochrome P-450 enzymes in rat and
human liver microsomes. Cancer Res.
51:528491
Manabe S, Tohyama K, Wada O, Aramaki T. 1991. Detection of a carcinogen,
2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), in cigarette smoke
condensate. Carcinogenesis 12:194547
Svensson CK. 2003. Do arylhydroxylamine metabolites mediate idiosyncratic
reactions associated with sulfonamides?
Naisbitt DJ. 2004. Drug hypersensitivity
reactions in skin: understanding mechanisms and the development of diagnostic
and predictive tests. Toxicology 194:179
96
Gorlewska-Roberts K, Green B, Fares
M, Ambrosone CB, Kadlubar FF. 2002.
Carcinogen-DNA adducts in human
breast epithelial cells. Environ. Mol. Mutagen. 39:18492
3 Dec 2004
20:35
AR
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE


29. Turesky RJ, Freeman JP, Holland RD,
Nestorick DM, Miller DW, et al. 2003.
Identification of aminobiphenyl derivatives in commercial hair dyes. Chem. Res.
Toxicol. 16:116273
30. Gago-Dominguez M, Bell DA, Watson
MA, Yuan JM, Castelao JE, et al. 2003.
Permanent hair dyes and bladder cancer: risk modification by cytochrome
P4501A2 and N-acetyltransferases 1 and
2. Carcinogenesis 24:48389
31. Kadlubar FF, Hammons GJ. 1987.
The role of cytochrome P-450 in the
metabolism of chemical carcinogens. In
Mammalian Cytochromes P-450, ed. FP
Guengerich, 2:81130. Boca Raton, FL:
CRC Press
32. Frederick CB, Mays JB, Ziegler DM,
Guengerich FP, Kadlubar FF. 1982. Cytochrome P-450 and flavin-containing
monooxygenase-catalyzed formation of
the carcinogen N-hydroxy-2-aminofluorene, and its covalent binding to nuclear
DNA. Cancer Res. 42:267177
33. Hammons GJ, Guengerich FP, Weis CC,
Beland FA, Kadlubar FF. 1985. Metabolic
oxidation of carcinogenic arylamines by
rat, dog, and human hepatic microsomes
and by purified flavin-containing and cytochrome P-450 monooxygenases. Cancer Res. 45:357885
34. Yamazoe Y, Miller DW, Weis CC, Dooley
KL, Zenser TV, et al. 1985. DNA adducts
formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin
H synthase in vitro and in the dog urothelium in vivo. Carcinogenesis 6:137987
35. Yamazoe Y, Zenser TV, Miller DW, Kadlubar FF. 1988. Mechanism of formation and structural characterization of
DNA adducts derived from peroxidative
activation of benzidine. Carcinogenesis
9:163541
36. Butler MA, Guengerich FP, Kadlubar
FF. 1989. Metabolic oxidation of the
carcinogens 4-aminobiphenyl and 4,4 methylene-bis(2-chloroaniline) by human
hepatic microsomes and by purified rat
37.
38.
39.
40.
41.
42.
43.
44.
41
hepatic cytochrome P-450 monooxygenases. Cancer Res. 49:2531

Butler MA, Iwasaki M, Guengerich FP,
Kadlubar FF. 1989. Human cytochrome
P-450PA (P-450IA2), the phenacetin Odeethylase, is primarily responsible for
the hepatic 3-demethylation of caffeine
and N-oxidation of carcinogenic arylamines. Proc. Natl. Acad. Sci. USA 86:
7696700
Wallin H, Mikalsen A, Guengerich FP,
Ingelman-Sundberg M, Solberg KE, et al.
1990. Different rates of metabolic activation and detoxication of the food mutagen
2-amino-1-methyl-6-phenylimidazo(4,5f)pyridine by different cytochrome P-450
enzymes. Carcinogenesis 11:48992
Seto Y, Guengerich FP. 1993. Partitioning between N-dealkylation and Noxygenation in the oxidation of N,N-dialkylarylamines catalyzed by cytochrome
P450 2B1. J. Biol. Chem. 268:998697
Kato R. 1986. Metabolic activation of
mutagenic heterocyclic aromatic amines
from protein pyrolysates. CRC Crit. Rev.
Toxicol. 16:30748
Kato R, Kamataki T, Saito K, Shinohara A. 1986. Acetyl-CoA dependent Oacetylation of N-hydroxyarylamines in
bacterial and mammalian cellsthe significance for mutagenesis and carcinogenesis. In Genetic Toxicology of Environmental Chemicals, Part A: Basic
Principles and Mechanisms of Action, pp.
50714. New York: Alan R Liss
Chou HC, Lang NP, Kadlubar FF.
1995. Metabolic activation of the Nhydroxy derivative of the carcinogen 4aminobiphenyl by human tissue sulfotransferases. Carcinogenesis 16:41317
Schut HAJ, Snyderwine EG. 1999. DNA
adducts of heterocyclic amine food mutagens: implications for mutagenesis and
carcinogenesis. Carcinogenesis 20:353
68
Snyderwine EG, Wirth PJ, Roller PP,
Adamson RH, Sato S, et al. 1988. Mutagenicity and in vitro covalent DNA
3 Dec 2004
20:35
42

45.
46.
47.
48.
49.
50.
AR
KIM
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
GUENGERICH
binding of 2-hydroxyamino-3-methylimidazolo[4,5-f]quinoline. Carcinogenesis 9:41118

Yamazaki H, Oda Y, Shimada T. 1992.
Use of a newly developed tester strain
Salmonella typhimurium NM2009 for the
study of metabolic activation of carcinogenic aromatic amines by rat liver microsomal cytochrome P-450 enzymes. Mutat.
Res. 272:18392
Snyderwine EG, Davis CD, Nouso
K, Roller PP, Schut HAJ. 1993. 32PPostlabeling analysis of IQ, MeIQx and
PhIP adducts formed in vitro in DNA
and polynucleotides and found in vivo
in hepatic DNA from IQ-, MeIQx- and
PhIP-treated monkeys. Carcinogenesis
14:138995
Oda Y, Yamazaki H, Watanabe M, Nohmi
T, Shimada T. 1995. Development of
high sensitive umu test system: rapid
detection of genotoxicity of promutagenic aromatic amines by Salmonella
typhimurium strain NM2009 possessing
high O-acetyltransferase activity. Mutat.
Res. 334:14556
Oda Y, Aryal P, Terashita T, Gillam EMJ,
Guengerich FP, et al. 2001. Metabolic
activation of heterocyclic amines and
other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P450 1A1, 1A2,
1B1, 2C9, 2D6, 2E1, and 3A4 and human
NADPH-P450 reductase and bacterial Oacetyltransferase. Mutat. Res. 492:8190
Miller JA, Surh YJ. 1994. Sulfonation
in chemical carcinogenesis. In Handbook
of Experimental Pharmacology.Vol. 112:
Conjugation-Deconjugation Reactions in
Drug Metabolism and Toxicity, ed. FC
Kauffman, pp. 42957. Berlin: SpringerVerlag
Kadlubar FF, Unruh LE, Beland FA,
Straub KM, Evans FE. 1980. In vitro reaction of the carcinogen N-hydroxy-2naphthylamine with DNA at the C-8 and
N2 atoms of guanine and at the N6 atom
of adenine. Carcinogenesis 1:13950
51. Beland FA, Kadlubar FF. 1990. Metabolic

activation and DNA adducts of aromatic
amines and nitroaromatic hydrocarbons.
In Handbook of Experimental Pathology.
Carcinogenesis and Mutagenesis, ed. CS
Cooper, PL Grover, Vol. 94/I, pp. 267
325. Heidelberg: Springer-Verlag
52. Wild D, Dirr A, Fasshauer I, Henschler D.
1989. Photolysis of arylazides and generation of highly electrophilic DNA-binding
and mutagenic intermediates. Carcinogenesis 10:33541
53. Guengerich FP. 2002. N-Hydroxyarylamines. Drug Metab. Rev. 34:60723
54. Novak M, Xu LL, Wolf RA. 1998. Nitrenium ions from food-derived heterocyclic
arylamine mutagens. J. Am. Chem. Soc.
120:164344
55. Humphreys WG, Kadlubar FF, Guengerich FP. 1992. Mechanism of C8 alkylation of guanyl residues by activated arylamines: evidence for a role of initial
attack at the N7 atom. Proc. Natl. Acad.
Sci. USA 89:827882
56. Guengerich FP, Mundkowski RG, Voehler
M, Kadlubar FF. 1999. Formation and
reactions of N7-aminoguanosine and
derivatives. Chem. Res. Toxicol. 12:906
16
57. Hashimoto Y, Shudo K, Okamoto T. 1982.
Modification of nucleic acids with mutacarcinogenic heteroaromatic amines
in vivo. Identification of modified bases in
DNA extracted from rats injected with 3amino-1-methyl-5H-pyrido[4,3-b]indole
and 2-amino-6-methyldipyrido[1,2-a3:3 ,
2 -d]imidazole. Mutat. Res. 105:913
58. Snyderwine EG, Roller PP, Adamson RH,
Sato S, Thorgeirsson SS. 1988. Reaction
of N-hydroxylamine and N-acetoxy
derivatives of 2-amino-3-methylimidazolo[4,5-f]quinoline with DNA. Synthesis and identification of N-(deoxyguanosin-8-yl)-IQ. Carcinogenesis 9:
106165
59. Turesky RJ, Rossi SC, Welti DH, Lay JO
Jr, Kadlubar FF. 1992. Characterization of DNA adducts formed in vitro
3 Dec 2004
20:35
AR
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE

60.
61.
62.
63.
64.
65.
by reaction of N-hydroxy-2-amino-3methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5f]quinoxaline at the C-8 and N2 atoms of

guanine. Chem. Res. Toxicol. 5:47990
Frandsen H, Grivas S, Andersson R,
Dragsted L, Larsen JC. 1992. Reaction of
the N2-acetoxy derivative of 2-amino-1methyl - 6 - phenylimidazo[4,5-b]pyridine
(PhIP) with 2 -deoxyguanosine and
DNA. Synthesis and identification of N2(2 -deoxyguanosin-8-yl)PhIP. Carcinogenesis 13:62935
Ochiai M, Nagaoka H, Wakabayashi K,
Tanaka Y, Kim SB, et al. 1993. Identification of N2-(deoxyguanosin-8-yl)-2amino-3,8-dimethylimidazo[4,5-f]quinoxaline 3 ,5 -diphosphate, a major DNA
adduct, detected by nuclease P1 modification of the 32P-postlabeling method, in the
liver of rats fed MeIQx. Carcinogenesis
14:216570
Tada A, Ochiai M, Wakabayashi K,
Nukaya H, Sugimura T, et al. 1994. Identification of N-(deoxyguanosin-8-yl)-2amino-3,4-dimethylimidazo[4,5-f]quinoline (dG-C8-MeIQ) as a major adduct
formed by MeIQ with nucleotides in
vitro with DNA in vivo. Carcinogenesis
15:127578
Frandsen H, Grivas S, Turesky RJ,
Andersson R, Dragsted LO, et al. 1994.
Formation of DNA adducts by the food
mutagen 2-amino-3,4,8-trimethyl-3Himidazo[4,5-f]quinoxaline (4,5-DiMeIQx) in vitro and in vivo. Identification
of a N2-(2 -deoxyguanosin-8-yl)-4,8-DiMeIQx adduct. Carcinogenesis 15:2553
58
Shibutani S, Fernandes A, Suzuki N, Zhou
L, Johnson F, et al. 1999. Mutagenesis of
the N-(deoxyguanosin-8-yl)-2-amino-1methyl - 6 - phenylimidazo[4,5-b]pyridine
DNA adduct in mammalian cells. Sequence context effects. J. Biol. Chem.
274:2743338
Wang Z, Rizzo CJ. 2001. Synthesis of
the C8-deoxyguanosine adduct of the
66.
67.
68.
69.
70.
71.
72.
73.
74.
43
food mutagen IQ. Org. Lett. 3:565

68
Ames BN, Durston WE, Yamasaki E, Lee
FD. 1973. Carcinogens are mutagens: a
simple test system combining liver homogenates for activation and bacteria for
detection. Proc. Natl. Acad. Sci. USA 70:
228185
Ames BN, Kammen HO, Yamasaki E.
1975. Hair dyes are mutagenic: identification of a variety of mutagenic ingredients.
Nebert DW, Bigelow SW, Okey AB,
Yahagi T, Mori Y, et al. 1979. Pyrolysis
products from amino acids and protein:
highest mutagenicity requires cytochrome
P1-450. Proc. Natl. Acad. Sci. USA 76:
592933
Fuscoe JC, Wu R, Shen NH, Healy SK,
Felton JS. 1988. Base-change analysis of revertants of the hisD3052 allele
in Salmonella typhimurium. Mutat. Res.
201:24151
Sugimura T. 1997. Overview of carcinogenic heterocyclic amines. Mutat. Res.
376:21119
Kosakarn P, Halliday JA, Glickman BW,
Josephy PD. 1993. Mutational specificity of 2-nitro-3,4-dimethylimidazo[4,5-f]
quinoline in the lacI gene of Escherichia
coli. Carcinogenesis 14:51117
Watanabe M, Ohta T. 1993. Analysis of
mutational specificity induced by heterocylic amines in the lacZ gene of Escherichia coli. Carcinogenesis 14:1149
53
Josephy PD, DeBruin LS, Lord HL, Oak
J, Evans DH, et al. 1995. Bioactivation
of aromatic amines by recombinant human cytochrome P450 1A2 expressed in
bacteria: a substitute for mammalian tissue preparations in mutagenicity testing.
Cancer Res. 55:799802
Josephy PD, Evans DH, Parikh A, Guengerich FP. 1998. Metabolic activation of
aromatic amine mutagens by simultaneous expression of human cytochrome
P450 1A2, NADPH-cytochrome P450
3 Dec 2004
20:35
44

75.
76.
77.
78.
79.
80.
81.
82.
83.
AR
KIM
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
GUENGERICH
reductase, and N-acetyltransferase in Escherichia coli. Chem. Res. Toxicol. 11:70

74
Constable A, Varga N, Josephy PD, Guy
P, Turesky RJ. 1999. Evaluation of Escherichia coli DJ4309 expressing human
P450 1A2 in mutagenicity testing of complex food mixtures. Mutat. Res. 442:79
87
Josephy PD. 2000. The Escherichia coli
lacZ reversion mutagenicity assay. Mutat.
Res. 455:7180
Marwood TM, Meyer D, Josephy PD.
1995. Escherichia coli lacZ strains engineered for detection of frameshift mutations induced by aromatic amines and
nitroaromatic compounds. Carcinogenesis 16:203743
Watanabe M, Nohmi T, Ishidate M Jr.
1987. New tester strains of Salmonella typhimurium highly sensitive to mutagenic
nitroarenes. Biochem. Biophys. Res. Commun. 147:97479
Suzuki A, Kushida H, Iwata H, Watanabe
M, Nohmi T, et al. 1998. Establishment of
a Salmonella tester strain highly sensitive
to mutagenic heterocyclic amines. Cancer
Res. 58:183338
Fujita K-I, Kamataki T. 2002. Genetically
engineered bacterial cells co-expressing
human cytochrome P450 with NADPHcytochrome P450 reductase: prediction of
metabolism and toxicity of drugs in humans. Drug Metab. Pharmacokin. 17:1
22
Parikh A, Josephy PD, Guengerich FP.
1999. Selection and characterization of
human cytochrome P450 1A2 mutants
with altered catalytic properties. Biochemistry 38:528389
Josephy PD, Bibeau KL, Evans DH.
2000. Activation of MeIQ (2-amino-3,4dimethylimidazo-[4,5-f]quinoline) by sequence variants of recombinant human
cytochrome P450 1A2. Environ. Mol. Mutagen. 35:32835
Kim D, Guengerich FP. 2004. Selection of human cytochrome P450 1A2
84.
85.
86.
87.
88.
89.
90.
91.
92.
mutants with selectivity enhanced catalytic activity for heterocyclic amine

N-hydroxylation. Biochemistry 43:981
88
Zhou H, Josephy PD, Kim D, Guengerich FP. 2004. Functional characterization of four allelic variants of human
cytochrome P450 1A2. Arch. Biochem.
Biophys. 422:2330
Chun Y-J, Kim S, Kim D, Lee S-K, Guengerich FP. 2001. A new selective and potent inhibitor of human cytochrome P450
1B1 and its application to antimutagenesis. Cancer Res. 61:816470
Langouet S, Furge LL, Kerriguy N, Nakamura K, Guillouzo A, et al. 2000. Inhibition of human cytochrome P450 enzymes
by 1,2-dithiole-3-thione, oltipraz and its
derivatives, and sulforaphane. Chem. Res.
Toxicol. 13:24552
Poirier MC, Beland FA. 1992. DNA
adduct measurements and tumor incidence during chronic carcinogen exposure in animal models: implications for
DNA adduct-based human cancer risk assessment. Chem. Res. Toxicol. 5:74955
Hemminki K. 1993. DNA adducts,
mutations and cancer. Carcinogenesis
14:200712
Nagao M, Ushijima T, Toyota M, Inoue
R, Sugimura T. 1997. Genetic changes induced by heterocyclic amines. Mutat. Res.
376:16167
Fujimoto Y, Hampton LL, Snyderwine
EG, Nagao M, Sugimura T, et al.
1994. p53 gene mutation in hepatocellular carcinoma induced by 2-amino-3methylimidazo[4,5-f]quinoline in nonhuman primates. Jpn. J. Cancer Res. 85:
5069
Okonogi H, Stuart GR, Okochi E, Ushijima T, Sugimura T, et al. 1997. Effects
of gender and species on spectra of mutation induced by 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine in the lacI
transgene. Mutat. Res. 395:9399
Randerath K, Randerath E, Agrawal HP,
Gupta RC, Schurdak ME, et al. 1985.
3 Dec 2004
20:35
AR
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
93.

94.
95.
96.
97.
98.
99.
100.
101.
Postlabeling methods for carcinogenDNA adduct analysis. Environ. Health

Perspect. 62:5765
Turesky RJ, Vouros P. 2004. Formation
and analysis of heterocyclic aromatic
amine-DNA adducts in vitro and in vivo.
J. Chromatog. B 803:15566
Turesky RJ, Markovic J, Bracco-Hammer
I, Fay LB. 1991. The effect of dose
and cytochrome P450 induction on the
metabolism and disposition of the foodborne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in
the rat. Carcinogenesis 12:184755
Turesky RJ, Constable A, Richoz J, Varga
N, Markovic J, et al. 1998. Activation of
heterocyclic aromatic amines by rat and
human liver microsomes and by purified
rat and human cytochrome P450 1A2.
Kadlubar FF, Miller JA, Miller EC. 1976.
Microsomal N-oxidation of the hepatocarcinogen N-methyl-4-aminoazobenzene
and the reactivity of N-hydroxy-Nmethyl-4-aminoazobenzene. Cancer Res.
36:1196206
Hueper WC, Wiley FH, Wolfe HD. 1938.
Experimental production of bladder tumors in dogs by administration of betanaphthylamine. J. Indust. Hygiene Toxicol. 20:4684
Sugimura T. 1985. Carcinogenicity of
mutagenic heterocyclic amines formed
during the cooking process. Mutat. Res.
150:3341
Kato T, Ohgaki H, Hasegawa H, Sato S,
Takayama S, et al. 1988. Carcinogenicity
in rats of a mutagenic compound, 2amino-3,8-dimethylimidazo[4,5-f ]quinoxaline. Carcinogenesis 9:7173
Nagao M, Sugimura T. 1993. Carcinogenic factors in food with relevance to
colon cancer development. Mutat. Res.
290:4351
Mueller GC, Miller JA. 1948. The
metabolism of 4-dimethylaminoazobenzene by rat liver homogenates. J. Biol.
Chem. 176:53544
45
102. Cramer JW, Miller JA, Miller EC. 1960.

N-Hydroxylation: a new metabolic reaction observed in the rat with the carcinogen 2-acetylaminofluorene. J. Biol. Chem.
235:88588
103. Thorgeirsson SS, Jollow DJ, Sasame HA,
Green I, Mitchell JR. 1973. The role of
cytochrome P-450 in N-hydroxylation of
2-acetylaminofluorene. Mol. Pharmacol.
9:398404
104. Felton JS, Nebert DW, Thorgeirsson
SS. 1976. Genetic differences in 2acetylaminofluorene mutagenicity in vitro
associated with mouse hepatic arylhydrocarbon hydroxylase activity induced
by polycyclic aromatic compounds. Mol.
Pharmacol. 12:22533
105. Dybing E, von Bahr C, Aune T, Glaumann H, Levitt DS, et al. 1979. In
vitro metabolism and activation of carcinogenic aromatic amines by subcellular fractions of human liver. Cancer Res.
39:420611
106. McManus ME, Minchin RF, Sanderson N, Schwartz D, Johnson EF, et al.
1984. Metabolic processing of 2-acetylaminofluorene by microsomes and six
highly purified cytochrome P-450 forms
from rabbit liver. Carcinogenesis 5:1717
23
107. Wang PP, Beaune P, Kaminsky LS, Dannan GA, Kadlubar FF, et al. 1983. Purification and characterization of six cytochrome P-450 isozymes from human
liver microsomes. Biochemistry 22:5375
83
108. Distlerath LM, Reilly PEB, Martin MV,
Davis GG, Wilkinson GR, et al. 1985.
Purification and characterization of the
human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation
and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem.
260:905767
109. Shimada T, Iwasaki M, Martin MV,
Guengerich FP. 1989. Human liver microsomal cytochrome P-450 enzymes
3 Dec 2004
20:35
46

110.
111.
112.
113.
114.
115.
116.
117.
118.
AR
KIM
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
GUENGERICH
involved in the bioactivation of procarcinogens detected by umu gene

response in Salmonella typhimurium
TA1535/pSK1002. Cancer Res. 49:3218
28
Pantuck EJ, Hsiao K-C, Maggio A, Nakamura K, Kuntzman R, et al. 1974. Effect of cigarette smoking on phenacetin
metabolism. Clin. Pharmacol. Therapeut.
15:917
Butler MA, Lang NP, Young JF, Caporaso NE, Vineis P, et al. 1992. Determination of CYP1A2 and NAT2 phenotypes
in human populations by analysis of caffeine urinary metabolites. Pharmacogenetics 2:11627
Shimada T, Guengerich FP. 1990. Inactivation of 1,3-, 1,6-, and 1,8-dinitropyrene
by human and rat microsomes. Cancer
Res. 50:203643
Guengerich FP, Shimada T. 1991. Oxidation of toxic and carcinogenic chemicals
by human cytochrome P-450 enzymes.
Yun C-H, Shimada T, Guengerich FP.
1992. Contributions of human liver cytochrome P-450 enzymes to the N-oxidation
of 4,4 -methylene-bis(2-chloroaniline).
Carcinogenesis 13:21722
Shimada T, Yamazaki H, Guengerich FP.
1992. Roles of rat pulmonary microsomal
cytochrome P-450 enzymes involved in
the activation of procarcinogens. Mutat.
Res. 283:23341
Shimada T, Gillam EMJ, Sandhu P, Guo Z,
Tukey RH, et al. 1994. Activation of procarcinogens by human cytochrome P450
enzymes expressed in Escherichia coli.
Simplified bacterial systems for genotoxicity assays. Carcinogenesis 15:252329
Boobis AR, Lynch AM, Murray S, de la
Toore R, Solans A, et al. 1994. CYP1A2catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in
humans. Cancer Res. 54:8994
Shimada T, Hayes CL, Yamazaki H, Amin
S, Hecht SS, et al. 1996. Activation of
119.
120.
121.
122.
123.
124.
125.
126.
chemically diverse procarcinogens by human cytochrome P450 1B1. Cancer Res.

56:297984
Josephy PD, Batty SM, Boverhof DR.
2001. Recombinant human P450 forms
1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens
in Escherichia coli lacZ strains. Environ.
Mol. Mutagen. 38:1218
Kitada M, Taneda M, Itahashi K, Kamataki T. 1991. Four forms of cytochrome
P-450 in human fetal liver: purification and their capacity to activate promutagens. Jpn. J. Cancer Res. 82:426
32
Ayesh R, Idle JR, Ritchie JC, Crothers
MJ, Hetzel MR. 1984. Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature 312:169
70
Wolff T, Distlerath LM, Worthington
MT, Groopman JD, Hammons GJ, et al.
1985. Substrate specificity of human
liver cytochrome P-450 debrisoquine 4hydroxylase probed using immunochemical inhibition and chemical modeling.
Langouet S, Paehler A, Welti DH, Kerriguy N, Guillouzo A, et al. 2002. Differential metabolism of 2-amino-1-methyl6-phenylimidazo[4,5-b]pyridine in rat
and human hepatocytes. Carcinogenesis
23:11522
Richardson HL, Stier AR, BorsosNachtnebel E. 1952. Liver tumor inhibition and adrenal histologic responses in
rats to which 3 -methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene were simultaneously administered.
Snyderwine EG, Yu M, Schut HA,
Knight-Jones L, Kimura S. 2002. Effect
of CYP1A2 deficiency on heterocyclic
amine DNA adduct levels in mice. Food
Chem. Toxicol. 40:152933
Kimura S, Kawabe M, Yu A, Morishima
H, Fernandez-Salguero P, et al. 2003. Carcinogenesis of the food mutagen PhIP in
3 Dec 2004
20:35
AR
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE

127.
128.
129.
130.
131.
132.
133.
mice is independent of CYP1A2. Carcinogenesis 24:58387

Turesky RJ, Constable A, Richoz J, Vargu
N, Markovic J, et al. 1998. Differences
in activation of heterocyclic aromatic
amines by rat and human liver microsomes and by rat and human cytochromes
P450 1A2. Chem. Res. Toxicol. 11:925
36
Turesky RJ, Constable A, Fay LB, Guengerich FP. 1999. Interspecies differences
in metabolism of heterocyclic aromatic
amines by rat and human P450 1A2. Cancer Lett. 143:10912
Guengerich FP. 1997. Comparisons of catalytic selectivity of cytochrome P450 subfamily members from different species.
Chem Biol. Interact. 106:16182
Langouet S, Welti DH, Kerriguy N, Fay
LB, Markovic J, et al. 2001. Metabolism
of 2-amino-3,8-dimethylimidazo [4,5f]quinoxaline in human hepatocytes:
2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline-8-carboxylic acid is a major
detoxication pathway catalyzed by cytochrome P450 1A2. Chem. Res. Toxicol.
14:21121
Turesky RJ, Parisod V, Huynh-Ba T, Langouet S, Guengerich FP. 2001. Regioselective differences in C8 and N-oxidation
of 2-amino-3,8-dimethylimidazo[4,5-f]
quinoxaline by human and rat liver
microsomes and cytochromes P450 1A2.
Adamson
RH,
Snyderwine
EG,
Thorgeirsson UP, Schut HA, Turesky RJ,
et al. 1990. Metabolic processing and
carcinogenicity of heterocyclic amines
in nonhuman primates. In Heterocyclic
Amines and Human Cancer, ed. RH
Gustafsson, N Ito, M
Adamson, J-A,
Nagao, K Wakabayashi, 21:289301.
Princeton, NJ: Princeton Sci. Publ.
Snyderwine EG, Turesky RJ, Turteltaub
KW, Davis CD, Sadrieh N, et al. 1997.
Metabolism of food-derived heterocyclic
amines in nonhuman primates. Mutat.
Res. 376:20310
47
134. Adamson RH. 2000. Carcinogenicity in

animals and specific organs. In Food
Borne Carcinogens Heterocyclic Amines,
ed. M Nagao, T Sugimura, pp. 22940.
West Sussex, UK: Wiley
135. Wild D, Degen GH. 1987. Prostaglandin
H synthase-dependent mutagenic activation of heterocyclic aromatic amines
of the IQ-type. Carcinogenesis 8:541
45
136. Wolz E, Pfau W, Degen GH. 2000.
Bioactivation of the food mutagen 2amino-3-methyl-imidazo[4,5-f]quinoline
(IQ) by prostaglandin-H synthase and by
monooxygenases: DNA adduct analysis.
Food Chem. Toxicol. 38:51322
137. Degen GH, Gerber MM, Obrecht-Pflumio
S, Dirheimer G. 1997. Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures. Arch. Toxicol.
71:36571
138. Guengerich FP. 2001. Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity.
139. Ortiz de Montellano PR. 1995. Oxygen
activation and reactivity. In Cytochrome
P450: Structure, Mechanism, and Biochemistry, ed. PR Ortiz de Montellano,
pp. 245303. New York: Plenum Press
140. Kim D, Kadlubar FF, Teitel CH, Guengerich FP. 2004. Formation and reduction of aryl and heterocyclic nitroso
compounds and significance in the flux
of hydroxylamines. Chem. Res. Toxicol.
17:52936
141. Morrison LD, Eling TE, Josephy
PD. 1993. Prostaglandin H synthasedependent formation of the direct-acting
mutagen 2-nitro-3-methylimidazo[4,5f]quinoline (nitro-IQ) from IQ. Mutat.
Res. 302:4552
142. Wolz E, Wild D, Degen GH. 1995.
Prostaglandin-H synthase mediated
metabolism and mutagenic activation of
2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Arch. Toxicol. 69:17179
143. El-Bayoumy K. 1992. Environmental
3 Dec 2004
20:35
48
144.

145.
146.
147.
148.
149.
150.
151.
AR
KIM
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
P1: GCE
GUENGERICH
carcinogens that may be involved in human breast cancer etiology. Chem. Res.
Toxicol. 5:58590
Klehr H, Eyer P, Schafer W. 1987. Formation of 4-ethoxy-4 -nitrosodiphenylamine
in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione.
Biol. Chem. Hoppe-Seyler 368:895902
Eyer P. 1988. Detoxication of Noxygenated arylamines in erythrocytes.
An overview. Xenobiotica 18:132733
Mandell GL, Sande MA. 1985. Antimicrobial agents: sulfonamides, trimethoprim-sulfamethoxazole and agents for
urinary tract infections. In The Pharmacological Basis of Therapeutics, ed.
AG Gillam, LS Goodman, TW Rall,
R Marud, pp. 1095114. New York:
Macmillan
Cribb AE, Spielberg SP, Griffin GP.
1995. N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine
in human and rat hepatic microsomes.
Naisbitt DJ, Hough SJ, Gill HJ, Pirmohamed M, Kitteringham NR, et al. 1999.
Cellular disposition of sulphamethoxazole and its metabolites: implications
for hypersensitivity. Br. J. Pharmacol.
126:1393407
Cribb AE, Nuss CE, Alberts DW, Lamphere DB, Grant DM, et al. 1996. Covalent binding of sulfamethoxazole reactive
metabolites to human and rat liver subcellular fractions assessed by immunochemical detection. Chem. Res. Toxicol. 9:500
7
Carr A, Tindall B, Penny R, Cooper DA.
1993. In vitro cytotoxicity as a marker of
hypersensitivity to sulphamethoxazole in
patients with HIV. Clin. Exp. Immunol.
94:2125
Meekins CV, Sullivan TJ, Gruchalla
RS. 1994. Immunochemical analysis of
sulfonamide drug allergy: identification
of sulfamethoxazole-substituted human
152.
153.
154.
155.
156.
157.
158.
159.
160.
serum proteins. J. Allergy Clin. Immunol.

94:101724
Rieder MJ, Krause R, Bird IA. 1995.
Time-course of toxicity of reactive sulfonamide metabolites. Toxicology 95:141
46
Bourdi M, Larrey D, Nataf J, Berunau J,
Pessayre D, et al. 1990. A new anti-liver
endoplasmic reticulum antibody directed
against human cytochrome P-450 IA2: a
specific marker of dihydralazine-induced
hepatitis. J. Clin. Invest. 85:196773
Reilly TP, Woster PM, Svensson CK.
1999. Methemoglobin formation by hydroxylamine metabolites of sulfamethoxazole and dapsone: implications for differences in adverse drug reactions. J.
Pharmacol. Exp. Ther. 288:95159
Fleming CM, Branch RA, Wilkinson GR,
Guengerich FP. 1992. Human liver microsomal N-hydroxylation of dapsone by
cytochrome P-450 3A4. Mol. Pharmacol.
41:97580
Gill HJ, Tingle MD, Park BK. 1995. NHydroxylation of dapsone by multiple enzymes of cytochrome P450: implications
for inhibition of haemotoxicity. Br. J. Clin.
Pharmacol. 40:53138
Mitra AK, Thummel KE, Kalhorn TF,
Kharasch ED, Unadkat JD, et al. 1995.
Metabolism of dapsone to its hydroxylamine by CYP2E1 in vitro and in vivo.
Clin. Pharmacol. Ther. 58:55666
Winter HR, Wang Y, Unadkat JD.
2000. CYP2C8/9 mediate dapsone Nhydroxylation at clinical concentrations
of dapsone. Drug Metab. Dispos. 28:865
68
Skog KI, Johansson MA, Jagerstad MI.
1998. Carcinogenic heterocyclic amines
in model systems and cooked foods: a review on formation, occurrence and intake.
Food Chem. Toxicol. 36:87996
Talaska G, Al-Juburi AZSS, Kadlubar FF.
1991. Smoking related carcinogen-DNA
adducts in biopsy samples of human
urinary bladder: identification of N(deoxyguanosin - 8 - yl)-4-aminobiphenyl
3 Dec 2004
20:35
AR
AR232-PA45-02.tex
AR232-PA45-02.sgm
LaTeX2e(2002/01/18)
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161.
162.
163.
164.
as a major adduct. Proc. Natl. Acad. Sci.

USA 88:535054
Lin D, Lay JO Jr, Bryant MS, Malaveille
C, Friesen M, et al. 1994. Analysis
of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas
chromatography-mass spectrometry. Environ. Health Perspect. 102 (Suppl. 6):11
16
Culp SJ, Roberts DW, Talaska G, Lang
NP, Fu PP, et al. 1997. Immunochemical,32P-postlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts
in human peripheral lung in relation
to metabolic activation pathways involving pulmonary N-oxidation, conjugation,
and peroxidation. Mutat. Res. 378:97
112
Thompson PA, Seyedi F, Lang NP,
MacLeod SL, Wogan GN, et al. 1999.
Comparison of DNA adduct levels associated with exogenous and endogenous
exposures in human pancreas in relation to metabolic genotype. Mutat. Res.
424:26374
Friesen MD, Kaderlik K, Lin D, Garren L, Bartsch H, et al. 1994. Analysis
49
of DNA adducts of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine in rat and

human tissues by alkaline hydrolysis
and gas chromatography/electron capture
mass spectrometry: validation by comparison with 32P-postlabeling. Chem. Res.
Toxicol. 7:73339
165. Totsuka Y, Fukutome K, Takahashi M,
Takahashi S, Tada A, et al. 1996. Presence
of N2-(deoxyguanosin-8-yl)-2-amino-3,
8-dimethylimidazo[4,5-f]quinoxaline (d
G-C8-MeIQx) in human tissues. Carcinogenesis 17:102934
166. Zhu J, Chang P, Bondy ML, Sahin AA,
Singletary SE, et al. 2003. Detection of
2-amino-1-methyl-6-phenylimidazo[4,5b]-pyridine-DNA adducts in normal
breast tissues and risk of breast cancer.
Cancer Epidemiol. Biomarkers Prev. 12:
83037
167. Lang NP, Butler MA, Massengill J, Lawson M, Stotts RC, et al. 1994. Rapid
metabolic phenotypes for acetyltransferase and cytochrome P4501A2 and putative exposure to food-borne heterocyclic
amines increase the risk for colorectal cancer or polyps. Cancer Epidemiol.
Biomarkers Prev. 3:67582
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Copyright
GLUTATHIONE TRANSFERASES

John D. Hayes, Jack U. Flanagan, and Ian R. Jowsey

Biomedical Research Center, Ninewells Hospital & Medical School, University of
Dundee, Dundee DD1 9SY, Scotland, United Kingdom; email: john.hayes@cancer.org.uk,
j.flanagan@imb.uq.edu.au, i.r.jowsey@dundee.ac.uk
Key Words antioxidant response element, oxidative stress,

15-deoxy-12,14-prostaglandin J2, prostaglandin E2, Nrf2, 4-hydroxynonenal,
maleylacetoacetate, glutathione peroxidase, leukotriene C4
Abstract This review describes the three mammalian glutathione transferase
(GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now
designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases
inactivate endogenous ,-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are
also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-12,14-prostaglandin J2
(15d-PGJ2) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor (PPAR ) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through
metabolism of 15d-PGJ2, GST may enhance gene expression driven by nuclear factorB (NF-B). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and
increased risk of myocardial infarction and stroke. Targeted disruption of murine
genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective,
whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of
mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu,
and Pi classes, an observation suggesting they are part of an adaptive mechanism that
responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and
MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative
stress.
0362-1642/05/0210-0051$14.00
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INTRODUCTION
The glutathione transferases (EC 2.5.1.18) have historically also been called glutathione S-transferases, and it is this latter name that gives rise to the widely
used abbreviation, GST. These enzymes catalyze nucleophilic attack by reduced
glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon,
nitrogen, or sulphur atom. Their substrates include halogenonitrobenzenes, arene
oxides, quinones, and ,-unsaturated carbonyls (15). Three major families of
proteins that are widely distributed in nature exhibit glutathione transferase activity.
Two of these, the cytosolic and mitochondrial GST, comprise soluble enzymes that
are only distantly related (6, 7). The third family comprises microsomal GST and
is now referred to as membrane-associated proteins in eicosanoid and glutathione
(MAPEG) metabolism (8). A further distinct family of transferases exists, represented by the bacterial fosfomycin resistance proteins FosA and FosB (9); this
family is not discussed further.
Cytosolic and mitochondrial GST share some similarities in their three-dimensional fold (6) but bear no structural resemblance to the MAPEG enzymes (10).
However, all three families contain members that catalyze the conjugation of
GSH with 1-chloro-2,4-dinitrobenzene (CDNB) and exhibit glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); these reactions are shown in
Figure 1. The cytosolic GST and MAPEG enzymes catalyze isomerization of various unsaturated compounds (8, 11, 12) and are intimately involved in the synthesis
of prostaglandins and leukotrienes (4, 8).
Cytosolic GSTs represent the largest family of such transferases and have activities that are unique to this group of enzymes. They catalyze thiolysis of 4nitrophenyl acetate; display thiol transferase activity; reduce trinitroglycerin, dehydroascorbic acid, and monomethylarsonic acid; and catalyze the isomerization
of maleylacetoacetate and 5-3-ketosteroids (Figure 1) (1, 1317).
Glutathione transferases are of interest to pharmacologists and toxicologists
because they provide targets for antiasthmatic and antitumor drug therapies (18
21), and they metabolize cancer chemotherapeutic agents, insecticides, herbicides, carcinogens, and by-products of oxidative stress. Overexpression of GST
in mammalian tumor cells has been implicated with resistance to various anticancer agents and chemical carcinogens (2). Furthermore, elevated levels of GST
have been associated with tolerance of insecticides and with herbicide selectivity
(22, 23).
In microbes, plants, flies, fish, and mammals, expression of GSTs are upregulated by exposure to prooxidants (2430). Increase in transferase activity is also
observed in animals that undergo prolonged torpor or hibernation when comparisons are made between their estivated state and their wakeful condition (31). It
is similarly observed during transition of the common toad Bufo bufo from an
aquatic environment to the land (32). Collectively, these findings indicate that
induction of GST is an evolutionarily conserved response of cells to oxidative
stress.
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Figure 1 Reactions catalyzed by GST. Example of conjugation, reduction, thiolysis, and isomerization reactions catalyzed by GST. The
following substrates are shown: (a) CDNB, (b) sulforaphane, (c) CuOOH, (d) 4-nitrophenyl acetate, (e) trinitroglycerin, ( f ) maleylacetoacetate, and (g) PGH2 (conversion to PGD2 is depicted).

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METABOLISM OF XENOBIOTICS BY GST

Detoxification through the Mercapturic Acid Pathway

Glutathione transferases catalyze the first of four steps required for the synthesis
of mercapturic acids (1). Subsequent reactions in this pathway entail sequential
removal of the -glutamyl moiety and glycine from the glutathione conjugate,
followed finally by N-acetylation of the resulting cysteine conjugate. It is important
to recognize that GST enzymes are part of an integrated defense strategy, and
their effectiveness depends on the combined actions of, on one hand, glutamate
cysteine ligase and glutathione synthase to supply GSH and, on the other hand, the
actions of transporters to remove glutathione conjugates from the cell (4). Once
formed, these conjugates are eliminated from the cell by the trans-membrane MRP
(multidrug resistance-associated protein). Nine MRP proteins exist (33), and these
are all members of the C family of ABC transporters. Among these, MRP1 and
MRP2 can export glutathione conjugates and compounds complexed with GSH
(34, 35). The dinitrophenol-glutathione ATPase called RLIP76 promotes efflux of
glutathione conjugates from cells (36), but as it is not a trans-membrane protein
the mechanism is probably indirect.
Exogenous substrates for soluble GST include drugs, industrial intermediates, pesticides, herbicides, environmental pollutants, and carcinogens. The cancer chemotherapeutic agents adriamycin, 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU), busulfan, carmustine, chlorambucil, cis-platin, crotonyloxymethyl-2cyclohexenone (COMC-6), cyclophosphamide, ethacrynic acid, melphalan, mitozantrone, and thiotepa are detoxified by GST (2, 37, 38). Environmental chemicals and their metabolites detoxified by GST include acrolein, atrazine, DDT,
inorganic arsenic, lindane, malathion, methyl parathion, muconaldehyde, and tridiphane (2, 39, 40).
A large number of epoxides, such as the antibiotic fosfomycin and those derived
from environmental carcinogens, are detoxified by GST. The latter group includes
epoxides formed from aflatoxin B1, 1-nitropyrene, 4-nitroquinoline, polycyclic
aromatic hydrocarbons (PAHs), and styrene by the actions of cytochromes P450
in the liver, lung, gastrointestinal tract, and other organs. Conjugation of aflatoxin
B1-8,9-epoxide with GSH is a major mechanism of protection against the mycotoxin, at least in rodents (41). The PAHs are ubiquitous, found in cigarette smoke
and automobile exhaust fumes, and represent an ever-present threat to health.
Those that are metabolized by GST include ultimate carcinogenic bay- and fjordregion diol epoxides produced from chrysene, methylchrysene, benzo[c]chrysene,
benzo[g]chrysene, benzo[c]phenanthrene, benzo[a]pyrene, dibenz[a,h]anthracene,
and dibenzo[a,l]pyrene (4244).
Heterocyclic amines, produced by cooking protein-rich food, represent another
important group of carcinogens. One of the major heterocyclic amines found in
cooked food is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and cytosolic GST isoenzymes have been shown to detoxify the activated metabolite,
N-acetoxy-PhIP (45).
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Bioactivation of Xenobiotics by GST

Conjugation of foreign compounds with GSH almost always leads to formation
of less reactive products that are readily excreted. In a few instances, however,
the glutathione conjugate is more reactive than the parent compound. Examples of
this phenomenon are short-chain alkyl halides that contain two functional groups.
Conjugation of GSH with the solvent dichloromethane results in the formation of
the highly unstable S-chloromethylglutathione, which still contains an electrophilic
center capable of modifying DNA (46, 47). The 1,2-dihaloethanes are another
group of GST substrates that are activated by conjugation with GSH to genotoxic
products. However, in this instance, the glutathione conjugate rearranges to form
an episulfonium intermediate that is responsible for modifying DNA (47).
Allyl-, benzyl-, phenethyl-isothiocyanates, and sulforaphane are moderately
toxic compounds that are formed from plant glucosinolates. They are reversibly
conjugated by GST with GSH to yield thiocarbamates (Figure 1). Following export from cells via MRP1 or MRP2, thiocarbamates spontaneously degrade to their
isothiocyanates, liberating GSH. Thereafter, the isothiocyanate may be taken up
again by the cell and reconjugated with GSH, only to be reexported as the thiocarbamate and revert to the isothiocyanate. This cyclical process results in depletion
of intracellular GSH and assists distribution of isothiocynates throughout the body.
Should isothiocyanates be taken up by cells that have a low GSH content, they
may not be conjugated with GSH, but rather are more likely to thiocarbamylate
proteins, a process that can result in cell death (48).
Conjugation of haloalkenes with GSH, which occurs primarily in the liver, can
lead ultimately to the generation in the kidney of reactive thioketenes, thionoacylhalides, thiiranes, and thiolactones through the actions of renal cysteine conjugate
-lyase (49).
In cancer chemotherapy, the ability of GST to produce reactive metabolites
has been exploited to target tumors that overexpress particular transferases (50).
The latent cytotoxic drug TER286 (now called TLK286) is activated by GST
through a -elimination reaction to yield an active analogue of cyclophosphamide
(51, 51a). More recently, the prodrug PABA/NO (O2-[2,4-dinitro-5-(N-methyl-N4-carboxyphenylamino)phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate)
has been designed to generate cytolytic nitric oxide upon metabolism by GST (52).
METABOLISM OF ENDOGENOUS COMPOUNDS BY GST

Detoxification of Products of Oxidative Stress
The production of reactive oxygen species, the superoxide anion O
2 , hydrogen
peroxide H2O2, and the hydroxyl radical HO, from partially reduced O2 is an
unavoidable consequence of aerobic respiration. Free radicals primarily arise
through oxidative phosphorylation, although 5-lipoxygenase-, cyclooxygenase-,
cytochrome P450-, and xanthene oxidasecatalyzed reactions are also a source
(4). Such species are scavenged by the catalytic activities of superoxide dismutase,
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catalase, and glutathione peroxidase and nonenzymatically by -tocopherol, ascorbic acid, GSH, and bilirubin. Despite these antioxidant defenses, reactive oxygen
species inflict damage on membrane lipid, DNA, protein, and carbohydrate. Oxidation of these macromolecules gives rise to cytotoxic and mutagenic degradation
products (53). Thus, although O
2 can damage DNA directly, it can also damage
DNA indirectly through the production of these reactive secondary metabolites.
Aldehyde dehydrogenase, alcohol dehydrogenase, aldo-keto reductase, GST, and
Se-dependent glutathione peroxidase (GPx) are some of the enzyme systems that
protect against the by-products of oxidative stress.
Free radical-initiated peroxidation of polyunsaturated fatty acids in membranes
is a particular problem as it results in chain reactions that serve to amplify damage to lipids. The process produces short-lived lipid hydroperoxides that breakdown to yield secondary electrophiles, including epoxyaldehydes, 2-alkenals,
4-hydroxy-2-alkenals, and ketoaldehydes, some of which are genotoxic (53). GST
isoenzymes exhibit modest Se-independent glutathione peroxidase activity toward
1-palmitoyl-2 - (13 - hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3-phosphatidylcholine and phosphatidylcholine hydroperoxide, indicating they may reduce
lipid hydroperoxides within membranes (5456). The transferases can also reduce
cholesteryl hydroperoxides (57) and fatty acid hydroperoxides, including (S)-9hydroperoxy-10,12-octadecadieonic acid and (S)-13-hydroperoxy-9,11-octadecadieonic acid (56). Presumably, reduction of phospholipid, fatty acid, and cholesteryl
hydroperoxides curtails formation of downstream epoxides and reactive carbonyls
arising from oxidation of membranes. Among the end-products of lipid peroxidation, GSTs conjugate GSH with the 2-alkenals acrolein and crotonaldehyde (2, 4),
as well as 4-hydroxy-2-alkenals of between 6 and 15 carbon atoms in length (58)
(Figure 2); conjugation of GSH with the (S) enantiomer of 4-hydroxynonenal
is favored over the (R) enantiomer (59). Further, GSTs catalyze the conjugation of cholesterol-5,6-oxide, epoxyeicosatrienoic acid, and 9,10-epoxystearic
acid with GSH (2). These findings indicate that GST, along with other antioxidant enzymes, such as Se-dependent GPx1, provide the cell with protection
against a range of harmful electrophiles produced during oxidative damage to
membranes (4).
The 1-cys peroxiredoxin, Prx VI, defends against cellular membrane damage
by reducing phospholipid hydroperoxides to their respective alcohols. Reduction
of these substrates results in oxidation of Cys-47 in Prx VI to sulfenic acid. It
has been proposed that GST reactivates oxidized Prx VI through glutathionylation
followed by reduction of the mixed disulfide (60). Through this process, GST may
indirectly combat oxidative stress by restoring the activity of oxidized Prx VI.
Oxidation of nucleotides yields base propenals, such as adenine propenal, and
hydroperoxides that are detoxified by GST (Figure 2). Oxidation of catecholamines
yields aminochrome, dopachrome, noradrenochrome, and adrenochrome that are
harmful because they can produce O
2 by redox cycling. These quinone-containing
compounds can be conjugated with GSH through the actions of GST, a
reaction that prevents redox-cycling (61). O-quinones formed from dopamine can
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Figure 2 GST-catalyzed conjugation of ,-unsaturated carbonyls and o-quinones with GSH. Reactions catalyzed by GST on the
following substrates are shown: (a) acrolein, (b) crotonaldehyde, (c) 4-hydroxynonenal, (d) adenine propenal, (e) dopa-o-quinone, and ( f )
aminochrome.

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also be conjugated with GSH by GST, and this reaction is similarly thought
to combat degenerative processes in the dopaminergic system in human brain
(Figure 2).

Degradation of Aromatic Amino Acids

In mammals, phenylalanine is degraded to acetoacetate and fumaric acid. The five
intermediates are tyrosine, 4-hydroxyphenylpyruvate, homogentisate, maleylacetoacetate, and fumarylacetoacetate. The cytosolic class Zeta GST has been identified as a maleylacetoacetate isomerase (14), and therefore catalyzes the penultimate
step in the catabolism of phenylalanine and tyrosine (shown in Figure 1).
GST and Synthesis of Steroid Hormones

Both testosterone and progesterone are synthesized from the cholesterol metabolite
3-hydroxy-5-pregnene-20-one. This compound undergoes side-chain cleavage
and oxidation of the 3-hydroxyl group in the A steroid ring to yield
5-androstene-3,17-dione as an intermediate in the testosterone pathway. Alternatively, it can undergo oxidation of the 3-hydroxyl to form 5-pregnene3,20-dione as an intermediate in the progesterone pathway. These two 3-keto5-steroids are converted to their 3-keto-4-steroid isomers by cytosolic GST
(62). The 3-keto-5-steroids are generated by actions of a 3-hydroxysteroid dehydrogenase that also exhibits keto-steroid isomerase activity and could therefore
be responsible for the isomerization step. However, Johansson & Mannervik (62)
have shown that a class Alpha GST isoenzyme present only in steroidogenic tissues
has a 230-fold higher catalytic efficiency in the isomerization of 3-keto-steroids
than the 3-hydroxysteroid dehydrogenase. It therefore seems most likely that
GST catalyzes this step in vivo.
GST and Eicosanoids: Synthesis and Inactivation

Glutathione transferases contribute to the biosynthesis of pharmacologically important metabolites of arachidonic acid. Although early studies suggested that
many GST catalyze the isomerization of PGH2 to a mixture of PGD2 and PGE2,
or reduce it to PGF2, it is now clear that certain transferases exhibit remarkable specificity for some of these reactions. The identification of mammalian
GSH-dependent prostaglandin D2 synthase as a cytosolic GST serves as an excellent paradigm in this regard (63, 64). This observation is of particular interest
as the enzyme contributes not only to PGD2 production but also to formation of
the downstream cyclopentenone prostaglandin, 15-deoxy-12,14-prostaglandin J2
(15d-PGJ2), which possesses distinct biological activities. Cytosolic transferases
expressed in human brain exhibit PGE2 synthase activity (65). In addition to the
cytosolic GST, members of the MAPEG family make major contributions to production of PGE2 (8), whereas a membrane-bound GSH-activated enzyme has been
shown to possess PGF2 synthase activity (66).
Prostaglandins and isoprostanes containing a cyclopentenone ring also represent GST substrates in glutathione-conjugation reactions (67). This modification
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facilitates the elimination of these eicosanoids from the cell via MRP1 and MRP3
transporters (68).
Leukotrienes (LTs) are another group of eicosanoids formed from arachidonic
acid. MAPEGs are critically involved in their synthesis because one member
uniquely activates 5-lipoxygenase, whereas several others catalyze the formation
of LTC4.
Modulation of Signaling Pathways by GST

As endogenous lipid mediators influence diverse signaling pathways, their metabolism by GST has many biological consequences. Although the effects of the
classical prostaglandins (PGD2, PGE2, and PGF2) are mediated through specific
G proteincoupled receptors, cyclopentenone prostaglandins exert their effects
through a separate mechanism. Undoubtedly the most widely studied of these is
15d-PGJ2, a downstream metabolite of PGD2. The ability of different transferases
to affect either synthesis or elimination of this eicosanoid places GST as central
regulators in this arena. Perhaps the most significant property of 15d-PGJ2 is its
ability to serve as an activating ligand for the peroxisome proliferator-activated
receptor (PPAR ). This transcription factor is a critical regulator of adipocyte
differentiation and also represents the molecular target of the thiazolidinedione
class of insulin sensitizing drugs. Over-expression of GST can diminish transactivation of gene expression by 15d-PGJ2 mediated by PPAR through conjugation
of the prostanoid with GSH (69).
15-Deoxy-12,14-prostaglandin J2 can stimulate nuclear factor-erythroid 2 p45related factor 2 (Nrf2)-mediated induction of gene expression through the antioxidant response element (ARE) (70, 71). This occurs because 15d-PGJ2 is able to
modify cysteine residues in the cytoskeleton-associated protein Keap1 (Kelchlike ECH-associated protein 1), and thus overcomes the ability of Keap1 to target
Nrf2 for proteasomal degradation (7173). Conjugation of 15d-PGJ2 with GSH
abolishes its ability to modify Keap1. A similar mechanism appears to underlie
the ability of 15d-PGJ2 to inactivate the subunit of the inhibitor of B kinase
(IKK) and inhibit nuclear factor B (NF-B)-dependent gene expression (74).
The extent to which GST-catalyzed synthesis and/or metabolism of 15d-PGJ2 impinges on these signaling pathways is an important area that warrants further study
(Figure 3).
The endogenous lipid peroxidation product 4-hydroxynonenal (4-HNE) is believed to act as an intracellular signaling molecule (7577), and therefore its conjugation with GSH will influence a number of pathways. Like 15d-PGJ2, this
2-alkenal is an ,-unsaturated carbonyl that can stimulate gene expression through
the ARE (78). In common with 15d-PGJ2 it is probable that Nrf2 mediates induction of ARE-driven genes by 4-HNE (79, 80). The aldehyde also prevents
activation of NF-B by inhibiting IB phosphorylation. It has been reported to
modulate several cell-surface receptors, activate epithelial growth factor receptor and platelet-derived growth factor- receptor, and upregulate transforming
growth factor receptor 1. Also, 4-HNE stimulates several components in signal
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Figure 3 Attenuation of 15-deoxy-12,14-prostaglandin J2 signaling by

GST. This figure shows the synthesis of 15d-PGJ2 and the various transcription factors whose activity may be influenced by the prostaglandin (6974).
transduction pathways, such as JNK, p38, and protein kinase C, as well as increasing p53 protein and promoting apoptosis (77). It is anticipated that conjugation of
4-HNE with GSH will influence many signal transduction pathways and modulate
the activity of transcription factors, including NF-B, c-Jun, and Nrf2.
GST FAMILIES
Cytosolic Enzymes
Mammalian cytosolic GSTs are all dimeric with subunits of 199244 amino acids
in length. Based on amino acid sequence similarities, seven classes of cytosolic GST are recognized in mammalian species, designated Alpha, Mu, Pi, Sigma,
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Theta, Omega, and Zeta (25). Other classes of cytosolic GST, namely Beta, Delta,
Epsilon, Lambda, Phi, Tau, and the U class, have been identified in nonmammalian species (5, 23, 81). In rodents and humans, cytosolic GST isoenzymes
within a class typically share >40% identity, and those between classes share
<25% identity.
At least 16 cytosolic GST subunits exist in the human. As those in the Alpha and
Mu classes can form heterodimers (2), a significantly larger number of isoenzymes
can be generated from these subunits. The total of 16 homodimers listed in Table 1
includes the relatively poorly characterized GSTM4-4 (82) and GSTM5-5 (83), as
well as a transferase, GSTA5-5, that has been identified by genomic cloning but
has not been characterized at the protein level (84). An additional human enzyme,
hGST5.8, with high activity toward 4-HNE, has been reported and is presumed to
be a class Alpha transferase (85). This enzyme seems to be distinct from GSTA1-1,
GSTA2-2, GSTA3-3, and GSTA4-4 but it is not included in Table 1 as its primary
structure has not been described. The transferases display overlapping substrate
specificities, a feature that makes it difficult to identify isoenzymes solely on their
catalytic properties. Substrates identified for each of the human cytosolic GST are
listed in Table 1 (some examples are illustrated in Figures 1 and 2).
Besides catalyzing conjugation, reduction, and isomerization reactions, cytosolic GST also bind, covalently and noncovalently, hydrophobic nonsubstrate ligands (2). This type of activity contributes to intracellular transport, sequestration,
and disposition of xenobiotics and hormones. Such compounds include azo-dyes,
bilirubin, heme, PAHs, steroids, and thyroid hormones; it is the nonsubstrate binding activity that led originally to class Alpha GST being called Ligandin (2).
Affinity labeling of rat class Alpha GST has revealed a high-affinity nonsubstrate binding site within the cleft between the two subunits (86), indicating that
there are two distinct xenobiotic-binding sites in certain isoenzymes. The second
nonsubstrate binding site formed in heterodimers will be distinct from those in
homodimers, and it may provide an evolutionary reason why it is beneficial for
members within the Alpha and Mu classes to heterodimerize.
Class Mu and Pi GST have been reported to inhibit Ask1 and JNK during
nonstressed conditions through physical interactions with the kinases (8789). It
has been shown that GSTM1 dissociates from Ask1 by heat shock (88), whereas
GSTP1 dissociates from JNK in response to oxidative stress (89). As described
above, GSTP1 also physically interacts with Prx VI, a process that leads to recovery of peroxiredoxin enzyme activity through glutathionylation of the oxidized
protein (60).
The majority of cytosolic GST isoenzymes are found in the cytoplasm of the
cell. However, mouse and human Alpha-class GSTA4-4 can associate with mitochondria and membranes (9092), as can mouse GSTM1-1 (91). In the case
of GSTA4-4, this entails phosphorylation of the transferase, and targeting is dependent on the Hsp70 chaperone (92). Using monkey COS cells, treatment with
4-HNE increases the amount of GSTA4-4 associated with the mitochondria (92).
A human transferase that is closely related to GSTA1-1 has been purified from
liver microsomes (56), and it appears that certain class Alpha enzymes have a
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Substrate preferences of human glutathione transferases
Family
Class, enzyme
Substrates or reaction
Cytosolic
Alpha, A1-1
5-ADD, BCDE, BPDE, Busulfan, Chlorambucil,

DBADE, DBPDE, BPhDE, N-a-PhIP
CuOOH, DBPDE, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole
5-ADD, 5-pregnene-3,20-dione, DBPDE
COMC-6, EA, 4-hydroxynonenal, 4-hydroxydecenal
not done
Alpha, A2-2
Alpha, A3-3
Alpha, A4-4
Alpha, A5-5
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Cytosolic
Mu, M1-1
Mu, M2-2
Mu, M3-3
Mu, M4-4
Mu, M5-5
trans-4-phenyl-3-buten-2-one, BPDE, CDE, DBADE,

trans-stilbene oxide, styrene-7,8-oxide
COMC-6, 1,2-dichloro-4-nitrobenzene, aminochrome,
dopa O-quinone, PGH2 PGE2
BCNU, PGH2 PGE2
CDNB
low for CDNB
Cytosolic
Pi, P1-1
acrolein, base propenals, BPDE, CDE, Chlorambucil,

COMC-6, EA, Thiotepa
Cytosolic
Sigma, S1-1
PGH2 PGD2
Cytosolic
Theta, T1-1
BCNU, butadiene epoxide, CH2Cl2, EPNP,

ethylene oxide
Theta, T2-2
CuOOH, menaphthyl sulfate
Cytosolic
Zeta, Z1-1
dichloroacetate, fluoroacetate, 2-chloropropionate,

malelyacetoacetate
Cytosolic
Omega, O1-1
Omega, O2-2
monomethylarsonic acid, dehydroascorbic acid

dehydroascorbic acid
Mitochondrial
Kappa, K1-1
CDNB, CuOOH, (S)-15-hydroperoxy-5,8,11,

13-eicosatetraenoic acid
MAPEG
gp I, MGST2
CDNB, LTA4 LTC4, (S)-5-hydroperoxy-8,11,

14-cis-6-trans-eicosatetraenoic acid
nonenzymatic binding of arachidonic acid
LTA4 LTC4
gp I, FLAP
gp I, LTC4S
MAPEG
gp II, MGST3
CDNB, LTA4 LTC4, (S)-5-hydroperoxy-8,11,

14-cis-6-trans-eicosatetraenoic acid
MAPEG
gp IV, MGST1
gp IV, PGES1
CDNB , CuOOH, hexachlorobuta-1,3-diene

PGH2 PGE2
Activity increased by treating enzyme with N-ethylmaleimide.
A systematic study of all these enzymes toward substrates has not been undertaken, and therefore it is not possible to
define relative activities toward the compounds listed. These data are taken from papers cited in the text.
Abbreviations: 5-ADD, 5-androstene-3,17-dione; BCDE, benzo[g]chrysene diol epoxide; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BPDE, benzo[a]pyrene diol epoxide; BPhDE, benzo[c]phenanthrene diol epoxide; CDE, chrysene1,2-diol 3,4-epoxide; COMC-6, crotonyloxymethyl-2-cyclohexenone; DBADE, dibenz[a,h]anthracene diol epoxide;
DBPDE, dibenzo[a,l]pyrene diol epoxide; EA, ethacrynic acid; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane; N-a-PhIP,
N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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propensity to associate with membranes. Mouse GSTO1-1 can be targeted to the

nucleus following TPA treatment (93), and rat GSTT2-2 can be found in the nucleus
following treatment with Oltipraz (4).
In addition to these GST classes, CLICs (chloride intracellular channels) (94,
95) and elongation factor 1B adopt the same crystal structure as cytosolic GST
(96). Other proteins, including ganglioside-induced differentiation-associated
protein-1 (97), have also been proposed to occupy the GST fold, but this remains
to be proven.
Mitochondrial GST
The mammalian mitochondrial class Kappa GST isoenzymes are dimeric and
comprise subunits of 226 amino acids. Mouse, rat, and human possess only a
single Kappa GST (6, 7, 98, 99). Molecular cloning and crystallography of the
mitochondrial GST have provided definitive evidence that it represents a distinct
type of transferase (6, 7). The three-dimensional fold of Kappa is more similar
to bacterial 2-hydroxychromene-2-carboxylate isomerase, a GSH-dependent oxidoreductase that catalyzes conversion of 2-hydroxy-chromene-2-carboxylate to
trans-O-hydroxy-benzylidenepyruvate, and to prokaryotic disulfide-bond-forming
DsbA and TcpG oxidoreductases, than to any of the cytosolic GST isoenzymes.
As such, it has provided a new insight into the evolution of GST.
GST class Kappa has high activity for aryl halides, such as CDNB, and can
reduce CuOOH and (S)-15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (99). In
view of its homology with 2-hydroxychromene-2-carboxylate isomerase, it will
be interesting to establish whether GST Kappa can metabolize aromatic hydrocarbons, such as naphthalene.
In the mouse, GST Kappa is present in large amounts in liver, kidney, stomach,
and heart, and electron microscopy has confirmed that it is associated with liver
and kidney mitochondria (100). Its tissue distribution in the rat seems similar to
that in the mouse (98). By contrast, GST Kappa appears to be more widely and
uniformly expressed in human tissues (99).
Although this transferase was originally isolated from mitochondria and is not
present in cytoplasm (98), it has also been shown to be located in peroxisomes
(99). The presence of GSTK1-1 in both organelles suggests it may be specifically
involved in -oxidation of fatty acids, either through its catalytic activity, some
transport function, or interaction with a membrane pore. The process of targeting
GST Kappa to mitochondria is unclear. It has been reported to associate with the
Hsp60 chaperone (7), and a possible cleavage site for a mitochondrial presequence
exists at the N-terminus (99). A peroxisomal targeting sequence (tripeptide ARL)
has been identified in the C-terminus of the human GSTK1 subunit (99).
Evolution of the GST Fold

Based on similarity of the tertiary structure of the N-terminal domain of cytosolic
transferases, the canonical GST fold is thought to have evolved from a thioredoxin/
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Figure 4 Schematic diagram showing evolution of the GST fold. The secondary structure arrangements of thioredoxin, the canonical cytosolic GST fold, and that of mitochondrial GSTK1 [the latter is predicted to be closely similar to the secondary structure
of bacterial DsbA (6, 7)] are illustrated. Arrows represent -sheets; rectangles represent
-helices. The regions corresponding to the core thioredoxin structure are shown for
the cytosolic GST fold and GSTK1. The positions of helical domain insertion that result in either fold are also shown, and they clearly illustrate two sites in the thioredoxin
fold that appear to have less evolutionary constraint. The differences in architecture
also provide substantial evidence that soluble GSTs have evolved through two differing
pathways.
glutaredoxin progenitor (3). Evolution of the cytosolic enzymes appears to be

through the addition of an all-helical domain after the thioredoxin
structure. By contrast, the crystal structure of the mitochondrial isoform, GSTK1-1,
provides clear evidence of a parallel evolutionary pathway (illustrated in Figure
4), as the all-helical domain responsible for binding of the second, electrophilic
substrate appears to have been inserted within the core after the
motif (7). The resulting Kappa isoform is more similar in its secondary structure
organization to the bacterial protein disulphide isomerase DsbA than to the cytosolic isomerases (6, 7). Moreover, the different mechanisms used to achieve the
common N- and C-terminal domains of cytosolic GST illustrate two regions in the
thioredoxin/glutaredoxin fold that are under less evolutionary constraint.
The cytosolic GSTs are catalytically active as dimers, with the dimer interface providing a noncatalytic site for ligand binding. A limited number of studies
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indicate that mammalian GSTM1 and GSTP1 can probably exist as monomers
through interactions with other proteins, such as Ask1, JNK, and Prx IV (60, 87
89). It is interesting to note that Pettigrew & Colman (101) have reported that
heterodimers can be formed between class Mu and class Pi polypeptides in vitro
without the need for denaturants, an observation that might reflect some promiscuity in the subunit dimerization in these two classes of GST. Monomeric forms
of cytosolic GST have been demonstrated convincingly in nonmammalian species
(102). The recent identification of a structural relationship between the cytosolic
GSTs and isoforms of the CLICs (94, 95, 102, 103) has revealed the potential for
proteins possessing the canonical GST fold to exist as soluble monomers when
purified in a functionally active state, in this case forming chloride ion channels.
It has also been shown that these monomers can undergo structural rearrangement
under oxidizing conditions to form dimers (103). Whether CLIC adopts this form
in the membrane is at this point unknown, but it has been proposed that a large
conformational rearrangement occurs, facilitating membrane insertion (102).
Identification of the canonical cytosolic GST fold in proteins involved in nondetoxication processes illustrates that this structure is amenable to many different
functions, yet it is not clear whether these proteins represent pathways of convergent evolution or the continued evolution of the cytosolic GST.
MAPEG Enzymes
These members of the GST superfamily constitute a unique branch where most of
the proteins are involved in the production of eicosanoids. Throughout nature, a
total of four MAPEG subgroups (IIV) have been described, with proteins within
a subgroup sharing >20% sequence identity. Six human MAPEGs have been
identified, and these fall within subgroups I, II, and IV (8).
The founding member of the MAPEG family, MGST1, was initially identified
as a microsomal CDNB-metabolizing enzyme that, in contrast to most cytosolic
GST, can be activated by treatment with N-ethylmaleimide (2, 8). Three further
MAPEG members with roles in eicosanoid synthetic pathways were subsequently
identified as leukotriene C4 synthase (LTC4S), a microsomal transferase that conjugates leukotriene A4 with GSH; 5-lipoxygenase-activating protein (FLAP), an
arachidonic acid-binding protein required for 5-lipoxygenase to exhibit full activity; and prostaglandin E2 synthase 1 (PGES1), which catalyses GSH-dependent
isomerization of PGH2 to PGE2 (8). Following the discovery of MGST1, FLAP,
and LTC4S, bioinformatic approaches were used to isolate cDNAs for MGST2
and MGST3, encoding enzymes that reduce (S)-5-hydroperoxy-8,11,14-cis-6trans-eicosatetraenoic acid (104). According to sequence-based subdivision of
the MAPEG family, subgroup I consists of FLAP, LTC4S, and MGST2; the only
member of subgroup II is MGST3; and MGST1 and PGES1 make up subgroup IV.
Subgroup III contains microsomal GST-like proteins from Escherichia coli and
Vibrio cholera.
Evidence suggests MGST1 functions solely as a detoxication enzyme. By
contrast, human MGST2 and MGST3 are capable of both detoxifying foreign
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compounds and synthesizing LTC4 (104); in the rat, MGST3 is apparently unable
to synthesize LTC4 (105). FLAP does not have catalytic activity but binds arachidonic acid and appears to be essential for the synthesis of all leukotrienes formed
downstream of 5-lipoxygenase. LTC4S and PGES1 seem to make no contribution
to detoxification, their catalytic actions being restricted to synthesis of LTC4 and
PGE2, respectively (see Table 1).
crystal structure for MGST1 has illustrated the hoDetermination of a 6 A
motrimeric quaternary structure of the enzyme (10), a quaternary structure also
observed for the other subgroup IV enzyme PGES1 (106). By contrast with the
trimeric structure of these enzymes, subgroup I contains members that either
form monomers or more complex aggregates. For example, FLAP can exist in
monomeric, dimeric and trimeric forms, and LTC4S can similarly form multimeric
complexes (107). FLAP and LTC4S can also form heterodimers and heterotrimers
with each other (107). More research is required to understand the stoichiometry
and membrane topology of these proteins.
GENETIC VARIATION IN HUMAN GLUTATHIONE

TRANSFERASES
Polymorphisms in Cytosolic GST
Cytosolic GST display polymorphisms in humans (Table 2, reviewed in 108
110), and this is likely to contribute to interindividual differences in responses to
xenobiotics. The earliest studies in this area addressed the question of whether individuals lacking GSTM1-1 and/or GSTT1-1 (i.e., are homozygous for GSTM1 0
and/or GSTT1 0 alleles) have a higher incidence of bladder, breast, colorectal,
head/neck, and lung cancer. Following the discovery of allelic variants of GSTP1
that encode enzymes with reduced catalytic activity, the hypothesis that combinations of polymorphisms in class Mu, Pi, and Theta class GST contribute to diseases
with an environmental component was examined by many researchers. In general,
it has been found that individual GST genes do not make a major contribution to
susceptibility to cancer, although GSTM1 0 has a modest effect on lung cancer,
GSTM1 0 and GSTT1 0 have a modest effect on the incidence of head and neck
cancer, and GSTP1 B influences risk of Barretts esophagus and esophageal carcinoma (111, 112, 112a). It is worth noting that a possible shortcoming of many
studies into the biological effects of GSTM1 0 and GSTT1 0 is that only individuals who are homozygous nulled for these genes (/) have been identified.
Invariably, individuals who are heterozygous (/+) or homozygous (+/+) for the
functional allele are not distinguished and analyzed separately. As a consequence,
the significance of being homozygous wild type for GSTM1 and GSTT1 is seldom addressed. The benefit of such a genotype is probably underestimated in the
literature because it is grouped together with the heterozygote genotype. A study
that uses a novel assay to distinguish between /, /+, and +/+ genotypes
at the GSTM1 locus has revealed significant protection against breast cancer in
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TABLE 2
Polymorphic human cytosolic GST
Class
Allele
Nucleotide(s) in gene at
variable position(s)
Protein affected
Alpha
GSTA1 A
GSTA1 B
GSTA2 A
GSTA2 B
GSTA2 C
GSTA2 D
GSTA2 E
631T/G, 567T, 69C, 52G

631G, 567G, 69T, 52A
328C, 335G, 588G, 629A
328C, 335G, 588G, 629C
328C, 335C, 588G, 629A
328C, 335G, 588T, 629C
328T, 335G, 588G, 629A
Reference protein levels

Low protein levels
Pro110, Ser112, Lys196, Glu210
Pro110, Ser112, Lys196, Ala210
Pro110, Thr112, Lys196, Glu210
Pro110, Ser112, Asn196, Ala210
Ser110, Ser112, Lys196, Glu210
Mu
GSTM1 A
GSTM1 B
GSTM1 0
GSTM1 1x2
GSTM3 A
GSTM3 B
GSTM4 A
GSTM4 B
519G
519C
gene deletion
gene duplication
wild-type
3 bp deletion in intron 6
wild-type
T2517C change in intron
Lys173
Asn173
No protein
Overexpression of M1 protein
Protein unchanged
Protein unchanged
Pi
GSTP1 A
GSTP1 B
GSTP1 C
GSTP1 D
313A, 341C, 555C

313G, 341C, 555T
313G, 341T, 555T
313A, 341T
Ile105, Ala114, Ser185

Val105, Ala114, Ser185
Val105, Val114, Ser185
Ile105, Val114
Sigma
GSTS1 A
GSTS1 B
IVS2 + 11 A
IVS2 + 11 C

Protein unchanged
Theta
GSTT1 A
GSTT1 0
GSTT2 A
GSTT2 B
wild-type gene
gene deletion
415A
415G

No protein
Met139
Ile139
Zeta
GSTZ1 A
GSTZ1 B
GSTZ1 C
GSTZ1 D
94A, 124A, 245C

94A, 124G, 245C
94G, 124G, 245C
94G, 124G, 245T
Lys32, Arg42, Thr82

Lys32, Gly42, Thr82
Glu32, Gly42, Thr82
Glu32, Gly42, Met82
Omega
GSTO1 A
GSTO1 B
GSTO1 C
GSTO1 D
GSTO2 A
GSTO2 B
419C, 464-IVS4 + 1 AAG

419C, 464 deleted
419A, 464-IVS4 + 1 AAG
419A, 464 deleted
424A
424G
Ala140, Glu155
Ala140, Glu155 deleted
Asp140, Glu155
Asp140, Glu155 deleted
Asn142
Asp142
Numbering of amino acids includes initiator methionine. Adapted from Reference 108.
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homozygous +/+ individuals (113). An assay has been developed that can identify heterozygotes at the GSTT1 locus (113a) though useful medical applications
remain to be established.
Besides influencing susceptibility to carcinogenesis, GSTP1 polymorphisms
are modifiers of response to chemotherapy in patients with metastatic colorectal
cancer (114) and those with multiple myeloma (115). It also influences risk of
therapy-related acute myeloid leukemia in patients successfully treated for breast
cancer, non-Hodgkins lymphoma, ovarian cancer, and Hodgkins disease (116).
By contrast with the weak effect that class Mu, Pi, and Theta GST polymorphisms have on tumorigenesis, a number of studies indicate that loss of these
genes increase susceptibility to inflammatory diseases, such as asthma and allergies, atherosclerosis, rheumatoid arthritis, and systemic sclerosis (117119).
In addition to allelic variants in class Mu, Pi, and Theta GST, polymorphisms
have also been identified in all the other classes of cytosolic GST (120122). Class
Alpha represents quantitatively a major group of transferases in the liver and these
enzymes presumably influence substantially detoxification processes. It has been
shown that both GSTA1 and GSTA2 are polymorphic, and the various alleles either
influence the amount of protein synthesized or the activity of the encoded proteins
(84, 123, 124). Further, GSTM4 and GSTT2 exhibit promoter polymorphisms that
are of functional significance (125). It will be interesting to know whether polymorphisms in these genes influence not only susceptibility to degenerative disease
but also efficacy of therapeutic drugs or adverse drug reactions.
Polymorphisms Among MAPEG Members

Several of the MAPEG genes have been reported to show variations in the population. As many as 46 single-nucleotide polymorphisms (SNPs) in MGST1 have
been reported in 48 healthy Japanese volunteers (126), and 25 diallelic variants in
MGST3 have been reported in Pima Indians (127); however, the number of true
alleles these SNPs reflect, and their biological significance, still requires evaluation in larger populations and in other ethnic groups. Promoter polymorphisms
have been reported in the LTC4S gene, 1072G/A, and 444A/C, and these appear to influence lung function (128). In the FLAP gene, also called ALOX5AP,
48 out of a possible 144 SNPs have been verified in 186 individuals from Iceland
(129). Among a population of 779 Icelandic individuals, a four-SNP haplotype was
found to associate with myocardial infarction and stroke, and this was attributed
to increased production of LTB4 (129).
CONSEQUENCE OF KNOCKOUT OF GST GENES

Disruption of Mouse Cytosolic GST Genes
Table 3 lists the mouse glutathione transferase genes (data taken from 130132).
A number of these have been disrupted by homologous recombination. The gene
knockout (KO) mice often show altered sensitivity to xenobiotics, and they reveal
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TABLE 3
Mouse GST genes

Gene
name
Previous designations
for subunits
Accession
number
Chromosomal
location
GSTA1
GSTA2
GSTA3
GSTA4
GSTA5
Ya
Ya2
GT10.6, Ya3, Yc
Yk, GST5.7
5
9
9
1
9
GSTM1
GSTM2
GSTM3
GSTM4
GSTM5
GSTM6
GSTM7
GT8.7, Yb1
Yb2
GT9.3, 4
Yb5, 7
Fsc2, mGSTM5
(also called mGSTM5)
3
GSTP1
GSTP2
Yf, piB
Yf, piA
Sigma
Ptgds2
Theta
GSTT1
GSTT2
GSTT3
5
Yrs
NP 032211
n
NM 010361
n
NM 133994
10
10
10
Zeta
GSTZ1
MAAI
12
GSTO1
GSTO2
p28
p
p
19
19
GSTK1
MGST2
FLAP
LTC4S
n
n
3
5
11
MAPEG, subgroup II
MGST3
MAPEG, subgroup IV
MGST1
Ptges1
6
2
Class or family
Alpha

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Mu
Pi
Omega
Kappa
MAPEG, subgroup I
NP 032207
NP 032208
p
CAA46155
p
NP 034487
NP 034488
AF319526
p
P19639
p
NP 081040
p
NP 034490
n
AJ000413
n
AK002213
n
NP 038569
NP 861461
NP 062328
NP 034493
NP 034492
NP 080895
AAP20655
BC028535
BC026209
n
NM 008521
NM 025569
NM 019946
n
NM 022415
3
3
3
3
3
3
3
19
19
Superscript prefix n = accession number for nucleotide sequence, superscript prefix p = accession number for protein
sequence.
The genes encoding the cytosolic class Sigma GSTS1 and the MAPEG PGES1 are called Ptgds2 and Ptges1, respectively.
This is adapted from the Web site established by Dr. William Pearson on mouse GST (132). The nomenclature for Mu-class
GST differs from that of Andorfer et al. (162): The subunit they called 3 is GSTM7, the subunit they called 4 is GSTM3,
and the subunit they call 7 is GSTM4.
that loss of certain GST isoenzymes causes an upregulation of the remaining

transferases.
Homozygous nulled GSTA4 mice appear normal but are more
susceptible to bacterial infection and display increased sensitivity to paraquat
(133). The GSH-conjugating activity toward 4-HNE in this mouse line was
CLASS ALPHA GST
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reduced to between 23% and 64% of wild-type levels in the tissues examined,
but it was particularly marked in brain, heart, kidney, and lung. Substantial increases in 4-HNE and malondialdehyde were found in the livers of KO animals
(133). The livers and brain of GSTA4/ mice contained increases in mRNA for
GSTA1/2, GSTA3, GSTM1, catalase, superoxide dismutases 1 and 2, and GPx1.
Activation of ARE-driven gene expression (78) appears to be one of the mechanisms by which these genes are upregulated in GSTA4 KO mice. Certainly, 4-HNE
is a Michael reaction acceptor (75) and many cancer chemopreventive blocking
agents that induce GST can be included in this category of compound (134). It is
therefore presumed that induction of transferases and antioxidant proteins in the
mutant mice represents a compensatory response to increases in the intracellular
levels of reactive aldehydes resulting from loss of GSTA4-4.
The GSTA4 subunit is induced in mice fed on diets containing the cancer
chemopreventive agents -angelicalactone, butylated hydroxyanisole, ethoxyquin,
indole-3-carbinol, limettin, oltipraz, or sulforaphane (135). These data suggest the
mouse GSTA4 gene contains an ARE. Consistent with this hypothesis, we have
found, using a bioinformatic search, that the 5 -upstream region of mouse GSTA4
contains the sequence 5 -TGAGTCAGC-3 . This sequence closely resembles the
5 -TGAGTCGGC-3 ARE in mouse NAD(P)H:quinone oxidoreductase 1 (136);
both differ from the prototypic core ARE, 5 -TGACnnnGC-3 (137), in having
a G rather than a C at nucleotide position 4 (shown underlined). Assuming this
putative ARE in GSTA4 is functional, induction of the gene by 4-HNE is likely
to be mediated by Nrf2. It is envisaged that increased concentrations of 4-HNE
lead to modification of cysteine residues in Keap1, stabilization and nuclear accumulation of Nrf2, and increased GSTA4-4 and glutathione levels, resulting in
increased capacity to metabolize 4-HNE (see Figure 5, color insert). According
to these predictions, mouse GSTA4-4 appears to comprise part of an autoregulatory homeostatic defense mechanism against lipid peroxidation products. Another
characteristic of the putative ARE in GSTA4 is that it contains an embedded 12-Otetradecanoylphorbol-13-acetate (TPA) response element and may therefore also
be regulated by the c-Jun transcription factor; for a review of transcriptional regulation of genes through the ARE and related enhancers, see References 138 and 139.
A mouse line lacking GSTM5, which encodes the brain/testisspecific transferase, has been generated, but no clear phenotype has been reported
to date (140).
CLASS Mu GST
Mice lacking both GSTP1 and GSTP2 have been generated (141).
Under normal conditions, the double gene knockout on 129MF1 or C57/BL6
backgrounds had no obvious phenotype. At a biochemical level, the mutant mice
demonstrated a complete lack of transferase activity toward ethacrynic acid in the
liver (141). Although GSTP1-1 is quantitatively the principal transferase in male
mouse liver, Western blotting failed to demonstrate compensatory increases in
expression of hepatic GSTA1/2, GSTA3, and GSTM1 subunits in the double gene
CLASS Pi GST
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KO animals (141). However, livers from GSTP1/P2/ mice have been reported
to contain a higher activator protein-1 activity than livers from GSTP1/P2+/+ mice
(142), a finding that is consistent with the hypothesis that class Pi GST inhibits
JNK (21, 89).
In a skin tumorigenesis regimen, GSTP1/P2/ mice yielded approximately
threefold more papillomas using 7,12-dimethylbenzanthracene as initiator and
TPA as promoter (141), demonstrating a role for GSTP1-1 in xenobiotic defense.
Surprisingly, GSTP1/P2/ mice are more resistant than wild-type mice to liver
toxicity caused by the analgesic acetaminophen, and this is attributed to faster
regeneration of hepatic GSH in the double gene KO animals (143). It was proposed that while Pi-class GST does not catalyze the conjugation of acetaminophen
with GSH, it contributes to oxidative stress by facilitating redox-cycling of the
acetaminophen metabolite NAPQI, possibly through formation of labile ipso
adducts with intracellular thiol groups (143). It is postulated that the absence of Pi
class GST lessens the ability of NAPQI to redox-cycle and thus deplete GSH.
This class of GST encodes the hematopoietic, or GSHdependent, prostaglandin D2 synthase. Knockout of the gene for this enzyme results in generation of mice with an allergic reaction that is weaker than wild-type
mice (144).
CLASS SIGMA GST
The murine GSTZ1 gene, encoding maleylacetoacetate (MAA)

isomerase (MAAI) has been disrupted on C57/BL6, 129SvJ, and BALB/c genetic
backgrounds. Under normal dietary conditions, the GSTZ1/ mice on C57/BL6
and 129SvJ backgrounds appeared healthy. However, rapid weight loss occurred
when the mutant mice were provided with drinking water containing 2% phenylalanine, resulting in death between 5 and 50 days (145). By contrast, under normal
dietary conditions, GSTZ1/ mice on a BALB/c background showed enlargement of liver and kidney as well as splenic atrophy (146). When administered 3%
phenylalanine in the drinking water, the adult mutant BALB/c mice developed
liver necrosis, macrovesicular steatosis, and a loss of circulating leucocytes.
At a biochemical level, livers from GSTZ1/ mice lacked activity toward maleylacetone and chlorofluoroacetic acid, suggesting there is no enzymatic redundancy for GSTZ1-1/MAAI activity. Large increases in fumarylacetoacetate, and
modest increases in succinylacetone were observed in the urine of mutant mice
(145). The latter metabolite was also observed in blood of GSTZ1/ mice (146).
The presence of fumarylacetoacetate in the urine of the KO mice suggests that this
MAA metabolite can be formed in extrahepatic tissue by an alternative catabolic
pathway (145). The pathophysiological effects observed in the GSTZ1/ animals
were attributed to failure to eliminate either succinylacetone or other MAA-derived
metabolites (146). The phenotype observed in the mutant mice was exacerbated
by inclusion of phenylalanine in the diet.
Hepatic detoxication and antioxidant enzymes are induced as a consequence
of perturbations in tyrosine degradation in the GSTZ1/ mice. The GSTA1/2,
CLASS ZETA GST
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GSTM1, and GSTP1/2 subunits, as well as NAD(P)H:quinone oxidoreductase

(NQO1), are increased in the livers of GSTZ1/ mice fed on a control diet (145,
146). It appears likely that succinylacetone, and possibly MAA or succinylacetoacetate, are responsible for enzyme induction in these mice. It is noteworthy that
certain metabolites that accumulate in the GSTZ1/ mice are capable of modifying protein thiol groups (147). This feature infers that enzyme induction is a
response to redox stress (Figure 6). It is not known whether the metabolite(s) that
affects gene induction is also responsible for the pathological changes.
Disruption of Mouse MAPEG Genes

The MAPEG genes in subgroups I and IV have been disrupted. The resulting mice
clearly show MAPEG genes are involved in allergic and inflammatory processes.
No evidence has been reported that they combat oxidative stress in vivo, although
this is anticipated from their Se-independent glutathione peroxidase activity.
Mice lacking the FLAP gene are unable to make leukotrienes.
Following stimulation with the calcium ionophore A23187, primary cultures of
peritoneal macrophages from FLAP/ mice did not synthesize LTC4 (148). However, production of PGE2 and thromboxane B2 was increased by stimulated peritoneal macrophages from FLAP/ mice to a level beyond that seen in wild-type
macrophages. In experimental peritonitis affected by Zymosan A, analyses of
peritoneal lavage fluid revealed no LTC4 synthesis in mutant mice but significant
amounts of LTC4 synthesis in wild-type mice. Importantly, no metabolites of the
5-lipoxygenase pathway, such as 5-HETE and LTA4, were found in lavage of the
FLAP/ mice, suggesting FLAP is essential for the synthesis of all leukotrienes.
Topical application of arachidonic acid to the ears of mutant mice elicited a reduced
inflammatory response as measured by edema.
Mice with the LTC4S gene disrupted develop normally and are fertile. In vitro
conjugation of LTA4 methyl ester with GSH in colon, spleen, lung, brain, and
tongue prepared from LTC4S/ mice was reduced to 10% of that in wild-type
mice (149). By contrast, in testis of the KO animals conjugation of LTA4 methyl
ester with GSH was only reduced to about 65% of the level in wild-type mice,
and possibly cytosolic class Mu GST contribute to LTC4 synthase activity in this
organ. Stimulation of LTC4 production by IgE was abolished in bone marrow
derived mast cells (BMMC) from mutant mice. Also, there was no evidence of
production of the LTC4 metabolites, LTD4 and LTE4, in IgE-stimulated BMMC
from LTC4S/ mice. By contrast, LTB4, 5-HETE, and PGD2 were produced by
BMMC from LTC4S/ mice (149). Examination of an acute inflammatory response in LTC4S/ mice by intraperitoneal injection with Zymosan A revealed
that protein extravasation was significantly reduced in the mutant mice, and this was
associated with failure to produce LTE4. The ear-swelling anaphylactic response
of LTC4S/ mice was reduced to about 50% of the response seen in LTC4S+/+
mice.
MAPEG SUBGROUP I
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Mice with disruption of the Ptges gene appear normal.

Macrophages from Ptges/ mice cultured in the presence of lipopolysaccharide
(LPS) for 16 h did not synthesize PGE2 but did produce IL-6, whereas macrophages
from wild-type mice produced both PGE2 and IL-6 (150). In vivo examination of
the arthritic response to immunization with chicken type II collagen showed that the
null mouse was protected against fibroplasias, inflammation, proteoglycan damage,
cellular infiltration, and cartilage damage associated with the disease (150).
Fever that occurs during inflammatory processes and infection arises in part
from PGE2 synthesis in the brain that acts on EP3 receptor-expressing neurons in
the hypothalamus. Following challenge with LPS, little increase above basal levels
of PGE2 was observed in CSF from Ptges/ mice, whereas substantial increases
were observed in CSF from wild-type mice (151). Thus, Ptges1 partly controls
fever that accompanies inflammatory disease.

MAPEG SUBGROUP IV
Knockout of Non-Mammalian GST Genes

In Proteus mirabilis, the cytosolic class Beta GST gene has been knocked out,
and the resulting bacterial strain was found to be more sensitive to H2O2, CDNB,
fosfomycin, and minocycline (24). In Drosophila melanogaster, a gene encoding
a protein homologous to mammalian MGST1 has been disrupted, and the resulting
fly line had a reduced life span (152).
REGULATION OF GST BY ENDOGENOUS ELECTROPHILES

THROUGH THE Keap1/Nrf2 PATHWAY
The fact that a significant number of cytosolic GST subunits are upregulated in
GSTA4/ and GSTZ1/ mice indicates that the expression levels of these transferases is dictated in part by endogenous substrates. This is consistent with the
proposal that GST isoenzymes detoxify endogenous carbonyl-containing compounds in vivo. In the case of GSTA4/ mice, the principal regulatory endobiotic
is probably 4-HNE (Figure 5). In the case of GSTZ1/ mice, it is likely that upregulation of class Alpha, Mu, and Pi transferases is stimulated by the tyrosine
catabolites MAA, succinylacetoacetate, or succinylacetone (Figure 6).
Conditional disruption of the selenocysteine tRNA[Ser]Sec (Trsp/) in the livers
of mice, by crossing onto an albumin-Cre transgenic background, leads to a loss of
the Se-dependent GPx1 and a marked increase in class Mu GST (153). Se-deficient
rats, which like Trsp/ mice have an impaired ability to synthesize selenoproteins,
possess large increases in hepatic class Alpha, Mu, and Theta GST, as well as aldoketo reductase 7A1 (154). This observation suggests that the Trsp/ mice almost
certainly overexpress many antioxidant enzymes besides class Mu GST. In the
mutant mice and Se-deficient rats, the stimulus for GST induction is presumed to
be increases in intracellular levels of hydroperoxides and H2O2.
It is postulated that as 4-HNE, tyrosine breakdown products, hydroperoxides,
and H2O2 can all modify protein thiol groups, the Keap1/Nrf2 pathway mediates
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induction of GST genes in the KO animals described above. According to this proposal, increased levels of 4-HNE, either MAA or its metabolites, and peroxides
in the GSTA4/, GSTZ1/, and Trsp/ mice modify Keap1, causing accumulation of Nrf2 and its translocation to the nucleus. Thereafter, Nrf2 is recruited
to ARE enhancers in the promoters of inducible genes. A substantial number of
GST genes have been found to contain an ARE or related sequences. Table 4
provides a compilation of those GST, NQO1, and SOD1 genes that contain such
elements (136, 137, 139, 155162) and could therefore be regulated by this mechanism; it also contains inducible GST genes that have ARE-like sequences that have
yet to be shown to be functional enhancers (these uncharacterized enhancers are
TABLE 4 Comparison between antioxidant response elements in GST, NQO1, and

SOD1 genes
Species
Gene
Enhancer
5 -USR
Rat
GSTA2
ARE
gctaa TGg TGACaaAGCA
Enhancer
687
Rat
GSTA5
ARE
gacac gGC TGACagAGCg
470
Rat
GSTP1
GPEI
agtca cta TGAtTCAGCA
2430
Mouse
GSTA1
EpRE
gctaa TGg TGACaaAGCA
719
Mouse
GSTA3
ARE
ctcag gca TGACattGCA
138
Mouse
GSTA4
n.c.
ctcag Taa TGAgTCAGCg
147
Mouse
GSTM1
n.c.
tgaac Ttg TGACagtGCA
1643
Mouse
GSTM2
n.c.
ggagt TGC TGACaCAGgt
202
Mouse
GSTM3
n.c.
tgaac Ttg TGACagtGCA
2315
Mouse
GSTP1
ARE
caacg TGt TGAgTCAGCA
50
Mouse
GSTP2
n.c.
caacg TGt TGAgTCAGCA
61
Human
MGST1
EpRE
ggaca Tcg TGACaaAGCA
490
Rat
NQO1
ARE
agtca cag TGACTtgGCA
412
Mouse
Nqo1
ARE
agtca cag TGAgTCgGCA
426
Human
NQO1
ARE
agtca cag TGACTCAGCA
460
Human
SOD1
ARE
ataac Taa TGACatttCt
323
ARE core
T-MARE
TGACnnnGC
TGC TGACTCAGCA
The mouse GSTM3 gene was called GSTM4 and 4 in Reference 162.
The core ARE required for gene induction is usually regarded as 5 -TGACnnnGC-3 , based on mutational analysis
of the promoter of rat GSTA2 (137). The nucleotides located in the 5 -upstream region (5 -USR) of the GSTA2-ARE
have been found to influence basal expression without altering the relative magnitude of induction, and therefore this
region is included in the line-up. Nucleotides in capital bold print are those that share identity with the Maf recognition
element (MARE); this contains an embedded TPA-response element, denoted by the abbreviation T-MARE (138).
The numbering in the right-hand column is the position of the 3 A nucleotide with respect to the transcriptional start
site; in the cases of rat GSTA5 and mouse GSTA4 this nucleotide is a G, and in the cases of GSTM2 and SOD1 this
nucleotide is a T. Data are taken from References 136, 137, 155162. The abbreviation n.c. stands for not characterized.
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indicated by the abbreviation n.c.). The observation that disruption of GSTA4 and
GSTZ1 genes upregulates the ARE-gene battery supports the hypothesis that the
transferases encoded by these genes not only make a major functional contribution to an antioxidant and electrophile defense network but that their substrates are
endogenous activators of Nrf2.
The notion that Nrf2 mediates basal expression of GST by endogenous thiolactive endobiotics is supported by the fact that in mice nulled for this transcription
factor the normal homeostatic levels of many class Alpha, Mu, and Pi transferases
are reduced (163). For example, the levels of mRNA encoding GSTA1, GSTA2,
GSTM1, and GSTM3 in the livers of Nrf2/ mice fed on a normal diet have
been reported to be less than 20% of the levels observed in Nrf2+/+ mice (131).
In addition to changes in expression of cytosolic GST, microarray analyses have
shown that expression of MAPEG genes is also affected in Nrf2 KO mice (164,
165). Further work is required to establish how important Nrf2 is in regulating
GST in species other than the mouse.
It should be appreciated that Nrf2 is not the only transcription factor involved
in regulating GST through the ARE. The 5 -upstream region immediately adjacent
to the core ARE in genes such as rat GSTA2, mouse GSTA1, mouse GSTM2,
mouse GSTP1, and mouse GSTP2 conforms more closely to a TRE-containing
Maf recognition element (i.e., T-MARE) than does the same region in rat GSTP1,
mouse GSTA3, or any of the NQO1 genes; for a review of transcriptional regulation
of AREs and MAREs, see Reference 138. It appears that some of these GST genes
may be regulated entirely by Nrf2-small Maf heterodimers, whereas others may
be regulated not only by Nrf2-small Maf heterodimers but also by small and large
Maf homodimers. The positive and negative regulation of ARE-driven genes is an
area that needs further study.
OVEREXPRESSION OF GSTs DURING TUMORIGENESIS

Expression of GST isoenzymes increases during the development of cancer. The
classic Solt-Farber liver chemical carcinogenesis model has been widely studied in this context. This model is established by subjecting rats to the following
three-step procedure: (a) initiation with diethylnitrosamine, (b) selective growth
inhibition of noninitiated hepatocytes with 2-acetylaminofluorene, and (c) stimulation of liver growth by partial hepatectomy (165a). Examination of this cancer
model has revealed that GSTP1 is upregulated >20-fold in both rat preneoplastic
nodules and hepatocellular carcinomas (2). This elevation occurs by transcriptional
activation through GPEI (155), and recent work has revealed that this is in part
mediated by Nrf2 (165b). It appears that sequences immediately 5 to the GPEI
element are required for strong enhancer activity, but the factor(s) involved has
not been identified. Members of the ARE-gene battery are often overexpressed
during carcinogenesis, and it seems likely that Nrf2 may be responsible for this
phenotype.
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CONCLUDING REMARKS
This review describes recent advances in knowledge about the transferases. The
availability of gene KO models has given unprecedented insights into the in vivo
functions of GST and MAPEG proteins. These studies have demonstrated that
cytosolic GST are an integral part of a dynamic and interactive defense mechanism
that protects against cytotoxic electrophilic chemicals and allows adaptation to
exposure to oxidative stress. They have antioxidant and antiinflammatory activities.
Similar investigations have shown MAPEG members contribute to inflammatory
responses, although it is likely that some are also involved in antioxidant defenses.
Further work is required to elucidate the biological functions of the mitochondrial
class Kappa GST.
Evidence suggests cytosolic GST metabolize many endogenous and foreign
compounds that stimulate expression of the ARE-gene battery. Their catalytic actions therefore negatively regulate Nrf2 by protecting Keap1 from modification of
those cysteines (Cys-273 and Cys-288) that are required to capture and destabilize
the transcription factor. A most important consequence of this conclusion is that
GST indirectly control the levels of other antioxidant and drug-metabolizing enzymes that are regulated through the Keap1/Nrf2 pathway. In addition to phase I,
phase II, and phase III detoxication proteins, GST will negatively regulate chaperones, ubiquitin-proteasome components, inflammation-associated proteins, and
apoptosis-associated proteins (165, 166).
The gene KO mouse models have revealed the importance of GST in detoxifying 4-HNE and tyrosine catabolites. It is predicted that glutathione transferases
similarly contribute to the elimination of 15d-PGJ2 in vivo. Thus, knockout of
certain GST genes will cause relative accumulation of 15d-PGJ2 and constitutive
upregulation of PPAR -driven gene expression and a decrease in expression of
NF-B-driven genes. A possible candidate for this function is GSTA3-3 because its
levels increase markedly in mouse 3T3-L1 cells during adipogenesis (70). It can be
hypothesized that induction of GSTA3 reflects a cellular response to accumulation
of 15d-PGJ2 designed to metabolize and eliminate the prostanoid.
A possibility that remains to be explored is whether polymorphisms in human
GST genes influence the activity of Nrf2, PPAR or NF-B.
ACKNOWLEDGMENTS
We are enormously grateful to the many colleagues in the GST field who have
generously given advice and details of their ongoing work. We can only apologize
to this community that space constraints have prevented us from citing many
excellent papers from our fellow researchers. We particularly thank Drs. Philip
Board, Irving Listowsky, and Bill Pearson for critical comments about the mouse
GST nomenclature. The work from the Hayes laboratory is funded by the Medical
Research Council (G0000281), the Association for International Cancer Research
(02049, 03074), and the World Cancer Research Fund (2000/11).
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LITERATURE CITED
1. Keen JH, Jakoby WB. 1978. Glutathione
transferases. Catalysis of nucleophilic
reactions of glutathione. J. Biol. Chem.
253:565457
2. Hayes JD, Pulford DJ. 1995. The glutathione S-transferase supergene family:
regulation of GST and the contribution
of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev.
Biochem. Mol. Biol. 30:445600
3. Armstrong RN. 1997. Structure, catalytic mechanism, and evolution of
the glutathione transferases. Chem. Res.
Toxicol. 10:218
4. Hayes JD, McLellan LI. 1999. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress.
Free Radic. Res. 31:273300
5. Sheehan D, Meade G, Foley VM, Dowd
CA. 2001. Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient
enzyme superfamily. Biochem. J. 360:1
16
6. Ladner JE, Parsons JF, Rife CL, Gilliland
GL, Armstrong RN. 2004. Parallel evolutionary pathways for glutathione transferases: structure and mechanism of
the mitochondrial class Kappa enzyme
rGSTK1-1. Biochemistry 43:35261
7. Robinson A, Huttley GA, Booth
HS, Board PG. 2004. Modelling and
bioinformatics studies of the human
Kappa class glutathione transferase
predict a novel third transferase family with homology to prokaryotic
2-hydroxychromene-2-carboxylate isomerases. Biochem. J. 379:54152
8. Jakobsson P-J, Morgenstern R, Mancini
J, Ford-Hutchinson A, Persson B.
9.
10.
11.
12.
13.
14.
15.
1999. Common structural features of

MAPEGa widespread superfamily of
membrane associated proteins with
highly divergent functions in eicosanoid
and glutathione metabolism. Protein Sci.
8:68992
Armstrong RN. 2000. Mechanistic diversity in a metalloenzyme superfamily.
Biochemistry 39:1362532
Holm PJ, Morgenstern R, Hebert H.
2002. The 3-D structure of microsomal
glutathione transferase 1 at 6 A resolution as determined by electron crystallography of p22121 crystals. Biochim.
Biophys. Acta 1594:27685
Khojasteh-Bakht SC, Nelson SD, Atkins
WM. 1999. Glutathione S-transferase
catalyzes the isomerization of (R)-2hydroxymenthofuran to mintlactones.
65
Dixon DP, Cole DJ, Edwards R. 2000.
Characterization of a Zeta class glutathione transferase from Arabidopsis
thaliana with a putative role in tyrosine catabolism. Arch. Biochem. Biophys. 384:40712
Singhal SS, Piper JT, Srivastava SK,
Chaubey M, Bandorowicz-Pikula J,
et al. 1996. Rabbit aorta glutathione Stransferases and their role in bioactivation of nitroglycerin. Toxicol. Appl.
Pharmacol. 140:37886
Fernandez-Cano n JM, Penalva MA.
1998. Characterization of a fungal maleylacetoacetate isomerase gene and
identification of its human homologue.
J. Biol. Chem. 273:32937
Board PG, Coggan M, Chelvanayagam
G, Easteal S, Jermiin LS, et al.
2000. Identification, characterization,
and crystal structure of the Omega class
7 Jan 2005
13:55
78

16.
17.
18.
19.
20.
21.
22.
23.
AR
HAYES
AR232-PA45-03.tex
AR232-PA45-03.sgm
JOWSEY
FLANAGAN
glutathione transferases. J. Biol. Chem.

275:24798806
Caccuri AM, Antonini G, Allocati N, Di
Ilio C, De Maria F, et al. 2002. GSTB11 from Proteus mirabilis. A snapshot
of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme. J. Biol. Chem. 277:18777
84
Zakharyan RA, Sampayo-Reyes A,
Healy SM, Tsaprailis G, Board PG, et al.
2001. Human monomethylarsonic acid
(MMA(V)) reductase is a member of
the glutathione S-transferase superfamily. Chem. Res. Toxicol. 14:105157
Evans JF, Leville C, Mancini JA, Prasit P,
Therien M, et al. 1991. 5-Lipoxygenaseactivating protein is the target of a quinoline class of leukotriene synthesis inhibitors. Mol. Pharmacol. 40:2227
Matsushita N, Aritake K, Takada A,
Hizue M, Hayashi K, et al. 1998. Pharmacological studies on the novel antiallergic drug HQL-79: II. Elucidation
of mechanisms for antiallergic and antiasthmatic effects. Jpn. J. Pharmacol. 78:
1122
Jakobsson P-J, Thoren S, Morgenstern
R, Samuelsson B. 1999. Identification
of human prostaglandin E synthase: a
microsomal, glutathione-dependent, inducible enzyme, constituting a potential
novel drug target. Proc. Natl. Acad. Sci.
USA 96:722025
Ruscoe JE, Rosario LA, Wang T, Gate
L, Arifoglu P, et al. 2001. Pharmacologic or genetic manipulation of glutathione S-transferase P1-1 (GST) influences cell proliferation pathways. J.
Ranson H, Rossiter L, Ortelli F, Jensen
B, Wang X, et al. 2001. Identification
of a novel class of insect glutathione
S-transferases involved in resistance to
DDT in the malaria vector Anopheles
gambiae. Biochem. J. 359:295304
Edwards R, Dixon DP. 2004. Metabolism of natural and xenobiotic sub-
24.
25.
26.
27.
28.
29.
30.
31.
32.
LaTeX2e(2002/01/18)
P1: GCE
strates by the plant glutathione Stransferase superfamily. In Molecular

Ecotoxicology of Plants, ed. H Sandermann, Ecological Studies Vol. 170, pp.
1750. Heidelberg: Springer Verlag
Allocati N, Favaloro B, Masulli M,
Alexeyev MF, Di Ilio C. 2003. Proteus
mirabilis glutathione S-transferase B11 is involved in protective mechanisms
against oxidative and chemical stress.
Biochem. J. 373:30511
Veal EA, Toone WM, Jones N, Morgan
BA. 2002. Distinct roles for glutathione
S-transferases in the oxidative stress response in Schizosaccharomyces pombe.
J. Biol. Chem. 277:3552331
Leiers B, Kampkotter A, Grevelding CG,
Link CD, Johnson TE, Henkle-Duhrsen
K. 2003. A stress-responsive glutathione
S-transferase confers resistance to oxidative stress in Caenorhabditis elegans. Free Radic. Biol. Med. 34:1405
15
An JH, Blackwell TK. 2003. SKN-1
links C. elegans mesendodermal specification to a conserved oxidative stress
response. Genes Dev. 17:188293
Desikan R, A-H-Mackerness S, Hancock
JT, Neill SJ. 2001. Regulation of the
Arabidopsis transcriptome by oxidative
stress. Plant Physiol. 127:15972
Wang MC, Bohmann D, Jasper H.
2003. JNK signaling confers tolerance
to oxidative stress and extends lifespan in Drosophila. Develop. Cell 5:
81116
Kobayashi M, Itoh K, Suzuki T, Osanai
H, Nishikawa K, et al. 2002. Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1
system. Genes Cells 7:80720
Grundy JE, Storey KB. 1998. Antioxidant defences and lipid peroxidation
damage in estivating toads, Scaphiopus
couchii. J. Comp. Physiol. B 168:132
42
Amicarelli F, Falone S, Cattani F, Alamanou MT, Bonfigli A, et al. 2004.
7 Jan 2005
13:55
AR
AR232-PA45-03.tex
AR232-PA45-03.sgm
LaTeX2e(2002/01/18)
P1: GCE

33.
34.
35.
36.
37.
38.
39.
Amphibian transition to the oxidant terrestrial environment affects the expression of glutathione S-transferases isoenzymatic pattern. Biochim. Biophys. Acta
1691(23):18192
Haimeur A, Conseil G, Deeley RG,
Cole SP. 2004. The MRP-related and
BCRP/ABCG2 multidrug resistance
proteins: biology, substrate specificity
and regulation. Curr. Drug Metab. 5:
2153
Paumi CM, Ledford BG, Smitherman
PK, Townsend AJ, Morrow CS. 2001.
Role of multidrug resistance protein 1
(MRP1) and glutathione S-transferase
A1-1 in alkylating agent resistance.
Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil
versus melphalan toxicity. J. Biol. Chem.
276:795256
Morrow CS, Smitherman PK, Townsend
AJ. 2000. Role of multidrug-resistance
protein 2 in glutathione S-transferase
P1-1-mediated resistance to 4-nitroquinoline 1-oxide toxicities in HepG2
cells. Mol. Carcinog. 29:17078
Awasthi S, Sharma R, Singhal SS,
Zimniak P, Awasthi YC. 2002. RLIP76,
a novel transporter catalysing ATPdependent efflux of xenobiotics. Drug
Hamilton DS, Zhang X, Ding Z, Hubatsch I, Mannervik B, et al. 2003. Mechanism of the glutathione transferasecatalyzed conversion of antitumor
2-crotonyloxymethyl-2-cycloalkenones
to GSH adducts. J. Am. Chem. Soc. 125:
1504958
Lien S, Larsson A-K, Mannervik B.
2002. The polymorphic human glutathione transferase T1-1, the most efficient glutathione transferase in the
denitrosation and inactivation of the
anticancer drug 1,3-bis(2-chloroethyl)1-nitrosourea. Biochem. Pharmacol. 63:
19197
Abel EL, Bammler TK, Eaton DL. 2004.
40.
41.
42.
43.
44.
45.
46.
79
Biotransformation of methyl parathion

by glutathione S-transferases. Toxicol.
Sci. 79:22432
Abel EL, Opp SM, Verlinde CLMJ,
Bammler TK, Eaton DL. 2004. Characterization of atrazine biotransformation
by human glutathione S-transferases.
Toxicol. Sci. 80:23036
Kelly VP, Ellis EM, Manson MM,
Chanas SA, Moffat GJ, et al. 2000.
Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer
of aflatoxin B1-aldehyde reductase, the
glutathione S-transferase A5 and P1
subunits, and NAD(P)H:quinone oxidoreductase in rat liver. Cancer Res. 60:
95769
Sundberg K, Widersten M, Seidel A,
Mannervik B, Jernstrom B. 1997. Glutathione conjugation of bay- and fjordregion diol epoxides of polycyclic hydrocarbons by glutathione transferases
M1-1 and P1-1. Chem. Res. Toxicol. 10:
122127
Hu X, Pal A, Krzeminski J, Amin
S, Awasthi YC, et al. 1998. Specificities of human glutathione S-transferase
isoenzymes toward anti-diol epoxides of methylchrysenes. Carcinogenesis
19:168589
Dreij K, Sundberg K, Johansson A-S,
Nordling E, Seidel A, et al. 2002. Catalytic activities of human Alpha class
glutathione transferases towards carcinogenic dibenzo[a,l]pyrene diol epoxides. Chem. Res. Toxicol. 15:82531
Coles B, Nowell SA, MacLeod SL,
Sweeney C, Lang NP, Kadlubar FF.
2001. The role of human glutathione
S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the
effect of a polymorphism in hGSTA1
on colorectal cancer risk. Mutat. Res.
482:310
Wheeler JB, Stourman NV, Their R,
Dommermuth A, Vuilleumier S, et al.
7 Jan 2005
13:55
80

47.
48.
49.
50.
51.
51a.
52.
53.
AR
HAYES
AR232-PA45-03.tex
AR232-PA45-03.sgm
JOWSEY
FLANAGAN
2001. Conjugation of haloalkanes by

bacterial and mammalian glutathione
transferases: mono- and dihalomethanes.
Guengerich FP, McCormick WA,
Wheeler JB. 2003. Analysis of the
kinetic mechanism of haloalkane conjugation by mammalian Theta-class
glutathione transferases. Chem. Res.
Toxicol. 16:149399
Xu K, Thornalley PJ. 2001. Involvement of glutathione metabolism in the
cytotoxicity of the phenethyl isothiocyanate and its cysteine conjugate to human leukaemia cells in vitro. Biochem.
Pharmacol. 61:16577
Anders MW, Dekant W. 1998. Glutathione-dependent bioactivation of haloalkenes. Annu. Rev. Pharmacol. Toxicol.
38:50137
Lyttle MH, Satyan A, Hocker MD, Bauer
KE, Caldwell CG, et al. 1994. Glutathione S-transferase activates novel
alkylating agents. J. Med. Chem. 37:
15017
Morgan AS, Sanderson PE, Borch RF,
Tew KD, Niitsu Y, et al. 1998. Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by
glutathione S-transferase. Cancer Res.
58:256875
Rosen LS, Brown J, Laxa B, Boulos L,
Reiswig L, et al. 2003. Phase I study
of TLK286 (glutathione S-transferase
P1-1 activated glutathione analogue) in
advanced refractory solid malignancies.
Clin. Cancer Res. 9:162838
Findlay VJ, Townsend DM, Saavedra
JE, Buzard GS, Citro ML, et al. 2004.
Tumor cell responses to a novel glutathione S-transferase-activated nitric
oxide-releasing prodrug. Mol. Pharmacol. 65:107079
Marnett LJ, Riggins JN, West JD. 2003.
Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 111:58393
LaTeX2e(2002/01/18)
P1: GCE
54. Hurst R, Bao Y, Jemth P, Mannervik B,

Williamson G. 1998. Phospholipid hydroperoxide glutathione peroxidase activity of human glutathione transferases.
Biochem. J. 332:97100
55. Yang Y, Sharma R, Zimniak P, Awasthi
YC. 2002. Role of class glutathione
S-transferases as antioxidant enzymes in
rodent tissues. Toxicol. Appl. Pharmacol.
182:10515
56. Prabhu KS, Reddy PV, Jones EC,
Liken AD, Reddy CC. 2004. Characterization of a class alpha glutathione
S-transferase with glutathione peroxidase activity in human liver microsomes. Arch. Biochem. Biophys. 424:72
80
57. Hiratsuka A, Yamane H, Yamazaki S,
Ozawa N, Watabe T. 1997. Subunit Yaspecific glutathione peroxidase activity toward cholesterol 7-hydroperoxides
of glutathione S-transferases in cytosols
from rat liver and skin. J. Biol. Chem.
272:476369
58. Hubatsch I, Ridderstrom M, Mannervik
B. 1998. Human glutathione transferase
A4-4: an Alpha class enzyme with high
catalytic efficiency in the conjugation of
4-hydroxynonenal and other genotoxic
products of lipid peroxidation. Biochem.
J. 330:17579
59. Hiratsuka A, Hirose K, Saito H, Watabe
T. 2000. 4-Hydroxy-2(E)-nonenal enantiomers: (S)-selective inactivation of
glyceraldehyde-3-phosphate dehydrogenase and detoxification by rat glutathione S-transferase A4-4. Biochem. J.
349:72935
60. Manevich Y, Feinstein SI, Fisher AB.
2004. Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pGST. Proc. Natl. Acad. Sci.
USA 101:378085
61. Dagnino-Subiabre A, Cassels BK, Baez
S, Johansson A-S, Mannervik B, SeguraAguilar J. 2000. Glutathione transferase
M2-2 catalyzes conjugation of dopamine
7 Jan 2005
13:55
AR
AR232-PA45-03.tex
AR232-PA45-03.sgm
LaTeX2e(2002/01/18)
P1: GCE
62.

63.
64.
65.
66.
67.
68.
and dopa o-quinones. Biochem. Biophys.

Johansson A-S, Mannervik B. 2001.
Human glutathione transferase A3-3, a
highly efficient catalyst of double-bond
isomerization in the biosynthetic pathway of steroid hormones. J. Biol. Chem.
276:3306165
Kanaoka Y, Ago H, Inagaki E,
Nanayama T, Miyano M, et al. 1997.
Cloning and crystal structure of haematopoietic prostaglandin D synthase. Cell
90:108595
Jowsey IR, Thomson AM, Flanagan JU,
Murdock PR, Moore GBT, et al. 2001.
Mammalian class Sigma glutathione
S-transferases: catalytic properties and
tissue-specific expression of human
and rat GSH-dependent prostaglandin
D2 synthases. Biochem. J. 359:507
16
Beuckmann CT, Fujimori K, Urade Y,
Hayaishi O. 2000. Identification of muclass glutathione transferases M2-2 and
M3-3 as cytosolic prostaglandin E synthases in the human brain. Neurochem.
Res. 25:73338
Nakashima K, Ueno N, Kamei D,
Tanioka T, Nakatani Y, et al. 2003.
Coupling between cyclooxygenases and
prostaglandin F2 synthase. Detection
of an inducible, glutathione-activated,
membrane-bound prostaglandin F2synthetic activity. Biochim. Biophys.
Acta 1633:96105
Bogaards JJ, Venekamp JC, van
Bladeren PJ. 1997. Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalysed
by the human glutathione S-transferases
A1-1, A2-2, M1a-1a. and P1-1. Chem.
Res. Toxicol. 10:31017
Paumi CM, Wright M, Townsend AJ,
Morrow CS. 2003. Multidrug resistance
protein (MRP) 1 and MRP3 attenuate
cytotoxic and transactivating effects
of the cyclopentenone prostaglandin,
15-deoxy-12,14-prostaglandin J2 in
69.
70.
71.
72.
73.
74.
75.
81
MCF7 breast cancer cells. Biochemistry

42:542937
Paumi CM, Smitherman PK, Townsend
AJ, Morrow CS. 2004. Glutathione Stransferases (GSTs) inhibit transcriptional activation by the peroxisomal proliferator-activated receptor (PPAR )
ligand, 15-deoxy-12,14-prostaglandin J2
(15-d-PGJ2). Biochemistry 43:2345
52
Jowsey IR, Smith SA, Hayes JD.
2003. Expression of the murine glutathione S-transferase 3 (GSTA3)
subunit is markedly induced during
adipocyte differentiation: activation of
the GSTA3 gene promoter by the proadipogenic eicosanoid 15-deoxy-12,14prostaglandin J2. Biochem. Biophys. Res.
Commun. 312:122635
Itoh K, Mochizuki M, Ishii Y, Ishii T,
Shibata T, et al. 2004. Transcription factor Nrf2 regulates inflammation by mediating the effect of 15-deoxy-12,14prostaglandin J2. Mol. Cell. Biol. 24:36
45
McMahon M, Itoh K, Yamamoto M,
Hayes JD. 2003. Keap1-dependent proteasomal degradation of transcription
factor Nrf2 contributes to the negative regulation of antioxidant response
element-driven gene expression. J. Biol.
Chem. 278:21592600
Wakabayashi N, Dinkova-Kostova AT,
Holtzclaw WD, Kang MI, Kobayashi
A, et al. 2004. Protection against electrophile and oxidant stress by induction
of the phase 2 response: fate of cysteines of the Keap1 sensor modified by
inducers. Proc. Natl. Acad. Sci. USA
101:204045
Rossi A, Kapahl P, Natoli G, Takahashi
T, Chen Y, et al. 2000. Anti-inflammatory
cyclopentenone prostaglandins are direct
inhibitors of IB kinase. Nature 403:
1038
Uchida K. 2003. 4-Hydroxy-2-nonenal:
a product and mediator of oxidative
stress. Prog. Lipid Res. 42:31843
7 Jan 2005
13:55

82
AR
HAYES
AR232-PA45-03.tex
AR232-PA45-03.sgm
JOWSEY
FLANAGAN
76. Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, et al. 2003. A signalling role for 4-hydroxy-2-nonenal in
regulation of mitochondrial uncoupling.
EMBO J. 22:410310
77. Awasthi YC, Sharma R, Cheng JZ,
Yang Y, Sharma A, et al. 2003. Role
of 4-hydroxynonenal in stress-mediated
apoptosis signalling. Mol. Aspects Med.
24:21930
78. Tjalkens RB, Luckey SW, Kroll DJ, Petersen DR. 1999. ,-Unsaturated aldehydes mediate inducible expression of
glutathione S-transferase in hepatoma
cells through activation of the antioxidant response element (ARE). Adv. Exp.
Med. Biol. 463:12331
79. Ishii T, Itoh K, Ruiz E, Leake DS, Unoki
H, et al. 2004. Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages. Activation by oxidatively modified LDL and
4-hydroxynonenal. Circ. Res. 94:60916
80. Levonen A-L, Landar A, Ramachandran
A, Ceaser EK, Dickinson DA, et al. 2004.
Cellular mechanisms of redox cell signalling: the role of cysteine modification in controlling antioxidant defences
in response to electrophilic lipid oxidation products. Biochem. J. 378:373
82
81. Ding Y, Ortelli F, Rossiter LC, Hemingway J, Ranson H. 2003. The Anopheles
gambiae glutathione transferase supergene family: annotation, phylogeny and
expression profiles. BMC Genomics 4:35
82. Patskovsky YV, Patskovska LN, Listowsky I. 2000. The enhanced affinity for
thiolate anion and activation of enzymebound glutathione is governed by an
arginine residue of human Mu class glutathione S-transferase. J. Biol. Chem.
275:3296304
83. Patskovsky YV, Patskovska LN, Listowsky I. 1999. An asparagine-phenylalanine substitution accounts for catalytic differences between hGSTM3-3
and other human class Mu glutathione
84.
85.
86.
87.
88.
89.
90.
91.
LaTeX2e(2002/01/18)
P1: GCE
S-transferases. Biochemistry 38:16187

94
Morel F, Rauch C, Coles B, Le Ferrec E, Guillouzo A. 2002. The human
glutathione transferase alpha locus: genomic organization of the gene cluster
and functional characterization of the genetic polymorphism in the hGSTA1 promoter. Pharmacogenetics 12:27786
Cheng J-Z, Yang Y, Singh SP, Singhal SS, Awasthi S, et al. 2001. Two
distinct 4-hydroxynonenal metabolising
glutathione S-transferase isoenzymes are
differentially expressed in human tissues. Biochem. Biophys. Res. Commun.
282:126874
Vargo MA, Colman RF. 2001. Affinity
labelling of rat glutathione S-transferase
isozyme 1-1 by 17-iodoacetoxy-estradiol-3-sulfate. J. Biol. Chem. 276:2031
36
Cho S-G, Lee YH, Park H-S, Ryoo
K, Kang KW, et al. 2001. Glutathione
S-transferase Mu modulates the stressactivated signals by suppressing apoptosis signal-regulating kinase 1. J. Biol.
Chem. 276:1274955
Dorion S, Lambert H, Landry J. 2002.
Activation of the p38 signaling pathway
by heat shock involves the dissociation of
glutathione S-transferase Mu from Ask1.
J. Biol. Chem. 277:3079297
Adler V, Yin Z, Fuchs SY, Benezra M,
Rosario L, et al. 1999. Regulation of JNK
signalling by GSTp. EMBO J. 18:1321
34
Gardner JL, Gallagher EP. 2001. Development of a peptide antibody specific
to human glutathione S-transferase Alpha 4-4 (hGSTA4-4) reveals preferential
localization in human liver mitochondria. Arch. Biochem. Biophys. 390:19
27
Raza H, Robin M-A, Fang J-k, Avadhani
NG. 2002. Multiple isoforms of mitochondrial glutathione S-transferases and
their differential induction under oxidative stress. Biochem. J. 366:4555
7 Jan 2005
13:55
AR
AR232-PA45-03.tex
AR232-PA45-03.sgm
LaTeX2e(2002/01/18)
P1: GCE

92. Robin M-A, Prabu SK, Raza H, Anandatheerthavarada HK, Avadhani NG.
2003. Phosphorylation enhances mitochondrial targeting of GSTA4-4 through
increased affinity for binding to cytoplasmic Hsp70. J. Biol. Chem. 278:1896070
93. Kodym R, Calkins P, Story M. 1999.
The cloning and characterization of a
new stress response protein. A mammalian member of a family of class
glutathione S-transferase-like proteins.
J. Biol. Chem. 274:513137
94. Harrop SJ, DeMaere MZ, Fairlie WD,
Reztsova T, Valenzuela SM, et al. 2001.
Crystal structure of a soluble form of the
intracellular chloride ion channel CLIC1
(NCC27) at 1.4-A resolution. J. Biol.
Chem. 276:449935000
95. Dulhunty A, Gage P, Curtis S, Chelvanayagam G, Board P. 2001 The glutathione transferase structural family includes a nuclear chloride channel and a
ryanodine receptor calcium release channel modulator. J. Biol. Chem. 276:3319
23
96. Jeppesen MG, Ortiz P, Shepard W, Kinzy
TG, Nyborg J, Andersen GR. 2003.
The crystal structure of the glutathione
S-transferase-like domain of elongation factor 1B from Saccharomyces
cerevisiae. J. Biol. Chem. 278:47190
98
97. Marco A, Cuesta A, Pedrola L, Palau
F, Marn I. 2004. Evolutionary and
structural analyses of GDAP1, involved
in Charcot-Marie-Tooth disease, characterize a novel class of glutathione
transferase-related genes. Mol. Biol.
Evol. 21:17687
98. Jowsey IR, Thomson RE, Orton TC,
Elcombe CR, Hayes JD. 2003. Biochemical and genetic characterization of
a murine class Kappa glutathione Stransferase. Biochem. J. 373:55969
99. Morel F, Rauch C, Petit E, Piton A,
Theret N, et al. 2004. The human glutathione transferase Kappa: gene and
protein characterization and evidence
100.
101.
102.
103.
104.
105.
106.
107.
83
for a peroxisomal localization. J. Biol.

Chem. 279(16):1624653
Thomson RE, Bigley AL, Foster JR,
Jowsey IR, Elcombe CR, et al. 2004.
Tissue-specific expression and subcellular distribution of murine glutathione Stransferase class Kappa. J. Histochem.
Cytochem. 52(5):65362
Pettigrew NE, Colman RF. 2001. Heterodimers of glutathione S-transferase
can form between isoenzyme classes
pi and mu. Arch. Biochem. Biophys.
396:22530
Cromer BA, Morton CJ, Board PG,
Parker MW. 2002. From glutathione
transferase to pore in a CLIC. Eur. Biophys. J. 31:35664
Littler DR, Harrop SJ, Fairlie WD,
Brown LJ, Pankhurst GJ, et al. 2004.
The intracellular chloride ion channel protein CLIC1 undergoes a redoxcontrolled structural transition. J. Biol.
Chem. 279:9298305
Jakobsson P-J, Mancini JA, Riendeau
D, Ford-Hutchinson AW. 1997. Identification and characterization of a novel
microsomal enzyme with glutathionedependent transferase and peroxidase
activities. J. Biol. Chem. 272:22934
39
Schroder O, Sjostrom M, Qiu H, Stein
J, Jakobsson P-J, Haeggstrom JZ. 2003.
Molecular and catalytic properties of
three rat leukotriene C4 synthase homologs. Biochem. Biophys. Res. Commun. 312:27176
Thoren S, Weinander R, Saha S,
Jegerschold C, Pettersson PL, et al. 2003.
Human microsomal prostaglandin E
synthase-1. Purification, functional characterization, and projection structure determination. J. Biol. Chem. 278:22199
209
Mandal AK, Skoch J, Bacskai BJ, Hyman BT, Christmas P, et al. 2004. The
membrane organization of leukotriene
synthesis. Proc. Natl. Acad. Sci. USA
101:658792
7 Jan 2005
13:55

84
AR
HAYES
AR232-PA45-03.tex
AR232-PA45-03.sgm
JOWSEY
FLANAGAN
108. Hayes JD, Strange RC. 2000. Glutathione S-transferase polymorphisms

and their biological consequences. Pharmacology 61:15466
109. Townsend DM, Tew KD. 2003. Cancer drugs, genetic variation and the glutathione S-transferase gene family. Am.
J. Pharmacogenomics 3:15772
110. Coles BF, Kadlubar FF. 2003. Detoxification of electrophilic compounds by
glutathione S-transferase catalysis: determinants of individual response to
chemical carcinogens and chemotherapeutic drugs? BioFactors 17:11530
111. Benhamou S, Lee WJ, Alexandrie AK, Boffetta P, Bouchardy C, et al. 2002.
Meta- and pooled analyses of the effects
of glutathione S-transferase M1 polymorphisms and smoking on lung cancer
risk. Carcinogenesis 23:134350
112. Hashibe M, Brennan P, Strange RC,
Bhisey R, Cascorbi I, et al. 2003. Metaand pooled analyses of GSTM1, GSTT1,
GSTP1, and CYP1A1 genotypes and risk
of head and neck cancer. Cancer Epidemiol. Biomarkers Prev. 12:150917
112a. van Lieshout EMM, Roelofs HMJ,
Dekker S, Mulder CJJ, Wobbes T, et al.
1999. Polymorphic expression of the
glutathione S-transferase P1 gene and
its susceptibility to Barretts esophagus
and esophageal carcinoma. Cancer Res.
59:58689
113. Roodi N, Dupont WD, Moore JH, Parl
FF. 2004. Association of homozygous
wild-type glutathione S-transferase M1
genotype with increased breast cancer
risk. Cancer Res. 64:123336
113a. Sprenger R, Schlagenhaufer R, Kerb R,
Bruhn C, Brockmoller J, et al. 2000.
Characterization of the glutathione Stransferase GSTT1 deletion: discrimination of all genotypes by polymerase
chain reaction indicates a trimodular
genotype-phenotype correlation. Pharmacogenetics 10:55765
114. Stoehlmacher J, Park DJ, Zhang W,
Groshen S, Tsao-Wei DD, et al. 2002.
115.
116.
117.
118.
119.
120.
121.
LaTeX2e(2002/01/18)
P1: GCE
Association between glutathione Stransferase P1, T1, and M1 genetic polymorphism and survival of patients with
metastatic colorectal cancer. J. Natl.
Cancer Inst. 94:93642
Dasgupta RK, Adamson PJ, Davies FE,
Rollinson S, Roddam PL, et al. 2003.
Polymorphic variation in GSTP1 modulates outcome following therapy for multiple myeloma. Blood 102:234550
Allan JM, Wild CP, Rollinson S, Willett EV, Moorman AV, et al. 2001. Polymorphism in glutathione S-transferase
P1 is associated with susceptibility to
chemotherapy-induced leukaemia. Proc.
Palmer CAN, Young V, Ho M, Doney A,
Belch JJF. 2003. Association of common
variation in glutathione S-transferase
genes with premature development of
cardiovascular disease in patients with
systemic sclerosis. Arthritis Rheumatism
48:85455
Gilliland FD, Li Y-F, Saxon A, DiazSanchez D. 2004. Effect of glutathione
S-transferase M1 and P1 genotypes
on xenobiotic enhancement of allergic responses: randomised, placebocontrolled crossover study. Lancet 363:
11925
Romieu I, Sienra-Monge JJ, RamrezAguilar M, Moreno-Macas H, ReyesRuiz NI, et al. 2004. Genetic polymorphism of GSTM1 and antioxidant supplementation influence lung function in
relation to ozone exposure in asthmatic
children in Mexico City. Thorax 59:810
Noguchi E, Shibasaki M, Kamioka M,
Yokouchi Y, Yamakawa-Kobayashi K,
et al. 2002. New polymorphisms of
haematopoietic prostaglandin D synthase and human prostanoid DP receptor
genes. Clin. Exp. Allergy 32:9396
Board PG, Anders MW, Blackburn AC.
2005. Catalytic function and expression
of glutathione zeta. In Drug Metabolism
and Transport: Molecular Methods and
Mechanisms, ed. LH Lash, Methods
7 Jan 2005
13:55
AR
AR232-PA45-03.tex
AR232-PA45-03.sgm
LaTeX2e(2002/01/18)
P1: GCE
122.

123.
124.
125.
126.
127.
128.
129.
Pharmacol. Toxicol., pp. 85107. Totowa, NJ: Humana Press

Whitbread AK, Tetlow N, Eyre HJ,
Sutherland GR, Board PG. 2003. Characterization of the human Omega class
glutathione transferase genes and associated polymorphisms. Pharmacogenetics
13:13144
Tetlow N, Board PG. 2004. Functional
polymorphism of human glutathione
transferase A2. Pharmacogenetics 14:1
6
Ning B, Wang C, Morel F, Nowell S,
Ratnasinghe DL, et al. 2004. Human
glutathione S-transferase A2 polymorphisms: variant expression, distribution
in prostate cancer cases/controls and a
novel form. Pharmacogenetics 14:35
44
Guy CA, Hoogendoorn B, Smith SK,
Coleman S, ODonovan MC, Buckland
PR. 2004. Promoter polymorphisms in
glutathione S-transferase genes affect
transcription. Pharmacogenetics 14:45
51
Iida A, Saito S, Sekine A, Harigae S, Osawa S, et al. 2001. Catalog of 46 singlenucleotide polymorphisms (SNPs) in the
microsomal glutathione S-transferase 1
(MGST1) gene. J. Hum. Genet. 46:590
94
Thameem F, Yang X, Permana PA, Wolford JK, Bogardus C, Prochazka M.
2003. Evaluation of the microsomal glutathione S-transferase 3 (MGST3) locus
on 1q23 as a Type 2 diabetes susceptibility gene in Pima Indians. Hum. Genet.
113:35358
Sayers I, Barton S, Rorke S, Beghe B,
Hayward B, et al. 2003. Allelic association and functional studies of promoter
polymorphism in the leukotriene C4 synthase gene (LTCS) in asthma. Thorax
58:41724
Helgadottir A, Manolescu A, Thorleifsson G, Gretarsdottir S, Jonsdottir H, et al.
2004. The gene encoding 5-lipoxygenase
activating protein confers risk of myocar-
130.
131.
132.
133.
134.
135.
136.
85
dial infarction and stroke. Nat. Genet.

36:23339
Pearson WR, Reinhart J, Sisk SC, Anderson KS, Adler PN. 1988. Tissuespecific induction of murine glutathione
transferase mRNAs by butylated hydroxyanisole. J. Biol. Chem. 263:1332432
Chanas SA, Jiang Q, McMahon M,
McWalter GK, McLellan LI, et al. 2002.
Loss of the Nrf2 transcription factor
causes a marked reduction in constitutive and inducible expression of the
glutathione S-transferase Gsta1, Gsta2,
Gstm1, Gstm2, Gstm3 and Gstm4 genes
in the livers of male and female mice.
Biochem. J. 365:40516
Mouse Glutathione Transferase Nomenclature. http://www.people.virginia.edu/
wrp/gst mouse.html
Engle MR, Singh SP, Czernik PJ,
Gaddy D, Montague DC, et al. 2004.
Physiological role of mGSTA4-4, a
glutathione S-transferase metabolising
4-hydroxynonenal: generation and analysis of mGsta4 null mouse. Toxicol.
Appl. Pharmacol. 194(3):296308
Dinkova-Kostova A, Massiah MA,
Bozak RE, Hicks RJ, Talalay P. 2001.
Potency of Michael reaction acceptors as
inducers of enzymes that protect against
carcinogenesis depends on their reactivity with sulfhydryl groups. Proc. Natl.
McMahon M, Itoh K, Yamamoto M,
Chanas SA, Henderson CJ, et al. 2001.
The Cap n collar basic leucine zipper transcription factor Nrf2 (NF-E2
p45-related factor 2) controls both
constitutive and inducible expression
of intestinal detoxification and glutathione biosynthetic enzymes. Cancer
Res. 61:3299307
Nioi P, McMahon M, Itoh K, Yama
moto M, Hayes JD. 2003. Identification of a novel Nrf2-regulated antioxidant response element (ARE) in
the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the
7 Jan 2005
13:55
86
137.

138.
139.
140.
141.
142.
143.
144.
145.
AR
HAYES
AR232-PA45-03.tex
AR232-PA45-03.sgm
JOWSEY
FLANAGAN
ARE consensus sequence. Biochem. J.

374:33748
Rushmore TH, Morton MR, Pickett CB.
1991. The antioxidant responsive element. Activation by oxidative stress and
identification of the DNA consensus sequence required for functional activity.
J. Biol. Chem. 266:1163239
Motohashi H, OConnor T, Katsuoka F,
Engel JD, Yamamoto M. 2002. Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors. Gene 294:1
12
Nguyen T, Sherratt PJ, Pickett CB.
2003. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev.
Pharmacol. Toxicol. 43:23360
Tchaikovskaya T, Fraifeld V, Asraf I,
Sagi O, Wolfson M, Listowsky I. 2002.
mGSTM5 KO mice as a potential model
for brain studies. Neural Plast. 9:119
Henderson CJ, Smith AG, Ure J, Brown
K, Bacon EJ, Wolf CR. 1998. Increased
skin tumorigenesis in mice lacking pi
class glutathione S-transferases. Proc.
Elsby R, Kitteringham NR, Goldring CE,
Lovatt CA, Chamberlain M, et al. 2003.
Increased constitutive c-Jun N-terminal
kinase signalling in mice lacking glutathione S-transferase Pi. J. Biol. Chem.
278:2224249
Henderson CJ, Wolf CR, Kitteringham
N, Powell H, Otto D, Park BK. 2000.
Increased resistance to acetaminophen
hepatotoxicity in mice lacking glutathione S-transferase Pi. Proc. Natl.
Urade Y, Eguchi N, Aritake K,
Hayaishi O. 2004. Functional analyses
of lipocalin-type and hematopoietic
prostaglandin synthases. Folia Pharmacol. Jpn. 123:513
Fernandez-Cano n JM, Baetscher MW,
Finegold M, Burlingame T, Gibson KM,
Grompe M. 2002. Maleylacetoacetate
146.
147.
148.
149.
150.
151.
152.
153.
LaTeX2e(2002/01/18)
P1: GCE
isomerase (MAAI/GSTZ)-deficient mice

reveal a glutathione-dependent nonenzymatic bypass in tyrosine catabolism.
Mol. Cell. Biol. 22:494351
Lim CEL, Matthaei KI, Blackburn AC,
Davis RP, Dahlstrom JE, et al. 2004.
Mice deficient in glutathione transferase
zeta/maleylacetoacetate isomerase exhibit a range of pathological changes
and elevated expression of alpha, mu and
pi class glutathione transferases. Am. J.
Pathol. 165:37993
Lantum HB, Liebler DC, Board PG,
Anders MW. 2002. Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone. Chem. Res.
Toxicol. 15:70716
Byrum RS, Goulet JL, Griffiths RJ,
Koller BH. 1997. Role of the 5lipoxygenase-activating protein (FLAP)
in murine acute inflammatory responses.
J. Exp. Med. 185:106575
Kanaoka Y, Maekawa A, Penrose JF,
Austen KF, Lam BK. 2001. Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent
passive cutaneous anaphylaxis in mice
lacking leukotriene C4 synthase. J. Biol.
Chem. 276:2260813
Trebino CE, Stock JL, Gibbons CP,
Naiman BM, Wachtmann TS, et al.
2003. Impaired inflammatory and pain
responses in mice lacking an inducible
prostaglandin E synthase. Proc. Natl.
Engblom D, Saha S, Engstrom L, Westman M, Audoly LP, et al. 2003. Microsomal prostaglandin E synthase-1 is the
central switch during immune-induced
pyresis. Nat. Neurosci. 6:113738
Toba G, Aigaki T. 2000. Disruption of the
microsomal glutathione S-transferaselike gene reduces life span of Drosophila
melanogaster. Gene 253:17987
Carlson BA, Novoselov SV, Kumaraswamy E, Lee BJ, Anver MR, et al. 2004.
Specific excision of the selenocysteine
7 Jan 2005
13:55
AR
AR232-PA45-03.tex
AR232-PA45-03.sgm
LaTeX2e(2002/01/18)
P1: GCE

154.
155.
156.
157.
158.
159.
160.
tRNA[Ser]Sec (Trsp) gene in mouse liver

demonstrates an essential role of selenoproteins in liver function. J. Biol. Chem.
279:801117
McLeod R, Ellis EM, Arthur JR, Neal
GE, Judah DJ, et al. 1997. Protection
conferred by selenium deficiency against
aflatoxin B1 in the rat is associated with
the hepatic expression of an aldo-keto reductase and a glutathione S-transferase
subunit that metabolise the mycotoxin.
Okuda A, Imagawa M, Sakai M, Muramatsu M. 1990. Functional cooperativity
between two TPA responsive elements
in undifferentiated F9 embryonic stem
cells. EMBO J. 9:113135
Pulford DJ, Hayes JD. 1996. Characterization of the rat glutathione S-transferase
Yc2 subunit gene, GSTA5: identification
of a putative antioxidant-responsive element in the 5 -flanking region of rat
GSTA5 that may mediate chemoprotection against aflatoxin B1. Biochem. J.
318:7584
Kelner MJ, Bagnell RD, Montoya MA,
Estes LA, Forsberg L, Morgenstern R.
2000. Structural organization of the microsomal glutathione S-transferase gene
(MGST1) on chromosome 12p13.1
13.2. Identification of the correct promoter region and demonstration of transcriptional regulation in response to
oxidative stress. J. Biol. Chem. 275:
130006
Kumar A, Reddy EP. 2001. Genomic
organization and characterization of the
promoter region of murine GSTM2 gene.
Gene 270:22129
Ikeda H, Serria MS, Kakizaki I,
Hatayama I, Satoh K, et al. 2002. Activation of mouse Pi-class glutathione Stransferase gene by Nrf2 (NF-E2-related
factor 2) and androgen. Biochem. J.
364:56370
Park EY, Rho HM. 2002. The transcriptional activation of the human
copper/zinc superoxide dismutase gene
161.
162.
163.
164.
165.
165a.
165b.
87
by 2,3,7,8-tetrachlorodibenzo-p-dioxin
through two different regulator sites,
the antioxidant responsive element and
xenobiotic response element. Mol. Cell.
Biochem. 24:4755
Jowsey IR, Jiang Q, Itoh K, Yamamoto M, Hayes JD. 2003. Expression
of the aflatoxin B1-8,9-epoxide-metabolizing murine glutathione S-transferase A3 subunit is regulated by the Nrf2
transcription factor through an antioxidant response element. Mol. Pharmacol.
64:101828
Andorfer JH, Tchaikovskaya T, Listowsky I. 2004. Selective expression of
glutathione S-transferase genes in the
murine gastrointestinal tract in response
to dietary organosulfur compounds. Carcinogenesis 25:35967
Hayes JD, Chanas SA, Henderson CJ,
McMahon M, Sun C, et al. 2000. The
Nrf2 transcription factor contributes both
to the basal expression of glutathione Stransferases in mouse liver and to their
induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem. Soc.
Trans. 28:3341
Thimmulappa RK, Mai KH, Srisuma S,
Kensler TW, Yamamoto M, Biswal S.
2002. Identification of Nrf2-regulated
genes induced by the chemopreventive
agent sulforaphane by oligonucleotide
microarray. Cancer Res. 62:5196
203
Kwak M-K, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, Kensler TW.
2003. Modulation of gene expression by
cancer chemopreventive dithiolethines
through the Keap1-Nrf2 pathway. Identification of novel gene clusters for
cell survival. J. Biol. Chem. 278:8135
45
Solt DB, Farber E. 1976. New principle
for the analysis of chemical carcinogenesis. Nature 263:7013
Ikeda H, Nishi S, Sakai M. 2004. Transcription factor Nrf2/MafK regulates rat
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placental glutathione S-transferase gene

during hepatocarcinogenesis. Biochem.
J. 380:51521
166. Lee J-M, Calkins MJ, Chan K, Kan YW,
Johnson JA. 2003. Identification of the
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conferring protection against oxidative
stress in primary cortical astrocytes using oligonucleotide microarray analysis.
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Figure 5 Negative regulation of the ARE-gene battery by GSTA4-4. This cartoon

shows how 4-HNE, produced through membrane damage by reactive oxygen species
(53), might modify cysteine residues in the cytoskeleton-binding protein Keap1 (73).
Such posttranslational modification of Keap1 allows the Nrf2 transcription factor to
accumulate and translocate into the nucleus. Once in the nucleus, Nrf2 forms heterodimers with small Maf proteins that are recruited to antioxidant response elements (AREs) in the promoters of antioxidant and detoxication genes. Trans-activation of ARE-driven genes by Nrf2 increases the production of many proteins, including the GSTA4, glutamate cysteine ligase catalytic, and glutamate cysteine modulatory subunits; the latter two comprise the subunits of GCL, which catalyzes the ratelimiting step in the synthesis of GSH. The resulting elevation in amounts of GSTA44 and GSH allow increased metabolism of 4-HNE and its elimination from the cell
via MRP.
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Figure 6 Induction of GST and NQO1 by tyrosine catabolites. Degradation products

of tyrosine that accumulate in GSTZ1 knockout mice stimulate upregulation of class
Alpha, Mu, and Pi GST, as well as NQO1 (145, 146). As shown in the figure, the
potential inducing agents include maleylacetoacetate, succinylacetone, and succinylacetoacetate. Certain of these tyrosine metabolites are thiol-active (147) and probably
induce gene expression through the Keap1/Nrf2 pathway.
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Copyright
PLEIOTROPIC EFFECTS OF STATINS

James K. Liao
Vascular Medicine Research, Brigham & Womens Hospital, Cambridge,
Massachusetts 02139; email: jliao@rics.bwh.harvard.edu
Ulrich Laufs
Klinik Innere Medizin III, Universitat des Saarlandes, 66421 Homburg, Germany;
email: ulrich@laufs.com
Key Words HMG-CoA reductase inhibitor, cholesterol, isoprenoids,

atherosclerosis, inflammation
Abstract Statins are potent inhibitors of cholesterol biosynthesis. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. However, the overall benefits observed with statins appear to be greater than what
might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent
or pleiotropic effects of statins involve improving endothelial function, enhancing
the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation,
and inhibiting the thrombogenic response. Furthermore, statins have beneficial extrahepatic effects on the immune system, CNS, and bone. Many of these pleiotropic
effects are mediated by inhibition of isoprenoids, which serve as lipid attachments
for intracellular signaling molecules. In particular, inhibition of small GTP-binding
proteins, Rho, Ras, and Rac, whose proper membrane localization and function are
dependent on isoprenylation, may play an important role in mediating the pleiotropic
effects of statins.
INTRODUCTION
Cardiovascular disease, in particular coronary heart disease (CHD), is the principal cause of mortality in developed countries. Among the causes of cardiovascular
disease, atherosclerosis is the underlying disorder in the majority of patients. Although the development of atherosclerosis is dependent on a complex interplay
between many factors and processes (1), a clear association has been established
between elevated levels of plasma cholesterol and increased atherosclerotic disease (2, 3). Indeed, several landmark clinical trials, such as the Scandinavian
Simvastatin Survival Study (4S) (4), Cholesterol and Recurrent Events (CARE)
(5), Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) (6),
West of Scotland Coronary Prevention Study (WOSCOPS) (7), Air Force/Texas
0362-1642/05/0210-0089$14.00
89
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Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS) (8), Heart Protection Study (HPS) (9), and the Anglo-Scandinavian Cardiac Outcome Trial
Lipid-lowering Arm (ASCOT-LLA) (10), have demonstrated the benefit of lipid
lowering with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
inhibitors or statins for the primary and secondary prevention of CHD.

PHARMACOKINETIC PROPERTIES OF STATINS

Cholesterol is an essential component of cell membranes and is the immediate
precursor of steroid hormones and bile acids (11). However, in excessive amounts,
cholesterol becomes an important risk factor for cardiovascular disease, as demonstrated in clinical trials from the Framingham Heart Study (3, 12) and the Multiple
Risk Factor Intervention Trial (13, 14). Although dietary cholesterol can contribute to changes in serum cholesterol levels, more than two thirds of the bodys
cholesterol is synthesized in the liver. Therefore, inhibition of hepatic cholesterol
biosynthesis has emerged as the target of choice for reducing serum cholesterol
levels (15).
The rate-limiting enzyme in cholesterol biosynthesis in the liver is HMGCoA reductase (11), which catalyzes the conversion of HMG-CoA to mevalonic
acid (16). Inhibitors of HMG-CoA reductase, or statins, were originally identified as secondary metabolites of fungi (17). HMG-CoA reductase catalyses the
rate-limiting step of cholesterol biosynthesis, a four-electron reductive deacylation of HMG-CoA to CoA and mevalonate. One of the first natural inhibitors
of HMG-CoA reductase was mevastatin (compactin, ML-236B), which was isolated from Penicillium citrinium by A. Endo et al. in 1976 (18). In its active
form, mevastatin resembles the cholesterol precursor, HMG-CoA. When mevastatin was initially administered to rats, it inhibited cholesterol biosynthesis with
a Ki of 1.4 nM. Unfortunately, it also caused unacceptable hepatocellular toxicity
and further clinical development was discontinued. Subsequently, a more active
fungal metabolite, mevinolin or lovastatin, was isolated from Aspergillus terreus
by Hoffman and colleagues in 1979 (19, 20). Lovastatin differs from mevastatin
in having a substituted methyl group. Compared to mevastatin, lovastatin was a
more potent inhibitor of HMG-CoA reductase, with a Ki of 0.6 nM, but did not
cause hepatocellular toxicity when given to rats. Lovastatin, therefore, became the
first of this class of cholesterol-lowering agents to be approved for clinical use in
humans. Since then, several new statins, both natural and chemically modified,
have become commercially available, including pravastatin, simvastatin, fluvastatin, atorvastatin, cerivastatin, and most recently, pitavastatin and rosuvastatin
(21). Indeed, statins have emerged as one of the most effective class of agents for
reducing serum cholesterol levels.
Statins work by reversibly inhibiting HMG-CoA reductase through side chains
that bind to the enzymes active site and block the substrate-product transition
state of the enzyme (22). Thus, all statins share an HMG-like moiety and inhibit
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NONCHOLESTEROL EFFECTS OF STATINS
91
the reductase by similar mechanism (Figure 1). Recently, the structure of the catalytic portion of human HMG-CoA reductase complexed with different statins was
determined (22). The bulky, hydrophobic compounds of statins occupy the HMGbinding pocket and block access of the substrate HMG. The tight binding of statins
is due to the large number of van der Waals interactions between statins and HMGCoA reductase. The structurally diverse, rigid, hydrophobic groups of the different
statins are accommodated in a shallow nonpolar groove that is present only when
COOH-terminal residues of HMG-CoA reductase are disordered. There are subtle
differences in the modes of binding between the various statins, with the synthetic
compounds atorvastatin and rosuvastatin having the greatest number of bonding
interactions with HMG-CoA reductase (22). Statins bind to mammalian HMGCoA reductase at nanomolar concentrations, leading to effective displacement
of the natural substrate, HMG-CoA, which binds at micromolar concentrations
(23).
Oral administration of statins to rodents and dogs showed that these drugs
are predominantly extracted by the liver and resulted in >30%50% reduction in
plasma total cholesterol levels and substantial decrease in urinary and plasma levels
of mevalonic acid, the end product of the HMG-CoA reductase reaction. Similar
reduction in cholesterol synthesis and decrease in circulating total and low-density
lipoprotein (LDL)-containing cholesterol (LDL-C) by these agents have been subsequently confirmed in humans. Because hepatic LDL-C receptors are the major
mechanism of LDL-C clearance from the circulation, the substantial declines in
serum cholesterol levels are accompanied by an increase in hepatic LDL-C receptor activity. Statins, therefore, effectively reduce serum cholesterol levels by two
separate mechanisms. They not only inhibit endogenous cholesterol biosynthesis
via HMG-CoA reductase inhibition but also increase cholesterol clearance from
the bloodstream via increases in LDL-C receptor.
The rank order of potency for HMG-CoA reductase inhibition among the
second-generation statins is simvastatin > pravastatin > lovastatin
= mevastatin,
with tissue IC50 values of simvastatin and mevastatin being approximately 4 nM
and 20 nM, respectively (24). The IC50 values for these statins correspond to their
relative potency for lowering serum cholesterol levels in vivo (i.e., simvastatin >
lovastatin) (25). The newer third-generation synthetic statins, which include fluvastatin, cerivastatin, the penta-substituted pyrrole atorvastatin, pitavastatin (NK104), and rosuvastatin, are much more potent than the mevastatin derivatives.
These newer statins are active compounds, which share some physico-chemical
properties with pravastatin, but have greater lipophilicity and half-life (26). Consequently, these statins, especially atorvastatin, pitavastatin, and rosuvastatin, appear
to be quite effective in lowering serum cholesterol levels, perhaps, in part, owing
to their ability to bind hepatic HMG-CoA reductase at higher affinity and inhibit
the enzyme for a longer duration.
Because statins differ in their tissue permeability and metabolism, they possess
different potencies for extrahepatic HMG-CoA reductase inhibition. These differences in tissue permeability and metabolism may account for some of the observed
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differences in their peripheral side effects (27). Lipophilic statins, such as simvastatin, are considered more likely to enter endothelial cells by passive diffusion
than hydrophilic statins, such as pravastatin and rosuvastatin, which are primarily
targeted to the liver. However, lipophilicity does not entirely predict the ability
of statins to exert extrahepatic effects in animal and human studies, and so other
unidentified factors may play a role. It may be that there are specific mechanisms
for hydrophilic statins to enter extrahapetic cells, such as endothelial cells. Such a
mechanism is present in the liver, where the organic anion transporter (OATP-C)
enables hydrophilic statins to enter hepatocytes (28).
Until recently, all cholesterol-independent or pleiotropic effects of statins
were believed to be mediated by inhibition of mevalonate synthesis. However,
statins can reportedly bind to a novel allosteric site within the 2 integrin functionassociated antigen-1 (LFA-1), independent of mevalonate production (29). LFA-1
belongs to the integrin family and plays an important role in leukocyte trafficking
and in T cell activation. Random screening of chemical libraries identified the
HMG-CoA reductase inhibitor, lovastatin, as an inhibitor of the LFA-1/intercellular
adhesion molecule (ICAM)-1 interaction.
STATINS AND ISOPRENYLATED PROTEINS

By inhibiting L-mevalonic acid synthesis, statins also prevent the synthesis of
other important isoprenoid intermediates of the cholesterol biosynthetic pathway,
such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)
(11) (Figure 2). These intermediates serve as important lipid attachments for the
posttranslational modification of a variety of proteins, including the subunit of
heterotrimeric G-proteins; Heme-a; nuclear lamins; and small guanosine triphosphate (GTP)-binding protein Ras; and Ras-like proteins, such as Rho, Rab, Rac,
Ral, or Rap (30). Thus, protein isoprenylation permits the covalent attachment,
subcellular localization, and intracellular trafficking of membrane-associated proteins. Members of the Ras and Rho GTPase family are major substrates for posttranslational modification by prenylation (30, 31). Both Ras and Rho are small
GTP-binding proteins, which cycle between the inactive GDP-bound state and
active GTP-bound state (Figure 3). In endothelial cells, Ras translocation from the
cytoplasm to the plasma membrane is dependent on farnesylation, whereas Rho
translocation is dependent on geranylgeranylation (32, 33). Statins inhibit both
Ras and Rho isoprenylation, leading to the accumulation of inactive Ras and Rho
in the cytoplasm.
Because Rho is major target of geranylgeranylation, inhibition of Rho and
its downstream target, Rho-kinase, is a likely mechanism mediating some of the
pleiotropic effects of statins on the vascular wall (34, 35). Each member of the
Rho GTPase family, which consists of RhoA, Rac, and Cdc42, serves specific
functions in terms of cell shape, motility, secretion, and proliferation, although
overlapping functions between the members could be observed in overexpressed
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93
Figure 2 Biological actions of isoprenoids. Diagram of cholesterol biosynthesis pathway

showing the effects of inhibition of HMG-CoA reductase by statins. Decrease in isoprenylation of signaling molecules, such as Ras, Rho, and Rac, leads to modulation of various
signaling pathways. BMP-2: bone morphogenetic protein-2; eNOS: endothelial nitric oxide
synthase; t-PA: tissue-type plasminogen activator; ET-1: endothelin-1; PAI-1: plasminogen
activator inhibitor-1.
systems. The activation of RhoA in Swiss 3T3 fibroblasts by extracellular ligands, such as platelet-derived lysophosphatidic acid, leads to myosin light chain
phosphorylation and formation of focal adhesion complexes (30, 31, 36). Indeed,
Rho-associated protein kinase increases the sensitivity of vascular smooth muscle
to calcium in hypertension (37) and coronary spasm (38). In contrast, activation
of Rac1 leads to the formation of lamellipodia and membrane ruffles, whereas
activation of Cdc42 induces actin-rich surface protrusions called filopodia.
These distinct but complementary functions of Rho family members also extend to their effects on cell signaling. When cells undergo reorganization of their
actin cytoskeleton in response to extracellular signals, such as growth factors, or
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Figure 3 Regulation of Rho GTPase by isoprenylation. Rho proteins change between a

cytosolic, inactive, GDP-bound state and an active, membrane, GTP-bound state. This cycle
is controlled by several cofactors, including guanine nucleotide exchange factors (GEF),
GTPase-activating proteins (GAP), and guanine nucleotide dissociation inhibitors (GDI). An
important step in the activation of Rho GTPases is the posttranslational isoprenylation, which
allows the translocation of Rho to the cell membrane and the subsequent activation.
during cell movement and mitosis, they alter the three-dimensional colocalization
of intracellular proteins (30, 31). Thus, changes in Rho-induced actin cytoskeleton can affect intracellular transport, membrane trafficking, mRNA stability, and
gene transcription. It is therefore not surprising to find that Rho-induced changes
in the actin cytoskeleton and gene expression are related. Indeed, experimental
evidence suggests that inhibition of Rho isoprenylation mediates many of the
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cholesterol-independent effects of statins not only in vascular wall cells (32, 39),
but also in leukocytes (40) and bone (41).

STATINS AND ENDOTHELIAL FUNCTION

The vascular endothelium serves as an important autocrine and paracrine organ
that regulates vascular wall contractile state and cellular composition. Hypercholesterolemia impairs endothelial function, and endothelial dysfunction is one of
the earliest manifestations of atherosclerosis, occurring even in the absence of angiographic evidence of disease (42, 43). An important characteristic of endothelial
dysfunction is the impaired synthesis, release, and activity of endothelial-derived
nitric oxide (NO). Endothelial NO has been shown to inhibit several components of
the atherogenic process. For example, endothelium-derived NO mediates vascular
relaxation (44) and inhibits platelet aggregation (45), vascular smooth muscle proliferation (46), and endothelial-leukocyte interactions (47, 48). Inactivation of NO
by superoxide anion (O2 ) limits the bioavailability of NO and leads to nitrate
tolerance, vasoconstriction, and hypertension (49, 50).
Acute plasma LDL-C apheresis improves endothelium-dependent vasodilatation (51), suggesting that statins could restore endothelial function, in part, by
lowering serum cholesterol levels. However, in some studies with statins, restoration of endothelial function occurs before significant reduction in serum cholesterol levels (5254), suggesting that there are additional effects on endothelial
function beyond that of cholesterol reduction. Indeed, statins increase endothelial
NO production by stimulating and upregulating endothelial NO synthase (eNOS)
(32, 55). Furthermore, statins have been shown to restore eNOS activity in the
presence of hypoxia (56) and oxidized LDL (ox-LDL-C) (32), conditions which
lead to endothelial dysfunction. Statins also increase the expression of tissue-type
plasminogen activator (t-PA) (57) and inhibit the expression of endothelin-1, a
potent vasoconstrictor and mitogen (58). Statins, therefore, exert many favorable
effects on the endothelium and attenuate endothelial dysfunction in the presence
of atherosclerotic risk factors.
Although the effects of statins on Ras and Rho isoprenylation are reversed in the
presence of FPP and GGPP, respectively, the effects of statins on eNOS expression
is only reversed by GGPP and not by FPP or LDL-C (33). Indeed, direct inhibition of geranylgeranyltransferase or RhoA leads to increases in eNOS expression
(33, 35, 59). These findings are consistent with a noncholesterol-lowering effect
of statins and suggest that inhibition of RhoA by statins mediates the increase
in eNOS expression. Indeed, statins upregulate eNOS expression by prolonging
eNOS mRNA half-life but not eNOS gene transcription (33). Because hypoxia,
ox-LDL-C, and cytokines such as TNF- decrease eNOS expression by reducing eNOS mRNA stability, the ability of statins to prolong eNOS half-life may
make them effective agents in counteracting conditions that downregulate eNOS
expression.
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Additional important effects of statin treatment on eNOS function include inhibition of caveolin (60, 61). Statins also increase eNOS activity via posttranslational
activation of the phosphatidylinositol 3-kinase/protein kinase Akt (PI3K/Akt) pathway (55). Phosphorylation of Akt is an important event in several cellular activities.
Indeed, production of NO by the endothelium can be regulated by phosphorylation
and activation of eNOS by Akt, which is promoted in the presence of statins (62,
63). Caveolin-1 binds to eNOS in caveolae, thereby negatively regulating the enzyme (64). Exposure of cultured endothelial cells to hypercholesterolemic serum
upregulates caveolin-1 abundance and promotes association of caveolin-1 and
eNOS into inhibitory complexes, thereby decreasing NO production (65). Statins
have been shown to reduce caveolin-1 abundance and decrease its inhibitory action
on both basal and agonist-stimulated eNOS activity.
Another potential mechanism by which statins may improve endothelial function is through their antioxidant effects. For example, statins enhance endotheliumdependent relaxation by inhibiting the production of reactive oxygen species
(ROS), such as superoxide and hydroxy radicals, from aortas of cholesterol-fed rabbits (66). Although lipid lowering by itself can lower vascular oxidative stress (67),
some of these antioxidant effects of statins appear to be cholesterol-independent.
For example, statins attenuate angiotensin IIinduced free radical production in
vascular smooth muscle cells (SMCs) by inhibiting Rac1-mediated NADH oxidase
activity and downregulating angiotensin AT1 receptor expression (68) (Figure 4).
Because NO is scavenged by ROS, these findings indicate that the antioxidant
properties of statins may also contribute to their ability to improve endothelial
function (49, 50).
STATINS AND ENDOTHELIAL PROGENITOR CELLS

Statins have also been found to increase the number of circulating endothelial
progenitor cells (EPCs) (69). EPCs augment ischemia-induced neovascularization
(70), accelerate re-endothelialization after carotid balloon injury (71, 72) and improve postischemic cardiac function (73). Indeed, statins induce angiogenesis by
promoting the proliferation, migration, and survival of circulating EPCs (74). In
patients with stable coronary artery disease, administration of statins for four weeks
augmented the number of circulating EPCs and enhanced functional capacity in
patients with stable coronary artery disease (75). These findings agree with earlier
data showing that statins rapidly mobilize EPCs from the bone marrow and accelerate vascular structure formation via activation of phosphatidylinositol 3-kinase
(PI3K)/protein kinase Akt and eNOS (55, 74, 76). These angiogenic effects were
observed at lower concentrations of statins and were cholesterol-independent. At
higher concentrations, statins appear to have an anti-angiogenic effect (77, 78),
suggesting a biphasic effect of statins on angiogenesis (79). However, this suggestion remains controversial because higher doses of statins have also been shown
to be angiogenic (80).
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Figure 4 Antioxidative mechanisms of statins. The core NAD(P)H oxidase comprises five
components: p40phox (PHOX for phagocyte oxidase), p47phox, p67phox, p22phox, and gp91phox.
In the resting cell (left), three of these five components, p40phox, p47phox, and p67phox, exist in
the cytosol as a complex. The other two components, p22phox and gp91phox, are located in the
membranes. When it is stimulated by angiotensin, the cytosolic component becomes heavily
phosphorylated and the entire cytosolic complex migrates to the membrane. Activation requires the participation not only of the core subunits but also of two low-molecular-weight
guanine nucleotide-binding proteins, Rac and Rap. During activation, Rac binds GTP and
migrates to the membrane along with the core cytosolic complex. Treatment with statin downregulates AT1-receptor expression and inhibits Rac1 GTPase, a necessary component of the
NAD(P)H oxidase complex.
Statins and Smooth Muscle Proliferation

The proliferation of vascular SMCs is a central event in the pathogenesis of vascular lesions, including post-angioplasty restenosis, transplant arteriosclerosis, and
veinous graft occlusion (81). Recent studies have shown that statins attenuate
vascular proliferative disease, such as transplant-associated arteriosclerosis (81).
In contrast to atherosclerosis, transplant-associated arteriosclerosis is more dependent on immunological mechanisms as opposed to lipid disorders, although hypercholesterolemia exacerbates the immunologic process (82). Inhibition of isoprenoid but not cholesterol synthesis by statins decreased PDGF-induced DNA
synthesis in vascular SMCs (39, 83). Treatment with statins decreased PDGFinduced Rb hyperphosphorylation and cyclin-dependent kinases (cdk)-2, -4, and
-6 activities. This correlated with increases in the level of Cdk inhibitor, p27Kip1,
without concomitant changes in p16INK4, p21Waf1, or p53 levels. These findings
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indicate that statins inhibit vascular SMC proliferation by arresting cell cycle
between the G1/S phase transition. It remains to be determined whether the upregulation of p27Kip1 is responsible for the cell cycle arrest and whether there are
differences between different statins in terms of p27Kip1.
Because the small GTP-binding proteins, Ras and Rho, require posttranslational modification for membrane localization and activity and are implicated in
cell cycle regulation, they are likely targets for the direct antiproliferative vascular effects of statins. Ras can promote cell cycle progression via activation
of the MAP kinase pathway (84), whereas RhoA causes cellular proliferation
through destabilizing p27Kip1 protein (85). Interestingly, inhibition of vascular
SMC proliferation by statins was reversed by GGPP, but not FPP or LDL-C (39).
Indeed, direct inhibition of RhoA by Clostridium botulinum C3 transferase, which
ADP-ribosylates and inactivates RhoA, or by a dominant-negative RhoA mutant increased p27Kip1 and inhibited Rb hyperphosphorylation and SMC proliferation following PDGF stimulation. Taken together, these findings indicate that
RhoA mediates PDGF-induced SMC proliferation and that inhibition of RhoA
by statins is the predominant mechanism by which statins inhibit vascular SMC
proliferation.
STATINS AND PLATELET FUNCTION

Platelets play a critical role in the development of acute coronary syndromes
(86). Circulating platelets are associated with mural thrombus formation at the
site of plaque rupture and vascular injury (87, 88). Hypercholesterolemia is associated with increases in platelet reactivity (89). These abnormalities are linked
to increases in the cholesterol/phospholipid ratio in platelets. Other potential
mechanisms include increases in thromboxane A2 (TXA2) biosynthesis (90),
platelet 2-adrenergic receptor density (91), and platelet cytosolic calcium
(92).
Statins have been shown to influence platelet function, although the precise
mechanisms involved are not fully understood (93, 94). One of the well-characterized effects of endothelial NO is the inhibition of platelet aggregation (45).
Statin-mediated upregulation of eNOS has been shown to be associated with
downregulation of markers of platelet reactivity (95). Potential additional mechanisms include a reduction in the production of TXA2 and modifications in the
cholesterol content of platelet membranes (96, 97). The cholesterol content of
platelet and erythrocyte membranes is reduced in patients taking statin therapy.
This may lead to a decrease in the thrombogenic potential of these cells. Indeed, animal studies suggest statin therapy inhibits platelet deposition on damaged vessels and reduces platelet thrombus formation (87, 98). Furthermore,
in vitro experiments have demonstrated that statins inhibit tissue factor expression
by macrophages, thereby potentially reducing thrombotic events in the vascular
wall (99).
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STATINS AND PLAQUE STABILITY

Plaque rupture is a major cause of acute coronary syndromes (43, 100, 101). The
atherosclerotic lesion contains highly thrombogenic materials in the lipid core that
are separated from the bloodstream by a fibrous cap (102). Fissuring, erosion,
and ulceration of the fibrous cap eventually lead to plaque rupture and ensuing
thrombosis (101). Collagen is the main component of fibrous caps and is responsible for their tensile strength. Because macrophages are capable of degrading the
collagen-containing fibrous cap, they play an important role in the development
and subsequent stability of atherosclerotic plaques (103, 104). Indeed, degradation of the plaque matrix appears to be most active in macrophage-rich regions
(43, 100). Secretion of proteolytic enzymes, such as metalloproteinases (MMPs),
by activated macrophages may weaken the fibrous cap, particularly at the vulnerable shoulder region where the fibrous cap joins the arterial wall (105, 106).
Weakened fibrous caps lead to plaque instability, rupture, and ensuing thrombosis,
which ultimately present as acute coronary syndromes (43, 101, 107).
Lipid lowering by statins may contribute to plaque stability by reducing plaque
size or by modifying the physiochemical properties of the lipid core (108, 109).
However, changes in plaque size by lipid lowering tend to occur over extended
time and are quite minimal as assessed by angiography. Rather, the clinical benefits from lipid lowering are probably due to decreases in macrophage accumulation in atherosclerotic lesions and inhibition of MMP production by activated
macrophages (99). Indeed, statins inhibit the expression of MMPs and tissue factor
by cholesterol-dependent and -independent mechanisms (99, 108, 110), with the
cholesterol-independent or direct macrophage effects occurring at a much earlier
time point. The plaque-stabilizing properties of statins, therefore, are mediated
through a combined reduction in lipids, macrophages, and MMPs (111). These
effects of statins may reduce the incidence of acute coronary syndromes by lessening the propensity for plaque to rupture and may explain the rapid time course
of event reduction in patients at high risk for recurrent coronary ischemia in the
MIRACL (112) and PROVE-IT trials (113).
STATINS AND VASCULAR INFLAMMATION

Atherosclerosis is a complex inflammatory process that is characterized by the
presence of monocytes or macrophages and T lymphocytes in the atheroma (114,
115). Inflammatory cytokines secreted by these macrophages and T lymphocytes
can modify endothelial function, SMC proliferation, collagen degradation, and
thrombosis (43). An early step in atherogenesis involves monocyte adhesion to
the endothelium and penetration into the subendothelial space (115). Recent studies suggest that statins possess antiinflammatory properties owing to their ability
to reduce the number of inflammatory cells in atherosclerotic plaques (96). The
mechanisms have yet to be fully elucidated but may involve inhibition of adhesion
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molecules, such as intercellular adhesion molecule-1 (ICAM-1), which are involved in the recruitment of inflammatory cells (116). Furthermore, statins attenuate P-selectin expression and leukocyte adhesion in normocholesterolemic animals
by increasing endothelial NO production (117, 118). This cholesterol-independent
effect of statins was absent in eNOS-deficient mice, suggesting that eNOS mediated the vascular protective effects of statins (119).
The activation of T-lymphocytes and the control of the immune response are
mediated by the major histocompatibility complex class II (MHC-II) and CD40/
CD40L. Under physiological conditions, antigen-presenting cells express MHC-II
constitutively, whereas the induction of interferon gamma (INF- ) leads to an increase of MHC-II expression in numerous cells, including human endothelial cells
and monocytes. An important regulator in this pathway is the transactivator CIITA.
Statins inhibit MHC-II expression on endothelial cells and monocyte-macrophages
via inhibition of the promotor IV of the transactivator CIITA and thereby repress
MHC-II-mediated T cell activation (120). In addition, statins have been shown to
decrease CD40 expression and CD40-related activation of vascular cells (121).
A clinical marker of inflammation is high-sensitivity C-reactive protein (hsCRP) (122). hs-CRP is an acute phase reactant that is produced by the liver in
response to proinflammatory cytokines, such as interleukin-6 (IL-6), and reflects
low-grade systemic inflammation (123). Elevated levels of hs-CRP have been
shown to be predictive of increased risk for coronary artery disease (CAD) in apparently healthy men and women (124, 125). hs-CRP is elevated in patients with
CAD, coronary ischemia and myocardial infarction compared with normal subjects
(126). It has been suggested that CRP could also contribute to the development
of atherosclerosis by binding to modified LDL-C within atherosclerotic plaques
(127, 128). Once CRP becomes bound, it activates complement, which has been
shown to play a role in promoting atherosclerotic lesion progression (129). Furthermore, CRP has been shown to induce plasminogen activator inhibitor (PAI)-1
expression and complement activation, increase the expression of cellular adhesion
molecules, and decrease eNOS expression, leading to propensity for thrombosis,
inflammation, and endothelial dysfunction. Indeed, transgenic overexpression of
human CRP in transgenic mice leads to increased thrombosis and vascular inflammation following arterial injury (130). However, further studies are needed to fully
elucidate the role CRP plays in atherosclerosis and cardiovascular risk.
Statin therapy lowers hs-CRP levels in hypercholesterolemic patients (122, 131,
132). In the CARE trial, statins significantly decreased plasma hs-CRP levels over
a five-year period in patients who did not experience recurrent coronary events
(133, 134). Similarly, an analysis of baseline and one-year follow-up from the AFCAPS/TexCAPS demonstrated that hs-CRP levels were reduced in statin-treated
patients who were free of acute major coronary events (122). Furthermore, preliminary data from the Pravastatin Inflammation/CRP Evaluation (PRINCE) study
confirm that statin therapy can significantly reduce serum hs-CRP levels in primary and secondary prevention populations (135). Following 24 weeks of therapy
with a statin, the hs-CRP level was reduced by approximately 13% in primary and
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secondary prevention populations, whereas placebo treatment of subjects in the

primary prevention arm of the study had no effect. These studies, therefore, indicate
that statins are effective in decreasing systemic and vascular inflammation. However, any potential clinical benefits conferred by the lowering hs-CRP are difficult
to separate from that of the lipid-lowering effects of statins without performing further clinical studies. Perhaps the ongoing randomized placebo-controlled Jupiter
Trial, which is enrolling patients with modest LDL-C (<130 mg/dl) and elevated
hs-CRP (>2 mg/dl), will help address the question of whether CRP is an additional nonlipid-associated cardiovascular risk factor that can be modified by statin
therapy.
EFFECTS OF STATINS ON THE MYOCARDIUM

Cardiac hypertrophy is an adaptive response of the heart to pressure overload. In the
myocardium, the small GTP-binding proteins, Ras, Rho, and Rac, and oxidative
stress are involved in the hypertrophic response (136, 137). Indeed, recent animal
studies suggest that a phagocyte-type NADPH oxidase may be a relevant source of
ROS in the myocardium (138140). NADPH oxidase-dependent ROS production
appears to be involved in cardiac hypertrophy in response to pressure overload (140,
141), stretch (142), angiotensin II-infusion (139, 143), and -adrenergic stimulation (144). In the cardiomyocytes, three of its five components, p40phox(PHOX for
phagocyte oxidase), p47phox, and p67phox, exist in the cytosol, forming a complex
(Figure 4). The other two components, p22phox and gp91phox, are bound to the membranes. Various stimuli lead to the phosphorylation of the cytosolic components,
and the entire cytosolic complex then migrates to the membrane. Importantly, not
only the core subunits but also two low-molecular-weight guanine nucleotidebinding proteins, Rac1 and Rap, are required for activation. During activation,
Rac1 binds GTP and migrates to the membrane with the core cytosolic complex.
Therefore, Rac1 is critically involved in the activation of cardiovascular NADPH
oxidase. Recent evidence both from animal and from human studies indicates that
in failing myocardium, upregulation of Rac1 and p47phox membrane protein expression, as well as increased Rac1-GTPase activity, may resemble the underlying
mechanisms for increased oxidase activity and may represent a novel therapeutic
target for statin therapy.
Although the main impact of statin therapy in cardiovascular disease appears to
be predominantly vascular, recent animal and human studies suggest that statins
may also have direct beneficial effects on the myocardium. Because Rac1 is required for NADPH oxidase activity and cardiac hypertrophy is mediated, in part,
by myocardial oxidative stress, it is likely that statins could inhibit cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1 geranylgeranylation. Indeed, statins inhibit angiotensin IIinduced oxidative stress and
cardiac hypertrophy in rodents (145). This has also been observed in clinical studies where statins inhibit cardiac hypertrophy in humans with hypercholesterolemia
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(146). NADPH-oxidase-mediated ROS are increased in left ventricular myocardium

from patients with heart failure and correlate with an increased activity of Rac1
GTPase, and oral statin treatment is able to decrease Rac1 function in the human
heart (147).
The development of congestive heart failure (CHF), a common sequela of decompensated cardiac hypertrophy, is a major cause of death and morbidity in the
Western world. Several lines of evidence suggest that statins may emerge as a
novel treatment option for patients with CHF. Retrospective analysis of the large
statin trials, such as the 4S, suggests that statins reduce the incidence and morbidity of heart failure (148). Second, patients with heart failure are characterized
by increased vascular tone and endothelial dysfunction (149), which may be improved by statin therapy, irrespective of serum cholesterol levels. Third, statins
have proven to preserve cardiac function in animal models of myocardial hypertrophy and heart failure, such as aortic banding, myocardial infarction, and several
transgenic models (145, 150152).
In a recent prospective, double blind, placebo-controlled study, patients with
symptomatic, nonischemic, dilated cardiomyopathy were randomly divided into
two groups receiving statin or placebo for 14 weeks (153). Although patients receiving statins exhibited a modest reduction in serum cholesterol level compared
to patients receiving placebo, these patients demonstrated a significant improvement in exercise endurance, as exhibited by a lower New York Heart Association
functional class compared to patients receiving placebo. This corresponded to improved left ventricular ejection fraction in the statin group (33 4 to 41 4%,
P < 0.01), but not in the placebo group. The improvements in their exercise endurance and heart function were in addition to the improvements already observed
with two current treatments for heart failure, beta-blockers and ACE inhibitors.
Furthermore, plasma concentrations of tumor necrosis factor alpha (TNF-), IL-6,
and brain natriuretic peptide (BNP) were lower in the statin group compared to
the placebo group. This study indicates that short-term statin therapy improves
cardiac function, neurohormonal imbalance, and symptoms associated with idiopathic dilated cardiomyopathy. These observations were confirmed in a second
study using cerivastatin (154). These findings suggest that statins may have therapeutic benefits in patients with heart failure irrespective of serum cholesterol levels
or atherosclerotic heart disease.
STATINS AND ISCHEMIC STROKE

Although myocardial infarction is closely associated with serum cholesterol levels, neither the Framingham Heart Study nor the Multiple Risk Factor Intervention Trial (MRFIT) demonstrated significant correlation between ischemic stroke
and serum cholesterol levels (12, 13). An intriguing result of large clinical trials
with statins is the reduction in ischemic stroke (155). For example, the recent
Heart Protection Study (HPS) shows a 28% reduction in ischemic strokes in over
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20,000 people with cerebrovascular disease or other high-risk conditions (156). The
proportional reductions in stroke were approximately one quarter in all subcategories studied, including those aged over 70 years at entry and those presenting
with different levels of blood pressure or lipids, even when the pretreatment LDLC was below 3.0 mmol/L (116 mg/dl). Thus, the findings of these large statin trials
raise the interesting question of how a class of cholesterol-lowering agents can
reduce ischemic stroke when ischemic stroke is not related to cholesterol levels. It
appears likely that there are cholesterol-independent effects of statins, which are
beneficial for ischemic stroke. Some of these beneficial effects may relate to the
effects of statins on endothelial and platelet function.
Cerebrovascular tone and blood flow are regulated by endothelium-derived
NO (157). Mutant mice lacking eNOS (eNOS/) are relatively hypertensive and
develop greater proliferative and inflammatory response to vascular injury (158).
Indeed, eNOS/ mice develop larger cerebral infarcts following cerebrovascular
occlusion (159). Thus, the beneficial effects of statins in ischemic stroke may, in
part, be due to their ability to upregulate eNOS expression and activity (32, 55). For
example, mice that were prophylactically treated with statins for up to two weeks,
have 25%30% higher cerebral blood flow and 50% smaller cerebral infarct sizes
following cerebrovascular occlusion (160). No increase in cerebral blood flow or
neuroprotection was observed in eNOS/ mice treated with statins, indicating
that the upregulation of eNOS accounts for most, if not all, of the neuroprotective
effects of these agents. Interestingly, treatment with statins did not affect blood
pressure or heart rate before, during, or after cerebrovascular ischemia and did not
alter serum cholesterol levels in mice, consistent with the cholesterol-independent,
neuroprotective effects of statins.
In addition to increases in cerebral blood flow, other beneficial effects of statins
are likely to occur that can impact on the severity of ischemic stroke. For example, statins attenuate P-selectin expression and leukocyte adhesion via increases
in NO production in a model of cardiac ischemia and reperfusion (161, 162). Others have reported that statins upregulate tissue-type plasminogen activator (t-PA)
and downregulate plasminogen activator inhibitor (PAI)-1 expression through a
similar mechanism involving inhibition of Rho geranylgeranylation (57). Thus,
the absence of neuroprotection in eNOS-deficient mice emphasizes the importance of endothelium-derived NO in not only augmenting cerebral blood flow
but also, potentially, in limiting the impact of platelet and white blood cell accumulation on tissue viability following ischemia. In humans, atherosclerosis of
precerebral arteries causes stroke through plaque disruption and artery-to-artery
thromboembolism, and, in contrast to the mouse models, statins exert additional
stroke-protective effects in humans through their anti-atherosclerotic and plaquestabilizing effects. Furthermore, the antiinflammatory actions and mobilization of
endothelial progenitor cells of statins may also contribute to neuroprotection. It
is therefore possible that statins have contributed to the decrease in the incidence
of ischemic strokes in clinical trials, in part, by reducing cerebral infarct size to
levels that were clinically unappreciated.
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STATINS AND DEMENTIA

Recent epidemiological reports suggest that statins might be protective for Alzheimers disease, and for other types of dementia (163). Dementia is a syndrome of
chronic or progressive nature with multiple disturbances of higher cortical functions. This syndrome occurs in Alzheimers disease, in cerebrovascular disease
(i.e., multi-infarct dementia), and in other conditions primarily or secondarily affecting the brain. Alzheimers disease is related to the effects of -amyloid, a peptide that accumulates in the brain, causing neurotoxicity and neurodegeneration.
Experimental and clinical studies suggest that there is a pathophysiologic relation
between -amyloid and cholesterol levels. Elevated -amyloid levels and the 4
allele of the apolipoprotein E (APOE4) are risk factors for Alzheimers disease
(164). In addition, APOE4 is correlated with increased risk for atherosclerosis and
amyloid plaque formation (165, 166). Observational studies revealed that an elevated serum cholesterol level is a risk factor for Alzheimers disease (167). Statins,
regardless of their brain availability, have been suggested to induce alterations in
cellular cholesterol distribution in the brain. Such cholesterol-independent effects
of statins might be mediated via NO or ApoE (168, 169). A cross-sectional analysis
of three hospital databases by Wolozin and colleagues suggested that the prevalence of Alzheimers disease in patients taking statins is 60% lower in comparison
to patients taking other medications used in the treatment of cardiovascular diseases (170). A nested case control study based on the UK-based General Practice
Research Database showed that among individuals 50 years and older with a statin
therapy, the risk for developing dementia was significantly reduced, independent
of their lipid status (171). Furthermore, other lipid-lowering agents had no influence on the risk of developing dementia in this population. The systemic vascular
protective effects of statin treatment are very likely to contribute to their beneficial effects, especially on vascular forms of the dementia syndrome. However, the
precise underlying molecular mechanisms are poorly understood. Indeed, the results of the recent HPS and Prospective Study of Pravastatin in the Elderly at Risk
(PROSPER) trials do not demonstrate the efficacy of statins in slowing cognitive
decline and dementia (9, 172).
Recent evidence has stimulated the discussion of statins as potential novel antiinflammatory and vascular protective agents for the treatment of other cerebral
diseases, such as multiple sclerosis and depression. Oral atorvastatin was shown
to prevent chronic and relapsing experimental autoimmune encephalomyelitis,
a CD4(+) Th1-mediated central nervous system (CNS) demyelinating disease
model of multiple sclerosis (173). Statin induced STAT6 phosphorylation and secretion of Th2 cytokines and transforming growth factor (TGF)-. Conversely,
STAT4 phosphorylation was inhibited and secretion of Th1 cytokines was suppressed. Statin promoted the differentiation of Th0 cells into Th2 cells. In adoptive
transfer, these Th2 cells protected recipient mice from induction of autoimmune
encephalomyelitis. Statin reduced CNS infiltration and mMHC-II expression.
Treatment of microglia inhibited IFN- -inducible transcription at multiple
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MHC-II transactivator (CIITA) promoters and suppressed MHC-II upregulation.

Statin suppressed IFN- -inducible expression of CD40, CD80, and CD86 costimulatory molecules as well as antigen-specific T cell activation. Similarly, a
second study shows that oral statin inhibited the development of actively induced
chronic CD4+T cellmediated experimental autoimmune encephalomyelitis in a
preventive and therapeutic fashion and significantly reduced the inflammatory
infiltration into the CNS (174). This potentially therapeutic effect was associated
with downregulation of Th1 immune response. In addition, similar to the effects of
statins in vascular SMCs, statins inhibited the cell cycle of human antigenspecific
T cells. Thus, statins exert pleiotropic immunomodulatory effects involving both
antigen presenting cells (APC) and T cell compartments, and they may be beneficial for multiple sclerosis and other Th1-mediated autoimmune diseases.
A possible association between lipid-lowering drug therapy and psychological well-being has been an issue of debate. A recent nested case-control analysis
of the United Kingdom General Practice Research Database revealed that the
use of statins and other lipid-lowering drugs is not associated with an increased
risk of depression or suicide (175). On the contrary, individuals with current statin
use may have a lower risk of developing depression, an effect that could be explained by improved quality of life owing to decreased risk of cardiovascular events
or more health consciousness in patients receiving long-term treatment. Furthermore, another comparison of patients who had continuous use of statins with the
patients who did not use any cholesterol-lowering drugs showed that statin use
was associated with lower risk of abnormal depression scores, anxiety, and hostility, after adjustment for the propensity for statin use and potential confounders.
Interestingly, the beneficial psychological effects of the statins appeared to be
independent of the drugs cholesterol-lowering effects (176).
CLINICAL TRIALS WITH STATINS: EVIDENCE

FOR PLEIOTROPY
Because serum cholesterol level is strongly associated with coronary heart disease, it has been generally assumed that cholesterol reduction by statins is the
predominant, if not the only mechanism, underlying their beneficial effects. Data
from a meta-analysis of lipid-lowering trials suggest lipid modification alone accounts for the clinical benefits associated with statin therapy. Indeed, the slope of
the relationship between cholesterol reduction and mortality risk reduction was
the same for statins and nonstatins, whereas the mortality risk reductions realized
over statin treatment periods of two years and longer were found to be a consequence of cholesterol reduction alone (Figure 5, left panel). However, this type of
meta-analysis does not take into account the differences in terms of the length of
the individual trials with respect to cardiovascular benefits. Some of the nonstatin
lipid-lowering trials, such as the Lipid Research ClinicCoronary Primary Prevention Trial (LRC-CPPT) using the bile acid resin cholestyramine (177), or the
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Figure 5 Relationship between LDL-C reduction and risk of cardiovascular events. (Left
panel) Decrease in LDL-C (% reduction) is correlated with reduction in risk of nonfatal
myocardial infarctions (MI) or coronary heart disease (CHD) among statin (WOSCOPS,
CARE, and 4S) and nonstatin (LRC-CPPT and POSCH) trials. Note that the relationship
(slope) holds between statin and nonstatin trials, suggesting that the beneficial effects of
statins are likely due to only cholesterol lowering. (Right panel) Decrease in LDL-C (%
reduction) is correlated with reduction in risk of nonfatal myocardial infarctions (MI) or
coronary heart disease (CHD) among statin (WOSCOPS, CARE, and 4S) and nonstatin
(LRC-CPPT and POSCH) trials after 4.5 years of treatment. Note that the nonstatin trials
(LRC-CPPT and POSCH; dashed lines) show less cardiovascular benefits than statin trials
(WOSCOPS, CARE, and 4S) and they no longer fall on the same slope (solid line).
Program on the Surgical Control of the Hyperlipidemias (POSCH) using partial

ileal bypass surgery (178), reported benefits after 7.4 and 9.7 years, respectively,
whereas most of the statin trials showed benefits at much earlier time points (e.g.,
within 5 years). Thus, if one compares the benefits after five years for all lipidlowering trials, one finds that the nonstatin trials no longer fall on the same slope
of cholesterol:mortality risk reduction as do all of the statin trials (Figure 5, right
panel). In fact, the benefits of cholesterol lowering after ileal bypass surgery in the
POSCH study were not realized at 4.5 years, despite significant LDL-C reduction
of 34% within the first 3 months after the surgical procedure. These results suggest
that the beneficial effects of statins occur more rapidly and may not be entirely
dependent on cholesterol reduction.
Despite the rapidity of benefits of statin therapy compared to other nonstatin
lipid-lowering therapies, it is still difficult to prove that pleiotropic effects of statins
are real. First, patients receiving statin therapy invariably will have reduced lipid
levels and it is often difficult to separate the lipid- from the nonlipid-lowering
effects of statins in clinical trials. Second, many effects of statins, such as improvement in endothelial function, decreased inflammation, increased plaque stability, and reduced thrombogenic response, could all be accounted for, to some
extent, by lipid lowering. Third, the concentrations used to demonstrate the biological effects of statins in cell culture and animal experiments, especially with
regards to inhibition of Rho geranylgeranylation but not PI3-kinase/Akt activation, appear to be much higher than what is prescribed clinically. Finally, both hydrophilic and lipophilic statins, which inhibit hepatic HMG-CoA reductase, appear
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to exert similar cholesterol-independent effects, despite the relative impermeability

of hydrophilic statins in vascular tissues. Thus, it appears that statins are very potent cholesterol-lowering agents and that reduction in cholesterol levels by statins
contributes to many of their clinical benefits.
However, in the recent HPS and ASCOT trials, the relative risk reduction conferred by statin treatment was independent of the pretreatment lipid levels (9, 10).
These large prospective trials raise the question of whether individuals with CHD
could benefit from statin drugs independently of cholesterol levels. Interestingly,
subgroup analyses of previous clinical trials suggested that the beneficial effects
of statins could extend to mechanisms beyond cholesterol reduction. For example, subgroup analysis of the WOSCOPS and CARE studies indicate that despite
comparable serum cholesterol levels among the statin-treated and placebo groups,
statin-treated individuals have significantly lower risks for coronary heart disease
compared to age-matched placebo-controlled individuals (5, 7). Indeed, when the
statin treatment group was divided into quintiles of percentage LDL-C reduction,
it was found that there was no difference in the 4.4-year coronary event rate for
quintiles 2 through to 5 (LDL-C reductions of 23%41%). Hence, there was no
apparent association between coronary event rate and the level of LDL-C reduction. Furthermore, meta-analyses of cholesterol-lowering trials suggest that the
risk of myocardial infarctions in individuals treated with statins is significantly
lower compared to individuals treated with other cholesterol-lowering agents or
modalities despite comparable reduction in serum cholesterol levels in both groups
(179). For example, application of the Framingham risk score to WOSCOPS produced a coincidence between predicted and observed risk in the placebo group but
underestimated the benefit of the pravastatin group by 31% (180).
TABLE 1
Statin pleiotropy
Effect
Benefit
Increased synthesis of nitric oxide
Improvement of endothelial dysfunction
Inhibition of free radical release

Decreased synthesis of endothelin-1
Inhibition of LDL-C oxidation
Upregulation of endothelial progenitor cells
Reduced number and activity of inflammatory cells
Reduced inflammatory response
Reduced levels of C-reactive protein

Reduced macrophage cholesterol accumulation
Stabilization of atherosclerotic plaques
Reduced production of metalloproteinases

Inhibition of platelet adhesion/aggregation
Reduced fibrinogen concentration
Reduced blood viscosity
Reduced thrombogenic response
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Finally, the lipophilic statins would be expected to penetrate cell membranes

more effectively than the more hydrophilic statins, causing more side effects but
at the same time eliciting more pleiotropic effects. However, the observation that
hydrophilic statins have similar pleiotropic effects as lipophilic statins puts into
question whether there are really any cholesterol-independent effects of statins.
Indeed, recent evidence suggests that some of the cholesterol-independent effects
of these agents may be mediated by inhibition of hepatic HMG-CoA reductase
leading to subsequent reduction in circulating isoprenoid levels (28). This hypothesis may help explain why hydrophilic statins, such as pravastatin and rosuvastatin,
are still able to exert cholesterol-independent benefits on the vascular wall without
directly entering vascular wall cells. In this respect, the word pleiotropic probably
does not reflect the hepatic versus nonhepatic effects of these agents.
SUMMARY
Statins exert many pleiotropic effects in addition to the lowering of serum cholesterol levels. These additional properties include beneficial effects on endothelial
function and blood flow, decreasing LDL-C oxidation, enhancing the stability
of atherosclerotic plaques, inhibiting vascular smooth muscle proliferation and
platelet aggregation, and reducing vascular inflammation (Table 1). Recent evidence suggest that most of these effects are mediated by statins inhibitory effect
on isoprenoid synthesis. In particular, inhibition of Rho GTPases in vascular wall
cells by statins leads to increased expression of atheroprotective genes and inhibition of vascular SMC proliferation. It remains to be determined which of and
to what extent these pleiotropic effects account for the clinical benefits of statin
therapy beyond cholesterol lowering.
ACKNOWLEDGMENTS
The work described in this paper was supported in part by the National Institutes of
Health (HL-52233, HL-48743, and NS-10828), the American Heart Association
Bugher Foundation Award, and the Deutsche Forschungsgesellschaft. Dr. Liao is
an Established Investigator of the American Heart Association.
LITERATURE CITED
1. Libby P, Sukhova G, Lee RT, Liao JK.
1997. Molecular biology of atherosclerosis. Int. J. Cardiol. 62 (Suppl 2):S2329
2. Sytkowski PA, Kannel WB, DAgostino
RB. 1990. Changes in risk factors and

the decline in mortality from cardiovascular disease. The Framingham Heart Study.
New Engl. J. Med. 322:163541
7 Jan 2005
13:56
AR
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE


3. Gordon T, Kannel WB. 1971. Premature
mortality from coronary heart disease.
The Framingham Study. JAMA 215:
161725
4. 1994. Randomised trial of cholesterol
lowering in 4444 patients with coronary
heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 344:
138389
5. Sacks FM, Pfeffer MA, Moye LA,
Rouleau JL, Rutherford JD, et al. 1996.
The effect of pravastatin on coronary
events after myocardial infarction in patients with average cholesterol levels.
Cholesterol and Recurrent Events Trial investigators. New Engl. J. Med. 335:1001
9
6. 1998. Prevention of cardiovascular events
and death with pravastatin in patients with
coronary heart disease and a broad range
of initial cholesterol levels. The LongTerm Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group.
New Engl. J. Med. 339:134957
7. Shepherd J, Cobbe SM, Ford I, Isles CG,
Lorimer AR, et al. 1995. Prevention of
coronary heart disease with pravastatin
in men with hypercholesterolemia. West
of Scotland Coronary Prevention Study
Group. New Engl. J. Med. 333:13017
8. Downs JR, Clearfield M, Weis S, Whitney E, Shapiro DR, et al. 1998. Primary
prevention of acute coronary events with
lovastatin in men and women with average cholesterol levels: results of AFCAPS/TexCAPS. Air Force/Texas Coronary Atherosclerosis Prevention Study.
JAMA 279:161522
9. 2002. MRC/BHF Heart Protection Study
of cholesterol lowering with simvastatin
in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet
360:722
10. Sever PS, Dahlof B, Poulter NR, Wedel H,
Beevers G, et al. 2003. Prevention of coronary and stroke events with atorvastatin in
hypertensive patients who have average
or lower-than-average cholesterol con-
11.
12.
13.
14.
15.
16.
17.
18.
19.
109
centrations, in the Anglo-Scandinavian

Cardiac Outcomes TrialLipid Lowering Arm (ASCOT-LLA): a multicentre
randomised controlled trial. Lancet 361:
114958
Goldstein JL, Brown MS. 1990. Regulation of the mevalonate pathway. Nature
343:42530
Kannel WB, Castelli WP, Gordon T, McNamara PM. 1971. Serum cholesterol,
lipoproteins, and the risk of coronary heart
disease. The Framingham study. Ann. Intern. Med. 74:112
1982. Multiple risk factor intervention
trial. Risk factor changes and mortality
results. Multiple Risk Factor Intervention
Trial Research Group. JAMA 248:1465
77
Iso H, Jacobs DR Jr., Wentworth D,
Neaton JD, Cohen JD. 1989. Serum
cholesterol levels and six-year mortality
from stroke in 350,977 men screened for
the multiple risk factor intervention trial.
New Engl. J. Med. 320:90410
Panel NCEPE. 2002. Third Report of
the National Cholesterol Education Program (NCEP) Expert Panel on Detection,
Evaluation, and Treatment of High Blood
cholesterol in Adults (Adult Treatment
Panel III). Natl. Heart Lung Blood Inst.
Natl. Inst. Health 02-5215:3143421
Rodwell VW, Nordstrom JL, Mitschelen
JJ. 1976. Regulation of HMG-CoA reductase. Adv. Lipid Res. 14:174
Alberts AW. 1988. Discovery, biochemistry and biology of lovastatin. Am. J. Cardiol. 62:10J15
Endo A, Kuroda M, Tsujita Y. 1976. ML236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by
Penicillium citrinium. J. Antibiot. (Tokyo)
29:134648
Alberts AW, Chen J, Kuron G, Hunt V,
Huff J, et al. 1980. Mevinolin: a
highly potent competitive inhibitor of
hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent.
7 Jan 2005
13:56

110
AR
LIAO
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAUFS
20. Alberts AW. 1990. Lovastatin and

simvastatininhibitors of HMG CoA
reductase and cholesterol biosynthesis.
Cardiology 77(Suppl. 4):1421
21. Illingworth DR, Tobert JA. 2001. HMGCoA reductase inhibitors. Adv. Protein
Chem. 56:77114
22. Istvan ES, Deisenhofer J. 2001. Structural
mechanism for statin inhibition of HMGCoA reductase. Science 292:116064
23. Moghadasian MH. 1999. Clinical pharmacology of 3-hydroxy-3-methylglutaryl
coenzyme A reductase inhibitors. Life Sci.
65:132937
24. Blum CB. 1994. Comparison of properties of four inhibitors of 3-hydroxy3-methylglutaryl-coenzyme A reductase.
Am. J. Cardiol. 73:3D11
25. Dansette PM, Jaoen M, Pons C. 2000.
HMG-CoA reductase activity in human
liver microsomes: comparative inhibition
by statins. Exp. Toxicol. Pathol. 52:145
48
26. McTaggart F, Buckett L, Davidson R,
Holdgate G, McCormick A, et al. 2001.
Preclinical and clinical pharmacology
of Rosuvastatin, a new 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor. Am. J. Cardiol. 87:28B32
27. Germershausen JI, Hunt VM, Bostedor
RG, Bailey PJ, Karkas JD, Alberts AW.
1989. Tissue selectivity of the cholesterollowering agents lovastatin, simvastatin
and pravastatin in rats in vivo. Biochem.
28. Corsini A, Bellosta S, Baetta R, Fumagalli R, Paoletti R, Bernini F. 1999.
New insights into the pharmacodynamic
and pharmacokinetic properties of statins.
Pharmacol. Ther. 84:41328
29. Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, et al. 2001.
Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat. Med. 7:68792
30. Van Aelst L, DSouza-Schorey C. 1997.
Rho GTPases and signaling networks.
Genes Dev. 11:2295322
31. Hall A. 1998. Rho GTPases and the actin

cytoskeleton. Science 279:50914
32. Laufs U, La Fata V, Plutzky J, Liao
JK. 1998. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 97:1129
35
33. Laufs U, Liao JK. 1998. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability
by Rho GTPase. J. Biol. Chem. 273:
2426671
34. Takemoto M, Sun J, Hiroki J, Shimokawa
H, Liao JK. 2002. Rho-kinase mediates
hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation 106:5762
35. Laufs U, Endres M, Stagliano N, AminHanjani S, Chui DS, et al. 2000. Neuroprotection mediated by changes in the
endothelial actin cytoskeleton. J. Clin. Invest. 106:1524
36. Hall A. 1994. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10:31
54
37. Uehata M, Ishizaki T, Satoh H, Ono T,
Kawahara T, et al. 1997. Calcium sensitization of smooth muscle mediated by a
Rho-associated protein kinase in hypertension. Nature 389:99094
38. Katsumata N, Shimokawa H, Seto M,
Kozai T, Yamawaki T, et al. 1997. Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm in a swine model with
interleukin-1beta. Circulation 96:4357
63
39. Laufs U, Marra D, Node K, Liao JK.
1999. 3-Hydroxy-3-methylglutaryl-CoA
reductase inhibitors attenuate vascular
smooth muscle proliferation by preventing rho GTPase-induced down-regulation
of p27(Kip1). J. Biol. Chem. 274:21926
31
40. Singh R, Wang B, Shirvaikar A, Khan S,
Kamat S, et al. 1999. The IL-1 receptor
and Rho directly associate to drive cell
7 Jan 2005
13:56
AR
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
41.

42.
43.
44.
45.
46.
47.
48.
49.
50.
activation in inflammation. J. Clin. Invest.

103:156170
Mundy G, Garrett R, Harris S, Chan
J, Chen D, et al. 1999. Stimulation of
bone formation in vitro and in rodents by
statins. Science 286:194649
Liao JK, Bettmann MA, Sandor T, Tucker
JI, Coleman SM, Creager MA. 1991.
Differential impairment of vasodilator
responsiveness of peripheral resistance
and conduit vessels in humans with
atherosclerosis. Circ. Res. 68:102734
Libby P. 1995. Molecular bases of the
acute coronary syndromes. Circulation
91:284450
Ignarro LJ, Buga GM, Wood KS, Byrns
RE, Chaudhuri G. 1987. Endotheliumderived relaxing factor produced and
released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84:9265
69
Radomski MW, Rees DD, Dutra A, Moncada S. 1992. S-Nitroso-glutathione inhibits platelet activation in vitro and in
vivo. Br. J. Pharmacol. 107:74549
Garg UC, Hassid A. 1989. Nitric oxidegenerating vasodilators and 8-bromocyclic guanosine monophosphate inhibit
mitogenesis and proliferation of cultured
rat vascular smooth muscle cells. J. Clin.
Invest. 83:177477
Gauthier TW, Scalia R, Murohara T,
Guo JP, Lefer AM. 1995. Nitric oxide
protects against leukocyte-endothelium
interactions in the early stages of hypercholesterolemia. Arterioscler. Thromb.
Vasc. Biol. 15:165259
Kubes P, Suzuki M, Granger DN. 1991.
Nitric oxide: an endogenous modulator of
leukocyte adhesion. Proc. Natl. Acad. Sci.
USA 88:465155
Harrison DG. 1997. Cellular and molecular mechanisms of endothelial cell
dysfunction. J. Clin. Invest. 100:2153
57
Munzel T, Sayegh H, Freeman BA, Tarpey
MM, Harrison DG. 1995. Evidence for
enhanced vascular superoxide anion pro-
51.
52.
53.
54.
55.
56.
57.
58.
111
duction in nitrate tolerance. A novel

mechanism underlying tolerance and
cross-tolerance. J. Clin. Invest. 95:187
94
Tamai O, Matsuoka H, Itabe H, Wada
Y, Kohno K, Imaizumi T. 1997. Single
LDL apheresis improves endotheliumdependent vasodilatation in hypercholesterolemic humans. Circulation 95:7682
Anderson TJ, Meredith IT, Yeung AC,
Frei B, Selwyn AP, Ganz P. 1995. The
effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent
coronary vasomotion. New Engl. J. Med.
332:48893
Treasure CB, Klein JL, Weintraub WS,
Talley JD, Stillabower ME, et al. 1995.
Beneficial effects of cholesterol-lowering
therapy on the coronary endothelium in
patients with coronary artery disease. New
Engl. J. Med. 332:48187
ODriscoll G, Green D, Taylor RR. 1997.
Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 95:1126
31
Kureishi Y, Luo Z, Shiojima I, Bialik A,
Fulton D, et al. 2000. The HMG-CoA reductase inhibitor simvastatin activates the
protein kinase Akt and promotes angiogenesis in normocholesterolemic animals.
Nat. Med. 6:100410
Laufs U, Fata VL, Liao JK. 1997. Inhibition of 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase blocks hypoxiamediated down-regulation of endothelial nitric oxide synthase. J. Biol. Chem.
272:3172529
Essig M, Nguyen G, Prie D, Escoubet
B, Sraer JD, Friedlander G. 1998. 3Hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors increase fibrinolytic
activity in rat aortic endothelial cells. Role
of geranylgeranylation and Rho proteins.
Circ. Res. 83:68390
Hernandez-Perera O, Perez-Sala D,
Navarro-Antolin J, Sanchez-Pascuala R,
Hernandez G, et al. 1998. Effects of the
7 Jan 2005
13:56
112

59.
60.
61.
62.
63.
64.
65.
66.
AR
LIAO
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAUFS
3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1

and endothelial nitric oxide synthase in
vascular endothelial cells. J. Clin. Invest.
101:271119
Laufs U, Endres M, Custodis F, Gertz K,
Nickenig G, et al. 2000. Suppression of
endothelial nitric oxide production after
withdrawal of statin treatment is mediated by negative feedback regulation of
rho GTPase gene transcription. Circulation 102:310410
Plenz GA, Hofnagel O, Robenek H. 2004.
Differential modulation of caveolin-1 expression in cells of the vasculature by
statins. Circulation 109:e78
Brouet A, Sonveaux P, Dessy C, Moniotte
S, Balligand JL, Feron O. 2001. Hsp90
and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of
statins. Circ. Res. 89:86673
Fulton D, Gratton JP, McCabe TJ, Fontana
J, Fujio Y, et al. 1999. Regulation of
endothelium-derived nitric oxide production by the protein kinase Akt. Nature
399:597601
Dimmeler S, Fleming I, Fisslthaler B,
Hermann C, Busse R, Zeiher AM. 1999.
Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:6015
Bucci M, Gratton JP, Rudic RD, Acevedo
L, Roviezzo F, et al. 2000. In vivo delivery of the caveolin-1 scaffolding domain
inhibits nitric oxide synthesis and reduces
inflammation. Nat. Med. 6:136267
Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL. 1999. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of
caveolin and endothelial nitric oxide synthase. J. Clin. Invest. 103:897905
Rikitake Y, Kawashima S, Takeshita S,
Yamashita T, Azumi H, et al. 2001.
Anti-oxidative properties of fluvastatin,
an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis
67.
68.
69.
70.
71.
72.
73.
74.
in cholesterol-fed rabbits. Atherosclerosis

154:8796
Cai H, Harrison DG. 2000. Endothelial
dysfunction in cardiovascular diseases:
the role of oxidant stress. Circ. Res.
87:84044
Wassmann S, Laufs U, Baumer AT,
Muller K, Ahlbory K, et al. 2001.
HMG-CoA reductase inhibitors improve endothelial dysfunction in normocholesterolemic hypertension via reduced
production of reactive oxygen species.
Hypertension 37:145057
Llevadot J, Murasawa S, Kureishi Y,
Uchida S, Masuda H, et al. 2001. HMGCoA reductase inhibitor mobilizes bone
marrowderived endothelial progenitor
cells. J. Clin. Invest. 108:399405
Murohara T, Ikeda H, Duan J, Shintani S,
Sasaki K, et al. 2000. Transplanted cord
blood-derived endothelial precursor cells
augment postnatal neovascularization. J.
Clin. Invest. 105:152736
Walter DH, Rittig K, Bahlmann FH,
Kirchmair R, Silver M, et al. 2002. Statin
therapy accelerates reendothelialization: a
novel effect involving mobilization and
incorporation of bone marrow-derived
endothelial progenitor cells. Circulation
105:301724
Werner N, Priller J, Laufs U, Endres M,
Bohm M, et al. 2002. Bone marrowderived progenitor cells modulate
vascular reendothelialization and neointimal formation: effect of 3-hydroxy3-methylglutaryl coenzyme a reductase
inhibition. Arterioscler. Thromb. Vasc.
Biol. 22:156772
Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, et al. 2001. Therapeutic potential of ex vivo expanded
endothelial progenitor cells for myocardial ischemia. Circulation 103:634
37
Dimmeler S, Aicher A, Vasa M, MildnerRihm C, Adler K, et al. 2001. HMGCoA reductase inhibitors (statins) increase endothelial progenitor cells via the
7 Jan 2005
13:56
AR
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
75.

76.
77.
78.
79.
80.
81.
82.
83.
PI 3-kinase/Akt pathway. J. Clin. Invest.

108:39197
Vasa M, Fichtlscherer S, Adler K,
Aicher A, Martin H, et al. 2001. Increase in circulating endothelial progenitor cells by statin therapy in patients with
stable coronary artery disease. Circulation 103:288590
Aicher A, Heeschen C, Mildner-Rihm C,
Urbich C, Ihling C, et al. 2003. Essential
role of endothelial nitric oxide synthase
for mobilization of stem and progenitor
cells. Nat. Med. 9:137076
Vincent L, Soria C, Mirshahi F, Opolon
P, Mishal Z, et al. 2002. Cerivastatin, an
inhibitor of 3-hydroxy-3-methylglutaryl
coenzyme A reductase, inhibits endothelial cell proliferation induced by angiogenic factors in vitro and angiogenesis
in in vivo models. Arterioscler. Thromb.
Vasc. Biol. 22:62329
Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D, Galper JB. 2002. 3Hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA. Circ. Res. 91:14350
Weis M, Heeschen C, Glassford AJ,
Cooke JP. 2002. Statins have biphasic effects on angiogenesis. Circulation
105:73945
Sata M, Nishimatsu H, Suzuki E, Sugiura
S, Yoshizumi M, et al. 2001. Endothelial
nitric oxide synthase is essential for the
HMG-CoA reductase inhibitor cerivastatin to promote collateral growth in response to ischemia. FASEB J. 15:253032
Braun-Dullaeus RC, Mann MJ, Dzau VJ.
1998. Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation 98:8289
Kobashigawa JA, Katznelson S, Laks H,
Johnson JA, Yeatman L, et al. 1995. Effect of pravastatin on outcomes after cardiac transplantation. New Engl. J. Med.
333:62127
Yang Z, Kozai T, van der Loo B,
Viswambharan H, Lachat M, et al. 2000.
84.
85.
86.
87.
88.
89.
90.
91.
92.
113
HMG-CoA reductase inhibition improves

endothelial cell function and inhibits
smooth muscle cell proliferation in human saphenous veins. J. Am. Coll. Cardiol. 36:169197
Hughes DA. 1995. Control of signal
transduction and morphogenesis by Ras.
Semin. Cell Biol. 6:8994
Hengst L, Reed SI. 1996. Translational
control of p27Kip1 accumulation during
the cell cycle. Science 271:186164
Fitzgerald DJ, Roy L, Catella F, FitzGerald GA. 1986. Platelet activation in unstable coronary disease. New Engl. J. Med.
315:98389
Lacoste L, Lam JY, Hung J, Letchacovski G, Solymoss CB, Waters D. 1995.
Hyperlipidemia and coronary disease.
Correction of the increased thrombogenic potential with cholesterol reduction.
Circulation 92:317277
Willerson JT, Golino P, Eidt J, Campbell WB, Buja LM. 1989. Specific platelet
mediators and unstable coronary artery
lesions. Experimental evidence and potential clinical implications. Circulation
80:198205
Opper C, Clement C, Schwarz H, Krappe
J, Steinmetz A, et al. 1995. Increased number of high sensitive platelets in hypercholesterolemia, cardiovascular diseases,
and after incubation with cholesterol.
Atherosclerosis 113:21117
Notarbartolo A, Davi G, Averna M,
Barbagallo CM, Ganci A, et al. 1995.
Inhibition of thromboxane biosynthesis and platelet function by simvastatin
in type IIa hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 15:24751
Baldassarre D, Mores N, Colli S, Pazzucconi F, Sirtori CR, Tremoli E. 1997.
Platelet alpha 2-adrenergic receptors in
hypercholesterolemia: relationship between binding studies and epinephrineinduced platelet aggregation. Clin. Pharmacol. Ther. 61:68491
Le Quan Sang KH, Levenson J, Megnien JL, Simon A, Devynck MA. 1995.
7 Jan 2005
13:56
114

93.
94.
95.
96.
97.
98.
99.
100.
AR
LIAO
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAUFS
Platelet cytosolic Ca2+ and membrane

dynamics in patients with primary hypercholesterolemia. Effects of pravastatin. Arterioscler. Thromb. Vasc. Biol. 15:759
64
Huhle G, Abletshauser C, Mayer N, Weidinger G, Harenberg J, Heene DL. 1999.
Reduction of platelet activity markers in
type II hypercholesterolemic patients by a
HMG-CoA-reductase inhibitor. Thromb.
Res. 95:22934
Hale LP, Craver KT, Berrier AM,
Sheffield MV, Case LD, Owen J. 1998.
Combination of fosinopril and pravastatin
decreases platelet response to thrombin
receptor agonist in monkeys. Arterioscler.
Thromb. Vasc. Biol. 18:164346
Laufs U, Gertz K, Huang P, Nickenig G,
Bohm M, et al. 2000. Atorvastatin upregulates type III nitric oxide synthase
in thrombocytes, decreases platelet activation, and protects from cerebral ischemia in normocholesterolemic mice.
Stroke 31:244249
Vaughan CJ, Gotto AM Jr., Basson CT.
2000. The evolving role of statins in the
management of atherosclerosis. J. Am.
Coll. Cardiol. 35:110
Lijnen P, Echevaria-Vazquez D, Petrov
V. 1996. Influence of cholesterol-lowering
on plasma membrane lipids and function. Methods Find. Exp. Clin. Pharmacol. 18:12336
Alfon J, Royo T, Garcia-Moll X, Badimon
L. 1999. Platelet deposition on eroded
vessel walls at a stenotic shear rate is inhibited by lipid-lowering treatment with
atorvastatin. Arterioscler. Thromb. Vasc.
Biol. 19:181217
Aikawa M, Rabkin E, Sugiyama S, Voglic
SJ, Fukumoto Y, et al. 2001. An HMGCoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue
factor in vivo and in vitro. Circulation
103:27683
Fuster V. 1995. Elucidation of the role
of plaque instability and rupture in acute
101.
102.
103.
104.
105.
106.
107.
108.
coronary events. Am. J. Cardiol. 76:24C

33
Fuster V, Stein B, Ambrose JA, Badimon L, Badimon JJ, Chesebro JH. 1990.
Atherosclerotic plaque rupture and thrombosis. Evolving concepts. Circulation
82:II4759
Fernandez-Ortiz A, Badimon JJ, Falk E,
Fuster V, Meyer B, et al. 1994. Characterization of the relative thrombogenicity of atherosclerotic plaque components:
implications for consequences of plaque
rupture. J. Am. Coll. Cardiol. 23:1562
69
Moreno PR, Falk E, Palacios IF, Newell
JB, Fuster V, Fallon JT. 1994. Macrophage
infiltration in acute coronary syndromes.
Implications for plaque rupture. Circulation 90:77578
Shah PK, Falk E, Badimon JJ, FernandezOrtiz A, Mailhac A, et al. 1995. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps
of atherosclerotic plaques. Potential role
of matrix-degrading metalloproteinases
and implications for plaque rupture. Circulation 92:156569
Henney AM, Wakeley PR, Davies MJ,
Foster K, Hembry R, et al. 1991. Localization of stromelysin gene expression
in atherosclerotic plaques by in situ hybridization. Proc. Natl. Acad. Sci. USA 88:
815458
Richardson PD, Davies MJ, Born GV.
1989. Influence of plaque configuration
and stress distribution on fissuring of
coronary atherosclerotic plaques. Lancet
2:94144
Davies MJ. 1995. Acute coronary
thrombosisthe role of plaque disruption and its initiation and prevention. Eur.
Heart J. 16(Suppl. L):37
Fukumoto Y, Libby P, Rabkin E, Hill
CC, Enomoto M, et al. 2001. Statins alter smooth muscle cell accumulation and
collagen content in established atheroma
of watanabe heritable hyperlipidemic rabbits. Circulation 103:99399
7 Jan 2005
13:56
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109. Koh KK. 2000. Effects of statins on vascular wall: vasomotor function, inflammation, and plaque stability. Cardiovasc.
Res. 47:64857
110. Bourcier T, Libby P. 2000. HMG CoA
reductase inhibitors reduce plasminogen
activator inhibitor-1 expression by human
vascular smooth muscle and endothelial
cells. Arterioscler. Thromb. Vasc. Biol. 20:
55662
111. Crisby M, Nordin-Fredriksson G, Shah
PK, Yano J, Zhu J, Nilsson J. 2001.
Pravastatin treatment increases collagen
content and decreases lipid content, inflammation, metalloproteinases, and cell
death in human carotid plaques: implications for plaque stabilization. Circulation
103:92633
112. Schwartz GG, Olsson AG, Ezekowitz
MD, Ganz P, Oliver MF, et al. 2001. Effects of atorvastatin on early recurrent
ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. JAMA 285:1711
18
113. Cannon CP, Braunwald E, McCabe CH,
Rader DJ, Rouleau JL, et al. 2004. Intensive versus moderate lipid lowering with
statins after acute coronary syndromes.
New Engl. J. Med. 350:1495504
114. Ross R. 1993. The pathogenesis of
atherosclerosis: a perspective for the
1990s. Nature 362:8019
115. Ross R. 1999. Atherosclerosis is an inflammatory disease. Am. Heart J. 138:
S41920
116. Niwa S, Totsuka T, Hayashi S. 1996. Inhibitory effect of fluvastatin, an HMGCoA reductase inhibitor, on the expression of adhesion molecules on human
monocyte cell line. Int. J. Immunopharmacol. 18:66975
117. Lefer AM, Campbell B, Shin YK, Scalia
R, Hayward R, Lefer DJ. 1999. Simvastatin preserves the ischemic-reperfused
myocardium in normocholesterolemic rat
hearts. Circulation 100:17884
118. Scalia R, Gooszen ME, Jones SP,
119.
120.
121.
122.
123.
124.
125.
126.
127.
115
Hoffmeyer M, Rimmer DM 3rd, et al.

2001. Simvastatin exerts both antiinflammatory and cardioprotective effects
in apolipoprotein E-deficient mice. Circulation 103:2598603
Stalker TJ, Lefer AM, Scalia R. 2001. A
new HMG-CoA reductase inhibitor, rosuvastatin, exerts anti-inflammatory effects on the microvascular endothelium:
the role of mevalonic acid. Br. J. Pharmacol. 133:40612
Kwak B, Mulhaupt F, Myit S, Mach F.
2000. Statins as a newly recognized type
of immunomodulator. Nat. Med. 6:1399
402
Mulhaupt F, Matter CM, Kwak BR, Pelli
G, Veillard NR, et al. 2003. Statins (HMGCoA reductase inhibitors) reduce CD40
expression in human vascular cells. Cardiovasc. Res. 59:75566
Ridker PM, Rifai N, Clearfield M, Downs
JR, Weis SE, et al. 2001. Measurement
of C-reactive protein for the targeting of
statin therapy in the primary prevention of
acute coronary events. New Engl. J. Med.
344:195965
Baumann H, Gauldie J. 1994. The acute
phase response. Immunol. Today 15:74
80
Ridker PM, Cushman M, Stampfer MJ,
Tracy RP, Hennekens CH. 1997. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men.
Ridker PM, Hennekens CH, Buring JE,
Rifai N. 2000. C-reactive protein and
other markers of inflammation in the
prediction of cardiovascular disease in
women. New Engl. J. Med. 342:836
43
Liuzzo G, Biasucci LM, Gallimore JR,
Grillo RL, Rebuzzi AG, et al. 1994. The
prognostic value of C-reactive protein and
serum amyloid a protein in severe unstable angina. New Engl. J. Med. 331:417
24
Bhakdi S, Torzewski M, Klouche M,
Hemmes M. 1999. Complement and
7 Jan 2005
13:56
116

128.
129.
130.
131.
132.
133.
134.
135.
136.
AR
LIAO
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAUFS
atherogenesis: binding of CRP to degraded, nonoxidized LDL enhances complement activation. Arterioscler. Thromb.
Vasc. Biol. 19:234854
Zhang YX, Cliff WJ, Schoefl GI, Higgins
G. 1999. Coronary C-reactive protein distribution: its relation to development of
atherosclerosis. Atherosclerosis 145:375
79
Torzewski J, Bowyer DE, Waltenberger
J, Fitzsimmons C. 1997. Processes in
atherogenesis: complement activation.
Atherosclerosis 132:13138
Danenberg HD, Szalai AJ, Swaminathan
RV, Peng L, Chen Z, et al. 2003. Increased
thrombosis after arterial injury in human
C-reactive protein-transgenic mice. Circulation 108:51215
Musial J, Undas A, Gajewski P, Jankowski
M, Sydor W, Szczeklik A. 2001. Antiinflammatory effects of simvastatin in
subjects with hypercholesterolemia. Int. J.
Cardiol. 77:24753
Ridker PM, Rifai N, Lowenthal SP. 2001.
Rapid reduction in C-reactive protein with
cerivastatin among 785 patients with primary hypercholesterolemia. Circulation
103:119193
Ridker PM, Rifai N, Pfeffer MA, Sacks
F, Braunwald E. 1999. Long-term effects
of pravastatin on plasma concentration of
C-reactive protein. The Cholesterol and
Recurrent Events (CARE) Investigators.
Ridker PM, Rifai N, Pfeffer MA, Sacks
FM, Moye LA, et al. 1998. Inflammation, pravastatin, and the risk of
coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent
Events (CARE) Investigators. Circulation
98:83944
Albert MA, Staggers J, Chew P, Ridker
PM. 2001. The pravastatin inflammation
CRP evaluation (PRINCE): rationale and
design. Am. Heart J. 141:89398
Wang SM, Tsai YJ, Jiang MJ, Tseng YZ.
1997. Studies on the function of rho A pro-
137.
138.
139.
140.
141.
142.
143.
144.
145.
tein in cardiac myofibrillogenesis. J. Cell

Biochem. 66:4353
Thorburn J, Xu S, Thorburn A. 1997.
MAP kinase- and Rho-dependent signals
interact to regulate gene expression but
not actin morphology in cardiac muscle
cells. EMBO J. 16:1888900
Aikawa R, Nawano M, Gu Y, Katagiri
H, Asano T, et al. 2000. Insulin prevents
cardiomyocytes from oxidative stressinduced apoptosis through activation of
PI3 kinase/Akt. Circulation 102:2873
79
Bendall JK, Cave AC, Heymes C, Gall
N, Shah AM. 2002. Pivotal role of a
gp91(phox)-containing NADPH oxidase
in angiotensin II-induced cardiac hypertrophy in mice. Circulation 105:293
96
MacCarthy PA, Grieve DJ, Li JM, Dunster
C, Kelly FJ, Shah AM. 2001. Impaired
endothelial regulation of ventricular relaxation in cardiac hypertrophy: role of
reactive oxygen species and NADPH oxidase. Circulation 104:296774
Li JM, Gall NP, Grieve DJ, Chen M, Shah
AM. 2002. Activation of NADPH oxidase
during progression of cardiac hypertrophy
to failure. Hypertension 40:47784
Aikawa R, Komuro I, Yamazaki T, Zou Y,
Kudoh S, et al. 1999. Rho family small
G proteins play critical roles in mechanical stress- induced hypertrophic responses
in cardiac myocytes. Circ. Res. 84:458
66
Nakagami H, Takemoto M, Liao JK. 2003.
NADPH oxidase-derived superoxide anion mediates angiotensin II-induced cardiac hypertrophy. J. Mol. Cell Cardiol.
35:85159
Xiao L, Pimentel DR, Wang J, Singh K,
Colucci WS, Sawyer DB. 2002. Role of
reactive oxygen species and NAD(P)H
oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am. J.
Physiol. Cell Physiol. 282:C92634
Takemoto M, Node K, Nakagami H, Liao
Y, Grimm M, et al. 2001. Statins as
7 Jan 2005
13:56
AR
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE

146.
147.
148.
149.
150.
151.
152.
153.
antioxidant therapy for preventing cardiac myocyte hypertrophy. J. Clin. Invest.

108:142937
Lee TM, Chou TF, Tsai CH. 2002. Association of pravastatin and left ventricular mass in hypercholesterolemic patients:
role of 8-iso-prostaglandin f2alpha formation. J. Cardiovasc. Pharmacol. 40:868
74
Maack C, Kartes T, Kilter H, Schafers HJ,
Nickenig G, et al. 2003. Oxygen free radical release in human failing myocardium
is associated with increased activity of
rac1-GTPase and represents a target for
statin treatment. Circulation 108:1567
74
Kjekshus J, Pedersen TR, Olsson AG,
Faergeman O, Pyorala K. 1997. The effects of simvastatin on the incidence of
heart failure in patients with coronary
heart disease. J. Card. Fail. 3:24954
Drexler H. 1998. Endothelium as a therapeutic target in heart failure. Circulation
98:265255
Bauersachs J, Galuppo P, Fraccarollo D,
Christ M, Ertl G. 2001. Improvement of
left ventricular remodeling and function
by hydroxymethylglutaryl coenzyme A
reductase inhibition with cerivastatin in
rats with heart failure after myocardial infarction. Circulation 104:98285
Dechend R, Fiebeler A, Park JK, Muller
DN, Theuer J, et al. 2001. Amelioration of
angiotensin II-induced cardiac injury by a
3-hydroxy-3-methylglutaryl coenzyme A
reductase inhibitor. Circulation 104:576
81
Laufs U, Kilter H, Konkol C, Wassmann
S, Bohm M, Nickenig G. 2002. Impact of
HMG CoA reductase inhibition on small
GTPases in the heart. Cardiovasc. Res.
53:91120
Node K, Fujita M, Kitakaze M, Hori M,
Liao JK. 2003. Short-term statin therapy improves cardiac function and symptoms in patients with idiopathic dilated
cardiomyopathy. Circulation 108:839
43
117
154. Laufs U, Wassmann S, Schackmann S,

Heeschen C, Bohm M, Nickenig G. 2004.
Beneficial effects of statins in patients
with non-ischemic heart failure. Z. Kardiol. 93:1038
155. Crouse JR 3rd, Byington RP, Furberg CD.
1998. HMG-CoA reductase inhibitor therapy and stroke risk reduction: an analysis of clinical trials data. Atherosclerosis
138:1124
156. Collins R, Armitage J, Parish S, Sleight
P, Peto R. 2004. Effects of cholesterollowering with simvastatin on stroke and
other major vascular events in 20536 people with cerebrovascular disease or other
high-risk conditions. Lancet 363:75767
157. Dalkara T, Yoshida T, Irikura K,
Moskowitz MA. 1994. Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 33:144752
158. Huang PL, Huang Z, Mashimo H, Bloch
KD, Moskowitz MA, et al. 1995. Hypertension in mice lacking the gene for
endothelial nitric oxide synthase. Nature
377:23942
159. Huang Z, Huang PL, Ma J, Meng W, Ayata C, et al. 1996. Enlarged infarcts in endothelial nitric oxide synthase knockout
mice are attenuated by nitro-L-arginine.
J. Cereb. Blood Flow Metab. 16:98187
160. Endres M, Laufs U, Huang Z, Nakamura
T, Huang P, et al. 1998. Stroke protection
by 3-hydroxy-3-methylglutaryl (HMG)CoA reductase inhibitors mediated by
endothelial nitric oxide synthase. Proc.
161. Scalia R, Lefer DJ, Lefter AM. 1999. Simvastatin inhibits leukocyte-endothelium
interaction in vivo under normocholesterolemic conditions: essential role of
endothelial nitric oxide synthase. Circulation 100(Suppl.):I409
162. Lefer AM, Scalia R, Lefer DJ. 2001. Vascular effects of HMG CoA-reductase inhibitors (statins) unrelated to cholesterol
lowering: new concepts for cardiovascular disease. Cardiovasc. Res. 49:28187
163. Vaughan CJ. 2003. Prevention of stroke
7 Jan 2005
13:56
118
164.

165.
166.
167.
168.
169.
170.
171.
172.
AR
LIAO
AR232-PA45-04.tex
AR232-PA45-04.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAUFS
and dementia with statins: effects beyond

lipid lowering. Am. J. Cardiol. 91:23B29
Corder EH, Saunders AM, Strittmatter
WJ, Schmechel DE, Gaskell PC, et al.
1993. Gene dose of apolipoprotein E type
4 allele and the risk of Alzheimers disease
in late onset families. Science 261:92123
Bales KR, Verina T, Dodel RC, Du Y, Altstiel L, et al. 1997. Lack of apolipoprotein E dramatically reduces amyloid betapeptide deposition. Nat. Genet. 17:263
64
Hofman A, Ott A, Breteler MM, Bots ML,
Slooter AJ, et al. 1997. Atherosclerosis,
apolipoprotein E, and prevalence of dementia and Alzheimers disease in the
Rotterdam Study. Lancet 349:15154
Jarvik GP, Wijsman EM, Kukull WA,
Schellenberg GD, Yu C, Larson EB. 1995.
Interactions of apolipoprotein E genotype, total cholesterol level, age, and sex
in prediction of Alzheimers disease: a
case-control study. Neurology 45:1092
96
Kirsch C, Eckert GP, Koudinov AR,
Muller WE. 2003. Brain cholesterol,
statins and Alzheimers disease. Pharmacopsychiatry 36(Suppl. 2):S11319
Fassbender K, Simons M, Bergmann C,
Stroick M, Lutjohann D, et al. 2001.
Simvastatin strongly reduces levels of
Alzheimers disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and
in vivo. Proc. Natl. Acad. Sci. USA
98:585661
Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G. 2000. Decreased
prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl
coenzyme A reductase inhibitors. Arch.
Neurol. 57:143943
Jick H, Zornberg GL, Jick SS, Seshadri S,
Drachman DA. 2000. Statins and the risk
of dementia. Lancet 356:162731
Shepherd J, Blauw GJ, Murphy MB,
Bollen EL, Buckley BM, et al. 2002.
Pravastatin in elderly individuals at
173.
174.
175.
176.
177.
178.
179.
180.
risk of vascular disease (PROSPER):

a randomised controlled trial. Lancet
360:162330
Youssef S, Stuve O, Patarroyo JC, Ruiz
PJ, Radosevich JL, et al. 2002. The HMGCoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in
central nervous system autoimmune disease. Nature 420:7884
Aktas O, Waiczies S, Smorodchenko A,
Dorr J, Seeger B, et al. 2003. Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1
cells through atorvastatin. J. Exp. Med.
197:72533
Yang CC, Jick SS, Jick H. 2003. Lipidlowering drugs and the risk of depression
and suicidal behavior. Arch. Intern. Med.
163:192632
Young-Xu Y, Chan KA, Liao JK, Ravid
S, Blatt CM. 2003. Long-term statin use
and psychological well-being. J. Am. Coll.
Cardiol. 42:69097
1984. The Lipid Research Clinics Coronary Primary Prevention Trial results. II.
The relationship of reduction in incidence
of coronary heart disease to cholesterol
lowering. JAMA 251:36574
Buchwald H, Varco RL, Matts JP, Long
JM, Fitch LL, et al. 1990. Effect of partial
ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report
of the Program on the Surgical Control
of the Hyperlipidemias (POSCH). New
Engl. J. Med. 323:94655
Pekkanen J, Linn S, Heiss G, Suchindran CM, Leon A, et al. 1990. Ten-year
mortality from cardiovascular disease in
relation to cholesterol level among men
with and without preexisting cardiovascular disease. New Engl. J. Med. 322:1700
7
1998. Influence of pravastatin and plasma
lipids on clinical events in the West
of Scotland Coronary Prevention Study
(WOSCOPS). Circulation 97:144045

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Figure 1 Structural basis of HMG-CoA reductase inhibition by statins. The active

forms of statins resemble the cholesterol precursor, HMG-CoA (right panels). All
statins share the HMG-like moiety and competitively inhibit the reductase by the
similar mechanism but have distinct pharmacologic and pharmacodynamic properties related to their chemical structures (left panels, ball and stick graphs: black, carbon; red, oxygen; light blue, hydrogen; dark blue, nitrogen).
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Copyright
First published online as a Review in Advance on September 7, 2004
FAT CELLS: Afferent and Efferent Messages

Define New Approaches to Treat Obesity

Max Lafontan
Obesity Research Unit, French Institute of Health and Medical Research
(Inserm-UPS-Unit 586), Universite Paul Sabatier, Institut Louis Bugnard,
Hopital Rangueil, TSA50032, 31059 Toulouse cedex 9, France;
email: max.lafontan@toulouse.inserm.fr
Key Words lipolysis, catecholamines, leptin, adiponectin, adipocyte

Abstract For a long time neural and endocrine messages were studied for their
impact on adipocyte metabolism and control of storage/release of fatty acids. In fact,
bidirectional communication exists between adipocytes and other tissues. Several
molecules secreted from adipocytes are involved in fat cell signaling to other tissues.
Adipocyte products could initiate antagonistic effects on target tissues. Fat cells produce
peptides that can elicit insulin resistance, such as tumor necrosis factor- and resistin,
as well as hormones that can improve insulin resistance, such as leptin and adiponectin.
Secretion of complement proteins, proinflammatory cytokines, procoagulant, and acute
phase reactant proteins have also been observed in adipocytes. There is much to learn
about how these signals function. It is unlikely that all the adipocytes endocrine and
paracrine signals have been identified. Putative pharmacological strategies aiming at
modulation of afferent and efferent fat cell messages are reviewed and discussed.
INTRODUCTION
Obesity can be viewed as an energy storage disorder where weight gain results
from an energy imbalance (i.e., energy input exceeding output), with most of the
excess calories stored as triglyceride in adipose tissue (AT). A strong correlation
exists between the prevalence of obesity and the prevalence of type 2 diabetes.
Excessive AT accumulation is a key pathological contributor to the metabolic
syndrome characterized by insulin resistance and dyslipidemia that leads to type
2 diabetes and an increased risk for cardiovascular diseases (1). What causes this
association between obesity, insulin resistance, and the development of type 2
diabetes? Although skeletal muscle, liver, and pancreas dysfunctions have been
implicated as the major sites for development of insulin resistance, a number of
recent results focus attention on AT as being a primary site (2, 3).
ATs represent complex organs, and a preliminary definition is useful to delineate
the topic covered in the present review. The major distinctive characteristics that
distinguish white AT (WAT) and brown AT (BAT) are detailed in a number of
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reviews (46). BAT is specialized in adaptative thermogenesis; its thermogenic

capacity is related to an original mitochondrial function related to the expression
of the uncoupling protein-1 (UCP-1) (7).
Our knowledge about the biology of the adipocyte, the physiology of AT, and
its involvement in obesity-related diseases has undergone major expansion during
the past decade. The striking point is that the adipocyte has gained the status of
an endocrine cell with the ability to regulate production of numerous secreted
products of various natures.
In this review, attention is focused on afferent messages converging on the white
fat cell and contributing to its functional control. Efferent messages originating in
the fat cell and directed toward other organs and cells also are considered. Putative
pharmacological strategies aiming at modulation of afferent and efferent fat cell
messages are reviewed and discussed.
GENERAL CONSIDERATIONS ABOUT THE FUNCTIONAL

ROLES OF ADIPOCYTES
Adipocytes allow surplus fuel to be stored as triacylglycerol (TAG) during caloric
abundance for retrieval during periods of food shortage and calorie debt (e.g., fasting, starvation, long-term exercise). Nonesterified fatty acids (NEFAs) appearing
as a result of lipolysis of TAG stores are released into the circulation and mainly
oxidized in skeletal muscle to provide energy. This fat-storing capacity remains
an important function of the fat cells, one that suffers some striking alterations
in physically inactive and overfed persons. In normal conditions, the adipocyte
is able to fine-tune a number of nervous and hormonal signals to precisely adapt
the balance between the pathways of synthesis (TAG synthesis) and catabolism
(lipolysis) of TAG to physiological needs. Through its TAG-storing capacity, involving a balanced lipogenic/lipolytic drive, the adipocyte could limit an abnormal
increase in plasma NEFAs. NEFAs are widely viewed as an important etiologic
factor in the initiation of insulin resistance and metabolic syndrome in the obese.
They are elevated in obesity and represent a risk factor for the development of type
2 diabetes (8).
The major physiological importance of the fat-storage capacity of the adipocytes
is evident when considering some situations when fat mass is reduced or lacking
(lipoatrophy) (9). In lipoatrophic mice, a lack of fat is associated with insulin resistance, hyperglycemia, and liver steatosis (10). Adipocytes exert, through their
fat-storing capabilities, a protective action against the occurrence of lipotoxic damage to lean tissues, which is referred to as lipotoxicity (lipid-induced dysfunction)
or as lipoapoptosis (lipid-induced programmed cell death) (11).
One of the major features in adipocyte biology is the discovery of its complex secretory activities. A number of peptide hormones and proinflammatory
cytokines, termed adipokines, secreted by the adipocyte exert endocrine effects
(1215). These adipokines allow the adipocyte to initiate potent feedback actions
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AFFERENT AND EFFERENT MESSAGES IN FAT CELLS
121
in the regulation of appetite, food intake, glucose disposal, and energy expenditure.
They are able to protect against the establishment of insulin resistance by actions on
liver, skeletal muscle, and pancreatic function. They also contribute to the prevention or worsening of atherogenic processes. In addition, some of the factors secreted
by the adipocyte exert local autocrine and paracrine actions mainly affecting AT
remodeling, adipogenesis, and angiogenesis and are not found in the circulation.
The topography of fat distribution plays an important role in the appearance
of health risks. Abdominal visceral fat extent is an important link between the
many facets of the metabolic syndrome: glucose intolerance, hyperinsulinemia,
hypertriglyceridemia, and other features such as hypertension and altered HDL
and VLDL levels (1618). Although it is quite well accepted that upper body
obesity, with increased visceral fat, should be considered as a factor that initiates
or exacerbates an individuals susceptibility to the components of the metabolic
syndrome, the causality is poorly understood. What causes this consistent association between obesity and the development of type 2 diabetes? What are the factors
interfering with the adipocytes from various depots that could explain the appearance of metabolic disorders? What is the contribution of adipocyte secretions to
the generation of diseases related to the development of obesity?
AFFERENT MESSAGES CONTROLING WHITE

FAT CELL FUNCTION
Physiological Features of White Adipose Tissue Innervation
The fat cell is under multiple influences, including that of the autonomous sympathetic nervous system [sympathetic (SNS) and parasympathetic (PSNS)], local
blood flow variations and various hormones, and factors delivered from the plasma
or produced locally by the various cell types existing in the fat pad (19). WAT is
innervated by the nerve endings of the autonomic nervous system. Nerve terminals run along blood vessels and a limited number of adipocytes seem to be in
direct contact with nerve varicosities. The direct links existing between SNS and
WAT deposits have been revealed by experimental interventions. Surgical sympathectomy reduces lipolysis in the denervated WAT depot, whereas electrical
stimulation of SNS nerve endings stimulates lipolysis in animals (20) and also in
humans (21). PSNS innervation has been shown in WAT in rats (22). PSNS stimulation increases insulin sensitivity in peritoneal fat. The physiological relevance
of these observations has been recently discussed (20, 23).
Adrenergic Regulation of Lipolysis and

Human Fat Cell Function
Following SNS stimulation, norepinephrine and neuropeptide Y (NPY) are released from sympathetic nerve terminals, whereas adrenal medulla cells secrete
epinephrine. The major elements involved in the regulation of the lipolytic
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pathways are depicted in Figure 1. Rodents possess abundant 3-adrenergic receptors ( 3-ARs) in the white fat cells, whereas in human fat cells, the role of the
3-AR remains quite puzzling and controversial (24). In human fat cells, both 1and 2-ARs initiate the activation of the lipolytic cascade by stimulation of cyclic
AMP (cAMP) production, activation of cAMP-dependent protein kinase A (PKA)
leading to phosphorylation of perilipin and hormone-sensitive lipase (HSL), and
promotion of lipolysis in vitro. The originality of the human fat cell is related to the
presence of abundant 2-adrenergic receptors ( 2-ARs): their stimulation inhibits
cAMP production and lipolysis (24, 25).
Differences exist in the adrenergic regulation of lipolysis in AT from different
sites in normal-weight subjects and in obese subjects (18, 26, 27). The lipolytic
response of isolated fat cells to catecholamines is weaker in the subcutaneous
gluteal/femoral and abdominal AT than in visceral AT. Lipolytic defects are explainable by the reduced expression or function of HSL and/or of proteins that interact with HSL [e.g., adipocyte lipid binding protein (ALBP)] or the lipid droplet,
such as perilipin. Alterations of the signaling pathways, such as reduced 12-AR
responsiveness or increased 2-AR responsiveness (and a possible association or
combination of defects), are also important. These site-related differences are more
noticeable in women than in men (24, 2729). An enhanced 2-AR responsiveness
associated with a concomitant decrease in -AR responsiveness explains the lower
lipolytic effect of catecholamines in gluteal/femoral fat cells of normal and obese
women and abdominal fat cells of obese men. Reduced lipid mobilization occurs
during exercise in subcutaneous AT of obese subjects (30). Functional changes in
12/ 2-AR balance appear with the extent of the fat mass and are related to fat cell
hypertrophy. Hypertrophic subcutaneous (abdominal, femoral) fat cells are known
to be the least responsive to the lipolytic action of catecholamines; they exhibit
the highest amount of 2-ARs and the lowest amount of 12-ARs. Increased expression of the 2-AR (and the concomitant decrease of -responsiveness) with
fat cell hypertrophy could be a physiological adaptation leading to a reduction of
the lipolytic responsiveness of the hypertrophied adipocytes of some fat deposits.
The mechanisms leading to the opposite regulation of the expression of 12- and
2-ARs as cells become hypertrophied are unknown.
Whatever the mechanism controlling AR expression, limitation of basal and
SNS-dependent lipolysis avoids excessive NEFA release from some fat deposits.
A recent study aimed at the direct assessment of fasting AT metabolism using
arterio-venous differences in defined depots has shown that the buttock is metabolically silent in terms of fatty acid release compared with the abdomen (31). The
buffering action of NEFAs by AT is an important phenomenon (32). When the
NEFA buffering system is inadequate, other tissues are exposed to elevated NEFA
concentrations. A role for the 2-AR gene in determining the propensity to store
fat in the abdominal area, independent of total body fatness, has recently been
reported (33).
Profound unresponsiveness of the subcutaneous AT to neurally stimulated lipolysis has been described in obese subjects (34). Reduced 2-adrenergic lipolytic
responsiveness has been reported in fat cells from obese subjects or subjects with
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123
a reduced isoproterenol sensitivity (35). In addition, an increased antilipolytic responsiveness linked to 2-AR stimulation has also been found in subcutaneous
adipocytes of obese individuals of both sexes. The lipolytic defects revealed in
fat cells have been confirmed during in vivo studies (36, 37). Using in situ microdialysis, a specific impairment in the capacity of 2-AR agonists to promote
lipolysis has been reported in the subcutaneous abdominal AT of obese adolescent girls (38). Moreover, when selective 1- and 2-AR-agonists are administered
intravenously, the increase in lipolysis and thermogenesis promoted by selective
2-adrenergic stimulation (salbutamol) was reduced in obese subjects. Conversely,
1-AR-mediated (dobutamine) metabolic processes (i.e., lipolysis, thermogenesis, and lipid oxidation) were similar in obese and lean men. In conclusion, 2adrenergic-mediated increases in thermogenesis and lipid oxidation are impaired
in the obese (39).
The putative role of 2- and 3-AR polymorphisms in the etiology of lipolysis disturbances and obesity has recently been reviewed (35, 40). To summarize, polymorphisms in the coding and noncoding sequences in the human 2-AR
gene could be of major importance for obesity, energy expenditure, and 2-ARdependent lipolytic function. Full -adrenergic activation of the human fat cell
usually requires synergistic activation of 1- and 2-ARs. A 2-adrenergic defect
could be sufficient to alter normal -adrenergic responsiveness. In addition, in human fat cells, any reduction of 2-AR-mediated lipolytic response will disturb the
normal functional balance existing between 2- and -AR-mediated effects and
amplify the reduction of the lipolytic responsiveness initiated by the physiological
amines in stressful situations. All the discussions related to the adrenergic regulation of lipolysis must be expanded in terms of regulation to all cAMP-related
events existing in fat cells.
Insulin Signaling in the Adipocyte: Heterogeneity of Responses

Insulin plays a major role in the control of AT development and function. Conditional ablation of the insulin receptor in adipocytes (FIRKO mice) has shown
how insulin affects the development of the common metabolic alterations arising from obesity (41). Insulin not only regulates lipogenesis but also the rate of
lipolysis and NEFA efflux. Insulin controls glucose uptake and causes fatty acid
transport protein translocation and enhanced fatty acid uptake in adipocytes (42).
The cascade of signaling events initiated by insulin binding to its receptor are
conserved across tissues and species (43). In fat cells, the Ser/Thr protein kinase
B (PKB/Akt) mediates the metabolic effects of insulin (44). Insulin inhibits basal
and catecholamine-stimulated lipolysis through phosphorylation (via PKB/Aktdependent action) and activation of type 3-B phosphodiesterase (PDE-3B), leading
to decreased cAMP levels that prevent HSL activation. Insulin-induced antilipolysis and activation of NEFA reesterification are blunted in omental compared with
subcutaneous fat cells. Various functional differences have been identified at the insulin receptor level and the postreceptor level of the insulin-signaling cascade (45).
Other partners such as PDE-3B, which is responsible for the antilipolytic action,
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and protein-tyrosine phosphatases, which are involved in the dephosphorylation

of the insulin receptor, could also play a role. Endogenous PTPase1B (PTP1B) is
increased in omental AT and may contribute to the relatively high insulin resistance of this fat depot (46). The role and regulation of this enzyme merit deeper
investigations in human fat cells. Clinical studies have confirmed the regional heterogeneity of insulin-regulated NEFA release in vivo. Visceral AT is more resistant
to insulins antilipolytic effects than are leg and nonsplanchnic body fat. Nevertheless, visceral fat may be a marker for, but not the source of, excess postprandial
NEFAs in obesity because the increased postprandial NEFA release observed in
upper body obese women and type 2 diabetics originates from the nonsplanchnic
upper body fat, not visceral fat (47).
Other Afferent Signals and Lipolytic Pathways

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) stimulate lipolysis as much as a nonselective -AR agonist
in isolated human fat cells (48). High levels of ANP receptors (NPR-A and NPRC subtypes) are found in human adipocytes. Natriuretic peptides operate via a
cyclic GMP (cGMP)-dependent pathway that does not involve PDE-3B inhibition or cAMP production. ANP stimulation of human fat cells activates a cGMPdependent protein kinase (cGK-I type), which phosphorylates perilipin and HSL,
thus explaining the lipolytic action (49). Intravenous administration of h-ANP in
humans promotes a striking increment in plasma levels of NEFAs and glycerol
(50). ANP is a relevant physiological activator of fat mobilization that contributes
significantly to exercise-induced lipid mobilization (51).
ATRIAL NATRIURETIC PEPTIDES
Although growth hormone (GH) treatments in adults reduce

abdominal obesity and affect insulin sensitivity, the physiological contribution of
GH to the control of human AT lipid mobilization has remained elusive. GH stimulates lipolysis in human adipocytes; the effect is delayed (23 h) when compared
with that of catecholamines. Contribution of cAMP-/PKA-dependent pathways
as those used by catecholamines is suspected. GH-dependent modification of the
relationships between adenylyl cyclase and Gi 2 protein removes inhibition of
cAMP production and consequently increases lipolysis (52). GH administration
promotes a significant increase in NEFA after 23 h, reflecting stimulation of
lipolysis and ketogenesis (53). Small physiological GH pulses increase interstitial
glycerol concentrations in both femoral and abdominal AT (54). Normal nocturnal
rise in plasma GH concentrations also leads to site-specific regulation of lipolysis in AT (55). A small synthetic peptide sequence of human GH (AOD-9041)
has been shown to increase human and rodent fat cell lipolysis in vitro and lipid
mobilization in rodents (56).
GROWTH HORMONE
In humans, the lipolytic peptides

(adrenocorticotropic hormone, -melanocyte-stimulating hormone, lipotropin)
OTHER LIPOLYTIC PEPTIDES AND CORTISOL
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commonly acting in rodent fat cells have no effect on human fat cells. Glucagon
and glucagon-like peptide-1 (GLP-1) do not stimulate in vitro lipolysis. Moreover, no significant effect of either GLP-1 or glucagon on either lipolysis rate or
blood flow was detected in muscle or AT during local or experimental i.v. hyperglucagonemia (57, 58). Parathyroid hormone (59) stimulates lipolysis in human fat
cells at rather high extraphysiological concentrations. Human adipocytes express
both IL-6 and its receptor system consisting of the IL-6 receptor and the signal
transducing protein gp130. IL-6 administered in the normal physiological concentration range elicits lipolytic effects in human subcutaneous AT in vivo (60, 61).
IL-6 stimulates lipolysis in human adipocytes and exerts anti-insulin actions (62).
IL-6 also induces the expression of SOCS-3, a potential inhibitor of insulin signaling (63, 64). Whatever the results, it could be premature to include IL-6 inside
a physiological loop of lipid mobilization regulation. Leptin induces a novel form
of lipolysis in which glycerol is released without proportional release of NEFA
and increase in peroxisome proliferator-activated receptor- (PPAR-) and NEFA
oxidation in rat fat cells (65). It is unknown if the same leptin-dependent action
operates in human fat cells.
Cortisol is less potent than catecholamines in the stimulation of lipolysis; the
lipolytic effect is delayed and in vivo action is counteracted by corticoid-promoted
insulin release (66). Cortisol-induced lipid mobilization is observed when cortisolinduced insulin increment is prevented (67). Short-term treatment with a standard
dose of corticosteroids (i.e., prednisolone) induces increased abdominal adipose
tissue lipolysis, hyperglucagonemia and insulin resistance, whereas GH levels are
unaffected (68).
Stimulation of lipolysis by tumor necrosis factor-
(TNF-) is not direct because it becomes apparent only after long-lasting exposure
of human and rodent adipocytes to the cytokine (69). TNF--induced lipolysis,
as well as inhibition of insulin-stimulated glucose transport, is predominantly
mediated by the receptor TNFR1 (70, 71). TNF- could regulate lipolysis, in part,
by decreasing perilipin protein levels at the lipid droplet surface and activating the
extracellular signal-related kinase (ERK) pathway (72). Blunting the endogenous
inhibition of lipolysis through Gi protein downregulation is also another possible
mechanism (73). In human fat cells, TNF- activates the three mammalian mitogen
activated protein kinases (MAPK) in a distinct time- and concentration-dependent
manner. TNF--induced lipolysis is mediated by only p44/42 and Jun kinase but
not by p38 kinase (74).
TUMOR NECROSIS FACTOR-
Nitric oxide (NO) or related redox species such as

NO+/NO have been proposed as potential regulators of lipolysis in rodent and
human fat cells (75, 76). Cachexia-inducing tumors produce a lipid-mobilizing factor (LMF) that causes immediate release of glycerol when incubated with murine
adipocytes. Induction of lipolysis by LMF was associated with an increase in intracellular cAMP levels (77). Zinc- 2-glycoprotein (ZAG), a protein of 43-kDa and a
MISCELLANEOUS AGENTS
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tumor-related LMF, was detected in the major fat deposits of mice and in 3T3-L1
cells. ZAG expression and protein was also found in human fat cells (visceral and
subcutaneous AT). ZAG is a new adipose tissue protein factor that may be involved
in the modulation of lipolysis in adipocytes (78).
Various hormones and
autacoid agents are known to negatively control adenylyl cyclase activity and
inhibit cAMP production and lipolysis in fat cells. The effects are mediated by
plasma membrane receptors, the stimulation of which inhibits adenylyl cyclase
and cAMP production (Figure 1). In addition, the stimulation of leptin secretion
was also observed with various agonists (A1-adenosine, 2-AR, and NPY-Y1 receptor agonists) (79, 80). The receptor of nicotinic acid (niacin), a well-known
lipid-lowering drug, has recently been discovered. The orphan G proteincoupled
receptor protein up-regulated in macrophages by interferon (mouse PUMAG, human HM74), which is highly expressed in adipose tissue, is a nicotinic acid
receptor that mediates the antilipolytic and lipid-lowering effect of nicotinic acid
in vivo (81). Agonists leading to activation of Gi protein-coupled receptors of the
adipocytes limit NEFA release and represent putative antihyperlipidemic drugs.
All these antilipolytic agents will also exert leptin-secreting effects. Antagonists
of such receptors, relieving inhibition of cAMP production promoted by the endogenous ligands, enhance the lipolytic activity of the fat cell. The physiological
relevance of all these in vitro investigations is yet to be established.

ADENYLYL CYCLASE INHIBITORSANTILIPOLYTIC AGENTS
ADIPOCYTE SECRETIONS AND EFFERENT

ENDOCRINE MESSAGES
In addition to the metabolic disturbances resulting from altered NEFA handling
(8, 32), AT can exert a substantial impact on systemic glucose homeostasis, insulin
resistance, and initiation of cardiovascular disorders through production and release of adipokines, the bioactive molecules originating from adipocytes. Some of
these molecules are secreted in the bloodstream in an AT deposit-related manner.
Leptin, adiponectin (also called Acrp30, AdipoQ, and apM1), interleukin-6 (IL-6),
TNF-, and resistin are candidates of great interest among the growing number of
factors found to be secreted by the adipocyte (14, 15).
Leptin
It is clear that leptin is an important part of the lipostatic system because it signals
the size of the energy reserves existing in the body and controls fuel mobilization
and utilization (82). Leptin crosses the blood-brain barrier, enters the central nervous system (CNS), and stimulates the long form of its receptor (Ob-Rb) located
in the arcuate nucleus of the hypothalamus. A complex set of leptin receptor isoforms exist (83). Results in rodents fit with the lipostatic paradigm, postulating
that the adipocyte, through leptin, is able to provide a peripheral message of fat
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mass repletion to the CNS areas involved in the regulation of energy balance to
regulate energy intake and energy expenditure and limit fat deposition (82, 84).
Leptin is a pleiotropic molecule that may regulate a number of other biological
processes, as recently reviewed (15, 85, 86). Understanding of metabolic effects
of leptin expanded after the discovery of its mechanism of action in liver and muscle. Leptin directly stimulates 5 -AMP-activated protein kinase (AMPK), which
increases ATP-producing catabolic pathways, such as beta-oxidation, glycolysis,
and mitochondrial biogenesis, and concomitantly decreases ATP-consuming anabolic pathways (87).
Under experimental adenovirus-induced hyperleptinemia in rats, the fatty acids
inside the white adipocytes appear to be oxidized (88, 89). Leptin confines storage
of excess calories to adipocytes and spares the appearance of chronic steatosis in
nonadipocyte cells. It was proposed that in humans the metabolic syndrome might
be the equivalent of the lipotoxic syndrome described in rodents (90). It will be
essential to evaluate whether these novel effects appear at leptin concentrations
that are found in a physiological context in humans. Some actions of leptin at high
concentrations could be associated to effects independent of Ob-Rb and recruit
additional transducing pathways. Cross talk of leptin pathways with other cytokinerelated pathways cannot be excluded because leptin has similarities to the family of
long-chain helical cytokines that includes IL-6, IL-11, ciliary neurotrophic factor,
and leukemia inhibitory factor.
The subcutaneous fat depot is the major source of leptin, owing to the combination of a mass effect (subcutaneous fat being the major depot in men and women)
and the higher secretion rate in the subcutaneous than in the visceral depots (91).
Adipocyte size and anatomical location appear to be the major determinants of leptin mRNA expression. In vivo, overfeeding and obesity, glucocorticoid treatments,
glucose, and insulin administration increase circulating leptin levels, whereas fasting, sustained exercise, cold exposure, and SNS activation reduce leptin levels.
In vitro, positive effectors of leptin production include glucose, insulin, glucocorticoids, TNF-, estrogens, IL-1, agents acting through Gi protein-dependent
pathways, and melanin concentrating hormone. Conversely, catecholamines and
cAMP agonists, -adrenergic agonists, androgens, polyunsaturated fatty acids,
peroxisome proliferator-activated receptor agonists, and phorbol esters negatively regulate leptin production. Insulin and glucocorticoids affect transcriptional
mechanisms that increase leptin mRNA levels but also increase the traffic out of
the adipocyte, which involves constitutive and regulated secretory pathways (14,
86, 92).
Interleukin-6
Plasma IL-6 levels are increased in obese subjects and correlate with fat mass and
body mass index (BMI). High levels of plasma IL-6 are found in type 2 diabetes
and also correlate with fasting insulin levels. The capacity of human adipocytes
to release IL-6 was observed in AT explants and freshly isolated fat cells (93) and
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human adipocytes differentiated in vitro (62). In vivo studies have shown that the
cytokine IL-6 is secreted by subcutaneous fat in humans (94). In subcutaneous
AT, IL-6 secretion increases tenfold during the postexercise rest period following
a one-hour endurance exercise, and a concomitant increment of NEFA output was
observed; this suggests a postexercise lipid mobilizing contribution of the cytokine
(95). The IL-6 receptor and the gp-130 protein of cytokine pathways are expressed
in human fat cells, suggesting that a direct paracrine action of IL-6 on the human
fat cell is possible (96). IL-6 secretion is strongly stimulated by -AR activation
and mildly suppressed by glucocorticoids. To conclude, a hormonally regulated
IL-6 secretion occurs in mature human fat cells and it is probable that a local
paracrine action of IL-6 on adipocytes exists.
Adiponectin: An Adipocyte-Derived Insulin-Sensitizing

Hormone
Adiponectin (Acrp30) is an abundant 30-kDa adipocyte-specific protein secreted
in high concentrations in the serum. Adiponectin serum concentrations are reduced in a variety of obese and insulin-resistant states. Detailed information is
provided in recent reviews (9799). Genetic data has provided arguments showing
that polymorphism within the adiponectin locus is linked to increased risk of type
2 diabetes (100). Altered multimerization of adiponectin and/or consequently impaired secretion could be among the causes of the hypoadiponectinemia described
in subjects affected by mutations of the adiponectin gene (101, 102). Moreover,
oligomerization of adiponectin is important for at least some of its biological activities. Hexameric and larger isoforms of adiponectin activate the nuclear factor-B
(NF-B) pathway, whereas trimeric or globular forms could not activate NF-B.
Changes in the relative abundance of each oligomeric isoform in plasma may
regulate adiponectin activity.
Globular adiponectin protects ob/ob mice from diabetes and apolipoprotein
E (ApoE)-deficient mice from atherosclerosis (103). Adiponectin knockout mice
exhibit severe diet-induced insulin resistance, glucose intolerance, and vascular
defects such as neointimal formation (104, 105). Conversely, adiponectin expression in transgenic mice ameliorates insulin resistance and diabetes as well
as vascular defects, even in ApoE-deficient mice that have a propensity to develop atherosclerosis. In nonalcoholic steato-hepatitis, adiponectin ameliorates
hepatomegaly, steatosis, and alanine aminotransferase abnormality in ob/ob mice
(106). Relationships between plasma adiponectin and insulin sensitivity for glucose disposal suggest that adiponectin also exerts pleiotropic insulin-sensitizing
effects in humans (107). Hypoadiponectinemia is closely linked to impaired vasoreactivity and endothelial dysfunction in man. Adiponectin may play a protective
role against atherosclerotic vascular change in humans (108110).
Adiponectin administration enhanced hepatic insulin action and reduced liver
gluconeogenesis and lipid accumulation in nonadipose tissues (97, 111). Glucose
uptake and fatty acid oxidation were increased, whereas lipid accumulation was
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decreased in skeletal muscle (112). Adiponectin effects are mediated by AMPK,

which has been reported to increase fatty acid oxidation during muscle contraction
and repress key enzymes of gluconeogenesis in hepatocytes. AMPK is known to
mediate the insulin sensitizing action of exercise, some antidiabetic effects of metformin, and leptin action on skeletal muscle (113). Adiponectin most likely exerts
its actions on muscle fatty acid oxidation by inactivating acetyl-CoA carboxylase1 (ACC-1) via activation of AMPK and perhaps other signal transduction proteins
(114). The effects of adiponectin are mediated by two receptor isoforms (AdipoR1
and AdipoR2) that have recently been cloned (115). The nature of the transducing
elements linking receptors and AMPK activation has not been clarified. AdipoR1,
which is mainly expressed in skeletal muscle but which is also expressed in many
other tissues, has a higher affinity for the globular form of adiponectin. AdipoR2
predominates in liver and is less selective for adiponectin isoforms. The discovery of the receptors and of their distribution will help in the understanding of
the molecular mechanisms of adiponectin action on various target cells. A number of hormones (insulin, catecholamines, and glucocorticoids) and other factors
and pharmacological agents (pharmacological stimulators or inhibitors of cAMP
production, TNF-, ionomycine, and thiazolidinediones) have been shown to modulate adiponectin expression and secretion in vitro (116, 117). Nevertheless, the
mechanisms of the secretory processes in the adipocyte have not been investigated in depth. Unlike other adipokines, adiponectin is decreased in adiposity and
increases after weight reduction. The mechanisms that determine interindividual
variability of adiponectin secretion, hence affecting body fatness, remain to be
clarified.
Resistin
Resistin, for resistance to insulin, is 10-kDa adipocyte-secreted protein that possesses hormonal properties and has been claimed to represent an important link
between obesity and insulin resistance. In the original paper, resistin administration
was reported to cause glucose intolerance and insulin resistance in mice, whereas
resistin antibody administration improved glucose intolerance. Moreover, serum
levels of resistin were higher in mouse models of obesity and decreased after peroxisome proliferator-activated receptor (PPAR ) agonist (e.g., thiazolidinedione)
treatment. WAT resistin mRNA and serum protein levels dropped during fasting
and increased during refeeding (118). Other groups using different methods have
confirmed the existence of resistin (119121). Resistin has a rapid effect on hepatic, but not peripheral, insulin sensitivity (119). Mice lacking resistin (rstn/)
exhibit low blood glucose levels after fasting owing to reduced hepatic glucose
production. This is partly mediated by AMPK activation and decreased expression
of gluconeogenic enzymes in the liver (122). The original concept is still open to
discussion after major controversial results concerning resistin expression in obese
rodents and thiazolidinedione effects. The role of resistin in human insulin resistance remains quite controversial. Very low levels of resistin mRNA were found in
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human adipocytes, whereas it is expressed at higher levels in macrophages (123

125). Are there divergences in the major sites of resistin production in humans
(e.g., macrophages) and rodents (e.g., adipocytes)?

Proinflammatory, Procoagulant, and

Acute Phase Molecules
AT of the obese expresses several proinflammatory cytokines, such as TNF-, IL-1,
IL-6, inducible nitric oxide synthase (iNOS), transforming growth factor-1, and
monocyte chemotactic protein (MCP-1) (126). Biologically active procoagulant
molecules, such as plasminogen activator inhibitor-1 (PAI-1), Factor VII, and tissue
factor, are also produced in direct proportion to adiposity (127). AT also expresses
a number of acute phase reactants at high levels, including serum amyloid A3
(SAA3), 1-acid glycoprotein, and the lipocalin 24p3. SAA3 expression is highly
expressed in the diabetic state. Pro-inflammatory stimuli and high glucose can
lead to the induction of SAA3 in adipose tissue in vivo as well as in the 3T3-L1
adipocytes (128). Cytokines within adipose tissue could originate from adipocytes,
preadipocytes, and other cell types such as macrophages and endothelial cells of
the stroma-vascular fraction. Expression studies with mRNA determinations have
shown that the adipocyte is able to synthesize several interleukins (IL-6, IL-1,
and IL-8), TNF-, macrophage colony-stimulating factor, various proteins of the
complement system, and molecules of the acute phase reactants (129131). Fat
cell isolation procedures per se trigger the induction of many genes encoding
inflammatory mediators, including TNF-, IL-1, IL-6, multiple chemokines, cell
adhesion molecules, acute phase proteins, Type-I IL-1 receptors, and transcription
factors involved in the inflammatory response (132). TNF- has been considered
as a key component in the obesity-diabetes link, at least in rodents (133, 134).
TNF- secretion has been correlated with insulin resistance measured in vitro
and in vivo in humans (94, 135). However, the relation between AT TNF- and
insulin resistance remains an open question in humans. Its mechanisms of action
have been extensively analyzed in recent reviews (136, 137). TNF--deficient
obese mice have lower levels of circulating free fatty acids and are protected
from the obesity-related reduction in insulin receptor signaling in muscle and fat
tissues (138). Expression and secretion of TNF- is increased in fat cells of obese
subjects. However, there is no clear agreement as to which cytokines derived from
adipose tissue act as hormonal regulators. IL-6 is systemically released, whereas
TNF- is not (94). To sum up recent literature, TNF- is increased in adipocytes
in obesity and -adrenergic receptor stimulation is a positive regulator of TNF-
expression, whereas GH and PPAR activators (e.g., thiazolidinediones) suppress
its expression. Regulators of TNF- production in fat cells might modulate insulin
sensitivity via this cytokine.
Two recent studies have led to a major breakthrough in our understanding
of the origin and the role of TNF- and other cytokines in obesity. They have
shown that macrophages accumulate in the adipose tissue of obese mouse strains
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and in human adipose tissue. Macrophage accumulation occurs in proportion to

adipocyte size. This adipocyte sizerelated accumulation probably increases the
capacity for production of proinflammatory and acute phase molecules that contribute to obesity-related disorders. Thus the AT macrophages could be largely
responsible for the major part of adipose tissue TNF-, IL-1, IL-6, MCP-1, and
iNOS expression. Release of macrophage TNF- and IL-6 may contribute to the
local decrease in insulin sensitivity of fat cells and to all the other TNF-/IL-6related disturbances (139, 140). What could attract macrophages to the adipose
tissue, as opposed to other locations in obesity? It is proposed that an influx of
bone-marrow-derived precursors into adipose tissue occurs, followed by their subsequent differentiation into macrophages. Adipocytes secrete MCP-1, which is
considered a specific chemoattractant for monocytes and macrophages (141, 142),
and colony stimulating factor-1 (CSF-1), a regulator of macrophage differentiation and survival (130). Leptin, released by the hypertrophied adipocyte, could
also play a role in the process of chemoattraction because it has been shown to
promote MCP-1 expression and secretion by endothelial cells (143, 144). Resistin
upregulates adhesion molecules and chemokines. It also downregulates TRAF3
(tumor necrosis factor receptor-associated factor 3), an inhibitor of CD40 ligand
signaling (145). It is of interest to establish if leptin, resistin, and other cytokines
and active molecules secreted by the fat cell are capable of initiating the homing
of various bone-marrow-derived precursors/progenitors toward the microvascular
endothelial cells of adipose tissue to create permissive sites for the monocytes to
enter adipose tissue and differentiate.
Miscellaneous Adipocyte Productions

As shown in Figure 2, some adipocyte products cannot be easily classified inside
a functional box. In addition to the various factors cited previously, adipocytes secrete many other bioactive molecules, the roles of which are not fully delineated.
Acylation stimulating protein (ASP) production, which results from the conversion
of complement C3 into C3adesArg, involves three proteins of the alternate complement system synthesized and secreted by the adipocyte: C3, factor B, and adipsin
(e.g., factor D). The ASP protein increases triglyceride synthesis in fat-storing cells
(i.e., it increases glucose transport and fatty acid incorporation into TAG). ASP
also inhibits hormone-sensitive lipase-mediated lipolysis (146). PAI-1 is a fibrinolytic inhibitor whose increased plasma concentrations are thought to contribute
to the increased susceptibility to atherogenesis described in obese insulin-resistant
patients. PAI-1 has been reported to be upregulated in AT from obese mice and humans concomitantly with increased plasma PAI-1 levels (147). Metalloproteases
(148), metallothionein (149), and autotaxin, a phospholipase D secreted by the
adipocytes that controls lysophosphatidic acid production in the adipose tissue
(150, 151), have been clearly identified in fat cells. They are all secreted products.
Angiotensinogen is expressed and released by mature white fat cells from rodents
and humans and is involved in AT growth and blood pressure regulation (152).
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WAT and adipocytes contain the components of the renin-angiotensin system,

giving rise to angiotensin II (Ang. II) from angiotensinogen. Ang. IIstimulated
adipocytes release prostacyclin, which is able to favor adipocyte formation. Ang.
II could be considered as a trophic factor involved locally in AT development
(153, 154). It is likely that many other secreted proteins have yet to be identified and their hormonal or local actions delineated. Genomic approaches reveal
genes encoding newly discovered factors. For example, a factor regulated by fasting and insulin [fasting-induced adipose factor (FIAF)] (155) and activated by
PPAR agonists (PGAR for PPAR angiopoietin related) (156) has been shown
to be angiopoietin-like 4, in that it promotes angiogenesis and is produced during
ischemia (157). Proteomic approaches will certainly be expanded for identification of secreted proteins in a high throughput and automatable fashion. Several
new molecules involved in AT function have been identified using such methods
(121).
Cortisol has been proposed to be involved in the development of visceral adiposity. Enhanced reactivation of cortisone to cortisol in AT may exacerbate obesity
(158). An 11-hydroxysteroid dehydrogenase1 (11-HSD1) is synthesized in AT
(in stromal preadipocytes and adipocytes) and exerts bidirectional effects; it has
both dehydrogenase (cortisol to corticosterone) and reductase (cortisone to cortisol) potencies. The enzyme could contribute to local cortisol production in AT and
exert differentiating effects on preadipocytes. Increased adipocyte 11-HSD1 activities may be a common molecular etiology for visceral obesity and the metabolic
syndrome (159).
NEW APPROACHES TO TREAT OBESITY

The history of obesity treatment is tarnished by limited long-lasting success,
rebound recovery of weight after cessation of treatment, and some therapeutic
disasters. Cure of obesity is rare and obesity is not, as previously discussed, a
single entity. Nevertheless, palliation of obesity-related disorders remains a realistic clinical goal. Pharmacological treatment of obesity is still a matter of debate. In view of the evidence linking obesity to increased morbidity and mortality, recent recommendations suggest that safe and effective therapeutics must
be employed to treat those overweight patients exhibiting symptoms of the metabolic syndrome to reduce their risk of type 2 diabetes, hypertension, and
dyslipidemias.
Currently, orlistat is the only drug acting peripherally to limit fat absorption.
Some beneficial effects have been reported in the prevention and management
of diabetes mellitus. Discoveries in the neurosciences have been oriented toward
drugs expected to modify the monoaminergic and peptidergic control of food intake
(160). The strategies and drugs targeting fat cell responsiveness and secretion as
well as some of the agents secreted by the fat cell, which could represent future
therapeutic agents, are briefly considered.
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Drugs Affecting NEFA Handling by the Fat Cell

and Energy Expenditure
Obesity in humans is related to reduced energy expenditure and lipid mobilizing
defects in some fat deposits. Owing to the lack of brown fat (6), skeletal muscle
is the essential site of thermogenesis and NEFA utilization in humans. Stimulation of lipolysis in WAT without use of released fatty acids might be detrimental
owing to the incidence of NEFA excess (8); energy expenditure must be activated in parallel. Sympathomimetic agents, by their ability to increase lipolysis
and energy expenditure, have been considered as possible tools to treat human
obesity. A marked thermogenic response to selective 3-adrenergic agonists, leading to antidiabetic and antiobesity effects, was found in rodents (161). Selective
3-adrenergic agonists were expected to limit adverse reactions, such as tremor
and tachycardia, observed with early and less-selective -agonists. Nevertheless,
results concerning the action on body weight and energy expenditure of orally
utilizable 3-adrenoceptor agonists are rather disappointing in humans and likely
explained by insufficient levels of expression and recruitment of 3-ARs in 3responsive tissues in humans (162, 163). In the absence of any new and convincing
input, this pharmacological strategy will become highly questionable in humans.
Blockade of fat cell 2-ARs, which are abundant and exceed 1/2-ARs in human fat cells, could promote lipid mobilization in fat deposits resistant to lipid
mobilization and increase energy expenditure by activation of the SNS. The antilipolytic drive of 2-ARs in the fat cell could be enhanced in patients with altered
-adrenergic responsiveness (18). Sustained lipid mobilization and increment of
energy expenditure was observed after 2-antagonist administration in dogs and
humans. The interest in and limitations of 2-antagonist use in obesity treatment
has already been extensively discussed. However, reliable clinical studies have
never been performed owing to the lack of suitable selective agents in humans (24,
164, 165).
Among possible agents, although GH is recognized to possess lipid-mobilizing
properties, its undesirable effects on glucose metabolism (i.e., a diabetogenic effect) limit its potential use as a weight lossinducing agent (166, 167). A domain
of human GH (hGH177-191), which appears to act through a site distinct from the
hGH receptor, promoted lipolysis in rodent and human fat cells. It also increased
fat oxidation and decreased body weight in ob/ob mice. This active GH fragment
has not been identified in human plasma; it is unknown if its administration would
have an effect on lipid mobilization and weight loss in humans. Atrial natriuretic
peptides exert potent lipid-mobilizing effects that are apparently independent of
SNS stimulation and insulin effect modulation (50). It remains to be established
whether the plasma NEFA increase promoted by the natriuretic peptides enhances
fat oxidation and energy expenditure in humans. Because the action of natriuretic
peptides is completely independent of that of insulin, it could be of interest to verify if such compounds and related agents are usable in weight loss management
in obese subjects with lipid mobilizing defects and alterations of the adrenergic
regulation of lipid mobilization.
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NEFA release by fat cells is also under the control of all the antilipolytic agents,
including insulin, but also hormones and agents acting via Gi protein-coupled receptors. Enhancement of antilipolytic effects will reduce plasma NEFA levels in
subjects with increased NEFA (e.g., insulin-resistant subjects and patients with
type 2 diabetes). Antilipolytic strategies based on nicotinic acid derivatives and
adenosine A1-receptor agonists have been proposed to reduce plasma NEFA and
TAG. Insulin sensitization of fat cells contributes to reducing the release of NEFAs
by fat cells. Insulin sensitizers that enhance insulin action by modulating the events
following the binding of insulin to its receptor and/or by activating transcription
factors affecting the expression of the genes involved in the action of insulin in
insulin-sensitive tissues are of interest to improve insulin action in the obese. Inhibitors of the protein tyrosine phosphatase 1B activity as well as of other putative
negative regulators of insulin signaling are candidates to reduce insulin resistance
and improve insulin action and may have potential in the future as antiobesity
agents (168). Several agonists active at both PPAR and PPAR represent promising tools with potential antidiabetic and lipid-lowering properties (3, 169). PPARs,
widely distributed in tissues and cell types, constitute multiple therapeutic targets
(168). Ideally, drugs possessing both PPAR/ agonist potencies are expected to
provide the best means to decrease multiple risk factors for morbidity and mortality existing in diabetic patients by acting on fat cells and liver (170). PPAR is
predominantly expressed in adipocytes, and the various beneficial metabolic effects reported for PPAR agonists are thought to result from direct actions on AT
along with secondary impact in skeletal muscle and liver. The beneficial actions
of PPAR agonists on muscle, liver, and vessels (i.e., atherosclerosis risk) are mediated by their ability (a) to improve insulin-mediated uptake and metabolism of
glucose and NEFA in the adipocyte; (b) to induce the production of adiponectin by
adipocytes (e.g., adiponectin is a relatively early and specific response to activation of PPAR ); and (c) to reduce production of adipocyte-derived factors leading
to insulin resistance, such as resistin, inflammatory molecules, and TNF-. The
insulin-sensitizing potency of PPAR agonists could be related to their antiinflammatory action because they inhibit TNF- action on adipocytes and limit production of inflammatory molecules by fat cells and monocytes/macrophages, which
have been found to be abundant in obese AT. In view of the multiple metabolic
and vascular actions of adiponectin, it is possible that a number of ameliorations
of metabolic disturbances related to the metabolic syndrome attributable to the effects of PPAR agonists could be related to their action on adiponectin production
and release by fat cells. Investigations in adiponectin-deficient mice will facilitate
the answer to the question.
Role of Major Adipocyte Hormones in the Partitioning of Fat

Defective leptin production and action have been proposed to be an important
element of the metabolic syndrome (90). Hyperleptinemia in the obese is considered to protect non-ATs from lipotoxicity (171). Leptin treatment of severely
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diabetic lipodystrophic rodents and humans improves insulin-stimulated hepatic

and peripheral glucose metabolism and promotes a reversal of insulin resistance
and hepatic and muscle triglyceride content (172, 173). Leptin operates through
activation of AMPK, which improves fatty acid oxidation in muscle and downregulates sterol regulatory element binding protein-1c (SREBP-1c) that reduces lipogenesis in liver. Recombinant leptin derivatives could be envisaged as new therapeutic
agents. Therapeutic use of leptin could be proposed in hypoleptinemic patients with
metabolic syndrome when its normal production by fat cells is defective. However,
interference of various pathways [e.g., suppressor of cytokine signaling (SOCS)-3
protein, PTP-1B activity, protein inhibitor of activated signal transducer and activator of transcription-3 (STAT-3), and 11--HSD-1 activity] has been proposed to
explain impairment of leptin action (e.g., leptin resistance) and could oppose the
therapeutic action. Adenovirus-induced hyperleptinemia promotes a dramatic reduction of white fat cell size in rats, and the fatty acids are oxidized directly inside
the white adipocytes that become able to burn fat. Because high circulating levels
of leptin are obtained in such conditions, it is highly questionable if this provocative
observation could have some kind of physiological or therapeutic relevance (89).
Adiponectin is an adipocyte product of major therapeutic interest with protective
actions on the major tissues affected by the metabolic syndrome. Like leptin, it
plays a major role in fat partitioning because it also prevents steatosis in various
nonadipose tissues. Its production exhibits regulation opposite to that of leptin.
Although plasma leptin levels increase with accumulation of fat and adipocyte
hypertrophy, plasma adiponectin levels decrease with fat cell size increment and
obesity. The physiological significance of the opposite regulation of two major
adipocyte hormones involved in the control of lipid deposition/utilization balance
requires further studies to be fully understood. Like leptin, adiponectin, via its
receptors, stimulates AMPK-related metabolic pathways. AMPK activation and
consequent ACC-1 inactivation will result in reduced lipid synthesis and increased
fat oxidation. Does leptin become operative when major adiponectin effects have
been reduced in the obese owing to a lack of adiponectin? It seems reasonable
to propose that either recombinant adiponectin derivatives or adiponectin-mimetic
compounds acting less or more specifically on adiponectin receptor subtypes could
be suggested as new therapeutic approaches. Owing to the size of the protein and
its various circulating forms, it would probably be better to use strategies aimed at
the promotion of adiponectin production by the adipocyte. PPAR agonists might
play a key role in such an enterprise because they have been shown to promote
adiponectin production in fat cells of humans and rodents (174, 175).
Control of Adipose Tissue Development and

Remodeling-Interconversion of White Adipocytes
into Fat Burning Cells
As is well known by investigators working on fat differentiation, nuclear receptors,
such as PPAR , play a crucial role in the regulation of adipocyte differentiation.
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Although they are on the market and of interest for the treatment of type 2 diabetes,
PPAR agonists, such as thiazolidinediones, are not effective against obesity because they are known to promote recruitment/differentiation of new fat cells while
improving insulin sensitivity. Moreover, adipogenesis could be induced in bone
marrow stromal cells. It is a major negative side effect; PPAR agonists might
promote weight gain in patients still having serious metabolic disorders, and their
short-term benefits could be reduced in the long term. Important efforts are being made to identify new PPAR modulators having antidiabetic action without
promoting fat cell differentiation and weight increase. In addition to adipogenesis, angiogenic processes play an important role in the development of AT mass
(176). Adipocyte productions, such as leptin and vascular endothelial growth factor (VEGF), are known to exert proangiogenic effects and contribute to vascular
development in AT. The extent of AT mass is sensitive to angiogenesis inhibitors
(177); strategies aimed at the limitation of vascular supply in fat are opening new
perspectives that merit future attention.
In humans, in whom there are no BAT depots in adults, conversion of white
adipocytes into brown-like fat cells could be a great challenge. Appearance of
brown adipocytes is possible in certain conditions in adults with pheochromocytoma. In a recent study using adenovirus expressing human PGC-1, a PPAR
coactivator has demonstrated that it is possible to promote a metabolic shift in human white fat cells from lipid storage to fatty acid utilization with a concomitant
induction of UCP-1, mitochondria respiratory chain proteins, and fatty acid oxidation enzymes. Palmitate oxidation was indeed elevated in such modified adipocytes
(178). Such a study suggests that future strategies aimed at altering the phenotype
of human white adipocytes could be envisaged for the treatment of obesity.
ISSUES AND TRENDS

The remarkable progress in our understanding of the regulation of fat cell function
and the identification of a large number of hormonal and paracrine agents secreted
by the adipocyte has prepared the ground for important reconsiderations of the role
of this underestimated cell type. Although it is well recognized that abnormalities
of fatty acid metabolism represent key components of the metabolic syndrome
and type 2 diabetes, a number of adipocyte-secreted hormones considered in the
present review are major contributors to the diseases affecting a large part of the
obese population. Moreover, the discovery of such products has revealed original
regulatory processes and offered new putative drug targets for pharmacological
intervention. Nevertheless, a number of secreted compounds do not as yet have a
clear functional status and will gain validation in the future.
Intensive DNA microarray utilization and gene knockouts will probably enable
the identification of components that will lead to a wider array of potential interventions. Obesity-related diseases are of a multifactorial nature with a number
of genetic and environmental factors leading to the final outcome. It is expected
that human genomic studies will identify subpopulations of patients and allow
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early and better targeting. Tailored treatment, adapted to a given pathophysiology,

is certainly important in complex metabolic diseases. The search for antiobesity
agents remains inherently difficult. The ultimate therapeutic goal is not necessarily
weight loss but reduction of related cardiovascular and metabolic morbidities. It
must be noted that only a very small number of effective compounds results from
the large number tested in preclinical research. It is difficult to know which of the
centrally or peripherally acting agents will be the most efficient and safe. Whatever
the research outcomes and discoveries, it is probable that the magic bullet that
promotes slimming despite excessive food intake and inactivity will never exist.
Antiobesity drugs should only be prescribed as adjuncts to dieting and exercise;
prevention remains the major goal.
LITERATURE CITED
1. Reaven GM, Abbasi F, McLaughlin T.
2004. Obesity, insulin resistance and cardiovascular disease. Rec. Prog. Horm.
Res. 59:20723
2. Hotasmiligil GS. 2000. Molecular mechanisms of insulin resistance and the role of
the adipocyte. Int. J. Obes. Relat. Metab.
Disord. 24:S2327
3. Arner P. 2003. The adipocyte in insulin
resistance: key molecules and the impact of the thiazolidinediones. Trends Endocrinol. Metab. 14:13745
4. Lafontan M, Berlan M. 1993. Fat cell
adrenergic receptors and the control of
white and brown fat cell function. J. Lipid
Res. 34:105791
5. Robidoux J, Martin TL, Collins S. 2004.
b-Adrenergic receptors and regulation of
energy expenditure: a family affair. Annu.
Rev. Pharmacol. Toxicol. 44:297323
6. Cannon B, Nedergaard J. 2004. Brown
adipose tissue: function and physiological
significance. Physiol. Rev. 84:277359
7. Rousset S, Alves-Guerra M, Mozo J,
Miroux B, Cassard-Doulcier A-M, et al.
2004. The biology of mitochondrial uncoupling proteins. Diabetes 53(Suppl. 1):
S13035
8. McGarry JD. 2002. Dysregulation of fatty
9.
10.
11.
12.
13.
14.
15.
16.
acid metabolism in the etiology of type 2

diabetes. Diabetes 51:718
Reitman ML, Arioglu E, Gavrilova O,
Taylor SI. 2000. Lipoatrophy revisited.
Trends Endocrinol. Metab. 11:41016
Gavrilova O, Marcus-Samuels B, Graham
D, Kim JK, Shulman GI, et al. 2000. Surgical implantation of adipose tissue reverses diabetes in lipoatrophic mice. J.
Unger R. 2003. Lipid overload and overflow: metabolic trauma and the metabolic
syndrome. Trends Endocrinol. Metab. 14:
398403
Ahima RS, Flier JS. 2000. Adipose tissue
as an endocrine organ. Trends Endocrinol.
Metab. 11:32732
Ailhaud G. 2000. Adipose tissue as an endocrine organ. Int. J. Obes. Relat. Metab.
Disord. 24(Suppl. 2):S13
Trayhurn P, Beattie JH. 2001. Physiological role of adipose tissue: white adipose
tissue as an endocrine and secretory organ.
Proc. Nutr. Soc. 60:32939
Havel P. 2004. Update on adipocyte hormones: regulation of energy balance and
carbohydrate/lipid metabolism. Diabetes
53(Suppl. 1): S14351
Montague CT, ORahilly S. 2000. The
7 Jan 2005
13:57
138
17.

18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAFONTAN
perils of portliness. Causes and consequences of visceral adiposity. Diabetes
49:88388
Wajchenberg BL. 2000. Subcutaneous
and visceral adipose tissue: their relation
to the metabolic syndrome. Endocr. Rev.
21:697738
Lafontan M, Berlan M. 2003. Do regional
differences in adipocyte biology provide
new pathophysiological insights? Trends
Pharmacol. Sci. 24:27683
Frayn KN, Karpe F, Fielding BA, McDonald IA, Coppack SW. 2003. Integrative
physiology of human adipose tissue. Int.
J. Obesity 27:87588
Bartness TJ. 2002. Dual innervation of
white adipose tissue: some evidence for
parasympathetic nervous system involvement. J. Clin. Invest. 10:123537
Dodt C, Lonnroth P, Fehm HL, Elam
M. 1999. Intraneural stimulation elicits
an increase in subcutaneous interstitial
glycerol levels in humans. J. Physiol.
521:54552
Kreier F, Fliers E, Voshol PJ, Eden
CGV, Havekes LM, et al. 2002. Selective
parasympathetic innervation of subcutaneous and intra-abdominal fat: functional
implications. J. Clin. Invest. 110:1243
50
Fliers E, Kreier F, Voshol J, Havekes LM,
Sauerwein HP, et al. 2003. White adipose tissue: getting nervous. J. Neuroendocrinol. 15:100510
Lafontan M, Berlan M. 1995. Fat cell
2-adrenoceptors: the regulation of fat
cell function and lipolysis. Endocr. Rev.
16:71638
Lafontan M, Berlan M. 1980. Evidence for the alpha2-nature of the alphaadrenergic receptor inhibiting lipolysis in
human fat cells. Eur. J. Pharmacol. 66:87
93
Arner P. 1988. Control of lipolysis and
its relevance to development of obesity in
man. Diabetes Metab. Rev. 4:50715
Langin D, Lucas S, Lafontan M. 2000.
Millenium fat-cell lipolysis reveals unsus-
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
pected novel tracks. Horm. Metab. Res.

32:44352
Jensen MD. 1997. Lipolysis: contribution from regional fat. Annu. Rev. Nutr.
17:12739
Arner P. 1999. Catecholamine-induced
lipolysis in obesity. Int. J. Obes. Metab.
Disord. 23 (Suppl. 1):1013
Stich V, de Glisezinsky I, Crampes F,
Hejnova J, Cottet-Emard J-M, et al. 2000.
Activation of alpha2-adrenergic receptors impairs exercise-induced lipolysis in
SCAT of obese subjects. Am. J. Physiol.
279:R499504
Tan GD, Goossens GH, Humphreys SM,
Vidal H, Karpe F. 2004. Upper and lower
body adipose tissue function: a direct
comparison of fat mobilization in humans.
Obes. Res. 12:11418
Frayn KN. 2002. Adipose tissue as a
buffer for daily lipid flux. Diabetologia
45:120110
Garenc C, Perusse L, Chagnon YC, Rankinen T, Gagnon J, et al. 2002. The alpha2adrenergic receptor gene and body fat
content and distribution: the HERITAGE
family Study. Mol. Med. 8:8894
Dodt C, Lonnroth P, Fehm HL, Elam
M. 2000. The subcutaneous lipolytic response to regional neural stimulation is
reduced in obese women. Diabetes 49:
187579
Arner P, Hoffstedt J. 1999. Adrenoceptor
genes in human obesity. J. Intern. Med.
245:66772
Bougnères P, Stunff CL, Pecqueur C,
Pinglier E, Adnot P, Ricquier D. 1997. In
vivo resistance of lipolysis to epinephrine.
A new feature of childhood onset obesity.
J. Clin. Invest. 99:256873
Horowitz JF, Klein S. 2000. Whole body
and abdominal lipolytic sensitivity to
epinephrine is suppressed in upper body
obese women. Am. J. Physiol. Endocrinol.
Metab. 278: E114452
Enoksson S, Talbot M, Rife F, Tamborlane WV, Sherwin RS, Caprio S. 2000.
Impaired in vivo stimulation of lipolysis in
7 Jan 2005
13:57
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE

39.
40.
41.
42.
43.
44.
45.
46.
47.
adipose tissue by selective 2-adrenergic

agonist in obese adolescent girls. Diabetes
49:214953
Schiffelers SLH, Saris WHM, Boomsma
F, Baak MAv. 2001. Beta1- and Beta2adrenoceptor-mediated thermogenesis
and lipid utilization in obese and lean
men. J. Clin. Endocrinol. Metab. 86:
219199
Small KM, McGraw D, Ligget SB. 2003.
Pharmacology and physiology of human
adrenergic receptor polymorphism. Annu.
Kitamura T, Kahn CR, Acili D. 2003. Insulin receptor knockout mice. Annu. Rev.
Physiol. 65:31332
Stahl A, Evans JG, Pattel S, Hirsch D,
Lodish HF. 2002. Insulin causes fatty acid
transport protein translocation and enhanced fatty acid uptake in adipocytes.
Dev. Cell 2:47788
Kido Y, Nakae J, Accili D. 2001. The insulin receptor and its cellular targets. J.
Clin. Endocrinol. Metab. 86:97279
Whiteman EL, Cho H, Birnbaum MJ.
2002. Role of Akt/protein kinase B in
metabolism. Trends Endocrinol. Metab.
13:44451
Zierath J, Livingston J, Thorne A, Bolinder J, Reynisdottir S, et al. 1998. Regional difference in insulin inhibition
of non-esterified fatty acid release from
human adipocytes: relation to insulin
receptor phosphorylation and intracellular signalling through the insulin receptor substrate-1 pathway. Diabetologia
41:134354
Wu X, Hoffstedt J, Deeb W, Singh R, Sedkova N, et al. 2001. Depot-specific variation in protein-tyrosine phosphatase activities in human omental and subcutaneous
adipose tissue: a potential contribution to
differential insulin sensitivity. J. Clin. Endocrinol. Metab. 86:597380
Basu A, Basu R, Shah P, Vella A, Rizza
RA, Jensen MD. 2001. Systemic and regional free fatty acid metabolism in type
2 diabetes. Am. J. Physiol. 280:E10006
139
48. Sengenes C, Berlan M, de Glisezinski I,

Lafontan M, Galitzky J. 2000. Natriuretic
peptides: a new lipolytic pathway in human adipocytes. FASEB J. 14:134551
49. Sengenes C, Bouloumie A, Hauner H,
Berlan M, Busse R, Lafontan M. 2003. Involvement of a cGMP-dependent pathway
in natriuretic peptide-mediated hormonesensitive lipase phosphorylation in human
adipocytes. J. Biol. Chem. 278:48617
26
50. Galitzky J, Sengenes C, Thalamas C, Marques M-A, Senard J-M, et al. 2001. The
lipid mobilizing effect of atrial natriuretic
peptides is unrelated to sympathetic nervous system activation or obesity in young
men. J. Lipid Res. 42:53644
51. Moro C, Crampes F, Sengenes C, de
Glisezinski I, Galitzky J, et al. 2004. Atrial
natriuretic peptide contributes to the physiological control of lipid mobilization in
humans. FASEB J. 18(7):90810
52. Yip RG-C, Goodman HM. 1999. Growth
hormone and dexamethasone stimulate
lipolysis and activate adenylyl cyclase in
rat adipocytes by selectively shifting Gi2
to lower density membrane fractions. Endocrinology 140:121927
53. Moller N, Gjedsted J, Gormsen L,
Fuglsang J, Djurhuus C. 2003. Effects
of growth hormone on lipid metabolism
in humans. Growth Horm. IGF Res.
13(Suppl. A):S1821
54. Gravholt C, Schmitz O, Simonsen L, Bulow J, Christiansen J, Moller N. 1999. Effects of a physiological GH pulse on interstitial glycerol in abdominal and femoral
adipose tissue. Am. J. Physiol. 277:E848
54
55. Samra JS, Clark ML, Humphreys SM,
MacDonald IA, Bannister PA, et al.
1999. Suppression of the nocturnal rise
in growth hormone reduces subsequent
lipolysis in subcutaneous adipose tissue.
Eur. J. Clin. Invest. 29:104552
56. Hefferman MA, Jiang WJ, Thorburn AW,
Ng FM. 2000. Effects of oral administration of a synthetic fragment of human
7 Jan 2005
13:57
140
57.

58.
59.
60.
61.
62.
63.
64.
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAFONTAN
growth hormone on lipid metabolism. Am.
J. Physiol. 279:E5017
Bertin E, Arner P, Bolinder J, HagstromToft E. 2001. Action of glucagon and
glucagon-like peptide-1-(7-36) amide on
lipolysis in human subcutaneous adipose
tissue and skelatal muscle in vivo. J. Clin.
Endocrinol. Metab. 86:122934
Gravholt C, Moller N, Jensen M, Christiansen J, Schmitz. 2001. Physiological
levels of glucagon do not influence lipolysis in abdominal adipose tissue as assessed
by microdialysis. J. Clin. Endocrinol.
Metab. 86:208589
Taniguchi A, Kataoka K, Kono T, Oseko F, Okuda H, et al. 1987. Parathyroid hormone-induced lipolysis in human adipose tissue. J. Lipid Res. 28:490
94
Lyngso D, Simonsen L, Bulow J. 2002.
Metabolic effects of interleukin-6 in human splanchnic and adipose tissue. J.
Physiol. 543:37986
van Hall G, Steenberg A, Sacchetti M,
Fisher C, Keller C, et al. 2003. Interleukin6 stimulates lipolysis and fat oxidation
in humans. J. Clin. Endocrinol. Metab.
88:300510
Path G, Bornstein SR, Gurniak M,
Chrousos GP, Scherbaum WA, Hauner
H. 2001. Human breast adipocytes express interleukin-6 (IL-6) and its receptor system: increased IL-6 production by
beta-adrenergic activation and effects of
IL-6 on adipocyte function. J. Clin. Endocrinol. Metab. 86:228188
Lagathu C, Bastard J-P, Auclair M,
Maachi M, Capeau J, Caron M. 2003.
Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention
by rosiglitazone. Biochem. Biophys. Res.
Commun. 311:37279
Rotter V, Nagaev I, Smith U. 2003.
Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like
IL-8 and tumor necrosis factor-, overexpressed in human fat cells from in-
65.
66.
67.
68.
69.
70.
71.
72.
73.
sulin resistant subjects. J. Biol. Chem.

278:4577784
Wang MY, Lee Y, Unger RH. 1999. Novel
form of lipolysis induced by leptin. J. Biol.
Chem. 274:1754144
Samra JS, Clark ML, Humphreys SM,
MacDonald IA, Bannister PA, Frayn KN.
1998. Effects of physiological hypercortisolemia on the regulation of lipolysis in
subcutaneous adipose tissue. J. Clin. Endocrinol. Metab. 83:62631
Djuurhus CB, Gravholt CH, Nielsen S,
Pedersen SB, Moller N, Schmitz O. 2003.
Additive effects of cortisol and growth
hormone on regional and systemic lipolysis in humans. Am. J. Physiol. Endocrinol.
Metab. 286:E48894
Gravholt CH, Dahl R, Christiansen JS,
Moller N, Schmitz O. 2002. Preferential stimulation of subcutaneous lipolysis
after prednisolone exposure in humans.
Obes. Res. 10:77481
Hauner H, Petruschke T, Russ M, Rohrig
K, Eckel J. 1995. Effects of tumor necrosis
factor alpha (TNF) on glucose transport
and lipid metabolism of newly differentiated human fat cells in culture. Diabetologia 38:76471
Sethi J, Xu H, Uysal K, Wiesbrock S,
Scheja L, Hotamisligil G. 2000. Characterisation of receptor-specific TNF functions in adipocyte cell lines lacking type 1
and 2 TNF receptors. FEBS Lett. 469:77
82
Xu H, Hotamisligil GS. 2001. Signaling
pathways utilized by tumor necrosis factor receptor 1 in adipocytes to suppress
differentiation. FEBS Lett. 506:97102
Souza SC, Palmer HJ, Kang Y-H, Yamamoto MT, Muliro KV, et al. 2003. TNF induction of lipolysis is mediated
through activation of the extracellular signal related kinase pathway in 3T3-L1
adipocytes. J. Cell. Biochem. 89:1077
86
Gasic S, Tian B, Green A. 1999. Tumor
necrosis factor alpha stimulates lipolysis
in adipocytes by decreasing Gi protein
7 Jan 2005
13:57
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
74.

75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
concentrations. J. Biol. Chem. 274:6770

75
Ryden M, Dicker A, van Harmelen V,
Hauner H, Brunnberg M, et al. 2002. Mapping of early signaling events in tumor
necrosis-alpha-mediated lipolysis in human fat cells. J. Biol. Chem. 277:108591
Gaudiot N, Jaubert A-M, Charbonnier E,
Sabourault D, Lacasa D, et al. 1998. Modulation of white adipose tissue lipolysis by
nitric oxide. J. Biol. Chem. 273:1347581
Andersson K, Gaudiot N, Ribière C,
Elizalde M, Giudicelli Y, Arner P. 1999.
A nitric oxide-mediated mechanism regulates lipolysis in human adipose tissue in
vivo. Br. J. Pharmacol. 126:163945
Tisdale MJ. 2002. Cachexia in cancer patients. Nat. Rev. Cancer 2:86271
Bing C, Bao Y, Sanders P, Manieri M,
Cinti S, et al. 2004. Zinc- 2-glycoprotein,
a lipid mobilizing factor, is expressed in
adipocytes and is up-regulated in mice
with cancer cachexia. Proc. Natl. Acad.
Sci. USA 101:25005
Rice AM, Fain JN, Rivkees SA. 2000. A1
adenosine receptor activation increases
adipocyte leptin secretion. Endocrinology
141:144245
Serradeil-Le Gal C, Lafontan M, Raufaste
D, Marchand J, Pouzet B, et al. 2000.
Characterization of NPY receptors controlling lipolysis and leptin secretion in
human adipocytes. FEBS Lett. 475:150
56
Tunaru S, Kero J, Schaub A, Wufka C,
Blaukat A, et al. 2003. PUMA-G and
HM74 are receptors for nicotinic acid and
mediate its anti-lipolytic effect. Nat. Med.
9:35255
Zigman JM, Elmquist JK. 2003. Minireview: from anorexia to obesitythe yin
and yang of body weight control. Endocrinology 144:374956
Tartaglia LA. 1997. The leptin receptor. J.
Biol. Chem. 272:609396
Friedman JM, Halaas JL. 1998. Leptin and
the regulation of body weight in mammals. Nature 395:76370
141
85. Faggioni R, Feingold KR, Grunfeld C.

2001. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J. 15:256571
86. Margetic S, Gazzola C, Pegg GG, Hill RA.
2002. Leptin: a review of its peripheral
actions and interactions. Int. J. Obesity
26:140733
87. Minokoshi Y, Kim YB, Peroni OD, Fryer
LG, Muller C, et al. 2002. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature
415:33943
88. Shimabukuro M, Koyama K, Chen G,
Wang M-Y, Trieu F, et al. 1997. Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc. Natl.
89. Orci L, Cook WS, Ravazzola M, Wang
M-Y, Park B-H, et al. 2004. Rapid transformation of white adipocytes into fat oxidizing machines. Proc. Natl. Acad. Sci.
USA 101:205863
90. Unger R. 2003. Minireview: weapons of
lean body mass destruction: the role of
ectopic lipids in the metabolic syndrome.
Endocrinology 144:515965
91. van Harmelen V, Reynisdottir S, Eriksson
P, Thorne A, Hoffstedt J, et al. 1998. Leptin secretion from subcutaneous and visceral adipose tissue in women. Diabetes
47:91317
92. Bradley RL, Cleveland KA, Cheatham B.
2001. The adipocyte as a secretory organ: mechanisms of vesicle transport and
secretory pathways. Recent Prog. Horm.
Res. 56:32958
93. Fried SK, Bunkin DA, Greenberg AS.
1998. Omental and subcutaneous adipose tissues of obese subjects release
interleukin-6: depot difference and regulation by glucocorticoids. J. Clin. Endocrinol. Metab. 83:84750
94. Mohamed-Ali V, Goodrick S, Rawesh A,
Katz DR, Miles JM, et al. 1997. Subcutaneous adipose tissue releases interleukin6 but not tumor necrosis factor-, in vivo.
J. Clin. Endocrinol. Metab. 82:4196200
7 Jan 2005
13:57

142
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAFONTAN
95. Lyngso D, Simonsen L, Bulow J. 2002.

Interleukin-6 production in human subcutaneous abdominal adipose tissue: the
effect of exercise. J. Physiol. 543:373
78
96. Bastard J-P, Maachi M, Nhieu JTV, Jardel
C, Bruckert E, et al. 2002. Adipose tissue
IL-6 content correlates with resistance to
insulin activation of glucose uptake both
in vivo and in vitro. J. Clin. Endocrinol.
Metab. 87:208489
97. Berg AH, Combs TP, Du X, Brownlee
M, Scherrer PE. 2001. The adipocytesecreted protein Acrp30 enhances hepatic
insulin action. Nat. Med. 7:94753
98. Ukkola O, Santaniemi M. 2002. Adiponectin: a link between excess adiposity and associated comorbidities? J. Mol.
Med. 80:696702
99. Kadowaki T, Hara K, Yamauchi T, Terauchi Y, Tobe K, Nagai R. 2003. Molecular mechanisms of insulin resistance and
obesity. Exp. Biol. Med. 228:111117
100. Vasseur F, Lepretre F, Lacquemant C,
Froguel P. 2003. The genetics of adiponectin. Curr. Diab. Rep. 3:15158
101. Waki H, Yamauchi T, Kamon J, Ito Y,
Uchida S, et al. 2003. Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of
adiponectin. J. Biol. Chem. 278:40352
63
102. Tsao T-S, Tomas E, Murrey HE, Hug
C, Lee DH, et al. 2003. Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity: different oligomers activate different signal
transduction pathways. J. Biol. Chem.
278:5081017
103. Yamauchi T, Oike Y, Kamon J, Waki
H, Komeda K, et al. 2002. Increased insulin sensitivity despite lipodystrophy in
Crebbp heterozygous mice. Nat. Genet.
30:22126
104. Maeda N, Shimomura I, Kishida K,
Nishizawa H, Matsuda M, et al. 2002.
Diet-induced insulin resistance in mice
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
lacking adiponectin/ACRP30. Nat. Med.

8:73137
Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M, et al. 2002. Disruption
of adiponectin causes insulin resistance
and neointimal formation. J. Biol. Chem.
277:2586366
Xu A, Wang Y, Keshaw H, Xu LY, Lam
KSL, Cooper GJS. 2003. The fat-derived
hormone adiponectin alleviates alcoholic
and nonalcoholic fatty liver disease in
mice. J. Clin. Invest. 112:91100
Tschritter O, Fritsche A, Thamer C, Haap
M, Shirkavand F, et al. 2003. Plasma
adiponectin concentrations predict insulin
sensitivity of both glucose and lipid
metabolism. Diabetes 52:23943
Ouchi N, Ohishi M, Kihara S, Funahashi
T, Nakamura T, et al. 2003. Association of
hypoadiponectinemia with impaired vasoreactivity. Hypertension 42:23134
Shimabukuro M, Higa N, Asahi T, Oshiro Y, Takasu N, et al. 2003. Hypoadiponectinemia is closely linked to endothelial dysfunction in man. J. Clin.
Matsuzawa Y, Funahashi T, Kihara S,
Shimomura I. 2004. Adiponectin and
metabolic
syndrome.
Arterioscler.
Thromb. Vasc. Biol. 24:2933
Combs TP, Berg AH, Obici S, Scherer
PE, Rossetti L. 2001. Endogenous glucose
production is inhibited by the adiposederived protein Acrp30. J. Clin. Invest.
108:187581
Yamauchi T, Kamon J, Minokoshi Y, Ito
Y, Waki H, et al. 2002. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat. Med. 8:128895
Hardie DG. 2003. Minireview: the AMPactivated protein kinase cascade: the key
sensor of cellular energy status. J. Clin.
Tomas E, Tsao T-S, Saha AK, Murrey
HE, Zhang CC, et al. 2002. Enhanced muscle fat oxidation and glucose
transport by ACRP30 globular domain:
7 Jan 2005
13:57
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
115.

116.
117.
118.
119.
120.
121.
122.
123.
124.
acetyl-CoA carboxylase inhibition and

AMP-activated protein kinase activation.
Yamauchi T, Kamon J, Ito Y, Tsuchida
A, Yokomizo T, et al. 2003. Cloning of
adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762
69
Berg AH, Combs TP, Scherrer PE. 2002.
ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism.
Stefan N, Stumvoll M. 2002. Adiponectinits role in metabolism and beyond. Horm. Metab. Res. 34:46974
Steppan C, Bailey ST, Bath S, Brown EJ,
Banerjee RR, et al. 2001. The hormone
resistin links obesity to diabetes. Nature
409:30712
Rajala MW, Obici S, Scherrer PE, Rossetti
L. 2003. Adipose tissue-derived resistin
and gut-derived resistin-like molecule-
selectively impair insulin action on glucose production. J. Clin. Invest. 111:225
30
Kim KH, Lee K, Moon YS, Sul HS. 2001.
A cysteine-rich adipose tissue-specific
secretory factor inhibits adipocyte differentiation. J. Biol. Chem. 276:11252
56
Kratchmarova I, Kalume DE, Blagoev
B, Scherer PE, Podtelejnikov AV, et al.
2002. A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to
adipocytes. Mol. Cell. Proteomics 1:213
22
Banerjee RR, Rangwala SM, Shapiro JS,
Rich AS, Rhoades B, et al. 2004. Regulation of fasted blood glucose by resistin.
Science 303:119598
Savage DB, Sewter CP, Klenk ES, Segal DG, Vidal-Puig A, et al. 2001. Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated
receptor- action in humans. Diabetes
50:2199202
Nagaev I, Smith U. 2001. Insulin resis-
125.
126.
127.
128.
129.
130.
131.
132.
133.
143
tance and type 2 diabetes are not related

to resistin expression in human fat cells or
skeletal muscle. Biochem. Biophys. Res.
Commun. 285:56164
McTernan PG, Fisher FM, Valsamakis G,
Chetty R, Harte A, et al. 2003. Resistin
and type 2 diabetes: regulation of resistin
expression by insulin and rosiglitazone
and the effects of recombinant resistin
on lipid and glucose metabolism in human differentiated adipocytes. J. Clin. Endocrinol. Metab. 88:6098106
Rajala MW, Scherer PE. 2003. Minireview: the adipocyteat the crossroads
of energy homeostasis, inflammation and
atherosclerosis. J. Clin. Endocrinol. Metab. 144:376573
Samad F, Pandey M, Loskutoff DJ. 1998.
Tissue factor gene expression in the adipose tissues of obese mice. Proc. Natl.
Lin Y, Rajala MW, Berger JP, Moller
DE, Barzilai N, Scherer PE. 2001.
Hyperglycemia-induced production of
acute phase reactants in adipose tissue. J.
Biol. Chem. 276:4207783
Mohamed-Ali V, Pinkey JH, Coppack
SW. 1998. Adipose tissue as an endocrine and paracrine organ. Int. J. Obesity 22:114558
Levine JA, Jensen MD, Eberhardt NL,
OBrien T. 1998. Adipocyte macrophage
colony-stimulating factor is a mediator
of adipose tissue growth. J. Clin. Invest.
101:155764
Coppack SW. 2001. Pro-inflammatory cytokines and adipose tissue. Proc. Nutr.
Soc. 60:34956
Ruan H, Zarnowski MJ, Cushman SW,
Lodish HF. 2003. Standard isolation of
primary adipose cells from mouse epididymal fat pads induces inflammatory
mediators and down regulates adipocyte
genes. J. Biol. Chem. 278:4758593
Hotamisligil GS, Spiegelman BM. 1994.
Tumor necrosis factor : a key component of the obesity-diabetes link. Diabetes
43:127178
7 Jan 2005
13:57

144
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAFONTAN
134. Moller DE. 2000. Potential role of TNF in the pathogenesis of insulin resistance
and type 2 diabetes. Trends Endocrinol.
Metab. 11:21217
135. Lofgren P, van Harmelen V, Reynisdottir
S, Naslund E, Ryden M, et al. 2000. Secretion of tumor necrosis factor- shows
a strong relationship to insulin-stimulated
glucose transport in human adipose tissue.
Diabetes 49:68892
136. Ruan H, Lodish HF. 2003. Insulin resistance in adipose tissue: direct and indirect effects on tumor necrosis factor-.
Cytokine Growth Factor Rev. 14:44755
137. Fasshauer M, Paschke R. 2003. Regulation of adipokines and insulin resistance.
Diabetologia 46:1594603
138. Uysal KT, Wiesbrock SM, Marino MW,
Hotamisligil GS. 1997. Protection from
obesity-induced insulin resistance in mice
lacking TNF-a function. Nature 389:610
14
139. Weisberg SP, McCann D, Desai M,
Rosenbaum M, Leibel RL, Ferrante AW
Jr. 2003. Obesity is associated with
macrophage accumulation in adipose tissue. J. Clin. Invest. 112:1796608
140. Xu H, Barnes GT, Yang Q, Tan G, Yang
D, et al. 2003. Chronic inflammation in fat
plays a crucial role in the development of
obesity-related insulin resistance. J. Clin.
Invest. 112:182130
141. Takahashi K, Mizuarai S, Araki H,
Mashiko S, Ishihara A, et al. 2003.
Adiposity elevates plasma MCP-1 levels leading to the increased CD11bpositive monocytes in mice. J. Biol. Chem.
278:4665460
142. Sartipy P, Loskutoff DJ. 2003. Monocyte
chemoattractant protein-1 in obesity and
insulin resistance. Proc. Natl. Acad. Sci.
USA 100:726570
143. Bouloumie A, Marumo T, Lafontan M,
Busse R. 1999. Leptin induces oxidative
stress in human endothelial cells. FASEB
J. 13:123138
144. Yamagishi S-I, Edelstein D, Du XL, Kaneda Y, Guzman M, Brownlee
145.
146.
147.
148.
149.
150.
151.
152.
M. 2001. Leptin induces mitochondrial

superoxide production and monocyte
chemoattractant protein-1 expression in
aortic endothelial cells by increasing fatty
acid oxidation via protein kinase A. J.
Biol. Chem. 276:25096100
Verma S, Li S-H, Wang C-H, Fedak
PWM, Li R-R, et al. 2003. Resistin promotes endothelial cell activation: further
evidence of adipokine-endothelial interaction. Circulation 108:73640
Cianflone K, Xia Z, Chen LY. 2003. Critical review of acylation-stimulating protein physiology in humans and rodents.
Biochim. Biophys. Acta 1609:12743
Juhan-Vague I, Alessi MC. 1999. Regulation of fibrinolysis in the development of
atherothrombosis: role of adipose tissue.
Thromb. Haemost. 82:83236
Bouloumie A, Sengenes C, Portolan G,
Galitzky J, Lafontan M. 2001. Adipocyte
produces matrix metalloproteinases 2 and
9. Involvement in adipose differentiation.
Diabetes 50:208086
Trayhurn P, Duncan JS, Wood AM, Beattie JH. 2000. Metallothionein gene expression and secretion in white adipose
tissue. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 279:R232935
Gesta S, Simon MF, Rey A, Sibrac D,
Girard A, et al. 2002. Secretion of
a lysophospholipase D activity by
adipocytes: involvement in lysophosphatidic acid synthesis. J. Lipid Res.
43:90410
Ferry G, Tellier E, Try A, Grès S, Naime
I, et al. 2003. Autotaxin is released
from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates
preadipocyte proliferation. Up-regulated
expression with adipocyte differentiation
and obesity. J. Biol. Chem. 278:18162
69
Massiera F, Bloch-Faure M, Ceiler D,
Marukami K, Fukamizu A, et al. 2001.
Adipose angiotensinogen is involved in
adipose tissue growth and blood pressure
regulation. FASEB J. 15:272729
7 Jan 2005
13:57
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE


153. Saint-Marc P, Kozak LP, Ailhaud G, Darimont C, Negrel R. 2001. Angiotensin II
as a trophic factor of white adipose tissue:
stimulation of adipose cell formation. Endocrinology 142:48792
154. Engeli S, Schling P, Gorzelniak K,
Boschmann M, Janke J, et al. 2003.
The adipose-tissue renin-angiotensinaldosterone system: role in the metabolic
syndrome. Int. J. Biochem. Cell Biol. 35:
80725
155. Kersten S, Mandard S, Tan NS, Escher
P, Metzger D, et al. 2000. Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferatoractivated receptor target gene. J. Biol.
Chem. 275:2848893
156. Yoon JC, Chickering TW, Rosen ED, Dussault B, Qin Y, et al. 2000. Peroxisome
proliferator-activated receptor gamma target gene encoding a novel angiopoietinrelated protein associated with adipose
tissue differentiation. Mol. Cell. Biol.
20:534349
157. Le Jan S, Amy C, Cazes A, Monnot C,
Lamande N, et al. 2003. Angiopoietin-like
4 is a proangiogenic factor produced during ischemia and in conventional renal cell
carcinoma. Am. J. Pathol. 162:152128
158. Rask E, Olsson T, Soderberg S, Andrew R, Livingstone DE, et al. 2001.
Tissue-specific dysregulation of cortisol
metabolism in human obesity. J. Clin. Endocrinol. Metab. 86:141821
159. Masuzaki H, Paterson J, Shinyama H,
Morton NM, Mullins JJ, et al. 2001.
A transgenic model of visceral obesity
and the metabolic syndrome. Science
294:216670
160. Clapham JC, Arch JRS, Tadayyon M.
2001. Anti-obesity drugs: a critical review
of current therapies and future opportunities. Pharmacol. Ther. 89:81121
161. Arch JRS, Wilson S. 1996. Prospects for
3-adrenoceptor agonists in the treatment
of obesity and diabetes. Int. J. Obesity
20:19199
162. Weyer C, Tataranni PA, Snitker S, Dan-
163.
164.
165.
166.
167.
168.
169.
170.
171.
145
forth E Jr, Ravussin E. 1998. Increase in

insulin action and fat oxidation after treatment with CL316,243, a highly selective
3-adrenoceptor agonist in humans. Diabetes 47:155561
Larsen TM, Toubro S, Baak MAv, Gottesdiener KM, Larson P, et al. 2002. Effect
of a 28-day treatment with L-796568, a
novel 3-adrenergic receptor agonist, on
energy expenditure and body composition
in obese men. Am. J. Clin. Nutr. 76:780
88
Lafontan M, Berlan M, Galitzky J, Montastruc J-L. 1992. Alpha2-adrenoceptors
in lipolysis: alpha2-antagonists and lipid
mobilizing strategies. Am. J. Clin. Nutr.
55:219S27
Berlan M, Montastruc J-L, Lafontan
M. 1992. Pharmacological prospect for
alpha2-adrenoceptor antagonist therapy.
Trends Pharmacol. Sci. 13:27782
Hansen TK. 2002. Pharmacokinetics and
acute lipolytic actions of growth hormone.
Impact of age, body composition, binding proteins, and other hormones. Growth
Horm. IGF Res. 12:34258
Bramnert M, Segerlantz M, Laurila E,
Daugaard JR, Manhem P, Groop L. 2003.
Growth hormone replacement therapy induces insulin resistance by activating the
glucose-fatty acid cycle. J. Clin. Endocrinol. Metab. 88:145563
Moller DE. 2001. New drug targets for
type 2 diabetes and the metabolic syndrome. Nature 414:82127
Wagner JA. 2002. Early clinical development of pharmaceuticals for type 2 diabetes mellitus: from preclinical models to
human investigation. J. Clin. Endocrinol.
Metab. 87:536266
Guo Q, Sahoo SP, Wang P-R, Milot DP,
Ippolito MC, et al. 2004. A novel peroxisome proliferator-activated receptor /
dual agonist demonstrate favorable effects on lipid homeostasis. Endocrinology
145:164048
Unger RH, Zhou Y-T, Orci L. 1999. Regulation of fatty acid homeostasis in cells:
7 Jan 2005
13:57
146
172.

173.
174.
175.
AR
AR232-PA45-05.tex
AR232-PA45-05.sgm
LaTeX2e(2002/01/18)
P1: GCE
LAFONTAN
novel role of leptin. Proc. Natl. Acad. Sci.
USA 96:232732
Shimomura I, Hammer RE, Ikemoto S,
Brown MS, Goldstein JL. 1999. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 401:7376
Petersen KF, Oral EA, Dufour S, Befroy
D, Ariyan C, et al. 2002. Leptin reverses
insulin resistance and hepatic steatosis
in patients with severe lipodystrophy. J.
Combs T, Wagner J, Berger J, Doebber T, Zhang B, et al. 2002. Induction
of Acrp30 levels by PPAR agonists: a
mechanism of insulin sensitization. Endocrinology 143:9981007
Moller DE, Berger JP. 2003. Role of
PPARs in the regulation of obesity-related

insulin sensitivity and inflammation. Int.
J. Obesity 27:S1721
176. Fukumura D, Ushiyama A, Duda DG, Xu
L, Tam J, et al. 2003. Paracrine regulation
of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ.
Res. 93:e8897
177. Rupnick MA, Panigrahy D, Zhang C-Y,
Dallabrida SM, Lowell BB, et al. 2002.
Adipose tissue mass can be regulated
through the vasculature. Proc. Natl. Acad.
Sci. USA 99:1073035
178. Tiraby C, Tavernier G, Lefort C, Larrouy
D, Bouillaud F, et al. 2003. Acquirement
of brown fat cell features by human white
adipocytes. J. Biol. Chem. 278:33370
76
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Figure 1 Control of human fat cell lipolysis: signal transduction pathways for catecholamines via - and 2-adrenergic receptors, atrial natriuretic peptide via type A receptor
(NPR-A), and insulin. Catecholamines, insulin, and various inhibitory receptors negatively coupled to adenylyl cyclase control cAMP production, whereas atrial and brain natriuretic peptides (ANP and BNP, respectively) control cGMP production. cAMP and cGMP
both contribute to the protein-kinase [PKA and PKG (cGK-I)]-dependent phosphorylation
of HSL and perilipin. Perilipin phosphorylation induces an important physical alteration
of the droplet surface that facilitates the action of HSL on triglyceride lipolysis. HSL phosphorylation promotes its translocation from the cytosol to the surface of the lipid droplet.
Docking of ALBP to HSL favors the efflux of NEFA released by the hydrolysis of triglycerides. PKA and PKG (cGK-I) phosphorylate a number of other substrates that are not
shown in the diagram and can influence the secretion of various adipocyte products such
as leptin, adiponectin, and interleukin-6. Question marks show pathways that are still
hypothetical or the relevance of which has not been fully demonstrated. AC, adenylyl
cyclase; ALBP, adipocyte lipid binding protein; AR, adrenergic receptor; EP3-R, EP3prostaglandin receptor; adenosine-A1-R, type A1 adenosine receptor; NPY-Y1-R, type Y1
neuropeptide Y receptor; GC, guanylyl cyclase; Gi, inhibitory GTP-binding protein; Gs,
stimulatory GTP-binding protein; HSL, hormone-sensitive lipase; IRS, insulin receptor
substrate; PDE-3B, phosphodiesterase 3B; PI3-K, phosphatidylinositol-3-phosphate
kinase; PKA, protein kinase A; PKB, protein kinase B/Akt; PKG (cGK-I), protein kinase
G; NEFA, nonesterified fatty acid; ALBP, adipocyte lipid binding protein; (), inhibition;
(), stimulation.
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Figure 2 Overview of the major functions under the control of products secreted by the
adipocyte. All the hormones and secretions (and abbreviations) are defined in the text.
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Copyright

FORMATION AND TOXICITY OF ANESTHETIC

DEGRADATION PRODUCTS
M.W. Anders
Department of Pharmacology and Physiology, University of Rochester Medical Center,
Rochester, New York 14642; email: mw anders@urmc.rochester.edu
Key Words trichloroethylene, dichloroacetylene, halothane,

1,1-difluoro-2-bromo-2-chloroethylene, sevoflurane, Compound A, desflurane,
enflurane, isoflurane, carbon monoxide
Abstract Toxic degradation products are formed from a range of old and modern
anesthetic agents. The common element in the formation of degradation products is
the reaction of the anesthetic agent with the bases in the carbon dioxide absorbents
in the anesthesia circuit. This reaction results in the conversion of trichloroethylene
to dichloroacetylene, halothane to 2-bromo-2-chloro-1,1-difluoroethylene, sevoflurane
to 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A), and desflurane,
isoflurane, and enflurane to carbon monoxide. Dichloroacetylene, 2-bromo-2-chloro1,1-difluoroethylene, and Compound A form glutathione S-conjugates that undergo
hydrolysis to cysteine S-conjugates and bioactivation of the cysteine S-conjugates by
renal cysteine conjugate -lyase to give nephrotoxic metabolites. The elucidation of
the mechanisms of formation and bioactivation of degradation products has allowed
for the safe use of anesthetics that may undergo degradation in the anesthesia circuit.
INTRODUCTION
The hazards associated with human exposure to degradation products of volatile
anesthetics have long been recognized. The effects of light, air, and heat on chloroform to produce phosgene, along with other degradation products, are well known.
The interaction of volatile anesthetic agents within the anesthetic circuit itself
was also a concern. The most notable interaction was that of trichloroethylene
with soda lime to produce dichloroacetylene. This highly toxic compound produced considerable morbidity and, perhaps, mortality, and it is discussed in this
review because of its historical importance. As these problems were identified and
corrected, concern shifted to the toxicity associated with the metabolism of inhaled anesthetics. Fluoride-induced nephropathy associated with methoxyflurane
and the halothane-associated hepatoxicity were of concern to anesthesiologists.
Recently, however, there has been renewed interest in the formation and toxicity of degradation products of volatile anesthetics, particularly the formation of
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ANDERS

Compound A from sevoflurane and the formation of carbon monoxide from anesthetics with -CHF2 groups, i.e., desflurane, enflurane, and isoflurane.
This review addresses the formation, fate, and animal and human toxicity of
dichloroacetylene (from trichloroethylene); 2-bromo-2-chloro-1,1-difluoroethylene (from halothane); 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene or
Compound A (from sevoflurane); and carbon monoxide (from desflurane, isoflurane, and enflurane). All of these degradation products have known or suspected
toxic potential for humans.
DICHLOROACETYLENE FORMATION FROM

TRICHLOROETHYLENE
Introduction
The successful human use of trichloroethylene as a general anesthetic and analgesic
was first reported by Striker et al. in 1935 (1). Trichloroethylene was introduced as
an alternative to other inhaled anesthetics, such as diethyl ether and cyclopropane,
because it possessed several advantages, including nonflammability, maintenance
of cardiovascular stability, and general lack of postoperative side effects. It remained popular in some countries as a general anesthetic and analgesic well into
the 1970s and is still used today in some parts of the world. Soon after its introduction, however, there appeared reports that its use was occasionally associated
with cranial nerve neuropathies, particularly of the trigeminal nerve (2, 3). Subsequently, the formation of dichloroacetylene was implicated in the observed toxicity
of trichloroethylene (4, 5).
Formation and Fate of Dichloroacetylene

Dichloroacetylene 2 is formed by the base-catalyzed elimination of HCl from
trichloroethylene 1 (Figure 1) (6); this reaction is dependent on temperature and
on the base-content of the soda lime (5). Dichloroacetylene is highly unstable and decomposes to give phosgene (the most abundant degradation product)
and several other compounds (7), but their formation has not been implicated in
dichloroacetylene-induced cranial nerve damage. Moreover, dichloroacetylene is
Figure 1 Base-catalyzed conversion of trichloroethylene 1 to dichloroacetylene 2.
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ANESTHETIC DEGRADATION PRODUCTS
149
stabilized by high concentrations of trichloroethylene, which may limit its decomposition during anesthesia.
The metabolic fate of dichloroacetylene has been investigated in experimental
animals. In rats exposed by inhalation to [14C]dichloroacetylene (40 ppm, 1 h), S(1,2-dichlorovinyl)-N-acetyl-L-cysteine 7 (Figure 2) (61.8%), 2,2-dichloroethanol
(12.2%), 2,2-dichloroethyl glucuronide (4.5%), dichloroacetic acid (8.9%), chloroacetic acid (4.7%), and oxalic acid (8.3%) were excreted in the urine over 96 h (8).
The finding that mercapturate 7 is the major metabolite of dichloroacetylene indicates that glutathione-dependent metabolism is the major pathway of metabolism.
The biotransformation and bioactivation of a range of nephrotoxic and cytotoxic
haloalkenes is dependent on glutathione S-conjugate formation and activation of
cysteine S-conjugates by cysteine conjugate -lyase. This pathway includes glutathione transferase-catalyzed glutathione S-conjugate formation, hydrolysis of the
conjugates by -glutamyltransferase and dipeptidases to give the corresponding
cysteine S-conjugates, active uptake of the cysteine S-conjugates by the kidney, and
bioactivation by cytosolic and mitochondrial -lyases. Reviews about the -lyase
pathway have appeared (9, 10).
Dichloroacetylene 2 undergoes bioactivation by the -lyase pathway (Figure 2).
The reaction of dichloroacetylene 2 with glutathione is catalyzed by rat hepatic and renal glutathione S-transferases to give S-(1,2-dichlorovinyl)glutathione 3
(11). The bioactivation mechanism of S-(1,2-dichlorovinyl)glutathione 3 has been
elucidated (12): S-(1,2-dichlorovinyl)glutathione 3 is hydrolyzed by -glutamyltransferase and dipeptidases to give S-(1,2-dichlorovinyl)-L-cysteine 4, which
undergoes bioactivation by renal cysteine conjugate -lyase or detoxication by
N-acetylation to give S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine 7, which is the
major urinary metabolite. S-(1,2-Dichlorovinyl)-L-cysteine 4 undergoes a -lyasecatalyzed -elimination reaction to give 1,2-dichloroethenethiolate 5, pyruvate,
and ammonia. Thiolate 5 may lose chloride to give chlorothioketene 6 or may tautomerize to give chlorothionoacetyl chloride 8. Both thioketene 6 and thionoacyl
chloride 8 may contribute to the toxicity of S-(1,2-dichlorovinyl)-L-cysteine 4, but
the finding that thioketene 6 is highly unstable in aqueous environments favors a
role for thionoacyl chloride 8 (13). The formation of 1,2-dichloroethenethiolate 5
and chlorothioketene 6 has been demonstrated by Fourier-transform ion cyclotron
resonance mass spectrometry (14).
Toxicity
The toxicity of dichloroacetylene and its glutathione and cysteines S-conjugates has
been investigated in experimental animals and in a range of in vitro systems. The
high reactivity of dichloroacetylene has prevented investigation of its cytotoxicity.
Dichloroacetylene is nephrotoxic,
nephrocarcinogenic, neurotoxic, and hepatotoxic in laboratory animals, but nephrotoxicity is the prominent feature of dichloroacetylene-induced toxicity (1518).
ANIMAL TOXICITY AND IN VITRO STUDIES
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Figure 2 Glutathione S-transferase- and cysteine conjugate -lyase-dependent

bioactivation of dichloroacetylene 2. 3, S-(1,2-dichlorovinyl)glutathione; 4, S(1,2-dichlorovinyl)-L-cysteine; 5, 1,2-dichloroethenethiolate; 6, chlorothioketene;
7, S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine; 8, chlorothionoacetyl chloride. GSH,
glutathione; GST, glutathione S-transferase; -GT, -glutamyltransferase; NAT,
N-acetyltransferase.
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Rabbits exposed to dichloroacetylene show extensive tubular and focal necrosis in the collecting tubules and accompanying clinical-chemical indices of renal
damage, including marked increases in blood urea nitrogen concentrations. As indicated above, the nephrotoxicity of dichloroacetylene is associated with -lyasedependent bioactivation (11). Dichloroacetylene is a potent nephrocarcinogen in
rats and mice: Cystadenomas and adenocarcinomas of the proximal tubules were
observed in all animals exposed to dichloroacetylene (18). The hepatotoxicity of
dichloroacetylene is characterized by fatty degeneration of parenchymal cells, but
only transient elevations in serum transaminases are observed (17).
The neurotoxicity of dichloroacetylene in rabbits is characterized by morphological changes in the sensory and motor trigeminal nuclei and in the facial and
oculomotor nerves and by functional neurological deficits that are manifested
as decreased thermal sensitivity (16). In mice, damage to the Purkinje layer of
the cerebellum is also observed (15). The mechanism of the neurotoxicity of
dichloroacetylene has not been elucidated. Both S-(1,2-dichlorovinyl)glutathione
3 and S-(1,2-dichlorovinyl)-L-cysteine 4 are efficiently taken up by the brain, and
-lyase activity is present in the brain, indicating a possible role for the -lyase
pathway in dichloroacetylene-induced neurotoxicity in rodents (19, 20).
The cytotoxicity of dichloroacetylene itself has apparently not been reported.
The dichloroacetylene-derived conjugates S-(1,2-dichlorovinyl)glutathione and S(1,2-dichlorovinyl)-L-cysteine are, however, cytotoxic in isolated rat renal proximal tubular cells (21). The cytotoxicity of S-(1,2-dichlorovinyl)glutathione is
blocked by the -glutamyltransferase inhibitor acivicin and by the dipeptidase inhibitors 1,10-phenanthroline and phenylalanylglycine, indicating that hydrolysis
of the glutathione S-conjugate to the cysteine S-conjugate is required for toxicity.
The -lyase inhibitor (aminooxy)acetic acid blocks the cytotoxicity of both the
glutathione and cysteine S-conjugates. S-Conjugate-induced mitochondrial dysfunction plays an important role in S-(1,2-dichlorovinyl)-L-cysteine-induced cytotoxicity (22). Similarly, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine are cytotoxic in pig kidney-derived cultured LLC-PK1 cells, and
their cytotoxicity is blocked by (aminooxy)acetic acid (23).
Pure dichloroacetylene is mutagenic in Salmonella typhimurium strain TA100
but not in strain TA98 (24). The glutathione and cysteine S-conjugates of dichloroacetylene are also mutagenic in the Ames test with S. typhimurium strain TA2638
(25). The -lyase inhibitor (aminooxy)acetic acid blocks the mutagenicity of both
S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)glutathione, indicating
a role for -lyase in S-conjugate-induced mutagenicity. -Glutamyltransferase,
which catalyzes the hydrolysis of glutathione S-conjugates to cysteine S-conjugates,
is present in extracts of S. typhimurium and converts the glutathione S-conjugates
to cysteine S-conjugates, which undergo -lyase-dependent bioactivation. Finally,
S-(1,2-dichlorovinyl)--methyl-DL-cysteine, which cannot undergo bioactivation
by the pyridoxal phosphate-dependent -lyase, is not mutagenic. S-(1,2-Dichlorovinyl)-L-cysteine induces unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts and expression of c-fos and c-myc in
LLC-PK1 cells (26, 27).
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Damage to cranial nerves is a distinctive feature of dichloroacetylene poisoning in man and is often associated with symptoms such as skin
irritation, headache, nausea, dizziness, and confusion (28). This unusual pattern of symptoms was described in one of the early reports of toxicity after
trichloroethylene anesthesia (4, 29). Patients given trichloroethylene through sodalime-containing circuits showed neurological symptoms that ranged from mild
trigeminal anesthesia to general encephalitis and death. The most striking feature
in all patients was trigeminal neuropathy, but many patients also showed involvement of other cranial nerves. Although the investigators did not establish the exact
cause of the toxicity, they believed that the most likely cause was dichloroacetylene
formed by a chemical reaction of trichloroethylene with soda lime. Accordingly,
they recommended that soda lime not be used during anesthesia with trichloroethylene. Detailed studies of the conditions required to produce dichloroacetylene from
trichloroethylene in soda lime revealed that not only were the temperature and base
content of the soda lime important, but also its degree of hydration: Only dry soda
lime produced significant amounts of dichloroacetylene (5).
Interestingly, nephrotoxicity, which is a prominent feature of dichloroacetyleneinduced toxicity in rodents, was apparently not observed in human subjects anesthetized with trichloroethylene. Although the evidence strongly indicates that
dichloroacetylene undergoes -lyase-dependent bioactivation in rodents, the failure to observe nephrotoxicity in human subjects may be attributed to the low
-lyase activities present in human kidney tissue (3032). The possible role of lyase-dependent bioactivation in the observed neurotoxicity of dichloroacetylene
merits further investigation.
Serious toxicity after trichloroethylene anesthesia ceased to be a problem once
the cause was identified. Anesthesia circuits that lacked carbon dioxide absorbents
were used to deliver trichloroethylene. Additionally, the base content of absorbents
was reduced and their formulations were changed to minimize the temperature
increase during use so that if they were used in error during trichloroethylene
anesthesia, risks would be minimized. Despite the apparent safety of modern absorbents, they have been implicated in the production of all of the other degradation
products of currently used anesthetics described in this review.

HUMAN TOXICITY
2-BROMO-2-CHLORO-1,1-DIFLUOROETHYLENE
FROM HALOTHANE
Introduction
Halothane was introduced into clinical anesthesia practice in 1956 (33) and soon
became the most commonly used volatile anesthetic because of its lack of flammability and its desirable anesthetic properties. By the early 1960s, however, reports
appeared that its use was occasionally associated with a type of fulminant hepatitis.
Although rare, approximately 1 case in 35,000 administrations, concern about this
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so-called halothane-associated hepatitis eventually led to its decline in popularity,

especially after the introduction of isoflurane. Nevertheless, halothane is still used
in many parts of the world and finds limited clinical use in the United States, most
notably for pediatric anesthesia. Halothane-associated hepatitis is now believed to
be associated with its cytochrome P450-dependent metabolism to trifluoroacetyl
chloride, which trifluoroacetylates lysine residues in liver proteins to give neoantigens that result in a drug-induced allergic hepatitis (34, 35).
2-Bromo-2-chloro-1,1-difluoroethylene, which has not been implicated in the
pathogenesis of halothane-associated hepatitis, was identified as a minor (<0.005%
w/w) impurity in halothane (3638). Later studies also showed that 2-bromo-2chloro-1,1-difluoroethylene is present in the breath of human subjects anesthetized
with halothane (39). S-(2-Bromo-2-chloro-1,1-difluoroethyl)-N-acetyl-L-cysteine
had previously been identified as a urinary metabolite of halothane (40), and its
formation from 2-bromo-2-chloro-1,1-difluoroethylene is discussed below.
Formation and Fate of 2-Bromo-2-chloro-1,1-difluoroethylene

2-Bromo-2-chloro-1,1-difluoroethylene 10 is formed by the base-catalyzed elimination of HF from halothane 9 in the presence of soda lime (Figure 3) (36,
39). Inhaled 2-bromo-2-chloro-1,1-difluoroethylene presumably undergoes rapid
metabolism in the body because it is not detectable in the expired gases of patients
within minutes of being disconnected from the anesthetic circuit (39).
2-Bromo-2-chloro-1,1-difluoroethylene reacts readily and nonenzymatically
with sulfur nucleophiles, such as glutathione and cysteine (41). The pseudo firstorder rate constants for the reaction of 2-bromo-2-chloro-1,1-difluoroethylene
with cysteine and glutathione are 1.7 0.4 104 sec1 and 1.77 0.20 to
2.02 0.22 104 sec1, respectively. The reaction of 2-bromo-2-chloro-1,1difluoroethylene with sulfhydryl groups is about 50 times faster than its rate of
hydrolysis.
The glutathione S-transferase-dependent metabolism of 2-bromo-2-chloro-1,1difluoroethylene 10 has been described. S-(2-Bromo-2-chloro-1,1-difluoroethyl)N-acetyl-L-cysteine 17 (Figure 4) is present in the urine of human subjects
Figure 3 Base-catalyzed conversion of halothane 9 to

2-bromo-2-chloro-1,1-difluoroethylene 10.
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Figure 4 Glutathione S-transferase- and cysteine conjugate -lyase-dependent

bioactivation of 2-bromo-2-chloro-1,1-difluoroethylene 10. 11, S-(2-bromo-2-chloro1,1-difluoroethyl)glutathione; 12, S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine;
13, 2-bromo-2-chloro-1,1-difluoroethanethiolate; 14, 2-chloro--thiolactone; 15,
2,2-difluoro-3-chlorothiirane; 16, glyoxylic acid; 17, S-(2-bromo-2-chloro-1,1difluoroethyl)-N-acetyl-L-cysteine. GST, glutathione S-transferase; GSH, glutathione;
-GT, -glutamyltransferase; NAT, N-acetyltransferase.
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anesthetized with halothane (40, 42). Its formation can be rationalized by the
addition of glutathione to 2-bromo-2-chloro-1,1-difluoroethylene 10 to give S-(2bromo-2-chloro-1,1-difluoroethyl)glutathione 11, which may undergo -glutamyltransferase- and dipeptidase-catalyzed hydrolysis to give (2-bromo-2-chloro-1,
1-difluoroethyl)-L-cysteine 12 (Figure 4). N-Acetylation would give the observed
mecapturate S-(2-bromo-2-chloro-1,1-difluoroethyl)-N-acetyl-L-cysteine 17.
Cysteine S-conjugate 12 undergoes -lyase-dependent bioactivation: Glyoxylic
acid 16 was identified as a terminal metabolite of S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine 12 (43). This was an unexpected finding, because other
bromine-lacking cysteine S-conjugates afford dihaloacetic acids as terminal products. Detailed mechanistic studies showed that 2-chloro--thiolactone 14 may
be an intermediate in the bioactivation of S-(2-bromo-2-chloro-1,1-difluoroethyl)L-cysteine (Figure 4). Subsequent experiments also showed, however, that
3-chloro-2,2-difluorothiirane 15 is also formed as an intermediate in the -lyasecatalyzed bioactivation of S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine 12
(44) (Figure 4). Hydrolysis of thiirane 15 would give glyoxylic acid 16. Subsequent
computational chemistry studies indicated that a role for 2-chloro--thiolactone 14
is unlikely and that 3-bromo-2,2-difluorothiirane 14 may be more important (45).
The finding that 3-chloro-2,2-difluorothiirane 15 is formed in the biotransformation of cysteine S-conjugate 12 marked the first demonstration of 2,2,
3-trihalothiirane formation, although 2,2,3-trihalothiiranes had earlier been suggested, but not identified, as possible intermediates in the bioactivation of cysteine
S-conjugates (46, 47). 2,2,3-Trihalothiirane formation from cysteine S-conjugates
was later confirmed by Commandeur et al. (48).
Toxicity
The toxicity of 2-bromo-2-chloro-1,1-difluoroethylene and its glutathione and cysteines S-conjugates has been investigated in experimental animals and in vitro
systems.
2-Bromo-2-chloro-1,1-difluoroethylene is nephrotoxic in mice: Its LC50 is approximately 0.025% (v/v) (36). Animals
exposed to 2-bromo-2-chloro-1,1-difluoroethylene show kidney damage characterized by intense renal tubular degeneration. To determine whether the formation
of 2-bromo-2-chloro-1,1-difluoroethylene in the anesthetic circuit might lead to
kidney damage, monkeys were anesthetized with halothane, but no abnormalities
were found on postmortem examination. In dogs anesthetized with halothane, concentrations of 0.00005 to 0.001% (v/v) 2-bromo-2-chloro-1,1-difluoroethylene are
found in the reservoir bag, but no macroscopic or microscopic changes are observed
on postmortem examination.
Detailed studies on the mechanism of 2-bromo-2-chloro-1,1-difluoroethyleneinduced kidney damage have been conducted and were designed to test the hypothesis that 2-bromo-2-chloro-1,1-difluoroethylene 10 undergoes glutathione
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S-transferase- and cysteine conjugate -lyase-dependent bioactivation. S-(2Bromo-2-chloro-1,1-difluoroethyl)glutathione 11 and S-(2-bromo-2-chloro-1,

1-difluoroethyl)-L-cysteine 12, the glutathione and cysteine conjugates of 2-bromo2-chloro-1,1-difluoroethylene 10, are nephrotoxic in Fischer 344 rats (49). The
nephrotoxicity of the S-conjugates is characterized by diuresis and increases in
urine glucose and protein concentrations, in blood urea nitrogen concentrations,
in kidney/body weight percentages, and in serum glutamate-pyruvate transaminase activities. Morphological examination of the kidneys of rats given either
S-conjugate showed severe damage to the proximal tubules. Hepatic lesions were
seen in some rats given the highest concentration studied (500 mol/kg).
Both S-(2-bromo-2-chloro-1,1-difluoroethyl)glutathione and S-(2-bromo-2chloro-1,1-difluoroethyl)-L-cysteine are cytotoxic in cultured LLC-PK1 cells (49).
The cytotoxicity of S-(2-bromo-2-chloro-1,1-difluoroethyl)glutathione is blocked
by the -glutamyltransferase inhibitor acivicin, and the cytotoxicity of both S-(2bromo-2-chloro-1,1-difluoroethyl)glutathione and S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine is inhibited by the -lyase inhibitor (aminooxy)acetic acid.
Also, S-(2-bromo-2-chloro-1,1-difluoroethyl)-DL--methylcysteine, which cannot undergo -lyase-catalyzed bioactivation, is not cytotoxic. These data demonstrate that the observed nephrotoxicity of 2-bromo-2-chloro-1,1-difluoroethylene
is attributable to the formation and bioactivation of S-(2-bromo-2-chloro-1,1difluoroethyl)glutathione and S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine
by the -lyase pathway.
The mutagenicity of 2-bromo-2-chloro-1,1-difluoroethylene has also been
investigated. In studies with the Ames Salmonella auxotroph reversion test, 2bromo-2-chloro-1,1-difluoroethylene induced both base-substitution and frameshift mutations in S. typhimurium strains TA92, TA98, and TA100 (50). With a
transformable strain of Bacillus subtilis, the induction of Spo mutants was observed. These experiments were conducted in the absence of hepatic microsomal
fractions and indicate that 2-bromo-2-chloro-1,1-difluoroethylene may be a directacting mutagen.
Further studies showed that 2-bromo-2-chloro-1,1-difluoroethylene is not mutagenic in the Ames test conducted in liquid culture in the absence or presence of
a microsomal activating system (51). When cells growing in enriched media were
used, 2-bromo-2-chloro-1,1-difluoroethylene induced an increase in revertants,
and the addition of S-9 fractions decreased the number of revertants. 2-Bromo2-chloro-1,1-difluoroethylene purified by preparative gas chromatography is not
mutagenic in the Ames test, whereas an unpurified commercial preparation is
mutagenic (52).
The mercapturate S-(2-bromo-2-chloro-1,1-difluoroethyl)-N-acetyl-L-cysteine
17 is not mutagenic in the Ames test with S. typhimurium strains TA1535 and
TA100 in the absence or presence of S-9 fractions, whereas mercapturate 17 inhibited growth of the recombination repair-deficient strain M45 in the B. subtilis
rec assay (53).
Other studies showed that S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine
12 is mutagenic in the Ames test with S. typhimurium strain TA2638 (54). The
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mutagenicity of S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine is inhibited by

(aminooxy)acetic acid, indicating a role for -lyase, which is present in S. typhimurium (55); furthermore, S-(2-bromo-2-chloro-1,1-difluoroethyl)-DL-methylcysteine, which is not a -lyase substrate, is not mutagenic. The finding that
mercapturate 17 is not mutagenic (see above) may indicate that S. typhimurium
lacks aminoacylase activity that catalyzes the hydrolysis of the mercapturates to
the cysteine S-conjugates.
The formation of 2-bromo-2-chloro-1,1-difluoroethylene under clinical conditions was established by Sharp et al. (39). 2-Bromo-2-chloro1,1-difluoroethylene was detectable in patients connected to a rebreathing circuit
containing soda lime, but not in patients connected to a nonrebreathing Bain circuit. If a semiclosed system was used to deliver 1% halothane at a 5 liter min1
total flow, the concentration of 2-bromo-2-chloro-1,1-difluoroethylene was less
than 1 ppm, whereas it rose to approximately 5 ppm if a completely closed system
was used to deliver 1% halothane at a 0.5 liter min1 total flow. 2-Bromo-2chloro-1,1-difluoroethylene disappeared from the patients breath within minutes
after disconnection from the anesthetic circuit, indicating that it may be rapidly
metabolized.
The study by Sharp et al. was not designed to determine the toxicity of 2bromo-2-chloro-1,1-difluoroethylene in man (39). Even so, the authors suggested
that the presence of up to 5 ppm 2-bromo-2-chloro-1,1-difluoroethylene was a
cause for concern. Although this is much less than the LC50 of 250 ppm in mice,
they pointed out that the lethal concentration and the concentrations that produce
nonlethal tissue damage in man are unknown.
Additional studies on the toxicity of 2-bromo-2-chloro-1,1-difluoroethylene in
man have apparently not been reported and, thus, any implication that toxic concentrations may be achieved during halothane anesthesia remains speculative. Indeed,
other than the rare cases of massive liver damage seen postoperatively, halothane is
a remarkably nontoxic drug and, in particular, does not cause nephrotoxicity, a characteristic feature of 2-bromo-2-chloro-1,1-difluoroethylene toxicity in animals.
HUMAN TOXICITY
2-(FLUOROMETHOXY)-1,1,3,3,3-PENTAFLUORO-1-PROPENE
(COMPOUND A) FORMATION FROM SEVOFLURANE
[FLUOROMETHYL 1-(TRIFLUOROMETHYL)2,2,2-TRIFLUOROETHYL ETHER]
Introduction
Sevoflurane is a fluorinated volatile anesthetic agent that is approved for use
in over 40 countries, including the United States. Its low blood-gas partition
coefficient allows rapid induction and awakening (56). In addition, sevoflurane
is nonirritating to the airways and is, therefore, useful for inhaled induction.
Although sevoflurane underwent clinical trials in the United States in the 1970s and
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was considered an excellent anesthetic, two concerns about its potential toxicity
have been raised: First, sevoflurane undergoes metabolism to inorganic fluoride,
which has the potential to induce nephrotoxicity. Second, sevoflurane undergoes
Baralyme- and soda lime-dependent degradation to the fluoroalkene Compound
A, which is nephrotoxic in rats.

Formation and Fate of Compound A from Sevoflurane

Sevoflurane 18 undergoes a base-catalyzed dehydrofluorination reaction in the
anesthetic circuit to form Compound A 19 (Figure 5). The degradation of sevoflurane to Compound A is catalyzed by the bases (NaOH, KOH) present in soda
lime and Baralyme (5759). Compound A and 1-(methoxy)-2-(fluoromethoxy)1,1,3,3,3-pentafluoropropane (Compound B) are the major degradation products
of sevoflurane that are formed by reaction of sevoflurane with soda lime (60).
The concentrations of Compound A found in anesthesia circuits are usually less
than 20 ppm, although higher concentrations have been reported (6066). Compound A concentrations are higher when Baralyme rather than soda lime is used
and when dry rather than wet absorbents are used (61, 64, 65). Compound A
formation is also greater at low (0.5 to 1 liter) fresh gas flows than at higher
(2 to 6 liters) fresh gas flows, perhaps because of the higher canister temperatures generated at low flow rates (64, 67). 1-Methoxy-2-(fluoromethoxy)-1,1,3,3,3pentafluoropropane (Compound B) is also formed in anesthesia circuits, but its
concentration is considered to be too low to be of toxicological concern: No toxicity was observed in rats exposed for 3 h to 2400 ppm Compound B (60, 62).
1-Methoxy-2-(fluoromethoxy)-1,1,3,3-tetrafluoro-2-propene (Compound C) and
(E)- and (Z)-1-methoxy-2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propene (Compounds D and E) are also formed as degradation products of sevoflurane (59, 68),
but no information about their fate or toxicity is apparently available.
The biotransformation of Compound A has been studied in human hepatic
microsomal fractions (69). Human cytochrome P450 2E1 catalyzes the defluorination of Compound A, although significant NADPH-independent defluorination
of Compound A is also observed. The enzymatic defluorination of Compound A
was significantly inhibited by sevoflurane, which is also a substrate for cytochrome
P450 2E1 (70).
Figure 5 Base-catalyzed conversion of sevoflurane

18 to 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene
(Compound A) 19.
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Compound A also undergoes glutathione-dependent metabolism. In rats given

Compound A intraperitoneally, diastereomeric S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]glutathione 20 and (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]glutathione 21 (Figure 6) are excreted in the bile, and
the corresponding mercapturates, diastereomeric S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]-N-acetyl-L-cysteine 26 and (E)- and (Z)-S-[2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine 27 (Figure 7), respectively, are
excreted in the urine (71, 72). Moreover, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid 24, the expected product of the -lyase-catalyzed metabolism of both cysteine S-conjugates 22 and 23, is excreted in the urine of rats given
Compound A 19 (73). These findings show that Compound A is metabolized by the
-lyase pathway. Cysteine S-conjugates 22 and 23 undergo -lyase-catalyzed biotransformation in rat, human, and nonhuman primate renal cytosol and mitochondria, and -lyase activity is lower in human kidney tissue than in rat or nonhuman
primate kidney tissue (32). Also, cysteine S-conjugate 22 is biotransformed by rat
renal cytosol to pyruvate, fluoride, and 2-fluoromethoxy-3,3,3-trifluoropropanoic
acid 24, which undergoes degradation to 3,3,3-trifluorolactic acid 25 (Figure 6)
(74). In addition, although cysteine S-conjugate 23 is a substrate for -lyase, it
also undergoes a rapid (t1/2 5 min) intramolecular cyclization reaction to give
the thiazole 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazole4-carboxylic acid, which, because it lacks a free amino group, cannot serve as
a -lyase substrate (74). Hence, these data show that Compound A undergoes
-lyase-dependent metabolism.
Studies designed to quantify relative metabolite excretion (mercapturates 26
and 27 versus 2-fluoromethoxy-3,3,3-trifluoropropanoic acid 24) in rats given
Compound A showed that the formation and excretion of 2-(fluoromethoxy)3,3,3-trifluoropropanoic acid 24 is greater than the formation and excretion of
mercapturates 26 and 27, again demonstrating the predominance of the -lyase
pathway in the metabolism of Compound A (75).
Compound Aderived mercapturates 26 and 27 (Figure 7) undergo little metabolism in rats (76). When [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine and [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine were given to rats, approximately 15% and
5%, respectively, were excreted as the unlabeled compounds, indicating minimal
hydrolysis and acetylation of the released cysteine S-conjugates. The observed
hydrolysis of Compound Aderived mercapturates is catalyzed by human and rat
kidney cytosol and by acylases I and III. Mercapturate 26, but not mercapturate
27, was mildly nephrotoxic in rats, indicating hydrolysis and bioactivation by the
-lyase pathway.
The metabolism of Compound A formed from sevoflurane in the anesthetic
circuit of human subjects anesthetized with sevoflurane has also been studied (77).
The human subjects were anesthetized with sevoflurane (1.25 minimum alveolar
concentration, 3%, 2 liter min1, 8 h), and urine was collected for 72 h after anesthesia. Analysis of the urine samples by 19F NMR spectroscopy and GC-MS showed
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Figure 6 Glutathione S-transferase- and cysteine conjugate -lyase-dependent bioactivation of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A) 19.
20, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione; 21, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione; 22, S-[2-(fluoromethoxy)-1,1,
3,3,3-pentafluoropropyl]-L-cysteine; 23, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1propenyl]-L-cysteine; 24, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; 25,
trifluorolactic acid. GST, glutathione S-transferase; GSH, glutathione; -GT, glutamyltransferase; -lyase, cysteine conjugate -lyase.
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Figure 7 N-Acetylation and sulfoxidation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine 22 and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine 23. 26, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetylL-cysteine; 27, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine; 28, S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine
sulfoxide; 29, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-Lcysteine. NAT, N-acetyltransferase; P450, cytochrome P450.
the presence of the Compound A metabolites S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]-N-acetyl-L-cysteine 26, (E)- and (Z)-S-[2-(fluoromethoxy)-1,
3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine 27, 2-(fluoromethoxy)-3,3,3trifluoropropanoic acid 24, 3,3,3-trifluorolactic acid 25, and inorganic fluoride,
indicating metabolism of Compound A by the -lyase pathway. Similar results
were found in human subjects anesthetized with sevoflurane under conditions
designed to maximize Compound A formation (75). The inspired Compound A
concentrations were 29 14 ppm (range 1067 ppm). Mercapturates 26 and
27 were identified along with 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid
24.
In vitro studies on the N-acetylation, N-deacetylation, and -lyase-catalyzed biotransformation of Compound Aderived cysteine S-conjugates and
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mercapturates by human kidney microsomes and cytosol showed significant interindividual variability (78). In human kidney cytosol, the rates of N-acetylation
of cysteine S-conjugates 22 and 23 were 0.024 0.01 (range 0.0080.045) nmol
mercapturate mg1 min1 and 0.024 0.02 (range 0.0010.07) nmol mercapturate mg1 min1, respectively. Similar results were obtained in human kidney
microsomes: The rates of N-acetylation of cysteine S-conjugates 22 and 23 were
0.025 0.02 (range 0.0050.055) nmol mercapturate mg1 min1 and 0.030
0.02 (range 0.0010.06) nmol mercapturate mg1 min1, respectively. The lyase-catalyzed biotransformation of cysteine S-conjugates 22 and 23 amounted
to 0.051 0.04 (range 0.0040.14) nmol pyruvate mg1 min1 and 0.26 0.08
(range 0.100.40) nmol pyruvate mg1 min1, respectively. The rates of hydrolysis of the mercapturates 26 and 27 were 1.25 0.57 (range 0.82.5) nmol mg1
min1 and 0.17 0.10 (range 0.050.37) nmol mg1 min1, respectively. These
data show that rates of -lyase-catalyzed bioactivation of Compound Aderived
cysteine S-conjugates in human kidney tissue were greater than the rates of Nacetylation of the cysteine S-conjugates and that the rates of N-deacetylation of
Compound Aderived mercapturates were greater than the rates of N-acetylation
of Compound Aderived cysteine S-conjugates. Hence, rates of bioactivation (lyase and N-deacetylation) of cysteine S-conjugates of Compound A exceed rates
of detoxication (N-acetylation) in human kidney tissue.
Recent studies also show that Compound Aderived cysteine S-conjugates and
mercapturates undergo biotransformation to the corresponding sulfoxides (79).
The sulfoxidation of (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-Nacetyl-L-cysteine 26 and S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-Nacetyl-L-cysteine 27 to give sulfoxides 28 and 29, respectively (Figure 7), is
catalyzed by rat liver microsomal fractions; little sulfoxidation is observed in
renal microsomal fractions. In contrast to the mercapturates, S-[2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl]-L-cysteine 22 underwent nonenzymatic sulfoxidation. Although both cytochromes P450 and flavin-containing monoxygenases
catalyze sulfoxidation reactions, P450 3A isoforms are the major enzymes responsible for the sulfoxidation of Compound Aderived mercapturates. Finally,
the sulfoxidation of mercapturates 26 and 27 is significantly greater in rat than in
human liver microsomes.
Toxicity
The toxicity of Compound A and its glutathione and cysteine S-conjugates has
been investigated in experimental animals and in vitro systems.
Compound A is nephrotoxic in Wistar
rats exposed by inhalation for 1 h to 700 to 1400 ppm Compound A or for 3 h to 110
to 460 ppm Compound A (60). The LC50s for 1-h exposures of male and female
rats are 1090 ppm and 1050, respectively, and, for 3-h exposures, 420 ppm and 400
ppm, respectively. The toxicity of Compound A is characterized by renal tubular
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necrosis, increased urine glucose and protein concentrations, and increased blood
urea nitrogen concentrations. Lung congestion and hyperemia are also observed,
but hepatotoxicity was not reported.
A more detailed investigation of the toxicity of Compound A reported a LC50 of
331 ppm for a 3-h exposure in Wistar rats (80). Compound Ainduced nephrotoxicity is characterized by corticomedullary necrosis; significant evidence of corticomedullary necrosis was not seen in 10 rats exposed to 50 ppm Compound A, but
was seen in rats exposed to concentrations of Compound A greater than 100 ppm.
Liver and brain injury, but not lung injury, were observed in some animals. The
effect of exposure time on Compound Ainduced toxicity has also been studied
(81). The LC50s in Wistar rats exposed to Compound A for 6 or 12 h were 203 or
127 ppm, respectively. As in other studies, the nephrotoxicity is characterized by
corticomedullary necrosis. Proliferating cell nuclear antigen, which is an indicator
of cell proliferation, increased with increasing exposure concentration. Lung injury
was seen only at near-lethal concentrations of Compound A. Additional studies on
Compound Ainduced toxicity showed a threshold for nephrotoxicity, as measured
by histopathological examination, of 150 to 200 ppm for a 1-h exposure (82).
The toxicity of Compound A was studied in Sprague-Dawley rats exposed by
nose-only inhalation to 0, 30, 61, 114, or 202 ppm Compound A (83). Increases in
blood urea nitrogen and creatinine concentrations are observed in male and female
rats exposed to 202 ppm Compound A, and renal tubular necrosis is observed in
rats exposed to 114 or 202 ppm Compound A.
The mechanism of Compound Ainduced nephrotoxicity has not been fully resolved, but most evidence implicates the -lyase pathway. A range of 1,
1-difluoroalkenes undergo -lyase-dependent bioactivation, including 2-bromo2-chloro-1,1-difluoroethylene (49, 54), bromotrifluoroethylene (54), chlorotrifluoroethylene (84), 1,1-dichloro-2,2-difluoroethylene (54), hexafluoropropene (85),
and tetrafluoroethylene (86).
Evidence for a role for the -lyase pathway in Compound Ainduced nephrotoxicity has been presented: (Aminooxy)acetic acid, a -lyase inhibitor (12), partially blocks Compound Ainduced nephrotoxicity and reduces the excretion of
2-fluoromethoxy-3,3,3-trifluoropropanoic acid 24 in rats given Compound A (71,
73). Also, the Compound Aderived cysteine S-conjugates S-[2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl]-L-cysteine 22 and S-[2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]-L-cysteine 23 are substrates for rat, human, and nonhuman primate renal -lyase (32). Finally, the Compound Aderived glutathione
S-conjugates S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione 20
and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione 21 and cysteine S-conjugate S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine
22 and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine 23 are
nephrotoxic in rats in vivo, and their nephrotoxicity is partially blocked by (aminooxy) acetic acid (87).
Martin et al. purported to show that the -lyase pathway is not involved in
the mechanism of Compound Ainduced nephrotoxicity (88, 89). These workers
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reported that the -lyase inhibitor (aminooxy)acetic acid and the -glutamyltransferase inhibitor acivicin either failed to block or increased Compound Ainduced
nephrotoxicity. The interpretation of these studies is limited, however, by the experimental design: In one study (88), only one exposure concentration (150 ppm)
and one exposure time (3 h) were used. In a second study (89), the concentrations
of Compound A used (600 and 800 ppm for 1 h) are greater than one half of
the observed LC50 of Compound A in Wistar rats (60). Moreover, in both studies
nephrotoxicity was assessed only by histopathological studies; no clinical chemical parameters were reported. Subsequent studies showed that (aminooxy)acetic
acid and acivicin also potentiate the nephrotoxicity of Compound Aderived glutathione and cysteine S-conjugates (90). The observation that acivicin potentiates
the toxicity of Compound A has been confirmed by others (91, 92). The mechanism
by which acivicin may increase the nephrotoxicity of Compound A is not apparent.
Acivicin also increases the nephrotoxicity of hexachlorobutadiene in rats (93), but
blocks hexachlorobutadiene-induced nephrotoxicity in mice (94); hexachlorobutadiene undergoes -lyase-dependent bioactivation in rats (95). Lantum et al. (96)
tested the hypothesis that acivicin may reduce renal glutathione concentrations and,
thereby, render the kidney susceptible to injury. It was found, however, that acivicin
significantly increases renal glutathione concentrations. Finally, probenecid blocks
the nephrotoxicity of Compound A, perhaps by preventing the renal uptake of glutathione and cysteine S-conjugates of Compound A (91, 92). Additional studies
are needed to clarify fully the mechanism of Compound Ainduced nephrotoxicity
in rats and, particularly, the effects of acivicin. Nevertheless the weight of the evidence supports a role for the -lyase pathway in the nephrotoxicity of Compound
A in rats.
The cytotoxicity of Compound A and several of its metabolites has been studied
in human-kidney-derived HD-2 cells (97). Compound A was cytotoxic only at concentrations greater than 0.9 mM, which is higher than would be achieved during the
clinical use of sevoflurane. Glutathione S-conjugates 20 and 21 of Compound A
were not cytotoxic in HK-2 cells. The Compound Aderived cysteine S-conjugates
22 and 23 were cytotoxic in HK-2 cells, but were much less cytotoxic than
S-(1,2-dichlorovinyl)-L-cysteine 4. The mercapturate (Z)-S-[2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine 27 was cytotoxic at the highest
concentration tested (2.7 mM), but S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L-cysteine 26 was not cytotoxic.
The Compound Aderived sulfoxides 28 and 29 (Figure 7) are also cytotoxic in HK-2 cells (97); indeed, these sulfoxides are more cytotoxic than the
corresponding cysteine S-conjugates and mercapturic acids. Several sulfoxides
of haloalkene-derived cysteine S-conjugates or mercapturates are nephrotoxic in
vivo or cytotoxic in vitro, or both, including S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (98), S-(1,2-dichlorovinyl)-N-acetyl-L-cysteine sulfoxide (99), S-(1,2,3,4,
4-pentachlorobutadienyl)-N-acetyl-L-cysteine sulfoxide (100), S-(trichlorovinyl)N-acetyl-L-cysteine sulfoxide (99), and (cis-3-chloro-2-propenyl)-N-acetyl-L-cysteine and (trans-3-chloro-2-propenyl)-N-acetyl-L-cysteine (101). These findings
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raise the question of whether sulfoxidation of cysteine S-conjugates or mercapturates is also a bioactivation pathway in addition to the -lyase pathway. As
expected, the nephrotoxicity of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, S-(1,
2-dichlorovinyl)-N-acetyl-L-cysteine sulfoxide, and S-(trichlorovinyl)-N-acetyl-Lcysteine sulfoxide is not blocked by the -lyase inhibitor (aminooxy)acetic acid
(98). Moreover, S-(1,2-dichlorovinyl)-L-cysteine sulfoxide is more cytotoxic in rat
renal distal tubular cells than in proximal tubular cells, but S-(1,2-dichlorovinyl)L-cysteine-induced nephrotoxicity is characterized by necrosis of renal proximal tubular cells rather than distal tubular cells (102). Finally, the -methyl
analogs of S-(1,2-dichlorovinyl)-L-cysteine and S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]-L-cysteine are not nephrotoxic in rats (12, 87). These compounds cannot undergo -lyase-catalyzed bioactivation but could presumably
undergo sulfoxidation. Hence, studies on the sulfoxidation of -methyl analogs of
cysteine S-conjugates and the toxicity of the sulfoxides are needed to resolve this
point.
Mutagenicity studies showed that Compound A does not induce reverse mutations, either in the absence or presence of a S-9 activating system, with S. typhimurium strains TA98, TA100, TA1535, and TA1537 or with Escherichia coli
strain WP2uvrA (60). Also, Compound A did not induce chromosome aberrations
or increase the number of micronuclei in bone-marrow cells (60).
The human toxicity of Compound A has been reviewed (103,
104). As discussed above, Compound A is produced when sevoflurane is delivered through absorbent-containing circuits. Concern has been expressed that
sevoflurane-derived Compound A may place patients at increased risk for
Compound Ainduced kidney damage. First, the relatively low safety factor of
Compound A concentrations produced in anesthetic circuits compared with concentrations that produce toxicity in rats is a potential concern. The safety factor is
hard to assess but is estimated to be in the range of 2 to 8, which is far less than
the safety factor of 10 to 100 that is commonly accepted by many toxicologists
as providing an adequate margin of safety (105). Second, evidence of renal injury
in human volunteers anesthetized with sevoflurane has been reported (106, 107).
Other workers have, however, failed to find evidence of renal injury or have observed mild and transient evidence of renal injury in human subjects anesthetized
with sevoflurane compared with reference anesthetics (see, for example, 62, 63,
108113). Also, a retrospective evaluation of the effect of sevoflurane on renal
function in adult surgical patients (1941 patients received sevoflurane and 1495
received control agents) found no evidence that sevoflurane administration is associated with renal injury (114).
There are well-documented scientific reasons why the nephrotoxicity of Compound A in man would be expected to be less than that in rats: If -lyase-dependent
bioactivation is the basis of the observed nephrotoxicity of Compound A, human
kidney tissue has much lower -lyase activities than does rat kidney (3032, 115).
Allometric scaling, which does not account for metabolic differences, indicates
HUMAN TOXICITY
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that the human threshold for Compound Ainduced nephrotoxicity would be approximately 9000 ppm h1, which is approximately 20 times the highest reported
human exposure (113). To date, it is estimated that more than 120 million human
subjects worldwide have been anesthetized with sevoflurane and no established
cases of Compound Aassociated renal damage have been reported (personal communication, R.D. Ostroff, Abbott Laboratories, Abbott Park, IL).
CARBON MONOXIDE FORMATION FROM DESFLURANE,

ISOFLURANE, AND ENFLURANE
Introduction
Although the toxicity of carbon monoxide is well established, significant intraoperative exposure of patients to carbon monoxide has been thought to be unlikely.
Thus, the reports by Moon et al. that patients anesthetized with isoflurane or enflurane had elevated blood carboxyhemoglobin (COHb) saturations were unexpected
(116, 117). As discussed below, it was later found that the source of the carbon
monoxide was a reaction between anesthetics bearing a -CHF2 moiety, e.g., isoflurane and enflurane, and the carbon dioxide absorbent (118).
The toxicity and biological effects of carbon monoxide have been summarized,
as has the issue of xenobiotic-derived carbon monoxide toxicity (119, 120). Because the carbon monoxide toxicity is well understood, this section focuses on the
mechanism and conditions of the formation of carbon monoxide from anesthetic
agents.
Formation and Fate of Carbon Monoxide

The observation of elevated COHb saturations in anesthetized patients led to an
attempt to determine the causes and conditions under which carbon monoxide is
produced in anesthesia circuits. Moon et al. showed increases in carbon monoxide
concentrations if enflurane or isoflurane was allowed to stand in cartridges of soda
lime (116). In most samples, the carbon monoxide concentrations were less than
20 ppm, which is well below the concentrations that had been observed under clinical conditions. In some (5%) samples, however, carbon monoxide concentrations
greater than 100 ppm were found, and the concentrations exceeded 1000 ppm in
a few of the samples.
Investigation of the degradation of several anesthetics in soda lime and Baralyme showed that significant amounts of carbon monoxide were generated from
desflurane, enflurane, and isoflurane, which contain the -CHF2 moiety, whereas
insignificant amounts were generated from halothane and sevoflurane, which lack
the -CHF2 group (121). Carbon monoxide production is dependent on the dryness
and type of absorbent: Carbon monoxide formation increases as the absorbent
water content decreases (122), and generation of carbon monoxide is greater with
barium hydroxide lime than with soda lime (121). The use of absorbents that lack
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strong bases, such as Amsorb, prevents the formation of carbon monoxide (and
the formation of Compound A from sevoflurane) (123).
The mechanisms by which desflurane and isoflurane are converted to carbon
monoxide have been elucidated (124). Their studies, based on well-established carbene and elimination chemistry, describe a mechanism for the formation of carbon
monoxide from anesthetics bearing a -CHF2 moiety. Base-catalyzed abstraction of
a proton from desflurane 30a or isoflurane 30b (Figure 8) affords the intermediate
carbanions 31a,b. Elimination of halide from the intermediate carbanions gives
difluorocarbene 32 and trifluoroacetaldehyde 34. Hydrolysis of carbene 32 affords
carbon monoxide 33. The hydrolysis of methylene carbenes to carbon monoxide is
known (125). Difluorocarbene 32 was trapped by reaction with -methylstyrene to
give 1,1-difluoro2-methyl-2-phenylcyclopropane, thereby establishing its formation as an intermediate. Moreover, the oxygen atom in carbon monoxide is derived
from water: When [18O]H2O was incorporated into barium hydroxide, [18O]carbon
monoxide was formed. Deuterium substitution in the -CHF2 group of desflurane
and isoflurane resulted in decreased carbon monoxide formation. Although mechanistic studies on the formation of carbon monoxide from enflurane have apparently
not been reported, a pathway similar to that described for desflurane and isoflurane
can be envisioned.
The biological fate of carbon monoxide is largely determined by its elimination
via the respiratory tract, although a small fraction of the body burden of carbon
monoxide is oxidized to carbon dioxide (126, 127).
Toxicity
The toxicity of carbon monoxide has been thoroughly investigated in animal studies and in cases of accidental or intentional human exposure; hence, only brief
comments about human carbon monoxide toxicity are warranted.
Moon et al. reported 28 cases of unexplained elevations of
COHb saturations during anesthesia (116, 117). Eight cases had COHb saturations
greater than 27%, and three cases had saturations of 30% or greater. To determine
the source of the carbon monoxide, gas samples were collected from inside the
soda lime canisters in idle anesthesia machines on 320 occasions. Carbon monoxide
concentrations were less than 20 ppm in 271 of the samples, between 100 and 1000
ppm in 10, and greater than 1000 ppm in 6.
The clinical significance of exposure to carbon monoxide concentrations in this
range and the physical properties and toxicity of carbon monoxide are well known.
The Haldane equation describes quantitatively the competition between oxygen
and carbon monoxide for the same ferrous heme binding sites on hemoglobin:
HUMAN TOXICITY
[Hb(CO)4 ]
[Pco ]
= 245
.
[Hb(O2 )4 ]
[Po2 ]
The constant, 245 at pH 7.4, indicates that if Pco = 1/245PO2 , then blood
will be half-saturated with oxygen and half with carbon monoxide at equilibrium.
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Figure 8 Base-catalyzed conversion of desflurane 30a and isoflurane 30b to carbon

monoxide. 31a,b, intermediate carbanions; 32, difluorocarbene; 33, carbon monoxide;
34, trifluoroacetaldehyde; 35, trifluoromethane; 36, formic acid.
For a human breathing room air containing only approximately 0.085% (850 ppm)
carbon monoxide at sea level, the COHb saturation will be 50% at equilibrium. This
relationship accounts for the dangerous toxicity of low concentrations of carbon
monoxide. The toxicological effects of carbon monoxide depend on a range of
factors, including preexisting anemia and cardiovascular disease. In general, COHb
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saturations between 30% and 50% result in tachycardia, hypoxic ECG changes,
headache, weakness, nausea, dizziness, and failing vision. Saturations between
50% and 80% result in coma, convulsions, and death.

CONCLUSIONS
The degradation of anesthetic agents to toxic products has been associated with
both old and modern agents. The common element that leads to the formation of
degradation products is the bases in carbon dioxide absorbents in the anesthetic
circuit. With some agents, e.g., sevoflurane, the degradation of an anesthetic to a
toxic product was discovered during manufacture or early in its clinical use. With
other agents, e.g., desflurane, isoflurane, and enflurane, however, the degradation
products were discovered after many years of clinical use. The elucidation of the
mechanisms of anesthetic degradation also provides a means for minimizing the
formation of toxic degradation products by use of absorbents that lack strong bases,
e.g., Amsorb, rather than Baralyme or soda lime.
ACKNOWLEDGMENTS
Research in the authors laboratory was supported by Abbott Laboratories and
by the National Institute of Environmental Health Sciences grant ES03127. The
author has served as a consultant to Abbott Laboratories.
LITERATURE CITED
1. Striker C, Goldblatt S, Warm IS, Jackson
DE. 1935. Clinical experiences with the
use of trichloroethylene in the production
of over 300 analgesias and anesthesias.
Curr. Res. Anesth. 14:6871
2. Hewer CL. 1943. Further observations
on trichloroethylene. Proc. R. Soc. Med.
36:46365
3. McAuley J. 1943. Trichloroethylene and
trigeminal anaesthesia. Br. Med. J. 2:713
14
4. Carden S. 1944. Hazards in the use of the
closed-circuit technique for trilene anaesthesia. Br. Med. J. 1:31920
5. Firth JB, Stuckey RE. 1945. Decomposition of trilene in closed circuit anaesthesia.
Lancet 1:81416
6. Ott E, Ottemeyer W, Packendorff K. 1930.
Uber
das Dichlor-acetylen. Chem. Ber.
63:194144
7. Reichert D, Metzler M, Henschler D.
1980. Decomposition of the neuro- and
nephrotoxic compound dichloroacetylene
in the presence of oxygen: separation and identification of novel products. J. Environ. Pathol. Toxicol. 3:525
32
8. Kanhai W, Koob M, Dekant W, Henschler D. 1991. Metabolism of 14C-dichloroethyne in rats. Xenobiotica 21:905
16
9. Dekant W, Vamvakas S, Anders MW.
1994. Formation and fate of nephrotoxic
and cytotoxic glutathione S-conjugates:
cysteine conjugate -lyase pathway. Adv.
Pharmacol. 27:11562
3 Dec 2004
20:38

170
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE
ANDERS
10. Anders MW, Dekant W. 1998. Glutathione-dependent bioactivation of haloalkenes. Annu. Rev. Pharmacol. Toxicol.
38:50137
11. Kanhai W, Dekant W, Henschler D.
1989. Metabolism of the nephrotoxin
dichloroacetylene by glutathione conjugation. Chem. Res. Toxicol. 2:5156
12. Elfarra AA, Jakobson I, Anders MW.
1986. Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity. Biochem. Pharmacol. 35:28388
13. Volkel W, Dekant W. 1998. Chlorothioketene, the ultimate reactive intermediate formed by cysteine conjugate
-lyase-mediated cleavage of the trichloroethene metabolite S-(1,2-Dichlorovinyl)-L-cysteine, forms cytosine adducts
in organic solvents, but not in aqueous solution. Chem. Res. Toxicol. 11:108288
14. Zhang T-L, Wang L, Hashmi M, Anders MW, Thorpe C, Ridge DP. 1995.
Fourier-transform ion cyclotron resonance mass spectrometric evidence for the
formation of -chloroethenethiolates and
thioketenes from chloroalkene-derived,
cytotoxic 4-thiaalkanoates. Chem. Res.
Toxicol. 8:90710
15. Reichert D, Ewald D, Henschler D. 1975.
Generation and inhalation toxicity of
dichloroacetylene. Food Cosmet. Toxicol.
13:51115
16. Reichert D, Liebaldt G, Henschler D.
1976. Neurotoxic effects of dichloroacetylene. Arch. Toxicol. 37:2338
17. Reichert D, Henschler D. 1978. Nephrotoxic and hepatotoxic effects of dichloroacetylene. Food Cosmet. Toxicol. 16:
22735
18. Reichert D, Spengler U, Romen W,
Henschler D. 1984. Carcinogenicity of
dichloroacetylene: an inhalation study.
19. Patel NJ, Fullone JS, Anders MW.
1993. Brain uptake of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione and
cysteine S-conjugates of the neuro-
20.
21.
22.
23.
24.
25.
26.
27.
toxin dichloroacetylene. Mol. Brain Res.

17:5358
Patel N, Birner G, Dekant W, Anders MW.
1994. Glutathione-dependent biosynthesis and bioactivation of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione
and cysteine S-conjugates of dichloroacetylene, in rat tissues and subcellular
fractions. Drug Metab. Dispos. 22:
14347
Lash LH, Anders MW. 1986. Cytotoxicity of S-(1,2-dichlorovinyl)glutathione
and S-(1,2-dichlorovinyl)-L-cysteine in
isolated rat kidney cells. J. Biol. Chem.
261:1307681
Lash LH, Anders MW. 1987. Mechanism
of S-(1,2-dichlorovinyl)-L-cysteine- and
S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity. Mol.
Pharmacol. 32:54956
Stevens J, Hayden P, Taylor G. 1986. The
role of glutathione conjugate metabolism
and cysteine conjugate -lyase in the
mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells. J. Biol. Chem. 261:
332532
Reichert D, Neudecker T, Spengler U,
Henschler D. 1983. Mutagenicity of
dichloroacetylene and its degradation
products trichloroacetyl chloride, trichloroacryloyl chloride, and hexachlorobutadiene. Mutat. Res. 117:2129
Vamvakas S, Elfarra AA, Dekant W,
Henschler D, Anders MW. 1988. Mutagenicity of amino acid and glutathione Sconjugates in the Ames test. Mutat. Res.
206:8390
Vamvakas S, Dekant W, Schiffmann
D, Henschler D. 1988. Induction of
unscheduled DNA synthesis and micronucleus formation in Syrian hamster
embryo fibroblasts treated with cysteine
S-conjugates of chlorinated hydrocarbons. Cell Biol. Toxicol. 4:393403
Vamvakas S, Koster U. 1993. The nephrotoxin dichlorovinylcysteine induces expression of the protooncogenes c-fos and
3 Dec 2004
20:38
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE

28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
c-myc in LLC-PK1 cellsa comparative

investigation with growth factors and 12O-tetradecanoylphorbolacetate. Cell Biol.
Toxicol. 9:113
Greim H, Wolff T, Hofler M, Lahaniatis E. 1984. Formation of dichloroacetylene from trichloroethylene in the presence of alkaline materialpossible cause
of intoxication after abundant use of
chloroethylene-containing solvents. Arch.
Toxicol. 56:7477
Humphrey JH, McClelland M. 1944.
Cranial-nerve palsies with herpes following general anaesthesia. Br. Med. J. 1:315
18
Lash LH, Nelson RM, Van Dyke RA, Anders MW. 1990. Purification and characterization of human kidney cytosolic
conjugate -lyase activity. Drug Metab.
Dispos. 18:5054
McCarthy RI, Lock EA, Hawksworth
GM. 1994. Cytosolic C-S lyase activity
in human kidney samplesrelevance for
the nephrotoxicity of halogenated alkenes
in man. Toxicol. Indust. Health 10:103
12
Iyer RA, Anders MW. 1996. Cysteine
conjugate -lyase-dependent biotransformation of the cysteine S-conjugates of
the sevoflurane degradation product Compound A in human, nonhuman primate,
and rat renal cytosol and mitochondria.
Anesthesiology 85:145461
Raventos J. 1956. The action of
fluothanea new volatile anesthetic. Br.
J. Pharmacol. 11:394409
Pohl LR, Kenna JG, Satoh H, Christ D,
Martin JL. 1989. Neoantigens associated
with halothane hepatitis. Drug Metab.
Rev. 20:20317
Pohl LR. 1990. Drug-induced allergic
hepatitis. Semin. Liver Dis. 10:30515
Raventos J, Lemon PG. 1965. The impurities in Fluothane: their biological properties. Brit. J. Anaesth. 37:71637
Chapman J, Hill R, Muir J, Suckling CW,
Viney DJ. 1967. Impurities in halothane:
their identities, concentrations and deter-
38.
39.
40.
41.
42.
43.
44.
45.
46.
171
mination. J. Pharm. Pharmacol. 19:231

39
Kodama G. 1973. Halothane impurities
and decomposition of halothane by sodalime. II. Analysis of halothane impurities.
Hiroshima J. Anesth. 9:7589
Sharp JH, Trudell JR, Cohen EN. 1979.
Volatile metabolites and decomposition
products of halothane in man. Anesthesiology 50:28
Cohen EN, Trudell JR, Edmunds HN,
Watson E. 1975. Urinary metabolites
of halothane in man. Anesthesiology
43:392401
Denson DD, Ford DJ. 1979. How reactive are the reductive metabolites of
halothane? Anesthesiology 51:S243
Wark H, Earl J, Chau DD, Overton J,
Cheung HTA. 1990. A urinary cysteinehalothane metabolite: validation and measurement in children. Br. J. Anaesth.
64:46973
Finkelstein MB, Dekant W, Kende AS,
Anders MW. 1995. -Thiolactones as
novel intermediates in the cysteine
conjugate -lyase-catalyzed bioactivation of bromine-containing cysteine Sconjugates. J. Am. Chem. Soc. 117:9590
91
Finkelstein MB, Dekant W, Anders
MW. 1996. Cysteine conjugate -lyasecatalyzed bioactivation of brominecontaining cysteine S-conjugates: stoichiometry and formation of 2,2-difluoro3-halothiiranes. Chem. Res. Toxicol. 9:
22731
Shim J-Y, Richard AM. 1997. Theoretical evaluation of two plausible routes
for bioactivation of S-(1,1-difluoro-2,2dihaloethyl)-L-cysteine conjugates: thiirane vs thionoacyl fluoride pathway.
Dohn DR, Quebbemann AJ, Borch RF,
Anders MW. 1985. Enzymatic reaction
of chlorotrifluoroethene with glutathione:
19
F NMR evidence for stereochemical
control of the reaction. Biochemistry 24:
513743
3 Dec 2004
20:38

172
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE
ANDERS
47. Commandeur JNM, Brakenhoff JPG,

De Kanter FJJ, Vermeulen NPE. 1988.
Nephrotoxicity of mercapturic acids of
three structurally related 2,2-difluoroethylenes in the rat. Biochem. Pharmacol.
37:4495504
48. Commandeur JNM, King LJ, Koymans
L, Vermeulen NPE. 1996. Bioactivation
of
S-(2,2-dihalo-1,1-difluoroethyl)-Lcysteines and S-(trihalovinyl)-L-cysteines
by cysteine S-conjugate -lyase: indications for formation of both thionoacylating species and thiiranes as reactive
intermediates. Chem. Res. Toxicol. 9:
1092102
49. Finkelstein MB, Baggs RB, Anders MW.
1992. Nephrotoxicity of the glutathione
and cysteine conjugates of 2-bromo-2chloro-1,1-difluoroethene. J. Pharmacol.
Exp. Ther. 261:124852
50. Garro AJ, Phillips RA. 1978. Mutagenicity of the halogenated olefin, 2-bromo-2chloro-1,1-difluoroethylene, a presumed
metabolite of the inhalation anesthetic
halothane. Mutat. Res. 54:1722
51. Edmunds HN, Baden JM, Simmon VF.
1979. Mutagenicity studies with volatile
metabolites of halothane. Anesthesiology
51(5):42429
52. Waskell L. 1979. Lack of mutagenicity
of two possible metabolites of halothane.
53. Sachdev K, Cohen EN, Simmon VF.
1980. Genotoxic and mutagenic assays of
halothane metabolites in Bacillus subtilis
and Salmonella typhimurium. Anesthesiology 53:3139
54. Finkelstein MB, Vamvakas S, Bittner
D, Anders MW. 1994. Structure-mutagenicity and structure-cytotoxicity studies on bromine-containing cysteine Sconjugates and related compounds. Chem.
55. Dekant W, Vamvakas S, Berthold K,
Schmidt S, Wild D, Henschler D.
1986. Bacterial -lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcino-
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
genic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene.

Chem. Biol. Interact. 60:3145
Wallin RF, Regan BM, Napoli MD, Stern
IJ. 1975. Sevoflurane: a new inhalational
anesthetic agent. Anesth. Analg. 54:758
66
Eger EI II. 1987. Stability of I-653 in soda
lime. Anesth. Analg. 66:98385
Strum DP, Johnson BH, Eger EI II. 1987.
Stability of sevoflurane in soda lime.
Hanaki C, Fujii K, Morio M, Tashima T.
1987. Decomposition of sevoflurane by
sodalime. Hiroshima J. Med. Sci. 36:61
67
Morio M, Fujii K, Satoh N, Imai M,
Kawakami T, et al. 1992. Reaction of
sevoflurane and its degradation products
with soda lime. Toxicity of the byproducts. Anesthesiology 77:115564
Frink EJ Jr, Malan TP, Morgan SE, Brown
EA, Malcomson M, Brown BR Jr. 1992.
Quantification of the degradation products of sevoflurane in two CO2 absorbants
during low-flow anesthesia in surgical patients. Anesthesiology 77:1064
69
Bito H, Ikeda K. 1994. Closed-circuit
anesthesia with sevoflurane in humans.
Effects on renal and hepatic function
and concentrations of breakdown products with soda lime in the circuit. Anesthesiology 80:7176
Bito H, Ikeda K. 1994. Plasma inorganic fluoride and intracircuit degradation
product concentrations in long-duration,
low-flow sevoflurane anesthesia. Anesth.
Analg. 79:94651
Fang ZX, Eger EI II. 1995. Factors affecting the concentration of Compound A
resulting from the degradation of sevoflurane by soda lime and Baralyme in a
standard anesthetic circuit. Anesth. Analg.
81:56468
Fang ZX, Kandel L, Laster MJ, Ionescu P,
Eger EI II. 1996. Factors affecting production of Compound A from the interaction
3 Dec 2004
20:38
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE
66.

67.
68.
69.
70.
71.
72.
of sevoflurane with Baralyme and soda

lime. Anesth. Analg. 82:17
Munday IT, Ward PM, Foden ND, Jones
RM, Van Pelt FNAM, Kenna JG. 1996.
Sevoflurane degradation by soda lime in
a circle breathing system. Anaesthesia
51:62226
Bito H, Ikeda K. 1995. Effect of total
flow rate on the concentration of degradation products generated by the reaction
between sevoflurane and soda lime. Brit.
J. Anaesth. 74:66769
Huang C, Venturella VS, Cholli AL,
Venutolo FM, Silbermann AT, Vernice
GG. 1989. Detailed investigation of fluoromethyl 1,1,1,3,3,3-hexafluoro-2-propyl
ether (Sevoflurane) and its degradation
products. Part I: synthesis of fluorinated,
soda lime induced degradation products.
J. Fluorine Chem. 45:23953
Kharasch ED, Hankins DC. 1996.
P450-dependent and nonenzymatic human liver microsomal defluorination
of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (Compound A), a
sevoflurane degradation product. Drug
Kharasch ED, Thummel KE. 1993. Identification of cytochrome P450 2E1 as
the predominant enzyme catalyzing human liver microsomal defluorination of
sevoflurane, isoflurane, and methoxyflurane. Anesthesiology 79:795807
Jin L, Baillie TA, Davis MR, Kharasch
ED. 1995. Nephrotoxicity of sevoflurane
Compound A [fluoromethyl-2,2-difluoro1-(trifluoromethyl)vinyl ether] in rats:
evidence for glutathione and cysteine conjugate formation and the role of renal cysteine conjugate -lyase. Biochem. Biophys. Res. Commun. 210:498506
Jin L, Davis MR, Kharasch ED, Doss GA,
Baillie TA. 1996. Identification in rat bile
of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl
ether, a nephrotoxic degradate of the anesthetic agent sevoflurane. Chem. Res. Toxicol. 9:55561
173
73. Spracklin DK, Kharasch ED. 1996. Evidence for metabolism of fluoromethyl 2,
2-difluoro-1-(trifluoromethyl)vinyl ether
(Compound A), a sevoflurane degradation
product, by cysteine conjugate -lyase.
74. Iyer RA, Anders MW. 1997. Cysteine
conjugate -lyase-dependent biotransformation of the cysteine S-conjugates
of the sevoflurane degradation product
2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene (Compound A). Chem. Res.
Toxicol. 10:81119
75. Kharasch ED, Jubert C. 1999. Compound A uptake and metabolism to
mercapturic acids and 3,3,3-trifluoro-2fluoromethoxypropanoic acid during lowflow sevoflurane anesthesia: biomarkers for exposure, risk assessment, and
interspecies comparison. Anesthesiology
91:126778
76. Uttamsingh V, Iyer RA, Baggs RB,
Anders MW. 1998. Fate and toxicity of
2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene (Compound A)-derived mercapturates in male, Fischer 344 rats.
77. Iyer RA, Frink EJ Jr, Ebert TJ, Anders MW. 1998. Cysteine conjugate
-lyase-dependent metabolism of Compound A (2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in
rats given Compound A. Anesthesiology
88:61118
78. Altuntas TG, Kharasch ED. 2002. Biotransformation of L-cysteine S-conjugates
and N-acetyl-L-cysteine S-conjugates
of the sevoflurane degradation product
fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (Compound A) in
human kidney in vitro: interindividual variability in N-acetylation, N-deacetylation, and -lyase-catalyzed metabolism. Drug Metab. Dispos. 30:148
54
79. Altuntas TG, Park SB, Kharasch ED.
2004. Sulfoxidation of cysteine and
3 Dec 2004
20:38
174

80.
81.
82.
83.
84.
85.
86.
87.
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE
ANDERS
mercapturic acid conjugates of the
sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A). Chem. Res.
Toxicol. 17:43545
Gonsowski CT, Laster MJ, Eger EI II,
Ferrell LD, Kerschmann RL. 1994. Toxicity of compound A in rats. Effect of
a 3-hour administration. Anesthesiology
80:55665
Gonsowski CT, Laster MJ, Eger EI II,
Ferrell LD, Kerschmann RL. 1994. Toxicity of compound A in rats. Effect of increasing duration of administration. Anesthesiology 80:56673
Kandel L, Laster MJ, Eger EI II, Kerschmann RL, Martin J. 1995. Nephrotoxicity in rats undergoing a one-hour exposure to Compound A. Anesth. Analg.
81:55963
Keller KA, Callan C, Prokocimer P,
Delgado-Herrera L, Friedman MB, et al.
1995. Inhalation toxicity study of a
haloalkene degradant of sevoflurane,
Compound A (PIFE), in Sprague-Dawley
rats. Anesthesiology 83:122032
Dohn DR, Leininger JR, Lash LH,
Quebbemann AJ, Anders MW. 1985.
Nephrotoxicity of S-(2-chloro-1,1,2-trifluoroethyl)glutathione and S-(2-chloro1,1,2-trifluoroethyl)-L-cysteine, the glutathione and cysteine conjugates of
chlorotrifluoroethene. J. Pharmacol. Exp.
Ther. 235:85157
Koob M, Dekant W. 1990. Metabolism of
hexafluoropropene. Evidence for bioactivation by glutathione conjugate formation in the kidney. Drug Metab. Dispos.
18:91116
Odum J, Green T. 1984. The metabolism
and nephrotoxicity of tetrafluoroethylene
in the rat. Toxicol. Appl. Pharmacol.
76:30618
Iyer RA, Baggs RB, Anders MW. 1997.
Nephrotoxicity of the glutathione and
cysteine conjugates of the sevoflurane
degradation product 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene (Com-
88.
89.
90.
91.
92.
93.
94.
95.
96.
pound A) in male, Fischer 344 rats. J.

Martin JL, Laster MJ, Kandel L, Kerschmann RL, Reed GF, Eger EI II. 1996.
Metabolism of Compound A by renal
cysteine-S-conjugate -lyase is not the
mechanism of Compound A-induced renal injury in the rat. Anesth. Analg. 82:
77074
Martin JL, Kandel L, Laster MJ, Kerschmann RL, Eger EI II. 1997. Studies on
the mechanism of nephrotoxicity of compound A in rats. J. Anesthesiol. 11:3237
Njoku DB, Pohl LR, Sokoloski EA,
Marchick MR, Borkowf CB, Martin JL.
1999. Immunochemical evidence against
the involvement of cysteine conjugate
beta-lyase in compound A nephrotoxicity
in rats. Anesthesiology 90:45869
Kharasch ED, Thorning D, Garton K,
Hankins DC, Kilty GC. 1997. Role of
renal cysteine conjugate -lyase in the
mechanism of Compound A nephrotoxicity in rats. Anesthesiology 86:16071
Kharasch ED, Hoffman GM, Thorning D,
Hankins DC, Kilty CG. 1998. Role of the
renal cysteine conjugate -lyase pathway
in inhaled Compound A nephrotoxicity in
rats. Anesthesiology 88:162433
Davis ME. 1988. Effects of AT-125
on the nephrotoxicity of hexachloro-1,3butadiene in rats. Toxicol. Appl. Pharmacol. 95:4452
de Ceaurriz J, Ban M. 1990. Role of
-glutamyltranspeptidase and -lyase in
the nephrotoxicity of hexachloro-1,3butadiene and methyl mercury in mice.
Toxicol. Lett. 50:24956
Nash JA, King LH, Lock EA, Green T.
1984. The metabolism and disposition of
hexachloro-1:3-butadiene in the rat and its
relevance to nephrotoxicity. Toxicol. Appl.
Pharmacol. 73:12437
Lantum HB, Iyer RA, Anders MW.
2004. Acivicin-induced alterations in renal and hepatic glutathione concentrations and in g-glutamyltransferase activities. Biochem. Pharmacol. 67:142126
3 Dec 2004
20:38
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE


97. Altuntas TG, Zager RA, Kharasch ED.
2003. Cytotoxicity of S-conjugates of
the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)
vinyl ether (Compound A) in a human
proximal tubular cell line. Toxicol. Appl.
Pharmacol. 193:5565
98. Lash LH, Sausen PJ, Duescher RJ,
Cooley AJ, Elfarra AA. 1994. Roles
of cysteine conjugate -lyase and Soxidase in nephrotoxicity: studies with
S-(1,2-dichlorovinyl)-L-cysteine and S(1,2-dichlorovinyl)-L-cysteine sulfoxide.
J. Pharmacol. Exp. Ther. 269:37483
99. Werner M, Birner G, Dekant W. 1996.
Sulfoxidation of mercapturic acids derived from tri- and tetrachloroethene by
cytochromes P450 3A: a bioactivation reaction in addition to deacetylation and
cysteine conjugate -lyase mediated
cleavage. Chem. Res. Toxicol. 9:41
49
100. Birner G, Werner M, Ott MM, Dekant W.
1995. Sex differences in hexachlorobutadiene biotransformation and nephrotoxicity. Toxicol. Appl. Pharmacol. 132:203
12
101. Park SB, Osterloh JD, Vamvakas S,
Hashmi M, Anders MW, Cashman JR.
1992. Flavin-containing monooxygenasedependent stereoselective S-oxygenation
and cytotoxicity of cysteine S-conjugates
and mercapturates. Chem. Res. Toxicol.
5:193201
102. Terracini B, Parker VH. 1965. A pathological study on the toxicity of Sdichlorovinyl-L-cysteine. Food Cosmet.
Toxicol. 3:6774
103. Reichle FM, Conzen PF, Peter K. 2002.
Nephrotoxicity of halogenated inhalational anaesthetics: fictions and facts. Eur.
Surg. Res. 34:18895
104. Reichle FM, Conzen PF. 2003. Halogenated inhalational anaesthetics. Best
Pract. Res. Clin. Anaesthesiol. 17:2946
105. Mazze RI. 1992. The safety of sevoflurane. Anesthesiology 77:106263
106. Eger EI II, Koblin DD, Bowland T,
107.
108.
109.
110.
111.
112.
113.
114.
175
Ionescu P, Laster MJ, et al. 1997. Nephrotoxicity of sevoflurane versus desflurane

anesthesia in volunteers. Anesth. Analg.
84:16068
Eger EI II, Gong D, Koblin DD, Bowland T, Ionescu P, et al. 1997. Dose-related
biochemical markers of renal injury after sevoflurane versus desflurane anesthesia in volunteers. Anesth. Analg. 85:1154
63
Ebert TJ, Frink EJ Jr, Kharasch ED.
1998. Absence of biochemical evidence
for renal and hepatic dysfunction after 8
hours of 1.25 minimum alveolar concentration sevoflurane anesthesia in volunteers. Anesthesiology 88:60110
Ebert TJ, Frink EJ, Kharasch ED. 1998.
Absence of renal and hepatic toxicity following 8 hours of 1.25 MAC sevoflurane
anesthesia in volunteers. Anesthesiology
100:12
Higuchi H, Sumita S, Wada H, Ura T, Ikemoto T, et al. 1998. Effects of sevoflurane
and isoflurane on renal function and on
possible markers of nephrotoxicity. Anesthesiology 89:30722
Obata R, Bito H, Ohmura M, Moriwaki
G, Ikeuchi Y, et al. 2000. The effects of
prolonged low-flow sevoflurane anesthesia on renal and hepatic function. Anesth.
Analg. 91:126268
Groudine SB, Fragen RJ, Kharasch ED,
Eisenman TS, Frink EJ, et al. 1999.
Comparison of renal function following
anesthesia with low-flow sevoflurane and
isoflurane. J. Clin. Anesth. 11:2017
Kharasch ED, Frink EJ Jr, Artru A,
Michalowski P, Rooke GA, Nogami W.
2001. Long-duration low-flow sevoflurane and isoflurane effects on postoperative renal and hepatic function. Anesth.
Analg. 93:151120
Mazze RI, Callan CM, Galvez ST,
Delgado-Herrera L, Mayer DB. 2000. The
effects of sevoflurane on serum creatinine and blood urea nitrogen concentrations: a retrospective, twenty-two-center,
comparative evaluation of renal function
3 Dec 2004
20:38
176
115.

116.
117.
118.
119.
120.
121.
AR
AR232-PA45-06.tex
AR232-PA45-06.sgm
LaTeX2e(2002/01/18)
P1: GCE
ANDERS
in adult surgical patients. Anesth. Analg.
90:68388
Green T, Odum J, Nash JA, Foster JR.
1990. Perchloroethylene-induced rat kidney tumors: an investigation of the mechanisms involved and their relevance to humans. Toxicol. Appl. Pharmacol. 103:77
89
Moon RE, Ingram C, Brunner EA, Meyer
AF. 1991. Spontaneous generation of carbon monoxide within anesthetic circuits.
Anesthesiology 75:A873
Moon RE, Meyer AF, Scott DL, Fox
E, Millington DS, Norwood DL. 1990.
Intraoperative carbon monoxide toxicity.
Anesthesiology 73:A1049
Fang ZX, Eger EI II. 1994. Source of toxic
CO explained: -CHF2 anesthetic + dry
absorbent. Anesthesia Patient Saf. Found.
Newsl. 9:2530
National Research Council CoMaBEoEP.
1977. Carbon Monoxide. Washington,
DC: Natl. Acad. Sci.
Pankow D. 1996. Carbon monoxide formation due to metabolism of xenobiotics.
In Carbon Monoxide, ed. DG Penney, pp.
2543. Boca Raton: CRC Press
Fang ZX, Eger EI II, Laster MJ, Chortkoff
BS, Kandel L, Ionescu P. 1995. Car-
122.
123.
124.
125.
126.
127.
bon monoxide production from degradation of desflurane, enflurane, isoflurane,

halothane, and sevoflurane by soda lime
and Baralyme. Anesth. Analg. 80:1187
93
Baxter PJ, Kharasch ED. 1997. Rehydration of desiccated Baralyme prevents carbon monoxide formation from desflurane
in an anesthesia machine. Anesthesiology
86:106165
Kharasch ED, Powers KM, Artru AA.
2002. Comparison of Amsorb, sodalime,
and Baralyme degradation of volatile
anesthetics and formation of carbon
monoxide and Compound A in swine
in vivo. Anesthesiology 96:17382
Baxter PJ, Garton K, Kharasch ED. 1998.
Mechanistic aspects of carbon monoxide
formation from volatile anesthetics. Anesthesiology 89:92941
le Noble WJ. 1974. Highlights of Organic
Chemistry. New York: Marcel Dekker
Tzagoloff A, Wharton DC. 1965. Studies on the electron transport system.
LXII. The reaction of cytochrome oxidase
with carbon monoxide. J. Biol. Chem.
240:262833
Fenn WO. 1970. The burning of CO in
tissues. Ann. NY Acad. Sci. 174:6471
3 Dec 2004
20:38
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AR232-PA45-07.tex
AR232-PA45-07.sgm
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Copyright

THE ROLE OF METABOLIC ACTIVATION IN

DRUG-INDUCED HEPATOTOXICITY
B. Kevin Park, Neil R. Kitteringham, James L. Maggs,
Munir Pirmohamed, and Dominic P. Williams
Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington
Buildings, Liverpool, Merseyside L69 3GE, United Kingdom; email: bkpark@liv.ac.uk,
neilk@liv.ac.uk, j.l.maggs@liv.ac.uk, munirp@liv.ac.uk, dom@liv.ac.uk
Key Words bioactivation, chemically reactive metabolite, critical protein,

acetaminophen, diclofenac, tamoxifen, troglitazone
Abstract The importance of reactive metabolites in the pathogenesis of druginduced toxicity has been a focus of research interest since pioneering investigations
in the 1950s revealed the link between toxic metabolites and chemical carcinogenesis. There is now a great deal of evidence that shows that reactive metabolites are
formed from drugs known to cause hepatotoxicity, but how these toxic species initiate and propagate tissue damage is still poorly understood. This review summarizes
the evidence for reactive metabolite formation from hepatotoxic drugs, such as acetaminophen, tamoxifen, diclofenac, and troglitazone, and the current hypotheses of
how this leads to liver injury. Several hepatic proteins can be modified by reactive
metabolites, but this in general equates poorly with the extent of toxicity. Much more
important may be the identification of the critical proteins modified by these toxic
species and how this alters their function. It is also important to note that the toxicity of
reactive metabolites may be mediated by noncovalent binding mechanisms, which may
also have profound effects on normal liver physiology. Technological developments in
the wake of the genomic revolution now provide unprecedented power to characterize
and quantify covalent modification of individual target proteins and their functional
consequences; such information should dramatically improve our understanding of
drug-induced hepatotoxic reactions.
INTRODUCTION
Adverse drug reactions (ADRs) are significant health problems that contribute to
patient morbidity and mortality. There are many different types of ADRs, affecting
every organ system in the body. However, drug-induced liver injury is the most
frequent reason for the withdrawal of an approved drug from the market, and
it also accounts for more than 50% of cases of acute liver failure in the United
States today (1). More than 600 drugs have been associated with hepatotoxicity.
The clinical picture is diverse, even for the same drug when given to different
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patients. The manifestations range from mild, asymptomatic changes in serum

transaminases, which occur at a relatively high frequency with a number of drugs,
to fulminant hepatic failure, which although rare, is potentially life threatening
and may necessitate a liver transplant.
Most drug-induced hepatic injuries that occur in humans are unpredictable and
poorly understood. Although the asymptomatic rises in transaminases are common, the more severe forms of liver damage are fortunately rare, generally occurring with a frequency between 1 in 1000 and 1 in 10,000. The patients present
with a pattern of liver injury that is consistent for each drug and may therefore
be termed idiosyncratic, a term that does not imply any particular mechanism.
Drug-induced liver toxicity mimics natural disease, and therefore lessons learned
from the study of drug-induced hepatotoxicity should not only enhance drug
safety but also provide new pharmacological strategies for the treatment of liver
disease.
The major advances in molecular toxicology over the past decade have provided
a conceptual framework for the mechanism of action of model hepatotoxins at the
chemical, molecular, biochemical, and cellular levels. In particular, we now have a
better understanding of the events that link drug metabolism and the formation of
toxic metabolites to changes in liver function and the evolution of liver pathology.
In this review, we relate recent advances in molecular toxicology to the clinical
problem of drug-induced hepatotoxicity.
HEPATOTOXICITY AND DRUG METABOLISM

The biotransformation of lipophilic compounds into water-soluble derivatives that
are more readily excreted is a physiological role of the liver. The liver receives
more than 80% of its blood flow from the gastrointestinal tract and has a high
capacity for both phase I and phase II biotransformations. Cytochrome P450
enzymes play a primary role in the metabolism of an incredibly diverse range
of foreign compounds, including therapeutic agents. Such compounds may undergo concentration in the liver by various processes, including active transport
systems.
Although the major role of drug metabolism is detoxication, it can also act
as an intoxication process. Thus, foreign compounds can undergo biotransformation to metabolites that have intrinsic chemical reactivity toward cellular
macromolecules (Figure 1). The propensity of a molecule to form such chemically
reactive metabolitesusually electrophilesis simply a function of its chemistry,
and structural alerts are now well defined. A number of enzymes, and in particular the cytochromes P450, can generate, and in many instances release, reactive
metabolites. The versatility of P450 together with the reactivity of their oxygen intermediates enables them to functionalize even relatively inert substrates, leading
to the direct formation of diverse chemically reactive species. Such metabolites are
short-lived, with half-lives of generally less than one minute, and are not usually
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179
Figure 1 Relationship between drug metabolism and toxicity. Toxicity may accrue
through accumulation of parent drug or, via metabolic activation, through formation
of a chemically reactive metabolite, which, if not detoxified, can effect covalent modification of biological macromolecules. The identity of the target macromolecule and
the functional consequence of its modification will dictate the resulting toxicological
response.
detectable in plasma. Their intracellular formation can be inferred from endogenous trapping reactions or physico-chemical techniques. Their formation may be
modulated by enzyme induction, enzyme inhibition, and gene deletion in animals.
However, none of these experimental procedures is directly applicable to man.
Hence, human exposure to chemically reactive metabolites in the liver is almost
impossible to quantify.
The concept that small organic molecules can undergo bioactivation to electrophiles and free radicals and elicit toxicity by chemical modification of cellular
macromolecules has its basis in chemical carcinogenicity and the pioneering work
of the Millers (2, 3). The application of such concepts to human drug-induced hepatotoxicity was established through the studies of Brodie, Gillette, and Mitchell
(4, 5) on the covalent binding to hepatic proteins of toxic (over) doses of the widely
used analgesic acetaminophen.
However, the relationship between bioactivation and the occurrence of hepatic
injury is not simple. For example, many chemicals undergo bioactivation in the
liver but are not hepatotoxic. The best example is the lack of hepatotoxicity with
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therapeutic doses of acetaminophen. Tight coupling of bioactivation with bioinactivation may be one reason for this. Many enzymic and nonenzymic pathways
of bioinactivation are present in the liver, which is perhaps the best equipped
of all the organs in the body to deal with toxins. Typical examples of bioinactivation pathways include glutathione conjugation of quinones by glutathione
S-transferases (GSTs) and hydration of arene oxides to dihydrodiols by epoxide
hydrolases. It is only when a reactive metabolite is a poor substrate for such enzymes that it can escape bioinactivation and thereby damage proteins and nucleic
acids.
Moreover, covalent binding per se does not necessarily lead to drug hepatotoxicity. The regioisomer of acetaminophen, 3-hydroxyacetanilide, becomes covalently bound to hepatic proteins in rodents without inducing hepatotoxicity (6).
It is therefore necessary to identify the subset of targets, i.e., covalently modified
macromolecules, that is critical to the toxicological process. Hard electrophiles
generally react with hard nucleophiles, such as functional groups in DNA and
lysine residues in proteins. Soft electrophiles react with soft nucleophiles, which
include cysteine residues in proteins and in glutathione, which has a concentration
of approximately 10 mM in the liver. Free radicals can also react with lipids and initiate lipid peroxidative chain reactions. Unfortunately, there are no simple rules to
predict the target macromolecule(s) for a particular chemically reactive metabolite
or the biological consequences of a particular modification. Furthermore, noncovalent interactions also play a role because covalent binding of hepatotoxins is not
indiscriminate with respect to proteins. Even within a single protein there can be
selective modification of an amino acid side-chain found repeatedly in the primary
structure. Thus, the microenvironment (pKa, hydrophobicity, etc.) of the amino
acid in the tertiary structure appears to be the crucial determinant of selective
binding, and therefore the impact of covalent binding on protein function. The
extent of binding and the biochemical role of the protein will in turn determine
the toxicological insult of drug bioactivation. The resulting pathological consequences will be a balance between the rates of protein damage and the rates of
protein replacement and cellular repair.
It is therefore not surprising that irreversible chemical modification of a protein, which has a profound effect on function, is a mechanism of drug-induced
hepatotoxicity. However, it is also important to note that a number of drugs (e.g.,
penicillins, aspirin, omeprazole) rely on covalent binding to proteins for their efficacy, and thus prevention of covalent binding through chemical modification of
the compound may also inadvertently lead to loss of efficacy. Similarly, endogenous compounds, such as cyclopentenone prostaglandins, are Michael acceptors,
which react with specific cysteine residues in transcription factors to elicit their
physiological effects in cell signaling (7).
The considerable task therefore facing the molecular toxicologist and drug
metabolist is to differentiate between those protein modifications that are critical
for a particular type of drug toxicity (and drug efficacy) and the white noise of
noncritical, background covalent binding.
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BIOACTIVATION AND HEPATOCARCINOGENESIS

The relationship between bioactivation, bioinactivation, and DNA adduct formation has been well established for a number of hepatocarcinogens. Aflatoxin, which
is a hepatocarcinogen and a hepatotoxin, is converted into an epoxide, which is
more readily detoxified by GST enzymes than by epoxide hydrolase. The balance
of these reactions explains the greater DNA damage in humans compared with
rodents because human forms of GST are less able to catalyze the conjugation
of aflatoxin than their rodent counterparts (8, 9). Transgenic knockout mice have
been used to establish the role of bioactivation by P450 (for review see 10) and
bioinactivation by GSTs (11) for a number of carcinogenic polyaromatic hydrocarbons.
An important safety issue with respect to a therapeutic agent arose with the
discovery that tamoxifen is a genotoxic hepatocarcinogen in the rat (12). Tamoxifen
is a nonsteroidal antiestrogen used for the treatment of breast cancer (13). It has
contributed to the reduction of deaths from breast cancer in the United States and
the United Kingdom. There is now sufficient human experience to indicate that
tamoxifen does not cause hepatic tumors in women after either prophylaxis or
treatment. A consideration of the relative rates of bioactivation and bioinactivation
provides a metabolic rationale for the safety of the drug in women.
The major route of bioactivation of tamoxifen to a genotoxic metabolite is
known to be by sequential -hydroxylation and sulphonation to a sulphate ester
that collapses to a reactive carbocation and forms DNA adducts (14). Importantly,
we observed that the corresponding glucuronide of -hydroxytamoxifen is chemically very stable, and thus this biotransformation represents bioinactivation. There
is no glutathione conjugate formed because the carbocation is a hard electrophile.
A comparison of the relative rates of hydroxylation, sulphonation, and glucuronylation was performed in vitro between human and rodent enzymes. Rats had a
greater propensity for sulphonation (bioactivation), whereas human liver had a
much greater ability to effect glucuronylation (bioinactivation) (15, 16). An overall analysis of risk based on dose and the relative rates of metabolism suggested a
150,000-fold safety factor for the development of liver cancer from tamoxifen in
humans when compared with rats (Figure 2).
BIOACTIVATION AND HEPATOTOXINS

A number of simple chemical compounds that produce selective hepatotoxicity
after a single dose have been widely studied. These compounds are generally toxic
in all species studied and include carbon tetrachloride, bromobenzene, furosemide,
and acetaminophen. For each compound, there is compelling evidence that bioactivation is essential for hepatotoxicity. The use of transgenic null mice for certain
P450 isoforms has been definitive in this regard. However, even for such simple compounds, the structure of the ultimate toxic metabolite is not known with
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Figure 2 Metabolic bioactivation of tamoxifen. Tamoxifen undergoes sequential

oxidation and sulphonation to form a carbocation that reacts covalently with DNA.
certainty. This information is essential if one is to relate global changes in gene expression, proteomics, and metabonomics in a way that can be used by the medicinal
chemist in drug design.
Acetaminophen
Acetaminophen is a major cause of drug-related morbidity and mortality in humans, producing massive hepatic necrosis after a single toxic dose. A similar
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pathological picture is observed in rodents. Toxicity is essentially dose-dependent,

but there is interindividual variability in susceptibility, with alcoholics and patients
on enzyme-inducing drugs perhaps being more susceptible. At therapeutic doses,
acetaminophen is deactivated by glucuronylation and sulphation to metabolites,
which are rapidly excreted in urine. However, a proportion of the drug undergoes
bioactivation to N-acetyl-p-benzoquinoneimine (NAPQI) by CYP2E1, CYP1A2,
and CYP3A4 (17, 18) (Figure 3).
NAPQI is rapidly quenched by a spontaneous reaction with hepatic glutathione
after a therapeutic dose of acetaminophen. After a toxic (over) dose, glutathione
Figure 3 Bioactivation of acetaminophen. Acetaminophen can undergo conversion

to the chemically reactive species N-acetyl-p-benzoquinoneimine, which can oxidize
and covalently modify proteins. The toxicological and pharmacological properties of
the molecule are a function of the redox potential of the molecule.
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depletion occurs, which is an obligatory step for covalent binding and toxicity
(19). The standard treatment for acetaminophen intoxication is N-acetylcysteine,
which replaces hepatic glutathione and prevents toxicity. N-acetylcysteine is most
beneficial if given within 16 h of the overdose. The early signs of cellular disruption
in isolated hepatocytes can be reversed by a disulphide reductant, dithiothreitol
(20, 21).
The massive chemical stress mediated by an acetaminophen overdose leads to
an immediate adaptive defense response in the hepatocyte. This involves various
mechanisms, including the nuclear translocation of redox-sensitive transcription
factors such as Nrf-2, which sense chemical danger and orchestrate cell defense
(Figure 4). Thus, with respect to acetaminophen, Nrf-2 genes of immediate significance are those involved in glutathione synthesis such as -glutamylcysteine
Figure 4 Activation of Nrf2 in hepatocytes in response to paracetamol exposure.

Generation of NAPQI in the hepatocytes results in GSH depletion, protein adducts,
and oxygen free radical formation. Each of these contributes to the release of Nrf2
from its cytoplasmic inhibitor, Keap1, and translocation to the nucleus. In the nucleus,
Nrf2 heterodimerizes with small Maf or other proteins and activates the antioxidant
response element (ARE), resulting in enhanced transcription of a battery of genes
encoding antioxidant proteins and phase II drug metabolizing enzymes.
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synthetase ( -GCS), GSTs, glucuronyltransferases, and heme oxygenase (22). Importantly, it has been observed that nuclear translocation occurs at nontoxic doses
of acetaminophen and at time-points before overt toxicity is observed. However,
with increasing doses of acetaminophen, there is progressive dislocation of nuclear
translocation, transcription, translation, and protein activity (23) as the rate of drug
bioactivation overwhelms cell defense through the destruction of critical proteins.
Since the initial discovery that covalent
binding of acetaminophen to hepatic proteins was associated with hepatotoxicity, there has been a progression of techniques that have been used to identify the
protein targets. Thus, radiolabeled drugs and Western blotting enable the detection
and quantification of adduct formation, whereas more recently proteomics has
allowed the simultaneous identification of several adducted proteins. The latter
technique offers the possibility of determining the amino acids modified and the
nature of that modification. This in turn allows a molecular rationale for the change
in activity of that protein.
At least 17 liver enzymes that show a loss of activity ex vivo after administration
of a toxic dose of acetaminophen to a rodent species have now been investigated;
these are listed in Table 1. An additional 14 liver enzymes are known to be adducted
by paracetamol in vivo and in vitro but have yet to be shown to be inhibited.
It is notable that modification of proteins can occur in most intracellular compartments of the hepatocyte, e.g., endoplasmic reticulum (ER), cytosol, mitochondria, and plasma membrane, which is an indication of the intracellular mobility of
the reactive metabolite once glutathione is depleted (Figure 5). The loss of hepatocyte viability is likely to be a function of the summation and extent of inhibition
of protein activity. Thus, inhibition of -GCS, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and Ca2+/Mg2+ ATPase will severely impair hepatocyte
function by uncoupling mitochondria, depleting glutathione and ATP, and disturbing Ca2+ homeostasis, which could lead to the expression of TNF and Fas receptors
on cell membranes. -GCS catalyses the rate-limiting step in glutathione synthesis,
the primary biochemical defense of the hepatocyte against NAPQI. GAPDH,
which, as a component of the glycolytic pathway, contributes to ATP production,
is more than 80% inhibited at 2 h after a toxic dose of acetaminophen. On the basis
of reaction with NAPQI in vitro, inhibition is thought to be due to modification of
a critical cysteine (cys-149) within the active site of the enzyme (24). The loss of
calcium homeostasis is one of the first pathological features of acetaminophen
toxicity. It is clear that as NAPQI diffuses from its site of formation, a number
of enzymes are chemically modifiedusually at cysteine or lysine residuesbut
there is a degree of protein selectivity and variation in amino acid modification:
Acetaminophen appears to react with lysine residues of three intraluminal ER
proteins (25). Presumably, noncovalent interactions and the microenvironment of
amino acid residues determine the precise structure of modified proteins.
The rapid inactivation of several proteins suggests that cellular failure is a consequence of multiple parallel events rather than a simple cascade or signaling

THE CRITICAL PROTEIN HYPOTHESIS
Mouse
Mouse
Mouse
Rat
Rat
Rat
ADP-ribose pyrophosphatase-1 (78)

-Glutamylcysteinyl synthetase (23)
GAPDH (24)
Glutathione S-transferase (79)
Methionine adenosyltransferase (80)
MIF tautomerase (81)
N-10-formyl-H4folate dehydrogenase (82)
Protein phosphatase (hepatocyte) (83)
Proteasome (84)
Tryptophan-2,3-dioxygenase (85)
Aldehyde dehydrogenase (86)
Carbamyl phosphate synthase-1 (76)
Glutamate dehydrogenase (87)
Mg2+ ATPase (88)
Ca2+/Mg2+-ATPase (89)
Na+/K+-ATPase (90)
Cytosol
Mitochondria
Cell membrane
32%, 2.5 h
52%, 3 h
Covalent?
?
Covalent
Covalent?
Covalent
Covalent
Noncovalent
?
Covalent
Covalent
Covalent
Covalent
Covalent
Noncovalent?
Covalent
?
?
?
?
Cysteine?g
?
?
?
?
0.71%
4.3%
1.4%
2%
?
?
2.05%
1.53.3
0.2%
?
0.55%
?
?
?
?
Cysteine?f
?
?
Proline
?
?
?
?
Cysteine?e
Abundanced
Modified
amino acid
Measured ex vivo.
The inhibited enzymes protein band was less extensively labeled by a thiol-modifying fluorescent reagent (monobromobimane).
Cys-149 of GAPDH is modified by NAPQI in vitro.
Relative expression levels in livers of male mice (92).
Enzyme inhibition in vivo was reversible ex vivo with dithiothreitol.
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Taken to be covalent modification (arylation) if radiolabel or immunologically reactive drug moiety coincided with protein on gel electrophoretogram except for the MIF tautomerase
adduct, which was characterized by MALDI-TOF analysis of the adducted protein isolated from mouse liver.
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a
These enzymes have been reported to be inhibited in animals or hepatocytes exposed to acetaminophen. A number of hepatic enzymes known to be covalently modified by acetaminophen
in vitro or in vivo have yet to be reported to be inhibited by the drug: calreticulin and two protein disulfide isomerases (25); aryl sulfotransferase, carbonic anhydrase III, 2,4-dienoyl-CoA
reductase, glutathione peroxidase, glycine N-methyltransferase, 3-hydroxyanthranilate 3,4-dioxygenase, inorganic pyrophosphatase, protein synthesis initiation factor 4A, sorbitol
dehydrogenase, thioether S-methyltransferase, urate oxidase (not found in primates) (91).
2.5 g/kg
850 mg/kg
40%, 4 h
65%, 6 h
35%, 1 h
35%, 24 h
ca. 50%, ? h
31%, 1 h
83%, 2 h
46%, 6 h
45%, 6 h
72%, 8 h
25%, 2 h
18%, 4 h
50%, 2 h
44%, 3 h
Covalent
Modificationc
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600 mg/kg
400 mg/kg
600 mg/kg
650 mg/kg
800 mg/kg
530 mg/kg
500 mg/kg
500 mg/kg
400 mg/kg
200 mg/kg
400 mg/kg
10 mM
600 mg/kg
3 100 mg/kg
65%, 6 h
Inhibitionb
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Rat
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Rat
400 mg/kg
Dose/
concentration
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Glutamine synthase (76, 77)
Microsomes
Species
Enzyme
Hepatic enzymes inhibited by acetaminophena
186
Fraction
TABLE 1

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Figure 5 Levels of interaction of the chemically reactive metabolite of acetaminophen, NAPQI, with cellular function. The cellular locations of specific proteins
involved in cell defense and cell damage, whose functions are known to be modified
by NAPQI following exposure to acetaminophen, are indicated.
mechanism. It is well established that one of the main events in isolated hepatocytes is overall energy failure (26, 27), which is accompanied by the generation of
megamitochondria that are apparently ATP-depleted and nonfunctional (28). The
execution of hepatocytes involves interplay between hepatocyte damage mediated
by chemical stress and the activation of nonparenchymal cells and the subsequent
release of various mediators. The role of Kupffer cells has been demonstrated by the
fact that mice treated with dichloromethylene diphosphonate (DMDP), which depletes 99% of macrophages from the liver, were protected against acetaminophen
toxicity (29). Indeed, acetaminophen-treated rats have four- to sevenfold more
infiltrating macrophages than resident Kupffer cells (30). Furthermore, neutralization of Fas ligand (31) and TNF (32) affords a degree of protection against the early
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apoptotic processes and the final overwhelming necrosis, which is the overriding
feature of acetaminophens hepatotoxicity.
Nitric oxide has a dual role in the hepatic response to acetaminophen. Nitric
oxide derived from iNOS contributes to acetaminophen-induced parenchymal cell
injury and to microvascular disturbances, whereas nitric oxide derived from constitutive NOS exerts a protective role in liver microcirculation and thereby minimizes
liver injury. In this context, it is of interest to note that glutathione depletion can
lead to oxidative deactivation of nitric oxide and thus produce hypertension (33).
It has also been suggested that liver blood flow is an important determinant
of toxicity. Consistent with this, it has been demonstrated that alpha-blockers,
which mediate vasodilatation, protect against acetaminophen toxicity even when
given after bioactivation and covalent binding of the drug has occurred (L. Randle,
unpublished data).
THE ASSOCIATION BETWEEN DRUG BIOACTIVATION

AND HEPATOTOXICITY IN MAN
Acetaminophen-induced hepatic necrosis is the best-described form of injury induced by reactive metabolites, but this type of toxicity is unusual in that it is caused
by a single dose as well as being clearly dose-dependent. In most instances, druginduced injury in man is an infrequent and variable event and a number of general
mechanisms have been proposed (1). Chemically reactive metabolites have been
proposed as being responsible for most types of drug-induced injury, but direct
evidence for the role of such metabolites is difficult to obtain because of the lack
of suitable in vitro and in vivo models.
Some drug reactions have all the clinical hallmarks of an immunological mechanism, which include time of presentation, general clinical features, greatly
enhanced reaction on reexposure to the drug, and some laboratory evidence of
drug-induced immunological perturbation. In such cases, the liver alone may be
involved, or liver injury may be part of a more complex hypersensitivity syndrome, as has been observed for anticonvulsants. Thus, for a series of drugs, there
is chemical evidence for bioactivation, based largely on in vitro or animal studies,
and some evidence of drug-induced antibody formation or a drug-related T cell
response (Table 2). The question is whether the association between bioactivation
and an immune response is coincidental or consequential.
Halothane
Halothane is the best-studied drug with respect to immunoallergic hepatitis. A
significant proportion of patients exposed to this inhalation anesthetic develop
asymptomatic rises in transaminases. Fulminant irreversible hepatitis is a rare but
life-threatening phenomenon. Most of the patients recorded in the literature with
immunoallergic hepatitis had more than one exposure (34). Antibodies have been
detected in such patients that recognize autoantigens and neoantigens created by
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TABLE 2
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Chemical and immunological basis of drug-induced immunoallergic hepatitis
Drug
Bioactivation
Immune response
Reference
Halothane
Oxidative dehalogenation
Drug metabolite IgG

Anti-CYP2E1 IgG
Autoantibodies
(39, 45)
Tienilic acid
Thiophene sulphoxidation
Anti-CYP2C9 IgG
(42)
Dihydralazine
Anti-CYP1A2 IgG
(44)
Sulphamethoxazole
N-hydroxylation
IgG antibodies
Drug and metabolite
T cells
(93)
(94)
Carbamazepine
Arene oxidation
Drug T cell
(46, 95)
Nevirapine
Arene oxidation
Drug T cell
Unpublished data
trifluoroacetylation of hepatic proteins (Figure 6). Preincubation of halothanepretreated, but not of control, rabbit hepatocytes with sera from patients with
halothane-induced fulminant hepatic failure rendered the hepatocytes susceptible
to the cytotoxic effects of normal lymphocytes in vitro (35). It is thus likely that
drug-specific T cells may play a role in the pathogenesis of hepatocyte injury, but
direct evidence for this is lacking.
It is likely that the common chemical trigger for both the mild and severe
forms of hepatocyte injury is drug bioactivation to an acyl halide. Bioactivation
of halothane is substantial and is a consequence of the presence of a vulnerable
proton alpha to halide groups, which are effective leaving groups. In this sense, the
only metabolic route available to the molecule is bioactivation. There is direct and
indirect evidence for this concept. First, the detection of drug metabolite-specific
antibodies in affected patients. Second, a global evaluation of the relationship between the metabolism and toxicity of inhalation anesthetics reveals that the newer,
metabolically inert anesthetics such as enflurane and isoflurane are rarely associated with hepatotoxicity in man. Pohl and colleagues (3639) have identified
a number of target proteins modified by halothane; trifluoroacetylation of lysine
residues is believed to be the principal chemical modification. Precisely how such
chemical modifications trigger an immune response and what is the immunological
mechanism of cell killing is still very much a matter of debate. Animal models of
experimental autoimmune hepatitis indicate that T cells rather than immunoglobulins provide the immunological trigger for cell death (40, 41). A feature of such
animal models is the minimal level of tissue injury, with protection partly afforded
by the presence of T suppressor cells. In man, therefore, the balance between the
different T cell subsets with different functions may be crucial in determining not
only individual susceptibility but also the severity of the injury.
A further clue to the mechanisms involved in such reactions was the discovery
of antibodies directed against the P450 enzymes responsible for the bioactivation
of tienilic acid, dihydralazine, and halothane (4244). In the case of halothane,
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Figure 6 Bioactivation of halothane. Halothane is metabolized by cytochrome P450

2E1 to a chemically reactive trifluoroacetyl radical, which has been shown to covalently
modify lysine residues in a range of target proteins, including CYP2E1 itself (39).
Chemical modification of protein(s) leads to the immune response associated with
halothane hepatitis.
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autoantibody generation was extensive (45). Collectively, these data provide evidence for a loss of tolerance to autologous proteins chemically modified as a
consequence of drug bioactivation. Further studies are required to examine patients with immunoallergic hepatitis for drug-specific T cells (46) and for genetic
variants in drug metabolism and immune responsiveness, which might provide the
key to understanding the idiosyncratic nature of such reactions.
Studies from our laboratories have already shown that patients with deranged
liver function as a consequence of taking carbamazepine or nevirapine have circulating T cells that recognize the drug (D.J. Naisbitt, unpublished data). Thus
most of the available information is compatible with the hapten mechanism of
drug-induced immunoallergic toxicity outlined in Figure 7.
Figure 7 Proposed mechanism for the role of reactive metabolites in immunoallergic

hepatitis. The drug undergoes bioactivation in the hepatocytes leading to drug-protein
conjugate formation in the liver. The resulting modified protein is internalized by
Kupffer cells and presented to cognate T cells that recognize modified peptide and
native peptide. This in turn can lead to the generation of cytotoxic T cells and B
lymphocytes producing antibody. In theory, such an unregulated response could explain
the severe idiosyncratic hepatotoxicity associated with halothane.
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Isoniazid
Isoniazid (INH) is still the most widely used drug in the treatment of tuberculosis (TB) and has high activity against Mycobacterium tuberculosis, although
resistant strains have emerged. INH is used in combination with drugs such as
rifampicin and pyrazinamide to reduce the chance of inducing resistant strains of
the mycobacterium.
INH causes two major adverse reactions: hepatitis and peripheral neuropathy.
The incidence and severity of the adverse drug reactions are related to dose and
duration of therapy. Toxicity may be delayed by several weeks. A minor asymptomatic increase in liver aminotransferases (less than threefold) is seen in 10%
20% of patients within the first two months of therapy, whereas fatal hepatitis is
seen in less than 1% of patients. Mortality is greater than 10% in patients with
jaundice (47, 48). INH typically produces diffuse massive necrosis or chronic hepatitis. Clinical features resemble acute viral-induced hepatitis. Anorexia, fatigue,
nausea, and vomiting are the usual prodromal features, but jaundice and dark urine
may be the first evidence of injury (49). Combination therapy is a risk factor for
hepatitis, although formal studies evaluating the mechanisms of this have not been
undertaken. Interestingly, of the three anti-TB compounds, it has been suggested
that pyrazinamide is the most hepatotoxic, with a rate of hepatitis three and five
times higher than that of rifampicin and INH, respectively (5052).
Studies in the rat (53) and rabbit (54), along with in vitro studies, indicate that
INH undergoes acetylation to give N-acetylisoniazid. This is then hydrolyzed to
acetylhydrazine, which undergoes bioactivation by P450 enzymes to give an acetyl
radical, a reactive species identified by trapping as a glutathione conjugate (53).
Precisely how such a reactive intermediate induces hepatocyte damage remains
to be elucidated, as do the reasons for the increased incidence of hepatotoxicity
when combination therapy is used. Target proteins have not been identified for the
reactive metabolite formed from INH. To date, there is no convincing clinical or
laboratory evidence to suggest an immunological mechanism.
Interestingly, bioactivation plays a role in the pharmacology of INH, with elimination of nitrogen being the driving force for the formation of an isonicotinoyl
radical intermediate (Figure 8). INH can thus be considered a prodrug, which is
activated by the mycobacterial catalase-peroxidase enzyme KatG. The product
of bioactivation forms a covalent adduct with NADH, which is an inhibitor of
InhA, an enoyl-acyl carrier protein reductase that is involved in the biosynthesis
of mycolic acids present in the mycobacterium cell wall (55, 56).
Diclofenac
The nonsteroidal antiinflammatory drugs (NSAIDs) as a class have a strong
association with hepatotoxicity. Several NSAIDs have been withdrawn after obtaining approval for a license, the most recent being bromfenac (57). The mechanism of hepatotoxicity appears to be complex and multifactorial, involving both
pharmacological and metabolic mechanisms. For example, inhibition of the COX
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Figure 8 Metabolic bioactivation of isoniazid. Reactive metabolites are responsible

for the pharmacology and toxicology of isoniazid. In Mycobacterium tuberculosis,
generation of the isonicotinoyl radical leads to the formation of an adduct with NADH,
which in turn inhibits an enoyl-acyl carrier protein reductase (InhA) (53, 56).
enzymes may lead to a reduction in cytoprotective prostaglandins, whereas bioactivation may occur by both oxidation and conjugation. This metabolic complexity
is illustrated with reference to diclofenac, which undergoes acyl glucuronylation
(58), acyl thiolation (59), and multiple P450-catalyzed oxidations producing two
p-benzoquinoneimines via phenols and an as yet uncharacterized intermediate
possibly an epoxideof mechanism-based inhibition (6062). The relative contributions of these metabolites to protein adduction, cytotoxicity, and hepatotoxicity
in vivo remains to be determined. In isolated rat hepatocytes, although the binding
of diclofenac to protein appears to derive principally from reactions of the acyl
glucuronide, the cytotoxicity has been attributed to products of oxidative pathways (63). However, diclofenac-protein adduct formationand especially on the
cell surfacemight be causally relevant to the expression of immune-mediated
hepatitis.
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Figure 9 Metabolic bioactivation of diclofenac. Diclofenac can form electrophilic

metabolites by either oxidation or glucuronylation. The precise role of such metabolites
in the rare hepatotoxicities associated with diclofenac remains to be elucidated (96).
Immunological and nonimmunological mechanisms have been proposed for

diclofenac toxicity. The acyl glucuronide can achieve concentrations in bile up to
5000-fold higher than those in peripheral blood because of a potent export pump
located in the canalicular membrane of hepatocytes (64). The acyl glucuronide is
protein reactive and forms covalent adducts with circulating proteins and hepatic
proteins (Figure 9). A particular target is the canalicular ectoenzyme dipeptidyl
peptidase (DPP) IV (CD26), which shows a decrease in activity following administration of diclofenac to rats (65).
Thiazolidinedione Antidiabetics
Chemically reactive metabolites have also been described for a number of drugs
that cause idiosyncratic hepatotoxicity, but for which no mechanistic studies are
available. An important example is troglitazone, a 2,4-thiazolidinedione, which
was the first of a new class of drugs for type 2 diabetes. Troglitazone was associated with a significant frequency of reversible increases in serum transaminases. Reports of severe and fatal liver injury finally led to the withdrawal of this
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important new drug (66). Fortunately, it could be replaced by newer and safer
2,4-thiazolidinediones (glitazones): pioglitazone and rosiglitazone (Figure 10).
The mechanism of troglitazone-induced hepatic injury is not known. The drug
undergoes oxidative bioactivation at both the chroman ringwhich is unique
to troglitazoneand the thiazolidinedione ring in rats, forming several reactive
metabolites that are eliminated as thioether and thioester conjugates of glutathione
(67, 68). It is also bioactivated in human hepatocytes (69, 70) and is cytotoxic (71).
An association of troglitazone hepatotoxicity in diabetic patients with a glutathione
S-transferase double null genotype provides indirect evidence for the importance
of bioactivation and bioinactivation in its pathogenesis (72). However, the less
hepatotoxic and cytotoxic glitazonespioglitazone and rosiglitazoneas well as
troglitazone undergo NADPH-dependent covalent binding to human microsomal
protein (73). At present, the toxicological significance of troglitazones metabolic
activation remains an open question; even the relative extents of the glitazones
bioactivation in vitro is unquantified. Finally, it is important to note that the heterogeneous clinical picture of troglitazone hepatotoxicity has prompted the suggestion
that this may be a reflection of interindividual variation in the balance of different
mechanisms of drug toxicity as well as varying patient characteristics (74).
CONCLUSIONS AND SOLUTIONS

There is overwhelming evidence that chemically reactive metabolites derived from
simple organic molecules, including therapeutic agents, can cause a wide range of
hepatic injuries. There are short- and long-term solutions to the problem.
In the short term, the drug metabolist can determine the propensity of a novel
chemical entity to undergo bioactivation in model systems ranging from expressed
enzymes, through genetically engineered cells, to whole animals. Bioactivation can
be assessed by trapping experiments with model nucleophiles in vitro or by measurement of uncharacterized covalent binding to endogenous proteins in vitro and
in vivo. The chemistry of the process needs to be defined, and the medicinal chemist
can then address the issue by seeking a metabolically stable pharmacophore to replace the potential toxicophore. Such an approach will minimize chemical hazard,
but cannot give any insight into biological risk in man, or in any other species
for that matter. Evans et al. (75) have provided an industrial perspective on this
topic and adopted a pragmatic approach to minimize reactive metabolite formation at an early stage in drug development. A decision tree has been designed
based on a target covalent binding value of 50 pmol drug equivalent /mg protein in
vitro and in vivo. The standard method measures covalent binding of radiolabeled
drug to hepatic microsomes. Such an approach seems to suggest that there may
be a threshold level of covalent binding, above which critical proteins necessarily become damaged. Appropriate dose-ranging studies are, however, required to
validate this concept.
In the long term, we require a more fundamental understanding of the role of
chemically reactive metabolites in human hepatotoxicity. We need to know how
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Figure 10 Metabolism and toxicity of glitazones. Troglitazone, a novel antidiabetic

agent, was withdrawn because of rare but serious hepatotoxicity. Rosiglitazone and
pioglitazone are now firmly established in the treatment of diabetes. It has been established that troglitazone undergoes bioactivation to several chemically reactive metabolites. Novel test systems are required to define the possible role of such metabolites in
hepatotoxicity (97, 98).
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the ultimate hepatotoxin interferes with signaling, and the sequence of molecular
events that impair cell defense, which ultimately lead to hepatocyte destruction. It
is only when such a mechanistic framework is established that we will be in position
to understand the time-course of the toxicity, the nature of the toxicity, and the
direction that the toxicity takes in a particular patient. It is therefore imperative that
such studies begin at the clinical level, but are then translated into molecular studies
in the laboratory with the design of appropriate in vitro and in vivo model systems
to fully exploit the molecular technology now available in the post genomic era.
ACKNOWLEDGMENT
The authors wish to acknowledge the generous and continuing support of the
Wellcome Trust.
LITERATURE CITED
1. Lee WM. 2003. Drug-induced hepatotoxicity. New Engl. J. Med. 349:47485
2. Miller E, Miller J. 1947. The presence
and significance of bound aminoazo
dyes in the livers of rats fed p-dimethylaminobenzene. Cancer Res. 7:468
80
3. Miller E, Miller J. 1952. In vivo combinations between carcinogens and tissue constituents and their possible role in carcinogenesis. Cancer Res. 12:54756
4. Brodie BB, Reid WD, Cho AK, Sipes
G, Krishna G, Gillette JR. 1971. Possible mechanism of liver necrosis caused
by aromatic organic compounds. Proc.
5. Gillette JR, Mitchell JR, Brodie BB. 1974.
Biochemical mechanisms of drug toxicity.
Annu. Rev. Pharmacol. 14:27188
6. Tirmenstein MA, Nelson SD. 1989. Subcellular binding and effects on calcium
homeostasis produced by acetaminophen
and a nonhepatotoxic regioisomer, 3 hydroxyacetanilide, in mouse liver. J.
Biol. Chem. 264:981419
7. Kawamoto Y, Nakamura Y, Naito Y, Torii
Y, Kumagai T, et al. 2000. Cyclopen-
8.
9.
10.
11.
tenone prostaglandins as potential inducers of phase II detoxification enzymes.

15-deoxy-delta(12,14)-prostaglandin j2induced expression of glutathione Stransferases. J. Biol. Chem. 275:11291
99
Guengerich FP, Johnson WW, Ueng YF,
Yamazaki H, Shimada T. 1996. Involvement of cytochrome P450, glutathione
S-transferase, and epoxide hydrolase in
the metabolism of aflatoxin B1 and relevance to risk of human liver cancer.
Environ. Health Perspect. 104(Suppl. 3):
55762
Wilson AS, Williams DP, Davis CD, Tingle MD, Park BK. 1997. Bioactivation and
inactivation of aflatoxin B1 by human,
mouse and rat liver preparations: effect on
SCE in human mononuclear leucocytes.
Mutat. Res. 373:25764
Gonzalez FJ, Kimura S. 1999. Role of
gene knockout mice in understanding the
mechanisms of chemical toxicity and carcinogenesis. Cancer Lett. 143:199204
Henderson CJ, Smith AG, Ure J, Brown K,
Bacon EJ, Wolf CR. 1998. Increased skin
tumorigenesis in mice lacking pi class
3 Dec 2004
20:38
198

12.
13.
14.
15.
16.
17.
18.
19.
AR
AR232-PA45-07.tex
AR232-PA45-07.sgm
LaTeX2e(2002/01/18)
P1: GCE
PARK ET AL.
glutathione S-transferases. Proc. Natl.
Carthew P, Rich KJ, Martin EA, De Matteis F, Lim CK, et al. 1995. DNA damage
as assessed by 32P-postlabelling in three
rat strains exposed to dietary tamoxifen:
the relationship between cell proliferation
and liver tumour formation. Carcinogenesis 16:1299304
Group EBCTC. 1998. Tamoxifen for
early breast cancer: an overview of
the randomised trials. Lancet 351:1451
67
Martin EA, Rich KJ, White IN, Woods
KL, Powles TJ, Smith LL. 1995. 32Ppostlabelled DNA adducts in liver obtained from women treated with tamoxifen. Carcinogenesis 16:165154
Boocock DJ, Maggs JL, White IN, Park
BK. 1999. Alpha-hydroxytamoxifen, a
genotoxic metabolite of tamoxifen in
the rat: identification and quantification
in vivo and in vitro. Carcinogenesis 20:
15360
Boocock DJ, Maggs JL, Brown K,
White INH, Park BK. 2000. Major
inter-species differences in the rates of
O-sulphonation and O-glucuronylation
of alpha-hydroxytamoxifen in vitro: a
metabolic disparity protecting human
liver from the formation of tamoxifenDNA adducts. Carcinogenesis 21:1851
58
Raucy JL, Lasker JM, Lieber CS,
Black M. 1989. Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2. Arch. Biochem. Biophys. 271:27083
Thummel KE, Lee CA, Kunze KL, Nelson SD, Slattery JT. 1993. Oxidation
of acetaminophen to N-acetyl-p-aminobenzoquinone imine by human CYP3A4.
Biochem. Pharmacol. 45:156369
Davis DC, Potter WZ, Jollow DJ, Mitchell
JR. 1974. Species differences in hepatic
glutathione depletion, covalent binding
and hepatic necrosis after acetaminophen.
Life Sci. 14:2099109
20. Albano E, Rundgren M, Harvison PJ, Nelson SD, Moldeus P. 1985. Mechanisms of
N-acetyl-p-benzoquinone imine cytotoxicity. Mol. Pharmacol. 28:30611
21. Rafeiro E, Barr SG, Harrison JJ, Racz
WJ. 1994. Effects of N-acetylcysteine
and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated
mouse hepatocytes. Toxicology 93:209
24
22. Goldring C, Kitteringham N, Elsby R,
Randle L, Clement Y, et al. 2004. Activation of hepatic Nrf2 in vivo by acetaminophen in CD-1 mice. Hepatology
39:126776
23. Kitteringham NR, Powell H, Clement
YN, Dodd CC, Tettey JN, et al. 2000. Hepatocellular response to chemical stress
in CD-1 mice: induction of early genes
and gamma-glutamylcysteine synthetase.
Hepatology 32:32133
24. Dietze EC, Schafer A, Omichinski JG,
Nelson SD. 1997. Inactivation of glyceraldehyde-3-phosphate dehydrogenase by a
reactive metabolite of acetaminophen and
mass spectral characterization of an arylated active site peptide. Chem. Res. Toxicol. 10:1097103
25. Zhou L, McKenzie BA, Eccleston ED Jr,
Srivastava SP, Chen N, et al. 1996. The
covalent binding of [14C]acetaminophen
to mouse hepatic microsomal proteins:
the specific binding to calreticulin and
the two forms of the thiol:protein disulfide oxidoreductases. Chem. Res. Toxicol.
9:117682
26. Andersson BS, Rundgren M, Nelson SD,
Harder S. 1990. N-acetyl-p-benzoquinone
imine-induced changes in the energy
metabolism in hepatocytes. Chem. Biol.
Interact. 75:20111
27. Burcham PC, Harman AW. 1991. Acetaminophen toxicity results in sitespecific mitochondrial damage in isolated
mouse hepatocytes. J. Biol. Chem. 266:
504954
28. Karbowski M, Kurono C, Wozniak M,
3 Dec 2004
20:38
AR
AR232-PA45-07.tex
AR232-PA45-07.sgm
LaTeX2e(2002/01/18)
P1: GCE

29.
30.
31.
32.
33.
34.
35.
36.
37.
Ostrowski M, Teranishi M, et al. 1999.

Free radical-induced megamitochondria
formation and apoptosis. Free Radic. Biol.
Med. 26:396409
Goldin RD, Ratnayaka ID, Breach CS,
Brown IN, Wickramasinghe SN. 1996.
Role of macrophages in acetaminophen
(paracetamol)-induced hepatotoxicity. J.
Pathol. 179:43235
Laskin DL, Pilaro AM. 1986. Potential role of activated macrophages in
acetaminophen hepatotoxicity. I. Isolation and characterization of activated
macrophages from rat liver. Toxicol. Appl.
Pharmacol. 86:20415
Zhang H, Cook J, Nickel J, Yu R, Stecker
K, et al. 2000. Reduction of liver Fas expression by an antisense oligonucleotide
protects mice from fulminant hepatitis.
Nat. Biotechnol. 18:86267
Blazka ME, Elwell MR, Holladay SD,
Wilson RE, Luster MI. 1996. Histopathology of acetaminophen-induced liver
changesrole of interleukin-1-alpha
and tumor-necrosis-factor-alpha. Toxicol.
Pathol. 24:18189
Ito Y, Abril ER, Bethea NW, McCuskey
RS. 2004. Role of nitric oxide in hepatic microvascular injury elicited by acetaminophen in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 286:G6067
Kenna JG. 1997. Immunoallergic druginduced hepatitis: lessons from halothane.
J. Hepatol. 26(Suppl. 1):512
Vergani D, Mieli-Vergani G, Alberti A,
Neuberger J, Eddleston AL, et al. 1980.
Antibodies to the surface of halothanealtered rabbit hepatocytes in patients
with severe halothane-associated hepatitis. New Engl. J. Med. 303:6671
Martin JL, Kenna JG, Martin BM,
Thomassen D, Reed GF, Pohl LR. 1993.
Halothane hepatitis patients have serum
antibodies that react with protein disulfide
isomerase. Hepatology 18:85863
Martin JL, Pumford NR, LaRosa AC,
Martin BM, Gonzaga HM, et al. 1991.
A metabolite of halothane covalently
38.
39.
40.
41.
42.
43.
44.
45.
199
binds to an endoplasmic reticulum protein that is highly homologous to

phosphatidylinositol-specific phospholipase C-alpha but has no activity. Biochem.
Hayden PJ, Ichimura T, McCann DJ,
Pohl LR, Stevens JL. 1991. Detection
of cysteine conjugate metabolite adduct
formation with specific mitochondrial
proteins using antibodies raised against
halothane metabolite adducts. J. Biol.
Chem. 266:1841518
Bourdi M, Chen W, Peter RM, Martin JL,
Buters JT, et al. 1996. Human cytochrome
P450 2E1 is a major autoantigen associated with halothane hepatitis. Chem. Res.
Toxicol. 9:115966
Lohse AW, Manns M, Dienes HP, Meyer
zum Buschenfelde KH, Cohen IR. 1990.
Experimental autoimmune hepatitis: disease induction, time course and T-cell reactivity. Hepatology 11:2430
Lohse AW, Brunner S, Kyriatsoulis A,
Manns M, Meyer zum Buschenfelde KH.
1992. Autoantibodies in experimental autoimmune hepatitis. J. Hepatol. 14:48
53
Lecoeur S, Andre C, Beaune PH. 1996.
Tienilic acid-induced autoimmune hepatitis: anti-liver and -kidney microsomal type
2 autoantibodies recognize a three-site
conformational epitope on cytochrome
P4502C9. Mol. Pharmacol. 50:32633
Eliasson E, Kenna JG. 1996. Cytochrome
P450 2E1 is a cell surface autoantigen
in halothane hepatitis. Mol. Pharmacol.
50:57382
Belloc C, Gauffre A, Andre C, Beaune
PH. 1997. Epitope mapping of human
CYP1A2 in dihydralazine-induced autoimmune hepatitis. Pharmacogenetics 7:
18186
Kitteringham NR, Kenna JG, Park BK.
1995. Detection of autoantibodies directed against human hepatic endoplasmic reticulum in sera from patients with
halothane-associated hepatitis. Br. J. Clin.
Pharmacol. 40:37986
3 Dec 2004
20:38

200
AR
AR232-PA45-07.tex
AR232-PA45-07.sgm
LaTeX2e(2002/01/18)
P1: GCE
PARK ET AL.
46. Naisbitt DJ, Britschgi M, Wong G, Farrell J, Depta JP, et al. 2003. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype,
and cytokine profile of drug-specific T cell
clones. Mol. Pharmacol. 63:73241
47. Mitchell JR, Zimmerman HJ, Ishak KG,
Thorgeirsson UP, Timbrell JA, et al. 1976.
Isoniazid liver injury: clinical spectrum,
pathology, and probable pathogenesis.
Ann. Intern. Med. 84:18192
48. Zimmerman HJ. 1990. Update of hepatotoxicity due to classes of drugs in common
clinical use: non-steroidal drugs, antiinflammatory drugs, antibiotics, antihypertensives, and cardiac and psychotropic
agents. Semin. Liver Dis. 10:32238
49. Zimmerman HJ, Ishak KG. 1995. Morphologic spectrum of drug-induced hepatic disease. Gastroenterol. Clin. North
Am. 24:75986
50. Durand F, Bernuau J, Pessayre D, Samuel
D, Belaiche J, et al. 1995. Deleterious influence of pyrazinamide on the outcome
of patients with fulminant or subfulminant liver failure during antituberculous
treatment including isoniazid. Hepatology 21:92932
51. Ormerod LP, Skinner C, Wales J. 1996.
Hepatotoxicity of antituberculosis drugs.
Thorax 51:11113
52. Schaberg T, Rebhan K, Lode H. 1996.
Risk factors for side-effects of isoniazid,
rifampin and pyrazinamide in patients
hospitalized for pulmonary tuberculosis.
Eur. Respir. J. 9:202630
53. Nelson SD, Mitchell JR, Timbrell JA,
Snodgrass WR, Corcoran GB 3rd. 1976.
Isoniazid and iproniazid: activation of
metabolites to toxic intermediates in man
and rat. Science 193:9013
54. Sarich TC, Zhou T, Adams SP, Bain AI,
Wall RA, Wright JM. 1995. A model
of isoniazid-induced hepatotoxicity in
rabbits. J. Pharmacol. Toxicol. Methods
34:10916
55. Rozwarski DA, Grant GA, Barton DH,
Jacobs WR Jr, Sacchettini JC. 1998. Mod-
56.
57.
58.
59.
60.
61.
62.
63.
ification of the NADH of the isoniazid

target (InhA) from Mycobacterium tuberculosis. Science 279:98102
Rawat R, Whitty A, Tonge PJ. 2003.
The isoniazid-NAD adduct is a slow,
tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase:
adduct affinity and drug resistance. Proc.
Fontana RJ, McCashland TM, Benner
KG, Appelman HD, Gunartanam NT,
et al. 1999. Acute liver failure associated
with prolonged use of bromfenac leading
to liver transplantation. The Acute Liver
Failure Study Group. Liver Transpl. Surg.
5:48084
Kenny JR, Maggs JL, Meng X, Sinnott
D, Clarke SE, et al. 2004. Syntheses and
characterisation of the acyl glucuronide
and hydroxy metabolites of diclofenac. J.
Med. Chem. 47:281625
Grillo MP, Hua F, Knutson CG, Ware JA,
Li C. 2003. Mechanistic studies on the
bioactivation of diclofenac: identification
of diclofenac-S-acyl-glutathione in vitro
in incubations with rat and human hepatocytes. Chem. Res. Toxicol. 16:141017
Shen S, Marchick MR, Davis MR, Doss
GA, Pohl LR. 1999. Metabolic activation
of diclofenac by human cytochrome P450
3A4: role of 5-hydroxydiclofenac. Chem.
Tang W, Stearns RA, Bandiera SM, Zhang
Y, Raab C, et al. 1999. Studies on cytochrome P-450-mediated bioactivation
of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites. Drug Metab. Dispos.
27:36572
Masubuchi Y, Ose A, Horie T. 2002.
Diclofenac-induced
inactivation
of
CYP3A4 and its stimulation by quinidine. Drug Metab. Dispos. 30:114348
Kretz-Rommel A, Boelsterli UA. 1993.
Diclofenac covalent protein binding is
dependent on acyl glucuronide formation and is inversely related to P450mediated acute cell injury in cultured rat
3 Dec 2004
20:38
AR
AR232-PA45-07.tex
AR232-PA45-07.sgm
LaTeX2e(2002/01/18)
P1: GCE
64.

65.
66.
67.
68.
69.
70.
71.
72.
hepatocytes. Toxicol. Appl. Pharmacol.

120:15561
Boelsterli UA. 2003. Mechanistic Toxicology. Chapter 9:202. London: Taylor &
Francis
Hargus SJ, Martin BM, George JW, Pohl
LR. 1995. Covalent modification of rat
liver dipeptidyl peptidase IV (CD26) by
the nonsteroidal anti-inflammatory drug
diclofenac. Chem. Res. Toxicol. 8:993
96
Tolman KG, Chandramouli J. 2003. Hepatotoxicity of the thiazolidinediones. Clin.
Liver Dis. 7:36979, vi
Kassahun K, Pearson PG, Tang W, McIntosh I, Leung K, et al. 2001. Studies on
the metabolism of troglitazone to reactive
intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission.
Tettey JN, Maggs JL, Rapeport WG, Pirmohamed M, Park BK. 2001. Enzymeinduction dependent bioactivation of
troglitazone and troglitazone quinone in
vivo. Chem. Res. Toxicol. 14:96574
Satoh H, Dilone E, Nangia A, Zhao S,
Frank K. 2000. Covalent adducts in human primary hepatocytes cultured with
[14C]-troglitazone. Drug Metab. Rev.
32(Suppl. 2):204
Prabhu S, Fackett A, Lloyd S, McClellan
HA, Terrell CM, et al. 2002. Identification
of glutathione conjugates of troglitazone
in human hepatocytes. Chem. Biol. Interact. 142:8397
Haskins JR, Rowse P, Rahbari R, de
la Iglesia FA. 2001. Thiazolidinedione
toxicity to isolated hepatocytes revealed
by coherent multiprobe fluorescence microscopy and correlated with multiparameter flow cytometry of peripheral leukocytes. Arch. Toxicol. 75:42538
Watanabe I, Tomita A, Shimizu M, Sugawara M, Yasumo H, et al. 2003. A study
to survey susceptible genetic factors responsible for troglitazone-associated hep-
73.
74.
75.
76.
77.
78.
79.
80.
201
atotoxicity in Japanese patients with type

2 diabetes mellitus. Clin. Pharmacol.
Ther. 73:43555
Jayyosi Z, Muc M, Gallagher M, Toutain
H, Stevens J, Kelley M. 1999. Covalent binding of troglitazone and other
members of the thiazolidinedione family
to human liver microsomes, implications
for cases of idiosyncratic hepatotoxicity.
Proc. N. Am. ISSX Meet., 9th, Nashville,
Oct. 2428, p. 200
Smith MT. 2003. Mechanisms of troglitazone hepatotoxicity. Chem. Res. Toxicol.
16:67987
Evans DC, Watt AP, Nicoll-Griffith DA,
Baillie TA. 2004. Drug-protein adducts:
an industry perspective on minimizing the
potential for drug bioactivation in drug
discovery and development. Chem. Res.
Toxicol. 17:316
Gupta S, Rogers LK, Taylor SK,
Smith CV. 1997. Inhibition of carbamyl
phosphate synthetase-I and glutamine
synthetase by hepatotoxic doses of acetaminophen in mice. Toxicol. Appl. Pharmacol. 146:31727
Bulera SJ, Birge RB, Cohen SD, Khairallah EA. 1995. Identification of the mouse
liver 44-kDa acetaminophen-binding
protein as a subunit of glutamine synthetase. Toxicol. Appl. Pharmacol. 134:
31320
Ribeiro JM, Costas MJ, Cameselle JC.
1999. ADP-ribose pyrophosphatase-I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro
by dithiothreitol. J. Biochem. Mol. Toxicol. 13:17177
Ozdemirler G, Aykac G, Uysal M, Oz
H. 1994. Liver lipid peroxidation and
glutathione-related defence enzyme systems in mice treated with paracetamol. J.
Appl. Toxicol. 14:29799
Shirota FN, DeMaster EG, Shoeman DW,
Nagasawa HT. 2002. Acetaminopheninduced suppression of hepatic AdoMet
synthetase activity is attenuated by
3 Dec 2004
20:38
202

81.
82.
83.
84.
85.
86.
87.
88.
89.
AR
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PARK ET AL.
prodrugs of L-cysteine. Toxicol. Lett. 132:
18
Senter PD, Al-Abed Y, Metz CN, Benigni F, Mitchell RA, et al. 2002. Inhibition of macrophage migration inhibitory
factor (MIF) tautomerase and biological activities by acetaminophen metabolites. Proc. Natl. Acad. Sci. USA 99:144
49
Pumford NR, Halmes NC, Martin BM,
Cook RJ, Wagner C, Hinson JA. 1997. Covalent binding of acetaminophen to N-10formyltetrahydrofolate dehydrogenase in
mice. J. Pharmacol. Exp. Ther. 280:5015
Bruno MK, Khairallah EA, Cohen SD.
1998. Inhibition of protein phosphatase
activity and changes in protein phosphorylation following acetaminophen exposure in cultured mouse hepatocytes. Toxicol. Appl. Pharmacol. 153:11932
Pierce RH, Franklin CC, Campbell JS,
Tonge RP, Chen W, et al. 2002. Cell
culture model for acetaminophen-induced
hepatocyte death in vivo. Biochem. Pharmacol. 64:41324
Daya S, Anoopkumar-Dukie S. 2000. Acetaminophen inhibits liver trytophan-2,3dioxygenase activity with a concomitant
rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic
acid. Life Sci. 67:23540
Landin JS, Cohen SD, Khairallah EA.
1996. Identification of a 54-kDa mitochondrial acetaminophen-binding protein as aldehyde dehydrogenase. Toxicol.
Appl. Pharmacol. 141:299307
Halmes NC, Hinson JA, Martin BM, Pumford NR. 1996. Glutamate dehydrogenase
covalently binds to a reactive metabolite
of acetaminophen. Chem. Res. Toxicol.
9:54146
Parmar DV, Ahmed G, Khandkar MA,
Katyare SS. 1995. Mitochondrial ATPase:
a target for paracetamol-induced hepatotoxicity. Eur. J. Pharmacol. 293:22529
Tsokos-Kuhn JO, Hughes H, Smith CV,
Mitchell JR. 1988. Alkylation of the
liver plasma membrane and inhibition
90.
91.
92.
93.
94.
95.
96.
97.
98.
of the Ca2+ ATPase by acetaminophen.

Corcoran GB, Chung SJ, Salazar DE.
1987. Early inhibition of the Na+/K+ATPase ion pump during acetaminopheninduced hepatotoxicity in rat. Biochem.
Qiu Y, Benet LZ, Burlingame AL. 1998.
Identification of the hepatic protein targets
of reactive metabolites of acetaminophen
in vivo in mice using two-dimensional gel
electrophoresis and mass spectrometry. J.
Biol. Chem. 273:1794053
Fountoulakis M, Berndt P, Boelsterli
UA, Crameri F, Winter M, et al. 2000.
Two-dimensional database of mouse
liver proteins: changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3-acetamidophenol. Electrophoresis
21:214861
Gruchalla RS, Sullivan TJ. 1991. Detection of human IgE to sulfamethoxazole
by skin testing with sulfamethoxazoylpoly-L-tyrosine. J. Allergy Clin. Immunol.
88:78492
Farrell J, Naisbitt DJ, Drummond NS,
Depta JP, Vilar FJ, et al. 2003. Characterization of sulfamethoxazole and
sulfamethoxazole metabolite-specific Tcell responses in animals and humans. J.
Madden S, Maggs JL, Park BK. 1996.
Bioactivation of carbamazepine in the rat
in vivo. Evidence for the formation of reactive arene oxide(s). Drug Metab. Dispos. 24:46979
Boelsterli UA. 2003. Diclofenac-induced
liver injury: a paradigm of idiosyncratic
drug toxicity. Toxicol. Appl. Pharmacol.
192:30722
Scheen AJ. 2001. Hepatotoxicity with thiazolidinediones: is it a class effect? Drug
Saf. 24:87388
Graham DJ, Green L, Senior JR, Nourjah
P. 2003. Troglitazone-induced liver failure: a case study. Am. J. Med. 114:299
306
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c 2005 by Department of Health, Canada
Copyright

NATURAL HEALTH PRODUCTS AND DRUG

DISPOSITION
Brian C. Foster,1,2 J. Thor Arnason,2 and Colin J. Briggs3
1
Therapeutic Products Directorate, Health Canada, Holland Cross 3102C3, Ottawa,

Ontario, Canada, K1A 1B6; email: brian foster@hc-sc.gc.ca
2
Center for Research in Biopharmaceuticals and Biotechnology, University of Ottawa,
Ottawa, Ontario, Canada, K1N 6N5; email: jarnason@science.uottawa.ca
3
Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2;
email: cbriggs@umanitoba.ca
Key Words herbal, metabolism, transport, cytochrome P450

Abstract Botanicals such as herbal products (HPs) and nutraceuticals (NCs) are
often regarded as low risk because of their long history of human use. Anecdotal and
literature reports of adverse drug events (ADEs) and clinical studies with HPs are
increasing, but many of the reports are incomplete and contradictory. These reports
need to identify confounding factors and explain contradictory findings if they are to
help health care professionals or patients understand what risks are involved. HPs are
complex botanicals, not single-active ingredient (SAI) products. Studies can be confounded by different manufacturing processes and formulations, including cosmetics
and food supplements; environment; chemotypes; misidentification or adulteration;
and factors associated with the patient or user population such as use, total drug
load, and genetics. Future studies need to be conducted with characterized product
that includes all commercially available related products. Clinical trials should be relevant to the user population and take into account the confounding factors that may
influence the interpretation of the findings.
OVERVIEW
Plant products contain bioactive phytochemicals that are finding increasing importance in foods as NCs and in HPs as medicinal principles. HPs are a very
diverse category of plant products and extracts; for example, they are known as
Abbreviations used in text: ADE, adverse drug event; AUC, area-under-the-plasmaconcentration-time curve; CAM, complementary and alternative medicine; Cmax, maximum concentration; Cmin, minimum concentration; CYP, cytochrome P450; EROD,
7-ethoxyresorufin O-deethylation; GST, glutathione S-transferase; HPs, herbal products;
MIC, minimal inhibitory concentration; NCs, nutraceuticals; NHPs, natural health products; Pgp, P-glycoprotein; PK, pharmacokinetic; P450, cytochrome P450; TM, traditional
medicine; SAI, single-active ingredient; SJW, St. Johns wort; UGT, uridine diphosphoglucuronosyl transferase.
203
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dietary supplements (United States), NHPs (Canada), phytomedicines (Europe),

and traditional medicines (developing countries). HPs and NCs present many
unique challenges that can confound pharmaceutical scientists and others alike
these are complex mixtures, not SAI products, having multiple pharmacological
properties. The different regulatory processes in different jurisdictions that lead to
different types of products available commercially confound the complexity. In the
United States, HPs are sold as largely unregulated and untested supplements, and
only structure function information but no therapeutic claims are permitted under
the Dietary Supplements Health and Education Act (DSHEA) legislation of 1994.
In Canada and under a somewhat similar system in Australia, NHPs are given a Natural Product Number for limited therapeutic claims for over-the-counter use based
on established traditional use or supportive data and will be formally regulated
with stricter controls on manufacturing and labeling. In Europe, phytomedicines
are strictly regulated as drugs under the European Scientific Cooperative on Phytotherapy (ESCOP). Japan has a system in which some products are regulated as
foods and others as drugs. In developing countries, the World Health Organization
reports that approximately 80% of the world populations rely on TMs, mainly of
herbal sources, in their primary healthcare (1). Indications for TMs in developing
countries include more serious conditions (malaria, AIDS, parasitic diseases, etc.)
than HPs in the developed countries, which are usually indicated as self-care products. The popularity of over-the-counter HPs, NCs, and medicinal products from
plants or other natural sources has increased dramatically in developed countries
and is one of the reasons for the present review. Whereas many purified botanical
marker substances have been used or examined as potential pharmaceuticals, these
SAIs are neither HPs nor NCs and will not be considered here.
Despite the popular believe that NPs are safe, these products are pharmacologically active and have inherent risk. Although the risk may be low in many cases
where the product is used alone, of particular interest here are the many interactions
that have been reported with enzymes affecting drug disposition. These include
CYP 3A4 (28), 1A1 (913), 1A2 (4, 8, 1215), 1B1 (16), 2A1, 2B (1213),
2C (8, 13, 1720), 2D6 (8, 1721), 2E1 (4, 8, 21, 2324), 3A1 (13), 3A5/7 (18
20), 4A/F (15, 25), 19 (26), P-glycoprotein (MDR1, ABCB1: 2733), MRP1 (33),
MRP2 (33), cyclooxygenase I and II (34), flavin-containing monooxygenease (35),
glutathione S-transferase P1-1 (12, 14, 36), N-acetyltransferase (37), monoamine
oxidase B (38), steroid X receptor (39), and uridine diphosphoglucuronosyl transferase (12, 40). This review provides a better understanding of what constitutes
representative products and confounding factors affecting interpretation of these
interactions.
Adverse effects, sometimes life threatening, have been associated with HPs or
traditional medicines contaminated with excessive or banned pesticides, microbial
contaminants, heavy metals, chemical toxins, or adulterated with orthodox drugs
(1, 6, 41). Contamination may be related to the source of these herbal materials.
Mycotoxins may arise during growth of fungi from unfavorable or improper storage
conditions. Many examples of adulteration and substitution exist. For example, the
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substitution of Eleutherococcus senticoussus by Chinese silk vine led to a case of

neonatal androgenization (42). In recent years, a serious situation occurred with
the adulteration of Stephania tetrandra with Aristolochia fangchi, which contains
nephrotoxic and carcinogenic aristolochic acids.

Natural Product Variation

Except perhaps in jurisdictions such as Europe and Japan, or more recently in
Canada and Australia, HPs generally lack the stringent quality assurance and
regulatory oversight of therapeutic products. Unlike SAIs, botanical raw material
may be sourced from several regions or countries and may have unique genotypic
and phenotypic characteristics that may confound product selection for clinical
examination. Examination of one or even a few samples may inadvertently lead to
testing of a single chemotype. A chemotype is a population of plants belonging to
a particular species that differs chemically from others of that species, although the
plants look identical. For example, Binns et al. (43) recently identified a distinct
chemotype of the popular herb Echinacea angustifolia. The clinical effects of
products containing different chemotypes (4446) may vary.
HPs and NCs may contain constituents from many biosynthetic classes of phytochemicals (Table 1). Phytochemical diversity and redundancy can result in plant
species having several different classes of phytochemicals (diversity) and multiple analogs of each biosynthetic type (redundancy). An example is valerian
root, used as a mild sedative, tranquilizer, and sleep inducer (47). The major constituents include approximately 0.4%1.4% monoterpenes; sesquiterpenes, including -bisabolene, caryophyllene, valerianol, valeranone, pacifigorgiol; patchouli
alcohol; valerenol; valerenyl esters; valerenal; valerenic acid (with acetoxy and
hydroxy derivatives); caffeic acid; gamma-aminobutyric acid; chlorogenic acid;
-sitosterol, methyl 2-pyrrolketone; choline; hydroxypinoresinol tannins; gum;
volatile oils; and resin. Shohet et al. (48) examined 31 commercial valerian preparations available in Australia and found substantial product heterogeneity of the
marker phytochemicals, valerenic acid and its derivatives, ranging from <0.01
to 6.32 mg/g of product. Powdered capsules, on average, contained the highest
concentration (2.46 mg/g) and liquids the lowest concentration (0.47 mg/ml). The
mean concentration of these markers in 5 standardized products (3.56 mg/g) was
significantly higher than in the 26 nonstandardized products (0.89 mg/g). Valepotriates were found at low levels (<1 mg/g) in some teas but were not detected in
any of the finished products.
Other sources of inherent variation include environmental conditions during
growth, harvest and storage conditions, and manufacturing and compounding
processes. The presence of nonactive conjugates that are converted to an active
moiety is another source of variation. Seasonal and environmental variation has
been shown to affect the essential oil extracts from the aerial parts of Santolina
rosmarinifolia (49) and Hypericum perforatum accessions grown at three experimental sites in Switzerland followed over a two-year period (19951996) (50).
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TABLE 1
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Botanical sources of chemicals implicated in interactions
Chemotaxonomic
category
Typical interacting
compounds
Monoterpenes
Limonene, sobrerol, geraniol
Green and yellow vegetables,

cereals, grains, citrus peel oils,
celery seed oil, herb extracts
Organosulfur
compounds
Diallyl sulfides
Onions, garlic, leeks, shallots
Isothiocyanates
Cruciferous vegetables,
horseradish, radishes
Flavonoids (genistein,
naringenin)
Green and yellow vegetables and

fruits, soy products, berries,
onions, garlic, citrus fruits,
licorice, spices
Theaflavins
Catechins
Curcuminoids (curcumin)
Gingerols and diarylheptanoids
Hydroxycoumarins
Black tea leaves

Green tea leaves, berries
Turmeric root, curry products
Ginger
Umbelliferae, horse chestnut,
chamomile
Grapefruit, Earl Gray tea, rue,
celery
Cinnamon, coffee beans, soybeans,
grapes, strawberries, raspberries
Cruciferous vegetables
Sesame seeds and oil
All plants and vegetables
Phenolics
polyphenols
Furanocoumarins
Acids (cinnamic, ellagic, sinapic)
Indoles
Lignans (sesamol, sesaminol)
Chlorophyll derivatives
(chlorophyllin)
Hypericin, hypericum
Botanical source
St. Johns wort
Tocopherols
tocotrienols
-Tocopherol (vitamin E),

-tocotrienol

cereals, grains, citrus peel oils,
celery seed oil, herb extracts,
mint oil
Triterpenes
Liminoids (limonene, ichangin)

Plant sterols (phytosterols)
Saponins/sapogenins
Glycyrrhetic acid and derivatives
Citrus fruits
fruits, grains and cereals,
soybeans
Ginseng (leaves, roots), soybeans
Licorice root
Gluten, fiber
Protease inhibitors, phytic acid
Phthalides
Whole grains
Soybeans
Celery, parsley, carrots
Others
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Differences were found in constituents in H. perforatum aerial parts (51). Some

plant constituents are sugar conjugates (glycosides) that are generally unable to
affect drug metabolism. Soybeans were analyzed by HPLC for the isoflavones
daidzein and genistein and their respective glycoside derivatives to determine if
amounts of these compounds could reliably predict the activity of the variety or
year (20). Genistein ranged from 5.8 to 28.7 g/g and daidzein from 0 to 42.6 g/g.
The glycosides daidzin and genistin were present in much larger amounts, ranging
from 198 to 792 and 458 to 1261 g/g, respectively. The free aglycones accounted
for less than 7% of the total in these samples. Neither the concentration of the individual compounds nor their total correlated with inhibition of 3A4 across genotypes
and years. In addition, there are individual variations in the amount taken, dosage
form, preparation, length of use (first time or repeated use), combination with other
products, genotype, and health status of the user.
HPs and NCs may be fresh or prepared botanicals, distillates, or extracts. For
example, the processing procedures for garlic can be broadly classified into four
categories: dried or dehydrated without enzyme deactivation, aqueous or oil extraction, distillation, and heating including frying and boiling (52). The products
are formulated as oils of steam-distilled garlic, garlic macerated in vegetable oils,
garlic powder, or gelatinous suspensions. The variation in composition as well as
the instability of some constituents poses serious problems for standardization and
comparison between related products.
Labeling Information
Information printed on some labels for products such as Echinacea, SJW, and
valerian root state that the products were standardized. SJW may be standardized to 0.3% hypericin or 4% hyperforin. Some labels mention hypericins. Wide
variation in hyperforin (0.006%2.64%), hypericin (0.008%0.08%), and pseudohypericin (0.014%0.19%) content was observed in tested products (52a). In
these products, the 0.3% hypericin standard was only approached (0.26%) when
total hypericin content was determined. Valerian root labels mention valerenic
acid, valerenic acids, and valeric acid (52b). This is indicative of the confusion created by the industry, as valeric acid is a five-carbon molecule that is
not related to the much larger valerenic acids. Silymarin is considered the active constituent of the milk thistle seed. It is a mixture of several flavonolignans, including silibinin (silybin A and B), isosilybinin, silichristin (silychristin),
and silidianin. Constituent analysis of five milk thistle extract products identified six major constituents: 3.3% taxifolin, 23.6% silychristin, 5.3% silidianin,
20% silybin A, 30.7% silybin B, and 17.3% isosilybin (B.C. Foster, C.E. Drouin,
J.F. Livesey, J.T. Arnason & E. Mills, unpublished findings). The total amounts
of silybin A and B ranged from 45.7 to 61%. The biological effect of each constituent is not known. Spectrophotometric analysis as used by many producers and
investigators for quality control would not provide sufficient information for a
critical comparison of these products.
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Another example of how labels may be misleading or confusing is the term

Echinacea. E. angustifolia (syn E. pallida var. angustifolia) and E. purpurea are
widely used as botanical medicines (43). A third species E. pallida (E. pallida var.
pallida) has been widely used in Europe. A second example is Echinacea where
the whole plant has been used for therapeutic purposes, but products can consist of
E. purpurea herb extracts; combination root and herb extracts; root extracts of the
three-species, single-entity, blended herbal teas with other botanicals; and blends
of E. purpurea and E. angustifolia leaves, stems, and flowers plus a dry extract
of E. purpurea roots. The constituents of Echinacea include alkamides, caffeic
acid derivatives, glycoproteins/polysaccharides, and ketoalkenynes (43, 5354),
but label information indicated that standardization is focused on phenols (4%) or
4% echinacoside. Echinacoside is only a marker for E. angustifolia. Echinacoside
and cichoric acid (Table 2) were not detected in some products (R.K. Drobitch,
A. Krantis, M. Panahi, J.T. Arnason, K. Kramp, F.J. Burczynski, C. Briggs, P. Jiang
& B.C. Foster, unpublished data). All extracts markedly inhibited CYP-mediated
metabolism (Table 2). The findings with soft gel products were the most variable.
Aliquots of the four soft gel products had moderate to high activity toward CYP2D6
and 3A4, but only NRP 69 and 72 had an inhibitory effect against CYP2C9. In
addition, NRP 71 did not inhibit CYP2C19-mediated metabolism.
Confounding Factors in Experimental Evaluations

Small changes in the lipophilic (or polar) nature of the extraction solvents used in
assays can greatly alter the results of the assays. A garlic product was extracted
with a sequential series of solvents ranging in lipophilicity from hexane (yellowgreen extract) followed by chloroform (brown-green), ethyl acetate (bright red),
methanol (orange-red), 55% ethanol (light peach color), and finally water (very
faint peach color) (18). Results suggesting the presence of fluorescent substances
were observed when testing the aliquots of ethyl acetate (169.9%) and hexane
(157.0%) extracts against 3A4. The chloroform and methanol extracts also had
high inhibition with values of 97.6% and 87.5%, respectively, but the weaker
solvents in this sequential extraction protocol, 55% ethanol and water, were less
inhibitory (20.6% and 6.3%, respectively). A series of nonsequential extracts also
gave high activity in all extracts. As differences in the inhibitory effect of aqueous and methanolic extracts of fresh and aged garlic cloves on 3A4-mediated
metabolism were noted previously, the three varieties were extracted under four
different conditions. Results varied with variety, but in general, distilled water and
phosphate buffer extracts gave the strongest overall suppression effect in isoformmediated metabolism of marker substrates.
Intrinsic (natural) fluorescence and quenching are confounding variables in
fluorescence-based enzyme inhibition assays of natural products. Zou et al. (55)
measured the fluorescence and quenching properties of 25 components of popular herbal products. These analyses were performed under conditions typically
Tea
Tablet
Tablet
Tincture
Softgel
Softgel
62
73a
74
29
69
71a
Not detected.
5 mg/ml stock solution.
652 g/ml stock solution.
2299
1920
793
ND
ND
ND
NDb
ND
32,423
258
ND
1852
8916
5895
953
5.9 10.19d
54.9 4.54
49.4 6.88
71.9 7.07d
ND
59.0 3.45
52.5 7.73
ND
16.2 2.03
61.2 6.13
85.3 4.93d
65.0 2.01
ND
65.5 1.67
77.1 7.35
52.4 6.42
68.5 0.75
91.4 2.22
49.8 0.75
48.1 1.49
9.3 3.52
41.5 4.03
66.1 0.73
79.9 0.49
64.5 0.38c
3A4
98.2 0.55
60.7 1.23
66.8 1.46
80.0 0.61
91.4 1.17
73.4 3.13
72.6 5.86
2D6
2C19
2C9
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Tea
27
2572
Echinacoside
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10B
Cichoric
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TABLE 2 Biomarker analysis and percentage inhibition of human cytochrome P450-mediated metabolism by
aliquots of 1.25 mg/ml extracts from products containing Echinacea (n 6 SD)

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employed in drug-drug interaction studies using c-DNA-derived P450 isoforms and

surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin,
quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere
with these assays. Intrinsic fluorescence had a greater effect on these assays than
quenching, and for one compound, yangonin, it was sufficient to mask inhibition and potentially produce a false negative result. Quenching was sufficient with
quercetin, to mimic weak inhibition. The intrinsic fluorescence or quenching
capacity of HPs may confound certain fluorometric assays, making it imperative
that proper controls are included in the evaluation studies on these products. The
degree of interference may preclude the assay or require supporting evidence from
chromatographic assays where this factor can be separated from the test substrate
and metabolite.
Dissolution of constituents from teas is an important consideration (5658).
Dissolution rates are routinely performed with synthetic drugs; however, with HPs
this crucial property is often not investigated. Using procedures of the European
Pharmacopoeia, Taglioli et al. (58) evaluated the dissolution behavior of capsules
containing various herbal drugs (Passira, Senna, Ginkgo) manufactured by different methods. Active components or marker constituents were analyzed. Adequate
dissolution behaviors of the flavonoids of Ginkgo were obtained for all preparations, whereas for Passiflora and Senna only the extracts showed a complete
dissolution of the marker flavones and sennosides, respectively. Three different
patterns were noted when four HPs were examined using tea bag infusions (20).
The initial 10-min values for Echinacea Special tea were higher than the other
NHPs, but the inhibition curve only increased slightly with time. Goldenseal herb,
Echinacea and Goldenseal had low initial values that nearly doubled after a second 10-min incubation. The third pattern with Feverfew showed a linear increase
in marker extraction through the incubation period. Visual examination of these
products found inter- and intraproduct differences in particulate size, ranging from
fine powder to substantially intact leaves and stems. As product dissolution varies
between products, it is expected that there will be clinical differences based on
the amount and temperature of the water, extent of agitation, and time the teas are
allowed to brew.
Stability
Bilia et al. (5960) investigated the stability of 40% and 60% v/v tinctures of
artichoke, SJW, Calendula flower, Milk thistle fruit, and Passionflower. Stability
was related both to the class of flavonoids and water content of the tinctures. Shelf
life at 25 C of the most stable tincture (Passionflower 60% v/v) was approximately
six months, whereas that of the Milk thistle tinctures was approximately three
months. Budzinski et al. (61) examined 21 tinctures and noted that there was
marked variation in the ability of these products to inhibit CYP3A4-mediated
metabolism. Several clinical trials have subsequently been conducted on some
of the products examined in this study. Based on the stability concerns (5960)
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it would be interesting to revisit this area to examine freshly manufactured and

expired commercial products.
Thermal and photostability of a commercial dried extract and capsules of SJW
were also evaluated (59). Photostability testing showed all the constituents to be
photosensitive in the tested conditions. However, different opacity agents and pigments influenced stability. Amber containers had little effect on the photostability
of the investigated constituents. Assays should be performed under reduced or F40
gold fluorescent lighting to minimize potential for photodecomposition or activation (18). Long-term thermal stability testing showed a shelf life of less than four
months for hyperforins and hypericins, even when ascorbic and citric acids were
added to the formulation.
SELECTED EXAMPLES
Citrus
The potential for some members of the Rutaceae family, such as grapefruit, lime,
and Seville orange, to interact with 3A4 and Pgp and affect PK has been extensively
examined (2, 48, 10, 6267). The mechanisms involved are understood in part.
Activity was initially attributed to bioflavonoids and then to furanocoumarins. Furanocoumarin derivatives can be characterized into two main groups: angular with
a furan ring attached to 7,8-position or linear with the furan ring at 6,7-position with
methoxy, prenyloxy, and geranlyoxy substitution at 5- and/or 8-position. Some, but
not all are inhibitory (10). In grapefruit, geranyloxy derivatives of furanocoumarins
(psoralens) are thought to be involved in competitive or mechanism-based inhibition (10).
What is generally poorly understood is that furanocoumarins, natural lightactivated toxins that protect plants from herbivores such as insects and microbes,
are also present in plants belonging to the Fabaceae (legumes), Moraceae (fig and
mulberry), and Apiaceae (carrot, celery) families (10). Hot water decoctions or
40% ethanol infusions from Apiaceae: Baizhi (Angelica dahurica and varieties),
Qianghuo (Notopterygium incisum or N. forbesii), Duhuo (Angelica biserrata),
Fangfeng (Saposhnikovia divaricata), Danggui (Angelica sinensis), and Rutaceae:
Zhishi or Zhiqiao (Citrus aurantium) resulted in various degrees of human CYP3A
inhibition as determined by microsomal testosterone 6-hydroxylation (67). The
inhibitory potency was consistent with the abundance of the hydrophobic components for each sample. Some products showed increased inhibition after preincubation, suggesting mechanism-based inhibition. Some formulated prescriptions,
however, showed intense inhibition with their hydrophilic fractions rather than
with their hydrophobic fractions, suggesting that components other than furanocoumarins in herbal prescriptions may also cause CYP3A inhibition. Studies
suggest that furanocoumarins can inhibit or induce a wide range of P450s in
addition to CYP3A4, such as CYP1a1, CYP1A2, Cyp1b1, Cyp2a5, CYP2A6,
CYP2B1, CYP6B1/3, CYP6B4, and CYP6D1 (10).
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Cranberry Juice

Cranberry juice (Vaccinium macrocarpon) is a popular drink that has been used
to reduce or prevent urinary infections. Five reports suggesting changes in the
International Normalized Ratio (INR) values for the Prothrombin Time have been
received by the Committee on Safety of Medicines, indicating an interaction between cranberry juice and warfarin, including one fatal case (68).
Garlic
Garlic (Allium sativum L.) and garlic products generally have been regarded as
safe, but conflicting reports in the literature make it difficult to unequivocally
establish the clinical efficacy and safety of these products either alone or in the
presence of therapeutic products. One case report identified two HIV-infected persons taking garlic or garlic supplements for more than two weeks who developed
severe gastrointestinal toxicity after beginning ritonavir-containing antiretroviral
therapy (400 or 600 mg twice daily) (69). The symptoms, including nausea, vomiting, and diarrhea, resolved with discontinuation of garlic or ritonavir. A recent
study analyzed 24 representative garlic products, including three fresh garlic bulbs
(P.S. Ruddock, M. Liao, B.C. Foster, L. Lawson, J.T. Arnason & J.R. Dillon, unpublished data). Interestingly, within the odorless garlic entities, the range for the
allicin/alliin ratio varied from 0 to 4.7. The major biomarker varied with manufactured product, and the constituent content did not correlate with the in vitro
inhibition of CYP-mediated metabolism. The results were consistent with earlier findings on their inhibitory effect on CYP 2C9 1, 2C9 2, 2C19, 2D6, and
3A-mediated metabolism (45). Extracts from garlic exhibited a similar inhibitory
effect on all 3A isoforms. Chinese and elephant garlic (Allium ampeloprasum) had
a lesser inhibitory effect on 3A7; Chinese garlic extracts also had a lesser effect
on the 3A5 isoform studied. All fresh varieties had a slight inhibitory effect on
2C9 1-mediated metabolism but highly stimulated metabolism of the marker substrate with the 2C9 2 isoform. The extracts had negligible to no effect on 2C19- and
2D6-mediated metabolism. However, all extracts strongly inhibited 3A4-mediated
metabolism. The effects of aqueous extracts from aged garlic capsules and the three
fresh varieties were examined for their ability to interact with human Pgp. Relative to 20 M verapamil as the positive control, the phosphate buffer extracts
of aged, common, and Chinese garlic had moderate levels of product-stimulated
vanadate-sensitive ATPase activity. Elephant garlic was inactive.
The effect of odorless garlic on single-dose pharmacokinetics of ritonavir was
examined in ten healthy volunteers (five male, five female) who received 400 mg
of a single dose of ritonavir either alone or with 10 mg of odorless garlic in a
randomized crossover design (70, 71). Coadministration of garlic decreased the
AUC by 17% (90% CI, 31% to 0%; range 46% to 68%) and decreased peak
plasma concentration of ritonavir by 1% (90% CI, 25% to 31%; range 51% to
136%). Although the trend was toward lower levels of ritonavir, the effect was not
significant. In a longer study, 10 healthy volunteers received 10 doses of saquinavir
at a dosage of 1200 mg 3 times daily with meals for 4 days on study days 14,
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2225, and 3639, and they received a total of 41 doses of garlic caplets taken 2
times daily on study days 525 (72). In the presence of garlic, the mean saquinavir
AUC during the 8-h dosing interval decreased by 51%, trough levels at 8 h after dosing decreased by 49%, and the Cmax decreased by 54%. After the 10-day washout
period, the AUC, trough, and Cmax values returned to 60%70% of their values
at baseline. Markowitz et al. (73) reported contradictory findings with no effect on
2D6-mediated metabolism of dextromethorphan and 3A4-mediated metabolism
of alprazolam with no significant differences in pharmacokinetic parameters at
baseline and after garlic extract treatment.
Foster et al. (74) demonstrated that garlic and SJW could have an antagonistic
or synergistic effect on antibiotics, indicating that herbal effects on host drug
disposition mechanisms may also affect response to antibiotics. Ward et al. (75),
using Staphylococcus aureus ATCC 29,213 or Escherichia coli ATCC 25,922 as
the indicator organisms, showed a general increase in the MIC of ampicillin by the
products they studied. There were 13 product-related increases in the MIC and 2
decreases. All garlic products increased the MIC of norfloxacin-sensitive organism
to greater than fourfold above baseline. With Escherichia coli ATCC 25922, the
greatest product-antibiotic interaction was with the ampicillin-sensitive organism.
Garlic, Echinacea, and zinc products all caused large increases in the MIC to
ampicillin over baseline values.
Ginkgo biloba
The effects of Ginkgo biloba leaf extract on the pharmacokinetics of diltiazem
were examined in rats (76). The simultaneous addition of extract to small intestine
and liver microsomes inhibited the formation of the active N-demethyl metabolite by CYP3A in a concentration-dependent manner with an IC (50) of approximately 50 and 182 g/ml, respectively. After a single oral pretreatment with extract
(20 mg/kg), both the rate of formation of the metabolite and total amount of CYP in
intestinal or hepatic microsomes decreased transiently. Pretreatment significantly
decreased the terminal elimination rate constant and increased the mean residence
time after intravenous administration of diltiazem (3 mg/kg). Furthermore, it significantly increased the AUC and absolute bioavailability after oral administration
of 30 mg/kg. These results indicated that the concomitant use of Ginkgo extract
in rats increased the bioavailability of diltiazem by inhibiting both intestinal and
hepatic metabolism, at least in part, via a mechanism-based inhibition for CYP3A.
A study in healthy volunteers phenotyped as CYP2D6 extensive metabolizers
with Ginkgo biloba (77) concluded that the products used in these studies at the
recommended dose was unlikely to significantly alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathways
for elimination.
Goldenseal
Goldenseal (Hydrastis canadensis Ranunculaceae), a popular herbal supplement
for gastrointestinal ailments, has been shown to affect CYP3A4-mediated
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metabolism and ATPase activity (27, 61). It contains the alkaloids berberine
and hydrastine, hydrastinine, and canadine. Extracts of goldenseal containing
approximately equal concentrations (approximately 17 mM) of two methylenedioxyphenyl alkaloids, berberine and hydrastine, inhibited with increasing potency
(CYP2C9) diclofenac 4 -hydroxylation, (CYP2D6) bufuralol 1 -hydroxylation,
and (CYP3A4) testosterone 6-hydroxylation activities in human hepatic microsomes (17). The inhibition of testosterone 6-hydroxylation activity was noncompetitive, with an apparent Ki of 0.11% extract. Of the methylenedioxyphenyl
alkaloids, berberine (IC50 = 45 M) was a better inhibitor toward bufuralol
1 -hydroxylation and hydrastine (IC50 approximately 350 M for both isomers),
and was an inhibitor toward diclofenac 4 -hydroxylation. For testosterone 6hydroxylation, berberine was the least inhibitory component (IC50 approximately
400 M). Hydrastine inhibited testosterone 6-hydroxylation with IC50 values
for the (+)- and ()-isomers of 25 and 30 M, respectively. For ()-hydrastine,
an apparent Ki value of 18 M without preincubation and an NADPH-dependent
mechanism-based inhibition with a kinactivation of 0.23 min1 and a KI of approximately 110 M were determined. CYP metabolic-intermediate complex formation
could be demonstrated for both hydrastine isomers. Hydrastine formed a CYP complex with CYP2C9, CYP2D6, and CYP3A4. Coexpression of cytochrome b5 with
the CYP isoforms enhanced the rate but not the extent of complex formation.
The pharmacokinetics of indinavir in 10 healthy volunteers before and after
14 days of treatment with goldenseal root (1140 mg twice daily) were not altered
significantly (78).
Three other herbals, barberry (Berberris vulgaris), Oregon grape (Mahonia
aquifolium), and Goldenthread (Coptis groenlandica), also contain measurable
amounts of these compounds (Table 3) and have a long ethnobotanical record in
TABLE 3 The chemical characterization and percentage inhibition of four berberinecontaining botanicals on cytochrome P450-mediated metabolism of three isozymes
Product
BERa
g/ml
HSb
g/ml
HSNc
g/ml
CDNd
g/ml
CYP
3A4
CYP
2C19
CYP
19
Mahonia aquifolium
124
NDe
ND
27.0
13.6
31.7
Coptis trifolia var

groenlandica
ND
135
ND
ND
46.2
24.7
41.3
Hydrastis canadensis
9000
4801
209
46.3
54.0
49.4
60.1
Berberris vulgaris
790
ND
ND
ND
ketoconazole
a
Berberine.
b
c
d
e
Hydrastine.
Hydrastinine.
Canadine.
Not detected.
47.5
25.4
45.5
84.2
40.6
33.9
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North America (R. Leduc, I. Scott, R. Marles, J. Dillon, J.T. Arnason & B.C.
Foster, unpublished findings). The relative amounts of alkaloids varied greatly in
the four species tested, with H. canadensis having considerably higher concentrations of the marker compounds. Extracts of H. canadensis were inhibitory to CYP
3A4, 2C19 and 2C19. C. groenlandica and B. vulgaris were less active, whereas
M. aquifolium extracts were the least inhibitory. These botanicals have the potential
to affect human drug and intermediary metabolism independent of the concentration of the major biomarkers for these botanicals.
Herbal Teas
Herbal and black teas were analyzed for their capacity to inhibit in vitro metabolism
of drug marker substrates by human CYP isoforms (20). Aliquots and infusions of
all products inhibited 3A4 metabolism. Of the aliquots from teas tested with 2C9,
2C19, and 2D6, many demonstrated inhibitory activity. Black teas and herbal tea
mixtures were generally more inhibitory than single-entity herbal teas. Maliakal &
Wanwimolruk (41) investigated the effect of herbal teas (peppermint, chamomile,
and dandelion) on the activity of hepatic Phase I and II metabolizing enzymes using
female rat liver microsomes. After four weeks of pretreatment, CYP isoforms and
Phase II enzyme activities were determined by incubation of liver microsomes or
cytosol with appropriate substrates. Activity of CYP1A2 in the liver microsomes of
rats receiving dandelion, peppermint, or chamomile tea was significantly decreased
(P < 0.05) to 15%, 24%, and 39% of the control value, respectively. CYP1A2
activity was significantly increased by pretreatment with caffeine solution. No
alterations were observed in the activities of CYP2D and CYP3A in any group of
pretreated rats. Activity of CYP2E in rats receiving dandelion or peppermint tea
was significantly lower than in the control group, 48% and 60% of the control,
respectively. There was a dramatic increase (244% of control) in the activity of
UGT in the dandelion teapretreated group. There was no change in the activity of
GST. The results suggested that certain herbal teas can cause modulation of Phase
I and II drug metabolizing enzymes.
Kava
Inhibition of CYP enzymes by kava extract was investigated (15, 79). Whole
kava extract (normalized to 100 M total kavalactones) caused concentrationdependent decreases in P450 activities, with significant inhibition of the activities
of CYP1A2 (56% inhibition), 2C9 (92%), 2C19 (86%), 2D6 (73%), 3A4 (78%),
and 4A9/11 (65%) following preincubation for 15 min; CYP2A6, 2C8, and 2E1
activities were unaffected. These data indicate that kava has a high potential for
causing drug interactions through inhibition of P450 enzymes.
Milk Thistle
Venkataramanan et al. (40) evaluated the effect of silymarin on the activity of
hepatic drug-metabolizing enzymes in human hepatocyte cultures. Treatment with
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silymarin significantly reduced the activity of CYP3A4 enzyme (by 50% and
100%, respectively) as determined by the formation of 6--hydroxy testosterone
and the activity of UGT1A6/9 by 65% and 100%, respectively, as measured by
the formation of 4-methylumbelliferone glucuronide. Silymarin also significantly
decreased mitochondrial respiration in human hepatocytes.
At least three studies have been conducted with products containing milk thistle
(Silybum marianum) extract to characterize the pharmacokinetics of indinavir in
healthy subjects. Piscitelli et al. (80) and DiCenzo et al. (81) concluded that these
silymarin products had no apparent effect on indinavir plasma concentrations. In
a third study with 16 subjects lasting 28 days by Mills et al. (83), the AUC0-8
indinavir was reduced by a mean 4.4% (90% CI, 26% to 27.5%, P = 0.6) from
Phase I to Phase II in the active group, rebounding to a Phase III reduction of 17.3%
(90% CI, 9% to 37.3%, P = 0.6) of baseline. Control AUC0-8 reduced by 21.5%
(90% CI, 8 to 43%, P = 0.2) from Phase I to Phase II and rebounded to a further
reduction at Phase III of 38.5% (90% CI, 15.3% to 55.3%, P < 0.01) of baseline.
This study has important implications for the conduct and design of herb-drug
interaction trials. The significant decline of AUC0-8 in the control group indicates
that factors other than the exposure of interest may affect drug metabolism.
St. Johns wort

The in vitro and clinical effects of SJW on drug disposition and safety have also
been extensively examined (38, 21, 31, 35, 5657, 59, 61, 8395). Additional
studies have shown that SJW can affect CYP-mediated metabolism, transport, cell
viability, and modulate induction of nitric oxide. Ruschitzka et al. (87) clearly
established that concomitant use of SJW with cyclosporin could cause serious
ADEs. Piscitelli (8889) showed that SJW reduced the AUC of indinavir by a
mean of 57% and decreased the extrapolated 8-h indinavir trough by 81% in
healthy volunteers. This could lead to drug resistance and treatment failure. As
with other HPs, there have been supportive (88, 91, 93, 94) and contradictory (90,
92) reports.
Markowitz et al. (94) assessed the potential of SJW with 12 healthy extensive
metabolizers of CYP 2D6 in a 14-day study. A twofold decrease in AUC for
alprazolam plasma concentration versus time (P < 0.001) and a twofold increase
in alprazolam clearance (P < 0.001) were observed following SJW administration.
Alprazolam elimination half-life was shortened from a mean of 12.4 h to 6.0 h
(P < 0.001). The mean urinary ratio of dextromethorphan to its metabolite was
0.006 at baseline and 0.014 after SJW administration (P = 0.26).
The effect of SJW on P-glycoprotein activity was examined with use of fexofenadine as selective probe drug (93). A single dose of SJW significantly (P < 0.05)
increased the maximum plasma concentration of fexofenadine by 45% and significantly (P < 0.05) decreased the oral clearance by 20%, with no change in half-life
or renal clearance. Fourteen-day administration of SJW did not cause a significant
change in fexofenadine disposition relative to the untreated phase. Compared with
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the single-dose treatment phase, SJW caused a significant 35% decrease (P < 0.05)
in maximum plasma concentration and a significant 47% increase (P < 0.05) in
fexofenadine oral clearance.
The effect of SJW on CYP activity was examined with a probe drug cocktail (91). Twelve healthy subjects (five female, seven male) completed this threeperiod, open-label, fixed-schedule study. Tolbutamide (CYP2C9), caffeine
(CYP1A2), dextromethorphan (CYP2D6), oral midazolam (intestinal wall and
hepatic CYP3A), and intravenous midazolam (hepatic CYP3A) were administered
before short-term SJW dosing (900 mg), and after two weeks of intake (300 mg
tid) to determine CYP activities. Short-term administration of SJW had no effect on CYP activities. Fourteen-day administration caused a significant (P < .05)
increase in oral clearance of midazolam from 121.8 70.7 to 254.5 127.8 and a
corresponding significant decline in oral bioavailability from 0.28 0.15 to 0.17
0.06. In contrast to the >50% decrease in the AUC when midazolam was administered orally, 14-day administration caused a 20% decrease in AUC when
midazolam was given intravenously. Fourteen-day SJW administration resulted
in a significant and selective induction of CYP3A activity in the intestinal wall.
SJW did not alter the CYP2C9, CYP1A2, or CYP2D6 activities in these healthy
subjects.
Spices
Ground fancy clove (Sri Lanka), ground ginger (China or India), oregano leaf
(Turkey), ground sage (Turkey), thyme leaf (Spain), and ground turmeric (India) extracts were found to inhibit CYP2C9, CYP2C19, CYP2D6, and CYP3A4mediated metabolism (20).
Traditional Medicine Plants

Deferme et al. (29) examined extracts of 43 Tanzanian medicinal plants for their
potential inhibitory effect on Pgp using the secretory transport of cyclosporin in
the Caco-2 system as a measure of the functionality of Pgp efflux. Extracts of
Annickia kummeriae and Acacia nilotica had a significant effect. In the presence
of the extract of A. kummeriae, a concentration-dependent decrease in transport of
cyclosporin was observed that was comparable to that of valspodar, a known Pgp
inhibitor.
Traditional Chinese medicine includes both crude Chinese medicinal materials
(plants, animal parts, and minerals) and Chinese proprietary medicine. They are
believed by many to be safe and are used for self-medication. Although the risk
appears to be low, certain products have been associated with a number of serious
ADEs. A study with 12 traditional products, including one subsequently shown to
contain three proprietary drugs, found most aqueous extracts inhibited CYP450mediated metabolism of at least three isozymes (19). All liquid samples markedly
inhibited the metabolism of 2C9, 2C19, 2D6, and 3A4. De le ke chuan kang and
Rensheng dao were the strongest CYP inhibitors.
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Ueng et al. (12) examined the effects of methanol and aqueous extracts of
Evodia rutaecarpa on CYP, UGT, and GST in C57BL/6J mice. Methanol extract
caused a dose-dependent increase of liver microsomal EROD activity. In liver,
methanol extract increased benzo(a)pyrene hydroxylation, 7-methoxyresorufin Odemethylation (MROD), 7-ethoxycoumarin O-deethylation (ECOD), benzphetamine N-demethylation, and N-nitrosodimethylamine N-demethylation activities.
Aqueous extract increased EROD, O-demethylation, and O-deethylation activities
68%, twofold, and 83%, respectively. For conjugation activities, methanol extract
elevated UGT and GST activities. Aqueous extract elevated UGT activity without
affecting GST activity. Immunoblot analyses showed that methanol extract increased the levels of CYP1A1, CYP1A2, CYP2B-, and GSTYb-immunoreactive
proteins. Aqueous extract increased CYP1A2 protein level. In kidney, neither extract had any effect on most activities. Rutaecarpine, evodiamine, and dehydroevodiamine contributed, at least in part, to the increase of hepatic EROD activity.
Ge-gen, the root of a wild leguminous creeper, Pueraria lobata (Willd.) Ohwi
(13), possesses a high content of flavonoid derivatives, the most abundant of which
is puerarin. Puerarin and Ge-gen crude extracts inhibited the steady-state chemiluminescent reaction in a dose-dependent fashion. Although both CYP content and
NADPH-(CYP)-c-reductase activity were significantly increased in all situations,
a complex pattern of CYP modulation was observed, including both induction
(puerarin: CYP2A1, 1A1/2, 3A1, 2C11; Ge-gen: CYP1A2, 3A1, 2B1) and inactivation (Ge-gen and puerarin: CYP3A, 2E1, 2B1). The latter are due to either
parental agents or metabolites, as demonstrated by in vitro studies. Ge-gen contains compounds with potent antioxidant activity, which impair CYP-catalyzed
drug metabolism.
Ohnishi et al. (96) examined the possibility of pharmacokinetic interactions
between Sho-saiko-to extract powder, a widely used traditional Japanese herbal
(Kampo) medicine and carbamazepine in rats. Sho-saiko-to inhibited hepatic microsome 10,11-epoxylase activity in a concentration-dependent manner. Liver
weight, amounts of CYP and cytochrome b(5) in hepatic microsomes, and the
formation of the 10,11-epoxide by microsomes were not influenced by two-week
repeated oral pretreatment, although pretreatment with phenobarbital (80 mg/kg/d,
i.p.) significantly increased these parameters. Simultaneous oral administration of
Sho-saiko-to significantly decreased Cmax of carbamazepine and the AUC of the
epoxide and lengthened the time to reach Cmax. Two-week repeated oral pretreatment with Sho-saiko-to, however, did not affect the plasma concentration-time
profile or any pharmacokinetic parameter of carbamazepine. A single oral administration of Sho-saiko-to (1 g/kg) significantly delayed gastric emptying and simultaneous oral administration of TJ-9 with carbamazepine CBZ to rats decreased the
gastrointestinal absorption of carbamazepine, at least in part, by delaying gastric
emptying without affecting the metabolism.
Ueng et al. (97) examined the effects of Wu-chu-yu-tang on hepatic and renal
CYP, UGT, and GST in C57BL/6J mice. Treatment of mice with 5 g/kg per day Wuchu-yu-tang for 3 days caused 2.5-fold and 2.9-fold increases of liver microsomal
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EROD and 7-methoxyresorufin O-demethylation activities, respectively. CYP activities toward 7-ethoxycoumarin, benzphetamine, N-nitrosodimethylamine, erythromycin, and nifedipine, and conjugation activities of UGT and GST were not
affected. In kidney, Wu-chu-yu-tang treatment had no effects on CYP, UGT, and
GST activities. Among the four component herbs of Wu-chu-yu-tang, only Evodiae fructus (Wu-chu-yu) extract increased EROD activity and CYP1A2 protein
level. The main active alkaloids in E. fructus are rutaecarpine, evodiamine, and
dehydroevodiamine. At doses corresponding to their contents in Wu-chu-yu-tang,
rutaecarpine treatment increased hepatic EROD activity, whereas evodiamine and
dehydroevodiamine had no effects. These results demonstrate that ingestion of
Wu-chu-yu-tang increases mouse hepatic Cyp1a2 activity and protein level.
PRODUCT SELECTION FOR CLINICAL STUDIES

The number of botanical varieties, dosage forms, and formulations in combination
with variability in botanical material make it impossible to evaluate all of these
products in animal models or clinical trials. How should a product be selected?
Should one choose an average or superior product, as the results of the study
will subsequently be viewed as representative of all related products? As an example, four SJW products with similar inhibitory activity against CYP-mediated
metabolism were evaluated further for their effects on cell viability, potential to
modulate induction of nitric oxide, and 1A1/2-mediated EROD activity in glial
cell cultures (86). SJW A failed to induce EROD activity or nitric oxide over
the concentration range studied. SJW B and C produced the highest nitric oxide
levels, which could be cause for concern for CNS toxicity. SJW C produced the
highest levels of resorufin, whereas SJW B and D showed minor induction of
EROD activity. SJW A and D both produced significant cell toxicity as measured
by LDH-release. Which product should be studied? As a minimum, several products used by the patient community should be obtained and authenticated. The
selection criteria should include multiple lot testing, cost, product availability, and
chromatographic separation and quantification of representative biomarker constituents and bioassay testing for relevant activities. Once a product is selected it
needs to be thoroughly characterized so that future commercial products can be
manufactured in a comparable way.
SUMMARY AND FUTURE PERSPECTIVES

Botanicals such as HPs and NCs are often regarded as low risk because of the
long history of human use, their natural origin, or simply because the concentration of active principles is lower than conventional drugs. All products have risk
when combined with other products, even those that when used traditionally may
be considered safe. There is a tendency to relate the pharmacological activity of
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an HP to a SAI. In the same fashion, there is a tendency to relate the effect of

a SAI on drug disposition parameters to the combined total HP, even when the
SAI may only account for a small fraction of the total weight. HPs are complex
products where synergistic pharmacokinetic and/or pharmacodynamic interactions
are of vital importance (98, 99). Many processes, from absorption, metabolism,
distribution, excretion and receptor binding may be affected. The purported pharmacological effect of the NHP must be separated from the potential effect on drug
disposition, particularly if it is a negative finding from an in vitro assay.
Anecdotal and literature reports of ADEs and clinical studies with HPs are increasing. Many of these reports are incomplete and contradictory. These reports
need to be standardized for clarity to appreciate the confounding factors and in
some cases contradictory findings. Average PK data obtained from clinical trials in
healthy subjects, with stringent exclusion criteria or when subjects with potentially
confounding polymorphisms have been excluded, may not show what is relevant
in the patient, regardless of what occurred in the healthy test subject. Studies with
HPs can be confounded by products from different manufacturing processes and
formulations, presence of these HPs in other products including cosmetics and
food supplements, total drug (and xenobiotic) load (100), environmental effects
on the plant, chemotypes, misidentification or adulteration of products, and factors associated with the patient or user population. When a HP has reported ADEs,
demonstrated in vitro or has the clinical potential to affect drug disposition, the
principal of caution should guide further use. Studies that attempt to extrapolate
negative findings with HPs, particularly with SAIs, are meaningless if the confounding factors are not taken into consideration. If there is wide variance in PK
ranges, this may suggest that some individuals would be at risk, particularly when
product is being used off label or in subjects who would not meet the inclusion criteria of purportedly definitive studies. Contradictory findings need to be explained
if they are to help regulatory agencies, health care professionals, or the patient
understand what risks are involved.
Future clinical studies need to be conducted with a fully characterized product
that includes comparisons to a number of commercially available related products.
Clinical trials should identify a representative product and take into account the
confounding factors which may influence the interpretation of the findings and be
consistent with how the product is usedin some cases a 1421 day study may be
insufficient. In addition to PK information on the drug, the PK of the main markers
should also be examined. In vitro studies should evaluate the effects of different
solvent extracts on drug-metabolizing enzymes beyond the major human CYP 13
isoforms to examine other Phase I enzymes, including Phase II drug metabolism
enzymes and transporters. Wider CYP screening with isoforms such as CYP 4
and 19 is required to determine if other crucial endogenous pathways are also
affected.
All products have risk, with risk generally increasing in patients who have
confounding health, genetic, and environmental factors, including polypharmacy.
Health care professionals and their patients need relevant information on both the
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benefits and the limitations of in vitro and clinical PK studies to determine what
risk, if any, may be associated with their combined drug and HP exposure.
ACKNOWLEDGMENTS

The Ontario HIV Treatment Network and Health Canada supported our work cited
in this article.
LITERATURE CITED
1. Chan K. 2003. Some aspects of toxic contaminants in herbal medicines. Chemosphere 52:136171
2. Ameer B, Weintraub RA. 1997. Drug
interactions with grapefruit juice. Clin.
Pharmacokinet. 33:10321
3. Barnes J, Anderson LA, Phillipson JD.
2001. St. Johns wort (Hypericum perforatum L.): a review of its chemistry,
pharmacology and clinical properties. J.
Pharm. Pharmacol. 53:583600
4. Harris R, Jang G, Tsunoda S. 2003. Dietary effects on drug metabolism and
transport. Clin. Pharmacokinet. 42:1071
88
5. Huang SM, Hall SD, Watkins P, Love LA,
Serabjit-Singh C, et al. 2004. Drug interactions with herbal products and grapefruit juice: a conference report. Clin.
6. Ioannides C. 2002. Pharmacokinetic interactions between herbal remedies and
medicinal drugs. Xenobiotica 32:45178
7. Izzo AA. 2004. Drug interactions with St.
Johns Wort (Hypericum perforatum): a
review of the clinical evidence. Int. J. Clin.
8. Zhou S, Gao Y, Jiang W, Huang M, Xu
A, Paxton JW. 2003. Interactions of herbs
with cytochrome P450. Drug Metab. Rev.
35:3598
9. Sun M, Sakakibara H, Ashida H, Danno
G, Kanazawa K. 2000. Cytochrome
P4501A1-inhibitory action of antimuta-
10.
11.
12.
13.
14.
15.
16.
genic anthraquinones in medicinal plants

and the structure-activity relationship.
Biosci. Biotechnol. Biochem. 64:137378
Guo LQ, Yamazoe Y. 2004. Inhibition
of cytochrome P450 by furanocoumarins
in grapefruit juice and herbal medicines.
Acta Pharmacol. Sin. 25:12936
Gorski JC, Huang SM, Pinto A, Hamman
MA, Hilligoss JK, et al. 2004. The effect
of echinacea (Echinacea purpurea root)
on cytochrome P450 activity in vivo. Clin.
Ueng YF, Ko HC, Chen CF, Wang JJ,
Chen KT. 2002. Modulation of drugmetabolizing enzymes by extracts of a
herbal medicine Evodia rutaecarpa in
C57BL/6J. mice. Life Sci. 71:126777
Guerra MC, Speroni E, Broccoli M,
Cangini M, Pasini P, et al. 2000. Comparison between Chinese medical herb Pueraria lobata crude extract and its main
isoflavone puerarin antioxidant properties
and effects on rat liver CYP-catalysed
drug metabolism. Life Sci. 67:29973006
Maliakal PP, Wanwimolruk S. 2001. Effect of herbal teas on hepatic drug metabolizing enzymes in rats. J. Pharm. Pharmacol. 53:132329
Mathews JM, Etheridge AS, Black
SR. 2002. Inhibition of human cytochrome P450 activities by kava extract
and kavalactones. Drug Metab. Dispos.
30:115357
Chun YJ, Kim S. 2003. Discovery of
3 Dec 2004
20:40
222

17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
AR
FOSTER
AR232-PA45-08.tex
ARNASON
AR232-PA45-08.sgm
LaTeX2e(2002/01/18)
P1: GCE
BRIGGS
cytochrome P450 1B1 inhibitors as new

promising anti-cancer agents. Med. Res.
Rev. 23:65768
Chatterjee P, Franklin MR. 2003. Human cytochrome P450 inhibition and
metabolic-intermediate complex formation by goldenseal extract and its methylenedioxyphenyl components. Drug Metab. Dispos. 31:139197
Foster BC, Foster MS, Vandenhoek S,
Budzinski JW, Gallicano KD, et al. 2001.
An in vitro evaluation of human cytochrome P450 3A4 and P-glycoprotein
inhibition by garlic. J. Pharm. Pharmaceut. Sci. 4:17684
Foster BC, Vandenhoek S, Li KY, Tang
R, Krantis A. 2002. Effect of Chinese
herbal products on cytochrome P-450
drug metabolism. J. Pharm. Pharmaceut.
Sci. 5:18589
Foster BC, Vandenhoek S, Hanna J,
Budzinski JW, Akhtar MH, et al. 2003.
Effects of natural health products on cytochrome P-450 drug metabolism. Phytomedicine 10:33442
Wang LS, Zhou G, Zhu B, Wu J, Wang
JG, et al. 2004. St Johns wort induces
both cytochrome P450 3A4-catalyzed sulfoxidation and 2C19-dependent hydroxylation of omeprazole. Clin. Pharmacol.
Ther. 75:19197
Zuber R, Modriansky M, Dvorak Z, Rohovsky P, Ulrichova J, et al. 2002. Effect of
silybin and its congeners on human liver
microsomal cytochrome P450 activities.
Phytother. Res. 16:63238
Brady JF, Ishizaki H, Fukuto JM, Lin
MC, Fadel A, et al. 1991. Inhibition of
cytochrome P-450 2E1 by diallyl sulfide
and its metabolites. Chem. Res. Toxicol.
4:64247
Kwak MK, Kim SG, Kim ND. 1995. Effects of garlic oil on rat hepatic P4502E1
expression. Xenobiotica 25:102129
Brigelius-Flohe R. 2003. Vitamin E and
drug metabolism. Biochem. Biophys. Res.
Commun. 305:73740
Hodek P, Trefil P, Stiborova M. 2002.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Flavonoidspotent and versatile biologically active compounds interacting with

cytochromes P450. Chem. Biol. Interact.
139:121
Budzinski JW, Arnason JT, Trudeau V,
Krantis A, Foster BC. 2001. Effects of
Goldenseal and milk thistle on the modulation of cytochrome P450 3A4 and Pglycoprotein. Drug Metab. Rev. 33(S1):86
Choi SU, Park SH, Kim KH, Choi EJ, Kim
S, et al. 1998. The bisbenzylisoquinoline
alkaloids, tetrandine and fangchinoline,
enhance the cytotoxicity of multidrug
resistance-related drugs via modulation of
P-glycoprotein. Anticancer Drugs 9:255
61
Deferme S, Kamuhabwa A, Nshimo C, de
Witte P, Augustijns P. 2003. Screening of
Tanzanian plant extracts for their potential
inhibitory effect on P-glycoprotein mediated efflux. Phytother. Res. 17:45964
Deferme S, Van Gelder J, Augustijns P.
2002. Inhibitory effect of fruit extracts on
P-glycoprotein-related efflux carriers: an
in-vitro screening. J. Pharm. Pharmacol.
54:121319
Dresser GK, Schwarz UI, Wilkinson GR,
Kim RB. 2003. Coordinate induction of
both cytochrome P4503A and MDR1 by
St Johns wort in healthy subjects. Clin.
Lin HL, Liu TY, Wu CW, Chi CW. 1999.
Berberine modulates expression of mdr1
gene product and the responses of digestive track cancer cells to Paclitaxel. Br. J.
Cancer. 81:41622
Wortelboer HM, Usta M, van der Velde
AE, Boersma MG, Spenkelink B, et al.
2003. Interplay between MRP inhibition
and metabolism of MRP inhibitors: the
case of curcumin. Chem. Res. Toxicol. 16:
164251
Wu D, Nair MG, DeWitt DL. 2002. Novel
compounds from Piper methysticum Forst
(Kava Kava) roots and their effect on
cyclooxygenase enzyme. J. Agric. Food
Chem. 50:7015
Foster BC, Sockovie ER, Bellefeuille NC,
3 Dec 2004
20:40
AR
AR232-PA45-08.tex
AR232-PA45-08.sgm
LaTeX2e(2002/01/18)
P1: GCE

36.
37.
38.
39.
40.
41.
42.
43.
44.
Vandenhoek S, Krantis A, et al. 2001b. Effect of St. Johns wort on cytochrome P450 and flavin monooxygenase enzymes
and on P-glycoprotein. Can. J. Infect. Dis.
12(Suppl. B):132P
van Zanden JJ, Ben Hamman O, van
Iersel ML, Boeren S, Cnubben NH, et al.
2003. Inhibition of human glutathione
S-transferase P1-1 by the flavonoid
quercetin. Chem. Biol. Interact. 145:139
48
Ferreira T, Drouin CE, Krantis A, Arnason JT, Foster BC. 2003. Effect of a natural health product containing echinacea
on human N-acetyltransferase expression.
Can. J. Infect. Dis. 14(Suppl. A):20A
Lin RD, Hou WC, Yen KY, Lee MH.
2003. Inhibition of monoamine oxidase B
(MAO-B) by Chinese herbal medicines.
Phytomedicine 10:65056
Wentworth JM, Agostini M, Love J,
Schwabe JW, Chatterjee VK. 2000. St
Johns wort, a herbal antidepressant, activates the steroid X receptor. J. Endocrinol.
166:R1116
Venkataramanan R, Ramachandran V,
Komoroski BJ, Zhang S, Schiff PL, Strom
SC. 2000. Milk thistle, a herbal supplement, decreases the activity of CYP3A4
and uridine diphosphoglucuronosyl transferase in human hepatocyte cultures. Drug
Koh HL, Woo SO. 2000. Chinese proprietary medicine in Singapore: regulatory
control of toxic heavy metals and undeclared drugs. Drug Saf. 23:35162
Awang DV. 1991. Maternal use of ginseng and neonatal androgenization. J. Am.
Med. Assoc. 266:363
Binns SE, Livesey JF, Arnason JT, Baum
BR. 2002. Phytochemical variation in
echinacea from roots and flowerheads of
wild and cultivated populations. J. Agric.
Food Chem. 50(13):367387
Saez F. 2001. Volatile oil variability
in Thymus serpylloides ssp. gadorensis growing wild in Southeastern Spain.
Biochem. Syst. Ecol. 29:18998
223
45. Lota ML, de Rocca Serra D, Tomi F,

Jacquemond C, Casanova J. 2002. Volatile
components of peel and leaf oils of lemon
and lime species. J. Agric. Food Chem.
50:796805
46. Russell MF, Southwell IA. 2003.
Monoterpenoid accumulation in 1,8cineole, terpinolene and terpinen-4-ol
chemotypes of Melaleuca alternifolia
seedlings. Phytochemistry 62:68389
47. Houghton PJ. 1999. The scientific basis
for the reputed activity of Valerian. J.
Pharm. Pharmacol. 51:50512
48. Shohet D, Wills RB, Stuart DL. 2001.
Valepotriates and valerenic acids in commercial preparations of valerian available in Australia. Pharmazie 56:860
63
49. Pala-Paul J, Perez-Alonso MJ, VelascoNegueruela A, Pala-Paul R, Sanz J,
Conejero F. 2001. Seasonal variation in
chemical constituents of Santolina rosmarinifolia L. ssp. rosmarinifolia. Biochem. Syst. Ecol. 29:66372
50. Buter B, Orlacchio C, Soldati A, Berger
K. 1998. Significance of genetic and environmental aspects in the field cultivation
of Hypericum perforatum. Planta Med.
64:43137
51. Southwell IA, Bourke CA. 2001. Seasonal variation in hypericin content of Hypericum perforatum L. (St. Johns Wort).
Phytochemistry 56:43741
52. Matsuura H. 1997. Phytochemistry of
garlic horticulture and processing procedures. In Nutraceuticals: Designer Foods
III Garlic, Soy and Licorice, ed. Paul
P. Lanchance, pp. 5569. Trumbell, CT:
Food & Nutr. Press
52a. Foster BC, Foster MS, Vandenhoek S,
Budzinski J, Krantis A, Arnason JT. St.
Johns wort-effects on cytochrome P450
and P-glycoprotein. Pharmaceut. Biol. In
press
52b. Lefebvre T, Foster BC, Drouin CE,
Krantis A, Arnason JT, et al. 2004.
In vitro activity of valerian against human cytochrome P450 3A4-mediated
3 Dec 2004
20:40
224
53.

54.
55.
56.
57.
58.
59.
60.
61.
AR
FOSTER
AR232-PA45-08.tex
ARNASON
AR232-PA45-08.sgm
LaTeX2e(2002/01/18)
P1: GCE
BRIGGS
metabolism. J. Pharm. Pharmaceut. Sci.

7:26573
Bauer R. 1996. Echinacea drugseffects
and active ingredients. Z Arztl Fortbild
(Jena) 90:11115
Bergeron C, Livesey JF, Awang DVC, Arnason JT, Rana J, et al. 2000. A quantitative method for identity and quality assurance of Echinacea products on
the North American market. Phytochem.
Anal. 11:20715
Zou L, Harkey MR, Henderson GL.
2002. Effects of intrinsic fluorescence and
quenching on fluorescence-based screening of natural products. Phytomedicine 9:
26367
Westerhoff K, Kaunzinger A, Wurglics
M, Dressman J, Schubert-Zsilavecz M.
2002. Biorelevant dissolution testing of St
Johns wort products. J. Pharm. Pharmacol. 54:161521
Jurgenliemk G, Nahrstedt A. 2003. Dissolution, solubility and cooperativity of phenolic compounds from Hypericum perforatum L. in aqueous systems. Pharmazie
58:2003
Taglioli V, Bilia AR, Ghiara C, Mazzi G,
Mercati V, Vincieri FF. 2001. Evaluation
of the dissolution behaviour of some commercial herbal drugs and their preparations. Pharmazie 56:86870
Bilia AR, Bergonzi MC, Morgenni F,
Mazzi G, Vincieri FF. 2001. Evaluation of
chemical stability of St. Johns wort commercial extract and some preparations.
Int. J. Pharm. 213(12):199208
Bilia AR, Bergonzi MC, Gallori S, Mazzi
G, Vincieri FF. 2002. Stability of the constituents of Calendula, milk-thistle and
passionflower tinctures by LC-DAD and
LC-MS. J. Pharm. Biomed. Anal. 30:613
24
Budzinski J, Foster BC, Vandenhoek S,
Arnason JT. 2000. An in vitro evaluation
of human cytochrome CYP450 3A4 inhibition by selected commercial herbal
extracts and tinctures. Phytomedicine
7:27382
62. Bailey DG, Spence JD, Edgar B, Bayliff

CD, Arnold JM. 1989. Ethanol enhances
the hemodynamic effects of felodipine.
Clin. Invest. Med. 12:35762
63. Bailey DG, Dresser GK, Kreeft JH,
Munoz C, Freeman DJ, Bend JR. 2000.
Grapefruit-felodipine interaction: effect
of unprocessed fruit and probable active ingredients. Clin. Pharmacol. Ther.
68:46877
64. Malhotra S, Bailey DG, Paine MF,
Watkins PB. 2001. Seville orange juicefelodipine interaction: comparison with
dilute grapefruit juice and involvement
of furocoumarins. Clin. Pharmacol. Ther.
69:1423
65. Penzak SR, Acosta EP, Turner M, Edwards DJ, Hon YY, et al. 2002. Effect of
Seville orange juice and grapefruit juice
on indinavir pharmacokinetics. J. Clin.
66. Bailey DG, Dresser GK, Bend JR. 2003.
Bergamottin, lime juice, and red wine as
inhibitors of cytochrome P450 3A4 activity: comparison with grapefruit juice.
67. Guo LQ, Taniguchi M, Xiao YQ, Baba K,
Ohta T, Yamazoe Y. 2000. Inhibitory effect of natural furanocoumarins on human
microsomal cytochrome P450 3A activity.
Jpn. J. Pharmacol. 82:12229
68. Committee on Safety of Medicines. 2003.
Possible interaction between warfarin and
cranberry juice. Curr. Prob. Pharmacovigil. 29:8
69. Laroche M, Choudhri S, Gallicano K, Foster BC. 1998. Severe gastrointestinal toxicity with concomitant ingestion of ritonavir and garlic. Can. J. Infect. Dis. Supp.
9(B):471P
70. Choudri S, Gallicano KD, Foster BC.
2000. Effect of garlic formulation on the
single dose pharmacokinetics of ritonavir.
Presented at Intersci. Conf. Antimicrob.
Ag. Chemother. (ICAAC), Sept., Toronto
71. Gallicano K, Choudhri S, Leclaire T,
Foster BC. 2003. Effect of short-term
administration of garlic supplements on
3 Dec 2004
20:40
AR
AR232-PA45-08.tex
AR232-PA45-08.sgm
LaTeX2e(2002/01/18)
P1: GCE
72.

73.
74.
75.
76.
77.
78.
79.
single-dose ritonavir pharmacokinetics in

healthy volunteers. Br. J. Clin. Pharmacol. 55:199202
Piscitelli SC, Burstein AH, Welden N,
Gallicano KD, Falloon J. 2002. The effect of garlic supplements on the pharmacokinetics of saquinavir. Clin. Infect. Dis.
34:23438
Markowitz JS, Devane CL, Chavin KD,
Taylor RM, Ruan Y, Donovan JL. 2003.
Effects of garlic (Allium sativumL.) supplementation on cytochrome P450 2D6
and 3A4 activity in healthy volunteers.
Foster MS, Vandenhoek S, Drouin CE,
Budzinski JW, Krantis A, et al. 2001.
In vitro evaluation of human cytochrome
P450, P-glycoprotein and antibiotic interactions by garlic (Allium sativum). Can.
J. Infect. Dis. 12(Suppl. B):131
Ward PM, Fasitsas S, Katz SE. 2002. Inhibition, resistance development, and increased antibiotic and antimicrobial resistance caused by nutraceuticals. J. Food
Prot. 65:52833
Ohnishi N, Kusuhara M, Yoshioka M,
Kuroda K, Soga A, et al. 2003. Studies
on interactions between functional foods
or dietary supplements and medicines. I.
Effects of Ginkgo biloba leaf extract on
the pharmacokinetics of diltiazem in rats.
Biol. Pharm. Bull. 26:131520
Markowitz JS, Donovan JL, Lindsay DeVane C, Sipkes L, Chavin KD. 2003.
Multiple-dose administration of Ginkgo
biloba did not affect cytochrome P-450
2D6 or 3A4 activity in normal volunteers.
J. Clin. Psychopharmacol. 23:57681
Sandhu RS, Prescilla RP, Simonelli TM,
Edwards DJ. 2003. Influence of goldenseal root on the pharmacokinetics of
indinavir. J. Clin. Pharmacol. 43:1283
88
Unger M, Holzgrabe U, Jacobsen W,
Cummins C, Benet LZ. 2002. Inhibition
of cytochrome P450 3A4 by extracts and
kavalactones of Piper methysticum (KavaKava). Planta Med. 68:105558
225
80. Piscitelli SC, Formentini E, Burstein AH,

Alfaro R, Jagannatha S, Falloon J. 2002.
Effect of milk thistle on the pharmacokinetics of indinavir in healthy volunteers.
Pharmacotherapy 22:55156
81. DiCenzo R, Shelton M, Jordan K, Koval
C, Forrest A, et al. 2003. Coadministration of milk thistle and indinavir in healthy
subjects. Pharmacotherapy 23:86670
82. Mills E, Wilson K, Clarke C, Foster BC,
Walker S, et al. 2004. Milk thistle and indinavir: a randomized controlled pharmacokinetics study. Int. J. Naturopath. Med.
In press
83. Obach RS. 2000. Inhibition of human cytochrome P450 enzymes by constituents
of St. Johns Wort, an herbal preparation
used in the treatment of depression. J.
84. Moore LB, Goodwin B, Jones SA,
Wisely GB, Serabjit-Singh CJ, et al.
2000. St. Johns wort induces hepatic
drug metabolism through activation of the
pregnane X receptor. Proc. Natl. Acad.
Sci. USA 97:75002
85. Noldner M, Chatterjee S. 2001. Effects
of two different extracts of St. Johns
wort and some of their constituents
on cytochrome P450 activities in rat
liver microsomes. Pharmacopsychiatry
34(Suppl. 1):S10810
86. Grenier M, Sun P, Drobitch R. 2002. Effect of St. Johns wort preparations on
cell viability, induction of nitric oxide and
ethoxyresorufin O-deethylase activity in
glial cell culture. J. Pharm. Pharmaceut.
Sci. 5:106
87. Ruschitzka F, Meier PJ, Turina M,
Luscher TF, Noll G. 2000. Acute heart
transplant rejection due to St. Johns Wort.
Lancet 355:54859
88. Piscitelli SC, Burstein AH, Chaitt D, Alfaro RM, Falloon J. 2000. Indinavir concentrations and St. Johns Wort. Lancet
355:54748
89. Piscitelli SC, Burstein AH, Chaitt D,
Alfaro RM, Falloon J. 2001. Indinavir concentrations and St Johns
3 Dec 2004
20:40
226
90.

91.
92.
93.
94.
95.
AR
FOSTER
AR232-PA45-08.tex
ARNASON
AR232-PA45-08.sgm
LaTeX2e(2002/01/18)
P1: GCE
BRIGGS
wort. Lancet 355(9203):54748. Erratum.

Lancet 357(9263):1210
Burstein AH, Horton RL, Dunn T, Alfaro RM, Piscitelli SC, Theodore W. 2000.
Lack of effect of St. Johns Wort on carbamazepine pharmacokinetics in healthy
volunteers. Clin. Pharmcol. Ther. 68:605
12
Wang Z, Gorski JC, Hamman MA, Huang
SM, Lesko LJ, Hall SD. 2001. The effects
of St Johns wort (Hypericum perforatum)
on human cytochrome P450 activity. Clin.
Bray BJ, Brennan NJ, Perry NB, Menkes
DB, Rosengren RJ. 2002. Short term treatment with St. Johns wort, hypericin or
hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse. Life
Sci. 70:132535
Wang Z, Hamman MA, Huang SM, Lesko
LJ, Hall SD. 2002. Effect of St Johns wort
on the pharmacokinetics of fexofenadine.
Markowitz JS, Donovan JL, DeVane CL,
Taylor RM, Ruan Y, et al. 2003. Effect of
St Johns wort on drug metabolism by induction of cytochrome P450 3A4 enzyme.
JAMA 290:15004
Wang LS, Zhou G, Zhu B, Wu J, Wang
96.
97.
98.
99.
100.
JG, et al. 2004. St Johns wort induces

both cytochrome P450 3A4-catalyzed sulfoxidation and 2C19-dependent hydroxylation of omeprazole. Clin. Pharmacol.
Ther. 75:19197
Ohnishi N, Okada K, Yoshioka M, Kuroda
K, Nagasawa K, et al. 2002. Studies on interactions between traditional
herbal and western medicines. V. Effects
of Sho-saiko-to (Xiao-Cai-hu-Tang) on
the pharmacokinetics of carbamazepine
in rats. Biol. Pharm. Bull. 25:1461
66
Ueng YF, Don MJ, Peng HC, Wang SY,
Wang JJ, Chen CF. 2002. Effects of Wuchu-yu-tang and its component herbs on
drug-metabolizing enzymes. Jpn. J. Pharmacol. 89:26773
Williamson EM. 2001. Synergy and
other interactions in phytomedicines.
Phytomedicine 8:4019
Spinella M. 2002. The importance of
pharmacological synergy in psychoactive herbal medicines. Altern. Med. Rev.
7:13037
Deckers CL, Hekster YA, Keyser A,
Meinardi H, Renier WO. 1997. Drug load
in clinical trials: a neglected factor. Clin.
3 Dec 2004
20:41
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Copyright
BIOMARKERS IN PSYCHOTROPIC DRUG

DEVELOPMENT: Integration of Data across
Multiple Domains
Peter R. Bieck1 and William Z. Potter
1
Eli Lilly & Company, Neuroscience Therapeutic Area, Lilly Corporate Center,
Indianapolis, Indiana 46285; email: bieck@lilly.com
Key Words data integration, CNS access, neuroimaging, CSF, proteomics,

psychotropic drug biomarkers
Abstract This review focuses on the current status of biomarkers and/or approaches critical to assessing novel neuroscience targets with an emphasis on new
paradigms and challenges in this field of research. The importance of biomarker data
integration for psychotropic drug development is illustrated with examples for clinically used medications and investigational drugs. The question remains how to verify
access to the brain. Early imaging studies including micro-PET can help to overcome
this. However, in case of delayed tracer development or because of no feasible application of brain imaging effects of the molecule, using CSF as a matrix could fill this gap.
Proteomic research using CSF will hopefully have a major impact on the development
of treatments for psychiatric disorders.
INTRODUCTION
The drug development process has multiple phases and decision points that require
development of stage-specific biomarkers (1, 2). Biomarker assays have to be
validated for criteria such as reference range, accuracy (sensitivity, selectivity,
specificity), and stability (3, 4).
There is general agreement that the main utilities of biomarkers in drug development are the following:
Discovery and selection of lead compounds

Generation of pharmacokinetic (PK) and pharmacodynamic (PD) models
Aid in clinical trial design and expedite drug development
Serving as surrogates for clinical or mortality endpoints

Optimizing drug therapy based on genotypic or phenotypic factors
Definition of patient enrollment in studies and help with stratification
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Psychotropic Drug Development

A principal question in psychotropic drug development is whether there are better
ways to predict therapeutic and unwanted effects of novel compounds targeting
the central nervous system (CNS).
In the absence of clear answers to this question or better means of prediction,
it is still possible to find better ways of testing novel compounds, especially if one
views them as reagents for evaluating a hypothesis. In other words, application
of multiple technologies may allow us to show that a molecule is acting on specific biochemical processes in humans. That, in turn, allows one to formally test
hypotheses on whether a particular biochemical effect in patients is or is not associated with clinical change and/or physiological side effects. This approach goes
beyond the generally recognized need to conduct early evaluations of drugs in humans more effectively and more rapidly with clinical pharmacological assessment
of CNS using batteries of objective and subjective measures that must be valid and
reliable. Independent of drug development, there has been great interest in finding
biological markers of psychiatric disorders not only for elucidating underlying
pathophysiology but also to serve as diagnostic tools or predicting treatment responses. The majority of biologic and laboratory markers and surrogate endpoints
that have demonstrated an association between the marker and the underlying condition come from other therapeutic fields. Biomarkers currently being investigated
in psychiatry and neurology encompass a wide variety of procedures (Table 1) (5,
6). None of these markers are useful for routine clinical practice. As cited in a
textbook, biological marker research in psychiatry often takes on the character
of a fishing expedition with better fishing spots suggested by earlier encouraging
findings or intriguing hypotheses (7).
In what follows, we review the factors that determine the relationship between
drug dosage and effect in light of the application of potential biomarkers of the
latter. We provide a number of examples of how these domains of investigation
can be integrated from discovery to the clinic.
To integrate the knowledge on biomarkers, a biomarker database is urgently
needed that can accept data from the scientific community at large. Very recently,
an information technology site for life sciences (http://www.integromics.com/) has
started offering help to pharmaceutical companies and academics for integrating
biomarker data using SpotFire (http://spotfire.com/).
This need for integration of data is similar in other areas of research. At a recent conference on Data Integration for the Pharmaceutical Industry, the term
integromics in drug discovery was used. Weinstein questioned the biological
and pharmacological meaning of the results of genomics and proteomics, but
showed how one can integrate them by using bioinformatics and chemoinformatics (http://discover.nci.nih.gov/). His group is using so-called micro array
tools, such as MedMiner, MatchMiner, GoMiner, and CIMMiner for integration of literature, gene identifiers, gene visualizations, and expression maps
(8).
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PSYCHOTROPIC DRUG BIOMARKERS

TABLE 1
229
Examples for biomarkers in psychotropic drug development

Biomarker procedures
Brain imaging technique
Computed tomography (CT), regional

cerebral blood flow (rCBF), magnetic
resonance imaging (MRI), positron
emission tomography (PET), single photon
emission computed tomography (SPECT),
magnetic resonance spectroscopy (MRS),
magnetoencephalography (MEG)
Cell-based imaging
Fluorescent resonant energy transfer,a

confocal imaging in brain slicesb
Electrophysiological marker
Electroencephalogram (EEG),
pupillometry, saccadic eye movements
Laboratory-based markerc
Concentrations of catecholamines,
hormones, enzymes, proteins, drugs, and
drug metabolites
Psycho-immunological marker
Immunoglobulin, lymphocyte responses,

lymphokine, cytokine, interleukin,
interferon; viral serology; Alz-50;
anticardiolipin antibodies (ACA)
Neuroendocrine marker
Dexamethasone-suppression test (DST),

thyrotropin-releasing hormone stimulation
test (TRHST), growth hormone (GH)
challenge test
Provocative anxiety tests
Lactate infusion, carbon dioxide (CO2)

challenge, cholecystokinin (CCK)
challenge
Genetic markers
DNA banking, genotyping, restriction

fragment length polymorphisms (RFLPs)
Nuclear magnetic resonance (NMR),
lipoprotein fractions and subfractions,
matrix assisted laser desorption/ionizationmass spectrometry (MALDI-MS)
Proteomic identification
Reference 5.
b
c
Reference 6.
Matrices mostly from plasma, urine, CSF, tissue, saliva, and hair.
Altman has reviewed the challenges regarding the integration and analysis of
genomic, molecular, cellular, and clinical data and has merged at Stanford University what was called Clinical Informatics and Bioinformatics to Biomedical
Informatics (9).
The publicly available internet research tool Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB at http://www.pharmgkb.org/)
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demonstrates how a database can help researchers in understanding the contribution of genetic variation among individuals to differences in reactions to drugs. It is
an integrated resource and can be researched for drugs, diseases, clinical outcome,
pharmacodynamics, pharmacokinetics, molecular/cellular functional assays, and
for genotype (10). As more and more measures of CNS during drug action become
available, integration of genetic, proteomic, metabolic, and pharmacokinetic data
can utilize these tools that are being developed.
CNS ACCESS TO THE SITE OF ACTION

Medications that affect the CNS have to be transported to the site of action. The
blood brain barrier (BBB) often prevents sufficient exposure to this site: 98% of
small-molecule drugs do not cross the BBB (11). With the recent exception of
application of PET ligands, human studies do not specifically measure the extent
of CNS penetration. A multitude of biological factors underlie what Spector calls
the phenomenology of CNS transport: drug concentrations in plasma, CSF, brain,
and extracellular fluid (Table 2). This author has stressed the importance and lack
of systematic analysis of CNS transport in preclinical and clinical studies (12). To
TABLE 2
Systematic analysis of CNS transporta
Type of study
Example
Phenomenology
Concentrations in plasma, CSF, brain,

and ECF
Physiology/Pharmacology
In vivo: Ventriculocisternal perfusion

Brain uptake index (BUI)
In situ intra-arterial brain perfusion
Brain efflux index (BEI)
Intravenous injection
Intraventricular injection
In vitro: Chorioid plexus (CP) preparation
Brain capillary preparation
Biochemical Pharmacology,
Anatomy, Histology
Purification of enzymes and receptors

Specificity of transport
Receptor analysis
Affinity of ligands (ex vivo binding)
Monolayer of cerebral capillary cells
CP epithelial cells in vitro
Histological localization of receptors
Molecular biology
Cloning and expressing genes knockout

mice
Analogy
Kidney, gut, and liver systems
Adapted from Reference 12.
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date, the underlying physiological processes regulating a drugs access to brain are
generally ignored in a still empirical approach with focus on the phenomenological
level.
Cerebrospinal fluid (CSF) sampling is a tool that allows access to the central
compartment, and ideally it would provide a better matrix than plasma to assess
drug concentration close to the site of action. Early studies showed that sporadic
simultaneous measurement of the CSF to plasma concentration ratio usually is
inadequate to describe CSF penetration. CSF concentration-time curves lag behind
those in plasma. The areas under the concentration-time curves in CSF and plasma
at steady state or after a short-term infusion are accepted as measures of CSF
passage (13). Continuous CSF collections for 12 h or longer provide concentrationtime curves for drug and or biomarkers and serve as dynabridge study (14).
This term was introduced to describe a type of exploratory study in which drug
concentrations and activities in the central compartment of patients are measured.
The techniques have been described as safe and well tolerated (15). The question
remains concerning the extent to which CSF concentration is representative of that
at the site of action.
One approach to systematically categorize intercellular communication in the
brain is based on the concepts of wiring transmission (WT) and volume transmission (VT) (16). Morphological and functional observations suggest that CSF might
represent an important vector for convection of VT signals, especially to peri- and
paraventricular areas. Any brain cell can participate in VT, and any kind of substance, such as ions, drugs, classical transmitters, peptides, and neurosteroids, can
be a signal (17, 18). MRI studies have indicated the existence of fluid movements
from the CSF via the paravascular space and the extracellular space into the brain
capillaries (19). Despite these supportive factors, it still remains to be shown when
and if CSF concentrations reflect target drug concentrations.
Direct measures of drug concentrations in human brain tissue can be obtained
in vivo using imaging techniques (20) or measured in postmortem brain (21, 22).
Neurosurgeons apply microdialysis and voltammetry/spectrophotometry for continuous monitoring of substrates, metabolites, or neurotransmitters in the human
brain with the disadvantage that these probing methods are invasive and focal (23).
Ahmed et al. have previously summarized the advantages and limitations of
selected biomarker technologies for assessing CNS access (Table 3) (24). The
main difference between the use of CSF and imaging consists in the ability for
continuous monitoring versus the intermittent snap-shot imaging. We later discuss
examples comparing studies utilizing these different methods.
TARGET DRUG-RECEPTOR INTERACTIONS

The most obvious measure of a drug interacting with its target is via receptor occupancy, a measure that is now feasible for a limited number of targets
for which validated PET or SPECT ligands are available (reviewed in 24a).
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Advantages and limitations of selected biomarker technologiesa
Technology
Advantages
Limitations
CSFb,c
Measurement of drug PK in
central compartment
Several surrogate markers
available
Possible to combine PK and
PD measures in one
protocol
Invasive
Sensitive assay required
EEGc
Unequivocal positive results

provide evidence of CNS
effects of intervention
Convenient for repeated
measures within subjects
Observed effects generally

not easily linked to specific
mechanism of interaction
Prone to artifacts and not
fully standardized
MRI
b,c
MRS
c
fMRI
c
sMRI
Pharmacological doses of
certain drugs can be
accurately measured
Structural and functional
modalities can be combined
to enhance overall signal
detection
Motion artifacts in agitated

patients
More validation necessary
for surrogate marker use
Expensive
PET/SPECT
b
Tracer techniques
c
Functional methods
c
Occupancy studies
Highly sensitive detection

of drugs that can be
radiolabeled
Direct evidence of effect of
drug at site of action
Exposure to ionizing
radiation
Tracer not available for
every application
Expensive
Reproduced with permission from the American Journal of Geriatric Psychiatry (Copyright 2002). American
Psychiatric Publishing, Inc. Reference 24.
b
c
Modality can demonstrate central penetration of a drug.
Method can show central PD effect of a drug and/or serve as a surrogate marker in selected situations.
PK: pharmacokinetic(s); PD: pharmacodynamic(s); CSF: cerebrospinal fluid; EEG: electroencephalography; MRI: magnetic resonance imaging; MRS: magnetic resonance spectroscopy; fMRI: functional MRI;
sMRI: structural MRI; PET: positron emission tomography; SPECT: single photon emission computed tomography.
Because this technology is generally limited to robust blockade and displacement of antagonist binding, other measures are required to establish the interaction of a drug with a target. These fall into the general class of functional
measures, the most generalizable and promising of which may prove to be
proteomics.
Proteomics is a research field aiming to characterize molecular and cellular
dynamics in protein expression and function on a global level (25, 26). Clinical
proteomics is a new subdiscipline that involves the application of these technologies at the bedside. Most advanced is the analysis of serum proteomic patterns
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to provide diagnostic end points for cancer detection (27). Proteomic approaches
to CNS disorders are progressing with the hope of establishing a CNS proteome
database derived from primary human tissues (28). Areas of research are encompassing proteomes of nerve cells (29, 30), proteomic profiling of autopsy brain
tissue from patients with Alzheimers and Parkinsons disease (compared with
control specimen from healthy subjects) (31, 32), and proteomic analysis of the
CSF of patients with schizophrenia (33) or Alzheimers disease (34). Multinational
pharmaceutical and smaller private biotechnology-based companies show a huge
interest in using proteomic techniques for new discoveries in psychotropic drug
development (antipsychotics, anxiolytics, depression, schizophrenia) (25, 35).
Application of proteomics as a read-out of target-drug interactions is just
beginning to be explored. Its success depends on whether there really are discretely
identifiable patterns of changes in proteins associated with specific drug-target
molecular interactions.
From a practical experimental viewpoint, the combination of CSF sampling
with proteomics enables access to a body fluid in close contact with brain cells.
CSF has only minimal protein content, which makes analyses less complicated
compared with serum proteomic patterns. We are currently exploring the best
means of achieving standardization of CSF collection, which is mandatory for
its use in proteomic studies. In a parallel effort, because blood contamination
cannot be entirely avoided during lumbar puncture, methods to correct for the
variable contamination-associated changes in the CSF proteomic profile are being
developed (36). A large survey in Europe confirmed that CSF/serum quotients of
proteins represent method-independent values approaching the quality of reference
values (37, 38).
DOWNSTREAM PHARMACOLOGICAL EFFECTS

Here we make a distinction between biochemical effects detectable in a matrix
accessible to the site of action, which are specific to a particular molecular event
(e.g., specific protein pattern changes in CSF), and biochemical or physiological effects, which are consistent with, but not necessarily specific to, a particular
pharmacologic action. There is a four-decade history of measuring biochemical
changes in CSF, blood, and urine as indices of such drug effects (24; reviewed in
24a). But, for instance, there are no established neuroimaging techniques available for determination of norepinephrine transporter (NET) inhibition in humans,
which could be used in drug development, although promising first results in humans studying NET receptor occupancy following treatment with clinical doses of
reboxetine have been reported (39). This latter study has utilized the specific PET
ligand, (S, S)-[11C] MeNER, an O-methyl analog of the selective and potent NET
inhibitor, (S, S)-reboxetine. For decades, however, peripheral measures that show
a decrease in NE turnover after NET inhibition have been used to and criticized
as reflecting changes in the peripheral sympathetic nervous system, which might
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not be surrogates for effects in the brain. Nonetheless, because there is only one
type of NET in the nervous system (peripheral and central), and metabolites of
NE formed in the brain are excreted, some of the peripheral biomarkers might
reflect changes in the CNS. Past experience has shown that peripheral biomarkers
obtained early during clinical pharmacological evaluation of MAO inhibitors (40)
can be validated at a later stage with PET imaging in peripheral organs (41) and
in brain (42).
Interestingly, investigations of clinical syndromes can also provide support for
an array of measures as indices of drug action. Recently, a functional polymorphism in the human NET has been discovered (43) in patients with orthostatic
intolerance taking the form of a NET deficiency that can be assessed simultaneously with a multitude of measures (44). The same type of measures should also be
applicable for testing drug-induced NET deficiency. In a study with duloxetine, a
5-HT and NE reuptake inhibitor, the following measures were applied: vital signs,
tyramine pressor test, posture test, NE and its metabolite DHPG in plasma and
urine, plasma melatonin, plasma inhibition of [3H]-nisoxetine binding (ex vivo
ligand), and plasma duloxetine. Selected results, consistent with a dose response
on measures related to NET inhibition, are shown in Figure 1ad. The findings of
this study suggest that a portfolio of biomarkers is useful for the assessment of
NET inhibition because there were substantial differences in the sensitivity with
which the different downstream biomarkers were affected: ex vivo binding =
DHPG: NE ratio > tyramine pressor test > heart rate (45). Assessment of such
biomarkers in CSF, plasma, and urine during treatment with the potent NET reuptake inhibitor atomoxetine (46) is presently ongoing. In the future, it will be
possible to validate such peripheral NET reuptake biomarkers with the highly sensitive (but expensive!) PET imaging as well as by comparing them with proteomic
patterns in the CSF.
CLINICAL RESPONSE
Evidence of drug effect in a model experimental paradigm can also be considered
as a type of biomarker. Historically, provocative anxiety tests have been given to
diagnose patients with panic disorders, but they also have been used in studies
on healthy subjects for psychotropic drug development. The scope in drug development has been to delineate the mechanism of action of potential antianxiety
agents and to determine a minimum effective dose and the duration of effect. However, these tests have not proved predictive of efficacy and are therefore limited
to providing evidence of some drug effect (47). Pharmacological challenges with
lactate, carbon dioxide, or cholecystokinin (CCK) can produce anxiety in healthy
human subjects (4852). They are usually regarded as models of unconditioned
anxiety, comparable to panic disorder. Their use requires careful consideration of
the experimental setting and the physiological changes. For example, Na-lactate
requires a 20-min infusion and CO2 needs several inhalations for 20 min each (53).
CCK can be given as i.v. bolus.
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Figure 1 (a) Quantal dose-response curves of DHPG/NE ratio in plasma before and
during duloxetine treatment. (b) Quantal dose-response curves of DHPG/NE ratio in
urine before and during duloxetine treatment. (c) Effect of duloxetine on ex vivo [3H]
nisoxetine binding to NE transporters. (d) Effect of duloxetine on tyramine pressor
dose to raise systolic blood pressure by 30 mm Hg (PD30). From Reference 45.

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There are major differences between challenges with the endogenous occurring
CCK, Na-lactate, and CO2. Patients with panic disorders show a left-shifted dose
response (i.e., are more sensitive) to CCK in comparison to healthy subjects (54). It
has been argued that CCK fulfills the criteria of an ideal anxiety-challenging model
(55). CCK not only elicits symptoms of anxiety but also produces physiological
and hormonal changes through activation of the HPA axis (48, 51, 52). This is not
the case during lactate or CO2 challenge. Thus, there is a wider range of acute CCK
effects that can be affected by drugs of multiple classes, such as imipramine (56),
benzodiazepines (57), and vigabatrine (58), all of which antagonize CCK effects
on panic symptoms.
EXAMPLES OF VALIDATED VERSUS EXPLORATORY

BIOMARKERS
Retrospective Biomarker Collection
Few biomarkers assessing CNS drug effects have been validated, most are of the
types already presented, and many are highly exploratory when a novel target is
in question. Perhaps the most widely utilized biomarker in neuropsychiatric drug
development is striatal dopamine-2 (D2) receptor binding, usually determined
by assessing displacement of the PET ligand [11C] raclopride. High occupancy
is usually associated with Parkinson-like side effects, whereas efficacy with socalled atypical antipsychotics can be achieved at lower levels of binding (59). An
associated biomarker for atypical antipsychotics is to look for high 5-HT2 receptor
occupancy in cortical areas assessed by displacement of spiperone or N-methyl
spiperone (60). The same approach has recently been applied to find clinical trial
doses for a selective 5-HT2A antagonist (61).
A particularly strong case for a validated marker depends on a retrospective
analysis of cumulative data on fluoxetine. Table 4 (upper part) lists studies spanning more than a decade, which bridge from the in vitro 5-HT transporter Ki of
fluoxetine to blood, CSF, brain (MRS) concentrations, receptor occupancy (PET),
and biochemical effects (decreased platelet 5-HT uptake and CSF concentrations
of 5-HIAA, the major metabolite of 5-HT). These measures, in turn, can be related
to clinical effects (6268). Prospective use of biomarkers to expedite CNS drug
development is our goal and is beginning to have an impact (6973). Perhaps the
most striking current example of application of a biomarker to establish doses for
large trials involves a NK-1 antagonist.
Recently, the FDA approved the NK-1 antagonist aprepitant based on the results
of two well-controlled studies that included more than 1000 cancer patients receiving chemotherapy that induced severe nausea and vomiting (CINV) (74, 75). In
these studies, fewer patients had symptoms of nausea and vomiting when aprepitant
was part of their treatment (combination with ondansetron and dexamethasone)
compared to patients who received standard antiemetic medicines. Human PET
studies had shown that aprepitant crosses the BBB and occupies brain NK-1 receptors (76, 77). The relationship of dose and plasma concentration of aprepitant to
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Time course of biomarker collection
Drug
Fluoxetine SSRI

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Biomarker
Retrospective biomarker collection

1978
Platelet [3H] serotonin uptake
1989
Rx scale: MADRS
PD: 5-HIAA
PK: plasma and CSF
19
1993
F MRS brain
2000
PET
2002
5-HT transporter Ki
19
2003
F MRS brain
Prospective biomarker collection

LY354740
Metabotropic
glutamate receptor
agonist
1998
2002
2003
2003
2004
2003
mGlu2/3 receptor Ki
PK: plasma and CSF
PK: plasma and CSF
CSF proteomics
Anxiety model: CO2
Anxiety model: CCK
Clinical studies
Reference
(62)
(64)
(65)
(66)
(63, 67)
(68)
(69)
(70)
(71)
(72)
(73)
Rx scale: treatment scale; MADRS: Montgomery and Asberg

Depression Rating Scale (Reference Montgomery
& Asberg:
Brit. J. Psychiat. (1979), 134, 382-9). 5-HIAA: 5-hydroxy indole acetic acid; PD: pharmacodynamic(s); PK: pharmacokinetic(s); 19F MRS: Fluorine 19 magnetic resonance spectroscopy; PET: positron
emission tomography; 5-HT: 5-hydroxytryptamine; Ki: inhibitory constant; mGlu: metabotropic glutamate;
CCK: cholecystokinin.
CNS receptor occupancy was defined in healthy subjects to predict the occupancy
of central NK-1 receptors. The effective doses of aprepitant in patients with CINV
were 125 mg and 375 mg (78), doses that lead to a receptor occupancy of >90%
in healthy subjects. In a Phase II trial, therapy with aprepitant was associated with
improvements in depression and anxiety symptoms that were quantitatively comparable with those seen with selective serotonin reuptake inhibitors (SSRIs) and
significantly greater than those seen with placebo (79). But, in 2003 the Phase III
clinical program was halted because the compound failed to demonstrate efficacy
for the treatment of depression despite being used at doses producing >90% NK-1
receptor occupancy in the brain. Thus, the hypothesis that antagonism of NK-1
receptors produces antidepressant effects was properly tested and not supported.
If trials with other NK-1 antagonists also fail to show sustained antidepressant
effects, this will stand as the first example in antidepressant research of using a
biomarker to show that a drug really did engage its target and thereby reject a
hypothesis, not just a compound.
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Prospective Biomarker Collection

The more common situation in this era of novel targets of unknown function in
humans is not having any convincing method for showing that effects of a new
compound result from engaging the stated biochemical target. Take the current case
of LY354740, an analog of glutamate (Table 4, lower part). It is nanomolar potent,
highly selective, and orally active at Group II cAMP-coupled metabotropic glutamate receptors (mGlu). Preclinical studies showed significant anxiolytic activity,
comparable to diazepam. However, anxiolytic doses did not cause any of the unwanted secondary pharmacology associated with diazepam (sedation, neuromuscular coordination deficit, interaction with CNS depressants, memory impairment,
or changing convulsive thresholds) (80). The affinity for recombinant human brain
mGlu2/3 receptors, measured as displacement of 3H-LY341495 binding, shows Ki
values of 85 and 125 nM. Agonist activity on human cloned metabotropic glutamate receptors, measured as decreases of forskolin-stimulated cAMP, shows EC50
values of 5 and 24 nM (81). Preclinical studies with this agonist have not, however, been able to relate the degree of mGlu2/3 receptor occupancy to behavioral
or functional changes.
In the absence of any means of assessing receptor occupancy or any measurable
physiologic effects in humans of doses up to 200 mg twice daily (Eli Lilly, unpublished results), we investigated the penetration of LY354740 into the CSF at steady
state following BID dosing for two weeks to see if drug was present in the CNS
compartment. The exposure of LY354740 in the CSF was approximately 5% of that
measured in plasma, and the median CSF concentrations over 12 h at steady state
were in the range of the in vitro potency for human mGlu2/3 receptors (70, 71). In
a highly exploratory approach, the CSF proteome is being assessed for LY354740
treatmentrelated changes as evidence of a functional effect. Even if positive, this
still cannot provide direct evidence that the predictions from in vitro models translate into a functional effect in living human brain without an analogous preclinical
in vivo proteomic study. In addition to looking for direct biomarkers of functional
effects, human anxiety models were applied in two studies (panic provocation by
CO2 and CCK challenge) of LY354740. Ten of 12 subjects reported significantly
fewer CCK-4-induced panic symptoms, had lower subjective anxiety ratings, and
had lower CCK-4-elicited ACTH release following one week of treatment with a
dose known to be in the range of the in vitro receptor potency (73). Collectively,
the data support the utility of a multimodal biomarker development strategy with
the mGlu2/3 receptor agonist for identifying biologically active doses of the compound to be used in large trials in anxiety-related conditions (72). The question
remains open whether it will ever be technically feasible for many receptor agonists and potentiators to relate occupancy to effect leaving one dependent on an
array of functional biomarkers.
In Table 5, selected CNS medications are summarized in context with assessed
biomarkers. It becomes clear that access to the brain and the fluid surrounding it
are least well known. Imaging studies usually become available later after tracer
development has been successful.
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Accessible biomarkers in humans for selected drugs

Plasma
PKa
Drug

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CSF
PKa
Proteomics
CSF
Brain
access
Receptor
[Ki]b
PDc
effects
Rxd
effects
Atomoxetine
Strattera
ADHDe
Fluoxetine
Prozac
Depression
Duloxetine
Cymbalta
Depression
LY354740
Anxiety
(+)h
Aprepitant
Emend
CINVf
Ki: Inhibitory constant.
PD: Pharmacodynamic(s).
d
e
+ (I)g
PK: Pharmacokinetic(s).
b
c
+ (I)g
Rx: Treatment.
ADHD: Attention-deficit/hyperactivity disorder.
CINV: Chemotherapy induced nausea and vomiting.
(I): Brain image.
(+): Unpublished results.
CONCLUSION
Many extensive reviews on biomarkers in drug development have been published
(24, 82, 83). This review focuses on the current status of biomarkers and/or approaches critical to assessing novel neuroscience targets with an emphasis on new
paradigms and challenges in this field of research. Nevertheless, some old questions
remain, such as how to verify access to the brain. Early imaging studies including
micro-PET (84, 85) can help to overcome this, at least for those compounds that
can be labeled and/or shown to affect another ligand. However, in case of delayed
tracer development or because of no feasible application of brain imaging effects
of the molecule, using CSF as a matrix could fill this gap. It is hoped that proteomic
research using CSF will have a major impact on the development of treatments for
psychiatric disorders.
In this review, the importance of biomarker data integration for psychotropic
drug development has been illustrated with examples for both clinically used medications and investigational drugs. The combination of biomarker development with
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current biomedical technologies applied to drug discovery can improve the level of
innovation and efficiency of drug discovery and developmental programs because
whether or not a drug engages its prestated target can be formally tested.

ACKNOWLEDGMENTS
The authors would like to acknowledge Robert A. Padich, Ph.D. of Lilly Global
Scientific Information and Communications for assisting with the preparation of
this manuscript. The authors are full-time employees of Eli Lilly and Company,
but the views expressed in this review are those of the authors.
APPENDIX
Definitions
Generally accepted definitions defined by working groups (86):
Biological marker (Biomarker): A characteristic that is objectively measured

and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
Pharmacologic marker (effect or response): A change representing a molecular interaction between drug and body constituent or the observable output.
Clinical end point: A characteristic or variable that reflects how a patient

feels, functions, or survives.
Surrogate end point: A biomarker intended to substitute for a clinical end

point. A surrogate end point is expected to predict clinical benefit (or harm
or lack of benefit) based on epidemiologic, therapeutic, pathophysiologic, or
other scientific evidence.
Proteomics: Proteomics represents the effort to establish the identities, quantities, structures, and biochemical and cellular functions of all proteins in an
organism, organ, or organelle, and how these properties vary in space, time,
or physiological state (87).
LITERATURE CITED
1. Bailey WJ, Ulrich R. 2004. Molecular profiling approaches for identifying
novel biomarkers. Expert Opin. Drug Saf.
3:13751
2. Colburn WA. 2003. Biomarkers in drug
discovery and development: from target
identification through drug marketing. J.

Clin. Pharmacol. 43:32941
3. Swanson BN. 2002. Delivery of highquality biomarker assays. Dis. Markers
18:4756
4. Colburn WA, Lee JW. 2003. Biomarkers,
3 Dec 2004
20:41
242
5.

6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
AR
BIECK
AR232-PA45-09.tex
AR232-PA45-09.sgm
LaTeX2e(2002/01/18)
P1: GCE
POTTER
validation and pharmacokinetic-pharmacodynamic modelling. Clin. Pharmacokinet. 42:9971022

Niggli E, Egger M. 2004. Applications of
multi-photon microscopy in cell physiology. Front. Biosci. 9:1598610
Oertner TG. 2002. Functional imaging of
single synapses in brain slices. Exp. Physiol. 87:73336
Rosse RB, Deutsch SI, Deutsch LH. 2000.
Medical assessment and laboratory testing in psychiatry. In Kaplan & Sadocks
Comprehensive Textbook of Psychiatry,
ed. BJ Sadock, VA Sadock. Philadelphia,
PA: Lippincott Williams & Wilkins. 750
pp.
Weinstein JN. 2001. Searching for pharmacogenomic markers: the synergy between omic and hypothesis-driven research. Dis. Markers 17:7788
Altman RB, Klein TE. 2002. Challenges
for biomedical informatics and pharmacogenomics. Annu. Rev. Pharmacol. Toxicol. 42:11333
Klein TE, Altman RB. 2004. PharmGKB:
the pharmacogenetics and pharmacogenomics knowledge base. Pharmacogenomics J. 4:1
Pardridge WM. 2003. Blood-brain barrier drug targeting: the future of brain
drug development. Mol. Intervent. 3:90
105
Spector R. 2000. Drug transport in the
mammalian central nervous system: multiple complex systems. A critical analysis
and commentary. Pharmacology 60:58
73
Nau R, Zysk G, Thiel A, Prange HW.
1993. Pharmacokinetic quantification of
the exchange of drugs between blood and
cerebrospinal fluid in man. Eur. J. Clin.
Pharmacol. 45:46975
Cutler NR, Sramek JJ. 1998. Exploratory
studies: implications for drug development in Alzheimers disease. Rev. Neurol.
(Paris) 154(Suppl. 2):S13136
Jhee SS, Zarotsky V. 2003. Safety and
tolerability of serial cerebrospinal fluid
16.
17.
18.
19.
20.
21.
22.
23.
24.
24a.
(CSF) collection during pharmacokinetic/pharmacodynamic studies: 5 years

of experience. Clin. Res. Regul. Aff. 20:
35763
Zoli M, Jansson A, Sykova E, Agnati
LF, Fuxe K. 1999. Volume transmission
in the CNS and its relevance for neuropsychopharmacology. Trends Pharmacol. Sci. 20:14250
Duggan AW. 2000. Neuropeptide spread
in the brain and spinal cord. Prog. Brain
Res. 125:36980
Hoeistad M, Jacobsen K, Olsson A,
Brodin E, Hansson H-A, et al. 2003. The
cerebrospinal fluid as a migration route
for neuropeptides in the rat brain: longdistance migration of beta-endorphin.
Presented at Int. Conf. In Vivo Methods, 10th, Karolinska Institutet, Stockholm, Sweden
Greitz D, Franck A, Nordell B. 1993. On
the pulsatile nature of intracranial and
spinal CSF-circulation demonstrated by
MR imaging. Acta Radiol. 34:32128
Christensen JD, Yurgelun-Todd DA, Babb
SM, Gruber SA, Cohen BM, Renshaw
PF. 1999. Measurement of human brain
dexfenfluramine concentration by 19F
magnetic resonance spectroscopy. Brain
Res. 834:15
Kornhuber J, Schultz A, Wiltfang J,
Meineke I, Gleiter CH, et al. 1999. Persistence of haloperidol in human brain tissue.
Am. J. Psychiatry 156:88590
Riederer P, Laux G. 1992. Therapeutic
drug monitoring of psychotropics: report
of a consensus conference. Pharmacopsychiatry 25:27172
Hutchinson PJ, OConnell MT, Kirkpatrick PJ, Pickard JD. 2002. How can we
measure substrate, metabolite and neurotransmitter concentrations in the human
brain? Physiol. Meas. 23:R75109
Ahmed S, Mozley PD, Potter WZ. 2002.
Biomarkers in psychotropic drug development. Am. J. Geriatr. Psychiatry 10:678
86
Wong DF, Potter WZ, Brasic JR. 2002.
3 Dec 2004
20:41
AR
AR232-PA45-09.tex
AR232-PA45-09.sgm
LaTeX2e(2002/01/18)
P1: GCE

25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Proof of concept: functional models for

drug development in humans. In Neuropsychopharmacology. The Fifth Generation of Progress, ed. KL Davis, D Charney, JT Coyle, C Nemeroff, pp. 457
73. Philadelphia: Lippincott Williams &
Wilkins
Marsden CA, Stanford SC. 2000. CNS
drugs III: psychotherapeutics. Expert
Opin. Investig. Drugs 9:192329
He QY, Chiu JF. 2003. Proteomics in
biomarker discovery and drug development. J. Cell. Biochem. 89:86886
Petricoin EF, Liotta LA. 2004. Clinical
proteomics: application at the bedside.
Contrib. Nephrol. 141:93103
Rohlff C, Hollis K. 2003. Modern proteomic strategies in the study of complex
neuropsychiatric disorders. Biol. Psychiatry 53:84753
Myung JK, Krapfenbauer K, Weitzdoerfer R, Peyrl A, Fountoulakis M, Lubec G.
2003. Expressional pattern of chaperones
in neuronal, glial, amnion, mesothelial,
and bronchial epithelial cell lines. Mol.
Genet. Metab. 80:44450
Peyrl A, Krapfenbauer K, Slavc I, Strobel
T, Lubec G. 2003. Proteomic characterization of the human cortical neuronal cell
line HCN-2. J. Chem. Neuroanat. 26:171
78
Tsuji T, Shimohama S. 2001. Analysis
of the proteomic profiling of brain tissue in Alzheimers disease. Dis. Markers
17:24757
Basso M, Giraudo S, Lopiano L, Bergamasco B, Bosticco E, et al. 2003. Proteome analysis of mesencephalic tissues:
evidence for Parkinsons disease. Neurol.
Sci. 24:15556
Jiang L, Lindpaintner K, Li HF, Gu NF,
Langen H, et al. 2003. Proteomic analysis of the cerebrospinal fluid of patients
with schizophrenia. Amino Acids 25:49
57
Puchades M, Hansson SF, Nilsson CL,
Andreasen N, Blennow K, Davidsson
P. 2003. Proteomic studies of poten-
35.
36.
37.
38.
39.
40.
41.
42.
43.
243
tial cerebrospinal fluid protein markers

for Alzheimers disease. Brain Res. Mol.
Brain Res. 118:14046
Voshol H, Glucksman MJ, van Oostrum J.
2003. Proteomics in the discovery of new
therapeutic targets for psychiatric disease.
Curr. Mol. Med. 3:44758
You J-S, Gelfanova V, Knierman MD,
Witzmann FA, Wang M, Hale JE. 2004.
The impact of blood contamination on
the proteome of cerebrospinal fluid. Proteomics. In press
Reiber H. 1995. External quality assessment in clinical neurochemistry: survey of
analysis for cerebrospinal fluid (CSF) proteins based on CSF/serum quotients. Clin.
Chem. 41:25663
Reiber H, Thompson EJ, Grimsley G,
Bernardi G, Adam P, et al. 2003. Quality assurance for cerebrospinal fluid protein analysis: international consensus by
an Internet-based group discussion. Clin.
Chem. Lab. Med. 41:33137
Schou M, Halldin C, Sovago J, Pike
VW, Gulyas B, et al. 2003. Specific in
vivo binding to the norepinephrine transporter demonstrated with the PET radioligand, (S,S)-[11C]MeNER. Nucl. Med.
Biol. 30:70714
Bieck PR, Antonin K-H, Schulz R.
1993. Clinical pharmacology of MAO inhibitors. In Monoamine Oxidase: Basic
and Clinical Aspects, ed. H Yasuhara, SH
Parvez, K Oguchi, M Sandler, T Nagatsu,
pp. 17796. Utrecht: VSP
Fowler JS, Logan J, Wang GJ, Franceschi
D, Volkow ND, et al. 2003. Monoamine
oxidase A imaging in peripheral organs in
healthy human subjects. Synapse 49:178
87
Fowler JS, Logan J, Volkow ND, Wang
GJ, MacGregor RR, Ding YS. 2002.
Monoamine oxidase: radiotracer development and human studies. Methods 27:
26377
Robertson D, Flattem N, Tellioglu T,
Carson R, Garland E, et al. 2001. Familial orthostatic tachycardia due to
3 Dec 2004
20:41
244
44.

45.
46.
47.
48.
49.
50.
51.
52.
AR
BIECK
AR232-PA45-09.tex
AR232-PA45-09.sgm
LaTeX2e(2002/01/18)
P1: GCE
POTTER
norepinephrine transporter deficiency.

Ann. NY Acad. Sci. 940:52743
Shannon JR, Flattem NL, Jordan J, Jacob
G, Black BK, et al. 2000. Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. New Engl. J. Med. 342:54149
Vincent S, Bieck PR, Garland EM, Loghin
C, Bymaster FP, et al. 2004. Clinical assessment of norepinephrine transporter
blockade through biochemical and pharmacologic profile. Circulation 109:3202
7
Bymaster FP, Katner JS, Nelson DL,
Hemrick-Luecke SK, Threlkeld PG, et al.
2002. Atomoxetine increases extracellular levels of norepinephrine and dopamine
in prefrontal cortex of rat: a potential mechanism for efficacy in attention
deficit/hyperactivity disorder. Neuropsychopharmacology 27:699711
Shlik J, Aluoja A, Vasar V, Vasar E, Podar
T, Bradwejn J. 1997. Effects of citalopram
treatment on behavioural, cardiovascular
and neuroendocrine response to cholecystokinin tetrapeptide challenge in patients
with panic disorder. J. Psychiatry Neurosci. 22:33240
Klein DF. 1993. False suffocation alarms,
spontaneous panics, and related conditions. An integrative hypothesis. Arch.
Gen. Psychiatry 50:30617
Bell CJ, Malizia AL, Nutt DJ. 1999. The
neurobiology of social phobia. Eur. Arch.
Psychiatry Clin. Neurosci. 249(Suppl.
1):S1118
Bourin M, Baker GB, Bradwejn J. 1998.
Neurobiology of panic disorder. J. Psychosom. Res. 44:16380
Kellner M, Yassouridis A, Hua Y, Wendrich M, Jahn H, Wiedemann K. 2002. Intravenous C-type natriuretic peptide augments behavioral and endocrine effects
of cholecystokinin tetrapeptide in healthy
men. J. Psychiatr. Res. 36:16
Wiedemann K, Jahn H, Yassouridis A,
Kellner M. 2001. Anxiolyticlike effects
of atrial natriuretic peptide on chole-
53.
54.
55.
56.
57.
58.
59.
60.
cystokinin tetrapeptide-induced panic attacks: preliminary findings. Arch. Gen.

Psychiatry 58:37177
Kent JM, Papp LA, Martinez JM, Browne
ST, Coplan JD, et al. 2001. Specificity
of panic response to CO(2) inhalation in
panic disorder: a comparison with major depression and premenstrual dysphoric disorder. Am. J. Psychiatry 158:5867
Bradwejn J, Koszycki D, Annable L, Couetoux du Tertre A, Reines S, Karkanias C.
1992. A dose-ranging study of the behavioral and cardiovascular effects of CCKtetrapeptide in panic disorder. Biol. Psychiatry 32:90312
Guttmacher LB, Murphy DL, Insel TR.
1983. Pharmacologic models of anxiety.
Compr. Psychiatry 24:31226
Bradwejn J, Koszycki D. 1994. Imipramine antagonism of the panicogenic effects of cholecystokinin tetrapeptide in
panic disorder patients. Am. J. Psychiatry
151:26163
Zwanzger P, Eser D, Aicher S, Schule
C, Baghai TC, et al. 2003. Effects of
alprazolam on cholecystokinin-tetrapeptideinduced panic and hypothalamic-pituitary-adrenal-axis activity: a
placebo-controlled study. Neuropsychopharmacology 28:97984
Zwanzger P, Baghai TC, Schuele C,
Strohle A, Padberg F, et al. 2001.
Vigabatrin decreases cholecystokinintetrapeptide (CCK-4) induced panic in
healthy volunteers. Neuropsychopharmacology 25:699703
Kapur S, Zipursky R, Jones C, Remington G, Houle S. 2000. Relationship between dopamine D(2) occupancy, clinical
response, and side effects: a double-blind
PET study of first-episode schizophrenia.
Nyberg S, Eriksson B, Oxenstierna G,
Halldin C, Farde L. 1999. Suggested minimal effective dose of risperidone based
on PET-measured D2 and 5-HT2A receptor occupancy in schizophrenic patients.
3 Dec 2004
20:41
AR
AR232-PA45-09.tex
AR232-PA45-09.sgm
LaTeX2e(2002/01/18)
P1: GCE


61. Mamo D, Sedman E, Tillner J, Sellers
EM, Romach MK, Kapur S. 2004. EMD
281014, a specific and potent 5HT(2)
antagonist in humans: a dose-finding
PET study. Psychopharmacology (Berl.)
http: / / www.springerlink.com/app/home/
contribution.asp?wasp = 320lvlyqlq4u1y
32wmf0&referrer=parent&backto=issue,
149,162;journal,1,183;linkingpublication
results,1:100390
62. Lemberger L, Rowe H, Carmichael R,
Oldham S, Horng JS, et al. 1978. Pharmacologic effects in man of a specific serotonin-reuptake inhibitor. Science
199:43637
63. Bymaster FP, Zhang W, Carter PA,
Shaw J, Chernet E, et al. 2002.
Fluoxetine, but not other selective serotonin uptake inhibitors, increases norepinephrine and dopamine extracellular
levels in prefrontal cortex. Psychopharmacology (Berl.) 160:35361
64. Martensson B, Nyberg S, Toresson G,
Brodin E, Bertilsson L. 1989. Fluoxetine treatment of depression. Clinical effects, drug concentrations and monoamine
metabolites and N-terminally extended
substance P in cerebrospinal fluid. Acta
Psychiatr. Scand. 79:58696
65. Karson CN, Newton JE, Livingston R,
Jolly JB, Cooper TB, et al. 1993. Human
brain fluoxetine concentrations. J. Neuropsychiatry Clin. Neurosci. 5:32229
66. Mayberg HS, Brannan SK, Tekell JL,
Silva JA, Mahurin RK, et al. 2000. Regional metabolic effects of fluoxetine in
major depression: serial changes and relationship to clinical response. Biol. Psychiatry 48:83043
67. Owens MJ, Morgan WN, Plott SJ, Nemeroff CB. 1997. Neurotransmitter receptor and transporter binding profile of
antidepressants and their metabolites. J.
68. Henry ME, Kaufman MJ, Hennen J,
Michelson D, Schmidt ME, et al. 2003.
Cerebral blood volume and clinical
changes on the third day of placebo substi-
69.
70.
71.
72.
73.
74.
75.
76.
245
tution for SSRI treatment. Biol. Psychiatry

53:1005
Mutel V, Adam G, Chaboz S, Kemp JA,
Klingelschmidt A, et al. 1998. Characterization of (2S,2 R,3 R)-2-(2 ,3 -[3H]dicarboxycyclopropyl)glycine binding in
rat brain. J. Neurochem. 71:255864
Bieck PR, Geracioti TD, DSouza B,
Ledent E, Perkins EJ, et al. 2002. Penetration of orally administered LY354740 into
the human CSF. Int. J. Neuropsychopharmacology 5(Suppl. 1):S150
Bieck PR, Jhee SS, Ledent E, Mackie
AE, Patil S, et al. 2003. Application of
continuous cerebrospinal fluid (CSF) collection in man to assess the glutamate
receptor antagonist LY354740 pharmacokinetics and its effect on biomarker
and proteomics. Presented at Monitoring
Molecules in Neuroscience, Int. Conf. on
In Vivo Methods, 10th, Stockholm, Sweden
Schoepp DD, Wright RA, Levine LR,
Gaydos B, Potter WZ. 2003. LY354740,
an mGlu2/3 receptor agonist as a novel
approach to treat anxiety/stress. Stress
6:18997
Kellner M, Muhtz C, Yassouridis A, Stark
K, Arlt J, et al. 2004. Effects of the
metabotropic glutamate type II agonist
LY544344 on panic and anxiety induced
by cholecystokinin tetrapeptide (CCK4).
Int. J. Neuropsychopharmacol. 7:S369
Van Belle S, Lichinitser MR, Navari RM,
Garin AM, Decramer ML, et al. 2002.
Prevention of cisplatin-induced acute and
delayed emesis by the selective neurokinin-1 antagonists, L-758,298 and MK869. Cancer 94:303241
Campos D, Pereira JR, Reinhardt RR, Carracedo C, Poli S, et al. 2001. Prevention of cisplatin-induced emesis by the
oral neurokinin-1 antagonist, MK-869, in
combination with granisetron and dexamethasone or with dexamethasone alone.
J. Clin. Oncol. 19:175967
Hargreaves R. 2002. Imaging substance P
receptors (NK1) in the living human brain
3 Dec 2004
20:41
246
77.

78.
79.
80.
81.
AR
BIECK
AR232-PA45-09.tex
AR232-PA45-09.sgm
LaTeX2e(2002/01/18)
P1: GCE
POTTER
using positron emission tomography. J.

Clin. Psychiatry 63(Suppl. 11):1824
Bergstrom M, Hargreaves RJ, Burns DH,
Goldberg MR, Sciberras D, et al. 2004.
Human positron emission tomography
studies of brain neurokinin 1 receptor occupancy by aprepitant. Biol. Psychiatry
55:100712
Chawla SP, Grunberg SM, Gralla RJ,
Hesketh PJ, Rittenberg C, et al. 2003.
Establishing the dose of the oral NK1 antagonist aprepitant for the prevention of
chemotherapy-induced nausea and vomiting. Cancer 97:2290300
Ranga K, Krishnan R. 2002. Clinical experience with substance P receptor (NK1)
antagonists in depression. J. Clin. Psychiatry 63(Suppl. 11):2529
Helton DR, Tizzano JP, Monn JA,
Schoepp DD, Kallman MJ. 1998. Anxiolytic and side-effect profile of LY354740:
a potent, highly selective, orally active
agonist for group II metabotropic glutamate receptors. J. Pharmacol. Exp. Ther.
284:65160
Schoepp DD, Johnson BG, Wright RA,
Salhoff CR, Mayne NG, et al. 1997.
LY354740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human
82.
83.
84.
85.
86.
87.
glutamate receptors. Neuropharmacology

36:111
Frank R, Hargreaves R. 2003. Clinical
biomarkers in drug discovery and development. Nat. Rev. Drug Discov. 2:566
80
Wong ML, Licinio J. 2004. From monoamines to genomic targets: a paradigm
shift for drug discovery in depression. Nat.
Rev. Drug Discov. 3:13651
Weber DA, Ivanovic M. 1999. Ultrahigh-resolution imaging of small animals:
implications for preclinical and research
studies. J. Nucl. Cardiol. 6:33244
Acton PD, Choi SR, Plossl K, Kung HF.
2002. Quantification of dopamine transporters in the mouse brain using ultra-high
resolution single-photon emission tomography. Eur. J. Nucl. Med. Mol. Imaging
29:69198
De Gruttola VG, Clax P, DeMets DL,
Downing GJ, Ellenberg SS, et al. 2001.
Considerations in the evaluation of surrogate endpoints in clinical trials. Summary of a National Institutes of Health
workshop. Control. Clin. Trials 22:485
502
Report W. 2002. Defining the Mandate
Of Proteomics in the Post-Genomics Era.
Washington, DC: The Natl. Acad. Press
7 Jan 2005
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Copyright
NEONICOTINOID INSECTICIDE TOXICOLOGY:

Mechanisms of Selective Action

Motohiro Tomizawa and John E. Casida
Environmental Chemistry and Toxicology Laboratory, Department of Environmental
Science, Policy and Management, University of California, Berkeley,
California 94720-3112; email: tomizawa@nature.berkeley.edu,
ectl@nature.berkeley.edu
Key Words binding site specificity, biotransformation, imidacloprid, nicotinic

receptor, selective toxicity
Abstract The neonicotinoids, the newest major class of insecticides, have outstanding potency and systemic action for crop protection against piercing-sucking
pests, and they are highly effective for flea control on cats and dogs. Their common
names are acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam. They generally have low toxicity to mammals (acute and
chronic), birds, and fish. Biotransformations involve some activation reactions but
largely detoxification mechanisms. In contrast to nicotine, epibatidine, and other ammonium or iminium nicotinoids, which are mostly protonated at physiological pH, the
neonicotinoids are not protonated and have an electronegative nitro or cyano pharmacophore. Agonist recognition by the nicotinic receptor involves cation- interaction
for nicotinoids in mammals and possibly a cationic subsite for interaction with the nitro
or cyano substituent of neonicotinoids in insects. The low affinity of neonicotinoids
for vertebrate relative to insect nicotinic receptors is a major factor in their favorable
toxicological profile.
INTRODUCTION
Pest insect control, an essential component of crop protection and public health,
has evolved over a recorded history of three millennia (1, 2). Sulfur was first referred to by Homer in 1000 BC as a fumigant for pest control, and, in California,
it is still used in larger amounts than any other pesticide. Nicotine in the form of
tobacco extracts was reported in 1690 as the first plant-derived insecticide, followed by the pyrethrins from pyrethrum flowers and rotenone from derris roots
in the early 1800s. Synthetic organics in the 1940s to the 1970s largely replaced
inorganics and botanicals with the introduction of organophosphates, methylcarbamates, organochlorines, and pyrethroids. With each new chemical class, resistant
strains were soon selected to limit their effectiveness. Genetically modified crops
expressing Bacillus thuringiensis (Bt) -endotoxin were introduced for pest insect
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control in 1995. Many of the remaining gaps in pest control capabilities were filled
recently by the neonicotinoids (Figure 1), which combine outstanding effectiveness
with relatively low toxicity to vertebrates (37).

NEONICOTINOIDS
The current synthetic organic insecticides were discovered by modifying natural products, e.g., using the pyrethrins as a prototype for synthetic pyrethroids,
or by screening hundreds of thousands of structurally diverse compounds for
novel leads. Nicotine is still used as a minor insecticide, particularly in China,
but attempts to improve its insecticidal activity, e.g., 3 ,4 -dehydronicotine and
3-(alkylaminomethyl)-pyridines (8), were not successful. The lead for the neonicotinoids, 2-(dibromonitromethyl)-3-methylpyridine, was discovered in 1970 by
Shell Development Company in California to have modest activity against house
flies and pea aphids (911). Molecular modifications to achieve optimal potency
on corn earworm larvae (a major lepidopterous pest) culminated with nithiazine,
but unfortunately it could not be commercialized for crop protection due to photoinstability (10, 12). This shortcoming relegated nithiazine to a niche market for
fly abatement in poultry and animal husbandry (11). A major improvement of its
structure was made by Nihon Tokushu Noyaku Seizo in Japan (presently Bayer
Crop Science Japan) by introducing a chloropyridinylmethyl group, leading to
a nitromethylene prototype of outstanding potency on green rice leafhopper (a
major pest of rice and vegetables). However, photoinstability again prevented its
use for crop protection. Further structure-activity studies established that good
activity was retained on replacement of the imidazolidine by thiazolidine or oxadiazinane or acylic counterpart, and the chloropyridinylmethyl by chlorothiazolylmethyl or tetrahydrofuranmethyl. Changing the nitromethylene to nitroguanidine
or cyanoamidine afforded photostability and produced highly effective compounds
in field conditions (3, 13, 14). The current neonicotinoids and their year of patent are
the heterocyclics nithiazine (1977), imidacloprid (IMI) (1985), thiacloprid (1985),
and thiamethoxam (1992); and the acyclics nitenpyram (1988), acetamiprid (1989),
clothianidin (1989), and dinotefuran (1994). The physical properties of the neonicotinoids and nicotine are compared in Table 1. Molecular weights range from 160
to 292 and log P values from 0.66 to 1.26. The compounds vary in water solubility
from 0.1850.61 g/l for clothianidin, IMI, and thiacloprid, to infinite for nicotine.
The neonicotinoids are the only major new class of insecticides developed in the
past three decades. Worldwide annual sales of neonicotinoids are approximately
one billion dollars, accounting for 11%15% of the total insecticide market. They
are readily absorbed by plants and act quickly, at low doses, on piercing-sucking insect pests (aphids, leafhoppers, and whiteflies) of major crops. The neonicotinoids
are poorly effective as contact insecticides and for control of lepidopterous larvae.
They are used primarily as plant systemics; when applied to seeds, soil, or foliage
they move to the growing tip and afford long-term protection from piercing-sucking
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NEONICOTINOID TOXICOLOGY
Figure 1 Nine neonicotinoid insecticides and four nicotinoids. The neonicotinoids

=CHNO2), nitroguanidines (C=
=NNO2), and cyanoamidines
are nitromethylenes (C=
=
=
(C NCN). Compounds with 6-chloro-3-pyridinylmethyl, 2-chloro-5-thiazolylmethyl,
and 3-tetrahydrofuranmethyl moieties are referred to as chloropyridinyls (or chloronicotinyls), chlorothiazolyls (or thianicotinyls), and tefuryl, respectively. The nicotinoids
are naturally occurring [()-nicotine and ()-epibatidine] and synthetics (ABT-594
and desnitroimidacloprid).
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Physical properties of the neonicotinoids and nicotine
Compound
Molecular weight
Water solubility (g/l)
Log Pa
222.7
249.7
202.2
255.7
270.7
160.1
252.7
291.7
4.25
0.300.34
54.3
0.61
>590
200
0.185
4.1
0.80
0.7
0.64
0.57
0.66
0.60
1.26
0.13
162.2

Neonicotinoids
Acetamiprid
Clothianidin
()-Dinotefuran
Imidacloprid
Nitenpyram
Nithiazine
Thiacloprid
Thiamethoxam
Nicotinoid
()-Nicotine
0.93 (free base)
Data from References 1517.

a
P = 1-octanol/water partition.
insects, e.g., for 40 days in rice. Some organophosphates and methylcarbamates

also have good systemic activity but their use is declining due to selection of
resistant insect strains and increasing restrictions based on human safety considerations. The expanding importance of crops expressing Bt -endotoxin encourages
neonicotinoid use because the types of pests not controlled by the endotoxin are
often those highly sensitive to neonicotinoids. Although crop protection is the major use for neonicotinoids, pest insect control on pets or companion animals is also
a significant market. IMI and nitenpyram are highly effective flea control agents
on cats and dogs, and are administered as oral tablets or topical spot treatments
(see, for example, Reference 18).
While the nicotinoids are structurally similar to the neonicotinoids, they primarily differ by containing an ionizable basic amine or imine substituent (Figure 1).
Notable nicotinoids other than nicotine are two very potent candidate analgesic
agents, i.e., epibatidine, which was isolated from the skin of an Ecuadoran frog
(19, 20), and ABT-594 (21, 22). Interestingly, the same chloropyridinyl substituent
appears in both these nicotinoids and the optimized neonicotinoid insecticides.
Desnitro-IMI, an iminium metabolite of IMI, fits the nicotinoid category (23).
TOXICOLOGY
The neonicotinoids have unique physical and toxicological properties as compared
with earlier classes of organic insecticides (Table 2). They generally have the lowest log P values, which is consistent with their outstanding plant systemic activity
shared by some organophosphates and methylcarbamates but not by the more
lipophilic organochlorines and pyrethroids. The neonicotinoids and pyrethroids
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TABLE 2
Comparison of neonicotinoids with other classes of insecticidesa

Potency
(LD50 , mg/kg)c
Systemic
action
Nerve targetb Insects Rats
Class
Log P
Neonicotinoids
0.7 to 1.3 +
Selectivity
factor
nAChR
2.0
912
456
Organophosphates 1 to 5.5
AChE
2.0
67
33
Methylcarbamates 1 to 3
AChE
2.8
45
16
Organochlorines
5.5 to 7.5
Na+ or Cl
channels
2.6
230
91
Pyrethroids
4 to 9
Na+ channel
0.45
2000
4500
Data from Reference 24 except for neonicotinoids.
Insecticide examples are the organophosphates parathion and malathion (as their oxon metabolites) and
methylcarbamates carbaryl and aldicarb inhibiting AChE, the organochlorine DDT and the pyrethroid deltamethrin
acting on the voltage-sensitive sodium channel, and the organochlorines endosulfan and lindane blocking the
-aminobutyric acid (GABA)-gated chloride channel.
Geometric means of large data sets (11 to 83 items each) for rat acute oral and insect topical (principally four
species) LD50 values for all classes of compounds except neonicotinoids (24). Values for the neonicotinoids are
geometric means for rat oral LD50 data in Table 3 and arbitrary for insects to reflect similar potency of neonicotinoids
and organophosphates on the same target insects.
have higher selectivity factors for insects versus mammals than the organophosphates, methylcarbamates, and organochlorines. This is attributable to both target
site specificity and detoxification, which are considered later. The neonicotinoids
act as agonists at the nicotinic acetylcholine receptors (nAChRs) of insects and
mammals (particularly the 42 subtype) (7).
The toxicological profiles of the individual neonicotinoids and nicotine are compared in Table 3. The acute oral LD50 values (mg/kg) for rats range from 5060 for
nicotine to >5000 for clothianidin. When ranked on the basis of chronic toxicity
to rats, reported as no-observed-adverse-effect-level (NOAEL; the principal toxicological parameter used in risk assessment), thiacloprid and thiamethoxam have
the lowest values (0.61.2 mg/kg/day) and are rated as likely human carcinogens.
Intermediate values (5.79.8 mg/kg/day) are observed for acetamiprid, clothianidin, and IMI, whereas dinotefuran has the highest value. The EPA has not followed
a cumulative risk approach in determining pesticide tolerances for neonicotinoids
and has not assumed that each neonicotinoid has a common mechanism of toxicity
with other substances (2530). Of the commercial neonicotinoids, acetamiprid,
IMI, and thiacloprid are the most toxic to birds, and thiacloprid to fish. Several
neonicotinoids are harmful to honeybees, either by direct contact or ingestion, but
potential problems can be minimized or avoided by treating seeds and not spraying
flowering crops (15).
The mammalian toxicity of neonicotinoids is considered to be centrally mediated because the symptoms of poisoning are similar to those of nicotine. Toxicity
correlates with agonist action and binding affinity at the vertebrate 42 nAChR,
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Toxicological profiles of the neonicotinoids and nicotinea

Mammalb

P1: GCE
Birdf
Fishg
Compound
Acute oralc
LD50 (mg/kg)
NOAELd
(mg/kg/day)
Carcinogene
Acute oral
LD50 (mg/kg)
LC50
(ppm)
Neonicotinoids
Acetamiprid
Clothianidin
()-Dinotefuran
Imidacloprid
Nitenpyram
Nithiazine
Thiacloprid
Thiamethoxam
182
>5000
2400
450
1628
300
640
1563
7.1
9.8
127
5.7
1.2
0.6
No
No
No
No
Yes
Yes
180
>2000
>2000
31
>2250
49
1552
>100
>100
>40
211
>1000
150
31
>100
Nicotinoid
()-Nicotine
5060
Toxic
Data from References 9, 15, 2530.
b
c
Dermal LD50 values of neonicotinoids are >2000 to >5000 mg/kg (rat) except for ()-nicotine 50 mg/kg (rabbit).
Average data for male and female rats with sex difference less than twofold.
No-observed-adverse-effect-level (NOAEL) for chronic toxicity studies in rats. This value also applies to all adverse effects
in chronic toxicity studies with mice and dogs.
Thiacloprid gives thyroid and uterine tumors in rats and ovary tumors in mice. Thiamethoxam gives hepatocellular adenomas
and carcinomas in male and female mice. They are considered to be likely human carcinogens.
Japanese or bobwhite quail.
Rainbow trout or carp.
the primary target in brain (31). Chronic exposure to neonicotinoid insecticides,

and certain metabolites as well as nicotine, upregulates 42 nAChR levels without altering the sensitivity of the binding site. This upregulation in M10 cells is
initiated by receptor-insecticide interaction (32). Neonicotinoids and metabolites
also elicit acute intracellular responses, particularly in relation to signal integration
pathways in mammalian cells. In mouse neuroblastoma cells, low levels of these
compounds activate the extracellular-regulated (also called mitogen-activated) protein kinase cascade via the nAChR and intracellular calcium mobilization, leading
to possible attenuation of neuronal functions (33). Nicotine and other nicotinoids
are candidate therapeutic agents as analgesics and for treatment of neurodegenerative diseases (21, 22, 34). The potential activity of neonicotinoids is therefore of
interest. A nitromethylene neonicotinoid, with modest agonist action on the 42
nAChR, is as potent as nicotine in inducing antinociceptive activity in preclinical
pain models in mice. This effect persists longer than that of nicotine or epibatidine and appears to involve a different mechanism of action (31). However, other
neonicotinoid insecticides and metabolites (even with high agonist potency at the
42 nAChR) fail to induce analgesia, perhaps owing to adverse nociceptive and
toxic effects (21, 35, 36, 37) or insufficient subtype selectivity (23, 38).
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There are no specific antidotes for neonicotinoid poisoning in mammals (39).

Treatment with an acetylcholinesterase (AChE)-reactivating oxime (e.g., pralidoxime important in organophosphate poisoning) or a nicotinic antagonist might
be either ineffective or contraindicated. Symptomatic treatment is recommended
for any possible acute poisoning case.

BIOTRANSFORMATIONS
Metabolism of the commercial neonicotinoids has been extensively studied in
crops, rats, lactating goats, and laying hens (15, 40). These studies are part of
the EPA registration requirements for approved uses. Extensive data has been
published for IMI (28, 41), thiacloprid (29, 42), clothianidin (26, 43, 44), and to
a lesser extent, nithiazine (45), nitenpyram (40), acetamiprid (40), thiamethoxam
(30), and dinotefuran (27). Owing to their relatively high water solubility and slow
metabolism in mammals, some (IMI and thiacloprid) to almost all (clothianidin,
dinotefuran, and nitenpyram) of an oral neonicotinoid dose is excreted unchanged
in urine. The chemical fate of neonicotinoids in and on crops is governed both by
metabolic and photochemical reactions. These processes may produce identical or
different products depending on the mechanisms involved.
Analysis of neonicotinoid residues to enforce crop tolerances and registered
uses involves the parent compound and toxic metabolites. IMI residues are determined as the parent compound plus metabolites with the chloropyridinyl moiety.
Thiacloprid is combined with an amide and a hydroxy derivative in evaluating
residues. Clothianidin and acetamiprid residues are regulated as the parent compounds. Thiamethoxam residues are considered along with those of its principal
metabolite clothianidin. Dinotefuran residues are combined with those of its guanidine and urea metabolites. The analyses are achieved by various combinations of
high-performance liquid chromatography or gas chromatography with UV, electron capture, or mass spectrometry for detection and characterization.
Most neonicotinoids undergo metabolic alterations at multiple sites (Figure 2).
For convenience, different parts of the molecules are considered separately, indicating the known or presumed effects of the reactions on bioactivity. In Figure 2A,
oxidation of the nitromethylene carbon of nithiazine is likely a detoxification
mechanism (45). IMI is hydroxylated in the imidazolidine moiety at either one of
the two methylene substituents, which is followed by conjugation or dehydration
to form the olefin, apparently with little or no ring opening; these unconjugated
metabolites retain insecticidal activity or insect nAChR potency (4648). Some
N-demethylation is observed in each case with compound-dependent effects on
product potency. It greatly increases insecticidal and/or receptor activity for Nmethyl-IMI (a model compound) (16), nitenpyram (49), and thiamethoxam (50,
51). Thiamethoxam is readily converted to clothianidin by ring methylene hydroxylation in insects and plants (52), whereas clothianidin undergoes N-demethylation
(43, 44). The thiazolidine ring of thiacloprid is opened and the sulfur oxidized
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Figure 2 Neonicotinoid biotransformations shown by arrows as sites of metabolic attack

(A and B) and substituent modifications (C) on three moieties (see Figure 1 for full structures
and text for references). Asterisks designate sites of change leading to active metabolites
based on nAChR potency or toxicity, whereas all other sites yield or are presumed to give low
activity or inactive metabolites. The biotransformation reactions shown are in mammalian
systems unless indicated otherwise in the text.
and methylated (42). Acetamiprid undergoes N-demethylation and cleavage of the

N-cyanoacetamidine linkage in plants (40). The chloropyridinylmethyl, chlorothiazolylmethyl, and tetrahydrofuranmethyl substituents (Figure 2B) undergo Nmethylene hydroxylation and cleavage, followed by aldehyde oxidation to the
corresponding carboxylic acids, which are commonly excreted as glycine derivatives following conjugation. The chloro substituent is displaced presumably by
glutathione and ultimately leads (via cysteine and -SH derivatives) to the methylsulfide. With dinotefuran, hydroxylation of the tetrahydrofuran moiety leads to
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ring opening and liberation of an aldehyde that forms cyclic derivatives (27). The
=N NO2 (nitroguanidine) moiety (Figure 2C) of IMI is reduced to C=
=N NO
C=
=N NH2 (aminoguanidine), and cleaved to the C=
=NH
(nitrosoguanidine) and C=
=O (urea) derivatives. The aminoguanidine of clothianidin is
(guanidine) and C=
also conjugated with pyruvic or acetic acid, followed by cyclization (26). The
nitrosoguanidine metabolite of IMI has moderate to high insecticidal activity and
insect nAChR potency (53), whereas the guanidine metabolite is highly activated
against mammalian but deactivated against insect nAChRs (23, 38, 54).
Cytochrome P450 (CYP450) isozymes are involved in oxidative IMI metabolism. Based on studies with individual recombinant enzymes, human CYP3A4
is the predominant IMI N-methylene hydroxylase (55). The insecticidal activity
of many neonicotinoids is strongly synergized by CYP450 inhibitors, such as
piperonyl butoxide and O-propyl O-propynyl phenylphosphonate, suggesting that
these enzymes limit their efficacy (16, 56). Fruit flies (Drosophila melanogaster)
overexpressing CYP6G1 are resistant to IMI, probably owing to enhanced detoxification (57, 58). Several human P450s also reduce IMI to the nitroso derivative in
an oxygen-sensitive manner (55). A relatively oxygen-insensitive neonicotinoid
nitro reductase of rabbit liver cytosol (59) was recently identified as aldehyde
oxidase and readily converts IMI to nitrosoguanidine and aminoguanidine (60).
The aminoguanidine of IMI (a hydrazone) is further derivatized to an acetaldehyde
imine upon incubation with mammalian liver cytosol (60) and to a triazinone and
=N CN moiety of thiaother conjugates in vitro and in vivo (59, 60a). The C=
=NC(O)NH2] and also undergoes N CN
cloprid is hydrolyzed to the amide [C=
cleavage. Descyanothiacloprid (a plant metabolite) (42) is a particularly potent
mammalian nAChR agonist (23).
Toxicokinetic studies in mammals, so important in pharmaceutical research, are
often given lower priority in pesticide investigations. As an example, it is not clear
if the toxicity of IMI in mammals is due to the parent compound or the desnitro
metabolite (which enters the brain following direct intraperitoneal administration
in mice) (54). IMI is highly absorbed in a human intestinal cell model, suggesting
potential effects in mammals following ingestion (61).
NICOTINIC RECEPTORS
The vertebrate nAChR is an agonist-gated ion channel responsible for rapid excitatory neurotransmission. It is a pentameric transmembrane complex in the superfamily of neurotransmitter-gated ion channels, including -aminobutyric acid
(GABAA and GABAC), glycine, and 5-HT3 serotonin receptors. The nAChR consists of diverse subtypes assembled in combinations from ten , four , , , and
subunits. The skeletal muscle or electric ray (Torpedo) subtype is made up of
two 1 subunits and one each of 1, , and (or in adult muscle) subunits.
Neuronal nAChR subtypes expressed in vertebrate brain and ganglia are assembled in combinations of 210 and 24, and are pharmacologically classified
into two groups based on sensitivity to -bungarotoxin (-BGT). The 26 and
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24 subunits are involved in assembling the -BGT-insensitive subtypes, whereas

710 subunits are responsible for -BGT-sensitive receptors. Of these, the most
abundant subtypes in vertebrate brain are 42 and 7 (-BGT-insensitive and
-sensitive, respectively). The 42 subtype consists of two 4 and three 2 subunits (heteropentameric) and the 7 subtype is considered to be a homopentameric
structure (34, 62, 63). The agonist or drug-binding site is localized at the interface region between subunits. Specific subunit combinations confer differences
in sensitivity to acetylcholine (ACh) and/or pharmacological profiles among the
nAChR subtypes. The ligand-binding site in all subtypes consists of a conserved
core of aromatic amino acid residues (6467). Neighboring variable residues are
considered to confer individual pharmacological properties to each subtype (62).
The mammalian nAChR is a potential target for therapeutic agents for analgesia,
neurodegenerative diseases, cognitive dysfunction, schizophrenia, depression, and
anxiety (22). The most potent nicotinic agonist is epibatidine (20). An important
aspect of nicotinic drug development is the discovery of highly subtype-selective
agents (e.g., ABT-594; Figure 1) (21, 22, 6870).
Neonicotinoids have little or no effect on the vertebrate peripheral nAChR
1 11 subtype (23, 7173) or some neuronal subtypes [32 (and/or 4)5,
42, and 7] (16, 23, 54, 72, 7477). Minor structural modifications of neonicotinoids confer differential subtype selectivity in vertebrate nAChRs. Nitromethylene
analogs with high insecticidal activity display comparable or higher affinity than
that of nicotine to the 3245 or 7 subtype (Table 4). Toxicological evaluations of insecticide safety should consider both the nAChRs as a whole and as
major subtypes (23, 38).
The insecticidal activity of the neonicotinoids is due to their action as insect
nAChR agonists. This was first demonstrated by electrophysiological and [125I]BGT binding studies with nithiazine and the cockroach nerve cord (78, 79). It was
verified with IMI using binding studies with insect brain membranes and [3H]- or
[125I]-BGT (8083). More definitive confirmations were obtained with [3H]IMI
(84) by structure-activity correlations for displacement of binding potency with
knockdown activity (56, 85) and electrophysiological responses (86).
Insect nAChRs are less well understood than their vertebrate counterparts as to
functional architecture and diversity (87), as illustrated here with Drosophila. They
are widely distributed in the synaptic neuropil regions of the insect central nervous
system. In Drosophila, genes for four (D14) and three (D13) subunits
have been identified, and several additional candidate genes for nAChR subunits
are predicted from genome data (7, 8789). On expression in Xenopus oocytes, human embryonic kidney 293 cells, or Drosophila S2 cells, the four subunit genes
(D14) and three subunit genes (D13), alone or in various combinations,
never produce an electrophysiological response or [3H]epibatidine binding. However, functional ion channel property or [3H]epibatidine binding is clearly observed
when any of the four subunits is coexpressed with chick or rat 2 or with rat 4
subunit (9093). Also, D1/D2/chick 2 ternary receptor can be coassembled
within a single receptor complex, although functional channel property is unclear
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TABLE 4 Specificity of neonicotinoids and nicotinoids for insect and vertebrate

42 nicotinic receptors
IC50, nM

Compound
Insect
Vertebrate 42b,c
Selectivity ratio
Neonicotinoids
Acetamiprid
Clothianidin
()-Dinotefuran
Imidacloprid
Nitenpyram
Nithiazine
Prototype
Thiacloprid
Thiamethoxam
8.3
2.2
900
4.6
14
4800
0.24
2.7
5000
700
3500
>100,000
2600
49,000
26,000
210
860
>100,000
84
1591
>111
565
3500
5.4
875
319
>20
Nicotinoids
Desnitroimidacloprid
Descyanothiacloprid
()-Nicotine
()-Epibatidine
1530
200
4000
430
8.2
4.4
7.0
0.04
0.005
0.022
0.002
0.0001
For structures see Figure 1.
IC50 values for displacing [3H]imidacloprid binding to the house fly (Musca domestica) (acetamiprid),
aphid (Myzus persicae) (thiamethoxam), and fruit fly (the other neonicotinoids) receptor, and [3H]nicotine
binding to the vertebrate 42 nAChR.
IC50 values (M) for the vertebrate 7 nAChR subtype (assayed by [125I]-BGT binding) are acetamiprid,
290; clothianidin, 190; ()-dinotefuran, >1000; imidacloprid, 270; nitenpyram, >300; nithiazine, >300;
prototype neonicotinoid, 6.1; thiacloprid, 100; thiamethoxam, >300; desnitroimidacloprid, 9.9;
descyanothiacloprid, 6.0; ()-nicotine, 21; and ()-epibatidine, 0.031.
(94). Three Drosophila subunits (D13), each coassembled within D3/rat

2 or rat 42 receptor hybrid complexes, modulate [3H]epibatidine binding activity (95). Thus, coexpression of any Drosophila subunit with a vertebrate
subunit constitutes the best available model at present. However, these hybrid
receptors do not faithfully reflect native insect nAChRs. [3H]Epibatidine is generally not useful as a radioligand for native insect receptors (5), except for that of the
American cockroach (96). As expected, epibatidine is a weak displacer of [3H]IMI
binding to the Drosophila receptor and shows very low toxicity to insects (23). Immunological approaches suggest that two Drosophila subtypes exist consisting of
D1/D2/D2 and D3/D1 (97, 98). Protein biochemical approaches to native
Drosophila nAChR subunits, involving neonicotinoid affinity chromatography and
photoaffinity labeling, reveal the existence of several subunits, including D2 as
the main neonicotinoid-binding component (7, 49, 98100).
Distinctive pharmacological profiles are observed for hybrid nAChRs consisting
of various combinations of Drosophila and vertebrate 2 subunits (7, 87, 89). For
example, ACh-evoked current is blocked by -BGT in hybrid nAChRs consisting
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of Drosophila D1 or D3 and chick 2 subunits expressed in Xenopus oocytes;

however, the electrophysiological response is not blocked by -BGT in a hybrid
receptor of D2 and chick 2 subunits (90, 92). Similarly, [125I]-BGT recognizes
either D1/rat 2 or D3/rat 2, but not D2/rat 2 hybrid receptor expressed
in a Drosophila S2 clonal cell line, yet [3H]IMI and [3H]epibatidine bind to all
three of these hybrid receptors with high affinities (101). In contrast, IMI is totally
ineffective in generating an electrophysiological response with the D1/chick 2
hybrid receptor expressed in Xenopus oocyte (77). A D4/rat 2 hybrid receptor
demonstrates [3H]epibatidine but not [125I]-BGT binding activity (93). Native
Drosophila nAChRs contain distinct binding sites for IMI and -BGT, but it is
not clear if they are on the same or different receptors (N. Zhang, M. Tomizawa,
J.E. Casida, unpublished observations).
MOLECULAR FEATURES OF NICOTINIC AGONISTS

Neonicotinoid insecticides display excellent selectivity profiles that are largely
attributable to specificity for insect versus mammalian nAChRs (7) (Table 4).
Neonicotinoids and nicotinoids have common structural features (Figure 1) but
different protonation states at physiological pH. The neonicotinoids (e.g., IMI) are
not protonated and selective for the insect nAChR, whereas the nicotinoids (e.g.,
nicotine) are cationic in nature and consequently selective for the mammalian
nAChR. Therefore, neonicotinoids and their analogs are excellent probes to help
define the mechanisms of selectivity and ultimately the topological divergence
between insect and vertebrate binding sites.
The nicotinic pharmacophore model for mammals is derived from nicotinoid
structure-activity relationships as compared with ACh, the endogenous agonist.
The pKa of nicotine (pyrrolidinyl nitrogen) is 7.90; therefore, at physiological pH,
89% will be protonated (8). ()-Nicotine and ACh share three structural elements:
a quaternary (sp3) nitrogen atom, a hydrogen bond acceptor (the pyridine nitrogen
of nicotine and the carboxyl oxygen of ACh), and a dummy point (a receptorrelated feature that imposes directionality to the pyridine nitrogen of nicotine or
the corresponding oxygen of ACh). The center of the sp3 nitrogen atom is situated
from the van der Waals surface of the hydrogen-bond acceptor
approximately 5.9 A
does not
(102, 103). The internitrogen (N-N) distance of ()-epibatidine (5.5 A)
coincide with that of ()-nicotine (4.8 A) (104, 105). This is rationalized by placing
an additional directional requirement on the ammonium nitrogen atom, which, in
modeling studies, confers reasonable overlap of ()-nicotine and ()-epibatidine
by orienting the pyridine nitrogen atom proximal to the sp3 nitrogen atom (N N
(68, 106). The iminium (+C NH2 C=
=+NH2) metabolites
distance of 4.79 A)
of IMI and thiacloprid (desnitro and descyano, respectively) are mostly protonated
at physiological pH (23).
Neonicotinoids have, instead of an easily protonated nitrogen, the nitro or cyano
or equivalent electronegative pharmacophore, and they demonstrate a coplanarity
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between this tip and the substituted guanidine/amidine moiety (Figure 3) (23, 53,
107, 108). The guanidine moiety of IMI has pKa values of 1.56 and 11.12 for
protonation and deprotonation, respectively (109), indicating less than 0.0002%
protonation at physiological pH. The coplanarity allows a conjugated electronic
system that facilitates negative charge flow toward the electronegative tip to consolidate the binding. Interestingly, high affinity and insect receptor selectivity are
=N NO) analogs of neonicotinoids, suggesting that only
retained in nitroso (C=
one (O1) of the electronegative oxygen atoms is essential (53). The role of N1
(Figure 3) in neonicotinoid action is of particular interest. The distance between
the van der Waals surfaces of the two nitrogen atoms in nicotine (102) is the same
as that between the pyridinyl and N1 nitrogen atoms in the neonicotinoids (5.45
Additionally, a partial positive charge ( +) for the N1 nitrogen atom is
6.06 A).
conferred by the electron-withdrawing nitro or cyano substituent (74, 83, 107)
as supported by comparative molecular field analysis (110) and semiempirical
molecular orbital theory (PM3) calculation (111). The same relationship in the
above distance is also observed between N1 and O1 of the neonicotinoids (107).
However, the N1 atom does not have a significant positive charge in PM3 and
high-level ab initio calculations (17, 53, 112), and it can be replaced by a carbon
atom with retention of significant binding activity to the Drosophila receptor and
toxicity to insects (108, 113). The pyridin-3-ylmethyl substituent or equivalent
moiety greatly enhances the binding affinity (82, 108). Thus, in terms of binding,
there is a crucial role for the nitro or cyano group, an important contribution from
the pyridine nitrogen, and a complementary role for N1 of the neonicotinoids (23,
53). Other molecular features also affect the selectivity. The N-methyl group of
thiamethoxam favors a specific receptor interaction particularly at low temperature
with Myzus compared with Drosophila or other insects (114, 114a); the preference
of insect nAChRs for chloropyridinyl versus chlorothiazolyl moieties depends on
the rest of the molecule (Figure 1) (51); introducing azido or amino at the 5-position
of the 6-chloropyridin-3-yl moiety of neonicotinoids and epibatidine reduces the
potency for Drosophila but not for 42 and 7 nAChRs (115, 116).
BINDING SITE AND SUBSITE SPECIFICITY

Agonist ligands acting at vertebrate neurotransmitter-gated ion channels are characteristically cationic in nature. The iminium cation of the N-unsubstituted imine
analogs of neonicotinoids (e.g., desnitro-IMI, Figures 3 and 4), or ammonium
nitrogen of nicotine, epibatidine, or ACh, binds to a -electron-rich subsite composed of aromatic residues, including the critical tryptophan in loop B of the
subunit. The cation makes van der Waals contact with the -electrons ( ) of
the aromatic residues (62, 6466, 117119). A supplementary role is proposed
for aspartate 152 and/or 200 as an anionic residue from loop B and/or loop C of
the 1 subunit (120, 121). These structural features are also conserved in a snail
ACh-binding protein (122, 123). The crystal structure of the snail ACh-binding
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protein as a complex with nicotine reveals the following molecular features: the
carbonyl oxygen of a tryptophan in loop B contacts through a hydrogen bond with
the ammonium nitrogen of nicotine; the carbonyl oxygen of a leucine and amide
nitrogen of a methionine (both from complementary loop E) make hydrogen bonds
with the pyridine nitrogen of nicotine through a bridging water molecule (123a).
The neonicotinoids are an anomaly for the nicotinoid cation- interaction model
(53). The electronegative pharmacophore, crucial for optimum potency of the
neonicotinoids, is proposed to associate with a cationic subsite (possibly lysine,
arginine, or histidine) in the insect nAChR (Figure 4) (23, 53, 108). Lysine and arginine are prominent (and histidine minor) in the extracellular domain of D2, the
main neonicotinoid-binding subunit (94, 98100). Although no direct information
is available on the actual location of the relevant residue(s), photoaffinity labeling
with a suitable neonicotinoid ligand (115) coupled with computer-assisted docking
simulation (118) may help define the orientation of the neonicotinoid electronegative tip in the binding domain. A point mutation (glutamine to arginine or lysine)
on the avian 7 subunit confers enhanced electrophysiological response for IMI
at 3 mM compared to that in the wild type (although the affinity of IMI on this
Figure 4 Binding subsite specificity shown as hypothetical schematic models for neonicotinoid imidacloprid binding in the insect nAChR and nicotinoid desnitroimidacloprid binding
in the mammalian nAChR, each at the ACh agonist site. The positioning of desnitroimidacloprid and the interacting amino acids in the mammalian site is based on earlier modeling with
ACh and nicotinoids (23, 53, 62, 65, 117, 118). In the mammalian binding site, loops A-C are
from an subunit and loop D from a complementary subunit. The insect (Drosophila) nAChR
subunits conserve the aromatic and vicinal cysteine residues suitably positioned from homology modeling. Imidacloprid is arbitrarily placed in the same way as desnitroimidacloprid and
a lysine (or alternatively arginine or histidine) cationic residue is introduced to interact with
the negatively charged ( ) tip important in selectivity for insect versus mammalian nicotinic
receptors.
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mutated receptor remains unchanged) (124), providing possible support for a binding model featuring the role of the neonicotinoid electronegative pharmacophore
(23, 53). These relationships provide a testable model for the hypothesis that
specific subsite differences between insect and vertebrate receptors confer neonicotinoid selectivity.

MECHANISMS OF SELECTIVE ACTION

The neonicotinoids are the newest major class of insecticides. They are structurally distinct from all other synthetic and botanical pesticides and exhibit favorable selectivity (7, 39, 125). As plant systemics they are increasingly replacing
organophosphates and methylcarbamates to control piercing-sucking insect pests,
and are also highly effective flea control agents for cats and dogs. They generally
have low acute toxicity to mammals, birds, and fish, but display some chronic
toxicities in mammals. Biotransformations involve initial oxidation or reduction
as both activation and detoxification mechanisms. The neonicotinoids are nicotinic agonists that interact with the nAChR in a very different way than nicotine,
which confers selectivity to insects versus mammals. The neonicotinoids are not
protonated but instead have an electronegative tip consisting of a nitro or cyano
pharmacophore that imparts potency and selectivity, presumably by binding to a
unique cationic subsite of the insect receptor. This is in marked contrast to the
action of protonated nicotinoids, which require a cation- interaction for binding to the vertebrate receptor. These differences provide the neonicotinoids with
favorable toxicological profiles.
ACKNOWLEDGMENTS
Neonicotinoid research in the Environmental Chemistry and Toxicology Laboratory at Berkeley is supported by grant R01 ES08424 from the National Institute of
Environmental Health Sciences (NIEHS), the National Institutes of Health (NIH).
The contents of this review are solely the responsibility of the authors and do not
necessarily represent the official views of NIEHS, NIH. We are greatly indebted
to our former or current laboratory colleagues Nanjing Zhang, Ryan Dick, David
Kanne, and Gary Quistad for valuable advice and assistance.
LITERATURE CITED
1. Ware GW. 2000. The Pesticide Book.
Fresno, CA: Thomson Publ. 418 pp.
2. Casida JE, Quistad GB. 1998. Golden
age of insecticide research: past, present,
or future? Annu. Rev. Entomol. 43:1

16
3. Kagabu S. 1997. Chloronicotinyl insecticidesdiscovery, application and
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4.

5.
6.
7.
8.
9.
10.
11.
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future perspective. Rev. Toxicol. 1:75

129
Yamamoto I, Casida JE, eds. 1999.
Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor. Tokyo:
Springer-Verlag. 300 pp.
Nauen R, Ebbinghaus-Kintscher U, Elbert A, Jeschke P, Tietjen K. 2001.
Acetylcholine receptors as sites for developing neonicotinoid insecticides. In
Biochemical Sites of Insecticide Action
and Resistance, ed. I Ishaaya, pp. 77
105. Berlin, Heidelberg: Springer
Kagabu S. 2003. Molecular design of
neonicotinoids: past, present and future. In Chemistry of Crop Protection,
Progress and Prospects in Science and
Regulation, ed. G Voss, G Ramos, pp.
193212. Weinheim, Ger.: Wiley-VCH
Tomizawa M, Casida JE. 2003. Selective
toxicity of neonicotinoids attributable
to specificity of insect and mammalian
nicotinic receptors. Annu. Rev. Entomol.
48:33964
Yamamoto I. 1965. Nicotinoids as insecticides. In Advances in Pest Control Research, ed. RL Metcalf, vol. 6, pp. 231
60. New York: Wiley
Soloway SB, Henry AC, Kollmeyer WD,
Padgett WM, Powell JE, et al. 1978.
Nitromethylene heterocycles as insecticides. In Pesticide and Venom Neurotoxicity, ed. DL Shankland, RM Hollingworth, T Smyth Jr, pp. 15358. New
York: Plenum
Soloway SB, Henry AC, Kollmeyer WD,
Padgett WM, Powell JE, et al. 1979. Nitromethylene insecticides. In Advances
in Pesticide Science, ed. H Geissbuehler,
vol. 2, pp. 20617. Oxford: Pergamon
Kollmeyer WD, Flattum RF, Foster JP,
Powell JE, Schroeder ME, Soloway SB.
1999. Discovery of the nitromethylene
heterocycle insecticides. In Nicotinoid
Insecticides and the Nicotinic Acetylcholine Receptor, ed. I Yamamoto, JE
Casida, pp. 7189. Tokyo: SpringerVerlag.
12. Kleier D, Holden I, Casida JE, Ruzo

LO. 1985. Novel photoreactions of an insecticidal nitromethylene heterocycle. J.
Agric. Food Chem. 33:9981000
13. Kagabu S, Medej S. 1995. Stability comparison of imidacloprid and related compounds under simulated sunlight, hydrolysis conditions, and to oxygen. Biosci.
Biotechnol. Biochem. 59:98085
14. Kagabu S, Akagi T. 1997. Quantum
chemical consideration of photostability
of imidacloprid and related compounds.
J. Pestic. Sci. 22:8489
15. Tomlin CDS, ed. 2003. The Pesticide
Manual. Alton, Hampshire, UK: Br.
Crop Protection Council. 1344 pp. 13th
ed.
16. Yamamoto I, Tomizawa M, Saito T,
Miyamoto T, Walcott EC, Sumikawa
K. 1998. Structural factors contributing
to insecticidal and selective actions of
neonicotinoids. Arch. Insect Biochem.
Physiol. 37:2432
17. Jeschke P, Moriya K, Lantzsch R, Seifert
H, Lindner W, et al. 2001. Thiacloprid
(Bay YRC 2894)a new member of the
chloronicotinyl insecticide (CNI) family.
Pflanzenschutz-Nachr. Bayer 54:14760
18. Schenker R, Tinembart O, HumbertDroz E, Cavaliero T, Yerly B. 2003.
Comparative speed of kill between
nitenpyram, fipronil, imidacloprid, selamectin and cythioate against adult
Ctenocephalides felis (Bouche) on cats
and dogs. Vet. Parasitol. 112:24954
19. Spande TF, Garraffo HM, Edwards
MW, Yeh HJC, Pannell L, Daly JW.
1992. Epibatidine: a novel (chloropyridyl)azabicycloheptane with potent
analgesic activity from an Ecuadoran
poison frog. J. Am. Chem. Soc. 114:
347578
20. Badio B, Daly JW. 1994. Epibatidine, a
potent analgesic and nicotinic agonist.
Mol. Pharmacol. 45:56369
21. Decker MW, Meyer MD. 1999. Therapeutic potential of neuronal nicotinic acetylcholine receptor agonists as
7 Jan 2005
13:58
AR
AR232-PA45-10.tex
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
22.

23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
novel analgesics. Biochem. Pharmacol.

58:91723
Lloyd GK, Williams M. 2000. Neuronal nicotinic acetylcholine receptors as
novel drug targets. J. Pharmacol. Exp.
Ther. 292:46167
Tomizawa M, Lee DL, Casida JE. 2000.
Neonicotinoid insecticides: molecular
features conferring selectivity for insect
versus mammalian nicotinic receptors. J.
Agric. Food Chem. 48:601624
Elliott M. 1977. Synthetic pyrethroids.
In Synthetic Pyrethroids. ACS Symposium Series 42, ed. M Elliott, pp. 128.
Washington, DC: Am. Chem. Soc.
Environmental Protection Agency. 2003.
Acetamiprid; pesticide tolerance. Fed.
Regist. 68:5234353
Clothianidin; pesticide tolerance. Fed.
Regist. 68:32390400
Dinotefuran; notice of filing a pesticide
petition to establish a tolerance for a certain pesticide chemical in or on food.
Fed. Reg. 68:3954754
Imidacloprid; pesticide tolerances. Fed.
Reg. 68:3530315
Thiacloprid; pesticide tolerances. Fed.
Reg. 68:5550313
Thiamethoxam; pesticide tolerance. Fed.
Reg. 67:6656171
Tomizawa M, Cowan A, Casida JE.
2001. Analgesic and toxic effects of
neonicotinoid insecticides in mice. Toxicol. Appl. Pharmacol. 177:7783
Tomizawa M, Casida JE. 2000. Imidacloprid, thiacloprid, and their imine
derivatives up-regulate the 42 nicotinic acetylcholine receptor in M10 cells.
Toxicol. Appl. Pharmacol. 169:11420
Tomizawa M, Casida JE. 2002. Desnitroimidacloprid activates the extracellular
signal-regulated kinase cascade via the
nicotinic receptor and intracellular cal-
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
263
cium mobilization in N1E-115 cells. Toxicol. Appl. Pharmacol. 184:18086

Cordero-Erausquin M, Marubio LM,
Klink R, Changeux J-P. 2000. Nicotinic
receptor function: new perspectives from
knockout mice. Trends Pharmacol. Sci.
21:21117
Hamann SR, Martin WR. 1992. Opioid
and nicotinic analgesic and hyperalgesic
loci in the rat brain stem. J. Pharmacol.
Exp. Ther. 261:70715
Khan IM, Yaksh TL, Taylor P. 1997. Epibatidine binding sites and activity in the
spinal cord. Brain Res. 753:26982
Khan IM, Stanislaus S, Zhang L, Taylor
P, Yaksh TL. 2001. A-85380 and epibatidine each interact with disparate spinal
nicotinic receptor subtypes to activate
analgesia and nociception. J. Pharmacol.
Exp. Ther. 297:23039
Tomizawa M, Casida JE. 1999. Minor
structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors. Br. J. Pharmacol.
127:11522
Sheets LP. 2002. The neonicotinoid insecticides. In Handbook of Neurotoxicology, ed. EJ Massaro, vol. 1, pp. 7987.
Totowa, NJ: Humana
Roberts TR, Hutson DH, eds. 1999.
Metabolic Pathways of Agrochemicals.
Part. 2: Insecticides and Fungicides.
Cambridge, UK: R. Soc. Chem. 1475 pp.
Thyssen J, Machemer L. 1999. Imidacloprid: toxicology and metabolism. In
Nicotinoid Insecticides and the Nicotinic
Acetylcholine Receptor, ed. I Yamamoto,
JE Casida, pp. 21322. Tokyo: SpringerVerlag
Klein O. 2001. Behaviour of thiacloprid (YRC 2894) in plants and animals.
Klein O. 2003. Behaviour of clothianidin (TI-435) in plants and animals.
Yokota T, Mikata K, Nagasaki H, Ohta
K. 2003. Absorption, tissue distribution,
7 Jan 2005
13:58
264

45.
46.
47.
48.
49.
50.
51.
52.
AR
AR232-PA45-10.tex
TOMIZAWA
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
CASIDA
excretion, and metabolism of clothianidin in rats. J. Agric. Food Chem. 51:

706672
Reed WT, Erlam GJ. 1978. The house
fly metabolism of nitromethylene insecticides. In Pesticide and Venom Neurotoxicity, ed. DL Shankland, RM Hollingworth, T Smyth Jr, pp. 15969. New
York: Plenum
Nauen R, Tietjen K, Wagner K, Elbert
A. 1998. Efficacy of plant metabolites of
imidacloprid against Myzus persicae and
Aphis gossypii (Homoptera: Aphididae).
Pestic. Sci. 52:5357
Nauen R, Reckmann U, Armborst S,
Stupp H-P, Elbert A. 1999. Whiteflyactive metabolites of imidacloprid: biological efficacy and translocation in cotton plant. Pestic. Sci. 55:26571
Nauen R, Ebbinghaus-Kintscher U,
Schmuck R. 2001. Toxicity and nicotinic
acetylcholine receptor interaction of imidacloprid and its metabolites in Apis
mellifera (Hymenoptera: Apidae). Pest
Manag. Sci. 57:55786
Tomizawa M, Latli B, Casida JE. 1996.
Novel neonicotinoid-agarose affinity
column for Drosophila and Musca nicotinic acetylcholine receptors. J. Neurochem. 67:166976
Wiesner P, Kayser H. 2000. Characterization of nicotinic acetylcholine receptors from the insects Aphis craccivora,
Myzus persicae, and Locusta migratoria
by radioligand binding assays: relation to
thiamethoxam action. J. Biochem. Mol.
Toxicol. 14:22130
Zhang A, Kayser H, Maienfisch P, Casida
JE. 2000. Insect nicotinic acetylcholine
receptor: conserved neonicotinoid specificity of [3H]imidacloprid binding site. J.
Neurochem. 75:1294303
Nauen R, Ebbinghaus-Kintscher U, Salgado VL, Kaussmann M. 2003. Thiamethoxam is a neonicotinoid precursor
converted to clothianidin in insects and
plants. Pestic. Biochem. Physiol. 76:55
69
53. Tomizawa M, Zhang N, Durkin KA,

Olmstead MM, Casida JE. 2003. The
neonicotinoid electronegative pharmacophore plays the crucial role in the high
affinity and selectivity for the Drosophila
nicotinic receptor: an anomaly for the
nicotinoid cation- interaction model.
54. Chao SL, Casida JE. 1997. Interaction
of imidacloprid metabolites and analogs
with the nicotinic acetylcholine receptor
of mouse brain in relation to toxicity. Pestic. Biochem. Physiol. 58:7788
55. Schulz-Jander DA, Casida JE. 2002. Imidacloprid insecticide metabolism: human cytochrome P450 isozymes differ
in selectivity for imidazolidine oxidation versus nitroimine reduction. Toxicol.
Lett. 132:6570
56. Liu M-Y, Lanford J, Casida JE. 1993.
Relevance of [3H]imidacloprid binding
site in house fly head acetylcholine
receptor to insecticidal activity of 2nitromethylene- and 2-nitroimino-imidazolidines. Pestic. Biochem. Physiol.
46:2006
57. Daborn P, Boundy S, Yen J, Pittendrigh B, ffrench-Constant R. 2001.
DDT resistance in Drosophila correlates with Cyp6g1 over-expression and
confers cross-resistance to the neonicotinoid imidacloprid. Mol. Genet. Genomics 266:55663
58. Le Goff G, Boundy S, Daborn PJ, Yen
JL, Sofer L, et al. 2003. Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila. Insect
59. Schulz-Jander DA, Leimkuehler WM,
Casida JE. 2002. Neonicotinoid insecticides: reduction and cleavage of
imidacloprid nitroimine substituent by
liver microsomal and cytosolic enzymes. Chem. Res. Toxicol. 15:1158
65
60. Dick RA, Kanne DB, Casida JE. 2004.
Aldehyde oxidase catalyzes the reduction of the nitroimine moiety of the
7 Jan 2005
13:58
AR
AR232-PA45-10.tex
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
60a.

61.
62.
63.
64.
65.
66.
67.
68.
69.
neonicotinoid insecticide imidacloprid.

Drug Metab. Rev. 36 (Suppl. 1):270
Klein O. 1994. The metabolism of imidacloprid in animals. IUPAC Int. Congr.
Pestic. Chem., 8th, Washington, DC
Abstr. 367
Brunet J-L, Maresca M, Fantini J,
Belzunces LP. 2004. Human intestinal
absorption of imidacloprid with Caco-2
cells as enterocyte model. Toxicol. Appl.
Pharmacol. 194:19
Corringer J-P, Le Novère N, Changeux
J-P. 2000. Nicotinic receptors at the
amino acid level. Annu. Rev. Pharmacol.
Toxicol. 40:43158
Elgoyhen AB, Vetter DE, Katz E, Rothlin
CV, Heinemann SF, Boulter J. 2001. 10:
a determinant of nicotinic cholinergic receptor function in mammalian vestibular
and cochlear mechanosensory hair cells.
6
Dougherty DA. 1996. Cation- interactions in chemistry and biology: a new
view of benzene, Phe, Tyr, and Trp. Science 271:16368
Zhong W, Gallivan JP, Zhang Y, Li L,
Lester HA, Dougherty DA. 1998. From
ab initio quantum mechanics to molecular neurobiology: a cation- binding
site in the nicotinic receptor. Proc. Natl.
Dougherty DA, Lester HA. 2001. Snails,
synapses and smokers. Nature 411:252
55
Zacharias N, Dougherty DA. 2002.
Cation- interactions in ligand recognition and catalysis. Trends Pharmacol.
Sci. 23:28187
Holladay MW, Dart MJ, Lynch JK. 1997.
Neuronal nicotinic acetylcholine receptors as targets for drug discovery. J. Med.
Chem. 40:417094
Romanelli MN, Gualtieri F. 2003.
Cholinergic nicotinic receptors: competitive ligands, allosteric modulators, and
their potential applications. Med. Res.
Rev. 23:393426
265
70. Bunnelle WH, Dart MJ, Schrimpf MR.

2004. Design of ligands for the nicotinic
acetylcholine receptors: the quest for selectivity. Curr. Top. Med. Chem. 4:299
334
71. Methfessel C. 1992. Effect of imidacloprid on the acetylcholine receptor of
rat muscle. Pflanzenschutz-Nachr. Bayer
45:36980
72. Zwart R, Oortgiesen M, Vijverberg
HPM. 1994. Nitromethylene heterocycles: selective agonists of nicotinic receptors in locust neurons compared to
mouse N1E-115 and BC3H1 cells. Pestic. Biochem. Physiol. 48:20213
73. Tomizawa M, Otsuka H, Miyamoto T,
Yamamoto I. 1995. Pharmacological effects of imidacloprid and its related compounds on the nicotinic acetylcholine
receptor with its ion channel from the
Torpedo electric organ. J. Pestic. Sci. 20:
4956
74. Yamamoto I, Yabuta G, Tomizawa M,
Saito T, Miyamoto T, Kagabu S. 1995.
Molecular mechanism for selective toxicity of nicotinoids and neonicotinoids. J.
75. Nagata K, Aistrup GL, Song JH, Narahashi T. 1996. Subconductance-state currents generated by imidacloprid at the
nicotinic acetylcholine receptor of PC 12
cells. NeuroReport 7:102528
76. DAmour KA, Casida JE. 1999. Desnitroimidacloprid and nicotine binding
site in rat recombinant 42 neuronal
nicotinic acetylcholine receptor. Pestic.
Biochem. Physiol. 64:5561
77. Ihara M, Matsuda K, Otake M, Kuwamura M, Shimomura M, et al. 2003.
Diverse actions of neonicotinoids on
chicken 7, 42 and Drosophilachicken SAD2 and ALS2 hybrid
nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. Neuropharmacology 45:13344
78. Schroeder ME, Flattum RF. 1984. The
mode of action and neurotoxic properties of the nitromethylene heterocyclic
7 Jan 2005
13:58
266

79.
80.
81.
82.
83.
84.
85.
86.
87.
AR
AR232-PA45-10.tex
TOMIZAWA
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
CASIDA
insecticides. Pestic. Biochem. Physiol.

22:14860
Sattelle DB, Buckingham SD, Wafford
KA, Sherby SM, Bakry NM, et al.
1989. Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on
insect and vertebrate nicotinic acetylcholine receptors. Proc. R. Soc. London
Ser. B 273:50114
Abbink K. 1991. The biochemistry
of imidacloprid. Pflanzenschutz-Nachr.
Bayer 44:183195
Bai D, Lummis SCR, Leicht W, Breer
H, Sattelle DB. 1991. Actions of imidacloprid and a related nitromethylene on
cholinergic receptors of an identified insect motor neurone. Pestic. Sci. 33:197
204
Tomizawa M, Yamamoto I. 1992. Binding of nicotinoids and the related compounds to the insect nicotinic acetylcholine receptor. J. Pestic. Sci. 17:231
36
Tomizawa M, Yamamoto I. 1993.
Structure-activity relationships of nicotinoids and imidacloprid analogs. J. Pestic.
Sci. 18:9198
Liu M-Y, Casida JE. 1993. High affinity
binding of [3H]imidacloprid in the insect
acetylcholine receptor. Pestic. Biochem.
Physiol. 46:4046
Tomizawa M, Latli B, Casida JE. 1999.
Structure and function of insect nicotinic acetylcholine receptors studied with
nicotinoid insecticide affinity probes. In
Nicotinoid Insecticides and the Nicotinic
Acetylcholine Receptor, ed. I Yamamoto,
JE Casida, pp. 27192. Tokyo: SpringerVerlag
Nishimura K, Kanda Y, Okazawa A,
Ueno T. 1994. Relationship between insecticidal and neurophysiological activities of imidacloprid and related compounds. Pestic. Biochem. Physiol. 50:
5159
Gundelfinger ED, Schulz R. 2000. Insect nicotinic acetylcholine receptors:
genes, structure, physiological and phar-
88.
89.
90.
91.
92.
93.
94.
95.
macological properties. In Handbook of

Experimental Pharmacology. Vol. 144:
Neuronal Nicotinic Receptors, ed. F
Clementi, D Fornasari, C Gotti, pp. 497
521. Berlin: Springer
Littleton JT, Ganetzky B. 2000. Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron
26:3543
Tomizawa M, Casida JE. 2001. Structure and diversity of insect nicotinic
acetylcholine receptors. Pest Manag. Sci.
57:91422
Bertrand D, Ballivet M, Gomez M,
Bertrand S, Phannavong B, Gundelfinger ED. 1994. Physiological properties
of neuronal nicotinic receptors reconstituted from the vertebrate 2 subunit and
Drosophila subunits. Eur. J. Neurosci.
6:86975
Lansdell SJ, Schmitt B, Betz H, Sattelle DB, Millar NS. 1997. Temperaturesensitive expression of Drosophila neuronal nicotinic acetylcholine receptors.
J. Neurochem. 68:181219
Schulz R, Sawruk E, Mulhardt C,
Bertrand S, Baumann A, et al. 1998.
D3, a new functional subunit of
nicotinic acetylcholine receptors from
Drosophila. J. Neurochem. 71:85362
Lansdell SJ, Millar NS. 2000. Cloning
and heterologous expression of D4,
a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification
of an alternative exon influencing the
efficiency of subunit assembly. Neuropharmacology 39:260414
Schulz R, Bertrand S, Chamaon K,
Smalla K-H, Gundelfinger ED, Bertrand
D. 2000. Neuronal nicotinic acetylcholine receptors from Drosophila: two
different types of subunits coassemble within the same receptor complex. J.
Neurochem. 74:253746
Lansdell SJ, Millar NS. 2002. D3, an
atypical nicotinic acetylcholine receptor subunit from Drosophila: molecular cloning, heterologous expression and
7 Jan 2005
13:58
AR
AR232-PA45-10.tex
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
96.

97.
98.
99.
100.
101.
102.
103.
104.
coassembly. J. Neurochem. 80:1009

18
Orr N, Shaffner AJ, Watson GB. 1997.
Pharmacological characterization of an
epibatidine binding site in the nerve
cord of Periplaneta americana. Pestic.
Biochem. Physiol. 58:18392
Chamaon K, Schulz R, Smalla K-H,
Seidel B, Gundelfinger ED. 2000. Neuronal nicotinic acetylcholine receptors of
Drosophila melanogaster: the -subunit
D3 and the -type subunit ARD coassemble within the same receptor complex. FEBS Lett. 482:18992
Chamaon K, Smalla K-H, Thomas U,
Gundelfinger ED. 2002. Nicotinic acetylcholine receptors of Drosophila: three
subunits encoded by genomically linked
genes can co-assemble into the same receptor complex. J. Neurochem. 80:149
57
Tomizawa M, Casida JE. 1997. [125I]Azidonicotinoid photoaffinity labeling of
insecticide-binding subunit of Drosophila nicotinic acetylcholine receptor.
Neurosci. Lett. 237:6164
Tomizawa M, Wen Z, Chin H-L, Morimoto H, Kayser H, Casida JE. 2001.
Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel
[3H]azidoneonicotinoid. J. Neurochem.
78:135966
Lansdell SJ, Millar NS. 2000. The influence of nicotinic receptor subunit composition upon agonist, -bungarotoxin
and insecticide (imidacloprid) binding
affinity. Neuropharmacology 39:67179
Beers WH, Reich E. 1970. Structure and
activity of acetylcholine. Nature 228:
91722
Sheridan RP, Nilakantan R, Dixon JS,
Venkataraghavan R. 1986. The ensemble
approach to distance geometry: application to the nicotinic pharmacophore. J.
Med. Chem. 29:899906
Glennon RA, Herndon JL, Dukat M.
1994. Epibatidine-aided studies toward
definition of a nicotinic receptor phar-
105.
106.
107.
108.
109.
110.
111.
112.
267
macophore. Med. Chem. Res. 4:461

73
Abreo MA, Lin N-H, Garvey DS, Gunn
DE, Hettinger A-M, et al. 1996. Novel 3pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors. J. Med. Chem. 39:
81725
Meyer MD, Decker MW, Rueter LE, Anderson DJ, Dart MJ, et al. 2000. The
identification of novel structural compound classes exhibiting high affinity for
neuronal nicotinic acetylcholine receptors and analgesic efficacy in preclinical models of pain. Eur. J. Pharmacol.
393:17177
Kagabu S, Matsuno H. 1997. Chloronicotinyl insecticides. 8. Crystal and
molecular structures of imidacloprid and
analogous compounds. J. Agric. Food
Chem. 45:27681
Zhang N, Tomizawa M, Casida JE. 2004.
-Nitro ketone as an electrophile and nucleophile: synthesis of 3-substituted 2nitromethylenetetrahydrothiophene and
-tetrahydrofuran as Drosophila nicotinic
receptor probes. J. Org. Chem. 69:876
81
Chamberlain K, Evans AA, Bromilow
RH. 1996. 1-Octanol/water partition coefficient (Kow) and pKa for ionizable pesticides measured by a pH-metric method.
Okazawa A, Akamatsu M, Ohoka A,
Nishiwaki H, Cho W-J, et al. 1998. Prediction of the binding mode of imidacloprid and related compounds to house-fly
head acetylcholine receptors using threedimensional QSAR analysis. Pestic. Sci.
54:13444
Matsuda K, Buckingham SD, Kleier D,
Rauh JJ, Grauso M, Sattelle DB. 2001.
Neonicotinoids: insecticides acting on
insect nicotinic acetylcholine receptors.
Nakayama A, Sukekawa M. 1998. Quantitative correlation between molecular
similarity and receptor-binding activity
7 Jan 2005
13:58
268
113.

114.
114a.
115.
116.
117.
118.
119.
AR
AR232-PA45-10.tex
TOMIZAWA
AR232-PA45-10.sgm
LaTeX2e(2002/01/18)
P1: GCE
CASIDA
of neonicotinoid insecticides. Pestic. Sci.

52:10410
Boelle J, Schneider R, Gerardin P, Loubinoux B, Maienfisch P, Rindlisbacher
A. 1998. Synthesis and insecticidal
evaluation of imidacloprid analogs. Pestic. Sci. 54:3047
Kayser H, Lee C, Decock A, Baur
M, Haettenschwiler J, Maienfisch P.
2004. Comparative analysis of neonicotinoid binding to insect membranes: I.
A structure-activity study of the mode of
[3H]imidacloprid displacement in Myzus
persicae and Aphis craccivora. Pest
Manag. Sci. 60:94558
Wellmann H, Gomes M, Lee C, Kayser
H. 2004. Comparative analysis of neonicotinoid binding to insect membranes:
II. An unusual high affinity site for
[3H]thiamethoxam in Myzus persicae
and Aphis craccivora. Pest Manag. Sci.
60:95970
Zhang N, Tomizawa M, Casida JE.
2002. Structural features of azidopyridinyl neonicotinoid probes conferring
high affinity and selectivity for mammalian 42 and Drosophila nicotinic
receptors. J. Med. Chem. 45:283240
Zhang N, Tomizawa M, Casida JE.
2003. 5-Azidoepibatidine: an exceptionally potent photoaffinity ligand for neuronal 42 and 7 nicotinic acetylcholine receptors. Bioorg. Med. Chem.
Lett. 13:52527
Foucaud B, Perret P, Grutter T, Goeldner
M. 2001. Cysteine mutants as chemical
sensors for ligand-receptor interactions.
Le Novère N, Grutter T, Changeux J-P.
2002. Models of the extracellular domain
of the nicotinic receptors and of agonistand Ca2+-binding sites. Proc. Natl. Acad.
Sci. USA 99:321015
Schmitt JD, Sharples CGV, Caldwell
120.
121.
122.
123.
123a.
124.
125.
WS. 1999. Molecular recognition in

nicotinic acetylcholine receptors: the importance of -cation interactions. J.
Med. Chem. 42:306674
OLeary ME, White MM. 1992. Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor. J. Biol. Chem. 267:836065
Sugiyama N, Boyd AE, Taylor P. 1996.
Anionic residue in the -subunit of
the nicotinic acetylcholine receptor contributing to subunit assembly and ligand
binding. J. Biol. Chem. 271:2657581
Brejc K, van Dijk WJ, Klaassen RV,
Schuurmans M, van der Oost J, et al.
2001. Crystal structure of an AChbinding protein reveals the ligandbinding domain of nicotinic receptors.
Nature 411:26976
Sixma TK, Smit AB. 2003. Acetylcholine binding protein (AChBP): a
secreted glial protein that provides a
high-resolution model for extracellular
domain of pentameric ligand-gated ion
channels. Annu. Rev. Biophys. Biomol.
Struct. 32:31134
Celie PHN, van Rossum-Fikkert SE, van
Dijk WJ, Brejc K, Smit AB, Sixma
TK. 2004. Nicotine and carbamylcholine
binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41:90714
Shimomura M, Okuda H, Matsuda K,
Komai K, Akamatsu M, Sattelle DB.
2002. Effects of mutations of a glutamine residue in loop D of the 7
nicotinic acetylcholine receptor on agonist profiles for neonicotinoid insecticides and related ligands. Br. J. Pharmacol. 137:16269
Sheets LP. 2001. Imidacloprid: a neonicotinoid insecticide. In Handbook of Pesticide Toxicology, ed. R Krieger, Vol. 2,
pp. 112330. San Diego: Academic
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Figure 3 Molecular features of nicotinic agonists shown as electrostatic potential (ESP)

mapping on the molecular surfaces of insect-selective imidacloprid and mammalianselective desnitroimidacloprid (protonated at physiological pH) obtained in the gas phase
by high-level ab initio calculation (53). ESP surfaces are shown as red for negative graded through orange, yellow, and green to blue for positive with an overall energy range of
60 to 160 kcal/mol. The strong electronegative tip illustrated for the nitro moiety of
imidacloprid is also evident for the cyano substituent of thiacloprid (17).
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GLYCERALDEHYDE-3-PHOSPHATE
DEHYDROGENASE, APOPTOSIS, AND
NEURODEGENERATIVE DISEASES
De-Maw Chuang,1 Christopher Hough,2
and Vladimir V. Senatorov3
1
Molecular Neurobiology Section, Mood and Anxiety Disorders Program,

National Institute of Mental Health, National Institutes of Health, Bethesda,
Maryland, 20892-1363; email: chuang@mail.nih.gov
2
Department of Psychiatry, Uniformed Services University of Health Sciences,
Bethesda, Maryland, 20814-4799; email: chough@usuhs.mil
3
Laboratory of Molecular and Developmental Biology, National Eye Institute, National
Institutes of Health, Bethesda, Maryland, 20892-0704; email: Senatorovv@nei.nih.gov
Key Words GAPDH, overexpression, nuclear accumulation, intracellular sensor,

neurodegeneration
Abstract Increasing evidence supports the notion that glyceraldehyde-3phosphate dehydrogenase (GAPDH) is a protein with multiple functions, including its
surprising role in apoptosis. GAPDH is overexpressed and accumulates in the nucleus
during apoptosis induced by a variety of insults in diverse cell types. Knockdown of
GAPDH using an antisense strategy demonstrates its involvement in the apoptotic cascade in which GAPDH nuclear translocation appears essential. Knowledge concerning
the mechanisms underlying GAPDH nuclear translocation and subsequent cell death
is growing. Additional evidence suggests that GAPDH may be an intracellular sensor
of oxidative stress during early apoptosis. Abnormal expression, nuclear accumulation, changes in physical properties, and loss of glycolytic activity of GAPDH have
been found in cellular and transgenic models as well as postmortem tissues of several
neurodegenerative diseases. The interaction of GAPDH with disease-related proteins
as well as drugs used to treat these diseases suggests that it is a potential molecular
target for drug development.
INTRODUCTION
Recent research has revealed a small class of proteins whose members are endowed with multiple functions (for review, see 1). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) is a typical example. Historically, GAPDH has been
The U.S. Government has the right to retain a nonexclusive, royalty-free license in and to
any copyright covering this paper.
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considered a glycolytic enzyme with a key role in energy production. It has also
been regarded as a product of a housekeeping gene whose transcript level remains
constant under most experimental conditions, and it has been frequently used as an
internal control in studying the regulation of gene expression. Mounting evidence,
however, supports the view that the expression of GAPDH is regulated and that
GAPDH is a protein with multiple intracellular localizations and diverse activities independent of its traditional role in glycolysis (for review, see 2, 3). These
new activities include regulation of the cytoskeleton (4, 5), membrane fusion and
transport (68), glutamate accumulation into presynaptic vesicles (9), and binding
to low-molecular-weight G proteins (10). A role of GAPDH in nuclear function
is also suggested by its ability to activate transcription in neurons (11), to export
nuclear RNA (12), and to effect DNA repair (13). Particularly intriguing are the
increasing reports that GAPDH is an integral part of various forms of apoptosis and
may participate in neuronal death in some neurodegenerative diseases. The aim of
this review is to provide evidence for the involvement of GAPDH in the apoptotic
cascade, to discuss potential underlying mechanisms, and to evaluate the roles of
GAPDH in preclinical models of neurodegeneration and human disease and in
the pharmacological treatments of neurodegenerative diseases. Several reviews in
these areas have appeared (2, 1420).
EVIDENCE FOR A ROLE OF GAPDH IN APOPTOSIS

GAPDH Overexpression
Apoptosis, or programmed cell death, results from the actions of a genetically
encoded suicide program that normally occurs in response to physiological or
relatively mild stimuli (for review, see 2123). Apoptotic cells display chromatin
condensation, internucleosomal DNA cleavage, cytoplasmic shrinking, and plasma
membrane blebbing, and they are phagocytized by microglia. This contrasts with
necrosis, where cells swell and rupture, eliciting an inflammatory response. Mitochondria play a pivotal role in the genesis and propagation of apoptosis via
events such as mitochondrial calcium accumulation, generation of free radicals,
and, perhaps, activation of the permeability transition pore (for review, see 24).
To date, four mitochondrial molecules mediating downstream cell-death pathways
have been identified: cytochrome c, Smac/Diablo, apoptosis-inducing factor, and
endonuclease G. Cytochrome c binds to Apaf-1, which, together with procaspase9, forms apoptosomes that, in turn, cause activation of caspase-9, caspase-3, and
others. Smac/Diablo binds to inhibitors of activated caspases, resulting in further
caspase activation. Apoptosis-inducing factor and endonuclease G act via caspaseindependent pathways to trigger cell death (for review, see 22, 2527).
The involvement of GAPDH in apoptosis was first demonstrated in cultured
cerebellar granule cells and cortical neurons undergoing spontaneous apoptosis
(28, 29). A 38-kDa protein was found to be overexpressed prior to apoptosis and
was soon identified as GAPDH by N-terminal sequencing. Direct evidence for its
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role in cell death came from studies using an antisense oligonucleotide knockdown
strategy. Antisense oligonucleotides directed against either the translation initiation
site or a segment of the coding region of GAPDH mRNA delay cell death; in
contrast, the corresponding sense and scrambled oligonucleotides are ineffective
(28, 29). Additionally, antisense, but not sense, oligonucleotides block the increase
in GAPDH mRNA and protein preceding the apoptotic death with little or no effect
on basal levels. Using similar approaches, GAPDH was found to be associated with
apoptosis, but not necrosis, of cerebellar granule cells grown in culture medium
with reduced concentrations of KCl (30).
The paradigm of cytosine arabinoside (AraC)-induced cell death has been used
to further investigate the generality of the role of GAPDH in neuronal apoptosis and the molecular events associated with this process. AraC is a pyrimidine
antimetabolite that has been used clinically for the treatment of acute leukemia.
Freshly plated cerebellar granule cells exposed to AraC undergo rapid apoptotic
neuronal death that is preceded by an upregulation of the tumor suppressor protein
p53 followed by an increase in levels of GAPDH and Bax, another proapoptotic
protein (31). Again, the role of GAPDH in AraC-induced apoptosis was confirmed
by antisense experiments (31, 32). Interestingly, a p53 antisense oligonucleotide
not only suppresses apoptosis and decreases p53 and Bax mRNA induced by AraC,
but also reduces the levels of upregulated GAPDH mRNA and protein. In the same
study, it was shown that neurons prepared from p53-deficient mice are resistant
to AraC neurotoxicity, and that p53 gene knockout also suppresses AraC-induced
GAPDH expression. Moreover, infection of PC12 pheochromocytoma cells with
an adenoviral vector containing the wild-type p53 gene dramatically increases
GAPDH expression and triggers cell death (31). Taken together, these results lend
support for the view that GAPDH is a novel target of p53, directly or indirectly
regulated by this proapoptotic transcription factor, and could be one of the downstream apoptotic mediators. A scan of the rat GAPDH promoter reveals three
potential p53 consensus binding sequences at positions 2008 to 1989, 1184 to
1165, and 1087 to 1068 from the ATG sequence and start site of translation,
and suggests that p53 induces GAPDH expression directly.
GAPDH Nuclear Accumulation

Because upregulated GAPDH protein is present in the particulate (200 103 g
pellet) fraction (28, 29, 32), subcellular fractionation studies using cultured neurons
treated with AraC were performed to identify the sites of GAPDH accumulation
(33). Immunoreactive GAPDH protein levels are markedly increased in the crude
nuclear fraction and to a lesser extent in the crude mitochondrial fraction, but are
unchanged in the crude fraction containing the endoplasmic reticulum. A similar
conclusion concerning GAPDH nuclear translocation was reached in an independent study using confocal immunocytochemistry and subcellular fractionation
of nonneuronal and neuronal cells subjected to various stresses, including dexamethasone treatment, nerve growth factor withdrawal, and aging of the cultures
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(34). Treatment with GAPDH antisense oligonucleotides abolished insult-induced

GAPDH nuclear accumulation. Subsequent electron microscopic immunocytochemistry confirmed the accumulation of GAPDH in neurons undergoing apoptosis (35). Experiments using a GAPDH-green fluorescent protein (GFP) construct
also showed movement of the GAPDH-GFP fusion protein from the cytosol to
the nucleus shortly after exposure of cells to apoptosis-stimulating agents (36).
A further study using highly purified nuclei from cerebellar granule cells treated
with AraC showed that nuclear GAPDH accumulates over time with a concomitant
degradation of lamin B1, a nuclear membrane protein, and caspase substrate (37).
Nuclear accumulation is associated with a progressive decrease in the activity of
uracil-DNA glycosylase, one of the nuclear functions of GAPDH (13). The nuclear
glycolytic dehydrogenase activity is initially increased after AraC treatment and
then decreases parallel to the DNA glycosylase activity. The same study shows that
six isoforms of GAPDH with a similar molecular weight are present in the purified
nuclei of cerebellar granule cells, and that all these nuclear isoforms are increased
after AraC treatment, but only the more acidic isoforms are rapidly translocated.
These GAPDH isoforms could be the results of posttranslational modifications,
variants of alternative splicing, or products of distinct genes. Future studies using
selective gene silencing, knockdown, or knockout may shed light on the differential
roles of GAPDH isoforms in apoptosis.
There is a growing list of studies showing the involvement of GAPDH in cell
death in various paradigms. These include androgen depletion-induced apoptosis
of prostate epithelial cells (38); 1-methyl-4-phenylpyridinium (MPP+)-induced
death of mesencephalic dopaminergic neurons (39) and human neuroblastoma SKN-SH cells (40); apoptosis induced by an endogenous dopaminergic neurotoxin,
N-methyl-(R)-salsolinol, in human dopaminergic SH-SY5Y cells (41); induction
of apoptosis (by staurosporine or MG 132) and oxidative stress (by H2O2 or FeCN,
which is presumed to be ferricyanide) in neuroblastoma NB41A3 and nonneuronal
cells (R6 fibroblasts) (42); and etoposide-induced apoptosis in cerebellar granule
cells (43). Transfection of GAPDH into cells has been shown to induce cell death
(44, 45) or facilitate cell death induced by apoptotic insults or oxidative stress
(42). The latter study shows that nuclear localization of GAPDH is insufficient to
induce apoptosis in NB41A3 cells. This can be explained by the fact that GAPDH
also performs functions in the nucleus that are nonapoptotic (see below).
PERSPECTIVES CONCERNING GAPDH NUCLEAR

TRANSLOCATION AND SUBSEQUENT APOPTOSIS
The mechanisms underlying the transport of GAPDH from the cytosol to the
nucleus resulting in apoptotic death are poorly understood. In GT1-7 hypothalmic
neurosecretory cells, treatment with thapsigargin, a selective inhibitor of calciumATPases of the endoplasmic reticulum, or buthionine sulfoximine, a specific glutamyl-cysteine synthetase inhibitor, triggers GAPDH overexpression, nuclear
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translocation, and subsequent apoptosis; all three events are blocked by Bcl-2
overexpression (46). In human neuroblastoma cells, transfection-mediated Bcl-2
overexpression completely suppresses nuclear accumulation of GAPDH induced
by N-methyl-(R)-salsolinol (41). In R6 cells overexpressing Bcl-2, the nuclear
levels of endogenous GAPDH are markedly reduced compared with the wildtype control (42). These observations suggest that Bcl-2 may participate in the
regulation of GAPDH nuclear translocation and this effect may be part of the
protective mechanisms of Bcl-2 against apoptosis. However, Bcl-2 has no effect
on transfection-induced nuclear accumulation of GAPDH-GFP fusion protein in
R6 cells, indicating the complexity of the translocation process. The observation
that GAPDH first localizes to the Golgi before proceeding to the nucleus following
6-hydroxydopamine treatment in neuroblastoma cells may be an indication of how
GAPDH is transported to the nucleus (47).
It has been reported that GAPDH binds to a nuclear localization signal (NLS)containing protein, Siah, to initiate the GAPDH translocation to the nucleus (48).
GAPDH appears to stabilize Siah following their association, and thereby enhances
Siah-mediated proteolytic cleavage of its nuclear substrates, such as N-CoR, to
trigger apoptosis. Siah is also a target of p53 induction (49, 50) and can trigger
apoptosis either alone (49) or in conjunction with Pw1/Peg3 (51). In addition,
Siah acts as an E3 ubiquitin-ligase in the ubiquitination/proteasome degradation
pathway for Numb and DCC (52). Although GAPDH is known to interact with
microtubules and microfilaments (53) and binds to a variety of proteins linked to
neurodegenerative diseases (see below), there is no evidence yet supporting the
notion that these proteins mediate the transport of GAPDH to the nucleus. In NIH
3T3 fibroblast cells, serum depletion-induced GAPDH import to the nucleus is a
reversible process; readdition of serum or stimulation with growth factors causes
GAPDH export from the nucleus (54). The GAPDH nuclear export requires the
activity of phosphatidylinositol 3-kinase but is not mediated by exportin 1 (54, 55).
However, a novel exportin1-dependent nuclear export signal (NES) comprising 13
amino acids of the C-terminal domain of GAPDH has been identified (56). Truncation or mutation of this NES abrogates exportin1 binding and causes nuclear
accumulation of GAPDH-GFP fusion protein expressed in colorectal adenocarcinoma cells. This suggests that GAPDH would be a nuclear protein were it not for
its NES, and that the cytoplasmic localization of GAPDH is an active rather than
a passive process. Reversible GAPDH nuclear translocation following its overexpression has also been found in human monocytes infected with vaccinia virus
(57).
It appears that there is a change in the structure of the GAPDH protein following its nuclear translocation. For example, sodium nitropruside (an NO donor)
-induced NAD labeling of nuclear GAPDH is decreased by 60% in cerebellar
granule cells after AraC treatment (37), suggesting that the active site of GAPDH
may be covalently modified, denatured, or improperly folded. GAPDH is present
as a tetramer in the cytoplasm where the enzyme catalyzes its glycolytic activity. However, the uracil glycosylase activity of GAPDH is associated with the
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monomer form of GAPDH in the nucleus (58). It is still unresolved as to whether

a change in the oligomeric state is required for GAPDH nuclear translocation.
In cerebellar granule cell cultures treated with SYM-2081, a purported glutamate
uptake inhibitor and receptor agonist, GAPDH is rapidly accumulated in the nucleus, and chromatin immunoprecipitation reveals that GAPDH complexes with
acetylated histone H3, including Lys9 acetylated histone (59). Pretreatment with
valproate causes a reduction in levels of nuclear GAPDH with a concomitant increase in acetylated histone in the immuno-complex and neuroprotection against
SYM-2081-induced excitotoxicity. These results suggest that valproate suppresses
excitotoxicity-induced GAPDH nuclear accumulation by weakening the interactions between GAPDH and histone through its hyperacetylation. A recent study
shows that GAPDH is a key component of the coactivator complex OCA-S that
is essential for S phasedependent histone H2B transcription (60). GAPDH binds
directly to Oct-1, exhibits potent transactivation potential, and is essential for S
phasespecific H2B transcription in vivo and in vitro. The binding of GAPDH
to Oct-1 is stimulated by NAD+ but inhibited by NADH, raising the possibility
that redox regulation could be important for the nuclear function of GAPDH (see
below). Taken together, it is conceivable that the structural and functional changes
of GAPDH following nuclear translocation result in alteration in nuclear function
in a gain-of-toxicity manner, thereby contributing to its proapoptotic activities.
GAPDH is also found in a nuclear protein complex involved in DNA repair of
mismatched base pairs, where GAPDH, through its ability to bind DNA, acts as a
sensor of altered base pairing in mismatched base pairs (61).
IS GAPDH AN INTRACELLULAR SENSOR

OF OXIDATIVE STRESS?
It has been hypothesized that GAPDH serves as an intracellular sensor of oxidative
stress and may play an early and pivotal role in the cascade leading to cell death
(43). As described in the preceding section, the binding of Siah to GAPDH stabilizes Siah, and this event appears to be crucial for GAPDH nuclear translocation.
The group reported that the GAPDH-Siah interaction is influenced by the oxidative
modification on GAPDH at Cys149. The addition of antioxidants to the culture
media inhibits GAPDH-Siah binding and GAPDH-dependent apoptotic death of
neurons (43). Transfection with wild-type GAPDH increases Siah levels, whereas
the GAPDH C149S mutant does not. This is an attractive hypothesis for several
reasons. Active site Cys149 in GAPDH is made more reactive by the removal
of its sulfhydryl proton to form a highly reactive thiolate group (cys-S) that is
essential for its enzymatic activity and its subsceptibility to modification by electrophiles (oxidants). These include oxidation by either reduced glutathione or Snitrosylated glutathione to form mixed disulfides such as enzyme-S-S-glutathione
(62), S-nitrosylation to form enzyme-S-NO (62), transition metal bonding to form
enzyme-S-Metal (63), attack of bound NADH by nitrosonium ion (NO+) to form an
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enzyme-NADH adduct (64), and mono-ADP-ribosylation by an alternate route of

the latter mechanism (64, 65). This mono-ADP-ribosylation is not to be confused
with the enzymatic ADP-ribosylation of an arginine of GAPDH in the presence
of ARF-1 catalyzed by Brefeldin A or stimulated by ER stress (66). Additionally,
direct and indirect reactions of reactive oxygen species to form carbonyls are also
possible (67).
In vivo, active site modification of GAPDH is most likely to arise from nitric oxidederived products produced in response to oxidative stress (68). The
formation of mixed disulfides, S-nitrosylation, and transition metal bonding are
reversible and protective. The remaining modifications are irreversible. In either
case, the enzyme activity is inhibited (69). Glucose metabolism continues, however, through the pentose phosphate shunt, which produces the NADPH used by
glutathione reductase to recycle oxidized glutathione to its reducing form. It also
uncouples glucose metabolism from the production of ATP and oxidative intermediates (70, 71). Thus, under oxidative conditions, GAPDH can act as a switch to
redirect glucose metabolism to a more appropriate pathway. Under mild conditions,
GAPDH activity can be recovered with the return of a reducing redox potential
aided by the presence of reduced glutathione. Irreversibly modified GAPDH is
ubiquitinated and degraded by proteasomes (72, 73). GAPDH, like histone H2A,
actin, -crystallin, and -lactalbumin, requires a non-N-end rule E2 and HSC 70
for this process (74, 75). Oxidized active site cysteine also alters the affinity of
GAPDH for its cofactors (76). The normal high affinity of GAPDH for NAD+ is
dramatically decreased, whereas the affinity for NADH is increased. The active
site lies at the end of a twofold axis fold in the tertiary structure of GAPDH that
accommodates the binding of the cofactor NAD+. This is known as the Rossmann
fold and is shared by a number of NAD+-linked dehydrogenases (77). The negatively charged thiolate ion forms an ionic bond with the positively charged NAD+
and stabilizes the GAPDH structure. This fold also accommodates the binding of
nucleic acids, as bound NAD+ or NADH blocks the binding of nucleic acids (78).
Because the binding of NAD+ stabilizes GAPDH tertiary structure, oxidation of
the active site cysteine destabilizes the normal tetrameric form of the enzyme, and
dimers, monomers, and denatured protein can be detected in the nucleus, as was
shown in HeLa cells (76).
In addition, GAPDH has been found to associate with Nm23-H1, a nucleoside diphosphate kinase, to form an enzyme exhibiting dehydrogenase, nucleoside diphosphate kinase, and phosphotransferase activities (79). The complex is
a tetramer composed of dimeric GAPDH and dimeric Nm23-H1. The separate
enzymatic activities of the two proteins are unaffected by the association, as is the
binding of NADH to GAPDH. Both Nm23-H1 and GAPDH are also components
of the OCA-S complex involved in activation of histone H2B transcription mentioned above (60). In this complex, NAD+ enhances association of the complex to
the H2B promoter, whereas NADH is inhibitory. Thus, normal enzymatic activity, cofactor binding, and tetrameric quaternary structure do not exclude GAPDH
from the nucleus, nor does the enhanced binding of nucleic acids resulting from
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active site oxidation appear to be required for at least three nuclear functions of
GAPDH. This does not, however, exclude all mechanisms of nuclear translocation
involving active site oxidation of GAPDH, and an explanation and function for
GAPDH translocation to the nucleus during oxidative stress remains to be found.
The function of a sensor is to detect and signal changes in intracellular conditions. In addition to nuclear translocation, increased GAPDH gene expression
with oxidation of the GAPDH active site cysteine has been observed (80). Could
the oxidation of GAPDH Cys149 signal transcriptional activation of its own gene?
Increased NADPH levels resulting from inhibition of GAPDH enable thioredoxin
to become reduced, which in turn, through Ref-1, reduces a putative Cys275Cys135 bridge within p53, enhances DNA binding, and activates the transcription
of several proteins, including, presumably, GAPDH (31, 81). Reduced thioredoxin,
through Ref-1, also enhances the function of NF-B, AP-1, and HIF-1. It has long
been thought that p53 mediates oxidative stress-induced apoptosis (82). Recently,
however, it has become apparent that p53 acts through another protein, p66Shc,
to mediate apoptosis (83). Overexpression of p66Shc potentiates overexpressed
p53-mediated apoptosis in DLD-1 colorectal cancer cells, whereas overexpressed
p53 induces no apoptosis in p66Shc/ mouse embryonic fibroblast cells. Overexpressed p66Shc alone has little effect and hence acts downstream of p53. Under
high oxidative stress, p66Shc plays a permissive role in cytochrome c release and
collapse of the mitochondrial membrane potential (84). Exactly how p53 and/or
GAPDH interact with p66Shc is still unclear.
The one property of GAPDH that ties the multiple functions, locations, and
ligands of this protein with its putative role as a sensor of oxidative stress is
its capacity to bind NAD+. Direct induction of GAPDH expression by p53 suggests that GAPDH plays a role in the function of p53, but the details of this role
are unknown. Perhaps GAPDH provides NAD+ to the NAD+-dependent protein
deacetylase, Sirt1. The human homologue of yeast Sir2a, Sirt1, inhibits p53 by
removing the acetyl group from Lys382 in the C-terminal tail of p53 (85). This action results in resistance to stress and enhanced survival (86). Because irreversible
oxidation of GAPDH Cys149 dramatically reduces its affinity for NAD+, it could
prevent delivery of the cofactor to Sirt1 and result in unchecked p53 activity toward
apoptosis. This function could explain why GAPDH is translocated to the nucleus
under conditions of cellular stress.
POTENTIAL ROLES OF GAPDH IN THE PATHOPHYSIOLOGY

OF NEURODEGENERATIVE DISEASES
There is a general consensus that the pathophysiology of a variety of neurodegenerative diseases involves excessive apoptosis in distinct brain areas. For example,
cytochrome c release, caspase activation, and apoptosis in the striatum have been
documented in Huntingtons disease (HD), an autosomal dominant neurodegenerative disease caused by an expansion of a sequence of repeated CAG triplets
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(coding for glutamine) in the gene of huntingtin, (reviewed in 87). The hallmarks
of apoptosis, such as DNA fragmentation, caspase activation, and induction of
apoptosis-related genes, have been found in brain neurons associated with the
deposits of -amyloid peptide in Alzheimers disease (AD) (for review, see 27).
Growing evidence also links Parkinsons disease (PD) with apoptotic death of
dopaminergic neurons in the substantia nigra (reviewed in 20, 27, 87). In brain
ischemia, the activation of caspases 1, 3, 8, 9, and 11 and apoptosis have been
reported in the ischemic penumbra, where hypoxia and energy depletion are not
as severe (reviewed in 8789). The participation of GAPDH in apoptosis in vitro
suggests significant roles in human pathophysiology. This section reviews literature reporting abnormal expression and intracellular localization of GAPDH and
implicating the involvement of GAPDH in the apoptosis and neurodegeneration
observed in HD, AD, PD, and ischemia.
Huntingtons Disease and Other Polyglutamine-Connected

Brain Disorders
Several pioneering studies have shown that GAPDH binds to proteins containing
polyglutamine tracts associated with several neurodegenerative diseases. These
include huntingtin for HD (90), atrophin for dentatorubral-pallidoluysian atrophy
(DRPLA) (90), ataxin for spinocerebellar ataxia type-1 (SCA-1) (91), and androgen receptor for spinobulbar muscular atrophy (91). Specific binding of GAPDH
to the polyglutamine stretch of huntingtin and atrophin depends on the number
of glutamines in their polyglutamine tracts (90). GAPDH binds in vitro to both
normal and mutant huntingtin with a preference for cleaved fragments of the protein. GAPDH binding sites for both ataxin-1 and androgen receptors are located in
the N-terminal polyglutamine-containing domain but do not depend on the length
of the polyglutamine tract (91). Although of potentially great importance, the
functional significance of these interactions with GAPDH remain unelucidated.
A recent postmortem study shows that the extent of cerebral white matter damage in DRPLA correlates with GAPDH immunoreactivity (92). An increase in
GAPDH immunostaining of endothelial cells, astrocytes, and oligodendrocytes
has been observed in this disease, and the abnormal staining appears to depend on
the severity of cerebral white matter damage. It has also been reported that profuse
GAPDH granular deposits were found in neuronal nuclei in the pontine region
of the postmortem brain of patients with spinocerebellar ataxia type-3 (MachadoJoseph disease), but only weak and diffuse GAPDH staining was detected in the
cytoplasm of neurons in control brain (19). Although not described in detail in that
report, similar granule nuclear GAPDH deposits were also found in the DRPLA
brain.
The interaction of huntingtin with GAPDH, resulting in either loss of normal
GAPDH function or gain of toxic function, is one possible cause of the pathology of HD, which is characterized by hyperkinetic involuntary movement, cognitive impairment, and depression. Several attempts have been made to investigate
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whether there is a change in the glycolytic activity of GAPDH in the HD state. In

the initial study, GAPDH was not significantly altered in homogenates of frontal
and parietal cortex, caudate putamen, or cerebellum of human postmortem HD
brain samples (93). A subsequent study showed, however, a small but significant
decrease (12%) in GAPDH glycolytic activity in homogenates of the caudate
nucleus of HD patients (94). As discussed in a previous section, nuclear translocation of GAPDH in vitro is associated with decreased glycolytic activity of nuclear
fractions (37). Thus, in future studies, the measurements of GAPDH activity in
nuclear preparations rather than whole homogenates from postmortem HD brains
are warranted. HD fibroblasts subjected to metabolic stress by withholding fresh
medium, on the other hand, increased their GAPDH glycolytic activity by only
threefold, compared to an eightfold increase in control fibroblasts (95). In another
study, GAPDH activity of HD fibroblasts decreased by 33% in the nuclear fraction
but not in the postnuclear fraction, when compared with age-matched controls
(96). This decrease in nuclear glycolytic activity of HD fibroblasts is associated
with the nuclear appearance of a high-molecular-weight GAPDH-immunopositive
species (97). This high-molecular-weight GAPDH-immunopositive species was
not found in whole-cell sonicates of HD fibroblasts, which have normal glycolytic
activity. It is unknown whether these changes are related to the transglutaminasecatalyzed, polyglutamine domain-dependent inactivation of GAPDH reported in
a cell-free study (98). Striatal neurodegeneration in HD patients is accompanied
by the appearance of nuclear inclusions of mutant huntingtin (for review, see 23,
87). It has been proposed that, through binding to the polyglutamine stretch of
this disease-causing protein, GAPDH functions as a molecular chaperon in the nuclear translocation of huntingtin (34, 35). However, there are not, as yet, sufficient
experimental data to substantiate this interesting hypothesis.
An increasing number of reports demonstrate that the expression in mice of
mutant huntingtin with expanded polyglutamine tracts leads to neuronal loss and
shows phenotypes of neurological disorders similar to those found in HD (99104).
Studies of abnormal GAPDH expression and localization have been conducted utilizing the brains of transgenic mice that express full-length huntingtin cDNA with
89 CAG repeats and display neurodegeneration in brain areas, including the striatum and cerebral cortex (100). The majority of the transgenic mice show a strong
increase in GAPDH immunofluorescence that increases with age, compared with
wild-type mice. The wild-type mice show an even and predominantly cytoplasmic
distribution of GAPDH (105). Increased GAPDH immunostaining in transgenic
mice occurs in cells of specific brain regions such as the caudate putamen, globus
pallidus, neocortex, and hippocampus. Double-staining experiments revealed that
GAPDH overexpression occurs in neurons but not glial cells. Subcellular fluorescence microscopy demonstrated that GAPDH accumulates in the nuclei of neurons
in these brain regions (Figure 1). Nuclear accumulation is associated with the loss
of medium-sized and small neurons in the caudate putamen and neurons in layers
V and VI of the neocortex (105). The marked increase of GAPDH in the cytoplasm
and nuclei of neurons suggests that GAPDH is involved in the apoptotic cascade
in the transgenic mouse model of HD.
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Alzheimers Disease
AD is the most common cause of progressive neurodegeneration and is characterized by dementia and memory loss. The deposition of -amyloid protein in
the cerebral cortex and hippocampus is one of the most distinct morphological
features in AD (reviewed in 106). -Amyloid peptide derives from irregular proteolytic processing of -amyloid precursor protein (-APP). The deposition of
-amyloid peptide is associated with neuronal loss in these brain regions and is,
at least in part, due to apoptosis. Initial in vitro studies found GAPDH binding to
the cytoplasmic carboxyl terminal of -amyloid precursor protein (-APP) (107).
Such binding could alter the normal processing of -APP to produce -amyloid
protein. A significant nuclear role for the C terminus of -APP has also been suggested (108, 109). The recognition of GAPDH by a monoclonal antibody raised
against amyloid plaques from the brains of patients with AD indicates the presence of GAPDH in these plaques and suggests an interaction between GAPDH and
-APP (110, 111). A more recent preliminary report suggests that cotransfection
of COS-7 cells with GAPDH and wild-type -APP cDNAs induces synergistic
cytotoxicity (112).
The initial postmortem study showed a significant (19%) reduction in GAPDH
glycolytic activity in the homogenates of the temporal cortex of AD patients (94).
However, a subsequent study failed to detect a change in GAPDH activity in homogenates of the frontal, temporal, parietal, and occipital lobes of AD brains,
although there was a significant increase in activity in the same brain regions in
Downs syndrome patients (113). Such a discrepancy could be related to the fact
that whole-cell homogenates, rather than subcellular fractions, were used in the
activity measurements. The presence of multiple cell types in a given brain region might have also masked potential changes in a specific cell population. Using
fibroblasts from AD patients, it has been reported that GAPDH glycolytic activity is decreased by approximately 27% in both postnuclear and nuclear fractions
compared with age-matched controls (58, 96). A high-molecular-weight species
of GAPDH-immunoreactivity was detected exclusively in the postnuclear fraction
of AD fibroblasts (58). The latter displayed reduced GAPDH activity and was
not present in postnuclear fractions from control subjects. Whether the shift in
molecular weight reflects GAPDH binding to -APP is unknown. Interestingly,
the association of GAPDH with a high-molecular-weight species was not detected
in sonicated AD whole-cell extracts, which exhibited normal levels of GAPDH
activity. This suggests that GAPDH is weakly bound to the high-molecular-weight
protein complex and that dissociation of GAPDH from the high-molecular-weight
complex restores its glycolytic activity. In the postmortem AD brain, nuclear aggregated GAPDH in neurons of affected areas has been found (114). It is also
noteworthy that potential drugs for treating AD, such as tetrahydroaminoacridine
(THA) and ONO-1603, effectively suppress GAPDH overexpression and nuclear
translocation in rat brain neurons undergoing apoptosis in cultures (115, 116).
Moreover, THA and another antidementia drug, donepezil, inhibit AraC-induced
increase in GAPDH promoter activity (114).
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Parkinsons Disease
PD is characterized by the loss of catecholaminergic neurons, including those in
the substantia nigra compacta and noradrenergic neurons in the locus coeruleus.
The first implication that GAPDH is involved in the pathogenesis of PD is from the
observation that CGP 3466 (also known as TCH 346), a structurally related analog
of R-(-)-deprenyl, protects human neuroblastoma PAJU cells from apoptosis induced by rotenone, a toxin producing PD-like neuropathology (44). The MAO-B
inhibitor, (-)-deprenyl, slows neurodegeneration and reduces the clinical deficits
of PD, HD, and AD. Unlike (-)-deprenyl, CGP 3466 does not inhibit MAO-B activity, but like (-)-deprenyl, binds to GAPDH. These results are compatible with
the view that GAPDH, rather than MAO-B, is the target of deprenyl-like compounds effective against PD neuropathology. An independent study suggested that
deprenyl-like compounds inhibit apoptosis by inducing GAPDH to dissociate from
its usual tetrameric form to a dimer, and thereby interfere with GAPDH nuclear
translocation (117). Nuclear localization of GAPDH monomers and dimmers were
readily detected in HeLa cells, however, following GAPDH active site oxidation
(76). R-2HMP (R-2-heptylmethyl-pargylamine) and other aliphatic pargylamines
bind GAPDH, prevent GAPDH overexpression, and block p53-dependent apoptosis (16, 17, 118). Other studies show that apoptosis of neurons or related cell-lines
induced by dopaminergic toxins such as MPP+, 6-hydroxydopamine, or N-methyl(R)-salsolinol involves GAPDH overexpression and nuclear translocation, and
these effects are prevented by GAPDH antisense oligonucleotides and anti-PD
drugs (3941, 47). In total, these observations suggest that GAPDH is a potential
molecular target of drugs used to treat PD and other neurodegenerative diseases.
Despite the number of cell culture studies of the role of GAPDH in apoptosis,
knowledge concerning GAPDH changes in the PD brain is limited. This may
be because there is still no clear consensus that apoptosis contributes to the loss
of dopaminergic neurons in PD. Interestingly, an accumulation of GAPDH was
found in the nuclei of melanized neurons of the nigra in postmortem brain sections
from PD patients, whereas GAPDH was found only in the cytoplasm of melanized
cells of age-matched control sections (119). Nuclear inclusion bodies, known as
Marinescos bodies, are immunoreactive for GAPDH in numerous nigral neurons
from PD, but not control brains. Many cytoplasmic inclusion bodies, known as
Lewy bodies, in the melanized neurons of PD brain are also immunopositive for
GAPDH, although it is unknown whether all Lewy bodies are immunopositive.
Moreover, GAPDH appears to be colocalized with Bax and caspase3 in melanized
neurons of the PD nigra. Although a direct link between PD and GAPDH-mediated
apoptosis is still undetermined, these results suggest a potential role of GAPDH
nuclear accumulation in dopaminergic cell death in the PD brain.
Stroke and Hypoxia

Stroke is a major cause of mortality and morbidity worldwide, and is one of
the neurodegenerative diseases linked to glutamate excitotoxicity. The significant
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increase in extracellular glutamate in the brain following ischemia and the brain
damage that occurs as a result are well recognized (for review, see 87, 89, 120).
Few studies address the involvement of GAPDH in the ischemic brain. In a rat focal ischemia stroke model, nuclear accumulation of GAPDH has been found in the
ischemic core of the parietal cortex of rats subjected to 2 h of middle cerebral artery
occlusion without reperfusion (121). During subsequent reperfusion, GAPDH immunostaining in the ischemic core decreases but cytoplasmic and nuclear staining
in the penumbra becomes detectable. The increase in nuclear GAPDH immunoreactivity persists up to 48 h with a concomitant decrease in cytoplasmic reactivity.
Double labeling of GAPDH-positive cells with TUNEL suggests an association of
GAPDH overexpression/nuclear accumulation with excitotoxicity-induced apoptotic death. Cell culture studies have provided insights into mechanisms by which
GAPDH is induced by hypoxia. A pioneering study has shown that hypoxia stimulates GAPDH overexpression in the cytoplasm and nucleus of endothelial cells
at both transcriptional and posttranscriptional levels (122). Subsequent studies
identified at least two hypoxia-responsive elements in the GAPDH gene promoter
(123, 124). Hypoxia induces an elevation of GAPDH protein in the cytosolic, nuclear, and particulate fractions by 4.0-, 2.3-, and 4.2-fold, respectively, in a mouse
brain capillary endothelial cell line (125). Little or no increase in GAPDH glycolytic activity was found in these fractions, however, suggesting a dynamic steady
state or an inactivation of newly induced GAPDH. The same study also shows that
GAPDH expression is suppressed by inhibiting the activation of nonselective Ca2+
channels, Na+/Ca2+ exchanger, Ca2+/calmodulin-dependent kinase, and c-Jundependent AP-1 binding. GAPDH has been shown to interact directly with heat
shock proteins (HSP), and in particular, with HSP70 (61, 126). HSP70 has a major
role in protection against ischemia-induced brain damage (88, 127, 128). Therefore, if GAPDH is involved in the apoptotic death induced by ischemia/hypoxia,
then GAPDH binding-induced inactivation of cytoprotective HSP70 and nuclear
translocation may be involved as well.
CONCLUSIONS AND FUTURE DIRECTIONS

The involvement of GAPDH in apoptosis was first demonstrated in primary cultures of brain neurons, and this finding was soon expanded to numerous apoptotic
paradigms in diverse cell types, including neurons and nonneuronal cells. Several lines of evidence also suggest that GAPDH may be an intracellular sensor
of oxidative stress during the early phase of the apoptotic cascade. Irreversible
oxidation of the active site cysteine of GAPDH triggers major changes in cellular
homeostasis. Enhanced expression, nuclear accumulation, changes in the apparent
molecular size, and a decrease in the glycolytic activity of GAPDH have also been
observed in some cellular, rodent, and transgenic mouse models as well as postmortem brain tissues of several neurodegenerative diseases, including HD, AD,
PD, and stroke/hypoxia. Table 1 lists cellular and rodent models as well as neurodegenerative diseases that have been linked to GAPDH abnormalities. Interactions
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TABLE 1 Changes in expression, intracellular localization, and glycolytic activity of GAPDH

are associated with diverse apoptotic paradigms in multiple cell types, as well as rodent models
and postmortem brain tissues of neurodegenerative diseases
Apoptotic stimuli or disease
Cell types or tissues
Spontaneous cell death, AraC toxicity,

K+ depletion, excitotoxicity, etoposide
Cerebellar granule cells, cerebral cortical neurons
Nerve growth factor withdrawal, serum

deprivation, dexamethasone stimulation
PC 12 cells, HEK 293 cells, S49 lymphoma cells
Androgen deprivation
Prostate epithelial cells
Thapsigargin, buthionine sulfoximine
GT1-7 hypothalmic neurosecretory cells
Dopaminergic toxins (MPP+, rotenone,

6-hydroxydopamine, N-methyl(R)-salsolinol)
Mesencephalic dopaminergic neurons; human

neuroblastoma PAJU, SK-N-SH, and
SH-SY5Y cells
Staurosporine, oxidative stress
Neuroblastoma NB41A3, R6 fibroblasts
Hypoxia, middle cerebral artery

occlusion (a rat stroke model)
Bovine and rodent endothelial cells,

rat brain penumbra
HD, DRPLA, Machado-Joseph disease
Postmortem brains, fibroblasts, transgenic

mouse brain of HD
AD
Postmortem brains, fibroblasts
PD
Postmortem brains, cellular models
See text for references.
of GAPDH with some disease-related proteins such as polyglutamine-containing

mutant disease proteins and -APP have been reported. Whether such interactions
occur in the affected brain areas during pathological states and play a role in nuclear
transport of the GAPDH complex need to be rigorously demonstrated. Drugs used
to treat PD, HD, and AD can bind GAPDH and/or suppress its overexpression, and
these actions correlate with their neuroprotective effects, suggesting that GAPDH
may be a therapeutic target for disease interventions and future drug design and
development.
Despite the advancement in knowledge concerning the proapoptotic effects of
GAPDH and the crucial role of GAPDH nuclear translocation in the apoptotic
process, their precise molecular mechanisms remain to be defined. The observations that GAPDH is positively regulated by p53 and is colocalized with Bax and
caspases and that Bcl-2 overexpression blocks GAPDH nuclear translocation suggest that GAPDH may act by perturbing the p53-Bax-Bcl-2-caspase pathway. It
appears that GAPDH nuclear translocation is integral to the apoptotic cascade and
that protein-protein interactions may be crucial in mediating this nuclear transport.
Although some GAPDH chaperone proteins have been proposed, future studies
are necessary to substantiate their roles in nuclear translocation. GAPDH exists
in multiple isoforms that are differentially translocated to the nucleus following
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apoptotic insults. Selective gene silencing and/or knockout/knockdown of these

isoforms will shed light on their potentially distinct involvements in apoptosis.
It seems possible that GAPDH translocation to the nucleus is not the sole mechanism whereby its proapoptotic effect is mediated. Translocation of GAPDH to
other intracellular organelles such as the mitochondria warrants future investigation, particularly considering that this organelle plays a pivotal role in apoptosis
and that GAPDH is also enriched in the crude mitochondrial fraction of neurons
undergoing apoptosis. The apparent contradictions as to whether GAPDH glycolytic activity is decreased in the postmortem HD and AD brains could be related
to the fact that whole-cell homogenates rather than subcellular fractions were used
in the analysis. It would seem necessary to reexamine changes in glycolytic activity using various subcellular fractions derived from the postmortem brains of
patients with these diseases. The success in demonstrating dramatic changes in
GAPDH overexpression and nuclear accumulation in distinct brain neurons of HD
transgenic mice raises the possibility that the transgenic disease model may be a
useful tool to elucidate the role of GAPDH in mediating neurodegeneration and
the pathophysiology of human diseases.
ACKNOWLEDGMENTS
The authors wish to thank Peter Leeds in the NIMH, NIH for assistance in editing the review and Dr. Akira Sawa of Johns Hopkins University for providing
information of his unpublished results.
LITERATURE CITED
1. Jeffery CJ. 1999. Moonlighting proteins.
Trends Biochemical Sci. 24:811
2. Sirover MA. 1999. New insights into an
old protein: the functional diversity of
mammalian glyceraldehyde-3-phosphate
dehydrogenase. Biochim. Biophys. Acta
1432:15984
3. Alderete JF, Millsap KW, Lehker MW,
Benchimol M. 2001. Enzymes on microbial pathogens and Trichomonas vaginalis: molecular mimicry and functional
diversity. Cell. Microbiol. 3:35970
4. Huitorel P, Pantaloni D. 1985. Bundling
of microtubules by glyceraldehyde-3phosphate dehydrogenase and its modulation by ATP. Eur. J. Biochem. 150:26569
5. Fuchtbauer A, Jockusch BM, Leberer
E, Pette D. 1986. Actin-severing activity copurifies with phosphofructokinase.

6. Glaser PE, Gross RW. 1995. Rapid
plasmenylethanolamine-selective fusion
of membrane bilayers catalyzed by
an isoform of glyceraldehyde-3-phosphate dehydrogenase-discrimination between glycolytic and fusogenic roles
of individual isoforms. Biochemistry 34:
12193203
7. Robbins AR, Ward RD, Oliver C. 1995. A
mutation in glyceraldehyde-3-phosphate
dehydrogenase alters endocytosis in CHO
cells. J. Cell. Biol. 130:1093104
8. Tisdale EJ. 2001. Glyceraldehyde-3phosphate dehydrogenase is required for
7 Jan 2005
13:59
284
9.

10.
11.
12.
13.
14.
15.
16.
17.
18.
AR
AR232-PA45-11.tex
CHUANG
HOUGH
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
SENATOROV
vesicular transport in the early secretory

pathway. J. Biol. Chem. 276:248086
Ikemoto A, Bole DG, Ueda T. 2003.
Glycolysis and glutamate accumlation
into synaptic vesiclesrole of glyceraldehyde phosphate dehydrogenase and
3-phosphogylcerate kinase. J. Biol. Chem.
278:592940
Doucet JP, Tuana BS. 1991. Identification of low-molecular-weight GTPbinding proteins and their sites of interaction in subcellular-fractions from skeletalmuscle. J. Biol. Chem. 266:1761320
Morgenegg G, Winkler GC, Hubscher
U, Heizmann CW, Mous J, Kuenzle CC.
1986. Glyceraldehyde-3-phosphate dehydrogenase is a nonhistone protein and a
possible activator of transcription in neurons. J. Neurochem. 47:5462
Singh R, Green MR. 1993. Sequencespecific binding of transfer-RNA by
glyceraldehyde-3-phosphate dehydrogenase. Science 259:36568
Meyer-Siegler K, Mauro DJ, Seal G,
Wurzer J, de Riel JK, Sirover MA. 1991.
A human nuclear uracil DNA glycosylase
is the 37-kDa subunit of glyceraldehyde3-phosphate dehydrogenase. Proc. Natl.
Chuang D-M, Ishitani R. 1996. A role for
GAPDH in apoptosis and neurodegeneration. Nat. Med. 2:60910
Sirover MA. 1997. Role of the glycolytic
protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function
and in cell pathology. J. Cell. Biochem.
66:13340
Berry MD, Boulton AA. 2000. Glyceraldehyde-3-phosphate dehydrogenase and
apoptosis. J. Neurosci. Res. 60:15054
Berry MD, Boulton AA. 2002. Aliphatic
propargylamines as symptomatic and
neuroprotective treatments for neurodegenerative diseases. Neurotoxicol. Teratol. 24:66773
Tatton W, Chalmers-Redman R, Tatton N.
2003. Neuroprotection by deprenyl and
other propargylamines; glyceraldhyde-
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
3-phosphate dehydrogenase rather than

monoamine oxidase B. J. Neural Transm.
110:50915
Ishitani R, Tajima H, Takata H, Tsuchiya
K, Kuwae T, et al. 2003. Proapoptotic
protein glyceraldehydes-3-phosphate dehydrogenase; a possible site of action
of antiapoptotic drugs. Prog. Neuropsychopharmacol. Biol. Psychiatry 27:291
301
Waldmeier PC, Tatton WG. 2004. Interrupting apoptosis in neurodegenerative
disease: potential for effective therapy?
Drug Discov. Today 9:21018
Margolis RL, Chuang D-M, Post RM.
1994. Programmed cell death: implications for neuropsychiatric disorders. Biol.
Psychiatry 35:94656
Horvitz HR. 1999. Genetic control of
programmed cell death in the nematode Caenorhabditis elegans. Canc. Res.
59(Suppl. S):1701S6S
Hickey MA, Chesselet MF. 2003. Apoptosis in Huntingtons disease. Progr. NeuroPsychopharm. Biol. Psychiatry 27:255
65
Reynolds IJ. 1999. Mitochondrial membrane potential and the permeability transition in excitotoxicity. Ann. NY Acad. Sci.
893:3341
Hengartner MO. 2000. The biochemistry
of apoptosis. Nature 407:77076
Wang X. 2001. The expanding role of
mitochondria in apoptosis. Genes Dev.
15:292233
Mattson MP, Kroemer G. 2003. Mitochondria in cell death: novel targets
for neuroprotection and cardioprotection.
Trends Mol. Med. 9:196205
Ishitani R, Sunaga K, Hirano A, Saunders
P, Katsube N, Chuang D-M. 1996. Evidence that glyceraldehyde-3-phosphate
dehydrogenase is involved in age-induced
apoptosis in mature cerebellar granule
neurons in culture. J. Neurochem. 66:928
35
Ishitani R, Kimura M, Sunaga K, Katsube N, Tanaka M, Chuang D-M. 1996.
7 Jan 2005
13:59
AR
AR232-PA45-11.tex
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE

30.
31.
32.
33.
34.
35.
36.
37.
An antisense oligodeoxynucleotide to
glyceraldehyde-3-phosphate dehydrogenase blocks age-induced apoptosis of mature cerebrocortical neurons in culture. J.
Ishitani R, Sunaga K, Tanaka M, Aishita
H. Chuang D-M. 1997. Over-expression
of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced
apoptosis, but not necrosis, of cultured
cerebellar granule cells. Mol. Pharmacol.
51:54250
Chen R-W, Saunders PA, Wei H, Li Z,
Seth P, Chuang D-M. 1999. Involvement
of GAPDH and p53 in neuronal apoptosis: evidence that GAPDH is upregulated
by p53. J. Neurosci. 19:965462
Ishitani R, Chuang D-M. 1996. Glyceraldehyde-3-phosphate dehydrogenase
antisense oligodeoxynucleotides protect
against cytosine arabinoside-induced
apoptosis in cultured cerebellar neurons.
Saunders PA, Chalecka-Franaszek E,
Chuang D-M. 1997. Subcellular distribution of GAPDH in cerebellar granule cells
undergoing cytosine arabinoside-induced
apoptosis. J. Neurochem. 69:182028
Sawa A, Khan AA, Hester LD, Snyder
SH. 1997. Glyceraldehyde-3-phosphate
dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell
death. Proc. Natl. Acad. Sci. USA 94:
1166974
Ishitani R, Tanaka M, Sunaga K, Katsube
N, Chuang D-M. 1998. Nuclear localization of over-expressed glyceraldehyde3-phosphate dehydrogenase in cultured
cerebellar neurons undergoing apoptosis.
Shashidharan P, Chalmers-Redman RME,
Carlile GW, Rodic V, Gurvich N, et al.
1999. Nuclear translocation of GAPDHGFP fusion protein during apoptosis. NeuroReport 10:114953
Saunders PA, Chen R-W, Chuang D-M.
1999. Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase iso-
38.
39.
40.
41.
42.
43.
44.
45.
285
forms during neuronal apoptosis. J. Neurochem. 72:92532

Epner DE, Sawa A, Isaacs JT. 1999.
Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate. Biol.
Reprod. 61:68791
Fukuhara Y, Takeshima T, Kashiwaya Y,
Shimoda K, Ishitani R, Nakashima K.
2001. GAPDH knockdown rescues mesencephalic dopaminergic neurons from
MPP+-induced apoptosis. NeuroReport
12:204952
Sharma SK, Carlson EC, Ebadi M. 2003.
Neuroprotective actions of Selegiline in
inhibiting 1-methyl, 4-phenyl, pyridinium
ion (MPP+)-induced apoptosis in SK-NSH neurons. J. Neurocytol. 32:32943
Maruyama W, Akao Y, Youdim MBH,
Davis BA, Naoi M. 2001. Transfectionenforced Bcl-2 over-expression and an
anti-Parkinson drug, rasagiline, prevent
nuclear accumulation of glyceraldehyde3-phosphate dehydrogenase induced by
an endogenous dopaminergic neurotoxin,
N-methyl-(R)-salsolinol. J. Neurochem.
78:72735
Dastoor Z, Dreyer JL. 2001. Potential
role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase
in apoptosis and oxidative stress. J. Cell
Sci. 114:164353
Hara M, Agarawal N, Luo X, Fujii K, Snyder SH, Sawa A. 2001. Oxidative modification of GAPDH and neuronal cell death;
a trigger to initiate a death cascade via
Siah and N-coR. Annu. Meet. Soc. Neurosci. Abstr. No. 19.11
Kragten E, Lalande I, Zimmermann K,
Roggo S, Schindler P, et al. 1998.
Glyceraldehyde-3-phosphate dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466 and R-(-)deprenyl. J. Biol. Chem. 273:582128
Tajima H, Tsuchiya K, Yamada M, Kondo
K, Katsube N, Ishitani R. 1999. Overexpression of GAPDH induces apoptosis in COS-7 cells transfected with cloned
7 Jan 2005
13:59
286

46.
47.
48.
49.
50.
51.
52.
AR
AR232-PA45-11.tex
CHUANG
HOUGH
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
SENATOROV
GAPDH cDNAs. NeuroReport 10:2029

33
Chen R-W, Wei H, Leng Y, Wei W,
Saunders PA, et al. 1999. Bcl-2 suppresses nuclear levels of glyceraldehyde3-phosphate dehydrogenase and protects
against apoptosis induced by thapsigargin
and buthionine sulfoximine. Annu. Meet.
Soc. Neurosci. Abstr. No. 606.19
Maruyama W, Oya-Ito T, ShamotoNagai M, Osawa T, Naoi M. 2002.
Glyceraldehyde-3-phosphate dehydrogenase is translocated into nuclei through
Golgi apparatus during apoptosis induced by 6-hydroxydopamine in human
dopaminergic SH-SY5Y cells. Neurosci.
Lett. 321:2932
Sawa A, Agrawal N, Hara M, Luo X, Snyder SH. 2001. Roles of N-CoR (nuclear
receptor corepressor) degradation in neuronal cell death: a main target of apoptotic
cascade by GAPDH/Siah. Annu. Meet.
Soc. Neurosci. Abstr. No. 19.10
Roperch JP, Lethrone F, Prieur S, Piouffre
L, Israeli D, et al. 1999. SIAH-1 promotes
apoptosis and tumor suppression through
a network involving the regulation of protein folding, unfolding, and trafficking:
identification of common effectors with
p53 and p21(Waf1). Proc. Natl. Acad. Sci.
USA 96:807073
Fiucci G, Beaucourt S, Duflaut D, Lespagnol A, Stumptner-Cuvelette P, et al. 2004.
Siah-1b is a direct transcriptional target of p53: Identification of the functional p53 responsive element in the siah1b promoter. Proc. Natl. Acad. Sci. USA
101:351015
Relaix F, Wei XJ, Li W, Pan JJ, Lin
YH, et al. 2000. Pw1/Peg3 is a potential
cell death mediator and cooperates with
Siah1a in p53-mediated apoptosis. Proc.
Susini L, Passer BJ, Amzallag-Elbaz N,
Juven-Gershon T, Prieur S, et al. 2001.
Siah-1 binds and regulates the function
of Numb. Proc. Natl. Acad. Sci. USA
98:1506772
53. Rogalski AA, Steck TL, Waseem A.

1989. Association of glyceraldehyde3-phosphate dehydrogenase with the
plasma-membrane of the intact human red
blood-cell. J. Biol. Chem. 264:643846
54. Schmitz HD. 2001. Reversible nuclear translocation of glyceraldehyde-3phosphate dehydrogenase upon serum depletion. Eur. J. Cell Biol. 80:41927
55. Schmitz HD, Dutine C, Bereiter-Hahn J.
2003. Exportin 1-independent nuclear export of GAPDH. Cell Biol. Int. 27:51117
56. Brown VM, Krynetski EY, Krynetskaia
NF, Grieger D, Mukatira ST, et al. 2004.
A novel CRM1-mediated nuclear export
signal governs nuclear accumulation of
glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress. J. Biol.
Chem. 279:598492
57. Nahlik KW, Mleczko AK, Gawlik MK,
Rokita HB. 2003. Modulation of GAPDH
expression and cellular localization after vaccinia virus infection of human
adherent monocytes. Acta Biochim. Pol.
50:66776
58. Mazzola JL, Sirover MA. 2003. Subcellular alteration of glyceraldehyde-3phosphate dehydrogenase in Alzheimers
disease fibroblasts. J. Neurosci. Res. 71:
27985
59. Kanai H, Sawa A, Chen R-W, Leeds P,
Chuang D-M. 2004. Valproic acid inhibits
histone deacetylase activity and suppresses excitotoxicity-induced GAPDH
nuclear accumulation and apoptotic death
in neurons. Pharmagenomics J. 4:33644
60. Zheng L, Roeder RG, Luo Y. 2003. S
phase activation of the histone H2B promoter by OCA-S, a coactivator complex
that contains GAPDH as a key component. Cell 114:25566
61. Krynetski EY, Krynetskaia NF, Bianchi
ME, Evans WE. 2003. A nuclear protein
complex containing high mobility group
proteins B1 and B2, heat shock cognate
protein 70, ERp60, and glyceraldehyde3-phosphate dehydrogenase is involved in
the cytotoxic response to DNA modified
7 Jan 2005
13:59
AR
AR232-PA45-11.tex
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
62.

63.
64.
65.
66.
67.
68.
69.
70.
by incorporation of anticancer nucleoside

analogues. Cancer Res. 63:1006
Mohr S, Hallak H, de Boitte A, Lapetina
EG, Brune B. 1999. Nitric oxide-induced
S-glutathionylation and inactivation of
glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 274:942730
Krotkiewska B, Banas T. 1992. Interaction of Zn2+ and Cu2+ ions with
glyceraldehyde-3-phosphate dehydrogenase from bovine heart and rabbit muscle.
Int. J. Biochem. 24:15015
Mohr S, Stamler JS, Brune B. 1996. Posttranslational modification of glyceraldehyde-3-phosphate dehydrogenase by
S-nitrosylation and subsequent NADH
attachment. J. Biol. Chem. 271:4209
14
Pancholi V, Fischetti VA. 1993. Glyceraldehyde-3-phosphate dehydrogenase on
the surface of group A streptococci is also
an ADP-ribosylating enzyme. Proc. Natl.
Zolkiewska A, Nightingale MS, Moss
J. 1992. Molecular characterization
of NAD:arginine ADP-ribosyltransferase
from rabbit skeletal muscle. Proc. Natl.
Cahuana GM, Tejedo JR, Jimenez J,
Ramirez R, Sobrino F, Bedoya FJ. 2004.
Nitric oxide-induced carbonylation of
Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F
cells. Exp. Cell. Res. 293:2230
Brune B, Mohr S. 2001. Protein thiol modification of glyceraldehyde-3-phosphate
dehydrogenase and caspase-3 by nitric oxide. Curr. Protein Pept. Sci. 2:6172
Yasuda M, Fujimori H, Panhou H. 1998.
NO depletes cellular ATP contents via inactivation of glyceraldehyde-3-phosphate
dehydrogenase in PC12 cells. J. Toxicol.
Sci. 23:38994
Albina JE, Mastrofrancesco B, Reichner JS. 1999. Acyl phosphatase activity of NO-inhibited glyceraldehyde-3phosphate dehydrogenase (GAPDH): a
potential mechanism for uncoupling gly-
71.
72.
73.
74.
75.
76.
77.
78.
287
colysis from ATP generation in NOproducing cells. Biochem. J. 341:59

Arutyunov DY, Muronetz VI. 2003. The
activation of glycolysis performed by
the non-phosphorylating glyceraldehyde3-phosphate dehydrogenase in the model
system. Biochem. Biophys. Res. Commun.
300:14954
Buchczyk DP, Briviba K, Harti FU, Sies
H. 2000. Responses to peroxynitrite in
yeast: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a sensitive intracellular target for nitration and enhancement of chaperone expression and
ubiquitination. Biol. Chem. 381:12126
Buchczyk DP, Grune T, Sies H, Klotz LO.
2003. Modifications of glyceraldehyde3-phosphate dehydrogenase induced by
increasing concentrations of peroxynitrite: early recognition by 20S proteasome. Biol. Chem. 384:23741
Blumenfeld N, Gonen H, Mayer A, Smith
CE, Siegel NR, et al. 1994. Purification
and characterization of a novel species
of ubiquitin-carrier protein, E2, that is
involved in degradation of non-N-end
rule protein substrates. J. Biol. Chem.
269:957481
Bercovich B, Stancovski I, Mayer A,
Blumenfeld N, Laszlo A, et al. 1997.
Ubiquitin-dependent degradation of certain protein substrates in vitro requires
the molecular chaperone Hsc70. J. Biol.
Chem. 272:900210
Arutyunova EI, Danshina PV, Domnina
LV, Pleten AP, Muronetz VI. 2003. Oxidation of glyceraldehyde-3-phosphate dehydrogenase enhances its binding to nucleic
acids. Biochem. Biophys. Res. Commun.
307:54752
Moras D, Olsen KW, Sabesan MN,
Buehner M, Ford GC, Rossmann MG.
1975. Studies of asymmetry in the
three-dimensional structure of lobster Dglyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 250:913762
Yi M, Schultz DE, Lemon SM. 2000.
Functional significance of the interaction
7 Jan 2005
13:59
288

79.
80.
81.
82.
83.
84.
85.
86.
87.
AR
AR232-PA45-11.tex
CHUANG
HOUGH
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
SENATOROV
of hepatitis A virus RNA with glyceraldehyde 3-phosphate dehydrogenase

(GAPDH): opposing effects of GAPDH
and polypyrimidine tract binding protein
on internal ribosome entry site function.
J. Virol. 74:645968
Engel M, Seifert M, Theisinger B, Seyfert
U, Welter C. 1998. Glyceraldehyde-3phosphate dehydrogenase and Nm23H1/nucleoside diphosphate kinase A. Two
old enzymes combine for the novel Nm23
protein phosphotransferase function. J.
Biol. Chem. 273:2005865
Ito Y, Pagano PJ, Tornheim K, Brecher
P, Cohen RA. 1996. Oxidative stress increases glyceraldehyde-3-phosphate dehydrogenase mRNA levels in isolated
rabbit aorta. Am. J. Physiol. Heart Circ.
Physiol. 270:H8187
Hainaut P, Mann K. 2001. Zinc binding
and redox control of p53 structure and
function. Antioxid. Redox. Signal 3:611
23
Vogelstein B, Lane D, Levine AJ. 2000.
Surfing the p53 network. Nature 408:307
10
Trinei M, Giorgio M, Cicalese A, Barozzi
S, Ventura A, et al. 2002. A p53-p66Shc
signalling pathway controls intracellular
redox status, levels of oxidation-damaged
DNA and oxidative stress-induced apoptosis. Oncogene 21:387278
Orsini F, Migliaccio E, Moroni M, Contursi C, Raker VA, et al. 2004. The
life span determinant p66Shc localizes
to mitochondria where it associates with
mtHsp70 and regulates trans-membrane
potential. J. Biol. Chem. 279:25689
95
Luo J, Li M, Tang Y, Laszkowska M,
Roeder RG, Gu W. 2004. Acetylation of
p53 augments its site-specific DNA binding both in vitro and in vivo. Proc. Natl.
Smith JS. 2002. Human Sir2 and the silencing of p53 activity. Trends Cell Biol.
12:4046
Friedlander RM. 2003. Apoptosis and cas-
88.
89.
90.
91.
92.
93.
94.
95.
96.
pases in neurodegenerative diseases. New

Engl. J. Med. 348:136575
Sharp FR, Lu AG, Tang Y, Millhorn DE.
2000. Multiple molecular penumbras after
focal cerebral ischemia. J. Cerebr. Blood
Flow Met. 20:101132
Love S. 2003. Apoptosis and brain ischaemia. Prog. NeuroPsychopharmacol.
Biol. Psychiatry 27:26782
Burke JR, Enghild JJ, Martin ME, Jou YS,
Myers RM, et al. 1996. Huntington and
DRPLA proteins selectively interact with
the enzyme GAPDH. Nat. Med. 2:347
50
Koshy B, Matilla T, Burright EN, Merry
DE, Fischbeck KH, et al. 1996. Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with
glyceraldehyde-3-phosphate dehydrogenase. Hum. Mol. Genet. 5:131118
Shiozawa M, Fukutani Y, Arai N, Cairns
NJ, Mizutani T, et al. 2003. Glyceraldehyde 3-phosphate dehydrogenase and
endothelin-1 immunoreactivity is associated with cerebral white matter damage in dentatorubral-pallidoluysian atrophy. Neuropathology 23:3643
Browne SE, Bowling AC, MacGarvey U,
Baik MJ, Berger SC, et al. 1997. Oxidative damage and metabolic dysfunction in
Huntingtons disease: selective vulnerability of the basal ganglia. Ann. Neurol.
41:64653
Kish SJ, Lopes-Cendes I, Guttman M,
Furukawa Y, Pandolfo M, et al. 1998.
Brain glyceraldehyde-3-phosphate dehydrogenase activity in human trinucleotide
repeat disorders. Arch. Neurol. 55:1299
304
Cooper AJL, Sheu KFR, Burke JR,
Strittmatter WJ, Blass JP. 1998. Glyceraldehyde 3-phosphate dehydrogenase abnormality in metabolically stressed Huntington disease fibroblasts. Dev. Neurosci.
20:46268
Mazzola JL, Sirover MA. 2001. Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimers disease
7 Jan 2005
13:59
AR
AR232-PA45-11.tex
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
97.

98.
99.
100.
101.
102.
103.
104.
and in Huntingtons disease fibroblasts. J.

Neurochem. 76:44249
Mazzola JL, Sirover MA. 2002. Alteration
of nuclear glyceraldehyde-3-phosphate
dehydrogenase structure in Huntingtons
disease fibroblasts. Mol. Brain Res. 100:
95101
Cooper AJL, Sheu KFR, Burke JR, Onodera O, Stritmatter WJ, et al. 1997.
Transglutaminase-catalyzed inactivation
of glyceraldehyde 3-phosphate dehydrogenase and alpha-ketoglutarate dehydrogenase complex by polyglutamine
domains of pathological length. Proc.
Mangiarini L, Sathasivam K, Seller M,
Cozens B, Harper A, et al. 1996. Exon 1
of the HD gene with an expanded CAG
repeat is sufficient to cause a progressive neurological phenotype in transgenic
mice. Cell 87:493506
Reddy PH, Williams M, Charles V, Garrett L, Pike-Buchanan L, et al. 1998.
Behavioural abnormalities and selective
neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA.
Nat. Genet. 20:198202
Hodgson JG, Agopyan N, Gutekunst CA,
Leavitt BR, LePiane F, et al. 1999. A
YAC mouse model for Huntingtons disease with full-length mutant huntingtin,
cytoplasmic toxicity, and selective striatal
neurodegeneration. Neuron 23:18192
Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, et al. 1999. Intranuclear inclusions and neuritic aggregates
in transgenic mice expressing a mutant
N-terminal fragment of huntingtin. Hum.
Mol. Genet. 8:397407
Laforet GA, Sapp E, Chase K, McIntyre
C, Boyce FM, et al. 2001. Changes in
cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model
of Huntingtons disease. J. Neurosci. 21:
911223
Slow EJ, van Raamsdonk J, Rogers D,
Coleman SH, Graham RK, et al. 2003. Se-
105.
106.
107.
108.
109.
110.
111.
112.
289
lective striatal neuronal loss in a YAC128

mouse model of Huntington disease.
Hum. Mol. Genet. 12:155567
Senatorov VV, Charles V, Reddy PH,
Tagle DA, Chuang D-M. 2003. Overexpression and nuclear accumulation of
glyceraldehyde-3-phosphate dehydrogenase in a transgenic mouse model of
Huntingtons disease. Mol. Cell. Neurosci. 22:28597
Selkoe DJ. 2001. Alzheimers disease:
genes, proteins, and therapy. Physiol. Rev.
81:74166
Schulze H, Schuler A, Stuber D, Dobeli
H, Langer H, Huber G. 1993. Rat-brain
glyceraldehyde-3-phosphate dehydrogenase interacts with the recombinant cytoplasmic domain of Alzheimers betaamyloid precursor protein. J. Neurochem.
60:191522
Cupers P, Orlans I, Craessaerts K, Annaert W, De Strooper B. 2001. The amyloid precursor protein (APP)-cytoplasmic
fragment generated by gamma-secretase
is rapidly degraded but distributes partially in a nuclear fraction of neurones in
culture. J. Neurochem. 78:116878
Kimberly WT, Zheng JB, Guenette SY,
Selkoe DJ. 2001. The intracellular domain
of the beta-amyloid precursor protein is
stabilized by Fe65 and translocates to the
nucleus in a Notch-like manner. J. Biol.
Chem. 276:4028892
Sunaga K, Takahashi H, Chuang D-M,
Ishitani, R. 1995. Glyceraldehyde-3phosphate dehydrogenase is over-expressed during apoptotic death of neuronal
cultures and is recognized by a monoclonal antibody against amyloid plaques
from Alzheimers brain. Neurosci. Lett.
200:13336
Tamaoka A, Endoh R, Shoji S, Takahashi
H, Hirokawa K, et al. 1996. Antibodies
to amyloid beta protein (A beta) crossreact with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Neurobiol. Aging 17:40514
Kuwae T, Yamada M, Takahashi H,
7 Jan 2005
13:59
290

113.
114.
115.
116.
117.
118.
119.
120.
121.
AR
AR232-PA45-11.tex
CHUANG
HOUGH
AR232-PA45-11.sgm
LaTeX2e(2002/01/18)
P1: GCE
SENATOROV
Tsuchiya K, Tajima H, et al. 2002. Proapoptotic protein GAPDH: a possible site

of action of anti-dementia drugs. Annu.
Meet. Soc. Neurosci. Abstr. No. 884.4
Lubec G, Labudova O, Cairns N, Fountoulakis M. 1999. Increased glyceraldehyde 3-phosphate dehydrogenase levels
in the brain of patients with Downs syndrome. Neurosci. Lett. 260:14145
Tsuchiya K, Tajima H, Yamada M, Takahashi H, Kuwae T, et al. 2004. Disclosure
of a pro-apoptotic glyceraldehyde-3phosphate dehydrogenase promoter; antidementia drugs depress its activation in
apoptosis. Life Sci. 74:324558
Katsube N, Sunaga K, Chuang D-M, Ishitani R. 1996. ONO-1603, a potential antidementia drug, shows neuroprotective
effects and increases m3-muscarinic receptor mRNA levels in differentiating
cerebellar granule cells. Neurosci. Lett.
214:15154
Katsube N, Sunaga K, Aishita H, Chuang
D-M, Ishitani R. 1999. ONO-163, a potential antidementia drug, protects against
age-induced apoptosis and suppresses
over-expression of GAPDH in cultured
CNS neurons. J. Pharmacol. Exp. Ther.
288:613
Carlile GW, Chalmers-Redman RME,
Tatton NA, Pong A, Borden KE, Tatton
WG. 2000. Reduced apoptosis after nerve
growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3phosphate dehydrogenase to a dimmer.
Berry MD. 1999. An orally active agent
combining independent anti-apoptotic
and MAO-B-inhibiting activities. CNS
Drug Rev. 5:10524
Tatton NA. 2000. Increased caspase 3
and Bax immunoreactivity accompany
nuclear GAPDH translocation and neuronal apoptosis in Parkinsons disease.
Exp. Neurol. 166:2943
Lipton P. 1999. Ischemic cell death in
brain neurons. Physiol. Rev. 79:143168
Tanaka R, Mochizuki H, Suzuki A, Kat-
122.
123.
124.
125.
126.
127.
128.
sube N, Ishitani R, et al. 2002. Induction

of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in rat brain
after focal ischemia/reperfusion. J. Cereb.
Blood Flow Metab. 22:28088
Graven KK, Troxler RF, Kornfeld H,
Panchenko MV, Farber HW. 1994. Regulation of endothelial-cell glyceraldehyde3-phosphate dehydrogenase expression
by hypoxia. J. Biol. Chem. 269:2444653
Graven KK, Yu Q, Pan D, Roncarati
JS, Farber HW. 1999. Identification of
an oxygen responsive enhancer element
in the glyceraldehyde-3-phosphate dehydrogenase gene. Biochim. Biophys. Acta
Gene Struct. Expres. 1447:20818
Lu S, Gu X, Hoestje S, Epner DE. 2002.
Identification of an additional hypoxia responsive element in the glyceraldehyde-3phosphate dehydrogenase gene promoter.
Biochim. Biophys. Acta Gene Struct. Expres. 1574:15256
Yamaji R, Fujita K, Takahashi S, Yoneda
H, Nagao K, et al. 2003. Hypoxia
up-regulates glyceraldehyde-3-phosphate
dehydrogenase in mouse brain capillary endothelial cells: involvement of
Na+/Ca2+ exchanger. Biochim. Biophys.
Acta Mol. Cell Res. 1593:26976
Nakamura T, Hinagata J, Tanaka T, Imanishi T, Wada Y, et al. 2002. HSP90,
HSP70, and GAPDH directly interact
with the cytoplasmic domain of macrophage scavenger receptors. Biochem. Biophys. Res. Comm. 290:85864
Ren M, Senatorov VV, Chen RW, Chuang
D-M. 2003. Postinsult treatment with
lithium reduces brain damage and facilitates neurological recovery in a rat
ischemia/reperfusion model. Proc. Natl.
Ren M, Leng Y, Jeong MR, Leeds PR,
Chuang D-M. 2004. Valproic acid reduces
brain damage induced by transient focal
cerebral ischemia in rats: potential roles
of histone deacetylase inhibition and heat
shock protein induction. J. Neurochem.
89:135867
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Figure 1 Overexpression and nuclear accumulation of GAPDH in the brain of

transgenic (Tg) mice expressing huntingtin gene with 89 CAG repeats. (A)
Relatively weak GAPDH immunostaining in the caudate putamen region of a wildtype (WT) mouse. (B, C) An increase in GAPDH immunofluorescent expression in
selective populations in the caudate putamen of a Tg mouse, as compared with WT
age-matched control in (A). The boxed area in (B) is expanded in (C). Note the
nuclear accumulation of immunofluorescence in Tg mouse cells. (D, E) Double
immunostaining of GAPDH-reactive cells in red (D) with the neuronal marker
NeuN in green (E). (F, G) Immunofluorescent staining shows an increase in
GAPDH expression in GAPDH immunoreactivity in specific cell populations in the
neocortex of Tg mice (G), as compared with the control (F). Note the accumulation
of immunofluorescence in the nuclei of Tg mouse cells. (H, I) Double immunostaining of GAPDH-reactive cells (H) and for the neuronal marker NeuN (I).
Arrowheads indicate cells double-immunostained for GAPDH and NeuN. (J, K)
Representative images of subcellular distributions of GAPDH immunoreactivity at
the level of neocortical layer V in WT (J) and Tg (K) mice, respectively. Bottom
panels show the fluorescent intensity profiles of GAPDH-expressing neurons in the
top panels. Note that the highest fluorescent intensity occurs in the nuclear portion
of the intensity profile as delineated by arrows. Results are modified from
Senatorov et al. 2003 (105). Reprinted with permission of the publisher.
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Copyright

NON-MICHAELIS-MENTEN KINETICS IN
CYTOCHROME P450-CATALYZED REACTIONS
William M. Atkins
Department of Medicinal Chemistry, University of Washington, Seattle,
Washington 98195-7610; email: winky@u.washington.edu
Key Words allosterism, drug metabolism, enzyme kinetics, CYP

Abstract The cytochrome P450 monooxygenases (CYPs) are the dominant enzyme system responsible for xenobiotic detoxification and drug metabolism. Several
CYP isoforms exhibit non-Michaelis-Menten, or atypical, steady state kinetic patterns. The allosteric kinetics confound prediction of drug metabolism and drug-drug
interactions, and they challenge the theoretical paradigms of allosterism. Both homotropic and heterotropic ligand effects are now widely documented. It is becoming apparent that multiple ligands can simultaneously bind within the active sites of individual
CYPs, and the kinetic parameters change with ligand occupancy. In fact, the functional
effect of any specific ligand as an activator or inhibitor can be substrate dependent. Divergent approaches, including kinetic modeling and X-ray crystallography, are providing new information about how multiple ligand binding yields complex CYP kinetics.
OVERVIEW
The cytochrome P450 monooxygenases (CYPs) are ubiquitous heme-containing
enzymes that catalyze an immense range of chemical reactions in prokaryotes,
plants, and animals (1, 2). CYPs participate in the biosynthesis of hormones,
second messengers, and other natural products. CYPs also dominate xenobiotic
detoxification and human drug metabolism. As a result, CYPs are of primary
importance in the pharmaceutical industry (36). In fact, characterization of the
interactions between new drugs and human CYPs is now a routine component of
early drug development. An enigmatic behavioral characteristic of CYPs, which
has only recently been appreciated fully, is their tendency to exhibit atypical
steady-state kinetic patterns in vitro, and possibly in vivo. In fact, several excellent
recent reviews have focused on this atypical behavior, also referred to as allosterism, thus highlighting its perceived importance (711). This review explores some
recent observations, while minimizing duplication with the previous reviews, and
it considers mechanistic aspects of the atypical kinetics in the context of recently
determined X-ray structures.
From a historical perspective, it is interesting that nonhyperbolic CYP kinetics
were documented as early as the 1980s (1214), but this received little attention.
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Subsequently, Korzekwa et al. (15, 16) provided thoughtful accounts of the relationship between atypical kinetics observed with CYP3A4 and the possibility that
multiple ligands could occupy the active site simultaneously. Today, it is widely
accepted that several CYP isoforms, including 3A4, 1A2, 2E1, 2D6, and 2C9, can
exhibit nonartefactual atypical kinetics in vitro. Furthermore, it is highly likely that
the kinetic behavior is related in some cases to simultaneous binding of multiple
ligands to a single active site, as elaborated here. Also, it is notable that experimental evidence for multiple ligand binding to CYP101 (P450cam) was provided
by Sligar and coworkers as early as 1994, based on NMR approaches (17).
In contrast to the widespread acceptance of this behavior in vitro, examples
of in vivo kinetics that deviate from Michaelis-Menten kinetics are sparse. Examples include interactions between diclofenac and quinidine in monkeys (18),
carbamezepine and felbamate in humans (19), and a marginal effect between flurbiprofen and dapsone in humans (20). Although examples of in vivo allosteric CYP
interactions are limited, they are likely to become more widespread as awareness of
their possibility increases and with improved analytical methods. Regardless, the
apparent universality of allosteric effects across several CYP isoforms and many
drugs in vitro (2126) demands a mechanistic understanding that could dramatically enhance in vitro predictability of drug-drug interactions. Presumably, this
understanding would translate directly into increased predictive power in vivo.
The behavior of CYPs also is extremely important from an academic perspective
because it demands significant revision of the paradigms of traditional allosteric
enzymes. Nearly all allosteric proteins are multisubunit oligomers (2729). Moreover, allosteric behavior of normal enzymes can be rationalized within their biological niche as a mechanism for achieving metabolic control through highly
specific molecular recognition. In contrast, although CYPs may sample several
aggregation states (30, 31), they can exhibit non-Michaelis-Menten kinetics under conditions in which they are predominantly monomeric. Although CYP-CYP,
CYP-reductase, and CYP-Cyt b5 interactions may provide an additional mechanism by which allosteric effects occur, they are considered only briefly here.
Also, according to traditional paradigms, allostery requires specificity. However, as detoxification enzymes, CYPs do not utilize specific molecular recognition. Rather, they are extraordinarily substrate diverse. The resulting nonspecific
allosterism is also of academic interest because it deviates from well-understood
allosterism of substrate-specific enzymes. It is not clear what biological advantage,
if any, is gained from the allosterism of CYPs, wherein some toxic substrates are
metabolized more efficiently and others less efficiently in the presence of allosteric
effectors. Both the mechanism and the biological purpose of CYP allosterism are
challenging (32).
What Are Atypical Kinetics and Why Do They Matter?

At the simplest level, atypical has become synonymous with a wide range of situations wherein nonhyperbolic plots of velocity versus [S] are obtained. Common
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ATYPICAL KINETICS OF CYPs
293

types of CYP allosterism are summarized below. Throughout this review, the term
allosterism is used interchangeably with atypical kinetics because both require
formation of a ternary complex, [CYPSS] or [CYPSE], where S and E are
substrate and effector, respectively, and these complexes have kinetic properties
that differ from [CYPS].
The implication of nonhyperbolic kinetics is that the Michaelis-Menten steady
state model is insufficient to describe drug clearancc, CLint. The Michaelis-Menten
model describes the velocity of product formation, v, as
v=
Vmax K M
,
[S] + K M
where Vmax and KM have their usual meanings. When the Michaelis-Menten relationship does apply, the clearance of a drug may accurately be estimated, in
principle, from the Vmax/KM. This parameter, approaches the intrinsic drug clearance (Clint = v/[S]) or the slope of a hyperbolic Michaelis-Menten plot at low
[S]. Furthermore, the in vitro clearance is frequently used to estimate in vivo clearance, after appropriate scaling for the CYP capacity of the liver or other tissue.
Obviously, the accuracy of the in vivo prediction is limited by the accuracy of the
model used to extract metabolic velocities from the in vitro data (6, 9).
TYPES OF ALLOSTERIC KINETICS

Homotropic Effects
Allosteric effects may result from homotropic substrate interactions in which the
[substrate] versus velocity curve is nonhyperbolic, as summarized previously by
others (7, 9, 11, 16) and as schematized in Figure 1. Homotropic effects may yield
velocity versus [substrate] curves that are either sigmoidal (also called autoactivation), biphasic with continuously increasing velocity at high [substrate] (implying
a low-affinity second substrate site and referred to as biphasic), or concave downward with a decrease in velocity at high [substrate] after an initial hyperbolic
increase (substrate inhibition). Apparent biphasic kinetics with decreasing rate at
high [substrate] may be observed also with product inhibition, but this does not represent allosteric kinetics by any definition, because it does not require simultaneous
binding of multiple ligands. Without quantitative models of homotropic effects,
in vitro kinetics will be inaccurately parameterized and in vivo drug clearance may
be estimated incorrectly.
Heterotropic Effects
Alternatively, heterotropic effects occur when one drug alters the CYP interactions
with a second drug, either activating or inhibiting the rate of product formation
(33, 34). Here, the drug acting as substrate may yield classic hyperbolic velocity
versus [substrate] curves, but the second drug changes the parameters Vmax or
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Figure 1 Velocity versus [substrate] plots depicting possible kinetic profiles with
homotropic effects. Top left: hyperbolic kinetics with no allosterism. Bottom left:
sigmoidal kinetics resulting from homotropic activation. Top right: biphasic kinetics
resulting from a low-affinity second ligand site. Bottom right: substrate inhibition,
wherein binding of the second substrate decreases Vmax. In each case, the inset depicts
an Eadie-Hoffstee plot (V versus V/[S]) corresponding to the velocity curves.
KM, or it induces nonhyperbolic behavior. Alternatively, if the substrate alone

exhibits nonhyperbolic kinetics, the heterotropic effector may restore hyperbolic
kinetics or maintain them but change the shape of the velocity versus [S] curve or
shift it along the [S] axis. A further case, which can occur through heterotropic
interactions when there is either hyperbolic or atypical kinetics, is partial inhibition,
wherein an effector bound at the same time as substrate may partially inhibit the
enzymatic reactions. Partial inhibition may also be observed for the homotropic
substrate inhibition mentioned above. Both the heterotropic and homotropic partial
inhibition cases are incompatible with simple competitive inhibition and require
allosteric interactions of some type.
Substrate and Effector Dependence

A particularly interesting aspect of CYP allosterism is the context dependence
of heterotropic effects. Any individual compound may activate CYP-dependent
metabolism of one drug, yet inhibit or have no effect on the metabolism of a
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second drug cleared by the same CYP isoform. Equally important, a single effector molecule may change from an activator at low concentration to an inhibitor
at higher concentration. Thus, the behavior of any effector compound depends
on the substrate that is being metabolized, as well as on the concentrations of
effector and substrate (35, 36). For example, testosterone inhibits with different
apparent potencies the metabolism of terfenadine and midazolam by CYP3A4.
In contrast, testosterone does not inhibit metabolism of nifedipine, but terfandine
does. Moreover, testosterone itself is a substrate for CYP3A4, and its metabolism is
partially inhibited by nifedipine (3537). Similar nonreciprocal effects have been
observed with CYP3A4-dependent interactions between -napthoflavone (-NF)
and aflatoxin B1. The -NF activates metabolism of the aflatoxin, but the latter
has no effect on the metabolism of aflatoxin B1 (38). Houston and coworkers have
initiated the categorization of various CYP3A4 substrates into subgroups based
on kinetic traits and heterotropic effects in which they participate (37). Clearly,
the behavior of any substrate or inhibitor depends on what other compounds are
present, and this is a major challenge for describing CYP allosterism. Moreover,
the heterotropic effects of any ligand pair are CYP isoform dependent. For example, the highly homologous CYPs 3A4 and 3A5 exhibit different heterotropic
interactions for several ligand pairs (39).
At least two molecular mechanisms may contribute to context-dependent ligand
effects. The first is ligand-dependent conformational change, wherein the enzyme
is sufficiently flexible that each combination of ligands induces a different enzyme conformation with different kinetic properties. This contrasts the case with
substrate-specific enzymes in which only a few specific conformations are coupled
to a few specific ligands (27, 28). If a wide range of ligand-dependent conformational space is available to the enzyme, this will promote context-dependent ligand
effects (40).
Based on flash photolysis and CO recombination experiments, it was proposed
that slowly equilibrating conformations of a single CYP isoform could differentially interact with ligands (4143). This possibility has been reconsidered based on
studies using hydrostatic pressure (44). Such persistent conformations could cause
allosteric kinetics, even in the absence of multiple ligand binding, just as mixtures
of isoforms can yield non-Michaeles-Menten kinetics. In contrast, ligand-induced
conformational changes, in the absence of nonequilibrating conformational states,
cannot cause allosteric kinetics. In the absence of persistent conformations with different properties, ligand-dependent conformational change is neither a necessary
nor sufficient condition for allosteric kinetics. Multiple ligand binding, however, is
a necessary but not a sufficient condition for allosterism. Conformational change
provides one mechanism by which multiple ligand binding can yield complex
kinetics (40).
Conformational changes induced by nonactive site ligands may also contribute.
For example, Schrag & Wienkers (45) found that addition of Mg2+ to CYP3A4
incubations with the substrate pyrene resulted in the conversion from positive
homotropic kinetics to hyperbolic patterns, and this correlated with a change in
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reioselectivity of heme adduction by phenyldiazene. Similarly, carbonate anion,

but not other common buffer salts, altered the O-dealkylation activity of CYP2D6,
but not its N-dealkylation activity (46). Possibly, different oxidative intermediates, either iron-peroxy or iron-oxo [FeO]3+, are differentially populated owing
to subtle conformational changes induced by carbonate. CYP3A4 is particularly
sensitive to nonactive site ligands and buffer conditions (47, 48). As noted above,
cytochrome b5 (Cyt b5; 49), possibly apo-Cyt b5 (5053), or possibly the CYP
reductase (54, 55) may also contribute to the conformational landscape of CYPs,
and thus provide additional mechanisms of allosterism. In fact, each of these effects may be ligand- and CYP isoform-dependent, as well (56). For example, Cyt
b5-CYP4B7 interactions are modulated differently by various CYP ligands.
A second mechanism for context-dependent effector behavior is direct ligandligand interactions. Few proteins allow multiple ligands to bind in a single active
site that promotes direct hydrophobic bonds, electrostatic effects, or hydrogen
bonds between the ligands. As elaborated further below, X-ray crystal structures
of CYPeryF clearly support this possibility for CYPs (57). Also, a recent computational docking study based on a homology model for CYP3A4 suggests the
possibility of hydrogen bonds between the amide groups of two carbamazepine
molecules simultaneously bound (58). When one molecule is bound, it may directly contribute to the binding site for a second ligand, even if no significant
protein conformational change takes place. Each ligand can change the active
site constraints directly, wherein the second ligand can exploit handles presented
by the first ligand. If ligand-ligand interactions are stronger than ligand-protein
interactions, they may control orientation of the complex within the active site.
Evidence for strong ligand-ligand interactions is limited, but one example is the
aromatic stacking of pyrenes simultaneously bound to CYP3A4 (59). The important point is that direct ligand-ligand contacts might provide a mechanism for
context-dependent allosteric effects.
THE PROGRESSION OF KINETIC MODELS

To improve in vitro-in vivo correlations, several steady-state kinetic models have
been developed that account for homotropic interactions and the possibilities that
(a) identical substrate molecules may have different affinities for free CYP versus
[CYPS], thus yielding two KS values, and (b) the [CYPS] and [CYPSS]
complexes may yield product at different rates, thus yielding different Vmax values
for each complex. Similarly, for heterotropic interactions the effector may have
different affinities for CYP versus [CYPS], thus yielding two KI or KA values. It
is beyond the scope of this review to summarize all of the possible models that may
describe CYP atypical kinetics, but it is instructive to consider a few as a means
to highlight the strengths and weaknesses of kinetic modeling in general. For a
more comprehensive survey of multisite kinetic models, the reader is referred to
other recent reviews (710) or to Segels classic book (60), which has become a
standard reference for those doing CYP research.
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Scheme 1 depicts the simplest generic model for homotropic effects, which has
been used by numerous investigators. Here S is substrate; P is product; Ks is the
affinity of the CYP for substrate; kcat is the rate of formation of product from the
[CYPS] complex; and Vmax is defined as 2kcat/[E]t, where [E]t is the total enzyme
concentration. The equation describing the fraction of maximal velocity at any
[S] is
Scheme 1
In this model, the substrate can bind in either of two sites, as indicated by
[SCYP] versus [CYPS], and these complexes have identical dissociation constants for substrate, KS, and identical kcat values for product formation. The widespread use of the two-site model in Scheme 1 in the CYP literature, or variations
of it, reflects the popular belief that at low occupancy, the bound ligand is localized in a discrete binding site, rather than sampling all parts of the active site,
i.e., that [CYPS] and [SCYP] are two different molecular species that can only
interconvert via substrate dissociation and rebinding, but neither is preferred thermodynamically. Regardless, binding of the second substrate leads to a complex
[CYPSS] with different kinetic properties, KS and kcat. Here, is the effect
that the first substrate has on the KS for the second substrate, and is the effect
that the presence of the first substrate has on the kcat for the second. Thus when
either < 1 or > 1, positive homotropic cooperativity may be apparent and
velocity curves will be sigmoidal. Alternatively, if > 1 the curves may appear
biphasic, and if < 1 substrate inhibition will be evident. The detailed shape of
the corresponding velocity versus [S] plot will be determined by KS, kcat, , and
. Although this model has been extremely useful for conceptualizing homotropic
allosteric kinetics for CYPs, it is inherently oversimplified because of the kinetic
equivalence of [SCYP] and [CYPS], which form with equal apparent affinities
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and generate product at equivalent velocities. Such kinetic symmetry is useful for
reducing the complexity of the system, but in the absence of any structural symmetry of the CYP enzymes, it is likely to be an inaccurate depiction of what really
occurs at the molecular level. This model is more suitable for normal allosteric
enzymes with multiple copies of identical active sites.
A more likely scenario, possibly, is that multiple [CYPS] complexes are
formed, with multiple orientations of S in rapid equilibrium, [SCYP] and [CYPS],
which form with different affinities and different kcat values associated with them,
as in Scheme 2. In this case, the system behaves like a mixture of enzyme-substrate
complexes, with the fractional contribution of [SCYP] versus [CYPS] determined by Ks1/Ks2, and with the reaction velocity equation shown. Here Vmax1 =
kcat[ET], Vmax2 = kcat[ET], and [ET] is the total enzyme concentration, and , ,
and are scaling factors that modulate the KS1, KS2, and kcat , respectively. The
parameters Vmax1 and Vmax2 are virtual parameters that represent the rate of product
formation if all of the enzyme could be forced into the [CYPS] or the [SCYP]
states; however, this cannot actually occur. Note that the number of fitting parameters has increased to eight (Ks1, Ks2, kcat, kcat , , , , ). This model was used
recently to explore the metabolism of verapamil by CYP3A4 (61). It was found
that formation of several metabolites could be described by Scheme 2, wherein
negative cooperativity was associated with and values less than 1 and and
values greater than 1.
Scheme 2
Comparison of Schemes 1 and 2 reveals the compromise that must accompany a choice between models. The model in Scheme 1 suffers from potentially
unrealistic features, such as the existence on a single unsymmetrical CYP enzyme
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with physically distinguishable, kinetically indistinguishable, binding sites. The

more realistic and general model in Scheme 2 suffers from the possibility of overparameterization if a sufficient number of data points are not included. With two
experimental observables (velocity and [S]) and ten fitting parameters, the recovered parameters for the best fit may not represent a unique solution. There are
likely to be other combinations of parameters that yield a fit that is very nearly as
good based on standard statistical criteria. Although the curve-fitting procedures
yield standard errors or standard deviations for each individual parameter, they
do not indicate how the overall goodness of fit for the model varies with each
parameter. To date, no kinetic models have included a rigorous analysis of the
uniqueness of the best fit or the sensitivity of the fit to parameter changes as is
routinely performed with complex fluorescence decay data (62).
For the case of homotropic effects, Shou and coworkers have used a variation of
these schemes to provide a detailed survey of several examples of substrate inhibition, including CYP1A2-catalyzed O-deethylation of ethoxyresorufin, CYP2C9dependent hydroxylation of celecoxib, O-demethylation of dextromethorphan by
CYP2D6, and other CYP-drug combinations (63). This analysis provides an extended description of substrate inhibition, which is observed with many combinations of CYPs and substrates.
Heterotropic effects are significantly more problematic to model owing to the
additional parameters required to describe the effector interactions with the enzyme
in multiple states, in addition to the substrate-enzyme interactions. For example,
the simplest general heterotropic model that allows for multiple binding of both
substrate and effector is shown in Scheme 3, along with the velocity equation.
Scheme 3
The model in Scheme 3 accounts for multiple substrate binding with homotropic
effects, heterotropic binding, and multiple effector (E) binding with homotropic
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effects. Differential affinity of both substrate and effector is caused by the presence of another effector or inhibitor, and differential effects on kcat are allowed.
This model, and variations, have been applied to examine context-dependent heterotropic effects (7, 36, 37, 64, 65).
The steady-state kinetics provide a powerful tool for conceptualizing the possible mechanisms responsible for the variety of atypical kinetics observed, and
they accurately predict metabolism rates. The examples described above demonstrate the necessary compromise between a level of complexity adequate to describe the experimental data and the need to use many data points to avoid
overparameterization.
How Many Ligands Bind?

Several investigators have suggested that more than two ligands can bind simultaneously with the active site of CYP3A4 (23, 25, 37, 6668). This is based on
inhibition studies and site-directed mutagenesis approaches. For example, it was
found that a peptide inhibitor of CYP3A4 yields differential KI values with respect to different products from midazolam, which is presumed to bind at two
different subsites. The authors propose two separate binding sites for midazolam, a site for testosterone that overlaps one of the midazolam sites and a site
for -NF in the active site of CYP3A4 (68). Also with CYP3A4, kinetic modeling suggests the presence of three sites wherein diazepam and testosterone
each bind to specific sites and both can bind to the third site (66, 69, 70). Perhaps the strongest support for a third binding site on a single CYP molecule
comes from inhibitor studies in which plots of fractional inhibition versus [S]
change slope with changing [I]. That is, with increasing inhibitor concentration the
slopes of relative velocity versus substrate, for example, become greater (37). The
change in slope indicates a cooperative interaction between inhibitor molecules
owing to simultaneous binding of inhibitors. However, the inhibition is not purely
competitive, implying that two inhibitors and one substrate can simultaneously
bind, [CYPSII]. Similarly, a scenario diagnostic for two S molecules bound
simultaneously with an inhibitor is the persistence of sigmoidal kinetics (positive homotropic) even at saturating concentrations of inhibitor. If the inhibitor
shifts the curve to higher [S] without converting it to a hyperbolic curve, then
a [CYPSSI] complex is implied. It will be particularly interesting to search
for direct evidence of three ligands simultaneously binding within a CYP active
site.
STRUCTURAL AND MECHANISTIC ASPECTS

Although the kinetic models can accurately predict rates of product formation, they
do not address directly specific mechanistic aspects of multiple ligand binding. This
is because KM, kcat, , , etc., are nearly impossible to interpret in molecular terms
given the complexity of the CYP reaction cycle. For example, there may be no
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cooperativity or negative cooperativity at the level of ligand binding, per se, and
positive cooperitivity on the ligand-dependent spin state shift (69, 70). It is unclear
how these results relate mechanistically to changes in the Ks parameter used in
kinetic models, as described above.
Several conceptual models that are based on multiple ligand binding to a single
active site remain viable. It is possible that there are discrete and static subsites
within the large active site, and each subsite has its own personality. Each subsite may have a characteristic affinity for each ligand and hold it in a preferred
orientation, which is static on the timescale of oxidative turnover (66, 68, 71,
72). In this extreme case, multiple ligands bind sequentially to the highest affinity available subsite and then to the lowest affinity site. From their respective
binding sites, ligands may alter the metabolism of other ligands by inducing conformational changes, causing minor shifts in the distances between oxidizable
sites on the drug and the heme iron-oxo species, or by altering relative uncoupling rates to nonproductive formation of superoxide. Support for discrete static
subsites has come, partly, from mutagenesis studies as championed by Halpert
and coworkers (6972). For example, midazolam appears to be an example of
this type of ligand wherein distinct subsites within the active site are responsible for the formation of the 1 -hydroxy- versus 4-hydroxy-midazolam products.
The results with midazolam support the unlikely suitability of Scheme 1 as a
model; the different binding subsites for midazolam, if they exist, are proposed
to have different Vmaxs and substrate affinities, and to even generate different
products.
Alternatively, the large active site may be fluid and multiple bound ligands may
sample several subsites within the large active site, either dynamically or through
a static heterogeneity. Evidence for a fluid active site has come mainly from kinetic deuterium isotope effects. Trager and coworkers have provided numerous
examples, and appropriate theory, to understand CYP substrates as moving within
the active site and presenting several points of oxidation on a single molecule to
the [FeO]3+ intermediate (7375). In effect, they have varied distances between
deuterium- and hydrogen-bearing benzylic methyl groups on ring systems of increasing size. The substrate size could be correlated to the extent of masking
of the isotope effect (kH/kD), as expected if smaller substrates rapidly reorient
within the active site. In principle, this approach could be used to determine the
effect of multiple ligand binding on substrate dynamics. Both homotropic and
heterotropic effects should modulate the magnitude of the observed isotope effects if they change the effective size of the active site. In fact, deuterium isotope
effects have already been used to observe multiple ligand binding to CYP BM3
(CYP102), wherein deuteration of laurate caused a change in the regioselectivity
of hydroxylation of palmitate (76). This could only occur if both ligands bound
simultaneously. As with the kinetic modeling, the mechanism of multiple ligand
binding within CYP active sites may be context-dependent. Some ligands may
dynamically or statically sample several parts of the active site, whereas others
may occupy well-defined subsites.
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An additional mechanistic complexity arises from the possibility that uncoupling may occur. Many [CYPsubstrate] complexes generate superoxide anion,
hydrogen peroxide, or water as the reduction products of O2 at the expense of
substrate oxidation (7779). As competing processes, these pathways decrease
the apparent kcat for product (oxidized substrate) formation. Hutzler and coworkers recently demonstrated that the branching ratios between substrate oxidation
and uncoupling could be altered by the addition of an effector (80). Specifically,
dapsone caused a decrease in the uncoupling of [CYP2C9flurbiporfen], thus explaining the activation by dapsone of flurbiprofen metabolism. Allosteric effects
on coupling are likely to be common.
Recent elegant strategies, and nearly heroic efforts, have led to the successful
solubilization of several mammalian CYP isoforms by engineering the membrane
binding regions (8185). Specifically, the N-terminal membrane anchor has been
partially truncated, and the F-G region, thought to be a peripheral membranebinding patch, has been mutated or chimerized in several ways. The resulting soluble proteins have been crystallized and they have afforded X-ray structural models.
The structures provide an obvious tool to look for mechanistic clues concerning
the atypical kinetic behavior described above, so they are briefly discussed here.
First, it is useful to highlight an important relevant aspect of the crystal structure of the bacterial CYPeryF as it relates to atypical kinetics. Cupp-Vickery
and coworkers provided a crystal structure of CYPeryF complexed with either
androstenedione or 9-amino-phenanthrene (57). For both, clear electron density
revealed the simultaneous presence of two molecules in the active site cavity proximal to the heme. Interestingly, for both complexes, direct ligand-ligand interactions
were observed, suggesting a possible contribution to positive homotropic cooperativity as noted above. Also, for both cases, only one of the bound ligands appears to
be in a location that would allow metabolism (of course, the 9-aminophenanthrene
forms a 6-coordiante nitrogen-liganded complex that is not expected to be metabolized, but if the exocyclic amine were not present, only one of the phenanthrene
rings would be sterically accessible to the heme iron-oxo complex). In short, the
structures demonstrated for the first time the possibility that two ligands could
simultaneously occupy a CYP active site, although only one could be a target for
oxidation with reorientation on the timescale of turnover.
The first X-ray structure of a mammalian CYP was that of rabbit CYP2C5 (86).
Perhaps the single most important conclusion resulting from this structure was
that mammalian CYPs are structural homologs of the bacterial CYPs, for which a
wealth of structural data already exists (8791). This was not a surprising conclusion, but its experimental validation was comforting and important. Subsequent
structures of the engineered rabbit CYP2C5 have allowed for a comparison of
ligand-free enzyme with complexes of diclofenac (92) or a benzenesulfonamide
(DPZ) derivative (93). This comparison reveals the likely ligand-dependent conformations of the protein in the B -C loop and the F-G loop, and it has prompted
the use of induced fit models for discussing CYP dynamics. These results extend
to the mammalian CYPs the notion that ligand binding alters the conformational
dynamics, particularly in these regions, as expected from the bacterial structures.
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In addition, the complex with DPZ suggests the possibility that the substrate binds
in two different orientations, with each providing electron density in partially occupied complexes. Interestingly, neither orientation, including the one with substrate
several angstroms from the heme iron and expected to yield less product, revealed
water bound to the iron. Thus, the crystallographic models are at odds with solution
spectroscopy, wherein addition of diclofenac or DPZ to 2C5 does not induce a high
spin transition (92, 93). Crystallographic evidence for multiple binding orientations
of a single substrate has also been provided with a [CYP101 nicotine complex]
(94). Here the major substrate orientation is nonproductive, with a substrate-heme
coordinate bond. Upon reduction and stabilization with CO, the nicotine orientation changes to a productive one, consistent with the metabolism of nicotine. This
clearly demonstrates the complexity of simple ligand binding with CYPs.
A structure of human CYP2C9 was recently solved, and reveals a striking behavior that is particularly relevant to CYP allosterism (95). The [CYP2C9warfarin]
complex positions warfarin in a corner of the active site, far from the heme iron, and
in an orientation inconsistent with the experimentally established regiospecificity
of warfarin hydroxylation (96). Based on this complex, the authors performed
docking experiments to demonstrate that there is ample room within the active
site for two ligands, suggesting that productive and nonproductive binding modes
may be available for any substrate, and that the relative population of these modes
will change with single occupancy versus multiple occupancy, i.e., [CYPS] versus [CYPSS]. For example, it is tempting to speculate that the first ligand can
merely take up space, without being a good target for oxidation, as suggested by
the crystal structure. Williams et al. note (see 95 for supporting information) that
in their attempt to obtain a ligand-free structure they observed undefined electron
density in the active site directly adjacent to the heme iron. Although they were
unable to identify the species yielding this density, it demonstrates that this part
of the active site is accessible to ligands, as required for metabolism. This amplifies
the possible preference of warfarin to not bind near the heme iron. In this case,
the bound warfarin may only occasionally sample portions of the active site closer
to the heme iron. However, at higher occupancy [CYPSS], the second ligand is
forced into more productive binding modes, thus providing a structural model for
positive homotropic or positive heterotropic effects. However, it should be emphasized that warfarin does not demonstrate atypical kinetics when metabolized by
CYP2C9, so this intuitive model is either incorrect or oversimplified.
A structure of CYP2B4 provides evidence for the possible contribution of conformational dynamics in atypical kinetics (97). Owing to structural rearrangements
in the B region and the F-G loops, an open conformation is captured in the crystal
state and stabilized by dimerization with a second CYP2B4. In fact, the cleft is
sufficiently pronounced to allow heme ligation by His-226 of the other CYP2B4
molecule of the dimer. Importantly, evidence for this dimer existing in solution
as well is presented. Comparison with the CYP2C5 structures suggests a range
of conformations in the B -C and F-G regions, including a significantly altered
conformation with a very large solvent-exposed crevice above the heme. Speculatively, the two structures, CYP2B4 and CYP2C5, may provide benchmarks for
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the range of conformations accessible to any single isoform and underscore the
extensive flexibility of the protein in these regions.
No structure of mammalian CYPs has revealed two ligands simultaneously
bound at the active site, so these structures have not provided any direct clues
about the mechanisms of atypical kinetics. In fact, a recent structure of human
2C8 reveals a nonsubstrate palmitic acid binding site that is peripheral to the active site, which could modulate catalytic properties (98). The apparent fatty acid
binding site includes determinants within the F , G , and G helices, which are
contiguous with ligand-sensitive regions of other isoforms, and this site communicates with substrate binding regions. At one level, this may be taken as evidence
for a true allosteric binding site remote from the heme iron and the active site
per se. However, without significant rearrangement, it is not obvious that this
site could accommodate hydrophobic drugs, and it is unlikely to be responsible
for the heterotropic effects discussed above. Rather, it supports the importance
of nonsubstrate ligand-dependent conformational effects (4754). Most recently,
crystal structures of CYP3A4 have been solved, and they further complicate the
existing paradigms (99, 100). Specifically, a structure of CYP3A4 complexed with
progesterone indicates that this ligand also binds at a site remote from the active
site, which suggests a separate allosteric site (99). Interestingly, with either progesterone at this remote site or with metyrapone coordinated to the heme iron,
no dramatic ligand-induced conformational changes are evident, compared to the
ligand-free CYP3A4 (99). However, the dimensions of the active site cavity are
significantly greater for CYP3A4 than the 2C isoforms in the immediate vicinity of
the heme (100). Thus, the possibility of mulitple ligand binding within the active
site remains. The available structures have not proven the central assumption of
current models for mammalian CYP allosterism: multiple ligand binding within a
single active site. However, collectively the available structures provide fundamentally important insights. For example, the presence of warfarin in a nonproductive
binding mode that limits the space available for a second ligand on CYP2C9, if
it binds, clearly demonstrates that each ligand can present new surfaces and handles to a second ligand, and direct ligand-ligand interactions can contribute, as
suggested for pyrene binding to CYP3A4 (59).
CONCLUSIONS
Atypical steady-state kinetics are now commonly observed among CYPs directly
involved in xenobiotic and drug metabolism for a wide range of drug structures.
In the past few years, the notion that multiple ligands bind within a single active
site of mammalian CYPs has evolved from an interesting speculation to a likely
possibility for many CYP-drug combinations. Of the experimental approaches used
to understand complex CYP kinetics, including kinetic modeling, crystallography,
and spectroscopic approaches, none alone are likely to reveal the mechanism of
CYP allosterism. Rather, there are likely to be multiple mechanisms spanning
different combinations of CYP isoform, substrate, and effector. An understanding
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of both ligand and protein dynamics will be necessary to fully understand CYP
kinetics. The combination of these approaches may be required to learn any general
rules of CYP allosterism, if they exist.

LITERATURE CITED
1. Guengerich FP. 2002. Cytochrome P450
enzymes in the generation of commercial
products. Nat. Rev. Drug Discov. 1:359
66
2. Coon MJ, Ding XX, Pernecky SJ, Vaz
AD. 1992. Cytochrome P450: progress
and predictions. FASEB J. 6:66973
3. Wienkers LC. 2001. Problems associated
with in vitro assessment of drug inhibition
of CYP3A4 and other P-450 enzymes and
its impact on drug discovery. J. Pharmacol. Toxicol. Methods 45(1):7984
4. Szklarz GD, Halpert JR. 1998. Molecular basis of P450 inhibition and activation: implications for drug development
and drug therapy. Drug Metab. Dispos.
26(12):117984
5. MacCoss M, Baillie TA. 2004. Organic
chemistry in drug discovery. Science
303(5665):181013
6. Venkatakrishnan K, Von Moltke LL,
Greenblatt DJ. 2001. Human drug
metabolism and the cytochromes P450:
application and relevance of in vitro models. J. Clin. Pharmacol. 41(11):114979
7. Shou M. 2002. Kinetic analysis for multiple substrate interaction at the active site
of cytochrome P450. Methods Enzymol.
357:26176
8. Shou M, Lin Y, Lu P, Tang C, Mei
Q, et al. 2001 Enzyme kinetics of cytochrome P450-mediated reactions. Curr.
Drug Metab. 2:1736
9. Houston JB, Kenworthy KE. 2000.
In vitro-in vivo scaling of CYP kinetic
data not consistent with the classical
Michaelis-Menten model. Drug Metab.
Dispos. 28:24654
10. Hlavica P, Lewis DF. 2001. Allosteric phenomena in cytochrome P450-catalyzed

monooxygenations. Eur. J. Biochem. 268:
481732
11. Tracy TS. 2003 Atypical enzyme kinetics: their effect on in vitro-in vivo
pharmacokinetic predictions and drug interactions. Curr. Drug Metab. 4:341
46
12. Lasker JM, Huang M-T, Conney AH.
1982. In vivo activation of zoxazolamine metabolism by flavone. Science
216:141921
13. Huang MT, Chang RL, Fortner JG,
Conney AH. 1981. Studies on the
mechanism of activation of microsomal
benzo[a]pyrene hydroxylation by flavonoids. J. Biol. Chem. 256:682936
14. Schwab GE, Raucy JL, Johnson EF. 1988.
Modulation of rabbit and human hepatic cytochrome P-450-catalyzed steroid
hydroxylations by alpha-naphthoflavone.
15. Shou M, Grogan J, Mencewicz JA, Krausz
KW, Gonzalez FJ, et al. 1994. Activation
of CYP3A4: evidence for the simultaneous binding of two substrates in a cytochrome P450 active site. Biochemistry
33:645055
16. Korzekwa KR, Krishnamachary N, Shou
M, Ogai A, Parise RA, et al. 1998. Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence
that multiple substrates can simultaneously bind to cytochrome P450 active
sites. Biochemistry 37:413747
17. Banci L, Bertini I, Marconi S, Pierattelli
R, Sligar SG. 1994. Cytochrome P450 and
4 Dec 2004
13:28
306
18.

19.
20.
21.
22.
23.
24.
25.
26.
AR
AR232-PA45-12.tex
AR232-PA45-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
ATKINS
aromatic bases: a 1H NMR study. J. Am.
Chem. Soc. 116(11):486673
Tang W, Stearns RA, Kwei GY, Iliff SA,
Miller RR, et al. 1999. Interaction of diclofenac and quinidine in monkeys: stimulation of diclofenac metabolism. J. Pharmacol. Exp. Ther. 291:106874
Egnell AC, Houston B, Boyer S. 2003.
In vivo CYP3A4 heteroactivation is a possible mechanism for the drug interaction
between felbamate and carbamazepine. J.
Hutzler JM, Frye RF, Korzekwa KR,
Branch RA, Huang SM, Tracy TS. 2001.
Minimal in vivo activation of CYP2C9mediated flurbiprofen metabolism by dapsone. Eur. J. Pharm. Sci. 14:4757
Ekins S, Ring BJ, Binkley SN, Hall SD,
Wrighton SA. 1998. Autoactivation and
activation of the cytochrome P450s. Int.
J. Clin. Pharmacol. Ther. 36:64251
Tracy TS, Hutzler JN, Haining RL, Rettie AE, Hummel MA, Dickmann LJ.
2002. Polymorphic variants (CYP2C9 3
and CYP2C9 5) and the F114L active site
mutation of CYP2C9: effect on atypical
kinetic metabolism profiles. Drug Metab.
Dispos. 30:38590
He YA, Roussel F, Halpert JR. 2003.
Analysis of homotropic and heterotropic
cooperativity of diazepam oxidation by
CYP3A4 using site-directed mutagenesis and kinetic modeling. Arch. Biochem.
Biophys. 409:92101
Hutzler JM, Hauer MJ, Tracy TA. 2001.
Dapsone activation of CYP2C9-mediated
metabolism: evidence for activation of
multiple substrates and a two-site model.
Khan KK, He YA, Domanski TL, Halpert
JR. 2002. Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding
sites and of the metabolic pathway that
leads to enzyme inactivation. Mol. Pharmacol. 61:495506
Ngui JS, Chen Q, Shou M, Wang RW,
Stearns RA, et al. 2001. In vitro stimu-
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
lation of warfarin metabolism by quinidine: increases in the formation of 4 - and

10-hydroxywarfarin. Drug Metab. Dispos. 29:87786
Edelstein SJ, Changeux JP. 1998. Allosteric transitions of the acetylcholine
receptor. See Ref. 99, pp. 12184
Ackers GK. 1998. Deciphering the molecular code of hemoglobin allostery. See
Ref. 99, pp. 185253
Wyman J, Gill SJ. 1990. Allosteric systems. In Binding and Linkage: Functional
Chemistry of Biological Macromolecules,
pp. 12363. Mill Valley, CA: Univ. Sci.
Books
Szczesna-Skorupa E, Mallah B, Kemper
B. 2003. Fluorescence resonance energy
transfer analysis of cytochromes P450
2C2 and 2E1 molecular interactions in
living cells. J. Biol. Chem. 278:31269
76
Myasoedova KN, Tsuprun VL. 1993.
Cytochrome P-450: hexameric structure
of the purified LM4 form. FEBS Lett.
325:25154
Atkins WM, Lu WD, Cook DL. 2002.
Is there a toxicological advantage for
non-hyperbolic kinetics in cytochrome
P450 catalysis? Functional allostery from
distributive catalysis. J. Biol. Chem.
277(36):3325866
Galetin A, Clarke SE, Houston JB. 2002.
Quinidine and haloperidol as modifiers of
CYP3A4 activity: multisite kinetic model
approach. Drug Metab. Dispos. 30:1512
22
Tang W, Stearns RA. 2001. Heterotropic
cooperativity of cytochrome P450 3A4
and potential drug-drug interactions.
Curr. Drug Metab. 2(2):18598
Wang RW, Newton DJ, Scheri TD, Lu
AY. 1997. Human cytochrome P450
3A4-catalyzed testosterone 6 betahydroxylation and erythromycin Ndemethylation. Competition during catalysis. Drug Metab. Dispos. 25(4):5027
Wang RW, Newton DJ, Liu N, Atkins
WM, Lu AY. 2000. Human cytochrome
4 Dec 2004
13:28
AR
AR232-PA45-12.tex
AR232-PA45-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
37.

38.
39.
40.
41.
42.
43.
44.
P-450 3A4: in vitro drug-drug interaction

patterns are substrate-dependent. Drug
Galetin A, Clarke SE, Houston JB. 2003.
Multisite kinetic analysis of interactions
between prototypical CYP3A4 substrates:
midazolam, testosterone, and nifedipine.
Drug. Metab. Dispos. 31(9):110816
Ueng YF, Kuwabara T, Chun YJ, Guengerich FP. 1997. Cooperativity in oxidations catalyzed by cytochrome P450 3A4.
Patki KC, Von Moltke LL, Greenblatt DJ.
2003. In vitro metabolism of midazolam,
triazolam, nifedipine, and testosterone by
human liver microsomes and recombinant
cytochromes p450: role of cyp3a4 and
cyp3a5. Drug Metab. Dispos. 31(7):938
44
Atkins WM, Wang RW, Lu AY. 2001.
Allosteric behavior in cytochrome p450dependent in vitro drug-drug interactions:
a prospective based on conformational
dynamics. Chem. Res. Toxicol. 14:338
47
Koley AP, Buters JTM, Robinson RC,
Markowitz A, Friedman FK. 1997.
Differential mechanisms of cytochrome
P450 inhibition and activation by
alpha-naphthoflavone J. Biol. Chem. 272:
314952
Koley AP, Robinson RC, Markowitz A,
Friedman FK. 1997. Drug-drug interactions: effect of quinidine on nifedipine
binding to human cytochrome P450 3A4.
Koley AP, Buters JT, Robinson RC,
Markowitz A, Friedman FK. 1995. CO
binding kinetics of human cytochrome
P450 3A4. Specific interaction of substrates with kinetically distinguishable
conformers. J. Biol Chem. 270(10):5014
18
Davydov DR, Halpert JR, Renaud JP, Hui
Bon Hoa G. 2003. Conformational heterogeneity of cytochrome P450 3A4 revealed
by high pressure spectroscopy. Biochem.
307
45. Schrag ML, Wienkers LC. 2000. Topological alteration of the CYP3A4 active
site by the divalent cation Mg(2+). Drug
Metab. Dispos. 28(10):1198201
46. Hutzler JM, Powers FJ, Wynalda MA,
Wienkers LC. 2003. Effect of carbonate
anion on cytochrome P450 2D6-mediated
metabolism in vitro: the potential role
of multiple oxygenating species. Arch.
Biochem. Biophys. 417(2):16575
47. Maenpaa JVA, Hall SD, Ring BJ, Strom
SC, Wrighton SA. 1998. Human cytochrome P450 3A (CYP3A) mediated
midazolam metabolism: the effect of assay conditions and regioselective stimulation by -naphthoflavone, terfenadine and
testosterone. Pharmacogenetics 8:137
55
48. Guengerich FP. 1999. Cytochrome P450 3A4: regulation and role in drug
metabolism. Annu. Rev. Pharmacol. Toxicol. 39:117
49. Schenkman JB, Jansson I. 2003. The
many roles of cytochrome b5. Pharmacol.
Ther. 97(2):13952
50. Yamazaki H, Johnson WW, Ueng YF,
Shimada T, Guengerich FP. 1996. Lack
of electron transfer from cytochrome
b5 in stimulation of catalytic activities
of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450
3A4/NADPH cytochrome P450 reductase
system and studies with apo-cytochrome
b5. J. Biol. Chem. 271(44):2743844
51. Gilep AA, Guryev OL, Usanov SA, Estabrook RW. 2001 Apo-cytochrome b5
as an indicator of changes in heme accessability: preliminary studies with cytochrome P450 3A4. J. Inorg. Biochem.
87(4):23744
52. Yamazaki H, Nakamura M, Komatsu T,
Ohyama K, Hatanaka N, et al. 2002. Roles
of NADPH-P450 reductase and apo- and
holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human
cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr.
Purif. 24(3):32937
4 Dec 2004
13:28

308
AR
AR232-PA45-12.tex
AR232-PA45-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
ATKINS
53. Guryev OL, Gilep AA, Usanov SA, Estabrook RW. 2001. Interaction of apocytochrome b5 with cytochromes P4503A4 and P45017A: relevance of heme
transfer reactions. Biochemistry 40(16):
501831
54. Modi S, Gilham DE, Sutcliffe MJ, Lian
LY, Primrose WU, et al. 1997. 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine as a
substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome
P450 reductase. Biochemistry 36(15):
446170
55. Hanna IH, Krauser JA, Cai H, Kim MS,
Guengerich FP. 2001. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric
role of NADPH-cytochrome P450 reductase in catalytic regioselectivity. J. Biol.
Chem. 276(43):3955361
56. Loughran PA, Roman LJ, Miller RT, Masters BS. 2001. The kinetic and spectral
characterization of the E. coli-expressed
mammalian CYP4A7: cytochrome b5 effects vary with substrate. Arch. Biochem.
Biophys. 385(2):31122
57. Cupp-Vickery J, Andersen R, Hatziris Z.
2000. Crystal structures of ligand complexes of P450eryF exhibiting homotropic
cooperativity. Proc. Natl. Acad. Sci. USA
97(7):305055
58. Torimoto N, Ishii I, Hata M, Nakamura H,
Imada H, et al. 2003. Direct interaction between substrates and endogenous steroids
in the active site may change the activity
of cytochrome P450 3A4. Biochemistry
42(51):1506877
59. Dabrowski MJ, Schrag ML, Wienkers LC,
Atkins WM. 2000. Pyrene-pyrene complexes at the active site of cytochrome
P4503A4: evidence for a multiple substrate binding site. J. Am. Chem. Soc.
124(40):1186667
60. Segel IH. 1993. Multisite and allosteric
enzymes. In Enzyme Kinetics. pp. 346
404. New York: Wiley
61. Shen L, Fitzloff JF, Cook CS. 2004. Differential enantioselectivity and product-
62.
63.
64.
65.
66.
67.
68.
69.
dependent activation and inhibition in

metabolism of verapamil by human
CYP3As. Drug Metab. Dispos. 32:186
96
Beechem JM, Gratton E, Ameloot M,
Knutson JR, Brand L. 1991. The global
analysis of fluorescence and intensity and
anisotropy decay data: second generation
theory and program. In Topics in Fluorescence Spectroscopy, ed. JR Lakowicz,
vol. 2, pp. 24198. New York: Plenum
Press
Lin Y, Lu P, Tang C, Mei Q, Sandig G,
et al. 2001. Substrate inhibition kinetics
for cytochrome P450-catalyzed reactions.
Kenworthy KE, Clarke SE, Andrews J,
Houston JB. 2001. Multisite kinetic models for CYP3A4: simultaneous activation
and inhibition of diazepam and testosterone metabolism. Drug Metab. Dispos.
29:164451
Shou M, Dai R, Cui D, Korzekwa KR,
Baillie TA, Rushmore TH. 2001. A kinetic
model for the metabolic interaction of two
substrates at the active site of cytochrome
P450 3A4. J. Biol. Chem. 276(3):2256
62
Hosea NA, Miller GP, Guengerich FP.
2000. Elucidation of distinct ligand binding sites for cytochrome P450 3A4. Biochemistry 39:592939
Domanski TL, He YA, Khan KK, Roussel F, Wang Q, Halpert JR. 2001. Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition
site residues and effect on substrate oxidation and cooperativity. Biochemistry
40(34):1015060
Khan KK, He YQ, Domanski TL, Halpert
JR. 2002. Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding
sites and of the metabolic pathway that
leads to enzyme inactivation. Mol. Pharmacol. 61(3):495506
Davydov DR, Kumar S, Halpert JR.
2002. Allosteric mechanisms in P450eryF
4 Dec 2004
13:28
AR
AR232-PA45-12.tex
AR232-PA45-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
70.

71.
72.
73.
74.
75.
76.
77.
probed with 1-pyrenebutanol, a novel

fluorescent substrate. Biochem. Biophys.
Res. Commun. 294(4):80612
Yoon MY, Campbell AP, Atkins WM.
2004. Allosterism in the elementary
steps of the cytochrome P450 reaction cycle. Drug Metab. Rev. 36:112
Harlow GR, Halpert JR. 1998. Analysis of
human cytochrome P450 3A4 cooperativity: construction and characterization of a
site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. Proc.
Natl. Acad. Sci. USA 95(12):663641
Domanski TL, He YA, Harlow GR,
Halpert JR. 2000. Dual role of human cytochrome P450 3A4 residue Phe-304 in
substrate specificity and cooperativity. J.
Pharmacol. Exp. Ther. 293(2):58591
Iyer KR, Jones JP, Darbyshire JF, Trager
WF. 1997. Intramolecular isotope effects
for benzylic hydroxylation of isomeric
xylenes and 4,4 -dimethylbiphenyl by cytochrome P450: relationship between distance of methyl groups and masking of
the intrinsic isotope effect. Biochemistry
36(23):713643
Korzekwa KR, Trager WF, Gillette JR.
1989. Theory for the observed isotope effects from enzymatic systems that form
multiple products via branched reaction
pathways: cytochrome P-450. Biochemistry. 28(23):901218
Nelson SD, Trager WF. 2003. The use
of deuterium isotope effects to probe
the active site properties, mechanism
of cytochrome P450-catalyzed reactions,
and mechanisms of metabolically dependent toxicity. Drug Metab. Dispos.
31(12):148198
Rock DA, Perkins BN, Wahlstrom J, Jones
JP. 2003. A method for determining two
substrates binding in the same active site
of cytochrome P450BM3: an explanation
of high energy omega product formation.
Arch. Biochem. Biophys. 416(1):916
Reed JR, Hollenberg PF. 2003. Comparison of substrate metabolism by cytochromes P450 2B1, 2B4, and 2B6: re-
78.
79.
80.
81.
82.
83.
84.
85.
86.
309
lationship of heme spin state, catalysis,

and the effects of cytochrome b5. J. Inorg. Biochem. 93(34):15260
Gorsky LD, Koop DR, Coon MJ. 1984.
On the stoichiometry of the oxidase
and monooxygenase reactions catalyzed
by liver microsomal cytochrome P-450.
Products of oxygen reduction. J. Biol.
Chem. 259(11):681217
Atkins WM, Sligar SG. 1987. Metabolic
switching in cytochrome p450cam: deuterium isotope effects on regiospecificity
and the monooxygenase/oxidase ratio. J.
Am. Chem. Soc. 109:375460
Hutzler JM, Wienkers LC, Wahlstrom
JL, Carlson TJ, Tracy TS. 2003. Activation of cytochrome P450 2C9-mediated
metabolism: mechanistic evidence in
support of kinetic observations. Arch.
Wester MR, Stout CD, Johnson EF. 2002.
Purification and crystallization of Nterminally truncated forms of microsomal
cytochrome P450 2C5. Methods Enzymol.
357:7379
Scott EE, Spatzenegger M, Halpert JR.
2001. A truncation of 2B subfamily
cytochrome P450 yields increased expression levels, increased solubility, and
decreased aggregation while retaining
function. Arch. Biochem. Biophys. 395(1):
5768
Cosme J, Johnson EF. 2000. Engineering microsomal cytochrome P450 2C5 to
be a soluble, monomeric enzyme. Mutations that alter aggregation, phospholipid
dependence of catalysis, and membrane
binding. J. Biol. Chem. 275(4):254553
Hsu PY, Wang LH. 2003. Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form.
Arch. Biochem. Biophys. 416(1):3846
Sueyoshi T, Park LJ, Moore R, Juvonen RO, Negishi M. 1995. Molecular
engineering of microsomal P450 2a-4
to a stable, water-soluble enzyme. Arch.
Tennant M, McRee DE. 2001. The
4 Dec 2004
13:28
310
87.

88.
89.
90.
91.
92.
93.
AR
AR232-PA45-12.tex
AR232-PA45-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
ATKINS
first structure of a microsomal P450
implications for drug discovery. Curr.
Opin. Drug Discov. Dev. 4(5):67177
Lepesheva GI, Waterman MR. 2004.
CYP51the omnipotent P450. Mol. Cell
Endocrinol. 215(12):16570
Li H, Poulos TL. 1996. Conformational
dynamics in cytochrome P450-substrate
interactions. Biochimie 78(89):69599
Poulos TL. 1988. Cytochrome P450:
molecular architecture, mechanism, and
prospects for rational inhibitor design.
Pharm. Res. 5(2):6775
Schlichting I, Berendzen J, Chu K,
Stock AM, Maves SA, et al. 2000.
The catalytic pathway of cytochrome
p450cam at atomic resolution. Science
287(5458):161522
Ravichandran KG, Boddupalli SS, Hasermann CA, Peterson JA, Deisenhofer J.
1993. Crystal structure of hemoprotein
domain of P450BM-3, a prototype for
microsomal P450s. Science 261(5122):
73136
Wester MR, Johnson EF, Marques-Soares
C, Dijols S, Dansette PM, et al. 2003.
Structure of mammalian cytochrome
P450 2C5 complexed with diclofenac at
2.1 A resolution: evidence for an induced
fit model of substrate binding. Biochemistry 42(31):933545
C, Dansette PM, Mansuy D, Stout CD.
2003. Structure of a substrate complex
of mammalian cytochrome P450 2C5 at
2.3 A resolution: evidence for multiple
substrate binding modes. Biochemistry
42(21):637079
94. Strickler M, Goldstein BM, Maxfield K,

Shireman L, Kim G, et al. 2003. Crystallographic studies on the complex behavior of nicotine binding to P450cam
(CYP101). Biochemistry 42(41):11943
50
95. Williams PA, Cosme J, Ward A, Angove
HC, Matak Vinkovic D, Jhoti H. 2003.
Crystal structure of human cytochrome
P450 2C9 with bound warfarin. Nature
424(6947):46468
96. Rettie AE, Eddy AC, Heimark LD,
Gibaldi M, Trager WF. 1989. Characteristics of warfarin hydroxylation catalyzed
by human liver microsomes. Drug Metab.
Dispos. 17(3):26570
97. Scott EE, He YA, Wester MR, White MA,
Chin CC, et al. 2003. An open conformation of mammalian cytochrome P450 2B4
at 1.6-A resolution. Proc. Natl. Acad. Sci.
USA 100(23):13196201
98. Schoch GA, Yano JK, Wester MR, Griffin
KJ, Stout CD, Johnson EF. 2004. Structure of human microsomal cytochrome
P450 2C8: Evidence for a peripheral
fatty acid binding site. J. Biol. Chem.
279:9497503
99. Williams PA, Cosme J, Vinkovic DM,
Ward A, Angove HC, et al. 2004. Crystal structures of human cytochrome P450
3A4 bound to metyrapone and progesterone. Science 305(5684):68386
100. Yano JK, Wester MR, Schoch GA, Griffin KJ, Stout CD, Johnson EF. 2004.
The structure of human microsomal cytochrome P450 3A4 determined by Xray crystallography to 2.05 resolution. J.
Biol. Chem. In press
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Copyright
EPOXIDE HYDROLASES: Mechanisms, Inhibitor

Designs, and Biological Roles

Christophe Morisseau and Bruce D. Hammock
Department of Entomology and U.C. Davis Cancer Center, University of California,
Davis, California 95616; email: bdhammock@ucdavis.edu
Key Words /-hydrolase fold family, hydroxyl-alkyl-enzyme intermediate,

N,N -dialkyl-ureas, epoxy-eicosatrienoic acids, hypertension
Abstract Organisms are exposed to epoxide-containing compounds from both
exogenous and endogenous sources. In mammals, the hydration of these compounds by
various epoxide hydrolases (EHs) can not only regulate their genotoxicity but also, for
lipid-derived epoxides, their endogenous roles as chemical mediators. Recent findings
suggest that the EHs as a family represent novel drug discovery targets for regulation
of blood pressure, inflammation, cancer progression, and the onset of several other
diseases. Knowledge of the EH mechanism provides a solid foundation for the rational
design of inhibitors, and this review summarizes the current understanding of the
catalytic mechanism of the EHs. Although the overall EH mechanism is now known,
the molecular basis of substrate selectivity, possible allosteric regulation, and many fine
details of the catalytic mechanism remain to be solved. Finally, recent development in
the design of EH inhibitors and the EH biological role are discussed.
INTRODUCTION
Epoxide-containing compounds are ubiquitously found in the environment from
both natural and man-made sources, and a large variety of aromatic and alkenic
compounds are also metabolized to epoxides endogenously (1, 2). An epoxide (or
oxirane) is a three-membered cyclic ether that has specific reactivity patterns owing
to the highly polarized oxygen-carbon bonds in addition to a highly strained ring
(3). Some reactive epoxides are responsible for electrophilic reactions with critical
biological targets such as DNA and proteins, leading to mutagenic, toxic, and
carcinogenic effects (4, 5). Although most epoxides are of intermediate reactivity,
relatively stable at physiological pHs, and do not present acute dangers to cells,
they still need to be transformed in a controlled manner (6). The catalytic addition
of water to epoxides or arene oxides by epoxide hydrolases (EHs, E.C.3.3.2.3) to
yield the corresponding 1,2-diols, or glycols (7), is only one of several ways that
cells transform oxiranes. However, EHs are ubiquitous and hydration seems to be a
common route of epoxide transformation. The reaction is energetically favorable,
with water as the only cosubstrate.
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The role of epoxide hydrolases seems to differ profoundly from organism

to organism. Overall, these enzymes have three main functions: detoxification,
catabolism, and regulation of signaling molecules. For microorganisms, EHs seem
important in the catabolism of specific carbon sources from natural sources, such
as tartaric acid or limonene (8, 9), as well as environmental contaminants such as
epichlorohydrin (10). However, microbial EHs are mainly studied for their potential uses in chiral chemistry (11, 12). In plants, EHs have been characterized from
several organisms (1319), and the enzymes seem important in cuticle formation,
responses to stresses, and pathogen defenses (15, 16, 2022). EHs have also been
characterized in several insects (2327). Their roles in the catabolism of a key
developmental chemical mediator, juvenile hormone (28), and in the detoxification of many plant chemical defenses have been studied (27, 29). In the vertebrate
branch of the evolutionary tree, EHs have been mostly studied in mammals, which
are emphasized in this review.
In mammals, there are several EHs, including cholesterol epoxide hydrolase
(chEH), which hydrates the 5,6-oxide of cholesterol and other 5-epoxy steroids
(30) and hepoxilin hydrolase (31). This review concentrates mostly on the soluble
epoxide hydrolase (sEH) and microsomal epoxide hydrolase (mEH), which have
been the most studied EHs over the past 30 years. These two enzymes were first
distinguished by their subcellular localization, but they also have distinct and complementary substrate specificity (6, 32). Although these two enzymes are highly
concentrated in the liver, they are found in nearly all tissues that were assayed for
EH activity (6). These two enzymes are described to complement each other in
detoxifying a wide array of mutagenic, toxic, and carcinogenic xenobiotic epoxides (6, 33); however, recent findings clearly implicate the sEH in the regulation of
blood pressure and inflammation (3440), and the mEH in xenobiotic metabolism
and the onset of several diseases (4145). Interestingly, inhibition of the sEH appears to be a potential therapeutic treatment for several diseases, including high
blood pressure, atherosclerosis, and kidney failure (35, 38, 39, 46). A prerequisite
for the development of potent inhibitors is an understanding of EHs mechanism
of action. This mechanism has been studied since these enzymes were discovered
more than 30 years ago; however, major breakthroughs were achieved in the past
10 years owing to the availability of recombinant EHs (4749). Reviews have summarized the progress in unraveling EH mechanism several times during the past
decade (6, 5052). In this review, we outline our current understanding of EHs,
catalytic mechanism. Furthermore, we focus on the development in the design of
EH inhibitors and their use to understand the biological role of EHs in mammals.
MECHANISM
Formation of a Hydroxyl Alkyl-Enzyme Intermediate
The observation that both the mammalian mEH and sEH sequences are similar to a
bacterial haloalkane dehalogenase and other related proteins was key in suggesting
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EPOXIDE HYDROLASE MECHANISMS
313
Figure 1 Proposed mechanism for epoxide hydrolase. (A) Two-step

mechanism with the formation of a hydroxyl-alkyl-enzyme intermediate;
(B) general-based-catalyzed direct hydration of the epoxide.
that both EHs have a similar mechanism to the bacterial enzyme (53) and that they
are members of the /-hydrolase fold family of proteins (54). All the enzymes
in this family are characterized by a nucleophile-histidine-acid catalytic triad and
have a two-step mechanism involving the formation of a covalent intermediate
(55, 56). This suggested that these EHs hydrolyze epoxides through the formation
of a hydroxyl alkyl-enzyme intermediate as described in Figure 1A. Before this
time, the generally accepted mechanism of EHs involved a general-based-catalyzed
direct attack of water on the epoxide ring (Figure 1B; 5759). Around the same
time that the sequence analysis was done (54), Lacourciere & Armstrong (60)
demonstrated the formation of a covalent intermediate for the mEH with a single
turnover experiment (excess of enzyme) in H218O showing that the 18O was not
incorporated in the formed glycol but rather in the protein. A second step was
shown to incorporate the 18O in the product, even in H216O. Further evidence was
gained through the isolation of the covalent intermediates for the sEH and mEH
(61, 62). Chemical characterization of the enzyme-product intermediate indicated
a structure consistent with an -hydroxyl alkyl-enzyme (61).
The Catalytic Components

The amino acid residues forming the catalytic triad of the EHs were first identified
from sequence alignment with the sequence of haloalkane dehalogenase (54, 63).
Electrospray mass spectrometric analysis of the tryptic digestion of murine sEH
incubated with susbtrate in H218O showed that the 18O was incorporated on a
peptide containing Asp333. This confirmed the role of this residue as the nucleophile
attacking the epoxide ring (64). Furthermore, the site-directed mutagenesis of this
amino acid to a serine resulted in a total loss of activity, whereas its mutation to
an asparagine yielded a mutant enzyme that reverted to the aspartate over time
and therefore regained the activity (65). This observation was later measured for
the mEH (66) and suggested the presence of a very basic water molecule near the
catalytic site. In other /-hydrolase fold enzymes, the water molecule responsible
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Principal catalytic amino acids in human epoxide hydrolases.

Nucleophilic
acid
Basic
histidine
Orienting
acid
Polarizing
tyrosines
Human mEH
Asp226
His431
Glu404
Tyr299 and Tyr374
Human sEH
Asp334
His523
Asp495
Tyr382 and Tyr465
for the hydrolysis of the covalent intermediate is activated by a histidine/acid pair

in a charge relay (55, 56). A histidine residue had been implicated in the catalytic
mechanism of EHs years before (67). Site-directed mutation of histidine 431 for
rat mEH and 523 for mouse sEH resulted in total loss of activity, indicating that
these residues may function as a general base (58, 65). Furthermore, the rat mEH
H431S mutant is still able to form the covalent intermediate but cannot hydrolyze
it, showing that this histidine plays a role in activating the molecule of water
(68). Based on the sequence alignments, the identification of the orientating acid
residue of the catalytic triad was more speculative (54, 63, 69). The preparation
of numerous site direct mutants of acid residues (66, 70, 71) has allowed the
identification of Asp495 and Glu404 as the orientating acid for the rat sEH and
mEH, respectively. Interestingly, for the rat mEH, the replacement of Glu404 with
an aspartic acid results in a dramatically increased turnover rate (71). In the recent
literature, numerous papers have reported the presence of similar catalytic triads
(Asp/His/Asp or Glu) in other EHs from diverse origins by sequence alignments.
We report in Table 1 the catalytic triad residues number of the human mEH and
sEH active sites.
Early work indicated that both mEH and sEH catalyzed the trans-addition of
water to epoxides through a general base catalysis (see 6 and references therein).
Furthermore, the occurrence of a nucleophilic mechanism in the rate-determining
step was strongly supported by the observed positive correlation between the rate
of hydrolysis by mEH and the Hammet constant of substituted styrene oxides (57).
Although these early findings agreed with the two-step mechanism described in
Figure 1A, it raised the question of which step was rate limiting. Presteady-state
kinetic analysis of epoxide hydration catalyzed by mEH (72) revealed that k3, the
rate of hydrolysis of the hydroxyl alkyl-enzyme intermediate (E-I in Equation 1),
was far slower that the rate of its formation (k2).
KS
2
3
E+S
E-I E + P

ES
k
2
1.
Furthermore, it was found that the formation of the ester intermediate was reversible (k2 = 0), indicating that the enzyme could stabilize the oxyanion in the
alkylation reaction (first step). Other /-hydrolase fold enzymes have an oxyanion hole that stabilizes tetrahedral intermediates for the formation and hydrolysis
of the covalent bond between the enzyme and the substrate (55, 56). The presence
of such an oxyanion hole in sEH was proposed with a push-pull mechanism that
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315
activated the epoxide by protonation or hydrogen bonding of the oxygen atom

(69). Using chalcone oxides, which are substrates of sEH with a very low turnover
rate (k3 is very small), a slightly negative correlation between k2 and the Hammet
constant of para-substitutions was found, indicating that the formation of E-I was
driven by a slight acidic mechanism (73). Furthermore, the low magnitude of the
Hammet parameter suggested the presence of a weak charge that excluded the
formation of a true carbonium ion or oxyanion, implying a general acido-basiccatalyzed process in the formation of E-I. The residues composing the oxyanion
hole vary greatly inside the /-hydrolase fold family. For example, haloalkane
dehalogenases have tryptophans (53), whereas esterases have two glycines (74).
Over the years, based on other /-hydrolase fold enzymes, several residues were
proposed and tested for being part of the oxyanion hole, but without success (58,
65, 66, 68, 70). The breakthrough was obtained with the acquisition of X-ray crystal
structures of EHs (7578). As shown in Figure 2 for the murine sEH, two tyrosine
residues (381 and 465) located above the nucleophilic aspartate 333 (responsible
for the formation of the E-I complex) are the best candidates for supplying general
acid catalysis in the first half reaction (77). The mutation of either of these two
tyrosines to phenylalanine results in a 90% decrease in activity (79). Furthermore,
the kinetics of chalcone oxide hydroysis show that both mutations decrease the
binding (larger KS) and the rate of formation of an intermediate (lower k2), suggesting that both tyrosines affect epoxide polarization and facilitate ring opening
(79). However, these two tyrosines are not implicated in the hydrolysis of the covalent intermediate (no change in k3), suggesting that there is no intermediate to
be stabilized in the hydrolysis step. This is consistent with the observation that the
second half of the reaction is not reversible (60). Interestingly, these two tyrosines
are conserved in EHs through evolution (78, 79), and the mutations of the equivalent residues in the mEH also resulted in dramatic loss of activity, like for the
sEH (51, 79). The polarizing tyrosines of the human mEH and sEH are reported
in Table 1.
The Catalytic Cycle

The above information is summarized in Figure 3. In the first step of the catalytic
cycle of EHs, the epoxide quickly binds to the active site of the enzyme. Crystal
structures show that the mouse sEH has a 25-A-deep

L-shaped hydrophobic tunnel,
with the nucleophile aspartate located near the bend of the L and both ends
open to the solvent (76). It is not known if the substrate enters the active site by
crawling down the tunnel or if the cap of the active site opens to let the substrate
in then closes for the catalysis. The latter possibility is supported by the fact
that the fluorescence of mEH changed significantly upon substrate binding (60),
suggesting a large movement in the enzyme structure. However, there are other
possible explanations for the observed change. Examination of the crystal structure
of the murine sEH with the inhibitor N-cyclohexyl-N -decylurea bound (Figure 4)
shows that there are hydrophobic pockets on either side of the central catalytic
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Figure 3 Catalytic mechanism of EH. The amino acid residue numbers correspond to the human sEH.
residues (Asp333 and His523). This suggests that Van der Walls interactions make
a significant contribution to substrate binding (77). However, such an analysis
also indicates a number of potential hydrogen bonding sites primarily located on
the surface opposite of Asp333, which could be important in the formation of the
intermediate (top right of Figure 3).
The substrate epoxide is polarized by two tyrosine residues (382 and 465),
which hydrogen bond with the epoxide oxygen. At the same time, the nucleophilic
carboxylic acid of Asp334, present on the side of the catalytic cavity opposite to the
tyrosines, makes a backside attack on the epoxide, usually at the least sterically
hindered and most reactive carbon. The nucleophilic acid is oriented and activated
by His523, a second carboxylic amino acid (Asp495) and possibly other amino acids
in the catalytic site for this attack. A recent study based on molecular dynamics
simulations (80) suggests that the protonation of His523 is essential for the right
orientation of Asp334; however, no experimental proofs exist yet. Because the
mEH has a higher optimal pH for activity (pH 8.09.0) than the sEH (pH 7.07.5)
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(32), the corresponding His in mEH is probably not protonated (80). The opening
of the epoxide results in an ester bond between the enzyme carboxylic acid and
one alcohol functionality of the diol. This is termed the hydroxyl alkyl-enzyme
intermediate (bottom right of Figure 3). An important unanswered question is
where the oxygen of the epoxide catches the hydrogen to form a hydroxyl because it
was determined that an oxyanion is not formed (73). It was proposed that the proton
came from one of the tyrosine side chains (77), as it is shown in Figure 3, and that the
formed tyrosinate ion is stabilized by edge-to-face -interactions with surrounding
aromatic residues (79). However, the presence of a tyrosinate ion has yet to be
proven. Furthermore, it could be argued that the high pKa (10) of the tyrosine
side chain makes it difficult for the phenolate to form at the pH (7.4 for sEH and
8.09.0 for mEH) at which the reaction is catalyzed (51). It would be intellectually
satisfying if the hydrogen come from the protonated His523, especially because
this histidine should be a base (not protonated) for the second half of the catalytic
reaction (80). However, crystal structures show that this histidine residue is on the
wrong side of the catalytic site to directly donate its proton to the epoxide (77).
Therefore, a proton shuttle mechanism was proposed to transfer the proton from the
histidine to the tyrosine (80), but this proton transfer pathway has yet to be shown.
Once the covalent hydroxyl alkyl-enzyme is formed, the histidine moves far
enough from the nucleophilic acid (now ester) to allow a water molecule to be
activated by the acid-histidine pair (bottom left of Figure 3). This movement may
account for the fluorescence shift during the enzyme reaction (60). The activation
of the water can occur only if the histidine is not protonated (80). This very
basic water attacks the carbonyl of the ester, releasing the diol product and the
original enzyme. As we described above, a variety of lines of evidence support
this mechanism for both the mammalian microsomal and soluble EH and EHs from
numerous other organisms. Interestingly, a few reports suggest that the cholesterol
epoxide hydrolase has a different mechanism (see below).
The Cholesterol Epoxide Hydrolase

The chEH is the other EH located in the microsomal fraction (81, 82). Because this
enzyme has not been purified to homogeneity or been cloned (33), little is known
about it. Unlike mEH and sEH, which have a wide range of substrate specificities
(6), chEH is very specific for cholesterol 5,6-oxide (82). The enzyme shows a
fivefold preference for the - over the -diastereomer (83). The exact mechanism
of catalysis of the chEH is not well known, but several lines of evidence suggest
a mode of action different than the one described above for the sEH and mEH.
First, its size is too small (84) to be a classical /-hydrolase fold enzyme (52,
55, 56). Furthermore, unlike mEH or sEH, chEH appears to hydrolyze cholesterol
oxides via a positively charged transition state (85). Although covalent hydroxyl
alkyl-enzyme were isolated from sEH and mEH (see above), Muller and collaborators were unable to isolate any covalent intermediate for chEH (62). All these
results suggest that chEH hydrolyzes its substrate through a one-step general base
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mechanism similar to the one described in Figure 1B. This hypothesis is supported by the recent report of the structure for the limonene-1,2-epoxide hydrolase
from Rhodococcus erythropolis that has a one-step general-based-catalyzed direct
hydration of the epoxide (86).

Quaternary Structures
Analysis of the primary structure suggests that the sEH gene (EPXH2) was produced by the fusion of two primordial dehalogenase genes; the C-terminal sEH
domain has high homology to haloalkane dehalogenase, whereas the N-terminal
domain is similar to haloacid dehalogenase (HAD) (54, 69). This gene fusion hypothesis was recently supported by an X-ray crystal structure of the mouse and
human sEH that exhibit a domain-swapped architecture (Figure 5) where both
domains of each monomer are separated by a proline-rich linker (76, 87). Furthermore, the three-dimensional (3-D) structures confirmed that unlike the mEH, the
sEH is a homodimer with with a monomeric unit of 62.5 kDa as determined from
biochemical analysis (6, 32). The C-terminal domain of one subunit interacts with
both the C- and N-terminal domains of the other monomer. Beside the physical
interaction between the two C-terminal domains, which contain the EH activity,
no cooperative allosteric effects have been reported for the sEH activity (6). The
fact that the C-terminal catalytic cavities are not close to any interdomain or interprotein interface may explain the lack of cooperativity in epoxide hydrolysis
(76). Alternatively, it was found that in solution the monomer and dimer sEH are
active (88, 89), suggesting a natural equilibrium between the two forms of sEH. Although a dissociation constant for the dimer formation has yet to be measured, it is
possible that, under the conditions where sEH activity is generally measured (low
nanomolar), the enzyme could be mainly in its monomeric form, thus preventing
the detection of any possible allosteric effects.
Analysis of the sEH crystal structure reveals that while the C-terminal domain containing the EH activity adopts an /-hydrolase fold as expected, the
N-terminal domain adopts an /- fold similar to HAD with a 15-A-deep

catalytic
cavity with catalytic residues properly oriented for catalysis (76, 87). However, no
dehalogenase activity was detected, and the N-terminal domain was first thought
to only stabilize the formation of the dimer, and thus qualified as a vestigial domain
(76). This hypothesis was supported by the fact that sEH orthologues in plants lack
the N-terminal domain and are monomeric (69). However, the amino-terminal catalytic DXDX(T/V) motif of HAD has been used to describe an enzyme class that
includes numerous phosphatases (90, 91), suggesting a possible catalytic activity for the N-terminal domain. Recently, a magnesium-dependent hydrolysis of
a phosphate ester was associated with this domain of sEH (92, 93). The human
sEH crystal structure supports a mechanism for this phosphatase activity similar
to the one previously described for phosphatases of the HAD family (87). Interestingly, the sEH is found to hydrolyze the monophosphates of dihydroxy stearic
acid yielding 1,2-diols similar to those obtained from the hydrolysis of stearic acid
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epoxides by the sEH (93). Although sEH inhibitors do not influence the phosphatase activity, kinetic analysis revealed a positive cooperative Hill coefficient of
2 for the hydrolysis of the monophosphate of dihydroxy stearic acid, suggesting
an allosteric interaction between the two monomers (93). The recent 3-D structure
of the human sEH reveals a 14-A-long

hydrophobic tunnel sufficiently large to
accommodate the binding of an aliphatic substrate with one end at the active site
and the other end near the interface of the N- and C-terminal domains, supporting
the observed positive cooperative kinetics (87).
The mEH (EXPH1) does not have a large N-terminal domain similar to the
sEH, but instead possesses a strongly hydrophobic transmembrane domain of approximately 20 residues, which anchors the protein to cellular membranes (94,
95). Although mEH activity is not completely lost after the removal of this anchor,
the resulting protein is not soluble (95), suggesting a strong association of mEH
to the membrane. The mEH is found to be tightly associated with phospholipids
(96, 97), suggesting a complex binding between mEH and cellular membranes. At
this time, little is known beyond these findings about how this enzyme is bound to
the membrane. In liver, mEH is found to reside on both the smooth endoplasmic
reticulum (ER; 98) and the plasma membrane (99, 100). Complicating matters,
the topological orientation of mEH in the membranes appears to be different in
the ER (catalytic C-terminal domain facing the cytosol) and in the plasma membrane (C-terminal facing the exocellular medium) (101, 102). A recent study using
recombinant enzymes showed that mEH could associate nonspecifically with several P450s, resulting in an activation of the mEH activity; however, it is not known
at the molecular level how this association occurs (103). Finally, the mEH was
reported to be a subunit of two multiprotein complexes: a Na+-dependent bile acid
transport (99, 104) and an antiestrogen binding site (105); however, nothing is yet
known about how mEH interacts within these complexes and how it regulates their
activities.
INHIBITOR DESIGNS
Specific enzyme inhibitors are important research tools to help understand the
catalytic mechanism of an enzyme and the pathologies that may be associated with
dysfunctions of this enzyme. This statement has been particularly true over the past
few years for sEH, and the recent design of potent inhibitors for sEH has opened
the door to new therapies for hypertension and inflammation (3440). To start
with a historic point of view, the first inhibitors discovered for the sEH and mEH
were epoxide-containing compounds (Figure 6) (see 6 and references therein).
However, most of these compounds are in fact substrates of the corresponding EH
with a relatively low turnover that gives only a transient inhibition in vitro and
are inefficient in cell cultures and in vivo (6, 73, 106, 107). A widely used mEH
inhibitor, trichloropropene oxide, not only reacts with many proteins directly but
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Figure 6 Epoxide-containing inhibitors of (A) the mEH and (B) the sEH.
it is rapidly turned over by the mEH (108). The mEH and sEH activities are found
to be inhibited by Hg2+ and Zn2+, and the sEH is also inhibited by Cu2+ and Cd2+
(109). The inhibition of the human mEH by Zn2+ is found to be competitive with a
KI of 60 M, whereas the inhibition of the human sEH is noncompetitive with a
KI of 20 M (109). This divalent cation is found to also inhibit the phosphatase
activity of the sEH (93). One could hypothesize that the binding of Zn2+ at the Mg2+
site in the N-terminal domain resulted in loss of both activities through some as yet
unknown allosteric mechanism that is suggested by the sEH quaternary structure
(see above). During inflammation, the concentrations of divalent cation metals,
especially zinc, increase in the liver (110), suggesting that the binding of Zn2+
could be a simple way for the organism to naturally reduce the sEH activity that
was shown to be proinflammatory (34, 38).
Approximately a decade ago, valpromide treatment was reported to affect the
normal metabolism of carbamazepine by inhibiting the mEH in vivo (111, 112).
The anticonvulsant properties of this compound could cause undesirable secondary
effects in experimental systems if used as mEH inhibitor, but other unsubstituted
amides could be used (113). Recently, primary ureas, amides, and amines were
described as mEH inhibitors (Figure 7A; 114). The most potent inhibitor obtained,
elaidamide, has a mix of competitive and noncompetitive inhibition kinetics with
a KI of 70 nM. It is efficient in vitro (114); however, its fast turn over by amidases limits its use in vivo, underlying the need of new potent and stable mEH
inhibitors.
1,3-Disubstituted ureas, carbamates, and amides (Figure 7B) were recently
described as new potent and stable inhibitors of sEH (115). These compounds
are competitive tight-binding inhibitors with nanomolar Ki that interact stoichiometrically with purified recombinant sEH (115, 116). Crystal structures show
that the urea inhibitors establish hydrogen bonds and salt bridges between the
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Figure 7 Structure of nonepoxide-containing inhibitors of (A) the

mEH and (B) the sEH.
urea functionality of the inhibitor and residues of the sEH active site, mimicking
the intermediate formed during the enzymatic epoxide ring opening, as described
in Figure 3 (76, 77, 87). Furthermore, the side chains of the inhibitors (R and
R ) need to be hydrophobic to bind tightly in the hydrophobic catalytic site,
as shown in Figure 4 (76, 77). Interestingly, because of the presence of a methionine residue (Met337) pointing into the catalytic cavity, the orientation of
the urea inhibitors is reversed in the human sEH compared with the mouse enzyme (87). Using classical quantitative structure activity relationship (QSAR),
3-D-QSAR, and medicinal chemistry approaches, the structure of these inhibitors
were improved to yield compounds that have an order of magnitude better inhibition potency (116119). This new generation of sEH inhibitors display on
one side of the urea functionality secondary and tertiary pharmacophores at 5
and 11 atoms away from the urea carbonyl group, respectively (119). These inhibitors efficiently inhibit epoxide hydrolysis in several in vitro and in vivo models (3840, 115, 120). The beneficial biological effects observed are discussed
below.
BIOLOGICAL ROLES
The biological role of any enzyme is intimately linked to the substrates the enzyme
transforms. Substrate specificity is probably the best way to distinguish between
the mEH and sEH. These two enzymes have been found to hydrolyze a broad
and complementary range of substrates (6, 32). In general, mEH seems to prefer
mono- and cis-disubstituted epoxides, whereas the sEH prefers gem-di-, trans-di-,
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Structure of typical substrates hydrolyzed by (A) the mEH and (B)
cis-di-, tri-, and tetra substituted epoxides (32). The latter enzyme hydrates epoxide
on cyclic system very poorly (107). A few examples of mEH and sEH substrates
are shown in Figure 8.
mEH is a key hepatic enzyme involved in the metabolism of numerous xenobiotics, such as 1,3-butadiene oxide 1, styrene oxide 2, and benzo()pyrene 4,5-oxide
3 (6, 33, 52). In addition, mEH is likely involved in the extrahepatic metabolism
of these agents (33, 121, 122). Styrene 2 and cis-stilbene 4 oxides are widely used
as surrogate substrates for mEH (32). The mEH action is part of a detoxification
process for most of the substrates (6, 33). This detoxification action is illustrated
by the example of a man who had a defect in mEH expression and suffered from
acute and severe phenytoin toxicity (123). However, in some cases, such as for
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benzo()pyrene 4,5-oxide 5, diol formation can lead to the stabilization of a secondary epoxide, increasing the mutagenic and carcinogenic potential of the product
(124). The procarcinogenic role of mEH was illustrated in mEH knockout mice that
were less sensitive to the carcinogenic activity of 7,12-dimethylbenz[]anthracene
than control mice (125). Furthermore, in human populations, polymorphism in the
mEH gene is associated with the onset of numerous cancers (4244, 126, 127).
The role of mEH in xenobiotic metabolism is probably also linked to the observed
relationship between mEH polymorphism and emphysema (41) or Crohns disease
(45).
Despite the fact that mEH knockout mice do not present an obvious phenotype
(125), there are several new lines of evidence suggesting an endogenous role for
this enzyme. A potential role of mEH in sexual development is supported by the
fact that androstene oxide 5 is a very good mEH substrate (128), and that mEH
is an apparent subunit of the antiestrogen-binding-site (105). Such a role could
be related to the observed relation between mEH polymorphism and spontaneous
abortion (129) or preeclampsia (130). Furthermore, mEH is well expressed in
ovaries (131), especially in follicle cells (132). During the past decade, mEH was
also described as mediating the transport of bile acid in the liver (133, 134). The
mechanism by which mEH participates in this transport is not known. Potent mEH
inhibitors could provide new tools to better understand the multiple roles of this
enzyme.
sEH was thought to participate in the metabolism of xenobiotics, like the mEH;
however, there is no evidence supporting this hypothesis in vivo in mammals (6,
52). Radioactive aromatic epoxides, such as trans-diphenyl-propene oxide 6 and
trans-stilbene oxide 7, are classically used as surrogate substrates for this enzyme
in vitro (32, 135). On the other hand, the sEH is clearly involved in the metabolism
of arachidonic epoxides (8, also called epoxyeicosatrienoic acids or EETs) and
linoleic acid epoxides (9, also called leukotoxins) both in vitro and in vivo (34, 35,
136, 137). The sEH hydrates all of these epoxy-fatty acids with high VM and low
KM. The sEH-dependent transformation of EETs decline as the epoxide approaches
the carboxyl terminal (i.e., 14,15-EET is hydrolyzed 20-fold faster than 8,9-EET
and 5,6-EET is hydrolyzed very slowly), whereas both mono-epoxides of linoleic
acid are hydrolyzed at similar rates (138, 139). Although epoxy-fatty acids are
relatively poor substrates for mEH compared to sEH (138), the former enzyme
hydrolyzes them with a high enantioselectivity, whereas the latter shows little or
no enantiomeric preference (140, 141).
The EETs, which are endogenous chemical mediators (142), act at the vascular, renal, and cardiac levels to regulate blood pressure (143, 144). The vasodilatory properties of EETs are associated with an increased open-state probability
of calcium-activated potassium channels, which lead to hyperpolarization of the
vascular smooth muscle (145). Hydrolysis of the epoxides by sEH diminishes
this activity (146). The sEH-dependent hydrolysis of EETs also regulates their
incorporation into coronary endothelial phospholipids, suggesting a regulation of
endothelial function by sEH (147). Recently, blood pressure reduction was achieved
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in the spontaneous hypertensive rat (SHR) and in angiotensin IIinduced hypertension rat models with pharmacological sEH inhibition (35, 39). Additionally,
male knockout sEH mice have significantly lower blood pressure than wild-type
mice (36), further supporting the role of sEH in blood pressure regulation and sEH
inhibition as a potential new therapeutic treatment for hypertension.
The EETs also display antiinflammatory properties in endothelial cells (37,
148). In contrast, diols derived from epoxy-linoleate (leukotoxin) perturb membrane permeability and calcium homeostasis (34), which results in inflammation
that is modulated by nitric oxide synthase and endothelin-1 (149, 150). Micromolar concentrations of leukotoxin reported in association with inflammation and hypoxia (151) depress mitochondrial respiration in vitro (152) and cause mammalian
cardio-pulmonary toxicity in vivo (150, 153, 154). Leukotoxin toxicity presents
symptoms suggestive of multiple organ failure and acute respiratory distress syndrome (ARDS) (151). In both cellular and whole organism models, leukotoxinmediated toxicity is dependent on epoxide hydrolysis (34, 115), suggesting a role
for sEH in the regulation of inflammation. Treatment with sEH inhibitors increases
EET levels in cell cultures and reduces indicators of vascular inflammation (38,
155), suggesting that sEH is a potential therapeutic target for the treatment of
several vascular inflammatory diseases, including atherosclerosis and kidney failure (38, 46). Inhibition of the sEH seems to result in general antiinflammatory
properties in many systems.
ACKNOWLEDGMENTS
The authors want to particularly thank Dr. John Newman for his precious help
in the preparation of the figures and review of this manuscript. Figures 2, 4, and
5 were prepared using the Cn3D program version 4.1 produced by the National
Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This work
was supported in part by NIEHS Grant R37 ES02710, NIEHS Superfund Basic
Research Program Grant P42 ES04699, NIEHS Center for Environmental Health
Sciences Grant P30 ES05707, and NIH/NHLBI R01 HL59699-06A1.
LITERATURE CITED
1. Jerina DM. 1974. Biological formation
and disposition of arene oxides. Lloydia
37:21218
2. Oliw EH. 1994. Oxygenation of polyunsaturated fatty acids by cytochrome
P450 monooxygenases. Prog. Lipid Res.
33:32954
3. Parker RE, Isaacs NS. 1959. Mechanism of epoxide reactions. Chem. Rev.
264:931013
4. Szeliga J, Dipple A. 1998. DNA adducts
formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res.
Toxol. 11:111
7 Jan 2005
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5. Zheng J, Myung C, Jones AD, Hammock
BD. 1997. Evidence of quinone metabolites of naphthalene covalently bound to
sulfur nucleophiles of proteins of murine
clara cells after exposure to naphthalene.
6. Hammock BD, Grant D, Storms D. 1997.
Epoxide hydrolase. In Comprehensive
Toxicology, ed. I Sipes, C McQueen, A
Gandolfi, pp. 283305. Oxford: Pergamon
7. Oesch F. 1973. Mammalian epoxide hydrases: inducible enzymes catalyzing the
inactivation of carcinogenic and cytotoxic
metabolites derived from aromatic and
olefinic compounds. Xenobiotica 3:305
40
8. Allen RH, Jakoby WB. 1969. Tartaric acid
metabolism. IX. Synthesis with tartrate
epoxidase. J. Biol. Chem. 244:207884
9. Van der Werf MJ, Swarts HJ, Bont JA.
1999. Rhodococcus erythropolis DCL14
contains a novel degradation pathway
for limonene. Appl. Environ. Microbiol.
65:2095102
10. Jacobs MHJ, Wijngaard AJ, Pentenga M,
Janssen DB. 1991. Characterization of
the epoxide hydrolase from epichlorohydrin-degrading Pseudomonas sp. Eur.
J. Biochem. 202:121722
11. Archelas A, Furstoss R. 2001. Synthetic
applications of epoxide hydrolases. Curr.
Opin. Chem. Biol. 5:11219
12. de Vries EJ, Janssen DB. 2003. Biocatalytic conversion of epoxides. Curr. Opin.
Biotechnol. 14:41420
13. Blee E, Schuber F. 1992. Occurrence of
fatty acid epoxide hydrolases in soybean
(Glycine max). Biochem. J. 282:71114
14. Stapleton A, Beetham JK, Pinot F, Garbarino JE, Rockhold DR, et al. 1994.
Cloning and expression of soluble epoxide hydrolase from potato. Plant J. 6:251
58
15. Kiyosue T, Beetham JK, Pinot F, Hammock BD, Yamaguchi-Shinozaki K, et al.
1994. Characterization of an Aradibopsis cDNA for a soluble epoxide hydrolase
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
325
gene that is inducible by auxin and water

stress. Plant J. 6:25969
Guo A, Durner J, Klessig DF. 1998. Characterization of a tobacco epoxide hydrolase gene induced during the resistance
response to TMV. Plant J. 15:64756
Arahira M, Nong VH, Ukada K,
Fukazawa C. 2000. Purification, molecular cloning and ethylene-inducible expression of a soluble-type epoxide hydrolase
from soybean (Glycine max [L.] Merr.)
Eur. J. Biochem. 267:264957
Bellevik S, Zhang J, Meijer J. 2002.
Brassica napus soluble epoxide hydrolase
(BNSEH1). Eur. J. Biochem. 269:5295
302
Edqvist J, Farbos I. 2003. A germinationspecific epoxide hydrolase from Euphorbia lagascae. Planta 216:40312
Kato T, Yamaguchi Y, Uhehara T, Yokohama T, Namai T, et al. 1983. Defense mechanism of the rice plant against
rice blast disease. Naturwissenschaften
70:2001
Blee E, Schuber F. 1993. Biosynthesis of
cutin monomers: involvement of a lipoxygenase/peroxygenase pathway. Plant J.
4:144856
Pinot F, Skrabs M, Compagnon V, Salaun
JP, Benveniste I, et al. 2000. Omegahydroxylation of epoxy- and hydroxylfatty acids by CYP94A1: possible involvement in plant defense. Biochem. Soc.
Trans. 28:86770
Mullin CA, Wilkinson CF. 1980. Purification of an epoxide hydrolase from
the midgut of the southern army worm
(Spodoptera eridania). Insect Biochem.
10:68191
Debernard S, Morisseau C, Severson TF,
Feng L, Wojtasek H, et al. 1998. Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta. Insect
VanHook-Harris S, Marin-Thompson D,
Linderman RJ, Tomalski MD, Roe RM.
1999. Cloning and expression of a novel
7 Jan 2005
14:1
326

26.
27.
28.
29.
30.
31.
32.
33.
34.
AR
AR232-PA45-13.tex
MORISSEAU
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE
HAMMOCK
juvenile hormone-metabolizing epoxide

hydrolase during larval-pupal metamorphosis in the cabbage looper, Trichoplusia
ni. Insect Mol. Biol. 8:8596
Keiser KC, Brandt KS, Silver GM, Wisnewski N. 2002. Cloning, partial purification and in vivo developmental profile of
expression the juvenile hormone epoxide
hydrolase of Ctenocephalides felis. Arch.
Insect Biochem. Physiol. 50:191206
Taniai K, Inceoglu AB, Yukuhiro K,
Hammock BD. 2003. Characterization
and cDNA cloning of a clofibrateinducible microsomal epoxide hydrolase in Drosophila melanogaster. Eur. J.
Biochem. 270:4696705
Hammock BD. 1985. Regulation of juvenile hormone titer: degradation. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology, ed. GA Kerkut,
LI Gilbert, pp. 43172. New York: Pergamon Press
Mullin CA. 1988. Adaptative relationships of epoxide hydrolase in herbivorous
arthropods. J. Chem. Ecol. 14:186788
Levin W, Michaud DP, Thomas PE, Jerina
DM. 1983. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6-alpha-oxide and
certain xenobiotic alkene and arene oxides. Arch. Biochem. Biophys. 220:485
94
Pace-Asciak CR, Lee WS. 1989. Purification of hepoxilin epoxide hydrolase from
rat liver. J. Biol. Chem. 264:931013
Wixtrom RN, Hammock BD. 1985.
Membrane-bound and soluble-fraction
epoxide hydrolases: methodological aspects. In Biochemical Pharmacology and
Toxicology: Methodological Aspects of
Drug Metabolizing Enzymes, ed D Zakim,
DA Vessey, vol. 1, pp. 193. New York:
Wiley
Fretland AJ, Omiecinski CJ. 2000. Epoxide hydrolases: biochemistry and molecular biology. Chem Biol. Interact. 129:41
59
Moghaddam MF, Grant DF, Cheek JM,
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
Greene JF, Williamson KC, et al. 1997.

Bioactivation of leukotoxins to their toxic
diols by epoxide hydrolase. Nat. Med.
3:56266
Yu Z, Xu F, Huse LM, Morisseau C,
Draper AJ, et al. 2000. Soluble epoxide
hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Circ. Res.
87:99298
Sinal CJ, Miyata M, Tohkin M, Nagata
K, Bend JR, et al. 2000. Targeted disruption of soluble epoxide hydrolase reveals
a role in blood pressure regulation. J. Biol.
Chem. 275:4050410
Campbell WB. 2000. New role for
epoxyeicosatrienoic acids as anti-inflammatory mediators. Trends Pharmacol. Sci.
21:12527
Davis BB, Thompson DA, Howard LL,
Morisseau C, Hammock BD, et al. 2002.
Inhibitors of soluble epoxide hydrolase
attenuate vascular smooth muscle cell
proliferation. Proc. Natl. Acad. Sci USA
99:222227
Imig JD, Zhao X, Capdevila JH, Morisseau C, Hammock BD. 2002. Soluble
epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension 39:69094
Yu Z, Davis BB, Morisseau C, Hammock
BD, Olson JL, et al. 2003. Vascular localization of soluble epoxide hydrolase in human kidney. Am. J. Physiol. Renal. Physiol. 286:F72026
Smith CA, Harrison DJ. 1997. Association between polymorphism in gene for
microsomal epoxide hydrolase and susceptibility to emphysema. Lancet 350:
63033
Kiyohara C, Otsu A, Shirakawa T, Fukuda
S, Hopkin JM. 2002. Genetic polymorphisms and lung cancer susceptibility: a
review. Lung Cancer 37:24156
Baxter SW, Choong DYH, Campbell IG.
2002. Microsomal epoxide hydrolase and
susceptibility to ovarian cancer. Cancer
Lett. 187:7581
To-figueras J, Gene M, Gomez-Catalan J,
7 Jan 2005
14:1
AR
AR232-PA45-13.tex
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE

45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
Pique E, Borrego N, et al. 2002. Microsomal epoxide hydrolase and glutathione

S-transferase polymorphism in relation to
laryngeal carcinoma risk. Cancer Lett.
187:95101
De Jong JD, Van der Logt EMJ, Schaik
AV, Roelfs HMJ, Peters WHM, et al. 2003.
Genetic polymorphisms in biotransformation enzyme in Crohns disease: association with microsomal epoxide hydrolase.
Gut 52:54751
Zhao X, Yamamoto T, Newman JW, Kim
IH, Watanabe T, et al. 2004. Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage.
J. Am. Soc. Nephrol. 15:124453
Grant DE, Storms DH, Hammock BD.
1993. Molecular cloning and expression
of murine liver soluble epoxide hydrolase.
J. Biol. Chem. 268:1762833
Beetham JK, Tian T, Hammock BD. 1993.
cDNA cloning and expression of a soluble epoxide hydrolase from human liver.
Lacourciere GM, Vakharia VN, Tan CP,
Morris DI, Edwards GH, et al. 1993. Interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with a azarene
oxide and an aziridine substrate analogue.
Biochemstry 32:261016
Armstrong RN. 1999. Kinetic and chemical mechanism of epoxide hydrolase.
Drug Metab. Rev. 31:7186
Armstrong RN, Cassidy CS. 2000. New
structural and chemical insight into the
catalytic mechanism of epoxide hydrolases. Drug Metab. Rev. 32:32738
Arand M, Cronin A, Oesch F, Mowbray
SL, Jones TA. 2003. The telltale structures
of epoxide hydrolases. Drug Metab. Rev.
35:36583
Verschueren KHG, Seijee F, Rozeboom
HJ, Kalk KH, Dijkstra BW. 1993 Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase. Nature
363:69398
Arand M, Grant DF, Beetham JK, Fried-
55.
56.
57.
58.
59.
60.
61.
62.
63.
327
berg T, Oesch F, et al. 1994. Sequence similarity of mammalian epoxide hydrolases

to the bacterial haloalkane dehalogenase
and other related proteins: implication for
the potential catalytic mechanism of enzymatic epoxide hydrolysis. FEBS Lett.
338:25156
Ollis DL, Cheah E, Cygler M, Dijkstra B,
Frolow F, et al. 1992. The / hydrolase
fold. Protein Eng. 5:197211
Holmquist M. 2000. Alpha/beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr. Protein Pep.
Sci. 1:20935
Dansette PM, Makedonska VB, Jerina
DM. 1978. Mechanism of catalysis for the
hydration of substituted styrene oxides by
epoxide hydrase. Arch. Biochem. Biophys.
187:29098
Bell PA, Kasper CB. 1993. Expression of
rat microsomal hydrolase in Escherichia
coli: identification of a histidyl residue
essential for catalysis. J. Biol. Chem.
268:1401117
Hammock BD, Ratcliff M, Schooley DA.
1980. Hydration of an 180 epoxide by a
cytosolic epoxide hydrolase from mouse
liver. Life Sci. 27:163541
Lacourciere GM, Armstrong RN. 1993.
The catalytic mechanism of microsomal
epoxide hydrolase involves an ester intermediate. J. Am. Chem. Soc. 115:10466
67
Hammock BD, Pinot F, Beetham JK,
Grant DF, Arand ME, et al. 1994. Isolation of the putative hydroxyacylenzyme intermediate of an epoxide hydrolase. Biochem. Biophys. Res. Commun.
198:85056
Muller F, Arand M, Frank H, Seidel A,
Hinz W, et al. 1997. Visualization of a
covalent intermediate between microsomal epoxide hydrolase, but not cholesterol
epoxide hydrolase, and their substrates.
Lacourciere GM, Armstrong RN. 1994.
Microsomal and soluble epoxide hydrolases are members of the same family
7 Jan 2005
14:1
328
64.

65.
66.
67.
68.
69.
70.
71.
72.
AR
AR232-PA45-13.tex
MORISSEAU
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE
HAMMOCK
of C-X bond hydrolases enzymes. Chem.

Borhan B, Jones AD, Pinot F, Grant
DF, Kurth MJ, et al. 1995. Mechanism
of soluble epoxide hydrolase: formation
of an -hydroxy ester-enzyme intermediate through Asp-333. J. Biol. Chem.
270:2692330
Pinot F, Grant DF, Beetham JK, Parker
AG, Borhan B, et al. 1995. Molecular and
biochemical evidence for the involvement
of the Asp333-His523 pair in the catalytic
mechanism of soluble epoxide hydrolase.
J. Biol. Chem. 270:796874
Laughlin LT, Tzeng H-F, Lin S, Armstrong RN. 1998. Mechanism of microsomal epoxide hydrolase. Semifunctional
site-specific mutants affecting the alkylation half reaction. Biochemistry 37:2897
904
Dubois GC, Appella E, Levin W, Lu AYH,
Jerina DM. 1978. Hepatic microsomal
epoxide hydrase: involvement of a histidine residue essential for catalysis. J. Biol.
Chem. 268:1401117
Tzeng H-F, Laughlin LT, Armstrong RN.
1998 Semifunctional site-specific mutants
affecting the hydrolytic half-reaction of
microsomal epoxide hydrolase. Biochemistry 37:290511
Beetham JK, Grant D, Arand M, Garbarino J, Kuyosue T, et al. 1995. Gene
evolution of epoxide hydrolases and recommended nomenclature. DNA Cell Biol.
14:6171
Arand M, Wagner H, Oesch F. 1996.
Asp333, Asp 495, and His523 form the catalytic triad of rat soluble epoxide hydrolase. J. Biol. Chem. 271:422329
Arand M, Muller F, Mecky A, Hinz W,
Urban P, et al. 1999. Catalytic triad of
microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a
strongly increased turnover rate. Biochem.
J. 337:3743
Tzeng H-F, Laughlin LT, Lin S, Armstrong RN. 1996. The catalytic mechanism of microsomal epoxide hydrolase
73.
74.
75.
76.
77.
78.
79.
80.
involves reversible formation and rate limiting hydrolysis of the alkyl-enzyme intermediate. J. Am. Chem. Soc. 118:943637
Morisseau C, Du G, Newman JW, Hammock BD. 1998. Mechanism of mammalian soluble epoxide hydrolase inhibition by chalcone oxides derivatives. Arch.
Satoh T, Hosokawa M. 1998. The mammalian carboxylesterases: from molecules
to functions. Annu. Rev. Pharmacol. Toxicol. 38:25788
Nardini M, Ridder IS, Rozeboom HJ,
Kalk KH, Rink R, et al. 1999. The Xray structure of epoxide hydrolase from
Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides. J.
Biol. Chem. 274:1457986
Argiriadi MA, Morisseau C, Hammock
BD, Christianson DW. 1999. Detoxification of environmental mutagens and carcinogens: structure, mechanism, and evolution of liver epoxide hydrolase. Proc.
Argiriadi MA, Morisseau C, Goodrow
MH, Dowdy DL, Hammock BD, et al.
2000. Binding of alkylurea inhibitors to
epoxide hydrolase implicates active site
tyrosines in substrate activation. J. Biol.
Chem. 275:1526570
Zou J, Hallberg BM, Bergfors T, Oesch
F, Arand M, et al. 2000. Structure of Aspergillus niger epoxide hydrolase at 1.8 A
resolution: implications for the structure
and function of the mammalian microsomal class of epoxide hydrolases. Struct.
Fold Des. 8:11122
Yamada T, Morisseau C, Maxwell JE, Argiriadi MA, Christianson DW, et al. 2000.
Biochemical evidence for the involvement
of tyrosine in epoxide activation during
the catalytic cycle of epoxide hydrolase.
J. Biol. Chem. 275:2308288
Schitt B, Bruice TC. 2002. Reaction
mechanism of soluble epoxide hydrolase:
insights from molecular dynamics simulations. J. Am. Chem. Soc. 124:14558
70
7 Jan 2005
14:1
AR
AR232-PA45-13.tex
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE


81. Levin W, Michaud DP, Thomas PE, Jerina
DM. 1983. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6-alpha-oxide and
certain xenobiotic alkene and arene oxides. Arch. Biochem. Biophys. 220:485
94
82. Oesch F, Timms CW, Walker CH, Guenthner TM, Sparrow A, et al. 1984. Existence
of multiple forms of microsomal epoxide
hydrolases with radically different substrate specificities. Carcinogenesis 5:79
83. Sevanian A, McLeod LL. 1986. Catalytic properties and inhibition of hepatic
cholesterol-epoxide hydrolase. J. Biol.
Chem. 261:5459
84. Watabe T, Ozawa N, Ishii H, Chiba
K, Hiratsuka A. 1986. Hepatic microsomal cholesterol epoxide hydrolase: selective inhibition by detergents and separation from xenobiotic epoxide hydrolase.
63237
85. Nashed NT, Michaud DP, Levin W, Jerina DM. 1986. 7-Dehydrocholesterol 5,6
beta-oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase. J. Biol. Chem. 261:2510
13
86. Arand M, Hallberg BM, Zou J, Bergfors
T, Oesch F, et al. 2003. Structure of
Rhodococcus erythropolis limone-1,2epoxide hydrolase reveals a novel active
site. EMBO J. 22:258392
87. Gomez GA, Morisseau C, Hammock BD,
Christianson DW. 2004. Structure of human epoxide hydrolase reveals mechanistic interferences on bifunctional catalysis
in epoxide and phosphate ester hydrolysis. Biochemistry 43:471623
88. Gill SS. 1983. Purification of mouse liver
cytosolic epoxide hydrolase. Biochem.
89. Dietze EC, Magdalou J, Hammock BD.
1990. Human and murine cytosolic epoxide hydrolase: physical and structural
properties. Int. J. Biochem. 22:46170
90. Collet JF, Stroobant V, Schaftingen E.
91.
92.
93.
94.
95.
96.
97.
98.
99.
329
1999. Mechanistic studies of phosphoserine phosphatase, an enzyme related to Ptype ATPases. J. Biol. Chem. 274:33985
90
Ndubuisil MI, Kwok BH, Vervoort J, Koh
BD, Elofsson M, et al. 2002. Characterization of a novel mammalian phosphatase having sequence similarity to
Schizosaccharomyces pombe PHO2 and
Saccharomyces cerevisiae PHO13. Biochemistry 41:784148
Cronin A, Mowbray S, Durk H, Homburg S, Fleming I, et al. 2003. The Nterminal domain of mammalian soluble
epoxide hydrolase is a phosphatase. Proc.
Newman JW, Morisseau C, Harris TR,
Hammock BD. 2003. The soluble epoxide
hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate
phosphatase activity. Proc. Natl. Acad.
Sci. USA. 100:155863
Craft JA, Baird S, Lamont M, Burchell B.
1990. Membrane topology of epoxide hydrolase. Biochim. Biophys. Acta 1046:32
39
Friedberg T, Lollmann B, Becker R,
Holler R, Oesch F. 1994. The microsomal
epoxide hydrolase has a single membrane
signal anchor sequence which is dispensable for the catalytic activity of this protein. Biochem. J. 303:96772
Bulleid NJ, Graham AB, Craft JA. 1986.
Microsomal epoxide hydrolase of rat
liver. Purification and characterization of
enzyme fractions with different chromatographic characteristics. J. Biochem.
233:60711
Griffin M. 1999. Regulation of rat liver
epoxide hydrolase by tightly bound phosphoinositides. Proc. Okla. Acad. Sci. 79:
16
Galteau MM, Antoine B, Reggio H. 1985.
Epoxide hydrolase is a marker for the
smooth endoplasmic reticulum in rat liver.
EMBO J. 4:2793800
Von Dippe P, Amoui M, Alves C, Levy
D. 1993. Na(+)-dependent bile acid
7 Jan 2005
14:1
330

100.
101.
102.
103.
104.
105.
106.
107.
AR
AR232-PA45-13.tex
MORISSEAU
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE
HAMMOCK
transport by hepatocytes is mediated by a

protein similar to microsomal epoxide hydrolase. Am. J. Physiol.Gastrointest. Liver
Physiol. 264:G52834
De Berardinis V, Moulis C, Maurice M,
Beaune P, Pessayre D, et al. 2000. Human
microsomal epoxide hydrolase is the target of germander-induced autoantibodies
on the surface of human hepatocytes. Mol.
Pharmacol. 58:54251
Holler R, Arand M, Meckey A, Oesch F,
Friedberg T. 1997. The membrane anchor
of microsomal epoxide hydrolase from
human, rat, and rabbit displays an unexpected membrane topology. Biochem.
Zhu Q-S, Dippe PV, Xing W, Levy D.
1999. Membrane topology and cell surface targeting of microsomal epoxide hydrolase: evidence for multiple topological
orientations. J. Biol. Chem. 274:27898
904
Taura K-I, Yamada H, Naito E, Ariyoshi
N, Mori M-a, Oguri K. 2002. Activation of microsomal epoxide hydrolase
by interaction with cytochromes P450:
kinetic analysis of the association and
substrate-specific activation of epoxide
hydrolase function. Arch. Biochem. Biophys. 402:27580
Von Dippe P, Zhu Q-S, Levy D. 2003. Cell
surface expression and bile acid transport function of one topological form of
m-epoxide hydrolase. Biochem. Biophys.
Mesange F, Sebbar M, Kedjouar B,
Capdevielle J, Guillemot J-C, et al. 1998.
Microsomal epoxide hydrolase of rat liver
is a subunit of the anti-estrogen-binding
site. Biochem. J. 334:10712
Oesch F, Jerina DM, Daly JW, Rice JM.
1973. Induction, activation and inhibition
of epoxide hydrase: an anomalous prevention of chlorobenzene-induced hepatotoxicity by an inhibitor of epoxide hydrase.
Magdalou J, Hammock BD. 1988. 1,2Epoxycycloalkanes: substrates and in-
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
hibitors of microsomal and cytosolic epoxide hydrolases in mouse liver.

Hanzlik RP, Waslsh JS. 1980. Halogenated epoxides and related compounds
as inhibitors of epoxide hydrolase. Arch.
Draper AJ, Hammock BD. 1999. Inhibition of soluble and microsomal epoxide
hydrolase by zinc and other metals. Toxicol. Sci. 52:2632
Gaetke LM, McClain CJ, Talwalkar RT,
Shedlofsky SI. 1997. Effects of endotoxin on zinc metabolism in human volunteers. Am. J. Physiol. Endocrin. Metab.
272:E95256
Kerr BM, Rettie AE, Eddy AC, Loiseau
P, Guyot M, et al. 1989. Inhibition of human liver microsomal epoxide hydrolase
by valproate and valpromide: in vitro/in
vivo correlation. Clin. Pharmacol. Ther.
46:8293
Robbins D, Wedlund P, Kuhn R, Baumann
R, Levy R, et al. 1990. Inhibition of epoxide hydrolase by valproic acid in epileptic
patients receiving carbamazepine. Br. J.
Kerr BM, Levy RH. 1990. Unsubstituted
amides: new class of potent inhibitors
of human microsomal epoxide hydrolase.
Drug. Metab. Dispos. 18:54042
Morisseau C, Newman JW, Dowdy DL,
Goodrow MH, Hammock BD. 2001. Inhibition of microsomal epoxide hydrolases
by ureas, amides and amines. Chem. Res.
Toxicol. 14:40915
Morisseau C, Goodrow MH, Dowdy D,
Zheng J, Greene JF, et al. 1999. Potent
urea and carbamate inhibitors of soluble
epoxide hydrolases. Proc. Natl. Acad. Sci.
USA 96:884954
Morisseau C, Goodrow MH, Newman
JW, Wheelock CE, Dowdy DL, et al.
2002. Structural refinement of inhibitors
of urea-based soluble epoxide hydrolases.
Nakagawa Y, Wheelock CE, Morisseau
C, Goodrow MH, Hammock BG, et al.
7 Jan 2005
14:1
AR
AR232-PA45-13.tex
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE

118.
119.
120.
121.
122.
123.
124.
125.
2000. 3-D QSAR analysis of inhibition of murine soluble epoxide hydrolase (MsEH) by benzoylureas, arylureas,
and their analogues. Bioorg. Med. Chem.
8:266373
McElroy N, Jurs PC, Morisseau C, Hammock BD. 2003. QSAR and classification of murine and human soluble
epoxide hydrolase inhibition by urea-like
compounds. J. Med. Chem. 46:106680
Kim I-H, Morisseau C, Watanabe T,
Hammock BD. 2004. Design, biosynthesis, and biological activity of 1,3disubstituted ureas as potent inhibitors of
the soluble epoxide hydrolase of increased
water solubility. J. Med. Chem. 47:2110
22
Newman JW, Denton DL, Morisseau C,
Koger CS, Wheelock CE, et al. 2001.
Evaluation of fish models of soluble epoxide hydrolase inhibition. Environ. Health.
Perspect. 109:6166
Zheng J, Cho M, Brennan P, Chichester
C, Buckpitt AR, et al. 1997. Evidence for
quinone metabolites of naphthalene covalently bound to sulfur nucleophiles of
proteins of mouse Clara cell after exposure to naphthalene. Chem. Res. Toxicol.
10:100814
Teissier E, Fennrich S, Strazielle N, Daval
J-L, Ray D, et al. 1998. Drug metabolism
in in vitro organotypic and cellular models
of mammalian central nervous system: activities of membrane-bound epoxide hydrolase and NADPH-cytochrome P-450
(c) reductase. Neurotoxicology 19:34756
Yoo JH, Kang DS, Chun WH, Lee WJ, Lee
KH. 1999. Anticonvulsant hypersensitivity syndrome with an epoxide hydrolase
defect. Br. J. Dermatol. 140:18183
Szeliga J, Dipple A. 1998. DNA adducts
formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res.
Toxicol. 11:111
Miyata M, Kudo G, Lee Y-H, Yang TJ,
Gelboin HV, et al. 1999. Targeted disruption of the microsomal epoxide hydrolase
gene: microsomal epoxide hydrolase is
126.
127.
128.
129.
130.
131.
132.
133.
331
required for the carcinogenic activity of

7,12-dimethylbenz[]anthracene. J. Biol.
Chem. 274:2396368
Benhamou S, Reinkainen M, Bouchardy
C, Dayer P, Hirvonen A. 1998. Association between lung cancer and microsomal epoxide hydrolase genotypes. Cancer. Res. 58:529193
Wang L-D, Zheng S, Liu B, Zhou J-X,
Li Y-J, et al. 2003. Cyp1A1, GSTs and
mEH polymorphisms and susceptibility to
esophageal carcinoma: study of population from a high-incidence area in North
China. World J. Gastroenterol. 9:139497
Vogel-Bindel U, Bentley P, Oesch F.
1982. Endogenous role of microsomal
epoxide hydrolase: ontogenesis, induction, inhibition, tissue distribution, immunological behavior and purification of
microsomal epoxide hydrolase with 16,
17-epoxyandrostene-3-one as substrate.
Wang X, Wang M, Niu T, Chen C, Xu
X. 1998. Microsomal epoxide hydrolase
polymorphism and risk of spontaneous
abortion. Epidemiology 9:54044
Laasanen J, Romppanen E-L, Hiltunen M,
Helisalmi S, Mannermaa A, et al. 2002.
Two exonic single nucleotide polymorphisms in the microsomal epoxide hydrolase gene are jointly associated with
preeclampsia. Eur. J. Hum. Gene. 10:569
73
Mukhtar H, Philpot RM, Bend JR. 1978.
The postnatal development of microsomal
epoxide hydrase, cytosolic glutathione Stransferase, and mitochondrial and microsomal cytochrome P-450 in adrenals and
ovaries of female rats. Drugs Metab. Dispos. 6:57783
Cannady EA, Dyer CA, Christian PJ,
Sipes IG, Hoyer PB. 2002. Expression and
activity of microsomal epoxide hydrolase
in follicles isolated from mouse ovaries.
Toxicol. Sci. 68:2431
Alves C, von Dippe P, Amoui M, Levy D.
1993. Bile acid transport into hepatocyte
smooth endoplasmic reticulum vesicles is
7 Jan 2005
14:1
332

134.
135.
136.
137.
138.
139.
140.
141.
AR
AR232-PA45-13.tex
MORISSEAU
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE
HAMMOCK
mediated by microsomal epoxide hydrolase, a membrane protein exhibiting two

distinct topological orientations. J. Biol.
Chem. 268:2014855
Zhu Q, Xing W, Qian B, von Dippe
P, Shneider BL, et al. 2003. Inhibition
of human m-epoxide hydrolase gene expression in a case of hypercholanemia.
Biochem. Biophys. Act. 1638:20816
Borhan B, Mebrahtu T, Nazarian S, Kurth
MJ, Hammock BD. 1995. Improved radiolabeled substrates for soluble epoxide
hydrolase. Anal. Biochem. 231:188200
Chacos N, Capdevila J, Falck JR, Manna
S, Martin-Wixtrom C, et al. 1983. The
reaction of arachidonic acid epoxides
(epoxyeicosatrienoic acids) with a cytosolic epoxide hydrolase. Arch. Biochem.
Biophys. 223:63948
Halarnkar PP, Wixtrom RN, Silva MH,
Hammock BD. 1989. Catabolism of
epoxy fatty esters by the purified epoxide
hydrolase from mouse and human liver.
Zeldin DC, Wei S, Falck JR, Hammock
BD, Snapper JR, et al. 1995. Metabolism
of a epoxyeicosatrienoic acids by cytosolic epoxide hydrolase: substrate structural determinants of asymmetric catalysis. Arch. Biochem. Biophys. 316:44351
Greene JF, Williamson KC, Newman
JW, Morisseau C, Hammock BD. 2000.
Metabolism of monoepoxides of methyl
linoleate: bioactivation and detoxification. Arch. Biochem. Biophys. 376:420
32
Zeldin DC, Kobayashi S, Falck JR,
Winder BS, Hammock BD, et al. 1993.
Regio- and enantiofacial selectivity of
epoxyeicosatrienoic acid hydration by cytosolic expoxide hydrolase. J. Biol. Chem.
268:64027
Summerer S, Hanano A, Utsumi S,
Arand M, Schuber F, Blee E. 2002.
Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by
plant and mammalian epoxide hydrolases.
Biochem. J. 366:47180
142. Carroll MA, McGiff JC. 2000. A new

class of lipid mediators: cytochrome
P450 arachidonate metabolites. Thorax
55:S1316
143. Capdevila JH, Falck JR, Harris RC. 2000.
Cytochrome P450 and arachidonic acid
bioactivation: molecular and functional
properties on the arachidonate monooxygenase. J. Lipid. Res. 41:16381
144. Flemming I. 2001. Cytochrome P450 enzymes in vascular homeostasis. Circ. Res.
89:75362
145. Fisslthaler B, Popp R, Kiss L, Potente M,
Harder DR, et al. 1999. Cytochrome P450
2C is an EDHF synthase in coronary arteries. Nature 401:49397
146. Fang X, Kaduce TL, Weintraub NL, Harmon S, Teesch LM, et al. 2001. Pathways
of epoxyeicosatrienoic acid metabolism
in endothelial cells, implication for the
vascular effects of soluble epoxide hydrolase inhibition. J. Biol. Chem. 276:14867
74
147. Weintraub NL, Fang X, Kaduce TL, VanRollins M, Chatterjee P, et al. 1999.
Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phopholipids. Am. J.
Physiol. Heart Circ. Physiol. 277:H208
18
148. Node K, Huo Y, Ruan X, Yang B, Spiecker
M, et al. 1999. Anti-inflammatory properties of cytochrome P450 epoxygenasederived eicosanoids. Science 285:1276
79
149. Ishizaki T, Shigemori K, Nakai T, Miyabo
S, Ozawa T, et al. 1995. Leukotoxin,
9,10-epoxy-12-octadecenoate causes edematous lung injury via activation of vascular nitric oxide synthase. Am. J. Physiol.
Lung Cell Mol. Physiol. 269:L6570
150. Ishizaki T, Shigemori K, Nakai T, Miyabo
S, Hayakawa M, et al. 1995. Endothelin1 potentiates leukotoxin-induced edematous lung injury. J. Appl. Physiol. 79:
110611
151. Dudda A, Spiteller G, Kobelt F. 1996.
Lipid oxidation products in ischemic
7 Jan 2005
14:1
AR
AR232-PA45-13.tex
AR232-PA45-13.sgm
LaTeX2e(2002/01/18)
P1: GCE


porcine heart tissue. Chem. Phys. Lipids.
82:3951
152. Sakai T, Ishizaki T, Ohnishi T, Sasaki
F, Ameshima S, et al. 1995. Leukotoxin, 9,10-epoxy-12-octadecenoate inhibits mitochondrial respiration of isolated perfused rat lung. Am. J. Physiol.
Lung Cell Mol. Physiol. 269:L32631
153. Fukushima A, Hayakawa M, Sugiyama S,
Ajioka M, Ito T, et al. 1988. Cardiovascular effects of leukotoxin (9,10-epoxy12-octadecenoate) and free fatty acids in
dogs. Cardiovas. Res. 22:21318
333
154. Ishizaki T, Takahashi H, Ozawa T, Chang

SW, Voelkel NF. 1995. Leukotoxin,
9,10-epoxy-12-octadecenoate causes pulmonary vasodilatation in rats. Am. J.
Physiol. Lung Cell Mol. Physiol. 268:
L12328
155. Slim R, Hammock BD, Toborek M,
Robertson LW, Newman JW, et al. 2001.
The role of methyl-linoleic acid epoxide
and diol metabolites in the amplified toxicity of linoleic acid and polychlorinated
biphenyls to vascular endothelial cells.
Toxicol. Appl. Pharmacol. 171:18493
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Figure 2 Structure of the active site of the mouse sEH showing the presence of two
tyrosine residues, 381 and 465 (gray), positioned opposite of the catalytic triad
(Asp333 in red, His523 in blue, and Asp495 in red). Structure obtained from Reference
76.

C-2
MORISSEAU
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Figure 4 Hydrophobicity map of the mouse sEH substrate binding pocket cocrystalyzed with the inhibitor 1-cyclohexyl-3-dodecyl urea (77). Amino acid side chains
within 6 of the inhibitor are displayed as space-filling models. The residues shown
in bright red and blue are the urea oxygen and nitrogens, respectively. A color gradient of brown to blue indicates degrees of hydrophobicity. Panel A shows a view of
the catalytic pocket from the inside of the enzyme toward the outside, and panel B
shows the opposite view. A series of hydrophilic residues are observed on the top
side of the channel (Phe265, Pro266, Trp334, Val337, Pro363, Pro369, Ile373, Phe385,
Phe406, Ile427, Thr468, Trp472), whereas the bottom of the channel is very
hydrophobic (Thr359, Met361, Pro363, Val372, Phe379, Ile416,Val418, Val497, Lys498,
Trp524), with the exception of the catalytic aspartate and histidine (Asp333 and
His523). This structural analysis indicates that a number of potential hydrogen bonding sites (Tyr381, Gln382, Tyr465) are observed in the substrate binding pocket of the
soluble epoxide hydrolase, primarily located on the surface opposite Asp333.
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Figure 5 Structure of the mouse sEH dimer (76). The N-terminal domains (residues
Arg4-Gly218) are in yellow-orange, the C-terminal domains (residues Val235-Ala544)
are in blue-green, and the proline-rich linker (Thr219-Asp234) is in magenta. Catalytic
residues for both the C- and N-terminal domains are displayed as space-filling
residues with blue for positive charge, red for negative charge, and gray for neutral.
The alternating helices and the beta sheet floor typical of the /-hydrolase fold
enzymes is clearly shown in the C-terminal domain.
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Copyright
NITROXYL (HNO): Chemistry, Biochemistry,

and Pharmacology
Jon M. Fukuto,1 Christopher H. Switzer,2
Katrina M. Miranda,3 and David A. Wink4
1
Interdepartmental Program in Molecular Toxicology, UCLA School of Public Health,

Los Angeles, California 90095-1772; email: jfukuto@mednet.ucla.edu
2
Department of Chemistry and Biochemistry, University of California, Los Angeles,
California 90095-1569; email: cswitzer@ucla.edu
3
Department of Chemistry, University of Arizona, Tucson, Arizona 85721;
email: miranda@email.arizona.edu
4
Tumor Biology Section, Radiation Biology Branch, National Cancer Institute,
Bethesda, Maryland 20892; email: wink@box-w.nih.gov
Key Words nitric oxide, thiols, calcitonin gene-related peptide,

ischemia-reperfusion
Abstract Recent discoveries of novel and potentially important biological activity have spurred interest in the chemistry and biochemistry of nitroxyl (HNO). It has
become clear that, among all the nitrogen oxides, HNO is unique in its chemistry and
biology. Currently, the intimate chemical details of the biological actions of HNO are
not well understood. Moreover, many of the previously accepted chemical properties of
HNO have been recently revised, thus requiring reevaluation of possible mechanisms
of biological action. Herein, we review these developments in HNO chemistry and
biology.
INTRODUCTION
The biological activity and biological chemistry of nitrogen oxide species in mammalian systems have received considerable attention over the past 15 years. Interest
in this area is primarily the result of the discovery of endogenous generation of
nitric oxide (NO) by mammalian cells. Although the focus of much of the past
research has been NO, it is becoming increasingly clear that other nitrogen oxides
derived in vivo from NO may have significant physiological and/or pathophysiolgical functions. Although significant advances have been made in our understanding
of the chemical biology of NO and related/derived nitrogen oxides, such as nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), and peroxynitrite (ONOO),
nitroxyl (HNO) remains the least studied and least understood of all the biologically relevant nitrogen oxides. Despite the original description of HNO more
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than 100 years ago, understanding of the chemistry and biochemistry of HNO
has seriously lagged behind other redox nitrogen oxide congeners, even after the
discovery of endogenous mammalian nitrogen oxide generation. However, recent
reports have indicated that HNO has novel and potentially important biological activity (see below), which prompted numerous labs to investigate the physiological
and chemical properties and reactivity of HNO. Much of this recent work has led
to redefinition of the fundamental chemistry of this enigmatic species, which aided
in partial understanding of the chemistry responsible for the newly discovered biological properties of HNO. In this chapter, we review some of the physiologically
relevant chemical properties of HNO and discuss some of its recently discovered
biological/pharmacological properties.
FUNDAMENTAL CHEMICAL PROPERTIES OF NITROXYL

From both theoretical and experimental perspectives, nitroxyl has been the topic
of numerous studies for more than 100 years. In this review, we concentrate on
the chemistry that may be relevant to the biological actions of nitroxyl. For more
comprehensive treatments of the pure and applied chemistry of nitroxyl (and related
nitrogen oxide species), readers are referred to other excellent reviews (13).
Before discussing the details of HNO chemistry, a comment regarding nomenclature is warranted. The term nitroxyl is sometimes used to describe a stable
radical functional group otherwise referred to as a nitroxide (i.e., R2NO). However,
nitroxyl is also used in the literature to describe the chemical species commonly
written as HNO (along with the various spin-state and protonation congeners, see
below). Owing to the current widespread use of the term nitroxyl in the literature
when referring to HNO (or even NO), we will continue to use it in this regard.
Another, more appropriate, name for HNO is nitrosyl hydride (4).
One of the first references to HNO in the chemical literature was by Angeli (5),
who proposed it as a decomposition product of sodium trioxodinitrate (Na2N2O3,
Angelis salt) (Reaction 1):
Na2 N2 O3 (Angelis salt) + H+ [NaHN2 O3 ] HNO + NaNO2
1.
Since then, others have proposed HNO as an intermediate in a variety of chemical and biological processes. For example, HNO has been proposed to be generated
during bacterial denitrification (6), released from acid-catalyzed solvolysis of acinitroalkanes (Nef reaction) (7), the product of the reaction of NO with hydrogen
(8), and a product of the reaction of NO with hydroxylamine (9, 10). Direct observation of HNO was accomplished by Brown & Pimentel (11) when they trapped
it in an argon matrix during the photolysis of methyl nitrite. HNO has also been
generated by pulse radiolysis (12, 13), a technique that has led to the elucidation
of some of the fundamental chemical properties of HNO (although some of these
properties have been reevaluated and revised recently, see below).
One of the most unique and fascinating aspects of HNO chemistry involves its
simple deprotonation. The electronic ground state of HNO is the singlet where all
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electrons are spin-paired (unlike radical species such as NO). Deprotonation of

HNO generates nitroxyl anion [more appropriately termed oxonitrate (1-), NO].
This species is isoelectronic with dioxygen (O2) and can exist as an electronic
singlet (1NO) or triplet (3NO), which is analogous to the relationship between
singlet O2 (1O2) and triplet O2 (3O2) (14). The electronic ground state of NO is
the triplet (3NO), which is reported to be approximately 1720 kcal/mol lower
in energy than the first excited electronic singlet state, 1NO (1519). Thus, in the
acid-base equilibrium expression for HNO/NO, H+ (Reaction 2), is not straightforward because the electronic ground states of the acid and conjugate base are
different. Although it was earlier proposed that HNO deprotonates to the singlet
excited state anion (20), 1NO, which would be followed by intersystem crossing
to the ground state triplet species, 3NO, this is no longer considered the case.
Recent theoretical and experimental work indicates that the relevant equilibrium
in the acid-base chemistry of HNO/NO, H+ in both the gas phase and in solution
is between singlet HNO and triplet NO (19, 2125) (Reaction 2):
1
HNO
3 NO + H+
2.
Hence, the deprotonation of HNO requires a spin conversion of ground state

products to ground state reactants (and vice versa for protonation of the anion).
As might be expected, this spin conversion will considerably slow the rate of both
deprotonation of HNO and protonation of 3NO. However, the spin conversion in
HNO deprotonation plays only a minor role in the intersystem barrier, with nuclear
reorganization representing the majority of the activation barrier (25). Regardless,
it is clear that HNO deprotonation and 3NO protonation are considerably slower
than typical proton transfer processes. This slow process predicts 3NO generated at neutral pH will have a significant lifetime (milliseconds), even though its
existence relative to the protonated species is unfavorable (25).
Direct experimental determination of the pKa of HNO by measuring equilibrium
concentrations of HNO and/or NO is difficult owing to the propensity of HNO to
undergo dimerization to hyponitrous acid followed by dehydration to give nitrous
oxide (N2O) and water (26) (Reaction 3). The rate constant for dimerization was
originally reported to be near diffusion controlled (26) but has recently been revised
to be significantly lower (24).
HNO + HNO [HONNOH] N2 O + H2 O (8 106 M1 s1 )
3.
Thus, equilibrium between HNO and NO cannot be achieved at suprananomolar concentrations because HNO can be siphoned off to N2O and H2O via
Reaction 3. Regardless, in a 1970 study where NO was generated using pulse
radiolysis, a pKa for HNO of 4.7 was reported (12). This report did not specify
the spin states of the relevant equilibrium species, and until recently this was the
exclusively quoted value for the pKa of HNO. Theoretical reevaluation of the HNO
pKa led to a revision of 7.2 (19). Of particular note, this work specifically indicated
that the relevant equilibrium in solution, between HNO and 3NO, was the same as
that proposed by Janaway & Brauman for the gas phase (22). Further experimental
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and theoretical work by Shafirovich & Lymar (24) and Bartberger and coworkers
(23) provided a consensus agreement that the pKa of HNO is 11.4.
Bartberger and coworkers (19) noted that the revision of the HNO pKa required reevaluation of an aspect of NO chemistry as well. An often-quoted reduction potential for the NO/3NO couple was calculated to be 0.39 V (versus
NHE throughout) using the assumptions, among others, that the HNO pKa was 4.7
and the relevant acid-base equilibrium was between HNO and 1NO (20). Considering the dramatic revision in the pKa and the establishment that the relevant
equilibrium is between HNO and 3NO, recalculation of the NO/3NO couple
gives a value of 0.8 V (23). This recalculated reduction potential is consistent
with experimentally derived values (27, 28), and this previously irreconcilable difference between experiment and calculation can now be explained. Protonation of
3
NO to HNO will be highly favorable at physiological pH and therefore results
in a positive shift in the potential to approximately 0.5 to 0.6V as the pH is
lowered (23, 24). These negative values for the one-electron reduction potentials
for both the NO/3NO and NO,H+/HNO couples indicate that direct one-electron
reduction of NO to reduced species by an outer sphere electron transfer process
is thermodynamically unfavorable and not likely to occur under biological (mammalian) conditions. This idea has, however, been challenged on the basis that if
the intracellular concentrations of the reductants and oxidants are considered, a
much less negative potential will be realized (29). Moreover, it has been pointed
out that the reduction potentials in prokaryotic cells may be capable of reducing
NO (29) and may be part of an antipathogenic response of NO.
The discussion of nitroxyl chemistry thus far has focused on HNO and 3NO.
However, a triplet protonated species and a singlet anionic species have been
examined in previous studies. Protonation of 3NO has been proposed to occur
on the more electronegative oxygen atom, generating 3NOH (30, 31). This triplet
protonated species has been calculated to be approximately 2023 kcal/mol less
stable than 1HNO (32, 33). Thus, interconverison between 1HNO and 3NOH is
biologically inaccessible. As noted earlier, the singlet anionic nitroxyl, 1NO,
has been determined to be approximately 1720 kcal/mol above the ground state
triplet species, which agrees reasonably well with theoretical studies (19). From
a biological perspective, the only accessible nitroxyl species are HNO and 3NO
(which is the reason the chemistry of these has been the focus of discussion).
Figure 1 depicts the energy relationships between all of these protonation- and
spin-related species.
As is evident from the above discussion, fundamental nitroxyl chemistry is
conceptually distinct and, at times, requires one to suspend commonly held chemical dogma/beliefs when thinking about it. When addressing the chemistry of
nitroxyl in biological/physiological systems, it will be important to remember the
following:
1. The pKa of HNO is approximately 11 and therefore, if formed initially, HNO
will be the predominant species present at physiological pH.
T o x i c o l .
2 0 0 5 . 4 5 : 3 3 5 - 3 5 5 .
D o w n l o a d e d
f r
H e i d e l b e r g
o n
1 0 / 0 1 / 0 5 .
F o r
p e r s o n a l
u s e
A n n u .
R e v .
P h a r m a c o l .
b y
U n i v e r s i t a e t
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NITROXYL (HNO)
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Figure 1 Energetics of the various nitroxyl species.
2. The relevant acid-base equilibrium for nitroxyl is between the singlet protonated, 1HNO, and the triplet anion, 3NO.
3. The requirement for a spin-state change for nitroxyl protonation-deprotonation means these reactions are slow relative to all other protonation-deprotonation events, which are extremely fast.
4. If circumstances exist whereby 3NO is generated/formed, it will have a
significant lifetime (milliseconds) because protonation to HNO is slow.
5. The other protonation/spin-state congeners of nitroxyl, namely 1NOH and
1
NO, are biologically inaccessible and irrelevant to most all discussions of
biological nitroxyl activity.
6. Generation of HNO or 3NO via single-electron reduction of NO by an outer
sphere process is not favorable, although it may be possible.
REACTIVITY OF NITROXYL
As already mentioned, an important (and bothersome) reaction of HNO is dimerization with itself followed by dehydration to give N2O and H2O (Reaction 3).
This propensity to dimerize necessitates the use of donor molecules for most studies of HNO. The ground state triplet anion, 3NO, reacts with O2 to generate
peroxynitrite, OONO (34). This reaction (Reaction 4) is isoelectronic with the
well-studied reaction of NO with O
2 (Reaction 5), and both reactions occur at
near diffusion-controlled rates (24, 35, 36):
3
NO + 3 O2
OONO
(2.7 109 M1 s1 )
4.
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NO + O
2
OONO
(47 109 M1 s1 )
5.
Nitroxyl anion generated by pulse radiolytic reduction of NO (spin state not
reported) reacts sequentially with NO to give N2O

2 and N3O3 , the latter species
decomposing to N2O and NO2 (12, 13, 37) (Reactions 6, 7, and 8):
NO + NO N2 O
2
N2 O
2 + NO N3 O3
(k = 1.73.3 109 M1 s1 )
6.
(k = 34.9 106 M1 s1 )
7.
N3 O
3 N2 O + NO2
(k = 87330 s1 )
8.
The existence of Reactions 68 preclude the possibility of synthesizing salts of

NO via direct reduction of NO because any anion formed will rapidly react with
excess NO. Sequential reaction of HNO with NO has also been observed (Reactions
9 and 10) with eventual formation of N2O and HNO2 (12, 13) (Reaction 11). A rate
constant for the reaction of HNO with NO was originally reported to indicate a near
diffusion-controlled reaction (1.7 109 M1 s1). However, recent reevaluation
of the reaction of HNO with NO (Reaction 9), using flash photolysis of Angelis
salt (Na2N2O3) for in situ HNO generation, has reported a significantly lower rate
constant of 5.8 106 M1 s1 (24):
HNO + NO HN2 O2
HN2 O2 + NO HN3 O3
HN3 O3 N2 O + HNO2
(5.8 106 M1 s1 )
9.
(8 106 M1 s1 )
10.
1 1
11.
(1.6 10 M
4
s )
The catenation reactions of HNO/NO with NO may be of some biological/pharmacological interest because both species may be present simultaneously
under certain circumstances. Indeed, nitroxyl may be capable of attenuating the
actions of NO (and vice versa).
Formation of N2O
2 /HN2O2 (Reactions 6 and 9) has been hypothesized to lead
to the generation of the potent oxidant hydroxyl radical (HO) (13) (Reaction 12):
N2 O
2 N2 O + O ( HO)
(3.5 102 s1 )
12.
Although this reaction has been proposed to account for some of the oxidative
chemistry and/or toxicity associated with nitroxyl (3840), unequivocal demonstration of this reaction is lacking.
One of the most important and biologically relevant aspects of HNO chemistry
is its ability to react as an electrophile with thiols. In fact, this reaction has been
used to distinguish between the biology of HNO versus NO because HNO is
much more reactive toward thiols compared with NO (41). The electrophilicity
of HNO appears to be particularly high for thiols and much less so with oxygenbased nucleophiles (19). The reactivity of HNO with amine nucleophiles has been
calculated to be intermediate between thiols and oxygen-based nucleophiles and
can be favorable. The initial product of the reaction of HNO with a thiol is an
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N-hydroxysulfenamide (Reaction 13) (42). This intermediate can react further

with excess thiol to give hydroxylamine and the corresponding disulfide (Reaction
14) or rearrange to generate a sulfinamide (43, 44) (Reaction 15):
HNO + RSH RS-NHOH
13.
RS-NHOH + RSH RSSR + NH2 OH
14.
RS-NHOH [RS+ NH + HO ] RS+ (OH)NH RS(O)NH2
15.
Sulfinamides can hydrolyze to generate ammonia and the corresponding sulfinic

acid (43). HNO-mediated oxidation to the disulfide and hydroxylamine (Reactions
13 and 14) represents a biologically reversible process because disulfides are easily regenerated. However, sulfinamide or sulfinic acid formation may represent a
process that is either irreversible or more difficult to reverse. To date, there is only
one reported example of biological reduction of a sulfinic acid back to the thiol
oxidation state (45).
Direct reaction of HNO with thiols represents an HNO reduction process (i.e.,
HNO serving as an oxidant). Other reports indicate that HNO is a reasonable
oxidant, and, indeed, HNO reduction may be a primary fate of HNO in cells. For
example, HNO can oxidize NADPH (4649). This reaction was inhibited by the
presence of superoxide dismutase (which converts HNO to NO), indicating that
HNO/NO was the oxidant and not NO. Moreover, NADPH oxidation occurred
anaerobically, eliminating the possibility that HNO/O2 adducts were the oxidizing
species. The two-electron reduction potential for the 1HNO, 2H+/NH2OH couple
has been reported to be 0.3 V (versus NHE) (24). This favorable potential indicates
that reduction of HNO to NH2OH may be biologically facile and that nitrogen
oxide species generated from HNO reduction need to be considered as possible
participants in the overall biology of HNO.
Analogous to the reaction of HNO with thiols, reaction of HNO with amines
should generate a substituted N-hydroxyhydrazine as an unstable intermediate. It
may be expected that loss of water from this species will then lead to the formation
of an alkyl diazene (Reaction 16):
R-NH2 + HNO R-NH-NH-OH RNNH + H2 O
16.
Alkyl and aryl diazenes are known to be unstable with respect to dinitrogen (N2)
loss, and oxidative decomposition leads to the formation of alkyl radicals (50, 51).
The reaction of HNO with amines has not been extensively examined beyond
the recently published theoretical treatment (19). However, one study in 1965
reported that reaction of secondary amines with the HNO-donor Angelis salt led
to the generation of dinitrogen and products consistent with radical intermediates
(52). Whether HNO release from Angelis salt was required for this chemistry was
not determined, however.
The reaction of nitroxyl with dioxygen has become a topic of considerable
interest. As discussed above, there appears to be little doubt that the reaction
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of 3NO with O2 to give ONOO is a fast reaction. Of, course, the biological
relevance of this reaction is largely dependent on the existence/levels of 3NO.
The pKa of HNO is approximately 11, making equilibrium concentrations of
3
NO extremely small under physiological conditions. However, as noted above,
biological circumstances whereby 3NO is made directly (if they exist) could lead
to reaction with O2 because protonation is very slow and, therefore, 3NO can
have a significant (millisecond) lifetime (25). A more intriguing, provocative, and
biologically relevant process is the reaction of HNO with O2. Although this reaction
has been analyzed experimentally and theoretically, it remains a controversial and
contentious topic. Several studies by Miranda and coworkers indicate that the
reaction of HNO with O2 results in the generation of a potent two-electron oxidant
whose reaction profile is distinct from that of OONO and/or N2O3 (53, 54).
Interestingly, aerobic decomposition of the HNO-donor molecule Angelis salt
does not generate nitrate (NO

3 ), which would be expected if OONO were the
primary nitroxyl-O2 product (owing to the fact that OONO decomposes to give
predominantly NO
3 ) (2). Moreover, a direct and spin-forbidden reaction of HNO
with O2 to generate HOONO/OONO would be very slow and highly unlikely
(25). Thus, the chemistry and biology of the HNO/O2 reaction remains one of the
most significant and important enigmas in the field of HNO chemistry and biology.
As mentioned above, NO is very difficult to reduce to 3NO (indicated by the
very low reduction potential for the NO/3NO couple). This means that 3NO, if
formed, can be a one-electron reducing agent. An example of this is the reduction
of the cupric form of the enzyme superoxide dismutase (SOD) to the cuprous form
by NO (49, 5557) (Reaction 17):
NO + SODCuII NO + SODCuI
17.
Most of the studies examining the interaction of nitroxyl with SOD were performed prior to the understanding that the primary nitroxyl species present in
solution at physiological pH is HNO rather than 3NO. Thus, all reactions were
written as occurring through the deprotonated anionic species. Although this is possible, previously mentioned difficulties in generating significant concentrations of
NO under biological conditions indicate that coordination of HNO to the metals
followed by deprotonation may be an equally likely mechanism for these reactions. HNO can also react with oxidized hemoproteins, such as methemoglobin, to
generate the ferrous nitrosyl adducts via reductive nitrosylation (26, 42) (Reaction
18):
HNO + HbFeIII HbFeII -NO + H+
Hb = hemoglobin
18.
Interestingly, reduction of a myoglobin-NO adduct (MbFeII-NO) results in the

formation of a stable HNO-Fe(II) adduct (MbFeII-HNO) (58), and more recently
it has been demonstrated that HNO can directly ligate deoxyhemoglobin (59).
It is clear that 3NO is a strong reductant (NO/3NO E0 = 0.8 versus NHE).
The protonated species, HNO, can also be a reasonable reductant under appropriate
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NITROXYL (HNO)
343
conditions. The H-NO bond strength is only 4850 kcal/mol (see, for example,
19). This relatively low bond strength indicates that HNO will be a good hydrogen
atom donor, and, therefore, can be a good reducing agent for radical species. The
product of hydrogen atom abstraction from HNO is NO, which can also react
rapidly with other radical species. Thus, it may be expected that HNO can be
an efficient radical scavenger via H-atom donation with subsequent generation of
another radical scavenger, NO.
To accurately predict which nitrogen oxide species are relevant following biological HNO exposure and which biological targets are affected, it is imperative
that the kinetics of the reactions of HNO are known. To this end, Miranda and
coworkers (60) used competition kinetics to determine the relative rates of reaction of HNO with a variety of possible chemical/biological reactants and derive
approximate rate constants. This study showed that relative reactivity toward HNO
is oxymyoglobin (1 107 M1 s1) > glutathione (GSH), horseradish peroxidase
(2 106 M1 s1) > N-acetyl cysteine, CuZnSOD, MnSOD, metmyoglobin, catalase (310 105 M1 s1) > Tempol, ferricytochrome c (48 104 M1 s1) >
O2 (3 103 M1s1). Considering the high concentrations of GSH in cells, these
results indicate that reaction of HNO with GSH may be a primary fate for cytosolic HNO. However, HNO partitioned into membrane compartments may have a
considerably longer lifetime.
NITROXYL DONORS
Dimerization of HNO (Reaction 3) precludes convenient and direct accessibility
to HNO for chemical or biological studies. Therefore, most of the studies of HNO
chemistry and biology utilize HNO-donor molecules. The best studied, most established and most utilized HNO-donor is sodium trioxodinitrate (Na2N2O3), or
Angelis salt (1 and references therein) (Reaction 1). This inorganic salt is fairly
stable in base but will spontaneously release HNO between pH 48 with a firstorder rate constant of 4.6 104 s1 (61). Thermal degradation of Angelis salt
can never be used as a source of 3NO because conditions for significant HNO
deprotonation (strong base) inhibits the release of HNO. At low pH, Angelis salt
becomes an NO-donor, possibly owing to protonation of a relatively nonbasic site,
resulting in a different mechanism of decomposition (62). Owing to its ability
of release HNO at physiological pH with predictable kinetics, most of the novel
biological effects of HNO have been discovered using Na2N2O3. However, this
compound is limited in that its half-life is only 2.1 min at 37 C (61), making
prolonged HNO delivery difficult. Moreover, NO2 is released as a coproduct that
exhibits its own array of chemistry/biology (see, for example, 63).
Another possible source of nitroxyl is via the decomposition of compounds
with the N-hydroxysulfonamide functional group. The best known of these is
N-hydroxybenzenesulfonamide (Pilotys acid), which, under basic conditions, decomposes to nitroxyl and benzenesulfinate (Reaction 19):
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C5 H6 S(O)2 NHOH + HO C5 H6 -S(O)2 NHO C5 H6 -S(O)O + HNO

19.
The basic conditions required for HNO release allow HNO deprotonation to
occur, making these compounds possible sources of 3NO (unlike Angelis salt,
which cannot be used as a ready source of 3NO). Like Angelis salt, the spin
state of HNO initially generated from Pilotys acid is singlet (64). Biological
studies using N-hydroxysulfonamides can be troublesome because strongly basic
conditions are required for HNO release. N-Hydroxysulfonamides are also readily
oxidized by one electron to give the corresponding nitroxide intermediate, which
releases NO, not HNO (65). For these reasons, Angelis salt has been used more
extensively for biological studies.
Derivatives of N-hydroxysulfonamides have also been developed as HNOdonors. For example, the Nagasawa lab has synthesized a series of N- and/or
O-substituted N-hydroxysulfonamides, which could be activated in biological systems to release HNO (6671). Similarly, N-hydroxybenzenecarboximidic acid
derivatives have also been developed as HNO-donors (72).
HNO can also be generated via a retro-Diels-Alder reaction from acyl- or
phosphinoyl-nitroso-diene cycloadducts (7377). Water-soluble analogs of acylnitroso-anthracene adducts, which are amenable to biological studies, have been
shown to release the acylnitroso moiety, followed by hydrolysis to give HNO
(78).
NITROXYL TRAPS/DETECTION
Studies on the biological actions and biochemistry of HNO are severely hindered
by the lack of specific and convenient traps and/or detectors for HNO. Many previous studies relied on the detection of N2O, which is the ultimate product of HNO
dimerization (Reaction 3), as an indication of the intermediacy or existence of
HNO. This is, however, equivocal because mechanisms exist whereby N2O can be
generated without the intermediacy of free HNO (see, for example, 43). Moreover, HNO dimerization is a second-order process requiring high concentrations
of HNO to get significant reaction in the thiol and metalloprotein-rich environment
of a cell. Considering the existence of other more likely fates for HNO in biological systems (i.e., reaction with thiols), it is not likely that low levels of HNO in
biological systems can be detected via N2O.
Other traps/detectors for HNO exist. For example, one of the first described
traps for nitroxyl is via reaction with tetracyanonickelate [Ni(CN4)2)] to give the
nickel-nitrosyl species (79) (Reaction 20):
2
Ni(CN)2
4 + HNO/NO NiNO(CN)3 + HCN/CN
20.
However, this is a pH-dependent (only occurs at high pH) and inefficient process
and unlikely to be useful in biological systems.
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Deoxymyoglobin efficiently reacts with HNO to form the HNO-Fe(II) complex

(59) (Reaction 21). However, this complex decomposes in the presence of O2 to
give Fe(III) myoglobin, thus limiting its utility as an HNO detector in biological
systems. Further, the aerobic reaction of NO and deoxymyoglobin will also produce
Fe(III) myoglobin, complicating identification of the reacting nitrogen oxide:

MbFe2+ + HNO MbFe2+ -HNO
21.
HNO can also be trapped and detected via reaction with nitrosobenzene (44).
This reaction generates cupferron (N-nitroso-N-phenylhydroxylamine) that can
chelate the cupric ion to form a colored complex (Reaction 22):
Phenyl-NO + HNO Phenyl-NH(OH)NO chelates Cu2+
22.
Unfortunately, nitrosobenzene lacks sufficient water solubility for this assay to

be useful in biological systems. Although the generation of water-soluble analogs
of nitrosobenzenes may prove useful in the future for biological studies, the intrinsic activity of nitroso compounds must be carefully evaluated because, for
example, nitroso compounds are known to bind to and inhibit hemeproteins (80)
as well as being subject to redox processes.
There are other published reports of the trapping/detection of HNO using metal
complexes and metalloproteins. For example, HNO can be directly trapped using
synthetic ferric porphyrins (81) as well as ferric hemoglobin and myoglobin (see,
for example, 82) giving the ferrous nitrosyl complex (Reaction 23):
Porphyrin-Fe3+ + HNO Porphyrin-Fe2+ -NO + H+
23.
Although this represents an efficient trap for HNO, the products of these reactions can also be generated by reaction with NO via a more involved series of
reactions (see, for example, 83) (Reactions 24 and 25):
+
Porphyrin-Fe3+ + NO + H2 O Porphyrin-Fe2+ + NO
2 + 2H
24.
Porphyrin-Fe2+ + NO Porphyrin-Fe2+ -NO
25.
Thus, detection of a ferrous nitrosyl complex from a ferric detector species is

not an explicit indication of the presence of HNO.
BIOLOGICAL GENERATION OF NITROXYL

Thus far, there has been no unequivocal evidence for the endogenous generation of HNO in mammalian systems. This may be due, however, to inefficient or
nonspecific detection systems for this elusive species. Regardless, chemical and
biochemical processes have been characterized that allow for the possibility, if not
probability, of endogenous HNO formation. For example, the S-nitrosothiols can
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react with other thiols to generate HNO according to Reaction 26 (43, 84):

RS-NO + R SH RSSR + HNO
26.
Another source of endogenous nitroxyl is via the oxidative degradation of the

NO biosynthesis intermediate, N-hydroxy-L-arginine (NOHA), which can be released at high levels by some cells both in vitro (85) and in vivo (86). NOHA is
easily oxidized to give nitroxyl (see, for example, 8790). Nitroxyl may also be
generated from L-arginine and/or NOHA by the action of the nitric oxide biosynthesis enzymes (NOS) (91, 92), especially when it is deplete of one of its prosthetic
groups, tetrahydrobiopterin (93, 94). This work provides the possibility that NOS
is capable of generating HNO depending on the experimental/cellular conditions.
Nitroxyl generation has also been reported to occur via the interaction of NO with
elements of the electron transport system in mitochondria (95, 96) from reaction
with ubiquinol (97), cytochrome c (98), manganese superoxide dismutase (99), and
xanthine oxidase (100). Thus, biochemical events have been characterized that can
result in endogenous HNO generation. Although purely speculative at this time, it
is also possible that an HNO-synthase enzyme exists and remains to be discovered
if and when a specific and sensitive HNO-detection system is developed.
NITROXYL PHARMACOLOGY/TOXICOLOGY
It remains uncertain whether HNO is endogenously generated in mammalian cells.
Therefore, the question of whether HNO is an endogenous signaling/effector
species or simply a metabolite of NO remains open. However, numerous studies indicate that exogenous HNO administration results in interesting, novel, and
potentially important pharmacology and toxicology. Some of the earliest studies
of the biological activity of HNO reported that nitroxyl can be a potent vasorelaxant (see, for example, 101). Because NO is known to elicit vasodilation via
activation of the enzyme soluble guanylate cyclase (sGC) (see, for example, 102),
it is possible that HNO was being converted to NO in these experiments. This
seems especially likely because HNO itself has been reported to be incapable of
activating sGC (103). It should be noted that this study was performed using an
in vitro preparation of the enzyme in the presence of the thiol dithiothreitol. As
discussed above, thiols can react rapidly with HNO precluding interaction with
the enzyme. Thus, possible HNO-mediated sGC activation needs reinvestigation.
The ability of HNO to react with thiols predicts that it will be capable of
disrupting thiol proteins. The Nagasawa group was one of the first to demonstrate this as they observed inhibition of the enzymes aldehyde dehydrogenase and
glyceraldehyde-3-phosphate dehydrogenase when exposed to HNO donors (67,
104, 105). The finding that aldehyde dehydrogenase was inhibited by HNO was
the result of the elucidation of the mechanism of action of the alcohol deterrent
drug cyanamide by the Nagasawa lab. They found that cyanamide could be hydroxylated by the enzyme catalase, which resulted in the formation of an unstable
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347
N-hydroxycyanamide intermediate. Decomposition of the N-hydroxycyanamide

resulted in the release of HNO along with hydrogen cyanide (67, 105). Thus,
in vivo, cyanamide can serve as a prodrug for HNO, and indeed, the utility of
cyanamide as an antialcohol drug is due to its ability to release HNO after bioactivation. More recently, HNO was found to disrupt the activity of a copper-sensing
yeast transcription factor, presumably via disruption of metal binding thiolate moieties (106). It should be noted that the ability of HNO to disrupt the actions of
intracellular thiol proteins need not occur via direct interaction with the protein
thiols. HNO has been shown dramatically lower cellular thiol levels (i.e., GSH)
(46), an effect that will alter the redox status of the cell and, subsequently, may
alter the activity of redox-sensitive thiol proteins. Poly(ADP-ribose) polymerase
(PARP), a protein that contains two zinc-finger motifs and that is involved in initiating DNA repair, is also inhibited by HNO (107), as is mitochondrial respiration,
presumably via reaction with critical thiol residues present in complex I and II
(108).
The toxicity of HNO has been examined in a variety of systems. Using in vitro
clonogenic assays, HNO was found to be toxic to fibroblasts via mechanisms not
involving conversion to NO (46). In this study, HNO in the presence of O2 resulted
in dramatic decreases in GSH levels and DNA strand breakage. Although it is
possible that HNO (or NO) can react with O2 to generate the oxidant ONOO, as
mentioned previously, it is reported that the reaction of HNO with O2 generates an
oxidant that is not ONOO and possesses a slightly different oxidation profile (53,
54). This topic remains controversial. However, it is clear that the reaction of HNO
with O2 is capable of generating an oxidizing species that has the potential to react
with and alter biological macromolecules. In contrast to ONOO, the oxidant
generated from HNO and O2 is capable of readily entering cells and reacting
with intracellular species (109). These reports suggest that there are fundamental
differences in the reaction pattern between HNO/O2 and NO/O
2 . The Ohshima
lab has also described the ability of HNO to cause DNA damage (39, 40).
Nitroxyl greatly exacerbates ischemia reperfusion injury when administered
during reperfusion, whereas NO has the opposite effect (110). Interestingly, HNO
given prior to ischemia offers protection against subsequent reperfusion damage
(111). The exacerbation of ischemia reperfusion injury was shown to be due to
increased neutrophil infiltration (110). In a recent and related study, Takahira
and coworkers (112) proposed that neutrophil infiltration in ischemia/reperfusion
injury may actually be due to endogenous HNO generation and that the protective
effects of dexamethazone may be the result of an inhibition of HNO generation.
Nitroxyl has been reported to attenuate the activity of the NMDA receptor by
modifying a critical thiol residue, leading to a decrease in Ca2+ influx (113). This
process was proposed to provide protection from neuroexcitotoxicity. In another
study of the effect of HNO on the NMDA receptor, it was found that HNO blocked
glycine-independent desensitization of the receptor (114). This observation is in
contrast to the findings of Kim et al. (113), which may be partially explained by
differences in experimental design because the levels of O2 may be an important
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factor in the effect of HNO. That is, under hypoxic conditions, HNO appears able
to decrease steady-state ion currents, whereas under normoxic conditions blockade
of glycine-independent desensitization occurs, leading to ion current enhancement
(114). Thus, the ultimate effect of HNO on the NMDA receptor can be a function
of cellular/tissue O2 levels.
One of the most recent and provocative pharmacological actions of nitroxyl
appears to be as a unique cardiovascular agent. Paolocci and coworkers (115)
have reported that HNO increases left ventricular contractility while lowering
cardiac preload and diastolic pressure without an increase in arterial resistance.
This novel activity is likely responsible for much of the recent attention given
to HNO (see below). The actions of HNO on the vascular system have been
found to be mediated by calcitonin gene-related peptide (CGRP). Significantly,
the effects of HNO were unaffected by -receptor blockade and additive to those
of the 1-selective agonist dobutamine, indicating that the effects of HNO are
independent of -adrenergic signaling (60, 116). Also, HNO administration to
animals does not result in an increase in cGMP, indicating that the vascular effects
were not due to enhancement of levels of this second messenger. CGRP is a broadly
distributed neuropeptide found in many cardiovascular tissues and is the most
potent vasorelaxant currently known, with established positive inotropic effects
on the human heart (see, for example, 117, 119). The actions of CGRP occur
through activation of the calcitonin-receptor-like receptor (CRLP), which leads
to activation of adenylate cyclase and elevation of intracellular cAMP (see, for
example, 118, 119). Increases in cAMP results in PKA activation followed by
phosphorylation of L-type Ca2+ channels and, eventually, vasodilation. Thus, the
actions of HNO, at least with regard to the cardiovascular system, appear to occur
primarily through a cAMP-mediated pathway. This is in contrast to NO, whose
actions in the cardiovascular system are mediated by cGMP. This fundamental
difference in signaling indicates that HNO is not merely converted to NO and that
the two species have orthogonal signaling pathways (60).
The ability of nitroxyl to elicit CGRP-mediated responses in vivo makes it a
candidate for the treatment of heart failure because it will increase heart contractility while decreasing vascular resistance. As pointed out by Feelisch (119), with the
current lack of selective CGRP-mimetics and the increasing interest in inodilators,
the potential for HNO-donors to be developed as drugs to be used in heart failure
is significant. Of course, it needs to be realized that these ideas are in the early
stages of development; much more work needs to be done before the therapeutic
utility of HNO and HNO-donor drugs can be properly evaluated.
SUMMARY
Recent reevaluation of some of the fundamental chemical properties and reactivity of HNO provides a basis to begin to understand the chemistry responsible for some of its novel and potentially important biology. However, a clear
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NITROXYL (HNO)
349
understanding of the chemistry and specific biological targets associated with the
physiological/pharmacological actions of HNO remains to be established and is an
area of extreme interest/need. It is clear that nitroxyl possesses biological properties
unique from those of other nitrogen oxides and that may be of significant physiological/pharmacological importance. The idea that the actions of HNO are, in part,
mediated through cAMP, whereas NO regulates through cGMP is intriguing and
represents an interesting physiological paradigm whereby redox nitrogen oxide
congeners have orthogonal signaling properties. Whether this redox nitrogen oxide system is physiologically important is a question that remains and is contingent
upon, among other things, determining whether HNO is generated endogenously,
and, if so, under what conditions.
The utility of HNO as a possible therapeutic agent, for example, in the treatment of heart failure or as a preconditioning agent to prevent ischemia-reperfusion
injury needs to be reconciled with its possible toxicity. Of course, this is not an
uncommon situation, as the same issues are important for the development of NO
as a therapeutic agent. However, it is worth noting that although HNO donors at
millimolar levels have significant toxicity (46), animal studies have shown that
long-term administration of the HNO donor Angelis salt is very well tolerated,
with an LD50 well above 130 mg/kg with no observable carcinogenesis (120). This
concentration is greater than 10,000 times that which has been shown to demonstrate beneficial cardiovascular effects. Regardless, HNO can now take its place
among other nitrogen oxides and oxygen-derived species as an important signaling/effector species possessing novel and possibly useful pharmacology as well
as toxicological properties. Considering that many of the major discoveries of the
physiological chemistry and biology of nitroxyl are relatively recent, it may be
expected that many more interesting and important discoveries await.
LITERATURE CITED
1. Bonner FT, Hughes MN. 1988. The aqueous solution chemistry of nitrogen in low
positive oxidation states. Comments Inorg. Chem. 7:21534
2. Hughes MN. 1999. Relationships between nitric oxide, nitroxyl anion, nitrosonium cation and peroxynitrite. Biochim.
3. Miranda KM. 2004. The chemistry of nitroxyl (HNO) and implications in biology.
Coord. Chem. Rev. In press
4. Koppenol WH, Traynham JG. 1996. Say
NO to nitric oxide: nomenclature for
5.
6.
7.
8.
nitrogen- and oxygen-containing compounds. Methods Enzymol. 268:36

Angeli A. 1903. Gazz. Chim. Ital. 33(II):
245
Garber EA, Wehrli S, Hollocher TC.
1983. 15N-Tracer and NMR studies on the
pathway of denitrification. J. Biol. Chem.
258:358791
Hawthorne MF. 1956. aci-Nitroalkanes.
II. The mechanism of the Nef reaction.
J. Am. Chem. Soc. 79:251015
Kohout FC, Lampe FW. 1967. Massspectrometric study of the reactions of
4 Dec 2004
13:24
350
9.

10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE
FUKUTO ET AL.
deuterium and hydrogen atoms with nitric
oxide. J. Chem. Phys. 46:407584
Bonner FT, Dzelzkalns LS, Bonucci JA.
1978. Properties of nitroxyl as intermediate in the nitric oxide-hydroxylamine reaction and in trioxodinitrate decomposition. Inorg. Chem. 17:248794
Cooper JN, Chilton JE Jr, Powell RE.
1970. Reaction of nitric oxide with alkaline hydroxylamine. Inorg. Chem. 9:
23034
Brown HW, Pimentel GC. 1958. Photolysis of nitromethane and of methyl nitrite
in an argon matrix; infrared detection of
nitroxyl, HNO. J. Chem. Phys. 29:88388
Gratzel M, Taniguchi S, Henglein A.
1970. A pulse radiolytic study of shortlived byproducts on nitric oxide reduction in aqueous solution. Ber. Bunsenges.
Phys. Chem. 74:100310
Seddon WA, Fletcher JW, Sopchyshyn
FC. 1973. Pulse radiolysis of nitric oxide in aqueous solution. Can. J. Chem.
51:112330
Gorman AA, Rogers MA. 1989. CRC
Handbook of Organic Photochemistry,
ed. JC Scaiano, vol. II, pp. 22947. Boca
Raton: CRC Press
Tennyson J, Noble CJ. 1986. Low-energy
electron scattering by the NO molecule.
J. Phys. B. 19:402533
Tronc M, Huetz A, Landau M, Pichou F,
Reinhardt J. 1975. Resonant vibrational
excitation of the NO ground state by electron impact in the 0.10.3 eV energy
range. J. Phys. B. 8:116069
Burrow PD. 1974. Temporary negative
ion formation in NO and O2. Chem. Phys.
Lett. 26:26568
Szmytkowski C, Maciag K. 1991. Total
cross section for electron impact on nitrogen monoxide. J. Phys. B. 24:427379
Bartberger MD, Fukuto JM, Houk KN.
2001. On the acidity and reactivity of
HNO in aqueous solution and biological systems. Proc. Natl. Acad. Sci. USA
98:219498
Stanbury DM. 1989. Reduction potentials
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
involving inorganic free radicals in aqueous solution. Adv. Inorg. Chem. 33:69
138
Janaway GA, Zhong M, Gatev GG,
Chabinyc ML, Brauman JI. 1997.
[FHNO]: An intermediate in a spin
forbidden proton transfer reaction. J. Am.
Chem. Soc. 119:1169798
Janaway GA, Brauman JI. 2000. Direct observation of spin forbidden proton
transfer reactions: 3NO + HA 1HNO
+ A. J. Phys. Chem. A 104:179598
Bartberger MD, Liu W, Ford E, Miranda
KM, Switzer C, et al. 2002. The reduction
potential of nitric oxide (NO) and its importance to NO biochemistry. Proc. Natl.
Shafirovich V, Lymar SV. 2002. Nitroxyl
and its anion in aqueous solutions: spin
states, protic equilibria, and reactivities
toward oxygen and nitric oxide. Proc.
Shafirovich V, Lymar SV. 2003. Spinforbidden deprotonation of aqueous nitroxyl (HNO). J. Am. Chem. Soc. 125:
654752
Bazylinski DA, Hollocher TC. 1985. Evidence from the reaction between trioxodinitrate(II) and 15NO that trioxodinitrate(II) decomposes into nitrosyl hydride
and nitrite in neutral aqueous solution. Inorg. Chem. 24:428588
Ehman DL, Sawyer DT. 1968. Electrochemistry of nitric oxide and of nitrous
acid at a mercury electrode. J. Electroanal. Chem. 16:54149
Benderskii VA, Krienko AG, Ponomarev
EA. 1989. Homogenous and electrode reactions of NO2
2 ions. Sov. Electrochem.
25:15461
Liochev SI, Fridovich I. 2003. The
mode of decomposition of Angelis salt
(Na2N2O3) and the effects thereon of oxygen, nitrite, superoxide dismutase and glutathione. Free Radic. Biol. Med. 34:1399
404
Peslak J Jr, Klett DS, David CW. 1971.
An ab initio molecular orbital study of the
4 Dec 2004
13:24
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE
NITROXYL (HNO)
31.

32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
hydrogen, lithium and fluorine derivatives

of nitric oxide. J. Am. Chem. Soc. 93:
50015
Gallup GA. 1975. Self-consistent field
calculation of nitrosyl hydride and nitrogen hydroxide. Inorg. Chem. 14:56365
Luna A, Merchan M, Roos BO. 1995.
A theoretical analysis of the lowest excited states in HNO/NOH and HPO/POH.
Chem. Phys. 196:43745
Guandagnini R, Schatz GC, Walch SP.
1995. Global potential energy surfaces
for the lowest 1A , 3A and 1A states of
HNIO. J. Chem. Phys. 102:77483
Donald CE, Hughes MN, Thompson JM,
Bonner FT. 1986. Photolysis of the N = N
bond in trioxodinitrate: reaction between
triplet NO and O2 to form peroxynitrite.
Inorg. Chem. 25:267677
Huie RE, Padmaja S. 1993. The reaction
of NO with superoxide. Free Rad. Res.
Commun. 18:19599
Goldstein S, Czapski G. 1995. The reaction of NO with O
2 and HO2: a pulse
radiolysis study. Free Radic. Biol. Med.
19:50510
Seddon WA, Young MJ. 1970. Pulse radiolysis of nitric oxide in aqueous solution.
Can. J. Chem. 48:39394
Ohshima H, Yoshie Y, Auriol S, Gilibert
I. 1998. Antioxidant and prooxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite
and nitroxyl anion. Free Radic. Biol. Med.
25:105765
Ohshima H, Gilibert I, Bianchini F. 1999.
Induction of DNA strand breakage and
base oxidation by nitroxyl anion through
hydroxyl radical production. Free Radic.
Biol. Med. 26:130513
Chazotte-Aubert L, Oikawa S, Gilibert I,
Bianchini F, Kawanishi S, Ohshima H.
1999. Cytotoxicity and site-specific DNA
damage induced by nitroxyl anion (NO)
in the presence of hydrogen peroxide. J.
Biol. Chem. 274:2090915
Pino RZ, Feelisch M. 1994. Bioassay discrimination between nitric ox-
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
351
ide (NO) and nitroxyl (NO) using cysteine. Biochem. Biophys. Res. Commun.
201:5462
Doyle MP, Mahapatro SN, Broene RD,
Guy JK. 1988. Oxidation and reduction of
hemoproteins by trioxodinitrate(II). The
role of nitrosyl hydride and nitrite. J. Am.
Chem. Soc. 110:59399
Wong PSY, Hyun J, Fukuto JM, Shiroda FN, DeMaster EG, Nagasawa HT.
1998. The reaction between nitrosothiols
and thiols: generation of nitroxyl (HNO)
and subsequent chemistry. Biochemistry
37:536271
Shoeman DW, Nagasawa HT. 1998. The
reaction of nitroxyl (HNO) with nitrosobenzene gives cupferron (N-nitrosophenylhydroxylamine). Nitric Oxide Biol.
Chem. 2:6672
Woo HA, Chae HZ, Hwang SC, Yang KS,
Kim K, Rhee SG. 2003. Reversing the
inactivation of peroxiredoxins caused by
cysteine sulfinic acid formation. Science
300:59294
Wink DA, Feelisch M, Fukuto J,
Christodoulou D, Jourdheuil D, et al.
1998. The cytotoxicity of nitroxyl: possible implications for the pathophysiological role of NO. Arch. Biochem. Biophys.
351:6674
Reif A, Zecca L, Riederer P, Feelisch M,
Schmidt HHHW. 2001. Nitroxyl oxidizes
NADPH in a superoxide dismutase inhibitable manner. Free Radic. Biol. Med.
30:8038
Liochev SI, Fridovich I. 2001. Copper,
zinc superoxide dismutase as a univalent NO-oxidoreductase and as a dichlorofluorescin peroxidase. J. Biol. Chem.
38:3525357
Liochev SI, Fridovich I. 2002. Nitroxyl
(NO-): a substrate for superoxide dismutase. Arch. Biochem. Biophys. 402:166
71
Huang PC, Kosower EM. 1968. Diazenes.
III. Properties of phenyldiazene. J. Am.
Chem. Soc. 90:236776
Kosower EM. 1971. Monosubstituted
4 Dec 2004
13:24
352
52.

53.
54.
55.
56.
57.
58.
59.
60.
61.
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE
FUKUTO ET AL.
diazenes (diimides). Surprising intermediates. Acc. Chem. Res. 4:19398
Lemal DM, Rave TW. 1965. Diazenes
from Angelis salt. J. Am. Chem. Soc. 87:
39394
Miranda KM, Espey MG, Yamada K, Krishna M, Ludwick N, et al. 2001. Unique
oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion. J.
Biol. Chem. 276:172027
Miranda KM, Yamada K, Espey MG,
Thomas DD, DeGraff W, et al. 2002. Further evidence for distinct reactive intermediates from nitroxyl and peroxynitrite:
effects of buffer composition on the chemistry of Angelis salt and synthetic peroxynitrite. Arch. Biochem. Biophys. 401:134
44
Murphy ME, Seis H. 1991. Reversible
conversion of nitroxyl anion to nitric oxide by superoxide dismutase. Proc. Natl.
Fukuto JM, Hobbs AJ, Ignarro LJ. 1993.
Conversion of nitroxyl (HNO) to nitric
oxide in biological systems: the role of
physiological oxidants and relevance to
the biological activity of HNO. Biochem.
Hobbs AJ, Fukuto JM, Ignarro LJ. 1994.
Formation of free nitric oxide from Larginine by nitric oxide synthase: direct
enhancement of generation by superoxide dismutase. Proc. Natl. Acad. Sci. USA
91:1099296
Lin R, Farmer PJ. 2000. The HNO adduct
of myoglobin: synthesis and characterization. J. Am. Chem. Soc. 122:239394
Sulc F, Immoos CE, Pervitsky D, Farmer
PJ. 2004. Efficient trapping of HNO by
deoxyhemoglobin. J. Am. Chem. Soc.
126:1096101
Miranda KM, Paolocci N, Katori T,
Thomas DD, Ford E, et al. 2003. A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system. Proc. Natl. Acad. Sci.
USA 100:9197201
Bonner FT, Ravid B. 1975. Thermal de-
62.
63.
64.
65.
66.
67.
68.
69.
70.
composition of oxyhyponitrite (sodium

trioxodinitrate(II)) in aqueous solution.
Dutton AS, Fukuto JM, Houk KN. 2004.
Mechanisms of HNO and NO production from Angelis Salt: density functional
and CBS-QB3 theory predictions. J. Am.
Chem. Soc. 126:3795800
van der Vliet A, Eiserich JP, Halliwell
B, Cross CE. 1997. Formation of reactive nitrogen species during peroxidasecatalyzed oxidation of nitrite. J. Biol.
Chem. 272:761725
Bonner FT, Ko Y. 1992. Kinetic, isotopic, and 15N NMR study of N-hydroxybenzenesulfonamide decomposition: an
HNO source reaction. Inorg. Chem. 31:
251419
Zamora R, Grzesiok A, Weber H, Feelisch
M. 1995. Oxidative release of nitric oxide
accounts for guanylate cyclase stimulating. Vasodilator activity of Pilotys acid:
a comparison with Angelis salt. Biochem.
J. 312:33339
Lee MJC, Elberling JA, Nagasawa HT.
1992. N1-Hydroxylated derivatives of
chlorpropamide and its analogs as inhibitors of aldehyde dehydrogenase. J.
Med. Chem. 35:364147
Lee MJC, Nagasawa HT, Elberling JA,
DeMaster EG. 1992. Prodrugs of nitroxyl
as inhibitors of aldehyde dehydrogenase.
J. Med. Chem. 35:364852
Fukuto JM, Hszieh R, Gulati P, Chiang KT, Nagasawa HT. 1992. N,ODiacylated - N-hydroxyarylsulfonamides:
nitroxyl precursors with potent smooth
muscle relaxant properties. Biochem.
Conway TT, DeMaster EG, Lee MJC, Nagasawa HT. 1998. Prodrugs of nitroxyl
and nitrosobenzene as cascade latentiated
inhibitors of aldehyde dehydrogenase. J.
Med. Chem. 41:29039
Nagasawa HT, Kawle SP, Elberling JA,
DeMaster EG, Fukuto JM. 1995. Prodrugs of nitroxyl as potential aldehyde dehydrogenase inhibitors vis-à-vis vascular
4 Dec 2004
13:24
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE
NITROXYL (HNO)
71.

72.
73.
74.
75.
76.
77.
78.
79.
smooth muscle relaxants. J. Med. Chem.

38:186571
Conway TT, DeMaster EG, Goon DJW,
Shirota FN, Nagasawa HT. 1999. Diethylcarbamoylating nitroxylating agents
as dual action inhibitors of aldehyde
dehydrogenase: a disulfiram-cyanamide
merger. J. Med. Chem. 42:401620
Lee MJC, Shoeman DW, Goon DJW, Nagasawa HT. 2001. N-Hydroxybenzenecarboximidic acid derivatives: a new class
of nitroxyl-generating prodrugs. Nitric
Oxide Biol. Chem. 5:27887
Corrie JET, Kirby GW, Laird AE, MacKinnon LW, Tyler JK. 1978. Detection
by microwave spectroscopy of HNO produced by a retro-Diels-Alder reaction. J.
C. S. Chem. Comm. pp. 27576
Ensley HE, Mahadevan S. 1989. DielsAlder and ene reactions of nitrosyl hydride and nitrosoformaldehyde. Tett. Lett.
30:325558
Ware RW, King SB. 1999. N-Phosphinoylnitroso compounds: new asymmetric N-O heterodienophiles and nitroxyl delivery agents. J. Am. Chem. Soc.
121:676970
Ware RW, King SB. 2000. P-Nitrophosphate compounds: new N-O heterodienophiles and nitroxyl-delivery agents.
J. Org. Chem. 65:872529
Xu Y, Alavanja MM, Johnson VL, Yasaki
G, King SB. 2000. Production of nitroxyl (HNO) at biologically relevant temperatures from retro-Diels-Alder reaction
of N-hydroxyurea-derived acyl nitroso9,10-dimethytlanthracene cycloadducts.
Tett. Lett. 41:426569
Zeng B, King SB. 2003. Water soluble
anthracene-acyl nitrosocycloadducts as
nitroxyl precursors through retro-DielsAlder reactions. Southeast Regional
Meet. Am. Chem. Soc., 55th, Nov. 1619,
Atlanta, GA
Bonner FT, Akhtar MJ. 1981. Formation of nitrosotricyanonickelate (NiNO
(CN)2
3 ) in a direct displacement reaction.
353
80. Fukuto JM, Brady JF, Burstyn JN,

VanAtta RB, Valentine JS, Cho AK. 1986.
Direct formation of complexes between
cytochrome P450 and nitrosoarenes. Biochemistry 25:271419
81. Bari SE, Marti MA, Amorebieta VT, Estrin DA, Doctorovich F. 2003. Fast nitroxyl trapping by ferric porphyrins. J.
Am. Chem. Soc. 125:1527273
82. Bazylinski DA, Hollocher TC. 1985. Metmyoglobin and methemoglobin as efficient traps for nitrosyl hydride (nitroxyl)
in neutral aqueous solution. J. Am. Chem.
Soc. 107:798286
83. Wayland BB, Olson LW. 1974. Spectroscopic studies and bonding model for nitric oxide complexes of iron porphyrins.
J. Am. Chem. Soc. 96:603741
84. Arnelle DR, Stamler JS. 1995. NO+, NO
and NO donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch.
85. Buga GM, Singh R, Pervin S, Rogers NE,
Schmitz DA, et al. 1996. Arginase activity in endothelial cells: inhibition by
N-hydroxy-L-arginine during high output NO production. Am. J. Physiol. Heart
Circ. Physiol. 271:H198898
86. Hecker M, Schott C, Bucher B, Bussi R,
Stoclet J-C. 1995. Increase in serum Nhydroxy-L-arginine in rats treated with
bacterial lipopolysaccharide. Eur. J. Pharmacol. 275:R13
87. Yoo J, Fukuto JM. 1995. Oxidation of Nhydroxyguanidine by nitric oxide and the
possible generation of vasoactive species.
88. Fukuto JM, Wallace GC, Hszieh R,
Chaudhuri G. 1992. The chemical oxidation of N-hydroxyguanidine compounds:
release of nitric oxide, nitroxyl and the relevance to the mechanism of nitric oxide
biosynthesis. Biochem. Pharmacol. 43:
60713
89. Fukuto JM, Stuehr DJ, Feldman PL, Bova
MP, Wong P. 1993. Peracid oxidation
4 Dec 2004
13:24
354

90.
91.
92.
93.
94.
95.
96.
97.
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE
FUKUTO ET AL.
of N-hydroxyguanidine compounds: a
chemical model for the oxidation of Nhydroxy-L-arginine by nitric oxide synthase. J. Med. Chem. 36:266670
Cho JY, Dutton A, Miller T, Houk
KN, Fukuto JM. 2003. Oxidation of Nhydroxyguanidines by copper(II): model
systems for elucidating the physiological chemistry of the nitric oxide (NO)
biosynthetic intermediate N-hydroxyl-Larginine. Arch. Biochem. Biophys. 417:
6576
Hobbs AJ, Fukuto JM, Ignarro LJ. 1994.
Formation of free nitric oxide from Larginine by nitric oxide synthase and enhancement of generation by superoxide
dismutase. Proc. Natl. Acad. Sci. USA
91:1099296
Schmidt HHHW, Hofman H, Schindler U,
Shutenko ZS, Cunningham DD, Feelisch
M. 1996. No NO from NO synthase. Proc.
Rusche KM, Spiering MM, Marletta
MA. 1998. Reactions catalyzed by
tetrahydrobiopterin-free nitric oxide synthase. Biochemistry 37:1550312
Adak S, Wang Q, Stuehr DJ. 2000. Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric oxide
synthase. J. Biol. Chem. 275:3355461
Clarkson RB, Norby SW, Smirnov A,
Boyer S, Vahidi N, et al. 1995. Direct
measurement of the accumulation and
mitochondrial conversion of nitric oxide
within Chinese hamster ovary cells using
an intracellular electron paramagnetic resonance technique. Biochim. Biophys. Acta
1243:496502
Zhao X-J, Sampath V, Caughey WS. 1995.
Cytochrome c oxidase catalysis of reduction of nitric oxide to nitrous oxide.
105460
Poderoso JJ, Carreras MC, Schopfer F,
Lisdero CL, Riobo NA, et al. 1999. The
reaction of nitric oxide with ubiquinol:
kinetic properties and biological significance. Free Radic. Biol. Med. 26:92535
98. Sharpe MA, Cooper CE. 1998. Reactions

of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the
formation of nitroxyl anion and peroxynitrite. Biochem. J. 332:919
99. Niketic V, Stojanovic S, Nikolic A, Spasic M, Michelson AM. 1999. Exposure of
Mn and FeSODs, but not Cu/ZnSOD, to
NO leads to nitrosonium and nitroxyl ions
generation which cause enzyme modification and inactivation: an in vitro study.
Free Radic. Biol. Med. 27:99296
100. Saleem M, Ohshima H. 2004. Xanthine
oxidase converts nitric oxide to nitroxyl
that inactivates the enzyme. Biochem. Biophys. Res. Commun. 315:45562
101. Fukuto JM, Chiang K, Wong P, Chaudhuri
G. 1992. The pharmacological activity of
nitroxyl: a potent vasodilator with activity
similar to nitric oxide and/or endotheliumderived relaxing factor. J. Pharmacol.
Exp. Ther. 263:54651
102. Hobbs AJ. 1997. Soluble guanylate cyclase: the forgotten sibling. Trends Pharmacol. Sci. 18:48491
103. Dierks EA, Burstyn JN. 1996 Nitric oxide (NO), the only nitrogen monoxide redox form capable of activating soluble
guanylate cyclase. Biochem. Pharmacol.
51:1593600
104. Nagasawa HT, DeMaster EG, Redfern
B, Shirota FN, Goon DJW. 1990. Evidence for nitroxyl in the catalasemediated bioactivation of the alcohol deterrent agent cyanamide. J. Med. Chem.
33:312022
105. DeMaster EG, Redfern B, Nagasawa
HT. 1998. Mechanisms of inhibition of
aldehyde dehydrogenase by nitroxyl, the
active metabolite of the alcohol deterrent cyanamide. Biochem. Pharmacol. 56:
200715
106. Cook NM, Shinyashiki M, Jackson MI,
Leal FA, Fukuto JM. 2003. Nitroxyl
(HNO)-mediated disruption of thiol proteins: inhibition of the yeast transcription factor Ace1. Arch. Biochem. Biophys.
410:8995
4 Dec 2004
13:24
AR
AR232-PA45-14.tex
AR232-PA45-14.sgm
LaTeX2e(2002/01/18)
P1: GCE

NITROXYL (HNO)
107. Sidorkina O, Espey MG, Miranda KM,
Wink DA, Laval J. 2003. Inhibition of
poly(ADP-ribose) polymerase (PARP) by
nitric oxide and reactive nitrogen species.
108. Shiva S, Crawford JH, Ramachandran
A, Ceaser EK, Hillson T, et al. 2004.
Mechanisms of the interaction of nitroxyl with mitochondria. Biochem. J.
doi:10.1042/BJ20031758
109. Espey MG, Miranda KM, Thomas DD,
Wink DA. 2002. Ingress and reactive
chemistry of nitroxyl-derived species
within human cells. Free Radic. Biol.
Med. 33:82734
110. Ma XL, Cao F, Liu GL, Lopez BL,
Christopher TA, et al. 1999. Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury. Proc. Natl.
111. Pagliaro P, Mancardi D, Rastaldo R,
Penna C, Gattullo D, et al. 2003. Nitroxyl
affords thiol-sensitive myocardial protective effects akin to early preconditioning.
112. Takahira R, Yonemura K, Fujise Y,
Hishida A. 2001. Dexamethazone attenuates neutrophil infiltration in the rat kidney in ischemia/reperfusion injury: the
possible role of nitroxyl. Free Radic. Biol.
Med. 31:80915
113. Kim W-K, Choi Y-B, Rayudu PV, Das
P, Asaad W, et al. 1999. Attenuation of
NMDA receptor activity and neurotoxicity by nitroxyl anion. Neuron 24:46169
355
114. Colton CA, Gbadegesin M, Wink DA,

Miranda KM, Espey MG, Vicini S. 2001.
Nitroxyl anion regulation of the NMDA
receptor. J. Neurochem. 78:112634
115. Paolocci N, Saavedra WF, Miranda
KM, Martignani C, Isoda T, et al. 2001.
Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling.
68
116. Paolocci N, Katori T, Champion HC,
St. John ME, Miranda KM, et al. 2003.
Positive inotropic and lusitropic effects
of HNO/NO in failing hearts: independence from -adrenergic signaling. Proc.
Nat. Acad. Sci. USA 100:553742
117. Juaneda C, Dumont Y, Quirion R. 2000.
The molecular pharmacology of CGRP
and related peptide receptor subtypes.
118. Huang M-H, Knight PR, Izzo JL. 1999.
Ca2+-induced Ca2+ release involved in
positive inotropic effect mediated by
CGRP in ventricular myocytes. Am. J.
Physiol. Regul. Integr. Comp. Physiol.
276:R25964
119. Feelisch M. 2003. Nitroxyl gets to the
heart of the matter (commentary). Proc.
120. Keefer LK, Anderson LM, Diwan BA,
Driver CL, Haines DC, et al. 1995. Experimental tests of the mutagenicity and
carcinogenicity of nitric oxide and its progenitors. Methods 7:12130
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Copyright

TYROSINE KINASE INHIBITORS AND THE DAWN OF

MOLECULAR CANCER THERAPEUTICS
Raoul Tibes, Jonathan Trent, and Razelle Kurzrock
Division of Cancer Medicine, University of Texas M.D. Anderson Cancer Center,
Houston, Texas 77030; email: rkurzroc@mdanderson.org
Key Words imatinib mesylate, Bcr-Abl, KIT, kinase, cancer

Abstract The clinical application of tyrosine kinase inhibitors for cancer treatment represents a therapeutic breakthrough. The rationale for developing these compounds rests on the observation that tyrosine kinase enzymes are critical components
of the cellular signaling apparatus and are regularly mutated or otherwise deregulated
in human malignancies. Novel tyrosine kinase inhibitors are designed to exploit the
molecular differences between tumor cells and normal tissues. Herein, we will review
the current state-of-the-art using agents that target as prototypes Bcr-Abl, plateletderived growth factor receptor (PDGFR), KIT (stem cell factor receptor), and epidermal
growth factor receptor (EGFR). These compounds are remarkably effective in treating
diverse cancers that are highly resistant to conventional treatment, including various
forms of leukemia, hypereosinophilic syndrome, mast cell disease, sarcomas, and lung
cancer. It is now clear that the molecular defects underlying cancer can be targeted
with designer drugs that yield striking salutary effects with minimal toxicity.
INTRODUCTION
Cancer is the second most common cause of death in developed countries and
is a rising health problem in less developed parts of the world. The diagnosis
of cancer carries great physical and mental suffering for affected individuals and
poses a significant burden on the health care system. For many tumors, conventional
management strategies (surgery, radiation, chemotherapy) have high toxicity with
Abbreviations: ATF2: Activating transcription factor; ERK: Extracellular regulated kinase;

GCK: Glucokinase; Grb2: growth factor receptor-bound protein 2; JNK: Jun N-terminal kinase/Janus kinase; MAPK: Mitogen-activated protein kinase; MEK: Mitogen ERK; MEKK:
Mitogen ERK kinase; MLK: Mixed lineage kinase; PAK: p21 activated kinase; PI3K:
phosphatidylinositide-3-kinase; PKC: protein kinase c; Myc: avian myelocytomatosis viral
oncogene homologue; PLC : Phospholipase C ; RAF: murine leukemia viral oncogene
homologue; Ras: Rat sarcoma viral oncogene homologue; SH: src homology domain; S6K:
S6-kinase; Sos: Son of sevenless; STAT: Signal transducer and activator of transcription;
TPI2: thiol proteinase inhibitor 2.
0362-1642/05/0210-0357$14.00
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marginal efficacy. The consensus that has emerged among investigators is that
surmounting the cancer therapeutic problem will be greatly facilitated by an indepth understanding of the molecular genetics underlying individual malignancies.
Autonomous cell growth resulting in tissue invasion and metastases is the defining feature of all malignant neoplasms (1). Cancers do not necessarily arise solely
as a result of an accelerated rate of cell proliferation. Rather, they are the consequence of an imbalance between the rate of cell-cycle progression (cell division)
and cell growth (cell mass) on one hand and programmed cell death (apoptosis)
on the other. Researchers now recognize that aberrant cellular signal transduction pathways play a vital role in driving this imbalance and hence in malignant
transformation (1).
Perhaps one of the most critical groups of signaling molecules involved in normal and abnormal cellular regulation are the tyrosine kinases (2). These proteins
constitute a family of enzymes that catalyze the phosphorylation of the tyrosine
residues of various target molecules. This process controls fundamental cellular
processes including cell cycle, migration, metabolism, proliferation, differentiation, and survival (2). Importantly, several tyrosine kinases are aberrantly expressed
in malignancies. The underlying defects may include, but are not limited to, mutation, hybrid gene formation, amplification, and perturbation of transcriptional
machinery (3).
In this review, we will highlight the role of select tyrosine kinasesBcr-Abl,
KIT, and platelet-derived growth factor receptor (PDGFR)in the clinical setting.
A specific inhibitor (imatinib mesylate) has been developed against these kinases,
and this compound demonstrates definitive therapeutic activity. More recently,
other kinases, including epidermal growth factor receptor (EGFR) and the vascular
endothelial growth factor (VEGF) system, have also been targeted successfully.
On the basis of the knowledge gained in the emerging field of molecular cancer
therapeutics, scientists are now developing a wealth of new compounds.
STRUCTURE AND FUNCTION OF TYROSINE KINASES:

AN OVERVIEW
Tyrosine kinases, enzymes that add a phosphate group to a tyrosine residue in a
protein substrate, exist as receptor-coupled forms (the receptor tyrosine kinases)
and cytosolic forms (2). Some kinases, such as Abl, may also be nuclear (46).
Common features of all tyrosine kinases include a separate domain for substrate
binding, ATP binding, and catalysis (Figure 1) (2). The latter domain promotes
the transfer of the terminal phosphoryl group from ATP to a tyrosine amino group
acceptor in a substrate. Autophosphorylation may also occur.
Over 90 tyrosine kinases have been identified, more than half of which are the
transmembrane receptor type; the balance are the cytoplasmic nonreceptor type (3).
Tyrosine kinase receptors transduce signals from both outside and inside the cell
and function as relay points for signaling pathways inside the cell. The cytoplasmic
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TYROSINE KINASE INHIBITORS
359
nonreceptor tyrosine kinases lack a transmembrane segment and generally function

downstream of the receptor tyrosine kinases (7).
Receptor tyrosine kinases comprise an extracellular domain containing a ligandbinding site, a single hydrophobic transmembrane helix, and a cytosolic domain
that includes a region with protein-tyrosine kinase activity. Ligand binding causes
most receptor tyrosine kinases to dimerize. The protein kinase of the receptor
monomer phosphorylates a distinct set of tyrosine residues in the cytosolic domain of its dimer partner (autophosphorylation). Initially, tyrosine residues in the
phosphorylation lip near the catalytic site are phosphorylated. This leads to a conformational change that facilitates binding of ATP in some receptors (such as the
insulin receptor) and binding of protein substrates in other receptors (such as the
fibroblast growth factor receptor). Subsequently, the receptor kinase activity phosphorylates other sites in the cytosolic domain. The resulting phosphotyrosines serve
as docking sites for adapter proteins containing src homology 2 (SH2) domains.
These adapter proteins can either phosphorylate effector molecules themselves or,
if devoid of kinase activity, couple the activated receptors to other components of
the signal transduction pathway (2, 710) (Figure 1).
Altered tyrosine kinases drive the development of several malignancies. There
are four major mechanisms for oncogenic transformation by tyrosine kinases:
(a) retroviral transduction of a proto-oncogene corresponding to a tyrosine kinase, concomitant with deregulating structural changes (a common transforming
mechanism in animals) (11); (b) genomic rearrangements, such as chromosomal
translocations, which result in oncogenic fusion proteins containing a tyrosine
kinase catalytic domain and part of an unrelated protein (e.g., Bcr-Abl in Philadelphia chromosomepositive leukemias); (c) gain-of-function mutations or small
deletions in tyrosine kinases (e.g., KIT in gastrointestinal stromal tumors); and
(d) tyrosine kinase overexpression resulting from gene amplification (e.g., EGFR
in several solid tumors) (3). In general, the transforming effect can be ascribed
to enhanced or constitutive kinase activity that escapes normal cellular control
mechanisms and induces quantitatively or qualitatively altered downstream signaling. It is now apparent that aberrant kinases are excellent targets for therapeutic
intervention.
Development of Tyrosine Kinase Inhibitors:

Imatinib Mesylate as a Prototype
Initially, protein kinase enzymes were thought to be poor treatment targets because
of their ubiquitous nature and critical role in diverse physiologic processes. However, the advent of imatinib mesylate as a prototype of signal transduction inhibitors
(STI) demonstrated that designer tyrosine kinase inhibitors could be specific and
effective therapeutic tools (1242). This is because kinases are notably distinct
in how their catalysis is regulated, even though they share catalytic domains conserved in sequence and structure. The ATP binding pocket lies between the two
lobes of the kinase fold. This site, together with the less conserved surrounding
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pockets, has been the focus of inhibitor design that exploits differences in kinase
structure and pliability in order to achieve selectivity. Imatinib mesylate [also
known as Gleevec (USA), Glivec (Europe), STI 571, or CGP57148] has shown
remarkable clinical activity in Philadelphia chromosomepositive leukemias, in
gastrointestinal stromal tumors (GIST), and in several unusual tumors with alterations in the PDGF system.
Imatinib mesylate was developed from a lead compound identified in a highthroughput compound screening program searching for protein kinase C and PDGF
receptor inhibitors (13). The initial compound was a phenylaminopyrimidine that
was modified to increase cellular activity, solubility, and oral bioavailability (16).
Imatinib mesylate occupies the nucleotide-binding cleft of the Bcr-Abl protein
tyrosine kinase, preventing access of ATP to the substrate and thus competitively
inhibiting phosphorylation of downstream effector molecules (14).
In pioneering work, Druker and coworkers demonstrated that imatinib mesylate
suppressed proliferation of BCR-ABLpositive chronic myelogenous leukemia
(CML) cells in vitro (15). Normal hematopoietic progenitors were mostly unaffected (15). Imatinib mesylate also showed activity against Philadelphia chromosomepositive acute lymphoblastic leukemia (ALL) cells and in in vivo models
(17). This compound is also an effective inhibitor of the PDGF receptor tyrosine
kinase and kit (CD 117) (stem cell factor receptor) tyrosine kinases (18). Imatinib
mesylate is very specific, with 50% inhibiting concentrations (IC50s) of 188 nM
for c-Abl, 413 nM for c-Kit, and 386 nM for PDGFR-, as opposed to IC50s of
>10,000 nM for most of the other cellular tyrosine kinases (13). These observations laid the groundwork for the use of imatinib mesylate in the clinical setting,
with potential for killing tumor cells harboring the target kinases without harm to
normal host tissue. Imatinib mesylate shows striking antitumor effects in Bcr-Abl
positive (Philadelphia chromosomepositive) leukemias, GISTs, with activating
KITmutations, and in a variety of cancers with alterations in the PDGF system
(1942) (Tables 1 and 2).
Philadelphia ChromosomePositive BCR-ABLPositive

Leukemias: Clinical and Molecular Features
The Philadelphia chromosome is a shortened chromosome 22. It usually results
from a balanced translocate between chromosomes 9 and 22 [t(9:22) (q34;q11)]
(43, 44). Philadelphia chromosomepositive leukemias include CML and a subset
of acute leukemias, most commonly ALL. The Philadelphia translocation juxtaposes two genes, BCR and ABL, to form a chimeric BCR-ABL gene, located on
chromosome 22. The Bcr-Abl protein is a constitutively active tyrosine kinase. This
abnormal enzymatic activation is crucial to the oncogenic potential of BCR-ABL
(4547).
The natural history of CML exemplifies the process of stepwise tumor progression. There is an inevitable evolution from the early chronic phase to an accelerated
phase, which ultimately leads to blast crisis. Though CML is a stem cell disorder,
Bcr-Abl
Bcr-Abl
KIT
KIT or
PDGFR-
PDGF
PDGFR-
PDGFR-
Chronic phase CML
Blast phase CML and

Philadelphia chromosomepositive acute leukemia
Gastrointestinal stromal
tumor
Mast Cell Disease
Dermatofibrosarcoma
protuberans
Hypereosinophilic
syndrome
Chronic myelomonocytic
leukemia
Anecdotal cases
Rare subset of patients with

chronic myelomonocytic
leukemia
Patients with or without

aberrant PDGFR can respond,
suggesting that an unidentified,
imatinib-mesylate-susceptible
tyrosine kinase exists.
up to 90%
(35)
(3134)
(29, 30)
(2528)
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TEL-PDGFR
Molecular aberration involves

the PDGF ligand rather than
the receptor.
Anecdotal cases
Patients with FIP1L1-PDGFR or

KIT mutation [Phe522sys] respond
Patients with KIT mutation
AspP816Val are resistant
50%
(2224)
(1921)
(36, 39)
Reference
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FIP1L1PDGFR
Col1/PDGF
FIP1L1-PDGFR
KIT [Phe522sys]
Responses are durable
Responses short-lived
CHR 520%
p210Bcr-Abl
p190Bcr-Abl
4090%, depending
on criteria
Responses durable
Refer to hematologic response
CHR > 90%
p210Bcr-Abl
KIT mutation
(exons 9, 11)
Comment
Response rate
Molecular
aberration
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Abbreviations: CHR = complete hematologic remission.
Target
AR
Tumor type
Features of tumors successfully targeted by imatinib mesylate
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1015
90100
40% at
6 months
>20
Results from one larger

retrospective and one smaller
study. Numbers dependent
on stage of disease
Dose = 600 mg/day

superior to 400 mg/day
(41, 42)
(19, 20)
(19, 21)
(40)
Number of patients in most studies ranges from about 100 to more than 1000.
Abbreviations: ALL = acute lymphoblastic leukemia; CML = chronic myelogenous leukemia; CCR = complete cytogenetic response; CHR = complete hematologic remission.
2040
Not stated
Median response
duration 3 months
median response
duration 6 months
Dose = 600 mg/day

superior to 400 mg/day
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Blast crisis
40
10
520
Lymphoid Blast Crisis/

Philadelphia-positive ALL
4060
510
520
Myeloid Blast Crisis
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100
1520
3040
Accelerated
Phase CML
TRENT
(38, 39)
Dose = 400 mg per day
(36, 37)
Reference
TIBES
>95
Markedly superior to
standard interferon-
and cytarabine
Comment
AR232-PA45-15.tex
CML relapse post allogeneic

transplant
Chronic phase
90
40
95
Chronic Phase CML

(Interferon- failure)
>95
Survival at
18 mos (%)
AR
60
>95
75
>95
Chronic Phase
CML (previously untreated)
Progression free
survival at 12 mos (%)
CCR (%)
CHR (%)
Stage/Status of disease
362
Results of representative studies ofimatinib mesylate in Philadelphia-positive leukemias
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the chronic phase is characterized by neutrophilic leukocytosis and can be easily

managed. Managing the chronic phase, however, does not prevent the ineluctable
progression toward blast transformation, a stage that resembles an aggressive acute
leukemia. Once blast crisis occurs, patients succumb within 6 to 12 months. The
phenotype of blast crisis is myeloid in two thirds of patients and lymphoid in up
to one third. In contrast, Philadelphia-chromosome-positive ALL is characterized
by uncontrolled growth of immature lymphoid cells from the outset. Patients with
Philadelphia-chromosome-positive ALL responded poorly to chemotherapy when
compared to those ALL patients who do not have the Philadelphia chromosome
(48, 49).
The t(9:22) translocation appears to be the initial transforming event in CML
(50, 51). However, secondary molecular driving forces are needed for disease progression (52). The constitutively activated tyrosine kinase activity of BCR-ABL
generates constant activation of downstream signaling pathways, as opposed to
the closely regulated Abl tyrosine kinase (53, 54). In this way, Bcr-Abl perturbs
myriad cellular functions: (a) Ras and PI3K signaling; (b) cytoskeletal structures;
(c) adhesion molecules; (d) cell survival/apoptosis; (e) growth factor dependence;
and ( f ) DNA damage and response processes (47). Consequently, disturbed proliferation and survival of cells results in the chronic phase of CML, and the impact
of Bcr-Abl on genomic stability/integrity may underlie progression toward blast
crisis (55).
Imatinib Mesylate in Chronic Phase CML

Prior to the discovery of imatinib mesylate, standard treatment of chronic phase
CML was based on either interferon- or allogeneic bone marrow transplant (56,
57). Unfortunately, interferon- was ineffective in late chronic phase, accelerated
phase, and blast crisis. Even in early chronic phase, only a small fraction of patients (525%) achieved complete cytogenetic remission (defined as elimination
of the Philadelphia chromosome and return to a diploid status, as determined by
karyotype analysis of bone marrow metaphases). Allogeneic stem cell transplantation did provide curative therapy, but was limited by donor availability and its
significant morbidity/mortality.
A large trial of previously untreated patients with chronic phase CML randomized to either imatinib mesylate or to the prior standard therapy (interferon-
given together with cytosine arabinoside, the IRIS trial) revealed that imatinib
mesylate was superior in terms of complete hematological response rates (95%
versus 56%) and complete cytogenetic responses (76% versus 15%) (37). In addition, freedom from progression to accelerated phase or blast crisis was 97% in the
imatinib mesylate-treated group. A survival difference could not be demonstrated,
most likely because of the high crossover rate (58%) from the interferon- to the
imatinib mesylate group (37). Imatinib mesylate was also far better tolerated than
interferon- and cytarabine, with an overall discontinuation rate of only 14% in the
imatinib group versus 89% for the interferon group. Molecular remissions were
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assessed by the sensitive polymerase chain reaction test that can detect BCR-ABL
transcripts from one leukemic cell among 105 normal cells. Thirty-nine percent
of imatinib-treated patients achieved more than a 3 log reduction in BCR-ABL
transcripts. For these patients, the probability of progression-free survival at 24
months was 100% (36).
Results of imatinib mesylate in patients with chronic phase CML who had failed
interferon- were also impressive. Complete hematological responses were reported in 95% of patients. Major and complete cytogenetic response rates were approximately 60% and 40%, respectively. In general, hematologic responses were
observed within days to weeks. Progression-free survival after 18 months followup was 89% (39).
Imatinib mesylate is now used as front-line therapy for chronic-phase CML.
Complete hematologic remission is expected by three months with major (<35%
Philadelphia chromosomepositive metaphases) or complete cytogenetic response
by 612 months. In patients who do not achieve these milestones, the imatinib
mesylate dose can be increased or a different treatment strategy may be considered
(58).
Imatinib Mesylate in Accelerated Phase

and Blast Crisis of CML
Results of imatinib mesylate therapy in patients with accelerated phase of CML
and blast crisis are generally inferior to those observed with chronic phase disease.
In patients with accelerated CML, sustained (>4 weeks) complete hematological
responses were seen in only 34% of patients. Complete cytogenetic responses
were achieved in <20% of patients. Results were slightly better if a higher dose
of imatinib mesylate was given (600 mg rather than 400 mg per day) (40).
In myeloid blast crisis, the complete hematological response is about 10% to
20%. Major and complete cytogenetic response rates range from 5% to 16%.
Hematological responses are short lived, most patients relapse, and median response duration is only about six months. Higher doses (600 and 800 mg) of
imatinib mesylate achieve somewhat higher and longer lasting responses (19, 21).
Compared with cytosine arabinosidebased chemotherapy regimens in blast crisis,
imatinib mesylate produces similar response rates, but with lower toxicity, lower
induction mortality, and better survival (59).
In lymphoid blast crisis and Philadelphia chromosomepositive ALL, imatinib
mesylate given at doses of 400 or 600 mg daily yields complete hematologic
response rates of about 20% or less and complete cytogenetic remission rates
of about 10% (19, 20). Reduction of blast count commonly occurs early, often
within one week after treatment start. The duration of responses is unfortunately
brief, with a median time to progression of about three months. Most patients who
progress will succumb to their disease soon thereafter.
Side effects of imatinib mesylate, especially grade three or four neutropenia
and thrombocytopenia, are more frequent in accelerated phase and blast crisis
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(20, 21). This is likely a consequence of decreased marrow reserve and progression
of underlying disease. Combination therapies of imatinib mesylate with more
conventional chemotherapy or other investigational agents are being studied.

Imatinib Mesylate after Allogeneic Stem Cell Transplantation

Imatinib mesylate also exhibits activity in patients who relapse after allogeneic
stem cell transplantation. Complete hematological response rates are in the range
of 70%. Major and complete cytogenetic responses occur in about 55% and 40% of
patients, respectively. More importantly, complete molecular remissions have been
observed in about 25% of patients. Response rates in all categories were highest
in chronic phase CML and progressively decreased in accelerated and blastic
phase (41, 42). Estimated overall survival at two years was 12% in blast crisis
and 100% in early chronic phase (42). Thus, imatinib mesylate can be considered
an important treatment alternative for CML patients who relapse after allogeneic
stem cell transplantation.
Resistance to Imatinib Mesylate

Despite the dramatic success achieved by imatinib mesylate, the issue remains as
how to maximize response and defy resistance. Even in early chronic phase CML,
not all patients will attain cytogenetic remission. Furthermore, most individuals
with blast transformation or Philadelphia chromosomepositive acute leukemia
who respond will relapse quickly. Because imatinib mesylate is commonly used
as front-line therapy in CML, its impact on long-term survival remains to be seen,
and comparison to allogeneic stem cell transplantation warrants full study.
Recent research has revealed mechanisms that mediate resistance. These include upregulation of multi-drug resistance proteins, functional inactivation of
imatinib mesylate, BCR-ABL gene amplification or mutations, and loss of the
Bcr-Abl kinase target (6062). The most cogent evidence supports a role for mutations in the emergence of resistance. Indeed, mutations in the BCR-ABL kinase
have been detected in up to 90% of patients who relapsed after initial response
(6264). In some patients, mutations have been present prior to starting treatment
and thus, mutated clones were presumably selected by a growth advantage during
imatinib mesylate treatment, similar to selection of resistant bacterial clones with
antibiotic treatment (65). Alternative innovative approaches that directly interfere
with Bcr-Abl function or enhance imatinib mesylate efficacy have therefore been
proposed: (a) targeting BCR-ABL RNA with antisense oligonucleotides or with
ribozymes (66); (b) using Bcr fragments as therapy (on the basis of the observation
that high levels of Bcr attenuate Bcr-Abl kinase activity) (67); (c) treating with
molecules, such as tyrphostin, that affect the binding of peptide substrates (rather
than ATP) to Bcr-Abl; (d) combining imatinib mesylate with other suppressors of
signaling (Jak2, Ras) (68, 69); (e) administering interferon-, which has known activity in CML, together with imatinib mesylate; ( f ) using suppressors of nuclear
export to entrap Bcr-Abl in the nucleus, where it promotes apoptosis (70); and
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(g) using dual src-abl inhibitors (such as BMS-354825), which impose less stringent conformational requirements on ABL for kinase inhibition. Indeed, BMS354825 has proved effective in animal models and clinical trials are underway (72).
The efficacy of these strategies may depend on the mechanism of resistance,
which could vary among patients. Although a large body of data implicates BCRABL mutations in the emergence of imatinib resistance, cells with mutated BCRABL often do not make up the predominant population in resistant disease. Hence,
other mechanisms of resistance must be operative in some individuals. Loss of
the Bcr-Abl kinase target or activation of pathways that supplant the role of BcrAbl may play a role (71). Approaches that target Bcr-Abl function or levels may
be moot for persons in whom molecular pathways other than Bcr-Abl mediate
resistance to imatinib mesylate.
PDGFR and Imatinib Mesylate

Recently, imatinib mesylate has shown remarkable activity in certain other hematologic malignancies and solid tumors: idiopathic hypereosinophilic syndrome,
eosinophilia-associated chronic myeloid disorder, chronic myelomonocytic
leukemia, systemic mast cell disease, atypical CML and dermatofibrosarcoma
protuberans (Table 1). The generation of a constitutively active PDGFR tyrosine
kinase is the common feature in these responsive tumors. Activation of the receptor
can be caused by an aberration in the gene encoding the PDGF receptor or in the
gene encoding the PDGF ligand.
The PDGFR family consists of the PDGF- and - receptors, which are tyrosine
kinase receptors stimulated by several extracellular PDGF ligands. Both receptors
have many well-characterized functions and are involved in proliferation, intracellular organization, chemotaxis, apoptosis, as well as oncogenic transformation (for
review, see 10) (Figure 1). Imatinib mesylate inhibits the tyrosine kinase enzyme
activity of both PDGFR- and PDGFR- (18).
Idiopathic Hypereosinophilic Syndrome

Idiopathic hypereosinophilic syndrome and chronic eosinophilic leukemia comprise a spectrum of rare disorders characterized by eosinophil overproduction and
clinical symptoms arising from organ involvement. Some patients carry a fusion
gene designated FIP1L1-PDFGRA that activates PDGFR- (31) and is generated
by a cryptic interstitial chromosomal deletion on chromosome 4q12. Imatinib mesylate is effective in patients harboring this abnormality, regardless of whether they
are classified as hypereosinophilic syndrome or as chronic eosinophilic leukemia.
In addition, responders can lack FIP1L1-PDGFR, suggesting that another relevant
kinase is present (73). Sustained responses with imatinib mesylate are achieved at
doses lower than those needed in CML, i.e., 100 to 400 mg per day, rather than
400 or more mg per day. This finding is consistent with the low IC50 of imatinib mesylate for suppressing FIP1L1-PDGFR kinase (3.2 nM) (31) compared
with its IC50 for the Bcr-Abl kinase (200 nM). Response can be seen within
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four weeks. As in CML, new mutations in this fusion gene may lead to resistance.
Rarely, patients with systemic mast cell disease also carry a FIP1L1-PDFGR fusion gene; patients harboring this mutation show response to imatinib mesylate
treatment (26).

Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia is a myeloproliferative disorder characterized
by an increased number of monocytes and granulocytes as well as dysplasia. There
is no approved standard therapy for this illness. A small proportion of patients with
chronic myelomonocytic leukemia have aberrations involving the PDGFR-, with
constitutive activation of the receptor tyrosine kinase. This aberration is caused by
a translocation between chromosomes 5 and 12, t [5;12], creating a fusion gene
ETV6-PDGFRB (also called TEL-PDGFRB). Durable clinical, hematological, cytogenetic, and molecular responses can be achieved in these patients with the use
of imatinib mesylate (34, 35).
Atypical CML
In several cases of atypical CML, the BCR region is fused to PDGFR because of a
translocation between chromosomes 4 and 22 instead of the usual t(9:22). Rapid
responses to imatinib mesylate have been reported in patients with this variant,
demonstrating the activity against the PDGFR tyrosine kinase in vivo in these
individuals (74).
Dermatofibrosarcoma Protuberans
Dermatofibrosarcoma protuberans is an uncommon, low-grade, fibrohistiocytic
tumor of intermediate malignant potential. This neoplasm represents a unique
molecular situation where the PDGF ligand, rather than the PDGF receptor itself, is altered. Patients with dermatofibrosarcoma protuberans harbor a t(17, 22)
translocation that generates a Col1-PDGF fusion gene (75). Fusion to Col1 enhances PDGF action by allowing constitutive expression of Col1-PDGF ligand
and constitutive PDGFR kinase activation through autocrine stimulation. Exposure of primary cultures of dermatofibrosarcoma protuberans to imatinib mesylate
in vitro has been shown to inhibit cell growth (76). Further, patients with this tumor
respond to imatinib mesylate even in the case of inoperable, metastatic disease (29,
30).
KIT and Gastrointestinal Stromal Tumors (GIST)

GIST are rare mesenchymal tumors arising from the interstitial cells of Cajal in
the gastrointestinal tract and abdomen. They represent less than one percent of
gastrointestinal tract tumors and a minority of all sarcomas (77). The vast majority
of GIST express the type III receptor tyrosine kinase CD117 (KIT or stem cell factor
receptor) (78). KIT resides on chromosome 4 (4q11-q12) and is translated as a
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145-kD receptor tyrosine kinase (79, 80). KIT is similar in structure and function to
the PDFGR or Flt3-receptor. It is expressed by many cells including hematopoietic
progenitor cells, germ cells, and mast cells (81). Physiologic functions of KIT
include cell survival, proliferation, differentiation, adhesion, and apoptosis (82,
83).
Several activating mutations of KIT lead to constitutive, ligand-independent
activation of the receptor tyrosine kinase and intracellular downstream pathways
including STAT, PI3K and MAPK (82, 84). Therefore, KIT expression is thought to
represent a crucial step in tumorigenesis. Some GIST tumors lack a mutation in the
KIT gene but still possess an activated, phosphorylated KIT protein (85). Mechanisms that might account for this finding include other undetected KIT mutations,
KIT ligand up-regulation, KIT heterodimerization, or alteration of phosphatases
that inhibit KIT (85). The natural ligand of KIT is stem cell factor. Interestingly,
6070% of GIST also express the CD34 antigen, a hematopoietic stem cell marker
of unknown function (86).
Prior to the availability of imatinib mesylate, prognosis of GIST was grave.
Conventional chemotherapy had response rates close to zero. This changed dramatically with the use of imatinib mesylate. The first patient reported had rapidly
progressive GIST, resistant to chemotherapy but responded to imatinib mesylate
with a complete metabolic response and tumor shrinkage (87). In subsequent
large studies for recurrent or advanced GIST, imatinib mesylate exhibited overall response rates (stable disease or partial response) anywhere from >40% (by
conventional response criteria) to 85% meaningful clinical responses (2224, 88).
The value of conventional response criteria [assessed by imaging studies, usually
in the form of computerized tomography (CT) scans] is in doubt because >90% of
treated patients showed clinical benefit, as manifest by long-term relief of cancerrelated symptoms. Patients should, therefore, be kept on the medication if they do
well symptomatically. Clinical improvement often occurs within one to two days.
Remarkably, positron emission tomography (PET) scans demonstrate metabolic
turn off of the tumor within several days and are highly predictive of anatomic
response (Figure 2). Conventional CT scans, on the other hand, may be misleading. They may demonstrate stability of disease or even disease growth for months,
despite clear-cut PET responses. On this basis, Choi and colleagues (89) proposed
new CT response criteria, including a greater than 10% decrease in tumor size
(rather than the usual greater than 50% decrease) or a greater than 15% decrease
in Hounsfield units, a measurement of tumor density on CT scan. Of particular
importance is that responses continue to increase with duration of treatment (23).
This is in contrast to chemotherapy, in which lack of early response predicts the
futility of further treatment. In the large phase III trials, progression-free survival
at 12 months was close to 70%, and overall survival was about 85%. Until now,
there has been no established dose response difference between 400, 600, and 800
mg imatinib mesylate (22, 90). However in patients failing treatment with 400 mg
of imatinib mesylate per day, increase to 800 mg per day of imatinib mesylate can
still be effective in up to 7% of patients (J. Trent, unpublished data). Response rate
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Figure 2 PET scan of a patient with GIST before and after treatment with imatinib mesylate.
Positron emission tomography (PET) scan uses a small amount of radioactive glucose [(18F)
fluorodeoxyglucose (FDG, FDG-PET)] injected intravenously. This material enriches in areas
of increased metabolic activity, such as in tumors. Emitted gamma ray photons are detected
with a scanner reflecting cell/tumor metabolism and showing tumor distribution in vivo. PET
scans can be used for diagnosis, staging, and monitoring treatment of cancers. This scan
shows a GIST patient before and after treatment with imatinib mesylate. Dark areas represent
tumor (pretreatment). These areas disappear posttreatment.
appears to correlate with site of mutation. Mutations in exon 11 of KIT are more
favorable than mutations in exon 9. The least favorable prognostic group in GIST
lacks the KIT mutation and has no other identifiable mutations (91).
Taken together, these observations suggest that the molecular defect in GIST
is inextricably related to tumor response, and that evaluation of response with
new targeted therapies may require clinical and imaging endpoints that differ from
those established over the years for chemotherapy.
Mast Cell DiseaseA Disorder with KIT

or PDGFR Deregulation
Systemic mastocytosis is a clonal neoplasm of the mast cell hematopoietic progenitor. It is a clinically heterogenous disorder with accumulation of mast cells
limited to the skin (cutaneous mastocytosis) or involving one or more extracutaneous organs. Mast cell disease is often associated with gain-of-function mutations
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involving the tyrosine kinase domain of KIT (92). Pardanani et al. (28) prospectively treated 10 adults suffering from symptomatic systemic mast cell disease
with imatinib mesylate at a dose of either 100 mg or 400 mg per day. Five of
the patients had a measurable response to the drug, four of whom had important
mast cell cytoreduction and two of whom had complete clinical and histological
remission. Three of the five patients with eosinophilia had major responses. The
other two, who did not respond to treatment, were the only patients with the KIT
Asp816Val mutation. It appears that these KIT mutations confer resistance to imatinib mesylate by interfering with the binding of the drug to the enzymatic site of
the KIT molecule (27).
To date, two imatinib mesylatesensitive molecular genetic defects have been
identified in mast cell disease. Akin et al. (25) reported a point mutation within the
transmembrane segment of KIT that resulted in a substitution of a phenylalanine
residue by a cysteine at codon 522 in a patient who was amenable to treatment
with imatinib mesylate. Pardanani et al. (26) demonstrated that FIP1L1-PDGFRA
is the therapeutic target of imatinib mesylate in the specific subset of patients with
mast cell disease and associated eosinophilia and that virtually all of these patients
respond to imatinib.
Adverse Effects of Imatinib Mesylate

Imatinib mesylate is remarkably well tolerated. Predominant adverse effects are
usually mild and consist of thrombocytopenia, neutropenia, edema/fluid retention, musculoskeletal pain/muscle cramps, gastrointestinal complaints, fatigue, and
headache. Side effects are dose-related and have occurred more frequently with
advanced disease. More significant neutropenia and thrombocytopenia occurred
in advanced CML states and likely reflect effective inhibition of the leukemic cell
clone in the setting of depleted normal progenitors. Fluid retention affects almost
half of the patients and is unusual in its periorbital and central abdomen localization. Nausea and gastrointestinal discomfort is uncommon at the 400 mg daily
doses, but is frequent at doses of 800 mg per day. Almost one quarter of patients
experience myalgias or skin rashes. Overall, fewer than 5% have discontinued
treatment because of side effects (93, 94).
Pharmacology of Imatinib Mesylate

Imatinib mesylate has high oral bioavailability. Peak plasma concentrations occur within four hours. Its half-life in humans is from 13 to 16 hours. The drug is
metabolized in the liver (primarily via cytochrome P450-3A4). Sufficient plasma
concentrations to achieve IC50s can be attained at doses >400 mg once a day.
Serum levels of imatinib mesylate range from 1.464.6 M (95). The recommended dose of imatinib mesylate for patients with chronic phase CML is 400 mg
by mouth per day. The dose is 600 mg per day for patients in accelerated/blast
crisis phase of the disease.The dose can be increased to 800 mg per day based on
tolerance and response. Patients with GIST are treated at a recommended dose of
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TABLE 3
Tyrosine kinase inhibitors in the clinic
Agent

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Target
FDA-approved agents and best

established clinical use
Reference
Imatinib
mesylate
Bcr-Abl
PDGFR
Kit
Philadelphia chromosomepositive leukemia

Gastrointestinal stromal tumors
Hypereosinophilic syndrome
Mast cell disease
Dermatofibrosarcoma protuberans
Subset of chronic myelomonocytic leukemia
Gefitinib
EGFR
Non-small cell lung cancer

Head and neck cancer
(127)
Cetuximab
EGFR
Colorectal cancer
Head and neck cancer
(97, 104)
Colorectal cancer
Renal cell carcinoma
(99, 100,
133)
Bevacizumab VEGF
Trastuzumab
Erb-2 (Her2/neu) Breast cancer
(28, 35,
37, 73,
93, 94,
96)
(102, 103)
Abbreviations: EGFR = epidermal growth factor receptor; FDA = Federal Drug Administration; PDGFR = platelet-derived
growth factor receptor: VEGF = vascular endothelial growth factor.
Denotes FDA-approved uses.
400600 mg/day, but 800 mg/day are easily tolerated and may yield better results.
Pediatric doses are calculated by body surface area (93, 94).
Targeting Other Tyrosine KinaseRelated Molecules:

Compounds Approved for Clinical Use
Drugs are in development that target aspects of the kinase machinery, from the
receptor tyrosine kinases that initiate intracellular signaling, through second messengers involved in signaling cascades, to the kinases that control the cell cycle and
govern cellular fate. The vast majority of this plethora of compounds are still in
preclinical or early clinical development. However, others have reached the stage
of Federal Drug Administration (FDA) approval (Table 3) (28, 35, 37, 73, 93, 94,
96137). These include molecules that target the EGFR and the VEGF system.
The EGFR Story Unfolds

The EGFR is a family of receptor tyrosine kinases thought to contribute to the formation of many solid tumors (105). This family consists of four different receptor
members: (a) EGFR, also known as ErbB1 or HER1; (b) ErbB2 or Her2/Neu;
(c) ErbB2 (HER3); and (d) ErbB4 (HER4) (2, 106). The ErbB receptors share
a similar structure with homology in their kinase domain, but diverge in their
extracellular domains and carboxy-terminal end tails (2). The EGFR itself is a
170-kDa transmembrane protein with a single polypeptide chain of 1186 amino
acids. It has a hydrophobic transmembrane segment of 23 amino acids attached
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to the intracellular domain with tyrosine kinase activity (107, 108). Endogenous
activating ligands are EGF, TGF-alpha, heparin-binding EGF, amphiregulin, betacellulin, epiregulin, and many others (105, 109). Transactivation through G-protein
coupled receptors and cytokines plays a role (109). ErbB family receptors are
widely expressed in all tissues where they regulate diverse functions, including
mitogenesis, differentiation, and cell survival (105). Downstream target activation includes PLC , Ras-Raf-MEK-MAPK (gene transcription and proliferation),
phosphatidylinositol-3 kinase (PI3K)/Akt (cell survival), the tyrosine kinase Scr,
the stress-activated protein kinases, PAK-JNKK, JNK, and the signal transducers
and activators of transcription (STAT) (110, 111). Of particular importance for the
ErbB receptor family is the ability of Erb2/Her2Neu to form heterodimers with the
other receptor subunits. ErbB2 does not have an extracellular ligand but is the most
oncogenic ErbB receptor-dimer known (109). The EGFR degradation constitutes
an important regulatory mechanism. After ligand binding, the receptor is internalized, with signal termination often within seconds, and either further endocytotic
degradation or recycling of receptor components to the cell surface for repeated
signaling (112).
Mechanisms mediating transformation include receptor overexpression, gene
amplification, activating mutations, alterations in the dimerization process, and
structural rearrangements (reviewed and referenced in 105). Furthermore activation of autocrine growth factor loops (113) and deficiency of specific phosphatases
may be of importance, as well. Receptor and ligand overexpression and gene amplification are the common causes of oncogenic transformation (114). Indeed, the
rationale for targeting the EGF receptor tyrosine kinases is based on the receptor
overexpression discerned in many human malignancies including colorectal, head
and neck, esophageal, ovarian, cervical, breast, endometrial, and nonsmall cell
lung cancer (105, 115, 116).
Several strategies to interfere with aberrant EGFR signaling have emerged. The
most successful in the clinic are antibodies that block the extracellular ligandbinding site (preventing ligand activation of the receptor) and small molecule
tyrosine kinase inhibitors that suppress the intracellular tyrosine kinase. To date,
the small molecule tyrosine kinase inhibitors Gefitinib (ZD1839, Iressa) (117)
and the antibody Cetuximab (118) have been approved by the FDA for use in lung
and colorectal cancer, respectively. Trastuzumab, which targets Erb-2 (Her2/neu),
is approved for treatment of Her2/neu-positive breast cancer. Numerous other
inhibitors of the EGFR machinery are in development (reviewed in 119).
Initially, the modest response rates to some of these compounds were considered
disappointing. However, recent data demonstrate that it is possible to identify
subsets of patients whose tumors have mutations in the targeted kinase that confer
profound susceptibility to the inhibitor.
Trastuzumab (Herceptin)
The approval of Trastuzumab in 1998 for the treatment of breast cancer is a milestone in the field of EGFR-directed therapy. It is now used worldwide for breast
cancer management.
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Her2/neu acts as a major signaling partner for other EGFR family members
by forming heterodimers with potent signaling activity leading to proliferative
and antiapoptotic effects. Her2/neu is amplified/overexpressed in about 30% of
breast cancers, and correlates with a poor outcome (101, 120). Trastuzumab is an
anti-ErbB-2 receptor (Her2/neu) humanized monoclonal antibody with benefit in
Her2/neu-positive metastatic breast cancer as a single agent (102). Compared to
chemotherapy alone, it demonstrates statistically significant increases in response
rate, time to progression, and survival time when combined with chemotherapy
(103). Benefit from Trastuzunab is only derived in Her2/neu positive patients, however, so selection of the study population for new targeted agents is very important.
Gefitinib (Iressa)
Gefitinib is an oral, highly bioavailable, EGFR-specific, anilinoquinazoline, smallmolecule inhibitor. It binds to the ATP site of the EGFR tyrosine kinase domain with
approximately 100-fold increased affinity compared to other kinases and reversibly
inhibits autophosphorylation of the receptor by competitively blocking access of
ATP to the EGFR kinase domain (121, 122), which prevents downstream kinase
activation. The IC50 for the inhibition of autophosphorylation of the EGFR/Her1
receptor in intact cells is 0.033 M (123). In preclinical models, gefitinib inhibited
the growth of multiple cell lines and mouse tumor xenografts in a dose-dependent
manner and was synergistic and additive with chemotherapeutic agents (platinum,
taxanes, etoposide), radiation therapy, as well as with the monoclonal antibody
Trastuzumab (124126).
In patients with nonsmall cell lung cancer, there was no synergism when gefitinib was administered with cytotoxic agents, according to two large randomized
trials (INTACT-1 and INTACT-2) (134, 135). The combination of gefitinib with radiation therapy might be more promising (124). Even so, gefitinib is now approved
for single-agent, third-line therapy of patients with nonsmall cell lung cancer who
have failed chemotherapy, on the basis of a response rate of about 10% (127).
This modest response rate was initially considered disappointing, and the lack of
a correlation between response and EGFR overexpression was frustrating (128).
However, a recent discovery indicates that activating mutations in the ATP-binding
pocket of the EGFR kinase domain confers susceptibility to gefitinib in nonsmall
cell lung cancer, hence allowing identification of subgroups of responsive patients
(137).
Gefitinib is well tolerated. The most common side effects are skin rash and
gastrointestinal complaints. An oral dose of 250 mg of gefitinib per day is recommended. Steady-state concentrations are achieved in most patients at seven
days. Gefitinib can be used in extensively pretreated patients or those with poor
performance status, for whom a therapy with a low toxicity profile is needed.
Erlotinib (TarcevaTM)
A second small-molecule EGFR inhibitorerlotinib, TarcevaTM, OSI-774demonstrates response rates of 10% to 19% as a single agent in patients with
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nonsmall cell lung cancer who failed chemotherapy (129). However, large randomized trials testing erlotinib in combination with chemotherapy in this disease
did not show any benefit derived from the addition of erlotinib (R. Herbst, personal
communication). Single agent TarcevaTM in patients with advanced NSCLC failing standard therapy has recently shown to have a survival advantage in a study
with 751 patients from Canada (F. Shepherd, Proc. ASW, June 2004).

Cetuximab (C-225) (Erbitux)

Cetuximab is a chimeric, recombinant humanized monoclonal antibody to the external EGFR/ErbB1 domain. It prevents receptor autophosphorylation and leads to
EGFR internalization with subsequent receptor degradation (130). This antibody
has nonlinear pharmacokinetics, and clinically effective doses range between 200
and 400 mg/m2 given intravenously. Cetuximab was recently FDA approved for use
in combination with irinotecan for the treatment of patients with EGFR-expressing,
metastatic colorectal carcinoma who are refractory to irinotecan-based chemotherapy. Combinations of cetuximab with irinotecan yielded a 23% overall response
rate and median time to disease progression of four months (97). Cetuximab alone
resulted in about a 10% overall response rate (98). The most common side effects
of cetuximab were acneiform skin rash and folliculitis. Severe hypersensitivity
reactions were rare.
Angiogenesis: Targeting the Vascular System

Because tumor progression depends on the formation of new blood vessels, blocking angiogenesis is an appealing approach to anticancer therapy. VEGF is a key
angiogenic factor. It binds to the VEGF1 and 2 receptors on endothelial and other
cells. Binding activates the intracellular pathways necessary for physiologic and
tumor angiogenesis (131). Current research is directed to agents that target this
molecule or its receptor. Recently, Bevacizumab, which targets the VEGF ligand,
has been approved for clinical use in colorectal cancer.
Bevacizumab (Avastin)
Bevacizumab is a humanized monoclonal antibody. It binds and inhibits the VEGF
growth factor ligand. A phase III trial involving 815 patients established Bevacizumab as the first angiogenesis-targeted agent to improve overall survival in
patients with metastatic colorectal cancer and lead to FDA approval. The addition of Bevacizumab in the frontline setting to a regimen containing multiagent
chemotherapy (irinotecan, 5-fluoruracil, and leucovorin) improved overall survival
from 15.6 to 20.3 months and progression-free survival from 6.4 to 10.6 months
when compared to the chemotherapy arm (99). These results, although modest,
were statistically significant. Bevacizumab has also been tested alone and/or in
combination with different conventional cytotoxic agents in several malignancies
(i.e., breast cancer, renal cell cancer, and nonsmall cell lung cancer). Overall results were disappointing and few partial tumor regressions were observed (132).
Promising results have been seen in renal cell cancer, however (133).
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Bevacizumab is administered intravenously at doses of 510 mg/kg every two

weeks. It is well tolerated. Side effects include headache/migraine, proteinuria,
hypertension, and rare cases of hemorrhage, thromboembolic events, and gastrointestinal perforations (100).

SUMMARY: THE STATE OF THE ART

Certain classes of signaling proteins and pathways are frequently altered by oncogenic mutations. Molecules governing extracellular growth, differentiation, and
developmental signals, in particular, are often mutated in cancers. Tyrosine kinases are especially important in this respect. These enzymes are pivotal regulators of the signal transduction pathways that mediate development and intracellular
communication. Their activity is normally tightly controlled. Perturbation of tyrosine kinase signaling by mutations and other genetic alterations drive malignant
transformation.
In principle, for tyrosine kinases involved in cancer, oncogenic deregulation
results from alteration of one of several of the auto-control mechanisms that ensure the normal repression of catalytic domains. A little more than half of the
known tyrosine kinase receptors have been found repeatedly in either mutated or
overexpressed forms associated with (human) malignancies, including sporadic
cases. In addition, many of the cytoplasmic tyrosine kinases are also disturbed
in cancer, either directly through mutation or indirectly via other genetic driving
forces.
Imatinib mesylate, a potent inhibitor of the Bcr-Abl, Kit, and PDGFR kinases,
has enjoyed great success in treating tumors, including those that are not susceptible to standard chemotherapy. Response rates of more than 80%90% can be
achieved with little toxicity in some malignancies bearing the proper targets. On
the other hand, the use of imatinib mesylate in the treatment of neoplasms in which
the pathogenesis is not clearly dependent on activation of a susceptible tyrosine
kinase has been disappointing. Even so, there may be other imatinib-sensitive tumors (or subsets of tumors), in addition to CML, GIST, chronic myelomonocytic
leukemia, mast cell disease, hypereosinophilic syndrome, and dermatofibrosarcoma protuberans, in which occult activation of the Abl, KIT, or PDGFR tyrosine
kinases exists or in which there is activation of an undiscovered kinase. As an example, some patients with hypereosinophilic syndrome without an obvious target
kinase aberration can respond to imatinib mesylate in a manner similar to those
with PDGFR abnormalities.
Other inhibitors of specific tyrosine kinases have been approved for clinical use, albeit with initial response rates that pale in comparison with those of
imatinib mesylate. Antibodies and small-molecule inhibitors of the EGFR and
VEGF pathways are now applied in the management of patients suffering from
breast cancer, colorectal cancer, and lung cancer. The modest response rates attained with these drugs are almost certainly due to the complex molecular heterogeneity of many solid tumors. Indeed, recent discoveries indicate that the presence of activating mutations in the EGFR allows identification of a subset of
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patients with lung cancer in whom EGFR inhibitors can elicit dramatic responses
(137).
A synthesis of the knowledge gleaned from the clinical application of tyrosine
kinase inhibitors indicates that a new paradigm for cancer therapy is emerging.
Optimal exploitation of targeted designer drugs as part of the oncology arsenal
mandates that treatment should be based on the molecular fingerprint of the tumor
rather than its anatomic locale.
WHAT DOES THE FUTURE HOLD?

Several vital lessons have been learned during the process of developing the tyrosine kinase inhibitor, imatinib mesylate, and the EGFR inhibitors. First, it is now
clear that designer compounds can target even ubiquitously expressed families of
proteins in a specific manner. Second, tumors that are highly resistant to conventional agents can be exquisitely sensitive to a rationally targeted agent. Third, with
the use of such agents, dramatic and durable responses can be achieved without
significant host toxicity. Fourth, proteins considered predominantly in the context
of the hematologic system (i.e., PDGFR or stem cell factor receptor KIT) may have
a profound contribution to solid tumors and vice versa. Fifth, to date, it appears
that activating mutations in kinases, rather than simple overexpression, confers
susceptibility to kinase inhibitors.
Future clinical trials may need to categorize patients by their molecular genetics
(e.g., p53 mutation-positive cancer for a therapy directed against the p53 tumor
suppressor gene) rather than by the disease site of their illness (breast cancer or
colorectal cancer). Such a nosology would represent a significant paradigm shift
in that clinical trials could be designed so that patients are enrolled based exclusively on the molecular defect in their tumor. This paradigm also recognizes that
classification of tumors based on anatomic location (e.g., lung cancer or sarcoma)
belies a complicated underlying molecular heterogeneity that, if unrecognized,
may obscure response rates. For instance, GIST comprise only a minority of sarcomas, and these are the only sarcomas that demonstrate profound sensitivity to
imatinib mesylate. Similarly, the 10% response to EGFR inhibitors in lung cancer
was initially considered a failure or, at best, a marginal success, despite the fact
that some patients with chemotherapy-refractory disease demonstrated profound
responses. This changed with the discovery that the subset of responders could be
identified on the basis that their neoplasms harbor an activating EGFR mutation
(137). It is likely that many other cancers, including breast, colorectal, and others,
are comprised of numerous small-molecular subsets, and that successful treatment
will require precise pinpointing and exploitation of molecular defects as targets
for therapy.
The field of molecular cancer therapeutics is now advancing rapidly. Indeed,
the time from the discovery of the activating KIT mutation in GIST tumors (138)
to the remarkable reversal of the hopeless prognosis for this disease via treatment
with imatinib mesylate (24) was only three years. Even considering that some
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kinase inhibitors may have serious limitations in their salutary activity, our current
successes almost certainly represent the tip of the therapeutic iceberg for targeted
treatments.

LITERATURE CITED
1. Hanahan D, Weinberg RA. 2000. The
hallmarks of cancer. Cell 100:5770
2. Schlessinger J. 2000. Cell signaling by receptor tyrosine kinases. Cell 103:21125
3. Blume-Jensen P, Hunter T. 2001. Oncogenic kinase signalling. Nature 411:355
65
4. Van Etten RA, Jackson P, Baltimore D.
1989. The mouse type IV c-abl gene product is a nuclear protein, and activation of
transforming ability is associated with cytoplasmic localization. Cell 58:66978
5. Wetzler M, Talpaz M, Van Etten RA,
Hirsh-Ginsberg C, Beran M, Kurzrock
R. 1993. Subcellular localization of Bcr,
Abl, and Bcr-Abl proteins in normal and
leukemic cells and correlation of expression with myeloid differentiation. J. Clin.
Invest. 92:192539
6. Wetzler M, Talpaz M, Yee G, Stass SA,
Van Etten RA, et al. 1995. Cell cyclerelated shifts in subcellular localization of
BCR: association with mitotic chromosomes and with heterochromatin. Proc.
7. Hubbard SR, Till JH. 2000. Protein tyrosine kinase structure and function. Annu.
Rev. Biochem. 69:37398
8. Ullrich A, Schlessinger J. 1990. Signal
transduction by receptors with tyrosine kinase activity. Cell 61:20312
9. Heldin CH. 1995. Dimerization of cell
surface receptors in signal transduction.
Cell 80:21323
10. Heldin CH, Westermark B. 1999. Mechanism of action and in vivo role of
platelet-derived growth factor. Physiol.
Rev. 79:1283316
11. Miller WE, Raab-Traub N. 1999. The

EGFR as a target for viral oncoproteins.
Trends Microbiol. 7:45358
12. Druker BJ, Lydon NB. 2000. Lessons
learned from the development of an abl
tyrosine kinase inhibitor for chronic myelogenous leukemia. J. Clin. Invest. 105:37
13. Manley PW, Cowan-Jacob SW, Buchdunger E, Fabbro D, Fendrich G, et al.
2002. Imatinib: a selective tyrosine kinase
inhibitor. Eur. J. Cancer 38 (Suppl 5.):
S19S27
14. Savage DG, Antman KH. 2002. Imatinib
mesylatea new oral targeted therapy.
15. Druker BJ, Tamura S, Buchdunger E,
Ohno S, Segal GM, et al. 1996. Effects
of a selective inhibitor of the Abl tyrosine
kinase on the growth of Bcr-Abl positive
cells. Nat. Med. 2:56166
16. Schindler T, Bornmann W, Pellicena P,
Miller WT, Clarkson B, Kuriyan J. 2000.
Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science
289:185759
17. le Coutre P, Mologni L, Cleris L, Marchesi E, Buchdunger E, et al. 1999. In vivo
eradication of human BCR/ABL-positive
leukemia cells with an ABL kinase inhibitor. J. Natl. Cancer Inst. 91:16368
18. Buchdunger E, Cioffi CL, Law N, Stover
D, Ohno-Jones S, et al. 2000. Abl proteintyrosine kinase inhibitor STI571 inhibits
in vitro signal transduction mediated by
c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther.
295:13945
19. Druker BJ, Sawyers CL, Kantarjian H,
7 Jan 2005
14:2
378

20.
21.
22.
23.
24.
25.
26.
AR
AR232-PA45-15.tex
TIBES
TRENT
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE
KURZROCK
Resta DJ, Reese SF, et al. 2001. Activity

of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic
myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:103842
Ottmann OG, Druker BJ, Sawyers CL,
Goldman JM, Reiffers J, et al. 2002.
A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 100:196571
Sawyers CL, Hochhaus A, Feldman E,
Goldman JM, Miller CB, et al. 2002. Imatinib induces hematologic and cytogenetic
responses in patients with chronic myelogenous leukemia in myeloid blast crisis:
results of a phase II study. Blood 99:3530
39
Benjamin RS, Rankin C, Fletcher CD,
Demetri GD. 2003. Phase III doserandomized study of imatinib mesylate
(STI571) for GIST: Intergroup S0033
early results. Proc. Am. Soc. Clin. Oncol.
22:3271 (Abstr.)
Blanke CD, Joensuu H, Demetri GD,
Heinrich P, von Mehren M. 2003. Longterm follow up of advanced gastrointestinal stomal tumor (GIST) patients treated
with imatinib mesylate. Proc. Am. Soc.
Clin. Oncol. 22:2 (Abstr.)
Demetri GD, von Mehren M, Blanke CD,
Van den Abbeele AD, Eisenberg B, et al.
2002. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N. Engl. J. Med. 347:47280
Akin C, Fumo G, Yavuz AS, Lipsky PE,
Neckers L, Metcalfe DD. 2004. A novel
form of mastocytosis associated with a
transmembrane c-kit mutation and response to imatinib. Blood 103:322225
Pardanani A, Ketterling RP, Brockman
SR, Flynn HC, Paternoster SF, et al. 2003.
CHIC2 deletion, a surrogate for FIP1L1PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and
predicts response to imatinib mesylate
therapy. Blood 102:309396
27. Ma Y, Zeng S, Metcalfe DD, Akin C,

Dimitrijevic S, et al. 2002. The c-KIT
mutation causing human mastocytosis is
resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic
site mutations show different inhibitor
sensitivity profiles than wild-type kinases
and those with regulatory-type mutations.
Blood 99:174144
28. Pardanani A, Elliott M, Reeder T, Li CY,
Baxter EJ, et al. 2003. Imatinib for systemic mast-cell disease. Lancet 362:535
36
29. Maki RG, Awan RA, Dixon RH, Jhanwar S, Antonescu CR. 2002. Differential sensitivity to imatinib of 2 patients
with metastatic sarcoma arising from
dermatofibrosarcoma protuberans. Int. J.
Cancer 100:62326
30. Rubin BP, Schuetze SM, Eary JF, Norwood TH, Mirza S, et al. 2002. Molecular targeting of platelet-derived growth
factor B by imatinib mesylate in a patient
with metastatic dermatofibrosarcoma protuberans. J. Clin. Oncol. 20:358691
31. Cools J, DeAngelo DJ, Gotlib J, Stover
EH, Legare RD, et al. 2003. A tyrosine
kinase created by fusion of the PDGFRA
and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N. Engl. J. Med.
348:120114
32. Cortes J, Ault P, Koller C, Thomas D,
Ferrajoli A, et al. 2003. Efficacy of
imatinib mesylate in the treatment of
idiopathic hypereosinophilic syndrome.
Blood 101:471416
33. Gleich GJ, Leiferman KM, Pardanani A,
Tefferi A, Butterfield JH. 2002. Treatment
of hypereosinophilic syndrome with imatinib mesilate. Lancet 359:157778
34. Pardanani A, Tefferi A. 2004. Imatinib
therapy for hypereosinophilic syndrome
and eosinophilia-associated myeloproliferative disorders. Leuk. Res. 28 (Suppl.
1):4752
35. Apperley JF, Gardembas M, Melo JV,
Russell-Jones R, Bain BJ, et al. 2002.
7 Jan 2005
14:2
AR
AR232-PA45-15.tex
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE

36.
37.
38.
39.
40.
41.
42.
Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the plateletderived growth factor receptor beta. N.
Engl. J. Med. 347:48187
Hughes TP, Kaeda J, Branford S, Rudzki
Z, Hochhaus A, et al. 2003. Frequency of
major molecular responses to imatinib or
interferon alfa plus cytarabine in newly
diagnosed chronic myeloid leukemia. N.
Engl. J. Med. 349:142332
OBrien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, et al. 2003. Imatinib
compared with interferon and low-dose
cytarabine for newly diagnosed chronicphase chronic myeloid leukemia. N. Engl.
J. Med. 348:9941004
Cortes J, Giles F, OBrien S, Thomas D,
Garcia-Manero G, et al. 2003. Result of
high-dose imatinib mesylate in patients
with Philadelphia chromosome-positive
chronic myeloid leukemia after failure of
interferon-alpha. Blood 102:8386
Kantarjian H, Sawyers C, Hochhaus A,
Guilhot F, Schiffer C, et al. 2002. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous
leukemia. N. Engl. J. Med. 346:645
52
Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti-Passerini C, et al.
2002. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic
myeloid leukemia: results of a phase 2
study. Blood 99:192837
Kantarjian HM, OBrien S, Cortes JE,
Giralt SA, Rios MB, et al. 2002. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation
for chronic myelogenous leukemia. Blood
100:159095
Olavarria E, Ottmann OG, Deininger M,
Clark RE, Bandini G, et al. 2003. Response to imatinib in patients who relapse
after allogeneic stem cell transplantation
for chronic myeloid leukemia. Leukemia
17:170712
379
43. Rowley JD. 1973. A new consistent

chromosomal abnormality in chronic
myelogenous leukaemia identified by
quinacrine fluorescence and Giemsa
staining. Nature 243:29093
44. Nowell PC, Hungerford DA. 2004. A
minute chromosome in human chronic
granulocytic leukemia. Science 132:1497
(Abstr.)
45. Kloetzer W, Kurzrock R, Smith L, Talpaz M, Spiller M, et al. 1985. The human
cellular abl gene product in the chronic
myelogenous leukemia cell line K562 has
an associated tyrosine protein kinase activity. Virology 140:23038
46. Kurzrock R, Shtalrid M, Romero P, Kloetzer WS, Talpas M, et al. 1987. A novel
c-abl protein product in Philadelphiapositive acute lymphoblastic leukaemia.
Nature 325:63135
47. Kurzrock R, Kantarjian HM, Druker BJ,
Talpaz M. 2003. Philadelphia chromosome-positive leukemias: from basic
mechanisms to molecular therapeutics.
Ann. Intern. Med. 138:81930
48. Sawyers CL. 1999. Chronic myeloid
49. Garcia-Manero G, Faderl S, OBrien S,
Cortes J, Talpaz M, Kantarjian HM. 2003.
Chronic myelogenous leukemia: a review
and update of therapeutic strategies. Cancer 98:43757
50. Kelliher MA, McLaughlin J, Witte ON,
Rosenberg N. 1990. Induction of a chronic
myelogenous leukemia-like syndrome in
mice with v-abl and BCR/ABL. Proc.
51. Heisterkamp N, Jenster G, ten Hoeve
J, Zovich D, Pattengale PK, Groffen J.
1990. Acute leukaemia in bcr/abl transgenic mice. Nature 344:25153
52. Guerrasio A, Serra A, Gottardi E, Cilloni
D, Vischia F, et al. 1997. Molecular events
in chronic myeloid leukemia progression.
Leukemia 11 (Suppl. 3):51921
53. Konopka JB, Watanabe SM, Witte ON.
1984. An alteration of the human c-abl
protein in K562 leukemia cells unmasks
7 Jan 2005
14:2
380
54.

55.
56.
57.
58.
59.
60.
61.
62.
AR
AR232-PA45-15.tex
TIBES
TRENT
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE
KURZROCK
associated tyrosine kinase activity. Cell

37:103542
Davis RL, Konopka JB, Witte ON. 1985.
Activation of the c-abl oncogene by viral
transduction or chromosomal translocation generates altered c-abl proteins with
similar in vitro kinase properties. Mol.
Cell Biol. 5:20413
Laurent E, Mitchell DL, Estrov Z, Lowery M, Tucker SL, et al. 2003. Impact of p210(Bcr-Abl) on ultraviolet C
wavelengthinduced DNA damage and
repair. Clin. Cancer Res. 9:372230
Sawyers CL. 1999. Chronic myeloid
Talpaz M, Kantarjian HM, McCredie K,
Trujillo JM, Keating MJ, Gutterman JU.
1986. Hematologic remission and cytogenetic improvement induced by recombinant human interferon alpha A in chronic
myelogenous leukemia. N. Engl. J. Med.
314:106569
Kantarjian H, Talpaz M, OBrien S, Giles
F, Rios MB, et al. 2003. Prediction of initial cytogenetic response for subsequent
major and complete cytogenetic response
to imatinib mesylate therapy in patients
with Philadelphia chromosome-positive
chronic myelogenous leukemia. Cancer
97:222528
Kantarjian HM, Cortes J, OBrien S,
Giles FJ, Albitar M, et al. 2002. Imatinib
mesylate (STI571) therapy for Philadelphia chromosome-positive chronic myelogenous leukemia in blast phase. Blood
99:354753
Weisberg E, Griffin JD. 2000. Mechanism of resistance to the ABL tyrosine
kinase inhibitor STI571 in BCR/ABLtransformed hematopoietic cell lines.
Blood 95:3498505
Gorre ME, Mohammed M, Ellwood K,
Hsu N, Paquette R, et al. 2001. Clinical resistance to STI-571 cancer therapy caused
by BCR-ABL gene mutation or amplification. Science 293:87680
Branford S, Rudzki Z, Walsh S, Grigg
A, Arthur C, et al. 2002. High frequency
63.
64.
65.
66.
67.
68.
69.
70.
71.
of point mutations clustered within

the adenosine triphosphate-binding region of BCR/ABL in patients with
chronic myeloid leukemia or Ph-positive
acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood
99:347275D
Hochhaus A, Kreil S, Corbin AS, La
Rosee P, Muller MC, et al. 2002. Molecular and chromosomal mechanisms of
resistance to imatinib (STI571) therapy.
Leukemia 16:219096
Shah NP, Nicoll JM, Nagar B, Gorre
ME, Paquette RL, et al. 2002. Multiple BCR-ABL kinase domain mutations
confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571)
in chronic phase and blast crisis chronic
myeloid leukemia. Cancer Cell. 2:11725
Roche-Lestienne C, Soenen-Cornu V,
Grardel-Duflos N, Lai JL, Philippe N,
et al. 2002. Several types of mutations
of the Abl gene can be found in chronic
myeloid leukemia patients resistant to
STI571, and they can pre-exist to the onset
of treatment. Blood 100:101418
James HA, Gibson I. 1998. The therapeutic potential of ribozymes. Blood 91:371
82
Wu Y, Ma G, Lu D, Lin F, Xu HJ, et al.
1999. Bcr: a negative regulator of the BcrAbl oncoprotein. Oncogene 18:441624
Sun X, Layton JE, Elefanty A, Lieschke
GJ. 2001. Comparison of effects of the tyrosine kinase inhibitors AG957, AG490,
and STI571 on BCR-ABLexpressing
cells, demonstrating synergy between
AG490 and STI571. Blood 97:200815
Beaupre DM, Kurzrock R. 1999. RAS
and leukemia: from basic mechanisms
to gene-directed therapy. J. Clin. Oncol.
17:107179
Vigneri P, Wang JY. 2001. Induction
of apoptosis in chronic myelogenous
leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase. Nat.
Med. 7:22834
Kurzrock R, Talpaz M, Li N, Estrov
7 Jan 2005
14:2
AR
AR232-PA45-15.tex
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE

72.
73.
74.
75.
76.
77.
78.
79.
Z. 2003. Distinct biological impact of

dephosphorylation vs downregulation of
p210 BCR-Abl: Implication for imatinib
mesylate response and resistance. Am.
Soc. Hematol. 102:264 (Abstr.)
Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL. 2004. Overriding imatinib resistance with a novel ABL kinase
inhibitor. Science 305:399401
Gotlib J, Cools J, Malone JM III, Schrier
SL, Gilliland DG, Coutre SE. 2004. The
FIP1L1-PDGFR{alpha} fusion tyrosine
kinase in hypereosinophilic syndrome and
chronic eosinophilic leukemia: implications for diagnosis, classification, and
management. Blood 103:287991
Garcia JL, Font DM, Hernandez JM,
Queizan JA, Gutierrez NC, et al. 2003.
Imatinib mesylate elicits positive clinical response in atypical chronic myeloid
leukemia involving the platelet-derived
growth factor receptor beta. Blood 102:
2699700
Shimizu A, OBrien KP, Sjoblom T,
Pietras K, Buchdunger E, et al. 1999.
The dermatofibrosarcoma protuberansassociated collagen type Ialpha1/plateletderived growth factor (PDGF) B-chain fusion gene generates a transforming protein
that is processed to functional PDGF-BB.
Sjoblom T, Shimizu A, OBrien KP,
Pietras K, Dal Cin P, et al. 2001. Growth
inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived
growth factor receptor antagonist STI571
through induction of apoptosis. Cancer
Res. 61:577883
Berman J, OLeary TJ. 2001. Gastrointestinal stromal tumor workshop. Hum.
Pathol. 32:57882
Duffaud F, Blay JY. 2003. Gastrointestinal
stromal tumors: biology and treatment.
Oncology 65:18797
Blume-Jensen P, Claesson-Welsh L, Siegbahn A, Zsebo KM, Westermark B,
Heldin CH. 1991. Activation of the human
c-kit product by ligand-induced dimeriza-
80.
81.
82.
83.
84.
85.
86.
87.
88.
381
tion mediates circular actin reorganization

and chemotaxis. EMBO J. 10:412128
Majumder S, Brown K, Qiu FH, Besmer
P. 1988. c-kit protein, a transmembrane
kinase: identification in tissues and characterization. Mol. Cell Biol. 8:4896903
Turner AM, Zsebo KM, Martin F, Jacobsen FW, Bennett LG, Broudy VC. 1992.
Nonhematopoietic tumor cell lines express stem cell factor and display c-kit receptors. Blood 80:37481
Heinrich MC, Rubin BP, Longley BJ,
Fletcher JA. 2002. Biology and genetic
aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations. Hum. Pathol. 33:484495
Taylor ML, Metcalfe DD. 2000. Kit signal
transduction. Hematol. Oncol. Clin. North
Am. 14:51735
Sattler M, Salgia R. 2004. Targeting c-Kit
mutations: basic science to novel therapies. Leuk. Res. 28 (Suppl. 1):1120
Rubin BP, Singer S, Tsao C, Duensing A,
Lux ML, et al. 2001. KIT activation is a
ubiquitous feature of gastrointestinal stromal tumors. Cancer Res. 61:811821
Miettinen M, Virolainen M, Maarit SR.
1995. Gastrointestinal stromal tumors
value of CD34 antigen in their identification and separation from true leiomyomas
and schwannomas. Am. J. Surg. Pathol.
19:20716
Joensuu H, Roberts PJ, Sarlomo-Rikala
M, Andersson LC, Tervahartiala P, et al.
2001. Effect of the tyrosine kinase inhibitor STI571 in a patient with a
metastatic gastrointestinal stromal tumor.
N. Engl. J. Med. 344:105256
Verweij J, van Oosterom A, Blay JY, Judson I, Rodenhuis S, et al. 2003. Imatinib
mesylate (STI-571 Glivec, Gleevec) is an
active agent for gastrointestinal stromal
tumours, but does not yield responses in
other soft-tissue sarcomas that are unselected for a molecular target. Results from
an EORTC Soft Tissue and Bone Sarcoma Group phase II study. Eur. J. Cancer
39:200611
7 Jan 2005
14:2

382
AR
AR232-PA45-15.tex
TIBES
TRENT
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE
KURZROCK
89. Choi H, Charnsangavej C, Macapinlac

HA, Burgess MA, Patel SR, et al. 2003.
Correlation of computerized tomography
(CT) and proton emission tomography
(PET) in patients with metastatic GIST
treated at a single institution with imatinib mesylate. Proc. Am. Soc. Clin. Oncol. 22:3290 (Abstr.)
90. Verweij J, Casali J, Zalcberg A, LeCesne
P, Reichard P, et al. 2003. Early efficacy comparison of two doses of imatinib for the treatment of advanced gastrointestinal stromal tumors (GIST): Interim
results of a randomized phase III trial
from the EORTC-STBSG, ISG and AGITG. Proc. Am. Soc. Clin. Oncol. 22:3272
(Abstr.)
91. Heinrich MC, Corless CL, Demetri GD,
Blanke CD, von Mehren M, et al. 2003.
Kinase mutations and imatinib response
in patients with metastatic gastrointestinal
stromal tumor. J. Clin. Oncol. 21:434249
92. Metcalfe DD, Akin C. 2001. Mastocytosis: molecular mechanisms and clinical
disease heterogeneity. Leuk. Res. 25:577
82
93. Cohen MH, Williams G, Johnson JR,
Duan J, Gobburu J, et al. 2002. Approval
summary for imatinib mesylate capsules
in the treatment of chronic myelogenous
leukemia. Clin. Cancer. Res. 8:93542
94. Dagher R, Cohen M, Williams G, Rothmann M, Gobburu J, et al. 2002. Approval summary: imatinib mesylate in
the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. Clin. Cancer Res. 8:3034
38
95. Druker BJ, Talpaz M, Resta DJ, Peng B,
Buchdunger E, et al. 2001. Efficacy and
safety of a specific inhibitor of the BCRABL tyrosine kinase in chronic myeloid
96. Trempat P, Villalva C, Laurent G, Armstrong F, Delsol G, et al. 2003. Chronic
myeloproliferative disorders with rearrangement of the platelet-derived growth
factor alpha receptor: a new clinical target
97.
98.
99.
100.
101.
102.
for STI571/Glivec. Oncogene 22:5702

06
Cunningham D, Humblet Y, Siena S,
Khayat D, Bleiberg H, et al. 2003. Cetuximab (C225) alone or in combination with
irinotecan (CPT-11) in patients with epidermal growth factor receptor (EGFR)positive, irinotecan-refractory metastatic
colorectal cancer (MCRC). Proc. Am.
Soc. Clin. Oncol. 22:1012 (Abstr.)
Saltz LB, Meropol NJ, Loehrer PJ Sr, Needle MN, Kopit J, Mayer RJ. 2004. Phase II
trial of cetuximab in patients with refractory colorectal cancer that expresses the
epidermal growth factor receptor. J. Clin.
Oncol. 22:120108
Hurwitz H, Fehrenbacher L, Cartwright
T, Hainsworth W, Heim W, et al. 2003.
Bevacizumab (a monoclonal antibody to
vascular endothelial growth factor) prolongs survival in first-line colorectal cancer (CRC): Results of a phase III trial
of bevacizumab in combination with bolus IFL (irinotecan, 5-fluorouracil, leucovorin) as first-line therapy in subjects
with metastatic CRC. Proc. Am. Soc. Clin.
Oncol. 22:3646 (Abstr.)
Kabbinavar F, Hurwitz HI, Fehrenbacher
L, Meropol NJ, Novotny WF, et al.
2003. Phase II, randomized trial comparing bevacizumab plus fluorouracil
(FU)/leucovorin (LV) with FU/LV alone
in patients with metastatic colorectal cancer. J. Clin. Oncol. 21:6065
Slamon DJ, Godolphin W, Jones LA,
Holt JA, Wong SG, et al. 1989. Studies
of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science
244:70712
Cobleigh MA, Vogel CL, Tripathy D,
Robert NJ, Scholl S, et al. 1999. Multinational study of the efficacy and safety
of humanized anti-HER2 monoclonal
antibody in women who have HER2overexpressing metastatic breast cancer
that has progressed after chemotherapy
for metastatic disease. J. Clin. Oncol. 17:
263948
7 Jan 2005
14:2
AR
AR232-PA45-15.tex
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE


103. Slamon DJ, Leyland-Jones B, Shak S,
Fuchs H, Paton V, et al. 2001. Use of
chemotherapy plus a monoclonal antibody against HER2 for metastatic breast
cancer that overexpresses HER2. N. Engl.
J. Med. 344:78392
104. Kim ES, Vokes EE, Kies MS. 2004. Cetuximab in cancers of the lung and head
& neck. Semin. Oncol. 31:6167
105. Salomon DS, Brandt R, Ciardiello F, Normanno N. 1995. Epidermal growth factorrelated peptides and their receptors in
human malignancies. Crit. Rev. Oncol.
Hematol. 19:183232
106. Ullrich A, Coussens L, Hayflick JS, Dull
TJ, Gray A, et al. 1984. Human epidermal
growth factor receptor cDNA sequence
and aberrant expression of the amplified
gene in A431 epidermoid carcinoma cells.
Nature 309:41825
107. Carpenter G, Cohen S. 1990. Epidermal
growth factor. J. Biol. Chem. 265:7709
12
108. Schlessinger J. 1988. The epidermal
growth factor receptor as a multifunctional allosteric protein. Biochemistry 27:
311923
109. Yarden Y, Sliwkowski MX. 2001. Untangling the ErbB signalling network. Nat.
Rev. Mol. Cell Biol. 2:12737
110. Arteaga CL. 2002. Overview of epidermal
growth factor receptor biology and its role
as a therapeutic target in human neoplasia.
Semin. Oncol. 29:39
111. Wells A. 1999. EGF receptor. Int. J.
Biochem. Cell Biol. 31:63743
112. Yarden Y. 2001. The EGFR family and its
ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur.
J. Cancer 37 (Suppl. 4):S3S8
113. Shvartsman SY, Hagan MP, Yacoub A,
Dent P, Wiley HS, Lauffenburger DA.
2002. Autocrine loops with positive feedback enable context-dependent cell signaling. Am. J. Physiol. Cell Physiol.
282:C54559
114. Grandis JR, Sok JC. 2004. Signaling
through the epidermal growth factor re-
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
383
ceptor during the development of malignancy. Pharmacol. Ther. 102:3746

Gullick WJ. 1991. Prevalence of aberrant
expression of the epidermal growth factor
receptor in human cancers. Br. Med. Bull.
47:8798
Nicholson RI, Gee JM, Harper ME. 2001.
EGFR and cancer prognosis. Eur. J. Cancer 37 (Suppl. 4):S915
Cohen MH, Williams GA, Sridhara R,
Chen G, Pazdur R. 2003. FDA drug
approval summary: gefitinib (ZD1839)
(Iressa) tablets. Oncologist 8:30306
Kies MS, Harari PM. 2002. Cetuximab
(Imclone/Merck/Bristol-Myers Squibb).
Curr. Opin. Investig. Drugs 3:1092100
Khalil MY, Grandis JR, Shin DM. 2003.
Targeting epidermal growth factor receptor: novel therapeutics in the management
of cancer. Expert. Rev. Anticancer Ther.
3:36780
Slamon DJ, Clark GM, Wong SG, Levin
WJ, Ullrich A, McGuire WL. 1987. Human breast cancer: correlation of relapse
and survival with amplification of the
HER-2/neu oncogene. Science 235:177
82
Grunwald V, Hidalgo M. 2003. Development of the epidermal growth factor receptor inhibitor OSI-774. Semin. Oncol.
30:2331
Ranson M, Mansoor W, Jayson G. 2002.
ZD1839 (IRESSA): a selective EGFR-TK
inhibitor. Expert. Rev. Anticancer Ther. 2:
16168
Wakeling AE, Guy SP, Woodburn JR,
Ashton SE, Curry BJ, et al. 2002. ZD1839
(Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res.
62:574954
Bianco C, Tortora G, Bianco R, Caputo R,
Veneziani BM, et al. 2002. Enhancement
of antitumor activity of ionizing radiation by combined treatment with the selective epidermal growth factor receptortyrosine kinase inhibitor ZD1839 (Iressa).
7 Jan 2005
14:2

384
AR
AR232-PA45-15.tex
TIBES
TRENT
AR232-PA45-15.sgm
LaTeX2e(2002/01/18)
P1: GCE
KURZROCK
125. Ciardiello F, Caputo R, Bianco R, Damiano V, Pomatico G, et al. 2000. Antitumor effect and potentiation of cytotoxic
drugs activity in human cancer cells by
ZD-1839 (Iressa), an epidermal growth
factor receptor-selective tyrosine kinase
inhibitor. Clin. Cancer Res. 6:2053
63
126. Normanno N, Campiglio M, De LA,
Somenzi G, Maiello M, et al. 2002. Cooperative inhibitory effect of ZD1839
(Iressa) in combination with trastuzumab
(Herceptin) on human breast cancer cell
growth. Ann. Oncol. 13:6572
127. Fukuoka M, Yano S, Giaccone G, Tamura
T, Nakagawa K, et al. 2003. Multiinstitutional randomized phase II trial of
gefitinib for previously treated patients
with advanced non-small-cell lung cancer.
J. Clin. Oncol. 21:223746
128. Sirotnak FM. 2003. Studies with ZD1839
in preclinical models. Semin. Oncol. 30:
1220
129. Perez-Soler R CA, Huberman M. 2004.
Final Results from a phase II study of erlotinib (Tarceva) monotherapy in patients
with advanced non-small cell lung cancer following failure of platinum-based
chemotherapy. Lung Cancer 42 (Suppl.
2):246 (Abstr.)
130. Baselga J. 2001. The EGFR as a target for
anticancer therapyfocus on cetuximab.
Eur. J. Cancer 37 (Suppl. 4):S1622
131. Bergers G, Benjamin LE. 2003. Tumorigenesis and the angiogenic switch. Nat.
Rev. Cancer 3:40110
132. Miller KD, Rugo HS, Cobleigh MA. MPKCLI. 2002. Phase III trial of capecitabine
(Xeloda) plus bevacizumab (Avastin)
versus capecitabine alone in women
133.
134.
135.
136.
137.
138.
with metastatic breast cancer previously

treated with an anthracycline and taxane.
Breast Cancer Res. Treat. 76 (Suppl. 1):37
(Abstr.)
Yang JC, Haworth L, Sherry RM, Hwu
P, Schwartzentruber DJ, et al. 2003. A
randomized trial of bevacizumab, an antivascular endothelial growth factor antibody, for metastatic renal cancer. N. Engl.
J. Med. 349:42734
Giaccone G, Herbst RS, Manegold C,
Scagliotti G, Rosel R, et al. 2004. Gefitinib in cominbation with gemcitabine and
cisplatin in advanced non-small-cell lung
cancer: a phase III trialINTACT 1. J.
Clin. Oncol. 22:77784
Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, et al. 2004. Gefitinib
in combination with paclitaxel and carboplatin in advanced non-small-cell lung
cancer: a phase III trialINTACT 2. J.
Clin. Oncol. 22:78594
Basel, 26 April 2004. New cancer drug
Tarcevaa breakthrough with significant
survival benefit in patients with advanced
lung cancer. http://www.gene.com/gene/
news/press-releases/display.do?method=
detail&id=7387
Lynch Tomas J, Bell DW, Sordella R,
Gurubhagavatula S, Okimoto RA, et al.
2004. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small cell lung
cancer to gefitinib. New Engl. J. Med.
350:212939
Hirota S, Isozaki K, Moriyama Y,
Sashimoto K, Nishida T, et al. 1998. Gainof-function mutations of c-kit in human
gastrointestinal stromal tumors. Science
279:57780
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C-1
Figure 1 PDGFR signaling Cascade: PDGFR is used as an example. Binding of PDGF

ligand to extracellular receptor domains leads to dimerization of receptor subunits, followed
by receptor autophosphorylation creating docking sites for several molecules leading to
induction of parallel intracellular signaling pathways. This process mediates pleiotropic biologic effects, including regulation of proliferation, cell cycle progression, apoptosis, survival,
and cell migration. Coupling proteins, such as PI3K, PLC, Src and others, facilitate GDPGTP exchange and phosphorylation. Other molecules, such as Grb2, Shc, and Crk, are devoid
of enzymatic activity but link the receptor to downstream targets. PI3K activates AKT, which
promotes cell survival through effects on transcription factors and inhibition of apoptosis.
Rho/Rac are involved in cellular structure, actin organization and chemotaxis. The Ras/MAP
kinase pathway is initiated by the adapter molecule Grb2, which forms a complex with Sos.
Raf stimulation of the MAPK pathway results in proliferation mediated by nuclear transcription factors. PLC phosphorylates PKC, which is a Ras-independent activator of the MAPK
pathway. Recruitment of src tyrosine kinase activates various cascades including the transcription factor c-myc. Not depicted in the figure is the JAK/STAT pathway, in which STATs
translocate from the membrane to the nucleus and directly activate cytokine-responsive
genes. Insert: Structure of the PDGFR . The extracellular region of PDGFR has five
immunoglobulin chain-like sites. The intracellular sites most frequently autophosphorylated
are shown by the numbers.
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Copyright

ACTIONS OF ADENOSINE AT ITS RECEPTORS IN THE

CNS: Insights from Knockouts and Drugs
Bertil B. Fredholm,1 Jiang-Fan Chen,2 Susan A. Masino,3
and Jean-Marie Vaugeois4
1
Department of Physiology and Pharmacology, Karolinska Institutet,

S-17177 Stockholm, Sweden
2
Department of Neurology, Boston University School of Medicine,
Boston, Massachusetts 02118
3
Department of Psychology and Neuroscience Program, Trinity College,
Hartford, Connecticut 06106
4
CNRS FRE2735, IFRMP 23, Faculty of Medicine and Pharmacy, 76183 Rouen, France
Key Words A1 receptor, A2A receptor, caffeine, ischemia, Parkinsons disease,

pain
Abstract Adenosine and its receptors have been the topic of many recent reviews
(126). These reviews provide a good summary of much of the relevant literature
including the older literature. We have, therefore, chosen to focus the present review on
the insights gained from recent studies on genetically modified mice, particularly with
respect to the function of adenosine receptors and their potential as therapeutic targets.
The information gained from studies of drug effects is discussed in this context, and
discrepancies between genetic and pharmacological results are highlighted.
GENETICALLY MODIFIED MICE

Adenosine Receptor Knockouts
Mouse strains lacking the genes for three of the four adenosine receptors have been
generated (Table 1). Two groups have generated adenosine A1 receptor knockouts
(A1R KO) (27, 28). A1R KO mice develop normally. They are fertile, but appear to
have a smaller number of offspring per litter, perhaps because sperm capacitation
is compromised in these animals (29). Their body temperature is normal, but, as
expected, the hypothermia elicited by A1R agonists is absent in A1R KO mice.
Interestingly, A1R KO mice had reduced survival rates as compared to A1R wildtype (WT) mice (30), although the maximal life span was unaffected. The increased
mortality in midlife may be linked to disturbances in cardiovascular, hepatic, and
renal systems, where A1Rs are likely to play an important role in the normal
physiology.
0362-1642/05/0210-0385$14.00
385
No
129/Sv C57BL/6
In frame insertion at exon 5
Adenosine
deaminase
No
129/JEms C57BL/6
In frame insertion at exon

amino acid Gly169-Thr225
Adenosine kinase
No
No
129 C57BL/6
129 B6D2
Entire coding exon 1

+ 7.5 kb immediate intron seq
A3 receptor
CD1, N12
129/Sv, N = 1
C57BL/6 N = 6
129/Sv CD1
129/Sv C57BL/6
Entire coding exon 2

3 portion of coding exon 2
1.0 kb immediate intron seq
No
129/SvJ C57BL/6
3 portion of coding exon 1 + intron

5 portion of coding exon 2
(42)
(39)
(34)
(31)
(32, 104)
(28)
(27)
References
AR232-PA45-16.sgm
Perinatal death,
die at 3 weeks
after trophoblast
rescue
Die at P4
No
No
No
No
No
No
No
Lethality
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A2A receptor
No
129/OlaHsd C57BL/6
Major portion of coding exon 2

+ 5 kb adjacent 3 genomic seq
A1 receptor
Congenic
Parent strains
Disrupted portion of the gene
Target
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NERVE PHENOTYPES OF ADENOSINE KNOCKOUTS
387
Two groups of scientists have generated adenosine A2A receptor knockout

(A2AR KO) mice. In the line generated by Ledent et al. (31), the mice were on a
CD1 outbred background, whereas in the two lines generated by Chen et al. (32)
the mice were bred onto C57BL/6 and Sv-129 backgrounds. All three lines of
A2AR KO mice were viable and bred normally. Blood pressure and heart rate were
increased, as well as platelet aggregation, in mice on a CD1 background (31), but
blood pressure and heart rate were not affected in mice with a C57BL/6 or Sv-129
background (32, 33).
Adenosine A3 receptors have been implicated in a variety of peripheral organ
system functions, including the regulation of cellular components of the immune
system (34) and cardiovascular function (35). The A3R KO mouse also had significantly lower intraocular pressure, suggesting that these receptors might be a target
for the development of drugs against glaucoma (36). However, an understanding of
the functions of A3 receptors in the central nervous system (CNS) has been impeded
both by a lack of specific ligands and the low density of these receptors (37).
All the KO mice mentioned so far lack one adenosine receptor subtype from a
very early developmental stage. Recently, a mouse with LoxP elements flanking the
second coding exon of the A1 gene was reported, as well as the combination with
an adenovirus expressing Cre recombinase. This opens the interesting possibility
of time- and tissue-specific inactivation of the adenosine A1R (38).
Metabolic Pathways
In addition to mice lacking specific adenosine receptor subtypes, there are mouse
strains in which the metabolic pathways controlling the levels of adenosine have
been genetically modified (Table 1). It is well known that adenosine levels are
regulated by adenosine kinase (phosphorylates adenosine to AMP) and adenosine
deaminase (converts adenosine to inosine).
A mouse strain with a targeted disruption of the adenosine kinase gene was
recently reported (39). These mice developed normally until birth, but they died
soon after birth. Low levels of adenine nucleotides and high levels of S-adenosyl
homocysteine are signature features of this genetic manipulation (39). It has been
shown in studies on yeast KOs that adenosine kinase plays an important role in
methyl transfer reactions (40). The fatal outcome in adenosine kinase KO mice
may be due, in particular, to the high levels of S-adenosyl homocysteine and
the consequent depression of several transmethylation reactions. For this reason,
we have to await the generation of region- and time-dependent KOs for adenosine
kinase before we can get clear information about its roles in the CNS. It is also worth
noting that adenosine may play a role in the association between cardiovascular
morbidity and hyperhomocysteinemia (41).
An adenosine deaminase KO mouse has been generated and provides a model
for increased adenosine levels (23, 42). Lack of adenosine deaminase is classically
associated with immune deficiency, but this is probably due to an accumulation of
2-deoxy-adenosine and subsequent accumulation of dATP, and not to adenosine
accumulation (43). Indeed, blockade of adenosine kinase in adenosine deaminase
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KO mice, which is expected to massively increase adenosine accumulation, decreased thymocyte death in parallel with decreased dATP accumulation (44).
Deamination of adenosine to inosine largely, but not completely eliminates the
actions of adenosine on adenosine receptors: The A1 and particularly the A3R
can also respond to inosine, although inosine is not a full agonist (4547). Indeed,
studies on KO mice have shown that the A3 receptor can mediate some of the effects
of inosine in the immune system, but for other effects of administered inosine only
mice lacking both the A2A and the A3 receptor were unresponsive (48), suggesting
that A2A receptors are also involved in mediating the effects of inosine. This does
not necessarily mean that inosine acts on A2A receptors, however. It could mean
that inosine increases levels of adenosine, which in turn acts on A2A receptors. In
addition, inosine may influence energy levels and polyADP-ribosylation (49).
Use of Heterozygotes
Attention is most often paid to the phenotype of the homozygous KO. However,
detailed examination of heterozygotes (HZ) can also be very revealing.
a) How well adjusted is the receptor level? If receptor number is directly proportional to gene dosageas is the case in A1R and A2AR HZthis argues
against strong autoregulation of transcription. Therefore, it seems likely that
neither A1 nor A2AR levels are regulated to a major extent by the ongoing
signaling via these receptors.
b) Heterozygotes often provide a better model for the effects likely to be seen
with antagonists because it is only rarely the case that antagonists can be
given at a dose that will inhibit all the receptors all the time. An especially
relevant aspect is that caffeine in doses commonly consumed by humans
gives plasma concentrations very close to the KD for caffeine at human A1
and A2ARs (3). Because responses to adenosine are shifted to the right, and
because there are only half the normal number of receptors in heterozygous
mice, it seems possible that heterozygous mice can be used as a genetic
model for caffeine use.
c) Heterozygous mice have also been used to circumvent the problems associated with the developmental effects that can potentially confound studies
on homozygous KO mice (50). In this approach, pharmacological agents
are given to heterozygous mice at doses that are subthreshold in WT mice.
There is no biological effect in WT mice treated with a subthreshold dose of
the drug or in HZ mice treated with vehicle, but a subthreshold dose elicits
a biological effect when combined with heterozygous genetic inactivation
of the target molecule (51). This approach may be particularly useful in
examining some adenosine receptor functions where discrepancies between
the pharmacological and genetic approaches have been reported (such as the
psychostimulant effect of A2AR KO and A2AR antagonists; see below under
the section on striatum and dopamine receptors).
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d) Heterozygotes can also be used to examine aspects of coupling, e.g., the

so-called receptor reserve in different tissues (52).

Limitations and Alternatives to Genetic Knockout Approaches

As powerful as it may be, the genetic KO approach also has its intrinsic limitations that may confound the correct interpretation of the phenotypic analysis of
KO mice. The two major limitations are the confounding developmental effect
and the lack of tissue specificity. The genes for adenosine receptors or enzymes
affecting adenosine level are depleted from early development throughout life in
KO mice, resulting in a phenotype that represents a developmental effect, rather
than an immediate consequence of receptor inactivation. Reproduction of KO phenotypes by adenosine receptor antagonistsgiven acutely or long-termwould
rule out developmental confounding effects. If developmental confounding effects are strongly suspected, the development of inducible knockouts (5355) may
be worth the effort. However, differences between acute effects of a purportedly
selective drug and phenotype in a KO could have many other causes than developmental effects. One potentially useful method to test specificity of drugs, and
the consequences of incomplete blockade, is to administer subthreshold doses of
pharmacological antagonists to heterozygotes (as described above). To address
the issue of lack of tissue specificity, a LoxP strategy has been used to create a
brain-specific depletion of A1Rs (38), and a similar approach can be applied to
other adenosine receptor knockouts. Finally, many studies have demonstrated the
effects of genetic backgrounds on differential phenotypic expression (5658). An
example could be the differences in blood pressure and heart rate of A2AR KO
mice in CD1 versus C57BL/6 or Sv-129 backgrounds, but methodological differences might also explain the different results. Another interesting example is the
finding that the A1R KO mouse possesses the Ren-2 renin gene derived from the
129 strain, whereas WTs do not (59). This is related to the fact that the Ren-2 gene
is positioned relatively close to the A1R gene (some 850 kb apart) on chromosome
1. Thus it is imperative to employ appropriate breeding strategies to control for
potentially confounding genetic backgrounds and flanking genes (60).
PRE- AND POSTSYNAPTIC EFFECTS

As a neuromodulator, adenosine affects synaptic transmission in a number of brain
regions (see Reference 6). Thus far, studies manipulating adenosine receptors have
focused primarily on characterizing responses in brain regions where that receptor
subtype is known to be important; subtle alterations or compensations in these or
other brain regions may yet be revealed.
The A1R subtype tonically inhibits synaptic transmission both pre- and postsynaptically in brain regions with a high concentration of A1Rs, such as the
hippocampus (see Reference 6). Heterozygote mouse brain contains half of the
number of receptors, and the EC50 for adenosine in the hippocampus is exactly
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Figure 1 Critical role of A1R in mediating inhibition of excitatory neurotransmission

by adenosine and ATP (insert) in hippocampus. Note that neither adenosine nor ATP
had any clear effect in A1R KO mice, and that the dose-response curve is shifted to the
left in the heterozygous mice, with no change in the maximal effect. Redrawn from
data in (27, 62).
twice that of the WT, whereas EMAX is unaffected (see Figure 1). Despite the
loss of tonic inhibition, no compensatory responses were found in other receptor
subtypes mediating similar G proteincoupled presynaptic inhibition of synaptic
transmission (27), but a wide range of potential compensatory mechanisms remain
to be explored.
Slices from homozygous A1R KO show no evidence of any remaining endogenous inhibitory influence of adenosine in the Schaffer collateral pathway in the
CA1 region of the hippocampus or at the mossy fiber synapses in the CA3 region (61). Furthermore, there is no inhibition of synaptic transmission when large
concentrations of adenosine (100 M) are applied exogenously (27). Using the
Cre-loxP system and an adeno-associated viral vector, a targeted deletion of the
A1R was induced separately in the CA1 and in the CA3 region of the hippocampus
(38). This approach holds promise for dissecting out specific pre- and postsynaptic actions of adenosine and its synaptic interactions with other molecules and
neurotransmitters, but so far no major results have been reported. Similar to the
constitutive KO model, there was no response to adenosine in the targeted inducible
knockout. Application of adenine nucleotides such as ATP was also ineffective in
hippocampal slices from KO mice (62) (see Figure 1). Together, these data suggest that the inhibitory effects of both adenosine and adenine nucleotides in the
hippocampus are mediated ultimately through the adenosine A1 receptor (27, 38),
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391
or alternatively, that effects of the other receptors require the presence of A1Rs, as
suggested for A2ARs (63). It will be important to determine the potency of adenosine in regulating neurotransmission via A1R in mice lacking the other receptors
to determine if there is such an interaction.
Dunwiddie and coworkers, using pharmacological tools (64), reported some
role of A3Rs in modulating the responses to A1 stimulation. This question was
re-examined recently and no significant interactions between A1 and A3Rs were
discovered using a host of different methods, including binding studies and electrophysiological studies (65). Thus, if A3Rs do play a role it is likely to be small
and indirect.
ISCHEMIA
It is generally believed that adenosine can protect tissues against the negative consequences of hypoxia or ischemia (1, 66), and that A1Rs play a particularly important
role. Hence, survival after a hypoxic challenge may be reduced if A1Rs are absent
or blocked (27). One consequence is that use of caffeine or other methylxanthines
in doses that would completely block A1Rs may be hazardous in hypoxic human
newborns. In keeping with the proposed role for adenosine acting at A1Rs, hippocampal slices taken acutely from adult mice do show greater functional recovery
from both hypoxic and ischemic insults when A1Rs are intact (27, 67). Moreover,
acute administration of an A1R antagonist did enhance ischemic damage in vivo,
giving further evidence that compensatory mechanisms may be providing protection in the knockout (68).
However, the severity of ischemic damage either in vivo or in organotypic
hippocampal slice cultures is not increased in the A1R KO model (68). The lack
of any obvious difference between the WT and the KO after pathophysiological
insult where A1Rs are considered neuroprotective is surprising. In immature brain,
blockade of A1Rs in fact attenuated ischemic injury. For example, the loss of white
matter that is a typical consequence of hypoxia in the newborn actually appears
to be mediated by adenosine acting on A1Rs (69). Thus, blockade of adenosine
receptorseven incomplete blockade like that achieved by caffeinereduces such
white matter loss (69). In addition, the consequences of prenatal hypoxic ischemia
in rats are reduced if the dams have been given caffeine (70).
Brain damage after focal ischemia has been reported to be attenuated in adult
A2AR KO mice compared with WT mice (32). On the other hand, aggravated brain
damage is observed after hypoxic ischemia in immature seven-day-old A2AR KO
mice (71). These results suggest that, in contrast to the situation in adult animals,
A2ARs play an important protective role against hypoxic ischemic brain injury in
neonates. Interestingly, a recent study using a novel approach where A2AR KO is
combined with bone marrow transplantation demonstrated that selective reconstitution of the A2AR in bone marrowderived cells of A2AR KO mice abolished the
neuroprotection against ischemic brain injury afforded by global depletion of A2AR
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(72). Conversely, selective A2AR inactivation by transplantation of bone marrow

cells from A2AR KO mice into WT mice reduced the volume of MCAO-induced
infarct in brain. This neuroprotection did not relate to the number of infiltrating
neutrophils and macrophages, but was associated with reduced MCAO-induced
expression of IL-6, IL-1, and IL-12 in the ischemic brain after gene inactivation.
These findings reveal a critical role for A2ARs on bone marrowderived cells following transient focal ischemia and suggest that targeting peripheral A2ARs in
bone marrowderived cells may be therapeutic against ischemic brain injury.
The role of A3Rs is enigmatic. Part of the reason for this is that the drugs
used to test their importance are not very selective, especially on rodent receptors (7). Indeed, some of the purportedly selective antagonists have effects in A3R
KO mice (36). A3 receptors are, however, clearly implicated in ischemia in the
heart, where the knockout shows significantly improved tolerance (21, 35, 73; J.
Yang, H. Sommerschild, G. Valen & B.B. Fredholm, unpublished data). In particular, recovery after myocardial ischemia was improved (74). By contrast, in
a model of carbon monoxideinduced hypoxia, the hippocampal neuronal damage was increased in A3R KO mice (75). The histological changes, along with
possible cognitive consequences, were also observed after administration of the
A3R antagonist MRS 1523 [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)6-phenylpyridine-5-carboxylate 1 mg/kg i.p.]. These results, and the observation
that deletion of the A3R had a detrimental effect in a model of mild hypoxia, suggest the possible use of A3R agonists in the treatment of ischemic, degenerative
conditions of the CNS (75).
The effects observed in these models and in in vivo models for other diseases
are summarized in Tables 2 and 3.
CAFFEINE
One reason why studies of adenosine and its receptors attract interest is that adenosine receptors (A1, A2A, and A2B) are the targets for the most widely used of all
psychoactive drugs, caffeine. Studies on KO animals have provided compelling
evidence that the psychostimulant effects of caffeine require blockade of A2ARs.
Caffeine has a mild stimulant effect in A2AR WT mice, but becomes a depressant of
locomotor activity in A2AR KO mice (31). Thus, A2ARs appear to be required for the
stimulant effect of caffeine (see Figure 2). In fact, caffeine dependently decreases
locomotion in A2AR KO mice over a wide range of doses (76). This effect probably
results from the other biological effects of caffeine, the blockade of A1Rs being a
candidate. Examining immediate early gene expression in WT and A2AR KO mice,
the Schiffmann group also concluded that A1R blockade was important for some
of the high-dose effects of caffeine (77). However, the role of A1R in the effects of
caffeine on motor activity is less clear. Recently, Halldner et al. (78) showed that
the A1R is not crucial for the stimulatory effect of caffeine, although the effect is
facilitated in the A1R KO mice. The results also suggest that the inhibitory effects
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TABLE 2 Adenosine receptor knockouts show a variety of behavioral changes that

differ according to receptor subtype. The different phenotypes may help to define brain
functions of adenosine and to discover novel targets for drugs against neurologic and
psychiatric disorders
Receptor
knockout
Modifications
Aggressiveness
A1
A2A
A3
Increased
Increased
Not determined
Anxiety
A1
A2A
A3
Increased/no change
Increased
No change
Despair-like
A1
A2A
A3
Not determined
Decreased
Increased
Memory
A1
A2A
A3
No change
Not determined
Not determined
Motor activity
A1
A2A
A3
No change/decreased
Decreased/slightly increased
Slightly increased
Neuroprotection
A1
A2A
A3
No effect in adults, beneficial in newborns

Beneficial in adults, detrimental in newborns
Detrimental effect
Sensorimotor gating
A1
A2A
A3
Not determined
Reduced startle inhibition and prepulse inhibition
Not determined
Thermal nociception
A1
A2A
A3
Hyperalgesia
Hypoalgesia
Hyperalgesia/no change

Function
of higher doses of caffeine are not due to blockade of the A1R. Rather, this effect
is likely to be independent of adenosine receptors. Clearly, many more studies of
the actions of caffeine in single and double KOs are necessary to delineate which
effects are entirely due to adenosine receptor blockade and which are not.
SLEEP
One of the best-known effects of caffeine is its effect on sleep (3). There is also
considerable evidence that adenosine is an endogenous promoter of sleep (for
references see 79). Adenosine levels, particularly in the basal forebrain, increase
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Figure 2 Biphasic effects of caffeine on locomotor behavior in mice. Data redrawn

from (78) and (76). The studies using A1R mice (squares) used mice on a mixed
C57BL/6 129OlaHsd background; those on A2A mice (circles) used CD1 mice.
Furthermore, the exact experimental setup differs. Hence, the two sets of data are not
strictly comparable. Filled symbol, WT mice; open symbols, KO mice.
during wakefulness and become particularly high during prolonged wakefulness

(80). Part of the reason for these changes in adenosine levels could be changes in
adenosine kinase and 5 -nucleotidase activities in the basal forebrain (81), but it
remains unclear if the basal forebrain differs from other brain regions and if there
are any changes upon sleep deprivation (82).
Most of the pharmacological data implicate A1R in the regulation of sleep
(79). Thus, A1R agonists induce sleep and sleep-like EEG (83, 84), whereas
antagonists reduce sleep (85). There are several mechanisms by which A1R stimulation may induce sleep. First, there is evidence that A1Rs are present on the cell
bodies of long cholinergic neurons and reduce their firing rate tonically (86), presumably by increasing potassium conductances. In hypothalamic slices, adenosine
disinhibits the GABAergic input to ventrolateral preoptic neurons (87). One possible additional substrate are the orexin-containing neurons, which express A1R (88).
Further support for an important role of A1R was given by studies showing
that an A1R antisense construct infused into the basal forebrain could decrease the
amount of REM sleep and increase wakefulness (89). However, the A1R KO mouse
did not show any difference from controls in the amount of sleep or in rebound
after sleep deprivation, even though an A1R antagonist produced the expected
effect in the control animals (90). Thus, there is evidence that the A1R is important
in sleep regulation in normal animals, but also that it is not absolutely necessary
for sleep regulation. Thus, when A1Rs are eliminated (and presumably when they
are profoundly antagonized for long periods of time) other regulatory mechanisms
take over. The nature of these adaptive changes is not known, and it remains to be
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shown if some level of adaptation also occurs after long-term, high-dose exposure
to caffeine.
Despite the fact that most attention has been focused on the role of A1 receptors,
there is increasing evidence that A2A receptors also play a role. For example, in
fetal sheep there is evidence for a tonic role of A2ARs in regulating REM sleep
state (91), and administration of A2AR agonists into the subarachnoid space close
to the preoptic area increases sleep (92). The possibility exists that the abundant
A2ARs present in the nucleus accumbens (92) or tuberculum olfactorium (93)
play a role. Thus, there are changes in A2ARs and the corresponding mRNA in
tuberculum olfactorium following sleep deprivation (93). Furthermore, the sleepinducing effect of the A2AR agonist CGS 21680 was eliminated in A2AR KO mice
(94). It will be of considerable interest to examine sleep in mice that lack both A1
and A2ARs and to determine whether caffeine has any effect in such mice.
STRIATUM AND INTERACTIONS WITH DOPAMINE

Although A2ARs are commonly believed to couple to Gs proteins, it is now established that in striatum, A2ARs (as well as dopamine D1 receptors) signal via
Golf proteins instead (95, 96). Indeed, full activity of A2A and D1 ligands requires
the presence of the normal number of Golf molecules, as shown by the reduced
response in mice heterozygous for Golf deletion (96). In agreement with this, the
same authors also found that the disruption of A2A or D1 receptors led to an altered
expression of Golf.
There is excellent evidence that A2ARs and dopamine D2 receptors are coexpressed on striatopallidal neurons and that they are functionally antagonistic (97,
98). Thus, blockade of D2 receptors would be expected to increase activity mediated
by A2ARs, and vice versa. One might therefore expect adaptive changes in KOs.
Indeed, in dopamine D2 receptor KO mice there is a functional decrease in A2AR
signaling (99), and A2AR KO mice are somewhat hypodopaminergic (100). This
might explain why A2AR deficiency selectively attenuates amphetamine-induced
and cocaine-induced locomotor responses (101), even though A2A receptor agonists can also attenuate psychostimulant responses (101, 102).
Even though A2A and D2 receptors are colocalized, interact at the membrane
level, and may form functional heterodimers, studies using A2A and D2 KO mice
show that endogenous adenosine can act independently of D2 receptors on A2ARs
and exerts a tonic influence. This tonic A2A stimulation is opposed by dopamine
acting at D2 receptors (98, 103105).
Locomotion
Basal locomotion is marginally affected in A1R KO mice (30, 78). Thus, no differences in overall spontaneous motor activity were detected over a 23-h monitoring
period, but activity was reduced in some parts of the light-dark cycle.
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A2ARs are highly expressed in the dorsal and ventral striatum, where they
could be involved in the physiological control of motor activity, and a major role
of A2AR stimulation is to modulate locomotor activity. In most studies performed
so far (31, 32, 101, 106), the exploratory behavior of A2AR KO mice was reduced
as compared to A2AR WT mice. As expected, treatment with the A2A agonist CGS
21680 strongly reduced locomotor activity in A2AR WT mice and had no significant
effect on A2AR KO mice (31, 32). However, this reduction of locomotor activity
en et al. (71)
is not an invariable characteristic of A2AR KO mice because as Ad
observed there is a small increase in basal locomotor activity at four weeks of
age in A2AR KO mice compared with A2AR WT mice. Locomotor behavior was
reported to be increased in A3R KO mice (75).
Parkinsons Disease
Among new therapeutic approaches for Parkinsons disease, one possibility being
investigated is modulation of dopamine-mediated striatal functions through the
blockade of A2ARs. The past ten years have witnessed significant progress in the
development and characterization of a new generation of A2AR antagonists for
use in Parkinsons disease. The motor enhancement afforded by A2A antagonists
was well documented in early pharmacological studies (19), and activity at A2ARs
reduces motor responses in both normal and dopamine-depleted animals. The
multiple benefits of A2AR inactivation (seen either after genetic deletion, as in
A2AR KO mice, or after treatment with A2A antagonists) advance the prospects of
A2AR antagonists as a novel treatment strategy for Parkinsons disease (18, 107)
(see Table 3).
Proof of principle comes from large epidemiological studies that firmly establish
an inverse relationship between caffeine consumption and the risk of developing
Parkinsons disease (108110). Studies carried out in mice support these epidemiological findings, providing evidence for neuroprotective effects of caffeine and
specific A2AR antagonists, as well as genetic deletion of the A2AR (111). Several
other pharmacological studies employing various A2AR antagonists also support
this neuroprotective effect (112, 113).
A2AR antagonism also provides symptomatic relief of surgical lesion or druginduced motor dysfunction. Catalepsy, a state where the animals remain immobile
for long periods, even if they are placed in awkward postures, can be induced by
dopamine D1 or D2 receptor antagonists or a muscarinic acetylcholine receptor agonist. In A2AR KO mice, such catalepsy was reduced as compared with A2AR WT
mice (104, 114). These results suggest that A2ARs influence not only dopamine D1
and D2 receptormediated neurotransmission but also that mediated via muscarinic
acetylcholine receptors. Interestingly, caffeine and muscarinic antagonists act in
synergy to inhibit haloperidol-induced catalepsy (115). The results on catalepsy
show that deletion of the A2A receptor alleviates dysfunction of basal ganglia motor
circuitry caused by drugs acting at dopamine and acetylcholine receptors. These
preclinical studies led to the clinical trial of the A2AR antagonist KW6002 in patients with Parkinsons disease, and the initial results were encouraging (116, 117).
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TABLE 3 Therapeutic implications for neurological disorders as suggested by genetic

knockout and pharmacological analysis
Disorder
Animal models
Effect
References
Parkinsons
disease
MPTP
MPTP
6-OHDA
Haloperidol-catalepsy
D2R KO-induced
hypolocomotion
MPTP-bradykinesia
in monkey
Reduced neurotoxicity in A2AR KO mice

Reduced neurotoxicity by A2AR antagonists
Reduced SN neuron loss by A2AR antagonists
Enhanced locomotor activity by A2AR KO
(111)
(111, 153)
(112)
(111, 114)
Reversed by A2AR antagonists
(154, 155)
3-NP
3-NP
3-NP
Reduced striatal damage in A2AR KO

Reduced striatal damage by A2AR antagonists
Enhanced/reduced striatal damage
in A2AR KO
Reduced striatal damage by A1 agonists
(111)
(111)
(120)
Huntingtons
disease
3-NP
(156)
Multiple
sclerosis
Experimental
autoimmune
encephalomyelitis
(EAE)
Increased demyelination and axonal

degeneration in A1R KO
(132)
Stroke (ischemic
brain injury)
Hypoxia
No change in organotypic hippocampus

slices in A1R KO mice
Reduced white matter in neonate A1R KO
Aggravated damage in neonate A2AR KO
(27)
Reduced infarct volume and neurological

deficit score in A2AR KO mice
No effect on damage in A1R KO
Reduced infarct volume in chimeric
mice with selective depletion of
bone marrow-derived cells
Increased hippocampus neuronal
damage in A3R KO mice
(32)
Hypoxic-ischemia
Prenatal hypoxic
ischemia
MCAO
MCAO
MCAO
Carbon monoxide
Alzheimers
disease
Beta-amyloid
aggregation
Reduced neurotoxicity in cerebellum

neurons
(69)
(71)
(68)
(72)
(75)
(157)
Finally, recent studies with A2AR KO mice and pharmacological agents suggest the possibility of another potentially beneficial effect of A2AR antagonists,
namely prevention of the development of dyskinesia after repeated treatment with
L-DOPA (118, 119). Debilitating motor complications such as dyskinesia are the
major limiting factors of management in the later stages of Parkinsons disease.
Thus the finding that repeated administration of L-DOPA did not lead to behavioral
sensitization in A2AR KO mice indicates that the A2AR may be required for the development of maladaptive changes after long-term treatment with L-DOPA (113).
This notion is further supported by recent studies in MPTP-treated nonhuman
primates, showing that coadministration of KW6002 with the dopamine agonist
apomorphine completely abolished apomorphine-induced dyskinesia (119).
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Huntingtons Disease
The cellular localization of the A2AR in striatopallidal neurons suggests that the
A2AR may contribute to selective vulnerability to neurotoxins in Huntingtons
disease. Indeed, there is also evidence that A2ARs may play a role in Huntingtons
disease (16, 120) (Table 3). In a neurochemical model of Huntingtons disease,
pharmacological and genetic inactivation of the A2AR have been shown to attenuate
striatal damage induced by the mitochondrial toxin 3-nitropropionic acid (121) or
the excitotoxin quinolinic acid (122). However, the role of the receptors is complex,
and it is difficult at present to envision A2A antagonism as a therapy in this disorder.
The complex actions were interpreted as resulting from a balance between negative
effects owing to blockade of presynaptic A2A receptors regulating glutamate release
and positive effects owing to blockade of postsynaptic receptors (120). However,
glutamate levels are also regulated by glutamate transporters on glial cells, which
express A2AR. Therefore, the interpretation of glutamate changes is not yet clear.
Schizophrenia
Another pathological condition involving both adenosine and dopamine in the
striatumand where A2A agonists might be beneficialis schizophrenia. Patients
with schizophrenia show impaired sensorimotor gating. Normally, this gating prevents excessive irrelevant sensory stimuli from disturbing integrative mental processes in the brain. In schizophrenic patients, the impairment in sensorimotor
gating results in reduced prepulse inhibition (PPI) and reduced startle habituation.
In experimental animals, both parameters are modulated by dopaminergic and
adenosine receptor agonists and antagonists. Wang et al. (123) recently found that
startle amplitude, startle habituation, and PPI were significantly reduced in A2AR
KO mice, which provides evidence that this receptor may be involved in the regulation of these phenomena. In addition, responses to an NMDA antagonist and amphetamine were altered (123). These data suggest substances with A2A receptor agonist properties may be of interest in the development of antipsychotic drugs (124).
ACTIONS IN OTHER PARTS OF THE NERVOUS SYSTEM

Addictive Drugs
A popular belief is that coffee can antagonize the intoxicating effects of alcohol.
However, the molecular mechanisms that might underlie this offsetting action of
coffee remain poorly identified. To investigate the possible involvement of the
A2AR in the behavioral sensitivity to high doses of ethanol, the hypnotic effect
of ethanol on A2AR KO mice and A2AR WT mice has been assessed (125). The
righting reflex was lost following acute ethanol administration, but the effect lasted
longer in A2AR WT mice than in A2AR KO mice. The fall in body temperature was
not different between the two phenotypes. Dipyridamole, an inhibitor of adenosine
uptake, increased the sleep time observed following administration of ethanol in
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A2AR WT mice but not in A2AR KO mice. The selective A2AR antagonist SCH
58261, but not the selective A1 receptor antagonist DPCPX, shortened the duration
of the loss of righting reflex induced by ethanol, thus mimicking the lack of the
receptor in A2AR-deficient mice. Caffeine (25 mg/kg) also reduced ethanol-induced
hypnotic effects. These results indicate that the activation of A2A receptors plays
a role in the hypnotic effect of ethanol.
The cessation of chronic ethanol intake or ethanol withdrawal is an experimental procedure recognized to produce seizures in mice. This convulsant activity is associated with an increase in excitatory neurotransmission in the brain.
Whereas A2AR KO mice and controls ingested similar amounts of ethanol during
forced ethanol consumption, the severity of handling-induced convulsions during
withdrawal was significantly lower in the A2AR KO mice than in A2AR WT mice.
Because the selective A2AR antagonist ZM 241385 also attenuated the intensity
of withdrawal-induced seizures, it was suggested that selective A2AR antagonists
may be useful in the treatment of alcohol withdrawal (126). The role of A2ARs
in ethanol consumption and neurobiological responses to this drug of abuse was
further characterized by Naassila et al. (127). Male and female A2AR KO mice consumed more ethanol than WT mice. This slightly higher ethanol consumption was
also related to ethanol preference. Relative to A2AR WT mice, A2AR KO mice were
found to be less sensitive to the sedative and hypothermic effects of ethanol. No
major difference in the development of tolerance to ethanol-induced hypothermia
was found between the two phenotypes, although female A2AR KO mice showed
a lower tolerance-acquisition rate. These results suggest that activating the A2ARs
may play a role in suppressing alcohol-drinking behavior and be associated with
sensitivity to the intoxicating effects of acute ethanol administration.
There is also evidence that morphine dependence is modified by A2ARs. Opiate
withdrawal was enhanced in mice lacking A2A receptors, and this enhancement
was abolished when both the cannabinoid CB1 receptor and A2AR were eliminated
(106).
Because there is considerable evidence for interactions between adenosine receptors and central stimulants (see above), for a role of adenosine in some actions
of morphine (17), for various interactions between adenosine and ethanol (128),
and because adenosine receptors are very important in regulating dopaminergic
transmission in the reward pathways in nucleus accumbens, it is important to
further examine the effects of addictive drugs in AR KO mice.
Seizures
It has long been known that adenosine can suppress repetitive neuronal firing, and a
role of adenosine as an endogenous modifier of seizures has been suspected. This
notion recently received support (129) when it was found that seizure-inducing
lesions can increase the level of adenosine kinase in astrocytes, and that this,
by reducing adenosine levels, contributes to increased seizure susceptibility. This
raises the possibility that modifying the extracellular adenosine level in brain may
be of therapeutic value against seizures. Indeed, cells that generate adenosine have
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been transplanted into rat brain and this has led to decreased seizure susceptibility
(130, 131). In particular, activation of A1Rs appears to be an interesting target
for therapy in drug-resistant epilepsy (131). Unless there are major compensatory
mechanisms in effect, seizure thresholds would be expected to be lower in A1R
KO animals, but this awaits further investigation.
Multiple Sclerosis
There is some evidence that adenosine may play a role in multiple sclerosisat
least there are effects in an experimental model (132). Thus, in A1R KO mice the
demyelination and axonal degeneration was much more pronounced than in WT
littermates. There was also a stronger activation of microglia/macrophages. Furthermore, macrophages from A1R KO animals exhibited increased expression of
the proinflammatory genes IL-1 and matrix metalloproteinase-12 on immune activation compared to control cells from A1R WT animals (132). This would imply
first that A1Rs are very important in regulating macrophages/microglial cells. However, this is not immediately obvious from other data where these cells have been
examined (e.g., 133). Furthermore, the role of A1Rs in regulating oligodendrocyte
function and survival appears to differ between the adult spinal cord (132) and the
immature brain (69). This again emphasizes that the roles of adenosine receptors
may be complex, and that they could differ with age, location, and pathology.
Memory
Despite some hints from experiments with drugs that affect adenosine receptors,
the evidence from KO animals does not reveal any clear effect of the A1R KO
genotype on memory (30, 134). Minor effects in the water maze were suggested
(134) to be due to the altered emotional stability reported for these mice (27,
30). Long-term potentiation (LTP), an in vivo model of memory formation, has
generally been observed to be inhibited by A1R activation (135) and enhanced by
A2AR activation (136, 137). Deletion of adenosine A2ARs did not alter ongoing
synaptic transmission in either striatum (138) or nucleus accumbens (137), but
accumbens neurons showed significantly reduced LTP when the effects of the
A2AR were removed (137). LTP was reduced greatly in the mossy fiber pathway in
hippocampal slices from A1R KO mice as well as rat hippocampal slices pretreated
with an A1R antagonist (61), providing strong evidence that adenosine acting at
the A1R augments LTP in this pathway.
Anxiety
The neurobiology of anxiety, including the role of adenosine, was recently comprehensively reviewed (139). Interestingly, anxiety-related behavior in the classical
light/dark box test was increased in the A1R KO mice, as shown by a reduction
in the number of entries into as well as the total time spent in the lit compartment
compared with A1R WT mice (27, 30). The A1R KO mice also showed a decrease
in exploratory behavior in the open-field and in the hole-board, results that could
reflect an anxiogenic state in A1R KO mice. However, another strain of A1R KO
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mice with a similar genetic background displayed a normal overall level of motor
activity, with very modest behavioral changes in the direction of increased anxiety
(134). It is likely that different environmental conditions have contributed substantially to the behavioral discrepancies between the two lines. This might prompt
us to ask whether the increased sensitivity to caffeine reported in patients with
panic disorders (140) is indeed linked to a disorder of adenosine neuromodulation
at A1Rs in the brain.
A2AR KO mice showed higher rates of spontaneous anxiety-like responses
in two different anxiety-like behavioral tests, the elevated plus-maze and the
light/dark box (31, 106, 141). Thus, A2AR KO mice and at least one strain of
A1R KO mice exhibit increased anxiety, consistent with the well-known, pronounced, anxiogenic effects of high doses of caffeine. High doses of caffeine will
presumably block most of these adenosine receptor subtypes, but low doses will
not. Despite several studies using pharmacological tools and performed in rodent
models (141144) there is no clear consensus concerning the role of A1 and A2ARs
in anxiety. However, on the basis of screening tests, it has been proposed that A1R
agonists exert anxiolytic effects, whereas A1R antagonists in some cases, but not
consistently, exert anxiogenic effects. On the other hand, it is still unclear whether
the A2AR also plays a major role in anxiety states. Selective A2AR antagonists
seem to be devoid of effects in tests on rodents (141). However, recent data from
humans shed fresh light on the potential role of A2ARs in the anxiogenic effects
of caffeine. In a study conducted by Alsene et al. (145), the association between
variations in anxiogenic responses to caffeine and polymorphisms in the adenosine A1 and A2AR genes has been examined. They found a significant association
between self-reported anxiety after oral administration of 150 mg of caffeine and
two linked polymorphisms on the A2AR gene. Individuals with the 1976T/T and
the 2592Tins/Tins genotypes reported greater increases in anxiety after caffeine
administration than the other genotypic groups. Moreover, in patients with panic
disorder, a psychiatric condition characterized by recurrent panic attacks and anticipatory anxiety, a single-nucleotide polymorphism haplotype in the A2AR gene
was found to be associated with the disease (146). Alpha-melanocyte-stimulating
hormone (alpha-MSH) influences anxiety, aggressiveness, and motor activity, all
of which are also influenced by A2AR gene disruption. In A2AR KO mice, significantly increased alpha-MSH content was observed in the amygdala and cerebral
cortex. Plasma corticosterone concentration was significantly higher in A2AR KO
mice, revealing hyperactivity of their pituitary-adrenocortical axis. Results suggest
that A2ARs are involved in the control of POMC gene expression and biosynthesis
of POMC-derived peptides in pituitary melanotrophs and corticotrophs (147).
Aggression
Several studies have suggested that adenosine receptors are involved in the modulation of aggressive behavior. In agreement with the decrease of offensive behavior
induced by a selective stimulation of A1Rs (148), A1R KO mice isolated for the
resident-intruder aggression test showed enhanced aggressive behavior (27). A
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similar enhancement was also observed in isolated male A2AR KO mice in the
resident-intruder test (31). The increased aggressiveness observed in both A1R
KO mice and A2AR KO mice is in agreement with the increase of offensive behavior induced by selective blockade of either A1 or A2ARs (M. El Yacoubi &
J.M.Vaugeois, unpublished observations). These results suggest that both adenosine receptor subtypes are involved in the effect of adenosine on aggressiveness.
The link between these effects and the increase in nervousness and irritability reported in humans (3) after chronic administration of high doses of caffeine remains
a matter of speculation.
Depression
In behavioral procedures used to screen potential antidepressants, such as tail
suspension and forced swim tests, A2AR KO mice were found to be less sensitive
to depressant challenges than their WT littermates, which were less immobile
than A2AR WT mice in both tests (149). Consistently, A2AR blockers reduced
the immobility times in tail suspension and forced swim tests. Taken together,
the results support the hypothesis that blockade of the A2AR might be an interesting
target for the development of effective antidepressant agents. Although their mode
of action in potentially alleviating mood disorders is unknown, modulation of
dopamine transmission might play a role (149). Future clinical trials with selective
A2AR antagonists as potential therapeutic agents for major depressive episodes will
help to delineate the role of adenosine in the pathophysiology of mood disorders.
Whereas A2AR antagonists have been proposed as antidepressants (149), A3R KO
mice showed an increase in the amount of time spent immobile in the two tests of
behavioral depression, the forced swim test and the tail suspension test (75).
Pain
The role of adenosine as an endogenous analgesic substance has also been evaluated
(27). A1Rs are abundant in mouse spinal cord, with the highest levels in the outer
lamina of the dorsal horns, where the density of receptors was close to that observed
in the hippocampus. A1Rs are responsible for the analgesic effects of intrathecally
administered A1 agonists. A1R KO mice react faster to thermal pain than A1R
WT mice. However, this increase is not matched by an increased sensitivity to
mechanical stimulation. The authors suggested that endogenous adenosine acting
at A1Rs decreases nociception, mediated via C fibers. These results also suggest
that the A1R may be a target for the development of antinociceptive drugs.
The response of A2AR KO mice to acute pain stimuli is slower in the hot
plate and tail-flick tests compared to A2AR WT mice (31). Similar reduced pain
responses were also found when a tail-immersion test was used (106). This higher
nociceptive threshold suggests that the peripheral lack of A2ARs predominates over
the spinal defect. Thus, depending on the site of action and the receptor activated
(A1 or A2A), adenosine may exert very different effects on pain. This variety of
effects may explain why caffeine has analgesic effects against some, but not all,
types of pain (3).
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403
A3R KO mice show decreased sensitivity to some painful stimuli, as evidenced

by the increase in latency in the hot plate but not tail-flick test (75). Another study
(150) found no evidence for a role of A3R in nociception or in the antinociceptive
effect of the adenosine analog R-phenylisopropyl adenosine (R-PIA). Thus, no
difference was seen between A3R KO and A3R WT mice in nociceptive response
to mechanical or radiant heat stimuli. The antinociceptive response to intrathecal
R-PIA was also unchanged in the A3R KO mice. In contrast, heat hyperalgesia,
plasma extravasation, and edema following carrageenan-induced inflammation
in the hind paw were significantly reduced in A3R KO mice compared to the
A3R WT controls. Thus, mice lacking A3R had deficits in generating the localized
inflammatory response to carrageenan, supporting a proinflammatory role of A3Rs
in peripheral tissues.
CONCLUSIONS
Whereas deletion of genes for enzymes critically involved in adenosine metabolism
leads to lethal phenotypes, deletion of A1, A2A, and A3 receptors has rather subtle
effects and the mice are remarkably normal. This agrees well with the conclusion
drawn before, i.e., that adenosine receptors are involved in modulating physiological responses and that they are particularly important under pathophysiological
conditions. Thus, to determine the roles of the adenosine receptors, the genetically
modified mice must be subjected to various types of challenges.
The results obtained so far have both confirmed previous data and yielded some
surprises. The important role of the A1R in modulating excitatory transmission and
its role in pain transmission was expected, as was the critically important role of
A2ARs in striatal function. Among the major surprises were the noncritical role of
A1Rs in brain ischemia and in sleep and the finding that A2ARs mediate aggravated
brain damage mainly via peripheral receptors. Our examination of the literature has
also indicated studies that can and should be performed to further define the roles
of adenosine receptors in the nervous system. Because the goal of these studies is to
examine the possibility for novel drug therapies, the use of KO mice to determine
that the drugs are indeed selective is very important. Indeed, data obtained already
suggest that some of the drugs used to delineate adenosine receptor effects are not
as selective as previously hoped (36, 151, 152).
LITERATURE CITED
1. Fredholm BB. 1996. Adenosine and
neuroprotection. In Neuroprotective
Agents and Cerebral Ischemia, ed. AR
Green, AJ Cross, pp. 25980. London:
Academic
2. Svenningsson P, Le Moine C, Fisone G,

Fredholm BB. 1999. Distribution, biochemistry and function of striatal adenosine A2A receptors. Prog. Neurobiol. 59:
35596
4 Dec 2004
13:21

404
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
FREDHOLM ET AL.
3. Fredholm BB, Battig K, Holmen J, Nehlig

A, Zvartau E. 1999. Actions of caffeine
in the brain with special reference to factors that contribute to its widespread use.
Pharmacol. Rev. 51:83153
4. Cunha RA. 2001. Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles,
different sources and different receptors.
Neurochem. Int. 38:10725
5. Fredholm BB, Arslan G, Halldner L, Kull
B, Schulte G, Wasserman W. 2000. Structure and function of adenosine receptors
and their genes. Naunyn-Schmiedebergs
Arch. Pharmacol. 362:36474
6. Dunwiddie TV, Masino SA. 2001. The
role and regulation of adenosine in the
central nervous system. Annu. Rev. Neurosci. 24:3155
7. Fredholm BB, IJzerman AP, Jacobson
KA, Klotz K-N, Linden J. 2001. International Union of Pharmacology. XXV.
Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53:527
52
8. Linden J. 2001. Molecular approach to
adenosine receptors: receptor-mediated
mechanisms of tissue protection. Annu.
9. Latini S, Pedata F. 2001. Adenosine in
the central nervous system: release mechanisms and extracellular concentrations.
10. Dalpiaz A, Manfredini S. 2002. Adenosine A(1) receptor: analysis of the potential therapeutic effects obtained by its activation in the central nervous system. Curr.
Med. Chem. 9:192337
11. Fredholm BB. 2002. Adenosine receptors.
In Understanding G Protein-Coupled Receptors and Their Role in the CNS, ed.
MN Pangalos, CH Davies, pp. 191204.
Oxford: Oxford Univ. Press
12. Rorke S, Holgate ST. 2002. Targeting
adenosine receptors: novel therapeutic
targets in asthma and chronic obstructive
pulmonary disease. Am. J. Respir. Med.
1:99105
13. Thong FS, Graham TE. 2002. The putative roles of adenosine in insulin- and
exercise-mediated regulation of glucose
transport and glycogen metabolism in
skeletal muscle. Can. J. Appl. Physiol.
27:15278
14. Klinger M, Freissmuth M, Nanoff C.
2002. Adenosine receptors: G proteinmediated signalling and the role of accessory proteins. Cell. Signal. 14:99
108
15. Schwarzschild MA, Chen JF, Ascherio A.
2002. Caffeinated clues and the promise
of adenosine A(2A) antagonists in PD.
Neurology 58:115460
16. Blum D, Hourez R, Galas MC, Popoli
P, Schiffmann SN. 2003. Adenosine receptors and Huntingtons disease: implications for pathogenesis and therapeutics.
Lancet Neurol. 2:36674
17. Sawynok J, Liu XJ. 2003. Adenosine in
the spinal cord and periphery: release
and regulation of pain. Prog. Neurobiol.
69:31340
18. Chen JF. 2003. The adenosine A(2A) receptor as an attractive target for Parkinsons disease treatment. Drug News Perspect. 16:597604
19. Fredholm BB, Svenningsson P. 2003.
Adenosine-dopamine interactions: development of a concept and some comments on therapeutic possibilities. Neurology 61:S59
20. Dhalla AK, Shryock JC, Shreeniwas R,
Belardinelli L. 2003. Pharmacology and
therapeutic applications of A1 adenosine
receptor ligands. Curr. Top. Med. Chem.
3:36985
21. Headrick JP, Hack B, Ashton KJ. 2003.
Acute adenosinergic cardioprotection in
ischemic-reperfused hearts. Am. J. Physiol. Heart Circ. Physiol. 285:H1797
818
22. Schulte G, Fredholm BB. 2003. Signalling from adenosine receptors to
mitogen-activated protein kinases. Cell.
Signal. 15:81327
23. Blackburn MR. 2003. Too much of a good
4 Dec 2004
13:21
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
24.

25.
26.
27.
28.
29.
30.
31.
32.
thing: adenosine overload in adenosinedeaminase-deficient mice. Trends Pharmacol. Sci. 24:6670

Fredholm BB, Cunha R, Svenningsson
P. 2003. Pharmacology of adenosine A2A
receptors and therapeutic applications.
Curr. Top. Med. Chem. 3:41326
Hasko G, Cronstein BN. 2004. Adenosine: an endogenous regulator of innate
immunity. Trends Immunol. 25:3339
McCallion K, Harkin DW, Gardiner KR.
2004. Role of adenosine in immunomodulation: review of the literature. Crit. Care
Med. 32:27377
Johansson B, Halldner L, Dunwiddie TV,
Masino SA, Poelchen W, et al. 2001.
Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the
adenosine A1 receptor. Proc. Natl. Acad.
Sci. USA 98:940712
Sun D, Samuelson LC, Yang T, Huang
Y, Paliege A, et al. 2001. Mediation of
tubuloglomerular feedback by adenosine:
evidence from mice lacking adenosine 1
receptors. Proc. Natl. Acad. Sci. USA 98:
998388
Minelli A, Liguori L, Bellazza I, Mannucci R, Johansson B, Fredholm BB.
2004. Involvement of A1 adenosine receptors in the acquisition of fertilizing capacity. J. Androl. 25:28692
Gimenez-Llort L, Fernandez-Teruel A,
Escorihuela RM, Fredholm BB, Tobena
A, et al. 2002. Mice lacking the adenosine A1 receptor are anxious and aggressive, but are normal learners with reduced
muscle strength and survival rate. Eur. J.
Neurosci. 16:54750
Ledent C, Vaugeois JM, Schiffmann SN,
Pedrazzini T, El Yacoubi M, et al. 1997.
Aggressiveness, hypoalgesia and high
blood pressure in mice lacking the adenosine A2A receptor. Nature 388:67478
Chen JF, Huang Z, Ma J, Zhu J, Moratalla
R, et al. 1999. A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice.
J. Neurosci. 19:9192200
405
33. Day YJ, Huang L, McDuffie MJ, Rosin

DL, Ye H, et al. 2003. Renal protection
from ischemia mediated by A2A adenosine receptors on bone marrow-derived
cells. J. Clin. Invest. 112:88391
34. Salvatore CA, Tilley SL, Latour AM,
Fletcher DS, Koller BH, Jacobson MA.
2000. Disruption of the A(3) adenosine
receptor gene in mice and its effect on
stimulated inflammatory cells. J. Biol.
Chem. 275:442934
35. Cerniway RJ, Yang Z, Jacobson MA, Linden J, Matherne GP. 2001. Targeted deletion of A(3) adenosine receptors improves
tolerance to ischemia-reperfusion injury
in mouse myocardium. Am. J. Physiol.
Heart Circ. Physiol. 281:H175158
36. Avila MY, Stone RA, Civan MM. 2002.
Knockout of A3 adenosine receptors reduces mouse intraocular pressure. Invest.
Ophthalmol. Vis. Sci. 43:302126
37. Rivkees SA, Thevananther S, Hao H.
2000. Are A3 adenosine receptors expressed in the brain? NeuroReport 11:1025
30
38. Scammell TE, Arrigoni E, Thompson
MA, Ronan PJ, Saper CB, Greene RW.
2003. Focal deletion of the adenosine A1
receptor in adult mice using an adenoassociated viral vector. J. Neurosci. 23:
576270
39. Boison D, Scheurer L, Zumsteg V,
Rulicke T, Litynski P, et al. 2002. Neonatal hepatic steatosis by disruption of the
adenosine kinase gene. Proc. Natl. Acad.
Sci. USA 99:698590
40. Lecoq K, Belloc I, Desgranges C,
Daignan-Fornier B. 2001. Role of adenosine kinase in Saccharomyces cerevisiae:
identification of the ADO1 gene and study
of the mutant phenotypes. Yeast 18:335
42
41. Riksen NP, Rongen GA, Blom HJ, Russel
FG, Boers GH, Smits P. 2003. Potential
role for adenosine in the pathogenesis of
the vascular complications of hyperhomocysteinemia. Cardiovasc. Res. 59:271
76
4 Dec 2004
13:21

406
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
FREDHOLM ET AL.
42. Blackburn MR, Datta SK, Kellems RE.

1998. Adenosine deaminase-deficient
mice generated using a two-stage genetic engineering strategy exhibit a combined immunodeficiency. J. Biol. Chem.
273:5093100
43. Apasov SG, Blackburn MR, Kellems
RE, Smith PT, Sitkovsky MV. 2001.
Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling. J. Clin.
Invest. 108:13141
44. Van De Wiele CJ, Vaughn JG, Blackburn
MR, Ledent CA, Jacobson M, et al. 2002.
Adenosine kinase inhibition promotes
survival of fetal adenosine deaminasedeficient thymocytes by blocking dATP
accumulation. J. Clin. Invest. 110:395
402
45. Jin X, Shepherd RK, Duling BR, Linden
J. 1997. Inosine binds to A3 adenosine receptors and stimulates mast cell degranulation. J. Clin. Invest. 100:284957
46. Tilley SL, Wagoner VA, Salvatore CA, Jacobson MA, Koller BH. 2000. Adenosine
and inosine increase cutaneous vasopermeability by activating A(3) receptors on
mast cells. J. Clin. Invest. 105:36167
47. Fredholm BB, Irenius E, Kull B, Schulte
G. 2001. Comparison of the potency of
adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochem. Pharmacol.
61:44348
48. Gomez G, Sitkovsky MV. 2003. Differential requirement for A2a and A3 adenosine receptors for the protective effect of
inosine in vivo. Blood 102:447278
49. Hasko G, Sitkovsky MV, Szabo C. 2004.
Immunomodulatory and neuroprotective
effects of inosine. Trends Pharmacol. Sci.
25:15257
50. Ohno M, Frankland PW, Chen AP, Costa
RM, Silva AJ. 2001. Inducible, pharmacogenetic approaches to the study of learning and memory. Nat. Neurosci. 4:1238
43
51. Chapman PF. 2002. Giving drugs to
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
knockout mice: can they do that? Trends

Neurosci. 25:27779
Armstrong JM, Chen JF, Schwarzschild
MA, Apasov S, Smith PT, et al.
2001. Gene dose effect reveals no Gscoupled A2A adenosine receptor reserve
in murine T-lymphocytes: studies of cells
from A2A-receptor-gene-deficient mice.
Biochem. J. 354:12330
Kuhn R, Schwenk F, Aguet M, Rajewsky
K. 1995. Inducible gene targeting in mice.
Science 269:142729
Mayford M, Kandel ER. 1999. Genetic
approaches to memory storage. Trends
Genet. 15:46370
Iwasato T, Datwani A, Wolf AM,
Nishiyama H, Taguchi Y, et al. 2000.
Cortex-restricted disruption of NMDAR1
impairs neuronal patterns in the barrel cortex. Nature 406:72631
Threadgill DW, Dlugosz AA, Hansen LA,
Tennenbaum T, Lichti U, et al. 1995. Targeted disruption of mouse EGF receptor:
effect of genetic background on mutant
phenotype. Science 269:23034
Gerlai R. 1996. Gene-targeting studies of
mammalian behavior: is it the mutation
or the background genotype? Trends Neurosci. 19:17781
Schauwecker PE, Steward O. 1997. Genetic determinants of susceptibility to excitotoxic cell death: implications for gene
targeting approaches. Proc. Natl. Acad.
Sci. USA 94:41038
Schweda F, Wagner C, Kramer BK, Schnermann J, Kurtz A. 2003. Preserved macula densa-dependent renin secretion in A1
adenosine receptor knockout mice. Am. J.
Physiol. Renal Physiol. 284:F77077
Wolfer DP, Crusio WE, Lipp HP. 2002.
Knockout mice: simple solutions to the
problems of genetic background and
flanking genes. Trends Neurosci. 25:336
40
Moore KA, Nicoll RA, Schmitz D. 2003.
Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses. Proc.
4 Dec 2004
13:21
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE


62. Masino SA, Diao L, Illes P, Zahniser
NR, Larson GA, et al. 2002. Modulation
of hippocampal glutamatergic transmission by ATP is dependent on adenosine
A1 receptors. J. Pharmacol. Exp. Ther.
303:35663
63. Lopes LV, Cunha RA, Kull B, Fredholm BB, Ribeiro JA. 2002. Adenosine
A2A receptor facilitation of hippocampal
synaptic transmission is dependent on the
tonic A1 receptor inhibition. Neuroscience
112:31929
64. Dunwiddie TV, Diao LH, Kim HO, Jiang
JL, Jacobson KA. 1997. Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptormediated responses in rat hippocampus.
J. Neurosci. 17:60714
65. Lopes LV, Rebola N, Costenla AR, Halldner L, Jacobson M, et al. 2003. Adenosine A3 receptors in the rat hippocampus:
lack of interaction with A1 receptors. Drug
Dev. Res. 58:42838
66. Rudolphi KA, Schubert P, Parkinson FE,
Fredholm BB. 1992. Neuroprotective role
of adenosine in cerebral ischaemia. Trends
Pharmacol. Sci. 13:43945
67. Diao L, Masino SA, Fredholm BB.
2002. The role of adenosine in hypoxia, hypoglycemia and ischemia in normal and adenosine A1 receptor knockout mice. Presented at Annu. Meet. Soc.
Neurosci., 32nd, pp. 54910, Orlando,
FL
68. Olsson T, Cronberg T, Rytter A, Asztely
F, Fredholm BB, Wieloch T. 2004. Effects
of adenosine A1 receptor gene deletion in
neuronal damage following global cerebral ischemia in mouse in vivo and in vitro.
Eur. J. Neurosci. 20:1197204
69. Turner CP, Seli M, Ment L, Stewart W,
Yan H, et al. 2003. A1 adenosine receptors mediate hypoxia-induced ventriculomegaly. Proc. Natl. Acad. Sci. USA
100:1171822
en U, Fredholm BB, Hag70. Bona E, Ad
berg H. 1995. The effect of long term
caffeine treatment on hypoxic-ischemic
71.
72.
73.
74.
75.
76.
77.
78.
407
brain damage in the neonate. Pediatr. Res.

38:31218
en U, Halldner L, Lagercrantz H, DalAd
mau I, Ledent C, Fredholm BB. 2003.
Aggravated brain damage after hypoxic
ischemia in immature adenosine A2A
knockout mice. Stroke 34:73944
Yu L, Huang Z, Mariani J, Wang Y,
Moskowitz M, Chen J. 2004. Selective inactivation or reconstitution of adenosine
A2A receptors in bone marrow cells reveals
their significant contribution to the development of ischemic brain injury. Nat.
Med. In press
Guo Y, Bolli R, Bao W, Wu WJ, Black
RG, et al. 2001. Targeted deletion of the
A3 adenosine receptor confers resistance
to myocardial ischemic injury and does
not prevent early preconditioning. J. Mol.
Cell. Cardiol. 33:82530
Harrison GJ, Cerniway RJ, Peart J, Berr
SS, Ashton K, et al. 2002. Effects of A(3)
adenosine receptor activation and gene
knock-out in ischemic-reperfused mouse
heart. Cardiovasc. Res. 53:14755
Fedorova IM, Jacobson MA, Basile A, Jacobson KA. 2003. Behavioral characterization of mice lacking the A3 adenosine
receptor: sensitivity to hypoxic neurodegeneration. Cell. Mol. Neurobiol. 23:431
47
El Yacoubi M, Ledent C, Menard JF, Parmentier M, Costentin J, Vaugeois JM.
2000. The stimulant effects of caffeine on
locomotor behaviour in mice are mediated
through its blockade of adenosine A(2A)
receptors. Br. J. Pharmacol. 129:146573
Dassesse D, Ledent C, Parmentier M,
Schiffmann SN. 2001. Acute and chronic
caffeine administration differentially alters striatal gene expression in wild-type
and adenosine A(2A) receptor-deficient
mice. Synapse 42:6376
en U, Dahlberg V, JohansHalldner L, Ad
son B, Ledent C, Fredholm BB. 2004. The
adenosine A1 receptor contributes to the
stimulatory, but not the inhibitory effect
of caffeine on locomotion: a study in mice
4 Dec 2004
13:21
408
79.

80.
81.
82.
83.
84.
85.
86.
87.
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
FREDHOLM ET AL.
lacking adenosine A1 and/or A2A receptors. Neuropharmacology 46:100817
Porkka-Heiskanen T, Alanko L, Kalinchuk A, Stenberg D. 2002. Adenosine and
sleep. Sleep Med. Rev. 6:32132
Porkka-Heiskanen T, Strecker RE, Thakkar M, Bjorkum AA, Greene RW, McCarley RW. 1997. Adenosine: a mediator
of the sleep-inducing effects of prolonged
wakefulness. Science 276:126568
Alanko L, Heiskanen S, Stenberg D,
Porkka-Heiskanen T. 2003. Adenosine kinase and 5 -nucleotidase activity after prolonged wakefulness in the cortex and the
basal forebrain of rat. Neurochem. Int.
42:44954
Mackiewicz M, Nikonova EV, Zimmerman JE, Galante RJ, Zhang L, et al. 2003.
Enzymes of adenosine metabolism in the
brain: diurnal rhythm and the effect of
sleep deprivation. J. Neurochem. 85:348
57
Ticho SR, Radulovacki M. 1991. Role
of adenosine in sleep and temperature regulation in the preoptic area of
rats. Pharmacol. Biochem. Behav. 40:33
40
Benington JH, Kodali SK, Heller HC.
1995. Stimulation of A1 adenosine receptors mimics the electroencephalographic
effects of sleep deprivation. Brain Res.
692:7985
Virus RM, Ticho S, Pilditch M, Radulovacki M. 1990. A comparison of the
effects of caffeine, 8-cyclopentyltheophylline, and alloxazine on sleep in rats.
Possible roles of central nervous system
adenosine receptors. Neuropsychopharmacology 3:24349
Rainnie DG, Grunze HCR, McCarley
RW, Greene RW. 1994. Adenosine inhibition of mesopontine cholinergic neurons: implications for EEG arousal. Science 263:68992
Chamberlin NL, Arrigoni E, Chou TC,
Scammell TE, Greene RW, Saper CB.
2003. Effects of adenosine on gabaergic
synaptic inputs to identified ventrolateral
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
preoptic neurons. Neuroscience 119:913

18
Thakkar MM, Winston S, McCarley RW.
2002. Orexin neurons of the hypothalamus express adenosine A1 receptors.
Brain Res. 944:19094
Thakkar MM, Winston S, McCarley
RW. 2003. A1 receptor and adenosinergic homeostatic regulation of sleepwakefulness: effects of antisense to the A1
receptor in the cholinergic basal forebrain.
Stenberg D, Litonius E, Halldner L,
Johansson B, Fredholm BB, PorkkaHeiskanen T. 2003. Sleep and its homeostatic regulation in mice lacking the
adenosine A1 receptor. J. Sleep Res. 12:
28390
Koos BJ, Maeda T, Jan C. 2001. Adenosine A(1) and A(2A) receptors modulate
sleep state and breathing in fetal sheep. J.
Appl. Physiol. 91:34350
Scammell TE, Gerashchenko DY,
Mochizuki T, McCarthy MT, Estabrooke
IV, et al. 2001. An adenosine A2a agonist
increases sleep and induces Fos in ventrolateral preoptic neurons. Neuroscience
107:65363
Basheer R, Halldner L, Alanko L, McCarley RW, Fredholm BB, Porkka-Heiskanen
T. 2001. Opposite changes in adenosine
A1 and A2A receptor mRNA in the rat
following sleep deprivation. NeuroReport
12:157780
Urade Y, Eguchi N, Qu WM, Sakata M,
Huang ZL, et al. 2003. Sleep regulation in
adenosine A2A receptor-deficient mice.
Neurology 61:S9496
Kull B, Svenningsson P, Fredholm BB.
2000. Adenosine A2A receptors are colocalized with and activate Golf in rat striatum. Mol. Pharmacol. 58:7717
Herve D, Le Moine C, Corvol JC, Belluscio L, Ledent C, et al. 2001. Galpha(olf)
levels are regulated by receptor usage and
control dopamine and adenosine action in
the striatum. J. Neurosci. 21:439099
Ferre S, Fredholm BB, Morelli M, Popoli
4 Dec 2004
13:21
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE

98.
99.
100.
101.
102.
103.
104.
105.
P, Fuxe K. 1997. Adenosine-dopamine

receptor-receptor interactions as an integrative mechanism in the basal ganglia.
Trends Neurosci. 20:48287
Svenningsson P, Fourreau L, Bloch B,
Fredholm BB, Gonon F, Le Moine
C. 1999. Opposite tonic modulation of
dopamine and adenosine on c-fos mRNA
expression in striatopallidal neurons. Neuroscience 89:82737
Zahniser NR, Simosky JK, Mayfield RD,
Negri CA, Hanania T, et al. 2000. Functional uncoupling of adenosine A2A receptors and reduced response to caffeine in
mice lacking dopamine D2 receptors. J.
Neurosci. 20:594957
Dassesse D, Massie A, Ferrari R, Ledent
C, Parmentier M, et al. 2001. Functional
striatal hypodopaminergic activity in mice
lacking adenosine A(2A) receptors. J.
Neurochem. 78:18398
Chen JF, Beilstein M, Xu YH, Turner
TJ, Moratalla R, et al. 2000. Selective
attenuation of psychostimulant-induced
behavioral responses in mice lacking
A(2A) adenosine receptors. Neuroscience
97:195204
Rimondini R, Ferre S, Ogren

SO, Fuxe
K. 1997. Adenosine A2A agonists: a potential new type of atypical antipsychotic.
Neuropsychopharmacology 17:8291
Svenningsson P, Lindskog M, Ledent
C, Parmentier M, Greengard P, et al.
2000. Regulation of the phosphorylation
of the dopamine- and cAMP-regulated
phosphoprotein of 32 kDa in vivo by
dopamine D1, dopamine D2 and adenosine A2A receptors. Proc. Natl. Acad. Sci.
USA 97:185660
Chen JF, Moratalla R, Impagnatiello F,
Grandy DK, Cuellar B, et al. 2001.
The role of the D(2) dopamine receptor (D(2)R) in A(2A) adenosine receptor
(A(2A)R)-mediated behavioral and cellular responses as revealed by A(2A) and
D(2) receptor knockout mice. Proc. Natl.
Aoyama S, Kase H, Borrelli E. 2000. Res-
106.
107.
108.
109.
110.
111.
112.
113.
409
cue of locomotor impairment in dopamine

D2 receptor-deficient mice by an adenosine A2A receptor antagonist. J. Neurosci.
20:584852
Berrendero F, Castane A, Ledent C, Parmentier M, Maldonado R, Valverde O.
2003. Increase of morphine withdrawal
in mice lacking A2a receptors and no
changes in CB1/A2a double knockout
mice. Eur. J. Neurosci. 17:31524
Schwarzschild MA, Xu K, Oztas E, Petzer
JP, Castagnoli K, et al. 2003. Neuroprotection by caffeine and more specific A2A
receptor antagonists in animal models of
Parkinsons disease. Neurology 61:S55
61
Ross GW, Abbott RD, Petrovitch H,
Morens DM, Grandinetti A, et al. 2000.
Association of coffee and caffeine intake
with the risk of Parkinson disease. JAMA
283:267479
Ascherio A, Zhang SM, Hernan MA,
Kawachi I, Colditz GA, et al. 2001.
Prospective study of caffeine consumption and risk of Parkinsons disease in
men and women. Ann. Neurol. 50:56
63
Ascherio A, Chen H, Schwarzschild MA,
Zhang SM, Colditz GA, Speizer FE. 2003.
Caffeine, postmenopausal estrogen, and
risk of Parkinsons disease. Neurology
60:79095
Chen JF, Xu K, Petzer JP, Staal R, Xu
YH, et al. 2001. Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinsons disease.
J. Neurosci. 21:RC143
Ikeda K, Kurokawa M, Aoyama S,
Kuwana Y. 2002. Neuroprotection by
adenosine A2A receptor blockade in experimental models of Parkinsons disease.
Alfinito PD, Wang SP, Manzino L,
Rijhsinghani S, Zeevalk GD, Sonsalla
PK. 2003. Adenosinergic protection of
dopaminergic and GABAergic neurons
against mitochondrial inhibition through
receptors located in the substantia nigra
4 Dec 2004
13:21
410
114.

115.
116.
117.
118.
119.
120.
121.
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
FREDHOLM ET AL.
and striatum, respectively. J. Neurosci.
23:1098287
El Yacoubi M, Ledent C, Parmentier
M, Costentin J, Vaugeois JM. 2001.
Adenosine A2A receptor knockout mice
are partially protected against druginduced catalepsy. NeuroReport 12:983
86
Moo-Puc RE, Gongora-Alfaro JL, Alvarez-Cervera FJ, Pineda JC, ArankowskySandoval G, Heredia-Lopez F. 2003. Caffeine and muscarinic antagonists act in
synergy to inhibit haloperidol-induced
catalepsy. Neuropharmacology 45:493
503
Hauser RA, Hubble JP, Truong DD. 2003.
Randomized trial of the adenosine A(2A)
receptor antagonist istradefylline in advanced PD. Neurology 61:297303
Bara-Jimenez W, Sherzai A, Dimitrova T,
Favit A, Bibbiani F, et al. 2003. Adenosine
A(2A) receptor antagonist treatment of
Parkinsons disease. Neurology 61:293
96
Fredduzzi S, Moratalla R, Monopoli A,
Cuellar B, Xu K, et al. 2002. Persistent behavioral sensitization to chronic L-DOPA
requires A2A adenosine receptors. J. Neurosci. 22:105462
Bibbiani F, Oh JD, Petzer JP, Castagnoli N, Chen JF, et al. 2003. A2A
antagonist prevents dopamine agonistinduced motor complications in animal
models of Parkinsons disease. Exp. Neurol. 184:28594
Blum D, Galas MC, Pintor A, Brouillet E,
Ledent C, et al. 2003. A dual role of adenosine A2A receptors in 3-nitropropionic
acid-induced striatal lesions: implications for the neuroprotective potential of
A2A antagonists. J. Neurosci. 23:5361
69
Fink JS, Kalda A, Ryu H, Stack EC,
Schwarzschild MA, et al. 2004. Genetic
and pharmacological inactivation of the
adenosine A2A receptor attenuates 3nitropropionic acid-induced striatal damage. J. Neurochem. 88:53844
122. Popoli P, Frank C, Tebano MT, Potenza

RL, Pintor A, et al. 2003. Modulation
of glutamate release and excitotoxicity
by adenosine A2A receptors. Neurology
61:S6971
123. Wang JH, Short J, Ledent C, Lawrence
AJ, Buuse M. 2003. Reduced startle habituation and prepulse inhibition in mice
lacking the adenosine A2A receptor. Behav. Brain Res. 143:2017
124. Ferre S. 1997. Adenosine-dopamine
interactions in the ventral striatum. Implications for the treatment of schizophrenia.
Psychopharmacology 133:10720
125. El Yacoubi M, Ledent C, Parmentier M,
Costentin J, Vaugeois JM. 2003. Caffeine reduces hypnotic effects of alcohol
through adenosine A2A receptor blockade. Neuropharmacology 45:97785
126. El Yacoubi M, Ledent C, Parmentier M,
Daoust M, Costentin J, Vaugeois J-M.
2001. Absence of the adenosine A(2A)
receptor or its chronic blockade decrease
ethanol withdrawal-induced seizures
in mice. Neuropharmacology 40:424
32
127. Naassila M, Ledent C, Daoust M. 2002.
Low ethanol sensitivity and increased
ethanol consumption in mice lacking
adenosine A2A receptors. J. Neurosci. 22:
1048793
128. Diamond I, Gordon AS. 1994. The role
of adenosine in mediating cellular and
molecular responses to ethanol. Suppl.
Exp. 71:17583
129. Gouder N, Scheurer L, Fritschy JM, Boison D. 2004. Overexpression of adenosine kinase in epileptic hippocampus contributes to epileptogenesis. J. Neurosci.
24:692701
130. Huber A, Padrun V, Deglon N, Aebischer P, Mohler H, Boison D. 2001.
Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy. Proc.
131. Boison D, Huber A, Padrun V, Deglon N,
Aebischer P, Mohler H. 2002. Seizure suppression by adenosine-releasing cells is
4 Dec 2004
13:21
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
132.

133.
134.
135.
136.
137.
138.
139.
140.
141.
independent of seizure frequency. Epilepsia 43:78896

Tsutsui S, Schnermann J, Noorbakhsh
F, Henry S, Yong VW, et al. 2004. A1
adenosine receptor upregulation and activation attenuates neuroinflammation and
demyelination in a model of multiple sclerosis. J. Neurosci. 24:152129
Hammarberg C, Schulte G, Fredholm BB.
2003. Evidence for functional adenosine
A3 receptors in microglia cells. J. Neurochem. 86:105154
Lang UE, Lang F, Richter K, Vallon V,
Lipp HP, et al. 2003. Emotional instability
but intact spatial cognition in adenosine
receptor 1 knock out mice. Behav. Brain
Res. 145:17988
de Mendonca A, Ribeiro JA. 1997.
Adenosine and neuronal plasticity. Life
Sci. 60:24551
Almeida T, Rodrigues RJ, de Mendonca
A, Ribeiro JA, Cunha RA. 2003. Purinergic P2 receptors trigger adenosine release
leading to adenosine A2A receptor activation and facilitation of long-term potentiation in rat hippocampal slices. Neuroscience 122:11121
dAlcantara P, Ledent C, Swillens S,
Schiffmann SN. 2001. Inactivation of
adenosine A2A receptor impairs long
term potentiation in the accumbens nucleus without altering basal synaptic
transmission. Neuroscience 107:45564
Schiffmann SN, Dassesse D, dAlcantara
P, Ledent C, Swillens S, Zoli M. 2003.
A2A receptor and striatal cellular functions: regulation of gene expression, currents, and synaptic transmission. Neurology 61:S2429
Millan MJ. 2003. The neurobiology and
control of anxious states. Prog. Neurobiol.
70:83244
Boulenger JP, Uhde TW, Wolff EA, Post
RM. 1984. Increased sensitivity to caffeine in patients with panic disorders.
Arch. Gen. Psychiatry 41:106771
El Yacoubi M, Ledent C, Parmentier M,
Costentin J, Vaugeois JM. 2000. The
142.
143.
144.
145.
146.
147.
148.
149.
411
anxiogenic-like effect of caffeine in two

experimental procedures measuring anxiety in the mouse is not shared by selective A(2A) adenosine receptor antagonists. Psychopharmacology 148:15363
Imaizumi M, Miyazaki S, Onodera K.
1994. Effects of xanthine derivatives in
a light/dark test in mice and the contribution of adenosine receptors. Methods
Find. Exp. Clin. Pharmacol. 16:63944
Jain N, Kemp N, Adeyemo O, Buchanan
P, Stone TW. 1995. Anxiolytic activity of
adenosine receptor activation in mice. Br.
J. Pharmacol. 116:212733
Florio C, Prezioso A, Papaioannou A,
Vertua R. 1998. Adenosine A1 receptors modulate anxiety in CD1 mice. Psychopharmacology 136:31119
Alsene K, Deckert J, Sand P, de Wit
H. 2003. Association between A2a receptor gene polymorphisms and caffeineinduced anxiety. Neuropsychopharmacology 28:1694702
Hamilton SP, Slager SL, De Leon AB,
Heiman GA, Klein DF, et al. 2004. Evidence for genetic linkage between a polymorphism in the adenosine 2A receptor
and panic disorder. Neuropsychopharmacology 29:55865
Jegou S, El Yacoubi M, Mounien L,
Ledent C, Parmentier M, et al. 2003.
Adenosine A2A receptor gene disruption
provokes marked changes in melanocortin
content and pro-opiomelanocortin gene
expression. J. Neuroendocrinol. 15:1171
77
Navarro JF, Romero C, Maldonado E.
2000. Effects of N6-cyclohexyl adenosine
(CHA) on isolation-induced aggression
in male mice. Methods Find. Exp. Clin.
Pharmacol. 22:4346
El Yacoubi M, Ledent C, Parmentier M,
Bertorelli R, Ongini E, et al. 2001. Adenosine A2A receptor antagonists are potential antidepressants: evidence based on
pharmacology and A2A receptor knockout mice. Br. J. Pharmacol. 134:68
77
4 Dec 2004
13:21

412
AR
AR232-PA45-16.tex
AR232-PA45-16.sgm
LaTeX2e(2002/01/18)
P1: GCE
FREDHOLM ET AL.
150. Wu WP, Hao JX, Halldner-Henriksson

L, Xu XJ, Jacobson MA, et al. 2002.
Decreased inflammatory pain due to reduced carrageenan-induced inflammation
in mice lacking adenosine A3 receptors.
Neuroscience 114:52327
151. Lopes LV, Halldner L, Rebola N, Johansson B, Ledent C, et al. 2004. Binding of the
prototypical adenosine A2A receptor agonist, CGS 21680, to the cerebral cortex of
adenosine A1 and A2A receptor knockout
mice. Br. J. Pharmacol. 141:100614
152. Halldner L, Lopes LV, Dare E, Lindstrom
K, Johansson B, et al. 2004. Binding of
adenosine receptor ligands to brain of
adenosine receptor knock-out mice. Evidence that CGS 21680 binds to A1 receptors in hippocampus. Naunyn. Schmiedebergs Arch. Pharmacol. In press
153. Chen JF, Steyn S, Staal R, Petzer JP, Xu
K, et al. 2002. 8-(3-Chlorostyryl)caffeine
may attenuate MPTP neurotoxicity
through dual actions of monoamine
oxidase inhibition and A2A receptor antagonism. J. Biol. Chem. 277:3604044
154. Kanda T, Jackson MJ, Smith LA, Pearce

RK, Nakamura J, et al. 1998. Adenosine
A2A antagonist: a novel antiparkinsonian
agent that does not provoke dyskinesia
in parkinsonian monkeys. Ann. Neurol.
43:50713
155. Grondin R, Bedard PJ, Hadj Tahar A,
Gregoire L, Mori A, Kase H. 1999.
Antiparkinsonian effect of a new selective adenosine A2A receptor antagonist in MPTP-treated monkeys. Neurology
52:167377
156. Blum D, Gall D, Galas MC, dAlcantara P,
Bantubungi K, Schiffmann SN. 2002. The
adenosine A1 receptor agonist adenosine
amine congener exerts a neuroprotective
effect against the development of striatal
lesions and motor impairments in the 3nitropropionic acid model of neurotoxicity. J. Neurosci. 22:912233
157. Dalllgna OP, Porciuncula LO, Souza DO,
Cunha RA, Lara DR. 2003. Neuroprotection by caffeine and adenosine A2A
receptor blockade of beta-amyloid neurotoxicity. Br. J. Pharmacol. 138:12079
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Copyright

REGULATION AND INHIBITION OF ARACHIDONIC

ACID -HYDROXYLASES AND 20-HETE
FORMATION
Deanna L. Kroetz1,2 and Fengyun Xu1
1
Department of Biopharmaceutical Sciences and the 2Liver Center,
University of California, San Francisco, California 94143-2911;
email: deanna@itsa.ucsf.edu, fengyun@itsa.ucsf.edu
Key Words 20-HETE, CYP4A, CYP4F, vascular reactivity, renal function

Abstract Cytochrome P450catalyzed metabolism of arachidonic acid is an important pathway for the formation of paracrine and autocrine mediators of numerous
biological effects. The -hydroxylation of arachidonic acid generates significant levels of 20-hydroxyeicosatetraenoic acid (20-HETE) in numerous tissues, particularly
the vasculature and kidney tubules. Members of the cytochrome P450 4A and 4F
families are the major -hydroxylases, and the substrate selectivity and regulation
of these enzymes has been the subject of numerous studies. Altered expression and
function of arachidonic acid -hydroxylases in models of hypertension, diabetes, inflammation, and pregnancy suggest that 20-HETE may be involved in the pathogenesis
of these diseases. Our understanding of the biological significance of 20-HETE has
been greatly aided by the development and characterization of selective and potent
inhibitors of the arachidonic acid -hydroxylases. This review discusses the substrate
selectivity and expression of arachidonic acid -hydroxylases, regulation of these enzymes during disease, and the application of enzyme inhibitors to study 20-HETE
function.
OVERVIEW OF ARACHIDONIC ACID METABOLISM

Arachidonic acid comprises part of the membrane phospholipid pool and is released following activation of phospholipase A2 by various agonists, such as norepinephrine, angiotensin II, and bradykinin (1). Metabolism of free arachidonic
acid by cyclooxygenases and lipoxygenases leads to the formation of prostaglandins,
thromboxanes, and leukotrienes with important roles in the regulation of vascular
tone, inflammation, and renal and pulmonary function (2). Cyclooxygenase- and
lipoxygenase-catalyzed arachidonic acid metabolism is well characterized, and
both of these pathways are targets of approved drugs. In contrast, our knowledge
of metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes is more
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Figure 1 Major pathways of arachidonic acid metabolism catalyzed by cytochrome P450. Arachidonic acid is metabolized into epoxyeicosatrienoic acids
(EETs) by CYP2C and CYP2J epoxygenases and into 20-hydroxyeicosatetraenoic acid
(20-HETE) by CYP4A and CYP4F -hydroxylases. EETs can also be further metabolized by soluble epoxide hydrolase (sEH) into their corresponding dihydroxyeicosatrienoic acids (DHETs).
limited, although recent efforts in this area hold the promise that new drug targets
will also emerge from this pathway.
CYP enzymes can metabolize arachidonic acid into numerous eicosanoids with
the relative abundance dependent on the tissue and species (Figure 1). The major
products in most tissues are the -hydroxylated metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) and regio- and stereospecific epoxyeicosatrienoic acids
(EETs). Hydroxylation of arachidonic acid at the -1 and midchain (carbons 16
18) positions is less common. CYP4A and CYP4F enzymes catalyze the - and
-1 hydroxylation reactions, whereas members of the CYP2C and CYP2J families are responsible for epoxidation (35). A novel CYP isoform, CYP2U1, has
recently been identified as a human arachidonic acid -hydroxylase (6). EETs are
efficiently hydrated by soluble epoxide hydrolase (sEH) into the corresponding dihydroxyeicosatrienoic acids (DHETs) (7, 8). CYP eicosanoids can also be further
metabolized by CYPs, dehydrogenases, or cyclooxygenases (4, 911); -oxidized
(4, 10); or incorporated into membrane phospholipid pools (10, 11). The focus of
this review is on pathologic regulation and inhibition of the CYP -hydroxylase
pathway.
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ARACHIDONIC ACID -HYDROXYLASES
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BIOLOGICAL SIGNIFICANCE OF 20-HETE

The importance of understanding the molecular mechanisms regulating 20-HETE
synthesis is evident from the numerous biological effects attributed to this eicosanoid. Although not the focus of this review, the major biological properties of
20-HETE are summarized below. The reader is directed to several recent reviews
that have discussed these properties in detail (4, 5). The main sites of synthesis
and action of 20-HETE are the vasculature, kidney, and lung. The formation of
20-HETE has been documented in rat renal microvessels, and expression of CYP
-hydroxylases in renal, cerebral, pulmonary, mesenteric, and skeletal muscle microvascular beds is consistent with 20-HETE formation throughout the vasculature
(1216). 20-HETE inhibits a large conductance Ca2+-activated K+ channel, resulting in depolarization of the vascular smooth muscle cell, Ca2+ entry, and potent
vasoconstriction (17). An ongoing area of investigation focuses on the possibility
that the vasoconstrictive effect of 20-HETE is receptor-mediated. The myogenic
response to elevations in transmural pressure is also mediated by 20-HETE in
cerebral, renal, skeletal muscle, and mesenteric arterioles (13, 15, 18, 19), and 20HETE plays a role in the autoregulation of renal blood flow and tubuloglomerular
feedback in rats (20, 21). 20-HETE can also act as an oxygen sensor in skeletal muscle microcirculation (22). A role for 20-HETE in signaling the mitogenic
actions of growth factors and vasoactive agents (23) and in promoting angiogenesis (24, 25) suggests that this eicosanoid is important in regulating vascular cell
growth. Arachidonic acid -hydroxylation also occurs in the renal proximal tubule
and the thick ascending limb of Henle (2628). In the proximal tubule, 20-HETE
inhibits Na+-K+-ATPase, whereas in the thick ascending limb, 20-HETE blocks
a 70-pS K+ channel, which limits K+ availability for transport by a Na+-K+-2Cl
cotransporter (2931). In the lung microvasculature, 20-HETE has an opposite
effect as that seen in other vascular beds, vasodilating pulmonary vessels in an
endothelium- and cyclooxygenase-dependent manner (32, 33). In most species,
20-HETE also has bronchodilatory effects (34). The increasing biological properties associated with 20-HETE and the relative abundance of this eicosanoid in
the vasculature and renal tubules make it of great importance to understand the
molecular mechanisms that regulate 20-HETE synthesis, degradation, and action.
CYP ARACHIDONIC ACID -HYDROXYLASES

CYP -hydroxylases belong to the CYP4 family, which is evolutionarily one of
the oldest members of the CYP superfamily. Isoforms of the CYP4A and CYP4F
subfamilies catalyze the -, and to a lesser extent the -1, hydroxylation of arachidonic acid and other medium- to long-chain fatty acids. In the sections below, the
expression and catalytic function of the human, mouse, rat, and rabbit CYP4A and
CYP4F enzymes are discussed in more detail.
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Catalytic Function of CYP4A and CYP4F

Arachidonic Acid -Hydroxylases
Members of the CYP4A subfamily were the first characterized arachidonic acid
-hydroxylases and include four isoforms in rats (3538), two in humans (39
41), four in rabbits (4245), and three in mice (4648). Heterologous expression
of the rat CYP4A isoforms in Escherichia coli (49) and baculovirus (5052) revealed that CYP4A1 has the highest catalytic activity toward arachidonic acid
-hydroxylation, followed by CYP4A2 and CYP4A3 with similar activity. Estimates of kcat for 20-HETE formation were 6 min1 for CYP4A1, 2 min1 for
CYP4A2 and CYP4A3, and 1 min1 for CYP4A8 (49). The rat CYP4A enzymes
also catalyze the -1 hydroxylation of arachidonic acid, with :-1 ratios ranging
from 612:1 for CYP4A1 to 24:1 for CYP4A2 and CYP4A3 (49, 51). Interestingly, both CYP4A2 and CYP4A3 can also epoxidate arachidonic acid at the 11,12
position (50, 51); however, this effect is likely dependent on the experimental conditions of expression and functional reconstitution of the enzymes, as others have
failed to confirm this reaction.
Metabolism of arachidonic acid by recombinant rabbit CYP4A enzymes is
highly dependent on the presence of cytochrome b5. A 2:1 ratio of cytochrome b5 to
CYP catalyzes arachidonic acid -hydroxylation with kcat values of 155 min1 and
152 min1 for CYP4A4 and CYP4A7, respectively (53, 54). In contrast, CYP4A5
can efficiently catalyze the -hydroxylation of lauric acid but not arachidonic acid
(55). The kinetics of arachidonic acid -hydroxylation by CYP4A6 have not been
described in detail, although this isoform can catalyze 20-HETE formation (56).
The substrate selectivity of the mouse Cyp4a enzymes has not been characterized,
although high sequence similarity to other members of the CYP4A gene family
implicate them in the -hydroxylation of arachidonic acid (4648).
CYP4A11 is the major human CYP4A isoform and purified CYP4A11 from
liver and kidney can catalyze the - and -1 hydroxylation of arachidonic acid (57,
58). In both tissues, CYP4A11-catalzyed 20-HETE formation was quantitatively
less important than the corresponding CYP4F2 component, consistent with the
low kcat values of 0.40.55 min1 for heterologously expressed CYP4A11 (40,
49). In a direct comparison of the four rat CYP4A isoforms and human CYP4A11,
the latter had the lowest reported kcat for arachidonic acid -hydroxylation (49).
Recently, CYP4A22 has been identified as a second human CYP4A isoform (39,
59). Considering the 96% sequence identity with CYP4A11, it is highly likely that
CYP4A22 will also catalyze 20-HETE formation, although low expression of this
gene may limit its functional impact.
Rat CYP4F1 (60) and human CYP4F3 (61) were the first cloned members of
the CYP4F family. To date, there have been four CYP4F members identified in the
rat (60, 62), five in the human (61, 6366), and five in the mouse (67). Initially,
the focus of investigations on CYP4F catalytic activity was on -hydroxylation
of leukotriene B4 and its importance in controlling inflammation (61, 63, 66, 68
73). More recently, a comparison of catalytic function of the rat CYP4F isoforms
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expressed in E. coli revealed that CYP4F1 and CYP4F4 could -hydroxylate

arachidonic acid with kcat values similar to those of CYP4A1 (9 and 11 min1 for
CYP4F1 and CYP4F4, respectively; 74). Human CYP4F3B can also metabolize
arachidonic acid with a Km value similar to that for leukotriene B4 (73). CYP4F2
has been purified from both human liver and kidney and shown to be the major
source of 20-HETE in these organs (57, 58). CYP4F12 can also -hydroxylate
arachidonic acid but with a lower activity than that of CYP4F2 (66). The functional
activity of the mouse CYP4F isoforms remains to be studied.
Expression of CYP4A and CYP4F Arachidonic

Acid -Hydroxylases
Unlike most CYPs, many CYP4A isoforms have their highest level of expression
in the kidney. CYP4A mRNA has been localized along the rat nephron and renal
vasculature. CYP4A2, CYP4A3, and CYP4A8 can be detected in the glomerulus, proximal tubules, cortical collecting duct, and cortical thick ascending limb of
Henle (75). Expression of CYP4A2 and CYP4A3 is also apparent in the medullary
regions of the collecting duct and thick ascending limb (75). Consistent with low
levels of CYP4A expression by RNase protection assay (76), CYP4A1 mRNA
levels were too low to be detected in microdissected tubules (75). Expression of
CYP4A1 is more easily detected in the rat renal vasculature, where it is the only
CYP4A gene expressed in the aorta and renal artery (77). In contrast, CYP4A1,
CYP4A2, CYP4A3, and CYP4A8 were all expressed in interlobar, arcuate, and
interlobular arteries (77). In the mouse kidney, Cyp4a10 is ubiquitously expressed
throughout the nephron and vasculature, whereas Cyp4A12 and Cyp4A14 expression is limited to the proximal tubule (78).
The rat and human CYP4F genes implicated in arachidonic acid -hydroxylation
are also highly expressed in the kidney, similar to the corresponding CYP4A genes.
CYP4F1 was originally cloned from rat hepatic tumors (60) and was subsequently
shown to be expressed in liver, kidney, and brain (79). CYP4F4 is expressed at
similar levels in liver and kidney (79). CYP4F2, CYP4F3B, and CYP4F12 are
expressed at significant levels in the human kidney (57, 58, 65, 73, 80). In the
mouse, Cyp4f mRNA is detected throughout the renal tubule and vasculature (78),
making it of interest to characterize its role in 20-HETE formation.
The high degree of amino acid similarity between the rat CYP4A and CYP4F
proteins makes it difficult to detect their expression in an isoform-specific manner. Despite these limitations, the expression of CYP4A immunoreactive protein
in the proximal tubules, glomerulus, medullary thick ascending limb of Henle,
and renal microvessels shows a similar pattern as that of CYP4A mRNA (75,
77, 8183). In the vasculature, the expression of CYP4A protein is highest in the
smaller-diameter vessels, consistent with an important role for 20-HETE in maintaining renal vascular tone. CYP4F expression has been detected at the protein
level in the liver, lung, kidney, and brain, but the isoform distribution is unknown
(79).
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Functional CYP -hydroxylase activity has been documented in the nephron

and vasculature of the rat kidney, although it is not clear what percentage of this
activity is due to CYP4A versus CYP4F isoforms. Detection of 20-HETE following addition of arachidonic acid to homogenates from microdissected nephrons
indicates that functional arachidonic acid -hydroxylase activity is highest in the
proximal tubules, but also detected in the glomerulus (28, 84, 85). In the renal
vasculature, arachidonic acid -hydroxylase activity is highest in the interlobular artery, with much lower levels detected in the interlobar and arcuate arteries
(77). The correlation of arachidonic acid -hydroxylase activity with the pattern
of CYP4A expression in the vasculature supports a role for the CYP4A enzymes
in this catalytic function. Importantly, endogenous 20-HETE levels are detectable
in the proximal tubules of Sprague-Dawley rats (28). The detection of endogenous 20-HETE levels in these tissues supports an important biological role for this
eicosanoid in controlling renal vascular tone and ion transport.
In the human kidney, both CYP4A11 and CYP4F2 are highly expressed in
the proximal tubules, and both enzymes contribute to renal arachidonic acid
-hydroxylation (58). Immuno- and chemical inhibition studies are consistent
with CYP4F2 being the major human kidney microsomal arachidonic acid hydroxylase. A less significant role for CYP4A11 in hepatic 20-HETE formation
is suggested from similar inhibition studies in human liver microsomes (57).
Most of the interest in 20-HETE formation focuses on its role in regulating renal
vascular tone and ion transport. However, CYP4A and CYP4F isoforms are also
expressed outside the kidney and likely have pharmacological significance in these
tissues as well. CYP4A protein is found in the brain, prostate, intestine, and lungs
(14, 8688), and CYP4F1 and CYP4F4 are expressed in the brain (79). Alternative
splicing of the CYP4F3 gene determines its expression pattern, with CYP4F3B
expressed in the liver, kidney, trachea, and ileum (73). In contrast, the leukotriene
B4 -hydroxylase CYP4F3A is expressed in myeloid cells in peripheral blood and
bone marrow (89). In the rabbit lung, expression of CYP4A protein decreased
with increasing arterial size (88). CYP4F12 expression is detected in the small
intestine, colon, and urogenital epithelia (66, 80), although a role for 20-HETE
in gastrointestinal physiology is not clear. The novel fatty acid -hydroxylase
CYP2U1 has limited expression in the thymus and cerebellum where its role in
mediating 20-HETE synthesis is not yet characterized (6).
REGULATION OF CYP ARACHIDONIC ACID

-HYDROXYLASES AND 20-HETE SYNTHESIS
Much of what we know about the biological roles of 20-HETE stem from studies of
the regulation of the CYP arachidonic acid -hydroxylases and the use of disease
state and xenobiotic treatment models in which 20-HETE synthesis is altered. The
induction of the CYP4A genes by peroxisome proliferators, such as fibric acid hypolipidemic drugs, has been well characterized and is the topic of several reviews
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(90, 91). Peroxisome proliferators typically exert their effects through activation of
the isoform of the peroxisome proliferator-activated receptor (PPAR). Unfortunately, the use of these inducers to characterize 20-HETE function is complicated
by the fact that the CYP4A isoforms, CYP2C23, and soluble epoxide hydrolase
are induced (35, 37, 92), whereas CYP4F and CYP2C11 are downregulated (67,
93) following exposure to peroxisome proliferators. With effects on both the CYP
-hydroxylase and epoxygenase pathways, often in opposite directions, the interpretation of results using these chemicals should be made with caution. The
abundant cellular signaling molecule nitric oxide inhibits CYP4A -hydroxylase
expression and function (94) and a novel mechanism of regulation involving covalent attachment of the prosthetic heme group through an ester link at a glutamic
acid residue conserved in the I-helix of the active site of most CYP4 members
has recently been described (9598). Interestingly, in some cases covalent modification of the enzymes results in increased activity. Heme levels are highest in
the liver and vary throughout the body, suggesting that covalent heme binding
may influence tissue-specific regulation of CYP4 arachidonic acid -hydroxylase
activity, a novel mechanism of CYP regulation. Although each of these mechanisms is interesting and of value in the field of eicosanoid biology, the focus of the
sections below is on the regulation of arachidonic acid -hydroxylation in various
disease states and the use of chemical inhibitors to study 20-HETE biology.
Pathophysiological Regulation of Arachidonic

Acid -Hydroxylation
The modulation of CYP arachidonic acid -hydroxlase expression and function has
been described in numerous animal models of disease. In some cases, alterations
in 20-HETE formation are hypothesized to play important roles in the pathophysiology of the disease, whereas in other cases, changes in 20-HETE formation are
considered an adaptive response to pathologic stimuli. Changes in arachidonic
acid -hydroxylation in hypertension, pregnancy, inflammation, and diabetes are
described below.
The ability of 20-HETE to influence vascular reactivity and renal
tubular sodium and water transport led to interest in understanding the significance
of this eicosanoid in regulating blood pressure. Many studies in this area have
used the spontaneously hypertensive rat (SHR) model of essential hypertension.
Increased cortical arachidonic acid -hydroxylase activity has been documented in
the SHR kidney relative to the normotensive Wistar-Kyoto (WKY) rat, suggesting
that increased renal 20-HETE levels contribute to the hypertensive phenotype
in these rats (76, 99101). Similarly, endogenous 20-HETE levels are elevated in
the SHR mesenteric artery relative to the WKY rat (1.34 0.16 versus 0.27
0.09 pmol/mg; 102). Increased expression of CYP4A mRNA and immunoreactive
protein has also been documented in the SHR kidney and this is consistent with the
increased arachidonic acid -hydroxylase activity. Using differential hybridization
HYPERTENSION
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techniques, CYP4A2 was identified as one of three mRNAs differentially expressed

in the kidneys of four-week-old SHRs and WKY rats (103). Gene-specific RNA
probes were later used to demonstrate differential expression of both CYP4A3 and
CYP4A8 in the young SHR kidney (76). In both cases, the increases in CYP4A
mRNA were modest (1.4- to 2.0-fold) and only significant between one and four
weeks of age. A similar pattern of increased CYP4A immunoreactive protein and
arachidonic acid -hydroxylase has been found (76). The mechanism by which
CYP4A expression and function is altered in the SHR kidney is still not understood.
The function and expression of CYP4A is also altered in other models of hypertension. In contrast to the SHR, in the Dahl hypertension model CYP4A levels and
arachidonic acid -hydroxylase activity are decreased in the salt-sensitive strain
relative to the normotensive salt resistant or Lewis strains, and these differences
are restricted to the outer medulla (104106). Both high-salt and high-fat diets
decrease arachidonic acid -hydroxylase activity and CYP4A protein levels in the
renal tubules of Sprague-Dawley rats (107, 108), whereas induction of hypertension by Angiotensin II treatment decreases CYP4A expression exclusively in the
renal microvessels (107).
Genetic deletion of Cyp4a14 revealed a complex phenotype and evidence that
20-HETE does indeed play a role in the regulation of blood pressure (109). Mice deficient in Cyp4a14 exhibited an androgen-sensitive increase in blood pressure that
is normalized by castration. Interestingly, Cyp4a14/ mice have increased renal
arachidonic acid -hydroxylase activity that corresponds to androgen-mediated
induction of the Cyp4a12 isoform. The increased functional Cyp4a activity is consistent with higher renal levels of 20-HETE and the observed increases in blood
pressure. Unfortunately, the multiplicity of the Cyp4a and Cyp4f isoforms in the
mouse kidney will limit the usefulness of genetic deletion in understanding the
physiological and pathophysiological role of 20-HETE.
Although numerous studies in animal models of hypertension have held promise
that 20-HETE is important in the regulation of blood pressure in humans, evidence for such an effect has only recently been described. In human essential
hypertension, urinary 20-HETE excretion is regulated by salt intake, with distinct relationships between natriuresis and 20-HETE excretion in salt-sensitive
and salt-resistant patients (110). The importance of 20-HETE in regulating natriuresis in humans is also supported by studies showing a role for this eicosanoid
in mediating the natriuretic properties of furosemide (111). In a population of
obese patients with essential hypertension, the urinary excretion of 20-HETE was
negatively correlated with insulin levels (112). The negative correlation between
urinary 20-HETE levels and insulin suggests that insulin may decrease the expression and function of the CYP -hydroxylases, consistent with inhibitory effects
of insulin on the rat CYP4A isoforms (113). Although limited in number, these
recent studies suggest that regulation of CYP -hydroxylase activity in the human
kidney is an important determinant of natriuresis and that pharmacological manipulation of this activity may have therapeutic potential in the regulation of blood
pressure.
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The first reports of altered CYP4A expression during pregnancy

were reported prior to significant interest in the role of these enzymes in arachidonic acid -hydroxylation. CYP4A protein levels and increased PGE1, PGA1,
and PGF2 -hydroxylation were found in pregnant rabbit lungs, and this effect
was attributed to changes in hormonal levels during pregnancy (114). Despite extensive biochemical and cellular characterization of these functional changes, the
physiological significance of altered CYP4A protein and activity in the rabbit lung
is still not understood. In the rat, pregnancy-induced changes in CYP4A expression and arachidonic acid -hydroxylation show distinct patterns in the tubule and
microvessels (115). During early gestation, CYP4A immunoreactive protein levels
and arachidonic acid -hydroxylase activity in the medullary thick ascending limb
are similar to baseline and these values increase at 19 days of gestation. In contrast,
both the expression of CYP4A immunoreactive proteins and arachidonic acid hydroxylase activity in renal microvessels are elevated at 6 and 12 days of gestation
compared to control, but return to nonpregnant levels at 19 days of gestation. The
decrease in microvascular and increase in tubular arachidonic acid -hydroxylase
activity at 19 days gestation is accompanied by a decrease in blood pressure and
elevated urinary excretion of 20-HETE. The vasoconstrictive effects predicted
from the increased renal microvascular 20-HETE synthesis during early gestation
might act to buffer the increased synthesis of nitric oxide, a potent vasodilator,
during this period (116). In later gestation, the increased 20-HETE synthesis in
the medullary thick ascending limb may modulate natriuresis and contribute to the
decreased blood pressure observed at this time. It will be of interest to define the
cellular signals that mediate this unique site- and time-dependent expression of
CYP4 arachidonic acid -hydroxylases during pregnancy.

PREGNANCY
Inflammation and infection are often associated with decreased

CYP content and drug clearance (117). However, the CYP4A enzymes are induced
in response to inflammation in the rat. Both renal and hepatic levels of CYP4A
mRNA, CYP4A immunoreactive protein, and lauric acid -hydroxylase activity
are elevated following treatment of Fisher rats with lipopolysaccharide (LPS) (118,
119). Studies in PPAR/ mice indicate that the dependency of these changes
on PPAR are gene- and tissue-specific. Both the induction of Cyp4a10 in the
kidney and its downregulation in the liver following LPS treatment are PPARdependent (120). However, for Cyp4a14, LPS induction is only found in the kidney,
and this effect is also PPAR-dependent. An interesting hypothesis is that LPS
treatment increases the level of an endogenous eicosanoid or other compound that
can activate PPAR. The effects of these inflammatory stimuli on renal 20-HETE
synthesis are important to study.
The effects of inflammation on CYP4F expression and activity are also tissueand gene-specific. Treatment of Fisher rats with LPS decreased hepatic CYP4F4
mRNA levels by 50% and increased CYP4F5 mRNA levels to a similar degree, resulting in no net change in leukotriene B4 -hydroxylation or CYP4F immunoreactive protein levels (121). Differences are also noted between various inflammatory
INFLAMMATION
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stimuli, as barium sulfate increased hepatic CYP4F4 mRNA and protein levels as
well as corresponding enzyme activity. By contrast, hepatic CYP4F1 and CYP4F6
were unaffected by these inflammatory stimuli. In the kidney, LPS had no effect
on CYP4F mRNA levels, but barium sulfate induced CYP4F1 and CYP4F6 up to
threefold (121). PPAR plays some role in mediating the inductive effects of LPS
on Cyp4f15 in the kidney and the downregulation of Cyp4f15 and Cyp4f16 in the
liver (67). Interestingly, a traumatic brain injury model is associated with isoformdependent changes in both hepatic and renal CYP activity, including a twofold
increase in renal CYP4F expression and activity that is sustained for at least two
weeks (122). It is tempting to speculate that changes in renal 20-HETE levels resulting from CYP4F activity might contribute to the renal effects associated with
head trauma.
Alterations in CYP4A expression and function have been noted in
animal models of diabetes. Following the induction of diabetes with streptozotocin,
CYP4A expression and function are increased in liver and kidney microsomes
(113, 123126). The effect of diabetes can be reversed with insulin treatment
(113, 123, 125, 126) or correction of the hyperketonic state (127). An elevation of
intracellular fatty acids during diabetes contributes to the effects on CYP4A (128).
Activation of PPAR is a necessary step in mediating the effects of diabetes on
CYP4A transcription, as streptozotocin treatment has no effect in PPAR/ mice
(126). It is postulated that levels of an endogenous fatty acid activator of PPAR are
increased in the diabetic state, resulting in PPAR activation and CYP4A induction.
The effects of streptozotocin-induced diabetes on CYP4A expression appear to be
a direct result of the disease state, as similar findings are reported for the fa/fa
Zucker rat and the ob/ob mice (129). The recent report of a negative correlation
between insulin levels and urinary 20-HETE excretion in humans (112) suggests
that renal CYP4A expression and function is likely altered in human diabetics.
Lower renal 20-HETE production in diabetics would provide a protective effect
from the vasoconstrictive properties of this eicosanoid but may alter the pressurenatriuresis profile and contribute to the hypertensive complications of diabetes.
DIABETES
Inhibition of CYP -Hydroxylases

Important tools for studying the biological role of 20-HETE are potent and selective fatty acid -hydroxylase inhibitors. Both small molecules and antisense
oligonucleotides have been developed as inhibitors of CYP -hydroxylases, and
these inhibitors have been used both in vitro and in vivo. A description of the selectivity, potency, and application of mechanism-based and competitive inhibitors,
antisense oligonucleotides, and 20-HETE antagonists are described below.
One approach to specific inhibitors is the use of
mechanism-based inhibitors, also known as suicide substrates, which specifically
inactivate enzymes in a catalysis-dependent manner (130). The irreversible nature
MECHANISM-BASED INHIBITORS
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of mechanism-based inhibitors makes it easy to document inhibition in tissues

by measuring enzyme function following administration of these inhibitors. As
a result, mechanism-based inhibitors have been widely used in in vivo and in
vitro studies to characterize the biological function of 20-HETE and the substrate
specificity of various CYP isoforms.
One of the first such inhibitors to be characterized is 1-aminobenzotriazole
(ABT) (Table 1). Inhibition of microsomal CYP-dependent activity by ABT is
NADPH- and time-dependent and follows pseudo first-order kinetics, a characteristic of mechanism-based CYP inhibitors (131). Inactivation of CYP enzymes
by ABT requires catalytic formation of benzyne, which in turn alkylates the prosthetic heme group (132). Arachidonic acid metabolism is inhibited by ABT in both
rat renal cortical and hepatic microsomes. The inhibition is dose-dependent and
in initial studies showed a fair degree of selectivity for the CYP4A/CYP4F catalyzed formation of 19- and 20-HETE in the cortex (133). A single intraperitoneal
injection of ABT (50 mg/kg) to Sprague-Dawley rats selectively inhibits renal
cortical and outer medullary 20-HETE formation by 84% and 76%, respectively.
In contrast, there is no inhibition of renal epoxygenase activity at this dose (133).
However, others have reported that the same dose of ABT completely blocks the
formation of 20-HETE and EETs in the kidney within 2 h and reduces the 24-h
urinary excretion of 20-HETE by >50% (134). Chronic treatment with ABT (50
mg/kg/day, ip) for 5 days or 2 weeks inhibits renal 20-HETE and EET formation
by 80%90% and 50%76%, respectively, and urinary 20-HETE excretion falls
by 68%80% (135, 136). Interstudy differences in the selectivity of ABT might
reflect strain differences in enzyme sensitivity. Although ABT has broad substrate
specificity and at certain doses nonselectively blocks both the -hydroxylation
and epoxidation of arachidonic acid, its water solubility and lack of toxicity still
make it of value for characterizing the biological function of CYP eicosanoids.
Studies with ABT in vivo have focused on the role of arachidonic acid hydroxylation on blood pressure regulation. A single dose of ABT to seven-weekold SHRs causes an acute reduction in MAP of 1723 mm Hg during the 4- to 12-h
period after administration and almost complete inhibition of renal cortical and
outer medullary arachidonic acid -hydroxylase activity (133). Chronic treatment
with ABT inhibits renal arachidonic acid -hydroxylase and epoxygenase activity
by 80%90% and 50%76%, respectively, with corresponding changes in urinary
20-HETE excretion (135137). The effects of chronic ABT treatment on blood
pressure are dependent on the experimental model. Chronic treatment of SpragueDawley rats with ABT attenuates the angiotensin IIinduced rise in arterial blood
pressure by 40% (137) and reduces the blood pressure in rats fed a low-salt diet,
while promoting the development of hypertension in rats fed a high-salt diet (136).
A five-day course of ABT therapy attenuates pressure-natriuresis by preventing the
decrease in Na+-K+-ATPase activity and internalization of the sodium-hydrogen
exchanger from the brush border of the proximal tubule following an elevation in
renal perfusion pressure (135). Collectively, these studies support an important role
for 20-HETE in the regulation of renal function and blood pressure. Although acute
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Inhibitors of arachidonic acid -hydroxylases
Inhibitor
Structure
ABT

10-UDYA
11-DDYA
17-ODYA
DMDYA
10-SUYS
DBDD
DDMS
HET0016
6(Z),15(Z)-20-HEDE
ABT: 1-aminobenzotriazole; 10-UDYA: 10-undecynoic acid; 11-DDYA; 11-dodecynoic acid; 17-ODYA:

17-octadecynoic acid; DMDYA: 2,2-dimethyl-11-dodecynoic acid; 10-SUYS: 10-undecynyl sulfate; DBDD:
12,12-dibromododec-11-enoic acid; DDMS: N-methylsulfonyl-12,12-dibromododec-11-enamide; HET0016: Nhydroxy-N -(4-n-butyl-2-methylphenyl)formamidine; 6(Z),15(Z)-20-HEDE: 20-hydroxyeicosa-6(Z),15(Z)-dienoic
acid.
inhibition of CYP arachidonic acid -hydroxylase activity has apparent effects on

vascular reactivity, chronic inhibition suggests that 20-HETE is more important in
maintaining renal tubular transport function.
A series of terminal acetylenic monocarboxylic acid fatty acids varying in
length from 11 [10-undecynoic acid (10-UDYA)] to 18 [17-octadecynoic acid
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(17-ODYA)] (Table 1) carbons (138, 139) have also been synthesized and characterized as arachidonic acid -hydroxylase inhibitors. These compounds are
oxidized to ketenes that inactivate the CYP protein instead of alkylating the prosthetic heme group (140). They are highly selective inhibitors of rat liver CYP
isoforms that are active toward fatty acid substrates without affecting total P-450
levels or other P-450-dependent activities. 10-UDYA and 11-dodecynoic acid (11DDYA) (Table 1) specifically inhibit hepatic CYP enzymes that catalyze lauric acid
- and -1 hydroxylation (138). 11-DDYA and 17-ODYA inhibit the lauric acid
- and -1 hydroxylation by microsomes prepared from the lungs of pregnant
rabbits and reconstituted P-450 (141). 17-ODYA is a very potent inhibitor toward
arachidonic acid metabolism; however, the inhibition is nonspecific (142, 143).
It irreversibly inhibits both -hydroxylation and epoxidation of arachidonic acid
with IC50 values of 7 and 5 M, respectively (142). It also potently inhibits and -1 hydroxylation of arachidonic acid catalyzed by recombinant rat CYP4A1,
CYP4A2, CYP4A3, CYP4F1, and CYP4F4 with a similar IC50 for all isoforms
(51, 74).
Despite its lack of selectivity, 17-ODYA has been widely used in in vitro and
in situ studies to characterize the biological function of 20-HETE. For example,
17-ODYA has been used to establish the role of 20-HETE in the regulation of
renal blood flow and tubuloglomerular feedback and as a K+ channel inhibitor
in rat renal arterioles (20, 21, 143). It also has been used to demonstrate that
20-HETE mediates the vasoconstrictor response to angiotensin II in isolated renal
arterioles and the myogenic response of renal, cerebral, and skeletal muscle arteries
(19, 137, 144). Intrathecal administration of 17-ODYA prevents the acute fall in
cerebral blood flow after subarachnoid hemorrhage in the rat (145).
Unfortunately, the terminal acetylenic fatty acid inhibitors are of little value
for in vivo inactivation of fatty acid hydroxylases because of their rapid metabolic
degradation by -oxidation, their esterification and storage in the liver, and extensive protein binding (139). Introduction of two methyl groups vicinal to the
carboxylic acid group in 10-UDYA yields 2,2-dimethyl-11-dodecynoic acid
(DMDYA) (Table 1), and the replacement of the carboxyl group in 10-UDYA
with a sulfate yields sodium 10-undecynyl sulfate (10-SUYS) (Table 1). Both of
these compounds have in vitro activities similar to that of 10-UDYA and are resistant to -oxidation and storage and exhibit substantial in vivo activity (146).
10-SUYS selectively inhibits arachidonic acid -hydroxylation in rat cortical microsomes (74). The IC50 of 10-SUYS for inhibition of 20-HETE formation is 10
M, whereas epoxygenase activity was not affected at a concentration up to 50
M. 10-SUYS also shows isoform-specific inhibition of rat recombinant CYP4F1and CYP4F4-catalyzed 20-HETE formation (74). The IC50 of 10-SUYS for inhibition of CYP4F4-catalyzed 20-HETE formation is 25 M, whereas 10-SUYS
has only minimal inhibition toward CYP4F1-catalyzed 20-HETE formation.
Administration of 150 mg/kg of 10-SUYS intraperitoneally to SHRs results
in a dose-dependent and selective inhibition of renal cortical arachidonic acid hydroxylase activity (147). A single dose of 10-SUYS (5 mg/kg) causes an acute
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reduction in mean arterial blood pressure by 18 mm Hg in 8-week-old SHRs 6 h

after the treatment. Treatment with 10-SUYS is associated with 66% inhibition
of 20-HETE formation and a loss of CYP4A immunoreactive proteins in cortical
microsomes, a decrease in urinary 20-HETE formation, and attenuation of the
vasoconstrictor response of renal interlobar arteries to angiotensin II in vitro (147).
Chronic treatment with 8 mg/kg/day of 10-SUYS for 4 weeks via an osmotic pump
results in 51% inhibition of 20-HETE formation in renal cortex without affecting
the epoxygenase activity and with no apparent toxicity (F. Xu and & D.L. Kroetz,
unpublished data). To date, 10-SUYS is the most potent and selective mechanismbased inhibitor of CYP-mediated 20-HETE formation, with useful properties for
in vivo studies. It will no doubt prove beneficial in further characterizing the
biological significance of 20-HETE.
In addition to mechanism-based fatty acid CYP hydroxylase inhibitors, the olefinic compounds N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) (Table 1) and 12,12-dibromododec-11-enoic acid
(DBDD) (Table 1) have been described as competitive inhibitors of arachidonic
acid -hydroxylation. DDMS and DBDD exhibit a high degree of selectivity, inhibiting microsomal -hydroxylation of arachidonic acid with an IC50 value of
2 M, whereas the IC50 values for epoxidation are 60 and 51 M, respectively
(142). However, studies with baculovirus-expressed rat CYP4A isoforms show
no selectivity of DDMS between CYP4A1-, CYP4A2-, and CYP4A3-catalyzed
20-HETE formation. A similar IC50 (0.8 M) for DDMS is found for all three
isoforms (51).
In contrast to the mechanism-based inhibitors described above, these acyclic
dibromide derivatives exhibit a reversible time- and NADPH-independent inhibition. However, the fatty acid structure of these inhibitors imparts a fair degree of
selectivity for the CYP fatty acid -hydroxylases. DDMS and DBDD are only
effective at inhibiting the formation of 20-HETE when added to protein-free solutions in vitro or when directly applied to tissues in vivo. Modification of the
carboxyl group in DBDD to a methyl sulfonate in DDMS does not change the potency or selectivity of the inhibitory activity and renders the inhibitor resistant to
-oxidation and of greater utility in vivo (142). Administration of DDMS locally
into an isolated perfused renal arteriolar preparation and systemically into anesthetized rats demonstrates a high degree of selectivity for inhibition of 20-HETE
formation (148). DDMS has been widely used to selectively inhibit 20-HETE formation and therefore to characterize its biological effects, especially its effect on
regulation of vascular tone. DDMS has been used in in vitro studies to establish
a role for 20-HETE in the vasoconstrictor response of renal, cerebral, and mesenteric arteries (13, 149, 150); the myogenic response of skeletal muscle resistance
arteries (19, 144); and the vasoconstrictor response to elevated PO2 in skeletal muscle resistance arterioles (16, 151, 152). Chronic intravenous infusion with DDMS
(10 mg/kg/day) for five days in Sprague-Dawley rats attenuates the angiotensin
IIinduced rise in arterial blood pressure by 40% (137).
COMPETITIVE INHIBITORS
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The most potent and selective inhibitor of 20-HETE formation reported so

far is N-hydroxy-N -(4-n-butyl-2-methylphenyl)formamidine (HET0016) (Table
1) (153). The examination of structure-activity relationships reveals that the unsubstituted hydroxyformamidine moiety and the substituent at the para-position of the
N-hydroxyformamidine moiety are necessary for the potent activity of HET0016
(154). The IC50 value of HET0016 for the formation of 20-HETE by rat renal microsomes is 35 nM, whereas its IC50 value for inhibition of the formation of EETs
is 2800 nM. In human renal microsomes, HET0016 potently inhibits the formation
of 20-HETE with an IC50 value of 9 nM (153). The IC50 values of HET0016 for the
formation of 20-HETE by human recombinant CYP4A11, CYP4F2, and CYP4F3
enzymes are 42, 125, and 100 nM, respectively (145). HET0016 has very little
effect on the activities of cyclooxygenase or other CYP enzymes (153).
HET0016 has been applied in some in vivo and ex vivo studies to characterize
the biological effects of 20-HETE. Chronic treatment with HET0016 (10 mg/kg
per day iv) for 10 days in Sprague-Dawley rats potently and selectively inhibits
the formation of 20-HETE in renal cortical homogenates and the urinary excretion
of 20-HETE by 90%, whereas renal epoxygenase activity was not significantly
altered. However, chronic treatment with HET0016 had no effect on blood pressure
in the Sprague-Dawley rats fed a low-salt diet, and the blood pressure rose by 18 mm
Hg after the rats are fed a high salt diet (136). Chronic treatment with HET0016 also
blocks the increase in 20-HETE formation and angiogenesis induced by electrical
stimulation in skeletal muscle (24). The angiogenic activity in rat renal interlobar
arteries transduced with adenovirus expressing the CYP4A1 cDNA is fully blocked
by treatment with HET0016 and is reversed by addition of a 20-HETE agonist (25).
A single dose of HET0016 (10 mg/kg iv) reduces 20-HETE from 199 to 39 ng/ml
in the cerebrospinal fluid and prevents the acute fall in cerebral blood flow in the
rat following subarachnoid hemorrhage (145, 155).
Despite its promising pharmacological properties, the preparation of an injectable formulation of HET0016 is limited by its poor solubility under neutral conditions and instability under acidic conditions owing to the N-hydroxyformamidine
moiety, an essential feature for potent and selective activity. A more recent study
shows that the activity is maintained when the N-hydroxyformamidine moiety is
replaced by isoxazole or pyrazole, and these derivatives have improved stability
(156). The biologic effects of these second-generation HET0016 derivatives and
their potential side effects have yet to be characterized.
In addition to CYP inhibitors, another approach
to block the formation of 20-HETE has been the use of antisense cDNA oligonucleotides (ODNs). Antisense ODNs offer the possibility of blocking the expression
of a particular CYP gene without any changes in the function of other genes, provided there is enough difference in the sequence of the targeted region between
CYP isoforms. CYP4A1- and CYP4A2/4A3-specific antisense ODNs can inhibit
protein expression and the corresponding catalytic activity (15, 157). Daily intravenous injections of an antisense CYP4A1 and CYP4A2/4A3 ODN for five days
ANTISENSE OLIGONUCLEOTIDES
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reduces the expression of CYP4A-immunoreactive proteins and the production of

20-HETE by 52% and 48%, respectively, in renal arterioles of Sprague-Dawley
rats. Blockade of this pathway is associated with a reduction in arterial blood
pressure by 16 and 17 mm Hg, respectively (157). Administration of CYP4A1 antisense ODN for five days in the SHR also decreases the arterial blood pressure by
16 mm Hg. Treatment with CYP4A1 antisense ODN reduces the level of CYP4Aimmunoreactive proteins along with 20-HETE synthesis in mesenteric arteries in
the SHR. Mesenteric arteries from rats treated with CYP4A1 antisense oligonucleotides exhibit decreased sensitivity to the constrictor action of phenylephrine
and decreased intensity of myogenic constrictor response to elevation in transmural pressure (15). These studies suggest that CYP4A antisense ODNs can provide
the specificity needed for evaluating the contribution of each CYP4A isoform to
the endogenous production of 20-HETE and thereby can be used to examine the
physiological role of 20-HETE.
A series of 20-HETE derivatives have been synthesized
and examined to determine the structural requirements of the vasoconstrictor response to 20-HETE. In renal arterioles, 5(S)-, 15(S)-, and 19(S)-HETE; a C19 analog; and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid [6(Z),15(Z)-20-HEDE or WIT002] (Table 1) block the vasoconstrictor actions of 20-HETE (158). The strongest
antagonist of 20-HETE is 6(Z),15(Z)-20-HEDE, which completely blocks the vasoconstrictor response to 20-HETE in renal (158), cerebral (13, 159), and skeletal
muscle (152, 160) arterioles. These antagonists are increasingly important in investigating the role of 20-HETE in the regulation of vascular tone but are somewhat
limited by their high plasma protein binding and their actions as competitive inhibitors of the synthesis of 20-HETE and EETs at high concentrations. These
properties limit their use in vivo.
20-HETE ANTAGONISTS
SUMMARY AND FUTURE PERSPECTIVES

Significant progress has been made in the past decade in understanding the biological function of 20-HETE and the molecular mechanisms that determine the
intracellular levels of this eicosanoid. The increasing body of literature describing altered CYP arachidonic acid -hydroxylase expression and function in various disease models supports an important role for this metabolic pathway in
vascular reactivity, renal tubular ion and water transport, organ blood flow, cell
growth, and inflammation. Likewise, the development and characterization of selective and potent inhibitors of arachidonic acid -hydroxylase have greatly enhanced our understanding of the role of 20-HETE in physiology and pathophysiology. The multiplicity of subfamilies and individual members of CYP arachidonic
acid -hydroxylases remains a challenge and limits the use of genetic deletion
and antisense techniques. Distinct patterns and levels of expression, as well as
differences in functional activity between the various members of the CYP4A
and CYP4F families, suggest unique roles for individual enzymes in mediating
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429
20-HETE effects. To further delineate the role of individual -hydroxylases, additional isoform-specific inhibitors will need to be characterized. The plausibility of
such an approach is supported by our recent studies that indicate isoform-specific
inhibition of the CYP4F enzymes by 10-SUYS and DDMS (74). Another challenge will be the continued improvement of the biopharmaceutical properties of
these inhibitors. Stability and appropriate pharmacokinetic properties are essential
for the application of inhibitors in vivo.
Future studies will begin to focus more on the application of our current knowledge of 20-HETE effects and CYP arachidonic acid -hydroxylase regulation in
human biology and disease. Sensitive LC/MS/MS and GC/MS assays are becoming more widely available for the quantitation of 20-HETE in urine, plasma, and
other biological samples. This technology will no doubt prove useful in exploring
the hypothesis that arachidonic acid -hydroxylation is altered in human disease,
e.g., diabetes, as well as hypertension, pregnancy, and inflammation. The recent
reports of 20-HETE urinary excretion patterns correlating with insulin levels and
natriuresis (110112) provide promise that therapeutic modulation of CYP arachidonic acid -hydroxylase may prove useful in the management of human disease.
Another exciting area that should be explored is genetic variability in 20-HETE
synthesis and the importance of this variation in eicosanoid function and disease
susceptibility. The clinical significance of drug-induced alterations in CYP arachidonic acid -hydroxylase activity should also be studied. Although much remains
unknown about the role of 20-HETE in human disease, the wealth of information
in animal models will no doubt stimulate increasing interest in this field of study,
with the hope that new drug targets might be identified for the management of
vascular reactivity, renal function, and likely additional clinical conditions that
remain to be discovered.
ACKNOWLEDGMENTS
Work from the authors laboratory cited in this article was supported by a grant
from the National Institutes of Health (HL53994) and the UCSF Liver Core Center
Facility supported by National Institutes of Health Grant P30 DK26743.
LITERATURE CITED
1. Mukherjee AB, Miele L, Pattabiraman N.
1994. Phospholipase A2 enzymes: regulation and physiological role. Biochem.
Pharmacol. 48:110
2. Funk CD. 2001. Prostaglandins and
leukotrienes: advances in eicosanoid biology. Science 294:187175
3. Capdevila JH, Falck JR, Harris RC. 2000.

Cytochrome P450 and arachidonic acid
bioactivation. Molecular and functional
properties of the arachidonate monooxygenase. J. Lipid Res. 41:16381
4. Roman RJ. 2002. P-450 metabolites
of arachidonic acid in the control of
4 Dec 2004
13:20
430
5.

6.
7.
8.
9.
10.
11.
12.
13.
AR
AR232-PA45-17.tex
KROETZ
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE
XU
cardiovascular function. Physiol. Rev. 82:

13185
Kroetz DL, Zeldin DC. 2002. Cytochrome P450 pathways of arachidonic
acid metabolism. Curr. Opin. Lipidol. 13:
27383
Chuang SS, Helvig C, Taimi M, Ramshaw
HA, Collop AH, et al. 2004. CYP2U1, a
novel human thymus- and brain-specific
cytochrome P450, catalyzes - and (1)-hydroxylation of fatty acids. J. Biol.
Chem. 279:630514
Zeldin DC, Kobayashi J, Falck JR, Winder
BS, Hammock BD, et al. 1993. Regioand enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase. J. Biol. Chem.
268:64027
Zeldin DC, Moomaw CR, Jesse N, Tomer
KB, Beetham J, et al. 1996. Biochemical
characterization of the human liver cytochrome P450 arachidonic acid epoxygenase pathway. Arch. Biochem. Biophys.
330:8796
Cowart LA, Wei S, Hsu MH, Johnson EF, Krishna MU, et al. 2002. The
CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity
peroxisome proliferator-activated receptor ligands. J. Biol. Chem. 277:3510512
Spector AA, Fang X, Snyder GD, Weintraub NL. 2004. Epoxyeicosatrienoic
acids (EETs): metabolism and biochemical function. Prog. Lipid Res. 43:5590
Kaduce TL, Fang X, Harmon SD, Oltman CL, Dellsperger KC, et al. 2004. 20hydroxyeicosatetraenoic acid (20-HETE)
metabolism in coronary endothelial cells.
J. Biol. Chem. 279:264856
Imig JD, Zou AP, Stec DE, Harder DR,
Falck JR, et al. 1996. Formation and actions of 20-hydroxyeicosatetraenoic acid
in rat renal arterioles. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 270:R217
27
Gebremedhin D, Lange AR, Lowry TF,
Taheri MR, Birks EK, et al. 2000. Production of 20-HETE and its role in au-
14.
15.
16.
17.
18.
19.
20.
21.
toregulation of cerebral blood flow. Circ.

Res. 87:6065
Zhu D, Zhang C, Medhora M, Jacobs
ER. 2002. CYP4A mRNA, protein, and
product in rat lungs: novel localization
in vascular endothelium. J. Appl. Physiol.
93:33037
Wang MH, Zhang F, Marji J, Zand BA,
Nasjletti A, et al. 2001. CYP4A1 antisense oligonucleotide reduces mesenteric
vascular reactivity and blood pressure in
SHR. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 280:R25561
Kunert MP, Roman RJ, Alonso-Galicia
M, Falck JR, Lombard JH. 2001. Cytochrome P-450 -hydroxylase: a potential O2 sensor in rat arterioles and skeletal
muscle cells. Am. J. Physiol. Heart Circ.
Physiol. 280:H184045
Zou AP, Fleming JT, Falck JR, Jacobs ER,
Gebremedhin D, et al. 1996. 20-HETE
is an endogenous inhibitor of the largeconductance Ca2+-activated K+ channel
in renal arterioles. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 270:R22837
Ma YH, Gebremedhin D, Schwartzman
ML, Falck JR, Clark JE, et al. 1993. 20Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal
arcuate arteries. Circ. Res. 72:12636
Frisbee JC, Roman RJ, Falck JR, Krishna
UM, Lombard JH. 2001. 20-HETE contributes to myogenic activation of skeletal
muscle resistance arteries in Brown Norway and Sprague-Dawley rats. Microcirculation 8:4555
Zou AP, Imig JD, Ortiz de Montellano PR,
Sui Z, Falck JR, et al. 1994. Effect of P450 -hydroxylase metabolites of arachidonic acid on tubuloglomerular feedback.
Am. J. Physiol. Renal Physiol. 266:F934
41
Zou AP, Imig JD, Kaldunski M, Ortiz de
Montellano PR, Sui Z, et al. 1994. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal
blood flow. Am. J. Physiol. Renal Physiol. 266:F27582
4 Dec 2004
13:20
AR
AR232-PA45-17.tex
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE


22. Harder DR, Narayanan J, Birks EK, Liard
JF, Imig JD, et al. 1996. Identification of
a putative microvascular oxygen sensor.
Circ. Res. 79:5461
23. Muthalif MM, Benter IF, Karzoun N,
Fatima S, Harper J, et al. 1998.
20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated
protein kinase activation in vascular
smooth muscle cells. Proc. Natl. Acad.
Sci. USA 95:127016
24. Amaral SL, Maier KG, Schippers DN,
Roman RJ, Greene AS. 2003. CYP4A
metabolites of arachidonic acid and
VEGF are mediators of skeletal muscle
angiogenesis. Am. J. Physiol. Heart Circ.
Physiol. 284:H152835
25. Jiang M, Mezentsev A, Kemp R, Byun
K, Falck JR, et al. 2004. Smooth muscle
specific expression of CYP4A1 induces
endothelial sprouting in renal arterial microvessels. Circ. Res. 94:16774
26. Carroll MA, Sala A, Dunn CE, McGiff
JC, Murphy RC. 1991. Structural identification of cytochrome P450-dependent
arachidonate metabolites formed by rabbit medullary thick ascending limb cells.
J. Biol. Chem. 266:1230612
27. Escalante B, Erlij D, Falck JR, McGiff
JC. 1994. Cytochrome P-450 arachidonate metabolites affect ion fluxes in rabbit medullary thick ascending limb. Am.
J. Physiol. Cell Physiol. 266:C177582
28. Lin F, Abraham NG, Schwartzman ML.
1994. Cytochrome P450 arachidonic acid
-hydroxylation in the proximal tubule of
the rat kidney. Ann. NY Acad. Sci. 744:11
24
29. Schwartzman M, Ferreri NR, Carroll MA,
Songu-Mize E, McGiff JC. 1985. Renal cytochrome P450-related arachidonate metabolite inhibits Na+,K+-ATPase.
Nature 314:62022
30. Wang W, Lu M. 1995. Effect of arachidonic acid on activity of the apical K+
channel in the thick ascending limb of the
rat kidney. J. Gen. Physiol. 106:72743
431
31. Amlal H, Legoff C, Vernimmen C, Paillard M, Bichara M. 1996. Na+-K+(NH4+)2Cl cotransport in medullary thick ascending limb: control by PKA, PKC, and
20-HETE. Am. J. Physiol. Cell Physiol.
271:C45563
32. Birks EK, Bousamra M, Presberg K,
Marsh JA, Effros RM, et al. 1997. Human
pulmonary arteries dilate to 20-HETE,
an endogenous eicosanoid of lung tissue.
Am. J. Physiol. Lung Cell Mol. Physiol.
272:L82389
33. Zhu D, Birks EK, Dawson CA, Patel
M, Falck JR, et al. 2000. Hypoxic pulmonary vasoconstriction is modified by
P-450 metabolites. Am. J. Physiol. Heart
34. Jacobs ER, Zeldin DC. 2001. The lung
HETEs (and EETs) up. Am. J. Physiol.
35. Hardwick JP, Song BJ, Huberman E, Gonzalez FJ. 1987. Isolation, complementary DNA sequence, and regulation of
rat hepatic lauric acid -hydroxylase (cytochrome P-450LA). Identification of a
new cytochrome P-450 gene family. J.
Biol. Chem. 262:80110
36. Kimura S, Hanioka N, Matsunaga E,
Gonzalez FJ. 1989. The rat clofibrateinducible CYP4A gene subfamily. I.
Complete intron and exon sequence of
the CYP4A1 and CYP4A2 genes, unique
exon organization, and identification of a
conserved 19-bp upstream element. DNA
8:50316
37. Kimura S, Hardwick JP, Kozak CA,
Gonzalez FJ. 1989. The rat clofibrateinducible CYP4A subfamily. II. cDNA sequence of IVA3, mapping of the Cyp4a
locus to mouse chromosome 4, and coordinate and tissue-specific regulation of the
CYP4A genes. DNA 8:51725
38. Stromstedt M, Hayashi S, Zaphiropoulos
PG, Gustafsson JA. 1990. Cloning and
characterization of a novel member of the
cytochrome P450 subfamily IVA in rat
prostate. DNA Cell Biol. 9:56977
39. Bellamine A, Wang Y, Waterman MR,
4 Dec 2004
13:20
432

40.
41.
42.
43.
44.
45.
46.
47.
AR
AR232-PA45-17.tex
KROETZ
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE
XU
Gainer JV 3rd, Dawson EP, et al. 2003.

Characterization of the CYP4A11 gene,
a second CYP4A gene in humans. Arch.
Palmer CN, Richardson TH, Griffin KJ,
Hsu MH, Muerhoff AS, et al. 1993. Characterization of a cDNA encoding a human
kidney cytochrome P-450 4A fatty acid
-hydroxylase and the cognate enzyme
expressed in Escherichia coli. Biochim.
Biophys. Acta. 1172:16166
Imaoka S, Ogawa H, Kimura S, Gonzalez
FJ. 1993. Complete cDNA sequence and
cDNA-directed expression of CYP4A11,
a fatty acid -hydroxylase expressed in
human kidney. DNA Cell Biol. 12:893
99
Yokotani N, Bernhardt R, Sogawa K,
Kusunose E, Gotoh O, et al. 1989. Two
forms of -hydroxylase toward prostaglandin A and laurate. cDNA cloning
and their expression. J. Biol. Chem. 264:
2166569
Johnson EF, Walker DL, Griffin KJ, Clark
JE, Okita RT, et al. 1990. Cloning and expression of three rabbit kidney cDNAs encoding lauric acid -hydroxylases. Biochemistry 29:87379
Muerhoff AS, Griffin KJ, Johnson EF.
1992. Characterization of a rabbit gene
encoding a clofibrate-inducible fatty acid
-hydroxylase: CYP4A6. Arch. Biochem.
Biophys. 296:6672
Palmer CN, Griffin KJ, Johnson EF. 1993.
Rabbit prostaglandin -hydroxylase
(CYP4A4): gene structure and expression. Arch. Biochem. Biophys. 300:
67076
Bell DR, Plant NJ, Rider CG, Na L,
Brown S, et al. 1993. Species-specific induction of cytochrome P-450 4A RNAs:
PCR cloning of partial guinea pig, human
and mouse CYP4A cDNAs. Biochem. J.
294:17380
Henderson CJ, Bammler T, Wolf CR.
1994. Deduced amino acid sequence of
a murine cytochrome P-450 Cyp4a protein: developmental and hormonal regula-
48.
49.
50.
51.
52.
53.
54.
55.
tion in liver and kidney. Biochim. Biophys.

Acta 1200:18290
Heng YM, Kuo CS, Jones PS, Savory R,
Schulz RM, et al. 1997. A novel murine
P-450 gene, Cyp4a14, is part of a cluster
of Cyp4a and Cyp4b, but not of CYP4F,
genes in mouse and humans. Biochem. J.
325:74149
Hoch U, Zhang Z, Kroetz DL, Ortiz de
Montellano PR. 2000. Structural determination of the substrate specificities and regioselectivities of the rat and human fatty
acid -hydroxylases. Arch. Biochem. Biophys. 373:6371
Wang MH, Stec DE, Balazy M, Mastyugin V, Yang CS, et al. 1996. Cloning,
sequencing, and cDNA-directed expression of the rat renal CYP4A2: arachidonic acid -hydroxylation and 11,12epoxidation by CYP4A2 protein. Arch.
Nguyen X, Wang MH, Reddy KM,
Falck JR, Schwartzman ML. 1999. Kinetic profile of the rat CYP4A isoforms:
arachidonic acid metabolism and isoformspecific inhibitors. Am. J. Physiol. Renal
Physiol. 276:R1691700
Helvig C, Dishman E, Capdevila JH.
1998. Molecular, enzymatic, and regulatory characterization of rat kidney cytochromes P450 4A2 and 4A3. Biochemistry 37:1254658
Aitken AE, Roman LJ, Loughran PA, de
la Garza M, Masters BS. 2001. Expressed
CYP4A4 metabolism of prostaglandin E1
and arachidonic acid. Arch. Biochem. Biophys. 393:32938
Loughran PA, Roman LJ, Miller RT, Masters BS. 2001. The kinetic and spectral
characterization of the E. coli-expressed
mammalian CYP4A7: cytochrome b5
effects vary with substrate. Arch. Biochem. Biophys. 385:31121
Hosny G, Roman LJ, Mostafa MH,
Masters BS. 1999. Unique properties
of purified, Escherichia coli-expressed
constitutive cytochrome P4504A5. Arch.
4 Dec 2004
13:20
AR
AR232-PA45-17.tex
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE


56. Roman LJ, Palmer CN, Clark JE, Muerhoff AS, Griffin KJ, et al. 1993. Expression of rabbit cytochromes P4504A
which catalyze the -hydroxylation
of arachidonic acid, fatty acids, and
prostaglandins. Arch. Biochem. Biophys.
307:5765
57. Powell PK, Wolf I, Jin R, Lasker JM.
1998. Metabolism of arachidonic acid
to 20-hydroxy-5,8,11,14-eicosatetraenoic
acid by P450 enzymes in human liver: involvement of CYP4F2 and CYP4A11. J.
58. Lasker JM, Chen WB, Wolf I, Bloswick
BP, Wilson PD, et al. 2000. Formation of 20-hydroxyeicosatetraenoic acid,
a vasoactive and natriuretic eicosanoid,
in human kidney. Role of CYP4F2 and
CYP4A11. J. Biol. Chem. 275:411826
59. Kawashima H, Naganuma T, Kusunose E,
Kono T, Yasumoto R, et al. 2000. Human fatty acid -hydroxylase, CYP4A11:
determination of complete genomic sequence and characterization of purified recombinant protein. Arch. Biochem. Biophys. 378:33339
60. Chen L, Hardwick JP. 1993. Identification of a new P450 subfamily, CYP4F1,
expressed in rat hepatic tumors. Arch.
61. Kikuta Y, Kusunose E, Endo K, Yamamoto S, Sogawa K, et al. 1993. A
novel form of cytochrome P-450 family 4 in human polymorphonuclear leukocytes. cDNA cloning and expression of
leukotriene B4 -hydroxylase. J. Biol.
Chem. 268:937680
62. Kawashima H, Strobel HW. 1995. cDNA
cloning of three new forms of rat brain cytochrome P450 belonging to the CYP4F
subfamily. Biochem. Biophys. Res. Commun. 217:113744
63. Kikuta Y, Kusunose E, Kondo T, Yamamoto S, Kinoshita H, et al. 1994.
Cloning and expression of a novel form
of leukotriene B4 -hydroxylase from human liver. FEBS Lett. 348:7074
64. Cui X, Nelson DR, Strobel HW. 2000.
65.
66.
67.
68.
69.
70.
71.
72.
433
A novel human cytochrome P450 4F isoform (CYP4F11): cDNA cloning, expression, and genomic structural characterization. Genomics 68:16166
Bylund J, Bylund M, Oliw EH. 2001.
cDNA cloning and expression of
CYP4F12, a novel human cytochrome
P450. Biochem. Biophys. Res. Commun.
280:89297
Hashizume T, Imaoka S, Hiroi T, Terauchi Y, Fujii T, et al. 2001. cDNA cloning
and expression of a novel cytochrome
P450 (CYP4F12) from human small intestine. Biochem. Biophys. Res. Commun.
280:113541
Cui X, Kawashima H, Barclay TB, Peters
JM, Gonzalez FJ, et al. 2001. Molecular
cloning and regulation of expression of
two novel mouse CYP4F genes: expression in peroxisome proliferator-activated
receptor -deficient mice upon lipopolysaccharide and clofibrate challenges. J.
Kikuta Y, Kusunose E, Ito M, Kusunose
M. 1999. Purification and characterization
of recombinant rat hepatic CYP4F1. Arch.
Kikuta Y, Kusunose E, Kusunose M.
2000. Characterization of human liver
leukotriene B4 -hydroxylase P450
(CYP4F2). J. Biochem. (Tokyo) 127:
104752
Kikuta Y, Kusunose E, Sumimoto H,
Mizukami Y, Takeshige K, et al. 1998. Purification and characterization of recombinant human neutrophil leukotriene B4
-hydroxylase (cytochrome P450 4F3).
Bylund J, Harder AG, Maier KG, Roman RJ, Harder DR. 2003. Leukotriene B4
-side chain hydroxylation by CYP4F5
and CYP4F6. Arch. Biochem. Biophys.
412:3441
Jin R, Koop DR, Raucy JL, Lasker JM.
1998. Role of human CYP4F2 in hepatic
catabolism of the proinflammatory agent
leukotriene B4. Arch. Biochem. Biophys.
359:8998
4 Dec 2004
13:20

434
AR
AR232-PA45-17.tex
KROETZ
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE
XU
73. Christmas P, Jones JP, Patten CJ, Rock

DA, Zheng Y, et al. 2001. Alternative splicing determines the function of
CYP4F3 by switching substrate specificity. J. Biol. Chem. 276:3816672
74. Xu F, Falck JR, Ortiz de Montellano
PR, Kroetz DL. 2004. Catalytic activity
and isoform-specific inhibition of rat cytochrome P450 4F enzymes. J. Pharmacol. Exp. Ther. 308:88795
75. Ito O, Alonso-Galicia M, Hopp KA,
Roman RJ. 1998. Localization of cytochrome P-450 4A isoforms along the rat
nephron. Am. J. Physiol. Renal Physiol.
274:F395404
76. Kroetz DL, Huse LM, Thuresson A, Grillo
MP. 1997. Developmentally regulated expression of the CYP4A genes in the spontaneously hypertensive rat kidney. Mol.
Pharmacol. 52:36272
77. Marji JS, Wang MH, Laniado-Schwartzman M. 2002. Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries. Am. J.
78. Stec DE, Flasch A, Roman RJ, White JA.
2003. Distribution of cytochrome P-450
4A and 4F isoforms along the nephron
in mice. Am. J. Physiol. Renal Physiol.
284:F95102
79. Kalsotra A, Anakk S, Boehme CL,
Strobel HW. 2002. Sexual dimorphism
and tissue specificity in the expression of CYP4F forms in Sprague Dawley rats. Drug Metab. Dispos. 30:1022
28
80. Stark K, Schauer L, Sahlen GE, Ronquist G, Oliw EH. 2004. Expression of
CYP4F12 in gastrointestinal and urogenital epithelia. Basic Clin. Pharmacol. Toxicol. 94:17783
81. Schwartzman ML, da Silva JL, Lin F,
Nishimura M, Abraham NG. 1996. Cytochrome P450 4A expression and arachidonic acid -hydroxylation in the kidney of the spontaneously hypertensive rat.
Nephron 73:65263
82. Cummings BS, Zangar RC, Novak RF,
83.
84.
85.
86.
87.
88.
89.
90.
91.
Lash LH. 1999. Cellular distribution of

cytochromes P-450 in the rat kidney. Drug
Schaaf GJ, de Groene EM, Maas RF,
Commandeur JN, Fink-Gremmels J.
2001. Characterization of biotransformation enzyme activities in primary rat proximal tubular cells. Chem. Biol. Interact.
134:16790
Omata K, Abraham NG, Schwartzman
ML. 1992. Renal cytochrome P-450arachidonic acid metabolism: localization
and hormonal regulation in SHR. Am. J.
Physiol. 262:F59199
Ito O, Roman RJ. 1999. Regulation of
P-450 4A activity in the glomerulus of
the rat. Am. J. Physiol. Renal Physiol.
276:R174957
Stromstedt M, Warner M, Gustafsson
JA. 1994. Cytochrome P450s of the 4A
subfamily in the brain. J. Neurochem.
63:67176
Koike K, Kusunose E, Nishikawa Y,
Ichihara K, Inagaki S, et al. 1997. Purification and characterization of rabbit
small intestinal cytochromes P450 belonging to CYP2J and CYP4A subfamilies. Biochem. Biophys. Res. Commun.
232:64347
Zhu D, Effros RM, Harder DR, Roman
RJ, Jacobs ER. 1998. Tissue sources of
cytochrome P450 4A and 20-HETE synthesis in rabbit lungs. Am. J. Respir. Cell
Mol. Biol. 19:12128
Christmas P, Carlesso N, Shang H, Cheng
SM, Weber BM, et al. 2003. Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative
promoter. J. Biol. Chem. 278:2513342
Johnson EF, Palmer CN, Griffin KJ,
Hsu MH. 1996. Role of the peroxisome proliferator-activated receptor in
cytochrome P450 4A gene regulation.
FASEB J. 10:124148
Johnson EF, Hsu MH, Savas U, Griffin
KJ. 2002. Regulation of P450 4A expression by peroxisome proliferator activated
receptors. Toxicology 181182:2036
4 Dec 2004
13:20
AR
AR232-PA45-17.tex
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE


92. Muller DN, Theuer J, Shagdarsuren E,
Kaergel E, Honeck H, et al. 2004. A peroxisome proliferator-activated receptor-
activator induces renal CYP2C23 activity
and protects from angiotensin II-induced
renal injury. Am. J. Pathol. 164:52132
93. Corton JC, Fan LQ, Brown S, Anderson SP, Bocos C, et al. 1998.
Down-regulation of cytochrome P450 2C
family members and positive acute-phase
response gene expression by peroxisome
proliferator chemicals. Mol. Pharmacol.
54:46373
94. Alonso-Galicia M, Sun CW, Falck JR,
Harder DR, Roman RJ. 1998. Contribution of 20-HETE to the vasodilator actions of nitric oxide in renal arteries. Am.
J. Physiol. Renal Physiol. 275:F37078
95. Hoch U, Ortiz De Montellano PR. 2001.
Covalently linked heme in cytochrome
P4504A fatty acid hydroxylases. J. Biol.
Chem. 276:1133946
96. Henne KR, Kunze KL, Zheng YM, Christmas P, Soberman RJ, et al. 2001. Covalent linkage of prosthetic heme to
CYP4 family P450 enzymes. Biochemistry 40:1292531
97. LeBrun LA, Hoch U, Ortiz de Montellano PR. 2002. Autocatalytic mechanism
and consequences of covalent heme attachment in the cytochrome P4504A family. J. Biol. Chem. 277:1275561
98. LeBrun LA, Xu F, Kroetz DL, Ortiz de
Montellano PR. 2002. Covalent attachment of the heme prosthetic group in
the CYP4F cytochrome P450 family. Biochemistry 41:593137
99. Omata K, Abraham NG, Escalante B,
Schwartzman ML. 1992. Age-related
changes in renal cytochrome P-450
arachidonic acid metabolism in spontaneously hypertensive rats. Am. J. Physiol.
Renal Physiol. 262:F816
100. Imig JD, Falck JR, Gebremedhin D,
Harder DR, Roman RJ. 1993. Elevated
renovascular tone in young spontaneously
hypertensive rats. Role of cytochrome P450. Hypertension 22:35764
435
101. Stec DE, Trolliet MR, Krieger JE, Jacob

HJ, Roman RJ. 1996. Renal cytochrome
P4504A activity and salt sensitivity in
spontaneously hypertensive rats. Hypertension 27:132936
102. Zhang F, Wang MH, Krishna UM,
Falck JR, Laniado-Schwartzman M,
et al. 2001. Modulation by 20-HETE of
phenylephrine-induced mesenteric artery
contraction in spontaneously hypertensive and Wistar-Kyoto rats. Hypertension
38:131115
103. Iwai N, Inagami T. 1991. Isolation of
preferentially expressed genes in the kidneys of hypertensive rats. Hypertension
17:16169
104. Ito O, Roman RJ. 1999. Role of 20-HETE
in elevating chloride transport in the thick
ascending limb of Dahl SS/Jr rats. Hypertension 33:41923
105. Ma YH, Schwartzman ML, Roman RJ.
1994. Altered renal P-450 metabolism
of arachidonic acid in Dahl salt-sensitive
rats. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 267:R57989
106. Stec DE, Deng AY, Rapp JP, Roman
RJ. 1996. Cytochrome P4504A genotype
cosegregates with hypertension in Dahl S
rats. Hypertension 27:56468
107. Zhao X, Pollock DM, Inscho EW, Zeldin
DC, Imig JD. 2003. Decreased renal cytochrome P450 2C enzymes and
impaired vasodilation are associated with
angiotensin salt-sensitive hypertension.
Hypertension 41:70914
108. Wang MH, Smith A, Zhou Y, Chang HH,
Lin S, et al. 2003. Downregulation of renal CYP-derived eicosanoid synthesis in
rats with diet-induced hypertension. Hypertension 42:59499
109. Holla VR, Adas F, Imig JD, Zhao X,
Price E Jr, et al. 2001. Alterations in
the regulation of androgen-sensitive Cyp
4a monooxygenases cause hypertension.
110. Laffer CL, Laniado-Schwartzman M,
Wang MH, Nasjletti A, Elijovich F. 2003.
Differential regulation of natriuresis by
4 Dec 2004
13:20
436
111.

112.
113.
114.
115.
116.
117.
118.
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KROETZ
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20-hydroxyeicosatetraenoic acid in human salt-sensitive versus salt-resistant hypertension. Circulation 107:57478

Laffer CL, Laniado-Schwartzman M,
Wang MH, Nasjletti A, Elijovich F. 2003.
20-HETE and furosemide-induced natriuresis in salt-sensitive essential hypertension. Hypertension 41:7038
Laffer CL, Laniado-Schwartzman M,
Nasjletti A, Elijovich F. 2004. 20-HETE
and circulating insulin in essential hypertension with obesity. Hypertension
43:38892
Shimojo N, Ishizaki T, Imaoka S, Funae
Y, Fujii S, et al. 1993. Changes in amounts
of cytochrome P450 isozymes and levels
of catalytic activities in hepatic and renal microsomes of rats with streptozocininduced diabetes. Biochem. Pharmacol.
46:62127
Muerhoff AS, Williams DE, Leithauser
MT, Jackson VE, Waterman MR, et al.
1987. Regulation of the induction of
a cytochrome P-450 prostaglandin hydroxylase by pregnancy in rabbit lung.
Wang MH, Zand BA, Nasjletti A,
Laniado-Schwartzman M. 2002. Renal
20-hydroxyeicosatetraenoic acid synthesis during pregnancy. Am. J. Physiol.
89
Wang MH, Wang J, Chang HH, Zand BA,
Jiang M, et al. 2003. Regulation of renal
CYP4A expression and 20-HETE synthesis by nitric oxide in pregnant rats. Am. J.
Iber H, Sewer MB, Barclay TB, Mitchell
SR, Li T, et al. 1999. Modulation of drug
metabolism in infectious and inflammatory diseases. Drug Metab. Rev. 31:29
41
Sewer MB, Koop DR, Morgan ET. 1997.
Differential inductive and suppressive effects of endotoxin and particulate irritants on hepatic and renal cytochrome P450 expression. J. Pharmacol. Exp. Ther.
280:144554
119. Sewer MB, Koop DR, Morgan ET. 1996.

Endotoxemia in rats is associated with
induction of the P4504A subfamily and
suppression of several other forms of
cytochrome P450. Drug Metab. Dispos.
24:4017
120. Barclay T, Peters JM, Sewer MB, Ferrari L, Gonzalez F, Morgan ET. 1999.
Modulation of cytochrome P-450 gene
expression in endotoxemic mice is tissue specific and peroxisome proliferatoractivated receptor- dependent. J. Pharmacol. Exp. Ther. 290:125057
121. Kalsotra A, Cui X, Antonovic L, Robida AM, Morgan ET, et al. 2003. Inflammatory prompts produce isoform-specific
changes in the expression of leukotriene
B4 -hydroxylases in rat liver and kidney.
FEBS Lett. 555:23642
122. Kalsotra A, Turman CM, Dash PK, Strobel HW. 2003. Differential effects of traumatic brain injury on the cytochrome
P450 system: a perspective into hepatic
and renal drug metabolism. J. Neurotrauma 20:133950
123. Barnett CR, Gibson GG, Wolf CR, Flatt
PR, Ioannides C. 1990. Induction of cytochrome P450III and P450IV family proteins in streptozotocin-induced diabetes.
Biochem. J. 268:76569
124. Barnett CR, Rudd S, Flatt PR, Ioannides
C. 1993. Sex differences in the diabetesinduced modulation of rat hepatic cytochrome P450 proteins. Biochem. Pharmacol. 45:31319
125. Imaoka S, Nakamura M, Ishizaki T, Shimojo N, Ohishi N, et al. 1993. Regulation
of renal cytochrome P450s by thyroid hormone in diabetic rats. Biochem. Pharmacol. 46:2197200
126. Kroetz DL, Yook P, Costet P, Bianchi P,
Pineau T. 1998. Peroxisome proliferatoractivated receptor controls the hepatic
CYP4A induction adaptive response to
starvation and diabetes. J. Biol. Chem.
273:3158189
127. Ferguson NL, Donahue BS, Tenney
KA, Morgan ET. 1993. Pretranslational
4 Dec 2004
13:20
AR
AR232-PA45-17.tex
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE

128.
129.
130.
131.
132.
133.
134.
135.
induction of CYP4A subfamily gene

products in diabetic rats and reversal by
oral vanadate treatment. Drug Metab. Dispos. 21:74546
Zangar RC, Novak RF. 1997. Effects
of fatty acids and ketone bodies on cytochromes P450 2B, 4A, and 2E1 expression in primary cultured rat hepatocytes. Arch. Biochem. Biophys. 337:217
24
Enriquez A, Leclercq I, Farrell GC,
Robertson G. 1999. Altered expression of
hepatic CYP2E1 and CYP4A in obese, diabetic ob/ob mice, and fa/fa Zucker rats.
Biochem. Biophys. Res. Comm. 255:300
6
Walsh CT. 1984. Suicide substrates,
mechanism-based enzyme inactivators:
recent developments. Annu. Rev. Biochem. 53:493535
Oritz de Montellano PR, Correia MA.
1983. Suicidal destruction of cytochrome
P-450 during oxidative drug metabolism.
503
Ortiz de Montellano PR, Mathews JM.
1981. Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group
by 1-aminobenzotriazole. Isolation of an
NN-bridged benzyne-protoporphyrin IX
adduct. Biochem. J. 195:76164
Su P, Kaushal KM, Kroetz DL. 1998.
Inhibition of renal arachidonic acid hydroxylase activity with ABT reduces
blood pressure in the SHR. Am. J. Physiol.
38
Maier KG, Henderson L, Narayanan J,
Alonso-Galicia M, Falck JR, et al. 2000.
Fluorescent HPLC assay for 20-HETE
and other P-450 metabolites of arachidonic acid. Am. J. Physiol. Heart Circ.
Physiol. 279:H86371
Dos Santos EA, Dahly-Vernon AJ,
Hoagland KM, Roman RJ. 2004. Inhibition of the formation of EETs and 20HETE with 1-aminobenzotriazole attenuates pressure-natriuresis. Am. J. Physiol.
136.
137.
138.
139.
140.
141.
142.
143.
144.
437

68
Hoagland KM, Flasch AK, Roman RJ.
2003. Inhibitors of 20-HETE formation
promote salt-sensitive hypertension in
rats. Hypertension 42:66973
Alonso-Galicia M, Maier KG, Greene
AG, Cowley AW, Roman RJ. 2002. Role
of 20-hydroxyeicosatetraenoic acid in the
renal and vasoconstrictor actions of angiotensin II. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 283:R6068
Ortiz de Montellano PR, Reich NO. 1984.
Specific inactivation of hepatic fatty acid
hydroxylases by acetylenic fatty acids. J.
Biol. Chem. 259:413641
Reich NO, Ortiz de Montellano PR.
1986. Dissociation of increased lauric
acid -hydroxylase activity from the antilipidemic action of clofibrate. Biochem.
CaJacob CA, Chan WK, Shephard E, Ortiz de Montellano PR. 1988. The catalytic site of rat hepatic lauric acid hydroxylase. Protein versus prosthetic
heme alkylation in the -hydroxylation
of acetylenic fatty acids. J. Biol. Chem.
263:1864049
Muerhoff AS, Williams DE, Reich NO,
CaJacob CA, Ortiz de Montellano PR,
et al. 1989. Prostaglandin and fatty acid
- and (-1)-oxidation in rabbit lung.
Acetylenic fatty acid mechanism-based
inactivators as specific inhibitors. J. Biol.
Chem. 264:74956
Wang MH, Brand-Schieber E, Zand BA,
Nguyen X, Falck JR, et al. 1998. Cytochrome P450-derived arachidonic acid
metabolism in the rat kidney: characterization of selective inhibitors. J. Pharmacol. Exp. Ther. 284:96673
Zou AP, Ma YH, Sui ZH, Ortiz de Montellano PR, Clark JE, et al. 1994. Effects of
17-octadecynoic acid, a suicide-substrate
inhibitor of cytochrome P450 fatty acid
-hydroxylase, on renal function in rats.
Frisbee JC, Roman RJ, Krishna UM,
4 Dec 2004
13:20
438

145.
146.
147.
148.
149.
150.
151.
152.
AR
AR232-PA45-17.tex
KROETZ
AR232-PA45-17.sgm
LaTeX2e(2002/01/18)
P1: GCE
XU
Falck JR, Lombard JH. 2001. 20-HETE

modulates myogenic response of skeletal
muscle resistance arteries from hypertensive Dahl-SS rats. Am. J. Physiol. Heart
Kehl F, Cambj-Sapunar L, Maier KG,
Miyata N, Kametani S, et al. 2002. 20HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat. Am. J. Physiol. Heart
CaJacob CA, Ortiz de Montellano PR.
1986. Mechanism-based in vivo inactivation of lauric acid hydroxylases. Biochemistry 25:470511
Xu F, Straub WO, Pak W, Su P, Maier
KG, et al. 2002. Antihypertensive effect
of mechanism-based inhibition of renal
arachidonic acid -hydroxylase activity.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 283:R71020
Alonso-Galicia M, Drummond HA,
Reddy KK, Falck JR, Roman RJ. 1997.
Inhibition of 20-HETE production contributes to the vascular responses to nitric
oxide. Hypertension 29:32025
Quilley J, Qiu Y, Hirt J. 2003. Inhibitors of
20-hydroxyeicosatetraenoic acid reduce
renal vasoconstrictor responsiveness. J.
Looft-Wilson RC, Falck JR, Krishna UM,
Gisolfi CV. 2002. 20-HETE pathway
antagonists inhibit rat small mesenteric
artery tone. Microvasc. Res. 64:34952
Frisbee JC, Roman RJ, Falck JR, Linderman JR, Lombard JH. 2000. Impairment of flow-induced dilation of skeletal
muscle arterioles with elevated oxygen in
normotensive and hypertensive rats. Microvasc. Res. 60:3748
Frisbee JC, Krishna UM, Falck JR, Lombard JH. 2001. Role of prostanoids and
20-HETE in mediating oxygen-induced
constriction of skeletal muscle resistance
arteries. Microvasc. Res. 62:27183
153. Miyata N, Taniguchi K, Seki T, Ishimoto T, Sato-Watanabe M, et al. 2001.

HET0016, a potent and selective inhibitor
of 20-HETE synthesizing enzyme. Br. J.
154. Sato M, Ishii T, Kobayashi-Matsunaga Y, Amada H, Taniguchi K, et al.
2001. Discovery of a N -hydroxyphenylformamidine derivative HET0016 as a
potent and selective 20-HETE synthase
inhibitor. Bioorg. Med. Chem. Lett. 11:
299395
155. Cambj-Sapunar L, Yu M, Harder DR,
Roman RJ. 2003. Contribution of 5hydroxytryptamine1B receptors and 20hydroxyeiscosatetraenoic acid to fall in
cerebral blood flow after subarachnoid
hemorrhage. Stroke 34:126975
156. Nakamura T, Kakinuma H, Umemiya H,
Amada H, Miyata N, et al. 2004. Imidazole derivatives as new potent and selective 20-HETE synthase inhibitors. Bioorg.
Med. Chem. Lett. 14:33336
157. Wang MH, Guan H, Nguyen X, Zand BA,
Nasjletti A, et al. 1999. Contribution of cytochrome P-450 4A1 and 4A2 to vascular
20-hydroxyeicosatetraenoic acid synthesis in rat kidneys. Am. J. Physiol. Renal
Physiol. 276:F24653
158. Alonso-Galicia M, Falck JR, Reddy KM,
Roman RJ. 1999. 20-HETE agonists and
antagonists in the renal circulation. Am. J.
159. Yu M, Cambj-Sapunar L, Kehl F, Maier
KG, Takeuchi K, et al. 2004. Effects of
a 20-HETE antagonist and agonists on
cerebral vascular tone. Eur. J. Pharmacol.
486:297306
160. Frisbee JC, Roman RJ, Krishna UM,
Falck JR, Lombard JH. 2001. Relative
contributions of cyclooxygenase- and cytochrome P450 -hydroxylase-dependent
pathways to hypoxic dilation of skeletal
muscle resistance arteries. J. Vasc. Res.
38:30514
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Copyright
CYTOCHROME P450 UBIQUITINATION:

Branding for the Proteolytic Slaughter?

Maria Almira Correia, Sheila Sadeghi,
and Eduardo Mundo-Paredes
Department of Cellular and Molecular Pharmacology, University of California,
San Francisco, California 94143-0450; email: mariac@itsa.ucsf.edu,
sadeghi@itsa.ucsf.edu, eduardo@itsa.ucsf.edu
Key Words ERAD, 26S proteasome, Ubc7p, HRD/DER, CYP3A4, CYP2C11

Abstract The hepatic cytochromes P450 (P450s) are monotopic endoplasmic
reticulum (ER)-anchored hemoproteins engaged in the enzymatic oxidation of a wide
variety of endo- and xenobiotics. In the course of these reactions, the enzymes generate reactive O2 species and/or reactive metabolic products that can attack the P450
heme and/or protein moiety and structurally and functionally damage the enzyme. The
in vivo conformational unraveling of such a structurally damaged P450 signals its rapid
removal via the cellular sanitation system responsible for the proteolytic disposal of
structurally aberrant, abnormal, and/or otherwise malformed proteins. A key player
in this process is the ubiquitin (Ub)-dependent 26S proteasome system. Accordingly,
the structurally deformed P450 protein is first branded for recognition and proteolytic
removal by the 26S proteasome with an enzymatically incorporated polyUb tag. P450s
of the 3A subfamily such as the major human liver enzyme CYP3A4 are notorious
targets for this process, and they represent excellent prototypes for the understanding
of integral ER protein ubiquitination. Not all the participants in hepatic CYP3A ubiquitination and subsequent proteolytic degradation have been identified. The following
discussion thus addresses the various known and plausible events and/or cellular participants involved in this multienzymatic P450 ubiquitination cascade, on the basis of
our current knowledge of other eukaryotic models. In addition, because the detection of
ubiquitinated P450s is technically challenging, the critical importance of appropriate
methodology is also discussed.
INTRODUCTION
Ubiquitination (or ubiquitylation as it is now often called) is a process in which cellular proteins are covalently modified, posttranslationally, with a single molecule
(monoubiquitination) or chains (polyubiquitination) of ubiquitin (Ub) (1 and references therein). Ub is an evolutionarily highly conserved 76-residue polypeptide
(8565 Da) that, as implied by its name, is ubiquitously present in all eukaryotic cells either as free species (monomers or in preformed chains) or covalently
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bound to proteins. Such covalent protein decoration with Ub serves many important physiological functions. Monoubiquitination can serve as a sorting signal
in endocytic vesicular transport as well as a critical regulator of transcription,
replication, and DNA repair (2 and references therein). In contrast, polyubiquitination largely targets proteins for degradation via the 26S proteasome, thereby
critically regulating various essential cellular processes such as cell cycle progression, antigen presentation, apoptosis and stress response, in addition to the vital
function of quality control by cellular disposal of aberrant, misfolded, damaged,
and/or abnormal proteins (1, 310). Indeed, in the latter context, high molecular mass (HMM) ubiquitinated species of certain (but not all) cytochromes P450
(P450s) have been detected in liver cells after structural damage and/or blockade
of their normal physiological turnover by proteasomal inhibitors (11, 12). The
HMM profile of this P450 ubiquitination and the striking temporal relationship
between its detection and P450 proteasomal degradation are consistent with a role
for polyubiquitination as a targeting signal in this process. Although a biological
role for monoubiquitination in P450 regulation may exist, it remains obscure. Furthermore, although monoubiquitination of the various multiple P450 surface Lys
residues could yield a HMM profile similar to that generated by polyubiquitination, its plausibility has been excluded by our in vitro studies with methylated Ub
(MeUb) (13), the Ub analog incapable of polyubiquitination because of chemical
methylation of its Lys residues. For these combined reasons, this review will focus
on what is currently known about P450 polyubiquitination and its association with
the proteasomal destruction of these enzymes.
THE CELLULAR POLYUBIQUITINATION MACHINERY

Protein polyubiquitination entails the covalent attachment of a chain of multiple
(>4) Ub molecules most often to a Lys-NH2 and, albeit much less frequently, to
an -NH2 terminus of a proteolytic substrate through the C-terminal Gly76 residue
of the first Ub molecule in a concerted ATP-dependent process (1, 310; Figure 1).
Three distinct classes of enzymes operate sequentially to catalyze this coupling (1,
310). The first is the Ub-activating enzyme (E1), a 100-kDa protein abundantly
present in the cytosol and nuclei of eukaryotic cells. E1 contains the nucleotide
binding consensus sequence Gly-X-Gly-XX-Gly (1, 4). Although comparative
analysis of cDNA-derived amino acid sequences of plant, yeast, and mammalian
E1s reveals five conserved Cys residues, site-directed mutagenesis studies of the
wheat E1 isoform UBA1 reveal that only one of these (Cys626) is essential for its
activity (14). This Cys residue is believed to reside at the E1-active site and in the
presence of ATP, to be directly involved in Ub activation to a high energy ternary
Ub-thioester complex (E1-S-C = OUb) via linkage to Ub-Gly76. Although Ub is
thus activated, it cannot be transferred directly onto the proteolytic target without
the intermediacy of a second enzyme (E2), a member of a family of multiple Ubconjugating enzymes (Ubcs)/Ub-carriers, as well as a third enzyme, Ub-protein
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ligase (E3). Such sequential shuttling of the Ub-thioester from E1 to the target
protein entails an initial transthiolation of an E2-Cys residue, with or without a
subsequent similar transthiolation of an E3-Cys residue. This Ub relay via E2 and
E3 in this process apparently insures substrate specificity by preventing the attack
of random proteins by the E1 charged molecule.
E2s differ in size, usually with low molecular weights ranging between 14
and 36 kDa1, and containing a 1416 kDa core that is 35% conserved among
family members (69). The remainder of the protein may contain N- and/or Cterminal extensions that confer substrate and/or E3 specificity or promote physical
interactions between the three entities, often by serving as membrane anchors. Both
ER-bound and soluble E2s exist for the ubiquitination of luminal and integral
endoplasmic reticulum (ER) proteins such as the P450s (9, 15, 16; see below).
This E2 multiplicity apparently insures functional redundancy on one hand, and
substrate specificity on the other (9).
The E3 Ub-ligases, considerably more numerous than E2s, often exist as monomeric proteins or heteromeric multisubunit protein complexes. The multiplicity
and structural diversity of E3s contribute to their remarkable substrate diversity
and/or specificity in the recognition of proteolytic targets (9, 1721). Three general
classes of E3s are known: the HECT-E3s (Homologous to E6-AP C-terminus, the
first HECT-E3 identified), CHIP-E3s (C-terminus of Hsc70 interacting protein),
and the RING-finger E3s. The same E2 can apparently interact with either the
HECT or the RING-finger domain of an E3 (9). HECT-E3s contain a conserved
Cys-SH in their C-terminal domain for Ub-thioester relay from its cognate E2 to
the target protein (9). The N terminus of some but not all HECT-E3s contains a
WW domain (with 2 Trps, 2022 residues apart and an invariant Pro within a 40residue region) that interacts with Pro-rich sequences including those containing
phosphorylated Ser/Thr residues (9, 22).
The first CHIP-E3 prototype was identified as a cochaperone of Hsc70. A typical
CHIP-E3 contains a tetratricopeptide repeat (TPR) motif that interacts with both
Hsc70 and Hsp90; its U-box domain exhibits E3 Ub-ligase activity. CHIP-E3s
play an active role in quality control through recognition of chaperone-associated
aberrant proteins that are ubiquitinated before their removal by the proteasome
(17, 18). These E3s may play a similar role in the Ub-dependent proteasomal
degradation of unfolded and/or misfolded P450 proteins.
The increasingly numerous RING-finger E3s, on the other hand, exhibit an
interleaved or cross-braced ring pattern with eight conserved metal-binding Cys
and His residues that coordinate two Zn atoms (9, 1921). The three RING-finger
motifs (RING-CH, RING-HC, and RING-H2) are distinguished by whether one
or two His are the middle two conserved residues. These E3s may exist as single
subunits with both substrate recognition and RING-finger E2 docking domains
on the same polypeptide or as multisubunit protein scaffolds that include a small
1
Although Ubcs are known to possess low molecular weights, a larger (582 kDa) polytopic
membrane-anchored Ubc (BRUCE) has been reported (9).
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RING-finger protein for E2 docking. Additional known components of these E3

scaffolds include a member of the cullin family, an F-box protein for the recognition
of phosphorylated substrates (i.e., phosphorylated IB, Sic1, and -catenin), and
other protein subunits as intercomplex adapters (9, 23). By docking E2s, RINGfinger E3s can facilitate the transfer of the activated Ub onto one of its own subunits
in a regulated autoubiquitination process, or onto that of a heterologous substrate
as in the case of the yeast RING-H2 finger E3 complex (Hrd1p/Hrd3p)-mediated
ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; 24, 25; see
below). Thus, unlike HECT-E3s, RING-finger E3s mediate substrate ubiquitination
by bringing the E2s in sufficiently close proximity to the protein target rather than
by directly participating as an intermediary in the Ub-thioester relay.
Such E1/E2/E3-mediated coupling of Ub to one (or more) Lys residue(s) of
target proteins entails channeling of the high energy of Ub-thioester hydrolysis
into isopeptide bond formation. Similar linkage of the internal Lys48 of this Ub
to the C-terminal Gly76 of a fresh Ub molecule results in an ordered branched
chain (Ub-Ub homopolymer; PolyUb) wherein Gly76-COOH of one is coupled to
the Lys48-NH2 of another Ub. It remains to be elucidated whether the formation
of Ub-Ub chains involves an identical sequence of events as that involved in attaching the first Ub molecule to target substrate Lys-NH2 groups. Nevertheless,
such polyubiquitination consisting of 4 to 20 Ub molecules in Lys48-Gly76-linkage
not only gives the targeted protein its characteristic step-ladder appearance on
SDS-PAGE/immunoblotting2 but is also essential for targeting it to the 26S proteasome for degradation (3, 26, 27 and references therein; Figures 1 and 2). The
Ub molecule has at the least seven conserved Lys residues, and Ub-Ub linkages
of proteins via Lys6, Lys11, Lys29, Lys48 and Lys63 have been identified (26, 27).
Of these, only Lys48 linkages are known to function as proteolytic signals (3, 26,
27).
THE PROTEASOMAL SYSTEM FOR THE DEGRADATION

OF POLYUBIQUITINATED PROTEINS
The 26S proteasome is a very large, highly complex 2000 kDa multisubunit
chambered protease (2733). Its key component is a 20S proteolytic core
(750 kDa) consisting of 28 subunits arranged in four rings (2 and 2) each
containing seven similar but functionally distinct subunits, stacked together with
an outer -ring flanking the two inner -rings to form a pseudosymmetrical barrel
with a cylindrical cavity (27, 3033). The function and assembly of the eukaryotic
20S proteasome (previously known as the multicatalytic protease complex) is ATPindependent. Three of the -subunits (1, 2 and 5) harbor the protease catalytic
2
Frequently, this is detected as a smear rather than a distinct step-ladder. However, in the
case of P450s it is important to distinguish between the essential features of ubiquitination
and protein aggregation.
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sites with a total of six proteolytic sites/20S species (27, 3033). Each of these
three (1, 2, and 5) sites differs in its preferential cleavage after acidic, basic,
or hydrophobic residues, respectively, giving rise to the typical chymotrypsinlike, trypsin-like, peptidoglutamyl hydrolase, and caseinolytic activities of the
proteasome (34). These catalytic -subunits are unusual in exhibiting a unique
catalytically active N-terminal Thr residue, a critical nucleophile in peptide bond
hydrolysis, strategically lining the barrel cavity. This unusual characteristic qualifies the 20S proteasome as an amino-terminal nucleophile (NTN) hydrolase. The
active site Thr residue is the target of selectively designed proteasomal inhibitors
such as MG-132, lactacystin -lactone, epoxomycin, and others (3537).
In the eukaryotic 26S complex, either (or both) of the 20S -ring structures
is capped by a 900 kDa multisubunit complex variably termed PA700 (proteasome activator) or the 19S regulatory complex, an assembly that is ATP-dependent
(27, 3841). Thus the 26S proteasome may exist as heterooligomeric 20S barrels
capped at one or both ends with this 19S complex. The 19S cap complex is composed of 17 or more subunits arranged in two structurally and functionally distinct
assemblies: The base situated directly above the 20S -ring contains six functionally distinct AAA-family ATPase or Rpt (regulatory particle ATPase) subunits and
two non-ATPase Rpn (regulatory particle non-ATPase) subunits. The 19S base is
topped by a lid containing eight non-ATPase Rpn subunits. In the mammalian 26S
proteasome, these two structures are further linked together by the seventeenth subunit, Rpn10. High salt can dissociate the lid from the base-bound 20S proteasome,
leaving a catalytic species that is capable of ATP-dependent degradation of an
unfolded protein but incapable of degrading ubiquitinated substrates. This finding
implies that the lid contains polyUb-recognition elements and unfoldases in addition to deubiquitinating enzymes. More recently, additional proteasome-associated
proteins (including E3s) have been identified with the 19S complex, although it
is unclear whether they are adventitiously bound or bona fide lid components.
Together the multiple subunits of the 19S regulatory complex are responsible for
the initial acceptance of the polyUb-tagged target substrates as well as the coordination of the subsequent release of this polyUb-tag with their high energy-coupled
unfolding and translocation through the 20S proteolytic core to be digested (27
34, 3841). Accordingly, the polyUb tag is initially recognized by the Rpt5 and/or
Rpn10 (or any other) of the 19S base subunits, thereby tethering the substrate to
the 19S subcomplex and bringing its termini or any other loosely folded domain in
close proximity to one or more of the 19S base Rpt ATPase subunits for unfolding
and translocation of the unfolded substrate through the 19S base pore. This event,
requiring ATP hydrolysis, denatures the substrate to enable its onward movement
through the juxtaposed 20S -subunit pore into the adjacent 20S catalytic chamber where it is processively digested into short peptides that exit through the distal
20S axial pore. As the entire polyubiquitinated substrate is unfolded and strung
through the 20S catalytic chamber to be proteolyzed, its polyUb tag is released
intact by hydrolyses of the UbGly76-NH2 substrate isopeptide bond by the 19S lid
subunits Rpn11, a deubiquitinase with a Zn-metalloprotease-like domain (27, 42,
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43), and Ubp6, another 19S deubiquitinase. Additional 19S subunits containing
Ub-hydrolases, the cysteine proteases that disassemble the Lys48-linked polyUb
chain, may be subsequently recruited to regenerate Ub for fresh proteolytic cycles
(2730).
The enmeshed 20S -subunit N termini normally clog the two gates to the
adjacent 20S proteolytic -subunit chamber, keeping its pores closed and inaccessible to peptides and unfolded proteins and therefore catalytically inactive. Thus
an additional critical function of the 19S base ATPase subunits is to activate the
20S proteolytic chamber by physically lifting these -subunit tails to open its gate
pore diameter that is accessible to unfolded proteins and polypepto a 20 A
tides but not to normally folded proteins (27, 44, 45). Such restrictive gating thus
protects native cellular proteins from promiscuous/indiscriminate proteasomal attack. Higher eukaryotes also contain other hybrid proteasome species, including
the immunoproteasomes, that are specifically engaged in antigen processing for
presentation to the immune system on major histocompatibility complex (MHC)
class I molecules (35, 4649). Because antigenic peptides from several P450 enzymes have been reported (5053), these proteasome species are relevant to the
current discussion. Unlike the above described constitutive 20S species of the 26S
proteasome, that of the immunoproteasome contains interferon- inducible 1, 2
and 5 proteolytic subunits responsible for generating antigenic peptides (27, 49).
This 20S immunoproteasome species may be capped on either or both ends by the
11S activator complex (also known as the PA28 activator, which consists of two
alternating non-ATPase subunits, PA28 and PA28, in a concentric heptameric
complex of 200 kDa) (27, 49), which modulates the proteasome-catalyzed generation of antigenic peptides. The PA28 activator apparently activates the 20S
proteolytic species by regulating the gating into the 20S chamber in a mechanism
analogous to that of the 19S complex (27, 49).
POLYUBIQUITIN AS A 26S PROTEASOME

TARGETING SIGNAL
A multitude of structurally diverse proteins incur 26S proteasomal degradation
in a seemingly nonspecific process. The only structural feature that qualified 26S
proteasomal substrates [with the exception of ornithine decarboxylase (54, 55) and
p21Cip1 (56)] exhibit in common is a Lys48-Gly76-linked polyUb chain attached to
an -NH2 group of a Lys-residue (and less frequently to an -NH2) of the protein
target. Although preferential Lys residues exist for ubiquitination, in their absence
alternative Lys residues can substitute in some instances. Such protein tagging as
discussed above, albeit insufficient for degradation, is essential for its recognition
as a proteasomal substrate. And as long as this polyUb tag remains both viable as a
recognition signal and latched firmly onto the 19S cap to induce protein unfolding
and translocation into the proteolytic chamber, the ubiquitinated substrate, whether
monomeric or a subunit of a heteromeric complex, is irrevocably committed to 26S
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proteasomal degradation (Figure 2). Any specific substrate sorting for proteolysis
thus has to occur before the protein is polyubiquitinated.
Indeed, such specific substrate sorting does occur and E3s apparently play a
critical role in the selection of a substrate for Ub-dependent 26S proteasomal
degradation through the recognition of specific substrate-based structural determinants that partly or fully constitute destruction signals called degrons (9, 27). Such
critical structural determinants of substrate recognition by E3s may be intrinsic to
the proteins primary sequence or acquired through posttranslational processing.
Degrons encoded in a short discrete sequence include the Deg1 sequence of the
yeast MAT2 transcriptional regulator, the destruction box of mitotic cyclins, the
degradation motif of IB proteins, the stability regulating region of cMOS, specific
phosphorylatable Ser/Thr residues, PEST sequences, Pro-rich domains, and some
N-terminal residues of target proteins (9, 27; reviewed in Reference 57). On the
other hand, instead of a discrete modular degron, the structural information for
Ub-dependent 26S proteasomal degradation may be distributed over a considerably large protein domain, as in the case of the entire N-terminal 523-residue-long
transmembrane domain of HMGR (58, 59) as well as the IB N-terminal phosphorylatable (Ser32/Ser36) and ubiquitinatable (Lys21/Lys22) and C-terminal PEST
domains (6062). Although certain P450s are indeed phosphorylated (13, 6369)
and/or ubiquitinated (13, 69) before their proteolytic degradation, it remains to
be determined whether they similarly harbor any intrinsic modular or distributed
degrons that are either normally accessible or unmasked for this event.
HEPATIC P450 UBIQUITINATION

The first clue that P450s might be subject to Ub-dependent 26S proteasomal degradation was provided by two independent 1992 reports (11, 70). In the first, immunoblotting analyses of liver microsomes revealed the strikingly enhanced formation of HMM Ub-conjugated liver microsomal proteins within 30 min of CCl4
administration to mice compared with that seen in vehicle-treated controls (70).
This HMM microsomal ubiquitination profile was only slightly subdued at 1 h after
CCl4, but markedly attenuated after 5 h. This profile correlated well with the timedependent immunochemically and functionally detectable proteolytic loss of microsomal CYP2E1, a known target of CCl4-mediated inactivation. CCl4 is known
to inactivate CYP2E1 in a mechanism-based process that results in heme fragmentation and irreversible modification of the P450 protein by the ensuing heme
fragments (71), presumably at the active site. The close temporal association of the
two profiles led to the proposal that the observed HMM ubiquitinated microsomal
protein included CYP2E1 species, although no HMM anti-CYP2E1 immunoreactivity was concomitantly detected (70). The failure to detect any immunoreactive
HMM CYP2E1 species was rationalized by the low abundance of HMM microsomal CYP2E1 species and/or plausible masking of its epitopes by Ub-conjugation
(see below). It is relevant to note that the parallel microsomal CYP2E1 inactivation by the pan-P450 mechanism-based inactivator, 1-aminobenzotriazole (ABT),
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yielded no comparable proteolytic loss of the enzyme nor a similar HMM ubiquitination profile (70). ABT inactivates most P450s via heme-N-arylation rather
than heme-modification of the protein (72). However, although ABT rapidly and
effectively abolished CYP2E1 function, minimal CYP2E1 proteolysis was observed along with relatively minor ubiquitination of the microsomal protein detectable only after 9 h of ABT treatment (70). Thus, despite marked P450 functional loss, the protein moiety apparently escapes unscathed and remains relatively
stable.
The second report documented the time-dependent ubiquitination and proteolytic loss of rat liver microsomal CYPs 3A after their mechanism-based inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), a
4-ethyl analog of the calcium channel antagonist nitrendipine (11, 73, 74). DDEPinduced CYP3A inactivation also results in heme-modification of the protein that
is accompanied within 30 min by ubiquitination of the microsomal protein and
immunochemically detectable loss of these microsomal enzymes (11, 73, 74).
This ubiquitination is, as expected, enhanced by the coadministration of hemin,
a known 26S proteasome inhibitor (7577; see below). More importantly, when
the microsomal CYPs 3A were first immunoprecipitated with goat anti-CYP3A23
IgGs and then immunoblotted with rabbit anti-Ub IgGs, they exhibited the characteristic step-ladder ubiquitination profile discussed earlier (11; Figure 3a). This
finding unequivocally established that the DDEP-inactivated CYPs 3A were indeed ubiquitinated. This DDEP-induced ubiquitination of liver microsomal CYPs
3A in intact rats could be reproduced in DDEP-incubations of freshly isolated hepatocytes obtained from dexamethasone (DEX)-pretreated rats (12). This system
provided a convenient experimental model for the definitive mechanistic characterization of the proteolytic process. Accordingly, CYP3A immunoprecipitation
analyses revealed that incubation of these freshly isolated hepatocytes with DDEP
resulted in the ubiquitination of CYPs 3A within 15 min of their inactivation, an
event that preceded the onset of their proteolytic degradation detectable at 30 min
(12). Inclusion of the proteasomal inhibitors aclarubicin or MG-132 in these incubations, while blocking the DDEP-induced immunochemically detectable loss
of microsomal CYPs 3A, also intensified the CYP3A ubiquitination profile (12).
These findings in freshly isolated rat hepatocytes thus unequivocally established
that DDEP-inactivated, heme-modified CYPs 3A undergo Ub-dependent 26S proteasomal degradation (12).
Both ubiquitination and 26S proteasomal degradation of heme-modified CYPs
3A can also be documented in in vitro reconstituted systems (13) containing purified recombinant CYP3A4, the major human liver CYP3A ortholog. For this
purpose, 35S-labeled CYP3A4 either native or heme-modified by inactivation with
cumene hydroperoxide (CuOOH) and Fraction II (a rat liver cytosolic subfraction
containing the requisite soluble E1, E2 and E3 enzymes and the 26S proteasome)
were incubated in the presence of Ub, an ATP-generating system, protease inhibitors (to block the ubiquitous lysosomal protease contaminants), MgCl2, and
Ub-aldehyde (Ubal). The inclusion of Ubal, an inhibitor of Ub-hydrolases and
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isopeptidases, in these incubations is essential for blocking protein deubiquitination so that Ub-conjugation of a substrate can be detected. In the absence of other
proteins, thermally sensitive CYP3A4 tends to aggregate on incubation at 37 C,
thereby generating undesirable and confounding cross-linking artifacts (see below). To preclude such incubation-induced aggregation artifacts, liver microsomal
membranes from female rats that are devoid of appreciable CYP3A content and
exhaustively washed free of luminally trapped cytosolic ubiquitinating and proteolytic enzyme contaminants were included as a membrane platform for CYP3A4.3
Parallel immunoblotting analyses of these incubates with either anti-CYP3A or
anti-Ub IgGs revealed that only the heme-modified but not the native CYP3A4
exhibited a time-dependent ubiquitination profile which was enhanced by the inclusion of the proteasome inhibitor, Z-IE(OtBu)ALCHO (PSI) (78). No CYP3A4
ubiquitination profile was detected if Fraction II was omitted from the incubations (13). Moreover, no similar profile was observed when Ub was replaced by
MeUb (the methylated Ub-analog incapable of any Lys48-Gly76 polyubiquitination
linkages; see above) in the incubation (13). If appreciable CYP3A4 aggregation
were to have occurred, it should have been detected in the incubations with MeUb.
The absolute dependence of the observed CYP3A4 ubiquitination profile on both
Fraction II and Ub convincingly attests to its authenticity, while excluding the recently raised possibility that this finding represents a CYP3A4 aggregation artifact
(79, 80).
Two native rat liver P450s, CYP2B1 and CYP2C11, that have relatively long
half-lives and reportedly are degraded by the lysosomal pathway in vivo, are also
subject to Ub-dependent 26S proteasomal degradation when suicidally inactivated
(13, 57, 81). Accordingly, in an in vitro reconstituted system similar to the one
described above, CuOOH-inactivated heme-modified CYP2B1 was shown to be
ubiquitinated and degraded by the 26S proteasome-species (13). On the other
hand, DDEP incubation of freshly isolated hepatocytes from untreated male rats
also caused a time-dependent structural and functional inactivation of CYP2C11
that is associated with CYP2C11 protein ubiquitination and proteolytic loss (Z.J. Song & M.A. Correia, unpublished observations; 81). Although such DDEPinduced CYP2C11 inactivation is largely due to its prosthetic heme destruction
to an N-ethylporphyrin, with little or no heme modification of the protein, no
structural or functional restoration of the enzyme was observed when hemin was
included in the incubations (81). Instead, as revealed by CYP2C11 immunoprecipitation analyses, inclusion of hemin in the DDEP-incubations resulted in an
accumulation of ubiquitinated CYP2C11 species, consistent with hemin blockade of proteasomal function at a step beyond protein ubiquitination (Figure 3e).
Unlike the recent findings in primary hepatocytes in culture (79), incubation of
3
In retrospect, this strategy for preventing CYP3A4 aggregation was fortuitous, given that
ERAD substrates such as CYP3A4 apparently require integral and ER-associated enzyme
components for their ubiquitination. The inclusion of ER membranes inadvertently provided
the ER ubiquitination components later found to be essential (16, 113).
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hemin alone with otherwise untreated hepatocytes yielded no detectable P450

aggregation/cross-linking HMM artifacts (Figure 3e). CYP2C11 inactivation by
DDEP is mechanistically similar to that of CYP2E1 by ABT in that the heme moiety of both isoforms is N-modified by the inactivators. Yet, albeit intriguing, it is
unclear why the heme-stripped CYP2C11 is a target for ubiquitination whereas the
heme-stripped CYP2E1 is not. It is conceivable that this CYP2C11 susceptibility
to the ubiquitination machinery is determined by discrete modular degrons in its
protein sequence that are unmasked on prosthetic heme destruction.
Finally, CYP2E1 Ub-conjugation has been documented in two in vitro systems
(82, 83), which to our knowledge, are the only reported sightings of ubiquitinated
CYP2E1. The first used CYP2E1-enriched rat liver microsomes incubated at 37 C
for 1 h with untreated rat liver cytosol as the ubiquitination/proteolytic system
supplemented with leupeptin, aprotinin, 2-macroglobulin, MgCl2, ATP, NADPH,
and with or without CCl4 (20 mM) as the CYP2E1 suicide inactivator. Microsomes
were reisolated and probed by immunoblotting with either anti-CYP2E1 or anti-Ub
antibodies. These findings revealed that immunoreactive HMM CYP2E1 species
and Ub-conjugates were detected, particularly after CCl4-inactivation of CYP2E1
and to a lesser extent in its absence (82). However, several methodological concerns
render these findings somewhat unconvincing. The first is that no attempt was made
to isolate CYP2E1 by immunoprecipitation for verification that it was truly ubiquitinated. As previously indicated (70), the HMM ubiquitinated species detected
in microsomal immunoblots would reflect Ub-conjugates of myriad microsomal
proteins including CYP2E1. Second, because CCl4 is a notorious inducer of lipid
peroxidation and no attempt was made to block NADPH/CCl4-induced microsomal lipid peroxidation, P450 cross-linking with itself and/or other Ub-conjugated
microsomal proteins could also account for the HMM protein species as previously
reported (84), and recently confirmed with CYPs 3A (80). The latter possibility is
particularly likely given that the functionally robust hepatic deubiquitinases were
not blocked in these studies (82), a sine qua non for detectable hepatic protein
ubiquitination. Third, the principal author was unable to confirm these findings
in a subsequent report (85), invoking instead a major role for the Ub-independent
20S proteasomal species in CYP2E1 degradation.
The second report describes 35S-CYP2E1 ubiquitination after its translation
from in vitro transcribed RNA in a rabbit cell-free reticulocyte lysate translation/
ubiquitination system (83). This CYP2E1 ubiquitination was apparently enhanced
by the inclusion of the proteasome inhibitor MG-132 in this system. Through immunoinhibition and modeling studies, this ubiquitination was postulated to occur
on Lys317 and Lys324 residues in the putative cytosol-exposed CYP2E1 J-helix-J loop domain (83). No comparable CYP2E1 ubiquitination was observed when a
wheat germ lysate translation system that lacks the ubiquitination machinery was
employed. The in vitro ubiquitination studies of newly translated CYP2E1 reveal
that the protein is ubiquitinated on two of its cytosol-exposed Lys residues (83; see
above). However, even though a large protein domain is presumably exposed to the
cytosol, only minimal ubiquitination of native hepatic CYPs 3A is normally
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detected in vivo, even under conditions optimized for its maximal detection
(Figure 3a,c; see below). This ubiquitination is considerably augmented after
the protein is structurally unraveled by catalytic insults such as futile oxidative
cycling and/or chemically induced suicide inactivation. Such structural damage
may expose normally concealed Lys residues and/or other degron components
such as phosphorylatable residue PEST sequences and other targetable domains
to the ubiquitinating enzymes. Together, the above findings attest to the indisputable fact that when inactivated, misfolded, or otherwise structurally deformed,
certain hepatic P450s incur ubiquitination and/or 26S proteasomal degradation.
But are the native CYPs 3A also ubiquitinated in the course of their physiological
turnover?
NATIVE P450 UBIQUITINATION: THE CELLULAR LOCUS,

PARTICIPANTS, AND LESSONS FROM THE YEAST
Hepatic P450s are monotopic proteins, N-terminally anchored to the ER with their
catalytic domain facing the cytosol wherein a substantial cellular inventory of the
ubiquitination machinery and/or the 26S proteasome are located. As ER residents
and documented substrates of Ub-dependent 26S proteasomal degradation, CYPs
3A qualify as bona fide models for the mechanistic characterization of hepatic
ER-associated degradation (ERAD). In analogy to the ERAD of other cellular
proteins (8688), ER-associated P450 ubiquitination in hepatocytes most likely
involves hepatic ER-associated Ub-conjugating E2 enzymes and E3 Ub-ligases,
whose specific identities remain to be divulged. However, the dilemma was in
identifying a suitable experimental model wherein this physiological process could
be mechanistically dissected without confounding inherent artifacts. As discussed
previously (57), P450s are not very stable and turn over rapidly in cell-lines or
when hepatocytes are cultured (57, 8992). This led to the serious consideration
of yeast as a model.
Until very recently, most of our knowledge of the ERAD of integral and luminal
proteins (8688) was derived from genetic analyses of the yeast Saccharomyces
cerevisiae. Studies of the polytopic ER protein Hmg2p (the sterol-regulated yeast
isoform of HMGR, the rate-limiting enzyme in sterol synthesis) and of CPY
(a misfolded carboxypeptidase mutant retained in the ER-lumen) have uncovered
HRD (HMGR Degradation) and DER (Degradation in ER) genes, respectively (86
88). This HRD/DER machinery includes (a) the ER-associated Ub-conjugating
enzymes (Ubc1p, Ubc6p, and Ubc7p). Ubc6p and Ubc7p are key enzymes in the
ERAD of luminal and membrane-bound proteins in yeast proteins (8688, 93
98). Ubc6p is an integral C-terminally anchored ER-protein with its N-terminal
catalytic domain exposed to the cytosol, whereas Ubc7p is a cytosolic protein that
requires an integral membrane-anchored partner Cue1p for its catalytic participation in ERAD. Both Ubc6p and Ubc7p/Cue1p are required for the ubiquitination
of certain polytopic ER-proteins (15, 9398) except HMGR which requires only
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Ubc7p/Cue1p but not Ubc6p (93, 97, 98). The HRD/DER machinery also includes
(b) Hrd2p, a 19S protein subunit that is a functionally indispensable component of
the 26S proteasome (98), and (c) Hrd1p/Hrd3p complex, the ER-associated Ubligase (E3) composed of Hrd1p/Der3p and its partner, Hrd3p. Hrd1p is an integral
ER-membrane protein with two distinct domains: a multitransmembrane-spanning
N-terminal hydrophobic region and a cytosolic C-terminal hydrophilic RING-H2
motif (24, 25, 99, 100) that binds Ubc1p or Ubc7p. Hrd3p is an ER glycoprotein with a single C-terminal membrane-anchor and a large N-terminal domain
in the ER-lumen. Hrd3p is found to stabilize Hrd1p in the ER membrane (24).
The Hrd1p/Hrd3p complex catalyzes the Ubc7p-dependent ubiquitination of target substrates such as Hmg2p and CPY (24, 99101). The HRD/DER machinery
also includes Cdc48p (p97, an AAA ATPase required for cellular processes such
as cell division, protein degradation, and ER membrane fusion), Npl4/Hrd4p, an
ER-specific adapter of undefined function, and Ufd1p, a Cdc48p protein adapter
for polyUb chain recognition and/or Cdc48p-association with Hrd4p (102104).
The Cdc48p-Ufd1p-Hrd4p complex is apparently involved in the recognition of
polyubiquitinated luminal and integral ER proteins, their dislocation from the ER,
and their subsequent delivery to the 26S proteasome. Mammalian homologs of
the yeast HRD/DER machinery such as Ubc6p, Ubc7p, Cue1p, Hrd2p, Hrd1p
Ufd1p, Hrd4p and Cdc48p have been recently documented (102112), indicating
that ERAD is evolutionarily a highly conserved process.
Because of this high homology between yeast and mammalian ubiquitination
enzymes and the availability of validated genetic S. cerevisiae strains with defined
defects in the ubiquitination and proteasomal degradation of several integral ER
proteins including Hmg2p, the yeast model was used to identify and characterize
the enzymes participating in the ubiquitination of CYP3A4, the dominant human
liver P450 (113, 114). To identify the Ubc involved in CYP3A4 ubiquitination,
isogenic wild-type (wt) yeast strains and strains deficient in Ubc6p, Ubc7p, or
in both Ubc6p and Ubc7p were transformed with the CYP3A4 expression vector
pAAH5/NF25 or the control vector (113). At the early stages of logarithmic cell
growth, CYP3A4 was equivalently expressed in all four strains, indicating their
comparable transcriptional and translational efficiencies (113). At the later stages
of culture, CYP3A4 was greatly stabilized only in mutants deficient in Ubc7p and
Ubc6p/Ubc7p but not in Ubc6p alone, thereby revealing the relative importance
of Ubc7p-dependent ubiquitination in the ERAD of this native integral protein.
Thus the monotopic CYP3A4 and the polytopic Hmg2p are alike in that they
require Ubc7p-dependent ubiquitination for their ERAD. Presumably, such Ubc7pmediated CYP3A4 ubiquitination also requires the ER-protein adapter Cue1p.
To determine whether the RING-H2 Hrd1p/Hrd3p Ub-ligase complex and
Hrd2p involved in Hmg2p Ub-dependent proteasomal degradation were similarly
involved in that of CYP3A4, isogenic wt and mutant hrd1, hrd2-1, and hrd3 S.
cerevisiae strains were transformed as discussed above with a CYP3A4 expression
plasmid and corresponding vector control. CYP3A4 protein was equivalently expressed in all four yeast strains during the early logarithmic growth phase, but at the
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later stationary growth stage of culture, CYP3A4 protein was comparably reduced
in wt and hrd1-deficient yeast, consistent with its degradation predominating over
its de novo synthesis after transient expression of both strains (113). In contrast,
consistent with the role of 26S proteasome in CYP3A4 degradation, the microsomal CYP3A4 protein content was significantly stabilized in hrd2-deficient yeast.
These findings thus reveal that in yeast, the RING-H2 finger Ub-ligase Hrd1p
is not required for CYP3A4 Ub-dependent proteasomal degradation in contrast
to that of Hmg2p (113). It is unclear whether the lesser, albeit statistically significant, CYP3A4 protein stabilization observed in hrd3-deficient yeast reflects
the interaction of Hrd3p with an Ub-ligase partner other than Hrd1p. The yeast
Ub-ligase required for CYP3A4 ubiquitination currently remains to be identified. Any of the several ERAD-associated E3 ligases such as the ER-localized
yeast RING-CH Doa-10 or HECT-like Rsp5p (115, 116) remain plausible E3
candidates in CYP3A4 ubiquitination. Similarly, the recently identified human
Hrd1p homolog HRD1 (110) or its related mammalian E3 homolog gp78/AMFR
(autocrine motility factor receptor; 111) could be involved in CYP3A4 ubiquitination in the human liver. Because all these E3 ligases duly engage Ubc7p or its human
counterpart UBC7 in their CYP3A4 ubiquitination reactions, their E3 candidacy is
plausible.
It is noteworthy, however, that the rapid disposal of human liver CYP3A4 via
the Ub-dependent 26S proteasomal degradation in S. cerevisiae is not because
it is an alien protein. Corresponding expression of other mammalian P450s (rat
liver CYPs 2B1 and 2C11) in these same yeast strains (114, 117) results in their
vacuolar lysosomal degradation rather than proteasomal degradation. The latter
findings are not only consistent with similar observations in intact rats (57 and
references therein), but also confirm the validity of the yeast model for mammalian
P450 turnover analyses.
P450 UBIQUITINATION: THE TRAJECTORY TO

PROTEASOMAL DEGRADATION
CYP3A immunoprecipitation analyses of ER (microsomal) and cytosolic subfractions over the time course of their DDEP inactivation in isolated hepatocytes
revealed that the 35S-labeled CYPs 3A initially present in the ER were translocated into the cytosol as their proteasomal degradation progressed. Treatment
with the proteasomal inhibitor aclarubicin resulted not only in decreased CYP3A
proteolysis but also in the impaired translocation of the ubiquitinated ER-bound
CYP3A species into the cytosol (12). Consequently, ubiquitinated CYP3A species
accumulated in the ER, consistent with aclarubicin blockade at a step beyond the
ubiquitination of these enzymes. These findings thus indicate that the ERAD of
DDEP-inactivated CYPs 3A entails their initial polyubiquitination while still incorporated in the ER, with subsequent dislocation from the ER membrane and
translocation into the cytosol (12). These results also strongly suggest that the
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cytosolic rather than the ER-associated 26S proteasome species is involved in

CYP3A ERAD. Furthermore, given that the inhibition of the proteasome blocked
CYP3A dislocation from the ER, it appears that a functional proteasome species
is required for this event. How exactly the ubiquitinated P450 is delivered from
the ER to the cytosolic 26S proteasome is an issue that remains to be elucidated.
The characterization of the export into the cytosol of other mammalian ERAD
substrates such as the luminal unassembled heavy chain of secretory immunoglobulin M (IgM) and the integral MHC class I heavy chains provides excellent
paradigms (105109, 112). From these models several instructive details can be
gleaned: (a) polyubiquitination (with intrinsic Lys48-linkage) rather than monoubiquitination is required but insufficient for degradation; (b) subsequent to ubiquitination, ATP -phosphate hydrolysis is required for the release of the ER-bound
substrate into the cytosol; (c) the polyUb chain serves as a recognition signal rather
than as a ratcheting molecule that drives the protein out of the ER; (d) proteasome
function is not required for ER protein ubiquitination but may be required for protein dislocation; (e) the chaperone Bip (Hsp70) may facilitate this process; and most
importantly, ( f ) a downstream component, the mammalian cytosolic ERAD chaperone p97-Ufd1-Npl4 complex (the equivalent of yeast Cdc48p-Ufd1p-Hrd4p),
is involved in the polyUb recognition step and ATP-hydrolysis. Apparently, the
AAA ATPase p97 is responsible for the ATP hydrolysis, whereas the N-terminal
Ufd1 Ub-binding domain (rather than the Npl4-Ub binding domain) that specifically recognizes UbLys48-linkages is responsible for the Ub-recognition (109,
112). Although the exact role of p97 remains controversial, its participation in the
ER to cytosol translocation of these ERAD substrates is indisputable (109, 112).
Similarly, it is unclear whether ERAD involves just the 26S proteasomal subpopulation recruited to the ER by p97 or its entire cytosolic pool. A notable difference
between the translocation of these model ER proteins and that of CYPs 3A is that
very little (if any) of the P450 protein domain is luminally oriented, whereas only
a small 27-residue N-terminal tail is ER-membrane bound, with most of the protein already facing the cytosol and poised for ubiquitination and/or proteasomal
degradation. Thus it remains to be determined whether the protein is translocated
intact or dislocated from its N-terminal anchor before it is ubiquitinated and/or
delivered to the proteasome.
P450s: THE DEFIANT ONES

CYP2E1 normally exhibits a biphasic turnover, with the rapid turnover species undergoing degradation that is inhibited by proteasome inhibitors (82, 85, 91, 92, 118,
119) and the long turnover species being degraded by an autophagic-lysosomal
pathway, sensitive to lysosomal inhibitors (120, 121; reviewed in Reference 57).
However, the in vitro CYP2E1 ubiquitination reports discussed above notwithstanding, no HMM ubiquitinated CYP2E1 species could be immunoprecipitated
from hepatocytes or HepG, Tc-HeLa, and Fr-8a2 cell lines (92). Indeed, studies
on stably expressed CYP2E1 in E36ts20 (a cell line with a temperature-sensitive
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Ub-activating E1 enzyme) revealed no significant stabilization of CYP2E1 turnover

at the nonpermissive temperature when compared with that in the corresponding
parental E36 cells (92). In contrast, as expected, the ubiquitination of two known
substrates (oxidized RNase and c-Jun) was apparently impaired at the nonpermissive temperature in the E36ts20 but not parental cells (92). Furthermore, mutation
of CYP2E1 Lys317, Lys324, or of both these residues (reportedly ubiquitinated
in vitro; 83) had no appreciable effect on CYP2E1 turnover in COS-1 cells (92).
Together these findings confirmed that CYP2E1 turnover, irrespective of the cell
type, is Ub-independent. Thus, although CYPs 3A and 2E1 share many common
characteristics such as a propensity for futile oxidative cycling, relatively short
in vivo half-lives, and induction via substrate-mediated stabilization, they apparently differ in their susceptibility to Ub-dependent 26S proteasome degradation.
However, given its relative ER abundance, it is conceivable that the fraction of
ubiquitinated CYP2E1 is too miniscule for detection. Whatever the reason for its
recalcitrance to ubiquitination, the fact that CYP2E1 turnover was unequivocally
inhibited by proteasome inhibitors (85, 92, 119), suggests that the 20S rather than
the 26S proteasome species is involved. These findings raise the provocative issue
of how, in the absence of a polyUb targeting signal, this ER-anchored enzyme
is dislocated, shuttled to the 20S proteasome, unfolded, and threaded through its
catalytic barrel. The requirement for ATP and the chaperone Hsp90 (122) and
the superiority of cytosol versus the purified 20S proteasome species may reflect
the involvement of yet to be identified cytosolic ATP-dependent dislocases and/or
unfoldases. It is also plausible that the ER-associated proteasome subpopulation
is responsible for the observed CYP2E1 degradation.
P450 UBIQUITINATION: A METHODOLOGICAL

POSTSCRIPTUM
The detection of protein ubiquitination is technically tricky and requires careful methodological approaches. Protein ubiquitination is a dynamic process, with
polyUb chains being put on the protein and taken off both as the ubiquitinated protein is proteasomally degraded and/or the polyUb chains are disassembled by the
ubiquitous and avid deubiquitinases (1, 123). Thus only a small fraction of the ubiquitinated protein can be captured at any given time. Reliable capture of a detectable
fraction of ubiquitinated proteins therefore often requires inhibition not only of the
proteasome by specific inhibitors but also of the deubiquitinases by inhibitors such
as Ubal and N-ethylmaleimide (NEM) (1, 123125). This is particularly true of the
detection of hepatic ubiquitinated P450 proteins. Not only are the liver deubiquitinases abundant and highly robust (12, 70, 124), but the subfractionation process
to isolate the microsomal P450s also results in the release of undesirable lysosomal proteases that can inactivate the essential ubiquitinating enzymes as well as
degrade P450 proteins (126). Thus maximal trapping of the ubiquitinated P450s
from the liver requires homogenization buffers supplemented with general protease
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inhibitors and NEM. Furthermore, in vitro ubiquitination reactions with cytosolic

Fraction II require the presence of lysosomal protease inhibitors and Ubal4. Even
after all these precautions, it is often difficult to detect significant P450 ubiquitination by immunoblotting analyses, unless the immunoblots are hydrated by autoclaving (127). The use of 125I-labeled Ub and autoradiography/PhosphorImager
analyses can circumvent these problems and considerably improve the detection
sensitivity (128). Even so, definitive detection of ubiquitinated P450s requires that
the proteins be immunoprecipitated from the liver microsomes or ubiquitination
reactions. This is particularly critical when P450 ubiquitination is examined in
vitro, because preformed but unattached polyUb chains in the reaction mixture
can also yield the HMM profile characteristic of a ubiquitinated protein.
Immunoprecipitation from liver microsomes is also essential because liver
P450s, particularly CYPs 3A and CYP2E1, are known to be highly sensitive to
structural insults derived from futile oxidative cycling, lipid peroxidation, storage,
and thermal changes, all of which can result in aggregation and/or cross-linking of
the P450s intermolecularly and/or with other microsomal proteins (80, 84, 129
132). Thus when liver microsomes are used as the P450 source, such protein
aggregation and/or cross-linking artifacts can greatly confound the determination
of whether a P450 is ubiquitinated and/or degraded5 by immunoblotting analyses
and lead to flawed conclusions. However, as discussed below, the P450 aggregation
and ubiquitination profiles can be readily distinguished on careful inspection.
Figures 3 and 4 illustrate some of the critical methodological issues, such as
the importance of using freshly prepared liver microsomes, optimal storage conditions, and immunoprecipitation procedures, for maximal detection of ubiquitinated
P450s by immunoblotting analyses, with CYP3A as an example. For this purpose,
the hepatic microsomal CYP3A content was enriched by DEX-pretreatment of
rats, followed by DDEP administration to inactivate the P450s. Liver microsomes
obtained from DEX- or DEX/DDEP-treated rats were immunoprecipitated with
polyclonal goat antirat CYP3A23 IgGs (Figure 3a). Note the time-dependent rat
liver CYP3A ubiquitination maximally detected at 60 min after DDEP-treatment
(Figure 3a). The corresponding Western CYP3A immunoblots of these liver microsomes, deliberately overexposed to enhanced chemiluminescence (ECL) detection,
are shown in Figure 3b. These immunoblots reveal time-dependent DDEP-induced
CYP3A loss (at 55 kDa), without any concurrently detected CYP3A aggregates
(dimers, trimers, and/or oligomers), even on ECL overexposure (Figure 3b). The
relative importance of NEM inclusion during liver homogenization for an appreciable intensification of microsomal CYP3A ubiquitination is shown in Figure 3c.
Because the goat IgGs used for CYP3A immunoprecipitation were fractionated
4
NEM would inactivate the sulfhydryl groups of several proteins including the ubiquitinating
enzymes, and should therefore be used only at the termination of the ubiquitination reactions.
5
The upward migration of CYP3A due to the formation of dimers, trimers, and/or oligomers
effectively reduces the levels of the parent proteins detected at 55 kDa, thereby leading
to the erroneous conclusion that P450 is lost because of protein degradation.
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from immunized goat serum and thus were not fully purified, the corresponding
immunoblot background reflecting the presence of serum contaminants is also
shown with a mock immunoprecipitate from mixtures without liver microsomes
(Figure 3c; last lane). Densitometric assessment of CYP3A4 ubiquitination requires that this background be subtracted from the corresponding profiles in the
other four lanes. Figure 3d illustrates the absolute need for autoclaving in the
immunoblotting analyses of P450 ubiquitination, particularly if neither the Ub
nor P450 molecules are radiolabeled and thus undetectable by autoradiography or
PhosphoImager analyses. Autoclaving of the electroblotted membranes is found to
improve the immunodetection of polyUb epitopes by rehydration of the denatured
proteins (127).
The source of the confounding artifacts often encountered during in vitro P450
ubiquitination assays are illustrated in Figure 4. Freshly prepared liver microsomes
from DEX-pretreated rats (stored as pellets at 80 C for 1 wk) were used for
CYP3A immunoblotting analyses before or after incubation at 37 C for 2 h, under
conditions identical to those previously detailed (129), except that CaCl2, ZnCl2
or MgCl2 were individually included instead of the previously used Ca+2/Zn+2
combination. Note that in the nonincubated (0 h) microsomes, no CYP3A aggregation was detected even after sample overload (Figure 4a). Incubation of these
microsomes at 37 C for 2 h in the presence of CaCl2, ZnCl2, or MgCl2 only minimally increased the detection of CYP3A dimers and trimers, even after sample
overload (Figure 4a). Figure 4b depicts the corresponding protein ubiquitination
pattern of the nonincubated (0 h) microsomes. This profile was clearly enhanced
by the inclusion of NEM during homogenization of the liver (Figure 4b).
On the other hand, immunoblotting analyses of liver microsomes stored as
pellets at 80 C for longer periods (1 year) clearly documented the formation of CYP3A dimers and trimers even without incubation (Figure 4b). Furthermore, immunoblotting analyses of these stored microsomes after incubation at
37 C for 2 h as described (Figure 4a) clearly revealed the presence of CYP3A
oligomers/aggregates at the stacking/running gel interphase (Figure 4c; marked
with an asterisk) in addition to CYP3A dimers and trimers and irrespective of
the cation present. Corresponding immunoblotting analyses with anti-Ub IgGs
of incubations depicted in Figure 4c are shown in Figure 4d. Note the salient
differences in this profile and that seen in Figures 3a and 3c, particularly at the
stacking/running gel interphase (marked with an asterisk). Additional storage of
the nonincubated microsomal suspensions for just a week at 20 C resulted in
the dramatic CYP3A aggregation, which was further intensified by incubation at
37 C for 2 h (Figure 4e). Only slightly lesser aggregation was detected if instead
these suspensions were stored at 80 C for a week (Figure 4e).
Collectively, these findings clearly indicate that maximal detection of liver
P450 ubiquitination requires (a) addition of NEM during liver homogenization;
(b) minimal (<2 wk) storage of microsomal pellets at 80 C; (c) immunoprecipitation of the P450 under scrutiny; and (d) autoclaving of the electroblotted membranes for maximal immunochemical detection. Artifacts such as those depicted in
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Figure 4 CYP3A aggregation: Effects of microsomal storage, incubation, NEM

and/or divalent cations. (a) Freshly prepared liver microsomes (75 g) were incubated
at 37 C for 0 or 2 h, in the presence of 25 mM sucrose, 0.154 mM KCl, 1 M Ub, and
2 mM CaCl2, 3 M ZnCl2, or 10 mM MgCl2 in 50 mM Tris.HCl buffer, pH 7.5. Reactions were terminated with an equal volume of sample loading buffer [50 l; corrected
version (129)]. An aliquot (7.65 g protein) was loaded onto a 420% gradient TrisHCl ready gel (BioRad) for CYP3A immunoblotting analyses using a primary goat
antirat CYP3A23 antibody (1:10,000, v/v) overnight, followed by a secondary swine
antigoat AP-conjugated antibody (1:3000, v/v) for 1 h followed by color development. (b) Nonincubated freshly prepared microsomes (15.3 g) from DEX-pretreated
rat livers homogenized with (+) or without () 5 mM NEM were subjected to immunoblotting analyses against a goat anti-Ub antibody exactly as described (Figure
3a). (c) Liver microsomes from DEX-pretreated rat livers stored at 80 C as pellets overlaid with 10% glycerol/0.1M phosphate buffer, pH 7.4, for 1 year, were
incubated and CYP3A content analyzed by immunoblotting exactly as detailed in (a).
(d) Corresponding anti-Ub immunoblotting analyses of microsomes incubated as detailed in (c) were carried out exactly as detailed in Figure 3. (e) Liver microsomes (0 h)
used in (c) and (d) above were further stored as suspensions at either 20 C or 80 C
for 1 week before CYP3A immunoblotting analyses exactly as described in (a) above.
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Figure 4d,e are observed if microsomes are stored for prolonged periods as pellets
or even as suspensions for just a week. More importantly, comparative inspection
of the immunoblots in Figures 3 and 4 clearly distinguishes the authentic CYP3A
ubiquitination profiles (Figure 3a and c) from the CYP3A aggregation artifacts illustrated in Figure 4d by the absence of any immunochemically detectable CYP3A
at the stacking/running gel interphase and/or bottoms of the gel wells (marked by
an asterisk). A distinguishing feature of truly ubiquitinated CYP3A species is that
they predominantly migrate to a region well above the 55 kDa parent protein but
distinctly below the stacking/running gel interphase where CYP3A aggregates are
usually found.
ACKNOWLEDGMENTS
The authors acknowledge Ms. Zhi-Juan Song for the CYP2C11 ubiquitination
studies, as well as Ms. Suzanne Davoll and Drs. Katy Korsmeyer, Huifen (Faye
Wang) and Bernard Murray for their invaluable contributions to the CYP3A ubiquitination studies. We gratefully acknowledge the financial support of NIH grants
GM44037 and DK26506 that made these studies possible.
LITERATURE CITED
1. Peters JM, Harris JR, Finley D, eds . 1998.
Ubiquitin and the Biology of the Cell. New
York: Plenum
2. Hicke L. 2001. Protein regulation by
monoubiquitin. Nat. Rev. Mol. Cell Biol.
2:195201
3. Pickart CM. 1997. Targeting of substrates
to the 26S proteasome. FASEB J. 11:
105566
4. Haas AL, Siepmann TJ. 1997. Pathways of ubiquitin conjugation. FASEB J.
11:125768
5. Hershko A, Ciechanover A. 1998. The
ubiquitin system. Annu. Rev. Biochem.
67:42579
6. Scheffner M, Smith S, Jentsch S. 1998.
The ubiquitin conjugation system. See
Ref. 1, pp. 6598
7. Hochstrasser M. 2000. Evolution and
function of ubiquitin-like protein-conjugation systems. Nat. Cell Biol. 2:E153
57
8. Pickart CM. 2001. Mechanisms underlying ubiquitination. Annu. Rev. Biochem.

70:50333
9. Weissman A. 2001. Themes and variations on ubiquitylation. Nat. Rev. Mol.
Cell Biol. 2:16978
10. Finley D. 2002. Ubiquitin chained and
crosslinked. Nat. Cell Biol. 4:E12123
11. Correia MA, Davoll SH, Wrighton SA,
Thomas PE. 1992. Degradation of rat liver
cytochromes P-450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl4-ethyl-1,4-dihydropyridine: characterization of the proteolytic system. Arch.
12. Wang H, Figueiredo-Pereira ME, Correia
MA. 1999. CYP 3A degradation in isolated rat liver hepatocytes: 26S Proteasome inhibitors as probes. Arch. Biochem.
Biophys. 365:4553
13. Korsmeyer KK, Davoll S, FigueiredoPereira ME, Correia MA. 1999.
7 Jan 2005
14:4
458

14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
AR
AR232-PA45-18.tex
CORREIA
SADEGHI
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE
MUNDO-PAREDES
Proteolytic degradation of heme-modified hepatic cytochromes P450: a role

for phosphorylation, ubiquitination and
the 26S proteasome? Arch. Biochem.
Biophys. 365:3144
Hatfield PM, Vierstra RD. 1992. Multiple
forms of ubiquitin-activating enzyme E1
from wheat. Identification of an essential
cysteine by in vitro mutagenesis. J. Biol.
Chem. 267:14799803
Sommer T, Jentsch S. 1993. A protein
translocation defect linked to ubiquitin
conjugation at the endoplasmic reticulum.
Nature 365:17679
Tiwari S, Weissman A. 2001. Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitinconjugating enzymes (E2s). J. Biol.
Chem. 276:16193200
Murata S, Chiba T, Tanaka K. 2003.
CHIP: a quality-control E3 ligase collaborating with molecular chaperones. Int. J.
Biochem. Cell Biol. 35:57278
Cyr DM, Hohfeld J, Patterson C. 2002.
Protein quality control: U-box-containing
E3 ubiquitin ligases join the fold. Trends
Biochem. Sci. 27:36875
Freemont PS. 2000. RING for destruction? Curr. Biol. 10:R8487
Joazeiro CA, Weissman AM. 2000. RING
finger proteins: mediators of ubiquitin ligase activity. Cell 102:54952
Jackson PK, Eldridge AG, Freed E,
Furstenthal L, Hsu JY, et al. 2000. The
lore of the RINGs: substrate recognition
and catalysis by ubiquitin ligases. Trends
Cell Biol. 10:42939
Lu P, Zhou X, Shen M, Lu K. 1999. Function of WW domains as phosphoserine- or
phosphothreonine-binding modules. Science 283:132528
Orlicky S, Tang X, Willems A, Tyers M,
Sicheri F. 2003. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.
Cell 112:24356
Bays NW, Gardner RG, Seelig LP,
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Joazeiro CA, Hampton RY. 2001. Hrd1p/

Der3p is a membrane-anchored ubiquitin
ligase required for ER-associated degradation. Nat. Cell Biol. 3:2429
Gardner R, Shearer A, Hampton R. 2001.
In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol
regulation. Mol. Cell. Biol. 21:4276
91
Thrower JS, Hoffman L, Rechsteiner M,
Pickart CM. 2000. Recognition of the
polyubiquitin proteolytic signal. EMBO J.
19:94102
Pickart CM, Cohen RE. 2004. Proteasomes and their kin: proteases in the machine age. Nat. Rev. Mol. Cell Biol. 5:177
87
Tanaka K, Yoshimura T, Ichihara A, Ikai
A, Nishigai M, et al. 1988. Molecular
organization of a high molecular weight
multi-protease complex from rat liver. J.
Mol. Biol. 203:98596
Rechsteiner M, Hoffman L, Dubiel W.
1993. The multicatalytic and 26 S proteases. J. Biol. Chem. 268:606568
Coux O, Tanaka K, Goldberg AL. 1996.
Structure and functions of the 20S and
26S proteasomes. Annu. Rev. Biochem.
65:80147
Schmidt M, Kloetzel PM. 1997. Biogenesis of eukaryotic 20S proteasomes: the
complex maturation pathway of a complex enzyme. FASEB J. 11:123543
Baumeister W, Walz J, Zuhl F, Seemuller
E. 1998. The proteasome: paradigm of
a self-compartmentalizing protease. Cell
92:36780
Glickman MH, Rubin DM, Fried VA,
Finley D. 1998. The regulatory particle
of the Saccharomyces cerevisiae proteasome. Mol. Cell. Biol. 18:314962
Orlowski M, Cardozo C, Michaud C.
1993. Evidence for the presence of five
distinct proteolytic components in the pituitary multicatalytic proteinase complex.
Properties of two components cleaving
bonds on the carboxyl side of branched
7 Jan 2005
14:4
AR
AR232-PA45-18.tex
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE
35.

36.
37.
38.
39.
40.
41.
42.
43.
chain and small neutral amino acids. Biochemistry 32:156372

Rock KL, Gramm, C, Rothstein L, Clark
K, Stein R, et al. 1994. Inhibitors of the
proteasome block the degradation of most
cell proteins and the generation of peptides presented on MHC class I molecules.
Cell 78:76171
Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, et al. 1996. Mechanistic studies on the inactivation of the
proteasome by lactacystin: a central role
for clasto-lactacystin beta-lactone. J. Biol.
Chem. 271:727376
Dick LR, Cruikshank AA, Destree AT,
Grenier L, McCormack TA, et al. 1997.
Mechanistic studies on the inactivation of
the proteasome by lactacystin in cultured
cells. J. Biol. Chem. 272:18288
DeMartino GN, Moomaw CR, Zagnitko
OP, Proske RJ, Chu-Ping M, et al. 1994.
PA700, an ATP-dependent activator of the
20 S proteasome, is an ATPase containing
multiple members of a nucleotide-binding
protein family. J. Biol. Chem. 269:20878
84
Lam YA, DeMartino GN, Pickart CM, Cohen RE. 1997. Specificity of the ubiquitin isopeptidase in the PA700 regulatory
complex of 26 S proteasomes. J. Biol.
Chem. 272:2843846
Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, et al. 1998. Subcomplex
of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome
and eIF3. Cell 94:61523
Rubin DM, Glickman MH, Larsen CN,
Dhruvakumar S, Finley D. 1998. Active
site mutants in the six regulatory particle ATPases reveal multiple roles for ATP
in the proteasome. EMBO J. 17:4909
19
Tran HJ, Allen MD, Lowe J, Bycroft M.
2003. Structure of the Jab1/MPN domain
and its implications for proteasome function. Biochemistry 42:1146065
Ambroggio XI, Rees DC, Deshaies RJ.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
459
2004. JAMM: A metalloprotease-like zinc

site in the proteasome and signalosome.
PLoS Biol. 2:E2
Kohler A, Cascio P, Leggett DS, Woo KM,
Goldberg AL, Finley D. 2001. The axial
channel of the proteasome core particle
is gated by the Rpt2 ATPase and controls
both substrate entry and product release.
Mol. Cell 7:114352
Forster A, Hill CP. 2003. Proteasome
degradation: enter the substrate. Trends
Cell Biol. 13:55053
Goldberg AL, Rock KL. 1992. Proteolysis, proteasomes and antigen presentation.
Nature 357:3759
Michalek MT, Grant EP, Gramm C, Goldberg AL, Rock KL. 1993. A role for the
ubiquitin-dependent proteolytic pathway
in MHC class I-restricted antigen presentation. Nature 363:55254
Monaco JJ. 1995. Pathways for the processing and presentation of antigens to T
cells. J. Leukoc. Biol. 57:54347
Kloetzel P. 2001. Antigen processing by
the proteasome. Nat. Rev. Mol. Cell Biol.
2:17987
Riley RJ, Smith G, Wolf CR, Cook VA,
Leeder JS. 1993. Human anti-endoplasmic reticulum autoantibodies produced in aromatic anticonvulsant hypersensitivity reactions recognize rodent
CYP3A proteins and a similarly regulated
human P450 enzyme(s). Biochem. Biophys. Res. Commun. 191:3240
Leeder JS, Gaedigk A, Lu X, Cook VA.
1996. Epitope mapping studies with human anti-cytochrome P450 3A antibodies.
Beaune P, Dansette PM, Mansuy D, Kiffel L, Finck M, et al. 1987. Human antiendoplasmic reticulum autoantibodies
appearing in a drug-induced hepatitis are
directed against a human liver cytochrome
P-450 that hydroxylates the drug. Proc.
Bourdi M, Gautier JC, Mircheva J,
Larrey D, Guillouzo A, et al. 1992.
Anti-liver microsomes autoantibodies and
7 Jan 2005
14:4
460

54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
AR
AR232-PA45-18.tex
CORREIA
SADEGHI
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE
MUNDO-PAREDES
dihydralazine-induced hepatitis: specificity of autoantibodies and inductive capacity of the drug. Mol. Pharmacol. 42:
28085
Zhang M, Pickart CM, Coffino P.
2003. Determinants of proteasome recognition of ornithine decarboxylase, a
ubiquitin-independent substrate. EMBO
J. 22:148896
Ghoda L, Sidney D, Macrae M, Coffino
P. 1992. Structural elements of ornithine
decarboxylase required for intracellular degradation and polyamine-dependent
regulation. Mol. Cell. Biol. 12:217885
Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, et al. 2000. Proteasomal turnover of p21Cip1 does not require
p21Cip1 ubiquitination. Mol. Cell 5:403
10
Correia MA. 2003. Hepatic cytochrome
P450 degradation: mechanistic diversity
of the cellular sanitation brigade. Drug
Metab. Rev. 35:10743
Gardner R, Cronin S, Leader B, Rine J,
Hampton R, et al. 1998. Sequence determinants for regulated degradation of yeast
3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol. Biol. Cell
9:261126
Gardner RG, Hampton RY. 1999. A distributed degron allows regulated entry
into the ER degradation pathway. EMBO
J. 18:59946004
Gilon T, Chomsky O, Kulka R. 1998.
Degradation signals for ubiquitin system
proteolysis in Saccharomyces cerevisiae.
EMBO J. 17:275966
Chen ZJ, Hagler J, Palombella VJ, Melandri F, Scherer D, et al. 1995. Signalinduced site-specific phosphorylation targets IB to the ubiquitin-proteasome
pathway. Genes. Dev. 9:158697
Chen ZJ, Parent L, Maniatis T. 1996.
Site-specific phosphorylation of IB by
a novel ubiquitination-dependent protein
kinase activity. Cell 84:85362
Pyerin W, Taniguchi H, Horn F, Oesch
64.
65.
66.
67.
68.
69.
70.
F, Amelizad Z, et al. 1987. Isoenzymespecific phosphorylation of cytochromes

P-450 and other drug metabolizing enzymes. Biochem. Biophys. Res. Commun.
142:88592
Koch JA, Waxman DJ. 1989. Posttranslational modification of hepatic cytochrome
P-450. Phosphorylation of phenobarbitalinducible P-450 forms PB-4 (IIB1) and
PB-5 (IIB2) in isolated rat hepatocytes
and in vivo. Biochemistry 28:314552
Menez JF, Machu TK, Song BJ, Browning MD, Deitrich RA. 1993. Phosphorylation of cytochrome P4502E1 (CYP2E1)
by calmodulin dependent protein kinase,
protein kinase C and cAMP dependent
protein kinase. Alcohol Alcohol. 28:445
51
Lohr JB, Kuhn-Velten WN. 1997. Protein
phosphorylation changes ligand-binding
efficiency of cytochrome P450c17
(CYP17) and accelerates its proteolytic
degradation: putative relevance for
hormonal regulation of CYP17 activity.
4038
Eliasson E, Mkrtchian S, IngelmanSundberg M. 1992. Hormone- and
substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in
the endoplasmic reticulum membranes. J.
Biol. Chem. 267:1576569
Eliasson E, Mkrtchian S, Halpert JR,
Ingelman-Sundberg M. 1994. Substrateregulated, cAMP-dependent phosphorylation, denaturation, and degradation of
glucocorticoid-inducible rat liver cytochrome P450 3A1. J. Biol. Chem. 269:
1837883
Wang X, Medzihradszky KF, Maltby D,
Correia MA. 2001. Phosphorylation of
native and heme-modified CYP3A4 by
protein kinase C: A mass spectrometric characterization of the phosphorylated
peptides. Biochemistry 40:1131826
Tierney DJ, Haas AL, Koop DR. 1992.
Degradation of cytochrome P450 2E1:
7 Jan 2005
14:4
AR
AR232-PA45-18.tex
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE

71.
72.
73.
74.
75.
76.
77.
78.
Selective loss after labilization of the enzyme. Arch. Biochem. Biophys. 293:9
16
Davies HS, Britt SG, Pohl LR. 1986.
Carbon tetrachloride and 2-isopropyl-4pentenamide-induced inactivation of cytochrome P-450 leads to heme-derived
protein adducts. Arch. Biochem. Biophys.
244:35287
Ortiz de Montellano PR, Mathews JM.
1981. Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group
by 1-aminobenzotriazole. Isolation of an
NN-bridged benzyne-protoporphyrin IX
adduct. Biochem. J. 195:76164
Correia MA, Decker C, Sugiyama K,
Caldera P, Bornheim L, et al. 1987.
Degradation of rat hepatic cytochrome
P-450 heme by 3,5dicarbethoxy-2,6dimethyl-4-ethyl-1,4-dihydropyridine to
irreversibly bound protein adducts. Arch.
Correia MA, Rettie A, Wrighton SA,
Waxman DJ. 1992. Differential apoprotein loss of rat liver cytochromes P-450
after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: A case for distinct proteolytic
mechanisms? Arch. Biochem. Biophys.
294:493503
Etlinger JD, Goldberg AL. 1980. Control of protein degradation in reticulocytes
and reticulocyte extracts by hemin. J. Biol.
Chem. 255:456368
Haas AL, Rose IA. 1981. Hemin inhibits
ATP-dependent ubiquitin-dependent proteolysis: role of hemin in regulating ubiquitin conjugate degradation. Proc. Natl.
Vierstra RD, Sullivan ML. 1988. Hemin
inhibits ubiquitin-dependent proteolysis
in both a higher plant and yeast. Biochemistry 27:329095
Figueiredo-Pereira ME, Berg KA, Wilk
S. 1994. A new inhibitor of the chymotrypsin-like activity of the multicatalytic
proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
461
conjugates in a neuronal cell. J. Neurochem. 63:157881

Zangar RC, Kocarek TA, Shen S,
Bollinger N, Dahn MS, Lee DW. 2003.
Suppression of cytochrome P450 3A protein levels by proteasome inhibitors. J.
Kimzey AL, Weitz KK, Guengerich
FP, Zangar RC. 2003. Hydroperoxy10,12-octadecadienoic acid stimulates cytochrome P450 3A protein aggregation by
a mechanism that is inhibited by substrate.
Wang H, Correia MA. 2003. Hemesensitive hepatic CYP2C11 degradation
and PERK activation in isolated rat hepatocytes: causal or coincidental association? Drug Metab. Rev. 5:135
Roberts BJ, Song BJ, Soh Y, Park SS,
Shoaf SE. 1995. Ethanol induces CYP2E1
by protein stabilization. Role of ubiquitin conjugation in the rapid degradation
of CYP2E1. J. Biol. Chem. 270:29632
35
Banerjee A, Kocarek TA, Novak R.F.
2000. Identification of a ubiquitinationtarget/substrate-interaction domain of
cytochrome P-450 (CYP) 2E1. Drug
Kitada M, Komori M, Ohi H, Imaoka
S, Funae Y, Kamataki T. 1989. Formspecific degradation of cytochrome P450
by lipid peroxidation in rat liver microsomes. Res. Commun. Chem. Pathol.
Pharmacol. 63:17588
Roberts BJ. 1997. Evidence of
proteasome-mediated cytochrome P-450
degradation. J. Biol. Chem. 272:977178
Wilhovsky S, Gardner R, Hampton R.
2000. HRD gene dependence of endoplasmic reticulum-associated degradation. Mol. Biol. Cell 11:1697708
Plemper RK, Wolf DH. 1999. Endoplasmic reticulum degradation. Reverse protein transport and its end in the proteasome. Mol. Biol. Rep. 26:12530
Hampton RY. 2002. ER-associated degradation in protein quality control and
7 Jan 2005
14:4
462
89.

90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
AR
AR232-PA45-18.tex
CORREIA
SADEGHI
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE
MUNDO-PAREDES
cellular regulation. Curr. Opin. Cell Biol.

14:47682
Watkins PB, Bond JS, Guzelian PS. 1987.
Degradation of the hepatic cytochromes
P-450. In Mammalian Cytochromes P450,
ed. FP Guengerich, 2:17392. Boca Raton, FL: CRC Press
Correia MA. 1991. Cytochrome P450
turnover. Methods Enzymol. 206:31525
Barmada S, Kienle E, Koop DR. 1995.
Rabbit P450 2E1 expressed in CHO-K1
cells has a short half-life. Biochem. Biophys. Res. Commun. 206:6017
Huan J-Y, Streicher JM, Bleyle LA, Koop
DR. 2004. Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1
expressed intetracycline-regulated HeLa
cells. Toxicol. Appl. Pharmacol. In press
Hampton RY, Bhakta H. 1997. Ubiquitinmediated regulation of 3-hydroxy-3methylglutaryl-CoA reductase. Proc.
Chen P, Johnson P, Sommer T, Jentsch
S, Hochstrasser M. 1993. Multiple
ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast
MAT2 repressor. Cell 74:35769
Lenk U, Yu H, Walter J, Gelman MS, Hartmann E, et al. 2002. A role for mammalian
Ubc6 homologues in ER-associated protein degradation. J. Cell Sci. 115:300714
Sommer T, Wolf DH. 1997. Endoplasmic reticulum degradation: reverse protein flow of no return. FASEB J. 11:1227
33
Biederer T, Volkwein C, Sommer T. 1997.
Role of Cue1p in ubiquitination and
degradation at the ER surface. Science
278:18069
Hampton RY, Gardner RG, Rine J.
1996. Role of 26S proteasome and HRD
genes in the degradation of 3-hydroxy3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane
protein. Mol. Biol. Cell 7:202944
Bordallo J, Plemper RK, Finger A, Wolf
DH. 1998. Der3p/Hrd1p is required for
endoplasmic reticulum-associated degra-
100.
101.
102.
103.
104.
105.
106.
107.
108.
dation of misfolded lumenal and integral membrane proteins. Mol. Biol. Cell
9:20922
Gardner RG, Swarbrick GM, Bays NW,
Cronin SR, Wilhovsky S, et al. 2000. Endoplasmic reticulum degradation requires
lumen to cytosol signaling. Transmembrane control of hrd1p by hrd3p. J. Cell
Biol. 151:6982
Plemper RK, Bordallo J, Deak PM, Taxis
C, Hitt R, Wolf DH. 1999. Genetic interactions of Hrd3p and Der3p/Hrd1p with
Sec61p suggest a retro-translocation complex mediating protein transport for ER
degradation. J. Cell Sci. 112:412334
Bays N, Wilhovsky S, Goradia A,
Hodgkiss-Harlow K, Hampton R. 2001.
HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol. Biol. Cell 12:411428
Braun S, Matuschewski K, Rape M,
Thoms S, Jentsch S. 2002. Role of the
ubiquitin-selective CDC48(UFD1/NPL4)
chaperone (segregase) in ERAD of OLE1
and other substrates. EMBO J. 21:61521
Jarosch E, Taxis C, Volkwein C, Bordallo
J, Finley D, et al. 2002. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat.
Cell Biol. 4:13439
Meyer H, Shorter J, Seemann J, Pappin
D, Warren G. 2000. A complex of mammalian ufd1 and npl4 links the AAAATPase, p97, to ubiquitin and nuclear
transport pathways. EMBO J. 19:2181
92
Ye Y, Meyer H, Rapoport T. 2001. The
AAA ATPase Cdc48/p97 and its partners
transport proteins from the ER into the cytosol. Nature 414:65256
Rabinovich E, Kerem A, Frohlich K,
Diamant N, Bar-Nun S. 2002. AAAATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulumassociated protein degradation. Mol. Cell
Biol. 22:62634
Tsai B, Ye Y, Rapoport TA. 2002.
Retro-translocation of proteins from the
7 Jan 2005
14:4
AR
AR232-PA45-18.tex
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
P1: GCE

109.
110.
111.
112.
113.
114.
115.
endoplasmic reticulum into the cytosol. Nat. Rev. Mol. Cell Biol. 3:246
55
Flierman D, Ye Y, Dai M, Chau V,
Rapoport TA. 2003. Polyubiquitin serves
as a recognition signal, rather than a
ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane. J. Biol. Chem.
278:3477482
Kikkert M, Doolman R, Dai M, Avner R,
Hassink G, et al. 2004. Human HRD1 is
an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum. J. Biol. Chem. 279:3525
34
Fang S, Ferrone M, Yang C, Jensen JP,
Tiwari S, Weissman AM. 2001. The tumor autocrine motility factor receptor,
gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA
98:1442227
Elkabetz Y, Shapira I, Rabinovich E, BarNun S. 2004. Distinct steps in dislocation of luminal endoplasmic reticulumassociated degradation substrates: roles
of endoplamic reticulum-bound p97/Cdc
48p and proteasome. J. Biol. Chem. 279:
398099
Murray BP, Correia MA. 2001. Ubiquitindependent 26 S proteasomal pathway: A
role in the degradation of the native human liver CYP3A4 expressed in Saccharomyces cerevisiae? Arch. Biochem. Biophys. 393:10616
Murray BM, Zgoda VG, Correia MA.
2002 Native CYP2C11: Heterologous expression in Saccharomyces cerevisiae reveals a role for vacuolar proteases rather
than the ubiquitin-26S proteasome system in the degradation of this endoplasmic reticulum enzyme. Mol. Pharmacol.
61:114653
Swanson R, Locher M, Hochstrasser M.
2001. A conserved ubiquitin ligase of the
nuclear envelope/endoplasmic reticulum
that functions in both ER-associated and
116.
117.
118.
119.
120.
121.
122.
123.
124.
463
Matalpha2 repressor degradation. Genes

Dev. 15:266074
Haynes CM, Caldwell S, Cooper AA.
2002. An HRD/DER-independent ER
quality control mechanism involves
Rsp5p-dependent ubiquitination and
ER-Golgi transport. J. Cell Biol. 158:91
101
Liao M, Zgoda VG, Murray BP, Correia
MA. 2003. Endoplasmic reticulum associated degradation (ERAD) of native rat
hepatic CYP2B1 in S. cerevisiae? Drug
Metab. Rev. 35:131
Yang MX, Cederbaum AI. 1995. Characterization of cytochrome P4502E1
turnover in transfected HepG2 cells expressing human CYP2E1. Arch. Biochem.
Biophys. 341:2533
Yang MX, Cederbaum AI. 1996. Role of
the proteasome complex in degradation
of human CYP2E1 in transfected HepG2
cells. Biochem. Biophys. Res. Commun.
226:71116
Song BJ, Veech RL, Park SS, Gelboin HV,
Gonzalez FJ. 1989. Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J. Biol. Chem. 264:356872
Ronis MJ, Johansson I, Hultenby K,
Lagercrantz J, Glaumann H, IngelmanSundberg M. 1991. Acetone-regulated
synthesis and degradation of cytochrome
P450E1 and cytochrome P4502B1 in rat
liver. Eur. J. Biochem. 198:38389
Goasduff T, Cederbaum AI. 2000.
CYP2E1 degradation by in vitro reconstituted systems: role of the molecular
chaperone hsp90. Arch. Biochem. Biophys. 379:32130
Wilkinson KD. 1997. Regulation of
ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 11:1245
56
Haas AL, Murphy KE, Bright PM. 1985.
The inactivation of ubiquitin accounts
for the inability to demonstrate ATP,
ubiquitin-dependent proteolysis in liver
extracts. J. Biol. Chem. 260:4694703
7 Jan 2005
14:4

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AR
AR232-PA45-18.tex
CORREIA
SADEGHI
AR232-PA45-18.sgm
LaTeX2e(2002/01/18)
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125. Laney JD, Hochstrasser M. 2002. Assaying protein ubiquitination in Saccharomyces cerevisiae. Methods Enzymol. 351:
24857
126. Tsuji H, Akasaki K. 1994. Identification and characterization of lysosomal enzymes involved in the proteolysis
of phenobarbital-inducible cytochrome
P450. Biol. Pharm. Bull. 17:56871
127. Swerdlow PS, Finley D, Varshavsky A.
1986. Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal.
Biochem. 156:14753
128. Haas AL, Warms JVB, Hershko A, Rose
IA. 1982. Ubiquitin-activating enzyme:
Mechanism and role in protein-ubiquitin
conjugation. J. Biol. Chem. 257:2543
48
129. Zangar RC, Kimzey AL, Okita JR, Wunschel DS, Edwards RJ, et al. 2002. Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical
ubiquitination pathway. Mol. Pharmacol.

61:892904
130. Ingelman-Sundberg M, Johansson I.
1984. Mechanisms of hydroxyl radical formation and ethanol oxidation by
ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450.
J. Biol. Chem. 259:644758
131. Zhukov A, Ingelman-Sundberg M. 1999.
Relationship between cytochrome P450
catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome
P450 2E1 (CYP2E1) in hepatoma cells is
abolished by inactivation of its electron
donor NADPH-cytochrome P450 reductase. Biochem. J. 340:45358
132. Goasduff T, Cederbaum AI. 1999.
NADPH-dependent microsomal electron
transfer increases degradation of CYP2E1
by the proteasome complex: role of reactive oxygen species. Arch. Biochem. Biophys. 370:25870
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Figure 1 The cellular enzymatic machinery available for P450 ubiquitination. The roles of E1, E2, and E3 enzymes are discussed in the
text. E2-substrate Ub-thioester relay via an E3-Cys residue (a) or directly (b) is shown.

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Figure 2 Hepatic P450 ERAD: Putative cellular participants. The plausible events in the Ub-dependent 26S proteasomal degradation of an ER-bound DDEP-inactivated CYP3A are illustrated. See the text for specific details.

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Figure 3 Immunochemical detection of rat liver microsomal P450 ubiquitination

after DDEP-treatment: effects of NEM, membrane autoclaving, and/or hemin.
(a) Microsomes (500 g) from DEX-pretreated rats given DDEP (125 mg/kg, ip) for 0,
30, 60, and 120 min were prepared from livers homogenized with (+) 5 mM NEM and
stored at 80C as pellets overlaid with 10% glycerol/0.1 M phosphate buffer, pH 7.4,
until use within 2 weeks. Microsomes were resuspended and subjected to immunoprecipitation with goat polyclonal antirat CYP3A23 IgGs (3 mg) as described (12). The
immunoprecipitated protein was solubilized by boiling in sample loading buffer consisting of final concentrations of 5% SDS, 25% glycerol, 50 mM DTT, and 2% -mercaptoethanol in 150 mM Tris, pH 6.8 (100 l). The supernatant (45 l) containing the
released P450 was subjected to anti-Ub immunoblotting analyses (ubiquitination) onto
a 420% gradient Tris-HCl ready gel (BioRad). The electroblotted membranes were
autoclaved at 120C for 30 min, before blocking with 3% gelatin and overnight incubation with rabbit anti-Ub primary antibody (1:100, v/v; Sigma-Aldrich), followed by
goat antirabbit alkaline phosphatase (AP)-conjugated secondary antibody (1:3000, v/v;
BioRad). The membranes were extensively washed with hourly changes of Trisbuffered saline (TBS) for 5 h before color detection. (b) Liver microsomal aliquots
(10 g) obtained from the above treated rats were also subjected to immunoblotting
analyses onto a 9% Tris-HCl gel. The extent of CYP3A degradation was assessed using
a primary goat antirat CYP3A23 antibody (1:10,000, v/v) overnight, followed by a secondary rabbit antigoat horseradish peroxidase (HRP)-conjugated antibody (1:40,000,
v/v) for 1 h followed by enhanced chemiluminescence (ECL) detection (113).
(c) Livers of the rats given DDEP for 0 or 60 min [see (a)] were homogenized with (+)
or without () 5 mM NEM. Microsomal aliquots (500 g) were immunoprecipitated
and the extent of ubiquitination analyzed exactly as described in (a). For reasons discussed, a mock immunoprecipitation without liver microsomes is included. (d)
Identically electroblotted membranes were subjected to anti-Ub immunoblotting analyses without the autoclaving step. (e) Freshly isolated hepatocytes from untreated male
rats were incubated with or without DDEP (0.5 mM), with or without hemin (100 M)
at 37C for 0120 min, exactly as described (12). Liver microsomes (1 mg) were
immunoprecipitated with rabbit polyclonal antirat CYP2C11 antibodies. The
CYP2C11 immunoprecipitates were subjected to anti-Ub immunoblotting analyses
as in (a) above. Arrows indicate the absence of any P450 aggregates after hemin
incubation of hepatocytes.
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Copyright

PROTEASOME INHIBITION IN MULTIPLE

MYELOMA: Therapeutic Implication
Dharminder Chauhan, Teru Hideshima,
and Kenneth C. Anderson
The Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology,
Dana Farber Cancer Institute, Harvard Medical School, Boston,
Massachusetts 02115; email: kenneth anderson@dfci.harvard.edu
Key Words plasma call neoplasm, proteasomes, growth, survival, apoptosis,

drug-resistance
Abstract Normal cellular functioning requires processing of proteins regulating
cell cycle, growth, and apoptosis. The ubiquitin-proteasome pathway (UBP) modulates
intracellular protein degradation. Specifically, the 26S proteasome is a multienzyme
protease that degrades misfolded or redundant proteins; conversely, blockade of the
proteasomal degradation pathways results in accumulation of unwanted proteins and
cell death. Because cancer cells are more highly proliferative than normal cells, their
rate of protein translation and degradation is also higher. This notion led to the development of proteasome inhibitors as therapeutics in cancer. The FDA recently approved the
first proteasome inhibitor bortezomib (VelcadeTM), formerly known as PS-341, for the
treatment of newly diagnosed and relapsed/refractory multiple myeloma (MM). Ongoing studies are examining other novel proteasome inhibitors, in addition to bortezomib,
for the treatment of MM and other cancers.
INTRODUCTION
Multiple myeloma (MM) remains fatal despite all available therapies (1, 2), and
novel approaches that target mechanisms regulating MM cell growth, survival,
and apoptosis are urgently needed. Apoptosis is the primary means by which most
radio- and chemotherapy modalities kill cancer cells (3); conversely, resistance to
apoptosis is one potential mechanism whereby tumor cells evade cytotoxic druginduced and immune-mediated cell death (4). Our studies to date have delineated
apoptotic signaling triggered by various conventional and novel anti-MM agents
(5). Importantly, recent studies show remarkable anti-MM activity of the proteasome inhibitor PS-341/bortezomib (VelcadeTM) even in MM cells refractory to
multiple prior therapies, including dexamethasone (Dex), melphalan, and thalidomide (6, 7). In addition to directly inducing apoptosis of MM cells, multiple other
lines of evidence provided rationale for the use of proteasome inhibitors (PIs) to
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treat MM. First, adhesion of MM cells to bone marrow stromal cells (BMSCs)
triggers transcription and secretion of MM growth factors, such as interleukin-6
(IL-6) or IGF-1, which stimulate the growth of MM cells and also block the cytotoxic effects of chemotherapy (8, 9). Inhibition of proteasomes downregulates
adhesion molecules and secretion of cytokines, thereby abrogating bone marrow
(BM)-dependent growth of MM cells (1012). Second, angiogenesis plays a role
in MM pathogenesis (13, 14), and bortezomib is an antiangiogenic agent (1517).
Finally, in vitro studies showed that bortezomib adds to the cytotoxicity of conventional anti-MM agents including Dex- and DNA-damaging agents (6, 18). Indeed,
based on our preclinical (6) and phase II clinical studies (19), the FDA recently
approved bortezomib for the treatment of relapsed/refractory MM. This successful
development of bortezomib therapy for MM has established proteasome inhibition
as an effective therapeutic strategy for the treatment of cancer.
PROTEIN DEGRADATION VIA

UBIQUITIN-PROTEASOME PATHWAY
Major intracellular processes are regulated by transcription, translation, and protein degradation (20). Specifically, recent reports show that degradation of proteins
is critical not only for maintaining normal cell functions but also for response to
various chemotherapeutic agents (21, 22). Protein ubiquitination and degradation regulate various cellular processes, including cell cycle progression from G1
to S phase, tumor suppression, transcription, DNA replication, inflammation, and
apoptosis (2326); conversely, mutations or alterations in the ubiquitination and/or
proteasomal degradation cascades result in defective transition from G1 to S phase
(24, 27). The ubiquitin-proteasome pathway (UBP) degrades the majority of damaged/misfolded, short (half-lives less than three hours), or long-lived regulatory
proteins in the cell (28); conversely, blockade of protein degradation by proteasome
inhibitors causes accumulation of ubiquitin-bound misfolded/damaged proteins,
which in turn triggers heat-shock response and cell death (28, 29). Indeed, proteasome inhibitors do not target specific cellular proteins or associated functions, but
rather, affect a wide spectrum of proteins with diverse functions.
Proteasomal protein degradation occurs through these sequential events: Protein
is first marked with a chain of small polypeptides called ubiquitin; E1 ubiquitin
enzyme then activates ubiquitin and links it to the ubiquitin-conjugating enzyme E2
in an ATP-dependent manner; E3 ubiquitin ligase then links the ubiquitin molecule
to the protein; a long polypeptide chain of ubiquitin moieties is formed; and finally,
proteasomes degrade protein into small fragments and free ubiquitin for recycling
(29, 30).
Proteasomes are key regulators of protein degradation (31): The human cell contains approximately 30,000 proteasomes, each equipped with protein-digesting
proteases. Proteasomes regulate diverse cellular functions, including transcription, stress response, viral infection, cell cycle, oncogenesis, ribosome biogenesis,
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BORTEZOMIB FOR THE TREATMENT OF MYELOMA
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abnormal protein catabolism, neural and muscular degeneration, cellular differentiation, antigen processing, and DNA repair (31). The 26S proteasome complex,
which constitutes up to 2%3% of the total protein in cells, has two 19S units flanking a barrel-shaped 20S proteasome core (24, 29, 32) (Figure 1A). Four stacked
rings comprise the 20S structure: two central rings are surrounded by two
rings, each composed of seven proteins. Most action occurs at six sites located
in the rings: Two sites act like chymotrypsin, which cleaves after hydrophobic
residues; two trypsin-like sites cleave after basic residues; and two are like caspase,
cleaving after acidic residues (33, 34) (Figure 1B).
The 19S units regulate entry into the 20S core chamber of only those proteins
marked for degradation (29, 35). Each 19S unit contains binding sites for ubiquitinated protein, enzymes to depolymerize the ubiquitin chain, and six ATPases
that unfold the proteins, thereby preparing them for entry into the proteasome
(Figure 1C). Attachment of ubiquitin to a target protein is the principal mechanism whereby proteins are marked for degradation by the proteasome. Importantly,
blocking proteasome activity leads to stabilization of inhibitory proteins, thereby
abrogating growth, survival, and triggering apoptosis (Figure 1D).
Most proteasome inhibitors fall in three categories: peptide aldehydes, peptide
boronates, and nonpeptide inhibitors such as lactacystin. Peptide aldehydes (MG132, MG-115, ALLN, or PSI) potently, but reversibly, block the chymotrypsinlike activity; however, they also inhibit lysosmal cysteine and serine proteases and
calpains. The peptide boronates, such as bortezomib/PS-341, are reversible and
more potent and selective than peptide aldehydes. Finally, lactacystin is a natural,
irreversible, nonpeptide inhibitor that is more selective than peptide aldehydes but
less selective than peptide boronates.
RATIONALE FOR TARGETING THE PROTEASOME

FOR CANCER THERAPY
Given that the proteasome is involved in various distinct cellular functions, it was
difficult to predict whether proteasome inhibition could be used as a target for
chemotherapy with an acceptable therapeutic index. However, multiple lines of
evidence suggest that proteasome inhibitors are more cytotoxic to proliferating
malignant cells to quiescent normal cells: (a) Proteasome inhibitor lactacystin
triggers apoptosis even in gamma-radiation-resistant CLL cells without affecting
the viability of normal lymphocytes (36); (b) proteasome inhibitor induces cell
death in contact-inhibited primary endothelial cells but not quiescent cells (37);
(c) lactacystin induces apoptosis in oral squamous carcinoma cells but not in oral
epithelial cells (38); (d) HL60 leukemic cells are significantly more sensitive to
proteasome inhibitor than quiescent cells (39); and (e) bortezomib triggers apoptosis of MM cells at doses that do not affect the viability of normal lymphocytes
(6) (Figure 2). The mechanism whereby cancer cells are more susceptible to proteasome inhibitors than normal counterparts is unclear (12). One possibility is that
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Figure 2 Bortezomib targets various growth and survival signaling mechanisms in

MM cells and abrogates protection from bone marrow stroma cells (BMSCs).
malignant cells have altered or defective cell cycle proteins, which leads to an
increased proliferation rate. These cells therefore accumulate damaged proteins at
a much higher rate than do normal cells, which in turn increases dependency on the
proteasomal degradation. In contrast, another study showed that quiescent cancer
cells are more susceptible to proteasome inhibition than are normal counterparts
(40). NF-B is linked to proliferation and drug-resistance in cancer cells (41, 42),
and PIs downregulate NF-B activation, thereby enhancing the cytotoxic effects
of chemotherapy (43). Together, these findings suggest that the proteasome is a
valid target for chemotherapy with a tolerable therapeutic index.
BORTEZOMIB/PROTEASOME INHIBITOR PS-341

TARGETS NF-B IN MM CELLS
As noted above, one of the major mechanisms whereby proteasome inhibitors
exert their growth inhibitory effects in cancer cells is by blocking NF-B signaling (28). Multiple studies have linked constitutive activation of NF-B to
growth/proliferation and drug-resistance, thereby conferring differential sensitivity to proteasome inhibitors in cancer versus normal cells (43). Activation
of NF-B occurs via the following sequential events: activation of IB kinase
(IKK), IB phosphorylation, ubiquitination and degradation of IB, and nuclear
translocation of p50/65 NF-B (44, 45). Once in the nucleus, NF-B promotes the
production of cytokines (IL-6, TNF-), survival factors (IAPs, Bcl-Xl), and cell
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469
adhesion molecules [intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and E-selectin] (45).
In the context of MM, NF-B mediates key cellular functions, including immune responses, as well as growth, survival, and apoptosis (46, 47). Intrinsic
activation of NF-B is associated with growth and survival of MM cells. Specifically, adhesion of MM cells to BMSCs triggers NF-B-mediated transcription and
secretion of IL-6 and insulin-like growth factor-I (4648); both IL-6 and IGF-1
promote the survival of MM cells in the BM by blocking apoptosis triggered by
conventional agents such as Dex (49) (Figure 2). Patient MM-derived primary cells
and BMSCs have upregulated NF-B activity relative to normal cells (50). Furthermore, drug-sensitive MM cells show lower NF-B activity than drug-resistant
MM cells, suggesting that NF-B confers chemoresistance (50). Elevated NF-B
levels have also been reported in MM cells derived from patients relapsing after chemotherapy (47). These findings indicate that NF-B is a key regulator of
growth and survival of MM cells in the BM milieu. Importantly, treatment of MM
with bortezomib prevents degradation of IB, thereby blocking not only NF-B
activation but also related cytokine production and the survival advantage for MM
cells conferred by BMSCs (Figure 2).
Bortezomib downregulates NF-B; however, our recent work shows that NF-B
inhibition alone is unlikely to account for the total anti-MM activity of bortezomib
(51, 52). The evidence for this finding is derived from the experiments using a
specific inhibitor of IB, PS-1145. Both PS-1145 and bortezomib blocked TNF-induced NF-B activation by inhibiting phosphorylation and degradation of
IB-. Dex, a conventional anti-MM agent, increases IB- protein and thereby
enhances blockade of NF-B activation by PS-1145. Importantly, both bortezomib
and PS-1145 block NF-B activation; however, in contrast to bortezomib, PS-1145
only partially inhibits MM cell growth (20%40% inhibition by PS-1145 versus
80%90% inhibition by bortezomib) (51), suggesting that NF-B inhibition cannot
account for the overall anti-MM activity of bortezomib.
Multiple genomics and proteomic studies have now established that besides
modulating NF-B, bortezomib indeed affects various other signaling pathways.
For example bortezomib-induced apoptosis is associated with initiation of the
following additional events: (a) activation of classical stress response proteins
such as heat shock proteins, Hsp27, Hsp70, and Hsp90 (17, 53); (b) upregulation
of c-Jun-NH2-terminal kinase (JNK) (54) (Figure 3); (c) alteration of mitochondrial membrane potential and generation of reactive oxygen species (ROS) (55)
(Figure 3); (d) induction of intrinsic cell death pathway, i.e., the release of mitochondrial proteins cytochrome-c/Smac into cytosol and activation of caspase-9
> caspase-3 cascade (Figure 3) (49); (e) activation of extrinsic apoptotic signaling through Bid and caspase-8 cleavage (17) (Figure 3); ( f ) inactivation of
DNA-dependent protein kinase (DNA-PK) (18) (Figure 2), which is essential
for the repair of DNA double-strand breaks; (g) inhibition of MM to BMSCshost interaction, thereby blocking of associated MM growth factor transcription and secretion from BMSCs (56) (Figure 2); and (h) inhibition of MM cell
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growth factortriggered signaling: MAPK and PI3-kinase/Akt (57). Although

many of these cellular events may appear correlative and common to other apoptotic agents, our studies have directly established an obligatory role of JNK using
dominant-negative strategies or specific biochemical inhibitors of JNK (54). The
role of IB is under evaluation using dominant-negative constructs and/or IB
knockout cells. Bortezomib may have additional substrates that mediate normal
cell growth and survival. It is very likely that all the above signaling cascades
mutually interact and contribute toward the overall response to bortezomib in
MM cells.
MECHANISMS MEDIATING BORTEZOMIB-RESISTANCE

AND THERAPEUTIC STRATEGIES TO OVERCOME
BORTEZOMIB-RESISTANCE
Bortezomib kills MM cells; however, prolonged exposure is associated with toxicity and development of bortezomib-resistance. Mechanisms mediating bortezomibresistance have now been delineated. For example, our recent study showed that
treatment with bortezomib induces apoptosis in SUDHL6 (DHL6), but not SUDHL4 (DHL4), lymphoma cells (53). Microarray analysis showed high RNA levels
for heat shock protein-27 (Hsp27) in DHL4 versus DHL6 cells, which correlated
with Hsp27 protein expression. Importantly, blocking Hsp27 using an antisense
(AS) strategy restores the apoptotic response to bortezomib in DHL4 cells; conversely, ectopic expression of wild-type (WT) Hsp27 renders bortezomib-sensitive
DHL6 cells resistant to bortezomib. These findings provide the first evidence
that Hsp27 confers bortezomib resistance. Moreover, MM cells obtained from
patients refractory to bortezomib treatment also show high levels of Hsp-27 expression. The mechanism(s) whereby Hsp-27 mediates bortezomib-resistance are
unclear. We and others have shown that Hsp-27 negatively regulates the release
of mitochondrial protein cytochrome-c and Smac, thereby blocking the intrinsic
cell death-signaling pathway (5860). Further studies are required to determine
whether inhibition of Hsp-27 using clinical gradespecific inhibitors enhances
bortezomib anti-MM activity and overcomes drug-resistance. Besides Hsp-27,
Bcl2 protein family members also confer drug-resistance in many cell types (61),
and bortezomib-triggered apoptosis in MM cells is also partially abrogated by
Bcl2 expression (17). Upregulated expression of inhibitors of apoptosis proteins
(IAPs), such as XIAP, may also contribute to bortezomib resistance (17). Indeed, it
is unlikely that one specific mechanism confers bortezomib resistance, suggesting
that combinations of bortezomib with other conventional and/or novel agents will
be required to overcome drug resistance.
To address this issue, in vitro studies showed that combining bortezomib with
other conventional agents, such as Dex, doxorubicin, melphalan, or mitoxantrone,
triggers additive and/or synergistic anti-MM activity (6, 18, 50). Moreover, combined treatment of MM cells and of MM patient cells with bortezomib and novel
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agents, such as relvimid or triterpenoids CDDO-Im, induces synergistic anti-MM

activity (18, 62). For example, bortezomib + CDDO-Im triggers synergistic apoptosis, even in bortezomib-resistant MM cells from patients, thereby providing the
basis for clinical protocols using this treatment regimen (62). Besides MM, combined bortezomib and irinotecan treatment also induces apoptosis in pancreatic
tumor xenografts (63). Another study showed that bortezomib prevents irinotecaninduced NF-B activation, thereby increasing chemosensitivity and apoptosis in
colorectal cancer cells in a xenograft model (64). Together, these combination
strategies will reduce attendant toxicity and overcome and/or prevent the development of drug-resistance.
Bortezomib in Clinic
It is known that bortezomib mediates its effects by inhibiting cellular proteasomes;
however, whether proteasome inhibition is universally required for bortezomibtriggered apoptosis is unclear. Our findings showed that treatment with bortezomib
led to 82% and 88% inhibition of proteasome activity in bortezomib-resistant
SUDHL4 and bortezomib-sensitive SUDHL6 lymphoma cells, respectively (53).
Together, these data confirm that (a) the proteasome inhibition pathway is not
defective in bortezomib-resistant DHL4 cells, and (b) proteasome inhibition is not
correlated with apoptosis.
Direct determination of proteasome inhibition in patient blood and tissue samples was examined in phase I studies. Bortezomib was well tolerated at doses,
resulting in up to 80% proteasome inhibition (65). Furthermore, extended dosing
did not further reduce sensitivity to proteasome inhibition. These data suggest
that proteasome inhibition is the main function of the proteasome inhibitor, but
that proteasome blockade may not correlate with degree of cytotoxicity in cancer
cells.
PHASE I TRIALS OF BORTEZOMIB

Phase I trials of bortezomib in hematologic and solid tumors confirmed the antineoplastic activity of bortezomib observed in preclinical in vitro studies (66, 67).
During an initial dose-ranging trial in patients with refractory MM, lymphoma,
and leukemia, patients received bortezomib twice weekly for 4 weeks followed by
2 weeks of no therapy. The maximum tolerated dose (MTD) was 1.04 mg/m2 (66).
Dose-limiting toxicities (DLTs) were fatigue and malaise, electrolyte imbalances,
and thrombocytopenia. Patients with lower than normal platelet counts at study
entry were at higher risk for the development of thrombocytopenia. Even in phase
I studies, encouraging responses were observed in MM patients: one complete
response (CR), evidenced by immunofixation-negativity, and eight responses with
reduction in serum monoclonal protein and marrow plasmacytosis. Bortezomib
antitumor activity in these phase I studies was also noted in non-Hodgkins lymphoma (NHL).
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The efficacy of bortezomib was evaluated in another phase I trial in advanced

solid tumors, using a 3-week dose cycle (twice weekly for 2 weeks followed
by 1 week of no therapy) (67). The MTD was 1.56 mg/m2, indicating that the
3-week cycle may allow administration of higher doses than the 6-week cycle.
No hematologic DLT was observed; and nonhematologic DLTs included grade-3
diarrhea and neuropathy. Of note, grade-3 neuropathy was observed predominantly
in patients with preexisting neuropathy, and improved after drug discontinuation.
Bortezomib also showed antitumor activity in other malignancies including nonsmall cell lung cancer, nasopharyngeal carcinoma, malignant melanoma, and renal
cell carcinoma (67).
PHASE II STUDIES IN MM
A phase II bortezomib study was conducted in MM patients who had relapsed
and were refractory to multiple prior therapies. Each cycle of therapy included
bortezomib (1.3 mg/m2) administered twice weekly for 2 weeks, with 1 week off
(19). Oral dexamethasone was given to patients with a suboptimal response after
two cycles, and eight cycles of therapy were given to responders. Two hundred and
two patients were enrolled, all of whom received corticosteroids, 92% alkylating
agents, 81% anthracyclines, 83% thalidomide, and 64% stem-cell transplant; the
median number of prior therapies was six. Poor prognostic factors at enrollment included elevated beta-2-microglobulin and abnormal cytogenetics. Of 193 patients,
4% achieved a CR, evidenced by MM protein undetectable by both electrophoresis
and immunofixation; 6% achieved showed a near CR, evidenced by MM protein
detectable only by immunofixation. Partial response (PR) was noted in 18% and
minimal response (MR) in 7% patients. Overall response rate (CR + PR + MR)
was 35%.
Response to bortezomib was examined relative to prognostic factors, including
the patients age; gender; type of MM; beta-microgloblin levels; extent of disease,
i.e., plasma cells in BM; chromosomal abnormalities, i.e., deletion of chromosome
13; and intensity of prior therapies. Statistical analysis (univariate) indicated that
>50% plasma cells in BM significantly predicted a lower response rate. Multivariate analysis suggested that older age (65 years or older) and >50% plasma cells
in BM significantly predicted for lower response rate. Major responses (CR and
PR) were associated with improved hemoglobin and nonmyeloma immunoglobulin levels, decreased transfusion requirements, increased platelet counts, and improvements in global quality of life and disease symptoms. Median time to disease
progression (TTP) for all patients was 7 months, compared with TTP of 3 months
for the last treatment before enrollment, and 13 months of TTP for patients achieving a CR or PR. Responses were durable: Median response duration was 12 months
among patients achieving an objective response (CR + PR + MR) and 15 months
among those achieving a CR or near CR. The median survival for the entire population (n = 202) was 16 months and patients achieving a major response (CR + PR)
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survived significantly longer than those who did not (P = 0.007). Of 74 patients
who did not achieve at least a MR and therefore received dexamethasone in combination with bortezomib, 18% improved; these included 6 patients with steroidrefractory disease, indicating that bortezomib can overcome resistance to steroids.
Most commonly reported adverse events were nausea, vomiting, diarrhea, fatigue,
loss of appetite including anorexia, constipation, anemia, thrombocytopenia, peripheral neuropathy, and pyrexia.
CONCLUSIONS
The proteasome is a promising target in the treatment of cancer. Ongoing research
in this field will unveil the complex mechanisms whereby proteasome inhibitors
impact a wide array of cellular functions with a differential sensitivity of normal
versus cancer cells. More specific therapeutic targets may be the E2 and E3 ubiquitin enzymes, which target unique proteins. Proteasomes have six active sites, and
blocking the chymotrypsin-like site decreases protein degradation significantly,
whereas inhibition of trypsin- or caspase-like sites is less effective. Whether simultaneous inhibition of all three activities is more cytotoxic to cancer versus
normal cells remains to be examined. The proteasome inhibitor bortezomib/PS341 has shown potent preclinical activity in vitro as well as therapeutic activity
in hematologic malignancies, especially MM. Importantly, bortezomib is the first
treatment in more than a decade to be FDA approved for patients with MM, and
the European Commission also granted marketing authorization for bortezomib
for the treatment of patients with MM who have received at least two prior therapies and have demonstrated disease progression on their last therapy. Ongoing
clinical trials are examining its efficacy in other hematologic malignancies and
solid tumors.
LITERATURE CITED
1. Anderson KC. 2004. Bortezomib therapy
for myeloma. Curr. Hematol. Rep. 3:65
2. Anderson KC. 2004. Plasma cell tumors. In
Cancer Medicine, ed. JF Holland, E Frei III,
RC Bast, DW Kufe, DL Norton, RR Weichselbaum. Baltimore: Williams and Wilkins.
In press
3. Wyllie A. 1980. Glucocorticoid-induced
thymocyte apoptosis is associated with endogenous endonuclease activation. Nature
284:55556
4. Reed JC. 1999. Dysregulation of apoptosis

in cancer. J. Clin. Oncol. 17:294153
5. Chauhan D, Hideshima T, Anderson KC.
2003. Apoptotic signaling in multiple
myeloma: therapeutic implications. Int. J.
Hematol. 78:11420
6. Hideshima T, Richardson P, Chauhan D,
Palombella VJ, Elliott PJ, et al. 2001.
The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple
7 Jan 2005
14:5
474

7.
8.
9.
10.
11.
12.
13.
14.
15.
AR
AR232-PA45-19.tex
CHAUHAN
AR232-PA45-19.sgm
HIDESHIMA
LaTeX2e(2002/01/18)
P1: GCE
ANDERSON
myeloma cells. Cancer Res. 61:3071

76
Chauhan D, Li G, Podar K, Hideshima
T, Shringarpure R, et al. 2003. Bortezomib/proteasome inhibitor PS-341 and
triterpenoid CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome bortezomib resistance.
Blood 103(8):315866
Anderson KC. 2003. Moving disease biology from the lab to the clinic. Cancer
97:796801
Nefedova Y, Landowski TH, Dalton WS.
2003. Bone marrow stromal-derived soluble factors and direct cell contact contribute
to de novo drug resistance of myeloma
cells by distinct mechanisms. Leukemia
17:117582
Read MA, Neish AS, Luscinskas FW,
Palombella VJ, Maniatis T, Collins T.
1995. The proteasome pathway is required for cytokine-induced endothelialleukocyte adhesion molecule expression.
Immunity 2:493506
Damiano JS, Cress AE, Hazlehurst LA,
Shtil AA, Dalton WS. 1999. Cell adhesion
mediated drug resistance (CAM-DR): role
of integrins and resistance to apoptosis in
human myeloma cell lines. Blood 93:1658
67
Schenkein D. 2002. Proteasome inhibitors
in the treatment of B-cell malignancies.
Clin. Lymphoma 3:4955
Vacca A, Ribatti D, Presta M, Minischetti
M, Iurlaro M, et al. 1999. Bone marrow
neovascularization, plasma cell angiogenic
potential, and matrix metalloproteinase-2
secretion parallel progression of human
multiple myeloma. Blood 93:306473
Podar K, Tai YT, Davies FE, Lentzsch S,
Sattler M, et al. 2001. Vascular endothelial
growth factor triggers signaling cascades
mediating multiple myeloma cell growth
and migration. Blood 98:42835
Oikawa T, Sasaki T, Nakamura M, Shimamura M, Tanahashi N, et al. 1998. The
proteasome is involved in angiogenesis.
Biochem. Biophys. Res. 246:24348
16. Sunwoo JB, Chen Z, Dong G, Yeh N, Crowl

Bancroft C, et al. 2001. Novel proteasome inhibitor PS-341 inhibits activation
of nuclear factor-kappa B, cell survival, tumor growth, and angiogenesis in squamous
cell carcinoma. Clin. Cancer Res. 7:1419
28
17. Mitsiades N, Mitsiades CS, Poulaki V,
Chauhan D, Fanourakis G, et al. 2002.
Molecular sequelae of proteasome inhibition in human multiple myeloma cells.
18. Mitsiades N, Mitsiades CS, Richardson
PG, Poulaki V, Tai YT, et al. 2003. The
proteasome inhibitor PS-341 potentiates
sensitivity of multiple myeloma cells
to conventional chemotherapeutic agents:
therapeutic applications. Blood 101:2377
80
19. Richardson PG, Barlogie B, Berenson J,
Singhal S, Jagannath S, et al. 2003. A phase
2 study of bortezomib in relapsed, refractory myeloma. N. Engl. J. Med. 348:2609
17
20. Karin M, Ben-Neriah Y. 2000. Phosphorylation meets ubiquitination: the control of
NF-[kappa]B activity. Annu. Rev. Immunol.
18:62163
21. Lee JC, Peter ME. 2003. Regulation of
apoptosis by ubiquitination. Immunol. Rev.
193:3947
22. Brooks CL, Gu W. 2003. Ubiquitination, phosphorylation and acetylation: the
molecular basis for p53 regulation. Curr.
Opin. Cell Biol. 15:16471
23. Almond JB, Cohen GM. 2002. The proteasome: a novel target for cancer chemotherapy. Leukemia 16:43343
24. Elliott PJ, Ross JS. 2001. The proteasome:
a new target for novel drug therapies. Am.
J. Clin. Pathol. 116:63746
25. Yew PR, Kirschner MW. 1997. Proteolysis
and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle. Science
277:167276
26. Verma R, Feldman RM, Deshaies RJ.
1997. SIC1 is ubiquitinated in vitro by
a pathway that requires CDC4, CDC34,
7 Jan 2005
14:5
AR
AR232-PA45-19.tex
AR232-PA45-19.sgm
LaTeX2e(2002/01/18)
P1: GCE
27.

28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
and cyclin/CDK activities. Mol. Biol. Cell.

8:142737
Goebl MG, Yochem J, Jentsch S, McGrath
JP, Varshavsky A, Byers B. 1988. The yeast
cell cycle gene CDC34 encodes a ubiquitinconjugating enzyme. Science 241:133135
Adams J. 2004. The proteasome: a suitable antineoplastic target. Nat. Rev. Cancer
4:34960
Goldberg AL, Rock K. 2002. Not just research toolsproteasome inhibitors offer
therapeutic promise. Nat. Med. 8:33840
Pickart CM. 2004. Back to the future with
ubiquitin. Cell 116:18190
Ciechanover A, Schwartz AL. 1998. The
ubiquitin-proteasome pathway: the complexity and myriad functions of proteins
death. Proc. Natl. Acad. Sci. USA 95:2727
30
Ciechanover A. 2003. The ubiquitin proteolytic system and pathogenesis of human
diseases: a novel platform for mechanismbased drug targeting. Biochem. Soc. Trans.
31:47481
Elliott PJ, Pien CS, McCormack ID, Chapman ID, Adams J. 1999. Proteasome inhibition: a novel mechanism to combat asthma.
J. Allergy Clin. Immunol. 104:17
Lupas A, Koster AJ, Baumeister W. 1993.
Structural features of 26S and 20S proteasomes. Enzyme Protein 47:25273
Eytan E, Ganoth D, Armon T, Hershko
A. 1989. ATP-dependent incorporation of
20S protease into the 26S complex that
degrades proteins conjugated to ubiquitin.
Masdehors P, Omura S, Merle-Beral H,
Mentz F, Cosset J-M, et al. 1999. Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by
the proteasome-specific inhibitor lactacystin. Br. J. Haematol. 105:75257
Drexler HC, Risau W, Konerding MA.
2000. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J. 14:6577
Kudo Y, Takata T, Ogawa I, Kaneda T, Sato
S, et al. 2000. p27Kip1 accumulation by
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
475
inhibition of proteasome function induces

apoptosis in oral squamous cell carcinoma
cells. Clin. Cancer Res. 6:91623
Bogner C, Schneller F, Hipp S, Ringshausen I, Peschel C, Decker T. 2003. Cycling B-CLL cells are highly susceptible
to inhibition of the proteasome: involvement of p27, early D-type cyclins, Bax, and
caspase-dependent and -independent pathways. Exp. Hematol. 31:21825
Guzman ML, Swiderski CF, Howard DS,
Grimes BA, Rossi RM, et al. 2002. Preferential induction of apoptosis for primary
human leukemic stem cells. Proc. Natl.
Stancovski I, Baltimore D. 1997. NF-B
activation: the IB kinase revealed? Cell
91:299302
Haefner B. 2002. NF-kappa B: arresting a
major culprit in cancer. Drug Discov. Today
7:65363
Jeremias I, Kupatt C, Baumann B, Herr
I, Wirth T, Debatin KM. 1998. Inhibition
of nuclear factor kappaB activation attenuates apoptosis resistance in lymphoid cells.
Blood 91:462431
Alkalay I, Yaron A, Hatzubai A, Jung S,
Avraham A, et al. 1995. In vivo stimulation of I kappa B phosphorylation is not
sufficient to activate NF-kappa B. Mol. Cell
Biol. 15:1294301
Karin M, Yamamoto Y, Wang QM. 2004.
The IKK NF-kappa B system: a treasure
trove for drug development. Nat. Rev. Drug
Discov. 3:1726
Chauhan D, Uchiyama H, Akbarali Y,
Urashima M, Yamamoto K, et al. 1996.
Multiple myeloma cell adhesion-induced
interleukin-6 expression in bone marrow
stromal cells involves activation of NFkappa B. Blood 87:110412
Feinman R, Koury J, Thames M, Barlogie
B, Epstein J. 1999. Role of NF-kB in the
rescue of multiple myeloma cells from
glucocorticoids-induced apoptosis by Bcl2. Blood 93:304452
Ogawa M, Nishiura T, Oritani K, Yoshida
H, Yoshimura M, et al. 2000. Cytokines
7 Jan 2005
14:5
476

49.
50.
51.
52.
53.
54.
55.
56.
57.
AR
AR232-PA45-19.tex
CHAUHAN
AR232-PA45-19.sgm
HIDESHIMA
LaTeX2e(2002/01/18)
P1: GCE
ANDERSON
prevent dexamethasone-induced apoptosis via the activation of mitogen-activated

protein kinase and phosphatidylinositol
3-kinase pathways in a new multiple myeloma cell line. Cancer Res. 60:4262
69
Chauhan D, Anderson KC. 2003. Mechanisms of cell death and survival in multiple
myeloma (MM): therapeutic implications.
Apoptosis 8:33743
Ma MH, Yang HH, Parker K, Manyak S,
Friedman JM, et al. 2003. The proteasome
inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells
to chemotherapeutic agents. Clin. Cancer
Res. 9:113644
Hideshima T, Chauhan D, Richardson P,
Mitsiades C, Mitsiades N, et al. 2002. NFkappa B as a therapeutic target in multiple
myeloma. J. Biol. Chem. 28:28
Mitsiades N, Mitsiades CS, Poulaki V,
Chauhan D, Richardson PG, et al. 2002.
Biologic sequelae of nuclear factor-kappaB
blockade in multiple myeloma: therapeutic
applications. Blood 99:407986
Chauhan D, Li G, Shringarpure R, Podar K, Ohtake Y, et al. 2003. Blockade of
Hsp27 overcomes bortezomib/proteasome
inhibitor PS-341 resistance in lymphoma
cells. Cancer Res. 63:617477
Chauhan D, Li G, Hideshima T, Podar K,
Mitsiades C, et al. 2003. JNK-dependent release of mitochondrial protein, Smac, during apoptosis in multiple myeloma (MM)
Chauhan D, Guilan L, Sattler M,
Hideshima T, Podar K, et al. 2003. Superoxide-dependent and independent mitochondrial signaling during apoptosis in
multiple myeloma (MM) cells. Oncogene
22:6296300
Hideshima T, Anderson KC. 2002. Molecular mechanisms of novel therapeutic approaches for multiple myeloma. Nat. Rev.
Cancer 2:92737
Hideshima T, Mitsiades C, Akiyama M,
Hayashi T, Chauhan D, et al. 2003. Molecular mechanisms mediating antimyeloma
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
activity of proteasome inhibitor PS-341.

Blood 101:153034
Chauhan D, Li G, Hideshima T, Podar K,
Mitsiades C, et al. 2003. Hsp27 inhibits
release of mitochondrial protein Smac in
multiple myeloma cells and confers dexamethasone resistance. Blood 102:337986
Concannon CG, Gorman AM, Samali A.
2003. On the role of Hsp27 in regulating
apoptosis. Apoptosis 8:6170
Bruey JM, Ducasse C, Bonniaud P, Ravagnan L, Susin SA, et al. 2000. Hsp27 negatively regulates cell death by interacting
with cytochrome c. Nat. Cell Biol. 2:645
52
Cory S, Adams JM. 2002. The Bcl2 family: regulators of the cellular life-or-death
switch. Nat. Rev. Cancer 2:64756
Chauhan D, Li G, Podar K, Hideshima T,
Shringarpure R, et al. 2004. The bortezomib/proteasome inhibitor PS-341 and
triterpenoid CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome bortezomib resistance.
Blood 103:315866
Shah SA, Potter MW, McDade TP, Ricciardi R, Perugini RA, et al. 2001. 26S proteasome inhibition induces apoptosis and
limits growth of human pancreatic cancer.
J. Cell Biochem. 82:11022
Cusack JC Jr, Liu R, Houston M, Abendroth K, Elliott PJ, et al. 2001. Enhanced
chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for
systemic nuclear factor-kappaB inhibition.
Adams J. 2002. Development of the proteasome inhibitor PS-341. Oncologist 7:916
Orlowski RZ, Stinchcombe TE, Mitchell
BS, Shea TC, Baldwin AS, et al. 2002.
Phase I trial of the proteasome inhibitor PS341 in patients with refractory hematologic
malignancies. J. Clin. Oncol. 20:442027
Aghajanian C, Soignet S, Dizon DS, Pien
CS, Adams J, et al. 2002. A phase I trial
of the novel proteasome inhibitor PS341 in
advanced solid tumor malignancies. Clin.
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2:28 PM
Figure 1 (A) Structure and function of proteasomes; (B) cross-sectional view of 26S proteasome complex; (C) process
of degradation of ubiquitinated proteins by proteasome complex; and (D) bortezomib/velcade blocks the proteasomal
protein degradation resulting in accumulation of cytotoxic proteins.

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Figure 3 Apoptotic signaling triggered by bortezomib in MM cells.
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CLINICAL AND TOXICOLOGICAL RELEVANCE OF

CYP2C9: Drug-Drug Interactions and
Pharmacogenetics
Allan E. Rettie1 and Jeffrey P. Jones2
1
Department of Medicinal Chemistry, University of Washington, Seattle,

Washington 98195; email: rettie@u.washington.edu
2
Department of Chemistry, Washington State University, Pullman,
Washington 99164; email: jpj@wsu.edu
Key Words cytochrome P450, structure-function, structure-activity, gene

regulation
Abstract CYP2C9 is a major cytochrome P450 enzyme that is involved in the
metabolic clearance of a wide variety of therapeutic agents, including nonsteroidal antiinflammatories, oral anticoagulants, and oral hypoglycemics. Disruption of CYP2C9
activity by metabolic inhibition or pharmacogenetic variability underlies many of the
adverse drug reactions that are associated with the enzyme. CYP2C9 is also the first
human P450 to be crystallized, and the structural basis for its substrate and inhibitor
selectivity is becoming increasingly clear. New, ultrapotent inhibitors of CYP2C9 have
been synthesised that aid in the development of quantitative structure-activity relationship (QSAR) models to facilitate drug redesign, and extensive resequencing of the
gene and studies of its regulation will undoubtedly help us understand interindividual
variability in drug response and toxicity controlled by this enzyme.
OVERVIEW
Cytochrome P450s are a superfamily of oxygen-activating enzymes that carry out
an enormous range of metabolic reactions on endogenous and exogenous substrates
in processes both beneficial and deleterious to the organism (1, 2). Therapeutically
administered drugs, endogenous eicosanoids, steroids, and bile salts, as well as
carcinogens and environmental pollutants, are but a few important targets for these
versatile catalysts (3). Annotation of the human genome has revealed the presence
of some 57 human P450 genes (4), but less than a dozen of these play important
roles in the hepatic clearance of drugs (5).
CYP2C9 is a major human liver form of P450 that has drawn considerable
attention from researchers owing, in large part, to its role in causing adverse drug
reactions (ADRs). ADRs, which are projected to cause hundreds of millions of
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dollars in health care costs per year in the United States alone, often result from
unanticipated changes in P450 enzyme activity secondary to genetic polymorphisms or metabolically based drug-drug interactions (6, 7). Both mechanisms
are highly pertinent to ADRs involving CYP2C9. These can be especially serious
because several of the enzymes substrates exhibit a narrow therapeutic index,
therefore the resulting clinical consequences can be severe.
This review summarizes our current knowledge of the enzymes structurefunction relationships, drug-drug interactions, pharmacogenetics, and gene regulation, and it attempts to relate these base-line parameters to key clinical and
toxicological features of this important enzyme. Several earlier reviews of CYP2C
enzyme properties, function, and genetics may prove to be useful adjuncts to this
material (811).
INTRODUCTION
CYP2C9 is one of four functional human CYP2C genes located on the long arm
of chromosome 10. Within the CYP2C subfamily, which also comprises CYP2C8,
CYP2C18, and CYP2C19, CYP2C9 is arguably the most important member for
several reasons. First, it is the largest contributor among these four isoforms to
total human liver microsomal P450 content, with estimates of mean microsomal levels ranging from 40 10 pmol/mg (12) to as high as 89 9 pmol/mg
(13). Only CYP3A4 is a more quantitatively significant P450 enzyme in human
liver. Second, like CYP3A4, CYP2C9 metabolizes a host of clinically important
drugs (Table 1). Indeed, it has been estimated that CYP2C9 is responsible for the
metabolic clearance of up to 15% of all drugs that undergo Phase I metabolism (5).
Third, drug-drug interactions pose therapeutic management problems for several
CYP2C9 substrates, especially those involving low therapeutic index substrates,
such as (S)-warfarin, tolbutamide, and phenytoin. Fourth, CYP2C9 is subject to
significant genetic polymorphism, such that up to 40% of Caucasian populations
are carriers of alleles that encode partially defective functional forms of the enzyme. Such gene-drug interactions can exacerbate adverse drug reactions with the
same battery of CYP2C9 substrates that display an intrinsically low margin of
safety.
Substrate Specificity: In Vitro and In Vivo Probes for CYP2C9

Three of the most commonly employed substrate probes for determining CYP2C9
activity in crude human tissue fractions are (S)-warfarin (7-hydroxylation), tolbutamide (methylhydroxylation), and diclofenac (4 -hydroxylation). Diclofenac has
the advantage that CYP2C9 catalyzes its metabolism with a high turnover number (ca 30/min). Although this is beneficial in allowing for facile, economical HPLC-UV assays to be employed for routine screening in vitro, substrate
depletion can prove problematic if this probe is used for determining kinetic
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CYP2C9 AND ADVERSE DRUG REACTIONS

TABLE 1
Common substrates for CYP2C9 by therapeutic class

Antiinflammatories
Oral
hypoglycemics
Oral
anticoagulants
Diuretics
and uricosurics
Angiotensin II
blockers
Flurbiprofen
Diclofenac
Naproxen
Piroxicam
Suprofen
Ibuprofen
Mefenamic acid
Celecoxib
Tolbutamide
Glyburide
Glipizide
Glimepiride
(S)-Warfarin
(S)-Acenocoumarol
(Phenprocoumon)
Torsemide
Ticrynafen
Sulfinpyrazone
sulfide
Losartan
Irbesartan
Candesartan
Class
(cont.)
Antiasthmatics
Anticonvulsants
Anticancer agents
Substrates
(cont.)
Zafirlukast
(Zileuton)
Phenytoin
(Phenobarbital)
(Trimethadione)
Cyclophosphamide
(Tamoxifen)
Class
Substrates

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Endogenous
compounds
Arachidonic acid
5-Hydroxytryptamine
Linoleic acid
Miscellaneous
Mestranol
Fluvastatin
9-Tetrahydrocannabinol
(Benzopyrene)
(Pyrene)
(Fluoxetine)
(Sildenafil)
(Rosiglitazone)
This listing is not intended to be exhaustive.
Parentheses indicates that (where known) other P450s or metabolic pathways play a major role in clearance.
parameters. Conversely, turnover of (S)-warfarin by CYP2C9 is extremely slow,

with a kcat of only 0.2/min. However, 7-hydroxywarfarin fluoresces strongly,
and this specific metabolite is readily quantitated from microsomal incubations
by HPLC with fluorescence detection. Tolbutamide is a convenient compromise
between these extremes, and confidence in its use is enhanced by the observation
of excellent correlations between rates of tolbutamide methylhydroxylation and
several other prototypical CYP2C9 activities in human liver microsomes (14).
Two of the best in vivo probes for CYP2C9 activity are tolbutamide and flurbiprofen (15, 16). Importantly, the clearance of each of these biomarkers is affected
in a predictable fashion by carriers of the CYP2C9 3 allele (see sections below),
thereby providing a pharmacogenetic validation for the in vivo probe. Interestingly, diclofenac is not a useful in vivo probe for the enzyme. Glucuronidation
of the parent is an important component of clearance, and the acyl glucuronide
itself is a substrate for CYP2C8, which then forms the 4 -hydroxy acyl glucuronide
(17). Consequently, CYP2C9-dependent formation of total (free plus conjugated)
4 -OH diclofenac in urine appears to be a modest contributor to the overall clearance of the drug. Safety concerns initially prevented the use of racemic warfarin as
an in vivo probe in healthy individuals. However, concomitant administration of
warfarin with vitamin K abrogates its pharmacodynamic effect and has permitted
its use in cocktail form to evaluate global P450 activity when administered with
probes for the other major P450 forms (18).
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Inhibitor Specificity: Ligand-Based Models for CYP2C9

Dangerous drug-drug interactions can arise when a CYP2C9 inhibitor is added to
a therapeutic regime that includes low therapeutic index drugs like (S)-warfarin,
tolbutamide, or phenytoin (7). In cases like these, patients can risk life-threatening
bleeding episodes, hypoglycemia, and neurotoxicity as a result of the diminished
CYP2C9 enzyme activity. Although considerable efforts have been expended to
construct computational P450 models directed toward predicting active-site structure or sites of substrate metabolism (1921), an important rationale for developing
predictive inhibitor-based models of CYP2C9 is that new therapeutic agents with
significant potential for drug-drug interactions may be identified early in the discovery process and appropriate structural modification initiated. It is axiomatic to
state that the study of a drugs functional group characteristics that impart high
affinity for a target enzyme or receptor also reveal complementary information
about the proteins binding pocket, and so these types of studies perform a dual
function.
Even cursory examination of the structural features of the drugs listed in Table 1 demonstrates that CYP2C9 exhibits selectivity for acidic compounds, as
exemplified by the large number of arylacetic acid or arylpropionic acids that
are substrates for the enzyme. Early studies, reviewed in Reference 22, established the basic CYP2C9 pharmacophore of a hydrogen bond donor heteroatom
from the substrate metabolism
and/or anionic moiety in the ligand located 78 A
site and separated by an intervening hydrophobic zone (2325). As more substrates
and inhibitors for CYP2C9 have emerged, it is evident that neutral compounds also
bind to this enzyme with high affinity (8, 26). However, for two very similar compounds, such as warfarin (ring closed and not an anion in the CYP2C9 active site)
and phenprocoumon (an anion at physiological pH), the anion is the tighter binder
to CYP2C9 (27).
The first quantitative structure-activity relationship (QSAR) for CYP2C9 inhibitors overlaid 19 coumarin derivatives; 5 carboxylate-containing drugs, including a number of NSAIDs; 2 sulfonamides, and phenytoin with Ki values ranging
from 100 nM30 M (28). The resulting partial least squares model, based on
the Comparative Molecular Field Analysis (CoMFA) program, had a standard error of the estimate of 0.17 log units. Subsequently, this QSAR model was able
to predict the binding affinity of 14 structurally diverse compounds, with a mean
error of approximately 6 M (29). Other three-dimensional (3D)-QSAR efforts
have focused on alignment-independent techniques that facilitate examination of
more structurally diverse training sets. In this regard, CYP2C9 inhibitor models
developed with the program Catalyst generally predicted Ki within 1 log residual
(30), and a conformer- and alignment-independent method predicted Ki for 11 of
12 structurally unrelated compounds within 0.5 log units (31).
A new generation of very-high-affinity CYP2C9 inhibitors is based on a 2alkyl, 3-benzoyl benzofuran template (32) (see Figure 1). This core structure
is found in the antiarrythmic agent, amiodarone, one of the most commonly
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coprescribed drugs with warfarin in the treatment of atrial fibrillation. Amiodarone

exhibits a clinically important drug-drug interaction with warfarin by inhibiting
P450-mediated clearance of both its enantiomers (33). Much of this effect may be
attributable to the major metabolite, desethylamiodarone, which has a Ki against
CYP2C9 of 2.3 M compared with 95 M for amiodarone itself (34). Comedication with amiodarone generally results in a decrease of the maintenance dose
of warfarin by 25%30% (35). The prototype in this series of new inhibitors is
benzbromarone, a uricosuric agent used in Europe and Japan. Its ADR liability
was first revealed with reports of a drug-drug interaction that occurs between
warfarin and benzbromarone, wherein the anticoagulant effect of warfarin is potentiated (36). Follow-up studies found that benzbromarones Ki for inhibition of
(S)-warfarin 7-hydroxylation by human liver microsomes was 10 nM, and the in
vivo clearance of (S)-warfarin in humans was reduced by approximately 50% upon
coadministration of the two drugs (37). In retrospect, given the potency of CYP2C9
inhibition by benzbromarone, this drug-drug interaction is not unexpected.
Almost two dozen benzbromarone analogs have now been synthesized and 2methyl-3-(3 ,5 -diiodo-4 hydroxybenzoyl)benzofuran (Figure 1) has emerged as
the most potent inhibitor of CYP2C9, with a Ki of 1 nM (26). The pharmacophore
obtained from CoMFA analysis, using a structurally diverse training set (n = 58)
that straddled four orders of magnitude of inhibitor potency, retained a number
of the earlier models features, but also reflected important new interactions. The
most striking of these is the identification of a region near the 1 position of
the benzofuran ring (see Figure 1) that exhibits negative steric interactions. The
relative affinities of the various classes of CYP2C9 inhibitors, the benzbromarones,
acyclic warfarins, sulfaphenazoles, and the cyclic hemi-ketal warfarins, are all
well predicted based on a combination of this new steric interaction, the degree
of negative charge(s) at a specific location(s) in the substrate, and the lipophilicity
of the hydrophobic zone. The reciprocal interactions with the CYP2C9 active site
are considered below.
CYP2C9 Structure: Site-Directed Mutagenesis

and Crystallization Studies
Because CYP2C9 mainly selects for substrates and inhibitors that are lipophilic
and weakly acidic, it would be expected that complementary interactions with both
hydrophobic and hydrogen bond donor or acceptor amino acids would occur in
the active site of the enzyme. Site-directed mutagenesis efforts from many groups
have identified numerous amino acids important to CYP2C9 function, including
Arg97, Arg108, Phe114, Arg144, Asp293, Ser286, Asn289, Ile359, Ser365, and
Phe476 (3843).
In particular, there is a strong support for a role for F114 and F476 in substrate
orienting interactions with (S)-warfarin and diclofenac because removal of these
aromatic residues substantially alters product regioselectivity (39, 43). The crystal
structure of CYP2C9 with (S)-warfarin bound confirms a central role for these two
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Figure 1 High-affinity CYP2C9 inhibitors.
aromatic residues in binding of this ligand (44). In contrast, Arg97 plays a more
important role in binding heme, whereas Asp293 appears to have dual functions
in controlling product regioselectivity as well as maintaining holo-enzyme stability (42, 4446). Ser286 and Asn289 are I-helix residues important in conferring
substrate specificity toward the NSAIDs, ibuprofen, and diclofenac (38), whereas
Ser365 appears to be the target nucleophile adducted by activation of tienilic acid
(43). Arg144 and Ile359 together largely determine the genetic background that
confers a wild-type phenotype (see following section).
Although significant progress was made prior to the initial crystallization of
CYP2C9 in mapping hydrophobic active site residues of CYP2C9 by mutagenesis,
the nature of the anionic binding site in CYP2C9 remained elusive. Early homology
modeling efforts gave rise to several predictions for charged amino acids that could
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be involved in polar interactions with CYP2C9 ligands, including Arg97, Arg105,

Arg108, and Asp293 (19, 20, 29, 41, 47). Recent mutagenesis and crystallization
studies now identify Arg108, specifically, in the NSAID substrate selectivity of
CYP2C9 (46, 48).
Interestingly, the first crystal structure of CYP2C9, with (S)-warfarin bound,
does not implicate Arg108 in active-site interactions with this ligand (44), and the
second, with flurbiprofen bound, does not implicate F476 (48). However, with the
solution of several crystal structures for CYP2C5, it has become apparent that
the binding of different ligands to a mammalian P450 can involve multiple substrate
binding modes (49, 50). Moreover, it is speculated by the authors of the first crystal
structure for CYP2C9 that (S)-warfarin is not bound in a catalytically productive
orientation (44). If this is the case, cocrystallization of CYP2C9 with multiple
ligands representative of the multiple binding pockets inferred from atypical kinetic
studies (see below) will be required to provide a detailed picture of the CYP2C9
active site and the enzymes interactions with structurally diverse ligands.
Atypical CYP2C9 Kinetics

A recently appreciated complicating factor in the prediction of drug-drug interactions and drug clearance from in vitro data is the atypical kinetic behavior exhibited
by several mammalian P450 enzymes (51, 52). Although CYP3A4 is the most extensively studied human P450 in this regard, an increasing allosteric literature
has accumulated for CYP2C9 over the past five years (53). Indeed, a recent systematic study of some 1500 structurally diverse compounds identified more than
30 activators of CYP2C9 activity from which a heteroactivation pharmacophore
for the enzyme was generated (54). Just as -naphthoflavone has emerged as the
prototypical effector molecule for CYP3A4, dapsone is the best documented activator of CYP2C9exhibiting heterotropic and homotropic positive cooperativity
(55). Recent mechanistic studies suggest that dapsone activation is accompanied
by a change in the partition between flurbiprofen hydroxylation and uncoupling
(56). More efficient catalysis in the presence of the activator may reflect a closer
approach of the substrate to the heme iron in the presence of the effector, as revealed recently by NMR (57). Although many scenarios might be envisioned for
the molecular basis underlying these phenomena (58), P450 activation kinetics is
generally held to result from multiple ligand occupancy in the active-site of the
isoform involved. Strong support for this view is derived from structural studies of
the soluble enzyme P450eryF. Spectral analysis demonstrated cooperative binding
of androstenedione and 9-amino-phenanthrene to P450eryF (Hill coefficients of
1.3), and crystallization of the protein with either of the ligands bound showed
that two molecules were present in the active site at the same time (59). No such
direct structural data are available for mammalian P450s that exhibit cooperative
ligand binding based on analysis of steady-state kinetics. However, site-directed
mutagenesis of CYP3A4 designed to crowd the active site of the enzyme (inferred
from homology modeling) has been shown to abolish cooperativity (60) and the
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crystal structures of CYP2C9 are indicative of a large active site that might readily
accommodate more than one ligand (44, 48).
Although CYP2C9 activation is now a well-documented phenomenon in vitro,
it remains to be seen whether it has clinical relevance. Few studies have been
performed as yet, but Tracy and coworkers did report a modest, yet statistically
significant, increase (11%) in flurbiprofen clearance following cotreatment with
dapsone in vivo (61). It is worth noting that an in vivo significance for such allosteric
phenomena with P450s is not established even for the much more intensively
studied CYP3A4 enzyme. Therefore, for the foreseeable future CYP2C9 activation
may remain an in vitro curiosity, albeit one that promotes new ideas about the
elasticity of the P450 active site.
CYP2C9 Pharmacogenetics
The first indications of polymorphism in the CYP2C9 gene arose when multiple
cDNAs were cloned in the late 1980s and early 1990s. Subsequently, a systematic
investigation of possible sites of allelic variation confirmed the existence of the
CYP2C9 2 and CYP2C9 3 variants at significant frequencies (close to 10%) in
a Northern European population (62). Population studies by several other groups
extended these findings and it is now clear that up to 40% of Caucasians possess one
or more variant CYP2C9 allele (63). This high frequency has prompted numerous
studies aimed at determining the functional effects of these common CYP2C9
variants.
The CYP2C9 2 allele reflects a missense mutation in exon 3 that causes a
nonconservative ArgCys substitution at amino acid 144. The consensus view
from in vitro studies conducted with the recombinantly expressed CYP2C9.2 is
that this mutation causes a small decrement in Vmax (0%35%) and little or no
change in the Km for substrate catalysis (64). In vivo studies have generally been
difficult to interpret owing to the paucity of CYP2C9 2/ 2 homozygotes available
for study, but recent clinical investigations that did include this test group also
suggest modest decreases in drug clearance attributable to this mutation (15, 65).
Arg144 maps to helix C, which is located on the exterior of the protein and forms
part of the putative P450 reductase binding site (66). Loss of activity may reflect
altered affinity for the coenzyme P450 reductase, which appears to bind reversibly
to positively charged surface amino acids on P450s (67).
The CYP2C9 3 allele arises from a missense mutation in exon 7 that causes
an IleLeu substitution at amino acid 359. In vitro and in vivo experiments consistently demonstrate substantial loss of enzyme activity owing to this mutation
(64). In fact, we recently identified five 3/ 3 homozygotes in a Caucasian anticoagulation clinic population and were able to demonstrate that this mutation
is associated with low warfarin dose and increased risk of bleeding during the
warfarin stabilization phase (68). Loss of activity for CYP2C9.3 reflects a combination of decreased Vmax and increased Km for CYP2C9 substrates (40). Recent
(unpublished) studies from our group confirm that the spectral binding constant,
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Ks, is increased substantially for CYP2C9.3 relative to the wild-type enzyme. The
structural basis for diminished P450 activity as a consequence of the 3 allele is
not yet clear, as new 3D information for CYP2C9 places this residue outside the
active site of the enzyme, some distance from the heme (44, 48). Ile359 is situated
below the SRS-6 region (70), which contains the important orienting amino acid
F476. The physical proximity of the 3 locus and this active-site region could conceivably result in global conformational changes that secondarily diminish binding
affinity. However, more studies are needed to better explain the functional deficit
attributable to this important CYP2C9 polymorphism.
Extensive resequencing of CYP2C9 in ethnically diverse populations demonstrates that this gene is highly polymorphic (71, 105) (http://egp.gs.washington.
edu). At the time of writing (April, 2004), a total of 12 CYP2C9 coding-region alleles were listed on the P450 Allele Web Site (http://www.imm.ki.se/CYPalleles),
and to our knowledge, all but the 4 and 7 alleles have been independently verified
by multiple research groups. In our own laboratory, resequencing across 60 kb of
CYP2C9 in 192 warfarin patients of Caucasian origin revealed a total of 129 single
nucleotide polymorphism (SNP) sites (105). The prevalence of coding-region mutations in this study population (allele frequency in parentheses) decreased in the
following order: 2 (11%), 3 (6%), 11 (1%), and 12 and 9 (0.5%). Consideration of sequence variation in the CYP2C9 gene allowed us to infer 23 haplotypes,
10 of which are represented at a frequency of >3% (105). In another study, resequencing of DNA from 92 individuals across three different racial groups predicted
at least 21 haplotypes (71). The 2 and 3 alleles are each isolated on one major
haplotype background, and both appear to be more significant contributors to variability in warfarin maintenance dose than any of the other eight major haplotypes
(105). A qualitatively similar situation has been reported for the warfarin analog
acenocoumarol (72).
CYP2C9 polymorphisms vary dramatically between different ethnic populations. An early genotyping study by Goldsteins group demonstrated that the
CYP2C9 2 and CYP2C9 3 variants that are common in Caucasians were represented in African-Americans but at much lower allele frequencies (1%2%) (73).
CYP2C9 6 was detected by the same group in one African-American patient who
had an adverse reaction to phenytoin. This rare, null allele is devoid of activity
owing to a splicing mutation that results in a truncated protein (74). CYP2C9 5,
D360E, is selectively expressed in African-Americans at an allele frequency of
1% (75, 76). Recombinant CYP2C9.5 exhibited a large increase in Km for several
substrates, but little change in Vmax, and we have suggested that this was predictive of a decrease in the in vivo catalytic efficiency of the enzyme. However, the
infrequence of this variant complicates further in vivo studies. Genotyping studies
in Korean and East Asian populations have not detected the CYP2C9 2 polymorphism, although the CYP2C9 3 allele is present at low frequencies (1%2%)
(77). Similar to the situation with CYP2C9 6, a rare polymorphism CYP2C9 4
(I359T), has been reported in one Japanese patient who had an adverse reaction
to phenytoin (78). Not surprisingly, recombinant CYP2C9.4 exhibited defective
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metabolism of several substrates in vitro (79). Recently, four new CYP2C9 SNPs
were reported in a Hong Kong Chinese anticoagulation clinic population, I181L,
H184P, Q192P, and L208V, but on closer examination these appear to be
spurious and likely a consequence of improper primer design (80). Further studies
are required to determine the frequency of occurrence of the newer ethnic-specific
CYP2C9 alleles, establish haplotypes in these populations, and delineate their
functional consequences.
Gene Regulation of CYP2C9

Whereas the great majority of drug-drug and pharmacogenetic interactions involving CYP2C9 result in an exacerbated clinical response, induction of CYP2C9
is associated with enhanced metabolic clearance and possible loss or diminution
of therapeutic activity. Consideration of in vivo drug-drug interaction data indicates that CYP2C9 is significantly induced by rifampin (81), and to a lesser extent
by phenobarbital and phenytoin (8). These observations can be replicated at the
mRNA and protein levels in primary human hepatocytes (82), thereby providing
a platform for detailed studies of CYP2C9 induction.
Several mechanisms exist for the upregulation of P450 genes, but enzyme induction most often involves transcriptional activation by nuclear receptors that
bind to cis-regulatory elements in the genes promoter (83). The CYP2C9 promoter contains at least four important regulatory elements: an HNF4 site located
at 139 to 125, a glucocorticoid responsive element at 1676 to 1662, a PXR
site at 1818 to 1802, and a CAR/PXR element at 2898 to 2882 (8486). The
PXR responsive element at 1818 appears to be the major contributor to rifampin
induction of CYP2C9 (87). Several other putative regulatory pathways have been
suggested, including those involving C/EBP (88) and vitamin D (89), but the
extent to which these and other regulatory mechanisms contribute to constitutive
expression of the gene remains to be established.
5 -Flanking polymorphisms of CYP2C9 are increasingly well documented.
Eleven SNPs have been reported in the first 2 kB of the promoter region in Japanese
and Caucasians, some of which may to be associated with altered gene transcription (90, 91). Marked allele frequency differences were noted between these two
ethnic groups that did not explain the population difference in (S)-warfarin clearance reported between the two populations (92). Nineteen promoter SNPs out to
2.7 kB are listed on the Environmental Genome Project Web site (http://egp.gs.
washington.edu/data/cyp2C9), and a further 40 SNPs have been identified out to
10 kB, thereby permitting a high resolution description of the haplotype structure
of the gene (vide infra). The extent to which these 5 -flanking polymorphisms contribute to interindividual variability in CYP2C9 status in different ethnic groups is
currently an active area of research.
Finally, the developmental expression pattern of CYP2C9 has recently been
established (93). Levels of CYP2C9 were 1%2% of mature values in the first
trimester, increasing substantially in late fetal life to approximately 30% of adult
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values. As for constitutive expression of the gene, further studies are needed to
evaluate upstream regulatory sequences and establish basic mechanisms of ontogenic regulation.

CYP2C9 and Endogenous Metabolism

Although members of the CYP 13 families are predominantly involved in the
metabolic clearance of drugs and other xenobiotics, pronounced extrahepatic expression in many cases has stimulated questions about their role in the metabolism
of endogenous substances. CYP2C9 protein expression has been demonstrated in
a wide variety of human extrahepatic tissues, both by Western blotting of tissue
microsomes and in situ immunohistochemistry (94, 95). CYP2C9 has also been
demonstrated to metabolize the endogenous compounds 5-hydroxytryptamine and
linoleic acid in vitro (96, 97); however, it is arachidonic acid that has garnered most
interest as an endogenous substrate for the enzyme.
Cytochromes P450, together with cyclooxygenase and lipoxygenase enzymes,
convert arachidonic acid to a plethora of products that exhibit critical pharmacological effects. CYP2 enzymes, in particular, have been implicated in the formation
of vasoactive epoxyeicosatrienoic acids (EETs) within the vascular system. EETs
relax vascular smooth muscle by opening potassium channels and hyperpolarizing smooth muscle cells. As such, EETs are prime candidates for the endothelialderived hyperpolarization factor (EDHF), a vasodilation pathway that remains after
inhibition of nitric oxide and prostacyclin-mediated responses. CYP2C9 generates
primarily 14,15-EET and 11,12-EET (98), and the enzyme is clearly expressed at
the protein level in a variety of endothelial cells (95).
CYP2C9 has also been linked with the putative EDHF synthase on the basis of
the finding that sulfaphenazole inhibited the EDHF-mediated response in pig coronary arteries, as did antisense oligonucleotides against CYP2C8/9 (99). Moreover,
nifedipine, an inducer of CYP2C enzymes, enhanced both endothelial mRNA expression of CYP2C and 11,12-EET production, also in pig coronary arteries (100).
However, given the lack of specificity of the molecular probes used in these studies and species differences in isoform expression, identification of CYP2C9 as an
EDHF synthase in human coronary vascular beds cannot be made with certainty.
Nonetheless, examination of the potential for CYP2C(9)-dependent vasoactivity to modulate cardiovascular disease is now an active area of investigation, and
one that extends interest in this enzyme to the new arena of disease pathology.
Recently, Yasar et al. concluded that possession of the more common genetic variants of CYP2C9 and CYP2C8 was associated with a modest increase in the risk
of acute myocardial infarction, at least in females (101). In the same study, no statistically significant associations were made between different CYP2C genotypes
and hypertension. However, other P450 isoforms such as CYP2J2, as well as nonP450 enzymes, such as soluble epoxide hydrolase, are also strongly implicated
in determining EET levels in humans (102). Future pharmacogenetic association
studies aimed at evaluating the role of these drug-metabolizing enzymes in complex diseases states such as hypertension will need to be multivariate in design.
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In conclusion, in the 20 plus years since CYP2C9 was first identified in human
liver (103), this isoform has become one of the most studied human P450s owing largely to its quantitative significance in oxidative drug metabolism, role in
adverse drug reactions, and pharmacogenetic variability. CYP2C9 is also the first
human P450 enzyme crystallized, and this pivotal event can be expected to propel
future structural, biochemical, biophysical, and clinical studies aimed at a fuller
understanding of this enzymes role in xenobiotic and endobiotic disposition.
ACKNOWLEDGMENTS
This work was supported in part by NIH grants GM32165, GM68797, and ES009
122. The authors would like to thank Dr. T.S. Tracy for helpful comments and
criticism.
LITERATURE CITED
1. Guengerich FP. 2001. Uncommon P450catalyzed reactions. Curr. Drug Metab.
2:93115
2. Guengerich FP. 2003. Cytochrome P450
oxidations in the generation of reactive
electrophiles: epoxidation and related reactions. Arch. Biochem. Biophys. 409:59
71
3. Gonzalez FJ, Kimura S. 2003. Study of
P450 function using gene knockout and
transgenic mice. Arch. Biochem. Biophys.
409:15358
4. Nelson DR. 2002. Introductory remarks
on human CYPs. Drug Metab. Rev. 34:
15
5. Evans WE, Relling MV. 1999. Pharmacogenomics: translating functional genomics into rational therapeutics. Science
286:48791
6. Pirmohamed M, Park BK. 2003.
Cytochrome P450 enzyme polymorphisms and adverse drug reactions.
Toxicology 192:2332
7. Thummel KE, Kunze KL, Shen DS. 2000.
Metabolically-based drug-drug interactions: principles and mechanisms. See
Ref. 104, pp. 319
8. Miners JO, Birkett DJ. 1998. Cytochrome

P4502C9: an enzyme of major importance
in human drug metabolism. Br. J. Clin.
Pharmacol. 45:52538
9. Rettie AE, Koop DR, Haining RL. 2000.
CYP2C Enzymes. See Ref. 104, pp. 75
86
10. Goldstein JA. 2001. Clinical relevance
of genetic polymorphisms in the human
CYP2C subfamily. Br. J. Clin. Pharmacol. 52:34955
11. Xie HG, Prasad HC, Kim RB, Stein CM.
2002. CYP2C9 allelic variants: ethnic distribution and functional significance. Adv.
Drug Deliv. Rev. 54:125770
12. Richardson TH, Griffin KJ, Jung F, Raucy
JL, Johnson EF. 1997. Targeted antipeptide antibodies to cytochrome P450 2C18
based on epitope mapping of an inhibitory monoclonal antibody to P450
2C51. Arch. Biochem. Biophys. 338:157
64
13. Lasker JM, Wester MR, Aramsombatdee E, Raucy JL. 1998. Characterization of CYP2C19 and CYP2C9 from
human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and
4 Dec 2004
13:15
AR
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
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14.

15.
16.
17.
18.
19.
20.
21.
omeprazole hydroxylations. Arch. Biochem. Biophys. 353:1628

Hall SD, Hamman MA, Rettie AE,
Wienkers LC, Trager WF, et al. 1994.
Relationships between the levels of cytochrome P4502C9 and its prototypic catalytic activities in human liver microsomes. Drug Metab. Dispos. 22:97578
Kirchheiner J, Bauer S, Meineke I,
Rohde W, Prang V, et al. 2002. Impact of
CYP2C9 and CYP2C19 polymorphisms
on tolbutamide kinetics and the insulin
and glucose response in healthy volunteers. Pharmacogenetics 12:1019
Lee CR, Pieper JA, Frye RF, Hinderliter
AL, Blaisdell JA, et al. 2003. Differences
in flurbiprofen pharmacokinetics between
CYP2C9 1/ 1, 1/ 2, and 1/ 3 genotypes. Eur. J. Clin. Pharmacol. 58:79194
Kumar S, Samuel K, Subramanian R,
Braun MP, Stearns RA, et al. 2002. Extrapolation of diclofenac clearance from
in vitro microsomal metabolism data:
role of acyl glucuronidation and sequential oxidative metabolism of the acyl
glucuronide. J. Pharmacol. Exp. Ther.
303:96978
Chainuvati S, Nafziger AN, Leeder JS,
Gaedigk A, Kearns GL, et al. 2003.
Combined phenotypic assessment of cytochrome p450 1A2, 2C9, 2C19, 2D6,
and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the Cooperstown 5+1 cocktail. Clin. Pharmacol.
Ther. 74:43747
Payne VA, Chang YT, Loew GH. 1999.
Homology modeling and substrate binding study of human CYP2C9 enzyme.
Proteins 37:17690
De Groot MJ, Alex AA, Jones BC. 2002.
Development of a combined protein and
pharmacophore model for cytochrome
P450 2C9. J. Med. Chem. 45:198393
Zamora I, Afzelius L, Cruciani G. 2003.
Predicting drug metabolism: a site of
metabolism prediction tool applied to the
cytochrome P450 2C9. J. Med. Chem.
46:231324
489
22. Ekins S, de Groot MJ, Jones JP. 2001.

Pharmacophore and three-dimensional
quantitative structure activity relationship
methods for modeling cytochrome p450
active sites. Drug Metab. Dispos. 29:936
44
23. Mancy A, Broto P, Dijols S, Dansette PM,
Mansuy D. 1995. The substrate binding
site of human liver cytochrome P450 2C9:
an approach using designed tienilic acid
derivatives and molecular modeling. Biochemistry 34:1036575
24. Jones BC, Hawksworth G, Horne VA,
Newlands A, Morsman J, et al. 1996.
Putative active site template model for
cytochrome P4502C9 (tolbutamide hydroxylase). Drug Metab. Dispos. 24:260
66
25. Mancy A, Dijols S, Poli S, Guengerich
P, Mansuy D. 1996. Interaction of sulfaphenazole derivatives with human liver
cytochromes P450 2C: molecular origin of the specific inhibitory effects of
sulfaphenazole on CYP 2C9 and consequences for the substrate binding site
topology of CYP 2C9. Biochemistry
35:1620512
26. Locuson CW, Rock DA, Jones JP. 2004.
Quantitative binding models for CYP2C9
based on benzbromarone analogs. Biochemistry 43:694858
27. He M, Korzekwa KR, Jones JP, Rettie
AE, Trager WF. 1999. Structural forms
of phenprocoumon and warfarin that are
metabolized at the active site of CYP2C9.
28
28. Jones JP, He M, Trager WF, Rettie
AE. 1996. Three-dimensional quantitative structure-activity relationship for inhibitors of cytochrome P4502C9. Drug
29. Rao S, Aoyama R, Schrag M, Trager
WF, Rettie A, et al. 2000. A refined
3-dimensional QSAR of cytochrome
P450 2C9: computational predictions of
drug interactions. J. Med. Chem. 43:
278996
4 Dec 2004
13:15

490
AR
RETTIE
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
P1: GCE
JONES
30. Ekins S, Bravi G, Binkley S, Gillespie

JS, Ring BJ, et al. 2000. Three- and fourdimensional-quantitative structure activity relationship (3D/4D-QSAR) analyses
of CYP2C9 inhibitors. Drug Metab. Dispos. 28:9941002
31. Afzelius L, Zamora I, Masimirembwa
CM, Karlen A, Andersson TB, et al. 2004.
Conformer- and alignment-independent
model for predicting structurally diverse
competitive CYP2C9 inhibitors. J. Med.
Chem. 47:90714
32. Locuson CW, Wahlstrom JL, Rock DA,
Rock DA, Jones JP. 2003. A new class
of CYP2C9 inhibitors: probing specifcity
with high-affinity benzbromarone derivatives. Drug Metab. Dispos. 31:96771
33. Heimark LD, Wienkers L, Kunze K,
Gibaldi M, Eddy AC, et al. 1992. The
mechanism of the interaction between
amiodarone and warfarin in humans. Clin.
34. Ohyama K, Nakajima M, Suzuki M, Shimada N, Yamazaki H, et al. 2000. Inhibitory effects of amiodarone and its
N-deethylated metabolite on human cytochrome P450 activities: prediction of in
vivo drug interactions. Br. J. Clin. Pharmacol. 49:24453
35. Gage BF, Eby C, Milligan PE, Banet GA,
Duncan JR, et al. 2004. Use of pharmacogenetics and clinical factors to predict the
maintenance dose of warfarin. Thromb.
Haemost. 91:8794
36. Shimodaira H, Takahashi K, Kano K,
Matsumoto Y, Uchida Y, et al. 1996.
Enhancement of anticoagulant action by
warfarin-benzbromarone interaction. J.
37. Takahashi H, Sato T, Shimoyama Y, Shioda N, Shimizu T, et al. 1999. Potentiation of anticoagulant effect of warfarin
caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone. Clin. Pharmacol. Ther. 66:569
81
38. Klose TS, Ibeanu GC, Ghanayem BI, Pedersen LG, Li L, et al. 1998. Identifica-
39.
40.
41.
42.
43.
44.
45.
46.
tion of residues 286 and 289 as critical

for conferring substrate specificity of human CYP2C9 for diclofenac and ibuprofen. Arch. Biochem. Biophys. 357:24048
Haining RL, Jones JP, Henne KR, Fisher
MB, Koop DR, et al. 1999. Enzymatic determinants of the substrate specificity of
CYP2C9: role of B-C loop residues in
providing the pi-stacking anchor site for
warfarin binding. Biochemistry 38:3285
92
Rettie AE, Haining RL, Bajpai M, Levy
RH. 1999. A common genetic basis for
idiosyncratic toxicity of warfarin and
phenytoin. Epilepsy Res. 35:25355
Ridderstrom M, Masimirembwa C,
Trump-Kallmeyer S, Ahlefelt M, Otter
C, et al. 2000. Arginines 97 and 108 in
CYP2C9 are important determinants of
the catalytic function. Biochem. Biophys.
Flanagan JU, McLaughlin LA, Paine
MJI, Sutcliffe MJ, Roberts GCK, et al.
2003. Role of conserved Asp(293) of cytochrome P4502C9 in substrate recognition and catalytic activity. Biochem. J.
370:92126
Melet A, Assrir N, Jean P, Lopez-Garcia
MP, Marques-Soares C, et al. 2003. Substrate selectivity of human cytochrome
P4502C9: importance of residues 476,
365, and 114 in recognition of diclofenac
and sulfaphenazole and in mechanismbased inactivation by tienilic acid. Arch.
Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, et al. 2003.
Crystal structure of human cytochrome
P450 2C9 with bound warfarin. Nature
424:46468
Davies C, Witham K, Scott JR, Pearson A,
DeVoss JJ, et al. 2004. Assessment of arginine 97 and lysine 72 as determinants of
substrate specificity in cytochrome P450
2C9 (CYP2C9). Drug. Metab. Dispos. 32:
43136
Dickmann LJ, Locuson CW, Jones JP,
Rettie AE. 2004. Differential roles of
4 Dec 2004
13:15
AR
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
P1: GCE

47.
48.
49.
50.
51.
52.
53.
54.
Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of

CYP2C9. Mol. Pharmacol. 65:84250
Jung F, Griffin KJ, Song W, Richardson
TH, Yang M, et al. 1998. Identification
of amino acid substitutions that confer a
high affinity for sulfaphenazole binding
and a high catalytic efficiency for warfarin
metabolism to P450 2C19. Biochemistry
37:1627079
Wester MR, Yano JK, Schoch GA, Yang
C, Griffin KJ, et al. 2004 The structure of
human cytochrome P450 2C9 complexed
with flurbiprofen at 2.0 A resolution. J.
Biol. Chem. 279:3563037
C, Dansette PM, Mansuy D, et al. 2003.
Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate
binding modes. Biochemistry 42:6370
79
C, Dijols S, Dansette PM, et al. 2003.
Structure of mammalian cytochrome
P450 2C5 complexed with diclofenac at
2.1 A resolution: evidence for an induced
fit model of substrate binding. Biochemistry 42:933545
Houston JB, Kenworthy KE. 2000. In
vitro-in vivo scaling of CYP kinetic
data not consistent with the classical
Michaelis-Menten model. Drug Metab.
Dispos. 28:24654
Atkins WM, Wang RW, Lu AY. 2001.
Allosteric behavior in cytochrome p450dependent in vitro drug-drug interactions:
a prospective based on conformational dynamics. Chem. Res. Toxicol. 14:33847
Korzekwa KR, Krishnamachary N, Shou
M, Ogai A, Parise RA, et al. 1998. Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence
that multiple substrates can simultaneously bind to cytochrome P450 active
sites. Biochemistry 37:413747
Egnell AC, Eriksson C, Albertson N,
Houston B, Boyer S. 2003. Generation
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
491
and evaluation of a CYP2C9 heteroactivation pharmacophore. J. Pharmacol. Exp.

Ther. 307:87887
Hutzler JM, Tracy TS. 2002. Atypical kinetic profiles in drug metabolism reactions. Drug Metab. Dispos. 30:35562
Hutzler JM, Wienkers LC, Wahlstrom
JL, Carlson TJ, Tracy TS. 2003. Activation of cytochrome P450 2C9-mediated
metabolism: mechanistic evidence in
support of kinetic observations. Arch.
Hummel MA, Gannett PM, Aguilar JS,
Tracy TS. 2004. Effector-mediated alteration of substrate orientation in cytochrome P450 2C9. Biochemistry 42:
720714
Hlavica P, Lewis DF. 2001. Allosteric
phenomena in cytochrome P450-catalyzed monooxygenations. Eur. J. Biochem. 268:481732
Cupp-Vickery J, Anderson R, Hatziris Z.
2000. Crystal structures of ligand complexes of P450eryF exhibiting homotropic
cooperativity. Proc. Natl. Acad. Sci. USA
97:305055
Domanski TL, Halpert JR. 2001. Analysis
of mammalian cytochrome P450 structure
and function by site-directed mutagenesis.
Curr. Drug Metab. 2:11737
Hutzler JM, Frye RF, Korzekwa KR,
Branch RA, Huang SM, et al. 2001.
Minimal in vivo activation of CYP2C9mediated flurbiprofen metabolism by dapsone. Eur. J. Pharm. Sci. 14:4752
Stubbins MJ, Harries LW, Smith G, Tarbit MH, Wolf CR. 1996. Genetic analysis
of the human cytochrome P450 CYP2C9
locus. Pharmacogenetics 6:42939
Yasar U, Eliasson E, Dahl ML, Johansson I, Ingelman-Sundberg M, et al. 1999.
Validation of methods for CYP2C9 genotyping: frequencies of mutant alleles in
a Swedish population. Biochem. Biophys.
Lee CR, Goldstein JA, Pieper JA. 2002.
Cytochrome P450 2C9 polymorphisms: a
comprehensive review of the in-vitro and
4 Dec 2004
13:15
492
65.

66.
67.
68.
69.
70.
71.
72.
73.
AR
RETTIE
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
P1: GCE
JONES
human data. Pharmacogenetics 12:251

63
Kirchheiner J, Brockmoller J, Meineke I,
Bauer S, Rohde W, et al. 2002. Impact of
CYP2C9 amino acid polymorphisms on
glyburide kinetics and on the insulin and
glucose response in healthy volunteers.
Scott EE, White MA, He YA, Johnson EF,
Stout CD, et al. 2004. Structure of mammalian cytochrome P450 2B4 complexed
with 4-(4-chlorophenyl)imidazole at 1.9
{angstrom} resolution: insight into the
range of P450 conformations and coordination of redox partner binding. J. Biol.
Chem. 279:27294301
Crespi CL, Miller VP. 1997. The R144C
change in the CYP2C9 2 allele alters interaction of the cytochrome P450 with
NADPH:cytochrome P450 oxidoreductase. Pharmacogenetics 7:20310
Higashi MK, Veenstra DL, Kondo LM,
Wittkowsky AK, Srinouanprachanh SL,
et al. 2002. Association between CYP2C9
genetic variants and anticoagulationrelated outcomes during warfarin therapy.
JAMA 287:169098
Deleted in proof
Gotoh O. 1992. Substrate recognition sites
in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses
of amino acid and coding nucleotide sequences. J. Biol. Chem. 267:8390
Blaisdell J, Jorge-Nebert LF, Coulter S,
Ferguson SS, Lee SJ, et al. (2004). Discovery of new potentially defective alleles
of CYP2C9. Pharmacogenetics 14:527
37
Morin S, Bodin L, Loriot MA, Thijssen
HH, Robert A, et al. 2004. Pharmacogenetics of acenocoumarol pharmacodynamics. Clin. Pharmacol. Ther. 75:403
14
Sullivan-Klose TH, Ghanayem BI, Bell
DA, Zhang ZY, Kaminsky LS, et al. 1996.
The role of the CYP2C9-Leu359 allelic
variant in the tolbutamide polymorphism.
Pharmacogenetics 6:34149
74. Kidd RS, Curry TB, Gallagher S, Edeki

T, Blaisdell J, et al. 2001. Identification of
a null allele of CYP2C9 in an AfricanAmerican exhibiting toxicity to phenytoin. Pharmacogenetics 11:8038
75. Dickmann LJ, Rettie AE, Kneller MB,
Kim RB, Wood AJ, et al. 2001. Identification and functional characterization of
a new CYP2C9 variant (CYP2C9 5) expressed among African Americans. Mol.
Pharmacol. 60:38287
76. Yasar U, Aklillu E, Canaparo R, Sandberg M, Sayi J, et al. 2002. Analysis of
CYP2C9 5 in Caucasian, Oriental and
Black-African populations. Eur. J. Clin.
Pharmacol. 58:55558
77. Yoon YR, Shon JH, Kim MK, Lim YC,
Lee HR, et al. 2001. Frequency of cytochrome P450 2C9 mutant alleles in a
Korean population. Br. J. Clin. Pharmacol. 51:27780
78. Imai J, Ieiri I, Mamiya K, Miyahara S,
Furuumi H, et al. 2000. Polymorphism of
the cytochrome P450 (CYP) 2C9 gene in
Japanese epileptic patients: genetic analysis of the CYP2C9 locus. Pharmacogenetics 10:8589
79. Ieiri I, Tainaka H, Morita T, Hadama
A, Mamiya K, et al. 2000. Catalytic activity of three variants (Ile, Leu, and
Thr) at amino acid residue 359 in human
CYP2C9 gene and simultaneous detection
using single-strand conformation polymorphism analysis. Ther. Drug Monit.
22:23744
80. Rettie AE, Tai G, Veenstra DL, Farin FM,
Srinouanprachan S, et al. 2003. CYP2C9
exon 4 mutations and warfarin dose phenotype in Asians. Blood 101:289697
81. Heimark LD, Gibaldi M, Trager WF,
OReilly RA, Goulart DA. 1987. The
mechanism of the warfarin-rifampin drug
interaction in humans. Clin. Pharmacol.
Ther. 42:38894
82. Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, et al. 2003. Effects
of prototypical microsomal enzyme inducers on cytochrome P450 expression in
4 Dec 2004
13:15
AR
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
P1: GCE
83.

84.
85.
86.
87.
88.
89.
90.
91.
cultured human hepatocytes. Drug Metab.

Dispos. 31:42131
Honkakoski P, Sueyoshi T, Negishi M.
2003. Drug-activated nuclear receptors
CAR and PXR. Ann. Med. 35:172
82
Ibeanu GC, Goldstein JA. 1995. Transcriptional regulation of human CYP2C
genes: functional comparison of CYP2C9
and CYP2C18 promoter regions. Biochemistry 34:802836
Gerbal-Chaloin S, Daujat M, Pascussi JM,
Pichard-Garcia L, Vilarem MJ, et al. 2002.
Transcriptional regulation of CYP2C9
gene. Role of glucocorticoid receptor and
constitutive androstane receptor. J. Biol.
Chem. 277:20917
Ferguson SS, LeCluyse EL, Negishi M,
Goldstein JA. 2002. Regulation of human
CYP2C9 by the constitutive androstane
receptor: discovery of a new distal binding
site. Mol. Pharmacol. 62:73746
Chen Y, Ferguson SS, Negishi M, Goldstein JA. 2004. Induction of human
CYP2C9 by rifampicin, hyperforin, and
phenobarbital is mediated by the pregnane X receptor. J. Pharmacol. Exp. Ther.
308:495501
Jover R, Bort R, Gomez-Lechon MJ,
Castell JV. 1998. Re-expression of C/EBP
alpha induces CYP2B6, CYP2C9 and
CYP2D6 genes in HepG2 cells. FEBS
Lett. 431:22730
Drocourt L, Ourlin JC, Pascussi JM, Maurel P, Vilarem MJ. 2002. Expression of
CYP3A4, CYP2B6 and CYP2C9 is regulated by the vitamin D receptor pathway
in primary human hepatocytes. J. Biol.
Chem. 277:2512532
Shintani M, Ieiri I, Inoue K, Mamiya K,
Ninomiya H, et al. 2001. Genetic polymorphisms and functional characterization of the 5 -flanking region of the human
CYP2C9 gene: in vitro and in vivo studies.
Takahashi H, Ieiri I, Wilkinson GR, Mayo
G, Kashima T, et al. 2004. 5 -Flanking region polymorphisms of CYP2C9 and their
92.
93.
94.
95.
96.
97.
98.
99.
100.
493
relationship to S-warfarin metabolism

in white and Japanese patients. Blood
103:305557
Takahashi H, Wilkinson GR, Caraco
Y, Muszkat M, Kim RB, et al. 2003.
Population differences in S-warfarin
metabolism between CYP2C9 genotypematched Caucasian and Japanese patients.
Koukouritaki SB, Manro JR, Marsh SA,
Stevens JC, Rettie AE, et al. 2004. Developmental expression of human hepatic
CYP2C9 and CYP2C19. J. Pharmacol.
Exp. Ther. 308:96574
Klose TS, Blaisdell JA, Goldstein JA.
1999. Gene structure of CYP2C8 and
extrahepatic distribution of the human
CYP2Cs. J. Biochem. Mol. Toxicol. 13:
28995
Enayetallah AE, French RA, Thibodeau
MS, Grant DF. 2004. Distribution of soluble epoxide hydrolase and of cytochrome
P450 2C8, 2C9, and 2J2 in human tissues.
J. Histochem. Cytochem. 52:44754
Draper AJ, Hammock BD. 2000. Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase. Arch.
Fradette C, Yamaguchi N, Du Souich P.
2004. 5-Hydroxytryptamine is biotransformed by CYP2C9, 2C19 and 2B6 to
hydroxylamine, which is converted into
nitric oxide. Br. J. Pharmacol. 141:407
14
Daikh BE, Lasker JM, Raucy JL, Koop
DR. 1994. Regioselective and stereoselective epoxidation of arachidonic-acid by
human cytochromes P450 2c8 and 2c9. J.
Fleming I, Michaelis UR, Bredenkotter D, Fisslthaler B, Dehghani F, et al.
2001. Endothelium-derived hyperpolarizing factor synthase (cytochrome P450
2C9) is a functionally significant source
of reactive oxygen species in coronary arteries. Circ. Res. 88:4451
Fisslthaler B, Hinsch N, Chataigneau T,
Popp R, Kiss L, et al. 2000. Nifedipine
4 Dec 2004
13:15

494
AR
RETTIE
AR232-PA45-20.tex
AR232-PA45-20.sgm
LaTeX2e(2002/01/18)
P1: GCE
JONES
increases cytochrome P4502C expression and endothelium-derived hyperpolarizing factor-mediated responses in

coronary arteries. Hypertension 36:270
75
101. Yasar U, Bennet AM, Eliasson E, Lundgren S, Wiman B, et al. 2003. Allelic variants of cytochromes P450 2C modify the
risk for acute myocardial infarction. Pharmacogenetics 13:71520
102. Kroetz DL, Zeldin DC. 2002. Cytochrome P450 pathways of arachidonic
acid metabolism. Curr. Opin. Lipidol. 13:
27383
103. Wang PP, Beaune P, Kaminsky LS, Dan-
nan GA, Kadlubar FF, et al. 1983.

Purification and characterization of six
cytochrome P-450 isozymes from human
liver microsomes. Biochemistry 22:5375
83
104. Levy RH, Thummel KE, Trager WF,
Hansten PD, Eichelbaum M, eds. 2000.
Metabolic Drug Interactions. Philadelphia: Lippincott Williams & Wilkins
105. Tai G, Rieder MJ, Dreisbach AW, Farin
FM, Srinouanpranach S, et al. 2004.
CYP2C9 sequence diversity, haplotype
analysis and genotype-phenotype correlations of warfarin dose. Drug Metab. Rev.
36(Suppl. 1):123
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Copyright

CLINICAL DEVELOPMENT OF HISTONE

DEACETYLASE INHIBITORS AS ANTICANCER
AGENTS
Daryl C. Drummond,1 Charles O. Noble,2,3
Dmitri B. Kirpotin,1 Zexiong Guo,2 Gary K. Scott,4
and Christopher C. Benz4
1
Hermes Biosciences, Inc., South San Francisco, California 94080

California Pacific Medical Center-Research Institute, San Francisco,
California 94115
3
University of California at San Francisco, San Francisco, California 94143
4
Buck Institute for Age Research, Novato, California 94945
2
Key Words HDAC inhibitors, targeting chromatin structure and epigenetic

mechanisms, transcription regulation, hydroxamic acids
Abstract Acetylation is a key posttranslational modification of many proteins responsible for regulating critical intracellular pathways. Although histones are the most
thoroughly studied of acetylated protein substrates, histone acetyltransferases (HATs)
and deacetylases (HDACs) are also responsible for modifying the activity of diverse
types of nonhistone proteins, including transcription factors and signal transduction
mediators. HDACs have emerged as uncredentialed molecular targets for the development of enzymatic inhibitors to treat human cancer, and six structurally distinct drug
classes have been identified with in vivo bioavailability and intracellular capability to
inhibit many of the known mammalian members representing the two general types of
NAD+-independent yeast HDACs, Rpd3 (HDACs 1, 2, 3, 8) and Hda1 (HDACs 4, 5, 6,
7, 9a, 9b, 10). Initial clinical trials indicate that HDAC inhibitors from several different
structural classes are very well tolerated and exhibit clinical activity against a variety
of human malignancies; however, the molecular basis for their anticancer selectivity
remains largely unknown. HDAC inhibitors have also shown preclinical promise when
combined with other therapeutic agents, and innovative drug delivery strategies, including liposome encapsulation, may further enhance their clinical development and
Nonstandard abbreviations: AOE, 2-amino-8-oxo-9,10-epoxy-decanoic acid; ATRA, alltrans-retinoic acid; CBHA, m-carboxylcinnamic acid bis-hydroxamide; CDK, cyclindependent kinase; DAC, 5-aza-2 -deoxycytidine; HAT, histone acetyltransferase; HDAC,
histone deacetylase; HMT, histone methyltransferase; IMID-1, immunomodulatory thalidomide derivative 1; PB, phenyl butyrate; SAHA, suberoylanilide hydroxamic acid; SB,
sodium butyrate; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TSA,
trichostatin A.
0362-1642/05/0210-0495$14.00
495
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DRUMMOND ET AL.
anticancer potential. An improved understanding of the mechanistic role of specific

HDACs in human tumorigenesis, as well as the identification of more specific HDAC
inhibitors, will likely accelerate the clinical development and broaden the future scope
and utility of HDAC inhibitors for cancer treatment.

INTRODUCTION
The packaging of DNA into the higher order and dynamic structure of chromatin
provides a pivotal point of control for gene expression by regulating access of
transcription factors to DNA. Chromatin is composed of multiple repeating units
termed nucleosomes, which are comprised of 146 base pairs of DNA wrapped
around a core of eight histone proteins composed of two copies each of H2A,
H2B, H3, and H4. Posttranslational modifications play a prominent role in the
regulation of gene expression and signal transduction pathways. Phosphorylation,
methylation, acetylation, ubquitination, and sumoylation are the known modifications thought to influence chromatin architecture and regulate gene transcription. The composition and consequences of these various histone modifications
are often referred to as the histone code, orchestrating an intricate regulation of
nucleosomal structure, DNA accessibility, and gene transcription (Figure 1). To
date, acetylation is the most thoroughly studied of these modifications; and while
the acetylation state of chromatin proteins is unquestionably very dynamic, it
seems to depend on the net local balance between histone acetyltransferase (HAT)
and histone deacetylase (HDAC) activities. Empirically observed is the fact that
HDAC activity is invariably increased in cancer cells, resulting in altered gene
transcription, impaired differentiation, increased cell survival, and dysregulated
proliferation (1).
Transcriptional Regulation and the Histone Code

Early models of how acetylation regulates transcription focused on the physical interactions of the basic histone proteins with negatively charged DNA. The addition
of charge-neutralizing acetyl groups to lysine residues on histones disrupts interactions with DNA, resulting in decompaction of chromatin, greater access of the
DNA to transcription factors, and the presence of a transcriptionally active genomic
locus. However, there is considerable evidence that these models are oversimplified. In cell culture studies, less than 10% of transcriptionally active genes appear
to be altered in response to treatment with HDAC inhibitors, with a near equal
proportion of these being induced as repressed (2, 3). This suggests that regulation
of gene expression by acetylation is more highly selective than would be expected
by a simple and unregulated physical disruption of histone-DNA structure, and
also likely involves chromatin-associated nonhistone proteins.
Nonetheless, the complex network of interdependent and site-specific histone
modifications associated with restricted and sequence-specific DNA binding by
transcription factors has resulted in a histone code hypothesis for gene-specific
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HDAC INHIBITORS FOR CANCER THERAPY
497
transcriptional control (4, 5). The code is set by a variety of histone tailderivatizing
enzymes, including HATs for the acetylation of lysine residues (6), histone methyltransferases (HMTs) for methylation of histone lysine and arginine residues (7,
8), serine kinases for the phosphorylation of specific histone serine residues (9),
ubiquitin ligase for the addition of the 76-amino-acid 9-kDa protein ubiquitin
to specific lysine residues (10), and the sumoylation of lysine by the 11-kDa
small ubiquitin-related modifier (SUMO) (11). In addition, the activities of modification destabilizing enzymes such as HDACs, methylases, phosphatases, and
ubiquitin and ULP-related proteases help shape the status of the code. The complexity of transcriptional regulation by histone modifications is further enhanced
by the interaction of HATs and HDACs with other proteins involved in chromatin modification, including methyl CpG-binding proteins and ATP-dependent
chromatin-remodeling complexes, which can lead to replication propagated and
more enduring epigentic modifications of DNA, such as the gene silencing cytosine
methylation of specific CpG dinucleotides (1214).
The setting of the histone code involves establishing defined patterns of histone
tail modifications, whereupon a particular modification in turn affects subsequent
modifications. For example, histone deacetylation has been shown to activate lysine
(K) methylation, resulting in relatively stable transcriptional silencing (15). In an
eloquent experiment demonstrating sequential histone modifications, Kouzarides
and colleagues showed that upon estrogen stimulation, H3 is acetylated initially
at K18, then at K23, and finally methylated at R17 (16). A specific set of histone modifications was proposed to direct DNA methylation (17). The reading of
the code can be accomplished through recognition of particular modifications or
groups of modifications (18, 19). The bromodomain of proteins such as BRG1 and
TAFII250 and the chromodomain of HP1 recognize acetylated lysines and methylated lysines, respectively (2022). Certain combinations of modifications can also
dictate the recruitment of various cis- or trans-acting regulatory proteins. The role
of the particular modification in transcriptional signaling may also be influenced
by the degree and stability of the modification. Lysine residues may be modified
with one, two, or three methyl groups, and the degree of methylation determines
if transcription of certain genes is activated or repressed (23, 24). The methylated
lysines, and more so the methylated cytosines in DNA, are more stable modifications than the relatively dynamic modifications of histone tail acetylation and
phosphorylation. Thus, with the lack of any known histone or DNA demethylases,
methylation may be more important in epigenetic memory, whereas the acetylation status of histones may be more of a switch that can be rapidly reset and allow
transcription to respond more rapidly to changes in the cells environment.
The histone code is just beginning to be deciphered and thus its complexity
and its role in carcinogenesis are far from understood. Although it is obvious that
a wide variety of posttranslational protein modifications are responsible for regulating transcription of any given gene and as such can play important roles in
human cancer cell behavior, the remainder of this review focuses specifically on
the preclinical and clinical development of HDAC inhibitors as potential anticancer
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agents. In this regard it is important to note that the activity of a wide variety of
nonhistone transcription factors and co-regulators of transcription are known to
be modified by acetylation, and both are structurally and functionally affected by
HDAC inhibitors. Acetylation may enhance or inhibit the function of transcriptional activators as well as transcriptional repressors; therefore, enhancing their degree of acetylation by cell treatment with an HDAC inhibitor can either increase or
repress the transcription of genes regulated by such nonhistone proteins (Table 1).
TFIIE (25), TFIIF (25), p53 (26), androgen receptor (27), estrogen receptor- (28),
and GATA-1 (29, 30) are promoter-binding and transcription-regulating proteins
shown to be acetylated in response to HDAC inhibition. In addition, other DNA
binding nonhistone proteins are functionally affected by acetylation. For example,
HMG-17 is a nucleosomal binding protein responsible for unfolding the higher order structure of chromatin and thus exerts indirect control over gene transcription;
and acetylation of HMG-17 has been shown to reduce its binding to chromatin
(31).
Classification of HDACs
There are three major groups or classes of mammalian HDACs based on their
structural homologies to the three distinct yeast HDACs: Rpd3 (class I), Hda1
(class II), and Sir2/Hst (class III). Class III HDACs consist of the large family of
sirtuins (SIRs) that are evolutionarily distinct, with a unique enzymatic mechanism dependent on the cofactor NAD+, and are virtually unaffected by all HDAC
inhibitors currently under development (32, 33). This review focuses on the NAD+independent class I and II HDACs (Figure 3), as they are evolutionarily similar,
contain an active site zinc as a critical component of their enzymatic pocket, have
been more thoroughly described in association with cancer, and are thought to
be comparably inhibited by most currently available HDAC inhibitors. The Rpd3
homologous class I HDACs include HDAC1, HDAC2, HDAC3, and HDAC8.
They are widely expressed in a variety of tissues and are primarily localized in
the nucleus. The Hda1 homologous class II HDACs include HDAC4, HDAC5,
HDAC6, HDAC7, HDAC9 (a and b isoforms), and HDAC10, and are structurally
much larger in size. Class II HDACs can shuttle between the nucleus and cytoplasm (3437), suggesting different functions and cellular substrates from Class I
HDACs. HDAC6 in particular is predominantly localized in the cytoplasm (38).
Class II HDACs also display a more limited tissue distribution (3941). HDACs
4, 8, and 9 are expressed to a greater extent in tumor tissues than in normal tissues,
with HDAC 4 demonstrating the greatest difference in this regard (39). Class II
enzymes have also been shown to be specifically involved in differentiation (40).
Finally, HDAC6 and HDAC10 are unique among class II HDACs in having two
catalytic domains (37, 40). Although there is some evidence that certain HDAC inhibitors display different degrees of HDAC specificity, considerable research must
still be performed to delineate differences in HDAC function, their roles in cancer,
and their sensitivities to drugs. Some of these differentiating features are reviewed
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TABLE 1 Nonhistone proteins whose acetylation may be increased by HDAC inhibitors

Protein
Intracellular function
Reference(s)
p53
Tumor suppressor
(26, 145, 146)
c-Myb
Protooncogeneregulates proliferation and

differentiation
(147)
GATA-1
Differentiation of blood cells
(29, 30)
Estrogen
receptor-
Stimulates growth of certain breast cancers
(28)
TFIIE
General transcription factor
(25)
TFIIF
General tanscription factor
(25)
Androgen
receptor
Androgen-dependent transcription factor
(27)
hsp90
Chaperonetargets proteins for degradation

by proteasome
(82)
-tubulin
Microtubule component
(61, 148)
HMG-17
Unfolds higher order chromatin structure
(31)
HMGI
Essential architectural component for enhancesome

assembly
(149)
TCF
Transcriptional regulator
(150)
PCNA
DNA repair and replication, cell cycle control,

chromatin remodeling
(151)
EKLF
Red cellspecific transcriptional activator
(152)
ACTR
Nuclear receptor coactivator, HAT
(153)
HNF-4
Transcriptional activation
(154)
Importin-
Nuclear import factor
(155)
NF-B
Regulates antiapoptotic responses
(156)
ER81
Downstream effector of HER2/neu and Ras
(157)
SF-1
Transcription factorexpression of steroidogenic

proteins
(158)
Ku70
Suppresses apoptosis
(159)
UBF
Structures DNA in ribosomal enhancesome
(160)
Sp3
Transcriptional activator or repressor
(161)
TAL1
Regulator of normal and leukemic hematopoiesis
(162)
YY1
Multifunction transcription factor
(163)
E2F1
Cell cycle activatorrequired for progression
(164)
MyoD
Stimulates cdk inhibitor p21
(165)
PCNA, proliferating cell nuclear antigen; SF-1, steroidogenic factor-1; UBF, architectural upstream binding factor.
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in detail elsewhere (39). Unraveling specific roles by these HDAC isozymes during
human tumorigenesis will further incentivize development of more specific HDAC
inhibitors (42), potentially enhancing their clinical activity as well as decreasing
their nonspecific toxicities, while also optimizing potential interactions with other
rationally designed and integrated therapeutic agents.
STRUCTURAL CLASSES AND MECHANISTIC

ACTIONS OF HDAC INHIBITORS
The six structurally distinct classes of HDAC inhibitors (Figure 2) act by binding to
various portions of the catalytic domains within class I and II HDACs (Figure 3A).
Although reviewed here briefly, a detailed examination of the medicinal chemistry
and activity relationships for these structurally varied inhibitors is beyond the
scope of this review, and the reader is directed to several excellent reviews on
this subject (4345). Hydroxamic acidtype chelators, including TSA, SAHA,
and LAQ824, have three basic components (Figure 3B): (a) a hydroxamic acid
moiety that chelates the active zinc in a bidentate manner, hydrogen bonds with
residues composing the charge relay systems, and displaces the nucleophilic water
molecule present in the active site; (b) a hydrophobic spacer that has a length
optimal for spanning the length of the hydrophobic pocket and dimensions capable
of navigating the narrowest segment of the cavity; and (c) a hydrophobic cap that
blocks the entrance to the active site. Design and understanding of the enzymatic
inhibitory mechanisms for various HDAC inhibitors was aided by solving the
crystal structure of an HDAC homologue that shares significant homology with
class I and class II HDACs, including all critical active site residues (46). The
active site of class I and class II HDACs includes critical zinc and water molecules;
two charge relay systems, where aspartate residues act to increase the basicity of
histidine residues by polarizing the epsilon nitrogen; and an active site tyrosine
residue that coordinates to the acetyl oxygen during the transition state (Figure 3C).
The zinc ion acts by polarizing the acetyl carbonyl to make the carbonyl carbon
a better electrophile for attack by the activated water molecule. Substitution of
other divalent cations, or chelation of the zinc cation by a small-molecular-weight
chelator, abolishes enzymatic activity. A hydrophobic pocket high in aromatic
and glycine residues leads to the active site, with the narrowest point having a
marked by two opposing phenylalanine residues. A depiction
distance of 7.5 A
of the predicted transition state interaction between HDAC1 and the hydroxamic
acidtype inhibitor LAQ824 is shown in Figure 3C. Hydroxamates with five or six
carbon spacers were found to be the most active inhibitors (47), and replacement of
the hydroxamic acid with a carboxylate was found to eliminate inhibitory activity
(48).
Epoxyketone-based HDAC inhibitors, such as trapoxin B, HC-toxin, or 2amino-8-oxo-9,10-epoxydecanoic acid (AOE), may act by chemically modifying an active site nucleophile with the epoxy group (49) and forming important
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Figure 2 Structural classes of HDAC inhibitors. Six basic classes of HDAC inhibitors
are shown: (a) small-molecular-weight carboxylates, including sodium butyrate, valproic
acid, and sodium phenylbutyrate; (b) hydroxamic acids, including CBHA, TSA, SAHA,
and LAQ824; (c) benzamides, including MS-275 and CI-994; (d) epoxyketones, including
AOE and trapoxin B; (e) cyclic peptides, including depsipeptide and apicidin; and ( f ) hybrid
molecules, such as CHAP31 and CHAP50.
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Figure 3 Structural basis for hydroxamic acid inhibition of HDACs. (A) Structural homology of class I and II HDACs showing hydroxamate-inhibiting catalytic domains.
(B) Functional components of hydroxamic acidtype HDAC inhibitors (LAQ824). Hydroxamic acidbased HDAC inhibitors are composed of four primary functional components: (a) a
zinc-chelating hydroxamic acid, (b) a linker region, (c) a polar site, and (d) a hydrophobic cap
that blocks the active site. (C) Predicted transition state inhibition of HDAC1 by LAQ824.
hydrogen bond contacts with the ketone. Elimination of the ketone, or reduction of
the ketone to an alcohol, abolishes the activity of these molecules (50). Trapoxin
B and HC-toxin also contain a five-carbon linker for transversing the cavity and a
cyclic tetrapeptide capable of acting as a hydrophobic cap for the cavity. Trapoxin
B is a hybrid molecule and can also be listed with the cyclic peptide HDAC inhibitors. The combination of cyclic peptide and epoxyketone resulted in nanamolar
HDAC inhibitory activity.
The carboxylates or short-chain fatty acids, including sodium butyrate, valproic
acid, and sodium phenylbutyrate, have much weaker HDAC inhibition constants
(Kis), commonly in the millimolar range. In spite of their weak activity, several of
these agents have been studied clinically (51) owing in part to their clinical use for
alternative medical indications. The most commonly studied members of this class
are simple molecules with alkyl or phenylalkyl carboxylates. The carboxylate is
thought to coordinate with the zinc ion in the active site, albeit more poorly than
in the case of hydroxamates.
Cyclic peptide HDAC inhibitors have been discovered or developed that either
contain an epoxyketone group (HC-toxin, trapoxin B) or are devoid of it (Apicidin,
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Depsipeptide). In general, these inhibitors have nanamolar HDAC inhibitory activity and can have either irreversible (epoxyketone-based) or reversible mechanisms
of action. The macrocyclic peptide portion of the inhibitor binds tightly to the rim
or opening of the channel to the active site, whereas an aliphatic linker navigates the
channel to the active site (44). Depsipeptide, also known as FK228, is a prodrug
that requires intracellular reduction to liberate a sulfhydryl-containing aliphatic
group that enters the active site and binds the active site zinc and water molecule
(44, 52). Hybrid molecules, including CHAP31 and CHAP50, that possess both
a cyclic peptide and an aliphatic hydroxamate have been prepared and shown to
have a reversible mechanism of action and remarkable inhibitory activity when
optimized in the range of 15 nanamolar (53, 54). The optimal linker in these
studies was found to have five methylenes, similar to that described previously for
other hydroxamates (47).
Inhibitors of the benzamide class, such as CI-994 (55) and MS-275 (56), are
in general less active than members of the hydroxamate or cyclic peptide classes,
with Kis in the micromolar range (44, 56). The mechanism of HDAC inhibition
for benzamides remains uncertain at present. In addition to the structural classes
of HDAC inhibitors described thus far, a variety of inhibitors have been prepared
that are not readily classified into one of the above mentioned five classes. Brosch
and colleagues have recently described 3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-Nhydroxy-2-alkylamides containing a range of different metal chelating groups with
IC50s in the micromolar range (57, 58). Another series of Psammaplin derivatives
containing novel metal chelating groups have demonstrated considerably greater
inhibitory activity, with the most active of these compounds having nanomolar Kis
(59).
Little is presently known about the potential selectivity of various HDAC class I
or II isoforms for structurally different inhibitors. HDAC6 and HDAC10 both possess two catalytic domains that appear to be differentially inhibited by drugs that
preferentially bind near the entrance of the catalytic site (37, 54, 55). These class
II HDAC isoforms appear relatively resistant to trapoxin when compared to class
I HDACs. Despeptide, MS-275, and several of the hybrid CHAP derivatives also
appear considerably more selective for HDAC1 over HDAC6 (54). TSA is generally considered a nonspecific HDAC inhibitor, as it has a similar Ki for all isoforms
examined. Recently, Schrieber and colleagues described an HDAC6-specific inhibitor, tubacin (Figure 2), responsible for the deacetylation of tubulin, as well as
another histacin, which appears to be a histone-selective deacetylase (60, 61).
The continued development of isoform-specific inhibitors will undoubtedly remain
a major emphasis of HDAC inhibitor development.
ANTITUMOR MECHANISMS OF HDAC INHIBITORS

There is an everexpanding body of evidence supporting the involvement of, as well
as structural alterations in, various HATs and HDACs with development of cancer (62, 63). Broadly speaking, this includes evidence for their genetic disruption
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(e.g., translocation, amplification, mutation, overexpression) in a subset of hematological and epithelial malignancies, as well as the aberrant genomic recruitment
of otherwise normal HDACs in conjunction with oncogenic transcription factors.
Such observations have led to the conclusion that defects and/or imbalances in the
genomes acetylation machinery accompany changes in local chromatin structure
and oncogenic dysregulation of genes controlling cell cycle progression, differentiation, and apoptosis. Despite these observations and conclusions, there are as yet
no specific HAT or HDAC measurements devised that can predict the sensitivity
of any given tumor to any class of HDAC inhibitor.
It has also been generally accepted that more actively transcribed chromatin
regions are associated with histone hyperacetylation and recruitment of HATs
(although HDACs are also known to be recruited), and histone deacetylation associated with recruitment of HDACs often restores these genomically active regions
to a more repressed and condensed chromatin state. Thus, an attractive paradigm
for the antitumor action of HDAC inhibitors has been the induction of histone
acetylation producing transcriptional activation of critical genes needed for tumor growth arrest (1, 43, 44, 6467). Unquestionably, HDAC inhibitors produce
a global increase in histone acetylation within hours of treatment of many different malignant and nonmalignant tissue types, including those showing little if
any biological consequences upon treatment with HDAC inhibitors. Thus, while a
global increase in the level of histone acetylation by itself cannot explain selective
changes in gene expression or specific patterns of antitumor activity following
HDAC inhibition, assaying for enhanced histone acetylation in readily sampled
cells or tissues (e.g., peripheral white blood cells) is being routinely employed
to demonstrate HDAC inhibitor bioavailability and activity. Greater attention is
currently being given to the expanding list of nonhistone proteins acetylated in direct response to HDAC inhibition (Table 1), especially because many of these are
tissue/development-specific (EKLF, GATA-1, ER, MyoD), oncogenic (c-Myb),
tumor-suppressing (p53), or even rather ubiquitous (TFIIE, TFIIF, TCF, HNF-4)
transcription factors.
Virtually all HDAC inhibitors currently in clinical development show some degree of preclinical activity against malignant cells proliferating in culture and also
tumors growing in animal models; this antitumor activity may be characterized as
either inducing cytostasis (cell cycle arrest), differentiation, or apoptosis. However, the HDAC-dependent mechanisms accounting for the observed and rather
selective modulation of gene expression, as well as specific patterns of antitumor
activity, remain poorly understood. Several studies have now revealed that fewer
than 10% of expressed genes in a given malignant cell population are affected
by an antitumor dose of an HDAC inhibitor, with a near equal number of transcriptionally active genes being repressed as those being stimulated; structurally
different HDAC inhibitors can similarly modulate expression of a relatively limited set of core genes (2, 3, 68). As shown in Table 2, among the commonly
up- and down-modulated gene transcripts identified in these expression microarray studies, as well as in numerous single-gene expression studies (6678), are
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TABLE 2 Tumor-associated proteins whose transcriptional expression is altered in response to

HDAC inhibitor treatment of cells
Regulated
protein
Function (oncogenic or antioncogenic/tumor

supressing)
Reference(s)

Downregulated by HDAC inhibitors (e.g., oncogenic)

HER2/neu
Growth factor receptor (EGFR class)
(81)
TGF-
Regulates cell proliferation and differentiation

through TGF- type II receptor
(166, 167)
Thioredoxin
Disulfide reductase, cytokine activity, can inhibit

apoptosis
(168)
Telomerase
Prevents telomere erosion
(97)
RECK
Regulates matrix metalloproteinases
(86)
VEGF
Angiogenic factor
(87, 169)
bFGF
Angiogenic factor
(87)
Myb/c-MyBL2
Oncogenic transcription factorregulation of

transformation and differentiation
(68)
raf-1
Effector of Ras
(68)
cyclin A
Cell cycle regulator
(111)
cyclin B
Cell cycle regulator
(111)
DAF
Complement inhibitory protein
(170)
abl
Growth factor receptor, component of bcr/abl chimeric

kinase
(68)
DEK
Putative role in regulating chromatin structure and

postsplicing events
(68)
Proteasome
Degradation of misfolded or oxidized proteins
(68)
Upregulated by HDAC inhibitors

Fas/Fas ligand
Proapoptotic
(76)
Bcl2
Proapoptotic
(78)
p53
Proapoptotic
(169)
Bak, Bax, Bim
Proapoptotic
(171)
c-myc
Inhibitor of differentiation
(100)
Caspase 3
Cysteine protease involved in apoptosis, proapoptotic
(125, 172)
Carboxypeptidase
A3 (CPA3)
Carboxypeptidase, putative role in regulating

differentiation
(173)
RECK
Negatively regulates matrix metalloproteinases
(86)
p21WAF1/Cip1
Gelsolin
Cell cycle regulation

Regulation of cell morphology
(66, 70)
(70)
(Continued)
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TABLE 2

AR232-PA45-21.tex
(Continued)
Regulated
protein
Function (oncogenic or antioncogenic/tumor

supressing)
ER
Estrogen-activated nuclear receptor regulates transcription

of estrogen responsive genes
(174)
TSSC3
Regulates Fas-mediated apoptosis
(68)
IGFBP-3
Augments IGF actions, promotes apoptosis, and inhibits

cell growth
(175)
TBP-2
Inhibits thiol-reducing activity of thioredoxin
(168)
Reference(s)
Bak, Bcl2 antagonist killer; Bax, Bcl2-associated X protein; DAF, decay-accelerating factor; TBP-2, thioredoxin binding
protein; TSSC3, tumor supressing subtransferable candidate.
those encoding known tumor-associated proteins that mediate proliferation and

cell cycle progression, survival factors, growth factor receptors, kinases and signal transduction intermediates, DNA synthesis/repair enzymes, shuttling proteins,
transcription factors, and proteases.
Some study has gone into the question of how HDAC inhibitors actually relieve
transcriptional repression and reverse the differentiation arrest in malignancies
such as acute leukemia, where differentiation arrest and the malignancy phenotype induced by such chimeric oncoproteins as PLZF-RAR, PLZF-RAR, or
AML1/ETO can be reversed, at least in part, by HDAC inhibitors (69, 79). In
other types of malignancies, HDAC inhibitors induce differentiation and/or apoptosis by activating transcription of CDKN1A through a p53-independent mechanism, producing increased levels of the cyclin-dependent kinase (CDK) inhibitor,
p21WAF1/CIP1 (66). Likewise, HDAC inhibitors have been observed to induce transcription of other tumor suppressor genes such as gelsolin and maspin (70, 71).
When administered in combination with DNA demethylating agents such as 5-aza2 -deoxycytidine, HDAC inhibition can fully restore transcriptional expression to
various genes, including MLH1, TIMP3, CDKN2A, and CDK2NB, that have been
epigenetically silenced by promoter methylation during the course of tumorigenesis (72, 73).
Apart from the upregulation of epigenetically silenced tumor suppressor proteins or induction of caspases and other proapoptotic proteins (26, 68, 7478),
there are emerging data showing HDAC-induced repression of critical transforming growth factor mechanisms, such as those involving oncogenic tyrosine kinases
like bcr/abl and ErbB2 (8082). We recently reported that HDAC inhibitors can
selectively repress ErbB2 transcript levels by two distinct HDAC-dependent mechanisms: repression of new ErbB2 transcript synthesis and the accelerated decay of
mature ErbB2 mRNA (81). The hydroxamic acid TSA was identified in a highthroughput cell-based chemical screen for its ability to repress ErbB2 promoter
activity (81). Figure 4 (panel A) compares the potency of TSA against several
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other HDAC inhibitors for their ability to inhibit ErbB2 promoter function. Of
interest, the rank order of potency for the HDAC inhibitors shown in this screening assay (LAQ824 > TSA > A1616906 > SAHA CI994) is comparable to
their relative antitumor activity against several ErbB2 overexpressing breast cancer cell lines. When evaluated further against SkBr3 and other ErbB2-dependent
breast cancer cell lines (e.g., BT-474, MDA-453), HDAC inhibitors were shown to
inhibit the synthesis and elongation of nascent ErbB2 transcripts as well as destabilize and accelerate the decay of mature cytoplasmic ErbB2 transcripts (Figure 4,
panels B and C). Although ongoing preclinical studies are confirming that ErbB2dependent cancers appear somewhat more sensitive to HDAC inhibitors than
ErbB2-independent cancers, molecular studies are attempting to define the drugsensitive HDAC-dependent nuclear and cytoplasmic mechanisms that differentially regulate ErbB2 transcription and ErbB2 transcript stability, respectively. The
presence of multiple distinct HDAC-dependent mechanisms capable of controlling ErbB2 transcript levels suggests that even among ErbB2-dependent cancers,
there will be differential sensitivity to structurally different classes of HDAC inhibitors. Other investigators have identified HDAC-dependent posttranslational
mechanisms that can also downregulate the expression of oncoprotein kinases like
ErbB2 and bcr/abl (80, 82). Acetylation of the chaperone protein, Hsp90, induced
by HDAC inhibition, results in the enhanced proteasomal degradation of ErbB2
and bcr/abl kinases. These examples of multiple mechanisms by which HDAC
inhibitors potentially downregulate critical oncogenic pathways also suggest new
combinatorial strategies for possible clinical evaluation, including HDAC inhibitor
treatment in conjunction with tyrosine kinase inhibitors (80, 8284) or Hsp90 antagonists (85).
Apart from directly affecting transformed cells, HDAC inhibitors have also been
shown to inhibit tumor angiogenesis, suggesting additional therapeutic mechanisms for the observed in vivo activity of these antitumor drugs (76, 8688).
Depsipeptide was shown to suppress the expression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor (bFGF) (87). VEGF and bFGF mRNA levels were significantly reduced
in prostate tumor xenografts sensitive to this cyclic peptide HDAC inhibitor. The
hydroxamic acid HDAC inhibitor TSA was shown to upregulate the RECK protein responsible in part for inhibiting tumor metastasis and angiogenesis through
its action on matrix metalloproteases (86). The carboxylate and short-chain fatty
acid HDAC inhibitor, valproic acid, was also shown to inhibit angiogensis both
in vitro and in vivo via a mechanism involving diminished expression of endothelial
nitric oxide synthase (88). Additional miscellaneous or less well-studied tumorassociated mechanisms may prove to be important in determining the ultimate
clinical utility of some HDAC inhibitors.
Last, various drug-resistance phenotypes have been shown to be modulated
by HDAC inhibitors (8995). Treatment of different multidrug-resistant cell lines
with TSA or SAHA was shown to downregulate P-glycoprotein (93), helping

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reverse the multidrug-resistant phenotype. SAHA and oxamflatin were shown

in separate studies to overcome multidrug resistance (89, 94), whereas in one
study depsipeptide was shown to be a substrate for P-glycoprotein (94). In another
study, depsipeptide was shown to inhibit cell growth in irinotecan-, etoposide-,
and cisplatin-resistant cell lines in conjunction with its ability to inhibit telomerase
expression and activity (90). Telomerase is responsible for adding telomeric repeats
to the ends of chromosomes and is required for the relative immortality of cancer
cells; other investigators have also shown an inhibitory effect of HDAC inhibitors
on telomerase activity (96, 97). These diverse examples illustrate the immense
need for further studies to understand the relative importance of the many potential
in vitro and in vivo mechanisms by which HDAC inhibitors can produce antitumor
responses.
Figure 4 Transcriptional repression of ErbB2 induced by hydroxamic HDAC inhibitors is caused by a combination of both ErbB2 promoter repression and transcript
destabilization. (A) Employing our previously described high-throughput screening assay (81), an ErbB2-independent subline of MCF-7 breast cancer cells (MCF/R06pGL4) bearing a chromatin-integrated ErbB2 promoter-driven luciferase construct was
used to compare the ErbB2 promoter repressing potency of four structurally different hydroxamic acidtype HDAC inhibitors (SAHA, A1616906, TSA, LAQ824) and
a benzamide-type HDAC inhibitor (CI994). After 24 h culture exposure to the indicated drug doses, cell viability as measured by MTT assay (squares) shows little
change, whereas specific repression of ErbB2 promoter activity is detected by luciferase expression (diamonds). The benzamide inhibitor (CI994) shows slight ErbB2
promoter stimulation with no evidence of promoter repression; in contrast, the hydroxamic acid inhibitors show ErbB2 promoter repression at different potencies as
indicated by the 25% luciferase inhibitory concentration (M IC25) values. (B) When
ErbB2-dependent SkBr3 breast cancer cells in culture are treated for 5 h with an
ErbB2 promoterrepressing dose of TSA, nascent ErbB2 transcript synthesis and
elongation, as measured by nuclear run-off assays (81), appears completely inhibited, whereas nascent transcript synthesis of the Ets transcription factor ESX appears
marginally increased. (C) Total RNA extracted and Northern blotted after 5-h treatment of cultured SkBr3 breast cancer cells shows treatment effects on mature longlived (8 h half-life) ErbB2 transcripts (4.8 kb) in comparison to short-lived (<2 h
half-life) ESX transcripts (2.2 kb). Treatment for 5 h with an RNA polymerase inhibiting dose of Actinomycin D (10 g/ml) demonstrates the expected absence of
ESX transcripts and partial decline in total ErbB2 transcripts. In contrast, and after 5-h treatment with comparable doses of the HDAC inhibitors TSA, CI994, and
LAQ824, ESX levels appear marginally increased, whereas ErbB2 transcript levels
are reduced below levels caused by Act D treatment, demonstrating the independent
ability of HDAC inhibitors to destabilize and accelerate the decay of mature ErbB2
transcripts.
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IN VIVO BIOLOGICAL AND CLINICAL CHARACTERISTICS

OF HDAC INHIBITORS

In Vivo Preclinical Antitumor Activity

Numerous animal model studies have demonstrated significant antitumor efficacy
for HDAC inhibitors from virtually every structural class (71, 87, 98106). One of
the newest clinical candidates, the hydroxamate JNJ1641199 (Figure 2), exhibited
nanomolar HDAC inhibition and antitumor activity against lung, ovarian, and
colon cancer xenograft models, along with excellent oral bioavailability (106).
Another hydroxamate now in clinical trials, NVP-LAQ824 (Figure 2), shows potent
antitumor activity against human colon (Figure 5A) and lung cancer xenograft
models at submicromolar concentrations when administered parenterally every
day and with a maximal tolerated dose (MTD) that exceeds 100 mg/kg (101).
Likewise, TSA, SAHA, and pyridoxamide hydroxamates were previously shown
to have in vivo antitumor activity with daily parenteral dosing associated with little
systemic toxicity (98, 104, 105).
The cyclic peptide prodrug depsipeptide (FK228) demonstrates efficacy in
leukemia and lymphoma models (100, 103), which can be further enhanced in
combination with the cell-differentiating retinoid, ATRA (103). Depsipeptide was
also recently shown to have clinical activity in treating T cell lymphoma in early
Phase I/II trials (107).
Although most of the carboxylated short-chain fatty acid HDAC inhibitors have
displayed limited potency in vivo owing to their lack of specificity and high drugconcentration requirements (51, 65), the prodrug AN-9 has shown good activity
Figure 5 In vivo antitumor efficacy of hydroxamate-type HDAC inhibitor, LAQ824,

is dependent on dose, schedule, and formulation. Insert arrows in both panels show
treatment points. In vivo antitumor activity of LAQ824 was determined in (A) a human
colon (HCT116) tumor xenograft model and (B) liposomal LAQ824 in an ErbB2dependent human breast (BT474) tumor xenograft model. The drugs in both studies
were administered intravenously. For the free LAQ824 study (A), treatments started
when HCT116 tumors reached a mean size of 50 mm3 and nude mice were injected
5 times per week for 3 weeks, for a total of 15 doses. The treatment groups were as
follows: (diamonds) 10% DMSO/D5W (control), (squares) 10 mg/kg/dose LAQ824,
(triangles) 25 mg/kg/dose LAQ824, (open circles) 50 mg/kg/dose LAQ824, and (closed
circles) 100 mg/kg/dose LAQ824. For the liposomal LAQ824 study (B), BT474 breast
tumor xenografts were allowed to grow to a size of approximately 250300 mm3. Mice
were then injected with either saline (open circles) or conventional liposomal LAQ824
(closed triangles) at a dose of 25 mg/kg weekly, for a total of 3 weeks, beginning
on day 27. The data are expressed as mean tumor volume standard error. (A) was
adapted from Remiszewski et al. (101) with permission from the American Chemical
Society.

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in several murine tumor and human tumor xenograft models (108), and has shown
some encouraging results in early clinical trials (109).
The benzamide HDAC inhibitors, MS-275 (56, 71, 110, 111) and CI-994 (55),
have also shown in vivo activity against various tumor models. MS-275 was shown
to inhibit the growth of three different orthotopic pediatric tumor xenografts (110).
In another study, MS-275 administered orally was shown to have potent antitumor
activity against a series of seven different human tumors xenografts (71). Unfortunately, antitumor doses of MS-275 in mice were also myelosuppressive, causing
decreases in red and white blood cells as well as platelets (111). A similar pattern
of toxicity was observed with CI-994 (112); and thrombocytopenia was a major
dose-limiting toxicity seen in Phase I and II clinical testing with CI-994 as well as
with the cyclic peptide depsipeptide (113, 114).
Clinical Toxicity and Antitumor Activity

Dose-limiting clinical toxicities and reported antitumor responses have been noted
in Phase I and II clinical trials for the limited number of structurally varied HDAC
inhibitors that have entered clinical testing to date. The carboxylate phenylbutyrate given by prolonged intravenous infusion has a dose-limiting toxicity (DLT)
of somnolence and confusion, which has not been reported for the benzamide or
hydroxamate HDAC inhibitors (115) or for the carboxylate prodrug AN-9 (109).
The carboxylate valproic acid has been in clinical use for more than two decades
as an anticonvulsant and thus has well-described pharmacologic properties and
a well-tolerated side effect profile; clinical trials are in progress evaluating the
antitumor potential of valproic acid as an HDAC inhibitor. Despite thrombocytopenia being a DLT for both CI-994 and depsipeptide, evidence for antitumor
clinical activity upon oral daily dosing of CI-994 has been noted in patients with
several epithelial types of advanced solid malignancies [including nonsmall cell
lung cancer (NSCLC), renal cell carcinoma, and bladder cancer]. Likewise, two
Phase I trials of depsipeptide have suggested that patients with T cell leukemia
or lymphoma, as well as other occasional cases of refractory malignancies, may
achieve clinical benefit from this HDAC inhibitor (113, 116). Curiously, depsipeptide is the only clinically tested HDAC inhibitor reported to date that is associated
with a significant incidence of cardiac dysrhythmias and nonspecific EKG abnormalities (113). Among the hydroxamates, daily infusions of pyroxamide and
LAQ824 are currently under Phase I clinical evaluation, whereas a trial of infusional SAHA was recently completed (115, 117). When given by 2-h infusions
daily five times, SAHA had a MTD of 300 mg/m2/day. Among treated patients
with advanced hematologic malignancies, myelosuppression (thrombocytopenia)
was the DLT. In those with advanced solid tumors, myelosuppression was observed
but was not a DLT; as well, nonspecific EKG changes without clinical signs or
symptoms were common. Fatigue was commonly observed with SAHA treatment
but was not dose-limiting and was similar to that previously reported for depsipeptide. Importantly, patients with renal cell carcinoma, head and neck squamous
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carcinoma, papillary thyroid carcinoma, mesothelioma, B and T cell lymphomas,

and Hodgkins disease all showed some degree of clinical improvement (115).

Pharmacokinetic Considerations
Although receiving less reported attention than studies elucidating the pharmacodynamics of various HDAC inhibitors, the pharmacokinetic characteristics and
limitations of different HDAC inhibitors are of critical interest and will likely
prove to be an important determinant of their ultimate clinical utility. Inhibition of
intracellular HDAC activity commonly requires continuous systemic circulation
and drug exposure to achieve maximal tumor cytostasis or apoptosis and clinical
response. Rapid clearance, a high degree of protein binding, rapid metabolism,
or rapid inactivation of reactive functional groups (i.e., epoxy groups) are factors
that can adversely affect HDAC inhibitor bioavailability and antitumor activity.
Although occasionally used in the clinic, prolonged or daily infusions of any
drug are generally undesirable. The requirement of constant systemic exposure
by parenteral administration to achieve an active antitumor drug concentration
will most likely limit the clinical development of any HDAC inhibitor that is not
orally bioavailable. For this reason, the clinical development of SAHA shifted
from Phase I evaluation of daily intravenous infusions to a more recently designed
oral formulation (115).
Novel drug delivery systems that allow for controlled drug release may help circumvent the clinical inconvenience of daily infusions as well as generally enhance
the therapeutic index of HDAC inhibitors. We have recently evaluated the potential
of liposomes for delivering the HDAC inhibitor LAQ824. When formulated properly, liposomes can entrap and concentrate amphipathic drugs (achieving >10,000
drug molecules per liposomal nanoparticle), releasing them slowly over time in
the plasma or delivering them specifically to solid tumors where they deposit their
drug in close proximity to the tumor, allowing for increased tumor accumulation
and drug exposure (118). In a pilot study administering liposomal LAQ824 on a
once-weekly schedule for three weeks, we observed significant growth arrest of
rapidly growing human breast tumor xenografts (Figure 5B). As shown in other
studies involving various tumor model systems, free LAQ824 requires daily injections of generally higher doses to slow tumor growth (Figure 5A). More recent
studies with optimized formulations and targeted liposomal constructs have shown
even greater efficacy (119).
HDAC INHIBITORS IN COMBINATION

WITH OTHER AGENTS
The greatest potential of HDAC inhibitors may lie in their ability to modulate
the activity of other therapeutic agents. A variety of different drug combinations
have demonstrated considerable promise in treating cancer. These are reviewed
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more extensively in a separate review of the field (65), and are summarized in
Table 3. The pretreatment or coadministration of HDAC inhibitors with a wide
range of agents has repeatedly been shown to additively or synergistically enhance
apoptosis of cancer cells in culture (68, 82, 85, 120125) as well as antitumor
efficacy in vivo (126128). Notably, enhancements in activity have been observed
when HDAC inhibitors are combined with a number of different commonly used
chemotherapeutics (82, 120, 121). Nuclear receptor ligands (123, 127, 129, 130),
Hsp90 antagonists (85), proteasome inhibitors (68, 84, 131), signal transduction
inhibitors (80, 82, 124, 125, 132136), and DNA demethylating agents (72, 73, 122,
128, 137) represent some of the more promising classes of agents. Demethylating
agents such as 5-aza-2 -deoxycytidine (DAC) are particularly interesting owing to
the interaction of DNA methylation with histone deacetylation in gene silencing of
tumor suppressor genes, as mentioned above. Combinations of DAC with TSA or
depsipeptide were shown to reactivate silenced tumor suppressor genes including
MLH1, TIMP3, CDKN2B, CDKN2A, ARHI, gelsolin, and maspin (72, 73, 137),
synergistically increasing the level of tumor cell apoptosis (122). Combinations
of nuclear receptor ligands, such as all-trans retinoic acid (ATRA), or vitamin
D analogs, such as 1,25-dihydroxyvitamin D, with HDAC inhibitors have been
shown to increase differentiation and apoptosis in cancer cells (123, 127, 130) and
also inhibit tumor growth in vivo (127, 130, 138). Small-molecule kinase inhibitors
may also be rationally combined with HDAC inhibitors. Imatinib (Gleevec) is
a specific inhibitor of Bcr/Abl with impressive clinical activity in the treatment
of chronic myeloid leukemia and selected other malignancies. Because LAQ824
has been shown to downregulate the expression of Bcr/Abl and also promote its
degradation through acetylation of Hsp90 (80), combinations of imatinib with
LAQ824 as well as other HDAC inhibitors, such as SAHA and apicidin, have
been tested and shown to dramatically increase the apoptosis of Bcr/Abl positive
leukemic cells (80, 83, 132, 133). A similar effect is seen when malignant cells
known to be transformed by oncogenic tyrosine kinases (ErbB2/HER2, Src/Abl,
PI3 kinase) are treated with HDAC inhibitors in combination with appropriate
kinase inhibitors like Herceptin, PD180970, or LY294002 (80, 82, 124).
As noted above, expression of the CDK inhibitor p21WAF1/CIP1 is regulated by
HDACs and plays a critical role in determining whether cells undergo differentiation or apoptosis in response to treatment with HDAC inhibitors (66, 70, 139).
Flavopiridol is a CDK inhibitor that results in a disruption of p21WAF1/CIP1 induction and induces apoptosis. Its combination with HDAC inhibitors (SAHA,
depsipetpide, sodium butyrate) has been shown to result in a disruption of p21
induction and an additive or synergistic increase in tumor cell apoptosis (125, 135,
139, 140).
Proteasome inhibitors and Hsp90 antagonists represent two other groups of
interesting agents that may be rationally combined with HDAC inhibitors. Hsp90
is a molecular chaperone that stabilizes and controls the intracellular trafficking of
important client proteins, including ErbB2/HER2, Bcr/Abl, EGF, cyclin D1, c-Raf,
and steroid receptors. The inhibition of Hsp90 with amsacrine antagonists, such as
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TABLE 3 Therapeutic agents used in combination with HDAC inhibitors in preclinical and
clinical studies
Combined
therapeutic agent
HDAC inhibitor
Standard chemotherapy
Gemcitabine
CI-994
VP-16 (etoposide)
cytarabine, etoposide, and
topotecan
Doxorubicin, melphalan,
chloroambucil, cisplatin,
carboplatin, fludarabine
Taxotere, gemcitabine,
epothilone B
Etoposide
Fludarabine
IMID-1, dexamethasone
Demethylating agents
DAC
Nuclear receptor ligands

ATRA
1,25-Dihydroxyvitamin D3
Signal transduction inhibitors
Imatinib mesylate (Gleevec)
Imatinib mesylate
or PD180970
Herceptin
LY-29, 4002
Flavopiridol
TRAIL
Combined
antitumor effects
Reference(s)
(176)
TSA, SAHA
PB
Phase II trial
increased toxicity
Synergistic
Synergistic
PB
Additive
(121)
LAQ824
Additive
(82)
TSA
MS-275
SAHA
Antagonistic
Synergistic
Synergistic
(177)
(178)
(68)
TSA, depsipeptide
Depsipeptide
PB
PB
Enhanced
Synergistic
Synergistic
Enhanced
(122)
(73)
(128)
(179)
CBHA
TSA
SB
Synergistic
Synergistic
(127)
(123)
Apicidin
SAHA
LAQ824
Synergistic
Synergistic
Synergistic
(83)
(132)
(80)
LAQ824
SAHA, SB, MS-275
SAHA
SB
Depsipeptide
SB
SB, SAHA
LAQ824
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
Enhanced
Synergistic
Enhanced
(82)
(124)
(125)
(135)
(136, 140)
(180)
(181)
(182)
Synergistic
Synergistic
Synergistic
Synergistic
Synergistic
(85)
(68)
(84)
(141)
(131)
Hsp90 antagonists and proteasome inhibitors

17-AAG
SAHA
Bortezomib (PS-341)
SAHA
SAHA, SB
SB
MG132
SB
(120)
(121)
ATRA, all-trans retinoic acid; CBHA, m-carboxylcinnamic acid bis-hydroxamide; DAC; 5-aza-2 -deoxycytidine; IMID-1,
immunomodulatory thalidomide derivative 1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
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17-AAG, results in proteasomal degradation of client proteins. Owing to the regulation of Hsp90 function by acetylation, the combination of HDAC inhibitors and
Hsp90 antagonists is a reasonable therapeutic strategy. Experimentally, 17-AAG
in combination with SAHA or sodium butyrate inhibited induction of p21WAF1/CIP1,
inducing Bcl-2 cleavage, and synergistically enhanced tumor cell apoptosis (85).
Proteasome inhibitors slow the degradation of many important and diverse cellular
proteins, and their combination with HDAC inhibitors results in more complete
inhibition of proteasome activity, which may synergistically enhance tumor cell
apoptosis (68, 84, 131, 141).
There is additional evidence that HDAC inhibitors may improve the efficacy
of radiation therapy (142). In this study, pretreatment with depsipeptide greatly
increased radiation-induced apoptosis. In another study, the HDAC inhibitors
phenylbutyrate, TSA, and valproic acid were able to reduce cutaneous radiation
toxicity following radiotherapy (143). This poorly understood interaction whereby
HDAC inhibitors potentially increase radiation-induced tumor cell death while decreasing normal host cell toxicity deserves further study, as it may lead to more
novel clinical indications for HDAC inhibitors.
Although these provocative combination regimens based on cell culture studies
have rational appeal, they must be explored more fully in vivo to assure that
they do not also lead to enhanced host toxicity. One recent Phase II study of the
combination of gemcitabine and CI-994 in patients with NSCLC demonstrated no
improvement in efficacy over gemcitabine alone, primarily because of increased
toxicity that limited dose intensity and reduced the net therapeutic index of the
two-drug combination (176).
CONCLUSIONS
The complexities of the histone code and the various other nuclear as well as
cytoplasmic nonhistone proteins whose functions are modulated by acetylation
underscore why HDAC inhibition was an empirically discovered, as well as novel,
form of cancer therapy. The biology of the various HDAC isoforms and their relationship to tumorigenesis is just beginning to be elucidated and is largely driven by
the perceived clinical potential of HDAC inhibitors. It remains to be seen if a more
detailed understanding of the specific roles played by various HDAC isoforms
during human tumorigenesis leads not only to development of isoform-specific
inhibitors but also to more effective or less toxic antitumor therapeutics, as compared to the multiclass HDAC inhibitors that are currently undergoing clinical
evaluation. Rationally designed combinations of HDAC inhibitors with various
other types of approved or investigational anticancer agents are showing promise
in tumor cell culture systems but must yet be proven in clinical trials. Of great interest to many cancer investigators is the potential ability to derepress the expression
of epigenetically silenced tumor suppressor genes by administering HDAC inhibitors in combination with inhibitors of DNA methyltransferases. There is a
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similar level of preclinical interest in combining inhibitors of oncogenic kinases

with HDAC inhibitors to strategically downregulate critical oncogenic pathways
at transcriptional and posttranslational levels. The potential ability of HDAC inhibitors to overcome various drug-resistance phenotypes is yet another preclinical
strategy warranting clinical evaluation. Finally, little is presently published on the
pharmacokinetics and biodistribution of various HDAC inhibitors now under clinical development. Owing to the preclinically determined need for constant drug
exposure to achieve in vivo tumor mass reduction by net inhibitory effects on tumor
cell proliferation and survival mechanisms, a more detailed study and comparison
of the pharmacokinetic profiles for various HDAC inhibitors is needed. Present
evidence suggests that more novel formulations and drug delivery strategies may
be able to significantly enhance the therapeutic index of even the most potent and
biologically active of currently available HDAC inhibitors. Although a clinical
role for HDAC inhibitors as novel cancer therapeutics seems almost inevitable
at present, their general clinical utility will likely depend greatly on the future
development of molecular or cellular predictors of their antitumor activity.
ACKNOWLEDGMENTS
We are grateful to the following for supplying their respective HDAC inhibitors: Dr.
Peter Atadja and Novartis Oncology for NVP-LAQ824, Dr. Alan Kraker and Pfizer
Oncology for CI-994, Dr. Victoria Richon and Aton Pharma for SAHA, and Dr.
Keith Glaser and Abbott for A-16,1906. We thank Crystal Berger and Cliff Amend
at the Buck Institute for their excellent technical assistance. Daryl Drummond was
supported in part by a New Investigator Award from the California Breast Cancer
Research Program of the University of California, Grant Number 7KB-0066A. This
work was supported in part by NIH grant R01-CA36773 (CCB) and a development
project award from the National Cancer Institute Specialized Programs of Research
Excellence (SPORE) in Breast Cancer (P50-CA 58207-01; CCB).
LITERATURE CITED
1. Cress WD, Seto E. 2000. Histone deacetylases, transcriptional control, and cancer.
J. Cell Physiol. 184:116
2. Glaser KB, Staver MJ, Waring JF, Stender
J, Ulrich RG, Davidsen SK. 2003. Gene
expression profiling of multiple histone
deacetylase (HDAC) inhibitors: defining
a common gene set produced by HDAC
inhibition in T24 and MDA carcinoma cell
lines. Mol. Cancer Ther. 2:15163
3. Van Lint C, Emiliani S, Verdin E. 1996.

The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expr. 5:245
53
4. Turner BM. 2002. Cellular memory and
the histone code. Cell 111:28591
5. Strahl BD, Allis CD. 2000. The language
of covalent histone modifications. Nature
403:4145
7 Jan 2005
14:6

518
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUMMOND ET AL.
6. Sterner DE, Berger SL. 2000. Acetylation

of histones and transcription-related factors. Microbiol. Mol. Biol. Rev. 64:43559
7. Jenuwein T. 2001. Re-SET-ting heterochromatin by histone methyltransferases. Trends Cell Biol. 11:26673
8. Lachner M, Jenuwein T. 2002. The many
faces of histone lysine methylation. Curr.
Opin. Cell Biol. 14:28698
9. Cheung WL, Ajiro K, Samejima K, Kloc
M, Cheung P, et al. 2003. Apoptotic phosphorylation of histone H2B is mediated
by mammalian sterile twenty kinase. Cell
113:50717
10. Jason LJ, Moore SC, Lewis JD, Lindsey
G, Ausio J. 2002. Histone ubiquitination:
a tagging tail unfolds? Bioessays 24:166
74
11. Nathan D, Sterner DE, Berger SL. 2003.
Histone modifications: now summoning
sumoylation. Proc. Natl. Acad. Sci. USA
100:1311820
12. Geiman TM, Robertson KD. 2002. Chromatin remodeling, histone modifications,
and DNA methylationhow does it all fit
together? J. Cell Biochem. 87:11725
13. Nephew KP, Huang TH. 2003. Epigenetic
gene silencing in cancer initiation and progression. Cancer Lett. 190:12533
14. Claus R, Lubbert M. 2003. Epigenetic
targets in hematopoietic malignancies.
Oncogene 22:648996
15. Czermin B, Imhof A. 2003. The sounds of
silencehistone deacetylation meets histone methylation. Genetica 117:15964
16. Daujat S, Bauer UM, Shah V, Turner B,
Berger S, Kouzarides T. 2002. Crosstalk
between CARM1 methylation and CBP
acetylation on histone H3. Curr. Biol. 12:
209097
17. Jenuwein T, Allis CD. 2001. Translating
the histone code. Science 293:107480
18. Beisel C, Imhof A, Greene J, Kremmer
E, Sauer F. 2002. Histone methylation by
the Drosophila epigenetic transcriptional
regulator Ash1. Nature 419:85762
19. Imhof A, Becker PB. 2001. Modifications of the histone N-terminal domains.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Evidence for an epigenetic code? Mol.

Biotechnol. 17:113
Jacobs SA, Taverna SD, Zhang Y, Briggs
SD, Li J, et al. 2001. Specificity of
the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J.
20:523241
Hassan AH, Prochasson P, Neely KE,
Galasinski SC, Chandy M, et al.
2002. Function and selectivity of bromodomains in anchoring chromatinmodifying complexes to promoter nucleosomes. Cell 111:36979
Agalioti T, Chen G, Thanos D. 2002.
Deciphering the transcriptional histone
acetylation code for a human gene. Cell
111:38192
Santos-Rosa H, Schneider R, Bannister
AJ, Sherriff J, Bernstein BE, et al. 2002.
Active genes are tri-methylated at K4 of
histone H3. Nature 419:40711
Zhang X, Yang Z, Khan SI, Horton JR,
Tamaru H, et al. 2003. Structural basis for
the product specificity of histone lysine
methyltransferases. Mol. Cell. 12:17785
Imhof A, Yang XJ, Ogryzko VV, Nakatani
Y, Wolffe AP, Ge H. 1997. Acetylation
of general transcription factors by histone
acetyltransferases. Curr. Biol. 7:68992
Juan LJ, Shia WJ, Chen MH, Yang WM,
Seto E, et al. 2000. Histone deacetylases specifically down-regulate p53dependent gene activation. J. Biol. Chem.
275:2043643
Fu M, Rao M, Wang C, Sakamaki T,
Wang J, et al. 2003. Acetylation of androgen receptor enhances coactivator binding
and promotes prostate cancer cell growth.
Mol. Cell Biol. 23:856375
Wang C, Fu M, Angeletti RH, SiconolfiBaez L, Reutens AT, et al. 2001. Direct
acetylation of the estrogen receptor alpha
hinge region by p300 regulates transactivation and hormone sensitivity. J. Biol.
Chem. 276:1837583
Yamagata T, Mitani K, Oda H, Suzuki
T, Honda H, et al. 2000. Acetylation
of GATA-3 affects T-cell survival and
7 Jan 2005
14:6
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
30.

31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
homing to secondary lymphoid organs.

EMBO J. 19:467687
Boyes J, Byfield P, Nakatani Y, Ogryzko
V. 1998. Regulation of activity of the transcription factor GATA-1 by acetylation.
Nature 396:59498
Herrera JE, Sakaguchi K, Bergel M, Trieschmann L, Nakatani Y, Bustin M. 1999.
Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes. Mol. Cell Biol.
19:346673
Imai S, Armstrong CM, Kaeberlein M,
Guarente L. 2000. Transcriptional silencing and longevity protein Sir2 is an NADdependent histone deacetylase. Nature
403:795800
Finnin MS, Donigian JR, Pavletich NP.
2001. Structure of the histone deacetylase
SIRT2. Nat. Struct. Biol. 8:62125
Kao HY, Verdel A, Tsai CC, Simon C,
Juguilon H, Khochbin S. 2001. Mechanism for nucleocytoplasmic shuttling of
histone deacetylase 7. J. Biol. Chem.
276:47496507
Wang AH, Yang XJ. 2001. Histone
deacetylase 4 possesses intrinsic nuclear
import and export signals. Mol. Cell Biol.
21:59926005
Gray SG, Ekstrom TJ. 2001. The human
histone deacetylase family. Exp. Cell Res.
262:7583
Guardiola AR, Yao TP. 2002. Molecular
cloning and characterization of a novel
histone deacetylase HDAC10. J. Biol.
Chem. 277:335056
Verdel A, Curtet S, Brocard MP, Rousseaux S, Lemercier C, et al. 2000. Active maintenance of mHDA2/mHDAC6
histone-deacetylase in the cytoplasm.
Curr. Biol. 10:74749
de Ruijter AJ, van Gennip AH, Caron
HN, Kemp S, van Kuilenburg AB. 2003.
Histone deacetylases (HDACs): characterization of the classical HDAC family.
Biochem. J. 370:73749
Grozinger CM, Hassig CA, Schreiber SL.
1999. Three proteins define a class of
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
519
human histone deacetylases related to

yeast Hda1p. Proc. Natl. Acad. Sci. USA
96:486873
Petrie K, Guidez F, Howell L, Healy
L, Waxman S, et al. 2003. The histone
deacetylase 9 gene encodes multiple protein isoforms. J. Biol. Chem. 278:16059
72
Hu E, Dul E, Sung CM, Chen Z, Kirkpatrick R, et al. 2003. Identification of
novel isoform-selective inhibitors within
class I histone deacetylases. J. Pharmacol. Exp. Ther. 307:72028
Jung M. 2001. Inhibitors of histone
deacetylase as new anticancer agents.
Curr. Med. Chem. 8:150511
Miller TA, Witter DJ, Belvedere S. 2003.
Histone deacetylase inhibitors. J. Med.
Chem. 46:5097116
Meinke PT, Liberator P. 2001. Histone
deacetylase: a target for antiproliferative and antiprotozoal agents. Curr. Med.
Chem. 8:21135
Finnin MS, Donigian JR, Cohen A,
Richon VM, Rifkind RA, et al. 1999.
Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401:18893
Breslow R, Belvedere S, Gershell L.
2000. Development of cytodifferentiating agents for cancer chemotherapy. Helv.
Chim. Acta 83:168592
Woo SH, Frechette S, Abou Khalil E,
Bouchain G, Vaisburg A, et al. 2002.
Structurally simple trichostatin A-like
straight chain hydroxamates as potent histone deacetylase inhibitors. J. Med. Chem.
45:287785
Yoshida M, Horinouchi S, Beppu T. 1995.
Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function.
Bioessays 17:42330
Shute RE, Dunlap B, Rich DH. 1987.
Analogues of the cytostatic and antimitogenic agents chlamydocin and HCtoxin: synthesis and biological activity
of chloromethyl ketone and diazomethyl
7 Jan 2005
14:6
520
51.

52.
53.
54.
55.
56.
57.
58.
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUMMOND ET AL.
ketone functionalized cyclic tetrapeptides. J. Med. Chem. 30:7178
Gore SD, Carducci MA. 2000. Modifying histones to tame cancer: clinical
development of sodium phenylbutyrate
and other histone deacetylase inhibitors.
Expert Opin. Invest. Drugs 9:292334
Furumai R, Matsuyama A, Kobashi N,
Lee KH, Nishiyama M, et al. 2002. FK228
(depsipeptide) as a natural prodrug that inhibits class I histone deacetylases. Cancer
Res. 62:491621
Komatsu Y, Tomizaki KY, Tsukamoto M,
Kato T, Nishino N, et al. 2001. Cyclic
hydroxamic-acid-containing peptide 31, a
potent synthetic histone deacetylase inhibitor with antitumor activity. Cancer
Res. 61:445966
Furumai R, Komatsu Y, Nishino N,
Khochbin S, Yoshida M, Horinouchi
S. 2001. Potent histone deacetylase inhibitors built from trichostatin A and
cyclic tetrapeptide antibiotics including
trapoxin. Proc. Natl. Acad. Sci. USA 98:
8792
el-Beltagi HM, Martens AC, Lelieveld P,
Haroun EA, Hagenbeek A. 1993. Acetyldinaline: a new oral cytostatic drug with
impressive differential activity against
leukemic cells and normal stem cells
preclinical studies in a relevant rat model
for human acute myelocytic leukemia.
Suzuki T, Ando T, Tsuchiya K, Fukazawa
N, Saito A, et al. 1999. Synthesis and histone deacetylase inhibitory activity of new
benzamide derivatives. J. Med. Chem. 42:
30013
Mai A, Massa S, Ragno R, Esposito M,
Sbardella G, et al. 2002. Binding mode
analysis of 3-(4-benzoyl-1-methyl-1H2-pyrrolyl)-N-hydroxy-2-propenamide: a
new synthetic histone deacetylase inhibitor inducing histone hyperacetylation,
growth inhibition, and terminal cell differentiation. J. Med. Chem. 45:177884
Ragno R, Mai A, Massa S, Cerbara I, Valente S, et al. 2004. 3-(4-Aroyl-1-methyl-
59.
60.
61.
62.
63.
64.
65.
66.
67.
1H-pyrrol-2-yl)-N-hydroxy-2-propenamides as a new class of synthetic histone

deacetylase inhibitors. 3. Discovery of
novel lead compounds through structurebased drug design and docking studies. J.
Med. Chem. 47:135159
Pina IC, Gautschi JT, Wang GY, Sanders
ML, Schmitz FJ, et al. 2003. Psammaplins
from the sponge Pseudoceratina purpurea: inhibition of both histone deacetylase and DNA methyltransferase. J. Org.
Chem. 68:386673
Haggarty SJ, Koeller KM, Wong JC,
Butcher RA, Schreiber SL. 2003. Multidimensional chemical genetic analysis of diversity-oriented synthesis-derived
deacetylase inhibitors using cell-based assays. Chem. Biol. 10:38396
Haggarty SJ, Koeller KM, Wong JC,
Grozinger CM, Schreiber SL. 2003. Domain-selective small-molecule inhibitor
of histone deacetylase 6 (HDAC6)mediated tubulin deacetylation. Proc.
Pandolfi PP. 2001. Transcription therapy
for cancer. Oncogene 20:311627
Timmermann S, Lehrmann H, Polesskaya
A, Harel-Bellan A. 2001. Histone acetylation and disease. Cell Mol. Life Sci. 58:
72836
Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. 2001. Histone deacetylases and cancer: causes
and therapies. Nat. Rev. Cancer 1:194
202
Rosato RR, Grant S. 2004. Histone
deacetylase inhibitors in clinical development. Expert Opin. Invest. Drugs 13:
2138
Richon VM, Sandhoff TW, Rifkind RA,
Marks PA. 2000. Histone deacetylase
inhibitor selectively induces p21WAF1
expression and gene-associated histone
acetylation. Proc. Natl. Acad. Sci. USA
97:1001419
Vigushin DM, Coombes RC. 2002. Histone deacetylase inhibitors in cancer treatment. Anticancer Drugs 13:113
7 Jan 2005
14:6
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE


68. Mitsiades CS, Mitsiades NS, McMullan
CJ, Poulaki V, Shringarpure R, et al.
2004. Transcriptional signature of histone deacetylase inhibition in multiple
myeloma: biological and clinical implications. Proc. Natl. Acad. Sci. USA
101:54045
69. Petti MC, Fazi F, Gentile M, Diverio D, De
Fabritiis P, et al. 2002. Complete remission through blast cell differentiation in
PLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and in vivo studies. Blood 100:106567
70. Han JW, Ahn SH, Park SH, Wang SY,
Bae GU, et al. 2000. Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of
p21WAF1/Cip1 and gelsolin. Cancer Res.
60:606874
71. Saito A, Yamashita T, Mariko Y, Nosaka
Y, Tsuchiya K, et al. 1999. A synthetic
inhibitor of histone deacetylase, MS-27275, with marked in vivo antitumor activity against human tumors. Proc. Natl.
72. Cameron EE, Bachman KE, Myohanen S,
Herman JG, Baylin SB. 1999. Synergy of
demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21:1037
73. Primeau M, Gagnon J, Momparler RL.
2003. Synergistic antineoplastic action
of DNA methylation inhibitor 5-AZA-2 deoxycytidine and histone deacetylase inhibitor depsipeptide on human breast carcinoma cells. Int. J. Cancer 103:17784
74. Henderson C, Brancolini C. 2003. Apoptotic pathways activated by histone
deacetylase inhibitors: implications for
the drug-resistant phenotype. Drug Resist.
Update 6:24756
75. Johnstone RW, Licht JD. 2003. Histone
deacetylase inhibitors in cancer therapy:
is transcription the primary target? Cancer Cell. 4:1318
76. Kwon SH, Ahn SH, Kim YK, Bae GU,
Yoon JW, et al. 2002. Apicidin, a histone deacetylase inhibitor, induces apop-
77.
78.
79.
80.
81.
82.
83.
521
tosis and Fas/Fas ligand expression in human acute promyelocytic leukemia cells.
J. Biol. Chem. 277:207380
Cao XX, Mohuiddin I, Ece F, McConkey
DJ, Smythe WR. 2001. Histone deacetylase inhibitor downregulation of bcl-xl
gene expression leads to apoptotic cell
death in mesothelioma. Am. J. Respir. Cell
Mol. Biol. 25:56268
Sawa H, Murakami H, Ohshima Y, Sugino T, Nakajyo T, et al. 2001. Histone
deacetylase inhibitors such as sodium butyrate and trichostatin A induce apoptosis through an increase of the bcl-2related protein Bad. Brain Tumor. Pathol.
18:10914
Slack JL. 1999. The biology and treatment
of acute progranulocytic leukemia. Curr.
Opin. Oncol. 11:913
Nimmanapalli R, Fuino L, Bali P, Gasparetto M, Glozak M, et al. 2003. Histone deacetylase inhibitor LAQ824 both
lowers expression and promotes proteasomal degradation of Bcr-Abl and
induces apoptosis of imatinib mesylatesensitive or -refractory chronic myelogenous leukemia-blast crisis cells. Cancer
Res. 63:512635
Scott GK, Marden C, Xu F, Kirk L,
Benz CC. 2002. Transcriptional repression of ErbB2 by histone deacetylase inhibitors detected by a genomically integrated ErbB2 promoter-reporting cell
screen. Mol. Cancer Ther. 1:38592
Fuino L, Bali P, Wittmann S, Donapaty
S, Guo F, et al. 2003. Histone deacetylase
inhibitor LAQ824 down-regulates Her-2
and sensitizes human breast cancer cells to
trastuzumab, taxotere, gemcitabine, and
epothilone B. Mol. Cancer Ther. 2:971
84
Kim JS, Jeung HK, Cheong JW, Maeng H,
Lee ST, et al. 2004. Apicidin potentiates
the imatinib-induced apoptosis of BcrAbl-positive human leukaemia cells by
enhancing the activation of mitochondriadependent caspase cascades. Br. J.
Haematol. 124:16678
7 Jan 2005
14:6

522
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUMMOND ET AL.
84. Yu C, Rahmani M, Conrad D, Subler M,

Dent P, Grant S. 2003. The proteasome
inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors
to induce apoptosis in Bcr/Abl+ cells
sensitive and resistant to STI571. Blood
102:376574
85. Rahmani M, Yu C, Dai Y, Reese E,
Ahmed W, et al. 2003. Coadministration
of the heat shock protein 90 antagonist
17-allylamino-17-demethoxygeldanamycin with suberoylanilide hydroxamic
acid or sodium butyrate synergistically
induces apoptosis in human leukemia
86. Liu LT, Chang HC, Chiang LC, Hung WC.
2003. Histone deacetylase inhibitor upregulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer
Res. 63:306972
87. Sasakawa Y, Naoe Y, Noto T, Inoue
T, Sasakawa T, et al. 2003. Antitumor efficacy of FK228, a novel histone deacetylase inhibitor, depends on
the effect on expression of angiogenesis factors. Biochem. Pharmacol. 66:897
906
88. Michaelis M, Michaelis UR, Fleming I,
Suhan T, Cinatl J, et al. 2004. Valproic
acid inhibits angiogenesis in vitro and
in vivo. Mol. Pharmacol. 65:520
27
89. Ruefli AA, Bernhard D, Tainton KM,
Kofler R, Smyth MJ, Johnstone RW.
2002. Suberoylanilide hydroxamic acid
(SAHA) overcomes multidrug resistance
and induces cell death in P-glycoproteinexpressing cells. Int. J. Cancer 99:292
98
90. Tsurutani J, Soda H, Oka M, Suenaga M,
Doi S, et al. 2003. Antiproliferative effects of the histone deacetylase inhibitor
FR901228 on small-cell lung cancer lines
and drug-resistant sublines. Int. J. Cancer
104:23842
91. Jing Y, Xia L, Waxman S. 2002. Targeted removal of PML-RARalpha protein is required prior to inhibition
92.
93.
94.
95.
96.
97.
98.
99.
of histone deacetylase for overcoming

all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia.
Blood 100:100813
Cote S, Zhou D, Bianchini A, Nervi
C, Gallagher RE, Miller WH Jr. 2000.
Altered ligand binding and transcriptional regulation by mutations in the
PML/RARalpha ligand-binding domain
arising in retinoic acid-resistant patients with acute promyelocytic leukemia.
Blood 96:32008
Castro-Galache MD, Ferragut JA, Barbera VM, Martin-Orozco E, GonzalezRos JM, et al. 2003. Susceptibility of
multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors. Int. J. Cancer 104:57986
Peart MJ, Tainton KM, Ruefli AA, Dear
AE, Sedelies KA, et al. 2003. Novel
mechanisms of apoptosis induced by histone deacetylase inhibitors. Cancer Res.
63:446071
Batova A, Shao LE, Diccianni MB, Yu
AL, Tanaka T, et al. 2002. The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia and drugresistant primary leukemia and cancer cell
lines. Blood 100:331924
Takakura M, Kyo S, Sowa Y, Wang Z,
Yatabe N, et al. 2001. Telomerase activation by histone deacetylase inhibitor in
normal cells. Nucleic Acids Res. 29:3006
11
Nakamura M, Saito H, Ebinuma H, Wakabayashi K, Saito Y, et al. 2001. Reduction of telomerase activity in human
liver cancer cells by a histone deacetylase inhibitor. J. Cell Physiol. 187:392
401
Vigushin DM, Ali S, Pace PE, Mirsaidi
N, Ito K, et al. 2001. Trichostatin A is a
histone deacetylase inhibitor with potent
antitumor activity against breast cancer
in vivo. Clin. Cancer Res. 7:971
76
Svechnikova I, Gray SG, Kundrotiene J,
Ponthan F, Kogner P, Ekstrom TJ. 2003.
7 Jan 2005
14:6
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE

100.
101.
102.
103.
104.
105.
106.
Apoptosis and tumor remission in liver

tumor xenografts by 4-phenylbutyrate.
Int. J. Oncol. 22:57988
Sasakawa Y, Naoe Y, Inoue T, Sasakawa T,
Matsuo M, et al. 2003. Effects of FK228,
a novel histone deacetylase inhibitor, on
tumor growth and expression of p21 and cmyc genes in vivo. Cancer Lett. 195:161
88
Remiszewski SW, Sambucetti LC, Bair
KW, Bontempo J, Cesarz D, et al. 2003.
N-hydroxy-3-phenyl-2-propenamides as
novel inhibitors of human histone
deacetylase with in vivo antitumor activity: discovery of (2E)-N-hydroxy-3-[4[[(2-hydroxyethyl)[2-(1H-indol-3-yl)eth
yl]amino]methyl]phenyl]-2-propenamide
(NVP-LAQ824). J. Med. Chem. 46:4609
24
Kuefer R, Hofer MD, Altug V, Zorn C,
Genze F, et al. 2004. Sodium butyrate and
tributyrin induce in vivo growth inhibition
and apoptosis in human prostate cancer.
Br. J. Cancer 90:53541
Kosugi H, Ito M, Yamamoto Y, Towatari
M, Ueda R, et al. 2001. In vivo effects of a
histone deacetylase inhibitor, FK228, on
human acute promyelocytic leukemia in
NOD/Shi-scid/scid mice. Jpn. J. Cancer
Res. 92:52936
Butler LM, Webb Y, Agus DB, Higgins
B, Tolentino TR, et al. 2001. Inhibition
of transformed cell growth and induction
of cellular differentiation by pyroxamide,
an inhibitor of histone deacetylase. Clin.
Cancer Res. 7:96270
Butler LM, Agus DB, Scher HI, Higgins
B, Rose A, et al. 2000. Suberoylanilide
hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of
prostate cancer cells in vitro and in vivo.
Arts J, Van Emelen K, Angibaud P,
Van Brandt S, Poncelet V, et al. 2003.
Small molecule inhibitors of histone
deacetylases (HDACs): identification of
JNJ16241199, a potent oral antitumoral
agent. Presented at AACR-NCI-EORTC
107.
108.
109.
110.
111.
112.
113.
114.
523
Int. Conf. Mol. Targets Cancer Ther.,

Boston
Piekarz RL, Robey R, Sandor V, Bakke
S, Wilson WH, et al. 2001. Inhibitor
of histone deacetylation, depsipeptide
(FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a
case report. Blood 98:286568
Siu LL, Von Hoff DD, Rephaeli A, Izbicka
E, Cerna C, et al. 1998. Activity of pivaloyloxymethyl butyrate, a novel anticancer
agent, on primary human tumor colonyforming units. Invest. New Drugs 16:113
19
Patnaik A, Rowinsky EK, Villalona MA,
Hammond LA, Britten CD, et al. 2002. A
phase I study of pivaloyloxymethyl butyrate, a prodrug of the differentiating
agent butyric acid, in patients with advanced solid malignancies. Clin. Cancer
Res. 8:214248
Jaboin J, Wild J, Hamidi H, Khanna C,
Kim CJ, et al. 2002. MS-27-275, an inhibitor of histone deacetylase, has marked
in vitro and in vivo antitumor activity
against pediatric solid tumors. Cancer
Res. 62:610815
Fournel M, Trachy-Bourget MC, Yan PT,
Kalita A, Bonfils C, et al. 2002. Sulfonamide anilides, a novel class of histone
deacetylase inhibitors, are antiproliferative against human tumors. Cancer Res.
62:432530
Volpe DA, LoRusso PM, Foster BJ, Parchment RE. 2004. In vitro and in vivo effects
of acetyldinaline on murine megakaryocytopoiesis. Cancer Chemother. Pharmacol. 54(1):8994
Sandor V, Bakke S, Robey RW, Kang MH,
Blagosklonny MV, et al. 2002. Phase I trial
of the histone deacetylase inhibitor, depsipeptide (FR901228, NSC 630176), in
patients with refractory neoplasms. Clin.
Cancer Res. 8:71828
Prakash S, Foster BJ, Meyer M, Wozniak
A, Heilbrun LK, et al. 2001. Chronic oral
administration of CI-994: a phase 1 study.
Invest. New Drugs 19:111
7 Jan 2005
14:6

524
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUMMOND ET AL.
115. Kelly WK, Richon VM, O Connor O, Curley T, MacGregor-Curtelli B, et al. 2003.
Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously.
116. Piekarz RL, Robey R, Sandor V, Bakke
S, Wilson WH, et al. 2001. Inhibitor
of histone deacetylation, depsipeptide
(FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a
case report. Blood 98:286568
117. Atadja P, Gao L, Kwon P, Trogani
N, Walker H, et al. 2004. Selective
growth inhibition of tumor cells by a
novel histone deacetylase inhibitor, NVPLAQ824. Cancer Res. 64:68995
118. Drummond DC, Meyer OM, Hong K,
Kirpotin DB, Papahadjopoulos D. 1999.
Optimizing liposomes for delivery of
chemotherapeutic agents to solid tumors.
Pharmacol. Rev. 51:691743
119. Benz C, Scott G, Berger C, Amend C,
Guo Z, et al. 2003. Liposome encapsulation of histone deacetylase inhibitor enhances in vivo activity against ErbB2positive breast cancers. Clin. Cancer Res.
9:6259s
120. Kim MS, Blake M, Baek JH, Kohlhagen
G, Pommier Y, Carrier F. 2003. Inhibition
of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA.
121. Witzig TE, Timm M, Stenson M, Svingen PA, Kaufmann SH. 2000. Induction of
apoptosis in malignant B cells by phenylbutyrate or phenylacetate in combination
with chemotherapeutic agents. Clin. Cancer Res. 6:68192
122. Zhu WG, Lakshmanan RR, Beal MD,
Otterson GA. 2001. DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors.
123. Rashid SF, Moore JS, Walker E, Driver
PM, Engel J, et al. 2001. Synergistic
growth inhibition of prostate cancer cells
by 1 alpha,25 dihydroxyvitamin D(3) and
124.
125.
126.
127.
128.
129.
130.
its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A. Oncogene 20:186072
Rahmani M, Yu C, Reese E, Ahmed
W, Hirsch K, et al. 2003. Inhibition of
PI-3 kinase sensitizes human leukemic
cells to histone deacetylase inhibitormediated apoptosis through p44/42 MAP
kinase inactivation and abrogation of
p21(CIP1/WAF1) induction rather than
AKT inhibition. Oncogene 22:623142
Almenara J, Rosato R, Grant S. 2002. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells
by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic
acid (SAHA). Leukemia 16:133143
Keen JC, Yan L, Mack KM, Pettit C,
Smith D, et al. 2003. A novel histone
deacetylase inhibitor, scriptaid, enhances
expression of functional estrogen receptor
alpha (ER) in ER negative human breast
cancer cells in combination with 5-aza 2 deoxycytidine. Breast Cancer Res. Treat.
81:17786
Coffey DC, Kutko MC, Glick RD, Butler LM, Heller G, et al. 2001. The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma
xenografts in vivo, alone and synergistically with all-trans retinoic acid. Cancer
Res. 61:359194
Belinsky SA, Klinge DM, Stidley CA,
Issa JP, Herman JG, et al. 2003. Inhibition
of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res. 63:708993
Kosugi H, Towatari M, Hatano S, Kitamura K, Kiyoi H, et al. 1999. Histone
deacetylase inhibitors are the potent inducer/enhancer of differentiation in acute
myeloid leukemia: a new approach to antileukemia therapy. Leukemia 13:131624
Zhou DC, Kim SH, Ding W, Schultz C,
Warrell RP Jr, Gallagher RE. 2002. Frequent mutations in the ligand-binding domain of PML-RARalpha after multiple relapses of acute promyelocytic leukemia:
7 Jan 2005
14:6
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE

131.
132.
133.
134.
135.
136.
137.
analysis for functional relationship to response to all-trans retinoic acid and histone deacetylase inhibitors in vitro and
in vivo. Blood 99:135663
Giuliano M, Lauricella M, Calvaruso G,
Carabillo M, Emanuele S, et al. 1999. The
apoptotic effects and synergistic interaction of sodium butyrate and MG132 in
human retinoblastoma Y79 cells. Cancer
Res. 59:558695
Yu C, Rahmani M, Almenara J, Subler M, Krystal G, et al. 2003. Histone
deacetylase inhibitors promote STI571mediated apoptosis in STI571-sensitive
and -resistant Bcr/Abl+ human myeloid
leukemia cells. Cancer Res. 63:211826
Nimmanapalli R, Fuino L, Stobaugh C,
Richon V, Bhalla K. 2003. Cotreatment
with the histone deacetylase inhibitor
suberoylanilide hydroxamic acid (SAHA)
enhances imatinib-induced apoptosis of
Bcr-Abl-positive human acute leukemia
cells. Blood 101:323639
Rosato RR, Almenara JA, Yu C, Grant
S. 2004. Evidence of a functional role
for p21WAF1/CIP1 down-regulation in
synergistic antileukemic interactions between the histone deacetylase inhibitor
sodium butyrate and flavopiridol. Mol.
Pharmacol. 65:57181
Rosato RR, Almenara JA, Cartee L,
Betts V, Chellappan SP, Grant S. 2002.
The cyclin-dependent kinase inhibitor
flavopiridol disrupts sodium butyrateinduced p21WAF1/CIP1 expression and
maturation while reciprocally potentiating apoptosis in human leukemia cells.
Mol. Cancer Ther. 1:25366
Nguyen DM, Schrump WD, Tsai WS,
Chen A, Stewart JHt, et al. 2003.
Enhancement of depsipeptide-mediated
apoptosis of lung or esophageal cancer
cells by flavopiridol: activation of the
mitochondria-dependent death-signaling
pathway. J. Thorac. Cardiovasc. Surg.
125:113242
Fujii S, Luo RZ, Yuan J, Kadota M, Oshimura M, et al. 2003. Reactivation of the
138.
139.
140.
141.
142.
143.
144.
525
silenced and imprinted alleles of ARHI

is associated with increased histone H3
acetylation and decreased histone H3 lysine 9 methylation. Hum. Mol. Genet.
12:1791800
Pili R, Kruszewski MP, Hager BW, Lantz
J, Carducci MA. 2001. Combination of
phenylbutyrate and 13-cis retinoic acid inhibits prostate tumor growth and angiogenesis. Cancer Res. 61:147785
Rosato RR, Wang Z, Gopalkrishnan RV,
Fisher PB, Grant S. 2001. Evidence of a
functional role for the cyclin-dependent
kinase-inhibitor p21WAF1/CIP1/MDA6
in promoting differentiation and preventing mitochondrial dysfunction and apoptosis induced by sodium butyrate in
human myelomonocytic leukemia cells
(U937). Int. J. Oncol. 19:18191
Nguyen DM, Schrump WD, Chen GA,
Tsai W, Nguyen P, et al. 2004. Abrogation of p21 expression by flavopiridol enhances depsipeptide-mediated apoptosis
in malignant pleural mesothelioma cells.
Denlinger CE, Keller MD, Mayo MW,
Broad RM, Jones DR. 2004. Combined
proteasome and histone deacetylase inhibition in non-small cell lung cancer. J.
Thorac. Cardiovasc. Surg. 127:107886
Zhang Y, Adachi M, Zhao X, Kawamura
R, Imai K. 2004. Histone deacetylase
inhibitors FK228, N-(2-aminophenyl)-4[N-(pyridin-3-yl-methoxycarbonyl) amino-methyl]benzamide and m-carboxycinnamic acid bis-hydroxamide augment
radiation-induced cell death in gastrointestinal adenocarcinoma cells. Int. J. Cancer 110:3018
Chung YL, Wang AJ, Yao LF. 2004. Antitumor histone deacetylase inhibitors suppress cutaneous radiation syndrome: implications for increasing therapeutic gain
in cancer radiotherapy. Mol. Cancer Ther.
3:31725
Spotswood HT, Turner BM. 2002. An increasingly complex code. J. Clin. Invest.
110:57782
7 Jan 2005
14:6

526
AR
AR232-PA45-21.tex
AR232-PA45-21.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUMMOND ET AL.
145. Gu W, Roeder RG. 1997. Activation of

p53 sequence-specific DNA binding by
acetylation of the p53 C-terminal domain.
Cell 90:595606
146. Sakaguchi K, Herrera JE, Saito S, Miki
T, Bustin M, et al. 1998. DNA damage
activates p53 through a phosphorylationacetylation cascade. Genes Dev. 12:2831
41
147. Tomita A, Towatari M, Tsuzuki S,
Hayakawa F, Kosugi H, et al. 2000.
c-Myb acetylation at the carboxylterminal conserved domain by transcriptional co-activator p300. Oncogene 19:
44451
148. Hubbert C, Guardiola A, Shao R,
Kawaguchi Y, Ito A, et al. 2002. HDAC6
is a microtubule-associated deacetylase.
Nature 417:45558
149. Munshi N, Merika M, Yie J, Senger K,
Chen G, Thanos D. 1998. Acetylation of
HMG I(Y) by CBP turns off IFN beta expression by disrupting the enhanceosome.
Mol. Cell. 2:45767
150. Waltzer L, Bienz M. 1998. Drosophila
CBP represses the transcription factor
TCF to antagonize Wingless signalling.
Nature 395:52125
151. Naryzhny SN, Lee H. 2004. The
post-translational modifications of proliferating cell nuclear antigen (PCNA):
acetylation, not phosphorylation, plays an
important role in the regulation of its function. J. Biol. Chem. 279:2019499
152. Zhang W, Bieker JJ. 1998. Acetylation
and modulation of erythroid Kruppellike factor (EKLF) activity by interaction with histone acetyltransferases. Proc.
153. Chen H, Lin RJ, Xie W, Wilpitz D,
Evans RM. 1999. Regulation of hormoneinduced histone hyperacetylation and
gene activation via acetylation of an acetylase. Cell 98:67586
154. Soutoglou E, Katrakili N, Talianidis I.
2000. Acetylation regulates transcription
factor activity at multiple levels. Mol.
Cell. 5:74551
155. Bannister AJ, Miska EA, Gorlich D,

Kouzarides T. 2000. Acetylation of
importin-alpha nuclear import factors by
CBP/p300. Curr. Biol. 10:46770
156. Chen LF, Greene WC. 2003. Regulation
of distinct biological activities of the NFkappaB transcription factor complex by
acetylation. J. Mol. Med. 81:54957
157. Goel A, Janknecht R. 2003. Acetylationmediated transcriptional activation of the
ETS protein ER81 by p300, P/CAF, and
HER2/Neu. Mol. Cell Biol. 23:6243
54
158. Jacob AL, Lund J, Martinez P, Hedin L.
2001. Acetylation of steroidogenic factor
1 protein regulates its transcriptional activity and recruits the coactivator GCN5.
J. Biol. Chem. 276:3765964
159. Cohen HY, Lavu S, Bitterman KJ,
Hekking B, Imahiyerobo TA, et al. 2004.
Acetylation of the C terminus of Ku70
by CBP and PCAF controls Bax-mediated
apoptosis. Mol. Cell 13:62738
160. Pelletier G, Stefanovsky VY, Faubladier
M, Hirschler-Laszkiewicz I, Savard J,
et al. 2000. Competitive recruitment of
CBP and Rb-HDAC regulates UBF acetylation and ribosomal transcription. Mol.
Cell. 6:105966
161. Ammanamanchi S, Freeman JW, Brattain MG. 2003. Acetylated sp3 is a
transcriptional activator. J. Biol. Chem.
278:3577580
162. Huang S, Qiu Y, Shi Y, Xu Z, Brandt SJ.
2000. P/CAF-mediated acetylation regulates the function of the basic helix-loophelix transcription factor TAL1/SCL.
EMBO J. 19:6792803
163. Yao YL, Yang WM, Seto E. 2001. Regulation of transcription factor YY1 by acetylation and deacetylation. Mol. Cell Biol.
21:597991
164. Martinez-Balbas MA, Bauer UM, Nielsen
SJ, Brehm A, Kouzarides T. 2000. Regulation of E2F1 activity by acetylation.
EMBO J. 19:66271
165. Sartorelli V, Puri PL, Hamamori Y,
Ogryzko V, Chung G, et al. 1999.
7 Jan 2005
14:6
AR
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AR232-PA45-21.sgm
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P1: GCE

166.
167.
168.
169.
170.
171.
172.
Acetylation of MyoD directed by PCAF is

necessary for the execution of the muscle
program. Mol. Cell. 4:72534
Park SH, Lee SR, Kim BC, Cho EA, Patel SP, et al. 2002. Transcriptional regulation of the transforming growth factor beta
type II receptor gene by histone acetyltransferase and deacetylase is mediated by
NF-Y in human breast cancer cells. J. Biol.
Chem. 277:516874
Lee BI, Park SH, Kim JW, Sausville EA,
Kim HT, et al. 2001. MS-275, a histone
deacetylase inhibitor, selectively induces
transforming growth factor beta type II receptor expression in human breast cancer
Butler LM, Zhou X, Xu WS, Scher HI,
Rifkind RA, et al. 2002. The histone
deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxinbinding protein-2, and down-regulates
thioredoxin. Proc. Natl. Acad. Sci. USA
99:117005
Kim MS, Kwon HJ, Lee YM, Baek JH,
Jang JE, et al. 2001. Histone deacetylases
induce angiogenesis by negative regulation of tumor suppressor genes. Nat. Med.
7:43743
Andoh A, Shimada M, Araki Y, Fujiyama Y, Bamba T. 2002. Sodium
butyrate enhances complement-mediated
cell injury via down-regulation of decayaccelerating factor expression in colonic
cancer cells. Cancer Immunol. Immunother. 50:66372
Zhang XD, Gillespie SK, Borrow JM,
Hersey P. 2004. The histone deacetylase inhibitor suberic bishydroxamate
regulates the expression of multiple apoptotic mediators and induces mitochondriadependent apoptosis of melanoma cells.
Mol. Cancer Ther. 3:42535
Bernhard D, Skvortsov S, Tinhofer I, Hubl
H, Greil R, et al. 2001. Inhibition of histone deacetylase activity enhances Fas
receptor-mediated apoptosis in leukemic
lymphoblasts. Cell Death Differ. 8:1014
21
527
173. Huang H, Reed CP, Zhang JS, Shridhar

V, Wang L, Smith DI. 1999. Carboxypeptidase A3 (CPA3): a novel gene highly
induced by histone deacetylase inhibitors
during differentiation of prostate epithelial cancer cells. Cancer Res. 59:298188
174. Yang X, Ferguson AT, Nass SJ, Phillips
DL, Butash KA, et al. 2000. Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition. Cancer Res.
60:689094
175. Walker GE, Wilson EM, Powell D, Oh
Y. 2001. Butyrate, a histone deacetylase
inhibitor, activates the human IGF binding protein-3 promoter in breast cancer cells: molecular mechanism involves
an Sp1/Sp3 multiprotein complex. Endocrinology 142:381727
176. Pawel JV, Shepherd F, Gatzmeier U, Natale RB, OBrien ME, et al. 2002. Randomized phase 2 study of the oral histone
deacetylase inhibitor CI-994 plus gemcitabine (Gem) vs placebo (PBO) plus
GEM in second-line nonsmall cell lung
cancer (NSCLC). Am. Soc. Clin. Oncol.
Annu. Meet. A1239, Orlando, Florida
177. Johnson CA, Padget K, Austin CA, Turner
BM. 2001. Deacetylase activity associates
with topoisomerase II and is necessary
for etoposide-induced apoptosis. J. Biol.
Chem. 276:453942
178. Maggio SC, Rosato RR, Kramer LB, Dai
Y, Rahmani M, et al. 2004. The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells.
179. Lemaire M, Momparler LF, Farinha NJ,
Bernstein M, Momparler RL. 2004. Enhancement of antineoplastic action of 5aza-2 -deoxycytidine by phenylbutyrate
on L1210 leukemic cells. Leuk. Lymphoma 45:14754
180. Hernandez A, Thomas R, Smith F, Sandberg J, Kim S, et al. 2001. Butyrate sensitizes human colon cancer cells to TRAILmediated apoptosis. Surgery 130:26572
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181. Rosato RR, Almenara JA, Dai Y, Grant

S. 2003. Simultaneous activation of the
intrinsic and extrinsic pathways by histone deacetylase (HDAC) inhibitors and
tumor necrosis factor-related apoptosisinducing ligand (TRAIL) synergistically
induces mitochondrial damage and apoptosis in human leukemia cells. Mol. Cancer Ther. 2:127384
LaTeX2e(2002/01/18)
182. Guo F, Sigua C, Tao J, Bali P, George

P, et al. 2004. Cotreatment with histone deacetylase inhibitor LAQ824 enhances Apo-2L/tumor necrosis factorrelated apoptosis inducing ligand-induced
death inducing signaling complex activity and apoptosis of human acute
leukemia cells. Cancer Res. 64:2580
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Figure 1 Histones can be modified in a variety of ways, primarily in the tails of core histones in a process that is referred to as the histone code. Acetylated lysine residues, methylated arginines, methylated lysines, phosphorylated serines, sumoylated lysines, and ubquitinated lysine residues all contribute to the histone code. The relative positions for each
modification on the various histone tails are depicted by symbols that are defined in the key.
The actual chemical modification of the various amino acids in the histone tails is shown with
the colored bonds indicating the modification and the amino acid residue shown in black.
This figure was adapted from Turner (4) and Spotswood & Turner (144) with permission.
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Copyright
First published online as a Review in Advance on October 7, 2004

THE MAGIC BULLETS AND TUBERCULOSIS

DRUG TARGETS
Ying Zhang
Department of Molecular Microbiology and Immunology, Bloomberg School of Public
Health, Johns Hopkins University, Baltimore, Maryland 21205; email: yzhang@jhsph.edu
Key Words antituberculosis agents, Mycobacterium tuberculosis, drug

development
Abstract Modern chemotherapy has played a major role in our control of tuberculosis. Yet tuberculosis still remains a leading infectious disease worldwide, largely
owing to persistence of tubercle bacillus and inadequacy of the current chemotherapy.
The increasing emergence of drug-resistant tuberculosis along with the HIV pandemic
threatens disease control and highlights both the need to understand how our current
drugs work and the need to develop new and more effective drugs. This review provides a brief historical account of tuberculosis drugs, examines the problem of current
chemotherapy, discusses the targets of current tuberculosis drugs, focuses on some
promising new drug candidates, and proposes a range of novel drug targets for intervention. Finally, this review addresses the problem of conventional drug screens based
on inhibition of replicating bacilli and the challenge to develop drugs that target nonreplicating persistent bacilli. A new generation of drugs that target persistent bacilli is
needed for more effective treatment of tuberculosis.
INTRODUCTION
Humankinds battle with tuberculosis (TB) dates back to antiquity. TB, which
is caused by Mycobacterium tuberculosis, was a much more prevalent disease
in the past than it is today, and it was responsible for the deaths of about one
billion people during the last two centuries (1). Improved sanitation and living
conditions significantly reduced the incidence of the disease even before the advent
of chemotherapy. The introduction of TB chemotherapy in the 1950s, along with
the widespread use of BCG vaccine, had a great impact on further reduction in TB
incidence. However, despite these advances, TB still remains a leading infectious
disease worldwide, especially in the third world countries.
M. tuberculosis is a particularly successful pathogen that latently infects about
2 billion people, about one third of world population (2). Each year, there are about
8 million new TB cases and 2 million deaths worldwide. TB is on the increase
in recent years, largely owing to HIV infection, immigration, increased trade,
and globalization (2). The increasing emergence of drug-resistant TB, especially
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multidrug-resistant TB (MDR-TB, resistant to at least two frontline drugs such

as isoniazid and rifampin), is particularly alarming. MDR-TB has already caused
several fatal outbreaks (2, 3) and poses a significant threat to the treatment and
control of the disease in some parts of the world, where the incidence of MDR-TB
can be as high as 14% (2). The standard TB therapy is ineffective in controlling
MDR-TB in high MDR-TB incidence areas (4, 5). Fifty million people have already
been infected with drug-resistant TB (2). There is much concern that the TB
situation may become even worse with the spread of HIV worldwide, a virus that
weakens the host immune system and allows latent TB to reactivate and makes
the person more susceptible to reinfection with either drug-susceptible or drugresistant strains. The lethal combination of drug-resistant TB and HIV infection
is a growing problem that presents serious challenges for effective TB control. In
view of this situation, the World Health Organization (WHO) in 1993 declared TB
a global emergency (6).
There is an urgent need to develop new TB drugs (7). However, no new TB drugs
have been developed in about 40 years. Although TB can be cured with the current
therapy, the six months needed to treat the disease is too long, and the treatment
often has significant toxicity. These factors make patient compliance to therapy
very difficult, and this noncompliance frequently selects for drug-resistant TB
bacteria. The current TB problem clearly demonstrates the need for a re-evaluation
of our knowledge of the current TB drugs and chemotherapy and the need for
new and better drugs that are not only active against drug-resistant TB but also,
more importantly, shorten the requirement for six months of therapy. This review
provides a brief overview of the history of TB drugs and chemotherapy, discuss
the targets of the current TB drugs, examine some promising drug candidates,
propose potential new targets for drug development, and finally address issues of
novel drug screens that target the nonreplicating persistent bacilli that currently
require lengthy therapy. Several recent reviews on TB drug discovery are available
(812).
HISTORY OF ANTITUBERCULOSIS DRUGS

The TB drugs in use today reflect their origins in two sources of antimicrobial
agents, i.e., chemical origin and antibiotic origin. Albert Schatz and Selman Waksman discovered the first effective TB drug streptomycin (Figure 1) from Streptomyces griseus in 1944 (17), a discovery that marked the beginning of modern TB
chemotherapy.
The modern chemotherapeutic treatment of TB also had its beginning in sulfa
drugs developed by Domagk for the treatment of gram-positive bacterial infections
(14). In 1938, Rich and Follis from Johns Hopkins University found that sulfanilamide at high doses significantly inhibited the disease pathology in experimental
TB infection in guinea pigs (18) but without significant effect in treatment of human TB in tolerable doses. This finding stimulated further effort to refine sulfa
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TUBERCULOSIS DRUG TARGETS
Figure 1
531
Structures of some commonly used TB drugs.
drugs for the treatment of TB and subsequently led to synthesis of thiosemicarbazones such as Conteben (also called amithiazone), which were more active than
sulfanilamide and had definite clinical value but were not as effective as streptomycin (19). In 1946, two years after the discovery of streptomycin, Lehmann
from Sweden discovered para-aminosalicylic acid (PAS) (Figure 1) as an effective
TB drug (20), a discovery based on a curious observation made by Bernheim that
salicylate and benzoate stimulated the oxygen consumption of tubercle bacillus
(21). This was quickly followed in 1952 by the sensational discovery of the highly
active TB drug isoniazid (INH) (Figure 1) simultaneously by three drug companies: Hoffman LaRoche, E.R. Squibb & Sons, and Bayer. The discovery of INH
was based on the nicotinamide activity against tubercle bacilli in the animal model
observed by Chorine in 1945 (22) and the reshuffling of chemical groups in the
thiosemicarbazone (2325). INH represented a major milestone in the chemotherapy of TB because it is highly active, inexpensive, and without significant side
effects (26). Remarkably, the nicotinamide lead also led to the discovery of pyrazinamide (PZA) (Figure 1) in 1952 by the Lederle Research Laboratories (27)
and ethionamide (ETH)/Prothionamide (PTH) (Figure 1) in 1956 (28). Ethambutol (EMB) was discovered in 1961 at Lederle on the basis of the observation that
polyamines and diamines had activity against tubercle bacilli; subsequent synthesis
of diamine analogs led to the identification of EMB (29). Further screening for antibiotics from soil microbes led to discovery of many other antituberculosis drugs:
cycloserine (30); kanamycin (31) and its derivative amikacin; viomycin (32); capreomycin (33); and rifamycins (34) and its derivative rifampin (RIF), developed at
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Dow-Lepetit Research Laboratories, Italy (35), which has been the drug of choice
for treatment of TB since the 1970s. The 1950s and 60s represent a golden era
of TB drug discovery. Most of the TB drugs in use today were discovered during
this period, except the broad-spectrum quinolone drugs, which were developed in
1980s on the basis of the antibacterial activity of nalidixic acid discovered in the
1960s (36). Although quinolone drugs were not initially used for TB treatment,
they were subsequently shown to have high activity against tubercle bacillus and
were used as second-line drugs for the treatment of drug-resistant TB since the
late 1980s (37, 38).
THE CURRENT TB THERAPY AND

THE PROBLEM OF PERSISTERS
The current TB chemotherapy evolved from numerous experimental and clinical
studies primarily conducted between the 1950s and 1970s (39). The current recommended standard TB chemotherapy, called DOTS (directly observed treatment,
short-course), is a six month therapy consisting of an initial two-month phase of
treatment with four drugs, INH, RIF, PZA, and EMB, followed by a continuation
phase of treatment with INH and RIF for another four months (2). DOTS is currently the best TB therapy; it has a cure rate of up to 95% and is recommended
by the WHO for treating every TB patient (2). However, DOTS alone may not
work in areas where there is high incidence of MDR-TB (4, 5), where its cure rate
is as low as 50%. In such situations, WHO recommends the use of DOTS-Plus,
which is DOTS plus second-line TB drugs (see next section) for the treatment of
MDR-TB and TB (2). However, treatment of MDR-TB with DOTS-Plus takes up
to 24 months and is not only costly but also has significant toxicity.
Although DOTS can cure TB, the lengthy six month therapy makes patient
compliance difficult, and noncompliance is a frequent source of drug-resistant
strains. Although the TB chemotherapy renders a patient noninfectious a few
weeks after the initiation of the therapy, the therapy has to be continued for a
considerable period to prevent relapse. Compared with treatment of other bacterial infections such as H. pylori and pneumococcal infections, which takes no
longer than one to two weeks, it is striking that treatment of TB requires at least
six months. Why is the TB therapy so long? This is a fundamental problem facing TB chemotherapy and deserves some in-depth analysis. Several factors may
be responsible. First, the nature of the disease pathology can influence the efficacy and duration of chemotherapy. For example, open cavities teeming with large
numbers of bacilli present a particular problem for eradication of the bacilli by
chemotherapy (40). Second, the phenotypic resistance in nonreplicating persisters
presents a major problem for the current TB therapy. Antibiotics are active against
growing bacteria but are ineffective against nongrowing bacteria. There are at least
three types of nongrowing bacteria that are phenotypically resistant to antibiotics:
(a) the stationary phase bacteria, (b) residual survivors or persisters not killed
during antibiotic exposure when a growing culture is treated with antibiotics, and
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(c) dormant bacteria. Although all three types of phenotypic resistance may share
some common mechanism, the mechanism of phenotypic resistance in M. tuberculosis is unknown. There is currently considerable interest in the study of
mycobacterial persistence and dormancy (4143), with the aim to better understand the basis of this phenomenon and devise therapeutic strategies that target
the persistent or dormant organisms for improved treatment of TB. Current TB
drugs are mainly active against growing bacilli, and except for RIF and PZA, they
are not good at killing persisters. Although RIF and PZA are important sterilizing
drugs that significantly reduce the number of bacilli in lesions and play an important role in shortening the therapy from 1218 months to 6 months, there are still
other persister populations that are not killed by RIF or PZA. TB is like a little
universe or a Russian Doll, consisting of layer after layer of different bacterial
populations within a large bacterial population. At this time, we have little knowledge about the biology of these persisters, despite significant interest in this area
(4143). The intracellular location of the bacilli could render some drugs such as
streptomycin inactive. However, most drugs do penetrate the necrotic tissues (40),
although they cannot effectively kill nonreplicating bacilli in the lesions. Third,
host immune system may not effectively eliminate tubercle bacilli in the lesions.
In many bacterial infections, small numbers of residual bacteria that remain after
antibiotic therapy can be effectively mopped up by the immune system. However,
in TB, it appears that the host immune system is not very effective in controlling
the residual bacteria not killed by TB chemotherapy. Thus, although achieving
a clinical cure, the current chemotherapy cannot achieve a bacteriological cure,
i.e., the therapy cannot completely eradicate all bacilli in the lesion (40). This
depressing fact underscores the need for developing better sterilizing drugs and
other interventions, such as improving host immune status, as adjunct treatment
for more effective therapy.
The varying types of lesions determine different metabolic status of tubercle
bacilli in vivo and are the basis for diverse bacterial populations. According to
Mitchison (44), tubercle bacilli in lesions consist of at least four different subpopulations: (a) those that are actively growing, which are killed primarily by INH
[but in case of INH resistance, are killed by RIF, SM (streptomycin), or inhibited by EMB]; (b) those that have spurts of metabolism, which are killed by RIF;
(c) those that are of low metabolic activity and reside in acid pH environment,
which are killed by PZA; and (d) those that are dormant, which are not killed
by any current TB drug. A modified version of the Mitchison hypothesis is shown
in Figure 2, where the speed of growth in the original Mitchison hypothesis is
replaced with metabolic status.
TARGETS AND MODE OF ACTION OF CURRENT TB DRUGS

The current TB drugs can be divided into two categories: bacteristatic and bactericidal drugs. The static drugs include EMB and PAS, whereas the cidal drugs
include INH, RIF, SM, and FQ (fluoroquinolones). However, the distinction
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Figure 2
Special bacterial populations and TB
chemotherapy.
between static and cidal drugs is only relative, because some static drugs can
be cidal under some conditions (such as with higher drug concentrations, smaller
inoculum, or change in bacterial physiological status). For example, PZA can show
cidal activity against small numbers of nongrowing bacilli at acid pH but primarily
shows static activity for growing bacilli with active metabolism (45). Cidal drugs
exhibit higher activity over static drugs in reducing the number of bacilli in the
lesions. The current TB drugs can also be categorized as either first-line drugs or
second-line drugs. The first-line drugs include INH, RIF, PZA, EMB, and SM; the
second-line drugs include kanamycin, amikacin, capreomycin, cycloserine (CS),
PAS, ETH/PTH, thiacetazone, and FQ. According to their specificity, TB drugs can
also be grouped as TB or mycobacteria-specific drugs such as INH, PZA, EMB,
PAS, ETH, and thiacetazone, and the broad-spectrum antibiotics such as RIF, SM,
kanamycin, amikacin, capreomycin, CS, and FQ. The mechanisms of action and
resistance to TB-specific drugs are specific to M. tuberculosis, whereas mechanisms of action and resistance of the broad-spectrum drugs in M. tuberculosis are
the same as in other bacterial species. The chemical structures and the targets of inhibition for the first-line and second-line TB drugs are shown in Figure 1 and Table
1, respectively. The mechanisms of action and resistance of TB drugs have been
reviewed recently (46, 47). For the purpose of comparison with new drug targets,
the mechanisms of action and resistance of the current TB drugs will be briefly reviewed here. These drugs can be grouped as cell wall synthesis inhibitors, nucleic
acid synthesis inhibitors, protein synthesis inhibitors, and energy inhibitors.
Inhibitors of Cell Wall Synthesis

INH is a prodrug that requires activation by M. tuberculosis catalase-peroxidase (KatG) (48) to generate a range of reactive oxygen species and reactive
INH
0.010.2
0.050.5
20100
pH 5.5 or 6.0
15
28
18
0.24
0.62.5
18
520
Isoniazid (1952)
Rifampin (1966)
Pyrazinamide (1952)
Ethambutol (1961)
Streptomycin (1944)
Kanamycin (1957)
Quinolones (1963)
Ethionamide (1956)
PAS (1946)
Cycloserine (1952)
Inhibition of peptidoglycan
synthesis
D-alanine
racemasec
alrA, Ddlc
Unknown
inhA
etaA/ethAb
Acyl carrier protein reductase

(InhA)
Unknown
gyrA
gyrB
rrs
rpsL, rrs
DNA gyrase
16S rRNA
Ribosomal S12 protein

and16S rRNA
embCAB
MIC is based on Inderlied & Salfinger (13).

KatG, PncA, and EtaA/EthA are enzymes involved in the activation of prodrugs INH, PZA, and ETH, respectively.
c
In fast growing M. smegmatis.
Bacteriostatic
Inhibition of folic acid and iron

metabolism?
Inhibition of mycolic acid

synthesis
Inhibition of DNA synthesis
Inhibition of protein synthesis
Inhibition of protein synthesis
Arabinosyl transferase
pncAb
rpoB
RNA polymerase subunit

Membrane energy metabolism
katGb
inhA
ndh
Multiple targets including acyl

carrier protein reductase
(InhA)
Bacteriostatic
Bacteriostatic
Bactericidal
Bactericidal
Bactericidal
Inhibition of cell wall

arabinogalactan synthesis
Disruption of membrane transport

and energy depletion
Inhibition of RNA synthesis
Inhibition of cell wall mycolic

acid synthesis and other multiple
effects on DNA, lipids,
carbohydrates, and
NAD metabolism
Targets
AR232-PA45-22.tex
Bacteriostatic
Bacteriostatic/
bactericidal
Bactericidal
Bactericidal
Mechanisms of action
Genes
involved in
resistance
AR
MICa (g/ml)
Effect on
bacterial cell
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Drug (year of
discovery)
Table 1 Commonly used TB drugs and their targets

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organic radicals, which then attack multiple targets in the tubercle bacillus. The
primary target of inhibition is the cell wall mycolic acid synthesis pathway (49),
where enoyl ACP reductase (InhA) was identified as the target of INH inhibition
(50). The active species for InhA inhibition has been found to be isonicotinic
acyl radical, which reacts with NAD to form INH-NAD adduct and then inhibits
the InhA enzyme (51, 52). The reactive species produced during INH activation
could also cause damage to DNA, carbohydrates, and lipids (53) and inhibit NAD
metabolism (54, 55). Changes in the NADH/NAD ratios caused by mutations in
NAD dehydrogenase II (ndh) could cause resistance to INH (56, 57). The cidal
activity of INH is very likely to be due to its effect on multiple targets in tubercle
bacillus (47). Mutations in KatG involved in INH activation (48), in the INH
target InhA (50), and Ndh II (NADH dehydrogenase II) (57) could all cause INH
resistance. KatG mutation is the major mechanism of INH resistance (46, 47).
ETH, structurally related to INH (Figure 1), is also a prodrug that is
activated by the enzyme EtaA (a monooxygenase, also called EthA) (58, 59) and
inhibits the same target InhA as INH (50) of the mycolic acid synthesis pathway.
PTH (prothionamide) shares almost identical structure and activity as ETH, where
the R group in ETH is C2H5 and the R group in PTH is C3H7 (Figure 1). EtaA is an
FAD-containing enzyme that oxidizes ETH to the corresponding S-oxide, which is
further oxidized to 2-ethyl-4-amidopyridine, presumably via the unstable oxidized
sulfinic acid intermediate (60). EtaA also activates thiacetazone, thiobenzamide,
and perhaps other thioamide drugs (60). Mutations in the drug-activating enzyme
EtaA/EthA and the target InhA cause resistance to ETA (61).
ETH/PTH
EMB [(S,S )-2,2 (ethylenediimino)di-1-butanol] (EMB) interferes with the

biosynthesis of arabinogalactan, a major polysaccharide of mycobacterial cell wall
(62). It inhibits the polymerization of cell wall arabinan of arabinogalactan and
of lipoarabinomannan (63) and induces accumulation of D-arabinofuranosyl-Pdecaprenol, an intermediate in arabinan biosynthesis (64). Arabinosyl transferase,
encoded by embB, an enzyme involved in synthesis of arabinogalactan, has been
proposed as the target of EMB in M. tuberculosis (65) and M. avium (66). In
M. tuberculosis, embB is organized into an operon with embC and embA in the
order embCAB. embC, embB, and embA share more than 65% amino acid identity with each other and are predicted to encode transmembrane proteins with 12
transmembrane-spanning domains (65). Mutations in embCAB operon are responsible for resistance to EMB and are found in approximately 65% of clinical isolates
of M. tuberculosis resistant to EMB (67).
EMB
CS inhibits the synthesis of cell wall peptidoglycan by blocking the action

of D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl) (68, 69). Alr is
involved in conversion of L-alanine to D-alanine, which then serves as a substrate
for Ddl. The D-alanine racemase encoded by alrA from M. smegmatis was cloned
and its overexpression in M. smegmatis and M. bovis BCG caused resistance to
CS
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cycloserine (70). Inactivation of alrA (71) or ddl (72) in M. smegmatis caused

increased sensitivity to CS. Overexpression of Alr conferred higher resistance to
CS than Ddl overexpression in M. smegmatis, suggesting Alr might be the primary
target of CS (73). Consistent with this finding, CS also preferentially inhibited Alr
over Ddl in M. smegmatis (73). However, the mechanism of resistance of CS in
M. tuberculosis remains to be identified.

Inhibitors of Nucleic Acid Synthesis

RIF is a broad-spectrum semisynthetic rifamycin B derivative that interferes
with RNA synthesis by binding to the bacterial DNA-dependent RNA polymerase
-subunit encoded by rpoB. An important feature of RIF is that it is active against
both actively growing and slowly metabolizing nongrowing bacilli. Its activity
against the latter is thought to be involved in shortening the TB therapy from 12
18 months to 9 months (74). Mutations in a defined 81-bp region of the rpoB are
found in about 96% of RIF-resistant M. tuberculosis isolates (75). Resistance to RIF
could confer cross-resistance to other rifamycins such as rifabutin and rifapentine.
Rifapentine, with a longer half-life and greater activity than RIF, is a new drug
approved by the FDA in 1998 for treatment of TB (76). Rifapentine can reduce
the frequency of drug dosage required, but it is not active against RIF-resistant M.
tuberculosis (77).
RIF
The first quinolone drug, nalidixic acid, was obtained as an impurity during
the manufacture of quinine in the early 1960s (36, 78). Since then, many FQ derivatives have been synthesized and evaluated for antibacterial activity. Ciprofloxacin
(Figure 1), ofloxacin, levofloxacin, and sparfloxacin are the best studied of these
agents and are highly active against M. tuberculosis (79). FQ inhibits DNA synthesis by targeting the DNA gyrase A and B subunits. FQ drugs are now used to
treat MDR-TB as second-line drugs but MDR-TB strains are becoming resistant
to FQ (80). An Indian study showed some promise of oxifloxacin in combination
with first-line drugs in ultra-short course of TB treatment in three months (81).
Strains of M. tuberculosis can develop resistance to FQ by mutations in GyrA or
GyrB subunit (82, 83).
FQ
Inhibitors of Protein Synthesis

SM, an aminoglycoside antibiotic, primarily interferes with protein synthesis by
inhibiting initiation of mRNA translation (84), facilitating misreading of the genetic code (85) and damaging the cell membrane (86). The site of action is in
the small 30S subunit of the ribosome, specifically at ribosomal protein S12
(rpsL) and 16S rRNA (rrs) in the protein synthesis (87). As in E. coli, mutations in rpsL and rrs are the major mechanism of SM resistance (88). Like SM,
kanamycin, amikacin, viomycin, and capreomycin are inhibitors of protein synthesis through modification of ribosomal structures at the 16S rRNA (89). Mutations
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at 16S rRNA position 1400 are associated with high-level resistance to kanamycin
and amikacin (9092). Cross-resistance may be observed between kanamycin and
capreomycin or viomycin (9092), but a recent study found little cross-resistance
between kanamycin and amikacin (93).

Inhibition and Depletion of Membrane Energy

PZA, a structural analog of nicotinamide, is a prodrug that requires conversion to its
active form, pyrazinoic acid (POA), by the PZase/nicotinamidase enzyme encoded
by the pncA gene of M. tuberculosis (94). Mutation in pncA is a major mechanism
of PZA resistance in M. tuberculosis (94, 95). PZA is an unconventional and
paradoxical drug that has high in vivo sterilizing activity involved in shortening
the TB therapy to six months (39, 74) but has no activity against the TB bacteria
at normal culture conditions near neutral pH (96). PZA is active against tubercle
bacilli at acid pH (97). It is more active against old cultures than young cultures (98)
and also more active at low oxygen or anaerobic conditions (99). Acid pH facilitates
the formation of uncharged protonated POA that permeates through the membrane
easily and causes accumulation of POA and reduces membrane potential in M.
tuberculosis (100, 101). The protonated POA brings protons into the cell and
can eventually cause cytoplasmic acidification and de-energize the membrane by
collapsing the proton motive force, which affects membrane transport (101). The
target of PZA is thus the membrane energy metabolism. For more details about
PZA, please see the review by Zhang & Mitchison (45).
PROMISING DRUG CANDIDATES

Numerous compounds have been found to have a varying degree of activity against
M. tuberculosis. Because this is a review of potential new drug targets, it is not
possible to cover all the literature on the compounds that have antimycobacterial
activity. Only the promising candidates that have passed preclinical development
and are close to entering clinical trials or those that are clinically used to treat
other disease conditions but happen to have antituberculous activity will be discussed here. A list of the drug candidates is shown in Figure 3. For a review of
natural products such as plants, fungi, and marine organisms that have significant
antimycobacterial activity, please see reference (102).
New Fluoroquinolones
The new C-8-methoxy-FQ moxifloxacin (MXF) (Figure 3) and gatifloxacin with
longer half-lives are more active against M. tuberculosis, with MIC of 0.125 and
0.06 g/ml, than are ofloxacin and ciprofloxacin, with MIC of 2 and 4 g/ml,
respectively (103, 104). MXF was active against M. tuberculosis comparable to
INH in a mouse model (105, 106). MXF appeared to kill a subpopulation of
tubercle bacilli not killed by RIF, i.e., RIF tolerant persisters in vitro (107). A
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Figure 3
539
Structures of some promising drug candidates.
recent study showed that MXF in combination with RIF and PZA killed the bacilli
more effectively than the INH +RIF +PZA in mice (108). This higher activity of
MXF-RIF-PZA regimen than INH-RIF-PZA combination could be due to MXF
killing a subpopulation of bacilli not killed by INH and RIF (107), or it could
be due to the absence of the curious antagonism between INH and PZA (109)
such that replacing INH with MXF relieved such antagonism and thus showed
better sterilizing activity of MXF and PZA. The higher activity of MXF-RIF-PZA
than INH-RIF-PZA has generated considerable excitement and raises the hope that
MXF may replace INH in combination with RIF and PZA to shorten the TB therapy
in humans. However, scientists are also concerned about the potential toxicity of
MXF-RIF-PZA combination in the absence of INH as seen in the treatment of
latent TB infections with RIF-PZA (110). MXF has early bactericidal activity
against tubercle bacilli comparable to INH in a preliminary human study (111)
and was well tolerated. Combination therapy with MXF seems to be as effective
as current standard drug combinations (112). MXF and gatifloxacin are currently
being evaluated in clinical treatment of TB in combination with RIF and PZA
(R. Chaisson, D. Mitchison, personal communication). The highly active MXF
or gatifloxacin may have the potential to be used as first-line drugs for improved
treatment of TB and MDR-TB.
New Rifamycin Derivatives

Rifalazil (RLZ) (KRM1648 or benzoxazinorifamycin), a new semisynthetic rifamycin with a long half-life, is more active than RIF and rifabutin against M.
tuberculosis both in vitro and in vivo in mice (113, 114). High-level RIF-resistant
strains (MIC > 32 g/ml) confer cross-resistance to all rifamycins; however, lowlevel resistant strains (MIC < 32 g/ml) are still susceptible to new rifamycins (77,
115, 116). A preliminary safety study in humans (117) showed that RLZ produced
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flu-like symptoms and transient dose-dependent decrease in white blood cell and
platelet counts and did not show any better efficacy than RIF (117). Further studies are needed to more definitively assess RLZ for treatment of TB in human
trials.

Oxazolidinones (Linezolid)
Oxazolidinones are a new class of antibiotics developed by Pharmacia which were
approved by the FDA for the treatment of drug-resistant gram-positive bacterial
infections (118). Oxazolidinones inhibit an early step of protein synthesis by binding to ribosomal 50S subunits, most likely within domain V of the 23S rRNA
peptidyl transferase and forming a secondary interaction with the 30S subunit
(118, 119). Oxazolidinones had significant activity against M. tuberculosis with
an MIC of 24 g/ml and were also active against tubercle bacilli in mice (120,
121). One derivative, PNU100480 (Figure 3) had activity against M. tuberculosis
comparable to that of INH and RIF in a murine model (122). Recently, a series
of 3-(1H-pyrrol-1-yl)-2-oxazolidinone analogues of PNU-10,0480 were synthesized and some of them were found to have significant activity against M. avium
in vitro (123). Oxazolidinones may have promising potential for the treatment of
mycobacterial infections. However, treatment of human TB with oxazolidinones
has not yet been reported.
Azole Drugs
The azole drugs that are used to treat fungal infections have been shown to have
activity against M. tuberculosis (124). The azole drugs miconazole (Figure 3) and
clotrimizole were quite active against growing M. tuberculosis with an MIC of
25 g/ml, and they were also active against stationary phase bacilli (124). The
subsequent identification of cytochrome P450 homologs, a target for azole drugs, in
the M. tuberculosis genome (125) provides an explanation for the activity of azole
drugs against M. tuberculosis and led to studies to examine the correlation between
the presence of P450 and susceptibility to azole drugs in M. tuberculosis (126
129). The M. tuberculosis cytochrome P450 enzyme has recently been crystallized
and is being pursued as a target for TB drug development (130). Further in vivo
studies are needed to assess whether azole drugs can be used for the treatment of
TB.
Nitro-Containing Drugs
M. tuberculosis is quite susceptible to nitro-containing compounds. For example, niclosamide, furazolidone, 2-nitroimidazole, and 4-nitroimidazole are active
against tubercle bacilli (124). The nitro-containing compounds are likely to be
prodrugs that require activation by nitroreductases in M. tuberculosis to produce
reactive species that can damage DNA. Nitrofuran was active against nonreplicating bacilli in the Wayne dormancy model (131). It is interesting to note that
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nitrofuran is more active against INH-resistant bacilli (132), which is probably a

reflection of the defect in KatG in INH-resistant strains such that they are more
sensitive to the reactive oxygen species generated during nitrofuran activation.
Some of nitro-containing compounds such as nitrofuran and furazolidone that are
currently used in clinics to treat other bacterial infections should have less safety
concern and could potentially be tested for the treatment of TB if proven to be
active against M. tuberculosis in animal models.
Riminophenazine Derivatives
Clofazimine (Figure 3) is a riminophenazine derivative originally developed in the
1950s from components in lichens active against M. tuberculosis (133). Clofazimine is commonly used to treat leprosy in combination with dapsone and RIF,
and it is also used to treat M. avium intracellulare infections (134). The emergence
of drug-resistant TB has stimulated renewed interest in developing phenazines as
TB drugs. The MIC of clofazimine and its derivative B669 for M. tuberculosis is
0.152.5 g/ml (134). The mode of action of riminophenazines is not clear, but
was proposed to induce mycobacterial phospholipase A2 activity, causing interference with bacterial potassium transport (135). However, a recent study failed
to confirm this proposition (136). Clofazimine at the maximum tolerated dose of
5 mg/kg had no effect on tubercle bacilli in mice (137), but the liposomal form of
clofazimine at 50 mg/kg reduced the bacterial numbers in infected organs by 23
logs (137). Novel tetramethylpiperidine (TMP)-substituted phenazines were found
to be more active than clofazimine against M. tuberculosis and MDR-TB strains in
vitro and also had higher activity against intracellular bacilli than clofazimine and
RIF in macrophages (138). No animal studies with TMP-substituted phenazines
are available.
Phenothiazines
Phenothiazines such as chlorpromazine (CPZ) (Figure 3), thioridazine, and trifluroperazine are antipsychotic drugs with antituberculosis activity (139). Phenothiazines are calmodulin antagonists and their antituberculous activity appears
to correlate with the presence of a calmodulin-like protein in mycobacteria (140).
Phenothiazines are also active against MDR-TB (141, 142), suggesting that they inhibit a novel target in M. tuberculosis. The MIC of trifluoperazine was 832 g/ml
in vitro (143). CPZ inhibited intracellular mycobacteria at lower concentrations
0.233.6 g/ml because of its accumulation inside macrophages (144). CPZ may
also enhance the effectiveness of TB drugs against intracellular mycobacteria
(144). However, because of significant side effects, CPZ is not recommended for
treating human TB but may be used along with other TB drugs to treat TB in psychiatric patients (139). Thioridazine, which has identical anti-TB activity as CPZ
but fewer side effects, has been proposed as a candidate for human testing (139,
141).
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Nitroimidazopyran (PA-824)
PA-824 (Figure 3) is a new nitroimidazole derivative developed by PathoGenesisChiron (145) on the basis of an earlier observation by Indian researchers that 5nitroimidazole had good in vitro and in vivo activity against M. tuberculosis (146,
147). PA-824 was highly active against M. tuberculosis with an MIC of 0.015
0.25 g/ml (145). PA-824 was also active against nonreplicating tubercle bacilli.
PA-824 is a prodrug that is activated by F420-dependent glucose-6-phosphate
dehydrogenase and a nitroreductase activity in the bacilli (145). The resulting active
metabolites interfere with cell wall lipid biosynthesis by inhibiting an enzyme
responsible for the oxidation of hydroxymycolic acid to ketomycolate (145). PA824 was also active against MDR-TB strains, suggesting that it inhibits a new
target in tubercle bacilli. PA-824 was as active as INH in animal models of TB
infection (145). A preliminary toxicity study indicated that mice tolerated a single
dose of PA-824 at 1000 mg/kg or 500 mg/kg daily for 28 days (145). However, no
safety and efficacy data in humans are available. PA-824 is being jointly developed
by the Global Alliance for TB Drug Development and Chiron.
Peptide Deformylase (PDF) Inhibitors

PDF is a metalloprotease enzyme essential for bacterial survival but is not vital
to human cells (148). PDF is a target for a new generation of broad-spectrum
antibiotics that has generated considerable recent interest. PDF inhibitor (Figure
3) NVP PDF-713 had activity against linezolid-resistant staphylococci (MIC =
0.252 g/ml), E. faecalis (MIC = 24 g/ml), E. faecium (MIC = 0.54 g/ml),
and quinupristin/dalfopristin-resistant E. faecium (MIC = 12 g/ml) (149). The
PDF inhibitor BB-3497 has recently been found to be active against M. tuberculosis
with MIC of 0.062 g/ml (150). The PDF inhibitor BB-83,698 was highly active
against drug resistant S. pneumoniae in a mouse model (151). BB-83,698 had a
favorable PK and PD profile. At 80 mg/kg, BB-83,698 had a peak concentration in
lung tissue of about 62 g/ml within 1 h (152). BB-83,698 is currently in clinical
trials in Europe (153) and may have good potential as a new candidate drug for
the treatment of TB.
POTENTIAL NEW DRUG TARGETS

Because of the drug-resistant TB problem, it is important to develop new drugs
that inhibit novel targets that are different from those of currently used drugs. To
avoid significant toxicity, the targets of inhibition should be present in bacteria
but not in the human host. Although modification of existing drugs for improved
half-life, bioavailability, or drug delivery may be of some use, agents obtained by
this approach may have a cross-resistance problem, as seen in the new rifamycins
or quinolones. Similarly, targeting existing TB drug targets for drug development
(154) may be of limited value because of potential cross-resistance. New drugs that
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inhibit novel targets are needed. In choosing targets for drug development, it is important that they be involved in vital aspects of bacterial growth, metabolism, and
viability. These targets could include cell wall synthesis, nucleic acid biosynthesis,
protein biosynthesis, and energy metabolism, resulting in either growth inhibition
or death of the bacteria. Recent developments in mycobacterial molecular genetic
tools such as transposon mutagenesis, signature-tagged mutagenesis, gene knockout, and gene transfer will facilitate the identification and validation of new drug
targets essential for the survival and persistance of tubercle bacilli not only in vitro
but also in vivo. Below is a list of potential targets whereby new drugs may be
developed for improved treatment of TB.
Targeting Mycobacterial Persistence

The two-component system DevR-DevS was initially identified as being preferentially expressed in virulent M. tuberculosis strain H37Rv
over that in avirulent strain H37Ra in a subtractive hybridization analysis (155).
In subsequent studies aimed at characterizing mycobacterial genes that are induced in the Wayne dormancy model, the same two-component system was
identified by microarray analysis and named Rv3133c/Rv3132c (156). Inactivation of DosR abolished the rapid induction of hypoxia-induced gene expression
(157, 158), suggesting that DosR is a key regulator in the hypoxia-induced mycobacterial dormancy response (158). The DosR mutant grew as well as the
wild-type strain initially in a five-day incubation, but it survived significantly less
well upon extended incubation up to 40 days in the Wayne model (158). A recent microarray study has found that DosR controls the expression of a 48-gene
dormancy regulon, which is induced under hypoxic conditions and by nitric
oxide (NO) (159). DosR could be a good target for developing drugs against
persisters.
DosR-Rv3133/DevR-DevS
In E. coli, the stringent response induced by starvation is mediated by the

signaling molecule hyperphosphorylated guanine (ppGpp) synthesized by RelA
(ppGpp synthase I) and SpoT (ppGpp synthase II) (160). In M. tuberculosis, however, there is only a single RelA homolog (125). RelA mutation in M. tuberculosis
caused significant defect in long-term survival in vitro and reduced ability to
survive at anaerobic conditions, although the mutant appeared to behave as the
parent strain in the initial growth phase and also survived inside macrophages
(161). Mice infected with RelA mutant had impaired ability to sustain chronic
infection compared with the wild-type strain H37Rv (162). Microarray analysis
showed that the RelA mutant had an altered transcriptional profile with specific
changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat
shock proteins, and secreted antigens that may change immune recognition of the
organism (162). These findings suggest that the M. tuberculosis RelA plays an important role in establishing persistent infection and could be a good target for drug
development.
RelA
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ICL catalyzes the conversion of isocitrate to glyoxylate

and succinate and is an essential enzyme for fatty acid metabolism in the glyoxylate
shunt pathway. Survival of M. tuberculosis in the adverse in vivo environment
requires utilization of C2 substrates (generated by -oxidation of fatty acids) as
the carbon source (163). ICL was induced in the Wayne dormancy model (164),
inside macrophages (165), and in the lesions of the human lung (166). ICL is
not essential for the viability of tubercle bacilli in normal culture or in hypoxic
conditions, but it is needed for long-term persistence in mice (163). The crystal
structure of ICL has been determined and is being pursued as a target for structurebased drug design (167).

ICL (ISOCITRATE LYASE)
Using a transposon
mutagenesis approach based on changes in colony morphology, a gene called
pcaA encoding a novel methyl transferase involved in the modification of mycolic
acids in mycobacterial cell wall was identified (168). Although the PcaA knockout
mutant grew normally in vitro and replicated in mice initially like the parent strain,
the mutant was defective in persisting in mice (168) and could be a target for drug
design against persistent bacilli.
PcaA (PROXIMAL CYCLOPROPANATION OF ALPHA-MYCOLATES)
Targeting Essential Genes

Essential genes are genes whose inactivation leads to nonviability or death of
the bacteria. Until recently when mycobacterial molecular genetic tools (transposon mutagenesis, gene knockout and gene transfer) became available (169171),
two approaches were used to identify essential genes in M. tuberculosis. One
approach is the random transposon mutagenesis approach, which relies on random transposon insertion into chromosomal genes followed by an analysis of the
genes in which the transposon is inserted. The genes in which no transposon has
been inserted are essential genes. A recent study using a transposon mutagenesis
and a statistical treatment of data indicated that one third of the M. tuberculosis genes are likely essential genes (172). Seven gene familiesaminoacyl tRNA
synthases, purine ribonucleotide biosynthesis, polyketide and nonribosomal peptide synthesis, fatty acid and mycolic acid synthesis, Ser/Thr protein kinases and
phosphotases, molybdopterin biosynthesis, and PE-PGRS repeatswere identified as essential genes (172). Conditionally lethal mutants, which are defective
in metabolic pathways and fail to grow on minimal medium, as well as genes
required for optimal in vitro growth, were also identified by transposon mutagenesis (173, 174). Another approach is to determine if a particular gene is essential
by gene knockout studies. If no mutant is recovered when the gene is inactivated but the mutant can be obtained when the gene is present on a plasmid, such
a gene is an essential gene. Many mycobacterial essential genes are identified
this way. The targets encoded by essential genes can be good targets for drug
design.
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Targeting Sigma Factors

Sigma factors bind to RNA polymerase to initiate transcription. There are 13
sigma factors present in the M. tuberculosis genome (125). For a recent review
of this topic, see Reference (175). Like other bacteria, M. tuberculosis has a general house-keeping sigma 70like principal sigma factor MysA or SigA (176),
as well as more specialized sigma factors such as RpoS-like sigma factor MysB
(SigB), SigC, SigE, SigH, SigF, which are induced under various stress conditions
(175). Increased SigA expression in M. tuberculosis and in transformed strains
caused faster growth inside macrophages and increased virulence in mice (177).
SigC, which controls the expression of virulence factors such as two-component
systems senX3-regX3, mtrA-mtrB and hspX (alpha-crystallin homolog), is also involved in virulence (178). Expression of SigB is dependent on SigE (179) and
SigH (180). SigE is involved in global gene expression, heat stress, oxidative
stress, exposure to SDS, and survival in macrophages (179181) and virulence
(182, 183). SigE is regulated by SigH, which plays a central role in regulation of heat and oxidative stress responses, and sigH mutants are more susceptible to these stresses (180). SigF is induced in stationary phase and a variety
of stress conditions such as nitrogen depletion, oxidative stress, cold shock, and
anaerobic conditions (184). Mutation in SigF did not affect in vitro growth or
survival in macrophages compared with the parent strain, but caused reduced
virulence in mice (185). Because of their importance in mycobacterial gene transcription and their absence in the host, sigma factors could be good targets for drug
design.
Targeting Virulence Factors

In recent years, scientists have become interested in developing antibacterial drugs
that target virulence factors in bacterial pathogens (186). Although the idea of targeting virulence factors and two-component systems (see below) is quite attractive,
it may have some potential drawbacks. For example, virulence factors may not be
essential viability genes, and inhibition of virulence factors may not be lethal for
the bacterial pathogen. Moreover, incomplete inhibition of virulence factors could
also have problems. The most worrying aspect of this approach is that such drugs
may be of little use for established infections. Although no drugs that target virulence factors have been developed so far, there is hope that such drugs may be
used in conjunction with conventional antibiotics to improve treatment of bacterial
infections (186). The recent developments in mycobacterial genetic tools have led
to the discovery of various virulence factors in M. tuberculosis. For a recent review
of mycobacterial virulence factors, see Reference (187). In addition, in a recent
study using transposon mutagenesis, 194 genes (about 5% of genome) in the M.
tuberculosis genome were identified as required for growth in mice (188). These
virulence factors could be potential drug targets.
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Targeting Two-Component Systems

Because of the important role of two-component systems in controlling bacterial
virulence genes, scientists are interested in developing inhibitors that target these
systems (189194). Several series of inhibitors have been found from chemical
library screens, including salicylanilides (190), diaryltriazole analogs (195), bisphenols, cyclohexenes, benzoxazines, and triphenylalkyl derivatives (192). However, most of these agents suffer from poor selectivity, excessive protein binding, or
limited bioavailability (191, 194). Researchers are pursuing alternate strategies to
identify inhibitors with more desirable properties; these strategies include design
of substrate-based inhibitors, generation of combinatorial libraries, and isolation of
natural products(192). The conserved domains of response regulators of different
two-component systems offer a common site of attack by inhibitors (194).
M. tuberculosis has 11 two-component system homologs in the genome (125).
Many of these homologs have now been characterized: MtrA-MtrB (197), SenX3RegX3 (198), the DevR (DosR)-DevS (158), PrrA-PrrB (199), MprA-MprB (200),
and PhoP/PhoR (201). Inactivation of the mtrA component of mtrA-mtrB of M.
tuberculosis H37Rv was possible only in the presence of plasmid-borne functional
mtrA, suggesting that this response regulator is essential for M. tuberculosis viability (200). Inactivation of either senX3 or regX3 caused attenuation of virulence in
mice (202, 203). DevR (DosR)-DevS was found to be expressed to higher levels in
virulent strain H37Rv than in avirulent strain H37Ra (204). Inactivation of DosR
(205), mprA (200), and phoP (201) caused attenuated virulence in animal studies.
These studies suggest that two-component systems in M. tuberculosis could be
important drug targets.
Targeting Cell Wall Synthesis

Because several TB drugs such as INH, ETH, and EMB target mycobacterial
cell wall synthesis, enzymes involved in this pathway have been preferred targets in drug development efforts. KasA and KasB, -ketoacyl-acyl-carrier protein
synthases, have been examined as potential targets for drug development. Thiolactomycin (TLM) targets KasA and KasB that belong to the fatty-acid synthase type
II (FASII) system involved in fatty acid and mycolic acid biosynthesis (206, 207).
TLM was also active against an MDR-TB clinical isolate. Several TLM derivatives
were found to be more potent than TLM in vitro in the fatty acid and mycolic acid
biosynthesis assays and against M. tuberculosis (208). No TLM-resistant mutants
of M. bovis BCG could be isolated, which could be a consequence of TLM inhibiting multiple enzymes of fatty acid synthesis in mycobacteria (207). Because TLM
inhibits the FASII enzyme in different bacterial species, it could be developed
into a broad-spectrum antibiotic for treating different bacterial infections including TB. Cerulenin inhibits KasA involved in mycolic acid synthesis with an MIC
of 1.512.5 g/ml against M. tuberculosis (209, 210). N-octanesulfonylacetamide
(OSA), an inhibitor of fatty acid and mycolic acid biosynthesis, was active against
M. tuberculosis and also MDR-TB strains with an MIC of 6.2512.5 g/ml (211).
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These inhibitors of fatty acid and mycolic acid synthesis could be good candidates
for further development. However, drugs that target cell wall synthesis are likely
to be active mainly against growing bacilli but not against persisters, and they may
not be able to shorten the lengthy therapy (212).

Targeting Unique Physiology of M. tuberculosis

Tubercle bacillus is generally thought to be a tough organism equipped with a
thick waxy cell envelope that provides a permeability barrier to a variety of agents
and many antibiotics that are effective against other bacterial pathogens. However,
recent studies have revealed that contrary to common beliefs, M. tuberculosis has
some surprising weaknesses that may be exploited in designing drugs against this
pathogen.
First, M. tuberculosis has a deficiency in efflux of POA. M. tuberculosis is
uniquely susceptible to PZA, whereas other mycobacteria and bacteria are naturally
resistant to it (100). The unique susceptibility to PZA is at least partly due to a
deficient POA efflux mechanism that allows POA to be increasingly accumulated
inside M. tuberculosis at acid pH (100). In contrast, naturally PZA-resistant M.
smegmatis and other bacteria such as E. coli have a highly active POA efflux
mechanism that does not allow accumulation of POA even at acid pH (100). The
M. tuberculosis POA efflux is at least 100 times slower than that of M. smegmatis
(100). Besides deficient POA efflux, M. tuberculosis appears to be defective in the
efflux of other compounds such as weak acids (Y. Zhang, unpublished data). New
TB drugs may be designed that take advantage of the deficient efflux mechanism
in M. tuberculosis.
Another defect is the poor ability of M. tuberculosis to maintain its energy
status. During our study of the mechanism of action of PZA, we found that in
addition to weak acid POA, M. tuberculosis is also more susceptible to many other
weak acids than other bacteria such as M. smegmatis or E. coli (213). This unique
weak acid susceptibility of M. tuberculosis seems to be related to its deficient
ability to maintain membrane potential and pH gradient (213), presumably caused
by its slow metabolism. It will be interesting to determine if weak acids or their
precursors can be developed into TB drugs.
A third defect of M. tuberculosis is its deficient ability to cope with endogenously generated reactive species. Studying the mechanisms of action of IHH,
researchers found that M. tuberculosis appears to be deficient in oxidative defense
and highly susceptible to endogenously produced oxygen radicals generated by
KatG-mediated INH activation (26). The unique susceptibility of M. tuberculosis
to INH is probably due to a combination of defective OxyR (214, 215) and poor
ability to remove or antagonize toxic reactive oxygen species and organic radicals
that have accumulated (26). In addition, M. tuberculosis appears to be particularly
susceptible to endogenously produced reactive nitrogen intermediates. For example, niclosamide (124), nitroimidazopyran PA-824 (145), and nitrofurans (131),
which presumably generate reactive nitrogen during their activation, are quite
active against M. tuberculosis, especially nongrowing bacilli. It will be interesting
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to see if compounds that generate reactive oxygen or nitrogen species inside bacilli
could be designed as TB drugs.

TB Genomics and Drug Targets

The first bacterial genome was sequenced by Fleischmann and colleagues at The
Institute for Genomic Research (TIGR) in 1995 (216). So far, more than 100
bacterial genomes have been sequenced (www.tigr.org). As bacterial genome sequences become available, there is increasing interest in developing new antibacterial agents using genomics-based approaches (217220). The available genome
sequence information, along with molecular genetic tools, allows researchers to
identify common essential targets among different bacterial species. The common
targets can then be overexpressed for biochemical assays in drug screens or structure determination, to be used in the drug design. So far, however, no company
has been successful in developing a drug using a genomics approach. The availability of the M. tuberculosis genome sequence (125) opens up a new opportunity
to understand the biology of the organism and provides a range of potential drug
targets (221). The recent developments in microarray technology (222), signaturetag mutagenesis (223), mycobacterial transposon mutagenesis (169), and gene
knock-out technology (170, 224) provide important tools to identify new drug targets. Microarray has been used to identify M. tuberculosis genes that are induced
by INH and ETH (225), and by INH, TLM, and triclosan (226). Microarray was
also used to identify genes that are switched on in the Wayne dormancy model
under hypoxic and nitric oxide stress conditions (156, 159), a discovery that led
to the identification of a 48-gene dormancy regulon controlled by DosR (159).
A proteomic approach was used to identify potential proteins that are induced in
starvation as an in vitro model of persistence (227). Two unique M. tuberculosis
proteins with homology to each other were identified: Rv2557 and Rv2558 (227).
Rv2557 was also induced inside granulomatous lesions in the human lung (166).
Genes identified by microarray analysis or proteins identified by a proteomic approach should be further validated as potential drug targets by gene knockout and
in vivo testing in mice before they are selected as targets for drug development.
STRUCTURE-BASED DRUG DESIGN

Structure-based drug design and combinatorial chemistry represent potentially
powerful and promising approaches for drug design. In the case of designing
antituberculous compounds, selection of targets usually involves identifying enzymes in pathways essential for the organism but not present or less important
in the human host. The number of three-dimensional structures of M. tuberculosis proteins has been increasing rapidly in recent years. This increase reflects an
awareness of the need for new targets for design of new antituberculosis drugs.
The Mycobacterium tuberculosis Structural Genomics Consortium (http://www.
doe-mbi.ucla.edu/TB/), consisting of 70 laboratories in 12 countries, was
established in 2000 and has contributed a significant number of structures of
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M. tuberculosis proteins (228). This consortium aims to crystallize 400 proteins

in five years. A list of 3D structures can be found in the Protein Data Bank
(http://www.rcsb.org/pdb/) and also in http://www.doe-mbi.ucla.edu/TB/EDIT/tb
structures in pdb.php?format=html. Many of these targets have not yet been validated as essential, and the structure-based drug design is only meaningful on
bacterial targets that have proven to be essential (see above). A list of crystal structures of mycobacterial enzymes with relevant properties as potential drug targets
have been recently reviewed (8) and will not be recounted here.
DRUG SCREENS
Because of the problem of drug-resistant TB and the need to shorten the lengthy TB
chemotherapy, there is currently a great deal of interest in TB drug development (7,
8, 11). NIH supports some antimycobacterial drug discovery research through the
NIAID Division of AIDS Opportunistic Infections Branch. The NIH-sponsored
consortium consists of in vitro screening facilities at the Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF) at Southern Research Institute
and at Hansen Disease Center (Baton Rogue), and at an animal testing facility at
Colorado State University. GlaxoSmithKline also has a program called Action TB
for TB drug discovery research. A private organization, the Global Alliance for
TB Drug Development, was recently established to facilitate TB drug development
(http://www.tballiance.org) and aims to have at least one TB drug registered by
2010 (229).
Both whole cell screens and cell-free target-based screens are used for antimicrobial drug discovery. The target-based screen is a relatively recent invention and
has so far been generally disappointing (233), except the recent development of
peptide deformylase inhibitors which represents the first success of the target-based
approach (148, 151, 152). However, all current TB drugs, with the exception of
PZA, were identified by in vitro whole cell screens. The current NIAID-sponsored
TB drug development effort is primarily based on screening of compounds active against growing bacilli using AlarMar Blue redox dye in a 96-well microtiter
plate format. About 70,000 compounds have been screened so far (R. Reynolds,
personal communication), and data for about 50,000 compounds were recently
published (230), where 11% (5251) had high activity against M. tuberculosis
in vitro. Of these, 53 were tested in vivo, and 9 were found to significantly reduce
bacterial numbers in the lungs of infected mice. A luciferase-reporter mycobacterial strain has also been used for screening more than 62,000 EMB analogs
generated by combinatorial chemistry for more active compounds (231). Twentysix compounds were identified; N-Geranyl-N -(2-adamantyl)ethane-1,2-diamine
(Compound 109), the most active of these diamines, was 14- to 35-fold more
active than EMB (231). Further development is required to assess its in vivo
activity. A green fluorescent protein based screening system utilizing acetamidase gene promoter was recently established for high throughput antimycobacterial compound screen (232). The combinatorial chemistry can be applied to
generate diverse compounds for screens in both whole cell and target-based screens.
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Although the whole cell screens are useful for TB drug development, we must
recognize the potential problem of developing drugs active against growing tubercle bacilli: drugs only active against growing bacilli are not going to be very
useful for killing nonreplicating persisters, which are the biggest stumbling block
for a more effective therapy. Although sterilizing drugs that can kill persisters and
shorten the TB therapy are desperately needed, it is not clear how this objective
can be effectively achieved. There is no good in vitro correlate of high sterilizing
activity against persisters in vivo. That is, we cannot infer from the MIC whether
the drug is going to be active against persistent bacilli or have high sterilizing activity. Low MIC does not mean the drug will have good sterilizing activity against
persistent bacilli in vivo. INH is a wonderful drug that is highly active against
growing tubercle bacilli with a very low MIC of 0.020.06 g/ml, but has no
activity against nonreplicating bacilli and therefore cannot effectively sterilize the
lesions (235). In contrast to INH, PZA is a paradoxical drug that has poor in vitro
activity against growing tubercle bacilli with a high MIC of 50100 g/ml at
pH 5.56.0 and is completely inactive against tubercle bacilli at normal culture
conditions near neutral pH, which is commonly used for whole cell MIC-based
screens. Unlike common antibiotics which are active against growing bacteria with
no activity against nonreplicating bacteria, PZA is exactly the opposite and is more
active against nonreplicating old bacilli (98) and under hypoxic conditions (99). It
is these properties that are responsible for its high sterilizing activity in vivo and
its ability to shorten the therapy from 912 months to 6 months. PZA was discovered by a serendipitous observation in 1940s that nicotinamide had activity against
mycobacteria in animal models; subsequent synthesis of nicotinamide analogs and
direct screen in mice without MIC testing identified PZA as the most active agent in
vivo (45). In a sense, we should feel fortunate that we have the wonderful sterilizing
drug PZA, which would have been missed altogether had the conventional MICbased screens been used. As we can see, the MIC-based approach does not work
here! If there is any lesson to be learned from the PZA story, it is that we cannot use
the MIC-based screens to identify drugs that have high sterilizing activity against
persisters.
To identify drugs that effectively kill nongrowing persisters and shorten the
therapy, we must design new and unconventional screens that mimic the persisters in vivo, such as using nonreplicating bacilli at low oxygen and acid pH
in the screen, a process that is more challenging. There are different persistence models that can potentially be used for screening for sterilizing drugs (41,
212). Stationary phase bacilli, old and starved bacilli, and persisting bacilli after drug treatment can all be used in such screens. Synergy screens with different agents should also be considered. Because of our limited understanding of
mycobacterial persisters, it is difficult to judge if one model is better than another. However, testing in animals will show which in vitro persistence model
is more relevant to the goal of shortening the therapy. In addition, potential targets involved in persistence (see above) could also be selected for target-based
screens.
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CONCLUDING REMARKS
The development of modern TB chemotherapy is indeed a remarkable achievement
of modern medicine and represents a major milestone in humankinds fight against
TB. Yet despite the availability of TB chemotherapy and the BCG vaccine, TB is
still a leading infectious disease worldwide. Along with the socio-economic and
host factors that underlie this problem, a fundamental problem that hinders more
effective TB control is the tenacious ability of M. tuberculosis to persist in the
host and to develop drug resistance, often as a consequence of poor compliance to
lengthy therapy. Novel screens targeting persisters are needed but such screens are
challenging. PZA represents a prototype model drug that can shorten TB therapy,
and improved understanding of PZA should help us to design drugs that are more
active against persisters. Although having another new drug like INH that only
kills growing bacilli may be useful for treating drug-resistant TB, it is unlikely
to improve the current TB therapy. The development of new sterilizing drugs that
target persisters and shorten the TB therapy must be a top priority. This represents
a paradigm shift from previous approaches, which focused on just finding another
drug, to beating mycobacterial persistence. In the big picture, we must recognize
that better control of TB extends beyond better chemotherapy; it requires a multifaceted approach, including improved socio-economic conditions and nutrition,
better management of adverse psychological factors, and improved host immunity
as adjunct treatment (41). The recent developments in mycobacterial genetic tools
and TB genomics, new technology of combinatorial chemistry and high throughput screening, structure-based drug design, and improved understanding of the
unique biology of tubercle bacillus provide an exciting opportunity to discover
new Magic Bullets that kill persisters and shorten the current TB treatment from
six months to a few weeks. A new era of TB chemotherapy will arrive when these
new Magic Bullets are identified.
ACKNOWLEDGMENTS
The support from NIH (AI44063 and AI/HL49485) is gratefully acknowledged.
LITERATURE CITED
1. Ryan F. 1993. The Forgotten Plague. How
the Battle against Tuberculosis Was Won
and Lost, p. 3. Boston: Little, Brown. 460
pp.
2. World Health Organization. 2003. The
World Health Organization Global
Tuberculosis Program. http://www.who.
int/gtb/
3. Center for Disease Control. 1993. Outbreak of multidrug-resistant tuberculosis

at a hospitalNew York City. 1991. Morb.
Mortal. Wkly. Rep. 42:427,43334
4. Kimerling ME, Kluge H, Vezhnina N,
Iacovazzi T, Demeulenaere T, et al.
1999. Inadequacy of the current WHO
re-treatment regiment in central Siberian
4 Dec 2004
18:50
552
5.

6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
prisons: treatment failure and MDR-TB.
Int. J. Tuberc. Lung Dis. 3:45153
De Cock KM, Chaisson RE. 1999. Will
DOTS do it? A reappraisal of tuberculosis control in countries with high rates of
HIV infection. Int. J. Tuberc. Lung Dis.
3:45765
Tuberculosis: a global emergency. 1993.
World Health Forum 14:438
Global Alliance for Tuberculosis Drug
Development. 2001. Scientific blueprint
for TB drug development. Tuberculosis
81(Suppl.):152
Smith CV, Sharma V, Sacchettini JC.
2004. TB drug discovery: addressing issues of persistence and resistance. Tuberculosis 84:4555
Duncan K. 2003. Progress in TB drug development and what is still needed. Tuberculosis 83:2017
Kremer LS, Besra GS. 2002. Current status and future development of antitubercular chemotherapy. Expert. Opin. Investig. Drugs 11:103349
Zhang Y, Amzel LM. Tuberculosis drug
targets. Curr. Drug Targets 3:13154
Barry CE 3rd. 2001. Preclinical candidates and targets for tuberculosis therapy.
Curr. Opin. Investig. Drugs 2:198201
Inderlied CB, Salfinger M. 1999. Antimycobacterial agents and susceptibility
tests. In Manual of Clinical Microbiology, ed. PR Murray, EJ Baron, MA Pfaller,
FC Tenover, RH Yolken, pp. 16011623.
Washington DC: ASM Press. 7th ed.
Domagk G. 1935. Ein Beitrag zur
Chemotherapie der bakteriellen Infektionen. Deutschemedizinische Wochenschrift 61:25053
Hatfull G, Jacobs WR Jr, eds. 2000.
Molecular Genetics of Mycobacteria.
Washington DC: ASM Press
Deleted in proof
Schatz AB, Bugie E, Waksman S. 1944.
Streptomycin, a substance exhibiting antibiotic activity against gram-positive and
gram-negative bacteria. Proc. Soc. Exp.
Biol. Med. 55:6669
18. Rich A, Follis RH. 1938. The inhibitory

effect of sulfanilamide on the development of experimental tuberculosis of the
guinea pig. Bull. Johns Hopkins Hosp.
62:7784
19. Hinshaw HC, McDermott W. 1950.
Thiosemicarbazone therapy of tuberculosis in human. Am. Rev. Tuberc. 61:145
57
20. Lehmann J. 1946. p-aminosalicylic acid
in the treatment of tuberculosis. Lancet
1:1516
21. Bernheim F. 1940. The effect of salicylate on the oxygen uptake of the tubercle
bacillus. Science 92:204
22. Chorine V. 1945. Action de lamide nicotinique sur les bacilles du genre Mycobacterium. C. R. Acad. Sci. 220:15051
23. Bernstein J, Lott WA, Steinberg BA,
Yale HL. 1952. Chemotherapy of experimental tuberculosis. V. Isonicotinic acid
hydrazide (Nydrazid) and related compounds. Am. Rev. Tuberc. 65:35764
24. Fox H. 1952. The chemical approach to
the control of tuberculosis. Science 116:
12934
25. Offe HA, Siefken W, Domagk G. 1952.
The tuberculostatic activity of hydrazine
derivatives from pyridine carboxylic acids
and carbonyl compounds. Z. Naturfobrsch. 7b:46268
26. Zhang Y. 2003. Isoniazid. In Tuberculosis, ed. W Rom, S Garay, pp. 73958. New
York: Lippincott. 2nd ed.
27. Malone L, Schurr A, Lindh H, McKenzie
D, Kiser JS, Williams JH.1952. The effect of pyrazinamide (Aldinamide) on experimental tuberculosis in mice. Am. Rev.
Tuberc. 65:51118
28. Liebermann D, Moyeux M, Rist N, Grumbach F. 1956. Sur la preparation de
nouveaux thioamides pyridiniques acitifs
dans la tuberculose experimentale. C. R.
Acad. Sci. 242:240912
29. Thomas JP, Baughn CO, Wilkinson
RG, Shepherd RG. 1961. A new synthetic compound with antituberculous
activity in mice: ethambutol (dextro-2,
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX
30.

31.
32.
33.
34.
35.
36.
37.
38.
39.
2-(ethylenediimino)-di-butanol). Am. Rev.

Respir. Dis. 83:89193
Kurosawa H. 1952. Studies on the antibiotic substances from actinomyces. XXIII.
The isolation of an antibiotic produced by
a strain of Streptomyces K 30. J. Antibiot. Ser. B 5:68288
Umezawa H, Ueda M, Maeda K, Yagishita
K, Kondo S, et al. 1957. Production and
isolation of a new antibiotic, kanamycin.
J. Antibiot. Jpn. Ser. A 10:18188
Bartz QR, Ehrlich J, Mold JD, Penner
MA, Smith RM. 1951. Viomycin, a new
tuberculostatic antibiotic. Am. Rev. Tuberc. 63:46
Herr EB Jr, Haney ME, Pittenger GE,
Higgens CE. 1960. Isolation and characterization of a new peptide antibiotic.
Proc. Indiana Acad. Sci. 69:134
Sensi P, Margalith P, Timbal MT. 1959.
Rifomycin, a new antibiotic. Preliminary
report. Il. Farmaco. Sci. 14:14647
Maggi N, Pasqualucci CR, Ballotta R,
Sensi P. 1966. Rifampicin: a new orally
active rifamycin. Farmaco. Sci. 21:6875
Deitz WH, Bailey JH, Froelich EJ.1963.
In vitro antibacterial properties of
nalidixic acid, a new drug active against
gram-negative organisms. Antimicrob.
Agents Chemother. 161:58387
Tsunekawa HMT, Nakamura E, Tsukamura M, Amano H. 1987. Therapeutic effect of ofloxacin on treatmentfailure pulmonary tuberculosis. Kekkaku
62:43539
Yew WW, Kwan SY, Ma WK, Khin
MA, Chau PY. 1990. In-vitro activity of
ofloxacin against Mycobacterium tuberculosis and its clinical efficacy in multiply
resistant pulmonary tuberculosis. J. Antimicrob. Chemother. 26:22736
Fox W, Ellard GA, Mitchison DA. 1999.
Studies on the treatment of tuberculosis
undertaken by the British Medical Research Council tuberculosis units, 19461986, with relevant subsequent publications. Int. J. Tuberc. Lung Dis. 3(10 Suppl.
2):S23179
553
40. Canetti G. 1955. The Tubercle Bacillus in

the Pulmonary Lesion of Man. New York:
Springer
41. Zhang Y. 2004. Persistent and dormant
tubercle bacilli and latent tuberculosis.
Front. Biosci. 9:113656
42. Gomez JE, McKinney J. 2003. Persistence and drug tolerance. In Tuberculosis,
ed. W Rom, S Garay, pp. 10114. New
York: Lippincott. 2nd ed.
43. Flynn JL, Chan J. 2001. Tuberculosis:
latency and reactivation. Infect. Immun.
69:4195201
44. Mitchison D. 1979. Basic mechanisms of
chemotherapy. Chest 76(6 Suppl.):771
81
45. Zhang Y, Mitchison DA. 2003. The curious characteristics of pyrazinamide: a review. Int. J. Tuberc. Lung Dis. 7:621
46. Wade MM, Zhang Y. 2004. Mechanisms
of drug resistance in Mycobacterium tuberculosis. Front. Biosci. 9:97594
47. Zhang Y, Jacobs WR Jr, Vilcheze C. 2004.
Mechanisms of drug resistance in Mycobacterium tuberculosis. In Pathogenesis of Tuberculosis. Washington, DC:
ASM Press. 2nd ed.
48. Zhang Y, Heym B, Allen B, Young D,
Cole S. 1992. The catalase-peroxidase
gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358:591
93
49. Winder FG, Collins PB. 1970. Inhibition
by isoniazid of synthesis of mycolic acids
in Mycobacterium tuberculosis. J. Gen.
Microbiol. 63:4148
50. Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, et al. 1994. inhA,
a gene encoding a target for isoniazid and
ethionamide in Mycobacterium tuberculosis. Science 263:22730
51. Rozwarski DA, Grant GA, Barton DH, Jacobs WR Jr, Sacchettini JC. 1998. Modification of the NADH of the isoniazid target
(InhA) from Mycobacterium tuberculosis.
Science 279:98102
52. Broussy S, Coppel Y, Nguyen M,
Bernadou J, Meunier B. 2003. 1H and
4 Dec 2004
18:50
554

53.
54.
55.
56.
57.
58.
59.
60.
61.
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
13C NMR characterization of hemiamidal isoniazid-NAD(H) adducts as possible
inhibitors of InhA reductase of Mycobacterium tuberculosis. Chemistry 9:2034
38
Winder F. 1982. Mode of action of the
antimycobacterial agents and associated
aspects of the molecular biology of mycobacteria. In The Biology of Mycobacteria, ed. C Ratledge, J Stanford, pp. 354
438. New York: Academic
Winder F, Collins P. 1968. The effect of
isoniazid on nicotinamide nucleotide levels in Mycobacterium bovis strain BCG.
Am. Rev. Respir. Dis. 97:71920
Sriprakash KS, Ramakrishnan T. 1969.
Isoniazid and nicotinamide adenine dinucleotide synthesis in M. tuberculosis. Indian J. Biochem. 6:4950
Miesel L, Weisbrod T, Marcinkeviciene
JA, Bittman R, Doshi P, et al. 1998.
NADH dehydrogenase defects confer resistance to isoniazid and conditional
lethality in Mycobacterium smegmatis. J.
Bacteriol. 180:245967
Lee AS, Teo AS, Wong SY. 2001. Novel
mutations in ndh in isoniazid-resistant
Mycobacterium tuberculosis isolates. Antimicrob. Agents Chemother. 45:215759
DeBarber AE, Mdluli K, Bosman M,
Bekker LG, Barry CE 3rd. 2000.
Ethionamide activation and sensitivity
in multidrug-resistant Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA
97:967782
Baulard AR, Betts JC, Engohang-Ndong
J, Quan S, McAdam RA, et al. 2000. Activation of the pro-drug ethionamide is
regulated in mycobacteria. J. Biol. Chem.
275:2832631
Vannelli TA, Dykman A, Ortiz de Montellano PR. 2002. The antituberculosis
drug ethionamide is activated by a flavoprotein monooxygenase. J. Biol. Chem.
277:1282429
Morlock GP, Metchock B, Sikes D, Crawford JT, Cooksey RC. 2003. ethA, inhA,
and katG loci of ethionamide-resistant
62.
63.
64.
65.
66.
67.
68.
69.
clinical Mycobacterium tuberculosis isolates. Antimicrob. Agents Chemother.

47:3799805
Takayama K, Kilburn JO. 1989. Inhibition
of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis. Antimicrob. Agents Chemother. 33:1493
99
Mikusov K, Slayden RA, Besra GS, Brennan P. 1995. Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob. Agents
Chemother. 39:248489
Wolucka BA, McNeil MR, de Hoffmann
E, Chojnacki T, Brennan PJ. 1994. Recognition of the lipid intermediate for arabinogalactan/arabinomannan biosynthesis and its relation to the mode of action
of ethambutol on mycobacteria. J. Biol.
Chem. 269:2332835
Telenti A, Philipp W, Sreevatsan S,
Bernasconi C, Stockbauer KE, et al. 1997.
The emb operon, a unique gene cluster
of Mycobacterium tuberculosis involved
in resistance to ethambutol. Nat. Med.
3:56770
Belanger AE, Besra GS, Ford ME,
Mikusov K, Belisle JT, et al. 1996. The
embA genes of Mycobacterium avium encode an arabinosyl transferase involved
in cell wall arabinan biosynthesis that is
the target for the antimycobacterial drug
ethambutol. Proc. Natl. Acad. Sci. USA
93:1191924
Sreevatsan S, Stockbauer KE, Pan X,
Kreiswirth BN, Moghazeh SL, et al. 1997.
Ethambutol resistance in Mycobacterium
tuberculosis: critical role of embB mutations. Antimicrob. Agents Chemother.
41:167781
David HL, Takayama K, Goldman DS.
1969. Susceptibility of mycobacterial
D-alanyl-D-alanine synthetase to Dcycloserine. Am. Rev. Respir. Dis. 100:
57981
Strych U, Penland RL, Jimenez M, Krause
KL, Benedik MJ. 2001. Characterization of the alanine racemases from two
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX
70.

71.
72.
73.
74.
75.
76.
77.
78.
79.
mycobacteria. FEMS Microbiol. Lett.

196:9398
Caceres NE, Harris NB, Wellehan JF,
Feng Z, Kapur V, et al. 1997. Overexpression of the D-alanine racemase gene
confers resistance to D-cycloserine in
Mycobacterium smegmatis. J. Bacteriol.
179:504655
Chacon O, Feng Z, Harris NB, Caceres NE, Adams LG, et al. 2002. Mycobacterium smegmatis D-alanine racemase mutants are not dependent on Dalanine for growth. Antimicrob. Agents
Chemother. 46:4754
Belanger AE, Porter JC, Hatfull GF. 2000.
Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization
of a ddlA mutant of Mycobacterium smegmatis. J. Bacteriol. 182:685456
Feng Z, Barletta RG. 2003. Roles of
Mycobacterium smegmatis D-alanine:Dalanine ligase and D-alanine racemase
in the mechanisms of action of and
resistance to the peptidoglycan inhibitor D-cycloserine. Antimicrob. Agents
Chemother. 47:28391
Mitchison DA. 1985. The action of
antituberculosis drugs in short course
chemotherapy. Tubercle 66:21925
Telenti A, Imboden P, Marchesi F,
Lowrie D, Cole ST, et al. 1993. Detection of rifampicin-resistance mutations
in Mycobacterium tuberculosis. Lancet
341:64750
Jarvis B, Lamb HM. 1998. Rifapentine.
Drugs 56:60716
Williams DL, Spring L, Collins L, Miller
LP, Heifets LB, et al. 1998. Contribution of rpoB mutations to development
of rifamycin cross-resistance in Mycobacterium tuberculosis. Antimicrob. Agents
Andersson MI, MacGowan AP. 2003. Development of the quinolones. J. Antimicrob. Chemother. 51(Suppl. 1):111
Jacobs M. 1999. Activity of quinolones
against mycobacteria. Drugs 58(Suppl.
2):1922
555
80. Grimaldo ER, Tupasi TE, Rivera AB,

Quelapio MI, Cardano RC, et al. 2001.
Increased resistance to ciprofloxacin
and ofloxacin in multidrug-resistant Mycobacterium tuberculosis isolates from
patients seen at a tertiary hospital in the
Philippines. Int. J. Tuberc. Lung Dis.
5:54650
81. Tuberculosis Research Centre C. 2002.
Shortening short course chemotherapy:
a randomised clinical trial for treatment
of smear positive pulmonary tuberculosis
with regimens using ofloxacin in the intensive phase. Indian J. Tuberc. 49:2738
82. Takiff HE, Salazar L, Guerrero C, Philipp
W, Huang WM, et al. 1994. Cloning and
nucleotide sequence of Mycobacterium
tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations.
Antimicrob. Agents Chemother. 38:773
80
83. Kocagoz T, Hackbarth CJ, Unsal I, Rosenberg EY, Nikaido H, et al. 1996. Gyrase mutations in laboratory selected,
fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra. Antimicrob. Agents Chemother. 40:176874
84. Spotts CR, Stanier RY. 1961. Mechanism
of streptomycin action on bacteria: a unitary hypothesis. Nature 192:63337
85. Davies J, Gorini L, Davis BD. 1965. Misreading of RNA codewords induced by
aminoglycoside antibiotics. Mol. Pharmacol. 1:93106
86. Anand N, Davis BD. 1960. Effect of
streptomycin on Escherichia coli. Nature
185:2223
87. Garvin RT, Biswas DK, Gorini L. 1974.
The effects of streptomycin or dihydrostreptomycin binding to 16S RNA or to
30S ribosomal subunits. Proc. Natl. Acad.
Sci. USA 71:381418
88. Finken M, Kirschner P, Meier A, Wrede
A, Bottger EC. 1993. Molecular basis
of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal
4 Dec 2004
18:50
556
89.

90.
91.
92.
93.
94.
95.
96.
97.
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
RNA pseudoknot. Mol. Microbiol. 9:
123946
Zhang Y, Telenti A. 2000. Genetics of
drug resistance in Mycobacterium tuberculosis. See Ref. 15, pp. 23554
Alangaden GJ, Kreiswirth BN, Aouad
A, Khetarpal M, Igno FR, et al. 1998.
Mechanism of resistance to amikacin
and kanamycin in Mycobacterium tuberculosis. Antimicrob. Agents Chemother.
42:129597
Suzuki Y, Katsukawa C, Tamaru A, Abe
C, Makino M, et al. 1998. Detection of
kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in
the 16S rRNA gene. J. Clin. Microbiol.
36:122025
Taniguchi H, Chang B, Abe C, Nikaido
Y, Mizuguchi Y, et al. 1997. Molecular
analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by
use of the conjugation system. J. Bacteriol. 179:4795801
Kruuner A, Jureen P, Levina K, Ghebremichael S, Hoffner S. 2003. Discordant resistance to kanamycin and
amikacin in drug-resistant Mycobacterium tuberculosis. Antimicrob. Agents
Scorpio A, Zhang Y. 1996. Mutations
in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to
the antituberculous drug pyrazinamide
in tubercle bacillus. Nat. Med. 2:662
67
Scorpio A, Lindholm-Levy P, Heifets
L, Gilman R, Siddiqi S, et al. 1997.
Characterization of pncA mutations in
pyrazinamide-resistant Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 41:54043
Tarshis MS, Weed WA. 1953. Lack of significant in vitro sensitivity of Mycobacterium tuberculosis to pyrazinamide on
three different solid media. Am. Rev. Tuberc. 67:39195
McDermott W, Tompsett R. 1954. Activation of pyrazinamide and nicotinamide
98.
99.
100.
101.
102.
103.
104.
105.
106.
in acidic environment in vitro. Am. Rev.

Tuberc. 70:74854
Zhang Y, Permar S, Sun Z. 2002. Conditions that may affect the results of
Mycobacterium tuberculosis susceptibility testing to pyrazinamide. J. Med. Microbiol. 51:4249
Wade MM, Zhang Y. 2004. Anaerobic incubation conditions enhances the activity
of anti-tuberculosis drug pyrazinamide. J.
Med. Microbiol. 53:76973
Zhang Y, Scorpio A, Nikaido H, Sun ZH.
1999. Role of acid pH and deficient efflux of pyrazinoic acid in the unique susceptibility of Mycobacterium tuberculosis
to pyrazinamide. J. Bacteriol. 181:2044
49
Zhang Y, Wade MM, Scorpio A, Zhang
H, Sun Z. 2003. Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid. J. Antimicrob.
Chemother. 52:79095
Okunade AL, Elvin-Lewis MP, Lewis
WH. 2004. Natural antimycobacterial
metabolites: current status. Phytochemistry 65:101732
Alvirez-Freites EJ, Carter JL, Cynamon
MH. 2002. In vitro and in vivo activities of gatifloxacin against Mycobacterium tuberculosis. Antimicrob. Agents
Rodriguez GM, Smith I. 2003. Mechanisms of iron regulation in mycobacteria:
role in physiology and virulence. Mol. Microbiol. 47:148594
Ji B, Lounis N, Maslo C, Truffot-Pernot
C, Bonnafous P, et al. 1998. In vitro
and in vivo activities of moxifloxacin
and clinafloxacin against Mycobacterium tuberculosis. Antimicrob. Agents
Miyazaki E, Miyazaki M, Chen JM,
Chaisson RE, Bishai WR. 1999. Moxifloxacin (BAY12-8039), a new 8methoxyquinolone, is active in a mouse
model of tuberculosis. Antimicrob. Agents
Chemother. 43:8589
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX


107. Hu Y, Coates AR, Mitchison DA. 2003.
Sterilizing activities of fluoroquinolones
against rifampin-tolerant populations of
Mycobacterium tuberculosis. Antimicrob.
108. Nuermberger EL, Yoshimatsu T, Tyagi
S, OBrien RJ, Vernon AN, et al. 2004.
Moxifloxacin-containing regimen greatly
reduces time to culture conversion in
murine tuberculosis. Am. J. Respir. Crit.
Care Med. 169:42126
109. Grosset J, Truffot-Pernot C, Lacroix C,
Ji B. 1992. Antagonism between isoniazid and the combination pyrazinamiderifampin against tuberculosis infection
in mice. Antimicrob. Agents Chemother.
36:54851
110. American Thoracic Society. 2001. Update: Fatal and severe liver injuries associated with rifampin and pyrazinamide
for latent tuberculosis infection, and revisions in American Thoracic Society/CDC
recommendationsUnited States, 2001.
Am. J. Respir. Crit. Care Med. 164:1319
20
111. Pletz MW, De Roux A, Roth A, Neumann KH, Mauch H, et al. 2004. Early
bactericidal activity of moxifloxacin in
treatment of pulmonary tuberculosis:
a prospective, randomized study. Antimicrob. Agents Chemother. 48:780
82
112. Valerio G, Bracciale P, Manisco V,
Quitadamo M, Legari G, et al. 2003.
Long-term tolerance and effectiveness of
moxifloxacin therapy for tuberculosis:
preliminary results. J. Chemother. 15:66
70
113. Hirata T, Saito H, Tomioka H, Sato
K, Jidoi J, et al. 1995. In vitro and
in vivo activities of the benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis. Antimicrob. Agents
114. Shoen CM, DeStefano MS, Cynamon
MH. 2000. Durable cure for tuberculosis:
rifalazil in combination with isoniazid in a
murine model of Mycobacterium tubercu-
115.
116.
117.
118.
119.
120.
121.
122.
557
losis infection. Clin. Infect. Dis. 30(Suppl.

3):S28890
Bodmer T, Zurcher G, Imboden P, Telenti A. 1995. Mutation position and type
of substitution in the beta-subunit of the
RNA polymerase influence in-vitro activity of rifamycins in rifampicin-resistant
Mycobacterium tuberculosis. J. Antimicrob. Chemother. 35:34548
Moghazeh SL, Pan X, Arain T, Stover
CK, Musser JM, et al. 1996. Comparative
antimycobacterial activities of rifampin,
rifapentine, and KRM-1648 against a collection of rifampin-resistant Mycobacterium tuberculosis isolates with known
rpoB mutations. Antimicrob. Agents
Dietze R, Teixeira L, Rocha LM, Palaci
M, Johnson JL, et al. 2001. Safety and
bactericidal activity of rifalazil in patients
with pulmonary tuberculosis. Antimicrob.
Barrett J. 2000. Linezolid Pharmacia
Corp. Curr. Opin. Investig. Drugs 1:181
87
Barbachyn MR, Hutchinson DK, Brickner SJ, Cynamon MH, Kilburn JO, et al.
1996. Identification of a novel oxazolidinone (U-100480) with potent antimycobacterial activity. J. Med. Chem.
39:68085
Oxazolidinones, a new class of synthetic
antituberculosis agent. In vitro and in vivo
activities of DuP-721 against Mycobacterium tuberculosis. Diagn. Microbiol. Infect. Dis. 14:46571
Zurenko GE, Yagi BH, Schaadt RD, Allison JW, Kilburn JO, et al. 1996. In vitro
activities of U-100592 and U-100766,
novel oxazolidinone antibacterial agents.
45
Cynamon MH, Klemens SP, Sharpe CA,
Chase S. 1999. Activities of several
novel oxazolidinones against Mycobacterium tuberculosis in a murine model.
91
4 Dec 2004
18:50

558
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
123. Sbardella G, Mai A, Artico M, Loddo

R, Setzu MG, et al. 2004. Synthesis
and in vitro antimycobacterial activity
of novel 3-(1H-pyrrol-1-yl)-2-oxazolidinone analogues of PNU-100480. Bioorg.
Med. Chem. Lett. 14:153741
124. Sun Z, Zhang Y. 1999. Antituberculosis activity of certain antifungal and
antihelmintic drugs. Tuberc. Lung Dis.
79:31920
125. Cole STR, Brosch J, Parkhill T, Garnier
C, Churcher D, et al. 1998. Deciphering
the biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature 393:53744
126. Aoyama Y, Horiuchi T, Gotoh O, Noshiro
M, Yoshida Y. 1998. CYP51-like gene
of Mycobacterium tuberculosis actually
encodes a P450 similar to eukaryotic
CYP51. J. Biochem. 124:69496
127. Guardiola-Diaz HM, Foster LA,
Mushrush D, Vaz AD. 2001. Azoleantifungal binding to a novel cytochrome
P450 from Mycobacterium tuberculosis: implications for treatment of
tuberculosis. Biochem. Pharmacol. 61:
146370
128. McLean KJ, Marshall KR, Richmond
A, Hunter IS, Fowler K, et al. Azole
antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and
bacterial growth in mycobacteria and
streptomycetes. Microbiology 148:2937
49
129. Bellamine A, Mangla AT, Nes WD, Waterman MR. 1999. Characterization and
catalytic properties of the sterol 14alphademethylase from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA
96:893742
130. Leys D, Mowat CG, McLean KJ, Richmond A, Chapman SK, et al. 2003.
Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 A reveals
novel features of cytochrome P450. J.
Biol. Chem. 278:514147
131. Murugasu-Oei B, Dick T. 2000. Bactericidal activity of nitrofurans against grow-
132.
133.
134.
135.
136.
137.
138.
139.
ing and dormant Mycobacterium bovis

BCG. J. Antimicrob. Chemother. 46:917
19
Wayne LG, Russell R. 1966. Selective effects of nitrofuraldoxime-anti on isoniazid resistant M. tuberculosis. Scand. J.
Respir. Dis. 47:1826
Barry VC, Belton JG, Conalty ML, Denney JM, Edward DW, et al. 1957. A new
series of phenazines (rimino-compounds)
with high antituberculosis activity. Nature
179:101315
Reddy VM, OSullivan JF, Gangadharam PR. 1999. Antimycobacterial activities of riminophenazines. J. Antimicrob. Chemother. 43:61523
Steel HC, Matlola NM, Anderson R.
1999. Inhibition of potassium transport
and growth of mycobacteria exposed to
clofazimine and B669 is associated with
a calcium-independent increase in microbial phospholipase A2 activity. J. Antimicrob. Chemother. 44:20916
Bopape MC, Steel HC, Cockeran R, Matlola NM, Fourie PB, et al. 2004. Antimicrobial activity of clofazimine is not
dependent on mycobacterial C-type phospholipases. J. Antimicrob. Chemother. 53:
97174
Adams LB, Sinha I, Franzblau SG,
Krahenbuhl JL, Mehta RT. 1999. Effective treatment of acute and chronic
murine tuberculosis with liposomeencapsulated clofazimine. Antimicrob.
van Rensburg CE, Joone GK, Sirgel FA,
Matlola NM, OSullivan JF. 2000. In
vitro investigation of the antimicrobial
activities of novel tetramethylpiperidinesubstituted phenazines against Mycobacterium tuberculosis. Chemotherapy
46:4348
Amaral L, Kristiansen JE, Viveiros M,
Atouguia J. 2001. Activity of phenothiazines against antibiotic-resistant
Mycobacterium tuberculosis: a review
supporting further studies that may elucidate the potential use of thioridazine as
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX
140.

141.
142.
143.
144.
145.
146.
147.
148.
anti-tuberculosis therapy. J. Antimicrob.

Chemother. 47:50511
Salih FA, Kaushik NK, Sharma P,
Choudary GV, Murthy PS, et al. 1991.
Calmodulin-like activity in mycobacteria.
Indian J. Biochem. Biophys. 28:49195
Ordway D, Viveiros M, Leandro C, Bettencourt R, Almeida J, et al. 2003. Clinical concentrations of thioridazine kill
intracellular multidrug-resistant Mycobacterium tuberculosis. Antimicrob.
Amaral L, Kristiansen JE, Abebe LS, Millett W. 1996. Inhibition of the respiration of multi-drug resistant clinical isolates of Mycobacterium tuberculosis by
thioridazine: potential use for initial therapy of freshly diagnosed tuberculosis. J.
Antimicrob. Chemother. 38:104953
Gadre DV, Talwar V. 1999. In vitro susceptibility testing of Mycobacterium tuberculosis strains to trifluoperazine. J.
Chemother. 11:2036
Crowle AJ, Douvas GS, May MH. 1992.
Chlorpromazine: a drug potentially useful for treating mycobacterial infections.
Chemotherapy 38:41019
Stover CK, Warrener P, VanDevanter DR,
Sherman DR, Arain TM, et al. 2000. A
small-molecule nitroimidazopyran drug
candidate for the treatment of tuberculosis. Nature 405:96266
Ashtekar DR, Costa-Perira R, Nagrajan
K, Vishvanathan N, Bhatt AD, et al.
1993. In vitro and in vivo activities of
the nitroimidazole CGI 17341 against
Mycobacterium tuberculosis. Antimicrob.
Nagarajan K, Shankar RG, Rajappa
S, Shenoy SJ, Costa-Pereira R. 1989.
Nitroimidazoles XXI 2,3-dihydro-6nitroimidazo [2,1-b] oxazoles with
antitubercular activity. Eur. J. Med.
Chem. 24:63133
Giglione C, Pierre M, Meinnel T. 2000.
Peptide deformylase as a target for new
generation, broad spectrum antimicrobial
agents. Mol. Microbiol. 36:1197205
559
149. Jones RN, Moet GJ, Sader HS, Fritsche

TR. 2004. Potential utility of a peptide deformylase inhibitor (NVP PDF713) against oxazolidinone-resistant or
streptogramin-resistant gram-positive organism isolates. J. Antimicrob. Chemother. 53:8047
150. Cynamon MH, Alvirez-Freites E, Yeo
AE. 2004. BB-3497, a peptide deformylase inhibitor, is active against Mycobacterium tuberculosis. J. Antimicrob.
Chemother. 53:4035
151. Waller AS, Clements JM. 2002. Novel approaches to antimicrobial therapy: peptide
deformylase. Curr. Opin. Drug Discov.
Dev. 5:78592
152. Azoulay-Dupuis E, Mohler J, Bedos JP.
2004. Efficacy of BB-83698, a novel peptide deformylase inhibitor, in a mouse
model of pneumococcal pneumonia. Antimicrob. Agents Chemother. 48:8085
153. Lofland D, Difuntorum S, Waller A,
Clements JM, Weaver MK, et al. 2004.
In vitro antibacterial activity of the peptide deformylase inhibitor BB-83698. J.
Antimicrob. Chemother. 53:66468
154. Chopra I, Hesse L, ONeill AJ. 2002. Exploiting current understanding of antibiotic action for discovery of new drugs. J.
Appl. Microbiol. 92(Suppl.):4S15S
155. Dasgupta N, Kapur V, Singh KK, Das
TK, Sachdeva S, et al. 2000. Characterization of a two-component system, devRdevS, of Mycobacterium tuberculosis. Tuber. Lung Dis. 80:14159
156. Sherman DR, Voskuil M, Schnappinger
D, Liao R, Harrell MI et al. 2001. Regulation of the M. tuberculosis hypoxic response gene alpha-crystallin. Proc. Natl.
157. Park HD, Guinn KM, Harrell MI, Liao R,
Voskuil MI, et al. 2003. Rv3133c/dosR
is a transcription factor that mediates
the hypoxic response of Mycobacterium
tuberculosis. Mol. Microbiol. 48:833
43
158. Boon C, Dick T. 2002. Mycobacterium
bovis BCG response regulator essential
4 Dec 2004
18:50
560
159.

160.
161.
162.
163.
164.
165.
166.
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
for hypoxic dormancy. J. Bacteriol.
184:676067
Voskuil MI, Schnappinger D, Visconti
KC, Harrell MI, Dolganov GM, Sherman
DR, Schoolnik GK. 2003. Inhibition of
respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198:70513
Cashel M, Gentry DR, Hernandez VJ,
Vinella D. 1996. The stringent response.
In Escherichia coli and Salmonella: Cellular and Molecular Biology, ed. FC Neidhardt, R Curtis, JL Ingraham, EC Lin,
KB Low, et al., pp. 148896. Washington, DC: ASM Press
Primm TP, Andersen SJ, Mizrahi V, Avarbock D, Rubin H, et al. 2000. The stringent
response of Mycobacterium tuberculosis
is required for long-term survival. J. Bacteriol. 182:488998
Dahl JL, Kraus CN, Boshoff HI, Doan B,
Foley K, et al. 2003. The role of RelMtbmediated adaptation to stationary phase in
long-term persistence of Mycobacterium
tuberculosis in mice. Proc. Natl. Acad.
Sci. USA 100:1002631
McKinney JD, Honer Zu, Bentrup K,
Munoz-Elias EJ, Miczak A, Chen B, et al.
2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:73538
Wayne LG, Lin KY. 1982. Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions. Infect. Immun.
37:104249
Graham JE, Clark-Curtiss JE. 1999. Identification of Mycobacterium tuberculosis
RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences
(SCOTS). Proc. Natl. Acad. Sci. USA
96:1155459
Fenhalls G, Stevens L, Moses L,
Bezuidenhout J, Betts JC, et al. 2002. In
situ detection of Mycobacterium tuberculosis transcripts in human lung granulo-
167.
168.
169.
170.
171.
172.
173.
174.
175.
mas reveals differential gene expression in

necrotic lesions. Infect. Immun. 70:6330
38
Sharma V, Sharma S, Hoener zu, Bentrup K, McKinney JD, Russell DG, et al.
2000. Structure of isocitrate lyase, a persistence factor of Mycobacterium tuberculosis. Nat. Struct. Biol. 7:66368
Glickman MS, Cox JS, Jacobs WR Jr.
2000. A novel mycolic acid cyclopropane
synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol. Cell 5:71727
Bardarov S, Kriakov J, Carriere C, Yu S,
Vaamonde C, et al. 1997. Conditionally
replicating mycobacteriophages: a system
for transposon delivery to Mycobacterium
tuberculosis. Proc. Natl. Acad. Sci. USA
94:1096166
Pelicic V, Jackson M, Reyrat JM, Jacobs
WR Jr, Gicquel B, et al. 1997. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc.
Jacobs WR Jr, Barletta R, Udani R, Chan
J, Kalkut G, et al. Genetic systems for mycobacteria. Methods Enzymol. 204:537
55
Lamichhane G, Zignol M, Blades NJ,
Geiman DE, Dougherty A, et al. 2003. A
postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium
tuberculosis. Proc. Natl. Acad. Sci. USA
100:721318
Sassetti CM, Boyd DH, Rubin EJ. 2001.
Comprehensive identification of conditionally essential genes in mycobacteria.
Sassetti CM, Boyd DH, Rubin EJ. 2003.
Genes required for mycobacterial growth
defined by high density mutagenesis. Mol.
Microbiol. 48:7784
Manganelli R, Proveddi R, Rodrigue S,
Beaucher J, Gaudreau L, et al. 2004.
Sigma factors and global gene regulation
in Mycobacterium tuberculosis. J. Bacteriol. 186:895902
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX


176. Doukhan L, Predich M, Nair G, Dussurget
O, Mandic-Mulec I, et al. 1995. Genomic
organization of the mycobacterial sigma
gene cluster. Gene 165:6770
177. Wu S, Howard ST, Lakey DL, Kipnis A,
Samten B, et al. 2004. The principal sigma
factor sigA mediates enhanced growth of
Mycobacterium tuberculosis in vivo. Mol.
Microbiol. 51:155162
178. Sun R, Converse PJ, Ko C, Tyagi S, Morrison NE, et al. 2004. Mycobacterium tuberculosis ECF sigma factor sigC is required
for lethality in mice and for the conditional
expression of a defined gene set. Mol. Microbiol. 52:2538
179. Wu QL, Kong D, Lam K, Husson RN.
1997. A mycobacterial extracytoplasmic
function sigma factor involved in survival
following stress. J. Bacteriol. 179:2922
29
180. Raman S, Song T, Puyang X, Bardarov S,
Jacobs WR Jr, et al. 2001. The alternative
sigma factor SigH regulates major components of oxidative and heat stress responses in Mycobacterium tuberculosis.
J. Bacteriol. 183:611925
181. Manganelli R, Voskuil MI, Schoolnik GK,
Smith I. 2001. The Mycobacterium tuberculosis ECF sigma factor sigmaE: role
in global gene expression and survival in
macrophages. Mol. Microbiol. 41:42337
182. Ando M, Yoshimatsu T, Ko C, Converse
PJ, Bishai WR. 2003. Deletion of Mycobacterium tuberculosis sigma factor E
results in delayed time to death with bacterial persistence in the lungs of aerosolinfected mice. Infect. Immun. 71:717072
183. Manganelli R, Fattorini L, Tan D, Iona
E, Orefici G, et al. 2004. The extra cytoplasmic function sigma factor sigma(E)
is essential for Mycobacterium tuberculosis virulence in mice. Infect. Immun.
72:303841
184. DeMaio J, Zhang Y, Ko C, Young DB,
Bishai WR. 1996. A stationary-phase
stress-response sigma factor from Mycobacterium tuberculosis. Proc. Natl.
561
185. Chen P, Ruiz RE, Li Q, Silver RF, Bishai

WR. 2000. Construction and characterization of a Mycobacterium tuberculosis
mutant lacking the alternate sigma factor
gene, sigF. Infect. Immun. 68:557580
186. Alksne L. 2002. Virulence as a target
for antimicrobial chemotherapy. Expert
Opin. Investig. Drugs 11:114959
187. Smith I. 2003. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev.
16:46396
188. Sassetti CM, Rubin EJ. 2003. Genetic
requirements for mycobacterial survival
during infection. Proc. Natl. Acad. Sci.
USA 100:1298994
189. Hoch J. 2000. Two-component and phosphorelay signal transduction. Curr. Opin.
Microbiol. 3:16570
190. Macielag MJ, Demers JP, Fraga-Spano
SA, Hlasta DJ, Johnson SG, et al. 1998.
Substituted salicylanilides as inhibitors
of two-component regulatory systems in
bacteria. J. Med. Chem. 41:293945
191. Macielag MJ, Goldschmidt R. 2000. Inhibitors of bacterial two-component signalling systems. Expert Opin. Investig.
Drugs 9:235169
192. Barrett JF, Goldschmidt RM, Lawrence
LE, Foleno B, Chen R, et al. 1998.
Antibacterial agents that inhibit twocomponent signal transduction systems.
22
193. Weidner-Wells MA, Ohemeng KA,
Nguyen VN, Fraga-Spano S, Macielag
MJ, et al. 2001. Amidino benzimidazole
inhibitors of bacterial two-component
systems. Bioorg. Med. Chem. Lett. 11:
154548
194. Stephenson K, Hoch JA. 2004. Developing inhibitors to selectively target
two-component and phosphorelay signal
transduction systems of pathogenic microorganisms. Curr. Med. Chem. 11:765
73
195. Sui Z, Guan J, Hlasta DJ, Macielag MJ,
Foleno BD, et al. 1998. SAR studies
4 Dec 2004
18:50
562

196.
197.
198.
199.
200.
201.
202.
203.
204.
AR
AR232-PA45-22.tex
P1: JRX
ZHANG
of diaryltriazoles against bacterial twocomponent regulatory systems and their
antibacterial activities. Bioorg. Med.
Chem. Lett. 8:92934
Deleted in proof
Via LE, Curcic R, Mudd MH, Dhandayuthapani S, Ulmer RJ, et al. 1996.
Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the
response regulator MtrA. J. Bacteriol.
178:331421
Supply P, Magdalena J, Himpens S, Locht
C. 1997. Identification of novel intergenic
repetitive units in a mycobacterial twocomponent system operon. Mol. Microbiol. 26:9911003
Ewann F, Locht C, Supply P. 2004.
Intracellular autoregulation of the Mycobacterium tuberculosis PrrA response
regulator. Microbiology 150:24146
Zahrt TC, Deretic V. 2001. Mycobacterium tuberculosis signal transduction
system required for persistent infections.
11
Perez E, Samper S, Bordas Y, Guilhot C,
Gicquel B, et al. 2001. An essential role
for phoP in Mycobacterium tuberculosis
virulence. Mol. Microbiol. 41:17987
Parish T, Smith DA, Roberts G, Betts
J, Stoker NG. 2003. The senX3-regX3
two-component regulatory system of
Mycobacterium tuberculosis is required
for virulence. Microbiology 149:1423
35
Rickman L, Saldanha JW, Hunt DM,
Hoar DN, Colston MJ, et al. 2004. A
two-component signal transduction system with a PAS domain-containing sensor is required for virulence of Mycobacterium tuberculosis in mice. Biochem.
Dasgupta N, Kapur V, Singh KK, Das
TK, Sachdeva S, et al. 2000. Characterization of a two-component system, devRdevS, of Mycobacterium tuberculosis. Tuber. Lung Dis. 80:14159
205. Malhotra V, Sharma D, Ramanathan VD,

Shakila H, Saini DK, et al. 2004. Disruption of response regulator gene, devR,
leads to attenuation in virulence of Mycobacterium tuberculosis. FEMS Microbiol. Lett. 231:23745
206. Slayden RA, Lee RE, Armour JW, Cooper
AM, Orme IM, et al. 1996. Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid
synthesis. Antimicrob. Agents Chemother.
40:281319
207. Kremer L, Douglas JD, Baulard AR,
Morehouse C, Guy MR, et al. 2000. Thiolactomycin and related analogues as novel
anti-mycobacterial agents targeting KasA
and KasB condensing enzymes in Mycobacterium tuberculosis. J. Biol. Chem.
275:1685764
208. Douglas JD, Senior SJ, Morehouse C,
Phetsukiri B, Campbell IB, et al. 2002.
Analogues of thiolactomycin: potential
drugs with enhanced anti-mycobacterial
activity. Microbiology 148:31019
209. Kremer L, Dover LG, Carrere S, Nampoothiri KM, Lesjean S, et al. 2002.
Mycolic acid biosynthesis and enzymic
characterization of the beta-ketoacyl-ACP
synthase A-condensing enzyme from Mycobacterium tuberculosis. Biochem. J.
364:42330
210. Parrish NM, Kuhajda FP, Heine HS,
Bishai WR, Dick JD. 1999. Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis. J. Antimicrob.
Chemother. 43:21926
211. Parrish NM, Houston T, Jones PB,
Townsend C, Dick JD. 2001. In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and
its effects on lipid and mycolic acid synthesis. Antimicrob. Agents Chemother.
45:114350
212. Mitchison D. 2004. The search for new
sterilizing anti-tuberculosis drugs. Front.
Biosci. 9:105972
213. Zhang Y, Zhang H, Sun Z. 2003. Susceptibility of Mycobacterium tuberculosis
4 Dec 2004
18:50
AR
AR232-PA45-22.tex
P1: JRX

214.
215.
216.
217.
218.
219.
220.
221.
222.
to weak acids. J. Antimicrob. Chemother.

52:5660
Deretic V, Philipp W, Dhandayuthapani
S, Mudd MH, Curcic R, et al. 1995. Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative stress
regulatory gene: implications for sensitivity to isoniazid. Mol. Microbiol. 17:889
900
Sherman DR, Sabo PJ, Hickey MJ, Arain
TM, Mahairas GG, et al. 1995. Disparate responses to oxidative stress in
saprophytic and pathogenic mycobacteria. Proc. Natl. Acad. Sci. USA 92:6625
29
Fleischmann RD, Adams MD, White O,
Clayton RA, Kirkness EF, et al. 1995.
Whole-genome random sequencing and
assembly of Haemophilus influenzae Rd.
Science 269:496512
Dougherty TJ, Barrett JF, Pucci MJ. 2002.
Microbial genomics and novel antibiotic
discovery: new technology to search for
new drugs. Curr. Pharm. Des. 8:1119
35
Chalker AF, Lunsford RD. 2002. Rational identification of new antibacterial drug
targets that are essential for viability using
a genomics-based approach. Pharmacol.
Ther. 95:120
Volker C, Brown JR. 2002. Bioinformatics and the discovery of novel antimicrobial targets. Curr. Drug Targets Infect. Disord. 2:27990
Haney SA, Alksne LE, Dunman PM,
Murphy E, Projan SJ. 2002. Genomics
in anti-infective drug discoverygetting
to endgame. Curr. Pharm. Des. 8:1099
118
Cole S. 2002. Comparative mycobacterial genomics as a tool for drug target
and antigen discovery. Eur. Respir. J.
36(Suppl.):78s86
Schena M, Shalon D, Davis RW, Brown
PO. 1995. Quantitative monitoring of
gene expression patterns with a complementary DNA microarray. Science
270:46770
563
223. Hensel M, Shea JE, Gleeson C, Jones

MD, Dalton E, et al. 1995. Simultaneous identification of bacterial virulence
genes by negative selection. Science 269:
4003
224. Balasubramanian V, Pavelka MS Jr, Bardarov SS, Martin J, Weisbrod TR, et al.
1996. Allelic exchange in Mycobacterium
tuberculosis with long linear recombination substrates. J. Bacteriol. 178:273
79
225. Wilson M, DeRisi J, Kristensen HH, Imboden P, Rane S, et al. 1999. Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis
by microarray hybridization. Proc. Natl.
226. Betts JC, McLaren A, Lennon MG, Kelly
FM, Lukey PT, et al. 2003. Signature gene
expression profiles discriminate between
isoniazid-, thiolactomycin-, and triclosantreated Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 47:290313
227. Betts JC, Lukey PT, Robb LC, McAdam
RA, Duncan K. 2002. Evaluation of a nutrient starvation model of Mycobacterium
tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol.
43:71731
228. Goulding CW, Apostol M, Anderson DH,
Gill HS, Smith CV, et al. 2002. The TB
structural genomics consortium: providing a structural foundation for drug discovery. Curr. Drug Targets Infect. Disord.
2:12141
229. Crabb C. 2002. A new TB drug by 2010
or sooner? Bull. World Health Organ.
80:51819
230. Orme I, Tuberculosis Drug Screening
Program. 2001. Search for new drugs
for treatment of tuberculosis. Antimicrob.
231. Lee RE, Protopopova M, Crooks E, Slayden RA, Terrot M, et al. 2003. Combinatorial lead optimization of [1,2]-diamines
based on ethambutol as potential antituberculosis preclinical candidates. J.
Comb. Chem. 5:17287
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232. Changsen C, Franzblau SG, Palittapongarnpim P. 2003. Improved green fluorescent protein reporter gene-based
microplate screening for antituberculosis
compounds by utilizing an acetamidase
promoter. Antimicrob. Agents Chemother.
47:368287
233. Projan S. 2002. New (and not so new) anAnnu. Rev. Pharmacol. Toxicol. 2005.45:529-564. Downloaded from arjournals.annualreviews.org
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tibacterial targetsfrom where and when

will the novel drugs come? Curr. Opin.
Pharmacol. 2:51322
234. Projan S. 2003. Why is big Pharma getting out of antibacterial drug discovery?
Curr. Opin. Microbiol. 6:42730
235. McDermott W. 1958. Microbial persistence. Yale J. Biol. Med. 30:25791
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MOLECULAR MECHANISMS OF RESISTANCE

IN ANTIMALARIAL CHEMOTHERAPY:
The Unmet Challenge

Ravit Arav-Boger1 and Theresa A. Shapiro2
1
Division of Infectious Diseases, Department of Pediatrics, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205; email: boger@jhmi.edu
2
Division of Clinical Pharmacology, Departments of Medicine and of Pharmacology
and Molecular Sciences, and The Johns Hopkins Malaria Research Institute, The Johns
Hopkins University, Baltimore, Maryland 21205; email: tshapiro@jhmi.edu
Key Words Plasmodium, malaria, drug resistance, mutations

Abstract The enormous public health problem posed by malaria has been substantially worsened in recent years by the emergence and worldwide spread of drugresistant parasites. The utility of two major therapies, chloroquine and the synergistic
combination of pyrimethamine/sulfadoxine, is now seriously compromised. Although
several genetic mechanisms have been described, the major source of drug resistance
appears to be point mutations in protein target genes. Clinically significant resistance to
these agents requires the accumulation of multiple mutations, which genetic studies of
parasite populations suggest arise focally and sweep through the population. Efforts to
circumvent resistance range from the use of combination therapy with existing agents
to laboratory studies directed toward discovering novel targets and therapies.
The prevention and management of drug resistance are among the most
important practical problems of tropical medicine and public health.
Leonard J. Bruce-Chwatt, 1972
INTRODUCTION
Malaria is one of the greatest of all infectious diseases, afflicting more than 500
million people and causing approximately 2 million deaths each year. Estimates
of the economic burden of malaria in terms of lost productivity are staggering
(1). Malaria is transmitted by mosquitoes and caused by intracellular protozoan
parasites from the genus Plasmodium. By far the most significant species is P. falciparum, which causes severe infections and death, enjoys widespread geographic
distribution, and is most likely to be drug resistant. In the years after World War II,
public health workers had ambitious plans to eradicate malaria by various means,
including DDT against mosquitoes and chloroquine against the parasite. These
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efforts unfortunately failed; among the reasons for failure was the appearance and
spread of chloroquine-resistant malaria, an event that is aptly considered a public
health crisis. Malaria now features prominently among the reemerging infectious
diseases (2).
Although much work is being done to develop malaria vaccines, estimates are
that it will be many years before these are suitable for use in humans, and drugs
are therefore required not only for treatment of established infections but also
for prevention of malaria in healthy travelers, tens of millions of whom go to
malarious countries every year (3). Malaria therapy is complicated by a number of
factors, including the considerable requirement for safety (huge numbers afflicted,
disproportionate severity in children and pregnant women, prophylaxis of healthy
travelers), the fact that selective toxicity may be more difficult to attain against these
eukaryotic pathogens, and by the inherent complexity of the parasites lifecycle
within the human host. Each lifecycle stage varies in its drug-sensitivity profile;
hence, for a given patient multiple drugs may be needed to eradicate the infection.
Infection begins with the bite of an infected Anopheline mosquito. Parasites first
invade hepatocytes and replicate there before bursting the cell. The released forms
then infect, replicate within, rupture, and reinfect red cells in a cycle that repeats
every 23 days. This asexual replication leads to tremendous amplification, with
parasite burdens that may reach 1012 organisms per patient. Drug-resistance genes
that arise and are selected in this setting are further spread through the gene pool
by the meiotic exchange that occurs during the sexual reproduction of Plasmodium
within the mosquito.
The recently available genome for P. falciparum provides powerful information
for understanding resistance mechanisms and opens exciting new avenues for
drug development (4). P. falciparum contains 14 chromosomes and approximately
5300 protein-encoding genes, almost two-thirds of which seem to be unique to
this organism. Newly recognized cellular pathways and organelles, such as the
apicoplast (a chloroplast-like structure with unique metabolism), provide novel
targets for the development of selectively toxic new therapies. Information on P.
falciparum genes and their expression is available on the PlasmoDB Web site
(http://www.plasmoDB.org).
In this review, we provide an overview of the problem of antimalarial drug
resistance, consider potential solutions, and refer interested readers to the many
excellent and detailed reviews that have appeared in recent years (512). Given its
clinical and public health importance, and because it is by far the most likely to
be drug resistant, the discussion focuses almost entirely on P. falciparum.
GENERAL ISSUES IN MALARIA PARASITE RESISTANCE

There are many definitions for drug resistance in malaria; indeed, classic textbooks
have been written on this subject (13). Definitions range from the earliest, which
were devised by the World Health Organization (WHO) to characterize clinical
drug failures (14), to those based on altered drug potency against parasites in vitro,
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and most recently to assays for known gene mutations. Each of these approaches
has its merits, but for many reasons they may not be concordant. The assessment of
antimalarial drug resistance, and the correlation of clinical and laboratory findings,
is confounded by many variables. These include the obvious generic issues: distinguishing genuine resistance from suboptimal therapy, immunity and nutritional
factors, and culturing parasites in conditions where key nutrients far exceed those
in blood. There are also confounding variables more particular to malaria. In the
field, resistant parasites may take weeks to recrudesce, at which point it becomes
difficult to distinguish drug failure from reinfection. Furthermore, patients may
harbor many clones of P. falciparum, each with a distinct set of mutations that
impart resistance. Thus, if two mutations in a single gene are detected in a patients blood sample, unless clonal parasites are isolated and assayed, it is difficult
to know whether both mutations are in one cell line or whether two cell lines each
have one mutation. Fluorogenic assays that distinguish between these possibilities
may provide a solution to this problem (15).
A rich variety of genetic mechanisms are exploited for drug resistance in bacteria and tumor cells (16, 17). These range from discrete point mutations to the
rearrangement of large blocks of DNA (e.g., inversion, duplication, insertion, deletion, transposition), and even to the acquisition of foreign DNA. Alterations in gene
transcription, in the posttranscriptional control of RNA, and in the posttranslational
modification of proteins, play important roles in drug resistance. By comparison,
relatively few mechanisms are recognized in malaria and, as described below, the
best understood of these are confined to point mutations and changes in steady-state
transcript levels. Point mutations provide a satisfying and consistent explanation
for many cases of antimalarial drug resistance. Almost certainly, however, there
are mechanisms at work in these parasites that remain to be found. The availability of the fully sequenced genome and proteome that follows will be key in this
discovery process.
DRUG-SPECIFIC RESISTANCE
4-Substituted Quinolines
The members of this largest class of antimalarial agents share obvious structural
analogy, which reflects their derivation from the natural product quinine (Figure 1).
As described below, they also have a common molecular mechanism of antimalarial activity. The preeminent agent in this class has been chloroquine, which in
retrospect has aptly been termed a wonder drug (18). The focus of some intrigue during the years of World War II (19), this fully synthetic antimalarial is
inexpensive, safe, and orally bioavailable. For decades, chloroquine provided reliable prophylaxis for travelers, therapy for those with established infection, and a
powerful tool for public health workers in their efforts to control malaria. The emergence in the early 1960s and subsequent spread of chloroquine-resistant parasites
created a tremendous therapeutic void, which has not yet been filled satisfactorily.
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Figure 1 Structures of antimalarial drugs. Chloroquine, quinine, and mefloquine

are 4-substituted quinolines that interfere with heme polymerization; sulfadoxine,
pyrimethamine, and cycloguanil are substrate analogs that interfere with folate
metabolism (Figure 2). In humans, proguanil is converted by CYP2C19 and CYP3A4 to
form cycloguanil. Newer antimalarials with novel structures and mechanisms include
atovaquone and artesunate.
Chloroquine resistance has resulted in demonstrably escalating mortality rates in

African children (20, 21); in Senegal, the emergence of resistance over a 12-year
period was associated with at least a doubling of the risk of death from malaria in
children under ten (22).
Chloroquine and the other 4-substituted quinolines kill malaria parasites by interfering with the detoxication of heme. During its intraerythrocytic development
and proliferation, hemoglobin is a major source of nutrition for the parasite (23).
Hemoglobin is transported into the acidic food vacuole and sequentially digested
into smaller peptide fragments by aspartic, cysteine, and metallo proteases. A toxic
byproduct of hemoglobin degradation is free heme. Unlike mammalian systems,
which detoxify heme by enzyme-mediated ring opening and glucuronidation, in
malaria parasites heme is polymerized to form an inert crystalline pigment called
hemozoin. Early studies with rodent malaria parasites revealed that chloroquine
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selectively disrupts the aggregation of malarial pigment within the food vacuole
(24), and more recent experiments have refined this picture to show that chloroquine
effectively blocks the sequestration of toxic heme into hemozoin (25). Chloroquine
accumulates in parasitized red cells, particularly in the acidic digestive vacuole,
to reach levels hundreds of times those in plasma, and the accumulation is reduced substantially in chloroquine-resistant cells (26). Subsequent studies with
chloroquine-resistant P. falciparum confirmed these findings; noted the lack of
cross-reactivity with quinine, mefloquine, or chloroquine analogs (27); described
a paradoxically increased sensitivity to some antimalarials (28); found that reduced steady-state levels were attributable to enhanced efflux, not reduced uptake
(29); and revealed that verapamil could partially restore the accumulation of, and
sensitivity to, chloroquine (30). Although these phenotypic characteristics have
been invaluable in suggesting and corroborating the molecular mechanisms of resistance, the definitive studies have been genetic. Despite heavy drug pressure, it
took many years for chloroquine-resistant P. falciparum to emerge in the field.
This observation, together with the fact that chloroquine resistance in the laboratory could only be generated in the presence of mutagens, led to the suspicion that
chloroquine-resistance might well be multigenic.
As described in detail below, two entirely distinct experimental approaches
have yielded two independent genetic sources of chloroquine resistance in P. falciparum. One of these, pfcrt (P. falciparum chloroquine-resistance transporter)
is now recognized to be both necessary and sufficient to impart chloroquine resistance. The other, pfmdr1 (P. falciparum multidrug-resistance 1), may further
modulate the degree of resistance.
One experimental approach was an undirected search for a gene(s) that would
sort with chloroquine resistance when sensitive and resistant parent lines were
crossed during sexual reproduction in the mosquito (31). The resulting progeny
were fully sensitive or resistant, consistent with changes at a single genetic locus in
these haploid forms. Some ten additional years of work were required to identify
the rather cryptic 13-exon pfcrt on chromosome 7 (32). This gene encodes a novel
45 kDa protein with ten predicted transmembrane domains that immunolocalizes
to the membrane of the digestive vacuole. It has no obvious homology to the
large family of ABC (ATP-binding cassette) transporters that pump drugs against
a concentration gradient at the expense of ATP (33, 34). The predicted protein
is thought to be a transporter or channel that reduces chloroquine levels in the
digestive vacuole, which in turn reduces the accumulation of free heme and relieves
cytotoxicity. The mechanism by which pfcrt affects chloroquine levels is not yet
clear but it may involve altered ion fluxes that change the acidity of the vacuole, or
alternatively, pfcrt may interact directly with chloroquine itself (35). Studies of this
process have been hampered by the difficulty in expressing this transmembrane
protein in heterologous systems.
Analysis of pfcrt in cell lines obtained from many geographic locations revealed
a consistent wild-type sequence in the sensitive lines, and a remarkable array of
mutations in chloroquine-resistant lines (32). Genes from resistant cells have at
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least five and up to eight mutations, all confined to ten positions that are clustered
within or near transmembrane domains. Common to all resistant lines are a K76
mutation, which now provides a valuable molecular marker in surveillance studies
and a predictor of chloroquine efficacy (36). The limited patterns of mutations
suggest that resistant lines originated in just a few discrete geographic locations
from which they then spread. This notion is strengthened by a more recent genomewide satellite marker analysis in dozens of strains of P. falciparum, which reveals
a striking lack of polymorphism surrounding pfcrt in chromosome 7, relative to all
other portions of the genome (37). This prominent aberration reflects the powerful
selective pressure that extensive chloroquine use has exerted on this parasites
evolution. The essential role of pfcrt was firmly established by allelic exchange of
the endogenous pfcrt in chloroquine-sensitive cells for mutant alleles from resistant
lines, which effectively conferred a chloroquine-resistant phenotype (38).
Mutations in pfcrt have now been shown to account for the recognized characteristics of chloroquine-resistant cells described above: reduced accumulation of
chloroquine (38, 39); lack of cross-resistance with quinine and mefloquine (35),
indeed a paradoxically increased sensitivity to some antimalarials (38); and an
acceptable fulfillment of the expectation for a multigenetic mechanism (e.g., multiple mutations required, although all in a single gene). Finally, although perhaps
not a consequence that should be expected, the chloroquine resistance imparted
by mutant PfCRT is partially reversible by verapamil (38, 39). The latter is a
well-recognized antagonist of drug efflux pumps (33, 34); however, its action is
confined to just one of the seven classes of ABC transporters, and PfCRT is not
even a member of the ABC transporter family.
A second independent experimental approach to understanding the genetic basis for chloroquine resistance actually preceded that described above, and was a
directed search for ABC transporter genes whose sequence or expression might be
altered in drug-resistant cells. This logical search was prompted by accumulating
evidence that upregulation of ABC transporter gene expression is associated with
multidrug resistance in tumor cells, and by the finding that chloroquine resistance
is partially reversed by verapamil (30). With the completion of the human genome,
the family of ABC transporters is now divided into seven different classes on the
basis of sequence homology (33, 34). All are membrane-spanning proteins and
have highly characteristic nucleotide-binding domains. Best studied of the ABC
transporters is ABCB1 (also termed Pgy1, MDR1, Pgp, or GP170), whose preferred substrates (hydrophobic, planar aromatic rings, with the presence of tertiary
amino groupscriteria all fulfilled by chloroquine; Figure 1) are pumped against
a concentration gradient at the expense of ATP hydrolysis and whose action is antagonized by verapamil. Notably, mutations in human ABCB1 are not associated
with recognizable disease or with altered drug transport; the latter is mediated by
upregulated expression.
Using phylogenetically conserved ABCB1 sequences, two laboratories identified mdr genes (termed pfmdr1 and pfmdr2) in P. falciparum (40, 41). Subsequent
studies implicated only pfmdr1 in drug resistance, although the association was
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imperfect and variably involved either gene amplification or point mutations. The
clearest data bearing on the role of pfmdrs in drug-resistant P. falciparum indicate that these genes do not sort with chloroquine resistance in genetic crosses of
sensitive and resistant cells (31), and that the introduction of mutant pfmdr1 into
cells with wild-type pfcrt has no effect on chloroquine sensitivity (42). Importantly, however, the addition of mutant pfmdr1 to cells already harboring mutant
pfcrt does enhance chloroquine resistance, indicating that mutations in this gene
may modulate the overall response to chloroquine (38, 42). Of considerable interest and distinct from pfcrt, mutations in pfmdr1 are associated with resistance to
mefloquine, quinine, and halofantrine (42).
Although the exposure of P. vivax and P. falciparum to chloroquine has been
similar, the appearance of chloroquine-resistant P. vivax took nearly 30 additional
years to appear. First reported from Papua New Guinea in 1989 (43), chloroquineresistant P. vivax has now spread through Southeast Asia and into South America.
Unexpected and intriguing is the finding that chloroquine resistance in P. vivax is
apparently not mediated by mutations in the vivax homolog of pfcrt (44). Despite
the interest and importance of this problem, the technical difficulties in studying
P. vivax seriously hamper definitive studies.
A number of new therapeutic approaches have been taken on the basis of lessons
learned from chloroquine and the mechanisms of chloroquine resistance. These
include the use of analogs that differ only in the length of the 4-aminoalkyl side
chain, which retain antimalarial activity but are not cross-resistant with chloroquine (45); coadministration of chloroquine with various chemosensitizers in an
effort to reverse the efflux mechanism (46), although for antitumor agents this
approach has met with very limited success (33); and use of chloroquine in combination with other antimalarials, most notably an artemisinin (47). The documented
reemergence of chloroquine-sensitive parasites when drug pressure is removed is
fascinating and may afford an opportunity to reintroduce chloroquine after years
of nonuse (48).
Folic Acid Antagonists

Malaria parasites were closely intertwined with the discovery of drugs that target folate biosynthesis. Two years after Domagks 1935 Nobel Prizewinning
description of sulfonamide activity against bacteria (49), a rather large clinical
trial established the efficacy of a sulfonamide in patients with malaria (50). Some
ten years later a concerted program of antimalarial drug discovery (51) yielded
proguanil (a prophetic name, given its prodrug nature; Figure 1). In a landmark
study reported in 1948, well before proguanils molecular mechanism had been
described, Greenberg showed for the first time that the combination of proguanil
with a sulfonamide was profoundly synergistic (52). His studies were on P. gallinaceum in chicks. This key observation had important and nearly immediate consequences. First, it led directly to the finding that proguanil also interferes with folate
metabolism in malaria parasites, but at a site distinct from that of sulfonamides
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(53); second, the structural analogy of proguanil to a series of antibacterial 2,4diaminopyrimidines was recognized by Hitchings, who then demonstrated potent
antimalarial activity in this new chemical class of antifolates (54); and third, it
provided an effective means to forestall the emergence of resistance, which even
in earliest experiments was recognized as a serious problem. The eventual consequence was an antifolate/sulfonamide combination of pyrimethamine/sulfadoxine
(Figure 1) that was carefully selected for matching pharmacokinetics, formulated
in fixed ratios to maximize synergy, and marketed as Fansidar . (The antibacterial trimethoprim/sulfamethoxazole was similarly developed.) The extraordinary
degree of synergism in these combinations, which allows some 20-fold reduction
in the dose of each component, is still attributed to multiple blockades in a single metabolic pathway, although evidence to support this widely cited mechanism
remains circumstantial and other mechanisms may contribute (5558).
Tetrahydrofolate is an essential cofactor in the methyl transfer reactions that
generate monomers for protein and nucleic acid synthesis (59). In several important respects, folate biosynthesis in malaria parasites is distinctly different from
that in other systems (pathways and key points of drug inhibition in Figure 2, see
color insert). First, from biochemical studies and the annotated genome it is now
clear that P. falciparum is unique in that both para-aminobenzoic acid (6062) and
dihydrofolic acid (58, 63, 64) can be synthesized de novo as well as salvaged from
the environment. The availability of these salvage pathways has severely complicated in vitro inhibition studies, and they clearly modulate antifolate efficacy in
patients, whose blood levels of para-aminobenzoic acid and dihydrofolate may
vary widely (65). A second dissimilar feature in Plasmodium folate metabolism is
that sequential reactions may be catalyzed by a single bifunctional protein. Thus,
dihydro-6-hydroxymethylpterin pyrophosphokinase and dihydropteroate synthase
are encoded by the same gene and contained within the same protein (66, 67). Dihydrofolate reductase and thymidylate synthase activities are similarly linked (68,
69). This structural organization may improve catalytic efficiency by channeling
substrates in a processive fashion through two sequential transformations; it may
also offer novel strategies for drug-mediated disruption. Finally, malaria parasites
are especially susceptible to inhibition of dihydrofolate reductase because (unlike
mammalian cells) transcriptional inhibition, mediated by the protein binding to its
own message, is not relieved by the accumulation of substrate that occurs in the
presence of inhibitor (70). This precludes the upregulation of protein synthesis as
a means to counter antifolate inhibitors and it contributes to the selective toxicity
of antifolates against the parasite.
Chloroquines efficacy, safety, and low cost made it the clear drug of choice
for many decades, but the advent of chloroquine-resistant parasites established
pyrimethamine/sulfadoxine as the next best option, despite the recognized propensity for resistance and the concern about antifolate teratogenicity (71). Malaria
parasite resistance to sulfonamides and antifolates has been known for more
than 50 years (7274). Although available mechanisms reportedly include gene
amplification, which is the only recognized mechanism associated with clinical
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resistance to antifolate therapy in cancer (17), a large body of evidence now indicates that in Plasmodium the major effector of resistance is point mutations in the
key target enzymes: dihydropteroate synthase and dihydrofolate reductase. Unlike
the transmembrane proteins that mediate chloroquine resistance, native and recombinant forms of the synthase and reductase are soluble and assayable; hence,
the findings in genetic studies have been bolstered by biochemical and structural
experiments.
Molecular epidemiology studies from South America and Africa provide multiple lines of evidence that application of pyrimethamine/sulfadoxine therapy leads
to the progressive and orderly accumulation of point mutations, first in dihydrofolate reductase and then in dihydropteroate synthase. The sequential addition of
new mutations is evident in field isolates collected over years of time (75, 76), in
pre- versus posttreated patients (77), and in correlation with the degree of clinical resistance for a given patient or geographic region (78). Evaluation of these
mutations in the context of surrounding polymorphisms in noncoding sequences
is consistent with focal origin of mutant strains followed by spread through the
population via gene flow (75, 76). Highest levels of clinical resistance result from
parasites with four mutations in dihydrofolate reductase and two in dihydropteroate
synthase, which may represent the maximum number of mutations that can be tolerated in competition with less-affected strains. The utility of these mutations as
predictors for therapeutic response is modulated by host immunity, as evidenced
by the persistent efficacy of pyrimethamine/sulfadoxine in holoendemic Malawi,
despite ongoing use of these agents in a population that has harbored highly mutant
parasites for at least five years (79).
Laboratory findings that corroborate these field data and underscore the central
importance of point mutations include the appearance of the appropriate drugresistant phenotype in genetic crosses or when mutant genes are introduced into
wild-type cells (8082) and analysis of the inhibition kinetics of recombinant wildtype versus mutant enzymes (83, 84). The recently available crystal structure for
dihydrofolate reductase-thymidylate synthase provides satisfying evidence that
the critical mutations mediating clinical drug resistance map to the dihydrofolate
reductase active site (85).
The well-studied and proven value of the folate synthetic machinery as an antimalarial target has prompted several ingenious research efforts to devise new
interventions against tetrahydrofolate production and use. These include inhibition of the shikimate pathway, which provides an intracellular source of paraaminobenzoic acid (Figure 2), alone or in combination with downstream inhibitors (61); dihydrofolate reductase inhibitors rationally designed and selected
for activity against the clinically important quadruple mutant malaria enzyme
but not the human reductase (86); identification of novel chemical classes by in
silico docking of large chemical libraries into the known dihydrofolate reductase three-dimensional (3-D) structure (87); and deployment of folate analogs
against thymidylate synthase (88). More immediate clinical efforts have focused
on using sulfonamide/antifolate combinations that are less cross-resistant and/or
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have a shorter plasma half-life (89, 90) and adding a third antimalarial to the
pyrimethamine/sulfadoxine dosing regimen (47, 91).

Mitochondrial Electron Transport Inhibitors

Although hydroxynaphthoquinones were the focus of considerable interest in the
1940s as a new class of synthetic antimalarials (92), they were upstaged first by
chloroquine and then pyrimethamine/sulfadoxine. However, by the early 1990s
the growing resistance to existing antimalarials and the activity of atovaquone (the
lead compound in this chemical class; Figure 1) against opportunistic Pneumocystis carinii in AIDS patients, spurred the clinical development of atovaquone
(93). Given its novel molecular mechanism of action (a ubiquinone analog that
blocks mitochondrial respiration at the cytochrome bc1 complex) and its potency
at low nanomolar concentrations in vitro, it came as an unexpected surprise that
atovaquone had a 30% failure rate in its first field trials (94). Remarkably, paired
isolates of P. falciparum obtained before treatment and after recrudescence showed
a more than 1000-fold reduction in sensitivity to atovaquone.
In a short time, an elegant series of studies confirmed the previously reported
molecular site of action (95) and provided a satisfying explanation for resistance
(96, 97). Atovaquone inhibits respiration and collapses the mitochondrial membrane potential in live intact malaria parasites. Sequence analysis of the mitochondrially encoded gene for cytochrome b from atovaquone-resistant P. yoelii
revealed a series of mutations that affect five amino acids clustered in a highly
conserved 15 amino acid sequence. Based on analogy to the crystal structure for
chicken cytochrome b, these residues all map to a cavity in the region of the
ubiquinol-oxidation site. Several factors were identified to help account for the
striking rapidity and magnitude of atovaquone resistance. First, 11 of the 12 mutations involved A:T to G:C changes, a lesion consistent with oxidative damage. By
disrupting the normal flow of electrons through the transport chain, atovaquone
may increase the formation of superoxide radicals, which in turn can damage mitochondrial DNA. Second, although there are approximately 100 copies of the 6kb
mitochondrial genome per parasite, sensitive methods failed to detect any evidence
of residual wild-type cytochrome b sequence. Thus, after a short period of time
under drug selection, every copy of the genome contained these advantageous mutations, perhaps as a result of the extensive recombination that accompanies mitochondrial DNA replication in malaria parasites. Analysis of P. falciparum isolated
from patients who failed atovaquone monotherapy confirmed the predilection for
mutations at the Y268 residue (98).
Fortunately, the clinical utility of atovaquone was salvaged by the timely discovery that its antimalarial activity is synergistically enhanced, in vitro and in
the clinic, by the simultaneous application of proguanil (94, 99). Proguanil is
classically regarded as an antifolate (Figure 1 and see above) and by itself has
no detectable effect on electron transport or mitochondrial membrane potential.
However, proguanil synergistically enhances atovaquones ability to depolarize
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the malarial mitochondrial membrane and inhibit respiration (100). Atovaquone

plus proguanil, now marketed as a fixed combination (Malarone ), generally provides safe and reliable prophylactic and therapeutic antimalarial activity (101,
102). Although there have been no published failures of atovaquone/proguanil
for prophylaxis, a handful of case reports document the recrudescence of P. falciparum after treatment of established infections. In all cases (some of which
include paired isolates), recrudescent parasites have a Y268N, or more commonly
a Y268S, mutation in cytochrome b (103, 104). The fact that just a single mutation
can significantly compromise the efficacy of this combination is worrisome and
underscores the need for careful selection of therapeutic indications to prolong its
useful lifetime.
MULTIDRUG-RESISTANT PARASITES
In cancer chemotherapy, resistance to structurally and mechanistically diverse
agents can be mediated by alterations in expression of a single ABC transporter
gene (17, 105). As we now understand it, multidrug resistance for P. falciparum
is different: It involves genetic alterations in at least two, and often more, proteins [the difficult problem of multidrug-resistant P. falciparum has been thoughtfully defined and reviewed recently (8)]. Typically, this means resistance to both
chloroquine and pyrimethamine/sulfadoxine, mediated by mutations in pfcrt, dihydropteroate synthase and dihydrofolate reductase, as described above. However,
strains resistant to chloroquine, sulfadoxine/pyrimethamine, mefloquine, and partially resistant to quinine and quinidine have been described (106). Malaria in
Southeast Asia is notorious for its propensity to develop early and multidrug resistance. This prompted an interesting experiment comparing the emergence of
resistance in a parasite clone from Africa (which was fully susceptible to conventional antimalarials) to that of a multidrug-resistant clone from Indochina (107).
Two compounds were selected that had novel killing mechanisms and had never
before been applied to these parasites. The Indochina clone acquired resistance
some 1000 times more frequently, suggesting these parasites may have an underlying accelerated mutator or hyperrecombination phenotype.
STRATEGIES TO COMBAT RESISTANCE

Artemisinins
The artemisinins are an important and exciting addition to the antimalarials (artemisinins reviewed in 108, 109; Figure 1). Hundreds of synthetic and semisynthetic analogs have been evaluated, and to date the most clinically successful
is artesunate. The essential pharmacophore is structurally and mechanistically
unique: an endoperoxide bridge that undergoes iron-catalyzed activation, probably
in the food vacuole, to form toxic free radicals. Recent studies suggest artemisinin
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may inhibit ATPase and alter intracellular calcium stores (110). As a class the
artemisinins are potent, fast-acting, and remarkably impervious to resistance, although recrudescence of fully sensitive parasites is common. Human safety for this
class is regularly claimed despite the unfortunate rarity of systematic safety evaluations available in the literature. The current recommended use for artemisinins
is in combination therapy, where they effect a rapid and massive decrease in parasite burden and their gametocytocidal activity may lessen transmission of resistant
parasites to the mosquito. As noted above, several large clinical trials have already demonstrated their meaningful contribution to efficacy (47, 111), and even
larger studies are underway as a likely prelude to national health policy recommendations (6).
Drugs Used in Other Diseases

It is a telling commentary on the state of antimalarial therapy that doxycycline,
an antibacterial, is among the agents now recommended for malaria prophylaxis.
Although intrinsically weak as antimalarials, clindamycin, azithromycin, and chloramphenicol also have some utility (112, 113), which may stem from their targeting
protein synthesis in the parasites apicoplast or mitochondrion. The antibacterial
quinolones act by inhibiting DNA gyrase, an enzyme also present in the P. falciparum genome; although fluoroquinolones have activity against parasites in vitro
(114), their clinical efficacy has been disappointing (115). The antifungal imidazoles are active against P. falciparum in vitro (116, 117). They form complexes
with heme (118), suggesting a mode of action that might be similar to chloroquines. Attractive features of this class are their good safety profile in children
and adults, oral bioavailability, and short half-life.
Combination Therapy
For both antitumor and antiinfective therapies, abundant laboratory and clinical
evidence attests to the fact that coadministration of drugs reduces the emergence
of resistance. As detailed above, this strategy has provided a useful antimalarial
therapeutic life span for pyrimethamine/sulfadoxine and atovaquone/proguanil,
agents that readily provoke resistance when used alone. To stem the further development and spread of antimalarial drug resistance, the combined use of three or
more drugs is under extensive study, and will likely succeed in reducing resistance
(119). Less easy to predict is how multiple agents will interact in terms of antimalarial potency and host toxicity, where the net effects may be additive, synergistic, or
even antagonistic. Distinguishing among these important outcomes requires careful attention to study design. Investigational combinations include coartemether,
a fixed dose of artemether and lumefantrine; the latter has structural similarities
to mefloquine and halofantrine. This combination originated in China and is in
advanced clinical development (120). The combination of dihydroartemisinin and
piperaquine has been evaluated in patients from Cambodia with uncomplicated falciparum malaria (121). Amodiaquine combined with sulfadoxine/pyrimethamine
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had substantial antimalarial activity in spite of preexisting resistance to each component drug (122).

New Molecular Targets

The discovery of new molecular entities is at once the most exciting and the
most risky approach to countering existing drug resistance. The handful of examples presented here is far from comprehensive and is intended just to illustrate possible avenues to new drug discovery [for more complete consideration of
experimental antimalarials, see (7, 123) and the Web site for Malaria Medicines
Venture, http://www.mmv.org/pages/page main.htm]. As noted above, the malaria
parasites apicoplast has its own distinctive genome and complement of proteins,
including a type II fatty acid synthesis pathway, which is unlike the pathway in
human cells and is inhibited by triclosan (124). Blood stage malaria parasites are
homolactate fermentors, an inefficient use of glucose that increases demand for its
transport. O-3 hexose derivatives selectively inhibit glucose transport in P. falciparum, kill parasites in vitro, and suppress P. berghei infection in mice (125). The
sequential proteolysis of globin is mediated by multiple proteases, which are all
potential therapeutic targets. Plasmepsin inhibitors have antimalarial effects (126);
falcipain inhibitors prevent hemoglobin hydrolysis and cure murine malaria (127
129). Glutathione metabolism offers several essential and vulnerable targets in
the parasite (130). Fosmidomycin blocks the synthesis of isopentenyl diphosphate
and the subsequent development of isoprenoids in P. falciparum (131), and it has
antimalarial activity in vitro and in a mouse model. An open label trial in Gabon
and Thailand showed that fosmidomycin is efficacious, although its use as a single
agent is associated with high recrudescence (132).
CONCLUDING REMARKS
In recent years the severe problem of drug-resistant malaria has been featured
extensively in the scientific and lay literature, leading to increased public awareness, new and better funding opportunities for research, and a growing sense that
the situation requires thoughtful public health policies to preserve the utility of
current therapies. Spurred by powerful genetic tools and availability of the fully
sequenced genome, effective new drugs will almost certainly be discovered. Less
certain is whether these agents will be inexpensive enough for widespread use in
developing countries. Also of obvious concern is the propensity for resistance,
which atovaquone has taught can appear immediately and at high levels. It is interesting to speculate that would-be new antimalarial drugs might better have a
nonprotein target (e.g., chloroquine against the growing hemozoin crystal) or an
irrational molecular mechanism (e.g., the artemisinins whose activated free radicals may pose a nonspecific oxidative stress). Although the pathway to design
such agents prospectively is less obvious than, for example, that for an enzyme
inhibitor, they may be inherently less affected by point mutations, which are the
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preferred molecular resistance mechanism in these pathogens. In any case, the

compelling medical problem of malaria, which captured the attention of some of
the finest scientific minds of the past century and led to seminal discoveries that
benefited all of chemotherapy, remains an urgent and unmet challenge.

ACKNOWLEDGMENTS
We apologize to our many colleagues whose work was not directly cited because
of strict space limitations, and thank Tom Kulikowicz and Rahul Bakshi for their
generous help with preparing the figures. Our work has been supported by the Johns
Hopkins Malaria Research Institute (TS), the Johns Hopkins Clinician Scientist
Award (RB), and the National Institutes of Health (RR-00052).
LITERATURE CITED
1. Krogstad DJ. 2000. Plasmodium species
(Malaria). See Ref. 133, pp. 281832
2. Nchinda TC. 1998. Malaria: a reemerging disease in Africa. Emerg. Infect. Dis.
4:398403
3. Wellems TE, Miller LH. 2003. Two
worlds of malaria. N. Engl. J. Med. 349:
149698
4. Gardner MJ, Hall N, Fung E, White
O, Berriman M, et al. 2002. Genome
sequence of the human malaria parasite Plasmodium falciparum. Nature
419:498511
5. White NJ. 2004. Antimalarial drug resistance. J. Clin. Invest. 113:108492
6. Talisuna AO, Bloland P, dAlessandro
U. 2004. History, dynamics, and public
health importance of malaria parasite resistance. Clin. Microbiol. Rev. 17:235
54
7. Rosenthal PJ. 2003. Antimalarial drug
discovery: old and new approaches. J.
Exp. Biol. 206:373544
8. Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR. 2002. Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2:20918
9. Hyde JE. 2002. Mechanisms of resistance
10.
11.
12.
13.
14.
15.
16.
of Plasmodium falciparum to antimalarial

drugs. Microbes. Infect. 4:16574
Winstanley P. 2001. Modern chemotherapeutic options for malaria. Lancet Infect.
Dis. 1:24250
Olliaro P. 2001. Mode of action and
mechanisms of resistance for antimalarial drugs. Pharmacol. Ther. 89:20719
Warhurst D. 2001. New developments:
chloroquine-resistance in Plasmodium
falciparum. Drug Resist. Updat. 4:141
44
Peters W. 1987. Chemotherapy and Drug
Resistance in Malaria. London: Academic Press
World Health Organization. 1973.
Chemotherapy of malaria and resistance
to antimalarials. WHO Tech. Rep. Ser.
529:1121
Decuypere S, Elinck E, Van Overmeir
C, Talisuna AO, dAlessandro U, et al.
2003. Pathogen genotyping in polyclonal infections: application of a fluorogenic polymerase-chain-reaction assay in
malaria. J. Infect. Dis. 188:124549
Opal SM, Mayer KH, Medeiros AA.
2000. Mechanisms of bacterial antibiotic
resistance. See Ref. 133, pp. 23653
4 Dec 2004
18:51
AR
AR232-PA45-23.tex
P1: JRX


17. Morrow CS, Cowan KH. 1997. Drug resistance and its clinical circumvention.
In Cancer Medicine, ed. JF Holland, pp.
799816. Baltimore: Williams & Wilkins
18. Hastings IM, Bray PG, Ward SA. 2002.
Parasitology. A requiem for chloroquine.
Science 298:7475
19. Coatney RG. 1963. Pitfalls in a discovery:
the chronicle of chloroquine. Am. J. Trop.
Med. Hyg. 12:12129
20. Greenwood BM, Bradley AK, Greenwood AM, Byass P, Jammeh K, et al.
1987. Mortality and morbidity from
malaria among children in a rural area of
The Gambia, West Africa. Trans. R. Soc.
Trop. Med. Hyg. 81:47886
21. Trape JF. 2001. The public health impact
of chloroquine resistance in Africa. Am.
J. Trop. Med. Hyg. 64:1217
22. Trape JF, Pison G, Preziosi MP, Enel C,
du Desgrees LA, et al. 1998. Impact of
chloroquine resistance on malaria mortality. C. R. Acad. Sci. III 321:68997
23. Francis SE, Sullivan DJ Jr, Goldberg
DE. 1997. Hemoglobin metabolism in the
malaria parasite Plasmodium falciparum.
Annu. Rev. Microbiol. 51:97123
24. Thurston JP. 1951. Morphological
changes in Plasmodium berghei following proguanil, sulphadiazine and
mepacrine therapy. Trans. R. Soc. Trop.
Med. Hyg. 44:7036
25. Sullivan DJ Jr, Gluzman IY, Russell DG,
Goldberg DE. 1996. On the molecular
mechanism of chloroquines antimalarial action. Proc. Natl. Acad. Sci. USA
93:1186570
26. Macomber PB, OBrien RL, Hahn FE.
1966. Chloroquine: physiological basis of
drug resistance in Plasmodium berghei.
Science 152:137475
27. Geary TG, Jensen JB. 1983. Lack of
cross-resistance to 4-aminoquinolines in
chloroquine-resistant Plasmodium falciparum in vitro. J. Parasitol. 69:97105
28. Geary TG, Divo AA, Jensen JB. 1987. Activity of quinoline-containing antimalarials against chloroquine-sensitive and
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
579
-resistant strains of Plasmodium falciparum in vitro. Trans. R. Soc. Trop. Med.

Hyg. 81:499503
Krogstad DJ, Gluzman IY, Kyle DE,
Oduola AM, Martin SK, et al. 1987.
Efflux of chloroquine from Plasmodium
falciparum: mechanism of chloroquine
resistance. Science 238:128385
Martin SK, Oduola AM, Milhous WK.
1987. Reversal of chloroquine resistance
in Plasmodium falciparum by verapamil.
Science 235:899901
Wellems TE, Panton LJ, Gluzman IY,
do Rosario V, Gwadz RW, et al. 1990.
Chloroquine resistance not linked to mdrlike genes in a Plasmodium falciparum
cross. Nature 345:25355
Fidock DA, Nomura T, Talley AK,
Cooper RA, Dzekunov SM, et al. 2000.
Mutations in the P. falciparum digestive
vacuole transmembrane protein PfCRT
and evidence for their role in chloroquine
resistance. Mol. Cell 6:86171
Ambudkar SV, Kimchi-Sarfaty C, Sauna
ZE, Gottesman MM. 2003. P-glycoprotein: from genomics to mechanism. Oncogene 22:746885
Borst P, Elferink RO. 2002. Mammalian
ABC transporters in health and disease.
Annu. Rev. Biochem. 71:53792
Waller KL, Muhle RA, Ursos LM, Horrocks P, Verdier-Pinard D, et al. 2003.
Chloroquine resistance modulated in vitro
by expression levels of the Plasmodium
falciparum chloroquine resistance transporter. J. Biol. Chem. 278:33593601
Djimde A, Doumbo OK, Cortese JF,
Kayentao K, Doumbo S, et al. 2001.
A molecular marker for chloroquineresistant falciparum malaria. N. Engl. J.
Med. 344:25763
Wootton JC, Feng X, Ferdig MT, Cooper
RA, Mu J, et al. 2002. Genetic diversity
and chloroquine selective sweeps in Plasmodium falciparum. Nature 418:320
23
Sidhu AB, Verdier-Pinard D, Fidock
DA. 2002. Chloroquine resistance in
4 Dec 2004
18:51
580

39.
40.
41.
42.
43.
44.
45.
46.
47.
AR
AR232-PA45-23.tex
ARAV-BOGER
P1: JRX
SHAPIRO
Plasmodium falciparum malaria parasites

conferred by pfcrt mutations. Science
298:21013
Cooper RA, Ferdig MT, Su XZ, Ursos
LM, Mu J, et al. 2002. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated
with chloroquine resistance and unique
stereospecific quinine and quinidine responses in Plasmodium falciparum. Mol.
Pharmacol. 61:3542
Wilson CM, Serrano AE, Wasley A,
Bogenschutz MP, Shankar AH, Wirth
DF. 1989. Amplification of a gene related to mammalian mdr genes in drugresistant Plasmodium falciparum. Science
244:118486
Foote SJ, Thompson JK, Cowman
AF, Kemp DJ. 1989. Amplification of
the multidrug resistance gene in some
chloroquine-resistant isolates of P. falciparum. Cell 57:92130
Reed MB, Saliba KJ, Caruana SR, Kirk
K, Cowman AF. 2000. Pgh1 modulates
sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403:9069
Rieckmann KH, Davis DR, Hutton DC.
1989. Plasmodium vivax resistance to
chloroquine? Lancet 2:118384
Nomura T, Carlton JM, Baird JK, del Portillo HA, Fryauff DJ, et al. 2001. Evidence for different mechanisms of chloroquine resistance in 2 Plasmodium species
that cause human malaria. J. Infect. Dis.
183:165361
De D, Krogstad FM, Byers LD, Krogstad
DJ. 1998. Structure-activity relationships
for antiplasmodial activity among 7substituted 4-aminoquinolines. J. Med.
Chem. 41:491826
van Schalkwyk DA, Walden JC, Smith PJ.
2001. Reversal of chloroquine resistance
in Plasmodium falciparum using combinations of chemosensitizers. Antimicrob.
Adjuik M, Babiker A, Garner P, Olliaro
P, Taylor W, et al. 2004. Artesunate com-
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
binations for treatment of malaria: metaanalysis. Lancet 363:917

Kublin JG, Cortese JF, Njunju EM,
Mukadam RA, Wirima JJ, et al. 2003.
Reemergence of chloroquine-sensitive
Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J.
Infect. Dis. 187:187075
Domagk G. 1935. Ein Beitrag zur
Chemotherapie der bakteriellen Infektionen. Dtsch. Med. Wochenschr. 61:25053
Hill RA, Goodwin MH Jr. 1937. Prontosil in treatment of malaria. South. Med.
J. 30:117072
Curd FHS, Davey DG, Rose FL. 1945.
Studies on synthetic antimalarial drugs. X.
Some biguanide derivatives as new types
of antimalarial substances with both therapeutic and causal prophylactic activity.
Ann. Trop. Med. Hyg. 39:20816
Greenberg J, Boyd BL, Josephson ES.
1948. Synergistic effect of chlorguanide
and sulfadiazine against Plasmodium gallinaceum in the chick. J. Pharmacol. Exp.
Ther. 94:6064
Greenberg J. 1949. The potentiation of the
antimalarial activity of chlorguanide by paminobenzoic acid competitors. J. Pharmacol. Exp. Ther. 97:23842
Falco EA, Hitchings GH, Russell PB,
VanderWerff H. 1949. Antimalarials as
antagonists of purines and pteroylglutamic acid. Nature 164:1078
Poe M. 1976. Antibacterial synergism:
a proposal for chemotherapeutic potentiation between trimethoprim and sulfamethoxazole. Science 194:53335
Sirawaraporn W, Yuthavong Y. 1986.
Potentiating effect of pyrimethamine
and sulfadoxine against dihydrofolate
reductase from pyrimethamine-sensitive
and pyrimethamine-resistant Plasmodium
chabaudi. Antimicrob. Agents Chemother.
29:899905
Wang P, Brobey RK, Horii T, Sims PF,
Hyde JE. 1999. Utilization of exogenous
folate in the human malaria parasite Plasmodium falciparum and its critical role in
4 Dec 2004
18:51
AR
AR232-PA45-23.tex
P1: JRX
58.

59.
60.
61.
62.
63.
64.
65.
66.
67.
antifolate drug synergy. Mol. Microbiol.

32:125462
Wang P, Wang Q, Aspinall TV, Sims
PF, Hyde JE. 2004. Transfection studies
to explore essential folate metabolism
and antifolate drug synergy in the human
malaria parasite Plasmodium falciparum.
Mol. Microbiol. 51:142538
Rosenblatt DS, Fenton WA. 2001. Inherited disorders of folate and cobalamin transport and metabolism. In The
Metabolic and Molecular Bases of Inherited Disease, ed. CR Scriber, AL Beaudet,
WS Sly, D Valle, pp. 3897929. New
York: McGraw-Hill
Dieckmann A, Jung A. 1986. Mechanisms of sulfadoxine resistance in Plasmodium falciparum. Mol. Biochem. Parasitol. 19:14347
Roberts F, Roberts CW, Johnson JJ, Kyle
DE, Krell T, et al. 1998. Evidence for the
shikimate pathway in apicomplexan parasites. Nature 393:8015
Kicska GA, Ting LM, Schramm VL, Kim
K. 2003. Effect of dietary p-aminobenzoic
acid on murine Plasmodium yoelii infection. J. Infect. Dis. 188:177681
Milhous WK, Weatherly NF, Bowdre JH,
Desjardins RE. 1985. In vitro activities of
and mechanisms of resistance to antifol
antimalarial drugs. Antimicrob. Agents
Chemother. 27:52530
Krungkrai J, Webster HK, Yuthavong Y.
1989. De novo and salvage biosynthesis
of pteroylpentaglutamates in the human
malaria parasite, Plasmodium falciparum.
Mol. Biochem. Parasitol. 32:2537
Wang P, Sims PF, Hyde JE. 1997. A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable
for investigating Fansidar resistance. Parasitology 115:22330
Ferone R. 1973. The enzymic synthesis of dihydropteroate and dihydrofolate
by Plasmodium berghei. J. Protozool.
20:45964
Triglia T, Cowman AF. 1994. Primary
structure and expression of the dihy-
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
581
dropteroate synthetase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci.

USA 91:714953
Bzik DJ, Li WB, Horii T, Inselburg J.
1987. Molecular cloning and sequence
analysis of the Plasmodium falciparum
dihydrofolate reductase-thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 84:
836064
Chen GX, Zolg JW. 1987. Purification
of the bifunctional thymidylate synthasedihydrofolate reductase complex from
the human malaria parasite Plasmodium
falciparum. Mol. Pharmacol. 32:723
30
Zhang K, Rathod PK. 2002. Divergent
regulation of dihydrofolate reductase between malaria parasite and human host.
Science 296:54547
Hernandez-Diaz S, Werler MM, Walker
AM, Mitchell AA. 2000. Folic acid antagonists during pregnancy and the risk of
birth defects. N. Engl. J. Med. 343:1608
14
Bishop A, Birkett B. 1947. Acquired resistance to paludrine in Plasmodium gallinaceum. Nature 159:88485
Plowe CV. 2001. Folate antagonists and
mechanisms of resistance. See Ref. 134,
pp. 17390
Yuthavong Y. 2002. Basis for antifolate action and resistance in malaria. Microbes. Infect. 4:17582
Cortese JF, Caraballo A, Contreras CE,
Plowe CV. 2002. Origin and dissemination of Plasmodium falciparum drugresistance mutations in South America. J.
Infect. Dis. 186:9991006
Roper C, Pearce R, Bredenkamp B,
Gumede J, Drakeley C, et al. 2003. Antifolate antimalarial resistance in southeast Africa: a population-based analysis.
Lancet 361:117481
Kublin JG, Dzinjalamala FK, Kamwendo
DD, Malkin EM, Cortese JF, et al.
2002. Molecular markers for failure
of
sulfadoxine-pyrimethamine
and
chlorproguanil-dapsone treatment of
4 Dec 2004
18:51
582

78.
79.
80.
81.
82.
83.
84.
AR
AR232-PA45-23.tex
ARAV-BOGER
P1: JRX
SHAPIRO
Plasmodium falciparum malaria. J.

Infect. Dis. 185:38088
Plowe CV, Cortese JF, Djimde A,
Nwanyanwu OC, Watkins WM, et al.
1997. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic
patterns of pyrimethamine-sulfadoxine
use and resistance. J. Infect. Dis. 176:
159096
Plowe CV, Kublin JG, Dzinjalamala
FK, Kamwendo DS, Mukadam RA,
et al. 2004. Sustained clinical efficacy of
sulfadoxine-pyrimethamine for uncomplicated falciparum malaria in Malawi after 10 years as first line treatment: five year
prospective study. BMJ 328:545
Peterson DS, Walliker D, Wellems TE.
1988. Evidence that a point mutation in
dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine
in falciparum malaria. Proc. Natl. Acad.
Sci. USA 85:911418
Wang P, Read M, Sims PF, Hyde JE.
1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional
factor associated with folate utilization.
Mol. Microbiol. 23:97986
Triglia T, Wang P, Sims PF, Hyde JE,
Cowman AF. 1998. Allelic exchange at
the endogenous genomic locus in Plasmodium falciparum proves the role of
dihydropteroate synthase in sulfadoxineresistant malaria. EMBO J. 17:380715
Sirawaraporn W, Sathitkul T, Sirawaraporn R, Yuthavong Y, Santi DV. 1997.
Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase.
29
Triglia T, Menting JG, Wilson C,
Cowman AF. 1997. Mutations in dihydropteroate synthase are responsible
for sulfone and sulfonamide resistance
in Plasmodium falciparum. Proc. Natl.
85. Yuvaniyama J, Chitnumsub P, Kamchonwongpaisan S, Vanichtanankul J,

Sirawaraporn W, et al. 2003. Insights into
antifolate resistance from malarial
DHFR-TS structures. Nat. Struct. Biol.
10:35765
86. Kamchonwongpaisan S, Quarrell R,
Charoensetakul N, Ponsinet R, Vilaivan T,
et al. 2004. Inhibitors of multiple mutants
of Plasmodium falciparum dihydrofolate
reductase and their antimalarial activities.
J. Med. Chem. 47:67380
87. Rastelli G, Pacchioni S, Sirawaraporn W,
Sirawaraporn R, Parenti MD, et al. 2003.
Docking and database screening reveal
new classes of Plasmodium falciparum dihydrofolate reductase inhibitors. J. Med.
Chem. 46:283445
88. Jiang L, Lee PC, White J, Rathod PK.
2000. Potent and selective activity of a
combination of thymidine and 1843U89,
a folate-based thymidylate synthase inhibitor, against Plasmodium falciparum.
50
89. Amukoye E, Winstanley PA, Watkins
WM, Snow RW, Hatcher J, et al.
1997. Chlorproguanil-dapsone: effective
treatment for uncomplicated falciparum
malaria. Antimicrob. Agents Chemother.
41:226164
90. Sulo J, Chimpeni P, Hatcher J, Kublin
JG, Plowe CV, et al. 2002. Chlorproguanil-dapsone versus sulfadoxinepyrimethamine for sequential episodes
of uncomplicated falciparum malaria in
Kenya and Malawi: a randomised clinical
trial. Lancet 360:113643
91. Dorsey G, Njama D, Kamya MR, Cattamanchi A, Kyabayinze D, et al. 2002.
Sulfadoxine/pyrimethamine alone or with
amodiaquine or artesunate for treatment
of uncomplicated malaria: a longitudinal
randomised trial. Lancet 360:203138
92. Fieser LF, Berlinger E, Bondhus FJ,
Chang FC, Dauben WG, et al. 1948.
Naphthoquinone antimalarials I. General
survey. J. Am. Chem. Soc. 70:315155
4 Dec 2004
18:51
AR
AR232-PA45-23.tex
P1: JRX


93. Hudson AT, Dickins M, Ginger CD, Gutteridge WE, Holdich T, et al. 1991.
566C80: a potent broad spectrum antiinfective agent with activity against
malaria and opportunistic infections in
AIDS patients. Drugs Exp. Clin. Res.
17:42735
94. Looareesuwan S, Viravan C, Webster HK,
Kyle DE, Hutchinson DB, et al. 1996.
Clinical studies of atovaquone, alone or
in combination with other antimalarial
drugs, for treatment of acute uncomplicated malaria in Thailand. Am. J. Trop.
Med. Hyg. 54:6266
95. Fry M, Pudney M. 1992. Site of
action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4 -chlorophenyl)
cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem. Pharmacol. 43:
154553
96. Srivastava IK, Rottenberg H, Vaidya AB.
1997. Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial
membrane potential in a malarial parasite.
J. Biol. Chem. 272:396166
97. Srivastava IK, Morrisey JM, Darrouzet E,
Daldal F, Vaidya AB. 1999. Resistance
mutations reveal the atovaquone-binding
domain of cytochrome b in malaria parasites. Mol. Microbiol. 33:70411
98. Korsinczky M, Chen N, Kotecka B,
Saul A, Rieckmann K, et al. 2000. Mutations in Plasmodium falciparum cytochrome b that are associated with
atovaquone resistance are located at a
putative drug-binding site. Antimicrob.
99. Canfield CJ, Pudney M, Gutteridge WE.
1995. Interactions of atovaquone with
other antimalarial drugs against Plasmodium falciparum in vitro. Exp. Parasitol.
80:37381
100. Srivastava IK, Vaidya AB. 1999. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob. Agents Chemother. 43:133439
101. Arguin P, Barber A, Kozarsky P, Mali S,
Newman R, et al. 2004. Malaria. http://
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
583
www.cdc.gov/travel/diseases/malaria/ind
ex.htm
Drugs for parasitic infections. 2004. Med.
Lett. Drugs Ther. August:112
Fivelman QL, Butcher GA, Adagu IS,
Warhurst DC, Pasvol G. 2002. Malarone
treatment failure and in vitro confirmation
of resistance of Plasmodium falciparum
isolate from Lagos, Nigeria. Malar. J. 1:1
Schwartz E, Bujanover S, Kain KC. 2003.
Genetic confirmation of atovaquoneproguanil-resistant Plasmodium falciparum malaria acquired by a nonimmune
traveler to East Africa. Clin. Infect. Dis.
37:45051
Lage H. 2003. ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int. J. Antimicrob. Agents 22:18899
Syafruddin D, Asih PB, Aggarwal SL,
Shankar AH. 2003. Frequency distribution of antimalarial drug-resistant alleles among isolates of Plasmodium falciparum in Purworejo district, Central Java
Province, Indonesia. Am. J. Trop. Med.
Hyg. 69:61420
Rathod PK, McErlean T, Lee PC. 1997.
Variations in frequencies of drug resistance in Plasmodium falciparum. Proc.
Meshnick SR. 2001. Artemisinins and its
derivatives. See Ref. 134, pp. 191202
Borstnik K, Paik IH, Shapiro TA, Posner
GH. 2002. Antimalarial chemotherapeutic peroxides: artemisinin, yingzhaosu A
and related compounds. Int. J. Parasitol.
32:166167
Eckstein-Ludwig U, Webb RJ, Van
Goethem ID, East JM, Lee AG, et al. 2003.
Artemisinins target the SERCA of Plasmodium falciparum. Nature 424:95761
Nosten F, Luxemburger C, ter Kuile FO,
Woodrow C, Eh JP, et al. 1994. Treatment of multidrug-resistant Plasmodium
falciparum malaria with 3-day artesunatemefloquine combination. J. Infect. Dis.
170:97177
Geary TG, Jensen JB. 1983. Effects of
4 Dec 2004
18:51
584
113.

114.
115.
116.
117.
118.
119.
120.
121.
AR
AR232-PA45-23.tex
ARAV-BOGER
P1: JRX
SHAPIRO
antibiotics on Plasmodium falciparum in

vitro. Am. J. Trop. Med. Hyg. 32:221
25
Clyde DF, Gilman RH, McCarthy VC.
1975. Antimalarial effects of clindamycin
in man. Am. J. Trop. Med. Hyg. 24:369
70
Mahmoudi N, Ciceron L, Franetich JF,
Farhati K, Silvie O, et al. 2003. In vitro
activities of 25 quinolones and fluoroquinolones against liver and blood stage
Plasmodium spp. Antimicrob. Agents
McClean KL, Hitchman D, Shafran SD.
1992. Norfloxacin is inferior to chloroquine for falciparum malaria in northwestern Zambia: a comparative clinical trial. J.
Infect. Dis. 165:9047
Pfaller MA, Krogstad DJ. 1981. Imidazole and polyene activity against
chloroquine-resistant Plasmodium falciparum. J. Infect. Dis. 144:37275
Chong CR, Sullivan DJ Jr. 2003. Inhibition of heme crystal growth by antimalarials and other compounds: implications
for drug discovery. Biochem. Pharmacol.
66:220112
Huy NT, Kamei K, Yamamoto T, Kondo
Y, Kanaori K, et al. 2002. Clotrimazole binds to heme and enhances
heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole. J.
Biol. Chem. 277:415258
Olliaro PL, Taylor WR. 2003. Antimalarial compounds: from bench to bedside. J.
Exp. Biol. 206:375359
Lefevre G, Looareesuwan S, Treeprasertsuk S, Krudsood S, Silachamroon U, et al.
2001. A clinical and pharmacokinetic trial
of six doses of artemether-lumefantrine
for multidrug-resistant Plasmodium falciparum malaria in Thailand. Am. J. Trop.
Med. Hyg. 64:24756
Denis MB, Davis TM, Hewitt S, Incardona S, Nimol K, et al. 2002. Efficacy and safety of dihydroartemisininpiperaquine (Artekin) in Cambodian
children and adults with uncomplicated
122.
123.
124.
125.
126.
127.
128.
129.
falciparum malaria. Clin. Infect. Dis.

35:146976
Schellenberg D, Kahigwa E, Drakeley
C, Malende A, Wigayi J, et al. 2002.
The safety and efficacy of sulfadoxinepyrimethamine, amodiaquine, and their
combination in the treatment of uncomplicated Plasmodium falciparum malaria.
Am. J. Trop. Med. Hyg. 67:1723
Guerin PJ, Olliaro P, Nosten F, Druilhe
P, Laxminarayan R, et al. 2002. Malaria:
current status of control, diagnosis, treatment, and a proposed agenda for research and development. Lancet Infect.
Dis. 2:56473
Surolia N, Surolia A. 2001. Triclosan offers protection against blood stages of
malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nat. Med.
7:16773
Joet T, Eckstein-Ludwig U, Morin C, Krishna S. 2003. Validation of the hexose
transporter of Plasmodium falciparum as
a novel drug target. Proc. Natl. Acad. Sci.
USA 100:747679
Moon RP, Tyas L, Certa U, Rupp K, Bur
D, et al. 1997. Expression and characterisation of plasmepsin I from Plasmodium
falciparum. Eur. J. Biochem. 244:552
60
Francis SE, Gluzman IY, Oksman A,
Banerjee D, Goldberg DE. 1996. Characterization of native falcipain, an enzyme involved in Plasmodium falciparum
hemoglobin degradation. Mol. Biochem.
Parasitol. 83:189200
Greenbaum DC, Baruch A, Grainger M,
Bozdech Z, Medzihradszky KF, et al.
2002. A role for the protease falcipain 1
in host cell invasion by the human malaria
parasite. Science 298:20026
Shenai BR, Lee BJ, Alvarez-Hernandez
A, Chong PY, Emal CD, et al. 2003.
Structure-activity relationships for inhibition of cysteine protease activity and
development of Plasmodium falciparum
by peptidyl vinyl sulfones. Antimicrob.
4 Dec 2004
18:51
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130. Becker K, Tilley L, Vennerstrom JL,
Roberts D, Rogerson S, et al. 2004.
Oxidative stress in malaria parasiteinfected erythrocytes: host-parasite interactions. Int. J. Parasitol. 34:16389
131. Jomaa H, Wiesner J, Sanderbrand S,
Altincicek B, Weidemeyer C, et al. 1999.
Inhibitors of the nonmevalonate pathway
of isoprenoid biosynthesis as antimalarial
drugs. Science 285:157376
132. Lell B, Ruangweerayut R, Wiesner J,
585
Missinou MA, Schindler A, et al. 2003.

Fosmidomycin, a novel chemotherapeutic agent for malaria. Antimicrob. Agents
Chemother. 47:73538
133. Mandell GL, Douglas RG, Bennett JE,
Dolin R, eds. 2000. Mandell, Douglas, and Bennetts Principles and Practice of Infectious Diseases. Philadelphia:
Churchill Livingstone
134. Rosenthal PJ, ed. 2001. Antimalarial Chemotherapy. Totowa, NJ: Humana Press
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Figure 2 Simplified scheme of therapeutically important variations in folate metabolism

in different organisms. Tetrahydrofolate cofactors are essential for biosynthetic reactions in
P. falciparum (green), bacteria (red), and mammalian cells (blue), and all three systems utilize a dihydrofolate reductase activity (reaction 2). Various antifolates inhibit the reductase
in Plasmodium (pyrimethamine, cycloguanil), bacteria (trimethoprim), or all three systems
(methotrexate). Dihydropteroate synthase (reaction 1) in parasites and bacteria has no
counterpart in human cells and is inhibited by sulfonamides. In malaria parasites, paraaminobenzoic acid from either salvage or the shikimate pathway (a multistep synthesis
from erythrose 4-phosphate, E4P, and phosphoenolpyruvate, PEP) can significantly reduce
the effectiveness of competitive sulfonamide inhibitors. In some P. falciparum strains, the
ability to import preformed dihydrofolate counters the efficacy of both sulfonamides and
antifolates. Large circle, cell membrane.
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Copyright
SIGNALING NETWORKS IN LIVING CELLS

Michael A. White and Richard G.W. Anderson

Department of Cell Biology, University of Texas, Southwestern Medical Center,
Dallas, Texas 75390-9039; email: Richard.Anderson@utsouthwestern.edu
Key Words scaffolding proteins, caveolae, compartmentalization, signal

transduction
Abstract Recent advances in cell signaling research suggest that multiple sets
of signal transducing molecules are preorganized and sequestered in distinct compartments within the cell. These compartments are assembled and maintained by specific
cellular machinery. The molecular ecology within a compartment creates an environment that favors the efficient and accurate integration of signaling information arriving
from humoral, mechanical, and nutritional sources. The functional organization of
these compartments suggests they are the location of signaling networks that naturally
organize into hierarchical interconnected sets of molecules through their participation in different classes of interacting units. An important goal is to determine the
contribution of the compartment to the function of these networks in living cells.
INTRODUCTION
Enormous progress has been made during the past decade identifying sets of molecular interactions that transmit information between different parts of the cell. The
increasing number of databases containing lists of interacting biomolecules has
sparked the development of the burgeoning field of network biology and with it
the realization that, to a first approximation, biological networks can be described
mathematically by scale-free power functions (1). These functions predict the hierarchical interconnectedness of molecules through their participation in different
classes of interacting units such as nodes, hubs, modules, and motifs. Power functions mathematically describe the organization of thermodynamically far from
equilibrium systems like living organisms. The network structure derived from
this type of abstract analysis is very useful for understanding the patterns created
by interconnecting the molecular constituents of different signaling pathways. Understanding exactly how these molecular interactions become determinants of cell
structure and function, however, remains a significant challenge. The molecular
interactions underlying biological networks take place in living cells, and network
analysis inherently is unable to consider the contribution of different intracellular
environments to signal transduction. Therefore, an important next step is to develop a high resolution map of signaling networks in living cells and the location
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of interacting signaling units (i.e., hubs, motifs, modules) relative to cell structures
like the plasma membrane, mitochondria, the nucleus, etc.
Cell biologists use many techniques to map the distribution of molecules in cells.
Cell fractionation as well as light and EM immunocytochemistry are the principal
methods that have been used to demonstrate that cell signaling molecules tend to
be concentrated in different cellular compartments. The compartmentalization of
interacting sets of signaling molecules has several implications for understanding
signaling networks in situ. First, these compartments often can be isolated in a
way that preserves the functionality of the resident signaling units. They contain
dynamic information about the behavior of molecules that make up specific signaling networks, and embedded in the pattern of molecular interactions are the codes
that govern cell behavior. Compartments also contain the molecular signature of
unknown signaling pathways that cannot be detected using ex vivo techniques.
For example, current estimates indicate that the human genome contains vastly
more signaling molecules than have been classified and assigned to pathways.
Determining the compartment where these molecules reside is a valuable first
step in identifying, mapping, and characterizing their function. Another important consideration is that similar sets of signaling units can be found in different
compartments, although the same class of compartments at other times contains
different sets of signaling units. Nothing is known about the rules that control the
compartmentalization of signaling units, nor how the spatial distribution of these
units and the environment created by the host compartment influences signal transduction. Deciphering the rules of compartmentalization can only be achieved by
studying the function of signaling units when they are in different host compartments. Each type of compartment is spatially restricted, so compartmentalization
also contributes to the spatial organization of signal transduction in the cell. A
final consideration is that the mechanism of action of a signaling molecule in a
compartment cannot be predicted simply by knowing all its interacting partners.
Compartmentalized sets of signaling molecules display emergent behavior that
can only be understood by studying the entire ensemble of molecules interacting
in their native environment. The fidelity of signal transduction depends as much
on the molecular ecology of the compartment as it does on the interaction between
individual signaling molecules.
ORGANIZATION OF A SIGNALING UNIT

Efforts to decipher the molecular nature of cell autonomous regulatory programs
often, by necessity, progress by collecting the protein components that appear
to play obligatory roles in the regulatory process and by determining the biochemical relationships among them. This approach has very successfully identified linear relationships between the interacting components of multiple signaling
pathways. Iteration of this process often reveals, however, that branch-points exist
in these pathways. These branch-points connect to other independently established
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pathways, creating a situation in which diverse inputs (S1, S2, S312, Figure 1A,
see color insert) can transmit through a limited number of core transducers (E1,
E2, E3, Figure 1A) to multiple outputs (R1, R2, R3, Figure 1A). The apparent
complexity of this organization raises questions about how these signaling units
are able to establish high fidelity coupling between diverse stimuli and discrete
physiological effects in the living cell. Moreover, the identity of interacting signaling units in a network does not reveal how these units are spatially and temporally
organized in the cell. The ERK1/2 MAP kinase cascade is a well-studied signaling
pathway that illustrates the dilemma.
The ERK1/2 protein kinases are a core signaling element implicated in the
regulation of assorted biological processes, ranging from cellular proliferation and
tumorigenesis to differentiation and cell specialization (2, 3). ERK1/2 apparently
function as the terminal kinase in a three-kinase cascade that includes the Mek MAP
kinase kinase (MAP2K) and the Raf MAP kinase kinase kinase (MAP3K). This
linked set of protein kinases is a signal propagation cassette (E1, E2, E3, Figure 1A)
typical of many signaling kinase families. ERK1/2 activation can be stimulated by
growth factor receptors, heterotrimeric G-protein coupled receptors, and integrins.
Many ERK1/2 effector proteins (R1, R2, R3, Figure 1A) have been identified,
including nuclear transcription factors like c-Fos and Elk-1 (4, 5), cytoplasmic
protein kinases like p90RSK (6, 7) and myosin light chain kinase (8), and lipases
like phospholipase A2 (9). Moreover, recent proteomic studies identified 20 new
targets for this kinase that are involved in such diverse activities as nuclear import,
nucleotide excision repair, membrane traffic, and cytoskeleton assembly (10). The
pleiotropic consequences of ERK1/2 activation imply that activated ERK1/2 is
directly connected to many different targets.
The ERK1/2 protein kinase cascade is functionally coupled to stimuli in part
by Ras family small GTPases (3). Ras proteins are tethered to membranes rich
in sensory receptors through carboxy-terminal lipidation (11). The bulk of the
kinase elements in the ERK1/2 kinase cascade, by contrast, are in the cytosol of
unstimulated cells. In response to stimulus, Ras proteins are activated through
GDP/GTP exchange as a consequence of receptor-driven association with guanyl
nucleotide exchange factors (GEFs). Ras-GTP adopts a conformation that favors
the direct interaction with downstream effector proteins, including Raf kinases
(11). Recent structural and biochemical analysis of Ras GEFs (12), together with
single-molecule imaging of activated Ras (13), suggests that these enzymes function processively by generating interactive surfaces in the activated state; these
surfaces form signaling platforms for combinatorial sets of protein-protein interactions. Normally, activation of Ras recruits Raf-1 to the plasma membrane, but Raf-1
artificially targeted to membranes causes activation of ERK1/2 independently of
Ras. Therefore, the current paradigm for Raf-Mek-Erk pathway activation suggests
that Ras-GTP acts as molecular flypaper that snares Raf kinases at the plasma
membrane where they are subsequently activated by other membrane-associated
components that have not yet been defined [reviewed in Kolch (14)]. Raf in turn
mediates activation of MEK and ERK through a linear cascade of kinase/substrate
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interactions (Figure 1A). Although this model partially explains how various
signals are linked to ERK1/2 activation, it does not clarify how activated ERK1/2
is tuned to the diverse signaling targets it controls.
A growing number of observations suggest that scaffolding proteins (M1, M2,
M3, Figure 1B) can selectively couple ERK1/2 activation to distinct regulatory
programs. Genetic screens for modifiers of the Ras-Raf-Mek-ERK cascade, together with protein/protein interaction studies, have identified proteins like KSR,
CNK, MP-1, and Sur-8 that possess no obvious intrinsic enzymatic activities but
physically interact with multiple core components of the ERK1/2 cascade (15).
These scaffolds appear to be obligate components of ERK1/2 signaling modules
(1618), are required for ERK1/2 activation in cells (1921), and contribute to
the functional coupling of the ERK1/2 cascade to selective stimuli (20, 22). For
example, CNK can interact directly with Raf kinases (17), is required for Raf activation in response to insulin in insect cells (21), and functions at least in part to
partition Raf into membrane compartments (21). In mammalian cells, the CNK
family member CNK2 may help neuronal precursor cells distinguish between neurotrophic and proliferative signals. For example, CNK2 is required for activation
of ERK1/2 by TrkA receptor signaling but not EGF receptor signaling (20). The
ERK1/2 scaffold Sur-8, by contrast, appears to be more important for EGF receptor signaling (22). These observations suggest that scaffold proteins mediate the
assembly of signaling modules that are selectively coupled to discrete receptor
inputs (Figure 1B).
An additional mechanism for establishing specific input-output connections for
these signaling modules may be to spatially segregate each module into a different
cell compartment. Thus, the higher-ordered molecular organization generated by
scaffold proteins may also function to target the kinases (E1, E2, E3) to a specific
location in the cell (Figure 1C). This hypothesis would require address information
on the scaffolding protein, or on some component of the module, that would target
it to a compartment, thereby creating spatially restricted signaling activity. There
is considerable evidence that the Raf-Mek-Erk1/2 modules can signal from multiple cellular compartments (Figure 1C), including the late endosomes (M1) (23),
caveolae (M2) (24), and the Golgi apparatus (M3) (25). Ras family GTPases may
control, in part, the localization of each module. These GTPases carry autonomous
address information at the carboxy-terminus specified by a pattern of methylation,
prenylation, and palmitoylation that controls the targeting of the protein to a distinct membrane domain (11). The scaffold associated with the kinase cascade may
also control compartmentalization. CNK contains a pleckstrin homology domain
that mediates association with membrane phosphoinositides, which may mediate
the association of insect Raf with membrane fractions (17, 26). KSR carries a
cysteine-rich motif that can mediate membrane association, perhaps through interactions with phosphatidylserine (27). Finally, MP-1, a potential scaffold for
MEK1 and MEK2, is required for the localization of ERK1/2 to late endosomes
via an interaction with the resident endosomal protein p14 (23).
From this brief analysis of signaling through the ERK1/2 kinase cascade, we
conclude that compartmentalization is a fundamental component of signaling
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network design. In these compartments, signaling is organized and local environmental factors exert control. An important strategy for understanding signal
transduction in cells, therefore, will be to devise methods to probe the functionality of signaling networks in the compartment where they normally reside.

PROFILE OF A SIGNALING COMPARTMENT

Many different compartments in the cell collect signaling units of one type or
another. One compartment that has received a lot of attention in the signaling field
is the plasmalemmal caveolae (28). Originally studied as regions of plasma membrane specialized for internalizing molecules in endothelial cells (29), caveolae are
enriched in a variety of signaling molecules that are functionally linked to specific
signaling cascades (30). We will use caveolae to illustrate how cellular compartments that contain signaling networks can be used to probe the functionality of
these networks as they exist in the cell.
Caveolae as a Compartment
Cell compartments have a distinctive morphology and dynamics that are important for understanding the functionality of the signaling units they contain. Like
all compartments, caveolae are constructed and maintained by specialized cellular
machinery and exhibit characteristic behaviors that define their lifetime functions.
They typically are recognized as flask-shaped membrane invaginations (31) decorated with a coat protein called caveolin-1 (32) and are best known as endocytic
organelles that internalize specific classes of molecules. There appear to be two
distinctive modes of internalization (Figure 2, see color insert). Some caveolae
(Type 1) invaginate much the same as clathrin-coated pits do and pinch off from
the membrane using dynamin to complete the fission step (33, 34). These caveolae
are able to travel to the interior of the cell. Other caveolae (Type 2) become deeply
invaginated to the point where they are functionally sealed off from the extracellular space but remain associated with the plasma membrane. These caveolae open
and close without ever leaving the vicinity of the cell surface. Type 1 and Type 2
caveolae can be distinguished by their ligand internalization patterns. For example, uptake of folate by the GPI-anchored folate receptor involves caveolae that
open and close in a 1 hr cycle, without ever leaving the vicinity of the plasma
membrane, by a process called potocytosis (35). Internalization of SV40 virus,
by contrast, depends on caveolae that pinch off from the plasma membrane and
travel to the cell interior (36). Inhibiting either PKC (36, 37) or tyrosine kinase
activity (36, 38) blocks both types of internalization, although uptake by coated
pits is unaffected.
The vesicles produced by caveolae (39) during endocytosis (cavicles) are impossible to identify by EM unless they are loaded with recognizable cargo. The
introduction of caveolin-GFP (green fluorescent protein) has made it possible for
the first time to study caveolae membrane traffic in detail. Unexpectedly, three
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different kinds of traffic can be detected in tissue culture cells. The most common
pattern (up to 75%) is a sessile behavior where the caveolin-GFP positive membrane appears to be firmly anchored at the cell surface (40). Sessile caveolin-GFP is
also concentrated at the cleavage furrow of dividing cells (41). Relatively immotile
caveolin-GFP is the expected behavior of a Type 2 caveola (Figure 2). A second
behavior (Figure 2) is a rapid bidirectional, microtubule-dependent movement of
caveolin-GFP positive vesicles (cavicles) between the center of the cell and the
cell surface (39). This movement most likely corresponds to Type 1 caveolae that
have budded from the membrane. In the case of CHO cells, cavicles appear to be
traveling to the recycling endosome (42), although it is not clear if they fuse with
this compartment. By contrast, cavicles carrying SV40 virus travel to a special
endocytic compartment called the caveosome (36). The third type of movement
detected with caveolin-GFP is the projection and retraction of fine tubular elements that can extend from the plasma membrane to the center of the cell (Figure
2). Recently these tubules have been captured in EM images of cells internalizing
protein A-gold bound to prions (43). Because prions are also concentrated in flaskshaped caveolae (44), the tubular elements, designated Type 3 caveolae (Figure 2),
may be derived from either Type 1 or 2 caveolae. To the extent that caveolin-GFP
marks caveolae and cavicles, there appears to be a high degree of plasticity to the
movement of caveolae-derived membranes. There are even instances in which entire sheets of caveolae membrane appear to internalize en masse to form internal,
endosome-like structures (39), which may be how certain bacterial pathogens are
internalized (45).
Isolating the Compartment

A principal tool for studying network organization in cellular compartments is
cell fractionation. In the case of caveolae, it is relatively easy to isolate them
from tissue culture cells. There are four methods in general use today. The first,
and by far the most widely used, takes advantage of the detergent insolubility
of caveolae membranes in combination with their light buoyant density on sucrose gradients (46). Triton X-100 insoluble, light membrane fractions can be
prepared either from isolated plasma membranes or from the whole cell. The
second method is an adaptation of the first in which the Triton X-100 is replaced with 500 mM sodium carbonate, pH 11 (47). Two other methods use
neither detergents nor carbonate. One depends on sonication to break isolated
plasma membrane into small pieces that are separated on the basis of their buoyant density (48). The other uses cationized silica to purify caveolae from isolated
plasma membranes by homogenization, density gradient centrifugation, and, in
some cases, immunoadsorption (49). The four methods do not yield exactly the
same fraction of membranes, which can be an important issue when studying signal
transduction.
To preserve the functionality of signaling units in their naturally organized
state, the isolation procedure has to produce a live compartment. Therefore,
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isolation needs to be rapid enough to retain the molecular composition of the

compartment at the time a signaling pathway may be activated. Neither the Triton
X-100 insolubility (50, 51) nor the pH 11, sodium carbonate method for isolating
caveolae meet this requirement, because each is known to extract molecules that are
native to caveolae. For example, Triton X-100 removes native prenylated proteins
(51), and carbonate removes GPI anchored proteins (47). Triton X-100 has the
added liability that it inactivates signaling molecules concentrated in caveolae (50).
No isolation procedure yields a pure compartment. Isolated caveolae fractions,
however, can yield real time in vivo and in vitro information about the dynamics
of signaling networks that congregate in this compartment (see below).
Compartmentalization of Signaling Molecules

The first step in studying signaling compartments is to identify the resident signaling units. The evidence that caveolae are enriched in signaling units comes from
three major sources. The first is cell fractionation. As soon as it was possible to obtain partially purified fractions of caveolae, many investigators found that signaling
proteins such as receptor and nonreceptor tyrosine kinases, PKC, heterotrimeric
G proteins, G-protein coupled receptors, eNOS, etc. were highly enriched relative
to the plasma membrane. These early studies also established that signaling lipids
like ceramide (52) and GM1 ganglioside (53) were enriched in caveolae. There
are now hundreds of reports in which cell fractionation has been used to document
that caveolae are enriched in a variety of different signaling molecules. Another
important source of information has been the identification of signaling molecules
that interact with caveolin-1. Using a combination of two-hybrid screen, immunoprecipitation, and various in vitro interaction techniques, more than 30 different
signaling proteins and lipids have been identified that interact with caveolin-1 (54).
Even though in many cases the exact function of these interactions remains to be
established, an interaction with caveolin-1 is a good indicator that the molecule
was in caveolae at the time of the experiment. Another source of information is
light and electron microscopy. Immunofluorescence and immunogold probes have
been very useful methods for showing that signaling molecules like Rho (55), Rac
(56), H-Ras (57), PDGF receptor (24), ERK1/2 (24), PKC (37), the PKC substrate SDR (58), and eNOS (59), to mention just a few, are concentrated in caveolae
relative to other regions of membrane. In addition, histochemical methods have
shown that second messengers like cAMP (60) and calcium (61) are concentrated
in caveolae, indicating that the molecular interactions involved in regulating these
signaling intermediates are functionally organized in this domain.
Functional Signaling Units in a Compartment

The molecular composition and known endocytic functions of caveolae originally
suggested four types of signaling activities that might originate from this compartment: a) activation of tyrosine kinases, b) transduction of mechanical signals, c)
regulation through second messengers, and d) the formation of chemical synapses
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between neuronal and non-neuronal cells (62). Here we focus the discussion on
three of these activities.
Receptor tyrosine kinases such as the PDGF receptor (24), the EGF receptor
(63), and insulin receptor (64) have been localized to caveolae using cell fractionation, immunocytochemistry, or caveolin-1 interaction. Investigators have used
several different experimental protocols to show that these tyrosine kinases are
linked to signaling units in caveolae. One successful approach demonstrates that
these receptors are coupled to other signaling molecules in caveolae in vivo. For
example, binding of PDGF to PDGFR in caveolae stimulates the phosphorylation of multiple caveolar substrates (65) and silences EGFR phosphorylation in
response to EGF (66). By contrast, EGF causes the recruitment of Raf-1 kinase to
caveolae where it is activated (67), and it stimulates the local generation of inositol trisphosphate from a pool of caveolar phosphatidylinositol 4,5-bisphosphate
(PtdIns 4,5-P2 ) (68). The general protocol for these experiments is to expose the
cells to the growth factor, prepare caveolae fractions, and assay for changes that
occur in this membrane fraction but not in noncaveolae fractions.
An extension of this method is to assay for the presence of functional signaling
units in isolated caveolae. The first test of this technique found that PDGFR was
functionally linked to the activation of the MAP kinase ERK1/2 in isolated caveolae (24). The interaction of as many as 11 different molecules (PDGF, PDGFR,
SOS, Ras, Raf-1, Grb2, SHC, 143-3, MEK-1, and ERK1/2) can be involved in
activating ERK, so all the members of this signaling unit must be preorganized in
the caveolae membrane, because nothing else was added to the preparation except
PDGF. Indeed, immunoblotting has documented that many of these molecules are
enriched in caveolae fractions (65).
Finding functional signaling units in caveolae fractions indicates that the isolated compartment can retain the cellular complexity necessary to study the natural
switching and branching that occurs between signaling units in the living cell. Recent studies on activation of eNOS support this reasoning (69). eNOS is targeted
to caveolae by an N-terminal acylation motif (59) and in this location can be activated by a number of different humoral and mechanical stimuli including estradiol,
bradykinin, VEGF, HDL, isometric vessel contraction, and shear stress. The linkage between the stimulus and the activation of eNOS depends on the interaction
of many cofactors and connectors, including nonreceptor tyrosine kinases, calcium, heterotrimeric G proteins, PI3 kinase, Akt kinase, ERK1/2, PKC, PKA,
calmodulin, and HSP90. Many of these molecules and ions have been localized to
caveolae. More important, the connectivity between these molecules is preserved
in isolated caveolae. Incubation of isolated endothelial cell caveolae in the presence
of estradiol (70), bradykinin (70), acetylcholine (70), or HDL (71) all stimulate
eNOS enzymatic activity (Figure 3A), although these ligands have no effect on
isolated noncaveolae membrane (Figure 3B). Thus, the natural organization of this
signaling pathway is preserved so well that caveolae eNOS can be activated by
four different ligands, each binding a receptor that is wired to eNOS through a
distinct set of connecting molecules (72).
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Figure 3
The eNOS signaling unit is located in caveolae. L-[3 H]-arginine conversion
to L-[3 H]-citrulline was measured in caveolae (A), and noncaveolae (B) membrane
fractions isolated from endothelial cells by the method of Smart et al. (48) and incubated
in the absence (B) or presence of 108 M estradiol (E2 ), 106 M acetylcholine (Ach),
or 106 M bradykinin (BK). Values are mean +/ SEM (n = 46), p < 0.05 relative
to basal. [Modified from (70).]
Probing the functionality of signaling units in isolated compartments will uncover unexpected molecular connections that will need to be verified in the living
cell. A major technical advance that makes this possible is the development of designer fluorescence resonance energy transfer (FRET) probes capable of detecting
specific signaling pathways in live cells (73). The typical probe is a chimeric protein consisting of a donor and an acceptor GFP, which have matched overlapping
excitation and emission spectra, connected together by a sensor that is designed to
bind a specific ionic or molecular intermediate in a signaling pathway (74). When
the sensor binds the molecule or ion of interest it undergoes a conformational
change that adjusts the distance between the two GFPs (e.g., cyan fluorescent
protein and yellow fluorescent proteins). When the two GFPs are close together,
intermolecular FRET occurs. Therefore, the emission ratio before and after cell
stimulation is a relative measure of how much of the signaling molecule or ion is
in the vicinity of the probe. If the probe is targeted to a cell compartment, then the
probe will record signal transduction at that location.
The calcium sensor yellow cameleon is an example of a FRET probe that has
been successfully used to monitor signal transduction from caveolae in living cells
(75). There is considerable evidence that caveolae contain the molecular machinery
for sensing [Ca2+ ] (76, 77). Yellow cameleon was used to monitor the dynamics
of [Ca2+ ] in endothelial cells. To do this, the cameleon was targeted either to the
cytoplasm, the plasma membrane, or caveolae. The internal ER Ca2+ stores were
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then depleted and the relative [Ca2+ ] detected by each cameleon from the respective
sites was recorded as the concentration of extracellular Ca2+ was increased. The
comparative response of the probe to being in the three different locations (i.e.,
compartments) indicates that caveolae are preferred sites of Ca2+ entry and that
the entering Ca2+ is linked to the activation of eNOS. FRET-based probes promise
to be extremely useful for mapping signaling networks in live cells.
Spatial and Temporal Organization of Signal Transduction

Compartments naturally restrict their special functions to the region of the cell
where they are located. Therefore, compartmentalization spatially organizes signal
units in the cell. Caveolae again provide a dramatic illustration of this point. In
addition to containing the molecular machinery that controls Ca2+ entry, caveolae
also contain the signaling molecules that regulate Ca2+ release from the ER (78).
Ca2+ is released from the ER when endothelial cells are incubated in the presence
of ATP (Figure 4, see color insert), and Ca2+ sensitive dyes show that sites of
release colocalize with a subpopulation of caveolae on the cell surface (arrows,
Figure 4A.1 and 4A.4). Because the ER is uniformly distributed beneath the plasma
membrane, the other caveolae either do not contain the same sets of signaling units
or the units they hold are inactive. Apparently not all the caveolae in the cell are the
same, which implies that signal transduction from caveolae is spatially restricted
both by the physical location of the caveolae and whether the signaling units are
active. Caveolae will relocate to the trailing edge of migrating cells (79, 80). These
caveolae contain active signaling machinery and ATP now stimulates Ca2+ release
exclusively from ER at the trailing edge of the cell (arrow, Figure 4B.1 and 4B.4).
Compartmentalization, therefore, is an important mechanism that cells use to carry
signaling units to different locations in the cell.
Three things are necessary for compartmentalized signal transduction. First, the
unit molecules must be in the right compartment; otherwise, they cannot connect
to the proper signaling molecules. Second, the molecular ecology of the compartment must be able to support the connectivity between unit molecules and their
downstream targets. Finally, the compartment must be in the right location at the
right time. Studies of signal transduction from caveolae experimentally verify each
of these principles.
Several different molecular addresses have been identified that direct molecules
to caveolae, including the acylation motif of eNOS (59, 81), the second cysteinerich region of the EGF receptor (82), and the transmembrane domain of influenza
HA (83). eNOS lacking the acylation motif does not localize to caveolae and is
disconnected from its normal signaling units (59, 70). Targeting to the proper
compartment, therefore, is essential for normal signaling. On the other hand, if
a signaling molecule is inappropriately targeted to caveolae, it may become connected to the wrong signaling units. In a recent test of this idea, oxytocin receptors
(OTR) expressed in MDCK cells that are excluded from caveolin-rich membrane
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domains inhibited cell growth in response to oxytocin. By contrast, when OTR

was targeted to caveolae, oxytocin stimulated cell proliferation (84). The apparent cause of the disparate downstream effects was a difference in the EGFR/Erk
activation patterns that occur in the two locations owing to OTR interacting with
different signaling intermediates (85). Moreover, stimulation of EGFR phosphorylation was transient when OTR was in caveolae but prolonged when in noncaveolae
membranes, which agrees with the finding that activated EGF receptors rapidly
move out of caveolae membranes (63). The results are also consistent with the
recent finding that TGF signaling depends on whether the TGF receptor is in
caveolae or clathrin coated pits (86).
Molecular ecology refers to the environment created by the collective interactions of compartmental ions, lipids, proteins, carbohydrates, etc. A significant
molecular component of a membrane compartment is the lipid bilayer, and for
caveolae, the operative lipid is cholesterol (32, 87). Removal of cholesterol leads
to the disintegration of caveola structure (87) and a loss of the ability of the domain
to internalize molecules (88). Numerous studies have documented that removal or
sequestration of cholesterol alters signal transduction from caveolae (57). Changes
in caveolae cholesterol sometimes enhance signal transduction (89) and other times
inhibit it (90). Presumably, cholesterol is required to maintain the characteristic
phase properties of the caveolae membrane (91), which is essential for the proper
organization and function of signaling units targeted to this compartment.
We discussed earlier how caveolae move around in cells and carry signaling
machinery to different locations. This behavior raises the possibility that migratory
compartments can acquire distinctive sets of signaling units at different locations
in the cell or use resident units to connect to new downstream targets. Differential signaling from mobile caveolae appears to occur in migrating fibroblasts (56,
92). Integrins are membrane receptors for extracellular matrix proteins, such as
fibronectin, that function to mediate cell adhesion and modulate signal transduction from growth factors (93). One of the signaling events that integrins control
is the movement of activated Rac1 to the plasma membrane. Rac1 binds preferentially to membranes from adherent cells compared to those from suspended
cells. Both Rac1 and Rho A are enriched in the caveolae fraction from unstimulated cells, but their concentration in the domain markedly increases when cells
are exposed to PDGF (55). Moreover, recruitment of Rac1 is blocked when membranes are depleted of cholesterol (56). Unexpectedly, integrins regulate Rac1
recruitment to the plasma membrane by controlling whether caveolae are at the
cell surface. Cells adhered to integrins have many caveolae on the surface, but
within minutes after they are released, the caveolae internalize and migrate to the
center of the cell. In response to the loss of caveolae from the plasma membrane,
Rac1 recruitment no longer occurs and the activation of Pak is blocked. Apparently the transfer of caveolae relocates the Rac1 binding sites, leading to inactivation of downstream signals. Therefore, the movement of a compartment changes
the response of a whole constellation of signaling molecules to their normal
stimulus.
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CONCLUSION
This analysis of caveolae illustrates how signal transduction is organized in a
compartment that controls important spatial and temporal parameters necessary
for fidelity in intracellular signaling. There are strong indications that other cellular
compartments attract specific sets of signaling molecules and modules, so most
likely, compartmentalization is a general way cells organize signaling networks.
Some of the questions that emerge from the current analysis include (a) How can
the same compartment contain operationally different sets of signaling molecules?
(b) How do the different molecular ecologies of compartments affect the input and
output signals of the same signaling unit? (c) What are the rules for targeting
signaling units to specific compartments? (d) Are interacting sets of signaling
units always in the same compartment of each cell type, or do they move around?
(e) What are the thermodynamic rules for how signaling networks are superimposed on to the architecture of the cell? Clearly, a systems biology approach to
understanding cell structure and function will require answers to these and many
similar questions.
ACKNOWLEDGMENTS
We would like to thank Brenda Pallares for administrative assistance. Some of
the work cited in this report was supported by grants from the National Institutes of Health, HL 20948, GM 52016 (RGWA), and CA71443 (MAW); Robert
Welch Foundation I-1414; the Perot Family Foundation; and the Cecil H. Green
Distinguished Chair in Cellular and Molecular Biology.
LITERATURE CITED
1. Barabasi AL, Oltvai ZN. 2004. Network biology: understanding the cells functional
organization. Nat. Rev. Genet. 5:10113
2. Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R. 2002. Ras and Rap control AMPA
receptor trafficking during synaptic plasticity. Cell 110:44355
3. Pearson G, Robinson F, Beers Gibson
T, Xu BE, Karandikar M, et al. 2001.
Mitogen-activated protein (MAP) kinase
pathways: regulation and physiological
functions. Endocr. Rev. 22:15383
4. Gille H, Kortenjann M, Thomae O,
Moomaw C, Slaughter C, et al. 1995.
ERK phosphorylation potentiates Elk-1mediated ternary complex formation and

transactivation. EMBO J. 14:95162
5. Janknecht R, Ernst WH, Pingoud V, Nordheim A. 1993. Activation of ternary complex factor Elk-1 by MAP kinases. EMBO
J. 12:5097104
6. Gavin AC, Ni Ainle A, Chierici E, Jones
M, Nebreda AR. 1999. A p90(rsk) mutant constitutively interacting with MAP
kinase uncouples MAP kinase from
p34(cdc2)/cyclin B activation in Xenopus
oocytes. Mol. Biol. Cell. 10:297186
7. Hsiao KM, Chou SY, Shih SJ, Ferrell
4 Dec 2004
18:52
AR
AR232-PA45-24.tex
P1: JRX

8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
JE, Jr., 1994. Evidence that inactive p42

mitogen-activated protein kinase and inactive Rsk exist as a heterodimer in vivo.
Nguyen DH, Catling AD, Webb DJ,
Sankovic M, Walker LA, et al. 1999.
Myosin light chain kinase functions downstream of Ras/ERK to promote migration
of urokinase-type plasminogen activatorstimulated cells in an integrin-selective
manner. J. Cell Biol. 146:14964
Lin LL, Wartmann M, Lin AY, Knopf JL,
Seth A, Davis RJ. 1993. cPLA2 is phosphorylated and activated by MAP kinase.
Cell 72:26978
Lewis TS, Hunt JB, Aveline LD, Jonscher
KR, Louie DF, et al. 2000. Identification
of novel MAP kinase pathway signaling
targets by functional proteomics and mass
spectrometry. Mol. Cell 6:134354
Reuther GW, Der CJ. 2000. The Ras branch
of small GTPases: Ras family members
dont fall far from the tree. Curr. Opin. Cell
Biol. 12:15765
Margarit SM, Sondermann H, Hall BE, Nagar B, Hoelz A, et al. 2003. Structural evidence for feedback activation by Ras.GTP
of the Ras-specific nucleotide exchange
factor SOS. Cell 112:68595
Murakoshi H, Iino R, Kobayashi T, Fujiwara T, Ohshima C, et al. 2004. Singlemolecule imaging analysis of Ras activation in living cells. Proc. Natl. Acad. Sci.
USA 101:731722
Kolch W. 2000. Meaningful relationships:
the regulation of the Ras/Raf/MEK/ERK
pathway by protein interactions. Biochem.
J. 351(Pt. 2):289305
Sternberg PW, Alberola-Ila J. 1998. Conspiracy theory: RAS and RAF do not act
alone. Cell 95:44750
Therrien M, Chang HC, Solomon NM,
Karim FD, Wassarman DA, Rubin GM.
1995. KSR, a novel protein kinase required
for RAS signal transduction. Cell 83:879
88
Therrien M, Wong AM, Rubin GM. 1998.
CNK, a RAF-binding multidomain protein
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
599
required for RAS signaling. Cell 95:343

53
Nguyen A, Burack WR, Stock JL, Kortum
R, Chaika OV, et al. 2002. Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo. Mol. Cell Biol. 22:3035
45
Roy F, Laberge G, Douziech M, FerlandMcCollough D, Therrien M. 2002. KSR
is a scaffold required for activation of the
ERK/MAPK module. Genes Dev. 16:427
38
Bumeister R, Rosse C, Anselmo A, Camonis J, White MA. 2004. CNK2 couples
NGF signal propagation to multiple regulatory cascades driving cell differentiation.
Curr. Biol. 14:43945
Anselmo AN, Bumeister R, Thomas JM,
White MA. 2002. Critical contribution of
linker proteins to Raf kinase activation. J.
Biol. Chem. 277:594043
Li W, Han M, Guan KL. 2000. The leucinerich repeat protein SUR-8 enhances MAP
kinase activation and forms a complex
with Ras and Raf. Genes Dev. 14:895
900
Teis D, Wunderlich W, Huber LA. 2002.
Localization of the MP1-MAPK scaffold
complex to endosomes is mediated by p14
and required for signal transduction. Dev.
Cell 3:80314
Liu P, Ying Y, Anderson RG. 1997.
Platelet-derived growth factor activates
mitogen-activated protein kinase in isolated caveolae. Proc. Natl. Acad. Sci. USA
94:1366670
Chiu VK, Bivona T, Hach A, Sajous JB,
Silletti J, et al. 2002. Ras signalling on the
endoplasmic reticulum and the Golgi. Nat.
Cell Biol. 4:34350
Yao I, Ohtsuka T, Kawabe H, Matsuura
Y, Takai Y, Hata Y. 2000. Association
of membrane-associated guanylate kinaseinteracting protein-1 with Raf-1. Biochem.
Zhou M, Horita DA, Waugh DS, Byrd RA,
Morrison DK. 2002. Solution structure and
4 Dec 2004
18:52
600
28.
29.

30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
AR
WHITE
AR232-PA45-24.tex
P1: JRX
ANDERSON
functional analysis of the cysteine-rich C1

domain of kinase suppressor of Ras (KSR).
J. Mol. Biol. 315:43546
Yamada E. 1955. The fine structure of the
gall bladder epithelium of the mouse. J.
Biophys. Biochem. Cytol. 1:44558
Palade GE. 1953. Fine structure of blood
capillaries. J. Appl. Physics 24:1424
Smart EJ, Graf GA, McNiven MA, Sessa
WC, Engelman JA, et al. 1999. Caveolins,
liquid-ordered domains, and signal transduction. Mol. Cell Biol. 19:7289304
Parton RG. 2003. Caveolaefrom ultrastructure to molecular mechanisms. Nat.
Rev. Mol. Cell Biol. 4:16267
Rothberg KG, Heuser JE, Donzell WC,
Ying YS, Glenney JR, Anderson RG. 1992.
Caveolin, a protein component of caveolae
membrane coats. Cell 68:67382
Henley JR, Krueger EW, Oswald BJ, McNiven MA. 1998. Dynamin-mediated internalization of caveolae. J. Cell Biol. 141:85
99
Oh P, McIntosh DP, Schnitzer JE. 1998.
Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma
membrane of endothelium. J. Cell Biol.
141:10114
Anderson RG, Kamen BA, Rothberg KG,
Lacey SW. 1992. Potocytosis: sequestration and transport of small molecules
by caveolae [Review]. Science 255:410
11
Pelkmans L, Kartenbeck J, Helenius A.
2001. Caveolar endocytosis of simian
virus 40 reveals a new two-step vesiculartransport pathway to the ER. Nat. Cell Biol.
3:47383
Smart EJ, Ying YS, Anderson RG. 1995.
Hormonal regulation of caveolae internalization. J. Cell Biol. 131:92938
Mineo C, Anderson RG. 2001. Potocytosis.
Robert Feulgen Lecture. Histochem. Cell
Biol. 116:10918
Mundy DI, Machleidt T, Ying YS, Anderson RG, Bloom GS. 2002. Dual control of caveolar membrane traffic by micro-
40.
41.
42.
43.
44.
45.
46.
47.
48.
tubules and the actin cytoskeleton. J. Cell

Sci. 115:432739
Thomsen P, Roepstorff K, Stahlhut M, van
Deurs B. 2002. Caveolae are highly immobile plasma membrane microdomains,
which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13:238
50
Kogo H, Fujimoto T. 2000. Concentration
of caveolin-1 in the cleavage furrow as
revealed by time-lapse analysis. Biochem.
Gagescu R, Demaurex N, Parton RG, Hunziker W, Huber LA, Gruenberg J. 2000. The
recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components. Mol. Biol.
Cell 11:277591
Peters PJ, Mironov A, Jr., Peretz D,
van Donselaar E, Leclerc E, et al. 2003.
Trafficking of prion proteins through a
caveolae-mediated endosomal pathway. J.
Cell Biol. 162:70317
Vey M, Pilkuhn S, Wille H, Nixon R,
DeArmond SJ, et al. 1996. Subcellular
colocalization of the cellular and scrapie
prion proteins in caveolae-like membranous domains. Proc. Natl. Acad. Sci. USA
93:1494549
Shin JS, Gao Z, Abraham SN. 2000. Involvement of cellular caveolae in bacterial
entry into mast cells. Science 289:78588
Lisanti MP, Tang ZL, Sargiacomo M. 1993.
Caveolin forms a hetero-oligomeric protein
complex that interacts with an apical GPIlinked protein: implications for the biogenesis of caveolae. J. Cell Biol. 123:595
604
Song KS, Li S, Okamoto T, Quilliam
LA, Sargiacomo M, Lisanti MP. 1996.
Co-purification and direct interaction of
Ras with caveolin, an integral membrane protein of caveolae microdomains.
Detergent-free purification of caveolae microdomains. J. Biol. Chem. 271:969097
Smart EJ, Ying YS, Mineo C, Anderson
RG. 1995. A detergent-free method for
purifying caveolae membrane from tissue
4 Dec 2004
18:52
AR
AR232-PA45-24.tex
P1: JRX

49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
culture cells. Proc. Natl. Acad. Sci. USA 92:

101048
Oh P, Schnitzer JE. 1999. Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin
cage. Toward understanding the basis of
purification. J. Biol. Chem. 274:2314454.
Erratum. 1999. J. Biol. Chem. 274(41):
29582
Huang C, Hepler JR, Chen LT, Gilman AG,
Anderson RG, Mumby SM. 1997. Organization of G proteins and adenylyl cyclase
at the plasma membrane. Mol. Biol. Cell
8:236578
Chang WJ, Ying YS, Rothberg KG, Hooper
NM, Turner AJ, et al. 1994. Purification
and characterization of smooth muscle cell
caveolae. J. Cell Biol. 126:12738
Liu P, Anderson RG. 1995. Compartmentalized production of ceramide at the cell
surface. J. Biol. Chem. 270:2717985
Parton RG. 1994. Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J. Histochem. Cytochem.
42:15566
Liu P, Rudick M, Anderson RG. 2002.
Multiple functions of caveolin-1. J. Biol.
Chem. 277:412958
Michaely PA, Mineo C, Ying YS, Anderson RG. 1999. Polarized distribution of
endogenous Rac1 and RhoA at the cell
surface. J. Biol. Chem. 274:2143036
del Pozo MA, Alderson NB, Kiosses WB,
Chiang HH, Anderson RG, Schwartz MA.
2004. Integrins regulate Rac targeting by
internalization of membrane domains. Science 303:83942
Roy S, Luetterforst R, Harding A, Apolloni A, Etheridge M, et al. 1999. Dominantnegative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma
membrane domains. Nat. Cell Biol. 1:98
105
Mineo C, Ying YS, Chapline C, Jaken S,
Anderson RGW. 1998. Targeting of Protein Kinase C alpha to Caveolae. J. Cell
Biol. 141:60110
Shaul PW, Smart EJ, Robinson LJ, Ger-
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
601
man Z, Yuhanna IS, et al. 1996. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J. Biol.
Chem. 271:651822
Rechardt L, Hervonen H. 1985. Cytochemical demonstration of adenylate cyclase activity with cerium. Histochemistry 82:501
5
Sugi H, Suzuki S, Daimon T. 1982. Intracellular calcium translocation during
contraction in vertebrate and invertebrate
smooth muscles as studied by the pyroantimonate method. Can. J. Physiol. Pharmacol. 60:57687
Anderson RG. 1993. Caveolae: where incoming and outgoing messengers meet.
Mineo C, Gill GN, Anderson RG. 1999.
Regulated migration of epidermal growth
factor receptor from caveolae. J. Biol.
Chem. 274:3063643
Saltiel AR, Pessin JE. 2003. Insulin signaling in microdomains of the plasma membrane. Traffic 4:71116
Liu P, Ying Y, Ko YG, Anderson RG. 1996.
Localization of platelet-derived growth
factorstimulated phosphorylation cascade
to caveolae. J. Biol. Chem. 271:10299303
Liu P, Anderson RG. 1999. Spatial organization of EGF receptor transmodulation by
PDGF. Biochem. Biophys. Res. Commun.
261:695700
Mineo C, James GL, Smart EJ, Anderson RG. 1996. Localization of epidermal growth factorstimulated Ras/Raf-1
interaction to caveolae membrane. J. Biol.
Chem. 271:1193035
Pike LJ, Casey L. 1996. Localization
and turnover of phosphatidylinositol 4,5bisphosphate in caveolin-enriched membrane domains. J. Biol. Chem. 271:26453
56
Shaul PW. 2002. Regulation of endothelial
nitric oxide synthase: location, location, location. Annu. Rev. Physiol. 64:74974
Chambliss KL, Yuhanna IS, Mineo C, Liu
P, German Z, et al. 2000. Estrogen receptor alpha and endothelial nitric oxide
4 Dec 2004
18:52
602
71.

72.
73.
74.
75.
76.
77.
78.
79.
80.
AR
WHITE
AR232-PA45-24.tex
P1: JRX
ANDERSON
synthase are organized into a functional

signaling module in caveolae. Circ. Res. 87:
E4452
Yuhanna IS, Zhu Y, Cox BE, Hahner
LD, Osborne-Lawrence S, et al. 2001.
High-density lipoprotein binding to scavenger receptorBI activates endothelial nitric oxide synthase. Nat. Med. 7:85357
Goligorsky MS, Li H, Brodsky S, Chen
J. 2002. Relationships between caveolae
and eNOS: everything in proximity and the
proximity of everything. Am. J. Physiol.
Renal Physiol. 283:F110
Tsien RY, Bacskai BJ, Adams SR. 1993.
FRET for studying intracellular signalling.
Trends Cell Biol. 3:24245
Siegel RM, Chan FK, Zacharias DA,
Swofford R, Holmes KL, et al. 2000.
Measurement of molecular interactions in
living cells by fluorescence resonance
energy transfer between variants of the
green fluorescent protein. Sci. STKE 2000:
PL1
Isshiki M, Ying YS, Fujita T, Anderson
RG. 2002. A molecular sensor detects signal transduction from caveolae in living
Hofer AM, Brown EM. 2003. Extracellular
calcium sensing and signalling. Nat. Rev.
Mol. Cell. Biol. 4:53038
Isshiki M, Anderson RG. 2003. Function of
caveolae in Ca2+ entry and Ca2+ -dependent
signal transduction. Traffic 4:71723
Isshiki M, Ando J, Korenaga R, Kogo H,
Fujimoto T, et al. 1998. Endothelial Ca2+
waves preferentially originate at specific
loci in caveolin-rich cell edges. Proc. Natl.
Isshiki M, Ando J, Yamamoto K, Fujita
T, Ying Y, Anderson RG. 2002. Sites of
Ca(2+) wave initiation move with caveolae to the trailing edge of migrating cells.
J. Cell Sci. 115:47584
Parat MO, Anand-Apte B, Fox PL. 2003.
Differential caveolin-1 polarization in endothelial cells during migration in two and
three dimensions. Mol. Biol. Cell 14:3156
68
81. Galbiati F, Volonte D, Meani D, Milligan

G, Lublin DM, et al. 1999. The dually
acylated NH2-terminal domain of gi1alpha
is sufficient to target a green fluorescent protein reporter to caveolin-enriched
plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha
subunits in vivo. J. Biol. Chem. 274:5843
50
82. Yamabhai M, Anderson RG. 2002. Second cysteine-rich region of EGFR contains
targeting information for caveolae/rafts. J.
Biol. Chem. 277:2484346
83. Scheiffele P, Roth MG, Simons K. 1997.
Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 16:55018
84. Guzzi F, Zanchetta D, Cassoni P, Guzzi V,
Francolini M, et al. 2002. Localization of
the human oxytocin receptor in caveolin1 enriched domains turns the receptormediated inhibition of cell growth into a
proliferative response. Oncogene 21:1658
67
85. Rimoldi V, Reversi A, Taverna E, Rosa P,
Francolini M, et al. 2003. Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its localization
in caveolin-1 enriched domains. Oncogene
22:605460
86. Di Guglielmo GM, Le Roy C, Goodfellow
AF, Wrana JL. 2003. Distinct endocytic
pathways regulate TGF-beta receptor signalling and turnover. Nat. Cell Biol. 5:410
21
87. Rothberg KG, Ying YS, Kamen BA, Anderson RG. 1990. Cholesterol controls
the clustering of the glycophospholipidanchored membrane receptor for 5methyltetrahydrofolate. J. Cell Biol. 111:
293138
88. Chang WJ, Rothberg KG, Kamen BA, Anderson RG. 1992. Lowering the cholesterol
content of MA104 cells inhibits receptormediated transport of folate. J. Cell Biol.
118:6369
4 Dec 2004
18:52
AR
AR232-PA45-24.tex
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89. Furuchi T, Anderson RG. 1998. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK). J. Biol. Chem. 273:21099104
90. Liu P, Wang P, Michaely P, Zhu M, Anderson RG. 2000. Presence of oxidized cholesterol in caveolae uncouples active plateletderived growth factor receptors from
tyrosine kinase substrates. J. Biol. Chem.
275:3164854
91. Ahmed SN, Brown DA, London E. 1997.
On the origin of sphingolipid/cholesterolrich detergent-insoluble cell membranes:
603
physiological concentrations of cholesterol and sphingolipid induce formation of

a detergent-insoluble, liquid-ordered lipid
phase in model membranes. Biochemistry
36:1094453
92. Palazzo AF, Eng CH, Schlaepfer DD, Marcantonio EE, Gundersen GG. 2004. Localized stabilization of microtubules by
integrin- and FAK-facilitated Rho signaling. Science 303:83639
93. Schwartz MA, Ginsberg MH. 2002. Networks and crosstalk: integrin signalling
spreads. Nat. Cell Biol. 4:E6568
1/7/05
2:31 PM
Figure 1 Schematic representation of how high fidelity signal transduction machines can be generated through modular organization of commonly engaged signaling proteins. (A) Representation of the biochemical relationships in a regulatory network
with multiple distinct inputs (stimuli S1, S2, and S3) and outputs (responses R1, R2, and R3) that propagate through a common
core enzymatic cascade (enzymes E1, E2, and E3). (B) Non-enzymatic accessory proteins (M1, M2, and M3) functionally segregate the core enzymatic cascade into separate modules with discrete input/output relationships. (C) Selective compartmentalization may restrict/facilitate coupling of spatially discrete stimuli to distinct signaling modules thereby generating fidelity
among stimulus/response pathways.

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C-2
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Figure 2 Multiple pathways of caveolae traffic. Type 1 caveolae are able to invaginate and bud from the membrane, probably in a dynamin-dependent process (33, 34).
The vesicles that form, called cavicles, are able to travel on microtubules to various
endosomal compartments. Cavicles also can travel from endosomes to other places
in the cell. Type 2 caveolae invaginate and seal off from the plasma membrane but
are retained at the surface by the actin cytoskeleton. We imagine that type 3 caveolae begin as membrane invaginations similar to the other types but then get caught on
microtubules and become stretched by microtubule motor activity into tubules.
(Diagram adapted from 39.)

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Figure 4 Polarization of caveolae leads to polarization of ATP-dependent release of

Ca2+ from the ER. Primary cultures of endothelial cells were either cultured on coverslips (A) or induced to migrate to the left by subjecting the cells to a fluid shear for
24 hr (B). Both sets of cells were loaded with the Ca2+ sensing dye Indo-1 (5 M)
before being incubated in the presence of either 0.5 M ATP (A) or 2 M ATP (B).
Images were taken at 0.38 sec intervals to visualize Ca2+ release (panel 4). At the end
of the recording, the coverslip was fixed and processed to localize caveolin-1 (panel
1) and actin (panel 2). The merge of 1 and 2 is shown in 3. Notice in A that not all
the caveolin-positive sites are active in ER Ca2+ release, indicating that caveolae are
heterogeneous in their ability to transmit signals to the ER. Arrows indicate areas of
caveolin-1 positive membrane that were active in stimulating Ca2+ release from the
ER. Bar, 20 M. See Reference 79 for details.
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Copyright
HEPATIC FIBROSIS: Molecular Mechanisms and

Drug Targets
Sophie Lotersztajn,1 Boris Julien,1 Fatima Teixeira-Clerc,1
Pascale Grenard,1 and Ariane Mallat1,2
1
Unite INSERM 581 Hopital Henri Mondor, 94010 Creteil, France;

Service dHepatologie et de Gastroenterologie, Hopital Henri Mondor, AP-HP, Creteil,
France; email: sophie.lotersztajn@im3.inserm.fr, boris.julien@im3.inserm.fr,
fatima.clerc@im3.inserm,fr, pascale.grenard@im3.inserm.fr,
ariane.mallat@hmn.ap-hop-paris.fr
2
Key Words liver, cirrhosis, myofibroblasts, hepatic stellate cells

Abstract Liver fibrosis is the common response to chronic liver injury, ultimately
leading to cirrhosis and its complications, portal hypertension, liver failure, and hepatocellular carcinoma. Efficient and well-tolerated antifibrotic drugs are currently lacking,
and current treatment of hepatic fibrosis is limited to withdrawal of the noxious agent.
Efforts over the past decade have mainly focused on fibrogenic cells generating the
scarring response, although promising data on inhibition of parenchymal injury and/or
reduction of liver inflammation have also been obtained. A large number of approaches
have been validated in culture studies and in animal models, and several clinical trials
are underway or anticipated for a growing number of molecules. This review highlights recent advances in the molecular mechanisms of liver fibrosis and discusses
mechanistically based strategies that have recently emerged.
INTRODUCTION
Chronic liver injury produces liver fibrosis, and its endstage, cirrhosis, is a major
public health problem worldwide owing to life-threatening complications of portal
hypertension and liver failure and to the risk of incident hepatocellular carcinoma.
A variety of adverse stimuli may trigger fibrogenesis, including viruses, toxins
such as alcohol, autoimmune diseases, chronic biliary stasis, metabolic disorders,
genetic defects, or hypoxia. In western countries, the prevailing causes of cirrhosis include chronic alcohol consumption, hepatitis C virus, and nonalcoholic
steatohepatitis. Current treatment of hepatic fibrosis is limited to withdrawal of
the noxious agent, which not only prevents fibrosis progression but may also induce its regression, as discussed below. Major advances have been made in this
respect during the past decade, with the advent of efficient antiviral treatments for
hepatitis B and C. Nevertheless, suppression of the cause of hepatic injury is not
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always feasible, and, therefore, numerous efforts are directed at the development
of liver-specific antifibrotic therapies. Although effective antifibrotic treatments
are not available as yet, several ongoing clinical trials are evaluating molecules
identified from the joint efforts of many researchers. In addition, recent advances
in the physiopathology of liver fibrosis are paving the way for the design of new
molecules interfering with regulatory pathways in fibrogenic cells. This review
highlights recent advances in the molecular mechanisms of liver fibrosis and discusses mechanistically based strategies that have emerged recently.
PROGRESSION AND REGRESSION OF LIVER FIBROSIS

Following acute liver injury, restoration of normal architecture results from an intricate inflammatory reaction and matrix remodeling process that combines matrix
synthesis and fibrolysis. In contrast, chronic liver injury is associated with prolonged and dysregulated wound healing, characterized by an imbalance between
excessive matrix synthesis and altered matrix degradation. This process leads to
a progressive three- to fivefold hepatic accumulation of a large variety of matrix proteins, including collagens, proteoglycans, and glycoproteins. Quantitative
changes are associated with qualitative alterations in the composition of matrix,
resulting in a predominance of type I and III fibrillar collagens, which accumulate
up to tenfold over time and build up a network resistant to fibrolysis following
crosslinking of collagen bundles (1). The cirrhotic endstage is characterized by
a distorted hepatic architecture associated with fibrotic septa surrounding regenerating hepatocyte nodules, with development of intrahepatic porto-hepatic and
arterio-venous shunts within the fibrotic septa.
Although traditionally seen as an irreversible process, advanced fibrosis, even at
the cirrhotic stage, may regress following control of the noxious stimulus. Hence,
in the rodent model of carbon tetrachloride-induced fibrosis, cessation of dosing is
followed by a reversal of fibrosis within four weeks (2). Similarly, fibrosis elicited
by bile duct ligation resolves following biliojejunal anastomosis (3). Regression of
fibrosis or cirrhosis has also been documented in patients by serial liver biopsies in
various settings, including autoimmune hepatitis controlled by immunosuppression (4), chronic hepatitis C responsive to antiviral treatment (5), chronic hepatitis
B under long-term treatment with lamivudine (6), or following biliary drainage
in patients with chronic pancreatitis or common bile duct stenosis (7). Although
older reports raised concerns as to possible false negatives of liver biopsy related
to sampling error, recent studies included larger numbers of patients and provided
large liver samples, yielding convincing results (79).
FIBROGENIC CELLS OF THE LIVER

The cellular source of fibrosis during chronic liver diseases has long been debated.
Accumulating data clearly indicate that matrix accumulation originates from different types of smooth muscle -actin myofibroblastic cells deriving from distinct
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LIVER FIBROSIS: ACTUAL PROSPECTS
607
cell populations, known as activated hepatic stellate cells and hepatic myofibroblasts (10, 11).
In the normal liver, hepatic stellate cells compose 5% to 10% of cells and
are located in the subendothelial space between hepatocytes and sinusoidal endothelial cells. Following acute or chronic liver diseases, they undergo phenotypic
changes, switching from a quiescent vitamin A-rich phenotype to a myofibroblastic phenotype (referred as to activated HSC) (12). Activated hepatic stellate cells
show de novo fibrogenic properties, including proliferation and accumulation in
areas of parenchymal cell necrosis, secretion of proinflammatory cytokines and
chemokines, and synthesis of a large panel of matrix proteins and of inhibitors of
matrix degradation, leading to progressive scar formation (Figure 1).
Hepatic myofibroblasts are another source of fibrogenic cells that derive from
fibroblasts of the portal connective tissue, perivascular fibroblasts of portal and
central veins, and periductular fibroblasts in close contact with bile duct epithelial
cells. Contribution of these cells to fibrogenesis was initially demonstrated in
experimental biliary cirrhosis by showing that myofibroblastic transformation of
Figure 1
Main properties of liver fibrogenic cells.
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portal and periductular fibroblasts precedes activation of hepatic stellate cells in

the lobule (1315).
Phenotypic and functional properties of hepatic myofibroblasts are grossly similar overall to those of activated hepatic stellate cells. However, culture studies have
clearly established that several phenotypic markers distinguish both cell types, including selective expression of fibulin-2 and interleukin-6 by hepatic myofibroblasts and protease P100 and reelin by activated hepatic stellate cells (10, 11, 16,
17). Cell-specific expression of these markers has also been described in experimental models (18) and suggests that hepatic myofibroblasts derived from portal
(myo) fibroblasts are present within fibrotic septa, whereas activated hepatic stellate cells are found in the subendothelial sinusoidal space close to portal tracts.
Regarding biological functions, activated hepatic stellate cells show minor functional differences with hepatic myofibroblasts, such as a short life span owing
to rapid apoptosis and low proliferative capacity (10). Further work is needed to
fully delineate the precise contribution of each cell type to the fibrogenic process,
and characterization of the fibrogenic cell lineage may provide useful information.
In this respect, recent studies indicate that as yet undefined bone marrow cells
constitute a significant source of hepatic stellate cells (19). In addition, bone marrow myofibroblasts represent a significant proportion of hepatic myofibroblasts in
cirrhosis of diverse etiologies (20).
ROLE OF MATRIX-PRODUCING CELLS

IN THE PATHOPHYSIOLOGY OF LIVER FIBROSIS
To identify targets for therapeutic intervention, numerous studies have extensively
investigated functional properties of fibrogenic cells and mechanisms involved in
their phenotypic activation. Selected illustrative examples are provided below.
Acquisition of the Myofibroblastic Phenotype

Mechanisms leading to the acquisition of the myofibroblastic phenotype have been
characterized extensively in hepatic stellate cells (for a review, see 21) and remain
ill-defined in portal fibroblasts. Briefly, activation of hepatic stellate cells is driven
by factors produced by neighboring cells and by remodeling of the surrounding
matrix. Thus, parenchymal injury promotes activation of Kupffer cells (resident
liver macrophages); endothelial cells and platelets; and an influx of leucocytes,
resulting in the generation of lipid peroxides, reactive oxygen species, and a number of cytokines such as TGF-, interleukin-1, TGF-, PDGF, and EGF. These
factors promote induction of specific sets of transcription factors in hepatic stellate cells within hours, resulting in induction or de novo expression of a variety of
cytokines and chemokines and of their receptors, which are involved in fibrogenesis. Transcription factors crucial at this step include ZF9, NFkB, and c-myb (21).
Remodeling of matrix also promotes activation of hepatic stellate cells. Thus,
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hepatic stellate cells cultured in a three-dimensional matrix of collagen I or matrigel retain a quiescent vitamin-A rich phenotype (22). In contrast, induction of
matrix degradation is rapidly associated with acquisition of the myofibroblastic
phenotype. Several lines of evidences also indicate that adhesion molecules are
important mediators of matrix-induced activation of hepatic stellate cells (23).

Synthesis of Cytokines and Chemokines

Fibrogenic cells produce a variety of proinflammatory chemokines and cytokines
with autocrine and paracrine effects (23). Thus, synthesis of TGF- and TGF-
promotes activation of neighboring quiescent hepatic stellate cells, whereas the
release of HGF stimulates regeneration of adjacent hepatocytes. In addition, production of MCP-1 and colony-stimulating factor contributes to the recruitment of
mononuclear leucocytes.
Proliferation and Increased Survival

Accumulation of fibrogenic cells during liver injury results from a high mitogenic
and an enhanced capacity to escape from apoptosis. Mitogenicity is stimulated
by a large variety of growth factors expressed during chronic liver injury, including PDGF, which displays the greater promitogenic effects (23); vasoconstrictors
such as thrombin (24); the metalloproteinase MMP-2 (25); or adhesion molecules
such as alphaVbeta3 integrins (26). Intracellular pathways governing mitogenicity include the ERK cascade, the PI3 kinase/Akt pathway, STAT 1, production of
phosphatidic acid, calcium influx, or acidification via the Na+ /H+ exchanger (23).
Mechanisms limiting proliferation of fibrogenic cells have also been the focus
of several studies. Typical examples include the vasodilating C-type natriuretic
peptide and prostaglandins, which elicit growth inhibitory effects via cGMP and
cAMP-dependent pathways, respectively (17, 24, 27, 28).
Survival factors protecting fibrogenic cells from apoptosis and enhancing their
accumulation during chronic liver disease have been identified. Tumor-necrosis
factor alpha and TGF- display antiapoptotic effects for activated hepatic stellate
cells in culture (29). Other examples include sphingolipid sphingosine-1-phosphate
(S1P) accumulation by a pathway involving ERK and PI3 kinase activation (30)
and type 1 tissue inhibitor of metalloprotinase (TIMP-1) (26, 31, 32). Finally,
interaction with matrix components such as collagen I and fibronectin also plays
a crucial role in survival of activated HSC, and interactions with alphaVbeta3
integrins are crucial in this process (26, 33).
Chemotaxis
Migration of fibrogenic cells toward injured areas may contribute to their accumulation at sites of injury. Migration is promoted by growth factors (e.g., PDGF,
FGF-2) or chemokines (MCP-1, CCl21) produced by inflammatory cells and involving the PI3 kinase pathway (23, 34).
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Fibrogenesis
The profibrogenic potential of activated hepatic stellate cells and hepatic myofibroblasts is due to their capacity to synthesize fibrotic matrix proteins and components that inhibit fibrosis degradation. Among the large number of factors identified
as activators of matrix production, TGF-, CTGF (35), and leptin (36) play a major
role.
Hepatic stellate cells express a wide range of metalloproteinases (MMPs) as well
as MMP activators that cleave pro-MMP into their active form. In addition, they
also produce specific tissue inhibitors of the metalloproteinase family (TIMPs).
Production of MMPs and TIMPs is tightly regulated according to the activation
state of hepatic stellate cells, and it reflects extracellular matrix remodeling during
chronic liver injury. At early stages, hepatic stellate cells express MMP-1, MMP-2,
MMP-3, and MMP-9 and their activators, but do not produce TIMPs; this allows degradation of normal matrix in the subendothelial space and its substitution
by fibrillar collagens. In contrast, fully activated hepatic stellate cells shut down
expression of MMPs and turn on expression of TIMPs, resulting in a dramatic
reduction of collagenolytic activity within the liver (37).
Strikingly, a number of cytokines simultaneously govern several functions of
fibrogenic cells. Thus, TGF-, interleukin-1, and leptin promote stellate cell activation, enhance collagen synthesis, and markedly induce TIMP-1. In addition,
TGF- also promotes cell survival (38).
EXPERIMENTAL MODELS AND ASSESSMENT

OF HEPATIC FIBROSIS
Development of antifibrotic drugs requires the availability of reliable experimental
systems for preclinical studies and the definition of accepted endpoints in clinical
trials.
Cell Culture Models

Rodent and human cultures of hepatic stellate cells and of hepatic myofibroblasts
are routinely used to define antifibrotic targets and to test potential antifibrotic
drugs. Isolation of hepatic stellate cells is based on enzymatic digestion of normal
liver (39), and purification of vitamin A-loaded cells through a density gradient
or by cell sorting (40). Within a few days, vitamin A-rich hepatic stellate cells
spontaneously acquire myofibroblastic features upon culture onto plastic. Hepatic
myofibroblasts are obtained from the culture of normal liver explants and do not
allow studies of the phenotypic transformation (41). Hepatic stellate cells and liver
myofibroblasts culture models display phenotypic properties similar to fibrogenic
cells in vivo. However, it should be stressed that several studies have used activated
hepatic stellate cells after several passages and these may in fact be largely contaminated by hepatic myofibroblasts, which progressively replace hepatic stellate
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cells that spontaneously undergo apoptosis (10). However, this hypothesis needs to
be explored by expression profiling of passaged cells. Therefore, in the following
sections, we refer to activated hepatic stellate cells or hepatic myofibroblasts, as
stated in the publications. Other culture models include rodent or human hepatic
stellate cell lines with myofibroblastic features obtained either spontaneously or
by transfection of the coding region of SV-40 (12). However, the relevance of these
models to the in vivo situation is questionable.
Animal Models
Rodent fibrosis models are widely used because of their convenient time frame.
Features of the fibrogenic process depend on the nature of liver injury. Compounds
such as carbon tetrachloride, dimethylnitrosamine, or galactosamine generate significant hepatocyte necrosis, associated with marked inflammation. In these models, antifibrotic effects of tested drugs may therefore result either from a direct
effect on fibrogenic cells or from nonspecific antiinflammatory effects. Therefore,
additional models with low degrees of cell damage and inflammation, such as bile
duct ligation or thioacetamide administration, should be used in parallel to validate efficiency of an expected antifibrotic molecule. It should also be stressed that
models of fibrosis recovery after cessation of chronic tetrachloride intoxication (2)
or following biliodigestive anastomosis in bile duct ligated rats (25) have proved
useful recently for the study of curative antifibrotic effects.
Fibrosis Staging in Humans

For years, liver biopsy has remained the gold standard for monitoring fibrosis in
clinical studies. Routine staging relies on several semiquantitative scores, such
as the widely used Knodell and Metavir scores. However, invasiveness of liver
biopsy limits serial repetition of the procedure. Quantification of the area of fibrosis by morphometry shows greater accuracy but carries a significant coefficient
of variation (42). Finally, sampling error related to the heterogeneous distribution
of fibrosis occurs in 15% to 25% of cases, particularly in advanced stages. These
limitations have stimulated the search for noninvasive sensitive and reliable serum
markers of fibrosis. Fragments of matrix constituents released in the circulation
during remodeling have not proved useful as yet, owing to inadequate diagnostic
specificity, particularly for intermediate fibrosis stages. Therefore, recent efforts
focused on indexes combining matrix protein markers or based on biochemical
and hematological parameters, and more recently, on glycomic serum analysis
(4346). In this expanding field, the Fibrotest combining five biochemical variables currently benefits from the larger experience (45). Finally, measurement of
liver elastometry also shows promising results that are currently being assessed
for validation in multicenter trials (47). Obviously, validation of noninvasive surrogate markers of fibrosis will be determinant for the rapid assessment of potential
antifibrotic therapies in large therapeutic trials.
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ANTIFIBROTIC STRATEGIES

An ideal antifibrotic drug should be liver specific to avoid adverse effects on

extrahepatic matrix proteins and should selectively attenuate excessive collagen
deposition without affecting normal extracellular matrix synthesis. Efforts over
the past decade have focused on fibrogenic cells generating the scarring response.
Recently, inhibition of parenchymal injury and of liver inflammation has also
proved of interest.
Inhibition of Parenchymal Injury

Several studies have shown that during chronic liver injury, hepatocyte and biliary
epithelial cells undergo apoptotic cell death. Interestingly, a direct link between
hepatocyte apoptosis and liver fibrogenesis has recently been demonstrated in several experimental models. Thus, Fas-deficient lymphoproliferation (lpr) mice show
decreased inflammation and fibrosis following bile duct ligation (48). Similarly,
immune-mediated liver fibrosis induced by repeated concanavalin A administration is strongly reduced by Fas-specific small interfering RNA (49). These data
therefore suggest that inhibiting hepatocyte apoptosis and thereby liver inflammation is an interesting approach for the prevention of liver fibrosis. Proof of concept
of this strategy is supported by the demonstration that IDN-6556, a general inhibitor of caspases currently undergoing phase II clinical studies (50), reduces
hepatocyte apoptosis and fibrosis in a mouse model of bile duct ligation (51). Although this approach appears promising, administration of molecules interfering
with hepatocyte apoptotic pathways may carry a high risk of carcinogenesis on
the long term, particularly at the cirrhotic stage, and therefore, this option should
be considered at early stages of chronic liver diseases.
Reduction of Liver Inflammation

Inflammation is commonly associated with progression of liver fibrosis during
chronic liver diseases. Moreover, leucocytes and Kupffer-derived products stimulate fibrogenic properties of activated hepatic stellate cells and hepatic myofibroblasts. These observations have stimulated studies investigating the effect of
antiinflammatory strategies. In this respect, beneficial effects have been observed
with inducers of Kupffer cell apoptosis, such as inhibitors of the 5-lipoxygenase
pathway, which reduce inflammation and liver fibrosis induced by carbon tetrachloride (52). Interleukin-10 has also been investigated, based on its beneficial
effect on the proinflammatory Th1 response. It was shown that IL-10 deficient
mice develop greater inflammation and fibrosis than wild-type mice (53, 54). In
keeping with these findings, a small pilot trial of interleukin-10 in -24 patients
with chronic hepatitis C showed improvement of inflammation and was associated
with a decrease in fibrosis (55).
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HEPATIC MYOFIBROBLASTS AS TARGETS

OF ANTIFIBROTIC DRUGS
Antifibrotic strategies based on inhibition of the scarring response have been extensively studied. Targets include (a) inhibition of hepatic stellate cell activation,
(b) reduction of fibrogenic cell accumulation by growth inhibitory or proapoptotic
compounds, and/or (c) reduction of extracellular matrix synthesis or enhancement of its degradation. Efficacy of these various strategies has been demonstrated
with several molecules in experimental models of liver fibrosis (see Table 1).
However, there are currently no molecules with demonstrated antifibrotic activity in humans. The following section depicts selected examples of promising
approaches.
Modulation of Cytokine Production and/or Activity

Inhibition of fibrogenic cytokines overproduced within the injured liver has been
extensively investigated. The most extensively studied strategy relates to inhibition
of TGF- signaling pathways.
TGF- is markedly overproduced by a variety of cells during chronic liver injury. The cytokine stimulates several steps of the
profibrogenic pathway, including phenotypic activation of hepatic stellate cells,
enhancement of survival, stimulation of matrix production, and overexpression
of TIMP-1 (38). The crucial role of TGF- is supported by studies showing that
overexpression of TGF- in transgenic animals induces spontaneous liver fibrosis
(56).
TGF--signaling pathways have been extensively characterized. The cytokine
is synthesized as a latent form (LAP) linked to a glycoprotein (latent TGF- binding
protein, LTBP), which anchors the complex to the extracellular matrix (ECM).
Proteolytic cleavage of LTBP by plasmin generates active TGF-, which binds
type I and type II receptors associated as heterodimers. Activation of TGF- RII
results in transphosphorylation of TGF- RI and subsequent phosphorylation of
cytoplasmic Smad transducers in cascade, leading to transcription of target genes.
Finally, several nuclear oncoproteins such as Smad 7 antagonize the cytoplasmic
Smad cascade and limit TGF- effects (57).
Several anti-TGF- strategies targeting various signaling steps have proved
effective. Thus, inhibition of activation of latent TGF- by the serine protease inhibitor camostat mesilate prevents and attenuates liver fibrosis induced by porcine
serum (58). Prevention of TGF- binding to type II receptor has also been achieved
either by administration of an adenovirus encoding dominant negative truncated
form of human TGF- RII (59) or by treatment with a soluble surrogate type II
receptor engineered by the fusion of the Fc portion of immunoglobulin G and the
ectodomain of TGF- RII (60). In both cases, liver fibrosis was strongly attenuated
in experimental models. Inhibition of intracellular signaling steps in the TGF-
TRANSFORMING GROWTH FACTOR-
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TABLE 1 Main potential antifibrogenic compounds
Compound
density of
fibrogenic
cells in vitro
fibrogenesis
and/or
fibrolysis
in vitro
Antifibrotic
effects in
animals
Reference(s)
Adiponectin
ND
(116)
Amiloride
(125)
Antiangiotensin
(86, 88, 91,

92)
Antioxidants (tocopherol,
resveratrol, sylimarin,
S-adenosylmethionine,
Sho-saiko-to. . .)
(6770)
Anti-TGF-
(5861)
Cannabinoid receptor 1
antagonism
ND
ND
(121)
Cannabinoid receptor 2
agonism
ND
(74)
Endothelin A receptor
antagonists
ND
ND
(84)
Endothelin B receptor
agonists
ND
ND
(28, 81, 83)
Gliotoxin
ND
(102, 103)
Halofuginone
(126)
Integrin antagonists
(26, 127)
Interleukin-10
ND
(53, 54)
Interferon-
(62)
Interferon-
(62)
Noradrenergic
antagonists
(128, 129)
Pentoxifylline
(130, 131)
15-D-prostaglandin J2
(73,
104107)
Prostaglandin E2
ND
(17, 24, 28,

83, 95)
Sphingosine-1 phosphate
ND
(17, 30)
Thiazolininediones
(104106,
108)
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signaling pathway may also reduce liver fibrogenesis, as shown by the beneficial
effects of an adenovirus carrying Smad 7 cDNA in the bile duct ligation model
(61).
Although attractive given their efficiency, systemic anti-TGF- strategies may
be limited by adverse effects, such as the risk of autoimmune disease secondary
to its prominent immunoregulatory properties.
Among antifibrogenic Th1 cytokines, interferons have been
the subject of extensive studies. Interferon- and interferon- inhibit activation,
proliferation, and collagen synthesis in cultures of activated hepatic stellate cells
and hepatic myofibroblasts (62); in addition, both cytokines directly inhibit collagen gene transcription in vivo and reduce progression of fibrosis, as shown in a
model of transgenic mice harboring the 2(I) collagen gene (63). In keeping with
these experimental findings, studies in patients with chronic hepatitis C suggest
that IFN- may improve the stage of fibrosis irrespective of virological response,
suggesting a direct inhibitory effect of the cytokine on fibrosis progression (5,
64). This hypothesis is being further evaluated in several ongoing clinical trials.
Beneficial effects of hepatocyte growth factor (HGF) delivered as a recombinant
protein or by gene therapy have also been reported following dimethylnitrosamine
administration (65). However, HGF being a promitogenic factor for parenchymal cells, long-term administration raises concern as to the risk of epithelial
tumors.

OTHER CYTOKINES
Reduction of Oxidative Stress

Oxidative stress has been detected in the vast majority of experimental and clinical chronic liver diseases (66). Several lines of evidence suggest that oxidative
stress modulates fibrogenic properties of activated hepatic stellate cells and hepatic
myofibroblasts. Thus, activation of hepatic stellate cells is associated to oxidative
stress and may be prevented by antioxidants, such as -tocopherol or resveratrol.
In addition, extracellular reactive oxygen species originating from Kupffer cells,
mononuclear cells, and polymorphonuclear cells stimulate transcription of collagen genes (66). In keeping with these observations, antioxidant compounds such
as tocopherol (67), the flavonoid sylimarin (68), the Japanese herbal medicine
Sho-saiko-to (69), and resveratrol (70) display antifibrogenic properties in cell
cultures and in experimental animal models (Table 1). However, data from clinical
trials are often conflicting or disappointing compared with results in experimental
models (67, 71, 72). Discrepancies are probably related to several factors, including the use of inadequate low dosages in clinical trials, the short time frame of
treatment, and the possible inefficiency of antioxidants at late stages of fibrosis.
Finally, the role of reactive oxidative stress may be more subtle than merely profibrogenic. Indeed, we recently showed that intracellular oxidative stress mediates
antifibrogenic properties of 15-D-PGJ2 and cannabinoids in hepatic myofibroblasts
(73, 74; see below). Therefore, future studies should further clarify the properties
of specific reactive intermediates.
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Modulation of Vasoactive Peptides

A number of vasoregulatory peptides are overproduced during liver fibrogenesis and show pro- or antifibrogenic properties. These observations have stimulated assessment of pharmacological activator or inhibitors of these compounds.
Endothelin-1, the angiotensin system, and prostaglandins have provided the most
convincing data.
Endothelin-1 is a potent vasoconstrictor that binds at least two G
proteincoupled receptors, ETA and ETB (7577). Investigation of the role of endothelins in liver fibrogenesis was stimulated by the finding that both endothelin-1
and its receptors are markedly induced in fibrogenic cells during chronic liver
diseases (78, 79) and by the previous demonstration of a profibrogenic role of the
peptide in kidney fibrogenesis (80). Culture studies have shown that endothelin1 displays dual pro- and antifibrogenic effects in the liver according to receptor
subtype: thus, binding of ETA receptors stimulates activation of hepatic stellate
cells and induces a weak mitogenic effect. In contrast, binding of ETB receptors
promotes marked growth inhibition (28, 81) by a mechanism involving the sequential generation of sphingosine-1-phosphate (S1P), cyclooxygenase-2 (COX2)-derived prostaglandins, and elevation of cAMP (28, 82, 83). Therefore, these
results suggested that antifibrotic effects may be achieved by selectively inhibiting
ETA receptors, whereas beneficial antifibrogenic effects of ETB receptors should
be protected, or even better enhanced. In keeping with these in vitro studies, administration of a selective ETA receptor antagonist prevents the development of
liver fibrosis in bile ductligated rats (84), whereas treatment with a nonselective
ETA/ETB receptor antagonist accelerates liver fibrosis in carbon tetrachloridetreated rats (85).
ENDOTHELIN-1
Angiotensin II is involved in cardiac and kidney fibrogenesis, and several recent studies support a significant role in liver fibrosis.
AT1 receptors are upregulated in fibrotic areas during experimental liver fibrosis
(86). Accordingly, cultured activated stellate cells express AT1 receptors and produce angiotensin II in response to growth factors via the renin angiotensin system
(87). Furthermore, activation of AT1 receptors stimulates secretion of TGF- and
proliferation of cultured activated stellate cells (88, 89). Finally, the relationship
between angiotensin II and liver fibrogenesis is supported by experimental and
clinical studies. Thus, mice invalidated for AT1 receptors show reduced liver fibrosis following administration of carbon tetrachloride (90). These observations
are corroborated by the beneficial effect of angiotensin antagonism in experimental
models of liver fibrosis, whether using angiotensin inhibitors or antagonists of AT1
receptors (88, 91, 92). In patients with chronic hepatitis C, there is a statistically
significant relationship between inheritance of a high angiotensinogen-producing
genotype and progression of hepatic fibrosis (93). Finally, a controlled pilot study
in hepatitis C recently showed that losartan reduces liver fibrosis as compared to
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untreated controls (94). Multicenter prospective trials assessing angiotensin antagonism in liver fibrosis are currently under way.
A number of studies have demonstrated antifibrogenic potential of prostaglandins. Thus, PGE2 reduces fibrosis progression in bile ductligated
rats (95). Beneficial effects are related to inhibition of proliferation and collagen
synthesis in hepatic myofibroblasts and activated hepatic stellate cells, as shown
in culture studies (95, 96). Interestingly, we have shown that growth inhibitory
effects of several factors, such as endothelin-1, TNF-, and S1P, involve induction of COX-2 and subsequent generation of PGE2 (17, 83, 96). Finally, we also
demonstrated that the mitogenic effects of PDGF-BB and thrombin result from a
balance between a promitogenic pathway and a parallel COX-2-dependent growth
inhibitory pathway (24). Together, these data point to COX-2 as a source of antifibrogenic prostaglandins in the liver.

PROSTAGLANDINS
Enhancement of Apoptosis
It has been demonstrated conclusively in experimental models that apoptosis of
hepatic fibrogenic cells is a key mandatory step in the recovery process following
fibrosis induction. Thus, available data indicate that during liver fibrogenesis, proliferation of fibrogenic cells predominates over spontaneous apoptosis, whereas
cessation of liver injury is associated with a reduction of proliferation and a marked
increase in apoptosis. Importantly, apoptosis of fibrogenic cells is accompanied
by a restoration of the collagenolytic capacities of MMP-1 and MMP-2 in the
liver, subsequent to a decrease in TIMP-1 and TIMP-2 expression, which allows
progressive matrix degradation (2, 97).
These observations have been strong incentives to characterize pathways regulating apoptosis and survival of fibrogenic cells. Available studies have been
performed mainly in cultures and have identified a number of apoptotic stimuli.
Classical apoptotic factors such as Fas-L, TRAIL 2, and TRAIL 5, and their receptors Fas and TRAIL, are upregulated during transition of hepatic stellate cells to
their activated myofibroblastic phenotype (98100). Other receptor-mediated stimuli include nerve growth factor and benzodiazepines (25, 101); however, expression
of the benzodiazepine receptor is transient and declines in activated hepatic stellate cells. Nonreceptor-mediated apoptosis of hepatic myofibroblasts also occurs
in response to a COX-2-derived prostaglandin, 15-deoxy 12,14 prostaglandin J2
(15-D-PGJ2) (73). Furthermore, we have also recently shown that hepatic myofibroblasts undergo apoptosis following exposure to sphingomyelinase metabolites,
including ceramide, sphingosine, and sphingosine-1-phosphate (S1P) (30). Investigation of the role of S1P arose from the findings that hepatic myofibroblasts express Edg receptors for the molecule (17, 30) and that sphingosine kinase activity
is increased in carbon tetrachloridetreated rats (P. Grenard, T. Levade, A. Mallat
& S. Lotersztajn, unpublished results). We found that S1P stimulates two parallel pro- and antiapoptotic pathways in human hepatic myofibroblasts, probably
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via distinct receptors. The apoptotic signal is mediated by caspase-3, whereas the
survival signal is conveyed by activation of ERK and PI3K (30).
Two experimental studies using the fungal toxin gliotoxin have documented
the potential efficiency of a proapoptotic strategy in vivo. It was shown that the
compound kills activated hepatic stellate cells in culture (102), and that in both carbon tetrachloride- and thioacetamide-treated rats, treatment with gliotoxin reduces
the number of fibrogenic cells and decreases fibrosis (102, 103). A major issue of
a proapoptotic strategy is that of cell specificity because nonselective effects may
result in life-threatening side effects, such as severe or fulminant hepatitis.
Emerging Therapeutic Targets

Potential new antifibrotic targets have been recently described. Selected examples
are described below.
Recent studies point to similar regulatory mechanisms in liver fibrogenic cells and in adipocytes.
LESSONS FROM ADIPOCYTES
PPAR Agonists Peroxisome proliferator activated receptor gamma (PPAR ),

a member of the nuclear receptor superfamily of ligand-dependent transcription
factors, is predominantly expressed in adipocytes and plays a key role in the regulation of adipogenesis. PPAR binds antidiabetic thioazelinediones compounds,
as well as eicosanoids (namely, 15-D-PGJ2), that display antiinflammatory, growth
inhibitory, and apoptotic properties. Expression of PPAR decreases during activation of hepatic stellate cells to almost undetectable levels (73, 104106), but is
reexpressed upon exposure to PPAR agonists. Moreover, thioazelinediones and
15-D-PGJ2 inhibit the main fibrogenic properties of activated hepatic stellate cells
and hepatic myofibroblasts via PPAR -dependent and independent mechanisms
(73, 105107). Finally, thiazolininediones decrease fibrosis progression in several experimental models (108), suggesting that these compounds may represent
a promising approach for the treatment of liver fibrosis.
Leptin Leptin, an obese gene product, is a potent adipocyte-derived hormone that
controls energy balance and food intake through widely expressed receptors (OBR). Leptin serum levels are increased in patients with alcoholic cirrhosis (109),
and in patients with chronic hepatitis C (110). In addition, leptin is an independent
predictor of the severity of fibrosis in alcoholic cirrhosis (110). Liver fibrogenesis
is reduced in mice with leptin deficiency (ob/ob) or bearing mutations in leptin
receptor (db/db and fa/fa), supporting a profibrogenic role of leptin. Accordingly,
the peptide is undetectable in the normal liver and is produced by activated hepatic
myofibroblasts in vitro and in vivo during fibrogenesis elicited by thioacetamide
(111, 112). The precise mechanism of action of leptin during liver fibrogenesis
is not clearly defined but may involve direct effect on matrix synthesis by myofibroblasts and upregulation of TGF- synthesis by liver cells (111, 112). These
observations suggest that antagonists of leptin receptors should be investigated as
antifibrotic agents.
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Adiponectin Adiponectin is also produced by adipocytes and acts as a major

insulin-sensitizing hormone by increasing glucose uptake and fat oxidation in
muscle and reducing fatty acid uptake and hepatic glucose production (113). Decreased circulating levels of adiponectin are found in patients with obesity, insulin
resistance, type 2 diabetes, and NASH, and administration of adiponectin causes
glucose-lowering effects, ameliorates insulin resistance in mice, and alleviates nonalcoholic steatohepatitis (113, 114). The peptide binds two receptors, R1, with an
ubiquitous distribution, and R2, which predominates in the liver (115). Several recent lines of evidence support an antifibrogenic role of adiponectin during chronic
liver diseases. Thus, mice knocked-out for adiponectin show enhanced liver fibrosis
following chronic administration of carbon tetrachloride, whereas treatment with
an adenovirus encoding adiponectin reduces liver fibrogenesis in wild-type mice
(116). Recent studies have partially elucidated targets of adiponectin in fibrogenic
cells and show that the peptide reduces proliferation and migration of activated
hepatic stellate cells as well as TGF-1-induced collagen synthesis. Unexpectedly,
serum adiponectin levels are elevated in patients with cirrhosis, suggesting that the
peptide may counteract progression of fibrosis at advanced stages (117). Although
promising, these results await confirmation when pharmacological agonists of
adiponectin receptors are available.
The cannabinoid 9-tetra-hydrocannabinol (THC) is the main
psychotropic constituent of Cannabis sativa and exerts a wide array of effects
via two G proteincoupled receptors, CB1 and CB2. Recently, THC has been
FDA-approved for the treatment of nausea following chemotherapy and the treatment of anorexia and weight loss in immunocompromised patients (118). There
is also growing interest in the use of pharmacological antagonists of cannabinoid
receptors, and the CB1 antagonist SR141716A (Rimonabant) is currently being
evaluated in phase III trials for the treatment of obesity and tobacco withdrawal
(119). Several studies also indicate that cannabinoids may also be potential antineoplastic agents owing to their ability to induce regression of various types of
tumors. These antineoplastic effects are mainly attributed to antiproliferative and
apoptotic properties of CB2 receptors (120).
We have recently demonstrated that the cannabinoid system may be a crucial
regulator of liver fibrogenesis. Thus, CB1 and CB2 receptors are marginally expressed in the normal liver and undergo marked upregulation in the cirrhotic liver,
predominating in smooth muscle -actin expressing cells within fibrotic septa (74).
Strikingly, functional studies show that CB1 and CB2 receptors display opposite
effects on liver fibrogenesis. Thus, in human hepatic myofibroblasts, selective activation of CB2 receptors triggers two antifibrogenic properties, growth inhibition
and apoptosis (74). Moreover, CB2 knock-out mice develop enhanced liver fibrosis
following chronic carbon tetrachloride treatment, demonstrating an antifibrogenic
role of CB2 receptors. In contrast, CB1 knock-out mice show reduced fibrosis following carbon tetrachloride administration, indicating a profibrogenic role of CB1
receptors (121). In keeping with these results, we have shown that daily cannabis
smoking is an independent predictor of fibrosis progression in patients with chronic
CANNABINOIDS
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hepatitis C (122). These promising results obviously warrant investigation of the

effects of pharmacological antagonists of CB1 receptors and of selective agonists
of CB2 receptors.
As outlined in this review, a number of antifibrotic approaches
are limited by the lack of cell and or tissue specificity, with a high risk of potentially severe adverse side effects. Recently, drug carriers have been designed that
specifically target liver fibrogenic cells. According to this approach, selected antifibrotic compounds are covalently linked to a cyclic peptide that selectively binds
receptors specifically expressed and upregulated in liver fibrogenic cells. Examples of carriers showing the desired cell specificity include the sugar mannose 6phosphate/insulin-like growth factor II (M6P/IGF II), which binds the M6P/IGFII
receptor, and a peptide selective for the PDGF-BB receptor and collagen VI receptor (123, 124). Such carriers appear promising for targeted delivery of antifibrotic
agents.

DRUG TARGETING
CONCLUSION
During the past decade, characterization of molecular mechanisms of liver fibrogenesis and resolution has revealed novel approaches for therapeutic intervention
based on interference with major pro- or antifibrogenic pathways in liver fibrogenic
cells. A large number of approaches have been validated in culture studies and in
animal models. Clinical trials are underway or anticipated for a growing number
of molecules, and will obviously be facilitated by the availability of noninvasive
methods for staging fibrosis. However, proof of effectiveness is still lacking in
humans. Combination of drugs with distinct antifibrogenic actions may result in
therapeutic benefits at low dosages and reduce the risk of unwanted side effects.
ACKNOWLEDGMENTS
P. Grenard was supported by INSERM and B. Julien by a fellowship from the
Ministère de la Recherche et de la Technologie. This work was supported by
the INSERM, the Universite Paris-Val-de-Marne, and by grants (to S.L.) of the
Association pour la Recherche sur le Cancer and the Ligue departementale du Val
de Marne de la Recherche contre le Cancer.
LITERATURE CITED
1. Benyon RC, Arthur MJ. 2001. Extracellular matrix degradation and the role of
hepatic stellate cells. Semin. Liver Dis.
21:37384
2. Iredale J, Benyon R, Pickering J, McCullen M, Northrop M, et al. 1998.

Mechanisms of spontaneous resolution
of rat liver fibrosis. Hepatic stellate cell
4 Dec 2004
19:4
AR
AR232-PA45-25.tex
P1: JRX
3.

4.
5.
6.
7.
8.
9.
10.
11.
apoptosis and reduced hepatic expression

of metalloproteinase inhibitors. J. Clin.
Invest. 102:53849
Abdel-Aziz G, Lebeau G, Rescan PY,
Clement B, Rissel M, et al. 1990. Reversibility of hepatic fibrosis in experimentally induced cholestasis in rat. Am
J. Pathol. 137:133342
Dufour JF, DeLellis R, Kaplan MM. 1997.
Reversibility of hepatic fibrosis in autoimmune hepatitis. Ann. Intern. Med.
127:98185
Duchatelle V, Marcellin P, Giostra E,
Bregeaud L, Pouteau M, et al. 1998.
Changes in liver fibrosis at the end of alpha interferon therapy and 6 to 18 months
later in patients with chronic hepatitis C:
quantitative assessment by a morphometric method. J. Hepatol. 29:2028
Dienstag JL, Goldin RD, Heathcote
EJ, Hann HW, Woessner M, et al.
2003. Histological outcome during longterm lamivudine therapy. Gastroenterology 124:10517
Hammel P, Couvelard A, OToole D, Ratouis A, Sauvanet A, et al. 2001. Regression of liver fibrosis after biliary drainage
in patients with chronic pancreatitis and
stenosis of the common bile duct. New
Engl. J. Med. 344:41823
Wanless IR, Nakashima E, Sherman M.
2000. Regression of human cirrhosis.
Morphologic features and the genesis of
incomplete septal cirrhosis. Arch. Pathol.
Lab. Med. 124:1599607
Pol S, Carnot F, Nalpas B, Lagneau JL,
Fontaine H, et al. 2004. Reversibility of
hepatitis C virus-related cirrhosis. Hum.
Pathol. 35:10712
Knittel T, Kobold D, Saile B, Grundmann A, Neubauer K, et al. 1999. Rat
liver myofibroblasts and hepatic stellate cells: different cell populations of
the fibroblast lineage with fibrogenic
potential. Gastroenterology 117:1205
21
Cassiman D, Libbrecht L, Desmet V,
Denef C, Roskams T. 2002. Hepatic stel-
12.
13.
14.
15.
16.
17.
18.
19.
621
late cell/myofibroblast subpopulations in

fibrotic human and rat livers. J. Hepatol.
36:2009
Geerts A. 2001. History, heterogeneity,
developmental biology, and functions of
quiescent hepatic stellate cells. Semin.
Liver Dis. 21:31135
Desmouliere A, Darby I, Costa AM,
Raccurt M, Tuchweber B, et al. 1997.
Extracellular matrix deposition, lysyl
oxidase expression, and myofibroblastic
differentiation during the initial stages of
cholestatic fibrosis in the rat. Lab. Invest.
76:76578
Milani S, Herbst H, Schuppan D, Kim
KY, Riecken EO, Stein H. 1990. Procollagen expression by nonparenchymal rat
liver cells in experimental biliary fibrosis.
Gastroenterology 98:17584
Kinnman N, Francoz C, Barbu V, Wendum D, Rey C, et al. 2003. The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells
is stimulated by platelet-derived growth
factor during liver fibrogenesis. Lab Invest. 83:16373
Kobold D, Grundmann A, Piscaglia F,
Eisenbach C, Neubauer K, et al. 2002.
Expression of reelin in hepatic stellate
cells and during hepatic tissue repair: a
novel marker for the differentiation of
HSC from other liver myofibroblasts. J.
Hepatol. 36:60713
Davaille J, Gallois C, Habib A, Li L,
Mallat A, et al. 2000. Antiproliferative
properties of sphingosine 1-phosphate in
human hepatic myofibroblasts. A cyclooxygenase-2 mediated pathway. J. Biol.
Chem. 275:3462833
Knittel T, Kobold D, Piscaglia F, Saile B,
Neubauer K, et al. 1999. Localization of
liver myofibroblasts and hepatic stellate
cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair. Histochem.
Cell Biol. 112:387401
Baba S, Fujii H, Hirose T, Yasuchika K,
Azuma H, et al. 2004. Commitment of
4 Dec 2004
19:4
622
20.

21.
22.
23.
24.
25.
26.
27.
28.
AR
AR232-PA45-25.tex
P1: JRX
LOTERSZTAJN ET AL.
bone marrow cells to hepatic stellate cells
in mouse. J. Hepatol. 40:25560
Forbes SJ, Russo FP, Rey V, Burra P,
Rugge M, et al. 2004. A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology 126:95564
Friedman SL. 2000. Molecular regulation
of hepatic fibrosis, an integrated cellular
response to tissue injury. J. Biol. Chem.
275:224750
Han YP, Zhou L, Wang J, Xiong S, Garner WL, et al. 2004. Essential role of
matrix metalloproteinases in interleukin1-induced myofibroblastic activation of
hepatic stellate cell in collagen. J. Biol.
Chem. 279:482028
Pinzani M, Marra F. 2001. Cytokine receptors and signaling in hepatic stellate
cells. Semin. Liver Dis. 21:397416
Mallat A, Gallois C, Tao J, Habib A,
Maclouf J, et al. 1998. Platelet-derived
growth factor-BB and thrombin generate
positive and negative signals for human
hepatic stellate cell proliferation. Role of
a prostaglandin/cyclic AMP pathway and
cross-talk with endothelin receptors. J.
Biol. Chem. 273:273005
Iredale JP. 2001. Hepatic stellate cell behavior during resolution of liver injury.
Semin. Liver Dis. 21:42736
Zhou X, Murphy FR, Gehdu N, Zhang
J, Iredale JP, Benyon RC. 2004. Engagement of alpha Vbeta 3 integrin regulates
proliferation and apoptosis of hepatic stellate cells. J. Biol. Chem. 24:24
Tao J, Mallat A, Gallois C, Belmadani S,
Mery PF, et al. 1999. Biological effects of
C-type natriuretic peptide in human myofibroblastic hepatic stellate cells. J. Biol.
Chem. 274:2376169
Mallat A, Preaux AM, Serradeil-Le Gal
C, Raufaste D, Gallois C, et al. 1996.
Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition
of both extracellular signal-regulated ki-
29.
30.
31.
32.
33.
34.
35.
36.
nase and c-Jun kinase and upregulation

of endothelin B receptors. J. Clin. Invest.
98:277178
Saile B, Matthes N, Knittel T, Ramadori
G. 1999. Transforming growth factor beta
and tumor necrosis factor alpha inhibit
both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology
30:196202
Davaille J, Li L, Mallat A, Lotersztajn
S. 2002. Sphingosine 1-phosphate triggers both apoptotic and survival signals
for human hepatic myofibroblasts. J. Biol.
Chem. 277:3732330
Yoshiji H, Kuriyama S, Yoshii J, Ikenaka
Y, Noguchi R, et al. 2002. Tissue inhibitor of metalloproteinases-1 attenuates
spontaneous liver fibrosis resolution in the
transgenic mouse. Hepatology 36:85060
Murphy FR, Issa R, Zhou X, Ratnarajah S,
Nagase H, et al. 2002. Inhibition of apoptosis of activated hepatic stellate cells by
TIMP-1 is mediated via effects on MMP
inhibition: implications for reversibility of
liver fibrosis. J. Biol. Chem. 277:11069
76
Issa R, Zhou X, Trim N, Millward-Sadler
H, Krane S, et al. 2003. Mutation in
collagen-1 that confers resistance to the
action of collagenase results in failure of
recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J. 17:4749
Bonacchi A, Petrai I, Defranco RM,
Lazzeri E, Annunziato F, et al. 2003. The
chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic
hepatitis C. Gastroenterology 125:1060
76
Paradis V, Dargere D, Bonvoust F, Vidaud
M, Segarini P, Bedossa P. 2002. Effects
and regulation of connective tissue growth
factor on hepatic stellate cells. Lab. Invest.
82:76774
Saxena NK, Saliba G, Floyd JJ, Anania FA. 2003. Leptin induces increased
alpha2(I) collagen gene expression in
4 Dec 2004
19:4
AR
AR232-PA45-25.tex
P1: JRX
37.
38.

39.
40.
41.
42.
43.
44.
45.
46.
cultured rat hepatic stellate cells. J. Cell

Biochem. 89:31120
Bataller R, Brenner DA. 2001. Hepatic
stellate cells as a target for the treatment of
liver fibrosis. Semin. Liver Dis. 21:43751
Gressner AM, Weiskirchen R, Breitkopf
K, Dooley S. 2002. Roles of TGF-beta in
hepatic fibrosis. Front Biosci. 7:d793807
Friedman SL, Roll FJ, Boyles J, Bissell
DM. 1985. Hepatic lipocytes: the principal collagen-producing cells of normal rat
liver. Proc. Natl. Acad. Sci. USA 82:8681
85
Geerts A, Niki T, Hellemans K, De Craemer D, Van Den Berg K, et al. 1998. Purification of rat hepatic stellate cells by side
scatter-activated cell sorting. Hepatology
27:59098
Win KM, Charlotte F, Mallat A, Cherqui
D, Martin N, et al. 1993. Mitogenic effect of transforming growth factor-beta 1
on human Ito cells in culture: evidence for
mediation by endogenous platelet-derived
growth factor. Hepatology 18:13745
Bedossa P, Dargere D, Paradis V. 2003.
Sampling variability of liver fibrosis in
chronic hepatitis C. Hepatology 38:1449
57
Forns X, Ampurdanes S, Llovet JM,
Aponte J, Quinto L, et al. 2002. Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive
model. Hepatology 36:98692
Wai CT, Greenson JK, Fontana RJ,
Kalbfleisch JD, Marrero JA, et al. 2003.
A simple noninvasive index can predict
both significant fibrosis and cirrhosis in
patients with chronic hepatitis C. Hepatology 38:51826
Poynard T, McHutchison J, Manns M,
Myers RP, Albrecht J. 2003. Biochemical surrogate markers of liver fibrosis and
activity in a randomized trial of peginterferon alfa-2b and ribavirin. Hepatology
38:48192
Callewaert N, Van Vlierberghe H, Van
Hecke A, Laroy W, Delanghe J, Contreras
R. 2004. Noninvasive diagnosis of liver
47.
48.
49.
50.
51.
52.
53.
54.
623
cirrhosis using DNA sequencer-based total serum protein glycomics. Nat. Med.
10:42934
Ziol M, Barget N, Sandrin L, Fournier C,
Mal F, et al. 2004. Correlation between
liver elasticiy measured by transient
elastography and liver fibrosis assessed
by morphometry in patients wih HCV
chronic hepatitis. J. Hepatol. 40(Suppl.
1):136
Canbay A, Higuchi H, Bronk SF, Taniai
M, Sebo TJ, Gores GJ. 2002. Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and
fibrosis. Gastroenterology 123:132330
Song E, Lee SK, Wang J, Ince N, Ouyang
N, et al. 2003. RNA interference targeting
Fas protects mice from fulminant hepatitis. Nat. Med. 9:34751
Schiff ER, Pockros P, Schiffman ML,
McHutchinson J, Gish R, et al. 2004. Oral
IDN-6556, an anti apoptotic caspase inhibitor lowers aminotransferases in HCV
patients. J. Hepatol. 40(Suppl. 1):24
Canbay A, Feldstein A, Baskin-Bey E,
Bronk SF, Gores GJ. 2004. The caspase
inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse. J. Pharmacol. Exp. Ther.
308:119196
Titos E, Claria J, Planaguma A, LopezParra M, Villamor N, et al. 2003. Inhibition of 5-lipoxygenase induces cell
growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis.
FASEB J. 17:174547
Louis H, Van Laethem JL, Wu W,
Quertinmont E, Degraef C, et al. 1998.
Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver
fibrosis induced by carbon tetrachloride in
mice. Hepatology 28:160715
Thompson K, Maltby J, Fallowfield J,
McAulay M, Millward-Sadler H, Sheron
N. 1998. Interleukin-10 expression and
function in experimental murine liver inflammation and fibrosis. Hepatology 28:
1597606
4 Dec 2004
19:4

624
AR
AR232-PA45-25.tex
P1: JRX
LOTERSZTAJN ET AL.
55. Nelson DR, Lauwers GY, Lau JY, Davis

GL. 2000. Interleukin 10 treatment reduces fibrosis in patients with chronic
hepatitis C: a pilot trial of interferon nonresponders. Gastroenterology 118:655
60
56. Sanderson N, Factor V, Nagy P, Kopp J,
Kondaiah P, et al. 1995. Hepatic expression of mature transforming growth factor
beta 1 in transgenic mice results in multiple tissue lesions. Proc. Natl. Acad. Sci.
USA 92:257276
57. Wells RG. 2000. Fibrogenesis. V. TGFbeta signaling pathways. Am. J. Physiol.
Gastrointest. Liver Physiol. 279:G84550
58. Okuno M, Akita K, Moriwaki H, Kawada
N, Ikeda K, et al. 2001. Prevention of rat
hepatic fibrosis by the protease inhibitor,
camostat mesilate, via reduced generation of active TGF-beta. Gastroenterology
120:1784800
59. Qi Z, Atsuchi N, Ooshima A, Takeshita
A, Ueno H. 1999. Blockade of type beta
transforming growth factor signaling prevents liver fibrosis and dysfunction in the
rat. Proc. Natl. Acad. Sci. USA 96:2345
49
60. George J, Roulot D, Koteliansky VE, Bissell DM. 1999. In vivo inhibition of rat
stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis. Proc. Natl. Acad. Sci. USA
96:1271924
61. Dooley S, Hamzavi J, Breitkopf K,
Wiercinska E, Said HM, et al. 2003.
Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 125:17891
62. Mallat A, Preaux A-M, Blazejewski S,
Rosenbaum J, Dhumeaux D, Mavier P.
1995. Interferon alpha and gamma inhibit proliferation and collagen synthesis
of human Ito cells in culture. Hepatology
21:100310
63. Inagaki Y, Nemoto T, Kushida M, Sheng
Y, Higashi K, et al. 2003. Interferon alfa
down-regulates collagen gene transcrip-
64.
65.
66.
67.
68.
69.
70.
71.
72.
tion and suppresses experimental hepatic fibrosis in mice. Hepatology 38:890

99
Manabe N, Chevallier M, Chossegros
P, Causse X, Guerret S, et al. 1993.
Interferon-alpha 2b therapy reduces liver
fibrosis in chronic non-A, non-B hepatitis: a quantitative histological evaluation.
Hepatology 18:134449
Ueki T, Kaneda Y, Tsutsui H, Nakanishi
K, Sawa Y, et al. 1999. Hepatocyte growth
factor gene therapy of liver cirrhosis in
rats. Nat. Med. 5:22630
Parola M, Robino G. 2001. Oxidative
stress-related molecules and liver fibrosis.
J. Hepatol. 35:297306
Houglum K, Venkataramani A, Lyche K,
Chojkier M. 1997. A pilot study of the
effects of d-alpha-tocopherol on hepatic
stellate cell activation in chronic hepatitis
C. Gastroenterology 113:106973
Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D.
1997. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 26:64349
Shimizu I, Ma YR, Mizobuchi Y, Liu F,
Miura T, et al. 1999. Effects of Sho-saikoto, a Japanese herbal medicine, on hepatic
fibrosis in rats. Hepatology 29:14960
Godichaud S, Krisa S, Couronne B,
Dubuisson L, Merillon JM, et al. 2000.
Deactivation of cultured human liver
myofibroblasts by trans-resveratrol, a
grapevine-derived polyphenol. Hepatology 31:92231
Pares A, Planas R, Torres M, Caballeria J,
Viver JM, et al. 1998. Effects of silymarin
in alcoholic patients with cirrhosis of the
liver: results of a controlled, double-blind,
randomized and multicenter trial. J. Hepatol. 28:61521
Ferenci P, Dragosics B, Dittrich H, Frank
H, Benda L, et al. 1989. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J. Hepatol. 9:10513
4 Dec 2004
19:4
AR
AR232-PA45-25.tex
P1: JRX


73. Li L, Tao J, Davaille J, Feral C, Mallat A, et al. 2001. 15-Deoxy-delta 12,14prostaglandin j2 induces apoptosis of human hepatic myofibroblasts. A pathway
involving oxidative stress independently
of peroxisome-proliferator-activated receptors. J. Biol. Chem. 276:3815258
74. Grenard P, Julien B, Tran-Van-Nhieu J, Li
L, Mallat A, Lotersztajn S. 2004. Activation of CB2 receptors reduces accumulation of human hepatic myofibroblasts: a
novel antifibrogenic strategy in the liver?
75. Mallat A, Lotersztajn S. 1996. Multiple hepatic functions of endothelin-1:
physiopathological relevance. J. Hepatol.
25:40513
76. Serradeil-Le Gal C, Jouneaux C, SanchezBueno A, Raufaste D, Roche B, et al.
1991. Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis. J. Clin. Invest.
87:13338
77. Jouneaux C, Mallat A, Serradeil-Le Gal
C, Goldsmith P, Hanoune J, Lotersztajn S.
1994. Coupling of endothelin B receptors
to the calcium pump and phospholipase C
via Gs and Gq in rat liver. J. Biol. Chem.
269:184551
78. Pinzani M, Milani S, De Franco R, Grappone C, Caligiuri A, et al. 1996. Endothelin 1 is overexpressed in human cirrhotic
liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology 110:53448
79. Leivas A, Jimenez W, Bruix J, Boix L,
Bosch J, et al. 1998. Gene expression of
endothelin-1 and ET(A) and ET(B) receptors in human cirrhosis: relationship
with hepatic hemodynamics. J. Vasc. Res.
35:18693
80. Hocher B, Thone-Reineke C, Rohmeiss
P, Schmager F, Slowinski T, et al.
1997. Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension. J. Clin. Invest. 99:138089
81. Mallat A, Fouassier L, Preaux AM,
82.
83.
84.
85.
86.
87.
88.
89.
625
Serradeil-Le Gal C, Raufaste D, et al.

1995. Growth inhibitory properties of endothelins in human hepatic myofibroblastic Ito cells: an ETB-mediated pathway. J.
Gallois C, Davaille J, Habib A, Mallat A,
Tao J, et al. 2000. Endothelin-1 stimulates
sphingosine kinase in human hepatic stellate cells. A novel role for sphingosine-1P as a mediator of growth inhibition. Ann.
NY Acad. Sci. 905:31114
Gallois C, Habib A, Tao J, Moulin S,
Maclouf J, et al. 1998. Role of NFkappaB in the antiproliferative effect of
endothelin-1 and tumor necrosis factoralpha in human hepatic stellate cells. Involvement of cyclooxygenase-2. J. Biol.
Chem. 273:2318390
Cho JJ, Hocher B, Herbst H, Jia JD, Ruehl
M, et al. 2000. An oral endothelin-A receptor antagonist blocks collagen synthesis and deposition in advanced rat liver
fibrosis. Gastroenterology 118:116978
Poo JL, Jimenez W, Maria Munoz
R, Bosch-Marce M, Bordas N, et al.
1999. Chronic blockade of endothelin
receptors in cirrhotic rats: hepatic and
hemodynamic effects. Gastroenterology
116:16167
Paizis G, Cooper ME, Schembri JM,
Tikellis C, Burrell LM, Angus PW. 2002.
Up-regulation of components of the reninangiotensin system in the bile duct-ligated
rat liver. Gastroenterology 123:166776
Bataller R, Sancho-Bru P, Gines P, Lora
JM, Al-Garawi A, et al. 2003. Activated human hepatic stellate cells express the renin-angiotensin system and
synthesize angiotensin II. Gastroenterology 125:11725
Yoshiji H, Kuriyama S, Yoshii J, Ikenaka
Y, Noguchi R, et al. 2001. AngiotensinII type 1 receptor interaction is a major
regulator for liver fibrosis development in
rats. Hepatology 34:74550
Bataller R, Gines P, Nicolas JM, Gorbig MN, Garcia-Ramallo E, et al. 2000.
Angiotensin II induces contraction and
4 Dec 2004
19:4
626
90.

91.
92.
93.
94.
95.
96.
97.
98.
AR
AR232-PA45-25.tex
P1: JRX
LOTERSZTAJN ET AL.
proliferation of human hepatic stellate
cells. Gastroenterology 118:114956
Kanno K, Tazuma S, Chayama K. 2003.
AT1A-deficient mice show less severe
progression of liver fibrosis induced by
CCl(4). Biochem. Biophys. Res. Commun.
308:17783
Croquet V, Moal F, Veal N, Wang J,
Oberti F, et al. 2002. Hemodynamic and
antifibrotic effects of losartan in rats with
liver fibrosis and/or portal hypertension.
J. Hepatol. 37:77380
Jonsson JR, Clouston AD, Ando Y,
Kelemen LI, Horn MJ, et al. 2001.
Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis. Gastroenterology 121:148
55
Powell EE, Edwards-Smith CJ, Hay JL,
Clouston AD, Crawford DH, et al. 2000.
Host genetic factors influence disease progression in chronic hepatitis C. Hepatology 31:82833
Terui Y, Saito T, Watanabe H, Togashi
H, Kawata S, et al. 2002. Effect of angiotensin receptor antagonist on liver fibrosis in early stages of chronic hepatitis
C. Hepatology 36(4 Pt. 1):1022
Beno DW, Espinal R, Edelstein BM,
Davis BH. 1993. Administration of
prostaglandin E1 analog reduces rat hepatic and Ito cell collagen gene expression
and collagen accumulation after bile duct
ligation injury. Hepatology 17:70714
Mallat A, Fouassier L, Preaux AM,
Mavier P, Lotersztajn S. 1995. Antiproliferative effects of ET-1 in human liver
Ito cells: an ETB- and a cyclic AMPmediated pathway. J. Cardiovasc. Pharmacol. 26(Suppl. 3):S13234
Preaux AM, DOrtho MP, Bralet MP,
Laperche Y, Mavier P. 2002. Apoptosis of
human hepatic myofibroblasts promotes
activation of matrix metalloproteinase-2.
Hepatology 36:61522
Gong W, Pecci A, Roth S, Lahme B, Beato
M, Gressner AM. 1998. Transformationdependent susceptibility of rat hepatic
99.
100.
101.
102.
103.
104.
105.
106.
stellate cells to apoptosis induced by

soluble Fas ligand. Hepatology 28:492
502
Saile B, Knittel T, Matthes N, Schott
P, Ramadori G. 1997. CD95/CD95Lmediated apoptosis of the hepatic stellate cell. A mechanism terminating
uncontrolled hepatic stellate cell proliferation during hepatic tissue repair. Am. J.
Pathol. 151:126572
Taimr P, Higuchi H, Kocova E, Rippe RA,
Friedman S, Gores GJ. 2003. Activated
stellate cells express the TRAIL receptor2/death receptor-5 and undergo TRAILmediated apoptosis. Hepatology 37:87
95
Fischer R, Schmitt M, Bode JG, Haussinger D. 2001. Expression of the
peripheral-type benzodiazepine receptor
and apoptosis induction in hepatic stellate
cells. Gastroenterology 120:121226
Wright MC, Issa R, Smart DE, Trim N,
Murray GI, et al. 2001. Gliotoxin stimulates the apoptosis of human and rat
hepatic stellate cells and enhances the
resolution of liver fibrosis in rats. Gastroenterology 121:68598
Dekel R, Zvibel I, Brill S, Brazovsky E,
Halpern Z, et al. 2003. Gliotoxin ameliorates development of fibrosis and cirrhosis in a thioacetamide rat model. Dig. Dis.
Sci. 48:164247
Miyahara T, Schrum L, Rippe R, Xiong
S, Yee HF Jr, et al. 2000. Peroxisome
proliferator-activated receptors and hepatic stellate cell activation. J. Biol. Chem.
275:3571522
Marra F, Efsen E, Romanelli RG,
Caligiuri A, Pastacaldi S, et al. 2000. Ligands of peroxisome proliferator-activated
receptor gamma modulate profibrogenic
and proinflammatory actions in hepatic
stellate cells. Gastroenterology 119:466
78
Galli A, Crabb D, Price D, Ceni E, Salzano
R, et al. 2000. Peroxisome proliferatoractivated receptor gamma transcriptional
regulation is involved in platelet-derived
4 Dec 2004
19:4
AR
AR232-PA45-25.tex
P1: JRX
107.

108.
109.
110.
111.
112.
113.
114.
115.
growth factor-induced proliferation of

human hepatic stellate cells. Hepatology
31:1018
Li L, Grenard P, Nhieu JT, Julien B,
Mallat A, et al. 2003. Heme oxygenase1 is an antifibrogenic protein in human
hepatic myofibroblasts. Gastroenterology
125:46069
Galli A, Crabb DW, Ceni E, Salzano R,
Mello T, et al. 2002. Antidiabetic thiazolidinediones inhibit collagen synthesis
and hepatic stellate cell activation in vivo
and in vitro. Gastroenterology 122:1924
40
Henriksen JH, Holst JJ, Moller S, Brinch
K, Bendtsen F. 1999. Increased circulating leptin in alcoholic cirrhosis: relation to release and disposal. Hepatology
29:181824
Piche T, Vandenbos F, Abakar-Mahamat
A, Vanbiervliet G, Barjoan EM, et al.
2004. The severity of liver fibrosis is associated with high leptin levels in chronic
hepatitis C. J. Viral. Hepat. 11:9196
Ikejima K, Takei Y, Honda H, Hirose
M, Yoshikawa M, et al. 2002. Leptin receptor-mediated signaling regulates
hepatic fibrogenesis and remodeling of
extracellular matrix in the rat. Gastroenterology 122:1399410
Saxena NK, Ikeda K, Rockey DC, Friedman SL, Anania FA. 2002. Leptin in
hepatic fibrosis: evidence for increased
collagen production in stellate cells and
lean littermates of ob/ob mice. Hepatology 35:76271
Berg AH, Combs TP, Scherer PE. 2002.
ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism.
Xu A, Wang Y, Keshaw H, Xu LY, Lam
KS, Cooper GJ. 2003. The fat-derived hormone adiponectin alleviates alcoholic and
nonalcoholic fatty liver diseases in mice.
J. Clin. Invest. 112:91100
Yamauchi T, Kamon J, Ito Y, Tsuchida
A, Yokomizo T, et al. 2003. Cloning of
adiponectin receptors that mediate antidi-
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
627
abetic metabolic effects. Nature 423:762

69
Kamada Y, Tamura S, Kiso S, Matsumoto
H, Saji Y, et al. 2003. Enhanced carbon tetrachloride-induced liver fibrosis in
mice lacking adiponectin. Gastroenterology 125:1796807
Tietge UJ, Boker KH, Manns MP,
Bahr MJ. 2004. Elevated circulating
adiponectin levels in liver cirrhosis are associated with reduced liver function and
altered hepatic hemodynamics. Am. J.
Physiol. Endocrinol. Metab. 287(1):E82
89
Piomelli D, Giuffrida A, Calignano A, Rodriguez de Fonseca F. 2000. The endocannabinoid system as a target for therapeutic drugs. Trends Pharmacol. Sci.
21:21824
Baker D, Pryce G, Giovannoni G, Thompson AJ. 2003. The therapeutic potential of
cannabis. Lancet Neurol. 2:29198
Bifulco M, Di Marzo V. 2002. Targeting the endocannabinoid system in cancer
therapy: a call for further research. Nat.
Med. 8:54750
Grenard P, Julien B, Tran-Van-Nhieu J, Li
L, Ledent C, et al. 2004. Reduced liver fibrosis in CB1 receptor knockout mice. J.
Hepatol. 40(Suppl. 1):8
Hezode C, Roudot-Thoraval F, Grenard
P, Julien B, Zafrani ES, et al. 2003. Daily
cannabis smoking as a risk factor for fibrosis progression in chronic hepatitis C.
Beljaars L, Molema G, Schuppan D,
Geerts A, De Bleser PJ, et al. 2000. Successful targeting to rat hepatic stellate
cells using albumin modified with cyclic
peptides that recognize the collagen type
VI receptor. J. Biol. Chem. 275:1274351
Beljaars L, Weert B, Geerts A, Meijer
DK, Poelstra K. 2003. The preferential
homing of a platelet derived growth factor receptor-recognizing macromolecule
to fibroblast-like cells in fibrotic tissue.
Benedetti A, Di Sario A, Casini A, Ridolfi
4 Dec 2004
19:4

628
AR
AR232-PA45-25.tex
P1: JRX
LOTERSZTAJN ET AL.
F, Bendia E, et al. 2001. Inhibition of the

NA(+)/H(+) exchanger reduces rat hepatic stellate cell activity and liver fibrosis:
an in vitro and in vivo study. Gastroenterology 120:54556
126. Bruck R, Genina O, Aeed H, Alexiev R,
Nagler A, et al. 2001. Halofuginone to
prevent and treat thioacetamide-induced
liver fibrosis in rats. Hepatology 33:379
86
127. Bruck R, Hershkoviz R, Lider O, Aeed
H, Zaidel L, et al. 1996. Inhibition of
experimentally-induced liver cirrhosis in
rats by a nonpeptidic mimetic of the extracellular matrix-associated Arg-Gly-Asp
epitope. J. Hepatol. 24:73138
128. Dubuisson L, Desmouliere A, Decourt B,
Evade L, Bedin C, et al. 2002. Inhibition
of rat liver fibrogenesis through noradrenergic antagonism. Hepatology 35:325

31
129. Oben JA, Roskams T, Yang S, Lin H,
Sinelli N, et al. 2004. Hepatic fibrogenesis requires sympathetic neurotransmitters. Gut 53:43845
130. Preaux AM, Mallat A, Rosenbaum J,
Zafrani ES, Mavier P. 1997. Pentoxifylline inhibits growth and collagen
synthesis of cultured human hepatic
myofibroblast-like cells. Hepatology 26:
31522
131. Windmeier C, Gressner AM. 1997. Pharmacological aspects of pentoxifylline
with emphasis on its inhibitory actions
on hepatic fibrogenesis. Gen. Pharmacol.
29:18196
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Copyright

ABERRANT DNA METHYLATION AS A

CANCER-INDUCING MECHANISM
Manel Esteller
Cancer Epigenetics Laboratory, Spanish National Cancer Center (CNIO),
Melchor Fernandez Almagro 3, 28029 Madrid, Spain; email: mesteller@cnio.es
Key Words tumor suppressor genes, CpG island, 5-methylcytosine,

hypomethylation, DNA demethylating agents
Abstract Aberrant DNA methylation is the most common molecular lesion of the
cancer cell. Neither gene mutations (nucleotide changes, deletions, recombinations)
nor cytogenetic abnormalities are as common in human tumors as DNA methylation
alterations. The most studied change of DNA methylation in neoplasms is the silencing of tumor suppressor genes by CpG island promoter hypermethylation, which
targets genes such as p16INK4a , BRCA1, and hMLH1. There is a profile of CpG island
hypermethylation according to the tumor type, and genes silent by methylation represent all cellular pathways. The introduction of bisulfite-PCR methodologies combined
with new genomic approaches provides a comprehensive spectrum of the genes undergoing this epigenetic change across all malignancies. However, we still know very
little about how this aberrant DNA methylation invades the previously unmethylated CpG island and how it is maintained through cell divisions. Furthermore, we
should remember that this methylation occurs in the context of a global genomic loss
of 5-methylcytosine (5mC). Initial clues to understand this paradox should be revealed
from the current studies of DNA methyltransferases and methyl CpG binding proteins.
From the translational standpoint, we should make an effort to validate the use of some
hypermethylated genes as biomarkers of the disease; for example, it may occur with
MGMT and GSTP1 in brain and prostate tumors, respectively. Finally, we must expect the development of new and more specific DNA demethylating agents that awake
these methyl-dormant tumor suppressor genes and prove their therapeutic values. The
expectations are high.
HISTORICAL INTRODUCTION
The field of DNA methylation is attracting the interest of many researchers and
clinicians around the world. Some of the best laboratories are gradually changing their old interests and are moving into the emerging fields of epigenetics
and, particularly, DNA methylation. Biotechnological and pharmaceutical companies are developing research programs specifically designed to develop new DNA
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demethylating drugs and to produce diagnostic kits based on DNA methylation.

This exciting new area of research combines questions about basic processes (How
are DNA methylation patterns established? What key molecules are involved in
the mechanism?) and extremely important clinical questions (Is the hypermethylation of this tumor suppressor gene a good marker of poor prognosis or good response to chemotherapy? Can we use DNA demethylating drugs in chemotherapy
regimens?).
The first observation of DNA methylation aberrations in human cancer cells
was the finding that tumors were globally hypomethylated (1) only one year after the first oncogene mutation was discovered in the H-ras in a human primary
tumor. The idea that the genome of the cancer cell undergoes a reduction of its
5-methylcytosine (5mC) content in comparison with the normal tissue has been
firmly corroborated (2, 3). However, genomic hypomethylation does not associate
with overexpression of oncogenes as originally thought, and it may be related to
the generation of chromosomal instability. Then, as a paradox, gene hypermethylation was also observed in human tumors. To the best of my knowledge, the first
discovery of methylation in a CpG island of a tumor suppressor gene in a human
cancer was that of the Retinoblastoma (Rb) gene in 1989 (4). Not until 1994 was
the idea that CpG island promoter hypermethylation could be a mechanism to
inactivate genes in cancer fully restored as a result of the discovery that the Von
Hippel-Lindau (VHL) gene also undergoes methylation-associated inactivation
(5). However, the true origin of the current period of research in cancer epigenetic
silencing was perhaps the discovery that CpG island hypermethylation was a common mechanism of inactivation of the tumor suppressor gene p16INK4a in human
cancer (68). The introduction of powerful and user-friendly techniques, such as
sodium bisulfite modification (9) and methylation-specific polymerase chain reaction (PCR) (10), were also of extreme relevance. From that time forward, the
list of candidate genes with putative aberrant methylation of their CpG islands has
grown exponentially (11) and it is time to prove the contribution of each gene to
tumorigenesis.
THE METHODOLOGY REVOLUTION IN DNA

METHYLATION
The emergence of a new technology for studying DNA methylation based on bisulfite modification coupled with PCR has been decisive in the expansion of the field
of DNA methylation. Until a few years ago, the study of DNA methylation was
almost entirely based on the use of enzymes that distinguished unmethylated and
methylated recognition sites. This approach had many drawbacks, from incomplete restriction cutting to limitation of the regions of study. Furthermore, it usually
involved Southern blot technologies, which required relatively substantial amounts
of DNA of high molecular weight. The popularization of the bisulfite treatment
of DNA (which changes unmethylated C to T, but maintains the methylated C as
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THE ROLE OF DNA METHYLATION IN CANCER
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a C), associated with amplification by specific PCR primers (methylation-specific

PCR), taqman, restriction analysis, and genomic sequencing (12), has made it
possible for every laboratory and hospital in the world to have a fair opportunity
to study DNA methylation, even using pathological material from old archives.
We call this change the universalization of DNA methylation. The techniques
described, which are ideal for studying biological fluids and the detailed DNA
methylation patterns of particular tumor suppressor genes, can also be coupled with
global genomic approaches for establishing molecular signatures of tumors based
on DNA methylation markers, such as CpG island microarrays, restriction landmark genomic scanning, and amplification of intermethylated sites (12) (Figures 1
and 2).
Moreover, we now have serious cause to believe that we can study the content and distribution of 5mC in the cellular nuclei and the whole genome thanks
to two new tools: the improved immunohistochemical staining of 5mC (13, 14),
which allows localization of the latter in the chromatin structure, and high performance capillary electrophoresis (HPCE), a reliable and affordable technique for
measuring total levels of 5mC (15) (Figures 1 and 2).
Figure 1 Protein occupancy and methylation status of a promoter CpG island of a tumor
suppressor gene in normal and cancer cells. Gray boxes, exons; white circles, unmethylated
CpGs; black circles, methylated CpGs.
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Figure 2 Techniques available for the study of DNA methylation according to the researcher
interests.
THE MOLECULAR PLAYERS IN DNA METHYLATION

ESTABLISHMENT AND SIGNALING
Methylation occurs at the 5 carbon of cytosine, a relatively unreactive position.
The catalytic mechanism of DNA (cytosine-5)-methyltransferases has been proposed as being similar to that of thymidylate synthetase, in which an enzyme
cysteine thiolate binds covalently to the 6-position. This pushes electrons to the
5-position to make the carbanion, which can then attack the methyl group of
N5,N10-methylenetetrahydrofolate. After methyl transfer, abstraction of a proton
from the 5-position may allow reformation of the 56 double bond and release of
the enzyme.
The first DNA cytosine-methyltransferase identified was revealed by purification and cloning. It remains the sole mammalian DNA methyltransferase to have
been identified by biochemical assay (16). This enzyme, now termed DNMT1, is
a protein that contains 1620 amino acids and exhibits a 5- to 30-fold preference
for hemimethylated substrates. This property led to the assignment of DNMT1 as
the enzyme responsible for maintaining the methylation patterns following DNA
replication (16). However, there is no direct evidence that DNMT1 is not also
involved in certain types of de novo methylation, and, in fact, DNMT1 is involved
in most of the de novo methylation activity in embryo lysates (16).
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The remaining DNA methyltransferases were identified by searches of expressed sequence tag (EST) databases. The first of these was DNMT2 (17). This
lacks the large N-terminal regulatory domain common to other eukaryotic methyltransferases and does not exhibit comparable DNA methyltransferase activity,
although it does seem to have some residual activity in vitro (18). DNMT3a and
DNMT3b were soon identified by searching EST databases (19) and were proposed to be the enzymes responsible for de novo methylation (20). Mutations in
the human DNMT3B gene are responsible for ICF syndrome. Figure 1A shows a
schematic representation of the DNMT family.
Although DNMTs were originally classified as maintenance or de novo DNA
methyltransferases, there are several strands of evidence that indicate that all three
DNMTs not only cooperate but also may possess both de novo and maintenance
functions in vivo (2125).
The information stored by methylation of CpGs has functional significance only
in the context of chromatin. Since its discovery, DNA methylation has been associated with a transcriptionally inactive state of chromatin; however, the mechanisms
by which DNA methylation is translated into transcriptionally silent chromatin
have only recently started to be unveiled.
Historically, several hypotheses have been proposed to explain the way by which
DNA methylation is interpreted by nuclear factors. The first possibility is that DNA
methylation inhibits the binding of sequence-specific transcription factors to their
binding sites that contain CpG (26). In this context, a protein with an affinity
for unmethylated CpGs has also been identified that is associated with actively
transcribed regions of the genome (27). In this case, methylation of CpGs would
result in release of this protein. An alternate model proposed that methylation may
have direct consequences for nucleosome positioning, for instance, by leading
to the assembly of specialized nucleosomal structures on methylated DNA that
silence transcription more effectively than conventional chromatin (28). The third
possibility is that methylation leads to the recruitment of specialized factors that
selectively recognize methylated DNA and either impede binding of other nuclear
factors or have a direct effect on repressing transcription (29).
Although there are examples that support all three possibilities, the active recruitment of methyl-CpG binding activities appears to be the most widespread
mechanism of methylation-dependent repression.
MeCP1 and MeCP2 were the first two methyl-CpG binding activities described
(29). Although MeCP1 was originally identified as a large multiprotein complex,
MeCP2 is a single polypeptide with an affinity for a single methylated CpG.
Characterization of MeCP2 in subsequent years led to the identification of the
minimum portion with affinity for methylated DNA, i.e., its methyl-CpG binding
domain (MBD) (30) and its transcriptional repression domain (TRD).
Database searches led to the identification of additional proteins harboring the
MBD, namely MBD1, MBD2, MBD3, and MBD4 (31). Whereas mammalian
MBD1 and MBD2 are bona fide methylated DNA binding proteins, MBD3 is able
to bind methylated DNA only in certain species (31, 32). In the case of MBD4, this
protein binds preferentially to m5CpG x TpG mismatches. The primary product
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of deamination at methyl-CpGs and the combined specificities of binding and

catalysis indicate that this enzyme functions to minimize mutation at methyl-CpGs.
In 1997, the laboratories of Drs. Adrian Bird and Alan Wolffe reported that
MeCP2 represses the transcription of methylated DNA through the recruitment of
a histone deacetylase-containing complex (33, 34). This finding established for the
first time a mechanistic connection between DNA methylation and transcriptional
repression by the modification of chromatin. Additional reports have established
the mechanism by which the remaining MBDs connect DNA methylation and gene
silencing (32, 35, 36). Ng et al. (35) reported that MBD2 is, in fact, a component
of the formerly identified MeCP1 complex, which exhibits histone deacetylase
activity. On the other hand, Wolffes laboratory identified MBD3 as a component
of the Mi-2/NURD complex, which exhibits both histone deacetylase and ATPasedependent nucleosome remodeling activities (32).
To understand the implications of the connections between DNA methylation
and histones, it is important to define the relevance of these posttranslational histone modifications to the determination of different chromatin states. Most histone modifications occur in their protruding N-terminal tails. This specificity in
the pattern of modifications under particular conditions led to the proposal of
the histone code hypothesis, in which histone modifications act sequentially or
in combination to form a code that may be read by nuclear factors (37). There
are several modifications that are compatible with gene silencing. In general, histone deacetylation leads to gene silencing. Furthermore, methylation of lysine 9
of histone H3 has been associated with gene silencing.
Following the finding of the coupling between DNA methylation and histone
deacetylation by MBDs, additional connections have been found. On one hand,
DNMTs are also known to recruit histone deacetylases (38, 39); on the other hand,
both DNMTs and MBDs have been reported to recruit histone methyltransferases
that modify lysine 9 of histone H3 (4042).
Therefore, multiple connections are established between hypermethylation of
the CpG islands of tumor-suppressor genes in cancer and their transcriptional silencing. The specificity of these connections and the special circumstances in which
these different elements participate for different genes remain to be determined.
In the case of MBD proteins, association with hypermethylated promoters and
their involvement in silencing their corresponding genes has now been demonstrated in a number of cases (4345). In fact, MBD proteins appear to be a common
feature of the methylated promoter of these genes and also display a remarkable
specificity in vitro (46) and in vivo (45). Thus, a MBD-specific profile for hypermethylated CpG islands is starting to be unveiled.
THE DNA METHYLATION SETTING OF HEALTHY CELLS

The inheritance of information based on gene expression levels is known as
epigenetics, as opposed to genetics, which refers to information transmitted on
the basis of gene sequence. The main epigenetic modification in humans is the
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methylation of the cytosine located within the dinucleotide CpG. 5mC in normal human tissue DNAs constitutes 0.75%1% of all nucleotide bases, and about
4%6% of all cytosines are methylated in normal human DNA (2, 3, 47).
CpG dinucleotides are not randomly distributed throughout the vast human
genome. CpG-rich regions, known as CpG islands (48), are usually unmethylated
in all normal tissues and frequently span the 5 -end region (promoter, untranslated region, and exon 1) of a number of genes; they are excellent markers of the
beginning of a gene. If the corresponding transcription factors are available, the
histones are in an acetylated and unmethylated state, and if the CpG island remains
in an unmethylated state, then that particular gene will be transcribed (Figure 3,
see color insert).
Of course, there are exceptions to the general rule. We can find certain normally
methylated CpG islands in at least four cases: imprinted genes, X-chromosome
genes in women, germline-specific genes, and tissue-specific genes (49). Genomic
or parental imprinting is a process involving acquisition of DNA hypermethylation in one allele of a gene early in the male and female germline that leads
to monoallelic expression (50). A similar phenomenon of gene-dosage reduction can also be invoked with regard to the methylation of CpG islands in one
X-chromosome in women, which renders these genes inactive to avoid redundancy. Finally, although DNA methylation is not a widely occurring system for
regulating normal gene expression, sometimes it does indeed accomplish this
purpose, as with the genes whose expression is restricted to the male or female
germline and not expressed later in any adult tissue, such as the MAGE gene
family. Finally, methylation has been postulated as a mechanism for silencing
tissue-specific genes in cell types in which they should not be expressed. However, it is still not clear whether this type of methylation is secondary to a lack of
gene expression owing to the absence of the particular cell typespecific transcription factor or whether it is the main force behind transcriptional tissue-specific
silencing.
What is the significance of the presence of DNA methylation outside the CpG
islands? One of the most exciting possibilities for the normal function of DNA
methylation is its role in repressing parasitic DNA sequences (51, 52). Our genome
is plagued with transposons and endogenous retroviruses acquired throughout the
history of the human species. We can control these imported sequences with direct
transcriptional repression mediated by several host proteins, but our main line of
defense against the large burden of parasitic sequence elements (more than 35%
of our genome) may be DNA methylation. Methylation of the promoters of our
intragenomic parasites inactivates these sequences and, over time, will destroy
many transposons.
The perfect epigenetic equilibrium of the previously described normal cell is
dramatically transformed in the cancer cell. The epigenetic aberrations observed
can be summarized as falling into one of two categories: transcriptional silencing
of tumor suppressor genes by CpG island promoter hypermethylation and global
genomic hypomethylation.
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GENOMIC HYPOMETHYLATION
OF TRANSFORMED CELLS
At the same time that certain CpG islands become hypermethylated, as discussed
below, the genome of the cancer cell undergoes dramatic global hypomethylation.
The malignant cell can have 20%60% less genomic 5mC than its normal counterpart (2, 3). The loss of methyl groups is accomplished mainly by hypomethylation
of the body (coding region and introns) of genes and through demethylation of
repetitive DNA sequences , which accounts for 20%30% of the human genome.
The degree of genomic DNA hypomethylation increases through all the tumorogenic steps, from the benign proliferations to the invasive cancers (14) (Figure 4,
see color insert).
How does global DNA hypomethylation contribute to carcinogenesis? Three
mechanisms can be invoked: chromosomal instability, reactivation of transposable
elements, and loss of imprinting. Undermethylation of DNA might favor mitotic recombination, leading to loss of heterozygosity as well as promoting karyotypically
detectable rearrangements. Additionally, extensive demethylation in centromeric
sequences is common in human tumors and may play a role in aneuploidy. It has
been reported that patients with germline mutations in DNA methyltransferase
3b (DNMT3b) have numerous chromosome aberrations (53). Hypomethylation of
malignant cell DNA can also reactivate intragenomic parasitic DNA, such as L1
(long interspersed nuclear elements, LINES) and Alu (recombinogenic sequence)
repeats (51, 52). These, and other previously silent transposons, may now be transcribed and even jump to other genomic regions where they can disrupt normal
cellular genes. Finally, the loss of methyl groups can affect imprinted genes. The
best-studied case concerns the effects of the H19/IGF-2 locus on chromosome
11p15 in certain childhood tumors (54, 55).
However, we still know very little about the real role of DNA hypomethylation
in the development of cancer cells. Is it really a causative factor? Or just a
modulator of cancer risk? Or only a bystander passenger? The studies in mouse
models are extremely interesting but puzzling: When the mouse deficient in DNA
methylation owing to a defect in DNMT1 is crossed with the colon adenomaprone Min mouse (with a genetic defect in APC), the resulting mouse has fewer
tumors (56); but another DNMT1 defective mouse may have an increased risk of
lymphomas (57). This paradox is an important question that needs to be addressed
in the near future.
METHYLATION-ASSOCIATED SILENCING OF TUMOR

SUPPRESSOR GENES
CpG islands located in the promoter region of tumor suppressor genes, normally
unmethylated at these regions like in all the other genes, undergo a dense hypermethylation in cancer cells leading to gene silencing (Figure 3). Not every gene is
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methylated in every tumor type, but strong specificity is apparent with respect to the
tissue of origin (11, 58). We have recently described the exquisite profile of hypermethylation that occurs in primary human tumors (11). Furthermore, the number
of hypermethylated genes increases with the malignant potential (14) (Figure 4).
We do not currently know why some genes became hypermethylated in certain
tumors, whereas others with similar properties (a typical CpG island, a history
of loss of expression in certain tumors, and the absence of mutations) remain
methylation-free. We can hypothesize, as researchers have done before with genetic
mutations, that a particular gene is preferentially methylated with respect to others
in certain tumor types because inactivation confers a selective advantage, in the
Darwinian sense, on the former. Another option is that aberrant DNA methylation is
directly targeted. It has been proposed that fusion proteins, such as PML-RAR, can
contribute to aberrant CpG-island methylation by recruiting DNMTs and HDACs
to aberrant sites (59). This latter activity is somewhat controversial but, in any
case, does not seem to be a general mechanism, at least in leukemia patients
(60). Selection and targeting are not exclusive events, and they are most probably
happening together in the generation and maintenance of hypermethylated CpG
islands of tumor suppressor genes.
The tumor suppressor genes, bona fide and look-alike, that undergo aberrant
CpG island methylation in human cancer affect all the cellular pathways and have
relevant consequences (49). A brief list of the most significant genes inactivated
by DNA hypermethylation is represented in Table 1 and includes the following:
(a) Cell cycle. The cell cycle inhibitor p16INK4a is hypermethylated in a wide
variety of human primary tumors and cell lines (68), allowing the cancer
cell to escape senescence and start proliferating. The Rb gene and the cell
cycle inhibitor p15INK4b can also suffer occasionally aberrant methylation
(4, 61).
(b) p53 network. p53 is the most frequently mutated tumor suppressor gene in
human cancer; nevertheless, half of human primary tumors are wild-type
p53. Another way to inactivate p53 is through the methylation-mediated silencing of the tumor suppressor gene p14ARF (6264) because in this way the
MDM2 oncogenic protein is not inhibited by p14ARF and is free to induce p53
degradation (64). p73, a gene that is a p53-homolog, is also hypermethylated
in leukemias (65).
(c) APC/-catenin/E-cadherin pathways. APC is commonly mutated in sporadic
colon tumors, but little was known about the relevance of this particular pathway in noncolorectal tumorogenesis until recently. Now it is recognized that
aberrant methylation of APC is a common lesion in other neoplasms of the
aerodigestive tract (66). E-cadherin, H-cadherin, and FAT tumor-suppressor
cadherin promoter hypermethylation is also important in the cancer biology
of breast, colon, and other tumor types (25, 67, 68). Finally, methylationassociated silencing of the genes encoding secreted frizzled-related proteins
(SFRPs), which possess a domain similar to one in the WNT-receptor frizzled
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Table 1

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A selected list of genes that undergo CpG island hypermethylation in human cancer
Gene
Function
Location Tumor profile
Consequences
p16INK4a
Cyclin-dependent kinase
inhibitor
9p21
Multiple types
Entrance in cell
cycle
p14ARF
MDM2 inhibitor
9p21
Colon,
stomach,
kidney
Degradation of p53
p15INK4b
Cyclin-dependent kinase
inhibitor
9p21
Leukemia
Entrance in cell
cycle
hMLH1
DNA mismatch repair
3p21.3
Colon,
endometrium,
stomach
Frameshift
mutations
MGMT
DNA repair of
06-alkyl-guanine
10q26
Multiple types
Mutations,
chemosentivity
GSTP1
Conjugation to
glutathione
11q13
Prostate, breast,
kidney
Adduct
accumulation?
BRCA1
DNA repair,
transcription
17q21
Breast, ovary
Double-strand
breaks?
p73
p53 homolog
1p36
Lymphoma
Unknown
LKB1/STK11 Serine/threonine kinase
19p13.3
Colon, breast,
lung
Unknown
ER
Estrogen receptor
6q25.1
Breast
Hormone
insensitivity
PR
Progesterone receptor
11q22
Breast
Hormone
insensitivity
AR
Androgen receptor
Xq11
Prostate
Hormone
insensitivity
PRLR
Prolactin receptor
5p13p12
Breast
Hormone
insensitivity
RAR2
Retinoic acid receptor

2
3p24
Colon, lung,
head, and neck
Vitamin
insensitivity?
RASSF1A
Ras effector homolog
3p21.3
Multiple types
Unknown
NORE1A
Ras effector homolog
1q32
Lung
Unknown
VHL
Ubiquitin ligase
component
3p25
Kidney, hemangioblastoma
Loss of hypoxic
response?
Rb
Cell cycle inhibitor
13q14
Retinoblastoma
Entrance in cell
cycle
THBS-1
Thrombospondin-1,
antiangiogenic
15q15
Glioma
Neovascularization
(Continued)
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Table 1 (Continued)
Gene
Function
Location Tumor profile
Consequences
CDH1
E-cadherin, cell
adhesion
16q22.1
Breast,
stomach,
leukemia
Dissemination
CDH13
H-cadherin, cell
adhesion
16q24
Breast, lung
Dissemination?
FAT
Cadherin, tumor
suppressor
4q34-35
Colon
Dissemination?
HIC-1
Transcription factor
17p13.3
Multiple types
Unknown
APC
Inhibitor of -catenin
5q21
Aerodigestive
tract
Activation
-catenin route
SFRP1
Secreted Frizzled-related
Protein 1
8p12p11
Colon
Activation WNT
signaling
COX-2
Cyclooxygenase-2
1q25
Colon, stomach
Antiinflammatory
resistance?
SOCS-1
Inhibitor of JAK/STAT
pathway
16p13.13 Liver, myeloma
JAK2 activation
SOCS-3
Inhibitor of JAK/STAT
pathway
17q25
Lung
JAK2 activation
GATA-4
8p23p22
Colon, stomach
Silencing of target
genes
GATA-5
20q13
Colon, stomach
Silencing of target
genes
SRBC
BRCA1-binding protein
1p15
Breast, lung
Unknown
SYK
Tyrosine kinase
9q22
Breast
Unknown
RIZ1
Histone/protein
methyltransferase
1p36
Breast, liver
Aberrant gene
expression?
DAPK
Pro-apoptotic
9q34.1
Lymphoma,
lung, colon
Resistance to
apoptosis
TMS1
Pro-apoptotic
16p11
Breast
Resistance to
apoptosis
2q33
Colon, bladder
Unknown
TPEF/HPP1 Transmembrane protein
proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development, has also been found in colorectal cancer (69).
(d) DNA repair. DNA methylation is one of the major players at this crossroads
of all cell pathways. Selected examples are the methylation-mediated silencing of the mismatch DNA repair gene hMLH1 in sporadic cases of colorectal
(70, 71), endometrial (72, 73), and gastric tumors (74) that cause the unusual
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phenotype known as microsatellite instability; the promoter hypermethylation of MGMT (75) that prevents the removal of groups at the O6 position of
the guanine and leads to the appearance of K-ras and p53 mutations (7678);
the hypermethylation of the mitotic checkpoint gene CHFR (79); and the somatic inactivation of BRCA1 by aberrant methylation in breast and ovarian
tumors (80), which alters its role in the repair of double-strand breaks in
the DNA and leads to the same global expression changes that occur in the
carriers of BRCA1 germ line mutations (81).
(e) Hormonal response. Aberrant methylation of the estrogen, progesterone,
androgen, and prolactin receptors occurs in breast and uterine tumors and
may render these cancer cells unresponsive to steroid hormones (45, 8284).
The differentiating action of the retinoids may also be abolished in tumors
that show promoter hypermethylation of the retinoic acid receptor-2 (60,
8587) and the cellular retinol-binding protein I (87).
(f) Cytokine signaling. The suppressor of cytokine signaling (SOCS) family of
proteins has been implicated in the negative regulation of several cytokine
pathways, particularly the receptor-associated tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathways of transcriptional
activation. SOCS-1 and SOCS-3 undergo methylation-associated silencing
in human cancer (8890).
(g) The remaining pathways. This is not an exhaustive list, but I would like to
emphasize that every imaginable molecular route can be affected by a candidate gene: the proapoptotic death-associated protein kinase (DAPK) (91) and
TMS1 (92); the kidney tumor and hemangioblastoma-related VHL gene (5);
the serine-threonine kinase LKB1/STK11 in hamartomatous neoplasms (93);
the ras-effector genes RASSF1A (94, 95) and NORE1A (96); the antiangiogenic factor thrombospondin-1 (THBS-1) (97); the prostaglandin generator
cyclooxygenase 2 (98); the TPEF gene that contains epidermal growth factor domains (99); the electrophilic detoxifier glutathione S-transferase P1
(GSTP1) in prostate, breast, and kidney tumors (100, 101); the transcription
factors GATA-4 and GATA-5 (102); and many more.
Finally, it is important to mention that as a consequence of the increasing
number of hypermethylated genes in human cancer, we need to demonstrate a
role for the methylation-associated silencing of the studied gene in tumor biology.
For example, we can check if the reintroduction of the gene in a deficient cancer
cell line reduces colony formation (25, 45, 103) or inhibits xenograft growth in
nude mice (95); if the hypermethylation of that gene correlates with a particular
molecular or clinical phenotype, as is the case with the MGMT methylation that
is associated with the appearance of transition mutations and chemosensitivity to
alkylating agents (78); if the methylation-mediated silencing has the same effects
as a frameshift mutation, as it has been shown for BRCA1 (81); or if mutations
for that gene are not described, generating a knockout mouse, as has been done
for HIC-1 (104).
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HOW TO FIND NEW HYPERMETHYLATED

GENES IN CANCER
Classical DNA methylation research concentrates on investigating the methylation
status of cytosines occurring in known (or partially known) DNA sequences. However, alternate ways of investigating genome-wide methylation by searching for
unidentified spots have been developed. They all rely on the distinctive properties
of the CpG islands to find new methylated sequences in the genome.
The restriction landmark genomic scanning (RLGS) technique is one of the
earliest ways reported for genome-wide methylation-specific searching (105).
DNA is radioactively labeled at methylation-specific cleavage sites and then sizefractionated in one dimension. The digestion products are then digested with any
restriction endonuclease that is specific for high-frequency targets. Fragments are
separated in the second dimension, yielding a number of scattered methylationrelated hot spots. The location and strength of a spot reveal its locus and the copy
number of the corresponding restriction site. This approach led to the discovery of
a number of CpG islands, for which there was no previous sequence knowledge
(58).
Another suitable tool for screening the genome for regions displaying altered
patterns of DNA is methylation-sensitive arbitrary primed PCR (AP-PCR), a simple DNA fingerprinting technique that relies on AP-PCR amplification followed
by digestion with restriction isoschizomers (106). Strain-specific arrays of DNA
fragments are generated by PCR amplification using arbitrary oligonucleotides to
prime DNA synthesis from genomic sites that accidentally or roughly match. Usually, two cycles of PCR are performed under low stringency conditions, followed
by PCR at high stringency with specific primers. DNA amplified in this manner
is digested with a couple of methylation-sensitive isoschizomers, and fragments
displaying differential methylation patterns are cloned and used as probes for
Southern analysis to corroborate differential methylation of such DNA regions.
Another approach is CpG island amplification (MCA) (107). DNA is digested
with restriction isoschizomers and restriction products are PCR-amplified after
end-adaptor ligation. Even though methylated CpG islands are preferably amplified, cloning of truly CpG-rich DNA regions is frequently a laborious task. A new
technique based on DNA arbitrary PCR enriched in methyl-sequences, amplification of intermethylated sites (AIMS) (108) has enormous potential to catch new
hypermethylated genes in human cancer (25).
Another original approach to isolated methylated CpG-rich regions has recently
been described (109). This method employs affinity chromatography of a fragment
of the methyl-CpG binding domain of MeCP2 to purify methylated CpG-rich fragments from mixtures obtained by digestion with methylation-specific restriction
endonucleases. Chosen fragments are then cloned into a lamda Zap II vector, and
fragments that are mostly rich in CpG dinucleotides are isolated by segregation
of partially melted molecules (SMP) in polyacrylamide gels containing a linear
gradient of chemical denaturant. Despite the advantages of this approach, the
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specificity of the methylated DNA binding column needs to be improved for it to

be a first-class method.
Undoubtedly, one of the most effective means of genome-wide searching for
CpG islands is the use of the novel CpG island arrays technology. The best proposed
array-based method, termed differential methylation hybridization (DMH), allows
the simultaneous determination of the methylation rate of >276 CpG island loci
(110). The CpG island library DNA fragments are gridded on high-density arrays.
Genomic DNA from the tissues of interest is digested with MseI, which yields a
large number of fragments containing intact CpG islands. Then, half of the subtracted DNA is digested with methylation-sensitive endonuclease BstU I, whose sequence target occurs frequently within CpG islands. Digestion products are used as
templates for linker PCR. Unmethylated targets are differentiated from methylated
ones because the former are cut and no PCR product is obtained, whereas the latter can be amplified by linker PCR. Resulting oligonucleotides, termed pretreated
amplicons, are used as probes for screening hypermethylated sequences within the
CpG island library. DMH has been applied to the screening of CpG methylation
in both cancer cell lines and breast cancer using CpG arrays of 300 and 1104
targets, respectively. Therefore, DMH can be used as a sophisticated screening
tool for selecting putative DNA fragments to be analyzed in greater depth by other
more specific methods. DMH technology has unveiled new hypermethylated genes
in colon cancer (25) and breast cancer, the latter using MBD-immunoprecipitated
DNA, the ChIP on CHIP approach (chromatin immunoprecipitation + microarray)
(45).
A modification of this method for the study of DNA methylation in cancer is
the methylation-specific oligonucleotide (MSO) microarray (111). After bisulfite
treatment and PCR amplification, products are array hybridized. MSO microarray
is designed to detect methylation at specific nucleotide positions. Quantitative
differences can be obtained by fluorescence detection.
Finally, one approach that it is becoming broadly used is the study of gene
expression by microarrays comparing RNA from cancer cell lines before and after
treatment with a demethylating drug (103, 112). This methodology has proven
to be very useful in identifying newly hypermethylated genes. However, not all
the genes that became reexpressed after the use of the demethylating agent are
going to be methylated: rigorous bisulfite genomic sequencing, expression, and
functional analysis are always required. However, this approach promises to be
very successful.
USING DNA METHYLATION FOR TRANSLATIONAL

PURPOSES
There is an increased necessity and obligation to translate these new results on
DNA methylation aberration in cancer from the basic research laboratory to the
clinic. There are several open fronts.
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CpG Island Hypermethylation as a Marker of Cancer Cells

In recent years, we and other groups have extensively mapped an increasing number
of gene CpG islands aberrantly hypermethylated in cancer (11, 49). These now
include examples from most classes of human neoplasia. Such DNA methylation
mapping has brought to light the existence of a unique profile of hypermethylated
CpG islands that define each neoplasia (11, 25, 49, 58). Following this lead, many
groups are currently providing us with the methylotype or DNA methylation
signature of each form of human cancer. Only those methylation markers that
are always unmethylated in normal healthy cells can be included in this panel.
Combining 34 methylation markers, we can reach an informativity of 100%
because hypermethylation events at different loci are unrelated (11). In some cases,
such as prostate cancer, a single hypermethylated marker, GSTP1, is informative
in 80%90% of the cases (100, 113).
If we wish to use these epigenetic markers in a clinical setting, we need to
use quick, easy, nonradioactive and sensitive ways of detecting hypermethylation
in CpG islands of tumor suppressor genes, such as the methylation-specific PCR
technique (MSP) (10). A careful design of the MS PCR primers with strong estringency conditions and the inclusion of positive and negative controls to avoid
false positive results are always strongly encouraged. In this spirit, CpG island
hypermethylation has been used as a tool to detect cancer cells in all types of biological fluids and biopsies: broncoalveolar lavage, lymph nodes, sputum, urine,
semen, ductal lavage, and saliva (114, 115). An exciting new line of research was
also initiated in 1999 when we demonstrated that it was possible to screen for
hypermethylated promoter loci in serum DNA from lung cancer patients (116).
Following our observation, many studies have corroborated the feasibility of detecting CpG island hypermethylation of multiple genes in the serum DNA of a
broad spectrum of tumor types (114, 115), some even using semiquantitative and
automated methodologies. Thus, DNA hypermethylation has proven its versatility
over a wide range of tumor types and environments.
Another aspect worth considering is whether promoter hypermethylation of the
CpG island of tumor suppressor genes occurs early on in tumorigenesis. Several
examples of this can be mentioned, such as the presence of p16INK4a , p14ARF ,
APC, and MGMT hypermethylation in colorectal adenomas and hMLH1 aberrant methylation in atypical endometrial hyperplasia (49). Thus, the presence of
aberrant CpG island methylation alone does not signal the presence of an invasive cancer. This may be the case, but premalignant or precursor lesions on
their way to full tumorigenesis can also carry this epigenetic culprit. In fact, this
finding may be useful in early detection screenings, especially in those people
with a high familiar risk of developing cancer because they have patterns of
CpG island hypermethylation similar to the sporadic cases (3). For those interested in cancer epidemiology, it should also be emphasized that aberrant DNA
methylation has been found up to three years before lung cancer diagnosis in subjects, such as uranium miners and smokers, who are overexposed to carcinogens
(117).
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CpG Island Hypermethylation as a Marker

for Tumor Behavior
All of us would like to look at a tumor and be able to predict its behavior. In recent
years, attempts have been made in the field of genetics to establish reliable tumor
prognosis, but they have faced a twofold problem: First, only a few genes are somatically mutated in human solid tumors (the oncogenes K-ras and Braf and the tumor
suppressor gene p53 are the most reliable) and, second, owing to the heterogeneous
cell population of a human primary neoplasm, no single marker can perfectly and
completely predict the behavior of the neoplasm. Although this second problem
cannot be solved by CpG island hypermethylation-based techniques, we can nevertheless take care of the first: Methylation-associated silencing affects many genes
in all existing cellular pathways (11, 49). From apoptosis to cell adherence, from
DNA repair to cell cycle, no route can escape from aberrant DNA methylation. As
examples of DNA methylation markers of poor prognosis, we can mention that the
DAPK and p16INK4a hypermethylation has been linked to tumor virulence in lung
and colorectal cancer patients (118, 119). Further candidates awaiting analysis to
determine their relation to enhanced metastasizing or angiogenic activity in primary tumors include the aberrant methylation of E-cadherin (CDH1), H-cadherin
(CDH13), FAT tumor suppressor-cadherin, and thrombospondin-1 (THBS-1).
CpG Island Hypermethylation as a Predictor

of Response to Treatment
The case for this concept needs to be made for each gene separately. The most compelling evidence is provided by the methylation-associated silencing of the DNA
repair MGMT in human cancer. The MGMT protein (O6 -methylguanine DNA
methyltransferase) is directly responsible for repairing the addition of alkyl groups
to the guanine (G) base of the DNA (78). This base is the preferred point of attack in
the DNA of several alkylating chemotherapeutic drugs, such as BCNU [1,3-bis(2chloroethyl)-1-nitrosourea], ACNU [1-(4-amino-2-methyl-5-pyrimidinyl)methyl3-(2-chloroethyl)-3-nitrosourea], procarbazine, streptozotocin, or temozolamide.
Thus, tumors that had lost MGMT owing to hypermethylation (75) would be more
sensitive to the action of these alkylating agents because their DNA lesions could
not be repaired in the cancer cell, leading to cell death. We gave proof of principle
for this hypothesis, and MGMT promoter hypermethylation effectively predicts a
good response to chemotherapy, greater overall survival, and longer time to progression in glioma patients treated with BCN (carmustine) (120). This study has
been followed up by other groups, who have obtained similar results (121). It is
important to note that MGMT hypermethylation alone, without treatment with
an alkylating agent, is not a good prognostic factor per se. In fact, it is a poor
prognostic factor, probably owing to the finding that patients with epigenetic silencing of MGMT accumulate more mutations, as demonstrated for p53 and K-ras
in colorectal, brain, and lung tumors (75). The potential of MGMT to predict the
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chemoresponse of human tumors to alkylating agents is not limited to BCNUlike alkylating agents; it also extends to other drugs such as cyclophosphamide
(122). This has been demonstrated in diffuse large cell lymphomas treated with
cyclophosphamide, where MGMT hypermethylation was the strongest predictor
of overall survival and time to progression and was far superior to classical clinical
factors such as the international prognostic index (122). Finally, we have extended
in gliomas the use of MGMT methylation as a marker of good clinical response
for another drug recently introduced, temozolomide (123).
Similar cases to that described for MGMT can be cited for other DNA repair and
detoxifier genes that also undergo aberrant DNA methylation. For example, the
response to cisplatin and derivatives may be a direct function of the methylation
state of the CpG island of hMLH1 (124), the response to adriamicine may be
related to the methylation status of GSTP1 (101), and the response to certain DNAdamaging drugs could be a function of the state of BRCA1 hypermethylation (80,
125).
Finally, gene inactivation by promoter hypermethylation may be the key to
understanding the loss of hormone response in many tumors. The inefficacy of
the antisteroids, estrogen-progesterone-androgen-related compounds such as tamoxifen, raloxifene, or flutemide, in certain breast, endometrial, and prostate cancer cases may be a direct consequence of the methylation-mediated silencing
of their respective cellular receptors (ER, PR, and AR genes). A similar picture
can be painted for the retinoids: Why has chemoprevention with these agents
not produced the results that we so desire and expect? A highly convincing explanation is that the tumors and the premalignant lesions become insensitive to
these compounds owing to epigenetic silencing of genes that are crucial in the
retinoid response, especially the retinoic acid receptor 2 (RAR2) (60, 8587)
and the cellular retinol binding protein I (CRBPI) (87). This is a dynamic process, and we have demonstrated that a suitable supply of dietary retinoids prevents the aberrant methylation of RAR2 and CRBPI in colorectal tumorigenesis
(87).
DNA DEMETHYLATING DRUGS IN CANCER THERAPY

A patient does not respond to drugs in the same manner as a cancer cell line
grown in vitro or a mouse with an implanted tumor. Since the mid-1980s, we
have been capable of reactivating hypermethylated genes in vitro. One obstacle to
the transfer of this technique to human primary cancers is the lack of specificity
of the drugs used (126). Demethylating agents such as 5-azacytidine or 5-aza2-deoxycytidine (Decitabine) inhibit DNA methyltransferases and cause global
hypomethylation, and we cannot reactivate solely the particular gene we would
wish to (126). Furthermore, the demethylating effect of 5-aza-2-deoxycytidine
seems to be universal, affecting all human cancer cell lines (127), although this
may not be the case for its cytotoxic capacity (128). New chemical inhibitors of
DNA methylation are being introduced, such as procainamide (129) and procaine
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(130), which give us more hope; however, the nonspecificity problem persists.
If only tumor suppressor genes were hypermethylated, this would not be a great
problem. However, we do not know if we have disrupted some essential methylation
at certain sites, and global hypomethylation may be associated with even greater
chromosomal instability. Another drawback is the toxicity to normal cells. Indeed,
this phenomenon was observed when higher doses were first used.
The first analog tested as a possible inhibitor of DNA methylation was 5azacytidine (131). This substance causes covalent arresting of DNMTs, resulting
in cytotoxicity, and tumors with increased levels of these enzymes are expected
to present higher sensitivity toward the drug (131). 5-azacytidine was tested as
an antileukemic drug before its demethylating activity was known (132, 133). It
is reported that 5-azacytidine has interference at very low concentrations (below
0.1 M) in RNA processing, tRNA methylation, and protein synthesis owing to its
incorporation preferential into RNA in vivo and in cultured cells. Treatment with
equimolar amounts of both cytidine and 5-azacytidine inhibits the incorporation
of the latter one in nucleic acids, resulting in no alteration of the cell cycle either in
vivo or ex vivo. 5-azacytidine is degradated by a nucleoside deaminase, so cells that
highly express this enzyme are not sensitive to this compound (132). Therefore, 5azacytidine is much less employed in studies related to methylation. However, more
recently, it has been concluded that irreversible cell cycle arresting at phases G1/G0,
G2, and S caused by this compound when used at micromolar concentrations is
due to its effects on DNA methylation and not on RNA metabolism (134). It is still
used in clinical trials (135, 136).
The analog 5-aza-2 -deoxycitidine (Decitabine) is one of the most used demethylating drugs for assays with cultured cells. It overcomes the major incorporation
of 5-azacytidine into RNA and reduces its side effects. Indeed, Decitabine is only
incorporated into DNA. However, it has been shown that cytidine deaminase can
degradate 5-aza-2 -deoxycytidine to 5-aza-2 -deoxyuridine (137), resulting in the
complete loss of DNMTs inhibition. The high level of cytidine deaminase in liver
and spleen may reduce the half-life of this compound to 1520 min when tested
in vivo (138). A Phase I clinical trial has suggested that deamination is the major
pathway for this compound (133).
In reference to new DNA demethylating agents, in addition to the previously
mentioned procaine and procainamide (129, 130), we should discuss zebularine [1(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one]. Zebularine is the first DNA
demethylating agent that can be administered orally and exhibits chemical stability
and minimal cytotoxicity both in vitro and in vivo (139, 140).
These compounds and their derivatives have been used in the clinic with some
therapeutic benefit, especially in hematopoietic malignancies such as myelodysplastic syndrome and acute myeloid leukemia (141143). Lower doses of 5azacytidine associated with inhibitors of histone deacetylases (such as trichostatin,
depsipeptide, SAHA, or sodium butyrate) may also reactivate tumor suppressor
genes (126). This was an encouraging discovery with respect to avoiding toxicity.
Hypermethylation of the CpG island is not a solitary phenomenon, but it occurs
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in the context of the action of methyl-binding proteins, histone hypoacetylation,

and histone methylation, which cooperate with DNA methylation in the cellular mechanisms that lead to a closed chromatin state and transcriptional silencing
(144). Several clinical trials to test this and other similar strategies in human cancer
patients are underway in the United States and Europe. It is extremely important
to define the parameters of response, clinically and molecularly. With respect to
the latter, the demethylation of the CpG islands of tumor suppressor genes, such
as that demonstrated for p15INK4b (145), and the quantitative measurement of the
5-methylcytosine DNA levels after treatment using high performance capillary
electrophoresis (HPCE) (15, 127) could be excellent surrogate markers. One of
the most promising models for testing some of these drugs is acute promyelocytic leukemia, where the transcriptional disruption induced by the PML-RAR
translocation is the main guilty party. In acute promyelocytic leukemia, treatments
that combine inhibitors of histone deacetylases, inhibitors of DNA methylation,
and differentiating factors (the rediscovered arsenic trioxide may have all three
functions) have met with success in several cases (146). On the other hand, 5-aza2 -deoxycytidine alone can induce, by mechanisms that are not fully understood,
the reexpression of certain silenced tumor suppressor genes that do not have apparent CpG island hypermethylation, such as the proapoptotic gene APAF-1 (147).
Furthermore, it is well known that 5-aza-2 -deoxycytidine has cytotoxic effects
in cancer cells over and above its DNA demethylation activity. These last two
activities expand the killing capabilities of these compounds, thus increasing their
power in cancer treatment.
These new findings have proven very attractive to several pharmacological and
biotechnology companies that are now studying how to achieve demethylation of
cancer cells using novel approaches such as antisense constructs, ribozymes, and
RNA interference against the DNA methyltransferases or other elements of the
DNA methylation machinery (methyl-CpG binding proteins) (45). Nevertheless,
we are left with the obstacle of nonspecificity. Other companies are tackling the
problem using gene therapy-like strategies whereby they specifically reactivate
a targeted methylated gene. These studies are still in their infancy, and we still
have the unsolved problem of achieving efficient delivery to the target tissue. The
classical demethylating agents are no strangers in this context: They all have to
be administered by injection as no oral compound is yet available. I hope that the
Phase II and III studies will answer some of our questions while we await better
epigenetic agents.
FINAL THOUGHTS
Cancer is a poligenetic disease, but it is also a poliepigenetic disease. We cannot understand the dynamics and plasticity of cancer cells if we do not invoke
epigenetic changes. This review has focused on DNA methylation alteration,
but the whole epigenetic setting of the transformed cell, including histone
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acetylation-methylation and chromatin remodeling factors, is disrupted. We know

that the silencing of tumor suppressor genes by CpG island promoter hypermethylation is one of the major epigentic culprits for human tumors. It affects genes
important for cell biology, such as p16INK4a , BRCA1, or hMLH1. The profile of
CpG island hypermethylation is specific to the tumor type, opening the avenue
for its use as a biomolecular marker of the disease. An issue strengthened by the
development of automatic PCR-based technologies is the easy detection of cancer
lesions. But more questions continue to arise: What is the real contribution of DNA
hypomethylation to tumorigenesis? Why are some tumor suppressor genes more
prone to be hypermethylated than others? How can DNA hypomethylation and
hypermethylation coexist in the same cancer cell? Are there any genetic disruptions prompting some of the DNA methylation changes observed or is it the other
way around? Will we ever find/create a DNA demethylating agent specific for the
hypermethylated tumor suppressor genes?
LITERATURE CITED
1. Feinberg AP, Vogeltein B. 1983. Hypomethylation distinguishes genes of
some human cancers from their normal
counterparts. Nature 301:8992
2. Ehrlich M. 2000. DNA hypomethylation
and cancer. In DNA Alterations in Cancer:
Genetic and Epigenetic Changes, ed. M
Ehrlich, pp. 27391. Natick, MA: Eaton
Publ.
3. Esteller M, Fraga MF, Guo M, GarciaFoncillas J, Hedenfalk I, et al. 2001. DNA
methylation patterns in hereditary human
cancers mimic sporadic tumorigenesis.
Hum. Mol. Genet. 10:30017
4. Greger V, Passarge E, Hopping W, Messmer E, Horsthemke B. 1989. Epigenetic
changes may contribute to the formation
and spontaneous regression of retinoblastoma. Hum. Genet. 83:15558
5. Herman JG, Latif F, Weng Y, Lerman MI,
Zbar B, et al. 1994. Silencing of the VHL
tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad.
Sci. USA 91:97004
6. Merlo A, Herman JG, Mao L, Lee DJ,
Gabrielson E, et al. 1995. 5 CpG island
7.
8.
9.
10.
methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers.
Nat. Med. 1:68692
Gonzalez-Zulueta M, Bender CM, Yang
AS, Nguyen T, Beart RW, et al. 1995.
Methylation of the 5 CpG island of the
p16/CDKN2 tumor suppressor gene in
normal and transformed human tissues
correlates with gene silencing. Cancer
Res. 55:453135
Herman JG, Merlo A, Mao L, Lapidus
RG, Issa JP, et al. 1995. Inactivation of
the CDKN2/p16/MTS1 gene is frequently
associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55:452530
Clark SJ, Harrison J, Paul CL, Frommer M. 1994. High sensitivity mapping of
methylated cytosines. Nucleic Acids Res.
22:299097
Herman JG, Graff JR, Myohanen S,
Nelkin BD, Baylin SB. 1996. Methylation-specific PCR: a novel PCR assay for
methylation status of CpG islands. Proc.
4 Dec 2004
19:7
AR
AR232-PA45-26.tex
P1: JRX


11. Esteller M, Corn PG, Baylin SB, Herman JG. 2001. A gene hypermethylation profile of human cancer. Cancer Res.
61:322529
12. Fraga MF, Esteller M. 2002. DNA methylation: a profile of methods and applications. Biotechniques 33:63249
13. Piyathilake CJ, Johanning GL, Frost AR,
Whiteside MA, Manne U, et al. 2000. Immunohistochemical evaluation of global
DNA methylation: comparison with in
vitro radiolabeled methyl incorporation
assay. Biotechnol. Histochem. 75:25158
14. Fraga MF, Herranz M, Espada J, Ballestar
E, Paz MF, et al. 2004. A mouse skin multistage carcinogenesis model reflects the
aberrant DNA methylation patterns of human tumors. Cancer Res. 64:552734
15. Fraga MF, Uriol E, Borja Diego L,
Berdasco M, Esteller M, et al. 2002. High
performance capillary electrophoretic
method for the quantification of 5-methyl
2 -deoxycytidine in genomic DNA: application to plant, animal and human cancer
tissues. Electrophoresis 23:167781
16. Bestor TH, Laudano A, Mattaliano R,
Ingram V. 1988. Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxylterminal domain of the mammalian
enzyme is related to bacterial restriction methyltransferases. J. Mol. Biol. 203:
97183
17. Yoder JA, Bestor TH. 1998. A candidate
mammalian DNA methyltransferase related to pmt1p of fission yeast. Hum. Mol.
Genet. 7:27984
18. Hermann A, Schmitt S, Jeltsch A. 2003.
The human Dnmt2 has residual DNA(cytosine-C5) methyltransferase activity.
J. Biol. Chem. 278:3171721
19. Okano M, Xie S, Li E. 1998. Cloning
and characterization of a family of novel
mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19:21920
20. Okano M, Bell DW, Haber DA, Li E.
1999. DNA methyltransferases Dnmt3a
and Dnmt3b are essential for de novo
21.
22.
23.
24.
25.
26.
27.
28.
29.
649
methylation and mammalian development. Cell 99:24757

Rhee I, Jair KW, Yen RW, Lengauer C,
Herman JG, et al. 2000. CpG methylation
is maintained in human cancer cells lacking DNMT1. Nature 404:10037
Rhee I, Bachman KE, Park BH, Jair
KW, Yen RW, et al. 2004. DNMT1 and
DNMT3b cooperate to silence genes in
human cancer cells. Nature 416:55256
Kim GD, Ni J, Kelesoglu N, Roberts
RJ, Pradhan S. 2002. Co-operation and
communication between the human maintenance and de novo DNA (cytosine-5)
methyltransferases. EMBO J. 21:4183
95
Chen T, Ueda Y, Dodge JE, Wang Z,
Li E. 2003. Establishment and maintenance of genomic methylation patterns in
mouse embryonic stem cells by Dnmt3a
and Dnmt3b. Mol. Cell Biol. 23:5594605
Paz MF, Wei S, Cigudosa JC, RodriguezPerales S, Peinado MA, et al. 2003.
Genetic unmasking of epigenetically silenced tumor suppressor genes in colon
cancer cells deficient in DNA methyltransferases. Hum. Mol. Genet. 12:2209
19
Tate PH, Bird AP. 1993. Effects of DNA
methylation on DNA-binding proteins
and gene expression. Curr. Opin. Genet.
Dev. 3:22631
Lee JH, Skalnik DG. 2002. CpGbinding protein is a nuclear matrix- and
euchromatin-associated protein localized
to nuclear speckles containing human
trithorax. Identification of nuclear matrix targeting signals. J. Biol. Chem. 277:
4225967
Kass SU, Pruss D, Wolffe AP. 1997. How
does DNA methylation repress transcription? Trends Genet. 13:44449
Lewis JD, Meehan RR, Henzel WJ,
Maurer-Fogy I, Jeppesen P, et al. 2002.
Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell
69:90514
4 Dec 2004
19:7

650
AR
AR232-PA45-26.tex
P1: JRX
ESTELLER
30. Meehan RR, Lewis JD, Bird AP. 1992.

Characterization of MeCP2, a vertebrate
DNA binding protein with affinity for
methylated DNA. Nucleic Acids Res.
20:508592
31. Hendrich B, Bird AP. 1998. Identification and characterization of a family
of mammalian methyl-CpG binding proteins. Mol. Cell. Biol. 18:653847
32. Wade PA, Gegonne A, Jones PL, Ballestar
E, Aubry F, Wolffe AP. 1999. Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat. Genet. 23:6266
33. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, et al. 1998. Methylated
DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet.
19:18791
34. Nan X, Ng HH, Johnson CA, Laherty CD,
Turner BM, et al. 1998. Transcriptional
repression by the methyl-CpG-binding
protein MeCP2 involves a histone
deacetylase complex. Nature 393:38689
35. Ng HH, Zhang Y, Hendrich B, Johnson
CA, Turne BM, et al. 1999. MBD2 is
a transcriptional repressor belonging to
the MeCP1 histone deacetylase complex.
Nat. Genet. 23:5861
36. Ng HH, Jeppesen P, Bird A. 2000. Active repression of methylated genes by the
chromosomal protein MBD1. Mol. Cell.
Biol. 20:1394406
37. Strahl BD, Allis CD. 2000. The language
of covalent histone modifications. Nature
403:4145
38. Fuks F, Burgers WA, Godin N, Kasai M, Kouzarides T. 2001. Dnmt3a
binds deacetylases and is recruited by
a sequence-specific repressor to silence
transcription. EMBO J. 20:253644
39. Robertson KD, Ait-Si-Ali S, Yokochi
T, Wade PA, Jones PL, Wolffe AP.
2000. DNMT1 forms a complex with Rb,
E2F1 and HDAC1 and represses transcription from E2F-responsive promoters.
Nat. Genet. 25:33842
40. Fuks F, Hurd PJ, Deplus R, Kouzarides
41.
42.
43.
44.
45.
46.
47.
48.
T. 2003. The DNA methyltransferases

associate with HP1 and the SUV39H1
histone methyltransferase. Nucleic Acids
Res. 31:230512
Fuks F, Hurd PJ, Wolf D, Nan X, Bird
AP, Kouzarides T. 2003. The methylCpG-binding protein MeCP2 links DNA
methylation to histone methylation. J.
Biol. Chem. 278:403540
Fujita N, Watanabe S, Ichimura T,
Tsuruzoe S, Shinkai Y, et al. 2003.
Methyl-CpG binding domain 1 (MBD1)
interacts with the Suv39h1-HP1 heterochromatic complex for DNA methylation-based transcriptional repression. J.
Biol. Chem. 278:2413238
Magdinier F, Wolffe AP. 2001. Selective
association of the methyl-CpG binding
protein MBD2 with the silent p14/p16 locus in human neoplasia. Proc. Natl. Acad.
Sci. USA 98:499095
Nguyen CT, Gonzales FA, Jones PA.
2001. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of
accessibility, methylation, MeCP2 binding and acetylation. Nucleic Acids Res.
29:4598606
Ballestar E, Paz MF, Valle L, Wei S, Fraga
MF, et al. 2003. Methyl-CpG binding proteins identify novel sites of epigenetic
inactivation in human cancer. EMBO J.
22:633545
Fraga MF, Ballestar E, Montoya G, Taysavang P, Wade PA, Esteller M. 2003. The
affinity of different MBD proteins for a
specific methylated locus depends on their
intrinsic binding properties. Nucleic Acids
Res. 31:176574
Paz MF, Avila S, Fraga MF, Pollan M,
Capella G, et al. 2002. Germ-line variants
in methyl-group metabolism genes and
susceptibility to DNA methylation in normal tissues and human primary tumors.
Bird AP. 1986. CpG-rich islands and
the function of DNA methylation. Nature
321:20913
4 Dec 2004
19:7
AR
AR232-PA45-26.tex
P1: JRX


49. Esteller M. 2002. CpG island hypermethylation and tumor suppressor genes: a
booming present, a brighter future. Oncogene 21:542740
50. Feinberg AP, Cui H, Ohlsson R. 2002.
DNA methylation and genomic imprinting: insights from cancer into epigenetic
mechanisms. Semin. Cancer Biol. 12:
38998
51. Yoder JA, Bestor TH. 1997. Cytosine
methylation and the ecology of intragenomic parasites. Trends Genet. 13:33540
52. Walsh CP, Chaillet JR, Bestor TH.
1998. Transcription of IAP endogenous
retroviruses is constrained by cytosine
methylation. Nat. Genet. 20:11617
53. Xu GL, Bestor TH, Bourchis D, Hsieh
CL, Tommerup N, et al. 1999. Chromosome instability and immunodeficiency
syndrome caused by mutations in a DNA
methyltransferase gene. Nature 402:187
91
54. Feinberg AP. 1999. Imprinting of a genomic domain of 11p15 and loss of
imprinting in cancer: an introduction.
Cancer Res. 59(7 Suppl.):1743s46
55. Plass C, Soloway PD. 2002. DNA methylation, imprinting and cancer. Eur. J. Hum.
Genet. 10:616
56. Laird PW, Jackson-Grusby L, Fazeli A,
Dickinson SL, Jung WE, et al. 1995. Suppression of intestinal neoplasia by DNA
hypomethylation. Cell 81:197205
57. Gaudet F, Hodgson JG, Eden A, JacksonGrusby L, Dausman J, et al. 2003. Induction of tumors in mice by genomic
hypomethylation. Science 300:48992
58. Costello JF, Fruhwald MC, Smiraglia
DJ, Rush LJ, Robertson GP, et al.
2000. Aberrant CpG-island methylation
has non-random and tumour-type-specific
patterns. Nat. Genet. 24:13238
59. Di Croce L, Raker VA, Corsaro M, Fazi F,
Fanelli M, et al. 2002. Methyltransferase
recruitment and DNA hypermethylation
of target promoters by an oncogenic transcription factor. Science 295:107982
60. Esteller M, Fraga MF, Paz MF, Campo E,
61.
62.
63.
64.
65.
66.
67.
68.
69.
651
Colomer D, et al. 2002. Cancer epigenetics and methylation. Science 297:18078

Herman JG, Jen J, Merlo A, Baylin SB.
1996. Hypermethylation-associated inactivation indicates a tumor suppressor role
for p15INK4B. Cancer Res. 56:72227
Robertson KD, Jones PA. 1998. The human ARF cell cycle regulatory gene promoter is a CpG island which can be
silenced by DNA methylation and downregulated by wild-type p53. Mol. Cell
Biol. 18:645773
Esteller M, Tortola S, Toyota M,
Capella G, Peinado MA, et al. 2000.
Hypermethylation-associated inactivation of p14(ARF) is independent of
p16(INK4a) methylation and p53 mutational status. Cancer Res. 60:12933
Esteller M, Cordon-Cardo C, Corn PG,
Meltzer SJ, Pohar KS, et al. 2001.
p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2. Cancer Res.
61:281621
Corn PG, Kuerbitz SJ, van Noesel MM,
Esteller M, Compitello N, et al. 1999.
Transcriptional silencing of the p73 gene
in acute lymphoblastic leukemia and
Burkitts lymphoma is associated with
5 CpG island methylation. Cancer Res.
59:335256
Esteller M, Sparks A, Toyota M, SanchezCespedes M, Capella G, et al. 2000.
Analysis of adenomatous polyposis coli
promoter hypermethylation in human
cancer. Cancer Res. 60:436671
Graff JR, Herman JG, Lapidus RG,
Chopra H, Xu R, et al. 1995. E-cadherin
expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res. 55:519599
Toyooka KO, Toyooka S, Virmani AK,
Sathyanarayana UG, Euhus DM, et al.
2001. Loss of expression and aberrant
methylation of the CDH13 (H-cadherin)
gene in breast and lung carcinomas. Cancer Res. 61:455660
Suzuki H, Watkins DN, Jair KW,
4 Dec 2004
19:7
652

70.
71.
72.
73.
74.
75.
76.
AR
AR232-PA45-26.tex
P1: JRX
ESTELLER
Schuebel KE, Markowitz SD, et al. 2004.
Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat. Genet. 36:41722
Kane MF, Loda M, Gaida GM, Lipman
J, Mishra R, et al. 1997. Methylation of
the hMLH1 promoter correlates with lack
of expression of hMLH1 in sporadic colon
tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 57:808
11
Herman JG, Umar A, Polyak K, Graff
JR, Ahuja N, et al. 1998. Incidence
and functional consequences of hMLH1
promoter hypermethylation in colorectal
carcinoma. Proc. Natl. Acad. Sci. USA 95:
687075
Esteller M, Levine R, Baylin SB, Ellenson LH, Herman JG. 1998. MLH1
promoter hypermethylation is associated
with the microsatellite instability phenotype in sporadic endometrial carcinomas.
Oncogene 17:241317
Esteller M, Catasus L, Matias-Guiu X,
Mutter GL, Prat J, et al. 1999. hMLH1
promoter hypermethylation is an early
event in human endometrial tumorigenesis. Am. J. Pathol. 155:176772
Fleisher AS, Esteller M, Wang S, Tamura
G, Suzuki H, et al. 1999. Hypermethylation of the hMLH1 gene promoter in
human gastric cancers with microsatellite
instability. Cancer Res. 59:109095
Esteller M, Hamilton SR, Burger PC,
Baylin SB, Herman JG. 1999. Inactivation of the DNA repair gene O6methylguanine-DNA methyltransferase
by promoter hypermethylation is a common event in primary human neoplasia.
Esteller M, Toyota M, Sanchez-Cespedes
M, Capella G, Peinado MA, et al.
2000. Inactivation of the DNA repair
gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation
is associated with G to A mutations in
K-ras in colorectal tumorigenesis. Cancer
Res. 60:236871
77. Esteller M, Risques RA, Toyota M,

Capella G, Moreno V, et al. 2001.
Promoter hypermethylation of the DNA
repair gene O(6)-methylguanine-DNA
methyltransferase is associated with the
presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis. Cancer Res. 61:468992
78. Esteller M, Herman JG. 2004. Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA
methyltransferase in human cancer. Oncogene 8:18
79. Mizuno K, Osada H, Konishi H, Tatematsu Y, Yatabe Y, et al. 2002. Aberrant
hypermethylation of the CHFR prophase
checkpoint gene in human lung cancers.
Oncogene 21:232833
80. Esteller M, Silva JM, Dominguez G,
Bonilla F, Matias-Guiu X, et al. 2000.
Promoter hypermethylation and BRCA1
inactivation in sporadic breast and ovarian tumors. J. Natl. Cancer Inst. 92:564
69
81. Hedenfalk I, Duggan D, Chen Y, Radmacher M, Bittner M, et al. 2001. Geneexpression profiles in hereditary breast
cancer. N. Engl. J. Med. 344:539
48
82. Ottaviano YL, Issa JP, Parl FF, Smith HS,
Baylin SB, Davidson NE. 1994. Methylation of the estrogen receptor gene CpG
island marks loss of estrogen receptor expression in human breast cancer cells.
83. Lapidus RG, Ferguson AT, Ottaviano YL,
Parl FF, Smith HS, et al. 1996. Methylation of estrogen and progesterone receptor
gene 5 CpG islands correlates with lack of
estrogen and progesterone receptor gene
expression in breast tumors. Clin. Cancer
Res. 2:80510
84. Jarrard DF, Kinoshita H, Shi Y, Hoff D,
Meisner LF, et al. 1998. Methylation of
the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells.
4 Dec 2004
19:7
AR
AR232-PA45-26.tex
P1: JRX


85. Cote S, Momparler RL. 1997. Activation
of the retinoic acid receptor beta gene by
5-aza-2 -deoxycytidine in human DLD-1
colon carcinoma cells. Anticancer Drugs
8:5661
86. Virmani AK, Rathi A, Zochbauer-Muller
S, Sacchi N, Fukuyama Y, et al. 2000.
Promoter methylation and silencing of
the retinoic acid receptor-beta gene in
lung carcinomas. J. Natl. Cancer Inst. 92:
13037
87. Esteller M, Guo M, Moreno V,
Peinado MA, Capella G, et al. 2002.
Hypermethylation-associated inactivation of the cellular retinol-binding-protein
1 gene in human cancer. Cancer Res.
62:59025
88. Yoshikawa H, Matsubara K, Qian GS,
Jackson P, Groopman JD, et al. 2001.
SOCS-1, a negative regulator of the
JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma
and shows growth-suppression activity.
Nat. Genet. 28:2935
89. Galm O, Yoshikawa H, Esteller M, Osieka
R, Herman JG. 2003. SOCS-1, a negative regulator of cytokine signaling, is
frequently silenced by methylation in
multiple myeloma. Blood 101:278488
90. He B, You L, Uematsu K, Zang K, Xu Z,
et al. 2003. SOCS-3 is frequently silenced
by hypermethylation and suppresses cell
growth in human lung cancer. Proc. Natl.
Acad. Sci. USA 100:1413338
91. Katzenellenbogen RA, Baylin SB, Herman JG. 1999. Hypermethylation of the
DAP-kinase CpG island is a common
alteration in B-cell malignancies. Blood
93(12):434753
92. Conway KE, McConnell BB, Bowring
CE, Donald CD, Warren ST, Vertino PM.
2000. TMS1, a novel proapoptotic caspase recruitment domain protein, is a
target of methylation-induced gene silencing in human breast cancers. Cancer
Res. 60:623642
93. Esteller M, Avizienyte E, Corn PG, Lothe
RA, Baylin SB, et al. 2000. Epigenetic
94.
95.
96.
97.
98.
99.
100.
101.
653
inactivation of LKB1 in primary tumors

associated with the Peutz-Jeghers syndrome. Oncogene 19:16468
Dammann R, Li C, Yoon JH, Bates S,
Pfeifer GP. 2000. Epigenetic inactivation
of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat. Genet. 25:31519
Burbee DG, Forgacs E, Zochbauer-Muller
S, Shivakumar L, Fong K, et al. 2001. Epigenetic inactivation of RASSF1A in lung
and breast cancers and malignant phenotype suppression. J. Natl. Cancer Inst.
93:69199
Irimia M, Sanchez-Cespedes M, Esteller
M. 2004. CpG island promoter hypermethylation of the Ras-effector gene
NORE1A occurs in the context of a wildtype K-ras in lung cancer. Oncogene 23:In
press
Li Q, Ahuja N, Burger PC, Issa JP.
1999. Methylation and silencing of the
thrombospondin-1 promoter in human
cancer. Oncogene 18:328489
Toyota M, Shen L, Ohe-Toyota M, Li
Q, Ohe-Toyota M, et al. 2000. Aberrant
methylation of the cyclooxygenase 2 CpG
island in colorectal tumors. Cancer Res.
60:404448
Liang G, Robertson KD, Talmadge C,
Sumegi J, Jones PA. 2000. The gene for
a novel transmembrane protein containing epidermal growth factor and follistatin
domains is frequently hypermethylated in
human tumor cells. Cancer Res. 60:4907
12
Lee WH, Morton RA, Epstein JI, Brooks
JD, Campbell PA, et al. 1994. Cytidine methylation of regulatory sequences
near the pi-class glutathione S-transferase
gene accompanies human prostatic carcinogenesis. Proc. Natl. Acad. Sci. USA
91:1173337
Esteller M, Corn PG, Urena JM, Gabrielson E, Baylin SB, Herman JG. 1998. Inactivation of glutathione S-transferase P1
gene by promoter hypermethylation in human neoplasia. Cancer Res. 58:451518
4 Dec 2004
19:7

654
AR
AR232-PA45-26.tex
P1: JRX
ESTELLER
102. Akiyama Y, Watkins N, Suzuki H, Jair

KW, van Engeland M, et al. 2003. GATA4 and GATA-5 transcription factor genes
and potential downstream antitumor target genes are epigenetically silenced in
colorectal and gastric cancer. Mol. Cell
Biol. 23:842939
103. Yamashita K, Upadhyay S, Osada M,
Hoque MO, Xiao Y, et al. 2002.
Pharmacologic unmasking of epigenetically silenced tumor suppressor genes
in esophageal squamous cell carcinoma.
Cancer Cell 2:48595
104. Chen WY, Zeng X, Carter MG, Morrell
CN, Chiu Yen RW, et al. 2003. Heterozygous disruption of Hic1 predisposes mice
to a gender-dependent spectrum of malignant tumors. Nat. Genet. 33:197202
105. Kawai J, Hirotsune SK, Hirose K, Fushiki
S, Watanabe S, Hayashizaki Y. 1993.
Methylation profiles of genomic DNA
of mouse developmental brain detected
by restriction landmark genomic scanning (RLGS) method. Nucleic Acids Res.
21:56048
106. Gonzalgo ML, Liang G, Spruck CH
3rd, Zingg JM, Rideout WM 3rd, Jones
PA. 1997. Identification and characterization of differentially methylated
regions of genomic DNA by methylationsensitive arbitrarily primed PCR. Cancer
Res. 57:59499
107. Toyota M, Ho C, Ahuja N, Jair KW, Li Q,
et al. 1999. Identification of differentially
methylated sequences in colorectal cancer
by methylated CpG island amplification.
108. Frigola J, Ribas M, Risques RA, Peinado
MA. 2002. Methylome profiling of cancer
cells by amplification of inter-methylated
sites (AIMS). Nucleic Acids Res. 30:
e28
109. Shiraishi M, Chuu YH, Sekiya T. 1999.
Isolation of DNA fragments associated
with methylated CpG islands in human
adenocarcinomas of the lung using a
methylated DNA binding column and
denaturing gradient gel electrophoresis.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.

18
Huang TH, Perry MR, Laux DE. 1999.
Methylation profiling of CpG islands in
human breast cancer cells. Hum. Mol.
Genet. 8:45970
Gitan RS, Shi H, Chen CH, Yan PS,
Huang TH. 2002. Methylation-specific
oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res. 12:15864
Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, et al. 2002.
A genomic screen for genes upregulated
by demethylation and histone deacetylase inhibition in human colorectal cancer.
Nat. Genet. 31:14149
Jeronimo C, Usadel H, Henrique R,
Oliveira J, Lopes C, et al. 2001.
Quantitation of GSTP1 methylation in
non-neoplastic prostatic tissue and organconfined prostate adenocarcinoma. J.
Natl. Cancer Inst. 93:174752
Esteller M. 2003. Relevance of DNA
methylation in the management of cancer.
Lancet Oncol. 4:35158
Laird PW. 2003. The power and the
promise of DNA methylation markers.
Nat. Rev. Cancer 3:25366
Esteller M, Sanchez-Cespedes M, Rosell
R, Sidransky D, Baylin SB, Herman JG.
1999. Detection of aberrant promoter hypermethylation of tumor suppressor genes
in serum DNA from non-small cell lung
cancer patients. Cancer Res. 59:6770
Palmisano WA, Divine KK, Saccomanno
G, Gilliland FD, Baylin SB, et al. 2000.
Predicting lung cancer by detecting aberrant promoter methylation in sputum.
Tang X, Khuri FR, Lee JJ, Kemp BL,
Liu D, et al. 2000. Hypermethylation of
the death-associated protein (DAP) kinase
promoter and aggressiveness in stage I
non-small-cell lung cancer. J. Natl. Cancer Inst. 92:151116
Esteller M, Gonzalez S, Risques RA, Marcuello E, Mangues R, et al. 2001. K-ras
4 Dec 2004
19:7
AR
AR232-PA45-26.tex
P1: JRX

120.
121.
122.
123.
124.
125.
126.
127.
and p16 aberrations confer poor prognosis

in human colorectal cancer. J. Clin. Oncol. 19:299304
Esteller M, Garcia-Foncillas J, Andion E,
Goodman SN, Hidalgo OF, et al. 2000. Inactivation of the DNA-repair gene MGMT
and the clinical response of gliomas
to alkylating agents. N. Engl. J. Med.
343:135054
Balana C, Ramirez JL, Taron M, Roussos
Y, Ariza A, et al. 2003. O6-methylguanine-DNA methyltransferase methylation in serum and tumor DNA predicts
response to 1,3-bis(2-chloroethyl)-1nitrosourea but not to temozolamide plus
cisplatin in glioblastoma multiforme.
Esteller M, Gaidano G, Goodman SN,
Zagonel V, Capello D, et al. 2002. Hypermethylation of the DNA repair gene
O(6)-methylguanine DNA methyltransferase and survival of patients with diffuse
large B-cell lymphoma. J. Natl. Cancer
Inst. 94:2632
Paz MF, Yaya-Tur R, Rojas-Marcos I,
Reynes G, Pollan M, et al. 2004. CpG
island hypermethylation of the DNA repair enzyme MGMT predicts response to
temozolomide in primary gliomas. Clin.
Plumb JA, Strathdee G, Sludden J, Kaye
SB, Brown R. 2000. Reversal of drug
resistance in human tumor xenografts by
2 -deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter.
Lafarge S, Sylvain V, Ferrara M,
Bignon YJ. 2001. Inhibition of BRCA1
leads to increased chemoresistance to
microtubule-interfering agents, an effect
that involves the JNK pathway. Oncogene
20:6597606
Villar-Garea A, Esteller M. 2003. DNA
demethylating agents and chromatinremodelling drugs: which, how and why?
Curr. Drug. Metab. 4:1131
Paz MF, Fraga MF, Avila S, Guo M, Pollan M, et al. 2003. A systematic profile
128.
129.
130.
131.
132.
133.
134.
135.
136.
655
of DNA methylation in human cancer cell

lines. Cancer Res. 63:111421
Nieto M, Samper E, Fraga MF, Gonzalez
de Buitrago G, et al. 2004. The absence of
p53 is critical for the induction of apoptosis by 5-aza-2 -deoxycytidine. Oncogene
23:73543
Lin X, Asgari K, Putzi MJ, Gage WR, Yu
X, et al. 2001. Reversal of GSTP1 CpG
island hypermethylation and reactivation of pi-class glutathione S-transferase
(GSTP1) expression in human prostate
cancer cells by treatment with procainamide. Cancer Res. 61:861116
Villar-Garea A, Fraga MF, Espada
J, Esteller M. 2003. Procaine is a
DNA-demethylating agent with growthinhibitory effects in human cancer cells.
Jones PA, Taylor SM. 1980. Cellular differentiation, cytidine analogs and DNA
methylation. Cell 20:8593
Li LH, Olin EJ, Buskirk HH, Reineke LM.
1970. Cytotoxicity and mode of action of
5-azacytidine on L1210 leukemia. Cancer
Res. 30:276069
Rivard GE, Momparler RL, Demers J,
Benoit P, Raymond R, et al. 1981. Phase
I study on 5-aza-2 -deoxycytidine in children with acute leukemia. Leukemia Res.
5:45362
Poot M, Koehler J, Rabinovitch PS,
Hoehn H, Priest JH. 1990. Cell kinetic disturbances induced by treatment of human
diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation
in the regulation of the chromosome cycle. Hum. Genet. 84:25862
Kornblith AB, Herndon JE 2nd, Silverman LR, Demakos EP, Odchimar-Reissig
R, et al. 2002. Impact of azacytidine on the
quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a cancer and leukemia
group B study. J. Clin. Oncol. 20:2441
52
Silverman LR, Demakos EP, Peterson
BL, Kornblith AB, Holland JC, et al. 2002.
4 Dec 2004
19:7
656

137.
138.
139.
140.
141.
142.
AR
AR232-PA45-26.tex
P1: JRX
ESTELLER
Randomized controlled trial of azacitidine
in patients with the myelodysplastic syndrome: a study of the cancer and leukemia
group B. J. Clin. Oncol. 20:242940
Laliberte J, Marquez VE, Momparler RL.
1992. Potent inhibitors for the deamination of cytosine arabinoside and 5aza-2 -deoxycytidine by human cytidine
deaminase. Cancer Chemother. Pharmacol. 30:711
Ho DH. 1973. Distribution of kinase and deaminase of 1-beta-D-arabinofuranosylcytosine in tissues of man and
mouse. Cancer Res. 33:281620
Cheng JC, Matsen CB, Gonzales FA, Ye
W, Greer S, et al. 2003. Inhibition of DNA
methylation and reactivation of silenced
genes by zebularine. J. Natl. Cancer Inst.
95:399409
Cheng JC, Weisenberger DJ, Gonzales
FA, Liang G, Xu GL, et al. Continuous
zebularine treatment effectively sustains
demethylation in human bladder cancer
cells. Mol. Cell Biol. 24:127078
Wijermans PW, Krulder JW, Huijgens
PC, Neve P. 1997. Continuous infusion
of low-dose 5-aza-2 -deoxycytidine in elderly patients with high-risk myelodysplastic syndrome. Leukemia 11:15
Schwartsmann G, Fernandes MS,
Schaan MD, Moschen M, Gerhardt
LM, et al. 1997. Decitabine (5-aza-2 -
143.
144.
145.
146.
147.
deoxycytidine; DAC) plus daunorubicin

as a first line treatment in patients with
acute myeloid leukemia: preliminary
observations. Leukemia 11(Suppl. 1):
S2831
Wijermans P, Lubbert M, Verhoef G,
Bosly A, Ravoet C, et al. 2000. Lowdose 5-aza-2 -deoxycytidine, a DNA hypomethylating agent, for the treatment
of high-risk myelodysplastic syndrome: a
multicenter phase II study in elderly patients. J. Clin. Oncol. 18:95662
Ballestar E, Esteller M. 2002. The impact of chromatin in human cancer: linking DNA methylation to gene silencing.
Daskalakis M, Nguyen TT, Nguyen
C, Guldberg P, Kohler G, et al.
2002. Demethylation of a hypermethylated P15/INK4B gene in patients
with myelodysplastic syndrome by 5-aza2 -deoxycytidine (decitabine) treatment.
Blood 100:295764
Lo Coco F, Zelent A, Kimchi A, Carducci
M, Gore SD, Waxman S. 2002. Progress
in differentiation induction as a treatment
for acute promyelocytic leukemia and beyond. Cancer Res. 62:561821
Soengas MS, Capodieci P, Polsky D,
Mora J, Esteller M, et al. 2001. Inactivation of the apoptosis effector Apaf-1 in
melanoma. Nature 409:20711
1/7/05
2:31 PM
Figure 3 Illustrative examples of DNA methylation analysis. (A) Chromatograms of the bisulfite genomic sequencing of a small fragment of a CpG island: left, unmethylated sequence (cytosines changed to thymines); right, methylated sequence (cytosines remined as
cytosines). (B) Methylation-specific PCR in primary tumors. The presence of a band under the M lanes represents hypermethylated neoplasms. (C) Staining for the 5-methylcytosine antibody in a cancer cell line.

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Figure 4 Cancer as a poligenetic and poliepigenetic disease: the model of mouse multistage skin carcinogenesis. Through all the tumoral
different stages (from benign lesions to invasive carcinomas) there is an accumulation of gene mutations, but also a double epigenetic
lesion: an increase in the number of genes undergoing methylation-associated silencing and in the degree of genomic hypomethylation
(14).

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Copyright
THE CARDIAC FIBROBLAST: Therapeutic Target in

Myocardial Remodeling and Failure

R. Dale Brown, S. Kelly Ambler, M. Darren Mitchell,
and Carlin S. Long
Division of Cardiology, University of Colorado Health Sciences Center, and Denver
Health Medical Center, Denver, Colorado 80262; email: Dale.Brown@uchsc.edu,
Kelly.Ambler@uchsc.edu, Darren.Mitchell@uchsc.edu, Carlin.Long@dhha.org
Key Words
fibrosis, myocardial infarction, heart failure, risk factors, clinical trials
Abstract Cardiac fibroblasts play a central role in the maintenance of extracellular

matrix in the normal heart and as mediators of inflammatory and fibrotic myocardial
remodeling in the injured and failing heart. In this review, we evaluate the cardiac
fibroblast as a therapeutic target in heart disease. Unique features of cardiac fibroblast cell biology are discussed in relation to normal and pathophysiological cardiac
function. The contribution of cardiac fibrosis as an independent risk factor in the outcome of heart failure is considered. Candidate drug therapies that derive benefit from
actions on cardiac fibroblasts are summarized, including inhibitors of angiotensinaldosterone systems, endothelin receptor antagonists, statins, anticytokine therapies,
matrix metalloproteinase inhibitors, and novel antifibrotic/anti-inflammatory agents.
These findings point the way to future challenges in cardiac fibroblast biology and
pharmacotherapy.
INTRODUCTION
As global human populations develop economically and technologically, human
physiological function is faced with challenges outside evolutionary experience.
The consequence is a paradigm shift in the causes of human mortality away from
extrinsic factors, such as infectious disease or nutritional insufficiency, and toward
failures of intrinsic physical function owing to longer life span or inherent genetic
abnormalities. These circumstances have resulted in a pandemic of heart disease.
In the United States alone, heart failure accounts for 400,000700,000 deaths
per year, $20$40 billion in yearly healthcare costs, and is the leading hospital
discharge diagnosis (1). These considerations provide the impetus for an ongoing
search for novel approaches to therapy.
Heart failure reflects the end result of a variety of primary or secondary causes,
including the hereditary and idiopathic cardiomyopathies, and the sequelae of
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hypertension, coronary artery disease, infectious myocarditis, and alcohol abuse

or other toxic insults (2). Irrespective of the underlying cause, heart failure is
defined functionally as an insufficient pumping capacity to meet the metabolic
needs of the tissues. This operational definition reflects the historical development
of cardiology, which until recent years has focused almost exclusively on the
muscular compartment of the heart, and at the cell and molecular level upon the
biology of the cardiac myocyte.
However, the nonmyocyte cell populations of the heart are increasingly appreciated to contribute to the performance of the normal and failing heart. In particular,
cardiac fibroblasts have been recognized to constitute the major nonmyocyte cell
type of the heart numerically and contribute importantly to multiple aspects of
myocardial function and pathophysiology.
In this review, we evaluate the cardiac fibroblast as a therapeutic target in heart
disease. We explore the biology of the cardiac fibroblast as a unique cell type,
distinct from fibroblasts in other organs and tissues. The role of the cardiac fibroblast in the function of the heart is discussed in the context of the etiology
and progression of heart failure. Established and emerging therapeutic agents that
derive benefit through actions on cardiac fibroblasts are summarized. Key unanswered questions in cardiac fibroblast biology are identified as they relate to novel
therapeutic targets in cardiac fibrosis and heart failure.
BIOLOGY OF THE CARDIAC FIBROBLAST

Fibroblast Function in Normal, Injured,
and Failing Myocardium
Cardiac fibroblasts are recognized as the cell type primarily responsible for homeostatic maintenance of extracellular matrix (ECM) in the normal heart. Cardiac
ECM is a highly differentiated structure (3; Figure 1). Myocytes are surrounded
by a basement membrane whose principal structural component is nonfilamentous type IV collagen. Collagen fibrils composed primarily of collagen I with
smaller amounts of collagen III are arranged in successive layers of organization.
The endomysium consists of a loose weave of fibrils wrapped around individual
myocytes, plus collagen struts that interconnect adjoining myocytes. Bundles of
myocytes are surrounded by a collagen sheath termed the perimysium. Perimysial
bundles are encased in a collagenous fascia, the epimysium. In addition, the intracoronary arterioles, which possess their own connective tissue tunica adventitia
around the outer vessel lamina, are integrated into the cardiac ECM by additional
collagen fibers.
In keeping with the structural and mechanical role of cardiac ECM, the major
constituents are the fibrillar collagens I (80%) and III (10%), with smaller
amounts of collagens IV, V, VI, elastin, laminin, proteoglycans, glycosaminoglycans, and others (4, 5). In addition, the ECM is decorated with a diverse assortment
of growth factors, proteases, and other molecules. These entities, many of which
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CARDIAC FIBROBLASTS IN HEART THERAPY
659
Figure 1
Structural organization of cardiac ECM. Schematic arrangement of fibrillar collagen in relation to cardiac myocytes and the coronary vasculature is shown.
Collagen weave surrounding individual myocytes and collagen struts tethering adjacent myocytes comprise the endomysium. Groups of myocytes are bundled within the
perimysium. The epimysium encloses groups of perimysial bundles. Capillaries and
coronary microvessels have free diffusion access to cardiac myocytes throughout the
ECM. Adapted from References 3 and 49.
are sequestered in inactive forms, serve important roles in regulating cell function
upon disruption of the ECM (6).
Extracellular matrix homeostasis involves ongoing cycles of synthesis and
degradation. Both synthetic and degradative aspects of collagen metabolism are
tightly regulated (5, 7). Fibrillar collagen is synthesized as a precursor polypeptide, exported from the cell, and proteolytically processed by removal of aminoand carboxy-terminal propeptides before insertion into nascent fibrils. Collagen
monomers are then cross-linked through hydroxyproline and hydroxylysine
residues to produce the mature structure. Mature fibrillar collagen is highly stable, with a turnover half-life of around 100 days in normal myocardium. Collagen biosynthesis is regulated transcriptionally by fibrogenic growth factors,
particularly TGF, and posttranscriptionally by the rate-limiting enzyme prolyl4-hydroxylase (5, 7). Collagen degradation is accomplished in stepwise fashion by members of the matrix metalloproteinase (MMP) superfamily, which are
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themselves under multiple levels of regulatory control. MMP production is regulated by three mechanisms: transcriptionally; posttranscriptionally by the activation of the proenzyme to the active form; and posttranslationally by endogenous
pseudosubstrate antagonists, the tissue inhibitors of metalloproteinases (TIMPs).
Of diagnostic significance, collagen metabolism has been monitored in vivo by
immunochemical measurement of key metabolic products and enzymes in serum
(Figure 2). Synthesis of collagens I or III produce stoichiometric equivalents of
the procollagen amino-terminal peptides (PINP, PIIINP) and procollagen carboxyterminal peptides (PICP and PIIICP), respectively, which are cleared from the circulation by the liver. Degradation of collagens I and III releases the corresponding
carboxy telopeptides, ICTP or IIICTP, which are excreted by the kidney (5, 7,
8). Serum concentrations of specific MMPs and TIMPs reflect the release of these
Figure 2
Extracellular metabolism of fibrillar collagen. Monomers of collagens I
and III are exported from the cell as propeptides and assembled. Amino- and carboxyterminal propeptides (P[I/III]NP and P[I/III]CP, respectively) are released into the
interstitial space during assembly. Mature collagen fibrils are further processed by
cross-linking through hydroxylated lysine and proline residues. Degradation of collagen fibrils by collagenase (MMP-1) releases stable carboxy-terminal telopeptides
([I/III]CTP).
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molecules into the interstitial space and are measured by ELISA techniques. These
methods have been validated to monitor altered ECM metabolism in the context
of heart disease (9).
Cardiac fibroblasts are activated in response to myocardial infarction (MI) or
injury and participate as key cells in the wound healing response. In common
with injury responses in other tissues, myocardial injury sets in motion a complex sequence of events involving coordinated interactions among multiple cell
types in the wound environment (10). The sequential steps in response to injury
include hemostasis, infiltration of immune and inflammatory cells, degradation
and phagocytosis of necrotic myocytes and cellular debris, repopulation of cardiac
fibroblasts within the zone of injury by chemotaxis and increased proliferation,
reconstruction of a granulomatous scar, and subsequent ECM remodeling to produce a mature scar. Thus, net ECM degradation, resulting from increased MMP
expression, dominates the initial phase of the injury response, whereas net ECM
deposition, arising from enhanced collagen synthesis, dominates the later phase
of healing.
Transitions of fibroblast phenotype and functional capabilities are regulated by
cytokines and growth factors released from other cell types and by the fibroblasts
themselves. Cardiac fibroblasts serve important roles as intermediate sensors and
amplifiers of signals from immune cells and myocytes, through production of autocrine and paracrine mediators such as cytokines, growth factors, prostaglandins,
and nitric oxide (NO) (reviewed in 10, 17). These agents are presumed to regulate the functional responses of cardiac fibroblasts through intracellular signaling networks, which converge at the level of transcription of coordinated gene
programs.
In the heart, as in other systems, termination of injury responses appears to
occur by apoptosis of activated cells (11). Chronic or repeated injury in the heart
ultimately overcomes the compensatory reactions of the myocardium. The ensuing progression to heart failure is accompanied by persistent inflammation and
fibrosis. The mechanisms that govern the resolution of acute injury responses,
versus the transition to chronic activation of cardiac fibroblasts, are not well
understood.
Myofibroblasts have been described as a specialized phenotype of activated
fibroblasts (12). These cells express contractile proteins, including smooth muscle -actin, vimentin, and desmin; effectively contract collagen gels in vitro;
and are postulated to be important for wound closure and structural integrity of
healing scars. In addition to normal wound healing, myofibroblasts are associated with hypertrophic fibrotic scars in injury models from multiple organ systems, and differentiation to the myofibroblast phenotype is strongly promoted by
the reference fibrogenic growth factor TGF. Myofibroblast apoptosis has been
associated with progression of granulomatous tissue to a mature scar, whereas
failure of myocyte apoptosis has been suggested to drive the progression to fibrosis (13). Cardiac myofibroblasts were shown to persist in mature infarct scars
(14).
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The Cardiac Fibroblast is a Unique Cell Type

Fibroblasts traditionally have been viewed as a uniform (and rather boring!) cell
type with equivalent functional capacity regardless of the tissue of origin. More
recently, this view has been challenged by experimental evidence suggesting
that intrinsic phenotypic heterogeneity exists among fibroblasts from different
tissues.
First, cardiac fibroblasts originate from a specific spatiotemporal locus in the
developing embryo and undergo a specific developmental sequence to acquire
their differentiated phenotype (15, 16). Following formation of the primitive heart
tube, extracardiac cells from the same coelomic splanchnopleura that produced
the heart tube migrate onto the external surface of the developing heart and
give rise to the proepicardium. These cells undergo an epithelial to mesenchymal transition (EMT) and migrate into the developing heart to form the coronary
vasculature and the cardiac fibroblasts. Differentiation to cardiac fibroblasts is
regulated by programmed sequences of growth factors, including FGF and PDGF
(16, 16a).
Additional evidence for phenotypic diversity among fibroblastic cells comes
from comparative studies on cellular responses to experimental stimuli in vitro.
In this regard, we reviewed the published literature on fibroblasts from skin, joint
synovium, lung, liver, and heart to compare responses to the proinflammatory
cytokines IL-1, TNF, IFN , and IL-6. We examined the functional endpoints
of fibroblast proliferation, chemotaxis, and extracellular matrix metabolism (17).
Superimposed on common themes of fibroblast function, we found significant
variations in responses to specific cytokines among individual tissues.
Work from our laboratory using cultured neonatal rat cardiac fibroblasts has
demonstrated unique features of cytokine responses in these cells. We find that IL1 strongly inhibits cardiac fibroblast proliferation (18) and enhances chemotaxis, in
concert with increased expression of cell adhesion molecules (19) and MMPs, but
diminished collagen biosynthesis (R.D. Brown, G.M. Jones, M. Atz, K. Spicka &
C.S. Long, unpublished data). In addition, IL-1 activates coordinate expression of
mRNAs for pro- and antiinflammatory cytokines and mediators, including TGF1
and inducible nitric oxide synthase (2022). IL-1 is by far the most robust and
multipotent agonist in these cells. TNF and IFN are less effective by themselves
but potentiate antiproliferative and pro-migratory responses of IL-1. The dominant
actions of IL-1, and its striking antiproliferative and pro-migratory effects contrast
with phenotypic responses in other fibroblast types and are likely to reflect specific
properties of the cardiac fibroblast.
Moreover, reports from kidney (23), lung (24), and skin (25) suggest that heterogeneities can be identified among fibroblasts even within a single tissue. These
observations argue that fibroblasts exist with specialized functional portfolios toward endpoint responses such as proliferation, migration, or ECM metabolism.
Further diversity has recently been recognized among fibroblasts regarding their
mobilization in response to injury. The conventional view holds that quiescent fibroblasts present in normal tissue undergo a phenotypic transition in response
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to injurious stimuli. However, emerging data from other systems suggest three
possible sources: (a) epithelial-to-mesenchymal transition from epithelial cells
(26, 27); (b) recruitment of circulating, collagen-secreting, bone marrowderived
hematopoietic precursor cells described as fibrocytes (28, 29); and (c) activation of
resident fibroblasts (28, 30). More than one route of recruitment may occur within
a single tissue (26, 28). In irradiation-induced pulmonary fibrosis, Hashimoto et al.
observed that myofibroblasts arose from resident fibroblasts, whereas the principal collagen-producing fibroblasts actually derived from bone marrow recruitment
(28). By contrast, peritoneal myofibroblasts were reported to derive from circulating precursor cells (31). These observations provide exciting new insights into the
biology of wound healing.
CLINICAL AND PATHOPHYSIOLOGICAL

CONSEQUENCES OF CARDIAC FIBROSIS
Changes in myocardial structure and function in response to injury, collectively
referred to as myocardial remodeling (32), may initially augment cardiac performance, but over the longer term may progress to a maladaptive response and heart
failure. In terms of the cardiac myocyte, these alterations include myocyte hypertrophy; disarray of myocyte organization; and increased wall thickness, which in a
majority of cases is followed by wall thinning and chamber dilation, with accompanying myocyte apoptosis or necrosis. Concomitant changes in cardiac fibroblasts
include increased fibroblast proliferation as well as accelerated and aberrant remodeling of extracellular matrix and net accumulation of ECM, resulting in cardiac
fibrosis (7, 33). This fibrosis may be reparative, replacing areas of myocyte loss
with structural scar, or reactive, involving diffuse increases in ECM deposition at
sites unrelated to focal injury. Perivascular fibrosis surrounding coronary arterioles
is also noted. Differences in the characteristics of fibrosis are observed depending
on the heart disease etiology.
Fibrosis has important functional consequences for the heart. First, increased
ECM content results in exaggerated mechanical stiffness and contributes to diastolic dysfunction. Progressive increases in fibrosis can cause systolic dysfunction and left ventricular hypertrophy (LVH). Second, increased collagen content
disrupts electrotonic connectivity between cardiac myocytes and provides an electrical substrate for reentrant arrhythmogenesis. Third, perivascular fibrosis surrounding intracoronary arterioles impairs myocyte oxygen availability, reduces
coronary reserve, and exacerbates myocyte ischemia.
Within this framework, heart failure is characterized by substantial heterogeneity of disease severity and progression even in cases of comparable heart failure
etiologies, presumably reflecting polygenic and environmental influences in the
disease phenotypes of individual patients. In this regard, we may consider the
hypothesis that properties of cardiac fibroblasts, and consequently fibrotic remodeling, act as disease modifiers, and more specifically, as independent predictive
risk factors in heart failure.
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Hypertrophic Cardiomyopathy
Hypertrophic cardiomyopathy (HCM; defined as increased septum and LV wall
thickness inappropriate for hemodynamic load) in affected individuals may be
asymptomatic or lead to alternate outcomes, including atrial fibrillation, symptomatic cardiac dysfunction progressing to heart failure, or sudden cardiac death
(SCD) from ventricular tachycardia (34). The latter result is encountered in young
competition athletes, where sudden death may be the first symptom of HCM. At
the cellular level, HCM is most commonly associated with myocyte hypertrophy
and myocyte disarray, accompanied by replacement fibrosis at foci of myocyte
death.
HCM in most cases has been shown to arise from hereditary or spontaneous
mutations in myocyte sarcomeric proteins responsible for force generation (35).
This observation provides proof of the principle that a single gene defect in the
cardiac myocyte is sufficient to drive the full syndrome of cardiac hypertrophy
and failure. However, the relationship between mutational genotype and disease
phenotype is highly variable and not well understood (36). These findings clearly
suggest the presence of additional modifiers of disease.
The association of cardiac fibrosis with diastolic dysfunction and SCD has been
studied in HCM. Both myocyte disarray and fibrosis are associated with diastolic
dysfunction and electrical instability in HCM patients (37, 38). Varnava et al.
(39), in examination of autopsy specimens from HCM-induced sudden death,
concluded that myocyte disarray correlated most strongly with SCD in young
patients, whereas fibrosis was associated with SCD in older patients. By contrast,
a second autopsy study by Shirani et al. (40) found morphologic abnormalities
and increased amounts of ECM in children and young-adult SCD victims, arguing
that expanded ECM is involved early in the disease process. These data clearly
suggest an association between cardiac fibrosis and HCM severity, although the
link to SCD needs clarification.
Dilated and Ischemic Cardiomyopathies

Dilated heart failure is the most commonly encountered end-stage of heart failure
progression, accounting for approximately 80% of cases, and may result from primary and secondary causes, including ischemic heart disease, hypertensive heart
disease, or the idiopathic dilated cardiomyopathies (reviewed in 41). Up to 30% of
dilated heart failure cases appear to be due to a diverse array of mutations in proteins
for force generation, force transmission, energy metabolism, and nuclear structure.
The variable penetrance of familial dilated and ischemic cardiomyopathies (DCM)
suggests the presence of disease modifier genes. Chamber dilation and wall thinning are associated with mild to moderate myocyte hypertrophy, but less myocyte
disarray than accompanies HCM. Diffuse interstitial and perivascular fibrosis are
often evident but vary substantially (42).
Significant associations of increased collagen deposition and elevated collagen
I:collagen III composition have been observed in autopsy specimens (43) or failing explanted hearts (44). Increased ECM remodeling on endomyocardial biopsy
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was associated with deterioration of LV performance on echocardiography (45).

Importantly, a prospective study on serum markers of collagen metabolism in idiopathic or ischemic DCM showed that patients at the upper strata of values for
serum collagen markers were at increased risk for advanced clinical stage of heart
failure, poor hemodynamic condition, transplantation, or death during follow-up
(46). These results suggest a definite association of DCM with fibrosis; more severe fibrotic reactions may act as an independent factor for risk stratification. It
should be noted that the effects of somatic mutations in noncontractile proteins
described in DCM have not been evaluated in cardiac fibroblasts.
Hypertensive Heart Failure

Hypertension exerts characteristic adverse effects on the heart, including LV hypertrophy, thickening of intracoronary arterioles, and cardiac fibrosis (47). The
causes of cardiac fibrosis in hypertensive heart disease have been attributed to
a combination of hemodynamic (pressure overload) and humoral factors (AngII,
ET-1, TGF; 48).
Using formalin fixation combined with alkaline digestion of cellular constituents
to selectively visualize the cardiac extracellular matrix, Rossi (49) obtained striking microscopic images showing changes in the ultrastructural organization of
collagen fibers relative to increasing degrees of LVH in autopsy heart specimens
with hypertensive heart disease. In mild hypertension, there was diffuse reactive
fibrosis with net increased collagen accumulation in endomysium and perimysium
and evidence of myocyte hypertrophy, but the overall structure of the ECM was
preserved. With increasing LVH, and particularly in the severely affected group,
greatly expanded and disorganized ECM deposition was observed, reflecting areas of myocyte death and replacement fibrosis interspersed with extreme myocyte
hypertrophy.
Previous studies have shown a correlation between fibrosis and diastolic dysfunction in hypertensive heart disease (50). These findings were extended by
Querejeta et al. (9) to show a predictive relationship between serum concentrations of PICP and endomyocardial fibrosis in hypertensive patients. In a second
study, these authors showed that hypertensives with LVH and renal fibrosis had
higher serum PICP than uncomplicated hypertensives or control subjects. Further,
six months of treatment with the angiotensin receptor blocker losartan revealed a
nonresponder subpopulation of these highly fibrotic patients whose serum markers were not improved by therapy, despite normalization of blood pressure. These
data indicate that fibrotic responses may contribute to hypertensive disease and
therapeutic outcomes independent of hemodynamic factors (33).
Cardiac Fibrosis and Arrhythmia

As alluded to above, fibrosis exerts adverse impacts on cardiac electrical properties
in addition to effects on the mechanical properties of the myocardium. The specific
relationship between fibrosis and arrhythmogenesis depends on the cardiomyopathy and the structural details of myocardial remodeling (reviewed in 51). Myocyte
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loss owing to apoptotic or necrotic mechanisms is followed by replacement fibrosis,

resulting in electrical isolation of myocytes, and introduction of alternate conductance pathways for reentrant arrhythmias. Arrhythmogenesis owing to reentrant
pathways commonly arise as a consequence of coronary artery disease resulting in
ischemic heart failure (52), MI (53), congestive (pressure-overload) heart failure
(54), or atrial fibrillation in elderly patients (55). These conductance disturbances
may be exacerbated by perivascular or reactive fibrosis, which impairs myocyte
oxygen availability. By contrast, arrhythmias may arise from focal mechanisms not
associated with sites of fibrosis, presumably arising from primary myocyte dysfunction and the resultant myocyte disarray, in idiopathic cardiomyopathy (56),
primary atrial fibrillation (57), or hypertrophic cardiomyopathy in young patients
(39). However, even in these cases fibrosis may provide adjunct sites for conduction
delay or block (56).
Arrhythmogenic right ventricular dysplasia (ARVD) is an extreme example
of a fibrotic cardiomyopathy and its consequences (41, 58). ARVD is at least
partly hereditary, of variable severity, characterized by fibrofatty replacement of
myocytes particularly in the right ventricle, and associated with a high frequency
of sudden cardiac death before middle age. Initial genetic mapping studies have
identified mutations in the ryanodine receptor, which is involved in intracellular
Ca release, and in desmoplakin, a constituent of myocyte desmosomes consistent
with a primary myocyte dysfunction leading to cell death. Arrhythmogenesis in
ARVD commonly appears to be associated with reentrant pathways arising from
replacement fibrosis at sites of myocyte loss.
In summary, cardiac fibrosis is clearly associated with altered myocardial mechanical performance and arrhythmogenesis. In some studies this association has
been extended to a statistical correlation of fibrosis severity with myocardial function. However, there are relatively few data that quantitate cardiac fibrosis as an
independent and predictive risk factor for heart disease outcome or therapeutic
effect.
THERAPIES DIRECTED AT THE CARDIAC FIBROBLAST

The importance of fibrosis as a determinant of myocardial performance and disease outcome is increasingly appreciated. Nevertheless, efforts to develop novel
therapies that specifically target the cardiac fibroblast are at a relatively early stage,
in common with approaches to fibrotic disease in other organ systems. This section discusses established and emerging therapies directed at cardiac fibroblasts in
heart disease. A summary of this discussion appears in Table 1.
Renin-Angiotensin System: ACE Inhibitors and

Angiotensin Receptor Antagonists
The renin-angiotensin system (RAS), through the production and actions of angiotensin II (AngII) at its receptors, plays a key role in the compensatory
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TABLE 1 Antifibrotic actions of current drug therapies

Agent class
Mechanism of action,
if known
Renin-angiotensin-aldosterone inhibitors
ACE
Enzymatic inhibitors of
inhibitors
angiotensin converting
enzyme, reduce Ang II
production
Angiotensin
AT1 receptor antagonists
receptor
blockers
Aldosterone
Mineralocorticoid receptor
antagonists
antagonists
Therapeutic status and

actions on cardiac fibrosis
References
Approved for heart failure;

probable beneficial actions
on cardiac fibrosis
(60, 61,
6365)

on cardiac fibrosis
on cardiac fibrosis
(6669)
(7173)
Endothelin receptor
antagonists
ETA -ETB endothelin

receptor antagonists
Failed Phase III trial for heart

failure
(77)
Statins
HMG-CoA reductase
inhibitors
Approved lipid lowering

drugs; investigational for
heart failure; effects on
fibrosis unknown
(8183)
Inhibit TNF availability
Approved for rheumatoid

arthritis; failed Phase III for
heart failure
Investigational, Phase III in
progress for pulmonary
fibrosis
Investigational, Phase III in
progress for
post-opthalmologic surgery
(84)
Cytokine therapies
Anti-TNF
IFN
Inhibit myofibroblasts,
collagen synthesis
Anti-TGF
Inhibit TGF availability or

action
(85)
(89)
MMP inhibitors
Enzyme inhibitors of MMPs,

prevent ECM remodeling
Failed Phase III trials for

different applications;
approved for periodontal
disease
(103, 123,
124)
Pirfenidone
Unknown; inhibits TGF1,

anti-inflammatory
Investigational for pulmonary

and renal fibrosis
(109, 110)
Tranilast
Unknown; antifibrotic and

antiinflammatory
Failed Phase III for

antiatherosclerosis
(111)
Nuclear receptor
agonists
PPAR agonists
Activate nuclear PPAR or

PPAR , respectively
Approved lipid lowering
drugs
Approved for Type II
diabetes
(114, 115)
PPAR agonists
(118)
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neurohumoral response to myocardial injury. The myocardium of animals and

humans contains an endogenous RAS independent of the renovascular RAS (reviewed in 7, 59). Stimulation with AngII results in cardiac fibroblast proliferation
and net accumulation of fibrillar collagen in vitro and cardiac fibrosis in vivo. These
responses are transduced by AT1 receptors, and their expression in cardiac fibroblasts far exceeds that in myocytes (20). An important aspect of AngII function
occurs through upregulation of additional fibrogenic growth factors, which mediate or augment the direct effects of AngII, including endothelin-1 (ET-1), TGF1,
and, as discussed below, aldosterone. In this context, Weber et al. have reported
upregulation of angiotensin production, angiotensin AT1 receptors, and increased
collagen mRNA in myofibroblasts associated with healing infarct scars (14).
Large clinical trials have shown that angiotensin-converting enzyme inhibitors
(ACE-I) reduce morbidity and mortality, slow the progression of established heart
failure (60), and reduce cardiovascular events in patients at risk but without symptomatic heart failure (61). A significant component of the therapeutic benefit has
been interpreted to be independent of blood pressurelowering effects, suggesting
actions on cardiac remodeling (but see 62).
Benefits of ACE-I therapy on cardiac fibrosis and myocardial performance have
been shown in limited populations of hypertensive patients with symptomatic heart
disease. In a prospective study, Brilla et al. found that six months of treatment with
the ACE-I lisinopril in patients with hypertensive heart disease reduced cardiac
fibrosis and improved left ventricular diastolic function, whereas the diuretic hydrochlorothiazide had comparable blood pressurelowering effects but did not
improve fibrosis or diastolic function (63). Schwartzkopff et al. reported that treatment of patients with hypertensive heart disease with the ACE-I perindopril for
12 months caused a significant regression of periarteriolar fibrosis and a marked
improvement in coronary blood flow reserve (64).
In addition to benefits in the treatment of chronic heart failure, ACE-I have
consistently been shown to improve survival when administered within the first
seven days following acute MI (65). Studies in experimental animals suggest that
ACE-I regulate both synthetic and collagenolytic aspects of ECM metabolism in
the early response to MI. However, AngII regulation of ECM metabolism in the
setting of acute MI is not understood. This will be an important area for continuing
research.
Advances in the molecular pharmacology of the AT1 receptor have led to the
development of angiotensin AT1 receptor blockers (ARB) as an alternate therapeutic approach to the ACE inhibitors. ARBs appear to offer clinical benefits similar
to ACE-I in heart failure therapy (66, 67). Antifibrotic actions of the ARB losartan
have also been studied in two small prospective studies. In a series of 37 patients
with hypertensive heart disease, 12 months of treatment with losartan reduced
cardiac fibrosis and serum collagen markers, whereas the calcium channel blocker
amlodipine had no effect despite similar hemodynamic normalization (68). In a
second series of patients with hypertensive heart disease, losartan treatment had
selective benefit to reduce collagen deposition and LV stiffness in more severely
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fibrotic patients than in patients with nonsevere fibrosis. These results suggest
heterogeneity of fibrosis responses to therapy (69).
In summary, inhibitors of the RAS clearly appear to derive a significant portion
of their therapeutic benefit from actions on cardiac fibroblasts and fibrotic remodeling of the heart. Despite a tremendous amount of research, however, the specific
mechanisms of these actions, and the underlying role of the RAS in myocardial remodeling and homeostasis remain enigmatic. Identifying the primary mechanisms
should provide further opportunities for therapeutic development.
Aldosterone Antagonists: Spironolactone, and Eplerenone

The mineralocorticoid aldosterone has been strongly implicated in the fibrogenic
response of the myocardium upon stimulation with AngII or through AngIIindependent mechanisms (70). The myocardium expresses enzymes for biosynthesis and metabolism of aldosterone as well as mineralocorticoid receptors (70).
Aldosterone infusion in animal models subjected to sodium overload resulted in
diffuse fibrosis of both RV and LV, combined with focal replacement fibrosis,
which appeared attributable to hyperkalemic myocyte loss. Aldosterone-induced
fibrosis was independent of blood pressure elevation but reversed by the receptor
antagonist spironolactone (7, 70).
This research came to fruition with the RALES clinical trial, which demonstrated that treatment with spironolactone in heart failure patients receiving an
ACE inhibitor and standard therapy produced marked reductions in all-cause cardiovascular mortality and improved NYHA classification (71). These findings
were confirmed and extended in a large-scale trial (EPHESUS) of the selective mineralocorticoid antagonist eplerenone in post-MI patients (72). A substudy arising from the RALES trial demonstrated that the serum collagen III
metabolic marker PIIINP predicts cardiovascular risk, and spironolactone therapy
normalizes ECM metabolism and the progression of LV dilatation (73). However,
only patients whose baseline PIIINP levels were above the median responded
to spironolactone therapy with improvement in event-free survival. In light of
the linkage between myocardial fibrosis and arrhythmogenesis, it is noteworthy
that both RALES and EPHESUS found reductions in sudden cardiac death from
aldosterone antagonist therapy. Additional trials are ongoing to compare the effectiveness of aldosterone antagonists in combination with ARBs versus ACE
inhibitors.
Despite the successful outcomes of these trials, fundamental questions remain
regarding the actions of aldosterone on cardiac fibroblasts. Attempts to identify
mineralocorticoid receptors and to elicit aldosterone-stimulated collagen synthesis
in cardiac fibroblasts in vitro have been problematic (7, 70). Induction of fibrosis
by aldosterone in experimental animal models exhibits slow onset (four weeks)
and is attenuated by treatment with endothelin antagonists or angiotensin receptor blockers, suggesting indirect rather than direct interaction with cardiac fibroblasts (7, 70). Regardless, mineralocorticoid antagonists represent an important new
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therapy that demonstrates the value of targeting cardiac fibrosis to improve cardiac
performance and prognosis in appropriately selected patients.

Endothelin Receptor Antagonists

Endothelin-1 (ET-1) regulates activities of cardiac fibroblasts in vitro and in vivo.
Cardiac fibroblasts express a mixed population of ETA and ETB receptor subtypes.
ET-1 increases cell proliferation, MMP activity, and net collagen synthesis in
cultured cardiac fibroblasts (74). AngII-elicited increases in ECM metabolism in
cultured fibroblasts were blunted by an ETA receptor antagonist (75), and complex
interactions among ET-1, AngII, and TGF occur in the myocardium as well. ET-1
is upregulated in failing LV of human heart failure patients, and elevations in serum
endothelin concentrations correlate with heart failure severity (76). Despite these
promising indications, ET-1 antagonists failed to show benefit following acute MI
and in chronic heart failure (reviewed in 77).
Statins
The proven clinical benefit of statins as cholesterol-lowering drugs in atherosclerotic disease was more recently complemented by the unexpected finding that these
agents exert pleiotropic effects on a variety of cell signaling pathways through inhibition of protein prenylation (78, 78a). These observations have fueled interest
in the potential utility of statins in heart failure. Studies in experimental animal
models provide support for beneficial actions of statins directed toward cardiac
fibroblasts. Treatment with statins reduces myocardial remodeling, fibrosis, and
collagen synthesis in models of myocardial injury including surgical infarction
(79), transgenic models of hypertrophic cardiomyopathy (79a) or NaCl-induced
pressure overload (80). In many of these studies, statins exert concordant antifibrotic and antiinflammatory actions. These results underscore that inflammation
and fibrosis represent aspects of a continuum of responses of cardiac fibroblasts
to myocardial injury.
In theory, statin therapy might confer either positive or negative impacts in
the setting of congestive heart failure (discussed in 81). Statins may act on cardiac fibroblasts to attenuate inflammatory signaling through reduced prenylation
of small GTPases. In this regard, elevated serum concentrations of the inflammatory marker C-reactive protein were shown to predict the likelihood of nonfatal
MI or fatal coronary events following an initial infarct. Treatment with pravastatin normalized serum concentrations of CRP and reduced the risks of cardiac
events equivalent to normal subjects (82). Moreover, amelioration of coronary
artery disease and ischemic events would relieve proapoptotic stress on cardiac
myocytes and indirectly diminish replacement fibrosis. The large-scale prospective CORONA trial is underway to explicitly test therapy with rosuvastatin in heart
failure (83). This trial does not include measurement of fibrotic or inflammatory
markers as a primary endpoint, but such a substudy would be of considerable
interest.
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Anticytokine Therapies: TNF, IFN , and TGF

Proinflammatory and profibrotic cytokines play central roles in coordinating the
activities of multiple cell types in the injured and failing myocardium. In light
of the importance of cardiac fibroblasts as cytokine effectors, these agents offer
a promising direction for therapeutic development. However, the complexity and
redundancy of cytokine signaling networks have posed a challenge for drug development. Initial efforts with anticytokine therapy in heart failure focused on TNF
(84). Strong experimental evidence indicates an important role for this cytokine in
the initial activation of MMPs requisite to myocardial remodeling following acute
MI and in the progression to chronic heart failure (17, 84). However, sequestration
of TNF with a receptor:antibody chimera (sTNFR1:Fc) was ineffective to ameliorate or regress symptomatic heart failure in a large-scale clinical trial (17, 84).
Promising results have been obtained with IFN in therapy of idiopathic pulmonary fibrosis. This cytokine exerts important actions to prevent the phenotypic
activation of myofibroblasts and to induce myofibroblast apoptosis in opposition
to the profibrotic actions of TGF. A randomized, double-blind trial in patients
with idiopathic pulmonary fibrosis demonstrated that IFN improved pulmonary
function, oxygenation, and symptoms, and decreased fibrogenic markers in lung
biopsies (85). A larger multicenter trial is near completion. This cytokine should
be investigated further in the context of myocardial fibrosis.
Considering the importance of TGF as a ubiquitous controller of fibrosis,
efforts have been undertaken to intervene directly on the production and activation of this cytokine. Experimental animal studies have offered promising results. Inhibition of TGF with neutralizing antibodies; soluble TGF receptor:
antibody chimeras (sTGFRII:IgG Fc); or adenoviral-mediated gene transfer of
decorin, a TGF binding protein, reduce fibrosis in rodent models of pressure overload cardiac hypertrophy, bleomycin-induced pulmonary fibrosis, or experimental
glomerulonephritis (86, 87). Inhibition of TGF with neutralizing antibodies or
antisense oligonucleotides reduces injury and scarring in ocular surgery in animals (88). Preliminary studies in patients undergoing glaucoma filtration surgery
indicate that a humanized neutralizing monoclonal antibody to TGF2 reduces
postoperative scarring (89). In this procedure, a single administration of TGF2
antibody at the time of surgery is sufficient to produce a favorable healing response.
A Phase III trial is in progress.
Systemic inhibition of TGF might be expected to lead to adverse side effects owing to the pleiotropic actions of this growth factor. Additional strategies
to inhibit TGF have targeted its interaction with binding partners in the ECM,
including latent TGF binding protein (90) or latency activated peptide (91), in
order to restrict therapeutic modulation of TGF to a specific tissue or physiological context. Further, the fibrogenic actions of TGF in many cases are mediated
through expression of connective tissue growth factor. Research is in progress to
target this effector molecule (92). Although these approaches are in a preliminary
stage, modulation of TGF production and actions is likely to remain a focus of
research in antifibrotic therapy.
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Inhibitors of ECM Metabolism: Collagen Synthesis, Matrix

Metalloproteinases, and Plasmin Systems
Drugs that act on the enzymatic steps of ECM metabolism present clear-cut therapeutic opportunities to modulate myocardial remodeling. These approaches intersect key functions of the cardiac fibroblast (17). Few studies have explored
inhibitors of collagen biosynthesis to limit cardiac fibrosis (5, 93), whereas extensive research focuses on MMP inhibitors (MMPI) in the context of pathologic
remodeling.
The rationale to develop inhibitors of MMPs to modulate ECM metabolism is
based on the demonstrated involvement of MMPs in at least three major aspects
of myocardial injury and failure (94, 95). First, activation of MMPs underlies myocyte slippage, ventricular wall thinning, and chamber dilation following acute
MI. Second, chronic MMP activation contributes importantly to the aberrant remodeling of ECM during the progression to chronic heart failure. Third, excess
MMP activation is a key contributor to instability and rupture of atherosclerotic
plaques. In this context, it is important to recognize that systemic inhibition of
MMPs might exert contravening effects on atherosclerotic plaque stability versus
cardiac fibrosis.
Studies in animals have investigated the effects of MMP inhibition with pharmacological agents or by gene deletion in transgenic animals in models of cardiac
injury and failure. Rohde et al. showed that a nonselective pharmacological MMP
inhibitor (CP-471 474) reduced LV dilatation four days following surgical infarction in mice (96). However, follow-up at later time intervals (15 days) post-MI
in MMP-9-deficient mice showed defective wound healing with diminished collagen accumulation in the infarct zone and decreased infiltration of macrophages
compared to wild type (97). These results emphasize the relationship between
degradation and repair of ECM.
Activation of MMPs by the plasmin system has been demonstrated in elegant
studies by Heymans et al. (98). Using surgical infarctions in transgenic mouse
strains, the authors showed that mice deficient in urokinase plasminogen activator (uPA) or MMP-9 were protected from acute post-MI cardiac rupture, but
subsequently exhibited reduced inflammatory infiltrates and defective healing.
Plasminogen-deficient mice failed to activate MMP-2 and -9 and exhibited similar
defects in infarct healing (94). Transient overexpression of plasminogen activator
inhibitor-1 (PAI-1), the principal physiological antagonist of plasmin, by adenoviral gene transfer following infarction in wild-type mice prevents cardiac rupture
but permits infarct healing to normalize as the expression of the adenoviral construct wanes (98). These findings have important clinical implications for the use
of lytic agents on ECM remodeling versus thrombosis and for potential drug interactions between lytic agents and MMP inhibitors in acute myocardial infarction. In
this context, PAI-1 is expressed by cardiac fibroblasts, upregulated by AngII, and
promotes cardiac fibrosis (98, 99). Additionally, therapy with thiazolidinedione
ligands of nuclear PPAR receptors is associated with reduced serum levels of
PAI-1 (reviewed in 99).
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The effects of MMP inhibitors in the progression to heart failure have also been
examined. Pacing-induced supraventricular tachycardia of three-weeks duration
in pigs produces congestive heart failure characterized by LV dilation; increased
activities of MMP-1, -2, and -3; and decreased collagen content. Pathological remodeling was attenuated and cardiac function was preserved by treatment with
the nonselective MMPI, PD-166 793 (100). Similar results were obtained more
recently with a newer generation MMPI, PGE 7113313, designed to spare inhibition of MMP-1, which is downregulated in chronic human heart failure (101).
It should be noted that inhibitors were administered prior to and throughout the
duration of pathological stimulus rather than after the establishment of congestive
heart failure.
Preliminary studies in humans lend further support for MMPs as targets in heart
disease (reviewed in 94). Gene polymorphisms have been identified in the promoters of MMP-1, -3, -9, and -12, which influence MMP expression. Polymorphisms
in the promoters for MMP-3, -9, and -12 were shown to confer susceptibility to
coronary artery disease and abdominal aortic aneurysm. Concentration ratios of
serum MMP/TIMP correlate with functional values of LV volume and ejection
fraction and predict clinical outcome of myocardial infarction. A Phase II trial
was recently completed to test the effect of MMP inhibitor PG 116800 to prevent adverse cardiac remodeling following a first myocardial infarction (102). The
tetracycline derivative Periostat (doxycycline) is the only MMP inhibitor currently
approved for clinical use, but its application is limited to periodontal disease. Treatment of coronary heart disease patients with Periostat reduced serum inflammatory
markers (C-Reactive Protein, IL-1, and IL-6) as well as circulating concentrations
of MMP-9 (103). Considering its safety, efficacy, and cost, clinical trials of doxycycline in myocardial remodeling also appear worth pursuing.
Novel Antifibrotic/Antiinflammatory Agents: Pirfenidone,

Tranilast, and Nuclear Receptor Ligands
Pirfenidone (PD), 5-methyl-1-phenyl-2(1H) pyridone, has been shown to exert
protective actions in animal models of tissue injury and fibrosis. The mechanism
of PD action is not fully understood but appears to involve inhibition of fibroblast
proliferation and collagen synthesis, potentially through disruption of TGF1 expression (106). PD regressed LVH and fibrosis in DOCA-salt hypertension (104)
and protected against doxorubicin-induced myocardial and renal oxidative injury
and resulting fibrosis (105). PD also reduced the fibrotic and inflammatory responses of bleomycin-induced pulmonary fibrosis (106) and LPS-induced toxic
shock in rodents (107). These results suggesting combined antifibrotic and antiinflammatory actions of PD were extended with in vitro studies, where PD demonstrated antifibrotic activities against smooth muscle leiomyoma cells and rat renal
myofibroblasts (108) and antiinflammatory actions in the RAW 246.7 macrophage
cell line (107). PD is under active investigation (Phase II) in idiopathic pulmonary
fibrosis (109) and in renal tubulointerstitial fibrosis (110). Based on its spectrum
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of activities, continued investigations of mechanisms of PD actions in cardiac

fibroblasts are warranted.
Tranilast [N(3,4-dimethoxy cinnamoyl)-anthranilic acid] is a second agent that
showed very promising antifibrotic and antiinflammatory characteristics in experimental studies and early phase clinical trials. Based on its abilities to inhibit
smooth muscle cell migration and proliferation, this agent was targeted for therapy
to prevent coronary vascular restenosis following angioplasty. However, a largescale clinical trial failed to show benefit (111). Nonetheless, recent studies in the
DOCA-salt hypertensive rat model show that tranilast blocks myocardial fibrosis
and suppresses inflammatory cell infiltrates (112). It should be emphasized that
tranilast, like PD, antagonizes production and activity of TGF (113).
Recent attention has focused on ligands of the peroxisome proliferator-activated
receptors, PPAR and PPAR , for their actions on the myocardium (reviewed in
114, 115). PPARs are nuclear receptors that regulate lipid storage and metabolism.
Activators of PPAR and PPAR are used clinically in dyslipidemias and in diabetes, respectively. The PPAR receptors are expressed by multiple cell types in the
cardiovascular system, including cardiac myocytes and fibroblasts. PPAR receptors share common properties to suppress production of inflammatory cytokines,
cellular adhesion proteins, and chemotactic peptides by inhibiting the transcription
factor NF-B. PPAR and PPAR ligands are cardioprotective in experimental
infarction (116), and they prevent interstitial fibrosis, preserve diastolic function,
and inhibit inflammatory activation in pressure overload cardiac hypertrophy (117).
These studies do not distinguish primary actions on cardiac fibroblasts from secondary actions via other cell types. Further, adverse effects of PPAR ligands in
congestive heart failure have been reported (118). Better understanding of PPAR
mechanisms in cardiac fibroblasts is important to complement ongoing research
in other cardiac cell types.
FUTURE CHALLENGES FOR THERAPY

The concepts that fibrosis and inflammation contribute to the myocardial response
to injury are generally accepted, as is the importance of the cardiac fibroblast as
a key cell type mediating these processes. From this perspective, the rationale for
development of therapies that target the cardiac fibroblast is clear. Indeed, we have
seen that established therapies that target the renin-angiotensin-aldosterone system actually may derive a significant part of their benefit from actions on cardiac
fibroblasts. However, fundamental unresolved issues stand between these generalizations and the development of effective new therapies. These may be broadly
divided between issues of fibroblast biology and issues of pharmacotherapy.
Unanswered Questions in Cardiac Fibroblast Biology

The ability to develop rational therapies targeted to the cardiac fibroblast ultimately
depends on a sound knowledge of the biology and the physiological role of this cell
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type in myocardial remodeling. Questions remain that bear on the therapeutic issues
discussed above and that may provide novel opportunities for drug development.
First, what are the fibroblast phenotypes of normal, injured, and failing myocardium? It will be important to resolve whether a single activated fibroblast
phenotype is capable of multiple functional responses, such as proliferation, migration, ECM metabolism, and production of autocrine/paracrine mediators, or
whether different subsets of fibroblasts subserve distinct endpoint responses. To
address this question, it will be necessary to define the cellular precursors of
activated fibroblasts; for example, whether activated fibroblasts derive from homogeneous or polyclonal populations of quiescent resident fibroblasts, from epithelialmesenchymal transitions of undifferentiated resident precursor cells, or from recruitment of circulating precursor cells. These same issues need to be addressed
for cardiac myofibroblasts.
These ideas lead to related thoughts about the mechanisms that underlie the termination of the myocardial injury response in normal wound healing compared to
the transition to maladaptive responses in fibrotic heart failure. One may speculate
that heart failure progression results from unresolved cardiac myocyte dysfunction.
Within this environment of chronic injury, cardiac fibrosis could reflect either a failure to terminate a normal injury phenotype, or alternatively, the de novo appearance
of a novel failure phenotype of cardiac fibroblasts. The role of fibroblast apoptosis
as a termination mechanism in adaptive myocardial healing versus the role of hyperplasia as a mechanism for fibroblast recruitment in fibrotic progression should
be a specific focus for investigation. Identification of populations of (myo)fibroblasts that are involved in distinct functional aspects or disease stages of myocardial
remodeling would offer obvious opportunities for therapeutic intervention.
Continued research on the signaling mechanisms that regulate cardiac fibroblast
phenotype in response to inflammatory and fibrotic stimuli represents a second major area of emphasis. The unique properties of the cardiac fibroblast relative to other
fibroblastic cells, the ability of cardiac fibroblasts to integrate many stimuli through
receptor-specific signaling pathways, and the diversity of endpoint responses all
point to a more complex regulatory organization than has been appreciated (Figure 3). The functional response of regulated gene expression reflects combinatorial
inputs in the evolving wound environment. These regulatory interactions underlie
the spatiotemporal sequencing of events that occurs in wound healing and provide
the basis for determining the intervals of opportunity for defined therapeutic targets. This model further suggests that genomic or proteomic pattern recognition
analyses will help to identify groups of genes that are expressed in concert, in
turn generating hypotheses for conserved regulatory motifs within the gene promoters and associated signaling pathways. Additional levels of complexity come
from the discovery that signaling molecules as well as transcriptional regulators
are spatially organized within the cell (119, 119a). In theory, these elements offer
a wealth of targets for therapy to intersect the inflammatory-fibrotic cascade, but
the dissection of key mechanisms will require acquisition of detailed molecular
information. Genomic analyses of myocardial injury models, and particularly of
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Figure 3
Signaling and transcriptional regulation of fibroblast function. Cardiac
fibroblasts respond to diverse humoral and mechanical inputs through cell surface
receptors. Environmental stimuli are integrated through receptor-mediated signaling
pathways leading to activation of nuclear transcription factors and altered gene expression. Integration of nuclear and cytoplasmic signaling networks controls fibroblast
phenotype. Abbreviations: ALDO, aldosterone; R, receptor; GPCR, G proteincoupled
receptor; FAK, focal adhesion kinase; IL-1 RA, IL-1 receptor antagonist; MAPK, mitogen activated protein kinase.
cardiac fibroblasts, are at an early stage, but a torrent of data is surely coming.
We are unaware of clinical trials utilizing agents that focus on cardiac fibroblast
intracellular signaling pathways. However, a number of agents are in preclinical or
early phase clinical trials for other applications, and it is likely that coming years
will see their evaluation in myocardial remodeling (120).
A third area for research will be to explore further the mechanisms of intercellular communication in the normal and failing heart. Homeostatic maintenance
and remodeling of the heart requires communication among cardiac myocytes, fibroblasts, and immune cells, as well as interactions with the coronary vasculature
(Figure 4). Mechanotransduction via integrins and the extracellular matrix, direct
cell-cell communication via gap junctions, or humoral transmission by diffusible
chemokines and cytokines all may contribute to this process. Therapies aimed at
intramyocardial signaling by AngII and aldosterone demonstrate the value of this
approach.
Conversely, cardiovascular exercise training has been shown to promote physiologically adaptive cardiac hypertrophy and nonfibrotic ECM remodeling, which
are associated with improved cardiac performance, and protect against the risk
of adverse cardiac events (121, 121a). These results raise questions of how the
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Figure 4 Intercellular communication in the myocardium. Cell types within the

myocardium respond to humoral and mechanical stimuli and, reciprocally, release
autocrine-paracrine mediators. Therapeutic modulation of intercellular signaling can
interrupt negatively reinforcing cycles of inflammation and fibrosis in failing myocardium (e.g., antagonism of RAS). Abbreviations: EC, endothelial cell; VSMC,
vascular smooth muscle cell; PG, prostaglandin; ROS, reactive oxygen species.
myocardium distinguishes the healthy stress of exercise from the pathologic stress
of injury. It is intriguing to speculate that exercise activates survival pathways in
cardiac myocytes, resulting in intercellular signals to cardiac fibroblasts, which are
distinct from signals of injury (121b). Knowledge of cardiac fibroblast responses
to exercise training may provide additional insight into approaches to oppose the
progression of fibrosis. An observational study is ongoing to evaluate cardiac fibrosis by serum collagen markers and MRI in relation to exercise tolerance in
hypertrophic cardiomyopathy (122).
Unresolved Issues for Pharmacotherapy

Despite strong physiologic rationales and abundant evidence from in vitro and
in vivo basic research, spectacular and costly failures have occurred in clinical
trials for novel agents in heart failure (see, for examples, the discussions above on
anti-TNF therapy, ET-1 receptor antagonists, and tranilast). A further cautionary
example comes from attempts to develop MMP inhibitors as therapeutic agents for
pathophysiologic ECM remodeling. Staggering investments of time and money by
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basic and pharmaceutic research sectors so far have yielded a single approved agent,
doxycycline (Periostat), for periodontal disease. Lessons from these experiences
have been thoughtfully reviewed and are relevant to cardiac fibroblasts (123, 124).
Cardiac fibrosis is not a primary cause, but rather a disease modifier, that affects
the progress, severity, and outcome of heart disease. Furthermore, fibrotic remodeling of the heart represents a spectrum of responses that may vary depending on
the specific etiology of myocardial injury; the stage of disease progression; and
differences in age, gender, ethnicity, and genetic polymorphisms between individual patients. It therefore is essential to develop algorithms for stratification of risk
in large patient populations. Preliminary results discussed above suggest that more
extensive fibrosis may confer measurable risk and that individuals differ in their
responses to therapy. However, these studies are in their initial stages compared to
the quantitative databases that have accumulated for prognostic indicators, such
as LV hypertrophy, serum cholesterol, or inflammatory status. Analysis of serum
collagen metabolites in archival samples from large-scale clinical trials in relation
to clinical outcome could offer a cost-effective starting point to obtain this type of
information.
Development and standardization of surrogate markers of fibroblast activation
or fibrosis are prerequisite to risk stratification and to evaluation of the efficacy
of pharmaceutical agents. The utility and economy of serum collagen metabolites
have been validated in this regard (5, 79). However, these measurements are
limited because of their lack of specificity for cardiac versus extracardiac ECM
remodeling, and the lack of sensitivity to detect key mechanistic steps in the
remodeling process. Analysis of coronary sinus blood may provide a means to
identify specific markers of cardiac ECM metabolism compared to general ECM
markers in the systemic circulation.
Selection of appropriate clinical endpoints is critical to assess therapeutic benefit
and to evaluate the relationship between target efficacy and clinical outcome.
Reduction in mortality may be the preferred endpoint in younger patients, whereas
reversal of disease progression or adverse events may assume greater importance
in the elderly.
Antifibrotic agents will likely be useful adjuncts for combination therapy in
selected patient populations, as is currently recommended for aldosterone antagonists. This approach offers the appeal of targeting complementary therapies for
cardiac myocytes and cardiac fibroblasts. The development of agents that combine
antifibrotic and antiinflammatory actions offers promise. However, issues of cost
and safety of polypharmacy, especially in older patients, must be considered.
Identification of the windows of opportunity for antifibrotic therapy is likely
to be crucial for effective intervention. Myocardial remodeling is progressive and
cumulative as the heart passes from the initial response to injury through the
transition to fibrosis and failure. It will be necessary to apply therapy at intervals
that are appropriate to the molecular target. Furthermore, it may be more feasible
to prevent fibrotic remodeling than to reverse it once it occurs. This is most likely
to be the case for replacement fibrosis following myocyte loss. On the other hand,
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reversal of reactive fibrosis may be more feasible as has been seen with regression
of LVH and fibrosis in hypertension.
Finally, choice of physiologically appropriate experimental models is important to extrapolate preclinical findings to positive results in human trials. Studies
with cultured fibroblasts in vitro typically examine responses to acute stimuli, and
animal studies examine the initial onset of myocardial remodeling and failure in
younger animals. By contrast, human heart failure is more commonly a progressive disease of the elderly, and clinical trials are aimed at therapy of established
disease. Transgenic mouse technology has provided powerful insights into the
roles of specific gene products in cardiovascular physiology, but the consequences
of transgene expression throughout the life span of the animal may not accurately reflect the sequential and coordinated activation of myocardial remodeling
in response to a pathological insult. Newer methodologies allowing conditional
transgenic expression will help address this disparity.
In conclusion, the available evidence provides a strong rationale for therapies
directed toward cardiac fibroblasts to improve outcomes in heart disease. Continued basic and translational research, and perseverance through the inevitable
setbacks, will be needed to bring this promise to fruition.
ACKNOWLEDGMENT
Work in the authors laboratories was supported by NIH (HL59428).
LITERATURE CITED
1. Graziano JM. 2001. Global burden of
cardiovascular disease. In Heart Disease: A Textbook of Cardiovascular
Medicine, ed. E Braunwald, DP Zipes,
P Libby, pp. 118. Philadelphia: WH
Saunders Co. 6th ed.
2. Richardson P, McKenna W, Bristow M,
Maisch B, Mautner B, et al. 1996. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the
definition and classification of cardiomyopathies. Circulation 93:84142
3. Eghbali M, Weber KT. 1990. Collagen
and the myocardium: fibrillar structure,
biosynthesis and degradation in relation
to hypertrophy and its regression. Mol.
Cell Biochem. 96:114
4. Bosman FT, Stamenkovic I. 2003. Functional structure and composition of the

extracellular matrix. J. Pathol. 200:423
28
5. Jugdutt BI. 2003. Remodeling of the myocardium and potential targets in the collagen degradation and synthesis pathways. Curr. Drug Targets Cardiovasc.
Haematol. Disord. 3:130
6. Maquart FX, Pasco S, Ramont L,
Hornebeck W, Monboisse JC. 2004. An
introduction to matrikines: extracellular
matrix-derived peptides which regulate
cell activity. Implication in tumor invasion. Crit. Rev. Oncol. Hematol. 49:199
202
7. Lijnen PJ, Petrov VV. 2003. Role of intracardiac renin-angiotensin-aldosterone
10 Dec 2004
17:42
680
8.

9.
10.
11.
12.
13.
14.
15.
16.
16a.
AR
AR232-PA45-27.tex
P1: JRX
BROWN ET AL.
system in extracellular matrix remodeling. Methods Find. Exp. Clin. Pharmacol. 25:54164
Lopez B, Gonzalez A, Varo N, Laviades
C, Querejeta R, et al. 2001. Biochemical assessment of myocardial fibrosis in
hypertensive heart disease. Hypertension
38:122226
Querejeta R, Varo N, Lopez B, Larman M, Artinano E, et al. 2000. Serum
carboxy-terminal propeptide of procollagen type I is a marker of myocardial fibrosis in hypertensive heart disease. Circulation 101:172935
Davidson JM. 1992. Wound repair. In
Inflammation: Basic Principles and
Clinical Correlates, ed. JI Gallin, RH
Goldstein, R Snyderman, pp. 809
19. New York: Raven Press. 2nd
ed.
Takemura G, Ohno M, Hayakawa Y,
Misao J, Kanoh M, et al. 1998. Role of
apoptosis in the disappearance of infiltrated and proliferated interstitial cells
after myocardial infarction. Circ. Res.
82:113038
Powell DW, Mifflin RC, Valentich JD,
Crowe SE, Saada JI, et al. 1999. Myofibroblasts. II. Intestinal subepithelial
myofibroblasts. Am. J. Physiol. Cell
Physiol. 277:C183201
Zhang H-Y, Phan SH. 1999. Inhibition
of myofibroblast apoptosis by transforming growth factor 1. Am. J. Respir. Cell
Mol. Biol. 21:65865
Sun Y, Weber KT. 2000. Infarct scar:
a dynamic tissue. Cardiovasc. Res. 46:
25056
Gittenberger-de Groot AC. 2003. Mannheimer lecture. The quintessence of the
making of the heart. Cardiol. Young
13:17583
Wessels A, Perez-Pomares JM. 2004.
The epicardium and epicardially derived
cells (EPDCs) as cardiac stem cells.
Anat. Rec. 276A:4357
Kalluri R, Neilson EG. 2003. Epithelialmesenchymal transition and its implica-
17.
18.
19.
20.
21.
22.
23.
24.
tions for fibrosis. J. Clin. Invest. 112:

177684
Brown RD, Mitchell MD, Long CS.
2004. Proinflammatory cytokines and
cardiac extracellular matrix: regulation
of fibroblast phenotype. In Interstitial Fibrosis in Heart Disease, ed. FJ Villarreal,
pp. 5781. New York: Springer
Koudssi F, Lopez JE, Villegas S, Long
CS. 1998. Cardiac fibroblasts arrest at
the G1/S restriction point in response
to interleukin (IL)-1. Evidence for IL1-induced hypophosphorylation of the
retinoblastoma protein. J. Biol. Chem.
273:25796803
Kacimi R, Karliner JS, Koudssi F, Long
CS. 1998. Expression and regulation of
adhesion molecules in cardiac cells by
cytokines: response to acute hypoxia.
Circ. Res. 82:57686
Gray MO, Long CS, Kalinyak JE, Li
HT, Karliner JS. 1998. Angiotensin II
stimulates cardiac myocyte hypertrophy
via paracrine release of TGF-1 and
endothelin-1 from fibroblasts. Cardiovasc. Res. 40:35263
Kacimi R, Long CS, Karliner JS.
1997. Chronic hypoxia modulates the
interleukin-1-stimulated inducible nitric oxide synthase pathway in cardiac myocytes. Circulation 96:1937
43
Yue P, Massie BM, Simpson PC, Long
CS. 1998. Cytokine expression increases
in nonmyocytes from rats with postinfarction heart failure. Am. J. Physiol. Heart Circ. Physiol. 275:H250
58
Okada H, Ban S, Nagao S, Takahashi H,
Suzuki H, et al. 2000. Progressive renal
fibrosis in murine polycystic kidney disease: an immunohistochemical observation. Kidney Int. 58:58797
Breen E, Falco VM, Absher M, Cutroneo
KR. 1990. Subpopulations of rat lung fibroblasts with different amounts of type
I and type III collagen mRNAs. J. Biol.
Chem. 265:628690
10 Dec 2004
17:42
AR
AR232-PA45-27.tex
P1: JRX


25. Jelaska A, Strehlow D, Korn JH. 1999.
Fibroblast heterogeneity in physiological conditions and fibrotic disease. Springer Semin. Immunopathol. 21:38595
26. Iwano M, Plieth D, Danoff TM, Xue C,
Okada H, et al. 2002. Evidence that fibroblasts derive from epithelium during
tissue fibrosis. J. Clin. Invest. 110:341
50
27. Yanez-Mo M, Lara-Pezzi E, Selgas
R, Ramirez-Huesca M, DominguezJimenez C, et al. 2003. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells. N. Engl. J.
Med. 348:40313
28. Hashimoto N, Jin H, Liu T, Chensue SW,
Phan SH. 2004. Bone marrow-derived
progenitor cells in pulmonary fibrosis. J.
29. Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R. 2004. Circulating fibrocytes: collagen-secreting cells of the
peripheral blood. Int. J. Biochem. Cell
Biol. 36:598606
30. Chai Q, Krag S, Chai S, Ledet T,
Wogensen L. 2003. Localisation and
phenotypical characterisation of collagen-producing cells in TGF-1induced renal interstitial fibrosis.
Histochem. Cell Biol. 119:26780
31. Campbell JH, Efendy JL, Han C, Girjes
AA, Campbell GR. 2000. Haemopoietic
origin of myofibroblasts formed in the
peritoneal cavity in response to a foreign
body. J. Vasc. Res. 37:36471
32. Swynghedauw B. 1999. Molecular
mechanisms of myocardial remodeling.
Physiol. Rev. 79:21562
33. Diez J, Lopez B, Gonzalez A, Querejeta
R. 2001. Clinical aspects of hypertensive
myocardial fibrosis. Curr. Opin. Cardiol.
16:32835
34. Maron BJ, McKenna WJ, Danielson
GK, Kappenberger LJ, Kuhn HJ, et al.
2003. American College of Cardiology/European Society of Cardiology
clinical expert consensus document on
hypertrophic cardiomyopathy. A report
35.
36.
37.
38.
39.
40.
41.
42.
43.
681
of the American College of Cardiology

Foundation Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology Committee for Practice Guidelines. J. Am. Coll.
Cardiol. 42:1687713
Maass AH, Leinwand LA. 2003. Mechanisms of the pathogenesis of troponin
T-based familial hypertrophic cardiomyopathy. Trends Cardiovasc. Med. 13:
23237
Arad M, Seidman JG, Seidman CE.
2002. Phenotypic diversity in hypertrophic cardiomyopathy. Hum. Mol.
Genet. 11:2499506
Kon-No Y, Watanabe J, Koseki Y,
Koyama J, Yamada A, et al. 2001. Microvolt T wave alternans in human cardiac hypertrophy: electrical instability
and abnormal myocardial arrangement.
J. Cardiovasc. Electrophysiol. 12:759
63
Lombardi R, Betocchi S, Losi MA,
Tocchetti CG, Aversa M, et al. 2003.
Myocardial collagen turnover in hypertrophic cardiomyopathy. Circulation
108:145560
Varnava AM, Elliott PM, Mahon N,
Davies MJ, McKenna WJ. 2001. Relation between myocyte disarray and outcome in hypertrophic cardiomyopathy.
Am. J. Cardiol. 88:27579
Shirani J, Pick R, Roberts WC, Maron
BJ. 2000. Morphology and significance
of the left ventricular collagen network
in young patients with hypertrophic cardiomyopathy and sudden cardiac death.
J. Am. Coll. Cardiol. 35:3644
Schonberger J, Seidman CE. 2001. Many
roads lead to a broken heart: the genetics
of dilated cardiomyopathy. Am. J. Hum.
Genet. 69:24960
Hsia HH, Marchlinski FE. 2002. Electrophysiology studies in patients with dilated cardiomyopathies. Card. Electrophysiol. Rev. 6:47281
Brooks A, Schinde V, Bateman AC, Gallagher PJ. 2003. Interstitial fibrosis in
10 Dec 2004
17:42
682
44.

45.
46.
47.
48.
49.
50.
51.
52.
AR
AR232-PA45-27.tex
P1: JRX
BROWN ET AL.
the dilated non-ischaemic myocardium.
Heart 89:125556
Marijianowski MM, Teeling P, Mann J,
Becker AE. 1995. Dilated cardiomyopathy is associated with an increase in the
type I/type III collagen ratio: a quantitative assessment. J. Am. Coll. Cardiol.
25:126372
Yokoseki O, Yazaki Y, Suzuki J, Imamura H, Takenaka H, et al. 2000. Association of matrix metalloproteinase expression and left ventricular function in
idiopathic dilated cardiomyopathy. Jpn.
Circ. J. 64:35257
Klappacher G, Franzen P, Haab D,
Mehrabi M, Binder M, et al. 1995. Measuring extracellular matrix turnover in
the serum of patients with idiopathic
or ischemic dilated cardiomyopathy and
impact on diagnosis and prognosis. Am.
J. Cardiol. 75:91318
Lip GY, Felmeden DC, Li-Saw-Hee FL,
Beevers DG. 2000. Hypertensive heart
disease. A complex syndrome or a hypertensive cardiomyopathy? Eur. Heart J.
21:165365
Kenchaiah S, Pfeffer MA. 2004. Cardiac remodeling in systemic hypertension. Med. Clin. North Am. 88:11530
Rossi MA. 1998. Pathologic fibrosis and
connective tissue matrix in left ventricular hypertrophy due to chronic arterial
hypertension in humans. J. Hypertens.
16:103141
Ohsato K, Shimizu M, Sugihara N, Konishi K, Takeda R. 1992. Histopathological factors related to diastolic function
in myocardial hypertrophy. Jpn. Circ. J.
56:32533
Eckardt L, Haverkamp W, Johna R,
Bocker D, Deng MC, et al. 2000.
Arrhythmias in heart failure: current
concepts of mechanisms and therapy.
J. Cardiovasc. Electrophysiol. 11:106
17
Pogwizd SM, Hoyt RH, Saffitz JE, Corr
PB, Cox JL, et al. 1992. Reentrant and
focal mechanisms underlying ventricu-
53.
54.
55.
56.
57.
58.
59.
60.
61.
lar tachycardia in the human heart. Circulation 86:187287

Chung MK, Pogwizd SM, Miller DP,
Cain ME. 1997. Three-dimensional
mapping of the initiation of nonsustained ventricular tachycardia in the human heart. Circulation 95:251727
Li D, Fareh S, Leung TK, Nattel S. 1999.
Promotion of atrial fibrillation by heart
failure in dogs: atrial remodeling of a different sort. Circulation 100:8795
Goette A, Juenemann G, Peters B, Klein
HU, Roessner A, et al. 2002. Determinants and consequences of atrial fibrosis in patients undergoing open heart
surgery. Cardiovasc. Res. 54:39096
Pogwizd SM, McKenzie JP, Cain ME.
1998. Mechanisms underlying spontaneous and induced ventricular arrhythmias in patients with idiopathic dilated
cardiomyopathy. Circulation 98:2404
14
Nattel S. 2002. New ideas about atrial
fibrillation 50 years on. Nature 415:219
26
Tabib A, Loire R, Chalabreysse L, Meyronnet D, Miras A, et al. 2003. Circumstances of death and gross and microscopic observations in a series of
200 cases of sudden death associated
with arrhythmogenic right ventricular
cardiomyopathy and/or dysplasia. Circulation 108:30005
Gonzalez A, Lopez B, Querejeta R, Diez
J. 2002. Regulation of myocardial fibrillar collagen by angiotensin II. A role in
hypertensive heart disease? J. Mol. Cell.
Cardiol. 34:158593
Sleight P. 2002. Angiotensin II and trials of cardiovascular outcomes. Am. J.
Cardiol. 89:11A16
Fox KM. 2003. Efficacy of perindopril in reduction of cardiovascular events
among patients with stable coronary
artery disease: randomised, doubleblind, placebo-controlled, multicentre
trial (the EUROPA study). Lancet
362:78288
10 Dec 2004
17:42
AR
AR232-PA45-27.tex
P1: JRX


62. Svensson P, de Faire U, Sleight P, Yusuf
S, Ostergren J. 2001. Comparative effects of ramipril on ambulatory and office blood pressures: a HOPE substudy.
Hypertension 38:E2832
63. Brilla CG, Funck RC, Rupp H. 2000.
Lisinopril-mediated regression of myocardial fibrosis in patients with hypertensive heart disease. Circulation
102:138893
64. Schwartzkopff B, Brehm M, Mundhenke
M, Strauer BE. 2000. Repair of coronary
arterioles after treatment with perindopril in hypertensive heart disease. Hypertension 36:22025
65. ACE Inhibitor Myocardial Infarction
Collaborative Group. 1998. Indications
for ACE inhibitors in the early treatment
of acute myocardial infarction: systematic overview of individual data from
100,000 patients in randomized trials.
66. Dimopoulos K, Salukhe TV, Coats AJ,
Mayet J, Piepoli M, et al. 2004. Metaanalyses of mortality and morbidity effects of an angiotensin receptor blocker
in patients with chronic heart failure already receiving an ACE inhibitor (alone
or with a beta-blocker). Int. J. Cardiol.
93:10511
67. Pfeffer MA, Swedberg K, Granger CB,
Held P, McMurray JJ, et al. 2003. Effects
of candesartan on mortality and morbidity in patients with chronic heart failure: the CHARM-Overall programme.
Lancet 362:75966
68. Lopez B, Querejeta R, Varo N, Gonzalez A, Larman M, et al. 2001. Usefulness
of serum carboxy-terminal propeptide of
procollagen type I in assessment of the
cardioreparative ability of antihypertensive treatment in hypertensive patients.
69. Diez J, Querejeta R, Lopez B, Gonzalez A, Larman M, et al. 2002. Losartandependent regression of myocardial fibrosis is associated with reduction of left
ventricular chamber stiffness in hyper-
70.
71.
72.
73.
74.
75.
76.
77.
683
tensive patients. Circulation 105:2512

17
Stowasser M. 2001. New perspectives on
the role of aldosterone excess in cardiovascular disease. Clin. Exp. Pharmacol.
Physiol. 28:78391
Pitt B, Zannad F, Remme WJ, Cody R,
Castaigne A, et al. 1999. The effect of
spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation
Study Investigators. N. Engl. J. Med.
341:70917
Pitt B, Remme W, Zannad F, Neaton
J, Martinez F, et al. 2003. Eplerenone,
a selective aldosterone blocker, in patients with left ventricular dysfunction
after myocardial infarction. N. Engl. J.
Med. 348:130921
Zannad F, Alla F, Dousset B, Perez A,
Pitt B. 2000. Limitation of excessive
extracellular matrix turnover may contribute to survival benefit of spironolactone therapy in patients with congestive heart failure: insights from the
randomized aldactone evaluation study
(RALES). Rales Investigators. Circulation 102:27006
Guarda E, Katwa LC, Myers PR, Tyagi
SC, Weber KT. 1993. Effects of endothelins on collagen turnover in cardiac
fibroblasts. Cardiovasc. Res. 27:2130
34
Cheng TH, Cheng PY, Shih NL, Chen
IB, Wang DL, et al. 2003. Involvement
of reactive oxygen species in angiotensin
II-induced endothelin-1 gene expression
in rat cardiac fibroblasts. J. Am. Coll.
Cardiol. 42:184554
Asano K, Bohlmeyer TJ, Westcott JY,
Zisman L, Kinugawa K, et al. 2002. Altered expression of endothelin receptors
in failing human left ventricles. J. Mol.
Cell. Cardiol. 34:83346
Rich S, McLaughlin VV. 2003. Endothelin receptor blockers in cardiovascular disease. Circulation 108:2184
90
10 Dec 2004
17:42

684
AR
AR232-PA45-27.tex
P1: JRX
BROWN ET AL.
78. Liao JK. 2002. Isoprenoids as mediators

of the biological effects of statins. J. Clin.
Invest. 110:28588
78a. Nakagami H, Jensen KS, Liao JK.
2003. A novel pleiotropic effect of
statins: prevention of cardiac hypertrophy by cholesterol-independent mechanisms. Ann. Med. 35:398403
79. Bauersachs J, Galuppo P, Fraccarollo D,
Christ M, Ertl G. 2001. Improvement of
left ventricular remodeling and function
by hydroxymethylglutaryl coenzyme A
reductase inhibition with cerivastatin in
rats with heart failure after myocardial infarction. Circulation 104:982
85
79a. Patel R, Nagueh SF, Tsybouleva N, Abdellatif M, Lutucuta S, et al. 2001. Simvastatin induces regression of cardiac
hypertrophy and fibrosis and improves
cardiac function in a transgenic rabbit
model of human hypertrophic cardiomyopathy. Circulation 104:31724
80. Hasegawa H, Yamamoto R, Takano H,
Mizukami M, Asakawa M, et al. 2003. 3Hydroxy-3-methylglutaryl coenzyme A
reductase inhibitors prevent the development of cardiac hypertrophy and heart
failure in rats. J. Mol. Cell. Cardiol.
35:95360
81. Krum H, McMurray JJ. 2002. Statins and
chronic heart failure: do we need a largescale outcome trial? J. Am. Coll. Cardiol.
39:156773
82. Ridker PM, Rifai N, Pfeffer MA, Sacks
FM, Moye LA, et al. 1998. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol
levels. Cholesterol and Recurrent Events
(CARE) Investigators. Circulation 98:
83944
83. Blenkinsopp J. 2003. The statin wars.
Lancet 362:1854
84. Feldman AM, Kadokami T, Higuichi Y,
Ramani R, McTiernan CF. 2003. The
role of anticytokine therapy in heart failure: recent lessons from preclinical and
85.
86.
87.
88.
89.
90.
91.
clinical trials? Med. Clin. North Am.

87:41940
Ziesche R, Hofbauer E, Wittmann K,
Petkov V, Block LH. 1999. A preliminary study of long-term treatment
with interferon -1b and low-dose prednisolone in patients with idiopathic
pulmonary fibrosis. N. Engl. J. Med.
341:126469
Isaka Y, Akagi Y, Ando Y, Tsujie M,
Sudo T, et al. 1999. Gene therapy by
transforming growth factor- receptorIgG Fc chimera suppressed extracellular matrix accumulation in experimental
glomerulonephritis. Kidney Int. 55:465
75
Kuwahara F, Kai H, Tokuda K, Kai M,
Takeshita A, et al. 2002. Transforming
growth factor- function blocking prevents myocardial fibrosis and diastolic
dysfunction in pressure-overloaded rats.
Cordeiro MF, Mead A, Ali RR, Alexander RA, Murray S, et al. 2003. Novel antisense oligonucleotides targeting TGF inhibit in vivo scarring and improve
surgical outcome. Gene Ther. 10:59
71
Siriwardena D, Khaw PT, King AJ, Donaldson ML, Overton BM, et al. 2002. Human antitransforming growth factor 2
monoclonal antibodya new modulator
of wound healing in trabeculectomy: a
randomized placebo controlled clinical
study. Ophthalmology 109:42731
Hori Y, Katoh T, Hirakata M, Joki N,
Kaname S, et al. 1998. Anti-latent TGF binding protein-1 antibody or synthetic
oligopeptides inhibit extracellular matrix
expression induced by stretch in cultured
rat mesangial cells. Kidney Int. 53:1616
25
Munger JS, Huang X, Kawakatsu H,
Griffiths MJ, Dalton SL, et al. 1999. The
integrin V6 binds and activates latent
TGF1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell
96:31928
10 Dec 2004
17:42
AR
AR232-PA45-27.tex
P1: JRX


92. Duncan MR, Frazier KS, Abramson S,
Williams S, Klapper H, et al. 1999. Connective tissue growth factor mediates
transforming growth factor -induced
collagen synthesis: down-regulation by
cAMP. FASEB J. 13:177486
93. Nwogu JI, Geenen D, Bean M, Brenner MC, Huang X, et al. 2001. Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitor improves left
ventricular function and alters the pattern of left ventricular dilatation after myocardial infarction. Circulation
104:221621
94. Creemers EE, Cleutjens JP, Smits JF,
Daemen MJ. 2001. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart
failure? Circ. Res. 89:20110
95. Li YY, McTiernan CF, Feldman AM.
2000. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac
matrix remodeling. Cardiovasc. Res. 46:
21424
96. Rohde LE, Ducharme A, Arroyo LH,
Aikawa M, Sukhova GH, et al. 1999. Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in
mice. Circulation 99:306370
97. Ducharme A, Frantz S, Aikawa M,
Rabkin E, Lindsey M, et al. 2000.
Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular
enlargement and collagen accumulation
after experimental myocardial infarction. J. Clin. Invest. 106:5562
98. Heymans S, Luttun A, Nuyens D,
Theilmeier G, Creemers E, et al. 1999.
Inhibition of plasminogen activators or
matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.
Nat. Med. 5:113542
99. Lyon CJ, Hsueh WA. 2003. Effect of
plasminogen activator inhibitor-1 in diabetes mellitus and cardiovascular dis-
100.
101.
102.
103.
104.
105.
106.
107.
108.
685
ease. Am. J. Med. 115(Suppl. 8A):62S

68
Spinale FG, Coker ML, Krombach SR,
Mukherjee R, Hallak H, et al. 1999. Matrix metalloproteinase inhibition during
the development of congestive heart failure: effects on left ventricular dimensions and function. Circ. Res. 85:36476
King MK, Coker ML, Goldberg A,
McElmurray JH III, Gunasinghe HR,
et al. 2003. Selective matrix metalloproteinase inhibition with developing heart
failure: effects on left ventricular function and structure. Circ. Res. 92:17785
Oral PG-116800 Study Group. 2004. A
multicenter, randomized, double-blind,
placebo-controlled study of oral PG116800 following a heart attack. http://
clinicaltrials.gov/show/NCT00067236
Brown PD. 2000. Ongoing trials with
matrix metalloproteinase inhibitors. Expert. Opin. Investig. Drugs 9:216777
Mirkovic S, Seymour AM, Fenning A,
Strachan A, Margolin SB, et al. 2002.
Attenuation of cardiac fibrosis by pirfenidone and amiloride in DOCA-salt
hypertensive rats. Br. J. Pharmacol. 135:
96168
Giri SN, Al Bayati MA, Du X, Schelegle E, Mohr FC, et al. 2004. Amelioration of doxorubicin-induced cardiac
and renal toxicity by pirfenidone in rats.
Cancer Chemother. Pharmacol. 53:141
50
Iyer SN, Gurujeyalakshmi G, Giri SN.
1999. Effects of pirfenidone on transforming growth factor- gene expression
at the transcriptional level in bleomycin
hamster model of lung fibrosis. J. Pharmacol. Exp. Ther. 291:36773
Nakazato H, Oku H, Yamane S, Tsuruta Y, Suzuki R. 2002. A novel antifibrotic agent pirfenidone suppresses tumor necrosis factor- at the translational
level. Eur. J. Pharmacol. 446:17785
Lee B-S, Margolin SB, Nowak RA.
1998. Pirfenidone: a novel pharmacological agent that inhibits leiomyoma cell
10 Dec 2004
17:42
686

109.
110.
111.
112.
113.
114.
115.
116.
AR
AR232-PA45-27.tex
P1: JRX
BROWN ET AL.
proliferation and collagen production.
J. Clin. Endocrinol. Metab. 83:21923
Raghu G, Johnson WC, Lockhart D,
Mageto Y. 1999. Treatment of idiopathic
pulmonary fibrosis with a new antifibrotic agent, pirfenidone: results of a
prospective, open-label phase II study.
Am. J. Respir. Crit. Care Med. 159:1061
69
Fukagawa M, Noda M, Shimizu T,
Kurokawa K. 1999. Chronic progressive interstitial fibrosis in renal disease
are there novel pharmacological approaches? Nephrol. Dial. Transplant.
14:279395
Holmes DR Jr, Savage M, LaBlanche
JM, Grip L, Serruys PW, et al. 2002. Results of prevention of REStenosis with
tranilast and its outcomes (PRESTO)
trial. Circulation 106:124350
Kagitani S, Ueno H, Hirade S, Takahashi T, Takata M, et al. 2004. Tranilast attenuates myocardial fibrosis in association with suppression of monocyte/
macrophage infiltration in DOCA/salt
hypertensive rats. J. Hypertens. 22:
100715
Pinto YM, Pinto-Sietsma SJ, Philipp T,
Engler S, Kossamehl P, et al. 2000.
Reduction in left ventricular messenger RNA for transforming growth factor 1 attenuates left ventricular fibrosis and improves survival without
lowering blood pressure in the hypertensive TGR(mRen2)27 rat. Hypertension
36:74754
Francis GA, Annicotte JS, Auwerx J.
2003. PPAR- effects on the heart and
other vascular tissues. Am. J. Physiol.
Schiffrin EL, Amiri F, Benkirane K,
Iglarz M, Diep QN. 2003. Peroxisome
proliferator-activated receptors: vascular
and cardiac effects in hypertension. Hypertension 42:66468
Wayman NS, Hattori Y, McDonald MC,
Mota-Filipe H, Cuzzocrea S, et al. 2002.
Ligands of the peroxisome proliferator-
117.
118.
119.
119a.
120.
121.
121a.
121b.
activated receptors (PPAR- and PPAR) reduce myocardial infarct size.

FASEB J. 16:102740
Diep QN, Benkirane K, Amiri F, Cohn
JS, Endemann D, et al. 2004. PPAR
activator fenofibrate inhibits myocardial
inflammation and fibrosis in angiotensin
II-infused rats. J. Mol. Cell. Cardiol.
36:295304
Nesto RW, Bell D, Bonow RO, Fonseca V, Grundy SM, et al. 2003. Thiazolidinedione use, fluid retention, and
congestive heart failure: a consensus
statement from the American Heart Association and American Diabetes Association. October 7, 2003. Circulation
108:294148
Manabe I, Shindo T, Nagai R. 2002.
Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy.
Circ. Res. 91:110313
Smith FD, Scott JD. 2001. Signaling
complexes: junctions on the intracellular
information super highway. Curr. Biol.
12:R3240
Chang F, Steelman LS, Lee JT, Shelton
JG, Navolanic PM, et al. 2003. Signal
transduction mediated by the Ras/Raf/
MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 17:126393
Powers SK, Quindry J, Hamilton K.
2004. Aging, exercise, and cardioprotection. Ann. NY Acad. Sci. 1019:462
70
Burgess ML, Terracio L, Hirozane T,
Borg TK. 2002. Differential integrin
expression by cardiac fibroblasts from
hypertensive and exercise-trained rat
hearts. Cardiovasc. Pathol. 11:7887
McMullen JR, Shioi T, Zhang L,
Tarnavski O, Sherwood MC, et al.
2003. Phosphoinositide 3-kinase (p110) plays a critical role for the induction of
physiological, but not pathological, cardiac hypertrophy. Proc. Natl. Acad. Sci.
USA 100:1235560
10 Dec 2004
17:42
AR
AR232-PA45-27.tex
P1: JRX

122. H/DCM Study Group. 2004. Family

studies of hypertrophic/dilated cardiomyopathy. http://clinicaltrials.gov/
show/NCT00045825
123. Coussens LM, Fingleton B, Matrisian
LM. 2002. Matrix metalloproteinase in-
687
hibitors and cancer: trials and tribulations. Science 295:238792

124. Peterson JT. 2004. Matrix metalloproteinase inhibitor development and the remodeling of drug discovery. Heart Fail.
Rev. 9:6379
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Copyright

EVALUATION OF DRUG-DRUG INTERACTION

IN THE HEPATOBILIARY AND RENAL
TRANSPORT OF DRUGS
Yoshihisa Shitara,1 Hitoshi Sato,1 and Yuichi Sugiyama2
1
School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555,
Japan; email: shitara@pharm.showa-u.ac.jp, satohito@pharm.showa-u.ac.jp
2
Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku,
Tokyo 113-0033, Japan; email: sugiyama@mol.f.u-tokyo.ac.jp
Key Words
interaction
transporter, pharmacokinetics, quantitative prediction of drug-drug
Abstract Recent studies have revealed the import role played by transporters in
the renal and hepatobiliary excretion of many drugs. These transporters exhibit a broad
substrate specificity with a degree of overlap, suggesting the possibility of transportermediated drug-drug interactions with other substrates. This review is an overview of
the roles of transporters and the possibility of transporter-mediated drug-drug interactions. Among the large number of transporters, we compare the Ki values of inhibitors
for organic anion transporting polypeptides (OATPs) and organic anion transporters
(OATs) and their therapeutic unbound concentrations. Among them, cephalosporins
and probenecid have the potential to produce clinically relevant OAT-mediated drugdrug interactions, whereas cyclosporin A and rifampicin may trigger OATP-mediated
ones. These drugs have been reported to cause drug-drug interactions in vivo with OATs
or OATP substrates, suggesting the possibility of transporter-mediated drug-drug interactions. To avoid adverse consequences of such transporter-mediated drug-drug
interactions, we need to be more aware of the role played by drug transporters as well
as those caused by drug metabolizing enzymes.
INTRODUCTION
The kidney and the liver play important roles in the elimination of drugs and xenobiotics from the body (15). Cumulative in vivo and in vitro studies have revealed
the importance of transporters in the renal and hepatobiliary excretion of many
drugs and other xenobiotics (15). Recent studies to investigate the molecular
mechanism of renal and hepatobiliary excretion have revealed that multiple transporters are expressed in the kidney and liver in animals and humans, as well as
revealing their function, tissue distribution, and intracellular localization (615).
These transporters exhibit broad substrate specificity with a degree of overlap.
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As each transporter accepts multiple drugs and/or xenobiotics as its substrates, it

may be competitively inhibited by other coadministered drugs and/or xenobiotics,
which may lead to drug-drug interactions involving the transporter (15, 16). In
this review, we summarize quantitatively the probability of drug-drug interactions
from in vitro and in vivo studies.
THE MECHANISM OF RENAL AND HEPATOBILIARY

EXCRETION OF DRUGS
The Mechanism of Renal ExcretionThe Role of Transporters
In the kidney, drugs are excreted in the urine as the net result of glomerular filtration,
tubular secretion, and reabsorption (4) (Figure 1). The mechanism of glomerular
filtration is simply ultrafiltration of drugs and xenobiotics, which do not bind
to macromolecules such as plasma proteins, and, therefore, transporters are not
involved in this process. Therefore, for drugs eliminated only by filtration, renal
excretion is not saturable and cannot be inhibited by other drugs. On the other
hand, in the case of tubular secretion, several active secretion mechanisms have
been reported in the proximal tubules, which are mainly mediated by transporters.
Figure 1
Mechanism of drug elimination in the kidney. Drug elimination in the
kidney takes place by glomerular filtration and secretion at the proximal tubules. However, they may return to the systemic circulation via a process of drug reabsorption.
Transporters are involved in drug secretion and reabsorption.
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Hence, this process is saturable and may be inhibited by coadministered drugs.

As with tubular secretion, the reabsorption process is sometimes mediated by
transporters in the proximal tubules. Therefore, the reabsorption process may be,
in part, saturable and inhibited by coadministered drugs. Renal clearance can be,
in general, described by the following equation:
CLR = (1 FR) (fu GFR + CLsec )

QR fu CLR,int
,
= (1 - FR) fu GFR +
QR + fu CLR,int
1.
where fu , GFR, FR, CLsec , QR , and CLR,int represent protein unbound fraction
in the blood, glomerular filtration rate [ml min1 ], the fraction reabsorbed, renal
secretion clearance, renal blood flow rate, and intrinsic clearance of tubular secretion, respectively (4). FR and CLR,int are partly saturable and can be inhibited,
suggesting the possibility of drug-drug interactions.
The Mechanism of Hepatobiliary ExcretionThe Role of

Transporters
In the liver, drugs are first taken up into hepatocytes, followed by metabolism
including oxidation (mediated by cytochrome P450, Phase I) and conjugation
(mediated by conjugation enzymes, Phase II), and excreted into the bile (Phase
III) (3) (Figure 2). Some drugs are excreted as intact drugs without metabolism.
In addition, drugs excreted in the intact form may pass into the blood again by
enterohepatic circulation. Drugs or their metabolites, once taken up into the liver,
may undergo secretion into the blood across the sinusoidal membrane, followed
by the hepatobiliary or renal excretion. To date, transporters have been shown to
play a role in hepatic uptake, biliary excretion, and the secretion into the blood
across the sinusoidal membrane (Figure 2). Hepatic clearance can be described by
the following equation (17, 18):
CLH =
QH fu CLH,int,all
,
QH + fu CLH,int,all
2.
where CLH , QH , and CLH,int,all represent the hepatic clearance; hepatic blood flow;
and overall intrinsic clearance of biliary excretion, including uptake, metabolism,
and biliary excretion, respectively. CLH,int,all can be described by the following
equation (3, 19):
CLH,int,all = PSinflux
CLH,int
,
PSefflux + CLH,int
3.
where PSinflux and PSefflux are the membrane permeability across the sinusoidal
membrane from the outside to the inside and from the inside to the outside of cells,
respectively, and CLH,int represents the exact intrinsic clearance for the metabolism
and/or biliary excretion of the unbound drugs. When CLH,int is negligibly low
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Figure 2 Mechanism of drug elimination in the liver. Drugs are taken up into hepatocytes via transporters and/or passive diffusion, followed by metabolism and/or
biliary excretion. Drugs are possibly effluxed into the circulation via sinusoidal
membrane.
compared with PSefflux (CLH,int PSefflux ), Equation 3 gives

CLH,int,all = PSinflux
CLH,int
.
PSefflux
4.
If PSefflux is much lower than CLH,int (CLH,int PSefflux ), Equation 3 gives

CLH,int,all = PSinflux .
5.
It should be noted that the uptake of drugs via the sinusoidal membrane (PSinflux ),
which is partly mediated by transporters, is a determinant of the net hepatic clearance regardless of the other processes, i.e., CLH,int and PSefflux . Therefore, hepatic clearance may be affected when the uptake clearance of drugs is altered,
even if the drug finally undergoes metabolism. On the other hand, the excretion of drugs via the bile canalicular membrane, which is partly mediated by
transporters, is a determinant of the net hepatic clearance, unless PSefflux is negligibly low compared with CLH,int . Therefore, except in this case, the change
in the biliary excretion may affect the net hepatic clearance. If PSefflux is much
lower than CLH,int , only a drastic reduction in the biliary excretion will affect
the net hepatic clearance, possibly leading to a transporter-mediated drug-drug
interaction.
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TRANSPORTERS IN THE KIDNEY AND LIVER

Recently, many types of transporters have been isolated in the kidney and liver
from animals and humans. Their substrate specificity has been characterized using
cRNA-injected oocytes and/or cDNA transfected cells. Generally, amphipathic
organic anions with relatively high molecular weights are eliminated from the liver
by metabolism and/or biliary excretion, whereas small and hydrophilic organic
anions are excreted into urine (7). In this section, the characteristics of transporters
expressed in the kidney and liver and their functions are summarized.
Transporters in the Kidney

Figure 3 shows transporters expressed in the kidney of rats and humans. Some
transporters are located on the basolateral membrane (blood side), whereas others
are located on the brush border membrane (luminal side), and these transporters
contribute to membrane transport, resulting in tubular secretion and/or reabsorption. In this section, the molecular aspects of renal transporters are summarized.
Figure 3
Transporters in the kidney. Transporters expressed in human and rodent kidney
are summarized in this figure. Some of the transporters in rodents are expressed only in
either rats or mice.
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The transport of organic anions is mainly mediated by organic anion transporters (OATs). Rat Oat1 has been isolated as a
renal transporter that is involved in the renal uptake of organic anions like paminohippuric acid (PAH) in an exchange of dicarboxylates (20). OAT15 have
been identified as human OAT family transporters (2124). Among them, OAT14
are expressed in the human kidney and OAT2 and 5 are expressed in the liver (21
24). In the kidney, OAT13 are localized on the basolateral membrane, whereas
OAT4 is localized on the brush border membrane (24). Each of these transporters
in the OAT family has a similar substrate specificity. These transporters accept organic anions with a relatively small molecular weight with some exceptions. They
accept PAH, methotrexate (MTX), nonsteroidal antiinflammatory drugs, and antiviral nucleoside analogues as substrates (2527). They also accept more lipophilic
organic anions, such as estrone 3-sulfate and ochratoxin A, and even an organic
cation, cimetidine (24, 25, 28).

ORGANIC ANION TRANSPORTERS
P-glycoprotein (P-gp) consists of two subclasses: MDR1 (MDR1

in humans, Mdr1a and 1b in rats and mice) and MDR2 [MDR2 (or 3) in humans
and Mdr2 in rats and mice] (8). The former is the well-known multidrug resistance
transporter, whereas the latter is a translocator for phospholipids (8). In the kidney,
P-gp is localized on the brush border membrane and acts as an efflux transporter
into the urine (8). P-gp is also expressed in the liver (8). In the liver, it is localized
on the bile canalicular membrane (8). P-gp was originally found as an overexpressing transporter in tumor tissues, and it acts as a multidrug resistance protein,
although it has also been identified in normal tissues such as kidney, liver, bloodbrain barrier, and intestine (8). P-gp substrates include anticancer drugs (such as
vincristine, vinblastine, doxorubicin, daunorubicin, etoposide, and paclitaxel), immunosuppresants (such as cyclosporin A), verapamil, digoxin, and steroids (such
as aldosterone and cortisole) (2936).
P-GLYCOPROTEIN
In the kidney, two isoforms of peptide transporters have

been identified: PEPT1 and PEPT2 (37). PEPT1 and 2 are localized on the brush
border membrane of the proximal tubule (38, 39). PEPT1 is expressed in the early
part of the proximal tubule (pars convoluta), whereas PEPT2 is expressed further
along the proximal tubule (pars recta) (38, 39). PEPT1 and 2 accept not only
di- or tri-peptides but also several therapeutic drugs. PEPT1 accepts therapeutic
drugs such as -lactam antibiotics (such as cephalexin, ceftibuten, cephradine),
ACE inhibitors (enalapril and temocapril), and valacyclovir (37, 40, 41). Although
there are few therapeutic drugs that have been reported to be substrates of PEPT2
(cephalexin), there are many drugs that interact with PEPT2 as inhibitors (37).
PEPTIDE TRANSPORTERS
OCT1 and OCT2 are expressed in the kidney, whereas only OCT1 is expressed in the liver (14, 42). OCT2 is highly expressed in the kidney (14, 42). In human kidney, these transporters are localized in
the basolateral membrane and are important organic cation transporters for renal
ORGANIC CATION TRANSPORTERS
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tubular secretion (14, 42). These are pH-independent, electrogenic, and polyspecific transporters (14, 42). These transporters accept organic cations with relatively
low molecular weight (type I cations), such as tetraethylammonium (TEA), as substrates (42, 43). Cimetidine, choline, dopamine, acyclovir, and zidovudine are also
reported to be substrates (4446).
OCTN1 is strongly expressed in the kidney but not in the adult liver (47).
In OCTN1-expressing HEK293 cells, pH-sensitive uptake of TEA has been observed (47). An inward proton concentration gradient stimulated the efflux of TEA
in OCTN1-expressing Xenopus leavis oocytes, indicating that OCTN1-mediated
transport couples with proton antiport (48). OCTN1 is considered to be localized on the brush border membrane of the kidney. Substrates include quinidine
and adriamycin as well as TEA (47, 48). OCTN2, an isoform of OCTN1, was
isolated from human placenta and it was also found to be expressed in the kidney
(49, 50). Although OCTN2 accepts TEA as its substrate, the transporter activity
is not as high as that of OCTN1. OCTN2 can also accept carnitine, a zwitterion
that is a cofactor essential for -oxidation of fatty acids, and several mutations in
mRNA encoding OCTN2 result in systemic carnitine deficiency owing to the poor
renal reabsorption of carnitine (51). This fact suggests that OCTN2 plays a role
in the renal reabsorption of carnitine. This transporter also accepts cephaloridine
and other cationic compounds, such as verapamil, quinidine, and phyrilamine, in
addition to TEA and carnitine, although it is not yet known whether this transporter
takes part in the renal reabsorption and/or excretion of these compounds together
with OCTN1 (52, 53).

OCTN
Transporters in the Liver

Figure 4 shows transporters in the liver. In the liver, uptake transporters are located
on the sinusoidal membrane (blood side) and efflux transporters are found on the
bile canalicular membrane, although some efflux transporters are on the sinusoidal
membrane and take part in the secretion into the blood.
In the liver, the uptake of many
organic anions is mediated by organic anion transporting polypeptides (OATPs),
although OAT2 and 5 are also reported to be expressed in the liver. In humans,
OATP-A, B, C, D, E, F, and 8 have been identified, and OATP-B, C, and 8 are
expressed in the liver (5458). In rats, the OATP family is also conserved and
Oatp1, 2, and 4 are expressed in the liver (5961). These transporters are localized
on the sinusoidal membrane of the liver. OATPs mainly accept bulky and amphipathic organic anions as substrates, although they also accept neutral compounds
such as digoxin. The substrates of OATP family transporters include therapeutic
drugs such as HMG-CoA reductase inhibitors, ACE inhibitors [enalapril and temocaprilat (an active form of temocapril)], and digoxin (55, 56, 58, 6265). Many
other therapeutic drugs also interact with OATP family transporters as inhibitors,
ORGANIC ANION TRANSPORTING POLYPEPTIDES
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Figure 4 Transporters in the liver. Transporters expressed in human and rodent liver are
summarized in this figure. Some of the transporters in rodents are expressed only in either
rats or mice.
suggesting there may be other drugs that are taken up into the liver via OATP
family transporters.
In TR and Eisai hyperbilirubinemic rats (EHBRs), which exhibit hyperbilirubinemia owing to a deficiency in
the biliary excretion of bilirubin glucuronide, mutations in the transporter, multidrug resistance-associated protein 2 (Mrp2), have been found (66, 67). This
finding and the comparison of the biliary excretion of several compounds between normal rats and Mrp2-deficient rats suggests that it plays an important
role in the biliary excretion of multispecific organic anions, including glucuronide
conjugates [bilirubin glucuronide, E3040 glucuronide, estradiol 17-D-glucuronide (E2 17G), grepafloxacin glucuronide, SN-38 glucuronide, glycyrrhizin, etc.],
glutathione conjugate [glutathione bimane (GSB), dinitrophenyl glutathione (DNPSG), leucotrienes (LTC4 , D4 and E4 ), etc.], grepafloxacine, MTX, pravastatin, SN38, and temocaprilat (8, 6872). This transporter is localized in the bile canalicular membrane of the liver (7375). Its human counterpart (MRP2) has been
isolated from the cisplatin-resistant tumor cells, KCP4 (76). This transporter is
MULTIDRUG RESISTANCE ASSOCIATED PROTEINS
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also localized on the bile canalicular membrane of the liver and accepts multiple organic anions, including glucuronides (bilirubin monoglucuronide, bilirubin
bisglucuronide, E3040 glucuronide, E2 17G, grepafloxacin glucuronide, LTC4 ,
etc.), glutathione conjugates (DNP-SG, GSB, glutathione-methylfluorescein, etc.),
pravastatin, MTX, vinblastine, vincristine, etoposide, etc. (33, 7788).
Human MRP3 is also a MRP family transporter, which is expressed in the liver.
However, this transporter is localized on the sinusoidal membrane and considered
to be involved in secretion into the blood. It also accepts many glucuronides,
glutathione conjugates, MTX, etc. (89, 90).
Breast cancer resistant protein (BCRP) has
been cloned from human MCF-7 breast cancer cells as a multidrug resistance
transporter (9193). This transporter also belongs to the ABC transporter family
(9193). However, its structure differs from that of other ABC transporters, such as
MDR1 and MRP, which contain two tandem repeats of transmembrane and ABC
domains. BCRP consists of only one ABC and one transmembrane domain, and,
therefore, it is referred to as a half-sized ABC transporter (9193). This transporter
is also expressed in normal tissues including the liver (94). In the liver, it is located
on the bile canalicular membrane (94). Many sulfated conjugates, such as estrone
3-sulfate (E1 S), dehydroepiandrosterone sulfate, 4-methylumbelliferone sulfate,
etc., are transported by BCRP (95). MTX, estradiol 17-D-glucuronide, and 2,4dinitrophenyl-S-glutathione are also transported but to a lesser extent compared
with E1 S (95). BCRP preferentially transports sulfate conjugates (95).
BREAST CANCER RESISTANT PROTEIN
METHODS FOR EVALUATING TRANSPORTER-MEDIATED

DRUG-DRUG INTERACTIONS IN THE KIDNEY AND
THE LIVER
In Vitro Transport Systems Using Tissues, Cells, Membrane
Vesicles, and Transporter-Expressing Systems
In vitro studies using tissues, cells, and membrane vesicles prepared from animals
have made it easy to characterize the mechanism of drug transport and estimate
the elimination rates of drugs via liver or kidney. Recently, these systems prepared from human sources have also become available, and they are likely to
be of great help in the drug discovery and other related research areas. Transporter cDNA-transfected cells or cRNA-injected oocytes are also available for
drug transport studies. Because of the scarcity of human tissue sources, transporterexpressing systems will be useful for predicting transporter-mediated drug-drug
interactions.
Kidney slices were used for the study to evaluate the renal uptake
of compounds/drugs from the basolateral side (9699). In rat kidney slices, the
KIDNEY SLICES
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uptake of compounds via Oct2, Oat1, Oat3, and a novel peptide transporter has been
observed (9699). The uptake of compounds into kidney slices is much lower than
the renal intrinsic uptake clearance in vivo, which may be partly due to diffusion
from the surface of the slices. Although extrapolation of the renal clearance using
kidney slices has not been reported, it may be used as a tool for the prediction of
transporter-mediated drug-drug interactions in the kidney. At the time of writing,
there have been no reports using human kidney slices; however, the use of this tool
will be useful for the prediction of transporter-mediated drug-drug interactions in
the kidney.
Isolated and cultured hepatocytes have
been used as an in vitro model of the liver (100, 101). The hepatic uptake of
peptidic endothelin antagonists using isolated rat hepatocytes showed that their in
vitro uptake clearance could be extrapolated to give their in vivo uptake clearance,
assuming a well-stirred model (100). Thus, isolated hepatocytes are a good tool
for the evaluation of drug uptake in the liver and transporter-mediated drug-drug
interactions in the liver. Recently, because of the progress in the techniques of
cryopreservation, it seems possible to preserve frozen human hepatocytes in such
a way that most of their enzymatic activity is retained (102). They have been used
to examine drug metabolism interactions, including induction of metabolic enzymes (103105). Recently, we have examined the uptake of taurocholate (TC)
and estradiol-17-D-glucuronide in freshly isolated and cryopreserved human hepatocytes (106). This study suggested that their active transports were retained even
in cryopreserved human hepatocytes, although the activity was decreased after
cryopreservation in some lots of hepatocytes (106). Therefore, cryopreserved human hepatocytes, at least, retain transporter function and they can be used as a
useful experimental system for examining the mechanism of the hepatic uptake of
drugs and interactions with other drugs (106).
ISOLATED AND CULTURED HEPATOCYTES
Liver slices are also used for the study of drug uptake in the liver
(107, 108). Olinga et al. examined the uptake of digoxin, a substrate of human
OATP8 [OATP1B3] and rat Oatp2 [Oatp1a4], and temperature-dependent uptake
was observed (107). Liver slices are supplemented with nonparenchymal cells,
and, therefore, the interaction between hepatocytes and other cells and the effect
of other cells on the function of hepatocytes can also be examined.
LIVER SLICES
Today, membrane vesicles prepared from the brush border

and basolateral membrane in the kidney and from the sinusoidal and bile canalicular
membrane in the liver are readily available for the study of renal and hepatobiliary
transport (109114). The advantages of using this system for transport studies are
(a) drug transport across the basolateral (sinusoidal) and apical (brush border or
bile canalicular) membrane can be measured separately, (b) intracellular binding
and/or metabolism can be ignored, and (c) buffers inside and outside vesicles can
be changed easily. On the other hand, using this system has limitations because it
MEMBRANE VESICLES
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requires a driving force for transport, so it is impossible to use this system without
prior characterization.
Using transporter expression systems,
the kinetic parameters for the target transporter can be obtained. Once the responsible transporter for the drugs in question has been identified, the possibility
of drug-drug interactions can be examined using the gene expression system,
i.e., without hepatocytes, membrane vesicles, and tissue slices. As human tissue
samples are scarcely distributed, transporter-expressing systems greatly help drug
transport studies. With the information of contributions of specific transporter(s)
to the total uptake of drugs in human liver or kidney, quantitative prediction of
drug uptake in human tissues is possible. The method to estimate the contributions of specific transporters is described below. cDNA-transfected cells and
cRNA-injected oocytes can be used as gene expression systems. More recently,
cultured cells stably transfected with both uptake and efflux transporters have
become available (85, 115). OATP-C/OATP2 [OATP1B1] and MRP2 transfected
cells and OATP8 [OATP1B3] and MRP2 transfected cells have been reported (Figure 5) (85, 115). Using them, hepatobiliary transport can be measured as vectorial
transcellular transport when these cells are cultured on a porous membrane. This
will make it easy to predict transporter-mediated drug-drug interactions in the
liver.

STUDIES USING GENE EXPRESSION SYSTEMS
The estimation of the contribution of specific transporter(s) is important for the quantitative prediction of the uptake in human tissues, including liver and kidney from the
in vitro data using transporter-expressing systems, and even for the quantitative
ESTIMATION OF THE CONTRIBUTION OF A SPECIFIC TRANSPORTER
Figure 5 Experimental system for the estimation of transcellular transport of drugs

mediated by OATP-C/OATP2 [OATP1B1] and MRP2. OATP-C/OATP2 [OATP1B1]
and MRP2 double-transfected MDCK cells are seeded in a membrane insert. The
basal-to-apical flux of drugs across the MDCK cell monolayer was examined to
estimate the transcellular transport mediated by OATP-C/OATP2 [OATP1B1] and
MRP2.
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prediction of transporter-mediated drug-drug interaction. Here, we show the method

to estimate it in in vitro assays.
First, injection of cRNA coding a transporter results in its expression on the
plasma membrane of Xenopus laevis oocytes that have been used for expression
cloning, functional analysis, or transport assays (117, 118). However, hybridization of mRNA with antisense oligonucleotide coding a specific sequence for the
target transporter specifically reduces the expression of the transporter (117, 118).
Comparison of the transporter activity in cRNA-injected oocytes in the presence
and absence of antisense nucleotides gives the contribution of each transporter to
the net transport (117, 118).
Kouzuki et al. have proposed a method using reference compounds (119, 120).
They measured the uptake of reference and test compounds at the same time in
transporter cDNA-transfected COS7 cells and rat hepatocytes and calculated the
contribution using the following equation (119, 120):
Contribution =
CLhep,ref /CLCOS,ref
,
CLhep,test /CLCOS,test
6.
where CLhep and CLCOS represent the uptake clearance of compounds into hepatocytes and transporter cDNA transfected cells, respectively. CLhep,ref and CLhep,test
represent the uptake clearance of the reference and test compounds, respectively.
The reference compounds should be specific substrates, otherwise the contribution
will be overestimated (119, 120). More recently, Hirano et al. proposed a method
to estimate the contributions of human transporters (OATP-C/OATP2 [OATP1B1]
and OATP8 [OATP1B3]) to the total hepatic uptake using estrone 3-sulfate and
cholecystokinine octapeptide (CCK8) as specific substrates, respectively, and actually estimated their contributions to the hepatic uptake of pitavastatin (121). They
also estimated their contributions by uptake in human hepatocytes and transporter
expression systems normalized by their transporter expression levels measured by
Western blot analysis (121). The contributions of OATP-C/OATP2 [OATP1B1] and
OATP8 [OATP1B3] estimated by these two different methods were comparable,
suggesting the validity of this method (121).
A specific inhibitor of a transporter also helps to estimate its contribution
to the total uptake. To identify a specific inhibitor, we examined the comparative inhibitory effects of many compounds on rat Oatp1 [Oatp1a1] and Oatp2
[Oatp1a4] (122). Among them, we found that digoxin specifically inhibited Oatp2
[Oatp1a4] with no effect on Oatp1 [Oatp1a1] (122). We also found several compounds which preferentially inhibited one of these transporters (122). These inhibitors may be used to estimate the contributions of Oatp1 [Oatp1a1] and Oatp2
[Oatp1a4] at appropriate concentrations (122). However, the selectivity of most
of the preferential inhibitors in this report was not very high, and inhibitors
that act as selective inhibitors over a wider range of concentrations are needed
(122).
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EVALUATION OF TRANSPORTER-MEDIATED
DRUG-DRUG INTERACTIONS

In this section, the inhibitory effects of therapeutic drugs and the possibility of
clinically relevant drug-drug interactions based on transporter-mediated processes
are described.
How to Evaluate the Extent of Transporter-Mediated

Drug-Drug Interactions
Previously, our group has suggested how to predict the extent of drug-drug interactions based on drug metabolism using in vitro studies (123, 124). This method
can also be applied to transporter-mediated drug-drug interactions (125). As transporter-mediated influx or efflux follows the Michaelis-Menten equation, the clearance can be described as follows:
CL =
Vmax
+ Pdif ,
Km + S u
7.
where CL is the influx or efflux clearance; Vmax , Km , and Pdif are the maximum
transport rate, Michaelis constant, and nonsaturable transport clearance, respectively; and Su is the protein-unbound substrate concentration. In the presence of
competitive inhibitors, it can be described as follows:
CL (+inhibitor) =
Vmax
+ Pdif ,
Km (1 + Iu /Ki ) + Su
8.
where Iu is the protein-unbound inhibitor concentration and Ki is the inhibition

constant. It should be noted that the Iu value is the protein-unbound inhibitor
concentration outside the cells for influx transporters, whereas it is that inside
the cells for efflux transporters. On the other hand, in the case of noncompetitive
inhibition, it can be described as follows:
CL (+inhibitor) =
Vmax /(1 + Iu /Ki )

+ Pdif .
Km + Su
9.
When the protein unbound substrate concentration is negligibly low compared

with the Km value, the influx or efflux clearance via transporters can be described
by the following equation, both for competitive and noncompetitive inhibition:
CL (+inhibitor) =
Vmax
+ Pdif .
Km (1 + Iu /Ki )
10.
Therefore, transporter-mediated influx or efflux clearance (i.e., net influx or

efflux clearance subtracted by the nonsaturable clearance) is decreased by the
following equation:
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CLtransporter (+inhibitor)
1
= R,
=
CLtransporter (control)
1 + Iu /Ki
11.
where CLtransporter represents transporter-mediated influx or efflux clearance.

When a transporter, which is a key determinant of the disposition of a drug, is
inhibited by a concomitantly administered drug, the area under the blood/plasma
concentration (AUC) after an oral administration will increase by at most R1 -fold
when the drug is predominantly excreted in the liver. In such cases, hepatic or
renal intrinsic clearances decrease by R-fold and, therefore, this R value is one of
the indicators of the severity of a drug-drug interaction. It should be particularly
useful for the evaluation of in vivo drug-drug interactions to avoid false negative
predictions.
For the liver transporters, the estimation of Iu should account for the inhibitors
in the portal vein as well as the hepatic artery when the inhibitor drug is orally
administered. In this case, Iu is not equal to the inhibitor concentration in the
circulating blood. Ito et al. have suggested a method to estimate the inhibitor
concentration at the inlet to the liver using the following equation (Figure 6) (123,
124):

vabs
Iu = fu (Isys + Ipv ) = fu Isys +
,
12.
QH
Figure 6
A model for estimating the inhibitor concentration at the inlet to the liver
after oral administration. Iinlet is the inhibitor concentration at the inlet to the liver. It
can be estimated from the inhibitor concentration in the hepatic artery (Ia ) plus that in
the portal vein (Ipv ).
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where Isys and Ipv are the inhibitor concentration in the circulating blood and portal
vein, respectively; fu is the blood protein unbound fraction; vabs is the absorption
rate from the intestine to the portal vein; and QH is the hepatic blood flow. When
the intestinal absorption is described by a first-order rate constant, this equation
becomes (123, 124)

F D ka ekat
F D ka
Iu = fu Isys +
fu Isys +
,
13.
QH
QH
where F is the fraction absorbed from the gastrointestinal tract, D is the dose, and
ka is the absorption rate constant. To avoid a false negative prediction, the unbound
a
inhibitor concentration should be estimated by fu (Isys + FDk
) for a drug-drug
QH
interaction based on a hepatic transporter-mediated process.
To date, there are many published inhibition studies of renal and hepatic uptake
transporters: OATs and OATPs. In this section, the inhibitory effects of therapeutic
drugs on these transporters are evaluated using Ki values, comparing them with
the therapeutic concentrations.
OAT-Mediated Drug-Drug Interactions

In the kidney, the OAT family transporters are involved in the uptake of organic
anions with relatively low molecular weights into the renal tubules, although OAT2
and 5 are localized in the liver and OAT4 is expressed in the brush border membrane of the kidney and may be involved in efflux from the renal tubules into the
urine (2124). These OAT family transporters are inhibited by several compounds,
including therapeutic drugs (Supplemental Table 1, Follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org).
Supplemental Table 1 gives a partial list of therapeutic drugs that interact with
OAT family transporters, together with their maximum plasma concentration and
maximum plasma unbound concentration in a clinical situation and R value.
The calculated R values suggest that many inhibitor drugs of OAT family transporters do not cause a serious drug-drug interaction because of the relatively low
plasma concentrations compared with their Ki values (Supplemental Table 1).
However, some cephalosporin antibiotics and probenecid exhibited low R values
and, therefore, may lead to clinically relevant drug-drug interactions (Supplemental Table 1). These results suggest that the concomitant use of these drugs with OAT
substrate drugs, which are mainly excreted in the urine, should be very carefully
monitored. Such use may cause at least a partial reduction in the intrinsic clearance
for renal secretion, possibly leading to an increase in plasma concentration.
OATP-Mediated Drug-Drug Interactions

Among OATP family transporters, OATP-B [OATP2B1], OATP-C/OATP2
[OATP1B1], and OATP8 [OATP1B3] are expressed in the human liver and are
involved in the hepatic uptake of several compounds, including therapeutic drugs
(5458). Although, in rats, some Oatp family transporters, such as Oatp1 [Oatp1a1],
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Oat-k1 [Oatp1a3], and k2, are reported to be expressed in the kidney (126130),
their human counterparts have not been characterized. As shown in Supplemental
Table 2 (Follow the Supplemental Material link from the Annual Reviews home
page at http://www.annualreviews.org), several therapeutic drugs are reported to
inhibit OATP family transporters. Because they are hepatic uptake transporters, R
values were calculated based on not only the maximum inhibitor unbound therapeutic concentration in the circulating blood but also that in the inlet to the
liver, calculated by Equation 13 (123, 124). Values calculated based on the unbound concentration in the inlet to the liver are given as R. Inhibitors of OATP
family transporters consist of bulky compounds, including anions, neutral compounds, and even cations (Supplemental Table 2). In Supplemental Table 2, only
cyclosporin A and rifampicin exhibited relatively low R and R values and may
lead to clinically relevant drug-drug interactions. On the other hand, pravastatin,
an HMG-CoA reductase inhibitor, is not a cause of a severe drug-drug interaction
based on OATP-mediated hepatic uptake because of its low plasma unbound concentration. As pravastatin is a potent HMG-CoA reductase inhibitor and is highly
distributed to the liver, its target organ, a low plasma concentration is sufficient
for its pharmacological effect, leading to a low risk of inhibition of transporter
function (132). A small number of inhibitors with relatively low R values may be
due to a lack of inhibition studies involving human OATP family transporters, and
further studies may provide other inhibitors that cause clinically relevant drug-drug
interactions. More inhibition studies on human OATP transporters are needed to
allow the quantitative prediction of transporter-mediated drug-drug interactions.
MDR-Mediated Drug-Drug Interactions

MDR1 is expressed in the liver and kidney (7, 8, 15). Therefore, MDR1-mediated
drug-drug interactions result in a reduction in renal and hepatobiliary excretion. It
is also expressed in the intestine and the blood-brain barrier and, therefore, MDR1mediated transport affects intestinal absorption and even distribution to the brain
(7). MDR1-mediated drug-drug interactions cause complex effects. MDR1 has a
broad substrate specificity and is inhibited by a large number of compounds. Quinidine is one MDR1 inhibitor (35). As the Km value of quinidine for ATP-dependent
efflux via MDR1 is approximately 5 M (32), its Ki value for MDR1 can be
assumed to be 5 M. The therapeutic steady-state concentration of quinidine is
approximately 4.5 M and its unbound concentration is 0.59 M. As MDR1 is
an efflux transporter, the R value should be calculated using the unbound concentration of inhibitor in the cell. However, it is practically impossible to measure
the intracellular unbound concentration of inhibitors in humans. Assuming the
cell-to-medium concentration ratio to be 10 as a safety margin, the R value can be
1
calculated to be 1+100.59/5
= 0.46, suggesting that renal efflux will be reduced
to at most 46% of the control. For hepatobiliary efflux, the blood concentration at
the inlet to the liver should be used. The plasma concentration of quinidine at the
inlet to the liver is calculated to be 4.6 M using QH = 1.6 liters min1 , Fa Fg =
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0.8, ka = 0.1 min1 , and fu = 0.13. Using this and assuming a cell-to-medium
1
concentration ratio of 10, the calculated R value is 1+104.6/5
= 0.098, suggesting
that hepatobiliary excretion will be reduced to at most 9.8% of the control. Actually, both the hepatobiliary and renal clearances of digoxin have been reported to
be reduced when concomitantly administered with quinidine (133).

MRP2-Mediated Drug-Drug Interactions

MRP2 also has a broad substrate specificity and is inhibited by a large number of
therapeutic drugs, including cyclosporin A, daunomycin, etoposide, probenecid,
and pravastatin (33, 134, 135). MRP2 functions as an efflux transporter for CPT11 and its metabolites, SN-38 and SN-38 glucuronide (SN38-glu) (136). CPT-11
is excreted into the bile mainly via MDR1 and, to a minor extent, via MRP2,
whereas SN-38 and SN38-glu are excreted via MRP2 (136). The biliary excretion
of its metabolites causes severe diarrhea as a side effect (137, 138). To prevent
this side effect, inhibition of MRP2-mediated transport by coadministration of its
inhibitor may be effective. Horikawa et al. have investigated the inhibitory effects
of several compounds on rat Mrp2 function (139). Among them, probenecid, sulfobromophthalein, and the glutathione-conjugate of sulfobromophthalein had potent
inhibitory effects (139). The inhibitory effects of probenecid were also confirmed
for the in vitro human biliary excretion of SN-38 with a Ki value of 42 M (139).
The same authors also confirmed these inhibitors of rat Mrp2 significantly reduced
the biliary excretion of CPT-11, SN-38, and SN38-glu (140). They suggested the
possibility of using MRP2 inhibitors such as probenecid to prevent the clinically
observed toxicity of diarrhea by CPT-11.
EXAMPLES OF CLINICALLY RELEVANT DRUG-DRUG

INTERACTIONS BASED ON RENAL AND
HEPATOBILIARY TRANSPORT
In this section, examples of clinically relevant drug-drug interactions based on
membrane transport in the kidney and the liver are described.
HMG-CoA Reductase Inhibitors Versus Cyclosporin A

As cerivastatin, a potent HMG-CoA reductase inhibitor (statin), is metabolized by
two different enzymes, cytochrome P450 2C8 (CYP2C8) and 3A4, the likelihood
of a severe drug-drug interaction was believed to be low (141). However, the plasma
concentration of cerivastatin was reported to be increased when coadministered
with cyclosporin A (142).
The plasma AUC and maximum plasma concentration of cerivastatin increased
by four- and fivefold, respectively, when concomitantly administered with cyclosporin A (142). Our group investigated the mechanism underlying this drugdrug interaction (62). We have shown that the transporter-mediated uptake of
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cerivastatin is inhibited by cyclosporin A at a low concentration (Ki was 0.3

0.7 M), whereas the in vitro metabolism of cerivastatin is inhibited with an IC50
value of more than 30 M, suggesting that this clinically relevant drug-drug interaction was caused by a transporter-mediated process rather than a metabolic
one (62). The unbound concentration of cyclosporin A in the circulating blood
and at the inlet to the liver, calculated by Equation 13, is, at most, 0.1 M and
0.6 M, respectively, which may explain the clinically relevant drug-drug interaction, although there may be other mechanisms involved (62). We also showed that
the OATP-C/OATP2 [OATP1B1]-mediated transport of cerivastatin was inhibited
by cyclosporin A with a Ki value of less than 0.2 M (Figure 7) (62).
In addition to cerivastatin, the plasma concentrations of pravastatin, pitavastatin, and HMG-CoA reductase inhibitory activity of atorvastatin are reported
to be affected by concomitantly administered cyclosporin A (143145). Among
them, pravastatin and pitavastatin undergo only minimal metabolism, and the likelihood of a drug-drug interaction owing to this is quite low. As these statins are
substrates of OATP-C/OATP2 [OATP1B1], interactions with cyclosporin A may
also be caused by a transporter-based mechanism (55, 56, 121). Interaction between atorvastatin and cyclosporin A may have occurred by a transporter-mediated
Figure 7
Transcellular transport of cerivastatin (CER) mediated by OATP-C/OATP2
[OATP1B1] and MRP2 and the inhibitory effect of cyclosporin A. (a) Transcellular transport of [14 C]CER in OATP-C/OATP2 [OATP1B1] and MRP2 double-transfected MDCK
cells (closed squares) and in vector-transfected cells (closed circles) was examined. Addition of cyclosporin A (10 M) inhibited OATP-C/OATP2 [OATP1B1]- and MRP2-mediated
transport of CER (open squares), whereas it did not change the transcellular transport in
vector transfected cells (open circles). (b) Cyclosporin A inhibited the transcellular transport
(PSB>A ) in a concentration-dependent manner. The IC50 value obtained in this experimental
system was 0.084 0.015 M. p < 0.01, p < 0.001.
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TABLE 1 Kinetic parameters of HMG-CoA reductase inhibitors coadministered with cyclosporin A

Cyclosporin A (+/)

HMGCoAreductase Cmax
inhibitors
[ng/mL]
Ratio
AUC
[ng h/mL]
Ratio
7.97
2.55
Simvastatin
18.9/2.5
20.6/9.9
7.56
2.08
78.1/9.8
101/39.6
Pravastatin
223/28.0
7.95
1300/
57.1
Fluvastatin
155/119
1.30
373/192
Cerivastatin
7.82/1.56
5.01
36.2/9.53
Atorvastatin
58.0/8.8#
6.59
Pitavastatin
179/27.6
6.49
Major
clearance
mechanism
Reference
CYP3A4
193
194
OATP-C
143
1.94
CYP2C9
195
3.80
CYP2C8/
3A4OATPC
142
595/79.9#
7.45
CYP3A4OATP-C
145
347/76.9
4.51
OATP-C
144
#ng eq./mL or ng eq. h/mL
p<0.05, p<0.01, p<0.001
and metabolism-based mechanism as atorvastatin is metabolized by CYP3A4 and

cyclosporin A inhibits CYP3A4-mediated metabolism (146). In Table 1, we summarize pharmacokinetic interactions between HMG-CoA reductase inhibitors and
cyclosporin A.
HMG-CoA Reductase Inhibitors Versus Gemfibrozil

Gemfibrozil also interacts with a wide range of statins (Table 2). In particular,
interactions with cerivastatin have been reported to cause the severe side effect
of myotoxicity, including lethal rhabdomyolysis (147). In addition, pharmacokinetic interaction between cerivastatin and gemfibrozil was reported (148, 149).
Although our group examined the inhibitory effects of gemfibrozil and its major metabolites on the OATP-C/OATP2 [OATP1B1]-mediated uptake of cerivastatin, we found gemfibrozil and its glucuronide inhibited it with IC50 values of
72 and 24 M, respectively, which were higher than their therapeutic unbound
concentrations, suggesting a low possibility of a transporter-mediated drug-drug
interaction (150). On the other hand, an interaction with rosuvastatin was reported to be caused by the inhibition of OATP-C/OATP2 [OATP1B1]-mediated
uptake by Schneck et al. (151). In their report, gemfibrozil inhibited the OATPC/OATP2 [OATP1B1]-mediated transport of cerivastatin with a low IC50 value
of 4 M (151). Although it is still higher than the therapeutic unbound concentration of cerivastatin, this value is lower than that we have obtained (150). This
gap may be partly due to the difference in the experimental system, i.e., we used
transporter-expressing MDCK cells, whereas Schneck et al. used cRNA-injected
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TABLE 2 Kinetic parameters of HMG-CoA reductase inhibitors coadministered with gemfibrozil

Gemfibrozil (+/)
HMG-CoA
reductase
inhibitors
Cmax
[ng/mL]
Lovastatin
Simvastatin
Pravastatin
Fluvastatin
Cerivastatin
2.38/2.69
6.15/6.87
120/66.3
54.3/48.4
8.0/3.2
Pitavastatin
Rosuvastatin
2.93/1.61 1.82
no data
1.30
109/49.5 2.20
Ratio
AUC
[ng h/mL]
Ratio
0.885
0.895
1.81
1.12
2.5
33.1/34.4
36.2/25.2
281/139
213/227
91.1/20.9
0.962
1.44
2.02
0.938
4.36
41.9/9.92
no data
771/410
4.22
1.45
1.88
Major
clearance
mechanism
CYP3A4
CYP3A4
OATP-C
CYP2C9
CYP2C8/3A4
OATP-C
OATP-C
CYP2C9
OATP-C
Reference
196
197
198
199
148
149
200
151
p<0.05, p<0.01, p<0.001
Xenopus laevis oocytes (150, 151). We also analyzed the inhibitory effects of
gemfibrozil and its metabolites on the P450-mediated metabolism of cerivastatin
and found that gemfibrozil and its glucuronide inhibited the CYP2C8-mediated
metabolism with IC50 values of 28 and 4 M, respectively (150). They are still
higher than the therapeutic unbound concentrations in the circulating blood. However, there are reports that, in rat perfusion studies, gemfibrozil-1-O-glucuronide
is actively taken up into the liver and accumulates there (152154). If this also
took place in human liver, the concentrated gemfibrozil-1-O-glucuronide might
act as an inhibitor of CYP2C8-mediated metabolism, leading to a drug-drug interaction. In this case, a transporter plays an important role, i.e., an inhibitor of the
metabolism leading to accumulation in the liver via transporter-mediated uptake.
Our hypothesis that interaction with gemfibrozil is not a transporter-mediated
one, but a metabolism-mediated one, is supported by the fact that gemfibrozil
does not cause a severe interaction with pravastatin and pitavastatin, which are
mainly cleared by the OATP-C/OATP2 [OATP1B1]-mediated hepatic uptake (Table 2). Therefore, we should also be more cautious about drug-drug interactions
when inhibitors of the metabolism are substrates of hepatic uptake transporters
(Figure 8).
Digoxin Versus Quinidine and Quinine

Digoxin undergoes biliary and renal excretion. Drug-drug interactions between
digoxin and quinidine and between digoxin and quinine (a stereoisomer of
quinidine) have been reported by Hedmann et al. (133). Quinidine reduced the
renal and biliary excretion of digoxin, whereas quinine reduced only the biliary
excretion of digoxin (133).
Because quinidine is a well-known P-gp inhibitor, its effect on biliary and
urinary excretion may be related to P-gp (MDR1)- mediated transport (35). As
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Figure 8 Possible mechanism of drug-drug interaction between cerivastatin and

gemfibrozil. Gemfibrozil-1-O-glucuronide is actively taken up via transporter(s) and
accumulates in the liver. In the liver, its concentration is hypothesized to be high enough
to inhibit the P450-mediated metabolism of cerivastatin.
described in MDR-Mediated Drug-Drug Interactions (above), the Ki value of quinidine for the MDR1-mediated efflux can be assumed to be 5 M. On the other hand,
the steady-state plasma concentration of quinidine in this study was 4.5 M, with
a protein unbound fraction of 0.13. Therefore, the protein unbound concentration
in the circulating blood is estimated to be 0.59 M. The unbound concentration of quinidine at the inlet to the liver estimated by Equation 13 is 4.6 M
using QH = 1.6 liters min1 , Fa Fg = 0.8, and ka = 0.1 min1 . With a safety
margin of 1 10 as a cell-to-medium concentration ratio, the estimated reduction
in the renal excretion of digoxin is 46% to 89% of the control, and the estimated
reduction in the hepatobiliary excretion of digoxin is 9.8% to 52% of the control. In
clinical situations, the hepatobiliary excretion was reduced to 42% of the control,
whereas the renal excretion was reduced to 60% of the control, which was within
the predicted range (133).
In rat hepatocytes, the inhibitory effect on the uptake of digoxin was more
potent for quinine than for quinidine, and the same tendency was observed using
the rat Oatp2 [Oatp1a4] expression system (122, 155). Therefore, the mechanism
of the drug-drug interaction between digoxin and quinine may be caused by the
inhibition of the transporter-mediated uptake. However, there is a study that shows
that both quinine and quinine had no inhibitory effects on the uptake of digoxin
into isolated human hepatocytes, although both of them inhibited the uptake of
digoxin into rat hepatocytes (156).
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Drug-Drug Interactions Between Cephalosporin

Antibiotics and Probenecid
There are many reports on the drug-drug interactions between cephalosporin antibiotics and probenecid (157). As both cephalosporins and probenecid interact
with OAT family transporters, some of these drug-drug interactions may be due
to an OAT-mediated uptake process. Most cephalosporins, which may be partly
mediated by OAT family transporters are excreted in the urine. The elimination
rates of cephazedone, cefazolin, cefalexin, cefradine, cefaclor, cefmetazole, cefoxitin, cefuroxime, cefmenoxime, ceftizoxime, and cedftriaxone were significantly
reduced by coadministration of probenecid, which may be partly caused by the
inhibition of their renal excretions (157).
Marino & Dominguez-Gil have shown that the pharmacokinetics of cefadroxil
is altered by coadministration of probenecid (158). In their report, the peak concentration and half-life of cefadroxil was increased 1.4- and 1.3-fold, respectively,
following coadministration of probenecid. Its urinary excretion rate constant falls
by 58%, supporting the possibility of drug-drug interaction at the renal excretion.
Supplemental Table 1 suggests that OAT1- and OAT3-mediated transport should
be decreased to at most 25%47% and 25%69% of the control, and, therefore, it
may be partly explained by the OAT-mediated drug-drug interaction.
Probenecid has also been shown to alter the plasma concentrations of cefamandole and ceftriaxone (159). The maximum plasma concentration and half-life of
cefamandole were increased 6- and 1.8-fold by coadministration of probenecid
(159). Also, 71% of cefamandole is excreted in the urine, and this was reduced to
66% of the control (159). The elimination of ceftriaxone was slightly affected by
coadministration of probenecid (160). Probenecid reduced the serum clearance of
ceftriaxone to 73% of the control (160). It reduced the renal and nonrenal clearance to 80% and 68% of the control, respectively, suggesting that this drug-drug
interaction is, to a minor extent, due to renal excretion (160).
Drug-Drug Interaction Between Methotrexate and NSAIDs

To date, there are reports that coadministration of MTX with penicillin, probenecid,
and NSAIDs cause drug-drug interactions and several potential sites for these DDI
have been reported: an increase in the protein unbound fraction of MTX, a decrease
in the urine flow rate resulting from the inhibition of prostaglandin synthesis, and
inhibition of the renal tubular secretion of MTX (161164). Nozaki et al. analyzed
the uptake mechanism of MTX in rat kidney slices and examined the effects of
NSAIDs on its uptake (165). They showed that rat Oat3 and reduced folate carrier
1 (RFC-1) equally contribute to the renal uptake (30% each), with the remaining
fraction being accounted for by passive diffusion and/or adsorption, whereas rOat1
makes only a limited contribution (165). Many NSAIDs inhibited both rOat3- and
RFC-1-mediated uptake of MTX, but the Ki value for Oat3 was lower than that
for RFC-1 (165). At their therapeutic concentrations, they inhibited only Oat3mediated uptake of MTX. Therefore, the affect of NSAIDs on the renal uptake of
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711
MTX is expected to be nonextensive and partial. Many NSAIDs also inhibit human
OAT3-mediated uptake of MTX with therapeutic relevant plasma concentrations
of unbound drugs (26). However, also in humans, the contribution of OAT3 to
the total renal uptake of MTX needs to be clarified for the identification of the
mechanism of the clinically relevant DDI.

CONCLUSION
In addition to phase I and phase II enzymes, transporters also play an important
role in drug elimination and distribution. Therefore, it is possible that transportermediated drug-drug interactions alter pharmacokinetics, and could result in severe
side effects.
A large number of transporters have been characterized in rodents and humans,
and the mechanism of the membrane transport of several compounds including
endogenous compounds and therapeutic drugs has been clarified. However, the
transport mechanism of most therapeutic drugs remains unknown. To predict a
transporter-mediated drug-drug interaction, the transporters involved in the membrane transport of the drug need to be characterized. As multiple transporters have
been characterized in the kidney and liver and their expression systems are available, it should be possible to predict a transporter-mediated drug-drug interaction
by using these systems with the information of the contribution made by each
transporter to the net transport in the kidney and liver.
We have estimated the possibility of a transporter-mediated drug-drug interaction from the R value, calculated using the maximum unbound concentration of
inhibitors. This method may avoid false negative predictions of drug-drug interactions. In conclusion, greater awareness of the possibility of transporter-mediated
drug-drug interactions is necessary.
http://pharmatox.annualreviews.org
LITERATURE CITED
1. Petzinger E. 1994. Transport of organic
anions in the liver. An update on bile
acid, fatty acid, monocarboxylate, anionic
amino acid, cholephilic organic anion,
and anionic drug transport. Rev. Physiol.
2. Oude Elferink RPJ, Meijer DKF, Kuipers
F, Jansen PLM, Groen AK, Groothis
GMM. 1995. Hepatobiliary secretion of
organic compounds; molecular mechanism of membrane transport. Biochem.
3. Yamazaki M, Suzuki H, Sugiyama Y.

1996. Recent advances in carrier-mediated hepatic uptake and biliary excretion of
xenobiotics. Pharm. Res. 13:497513
4. Okudaira N, Sugiyama Y. 1996. Use of
an isolated perfused kidney to assess renal
clearance of drugs: information obtained
in steady-state and non-steady-state experimental systems. In Models for Assessing Drug Absorption and Metabolism, ed
RT Borchard, PL Smith, G Wilson, pp.
21138. New York: Plenum Press
4 Dec 2004
19:14

712
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
5. Giacomini KM, Hsyu PH, Gisclon JG.

1988. Renal transport of drugs: an
overview of methodology with application to cimetidine. Pharm. Res. 5:46571
6. van Aubel RAMH, Masereeuw R, Russel FGM. 2000. Molecular pharmacology
of renal organic anion transporters. Am. J.
7. Kusuhara H, Sugiyama Y. 2002. Role of
transporters in the tissue-selective distribution and elimination of drugs: transporters in the liver, small intestine, brain
and kidney. J. Control. Release 78:4354
8. Kusuhara H, Suzuki H, Sugiyama Y.
1998. The role of P-glycoprotein and
canalicular multispecific organic anion
transporter in the hepatobiliary excretion
of drugs. J. Pharm. Sci. 87:102540
9. Suzuki H, Sugiyama Y. 2000. Transport
of drugs across the hepatic sinusoidal
membrane: sinusoidal drug influx and efflux in the liver. Semin. Liver Dis. 20:251
63
10. Inui K, Masuda S, Saito H. 2000. Cellular
and molecular aspects of drug transport in
the kidney. Kidney Int. 58:94458
11. Dresser MJ, Leabman MK, Giacomini
KM. 2001. Transporters involved in the
elimination of drugs in the kidney: organic
anion transporters and organic cation
transporters. J. Pharm. Sci. 90:397421
12. Sweet DH, Bush KT, Nigam SK. 2001.
The organic anion transporter family:
from physiology to ontogeny and the
clinic. Am. J. Physiol. Renal Physiol. 281:
F197205
13. Sekine T, Cha SH, Endou H. 2000. The
multispecific organic anion transporter
(OAT) family. Pflugers Arch. 440:337
50
14. Koepsell H, Gorboulev V, Arndt P.
1999. Molecular pharmacology of organic cation transporters in the kidney. J.
Membr. Biol. 167:10317
15. Mizuno N, Sugiyama Y. 2002. Drug transporters: their roles and importance in the
selection and development of new drugs.
Drug Metab. Pharmacokin. 17:93108
16. Meier PJ, Eckhardt U, Schroeder A, Hagenbuch B, Stieger B. 1997. Substrate

specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver. Hepatology 26:166777
17. Pang KS, Rowland M. 1977. Hepatic
clearance of drugs I: theoretical considerations of a well-stirred model and a
parallel-tube model. Influence of hepatic blood flow, plasma and blood cell
binding, and the hepatocellular enzymatic
activity on hepatic drug clearance. J.
Pharmacokin. Biopharm. 5:62553
18. Pang KS, Gillette R. 1978. Kinetics
of metabolite formation and elimination in the perfused rat liver preparation: difference between the elimination of preformed acetaminophen and
acetaminophen formed from phenacetin.
19. Miyauchi S, Sugiyama Y, Sawada Y,
Morita K, Iga T, et al. 1987. Kinetics
of hepatic transport of 4-methylumbelliferone in rats: analysis by multiple indicator dilution method. J. Pharmacokin.
Biopharm. 15:2538
20. Sekine T, Watanabe N, Hosoyamada M,
Kanai Y, Endou H. 1997. Expression
cloning and characterization of a novel
multispecific organic anion transporter. J.
Biol. Chem. 272:1852629
21. Hosoyamada M, Sekine T, Kanai Y,
Endou H. 1999. Molecular cloning and
functional expression of a multispecific
organic anion transporter from human
kidney. Am. J. Physiol. Renal Physiol.
276:F12228
22. Sun W, Wu RR, van Poelje PD, Erion
MD. 2001. Isolation of a family of organic
anion transporters from human liver and
kidney. Biochem. Biophys. Res. Comm.
283:41722
23. Race JE, Grassl SM, Williams WJ, Holtzman EJ. 1999. Molecular cloning and
characterization of two novel human renal
organic anion transporters (hOAT1 and
hOAT3). Biochem. Biophys. Res. Commun. 255:50814
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX


24. Cha SH, Sekine T, Kusuhara H, Yu E,
Kim JY, et al. 2000. Molecular cloning
and characterization of multispecific organic anion transporter 4 expressed in the
placenta. J. Biol. Chem. 275:450712
25. Cha SH, Sekine T, Fukushima JI, Kanai
Y, Kobayashi Y, et al. 2001. Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol. 59:127786
26. Takeda M, Khamdang S, Narikawa S,
Kimura H, Hosoyamada M, et al. 2002.
Characterization of methotrexate transport and its drug interactions with human
organic anion transporters. J. Pharmacol.
Exp. Ther. 302:66671
27. Takeda M, Khamdang S, Narikawa S,
Kimura H, Kobayashi Y, et al. 2002. Human organic anion transporters and human organic cation transporters mediate
renal antiviral transport. J. Pharmacol.
Exp. Ther. 300:91824
28. Jung KY, Takeda M, Kim DK, Tojo A,
Narikawa S, et al. 2001. Characterization of ochratoxin A transport by human organic anion transporters. Life Sci.
69:212335
29. Lecureur V, Sun D, Hargrove P, Schuetz
EG, Kim RB, et al. 2000. Cloning
and expression of murine sister of Pglycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol. Pharmacol. 57:2435
30. Yamazaki M, Neway WE, Ohe T, Chen
I, Rowe JF, et al. 2001. In vitro substrate
identification studies for p-glycoproteinmediated transport: species difference and
predictability of in vivo results. J. Pharmacol. Exp. Ther. 296:72335
31. Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, et al. 1992.
Partial purification and reconstitution of
the human multidrug-resistance pump:
characterization of the drug-stimulatable
ATP hydrolysis. Proc. Natl. Acad. Sci.
USA 89:847276
32. Adachi Y, Suzuki H, Sugiyama Y. 2001.
33.
34.
35.
36.
37.
38.
39.
40.
713
Comparative studies on in vitro methods

for evaluating in vivo function of MDR1
P-glycoprotein. Pharm. Res. 18:1660
68
Guo A, Marinaro W, Hu P, Sinko PJ.
2002. Delineating the contribution of
secretory transporters in the efflux of
etoposide using Madin-Darby canine kidney (MDCK) cells overexpressing Pglycoprotein (Pgp), multidrug resistanceassociated protein (MRP1), and canalicular multispecific organic anion transporter
(cMOAT). Drug Metab. Dispos. 30:457
63
Walle UK, Walle T. 1998. Taxol transport by human intestinal epithelial Caco-2
cells. Drug Metab. Dispos. 26:34346
Tanigawara Y, Okamura N, Hirai M, Yasuhara M, Ueda K, et al. 1992. Transport
of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell
line (LLC-PK1). J. Pharmacol. Exp. Ther.
263:84045
Ueda K, Okamura N, Hirai M, Tanigawara Y, Saeki T, et al. 1992. Human
P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J. Biol. Chem. 267:2424852
Ganapathy ME, Brandsch M, Prasad PD,
Ganapathy V, Leibach FH. 1995. Differential recognition of beta-lactam antibiotics by intestinal and renal peptide
transporters, PEPT 1 and PEPT 2. J. Biol.
Chem. 270:2567277
Smith DE, Pavlova A, Berger UV, Hediger MA, Yang T, et al. 1998. Tubular localization and tissue distribution of peptide transporters in rat kidney. Pharm. Res.
15:124449
Shen H, Smith DE, Yang T, Huang YG,
Schnermann JB, et al. 1999. Localization of PEPT1 and PEPT2 proton-coupled
oligopeptide transporter mRNA and protein in rat kidney. Am. J. Physiol. Renal
Physiol. 276:F65865
Tsuji A. 2002. Transporter-mediated drug
interactions. Drug. Metabol. Pharmacokin. 17:25374
4 Dec 2004
19:14

714
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
41. Han HK, Rhie JK, Oh DM, Saito G, Hsu

CP, et al. 1999. CHO/hPEPT1 cells overexpressing the human peptide transporter
(hPEPT1) as an alternative in vitro model
for peptidomimetic drugs. J. Pharm. Sci.
88:34750
42. Gorboulev V, Ulzheimer JC, Akhoundova
A, Ulzheimer-Teuber I, Karbach U, et al.
1997. Cloning and characterization of two
human polyspecific organic cation transporters. DNA Cell. Biol. 16:87181
43. Zhang L, Schaner ME, Giacomini KM.
1998. Functional characterization of an
organic cation transporter (hOCT1) in
a transiently transfected human cell
line (HeLa). J. Pharmacol. Exp. Ther.
286:35461
44. Dudley AJ, Bleasby K, Brown CD. 2000.
The organic cation transporter OCT2 mediates the uptake of beta-adrenoceptor antagonists across the apical membrane of
renal LLC-PK1 cell monolayers. Br. J.
Pharmacol. 131:7179
45. Busch AE, Karbach U, Miska D, Gorboulev V, Akhoundova A, et al. 1998.
Human neurons express the polyspecific cation transporter hOCT2, which
translocates monoamine neurotransmitters, amantadine, and memantine. Mol.
Pharmacol. 54:34252
46. Urakami Y, Akazawa M, Saito H, Okuda
M, Inui K. 2002. cDNA cloning, functional characterization, and tissue distribution of an alternatively spliced variant
of organic cation transporter hOCT2 predominantly expressed in the human kidney. J. Am. Soc. Nephrol. 13:170310
47. Tamai I, Yabuuchi H, Nezu J, Sai Y, Oku
A, et al. 1997. Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1. FEBS
Lett. 419:10711
48. Yabuuchi H, Tamai I, Nezu J, Sakamoto
K, Oku A, et al. 1999. Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent
transport of organic cations. J. Pharmacol. Exp. Ther. 289:76873
49. Tamai I, Ohashi R, Nezu J, Yabuuchi

H, Oku A, et al. 1998. Molecular and
functional identification of sodium iondependent, high affinity human carnitine transporter OCTN2. J. Biol. Chem.
273:2037882
50. Wu X, Huang W, Prasad PD, Seth P, Rajan
DP, et al. 1999. Functional characteristics
and tissue distribution pattern of organic
cation transporter 2 (OCTN2), an organic
cation/carnitine transporter. J. Pharmacol. Exp. Ther. 290:148292
51. Nezu J, Tamai I, Oku A, Ohashi R, Yabuuchi H, et al. 1999. Primary systemic carnitine deficiency is caused by mutations
in a gene encoding sodium ion-dependent
carnitine transporter. Nat. Genet. 21:91
94
52. Ohashi R, Tamai I, Yabuuchi H, Nezu JI,
Oku A, et al. 1999. Na+ -dependent carnitine transport by organic cation transporter (OCTN2): its pharmacological and
toxicological relevance. J. Pharmacol.
Exp. Ther. 291:77884
53. Ganapathy ME, Huang W, Rajan DP,
Carter AL, Sugawara M, et al. 2000.
-lactam antibiotics as substrates for
OCTN2, an organic cation/carnitine transporter. J. Biol. Chem. 275:1699707
54. Tamai I, Nezu J, Uchino H, Sai Y, Oku A,
et al. 2000. Molecular identification and
characterization of novel members of the
human organic anion transporter (OATP)
family. Biochem. Biophys. Res. Commun.
273:25160
55. Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, et al. 1999. A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liverspecific human organic anion transporting polypeptide and identification of rat
and human hydroxymethylglutaryl-CoA
reductase inhibitor transporters. J. Biol.
Chem. 274:3716168
56. Nakai D, Nakagomi R, Furuta Y, Tokui
T, Abe T, et al. 2001. Human liverspecific organic anion transporter, LST-1,
mediates uptake of pravastatin by human
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX
57.

58.
59.
60.
61.
62.
63.
64.
65.
hepatocytes. J. Pharmacol. Exp. Ther.

297:86167
Konig J, Cui Y, Nies AT, Keppler D. 2000.
A novel human organic anion transporting
polypeptide localized to the basolateral
hepatocyte membrane. Am. J. Physiol.
Gastrointest. Liver Physiol. 278:G15664
Konig J, Cui Y, Nies AT, Keppler D.
2000. Localization and genomic organization of a new hepatocellular organic anion
transporting polypeptide. J. Biol. Chem.
275:2316168
Jacquemin E, Hagenbuch B, Stieger B,
Wolkoff AW, Meier PJ. 1994. Expression
cloning of a rat liver Na+ -independent organic anion transporter. Proc. Natl. Acad.
Sci. USA. 91:13337
Noe B, Hagenbuch B, Stieger B, Meier
PJ. 1997. Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain. Proc. Natl. Acad.
Sci. USA 94:1034650
Cattori V, Hagenbuch B, Hagenbuch N,
Stieger B, Ha R, et al. 2000. Identification
of organic anion transporting polypeptide
4 (Oatp4) as a major full-length isoform
of the liver-specific transporter-1 (rlst-1)
in rat liver. FEBS Lett. 474:24245
Shitara Y, Itoh T, Sato H, Li AP,
Sugiyama Y. 2003. Inhibition of transporter-mediated hepatic uptake as a mechanism for drug-drug interaction between
cerivastatin and cyclosporin A. J. Pharmacol. Exp. Ther. 304:61016
Pang KS, Wang PJ, Chung AYK, Wolkoff
AW. 1998. The modified dipeptide
enalapril, an angiotensin-converting enzyme inhibitor, is transported by the rat
liver organic anion transport protein. Hepatology 28:134146
Ishizuka H, Konno K, Naganuma H,
Nishimura K, Kouzuki H, et al. 1998.
Transport of temocaprilat into rat hepatocytes: role of organic anion transporting polypeptide. J. Pharmacol. Exp. Ther.
287:3742
Kullak-Ublick GA, Ismair MG, Stieger
B, Landmann L, Huber R, et al. 2001.
66.
67.
68.
69.
70.
71.
72.
73.
74.
715
Organic anion-transporting polypeptide B

(OATP-B) and its functional comparison
with three other OATPs of human liver.
Gastroenterology 120:52533
Jansen PL, Peters WH, Lamers WH.
1985. Hereditary chronic conjugated hyperbilirunemia in mutant rats caused by
defective hepatic anion transport. Hepatology 5:57379
Hosokawa S, Tagaya O, Mikami T,
Nozaki Y, Kawaguchi Y, et al. 1992. A
new rat mutant with chronic conjugated
hyperbilirunemia and renal glomerular lesions. Lab. Animal Sci. 42:2734
Muller M, Jansen PL. 1997. Molecular
aspects of hepatobiliary transport. Am.
J. Physiol. Gastrointest. Liver Physiol.
272:G1285303
Oude Elferink RP, Meijer DK, Kuipers
F, Jansen PL, Groen AK, et al. 1995.
Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport. Biochim. Biophys. Acta
1241:21568
Stieger B, Meier PJ. 1998. Bile acid
and xenobiotic transporters in liver. Curr.
Opin. Cell. Biol. 10:46267
Suzuki H, Sugiyama Y. 1998. Excretion of GSSG and glutathione conjugates
mediated by MRP1 and cMOAT/MRP2.
Semin. Liver Dis. 18:35976
Konig J, Nies AT, Cui Y, Leier I, Keppler D. 1999. Conjugate export pumps of
the multidrug resistance protein (MRP)
family: localization, substrate specificity,
and MRP2-mediated drug resistance.
Biochim. Biophys. Acta 1461:37794
Paulusma CC, Bosma PJ, Zaman GJ,
Bakker CT, Otter M, et al. 1996. Congenital jaundice in rats with a mutation in
a multidrug resistance-associated protein
gene. Science 271:112628
Ito K, Suzuki H, Hirohashi T, Kume K,
Shimizu T, et al. 1997. Molecular cloning
of canalicular multispecific organic anion transporter defective in EHBR. Am.
272:G1622
4 Dec 2004
19:14

716
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
75. Buchler M, Konig J, Brom M, Kartenbeck J, Spring H, et al. 1996. cDNA

cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic
mutant rats. J. Biol. Chem. 271:15091
98
76. Taniguchi K, Wada M, Kohno K, Nakamura T, Kawabe T, et al. A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in
cisplatin-resistant human cancer cell lines
with decreased drug accumulation. Cancer Res. 56:412429
77. Borst P, Evers R, Kool M, Wijnholds
J. 1999. The multidrug resistance protein family. Biochim. Biophys. Acta
1461:34757
78. Kuwano M, Toh S, Uchiumi T, Takano
H, Kohno K, Wada M. 1999. Multidrug
resistance-associated protein subfamily
transporters and drug resistance. Anticancer Drug Des. 14:12331
79. Jedlitschky G, Leier I, Buchholz U,
Hummel-Eisenbeiss J, Burchell B, et al.
1997. ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte
canalicular isoform MRP2. Biochem. J.
327:30510
80. Niinuma K, Kato Y, Suzuki H, Tyson
CA, Weizer V, et al. 1999. Primary active transport of organic anions on bile
canalicular membrane in humans. Am.
276:G115364
81. Cui Y, Konig J, Buchholz JK, Spring H,
Leier I, et al. 1999. Drug resistance and
ATP-dependent conjugate transport mediated by the apical multidrug resistance
protein, MRP2, permanently expressed in
human and canine cells. Mol. Pharmacol.
55:92937
82. Niinuma K, Kato Y, Suzuki H, Tyson
CA, Weizer V, et al. 1999. Primary active transport of organic anions on bile
canalicular membrane in humans. Am.
83.
84.
85.
86.
87.
88.
89.
90.

276:G115364
Paulusma CC, van Geer MA, Evers R,
Heijn M, Ottenhoff R, et al. 1999. Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced
glutathione. Biochem. J. 338:393401
Ryu S, Kawabe T, Nada S, Yamaguchi A.
2000. Identification of basic residues involved in drug export function of human
multidrug resistance-associated protein 2.
J. Biol. Chem. 275:3961724
Sasaki M, Suzuki H, Ito K, Abe T,
Sugiyama Y. 2002. Transcellular transport of organic anions across a doubletransfected Madin-Darby canine kidney
II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and multidrug
resistance-associated protein 2 (MRP2/
ABCC2). J. Biol. Chem. 277:6497
503
Hooijberg JH, Broxterman HJ, Kool M,
Assaraf YG, Peters GJ, et al. 1999. Antifolate resistance mediated by the multidrug
resistance proteins MRP1 and MRP2.
Tang F, Horie K, Borchardt RT. 2002. Are
MDCK cells transfected with the human
MRP2 gene a good model of the human
intestinal mucosa? Pharm. Res. 19:773
79
Chen ZS, Kawabe T, Ono M, Aoki
S, Sumizawa T, et al. 1999. Effect of
multidrug resistance-reversing agents on
transporting activity of human canalicular
multispecific organic anion transporter.
Akita H, Suzuki H, Hirohashi T, Takikawa
H, Sugiyama Y. 2002. Transport activity of human MRP3 expressed in Sf9
cells: comparative studies with rat MRP3.
Pharm. Res. 19:3441
Zeng H, Liu G, Rea PA, Kruh GD. 2000.
Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res. 60:477984
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX


91. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, et al. 1998. A multidrug
resistance transporter from human MCF-7
breast cancer cells. Proc. Natl. Acad. Sci.
USA 95:1566570
92. Allikmets R, Schriml LM, Hutchinson A,
Romano-Spica V, Dean M. 1998. A human placenta-specific ATP-binding cassette gene (ABCP) on chromosome 4q22
that is involved in multidrug resistance.
93. Miyake K, Mickley L, Litman T, Zhan Z,
Robey R, et al. 1999. Molecular cloning of
cDNAs which are highly overexpressed in
mitoxantrone-resistant cells. Cancer Res.
59:813
94. Maliepaard M, Scheffer GL, Faneyte IF,
van Gastelen MA, Pijnenborg ACLM,
et al. 2001. Subcellular localization and
distribution of the breast cancer resistance
protein transporter in normal human tissues. Cancer Res. 61:345864
95. Suzuki M, Suzuki H, Sugimoto Y,
Sugiyama Y. 2003. ABCG2 transports
sulfated conjugates of steroids and xenobiotics. J. Biol. Chem. 278:22644
49
96. Urakami Y, Nakamura N, Takahashi K,
Okuda M, Saito H, et al. 1999. Gender differences in expression of organic cation
transporter OCT2 in rat kidney. FEBS
Lett. 461:33942
97. Hasegawa M, Kusuhara H, Sugiyama D,
Ito K, Ueda S, et al. 2002. Functional involvement of rat organic anion transporter
3 (rOat3; Slc22a8) in the renal uptake of
organic anions. J. Pharmacol. Exp. Ther.
300:74653
98. Hori R, Ishikawa Y, Takano M, Okano T,
Kitazawa S, et al. 1982. The interaction of
cephalosporin antibiotics with renal cortex of rats: accumulation to cortical slices
and binding to purified plasma membranes. Biochem. Pharmacol. 31:2267
72
99. Terada T, Sawada K, Ito T, Saito H,
Hashimoto Y, et al. 2000. Functional expression of novel peptide transporter in re-
100.
101.
102.
103.
104.
105.
106.
107.
717
nal basolateral membranes. Am. J. Physiol. Renal Physiol. 279:F85157

Akhteruzzaman S, Kato Y, Kouzuki H,
Suzuki H, Hisaka A, et al. 1999. Carriermediated hepatic uptake of peptidic endothelin antagonists in rats. J. Pharmacol.
Exp. Ther. 290:110715
Ishigami M, Tokui T, Komai T, Tsukahara
K, Yamazaki M, et al. 1995. Evaluation of
the uptake of pravastatin by perfused rat
liver and primary cultured rat hepatocytes.
Pharm. Res. 12:174145
Madan A, DeHaan R, Mudra D, Carroll
K, LeCluyse E, Parkinson A. 1999. Effect
of cryopreservation on cytochrome P-450
enzyme induction in cultured rat hepatocytes. Drug Metab. Dispos. 27:32735
Li AP, Maurel P, Gomez-Lechon MJ,
Cheng LC, Jurima-Romet M. 1997. Preclinical evaluation of drug-drug interaction potential: present status of the application of primary human hepatocytes in
the evaluation of cytochrome P450 induction. Chem. Biol. Interact. 107:516
Li AP, Lu C, Brent JA, Pham C, Fackett A, et al. 1999. Cryopreserved human
hepatocytes: characterization of drugmetabolizing enzyme activities and applications in higher throughput screening
assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.
Li AP, Gorycki PD, Hengstler JG, Kedderis GL, Koebe HG, et al. 1999. Present
status of the application of cryopreserved
hepatocytes in the evaluation of xenobiotics: consensus of an international expert
panel. Chem. Biol. Interact. 121:11723
Shitara Y, Li AP, Kato Y, Lu C, Ito
K, et al. 2003. Function of uptake transporters for taurocholate and estradiol 17D-glucuronide in cryopreserved human
hepatocytes. Drug Metab. Pharmacokin.
18:3341
Olinga P, Hof IH, Merema MT, Smit M,
de Jager MH, et al. 2001. The applicability
of rat and human liver slices to the study
of mechanisms of hepatic drug uptake.
4 Dec 2004
19:14
718
108.

109.
110.
111.
112.
113.
114.
115.
116.
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
J. Pharmacol. Toxicol. Methods 45:55

63
Shigematsu A, Motoji N, Momose Y, Iida
A, Higashi N. 2000. Viability of liver
slices exhibiting absorption, metabolism,
and elimination of substrates in culture
medium. Exp. Mol. Pathol. 69:11943
Pritchard JB, Miller DS. 1993. Mechanisms mediating renal secretion of organic
anions and cations. Physiol. Rev. 73:765
96
Murer H, Gmaj P, Steiger B, Hagenbuch B. 1989. Transport studies with
renal proximal tubular and small intestinal brush border and basolateral
membrane vesicles: vesicle heterogeneity,
coexistence of transport system. Methods
Enzymol. 172:34664
Boyer JL, Meier PJ. 1990. Characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles.
Methods Enzymol. 192:51733
Kinne-Saffran E, Kinne RK. Isolation of
luminal and cotralumenal plasma membrane vesicles from kidney. Methods Enzymol. 191:45069
Meier PJ, Boyer JL. 1990. Preparation
of basolateral (sinusoidal) and canalicular
plasma membrane vesicles for the study of
hepatic transport processes. Methods Enzymol. 192:53445
Meier PJ, St Meier-Abt A, Barrett C,
Boyer JL. 1984. Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier. J. Biol. Chem.
259:1061422
Cui Y, Konig J, Keppler D. 2001.
Vectorial transport by double-transfected
cells expressing the human uptake transporter SLC21A8 and the apical export
pump ABCC2. Mol. Pharmacol. 60:934
43
Schinkel AH, Mayer U, Wagenaar E,
Mol CA, van Deemter L, et al. 1997.
Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-
117.
118.
119.
120.
121.
122.
123.
124.
transporting) P-glycoproteins. Proc. Natl.

Hagenbuch B, Scharschmidt BF, Meier
PJ. 1996. Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes.
Biochem. J. 316:9014
Tamai I, Nakanishi T, Hayashi K, Terao
T, Sai Y, et al. 1997. The predominant
contribution of oligopeptide transporter
PepT1 to intestinal absorption of betalactam antibiotics in the rat small intestine. J. Pharm. Pharmacol. 49:796801
Kouzuki H, Suzuki H, Ito K, Ohashi
R, Sugiyama Y. 1998. Contribution
of sodium taurocholate co-transporting
polypeptide to the uptake of its possible
substrates into rat hepatocytes. J. Pharmacol. Exp. Ther. 286:104350
Kouzuki H, Suzuki H, Ito K, Ohashi R,
Sugiyama Y. 1999. Contribution of organic anion transporting polypeptide to
uptake of its possible substrates into rat
hepatocytes. J. Pharmacol. Exp. Ther.
288:62734
Hirano M, Maeda K, Shitara Y, Sugiyama
Y. 2004. Contribution of OATP2
(OATP1B1) and OATP8 (OATP1B3)
to the hepatic uptake of pitavastatin
in humans. J. Pharmacol. Exp. Ther.
311:13946
Shitara Y, Sugiyama D, Kusuhara H, Kato
Y, Abe T. 2002. Comparative inhibitory
effects of different compounds on rat oatpl
(Slc21a1)- and Oatp2 (Slc21a5)-mediated
transport. Pharm. Res. 19:14753
Ito K, Iwatsubo T, Kanamitsu S, Ueda K,
Suzuki H, et al. 1998. Prediction of pharmacokinetic alterations caused by drugdrug interactions: metabolic interaction
in the liver. Pharmacol. Rev. 50:387
412
Ito K, Iwatsubo T, Kanamitsu S, Nakajima Y, Sugiyama Y. 1998. Quantitative
prediction of in vivo drug clearance and
drug interactions from in vitro data on
metabolism, together with binding and
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX
125.

126.
127.
128.
129.
130.
131.
132.
133.
transport. Annu. Rev. Pharmacol. Toxicol.

38:46199
Ueda K, Kato Y, Komatsu K, Sugiyama
Y. 2001. Inhibition of biliary excretion
of methotrexate by probenecid in rats:
quantitative prediction of interaction from
in vitro data. J. Pharmacol. Exp. Ther.
297:103643
Saito H, Masuda S, Inui K. 1996. Cloning
and functional characterization of a novel
rat organic anion transporter mediating
basolateral uptake of methotrexate in the
kidney. J. Biol. Chem. 271:2071925
Masuda S, Saito H, Inui K. 1997. Interactions of nonsteroidal anti-inflammatory
drugs with rat renal organic anion transporter, OAT-K1. J. Pharmacol. Exp. Ther.
283:103942
Masuda S, Takeuchi A, Saito H,
Hashimoto Y, Inui K. 1999. Functional
analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane. FEBS
Lett. 459:12832
Masuda S, Ibaramoto K, Takeuchi A,
Saito H, Hashimoto Y, et al. 1999.
Cloning and functional characterization
of a new multispecific organic anion transporter, OAT-K2, in rat kidney. Mol. Pharmacol. 55:74352
Takeuchi A, Masuda S, Saito H, Abe
T, Inui K. 2001. Multispecific substrate
recognition of kidney-specific organic anion transporters OAT-K1 and OAT-K2. J.
Lu R, Kanai N, Bao Y, Wolkoff AW,
Schuster VL. 1996. Regulation of renal
oatp mRNA expression by testosterone.
Am. J. Physiol. Renal Physiol. 270:F332
37
Yamazaki M, Tokui T, Ishigami M,
Sugiyama Y.1996. Tissue-selective uptake of pravastatin in rats: contribution of
a specific carrier-mediated uptake system.
Biopharm. Drug Dispos. 17:77589
Hedman A, Angelin B, Arvidsson A,
Dahlqvist R, Nilsson B. 1990. Interactions in the renal and biliary elimination
134.
135.
136.
137.
138.
139.
140.
719
of digoxin: stereoselective difference between quinine and quinidine. Clin. Pharmacol. Ther. 47:2026
Bohme M, Jedlitschky G, Leier I, Buchler M, Keppler D. 1994. ATP-dependent
export pumps and their inhibition by cyclosporins. Adv. Enzyme Regul. 34:371
80
Versantvoort CH, Broxterman HJ,
Lankelma J, Feller N, Pinedo HM. 1994.
Competitive inhibition by genistein
and ATP dependence of daunorubicin
transport in intact MRP overexpressing
human small cell lung cancer cells.
Chu XY, Kato Y, Niinuma K, Sudo KI,
Hakusui H, et al. 1997. Multispecific organic anion transporter is responsible for
the biliary excretion of the camptothecin
derivative irinotecan and its metabolites in
rats. J. Pharmacol. Exp. Ther. 281:30414
Araki E, Ishikawa M, Iigo M, Koide T,
Itabashi M, et al. 1993. Relationship between development of diarrhea and the
concentration of SN-38, an active metabolite of CPT-11, in the intestine and the
blood plasma of athymic mice following
intraperitoneal administration of CPT-11.
Jpn. J. Cancer Res. 84:697702
Gupta E, Lestingi TM, Mick R, Ramirez
J, Vokes EE, et al. 1994. Metabolic fate of
irinotecan in humans: correlation of glucuronidation with diarrhea. Cancer Res.
54:372325
Horikawa M, Kato Y, Tyson CA,
Sugiyama Y. 2002. The potential for an
interaction between MRP2 (ABCC2) and
various therapeutic agents: probenecid
as a candidate inhibitor of the bibiary excretion of irinotecan metabolites. Drug Metab. Pharmacokin. 17:23
33
Horikawa M, Kato Y, Sugiyama Y.
2002. Reduced gastrointestinal toxicity
following inhibition of the biliary excretion of irinotecan and its metabolites by
probenecid in rats. Pharm. Res. 19:1345
53
4 Dec 2004
19:14

720
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
141. Muck W. 2000. Clinical pharmacokinetics of cerivastatin. Clin. Pharmacokinet.

39:99116
142. Muck W, Mai I, Fritsche L, Ochmann
K, Rohde G, et al. 1999. Increase in
cerivastatin systemic exposure after single and multiple dosing in cyclosporintreated kidney transplant recipients. Clin.
143. Regazzi MB, Campana IC, Raddato V,
Lesi C, Perani G, et al. 1993. Altered
disposition of pravastatin following concomitant drug therapy with cyclosporin A
in transplant recipients. Transplant. Proc.
25:273234
144. Hasunuma T, Nakamura M, Yachi T, Arisawa N, Fukushima K, et al. 2003. The
drug-drug interactions of pitavstatin (NK104), a novel HMG-CoA reductase inhibitor and cyclosporine. J. Clin. Ther.
Med. 19:38189
145. Asberg A, Hartmann A, Fjeldsa E,
Bergan S, Holdaas H. 2001. Bilateral
pharmacokinetic interaction between cyclosporine A and atorvastatin in renal
transplant recipients. Am. J. Transplant. 1:
38286
146. Lennernas H. 2003. Clinical pharmacokinetics of atorvastatin. Clin. Pharmacokinet. 42:114160
147. Bruce-Joyce J, Dugas JM, MacCausland
OE. 2001. Cerivastatin and gemfibrozilassociated rhabdomyolysis. Ann. Pharmacother. 35:101619
148. Mueck W, Frey R, Boix O, Voith B.
2001. Gemfibrozil/cerivastatin interaction. AAPS PharmSci. 3(Suppl.):3566
(Abstr.)
149. Backman JT, Kyrklund C, Neuvonen
M, Neuvonen PJ. 2002. Gemfibrozil
greatly increases plasma concentrations
of cerivastatin. Clin. Pharmacol. Ther.
72:68591
150. Shitara Y, Hirano M, Sato H, Sugiyama
Y. 2004. Gemfibrozil and its glucuronide
inhibit the OATP2(OATP1B1: SLC21A6)mediated hepatic uptake and CYP2C8mediated metabolism of cerivastatin
151.
152.
153.
154.
155.
156.
157.
158.
159.
analysis of the mechanism of the clinically

relevant drug-drug interaction between
cerivastatin and gemfibrozil. J. Pharmacol. Exp. Ther. 311:22836
Schneck DW, Birmingham BK, Zalikowski JA, Mitchell PD, Wang Y, et al.
2004. The effect of gemfibrozil on the
pharmacokinetics of rosuvastatin. Clin.
Sallustio BC, Fairchild BA, Shanahan K,
Evans AM, Nation RL. 1996. Disposition
of gemfibrozil and gemfibrozil acyl glucuronide in the rat isolated perfused liver.
Sabordo L, Sallutio BC, Evans AM, Nation RL. 1998. Hepatic disposition of the
acyl glucuronide 1-O-gemfibrozil -Dglucuronide: effects of dibromosulfophthalein on membrane transport and aglycone formation. J. Pharmacol. Exp. Ther.
288:41420
Sabordo L, Sallustio BC, Evans AM, Nation RL. 2000. Hepatic disposition of
the acyl glucuronide 1-O-gemfibrozil-D-glucuronide: effects of clofibric acid,
acetaminophen, and acetaminophen glucuronide. J. Pharmacol. Exp. Ther. 295:
4450
Hedman A, Meijer DK. 1998. Stereoselective inhibition by the diastereomers
quinidine and quinine of uptake of cardiac
glycosides into isolated rat hepatocytes. J.
Pharm. Sci. 87:45761
Olinga P, Merema M, Hof IH, Slooff MJ,
Proost JH, et al. 1998. Characterization of
the uptake of rocuronium and digoxin in
human hepatocytes: carrier specificity and
comparison with in vivo data. J. Pharmacol. Exp. Ther. 285:50610
Brown GR. 1993. Cephalosporin-probenecid drug interactions. Clin. Pharmacokinet. 24:289300
Marino EL, Dominguez-Gil A. 1981. The
pharmacokinetics of cefadroxil associated
with probenecid. Int. J. Clin. Pharmacol.
Ther. Toxicol. 19:5068
Griffith RS, Black HR, Brier GL, Wolny
JD. 1977. Effect of probenecid on
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX
160.

161.
162.
163.
164.
165.
166.
167.
the blood levels and urinary excretion of cefamandole. Antimicrob. Agents

Chemother. 11:80912
Stoeckel K, Trueb V, Dubach UC, McNamara PJ. 1988. Effect of probenecid on the
elimination and protein binding of ceftriaxone. Eur. J. Clin. Pharmacol. 34:151
56
Furst DE. 1995. Practical clinical pharmacology and drug interactions of lowdose methotrexate therapy in rheumatoid arthritis. Br. J. Rheumatol. 34(Suppl.
2):2025
Tracy TS, Krohn K, Jones DR, Bradley
JD, Hall SD, et al. 1992. The effects of
a salicylate, ibuprofen, and naproxen on
the disposition of methotrexate in patients
with rheumatoid arthritis. Eur. J. Clin.
Pharmacol. 42:12125
Kremer JM, Hamilton RA. 1995. The
effects of nonsteroidal antiinflammatory
drugs on methotrexate (MTX) pharmacokinetics: impairment of renal clearance
of MTX at weekly maintenance doses but
not at 7.5 mg. J. Rheumatol. 22:207277
Frenia ML, Long KS. 1992. Methotrexate
and nonsteroidal antiinflammatory drug
interactions. Ann. Pharmacother. 26:234
37
Nozaki Y, Kusuhara H, Endou H,
Sugiyama Y. 2004. Quantitative evaluation of the drug-drug interactions between methotrexate and nonsteroidal antiinflammatory drugs in the renal uptake
process based on the contribution of organic anion transporters and reduced folate carrier. J. Pharmacol. Exp. Ther. 309:
22634
Apiwattanakul N, Sekine T, Chairoungdua A, Kanai Y, Nakajima N, et al.
1999. Transport properties of nonsteroidal
anti-inflammatory drugs by organic anion
transporter 1 expressed in Xenopus laevis
oocytes. Mol. Pharmacol. 55:84754
Uwai Y, Saito H, Inui K. 2000. Interaction
between methotrexate and nonsteroidal
anti-inflammatory drugs in organic anion
transporter. Eur. J. Pharmacol. 409:3136
721
168. Lu R, Chan BS, Schuster VL. 1999.

Cloning of the human kidney PAH transporter: narrow substrate specificity and
regulation by protein kinase C. Am. J.
169. Ohtsuki S, Asaba H, Takanaga H, Deguchi
T, Hosoya K, et al. 2002. Role of bloodbrain barrier organic anion transporter 3
(OAT3) in the efflux of indoxyl sulfate,
a uremic toxin: its involvement in neurotransmitter metabolite clearance from the
brain. J. Neurochem. 83:5766
170. Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, et al. 1998. Identification of multispecific organic anion
transporter 2 expressed predominantly in
the liver. FEBS Lett. 429:17982
171. Morita N, Kusuhara H, Sekine T, Endou
H, Sugiyama Y. 2001. Functional characterization of rat organic anion transporter
2 in LLC-PK1 cells. J. Pharmacol. Exp.
Ther. 298:117984
172. Kusuhara H, Sekine T, Utsunomiya-Tate
N, Tsuda M, Kojima R, et al. 1999. Molecular cloning and characterization of a new
multispecific organic anion transporter
from rat brain. J. Biol. Chem. 274:13675
80
173. Jariyawat S, Sekine T, Takeda M, Apiwattanakul N, Kanai Y, et al. 1999. The interaction and transport of beta-lactam antibiotics with the cloned rat renal organic
anion transporter 1. J. Pharmacol. Exp.
Ther. 290:67277
174. Takeda M, Babu E, Narikawa S, Endou H.
2002. Interaction of human organic anion
transporters with various cephalosporin
antibiotics. Eur. J. Pharmacol. 438:137
42
175. Jung KY, Takeda M, Shimoda M,
Narikawa S, Tojo A, et al. 2002. Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: in comparison with
rOAT1. Life Sci. 70:186174
176. Uwai Y, Saito H, Inui K. 2002. Rat
renal organic anion transporter rOAT1
mediated transport of urinary-excreted
4 Dec 2004
19:14
722
177.

178.
179.
180.
181.
182.
183.
184.
185.
AR
AR232-PA45-28.tex
SHITARA
SATO
P1: JRX
SUGIYAMA
cephalosporins, but not of biliary excreted cefoperazone. Drug Metab. Pharmacokin. 17:12529
Babu E, Takeda M, Narikawa S,
Kobayashi Y, Enomoto A, et al. 2002.
Role of human organic anion transporter 4
in the transport of ochratoxin A. Biochim.
Babu E, Takeda M, Narikawa S,
Kobayashi Y, Yamamoto T, et al. 2002.
Human organic anion transporters mediate the transport of tetracycline. Jpn. J.
Pharmacol. 88:6976
Uwai Y, Saito H, Hashimoto Y, Inui K.
2000. Interaction and transport of thiazide diuretics, loop diuretics, and acetazolamide via rat renal organic anion transporter rOAT1. J. Pharmacol. Exp. Ther.
295:26165
Uwai Y, Saito H, Hashimoto Y, Inui K.
2000. Inhibitory effect of anti-diabetic
agents on rat organic anion transporter
rOAT1. Eur. J. Pharmacol. 398:19397
Nagata Y, Kusuhara H, Endou H,
Sugiyama Y. 2002. Expression and functional characterization of rat organic anion transporter 3 (rOat3) in the choroid
plexus. Mol. Pharmacol. 61:98288
Takeda M, Narikawa S, Hosoyamada M,
Cha SH, Sekine T, et al. 2001. Characterization of organic anion transport inhibitors using cells stably expressing human organic anion transporters. Eur. J.
Enomoto A, Takeda M, Shimoda M,
Narikawa S, Kobayashi Y, et al. 2002. Interaction of human organic anion transporters 2 and 4 with organic anion transport inhibitors. J. Pharmacol. Exp. Ther.
301:797802
Cvetkovic M, Leake B, Fromm MF,
Wilkinson GR, Kim RB. 1999. OATP and
P-glycoprotein transporters mediate the
cellular uptake and excretion of fexofenadine. Drug Metab. Dispos. 27:86671
Tokui T, Nakai D, Nakagomi R, Yawo H,
Abe T, et al. 1999. Pravastatin, an HMGCoA reductase inhibitor, is transported by
186.
187.
188.
189.
190.
191.
192.
193.
rat organic anion transporting polypeptide, oatp2. Pharm. Res. 16:9048

Kullak-Ublick GA, Fisch T, Oswald M,
Hagenbuch B, Meier PJ, et al. 1998. Dehydroepiandrosterone sulfate (DHEAS):
identification of a carrier protein in human
liver and brain. FEBS Lett. 424:17376
Bossuyt X, Muller M, Hagenbuch B,
Meier PJ. 1996. Polyspecific drug and
steroid clearance by an organic anion
transporter of mammalian liver. J. Pharmacol. Exp. Ther. 276:89196
Sugiyama D, Kusuhara H, Shitara Y, Abe
T, Meier PJ, et al. 2001. Characterization
of the efflux transport of 17-estradiolD-glucuronide from the brain across the
blood-brain barrier. J. Pharmacol. Exp.
Ther. 298:31622
Sugiyama D, Kusuhara H, Shitara Y, Abe
T, Sugiyama Y. 2002. Effect of 17estradiol-D-17-glucuronide on the rat
organic anion transporting polypeptide 2mediated transport differs depending on
substrates. Drug Metab. Dispos. 30:220
23
Abe T, Unno M, Onogawa T, Tokui
T, Kondo TN, et al. 2001. LST-2, a
human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers. Gastroenterology 120:168999
Vavricka SR, van Montfoort J, Ha HR,
Meier PJ, Fattinger K. 2002. Interactions
of rifamycin SV and rifampicin with organic anion uptake systems of human
liver. Hepatology 36:16472
Fattinger K, Cattori V, Hagenbuch B,
Meier PJ, Stieger B. 2000. Rifamycin SV
and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides, Oatp1 and Oatp2.
Hepatology 32:8286
Ichimaru N, Takahara S, Kokado Y, Wang
J-D, Hatori M, et al. 2001. Changes in
lipid metabolism and effect of simvastatin
in renal transplant recipients induced by
cyclosporine or tacrolimus. Atherosclerosis 158:41723
4 Dec 2004
19:14
AR
AR232-PA45-28.tex
P1: JRX


194. Arnadottir M, Eriksson LO, Thysell H,
Karkas JD. 1993. Plasma concentration profiles of simvastatin 3-hydroxy3-methyl-glutaryl-coenzyme A reductase
inhibitory activity in kidney transplant
recipients with and without cyclosporin.
Nephron 65:41013
195. Goldberg R, Roth D. 1996. Evaluation
of fluvastatin in the treatment of hypercholesterolemia in renal transplant recipients taking cyclosporine. Transplantation
62:155964
196. Kyrklund C, Backman JT, Kivisto KT,
Neuvonen M, Latila J, et al. 2001. Plasma
concentrations of active lovastatin acid
are markedly increased by gemfibrozil but
not by bezafibrate. Clin. Pharmacol. Ther.
69:34045
197. Backman JT, Kyrklund C, Kivisto KT,
Wang J-S, Neuvonen PJ. 2000. Plasma
723
concentrations of active simvastatin acid

are increased by gemfibrozil. Clin. Pharmacol. Ther. 68:12229
198. Kyrklund C, Backman JT, Neuvonen
M, Neuvonen PJ. 2003. Gemfibrozil increases plasma pravastatin concentrations
and reduces pravastatin renal clearance.
199. Spence JD, Munoz CE, Hendricks L, Latchinian L, Khouri HE. 1995. Pharmacokinetics of the combination of fluvastatin
and gemfibrozil. Am. J. Cardiol. 76: 80A
83
200. Mathew P, Cuddy T, Tracewell WG,
Salazar D. 2004. An open-label study
on the pharmacokinetics (PK) of pitavastatin (NK-104) when administered concomitantly with fenofibrate or gemfibrozil
in healthy volunteers. Clin. Pharmacol.
Ther. 75:P33
4 Dec 2004
19:16
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Annu. Rev. Med. 2005 . 45:72550

Copyright
DUAL SPECIFICITY PROTEIN PHOSPHATASES:

Therapeutic Targets for Cancer and Alzheimers

Disease
Alexander P. Ducruet1 , Andreas Vogt1 , Peter Wipf,2
and John S. Lazo1
1
Department of Pharmacology, the Combinatorial Chemistry Center and the Fiske Drug
Discovery Laboratory, University of Pittsburgh Cancer Institute, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261; email: lazo@pitt.edu
2
Department of Chemistry, the Combinatorial Chemistry Center and the Fiske Drug
Discovery Laboratory, University of Pittsburgh Cancer Institute, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
Key Words
Cdc25, Cdk, MKP, MAPK, drug discovery
Abstract The complete sequencing of the human genome is generating many

novel targets for drug discovery. Understanding the pathophysiological roles of these
putative targets and assessing their suitability for therapeutic intervention has become the major hurdle for drug discovery efforts. The dual-specificity phosphatases
(DSPases), which dephosphorylate serine, threonine, and tyrosine residues in the same
protein substrate, have important roles in multiple signaling pathways and appear to be
deregulated in cancer and Alzheimers disease. We examine the potential of DSPases
as new molecular therapeutic targets for the treatment of human disease.
INTRODUCTION
Cellular signaling networks are controlled by reversible covalent phosphorylation,
which depends on a precise balance between protein kinase and phosphatase activities (1). These signaling networks govern processes such as cell growth, cell
division, and cell death; perturbation of these pathways, whether by environmental
stresses or genetic defects, underlies the pathophysiology of many diseased states.
The sequencing of the human genome predicts approximately 428 protein kinases,
the majority of which catalyze serine and threonine phosphorylation (Figure 1) (2).
Although protein kinases were originally considered the prime regulators of signal
transduction-mediated events, it is now recognized that protein dephosphorylation
is an equally important component, playing a central role in cell cycle transitions
and other signal transduction mechanisms (3). Furthermore, protein phosphatase
activity critically regulates fundamental cellular processes that are perturbed in diseased states. The human genome is estimated to encode 99 protein phosphatases,
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Figure 1
The balance of protein kinases and phosphatases in the human genome.
This figure is based on DNA sequence and protein structural analyses described by
others (2, 3, 6a). The total predicted number of human protein tyrosine (Tyr), serine
(Ser), threonine (Thr), and dual-specificity kinases and phosphatases are indicated.
Catalytically inactive phosphatases and kinases and the phosphatases with lipid or
nucleic acid substrates are not included. See text for details.
approximately one quarter the number of protein kinases, suggesting functional redundancy and/or substrate promiscuity (Figure 1) (2, 3). Protein phosphatases are
classified according to their substrate specificity, either serine/threonine-specific
protein phosphatases (PS/TPases) or tyrosine-specific protein phosphatases (PTPases) (4), although there have been recent efforts to exploit structural information (3), which may result in some reassignments. Dual-specificity phosphatases
(DSPases) represent a subclass of the protein tyrosine phosphatase superfamily by
virtue of their highly conserved PTPase active site motif and because they employ
the PTPase catalytic mechanism, which proceeds via the formation of a transient
enzyme-phosphosubstrate intermediate [4; reviewed in Zhang (5)]. DSPases, however, are unique in their ability to dephosphorylate protein substrates containing
both phosphotyrosine and phosphoserine or phosphothreonine, either immediately adjacent or separated by one amino acid; such substrates are exemplified
by the cyclin-dependent kinases (Cdks) and the mitogen-activated protein kinases
(MAPKs), which play essential roles in the signaling pathways that regulate cell
division and cell growth (Figures 2 and 3). Recent structural analyses suggest the
human genome encodes 38 DSPases, including 11 MAPK phosphatases (MKPs),
17 atypical DSPases, 4 PRL phosphatases, 3 Cdc14 phosphatases, and 3 Cdc25
phosphatases (3) (Figure 1). The DSPases share the conserved PTPase active site
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727
and catalytic mechanism but they have a shallower active site cleft than PTPases,
presumably to accommodate the sterically less accessible phosphoserine and phosphothreonine residues (4, 6). The most widely studied DSPases are the Cdc25
phosphatases and the MKPs, two protein families that play central roles in the
biology of the cell.

CDC25 DSPASES
The first DSPases to be discovered were the Cdc25 phosphatases, which were
functionally defined as promoters of the cell division cycle in yeast (7). More
specifically, Cdc25 phosphatases dephosphorylate and activate the Cdks (Figures
2 and 4), which are key participants in the cellular division program induced
in response to extracellular signals including growth factors. Cdks coupled to
their cyclin partner are maintained in an inactive state by dual phosphorylation at
adjacent threonine and tyrosine (-T-Y-) residues near their amino terminus; these
inactivating phosphorylations are mediated by Wee1 and Myt1 protein kinases
(8, 9). Cdc25s activate Cdks by dephosphorylating both phosphothreonine and
phosphotyrosine residues (Figure 2); regulation of Cdk kinase activity remains an
Figure 2
Cdc25 phosphatases dephosphorylate and activate the cyclin-dependent
kinases. Mitogenic signal transduction cascades induce cell division. Progression
through cell cycle transitions is achieved by dephosphorylation and activation of the
cyclin-dependent kinases by Cdc25 phosphatases. In contrast to the MKPs, the Cdc25
phosphatases activate Cdks by dephosphorylating both residues in the Cdk -T-Y- motif
(see text for details).
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Figure 3
Mitogen-activated protein kinase phosphatases dephosphorylate and inactivate the mitogen-activated protein kinases. Growth factor receptor signal transduction
cascades, cellular stresses, and chemotherapeutics can activate mitogenic signaling
pathways, culminating in the activation of upstream mitogen-activated protein kinase
kinase kinases (MAPKKKs), which phosphorylate and activate mitogen-activated protein kinase kinases (MAPKKs), which phosphorylate and activate mitogen-activated
protein kinases (MAPKs) in the -T-x-Y- motif. Downregulation of mitogenic signaling through MAPKs is achieved by dephosphorylation of both residues in the -T-x-Ymotif, a process regulated by the dual-specificity MAPK phosphatases (DS-MKPs).
area of considerable investigation, and Cdks have emerged as a novel therapeutic

target for the treatment of cancer (10).
The human Cdc25 DSPases comprise a family of three genes originally identified by their ability to complement a temperature-sensitive Cdc25 yeast strain,
thus restoring a normal growth phenotype. The protein products of the three Cdc25
genes, Cdc25A, Cdc25B, and Cdc25C, possess a high degree of homology in
their carboxy-terminal domain, the location of the catalytic active site, whereas
their amino terminal domains are much less conserved, perform regulatory roles,
and possibly contribute to the diverse nature of their biological activities (see
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Figure 4
Cdc25 phosphatases promote mammalian cell cycle progression. Cdc25
phosphatases drive the cell division cycle by dephosphorylating and activating the Cdks.
The three human Cdc25 isoforms, Cdc25A, Cdc25B, and Cdc25C, have overlapping
roles in the cell cycle. Cdc25A exclusively promotes the G1/S transition and S phase
progression and contributes to the Cdc25 activity necessary for G2 phase progression,
the G2/M transition, and mitosis. Cdc25B contributes to G2 progression and is believed
to be the trigger for initiating the G2/M transition. Cdc25C activity is restricted to
mitosis. The Cdc25 phosphatases are targeted by the G1/S, intra-S, and G2/M cell cycle
checkpoints to inhibit their activity in response to genotoxic stress. Cdc25 activity is
influenced by Cdk activity in regulatory feedback loops: solid arrows indicate activation
by Cdc25 and dotted arrows represent known positive (+) or negative () feedback
loops. Cdk2 has both positive and negative effects on Cdc25A (+/). It is unclear
whether Cdk4/cyclin D is a bona fide substrate of Cdc25A in cells.
below). Cdc25C, the first human Cdc25 isoform identified, functions primarily in
mitosis and catalyzes mitotic progression by activating Cdk1/cyclin B; microinjection of anti-Cdc25C antibodies into HeLa cells prevented mitotic entry (1113).
Cdc25B also activates Cdk1/cyclin B, and microinjection of anti-Cdc25B antibodies inhibits mitotic entry, leading many to speculate that Cdc25B is functionally
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redundant to Cdc25C (14, 15). Nonetheless, Cdc25B and Cdc25C activities are
temporally distinct, with Cdc25B activity peaking prior to that of Cdc25C. More
recently, Cdc25B has been described as the trigger for the G2/M transition; Cdc25B
appears to initiate the mitotic transition by activating a particular pool of Cdk1/cyclin
B (1518). Cdc25B also contributes to the Cdk phosphatase activity necessary to
activate Cdk2/cyclin A in S phase and Cdk1/cyclin A in G2 (17, 19, 20). Cdc25A
promotes the G1/S cell cycle transition and S phase progression by activating
Cdk2/cyclin E (21, 22). Microinjection of anti-Cdc25A antibodies prevented S
phase entry in cells following serum induction, and overexpression of Cdc25A accelerated S phase entry with premature Cdk2 activation (2123). Cdc25A activity
is rate limiting for the G2/M transition and mitotic progression by contributing to
Cdk1/cyclin B activation (24, 25).
Although the emerging model for temporal and combinatorial contributions of
Cdc25A, Cdc25B, and Cdc25C activities to achieve precise control over cell cycle
progression is appealing, Cdc25B/ mice and Cdc25C/ mice are viable and
cells isolated from these mice undergo normal mitotic cell division, implying that
Cdc25A has the potential to drive the entire mitotic cell division cycle (26, 27).
The preeminence of Cdc25A is further illustrated by the prompt inactivation of
Cdk activity and cell cycle arrest seen with rapid Cdc25A degradation (24, 2830).
Cdc25A has, thus, been dubbed the master Cdk phosphatase (31), as it appears
to be responsible for Cdk activation to promote the G1/S cell cycle transition,
for maintaining Cdk activity throughout S phase and G2 progression, and for
contributing to the Cdk phosphatase activity necessary for the G2/M transition
and mitotic progression (Figure 4) (21, 22, 24, 25). It remains unclear why cells
have multiple Cdc25s to regulate mitotic cell division, although it is possible that
their combined activities ensure optimal Cdk activation to promote the irreversible
process of mitotic division. In such a model, the multiple Cdc25s would impose
a switch-like regulatory mechanism, consisting of a biological threshold of Cdk
activation, to achieve strict unidirectional control of cell division (32).
Cdc25 Regulation
As key controllers of cell division, Cdc25 DSPases are subject to precise regulation, including enzyme-substrate feedback loops involving specific Cdk/cyclin
complexes and their activating Cdc25. For example, Cdc25A activity is upregulated by Cdk2/cyclin E following its activation, and Cdc25A protein stability is
increased by Cdk1/cyclin B phosphorylation (21, 24); Cdk2 activity also appears
to negatively regulate Cdc25A protein stability (33). Cdc25B protein stability is
negatively regulated by Cdk1/cyclin A (16) and Cdc25C catalytic activity is upregulated by Cdk1/cyclin B (Figure 4) (34). In addition, the Cdc25 DSPases are
regulated by alternative gene splicing, which results in the expression of 12 splice
variants. The precise role of alternative splicing in Cdc25 biology remains unclear,
although the splice variants could have altered tissue or cell cycle phase activity
profiles, or they may have different specific catalytic activities as a result of loss
of consensus regulatory phosphorylation sites (35, 36).
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Throughout the cell cycle, Cdc25C protein expression does not appreciably fluctuate; however, Cdc25C remains inactive during interphase by 14-3-3-mediated
sequestration in the cytoplasm (37). Cdc25A and Cdc25B, on the other hand, are
labile proteins, most likely owing to their role as the major catalysts of the cell
cycle transitions (25, 38). Cdc25B protein levels accumulate throughout late S
and early G2, peaking at the G2/M transition (17, 39). Although a detailed understanding of Cdc25B protein turnover is lacking, its proteolysis requires prior
phosphorylation by Cdk1/cyclin A (16). Like Cdc25C, Cdc25B activity is also
regulated by its subcellular localization, which is facilitated by interactions with
14-3-3 (40). Cdc25A protein levels and activity remain elevated past S phase and
increase as cells enter mitosis. Cdc25A activity is primarily regulated by protein
turnover, although 14-3-3 can prevent the phosphatase from interacting with its
mitotic substrate, Cdk1/cyclin B. Furthermore, Cdc25A activity has been reported
to be upregulated by phosphorylation in response to mitogenesis (41, 42). Cdc25A
protein turnover is catalyzed by the ubiquitin-proteasome pathway; Cdc25A ubiquitination is catalyzed by the APC/CCdh1 ubiquitin ligase during mitotic exit and
early G1 and by the SCFTrCP ubiquitin ligase during interphase [reviewed in
Busino et al. (43)]. The subcellular localization of Cdc25A remains a matter of
some debate, as Cdc25A has been reported to localize in the nucleus, the cytoplasm, and the plasma membrane and to interact with proteins that reside in each
of these cellular compartments (21, 41, 42, 4446).
Cell Cycle Checkpoints

As major promoters of cell cycle progression and the main drivers of passage
through the cell cycle transitions, the Cdc25s are targets of cell cycle checkpoint
proteins, which are activated in response to genotoxic stress and terminate cell
cycle progression in an effort to preserve genomic integrity. The Cdc25-dependent
cell cycle checkpoints appear to be independent of p53 and serve as a rapid and primary response to genotoxic stresses (29). Whereas Cdc25B and Cdc25C are targets
of the G2/M cell cycle checkpoint, Cdc25A is targeted by the G1/S, intra-S, and
G2/M cell cycle checkpoints (24, 2831, 47). Cdc25s are inactivated at cell cycle
checkpoints by one or a combination of Chk1-, Chk2-, and p38 MAPK-mediated
phosphorylations (Figures 57); checkpoint-dependent Cdc25 regulation has been
the subject of several recent reviews (31, 43, 48, 49). In response to genotoxic
stress, checkpoint kinases phosphorylate Cdc25C, resulting in 14-3-3 binding and
cytoplasmic sequestration (Figure 5); in addition, checkpoint-mediated Cdc25C
inactivation has been reported to occur via APC/C-mediated ubiquitination and
proteolytic degradation, specifically in response to arsenite treatment (50). Although Cdc25B is a labile protein under physiologic conditions (see above), cell
cycle checkpoint-mediated inactivation is thought to be due to 14-3-3 binding (Figure 6) (31, 49, 51). In contrast, the cell cycle checkpoints targeting Cdc25A appear
to be independent of 14-3-3 binding and involve ubiquitin-mediated proteolytic
degradation [reviewed in Donzelli & Draetta (31); Busino et al. (43)]. In response
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Figure 5
Cdc25C inactivation by the G2/M cell cycle checkpoint. In response to
genotoxic stress, checkpoint kinases Chk1 and Chk2 phosphorylate Cdc25C, promoting its cytoplasmic sequestration by 14-3-3 binding.
to genotoxic stresses, Cdc25A is phosphorylated by Chk1, Chk2, and p38, which

promote its polyubiquitination catalyzed by the SCFTrCP ubiquitin ligase (Figure 7) (31, 43, 52, 53). However, neither Chk1, Chk2, nor p38 can phosphorylate
the Cdc25A serine residues necessary for recruitment to the SCFTrCP ubiquitin
ligase, indicating that other kinases are necessary for promoting Cdc25A turnover
(52, 53). Mutations in one or several of the components of these checkpoint pathways are common in cancers, resulting in a defective response to genotoxic stress
and promoting genetic instability (31, 54)
In addition to their role in cell cycle control (Figures 2 and 4), Cdc25s regulate
mitogenic and steroid receptor signal transduction pathways and the apoptotic
response to cellular stresses (see below) (Figure 8) [reviewed in Lyon et al. (55)].
MKP DSPASES
MKPs dephosphorylate and inactivate MAPKs on threonine and tyrosine residues
(Figure 3). MAPKs are widely studied protein kinases that play pivotal roles in
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Figure 6 Cdc25B inactivation by the G2/M cell cycle checkpoint. In response to

genotoxic stress (predominantly UV irradiation), p38 MAPK phosphorylates Cdc25B,
promoting its association with 14-3-3, which inhibits Cdc25B activity.
mitogenic signal transduction, survival, stress response, and programmed cell

death. There are currently three members of the MAPK family: extracellular
signal-regulated kinase (Erk), c-Jun terminal kinase/stress-activated protein kinase
(JNK/SAPK), and p38/high osmolarity glycerol response kinase (HOG) MAPK.
Although activation of Erk is most often associated with growth and survival,
JNK and p38 are thought to primarily mediate stress responses and programmed
cell death (apoptosis) [reviewed in Chang & Karin (56)]. Extensive studies addressing the activation of MAPK pathways by upstream kinases and cell-surface
receptor-mediated events have placed MAPK signal transduction cascades at the
heart of a sophisticated signaling network with multiple levels of complexity. In
contrast, the events that regulate termination of MAPK signaling are less well
understood, although it is clear that MKPs play a major role, and a large body of
evidence now demonstrates that the regulation of MAPKs at the level of the protein
phosphatases is as sophisticated as that mediated by the protein kinases [reviewed
in Tonks & Neel (57); 58]. MKPs have been grouped into three major categories:
dual-specificity MKPs (DS-MKPs), tyrosine-specific MKPs, and serine/threoninespecific MKPs (58). In this review, we have limited our discussion to the DS-MKP
family because of their similarities to the Cdc25 DSPases.
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Figure 7
Cdc25A inactivation by cell cycle checkpoints. Cdc25A is rapidly and
irreversibly inactivated by the G1/S, intra-S phase, and G2/M cell cycle checkpoints.
In response to genotoxic stress or interruptions to DNA synthesis, stress-responsive
p38 MAPK and checkpoint kinases Chk1 and Chk2 phosphorylate Cdc25A (at multiple
sites), promoting its association with ubiquitin ligases. Following polyubiquitination
(Ub), Cdc25A is degraded by the 26S proteasome; dashed outlined Cdc25A indicates
degraded protein.
To date, 12 bona fide human DS-MKPs have been cloned and characterized
(Table 1). Table 1 also contains two putative DS-MKPs, namely hVYH1, whose
substrate has not been identified, and JSP-1, which fails to dephosphorylate MAPK
in cells but nonetheless specifically activates the JNK pathway by an as of yet undetermined mechanism (59). The first MKP discovered was 3CH134/MKP-1 (60),
which was later found to have PTPase activity (61) and DSPase activity (62). The
human homolog of 3CH134/MKP-1, CL100 or DUSP1, was independently cloned
(63). Other DS-MKPs were subsequently discovered in a variety of organisms [a
comprehensive listing of DS-MKPs from various species was compiled by Farooq
& Zhou (64)].
The DS-MKPs have unique but overlapping MAPK substrate specificities, as
recently reviewed by Farooq & Zhou (64). For example, the Erk isoforms are
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Figure 8
Cdc25 phosphatases regulate multiple signaling pathways. In addition to
driving cell cycle transitions, Cdc25 phosphatases promote hormone-responsive gene
expression by affecting steroid receptor activity, downregulate apoptotic responses to
genotoxic stresses by blocking Ask1 homo-dimerization (which is necessary for Ask1
activation), and downregulate mitogenic signaling by dephosphorylating the epidermal
growth factor receptor (EGFR) and Raf-1, which can also have a cytoprotective effect.
selectively dephosphorylated by MKP-3, whereas M3/6 selectively dephosphorylates JNK. MKP-1 recognizes JNK, ERK, and p38, and MKP-2 recognizes Erk
and JNK. PAC-1, a DSPase from human T cells that is similar to MKP-3, is specific
for Erk. MKP-5 appears to be somewhat selective for p38. The prototype DSPase
VHR dephosphorylates Erk and JNK. There is also evidence for cross-talk between
the MAPK pathways. For example, MKP-7 interacts with Erk, JNK, and p38, but
shows substrate specificity for JNK and is phosphorylated in an Erk-dependent
manner (65).
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Table 1 Human DS-MKPs identified by DUSP nomenclature based
on analysis described in Reference 64

DUSP
Synonyms
GenBank
Accession Number
DUSP1
HVH1, CL100, MKP-1, PTPN10
NM 004417
DUSP2
PAC1, PAC-1
NM 004418
DUSP3
VHR
NM 004090
DUSP4
MKP-2, TYP, HVH2
NM 001394
DUSP5
HVH3
NM 004419
DUSP6
MKP-3, PYST1
NM 001946
DUSP7
MKP-X, PYST2
NM 001947
DUSP8
HB5, HVH8, HVH-5
NM 004420
DUSP9
MKP-4
NM 001395
DUSP10 MKP-5
NM 007207
DUSP14 MKP6, MKP-L
NM 007026
DUSP16 MKP-7
NM 030640
DUSP12 YVH1
NM 007240
DUSP22 JKAP, JSP1
NM 020185
DS-MKP Regulation
The DS-MKPs are regulated on multiple levels. The majority of DS-MKPs are
inducible genes, and basal levels of DS-MKPs are low in nonstressed or unstimulated cells [reviewed in Keyse (58)]. Some DS-MKPs are immediate early genes.
For example, MKP-1, MKP-2, MKP-X (PYST2), and PAC-1 are rapidly induced
in response to serum stimulation (6668). In contrast, MKP-3 (PYST1), MKP4, MKP-5, MKP-X, and M3/6 are not encoded by immediate early genes (58).
MKP-3 and VHR are constitutively expressed (67), and while MKP-3 is moderately inducible after several hours of stimulation (67, 69), VHR is not known to be
inducible. Different DS-MKPs respond to different stimuli: MKP-1 is inducible by
mitogens, oxidative stress, heat shock (63, 69), and hypoxia (7072). In contrast,
MKP-X is only moderately induced by serum but not by cellular stress (67).
Inducible expression of DS-MKPs is thought to be a mechanism for attenuation of mitogenic signaling. Induction of MKP-1 in NIH3T3 cells (62) and
CCL39 hamster lung fibroblasts temporally correlates with Erk inactivation and is
dependent on Erk activity (66). An additional mechanism by which Erk induces
MKP-1 is through stabilization of MKP-1 protein levels. This is achieved by direct
phosphorylation of MKP-1 by Erk, leading to reduced MKP-1 ubiquitination and
proteasomal degradation (73). Furthermore, some DS-MKPs are activated by activated forms of their respective substrates. MKP-3 experiences a 25-fold increase
in catalytic activity when complexed to its phosphorylated substrate, Erk2 (74).
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This activation is specific, as neither p38 nor JNK activated MKP-3, but they did
activate a nonspecific DS-MKP (MKP-4) (74). Taken together, the data indicate
that inactivation of the Erk cascade is regulated through induction and stabilization
of DS-MKPs in an inhibitory feedback loop.

ABERRANT DSPASE REGULATION IN DISEASED STATES

The pathogenic mechanisms underlying disease progression frequently involve
perturbations in molecular signaling pathways. Cdc25A and Cdc25B are overexpressed in multiple human tumors, and high levels correlate with a poor prognosis
(55, 75, 76). Cdc25A and Cdc25B have also been observed to be highly expressed
in the brains of patients with Alzheimers disease and may contribute to the pathology of neurodegeneration (77, 78). Although the mechanism by which Cdc25A
and Cdc25B are overexpressed in human cancers is poorly understood, their expression may be elevated by increased gene expression, increased protein stability
as a result of deficiencies in protein turnover, or both (31, 43, 55, 75, 76). Cdc25A
and Cdc25B have oncogenic activity and can transform normal cells in cooperation
with an activated Ras oncogene or inactivation of the Retinoblastoma (Rb) tumor
suppressor protein (79). Targeted overexpression of Cdc25B in transgenic mice
resulted in the formation of mammary gland tumors and an increased susceptibility
to carcinogen-induced tumor formation (80, 81). Cdc25C, on the other hand, has
not been found to be overexpressed in human tumors and does not transform cells
(79); induction of premature mitosis by ectopic overexpression of Cdc25C was
inefficient when compared to Cdc25B, providing a possible rationale for the lack
of Cdc25C-associated oncogenic activity (18). Deregulated Cdc25 expression may
contribute to the malignant phenotype by a combination of several mechanisms
(Figure 8). As major targets of cell cycle checkpoints, overexpression of Cdc25A
and Cdc25B may enable cell division in the presence of compromised genetic
material by overwhelming the cell cycle checkpoint machinery, promoting genetic
instability (24, 29, 51). Cdc25A and Cdc25B function as coactivators for steroid
hormone receptors, independent of catalytic activity, and Cdc25 overexpression
may promote expression of steroid hormone-responsive genes in the absence of
ordinarily required stimuli or lower the threshold for such gene expression (82).
Cdc25A functions as a liaison between mitogenic signaling pathways and the
cell cycle, and overexpression of Cdc25A may promote unwarranted cell cycle
activation in the absence of mitogenic stimuli, leading to a deregulated hyperproliferative state (41, 42). Furthermore, Cdc25A possesses antiapoptotic potential.
Cdc25A downregulates the proapoptotic kinase apoptosis signal-regulating kinase
1 (Ask1) through a noncatalytic protein-protein interaction mechanism; overexpression of Cdc25A may block Ask1 activation in response to apoptotic stimuli
(83). Cdc25A also downregulates Erk MAPK signaling by inactivating Raf1 and
the epidermal growth factor receptor (44, 45). Prolonged Erk activation has been
reported to promote cell cycle arrest and cytotoxicity in several cell types (84,
85); Cdc25A overexpression may thus provide a selective growth advantage by
downregulating the deleterious effects of prolonged Erk MAPK activation in cells
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transformed by upstream components of the Erk MAPK signaling cascade. Therefore, overexpression of Cdc25A and Cdc25B may contribute to the transformed
phenotype by endowing cells with a proliferative advantage or by generating resistance to genotoxic stress-induced cell cycle arrest and apoptosis.
The role of the Cdc25s in neurodegeneration remains unclear. Cdc25A and
Cdc25B are expressed and active in the brains of Alzheimers disease patients (77,
78), and there is increasing evidence that expression and activation of the cell cycle
machinery is associated with neurodegeneration in postmitotic neurons (8689).
Cell cycle activation appears to be a critical element of the apoptotic response
to DNA damage in postmitotic neurons, and Cdk activation is a precursor to the
neurodegeneration characteristic of Alzheimers disease (78, 90, 91); moreover,
inhibition of Cdk activity provides a neuroprotective effect, substantiating a role
for the cell cycle machinery in the pathophysiology of neurodegeneration (92).
The Cdc25 DSPases, therefore, constitute attractive potential targets for cancer
and neurodegenerative disease drug discovery.
DS-MKPs in Neoplastic Disease

The chromosomal locations for all the human DS-MKP genes have been mapped,
and many DS-MKPs reside in regions that are deleted in human tumors. For example, frequent loss of heterozygosity at 12q21 and 12q22-q23.1 has been observed
in primary pancreatic cancers, and DUSP6/MKP-3 gene expression is lost in the
majority of pancreatic cancer cell lines; MKP-3 maps to chromosome 12q22 (93).
Consequently, a tumor suppressor function has been proposed for MKP-3; consistent with this hypothesis, exogenous expression of MKP-3 induced apoptosis
in pancreatic cancer cells (93). Furthermore, MKP-X, MKP-5, and MKP-2 were
mapped to chromosomes 3p21, 1q41, and 8p11-p12, respectively, where frequent
deletions have been reported in multiple tumors (9499).
Although a tumor suppressor function might be intuitively expected for phosphatases that deactivate Erk (i.e., MKP-3 and MKP-X), which is conventionally
believed to promote growth and survival, phosphatases involved in JNK signaling
are also found in regions of the genome suspected to harbor tumor suppressors.
For example, hVH5, the human homolog of mouse M3/6, maps to 11p15 (100), a
locus deleted in non-small-cell lung cancer (101). MKP-7 maps to chromosome
12p1213 (102), where deletions have been found in several human tumors (103).
Functional evidence that MKP-7 may be a tumor suppressor comes from a study by
Hoornaert et al., who showed that BCR-Abl transformed cells reverted to a normal
phenotype following MKP-7 overexpression (102). MKP-1 maps to chromosome
5q35 (104), and 5q gains have been found in malignant glioma cell lines (105) and
in breast fibroadenomas (106), although there are also reports of 5q deletions in
testicular (107, 108) and ovarian germ cell cancers (109). Although it was initially
hypothesized that MKP-1 was a tumor suppressor (110), no evidence has been
found to support this hypothesis. On the contrary, initial reports indicate mice with
a targeted disruption of the MKP-1 gene developed normally and had no increased
frequency of malignancies compared to wild-type animals, even when the mice
were over 1-year-old (111, 112).
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A number of investigators have observed high basal levels of MKP-1 in human

tumors, including prostate (113), gastric (114), breast (115), and pancreatic cancer
(116). In ovarian cancer samples, MKP-1 expression was correlated with decreased
progression-free survival (117). High levels of MKP-1 expression were also found
in the early stages of prostate, colon, and bladder carcinogenesis (118). Evidence
that MKP-1 may actually support the transformed phenotype comes from a recent
study by Liao et al., who showed that PANC-1 human pancreatic cancer cells stably transfected with a full-length MKP-1 antisense construct had longer doubling
times, decreased ability to form colonies in soft agar, and were unable to form
tumors in nude mice (116). The precise mechanism by which loss of MKP-1 expression affected tumorigenicity, however, remains unknown. MKP-1 can protect
cells against UV irradiation-induced apoptosis (119) and can inhibit JNK activity
and AP-1-dependent gene expression in response to UV irradiation and the DNA
damaging agent methyl methane sulfonate (120). Ectopic expression of MKP1 also protects cells against cisplatin-induced apoptosis, whereas a catalytically
inactive mutant of MKP-1 enhanced cisplatin toxicity (121). Thus, MKP-1 may
have a cytoprotective role. It is interesting to note, however, that Liao et al. (116)
found that MKP-1 antisense expression did not affect apoptosis by actinomycin D,
which activates the JNK pathway. The MKP-1 antisense oligonucleotides also did
not increase JNK or p38 phosphorylation, but did increase basal Erk phosphorylation and prolonged Erk phosphorylation in response to epidermal growth factor
stimulation. This suggests that the primary mechanism by which MKP-1 supports
the transformed phenotype may be mediated by an Erk, but not JNK, dependent
process. Consistent with this hypothesis, several groups have shown that MKP-1
and activated Erk can coexist in malignant tissue (114, 115) and in cancer cells
(116). This has led to a model where cells balance mitogenic overstimulation by
expressing MKPs, the end result being a higher basal level of Erk signaling in
tumors than in normal tissues. Additional evidence suggesting a role for MKP-1
in cancer comes from DNA microarray experiments, where high levels of MKP-1
in recurrent acute myelogenous leukemia (AML) were found concomitant with an
activation of the Ras-Raf-Erk pathway (122). Furthermore, a recent report by Kang
et al. has identified MKP-1 as one of 53 genes that were upregulated (4.03-fold)
in highly metastatic breast cancer sublines compared to the parental MDA-MB
231 cells or cells with low metastatic potential (123). It should be noted, however,
that the functional significance of many of these observations remains unclear, and
more work needs to be done to precisely determine the roles that MKP-1 plays in
the context of neoplastic disease.

Protein kinases have been a major focus of recent molecular-targeted drug discovery efforts, producing drugs such as imatinib mesylate (Gleevec ) and gefitinib
(Iressa ), and the success of these drugs has prompted a substantial effort to target
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other kinases, such as the Cdks, mitogen-activated protein kinase kinase (MEK),
Raf, and mTOR (124). Based on their roles in multiple signaling pathways and
altered expression in diseased states, there has been increasing interest in identifying DSPase inhibitors that are more potent and selective than the general tyrosine
phosphatase inhibitor sodium orthovanadate (55). Such targeted agents may provide value as therapeutics for cancer and Alzheimers disease. The shallow nature
of the DSPase active site, combined with the conserved nature of the PTPase active
site cleft, has lead some investigators to believe that DSPase-selective inhibitors
may be difficult to identify. Although the three Cdc25 isoforms possess the common, highly conserved PTPase active site motif, the architecture of their active site
appears to be different. Thus, the Cdc25 phosphatases have a shallow catalytic do cleft (125,126). Indeed, several groups
main, whereas the PTPases have a deep 9 A
have identified lead compounds with favorable selectivity profiles, suggesting that
phosphatase-selective inhibition is plausible (55).
Structure-activity relationships of natural and synthetic inhibitors of DSPases
have been partially reviewed (55, 127). Representative members in this group include the natural products dnacin B1 (1), dysidiolide (2), menadione (3), and coscinosulfate (4), which inhibited the Cdc25 family with IC50 values in the 110 M
range (Figure 9) (128132). The biological activities of these natural products inspired total syntheses as well as the preparations of synthetic analogs and chemical
libraries (133138). Structurally most conspicuous among the small-molecule inhibitors discovered through combinatorial library and random screening are highly
lipophilic acids [e.g., 5 (139), 6 (140), 7 (141)] as well as annulated para-quinones
[e.g., 8 (142), 9 (143), 10 (144), 11 (145)] (Figure 9). In addition, moderately
potent heterocyclic [e.g., 12 (146), 13 (147)] and phenolic derivatives [14 (148)]
have also been identified (Figure 9).
To date, compounds with quinone moieties have demonstrated the highest potency as well as considerable specificity in DSPase screens. Specifically, 10 was
found to inhibit Cdc25B and VHR with IC50 values of 206 nM and 4.0 M, respectively. Compound 11 had IC50 values of 22, 125, and 57 nM for Cdc25A, B, and
C, respectively, and showed partial mixed-inhibitory kinetics. It is also interesting
to note that indolyldihydroxyquinone 9 was found to bind competitively with the
substrate at the active site of Cdc25 and yield a Ki of 470 nM (143). Molecular
modeling of enzyme-inhibitor complexes is possible (145, 149) because several
crystal structures of Cdc25 isoforms are available, but rational design has so far
met with limited success for the improvement of the binding characteristics of lead
structures. A recent report presents the homology-modeling of a Cdc25B-inhibitor
complex, which might provide a more suitable starting point for rational inhibitor
design (150).
Whether Cdc25 isoform specificity can be achieved is a challenging issue.
All three Cdc25 isoforms possess identical amino acids in their highly conserved
PTPase active site motif (HCEFSSER), and they share a high degree of sequence
homology outside of this catalytic loop, posing a high hurdle for selective targeting
of the individual Cdc25 isoforms. Nonetheless, selective protein kinase inhibition
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741
Figure 9
Natural product and synthetic inhibitors of the Cdc25 family of DSPases.
Representative examples for small-molecule inhibitors of this enzyme class are natural
products (14), lipophilic acids (57), quinones (811), heterocycles (12, 13), and
phenols (13, 14).
has been achieved with ATP competitive inhibitors, which target similar active
site structures and catalytic mechanisms (124), lending credence to the hypothesis
that selective Cdc25 inhibition may yet be achieved. It is worth mentioning that
Cdc25-specific inhibitors lacking isoform selectivity may have some theoretical
therapeutic appeal, but additional small-molecule inhibitors will be required to
fully test this hypothesis.
Although screening strategies for Cdc25 inhibitors have focused on identifying
active site inhibitors, an alternate mechanism for targeted inhibition of Cdc25
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DUCRUET ET AL.
phosphatases may exist, as exemplified by the naphthoquinone NSC 95397, which

inhibits Cdc25A activity by a bimodal mechanism. Although NSC 95397 was
identified as an inhibitor of Cdc25A phosphatase activity in a high-throughput in
vitro screen (145), treatment of prostate cancer cells (PC-3 and LNCap) with NSC
95397 resulted in decreased Cdc25A protein levels by stimulating its degradation
(46). Cdc25A degradation promoted by NSC 95397 was independent of genotoxic
stress, as p53, Chk1, and Chk2 were not affected (46) and, therefore, presumably
occurred through the ubiquitin-proteasome pathway that regulates physiological
Cdc25A protein turnover. NSC 95397 therefore represents a novel class of Cdc25
inhibitors that can inhibit Cdc25A activity via the combined mechanism of catalytic
inhibition and increased protein turnover (46). One advantage of such a novel class
of Cdc25 inhibitors would be that, in addition to inhibiting Cdc25 catalytic activity,
these compounds could also downregulate Cdc25 expression, thereby functioning
as inhibitors of the noncatalytic activities of Cdc25.
Like the Cdc25 DSPases, MKP-1 may be an important regulator of the malignant phenotype, and it thus represents a rational target for anticancer drug discovery; however, selective small-molecule inhibitors of MKP-1 are lacking. This has
been hampered, at least in part, by the lack of an available X-ray crystal structure
and the lack of definitive assays for detection of cellular DS-MKP inhibition. Recently, a high-content fluorescence-based cellular assay for detection of MKP-3
inhibition was published (151). This assay was used to identify novel inhibitors of
MKP-3 and might be applicable for MKP-1 in the future.
CONCLUSIONS
DSPases have critical roles in regulating cellular phosphorylation signaling networks and are deregulated in human cancer and Alzheimers disease. The uniqueness of their biochemical mechanism and the central role of their substrates make
DSPases an attractive target for further pharmacological studies. In recent years,
several natural products and novel small organic molecules have been identified
that can block phosphatase activity. Nonetheless, there continues to be a need
for more potent and selective inhibitors of DSPases to permit a further dissection
of their roles in biological systems and to clinically validate their potential as
anticancer targets.
LITERATURE CITED
1. Hunter T. 1995. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225
36
2. Venter JC, Adams MD, Myers EW, Li

PW, Mural RJ, et al. 2001. The sequence
of the human genome. Science 291:1304
51
4 Dec 2004
19:16
AR
AR232-PA45-29.tex
P1: JRX


3. Alonso A, Sasin J, Bottini N, Friedberg I,
Friedberg I, et al. 2004. Protein tyrosine
phosphatases in the human genome. Cell
117:699711
4. Denu JM, Stuckey JA, Saper MA, Dixon
JE. 1996. Form and function in protein
dephosphorylation. Cell 87:36164
5. Zhang ZY. 2002. Protein tyrosine phosphatases: structure and function, substrate
specificity, and inhibitor development.
34
6. Yuvaniyama J, Denu JM, Dixon JE, Saper
MA. 1996. Crystal structure of the dual
specificity protein phosphatase VHR. Science 272:132831
6a. Hanks SK. 2003. Genomic analysis of the
eukaryotic protein kinase superfamily: a
perspective. Genome Biol. 4:111.17
7. Millar JB, Russell P. 1992. The cdc25 Mphase inducer: an unconventional protein
phosphatase. Cell 68:40710
8. Honda R, Ohba Y, Yasuda H. 1992. The
cell cycle regulator, human p50weel, is a
tyrosine kinase and not a serine/tyrosine
kinase. Biochem. Biophys. Res. Commun.
186:133338
9. Liu F, Stanton JJ, Wu Z, Piwnica-Worms
H. 1997. The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic
reticulum and Golgi complex. Mol. Cell
Biol. 17:57183
10. Senderowicz AM, Sausville EA. 2000.
Preclinical and clinical development of
cyclin-dependent kinase modulators. J.
Natl. Cancer Inst. 92:37687
11. Sadhu K, Reed SI, Richardson H, Russell P. 1990. Human homolog of fission
yeast cdc25 mitotic inducer is predominantly expressed in G2. Proc. Natl. Acad.
Sci. USA 87:513943
12. Strausfeld U, Fernandez A, Capony JP,
Girard F, Lautredou N, et al. 1994. Activation of p34cdc2 protein kinase by
microinjection of human cdc25C into
mammalian cells. Requirement for prior
phosphorylation of cdc25C by p34cdc2
13.
14.
15.
16.
17.
18.
19.
20.
21.
743
on sites phosphorylated at mitosis. J. Biol.

Chem. 269:59896000
Millar JB, Blevitt J, Gerace L, Sadhu
K, Featherstone C, Russell P. 1991.
p55CDC25 is a nuclear protein required
for the initiation of mitosis in human cells.
Honda R, Ohba Y, Nagata A, Okayama
H, Yasuda H. 1993. Dephosphorylation
of human p34cdc2 kinase on both Thr14 and Tyr-15 by human cdc25B phosphatase. FEBS Lett. 318:33134
Lammer C, Wagerer S, Saffrich R,
Mertens D, Ansorge W, Hoffmann I.
1998. The cdc25B phosphatase is essential for the G2/M phase transition in human cells. J. Cell Sci. 111(Pt. 16):2445
53
Baldin V, Cans C, Knibiehler M, Ducommun B. 1997. Phosphorylation of human
CDC25B phosphatase by CDK1-cyclin A
triggers its proteasome-dependent degradation. J. Biol. Chem. 272:3273134
Gabrielli BG, De Souza CP, Tonks ID,
Clark JM, Hayward NK, Ellem KA. 1996.
Cytoplasmic accumulation of cdc25B
phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells.
J. Cell Sci. 109(Pt. 5):108193
Karlsson C, Katich S, Hagting A, Hoffmann I, Pines J. 1999. Cdc25B and
Cdc25C differ markedly in their properties as initiators of mitosis. J. Cell Biol.
146:57384
Sebastian B, Kakizuka A, Hunter T.
1993. Cdc25M2 activation of cyclindependent kinases by dephosphorylation
of threonine-14 and tyrosine-15. Proc.
Gabrielli BG, Clark JM, McCormack AK,
Ellem KA. 1997. Hyperphosphorylation
of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2
but not cyclin A/Cdk2. J. Biol. Chem.
272:2860714
Hoffmann I, Draetta G, Karsenti E. 1994.
Activation of the phosphatase activity
of human cdc25A by a cdk2-cyclin E
4 Dec 2004
19:16
744
22.

23.
24.
25.
26.
27.
28.
29.
30.
31.
AR
AR232-PA45-29.tex
P1: JRX
DUCRUET ET AL.
dependent phosphorylation at the G1/S
transition. EMBO J. 13:430210
Jinno S, Suto K, Nagata A, Igarashi M,
Kanaoka Y, et al. 1994. Cdc25A is a novel
phosphatase functioning early in the cell
cycle. EMBO J. 13:154956
Blomberg I, Hoffmann I. 1999. Ectopic
expression of Cdc25A accelerates the
G(1)/S transition and leads to premature activation of cyclin E- and cyclin
A-dependent kinases. Mol. Cell Biol.
19:618394
Mailand N, Podtelejnikov AV, Groth A,
Mann M, Bartek J, Lukas J. 2002. Regulation of G(2)/M events by Cdc25A
through phosphorylation-dependent modulation of its stability. EMBO J. 21:5911
20
Donzelli M, Squatrito M, Ganoth D, Hershko A, Pagano M, Draetta GF. 2002.
Dual mode of degradation of Cdc25 A
phosphatase. EMBO J. 21:487584
Chen MS, Hurov J, White LS, WoodfordThomas T, Piwnica-Worms H. 2001. Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase. Mol.
Lincoln AJ, Wickramasinghe D, Stein
P, Schultz RM, Palko ME, et al. 2002.
Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation. Nat. Genet. 30:44649
Falck J, Mailand N, Syljuasen RG,
Bartek J, Lukas J. 2001. The ATM-Chk2Cdc25A checkpoint pathway guards
against radioresistant DNA synthesis. Nature 410:84247
Mailand N, Falck J, Lukas C, Syljuasen
RG, Welcker M, et al. 2000. Rapid destruction of human Cdc25A in response
to DNA damage. Science 288:142529
Zhao H, Watkins JL, Piwnica-Worms H.
2002. Disruption of the checkpoint kinase
1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2
checkpoints. Proc. Natl. Acad. Sci. USA
99:14795800
Donzelli M, Draetta GF. 2003. Regulating
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
mammalian checkpoints through Cdc25

inactivation. EMBO Rep. 4:67177
Nash P, Tang X, Orlicky S, Chen Q,
Gertler FB, et al. 2001. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414:51421
Ducruet AP, Lazo JS. 2003. Regulation of
Cdc25A half-life in interphase by cyclindependent kinase 2 activity. J. Biol. Chem.
278:3183842
Hoffmann I, Clarke PR, Marcote MJ,
Karsenti E, Draetta G. 1993. Phosphorylation and activation of human cdc25-C
by cdc2cyclin B and its involvement in
the self-amplification of MPF at mitosis.
EMBO J. 12:5363
Forrest AR, McCormack AK, DeSouza
CP, Sinnamon JM, Tonks ID, et al. 1999.
Multiple splicing variants of cdc25B regulate G2/M progression. Biochem. Biophys. Res. Commun. 260:51015
Wegener S, Hampe W, Herrmann D,
Schaller HC. 2000. Alternative splicing in
the regulatory region of the human phosphatases CDC25A and CDC25C. Eur. J.
Cell Biol. 79:81015
Graves PR, Lovly CM, Uy GL, PiwnicaWorms H. 2001. Localization of human
Cdc25C is regulated both by nuclear export and 14-3-3 protein binding. Oncogene 20:183951
Nishijima H, Nishitani H, Seki T, Nishimoto T. 1997. A dual-specificity phosphatase Cdc25B is an unstable protein and
triggers p34(cdc2)/cyclin B activation in
hamster BHK21 cells arrested with hydroxyurea. J. Cell Biol. 138:110516
Nagata A, Igarashi M, Jinno S, Suto K,
Okayama H. 1991. An additional homolog of the fission yeast cdc25+ gene
occurs in humans and is highly expressed
in some cancer cells. New Biol. 3:95968
Davezac N, Baldin V, Gabrielli B, Forrest
A, Theis-Febvre N, et al. 2000. Regulation
of CDC25B phosphatases subcellular localization. Oncogene 19:217985
Galaktionov K, Jessus C, Beach D. 1995.
4 Dec 2004
19:16
AR
AR232-PA45-29.tex
P1: JRX

42.
43.
44.
45.
46.
47.
48.
49.
50.
Raf1 interaction with Cdc25 phosphatase

ties mitogenic signal transduction to cell
cycle activation. Genes Dev. 9:104658
Mochizuki T, Kitanaka C, Noguchi K,
Muramatsu T, Asai A, Kuchino Y. 1999.
Physical and functional interactions between Pim-1 kinase and Cdc25A phosphatase. Implications for the Pim-1mediated activation of the c-Myc signaling pathway. J. Biol. Chem. 274:18659
66
Busino L, Chiesa M, Draetta GF, Donzelli
M. 2004. Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and
proteolysis. Oncogene 23:205056
Xia K, Lee RS, Narsimhan RP,
Mukhopadhyay NK, Neel BG, Roberts
TM. 1999. Tyrosine phosphorylation of
the proto-oncoprotein Raf-1 is regulated
by Raf-1 itself and the phosphatase
Cdc25A. Mol. Cell Biol. 19:481924
Wang Z, Wang M, Lazo JS, Carr BI. 2002.
Identification of epidermal growth factor
receptor as a target of Cdc25A protein
phosphatase. J. Biol. Chem. 277:19470
75
Nemoto K, Vogt A, Oguri T, Lazo JS.
2004. Activation of the Raf-1/MEK/Erk
kinase pathway by a novel Cdc25 inhibitor
in human prostate cancer cells. Prostate
58:95102
Hassepass I, Voit R, Hoffmann I. 2003.
Phosphorylation at serine 75 is required
for UV-mediated degradation of human Cdc25A phosphatase at the S-phase
checkpoint. J. Biol. Chem. 278:2982429
Bartek J, Lukas J. 2001. Mammalian G1and S-phase checkpoints in response to
DNA damage. Curr. Opin. Cell Biol.
13:73847
Bulavin DV, Amundson SA, Fornace
AJ. 2002. p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony. Curr. Opin. Genet. Dev.
12:9297
Chen F, Zhang Z, Bower J, Lu Y,
Leonard SS, et al. 2002. Arsenite-induced
Cdc25C degradation is through the KEN-
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
745
box and ubiquitin-proteasome pathway.

Bulavin DV, Higashimoto Y, Popoff IJ,
Gaarde WA, Basrur V, et al. 2001. Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature
411:1027
Busino L, Donzelli M, Chiesa M, Guardavaccaro D, Ganoth D, et al. 2003.
Degradation of Cdc25A by beta-TrCP
during S phase and in response to DNA
damage. Nature 426:8791
Jin J, Shirogane T, Xu L, Nalepa G,
Qin J, et al. 2003. SCFbeta-TRCP links
Chk1 signaling to degradation of the
Cdc25A protein phosphatase. Genes Dev.
17:306274
Molinari M. 2000. Cell cycle checkpoints
and their inactivation in human cancer.
Cell Prolif. 33:26174
Lyon MA, Ducruet AP, Wipf P, Lazo
JS. 2002. Dual-specificity phosphatases
as targets for antineoplastic agents. Nat.
Rev. Drug Discov. 1:96176
Chang L, Karin M. 2001. Mammalian
MAP kinase signalling cascades. Nature
410:3740
Tonks NK, Neel BG. 2001. Combinatorial
control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol.
13:18295
Keyse SM. 2000. Protein phosphatases
and the regulation of mitogen-activated
protein kinase signalling. Curr. Opin. Cell
Biol. 12:18692
Shen Y, Luche R, Wei B, Gordon ML,
Diltz CD, Tonks NK. 2001. Activation of the Jnk signaling pathway by a
dual-specificity phosphatase, JSP-1. Proc.
Charles CH, Abler AS, Lau LF. 1992.
cDNA sequence of a growth factorinducible immediate early gene and characterization of its encoded protein. Oncogene 7:18790
Charles CH, Sun H, Lau LF, Tonks
NK. 1993. The growth factor-inducible
immediate-early gene 3CH134 encodes a
4 Dec 2004
19:16
746
62.

63.
64.
65.
66.
67.
68.
69.
70.
AR
AR232-PA45-29.tex
P1: JRX
DUCRUET ET AL.
protein-tyrosine-phosphatase. Proc. Natl.
Sun H, Charles CH, Lau LF, Tonks NK.
1993. MKP-1 (3CH134), an immediate
early gene product, is a dual specificity
phosphatase that dephosphorylates MAP
kinase in vivo. Cell 75:48793
Keyse SM, Emslie EA. 1992. Oxidative
stress and heat shock induce a human gene
encoding a protein-tyrosine phosphatase.
Nature 359:64447
Farooq A, Zhou MM. 2004. Structure and
regulation of MAPK phosphatases. Cell.
Signal. 16:76979
Masuda K, Shima H, Katagiri C, Kikuchi
K. 2003. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a
JNK specific phosphatase, at Ser-446. J.
Biol. Chem. 278:3244856
Brondello JM, Brunet A, Pouyssegur J,
McKenzie FR. 1997. The dual specificity mitogen-activated protein kinase
phosphatase-1 and -2 are induced by the
p42/p44MAPK cascade. J. Biol. Chem.
272:136876
Dowd S, Sneddon AA, Keyse SM. 1998.
Isolation of the human genes encoding
the pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dualspecificity MAP kinase phosphatase and
its catalytic activation by both MAP and
SAP kinases. J. Cell Sci. 111:338999
Grumont RJ, Rasko JE, Strasser A, Gerondakis S. 1996. Activation of the mitogenactivated protein kinase pathway induces
transcription of the PAC-1 phosphatase
gene. Mol. Cell. Biol. 16:291321
Groom LA, Sneddon AA, Alessi DR,
Dowd S, Keyse SM. 1996. Differential regulation of the MAP, SAP and
RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase. EMBO J.
15:362132
Bernaudin M, Tang Y, Reilly M, Petit E,
Sharp FR. 2002. Brain genomic response
following hypoxia and re-oxygenation
in the neonatal rat. Identification of
genes that might contribute to hypoxia-
71.
72.
73.
74.
75.
76.
77.
78.
induced ischemic tolerance. J. Biol.

Chem. 277:3972838
Laderoute KR, Mendonca HL, Calaoagan JM, Knapp AM, Giaccia AJ, Stork
PJ. 1999. Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression
is induced by low oxygen conditions
found in solid tumor microenvironments.
A candidate MKP for the inactivation
of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity. J. Biol. Chem. 274:12890
97
Seta KA, Kim R, Kim HW, Millhorn DE, Beitner-Johnson D. 2001.
Hypoxia-induced regulation of MAPK
phosphatase-1 as identified by subtractive suppression hybridization and cDNA
microarray analysis. J. Biol. Chem.
276:4440512
Brondello JM, Pouyssegur J, McKenzie FR. 1999. Reduced MAP kinase phosphatase-1 degradation after
p42/p44MAPK- dependent phosphorylation. Science 286:251417
Camps M, Nichols A, Gillieron C, Antonsson B, Muda M, et al. 1998. Catalytic
activation of the phosphatase MKP-3 by
ERK2 mitogen-activated protein kinase.
Science 280:126265
Aref S, Fouda M, El Dosoky E, Menessy
A, Mabed M, et al. 2003. c-Myc oncogene
and Cdc25A cell activating phosphatase
expression in non-Hodgkins lymphoma.
Hematology 8:18390
Loffler H, Syljuasen RG, Bartkova J,
Worm J, Lukas J, Bartek J. 2003. Distinct modes of deregulation of the protooncogenic Cdc25A phosphatase in human breast cancer cell lines. Oncogene
22:806371
Ding XL, Husseman J, Tomashevski A,
Nochlin D, Jin LW, Vincent I. 2000. The
cell cycle Cdc25A tyrosine phosphatase is
activated in degenerating postmitotic neurons in Alzheimers disease. Am. J. Pathol.
157:198390
Vincent I, Bu B, Hudson K, Husseman
4 Dec 2004
19:16
AR
AR232-PA45-29.tex
P1: JRX

79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
J, Nochlin D, Jin L. 2001. Constitutive

Cdc25B tyrosine phosphatase activity in
adult brain neurons with M phase-type alterations in Alzheimers disease. Neuroscience 105:63950
Galaktionov K, Lee AK, Eckstein J,
Draetta G, Meckler J, et al. 1995. CDC25
phosphatases as potential human oncogenes. Science 269:157577
Yao Y, Slosberg ED, Wang L, Hibshoosh
H, Zhang YJ, et al. 1999. Increased susceptibility to carcinogen-induced mammary tumors in MMTV-Cdc25B transgenic mice. Oncogene 18:515966
Ma ZQ, Chua SS, DeMayo FJ, Tsai
SY. 1999. Induction of mammary gland
hyperplasia in transgenic mice overexpressing human Cdc25B. Oncogene
18:456476
Ma ZQ, Liu Z, Ngan ES, Tsai SY. 2001.
Cdc25B functions as a novel coactivator
for the steroid receptors. Mol. Cell Biol.
21:805667
Zou X, Tsutsui T, Ray D, Blomquist JF,
Ichijo H, et al. 2001. The cell cycleregulatory CDC25A phosphatase inhibits
apoptosis signal-regulating kinase 1. Mol.
Nishikawa Y, Carr BI, Wang M, Kar S,
Finn F, et al. 1995. Growth inhibition of
hepatoma cells induced by vitamin K and
its analogs. J. Biol. Chem. 270:2830410
Pumiglia KM, Decker SJ. 1997. Cell cycle arrest mediated by the MEK/mitogenactivated protein kinase pathway. Proc.
Liu DX, Greene LA. 2001. Neuronal
apoptosis at the G1/S cell cycle checkpoint. Cell Tissue Res. 305:21728
McShea A, Wahl AF, Smith MA. 1999.
Re-entry into the cell cycle: a mechanism
for neurodegeneration in Alzheimer disease. Med. Hypotheses 52:52527
Nagy Z, Esiri MM, Smith AD. 1998. The
cell division cycle and the pathophysiology of Alzheimers disease. Neuroscience
87:73139
Smith MZ, Nagy Z, Esiri MM. 1999. Cell
90.
91.
92.
93.
94.
95.
96.
97.
98.
747
cycle-related protein expression in vascular dementia and Alzheimers disease.

Neurosci. Lett. 271:4548
Kruman II, Wersto RP, Cardozo-Pelaez F,
Smilenov L, Chan SL, et al. 2004. Cell
cycle activation linked to neuronal cell
death initiated by DNA damage. Neuron
41:54961
Vincent I, Jicha G, Rosado M, Dickson
DW. 1997. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimers disease brain.
Rideout HJ, Wang Q, Park DS, Stefanis
L. 2003. Cyclin-dependent kinase activity is required for apoptotic death but not
inclusion formation in cortical neurons after proteasomal inhibition. J. Neurosci.
23:123745
Furukawa T, Sunamura M, Motoi F,
Matsuno S, Horii A. 2003. Potential
tumor suppressive pathway involving
DUSP6/MKP-3 in pancreatic cancer. Am.
J. Pathol. 162:180715
Kok K, Osinga J, Carritt B, Davis MB,
van der Hout AH, et al. 1987. Deletion of
a DNA sequence at the chromosomal region 3p21 in all major types of lung cancer. Nature 330:57881
Kovacs G, Erlandsson R, Boldog F, Ingvarsson S, Muller-Brechlin R, et al.
1988. Consistent chromosome 3p deletion
and loss of heterozygosity in renal cell
carcinoma. Proc. Natl. Acad. Sci. USA
85:157175
Masuda K, Shima H, Kikuchi K, Watanabe Y, Matsuda Y. 2000. Expression
and comparative chromosomal mapping
of MKP-5 genes DUSP10/Dusp10. Cytogenet. Cell Genet. 90:7174
Schneider BG, Pulitzer DR, Brown RD,
Prihoda TJ, Bostwick DG, et al. 1995. Allelic imbalance in gastric cancer: an affected site on chromosome arm 3p. Genes
Chromosomes Cancer 13:26371
Vocke CD, Pozzatti RO, Bostwick DG,
Florence CD, Jennings SB, et al. 1996.
Analysis of 99 microdissected prostate
4 Dec 2004
19:16
748
99.

100.
101.
102.
103.
104.
105.
106.
AR
AR232-PA45-29.tex
P1: JRX
DUCRUET ET AL.
carcinomas reveals a high frequency of allelic loss on chromosome 8p1221. Cancer Res. 56:241116
Yokota J, Tsukada Y, Nakajima T, Gotoh
M, Shimosato Y, et al. 1989. Loss of heterozygosity on the short arm of chromosome 3 in carcinoma of the uterine cervix.
Theodosiou AM, Rodrigues NR, Nesbit MA, Ambrose HJ, Paterson H, et al.
1996. A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the
coding region. Hum. Mol. Genet. 5:675
84
Bepler G, Garcia-Blanco MA. 1994.
Three tumor-suppressor regions on chromosome 11p identified by high-resolution
deletion mapping in human non-smallcell lung cancer. Proc. Natl. Acad. Sci.
USA 91:551317
Hoornaert I, Marynen P, Goris J, Sciot
R, Baens M. 2003. MAPK phosphatase
DUSP16/MKP-7, a candidate tumor suppressor for chromosome region 12p12-13,
reduces BCR-ABL-induced transformation. Oncogene 22:772836
Montpetit A, Boily G, Sinnett D. 2002. A
detailed transcriptional map of the chromosome 12p12 tumour suppressor locus.
Eur. J. Hum. Genet. 10:6271
Martell KJ, Kwak S, Hakes DJ, Dixon
JE, Trent JM. 1994. Chromosomal localization of four human VH1-like proteintyrosine phosphatases. Genomics 22:462
64
Weber RG, Rieger J, Naumann U, Lichter
P, Weller M. 2001. Chromosomal imbalances associated with response to
chemotherapy and cytotoxic cytokines in
human malignant glioma cell lines. Int. J.
Cancer 91:21318
Ojopi EP, Rogatto SR, Caldeira JR,
Barbieri-Neto J, Squire JA. 2001. Comparative genomic hybridization detects
novel amplifications in fibroadenomas of
the breast. Genes Chromosomes Cancer
30:2531
107. Murty VV, Reuter VE, Bosl GJ, Chaganti RS. 1996. Deletion mapping identifies loss of heterozygosity at 5p15.1-15.2,
5q11 and 5q34-35 in human male germ
cell tumors. Oncogene 12:271923
108. Peng HQ, Liu L, Goss PE, Bailey D, Hogg
D. 1999. Chromosomal deletions occur in
restricted regions of 5q in testicular germ
cell cancer. Oncogene 18:327783
109. Faulkner SW, Friedlander ML. 2000.
Molecular genetic analysis of malignant
ovarian germ cell tumors. Gynecol. Oncol. 77:28388
110. Emslie EA, Jones TA, Sheer D, Keyse
SM. 1994. The CL100 gene, which encodes a dual specificity (Tyr/Thr) MAP
kinase phosphatase, is highly conserved
and maps to human chromosome 5q34.
Hum. Genet. 93:51316
111. Dorfman K, Carrasco D, Gruda M, Ryan
C, Lira SA, Bravo R. 1996. Disruption of
the erp/mkp-1 gene does not affect mouse
development: normal MAP kinase activity in ERP/MKP-1-deficient fibroblasts.
Oncogene 13:92531
112. Khosravi-Far R, White MA, Westwick
JK, Solski PA, Chrzanowska-Wodnicka
M, et al. 1996. Oncogenic Ras activation of Raf/mitogen-activated protein
kinase-independent pathways is sufficient
to cause tumorigenic transformation. Mol.
113. Magi-Galluzzi C, Mishra R, Fiorentino
M, Montironi R, Yao H, et al. 1997.
Mitogen-activated protein kinase phosphatase 1 is overexpressed in prostate cancers and is inversely related to apoptosis.
Lab. Invest. 76:3751
114. Bang YJ, Kwon JH, Kang SH, Kim JW,
Yang YC. 1998. Increased MAPK activity
and MKP-1 overexpression in human gastric adenocarcinoma. Biochem. Biophys.
115. Wang HY, Cheng Z, Malbon CC. 2003.
Overexpression of mitogen-activated protein kinase phosphatases MKP1, MKP2
in human breast cancer. Cancer Lett.
191:22937
4 Dec 2004
19:16
AR
AR232-PA45-29.tex
P1: JRX


116. Liao Q, Guo J, Kleeff J, Zimmermann
A, Buchler MW, et al. 2003. Downregulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells. Gastroenterology 124:183045
117. Denkert C, Schmitt WD, Berger S, Reles A, Pest S, et al. 2002. Expression of mitogen-activated protein kinase
phosphatase-1 (MKP-1) in primary human ovarian carcinoma. Int. J. Cancer
102:50713
118. Loda M, Capodieci P, Mishra R, Yao
H, Corless C, et al. 1996. Expression of mitogen-activated protein kinase
phosphatase-1 in the early phases of human epithelial carcinogenesis. Am. J.
Pathol. 149:155364
119. Franklin CC, Srikanth S, Kraft AS.
1998. Conditional expression of mitogenactivated protein kinase phosphatase-1,
MKP-1, is cytoprotective against UVinduced apoptosis. Proc. Natl. Acad. Sci.
USA 95:301419
120. Liu Y, Gorospe M, Yang C, Holbrook NJ.
1995. Role of mitogen-activated protein
kinase phosphatase during the cellular response to genotoxic stress. Inhibition of cJun N- terminal kinase activity and AP-1dependent gene activation. J. Biol. Chem.
270:837780
121. Sanchez-Perez I, Martinez-Gomariz M,
Williams D, Keyse SM, Perona R. 2000.
CL100/MKP-1 modulates JNK activation
and apoptosis in response to cisplatin.
Oncogene 19:514252
122. Staber PB, Linkesch W, Zauner D,
Beham-Schmid C, Guelly C, et al. 2004.
Common alterations in gene expression and increased proliferation in recurrent acute myeloid leukemia. Oncogene
23:894904
123. Kang Y, Siegel PM, Shu W, Drobnjak
M, Kakonen SM, et al. 2003. A multigenic program mediating breast cancer
metastasis to bone. Cancer Cell 3:537
49
124. Dancey J, Sausville EA. 2003. Issues and
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
749
progress with protein kinase inhibitors for

cancer treatment. Nat. Rev. Drug Discov.
2:296313
Fauman EB, Cogswell JP, Lovejoy B,
Rocque WJ, Holmes W, et al. 1998. Crystal structure of the catalytic domain of
the human cell cycle control phosphatase,
Cdc25A. Cell 93:61725
Reynolds RA, Yem AW, Wolfe CL,
Deibel MR Jr, Chidester CG, Watenpaugh
KD. 1999. Crystal structure of the catalytic subunit of Cdc25B required for
G2/M phase transition of the cell cycle.
J. Mol. Biol. 293:55968
Kristjansdottir K, Rudolph J. 2004. Cdc25
phosphatases and cancer. Chem. Biol.
11:104351
Gunasekera SP, McCarthy PJ, KellyBorges M, Lobkovsky E, Clardy J. 1996.
Dysidiolide: a novel protein phosphatase
inhibitor from the Caribbean sponge
Dysidea etheria de Laubenfels. J. Am.
Chem. Soc. 118:875960
Ham SW, Park HJ, Lim DH. 1997. Studies
on menadione as an inhibitor of the cdc25
phosphatase. Bioorg. Chem. 25:3336
Horiguchi T, Nishi K, Hakoda S, Tanida S,
Nagata A, Okayama H. 1994. Dnacin A1
and dnacin B1 are antitumor antibiotics
that inhibit cdc25B phosphatase activity.
Loukaci A, Le SI, Samadi M, Leclerc S,
Damiens E, et al. 2001. Coscinosulfate,
a CDC25 phosphatase inhibitor from the
sponge Coscinoderma mathewsi. Bioorg.
Med. Chem. 9:304954
Wipf P, Hopkins CR, Phillips EO, Lazo
JS. 2002. Separation of Cdc25 dual specificity phosphatase inhibition and DNA
cleaving activities in a focused library
of analogs of the antitumor antibiotic
Dnacin. Tetrahedron 58:636772
Brohm D, Waldmann H. 1998. Stereoselective synthesis of the core structure of
the protein phosphatase inhibitor dysidiolide. Tetrahedron Lett. 39:399598
Brohm D, Metzger S, Bhargava A, Muller
O, Lieb F, Waldmann H. 2001. Natural
4 Dec 2004
19:16
750

135.
136.
137.
138.
139.
140.
141.
142.
AR
AR232-PA45-29.tex
P1: JRX
DUCRUET ET AL.
products are biologically validated starting points in structural space for compound library development: Solid-phase
synthesis of dysidiolide-derived phosphatase inhibitors. Angew. Chem.-Int. Ed.
41:30711
Sodeoka M, Sampe R, Kojima S, Baba
Y, Usui T, et al. 2001. Synthesis of a
tetronic acid library focused on inhibitors
of tyrosine and dual-specificity protein
phosphatases and its evaluation regarding VHR and cdc25B inhibition. J. Med.
Chem. 44:321622
Kar S, Lefterov IM, Wang M, Lazo JS,
Scott CN, et al. 2003. Binding and inhibition of Cdc25 phosphatases by vitamin K
analogues. Biochemistry 42:1049097
Poigny S, Nouri S, Chiaroni A, Guyot
M, Samadi M. 2001. Total synthesis and
determination of the absolute configuration of coscinosulfate. A new selective inhibitor of Cdc25 protein phosphatase. J.
Org. Chem. 66:726369
Wipf P, Aslan DC, Luci DK, Southwick
EC, Lazo JS. 2000. Synthesis and biological evaluation of a targeted library of
protein phosphatase inhibitors. Biotechnol. Bioeng. 71:5870
Kitaide M, Nagai K, Terada T, Asao T,
Sugimoto Y, Yamada Y. 2001. JP Patent
No. 2003104964
Peng H, Xie W, Otterness DM, Cogswell
JP, McConnell RT, et al. 2001. Syntheses and biological activities of a novel
group of steroidal derived inhibitors for
human Cdc25A protein phosphatase. J.
Med. Chem. 44:83448
Fritzen EL, Brightwell AS, Erickson LA,
Romero DL. 2000. The solid phase synthesis of tetrahydroisoquinolines having
cdc25B inhibitory activity. Bioorg. Med.
Chem. Lett. 10:64952
Galcera Contour MO, Lavergne O, Brezak
143.
144.
145.
146.
147.
148.
149.
150.
151.
Pannetier MC, Prevost G. 2002. WO

Patent No. 2003055868
Sohn J, Kiburz B, Li Z, Deng L, Safi
A, et al. 2003. Inhibition of Cdc25 phosphatases by indolyldihydroxyquinones. J.
Med. Chem. 46:258088
Lazo JS, Aslan DC, Southwick EC, Cooley KA, Ducruet AP, et al. 2001. Discovery and biological evaluation of a new
family of potent inhibitors of the dual
specificity protein phosphatase Cdc25. J.
Med. Chem. 44:404249
Lazo JS, Nemoto K, Pestell KE, Cooley K,
Southwick EC, et al. 2002. Identification
of a potent and selective pharmacophore
for Cdc25 dual specificity phosphatase inhibitors. Mol. Pharmacol. 61:72028
Wipf P, Aslan DC, Southwick EC, Lazo
JS. 2001. Sulfonylated aminothiazoles as
new small molecule inhibitors of protein
phosphatases. Bioorg. Med. Chem. Lett.
11:31317
Pfahl M, Al shamma HA, Fanjul AN,
Pleynet DPM, Bao H, et al. 2002. WO
Patent No. 2003050098
Brezak MC, Quaranta M, Mondesert O,
Galcera MO, Lavergne O, et al. 2004.
A novel synthetic inhibitor of CDC25
phosphatases: BN82002. Cancer Res.
64:332025
Taylor NR, Borhani D, Epstein D,
Rudolph J, Ritter K, et al. 2001. U.S.
Patent No. 2002183249
Baurle S, Blume T, Gunther J, Henschel
D, Hillig RC, et al. 2004. Design and synthesis of macrocyclic inhibitors of phosphatase Cdc25B. Bioorg. Med. Chem.
Lett. 14:167377
Vogt A, Cooley KA, Brisson M, Tarpley MG, Wipf P, Lazo JS. 2003. Cellactive dual specificity phosphatase inhibitors identified by high-content screening. Chem. Biol. 10:73342

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