Nutrigenomics (Oxidative Stress and Disease) (PDFDrive)

Nutrigenomics
edited by
Gerald Rimbach
Jürgen Fuchs
Lester Packer
Boca Raton London New York Singapore
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Series Introduction
Oxygen is a dangerous friend. Through evolution, oxygen—itself a free radical—

was chosen as the terminal electron acceptor for respiration. The two unpaired
electrons of oxygen spin in the same direction; thus, oxygen is a biradical.
Other oxygen-derived free radicals, such as superoxide anion or hydroxyl rad-
icals, formed during metabolism or by ionizing radiation are stronger oxidants,
i.e., endowed with a higher chemical reactivity. Oxygen-derived free radicals
are generated during oxidative metabolism and energy production in the body
and are involved in regulation of signal transduction and gene expression,
activation of receptors and nuclear transcription factors, oxidative damage to
cell components, the antimicrobial and cytotoxic action of immune system
cells, neutrophils and macrophages, as well as in aging and age-related degenera-
tive diseases. Overwhelming evidence indicates that oxidative stress can lead to
cell and tissue injury. However, the same free radicals that are generated during
oxidative stress are produced during normal metabolism and, as a corollary, are
involved in both human health and disease.
In addition to reactive oxygen species, research on reactive nitrogen
species has been gathering momentum to develop an area of enormous import-
ance in biology and medicine. Nitric oxide or nitrogen monoxide (NO) is a
free radical generated by nitric oxide synthase (NOS). This enzyme modulates
physiological responses in the circulation such as vasodilation (eNOS) or signal-
ing in the brain (nNOS). However, during inflammation, a third isoenzyme is
induced, iNOS, resulting in the overproduction of NO and causing damage to tar-
geted infectious organisms and to healthy tissues in the vicinity. More worrisome,
however, is the fact that NO can react with superoxide anion to yield a strong
oxidant, peroxynitrite. Oxidation of lipids, proteins, and DNA by peroxynitrite
increases the likelihood of tissue injury.
iii
iv Series Introduction
Both reactive oxygen and nitrogen species are involved in the redox regu-
lation of cell functions. Oxidative stress is increasingly viewed as a major
upstream component in the signaling cascade involved in inflammatory responses
and stimulation of adhesion molecule and chemoattractant production. Hydrogen
peroxide decomposes in the presence of transition metals to the highly reactive
hydroxyl radical, which by two major reactions—hydrogen abstraction and
addition—accounts for most of the oxidative damage to proteins, lipids,
sugars, and nucleic acids. Hydrogen peroxide is also an important signaling mol-
ecule that, among others, can activate NF-kB, an important transcription factor
involved in inflammatory responses. At low concentrations, hydrogen peroxide
regulates cell signaling and stimulates cell proliferation; at higher concentrations
it triggers apoptosis and, at even higher levels, necrosis.
Virtually all diseases thus far examined involve free radicals. In most cases,
free radicals are secondary to the disease process, but in some instances free
radicals are causal. Thus, there is a delicate balance between oxidants and anti-
oxidants in health and disease. Their proper balance is essential for ensuring
healthy aging.
The term oxidative stress indicates that the antioxidant status of cells and
tissues is altered by exposure to oxidants. The redox status is thus dependent
on the degree to which a cell’s components are in the oxidized state. In general,
the reducing environment inside cells helps to prevent oxidative damage. In
this reducing environment, disulfide bonds (S –S) do not spontaneously form
because sulfhydryl groups are maintained in the reduced state (SH), thus prevent-
ing protein misfolding or aggregation. This reducing environment is maintained
by oxidative metabolism and by the action of antioxidant enzymes and sub-
stances, such as glutathione, thioredoxin, vitamins E and C, and enzymes such
as superoxide dismutases, catalase, and the selenium-dependent glutathione
reductase and glutathione and thioredoxin hydroperoxidases, which serve to
remove reactive oxygen species (hydroperoxides).
Changes in the redox status and depletion of antioxidants occur during oxi-
dative stress. The thiol redox status is a useful index of oxidative stress mainly
because metabolism and NADPH-dependent enzymes maintain cell glutathione
(GSH) almost completely in its reduced state. Oxidized glutathione (glutathione
disulfide, GSSG) accumulates under conditions of oxidant exposure and this
changes the ratio GSSG/GSH; an increased ratio is usually taken as indicating
oxidative stress. Other oxidative stress indicators are ratios of redox couples
such as NADPH/NADP, NADH/NAD, thioredoxinreduced/thioredoxinoxidized ,
dihydrolipoic acid/a-lipoic acid, and lactate/pyruvate. Changes in these ratios
affects the energy status of the cell, largely determined by the ratio ATP/
ADP þ AMP. Many tissues contain large amounts of glutathione, 2 –4 mM in
erythrocytes or neural tissues and up to 8 mM in hepatic tissues. Reactive
oxygen and nitrogen species can oxidize glutathione, thus lowering the levels
of the most abundant nonprotein thiol, sometimes designated as the cell’s
primary preventative antioxidant.
Series Introduction v
Current hypotheses favor the idea that lowering oxidative stress can have a
health benefit. Free radicals can be overproduced or the natural antioxidant
system defenses weakened, first resulting in oxidative stress, and then leading to oxi-
dative injury and disease. Examples of this process include heart disease, cancer,
and neurodegenerative disorders. Oxidation of human low-density lipoproteins is
considered an early step in the progression and eventual development of athero-
sclerosis, thus leading to cardiovascular disease. Oxidative DNA damage may
initiate carcinogenesis. Environmental sources of reactive oxygen species are also
important in relation to oxidative stress and disease. A few examples: UV radiation,
ozone, cigarette smoke, and others are significant sources of oxidative stress.
Compelling support for the involvement of free radicals in disease devel-
opment originates from epidemiological studies showing that an enhanced
antioxidant status is associated with reduced risk of several diseases. Vitamins
C and E and prevention of cardiovascular disease are a notable example. Elevated
antioxidant status is also associated with decreased incidence of cataracts, cancer,
and neurodegenerative disorders. Some recent reports have suggested an inverse
correlation between antioxidant status and the occurrence of rheumatoid arthritis
and diabetes mellitus. Indeed, the number of indications in which antioxidants
may be useful in the prevention and/or the treatment of disease is increasing.
Oxidative stress, rather than being the primary cause of disease, is more
often a secondary complication in many disorders. Oxidative stress diseases
include inflammatory bowel diseases, retinal ischemia, cardiovascular disease
and restenosis, AIDS, adult respiratory distress syndrome, and neurodegenerative
diseases such as stroke, Parkinson’s disease, and Alzheimer’s disease. Such
indications may prove amenable to antioxidant treatment (in combination with
conventional therapies) because there is a clear involvement of oxidative
injury in these disorders.
In this series of books, the importance of oxidative stress and disease
associated with organ systems of the body is highlighted by exploring the scien-
tific evidence and the medical applications of this knowledge. The series also
highlights the major natural antioxidant enzymes and antioxidant substances
such as vitamins E, A, and C, flavonoids, polyphenols, carotenoids, lipoic acid,
coenzyme Q10 , carnitine, and other micronutrients present in food and beverages.
Oxidative stress is an underlying factor in health and disease. More and more evi-
dence indicates that a proper balance between oxidants and antioxidants is
involved in maintaining health and longevity and that altering this balance in
favor of oxidants may result in pathophysiological responses causing functional
disorders and disease. This series is intended for researchers in the basic bio-
medical sciences and clinicians. The potential of such knowledge for healthy
aging and disease prevention warrants further knowledge about how oxidants
and antioxidants modulate cell and tissue function.
Lester Packer
Enrique Cadenas
Preface
Nutrition research commenced more than 200 years ago in the dawn of the chemi-
cal revolution. The “golden age of nutrition” began in the early 1910s and contin-
ued into the 1940s when nutritional sciences focused primarily on diseases
associated with single nutrient deficiencies. This led to the formulation of the
Recommended Daily Allowance (RDA) of each nutrient. After almost all of
the essential nutrients had been discovered, nutrition research focused on the
problem of multifactorial chronic diseases, many of which are caused not by nutri-
tional deficiency but by overnutrition. In the following years, the revolutionary
progress in recombinant DNA technology and genomics culminated in 2001 with
completion of the Human Genome Project and sequencing of the entire human
genome. As a result of all these developments, genomics, transcriptomics,
proteomics, and metabolomics are increasingly being used in nutritional research.
Nutritional genomics, also called nutrigenomics, is an emerging field in the
life sciences and is considered as one of the next frontiers in the postgenomic era.
Its fundamental concept is that a healthy phenotype can develop into a chronic
disease phenotype via alterations in gene expression or epigenetic phenomena
and that the diet contains substances having the potential to modify these pro-
cesses. Nutrigenomics focuses on the relationship between dietary nutrients
and gene expression using state-of-the-art technology.
The development of DNA microarrays and protein chips make large-scale
genomic and proteomic investigations possible by allowing simultaneously high
throughput monitoring of the expression of thousands of genes in response to
diet. The emerging knowledge will aid in the understanding of how nutrients
modify cancer risk, chronic diseases, and aging. It is generally recognized that
most human diseases are largely avoidable by lifestyle changes. This places nutri-
genomics at the forefront of preventive medicine.
vii
viii Preface
The present book was compiled to update the reader on recent advances
in nutrigenomics with special emphasis on the gene regulatory activity of oxi-
dants, antioxidants, phytochemicals, and micronutrients in human health and
disease.
Gerald Rimbach
Jürgen Fuchs
Lester Packer
About the Editors
GERALD RIMBACH is Professor of Food Science and Director of the Institute

of Human Nutrition and Food Science, Christian Albrechts University, Kiel,
Germany. Prior his appointment in Kiel, he worked at the University of
California, Berkeley, and was Lecturer at the University of Reading, United
Kingdom. Dr. Rimbach is a member of the German and British Nutrition
Society and the Society of Nutrition Physiology (GfE) and is the vice-president
of the German Society for Quality Research of Plant Food (DGQ). He is the
author, coauthor or coeditor of over 100 journal articles and book chapters.
Dr. Rimbach received the M.Sc. and Ph.D. degrees from the University
Giessen, Germany.
JÜRGEN FUCHS is Professor, Department of Dermatology, Johann Wolfgang

Goethe-Universität, Frankfurt, Germany. The author of approximately 250 pub-
lished articles and the coeditor of Environmental Stressors in Health and
Disease, Lipoic Acid in Health and Disease, Oxidative Stress in Dermatology,
Vitamin C in Health and Disease, and Vitamin E in Health and Disease
(all titles, Marcel Dekker, Inc.), he received the Ph.D. (1985) and M.D. (1986)
degrees from the University of Frankfurt, Germany.
LESTER PACKER is Adjunct Professor of Molecular Pharmacology and Toxi-

cology, University of Southern California School of Pharmacy, Los Angeles. He
is the author, coauthor, or coeditor of over 800 journal articles and book chapters,
and many books including Antioxidants in Diabetes Management; Environ-
mental Stressors in Health and Disease; Flavonoids in Health and Disease;
Free Radicals in Brain Pathophysiology; Handbook of Antioxidants, Second
Edition; Redox Regulation of Cell Signaling and Its Clinical Application;
ix
x About the Editors
Vitamin C in Health and Disease; and Vitamin E in Health and Disease (all titles,
Marcel Dekker, Inc.). Dr. Packer received the B.S. (1951) and M.S. (1952)
degrees from Brooklyn College, New York, and the Ph.D. degree (1956) from
Yale University, New Haven, Connecticut.
Contributors
R. Ambra National Institute for Food and Nutrition Research (INRAN), Rome,
Italy
Max O. Bingham Food Microbial Sciences Unit, School of Food Biosciences,

University of Reading, Reading, UK
Raymond K. Blanchard Nutritional Genomics Laboratory, Food Science and

Human Nutrition Department, Center for Nutritional Sciences, University of
Florida, Gainesville, Florida, USA
R. Canali National Institute for Food and Nutrition Research (INRAN), Rome,
Italy
Aedin Cassidy School of Medicine, Health Policy & Practice, University of

East Anglia, Norwich, UK
Rainer Cermak Institute of Animal Nutrition and Physiology, Christian

Albrechts University, Kiel, Germany
Kyung-Joo Cho Laboratory of Cell Biology, Korea Advanced Institute of

Science and Technology, Daejeon, South Korea
An-Sik Chung Department of Biological Sciences, Korea Advanced Institute

of Science and Technology, Daejeon, South Korea
Robert J. Cousins Nutritional Genomics Laboratory, Food Science and

Human Nutrition Department, Center for Nutritional Sciences, University of
Florida, Gainesville, Florida, USA
S. G. Cremers School of Animal and Microbial Sciences, University of

Reading, Reading, UK
xi
xii Contributors
Sonia De Pascual-Teresa Department of Plant Food Science and Technology,

Instituto del Frı́o, Consejo Superior de Investigaciones Cientificas, Madrid,
Spain
B. A. Ewins School of Food Biosciences, University of Reading, Reading, UK
Alexandra Fischer Department of Cardiovascular Medicine, Wellcome Trust

Centre for Human Genetics, University of Oxford, Oxford, UK
Glenn R. Gibson Food Microbial Sciences Unit, School of Food Biosciences,

R. D. Gill-Garrison Sciona, Ltd., Havant, UK
K. Grimaldi Sciona, Ltd., Havant, UK
Robert Francis Grimble Institute of Human Nutrition, Biomedical Sciences

Building, University of Southampton, Southampton, UK
O. Gulati Horphag Research Ltd., Geneva, Switzerland
Shuji Honda Aging Redox Regulation Research Group, Tokyo Metropolitan

Institute of Gerontology, Tokyo, Japan
Yoko Honda Aging Redox Regulation Research Group, Tokyo Metropolitan

Institute of Gerontology, Tokyo, Japan
John K. Lodge School of Biomedical and Molecular Sciences, University of

Surrey, Guildford, Surrey, UK
Silvia Mandel Eve Topf and USA National Parkinson Foundation Centers of
Excellence for Neurodegenerative Diseases Research and Department
of Pharmacology and Rappaport Family Research Institute, Technion-Faculty
of Medicine, Haifa, Israel
Anne M. Minihane Hugh Sinclair Unit of Human Nutrition, University of

Reading, Reading, UK
Ken Mills Department of Haematology, Wales College of Medicine, Cardiff

University, Cardiff, Wales, UK
Hadi Moini Department of Molecular Pharmacology and Toxicology, School

of Pharmacy, University of Southern California, Los Angeles, California, USA
B. A. Nier School of Animal and Microbial Sciences, University of Reading,

Reading, UK
Estibaliz Olano-Martin School of Food Biosciences, University of Reading,

Reading, UK
Contributors xiii
Lester Packer Department of Molecular Pharmacology and Toxicology,

School of Pharmacy, University of Southern California, Los Angeles, California,
USA
Josef Pallauf Institute of Animal Nutrition and Nutrition Physiology, Justus-
Liebig-University of Giessen, Giessen, Germany
Gerald Rimbach Institute of Human Nutrition and Food Science, Christian
Cristina Rota Department of Biomedical Sciences, University of Modena and
Reggio Emilia, Modena, Italy
Abulkalam M. Shamsuddin Professor of Pathology, The University of
Maryland School of Medicine, Baltimore, Maryland, USA
Jeremy P. E. Spencer School of Food Biosciences, University of Reading,
Reading, UK
Paul Sharp School of Health and Life Sciences, King’s College, London, UK
J. L. Slater Sciona, Ltd., Havant, UK
F. Virgili National Institute for Food and Nutrition Research (INRAN), Rome,
Italy
Johannes von Lintig Department of Animal Physiology and Neurobiology,
Institute for Biology I, University of Freiburg, Freiburg, Germany
Stefan Weber Klinik und Poliklinik für Anästhesiologie und Operative
Intensivmedizin, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms-
Universität, Bonn, Germany
Peter D. Weinberg Physiological Flow Studies Group, Department of Bio-
engineering, Imperial College, London, UK
Orly Weinreb Eve Topf and USA National Parkinson Foundation Centers
of Excellence for Neurodegenerative Diseases Research and Department of
Pharmacology and Rappaport Family Research Institute, Technion-Faculty of
Medicine, Haifa, Israel
Siegfried Wolffram Institute of Animal Nutrition and Physiology, Christian
Moussa B. H. Youdim Eve Topf and USA National Parkinson Foundation
Centers of Excellence for Neurodegenerative Diseases Research and Department
of Pharmacology and Rappaport Family Research Institute, Technion-Faculty of
Medicine, Haifa, Israel
Contents
Series Introduction ....................................... iii

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1. Application of Nutrigenomics Tools to Analyze the Role of

Oxidants and Antioxidants in Gene Expression . . . . . . . . . . . . . . . 1
Gerald Rimbach and Sonia De Pascual-Teresa
2. Oxidative Stress and Human Genetic Variation . . . . . . . . . . . . . . 13

R. D. Gill-Garrison, J. L. Slater, and K. Grimaldi
3. Analysis of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Ken Mills
4. Oxidative Stress, Gene Expression, and Lifespan . . . . . . . . . . . . . 67

Yoko Honda and Shuji Honda
5. Anti-Oxidant Modulation in Immune Function . . . . . . . . . . . . . . . 97

Robert Francis Grimble
6. Concentration-Dependent Gene and Protein Expressions of

Neuroprotective and Neurotoxic Activities of
Antioxidants, Including Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . 123
Orly Weinreb, Silvia Mandel, and Moussa B. H. Youdim
xv
xvi Contents
7. Effects of Antioxidants on Gene Expression in

Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
B. A. Nier, B. A. Ewins, S. G. Cremers,
and Peter D. Weinberg
8. Fatty Acids, Gene Expression, and

Coronary Heart Disease (CHD) . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Anne M. Minihane
9. Cell Regulatory Activity of Tocopherols

and Tocotrienols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Cristina Rota, Anne M. Minihane,
Peter D. Weinberg, Stefan Weber, John K. Lodge,
Lester Packer, and Gerald Rimbach
10. Molecular Analysis of the Vitamin A

Biosynthetic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Johannes von Lintig
11. Molecular Mechanisms Underlaying the Health

Promoting Activity of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . 241
Estibaliz Olano-Martin
12. Cellular Redox Activity and Molecular Functions

of Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
John K. Lodge
13. Cell Signaling Properties of a-Lipoic Acid:

Implications in Type 2 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Hadi Moini, Lester Packer, Kyung-Joo Cho,
and An-Sik Chung
14. Dietary Isoflavones and Coronary Artery

Disease—Proposed Molecular Mechanisms
of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Aedin Cassidy and Sonia De Pascual-Teresa
15. Anti-Carcinogenic Properties of Soy Isoflavones . . . . . . . . . . . . . . 327

Max O. Bingham and Glenn R. Gibson
16. Effect of Ginkgo biloba Extract EGb 761

on Differential Gene Expression in
the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Rainer Cermak and Siegfried Wolffram
Contents xvii
17. Interactions of Flavonoids and Their Metabolites

with Cell Signaling Cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Jeremy P. E. Spencer
18. Antioxidant and Gene Regulatory Properties

of Procyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
R. Canali, R. Ambra, O. Gulati, and F. Virgili
19. Cell Signaling Properties of Inositol

Hexaphosphate (IP6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Abulkalam M. Shamsuddin
20. Modulation of Gene Expression by Dietary Iron . . . . . . . . . . . . . . 421

Paul Sharp
21. Dietary Selenium and Gene Expression . . . . . . . . . . . . . . . . . . . . 441

Alexandra Fischer and Josef Pallauf
22. Modulation of Gene Expression by Dietary Zinc . . . . . . . . . . . . . 457

Raymond K. Blanchard and Robert J. Cousins
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
1
Application of Nutrigenomics Tools
to Analyze the Role of Oxidants
and Antioxidants in
Gene Expression
Gerald Rimbach
Christian Albrechts University, Kiel, Germany
Sonia De Pascual-Teresa
Instituto del Frı́o, Consejo Superior de Investigaciones Cientificas,
Madrid, Spain
What is Nutrigenomics? 1
Do Oxidants and Antioxidants Affect Gene Expression? 3
Methods and Applications in Nutrigenomics 4
References 9
WHAT IS NUTRIGENOMICS?
The rapid progress in the understanding of the human genome has opened up
new avenues to study interactions between diet, gene expression, genetic varia-
bility, health, and disease. Nutrigenomics considers the relationship between
specific nutrients or diet and gene expression (1), whereas nutrigenetics deter-
mines how genetic variability affects the response to diet (2). Nutrigenomics
is a modern discipline at the interface between genetics, molecular nutrition,
molecular biology, pharmacogenomics, and molecular medicine (Fig. 1.1).
1
2 Rimbach and De Pascual-Teresa
Figure 1.1 Nutrigenomics as a discipline at the interface between genetics, molecular

nutrition, molecular biology, pharmacogenomics, molecular medicine, and bioinformatics.
Nutrigenomics applies high-throughput molecular biology techniques inclu-

ding sequencing and genotyping (genomics), transcriptomics, proteomics, and
metabolomics.
Transcriptomics determines patterns of gene expression in response to a
nutrient, whereas proteomics studies the effect of nutrients on protein synthesis,
protein structure, and patterns of protein expression. The profile and function of
metabolites are analyzed by metabolomic techniques, whereas the comprehen-
sive data handling is finally accomplished by bioinformatic tools (Fig. 1.2). By
combining information from transcriptomics, proteomics, metabolomics, and
bioinformatics one can gain a comprehensive understanding of nutrient related
homeostasis.
Figure 1.2 Genomics, transcriptomics, proteomics, and metabolomics as analytical

tools in molecular nutrition.
Application of Nutrigenomics 3
DO OXIDANTS AND ANTIOXIDANTS AFFECT GENE EXPRESSION?

It has been clearly shown that the cellular oxidant/antioxidant equilibrium is a
key factor in determining redox-dependent signal transduction pathways both
in vitro and in vivo. Antioxidant nutrients interact with cell receptors (e.g., isofla-
vones bind to estrogen receptor alpha and beta) and modulate key enzymes such
as phosphatase and kinases. Changes in transcription factor activity leads to
changes in mRNA and protein levels as summarized in Fig. 1.3. Furthermore,
antioxidants can directly interact with enzymes (e.g., through protein-binding
properties), thereby changing their activity.
Molecular and cell biology has changed our understanding of how antiox-
idants can mediate their biological properties. A good example is vitamin E—the
most important lipid-soluble antioxidant. Since its discovery, studies of the con-
stituent tocopherols and tocotrienols have focused mainly on their antioxidant
properties. In 1991, Angelo Azzi’s group first described nonantioxidant, cell
signaling functions for vitamin E, demonstrating that vitamin E regulates
protein kinase C activity in smooth muscle cells (3). At the transcriptional level,
vitamin E modulates the expression of the hepatic a-tocopherol transfer protein
(TTP) (4) as well as the expression collagenase gene (5) and a-tropomyosin gene
(6). Recently, a tocopherol-dependent transcription factor (tocopherol associated
protein, TAP) has been discovered (7). In cultured cells, it has been demonstrated
that vitamin E inhibits inflammation, cell adhesion, platelet aggregation, and
smooth muscle cell proliferation (8). Many of these cellular functions of
vitamin E seem to be independent of its antioxidant properties. Thus, antioxidants
do not act only as scavengers of reactive oxygen and nitrogen species, thereby
Receptors
Phosphatase
Kinases
Transcription
Factors
Oxidants Antioxidants
Gene Expression
Protein levels
Enzyme Activity
Figure 1.3 Cell receptors, cellular key enzymes, and transcription factors as molecular
targets of oxidants and antioxidants.
preventing oxidative damage towards lipids, protein, and DNA, they are also
cell signaling molecules. Both the free-radical scavenging as well as the cell
signaling activity of antioxidants may contribute to their potential beneficial
effects in preventing atherogenesis, carcinogenesis, and neurodegneration
(Fig. 1.4).
Various transcription factors such as NF-kB, AP-1, Nrf-1, and SP-1 are
regulated by the cellular redox status. NF-kB controls the expression of different
genes involved in inflammatory and proliferative responses. A spectrum of key
genes known to be involved in the development of atherosclerosis have been
shown to be regulated by NF-kB, including those encoding for cytokines,
chemoattractants, and cell adhesion proteins (8). Several lines of evidence includ-
ing the inhibition caused by various antioxidants, suggest that NF-kB is subject to
redox regulation. Owing to its pivotal role in inflammation and atherogenesis, a
significant effort has focused on identifying nutrients that regulate NF-kB
activity. In this scenario, flavonoids may play an important role, either by directly
affecting key steps in the activation pathway of NF-kB, or by modulating the
intracellular redox status, which is, in turn, one of the major determinants of
NF-kB activation. Consistent experimental data is accumulating, which suggests
that the anti-inflammatory properties of flavonoids are in part due to their ability
to down-regulate NF-kB (9).
METHODS AND APPLICATIONS IN NUTRIGENOMICS

There is an increasing evidence indicating that antioxidants such as vitamin E,
carotenoids, ascorbic acid, lipoic acids, bioflavonoids, and ginkgo biloba as
well as trace elements (e.g., Zn, Fe, Se) affect differential gene expression in
Figure 1.4 Antioxidants as free-radical scavengers, metal chelators, and redox signaling
molecules—both prevention of oxidative damage towards lipids, proteins, and DNA as
well as redox signaling contributes to their potential beneficial effects.
Table 1.1 Studies on the Effect of Oxidants and Antioxidants on Differential Gene Expression in Cultured Cells, Laboratory Animals and
in Humans
Number of genes
Cell/Tissue Species monitored Reference
Oxidants
Cigarette smoke Swiss 3T3 Mouse 513 Bosio et al. (10)
Hydrogen peroxide, menadione Breast cancer cells Human 17,000 Chuang et al. (11)
t-butyl hydroperoxide
Hydrogen peroxide Retinal pigment epithelium cells Human 1,176 Weigel et al. (12)
Application of Nutrigenomics
4-hydroxynonenal
t-butyl hydroperoxide
Oxidized LDL Aortic smooth muscle cells Human 35,932 Sukhanov et al. (13)
Endothelial cells Human 588 Virgili et al. (14)
Ozone Lung Mouse 4,000 Gohil et al. (15)
UVB radiation Keratinocytes Human 6,000 Sesto et al. (16)
Antioxidants
Ascorbic acid Keratinocytes Human 588 Catani et al. (17)
Coenzyme Q10 Skeletal muscle Human 12,000 Linnane et al. (18)
Copper Macrophages Human 6,800 Svensson et al. (19)
Epigallocatechin-3-gallate Cervical cancer cells Human 384 Ahn et al. (20)
Lung cancer cells Human 588 Fujiki et al. (21)
Lung cancer cells Human 588 Okabe et al. (22)
Prostate carcinoma cells Human 250 Wang and Mukhtar (23)
Epigallocatechin-3-gallate, melatonin Neuroblastoma cells Human 25 Weinreb et al. (24)
Fish oil Brain Rat 3,200 Kitajka et al. (25)
Liver Mouse 6,521 Takahashi et al. (26)
5
(continued )
Table 1.1 Continued 6
Number of genes
Cell/Tissue Species monitored Reference
Folic acid Nasopharyngeal carcinoma cells Human 2,200 Jhaveri et al. (27)
Ginkgo biloba Brain Rat 8,000 Li et al. (28)
Brain Mouse 7,000 Watanabe et al. (29)
Genistein Prostate cancer cells Human 557 Suzuki et al. (30)
Indole-3-carbinol Prostate cancer cells Human 22,215 Li et al. (31)
Lycopene, Vitamin E Prostate Rat 7,000 Siler et al. (32)
Melatonin Retina Rat 24,000 Wiechmann (33)
Methylseleninic acid Premalignant breast cells Human 316 Dong et al. (34)
Proanthocyanidin extract Endothelial cells Human 2,400 Bagchi et al. (35)
from grape seed
Procyanidins from pine bark Keratinocytes Human 588 Rihn et al. (36)
Resveratrol Prostate cancer cells Human 2,400 Narayanan et al. (37)
Selenium Mammary epithelial organoids Rat 588 Dong et al. (38)
Intestine Mouse 6,347 Rao et al. (39)
Sulphoraphane Small intestine Mouse 6,000 Thimmulappa et al. (40)
Vitamin A Airway tissues Human 30,000 Soref et al. (41)
Vitamin A and E, selenium Skeletal muscle Rat 800 Sreekumar et al. (42)
Vitamin D3 Osteosarcoma cells Rat 5,000 Farach-Carson and Xu (43)
Prostate cancer cells Human 20,000 Krishnan et al. (44)
Kidney Mouse 12,422 Li et al. (45)
Vitamin E Liver Rat 7,000 Barella et al. (46)
Aortic smooth muscle cells Human 10,000 Villacorta et al. (47)
Vitamin E (Tocotrienol) Fetal brains Rat 8,000 Roy et al. (48)
Vitamin E and Selenium Liver Rat 465 Fischer et al. (49)
Zinc Mucosa cells of small intestine Rat 1,185 Blanchard et al. (50)
Rimbach and De Pascual-Teresa
Liver Rat 2,500 tom Dieck et al. (51)

cultured cells, in laboratory animals, and in humans. In addition, oxidative stress,

induced by oxidants such as ozone, cigarette smoke, UV radiation, and oxidized
LDL, is associated with changes in differential gene expression. Recent studies
on the effect of oxidants and antioxidants on differential gene expressions are
summarized in Table 1.1.
The potential applications of transcriptomics in the field of antioxidant and
free-radical research are manifold. Several methods have been developed for the
quantitative and comprehensive analyses of changes in mRNA expression such
as differential display, serial analysis of gene expression, DNA microarrays,
and gene chips (Fig. 1.5). Gene-arrays have been used in order to analyze
redox-sensitive signal transduction pathways, thereby getting more insights
into the molecular functions of oxidants and antioxidants. A novel application
of gene-array technology may be the development of new biomarkers of
oxidative stress, which is still an open issue. Furthermore, differences in the bio-
potency and bioavailability among different antioxidants may be detected by
gene-array technology. Finally, gene-arrays can be applied in order to study the
toxicity of oxidants and antioxidants as well as for screening new antioxidants.
Once candidate genes have been identified by array technology, the corre-
sponding mRNA sequence has to be obtained from a data base (e.g., gene bank),
and a blast of the sequence against the genome is performed in order to retrieve
the DNA sequence, chromosomal loci, as well as the upstream sequence. This
procedure is followed by a search for homology in regulatory elements and tran-
scription factors (52) as summarized in Fig. 1.6.
Figure 1.5 Analytical techniques and potential applications of transcriptomics in the

field of free-radical research.
Identify Candidate Genes by

Gene Chip Technology
Obtain mRNA Sequences from Data Base (e.g.

Gene Bank) and Blast Sequence against Genome
Retrieve DNA Sequences, Chromosomal Loci

and 10Kb Upstream Sequence
Search for Homology in Regulatory Elements and

Transcription Factors
Describe potential signal transduction pathways
Figure 1.6 Major steps in the identification of redox-sensitive signal transduction

pathways.
In order to obtain a comprehensive understanding of the molecular

mechanisms of action of oxidants and antioxidants, rigorous study designs and
statistical analysis are necessary. Time-point measurements need to be intro-
duced in order to elucidate whether differences in the gene expression profile
are manifested consistently over a prolonged period of time. In previous
studies, differential changes in gene expression in response to dietary treatments
Treatment Target tissue RNA

(e.g. antioxidant) (e.g. rat liver) Extraction
Gene chip Biotinylated cDN A

hybridization cRN A Synth esis
I mage An alysis/ Identification of Verification by

Bioinformatics C andidate Genes R T -PCR/
Nor thern blotting
Functional Verification by
Endpoint Western blotting/
Measurements ELISA/Proteomics
Figure 1.7 Schematic representation of the analytical steps involved in a gene chip
experiment.
were often monitored only at one time-point and in pooled samples. Furthermore,
differences in gene expression levels observed by gene chips technology should
always be confirmed by independent methods such as real-time PCR and northern
blotting, and then substantiated by functional parameters (Fig. 1.7). A set of
guidelines (Minimum Information About a Micorarray Experiment, MIAME)
have been established to outline the minimum information required for micro-
array experiments.
Overall, gene expression profiling using array technology is rapidly becom-
ing an important tool in nutrition and free-radical research. Array technology
enables to study nutrient gene interactions on large scale that would be imposs-
ible using conventional analysis. Nutrigenomics has the potential to validate and
to extend many of the strategies used in human nutrition on a molecular level,
thereby improving human health.
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2
Oxidative Stress and Human
Genetic Variation
R. D. Gill-Garrison, J. L. Slater, and K. Grimaldi

Sciona, Ltd., Havant, UK
Manganese Superoxide Dismutase 15

MnSOD Background 15
MnSOD Polymorphisms 16
MnSOD and Health Conditions 17
Late-Onset Neurological Disorders 17
Cancer 17
Copper, Zinc Superoxide Dismutase 18
SOD3 Background 19
SOD3 Polymorphisms 19
The Glutathione-S-Transferases 19
The GST Genes 20
GSTM1—Glutathione-S-Transferase M1 20
GSTM1 Background 20
GSTM1 Biomarkers 21
GSTM1 and Health Conditions 21
Glutathione-S -Transferase P1 22
GSTP1 Background 23
GSTP1 Biomarkers 23
GSTP1 and Health Conditions 23
Glutathione-S-Transferase T1 24
GSTT1 Biomarkers 24
13
14 Gill-Garrison, Slater, and Grimaldi
GSTT1 and Health Conditions 24

Cancer 24
Endothelial Nitric Oxide Synthase 25
eNOS Summary 25
Functional Studies of the 894G . T (Glu298Asp) Polymorphism 25
eNOS Glu298Asp (G . T) and Cardiovascular Disease 26
eNOS and Oxidative Stress of Smokers 26
Potential Dietary Interventions 26
Catalase 27
CAT Background 27
CAT Polymorphisms 27
NADPH Oxidase 28
NADPH Oxidase Background 28
NADPH Polymorphisms 28
NADPH Polymorphisms and Health Conditions 28
Glutathione Peroxidase 29
GPX1 Background 29
GPX1 Polymorphisms 29
GPX1 Polymorphisms and Health 29
Myeloperoxidase 30
MPO Background 30
MPO Polymorphisms 30
NADPH Dehydrogenase, Quinone 1 31
NADPH Background 31
NADPH Polymorphisms 31
Conclusion 31
References 32
Oxidative stress has been attributed to a host of human disorders ranging from
cancer to premature aging. Although the role of oxidative stress in damaging
tissues ultimately leading to disease or disrepair is widely accepted, science is
progressing rapidly in terms of understanding the impact of interindividual vari-
ation in genes responsible for dealing with oxidative stress in cells. As molecular
techniques have advanced and the technology has become more accessible, it has
become much easier to include analysis of genetic variation in epidemiological
studies as well as biochemical studies modeling particular genetic variations.
Single nucleotide polymorphisms (snps) are the most common variations that
exist in the human genome, occurring at 1500 bp intervals in the human
genome. Snps are considered distinct from rare mutations in that a snp by
definition must occur in at least 1% of the population. Many of these sequence
Oxidative Stress and Human Genetic Variation 15
alterations may occur in noncoding regions of the genome, and may have no
significance, others may lead to alterations in the amino acid sequence of the
gene product, leading to a functional change in the protein, still others may
alter the promoter region, thus having an impact on efficiency of transcription
of the gene. Often, snps are identified through molecular epidemiological
methods that do not have a clear functional effect, and these variations are
assumed to be in linkage disequilibrium with some other, yet unidentified func-
tional variation. Other variations that can occur include insertions and deletions,
in one common example, the complete sequence of the GSTM1 gene, for
example, is deleted in 50% of the Caucasian population. Useful online
resources for studying human variation include the Database of Single Nucleo-
tide Polymorphisms http://www.ncbi.nlm.nih.gov/SNP, GeneCards http://
bioinformatics.weizmann.ac.il/cards, the Human Gene Mutation Database
http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html, OMIM (Online Mendelian
Inheritance in Man) http://www.ncbi.nlm.nih.gov/OMIM, and the SNP consor-
tium http://snp.cshl.org.
This chapter is an overview of several of the genes associated with antiox-
idant activity and the most common variations found within those genes. Associ-
ations of the variations with disorders often attributed to oxidative stress are
included, as well as dietary interventions, where possible, which demonstrate
an altered response in the population in epidemiological studies.
MANGANESE SUPEROXIDE DISMUTASE
Gene: MnSOD—SOD2—Manganese superoxide dismutase

Polymorphisms: C(228)T—Ala(29)Val
T175C—Ile58Thr
. Function: Destroys radicals which are normally produced within the
cells and which are toxic to biological systems
. Biochemical activity: 2 superoxide þ 2Hþ ¼ O2 þ H2O2
. Cofactor: Manganese
. Structure: Homotetramer
. Location: Mitochondria
. Population frequency: C(228)T 50% Caucasian, 12% Japanese
MnSOD Background
MnSOD, also known as SOD2, is an enzyme that acts as a free-radical scavenger.
The SOD2 gene is located on chromosome 6 in humans (region 6q25), with
5 exons and 4 introns, spanning 11,091 bp (1,2) and the active enzyme is gene-
rally found within the mitochondria of cells. This is the site of many oxidative
reactions; thus, free radicals are generated here which can cause damage to the
DNA, proteins, cell membranes, and the mitochondrion itself. The MnSOD
enzyme converts the superoxide free radical into hydrogen peroxide at near dif-
fusion limiting rates at the catalytic manganese site (3). The active MnSOD
enzyme is a homotetramer with the four subunits each contributing to the for-
mation of the catalytic site (4). Knock-out mice, in which the mouse equivalent
gene was removed, died within 10 days of birth with dilated cardiomyopathy,
accumulation of lipid in the liver and skeletal muscle, and metabolic acidosis,
leading to the conclusion that the MnSOD is a vital enzyme, necessary for life (5).
MnSOD Polymorphisms
There have been two polymorphisms described for this gene, the Ala(29)Val
polymorphism and the Ile58Thr polymorphism. The Ala(29)Val polymorphism
was discovered by cloning the MnSOD gene derived from patients with diseases
of premature aging, progeria, Werner syndrome, and Cockayne syndrome; and
comparing them with normal samples. The polymorphism consists of a single
nucleotide change; a cytosine is replaced by a thymine, leading to the substitution
of a valine (Val) amino acid in place of an alanine (Ala) in the region of the gene
known as the signal sequence, which directs the enzyme into the mitochondria.
The authors hypothesized that these diseases of premature aging may be due to
a so-called disease of distribution, in which an essential enzyme is not targeted
to the proper location in the cell, leading to deficiencies of critical enzymes in
specific locations within the cell. In this case, the movement of the enzyme
within the cell, rather than its actual activity is defective (6). Further studies of
the signal sequence demonstrated that the Ala allele codes for the ideal helical
protein structure that would allow maximal transport into the mitochondria,
and this helical structure is disrupted when valine is present (7).
Most of the recent research on the MnSOD Ala(29)Val polymorphism has
focused on epidemiological data; few studies have measured function or bio-
markers associated with this polymorphism. However, one study has measured
a reduction of processing into the mitochondria of 11% when the Val residue
is present (8). Earlier work determined that the signal sequence is essential for
protection of mitochondria from ionizing radiation (9). One study has measured
the formation of 8-hydroxy-deoxyguanosine adducts, an indicator of oxidative
stress, in term pregnant women; the investigators found higher levels of
adducts associated with the Ala(29) allele (10).
The population distribution for the Ala(29)Val is random (50%) for the
Caucasian population of North America and Western Europe (6), whereas the
occurrence in the Japanese population is much lower at 12% (7). This has led
to some confusion in the literature, with some investigators referring to the poly-
morphism as Val(29)Ala; however, the convention used on the major human
sequence databases such as EMBL and OMIM is that of Ala(29)Val which
reflects the functionality of the polymorphism.
The second polymorphism identified, Ile58Thr, occurs much more rarely in
the population, and there are no studies to date that have measured the occurrence
in particular ethnic groups. This polymorphism occurs at a position that causes

two packing defects in the assembly of the four subunits into the active tetrameric
enzyme. The defect causes a reduction in stability of the enzyme; studies in vitro
have shown the polymorphic enzyme to exist in a dimeric form in solution (with
two subunits only) due to a lack of thermostability. Predictions are that the
enzyme would be even less stable at normal body temperatures (4). Further
studies of cells in culture demonstrated that the wild-type (Ile) enzyme had
three times the activity of the polymorphic (Thr) enzyme (11).
MnSOD and Health Conditions

Late-Onset Neurological Disorders
The Ala(29)Val polymorphism has been associated with increased risk of occur-
rence in several disorders, including idiopathic dilated cardiomyopathy (9) and
diabetic neuropathy. The Val allele has been implicated in early studies of
Parkinson’s disease (7) and progeria-type diseases (6); however, further studies
have not found a link with Parkinson’s disease (12,13). The contribution of this
polymorphism to these late-onset neurological diseases has been suggested to
be due to the accumulation of mitochondrial oxidative damage in the absence
of adequate levels of MnSOD enzyme activity (14). A Chinese study has reported
a synergistic effect of the Val allele together with the ser9 allele of the dopamine
receptor (DR3) ser9gly polymorphism in the development of tardive dyskinesia in
a population of patients with schizophrenia, suggesting a link between increased
mitochondrial oxididative stress and dopamine receptor function in the onset of
tardive dyskinesia (15). The Ala allele has been associated with increased risk
of other conditions including age-related macular degeneration with an odds
ratio of 10.14 (16), as well as motor neurone disease, with an odds ratio of 2.9
for the homozygous Ala allele, which increased to an odds ratio of 5.0 when
females alone were considered (14), and severe alcoholic liver disease, with a
6 –10-fold increase in cirrhosis and alcoholic hepatitis (17).
Cancer
The Ala allele has been associated with increased risk of developing certain
forms of cancer. This association is particularly interesting because it is the
Ala allele that has the signal sequence intact, and should have the most efficient
transport into the mitochondria. A study by Ambrosone et al. in 1999 demon-
strated an increased risk of breast cancer (odds ratio 4.3) in premenopausal
women with homozygous Ala alleles. In this study, levels of fruits and vegetables
in the diet were also measured, and the odds ratio for premenopausal women with
the lowest consumption of fruits and vegetables increased to 6.0, whereas the
odds ratio for those with the highest consumption was lower, at 3.2. When vitamin
supplementation was considered, the risk for premenopausal women with the
homozygous Ala genotype was only observed among women who did not take
supplements of vitamin C (odds ratio 4.8) and a-tocopherol (odds ratio 3.8).
Premenopausal women taking supplements did not show increased risk with the
homozygous Ala allele. (18). The link between breast cancer and the MnSOD
Ala allele has been repeated in another study, the odds ratio reported was 1.5;
however, there was no dietary information included in the study (19). A further
study carried out by Egan et al. (20) has given limited support to this association;
the odds ratio reported were much lower; however, there was no data reported on
antioxidant supplementation to contribute to the analysis, which prevents direct
comparison with the previous study. Further evidence has been presented which
demonstrated a link between the Ala allele, dietary consumption of antioxidants,
and risk of ovarian cancer. In this study, women with low intake of antioxidants
and the Ala allele were at increased risk of ovarian cancer, with an odds ratio of
2.6 (21). These studies have provided a potential link between consumption of anti-
oxidants and reduction of risk associated with a specific genotype.
A study of colorectal cancer in young individuals (below the age of 40)
found the frequency of the Ala allele was greatly increased in patients with the
disease, the authors calculated an odds ratio of 7.5 for the homozygous Ala
allele in individuals under 40, and an odds ratio of 1.5 for Ala/Val heterozygotes.
The authors suggest that this polymorphism may be a useful marker to identify
young individuals at a high risk of developing colorectal cancer (22) and have
filed a US patent covering the use of a genetic test of the MnSOD gene poly-
morphism to screen for colon cancer risk in this population (see http://www.
usc.edu/academe/otl/3015w.htm). Similar results were reported in a study of
colorectal cancer in a Hispanic population (23). The Ala allele has also been
associated with elevated prostate cancer risk (24,25).
Not all studies have shown positive correlations however, which may indi-
cate differences in tissue specificity, age-related differences, or other mechanistic
contributions to the etiology of cancer. Also, the studies rarely include dietary
information, which has been shown to have an impact on the incidence of breast
cancer in women (18). Negative results have been reported recently in distal color-
ectal adenomas in men aged 50–74 (26), and in lung cancer risk in Taiwan (27).
COPPER, ZINC SUPEROXIDE DISMUTASE
Gene: SOD3—CuZn SOD—SOD3—Copper, Zinc superoxide dismutase—

CuZn SOD
Polymorphisms: C760G Arg213Gly
. Function: Destroys free radicals
. Biochemical activity: 2 superoxide þ 2Hþ ¼ O2 þ H2O2
. Cofactors: Copper and zinc
. Location: Extracellular
. Population frequency of polymorphisms: 3 – 4% Caucasian
SOD3 Background
EC-SOD or SOD3, the extracellular form of SOD, is found in plasma, lymph, and
synovial fluid as well as in tissues. SOD3 is the major antioxidant enzyme system
of the vessel wall of the cardiovascular system. It is a tetrameric glycoprotein
with an apparent subunit molecular weight of 30,000 Da. Like the CuZn
SOD (SOD1), SOD3 contains one Cu2þ and one Zn2þ ion per subunit;
however, the amino acid compositions of SOD1 and SOD3 are quite different
and no cross-reactivity is observed in immunologic studies. SOD3 also has a
different tissue distribution than SOD1. The SOD3 enzyme is synthesized with
a putative 18-amino acid signal peptide preceding the 222 amino acids in the
mature enzyme, indicating that the enzyme is a secretory protein. The primary
characteristic distinguishing SOD3 from SOD1 and SOD2 is the heparin-
binding capacity of SOD3. SOD3 binds on the surface of endothelial cells
through the heparan sulfate proteoglycan and eliminates the oxygen radicals
from the NADP-dependent oxidative system of neutrophils (28,29).
SOD3 Polymorphisms
One SOD3 SNP that has been studied extensively, Arg213Gly, results in the
accelerated release of the enzyme from the tissue interstitium into the plasma
and is accompanied by reduced tissue SOD3 activities. The Arg21Gly SNP
was discovered by Adachi et al. (29), who developed an immunoassay system
for SOD3 in order to measure SOD3 levels in the serum of healthy subjects.
They found that 6% of these persons had an SOD3 level that was 10 –15-fold
higher than the mean SOD3 level in all subjects, which was familial in nature
(30). Sandstrom et al. (31) reported that 2% of the plasma donors in Sweden
had an 8– 10-fold higher SOD3 level and that a single base substitution of C to
G at position 760 of the cDNA was responsible for the high level in plasma.
The polymorphism is located in the region associated with the heparin affinity
of the enzyme. The resulting amino acid substitution may result in a decrease
of heparin affinity which favors the presence of SOD3 in the serum; thus, the
high plasma activity can be explained by an accelerated release from the tissue
interstitium heparan sulfate to the vasculature which is accompanied by signifi-
cantly reduced tissue SOD3 activities. Marklund (32) observed the frequency
of the Arg213Gly variant was 3.8% in the Northern Swedish population. Their
data also suggested that plasma levels of the wild-type form of SOD3 may be
modulated by lifestyle factors, such as smoking, and show a complex covariation
with many of the conventional cardiovascular risk factors.
THE GLUTATHIONE-S-TRANSFERASES
The enzymes belong to a super-family with broad and overlapping substrate spe-
cificities. The active enzymes exist as dimeric structures and catalyze conjugation
reactions between glutathione and aromatic radicals and epoxides. The active
enzymes contain a highly conserved G-site, which is the binding site for gluta-
thione, and a divergent H-site, which provides a site for binding of electrophiles
(33). The majority of the conjugation activity is believed to occur in the liver,
with a transfer of intermediates into the kidney for excretion. Glutathione-S-
transferases provide a major pathway of protection against chemical toxins and
carcinogens, as well as reactive oxygen species, and are thought to have
evolved as an adaptive response to environmental insult, thus accounting for
their wide substrate specificity (34). There are four family members: A, M, T,
and P. Polymorphisms have been identified in each family (35).
These enzymes catalyze reactions in which the products of Phase I metab-
olism are conjugated with glutathione, thus making them more water soluble and
more easily excreted from the body. When the activity of the enzymes is
enhanced, the products of Phase I metabolism should be excreted more rapidly
from the body, and therefore less likely to interact with the DNA and proteins
of the cells.
Individuals with polymorphisms in the GST Phase II detoxification genes
have a decreased rate of detoxification, with a corresponding increase in levels
of carcinogen—DNA adduct formation and also an increased level of chromoso-
mal aberrations (36,37). Cruciferous vegetables, such as broccoli, and members
of the allium family, such as garlic and onion, have been shown to be potent
inducers of these enzymes, which would be expected to increase clearance of
potential toxins from the body (38,39).
THE GST GENES

GSTM1—Glutathione-S-Transferase M1
Gene: GSTM1—Glutathione-S-transferase mu, M1

Polymorphisms: Gene deletion
. Function: Conjugation of glutathione to hydrophobic compounds
. Biochemical activity: RX þ glutathione ¼ HX þ R-S-glutathione
. Structure: Homodimer
. Location: Cytoplasm of the cell, in the liver
. Population frequency: 50% Caucasian
GSTM1 Background
The GSTM1 subtype has the highest activity of the four types of GSTs and is
predominately located in the liver (34). Approximately half of the Caucasian
population has a complete deletion of this gene (35,40). There have been
reports of two alleles in the literature and on public domain websites:
GSTM1A and GSTM1B reported to differ in position 172; however, this is an
error that has arisen due to homology with GSTM4 (41).
GSTM1 Biomarkers
The effects of diet on activity of the GST enzymes have been demonstrated in
several studies. For example, in a feeding study with healthy volunteers, activity
of GSTA and GST serum levels were measured in GSTM1(þ) and GSTM1(2/2)
subjects. Brassica vegetable diets increased GSTA by 26% and GST serum
activity by 7% in the GSTM1-null individuals, particularly in women. Among
the GSTM1(þ) women, GSTM activity was increased by both brassica (18%)
and the allium (26%) diets (42).
Palli et al. (43) have previously shown that that DNA adducts, a reliable
indicator of genotoxic damage and, possibly, of cancer risk, are modulated by
plasma levels of selected micronutrients. In a follow-up study, stratification by
GSTM1 genotype showed strong inverse associations of DNA adduct levels
with increasing consumption of vegetables, particularly raw and leafy vegetables,
as well as vitamins E and C. In contrast, no associations were found among 295
GSTM1 wild-type individuals. The authors conclude that the results suggest that
the role of a diet rich in antioxidants in preventing or reducing DNA adduct
formation is restricted to subjects lacking the detoxifying activity of GSTM1
isoenzyme (50% of the general population) (44).
Plasma autoantibodies (aAbs) against the oxidized DNA base derivative
5-hydroxymethyl-20 -deoxyuridine (5-HMdU) are potential biomarkers of cancer
risk and oxidative stress. Current smokers lacking GSTM1, particularly men,
have shown greater aAb titers compared with nonsmokers or persons with
intact GSTM1 (45).
GSTM1 and Health Conditions
Cancer: There have been a host of studies examining the association
between GSTM1-null individuals and increased cancer risk, with many studies report-
ing positive finding (46). The following is a brief synopsis of studies that have inves-
tigated cancer incidence, GSTM1 status, and consumption of isothiocyanates, which
are known to increase the activity of glutathione-S-transferase enzymes; however,
these compounds can also be conjugated and thus removed from the body by GST
enzymes. Isothiocyanates are contained in cruciferous vegetables, and have been
shown to have chemopreventive effects in laboratory animals. The investigators
found that isothiocyanates appeared to reduce the risk of lung cancer in Chinese
men, with reduction of risk more pronounced in individuals with homozygous
GSTM1-null genotype, odds ratio 0.36, and more so if both GSTM1 and GSTT1
were deleted, odds ratio 0.28 (47). Similar results were described in studies of lung
cancer (48), colorectal adenomas (49, 50), and head and neck cancer (51).
Lung disease: Ozone is a powerful oxidant associated with impairment
of pulmonary function and increased airway inflammation. Romieu et al.
studied 158 asthmatic children in Mexico city, an area with high ozone exposure
and looked at the effect of GSTM1 genotype and antioxidant supplementation
(vitamins C and E). The GSTM1-null genotype was present in 39% of the
children. In those given placebo, the deleterious effect of ozone on forced expira-
tory flow was worse in GSTM1-nulls than in those carrying the gene. In all chil-
dren receiving antioxidant supplementation, there was no decrease suggesting
that the supplementation abolished the effect of the null genotype (52). A
recent report found that individuals with GSTM1-null or GSTP1 Ile105 wild-
type genotypes showed enhanced nasal allergic responses to diesel-exhaust par-
ticles. The GSTM1-null individuals had a significantly larger increase in IgE and
histamine in nasal lavage fluid after challenge with diesel-exhaust particles or
allergen than children with a functional GSTM1 genotype (53). The data was
highly reproducible between individuals and provides additional evidence sup-
porting the concept of individual sensitivity to air pollution (54,55).
Other disorders: Focusing on the antioxidant activity of GSTM1 and
publications of the last year, one study has compared levels of 8-hydroxy
guanine adducts, GSTM1 and GSTT1 genotypes in glaucoma patients. The glau-
coma patients had higher levels of DNA adducts than healthy controls, and there
were more GSTM1-null primary open angle glaucoma patients than healthy con-
trols. The authors conclude that oxidative damage is increased in glaucoma
patients, particularly in the trabecular meshwork, and that the GSTM1-null gen-
otype predisposes glaucoma patients to more severe oxidative DNA damage (56).
Other reports include increased biomarkers of lung inflammation in GSTM1
individuals exposed to ozone (57), protection against loss of hearing owing to
oxidative stress in cochlear tissue of workers exposed to work-place noise for
GSTM1(þ) individuals (58), and increased levels of 8-OH guanine adducts in
pregnant GSTM1 women, also in MnSOD variant women (11).
A study of patients with acute myocardial infarction (AMI) found that, in con-
trast to the other disorders described in this chapter, the GSTM1 positive allele con-
ferred an increased risk, with significant differences more pronounced in smokers
(59). The GSTM1-null allele was also found to have a protective effect in the devel-
opment of cortical cataracts in an Estonian population. The GSTM1(þ) genotype
was shown to confer risk in this study, with an odds ratio of 1.88 (60). Further
study is required to determine the mechanism for this paradoxical effect.
Glutathione-S-Transferase P1
Gene: GSTP1—Glutathione-S-transferase P1, pi

Polymorphisms: A313G—Ile105Val
C341T—Ala113Val
. Location: Cytoplasm of the cell, located in the lungs
. Population frequency of polymorphisms: A313G, 50% Caucasian,
C341T, 10% Caucasian
GSTP1 Background
The GSTP1 gene is known to metabolize many carcinogenic compounds and is
the most abundant subtype in the lungs (34). Two snps have been reported to
date. The first is GSTP1 B (Ile/Val), with a single nucleotide change at position
313 in the DNA, leading to the substitution of a valine (Val) amino acid in place
of an isoleucine (Ile) at position 105 of the enzyme. The second is GSTP1 C, with
the previous change, together with a single nucleotide change at position 341 of
the gene leading to the substitution of a valine residue for an alanine at position
113 of the enzyme (Ala/Val). The latter polymorphism may also occur on its
own, the current designation is GSTP1 D. The enzymes of these polymorphic
genes have decreased activity compared with the wild-type due to changes in
the active pocket of the enzyme (61 – 63) and non-Hodgkin’s lymphomas (64).
GSTP1 Biomarkers
Vegetable juice has been shown to induce the expression of GSTP1 (65). The
results of five large intervention studies showed considerable induction of
GSTP activity after consumption of Brussels sprouts and red cabbage (66). A
further study has identified a component of Japanese horseradish wasabi,
6-methylsulfinylhexyl isothiocyanate (6-HITC), as potent inducer of GSTP
enzymes (67).
GSTP1 enzyme activity is reduced by the presence of the polymorphisms,
reduction in conjugation activity was 82% of wild-type for the heterozygous
Ile/Val(105), and 70% for the homozygous Val/Val(105) (68); these results cor-
respond with previous studies carried out in vitro (69). A study of DNA adducts
resulting from oxidative damage in breast cancer tissue found lower levels of
DNA adducts in patients with the wild-type Ile/Ile allele (70). A study that exam-
ined combinations of GSTM1 and GSTP1 found the highest level of DNA adducts
in smokers with one or two copies of the Ile/Val polymorphism, together with the
GSTM1(2/2) genotype (37).
GSTP1 and Health Conditions
Val allele associations: The GSTP1 Val polymorphism has been associ-
ated with an increased risk of developing prostate cancer (71,72), lung cancer
(27,73), and recurrent early pregnancy loss (74).
Asthma: The Val/Val genotype was found to be protective against
asthma (75). The Ile/Ile was more prevalent in cases of atopy with increasing
severity of airflow obstruction and bronchial hyper-responsiveness, implying a
protective effect of the Val/Val polymorphism (76).
Ile allele associations: The Ile allele was associated with greater risk of
severe disability in patients with muscular dystrophy of duration greater than 10
years (77), squamous cell carcinoma of the esophagus (78). A recent publication
which examined dietary patterns using cluster, as well as genetic polymorphisms
in both GSTP1 and GSTM1 alleles found that smokers that were heterozygous for
the Ile/Val allele and a “healthy” diet had a lower risk of lung cancer than indi-
viduals homozygous for the Ile allele with an “unhealthy” diet, OR ¼ 0.16 (79).
The diets were categorized using four major dietary constituents, including
carbohydrate, fat, protein, and fiber. Healthy diets were considered low in
protein and fat and high in carbohydrate and fiber, whereas unhealthy diets had
high levels of protein and fat, with low levels of carbohydrate and fiber.
GLUTATHIONE-S-TRANSFERASE T1
Gene: GSTT1— Glutathione-S-transferase theta, T1

Polymorphism: Gene deletion
. Location: Cytoplasm of the cell, located in variety of tissues
. Population frequency: 20% Caucasian, 80% Asian
GSTT1 is considered one of the most ancient forms of glutathione-S-

transferases. GSTTs are found in mammals, fish, plants, and bacteria (80). The
GSTT1 gene is deleted in 20% of the Caucasian population and 80% of the
Asian population (80). The enzyme is found in a variety of tissues, including
red blood cells, liver, and lung (81), and the liver and kidney have the highest
expression of GSTT1 (80).
GSTT1 Biomarkers
Dietary isothiocyanates have been shown to induce GSTT1 (82). A study of poly-
cyclic aromatic hydrocarbon-induced chromosomal breaks revealed a higher
levels of breaks associated with GSTT1(2/2) genotypes (83). Lymphocytes
from GSTT1(2/2) individuals are also more prone to strand breaks induced
by butadiene (84). A study of DNA adducts in smokers revealed higher levels
of DNA adducts in individuals with both GSTT1(2/2) and NAT2-slow genotype
(1.80) compared with GSTT1(þ)/NAT2 fast individuals (0.96) (85).
GSTT1 and Health Conditions

Cancer
The deletion is associated with an increased risk of lung, larynx, prostate, cervi-
cal, and bladder cancers (34,80,86 –89), and asthma (90). When broccoli con-
sumption was factored into a study of the incidence of colorectal adenomas, a
protective effect was observed in GSTT1(2/2) with individuals consuming
highest amounts of broccoli (91).
ENDOTHELIAL NITRIC OXIDE SYNTHASE
Gene: eNOS—Endothelial Nitric Oxide Synthase (NOS3)

Polymorphisms: G894T—Glu298Asp
. Function: Nitric oxide production which is implicated in relaxation
of vascular muscle
. Biochemical activity: L -Arginine þ N NADPH þ M O2 ¼ citrulline
þ nitric oxide þ N NADPþ
. Cofactors: Heme protein, FAD, FMN, BH4
. Location: Platelets
. Population frequency: 29% Caucasion, 9% Japanese
eNOS Summary
Endothelium-derived nitric oxide (NO) plays a key role in the regulation of
vascular tone, peripheral resistance and also has vasoprotective effects by
suppressing platelet aggregation, leukocyte adhesion, and smooth muscle cell
proliferation. As eNOS mediates basal vascular wall nitric oxide production,
and altered nitric oxide production has been implicated in the development of
coronary atherosclerosis, variants in the eNOS gene have been speculated to
promote atherosclerosis via altered eNOS function (92). Four polymorphisms:
2786T . C; Glu298Asp; Intron 4 VNTR, and Intron 13 CA repeat have been
included in a large number of genetic epidemiology studies (92).
Functional Studies of the 894G > T (Glu298Asp) Polymorphism

A study by Tesauro et al. (93) suggests that the Glu298Asp variant may affect
proteolytic cleavage of the enzyme. Because glutamate and aspartate are con-
sidered to be conservative replacements, the polymorphism was thought to be
a marker for a functional locus elsewhere in the gene. Using transfected cells,
primary human endothelial cells, and human hearts, Tesauro et al. showed that
eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting
in the generation of 100 and 35 kDa products. Thus, the eNOS gene with
polymorphisms at nucleotide 894 generates protein products with differing sus-
ceptibility to cleavage, suggesting that, in contrast to prior predictions, this
polymorphism has a functional effect on the eNOS protein. A recent report
examining the function of wild-type and polymorphic enzymes in a yeast
expression system did not find differences in the enzyme activity or cofactor
binding, so clearly further work is required to fully understand the impact of
the polymorphism (94).
eNOS Glu298Asp (G > T) and Cardiovascular Disease

Associations with the Glu298Asp polymorphism and cardiovascular disease have
been reported in many studies (95,96), The Asp298 polymorphism has been
shown to have significantly higher (P ¼ 0.001) median plasma NOx than those
without this mutation (97). How this reflects NO levels in the vasculature is
unclear.
Leeson et al. (98) studying vascular function by endothelium-dependent,
flow-mediated brachial artery dilatation (FMD) and endothelium-independent
dilatation response to glyceryl trinitrate observed that vascular function was
lower in Asp298 carriers, particularly in smokers and that n-3 fatty acids had a
positive effect in this group, but not in Glu298 homozygotes.
Not all studies have shown positive associations: Nassar et al. (99) exam-
ined the prevalence of this polymorphism in patients with early-onset coronary
artery disease (CAD) compared with those manifesting CAD later in life and
found no association of the eNOS Asp298 allele with premature CAD, no associ-
ation was found in a Taiwanese population (100), or a Finnish population (101).
eNOS and Oxidative Stress of Smokers

The effects of smoking on white matter lesions, such as lacunar infarction and
leukoaraiosis, are still controversial; recently the hypothesis that the eNOS
T-786C genotype was a modulating factor for the effect of smoking on cerebral
circulation was examined. Smokers were showed greater oxidative stress, as
estimated by urinary F(2)-isoprostane excretion. In smokers, 2786CC homozy-
gotes showed a significant decrease of cerebral blood flow and a significant
increase of cerebrovascular resistance, whereas the eNOS genotype did not
affect these parameters in nonsmokers (102). Further evidence for a role of
smoking in modulating the activity of eNOS was demonstrated in vitro using
luciferase reporter vectors with the various haplotype combinations of the
2786T . C and intron 4 repeat polymorphisms. Transcription efficiency in the
T promoter was lower than in the C promoter. Treatment of the constructs
with cigarette smoking extract increased the transcription efficiency significantly
in the T promoter (1.7-fold, P , 0.01), it reduced further the already lower C
promoter efficiency (by 10– 15%) (103).
Potential Dietary Interventions

A role for omega-3 fatty acids in modulating enzyme activity is provided by
Leeson et al. (98), in which there is a significant positive relationship between
the number of Asp alleles and flow-mediated dilation and omega-3 fatty acids
levels. Benefit of vitamin E supplementation is suggested by Hingorani et al.
(104). The authors state that the altered production of NO mediated by the
Glu298Asp polymorphism might explain the unexpectedly large benefit of
a-tocopherol in preventing MI in the CHAOS study (105). The authors
propose that a-tocopherol may act to prevent the accelerated NO destruction

caused by free oxygen radicals in the atherosclerotic vessel wall.
CATALASE
Catalase—CAT
Polymorphisms: C(2264)T, BstXI C . T
. Function: Decompose hydrogen peroxide into water and oxygen
. Biochemical activity: 2H2O2 ¼ O2 þ 2H2O
. Location: Cytoplasm of the cell, located in variety of tissues
. Population frequency of polymorphisms: 2262C/T 28% Caucasian
CAT Background
CAT is a ubiquitous enzyme found in all known organisms, it is most abundant in
liver, kidney, and erythrocytes. CAT has a very high turnover number, rapidly
decomposing H2O2 into O2 and H2O. Together with SOD and glutathione peroxi-
dases (GPX), CAT constitutes a primary defense against oxidative stress (106).
The human CAT gene consists of 13 exons and is located on chromosome
11p13. Several rare mutations/polymorphisms have been reported in the CAT
gene, most of them being associated with acatalasemia, an autosomal recessive
trait characterized by erythrocyte CAT levels 0.2– 4% of normal.
CAT Polymorphisms
A common functional polymorphism (28% in a Swedish population) in the pro-
moter region has been identified. This is a C . T substitution at position 2262
and the variant appears to bind different transcription factors. The T variant was
reported to be expressed at higher levels in HepG2 and K562 cell lines and it was
confirmed that in vivo levels in liver and blood cells were higher for the T-allele.
Individuals homozygous for TT and heterozygous CT have significantly higher
CAT concentrations compared with individuals homozygous for CC (107).
There are also other CAT polymorphisms that have been reported to affect
enzyme levels and be associated with disease states. For example, a recent report
has described the association of a T/C SNP in exon 9 of the CAT gene (BstX I)
with vitiligo susceptibility. Vitiligo susceptibility is a complex genetic trait that
may involve genes important for melanin biosynthesis, response to oxidative
stress, and/or regulation of autoimmunity, as well as environmental factors.
The authors chose the CAT gene as a candidate gene because of the reduction
of CAT enzyme activity and concomitant accumulation of excess hydrogen
peroxide observed in the entire epidermis of vitiligo patients. They observed
that T/C heterozygotes are more frequent among vitiligo patients than controls
and that the C allele is transmitted more frequently to patients than controls.
The authors suggest that the SNP or nearby variations may contribute to a quan-
titative deficiency of CAT activity in the epidermis and the accumulation of
excess hydrogen peroxide (H2O2) (108).
NADPH OXIDASE
NADPH oxidase
Polymorphisms: C242 p22phox subunit, BstXI C . T
. Function: Membrane bound enzymes that catalyze the one-electron
reduction of oxygen
. Location: Integral membrane protein
NADPH Oxidase Background

NAD(P)H oxidases are membrane-associated enzymes that catalyze the one-
electron reduction of oxygen using either NADH or NADPH as the electron
donor, and they are the major oxidases in vascular tissue. NAD(P)H oxidase
comprises several distinct subunits; gp91phox and p22phox are electron-transfer
proteins, and both are expressed in endothelial cells (109).
NADPH Polymorphisms
The C242T polymorphism results in substitution of Tyr for His at residue 72 of
p22phox modifying one of the two heme-binding sites that is thought to be essen-
tial for the stability of the protein (110). This polymorphism is associated with
significantly reduced superoxide production in patients carrying the 242T
allele, suggesting a role for genetic variation in modulating vascular superoxide
production (111).
NADPH Polymorphisms and Health Conditions

The role of this polymorphism and disease association is still controversial, with
reports of increased risk of cerebrovascular disease (112), whereas others have
reported reduced risk of artherosclerosis associated with reduction in superoxide
production (113) and reduced risk of CAD (114). Hayaishi-Okano looked at the
role of the variant in atherosclerosis in type 2 diabetic subjects, they also looked
at the level of 8-hydroxy-20 deoxyguanosine (8-OHdG) DNA adducts as an index
of oxidative DNA damage. The diabetic subjects with TC or TT genotypes dis-
played less vascular thickening than those with the CC genotype and insulin
levels were also affected. They concluded that the C242T polymorphism in the
p22 phox gene is associated with progression of asymptomatic atherosclerosis
in the subjects with type 2 diabetes and is also associated with insulin resistance
in nondiabetic subjects (115).
GLUTATHIONE PEROXIDASE
Glutathione peroxidase—GPX1
Polymorphisms: GCG repeats (Ala4, 5, 6), Pro198Leu
. Function: Reduce peroxides by coupled oxidation with GSH
. Biochemical activity: 2 glutathione þ H2O2 ¼ oxidized
glutathione þ 2H2O
. Cofactor: Selenium
. Location: Cytoplasm
GPX1 Background
Human cellular glutathione peroxidase 1 (hGPX1) is a selenium-dependent
enzyme that participates in the detoxification of hydrogen peroxide and a wide
range of organic peroxides by reducing organic peroxides and hydrogen per-
oxides through the coupled oxidation of reduced glutathione (GSH).
GPX1 Polymorphisms
An inframe GCG trinucleotide repeat was reported in 1994 by Shen et al. They
looked at the GPX1 gene in 55 individuals and the allele frequencies for 4, 5,
and 6 GCG repeats were 0.40, 0.35, and 0.25, respectively (116). A proline to
leucine polymorphism at position198 has been reported in Caucasians, but has
not been found in Asian populations (117).
GPX1 Polymorphisms and Health

Preliminary examination of the relationship of the GCG repeats with lung cancer
and DNA damage [specifically 8-hydroxydeoxyguanosine (8-OHdG) adducts]
indicated a trend towards lower 8-OHdG levels in normal lung tissue possessing
the ALA6 allele of the GPX1 gene, implying a possible functional change in
enzyme activity as a result of sequence characteristics affecting its protection
against DNA (118). In contrast, a recent study of association of five, six, or
seven alanine (ALA) repeats and CAD found a significant association between
individuals with at least one ALA6 allele and an increased risk of CAD (119).
The leucine polymorphism at codon 198 was reported to be associated with
an increased lung cancer risk (117) and breast cancer (120).
MYELOPEROXIDASE
Myeloperoxidase—MPO
Polymorphisms: 2129G/A, 2436G/A
. Function: Catalyzes formation of hypochlorous acid
. Biochemical activity: Donor þ H2O2 ¼ oxidized donor
þ2H2O
. Cofactor: Heme, iron(III), and calcium
. Structure: Tetramer
. Location: Lysosomes
MPO Background
MPO, an iron-containing protein, is found in neutrophils and in the lysosomes of
monocytes in humans, it is most abundant in the granules of neutrophils. MPO
plays a key role in immune activity within a cell. When neutrophils become acti-
vated, they undergo a process referred to as a respiratory burst which produces
superoxide, hydrogen peroxide, and other reactive oxygen derivatives. These
reactive oxygen species are released from the cell and are toxic to bacteria.
MPO catalyzes the conversion of hydrogen peroxide and chloride ions (Cl)
into hypochlorous acid, which is a potent agent for killing microbes, more so
than hydrogen peroxide. Other targets include fungi, parasites, protozoa,
viruses, tumor cells, natural killer cells, red cells, and platelets. The MPO –
hydrogen peroxide –Cl system is also believed to be involved in terminating
the respiratory burst and may be involved in down-regulating the inflammatory
response (121).
MPO Polymorphisms
Two polymorphisms have been identified in the promoter region of the MPO
gene, 463G/A and 129G/A, which reduce MPO enzyme activity in neutrophils,
with the 463G/A showing gender- and age-specific effects (122). Examination of
women on hormone replacement therapy revealed an association of the lower-
activity 463A allele and increased progression of atherosclerotic lesions (123),
and examination of men found increased fibrotic lesions associated with the
463A allele in men under the age of 53 years (124). In studies of brain infarction,
an association with outcome rather than cause was reported, with patients with
G-129 allele showing larger brain infarction, and patients with G-463 allele
demonstrating worse short term functional outcome (125).
In contrast, other studies have reported a protective effect associated with
the A allele. A study of CVD in end-stage renal disease, found reduced incidence
associated with the A allele, possibly due to lower production of reactive oxygen
species (126). A protective effect against the development of hepatoblastoma was
also reported recently (127) with the authors suggesting that the lower activity of
the A allele has a reduced rate of activation of potentially carcinogenic sub-
stances. Reports of reduction of risk of lung cancer have produced conflicting
results; however, analysis of tumor histological type may account for some of
the conflicting results (128).
NADPH DEHYDROGENASE, QUINONE 1
NADPH Dehydrogenase, Quinone 1—NQO1

Polymorphisms: C609T
. Function: Acts as two-electron quinone reductase in detoxification
and biosynthetic pathways
. Biochemical activity: NAD(P)H þ acceptor ¼ NAD(P)þ
þ reduced acceptor
. Cofactor: FAD
. Location: Cytoplasm
NADPH Background
NAD(P)H:quinone oxidoreductase (NQO1), also known as DT-diaphorase is an
enzyme catalyzes 2- or 4-electron reductions of quinones, both endogenous and
exogenous. The reduction reactions prevent redox cycling, which leads to the
generation of free radicals. Synthetic antioxidants and extracts of cruciferous
vegetables, including broccoli, are potent inducers of NQO1 (129).
NADPH Polymorphisms
A snp (C ! T) at position 609 of the NQO1 gene has been shown to reduce
activity of the gene. This polymorphism has been associated with susceptibility
to cancer (130 – 132), including lung cancer in smokers (27) and bladder cancer
in smokers (133). In a population of GSTM1-null children exposed to high
levels of ozone in Mexico, the NQO1 polymorphism was found to have a protec-
tive effect (134).
CONCLUSION
It is clear from the literature that there is significant interindividual variability in
genes which play a role in the body’s antioxidant defenses. The impact of this
variation on human health is being unraveled now as techniques of molecular epi-
demiology are more widely applied. We look forward for further improvements
in techniques and experimental design, so that variation in not only one or two
specific genes, but panels of hundreds to thousands of gene variations can be

examined during the course of a well-planned study. The burden will turn to
the existing data management and statistical analysis tools to accurately interpret
the results. Given the growing popularity of alternative health methods, which
often claim miraculous antioxidant or detoxification treatments, building a
credible body of evidence to understand reponse to dietary antioxidant and detox-
ification agents becomes an increasingly important goal, both from a consumer
protection and a public health standpoint. As future studies continue to
examine associations of many and various genetic polymorphisms with health
conditions, it is acknowledged that many initial positive associations may have
negative results, and the converse may occur, as well. Continuing to develop
the body of knowledge of individual variation, particularly in genes with antiox-
idant activity, should help to advance the understanding of the mechanisms in
which oxidative stress contribute to disease and aging; to turn the widely
accepted “truism” of the impact of oxidative stress on human health into a
well-proven mechanism which explains how oxidative damage sustained by a
cell can ultimately lead to aging or disease.
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3
Analysis of Microarray Data
Ken Mills
Cardiff University, Cardiff, Wales, UK
Introduction 44
Normalization Methods 44
Per Array Normalization 45
Normalize to a Median or Percentile 45
Normalize to Positive Control Genes 46
Normalize to a Constant Value 46
Per Gene Normalizations 46
Normalization to Specific Samples 46
Normalize to Median 47
Data Analysis 47
Fold Changes in Expression Levels 48
Class Discovery—Unsupervised Learning Methods 50
k-Means Clustering 50
Self-Organizing Maps 51
Principal Component Analysis 51
Hierarchical Trees (Clustering) 54
Class Prediction—Supervised Learning Methods 60
Class Prediction 60
Cross-Validation 61
Validation of Microarray Analysis 61
In silico Validation 61
Laboratory Based Validation 62
43
44 Mills
Microarray Analysis Software 62

Summary 63
References 64
INTRODUCTION
Microarray analysis of gene expression has become one of the most widely used
functional genomic tools, since its development in the mid 1990s (1,2). This is
reflected in the number of publications containing or arising from some aspect
of microarray technology. The development has allowed researchers from all
areas of biological research to simultaneously monitor the expression levels of
thousands of genes.
However, the efficient application of this powerful technique requires
robust and reproducible protocols to be developed. These protocols must not
only encompass all aspects of the technical processes but also include robust
strategies for data normalization and in silico data analysis (3).
As in any experiment, conventional or microarray, initial experimental
design should be considered, planned, and refined to ensure that the data pro-
duced is truly meaningful (4). For example, will comparison of control and
test samples produce data that reflects the biological question rather than
simply reflect a difference in maturation status of the cells. The production of
the raw intensity data is only the first step in a microarray experiment. The
post-technical stages can then be divided into two broad sections: normalization
steps and data analysis.
NORMALIZATION METHODS
Data normalization procedures are related to, and dependent, on the design of the
experiment. In microarray studies, large number of genes and samples are usually
analyzed producing amounts of data unimaginable 5 years ago. Some arrays
contain up to 30,000 gene probe sets (e.g., the Affymetrix# U133 arrays) and
have been used in experiments of up to 500 samples; this produces approximately
15,000,000 data points. Therefore, experiment normalizations are used to stan-
dardize microarray data and to differentiate between real (biological) variations
in gene expression levels and variations due to the measurement process. It must
also be noted that normalizing procedures also scale your data so that you can
compare relative gene expression levels between samples within an experi-
mental set.
Data produced from some cDNA arrays undergo some aspect of normali-
zation as a consequence of the technique—ratios of the fluorescent dye Cy3
and Cy5 labeled probes are used to produce raw data as image files that have
Analysis of Microarray Data 45
to be transformed into gene expression formats. This process requires raw data
manipulation, owing to differences in the chemistry of the dyes, before differ-
ences in transcript levels can be identified. It is necessary to normalize the
fluorescence ratios in order to compensate for systematic variations (5).
Most of the currently used normalization methods are linear; however, non-
linear methods have recently started to appear. One of these methods is that of
QQ Quantile Normalization with log centering, which can be used for the nor-
malization of microarray data (6).
In this section, we concentrate on the type of linear normalizations methods
available (Fig. 3.1).
Per Array Normalization

Per array normalizations are used to control array-wide variations in intensity.
Such variations may be due to inconsistent washing, inconsistent sample pre-
paration, or other microarray production or microfluidics imperfections.
Normalize to a Median or Percentile

With this option, all of the measurements on each array are divided by a specific
value; this is usually the median value of all the intensity values on the array.
Other values or percentiles usually can be specified depending on the overall
range of data, although these should be carefully considered before use.
Signal strength of gene A in sample X
Median signal strength of all genes in sample X
In any case, the method of global per array normalization is not recommended for
any experiment where .50% of the genes on the array are likely to be affected
similarly by the experimental conditions. For example, if a focused gene micro-
array containing only known growth factors were used to study gene expression
in malignant and benign tumors, it might be expected that a majority of the genes
Figure 3.1 A schematic example of microarray expression data. Int. 1,1 is the intensity
of gene 1 in array 1, Int. 1,2 is the intensity of gene 1 in array 2, whereas Int. 2,1 is the
intensity of gene 2 in array 1.
46 Mills
will be differentially expressed. In this case, applying a median normalization

would mask the changes in expression.
Normalize to Positive Control Genes

Many arrays have positive controls (mRNA from another genome or housekeep-
ing genes) as part of the array. These can be used to normalize intensity differ-
ences and are often used to control differences during drug treatment or time
course experiments. The formula for this difference is:
Median signal strength of the positive controls in sample X
Normalize to a Constant Value
Some gene array commercial technologies will calculate their own values for
normalization on the basis of a constant value. For example, if the Affymetrix
Global ScalingTM algorithm is used, which centers the data on a specific value,
usually 100, then the data would be normalized to 100 to center it around 1.
Constant value
Per Gene Normalizations

Per gene normalizations have the effect of normalizing each gene on the array
across all the samples within the experiment. If additional microarray sample
data are added to an experiment, per gene normalizations need to be recalculated.
Only one type of per gene normalization is required for an experiment, as they
address the same issue.
Normalization to Specific Samples

In normalization to specific samples, each gene is divided by the intensity of that
gene in a specific control sample or by the average intensity in several control
samples. The formula for this is:
Signal strength of gene A in control sample
or
Average signal strength of gene A in control samples
This type of normalization might be used, for example, during a time course
experiment; and changes in gene expression are related to the untreated or
time zero sample array. Different samples within an experiment may be normal-
ized to different control samples. For example, when comparing the effect of a
drug treatment in two different cell lines: time points for cell line A may be
related to untreated cell line A; whereas time points for cell line B may be
related to untreated cell line B. It should be noted that, although patterns of
gene expression during the time course of each cell line can be identified using
this method, it does not allow the direct comparison of expression levels of indi-
vidual genes between cell lines A and B.
Normalize to Median
Normalize to median is the most common type of normalization. Per gene nor-
malization is related to the median signal intensity of each gene across all of
the samples in an experiment.
Median of every measurement taken for gene A throughout experiment
It allows direct comparison of gene expression levels for each gene across all the
samples within an experiment. However, care should be taken that all other pro-
cedures within the microarray techniques are comparable; different RNA extrac-
tion procedure or even location of RNA extraction may result in step changes in
expression levels unrelated to biological variations.
DATA ANALYSIS
At the moment, analysis of microarray data is perhaps the most exciting area of
biology. It has the potential to identify changes in gene expression related to
disease classification, disease diagnosis, responses to pharmacological or toxico-
logical reagents, and in a multitude of basic biological science studies. As a
consequence, a new and developing field of bioinformatics and biostatistics
has been developed.
Expression databases that follow standardized methods for reporting array
data have been implemented, which allows more uniformity and confidence
when comparing expression levels. Standardized approaches such as that imp-
lemented by minimal information about a microarray experiment (MIAME)
(7,8) need to be applied to allow the comparison of data produced in different
laboratories and different array platforms. The microarray gene expression data
(MGED) (www.mged.org) organization provides well defined guidelines for
the cross-comparison of array data and the need for comprehensive records of
sample type, extraction processes, and labeling and hybridization techniques.
Other databases are also available (9 – 14).
All the questions asked of microarray data can probably be reduced to one
overall question: “How can microarray data be mined to identify hidden patterns
of gene expression?”. In particular, can we identify changes in genes expression
patterns between different samples within an experiment or can we identify
groups of genes that have similar expression patterns. These patterns will be
48 Mills
Figure 3.2 Schematic illustrating the differences between class discovery and class
prediction. Class discovery starts with no concept of groupings and the class discovery
clustering algorithms place samples into groups based on similarities. Training/Test
sets are used to identify genes that will be able to predict which known sub-groups an
unknown sample should be allocated.
complex and interpretation will be compounded by the gene array data from a
sample being associated with several parameters or factors. These parameters
will be related to the sample type, treatment, or origin; however, the quality of
sample, extraction procedures, and labeling methods should also be considered.
The identification of genes that are differentially expressed among pre-
defined set of samples with known parameters or classes within an experiment
is called class comparison. Class prediction is related to identifying a set of
genes that are capable of predicting which parameter group a specific sample
should belong (Fig. 3.2).
An increasing number of statistical methods have been, and continue to be,
developed to extract reliable biological information from the microarray data.
The analysis methods ranging from the basic to the more complex and some of
these will be considered in this section. Whichever method is used to identify
differentially expressed genes, further validation or selection is then required
(see section “Validation of Microarray Analysis”).
Fold Changes in Expression Levels

Although this is one of the more basic methods of mining relevant data from
microarrays, it can have disadvantages, which are usually associated with the
lack of comparison of sample type within the experiment. In general, the
Figure 3.3 Fold change in a series of 12 samples. (A) The normalized level of
expression of one gene selected to show at least 2-fold higher expression in series of 12
samples (A1– C4). (B) The same gene expression level averaged over the four samples
in each of three of discrete variables (A– C).
expression data is systematically analyzed for genes that show up or down

changes in expression when comparing samples with two or more different
parameters (Fig. 3.3). Often changes of 2-fold may be sufficient to identify a
list of candidate genes.
The accuracy of the candidate genes list will be increased when the issues of
replicates are also considered. Several replicates must be done in order to attach
statistical significance of the expression data. Ideally, at least three replicates
should be done for each condition or parameter; however, in the case of disease
analysis the replicates could also be biological replicates rather than technical repli-
cates. A simple type of analysis of microarrays, containing several thousand genes,
may result in a large list of genes, particularly, if a small number of samples are
also analyzed. This is because the “noise level” from the array data is reduced
when a greater number of samples, or replicates, are analyzed. For example, if
an array contains 10,000 genes, then in any one sample or replicate, it may be
that approximately 5000 genes will have either intensity values above the back-
ground threshold or are called “present.” A replicate of this sample may have a
similar number of genes, but only 95% of these genes are in common, and so
on as more samples or replicates are analyzed. By combining the data from
the replicates, the number of genes consistently over the threshold, or called
“present,” is rationalized. Furthermore, the intensity levels now have values statis-
tically associated with them (median and standard deviation). Therefore, the
identification of genes showing .2-fold change in expression is now statistically
relevant, although the list may still be relatively large.
The degree of fold change also has to be considered. Owing to the data nor-
malization methods, many genes may be identified that show 2-fold changes in
expression level, but when the raw intensity data are examined some of the genes
may have very low levels. These genes should probably be considered as absent
and removed from further consideration. In general, microarray data cannot be
50 Mills
used to identify gene expression changes with less than an overall 2-fold with
great confidence. This can be a major consideration if a sample does not contain
100% of the same cell type. For example, in a sample in which only 40% of the
cells show a response to a drug treatment, candidate gene expression levels would
need to change by at least 3.5-fold in all of those 40% of cells to result in an
overall 2-fold change in expression level.
Another method of identifying genes with significant changes within an
experiment is to use a one- or two-way analysis of variance (ANOVA) filter.
ANOVA is performed separately for each gene, only those genes that pass the
significance level are retained for further analysis and therefore the changes in
expression that are identified are probably likely to be due to the defined par-
ameter rather than random fluctuations.
If we assume that some genes are significantly identified during these analy-
sis, the gene list may still be number several hundred. These candidate genes can
be reduced by further by adding further significance levels. Tests such Bonferroni
correction or “significance analysis of microarrays (SAM)” can be used (15 – 17).
The former correction can often be restrictive and may result in no significant
genes being identified, the latter method is now becoming a standard statistical
technique for identifying genes lists of differentially expressed genes.
Class Discovery—Unsupervised Learning Methods

One of the major strengths of the gene microarrays is the ability to identify and
reveal hidden patterns of gene expression. Various methods of data reduction and
classification have been formulated that will link together groups of genes that
have similar patterns of gene expression throughout the whole experiment.
These methods reduce the initial main list of genes into smaller sub-groups or
clusters. These sub groups are based on similar patterns of gene expression in
the experiment. Basically, genes are clustered together because their expression
is similar; each gene will show an increased or decreased expression which
follows the same pattern as other gene members of the same sub-group. These
clusters are identified without prior knowledge or input into the group, parameter
or class that an individual samples is a member (Fig. 3.2). Gene expression sig-
natures have been used to classify disease outcome in several disease types. The
main successes have been reported within cancer research (18 – 21) using super-
vised learning algorithms have been developed to classify samples into distinct
specific sub-sets based on gene expression. Classical examples of this are the
first example of the feasibility of a cancer classification which was based
solely on gene expression in acute leukemia (20,21) and the identification of a
novel class of patients with diffuse large B-cell lymphomas (20).
k-Means Clustering
k-Means clustering divides genes into a (user) defined number of groups on the
basis of their expression patterns (22,23). This produces groups of genes with a
high degree of similarity within each group and a low degree of similarity between
k groups. k-Means clustering does not show the relationship between clusters, but
instead, k-means clusters are constructed so that the average behavior in each group
is distinct from any of the other groups. For example, in a time series experiment,
k-means clustering could be used to identify unique classes of genes that are
up-regulated or down-regulated in a time dependent manner (Fig. 3.4).
A k-means clustering algorithm will typically divide genes into a user-
defined number (k) of equal-sized groups on the basis of the order in the selected
gene list. It will then create centroids around the average location of each group
of genes. This is then done over several iterations and genes are reassigned to
the group with the closest centroid. When all the genes have been assigned, the
location of the centroids is recalculated and the process is repeated until the
number of user-defined iterations has been reached.
Self-Organizing Maps
Self-organizing maps (SOM) is a technique similar to k-means clustering. SOMs
show the relationship between groups by arranging them in a two-dimensional
map as result of dividing genes into groups on the basis of expression patterns
(Fig. 3.5). SOMs can visualize distinct expression patterns and determine
which of these patterns are variants of each other by Refs. (24,25). SOMs can
be used to analyze many kinds of data, and some applications to gene expression
analysis were described by Tamayo et al. (26).
SOM algorithms create a two-dimensional grid of nodes in the space of
gene expression. In each iteration, one gene is selected and all of the nodes
within a user-defined “neighborhood” are moved closer to it. This process is
repeated with each gene in the selected gene list until the maximum number of
iterations has been reached.
With each iteration, the “neighborhood radius” is incrementally reduced
and nodes are moved by smaller amounts to produce convergence. In this way,
the grid of nodes is stretched and wrapped to best represent the variability of
the data, while still maintaining similarity between adjacent nodes. After the iter-
ation is complete, genes are assigned to the nearest node, and a display grid of
gene expression graphs is generated, corresponding to the initial grid of nodes.
Principal Component Analysis
Principal component analysis (PCA) is a “data reduction” technique used to iden-
tity uniquely expressing genes (27). PCA is not a clustering technique, but is a
tool that will characterize the most abundant components that re-occur within
many genes in an experiment. PCA is a decomposition technique that produces
a set of expression patterns that are known as principal components. Diagonal
or 3D combinations of these patterns can be assembled to represent the behavior
of all of the genes in a given data set (28) (Fig. 3.6).
In this procedure, the expression data from the “genes X expression” space
are transformed to produce the principal components of a data set that are
52 Mills
Figure 3.5 Self-organizing maps using the same series of 12 samples (A1– C4) as in
Fig. 3.2 using the list of genes showing above background levels of normalized expression.
known as “eigenvectors,” which are obtained from an eigenvector – eigenvalue

decomposition of the covariance matrix of the data (29,30). The eigenvalue
corresponding to an eigenvector represents the amount of variability explained
by that eigenvector. The eigenvector of the largest eigenvalue produces the
first principal component. The eigenvector of the second largest eigenvalue
is the second principal component and so on. There are never more principal
components than there are conditions in the data.
PCA identifies the correlations between gene expression profiles and
attempts to explain a majority of the variance in the entire data set. Hierarchical
clusters will group together gene expression profiles with similar distances.
Figure 3.4 k-Means clustering using the same series of 12 samples as in Fig. 3.2.
(A) A 9 cluster k-means separation of the gene as showing above background levels of
normalized expression. (B) The same k-means cluster showing the averaged patterns of
gene expression in the three main populations (A – C).
54 Mills
Figure 3.6 Principal component analysis. (A) 2-Dimensional map of the principal com-
ponents showing distinct separation into three groups each comprising the four related
samples. (B) 3-Dimensional map of (A) showing three distinct groupings of related
samples. Note, however, that one sample (arrowed) lies in the same horizontal plane as
the other related samples, but is slightly removed in the Z-direction; this may indicate
that this sample has a slightly different phenotype from the other members of the group,
but distinct from the other groups.
Hierarchical Trees (Clustering)

The classification of organisms into phylogenetic trees is a central concept of
biology: organisms that share properties are clustered together. The tree branches
indicate how far close or diverse two sets of organisms are. The distance from two
organisms to a common branch can be considered as a measure of how different
the organisms are. Other concepts can also can be classified in a similar manner
(31,32); for example, cluster analysis will place genes whose expression patterns
are similar to the adjacent branches in a tree. Such mock-phylogenetic trees are
also called dendrograms (Fig. 3.7). Complex trees can be made from multiple
experiments or by tightly defining the types of data used. Cluster analysis is a
useful method of class discovery. This is because the clustering algorithms
do not depend on sample parameters—and as such are classified as unsuper-
vised analyses.
Figure 3.7 A typical dendrogram of 50 genes. Two main branches or clusters can be
seen, with various sub-branches. Genes with related expression features are placed
closest together, and the point where they join is the node, or junction, of the branches.
56 Mills
Measurements of similarity, distance, and separation: Hierarchical

analysis techniques are based on measures of gene similarity. Similarity or
“nearness” between genes is a measure of the degree of correlation between
the expression profiles of the two genes.
Measures of similarity. Measures of similarity take two expression pat-
terns and produces a number representing how similar the two genes are. Most
of the measures of similarity are correlation measures, and their value varies
from 21 (exactly opposite) to 1 (the same). For a measure of distance, the
result will vary from 0 (the same) to infinity (different). Measures of confidence
vary from 0 (no confidence) to 1 (perfect confidence). Both distance and confi-
dence can be considered as measures of dissimilarity: this means that “small”
relates to “close”; whereas “large” means “far away.”
Similarity definitions. The default correlation is the standard correlation,
Pn
i¼1 Ai Bi
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn Pn
2 2
i¼1 Ai i¼1 Bi
To make a tree, the correlation for each gene with every other gene in the set is
calculated, the pair of genes with the highest correlation is then taken, and their
expression profiles averaged. This new composite gene is then compared with all
of the other unpaired genes. This process is repeated until all of the genes have
been paired.
Separation ratio. The minimum distance and the separation ratio affect
the branching behavior of the tree. The separation ratio determines how large
the correlation difference between groups of clustered genes must be for them
to be considered discrete groups. This number should be between 0 and 1 and
determines how large the correlation difference between groups of clustered
genes has to be for the groups to be considered discrete groups and not be
joined together. Increasing separation increases the “branchiness” of the tree,
whereas a separation ratio of 0 indicates that all gene expression profiles can
be regarded as identical.
Minimum distance. The minimum distance deals with how far down the
tree discrete branches are depicted. A value ,0.001 has very little effect, this is
because most genes are not more closely related than that. A higher number tends
to add more genes to a group, making the groups less specific. The minimum dis-
tance deals with how far down the tree discrete branches are depicted. A higher
number tends to combine more genes to a group, making the groups less specific
(Fig. 3.8).
Experiment and gene clusters: It is becoming increasingly common for

clustering algorithms to be done on both the gene lists and the samples. The resul-
tant dendrograms is a graphical matrix of the relationship between genes and the
corresponding samples (Fig. 3.9). The extent of gene expression is usually shown
by changes in color. Typically, red depicts high expression and green depicts
Figure 3.8 The effect of different statistical methods for the calculation of hierarchical
clusters. (A) The sample dendrogram produced using a standard correlation of similarity
measurement. (B) The related dendrogram when distance is used of similarity measurement.
lower or absent expression. The gradient of color change between these two
colors is proportional to the signal intensity (Fig. 3.10) of an individual gene.
Cluster analysis can be a very powerful tool. Genes with similar expression
profiles across a series of samples are clustered together; often these genes do not
have functional similarity, which may lead to a re-examination of the biological
hypothesis. It should always be noted that similar expression levels or patterns
does not always mean that the genes are related in the same biological
pathway. This is because any genetic abnormality or external stimulus may
initiate several different, and separate, pathways that exhibit concordant gene
expression patterns, but these may be coincidental. A related strategy is to ident-
ify genes whose expression patterns are similar or opposite to that of a known
Figure 3.9 A two-way cluster analysis, or heat-map, of gene expression. Vertical

columns within the heat-map represent expression within each sample. Horizontal rows
show the expression level of individual genes across the samples.
58 Mills
Figure 3.10 The vertical color gradient represents the relative expression levels: white
at the bottom of the bar represent low or absent expression, whereas black are high levels
of normalized expression. The gradient between these two extremes correlates with the
expression levels. The horizontal axis of the color bar indicates a measure of reliability
of the data. The darker the region towards the right represents increasing trust or reliability
of the data. The trust is a representation of the (the median value of the chip) (the
median value of the gene).
candidate gene. These approaches will allow relationships between, and within,
molecular proliferation, differentiation, or apoptotic pathways.
Hierarchical clustering of samples within an experiment will usually group
together those samples that show distinct phenotypes. However, it should be
noted that the concept of “majority voting” is very important in the sample
clustering. This means that each biological parameter within an experimental
sample population should be equally represented as in majority voting, the
results of presenting a pattern to a number of networks are tallied, and the
majority classification is taken as correct.
QT clustering: QT clustering identifies clusters of genes so that each

gene in the cluster is within a specified distance metric of every other gene in
the cluster. In QT clustering, the “diameter” of a cluster refers to the largest dis-
tance between any two genes in the same cluster (Fig. 3.11). A cluster built by QT
clustering starts with a single gene. The diameter at that point is 0. It then adds the
Analysis of Microarray Data
Figure 3.11 Three of the 57 QT clusters, grouped on the basis of the criteria of minimum cluster size of 25 genes with
59
minimum similarity of 0.1 and standard correlation of similarity measurement.

60 Mills
gene that is closest to the starting gene. The diameter of the cluster is now equal to
the distance between the two genes. It continues adding genes one at a time,
always choosing a gene that will result in the smallest cluster diameter. Even-
tually, it reaches a point where no genes can be added without the diameter
growing beyond a defined cutoff. The cluster is then complete.
Importantly, the first cluster obtained is dependant on the initial gene is
chosen. Therefore, it independently builds clusters starting from each gene in the
user-selected gene list. The cluster with the most genes is kept, and is part of the
final classification. All others are discarded. That stage results in a single cluster.
The genes contributing to that cluster are removed from the overall gene list,
and the clustering process is begun again. Again, a new cluster is built from
every gene in the reduced gene list, the largest one is kept. This process is repeated
until the number of genes in the largest cluster is smaller than a user-defined cutoff.
Class Prediction—Supervised Learning Methods

In the earlier sections, methods are described which can be considered as “class
discovery.” These are used with out a preconception of classes or parameters
associated with any sample (Fig. 3.2) and k-means, SOMs, and hierarchical
cluster analysis are types of this analysis. Class prediction methods, or supervised
learning methods, use sample associated parameters or classes to identify gene
lists that can associate unknown samples with these parameters or classes
(33 – 35) (Fig. 3.2).
Class Prediction
The class predictor is designed to predict the value, or “class,” of an individual
parameter in an uncharacterized sample or set of samples. This is usually
performed on a split sample method. The number of samples is divided into
two groups: a training set (usually comprising two-third of the total sample
size); and a test set (the remaining one-third). In the training set, multiple
kinds of parameters can be defined; however, the parameters within the separate
test set are not used for the development of the prediction model. In the first of
two steps, the class predictor algorithm examines all genes in the training set indi-
vidually and ranks them on their power to discriminate each class from all the
others. In the next step, it uses the most predictive genes to classify the test set
(in this set the parameter value is unknown). For example, a disease type could
be predicted from the expression data from samples whose disease status is
known. Class predictor can also be used to identify genes whose behavior is
related to a given parameter by examining the list of predictor genes.
Ordering all the measurements for a given gene according to their normal-
ized expression levels assembles the list of predictor genes. For each parameter
value, the predictor places a mark in the list where the relative abundance of the
class on one side of the mark is the highest in comparison to the other side of the
mark. The genes that are most accurately segregated by these markers are
considered to be the most predictive. A list of the most predictive genes is made
for each class and an equal number of genes are taken from each list.
Cross-Validation
Cross validation, or “jack knifing” is an alternative to the split sample method of
class prediction (36 – 38). This employs a “leave one out” cross validation method
in which the test set consists of only one sample. The remainder form the training
set, which is used to develop the class prediction gene model. This model is then
used to predict the class of test sample on the basis of the expression profile. If the
model-test sample does not correlate, a new cycle of training-omitted sample is
run in which a different sample is omitted from the training set. At the end of
n models, where n is the number of samples, a cross-validated error rate is cal-
culated. This is an estimate of error rate that could be used for future samples,
if the relationships between class and expression profiles are the same.
VALIDATION OF MICROARRAY ANALYSIS

As indicated previously, microarrays represent an extremely powerful technique
for the analysis of gene expression. The range of experiments is endless and the
data produced from even a limited experiment using an array with only a limited
number of genes can be produced enough candidates for several new research
projects. However, it should be recognized that there are technical limitations
that may distort the data analysis. The size (in bp), the type (cDNA or oligo-
nucleotides), and the location in the target gene of microarray probes may all
contribute to efficiency of the hybridization reaction and thus the ability of
the technique to accurately detect differences in gene expression levels. One of
the easiest ways to reduce erroneous expression results is to ensure that array
experiments are fully optimized. Validation approaches can be divided into
two main areas: in silico or laboratory approaches.
In silico Validation
This is based on bioinformatics and allows data from microarray experiments to
be analyzed against publicly or privately available databases. Publicly available
expression data is often available as an appendix to published articles. However,
comparison of locally produced data with that within any other database should
be examined with several provisos, many of which relate to the technical pro-
cedure and arrays analyzed.
A wide range of information about candidate genes can also be mined via
bioinformatics databases. Gene annotations can be obtained from the National
Center for Bioinformation Technology (www.ncbi.nlm.nih.gov/) or The Institute
for Genome Research (TIGR) (www.tigr.org). The GeneCards is a database
of human genes, their products and their involvement in diseases (http://
bioinformatics.weizmann.ac.il/cards/). Other relevant databases include those
62 Mills
for literature information such as MEDLINE (http://ncbi.nlm.nih.gov/PubMed/),

genetic variations in sequence such as registered by dbSNP (http://ncbi.nlm.nih.
gov/SNP) or HGBase (http://hgbase/interactiva/de), or location to specific meta-
bolic pathways using KEGG (http://kegg.genome.ad.jp/kegg/).
The Gene Ontology (GO) Consortium (www.geneontology.org) produces a
controlled vocabulary that can be applied to all organisms as the knowledge of
genes and the role of their proteins accumulates. The ontologies relate genes
into three broad categories: molecular function; biological processes; or cellular
component. Numerous other databases of promoter function, structural motifs, or
phenotype data are becoming available.
Laboratory Based Validation

Laboratory-based validation of data provides a second level of independent,
experimental verification of gene expression levels. Methods that can be used
to confirm, and increase confidence, in microarray expression results include
northern blotting and quantitative RT-PCR (RQ-PCR), RNA protection assays,
in situ hybridization or immunohistochemistry using tissue microarrays (TMAs).
RQ-PCR allows the continuous measurement of products produced during
the course of PCR reaction. This can be done using the TAQman based fluor-
escent probe method or by using Sybr Green to detect double stranded DNA
products. Both methods rely on the detection of product molecule present
throughout the numerous cycles occurring during a complete PCR reaction.
The exponential growth in PCR products is related to the cycle threshold point
and from this crossing point, the number of targets present in the input sample
can be determined. TMA containing numerous microscopic sections of tissue
are another approach for the validation of array data in a large set of diverse
tissue specimens unrelated to the initial experimental samples.
Perhaps the most relevant, but also time-consuming method of validation
is to examine protein levels of the relevant candidate genes. Protein analysis
can be done on several levels depending on the resources and questions asked.
Proteomics can study of the function of all expressed proteins; and although
rapid progress has been made in the past few years in generating large-scale
data sets of protein profiles, the technique still requires further improvements
to achieve its potential. Protein analysis will allow post-translational modi-
fications to be detected—which microarray techniques will fail to identify.
Changes in protein levels can be done by western blot analysis, in combination
with immunoprecipitation, or by sensitive flow cytometry methods.
MICROARRAY ANALYSIS SOFTWARE

The statistical methodologies described earlier are complicated and involve very
large data sets and calculations. Numerous software packages have been designed
to analyze interpret, and analyze the images and intensity data obtained from
microarrays. Three general categories of software are available. The first group
could be called “stand-alone” packages and will accept data from several differ-
ent array scanners and imagers: ArrayGauge (Fujifilm Medical Systems) and
ImageMaster Array 2 (Amersham Pharmacia). A second group are designed to
interact with only with data produced from specific microarray scanners;
QuantArray (Packard BioChip Technologies) and GenePix Pro 3.0 Array Analy-
sis Software (Axon Instruments). The third group consists of software for the
analysis of array-specific systems. The best known, owing to the high use of
GeneChips is the Microarray Suite (MAS) Software (Affymetrix Inc.). Array-
ToolsTM (Incyte Genomics) allows the investigation of data generated by Life
ArrayTM microarrays; whereas Pathways 3 Microarray Analysis Software
(Research Genetics) of Huntsville, Ala., analyses GeneFilters.
There are also available a number of post-imaging analysis software
packages that use data mining tools. VistaLogic software has methods for visua-
lizing gene expression profiles, and has the novel approach of using 3-D plot with
peaks and valleys to depict up and down regulated genes. This plot rotates and
has a zoom feature designed to extract additional information from the data.
GeneSpringTM Expression Analysis software (Silicon Genetics) has a host of
visualization and analysis tools. The visualization options include graphics show-
ing the physical position of a gene on a chromosome and expression profile graphs.
Individual profiles are hyperlinked to annotations about the underlying gene and
can find genes with a similar expression profile based on a variety of similarity
measures (e.g., Pearson correlation or Euclidian distance). Spotfire Array Explorer
(Spotfire Inc.) contains numerous clustering packages, whilst BioDiscovery’s
GeneSight offers some 19 color maps for data visualization. A comprehensive
list of commercial microarray analysis software packages has been published
(http://www.the-scientist.com/images/yr2001/pdfs/microarray_010430.pdf).
However, new packages and databases are constantly being developed by aca-
demics (39 –44) and commercial companies.
SUMMARY
Microarray analysis of gene expression is still in an early phase of development.
Statistical algorithms can be used to identify numerous genes that change in
expression levels: may only be expressed in certain sub-groups of samples; or
can predict which type of group a specific sample should be placed. However,
perhaps the overall goal of any microarray experiment is to place changes in
gene expression within the concept of molecular networks and interactions. If
one gene shows, for example, a 3-fold increase in expression, what effect does
that have on other genes within the same pathway, on related pathways, or on
the cellular phenotype. The more the microarray data analyzed and validated,
the more we will understand of interactions and nodes of interactions. It is
only when these complex networks are identified and validated will we truly
understand how normal and abnormal cells function differently.
64 Mills
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4
Oxidative Stress, Gene Expression,
and Lifespan
Yoko Honda and Shuji Honda

Tokyo Metropolitan Institute of Gerontology,
Tokyo, Japan
Introduction 68
Lifespan Regulation in C. elegans 69
Aging of C. elegans 69
Stress, Hormesis, and Lifespan in C. elegans 69
Insulin/IGF-I Signaling and Lifespan Regulation 70
Insulin/IGF-I Signaling Pathway and
Stress Resistance 73
DAF-16 Transcription Target 76
Reproductive System and Lifespan 77
Nervous System and Lifespan 78
Mitochondrial Electron Transport
and Lifespan 78
Lifespan Regulation in Drosophila 81
Lifespan Regulation in Mammals 83
Replicative Lifespan and Oxidative Stress 85
Conclusion 86
References 86
67
68 Honda and Honda
INTRODUCTION
The lifespan of metazoans can be extended by environmental conditions: caloric
restriction (CR) in a wide range of organisms (1), low temperature in some
poikilothermic animals (2), and low oxygen concentrations in the nematode
Caenorhabditis elegans (3). The mechanisms by which each condition slows
the aging rate have not yet been fully elucidated. Function of CR has been pos-
tulated as hormonal changes, altered gene expression, lowered metabolic rate,
and a reduced generation rate of mitochondrial reactive oxygen species (ROS).
Lifespan could also be lengthened by environmental perturbations. Hormesis is
a phenomenon occurring when agents that are harmful at high doses or over
long periods, actually produce beneficial effects, such as lifespan extension,
when used at low doses or over short periods. C. elegans shows lifespan-
extension hormesis when exposed to low doses of radiation (4) or short-term
heat (5), hyperoxia (6), or hyperbaric oxygen (7). These treatments are associated
with adaptive resistance to lethal thermal or oxidative stress, and the gene
expression of stress-defense proteins (5,6,8).
Recently, lifespan-extension mutants of the nematode C. elegans have been
extensively isolated, and the gene network responsible for its longevity has been
unraveled [for a review see Ref. (9)]. Two main classes of lifespan-extension
mutants have been reported; one class is related to the activity of the mitochon-
drial electron transport chains, such as clk-1 (10) and isp-1 (11), and the other is
related to hormonal mechanisms, especially an insulin/IGF-I signaling pathway,
such as daf-2 (12,13) and age-1 (14,15). The insulin/IGF-I signaling pathway
regulates the activities of DAF-16, the fork head transcription factor (16,17)
and HSF-1, heat shock transcription factor 1 (18) to activate the transcription
of genes that have more direct effect on lifespan determination.
This pathway confers resistance to a variety of stresses, including heat (5),
UV (19), metal (20), and oxidative stress (21 –23) as well as lifespan extension in
C. elegans. Recent investigations suggest that an analogous pathway also regu-
lates stress resistance and longevity in Drosophila (24,25) and mammals
(26,27). The close association between stress resistance and longevity suggests
the possibility that the molecular mechanisms protecting against stress may
overlap to retard the aging process.
The free-radical theory of aging, first proposed by Harman (28), is attract-
ing considerable attention (29). According to this theory, aging is the result of
accumulated ROS-induced oxidative stress. Indeed, a large body of correlative
evidence is consistent with this hypothesis (30).
Recently, studies into the insulin/IGF-I signaling pathway that promotes
longevity and changes gene expression in C. elegans (31,32) have been initiated,
and the RNAi inactivation effect on lifespan of these genes has been systemati-
cally analyzed. The gene expression profiles of various tissues in calorically
restricted mammals or lifespan-extension mutant mammals have also been
studied (33,34). These studies will provide a molecular description of organismal
mechanisms regulating the aging processes.
Oxidative Stress, Gene Expression, and Lifespan 69
LIFESPAN REGULATION IN C. ELEGANS

Aging of C. elegans
The wild-type C. elegans lives for 3 weeks. As an animal ages, various senes-
cence symptoms become apparent (35). From studies of ultrastructural obser-
vation and the visualization of specific cell types with green fluorescent protein
(GFP), the nervous system is shown to be intact even in advanced old age,
however, a gradual and progressive deterioration of muscles becomes apparent
with increasing age resembling human sarcopenia (36). The stochastic features
in the aging processes were clearly shown.
Using full-genome microarrays, the gene expression changes during aging
were examined (37). One hundred and sixty four genes, ,1% of the genome,
show significant changes in expression as aging advances. Dynamic changes in
the expression of four insulin/IGF-I-like genes and two sir-2 homologues are
observed during aging. The expression of two heat shock proteins, HSP16 and
HSP70, also shows age-related changes.
Murakami and Johnson (38) showed that the expression of old-1 gene
increase with advancing age. The old-1 gene product, OLD-1, is a transmem-
brane tyrosine kinase protein that is upregulated in daf-2 and age-1 lifespan-
extension mutants in a daf-16-dependent manner. Various stresses, such as
heat, UV, and starvation induce old-1 expression. The overexpression of old-1
induces stress resistance and lifespan extension. In contrast, the genomic knock-
out (KO) of old-1 in wild-type animals causes lifespan shortening and stress
hypersensitivity. Furthermore, the genomic KO or RNAi inactivation of old-1
in lifespan-extension mutants restores the normal lifespan. This indicates that
OLD-1 is a positive regulator of stress resistance and longevity.
Stress, Hormesis, and Lifespan in C. elegans

We showed that the lifespan of C. elegans decreases with increasing environ-
mental oxygen concentration (39). On the other hand, low oxygen concentrations
extend the lifespan (3), indicating that environmental oxygen concentration is
one of lifespan determinants. Since ROS are thought to arise in organisms
depending on the oxygen concentration (40), these findings suggest that ROS
are involved in determination of lifespan. On the other hand, short-term exposure
to hyperoxia slightly lengthened lifespan (6), short-term exposure to hyperbaric
oxygen also increases lifespan (7). These effects are thought to be a form of
hormesis for lifespan. In response to oxidative stress, the level of SOD activity
and the expression of CuZnSOD, MnSOD, and catalase genes and oxidative
stress resistance (6,8) increase in C. elegans. Pretreatment with hyperbaric
oxygen or juglone (an intracellular generator of ROS) significantly increased sub-
sequent resistance to the same or reciprocal stressors (7). The most widespread
explanation is that an increased ability to remove ROS induced by oxidative
stress could reduce normally occurred oxidative stress that may cause aging.
70 Honda and Honda
High doses of ionizing radiation reduce the lifespan of C. elegans, but low
doses induce moderate lifespan extensions (4). We reproduced this result
(S. Honda, Y. Honda, and S. Suzuki, unpublished observation) but Cypser and
Johnson (7) found no lifespan extension by low-dose ionizing radiation as well
as UV. These hormetic effects may be weak and occasional.
Heat treatment at sublethal temperatures induces increased resistance to
subsequent lethal heat stress and modestly extends the lifespan of C. elegans
(5). Heat shock induces a small heat shock protein (SHSP), HSP-16, in wild-
type animals (41). Hsu et al. (42) also showed the increased expression of shsp
genes, hsp-16.1, hsp-16.49, hsp-12.6, and sip-1 by heat shock. Yokoyana et al.
(43) showed the induction of hsp70F and lifespan extension by heat shock.
The overexpression of hsp70F, predominantly in the muscles, induces lifespan
extension, and overexpression of the hsp16 gene induces thermal resistance
and extended lifespan (44). These HSPs may play an important role as the mol-
ecular chaperon for preventing improper protein associations accumulated in the
aging process.
Treatment of C. elegans worms with salen– manganese complexes, EUK-8
or EUK-134, synthetic SOD/catalase mimetics increased lifespan by 44% (45),
but other investigators could not reproduce their results (46). The extension of
lifespan by SOD/catalase mimetics may only occur under very particular
culture conditions. SOD/catalase mimetics was reported to have cytoprotective
activities in ischemic rat brain injury (47), and reverse age-related learning
impairment and brain protein oxidation in mice (48).
Insulin/IGF-I Signaling and Lifespan Regulation

One of the two main classes of the lifespan-extension mutations of C. elegans
is related to the insulin/IGF-I signaling. Insulin/IGF-I signaling is mediated
by the DAF-2 insulin/IGF-I receptor. The daf-2 mutants that reduce the activity
of DAF-2 remain youthful and active much longer than the wild-type animals and
live more than twice as long [reviewed in Ref. (9)]. The lifespan-extension
phenotype of the daf-2 is suppressed by mutations in daf-16, indicating that
daf-16 is negatively regulated by DAF-2 signaling and is the major downstream
effector. The daf-16 encodes a FOXO transcription factor (16,17).
Binding of insulin/IGF-I-like ligands to the DAF-2 insulin/IGF-I receptor
controls insulin/IGF-I signaling. There are at least 38 genes (ins) encoding
insulin/IGF-I-like peptides in C. elegans (49,50). Many of these genes are diver-
gent insulin superfamily members, and as the specific ligand has not yet been
identified, these members may be possible to have complex and redundant
roles. The daf-28 gene encodes insulin-like peptide. A dominant-negative
allele of the daf-28 mutant lives 10% longer than wild-type animals. A phenotype
of the daf-28 mutant is rescued by ins-4 or ins-6 transgene, suggesting a redun-
dant nature (51). Some ins genes are expressed in sensory neurons (50,51).
Environmental cues such as food, pheromones and temperature may affect
insulin/IGF signaling through different expression and the secretion of various

INS peptides.
The mutation of age-1, which encodes the PI3 (phosphoinositide-3-OH)
kinase catalytic subunit, doubles the lifespan in C. elegans (14,15).
The current model of insulin/IGF-I signaling is as follows (Fig. 4.1).
The DAF-2 insulin/IGF-I receptor transduces signals by activating AGE-1.
AGE-1 PI3 kinase phosphorylates PIP2 to generate the second messenger PIP3.
Environmental Cues
Food
Temperature
Pheromone cGMP Signaling
DAF-11 DAF-21
Guanyl Cyclase HSP90
Sensory Neurons
TGF-β Signaling
DAF-7 DAF-1 DAF-8 DAF-3 DAF-9 DAF-12

TGF-β DAF-4 DAF-14 SMAD P450
Nuclear
Receptor Hormone
Receptor
Serotonergic
Signaling Longevity
SOD-3
AKT-1/AKT-2/SGK-1 DAF-16 MTL-1
Insulin/IGF-I DAF-2 AGE-1
Receptor PI3K FOXO SCL-1
Transcription
Insulin/IGF-I PDK-1 Factor
Signaling PIP2 PIP3 SHSP
DAF-18
PTEN Stress HSF-1
Germ Line Heat Shock
Transcription
Factor
Reproductive
System
Gonad
Figure 4.1 The signaling pathways regulating lifespan in C. elegans. C. elegans senses
environmental cues including food, pheromones, and temperature by sensory organs. The
environmental information is transduced into at least four signaling pathways including
TGF-b, cGMP, serotonin, and insulin/IGF-I. The serotonergic signaling affects the
TGF-b and the insulin/IGF-I signaling. TGF-b is secreted by a pair of specific sensory
neurons. Insulin/IGF-I ligands appear also to be secreted by sensory neurons and are ren-
dered in neuroendocrine system to bind to the receptor DAF-2. This signal is finally trans-
duced to regulate the activity of the transcription factor DAF-16. TGF-b, cGMP, and
insulin/IGF-I signaling pathways converge in the regulation of the activity of P450
DAF-9 to synthesize lipophilic (steroid?) hormones. Those appear to circulate systemi-
cally and activate the nuclear hormone receptor DAF-12. The gonadal tissue and germ-
line cells send the signal that regulates to activate DAF-16 and DAF-12. Various stresses
also regulate to activate heat-shock transcription factor HSF-1 as well as DAF-16. These
transcription factors in concert regulate the aging rate and lifespan by controlling tran-
scription of the target genes.
72 Honda and Honda
On the other hand, DAF-18 PTEN dephosphorylates PIP3 and thus antagonizes
the action of AGE-1. Thus, the PIP3 level is determined by a balance between
generation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN. PIP3
activates PDK-1 (3-phosphoinositide-dependent kinase-1), which in turn phos-
phorylates and activates AKT-1/AKT-2/SGK-1 Ser/Thr kinase. AKT-1/
AKT-2/SGK-1 phosphorylates and inactivates the DAF-16 transcription factor
to be sequestered from the nucleus to the cytoplasm. In this state, adults age
rapidly. On the contrary, when DAF-2 signaling is reduced, DAF-16 is eventually
translocated to the nucleus to promote transcription of target genes. In fact, dis-
rupting AKT-consensus phosphorylation sites in DAF-16 causes nuclear
accumulation, although the nuclear accumulation is not sufficient for lifespan
extension (52,53).
C. elegans worms grow through four larval stages (L1 –L4) before reaching
maturity. However, when the food supply is limited and the population density is
high at the L1 stage, animals become dauer larvae after the L2 stage. The dauer
larva is a developmentally arrested dispersal stage and lives up to several months,
greatly exceeding the normal adult lifespan of about 3 weeks under stressful
environmental conditions (53). It seems that the dauer stage is nonaging,
because the post-dauer life span is not affected by a prolonged dauer stage of
up to 2 months (54). The dauer larva is more resistant to a variety of environ-
mental stresses, including hypoxia, heat, desiccation, and oxidative stress and
has increased levels of SOD and catalase (21,55). The expression of the
MnSOD gene (sod-3) is higher in the dauer larvae than in the adults (23). As
dauer larvae live much longer than adults, some genes expressing altered
levels in dauer state may be the key to longevity. By using serial analysis of
gene expression (SAGE), Jones et al. (56) found that the expression of tts-1 (tran-
scribed telomere-like sequence), a variant histone H1 and a nucleosome assembly
protein possibly relating to the structure or stability of chromatin is high in dauer
larvae. These results suggest that the chromatin structure may change to be more
stable in the dauer state than in the growing state. Holt and Riddle (57) examined
gene expression profiles of carbohydrate metabolism in dauer larvae by using
SAGE. A high gene expression of pyruvate kinase, alcohol dehydrogenase, a
putative cytosolic fumarate reductase, two pyruvate dehydrogenase components,
and a succinyl CoA synthetase a subunit implies that anaerobic metabolism is
prominent in dauer larvae.
By genetic analysis of mutants displaying “dauer larva formation abnor-
mal,” Daf phenotype, a number of genes that regulate dauer formation have
been identified (53). These genes have been assembled into four neuroendocrine
signaling pathways: TGF-b/SMAD, cGMP, serotonin (58) and insulin/IGF-I.
DAF-7, a TGF-b family member expressed in a pair of sensory neurons,
signals through transmembrane receptor kinases DAF-4 and DAF-1. These
receptors regulate the activities of DAF-8 and DAF-14, dwarfin/MAD/DPC-4,
and DAF-3, SMAD transcription factors. The cGMP pathway is composed of
DAF-11, transmembrane guanyl cyclase and DAF-21, HSP90. The mutant
adults in the TGF-b and cGMP pathway do not exhibit a lifespan-extension

phenotype. The serotonin pathway affects TGF-b and insulin/IGF-I signaling.
The mutant of the serotonin pathway has a longer reproductive period than
wild-type animals (58).
TGF-b, insulin/IGF-I, and cGMP signaling pathways converge on DAF-9,
a member of the cytochrome P450 hydroxylase family (59,60) that is implicated
in the synthesis of a lipophilic hormone acting upstream of DAF-12, nuclear
hormone receptor (61,62). Sterols may be the DAF-9 substrate and DAF-12
ligand because cholesterol deprivation displays daf-9 mutant phenotype.
DAF-12 is expressed within almost all cells, whereas DAF-9 is expressed
within two sensory neurons, hypodermal cells, and somatic gonadal cells
thought to be endocrine tissues.
In these pathways, the mutations in the insulin/IGF-I pathway mainly
affect the adult lifespan. The simplest interpretation of these observations is
that the dauer larvae have an efficient life-maintenance mechanism for long
dauer survival under stressful conditions, and that the insulin/IGF-I pathway is
closely related to this mechanism (12,63). Although the mutants in the TGF-ß
and cGMP pathways do not display a lifespan-extension phenotype, these path-
ways interact with the insulin/IGF-I pathway at daf-9 position to affect lifespan
(Fig 4.1). The daf-9 mutations extend lifespan at a certain temperature (59,60).
The daf-9 and daf-12 mutations enhance the lifespan-extension of certain daf-2
mutants (63,64).
Insulin/IGF-I Signaling Pathway and Stress Resistance

To investigate the relationship between lifespan-extension and oxidative-stress
resistance, we screened the oxidative-stress resistance phenotype in various
mutants (6,23). We examined the survival period of each mutant under exper-
imentally induced, acute oxidative stress. We used paraquat, an intracellular
superoxide O2 2 generator, under hyperoxia for acute oxidative stress. The daf-2
†
mutants with an extended lifespan, survived for a longer period of time than
wild-type animals in the presence of paraquat under hyperoxic or normoxic con-
ditions. The daf-2 mutant is also more resistance to menadione, another intra-
cellular O22 generator, under hyperoxia, than wild-type animals. The mutants
†
in the TGF-b pathway and cGMP pathway do not show oxidative-stress resist-
ance. The oxidative-stress resistance seen in the daf-2 mutants is suppressed by
mutations in daf-18 or daf-16 indicating that daf-16 and daf-18 act downstream
of daf-2 to confer oxidative-stress resistance, as well as extended lifespan.
Vanfleteren (22) and Larsen (21) showed that the lifespan-extension mutant
age-1 is more resistant to oxidative stress in old age than wild-type animals at
the same age. We showed that the age-1 mutants of young adults also display
the oxidative-stress resistance. The oxidative-stress resistance in age-1 mutants
is suppressed by daf-16 mutation, indicating that daf-16 is located downstream
of age-1 in the pathway for regulating oxidative-stress resistance.
74 Honda and Honda
On the other hand, oxidative-stress resistance in two alleles of age-1

mutants (m333 and mg44) is not fully suppressed by daf-18 mutation, indicating
that daf-18 does not act downstream of age-1. daf-16 and daf-18 act downstream
of daf-2 in the insulin/IGF-I signaling pathway for oxidative-stress resistance
(23). Taken together, we postulate the following pathway for oxidative-stress
resistance:
daf -2 ! daf -18 ! age-1 ! daf -16

! Oxidative-stress resistance
This pathway is essentially identical to the pathway regulating longevity (63),

suggesting a strong association between lifespan-extension and oxidative-stress
resistance. However, Dorman and Canyon (65) demonstrated that the daf-18
mutation suppressed the lifespan-extension phenotype of another allele of
age-1 (hx546) indicating that daf-18 acts downstream of age-1. Such differences
could be attributed to the differences in severity of the age-1 alleles used.
Two alleles of age-1 mutants (m333 and mg44) display the Daf phenotype,
which is completely suppressed by daf-18 or daf-16 mutations, indicating the
following pathway for dauer formation:
daf -2 ! age-1 ! daf -18 ! daf -16 ! dauer formation
Thus, oxidative-stress resistance is closely associated with longevity but not with
dauer formation. The PIP3 level is maintained under a balance between gener-
ation by AGE-1 PI3 kinase and degradation by DAF-18 PTEN, which could
determine the impact of this pathway. The loss or reduction of function mutations
in age-1 could reduce PI3 kinase activity to drop this second messenger level.
When DAF-18 is reduced, only the preexisting pool of the second messenger
may be insufficient to inhibit longevity and oxidative-stress resistance but
sufficient to inhibit dauer formation. Dillin et al. (66) found that the inactivation
of daf-2 during adulthood by RNAi extends lifespan and increases oxidative-
stress resistance. Since dauer formation is switched in the early larval stage,
the insulin/IGF-I pathway controls the dauer switch and oxidative-stress resist-
ance/longevity independently.
There are several genes in C. elegans that encode SOD enzymes: sod-1
encodes cytosolic CuZnSOD (21), sod-2 and sod-3 each encodes mitochondrial
MnSOD (67 – 69), and sod-4 encodes extracellular CuZnSOD (70). The level
of sod-3 mRNA in daf-2 mutants is higher than that in the wild-type animals
(23). The levels of mRNA transcripts of sod-1, sod-2, and catalase in the daf-2
are similar to those in the wild-type animals. The level of sod-3 mRNA in
daf-2 mutants increases as it develops from the egg to the L2 larval stage
coinciding with increased in oxidative-stress resistance. The elevated level of
sod-3 mRNA in the daf-2 mutants is suppressed by daf-16 and daf-18 mutation
(23). The level of sod-3 mRNA in the age-1 mutants is higher than that in
wild-type animals (6). The elevated level of sod-3 mRNA in age-1 mutants is
suppressed by daf-16 mutation but is not fully suppressed by daf-18 mutation.

These results provide further evidence that the insulin/IGF-I signaling
pathway regulates extended lifespan, oxidative-stress resistance and sod-3
expression in a similar way. These results suggest that the extended lifespan is
correlated with the efficient withdrawal of ROS generated in mitochondria
during normal metabolism.
Murakami and Johnson (19) showed that the insulin/IGF-I pathway
confers resistance to UV exposure. Lithgow (71) indicated that the insulin/
IGF-I pathway also confers increased Cd- and Cu-ion resistance. Metallothio-
neins are metal-binding proteins that are induced in response to a wide variety
of stresses including metal ions and oxidative stress. In C. elegans, there are
two isoforms of metallothioneins. Metallothionein-1 (MTL-1) is induced
by Cd and heat in intestinal cells. Levels of mtl-1 mRNA are high in daf-2
mutants compared with wild-type animals under normal conditions. Cd chal-
lenge induces mtl-1 and mtl-2 in daf-2 mutants more greatly than in wild-type
animals.
The daf-2 mutant is resistant to hypoxia. Scott et al. (72) showed that daf-2
is important for preventing hypoxic death in myocyte and neurons. The signaling
pathway for hypoxia resistance is somewhat distinct from insulin/IGF-I signal-
ing for longevity.
The age-1 and daf-2 mutants survive longer in acute thermal stress than
wild-type animals (5,64). The age-1 mutant has elevated levels of HSP-16 at
normal temperature, and when challenged by heat shock, accumulates greater
levels of HSP16 compared with wild-type animals (41). The hsp-16 transgene
induces heat-stress resistance and extended lifespan both in wild-type and
age-1 mutant animals. The DAF-16 transcription factor is essential for maximal
hsp-16 expression and for lifespan-extension induced by the hsp-16 transgene
(44). DAF-16 translocates into the nucleus upon heat and oxidative stress (73).
Taken together, these results suppose that molecular chaperons play an important
role in the extension of lifespan by preventing the accumulation of conformation-
ally altered protein associated with aging. Muñoz and Riddle (74) isolated ther-
motolerant mutants of C. elegans, and 80% of these mutants exhibit an extended
lifespan, suggesting a strong correlation between stress resistance and lifespan
extension.
From the overall screening of RNAi inactivation of chromosome I genes,
Garigan et al. (18) found that the inactivation of HSF-1, a transcription factor reg-
ulating the response to heat and oxidative stress, shortens the lifespan and causes
premature aging. These findings raise the possibility that the activation of thermal
and oxidative stress response mechanisms may slow down the rate of aging. Hsu
et al. (42) introduced extra-copies of the hsf-1 gene into animals, resulting in
resistance to heat and oxidative stress (paraquat) and lifespan extension. The life-
span extension by hsf-1 extra-copies requires daf-16, suggesting that DAF-16 and
HSF-1 may act together to promote longevity. DAF-16 also appears to act
independently of HSF-1, because hsf-1 RNAi does not prevent DAF-16 from
76 Honda and Honda
accumulating in the nucleus of daf-2 mutants or activating two known DAF-16-

downstream genes, mtl-1 and sod-3. The expression of several shsp genes,
hsp-16.1, hsp-16.49, hsp-12.6, and sip-1 is increased in daf-2 mutants and
decreased in daf-16 mutants. HSF-1 is required for increased shsp gene
expression in daf-2 mutants, thus, HSF-1 functions in the insulin/IGF-1
system. DAF-16 as well as HSF-1 is required to activate shsp expression after
heat shock. Furthermore, both the DAF-16 binding site (GTAAAc/tA) and
HSF-1 binding site (TTCTa/cGAA) are located at the regulatory regions of
the shsp genes. The RNAi inactivation of each shsp genes partly shortens the life-
span of daf-2 mutant and HSF-1 overexpressed animals. These results suggest
that DAF-16 from insulin/IGF-I signals and stress, and HSF-1 from stress, act
together to activate the transcription of a variety of genes inducing lifespan
extension (Fig. 4.1). The RNAi inactivation of shsp accelerates the onset of
polyglutamine-expansion protein aggregation in a C. elegans model for triplet
repeat disease. This result suggests that SHSPs may influence the aging rate
and polyglutamine aggregation coordinately by in part, preventing the improper
association of oxidized or abnormally folding proteins.
DAF-16 Transcription Target

Lee et al. (32) identified genes bearing the DAF-16 binding site, within the pro-
moter region in C. elegans and Drosophila. In addition to sod-3 (MnSOD) (23),
an FK506-binding protein and a nucleolar protein (75) previously known to be
regulated by DAF-16, they found several DAF-16 target genes that are regulated
by insulin/IGF-I signaling in a daf-16-dependent manner in C. elegans. The
expression of a ser/thr phosphatase (C25E10.12) gene is upregulated in daf-2
mutants in a daf-16-dependent manner. When C25E10.12 is inactivated by
RNAi, it shortens the lifespan of the age-1 mutant but does not alter the lifespan
of the wild-type animals.
Ookuma et al. (76) surveyed genes with the DAF-16 consensus-binding site
within the regulatory region in C. elegans. They found a candidate DAF-16 tran-
scriptional target gene, scl-1, that is a putative secretory protein with an SCP
domain and is homologous to the mammalian cysteine-rich secretory protein
(CRISP) family. The expression of scl-1 is upregulated in daf-2 and age-1
mutants. The inactivation of scl-1 by RNAi reduces both the lifespan and
stress resistance of these mutants.
Using DNA microarray analysis, Murphy et al. (31) identified genes whose
expression increases or decreases when insulin/IGF-I signaling activity is
reduced. Many daf-2 (2)-induced genes encoded proteins that may be involved
in the synthesis of downstream signaling molecules such as a steroid or lipid-
soluble hormone. The reduction of these gene activities with RNAi shortened
lifespan. These results suggest that insulin/IGF-I signaling produces a putative
secondary endocrine system that promotes longevity. In addition to sod-3
(23) and mtl-1 (17), they found that the expression of the catalase genes
(ctl-1: peroxisomal and ctl-2: cytosolic), the glutathione-S-transferase gene, and

the small heat-shock protein genes (hsp-12.6, hsp-16.1, hsp-16.2, hsp-16.11,
hsp-16.49, and sip-1) are all increased when DAF-2 activity is reduced. The inhi-
bition of the activities of these genes with RNAi generally shortens the lifespan of
daf-2 mutants. This supports the hypothesis that genes that increase resistance to
environmental stress contribute to longevity. They also found that some antimicro-
bial and metabolic genes are upregulated when DAF-2 activity is reduced. In
addition to old-1 (38), they found that reducing DAF-2 activity downregulates
specific life-shortening genes including guanyl cyclase and vitellogenin. These
results suggest that multiple effector genes, whose expression is regulated by the
insulin/IGF-I signaling pathway, act in a cumulative manner to influence longev-
ity. They found that the DAF-16 consensus site, GTAAAc/tA is present not only
in the promoter of daf-2 (2)-induced genes, but also in the promoters of daf-2
(2)-downregulated genes, indicating that DAF-16 may both directly repress and
activate gene expression. They also found a new site, CTTATCA, in the promoter
of many of these genes suggesting that DAF-16 regulates these genes with another
transcription factor.
Reproductive System and Lifespan

The somatic gonad and germ line can be selectively deleted by laser beam
irradiation of their precursor cells at the early larval stage in C. elegans. Hsin
et al. (77) ablated the germ line and left the somatic gonad intact resulting in a
large extension of lifespan in wild-type animals. Ablation of the somatic gonad
(entire gonad organ) has no effect on wild-type lifespan. In the case of certain
daf-2 mutants, ablation of the somatic gonad as well as the germ line promote
an extended lifespan. In daf-16 mutants, ablation of the germ line does not
extend lifespan and ablation of the somatic gonad rather shortens lifespan. One
model to explain these results is that the germ line produces a signal that inacti-
vates DAF-16 to shorten lifespan and that the somatic gonad emits a signal
reducing insulin/IGF-I signaling to extend lifespan (77) (Fig. 4.1). In fact, the
ablation of germ line induces DAF-16 translocation into the nucleus in the
intestine (52).
In daf-12 mutants, ablation of the germ line does not extend the lifespan but
ablation of the somatic gonad rather shortens lifespan. Gerisch et al. (59) showed
that ablation of the germ line does not extend lifespan in certain daf-9 mutants.
Thus, DAF-9 and DAF-12 are required for germ-line ablation to extend the life-
span. As DAF-9 is expressed within the somatic gonad, the lifespan-regulating
signal from the gonad may be a DAF-9-related hormone. The extended lifespan
in certain daf-2 mutants is promoted by daf-9 or daf-12 mutations (59,63,64).
This result suggests the existence of cross-talk between DAF-9/DAF-12 signal-
ing and insulin/IGF-I signaling.
Germ-line ablation induces heat and oxidative-stress (paraquat) resistance
and increased sod-3 expression as well as lifespan extension. The heat resistance
78 Honda and Honda
as well as lifespan-extension is reproduced by mutations in the mes-1 and glp-1

genes that lack germ line (78).
Nervous System and Lifespan

Apfeld and Kenyon (79) found, by mosaic analysis, that daf-2 controls lifespan in
a cell-nonautonomous manner in that the loss of daf-2 (þ) extrachromosomal
duplication from the neuron-generating cell lineage of daf-2 mutant restores
the extended lifespan (79). The expression of normal daf-2 (þ) or age-1 (þ)
genes in neurons but not muscle, or intestines restores the normal lifespan in
daf-2 or age-1 mutants, respectively. These findings suggest that insulin/IGF-I
signaling in neurons alone is sufficient to specify a normal lifespan (80).
C. elegans senses environmental signals including food, temperature,
pheromones, and osmotic pressure through ciliated sensory neurons located in
sensory organs in the head and tail. Mutations that disrupt the cilium structure
or signal transduction in sensory neurons extend the lifespan. This lifespan exten-
sion is suppressed by the daf-16 mutation (81). These results support the notion
that environmental cues are transduced into insulin/IGF-I signaling, which exerts
its function for regulating lifespan in the nervous system.
Mitochondrial Electron Transport and Lifespan

The other lifespan-extension mutants are those in a set of clk genes (clk-1, clk-2,
clk-3, gro-1, and isp-1), which display altered biological timing, including the
defecation cycle, pharyngeal pumping for food intake and cell cycle, and duration
of development (10). CLK-1 is involved in the biosynthesis of coenzyme-Q,
a substance that regulates energy production in the mitochondria. The clk
mutants do not display the Daf phenotype. The maximum life span is extended
in a clk-1 mutant is only 40%. However, the mean life span of the double
mutant, daf-2; clk-1, is over five times the normal lifespan (10). This synergistic
effect indicates that the insulin/IGF-I signaling pathway and the clk mechanism
interact in determining life span. Mutants in clk-1 lack coenzyme-Q9 and instead
accumulate the biosynthetic intermediate demethoxy-Q9, which does not seem to
function in electron transport (82). ATP levels are normal or slightly elevated in
clk-1 mutants (83). Withdrawal of coenzyme-Q from the diet extend the lifespan
of wild-type and daf-2 mutant animals (84). Mouse and human genes homolo-
gous to C. elegans clk-1 restore normal rhythmic movement and lifespan in
clk-1 mutants (85).
CLK-2 is homologous to Saccharomyces cerevisiae Tel2p, an essential
DNA-binding protein that regulates telomere length in yeast. The clk-2 gene is
found to be the same with rad-5, that is, DNA damage checkpoint gene (86).
Mutants in clk-2/rad-5 are hypersensitive to UV- and X-irradiation. CLK-2/
RAD-5 acts in a pathway that partially overlaps the MRT-2 checkpoint
pathway, in which genotoxic stress induces cell cycle arrest and apoptosis of
germ-line cells. In the mutant of mrt-2, which is homologous to the S. pombe
rad1 checkpoint gene, the germ line cannot be passed indefinitely (87). The mrt-2
mutant exhibits progressive telomere shortening, late-onset chromosome fusions
and x-ray hypersensitivity of the germ line. The mutant in cep-1, a homologue of
the mammalian p53 tumor suppressor gene, also shows resistance to DNA
damage-induced apoptosis of germ-line cells independent of the casparse
pathway (88). The effect of clk-2/rad-5 mutation on telomere length has not
yet been established (86,89,90) and the relationship between altered biological
timing and the DNA-damage checkpoint is not yet known.
GRO-1 is a tRNA-modifying enzyme in the mitochondria. Mutants in
isp-1, which encodes iron sulfur protein of mitochondrial Complex III, inhibit
mitochondrial respiration, show slow biological timing, and extend lifespan
(11). Mutants in mitochondrial leucyl-tRNA synthetase (lrs-2) that is predicted
to compromise mitochondrial electron transport through suppressing the trans-
lation of 12 mitochondrial polypeptides encoded by the mitochondrial genome,
show slow biological rhythms and lives 200% longer than wild-type animals.
The extended lifespan of lrs-2 does not require DAF-16 (91).
Mutants in clk-1 do not display oxidative-stress resistance or increased
expression of sod-3. However, clk-1 mutation largely promotes oxidative-stress
resistance and an increased expression of sod-3 in daf-2 mutants (23) as well
as extended lifespan(10). On the other hand, mutation in isp-1 cannot promote
an extended lifespan of daf-2 and itself causes oxidative-stress resistance and
increased expression of sod-3 (11). Insulin/IGF-I signaling seems to regulate
the expression of sod-3 in concert with mitochondrial energy metabolites.
Mammalian MnSOD has a dual role: one is as a protective function against
the damaging effects of ROS (92 –95), and another is as a regulator of levels of
ROS that mediate signal transduction (96 –99), [reviewed in Ref. (100)]. Interest-
ingly, there is an intriguing link between an insulin signaling, and the gene
expression of MnSOD in vertebrates: TNF-a, which interferes with insulin-
receptor signaling (101), induces the gene expression of MnSOD (102). The
FOXO3a transcription factor, a mammalian homologue of DAF-16, activates
MnSOD transcription and the subsequent reduction of ROS (103). ROS particu-
larly H2O2, has been found to be involved in insulin or IGF-I signaling (104). The
link between insulin signaling and ROS, may have been conserved among
diverse species. Further studies are needed to clarify the roles of sod-3 in
C. elegans aging. The functions of two MnSOD isoforms, sod-2 and sod-3, in
C. elegans are now under investigation.
From systematic RNAi screening of 5690 chromosome I and II genes in
C. elegans, Lee et al. identified genes that are related to lifespan. Fifteen percen-
tage of the genes influencing lifespan are specific for mitochondrial functions,
including mitochondrial carriers, electron-transport chain components, and a
mitochondrial ribosomal subunit. The responses to heat and oxidative stress
(paraquat and H2O2) of the RNAi inactivation of these genes are different from
each other, suggesting that the lifespan-extension by impaired mitochondrial
function is not simply due to reduction in ROS generation. Lifespan-extension
80 Honda and Honda
by RNAi inactivation of some genes is suppressed by daf-16 mutation, however,

DAF-16 translocation into the nucleus is not observed when any of these genes
influencing lifespan are RNAi inactivated (91).
Dillin et al. (105) reported the reduction of electron transport chain activity
by RNAi of NADH/ubiquinone oxidoreductase (nuo-1, complex I), cytochrome-c
reductase (cyc-1, complex III), cytochrome-c oxidase (cco-1, complex IV), and
ATP synthase (atp-3, complex V). These RNAi treatments all decreased ATP
levels and increased lifespan. Lifespan-extension by these RNAi treatments is
not suppressed by the daf-16 mutation, and is promoted by the daf-2 mutation
and germ-line ablation, but not by somatic – gonad ablation. In contrast to
these RNAi treatments, somatic – gonad ablation shortens the lifespan of clk-1
mutants to the wild-type level. Therefore, the mechanism of lifespan-extension
of these RNAi-treated animals is likely to differ from that of clk-1 mutation. In
fact, ATP levels are normal or slightly elevated in clk-1 mutants (83).
Interestingly, these RNAi treatments in early development are critical for
lifespan-extension, in contrast with insulin/IGF-1 signaling, which functions
exclusively in adulthood to influence lifespan, suggesting that a regulatory
system monitoring mitochondrial activity during development specifies the
aging rate. As animals with reduced ATP levels by these RNAi treatments
only in adulthood do not live longer than untreated animals, the mechanism of
lifespan-extension by CR in adulthood is not likely to reduce the respiratory
rate. Together, these findings indicate that mitochondrial electron transport is
generally a regulator of lifespan as well as behavioral rates. This regulator
seems to mainly function independently on insulin/IGF-I signaling pathway
but there seems to be possible cross-talk between them (Fig. 4.2).
Development Adult
Aging
Reproduction
Respiratory Chain Insulin / IGF-I

Activity Signaling
Caloric Restriction
Figure 4.2 Timing of lifespan regulator’s action in C. elegans. A regulatory system

monitoring respiratory chain activity during development specifies the aging rate. On
the other hand, insulin/IGF-I signaling and CR act in adulthood to influence the aging
rate. Conditions of reproductive system influence the aging rate mainly through insulin/
IGF-I signaling. There seems to be possible cross-talk between respiratory chain activity,
insulin/IGF-I signaling and CR.
Rea and Johonson (106) proposed a general model in which the utilization
of fermentative malate dismutation as alternative energy generation can induce
longevity in a variety of lifespan-extension mutants.
Sir-2 has been postulated to play a role in lifespan-extension by CR, and
mediates chromatin silencing through histone deacetylase activity that depends
on NAD (107). The transgene of sir-2.1, one of four in the C. elegans SIR-2
family extends the lifespan. This extended lifespan is suppressed by daf-16
mutation and is not promoted by daf-2 mutation, indicating that SIR-2.1 func-
tions upstream of daf-16 in the insulin/IGF-I signaling pathway (108). In
yeast, CR extends the lifespan by increasing the activity of Sir-2. Resveratrol,
a polyphenol found in red wine, stimulates the activity of SIRT1, a human
Sir-2 homologue, and increases DNA stability and extends the lifespan of
yeast, C. elegans, and Drosophila (109).
LIFESPAN REGULATION IN DROSOPHILA

The mutations of Drosophila melanogaster in the chico gene that encodes an
insulin receptor substrate in insulin/IGF-I signaling extend lifespan by up to
48% (24). The chico mutations increase resistance to starvation stress but not
to heat stress. Slight resistance to oxidative stress (paraquat) is seen in chico het-
erozygotes chi2/þ but not in homozygotes chi2/chi2. The effects on stress
resistance of chico mutations are not so marked as in the daf-2 and age-1
mutations of C. elegans. Total SOD activity is higher in chico homozygotes
than chico heterozygotes and wild-type flies (24). On the other hand, when the
food supply is ,65% of the control food, the chico mutant has a rather shorter
lifespan than wild-type flies, suggesting that CR and the insulin/IGF-I pathway
in Drosophila act through overlapping mechanisms (110).
A heteroallelic insulin-like receptor (InR) mutation of Drosophila results in
dwarf and extends lifespan by up to 85%. The InR mutation impairs juvenile
hormone (JH) synthesis. Treatment of the InR mutants with a JH analog metho-
prene, restores the lifespan to the normal level. Fruit flies overwinter as a reproduc-
tive diapause controlled through the downregulation of JH synthesis. Ovaries of
InR dwarf females morphologically resemble the ovaries of diapause wild-type
flies. Diapause flies do not age and are stress resistant. SOD activity of the InR
mutants increases 2-fold. Reduced JH may induce InR mutants to inappropriately
express an efficient survival diapause program resulting in extended lifespan (25).
A loss-of-function mutation in the gene, Methuselah, extends lifespan by
35% (111). The methuselah mutant is resistant to a number of different stresses,
including starvation, heat and oxidative stress (paraquat). The methuselah gene
encodes a G-protein-coupled seven-transmembrane-domain receptor suggesting
that a signal-transduction pathway regulates lifespan and stress resistance.
An insertional mutation in Indy, a homologue of a mammalian sodium
dicarboxylate cotransporter, a membrane protein that transports Krebs cycle
82 Honda and Honda
intermediates, extends lifespan. Indy may induce a metabolic state that mimics
CR (112).
Orr and Sohal (113) indicated that the simultaneous overexpression of
CuZnSOD and catalase extends the lifespan of Drosophila (113). Sun and
Tower (114) demonstrated that the overexpression of CuZnSOD extends life-
span, and that catalase overexpression has no lifespan-extension effect. Parkes
et al. (115) showed that the overexpression of human cytosolic CuZnSOD
(SOD1) in the motor neuron extends the lifespan of Drosophila and rescues
the lifespan of a short-lived SOD1 null mutant (115). They also showed that over-
expression of SOD1 and catalase combination in the motor neuron diminishes the
lifespan-extension effect of SOD1 overexpression (116). Mockett et al. (117)
showed that the overexpression of MnSOD in the mitochondria of Drosophila
increases heat tolerance but rather decreases lifespan. On the other hand, Sun
et al. (118) demonstrated that the overexpression of MnSOD extends lifespan
in a dose-dependent manner without decreasing the metabolic rate. The mito-
chondrial overexpression of catalase causes an increase in resistance to oxidative
stress (H2O2 and paraquat) and cold stress, but there is no lifespan-extension
effect (119).
The overexpression of glutathione reductase in Drosophila increases the
survival period under hyperoxia but no effect on lifespan under normoxia,
suggesting that glutathione reductase is critical for protecting against robust
oxidative stress but not against aging-associated damages (120).
Orr and Sohal (121) posed the question whether transgenes of these antiox-
idant enzymes could decrease the aging rate on the basis that the lifespan of the
controls was too short. They introduced a combination of antioxidant-enzyme
genes into relatively long-lived Drosophila strains and examined their lifespan.
The transgenes of various combinations of CuZnSOD, MnSOD, catalase, and
thioredoxin reductase, all have no lifespan-extension effect, although activities
of some enzymes actually increase above wild-type levels (122). Currently, it
may be difficult to assert the establishment of the free-radical theory of aging
from antioxidant-enzyme trangene experiments with Drosophila.
Methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized
methionine in proteins by reducing methionine sulfoxide back to methionine.
Overexpression of the msrA gene predominantly in the nervous system, increa-
ses resistance to oxidative stress (paraquat) and extends the lifespan of
Drosophila (123).
Using DNA microarray, Zou et al. (124) assessed age-related changes
in gene expression levels in Drosophila and compared these changes with
those induced by oxidative stress (paraquat). Age-related downregulation genes
are involved in reproduction, metabolism, detoxification, chaperone, and
protein turnover. Age-related downregulation genes functioning as chaperones
or detoxification agents are SHSP, Hsp26, alcohol dehydrogenase, a-tocopherol
transfer-related protein, and a homologue of microsomal epoxide hydrolase.
One-third of the age-related genes overlap the paraquat-regulated genes. Both
age-related and paraquat-regulated upregulation genes are homologues of

arsenite-translocating ATPase and glutathione S-transferase D1 that are involved
in detoxification (124).
Treatment of Drosophila with 4-phenylbutyrate (PBA), an inhibitor of
histone deacetylase, induces resistance to starvation and oxidative stress (para-
quat) and extends lifespan. PBA dramatically changes the gene expression
pattern associated with increased histone acetylation, and the SOD expression
is increased greatly (125).
LIFESPAN REGULATION IN MAMMALS

In mice, the constitutive and ubiquitous overexpression of CuZnSOD in various
tissues does not extend lifespan (126). However, ROS may not necessarily be
unrelated to aging in mammals. In fact, KO mice of the p66Shc gene that
show extended lifespan indicate resistance to oxidative stress in various
aspects. Cultured p66shc2/2 cells are resistant to H2O2- or UV-induced apop-
tosis (127). In p66Shc KO mice, systemic oxidative stress including plasma LDL
oxidation and arterial oxidative damage is reduced (128).
In mammals, the DAF-16 homologues regulate apoptosis and cell-cycle
progression in response to stress. Nemoto et al. (129) demonstrated that
mutations in p66shc2/2 activate the FOXO transcription factor, a mammalian
DAF-16 homologue, through the reduction in cellular ROS levels. FOXO tran-
scription factor regulates both H2O2 scavenging and oxidative stress resistance
in response to oxidative stress. The DAF-16 homologue activates MnSOD to
protect cells from apoptosis caused by glucose deprivation (103). It has been
found that DAF-16 homologues regulate transcription of the DNA-damage
response gene, Gadd45 that functions as a G2 – M checkpoint in response to
UV-induced damage (130) or oxidative stress (131). DAF-16 homologues down-
regulate D-type cyclins to promote cell-cycle progression (132).
Several single-gene mutants have been reported to have a long lifespan [for
review see Ref. (133)]. The Ames dwarf mouse (df/df), a mutant of the Pro-1
gene (134); and the Snell dwarf mouse (dw/dw), a mutant of the Pit-1 gene
(135), live more than 40 –60% longer than wild-type mice. Both genes are
required for the development of the pituitary cells that secrete growth hormone
(GH), thyroid stimulating hormone and prolactin. Furthermore, KO of GH recep-
tor, GHR-KO mice (Ralon model) are found to live longer than heterozygotes and
wild-type mice (136). GHR-KO decreases plasma IGF-I and insulin levels (137).
Thyroid hormone synthesis is also reduced in these dwarf mice. Hypothyroidism
induced by a thyroxin injection into neonatal rats actually extends lifespan (138).
Hypothyroidism in rats decreases heart mitochondrial H2O2 production and
suppresses oxidatively damaged DNA (8-oxodG) (139).
Heterozygous KO mice of Igf1r, an IGF-I receptor gene, live longer than
wild-type controls. Lifespan-extension is associated with increased resistance
to oxidative stress in vivo (paraquat) and in vitro (H2O2). Mutation in Igf1r
84 Honda and Honda
reduces the phosphorylation of p66Shc that is a substrate of IGF-IR (26). IGF-I

injection with an adeno-associated virus into motor neurons prolongs the lifespan
of amyotrophic lateral sclerosis (ALS) model mice with mutated CuZnSOD
(140). IGF-I may prevent ROS-mediated impairment (140), although how the
CuZnSOD mutations induce the neurodegeneration is not yet clearly understood.
Disruption of the insulin receptor gene in the fat tissue also extends the
lifespan of mice. These animals have reduced fat mass, which is often seen
upon CR (27). CR also leads to a decrease in plasma IGF-I levels early in
adult life, suggesting the involvement of IGF-I in CR-induced lifespan-extension
(141, 142). However, CR extends further the lifespan of Ames dwarf mice,
suggesting that the mechanisms regulating lifespan-extension in CR and dwarf
mice are not identical (143).
Bonafe et al. (144) examined the relationship between human longevity
and polymorphic variants of the IGF-I response pathway genes, namely, IGF-I
receptor, PI3-kinase, insulin receptor substrate-1, and FOXO1A. Free IGF-I
plasma levels and human longevity are co-regulated by a set of IGF-I signaling
pathway genes. Insulin/IGF-I signaling may regulate longevity in a wide range
of organisms from worms to humans.
Genome-wide searches for aging-, CR-, stress-, and lifespan-extension
mutation-associated gene expression changes in mammals have been conducted
using DNA microarray. The gene expression profile of the skeletal muscle
between adults and elderly mice and the effect of CR were examined (33).
Fifty-eight genes of 6347 genes, express more markedly in the elderly animals
than in the adults. Of the 58 genes upregulated with age, 16% were related to
stress responses, including the heat shock factors Hsp71 and Hsp27, protease
Do, and the DNA damage-inducible gene GADD45. The largest differential
expression between adult and elderly animals is the mitochondrial sarcomeric
creatine kinase, a critical target for ROS-induced inactivation. Most alterations
are prevented by CR. Transcriptional patterns of calorie-restricted animals
suggest that CR retards the aging process by inducing a metabolic shift toward
increased protein turnover and decreased macromolecular damage.
Lee et al. (145) further examined the gene-expression changes in the neo-
cortex and cerebellum of mice during aging and showed that aging induces a
gene-expression profile indicative of an inflammatory and oxidative stress
response. CR selectively attenuates the age-associated induction of genes related
to inflammatory and stress responses such as JunB, Fos, and DnaJ homologues.
Kayo et al. (146) examined the expression of 7070 genes in muscles
between young and aged rhesus monkeys and the effect of CR. Aging resulted
in the selective up-regulation of transcripts involved in inflammation and oxi-
dative stress, and a downregulation of genes involved in mitochondrial electron
transport. These age-related changes are not inhibited by adult-onset CR. The
retardation of age-related changes in gene expression may depend on the species.
Miller et al. (34) assessed CR-induced changes in the expression of 2352
genes in the liver and compared them with long-lived GHR KO mice. A total
of 352 genes are increased or decreased by CR. A parallel alteration in the

expression of 29 genes can be seen between the calorically restricted and GHR
KO mice. The gene expression profile of the intestine of Se(2)-diet fed mice
was examined. A low Se state actually reduces the activity of glutathione per-
oxidase, a Se-containing antioxidant enzyme, and upregulates the expression
of genes related to genetic instability and oxidative stress, including XP-E,
GADD34, GADD45, metallothionein-I, HSP27, and HSP40. A low Se state
appears to induce a stress response at the transcriptional level (147).
Edwards et al. (148) searched genes among a 9977-gene array that are dif-
ferentially expressed in the heart after treatment with paraquat, with variously
aged mice. They demonstrated that only young mice display a significant increase
in the expression of all three isoforms of GADD45, a DNA damage-responsive
gene. Additionally, the number of immediate early response genes found to be
induced by paraquat was considerably higher in the younger animals. These
results indicate that there is an age-related impairment of specific inducible path-
ways in response to oxidative stress in the murine heart.
REPLICATIVE LIFESPAN AND OXIDATIVE STRESS

Cultured normal cells generally have a finite replicative lifespan as originally
shown by Hayflick and Moorhead (149). Human diploid fibroblasts lose the
capacity to replicate after vigorous proliferation, and enter a viable but non-
proliferative state of senescence. Senescent cells are characterized by the upre-
gulation of cyclin-dependent kinase inhibitors, p21SDI1/WAF1/CIP1 (150), and
p16INK4a (151), and hypophosphorylated Rb (152). Human fibroblasts grown
in hypoxia have an extended replicative lifespan (153).
Mouse embryonic fibroblasts (MEFs) senesce after vigorous proliferation,
and then grow into an immortal cell in normoxia (20% oxygen), whereas human
normal fibroblasts never achieve immortality. Parrinello et al. (154) found that
MEFs do not senesce in hypoxia (3% oxygen) and that MEFs accumulate
more DNA damage in normoxia than hypoxia, and more damage than human
fibroblasts in normoxia. These results suggest that human cells have a superior
ability to prevent or repair oxidative DNA damage compared to murine cells.
Stress-induced premature senescence (SIPS) occurs when cells are grown
in hyperoxia (155), and treated with hyperbaric oxygen (156) and H2O2 (157),
with appearance of several biomakers of replicative senescence. Hyperoxia
(40% oxygen) increases the rate of telomere shortening from 90 bp per popu-
lation doubling (normoxia) to more than 500 bp per population doubling (hyper-
oxia) (158), suggesting that telomere shortening causes premature senescence.
On the other hand, Gorbunova et al. (159) indicated that the overexpression of
the catalytic subunit of telomerase cannot prevent SIPS but can protect from
stress-induced apoptosis and necrosis. This finding suggests that SIPS may not
be due to telomere shortening.
86 Honda and Honda
The addition of N-acetylcysteine, a cellular ROS scavenger, into culture

medium extends the replicative lifespan of human embryonic fibroblsts (160).
On the other hand, the addition of L -buthionine-(R, S)-sulfoximine, a specific
inhibitor of GSH synthetase, shortens the replicative lifespan (160). These
results suggest that cellular ROS levels are a determinant of replicative lifespan.
The inactivation of cytosolic CuZnSOD (SOD1) by RNAi induces prema-
ture senescence in human fibroblast depending on p53 induction (161). In human
fibroblasts with inactivated p53, the SOD1 RNAi is without effect suggesting that
oxidative DNA damage mediates premature senescence. The overexpression of
extracellular CuZnSOD decreases the intracellular peroxide content, slows the
telomere-shortening rate, and extends the replicative lifespan under normoxia
and hyperoxia (162).
The overexpression of an activated V12Ras induces premature senescence
in human fibroblasts and induces increased mitochondrial ROS generation (162).
In hypoxia, the overexpression of the V12Ras cannot induce premature senes-
cence or increase the level of p21 that is related to senescent phenotype (163).
These findings suggest that ROS play a role in the regulation of the replicative
lifespan.
CONCLUSION
In the last decades, gene-manipulation studies have revealed that gene networks
exist for the determination of lifespan in diverse species. There appear to be
common and different features among species. ROS appear at various points in
the aging processes, and play a variety of roles, including the manifestation of
oxidative stress and the mediation of signal transduction. The precise role of
ROS in the aging process is not yet clear. DNA microarray technology allows
us to show global gene expression profiles governing the lifespan and aging
rate, although these studies are just beginning. Using the data from DNA micro-
array analysis, painstaking studies are needed to clarify the aging mechanism to
analyze how various gene products work in concert to regulate the aging rate.
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5
Anti-Oxidant Modulation in
Immune Function
Robert Francis Grimble

University of Southampton, Southampton, UK
Introduction 98
The Immune Response to Infection Injury and Inflammatory Agents 98
The Function of Pro-Inflammatory Cytokines During the Normal
Response to Infection and Injury 99
Adverse Effects of Pro-Inflammatory Cytokines 101
Anti-Oxidant Defenses are Interlinked and Interdependent 101
A Decline in Anti-Oxidant Defenses and Increased Oxidant Damage
Follows Infection and Injury 102
Aging Increases Oxidative and Inflammatory Stress 103
Mechanisms Underlying Low-Grade Inflammation During Aging 105
Mechanisms of the Effects of Oxidants and Anti-Oxidants
on Inflammation and Immune Function 106
Effects of Anti-Oxidants on Immune Function 110
Effects of Vitamin E 110
Ascorbic Acid and Immune Function 111
Glutathione and Immune Function 111
Effects of Substances that Act as Precursors for GSH or Cofactors
in Enzyme Pathways Associated with GSH on Immune Function 112
Effects of Precursors of GSH on Immune Function 112
Effects of Vitamin B6 on Immune Function 114
97
98 Grimble
Effects of Folic Acid on Immune Function 115

Taurine and Immune Function 115
Conclusions 116
References 117
INTRODUCTION
The production of oxidant molecules is both an integral part of the immune
response and a major modulator of immune function. As a corollary to this
concept anti-oxidant defences play a pivotal role in immune function by poten-
tially interacting at both of these levels. In practice, however, anti-oxidants
exert only a modulatory influence on the effects of oxidants on immune function.
The reduction in anti-oxidant defenses that occurs during the normal process of
inflammation may be an attempt by the body to expose pathogens to the full
strength of oxidants produced by the immune system. This phenomenon is, as
will be seen later, not without risk to the host.
In this chapter, the circumstances under which oxidant production occurs
will be described, the effect of oxidants on immune function will be examined,
the mechanisms whereby oxidants produce these effects will be outlined, and
the interaction of dietary and endogenously produced anti-oxidants on immune
function will be discussed.
THE IMMUNE RESPONSE TO INFECTION INJURY

AND INFLAMMATORY AGENTS
The immune system has a large capability for immobilizing invading microbes,
creating a hostile environment for them, and bringing about their destruction (1).
The immune system may also become activated, in a similar way to the res-
ponse to microbial invasion, by a wide range of stimuli and conditions that
do not directly involve pathogens; these include burns, penetrating and
blunt injury, the presence of tumor cells, environmental pollutants, radiation,
exposure to allergens, and the presence of chronic inflammatory diseases.
The response of the immune system to this diverse range of agents and con-
ditions contains many common elements. These, however, vary in intensity
according to their impact on the body. The elements of the response include
the production of immunomodulatory proteins (cytokines), oxidant molecules
(hydrogen peroxide, superoxide, hypochlorous acid, and nitric oxide), anti-
inflammatory hormones (cortisol), natural antagonists (cytokine receptor
antagonists), and anti-oxidants enzymes (superoxide dismutase, catalase, and
glutathione peroxidase) (2).
Anti-Oxidant Modulation in Immune Function 99
THE FUNCTION OF PRO-INFLAMMATORY CYTOKINES DURING

THE NORMAL RESPONSE TO INFECTION AND INJURY
The pro-inflammatory cytokines, interleukin-1b (IL-1), interleukin-6 (IL-6), and
tumor necrosis factor-a (TNF-a), have widespread metabolic effects on the body
and bring about the process of inflammation. Many of the signs and symptoms
experienced after infection and injury, such as fever, loss of appetite, weight
loss, negative nitrogen, sulfur and mineral balance, and lethargy are caused
directly or indirectly by pro-inflammatory cytokines (Fig. 5.1). Indirect effects
of cytokines are mediated by neural actions on the adrenal glands and endocrine
pancreas resulting in increased secretion of the catabolic hormones adrenalin,
noradrenalin, glucocorticoids, and glucagon. Insulin insensitivity occurs in
addition to this “catabolic state.” The biochemistry of an infected individual is
thus fundamentally changed in a way which will ensure that the immune
system receives nutrients from within the body (Fig. 5.2). Muscle protein is cat-
abolized to provide amino acids for synthesizing new cells, glutathione (GSH),
and proteins for executing and controlling the immune response. Furthermore,
amino acids are converted to glucose (a preferred fuel, together with glutamine,
for the immune system (1). The extent of the rearrangement in protein metab-
olism is evident from changes in excretion of substances in the urine following
infection and injury. An increase in urinary nitrogen and sulfur excretion
Raised blood lipids Fever Glucose synthesis
Plasma copper
Production of Effects of
oxidant molecules cytokines TNF, Plasma Zn
IL1 and IL6
Plasma iron
Acute phase
Appetite Loss of lean
proteins
loss and tissue and
lethargy fat
Increased urinary
nitrogen sulphur
and mineral losses
Figure 5.1 Alterations in body function, composition, and metabolism that result from
production of pro-inflammatory cytokines following infection and injury and during
chronic inflammatory disease.
100 Grimble
Trauma/infection/burns
Immune system activation

Immuno-
Pro-inflammatory cytokines - nutrition
Feedback
systems
T and B cells
IL10, Heat
shock proteins
Oxidants Antioxidant
defence Glucose
- Nutrient
release from
Glutamine
Pathogen Tissue
killing host tissues
damage
Sulphur Glutathione
amino synthesis
Creation of a acids
Antioxidant
hostile environment defences
Appetite loss strengthened
Figure 5.2 Main metabolic events and their purpose during the response to infection
and injury.
occurs. There is an increase in urinary nitrogen excretion from 9 g/day in mild

infection to 20 –30 g/day following major burn or severe traumatic injury (3).
The loss of nitrogen from the body of an adult during bacterial infection, may
be equivalent to 60 g of tissue protein and in a period of persistent malarial
infection, equivalent to over 500 g of protein. However, during the response to
infection and injury the urinary excretion of sulfur increases to a lesser extent
than that of nitrogen (4), suggesting that sulfur amino acids are preferentially
retained and so “spared” from catabolism. Large decreases in plasma glycine,
serine, and taurine concentrations occur following infection and injury. These
compounds are metabolically related to sulfur amino acids. The changes may
reflect enhanced utilization of these amino acids. Many substances produced in
enhanced amounts in response to pro-inflammatory cytokines, are particularly
rich in these amino acids. These substances include GSH, which contains
glycine, glutamic acid, and cysteine; metallothionein (the major zinc-transport
protein), which contains glycine, serine, cysteine, and methionine to a composite
percentage of 56%; and a range of acute phase proteins that contain up to 25% of
these amino acids in their structure. Among these compounds, GSH, metallothio-
nein, and the acute phase protein ceruloplasmin are important endogenously
derived anti-oxidants. If an increased demand for sulfur and related amino
acids is created by the inflammatory response, then provision of additional
supplies of these amino acids may assist the response (2). Indeed whey protein,
which is rich in sulfur amino acids, has been shown to be beneficial in the
treatment of children suffering from burn injury (5). Children receiving whey
protein, rather than a standard high protein enteral formulation, had higher
plasma C3 complement and IgG concentrations and less bacteremic days and
better survival.
Infection with human immunodeficiency virus (HIV) has been shown to
cause substantial excretion of sulfate in the urine during the asymptomatic
phase of the disease (6). The losses reported were equivalent to 10 g of cysteine
per day, in contrast to losses of 3 g/day for healthy individuals on a “Wester-
nized diet.” As cysteine is the precursor for both sulfate and GSH, this finding
may be linked with the decline in tissue glutathione pools that has been observed
in HIV infection (7). Clearly, such a depletion of anti-oxidant defenses will have
serious effects.
ADVERSE EFFECTS OF PRO-INFLAMMATORY CYTOKINES

Although pro-inflammatory cytokines are essential for the normal operating of
the immune system, they play a major role in tissue damage during many inflam-
matory diseases such as rheumatoid arthritis, inflammatory bowel disease,
asthma, psoriasis, multiple sclerosis, and cancer (2,8). In these type of disease,
combating pro-inflammatory cytokine production with antibodies to TNF-a
has shown clinical benefit. Good examples of success with this strategy have
been observed in Crohn’s disease and rheumatoid arthritis (9,10). Pro-
inflammatory cytokines are also thought to be important in the development of
atheromatous plaques in cardiovascular diseases (11). In conditions such as cer-
ebral malaria, meningitis, and sepsis, pro-inflammatory cytokines are produced in
excessive amounts and are an important factor in increased mortality (8). Clearly,
in all of the diseases mentioned earlier, cytokines are being produced in the
wrong biological context. Cytokines exert an adverse influence on anti-oxidant
defenses, because in most of these diseases and conditions, pro-inflammatory
cytokines bring about a loss of lean tissue, which is associated with depleted
tissue-GSH content.
ANTI-OXIDANT DEFENSES ARE INTERLINKED

AND INTERDEPENDENT
Many of the components of antioxidant defense interact to maintain antioxidant
status (1). Glutathione and the enzymes that maintain it in its reduced form are
central to effective anti-oxidant status. For example, when oxidants interact
with cell membranes, the oxidized form of vitamin E that results is restored to
its reduced form by ascorbic acid. Dehydroascorbic acid formed in this process
is reconverted to ascorbic acid by interaction with the reduced form of gluta-
thione. Subsequently, oxidized glutathione formed in the reaction, is reconverted
102 Grimble
Methionine
Homocysteine
Vit B6
Cysteine
Dehydro
Vit E ascorbic Glutathione
Oxidants reduced acid GSH Glutathione
reductase
Riboflavin
Vit E Ascorbic Glutathione
oxidised acid GSSG
Figure 5.3 Main linkages and relationships between key components of the anti-oxidant
defenses system.
to the reduced form of glutathione by glutathione reductase (Fig. 5.3). Vitamins E

and C and glutathione are thus intimately linked in anti-oxidant defense. The
interdependence of the various nutritional component of anti-oxidant defense
is illustrated in a study in which healthy subjects were given 500 mg ascorbic
acid per day for 6 weeks (12). A 47% increase in glutathione content of red
blood cells occurred.
Nutrients that do not have anti-oxidant properties also contribute indirectly
to the robustness of anti-oxidant defenses. These include Vitamin B6 and ribofla-
vin. Vitamin B6 is the cofactor in the metabolic pathway for the biosynthesis of
cysteine (Fig. 5.3). Cellular cysteine concentration is rate limiting for glutathione
synthesis. Riboflavin is a cofactor for glutathione reductase, which maintains the
major part of cellular glutathione in the reduced form (Fig. 5.3).
A DECLINE IN ANTI-OXIDANT DEFENSES AND INCREASED

OXIDANT DAMAGE FOLLOWS INFECTION AND INJURY
There is a growing body of evidence that anti-oxidants suppress inflammatory
components of the response to infection and trauma and enhance components
related to cell-mediated immunity. The reverse situation applies when anti-
oxidant defenses become depleted.
Although the body strives to maintain them, observations in experimental
animals and patients indicate that anti-oxidant defenses become depleted during
infection and after injury. For example, in mice infected with influenza virus,
there were 27%, 42%, and 45% decreases in the vitamin C, vitamin E, and glu-
tathione contents of blood, respectively (13). In a further study on mice, given a
dose of endotoxin sufficient to cause septic shock, both GSH and ascorbic acid
were shown to fall precipitately in spleen, lymph nodes, and peritoneal macro-
phages (14). In asymptomatic HIV infection, substantial decreases in glutathione
concentrations in blood and lung epithelial lining fluid have been noted (15). In
patients undergoing elective abdominal operations, the glutathione content of
blood and skeletal muscle fell by over 10% and 42%, respectively, within 24 h
of the operation (16). Blood concentrations returned rapidly to preoperative
values; however, concentrations in muscle were still depressed 48 h after the
operations. A diverse range of clinical treatments and diseases, all of which
involve the inflammatory process, have been shown to lead to a decrease in
tissue anti-oxidant concentrations. These include hepatitis C, ulcerative colitis,
and cirrhosis. In patients with malignant melanoma, metastatic hypernephroma,
and metastatic colon cancer, plasma ascorbic acid concentrations fell from
normal to almost undetectable levels within 5 days of commencement of treat-
ment with IL-2 (17). In patients with inflammatory bowel disease, substantial
reductions in ascorbic acid concentrations occurred in inflamed gut mucosa
(18). As a general consequence of the weakening of anti-oxidant defenses,
during disease, oxidative damage is apparent in a wide range of clinical con-
ditions in which cytokines are produced. Lipid peroxides and increased thiobar-
bituric acid reactive substances are present in blood of patients with septic shock,
asymptomatic HIV infection, chronic hepatitis C, breast cancer, cystic fibrosis,
diabetes mellitus, and alcoholic liver disease. Peroxides also increase following
cancer chemotherapy, open heart surgery, bone marrow transplantation, and
hemodialysis (17).
There is evidence, from studies on experimental animals and patients, that
the decrease in strength of anti-oxidant defenses may exert a deleterious influ-
ence. When glutathione status was reduced in rats by injection of diethyl
maleate, which binds irreversibly to GSH rendering it inactive, a sublethal
dose of TNF became lethal (19), thus illustrating the importance of GSH in pro-
tection from the adverse effects of pro-inflammatory cytokines. A parallel
phenomenon was noted in patients with sepsis. The onset of sepsis in patients
led to a transient decrease in the total anti-oxidant capacity of blood plasma
(a functional measure of the total anti-oxidant content) (20). The capacity
returned to normal values over the following 5 days. However, this was not
the case for patients who subsequently died, in whom values remained well
below the normal range.
As well as increasing the risk of direct oxidant damage, a reduction in
the strength of anti-oxidant defenses also indirectly increases the risk of
damage to the host via transcription factor activation leading to upregulation of
pro-inflammatory cytokine production (see later).
AGING INCREASES OXIDATIVE AND INFLAMMATORY STRESS

Although inflammation is an integral part of the response to pathogens, it would
appear that the general level of inflammatory stress increases during aging
104 Grimble
irrespective of the presence of infections and inflammatory disease. It is inter-

esting to note that aging is also associated with a decline in immune function.
An age-related increase of IL-6 concentration has been found in serum,
plasma, and supernatants of mononuclear blood cell cultures from apparently
healthy elderly people and centenarians (21 –23). Increases in serum levels
of this cytokine have also been found as early as 30 –40 years of age (24),
particularly in men (25). Furthermore, population studies have shown that the
magnitude of increase in the concentration of IL-6 is a reliable marker for
functional disability and a predictor of disability and mortality in the elderly
(26,27).
An enhanced capacity for the release of pro-inflammatory cytokines by leu-
cocytes may contribute to the pathogenesis of ischaemic stroke. Grau et al. (28)
investigated the lipopolysaccharide (LPS)-induced release of IL-1, IL-6, IL-8,
and TNF-a in whole blood from 20 patients with a history of ischaemic stroke
under the age of 50, and 21 age- and sex-matched healthy control subjects.
Release of IL-8 was significantly higher and release of TNF-a and IL-6 tended
to be higher in young stroke patients than in control subjects. The authors con-
cluded that high inducible release of IL-8, TNF-a, and IL-6 may contribute to
the odds of suffering from ischaemic stroke in young adults (28).
The question of whether aging is associated with chronic elevation of
cytokine production or whether an increased capacity for cytokine production
develops during aging is an interesting matter for consideration. An insight
into this issue can be gained from the response to surgery where an inflammatory
stimulus is applied at a defined moment in time, making it easy to follow the sub-
sequent response, Ono et al. (29) investigated the age-related changes in the
inflammatory response to surgical stress in patients with gastric cancer, under-
going distal gastrectomy. Patients were divided into 2 groups: .75 years of
age (elderly group) and 75 years of age (young group). Serum IL-6 levels,
TNF-a production, and CD11b/CD18 expression by monocytes, and the post-
operative clinical course were compared between the 2 groups. The clinical
course, serum IL-6 levels, monocyte production of TNF-a, and monocyte
expression of CD11b/CD18 were used as markers of the systemic response:
TNF-a production by LPS-stimulated monocytes and CD11b/CD18 expression
on monocytes after the inflammatory stimulus of surgery were significantly
higher in the elderly than in the young group. Moreover, serum IL-6 levels on
the first postoperative day in the elderly group were significantly higher than
those in the young group. The incidence and duration of systemic inflammatory
response syndrome were significantly greater in the elderly than in the young
group. The authors concluded that activation of monocytes and raised blood con-
centrations of pro-inflammatory cytokines occur more readily in the elderly than
in young subjects.
Paradoxically, both loss of body weight and lean tissue and obesity are
found in elderly populations. Is there a link between this phenomenon and
increased levels of inflammation?
Mechanisms Underlying Low-Grade Inflammation During Aging

There are a number of potential mechanisms for the higher level of chronic inflam-
mation and hence oxidant stress, observed in the elderly than in younger subjects.
The first of these is that the elderly are experiencing a higher level of asympto-
matic bacteriuria. This possibility was studied in 40 consecutive patients (age
70 –91 years) admitted to hospital for functional disability. Patients were exam-
ined for the presence or absence of bacteria in urine. Twenty subjects had positive
urine culture and 20 sex- and age-matched subjects had negative urine culture.
Patients with asymptomatic bacteriuria had significantly increased levels of
circulating tumor necrosis factor receptors (sTNFR-I) and a higher number of neu-
trophils in the blood compared with the group without bacteriuria. Thus, the study
provides some support for the hypothesis that asymptomatic urinary infections are
associated with low-grade inflammatory activity in frail, elderly subjects (30).
A second potential mechanism resides in endocrine changes during aging. In
aging, dysregulation of secretion of hormones that come under the regulation of
the HPA axis may occur. This may have impact on the regulation of cortisol
secretion. Cortisol is important as an anti-inflammatory agent. The effect of
aging on glucocorticoid (GC) sensitivity of pro-inflammatory cytokine production
was examined in elderly men, testosterone-treated elderly men, and young con-
trols. Stress-induced increases in cortisol did not differ significantly between
experimental groups, but GC sensitivity increased significantly in young controls
and testosterone-treated elderly men, whereas a decrease was found in untreated
elderly men. As the increase in GC sensitivity after stress serves to protect the indi-
vidual from detrimental increases of pro-inflammatory cytokines, the disturbed
mechanism in elderly men may result in enhancement of inflammation. The
decrease in sensitivity is linked to decreased testosterone production during
aging as impaired sensitivity was partly restored by testosterone treatment (31).
Now, there is a large body of evidence suggesting that the decline in ovarian func-
tion with menopause is associated with spontaneous increases in pro-inflammatory
cytokines. Studies in men and postmenopausal women indicate a remarkable indi-
vidual constancy in the ability of PBMCs to produce TNF-a ex vivo. Genetic deter-
minants underlie this constancy. However, in premenopausal women, production
is highly variable at an individual level indicating how ovarian hormones are able
to override the influence of genotype (32). The exact mechanisms by which estro-
gen interferes with cytokine activity are still incompletely known but may poten-
tially include interactions of the estrogen receptor with other transcription factors,
modulation of nitric oxide activity, anti-oxidative effects, plasma membrane
actions, and changes in immune cell function. Experimental and clinical studies
also strongly support a link between the increased state of pro-inflammatory
cytokine activity and postmenopausal bone loss (33).
The third potential mechanism is the general increase in the incidence in
inflammatory disease that occurs with aging. However, the link between inflam-
mation and aging is of a two-way nature, as chronic inflammation may contribute
106 Grimble
to the pathogenesis of many diseases encountered with greater frequency in the

elderly population.
A fourth mechanism for the increase in inflammatory and oxidant
stress during aging may be the increased association with obesity in the
middle-aged and the elderly populations. It is well known that the adipocyte is
able to produce a wide range of pro-inflammatory cytokines. Furthermore, a
body mass index of 25 is associated with increased signs of inflammation
(raised plasma acute phase protein concentrations), which decline if a program
of weight loss is initiated [for review see Ref. (34)].
A fifth potential mechanism involving changes in peroxisome proliferator-
activated receptor-a (PPARalpha) activity has been revealed by recent studies
on aged mice (see next section).
MECHANISMS OF THE EFFECTS OF OXIDANTS

AND ANTI-OXIDANTS ON INFLAMMATION
AND IMMUNE FUNCTION
The oxidant molecules produced by the immune system to kill invading organ-
isms may activate at least two important families of proteins that are sensitive
to changes in cellular redox state. The families are nuclear transcription factor
kappa B (NFkB) and activator protein 1 (AP1). These transcription factors act
as “control switches” for biological processes, not all of which, as illustrated
earlier, are of advantage to the individual. NFkB is present in the cytosol in an
inactive form, by virtue of being bound to IkB. Phosphorylation and dissociation
of IkB renders the remaining NFkB dimer active. Activation of NFkB can be
brought about by a wide-range of stimuli including pro-inflammatory cytokines,
hydrogen peroxide, mitogens, bacteria and viruses and their related products, and
UV and ionizing radiations. The dissociated IkB is degraded and the active NFkB
is translocated to the nucleus where it binds to response elements in the enhancer
and promoter regions of genes. A similar translocation of AP1, a transcription
factor composed of the proto-oncogenes c-fos and c-jun, from cytosol to
nucleus, also occurs in the presence of oxidant stress. Binding of the transcription
factors is implicated in activation of a wide-range of genes associated with
inflammation and the immune response, including those encoding cytokines,
cytokine receptors, cell adhesion molecules, acute phase proteins, and growth
factors (35) (Fig. 5.4).
Unfortunately, NFkB also activates transcription of the genes of some
viruses, such as HIV. This sequence of events, in the case of HIV, accounts
for the ability of minor infections to speed the progression of individuals
who are infected with HIV towards AIDS because, if anti-oxidant defenses are
poor, each encounter with general infections results in cytokine and oxidant
production, NFkB activation, and an increase in viral replication. It is thus unfor-
tunate that reduced cellular concentrations of GSH are a common feature of
asymptomatic HIV infection (15).
Inflammatory stimuli
LPS, oxidants,stress
Transcription
AP1 NFkB Adhesion
factors
molecules
Acute HIV
IL2 IL1, replication
Cell phase IL6,
proliferation proteins IL8
TNF
GSH
synthesis
Figure 5.4 The influence of activation of AP1 and NFkB on gene transcription following
infection and injury.
Oxidant damage to cells will indirectly create a pro-inflammatory effect by

the production of lipid peroxides. This situation may also lead to upregulation of
NFkB activity, as the transcription factor has been shown to be activated in endo-
thelial cells cultured with linoleic acid, the main dietary n-6 poly-unsaturated
fatty acid, an effect inhibited by vitamin E and NAC (36). The interaction
between oxidant stress and an impaired ability to synthesize glutathione that
results in enhanced inflammation is clearly seen in cirrhosis, a disease that
results in high levels of oxidative stress and an impaired ability to synthesize
GSH (37). In this study, an inverse relationship between glutathione con-
centration and the ability of monocytes to produce IL-1, IL-8, and TNF-a was
observed. Furthermore, treatment of the patients with the GSH prodrug,
oxothiazalidine-4-carboxylate (procysteine), increased monocyte GSH content
and reduced IL-1, IL-8, and TNF-a production (Fig. 5.5). Thus, anti-oxidants
might act, to prevent NFkB activation, by quenching oxidants. However,
NFkB and AP1 may not respond to changes in cell redox state in the same
way. When rats were subjected to depletion of effective tissue GSH pools by
administration of diethyl maleate, there was a significant reduction in lymphocyte
proliferation in spleen and mesenteric lymph nodes (38). An increase in inflam-
matory stress would be expected in this study. In an in vitro study using HeLa
cells and cells from human embryonic kidney, both TNF and hydrogen peroxide
resulted in activation of NFkB and AP1 (39). Addition of the anti-oxidant sorbitol
108 Grimble
Methionine NAC OTZ

Glutamine
Vit B6
Folic
Cysteine Glutamic acid
acid
Glutathione synthesis Glycine
OTZ (Procysteine)L- 2- NAC n- acetyl

oxothiazolidine-4-carboxylate cysteine
Figure 5.5 Nutrients and drugs that may be used to improve glutathione synthesis during
treatment for infection and injury.
to the medium suppressed NFkB activation (as expected) but (unexpectedly) acti-
vated AP1. Thus, the anti-oxidant environment of the cell might exert opposite
effects upon transcription factors closely associated with inflammation (e.g.,
NFkB) and cellular proliferation (e.g., AP1). Evidence for this biphasic effect
was seen when glutathione was incubated with immune cells from young
adults (40). A rise in cellular glutathione content was accompanied by an increase
in IL-2 production, and lymphocyte proliferation, and a decrease in production of
the inflammatory mediators, PGE2 and LTB4. Modification of the glutathione
content of liver, lung, spleen, and thymus in young rats, by feeding diets con-
taining a range of casein (a protein with a low sulfur amino acid content)
concentrations, changed immune cell numbers in lung (41). It was found that
in unstressed animals, the number of lung neutrophils decreased as dietary
protein intake and tissue glutathione content fell. However, in animals, given
an inflammatory challenge (endotoxin) liver and lung GSH concentrations
increased directly in relation to dietary protein intake. Lung neutrophils,
however, became related inversely with tissue glutathione content. Addition of
methionine to the protein deficient diets normalized tissue glutathione content
and restored lung neutrophil numbers to those seen in unstressed animals fed a
diet of adequate protein content (Fig. 5.6).
Thus, it can be hypothesized that anti-oxidants exert an immuno-enhancing
effect by activating transcription factors that are strongly associated with cell pro-
liferation (e.g., AP1) and an anti-inflammatory effect by preventing activation of
NFkB by oxidants produced during the inflammatory response.
The molecular mechanisms that underlie the increased inflammatory and
oxidant stress that accompanies aging has been examined in aged mice. The
role of changes in PPAR-a activity in the process has been examined by
Poynter and Daynes (42). Their findings suggest a role for PPAR-a in the main-
tenance of redox balance during the aging process.
25
Influence of GSH
20 Rats injected with LPS and NF B activity?
Neutrophils/lung area*
15
Control rats injected
10 with sterile saline
Influence of GSH
5 and AP1 activity?
0
0 0.5 1 1.5 2 2.5
GSH (umol/g lung tissue)
Figure 5.6 The relationship between lung neutrophil and glutathione content in animals
fed diets with sulfur amino acid contents ranging from 2.2 to 6.5 g/kg and then given
either a control saline or lipopolysaccharide injection intraperitoneally. Note: Neutrophils
were counted in total lung sections and are expressed as the total number 100 observed
per subdivision of the graticule field.
In aged mice, the administration of agents capable of activating the alpha

isoform of the PPAR-a receptor was able to restore the cellular redox balance.
Evidence for this effect came from the observation that tissue lipid peroxidation
was decreased, NF-kB activity decreased, and spontaneous inflammatory
cytokine production was reduced. Aged animals bearing a null mutation in
PPAR-a failed to elicit these changes following treatment with PPAR-a activa-
tors, but remained responsive to vitamin-E supplementation, thereby highlighting
independent effects of anti-oxidants and PPAR-a on inflammation. Aged mice
were also found to express reduced transcript levels of PPAR-a and the
peroxisome-associated genes, acyl-CoA oxidase and catalase. Supplementation
of aged mice with PPAR-a activators or with vitamin E caused elevations in
these transcripts to levels seen in young animals. A number of natural endogen-
ous molecules have been found that are capable of activating PPARs. For
example, 15-deoxy-D12,14 prostaglandin J2 represents a natural PPAR-g ligand
(43,44). Many specific fatty acid species and their derivatives, especially poly-
unsaturated fatty acids (45 –48), the leukotriene B4 (49), and the eicosanoid
8(S)-hydroxyeicosatetraenoic acid (50), have been shown to be ligands for
PPAR-a. Activation of PPARs has been demonstrated to antagonize signaling
through an array of important pathways, including STATs, AP-1, and NF-kB
(45,51 – 55). Poynter and Daynes (42) demonstrated that NF-kB is present in
an active state in the macrophages and lymphocytes that reside in the spleens
of aged mice (56). This active NF-kB correlated with the expression of the
110 Grimble
NF-kB-regulated genes IL-6, IL-12, macrophage migration inhibitory factor,

cyclooxygenase-2, and TNF (56). The administration of specific PPAR-a activa-
tors, or vitamin E, to aged rodents effectively reduced the elevated levels of
active NF-kB, re-established control over pro-inflammatory cytokine production,
and reduced lipid peroxide levels in various tissues (56).
EFFECTS OF ANTI-OXIDANTS ON IMMUNE FUNCTION

Effects of Vitamin E
A number of studies in which anti-oxidant status has been raised by dietary sup-
plementation indicate that improvement of anti-oxidant status is associated with
an increase in cellular aspects of immune function. Vitamin E exerts modulatory
effects on both inflammatory and immune components of immune function. In
general, vitamin E deficiency and low tissue vitamin E content enhance com-
ponents of the inflammatory response and suppress components of the immune
response. Dietary vitamin E supplementation brings about the opposite effect.
Studies in animals have demonstrated that vitamin E deficiency impairs cellular
and humoral immunity and is associated with an increased incidence of disease.
Supplementation of the diet with vitamin E, at levels that are several fold greater
than requirements, increases resistance to a number of pathogens. Resistance
of chickens and turkeys to Escherichia coli and of mice to pneumococci, was
enhanced by vitamin E supplementation. A similar phenomenon may also
occur in humans, since epidemiological evidence shows lower incidence of
infectious disease in subjects with high plasma a-tocopherol concentrations.
Rats consuming diets that were deficient in vitamin E, and given injections of
endotoxin, showed a greater degree of anorexia and greater concentrations of
plasma a-1-acid glycoprotein and IL6, than animals consuming adequate
amounts of the vitamin. In smokers, a low intake of vitamin E was associated
with an increased intensity of the inflammatory response to cigarette smoke.
Plasma concentrations of a-1-acid glycoprotein was 50% higher in subjects in
the lowest tertile of intake compared with the values for subjects in the highest
tertile (57). Large doses of a-tocopherol 50 mg/kg given i.p. significantly
decreased the number of neutrophils in airspaces of rats given endotoxin aerosols;
however, there appeared to be no change in NFkB or AP1 activation (58).
Supplementation of the diet of healthy subjects, and smokers with 600 IU/
day a-tocopherol, for 4 weeks, suppressed the ability of PBMCs to produce
TNF-a (59). Intense exercise of healthy young and elderly subjects results in
the appearance of a mild inflammatory response characterized by raised blood
IL1, IL6, and acute phase protein concentrations. A twice daily supplement of
400 IU of a-tocopherol inhibited the response.
A dose of 600 IU/day a-tocopherol given to healthy elderly subjects,
for 235 days, increased delayed type hypersensitivity and raised antibody titers to
hepatitis B (60). When elderly subjects were supplemented with 800 IU of
a-tocopherol for 30 days, there was a 50% increase in the delayed type
hypersensitivity response, a 65% increase in IL-2 production, and a decrease in

oxidative stress as indicated by a major decrease in plasma thiobarbituric acid sub-
stances (TBARS). An enteral feed, enriched with vitamin E, vitamin C, and taurine,
given to intensive care patients decreased total lymphocyte and neutrophil content
in bronchio-alveolar lavage fluid (decreased inflammation) and resulted in a
reduction in organ failure rate, reduced requirement for artificial ventilation, and
a reduction of 5 days in the requirement for intensive care (61). These results high-
light that reduced inflammation and improved immune function are interrelated.
Ascorbic Acid and Immune Function

The observation that high concentrations of vitamin C are found in phagocytic cells
has underpinned the concept that ascorbic acid is an important nutrient for optimal
immune function. However, although the role of vitamin C as a key component of
antioxidant defense is well established, most studies have shown only minor effects
on a range of immune functions, except in cases where the vitamin may be acting
by interacting with GSH metabolism. Unlike deficiencies in vitamins B6 , E, and
riboflavin, deficiency of vitamin C does not cause atrophy of lymphoid tissue. In
a study of ultra marathon runners, dietary supplementation with 600 mg/day of
ascorbic acid reduced the incidence of upper respiratory tract infections after a
race by 50% (62). It is interesting to note that strenuous exercise has been
shown to deplete tissue glutathione content. The interrelationship between gluta-
thione and ascorbic acid may therefore play a role in the effect of exercise on
immune function.
When immunological parameters and anti-oxidant status were measured
in adult males fed 250 mg/day of vitamin C for 4 days followed by 5 mg/day
for 32 days, plasma ascorbic acid and glutathione decreased and impairment of anti-
oxidant status became evident from a doubling in semen 8-hydroxydeoxyguanosine
concentration (a measure of oxidative damage to nucleic acids) during the second
dietary period (63). A fall in vitamin content in peripheral blood mononuclear
cells was noted and the delayed type hypersensitivity reaction to seven recall anti-
gens was significantly reduced in intensity. These results again highlight that the
strength of inflammation and immune function is interrelated in an inverse manner.
A further potential facet of the effect of ascorbic acid on immune function,
other than by modulating anti-oxidant defenses, was shown in an in vitro study
that examined the effects of physiological concentrations of the vitamin on
binding of AP1 to its receptor in macrophages. The vitamin enhanced binding.
The effect was shown to be independent of the oxidation state of iron, which is
important in binding of AP1 to its receptor (64).
Glutathione and Immune Function

One of the first indications that glutathione influences aspects of immune func-
tion, which are related to T lymphocytes, came from a study in which the GSH
content of these cells was measured in a group of healthy volunteers (65). The
numbers of helper (CD4þ) and cytotoxic (CD8þ) T cells increased in parallel
112 Grimble
with intracellular GSH concentrations up to 30 nmol/mg of protein. However,

the relationship between cellular glutathione concentrations and cell numbers
was complex, with numbers of both subsets declining at intracellular glutathione
concentrations between 30 and 50 nmol/mg of protein. The study also revealed
that cell numbers were responsive to long-term changes in GSH content. When
the subjects engaged in a program of intensive physical exercise daily for
4 weeks, a fall in glutathione concentrations occurred. Individuals with gluta-
thione concentrations in the optimal range before exercise, who experienced a
fall in concentration after exercise, showed a 30% fall in CD4þ T cell numbers.
The decline in T cell number was prevented by administration of N-acetyl
cysteine (NAC is metabolized to cysteine, see later section). This study suggests
that immune cell function may be sensitive to a range of intracellular sulfhydryl
compounds including glutathione and cysteine. In HIVþ individuals and patients
with AIDS, a reduction in cellular and plasma glutathione has been noted (15). At
present, it is unclear whether the depletion in lymphocyte population that occurs
in these subjects is related to this phenomenon. However, in a large randomized,
double-blind, placebo-controlled trial, administration of 600 mg/day of NAC for
7 months, resulted in both anti-inflammatory and immunoenhancing effects (6).
A decrease in plasma IL-6 concentration occurred, together with an increase in
lymphocyte count and in the stimulation index of T lymphocytes in response
to tetanus toxin. The precise mechanism underlying the complex effects of
changes in cellular glutathione content are not clear, and whether they are
related to GSH function as an anti-oxidant or to some other property, such as
the effect of GSH on thioredoxin, is not apparent. However, a recent study
suggests that glutathione promotes IL-12 production by antigen-presenting
cells so driving T helper cells along the Th1 pathway of differentiation (66).
Paradoxically, when GSH is processed by glutathione-S-transferases (GST), in
pathogens, suppressive effect on immune function are achieved. The enzyme
in Faciola hepatica in rat is responsible for inhibition of T cell function and
downregulation of nitric oxide production thereby allowing the parasite to
evade the animals immune defenses. Likewise, the human parasites Onchocerca
volvulus and Trapanozoma cruzi may also, via GST, interfere with immune func-
tion in the host. On the basis of these observations, a hypothesis has been pro-
posed that some pathogens via release of GST interfere with the functions of
cells of the immune system through their ability to scavenge GSH (67).
EFFECTS OF SUBSTANCES THAT ACT AS PRECURSORS

FOR GSH OR COFACTORS IN ENZYME PATHWAYS
ASSOCIATED WITH GSH ON IMMUNE FUNCTION
Effects of Precursors of GSH on Immune Function
A number of strategies have evolved to raise GSH concentrations in depleted
individuals. As shown in Fig. 5.4, there are three potential ways of enhancing
cellular GSH content: administration of the three amino acids (cysteine, glutamic
acid, and glycine) that comprise the tri-peptide, either singly or in various
combinations; administration of cofactors for the metabolic pathways leading
to GSH production, that is, vitamin B6, riboflavin, and folic acid; administration
of synthetic compounds, which become converted to precursors of GSH.
Although cysteine supplies are the primary determinant of the ability to
synthesize GSH, in some circumstances an insufficiency in the other two
amino acids, from which it is made, might limit synthesis. Glutamine (a precursor
of glutamate), for example, has been shown to maintain hepatic GSH in animals
poisoned with acetaminophen, to enhance gut GSH synthesis in rats, when given
by gavage, and to enhance hepatic GSH synthesis when given intravenously to
rats (68). In human studies, a similar effect on gut GSH concentrations was
noted (69). Glycine supplements have been shown to raise hepatic GSH in rats
exposed to hemhorragic shock (70). In this condition, however, the metabolic
demand for glycine is increased as glycine is the sole nitrogen donor for haem
synthesis and, therefore, becomes rate limiting for GSH synthesis.
There are many studies that illustrate the ability of sulfur amino acid avail-
ability to influence tissue GSH concentrations (71). Studies using animal models
of inflammation have shown that a low protein diet will suppress glutathione syn-
thesis, a situation that is reversed by provision of cysteine or methionine (41,72).
Because cysteine is unstable in its reduced form, is toxic in high doses, and
is mostly degraded in the extracellular compartment, several compounds have
been used to deliver cysteine directly to cells. These are L -2-oxothiazalidine-4-
carboxylate (OTC) and NAC. OTC is an analog of 5-oxoproline in which the
4-methylene moiety has been replaced by sulpfur. It provides an excellent sub-
strate for 5-oxoprolinase (an intracellular enzyme). The enzyme converts OTC
to S-carboxy-L -cysteine, which is rapidly hydrolyzed to L -cysteine. NAC
rapidly enters the cell and is immediately deacylated to yield L -cysteine.
Recent animal and clinical trials with NAC and OTC have demonstrated the
ability of the compounds to enhance GSH status (7,73,74). In studies on patients
with sepsis, NAC infusion was shown to increase blood GSH, decrease plasma
concentrations of IL-8 and soluble TNF receptors (an index of TNF production),
improve respiratory function, and shorten the number of days needed in intensive
care (74,75). Although not affecting mortality rates, NAC shortened hospital
length of stay by .60%. OTZ increased whole blood GSH in peritoneal dialysis
patients, normalized tissue GSH in rats fed with sulfur amino acid deficient
diet, and decreased the extent of inflammation in a rat peritonitis model (74).
In a randomized double blind controlled study on asymptomatic HIV-infected
patients, oral OTC treatment increased GSH concentrations in whole blood (6).
Other randomized studies on asymptomatic HIV positive patients in the presence
and absence of anti-retroviral therapy (ART), have shown that NAC can raise
blood GSH, increase natural killer cell activity and enhance stimulation indices
of T cells incubated with mitogen or tetanus toxin (6,76). Interestingly, the rise
in T-cell function was accompanied by a fall in plasma IL-6 in subjects receiving
114 Grimble
ART as well as the drug. Furthermore, studies have shown that survival time was
improved in HIVþ patients who maintained high concentrations of GSH in
CD4þ T lymphocytes (77). It could therefore be surmised that improved
T-cell function and reduced inflammation are modulated by improvement on
anti-oxidant status in these patients. a-Lipoic acid provides a further means of
enhancing tissue GSH content (73). The compound is reduced to dihydrolipoic
acid, which converts cystine to cysteine. This change has functional significance
for glutathione status in lymphocytes, because the xc-transport system, which is
needed to take up cystine into the cells, is weakly expressed and is inhibited by
glutamate, whereas the neutral amino acid transport system that takes up cysteine
is functional. Cysteine, upon gaining entry to the immune cells is rapidly con-
verted to GSH. Flow cytometric analysis of freshly prepared human peripheral
blood lymphocytes shows that lipoic acid is able to normalize a subpopulation
of cells with severely compromized thiol status rather than increasing the level
in all cells above normal values (78). Therefore, lipoic acid may also prove to
be a useful clinical agent for restoring cellular GSH concentration in immuno-
compromised subjects.
Effects of Vitamin B6 on Immune Function

Vitamin B6 , although having no anti-oxidant properties, plays an important part
in anti-oxidant defenses because of its action in the metabolic pathway for the
formation of cysteine, which, as indicated earlier, is the rate limiting precursor
in glutathione synthesis. Vitamin B6 status has widespread effects on immune
function (79). Vitamin B6 deficiency causes thymic atrophy and lymphocyte
depletion in lymph nodes and spleen. Antigen processing is unaffected. However,
the ability to make antibodies to sheep red blood cells is depressed. In human
studies, the ability to make antibodies to tetanus and typhoid antigens is not
seriously affected. Various aspects of cell-mediated immunity are also influenced
by vitamin B6 deficiency. Skin grafts in rats and mice survive longer during
deficiency, and guinea-pigs exhibit decreased delayed hypersensitivity reactions
to bacille Calmete-Guerin (BCG) administration. Deficiency of vitamin B6 is rare
in humans but can be precipitated with the anti-TB drug isoniazid. However,
experimental deficiency in elderly subjects has been shown to reduce total
blood lymphocyte numbers and decrease the proliferative response of lympho-
cytes to mitogens (80). Similarly, IL-2 production is reduced by deficiency of
the vitamin. Restoration of vitamin B6 intake to normal by dietary supplements
restores immune function. It is unclear, at present, whether a similar situation
occurs in younger subjects.
One mechanism for the effect of vitamin B6 on immune function may be
due to the importance of the vitamin in cysteine synthesis, as outlined earlier.
Deficiency of the vitamin may limit the availability of cysteine for glutathione
synthesis. In rats, vitamin B6 deficiency resulted in decreases of 12% and 21%
in glutathione concentrations in plasma and spleen, respectively (81). In
healthy young women, large doses of vitamin B6 (27 mg/day for 2 weeks)
resulted in a 50% increase in plasma cysteine content (82), presumably by
increased flux through the transulfuration pathway. As cysteine is a rate-limiting
substrate for glutathione synthesis, these findings may have implications for the
response to pathogens because of the importance of glutathione in lymphocyte
proliferation and anti-oxidant defense. However, although vitamin B6 has cellular
effects on the immune system, evidence is lacking of any effect on the inflamma-
tory response.
Effects of Folic Acid on Immune Function

Folic acid plays a crucial role in DNA synthesis suggesting that every aspect
involving cell proliferation might be affected by deficiency in the vitamin.
Indeed, cell-mediated immunity is especially affected by deficiency in humans
and animals (83). Folate deficiency also impairs natural killer cell activity in
rats (84).
As the vitamin is also intimately involved in sulfur amino acid metabolism,
it might be expected that the vitamin would modulate anti-oxidant status and
immune function. However, there is evidence that only the second of these
two effects occurs. Indeed, oxidative stress may impair folate metabolism. In a
double-blind, placebo-controlled crossover intervention in healthy subjects, it
was found that although a folate rich diet and folate supplements caused a fall
in plasma homocysteine concentrations, there was no change in anti-oxidant
activity (plasma and red blood cell glutathione peroxidase activity and red cell
superoxide dismutase activity) or oxidant damage (plasma malonaldehyde)
(85). Infection and inflammation are often associated with hyperhomocysteine-
mia, an indicator of folate insufficiency (86). It has been hypothesized that the
underlying cause of this effect is that the active form of folate (tetrahydrofolate)
is susceptible to oxidation during the oxidative stress involved in infection and
inflammatory disease (86). Therefore, folate insufficiency is the result rather
than the cause of a weakening in anti-oxidant defenses during the immune
response.
Taurine and Immune Function

Taurine, together with sulfate, can be regarded as biochemical end products of
cysteine metabolism. However, it is apparent that taurine also plays a role in
immune function. It is the most abundant free nitrogenous compound (often
incorrectly classified as an amino acid) in cells. It is a membrane stabilizer and
regulates calcium flux thereby controlling cell stability. It has been shown to
possess anti-oxidant properties and to regulate the release of pro-inflammatory
cytokines in hamsters, rats, and humans (87 – 89).
The possibility that taurine might have immunomodulatory properties was
indicated in studies in obligate carnivores, such as cats, in which taurine is an
essential nutrient owing to an inability to synthesize the compound. In cats
116 Grimble
deprived of taurine, substantial impairment of immune function occurs (88). A

large decline in lymphocytes, an increase in mononuclear cells, and a decrease
in the ability of these cells to produce a “respiratory burst” and to phagocytose
bacteria, occurs. There was a rise in g-globulin concentrations in deficient
animals. Spleen and lymph nodes showed regression of follicular centers
and depletion of mature and immature B lymphocyte numbers. The changes
were reversed by inclusion of taurine in the diets. Studies in other species
have also reported effects of supplementation on immune system and function.
In mice, administration of taurine prevented the decline in T cell number that
occurs with aging and enhanced the proliferative responses of T cells in both
young and old mice (17). The effect was more marked in cells from old than
young animals. Taurine has been shown to ameliorate inflammation in trinitro-
benzene sulfonic acid-induced colitis.
Taurine interacts with hypochlorous acid, produced during the “oxidant
burst” of stimulated macrophages, to produce taurine chloramine (TauCl).
This compound may have important immunomodulatory properties and may
be responsible for properties that have been ascribed earlier to taurine. TauCl
has been shown to inhibit NO, PGE2, TNF-a, and IL-6 production from stimu-
lated macrophages in culture and to inhibit the ability of antigen-presenting
cells to process and present ovalbumin (17). In in vitro studies with murine
dendritic cells, the compound altered the balance of Th1 to Th2 cytokines
suggesting that it might play a role in maintaining the balance between the
inflammatory response and the acquired immune response.
CONCLUSIONS
Oxidant stress is both an integral part of the body’s response to invasion by patho-
gens and a modulator of immune function. The modulation may take the form of
an upregulation of inflammatory components of the response and downregulation
of cell-mediated immunity. This apparent paradoxical response is due in part to
the action of oxidant stress on the activity of transcription factors such as NFkB
and AP1. The former has an impact on inflammation and the latter on cell-
mediated immunity. In addition to these molecular factors, the age of the host
may influence the balance between inflammation and cell-mediated immunity.
As aging proceeds, inflammation increases in intensity, even in the absence of
pathogenic invasion of the body. Cell-mediated immunity may also weaken.
Studies on PPAR-a in mice indicate that the normal restraining influence of
this group of transcription factors on inflammation may weaken, thereby, contri-
buting to an inflammatory phenotype in the aged. Paradoxically, the inflamma-
tory response may deplete anti-oxidant defenses, an action that may lead to
upregulation of inflammation and impairment of cell-mediated immunity.
Many studies in humans and animals have shown that nutrients, which
contribute to anti-oxidant defenses within the body, in general have an anti-
inflammatory, immunostimulatory influence. For some nutrients such as ascorbic
acid, glutathione, and its precursors, the modulatory effect is via actions on
NFkB, for other nutrients, such as a-tocopherol, actions may be via an influence
on AP1 activity or by unknown mechanisms.
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6
Concentration-Dependent Gene
and Protein Expressions of
Neuroprotective and Neurotoxic
Activities of Antioxidants,
Including Nutrients
Orly Weinreb, Silvia Mandel, and Moussa B. H. Youdim

Technion-Faculty of Medicine, Haifa, Israel
Introduction 123
Cellular Viability in Response to Antioxidants 125
Expression of Apoptosis and Cell Survival-Related Genes and
Proteins in Response to Antioxidants 125
The Effect of the Antioxidants on Caspase-3 Protein Level and Activity 129
The Molecular Mechanism of Action of Antioxidants 134
Conclusion 137
Acknowledgment 137
References 137
INTRODUCTION
Parkinson’s disease (PD) is a progressive and age-dependent neurodegenerative
disease, characterized at cellular level by a depletion of dopamine (DA) in
DA neurons of substantia nigra pars compacta (SNPC). Although the etiology
123
124 Weinreb, Mandel, and Youdim
of the neuronal cell death is still unclear and no casual therapy is available yet, the
current view supports oxidative stress (OS) as a key factor of neurodegeneration
(1,2). Analysis of Parkinsonian brain samples demonstrated certain apoptotic cell
features in DA neurons of SNPC, but the reported results remain highly
controversial (3). However, in vitro and in vivo experiments with neurotoxins
such as 6-hydroxydopamine (6-OHDA) (4) and N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) (5) have shown OS-dependent apoptosis in death
of DA neurons and many of the neurochemical changes reported in substantia
nigra of Parkinsonian brains (6), and thus it may be a contributing pathway to
dopaminergic neuronal cell death in PD (7). Previous studies have shown that
high concentrations of antioxidants, such as DA, DA D1-D2-receptor agonist,
R-apomorphine (R-APO), green tea polyphenol (2)-epigallocatechine-3-gallate
(EGCG), and the pineal indoleamine hormone, melatonin, can be cytotoxic to
neuronal cells (8 – 12), raising the question whether long-term treatment with
these compounds will contribute to the degeneration of the dopaminergic
neurons of the substantia nigra in PD (13). Nonetheless, the few clinical
studies done with these compounds have not exhibited such a phenomenon.
Thus, the in vitro studies with these antioxidant agents are not compatible with
in vivo studies.
The neuroprotective therapy aimed to interfere with cytotoxic processes or
promote neural growth and function have been successfully demonstrated in cell
and animal models of PD with several antioxidants, but not in the clinic. The few
clinical neuroprotection so far done has been far more difficult to establish.
Several catechol derivatives, especially L-dopa, or the DA D1-D2-receptor
agonists, such R-APO, bromocriptine, and pramipexole, are employed for the
therapy of PD (14). Also, the antioxidant, melatonin, has been suggested to
have clinical potential in the treatment of PD (15). Previous studies have
observed that in vitro low concentrations of DA receptor agonists, such as
R-APO (16,17), tea extracts (18), the major polyphenol component of green
tea EGCG (10), and melatonin (19), protected neuronal cells from the toxic
effects of 6-OHDA and were able to protect against MPTP-induced neurotoxicity
in vivo in mice (20 – 23).
Iron chelators and radical scavengers such as R-APO, DA, EGCG, and mel-
atonin possess concentration-dependent biphasic actions in preventing and pro-
moting neuronal cell death in models of PD. Potential candidates possessing a
key role in cell survival/death are the conserved group of mitochondrial Bcl-2
family members. The apoptotic proteins, Bax and Bak, and the BH-3-only
proteins (e.g., Bad, Bid, Bim, Noxa, and Puma) may trigger the opening of the
mitochondrial megachannel, or a specific channel in the outer mitochondrial
membrane, both of which promote the fall in mitochondrial membrane potential,
leading to cytochrome c release. The anti-apoptotic members, Bcl-2 and Bcl-xL
proteins, prevent this, probably by inhibiting Bax or Bad translocation and
insertion into mitochondrial membrane, or via a direct interaction with the chan-
nels (24,25). Indeed, the anti-Parkinson neuroprotective anti-apoptotic drug,
Neuroprotective/Neurotoxic Activities of Antioxidants 125
rasagiline, was recently shown to prevent the fall in mitochondrial membrane

potential and the opening of mitochondrial voltage-dependent anion channel
via the increase in bcl-2 and bcl-xl mRNAs and their proteins (26,27).
An important aspect of neuroprotection in PD or other neurodegenerative
diseases [Alzheimer’s disease (AD), Huntington’s disease, amyotrophic lateral
sclerosis, Friedreich’s ataxia] or their animal models is the onset of OS resulting
from generation of reactive oxygen species. In vivo and cell culture studies with
antioxidant neuroprotective agents have revealed that these compounds have a
bell shaped, concentration-dependent pharmacological response and duration
of action, which would be translated into a complex set of gene alterations
(10,21,28). The significant questions are whether the concentration-dependent
pharmacological actions of antioxidant as associated with alteration in different
gene expressions and whether antioxidant neuroprotective drugs are engaged in a
common genetic program, since they exhibit similar neuroprotective activity, at
least in the PD models.
In this article, we discuss the molecular mechanisms involved in the cell
survival and cell death action of the catechol derivatives, DA and R-APO, and
the radical scavengers, EGCG and melatonin, at low and high concentrations,
since there are numerous reports that such compounds exhibited concentration-
dependent effects, where at low concentrations they are antioxidants and at
high concentrations they are prooxidants. We have therefore employed custo-
mized cDNA and proteomics to illustrate the dose-dependent actions of these
compounds. We have shown a considerable extent of similarity between DA,
R-APO, EGCG, and melatonin with respect to cell survival/cell cycle-related
gene and protein expression at their neuroprotective and neurotoxic concen-
trations. Furthermore, their concentration-dependent ability to induce cell survi-
val and death are related to expression of genes that promote such activities.
CELLULAR VIABILITY IN RESPONSE TO ANTIOXIDANTS

In order to examine whether the four antioxidants, DA, R-APO, EGCG, and
melatonin, display a biphasic mode of action, NB SH-SY5Y cell viability
studies were conducted with these compounds over a broad range of concen-
trations. The antioxidant agents demonstrated a dose-dependent effect on the
viability of NB SH-SY5Y cells. At low concentrations (1 – 10 mM) they had
no effect on cell survival, whereas at higher concentrations (.10 mM) they
induced a gradual decrease with rank order being R-APO . EGCG . DA .
melatonin (Fig. 6.1).
EXPRESSION OF APOPTOSIS AND CELL SURVIVAL-RELATED

GENES AND PROTEINS IN RESPONSE TO ANTIOXIDANTS
The advent of genomic tools such as cDNA microarrays and proteomics provide
an entirely new approach for molecular characterization of neurodegenerative
110
Cell Viability (% of control)
Dopamine
100 R-apomorphine
90 Melatonin
80 EGCG
70
60
50
40
30
20
10
0
1 10 100 1000
Concentration (mM)
Figure 6.1 The effect of DA, R-APO, EGCG, and melatonin on cellular viability. NB
SH-SY5Y cells were pretreated with increasing concentrations of DA, R-APO, EGCG,
and melatonin for 24 h. Cell viability was assessed by MTT test, and expressed as a
percent of untreated control without the compounds. The results are expressed as the
mean + SD. The experiments were repeated at least three times in duplicates.
diseases and their models. The combined implementation of these methods may
shed light on the extent of homology existing between the models of PD,
whether the models are relevant to the disease pathology and on the effect of
drugs reputed to exert neuroprotective activity in such models. It may contribute
to the characterization of novel genes implicated in the pathogenesis of neuro-
degeneration, or other pathways leading to cell death, for which novel neuropro-
tective drugs need to be developed. Employing a customized cDNA array
containing 25 human cDNA fragments of genes coding for protein related to
apoptosis and cell survival pathways, we have observed (28) that low concen-
trations of DA (10 mM), R-APO (1 mM), and melatonin (1 mM) had no effect
on gene expression and only EGCG (1 mM) decreased the expression of bad,
bax, and tumor necrosis factor ligand member 10 (TRAIL) mRNAs (Table 6.1).
However, similar induction of mRNAs coding for pro-apoptotic proteins was
observed with the high toxic concentrations of the antioxidants (Table 6.1).
DA (500 mM), R-APO (50 mM), and melatonin (50 mM) increased apoptosis-
related cysteine proteases (e.g., caspase-3, -10), tumor necrosis factor receptor
members fas and fas-ligand, nuclear factor kappa B (NF-kB p105 subunit),
and tumor suppressor protein p53. DA (500 mM), R-APO (50 mM), and EGCG
(50 mM), but not melatonin (50 mM), increased the expression of pro-apoptotic
Bcl-2 family members bad, bax, and caspase-6 mRNAs. Only DA and R-APO
affected the expression of TRAIL, TRAIL receptor DR5, and DNA-damage
inducible transcript gadd45.
Table 6.1 Apoptosis and Cell Survival Gene Expression Analysis Identified by the cDNA Microarray
Gene name DA DA R-APO R-APO

(Unigene) (10 mM) (500 mM) (1 mM) (50 mM)
bad (U66879) 0.897+ 0.032 1.292 + 0.045 " 0.865 + 0.038 1.377 + 0.020 "
bax (L22474) 0.899 + 0.050 1.211 + 0.010 " 0.871 + 0.073 1.463 + 0.039 "
bcl-2 (M14745) 0.964 + 0.208 0.940 + 0.019 0.946 + 0.225 0.986 + 0.031
bcl-xL (U59747) 0.896 + 0.023 1.029 + 0.005 0.881 + 0.046 1.146 + 0.245
caspase-3 (U13737) 0.911 + 0.098 1.385 + 0.054 " 0.996 + 0.210 1.483 + 0.139 "
caspase-6 (U20537) 0.896 + 0.028 1.303 + 0.007 " 0.863 + 0.009 1.593 + 0.135 "
caspase-10 (U60519) 0.942 + 0.174 1.370 + 0.087 " 0.996 + 0.210 1.739 + 0.037 "
DR5 (AF016266) 0.95 + 0.192 1.475 + 0.047 " 0.995 + 0.286 1.589 + 0.119 "
fas (X63717) 0.932 + 0.150 1.309 + 0.09 " 0.928 + 0.190 1.406 + 0.052 "
fas-ligand (U08137) 0.936 + 0.137 1.354 + 0.017 " 0.975 + 0.249 1.499 + 0.039 "
gadd45 (M60974) 0.897+ 0.033 1.266 + 0.006 " 0.903 + 0.154 1.383 + 0.030 "
Neuroprotective/Neurotoxic Activities of Antioxidants
gadd45b (AF078077) 0.896 + 0.028 1.033 + 0.007 0.871 + 0.065 1.346 + 0.012 "
NF-kB (M58603) 0.930 + 0.157 1.550 + 0.011 " 1.005 + 0.360 1.744 + 0.137 "
p53 (M14694) 0.900 + 0.055 1.281 + 0.040 " 0.886 + 0.096 1.344 + 0.056 "
TRAIL (U37518) 0.899 + 0.049 1.322 + 0.009 " 0.922 + 0.190 1.535 + 0.032 "
(continued )
127
128
Table 6.1 Continued
Gene name Melatonin Melatonin EGCG EGCG

bad (U66879) 1.034 + 0.032 1.088 + 0.019 0.395+ 0.058 # 1.693 + 0.114 "
bax (L22474) 1.135 + 0.076 1.065 + 0.032 0.795 + 0.008 # 1.594 + 0.119 "
bcl-2 (M14745) 1.198 + 0.127 1.125 + 0.037 1.001 + 0.097 1.005 + 0.091
bcl-xL (U59747) 0.917 + 0.109 1.090 + 0.098 0.873 + 0.072 0.547 + 0.096 #
caspase-3 (U13737) 1.045 + 0.093 1.383 + 0.023 " 1.024 + 0.096 1.109 + 0.054
caspase-6 (U20537) 1.070 + 0.051 1.120 + 0.012 0.877 + 0.035 1.663 + 0.072 "
caspase-10 (U60519) 0.926 + 0.134 1.331 + 0.017 " 0.987 + 0.119 1.187 + 0.078
DR5 (AF016266) 0.998 + 0.053 1.062 + 0.053 0.928 + 0.019 1.012 + 0.033
fas (X63717) 1.002 + 0.069 1.444 + 0.009 " 1.009 + 0.012 1.779 + 0.123 "
fas-ligand (U08137) 0.991 + 0.087 1.219 + 0.012 " 0.907 + 0.087 1.005 + 0.029
gadd45 (M60974) 1.009 + 0.122 1.164 + 0.098 1.003 + 0.061 1.105 + 0.032
gadd45b (AF078077) 1.028 + 0.076 1.367 + 0.045 " 0.528 + 0.200 1.694 + 0.173 "
NF-kB (M58603) 1.001 + 0.021 1.372 + 0.039 " 1.051 + 0.021 1.123 + 0.054
p53 (M14694) 1.011 + 0.121 1.218 + 0.099 " 1.018 + 0.045 1.009 + 0.041
TRAIL (U37518) 0.993 + 0.198 0.927 + 0.004 0.412 + 0.123 # 0.921 + 0.028
Note: NB SH-SY5Y cells were treated without or with DA (10 and 500 mM), R-APO (1 and 50 mM), melatonin (1 and 50 mM), and EGCG (1 and 50 mM) for 6 h.
cDNA probes were hybridized to a microarray, containing 25 genes related to cell survival and apoptotic pathways. The amount of each product was normalized to b-
actin and expressed as fold stimulation of untreated control cells, set arbitrarily as 1. The results are the mean of three separate experiments, performed in duplicates.
t-test: p , 0.05 vs. control. The arrows indicate alterations of gene expression.
Weinreb, Mandel, and Youdim
Verification of apoptosis-related gene changes (Table 6.2) was assessed

using quantitative real-time RT –PCR of RNA samples isolated from NB
SH-SY5Y cells treated with the antioxidants. Low neuroprotective concen-
trations of DA, R-APO, and EGCG (10, 1, and 1 mM, respectively) induced an
acute decrease of bax mRNA, evident as early as 1.5 h. A delayed and less
marked reduction in bax expression was observed with melatonin (1 mM),
being induced at 6 h of incubation.
In parallel, an immediate substantial reduction in Bax protein content was
induced by the low concentrations of the compounds after 1.5 h of exposure and
except for R-APO, subsequently declined to control values (Fig. 6.2). The
expression of the cell-cycle inhibitor gadd45 and fas-ligand mRNAs were
reduced by EGCG, R-APO, and DA at 1.5 h and both R-APO and DA treatments
accompanied by an increase in the expression of anti-apoptotic bcl-2 and bcl-xL
mRNAs. This effect was transiently manifested 1.5 h after drug administration,
and returned to control levels at 6 h. A strict correlation between bcl-2 gene
and protein expression was observed with R-APO and DA, since both of them
upregulated Bcl-2 protein already at 1.5 h of treatment, whereas R-APO
induced a sustained upregulation of protein levels up to the 6 h incubation
period. Low concentration (1 mM) of EGCG decreased the expression of bax
and caspase-6 mRNA at 6 h of treatment. The high toxic concentrations of
DA, R-APO, and melatonin (500, 50, and 50 mM, respectively) caused a contin-
ued increase in the expression of bax, gadd45, and fas-ligand mRNAs, which was
accompanied by an increase in Bax protein content. However, the effect of mel-
atonin was comparably lower than the other three compounds DA, R-APO, and
EGCG, markedly reduced bcl-2 and bcl-xL mRNA expression, whereas no effect
was obtained with melatonin (Table 6.2).
THE EFFECT OF THE ANTIOXIDANTS ON CASPASE-3 PROTEIN

LEVEL AND ACTIVITY
Caspase-3, -6, and -10 are members of a family of cysteine proteases and import-
ant components of the apoptosis machinery (29). DA, R-APO, EGCG, and
melatonin affected, either way, the mRNAs coding for these proteases (Tables 6.1
and 6.2). Caspase-3, an apoptotic marker, is activated by multiple proteolytic clea-
vage of its 32 kDa precursor form to generate an enzymatically active p17/p12
complex. In control-untreated NB SH-SY5Y cells or cells treated with the low
concentrations of DA (10 mM), R-APO (1 mM), or melatonin (1 mM), only the
32 kDa precursor was detected. However, exposure of NB SH-SY5Y cells to
the high concentrations of DA (200 and 500 mM), R-APO (20 and 50 mM), or
melatonin and EGCG (50 mM) for 1.5– 6 h induced the cleavage of caspase-3,
as demonstrated by the appearance of the 17 kDa fragment detected with specific
antibody for caspase-3 [Fig. 6.3(a)] and stimulation of caspase-3 enzymatic
activity by 1.5– 2.5-fold over the control at 3 and 6 h after exposure [Fig. 6.3(b)].
130
Table 6.2 Apoptosis and Cell Survival Gene Expression Analysis Identified by Quantitative Real-Time RT– PCR
Gene name DA DA R-APO R-APO

For 1.5 h
bax (L22474) 0.251 + 0.001 # 1.426 + 0.005 " 0.571 + 0.029 # 1.657 + 0.087 "
bcl-2 (M14745) 2.296 + 0.089 " 0.641 + 0.018 # 2.270 + 0.082 " 0.352 + 0.012 #
bcl-xL (U59747) 1.465 + 0.007 " 0.626 + 0.012 # 1.991 + 0.054 " 0.199 + 0.008 #
caspase-6 (U20537) 0.935 + 0.031 0.783 + 0.062 1.143 + 0.144 1.395 + 0.145
fas-ligand (U08137) 0.358 + 0.028 # 1.480 + 0.070 " 1.084 + 0.161 2.063 + 0.125 "
gadd45 (M60974) 0.220 + 0.01 # 1.853 + 0.011 " 0.33 + 0.01 # 2.218 + 0.231 "
For 6 h
bax (L22474) 0.851 + 0.242 1.360 + 0.005 " 0.971 + 0.045 1.321 + 0.049 "
bcl-2 (M14745) 1.071 + 0.083 1.074 + 0.010 1.81 + 0.132 " 1.293 + 0.200
bcl-xL (U59747) 1.030 + 0.003 0.773 + 0.004 0.973 + 0.012 0.645 + 0.002 #
caspase-6 (U20537) 0.962 + 0.011 1.510 + 0.024 " 1.051 + 0.060 1.620 + 0.012 "
fas-ligand (U08137) 0.572 + 0.011 # 1.410 + 0.024 " 1.390 + 0.160 1.593 + 0.050 "
gadd45 (M60974) 0.610 + 0.006 # 1.502 + 0.039 " 1.106 + 0.073 1.420 + 0.121 "
Table 6.2 Continued
Gene name Melatonin Melatonin EGCG EGCG

For 1.5 h
bax (L22474) 1.008 + 0.321 1.272 + 0.032 " 0.560 + 0.11 # 0.86 + 0.16
bcl-2 (M14745) 1.055 + 0.118 1.139 + 0.127 1.05 + 0.08 0.505 + 0.01 #
bcl-xL (U59747) 1.825 + 0.029 " 1.164 + 0.022 0.99 + 0.13 0.561 + 0.04 #
caspase-6 (U20537) 0.804 + 0.059 0.868 + 0.063 0.61 + 0.09 # 0.86 + 0.23
fas-ligand (U08137) 1.003 + 0.328 1.083 + 0.354 0.630 + 0.09 # 1.02 + 0.34
gadd45 (M60974) 1.081 + 0.110 2.330 + 0.143 " 0.470 + 0.03 # 0.69 + 0.23
For 6 h
bax (L22474) 0.870 + 0.013 1.031 + 0.069 0.603 + 0.13 # 1.52 + 0.105 "
bcl-2 (M14745) 0.864 + 0.112 0.845 + 0.040 0.819 + 0.002 0.453 + 0.09 #
Neuroprotective/Neurotoxic Activities of Antioxidants
bcl-xL (U59747) 0.901 + 0.021 0.863 + 0.002 1.065 + 0.07 0.491 + 0.09 #
caspase-6 (U20537) 0.863 + 0.021 3.271 + 0.142 " 0.473 + 0.04 # 1.101 + 0.05
fas-ligand (U08137) 1.061 + 0.021 1.310 + 0.001 " 1.001 + 0.071 1.010 + 0.01
gadd45 (M60974) 0.863 + 0.053 0.805 + 0.132 0.936 + 0.53 2.967 + 0.27 "
Note: NB SH-SY5Y cells were treated with DA (10 and 500 mM), R-APO (1 and 50 mM), melatonin (1 and 50 mM), and EGCG (1 and 50 mM) for 1.5 and 6 h. The
amount of each product was normalized to the housekeeping gene 18S-rRNA, and expressed as fold stimulation of untreated control, arbitrarily set as 1. The results
are the mean of three separate experiments, performed in duplicates. t-test: p , 0.05 vs. control. The arrows indicate alterations of gene expression.
131
Time of incubation: 1.5 h 3h 6h
132
(a) 2.5 2.5

2.5
Bcl-2
2.0 *
2.0 2.0
* * *
1.5 *
1.5 1.5
1.0 1.0 1.0

*
Relative expression
0.5 0.5 0.5
0.0 0.0 0.0

10 200 500 1 50 1 20 50 C 10 200 500 1 50 1 20 50 C 10 200 500 1 50 1 20 50
(mM) C
DA Melatonin R-APO DA Melatonin R-APO DA Melatonin R-APO
(b) 2.5 * 2.5
2.5 *
2.0 * Bax
* 2.0
2.0
1.5 * 1.5 *
1.5
1.0 1.0
* 1.0 *
*
Relative expression
0.5 * 0.5 0.5
0.0 0.0 0.0

(mM) C 10 200 500 1 50 1 20 50 C 10 200 500 1 50 1 20 50 C 10 200 500 1 50 1 20 50
DA Melatonin R-APO DA Melatonin R-APO DA Melatonin R-APO
Figure 6.2 The effect of DA, R-APO, and melatonin on protein levels: (a) Bcl-2 and (b) Bax. NB SH-SY5Y cells were treated with increas-
ing concentrations of DA (10, 200, and 500 mM), R-APO (1, 20, and 50 mM), and melatonin (1 and 50 mM), and harvested at different time
intervals (1.5, 3, and 6 h). Western blotting were probed with anti-Bax and anti-Bcl-2. An antibody for b-actin was used to normalize the
expression level of the proteins. The bands were quantified by densitometry and represented graphically. The results are the mean of
three independent experiments, performed in duplicates. t-test: p , 0.05 vs. control.

Figure 6.3 The effect of DA, R-APO, and melatonin on caspase-3 protein level and
activity. NB SH-SY5Y cells were exposed to DA, R-APO, EGCG, and melatonin, and har-
vested at different time intervals (1.5, 3, and 6 h). Activation of caspase-3 was analyzed in
cell lysates and analyzed with: (a) Immunoblotting using antibody against pro-caspase-3
(32 kDa) and the activated cleaved caspase-3 fragments (17 kDa and 12 kDa). An antibody
for b-actin was used to normalize the expression level of the proteins. The data are from
one representative experiment of three independent experiments that exhibited similar
results. (b) Caspase-3 like protease activity was measured with the caspase-3 substrate
colorimetric assay in accordance with the protocol supplied by the manufacturer
(Calbiochem, CA). The assay was preformed in 96-well microtiter plates. The colorimetric
substrate (Ac-DEVD-pNA) was added to 20 mg of cell lysates in 100 ml reaction buffer.
After 4 h incubation at 378C, absorbance was determined at 405 nm. The results are
expressed as the mean + SD. The experiments were repeated at least three times.

p , 0.01 vs. control.
THE MOLECULAR MECHANISM OF ACTION OF ANTIOXIDANTS

Antioxidants have neuroprotective, antioxidant anti-apoptotic activity at low
concentrations, whereas at high concentrations they induce pro-oxidant and
neurotoxic pro-apoptotic actions in cell cultures and in vivo models of neuro-
degenerative diseases. Our recent studies (10,28) provide new insights into the
molecular events involved in the dose-dependent anti- and/or pro-apoptotic
activities of catechol-derived and indoleamine compounds at low and high
concentrations. For this purpose, low neuroprotective and 50-fold higher con-
centrations were chosen. cDNA microarray has demonstrated a concentration-
dependent homology among antioxidants R-APO, DA, melatonin, and EGCG
for modulation of cell survival/cell death-related gene pathways. The gene
microarray analysis provides the first evidence for a selective, dose-dependent
regulation of a number of mRNAs by these drugs. One important aspect of our
study is the significant homology between the catechol-derived compounds and
differences observed with the indoleamine, melatonin (Fig. 6.4). The extremely
DA
NFkB, P53
Fas-ligand DR5,
Caspase-3,10 TRAIL
Bad
Bax, Caspase-6
Melatonin Fas, Gadd45 Bcl-2,
Bcl-xL R-APO
Gadd45b
EGCG
Cell Death Regulation

Figure 6.4 The significant gene expression homology between the catechol-derived
compounds, DA, R-APO, and EGCG, and differences observed with the indoleamine,
melatonin.
low pro-apoptotic activity of melatonin, when compared with DA, R-APO, and
EGCG, may be partially explained by its mild or lack of effect on the expression
of Bcl-2 family genes.
No significant gene changes were observed with the low concentrations of
R-APO, DA, and melatonin, previously reported (30 – 33) to induce effective
neuroprotection in both neuronal primary and cell line cultures. However,
when quantitative real-time RT-PCR method was applied, specific gene expres-
sion changes as a function of concentration and time were observed. This discre-
pancy may result from the sensitivity thresholds of both methods. Low DA,
R-APO, and melatonin concentrations induced an immediate expression of
anti-apoptotic bcl-xL and/or bcl-2 mRNAs, whereas bax mRNA was reduced.
The gene changes were correlated with alterations in protein levels of both
Bcl-2 and Bax. The increased bcl-2 or bcl-xL to bax ratio as a result of R-APO,
DA, and melatonin suggests the involvement of these pathways in their neuro-
protective and anti-apoptotic actions. Similarly, the potent antioxidant, EGCG,
at low concentration decreased pro-apoptotic genes bax and caspase-6 supporting
previous findings (10).
However, a pro-apoptotic pattern of gene expression was observed at high
concentrations of the antioxidants. A similar expression profile was obtained after
exposure to R-APO, DA, and melatonin, including upregulation of pro-apoptotic
caspases-3 and -10, tumor necrosis factor receptor fas and fas-ligand, NF-kB
p105 subunit, and tumor suppressor protein p53 mRNAs. DA and R-APO
displayed a greater homology of gene expression when compared with melatonin,
suggesting that these drugs may share a similar mechanism in their cell death
action. The wide range between the neuroprotective and the toxic concentrations
of melatonin may be of critical importance in its pharmacotherapy, since it may
provide a safer dosage window than R-APO and DA. One possible explanation
for the high cell viability in the presence of high melatonin concentration may
be that caspase-3 activation, per se, or its upstream/downstream effectors may
not be indispensable for onset of apoptosis, as has been previously suggested
(34). The observation that neither bcl-2 nor bcl-xL mRNA level was altered by
high concentration of melatonin emphasizes their pivotal role in cell survival.
This is in contrast to what was observed with high concentrations of R-APO,
DA, and EGCG. Schematic overview suggesting potential gene targets involved
in the pro-apoptotic and anti-apoptotic action of DA, R-APO, melatonin, and
EGCG is described in Fig. 6.5. The gene expressions are associated with regu-
lation of their proteins and these effects are concentration- and time-dependent.
In vitro cell culture studies have suggested that the neurotransmitter DA
serves as an endogenous neurotoxin, thereby participating in neurodegenerative
processes in PD (35 –37). This assumption is based on observations that high con-
centrations (200 –1000 mM) of DA induce apoptosis in neuronal cell culture but
not in vivo. They have implicated endogenous DA as a neurotoxic culprit in PD
(38). In vivo however, the highly active intraneuronal and extraneuronal (cells
such as glia, astrocytes) monoamine oxidase (MAO) never allows the build-up
Low Concentrations of DA, High concentrations of DA, High concentrations of DA

R-APO, EGCG and melatonin R-APO, EGCG and melatonin and R-APO
bcl-xL, bcl-2 bad, bax

High concentrations
p53 of melatonin
Mitochondria
gadd45 fas-ligand TRAIL
Cytochrome c fas DR5
caspase-9 caspase-3,-6 caspase-8
Apoptosis
Figure 6.5 Schematic overview indicating potential gene targets involved in the anti-
apoptotic and pro-apoptotic action of low and high concentrations of antioxidants in NB
SH-SY5Y cells. Solid arrows and dotted lines indicate induction and inhibition of gene
expression, respectively.
of such high, nonphysiological concentration of intraneuronal or extraneuronal

DA. Only when the brain MAO is inhibited, neuronal and synaptic DA levels
increase. Selective and nonselective MAO inhibitors (tranylcypramine, selegi-
line, moclobemide, and rasagiline) (39) have been employed in the treatment
of depressive illness and PD. There is no evidence for increased incidents of
PD or neurodegeneration in depressive illness or that such treatment aggravates
the neurodegeneration process in PD subjects. The present study has clearly
shown that DA, at low concentrations, which is relatively closer to its physiologi-
cal concentration, does not affect cell viability, but rather activates cell survival
genes and their associated proteins. This supports the most recent view that low
DA concentration may indeed have a beneficial effect on DA neuron plasticity
(40). This assumption may be also relevant for the dopaminergic receptor
agonist R-APO, since it may have a neuroprotective action in addition to its
favorable symptomatic action in PD patients. The phenomenon described for
DA may also apply to the action of other neurotransmitters and antioxidants.
Implementation of cDNA microarrays represents an invaluable tool for
the identification of gene alterations at the level of the mRNA, providing the
possibility of assessing the simultaneous expression of thousands of genes at a
specific time and conditions. As shown in the studies with antioxidant, the
activities of these compounds to alter gene expressions are concentration- and
time-dependent. Thus, they have a window of activities that need to be considered
when being applied in vitro, cell culture, in vivo, and clinical studies. The global
picture obtained with microarray and proteomic studies indicates a domino
cascade of events in drug action and drug response in neurodegenerative diseases

(41). This may explain why up to date single neuroprotective drug therapy has
failed in clinical neuroprotection, and thus serious consideration needs to be
given to their concentration-dependent action and multidrug cocktail, as currently
implemented in other diseases such as cancer and cardiovascular diseases. Future
in vivo studies with multiple antioxidant compounds as therapeutic agents will be
aimed to prevent changes in mitochondrial membrane potential involving gene
expression cascades associated with programed cell survival and cell death. It
will address the questions whether combined antioxidant agents would have a
synergistic action and can be more effective in clinical treatment of neurode-
generative diseases.
CONCLUSION
Significant evidence has been provided to support the hypothesis that OS and
inflammatory processes trigger a cascade of events leading to apoptotic/necrotic
cell death in neurodegenerative disorders such as PD, AD and Huntington’s
disease, stroke, and amyotrophic lateral sclerosis. The novel therapeutic
approaches aimed at neutralization of OS-induced neurotoxicity, support the
application of reactive oxygen species scavengers, transition metals (iron and
copper) chelators, and nonvitamin natural antioxidants, in monotherapy, or as
part of antioxidant cocktail formulation for these diseases. Recent studies
indicate that the radical scavenger property of antioxidant agents, such as DA,
R-APO, green tea polyphenol EGCG, and melatonin, is unlikely to be the sole
explanation to their neuroprotective effects in models of PD and AD, but a
wide spectrum of cellular signaling events may also account for their biological
actions. This article provides a new insight into the gene and protein mecha-
nisms involved in both the neuroprotective and anti-apoptotic activities of anti-
oxidant drugs, demonstrating a concentration- and time-dependent correlation
between R-APO, DA, EGCG, and melatonin in modulation of cell survival/
cell death-related gene pathways.
ACKNOWLEDGMENT
The authors acknowledge the support of National Parkinson Foundation (Miami),
Stein Foundation (Philadelphia) and Rappoport Family Research, Technion-
Israel Institute of Technology, and Friedman Fund for Parkinson’s Disease
(Technion).
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7
Effects of Antioxidants on Gene
Expression in Endothelial Cells
B. A. Nier, B. A. Ewins, and S. G. Cremers

Peter D. Weinberg
Imperial College, London, UK
Introduction 141
Oxidants, Antioxidants, and NF-kB Mediated Gene Expression
in the Endothelium 143
Oxidants, Antioxidants, and AP-1 Mediated Gene Expression
in the Endothelium 148
Vitamin E and Endothelial Gene Expression 152
Regulation of Transcription Through Changes in Chromatin Structure 157
Applicability to Physiological Conditions 160
Discussion 164
Conclusion 167
Acknowledgments 167
References 167
INTRODUCTION
Thirty years ago, the main roles of endothelial cells—the cells lining the inner
surface of all blood vessels—were thought to be the largely passive ones of
providing a nonthrombogenic surface and of keeping macromolecules within
141
142 Nier et al.
plasma, while allowing the exchange of smaller solutes with underlying tissue.
Since then, however, endothelial cells have been shown to play an active and
key role in a large number of physiological processes that are critical for vas-
cular homeostasis (1). The explosion of interest in these cells has undoubtedly
been fueled by their central involvement in the development of atherosclerosis.
This disease underlies most myocardial infarctions and cerebrovascular acci-
dents, and in many countries it constitutes the major cause of death. Several
aspects of its pathogenesis remain to be established but there is currently a con-
sensus that key events include the activation or perturbation of endothelial cells
by various mechanical, chemical, or biological stimuli, entry of lipoproteins
and inflammatory cells into the arterial wall, the migration of smooth muscle
cells (SMC) into the arterial intima and their subsequent proliferation, and
interactions between the atheromatous plaque and circulating platelets, all of
which are strongly influenced by the endothelium (2).
In parallel with the increasing interest in endothelial cells, there has been
increasing interest in the proatherogenic role of oxidative stress and, conse-
quently, in the possible atheroprotective role of dietary and other antioxidants.
Initially, this interest resulted from the probable involvement of oxidized low
density lipoprotein (LDL) in the development of the disease. A characteristic
of atherosclerotic lesions is the presence of foam cells containing large
numbers of cholesterol-rich lipid droplets within their cytoplasm. LDL is the
major carrier of cholesterol in the human circulation, but its uptake by cells is
tightly controlled: increasing uptake of cholesterol leads to downregulation of
LDL receptors in a classical negative feedback loop (3). However, uptake of
modified forms of LDL occurs by different routes, and is not regulated. It has
been shown that LDL oxidation can occur in vivo, which oxidized LDL is
present in lesions but rarely elsewhere, that oxidized LDL is taken up by scaven-
ger receptors rather than LDL receptors, and that incubation of macrophages with
oxidized LDL leads to the formation of foam cells (4).
More recently, other potentially important roles of oxidative stress have
emerged. These stem from a re-emphasis (5) of the role of inflammation in
atherogenesis, a concept dating back at least as far as Virchow, and from the
realization that oxidative stress, or reactive oxygen species (ROS), are potent
pro-inflammatory stimuli in endothelial cells (6,7). Hence, the balance between
ROS and dietary and other antioxidants might control the rate of atherogenesis
through pro- and anti-inflammatory actions, as well as by modifying the rate of
oxidation of LDL.
Sources of ROS within cells include mitochondrial respiration, NAD(P)H
oxidase, nitric oxide synthases (NOS), cyclo-oxygenases (COX), lipoxygenases,
xanthine oxidase, cytochrome P-450 mono-oxygenase, heme oxygenases,
peroxidases, and hemoproteins. There is evidence that NAD(P)H oxidases are
the primary source in cultured endothelium. The main enzymatic defenses are
superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) (6,7).
Of course, effects of exogenous ROS and antioxidants must also be considered.
Effects of Antioxidants on Gene Expression in Endothelial Cells 143
This review concerns the roles of ROS and antioxidants in modifying

expression of genes within endothelial cells. Although relatively novel, this
subject is already a large one. The review takes a critical approach to the current
orthodoxy, and it concentrates on (i) the control and actions of the two principal
redox-sensitive transcription factors, Nuclear Factor kappa B (NF-kB), and Acti-
vator Protein-1 (AP-1), (ii) the effects of the most important lipid-soluble anti-
oxidant in the diet, vitamin E, (iii) the possibility that oxidants and antioxidants
might affect gene expression other than through influences on transcription
factors, (iv) the likelihood that effects of oxidants and antioxidants observed in
culture are modified in vivo, particularly by shear stresses, and (v) whether the
putatively anti-atherogenic effects of antioxidants observed in vitro are supported
by results from dietary intervention trials.
OXIDANTS, ANTIOXIDANTS, AND NF-kB MEDIATED GENE

EXPRESSION IN THE ENDOTHELIUM
The biology of NF-kB in endothelial cells, and its possible modification by
dietary components, has attracted considerable attention because of accumulating
evidence that this transcription factor might play a major role in atherogenesis.
First, activated NF-kB is present in the atheromatous plaque—in SMC and
macrophages as well as endothelial cells—but is absent in cells of the healthy
arterial wall (8). Second, NF-kB is activated by an array of stimuli thought to
play a key role in the development of atherosclerotic lesions, including tumor
necrosis factor alpha (TNF-a), interleukin-1 (IL-1), bacterial lipopolysaccharide
(LPS), advanced glycation end products (AGEs), hyperglycemia, platelet activat-
ing factor, shear stress, oxidized lipids, oxidant stress, and hypoxia/reperfusion
(9). Third, NF-kB regulates genes responsible for key atherogenic proteins,
including pro-inflammatory cytokines (TNF-a), chemokines such as monocyte
chemoattractant protein-1 (MCP-1), cell adhesion molecules such as intercellular
adhesion molecule-1 (ICAM-1) and E-selectin, and inflammatory enzymes such
as inducible nitric oxide synthase (10,11)—a more complete list is presented
below. Consequently, regulation of the action of the transcription factor, or of
related upstream and downstream signaling events, might inhibit the formation
of lesions or aid in their regression.
The normal regulation and activation pathways of NF-kB are considered
first, with particular reference to studies in endothelial cells and to stimuli and
genes relevant to atherosclerosis. NF-kB is a ubiquitously expressed protein
(12). It belongs to a family of proteins that share a conserved central region of
300 amino acids called the Rel homology domain. Five members of the
NF-kB family have been described: p50, p65 (RelA), c-Rel, RelB, and
p52; p105 and p100 are the precursors of p50 and p52, respectively (12).
NF-kB usually exists as a dimer. The classical form is a heterodimer composed
of the p50 and p65 subunits.
144 Nier et al.
NF-kB exists in two states: in quiescent cells it is bound to an inhibitory

protein, inhibitory kappa B (IkB), but it dissociates from IkB when activated
(13,14). Binding to IkB keeps NF-kB in the cytoplasm and therefore incapable
of acting upon its target genes. The Rel domain plays an important role in this
interaction. Multiple mammalian forms of IkB exist. Those identified so far
are IkB-a, -b, -g (p105), d (p100), and 1. [The C-terminal regions of the p50
and p52 precursors, p105 and p100, have IkB-like segments; free C-terminal
segments are therefore termed IkB-g (p105) and IkB-d (p100).]
Removal of IkB requires activation of IkB-a, -b, and -g kinases (IKK-a,
-b, and -g, respectively). Their activation leads to phosphorylation of two specific
serine residues near the N-terminus of IkB-a (Ser32 and Ser36), an event that can
occur within minutes. IkB-b is phosphorylated on two equivalent serine residues
(Ser19 and Ser23) by both IKK-a and IKK-b (15,16). Homologous sites are found
at Ser157 and Ser161 in IkB-1 (17). Phosphorylation targets IkB for ubiquitination
and thus rapid degradation by the 26S proteasome (18).
The IKK belong to a larger, more complicated protein complex called the
IKK signalosome. This complex includes IKK complex-associated protein
(IKAP) and NF-kB essential modulator (NEMO, or IKK-g), which are essential
for NF-kB activation (19,20). In the endothelium, the activation of IKK in the
IKK signalosome, and the subsequent phosphorylation and degradation of IkB,
is triggered by extracellular stimuli that lead to the activation of intermediate
protein kinases such as mitogen-activated protein kinase (MAPK), protein
kinase C (PKC), protein tyrosine kinase, NF-kB-inducing kinase (NIK) (21),
and MAPK/ERK kinases 1, 2, and 3 (MEKK-1, -2, and -3) (22,23).
When the NF-kB heterodimer is released by these processes, its nuclear
localization sequence is unmasked and it rapidly enters the nucleus. Once it
enters, it binds to recognition elements, called kB sites, in the enhancer and
promoter regions of inducible genes, and regulates their expression. Since its dis-
covery in 1986 (24), NF-kB has been shown to play a key role in the regulation
of many genes, especially those related to inflammatory and immune responses.
A list of NF-kB-regulated proteins that are relevant to the vasculature can be
found in Table 7.1 and the activation process is summarized in Fig. 7.1.
Interestingly, NF-kB can bind to kB elements in the promoter region of its
own inhibitory protein, IkB-a. Hence, activated NF-kB levels are regulated by
negative feedback. This autoregulatory system ensures a transient activation of
NF-kB, with a subsequent return to a quiescent state. Positive feedback loops
also exist, since NF-kB is upregulated by some of the products of the genes
that contain NF-kB-binding domains, such as TNF-a.
Much recent research has attempted to elucidate further the molecular
mechanisms underlying activation of NF-kB. The majority of this research has
centred on the p50/p65 heterodimer and its association with IkB-a as this
appears to be the predominant form of NF-kB in mammalian cells. Although
many compounds are known to activate NF-kB, it is unclear what unified mech-
anism, if any, explains how such a diverse array of stimuli can have the same
Table 7.1 Proteins Regulated by NF-kB
Interleukins and growth factors IL-1, IL-6, IL-8, TNF-a, G-CSF, M-CSF,
GM-CSF, MIP1-k, MCP-1, RANTES
Cytokine and cell adhesion receptors E-selectin, ICAM-1, VCAM-1, MAdCAM-1,
Lox-1, RAGE, A20, A1, XIAP, c-IAP1,
c-IAP2
Immunomodulatory MHC-I, MHC-II, IRF-1
Others iNOS, COX-2, tissue factor, PLA2, IkBa,
MnSOD, MMP-2, MMP-9
Note: Interleukin, IL; tumor necrosis factor alpha, TNFa; colony stimulating facor, CSF; granulocyte
macrophage, GM; macrophage inflammatory protein-1a, MIP-1a; monocyte chemoattractant protein-
1, MCP-1; regulated upon activation normal T-cell expressed and secreted, RANTES; intercellular
adhesion molecule-1, ICAM-1; vascular adhesion molecule-1, VCAM-1; mucosal addressin cellular
adhesion molecule-1, MAdCAM-1; lipoxygenase, Lox; receptor for AGE, RAGE; x-linked inhibitor
of apoptosis, XIAP; interferon regulatory factor, IRF; inducible nitric oxide synthase, iNOS; cyclo-
oxygenase-2, COX-2; phospholipase A2, PLA2; inhibitory kappa B alpha, IkBa; superoxide dismu-
tase, SOD; matrix metalloproteinase, MMP.
Modified from De Martin et al. (25).
effect. It has been postulated that NF-kB is regulated by the redox status of the
cell (26,27). This view led to NF-kB being termed “the redox sensor of the
cell,” but recently met with controversy. Although pathways leading to the acti-
vation of NF-kB have been delineated, there have been few data to link ROS
directly to these mechanisms.
The first suggestion that ROS were involved in the modulation of NF-kB
came in 1990, just 4 years after the discovery of the transcription factor (28).
Shortly after, Schreck et al. put forward the hypothesis that diverse agents acti-
vated NF-kB through an increase in ROS and oxidative stress within the cell
(29,30). This hypothesis was based on four main lines of evidence. First,
NF-kB is activated in cells exposed to hydrogen peroxide (H2O2). Second,
ROS are found in increased levels in cells treated with agents that activate
NF-kB (30,31). Third, compounds with antioxidant properties are able to
inhibit the activation of NF-kB (30). Fourth, modulation of molecules known
to regulate intracellular levels of ROS, such as glutathione (32) and SOD (30),
can affect the activation of NF-kB.
These lines of evidence led to the widely accepted view that H2O2 acts as a
second messenger in the activation of NF-kB. However, it is becoming clear that
the role of H2O2, and perhaps of ROS in general, may have been overstated; its
involvement in the activation of NF-kB is restricted to certain cell lines, and its
role is well characterized only in lymphocytes (33,34). Thus, H2O2 is a potent
activator of NF-kB in Wurzburg subclone of T cells, L6 skeletal muscle myo-
tubes, human breast MCF-7 cells, and 70Z/3 pre-B cells (27,29,35). However,
activation in a number of cell types have been shown to be completely insensitive
to H2O2 . These include Jurkat cells (36), monocytic cell lines, EL4.NOB-1 T
146 Nier et al.
Figure 7.1 Activation pathway of NF-kB. Many external stimuli activate signal
transduction pathways ultimately leading to the activation of the IKK signalosome. This
in turn results in the phosphorylation of IkB, which is in turn polyubiquitinated by a
specific E3 ligase. Ubiquitinated IkB is then sent for degradation by the 26S proteasome.
(Mitogen activated protein kinase, MAPK; MAPK/ERK kinases 1, 2, and 3, MEKK-1, -2,
and -3; protein kinase C, PKC; NF-kB-inducing kinase, NIK; transforming growth factor
b-activated protein kinase 1, TAK-1.)
cells, KB epidermal cells, astrocytoma, J. Jhan lymphoblastoid T cells, and

human umbilical vein endothelial cells (HUVEC) (37,38).
It has already been mentioned that antioxidant compounds are able to
inhibit the activation of NF-kB. This is one of the most compelling arguments
for the involvement of ROS and oxidative stress. Several natural antioxidants,
such as curcumin (39), gallic acid (40), quercetin (41), vitamin C (42), and
vitamin E (43), as well as artificial compounds, have been reported to inhibit
the cytokine-mediated activation of NF-kB in endothelial and non-endothelial
cell lines. However, recent work suggests that they do not consistently do so,
that they may act through diverse pathways, and that they may exert influences
unrelated to their antioxidant properties. Examples are given as follows.
Two nondietary antioxidants widely studied with regard to NF-kB
activation are the glutathione precursor N-acetyl cysteine (NAC) and the thiol
pyrrolidine dithiocarbamate (PDTC). Both have been reported to inhibit

activation of NF-kB in many cell types (29,30,36,44). Indeed it was NAC, an
antioxidant that can increase intracellular levels of glutathione and therefore
directly scavenge ROS, that was used in the initial experiments of Schreck
et al. which led to the redox-regulation hypothesis. However, there are now
numerous reports of NAC- and PDTC-insensitive pathways of NF-kB regulation;
these demonstrate that the effect is not only cell specific, but also stimulus
specific within the same cell type (36,38,45). (Possible non-redox mediated
effects of thiol antioxidants such as PDTC are considered in the following
section.)
Different antioxidants often inhibit activation of NF-kB through different
mechanisms. The inhibition of cytokine-mediated NF-kB activation by salicy-
lates and by curcumin results from the inhibition of IkB phosphorylation and
degradation (39,46). IKK-a and IKK-b are inhibited by these antioxidants at
the phosphorylation level. These antioxidants also inhibit the activation of
additional kinases, such as MEKK-1, resulting in the inhibition of other
downstream transcription factors such as Fos and Jun. It can be therefore be pos-
tulated that these antioxidants act on early steps in the signaling cascade,
although their specific target has not yet been identified. Conversely, caffeic
acid phenethyl ester and gallates reduce NF-kB translocation and binding
without influencing IkB-a degradation (40,47). Similarly, Schubert et al. (48)
have shown that NAC and pomegranate wine both have antioxidant effects in
TNF-a-stimulated bovine aortic endothelial cells (BAEC) and both inhibit the
activation and nuclear translocation of NF-kB, but do not do so by reducing
Ser32 phosphorylation of IkB-a, or IkB-a degradation (which depends on such
phosphorylation).
Differences between NAC and pomegranate wine were also observed by
Schubert et al. (48). In the TNF-a-stimulated BAEC, NAC reduced p65 Ser536
phosphorylation, a recently postulated step in the cascade of events leading to
the activation of NF-kB (49), but the wine did not. Conversely, subsequent
de novo synthesis of IkB was inhibited by the wine but not by NAC.
Other pathways by which antioxidants inhibit NF-kB activation include
S-nitrosylation of cysteine residues on NF-kB subunits, which interferes with
their binding to DNA but not with nuclear translocation (50,51).
The most important dietary antioxidant that has been studied in detail with
respect to NF-kB and the endothelium is vitamin C. Vitamin C inhibits NF-kB
activation in the human endothelial cell line ECV304 and in HUVEC.
However, the effect may be unrelated to its antioxidant activity (42). HUVEC
were activated through multiple pathways using an array of stimuli including
IL-1 and TNF-a, whereas H2O2 was used to stimulate the ECV304 cells. The
inhibitory effects were independent of both the cell type and the stimulus used,
something not seen with either NAC or PDTC. In particular, vitamin C inhibited
NF-kB when the latter was activated through pathways previously shown to
be insensitive to other antioxidants, such as those stimulated by IL-1 (38).
148 Nier et al.
Furthermore, the effect of vitamin C was not potentiated by co-incubation with

vitamin E, even though vitamins C and E are known to have a cooperative
effect (42).
Experiments such as these provide evidence against the general hypothesis
that ROS are central to the activation of NF-kB in endothelial cells. If NF-kB
were universally controlled by ROS, less diversity in the inhibitory effects of
antioxidants would be expected. Nevertheless, they do suggest that dietary
antioxidants might exert an atheroprotective effect by inhibiting its activation.
Possible additional modes of action that require further investigation are
effects on the IKK complex, on the ubiquitination and degradation of IkB, on
NF-kB nuclear import/export, and on its interactions with DNA.
OXIDANTS, ANTIOXIDANTS, AND AP-1 MEDIATED GENE

EXPRESSION IN THE ENDOTHELIUM
Although NF-kB has been shown to play a pivotal role in EC activation (8,52,53),
increasing evidence indicates the importance of the transcription factor AP-1 in
endothelial homeostasis and dysfunction.
AP-1 can be either a homo- or heterodimer, involving members of the c-Jun
(c-Jun, Jun B, and Jun D) and c-Fos (c-Fos, Fra-1, Fra-2, and Fos B) proto-
oncogene families (54). The AP-1 subunits contain the basic-leucine zipper
(bZIP) motif, where the leucine zipper mediates dimerization and the basic
region is directly involved in DNA binding. Owing to the biochemical properties
of Fos and Jun leucine zippers, all of the Jun protein family can form homo- and
heterodimers, which are capable of binding to AP-1 DNA binding sites. Fos-
proteins do not associate with each other, but are capable of associating with
any member of the Jun family to form stable heterodimers that have higher
DNA-binding activity than Jun –Jun homodimers (55). Jun and Fos can also
form heterodimers with other bZIP transcription factors, such as members of
the cyclic adenosine monophosphate response element binding protein (CREB)
and activating transcription factor (ATF) family to form an AP-1 complex
(56,57).
Jun – Jun and Jun –Fos forms of AP-1 bind to a specific DNA sequence,
referred to as the 12-O-tetradecanoyl-13-phorbol acetate (TPA) response
element (TRE). This sequence is present in the promoters of many inducible
genes implicated in cell proliferation, differentiation, inflammation, and stress
response (58), including genes for ICAM-1, VCAM-1 (59), E-selectin (60)
MCP-1 (61), and tissue factor (TF) (62,63). c-Jun and c-Fos proteins also
contain several transcriptionally active regions distinct from the leucine zipper
and DNA basic region, including several autonomous transactivation domains,
a carboxyl-terminal transrepression domain (c-Fos), and a region that interacts
with the TATA box-binding protein (64,65). These domains in Fos and Jun func-
tion additively and perhaps cooperatively in transcriptional regulation. Further-
more, binding by Jun homodimers leads to DNA binding that is distinct from
binding by Fos– Jun heterodimers, which may result in highly specific protein –
protein interactions between AP-1 factors and other promoter-bound transcrip-
tion complexes (65). Therefore, the distinct AP-1 proteins can exhibit distinct
transcriptional properties, and the composition of the AP-1 complex may be
critical to its regulatory function with diverse biological consequences.
The activity of AP-1 can be controlled by transcriptional mechanisms,
leading to increased amounts of AP-1 protein, and by posttranslational mechan-
isms, acting on pre-existing AP-1 proteins, in response to a variety of extracellu-
lar stimuli, including mitogens, phorbol esters, and differentiation signals.
Expression of the various AP-1 dimers is differentially regulated temporally
during cell cycle progression and in response to many stimuli (66). The promoter
for the c-Jun gene itself contains a TRE region, so it can be activated by AP-1 in a
positive autoregulatory way (67,68). The c-Fos promoter lacks the TRE region
and thus is not subject to this type of autoregulation.
The transcriptional and posttranslational regulators of AP-1 activity are in
turn regulated through different signaling cascades, thereby explaining the versa-
tility of AP-1 in responding to a broad spectrum of stimuli (68 –70). In various
cell types, three different MAPK cascades are known to be involved in the induc-
tion of AP-1 activity. These are extracellular signal-regulated kinases (ERK-1
and -2), c-Jun-N-terminal protein kinases (JNKs)—also known as stress-
activated protein kinases (SAPKs)—and p38 kinase cascades (69,70) (Fig. 7.2).
In general, the ERK pathway is strongly activated by growth factors and
cytokines. Its activation is related to the stimulation of upstream tyrosine
kinase receptors, which start a signaling cascade involving Ras activation,
recruitment of Raf kinase to the plasma membrane, and sequential activation/
phosphorylation of MAPK/ERK kinase 1, 2 (MEK-1, -2) and ERK-1 and -2.
The major regulatory element of the c-Fos promotor, the serum response
element (SRE), is recognized by the ternary complex factor (TCF) member
Elk-1 and the dimeric serum responsive factor (SRF). When Elk-1 is phospho-
rylated by ERK-1 and -2, it combines with SRF to form a ternary complex
with SRE which leads to the transcriptional induction of c-Fos (71).
JNK and p38 cascades are only weakly activated by mitogens but are
highly stimulated by exposure to pro-inflammatory cytokines such as TNF-
alpha and IL-1 and a wide variety of environmental stress inducers such as
LPS. JNKs are phosphorylated by SAPK/ERK-1 (SEK1), also known as
MAPK kinase 4 (MKK4), which in turn is activated through phosphorylation
by MEKK-1. The activation/phosphorylation of MKK3/6 leads to the activation
of p38 (69,72). These cascades can also be activated by members of the Ras
superfamily of small GTPases, such as Rac and Cdc42, two Rho-like proteins
that in the case of the JNK cascade have been reported to act synergistically
with Ras. Ras therefore appears to be an alternative upstream component of
this pathway (69,73). JNK and p38 cascades are connected with the activation
of AP-1 in a similar fashion to the ERK cascade described earlier. Two
members of the TCF family, Elk-1 and Sap-1a, are substrates of JNK in activated
150 Nier et al.
Figure 7.2 Regulation of AP-1 by extracellular stimuli. Extracellular signals received

by membrane receptors are transduced into the nucleolus through three different MAPK
subgroups ERK 1/2, JNKs, and p38. The transcription factors ELK-1, Sap-1a, c-Jun,
and ATF-2 couple the activation of the different MAPKs with the transcriptional activation
of c-Fos and c-Jun promoters. (ERK, extracellular signal-regulated kinase; JNK, c-Jun-
N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/
ERK kinase; MEKK, MEK kinase; MKK, MAPK kinase; SEK, stress-activated protein
kinase (SAPK)/ERK; SRE, serum response element; SRF, serum responsive factor;
TCF, ternary complex factor; TRE, TPA (12-O-tetradecanoyl-13-phorbol acetate)
responsive element.)
cells, and p38 has been reported to phosphorylate Elk-1 (69,71 – 74). Therefore,
the three pathways converge at the TCF level and are potential regulators of the
c-Fos gene through the transcriptional activation of the SRE. However, ATF-2
and c-Jun are also substrates of JNK, and p38 has been reported to phosphorylate
ATF-2 (69,71,72). Thus, JNK and p38 are also involved in the transcriptional and
posttranslational regulation of c-Jun and/or ATF-2.
AP-1-binding activity is regulated in vitro by the redox status of a single
conserved cysteine residue in the DNA-binding domains of Jun and Fos, which
has to be in the reduced state for DNA binding to occur (75,76). The nuclear
redox factor Ref-1, which was initially identified in HeLa nuclear extracts, was
shown to stimulate DNA binding of AP-1 in vivo via reduction of the conserved
cysteine residues (77). Ref-1 activity is itself regulated by a redox mechanism
involving Thioredoxin (TRX), another pleiotropic cellular factor with thiol-
mediated redox activity that facilitates protein –nucleic acid interactions. TRX
enhances DNA-binding activity of Jun and Fos via direct association with
Ref-1, acting as a hydrogen donor (78). Although not formally demonstrated in
endothelial cells, the ubiquitous nature of Ref-1 and TRX makes it likely that
this redox cascade may modulate AP-1 activity in these cells.
In several cell types, AP-1 behaves as a redox-sensitive transcription factor
which is activated, to different extents, under pro-oxidant conditions generated
by treatment with agents such as H2O2 , UV light, g-radiation, or various cyto-
kines (55,68). In endothelial cells, agents such as H2O2 (79), oxidized LDL
(80), and native LDL (81), which has been demonstrated to increase the
amount of free radicals in ECs (82), have been reported to activate AP-1
DNA-binding activity. Regulation of the endothelial expression of genes like
MCP-1 and ICAM-1 by H2O2 is mediated through AP-1-binding elements in
the promoters of these genes (83,84). Overexpression of AP-1 components
suggested that AP-1 itself is sufficient to induce ICAM-1 and MCP-1 genes in
endothelial cells and that AP-1-induced gene expression is mediated through a
mechanism independent of NF-kB (85). Induction of VCAM-1 by LDL
through activation of the VCAM-1 promoter is concomitant with increased
AP-1-binding activity (81). Recently, Wang et al. (86) demonstrated for the
first time that the AP-1 signaling pathway and its cognate binding motif are of
major importance for ICAM-1 expression in endothelial cells activated by LDL.
Stimulation of cultured HUVEC with leptin, which is concomitant with
increased intracellular accumulation of ROS, led to enhanced AP-1 DNA-
binding activity and enhanced expression of MCP-1 (87).
The pathways of ROS-mediated AP-1 activation and involvement of differ-
ent MAPKs are less well characterized in endothelial cells than in other cell types.
Laminar shear stress on endothelial cells is associated with peroxynitrite-
dependent activation of JNK (88). Chen et al. (89) found that AP-1 activation
by H2O2 in porcine aortic endothelial cells was mediated by the activation of
the JNK pathway. This JNK activation by H2O2 was in turn mediated by
Src-dependent epidermal growth factor (EGF) receptor transactivation without
involvement of PKC. Activation of AP-1 by LDL in human endothelial cells
was shown to involve the JNK-c-Jun and the p38-ATF-2 pathways through Ras
activation (90), but not the ERK/c-Fos pathway (90,91). Bouloumie et al. (87)
showed that leptin activated the JNK pathway, as demonstrated by enhanced
JNK activity and AP-1 binding.
Unlike NF-kB, AP-1 is activated not only by oxidants but also, paradoxi-
cally, by a number of antioxidants, including dithiocarbamates, the antioxidant
enzyme TRX, and NAC. These have been shown to stimulate the DNA
152 Nier et al.
binding and transcriptional activity of AP-1 in several cell types (92). In endo-
thelial cells, ICAM-1 expression induced by PDTC correlated with increased
AP-1 binding to the PDTC-responsive region of the ICAM-1 promoter (93).
PDTC was also shown to augment cytokine-induced AP-1 activation and
ICAM-induction (94).
On the other hand, antioxidants have also been shown to block AP-1
transcriptional activity in the endothelium. Shau et al. (95) demonstrated that
the antioxidant enzyme TRX peroxidase-1 blocked AP-1 activation induced by
TNFa. Similarly, NAC and catalase prevented the cyclic strain- or H2O2-
induced AP-1 binding to an AP-1 element in the MCP-1 promoter and MCP-1
expression (84). Enhanced AP-1-binding activity and expression of MCP-1 in
leptin-activated HUVEC was also abolished by NAC (87).
The mechanisms by which certain kinds of antioxidants selectively activate
AP-1 in endothelial cells are poorly understood. Recently, JNK and ERK
pathways were revealed as upstream regulatory mechanisms in PDTC-induced
c-Jun and c-Fos activation, with concomitant ICAM-induction, in endothelial
cells (94). Whereas JNK and c-Jun activation was sustained, upregulation of
ERK and c-Fos was only transient.
The thiol-antioxidant PDTC may activate AP-1 through pro-oxidant effects
since it is known to act as a copper ionophore (96 –98). Recently Kim et al. (99)
demonstrated in bovine cerebral endothelial cells (BCEC) that the activation of
AP-1 and suppression of NF-kB by PDCT was mediated by zinc. This idea
was based on the observation that PDTC treatment enhances zinc influx into
BCEC (99). Activation of AP-1 and inhibition of NF-kB could be blocked by
zinc chelation with Ca2þ-EDTA, but not by Zn2þ-EDTA. Zinc sulfate mimicked
the action of PDTC in activating AP-1 and inhibiting NF-kB, and depleted the
cellular glutathione store in a manner that could be reversed by thiol antioxidants
like NAC but not nonthiol antioxidants like Trolox, ascorbic acid, and butylated
hydroxyanisole. Finally, thiol antioxidants, but not nonthiol antioxidants, could
reverse activation by zinc of AP-1 and inhibition of NF-kB (100). These
results suggest that thiol antioxidants prevent the reciprocal actions of PDTC
on AP-1 and NF-kB by acting as metal-chelators, rather than by scavenging
oxygen free radicals or replenishing the cellular glutathione content.
Thus, antioxidant-mediated AP-1 activation and gene expression remain a
complex field, and further studies will be necessary to reveal the diverse mech-
anisms. As only effects of synthetic antioxidants on AP-1 activation have so
far received extensive study, investigation of nutritionally relevant antioxidants
would be of great interest.
VITAMIN E AND ENDOTHELIAL GENE EXPRESSION

Vitamin E is the most important lipid-soluble antioxidant in the diet. It consists of
a group of tocopherol and tocotrienol compounds that have in common a
6-chromanol ring coupled to a C16 aliphatic side chain. For tocopherols, this
side chain is saturated, whereas for tocotrienols, it contains three double bonds.
The tocopherols (a, b, g, and d) are subdivided according to the number and
position of methyl groups—1, 2, or 3—on the phenolic ring. The antioxidant
properties are conferred by a hydroxyl group, also on the phenolic ring. The
ability of this group to donate an H atom is dependent on the number of
methyl groups. a-Tocopherol, with three methyl groups, is thought to be most
potent.
Antioxidant effectiveness in vivo also depends on factors other than the
number of methyl groups. For example, the mobility of the molecule within
lipid layers, which depends on the nature of the aliphatic side chain, seems
important (101,102). Additionally, bioavailability may differ between homologs.
Vitamin E circulates in the blood within lipoprotein particles. It is transferred
from the gut to the liver in chylomicrons, but in hepatocytes is transferred into
very low density lipoprotein by a process that involves a cytosolic a-tocopherol
transfer protein (a-TTP). Circulating and tissue vitamin levels are therefore
determined by the affinities of a-TTP for the different tocopherols. For the
b-, g-, and d-forms, affinities are 38%, 9%, and 2% that for a-tocopherol, respect-
ively (103). Finally, effectiveness is also determined by the presence of other
antioxidants. In particular, ascorbate can regenerate vitamin E from the tocopher-
oxyl radical that is formed when vitamin E donates an H atom (104).
The possibility that vitamin E might reduce the oxidation of LDL and
hence slow atherogenesis has motivated a large number of in vitro and in vivo
studies. However, in addition to the inhibition of such oxidation, vitamin E has
been identified as a favorable modulator of many other processes at the molecular
and cellular levels that may be of importance in atherogenesis (Table 7.2).
Several of these potentially beneficial effects may be mediated through
influences of the vitamin on gene expression. Thus, for example, the expression
of CD36, a-TTP, a-tropomyosin, and collagenase are affected by a-tocopherol at
Table 7.2 Potential Mechanisms by Which Vitamin E

Inhibits Atherosclerosis
# LDL oxidation, # macrophage uptake of oxLDL (105)

# Endothelial cell injury (106 –110)
# Adhesion molecule expression (107,109,111)
# Immune/endothelial cell adhesion (107,112)
# Inflammatory cytokines and chemokines (109)
# Smooth muscle cell proliferation (113– 115)
# Platelet aggregation (116,117)
" NO production, " arterial dilatation (118,119)
" PGI2 , # TXA2 (116)
Note: Low density lipoprotein, LDL; oxidized low density lipo-

protein, oxLDL; nitric oxide, NO; prostacyclin, PGI2; throm-
boxane A2 , TXA2 .
154 Nier et al.
the transcriptional level (120). The effects of vitamin E on expression of inflam-

matory genes has received particular attention. It inhibits the production of
cytokines (IL-1b, IL-6, IL-8) and suppresses the expression of adhesion
molecules (ICAM-1, VCAM-1, E-selectin) and chemoattractants (MCP-1) in
endothelial cells, and reduces their adhesive interaction with monocytes.
Table 7.3 gives further details of these studies of protein (and, where indicated,
mRNA) expression.
Although there is strong evidence for effects of vitamin E, the mechanisms
involved are less clear. The authors of many of these studies speculate that the
vitamin is altering gene expression by inhibiting NF-kB activation. However,
the evidence for this in endothelial cells is exceptionally limited. Of the
studies cited, only that by Islam et al. (123) showed, by gel shift assay, that
NF-kB activation actually decreased.
Several studies have shown that vitamin E modulates PKC activity. This
could explain any influence on NF-kB that does occur, since PKC affects the
IKK signalosome (see earlier). There is considerable recent evidence for a
direct effect of PKC on NF-kB activity. Ogata et al. (128), for example, have
demonstrated that ROS-induced NF-kB activation in human endothelial cells
was abolished by PKC inhibitors.
The influence of vitamin E on PKC was first demonstrated by Boscoboinik
et al. (129) in SMC, where the vitamin inhibits proliferation induced by various
stimuli. The effect, which occurs at physiological vitamin E levels, is not limited
to this cell type or response, however. For example, vitamin E inhibits platelet
aggregation by almost complete abrogation of the phosporylation of a 47 kDa
PKC substrate (130). Consistent with this, it causes a delay in intra-arterial
thrombus formation (131). In endothelial cells, vitamin E exerts an inhibitory
action on thrombin-induced PKC activation and endothelin secretion (132).
A putative mechanism for the influence of vitamin E on PKC is the
activation of protein phosphatase 2A (PP2A) (115,133,134), which causes the
dephosphorylation and hence inactivation of PKCa (115). The expression of
PKC is not affected. b-Tocopherol or Trolox, which have antioxidant capacity
similar to a-tocopherol, do not exert such an effect, showing that a-tocopherol
inhibits PKC activity in a specific manner (135). The phenolic hydroxyl group
on the chromanol ring does not appear to be involved. Freedman and Keaney
(130) found that blocking this group with an acetate ester does not alter the inhi-
bition of PKC. Furthermore, b-tocopherol (which has the same hydroxyl group)
does not appear to be active (129).
At least one study suggests that vitamin E modifies gene expression inde-
pendently of its antioxidant function and of its effect on PKC. Faruqi et al. (121)
demonstrated that vitamin E inhibits monocyte adhesion to the endothelium
induced with phorbol 12-myristate 13-acetate (PMA, a well-established activator
of PKC) and thrombin. However, it did not inhibit phosphorylation of the
myristoylated alanine-rich C kinase substrate protein, which is specifically phos-
phorylated by PKC and is a good indicator of PKC activity. In addition, it did not
Table 7.3 Effects of Vitamin E on Expression of Inflammatory Genes
Authors Cell type/Animal Experiment Genes/Proteins
Faruqi et al. (121) HUVEC a-Tocopherol (cells pretreated); E-selectin # (mRNA and surface
stimulaton with IL-1, thrombin, protein expression)
PMA
Devaraj et al. (107) Monocytes from healthy a-Tocopherol for 8 weeks IL-1b #, monocyte-endothelial cell
humans, HUVEC (1200 IU/d) adhesion #
Cominacini et al. (122) HUVEC 1. Pretreating cells with vitamin E 1. VCAM-1 #, ICAM-1 #, E-selectin
2. Pretreating LDL with vitamin E 2. VCAM-1 #, ICAM-1 #, E-selectin
(5 mM before oxidation) and
probucol (incorporation of
antioxidants into lipoproteins)
Martin et al. (61) HAEC Incubated with LDL, vitamin E sICAM-1 #, Prostacyclin I2 "
Islam et al. (123) HUVEC, U937 a-Tocopherol (25, 50, 100 mM) CD11b #, VLA-4 # , reduced agonist-
induced adhesion to HUVEC
Fruebis et al. (111) Normocholesterolemic rabbits Vitamin E (0.1%), Probucol (low: VCAM-1 (mRNA, Protein) #
0.04– 0.075%, high: 0.5%)
Wu et al. (109) HAEC, U937 1. Vitamin E (20, 40, 60 mmol/L) 1. IL-8 #
2. Cells stimulated with IL-1, 2. ICAM-1 #, VCAM-1 #,
Effects of Antioxidants on Gene Expression in Endothelial Cells
vitamin E (20, 40, 60 mmol/L) E-selectin #, MCP-1 #, IL-8 # (at 40

and 60 mmol/L), IL-6 #
(at 40 and 60 mmol/L)
(continued )
155
156
Table 7.3 Continued

Authors Cell type/Animal Experiment Genes/Proteins
Desideri et al. (124) Hypercholesterolemic patients Vitamin E (400 IU/d or 800 IU/d) sVCAM-1 #
Yoshida et al. (125) EC Treatment with LDL, pretreatment ICAM-1 #, VCAM-1 #
with vitamin E
Zapolska-Downar Human EC Cells activated with IL-1b, MCP-1 #
et al. (126) vitamin E
Peluzio et al. (127) Apo E knockout mice Vitamin E (control: 40 mg/kg diet, MCP-1 # (mRNA and protein)
suppl. group: 800 mg/kg diet)
Note: Endothelial cells, EC; human umbilical vein endothelial cells, HUVEC; human aortic endothelial cells, HAEC; human monocyte cell line, U937; phorbol
12-myristate 13-acetate, PMA; integrin alpha M or Mac-1 alpha (leukocyte) CD11b; very late antigen-4 (expressed by leukocytes), VLA-4; apolipoprotein E,
Apo E; interleukin, IL; monocyte chemoattractant protein-1, MCP-1; intracellular adhesion molecule-1, ICAM-1; vascular cell adhesion molecule-1, VCAM-1;
E-selectin, cell adhesion molecule; nuclear transcription factor-kappa B, NF-kB; protein kinase C, PKC; low density lipoprotein, LDL; real time polymerase
chain reaction, rtPCR.
Nier et al.
affect the activation of NF-kB at vitamin concentrations that caused marked

decreases in E-selectin mRNA and protein expression. Consistent with this
result are studies showing an absence of effect of vitamin E on NF-kB in non-
endothelial cells. Suzuki and Packer (43) showed that although vitamin E
acetate and a-tocopheryl succinate dose-dependently inhibit TNF-a-induced
NF-kB activation in Jurkat cells, a-tocopherol does not. Similarly, Nakamura
et al. (136) found that pretreatment with 50 mM a-tocopheryl succinate produced
a 43% inhibition of NF-kB binding to DNA when cells of the human macrophage
cell line THP-1 were activated with LPS, but a-tocopherol itself was without
effect.
Thus although it seems clear that vitamin E affects gene expression, the
transcription factors responsible have not been clearly identified. Whether or
not NF-kB is involved, perhaps through the influence of vitamin E on PKC,
there is evidence that the vitamin is not acting through an antioxidant effect.
REGULATION OF TRANSCRIPTION THROUGH CHANGES IN

CHROMATIN STRUCTURE
Changes in gene expression are mediated primarily through the action of tran-
scription factors. However, activation or inhibition of the transcription factors
themselves, discussed in the preceding sections, are not the only ways in
which their action is controlled; modification of chromatin structure affects
whether transcription factors are able to influence gene expression.
Chromatin is a filamentous complex of DNA, histones (the principal
protein), and other proteins, which are predominantly transcription factors.
Some is tightly coiled (“heterochromatin”) and transcriptionally inert, whereas
the remainder (“euchromatin”) is transcriptionally active in places. DNA is
divided between nucleosomes and linker segments. In the nucleosome, a DNA
segment 146 bp long is wrapped around an octomer of histones (two each of
types H2A, H2B, H3, and H4). Linker regions of DNA having variable length
separate the nucleosomes; the nucleosomes and the linker DNA are associated
with histone H1. Structural modifications of the histones and of the DNA itself
have been implicated in the control of gene expression. Such modifications are
enzymatically controlled, and can reflect dynamic changes in cellular environ-
ment. However, they can also be heritable (mitosis and meiosis). This leads to
the fascinating possibility that environmental effects in one generation may influ-
ence gene expression for several generations. Because the structural changes do
not modify the genome sequence itself, they have been termed “epigenetic.”
Electrostatic interactions between histones and DNA are regulated by
acetylation, methylation, and phosphorylation of the histones. Broadly speaking,
acetylation of histones allows transcriptional machinery to access DNA, whereas
deacetylation and methylation block it, preventing transcription factors from
recognizing their response elements. Histone phosphorylation also seems to
play a role. Chromatin remodeling complexes aid these processes by continually
158 Nier et al.
shuffling the positions of individual nucleosomes, which are transcriptionally

inactive, to expose random sequences for short periods (137,138). Thus, an inter-
play exists between chromatin remodeling and histone modification. For
example, expression of a gene may require disruption of nucleosomes positioned
at the promoter region by a chromatin remodeling complex before an enzyme
required for histone acetylation can be recruited (139). Alternatively, expression
of genes may require that histone acetylating enzymes and even RNA polymerase
bind to the promotor prior to recruitment of the chromatin remodeling complex
(140). Specific histone modifications and chromatin remodeling complexes have
also been implicated in silencing at some loci (137).
DNA methylation is the addition of methyl groups, after replication, to the
50 position of cytosine rings within guanine – cytosine (CpG) dinucleotides. This
results in alterations in the structure of DNA—specifically to the conformation of
the groove—and hence alters the binding of proteins to it (141). For example,
methylated CpG dinucleotide sites near a gene can recruit specific DNA-
binding proteins, which in turn recruit histone deacetylases, resulting in loss of
histone acetylation and silencing of gene expression (142). Approximately
70% of CpG pairs in the mammalian genome are constitutively methylated
(143). There are short DNA sequences, however, with an unusally high
guaninephosphate – cytosine content and a high frequency of unmethylated
CpG dinucleotides (144). These regions, called CpG islands, are primarily
located in 50 regulatory regions. They are present in all housekeeping and most
tissue-specific active genes (145) and have also been proposed to function as
replication origins (146). Table 7.4 lists genes of importance in atherogenesis
which are known to be at least partially regulated by DNA methylation.
Table 7.4 Examples of Genes Implicated in Atherogenesis Which Are At Least

Partially Regulated by DNA Methylation
Gene Name Reference
IFN-g Interferon-g White et al. (147)

PDGF-A Plateled-derived growth factor Lin et al. (148)
MMP-2 Matrix metalloproteinase-2 Sato et al. (149)
TIMP-3 Tissue inhibitor of metalloproteinase Wild et al. (150)
ICAM-1 Intracellular adhesion molecule Tanaka et al. (151)
p53 Estrogen receptor Schroeder and Mass (152),
Guevara et al. (153)
ERa Extracellular superoxide dismutase Post et al. (154),
Ying et al. (155)
EC-SOD Tumor suppressor Laukkanen et al. (156)
Source: From Hiltunen and Yla-Herttuala (157).

Direct evidence for the methylation of genes in atherogenesis is emerging.

To date, the evidence is strongest for the estrogen receptor a (ERa). This receptor
is activated by estrogen and regulates a variety of cellular activities of potential
importance in the disease. For example, activated ERa increases the expression
of eNOS, resulting in enhanced nitric oxide (NO) production that, in turn, is
known to inhibit SMC proliferation, platelet aggregation, leukocyte adhesion, etc.
Post et al. (154) found increased methylation of CpG islands in the ERa
gene in atherosclerotic human tissue compared with normal arterial tissue.
Furthermore, evidence for ERa gene methylation was obtained in contractile
vascular smooth muscle (VSMC) but not in proliferative VSMC in culture,
indicating that methylation had occurred during the phenotypic switch (155).
However, human aortic endothelial cells did not show differential methylation
of ERa in culture.
More limited evidence is available for extracellular superoxide dismutase
(EC-SOD), which protects arteries against deleterious effects of superoxide
anions and the development of atherosclerosis. Laukkanen et al. (156) cloned
and characterized the rabbit EC-SOD gene and showed it had a marked reduction
in the amount of methylated CpG dinucleotides in atherosclerotic arteries
compared with normal arteries, perhaps indicating some form of compensatory
upregulation of gene expression. However, the methylation was in the coding
region, and it is not established that such modification alters transcription.
There is an intriguing suggestion that DNA methylation may explain
why age is a risk factor for atherosclerosis (158). A number of studies have
demonstrated an increased methylation of specific genes, and a global drop in
methylation, with age. Consistent with this, the study by Post et al. (154)
found decreased methylation of the ERa gene with age in cardiac mycocytes.
As with aging, experimental atherosclerosis was found to decrease global methyl-
ation in rabbit aortas (156).
There is also a link with hyperhomocysteinaemia, which causes oxidative
stress (159), and is another risk factor for atherosclerosis. Homocysteine can be
methylated to methionine, which in turn is metabolized to products involved in
the methylation of DNA. One of the methyl donors for the conversion of
homocysteine to methionine is produced by the action of the enzyme methy-
lenetetrahydrofolate reductase. Mice deficient in this enzyme have elevated
homocysteine, global hypomethylation of DNA, and aortic lipid deposition
(160,161).
To date, there is no evidence for epigenetic modification by dietary antiox-
idants in endothelial cells. We speculate that such evidence will be obtained,
however, because a recent study has demonstrated that shear stress does lead
to modifications of this type; as explained in the following section, there is a
strong link between shear stress and oxidative stress. Illi et al. (162) have demon-
strated that shear stress induces histone H3 acetylation and phosphorylation and
cooperates with the histone deacetylase inhibitor trichostatin to enhance histone
H3 phosphoacetylation and histone H4 acetylation. These histone modifications,
160 Nier et al.
which lead to chromatin remodeling and DNA unwinding, may play a fundamen-
tal role in the shear stress-dependent regulation of gene expression.
APPLICABILITY TO PHYSIOLOGICAL CONDITIONS

The data discussed in the preceding sections have been obtained almost exclu-
sively by using endothelial cells in static culture. The use of cell culture, and
the absence of cyclic strain and shear stress (which are present in vivo as a
result, respectively, of pulsatile pressure and blood flow), could have had a
large influence on the results.
In culture, it is conventional to incubate cells under a gas mixture of 95%
air/5% CO2 . This gives an oxygen partial pressure of 150 mm Hg, whereas the
endothelium will be exposed to a partial pressure in systemic arteries—the site of
atherosclerosis—of 105 mm Hg and in veins to 40 mm Hg or less, depending
on tissue activity. The hyperoxic culture environment has definite effects on cell
biology. Cells grown under these conditions (20% O2) are preadapted and prese-
lected to survive oxidative stress (and have increased accumulation of oxidant
damage compared with cells grown at 3% oxygen) (163). The adaptation
appears to involve upregulation of heat shock protein (164) and other genes.
Halliwell (165) has recently reviewed the culture artifacts likely to affect inves-
tigations of ROS-mediated effects on cells. First, the high oxygen tension will
lead to elevated leakage of ROS from the electron transport chain. Then the
media themselves can be pro-oxidant, since they may contain iron or copper
ions, and can generate further ROS from substances added to them (including
ascorbate, polyphenolics, and thiols). Serum added to the media may also
release metal ions. Additionally, media are generally deficient in ascorbate and
vitamin E, and also in selenium which is a cofactor for antioxidant enzymes
such as TRX and GPx. Hence, effects of exogenous antioxidants may be
exaggerated in culture.
Cyclic strain is also known to influence oxidative stress and redox-sensitive
gene expression (84,166). Here, however, it is shear stress (the frictional force
acting per unit area on the endothelium, parallel to its surface) that is considered
in detail, since it has been more extensively studied. Although it is well known to
modify redox-sensitive signaling and gene expression, the effects are complex
and not fully understood. A key consideration is that shear varies substantially
in magnitude and temporal pattern from site to site within the vasculature.
Hence, the phenomena observed in static culture might be modified in different
ways at different locations in vivo. This is potentially important because athero-
sclerosis is a patchy disease—its prevalence varies substantially with location.
Paralleling the confusion concerning the influences of shear on redox signaling,
however, there has been confusion about the locations and hemodynamic
environment predisposing to atherosclerosis. This confusion arises from
neglect of effects of age and species on the location of disease, and because of
the technical difficulty in measuring wall shear stress with adequate spatial
resolution under physiological conditions (167,168).
It has been known for more than a decade that the application of fluid
dynamic shear stress to cultured endothelial cells can affect gene expression
(169 – 171). Recent studies have demonstrated that over 100 genes are regulated
by the level or type of shear (172). It has subsequently emerged that shear also
affects levels of ROS in endothelial cells. Most notably, Laurindo et al. (173)
demonstrated this in intact vessels. In rabbit aortas perfused with a spin trap,
radical adducts, detected by electron paramagnetic resonance spectroscopy,
increased with increase in flow rate. This effect was completely blocked by
endothelial denudation and by SOD, but not by NG-nitro-L -arginine methyl
ester (L-NAME) or indomethacin (inhibitors of NOS and COX, respectively).
Similarly, when flow was increased in iliac arteries in vivo by infusing saline
through an extracorporeal circuit or by pharmacological or physiological
means, levels of the ascorbyl radical, a stable oxidation product of ascorbate,
were increased. Again, this response was completely blocked by SOD, but
not by L-NAME, indomethacin, or catalase. Stopping the flow decreased
ascorbyl levels.
Shear alters many signaling pathways in endothelial cells (174,175), so the
increase in ROS need not necessarily explain the altered gene expression.
However, there is increasing evidence for a causal relation. Several experiments
have shown that shear increases ROS levels and putatively proatherogenic gene
expression in endothelial cells, and that antioxidants block the change in
expression. Thus, for example, Chiu et al. (176) exposed endothelial cells from
human umbilical cords to a shear stress of 20 dynes/cm2 and detected increased
superoxide concentrations after 15 min, with a peak at 30 min. Levels then
declined but were still elevated by 6 h if flow was maintained. Shear also
increased ICAM-1 mRNA at 3 or 6 h, an effect that was reduced by NAC and
abrogated by catalase. A reporter gene assay demonstrated altered transcription,
and surface ICAM-1 expression was also increased. A similar result was obtained
by Yeh et al. (177), who additionally obtained evidence for the involvement of
the glycosphingolipid, lactosylceramide. When an inhibitor of the enzymes
involved in its synthesis was applied, there was no shear-induced increase in
superoxide production or ICAM-1 expression. Hsieh et al. (178) showed an
increase in ROS in HUVEC exposed to 15 – 40 dynes/cm2 that was blocked by
NAC or catalase, and reduced by deferoxamine mesylate or a hydroxyl radical
scavenger. An increase in c-Fos gene expression was also seen; this increase
was reduced by NAC and catalase.
Although such experiments suggest that shear stress has effects similar to
cytokines, other studies have demonstrated greater complexity. As mentioned
above, Mohan et al. (179) exposed human aortic endothelial cells to a shear
stress of 2 dyne/cm2 for 6 h and detected increased NF-kB activation and
mRNA and protein expression for VCAM-1 (which has NF-kB-binding sites
in its promoter). The antioxidant PDTC blocked all three, but NAC only slightly
162 Nier et al.
decreased NF-kB, did not affect VCAM-1 mRNA levels and increased VCAM-1
protein expression. With cytokine stimulation, both PDTC and NAC inhibited all
three. In this case, therefore, shear and cytokines acted through different path-
ways. Other studies have shown that shear increases ROS but that this down-
regulates putatively pro-atherogenic genes. Thus, Masatsugu et al. (180) found
that endothelin converting enzyme-1 (ECE-1) and endothelin-1 (ET-1) mRNA
were downregulated in bovine carotid artery endothelial cells and in HUVEC
by shear (1.5 – 15 dyne/cm2) (and by H2O2). Shear stress increased intracellular
peroxide concentrations, and the downregulation of both mRNAs was almost
completely blocked by NAC.
An additional complication arises from studies demonstrating that shear
can oppose effects of ROS on gene expression. Tsao et al. (181) found, as
expected, that incubation with oxidized LDL or with LPS and TNF-a increased
superoxide production, NF-kB production, and VCAM-1 expression in human
aortic endothelial cells. However, these effects were inhibited by a 4 h pre-
exposure to shear, in complete contrast to the effects of shear alone described
earlier. The effects of flow were abolished by nitro-L -arginine (another NOS inhi-
bitor) and mimicked by an NO donor, so NO may have been influencing oxidant-
mediated transcription. Similarly, Hojo et al. (182) found that shear can block
signaling events induced by ROS. JNK activation (which is thought to be
pro-atherogenic through phosphorylation of c-Jun, activation of AP-1, and
stimulation of pro-inflammatory genes such as ICAM-1) was induced in bovine
lung microvascular endothelial cells by H2O2 ; this effect was reduced by a 10 min
pre-exposure to a shear stress of 12 dynes/cm2. Shear increased the activity of
glutathione reductase and increased the ratio of reduced to oxidized glutathione.
When glutathione reductase was inhibited, shear was ineffective. And shear
can also block ROS-induced apoptosis. Hermann et al. (183) induced apoptosis
in HUVEC by incubation with H2O2 or TNF-a. Concomitant shear stress
(15 dynes/cm2) completely inhibited this effect. The effect of shear was, in turn,
partly reduced by N G-monomethyl-L -arginine (L -NMMA, a further NOS inhibitor)
and by inhibition of the GSH pathway with buthionine sulfoximine (BSO).
L -NMMA and BSO together completely blocked the effect of shear. Dimmeler
et al. (184) additionally showed that the inhibitory effect of shear on apoptosis
in HUVEC could be reduced by an antisense oligonucleotide to Cu/Zn SOD.
Indeed, although it has been known for some time that shear upregulates
SOD gene and protein expression (185,186), recent studies, including those
using microarray technologies, show that shear induces a wide range of
antioxidant genes (187 – 190). Chen et al. (191) note that a large number of the
antioxidant defense genes upregulated by shear have an antioxidant response
element (ARE) or an ARE-like sequence in their promoters. These genes
include NAD(P)H:quinone oxidoreductase (NQO1), heme oxygenase-1
(HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, gluta-
thione S-transferase, and gamma-glutamylcysteine synthase. Shear appears to
activate ARE-mediated transcriptional activity in endothelial cells. For example,
mutation of ARE, expression of antisense Nrf2 (a transcription factor for ARE),

a dominant negative Nrf2, or an Nrf2 inhibitor all prevented shear upregu-
lating NQO1.
How can the contradictory effects of shear on redox status and its effects be
reconciled? This question cannot be answered at the moment, but a number of
possibilities are emerging. One is that both ROS and antioxidant defenses are
increased by shear—the latter may be a response to the former. Reference has
already been made to the study by Hsieh et al. (178) in which shear increased
ROS. The same study also found that shear increased antioxidant capacity:
when exogenous H2O2 was added to extracts of the endothelial cells, it was
decomposed more quickly in those cells that had been exposed to shear. This
effect was blocked by an inhibitor of catalase.
Another possibility gaining increasing support is that the type of shear is
itself important for the influence on redox status or redox-sensitive transcription
factors. (This explanation obviously fits well with the non-uniform localization of
atherosclerosis in the vasculature; all cells will be exposed to shear, but its level
and temporal pattern will vary from site to site.) Topper et al. (186) exposed
HUVEC to laminar shear stress or to turbulent shear stress of comparable
time-averaged magnitude (10 dynes/cm2) for 1 –24 h, and then examined
effects on Mn SOD. Protein levels and mRNA increased with laminar but not
with turbulent shear. Nuclear runoff analysis showed similar changes in tran-
scriptional rates. Comparable data were obtained for eNOS mRNA levels.
de Keulenaer et al. (192) compared effects on HUVEC of oscillatory and
steady shears, both at 5 dynes/cm2, for 1 – 24 h. The oscillatory shear caused a
sustained increase in NADH oxidase activity, superoxide levels, and gene and
protein expression of HO-1, whereas the steady shear only transiently increased
NADH oxidase activity and HO-1 expression, and gave lower superoxide levels.
However, steady shear, unlike oscillatory shear, did increase Cu/Zn SOD. Com-
parable data concerning superoxide levels were obtained in a similar experiment
by McNally et al. (193); use of a series of inhibitors and other interventions
demonstrated that the superoxide was generated by xanthine oxidase and that
this in turn was maintained by NAD(P)H oxidase. Nagel et al. (194) inserted a
bar across their culture dish to obtain regions of disturbed and then uniform
flow (12 dynes/cm2). Compared with culture in the absence of flow, the
nuclear concentration of the ROS-sensitive transcription factors NF-kB, Egr-1,
c-Jun and c-Fos, assessed by quantitative immunofluorescence, were all upregu-
lated more by the disturbed than the uniform shear. All these studies suggest that
steady laminar shear stress might have a more atheroprotective, less atherogenic
effect than turbulent, oscillatory, or disturbed flows.
Finally, we note that recent evidence directly supports the view that differ-
ent shear stresses have different influences on the response of endothelial cells to
antioxidants. de Nigris et al. (195) exposed human coronary endothelial cells
to shears of 1 or 15 dyne/cm2. eNOS protein expression increased more at
1 than at 15 dyne/cm2, and treatment with a-tocopherol and ascorbic acid
164 Nier et al.
(50 and 10 mM, respectively) substantially increased expression at 1 dyne/cm2,

but had little or no effect at 15 dyne/cm2. Conversely, expression of the redox-
sensitive transcription factors Elk-1 and p-CREB increased at 1 dyne/cm2 and
increased more at 15 dyne/cm2. The antioxidants reduced the effect at
15 dyne/cm2 but had no effect at 1 dyne/cm2.
Because effects of antioxidants on transcription factors and gene
expression are likely to be modulated in complex ways by shear stress, measure-
ment of net effects in vivo—preferably in lesion prone and protected sites—is
essential. Such studies are starting to emerge. One such trial was conducted
by de Nigris et al. (196). Levels of cMyc (an oxidant-sensitive transcription
factor) and its binding partner Max were examined in coronary arteries from
pigs fed a cholesterol-enhanced diet. Higher levels were found in lesion prone
areas than in protected vessels, and even higher levels where mild lesions were
present. The cMyc-regulated genes GAD4 and p53 were down- and up-regulated,
respectively, in the same distribution. All these changes were attenuated by
dietary antioxidant vitamins. Subsequently, the same group found that pigs
fed a cholesterol-enhanced diet had elevated activation of NF-kB, decreased
eNOS expression and decreased radical scavenging activity in coronary
artery tissue compared with animals on a normal diet. In a third group of
animals, administered 100 IU/kg day vitamin E and 1 g/day vitamin C con-
currently with the cholesterol, NF-kB activation and NO bioactivity were
normalized (197).
Along similar lines, de Nigris et al. (195) examined healthy vessels, areas
with small lesions and areas with large lesions from LDL-receptor knockout
(LDLR2/2 ) mice fed with high-fat diet for 6 months, with or without an
additional 1 week of dietary antioxidant supplementation. (Endothelial mor-
phology, which is flow-dependent, suggests that the areas with large lesions
experience low time-averaged levels of shear with complex patterns, whereas
the areas with small lesions experience high unidirectional shear.) eNOS levels
were lower in small lesions than in healthy vessels, and even lower in large
lesions, and were increased modestly by the antioxidants. Conversely, Elk-1
and p-CREB levels were increased, not reduced, in small lesions and more so
in large lesions, and were decreased by the antioxidants. No substantial differ-
ences were seen in p-JUN expression.
The complex and contradictory nature of the data emerging from studies of
cultured endothelial cells means that further studies of this type are urgently
required.
DISCUSSION
The widely held views that inflammation is a key event in atherogenesis and that
antioxidants suppress the expression of pro-inflammatory genes imply that
administration of antioxidants in vivo should be anti-atherogenic. The results
of antioxidant trials using animal models of atherosclerosis have generally tended

to support this idea. Trials of probucol (and its analogs), butylated hydro-
xytoluene (BHT), diphenylphenylenediamine, and coenzyme Q have shown
that these antioxidants cause large reductions in the extent of arterial lipid depo-
sition (198). The studies in which vitamin E has been administered to hypercho-
lesterolaemic rabbits have shown less consistency. Although several found a
protective effect (199 –201), others did not (202) and some showed an increase
in the extent and severity of lesions (203). Nevertheless, vitamin E does
appear to be effective in reducing lesions in knockout mice, either when used
alone (204) or in combination with other antioxidants (205,206). The broadly
similar effect of a wide range of structurally unrelated antioxidant compounds
suggests that it is their antioxidant activity per se rather than, for example, the
lipid-lowering effect of probucol or other as yet undetermined anti-atherogenic
properties of these compounds, which accounts for their beneficial effect in
animal studies.
The influence of dietary antioxidants on human disease is much less clear.
A substantial body of epidemiological evidence has shown that antioxidant
vitamin intake or supplementation is inversely associated with the risk of cardio-
vascular disease (207). However, the results of prospective clinical trials using
beta carotene, vitamin E, or vitamin E plus other antioxidant vitamins in subjects
with coronary heart disease, or at risk of it, have been disappointing. Ten such
double-blind, placebo-controlled trials have recently been reviewed by Steinberg
and Witztum (208), and 11 by Jialal and Devaraj (209). Of the 11, only 4 gave a
positive effect on the primary endpoint and of these one (CHAOS) showed no
effect on cardiovascular mortality, another (Transplant Associated Arterio-
sclerosis Study) was concerned with transplant arteriosclerosis, and a third
(SPACE) used subjects with end stage renal disease. The four largest studies
were all negative. No trials showed a beneficial influence of beta carotene, and
there was evidence in some trials (although not others) for possible adverse
effects (210 – 212). A recent review by Padayatty et al. (213) suggests similarly
negative results concerning effects of vitamin C on markers of oxidation or
clinical benefit.
The results of prospective clinical trials therefore seem to contradict the
idea that antioxidants could reduce atherosclerosis by inhibiting expression of
pro-inflammatory genes. However, proponents of the beneficial effects of anti-
oxidant therapy point to a number of doubts concerning the interpretation of
some trials. These views are controversial. Jialal and Devaraj noted that in three
of the seven negative studies, plasma antioxidant levels were not measured
(and in a fourth, they rose in the placebo group too), and that in four of them
there were no biomarkers of oxidative stress. They also note that the form of
a-tocopherol might be important. All four positive trials used RRR-a-tocopherol,
whereas only two of the seven negative trials did so; the remainder used all
racemic a-tocopherol, in which the RRR form is only one of eight stereoisomers.
Nevertheless, one of the two negative trials using the RRR form, the HOPE trial,
166 Nier et al.
was far larger than the four positive ones, and showed no hint of a bene-
ficial effect. Furthermore, uptake by endothelial cells appears stereoisomer-
independent (214).
Steinberg and Witztum (208) suggest that vitamin E might have been the
wrong antioxidant to use in the trials since it reacts only slowly with superoxide,
is much less potent than probucol (as judged by effects on LDL oxidation when
administered in vivo) and, in the absence of vitamin C or other co-antioxidants,
can become a pro-oxidant. They also suggest that subjects should have been
selected for the presence of oxidative stress; an analogy is drawn with trials of
antihypertensive or lipid-lowering drugs which used subjects selected for
hypertension and hyperlipidaemia, respectively. They consider the SPACE
trial, which used subjects with end stage renal disease, to be important in this
regard since such subjects are known to have high oxidative stress: this trial
showed a big reduction in end point. However, the trial was a small one
(15 vs. 33 primary endpoints). In the more recent and much larger Heart Protec-
tion Study, subjects with high plasma creatinine levels (indicative of renal
problems) had more incidents than those with lower levels, but received no
benefit from vitamin E. Furthermore, their analogy is not completely convincing
since lowering blood pressure and plasma cholesterol levels are beneficial even in
subjects who do not have hypertension or hyperlipidaemia. The various hypoth-
eses invoking a role of oxidative stress in atherogenesis have, in any case,
regarded this as being a nearly universal process.
Finally, Steinberg and Witztum (208) suggest that the typical 5-year dur-
ation of a clinical antioxidant trial may be too short to reveal effects on the
initial development of lesions, instead indicating only effects (or, rather, a lack
of them) on plaque rupture. They argue that the positive results obtained in the
even shorter-term animal studies may be misleading because of the rapid
disease development in these models and because of the use of endpoints, such
as area of lipid deposition, that correspond to early disease. Nevertheless, lipid
lowering and blood pressure lowering trials—addressing factors also thought
to influence the early stages of atherogenesis—have had positive outcomes
over such timescales; it seems unlikely that all these beneficial effects can be
explained in terms of pleiotropic effects of the drugs on plaque rupture. Further-
more, their comments were made in the context of using antioxidants to control
LDL oxidation. When considering the hypothesis that antioxidants might control
inflammation, it is not so plausible to argue that only early stages of the disease
should be affected: plaque rupture involves the activation of macrophages, lym-
phocytes, and mast cells which then degrade extracellular matrix and the fibrous
cap (215). It seems inconceivable that inflammation is not involved in the 5 years
preceding a clinical event. Hence, the discrepancy between the success of antiox-
idant trials in animals and the absence of such success in human trials might most
economically be explained by the hypothesis that oxidation is more important in
animal models of the disease. It is known that smaller animals generate more
ROS (which arise mainly from mitochondria) because of their higher metabolic
rate, and the very high cholesterol levels generally present in the animal models
will exacerbate this difference (208).
CONCLUSION
Antioxidants affect pro-inflammatory transcription factors in vitro, and evidence
is emerging for influences in animals, but the effects are not simple and not
always related to antioxidant activity per se. Evidence is lacking that antioxidants
affect atherosclerosis in people, despite the likely role of inflammation in this
disease. Although it is possible that a beneficial effect of antioxidants on athero-
genesis, mediated by regulation of pro- and anti-inflammatory genes, could be
obtained in the future through the development of suitable compounds and
protocols, the data obtained at a cellular level are currently too complex and
contradictory, and there are too many doubts about their relevance to in vivo
conditions, to be assured of this.
ACKNOWLEDGMENTS
The support of the BHF, BBSRC, and Gen Foundation, and the assistance of
Mrs. J. D. del Rio is gratefully acknowledged.
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8
Fatty Acids, Gene Expression, and
Coronary Heart Disease (CHD)
Anne M. Minihane
Introduction 182
Fatty Acid Structure and Tissue Sources 182
Metabolism of Fatty Acids 183
Absorption 183
Transport as Lipoproteins 184
Intracellular Metabolism 186
Fatty Acid Regulation of Gene Expression 187
PUFA and Hepatic Lipogenesis 187
PUFA Induction of Lipid Oxidation 189
Fatty Acids and Adipocytes Gene Expression 189
Fatty Acid and Arterial Wall Gene Expression 190
PUFA and Their Cellular Mechanisms of Action 191
Transcription Factors 192
Peroxisome Proliferator-Activated Receptors (PPAR) 192
PPAR Ligands 193
Other Families of Transcription Factors that Mediate the PUFA/PUFA
Derivative Effect on Gene Expression 193
Sterol Regulatory Element-Binding Protein 194
Hepatic Nuclear Receptor-4 (HNF-4) 194
Nuclear Factor-Y (NF-Y) and Nuclear Factor Kappa B (NF-kB) 194
181
182 Minihane
Fatty Acids, Gene Expression, and the Coordination of Glucose and

Insulin Homeostasis and Lipoprotein Metabolism 195
Summary 196
References 196
INTRODUCTION
Over 95% of fat in the diet and in the body is present as fatty acids. In addition to
meeting 30 – 40% of total body energy demands in Westernized societies, fatty
acids are an integral component of all biological membranes and serves as a
precursor for a number of essential compounds in the body such as the
hormone-like eicosanoids, which mediate inflammatory and thrombotic pro-
cesses. Furthermore, in recent years, it has become evident that fatty acids can
also act as signalling molecules by serving as ligands for transcriptional factors
which modulate gene expression.
Research in this area is in its relative infancy, and has for the most part
focussed on the expression of hepatic genes directly involved in fatty acid meta-
bolism or lipid transport as lipoproteins. However, data on the ability of fatty
acids to modulate gene expression in other tissues such as adipose tissue, endo-
thelial cells, and macrophages are beginning to emerge in the literature. Although
findings thus far have provided a valuable insight into the impact of dietary fat on
the human genome, it is likely that it only represents the “tip of the iceberg.”
Given the vast body of evidence implicating dietary fat composition in the path-
ology of many chronic diseases including coronary heart disease (CHD), such
nutrient –gene interaction information could provide us with valuable insights
into how fatty acids changes can be used as a measure to reduce the public
health burden of such diseases.
An understanding of the tissue specific metabolic effects of fatty acids
relies on knowledge of the basic structure and nomenclature of fatty acids, of
how they are absorbed and transported in the bloodstream, and upon reaching
the target tissue are either stored, incorporated into the membrane bi-layer, or
metabolized to active metabolites. Such information is detailed in the earlier
part of the chapter.
The chapter, which is by no means exhaustive, will then proceed to examine
some important fatty acid – gene interactions and their potential impact on cardio-
vascular health. For a more comprehensive examination of the area please refer to
a number of excellent recently published review articles (1 – 5).
FATTY ACID STRUCTURE AND TISSUE SOURCES

All fatty acids have a common basic structure, consisting of a hydrocarbon chain
and a terminal carboxyl group. About 21 are found in significant amounts in food
and they are characterized according to the length of the hydrocarbon chain and
Fatty Acids, Gene Expression, and CHD 183
the degree of saturation and the position of the double bonds, if present. The short
(SCFA) and medium chain (MCFA) C2 – C14 fatty acids are generally saturated
in nature, whereas the C16 – C22, long chain fatty acids (LCFA) may be saturated
or unsaturated. The most abundant monounsaturated fatty acid is oleic acid,
which is an 18 carbon fatty acid containing one double bond at carbon 9 from
the methyl end, and is therefore depicted by the notation C18:1 n-9. Polyunsatu-
rated fatty acids (PUFA), as the name suggests contain two or more double bonds,
with the two major PUFA classes the n-3 and n-6 having the first double bond at
C3 and C6, respectively. Alpha-linolenic (ALA, C18:3, n-3) and linoleic (LA,
C18:2, n-6) acid are the precursors for the n-3 and n-6 fatty acid families, respec-
tively, and are considered essential fatty acids as the mammalian body does not
contain the enzymatic machinery to insert double bonds beyond the C9 position,
therefore a dietary supply is necessary. The longer chain metabolic derivatives of
these fatty acids eicosapentaenoic acid (EPA, C20:5, n-3) and arachidonic acid
(AA, C20:4, n-6) often have opposing metabolic effects, in large part attributable
to the fact that they give rise to different families of eicosanoid end products. The
long chain n-3 PUFAs EPA, docosahexaenoic acid (DHA, C22:6, n-3) and doc-
osapentaenoic acid (DPA, C22:5, n-3) are currently almost exclusively ingested
as oily fish or fish oil capsules, although it is likely that genetic engineering will
lead to a vegetable oil source within the next 10 years. Recent publications
suggest that this series of LCFA may be the most significant fatty acid modulators
of gene expression.
Tissues EPA and DHA may be synthesized from ALA through a series of
elongation and desaturation reactions (Fig. 8.1). However, reaction efficiency is
generally low (6), with an average estimated 7 M of ALA required to produce
1 M of EPA. Furthermore, as the n-6 fatty acid metabolic pathways uses the
same desaturase enzymes, high dietary LA inhibits EPA formation (Fig. 8.1).
In the UK current dietary intakes of 10.0 g LA per day compared with 1.6 g
ALA (7) does not favor this metabolic conversion. Therefore, an increased con-
sumption of fatty fish represents a more effective means of increasing total
body content of LC n-3 PUFA. As currently fish is almost the exclusive dietary
source, nonfish eaters must rely on conversion from the precursor ALA, which
is naturally present in certain vegetable oils and green vegetables.
METABOLISM OF FATTY ACIDS

Absorption
Fatty acids are ingested mainly in the form of triglycerides (TAG), which are
hydrolyzed in the gut lumen under the action of lipases. The released fatty
acids and monoacyl glycerol are taken up into the enterocyte, where the LCFA
are re-esterified to form TAG. As fat is largely insoluble in the aqueous
medium of the blood stream the TAG, cholesterol, and phospholipids are pack-
aged into lipid – protein structures called chylomicrons, which contain apoB48
184 Minihane
n-3 PUFA pathway n-6 PUFA pathway
C18:3 (ALA) C18:2 (LA)

Delta-6 desaturase
C18:4 C18:3
El ongase
C20:4 C20:3
Delta-5 desaturase
C20:5 (EPA) C20:4 (AA)
El ongase
C22:5 (DPA, n-3) C22:4
El ongase
C24:5 C24:4
Delta-6 desaturase
C24:6 C24:5
Partial ß oxidation
C22:6 (DHA) C22:6 (DPA, n-6)
Figure 8.1 Long chain polyunsaturated fatty acid (LC-PUFA) biosynthetic pathways.
ALA, alpha-linolenic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid;
LA, linoleic acid; AA, arachidonic acid.
as its constitutive protein. Chylomicrons are secreted into the bloodstream via the
lymphatic duct. The SCFA and MCFA (C4 –C14) are secreted directly into the
bloodstream by the enterocyte and these nonesterified fatty acids (NEFA) are
transported in the circulation loosely attached to albumin.
Transport as Lipoproteins
Once in the circulation, chylomicrons are sequentially hydrolyzed by the action of
lipoprotein lipase, found attached to the luminal side of the capillary endothelium,
mainly in muscle and adipose tissue [Fig. 8.1(a)]. In muscle, the majority of fatty
acids are used as a source of energy for driving muscle action. Adipose tissue
serves as a reserve of fatty acids and release from this depot is under strict hormo-
nal control with insulin and catecholamines being centrally involved. In times of
need, for example, starvation or intensive exercise, hormone sensitive lipase
(HSL) hydrolyzes the TAG contained within the adipocytes fat droplet and
fatty acids are released into the circulation.
Following several passages through the capillary bed a lipid depleted chy-
lomicron particle results, which is removed by the liver by a receptor mediated
process. The fatty acid may be directly used by the hepatocyte or re-enter the cir-
culation as very low density lipoproteins (VLDL), the main carrier of lipids pro-
cessed or synthesized by the liver [Fig. 8.1(b)].
Postprandial lipoprotein metabolism is a highly orchestrated process in part
determined by the impact of fatty acids on the hepatic and extra-hepatic
expression of genes involved in lipoprotein clearance and VLDL secretion into
the circulation (see later). In the postprandial state, hepatic VLDL synthesis and
secretion are inhibited as the tissues utilize the dietary fatty acids supplied by
the chylomicrons. Postprandial lipoprotein metabolism is a major risk factor for
CHD and as will be discussed later the impact of dietary fat composition, in
particular PUFA intake, on CHD risk is in part mediated via alterations in this
metabolic pathway.
In addition to fatty acids of dietary origin, or derived from the elongation
and desaturation of essential fatty acids, fatty acids may also be synthesized
de novo from glucose, with adipose tissue and liver being the main lipogenic
tissues. Fatty acids are themselves important regulators of a number of key lipo-
genic enzymes as will be described later (8) [Fig. 8.2(a) and (b)].
Figure 8.2 Lipoprotein metabolism pathways: (a) exogenous pathway for the transport
of dietary derived lipids and (b; see pg. 186) endogenous pathway for the transport of
hepatic derived lipids. The chylomicrons and VLDL pass through the capillary bed
several times until chylomicrons and VLDL remnants remain. Apo, apolipoprotein;
LPL, lipoprotein lipase; HDL, high density lipoproteins; VLDL, very low density lipopro-
teins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; LCAT,
lecithin-cholesterol acyl transferase; LDL-R, low density lipoprotein receptor.
186 Minihane
Figure 8.2 Continued.
Intracellular Metabolism
Fatty acids are thought to gain access to cells by a receptor driven saturable
process. Although specific receptors and transporters have not as yet been fully
characterized, it is thought that an albumin receptor and one or more fatty acid
transporters (FAT) may be involved (9,10). The fatty acids are metabolized
into fatty acyl-CoA-thioesters (FA-CoA) and transported intracellularly bound
to fatty acid binding proteins (FABPs), where the fatty acids have a number of
potential metabolic fats including: (i) peroxisomal or mitochondrial b-oxidation;
(ii) incorporated into complex lipids such as phospholipids or TAG; (iii)
elongated and/or desaturated to form other fatty acids; (iv) metabolized into
lipid derivatives such as eicosanoids; and (v) following release from the
FABP, serve as a ligand for transcription factors that subsequently translocate
to the nucleus and impact on the expression of target genes.
Fatty acids directly released from biological membrane are also thought to
serve as ligands for these transcription factors. Furthermore, fatty acid oxidation
products such as specific eicosanoids or epoxy- or hydroxyl-fatty acids produced
during eicasonoid synthesis in microsomes are also known to interact with tran-
scription factors, often with a higher affinity than the parent PUFA (Fig. 8.3).
Figure 8.3 Cellular fatty acid metabolism including postulated mechanisms of action on
gene expression. FA, fatty acid; PL, phospholipids; TAG, triglycerides; FABP, fatty acid
binding protein; FA-CoA, fatty acyl-CoA.
FATTY ACID REGULATION OF GENE EXPRESSION

In order to maintain cellular homeostasis and in order to be adaptive to extracellu-
lar events, expression of the 30 – 40,000 genes that encode for all cellular proteins
needs to be carefully orchestrated. Over the last 30 –40 years, it has become
evident that the fatty acid composition within the cell has the ability to either
directly or indirectly regulate the expression of numerous genes. As this is reflec-
tive of whole body and dietary fat intake, this allows the cells to adapt its meta-
bolic processes in response to fat supply. The overall main effects of PUFA on
tissue gene expression is summarized in Fig. 8.4.
PUFA and Hepatic Lipogenesis

Over 35 years ago, the ability of PUFA to impact on macronutrient metabolism
was first recognized. It was observed that the ability of mouse liver to synthesize
long chain PUFA de novo from carbohydrate was greatly enhanced, and the con-
centration of a number of lipogenic enzymes [fatty acid synthetase (FAS), malic
enzyme and glucose-6-phosphate dehydrogenase (G6PDH)] increased when the
mouse diet was deficient in LA (11). Conversely, inclusion of LA in the diet
188 Minihane
Figure 8.4 Tissue specific effects of polyunsaturated fatty acids (PUFA). LPL,
lipoprotein lipase; apo, apolipoprotein; MTP, microsomal transfer protein; TNF, tumour
necrosis factor.
decreased fatty acid synthesis following a high carbohydrate diet. No such effects
were observed following palmitic acid (C16:0) or oleic acid (C18:1). In a study
conducted by Wahle and Radcliffe (12), the feeding of a sunflower enriched diet
rich in LA resulted in 40 –50% lower hepatic lipid accretion and steroyl-CoA
desaturase (SCD) 1 activity, in genetically obese (fa/fa) rats with an inherent
high lipogenic activity, in comparison to animals fed a standard or low sunflower
oil diet. A large number of subsequent studies have verified these earlier findings
with a range of dietary PUFAs shown to suppress lipogenesis by inhibiting the
concentration of enzymes involved in glucose metabolism and fatty acid biosyn-
thesis such as G6PDH, malic enzyme, S14, FAS, acyl co-carboxylase, stearoyl
CoA desaturase 1 (SCD1), L -pyruvate kinase (L -PK), glucose transporter-4 and
more recently D-5 and D-6 desaturases (1 –5,13 – 15).
Early work failed to identify the molecular target of the PUFA effect, with
modulation of gene transcription and translation and specific effects on enzyme
activity and protein integrity proposed. The cloning of the peroxisome prolifera-
tors receptors (PPARs) in 1990 (16), led quickly to the idea that this group of
nuclear receptors were the molecular target whereby PUFA co-ordinately sup-
pressed genes involved in lipid biosynthesis, whereas increased expression of
proteins involved in lipid transport (see following section). More recent studies
have identified other important transcription factors such as sterol regulatory
element (SREBP) and hepatic nuclear factor-4 (HNF-4) that modulate cellular
lipogenesis. The question of whether the PUFAs per se are the regulators of lipo-
genic gene expression remains controversial. For example, for the S14 gene, eico-
sanoid inhibitors did not affect gene expression (17) in hepatocytes suggesting
that these derivatives of PUFA were not involved. Because the effect of PUFA
on lipogenesis is largely localized to hepatic tissue where lipid peroxidation
occurs, it has been postulated that lipid peroxidation products may be responsible
for the altered lipogenic gene expression (18). However, it is likely that a range of
fatty acid derivatives can act as modulators of lipogenesis.
PUFA Induction of Lipid Oxidation

In addition to their role in lipogenesis, PUFA impact on metabolic fuel reparti-
tioning by increasing hepatic fatty acid oxidation. One of the initial targets for
the effect of PUFA is a reduction in the synthesis of hepatic malonyl coenzyme
A, which favors fatty acid entry into mitochondria and peroxisomes leading to
increased oxidation. Whether such a suppression in malonyl CoA levels is
observed in skeletal muscle following PUFA exposure remains controversial,
although this proposal will be consistent with the increased fatty acid oxidation
observed in human and animals following the feeding of PUFA rich diets (19,20).
The increased partitioning of fatty acids is accompanied by a PUFA
induced induction of proteins involved in fatty acid oxidation and ketogenesis
such as acyl-CoA oxidase (AOX) and CYP4A2 (21). Desaturation of LA and
ALA is required to alter metabolic fuel repartitioning (22) and LC n-3 PUFA
are thought to be more potent than the n-6 PUFA family. This partitioning
effect of PUFA, in particular, n-3 PUFA, has been observed in human as well
as animal models (1,19,20,23,24) and it is likely that part of the benefit of fish
oil feeding on cardiovascular risk is attributable to an effect of EPA and/or
DHA on lipid oxidation (see later). Much of the effect of PUFA on lipid oxidation
is mediated by PPARa as will be discussed.
Fatty Acids and Adipocytes Gene Expression

Advances in our understanding of lipid metabolism have identified the adipose
tissue as a key player. Storage and release of fatty acids from adipocytes
impact on both hepatic and peripheral lipid and glucose metabolism. Further-
more, although most of the early work on fatty acids as regulators of gene
expression focussed on hepatic tissue, it is now evident that the effects of
PUFA on macronutrient homeostasis is not tissue exclusive, with PUFA
having a significant effect on adipocyte metabolism, in particular, genes involved
in adipogenesis and adipose tissue lipogenesis. The molecular action of PUFA, in
190 Minihane
adipocyte maturation and lipid uptake is mediated by PPARg acting in concert

with other transcription factor families such as C/EBPs and SREBPs (25 – 27).
A number of adipocyte genes are induced by PUFA including adipocyte
FABP, fatty acid transport protein (FAT) and CD 36, lipoprotein lipase (LPL),
long chain acyl-Co synthase, phosphenoenoylpyruvate carboxykinase (PEPCK)
and a specific FABP aP2 (3,5,28,29). The upregulation of such genes is consistent
with adipocyte lipid accumulation and removal from the circulation. PPARg is
thought to be the main transcription factor that modulates the effects of fatty
acids in adipose tissue (30,31). Evidence for a role of PPARg agonists in modu-
lating insulin action is provided by the thiazolidiones series of antidiabetic drugs,
potent and selective PPARg-ligands, which effectively improve insulin sensi-
tivity and glucose homeostasis (32,33).
In addition to effecting the protein concentration of enzymes that function
within the adipocytes, PUFA is also thought to affect the levels of adipocyto-
kines. Upon secretion into the circulation, adipocyte derived signalling molecules
act in tissues such as the brain, liver, and skeletal muscles to regulate energy
homeostasis. One such molecule is leptin, a product of the ob gene, whose
specific site of action is the hypothalamus of the brain, where it binds to the
leptin receptor (34,35). This adipocytes derived hormone modulates food
intake and energy expenditure and its levels in the blood stream are controlled
by hormonal factors such as catecholamine (downregulates) and insulin (upregu-
lates) and nutritional signals such as the fat composition of the diet. In humans,
low fat diets and a reduction in the SFA:PUFA ratio of the diet attenuates plasma
leptin concentrations (36). Furthermore, in a series of rat models and cell culture
experiments, n-3 PUFA, in particular, reduces leptin concentration with an
associated reduction in ob mRNA levels (37).
The expression of other adipokines such as adiponectin and resistin, which
have been shown to be sensitive to the modulating effects of glitazones (34), are
also likely to be sensitive to the actions of lipid derived agonists although
substantial evidence is currently lacking.
Fatty Acid and Arterial Wall Gene Expression

The primary pathological features of CHD, atherosclerosis and thrombosis,
are now recognized to have a large inflammatory component in their etiology
(5,38). Recent evidence suggests that arterial inflammatory processes may be
altered by n-3 PUFA and conjugated linoleic acid (CLA, a group of structural
isomers of C18:2) (39 – 41).
In the arterial wall, the progression of atherogenesis involves the coordinated
alteration of the gene expression patterns of endothelial, blood mononuclear, and
smooth muscle cells. Various stimuli including damage to the endothelial lining
induced by either oxidative stress or hemodynamic blood flow, or locally produced
cytokines, induce the increased expression of adhesion molecules on the endo-
thelial surface, which facilitates the recruitment of monocytes into the vascular
wall. In the intima, monocytes rapidly accumulate oxidized LDL via a number of
scavenger receptors, including scavenger receptor A (SR-A), CD36, and CD38,
and develop into lipid laden macrophages called foam cells. Macrophages, in
addition to accumulating lipid, secrete a range of cytokines that further promote
monocyte recruitment, and contribute to the endothelial and smooth muscle cell
dysfunction.
As mentioned earlier, a number of studies have shown that n-3 PUFA can
attenuate the expression of inflammatory cytokines. A decrease in the expression
of vascular adhesion molecules such as vascular cell adhesion molecule 1
(vCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin has been
observed following supplementation of endothelial cells in culture with n-3
PUFA, with no effect evident following supplementation with n-6 PUFA
(42,43). Furthermore, the feeding of fish oils to human volunteers has been
shown to decrease the plasma levels of adhesion molecules such as vCAM-1
(44). Recent evidence indicates that CLA may also have the potential to modulate
adhesion molecule levels. In cell culture, a mixture of the two main isomers (cis-9,
trans-11, trans-10, cis-12) has been shown to attenuate adhesion molecule mRNA
and protein levels in endothelial and smooth muscle cells, respectively (41).
Dietary CLA has also been shown to reduce atherogenesis in rabbits fed a high-
cholesterol atherogenic diet, which has been suggested to be in part attributable
to a reduction in adhesion molecule expression (45). However, data from human
studies are distinctly lacking. In addition to effects on endothelial gene expression,
evidence is now emerging that PUFA may bring about positive changes in macro-
phages metabolism, such as a reduced expression of scavenger receptors and an
increased efflux of cholesterol from the cell.
PUFA AND THEIR CELLULAR MECHANISMS OF ACTION

The exact molecular mechanisms by which PUFA modulate the diverse but
highly coordinated effects on a number of body tissues is only partly understood
and is very much “work in progress.” It is likely that much of the effects are either
directly or indirectly modulated at the gene level, rather than a specific effect of
the fatty acid on protein integrity or activity. A comprehensive understanding of
fatty acid – gene interactions will greatly improve our understanding of how
dietary fat intake contributes to CHD pathology and maximizes our understand-
ing of how changes in dietary fat composition can contribute to reduced CHD
burden.
PUFA have the potential to alter gene expression in a number of ways.
1. Fatty acid changes to cell membranes can alter receptor activity and
cell signalling cascades. This in turn can result in alterations (e.g.,
phosphorylation) to specific transcription factors that are responsive
to specific cell surface receptor–ligand interactions and messenger
signal cascades (5).
192 Minihane
2. Native PUFA or their derivatives may impact on the gene expression

or mRNA stability of transcription factors or their specific co-factor
or inhibitory molecules.
3. PUFA, owing to their unsaturated nature have the potential to
decrease the antioxidant activity and increase the redox stress within
the cell, which may in turn influence the activity of redox sensitive
transcription factors such as nuclear factor kB (NF-kB) and its
inhibitor (I-kB).
4. PUFA or PUFA metabolites such as eicosanoids act as ligands and
bind with high affinity to specific transcription factors that sub-
sequently translocate to the nucleus and impact on the expression of
specific genes. The discovery of the PPAR family, and a subsequent
understanding of their function, activation, and nuclear effects has
led to the realization that much of the modulating effect of PUFA on
cellular metabolism is via this family of transcription factors.
TRANSCRIPTION FACTORS
Peroxisome Proliferator-Activated Receptors (PPAR)
In 1990, the identification of a ligand induced transcription factor that caused pro-
liferation of peroxisomes in the liver was discovered by Issemann and Green (16)
and named the peroxisome proliferator-activated receptor (PPAR). Although this
effect on increased peroxisome b-oxidation of fatty acids was found to be mainly
confined to rodents, subsequent research demonstrated that PPAR and their
ligands were important modulators of other genes involved in both hepatic and
extra-hepatic lipid metabolism (46 –48). PPARs have a similar structure to the
other members of the steroid/thyroid nuclear receptor superfamily, which has
over 2000 members including endocrine receptors such as the estrogen receptor
(ER). Similar to other nuclear receptors PPARs possess both a ligand and DNA
binding domain (2,5,49). Upon activation by ligands, PPARs translocate to the
nucleus and binds to a specific DNA sequence, referred to as a PPAR-response
element (PPRE) in the promoter region of target genes (PPRE), resulting in an
up- or down-regulation of gene expression. PPREs comprise imperfect direct
repeats of AGGTCA separated by a single nucleotide, with 50 extensions
portion of AACT (AACTAGGNCAAAGGTCA) (3,5), with PPAR binding to
the 50 end of the PPRE and retinoid X receptors (RXR) to the 30 end. These
PPRE sites are located on a number of genes involved in lipid metabolism and
adipocytes differentiation.
The PPAR family consists of three specific receptors, PPARa, PPARd, and
PPARg that differ in their tissue distribution and ligand specificity, but possess a
common ability to form a heterodimer with the RXR a, b, and g (50). It is the
PPAR – RXR complex that is active in altering gene expression (2,28,51).
PPARa is known to exert its effect mainly in liver and muscle cells where it
is centrally involved in glucose and fat homeostasis. PPARg modulates gene
expression in tumor and adipose tissues. In adipose tissue, PPARg activation is
associated with adipocyte maturation and lipid storage as TAG. Both PPARa
and PPARg are expressed in the arterial wall by both endothelial cells and
macrophages, and their activation in these tissues inhibits the expression of
inflammatory genes and induces the expression of genes involved in cholesterol
efflux from macrophages such as apoE and the ABCA1 genes (29). PPARb
appears to be expressed in a variety of tissues. However, its specific role is not
currently understood.
PPAR Ligands
Early studies on the effects of PPARs used synthetic agonists, such as the potent
hypolipidaemic fibrate drug group, which are PPARa ligands and the thiazoli-
diones anti-diabetic drugs that are potent PPARg agonists (28,52,53). In 1992,
Auwerx (54) demonstrated that fatty acids can serve as PPAR ligands and
since that early study it has become apparent that PPARs are activated either
directly by PUFA or by a diverse range of PUFA derivatives (5,29,49,55).
Numerous competitive assays using labeled ligands have demonstrated the
ability of PUFAs, including CLA and the cyclooxygenase and lipoxygenase
PUFA oxidation products (specific eicosanoids) to act as PPARa and PPARg
agonists (5).
The n-3 PUFA, in particular EPA and DHA, are thought to be more potent
than the n-6 PUFA as activators of PPARa (1,20), but neither family acts as a
particularly strong activator. In contrast, PUFA derivatives, including specific
eicosanoids and oxidized lipids such as 8S-hydroxyeicosatetraenoic acid bind
PPARa with 1– 2 order of magnitude higher affinity are far more potent activa-
tors of PPARa-dependent genes (56).
CLAs are also potent PPARa activators being able to displace synthetic
PPARa specific synthetic ligand at low concentrations. A 5-fold upregulation
of PPARa was evident at CLA concentrations as low as 5 mM in a rat hepatoma
cell line (57). CLA is also a recognized PPARg agonist resulting in a decreased
PPARg induced inflammatory response in CLA supplemented pigs and
RAW264.7 mouse macrophages (5).
Other Families of Transcription Factors that Mediate the PUFA/PUFA

Derivative Effect on Gene Expression
Although PPARs were originally thought as the mediator of all the PUFA effects
on gene expression, evidence quickly emerged to suggest that other transcription
factors may also be involved. The genes encoding for the delta-5 and delta-6
desaturases are downregulated by PUFA but unregulated by non-PUFA PPAR
activators (13,14). PUFA are known to suppress hepatic lipogenesis in
PPARa2/2 mice (1,23). These observations have been confirmed by other
194 Minihane
investigators, who observed that n-3 PUFA suppressed the expression of the lipo-
genic S14 and FAS genes in vivo or in hepatocytes derived from PPARa2/2
mice (4). In addition to PPARs, more recently identified and studied transcription
factors include, SREBP, hepatocyte nuclear factor 4 (HNF-4), nuclear factor-Y
(NF-Y), and nuclear factor kB (NF-kB).
Sterol Regulatory Element-Binding Protein
Many genes involved in glucose, cholesterol, and fatty acids metabolism contain
a sterol response element (SRE) and their expression is regulated in part by the
SREBP family of transcription factors. SREBP-1a, 1c, and 2 regulate genes
involved in both cholesterol and fatty acid biosynthesis, with 90% of the
total SREBP-1 found in vivo as the 1c form (58). The parent SREBP-1 molecule
is a 125 kDa protein attached to the endoplasmic reticulum in the cell. Following
proteolytic cleavage, the mature SREBP-1 translocates to the nucleus and binds
to the SRE of target genes, where it results in an increase in lipogenic gene
expression. Overexpression of SREBP-1 in liver is associated with high rates
of fatty acid biosynthesis. In rats, the feeding of diets rich in LA or EPA and
DHA were found to reduce hepatic nuclear SREBP-1 content by 60– 70%
(59), which was associated with a comparable decrease in the expression of
the hepatic FAS gene. Unlike PUFA, SFA or MUFA had no effect (1). The
pattern of the response of SREBP-1 to PUFA feeding suggests an effect at
both the transcriptional (mRNA) and post-translational (protein) level (2,5).
Hepatic Nuclear Receptor-4 (HNF-4)
Although PUFA and their metabolites are potent PPAR agonists, the expression
of PPAR-a in the human liver is relatively low (46) and it is likely that much of
the PUFA effects on hepatic metabolism are mediated through the HNF-4 orphan
receptors. In contrast to PPAR, which does not bind fatty acyl-CoA derivatives,
HNF-4 does not bind PUFA but is activated by fatty acyl-CoA (2,60). The HNF-4
binds to the DNA promoter region as a homodimer (61) and elicits changes in the
gene expression of proteins involved in fatty acid (PEPCK and L -pyruvate
kinase) and lipoprotein metabolism [apoC3, apoA1, microsomal transfer
protein (MTP)] (5,29).
Nuclear Factor-Y (NF-Y) and Nuclear Factor Kappa B (NF-kB)
Although the promoter region of the S14 gene (S14 being a primary lipogenic
protein) contains SRE and PPRE sequences, the expression of this gene is
though to be mainly via the NF-Y transcription factor (2).
The transcription factor NF-kB is an important regulator of the expression
of inflammatory mediators such as the proinflammatory cytokines. Studies in cell
lines have demonstrated strong upregulation of NF-kB translocation following
ARA and n-6 PUFA, both not EPA supplementation (62). This divergent effect
on NF-kB activity may in part explain the recognized differing effects of n-3
and n-6 PUFA on inflammatory processes (40,63).
FATTY ACIDS, GENE EXPRESSION, AND THE COORDINATION

OF GLUCOSE AND INSULIN HOMEOSTASIS AND
LIPOPROTEIN METABOLISM
As has been discussed, PUFA and, in particular, the long chain n-3 PUFA, EPA,
and DHA impact on lipid, glucose, and insulin metabolism in both hepatic and
extra-hepatic tissues.
In liver, PUFA modulates numerous genes encoding for proteins that play
central roles in lipogenesis, fatty acid oxidation, lipoprotein metabolism, and
cholesterol synthesis and secretion as bile salts. The dual action of PUFA on sup-
pressing lipogenic genes such as S14, FAS, and L -pyruvate kinase while increa-
sing the expression of genes involved in fatty acid oxidation such as AOX and
FABP, would result in a reduced hepatic fatty acid load. Such reduction in
liver fatty acid levels would lead to an improvement in insulin sensitivity and
a favorable shift in lipoprotein metabolism. Availability of fatty acid for TAG
synthesis is a major factor controlling the secretion of VLDL-TAG by the liver
and therefore circulating TAG levels. In addition to plasma TAG levels being
an independent marker of CHD risk, high TAG in the circulation results in a
pro-atherogenic shift in LDL and HDL metabolism (64). Furthermore, PUFA
impacts on lipoprotein metabolism via an effect on a number of proteins involved
in lipoprotein secretion, metabolism in the circulation, and subsequent clearance
by the liver. PUFA supplementation, and in particular n-3 PUFA, is associated
with increased expression of the LPL and apoA-1 genes and decreased expression
of the apoC3 gene. These alterations in gene expression would be expected to
result in a more effective hydrolysis and clearance of chylomicrons and VLDL
from the circulation and in higher HDL concentrations, lipoprotein changes
associated with reduced atherogenesis and improved insulin sensitivity.
In the adipose tissue, genes such as LPL, FAT, and acyl-co synthase, which
modulate lipogenesis, the leptin gene and the genes for insulin receptor substrate
2 (IRS-2), and pyruvate dehydrogenase kinase-4 (PDK-4) that are involved in
glucose metabolism are PUFA responsive via PPARg. The coordinated altera-
tions in adipocyte gene expression are thought to result in an increased fatty
acid flux into the white adipose tissue and away from muscle and liver. These
changes would be predicted to make an important contribution to improved
whole body insulin sensitivity by decreasing the exposure of this tissue to fat,
thereby increasing muscle cell glucose utilization. Although the benefits of n-3
PUFA on adipose tissue metabolism and whole-body glucose utilization have
shown promising results in rodent studies, n-3 PUFA supplementation studies
in humans have not as yet demonstrated any benefit with respect to insulin
sensitivity (65). However, relatively short supplementation periods and the use
of nonsensitive measures of insulin sensitivity may in part explain this apparent
lack of effect.
Although research is at a relatively early stage, it appears likely that n-3
PUFA also has positive effects on the arterial wall gene expression profile,
196 Minihane
resulting in a reduction in endothelial adhesion molecule expression and scaven-

ger receptor expression in macrophages. Such changes would be predicted to
decrease atherogenesis by reducing arterial monocyte recruitment and accumu-
lation of modified lipids by macrophages in the intima.
SUMMARY
It has long been recognized that the fatty composition of the diet and of body
tissue has significant effects on a range of metabolic processes such as lipid
synthesis and metabolism, glucose and insulin homeostasis and inflammation.
Over the last 30 years, with the discovery of an array of fatty acid sensitive
genes and transcription factors the molecular basis of this association is being
“unravelled.” Given the large body of epidemiological and clinical evidence
linking dietary fatty acid composition and the pathology of major chronic dis-
eases such as coronary heart disease and diabetes (which is currently reaching
epidemic levels), a fuller understanding of fatty acid– gene interaction will
help target nutritional advise to maximize the benefits of dietary change.
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9
Cell Regulatory Activity of Tocopherols
and Tocotrienols
Cristina Rota
University of Modena and Reggio Emilia, Modena, Italy
Anne M. Minihane
Peter D. Weinberg
Imperial College, London, UK
Stefan Weber
Universitätsklinikum Bonn,
Rheinische Friedrich-Wilhelms-Universität,
Bonn, Germany
John K. Lodge
University of Surrey, Guildford, Surrey, UK
Lester Packer
University of Southern California, Los Angeles, California, USA
Gerald Rimbach
Chemistry and Antioxidant Properties of VE 202

Absorption, Transport, and Metabolism 204
Molecular Targets of Alpha Tocopherol 207
Protein Kinase C 207
201
202 Rota et al.
Cell Adhesion Proteins, Chemokines, Scavenger Receptors,

and a-Tropomyosin 208
Cyclooxygenase 209
NO and Platelet Aggregation 210
VE Sensitive Genes In Vivo 211
Biological Properties of Tocotrienols 213
Anticarcinogenic Properties 213
Neuroprotection and Src Activity 213
References 214
Vitamin E (VE) is a potent lipid soluble antioxidant that prevents the propagation
of free-radical damage in biological membranes. Tocopherols and tocotrienols
are part of an interlinking set of antioxidant cycles, which has been termed the
antioxidant network. Besides its antioxidant properties, cell regulatory activities
of VE have been found. Advances in molecular and cell biology have led to the
discovery of VE sensitive genes and underlying signal transduction pathways. In
this review, antioxidant properties, absorption, and transport of VE have been
examined. Furthermore, important cell culture and animal studies related to the
antiantherogenic, anticarcinogenic and neuroprotective actions of VE, on a
molecular level, have been summarized.
CHEMISTRY AND ANTIOXIDANT PROPERTIES OF VE

VE occurs in nature in at least eight different isoforms: a-, b-, g-, and d-tocopherols
and a-, b-, g-, and d-tocotrienols. The four tocopherol homologs (a-, b-, g-, d-)
have a fully saturated 16-carbon phytol side chain, whereas tocotrienols have a
similar isoprenoid chain containing three double bonds. Individual tocopherols
are named according to the position and number of the methyl groups on the
phenol ring, with the a-, b-, g-, and d-vitamins containing three, two, two, and
one methyl groups, respectively (Fig. 9.1). These structural differences determine
biological activity, a-homologs being the most biologically active.
The majority of the functionality of VE is through its role as an antioxi-
dant, maintaining the structural integrity of virtually all cells in the body.
Its antioxidant function is mediated through the reduction of free radicals, thus
protecting the body against the deleterious effects of such highly reactive
oxygen species (ROS) and reactive nitrogen species (RNS), which have been
implicated in the pathophysiology of ageing and a number of chronic diseases
such as atherosclerosis, cancers, and rheumatoid arthritis (1 – 3). The ROS,
which include hydrogen peroxide (H2O2), the superoxide radical (O22), and
†
†
the highly reactive hydroxy radical ( OH), are by-products of normal aerobic
Cell Regulatory Activity of Tocopherols and Tocotrienols 203
R1
tocotrienol isoforms tocotrienol isoprenoid side chain
HO
R1 R2 R3
a: CH 3 CH 3 CH 3 R2 O
CH3
b: CH 3 H CH3 R3
g: H CH3 CH 3 H CH3 H CH3 H CH3
d: H H CH3 CH3
tocopherol phytyl side chain
Figure 9.1 Molecular structure of VE stereoisomers. Tocotrienols consist of a chromanol

nucleus and a liphiphilic isoprenoid chain. Tocopherols only differ in the side chain (phytyl).
The naturally occurring isoforms a, b, g, and d have methylation patterns as indicated.
metabolism formed during the respiratory and phagocytic processes and during
microsomal cytochrome P450 metabolism. The RNS include nitric oxide (NO)
and peroxynitrite, formed by the reaction of NO and O22.
†
The polyunsaturated fatty acids (PUFA) of biological membranes are par-

ticularly susceptible to free-radical attack owing to their high degree of unsatura-
†
tion. In brief, the process is initiated by a free-radical such as OH, which extracts
†
hydrogen from PUFA resulting in a PUFA radical. Following molecular
rearrangement to form a conjugated diene, the molecule is susceptible to attack
†
by molecular oxygen (O2) resulting in a peroxyl radical (PUFAOO ). Peroxyl
radicals are capable of extracting a hydrogen atom from adjacent PUFA, thus
propagating a chain reaction. Such auto-oxidation continues, severely affecting
the functionality of the tissue, unless the free radicals are scavenged. Owing to
its abundance, lipid-solubility and its efficacy with respect to radical quenching,
VE is considered to be the most important antioxidant in cell membranes (1,4,5).
The antioxidant property of VE is exerted through the phenolic hydroxyl
group, which readily donates its hydrogen to the peroxyl radical, resulting in
the formation of a stable lipid species. In donating the hydrogen atom, VE
becomes a relatively unreactive free radical as the unpaired electron becomes
delocalized into the aromatic ring. The efficiency of this protection depends on
two factors: first, the mobility of the molecule in membranes, which is deter-
mined by the aliphatic tail and second, the number of methyl species on the chro-
manol ring, with each methyl group conferring additional antioxidant capacity. In
addition, the proximity of the methyl species to the hydroxyl group is an import-
ant factor. Therefore, a-homologs, which have the greatest number of methyl
species, and in which these flank the hydroxyl group, are thought to be more
effective than the other homologs.
a-Tocotrienol has been shown to be more effective in protecting against
lipid peroxidation than a-tocopherol (6,7). A reason suggested for this is the
nature of the aliphatic tail. The isoprenoid chain of a-tocotrienol has a stronger
disordering effect on membranes than a-tocopherol. This leads to a greater
204 Rota et al.
mobility and more uniform distribution within the membrane. Nuclear magnetic
resonance studies have also shown that the chromanol ring of a-tocotrienol is
situated closer to the membrane surface. These factors contribute to a greater
ability of tocotrienols to interact with radicals and allow for quicker recycling
of the molecule to its active reduced form (6,7). Possible explanations for the
greater in vitro antioxidant activity of a-tocotrienol compared with a-tocopherol
are summarized in Fig. 9.2.
VE does not work in isolation from other antioxidants; rather it is part of an
interlinking set of redox antioxidants, which has been termed the “antioxidant
network” (8). It is hypothesized that VE acts catalytically, being efficiently
reduced from its free-radical (chromanoxyl) form, that arises after quenching
lipid radicals to return back to its reduced native state. This catalysis occurs
through the interactions between water- and lipid-soluble substances by both
nonenzymatic and enzymatic mechanisms that regenerate VE from its tocotrie-
noxyl or tocopheroxyl radical back to tocotrienol and tocopherol, respectively.
Vitamin C can regenerate VE directly and thiol antioxidants, such as glutathione
and lipoic acid, can regenerate VE indirectly via vitamin C. Under conditions
where these systems act synergistically to keep the steady-state concentration
of VE radicals low, the loss or consumption of VE is prevented.
ABSORPTION, TRANSPORT, AND METABOLISM

To date the majority of information available on VE absorption and transport is
based on tocopherol (9 –11). In the small intestine, tocopherol esters hydrolyzed
to free VE species are incorporated into mixed micelles owing to the action of
bile salts and pancreatic juices. Lack of these gastric secretions, as occurs in
Greater in vitro antioxidant activity

of α-tocotrienol compared with
α-tocopherol results from
More uniform Greater recycling

1.
distribution in activity of 4.
membrane bilayer chromanoxyl radicals
2. Stronger Recycling 5.
activity correlates
disordering of
with inhibition of
membrane lipids
lipid peroxidation
3. More effective
collision with radicals
Figure 9.2 Potential mechanisms by which a-tocotrienols exhibit greater antioxidant

activity compared to a-tocopherols.
individuals with conditions such as pancreatitis, cystic fibrosis, or choleostatic

liver disease, leads to VE malabsorption and resultant nutrient deficiency symp-
toms. The micelles enter the enterocyte via passive diffusion and the tocopherols
are packaged into chylomicrons along with the phospholipid, cholesterol, trigly-
ceride, and apolipoproteins moieties. Upon entry into the circulation via the
lymphatic system, the chylomicrons are sequentially hydrolyzed due to the
action of lipoprotein lipase attached to the capillary endothelium in the target
tissue, such as muscle and adipose tissue; a fraction of the tocopherol is released
and taken up by the endothelial cells. The resulting chylomicron remnants are
taken up by the liver by receptor mediated endocytotic processes.
In contrast to vitamins A and D, there does not appear to be a specific carrier
protein for VE in the circulation. Instead VE is incorporated into lipoprotein
particles in a nonspecific manner. In hepatic cells, tocopherol from chylomicron
remnants binds to cytosolic a-tocopherol transfer protein (a-TTP) (12,13),
which mediates its transfer to the site of VLDL synthesis (RER and Golgi appara-
tus). This 32 kDa protein is expressed almost exclusively in the liver, and recent
evidence from animal studies suggests that dietary a-tocopherol modulates
hepatic a-TTP mRNA levels (14). Unlike tocopherol absorption, which is
thought to be nonselective with respect to isomer, a-TTP displays stereoisomer
specificity, with almost exclusive incorporation of a-tocopherol into the nascent
VLDL particle. Relative affinities of tocopherol analogs for a-TTP, calculated
from competition studies, are as follows: a-tocopherol 100%, b-tocopherol 38%,
g-tocopherol 9%, and d-tocopherol 2% (13). The majority of non-a isomers are
excreted via the bile (15). a-TTP is now recognized to be the primary determinant
of plasma tocopherol levels. Mutations of the a-TTP gene lead to reduced plasma
and tissue a-tocopherol, which may ultimately lead to a severe condition known as
ataxia with VE deficiency (AVED), with associated neuronal and retinal damage
(16,17). In a recent study, a-TTP knockout mice (Ttpaþ/2 and Ttpa2/2)
were used as a model to examine the association between VE deficiency and ather-
osclerosis (18). Plasma and tissue a-tocopherols were reduced in a stepwise
manner from control through Ttpaþ/2 to Ttpa2/2, with an absence of liver
a-TTP in liver homogenates from Ttpa2/2 and a 50% reduction of protein
level in the Ttpaþ/2 animals. The VE deficiency was associated with increased
lesions in the proximal aorta and increased rates of isoprostanes, a marker of
lipid peroxidation. These findings further demonstrate the role of this transfer
protein on tocopherol metabolism and, ultimately, on CHD risk.
Approximately 50 –70% of total secreted VLDL is hydrolyzed to low
density lipoprotein (LDL) with associated transfer of tocopherols into the LDL
fraction (19). In the circulation, tocopherol exchanges rapidly between the lipo-
protein particles, although .90% is contained within the LDL and HDL fractions
(20). The 75 kDa plasma phospholipid transfer protein facilitates tocopherol
exchange between HDL and LDL (21).
The mechanisms of peripheral cellular uptake of VE are poorly understood
although simultaneous uptake of tocopherol, via receptor mediated lipoprotein
206 Rota et al.
endocytosis, or via fatty acid binding proteins, may be involved. However, recent
evidence suggests that specific membrane tocopherol binding proteins (TBPpm)
may also mediate tocopherol uptake (22).
Information on intracellular tocopherol transport is currently lacking. Owing
to its strong hydrophobicity, transfer to cellular sites requires a specific transfer
protein. However, it is still unclear how many other a-TBPbm exist and which
mechanisms regulate tocopherol transfer within peripheral cells. Recently, a
novel binding protein, tocopherol-associated protein (TAP) has been identified
(23–26). This 46 kDa protein, which displays significant sequence homology to
a-TTP, is ubiquitously expressed although the highest levels have been observed
in the liver, brain, and prostate (26). It is suggested that this protein plays a signifi-
cant general role in intracellular tocopherol metabolism. Structural analysis of TAP
suggested that it is a member of the widespread SEC14-like protein family, which
plays a role in phospholipid exchange in the cell. Recent ligand competition studies
suggest that TAP binds to a-tocopherol but not other tocopherol isomers (23).
Although research is at an early stage, it is likely that TAP will prove an important
molecule with respect to cellular tocopherol events.
Tocopherols are metabolized by cytochrome P450 (CYP) induced omega-
oxidation followed by consecutive beta-oxidation yielding carboxyethyl-
hydroxychromans (CEHC) as the final product, which have been found both in
the plasma (27) and in the urine (28). CYP4F2 appears to be the primary P450
isoform involved in the oxidation of a, and g-tocopherol (29), but the CYP3A
family has also been implicated (30,31). The recent observation shows that VE
can activate the pregnane X nuclear receptor (PXR) (32), which leads to
expression of CYPs, suggests that VE can regulate its own metabolism. A
scheme of VE absorption, transport, and metabolism is given in Fig. 9.3.
The antioxidant efficacy of tocotrienols in membranes is higher than that of
tocopherols, although its uptake and distribution after oral ingestion is less than
that of a-tocopherol. In hamsters fed with a mixture of VE isoforms containing
also tocotrienols, a-tocopherol was absorbed preferentially. However, tocotrie-
nols could still be detected in the postprandial plasma of humans and tocotrienols
were found in all classes of lipoproteins (33). Even though tocotrienols have a
higher radical scavenging activity than tocopherols, they are less bioavailable
after oral ingestion. It can be hypothesized that if similar tissue levels can be
achieved, tocotrienols would be more effective antioxidants than tocopherols.
There is some evidence supporting this hypothesis. When supplementation was
carried out in such a way that comparable tissue concentrations of a-tocopherol
and a-tocotrienol were reached in rat microsomes and mitochondria, tocotrienol-
supplemented heart tissues were more resistant to lipid peroxidation in vitro than
the tocopherol-supplemented counterparts (34). However, it has to be taken
into account that tocotrienols belong to a family of plant phenolic compounds
that have a brief and transient nature with respect to their metabolism, which
when compared to a-tocopherol is inferior with regard to tissue retention and
half life.
Figure 9.3 Schematic representation of the delivery and intracellular transport of VE in

various tissues. VE, vitamin E; CM, chylomikron; LPL, lipoprotein lipase; TTP, toco-
pherol transfer protein; TAP, tocopherol-associated protein; PXR, pregnane X receptor;
CYP, cytochrome P450 isoforms; CEHCs, carboxyethyl-hydroxychromans.
MOLECULAR TARGETS OF ALPHA TOCOPHEROL

Protein Kinase C
Since the discovery of tocopherols and tocotrienols, their antioxidant properties
have received most study. However, it is now clear, that the role of VE goes
beyond its antioxidant function. The first observation of a cell signaling role of
VE was the finding by Angelo Azzi’s group that smooth muscle cell (SMC) pro-
liferation and protein kinase C (PKC) activities are inhibited by a-tocopherol.
The inhibition of SMC proliferation was specific to a-tocopherol: trolox, phytol,
b-tocopherol, and a-tocopherol esters had no effect (35,36). As a-tocopherol
and b-tocopherol have very similar free-radical-scavenging activities, the mechan-
ism by which a-tocopherol acts on PKC is not related to its antioxidant properties.
Subsequent studies have shown that PKC is inhibited in a number of other cell
types, including monocytes (37), neutrophils (38), fibroblasts (39), and mesangial
cells (40). Most importantly, this inhibition of PKC by a-tocopherol occurs at con-
centrations close to those measured in human plasma (41). Antiproliferative effects
of VE were not seen for HeLA cells suggesting that there are different cell-specific
208 Rota et al.
pathways of cellular proliferation in which VE can act (42). In addition, the inhi-
bition of PKC was not related to a direct interaction of a-tocopherol with
the enzyme nor with a diminution of its expression. Instead, PKC inhibition by
a-tocopherol is linked to the activation of a protein phosphatase 2A, which
in turn dephosphorylates PKC-a and thereby inhibits its activity (43,44). An
inhibitory effect of a-tocopherol on PKC may be seen only at the cellular level
and is not evident with recombinant PKC.
Important milestones in experimental VE research are summarized in
Fig. 9.4.
Cell Adhesion Proteins, Chemokines, Scavenger Receptors,

and a-Tropomyosin
Activation of endothelial cells results in release of vascular cytokines such as
IL-1 and TNF-a. These cytokines in turn induce the expression of cell surface
adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and
intracellular adhesion molecule-1 (ICAM-1), which are centrally involved in
the endothelial recruitment of neutrophils (45). Focal expression of ICAM-1
and VACM-1 has been reported in arterial endothelium overlying early foam
cell lesions in both dietary and genetic models of atherosclerosis in rabbits
(46). This expression, together with the activation of monocyte chemoattractant
protein-1 (MCP-1) leads to infiltration of mononuclear cells into the wall and, it is
widely supposed, to a consequent increase in the oxidation and scavenging of
Figure 9.4 Milestones in experimental VE research.

LDL, formation of lipid-laden foam cells, and development or progression of

atherosclerotic plaques (47).
Transcription of ICAM-1, VCAM-1, and MCP-1 is dependent, at least in
part, on the activation of NF-kB. Cell culture studies have shown that treatment
of endothelial cells with oxidized LDL significantly increases expression of
mRNA and proteins levels of ICAM-1 and VCAM-1 (48). However, pre-
treatment with a-tocopherol reduced cell adhesion protein expression in a
dose-dependent manner. Consistent with this finding, adherence of polymorpho-
nuclear leukocytes (PMN) or mononuclear leukocytes (MNC) to endothelial cells
activated by oxidized LDL (which is much higher than adherence to unstimulated
endothelial cells) was reduced by supplementation of the endothelial cells with
a-tocopherol. Furthermore, IL-1 beta-induced production of MCP-1 was dose-
dependently suppressed by enrichment of human endothelial cells with VE
(49). From this and other studies, it is suggested that the putative antiatherogenic
effect of a-tocopherol may in part be due to a down-regulation of cell adhesion
proteins and chemokines. Despite evidence that VE down-regulates cell adhesion
proteins in vitro, in vivo evidence is currently lacking.
Ricciarelli et al. (50) have recently demonstrated that the CD36 scavenger
receptor, which transports oxidized LDL into the cytosol, is expressed in human
SMC. Interestingly, a-tocopherol inhibited the uptake of oxidized LDL by a
mechanism involving down-regulation of CD36 mRNA and protein expression.
It is hypothesized that beneficial cardiovascular effects of a-tocopherol are at
least in part mediated by lowering the uptake of oxidized LDL, which sub-
sequently results in a reduction of foam cell formation.
An involvement of tropomyosin in the progression of restenosis has been
suggested (51). Early after balloon injury, SMC of the media and those that
have migrated into the intima contain decreased amounts of tropomyosin, and
late after balloon injury tropomyosin returns toward normal values. In 1999,
Aratri et al. (52) discovered the induction of a-tropomyosin expression in rat
vascular SMC by a-tocopherol, using differential display techniques (52). No
significant changes in mRNA levels were observed when b-tocopherol was
used. The authors suggest that the overexpression of tropomyosin induced
by a-tocopherol may decrease the contractility of SMC and hence form the
molecular basis for the hypotensive effect of VE.
Cyclooxygenase
Cyclooxygenase has two isoforms, COX-1 and COX-2. COX-1 is constitutively
expressed in most cells, whereas COX-2 is regulated by growth factors, tumor
promoters, cytokines, glucocorticoids, and lipopolysaccharide (LPS). Cyclooxy-
genases convert arachidonic acid (AA) into prostaglandin E2 (PGE2), the precursor
of thromboxane and eicosanoid synthesis. High levels of COX-2 in epithelial cells
are associated with the inhibition of apoptosis, and overexpression of COX-2 has
been implicated in the pathogenesis of neoplastic diseases. An upregulation
210 Rota et al.
of COX-2 transcription has been shown in most human colorectal cancers (53).
Interestingly, changes in AA metabolism stimulate cell proliferation via activa-
tion of PKC, indicating that PKC might be one of the primary signaling path-
ways through which certain tumors are initiated or maintained. In recent years,
a role of COX-2 in atherogenesis has been identified. Immunocytochemical
studies using anti-COX-2 showed that COX-2 is localized to macrophages in
atherosclerotic lesions of patients with coronary artery disease (54).
In monocytes derived from aged mice, it has been shown that a VE-induced
decrease in PGE2 production is mediated via a decreased COX activity (55).
However, VE has no effect on COX mRNA and protein levels, indicating a
posttranslational regulation of the COX enzyme. Non-a-tocopherol homologs
were, like b-tocopherol, effective in inhibiting COX activity, but the degree of
inhibition varied in proportion to their antioxidant capacity, suggesting that an
antioxidant mechanism may be involved.
It has been shown in LPS stimulated RAW264.7 macrophages and IL-1beta
treated A549 human epithelial cells that gamma tocopherols inhibited the pro-
duction of PGE2 owing to a direct inhibition of COX-2 (56). Furthermore, the
major metabolite of dietary gamma tocopherols also exhibited an inhibitory
effect in these cells. In contrast, a-tocopherol at 50 mM slightly reduced (25%)
PGE2 formation in macrophages, but had no effect in epithelial cells. Similar
to the previously mentioned study, the inhibitory effect of g-tocopherol and
g-CEHC stemmed from their inhibition of COX-2 activity, rather than from
affecting protein expression or substrate availability and appeared to be indepen-
dent of their antioxidant activity.
NO and Platelet Aggregation

NO produced by the endothelial NO synthase (eNOS) is a pivotal molecule in the
regulation of vascular tone. In addition, its production suppresses expression of
proinflammatory cytokines, adhesion molecules (57), and MCP-1 (58). It also inhi-
bits platelet adhesion to the endothelium (59), can modify the permeability of the
arterial wall (60), suppresses vascular SMC proliferation and migration (61), and
can act as an antioxidant (62). The major risk factors for atherosclerosis—age
(63), hypercholesterolaemia (64), diabetes (65), hypertension (66), smoking (67),
and low birth weight (68)—are all associated with impaired NO activity, often
before appreciable disease develops. In rabbits, inhibition of NO production accel-
erates experimental atherosclerosis (69), whereas increase in NO synthesis reduces
it (70). Importantly, NO significantly inhibits NF-kB (71). This may account for
its influence on the transcription of genes for adhesion proteins, MCP-1 and
others. The postulation of key roles for both NO and NF-kB is therefore not
self-contradictory.
There is evidence from studies in rabbits that VE reverses the well
established deleterious effects of hypercholesterolaemia on NO activity.
Stewart-Lee et al. (72) found that relaxation in response to acetylcholine, an
NO-dependent phenomenon, in the carotid artery was reduced after 4 weeks of

diet-induced hypercholesterolaemia but was restored by the addition of 0.2%
a-tocopherol acetate to the diet. Andersson et al. (73) obtained a similar result for
the coronary circulation. The mechanisms underlying this effect are a matter of
controversy. It has been suggested that inactivation of NO by ROS is increased
during hypercholesterolaemia and reduced by VE (72,73). However, Böger et al.
(74) found that VE did not reduce ROS release by aortic tissue from cholesterol-
fed rabbits; instead, they suggested its protective effect on the NO pathway was
related to its inhibition of LDL oxidation. Since PKC inhibits NO (75), another
possible mechanism arises from the observation that hypercholesterolemia
increases PKC levels in rabbit aortic smooth muscle and this is reduced by VE (76).
Whatever the mechanism is, protective effects of VE on NO function might
be expected to reduce atherosclerosis in hypercholesterolemic rabbits. Although
many studies have found such an effect (74,77,78), others have shown an increase
in the extent and severity of lesions (79). Keaney et al. (80) obtained an interesting
result which may in part account for these discrepancies: although 1000 IU
a-tocopherol/kg diet protected against the inhibitory effect of hypercholesterole-
mia on the NO pathway, 10,000 IU a-tocopherol/kg diet markedly increased it,
and also increased the severity of lesions, despite the fact that the oxidizability
of LDL was still reduced. Possible mechanisms include pro-oxidant effects of
a-tocopherol, or reactions of a-tocopherol with NO to give the tocopheroxyl
radical.
Li et al. (81) studied the effect of different isoforms of VE on NO activity
and platelet aggregation in human platelet rich plasma. All three isoforms tested
(a-, b-, or d-tocopherol) markedly decreased ADP-induced platelet aggregation
and increased NO release in a dose-dependent manner. The isoforms did not
affect cNOS protein expression, but increased cNOS phosphorylation. Further-
more, it has been demonstrated in human subjects that oral supplementation
with a-tocopherol (400 – 1200 IU/day) resulted in an increase in platelet toco-
pherol concentration that correlated with marked inhibition of PMA-mediated
platelet aggregation (82). Platelets derived from these subjects also demonstrated
apparent complete inhibition of PKC. These findings represent another potential
mechanism by which tocopherol could prevent the development of coronary
artery disease. Several mechanisms by which VE may prevent coronary artery
disease are summarized in Fig. 9.5.
VE Sensitive Genes In Vivo

Barella et al. (83) monitored differential gene expression in rat liver over a period
of 9 months. Similar to findings in cultured cells, VE supplementation down-regu-
lated scavenger receptor CD36. Furthermore, coagulation factor IX and 5-alpha-
steroid reductase type 1 mRNA levels where down-regulated while hepatic
gamma glutamyl–cysteinyl synthetase was significantly up-regulated in response
to dietary VE. Measurement of the corresponding biological endpoints such as
212 Rota et al.
Figure 9.5 Molecular mechanisms for the anti-atherogenic activity of VE.
activated partial thromboplastin time, plasma dihydrotestosterone, and hepatic glu-

tathione substantiated the gene chip data, which indicated that dietary VE plays an
important role in a range of metabolic processes within the liver.
Gene-chip technology was also employed to study the effect of dietary VE
on gene expression in rat testes (84). Differential gene expression was monitored
at five individual time-points over a period of 14 months with all animals
individually profiled. Low VE intake resulted in the consistent up-regulation of
7-dehydrocholesterol reductase and GATA binding protein 4, both involved in
testosterone synthesis. Cyclin D3, important in cell cycle progression and
Wilms tumor 1, related to cancer development, was also up-regulated in the
VE deficient animals.
Furthermore, in the rat hippocampus, VE exhibited a significant impact on
the expression of genes associated with hormones and hormone metabolism (e.g.,
growth hormone, thyroid hormones, prolactin, and melatonin), nerve growth
factor, apoptosis, dopaminergic neurotransmission, and Abeta and AGEs clear-
ance. In particular, VE strongly affected the expression of an array of genes
encoding for proteins directly or indirectly involved in the clearance of
amyloid beta, changes that are consistent with a protective effect of VE on AD
progression, independent of its role as an antioxidant.
Gohil et al. (85) explored genome-wide changes in mRNAs from brain
cortex and liver of TTP deficient, a genetic model of VE deficiency. Selective
inductions of genes regulated by antioxidant response elements were detected
in liver of TTP2mice, suggesting increased oxidant stress. Cortex of TTP2
mice revealed repression of genes encoding synaptic proteins, PKC family
members, and myelin proteins. A significant decrease in the expression of reti-
noic acid receptor-related orphan receptor-alpha mRNA indicated staggerer-
like phenotype including ataxia and deficits of motor coordination of TTP-mice.
Overall, advances in microarray technology have enabled us to investigate
genes differentially expressed in response to tocopherols in cultured cell and
in vivo thereby offering the possibility of more insight into the biological
properties of VE. DNA arrays are important experimental platforms and might
help to address tocoperhols and tocotrienols more specifically with regard to
their molecular biological functions.
BIOLOGICAL PROPERTIES OF TOCOTRIENOLS

Anticarcinogenic Properties
Tocotrienols belong to a phytochemical class of isoprenoid molecules. These
compounds share a common precursor, mevalonic acid. Tocotrienols are
mixed isoprenoids, meaning that only a part, the lipophilic chain is derived via
the isoprenoid pathway. Isoprenoids have been shown to exhibit anticarcinogenic
properties. When different VE isoforms were analyzed, it could be demonstrated
that a-tocopherol and a-tocotrienol inhibited tumor promotion in Raji-cells most
effectively (86). Tocotrienols from TRF inhibited the proliferation of human
breast cancer cell lines (87,88). The inhibition was found to be independent of
the estrogen receptor status of the cell lines (89). Isoprenoids, among them
tocotrienols also suppressed the growth of murine B16 melanomas in vitro and
in vivo (90). Interestingly, correlations between the late stage tumor-suppressive
potency of diverse isoprenoids and their effect of HMG CoA reductase activity
approached unity. It is hypothesized that VE might exert antiproliferative
properties by interfering with signal transduction events involving PKC. It has
been shown that a-tocopherol inhibits the proliferation of SMC by the inhibition
of PKC (91). This effect was specific for a-tocopherol as opposed to the isoform
b-tocopherol (92). There is no information, however, on the potency of
a-tocotrienol on PKC-activity, which shares the structure of the chromanol
nucleus with a-tocopherol. Recently, it has been reported that isoprenoids,
including tocotrienols, induce cell-cycle arrest in G-1 phase and apoptosis in
human and murine tumor cells (93). As these effects can be observed with differ-
ent isoprenoid, which are not antioxidants, it is possible that the anticarcinogenic
effects of tocotrienols are not necessarily related to their antioxidant properties.
Neuroprotection and Src Activity

Elevated levels of glutamate have been implicated in a wide range of neurologi-
cal diseases, including epilepsy, cerebral ischemia, Huntington’s disease, and
Parkinson’s disease. Receptor-mediated glutamate exicitotoxicity is believed to
be a major mechanism of damage in these pathologies and induction of oxidative
stress by glutamate has been demonstrated to be the primary cytotoxic mechan-
isms in cell lines such as C6 glial cells (94), PC-12 neuronal cells (95), immature
cortical neuron cells (96), and oligodendroglia cells (97). It has been demon-
strated that high glutamate levels block cystine uptake via amino acid transporter
Xc2 resulting in a significant depletion of cellular GSH. A glutathione-depleted
state impairs cellular antioxidant defenses, followed by an increased vulnerability
214 Rota et al.
of the cell to ROS. The mitochondrial electron transport chain has been shown to
be a source of ROS production during glutamate-induced apoptosis (98).
Recently, VE isoforms were tested in a model of neuronal cell death, where
HT4 neuronal cells were challenged with glutamate (99). Tocotrienols counter-
acted glutamate-induced cell death at much lower concentrations than tocopher-
ols. Moreover, tocotrienols effectively inhibited the activation of pp60 c-Src
kinase, a kinase that is centrally involved in glutamate-induced cell death. It is
hypothesized that these protective effects of tocotrienols are probably indepen-
dent of their antioxidant activity, because tocopherols were effective only at
multi-fold higher concentration (99). The activity of Src kinase has also been
shown in the progression of breast cancer (100). Moreover, elevated levels of
Src kinase have been found in human skin tumors (101). Owing to the key
involvement of Src kinase in neurodegenerative diseases and oncogenesis, inhi-
bition of these kinases would seem to be a likely basis for developing a strategy to
create neuroprotective and anticancer drugs. It has been recently shown that
tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced
neurodegeneration (102). Interestingly, BL15, an inhibitor of 12-LOX, prevented
glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOX-
deficient mice were observed to be resistant to glutamate-induced death.
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10
Molecular Analysis of the Vitamin A
Biosynthetic Pathway
Johannes von Lintig

University of Freiburg, Freiburg, Germany
Carotenoids and Apocarotenoids: Colors with Functions 222

Vitamin A Functions in Animal Physiology 223
Molecular Identification of b,b-Carotene-15,150 -mono-oxygenases 226
In Vertebrates Three Different VP14-Homologs Exist 227
Molecular Analyses of the Vitamin A Biosynthetic Pathway in Model
Organism 229
What Can We Learn from Insects? 229
Molecular Analysis of the Vitamin A Biosynthetic Pathway in Vertebrates 231
Conclusions 235
References 236
Elucidating the physiological roles of lipids has long been a major concern in
both biology and medicine. Today, we know that lipids are not only a source
of energy, but are also essential for the growth and development of the organism.
Fat-soluble vitamins and other lipids serve as precursors for ligands that bind to
receptors in the nucleus. These receptors, in response to ligand binding, regulate a
complex network of gene activities (1). To become biologically active, dietary
221
222 von Lintig
lipids must first be absorbed by the intestine and transformed by metabolic

enzymes for delivery to their sites of action in the body. The co-ordination of
all these processes involves a large number of binding proteins, transporters,
and metabolizing enzymes, which contribute to lipid homeostasis.
Carotenoids are essential lipids in the human diet. They are not only the
natural precursors (provitamin A) for retinoids (vitamin A and its derivatives),
but also exert biological function per se. Despite their importance, the molecular
components involved in carotenoid metabolism have remained elusive for a long
time. This chapter will focus on recent advances in this field of research. The use
of model organisms such as the fruit fly Drosophila melanogaster provided first
insights into basic principles of this metabolism. The increasing amount of DNA
sequence information from different genomes then led to the molecular identifi-
cation and cloning of homologous genes in vertebrates including human. With
this tool in hand, old questions in carotenoid research could finally begin to be
addressed definitively on the molecular level, contributing to a mechanistic
understanding, for example, the regulation of vitamin A homeostasis or tissue
specificity of vitamin A formation, with impact on animal physiology and
human health.
CAROTENOIDS AND APOCAROTENOIDS: COLORS

WITH FUNCTIONS
We all are familiar with carotenoids because of the yellow to red colors of fruits,
flowers, and vegetables. These compounds are C40 isoprenoids synthesized in
plants, certain fungi, and bacteria. Besides pure carbohydrates, this class of
compounds comprises hydroxylated and epoxydated derivatives, the so-called
xanthophylls (2). The characteristic chemical and physical properties of caroten-
oids are responsible for light absorption as well as for the inactivation of free
radicals, properties which play a crucial role for their diverse biological func-
tions. Carotenoids are essential components of the photosynthetic apparatus by
being constituents of the reaction centers as well as of the light-harvesting com-
plexes (3). In addition, these pigments contribute to the color of several plant
tissues which accumulate carotenoids to a high extent. Well-known examples
are the fruits of pepper and tomato, the flowers of daffodils and dandelions, or
the roots of carrots. Among the various classes of pigments found in nature, caro-
tenoids are the most wide spread, with important functions not just restricted to
carotenoid-producing organisms. Some animals use dietary carotenoids for color-
ation. Well-known examples are the feathers of flamingos and the red color of
salmon. Because of their antioxidative properties, beneficial effects of caroten-
oids have been reported in the prevention of coronary heart disease, certain
kinds of cancer, and age-related macular degeneration in humans (4).
Besides the physiological functions exerted by carotenoids per se, they are
the precursors for apocarotenoid synthesis. These wide-spread carotenoid-
derivatives also exert key biological functions. In plants, they play roles as
Molecular Analysis of Vitamin A Biosynthetic Pathway 223
hormones, pigments, flavors, aromas, and defense compounds. Probably, the best
known example is the plant hormone abscisic acid (ABA)—a C15 compound
derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and
9-cis-neoxanthin (5). Plant apocarotenoids are also of some economic value,
for example, bixin (annatto) is commonly used as a natural food colorant and
crocin is the main pigment in saffron. In animals, retinoids (vitamin A and its
derivatives) are C20 apocarotenoids, which can exert vital functions in a pano-
ply of different physiological processes as diverse as vision, pattern formation
during embryonic development and metabolic control.
The diverse assortment of apocarotenoids found in nature arises from the
large number of carotenoids (.600), variations in the cleavage site, and modifi-
cations of the primary cleavage products. The pathways for apocarotenoid
formation have two characteristics in common: the starting compound is a C40
carotenoid and the first product is an aldehyde (Fig. 10.1). These commonalties
suggest that the corresponding enzymes catalyze the oxidative cleavage in a
common reaction mechanism. By analyzing the molecular basis of the ABA-
deficient phenotype of the maize vp14 mutant, Schwartz et al. (6) cloned for
the first time a gene encoding a carotenoid-cleaving enzyme. The heterologously
expressed and purified recombinant enzyme catalyzes the oxidative cleavage of
9-cis-epoxycarotenoids such as neoxanthin and violaxanthin to form xanthoxin,
the direct precursor of ABA. Maize vp14 belongs to an emerging gene family
of putative carotenoid-cleavage enzymes found in animals, plants, and bacteria
(7). The genomes of the plant Arabidopsis thaliana, the fruit fly D. melanogaster,
and humans were found to encode nine, one, and three homologs, respectively.
This led to the hypothesis that some of these homologues also catalyze the
oxidative cleavage of carotenoids. Indeed, we showed that the maize vp14
homologs of animals encode carotenoid-cleavage enzymes essentially involved
in vitamin A metabolism (8 –10).
VITAMIN A FUNCTIONS IN ANIMAL PHYSIOLOGY

In humans, vitamin A deficiency (VAD) leads to night blindness in milder forms,
whereas more severe progression results in corneal malformations, for example,
xerophthalmia. Besides visual defects, this deficiency affects the immune system,
leads to infertility, or causes malformations during embryogenesis. The molecu-
lar basis for these diverse effects is found in the dual role exerted by vitamin A
derivatives in animal physiology. In visual systems, retinal or closely related
compounds such as 3-hydroxy-retinal serve as the chromophores of the visual
pigments (rhodopsin) (11,12). In vertebrates, the vitamin A derivative retinoic
acid (RA) is also a major signal controlling a wide range of biological processes.
RA is the ligand of two classes of nuclear receptors, the retinoic acid receptors
(RARs) and the retinoid X receptors (RXRs) (13 –16). The active receptor
complex, involved in processes as diverse as pattern formation during embryonic
development, cell differentiation, and control of metabolic activity, is a
224 von Lintig
Figure 10.1 Proposed pathways for apocarotenoid biosynthesis (7 and references

cited herein). (A) ABA biosynthesis, (B) zeaxanthin oxidation in saffron styles, and
(C) vitamin A biosynthesis. VP14, 9-cis-violaxanthin-oxygenase; BCO, b,b-carotene-
15,150 -oxygenase; ZCD, zeaxanthin cleavage oxygenase.
RAR/RXR heterodimer that binds DNA regulatory sequences and regulates

gene transcription in response to RA-binding. RXR is not only the heterodimer
partner of the RAR receptor, but also an obligate partner for other nuclear
receptors (orphan receptors), controlling a wide range of activities in lipid
metabolism (1).
VAD is a major problem leading to blindness and childhood mortality,
particularly in developing countries (17). The demand for this vitamin can be
satisfied by the natural content of either animal or plant food sources. This
phenomenon was first explained by Moore in 1930 (18). He described a conver-
sion of b-carotene to vitamin A in the small intestine, providing the evidence that
a plant-derived carotenoid is the direct precursor for vitamin A in animals. Today,
we know that all naturally occurring vitamin A derives from carotenoids which
exert provitamin A activity and that the world’s population mainly relies on
plant carotenoids from staple foods to satisfy their vitamin A requirement.
For vitamin A formation, a central cleavage mechanism at the C-15,C-150
double bond for the conversion of b-carotene to vitamin A was initially proposed
by Karrer et al. (19). In 1954, Glover proposed an eccentric cleavage reaction and
a stepwise process, leading ultimately to only one mole vitamin A per mole
carotene consumed (20). Evidence for this eccentric cleavage was provided by
the observation that radioactive b-apocarotenals were converted in mammals
to vitamin A esters with the release of “small” radioactive fragments (21).
Goodman and Huang (22) and Olson and Hyaishi (23) first described an enzy-
matic activity in cell-free homogenates from rat’s small intestine which catalyzed
provitamin A conversion. These analyses showed that b-carotene is enzymati-
cally cleaved at the central C-15,C-150 double bond to yield two molecules of
vitamin A aldehyde (retinal). The enzymatic activity depended on molecular
oxygen and thus the enzyme was termed b,b-carotene-15,150 -oxygenase
(BCO). It was reported to be soluble, to have a slightly alkaline pH-optimum,
and to be inhibited by ferrous iron chelators and by sulfhydryl-binding com-
pounds, indicating that it contains a ferrous iron co-factor (24). Subsequently,
this enzyme was also characterized in different mammalian species (25,26)
and substrate specificity was determined for different b-carotene stereoisomers
(27). Recent analysis dealing with the mode of action of BCO provided strong
evidence that oxidative cleavage at the central (15,150 ) double bond is catalyzed
in a mono-oxygenase mechanism via a transient carotene epoxide (28). After the
description of this enzyme, it was generally assumed that the centric cleavage
reaction constitutes the major step in vitamin A synthesis. However, the eccentric
cleavage of b-carotene was subsequently also demonstrated in cell-free homo-
genates of mammals (29,30). Furthermore, it was shown that the resulting
long-chain apocarotenoids (.C20) are shortened to RA in a stepwise process
which is most probably mechanistically related to the b-oxidation of fatty
acids (31,32). Although several endeavors were undertaken, and highly enriched
enzyme fractions could be obtained, all attempts to purify these enzymes to
homogeneity failed over 40 years of research.
226 von Lintig
MOLECULAR IDENTIFICATION OF b,b-CAROTENE-

15,150 -MONO-OXYGENASES
In plants, the first carotenoid cleavage enzyme, VP14, was molecularly identified
and characterized by the analysis of an ABA-deficient maize mutant (6). Thus,
we pursued the hypothesis that a VP14-related enzyme catalyzes carotenoid-
cleavage in animals as well. For the cloning of an animal BCO, we employed
an expression cloning strategy using Escherichia coli cells genetically engin-
eered to synthesize b-carotene de novo. We searched the entire expression data-
base and found several expressed sequence tags in animals with some weak
sequence similarity to plant VP14. After cloning the full-length cDNAs, we
expressed the corresponding proteins in the E. coli test system. Upon expression
of a VP14 homolog from D. melanogaster, the recipient bacterial strain bleached,
thus indicating that retinoids were formed at the expense of orange b-carotene
and this was confirmed by HPLC-analyses. The enzymatic properties of the pur-
ified recombinant Drosophila enzyme revealed that it exclusively catalyzed the
centric cleavage of b-carotene (C40) to yield retinal (C20). Thus, we molecularly
identified and functionally characterized for the first time a BCO (8).
Confirming that this type of enzyme generally catalyzes in metazoans, the
first step in vitamin A metabolism was provided by Wyss et al. (33,34). In an
independent approach, they succeeded in the molecular cloning and functional
characterization of a BCO from chicken. Their approach relied on partial
protein purification and determination of peptide sequences, then using this infor-
mation to synthesize oligonucleotide primers to generate a partial cDNA and
screen a cDNA library derived from small intestine. Amino acid sequence com-
parison between the Drosophila and chicken BCOs showed an overall similarity
with several highly conserved regions and a significant similarity to some
domains of the plant carotenoid oxygenase VP14 (35).
By sequence similarity to the so far identified genes from Drosophila and
chicken, their counterparts from mouse and human were identified and func-
tionally characterized in several laboratories (36 – 39). Using the carotenoid-
accumulating E. coli test system or by in vitro assays for enzymatic activity
with the purified recombinant protein, it was shown that these mammalian homo-
logs catalyze exclusively the centric oxidative cleavage of b-carotene to yield
retinal. Expression of the murine BCO in various carotenoid-accumulating
E. coli revealed the cleavage of not only carotenoid substrates such as b- and
a-carotene but also lycopene, resulting in the last case in the formation of
acyclic retinoids (36). The purified recombinant BCO, however, catalyzed only
the cleavage of carotenoid substrates with at least one unsubstituted b-ionone
ring, such as b-carotene and b-cryptoxanthin, and there was no significant
cleavage of lycopene or zeaxanthin (39). The Km values for b-carotene were esti-
mated to be in the range of 1–10 mM for BCOs from the different species
(8,36,37,39). BCO exhibits a slightly alkaline pH-optimum, and enzymatic activity
is sensitive to chelating agents such as o-phenanthroline and a,a0 -bipyridyl,
indicating that it depends on ferrous iron (8,39). Thus, the purified recombinant
BCOs share biochemical properties which have been already described for the
native BCOs.
Purification of the recombinant BCO fusion proteins by affinity chromato-
graphy was achieved without the addition of detergents. This characteristic and
the predicted amino acid sequences of the various BCOs indicate that we are
dealing with hydrophilic, nonmembrane-bound proteins. Indeed, a cytosolic
localization of the native BCO was recently demonstrated for its human represen-
tative (39). Therefore, in vitro tests for enzymatic activity must be conducted in
the presence of detergents to mimic the interaction between the enzyme and its
insoluble substrate. However, in vivo, the cytosolic localization of BCO may
require specific binding proteins to deliver the carotenoid substrate as well as
to pick up the retinoid product, since both are highly lipophilic compounds.
On the product side, three different types of cellular retinoid-binding proteins
(CRBP I– III) have been characterized (40 and references therein). However,
no direct protein –protein interaction between a recombinant murine BCO –
GST fusion protein and CRBPs could be detected in pull-down experiments
(37). Even though these results argue against a tight protein –protein interaction
of CRBP with BCO, it seems likely that CRBPs may facilitate b-carotene clea-
vage by binding retinal. In mouse testis homogenates, a L -lactate dehydrogenase
C was identified and shown to interact specifically with BCO. So far, the exact
physiological role of this type of alcohol dehydrogenase is not known, and
there is no experimental evidence that this enzyme catalyzes either the oxidation
or reduction of aldehydes like retinal. Hence, it remains to be elucidated whether
BCO may interact tissue-specifically with a certain subset of proteins involved in
retinoid metabolism. Such proteins might control the metabolic flow of the
primary cleavage product retinal, either to retinol formation for vitamin A trans-
port and storage, or in the direction of RA formation for retinoid-signaling.
To sum up, this recent research led to the molecular identification of BCOs
from various metazoan species. The recombinant enzymes share common bio-
chemical properties with the native BCO from tissue homogenates. On the
basis of its structural and biochemical properties, BCO from animals belongs
to an ancient family of nonheme iron oxygenases, heretofore described only in
plants and micro-organisms.
IN VERTEBRATES THREE DIFFERENT VP14-HOMOLOGS EXIST

The dual functions of vitamin A in vision (retinal) as well as in development and
cell differentiation (RA) indicate that vertebrate retinoid metabolism is quite
complex. The need for a close co-ordination of the entire vitamin A metabolism
is reflected in the large number of different retinoid-modifying enzymes, such as
various retinoid-oxidizing enzymes, as well as intracellular and extracellular
retinoid-binding proteins.
228 von Lintig
In Drosophila, with vitamin A functions being restricted to vision, only one

family member of nonheme iron carotenoid cleavage enzymes is found in the
entire genome (10). In vertebrates, however, besides BCO, two additional
family members, RPE65 and b,b-carotene-90 ,100 -oxygenase (BCOII), exist
(9,41). In humans, bco, rpe65, and bcoII map to the genomic positions 16q21,
1q31, and 11q23, respectively. The existence of these bco homologs indicated
that they are related to specific carotenoid/retinoid functions of vertebrates.
RPE65 was first described as an abundant protein of the retinal pigment
epithelium (RPE) with a molecular mass of 65 kDa (41). This epithelium is cru-
cially involved in vitamin A metabolism of the eyes, namely in a process called
the visual cycle. In this multistep pathway, all-trans retinol is converted to 11-cis
retinal, which is the photosensitive vitamin A analog of the visual pigments.
Mutations in the RPE65 gene are responsible for a severe form of autosomal
recessive childhood-onset retinal dystrophy, Leber’s congenital amaurosis
(42 – 44). A direct involvement of RPE65 in the visual cycle was indicated by
the analysis of Rpe65-deficient mice which accumulate all-trans-retinyl esters
in the RPE, as intermediates of the visual cycle. All-trans-retinyl esters are sub-
strates for the isomerohydrolase reaction (45). This molecularly uncharacterized
enzyme(s) catalyzes 11-cis retinol formation from all-trans retinyl esters in a
combined hydrolase/isomerase reaction (46). Even though RPE65 seems to be
crucial for this reaction, the recombinant RPE65 lacks isomerohydrolase activity
in vitro. Recent biochemical studies suggest that RPE65 binds stereospecifically
all-trans retinyl ester and stimulates the intrinsic isomerohydrolase activity of
RPE membranes (47,48). Thus, it was proposed that RPE65 is an all-trans-
retinyl ester binding protein. Since RPE65 shares overall sequence similarities
to BCO and other members of this nonheme iron oxygenase family, it remains
to be elucidated whether this representative is just a binding protein or an enzy-
matically active component of the carotenoid/retinoid metabolism in the eyes.
Besides RPE65, an additional in silico predicted putative family member,
BCOII, was found in the database (9). Upon cloning the full-length cDNA of a
murine BcoII, sequence analyses revealed that it encodes a protein of 532
amino acids. The deduced amino acid sequence shares 40% sequence identity
with murine BCO. Expression in the E. coli test system revealed that this enzyme
specifically catalyzes the cleavage of b-carotene at the C-90 ,C-100 double bond,
resulting in the formation of one molecule of b-100 -apocarotenal and one
molecule of b-ionone (9). To establish the occurrence of this type of carotene-
oxygenase in other vertebrates, we manage to clone cDNAs encoding this
BCOII in human and zebrafish. The molecular identification and functional
characterization of BCOII in several vertebrate species provide strong evidence
that, besides centric (C-15,C-150 ), an additional eccentric (C-90 ,C-100 ) cleavage
pathway for b-carotene exists in vertebrates.
To sum up, in vertebrates a small gene family of VP14-homolog exists. On
the level of the deduced amino acid sequences, all three representatives in a given
vertebrate genome possess several common structural features: six histidine
residues at conserved positions, which may be involved in binding the co-factor

ferrous iron; and a well-conserved domain EDDGVVLSSXVVS close to the
C-terminus, which can be considered a family signature sequence. Furthermore,
sequence comparison revealed that the three different representatives from ver-
tebrates all have a higher degree of similarity to each other than to Drosophila
BCO. With the emerging number of sequences for carotenoid cleaving
enzymes not only from animals but also from plants now becoming available
in the public database, this information can be used to define their catalytic
domains and identify their active sites.
MOLECULAR ANALYSES OF THE VITAMIN A BIOSYNTHETIC

PATHWAY IN MODEL ORGANISM
In the past few years, a large number of different components of retinoid metab-
olism were molecularly identified (49). By reverse genetics, animal models were
established with mutations in these genes. This strategy proved to be extremely
powerful to learn more about single aspects of the pleiotropic effects of this
vitamin, for example, it could be demonstrated that a co-ordinated expression
of RA synthesizing and catabolizing enzymes is crucial to fine-tune RA-signaling
in the embryo. Furthermore, natural mutations in the genes necessary for the
metabolism of vitamin A (visual cycle) in the eyes have recently emerged as
an important class of genetic defects responsible for a wide range of retinal
dystrophies and dysfunctions in humans (50). The formal first step in retinoid
metabolism is the conversion of the provitamin to the actual vitamin. The recent
molecular cloning of carotene-oxygenases provided molecular tools to analyze
the impact of provitamin A conversion on retinoid metabolism in more functional
detail. In the following text, new results from genetically well-defined model
organisms will be discussed. The use of these models proved to be a promising
strategy to learn more about single aspects of the vitamin A biosynthetic
pathway. These analyses demonstrated that BCO catalyzes provitamin A conver-
sion in vivo and revealed the existence of a specific protein-mediated transport
mechanism for carotenoids in animals.
WHAT CAN WE LEARN FROM INSECTS?

The completed human and Drosophila genome projects have revealed that 60%
of the genes of Drosophila possess homologs in the human genome. For a
long time, the fruit fly has served as a model for functional genomics, and a mul-
titude of investigations led to the identification of genes involved in the visual
process and the elucidation of their functions (51). Furthermore, with vitamin A
functions being restricted to vision, Drosophila represents an excellent model
for the genetic dissection of the pathway leading from dietary carotenoids to
vitamin A. Among the various Drosophila mutants affected in their visual per-
formance, the phenotype of five blind mutants could be assigned because of
230 von Lintig
their characteristic electroretinograms to the so-called neither inactivation nor

after potential (nina) phenotype. This phenotype is caused by a lack of functional
visual pigments (rhodopsin) in the compound eyes of these fly mutants (52). Like
in all animals, these visual pigments consist of a protein moiety, the opsin,
to which an 11-cis-retinal derivative is covalently bound via a Schiff base
linkage. The molecular reason for this phenotype was already determined for
three (ninaA, ninaC, ninaE) of these five nina-mutants, which all affected differ-
ent aspects of the synthesis of the protein moiety of the visual pigments (53 – 55).
The ninaB and ninaD mutants, however, feature a characteristic not found in the
three other nina-mutants. Their visual performance can be rescued by feeding
these flies retinal. Thus, the ninaB and ninaD genes were promising candidates
to encode molecular components in the synthesis of the visual chromophore
from dietary carotenoids, the sole precursor for vitamin A, in Drosophila stan-
dard growth medium. The existence of two different mutants with a VAD pheno-
type indicated that at least two different genes were involved in the formation of
vitamin A in insects.
The ninaB mutation has been cytologically mapped on chromosome 3 to
the position 87E-F in the Drosophila genome (52), coinciding with the physical
location of the Drosophila bco gene. We performed detailed molecular analyses of
this gene locus and found mutations in the bco gene in two independent ninaB fly
stocks, which we showed to abolish the BCO function. Thus, the blind vitamin A
deficient phenotype of ninaB flies is caused by mutations in the bco gene, pro-
viding the first direct genetic evidence that BCO actually catalyzes vitamin A
synthesis in vivo (10). Since only this one representative of the nonheme iron
carotenoid-oxygenase gene family is encoded in the entire Drosophila genome,
centric cleavage of carotenoids may represent the universal pathway for the
synthesis of vitamin A in metazoans.
The ninaB gene is expressed exclusively in close spatial vicinity of the
photoreceptor cells, indicating that carotenoids must be transported and delivered
to BCO-expressing cells for vitamin A synthesis. Carotenoids with provitamin A
activity are highly lipophilic molecules, indicating that, as with other lipids,
specific binding/transport proteins may exist. In the second vitamin A-deficient
Drosophila mutant, ninaD, the carotenoid content was shown to be significantly
altered when compared with wild-type flies and ineffective in mediating visual
pigment synthesis (56). Our molecular analyses revealed that this phenotype is
caused by a defect in the uptake and body distribution of dietary carotenoids
(57). The ninaD gene encodes a cellular surface receptor with significant
sequence similarity to the mammalian class B scavenger receptor, SR-BI. This
receptor plays a key role in HDL-metabolism in mammals (58). In ninaD flies,
we found a nonsense mutation in the gene encoding this receptor, thus abolishing
its function (57). Direct functional evidence for the role of the ninaD receptor in
cellular carotenoid uptake was provided by P-element mediated transformation
of flies with a wild-type ninaD allele. Heat-shock induced expression of the
wild-type allele in the genetic background of ninaD flies restored carotenoid
uptake and visual pigment synthesis (57). These analyses provide genetic and
functional evidence that carotenoids are distributed to target tissues within the
body by protein-mediated transport processes. It has also been reported that in
mice deficient for SR-BI, the ninaD homologous receptor, vitamin E metabolism
is impaired, resulting in an elevated plasma concentration of this vitamin (59).
Together with the results from Drosophila, this finding may indicate that this
type of receptor is more generally involved in the metabolism of fat soluble
vitamins.
Insight into the molecular structure of a cellular carotenoid-binding protein
(CBP) comes from the silkworm Bombyx mori. Tabunoki et al. (60) purified a
lutein-binding protein from the silk gland of this insect and cloned the corre-
sponding cDNA. This insect CBP has an apparent molecular mass of 33 kDa
and binds carotenoids in a 1:1 molar ratio. Sequence comparison revealed that
CBP is a new member of the steroidogenic acute regulatory (StAR) protein
family. In mammals, these proteins are known as soluble protein carriers mediat-
ing the intracellular transport of lipids (61). An example is StAR/StarD1, which
delivers cholesterol to mitochondrial P450 side chain cleavage enzymes in ster-
oidogenic cells. The other family members are characterized by the 200 –300
amino acid StAR-related lipid transfer domain with homology to StarD1.
MLN64/StarD3 has been also shown in vitro to bind cholesterol, whereas
Star2 binds phosphatidylcholine. There are several more family members for
which the ligand is so far unknown (62). On the basis of the results from
B. mori, these family members are putative candidates for cellular CBPs.
These proteins may be needed for delivering the lipophilic carotenoid substrates
to BCO and/or for mediating the cellular transport of carotenoids in carotenoid-
accumulating tissues.
To sum up, these recent results with insects provide molecular insight into
basic principles in animal carotenoid metabolism. To become biologically active,
dietary provitamin A must be first absorbed, then delivered to the site of action in
the body to be metabolically converted to vitamin A (Fig. 10.2). The identifi-
cation of molecular players acting at the different levels of this process in
insects may provide the key toward a better understanding of this metabolism
in mammals as well.
MOLECULAR ANALYSIS OF THE VITAMIN A BIOSYNTHETIC

PATHWAY IN VERTEBRATES
In chicken, the tissue-specific expression patterns of BCO were analyzed by a
combination of Northern blot and in situ hybridization experiments. Its mRNA
was mainly localized in liver, in duodenal villi, as well as in tubular structures
of the lung and the kidney (34). In the mouse, BCO mRNA was detectable not
only in small intestine and liver but also in kidney, testes, uterine tissues, skin,
and skeletal muscle (9,36,37). Analyses of BCO mRNA expression in humans
revealed a comparable picture (39). Although Yan et al. (38) reported that
232 von Lintig
Insect LDL
(lipophorin)
Carotenoids
NinaD Extra-cellular
Target cell
CBP
NinaB Retinoids
Biological Functions
Figure 10.2 Schematic overview of the molecular players involved in insect carotenoid
metabolism. The cellular uptake of carotenoids is mediated by the class II scavenger recep-
tor ninaD from circulating lipophorins of the hemolymph. For the cellular transport,
specific carotenoid binding proteins (CBP) belonging to the StAR gene family exist. For
retinoid synthesis carotenoids are converted by the BCO function encoded by the ninaB
gene in Drosophila.
BCO is preferentially expressed in the RPE of the human eye and only at
much lower levels in other tissues, more recent results dealing with BCO
expression in the eye showed only low mRNA levels in the RPE of humans
and monkeys (63).
In mammals, a majority of provitamin A carotenoids is already converted
to vitamin A in epithelial cells of the intestinal mucosa and then transported to the
liver for storage. However, the surprising result of all these current investigations
is that BCO steady-state mRNA levels are quite high in peripheral nondigestive
tissues. Testes, for example, require retinoids for spermatogenesis, and vitamin A
is needed for retinoid-signaling in almost all tissues. Thus, BCO expression in
peripheral tissues indicates that, besides an external vitamin A supply via the cir-
culation, the provitamin A may contribute to satisfying local vitamin A demands.
This inference is in agreement with the fact that in the circulation of mammals
significant amounts of provitamin A carotenoids are present in addition to

vitamin A derivatives.
Direct evidence for such a role for provitamin A as an indispensable pre-
cursor for the formation of bioactive retinoids comes from the analysis of BCO
function in zebrafish embryos (Danio rerio) which have proven to be valuable
for the analysis of complex molecular processes in vertebrate biology. Using
this system, we addressed the question whether BCO is needed for embryonic
development. First, we demonstrated that BCO is expressed in clearly defined
spatial compartments and translated into protein in the zebrafish embryo (64).
Additionally, we demonstrated that the egg-yolk of zebrafish contains, besides
retinoids, significant amounts of b-carotene. To test whether there is an actual
requirement for BCO during zebrafish embryonic development, we performed
targeted gene knock-down experiments using morpholino antisense oligonucleo-
tides. Loss of BCO function resulted in abnormalities of the craniofacial skeleton,
pectoral fins, and eyes, which are impairments well known from mammalian and
zebrafish VAD embryos (64). Comparable impairments have been described in
RA-deficient fish, either with mutations in the retinal aldehyde dehydrogenase 2
(raldh2) gene or upon treatment of wild-type embryos with citral, an inhibitor
of RA-synthesis (65 – 67). Thus, these analyses suggest that provitamin A conver-
sion is the prerequisite for RA-signaling in several distinct developmental
processes in zebrafish embryos and reveal an unexpected vital role of the provi-
tamin in the development of a vertebrate species. The developmental use of the
nontoxic provitamin instead of preformed yolk vitamin A for RA-signaling pro-
cesses may provide an additional control mechanism to finely balance retinoid
levels at the cellular level in local tissue environments. Interestingly, embryonic
expression of BCO has been also reported in mice, suggesting that a develop-
mental function of provitamin A may exist in mammals as well (36).
Unlike vitamin A, high-dose supplementation of b-carotene in humans
causes no hypervitaminosis A, indicating that b-carotene cleavage to vitamin A
is tightly regulated. Several investigations with animal models showed that the
vitamin A status of the individual affects BCO enzymatic activity (68,69).
Recent analyses provided evidence that BCO regulation in the small intestine
is mediated on the transcriptional level, possibly via a negative feedback regu-
lation mechanism involving RA and its nuclear receptors (70). More detailed
analyses of the regulation of the murine Bco gene were provided by promoter
analysis by Boulanger et al. (71). These studies provided strong evidence that
the Bco promoter contains a PPRE (peroxisome proliferator response element)
as a key regulatory switch and is regulated by PPARg (peroxisome proliferator
activated receptor). PPARs constitute a subfamily of the steroid hormone super-
family (72). Most of the known naturally occurring ligands of PPARs are dietary-
derived fatty acids and their metabolites (73). PPARg activates genes involved in
anabolic pathways, particularly in adipose tissues, and is required for placental,
cardiac, and adipose tissue development (74). RXR is the obligate heterodimeric
partner of the PPAR transcription factors. Promoter analysis showed that Bco
234 von Lintig
expression is positively regulated by both the PPAR/RXR heterodimer and the

RXR/RXR homodimer, implying that the expression of the key enzyme for
vitamin A synthesis can be upregulated by 9-cis RA. A role of retinoid-signaling
in the positive regulation of retinoid-metabolizing enzymes on the transcriptional
level has also been demonstrated for the lecithin:retinol acyltransferase, LRAT
(75). Interestingly, the cellular retinol-binding protein II (CrbpII) gene is the
only other gene in carotenoid and retinoid metabolisms yet known to contain a
PPRE. As reported for Bco, this gene is not only upregulated by ligands of
PPAR, but also by 9-cis RA (76). CRBP-II is expressed in large amounts in
the small intestine of adult mammals, the major site of vitamin A synthesis. As
described earlier, CRBP-II may act downstream of BCO in binding retinal, the
primary cleavage product of provitamin A conversion. LRAT catalyzes the syn-
thesis of retinyl esters, the storage form for vitamin A in the liver. The expression
of LRAT is induced by feeding RA. However, details of its gene promoter
responsive elements are so far missing. Common mechanisms in the regulation
of the genes involved in the vitamin A biosynthetic pathway may contribute to
vitamin A homeostasis. The involvement of PPARs may interlink it to the regu-
lation of overall lipid metabolism. The controversial results coming from studies
on the Bco promoter level (positive regulation by 9-cis RA) and by analyzing
BCO enzymatic activity in the gut (negative regulation by RA) need further elu-
cidation. It may be speculated that the latter is the result of indirect effects due to
the influence of RA on additional genes involved in this process.
The role of BCO in vitamin A biosynthesis has been well established,
whereas the role of the second putative carotene-oxygenase, BCOII, still
remains somehow elusive. There has long been a controversy over centric vs.
eccentric cleavage of b-carotene in the synthesis of vitamin A. Evidence that
eccentric cleavage of carotenoids also occurs was provided by several investi-
gations. Napoli and Race (77), for example, showed that, besides the formation
of RA from retinal as the initial product of symmetric b-carotene cleavage, RA
is directly formed from b-carotene in cell-free homogenates. Furthermore, it
was shown that long-chain apocarotenoids (.C20) are shortened to RA in a step-
wise process which is most probably mechanistically related to the b-oxidation of
fatty acids (31,22). Thus, BCOII may catalyze the first step in an alternative
pathway for RA formation (Fig. 10.3). Direct evidence was provided in the
mouse embryo that, besides Raldh1, 2, and 3-dependent pathways, additional
ways exist for RA-generation involving so far uncharacterized RA-generating
enzymes (78,79). Recently, another Raldh (Raldh4) was molecularly identified
in the mouse, but its mRNA was mainly expressed in fetal liver (80).
We investigated the expression patterns of the eccentric carotenoid-
cleaving enzyme in the mouse (9). Here, the BcoII gene was expressed in the
same tissues as Bco. The mRNA expression of both types of carotene-oxygenases
in the same tissues, for example, small intestine and liver, confirms biochemical
investigations and explains the observation of both centric and eccentric cleavage
activity in cell-free homogenates of the same tissue. It is not yet clear whether
both enzymes are expressed in the same or different cell types of these tissues.
RE ROL
Adh
Sdh
RAL Raldh
BCO
RA RXR RAR
β-Carotene
?
BCOII AC cytochrom P450- TAT A
enzymes RARE
4-Oxo RA nucleus
4-OH RA
5,8-Epoxy RA
Figure 10.3 Schematic overview of the proposed pathways leading to RA signaling in

vertebrates. RE, retinyl ester; ROL, retinol; RA, retinoic acid; Adh, alcohol dehydrogenase;
Sdh, short chain reductase; Raldh, retinal dehydrogenase; BCO, b,b-carotene-15,150 -
oxygenase; BCOII, b,b-carotene-90 ,100 -oxygenase; RARE, retinoid receptor responsive
element; RAR, retinoic acid receptor; RXR, retinoid X receptor; 4-OXO RA, 4-oxo-
retinoic acid; 4-OH RA, 4-hydroxy retinoic acid; 5,8-Epoxy RA, 5,8-epoxy retinoic acid.
In addition, low-abundance steady-state mRNA levels of BCOII were present in

spleen, brain, lung, and heart (9). In the zebrafish, embryo bcoII expression was
also found in the developing heart. In the human fetal heart, BCOII mRNA was
found upon analyzing a commercial multitissue RNA panel (unpublished
results). These results and the existence of an alternative pathway for RA-
generation in the heart may indicate that BCOII plays a particular role for the
development of the cardiovascular system.
Biological activities of b-apocarotenoids different from retinoids have also
been reported by various studies in animals (81). In vitro, BCOII catalyzes,
besides b-carotene cleavage, the oxidative cleavage of lycopene (9). Favorable
effects of lycopene, for example, on certain kinds of cancers, have been repeat-
edly reported (82). Thus, besides being a putative precursor for RA-formation, in
the case of b-carotene cleavage, it may be speculated that apocarotenoids deriv-
ing from other carotenoids may represent biologically active substances.
Much further work needs to be done to understand fully the exact physio-
logical function of BCOII. This must include a detailed biochemical analysis of
its enzymatic properties, substrate specificity, subcellular localization, and the
elucidation of the fate of the primary cleavage products of the reaction. Further-
more, animal models with mutations in this gene must be established to unequi-
vocally address the impact of this type of carotene-oxygenase on vitamin A
metabolism.
CONCLUSIONS
The molecular identification of the different metazoan carotene-oxygenases
established the existence of an ancient family of nonheme iron oxygenases in
236 von Lintig
animals. Through these enzymes, animals have access to and can modulate their
retinoids as needed for biological processes as diverse as vision, cell differen-
tiation, and development. With the increasing number of carotene-oxygenases
in the database, sequence information can be used to predict common structural
features and to identify functional domains and active site residues. The identifi-
cation of proteins involved in the transport of carotenoids in insects demonstrated
that this process, as described for other lipids, is protein-mediated. The identifi-
cation of these genes provides a starting point to characterize analogous genes in
mammals. The advanced state of knowledge about the molecular components of
the vitamin A biosynthetic pathway gained in the past few years will surely help
in the fight against VAD and will open new avenues of research, dealing with bio-
chemical, physiological, developmental, and medical aspects of carotenoids and
their numerous derivatives. Furthermore, the identification of genes involved in
carotenoid metabolism provides molecular markers to analyze genetic aspects of
nutrient interactions and the basis to analyze genetic polymorphism in these
genes within the population. The establishment of suitable animal model sys-
tems such as knock-out mice in these genes may contribute to elucidating mecha-
nisms underlying the pathogenesis of diseases as well as providing starting
points for their prevention.
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11
Molecular Mechanisms Underlaying
the Health Promoting Activity
of Lycopene
Estibaliz Olano-Martin
Introduction 241
Antioxidant Activity 243
Modulation of Intracellular Communication 245
Inhibition of Cell Cycle Progression 246
Inhibition of Insulin-Like Growth Factor (IGF-1) Signaling 247
Inhibition of HMG-CoA Reductase 249
Conclusions 250
References 251
INTRODUCTION
Lycopene (Fig. 11.1) is an acyclic isomer of beta-carotene consisting of a
40-carbon atom open polyisoprenoid chain, which contains 11 conjugated and
2 unconjugated double bonds (1). It is a pigment that imparts red color to
tomatoes, rosehip, guava, apricots, watermelon, and pink grapefruit. Lycopene
is synthesized by plants and some micro-organisms, but not by animals. It
serves as an accessory light-gathering pigment and protects these organisms
against the toxic effects of oxygen and light. In plants, it tends to exist in an
241
242 Olano-Martin
Figure 11.1 Structural formula of lycopene.
all-trans configuration but easily undergoes cis/trans isomerization (1 –3). The

exact functions and relative activities of the different isomers are not fully under-
stood and investigations are currently underway to determine whether there are
biological differences between them (3).
Humans obtain lycopene from their diet and it is the predominant carote-
noid in human plasma (4), with a half-life of 2– 3 days. Lycopene levels in
blood do not differ significantly between men and women, but its level is affected
by age (2) and several biological and lifestyle factors. However, in contrast to
other carotenoids, lycopene serum values are not usually reduced by smoking
or alcohol consumption (5). Because of its lipophilic nature, lycopene concen-
trates in low-density and very low-density lipoprotein fractions of the serum
and it is present as a 50/50 cis/trans isomeric mixture (2,5). Lycopene is
found in higher concentrations in the adrenal, liver, testes, and prostate (6).
Unlike beta-carotene, lycopene cannot be converted to vitamin A in the
body, since it lacks a beta-ionone ring structure. Therefore it is not an essential
nutrient, and it attracted little attention until it was shown in vitro to have a
singlet-oxygen-quenching ability twice as high as that of beta-carotene and ten
times higher than that of alpha-tocopherol. This made lycopene the most
potent antioxidant of the common carotenoids (7).
In addition to its antioxidant properties, many epidemiological studies have
indicated an association between regular consumption of lycopene-rich foods and
decreased risk for a range of common cancers. In 1999, Giovannucci reviewed 72
human studies and found in 57 of these reports an inverse association between
tomato consumption or blood lycopene levels and risk of various types of
cancer (8). Thirty-five of these associations were significant. Evidence of protec-
tive properties of lycopene was highest for cancers of the prostate, lung, and
stomach. The fact that the data relating to lycopene and the risk of aggressive
prostate cancer are especially compelling (9,10) made this carotenoid one of
the most promising chemopreventive agents found in western diets. Indeed, con-
sumption of tomato-based foods has been associated with a 30– 40% lower risk of
prostate cancer, and it is lycopene rather than other carotenoids that is responsible
for this effect.
Health Promoting Activity of Lycopene 243
Most of the data in Giovannuccis’ review were from observational studies,

and therefore a cause-and-effect relationship could not be firmly established.
However, the consistently lower risk of cancer associated with higher consump-
tion of lycopene-containing tomatoes provided a strong foundation for further
research on lycopene.
Recently, more direct, though still preliminary, evidence has emerged,
which suggests not only protective effects of lycopene in prostate cancer, but
also in breast cancer (11) and cervical dysplasia (12). In addition to that, resear-
chers have found a statistically significant association between high dietary
lycopene and a 48% lower risk of heart disease (13). Studies have also investi-
gated the relationship of lycopene with many other diseases such as cataracts
(14), asthma, longevity (15), malaria (16), digestive-tract cancers (17,18),
immune modulation (19), Alzheimer’s disease (20), and preclampsia (21).
The mechanisms involved in the protective effects of lycopene are still
poorly understood, but it is thought that lycopene’s potent antioxidant activity
might confer protection against chronic diseases such as cancer, atherosclerosis,
and associated coronary heart disease. A number of other non-antioxidant mecha-
nisms have also been suggested.
ANTIOXIDANT ACTIVITY
The human body is constantly exposed to oxidative stress. Oxidative stress arises
partly from environmental parameters (air pollution, tobacco smoke, and radi-
ation) (22,23) and partly as a natural result of aerobic (oxygen dependent) meta-
bolism. The by-products of oxidation are highly reactive molecules and include
free radicals, as well as the highly reactive singlet form of oxygen (24). All these
molecules can react with various components of a living cell—proteins, DNA,
and lipids—leading to a number of pathological conditions including aging,
senile dementia, atherogenesis, ischemia-reperfusion injury, alcohol damage,
age-related macular degeneration, and cancer (25).
Even if the human body has evolved to fight oxidative stress with endogen-
ous antioxidant defenses, diet-derived antioxidants—including ascorbic acid,
alpha-tocopherol, and carotenoids—are still important in protecting against the
sum of oxidative stresses challenging the body. In healthy human subjects and
in vitro, lycopene has been shown to protect against lipid, lipoprotein, protein,
and DNA oxidation (25 – 27), to reduce the susceptibility of lymphocyte DNA
to oxidative damage (28,29), to inactivate hydrogen peroxide and nitrogen
dioxide (30), and to protect lymphocytes from nitrogen oxide-induced membrane
damage and cell death twice as efficiently as beta-carotene (31). Ingestion of a
lycopene free diet for 2 weeks resulted in 50% loss of serum lycopene with a
25% increase of in vivo lipid oxidation in healthy human subjects. Low lycopene
levels in serum have also been associated with increased risk of coronary heart
disease (13,25,32,33) and have been found in patients with cancer.
244 Olano-Martin
In vitro studies have shown that the lycopene prevents oxidation by

quenching singlet oxygen. Among the carotenoids, lycopene exhibits one of
the highest physical quenching rate constant (Kq ¼ 31 109 M-l S-1) (7).
This protective function is achieved by both physical (energy transfer) and
chemical processes [Eqs. (1) and (2)]. The physical quenching rate is much
higher than the chemical; and once produced, the triplet excited state of the
3
Lycopene can return to the ground state, dissipating the excess energy as
heat (34). Thus, lycopene acts as a catalyst in the deactivation of singlet oxygen.
Heat
!
1
O2 þ Lycopene ! O2 þ 3 Lycopene ! O2 þ Lycopene (1)
1
O2 þ Lycopene ! Lycopene-O2 (2)
A second major antioxidant role of carotenoids is the scavenging of free

radicals (35). Hydrogen peroxide, nitrogen dioxide, thyi and sulphonyl radicals
are some of the reactive oxygen species (ROS) that lycopene has been shown
to deactivate. As shown earlier (Fig. 11.1), lycopene has several conjugated
double bonds that enable the electrons to be delocalized over the whole system
and so be shared by many atoms, making the whole chain relatively electron-
rich. This means that the delocalized electrons may move around the whole
system resulting in an electron cloud, where the electrons do not specifically
belong to any one atom. Lycopene will stabilize a nearby free radical by
giving up an electron. Lycopene then becomes a free radical itself, but owing
to the conjugated bonds, it will be hundreds of times more stable than, for
example, hydroxyl radical (Fig. 11.2).
Although high concentrations of ROS are cytotoxic, low concentrations are
still needed in the cell to keep its intracellular redox state that regulates the
activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phos-
pholipases, and transcription factors (36–39). As lycopene is a potent antioxidant, it
could modulate the intracellular redox status by scavenging ROS and quenching
singlet oxygen inside the cell, affecting the expression of redox regulated genes
such as NF-kB and AP-1. NF-kB is involved in inflammatory and immune
responses, cell survival, and activation, and AP-1 is involved in cell growth, cell pro-
liferation, and differentiation. There are no published data showing the relationship
electron cloud (delocalised e– )
·
· –
OH OH
Figure 11.2 Lycopene free radical scavenging mechanism.

between lycopene supplementation and changes in the redox potential of the cell, but
previous studies have reported the role of oxidants and antioxidants in the regulation
of redox-sensitive transcription factors (38,40–43).
Therefore, the antioxidant activity of lycopene could protect cells from oxi-
dative damage by preventing arteriosclerosis and/or cancer, but also decrease
cell proliferation of cancer cells by interfering with redox-sensitive genes.
MODULATION OF INTRACELLULAR COMMUNICATION

Cells connect and communicate via transmembrane channels called gap junctions
(Fig. 11.3) that connect the cytoplasms of neighboring cells. The channels consist
of two hexamers of specific proteins, which belong to the gene family of connex-
ins. These channels permit small metabolites (,1000 Da), ions, and second mes-
sengers to pass from cell to cell. This intercellular signaling mechanism is very
important for coordinating biochemical functions in multicellular organisms,
and loss of this signaling activity often happens during malignant transformation
of cells. Both in vitro (44,45) and in vivo studies (46,47) have demonstrated that
inhibition of gap-junctional intracellular communication (GJIC), leading to a
long-lasting decrease in cell communication, appears to be a strong tumor-
promoting factor. Moreover, Zang et al. reported recently that over-expression
of connexin 43 was associated with suppressed proliferation of human osteosar-
coma U2OS cells through arresting cell cycle at G1/S phase (48).
The GJIC impairment seems to be reversible, and chemicals that increase
gap-junctions could be used as chemopreventive agents. Different publications
have demonstrated a correlation between the ability of diverse carotenoids to
inhibit chemically induced neoplastic transformation and their ability to stimulate
gap-communication by increasing connexin 43 levels (49 – 52).
In particular, lycopene has been shown to stimulate GJIC in mouse
embryonic fibroblast 10T1/2 (52), human dermal fibroblasts (53), human fetal
connexin
Gap junction
Intracellular gap
Figure 11.3 Scheme of a gap junction between two adjacent cells. It works as a window
between neighboring cells through which ions, current, and cytoplasm can flow.
246 Olano-Martin
skin fibroblast (54), and human oral cavity tumors as seen by scrape-loading dye
transfer and electron microscopy (55). In addition to lycopene, its oxidation pro-
ducts such as 2,7,11,-trimethyl-tetradecahexane-1,14dial have been found to
have similar activity (56). In many of these studies, an up-regulation of both
the transcription and expression of connexin 43 in cells was also observed.
In vivo experiments using rats also found enhanced GJIC after treatment with
50 mg/kg lycopene (47).
However, this chemopreventive mechanism may be only effective in early
stages of cancer as cells loose their growth inhibitory response to lycopene
through the selection pressures involved in metastasis. A recent study by
Forbes et al. shows how lycopene treatment at a physiological dose (1.0 mM)
increased connexin 43 expression and inhibited cell growth in PC-3, an estab-
lished human prostate cancer cell line, but not in metastatically passaged prostate
cells (PC-3MM2) (57).
INHIBITION OF CELL CYCLE PROGRESSION

In addition, lycopene at physiological concentrations has been shown to inhibit
proliferation of several types of human cell lines, including HL-60 leukemic
cells (58), human aortic smooth muscle cells (59), endometrial cells (60,61),
breast (60 –64), prostate (65 – 68), and lung (60). This is a very important trait
since cancer can be very briefly described as uncontrolled cell growth and pro-
liferation (as well as metastasis, or the invasiveness of cancerous cells into
other tissues). Therefore, the fact that lycopene can either stop or slow down
proliferation makes it a promising chemoprotective agent.
In proliferating cells, the cell cycle consists of four phases. Gap 1 (G1) is
the interval between mitosis and DNA replication that is characterized by cell
growth. The transition that occurs at the restriction point (R) in G1 commits
the cell to the proliferative cycle. If the conditions that signal this transition
are not present, the cell exits the cell cycle and enters G0, a nonproliferative
phase during which growth, differentiation, and apoptosis occur.
The G1 phase is characterized by gene expression and protein synthesis and
is really the only part of the cell cycle regulated primarily by extracellular stimuli
(like mitogens such as growth factors and adhesion). Late G1 phase is marked by
transcription of D-cyclins, which bind and activate cyclin kinases (Cdk4 and
Cdk6). Once bound, the CDK complex phosphorylates the retinoblastoma
protein pRB, which then allows cell cycle progression (Fig. 11.4). Unphosphory-
lated pRB normally masks E2F transcription activators (actually a family of
factors, E2F1-3) so that E2F target genes are not expressed. These target genes
are all related to DNA replication and cell division, so pRB acts as a general
repressor of cell proliferation (69).
It is speculated that lycopene may suppress carcinogen-induced phos-
phorylation of regulatory proteins, such as p53 and Rb antioncogenes, and stop
cell division at the G0 – G1 cell cycle phase. Research has shown (61,68) that
Figure 11.4 Mitogens drive cell cycle progression by induction of cyclin D and
inactivation of the retinoblastoma (Rb) protein. Inactivation of the Rb protein marks the
restriction point at which cell-cycle progression becomes independent of mitogens. Inac-
tivated Rb releases E2F transcription factors, which stimulate the expression of down-
stream cyclins and other genes that are required for DNA synthesis. (Figure reproduced
with permission of Dr W. Koch and Expert Reviews in Molecular Medicine.)
2 –5 mM of lycopene supplementation interferes with cell cycle progression at

G1 as observed by flow cytometry (Fig. 11.5), reducing the levels of cyclins
(mostly cyclin D) and Cdk activity. It is not clear whether lycopene acts directly
on cyclin D or interferes with mitogens. Alternatively, the decrease of cyclin D
levels may be related to lycopene effects on other sites of the cell cycle
machinery. However, this chain of events is responsible for a decrease in pRB
phosphorylation and a delay in G1/S transition.
Another important protein in regulating the cell cycle is the tumor suppres-
sor protein p53. In fact, p53 is the most frequently disrupted gene in cancer (70),
illustrating its importance in this disease, and it has a role in regulating the
expression of genes involved in cell cycle arrest and apoptosis. However, there
are currently no data that suggest that lycopene might have an impact on p53
levels.
INHIBITION OF INSULIN-LIKE GROWTH FACTOR (IGF-1)

SIGNALING
Lycopene may also reduce cellular proliferation induced by insulin-like growth
factors (IGFs). These growth factors are naturally secreted by the liver, but are
also produced in several other tissues (71). They are necessary for normal phys-
iological functions including promotion of cell survival (inhibition of apoptosis),
stimulation of metabolism, and proliferation of differentiating cells (72).
However, since IGFs are mitogenic and anti-apoptotic, it is thought that high
248 Olano-Martin
80
*
60
% of PrEC
40
*
20
*
0
Control 0 0.5 5.0
G0G1 48 56 56 71
G2M 15 14 11 9
S 37 31 32 22
Lycopene [µM]
Figure 11.5 Summary of the cell cycle analysis by flow cytometry based on DNA
content propidium iodide stained cells. When treated with 5 mM of lycopene, cells were
arrested at the beginning of S phase. Flow cytometry data were collected for each of
the treatments at least 6 times from 6 different experiments. Indicates a significant differ-
ence ( p , 0.05) compared with the control.
levels might enhance cell turnover, increasing the chances of cellular transfor-
mation that then could lead to cancer. Indeed, high IGF-1 levels in plasma have
been associated with an increased risk of breast cancer in premenopausal women
and prostate, colon, and lung cancer (73).
Research has shown that lycopene interferes with IGF signaling and cell
cycle progression specifically in breast and prostate cancer cells (62,74). Karas
et al. examined the effect of physiological concentrations of lycopene
(,1 mM) on IGF-1 induced growth of MCF7 mammary cancer cells (62).
Lycopene-treated cells significantly reduced the IGF-1 cell growth stimulation
(Fig. 11.6), slowing down cell proliferation. These effects were associated with
an inhibition of the IGF signaling in the cells and an increase in IGFBs. The sup-
pression of cell proliferation by IGF stimulated growth had been previously
reported in endometrial cells (60); and importantly, normal human cells were
found to be much less sensitive to lycopene than cancer cells. By interfering
with these growth factor-related cancer stimulators, lycopene may reduce both
the occurrence and the progression of breast and prostate cancers.
The exact mechanism in which lycopene interferes with IGF-1 signaling
pathway and slows down cellular growth might be also related with the
lycopene’s ability to suppress 3-hydroxy-3 methyl glutaryl coenzyme A
(HMG-CoA) reductase activity. It has been found that lycopene can inhibit
macrophage HMG-CoA reductase activity (32), which constitutes the key meta-
bolite in the biosynthesis of cholesterol and a variety of sterol and nonsterol
Figure 11.6 Possible effect of lycopene on IGF-1 signal transduction pathway.
isoprenoids such as dolichol. Because dolichol is a crucial isoprenoid during G1

for the expression of IGF-receptors, a decrease in the dolichol levels will then
interfere with IGF-1 signaling. Indeed, cells that cannot synthesize dolichol
experience an effective deficiency of IGF-1 activity (75,76). Therefore, lycopene
might induce a deficit, the isoprenol dolichol, that in turn impairs IGF-1 and cell
cycle progression.
INHIBITION OF HMG-CoA REDUCTASE

As mentioned earlier, it has been reported that lycopene supplementation
(10 mM) inhibits the de novo synthesis of cholesterol from [3H]acetate, but not
from [14C]mevalonate in the J-774A macrophage cell line (32). This effect has
been related with an inhibition of the enzyme HMG-CoA reductase activity by
lycopene, and could explain both its cancer prevention and hypocholesterolemia
properties.
It is well known that mevalonate is required for growth of mammalian cells
playing a key role in controlling cell proliferation through the Ras-mediated
signal transduction pathway (77 – 79). The Ras oncogenes (HRAS, KRAS, and
NRAS) encode 21 kDa proteins called p21s. To initiate eukaryotic cell prolifer-
ation, Ras requires the posttranslational attachment of a farnesyl group, an isopre-
noid lipid moiety derived from mevalonate, to the carboxyl-terminus of the
protein (Fig. 11.7). Therefore, an inhibition of HMG-CoA reductase activity
will lead to a mevalonate depletion, which in turn will interfere with the Ras
pathway causing G1 arrest in cycling mammalian cells and retarding cancer
induction and slowing cellular growth (80,81).
250 Olano-Martin
Figure 11.7 Inhibition of cell cycle progression from G1 to S phase by lycopene by

interfering with the Ras signaling pathway.
The effect of lycopene on HMG-CoA reductase activity may also be impor-

tant in cardiovascular disease. Recent epidemiological studies have reported
an inverse association between higher tissue and serum levels of lycopene and
the risk of coronary heart disease in women (82). It is thought that lycopene
might reduce LDL cholesterol in plasma by inhibiting HMG-CoA reductase
(an important step in cholesterol synthesis) and by up-regulating the LDL
receptor (32). However, more research is needed in this field since the protective
effects of lycopene in cardiovascular disease have been also related to the
reduction of homocysteine, platelet aggregation, and blood pressure (83).
CONCLUSIONS
There is extensive evidence that regular high consumption of fruits and vege-
tables decreases the risk of chronic diseases such as cancer or atherosclerosis
(84). Among them, tomatoes have gain lots of attention since epidemiological
studies have shown that high intake of tomato products were inversely associated
with the incidence of certain types of cancer (mostly prostate cancer) (8).
To understand the molecular mechanisms involved in the beneficial effects of
tomatoes, much research has focused on the carotenoid lycopene—the most
abundant phytochemical in tomatoes and the one thought to be responsible of
heath-related effects. Research has shown that lycopene interferes in many diffe-
rent pathways, changing the expression of proteins involved in cell to cell com-
munication, cycle, and cell growth. The high antioxidant potential of lycopene
might be responsible for some of the effects. However, there is enough evidence
that suggests other non-antioxidant mechanisms, such as modulating transcription
factors might be responsible for the reduction of the risk for chronic diseases of
lycopene.
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12
Cellular Redox Activity and Molecular
Functions of Ascorbic Acid
John K. Lodge
University of Surrey, Guildford, Surrey, UK
Properties of Ascorbic Acid 257

Nutritional Aspects 259
Ascorbic Acid and Redox Status 260
Ascorbate Recycling 260
Ascorbate Recycling and Metabolism 263
Molecular Functions of Ascorbic Acid 265
Ascorbate Modulation of Collagen Formation 265
Ascorbate Modulation of Cell Differentiation 267
Modulation of Transcription Factors 268
Ascorbate Modulation of Nitric Oxide Production 271
Ascorbate-Induced Modulation of DNA Repair 272
Ascorbate-Induced Modulation of Other Genes 272
Conclusion 273
References 273
PROPERTIES OF ASCORBIC ACID

Ascorbic acid is six carbon lactone structurally related to glucose. Indeed,
glucose provides the starting point of ascorbic acid synthesis in those animals
257
258 Lodge
capable of synthesis. Primates are one of the few mammalian species that lack the
ability to synthesize ascorbic acid, and must therefore obtain ascorbic acid from
the diet. Hence, it is also known as vitamin C. The term ascorbic acid derives
from experiments into the causes of scurvy (vitamin C deficiency disease)
where a “scorbutic factor” was used to describe a substance, later found to be
vitamin C, found in citrus fruits which prevents the onset of scurvy.
The structures of ascorbic acid, and its oxidation products, are shown in
Fig. 12.1. Owing to the hydroxyl at the C3 position having a pKa of 4.2, at phys-
iological pH ascorbic acid is present as the ascorbate ion and is hence referred to
as ascorbate in this text. The loss of one electron results in formation of the ascor-
bate free radical (AFR), also known as semi-dehydroascorbate. If not recycled
back to ascorbate, this will quickly dismute into dehydroascorbate (DHA). Simi-
larly, DHA can be formed from the two-electron oxidation of ascorbate. Thus, as
an electron donor, ascorbate can participate in both one and two electron transfer
reactions. Both ascorbate and AFR have relatively low one-electron reduction
potentials of 282 and 2174 mV, respectively (1), enabling these molecules to
react with and reduce a variety of species (2). This also makes ascorbate a power-
ful reducing agent and most, if not all, of the biochemical functions of ascorbate
can be attributed to these reducing properties. Ascorbate oxidation products
undergo recycling. However, if not recycled, the lactone ring of DHA breaks
down to form first 2,3-diketogulonic acid and finally oxalic acid which accounts
for the major metabolite and excretory product of ascorbate.
The functions of ascorbate have been reviewed elsewhere (3) and are only
summarized here. In mammals, ascorbate is required as an electron donor for
eight enzymes, which have either mono-oxygenase or di-oxygenase activity.
The mono-oxygenases are dopamine b-hydroxylase, which converts dopamine
to noradrenaline, and peptidyl-glycine a-mono-oxygenase, which converts
a peptide with a C-terminal glycine to a C-terminal amidated peptide. The
di-oxygenases are prolyly 4-hydroxylase, prolyl 3-hydroxylase, and lysyl
hydroxylase, which convert proline or lysine residues to the hydroxylated residue
during collagen biosynthesis; trimethyllysine hydroxylase and g-butyrobetaine
ascorbic acid ascorbate free radical dehydroascorbic acid

ASC AFR DHA
CH2OH CH2OH
CH2OH
HO CH HO CH
O O HO CH
O O O
O
HO OH HO O
O O
Figure 12.1 Ascorbic acid and its one- and two-electron oxidation products.
Cellular Redox Activity and Molecular Functions of Ascorbic Acid 259
hydroxylase, which convert trimethyllysine to hydroxythimethylysine and

trimethylaminobutyrate to carnitine, respectively; and hydroxyphenylpyruvate
dioxygenase, which converts hydroxyphenylpyruvate to homogentisic acid in
tyrosine catabolism. Ascorbate acts either as a direct source of electrons (as in
the copper containing mono-oxygenases), or to reduce the iron cofactor in the
active site of various di-oxygenases which undergo iron oxidation during the
catalytic cycle.
Ascorbate has a well-characterized antioxidant role in scavenging reactive
oxygen species (ROS) and reactive nitrogen species and recycling oxidized
a-tocopherol. Briefly, in vitro systems have demonstrated ascorbate as scaven-
ging superoxide, hydroxyl, hydrophilic peroxyl, thiyl, and nitroxide radicals, as
well as hypochlorous acid and hydrogen peroxide. These actions have been
reviewed in detail elsewhere (4,5).
Other functions for ascorbate are in iron metabolism, by maintaining iron in
the reduced state ascorbate promotes iron absorption, and can also mobilize iron
from ferritin deposits.
The classical symptoms of ascorbate deficiency, known as scurvy, can be
directly related to these functions of ascorbate and predominantly those in
enzyme action. For example, one of the first symptoms is fatigue and this can
be explained by a shortage of carnitine, required for shuttling of acyl groups
across the mitochondria for b-oxidation and energy production. The most charac-
teristic symptom of scurvy is the poor wound-healing, hemorrhaging, and joint
pains which are associated with a lack of collagen. Incomplete hydroxylation
of proline and lysine residues disrupts the normal triple-helix formation and
cross-linking of collagen fibres.
Nutritional Aspects
Ascorbate is predominantly found in fruits and vegetables with citrus fruits and
berries having the richest sources in fruits, whereas green vegetables such as
broccoli, cabbage, sprouts, and others such as peppers have the highest sources
in vegetables (6). The recommended daily intake for ascorbate in adult males
ranges from 40 mg/d in the United Kingdom to 90 mg/d in the United States
and is increased by a further 35 mg/d in cigarette smokers to take into account
increased turnover of ascorbate in smokers (7). As one serving of fruit or veg-
etables can contain 30 mg ascorbate, these intakes can be readily achievable
from a healthy diet. DHA is also present in food, accounting 10 –20% of all
vitamin C, whereas it can also be formed by oxidation of ascorbate in the GI tract.
Absorption of ascorbate occurs primarily by the sodium-dependent vitamin
C transporter (SVCT1) in the upper GI tract (8). This is a highly efficient and
specific process and requires two sodium ions for the transport of one ascorbate
molecule. DHA is absorbed by a sodium-independent process of facilitated diffu-
sion, by an as yet unknown mechanism. Following absorption DHA is presum-
ably reduced to ascorbate in the enterocyte. Using human repletion studies,
260 Lodge
Levine et al. have assessed ascorbate bioavailability at almost 100% at doses

between 15 and 200 mg (9,10). However, bioavailability decreases with increas-
ing dose (11) dropping to 50% with doses around 1 g, and 20% with doses of
5 g (12). In a cell culture intestinal model, chronic exposure to ascorbate
appeared to reduce the expression of the major intestinal ascorbate transporter
SVCT1 in CaCo-2 cells (13), which may help to explain decreased bioavailability
at higher doses. It is also likely that high doses of ascorbate will saturate the trans-
port system. Flavonoids such as quercitin have also recently been shown to
inhibit ascorbate uptake (14). The bioavailability of ascorbate from food
sources appears to be similar to that from the synthetic form (15). Factors
which are known to influence bioavailability include glucose which can inhibit
ascorbate absorption, but not DHA absorption (16), and in general anything
that could destroy ascorbate. There is no data regarding bioavailability of DHA.
Following enterocyte absorption, ascorbate is transported across the baso-
lateral membrane into the bloodstream via the sodium-dependent vitamin C
transporter 2 (SVCT2) (17). Ascorbate accounts for over 95% of vitamin C in
the blood, and in the absence of any transport proteins, is able to travel freely
and associate into extracellular spaces.
The ascorbate transporters SVCT1 and 2 are also responsible for cellular
uptake. Because certain tissues appear to accumulate ascorbate, transport must
be against a concentration gradient. This tissue-specific localization reflects the
main function of ascorbate in these tissues. Tissues that actively accumulate
ascorbate include the adrenals, leucocytes, mesenchymal cells (muscle, cartilage,
and bone cells), pituitary, lung, liver, and eye (18). DHA transport is sodium and
energy independent and occurs via the glucose transporters GLUT1 and GLUT3
(19). Transport of DHA was found to be at least 10-fold faster than that of ascor-
bate in neutrophils (20). Once transported, DHA is immediately reduced to ascor-
bate, thereby preventing reverse transport. Ascorbate release from cells is
negligible.
ASCORBIC ACID AND REDOX STATUS

Ascorbate Recycling
Ascorbate is oxidized to the AFR or DHA (Fig. 12.1). It is well established
that ascorbate undergoes recycling from both these oxidation products
(21 – 26). Ascorbate recycling is an important process in maintaining ascorbate
concentrations. The processes involved have been demonstrated in a number of
mammalian cell types, and proceed via different mechanisms either enzymati-
cally or non-enzymatically. The various processes are highlighted in Fig. 12.2.
Intracellularly, the AFR is produced following certain free radical scaven-
ging actions and recycling of the tocopheroxyl radical (oxidation product of
a-tocopherol formed during lipid peroxidation). Owing to electron delocalization
in the lactone ring, the AFR is relatively unreactive and will disproportionate to
.
α-T α-TH
ASC
oxidants ASC
1/2 NADH
ASC:AFR oxidants 1/2 NAD+
reductase
reduced AFR reductase
-1e- (cytochrome b5 reductase)
oxidants
AFR AFR ASC
thioredoxin reductase
-2e- AFR
GLUT
1/2 NADPH 1/2 NADP+
1 or 3
reduced
DHA ASC
oxidants DHA
glutaredoxin
-2e- GSH GSH
NADH
AFR GSSG GSSG
ASC ASC
-1e- NADH dependent glutathione reductase
AFR reductase
NADPH NADP+
NAD+
oxidants ASC
pentose phosphate
EXTRACELLULAR pathway INTRACELLULAR
Figure 12.2 Schematic representation of various pathways of ascorbate free radical

(AFR) and dehydroascorbate (DHA) recycling in human cell lines. Intracellular AFR is
reduced by enzymatic pathways requiring either NADH (e.g., mitochondrial AFR
reductase) or NADPH (e.g., TR). Extracellular AFR can also be reduced using electrons
donated intracellularly via either an ascorbate- or NADH-dependent AFR reductase.
DHA is predominantly reduced via GSH-dependent mechanisms which can be either
direct or enzymatic (glutaredoxin). Both these pathways, however, require the recycling
of GSH by GSH reductase which consumes NADPH. A decrease in the NADPH:NADPþ
ratio stimulates the pentose phosphate pathway.
DHA and ascorbate (27,28). AFR can be reduced back to ascorbate enzymatically
by both NADH- and NADPH-dependent mechanisms (23). A cytochrome b5
reductase in the outer mitochondrial membrane of rat liver mitochondria was
found to possess NADH-dependent AFR reductase activity (29), and this mech-
anism presumably accounts for NADH-dependent AFR reductase activity in
endothelial cells (23). NADPH-dependent reduction of AFR involves thioredoxin
reductase (TR), and this has been demonstrated in rat liver homogenates (21),
human erythrocytes (30), and endothelial cells (23). AFR reduction by NADH-
and NADPH-dependent mechanisms were compared in endothelial cells by
May et al. (23). They found that in whole lysates, NADH-dependent reduction
predominated, but following removal of particulate matter, NADPH-dependent
mechanisms predominanted, thus NADH-dependent AFR reductase activity is
largely confined to particulate fractions, whereas NADPH-dependent activity
was confined to the cytosol (23). This activity was identified as TR (23).
262 Lodge
Within cytosolic secretory vesicles, the AFR is produced during the ascorbate-
dependent mono-oxygenase reactions of either dopamine hydroxylase or pepti-
dylglycine mono-oxygenase. AFR is then reduced back to ascorbate utilizing
electrons transferred across the vesicle membranes from cytosolic ascorbate
via cytochrome b561 (31). Although cytosolic ascorbate is oxidized to AFR in
this process, this can then be recycled itself by mechanisms described earlier.
Extracellular AFR an also be reduced by various mechanisms that require
intracellular donors. It has been demonstrated in erythrocytes that intracellular
ascorbate can donate electrons to extracellular AFR via a plasma membrane
redox system (32), and that this process causes depolarization of the membrane
(33). AFR reductases are present in the plasma membrane of erythrocytes which
can potentially reduce AFR using intracellular NADH (34). Intracellular ascor-
bate can also supply electrons for transmembrane electron transport using
ferricyanide as an electron as the extracellular electron acceptor (27,35). An
ascorbate-dependent trans-plasma membrane oxidoreductase has been proposed
(27); however, more work is required in this area to fully elucidate this activity.
DHA is formed either by dismutation of the AFR, or by two electron oxi-
dation of ascorbate by reactive oxidizing species. Efficient AFR reductase activity
reduces the amount of AFR available for dismutation into DHA (23,28) and is such
an important process. It has been proposed that due to high capacity and affinity of
the AFR reductases, in the absence of severe oxidative stress, AFR reduction is
probably the most important mechanism of ascorbate recycling (23). Thus, the
majority of DHA must come from the antioxidant action of ascorbate following
sustained oxidative stress either intra- or inter-cellularly. DHA is formed during
the “oxidative burst” of mononuclear cells to destroy pathogens. Indeed, ascorbate
recycling in neutrophils can be induced by micro-organisms (24) which initiate the
oxidative burst and production of ROS. Similarly, HL-60 promyeloctic tumor cells
increased their rate of DHA uptake following activation (36).
DHA is also reduced back to ascorbate by enzymatic and non-enzymatic
mechanisms. In both circumstances, GSH and NADPH play an important role
as the electron donor. Direct chemical reduction by GSH has been demonstrated
(37,38); however, some of these experiments were performed in a “test-tube”
model. The products of this reaction are ascorbate and GSSG in equimolar
amounts, GSSG being formed stoicheometrically (37). Enzymatic reduction of
DHA is more efficient. The majority of GSH-dependent enzyme reduction can
be attributed (at least in neutrophils) to glutaredoxin (39). Inhibition of this
enzyme blocked DHA reduction by up to 80% in neutrophil lysates (39). This
reduction is 10 –20-fold faster than direct chemical reduction. In human endo-
thelial cells, DHA reduction was substantial even in the absence of lysate;
however, there was a linear increase in DHA reduction with increasing
amounts of lysate (23). This demonstrates that in this cell line both enzymatic
and non-enzymatic reductions are important. In human erythrocytes, DHA
reduction is also GSH-dependent (22,30). Interestingly, in the absence of
glucose, DHA reduction consumed NADH as well as NADPH; whereas in the
presence of glucose, only NADPH was consumed (22), indicating that cellular
metabolic pathways must also be taken into account. Although DHA reduction
in erythrocytes occurs substantially through a direct chemical reaction with
GSH, enzymes such as TR also appear important (30). Other GSH-dependent pro-
teins with DHA reductase activity include protein disulfide isomerase (40), gluta-
thione peroxidase (41), and an as yet unidentified protein isolated from human
erythrocytes (42,43). DHA can also be reduced back to ascorbate via a non-
GSH-dependent enzyme action. TR, as well as reducing the AFR, can reduce
DHA (30,44) and requires NADPH as an electron donor. The contribution of
TR though appears to be minor in certain cells, as a 3-fold increase in TR
enzyme activity following selenium supplementation did not increase the
ability of cultured hepatoma cells to reduce DHA (45). There was also little con-
tribution of TR to DHA reduction relative to that mediated by GSH in human
endothelial cells (23). Another NADPH-dependent DHA reductase was identified
from rat liver to be 3-a-hydroxysteroid dehydrogenase (46).
The importance of GSH-independent DHA reductases was demonstrated in
human leukemic cells (HL-60) depleted of GSH with the agent BSO, which were
still able to accumulate millimolar concentrations of ascorbate from DHA (47).
However, in contrast to these results, human erythrocytes treated with diethyl-
maleate to deplete GSH showed parallel decreases in the reduction of DHA,
and ascorbate-dependent ferricyanide reduction (22). Thus, the major mechanism
of DHA reduction may depend on the cell type. The physiological role for DHA
reduction is still a matter for debate, at least in the absence of sustained oxidative
stress (28).
Human cells can been shown to generate ascorbate via recycling of DHA
generated extracellularly by activated cells undergoing the oxidative burst, in
what was termed a “bystander effect” by the authors (25). Uptake of DHA into
cells is obviously a pre-requisite for recycling. However, reduction of DHA by
erythrocytes can occur extracellularly by the passage of electrons through the
plasma membrane (48).
Ascorbate recycling capacity has been investigated in severe deficiency of
both young and mature guinea pigs (49). The guinea pig is a good model for study
since, like humans, they lack the ability to synthesize ascorbic acid and hence
require it in the diet. Ascorbate deficiency resulted in significantly increased recy-
cling capacity of erythrocytes in young but not mature animals, whereas liver
homogenate ascorbate recycling was unaffected by age or diet (49). Consistent
with this data, a recent human study of ascorbate recycling in cigarette smokers
has demonstrated that smoking-induced oxidative stress enhances ascorbate recy-
cling in erythrocytes (50). Taken together these two studies indicate an adaptive
mechanism involving induction of DHA reductases during de novo erythrocyte
synthesis, in response to oxidative stress (50).
Ascorbate Recycling and Metabolism

The recycling of ascorbate utilizes reducing equivalents in the form of NAD(P)H.
This occurs either directly, via NAD(P)H-dependent reductases, or indirectly via
264 Lodge
GSH-dependent reduction which requires glutathione reductase to recycle the

oxidized GSH (GSSG), a reaction which requires NADPH. Ascorbate recycling
therefore decreases the intracellular concentration of NAD(P)H and GSH (22),
thereby directly influencing intracellular redox status. A consequence of ascor-
bate recycling is stimulation of the pentose phosphate pathway (22,51). This
pathway provides NADPH which is required for synthetic pathways, and main-
tains GSH concentrations via GSH reductase. Modulation of the pentose phosphate
pathway has been demonstrated in various mammalian cell lines (22,51 –53). In
Jurkat and human (H9) T-lymphocytes, DHA, both dose-dependently and time-
dependently, increased the activity of the pentose phosphate pathway enzymes
glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogen-
ase and transaldolase (51). An increase in G6PD protein levels was also observed,
indicating a stimulation of gene expression by DHA (51). At the same time, DHA
treatment (800 mM for 48 h) increased intracellular GSH levels by over 3-fold
(51) and is thus equal, if not more, potent than N-acetyl cysteine in increasing
intracellular GSH concentrations. Breakdown products of DHA include the
five carbon sugars L -lyxose and L -xylose, which can enter the pentose phosphate
pathway (54), thus providing a mechanism for both increased enzyme activity
and GSH levels induced by DHA. In ascorbate synthesizing tissues, ascorbate
has been shown to be catabolized and recycled through pentose phosphate and
gluconeogenic pathways (52). Ascorbate metabolism was investigated further
in cells that cannot synthesis ascorbate. Human erythrocytes were shown to effec-
tively metabolize ascorbate to lactate, with DHA being the better substrate (53);
whereas in human HepG2 cells, the end product of ascorbate metabolism was
glucose instead of lactate, again with DHA being the better substrate (53). The
authors suggest that in species that are unable to synthesize ascorbate there
exists a catabolic side to an inter-organ ascorbate cycle (53). Experiments in
growing cartilage cells demonstrated that ascorbate could maintain the energy
status of the cells (55). In ascorbate treated cells, there was a decreased rate of
lactate formation, coupled with a maintenance of mitochondrial energy gener-
ation (measured by the adenylate energy charge ratio) and isocitrate dehydrogen-
ase (NAD-dependent) activity (55). The authors suggest that ascorbate could
inhibit the utilization of pyruvate by anaerobic glycolysis, so promoting mito-
chondrial oxidative reactions (55).
Because ascorbate recycling can deplete glutathione, it has been suggested
that this could generate oxidative stress. This has been studied in several systems
(56 – 59). Ascorbate/DHA treatment depleted GSH and increased lipid per-
oxidation in brain slices (56), neuronal cells (57), pancreatic tissue (59), and
liver slices (58). These effects were prevented by inhibitors of GLUT transport
(57 – 59), implying that the uptake and recycling of DHA induces oxidative
stress. The dependence of ascorbate recycling on provision of reducing equiva-
lents via metabolic pathways was shown in experiments with human erythrocytes
in the presence or absence of glucose (22). In the presence of glucose, DHA
reduction was enhanced and resulted in oxidation of NADPH and activation of
the pentose phosphate pathway at the G6PD step, whereas GSH and NADH
levels were maintained (22). In the absence of glucose, although DHA reduction
still occurred, this was associated with a decrease in GSH and NADH levels (22).
This implies that in normal metabolizing cells with sufficient metabolic substrate,
reduction of DHA occurs via a NADPH- and GSH-dependent cycle, but when
metabolism is limited, DHA reduction is switched to a different mechanism
involving NADH which does not involve the pentose phosphate pathway.
MOLECULAR FUNCTIONS OF ASCORBIC ACID

Ascorbate Modulation of Collagen Formation
Maturation of certain mesenchymal tissues including bone and cartilage involves
both the synthesis and secretion of collagen forming an extracellular matrix. One
of the cellular functions of ascorbate involves the hydroxylation of proline and
lysine residues in procollagen. Without this ascorbate-requiring hydroxylation,
collagen fibrils cannot bind and form their triple helix structure and thus extra-
cellular matrix formation is impaired. In addition to these posttranslation
effects, it is well established that ascorbate can also modify collagen production
via pretranslational events.
An ascorbate-induced increase in collagen gene expression has been
demonstrated in a variety of cell lines including primary avian tendon cells
(60), human (61) and porcine (62) skin fibroblasts, human (63) and porcine (62)
smooth muscle cells, bovine chondrocytes (64), and even human dermis (65). On
the mechanism, studies have demonstrated that ascorbate induces an increase in
the rate-constant for procollagen translation and secretion (66) and this involved
an enhancement of procollagen gene transcription, accompanied by increased
stability of procollagen mRNA from a half-life of 10 to 20 h (67). A stabilization
of type I collagen mRNA was also the mechanism of ascorbate-induced collagen
expression in porcine smooth muscle cells and fibroblasts (62). Concentrations of
ascorbate used typically range from 3 to 300 mM, with effects demonstrated at
concentrations as low as 50 mM, and this typically induces a 2 –3-fold increase
in levels of procollagen mRNA. Although induction of type I collagen is fre-
quently associated with ascorbate, other forms of collagen have also been inves-
tigated. In human skin fibroblasts, types I and II procollagen mRNA are induced
by ascorbate (68). In bovine smooth muscle cells, type VI collagen is produced in
the absence of ascorbate, but the addition of ascorbate induces type I, decreases
type VI, with no change in the levels of type III (69). However, in porcine smooth
muscle cells, types I and III are induced by ascorbate (62). In chondrocytes type
X collagen (70) and types I, II, and X collagen (64) have been found to be induced
by ascorbate. Thus, ascorbate can modulate collagen synthesis not only by
hydroxylation of procollagen, but also by increasing both the level and
stability of procollagen mRNA. The ability of ascorbate to increase stability of
mRNA has been investigated independently. It was found that ascorbate
266 Lodge
(at ascorbate:RNA ratios .1:40) was able to interact directly with both G –C
and A –U base pairs of RNA via H-bonding through its anion CO and OH
groups (71), providing a potential mechanism of this enhanced stabilization
effect.
The induction of collagen mRNA by ascorbate appears to occur following a
lag period, which appears to depend on the cell type and conditions, but can be
from a few hours in L5 skeletal muscle cells (72), to 12 h in avian tendon cells
(66) to 1 or 2 days in chondrocytes (70). Such a response is indicative of prior
events which are required for transcription. One of these effects may involve
the production of lipid peroxides, as ascorbate was found to induce lipid per-
oxides in fibroblasts (73) and this step was suggested to be necessary for stimu-
lation of collagen gene expression (73). However, one study has demonstrated
that the ascorbate-induced generation of lipid peroxides and collagen are coin-
cidental, since in the presence of iron chelators lipid peroxidation was blocked
but collagen synthesis was unaffected (74). Also, multiple dosing is often
required for these ascorbate-induced effects (62,70), presumably to circumvent
the depletion of ascorbate in the culture medium. The use of stable ascorbate
analogs may aid in such studies. In preliminary studies reported by Davidson
et al. (62), multiple dosing and long exposure times were not necessary when
using ascorbate 2-phosphate. The augmentation of procollagen mRNA by ascor-
bate and the stable analog palmitoyl ascorbate was compared in human intestinal
smooth muscle cells (63). The increase in both procollagen mRNA and collagen
synthesis was more efficient and sensitive with palmitoyl ascorbate; however, the
response was similar with both species at higher concentrations (63). It has been
suggested that the requirement for longer incubation times with lower doses of
ascorbate may be related to the oxidation of ascorbate in the culture medium
(62). However, it is uncertain if it is related to the induction of cellular lipid
peroxidation, which can potentially influence gene expression.
In opposite to the effect of ascorbate on collagen formation, studies have
shown that ascorbate exerts a negative effect on elastin production (62,75 – 77).
When both collagen and elastin production were compared together in vascular
smooth muscle cells and skin fibroblasts, the differential effects of ascorbate
were found to result from a marked stabilization of collagen mRNA over
the lesser stabilization of elastin mRNA and the repression of elastin gene
transcription (62).
Collagen degradation is a symptom of aging. Both aging and UV irradi-
ation can induce levels of matrix metalloproteinases (MMP) which degrade
collagen and elastin. In addition to regulating type I and III collagen gene
transcription, ascorbate has been shown to downregulate MMP-2 in cultured
human amnion-derived cells (78), and reducing activity and expression of
MMP-1 and 2 in UV irradiated human keratinocytes (79). Conversely, ascorbate
was shown to induce expression of MMP-1 in human periodontal ligament cells
but not in MC3T3-E1 osteoblast-like cells (80), whereas MMP-13 expression
was upregulated during growth of osteoblastic MC3T3-E1 cells in the presence
of ascorbate 2-phosphate (81). Although human skin produces both MMP-1 and
MMP-8 following UV irradiation, it appears that MMP-1 may play the most
important role in collagen degradation (82).
Ascorbate concentration has shown to be lower in the epidermis and dermis
of photoaged skin (83) and when exposed to UV irradiation, levels of ascorbate
(as well as the other antioxidants a-tocopherol and glutathione) are depleted in
the skin of humans in vivo (84). These data coupled to the study of Nusgens
et al. (65) who demonstrated that topically applied ascorbate enhanced mRNA
levels of collagen types I and III as well as the mRNA of the MMP-1 inhibitor
TIMP-1 in human dermis (65) indicate that ascorbate treatment can potentially
delay the skin aging process.
Ascorbate Modulation of Cell Differentiation

Ascorbate is required for normal cell growth and differentiation. Through its pre-
and posttranslational modulation of collagen formation, ascorbate is important
for growth of mesenchyme-derived connective tissues such as muscle, cartilage,
and bone (85). The mechanisms of ascorbate-induced differentiation of collagen-
secreting cells appear to depend on the cell type. In osteoblasts, the effect of
ascorbate is dependent on its collagen-inducing properties (85 – 88); whereas in
chondrocytes, ascorbate modulation of gene expression is independent of col-
lagen secretion (70). In muscle cell cultures, ascorbate treatment also appears
to induce acetylcholine receptor (AchR) expression (72,89 –91). An investigation
into this effect revealed that it was a specific action on the a subunit of AchR and
involves mRNA synthesis (91). Induction of AchR expression occurred with a lag
of 24 h and followed collagen synthesis. Although collagen secretion is also
induced by ascorbate under these conditions, the process of AchR expression
and collagen secretion induced by ascorbate were found to be independent pro-
cesses (72,89). Cell maturation in mineralizing tissues, such as cartilage and
bone, is linked to the activity of the enzyme alkaline phosphatase. The activity
of this enzyme has been shown to be increased during the ascorbate-induced
differentiation of osteoblasts (87,88), chondrocytes (55,92), and ST2 stromal
cells (93). This effect also follows a lag period, but the increase in activity is
quite dramatic, being over 10-fold higher in chondrocyte cell lines (70,92). In
human fibroblasts, ascorbate induced alkaline phosphatase activity, but this
was dependent on the presence of an extracellular matrix of fibronectin (94).
Collagen matrix formation was also found to be essential for the inductive
effects of ascorbate in the differentiation of stromal cells, which included
increased alkaline phosphatase activity (93).
Ascorbate also appears to be important for growth and differentiation of
noncollagen-secreting cells, such as monocytes (95), and keratinocytes (96). It
has also been established that ascorbate can stimulate the differentiation of
leukemic cells induced by 1a,25-dihydroxyvitamin D3 (97 –100). The ascorbate
2-phosphate induced differentiation of keratinocytes was found to be mediated by
268 Lodge
protein kinase C (PKC), causing activation of PKC by translocating it from the

cytosol to the membrane (96). This PKC activation then led to activation of acti-
vator protein (AP)-1 (96) which mediates expression of genes associated with cell
growth and development. A further interesting observation in this study was that
ascorbate treatment also caused the upregulation of the ascorbate transporters
SVCT1 and SCVC2, which in turn resulted in accumulation of intracellular
ascorbate (96). The involvement of AP-1 has also been demonstrated in the ascor-
bate stimulation of 1a,25-dihydroxyvitamin D3-induced monocyte differen-
tiation (98). In these experiments, ascorbate increased AP-1 DNA binding in
non-induced cells, but inhibited DNA binding in induced cells even though
mRNA levels of AP-1 constituents (c-jun, junB, c-fos) were increased (98).
The modulation of transcription factors by ascorbate is discussed in the following
section. The influence of ascorbate on 1a,25-dihydroxyvitamin D3-induced cell
differentiation were caused by addition of ascorbate, but not of DHA or the
more stable analog ascorbate 2-phosphate, implying perhaps that oxidation of
ascorbate in the medium is a major contributing factor to the effects in such
studies. Indeed, in a study in human leukemic cells, AFR, but not DHA or the
ascorbate analog isoascorbate, increased cell growth (95). This suggests that
ascorbate oxidation and induction of lipid peroxidation are prerequisite for
these ascorbate-mediated effects, as in that proposed in collagen synthesis in
the previous section, and may therefore be an artifact of the conditions.
Modulation of Transcription Factors

Ascorbate has been shown to modulate the activities of the nuclear transcription
factors nuclear factor-kappa B (NF-kB), and AP-1. Both these factors are redox
sensitive and can be modulated by antioxidants (101,102).
NF-kB is involved in the regulation of many genes associated with the
inflammatory response. In the dormant form, NF-kB, homo- or heterodimers
of constituents of the Rel family, is bound to I-kB in the cytosol, but upon
activation, I-kB is phosphorylated leading to degradation and the release of
NF-kB, which translocates to the nucleus for DNA binding. Modulation of
NF-kB activation by ascorbate appears to be dependent on the conditions used.
In Jurkat T-cells, ascorbate (0.2 mM) potentiated the activation of NF-kB
induced by TNF-a (103). This was evidenced by increased DNA binding of
NF-kB, and enhanced I-kB degradation (103). In LPS and IFN-g activated
murine macrophages, ascorbate did not induce I-kB-a degradation or NF-kB
activation but did prolong the recovery of I-kB, which led indirectly to an
enhancement of NF-kB DNA binding (104). NF-kB activation can be induced
by UVA irradiation, and in HaCaT keratinocytes, ascorbate (1 mM) was found
to potentiate this UVA-induced NF-kB nuclear binding activity (105). Shang
et al. investigated the effects of both UVA irradiation and ascorbate on NF-kB
activation in melanocytes and keratinocytes and found contrasting results.
Although ascorbate (0.1, 0.25 mM) increased NF-kB binding activity in
HaCaT keratinocytes, ascorbate decreased NF-kB binding in melanocytes (106).

Carcamo et al. found that ascorbate inhibited TNF-a-induced activation of
NF-kB in the human cell lines HeLa, U937 monocytes, HL-60 myeloid leukemic,
breast MCF7 and HUVEC primary endothelial cells (107). Both NF-kB nuclear
translocation and I-kB phosphorylation were decreased in ascorbate treated cells
(107) and the authors postulated a mechanism whereby ascorbate inhibited the
TNF-a-induced activation of NF-kB-inducing kinase (NIK) and I-kB kinases
(IKK), independent of p38 MAP kinase (107). Bowie et al. also found that ascor-
bate (5 – 20 mM) inhibited the TNF-a- or IL-1-induced activation of NF-kB in
two endothelial cell lines (primary HUVEC and ECV304) (108). No effect on
DNA binding of NF-kB was found but ascorbate was found to inhibit phos-
phorylation (and therefore degradation) of I-kB through a sustained activation
of p38 MAP kinase (108). The p38 MAP kinase mediated this effect by inhibition
of IKK (108). It appears then that p38 has a dual role depending on the kinetics of
its activation; whereas p38 has a positive regulatory role at the transcriptional
level, a rapid and sustained activation can inhibit upstream I-kB degradation
and override the positive effect (108). The mechanisms of ascorbate-induced
inhibition of NF-kB activation are summarized in Fig. 12.3. Ascorbate has also
been shown to modulate NF-kB activation in an animal model of atherosclerosis
(109). Antioxidant supplementation (vitamin E 100 IU/kg plus 1 g ascorbic acid
per day) in hypercholesterolemic pigs normalized NF-kB activation and pre-
served endothelial function (109). In contrast to these reports, ascorbate appeared
to have no effect on NF-kB activity in HIV infected ACH-2 T cells (110), in
TNF-a activated human aortic endothelial cells (111), in LPS or IFN-g activated
rat skeletal muscle endothelial cells (112), and in TNF-a activated endothelial
cells (113). Downstream products of NF-kB activation have also been investi-
gated. In a cell culture model, ascorbate downregulated IL-1a mRNA expression
in both UVA irradiated and non-irradiated cells, whereas IL-6 mRNA expression
was unaffected (114). In an animal model, B and T lymphocytes isolated from
pigs with hereditary vitamin C deficiency with and without ascorbate supple-
mentation, showed decreased proliferative responses in the depleted group and
differential production of IL-2 and IL-6 (115).
AP-1 is involved in the expression of genes involved in cellular prolifer-
ation and the cell cycle. AP-1 is composed of a mixture of heterodimeric
protein complexes derived from the Fos (c-Fos, FosB, Fra-1, Fra-2) and Jun
(c-Jun, JunB, and JunD) families. Phosphorylation of these proteins by various
kinases, for example, Jun N-terminal Kinase (JNK), is required for transactiva-
tion activity. In a macrophage, cell line ascorbate was found to potentiate
PMA-induced AP-1 nuclear binding (116), and this involved the amplification
of p38 MAP kinase and JNK induction, and thus the authors proposed that ascor-
bate induces these effects via modulation of critical thiols in the MAP kinase
pathway (116). Opposing results were found by Kyaw et al. (117) who demon-
strated ascorbate inhibition of endothelin-induced AP-1 DNA binding activity
in rat aortic smooth muscle cells. This mechanism involved the inhibition of
270 Lodge
Figure 12.3 Representation of the ascorbate-induced inhibition of nuclear transcription

factor DNA binding. Ascorbate treatment has been shown to inhibit TNF-a and/or IL-1
induced activation of NF-kB in endothelial cells by an inhibitory effect on I-kB
phosphorylation either by inhibiting I-kB kinase directly (107) or mediated through an
activation of p38 MAP kinase (108). Ascorbate 2-phosphate treatment of keratinocytes
inhibited AP-1 activation by inhibiting the JNK-mediated phosphorylation of c-Jun
(preventing stable AP-1 binding complexes) and by inducing fra-1 which is a negative
regulator of AP-1. [This figure reproduced with permission from Catani et al. (118).]
JNK and p38 MAP kinase activation (117). Another investigation found that
ascorbate 2-phosphate can interfere with the activity of the JNK and AP-1
pathway in HaCaT keratinocytes (118). In this case, ascorbate 2-phosphate
modulation of AP-1 occurred via dual mechanisms. First, ascorbate 2-phosphate
was able to inhibit the phosphorylation (and hence activation) of JNK, which in
turn prevents phosphorylation of c-Jun (118), preventing the formation of stable
AP-1 transcription factor complexes. Secondly, ascorbate 2-phosphate caused the
induction of fra-1, which is a negative regulator of AP-1, such that ascorbate was
able to inhibit basal and UVB-induced AP-1 activity (118). These pathways are
shown in Fig. 12.3. A dual effect of ascorbate on AP-1 binding was demonstrated
in HL-60 cells (98). In non-induced cells, ascorbate treatment increased AP-1
DNA binding; however, if the cells were induced to differentiate with
1a,25-dihydroxyvitamin D3 , ascorbate treatment then inhibited AP-1 DNA
binding, even though ascorbate treatment increased mRNA levels of c-jun,
junB, and c-fos (98), which aid in the formation of stable AP-1 binding
complexes.
The modulation of NF-kB and AP-1 by ascorbate appears to be a matter for

controversy since both inhibition and potentiation of these transcription factors
has been demonstrated. A critical factor in these observations could be the con-
ditions used, and especially the cell line, mode of activation, and concentration of
ascorbate. For example, relatively low concentrations of ascorbate resulted in
potentiation of NF-kB activation, whereas millimolar concentrations result in
inhibition of activation. This in itself implies multiple mechanisms of ascorbate
modulation at various levels of cell signaling. It is interesting that both AP-1 and
NF-kB modulation by ascorbate do share a common mechanism in the blunting
of phosphorylation reactions by kinases. It is likely that ascorbate influences criti-
cal thiols in these kinases, as previously suggested (116). However, further work
is required to characterize this effect.
In regard to other transcription factors, ascorbate treatment was shown to
attenuate activation of the transcription factor IRF-1, which is involved in
iNOS expression, in endothelial cells (112), and had no inhibitory effect on
IFN-g-induced STAT1 DNA binding in endothelial cells (108).
Ascorbate Modulation of Nitric Oxide Production

Antioxidants such as ascorbate and vitamin E are known to protect and enhance
NO levels (119,120). This forms the basis of studies demonstrating an improve-
ment in endothelium-dependent dilation in patients with CHD (121) and essential
hypertension (122) following acute ascorbate supplementation. Various mechan-
isms have been postulated for the ascorbate prevention of endothelium dysfunc-
tion (123). In murine macrophages activated with LPS and IFN-g, a 40% increase
in NOx production was induced by ascorbate (124). The authors demonstrated
that this effect was due to an 2-fold increase in iNOS mRNA and protein
levels in cells in the presence of ascorbate (124). These studies were extended
by the same group who investigated the potential mechanism of ascorbate-
induced iNOS expression (104). Ascorbate was found to prolong the recovery
of I-kB which paralleled the elevated NF-kB binding to DNA (104). In contrast
to these effects, ascorbate was found to inhibit iNOS expression in rat skeletal
muscle endothelial cells (125) and septic rat skeletal muscle (112). Pretreatment
of endothelial cells with ascorbate inhibited the induction of iNOS protein and
NOx production induced by LPS and IFN-g (125). This was suggested to be an
antioxidant effect via blunting of superoxide and did not involve the NF-kB
pathway (125). Another interesting mechanism involves the ascorbate effect on
tetrahydrobiopterin (BH4) which acts as a cofactor for eNOS. In porcine aortic
endothelial cells, NO production was enhanced by ascorbate by up to 70%, con-
sistent with a stimulation of the catalytic activity (Vmax) of bovine eNOS by up to
50% (126). This effect was mediated through an increase in intracellular BH4
(126). The mechanism for this ascorbate action on BH4 in human umbilical
vein endothelial cells was found to involve chemical stabilization (127) since
ascorbate increased the half-life of BH4 and decreased biopterin oxidation
272 Lodge
products (127). Thus, the action of ascorbate on eNOS can be seen to be similar to
that with its di-oxygenase activity, in maintaining adequate levels of cofactor.
Ascorbate-Induced Modulation of DNA Repair

Several reports have demonstrated an ascorbate-induced modulation of pathways
involved in either DNA repair or apoptosis. Catani et al demonstrated that ascor-
bate 2-phosphate treatment of HaCaT cells resulted in up-regulation of Mut L
homolog-1 (MLH1) expression in spite of UVB irradiation (118) and went on
to investigate this regulation further (128). MLH1 allows the removal of mis-
matches which can occur during DNA replication and is thus part of the
DNA repair machinery of the cell. The MLH1 signal transduction pathway
involves c-Abl and p73 as downstream targets and is a critical factor in whether
the cells undergo either DNA mutation or apoptosis. Ascorbate-induced
up-regulation of MLH1 occurred following just 2 h incubation with ascorbate
2-phosphate (1 mM) and also resulted in the up-regulation of p73 gene
expression (128). The authors concluded that ascorbate can improve the cellular
response to DNA damage, and this was demonstrated by ascorbate increasing
cisplatin-induced apoptosis (128). The authors also suggest that both the anti-
carcinogenic and anti-cancer activities of ascorbate might be explained by this
modulation of MLH1 gene expression (128). An ascorbate-induced potentiation
of Fas-mediated apoptosis has also been demonstrated in Jurkat and human T
cells (129). In this study, DHA was able to enhance cell-surface expression of
the Fas receptor (129). However, a recent study has shown an opposite effect.
DHA treatment of U937 monocytes or fresh primary human monocytes resulted
in inhibition of Fas-mediated apoptosis (130). This effect was associated with
an inhibition of caspase-8 activation (130). A role for ascorbate in the regulation
of DNA repair enzymes has been previously postulated (131), as significant
decreases in specific DNA oxidation products (8-oxodG) of mononuclear cells
were found following ascorbate supplementation in humans (131). Taken
together these reports demonstrate that ascorbate can protect against oxidative
damage both as an antioxidant and as a regulator of DNA repair.
Ascorbate-Induced Modulation of Other Genes

In a search for ascorbate-regulated genes, Mizutani et al. (132) demonstrated that
ubiquitin mRNA levels in guinea pig tissues decreased under ascorbate-deficient
conditions and increased under ascorbate-replete conditions. However, the same
group failed to demonstrated any ascorbate-induced gene expression of ubiquitin
in activated macrophages (104). Ascorbate deficiency in guinea pigs also led to a
decreased expression of mRNA for the cytochrome P450 variants CYP1A1 and
CYP1A2 (133), leading to the authors to suggest that P450 transcription is regu-
lated by ascorbate in guinea pigs (133). Using cDNA microarray technology,
Catani et al. demonstrated that ascorbate 2-phosphate treatment of HaCaT cells
resulted in up-regulation of fra-1, glutathione S-transferase Pi (GSTpi) and
MLH1 expression inspite of UVB irradiation (118). This latter effect was
described in the preceding section. Ascorbate treatment of epithelial cells has
also been shown to increase mRNA for ferritin by 30% (134), consistent with
a close relationship between ascorbate and iron status.
CONCLUSION
Ascorbic acid is an essential micronutrient required for a number of cellular func-
tions. The importance of maintaining intracellular ascorbate is demonstrated in
the variety of potential mechanisms available to recycle ascorbate from its one
and two electron oxidation products AFR and DHA. Ascorbate recycling depletes
NAD(P)H and GSH, and in this way ascorbate can influence metabolic pathways.
For example, the pentose phosphate pathway is stimulated, which supplies
NADPH. It is becoming increasingly evident that ascorbate can also modulate
signaling pathways and influence gene expression. Such effects include increas-
ing collagen expression by stabilization of its mRNA, influencing cell maturation
and differentiation, modulation of the transcription factors NF-kB and AP-1,
and modulating NO formation. Although some of these actions are not well
characterized and may be highly dependent on the conditions used, it is clear
that ascorbate is more than just a reducing agent.
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13
Cell Signaling Properties of a-Lipoic Acid:
Implications in Type 2 Diabetes
Hadi Moini and Lester Packer

University of Southern California, Los Angeles, California, USA
Kyung-Joo Cho and An-Sik Chung
Korea Advanced Institute of Science and Technology, Daejeon, South Korea
Introduction 283
Diabetes and Current Strategies for Its Treatment 284
Targeting the Insulin-Signaling Pathway 285
a-Lipoic Acid Improves Glucose Metabolism in Type 2 Diabetes 287
a-Lipoic Acid Activates the Insulin-Signaling Pathway 288
a-Lipoic Acid Regulates Adipocyte Differentiation 290
Concluding Remarks 293
References 295
INTRODUCTION
The prevalence of diabetes is on the rise in the world population. It is estimated
that 6.2% of the US population is affected by diabetes. The number of diabetics
is expected to continue to increase by 4– 5% per year, potentially reaching a total
of 220 million cases in 2010 worldwide. Therefore, effective interventions are
needed to prevent or cure diabetes and/or attenuate its symptoms and compli-
cations. a-Lipoic acid is a disulfide derivative of octanoic acid that forms an
283
284 Moini et al.
intramolecular disulfide bond in its oxidized form (Fig. 13.1). High

electron density resulting from special position of the two sulfur atoms in the
1,2-dithiolane ring confers upon a-lipoic acid a high tendency for reduction
of other redox-sensitive molecules according to environmental condition (1).
a-Lipoic acid has been long used in the treatment of diabetic neuropathy (2).
Recent advances in our understanding of a-lipoic acid actions in muscle and
fat cells in vitro and in vivo established that a-lipoic acid enhances glucose
disposal and attenuates adipogenesis. These actions of a-lipoic acid are mediated
by an array of signaling molecules and transcription factors that now have
become attractive targets for design of new pharmacological agents to treat
diabetes. Recent findings suggest that a-lipoic acid may emerge as an effective
anti-diabetic agent that can be used as an adjunctive treatment in type 2 diabetes.
DIABETES AND CURRENT STRATEGIES FOR ITS TREATMENT

Diabetes is of two types. Type 1 diabetes is a multifactorial autoimmune disease,
which is characterized by T-cell-mediated destruction of the insulin secreting
b cells of the Langerhans islets in the pancreas. The destructive process leads
to severe insulin depletion, which results in hyperglycemia, owing to hepatic
overproduction of glucose and decreased cellular uptake of glucose from the
circulation (3). The pathogenesis of type 2 diabetes, however, is not fully under-
stood. The transition from normal glucose tolerance to type 2 diabetes in suscep-
tible individuals is punctuated by insulin resistance, dysregulated hepatic glucose
production, impaired glucose tolerance, and declining b cell function. Many
obese and insulin-resistant subjects escape diabetes by oversecreting insulin. It
is the failure of the pancreatic b cells to compensate for insulin resistance that
leads to hyperglycemia. Once type 2 diabetes has occurred, hyperglycemia
worsens the pre-existing b cell dysfunction. Approximately, 90 – 95% of individ-
uals diagnosed for diabetes have type 2 diabetes (4).
Type 1 and type 2 diabetes are associated with increased risk for develop-
ment of cardiovascular disease, neuropathy, retinopathy, and nephropathy.
Type 1 diabetes requires life-long treatment with exogenous insulin for survival.
The therapeutic goals in type 2 diabetes are attenuation of symptoms by normal-
izing fasting and postprandial blood glucose levels and prevention of acute and
long-term complications. It is now clear that aggressive control of hyperglycemia
in patients with type 2 diabetes can reduce the development of chronic compli-
cations such as retinopathy and nephropathy. Hence, current strategies for
(a) COOH
(b)
COOH
S S SH SH
Figure 13.1 Molecular structure of a-lipoic acid (a) and dihydrolipoic acid (b).
Cell Signaling Properties of a-Lipoic Acid 285
treatment of type 2 diabetes rely mainly on several approaches aimed to decrease

the hyperglycemia itself. These approaches include (i) increasing insulin release
from pancreatic islets by sulfonylureas, (ii) attenuating hepatic glucose pro-
duction by metformin, (iii) enhancing sensitivity of hepatic and peripheral
tissues to insulin by peroxisome proliferators-activated receptor-g (PPARg)
agonists (thiazolidinediones), (iv) interfering with gut glucose absorption by
a-glucosidase inhibitors, and (v) suppressing glucose production and augmenting
glucose utilization by administration of exogenous insulin (3,4).
TARGETING THE INSULIN-SIGNALING PATHWAY

Current therapeutic approaches were largely developed in the absence of a solid
understanding of the pathogenesis of type 2 diabetes. However, with the expan-
sion of our understanding of biochemical pathways involved in the development
of type 2 diabetes, several mechanistic sites, such as the insulin-signaling
pathway (Fig. 13.2), are now considered as a target for new therapeutic interven-
tions. Defects such as decreased tyrosine phosphorylation of the insulin receptor
(IR) and insulin receptor substrate-1 or -2 (IRS-1/2), attenuated association of
phosphoinositol-3-kinase (PI3-K) with IRS-1/2, and diminished PI3-K activity
were repeatedly demonstrated in muscle, fat, or liver tissues from type 2 diabetic
patients, individuals with abnormal oral glucose tolerance, or animal models of
insulin resistance (7 –13). In contrast, activation of mitogen-activated protein
kinases (MAPK) signaling pathway by insulin was not affected in type 2 diabetic
patients (14). Hence, it is now clear that type 2 diabetic patients display dimi-
nished signaling in the PI3-K axis, which might be overcome by interventions
aimed at augmenting activation of the insulin-signaling pathway.
One such approach involves developing pharmacological agents that are
capable of inhibiting the negative regulator(s) of the insulin-signaling pathway
and thereby potentiating actions of insulin. Protein tyrosine phosphatases
(PTP) regulate signal transduction pathways involving tyrosine phosphorylation.
General inhibition of PTP by nonselective inhibitors such as vanadyl sulfate
improved hepatic and peripheral insulin sensitivity in type 2 diabetic patients
(15,16). A large body of evidence from cellular, biochemical, and genetic
studies have identified protein tyrosine phosphatase 1B (PTP1B) as the major
phosphatase responsible for dephosphorylation and inactivation of the IR (17).
Deletion of PTP1B gene markedly enhanced insulin sensitivity in mice (18).
Furthermore, treatment of insulin-resistant mice with PTP1B antisense oligonu-
cleotide increased activity of the insulin-signaling pathway in liver and fat cells,
lowered plasma glucose and insulin levels, and enhanced insulin sensitivity
(19,20). These findings established PTP1B as a potential therapeutic target and
raised much promise for use of its selective inhibitors in the treatment of
type 2 diabetes and obesity (21 –23).
The insulin-signaling pathway may be also negatively regulated at the level
of IRS-1. Phosphorylation of IRS-1 on serine/threonine residues impairs the
286 Moini et al.
Figure 13.2 Signaling pathways of insulin. The IR is a tyrosine kinase that undergoes
autophosphorylation and catalyzes the phosphorylation of cellular proteins such as
members of IRS family, Shc, and Cbl. Upon tyrosine phosphorylation, these proteins inter-
act with signaling molecules through their Src homology 2 (SH2) domain, resulting in acti-
vation of diverse series of signaling pathways. Engagement of tyrosine-phosphorylated
IRS with the SH2 domains in the p85 regulatory subunit of type I phosphatidylinositol
3-kinase (PI 3-K) activates the catalytic p110 subunit, which catalyzes the phosphorylation
of PI 4,5-diphosphate on the 3 position, generating PI 3,4,5-triphosphate. PI 3,4,5-
triphosphate serves as an allosteric regulator of the phosphoinositide-dependent kinase
(PDK), which phosphorylate and activate protein kinase B (Akt) as well as the atypical
protein kinase C isoforms, PKCj and PKCl. Although PI 3-K activation was shown to
be necessary for insulin-stimulated glucose uptake, the precise identity of the physiologi-
cally relevant kinase that triggers translocation of glucose transporter-4 (GLUT4) contain-
ing vesicles to plasma membrane is unclear. However, in addition to PI3-K activity other
signals seem to be required for insulin-stimulated glucose uptake. This second pathway
appears to involve tyrosine phosphorylation of the Cbl proto-oncogene, which is in
complex with the adaptor protein CAP. Upon phosphorylation, the Cbl – CAP complex
translocates to lipid rafts domains in the plasma membrane and recruits the adaptor
protein CrkII through interaction of the SH2 domain of CrkII with phospho-Cbl. CrkII
forms a constitutive complex with guanyl nucleotide-exchange protein C3G, which acti-
vates the small GTP-binding protein TC10. Once activated, TC10 seems to provide a
second signal, in parallel with the activation of the PI3-K pathway, to the GLUT4 contain-
ing vesicles. Tyrosine phosphorylated Shc, however, interacts with the adaptor protein
Grb2 and recruits the son-of-sevenless exchange protein to the plasma membrane and
thereby activates Ras. Once activated, Ras initiates a cascade of serine phosphorylation,
which results in activation of Raf, MEK, and MAPK, such as JNK, ERK, and p38
MAPK. p38 MAPK were shown to be involved in enhancing of the intrinsic activity of
GLUT4 (5,6).
ability of IRS-1 to activate downstream PI3-K (24 – 26). Glycogen synthase

kinase-3 (GSK-3) and inhibitor kappaB kinase were demonstrated to serine phos-
phorylate IRS-1 and attenuate insulin signaling (27,28). Selective inhibition of
GSK-3 activity lowers blood glucose levels and augments insulin action in
insulin-resistant mice, whereas heterozygous deletion of inhibitor kappaB
kinase or its inhibition by salicylate prevents fat-induced insulin resistance in
rodents (29 – 32). Type-II SH2-domain-containing inositol 5-phosphatase
(SHIP2) dephosphorylates key phospholipids that are generated by insulin-
mediated PI3-K activation and hence negatively regulates the insulin-signaling
pathway (33). Heterozygous deletion of SHIP2 increased glucose tolerance and
insulin sensitivity, which was accompanied with an elevated recruitment of
glucose transporter-4 (GLUT4) and increased glycogen synthesis in mouse skel-
etal muscles (34). Thus, GSK-3, inhibitor kappaB kinase, and SHIP2 represent
potentially novel therapeutic targets for the treatment of type 2 diabetes.
An alternate approach to inhibiting the negative regulators would be iden-
tifying nonpeptide small molecules that can activate the insulin-signaling
pathway. Discovering mimetics of insulin has proven to be difficult. Through
extensive screening of over 50,000 mixtures of synthetic compounds and
natural products, a small molecule, L-783,281, was identified from a fungal
extract (Pseudomassaria sp.) that activated IR in vitro by altering conformation
of its kinase domain (35). Oral administration of L-783,281 lowered blood
glucose level, improved glucose tolerance, and suppressed elevated plasma
insulin levels in animal models of type 2 diabetes (35,36). a-Lipoic acid was
also demonstrated to mimic insulin action, activate IR, and enhance glucose
uptake into fat and muscle cells. A growing body of evidence suggests that
a-lipoic acid administration to type 2 diabetic patients may have potential thera-
peutic value in lowering elevated glucose levels and suppressing diabetic
complications.
a-LIPOIC ACID IMPROVES GLUCOSE METABOLISM IN

TYPE 2 DIABETES
a-Lipoic acid was first reported to enhance glucose utilization in isolated rat heart
and diaphragm (37,38). Subsequent studies demonstrated that chronic a-lipoic acid
administration increases GLUT4 protein level in muscle membranes, improves
insulin-stimulated 2-deoxyglucose uptake into isolated skeletal muscles, and
reduces blood glucose levels in animal models of diabetes (39–41). Furthermore,
a-lipoic acid in combination with exercise training additively increased insulin-
mediated glucose transport in isolated skeletal muscles, decreased the glucose–
insulin index, an indication of increased insulin sensitivity, and enhanced oral
glucose tolerance in insulin-resistant obese ( fa/fa) Zucker rats (42). This ben-
eficial interactive effect of a-lipoic acid and exercise training, however, was not
apparent in insulin-sensitive lean ( fa/2) Zucker rats (43). Oral or intravenous
administration of a-lipoic acid also increased insulin sensitivity in individuals
288 Moini et al.
with type 2 diabetes (44–46). Taken together, these studies show that a-lipoic acid
may enhance the capacity of insulin-stimulated glucose transport and utilization.
However, a-lipoic acid itself also enhanced glucose uptake into epitrochlearis
muscles isolated from both insulin-resistant obese ( fa/fa) and insulin-sensitive
lean ( fa/2) Zucker rats (47), suggesting that a-lipoic acid may mimic insulin
action on glucose transport and metabolism.
a-LIPOIC ACID ACTIVATES THE INSULIN-SIGNALING PATHWAY

Mechanistic studies conducted in L6 myotubes and 3T3-L1 adipocytes as a
model of muscle and fat cells in culture revealed that a-lipoic acid rapidly stimu-
lates tyrosine phosphorylation of IR and IRS-1, enhances PI3-K, Akt, and p38
MAPK activities, elevates GLUT4 content in the plasma membranes, and
increases glucose uptake into the cells (48 –50). These studies provided strong
evidence that stimulation of glucose uptake by a-lipoic acid is due to enhanced
translocation and intrinsic activity of GLUT4 through activation of Akt and p38
MAPK, respectively.
Subsequent studies investigated the basis for the ability of a-lipoic acid to
activate the elements of the insulin-signaling pathway. 3T3-L1 adipocytes were
found to have a low capacity to reduce a-lipoic acid to dihydrolipoic acid. Even
when added exogenously to the cell culture media, dihydrolipoic acid marginally
increased glucose uptake into the cells (51). However, a parallel trend was
observed between the intracellular oxidant levels, GSH levels, and glucose
uptake following incubation of cells with a-lipoic acid (Fig. 13.3). Glucose
uptake was increased by several folds reaching to the peak level at 6 h. Intracellu-
lar oxidant levels, measured as 20 ,70 -dichlorofluorescin fluorescence, was also
increased in parallel to the changes in the glucose uptake and simultaneously
reached to the peak level at 6 h. However, as GSH levels started to rise, the intra-
cellular oxidant levels and the glucose uptake declined (51). These findings
suggest that at the early time points (up to 6 h) a-lipoic acid increases glucose
uptake by changing the intracellular redox status toward an oxidizing condition,
whereas at later time points (.12 h) a-lipoic acid, by increasing GSH levels,
shifts the intracellular environment toward more reducing conditions, which
lowers the rate of glucose uptake.
Intracellular redox status is known to play an important role in the modu-
lation of insulin action. Treatment of IR-transfected Chinese hamster ovary cells
with antioxidants such as N-acetyl-cysteine or butylated hydroxyanisole inhibits
insulin responsiveness, whereas partial inhibition of glutathione metabolism,
which intracellularly induces a mild oxidative stress condition, stimulates IR
tyrosine phosphorylation in vitro (52). Moreover, oxidation of critical cysteine
residues in the IR b-subunit was found to result in an increase in its intrinsic tyro-
sine kinase activity, whereas low concentrations of dithiothreitol inactivated the
IR kinase, suggesting that the functional activity of the IR can be also modulated
by alteration of the redox state of cysteine residues present in IR b-subunit
6
Glucose uptake
5 GSH
compared to basal
∑ DCF fluorescence
Fold increase
0
0 6 12 18 24 30 36 42 48
-1
-2
Time (h)
Figure 13.3 Temporal kinetics of a-lipoic acid-induced changes in glucose uptake,

GSH levels, and oxidant levels in 3T3-L1 adipocytes. Glucose uptake, GSH levels, and
intracellular oxidant were determined following treatment of 3T3-L1 adipocytes with
250 mM a-lipoic acid for indicated times. Intracellular oxidants were detected by 20 ,70 -
dichlorofluorescin (DCF ) fluorescence.
(53,54). Furthermore, IR was demonstrated to couple, via Gai2 , to the NADPH-

dependent H2O2 generating system, which upon insulin stimulation produces
H2O2 in 3T3-L1 adipocytes (55). Insulin-dependent H2O2 production was associ-
ated with a decreased PTP activity (56) and was also found essential for the
activation of PI3-K (57). These findings suggest that redox signals are involved
in the regulation of both the early tyrosine phosphorylation cascade and the
downstream insulin-signaling events.
In parallel to the increase in the intracellular oxidant levels, a-lipoic acid
stimulated tyrosine phosphorylation of IR in vivo, which was accompanied by
a decrease in the thiol reactivity of IR b-subunit (Fig. 13.4) (58). a-Lipoic
acid also directly stimulated tyrosine phosphorylation of immunoprecipitated
IR in vitro (51). Furthermore, a-lipoic acid inhibited total PTP activity in vivo,
which was accompanied by a decrease in the thiol reactivity of PTP1B,
whereas N-acetyl-L -cysteine, a mild thiol antioxidant, increased total PTP
activity (Fig. 13.5) (58). The impact of inhibition of oxidant production on IR
tyrosine phosphorylation could not be assessed by using inhibitors of phagocyte
NADPH oxidase owing to poor characterization of the adipocyte NADPH
oxidase. However, these findings strongly suggest that a-lipoic acid directly or
indirectly by increasing intracellular oxidant levels may oxidize critical thiol
groups of IR b-subunit and PTPs and result in their activation or inactivation,
290 Moini et al.
Figure 13.4 a-Lipoic acid decreases thiol reactivity of the IR and enhances its tyrosine
phosphorylation. Free thiol (a) and phosphotyrosine (b) content of IR b-subunit was deter-
mined following treatment of 3T3-L1 adipocytes with a-lipoic acid (500 mM, 30 min) or
insulin (100 nM, 10 min).
respectively, and thereby enhance the early tyrosine phosphorylation cascade of

the insulin-signaling pathway (58).
a-LIPOIC ACID REGULATES ADIPOCYTE DIFFERENTIATION

Obesity is closely correlated with the prevalence of diabetes and cardiovascular
disease. Obesity is caused not only by hypertrophy of adipose tissue, but also by
adipose tissue hyperplasia, which triggers the transformation of preadipocytes
into adipocytes (59). Adipocyte differentiation is a complex process and is regu-
lated by several signaling pathway (60). The PI3-K pathway transduces the
proadipogenic effects of insulin by promoting activation of CCAAT element
binding protein (C/EBP) b and d and sterol response element binding protein
1. These transcription factors induce the expression and/or activity of PPARg,
a pivotal coordinator of adipocyte differentiation. Activated PPARg induces
exit from the cell cycle and in cooperation with C/EBPa stimulates the
expression of many metabolic genes such as GLUT4, lipoprotein lipase (LPL),
and adipocyte-specific fatty acid binding protein (aP2) (61,62), thus constituting
a functional lipogenic adipocyte. Besides these integral members of the
adipogenesis program, other transcription factors, such as AP-1 and cAMP-
responsive element binding protein (CREB), are also known to promote adipo-
genesis, whereas nuclear factor-kB (NF-kB) suppresses adipocyte differentiation
(63 – 65). In contrast to the PI3-K-signaling pathway, MAPK such as extracellular
Figure 13.5 a-Lipoic acid decreases thiol reactivity of PTPB1 and inactivates total
PTP activity. The level of thiol-biotinylated PTP1B (a) or total PTP activity (b) was
determined following treatment of 3T3-L1 adipocytes with a-lipoic acid, insulin,
N-acetyl-L -cysteine, or H2O2 .
signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) suppress the
process of adipocyte differentiation by phosphorylating and thereby attenuating
the transcriptional activity of PPARg (66,67).
3T3-L1 preadipocytes initiate their conversion to mature adipocytes 3 days
after addition of a hormonal cocktail consisting of insulin, dexamethasone, and
3-isobutyl-1-methylxanthine. By the day 6, the number of fully differentiated
adipocytes is increased by several folds. a-Lipoic acid displays a biphasic
effect on differentiation of preadipocytes to mature adipocytes (68). At low con-
centrations (100 mM) a-lipoic acid promotes whereas at higher concentrations
(250 mM) it attenuates the hormonal cocktail-, insulin-, or PPARg agonist
troglitazone-induced differentiation. The pro- and anti-adipogenic effects of
292 Moini et al.
Figure 13.6 a-Lipoic acid inhibits differentiation of preadipocytes induced by a hormo-

nal cocktail (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine) or insulin. 3T3-L1
preadipocytes were treated with 10 nM insulin or the hormonal cocktail for 3 days in the
absence or presence of indicated concentrations of a-lipoic acid. Cells were then main-
tained in cell culture media containing a-lipoic acid for additional 3 days in the
absence of insulin or the hormonal cocktail. (a) Morphological changes associated with
adipogenesis was photographed on the basis of staining cellular triglyceride deposition
by Oil Red O. (b) mRNA levels of LPL and aP2 were determined by northern blot analysis
and were expressed as fold increase compared with basal.
a-lipoic acid are accompanied with an increase or a decrease, respectively, in

proadipogenic proteins such as aP2 and LPL (Fig. 13.6). Analysis of transcription
factors involved in the process of adipocyte differentiation demonstrated that
a-lipoic acid diminished activities of proadipogenic transcription factors such
Figure 13.7 a-Lipoic acid modulates DNA-binding activities of pro- and anti-
adipogenic transcription factors in 3T3-L1 adipocytes. DNA-binding activities of
NF-kB, AP-1, CREB, C/EBP were analyzed by electrophoretic mobility shift assay fol-
lowing treatment of preadipocytes with 10 nM insulin (I) or the hormonal cocktail (C)
in the absence or presence of 500 mM a-lipoic acid for 2 h. Arrows indicate specific
binding of nuclear proteins to the labeled DNA.
as AP-1, C/EBP, CREB, and PPARg while enhancing the activity of the anti-
adipogenic transcription factor NF-kB (Fig. 13.7) (68).
a-Lipoic acid and insulin also display a differential effect on activation of
MAPK and PI3-K pathways in preadipocytes primarily due to the differences in
the potency and kinetics of the activation of these two pathways (Fig. 13.8). The
adipogenic hormone insulin strongly activated PI3-K pathway, which lasted up to
several hours but weakly and transiently activated MAPK-signaling pathway. In
contrast, a-lipoic acid weakly and transiently activated PI3-K pathway, whereas
more strongly activated MAPK-signaling pathway. Importantly, inhibitors of
ERK or JNK abolished the anti-adipogenic effect of a-lipoic acid on insulin-
or the hormonal cocktail-induced adipogenesis (68). These findings demonstrate
that insulin and a-lipoic acid oppositely regulate adipocyte differentiation and
that the MAPK-signaling pathway mediates actions of a-lipoic acid on adipo-
cytes differentiation by down- or up-regulating activities of the pro- or anti-
adipogenic transcription factors, respectively.
CONCLUDING REMARKS
In differentiated adipocytes, a-lipoic acid mimics insulin actions on glucose
uptake by modulating activities of several components of the insulin-signaling
pathway (Fig. 13.9). a-Lipoic acid may directly interact with IR b-subunit,
oxidize its critical thiol groups, and thereby facilitate its autophosphorylation.
Alternatively, a-lipoic acid may indirectly oxidize the cysteine residue of IR
b-subunit by increasing intracellular levels of oxidants. Whether a-lipoic acid
Figure 13.8 a-Lipoic acid and insulin differentially regulate PI3-K- and MAPK-
signaling pathways in preadipocytes.
294 Moini et al.
Figure 13.9 Molecular targets of a-lipoic acid in the insulin-signaling pathway.
stimulates adipocyte plasma membrane NADPH oxidase is not known. Neverthe-

less, the generated oxidants may also oxidize the cysteine residues of PTP such as
PTP1B, and inhibit its activity leading to enhancement of IR activation. Further-
more, a-lipoic acid enhances the intrinsic activity of GLUT4 by activating p38
MAPK. Hence, the short-term effect of a-lipoic acid in differentiated adipocytes
is the activation of proximal components of the insulin-signaling pathway and the
enhancement of glucose uptake. However, long-term incubation of adipocytes
with a-lipoic acid increases intracellular GSH, which scavenge H2O2 , and may
restore redox states of IR and PTP1B and thereby inactivate the insulin-signaling
pathway and attenuate glucose uptake.
In preadipocytes, a-lipoic acid and insulin differentially regulate PI3-K-
and MAPK-signaling pathways. In contrast to insulin, a-lipoic acid strongly
activates MAPK such as JNK and ERK. Activation of JNK and ERK leads to
opposite regulation of pro- and anti-adipogenic transcription factors and results
in diminished differentiation of preadipocytes. Hence, a-lipoic acid and insulin
exert opposite effects on adipocyte differentiation.
Decrease in the thiol reactivity of IR and PTPB1 and activation of MAPK
following a-lipoic acid treatment of adipocytes are all indicative of a shift in the
intracellular redox state toward an oxidizing condition. It is highly likely that
a-lipoic acid may directly or indirectly lead to oxidation of other thiol-containing
proteins such as Keap1. Thiol groups of Keap1 were recently demonstrated to
function as sensors that regulate induction of phase 2 enzymes (69). Oxidation
of critical thiol groups of Keap1 resulted in disruption of Keap1 – Nrf2
complex followed by migration of Nrf2 to nucleus where it forms heterodimers
with other transcription factors such as small Maf and binds to the antioxidant
response element regions of phase 2 genes and accelerates their transcription.
Induction of phase 2 enzymes is known to be an effective strategy to protect
cell against toxic effects of reactive oxygen species and many carcinogens by
detoxifying harmful molecules and augmenting cellular levels of antioxidants.
Whether the long-term antioxidant effect of a-lipoic acid is due to an initial
mild oxidative action that induces phase 2 enzymes is an intriguing possibility,
which remains to be investigated.
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14
Dietary Isoflavones and Coronary
Artery Disease—Proposed Molecular
Mechanisms of Action
Aedin Cassidy
University of East Anglia, Norwich, UK
Sonia De Pascual-Teresa
Instituto del Frı́o, Consejo Superior de Investigaciones Cientificas,
Madrid, Spain
Abstract 302
Introduction 302
Dietary Sources of Phytoestrogens 302
Absorption and Metabolism 303
Estrogen Receptor Mediated Mechanisms of Action 305
Anti-Oxidant Activity 306
Cardiovascular Effects 308
Lipid Metabolism 308
Animal Studies 308
Clinical Studies 309
Blood Pressure 310
Inflammation and Cell Adhesion 313
Platelet Aggregation and Endothelium Reactivity 317
Conclusion 318
References 319
301
302 Cassidy and De Pascual-Teresa
ABSTRACT
Experimental and epidemiological data are available to support the concept that
isoflavone-rich diets exert physiological effects in humans; however, to date most
research interest has focused on their potential hormonal activities. Dietary iso-
flavones are one of the major classes of phytoestrogens, and are currently receiv-
ing much attention because of their potential role in preventing coronary artery
and other chronic diseases. In the overall scheme of cardiovascular protection,
isoflavones appear to potentially have a more important role in conditioning
the vascular tree than on influencing cholesterol levels. The preferential
binding of isoflavones to the ERb and the increasing recognition of the role of
this receptor in the endothelial wall provide justification for increasing the aware-
ness of the heart-health effects of diets rich in these phytoestrogens. Furthermore,
such effects are not restricted to soy isoflavones but also apply to lignans and
many other flavonoid sub-classes, which are abundant in cereals, pulses, fruits,
and vegetables. Potential anti-atherogenic effects of isoflavones include a
reduction in LDL cholesterol, modulation of pro-inflammatory cytokines, cell
adhesion proteins, and nitric oxide (NO) formation, protection of LDL against
oxidation, inhibition of platelet aggregation, and an improvement in vascular
reactivity. However, further molecular work is required to elucidate the exact
mechanisms by which isoflavones affect these processes and to define the phys-
iological relevance of these mechanisms relative to human exposure from these
compounds. Although epidemiological data and laboratory studies allude to the
possible protective effects of soy isoflavones at specific target tissues, random-
ized placebo controlled clinical trials are necessary to further address the relative
importance of these compounds for cardiovascular health.
INTRODUCTION
Dietary phytoestrogens are a sub-class of flavonoids that are of particular interest in
relation to human health, which embody several groups of nonsteroidal estrogens
including isoflavones and lignans that are widely distributed within the plant
kingdom (1). Phytoestrogen-rich diets exert physiological effects, and preliminary
human studies suggest a potential role for dietary phytoestrogens in hormone-
dependent disease (2). In particular, a number of cardioprotective benefits have
been attributed to dietary isoflavones including a reduction in LDL cholesterol, an
inhibition of pro-inflammatory cytokine, cell adhesion protein, and nitric oxide
production, potential reduction in the susceptibility of the LDL particle to oxidation,
inhibition of platelet aggregation, and an improvement in vascular reactivity.
DIETARY SOURCES OF PHYTOESTROGENS

In relation to human health, research interest has to date concentrated on the
isoflavone and lignan subgroups of phytoestrogens. The isoflavones are the most
Dietary Isoflavones and Coronary Artery Disease 303
extensively studied of the phytoestrogen class; however, their occurrence in

foods is limited largely to soyabeans and a few other legumes (1). Lignans, in
contrast, are widely distributed but they have been relatively little studied due
in part to difficulties in their isolation and analysis (3,4).
The basic structural unit of the isoflavones comprises of two benzene rings,
linked via a heterocyclic pyrone ring. The chemical structures of commonly
occurring isoflavones are shown in Fig. 14.1.
Although nonsteroidal, it is the phenolic ring and in particular the 40 -OH
hydroxyl group of isoflavones that are the essential structural component for
interaction with estrogen receptors (ER). Although isoflavones have estrogenic
activity 100– 1000 times weaker than estradiol (1), some foods and dietary
supplements contain comparatively high amounts of these compounds so that
plasma levels may exceed endogenous estrogen levels by several orders of
magnitude and therefore these compounds have the potential to exert biological
effects in vivo. Daily dietary intake of isoflavones in western populations is
typically negligible (,1 mg/day), whereas recent estimates indicate intakes of
20 –50 mg/day in Japan, but this may vary between urban and rural areas, and
with other lifestyle factors (1).
Although all soyabean-derived protein extracts and foods available for
human consumption contain significant levels of isoflavones, there is a large
variability in concentration and profile among these products as factors such as
species, geographical, and environmental conditions, and the extent of industrial
processing of the soyabeans and all alter the levels of isoflavones present (5).
ABSORPTION AND METABOLISM

To date, 12 different isoflavone isomers have been identified. The primary isofla-
vones in soybeans are the glucosides, genistin, and daidzin and their respective
aglycones gensitein and daidzein. Typically, in soybeans and soy foods there
are higher gensitein levels than daidzein. Isoflavones are present in plants as
O O O
HO HO HO
A C
H3CO
O B OH O OH O
OH OH OH
Daidzein Genistein Glycitein
O O
HO HO
O OH O
OCH3 OCH3
Formononetin Biochanin A
Figure 14.1 A comparison of the chemical structures of isoflavones.

Small intestine Gut microflora
demethylation equol
malonylglucosides
daidzein dehydroxylation dehydrodaidzein
acetylglucosides Glucosidases
genistein reduction desmethylangolesin
b-glucosides
ring cleavage p-ethylphenol
Absorption
hepatic conjugation
Urinary excretion
(enterocyclic cycling)
Glucuronide conjugates
Sulphate conjugates
Liver
Figure 14.2 Absorption and metabolism isoflavones in humans.
glycoside conjugates, but following ingestion (Fig. 14.2) they are hydrolyzed by
intestinal glucosidases and the resulting aglycones may be absorbed or further
metabolized in the large gut to specific metabolites. Interest in gut metabolites
has increased in recent years, specifically in the daidzein metabolite, equol,
because of its stronger binding affinity to ERs and preliminary evidence to
suggest that it is a more potent modulator of hormonal status in healthy young
women (6). The role of the intestinal microflora in the metabolism of phytoestro-
gens has long been established (3,7) with early evidence showing that antibiotic
administration blocks metabolism and germ free animals do not excrete the
metabolites (7). However, to date it is still unclear what specific bacterial
species are responsible for the conversions.
Until recently, available data on the absorption and metabolism of dietary
phytoestrogens were of a qualitative nature; dietary phytoestrogens are meta-
bolized by intestinal bacteria, absorbed, conjugated in the liver, circulated in
plasma, and excreted in urine (Fig. 14.2). Recent studies have addressed quanti-
tatively what happens to isoflavones following ingestion—with pure compound
and stable isotope data to compliment recent pharmacokinetic data for soy
foods (8,9). Serum genistein and isoflavone levels increase in response to soy
or pure compound administration, although not always in a dose dependent
manner, and concentrations can readily reach the low micromolar level.
Plasma levels in free living Asian subjects are 500 nmol/L when measured
after an overnight fast, but because the half life of isoflavones is 6– 9 h, fasting
levels are much lower than postprandial levels. Isoflavones circulate in plasma
primarily in the conjugated form, mostly bound to glucuronic acid with 3%
circulating in the free form.
Knowledge of the pharmacokinetics of phytoestrogens is essential prior to
making recommendations regarding long-term efficacy in clinical studies, as
recent research suggests significant differences in bioavailability between foods

rich in phytoestrogens and supplements (8). In addition, dose administered,
food matrix, and the chemical form of the compound appear to exert effects on
the bioavailability (9). Maintenance of a steady state serum level should
be optimal for clinical effectiveness of these compounds and on the basis of
recent pharmacokinetic data, this would be best achieved by divided doses of
the soya food or supplement throughout the day, rather than by a single dose.
ESTROGEN RECEPTOR MEDIATED MECHANISMS OF ACTION

Isoflavones have a spatial configuration similar to that of mammalian estrogens,
bind to ERs and affect estrogen regulated gene products (10 –12).
The higher binding affinity of isoflavones for ERb, compared with ERa
and the different tissue distributions of these receptors (Fig. 14.3) suggest that
these compounds may be tissue selective, exerting estrogenic actions in some
tissues such as coronary vessels (13) but not in other tissues such as the endome-
trium (1,2,14). The estrogenic potency of isoflavones is low compared with
17-b-estradiol, with soy isoflavones having approximately one-third and
1/1000 of the affinity of 17-b-estradiol for the ERb and ERa, respectively
(15). Genistein has been shown to have a binding affinity for ERa and ERb of
4% and 87%, respectively, of estrogen, whereas daidzein is less potent having
affinities of 0.1% and 0.5%, respectively (15). Since genistein possesses an
order of 20 times higher binding affinity for ERb than for ERa, isoflavones
Activating stimuli:
Reactive Oxygen Species (ROS)
Cytokines
IkB
Inactive form
Inflamatory and inmune proteins

IkB kinase
IkB
Active form
Transcription
Translocation NF-kB
Target genes
Cytoplasm
Nucleus
Figure 14.3 Estrogen receptor mediated effects and tissue distribution.

can be regarded as a type of natural selective estrogen receptor modulator

(SERM) for this receptor. However, recent X-ray crystallographic studies exam-
ining the interaction of estrogens, raloxifene, and genistein with ERb suggest that
the orientation of raloxifene and genistein with ERb is different from that of
estradiol, in particular in the interaction with helix 12 of the receptor (1,8). Iso-
flavones lack specific lipophilic regions, which undoubtedly impact on their ERb
binding ability and the subsequent sequence in cellular events triggered by their
binding of the agonist. Therefore, isoflavones may be more correctly classified as
SERMs than “estrogens,” which suggests that isoflavones may possess the ben-
eficial physiological actions of natural estrogens, without the associated negative
effects, in particular in tissues such as the breast (8). Although the reported estro-
genic potency of isoflavones is weak, 100– 1000 times less compared with
17-b-estradiol, their biological potential cannot be ignored, as typical circulating
levels of isoflavones can exceed endogenous estradiol concentrations by 10,000-
fold following consumption of a diet containing soy foods (16,17). Genistein has
a 100-fold greater binding affinity than daidzein for the mouse uterine cytosolic
estrogen receptors. Furthermore, the formation of glucuronide conjugates
decreases the relative affinities of isoflavones to estrogen receptors (18).
However, binding affinity alone does not determine potency, because the result-
ing conformational change in the ligand (isoflavone – receptor complex varies
among ligand regardless of binding affinity (19). The isoflavone genistein is
also .1000-fold more potent at triggering transcriptional activity with ERb
than ERa (20) and this difference is far greater than the 30-fold greater
binding affinity for ERb than ERa (21). These data therefore suggest that the iso-
flavone genistein is a potent agonist for ERb and the divergent transcriptional
activities of estrogens and isoflavones results not only from their different
binding affinities but also from differences in their ability to recruit coregulators
and trigger transcriptional functions of ERa and ERb (20).
Anti-Oxidant Activity
Although there is significant interest in the anti-oxidant properties of isoflavones,
to date most of these investigations have focused solely on the anti-oxidant
effects of genistein (22). Proposed molecular mechanisms responsible for its
anti-oxidant potential include the ability to scavenge radicals, chelate metals,
inhibit hydrogen peroxide (H2O2) production, and stimulate anti-oxidant
enzymes, including catalase and superoxide dismutase.
In a liposomal system, it has been demonstrated that genistein is a more
effective anti-oxidant than daidzein, which is likely to be attributable to its
third hydroxyl group in the C-5 position. Moreover, the isoflavone precursors
biochanin A and formononetin showed very weak anti-oxidant capacities in
this in vitro system as they lack the C-40 hydroxyl group, which appears to be
an important determinant of the anti-oxidant properties of isoflavones (23).
Equol showed superior anti-oxidant actions compared to both the precursor
molecules and the parent isoflavones, suggesting that the absence of the 2,3-
double bond in conjunction with a loss of the 4-oxo group enhances anti-
oxidant properties (23). Antioxidant activity, assessed by the trolox equivalent
anti-oxidant capacity (TEAC) assay are consistent with these data that equol is
a more potent isoflavone compared with genistein and daidzein (24).
In an in vitro experimental system, genistein (IC50 ¼ 25 mM) was a more
potent inhibitor of the formation of H2O2 by 12-O-tetradecanoylphorbol-13-
acetate-activated HL-60 cells and the generation of superoxide anions by
xanthine/xanthine oxidase compared with daidzein (IC50 ¼ 150 mM), apigenin,
and biochanin A (22,25). Activities of anti-oxidant enzymes such as superoxide
dismutase, catalase, and glutathione peroxidase were also significantly increased
with gensitein (25). Furthermore, genistein has been shown to enhance anti-
oxidant enzyme activity in murine cells by the suppression of tumor promoter-
induced H2O2 formation (22).
The free radical-scavenging and anti-oxidant activities of various struc-
turally related isoflavones including genistein, daidzein, biochanin A, and
genistin in a cell-free and an endothelial cell model systems were investigated
(26). All isoflavones tested had no significant scavenging effects on these
radicals at concentrations up to 1.0 mM, suggesting that free radical scaven-
ging activities of some isoflavones may not substantially contribute to their
anti-oxidant properties. However, as physiologically achievable concentrations
(5 nM) increased intracellular-reduced glutathione levels, this ability may
make a more significant contribution to their biological action than their
scavenging activities.
Genistein and daidzein, the major isoflavone aglycones, have received
most attention; however, they undergo extensive metabolism in the gut and
liver, which may affect their anti-oxidant properties. Recently, the anti-oxidant
activity and, free radical-scavenging properties of the isoflavone metabolites
equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene
in comparison to their parent aglycones, genistein, and daidzein have been inves-
tigated, with electron spin resonance spectroscopy indicating that 8-hydroxydaid-
zein was the most potent scavenger of hydroxyl and superoxide anion radicals.
Isoflavone metabolites also exhibited higher anti-oxidant activity than parent
compounds in standard anti-oxidant (FRAP and TEAC) assays indicating that
the metabolism of isoflavones affects their free radical scavenging and anti-
oxidant properties (27).
Isoflavones have been shown to reduce LDL oxidation. Six healthy volun-
teers consumed soy protein (60 mg isoflavones per day) for 2 weeks. LDL
oxidation, as assessed by the lag time of copper induced oxidation, was shown
to be significantly prolonged compared with baseline measurements (28).
In vitro data are consistent with these findings, with Kapiotis et al. (29) demon-
strating that genistein inhibited the oxidation of LDL in the presence of copper
ions or superoxide and NO radicals as measured by thiobarbituric acid-reactive
substance formation (TBARS).
CARDIOVASCULAR EFFECTS
Although the mechanisms involved in the development of arteriosclerosis have
not been fully established, there is a consensus that the expression by endothelial
cells of inflammatory cytokines, adhesion molecules, and chemotactic proteins
plays a key role. Epidemiological studies suggest that differences in diet may
explain the lower incidence of CVD in Japan compared with other industrialized
countries such as the United States or the UK and the wide international variabil-
ity in intake of dietary isoflavones may play a role (1,30,31). Potential anti-
atherogenic effects of isoflavones (Table 14.1) include a reduction in LDL
cholesterol; modulation of pro-inflammatory cytokines; cell adhesion proteins,
and nitric oxide (NO) formation; protection of LDL against oxidation, inhibition
of platelet aggregation; and an improvement in vascular reactivity.
LIPID METABOLISM
Animal Studies
The hypocholesterolemic effect of soy protein has been known for many decades
(32). In many animal species, substituting soy protein for dietary animal protein
consistently reduces LDL-cholesterol and total cholesterol levels (33). Gerbils
fed soy-based diets have significantly lower levels of total cholesterol, LDL þ
VLDL cholesterol, and apolipoprotein B concentrations (34). Isoflavone con-
sumption led to a 30% decrease in plasma cholesterol levels and a 50% reduction
in atherosclerotic lesion area in a strain of mice with low HDL-cholesterol (35).
Table 14.1 Potential Mechanisms by Which Isoflavones Protect Against

Arteriosclerosis
Antioxidant properties
Inhibition of LDL oxidation
Stimulation of antioxidant enzymes
Induction of GSH synthesis
Gene regulatory activity
Inhibition of NF-kB dependent signal transduction pathways
Inhibition of protein tyrosine kinase activity
Inhibition of inducible nitric oxide production in macrophages
Down-regulation of cell adhesion and proinflammatory cytokine expression
Hypocholesterolaemic effects
Increased bile acid secretion
Increased LDL receptor activity
Reduced cholesterol absorption from gut
Platelet function and vascular effects
Inhibition of platelet aggregation
Improvement of vascular reactivity
Soy protein containing isoflavones decreased LDL-cholesterol and increased

HDL-cholesterol in a group of female monkeys fed a moderately atherogenic
diet (36,37) and when a state of menopause was experimentally established in
this animal model, by ovariectomy, soy protein consumption, as compared to
casein consumption, significantly improved plasma lipids, and lipoprotein
concentrations.
The key issue of whether the response to soy protein is mediated through
the presence of isoflavones has been the focus of much attention. It appears
that when isolated soy protein is alcohol-washed and most, but not all, of the iso-
flavones are removed, there is only a marginal reduction in plasma cholesterol.
The hypocholesterolemic effect, however, is restored when the isoflavones are
added back to the alcohol-washed isolated soy protein. Furthermore, isoflavones
alone have little or no cholesterol-lowering effect in cynomologus monkeys
(36,37).
Clinical Studies
Although the mechanism of action of the cholesterol lowering effect of soya is
still poorly understood, clinically it has been effectively used in the therapy of
patients with hypercholesterolemia for several decades (33). A meta-analysis
of 38 clinical studies concluded that the mean reduction in serum total cholesterol
was 9.3%, whereas LDL decreased by 12.9% with soya protein extracts (32).
Individuals with the highest initial cholesterol levels experienced the greatest
reduction.
An intake of 25 g/day of soya protein extract would be associated with a
0.23 mmol/L decrease in serum cholesterol (32). On the basis of this evidence
and further clinical studies, the FDA approved a health claim for cholesterol
reduction based on an intake of 25 g soya protein per day. This intake is
higher than the current daily intake in Japan and it is unknown whether life
time exposure to diets rich in these compounds accounts for the lower blood
cholesterol and CHD rates in the Asian populations. A Japanese health check-
up study of 3596 women observed a strong inverse relationship between daily
soy protein intake and serum cholesterol. The average soy protein intake for
women was 6.88 g (38), which is calculated as an isoflavone intake of 10 –
30 mg/day. The FDA drew no conclusion regarding the role of isoflavones in
the cholesterol lowering effect, but a recent study has shown that isoflavones
play a significant role in lowering plasma LDL and their absence from soya
renders the food ineffective in reducing cholesterol levels. They showed a
linear dose –response relationship between dietary isoflavone content and choles-
terol reduction, with no lowering effect observed when isoflavones were removed
from the soy protein (39). A group of 156 men and women with moderately
elevated total cholesterol and LDLc were randomized to receive a soy protein
beverage with differing amounts (0 –58 mg/day) of isoflavones (39) only
the isoflavone-containing beverages lowered total cholesterol and LDLc.
Potter and co-workers (40) randomly assigned 66 hypercholesterolemic, postme-

nopausal women to one of three diet groups: 40 g milk protein/day, 40 g isolated
soy protein/day with 1.39 mg isoflavones/g protein, or 40 g isolated soy protein/
day with 2.25 mg isoflavones/g. The women had been placed on a National
Cholesterol Education Program Step I diet, 2 weeks prior to randomization and
continued the diet throughout the course of the study. At the end of the
24-week diet period, HDLc had risen and non-HDLc had fallen in the two soy
groups, as compared with the milk protein group (40). In a study of 43 postme-
nopausal women, the HDL cholesterol:total cholesterol level was increased 5.5%
by a diet comprising 750 mL of soymilk or 30 g of soy nuts (containing 60 –
70 mg/day isoflavones) (41), whereas the resistance of LDL-cholesterol to oxi-
dation was significantly increased.
On the other hand, isoflavones alone (fed as pure compounds or extract)
have consistently been found to have no lipid-lowering effect (42 – 44) indicating
that the mechanism of action of isoflavones on lipids is complex and probably
involves an interaction with the food matrix (Table 14.2).
BLOOD PRESSURE
The endothelial wall of the blood vessel has been found to have almost equal pro-
portions of ERa and ERb (64,65), and these two receptors play an important role in
the vasoreactivity of the blood vessels. ERa rapidly activates endothelial derived
nitric oxide synthase (eNOS), the key enzyme responsible for the production of
nitric oxide-induced dilatation of the blood vessels (66). This is a nongenomic
and rapid event that is reduced in atherosclerotic arteries. Studies of the ERa and
ERb knock-out mouse attest to the important role that these two receptors play in
vascular events related to cardiovascular disease with data suggesting that ERb
deficient mice have several functional abnormalities in vascular smooth muscle
cells and blood vessels, providing strong evidence for the essential role for ERb
in the regulation of blood pressure and vascular function (67). Therefore, perhaps
the greatest benefits of a diet rich in isoflavones may be their effects on improving
the quality of blood vessels, rather than effects on blood cholesterol levels per se.
Vascular constriction is associated with increased risk of arteriosclerosis
and hypertension. The few animal and clinical studies conducted to date
suggest that isoflavones can improve vascular compliance (Table 14.3). Isofla-
vones increase blood vessel dilation and improve blood flow in rhesus monkeys
(68). Monkeys fed a diet with isoflavone-containing soy protein isolate had a
6% dilation in lumen diameter, compared with a 6% constriction in monkeys
fed soy protein isolate from which isoflavones had been removed. Soy isoflavones
were shown to promote arterial dilatation and inhibit constriction in a group of
female rhesus monkeys fed an atherogenic diet containing soy isoflavones (69).
In vitro platelet aggregation to thrombin and serotonin was also less when com-
pared with female monkeys consuming an atherogenic diet from which soy isofla-
vones had been removed. Most of these effects have also been confirmed in human
Table 14.2 Effects of Soy Isoflavones on Serum Lipids
Reference Design of the study Totalc HDLc LDLc TGA
Ashton Placebo-controlled crossover in # # nc #

et al. (45) healthy men (n ¼ 42).
120 mg/day for 1 month
Blakesmith Parallel-group in premenopausal nc nc nc nc
et al. (46) women (n ¼ 25). 0 or
86 mg/day for 12 weeks
Cassidy Intervention in #
et al. (6) normocholesterolemic
premenopausal women
(n ¼ 6). 45mg/day for 1 month
Crouse Parallel-group in healthy subjects # nc # nc
et al. (39) (n ¼ 156). 0, 3, 27, 37, or
Gardner Parallel-group in # nc # nc
et al. (47) postmenopausal women
(n ¼ 31). 80 mg/day for
12 weeks
Gardner-Thorpe Placebo-controlled crossover in nc nc nc nc
Gooderham Placebo-controlled crossover in nc nc
60 g/day soy protein
for 4 weeks
Hale et al. (50) Parallel-group in nc nc nc nc
postmenopausal women
(n ¼ 29). 80 mg/day for
2 weeks
Hodgson Placebo-controlled crossover in nc nc nc nc
et al. (51) healthy subjects (n ¼ 59, 46
men and 13 postmenopausal
women). 55 mg/day
for 8 weeks
Jayagopal Placebo-controlled crossover in # nc # nc
et al. (52) Postmenopausal diet-
controlled type 2 diabetes
women (n ¼ 32). 132 mg/day
for 12 weeks
Jenkins Placebo-controlled crossover in nc nc nc nc
et al. (53) hyperlipidemic men and
postmenopausal women
(n ¼ 41). 10 and 73 mg/day
for 4 weeks
(continued )
Table 14.2 Continued

Merz-Demlow Placebo-controlled crossover in # " # nc

et al. (54) premenopausal women
(n ¼ 13). 10, 65, and
Nestel Placebo-controlled crossover in nc nc nc nc
et al. (42) healthy women (n ¼ 21).
Potter Parallel-group in nc " nc
(n ¼ 66). 56 or 90 mg/day
for 6 months
Ridges Hypercholesterolaemic # nc # #
(n ¼ 18). 45 mg/day
for 8 weeks
Samman Placebo-controlled crossover nc " nc
et al. (56) in premenopausal women
(n ¼ 14). 86 mg/day
for 8 weeks
Sanders Placebo-controlled crossover in " nc
et al. (57) healthy subjects (n ¼ 22,
5 men). 2 (control) or
56 mg/day for 17 days
Scheiber Single group of postmenopausal # " nc #
et al. (41) women (n ¼ 42). 60 mg/day
for 12 weeks
Simons Placebo-controlled crossover in nc nc nc nc
(n ¼ 20). 80 mg/day
for 8 weeks
Teede Parallel-group in healthy subjects # nc # #
et al. (59) (n ¼ 179, 96 men).
Teixeira Parallel-group in men with # nc nc
et al. (60) moderate
hypercholesterolemia
(n ¼ 81). 38, 57, 76, and
Uesugi Placebo-controlled crossover in # nc nc #
et al. (61) perimenopausal women
(n ¼ 12). 61.8 mg/day
for 4 weeks
(continued )

Wangen Placebo-controlled crossover in # nc # nc

(n ¼ 18). 7.1 (control), 65, and
132 mg/day for 93 days
Washburn Placebo-controlled crossover in # nc # nc
et al. (63) perimenopausal women
(n ¼ 51) 34 mg for 6 weeks
# ¼ decrease, " ¼ increase, nc ¼ not changed.
studies. Isoflavones, 80 mg/day, containing 45 mg genistein, improved systemic

arterial compliance by 26%, as compared to placebo, in a group of 21 peri- and
postmenopausal women (43). A recent study of perimenopausal women fed
34 mg of isoflavones contained in 20 g of soy protein showed a reduction in dias-
tolic blood pressure (63). The mechanism of attenuated contractility in arteries by
a low dose of Genistein (2.5 mg/kg b.w.) in rats may be due to the tyrosine kinase
inhibitory property of genistein, because at this low dose there was no evidence of
estrogenic effects (70). In a study carried out in 41 hyperlipidemic men and
women (53), significant differences were observed for systolic blood pressure,
which was significantly lower in men following the soy diets than after the
control diet. In other human studies, a decrease in both systolic and diastolic
blood pressure has been observed in both men and women, following a long
period of consumption of soya milk, and with a significant correlation between
blood pressure reduction and urinary excretion of genistein (71). However,
since the existing data from clinical studies is inconsistent on the effects of isofla-
vones on blood pressure further studies are required to define the optimal dose and
duration for blood pressure lowering effects.
INFLAMMATION AND CELL ADHESION

The potential anti-inflammatory and cell adhesion properties of isoflavones have
been tested in several in vitro model systems. However, to date most of the
studies conducted have used concentrations of isoflavones that are unlikely to
be achievable at physiologically relevant concentrations.
Activation of the endothelium results in the release of vascular cytokines
such as interleukin-1 (IL-1b) and tumor necrosis factor alpha (TNF-a). These
cytokines in turn induce the cell surface expression of adhesion molecules
such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion
molecule-1 (ICAM-1), which are centrally involved in the endothelial recruit-
ment of leucocytes (75). Focal expression of ICAM-1 and VCAM-1 has been
reported in arterial endothelium overlying early foam cell lesions in both
Table 14.3 Effect of Soy Isoflavones on Blood Pressure
Change of
SBP/DBP
Reference Design of the study (mmHg)
Bloedon et al. (72) Single dose in postmenopausal women 216/213

(n ¼ 24). 2, 4, 8, or 16 mg/day
for 24 h
Chiechi et al. (73) Parallel-group in postmenopausal 23/0
women (n ¼ 187). 0, 40, or
Hale et al. (50) Parallel-group in postmenopausal nc

for 2 weeks
Hodgson et al. (51) Placebo-controlled crossover in healthy nc
subjects (n ¼ 59, 46 men and
13 postmenopausal women).
Jayagopal et al. (52) Placebo-controlled crossover in 22/21
postmenopausal diet-controlled
type 2 diabetes women (n ¼ 32).
Jenkins et al. (53) Placebo-controlled crossover in nc
hyperlipidemic men and
postmenopausal women (n ¼ 41).
10 and 73 mg/day for 4 weeks
Nestel et al. (41) Placebo-controlled crossover in healthy nc
for 5 weeks
Rivas et al. (71) Parallel-group in subjects with 218/216
hypertension (n ¼ 40). 143 mg/day
for 12 weeks
Simons et al. (58) Placebo-controlled crossover in nc
postmenopausal women (n ¼ 20).
Teede et al. (59) Parallel-group in healthy subjects 23.9/22.4
(n ¼ 179, 96 men). 118 mg/day
for 12 weeks
Vigna et al. (74) Parallel-group in postmenopausal 23/0
women in postmenopausal women
(n ¼ 77). 76 mg/day for 12 weeks
Washburn et al. (63) Placebo-controlled crossover in 0/25
peri-menopausal women (n ¼ 51).
34 mg for 6 weeks
# ¼ decrease, " ¼ increase, nc ¼ not changed.

dietary and genetic models of arteriosclerosis in rabbits (76). This expression,

together with the activation of monocyte chemoattractant protein-1 (MCP-1),
leads to infiltration of mononuclear cells into the artery wall (77). The uptake
of oxidized LDL by these cells leads to the formation of lipid-laden foam
cells, and the development or progression of atherosclerotic plaques (78).
Transcription of ICAM-1, VCAM-1, and MCP-1 is dependent, at least in
part, on the activation of NF-kB, a classical member of the Rel family of
transcription factors, and several observations suggest a key role for NF-kB in
atherogenesis. In unstimulated cells, NF-kB is inactivated by sequestration to
the I-kB family of inhibitor proteins. Agents that activate NF-kB induce degra-
dation of I-kB. Free NF-kB then enters the nucleus and binds to the regulatory
regions of its target genes (79) as shown in Fig. 14.4.
Its activity is controlled by the redox status of the cell, and generation of
reactive oxygen species may be a common step in all of the signaling pathways
that lead to I-kB degradation. NF-kB activation is inhibited by several chemically
distinct anti-oxidants, including N-acetylcysteine, dithiocarbamates, vitamin E
derivatives, GPx activators, and various metal chelators (80) and flavonoids (81).
There are now several pieces of evidence which suggest a key role for
NF-kB in atherogenesis; activated NF-kB has been identified in situ in human
atherosclerotic plaques (82) as well as in an arterial injury model (83), but not
in cells of normal vessels devoid of arteriosclerosis. Furthermore, NF-kB is acti-
vated by an atherogenic diet and by oxidized LDL (84) and advanced glycosated
end products (85).
In a human monocyte in vitro model system, lipopolysaccharide (LPS)
increased both NF-kB binding to DNA consensus sites and TNF-a release
ERa ERb Brain ERb

E2 100 100
Diethylstilbestrol 468 295 Vascular ERb
Breast ER a/b
17a-Estradiol 58 11
4-OH-Tamoxifen 178 339 Adrenal ERa
Uterus ER a/b
Genistein 5 36
Kidney ERa Ovary ER a/b
Coumestrol 94 185
b-Zearalanol 16 14
Prostate ERb Bladder ERb
Bisphenol A 0.05 0.33
Methoxychlor 0.01 0.13
Testes ERa Bone ERb
Figure 14.4 The transcription factor NF-kB as a potential molecular target of

isoflavones.
(86). At pharmacological concentrations, genistein (40 mM) attenuated both

NF-kB DNA binding and TNF-a release. Genistein is known to inhibit protein
tyrosine kinase (87), so these data suggest that LPS-induced NF-kB activation
and TNF-a release in human monocytes is protein tyrosine kinase-dependent
(86). At these high concentrations, genistein (30 mM) also inhibited NF-kB
DNA-protein binding in LPS-stimulated monocytes (RAW 264.7) by 50%
(88). Genistein (50 mM), but not daidzein, inhibited TNF-a induced NF-kB
activation in cultured human lymphocytes. Furthermore, the consumption of a
soy-based dietary supplement containing 50 mg of a isoflavone mixture (genis-
tein:daidzein:glycitin ratio ¼ 1.3:1:0.3) twice daily for 3 weeks was shown to
reduce ex vivo NF-kB activation induced by TNF-a in peripheral lymphocytes
in healthy male volunteers (89).
Following stimulation with the pro-inflammatory cytokines, IL-1 and
TNF-a, endothelial cells express the leukocyte adhesion molecules E-selectin,
VCAM-1, and ICAM-1. To date several studies have investigated the potential
intracellular signaling mechanisms of genistein that would result from expression
of these cytokines. Genistein dose-dependently inhibited the maximal E-selectin
expression induced by incubation of HUVEC for 4 h with TNF-a (100 U/mL)
and IL-1 (100 U/mL). Furthermore, VCAM-1 secretion was inhibited by genis-
tein after stimulation of HUVEC for 24 h with TNF-a or IL-1. In contrast, gen-
istein did not alter ICAM-1 secretion after a 24 h incubation of HUVEC with
either of the two cytokines (90). Weber et al. (91) investigated the role of tyrosine
phosphorylation in the induction of endothelial leukocyte adhesion molecule 1
(ELAM-1), VCAM-1, and ICAM-1 in HUVECs. Pretreatment of the cells with
genistein resulted in a dose-dependent inhibition of TNF-a-induced ELAM-1,
VCAM-1, and ICAM-1 surface expression (IC50 ¼ 30 mM). In a further study
examining the effects of genistein as a PTK inhibitor of leukocyte (neutrophils,
lymphocytes, and monocytes) adhesion to endothelial cells, IL-1 and TNF-a-
stimulated neutrophil and monocyte adhesion to HUVEC was significantly
inhibited by genistein compared with stimulated control cells (92).
Monocyte-derived macrophages are the principal inflammatory cell in the
atheromata. In early stages of artherosclerotic lesion formation, macrophages and
endothelial cells interact to trigger a cycle of events that exacerbates endothelial
dysfunction, resulting in a loss of homeostatic control (93). Activated macro-
phages generate large amounts of NO from L -arginine by the action of inducible
NO synthase (iNOS) and its overproduction has been associated with oxidative
stress and chronic inflammation (94). NO is an important intracellular and inter-
cellular regulator of many biological functions, including macrophage-mediated
cytotoxicity (95,96). Cytokines such as IFN-g and other inflammatory stimuli
such as bacterial lipopolysaccharide (LPS) regulate the activity of iNOS in
macrophages (97,98).
Genistein inhibited nitrite production in a dose-dependent manner when rat
mesangial cells were activated with IL-1b. This finding suggests a central role for
PTK in the signaling pathway of IL-1b, resulting in the activation of iNOS in rat
mesangial cells (99). Li et al. (100) showed that genistein had an inhibitory effect
on NO production (IC50 ¼ 72 mM) in an immortalized astrocyte cell line
(DITNC), which were activated using a three-cytokine mixture (TNF-a, IL-1,
and IFN-g) designed to maximally induce iNOS.
In a further study, Gottstein et al. (101) found that genistein
(IC50 ¼ 58 mM) and daidzein (IC50 ¼ 107 mM) significantly inhibited IFN-g
plus LPS induced NO production in RAW 264.7 macrophages. iNOS mRNA
levels remained unchanged by the isoflavone treatment, which suggests that
the inhibitory effect is posttranscriptional and this suppression of LPS activity
has also been shown for other plant phenolics (102). Sheu et al. (103) reported
an inhibitory effect of genistein and daidzein on LPS-induced expression of
the iNOS gene in macrophages, but their co-incubation with LPS and isoflavones
may have given rise to this effect. Thus inhibition of iNOS expression may have
been caused by a direct interaction of these compounds with the LPS molecule,
rather than a direct effect on the cell. In the Gottstein study (101), macrophages
were preincubated with genistein and daidzein for 24 h and were then washed
twice with PBS before the addition of IFN-g and LPS in order to avoid any
direct chemical interaction. Thus, the inhibition of NO production observed in
this study may reflect inhibition of TNF-a secretion by genistein and daidzein
as it has previously been demonstrated that TNF-a is crucial for the induction
of NO synthesis in IFN-g and/or LPS-stimulated macrophages (104,105).
MCP-1 is a CC-chemokine consisting of 76 amino acids. This molecule
may play a key role in atherogenesis, because it is involved in the recruitment
of monocytes and T-cells into the arterial wall. MCP-1 mRNA has been detected
in atherosclerotic lesions by in situ hybridization (106,107). Furthermore, a
decrease in atherosclerotic lesion size is seen in mice deficient of the MCP-1
receptor CCR-2, and fewer macrophages and monocytes are present in their
aortas (108). Therapeutic drugs and dietary factors targeting MCP-1 and/or its
receptor may prove useful in the prevention of atherosclerotic lesion develop-
ment. Recently, it has been shown that genistein (IC50 ¼ 29 mM) and daidzein
(IC50 ¼ 37 mM) dose-dependently down-regulated MCP-1 secretion (101), indi-
cating that both of these isoflavones may have the potential to inhibit monocyte
infiltration into the arterial wall. It is known that the expression of MCP-1 is regu-
lated at the transcriptional level (77). Therefore, it is hypothesized that genistein
and daidzein may regulate TNF-a-induced MCP-1 expression through transcrip-
tion factors such as nuclear factor kappa B and activator protein-1, present in the
promoter region of the MCP-1 gene.
PLATELET AGGREGATION AND ENDOTHELIUM REACTIVITY

Both genistein and daidzein exert anti-aggregatory activity in human platelets
in vitro (101). This finding is consistent with earlier reports that the consumption
of soy protein and its isoflavone-enriched fraction lowers platelet aggregation in
rats (109). The exact molecular mechanisms by which isoflavones affect platelet
aggregation are unclear and currently under investigation. Apart from protein
tyrosine kinase inhibition (110) within the cyclooxygenase pathway, several
other reported molecular effects of flavonoids could have influenced platelet
function in the present study. The modification of platelet cyclic-30 ,50 -adenosine
monophosphate (cAMP) via the inhibition of phosphodiesterase activity is the
most supported pathway for anti-aggregatory effects of flavonoids (111). Inhi-
bition of lipoxygenase activity, as demonstrated principally for myricetin and
quercetin (112), is another possible mechanism. Stimulation of adenylate
cyclase, leading to increased cAMP levels, has been proposed as a further anti-
aggregatory signal transduction pathway (113).
In addition, the anti-oxidant character of isoflavones may play a role in
inhibiting platelet aggregation. Pignatelli et al. (114) showed that collagen-
induced platelet aggregation was associated with the production of H2O2 ,
which acts as an important second messenger in platelets, stimulating both the
phospholipase C pathway and the arachidonic acid metabolism. Consistent
with this finding, platelets primed with nonactivating concentrations of arachido-
nic acid or collagen were activated by nanomolar concentrations of H2O2 (115).
Since isoflavones possess anti-oxidant properties (23,116) and can scavenge rad-
icals, this evidence that reactive oxygen species are involved in platelet stimu-
lation suggests another anti-aggregatory mechanism. In comparison with
daidzein, the genistein molecule contains an additional hydroxyl group in the
C-5 position, possibly resulting in a higher anti-oxidant activity (25). This
might explain why in the present study genistein has been demonstrated to be
a more potent inhibitor of platelet aggregation than daidzein.
CONCLUSION
In the overall scheme of cardiovascular protection, isoflavones appear to poten-
tially have a more important role in conditioning the vascular tree than on influ-
encing cholesterol levels. The preferential binding of isoflavones to the ERb and
the increasing recognition of the role of this new receptor in the endothelial wall
provides justification for increasing the awareness of the heart-health effects of
diets rich in these phytoestrogens. Furthermore, such effects are not restricted
to soy isoflavones but also apply to lignans, which are abundant in fruits and veg-
etables. Potential anti-atherogenic effects of isoflavones include a reduction in
LDL cholesterol, modulation of pro-inflammatory cytokines, cell adhesion pro-
teins and nitric oxide formation, protection of LDL against oxidation, inhibition
of platelet aggregation, and an improvement in vascular reactivity. To date, most
of the published in vitro data have reported beneficial effects only following
exposure to pharmacological doses of isoflavones; thus, further research is
required to understand the importance of this mechanism following exposure
to physiologically relevant levels of isoflavones; and, their corresponding liver
and gut metabolites. Although epidemiological data and laboratory studies
allude to the possible protective effects of soy isoflavones at specific target
tissues, randomized placebo controlled clinical trials are necessary to further

address the relative importance of these compounds for cardiovascular health.
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15
Anti-Carcinogenic Properties of
Soy Isoflavones
Max O. Bingham and Glenn R. Gibson

Introduction 327
Isoflavones 329
Isoflavones and Breast Cancer 329
Isoflavones and Prostate Cancer 332
Isoflavones and Colon Cancer 334
Isoflavones and Cancer—An Overview and the Future 335
References 336
INTRODUCTION
The incidence and mortality rates of several hormonally related tumors, including
breast, prostate, and colon cancer, have until recently been considered low in
Asian countries like China, Japan, and Indonesia when compared with western
countries (1). Additionally, cancer rates appear to differ between westernized
countries. For example, Italy and Finland have substantially lower rates than
other western countries. A number of studies have concluded that lifestyle and
dietary factors may be important in explaining these differences in incidence
rate. Asian diets, which are mainly vegetarian or semivegetarian, differ markedly
327
328 Bingham and Gibson
from western diets, which are rich in animal proteins and fats. This may affect
cancer incidence in a number of ways including alteration of the metabolism
and actions of a number of phytochemicals, some of which may have steroidal
effects. Of crucial importance is the role of the gut and more specifically the
microflora in the gut and its impact in mediating certain effects of diet on
disease patterns in western countries (2 –4).
The isoflavones, daidzein and genistein, which are a type of phytoestrogen
can be included in the group of phytochemicals. These compounds are found in
abundance in the plasma and urine of people living in areas of low cancer inci-
dence (5,6). They are either hormone like with inherent estrogenic activities, or
converted by the human gut microflora to estrogenic compounds. These com-
pounds have been shown to influence several biological phenomenon, including
production, metabolism, and biological activities of sex hormones and also many
intra-cellular steroid metabolizing enzymes (3,7,8). Evidence that soy products
help to prevent cancer existed many years before it was suggested that isofla-
vones may be important. Originally, it was thought that components such as pro-
tease inhibitors, phytic acid, or b-sitosterol were the active components (9). The
subsequent suggestion that isoflavones were more important has led to numerous
publications linking isoflavone consumption and cancer incidence. However, the
research has not always been positive and recent reports suggest, in animal exper-
iments at least, that isoflavones may have adverse effects with respect to carcino-
genesis and other biological functions. Given this conflicting evidence, we
review the benefits and risks associated with soy consumption and examine the
possible role soy may exert in the mechanisms of carcinogenesis.
The main phytoestrogens derived from the diet are genistein, daidzein, and
glycitin, which are isoflavones almost exclusively found in soy. They occur as
glycosidic conjugates or in unconjugated or conjugated forms in most soy protein
products, usually in very high concentrations. The two most estrogenic iso-
flavones are genistein and equol (10), the latter being a product of daidzein
metabolism in the gut by the activities of the human gut microflora. Recent
research in estrogen receptors (ERs) has clarified some of the conflicting findings
regarding phytoestrogen potency. The application of a wide variety of techniques
has tended to generate data indicating different relative potency values. Addition-
ally, since the identification and cloning of a second ER, ERb (11), it has become
apparent that isoflavones have different affinities for both ERa and ERb and are
found at different concentrations in organ systems within the body. Originally, it
was thought that the protective actions of isoflavones were due to anti-estrogenic
activities, whereby they blocked ERs to more potent mammalian estrogens such
as 17b-estradiol. However, this appears to be an oversimplification, and actions
are likely to occur on many different levels.
The relationship among health, protection against disease, and isoflavones
may in itself be an oversimplification. Anti-carcinogenic properties of soy are
more likely to be due to a concerted action of many types of phenolic and
other biologically active compounds, including those of isoflavones, lignans,
Anti-Carcinogenic Properties of Soy Isoflavones 329
and flavonoids. Given the myriad of research that has been completed over recent
years in the anti-carcinogenic properties of phenolic compounds in general, it
appears likely that the gross impact of such dietary components is more important
than the actions of singular compounds. However, to understand the actions, and
thus the potential of dietary components to protect against chronic disease devel-
opment, it is still worthwhile taking the reductionist approach.
ISOFLAVONES
Isoflavones are non-nutrient plant compounds which belong to the phytoestrogen
class, having a similar structure to mammalian estrogens (12). Genistein, one of
the predominant soy isoflavones, has been shown to inhibit the growth of cancer
cells through modulation of genes that are related to the homeostatic control of
cell cycle and apoptosis. It has also been found that genistein inhibits the acti-
vation of the nuclear transcription factor, NF-kB, and Akt-signaling pathway,
both of which are known to maintain a balance between cell survival and pro-
gramed cell death (apoptosis). It is known to have antioxidant and estrogenic
properties, which target estrogen- and androgen-mediated signaling pathways
in the processes of carcinogenesis. Genistein is also found to be a potent inhibitor
of angiogenesis and metastasis (13). One of the most important considerations in
its ADME sequence is its release from its precursor, genistin. This involves a
number of steps, and for a long time it was thought that the human gut microflora
was solely responsible for its release and subsequent metabolism. However,
recent reports demonstrate that the conversion of genistin to genistein begins
in the mouth and then continues in the small intestine suggesting that genistein
may be released along the entire length of the alimentary tract (14). However,
the mechanisms of genistein in cancer development are not all positive and
much disagreement exists within the research environment as to its specific
role in cancer development. Many examples have appeared indicating that gen-
istein may help to promote carcinogenesis. Indeed, recent reports have indicated
that genistein, daidzein, and biochanin A are capable of achieving chromosome
aberrations and aneuploidy, suggesting a possible involvement in the initiation of
carcinogenesis (15).
Isoflavones and Breast Cancer

Soya bean products are regularly consumed in Asian countries such as China,
Japan, Korea, and Indonesia. These countries have traditionally had low inci-
dences of breast, prostate, and colon cancer. However, rates are steadily increas-
ing because of changes in dietary habits and lifestyle (5). Traditional diets have
been low in fats and red meat, and often rich in fish—all elements that have been
independently linked to a decreased cancer risk. However, studies have also indi-
cated that soy bean products may be protective and anti-carcinogenic and this was
long before the beneficial effects of isoflavones were recognized (9,16). Thus, it
is likely that some, if not all, of these purported bioactive compounds from soy,
and indeed components from the rest of the diet, may contribute towards this
overall epidemiological observation.
Specific epidemiological studies of Japanese and White populations who
emigrated to Los Angeles, the United States, indicated that the later in life emi-
gration was completed, the lower the risk of breast cancer when compared with
those who emigrated earlier in life (17). Additionally, it has been observed that
the risk of breast cancer in Asian-American populations was inversely pro-
portional to intake of tofu (a soy protein food that contains high levels of isofla-
vones) (18). These findings support earlier observations that there is an increased
disease risk in populations following emigration from Asia to the United States.
Within 6 months of immigration, isoflavones excreted in urine by oriental immi-
grants were shown to be 10% the amount excreted in the urine of Japanese
women—indicating that the Japanese population may have a lower risk of devel-
oping diseases such as breast cancer (2).
Further evidence for this protective characteristic of isoflavones in breast
cancer includes that of some chimpanzees in captivity given experimental
breast cancer. These proved very resistant to developing the cancer and excreted
high levels of isoflavones, including equol, in urine (19). Moreover, women with
breast cancer tend to excrete small amounts of isoflavones, whereas those living
in areas of low incidence tend to excrete high levels of isoflavones. Additionally,
vegetarians exhibit much higher levels of isoflavones in plasma and urine than
omnivores (2,6,20). In summary, epidemiological evidence appears to be
supportive for a protective role in isoflavones in breast cancer and indicates
that diets high in soy may provide some protection against the development of
breast cancer. However, this type of evidence provides no insight into possible
mechanisms of action, nor whether isoflavones can conclusively afford protection
against cancer.
Studies with breast cancer cell lines in culture have shown that phytoestro-
gens, including daidzein and genistein, stimulate tumor growth at low concen-
trations but inhibit growth at high concentrations (21). However, the highest
concentrations of some of these studies were higher than physiologically relevant
levels observed in Japanese women (22 – 24). Recent reports have indicated that
genistein alone could inhibit mouse mammary adenocarcinoma, but when this
genistein was fed as soy extract, the magnitude of inhibition was much greater,
indicating that genistein along with other constituents was important in the sup-
pression of tumor growth in this study (25). In terms of genotoxicity, recent
reports suggest that genistein, but not daidzein, could protect against chemically
induced breast cancer by inhibiting certain phase-1 cytochrome P450 enzymes
involved in initiation (26). The effects of isoflavones on the human breast
cancer cell line MCF-7 have been studied and results indicated that they were
also capable of significantly inhibiting MCF-7 growth and additionally induced
cell apoptosis by regulating iNOS gene expression (27). Subsequent studies
have revealed further genes regulated by genistein and involved with breast
cancer cell proliferation, suggesting that the inhibitory action of genistein appears
to be complex and only partially mediated by the alteration of ER-dependent
pathways (28,29).
The possible association between phytoestrogens or phytoestrogen-rich
diets and breast cancer risk has been reviewed (30). Limited evidence suggests
that a diet containing soy bean products is chemoprotective. Indeed, a large pro-
spective study in Japan did not show any favorable effect of soy consumption on
breast cancer risk (31). The most promising data have originated from studies in
rodents on the impact of an isoflavone-rich diet during puberty and adolescence.
These demonstrated that an isoflavone-rich diet may only be significant at redu-
cing risks of breast cancer if consumption occurred during this early period of life
(32). Later studies (in Japanese women and in rodents) have supported this con-
clusion (33). Most of the experimental research suggests that prepubertal genis-
tein administration prevented the development of tumors by acting directly upon
the mammary gland to enhance maturation and by reducing cell proliferation
(34). Early breast-feeding has previously been shown to stimulate breast
lobular maturation and is also associated with a reduced risk of premenopausal
breast cancer in women (35). Isoflavone-induced breast differentiation could
mimic this effect.
The production of equol, a secondary metabolite of daidzein, in the gut has
been associated with a lower risk of breast cancer (36). It has been shown that equol
production tends to be high in people who consume high levels of plant-based
carbohydrates, fiber, and protein, but low in those that have a high-fat diet
(37,38). People who excrete high levels of equol have an increased ratio of
2-hydroyestrone to 16a-hydroxyestrone in their urine (39), which is thought to
lower the risk of breast cancer (40). However, high-equol producers have a slightly
higher concentration of sex-hormone binding globulin (SHBG). This increase
diminishes the availability of estradiol for 16a-hydroxylation in the liver. Also,
an increased load of isoflavones also increases estrogen 2-hydroxylation (41).
Thus, this increase in the ratio of 2-hydroyestrone to 16a-hydroyestrone, mediated
by levels of SHBG and isoflavone load, appears to be an important issue in explain-
ing a possible protective mechanism of isoflavones in breast cancer. Recent studies
support this finding with observations that equol excretion was positively corre-
lated with the ratio of 2-hydroyestrone to 16a-hydroyestrone (42). These obser-
vations suggest that the human gut microflora profile associated with equol
production may be involved in estrogen metabolism and may therefore also influ-
ence breast cancer risk. Interestingly, treatment with the anti-estrogen tamoxifen
decreased the ratio of 2-hydroyestrone to 16a-hydroyestrone (43), which accord-
ing to the theory increases the risk of breast cancer.
Other research has suggested negative effects of isoflavones in breast
cancer. High prenatal endogenous estrogen concentrations may increase risk in
women (34,44). Additionally, experimental evidence from rats supports the
view that isoflavones may negatively affect breast cells during pregnancy (34)
and increase the number of tumors developing later in life (45). It appears that
genistein acts as an estrogen agonist and stimulates cell division and proliferation
of existing breast cancer cells (46). This has additionally been shown in vivo in a
dose-dependent manner and the removal of genistin and genistein from the diet
caused tumors to regress (14). Limited evidence also indicates that daidzein
may have similar actions to genistein (47). However, given the fact that both
Japanese newborns and their mothers have high levels of isoflavones (48), this
would indicate that the incidence of breast cancer in this population would be
much higher—the opposite is true.
Overall, there is little convincing evidence to suggest that isoflavones from
soy consumed in adult life are protective against breast cancer for women living
in western countries. However, sustained intake throughout life may afford some
protection. Moderate intakes of soy products or supplements with isoflavones
may be beneficial in terms of cardiovascular health, osteoporosis, and meno-
pause, but negative effects on the breast cannot be ruled out.
Isoflavones and Prostate Cancer

Incidents of mortality from prostate cancer in western countries is much higher
than those in Asia, despite similar incidences of latent, small, or non-infiltrative
prostate carcinomas. Prostate cancer is hormone dependent and based upon early
studies in Japan, a hypothesis was formed suggesting that diets in countries with
low prostate cancer risk may contain high amounts of hormonally active and
cancer protective compounds such as isoflavones (2).
Plasma concentrations of isoflavones in Japanese men are high (23).
However, levels are higher still in prostatic fluid and possibly related to prostate
cancer incidence (24). Genistein and its precursor, and biochanin A inhibit cell
growth in prostate cancer cells at high concentrations (49). However, the most
effective isoflavone is 40 -methylequol and this has additionally been shown to
be effective at reducing prostate specific antigen (PSA) (50). This effect is
thought to be important because PSA is associated in many cases with a decrease
in cell proliferation.
A recent case-controlled clinical study (51) was carried out to compare cir-
culating levels of isoflavones in prostate cancer patients and control subjects.
Serum levels of genistein, daidzein, and equol were compared and it was
found that the group with prostate cancer contained significantly less patients
capable of converting daidzein to equol. This may be a significant risk indicator
given that it is generally thought that only 30% of western populations have the
human gut microflora profile capable of producing equol. Further recent studies
have added strength to this argument, because it has been shown that equol was
biologically active with potent anti-proliferative effects on benign and malignant
prostatic cells at concentrations that can be obtained naturally through dietary soy
consumption (52).
Animal studies investigating the effects of feeding soy and pure isoflavones
have indicated a generally positive response whereby prostate cancer growth was
inhibited (30). In two different rat models of prostate cancer, soybean isoflavones
inhibited the onset of cancer and delayed growth. However, increasing dietary fat
intake abolished these effects. In studies of nude mice with transplanted human
LNCaP prostate cancer cells, soy products or isoflavone concentrate inhibited
cancer growth. Studies of tumors revealed that apoptosis was increased and
angiogenesis was inhibited. Genistein is an inhibitor of angiogenesis—a
process that is necessary for extended growth and metastatic spread of tumors (7).
Studies in humans have revealed a similar relationship. The most recent
studies have all indicated that soy intake prevents prostate cancer. For
example, a prospective study revealed that consuming soymilk more than once
a day was protective against prostate cancer (53). Additionally, a very recent
case – control study evaluating the effect of soy food consumption, isoflavone
intake, and prostate cancer risk indicated a reduced risk associated with the con-
sumption of soy foods and isoflavones. The promise of subsequent longitudinal
follow-up studies will confirm these findings (54). To address issues surrounding
potential genistein genotoxicity, Miltyk et al. (55) investigated the potential of
this characteristic of genistein to cause genetic damage in men with prostate
cancer. They found that while genistein was capable of inducing genetic
damage in vitro, a similar effect was not observed in the subjects treated.
ERb receptors are present in the prostate and this has lead to increasing
interest in how isoflavones may be important in protection from prostate
cancer, because many isoflavones bind well to this receptor. The administration
of genistein to rats over their lifetime led to a downregulation of both ERs (ERa
and ERb) in the prostate (56). This observation indicates that genistein can regu-
late steroid receptor pathways and may help to prevent prostate cancer via this
mechanism.
The importance of dietary factors in prostate carcinogenesis has been
proven in many epidemiological studies of immigrants from Asia to the United
States. However, it appears as though the totality of diet is an important consider-
ation (57,58). A combination of higher meat intakes and the possibility of greater
levels of carcinogenic compounds (such as N-nitroso compounds) originating
from charcoal cooked red meat and fish increases the potential risk of developing
a wide range of cancers including prostate cancer. If this is combined with lower
intake of a wide range of protective dietary factors, risks must increase. However,
given the extent of factors that have been listed as protective (selenium, zinc,
carotenoids, lycopenes, vitamins E and D, and isoflavones to name but a few),
it appears as though concentrating upon single dietary factors as protective in
prostate cancer would lead to an underestimation of the contribution of the diet
as a whole. Indeed, a recent study has indicated that the polyphenols, quercitin
and kaempfaerol, and the isoflavones, genistein and biochanin A, originating
from both soy and tomato products could inhibit multiple intra-cellular pathways
involved in prostate cancer development (59). Furthermore, more holistic
research is likely to be needed to understand the complexity of diet and its
contribution in the condition.
Isoflavones and Colon Cancer

A previous review about soy foods, isoflavones, and colon cancer concluded that
the available data were conflicting and that evidence for the protective effects of
soy, in this instance, was limited (60). Since that time, further studies have indi-
cated that soy and isoflavones have either no effect on colon cancer or are mildly
procarcinogenic. ERs have been found in normal mucosal and cancer cells in the
colon (61), and while low, correspond to that in normal breast tissue (62). Estro-
gen replacement therapy in postmenopausal women reduces the mortality from
colon cancer (63), whereas anti-estrogenic treatments with tamoxifen may
enhance risk (64).
Unconjugated isoflavone concentrations in gut contents are high and it is
thought that they are more than likely to have biological effects on the
mucosal cells (7). Animal studies fairly consistently show that soy foods or iso-
flavones inhibit the formation of aberrant crypt foci, but these did not clearly
demonstrate an inhibitory effect on chemically induced colon cancer. Addition-
ally, case –control studies have suggested that soy food consumption may confer
a reduced risk of colon cancer, yet the findings are inconsistent (65). Recent
reports have indicated a possible role for the Bowman-Birk Inhibitor (BBI)
suggesting that it may be important in preventing colon cancer. This is one of
the constituents of soy originally thought to be important in observations that
soy was protective against cancer, before research interest focused upon isofla-
vones. Purified BBI and an extract of soy enriched with BBI reduced the inci-
dence and frequency of tumors in rats with chemically induced tumors.
However, soy molasses which contained soy isoflavones did not have any
effect on the colon carcinogenesis in rats. Additionally, no adverse effects
where observed in the rats treated with the purified BBI and the extract enriched
with BBI (66).
Bacterial fermentation is of considerable importance in the etiology of
colon cancer—germ-free animals have much lower rates of incidence of colon
cancer (67). However, research to date, although comprehensive, is highly con-
tradictory. Major products of bacterial fermentation include the short chain fatty
acids acetate, propionate, and butyrate. These decrease colonic pH and this has
been associated with a decreased risk of cancer. There has been considerable
interest in the role of butyrate in colon cancer. This is an important source of
energy for colonocytes and is thought to have a number of anti-carcinogenic
properties, including the ability to inhibit DNA synthesis and reduce cell prolifer-
ation (68 –70) fermentation rates in the colon. This may result in decreased
ammonia in feces and increases in breakdown and release of lignans (phytoestro-
gens of similar structure to isoflavones) (71).
However, bacterial fermentation has also been shown to produce a wide
range of potential carcinogens in the gut. The end products of certain colonic
bacteria have been described as carcinogenic or genotoxic: these include
nitrosamines, fecapentaenes, secondary bile acids, heterocyclic amines, various
aglycones of plant origin, phenolic and indolic compounds, nitrated polycyclic

aromatic hydrocarbons, diacylglycerol, some azo-based products, and ammonia.
Often, these sorts of compounds originate from fermentation of amino acids and
proteins or by the activities of microbial enzymes on dietary compounds and
xenobiotic compounds from the diet (72). Of course, similar pathways can also
release compounds that may be anticarcinogenic. Examples include flavonoids
and phytoestrogens such as isoflavones.
In general terms, it appears that dietary composition is important in the
overall activities and composition of the human gut microflora and this may be
important in the development and etiology of colonic cancer. Undoubtedly, the
human gut microflora is important in the bioavailability of isoflavones (particu-
larly the secondary metabolite, equol). However, what is far from clear is the role
that isoflavones might have in colon cancer. Given the multitude of activities of
the human gut microflora and the wide range of products generated that may be
important in carcinogenesis, it is vital that the specific gut microflora components
involved in colon cancer are identified.
ISOFLAVONES AND CANCER—AN OVERVIEW AND THE FUTURE

Many different diets appear to afford protection against cancer and the mechan-
isms are probably variable. The importance of the role of the human gut microflora
in mediation of the effects of plant phytochemicals in humans cannot be overem-
phasized. Diets rich in fiber and carbohydrate result in increased fermentation rate,
which may subsequently impact upon the production of butyrate and secondary
plant metabolites, such as equol. This appears to be a function of human gut micro-
flora composition because in certain populations this ability is downregulated or
even lacking. In turn, this may be important in modifying the risk of developing
breast, prostate, and colon cancer among other chronic diseases.
Whether soy isoflavones are important in protection against breast cancer is
not currently known. Available evidence suggests that the soybean complex is
protective but only in its entirety. Soy and isoflavones appear to be protective
in prostate cancer although further work needs to be completed before definitive
conclusions may be drawn. In colon cancer, the situation is very unclear, but cur-
rently evidence suggests that isoflavones and soy do not afford protection against
colon cancer. Parallel processes such as increased butyrate production in the
colon appear to be more important protective mechanisms.
Given the complexity of the research base currently available and the con-
tradictory evidence of whether isoflavones are anti-carcinogenic or carcinogenic,
some difficult questions arise. A significant driving force behind the volume of
research completed on these compounds is the potential for developing functional
food products that target, for example, cancer. Nutrition research has traditionally
focused on single issues (such as reducing the risk of developing particular
diseases) in “at risk” individuals. This approach is entirely valid and has provided
us with many valuable hypotheses. True “gold standard” approaches include

characterization of the extent and rate of absorption, tissue dispersal, site-specific
targeting of metabolically relevant compounds, and comprehensive studies of
time and dose effects—studies which have only recently been completed for
some compounds. For completeness, however, we need to address the question
of evaluating all the possible effects of specific food components in a genetically
heterogeneous population. This is especially important for determining unin-
tended risk as well as intended benefit.
Isoflavones clearly fit with this paradigm. Until the impact of these com-
pounds on health and disease is fully established, products developed from
these are unlikely to be popular with the consumer even if the smallest risk
exists. Therefore, there is a need for research on isoflavones to move toward
nutritional genomics, that is, the application of high-throughput genomics tools
in nutrition research (73). Careful application of this suite of technologies in
nutrition may help an increasing understanding of how nutrition influences meta-
bolic pathways and homeostatic control; how regulation (genomics) is disturbed
in early diet-related disease development; the extent to which individual sensitiz-
ing genotypes contribute toward such diseases (proteomics and transcriptomics)
and the impact on overall metabolism and output in disease status and develop-
ment (metabonomics). It is likely that the application of these technologies will
significantly contribute toward the goal of developing effective dietary interven-
tion strategies on the basis of isoflavones and other phytochemicals and the
prevention of diet-related diseases such as hormonally related cancers.
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16
Effect of Ginkgo biloba Extract
EGb 761 on Differential Gene
Expression in the Brain
Rainer Cermak and Siegfried Wolffram

Chemical Composition of EGb 761 341

In Vitro Effects of EGb 761 on Differential Gene Expression
in Neuronal Cells 344
In Vivo Effects of EGb 761 on Differential Gene Expression in the Brain 346
Conclusion 349
References 349
CHEMICAL COMPOSITION OF EGb 761

The leaves of Ginkgo biloba L. contain a variety of chemical compounds, some
of which are unique to this ancient tree. Various substances can be found in its
leaves, among which flavonoids and terpenoids are probably the most interesting
components (1 – 3). In dried Ginkgo leaves, the content of flavonoid glycosides
is 0.5– 1% w/w, whereas the content of terpenoids is often ,0.1% w/w (2).
The phenolic aglycone moiety of the flavonoid glycosides is made up pre-
dominantly of the flavonols quercetin or kaempferol, the occurrence of isorham-
netin being significantly smaller. Other flavonoids are found only in very small
341
342 Cermak and Wolffram
amounts. The glycoside moiety consists of mono- or diglycosides (mainly

D -glucose and L -rhamnose units) that are connected to the aglycone via
O-b-glycosidic bonds. Some of these glycosides are additionally acylated by
p-coumaric acid (Fig. 16.1). These latter compounds are distinctive to Ginkgo
biloba leaves and extracts (4,5).
In addition to flavonoids, compounds that belong to the group of terpenoids
are of particular interest. This group contains the ginkgolides with a diterpenoid
structure and the sesquiterpenoid bilobalide, these being unique to Ginkgo biloba
(Fig. 16.2). Five different ginkgolides have been isolated, of which ginkgolides
A, B, C, and J are found in the leaves; the fifth, ginkgolid M, occurs solely in
the roots. Ginkgolides are hexacyclic diterpenes possessing three lactone rings
forming electrophilic cage-like structures that enable them to interact with
cations and positively charged residues of other molecules. Bilobalide, a
C15-trilactone, is considered to be a degradation product of the ginkgolides. Its
concentration in Ginkgo biloba leaves is as high as that of all the ginkgolides
(1,2,5).
R2
OH
R1O O
OH
O
OH O
H3C O O
HO
HO O O
O
HO OH
OH
Coumaroyl flavonol glycoside R1 R2
Quercetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside] H OH
Kaempferol 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside] H H
Isorhamnetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside] H O-CH3
Quercetin 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]-
7-O-β-D-glucoside glucoside OH
Kaempferol 3-O-α-L-[6-p-coumaroyl-(β-D)-glucosyl-(1,2)-rhamnoside]-
7-O-β-D-glucoside glucoside H
Figure 16.1 Structures of some flavonol glycoside esters with coumaric acid, distinctive
compounds of Ginkgo biloba. The coumaroyl esters of the quercetin-rhamnoglucoside and
of the kaempferol-rhamnoglucoside are most common in Ginkgo leaves.
Effect of Ginkgo biloba Extract EGb 761 343
A O
O Ginkgolide R1 R2 R3
HO H
H H
O C(CH3)3 A OH H H
O
H R2 O H B OH OH H
H
H C OH OH OH
H3C R1 H
R3 J OH H OH
O O
M H OH OH
B O
O
HO
C(CH3)3
O OH
O
O O
Figure 16.2 Structures of ginkgolides (A) and bilobalide (B) found in leaves of Ginkgo
biloba. Ginkgolide M is only present in the roots.
In addition to the preceding substances, a large variety of polysaccharides,

long-chain hydrocarbons, alcohols, acids, and so on have been isolated (1,2). The
cytotoxic, mutagenic, and allergenic potential of some constituents, especially
anacardic or ginkgolic acids, limits the use of a crude leaf extract for therapeuti-
cal or prophylactic purposes in humans (6). Thus, several extraction processes
have been developed for the removal of toxic substances and concomitant enrich-
ment of desired compounds. The extract EGb 761, formerly patented by
Dr. W. Schwabe GmbH & Co, Karlsruhe, Germany, contains 22 – 27% flavone
glycosides, 5 –7% terpenoids (2.8 – 3.4% ginkgolides A, B, C and 2.6 – 3.2% bilo-
balide), and not more than 5 ppm ginkgolic acids (7). This has been the most
widely used extract of Ginkgo biloba leaves in clinical studies and is the basis
for many commercially available medicinal products of Ginkgo biloba.
In Germany and France, EGb 761 is widely prescribed in cases of cerebral
insufficiency. In many other countries, it is sold over-the-counter with the objec-
tive to improve brain performance and to counteract cerebral insufficiency as a
consequence of ageing. EGb 761 is thought to improve the memory of elderly
subjects. In addition, several clinical studies claim a therapeutic or prophylactic
effect against dementia, especially against Alzheimer’s disease [reviewed in
Refs. (8 –12)].
In an effort to search for mechanisms that could explain these clinical
observations at a cellular and molecular level, a multitude of effects have been
described for EGb 761 or for some of its constituents. It is well known that the
ginkgolides are high-affinity competitive platelet activator factor (PAF) receptor
antagonists. This enables them to potently inhibit PAF-induced platelet aggrega-
tion and could explain some of its neuroprotective activity (13,14). Another well
described feature of EGb 761 is its antioxidative activity. Several studies demon-
strated a protective effect of the extract on neuronal damage induced by oxidative
radicals (15 – 18). Although it is thought that the flavonoids are mainly respon-
sible for the antioxidative effects, ginkgolides and bilobalides are also potential
radical scavengers (19). In addition, EGb 761 acts vasodilatatory and, thus,
increases peripheral and cerebral blood flow, a quality that diminishes ischemic
brain damage (20 –23). Various other actions of EGb 761 have been described,
among them a prevention of age-related decreases in neurotransmitter density
and a decrease in the expression of the peripheral benzodiazepine receptor,
which are reviewed elsewhere (24 – 26).
In recent years, it has become obvious that the extract and some of its con-
stituents are also able to modify the expression of cellular proteins at the tran-
scriptional level. These observations open a new avenue for possible beneficial
actions and future applications of EGb 761 and will be described in the following
sections. In this review, only studies suggesting gene modulatory effects of
Ginkgo biloba in the brain or in neurons will be considered.
IN VITRO EFFECTS OF EGb 761 ON DIFFERENTIAL GENE

EXPRESSION IN NEURONAL CELLS
It is widely known that Ginkgo biloba extract EGb 761 has potent antioxidant
properties achieved by either scavenging free radicals or chelating prooxidant
transition metals (25). As reactive oxygen species (ROS) are involved in the regu-
lation of the expression of a large number of genes, it is likely that EGb 761
modulates genes that are under the control of redox-sensitive transcription
factors such as NF-kB, AP-1, and Nrf-1 (27,28).
Several studies have reported alterations of gene expression induced by the
Ginkgo biloba extract or by some of its constituents in neuronal-like PC12 cells
(pheochromocytoma cells differentiated with nerve growth factor). EGb 761 was
able to rescue PC12 cells submitted to oxidative stress induced by the toxin
1-methyl-4-phenyl-pyridine (MPPþ) (29). A flavonoid-containing fraction of
EGb 761, Cp 202, which is devoid of the terpenoids, was as effective as the
whole extract in counteracting MPP þ -induced cytotoxicity. Both EGb 761
and Cp 202 also decreased the protein expression of the high-affinity dopamine
transporter, which is responsible for uptake of MPPþ into dopaminergic
neurons. Although the reduced protein expression of this transporter can
explain the protective effect of EGb 761 and Cp 202 in PC12 cells, the mechan-
ism by which the plant extracts altered its expression remains unknown (29). In
this context, it is interesting to note that an inhibitory activity of EGb 761 on the
uptake of dopamine into synaptosome-enriched fractions of rat brain has been

reported (24).
EGb 761 also showed anti-apoptotic properties in PC12 cells grown in
serum-deprived medium in order to induce apoptosis (30). The extract inhibited
several steps of the apoptotic process like mitochondrial cytochrome c release,
caspase 3 activation, and DNA fragmentation. A DNA microarray analysis in
those cells demonstrated an increased amount of the transcript for an anti-
apoptotic Bcl-2-like protein after treatment with EGb 761. This was verified
on the protein level with Western blots showing that the expression of Bcl-2
protein was significantly up-regulated by the plant extract. Other transcripts of
genes involved in apoptosis were also altered in the microarray study. However,
these changes were only moderate and not verified by other methods. Thus, at
least a part of the anti-apoptotic activity of EGb 761 could be explained by its
effects on the transcription of relevant genes.
As complex I of the mitochondrial respiratory chain, the enzyme NADH
dehydrogenase plays a key role in energy metabolism and oxidative phosphoryl-
ation. In differentiated PC12 cells, EGb 761 and bilobalide increased the tran-
script for complex I (31). In these cells, bilobalide also increased the transcript
of another mitochondrial respiratory chain enzyme, namely of cytochrome c
oxidase subunit III (32). The latter observation was confirmed in gerbil brains
in vivo (discussed subsequently). These results suggest that EGb 761 and bilo-
balide could counteract defective energy metabolism and ATP depletion by an
increased expression of respiratory chain enzymes. This is of special relevance
for neurons owing to their high energy demand.
In primary cultures of cortical neurons obtained from mice embryos, EGb
761 increased the protein expression of heme oxygenase 1 (33). Heme oxygenase
acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide,
and biliverdin. Under physiological conditions, iron generated from heme degra-
dation is captured by ferritin. Thus, increasing heme degradation by increased
heme oxygenase expression could be a mechanism by which the concentration
of prooxidant iron is reduced and the generation of ROS is attenuated. Besides
the established antioxidant properties of EGb 761—achieved by scavenging
free radicals or chelating prooxidant transition metals like iron (25)—altering
the expression of antioxidant enzymes is a rather novel mechanism by which
the extract could counteract disturbances of cellular redox homeostasis.
An effect of EGb 761 on the expression of enzymes involved in antioxidant
defense was also demonstrated in a study investigating differential gene
expression in human hNT neurons using a cDNA macroarray (34). Cytosolic
superoxide dismutase 1 (CuZn SOD), which catalyzes the dismutation of the
oxygen radical superoxide to hydrogen peroxide, was upregulated. Surprisingly,
glutathione reductase (which regenerates reduced glutathione from diglutathione)
and glutathione S-transferase pi (which neutralizes electrophilic compounds able
to generate ROS) were downregulated. In accordance with the macroarray results,
the enzyme activities of glutathione reductase and glutathione S-transferase pi
were also lower after treatment with EGb 761. Whereas the mRNA levels of CuZn
SOD were increased, the activity of this enzyme was also lower in EGb 761 treated
cells. Thus, antioxidant defense was rather weakened by the Ginkgo biloba extract
in hNT neurons. In addition, the macroarray revealed an effect of EGb 761 on
genes encoding transcription factors (increase of NF-kB p65 subunit and zinc
finger protein 91 mRNAs, decrease of c-myc oncogene transcripts) and genes
involved in stress reponses (upregulation of 70 kDa heat shock protein 1).
However, the expression of those latter genes was not verified by RT-PCR or at
the protein level (34).
Compounds of Ginkgo biloba also affected gene expression in glial cells,
which account for the majority of cells in the brain. Altered expression of
genes in microglia and astrocytes have been described (35,36) Advanced glyca-
tion endproducts (AGE), which accumulate under neuroinflammatory conditions,
were shown to increase mRNA and protein levels of the inducible nitric oxide
synthase (iNOS) in the murine microglial cell line N-11, leading to increased pro-
duction of nitric oxide. EGb 761 prevented AGE-induced upregulation of iNOS
expression and nitric oxide production in these microglial cells. This suggests
that the extract might have anti-inflammatory activities in the brain (35).
In rat astrocytes, bilobalide induced the expression of glial cell line-derived
neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) at the
mRNA and protein level (36). VEGF is able to induce angiogenesis. GDNF has
the ability to promote survival of substantia nigra dopamine neurons. Thus, bilo-
balide may be an interesting drug in the treatment of Parkinson’s disease (37).
IN VIVO EFFECTS OF EGb 761 ON DIFFERENTIAL GENE

EXPRESSION IN THE BRAIN
The earlier mentioned effects were observed after direct treatment of neuronal
and glial cells with EGb 761 or its constituents in vitro. One has to bear in
mind, though, that these compounds have to cross the blood – brain barrier
(BBB) in order to act on central neurons in vivo. At present, hardly any data
exist that demonstrate transfer of EGb 761 constituents into brain tissue.
Recently, some studies have suggested that certain components of EGb 761,
namely flavonoids, might indeed be able to penetrate the BBB (38,39). More
importantly, some in vivo studies have demonstrated that EGb 761 has the poten-
tial to alter gene expression in central neurons, thereby indirectly proving that its
constituents or their metabolites reach the CNS.
In an animal model of global brain ischemia, EGb 761 and bilobalide
protected hippocampal neurons from death in gerbils (22). EGb 761 (25 –
100 mg/kg) or bilobalide (3 or 6 mg/kg) were administered orally to the
animals for 7 days before the gerbils were subjected to ischemia. The treatment
with EGb 761 and bilobalide prevented ischemia-induced reductions of cyto-
chrome c oxidase subunit III mRNA. A stimulatory effect of bilobalide on
mRNA expression of this enzyme was already observed in PC12 cells (32).
Thus, orally ingested bilobalide seems to protect neurons by an increased

expression of mitochondrial respiratory chain enzymes, thereby probably pre-
venting failure of energy production by oxidative phosphorylation in ischemic
brains.
Another study suggested that the Ginkgo biloba extract was able to alter the
mRNA levels of the glucose transporter subtype GLUT3 in rat hippocampal
neurons (40). When rats received 50 mg EGb 761 per day together with their
regular diet over a period of 19 days, the transcript of this glucose transporter
was upregulated in a subpopulation of rats designated as “poor performers”
because of their weak performance in memory tests. This was demonstrated by
in situ hybridization of GLUT3 mRNA in hippocampal brain sections. The
authors suggested that this effect of EGb 761 could increase the neuronal
glucose supply. The question remains open as to whether an improved energy
supply via an increased expression of glucose transporters is a mechanism by
which EGb 761 may affect hippocampal memory and learning processes.
In a study using bulbectomized mice, it was shown that treatment with EGb
761 also affects the expression of proteins involved in neurogenesis (41). In this
in vivo model, globose basal cells in the olfactory epithelium start to proliferate
after ablation of the olfactory bulb and their progeny differentiates to neurons.
Such proliferating neurons increase the expression of the proliferating cell
nuclear antigen protein (PCNA). The increase in PCNA expression occurred
2 days sooner in bulbectomized mice treated with EGb 761 in their drinking
water (100 mg/kg b.w.) compared with control animals. This accelerated
PCNA expression after bulbectomy indicated an earlier onset of proliferation.
Three weeks after bulbectomy, protein expression of the growth associated
protein 43 (GAP-43), which is a marker for postmitotic, differentiated neurons,
was also higher in EGb 761 treated mice. This could be linked to the enhanced
proliferation observed in these animals, yielding more cells to enter the differen-
tiation pathway. Thus, EGb 761 appears to modulate the balance between
proliferation and differentiation in neurons, which is visible in an altered
expression of proteins involved in these processes.
Exposure of rats to heat stress leads to leakage of the BBB, brain edema
formation, and cell injury with a concurrent increase in the protein expression
of constitutive NO synthase (cNOS) and causes expression of iNOS (42).
Increased synthesis of NO is thought to be involved in the brain injury after
hyperthermia. When EGb 761 (50 mg/kg) or ginkgolide B (2 mg/kg) was
orally applied to rats for 5 days prior to heat stress, brain edema formation and
BBB permeability were significantly attenuated. In addition, both treatments atte-
nuated the increase of cNOS and iNOS expression in several brain regions (42).
To gain further insight into the biochemical effects of Ginkgo biloba in the
CNS, a recent study profiled the transcriptional effects of the extract on the brains
of mice using oligonucleotide microarrays (43). The effects of EGb 761 on gene
transcription were measured in the hippocampus and cerebral cortex of adult
mice fed a diet with or without Ginkgo biloba extract (300 mg EGb 761/kg
diet) over a period of 4 weeks. Plasma concentrations of the flavonols quercetin,

kaempferol, and isorhamnetin were measured by HPLC (44,45). In EGb 761
treated mice, the concentrations of these flavonols were significantly higher
than those of the control group, confirming the absorption of at least some of
the major components of the extract into the systemic circulation.
The microarrays used represent all (6000) sequences in the Mouse
UniGene database that have been functionally characterized, as well as around
6000 expressed sequence tags (EST) clusters. Of the 12,000 combined genes
and ESTs represented on the array, only 10 changed in expression level by a
factor of three-fold or more and all were upregulated. In the cortex, mRNAs
for neuronal tyrosine/threonine phosphatase 1 and microtubule-associated
tau were significantly enhanced. These proteins are both associated with
formation –breakdown of intracellular neurofibrillary tangles, a hallmark lesion
of Alzheimer’s disease. Hyperphosphorylated tau has been found to be the
major protein of these neurofibrillary tangles, possibly because of an imbalance
in tau kinase and phosphatase activities in the affected neurons (46). Hyperpho-
sphorylated tau isolated from brains of patients with Alzheimer’s disease has
been shown to be efficiently dephosphorylated in vitro by protein phosphatases
1, 2A, and 2B. Thus, upregulation of neuronal phosphatase 1 by EGb 761
could play a neuroprotective role in the brain. Selective inhibition of protein
phosphatase 2A by okadaic acid in metabolically competent rat brain slices
has been shown to induce a hyperphosphorylation and accumulation of tau like
that in Alzheimer’s disease (47,48).
The expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-
acid-2 (AMPA-2), calcium and chloride channels, prolactin, and growth
hormone (GH) were also upregulated in the cortex. Within the past decade,
studies have revealed that GH may exert significant effects on the central
nervous system. Cognitive impairments are well documented hallmark features
of GH deficiency, and clinical studies have reported psychological improvements
(in mood and well being) and beneficial effects on certain functions including
memory, mental alertness, motivation, and working capacity in adults receiving
GH replacement therapy. Moreover, GH therapy in children deficient in this
protein has been reported to produce marked behavioral improvement (49).
In the hippocampus, only transthyretin mRNA was induced. Transthyretin
plays a role in the transport of tyroxine and retinol binding protein in the brain.
Thyroid hormones regulate neuronal proliferation and differentiation in discrete
regions of the brain during development and are necessary for normal cyto-
skeletal outgrowth (50). Transthyretin has also been shown in vitro to sequester
amyloid beta protein and, thus, to prevent amyloid beta aggregation and amyloid
formation (51). Moreover, transthyretin levels in cerebrospinal fluid have been
found to be significantly decreased in Alzheimer’s disease patients. Thus, one
mechanism whereby EGb 761 may exert neurological effects is the modulation
of transthyretin levels. This could have an impact on hormone transport or on
amyloid beta sequestration in the brain.
CONCLUSION
The mechanisms responsible for the beneficial effects of the Ginkgo biloba
extract EGb 761 are largely unknown. The observation that the extract influences
the expression of certain genes is a rather novel finding. Several studies have
shown that EGb 761 and some of its constituents affect gene expression in
neurons. Moreover, animal studies demonstrated the efficacy of the ingested
extract to alter in vivo gene expression in the brain. The consideration of the pro-
ducts of at least some of the genes over-expressed after EGb 761 treatment offer
new mechanistic explanations for the clinical effects of the extract. This is of
special relevance to certain neurological disorders like ischemic lesions or
Alzheimer’s disease. On the basis of these recent findings, future investigations
on the gene regulatory activities of Ginkgo biloba will provide a better under-
standing of the effects of this complex extract at the molecular level.
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17
Interactions of Flavonoids and
Their Metabolites with Cell
Signaling Cascades
Jeremy P. E. Spencer
Introduction 354
Potential Bioactive Forms of Flavonoids In Vivo 355
Metabolism in the GI Tract and Liver 356
Colonic Metabolism 356
Intracellular Metabolism 358
Modulation of Signaling Cascades by Flavonoids 359
MAP Kinase Signaling and Cell Function 360
Interactions of Flavonoids Within Signaling Pathways 361
Specific Actions of Flavanols 362
Specific Actions of Flavonols 365
Summary 367
References 367
Flavonoids have been proposed to act as beneficial agents in a multitude of

disease states, including cancer, cardiovascular disease, and neurodegenerative
disorders. The biological effect of these polyphenols will ultimately depend on
the cellular effects of their circulating metabolites, such as glucuronides and
353
354 Spencer
O-methylated forms and on the extent to which they interact with and/or associ-
ate with cells, either by interactions at the membrane or their uptake into the
cytosol. Following uptake, flavonoids and their metabolites may affect cells in
a number of ways: (i) by regulating the intracellular redox status, (ii) by effecting
enzyme function, (iii) by interacting with mitochondria, and (iv) by specific inter-
actions within intracellular signaling cascades vital to cell function. This review
will summarize the current knowledge on the role of flavonoids as modulators of
cell signaling pathways, such as the MAP kinase pathway, which are vital in
determining neuronal survival, regeneration, development, and death.
INTRODUCTION
Flavonoids have been proposed to act as beneficial agents in a multitude of
disease states (1,2), most commonly cancer (3–6), cardiovascular disease (6,7),
and neurodegenerative disorders (8 – 12). Epidemiological and dietary interven-
tion studies, in both humans and animals, have suggested that diet-derived
phenolics, in particular flavonoids, may play a beneficial role in the prevention
of neurodegeneration, age-related cognitive and motor decline (10,11), and
brain ischaemia/reperfusion injury (9). Much evidence also exists to support
the potential beneficial and neuromodulatory effects of flavonoid-rich Ginkgo
biloba extracts, such as EGb 761, in the CNS (13 –15). Clinical trials with
EGb 761, which is rich in the flavonoids kaempferol and quercetin, have
indicated beneficial effects on brain function, particularly in connection with
age-related dementias and Alzheimer’s disease (15,16). Furthermore, the neuro-
protective action of one of the green tea flavonoids, epigallocatechin gallate
(EGCG), has been shown in both oxidative stress- (17) and Ab-induced (18)
neuronal death models. Interestingly, the protective effects observed in both
systems were linked to a modulation in signaling through protein kinase C
and/or modulation of cell survival/cell cycle genes.
Much evidence has linked flavonoids to both beneficial and cytotoxic
effects. Such effects may be mediated in a number of ways: (i) by the regulation
of the intracellular redox status, (ii) by direct effects on enzyme function, (iii) via
the interaction with mitochondria, and (iv) by specific interactions within intra-
cellular signaling cascades vital to cell function. The latter has received much
interest over the last few years and this review will attempt to summarize
current knowledge on the role flavonoids play as modulators of cell signaling cas-
cades. One such pathway of interest is mitogen activated protein kinase (MAP
kinase) pathway, which is vital in determining cellular survival, regeneration,
development, and death. In doing so, however, it is essential to consider and inte-
grate knowledge on the potential bioactive forms of flavonoids in vivo. It is now
clear that dietary forms of flavonoids are not those found in the circulation and
indeed those exposed to tissues. Rather extensive modification of these polyphe-
nols occurs on uptake, which results in significant changes in their chemical and
biological properties. These circulating metabolites are thought to be important in
Interactions of Flavonoids 355
mediating cellular functions and consequently the initial part of this review will
summarize the processes that lead to the formation of in vivo metabolites from the
dietary forms.
POTENTIAL BIOACTIVE FORMS OF FLAVONOIDS IN VIVO

As mentioned earlier, it has become clear over the last few years that the bio-
active forms of flavonoids in vivo are not those forms found in plants, for
example, the aglycones or their various glycosides, but instead conjugates and
metabolites arising from these on absorption [reviewed in Refs. (19 – 23);
Fig. 17.1]. In particular, there is now strong evidence for the extensive phase I
de-glycosylation and phase II metabolism of the resulting aglycones such as
quercetin, hesperetin, naringenin, and epicatechin to glucuronides, sulfates, and
Neurons
glia
Dietary Flavonoid
Oligomers cells
Stomach
cleaved
Blood-brain
Oligomeric Monomeric barrier
Flavonoids units
O-methylated
A-ring glucuronides
jejunum
O-methylated Further
glucuronides metabolism
Sulphates
Small Intestine O-methylated Portal
vein glucuronides
ileum aglycone Liver
glucuronides
Colon
Kidney
Renal excretion
Flavonoid Phenolic acids of glucuronides
Gut microflora Urine
Figure 17.1 Summary of the formation of gastrointestinal tract and hepatic metabolites
and conjugates of flavonoids in humans. Cleavage of oligomeric flavonoids such as pro-
cyanidins may occur in the stomach in environments of low pH. All classes of flavonoids
undergo extensive metabolism in the jejunum and ileum of the small intestine and result-
ing metabolites enter the portal vein and undergo further metabolism in the liver. Colonic
microflora degrade flavonoids into smaller phenolic acids that may also be absorbed. The
fate of most of these metabolites is renal excretion, although, some may enter cells and
tissues.
356 Spencer
O-methylated forms during transfer across the small intestine (24) and then again
in the liver (Fig. 17.1). Further transformation has been reported in the colon
where the enzymes of the gut microflora degrade flavonoids to simple phenolic
acids (25), which may also be absorbed and subsequently further metabolized
in the liver.
Metabolism in the GI Tract and Liver

It is now well established that the gastrointestinal tract plays a significant role in
the metabolism and conjugation of these polyphenols before entry into the sys-
temic circulation and the liver (19,20,24,26 –28). Enterocytes in the jejunum
and ileum of the small intestine transfer flavonoids from the luminal side of
the gut to portal vein during which there is removal of sugar groups followed
by significant glucuronidation of nearly all flavonoids tested (Fig. 17.2) by the
action of b-glucosidase and UDP-glucuronosyltransferase enzymes (19 – 21). In
addition, in the case of catechol containing B-ring flavonoids, there is also exten-
sive O-methylation (Fig. 17.2) catalyzed by the action of COMT (24). For
example, the flavonoid glycosides, luteolin-7-glucoside, kaempferol-3-glucoside,
naringenin-7-glucoside, and quercetin-3-glucoside were all cleaved by rat jejunal
or ileal mucosa, before their efflux into the serosal fluid as glucuronides and/or
O-methylated metabolites (24,29). A full assessment of uptake and metabolism
of flavonoids has been well reviewed previously (19 –23). Interestingly, research
with O-methylated metabolites of flavonoids, such as those of epicatechin,
provided some of the initial evidence that flavonoids may not simply exert
cellular protection through antioxidant mechanisms (30 –32). Here, the protec-
tion elicited by 30 -O-methyl epicatechin against oxidative stress-induced cellular
damage was not significantly different from that of epicatechin, even though its
classical antioxidant nature is greatly reduced. These studies suggested the first
time that hydrogen-donating antioxidant activity is unlikely to be the sole
mechanism of protection against oxidative cellular damage and led to further
mechanistic investigations involving their actions within signaling cascades (33).
Colonic Metabolism
Evidence suggests that the extent of absorption of dietary polyphenols in the
small intestine is relatively low (10 –20%) (24,28,34). The implications of this
low absorption in the small intestine means that the majority of ingested polyphe-
nols, including those absorbed and conjugated in the enterocytes and/or the liver
and transported back into the lumen, either directly or via the bile (35), will reach
the large intestine where they encounter the colonic microflora. The colon con-
tains 1012 microorganisms/cm3, which has an enormous catalytic and hydro-
lytic potential, and this enzymatic degradation of flavonoids by the colonic
microflora results in a huge array of new metabolites. For example, bacterial
enzymes may catalyze reactions including hydrolysis, dehydroxylation, demethy-
lation, ring cleavage, and decarboxylation as well as rapid de-conjugation (25).
A OH B OH
OH O
CH3
HO O HO O
OH OH
OH OH
OH
C
OH D OH OH
E
OH OH
HO O
-
NaO3SO O
COO
OH
O
O OH O
OH
OH
OH O
OH
F G
O
O N O
N OH
NH HO OH OH OH
NH2 S
COOH
O HO O
OH
OH O
Figure 17.2 Structure of epicatechin and its major circulating metabolites: (A)
epicatechin, (B) 30 -O-methyl epicatechin, (C) epicatechin-5-O-b-D -glucuronide, (D) epi-
catechin-7-sulfate, (E) (2)-5-(30 ,40 -dihydroxyphenyl)-g-valerolactone, (F) hippuric acid,
(G) 8-glutathionyl quercetin. Glucuronide and sulfate conjugates are formed with the
majority of flavonoids in the small intestine and liver, whereas O-methylated forms are
only formed where the flavonoid has a catechol B-ring. (B)– (D) represent GI tract and
hepatic metabolites, whereas (E) and (F) derive from metabolism by the enzymes of the
colonic microflora and (G) may result from intracellular metabolism.
Unlike human enzymes, the microflora catalyze the breakdown of the flavonoid
backbone itself to simpler molecules such as phenolic acids. Specific metabolites
have been observed in urine after consumption of a variety of phenolics, for
example, the glycine conjugate of benzoic acid, hippuric acid (Fig. 17.2). This
metabolite is primarily derived from plant phenolics and aromatic amino acids
through the action of intestinal bacteria and, consequently, the level of hippuric
acid would be expected to increase in the circulation and urine of individuals
consuming diets rich in flavanols or polyphenols.
The 5,7,3,30 ,40 -hydroxylation pattern of flavanols is believed to enhance
ring opening after hydrolysis (25) and metabolism of flavanols by enzymes of
358 Spencer
the microflora of the large intestine results in many metabolites: 3,4-dihydro-

phenylacetic acid, 3-hydroxyphenylacetic acid, homovanillic acid, and their con-
jugates derived from the B-ring (25) and phenolic acids from the C-ring. Because
flavanols have no C-4 carbonyl group in their structures, they may also be
degraded by colonic bacteria to the specific metabolites phenylvalerolactones,
such as (2)-5-(30 ,40 -dihydroxyphenyl)-gamma-valerolactone (36) (Fig. 17.2).
Indeed, valerolactones derived from colonic metabolism are now believed to
be major in vivo metabolites of flavanol or tea ingestion in humans and
animals (37 – 41). These colonic metabolites, such as valerolactones, may rep-
resent novel bioactive metabolites of flavonoids in vivo, and should be considered
as possible mediators of flavonoid action in the body. Thus far, there is no data
describing the cellular effects of such metabolites, although interest is growing
in these somewhat forgotten in vivo forms.
Intracellular Metabolism
The cellular effects of flavonoid metabolites will ultimately depend on the
extent to which they associate with cells, either by interactions at the membrane
or uptake into the cytosol. Information regarding uptake of flavonoids and
their metabolites from the circulation into various cell types and whether they
are modified further by cell interactions has become increasingly important as
attention focuses on the new concept of flavonoids as potential modulators of
intracellular signaling cascades vital to cellular function.
The uptake of flavonoids and their in vivo metabolites is dependent on cell
type (30). However, this is most probably due to a greater level of intracellular
metabolism and faster rate of export from some cells rather than simply differing
levels of passive diffusion into the cell. Generally, flavonoids may undergo three
forms of intracellular metabolism: (i) conjugation with thiols, particularly GSH,
(ii) oxidative metabolism, and (iii) P450-related metabolism. For example, the
intracellular metabolism of quercetin in human dermal fibroblasts has been
shown to involve the formation of intracellular oxidation products, the generation
of 20 -glutathionyl quercetin (Fig. 17.2) and the demethylation of O-methylated
forms of quercetin (42). In contrast, epicatechin and its O-methylated metabolites
entered fibroblasts to a lesser extent and do not undergo measurable cellular
metabolism (30). Furthermore, though neurons also accumulate flavonoids and
O-methylated forms over time, this uptake is low compared to that in astrocytes
where there is also extensive intracellular metabolism detected. Cell-generated
metabolites, such as 20 -glutathionyl quercetin are of interest as they may be
capable of mediating cellular effects whether beneficial or toxic in cells.
Interestingly, no glucuronide metabolites of flavonoids tested thus far,
associate with, or are taken up by, cells (30,42), which is most probably because
of the hydrophilic nature of these metabolites. This clearly has implications for
flavonoid action in vivo as glucuronide metabolites appear as the most abundant
following flavonoid ingestion. It therefore appears that glucuronide metabolites
may only be able to influence cell processes via membrane interactions and not by
directly interacting with signaling proteins intracellularly. However, there is the
possibility that the glucuronides of flavonoids may be cleaved in vivo under
local conditions of neuroinflammation or during inflammatory processes per se
and the free aglycone go on to express cellular effects. Indeed, b-glucuronidases
are present in a number of tissues within the body (43) and may be released by
certain cells. For example, histamine causes rapid exocytosis of b-glucuronidase
from lung macrophages (44) and luteolin monoglucuronide is cleaved to free
luteolin by b-glucuronidase released from neutrophils stimulated with iono-
mycin (45,46).
MODULATION OF SIGNALING CASCADES BY FLAVONOIDS

The precise mechanisms by which flavonoids exert their beneficial or toxic
actions remain unclear. However, as mentioned earlier recent studies have specu-
lated that their classical hydrogen donating antioxidant activity (47 – 49) is unli-
kely to be the sole explanation for cellular effects (30,31,33). This premise is
based on a number of lines reasoning. First, as discussed in “Potential Bioactive
Forms of Flavonoids In vivo,” flavonoids are extensively metabolized in vivo
resulting in a significant alteration in their redox potentials. Indeed, circulating
metabolites of flavonoids, such as glucuronides or O-methylated forms, and intra-
cellular metabolites, for example flavonoid –GSH adducts, have a reduced ability
to donate hydrogen ions (30) and are less effective scavengers of reactive
oxygen and nitrogen species relative to their parent aglycone forms (Spencer
et al., unpublished observations). Indeed, studies have indicated that although
glucuronides, sulfates, and O-methylated metabolites may participate directly
in plasma antioxidant reactions and by scavenging reactive oxygen and nitrogen
species in the circulation, their effectiveness is reduced relative to their parent
aglycones (50 – 54).
Second, concentrations of flavonoids and their metabolite forms accumu-
lated in vivo, for example in the plasma or in organs such as the brain (55) are
lower than those recorded for well known small molecule antioxidants such as
ascorbic acid and a-tocopherol (56). Consequently, flavonoids are unlikely to
express beneficial action in vivo by out-competing antioxidants like vitamin C,
which are present at much higher concentrations. Recent experiments have indi-
cated that flavonoids are capable of protecting neurons against oxidative stress
more effectively than ascorbate even when the latter was used at 10-fold
higher concentrations (32).
Third, other biological activities of flavonoids have emerged which suggest
additional cellular mechanisms of action. Perhaps most prominently, these
include their ability to bind to ATP-binding sites of proteins (57), such as mito-
chondrial ATPase (58), calcium plasma membrane ATPase (59), protein kinase A
(60), protein kinase C (61 –65), and topoisomerase (66). In addition, binding to
the benzodiazapine binding sites of GABA-A and adenosine receptors (67,68)
360 Spencer
has been reported and is of interest with regards to their potential abilities to inter-
act within signaling pathways. They may also interact with mitochondria, directly
effect enzyme activity (69), interfere with pathways of intermediary metabolism,
and down-regulate the expression of adhesion molecules (70 – 72). An example of
such action is illustrated by the inhibition of protein kinases by resveratrol and
several flavonoids, including the citrus flavanones hesperetin and naringenin
(73 – 75). This inhibition is mediated via the binding of the polyphenols to the
ATP binding site, presumably causing 3D structural changes in the kinase and
consequently its inactivity.
Finally, recent evidence suggests that the cellular effects of flavonoids may
be mediated by their interactions with specific proteins central to intracellular
signaling cascades (8). In particular, investigation has indicated that they may
interact selectively within the MAP kinase signaling pathway (76,77) (Fig. 17.3).
The next sub-section details the pivotal position the MAP kinase signaling
pathway holds in determining the fate of a cell.
MAP Kinase Signaling and Cell Function

The modulation of the MAP kinase signaling pathway is significant as members
of the MAPK family such as the extracellular signal-related kinase (ERK) and
Signal Signal
Plasma
Small membrane
G-protein
MAPKKKs/ MAPKK PI3-kinase

Ca(II)
Pro-survival
MAPK
pathway
p38 JNK ERK Akt/PKB
Cytosolic Nuclear
Targets Targets
Neuronal Death/Survival
Figure 17.3 Diagrammatic representation of the MAP kinase and Akt/PKB signaling
pathways. Extracellular signal-related kinase (ERK) and c-jun amino-terminal kinase
(JNK) are involved in signaling to neuronal survival, regeneration, development, and
death. ERK and JNK are generally considered as having opposing actions on neuronal
apoptosis, with signaling through ERK usually regarded as pro-signaling and JNK pro-
apoptotic. The serine/threonine kinase, Akt/PKB, is one of the main downstream effectors
of phosphatidylinositol 3-kinase (PI3-kinase) and a pivotal kinase in neuronal survival.
c-jun amino-terminal kinase (JNK) are involved in signaling to neuronal survival,

regeneration, development, and death (78 –82) (Fig. 17.3). ERK and JNK are
generally considered as having opposing actions on neuronal apoptosis (83).
ERK1/2 are usually associated with pro-survival signaling (84 – 86) through
mechanisms that may involve activation of the cyclic AMP regulatory-binding
protein (CREB) (85,87), the up-regulation of the anti-apoptotic protein Bcl-2
and nontranscriptional inhibition of BAD (85,86). On the other hand, JNK has
been strongly linked to transcription-dependent apoptotic signaling (81,82), pos-
sibly through the activation of c-Jun (88) and other AP-1 proteins including JunB,
JunD, and ATF-2 (89).
Oxidative stress has a diverse effect of signaling pathways in cells, in
particular the MAP kinase cascade (90 –92). Changes in the cellular redox status
may result in the activation of pro-apoptotic signaling proteins such as JNK
(8,89,93– 95), which may initiate the apoptotic mechanism within cells (96).
Additionally, oxidative stress may affect mitochondria by influencing the mito-
chondrial transition pore (mPT) and/or release of cytochrome c (97,98). There
is strong evidence linking the activation of JNK to neuronal loss in response to
a wide array of pro-apoptotic stimuli both in developmental and degenerative
death signaling (81,89). In the context of oxidative insults in neurons, JNK has
been shown to be activated by dopamine (99), by 4-HNE (100 –102) and
through reduced expression of SOD1 (103). Alternatively, the modulation of
signaling through the serine/threonine kinase, Akt/PKB, one of the main down-
stream effectors of phosphatidylinositol 3-kinase (PI3-kinase) and a pivotal
kinase in neuronal survival (104 – 107), may also be important (Fig. 17.3).
Indeed, activation of Akt in some neurons has been shown to lead to an inhibition
of proteins central to the cell death machinery, such as the pro-apoptotic Bcl-2
family member, BAD (108), and members of the caspase family (85,104) that
specifically cleave poly(ADP-ribose) polymerase (105,109), thus promoting
cell survival. BAD itself is regulated by phosphorylation of two serine residues,
Ser112 and Ser136 (108), and several studies have revealed that the Ser136 site
can be specifically phosphorylated by Akt/PKB (110,111).
Interactions of Flavonoids Within Signaling Pathways

There are a number of potential sites within signaling pathways where flavonoids,
or their metabolites, may interact (Fig. 17.4). Flavonoid-mediated inhibition of
oxidative stress-induced apoptosis may occur by preventing the activation of
JNK, for example by influencing one of the many upstream MAPKKK activating
proteins that transduce signals to JNK (Fig. 17.3). Alternatively, they may act by
maintaining calcium homeostasis, which is important in MAP kinase activation
(112,113). They may also interact directly with mitochondria, for example by
modulating the mPT, which controls cytochrome c release during apoptosis
(114,115), or by modulating other mitochondrial associated pro-apoptotic
factors such as DIABLO/smac (116,117). Potential interactions with the mPT
362 Spencer
Oxidative Stress
Mitochondrial MAPK
dysfunction Signaling
Apoptosis
Necrosis
Degenerative
Diseases
Figure 17.4 Relationship between oxidative stress, signaling, mitochondrial function,

and cell death/survival. Evidence suggests that flavonoids and/or their in vivo metabolites
may protect cells against oxidative stress by influencing the intracellular redox status, by
modulating mitochondrial function or by interacting with signaling pathways. Specific
actions within signaling pathways are now believed to be partly responsible for both the
beneficial and/or cytotoxic effects of flavonoids on cells.
are especially interesting, as the transition pore possesses a benzodiazapine-

binding site where flavonoids may bind (67,68) and influence pore opening
and cytochrome c release during apoptosis.
Interestingly, pharmacological inhibitors of intracellular signaling cas-
cades have similar structures to flavonoids, such as quercetin. For example,
analogs of quercetin such as LY294002 have been developed as potent PI3-
kinase inhibitors (118 –120) and the MEK inhibitor, PD98059, is closely
related to the basic flavonoid structure (Fig. 17.5). Quercetin and other flavonoids
have also been shown to inhibit PI3-kinase (61,121,122) with inhibition directed
at the ATP-binding site of the kinase (118,123). The quercetin analog LY294002
completely and specifically abolishes PI3-kinase activity but not PI4-kinase or
other protein and lipid kinases (118). As PI3-kinase appears to be centrally
involved with growth factor signal transduction, the inhibition of this kinase
may be beneficial in the treatment of proliferative diseases as well as in elucida-
ting the biological role of the kinase in cellular proliferation and growth factor
response.
Specific Actions of Flavanols

Much interest has centered on the beneficial effects of flavanols, such as epicate-
chin, epicatechin gallate (EGC), and epigallocatechin gallate (EGCG) and there
is growing evidence that the cytoprotective nature of these polyphenols is based
on their interactions within signaling pathways. For example, epicatechin and one
OH
OH
O
HO O O N
OH
OH O O
Quercetin LY294002
OH
H2N
O PD98059
Figure 17.5 Structural similarity between quercetin and the PI3-kinase inhibitor,
LY294002, and the MEK inhibitor, PD98059. Both kinase inhibitors show structures
that are closely related to the basic flavonoid backbone structure.
of its major in vivo metabolites, 30 -O-methyl epicatechin, have been shown to

elicit strong cytoprotective effects against oxidative stress in fibroblasts and
neurons (30 – 32). Importantly, the cytoprotective action of these compounds
was found to involve both the inhibition caspase-3 activation (31,33) and acti-
vation of pro-apoptotic MAPK proteins (33). In particular, both epicatechin
and 30 -O-methyl-epicatechin acted to protect neurons against oxLDL-induced
activation of JNK, c-jun, and pro-caspase-3. The inhibition of JNK by flavonoids
would lead to removal of c-jun/AP-1 mediated regulation of pro-apoptotic
proteins, such as Bax, eventual mitochondrial dysfunction and activation of
caspases. Interestingly, both epicatechin and its O-methylated metabolite had
no effect on the ox-LDL-mediated increase in intracellular oxidative stress and
were more protective than 10-fold higher concentrations of ascorbate. These
studies strongly suggest that protection in this model is not mediated primarily
by antioxidant processes. In another study, the neuroprotective mechanism of
another flavanol, EGCG, against oxidative stress-induced cell death was also
found to involve modulations of signaling proteins. Here, EGCG caused a stimu-
lation of PKC and a modulation of cell survival/cell cycle genes, such as Bax,
Bad, Mdm2, Bcl-2, Bcl-w, and Bcl-x(L) (17). Together, these investigations
suggest that protection is likely to be partly mediated through specific actions
within signaling pathways, although at this time it remains unclear exactly
where such interactions occur within the pathway.
364 Spencer
There has been huge interest in the molecular mechanisms by which flavo-
noids may affect growth-related signal transduction pathways involved in the
progression of cancer (124). Their actions can be divided into two main cat-
egories: (i) their ability to stimulate apoptosis of cancer cells but not normal
cells (125) and (ii) their inhibition of signaling pathways involved in the pro-
gression of carcinogenesis. Many studies have tested for the ability of flavanols
such as EGCG to induce apoptosis and recently the mechanisms by which they do
this has begun to unravel. For example, the mechanism by which EGCG induces
apoptotic cell death in human leukemia U937 cells involves the ASK1, MKK,
and JNK/p38 cascade (95) and ERK activation plays an active role in mediating
EGCG plus vanadate-induced apoptosis of U937 cells (126). Furthermore, the
inhibition of ERK and JNK phosphorylation by EGCG has been proposed to
play a role in the inhibition of arsenite-induced apoptosis (127).
EGCG and other green tea polyphenols have been found to exert strong
inhibition of cell growth and AP-1 activity (128) and activation of ERK and
MEK1/2 (129) in cells transfected with a mutant H-ras gene to mimic carcino-
genesis in vitro (128). The ras gene mutation, which occurs frequently in many
cancer types, perpetually turns on the growth signal transduction pathway and
is associated with high endogenous levels of AP-1 (130). Other studies have pos-
tulated that the anti-cancer properties of EGCG may be mediated by the selective
inhibition of tyrosine phosphorylation of PDGF-Rb (131) or to the down-
regulation of NF-kappaB inducing kinase (NIK), death-associated protein
kinase 1, rhoB, and tyrosine-protein kinase genes and the up-regulation of the
gene for retinoic acid receptor alpha1 (132).
The treatment of breast cancer cells with epigallocatechin (EGC) has pro-
vided evidence that this flavanol may stimulate apoptosis by inducing a decrease
in the anti-apoptotic protein, Bcl-2, increases in the pro-apoptotic Bax, and
increases in caspase 8 and caspase 10 (125). Here, it was postulated that
EGC-triggered apoptosis in breast cancer cells might involve Fas death receptor
signaling. In another breast cancer cell line, EGCG was found to induce the
phosphorylation of JNK/SAPK and p38, which led to an inhibition of cdc2 phos-
phorylation, the regulated expression of cyclin A, cyclin B1, and cdk proteins
finally and G2 cell cycle arrest. EGCG has also been shown to inhibit the acti-
vation of the epidermal growth factor receptor (EGFR) and related signaling
pathways in head and neck squamous cell carcinoma (HNSCC) cells (133).
Data suggests that EGCG may inhibit both EGFR-related pathways of signal
transduction (134) and the activation of HER-2/neu and downstream signaling
pathways (135,136) in human head and neck and breast carcinoma cells. These
studies provide evidence that EGCG may be useful in treating cases of breast car-
cinoma, HNSCC, or other cancers where the activation of the signaling pathways
plays an important role in tumor survival and growth.
Many studies have suggested a role for EGCG and other flavanols in pre-
venting UV-induced skin damage. For example, EGCG has been found to be
effective in inhibiting UVB-induced oxidative stress-mediated phosphorylation
of ERK1/2, JNK, and p38 in normal human epidermal keratinocytes (137) and
mouse epidermal cells (138) and to markedly increase AP1 factor-associated
responses via a MAPK signaling mechanism (139). More recently, a cream
based formulation of green tea polyphenols, or EGCG alone, have been shown
to attenuate solar UVB light-induced oxidative stress-mediated ERK1/2, JNK
and p38 activation in SKH-1 hairless mouse skin (140). Interestingly, the
degree of protection exerted was greater when the flavanols were applied in
this topical form than when given orally, presumably because of their greater
bioavailability to skin target cells. These data help explain why there are a
growing number of commercial skincare products containing flavonoids or
flavonoid-extracts even though at this time, neither their mechanism of action
has been elucidated nor their beneficial effect has been critically evaluated. In
addition to modulations in the MAP kinase pathway, EGCG has also been
shown to protect against the adverse effects of UV radiation via modulations
in the NF-kappaB pathway (141). This pathway is known to play a critical role
in a variety of physiological functions and is involved in inflammation and the
development of skin cancer. These observations suggest that EGCG, a possibly
other flavanols may be useful in the attenuation of oxidative stress-mediated
and MAPK-induced skin disorders such as photo-carcinogenesis in humans.
The earlier studies provide evidence that flavanols such as epicatechin and
EGCG may exert beneficial and/or cytotoxic actions through their modulation of
the MAP kinase pathway and other signaling cascades. It has been postulated that
at the activation of pro-survival MAP kinase, signaling proteins may only occur
with relatively low concentrations of flavonoids (nM to low mM) and could lead
to antioxidant response element (ARE)-mediated gene expression, including that
of phase II detoxifying enzymes, such as b-glucuronosyltransferase. Increases in
such enzymes could lead to a more rapid and efficient removal of carcinogenic
agents in the body. In contrast, higher concentrations of flavonoids, may lead
to a sustained activation of MAP Kinases, such as JNK, which would result in
an induction of apoptosis (142). These mechanisms together with others, may
contribute to the overall chemopreventive function of EGCG and other flavanols
in vivo.
Specific Actions of Flavonols

Although the flavonol quercetin is one of the most frequently researched flavo-
noids, with both its beneficial (143,144) and deleterious effects (42,145 – 148)
on different cell types well described, its mechanism of action remains unclear.
Quercetin has been thoroughly investigated for its abilities to express antiproli-
ferative effects (149,150) and induce cell death predominantly by an apoptotic
mechanism in cancer cell lines (147,151 –153). For example, it has been
observed to induce caspase-3 activation in the malignant cell line HPB-ALL
(153) and activate caspase-3, caspase-9 and release cytochrome c in HL-60
cells (152) as well as induce chromatin and nuclear fragmentation in colonic
366 Spencer
cancer cells (150). On the other hand, quercetin treatment has been shown to
suppress JNK activity and apoptosis induced by hydrogen peroxide (154) and
4-hydroxy-2-nonenal (155). Furthermore, quercetin may evoke anti-apoptotic
effects via the suppression of the peroxide-induced JNK-c-Jun/AP-1 pathway
and the ERK-c-Fos/AP-1 pathway in cultured mesangial cells (156). The
ability of quercetin to inhibit both AP-1 activation and the JNK pathway (76)
has been shown to have relevance in both phorbol 12-myristate 13-acetate
(PMA)- and tumor necrosis factor-alpha (TNF-alpha)-induced ICAM-1 expres-
sion. As discussed earlier, it has been proposed that low concentrations of quer-
cetin, may activate the MAPK pathway (ERK2, JNK1, and p38) leading to
expression of survival genes (c-Fos, c-Jun) and defensive genes (Phase II detox-
ifying enzymes; glutathione-S-transferase, quinone reductase) resulting in survi-
val and protective mechanisms (homeostasis response). However, increasing the
concentrations of these compounds will additionally activate the caspase
pathway, leading to apoptosis (77).
Quercetin and its O-methylated metabolites have also been shown to be
toxic to primary cortical neurons via an inhibition of pro-survival protein
kinase cascades (146). Quercetin, and to a lesser extent its O-methylated metab-
olites, induced neuronal death via a mechanism involving direct inhibition of
survival signaling through Akt/PKB and ERK rather than by an induction of
the JNK-mediated death pathway. Prior to measurable losses of neuronal viability
and membrane integrity, quercetin was observed to stimulate a strong inhibi-
tion of basal Akt phosphorylation in cortical neurons that was both time- and
concentration-dependent. Furthermore, this inhibition of Akt phosphorylation
was apparent at both the regulatory serine 473 and catalytic threonine 308
sites, rendering it inactive. The inhibition of Akt/PKB phosphorylation by
quercetin may reflect potential inhibition of its upstream partner PI3-Kinase, as
has been described previously (122). The potent inhibition of both Akt/PKB
and ERK phosphorylation in this model was accompanied by a reduced phos-
phorylation of BAD and a strong activation of caspase-3 at later time points.
Interestingly, high quercetin concentrations (30 mM) led to sustained loss of
Akt phosphorylation and subsequent Akt cleavage by caspase-3, whereas at
lower concentrations (,10 mM) the inhibition of Akt phosphorylation was tran-
sient and eventually returned to basal levels.
In addition, quercetin also triggered CREB activation in neurons where
potent inhibition of Akt and ERK and inactivation of BAD was present. This indi-
cates that both pro-apoptotic and potentially anti-apoptotic pathways are acti-
vated in neurons in response to quercetin stimulus. However, as overall
neuronal death resulted from exposure, it appears that quercetin-induced inhi-
bition of the Akt/Bad survival pathway is dominant here in determining the
fate of the neurons. Thus, high concentrations of quercetin may produce a sus-
tained deactivation of Akt/PKB, which leads to extensive caspase-3 activation
and subsequent caspase-dependent cleavage of anti-apoptotic Akt/PKB, an
event that effectively turns-off the major survival signal and results in the
acceleration of apoptotic neuronal death. However, at lower concentrations,

reversible inhibition of Akt phosphorylation is observed and there is evidence
of an attempted survival response reflected in the increase in CREB phosphoryl-
ation. These data indicate that although flavonoids are generally regarded as ben-
eficial agents, consideration must be given to their potential toxic actions
especially as in vivo concentrations are unclear.
SUMMARY
Cellular signaling pathways, such as the MAP kinase cascade, are pivotal in the
sensing and translation of both extracellular and intracellular signals into specific
cellular responses. There is now convincing evidence to suggest that flavonoids
and more importantly their in vivo metabolites such as O-methylated forms may
interact with such pathways and that these interactions may mediate their cellular
effects. This proposal is strengthened by findings indicating that their antioxidant
activity is unlikely to be an explanation for their biological actions. Indeed, the
metabolism of flavonoids in the gastrointestinal tract, liver, and colon all act to
reduce their antioxidant potential and results in plasma concentrations well
below that of established physiological antioxidants such as ascorbate and
a-tocopherol.
It appears that flavonoids are capable of direct modulations of signaling
proteins making up important signaling pathways. On the one hand, they may
activate pro-apoptotic kinases, such as JNK, and inhibit pro-survival kinases,
such as Akt/PKB. On the other hand, they may activate pro-survival kinases,
such as ERK, and act to inhibit pro-apoptotic kinases activated by oxidative
stress, possibly explaining their beneficial effects against oxidative stress in
many cell models. However, the overall cellular effects of flavonoids are still
unclear and may be ultimately dependent on the concentration in vivo. To sub-
stantiate further a role for flavonoids as beneficial and/or cytotoxic agents
in vivo, advances are now needed with regards to the precise molecular targets
of action of flavonoids and to which flavonoids and/or metabolites have the
most profound effects.
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FEBS Lett 1999; 462:322 – 328.
154. Wang L, Matsushita K, Araki I, Takeda M. Inhibition of c-Jun N-terminal kinase
ameliorates apoptosis induced by hydrogen peroxide in the kidney tubule epithelial
cells (NRK-52E). Nephron 2002; 91:142 –147.
155. Uchida K, Shiraishi M, Naito Y, Torii Y, Nakamura Y, Osawa T. Activation of stress
signaling pathways by the end product of lipid peroxidation. 4-hydroxy-2-nonenal is
a potential inducer of intracellular peroxide production. J Biol Chem 1999;
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156. Ishikawa Y, Kitamura M. Anti-apoptotic effect of quercetin: intervention in the JNK-
and ERK-mediated apoptotic pathways. Kidney Int 2000; 58:1078– 1087.
18
Antioxidant and Gene Regulatory
Properties of Procyanidins
R. Canali*, R. Ambra*, and F. Virgili

National Institute for Food and Nutrition Research (INRAN), Rome, Italy
O. Gulati
Horphag Research Ltd., Geneva, Switzerland
Introduction 379
Sources and Bioavailability 380
Antioxidant Capacity 382
Modulation of Gene Expression 383
Cardiovascular Related Genes 384
Cancer Related Genes 384
Inflammation Related Genes 385
Application of cDNA Array Techniques 386
Other Avenues 389
Conclusion 389
References 390
INTRODUCTION
Polyphenols are attracting growing interest in nutrition and medicine for their
anti-inflammatory, anti-viral, or anti-allergic effects and for their protective
role in heart diseases, cancer, and different pathologies. Polyphenols are phenolic

Contributed equally to this study.
379
380 Canali et al.
compounds, naturally found in vegetables and responsible for their characteristic

colors. Nowadays, polyphenols have achieved such a big interest that plants have
been engineered for the production of new strains with increased levels of
selected polyphenols (1). Initially, polyphenols have been considered molecules
without any benefit for humans. Later, nutritional studies have shown that the
polyphenol component of vegetables, accounts for most of their antioxidant
and free radical-scavenging activities. More recently, their ability to affect
enzyme activity and cell response and to modulate gene expression have been
reported, providing a novel and different mechanistic perspective underlying
their biological activity. This chapter focuses on the recent advances and hypoth-
esis on the antioxidant activity and gene expression effects of procyanidins, a
class of polyphenols that is gaining interest within the scientific community.
SOURCES AND BIOAVAILABILITY

Chemically, procyanidins are oligomeric or polymeric chains of flavan-3-ols, that
is, catechins and epicatechins (Fig. 18.1). These compounds are naturally present
in plants as a complex mixture of polymers with an average degree of polymer-
ization between 4 and 10.
OH OH
OH OH
HO O HO O
OH OH
OH Catechin OH Epicatechin
OH
PROCYANIDIN dimer B4
OH
HO O
OH n
OH
OH
OH n = 2 (dimer)
HO O to 10 (decamer)
OH
OH
Figure 18.1 Structure of flavan-3-ols and procyanidin.

Properties of Procyanidins 381
The main nutritional sources of procyanidins are cocoa, apples,

grapes, tea, wine, and strawberries. The average intake, taking into account
different characteristics of dietary habits, has been estimated in the range of
30 mg/day (2). The role of procyanidins in human nutrition and disease preven-
tion depends on their bioavailability. The extent of absorption and metabolism
of polyphenols is determined largely by their chemical structure and by the rate
of glycosation, acylation, conjugation, polymerization, and solubility of the
compound.
The kinetic of absorption of monomeric units of procyanidins, flavanol-
3-ols, into plasma has been extensively elucidated (3 – 5). Catechins and
epicatechins, usually present in plants in an aglycone form, easily cross-biological
membranes, without any deconjugation or hydrolysis (6). Once entered the enter-
ocytes or the liver, monomer units are extensively metabolized to glucuronides,
O-methylglucuronides and O-methyl conjugates metabolites.
The results concerning the bioavailability of procyanidins polymers are
somehow contradictory. Procyanidin polymers, because of their size, are unlikely
to be absorbed at the level of the small intestine in their native form (7).
However, depolimerization of procyanidin oligomers to monomer and dimer
units has been demonstrated to occur at pH 2 (8), allowing the absorption of pro-
cyanidin monomer units through the small intestine. The absorption of procyani-
din dimer and trimer units has been demonstrated in vitro, in studies with CaCo-2
cells (9) and isolated small intestine perfused with dimer solution (10), and
in vivo, in plasma of healthy subjects after consumption of cocoa beverage
(11). Hydrolysis of procyanidin polymers to monomers and dimers in the
stomach represents a key event, improving procyanidin bioavailability. On the
other hand, results obtained by a direct analysis of procyanidin levels in
human stomach indicate that procyanidins are not hydrolized into monomers
(12). Similarly, Donovan et al. reported that no hydrolysis of oligomers occurred
in rat stomach. Moreover, no dimers absorption was detected, suggesting that
most of procyanidins ingested reached the small intestine unchanged (7). Procya-
nidin polymers that are not absorbed in the small intestine are metabolized by
the microflora in the large intestine into low molecular weight phenolic acids
(13). Bacterial enzyme may catalyze many reactions including hydrolysis, dehy-
droxylation, demethylation, ring cleavage, and decarboxylation. Such metab-
olites of procyanidins detected in rats and human urine are mainly derivatives
of phenylpropionic, phenylacetic, and benzoic acid (14 – 16). They are easily
absorbed and consequently transformed by conjugation with glycine, glucuronic
acid, or sulfate groups. The observation that procyanidin polymers are not
degraded in the stomach suggests that their healthy effects can be associated
to the metabolites generated by the microflora in the colon. Moreover, they
may interact directly with intestinal mucosa or interfere with digestion. Procya-
nidins may contribute in the prevention of various cancers of gastrointestinal
tract (17) or influence the bioavailability of metal ions forming stable complexes
with them (18).
382 Canali et al.
Although the mechanisms of procyanidin metabolism and absorption

still need some elucidations, most of the in vivo studies support the human
health benefits related to procyanidin consumption.
ANTIOXIDANT CAPACITY
Epidemiological studies have shown inverse association between dietary poly-
phenols and cardiovascular disease. Polyphenols are associated to the decrease
of some risk factors, including reduction of LDL oxidation and modulation of
cytokines and eicosanoids involved in the inflammatory response. The anti-
oxidant properties observed in chemical and biological systems associated to
polyphenols can contribute to the positive health effects related to polyphenols.
Procyanidins, characterized by multiple phenolic groups in their molecular struc-
ture, are potentially able to quench free radicals by forming more stable oxidized
products. In vitro models showed that procyanidins exhibited scavenging proper-
ties toward superoxide anion, hydroxyl radical and lipid peroxyl radicals (19 – 21)
and peroxynitrite (22), protecting LDL from oxidation (23,24). Oxidative modi-
fication of LDL is, in fact, known to play a key role in the initiation of atherogen-
esis (25). The antioxidant activity of procyanidins is based on their capacity to
trap radicals and to chelate transition metals (21), in contrast long-chain procya-
nidins lack the capacity to chelate metals, acting only as radical scavengers.
The antioxidant potential of procyanidin is influenced by the length of the
chain. Catechin monomers resulted to be the most effective in inhibiting iron/
ascorbate-dependent lipid oxidation (26). However, dimers and trimers were
associated to the highest protection against 2,20 -azobis(2-amidinopropane)
hydrocloride dependent lipid oxidation (27). Moreover, long-chain procyanidins
had the strongest effect against the UV-C dependent oxidation (27). Protection of
LDL from in vitro oxidation increases with oligomer chain length (28). This high
activity is possibly due to the fact that catechol units, the part of the structure
responsible for the antioxidant capacity, are in a tight configuration, that is,
close one another, therefore increasing the possibility of electron delocalization
of the phenoxyl radical formed (29). Accordingly, procyanidin oligomers were
more protective against peroxynitrite-dependent damage in comparison with
monomers (22). If procyanidins are absorbed and biologically active in vivo,
they may prevent free radicals mediated cytotoxicity and lipid peroxidation and
protect LDL from oxidation. While catechins and procyanidins have a powerful
antioxidant activity in vitro, it is not clear if the extent and the position of the
post-absorption modifications can have any influence on their reactive oxygen
and nitrogen scavenger activity. Spencer et al. reported that fibroblast protection
elicited by 30 -O-methyl epicatechin, the most common epicatechin metabolite in
circulation, against hydrogen peroxide damage, was not significantly different
from epicatechin. They proposed that metabolites interaction with cell signaling
cascades, and not a redox mechanism, is associated with the protective effects
(30). Studies in vivo are fundamental to assess the real antioxidant efficacy of
procyanidins. In vivo treatment with a grape seed procyanidin extract (GSPE,

containing 75 – 80% oligomeric proanthocyanidins and 3 –5% monomeric pro-
anthocyanidin) was associated with a decrease of reactive oxygen species pro-
duction in peritoneal macrophages and hepatic mitochondrial and microsomal
lipid peroxidation induced by 12-O-tetradecanoylphorbol-13 acetate (31). Diets
rich in catechin and procyanidin, abundant in cocoa, were associated to an
increase of plasma antioxidant capacity and a decrease of plasma TBARS (32).
LDL isolated from the plasma of subjects consuming cocoa has been reported
to be more resistant to ex vivo oxidation processes (28,33). Procyanidins, due
to their strong-binding protein properties, may bind LDL protecting them
against oxidative challenge.
Recently, several studies have focused on the antioxidant activity of
Pycnogenolw, a standardized phenolic extract from the bark of French Pinus
maritime. Pycnogenolw main constituents are monomers (catechin, epicatechin,
and taxifolin) and procyanidin, mainly oligomers of 5 –7 units. Pycnogenolw has
been shown to have a fundamental role in the antioxidant network recycling the
ascorbic radical and protecting vitamin E against oxidation (34). As shown for
isolated procyanidin, we demonstrated that the Pycnogenolw is characterized
by a strong-scavenging activity against reactive oxygen species (superoxide
anion and hydroxyl radical) measured by ESR techniques and in a dose-
dependent fashion against nitric oxide (NO). NO was indirectly measured
by nitrite generation produced by the reaction of NO (released from sodium
nitroprusside decomposition) with oxygen (35). In the same study, we reported
that pretreatment of a monocyte – macrophages cell line (RAW 264.7) with
Pycnogenolw for 24 h before the administration of lipopolysaccharide (LPS)
and interferon g (IFN-g), was associated to a dose dependent decrease of nitrite
and nitrate generation (35). Although, this effect could be the result of a direct
scavenger activity against NO, we observed a direct inhibitory action on the
inducible form of NO-synthase (iNOS) activity and a modulation of
iNOS – mRNA expression (35), giving a first indication that gene expression
modulatory effects play a key role in the protective mechanism associated to
Pycnogenolw.
MODULATION OF GENE EXPRESSION

Eventhough out of the strict matter of this chapter, one of the first indications of
polyphenols ability to modulate gene expression was reported by Nakane and
Ono on (2)-epigallocatechin gallate (EGCG), the main green tea polyphenol.
The authors reported that EGCG inhibited the activities of cellular DNA and
RNA polymerases and reverse transcriptase (36). After this first evidence by
Nakane and Ono, many studies have focused on polyphenols ability to modulate
gene expression and at present, there are more than 300 medline entries addres-
sing their effects on genome functions. On this line, Hofmann and Sonenshein,
utilizing dominant negative mutants, recently demonstrated that apoptosis and
384 Canali et al.
cell growth inhibition induced by EGCG, in aortic smooth muscle cells (SMC),
rely on the tumor suppressor p53 and the redox related transcription factor,
nuclear factor-kB (NF-kB) (37).
More adherent to the matter of this chapter, procyanidins have been
reported to modulate the expression of genes involved in physiological and
pathological processes correlated with inflammation, cardiovascular disease,
and cancer.
Cardiovascular Related Genes

Abou-Agag et al. observed that one hour incubation with catechin or epicatechin
increased the expression of genes coding for tissue-type plasminogen activator
and urokinase-type plasminogen activator in cultured human umbilical vein
endothelial cells (HUVECs), suggesting that cardioprotective benefits of red
wine could rely on procyanidins ability in regulating fibrinolytic activities (38).
Ardevol et al. observed a reduced expression of the hormone-sensitive
lipase as a response to wine-procyanidin extracts on 3T3-L1 cells, reporting
that the effect was only elicited by polimers and not by monomers (39).
Recently, Pal et al. proposed a feedback mechanism in which wine-
polyphenolics treatment in HepG2 cells lowers intracellular cholesterol levels
inducing the expression of hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase and LDL receptor (40).
Bagchi and coworkers studied the protective abilities of GSPE (discussed
earlier), focusing on its cardioprotective roles. They demonstrated that GSPE
inhibited the tumor necrosis factor a (TNF-a)-induced vascular cell adhesion
molecule-1 (VCAM-1) expression in HUVEC (41), and increased vascular endo-
thelial growth factor (VEGF) expression in HaCaT human keratinocytes follow-
ing hydrogen peroxide and TNF-a exposure (42). In a recent comprehensive
review of their previous papers on GSPE, Bagchi et al. reported that GSPE
inhibits CD36 TNF-a inducible gene expression in HUVEC assayed by micro-
arrays, indicating CD36 as a novel cardioregulatory protein and reinforcing
their hypothesis of GSPE as a potential therapeutic tool in promoting cardio-
vascular health (43).
Cancer Related Genes

Soleas et al. examined the ability of the main phenolic substances in red wine to
modulate the expression of the tumor suppressor protein p53 and found that a
24 h incubation with catechin increased p53 levels in MCF-7 breast cancer
cells. Even if at borderline statistical significance, their data may explain the
anticancer activities proposed for red wine polyphenols (44).
Bagchi et al. observed a protective effect of GSPE against a smokeless
tobacco-induced oxidative stress in human mouth keratinocytes, reporting that
GSPE exhibited better protection towards apoptotic cell death in comparison
with vitamins C and E (45). At molecular level, protection was associated with
the modulation of the expression of bcl-2 and p53 genes (46). Moreover, GSPE
administration protected mice from acetaminophen-induced liver injury and
animal lethality. Such protection was associated with increased expression of
bcl-XL in the liver (47). Protection against drug-induced cytotoxicity and modu-
lation of apoptosis was reported by Joshi et al. who demonstrated, utilizing a
different experimental model, that GSPE modulates the expression of bcl-2,
c-myc, and p53 in normal human liver Chang cells (48). Down regulation
of pro-apoptotic genes, that is, c-jun and jnk-1, has also been observed by
Sato et al. who observed a causative link between beneficial effects of
GSPE administration in rats towards ischemia/reperfusion and the ability of
GSPE in preventing the expression of anti-death signals such as jnk-1 and
c-jun genes (49).
Inflammation Related Genes

Bito et al. reported that epicatechin was able to modulate the IFN-g-induced
intercellular adhesion molecule-1 (ICAM-1) expression in HaCaT cells, although
its effectiveness was lower than other polyphenols (50). These results suggest a
possible role of procyanidins in modulating cell adhesion processes during
inflammation.
Cocoa procyanidins have been reported by Mao et al. to affect the transcrip-
tion of several genes coding for cytokines in mitogen-stimulated human peri-
pheral blood mononuclear cells but not in resting cells (51). Analyzing several
procyanidin fractions, they found that the most effective was the larger ones
(up to 10 units), inducing increased interleukin-1 (IL-1) b expression and
reduced IL-4 and IL-2 transcriptions (51).
Different reports addressed the molecular mechanism underlying the
anti-inflammatory properties of Pycnogenolw (discussed earlier). As already
mentioned, we observed a modulation of iNOS activity and expression by
Pycnogenolw in activated RAW 264.7 macrophages (35). We observed that
24 h preincubation of murine macrophages with Pycnogenolw was associated
with a significant reduction of NO generation, inhibition of iNOS activity, and
iNOS – mRNA expression in response to IFN-g and LPS. Using a dual-luciferase
reporter gene assay that reveals the NF-kB-dependent gene expression induced
by IFN-g in RAW 264.7, Park et al. demonstrated that 1 h incubation with pro-
cyanidins was associated with different effects toward gene expression, depend-
ing on the degree of polymerization: Pycnogenolw and trimeric procyanidin
enhanced the NF-kB-dependent gene expression, while monomers and dimers
repressed it (52).
Bito et al. reported that 12 h pretreatment with Pycnogenolw significantly
inhibited, in a dose-dependent manner, IFN-g-induced expression of ICAM-1
in the human immortalized keratinocytes cell line HaCaT. This observation
pairs with the reported modulatory effects of Pycnogenolw on the adhesion of
Jurkat T cells to activated keratinocytes in a co-culture assay (53). Using gel
386 Canali et al.
mobility shift assays (EMSA), Bito et al. identified Stat1 and IRF-1 as two factors
affected by Pycnogenolw, critical for IFN-g-dependent gene activation and sig-
naling pathway.
Cho et al. confirmed that Pycnogenolw pretreatment prevented the
activation of NF-kB and also of the activator protein-1 (AP-1) in activated
RAW 264.7 (54). They also found that Pycnogenolw pretreatment abolished
LPS-induced IkB (an inhibitory protein that associates with NF-kB) degradation
and down-regulated IL-1b gene expression in RAW 264.7 and IL-2 gene
expression in human T-cell line Jurkat E6.1 (55).
Pycnogenolw ability to inhibit NF-kB activation and VCAM-1 and ICAM-1
expression was reported by Peng et al. also in TNF-a-treated HUVEC (56).
Saliou et al. reported that Pycnogenolw treatment in HaCaT inhibited, in a
concentration-dependent manner, UVR-induced NF-kB-dependent gene expression
suggesting a role of the extract in protecting human skin against erythema (57).
Application of cDNA Array Techniques

Complementary DNA (CDNA) arrays are global expression analysis tools that
have been recently applied also to nutrition science and are providing insights
onto how nutrients act at molecular level, regulating gene functions, and
signal-transduction pathways. Application of arrays to nutrigenomics will accel-
erate discoveries on this field and, if possible, allow the treatment or the preven-
tion of diet-related diseases. cDNA arrays have been applied to the study of the
molecular effects of nutrients also in organisms ranging from bacteria (58),
plants (59), and Drosophila (60) and have been helpful tools in the identification
of pathways influenced by the intake of fatty acids (61), dietary protein (62),
short chain fatty acids (63), zinc (64), and heme (65) in mammalian cells.
Briefly, cDNA arrays consist in high-density nylon, plastic, or glass supports
bearing immobilized DNA sequences of thousands genes. Supports are hybri-
dized with labeled cDNA sequences obtained by reverse transcription of
mRNA prepared from cells or tissues and resulting hybridization signals are
compared to controls.
Rihn et al. reported that 24 h supplementation of Pycnogenolw is associated
to a significant modulation of 39 out of the 83 genes detected in a commercially
available human cDNA array bearing a total of 588 genes in the human cell
keratinocyte line HaCaT (66). Among modulated genes, are to be mentioned
two genes coding calgranulins A and B, members of the highly conserved
S100 family of low weight calcium-binding proteins, which are known to be
accumulated in psoriasis and various dermatitis.
Targeting to a better understanding of the anti-inflammatory and a putative
anti-cancer properties of Pycnogenolw, we recently applied a genomic analysis
method to the human tumor monocytic U937 cell line. U937 cells are model
for monocyte to macrophage differentiation studies and are currently used in
many laboratories in inflammatory and tumorigenicity studies. U937 cells
derive from a pleural effusion of a human caucasian histiocytic lymphoma and

although they exhibit a lymphoblastoid morphology, they own many of the
monocytic-like characteristics exhibited by cells of histiocytic origin. Prelimi-
nary, unpublished results of Pycnogenolw effects on U937 cells indicate a signifi-
cant down regulation of genes encoding for oncogenic hyper-proliferative
proteins and an up regulation of genes coding check-point proteins. Moreover,
we found that some of the genes affected by Pycnogenolw were modulated in
a similar fashion after treatment with purified catechin, demonstrating the contri-
bution of this component to the known biological effects of Pycnogenolw (67).
Among others, we found a down regulation of hCDC10 and an up regulation
of KOX15 genes in both Pycnogenolw and catechin-treated U937 cells.
The KOX15 (or zinc finger protein 22) cDNA was isolated by Wu et al.
who reported high expression of the gene in T-leukemia lines but not in nonlym-
phoid cells, indicating a role in differentiation within lymphoid cells (68). On the
other hand, CDC10 is a protein belonging to the septins family which are well-
conserved GTPases found in animals and fungi. CDC10 is a structural component
of the 10-nm filaments which are assembled in cytoplasmic membrane and are
essential for budding of yeasts. By differential display, Nagata et al. found that
the expression levels of hCDC10, the human homologue gene, were related to
the biological and clinical characteristics of neuroblastoma, that is, high
expression levels in tumors are associated with the good prognosis in patients
under 1 year of age (69). More recently, using an expression profile analysis,
Nishiu et al. identified high expression of hCDC10 in advanced stage malignant
lymphomas (70). Gene expression profiling of cancer cell lines challenged by
procyanidins is providing clues on the molecular pathways that are related to
the anti-cancer properties of Pycnogenolw. It is important to stress that these
observations, even though of interest, must be considered preliminary and confir-
mation is necessary before reaching any final conclusion about the effective role
of pine bark procyanidins on gene expression in human cancer cells. We already
reported that cardioprotective properties of procyanidins were at least in part
associated to their ability to modulate gene expression in HUVEC (38). The
study of the proliferative responses of endothelial cells is crucial for the identifi-
cation of molecular pathways involved in the response to specific diet or nutrient.
To this regard, Sartippour recently demonstrated that procyanidins inhibit in vitro
HUVEC proliferation and in vivo vessel formation (71,72). They observed that
the effects of procyanidins on proliferation are associated to decreased levels
of secreted VEGF and that the regulation occurs at the transcriptional level.
Oxidized LDL (ox-LDL) induces a proliferative and atherogenic response
in endothelial cells including the chemotaxis of inflammatory cells (monocytes/
macrophages) into the subendothelial space. The nonsaturable accumulation of
ox-LDL within macrophages, eventually leads to foam cell formation, cytokine
expression and fatty streak (73). NF-kB and AP-1 are well-known inflammation-
related transcription factors and have been recently reported to be involved
in endothelial dysfunction. As shown in Fig. 18.2, here we report that a 4 h
388 Canali et al.
Figure 18.2 Effect of Pycnogenolw preincubation on ox-LDL dependent NF-kB acti-

vation. HUVEC were treated 4 h with ox-LDL (20 mg protein/mL) and NF-kB activation
was assayed by EMSA. Pycnogenolw (5 mg/mL) was preincubated for 12 h and washed
out before ox-LDL treatment.
treatment of HUVEC with ox-LDL (20 mg protein/mL) induces a strong activa-

tion of NF-kB, assayed by EMSA. If HUVEC are preincubated with Pycnogenolw
(5 mg/mL) for 12 h before ox-LDL treatment, a sensible reduction of the NF-kB
activation occurs (Fig. 18.2), according to its anti-inflammatory effects (67).
To characterize the atherogenic response at the molecular level, we
recently applied a global analysis method to cultured HUVEC, obtaining a com-
prehensive database of candidate genes possibly involved in atherogenesis
induced by ox-LDL (74).
In the same series of experiments, we studied the effects of purified cate-
chin preincubation on ox-LDL-treated HUVEC for 16 h observing that the
monomer is able to prevent or even reverse about one fifth of the changes in
gene expression induced by ox-LDL. These data must be considered preliminary,
and they need further confirmation of the observations obtained by cDNA array.
However, these results can be of interest and provide suggestions for more inves-
tigation. We therefore present here some data regarding the genes which dis-
played the most significant regulation with respect to untreated cells and a
brief description of the known function, leaving to the reader the option to get
any possible conclusion. Half of the genes modulated by catechin treatment
encode transcription factors, whereas others are mainly related to adhesion
molecules and matrix metalloproteinases expression. For example, we observed
a twofold increase in mRNA levels of the GATA-2 transcription factor in
ox-LDL-treated HUVEC (74). This expression was switched to a 50% down

regulation in catechin-pretreated cells, compared with control untreated cells.
GATA-2 has a known function in the control of the expression of adhesion pro-
teins. Accordingly with this function, the messengers of some genes encoding for
these class of proteins (e.g., leukocyte adhesion glycoprotein p150, vinculin, and
cadherin 16) were altered in catechin preincubated cells, as compared to ox-LDL
treated HUVEC. Moreover, catechin preincubation strongly inhibited the pre-
viously observed (74) up-regulation of FOS-related antigen (FRA)-2, one of
the four members of the FOS gene family, leucine zipper proteins that dimerize
with proteins of the JUN family, thereby forming the transcription factor complex
AP-1 and playing an important role as regulators of cell proliferation, apoptosis,
and differentiation.
Other Avenues
In addition to their cardioprotective, anti-inflammatory, anti-cancer activities,
recent data suggest that polyphenols have anti-HIV effects. A grape seed
extract was able to interact with the expression of genes coding for several
co-receptors necessary for the internalization of HIV in mononuclear leukocytes,
possibly interfering with virus replication (75).
Moreover, Ratna and Simonelli demonstrated that intra-muscular injection
of catechin in livers of roosters increased the transcript of the estrogen-regulated
mRNA stabilizing factor (E-RmRNASF), which is necessary for the estrogen-
dependent stabilization apolipoprotein mRNA, indicating a possible therapeutic
use of procyanidins as estrogen-mimicking agents (76).
CONCLUSION
As shown, results on procyanidin bioavailability are somehow contradictory and
need further investigations. Moreover, it is not still clear if the postabsorption
modifications can affect their biological effects. However, in vivo studies
support the human health benefits of procyanidin consumption and in vitro
studies strongly suggest that health improvement depend on the ability of these
molecules to activate signaling pathways and modulate gene expression.
Figure 18.3 summarizes procyanidin fate in the digestive tract and gene effects
reported in this chapter, providing a “nutrigenomic picture” of components of
this important group of polyphenols.
The effect of diet on and nutrients on gene expression can be studied at differ-
ent levels of complexity. The first and simplest activity is at the level of single gene
expression. In this case, the attention of the investigator is focused to single genes
in order to establish whether a gene is differentially modulated by a specific exper-
imental treatment vs. the control. The major proportion of the research addressing
the biological activity of procyanidin is actually within this level of complexity.
Most of these studies, similar to other diet-genome-related studies, lack corrobora-
tion by inverse genetic approaches that unequivocally demonstrate a causative role
of a gene/protein as a response to in a dietary manipulation.
390 Canali et al.
Figure 18.3 Diet procyanidin fate and summary of effects on gene expression (see text).
On the other hand, the use of gene arrays is enabling the analysis of geno-
mics applied to nutrition at a higher levels. Firstly, at the level of multiple genes,
where these are grouped on clusters on the basis of arbitrary choices, usually
addressing a common putative or known feature, such as gene co-regulation,
similar protein function, or known interactions. For example, genes sharing
expression patterns as a response to a treatment with a specific nutrient are poss-
ibly co-regulated and participate in functionally related processes. Finally, when
all genes are taken in account “in chorus” (considering also unaffected genes), it
is possible to reach the ambitious level of “system biology” where all gene fluc-
tuations induced by the treatment with a specific nutrient are taken into account to
infer a network picture of the tangled relationships existing between all coded
proteins and their genes. Another possibility is simple identification of a “diet-
gene signature”—that is, a global gene response of a specific cell type or
tissue to one nutrient—without considering the gene functions and not even con-
sidering their names. Several applications of this signatures can be hypothesized
to be utilized for the certification of food or in order to compare different
matrixes. The concept of “normal nutrient signatures” in humans could also be
used as a tool for the identification and prevention of diet-related diseases. The
application of array technology to nutrition will help the comprehension of nutri-
ent effects, as well as the molecular mechanisms underlying them, on human
heath and how nutrition influences the normal homeostasis or may restore a
pathological condition.
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19
Cell Signaling Properties of Inositol
Hexaphosphate (IP6)
Abulkalam M. Shamsuddin
The University of Maryland School of Medicine, Baltimore, Maryland, USA
Introduction 398
Inositol Compounds in Cellular Signaling 399
IP3 and Its Receptor 399
IP6 and Its Receptor 400
IP6 as a Signal Molecule 400
In Mammalian Cells 400
Signal Transduction by IP3 and IP6 Along the Evolutionary Tree 401
Interactions of IP6 with Other Proteins and Macromolecules 402
Alterations in Levels of IP5 and IP6 402
Vesicle Trafficking: Exocytosis and Endocytosis 402
Cell Proliferation and Cell Cycle 405
Normalization of Cell Proliferation 405
DNA Synthesis 406
The Retinoblastoma Protein 406
Protein Kinase C 407
Ras Proteins 407
Induction of Cell Differentiation 408
Programmed Cell Death (Apoptosis) 409
PI 3-Kinase 409
Nuclear Inositol Signaling 410
mRNA Transport 410
397
398 Shamsuddin
Chromatin Remodeling 411

NF-kB (Nuclear Transcription Factor-kB) 411
Zinc-Finger Motif 412
DNA Repair 412
Role of IP6 in Energy Transduction 413
Anticancer Action of IP6: from the Laboratory to the Clinic 413
Other Biological Effects of IP6 415
Conclusion 415
References 415
INTRODUCTION
Inositol hexaphosphate (IP6, InsP6) is a polyphosphorylated carbohydrate, con-
tained in high concentrations (0.4 –6.4%) in cereals and legumes. IP6 known
more commonly as “phytic acid” is the major form of phosphorous in the
seeds, wherein it is found as deposits of mixed “phytate” salts of K, Mg, Ca,
Mn, and Zn (1 – 3). In the plant kingdom, it is also found in other plant tissues
and organs such as pollen, roots, tubers, and turions (4). IP6 accumulates
during seed development and is broken down into lower inositol phosphates
during germination. The cytosol of almost all mammalian cells contain IP6 and
its lower phosphorylated forms (IP1 – 5) as well as the dephosphorylated form,
the parent compound—inositol. myo-Inositol is a cyclic alcohol (cyclitol) deriva-
tive of glucose. The enzyme myo-inositol(3)P1 synthase (MIPS) converts
glucose-6-phosphate to inositol(3)phosphate1 [Ins(3)P1]. In general, IP6 is con-
verted from inositol through the various polyphosphate intermediates via differ-
ent pathways in different organisms; these pathways are being more similar than
different. In addition to being a part of the phospholipids and eventually con-
verted to IP6 and its pyrophosphates, in the plant cells inositol is also utilized
in cell wall polysaccharides and other cyclitols. To the surprise of many in the
field of nutritional biochemistry, it has been a relatively recent finding that IP6
is not only present, but also is the most abundant of intracellular inositol phos-
phates in eukaryotes (5).
In multicellular organisms, it is imperative that cells communicate with
each other and these communications are dependent on external signal mol-
ecules, which evoke a “chain reaction” mediated by a host of molecules within
the cell. Though there are different families of molecules (such as lipids, glyco-
lipids, carbohydrates, including inositol phosphates) that may serve the latter role
in conveying the signals; much of our present day knowledge is on intracellular
signaling proteins. At the end of the various signaling pathways are the target pro-
teins; depending on the signals, these proteins carryout specific functions such as
metabolism, gene regulation, and structural alterations.
Cell Signaling Properties of IP6 399
Signal molecule ! binding with cell surface receptor protein !

intracellular signaling molecules ! target molecules ðand=or !
nuclear signalingÞ ! effects: altered metabolism=gene expression=
structure, and so on:
As can be seen from the preceding, the first step is for the extracellular
signal molecule to bind to cell surface receptors. Interestingly, it appears that
there are binding (receptor) proteins for inositol phosphates including IP6, and
that they act as signal molecule themselves.
INOSITOL COMPOUNDS IN CELLULAR SIGNALING

IP3 and Its Receptor
Intracellular signaling proteins behave as switches that turn on or off depen-
ding on the signal, the commonest of these proteins are turned activated or
inactivated by the addition (by protein kinase) or deletion (by protein phos-
phatase) of phosphate groups. The other main class of molecular switches is
the guanosine 50 -triphosphate (GTP)-binding proteins. Of the two types of
GTP-binding proteins, the large trimeric GTP-binding proteins are commonly
called G proteins. The G proteins are attached to the cytoplasmic face of the
plasma membrane where they serve as relay molecules. A variety of extra-
cellular signals such as vasopressin, acetylcholine, and thrombin activate the
inositol –phospholipid signaling pathway by way of G proteins, specifically,
Gq via activation of phospholipase C-b (PLC-b), which is also on the
plasma membrane. In the inner half of the plasma membrane lipid bilayer is
the inositol –phospholipid phosphatidylinositol 4,5-biphosphate (PIP2). The
activated PLC-b cleaves PIP2 to generate diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP3). IP3 leaves the plasma membrane, diffuses rapidly
through the cytosol to bind to its receptor protein in the endoplasmic reticulum
causing release of Ca2þ stored within the endoplasmic reticulum. This may
result in increase of local Ca2þ concentration by 10 – 20-fold and trigger Ca2þ
responsive proteins in the cell (6). Inositol 1,3,4,5-tetrakisphosphate (IP4) is
also considered to be a signaling molecule, albeit to a lesser extent than its
more famous cousin IP3; it has been implicated in the movement of Ca2þ
into the cell and/or in maintaining levels of IP3-sensitive Ca2þ pools (6).
Interestingly, the level of IP3 itself can be affected during glucose metabolism:
an increase in IP3 secondary to an influx of extracellular Ca2þ following mem-
brane depolarization and activation of PLC.
PIP2 can also be converted to phosphatidylinositol 3,4,5-triphosphate by
PI-3 kinases that also play important roles in regulation of protein trafficking,
cell growth and cell survival, cytoskeletal organization, and so on. PIP2 itself
400 Shamsuddin
may also have roles in binding to, and regulation of vinculin, a-actinin, and
gelsolin; binding of PIP2 to specific sites on them influences their interactive
properties with actin.
IP6 and Its Receptor

Although much of the research in inositol phosphate-signal transduction centers
around IP3 and to a much lesser extent IP4, the role of IP6 as a signal molecule
received very little attention till recently. In the late 1980s and early 1990s, IP6
was found to stimulate 45Ca2þ uptake in cultured cerebellar neurons (7) and
anterior pituitary cells (8). Coupled with the fact that Law et al. (9) demonstrated
that low concentration of IP6 increases both intracellular free-Ca2þ and prolactin
secretion in perifused pituitary cells, the stage is set for IP6 to be a signal mol-
ecule by its own right. These and other investigators subsequently isolated
IP6-binding sites, the IP6 receptor (10 –13). [3H] IP6 bound to specific and satur-
able recognition sites in membranes prepared from cerebral hemispheres, anterior
pituitaries, and cultured cerebellar neurons. Interestingly, the specific binding
sites were present in high density in all subcellular fractions including the mito-
chondria. The presence of IP6-binding sites (or receptor) in the mitochondrial
fraction further enhanced the belief that IP6 may act also as an intracellular reg-
ulator of Ca2þ homeostasis just like IP3.
It appears that the binding affinity for IP6 receptor (or binding sites)
reported by different groups is different. For instance, Theibert et al. (12) reported
that the IP6 receptor comprises a protein complex of 115, 105, and 50 kDa sub-
units. On the other hand, the concept that the IP6-binding site in the membrane
may not necessarily be protein but nonprotein entities such as phospholipid has
been advanced by Poyner et al. (14).
IP6 AS A SIGNAL MOLECULE

In Mammalian Cells
Be that as it may, what does it mean to have IP6-binding sites (receptor) on cell
membranes in the brain? The discovery of high-affinity membrane binding sites
lends support to the assumption that IP6 may serve as a signal molecule. In the
central nervous system, IP6 acts as a potent neural stimulator. Experiments by
microinfusion of IP6 into the nucleus tractus solitarius or by iontophoretically
applied to the dorsal horn of the spinal cord have demonstrated that IP6
mimics the action of excitatory neurotransmitter glutamate receptor (15,16).
However, central nervous system and the anterior pituitary gland are not the
only place to have IP6-binding sites (receptors). Since IP6 and IP4 are produced
in response to stimulation of cardiac a1-adrenoreceptors, Rowley et al. (17)
investigated the IP6-binding sites (receptor) in the rat heart. [3H]-IP6 bound to
a single, high affinity site in sections of rat heart (KD ranging from
22 + 1.9 nM in right atria to 35 + 2.6 nM in the interventricular septum).
The maximal number of binding sites (Bmax) ranged from 5.1 + 0.48 to
12 + 1.8 pmol mg21 protein in left atrium and left ventricle, respectively.
Presence of IP6-binding sites in mitochondrial and sarcoplasmic reticulum frac-
tions of heart certainly points to the role(s) of IP6 and IP6-binding sites in the
heart muscle.
Reports of such IP6-binding sites or receptors in other cells have been stea-
dily emerging. O’Rourke et al. (18) report of IP6-binding sites in a low-density
membrane fraction from human platelets, and Kitchen et al. (19) report of
specific, albeit low affinity, IP6-binding site in human neutrophil (polymorpho-
nuclear leukocytes) membrane preparations. Interestingly, IP6 plays an important
role in the neutrophils. After priming by a number of different agents, human neu-
trophils may be stimulated to produce a greater respiratory burst than would be
elicited by the stimulus alone and some other neutrophil functions may be simi-
larly enhanced by pre-exposure to a priming agent. Eggleton et al. (20) reported
that pre-incubation of neutrophils with IP6 alone had no stimulatory effect upon
the basal production of reactive oxygen intermediates but the response to a sub-
sequent stimulus by phagocytic particles or N-formyl-Met-Leu-Phe (fMLP) is
substantially enhanced (20). An increase in IL-8 secretion by stimulated cells
also occurred in the presence of IP6 (21). Thus, the local release of IP6 could
have a physiologically important modulatory role on neutrophil functions and
confer more effective combat to invading microorganisms in our first line of
defense.
Signal Transduction by IP3 and IP6 Along the Evolutionary Tree

The role of inositol triphosphate as a signaling molecule has been conserved evo-
lutionarily, from the plant through the slime mold up to the mammalian cells. The
role of IP3 as a signaling molecule in plants was demonstrated by Samanta et al.
(22) when IP6-phytase was added after a definite time of hydrolysis (20 – 30 min)
coinciding with the optimal production of Ins(2,4,5)P3 bound to phytase, mobil-
ization of Ca2þ from microsomal/vacuolar fractions was detected. These inves-
tigators further demonstrated that Ins(1,4,5)P3 or Ins(2,4,5)P3 – phytase complex
constituted in vitro is also effective in releasing Ca2þ. Thus, the IP3 – phytase
complex is recognized by putative receptor (or binding site) associated with
microsomal fraction resulting in Ca2þ mobilization in plants (23).
The preceding has shown the conservation of the signaling role of IP3
through the different organisms. However, the signaling role of IP6 is also con-
served. In plants, IP6 plays a role in the processes by which the drought stress
hormone abscisic acid induces stomatal closure, conserving water and ensuring
plant survival. IP6 levels in Solanum tuberosum stomatal guard cells are elevated
in response to abscisic acid, and IP6 inactivates the plasma membrane inward Kþ
conductance in a cytosolic calcium-dependent manner. Lemtiri-Chlieh et al.
report that guard cell protoplasts release of IP6 from a caged precursor mobilizes
calcium. IP6-induced increase in cytoplasmic calcium does not result from
402 Shamsuddin
calcium influx but from IP6-triggered release of calcium from endomembrane

stores. These data define IP6 as an endomembrane-acting calcium-release
signal in guard cells (24). In an attempt to understand the intricacies of
IP6-induced differentiation and inhibition of cell proliferation of cancer cells,
pilot studies in our laboratories using HT-29 human colon cancer cell line demon-
strated a similar very transient increase in intracellular Ca2þ following IP6 treat-
ment. This increase in Ca2þ level by 3 –4-fold was within 10 s of exposure to IP6.
Along with this increased intracellular Ca2þ there was a concomitant down-
regulation of cyclin E (25).
Interactions of IP6 with Other Proteins and Macromolecules

Not only have IP6 binding proteins or receptors on cell surface have been ident-
ified, but also a variety of proteins within the cytosol may bind IP6 (26). But
before we discuss those, let us first see what happens to IP6 levels during the
normal physiological activities of various cells.
Alterations in Levels of IP5 and IP6
Fluctuations, particularly a substantial increase in the level of IP6 in various cells
during different functional or developmental stages have led to investigations
into its roles. The levels of IP5 and subsequently IP6 progressively increases in
Xenopus laevis oocyte over a 72-h-period (27) during which IP6 remained unme-
tabolized. During progression of oocyte maturation between stages 3 and 6 of
oogenesis, IP6 level rose by 6-fold. Thus, IP6 is intimately associated with, if
not directly regulating oogenesis and oocyte maturation.
Changes in the cellular mass and concentration of IP5 and IP6 were
observed during a complete cell cycle of proliferating rat thymocytes (28).
After a very early transient rise in both IP5 and IP6 at the beginning of the cell
cycle, a decrease of the intracellular concentration of both compounds occurred.
During cell division (between 48 and 72 h) a pronounced increase in IP5 and IP6
was observed indicating a role for these compounds during cell cycle pro-
gression. We are beginning to understand how some of these functions may be
brought about. The modulation of various cell cycle regulators by IP6 will be
discussed later in this chapter.
Vesicle Trafficking: Exocytosis and Endocytosis
The process by which eukaryotic cells package secretory molecules in plasma
membrane and extrude these is exocytosis. On the other hand, cells ingest macro-
molecules (in specialized cells even other cells) by invagination of the plasma
membrane—endocytosis.
Exocytosis: A transient and consistent elevation in IP6 concentration in
pancreatic insulin secreting b cells has been observed following glucose stimu-
lation and the temporal changes in IP6 concentration correlate well with the
initial rise in intracellular Ca2þ (29). This rise in IP6 level is above an extremely
stable baseline physiological level of IP6 in the unstimulated cells (40 –54 mM); a
10 mM rise in concentration is sufficient to inhibit the serine/threonine protein
phosphatase activity and increase the current through voltage-gated L-type
Ca2þ channel activity. IP6 has also been demonstrated to stimulate exocytosis
in pancreatic b cells and IP6 could recruit insulin secretory granules to the site
of exocytosis (30). Interestingly, the increased concentration of IP6 following
glucose-stimulation, to bring about this function is most pronounced at the site
of exocytosis and the vicinity of Ca2þ channels. Since L-type Ca2þ channels
have a key role in various tissues such as the myocardium, smooth muscles,
and the brain, this effect of IP6 clearly has a great physiological significance as
well as crucial in the pathogenesis of diseases in those. For instance, prolonged
opening of L-type Ca2þ channels mediate apoptosis of the b cells; thus, a sus-
tained increase in IP6 concentration could stimulate apoptosis. Indeed, it has
been known that a high level of IP6 or prolonged exposure can induce apoptosis
in various cancer cell lines (31,32). In the brain, the pyramidal cells of the
hippocampus associated with memory possess L-type Ca2þ channels. Long-
term disturbance in IP6 metabolism could result in the death of these cells and
therefore be instrumental in the pathogenesis of Alzheimer’s disease.
It is becoming clear from these experiments that the overall scheme of how
the functions of IP6 are concentration dependent: there is a normal physiological
level, then the level changes (usually a slight increase), and there are additional
functions. Not only the levels in the cells fluctuate, the levels of IP6 in the plasma
and other body fluids also vary.
The synaptic vesicle protein synaptotagmin I has been proposed to serve as
a Ca2þ sensor for rapid exocytosis. The two fragments of the large cytoplasmic
domain of synaptotagmin I are C2A and C2AB. IP6 binds to and induces a
conformational change in C2AB in the presence of lysosome. The IP6 bind-
ing notably weakens the Ca2þ-dependent C2AB –membrane interaction sug-
gesting that IP6 may act as a modulator of neurotransmitter by altering the
state of synaptotagmin – phospholipid interaction (33).
Endocytosis: The process of endocytosis involves PIP2, clathrin, and
clathrin adapters, the guanosine triphosphatase dynamin I, synaptojanin 1, and
the amphiphysin dimer. Dynamin, a high-affinity substrate for calcineurin is a
force-generating molecule responsible for membrane fission during endocytosis.
IP6 promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This
effect of IP6 is dependent on calcineurin-induced dephosphorylation and acti-
vation of protein kinase C and inhibition of the phosphoinositide phosphatase
synaptojanin (34).
Growth factors and receptors: Binding of ligands to the epidermal
growth factor receptors (EGFR or erbB) results in rapid disappearance of the recep-
tors from the cell surface. The process of activation of the receptor includes
receptor dimerization, activation of intrinsic receptor tyrosine kinase activity,
autophosphorylation of the receptor at the c-terminus and tyrosine phosphorylation
404 Shamsuddin
of intracellular signaling molecules such as Shc and PI3K (phosphatidylinositol

3-kinase). Ligand-induced endocytosis and degradation of the erbB receptor
cause down-regulation of the receptor. The ligands accelerate the endocytosis of
the receptors by promoting clustering of the receptors into clathrin-coated pits
on the plasma membrane. This is followed by receptor internalization into
clathrin-coated vesicles (35). An important structural component of coated pits is
the clathrin lattice anchored to cytoplasmic surface of the membrane by “plasma
membrane clathrin-associated protein complex 2” (AP2). Initial binding of erbB
receptors with AP2 is necessary for receptor-mediated endocytosis. From a
series of very elegant studies done at Professor Agarwal’s laboratory, Zi et al.
(36) report that IP6 impairs both receptor-mediated and fluid-phase endocytosis
resulting in inhibition of mitogenic signals associated with growth and prolifer-
ation of human prostate cancer cell line DU145. IP6 interacts with AP2 and inhibits
PI3K. IP6 also inhibits PI-30 -K-AKT signaling pathway as an upstream response in
its effects on the inhibition of fluid-phase endocytosis. IP6 completely inhibits
transforming growth factor-a [TGFa]-induced binding of activated erbB1 receptor
to AP2. IP6 treatment of DU145 human prostate cancer cells also results in a dose-
dependent increase in levels of activated erbB1 receptor. These are associated with
strong inhibition of ligand-induced Shc phosphorylation (36).
Interestingly, Voglmaier et al. (37) report that a partial amino acid sequence
from the purified IP6 receptor protein and a partial nucleotide sequence from a
cDNA clone of the gene are essentially identical to those of the a-subunit of
the clathrin assembly protein AP2. The IP6 receptor protein contains a series
of subunits, which are the same as those of AP2. In addition, antibodies to
AP2 react with the IP6 receptor protein (37).
Fibroblast growth factors. Fibroblast growth factors (FGF), which have
been implicated in tumor cell growth and angiogenesis, have biological activities
that appear to be mediated by both heparin-like extracellular matrix sites and
transmembrane tyrosine kinase receptor sites. IP6 inhibits basic FGF (bFGF)
binding to heparin. IP6 not only binds to bFGF, but also protects bFGF from
degradation by trypsin. In addition, IP6 inhibits the cellular binding of bFGF
and other fibroblast growth factor family members such as acidic FGF (aFGF)
and K-FGF in a saturable and dose-dependent manner. Concentrations as low
as 100 mM IP6 inhibits bFGF-induced DNA synthesis in AKR-2B fibroblasts, as
well as the growth of bFGF- and K-FGF-transfected NIH/3T3 cells (38).
Insulin-like growth factor. Insulin-like growth factor II/mannose-
6-phosphate (IGF-II/Man-6-P) receptors participate in the trafficking of
lysosomal enzymes. The transduction of the effects of IGF-II is also via trans-
membrane-anchored receptor protein. As in erbB described earlier, during
ligand-induced endocytosis, this receptor interacts with AP2, which can lead to
their assembly and subsequent transport in coated vesicles to the lysosomes.
Kar et al. (39) discovered that IP6-binding sites in rat brain are similarly distrib-
uted to IGF-II sites and that IP6 competes for IGF-II binding sites in the rat
brain. Our studies using HT-29 human colon cancer cells showed that the
precipitous increase in intracellular Ca2þ following IP6 treatment can be blocked

by prior pretreatment of cells with IGF-II, indicating a competition with the IGF-II
receptor (25).
Syndecan-4. Syndecan-4 is a transmembrane heparan sulfate proteogly-
can that can regulate cell – matrix interactions and is enriched in focal adhesions
(sites where cells form junctions with the extracellular matrix). Its cytoplasmic
domain contains a central region with a cationic motif that binds inositol phos-
pholipids. Highest affinity of the syndecan-4 cytoplasmic domain was seen
PIP2 and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligo-
merization. IP6 binds with high affinity to the syndecan-4 cytoplasmic domain
and is considered a potential down-regulator of syndecan-4 signaling. Cell
adhesion experiments showed that IP6 could block syndecan-4-dependent focal
adhesion and microfilament bundle formation in fibroblasts (40). Since metasta-
sis of cancer cells to distant sites depend initially on the ability of cancer cells to
attach to the extracellular matrix, these results explain at least in part, the molecu-
lar basis of decreased cell adhesion to extracellular matrix by IP6 as seen human
mammary cancer cell lines (41,42).
CELL PROLIFERATION AND CELL CYCLE

Normalization of Cell Proliferation
Uncontrolled proliferation is a hallmark of malignant cells, and IP6 reduces the
abnormally elevated and uncontrolled rate of cell proliferation of all the different
cell lines tested so far (43 –46). Since it does not affect the rate of proliferation of
normal cells either in vivo or in vitro, this function can best be called “normal-
ization” of cell proliferation. Although normal cells divide at a controlled and
limited rate, malignant cells escape from the control mechanisms that regulate
the frequency of cell multiplication and usually have lost the checkpoint controls
that prevent replication of defective cells. IP6 can regulate the cell cycle to block
uncontrolled cell division and force malignant cells either to differentiate or go
into apoptosis.
At the present time, aside from normalization of abnormal and uncontrolled
cell proliferation, the other fundamental processes are affected by IP6 induction
of cellular differentiation. It appears that IP6 also induces apoptosis, albeit in
large dosage. It is most likely that execution of these divergent and multiple sig-
naling pathways rather than a single signal and/or pathway brings about these
complex processes. Some of the cellular responses to extracellular signals may
be dose-dependent and abrupt, whereas other manifestations may not follow
any such rule. And the effect of extracellular signal may persist long after the
signal has disappeared. Data obtained from in vitro experiments show that the
cells’ response to IP6 insofar as cell proliferation and differentiation is concerned
follow this last pattern.
406 Shamsuddin
DNA Synthesis
Studies in my laboratory showed a suppression of DNA synthesis as measured by
3
H-thymidine incorporation and down-regulation of proliferation marker prolif-
erating cell nuclear antigen (PCNA) by IP6 (47). A marked decrease in the
expression of proliferation markers indicated that IP6 disengaged cells from
actively cycling. Using dual parameter flow cytometry and combined analysis
of the expression of cell cycle-related proteins, we also demonstrated that IP6
controls the progression of the cells through the cell cycle (48). IP6 treatment sig-
nificantly decreased the S-phase and arrested the human colon and breast cancer
cells in the G0 –G1 phase. Interestingly, the intracellular levels of IP6 are high in
G1 and G2 – M phases of cell cycle, but drop by 50– 75% during the S-phase
(28,49).
Studies of human leukemia cells at Professor Lambertenghi-Deliliers’ lab-
oratory at the University of Milan demonstrate that IP6 shows a dose-dependent
cytotoxic effect on human leukemia cell lines. The IP6-treated leukemia cells
accumulate in G2M phase of cell cycle (as opposed to G0 –G1 phase in breast
cancer cells (48)); once again arrest of cells in the cycle, albeit in a different
phase (50). cDNA microarray analysis showed an extensive down-modulation
of genes involved in transcription and cell cycle regulation (c-myc,
HPTPCAAX1, FUSE, cyclin H) and an up-regulation of cell cycle inhibitors
such as CKS2, p57, and Id-2. Genes such as STAT-6 and MAPKAP, involved
in important signal transduction pathways were also downregulated (50).
The Retinoblastoma Protein

One of the cell cycle controls disrupted in cancer cells resulting in unrestrained
entry into the cell cycle (hence uncontrolled cell proliferation) is the progression
of cells through G1 and entry to the S-phase. This progression requires hyperphos-
phorylation of retinoblastoma protein (pRb). The pRb was originally discovered
through studies of an inherited form of eye cancer in children—retinoblastoma.
The loss of both copies of the Rb gene results in excessive cell proliferation
of the immature retina. Thus, pRb was considered to be important in restraining
the cells from uncontrolled cell proliferation. A marked reduction in pRb
phosphorylation by IP6 has been reported in human mammary cancer cell
line (51).
The pRb-related proteins pRb/p107 and pRb2/p130 show considerable
homology with pRb and are considered to be a subfamily of pRb family of
proteins. They cooperate to regulate cell cycle progression through G1 phase.
These proteins bind to and modulate the E2F family of transcription factors
that induce the transcription of genes needed for progression to S-phase.
pRb2/p130 is the most abundant E2F complex found in quiescent cells in G0
phase. Singh et al. (32) reported that IP6 increases the level of hypophosphory-
lated Rb-related proteins pRb/107 and pRb2/p130 in DU145 human prostate
cancer cell line. IP6 moderately decreased E2F4, but increased its binding to both
pRb/p107 and pRb2/p130.
Protein Kinase C
Protein kinase C (PKC) signaling has an important role in diverse cellular pro-
cesses such as cell proliferation, differentiation, cell death, gene expression,
and tumor promotion. Thus, it is no surprise that at least some investigators
have directed their attention to the modulation of this family of serine/threonine
kinases by IP6.
IP6 stimulates insulin secretion and primes Ca2þ-induced exocytosis in
pancreatic b cells through activation of PKC (52). Intracellular application of
IP6 produces a dose-dependent stimulation of exocytosis, which is dependent
on PKC activity. Antisense oligonucleotides directed against specific PKC iso-
forms reveals the involvement of PKC-1 in IP6-induced exocytosis. Furthermore,
expression of dominant negative PKC-1 abolishes IP6-evoked exocytosis,
whereas expression of wild-type PKC-1 leads to a significant stimulation of
IP6-induced exocytosis (53).
Nickel and Belury (54) investigated the effect of IP6 on 12-O-tetradeca-
noylphorbol -13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity
in HEL-30 cells, a murine keratinocyte cell line, and SENCAR mouse skin. TPA-
induced ODC activity is an essential event in tumor promotion in mouse skin
model. ODC activity was significantly reduced by IP6. When mouse skin was
treated in vivo with IP6, ODC induction was also significantly inhibited. The
expression of TPA-induced c-mRNA was significantly inhibited by the same
IP6 treatments in HEL-30 cells and CD-1 mouse skin. No changes in PKC iso-
forms a and z expression and phorbol dibutyrate binding due to IP6 treatment
were found in HEL-30 cells. These results indicate that IP6 reduces TPA-
induced ODC activity independent of PKCa and z expression (54). While
Vucenik et al. (51) similarly report that treatment of human breast cancer cells
with IP6 resulted in no changes in the expression of PKCa, b, or j isomers.
However, there was a 3.1-fold increase in the expression of PKC. Along with
the increased activity, there was translocation of the enzyme from the cytosol
to the membrane (Vucenik et al., manuscript in preparation). Thus, not only
different isomers may be involved in different pathways, but IP6 may also differ-
entially modulate them.
Ras Proteins
One of the important family of protein molecules that helps broadcast the signal
from the cell surface to other parts of the cell is Ras family of monomeric
GTPases. There are two subfamilies: that involved in relaying the signal from
the cell-surface receptor to the actin cytoskeleton (Rho family) and that involved
in regulating the traffic of intracellular transport vesicle (Rab family). As in other
GTP-binding proteins, Ras is activated when bound to GTP as opposed to GDP,
which renders it inactive. Following activation of Ras, it stimulates various
408 Shamsuddin
signaling proteins downstream along different pathways, one of the most import-
ant being serine/threonine phosphorylation cascade. A critical component of this
cascade is mitogen-activated protein kinase (MAPK), which when activated
enters the nucleus. MAP kinases are usually activated transiently, which peaks
at 5 min followed by rapid decline and resulting in (as the name suggests) cell
division through activation of genes encoding G1 cyclins. Interestingly, full acti-
vation of MAPK requires phosphorylation of both a tyrosine and a threonine
residue, done by MAP-kinase – kinase also called MEK. MEK itself needs to
be activated via phosphorylation by Map-kinase –kinase – kinase also known as
Raf in mammalian system.
Since IP6 inhibits cell division, its effect on the cell cycle and these cell
cycle regulatory proteins and their genes have been looked at. Using DU145
human prostate cancer cell line, Singh et al. (32) studied the cell cycle
progression and apoptosis by flow cytometry. They also investigated the involve-
ment of G1 cell cycle regulators and their interplay, and end point markers of
apoptosis. A significant dose- and time-dependent growth inhibition of IP6-
treated cells was associated with an increase in cells in G1. IP6 strongly increased
the expression of cyclin-dependent kinase inhibitors (CDKIs)—Cip1/p21 and
Kip1/p27, without any noticeable changes in G1 CDKs and cyclins, except a
slight increase in cyclin D2. IP6 inhibited kinase activities associated with
CDK2, 4, and 6, and cyclin E and D1. Further studies showed the increased
binding of Kip1/p27 and Cip1/p21 with cyclin D1 and E. In down-stream of
CDKI–CDK/cyclin cascade, IP6 increased hypophosphorylated levels of Rb-
related proteins, pRb/p107 and pRb2/p130, and moderately decreased E2F4
but increased its binding to both pRb/p107 and pRb2/p130. At higher doses
and longer treatment times, IP6 caused a marked increase in apoptosis, which
was accompanied by increased levels of cleaved PARP and active caspase 3.
IP6 modulated CDKI – CDK – cyclin complex, and decreases CDK –cyclin kinase
activity, possibly leading to hypophosphorylation of Rb-related proteins and an
increased sequestration of E2F4. Higher doses of IP6 could induce apoptosis
and that might involve caspases activation.
Induction of Cell Differentiation

In general, cell differentiation depends on changes in gene expression resulting in
synthesis and accumulation of different sets of RNA and consequently protein
molecules; the latter often represent the differentiated features. Examples of
such markers of differentiation include hemoglobin for mature red blood cells,
prostate specific acid phosphatase for prostatic epithelial cells, lactalbumin for
mammary cells, and myoglobin for muscle cells. There are many steps in the
pathway leading from DNA to protein; in principle, all of them can be
regulated, affecting gene expressions. These steps include: transcriptional
control—when and how often a given gene is transcribed; RNA processing
control—regulating how the RNA transcript is spliced; RNA transport and
localization control—selecting the mRNAs to be exported from the nucleus to the

cytoplasm and where in the cytoplasm they are to be localized; translational
control—which mRNAs in the cytoplasm are translated; mRNA degradation
control—selectively inactivating certain mRNA molecules in the cytoplasm; or
protein activity control. For most genes, transcriptional control is the critical one.
These alterations in gene expressions can be (and often are) responses to
external cues or stimuli; the signal(s) switching the regulatory regions of DNA
near the site where transcription begins or by activating the gene regulatory
proteins that turn genes on or off.
IP6 has been demonstrated to induce differentiation of malignant cells of
divergent origins to the normal phenotype. It was first demonstrated in K-562
human erythroleukemia cells, which showed increased hemoglobin production
following IP6 treatment (55). Similar induction of tissue specific differentiation
was reported for human colon carcinoma HT-29 cells (47,56), prostate cancer
cells (57), breast cancer cells (58), and rhabdomyosarcoma cells (59). The mol-
ecular mechanisms involving these IP6-induced differentiation will be fascinating
to study. It is, however, known that PI 3-K (phosphoinositide 3-kinase) plays an
important role in granulocytic differentiation of HL-60 leukemia cells (60). Inter-
estingly, the intracellular concentration of IP6 (and IP5) is elevated by about two
orders of magnitude during chemotactic stimulation of HL-60 cells (61). Clearly,
an elevated level of IP6 plays a yet to be determined role in these differentiated
functions.
Programmed Cell Death (Apoptosis)

At concentrations up to 2 – 5 mM, IP6 inhibits cell proliferation of cancer cell
lines with concomitant induced differentiation, but without a substantial increase
in cell death in most cell lines. However, at higher dosage, or on prolonged treat-
ment it induces apoptosis or programmed cell death (32). HeLa cells on the other
hand appear to be more sensitive, undergoing apoptosis at IP6 concentrations
where very little apoptosis was observed in other cell lines (31). Be that as it
may, treatment of HeLa cells with tumor necrosis factor or insulin stimulates
the Akt-nuclear factor kB (NFkB) pathway. This is a cell survival signal invol-
ving the phosphorylation of Akt and IkB, nuclear translocation of NFkB, and
NFkB-luciferase transcription activity. IP6 blocks all these cellular events and
also causes mitochondrial permeabilization, followed by cytochrome c release
setting in motion the activation of the apoptotic machinery: caspase 9, caspase 3,
and poly(ADP-ribose) polymerase (PARP).
PI 3-Kinase
As one can gather from the preceding discussion, the enzyme PI 3-K has been
involved in a variety of cellular processes, including those affected by IP6;
indeed it is a crucial molecule in cellular signal transduction. PI 3-K causes
410 Shamsuddin
phosphorylation of the D-3 position of the inositol ring of phosphoinositides to

produce phosphatidylinositol-3-phosphate.
In mouse epidermal cell line JB6, IP6 markedly blocks epidermal growth
factor-induced PI 3-kinase activity in a dose-dependent manner. This blocking
of PI 3-K activity by IP6 profoundly impairs epidermal growth factor- or
phorbol ester-induced JB6 cell transformation and extracellular signal-regulated
protein kinases activation, as well as the transcription factor activator protein 1
(AP-1) activation (62).
NUCLEAR INOSITOL SIGNALING

mRNA Transport
Contrary to the fact that the genetic machinery of the cell lies in the nucleus,
detailed investigations of its structure has been relatively scanty. So it is no sur-
prise that the nuclear inositol signaling was virtually ignored, except for a notable
few (63). The nuclear skeleton, also called nuclear matrix or scaffold is a complex
structure of fibrogranular network and is contiguous with the intermediate fila-
ments of cytoskeleton. And then there are intranuclear channels, which derive
from both the layers of the nuclear envelope terminate at the nucleoli or pass
through the cytoplasm. Because these intranuclear channels are continuous
with the cytoplasm they have been speculated to be involved in nucleo-
cytoplasmic transport and that they would make the contact between the nucleoli
and the nuclear pores possible. There are no reasons to believe that external
stimuli working on the cell membrane would not influence the nucleus;
insulin-like growth factor (IGF), when bound to its receptor in the plasma
membrane rapidly activates nuclear PLC-b1. It was Cocco et al. (64) who first
demonstrated that the envelope-deprived nuclei could synthesize both phospha-
tidyl 4-phosphate and phosphatidyl 2-phosphate (PIP2). Thanks to this pioneering
work, it has been known (at least to a few investigators!) for some time now that
almost all, if not all of the enzymes and inositol derivatives that have been ident-
ified in the cytoplasm have also been found in the nucleus (63). So what do they
do there? It should not come as a surprise if they are involved in regulation of
gene expression.
The transcription of DNA to messenger RNA (mRNA) is carried out within
the nucleus, being segregated from the cytoplasm by the nuclear envelope; the
mRNA is then transported by a complex series of events through pores into
the cytoplasm. York et al. (65) demonstrated that the enzyme phospholipase C
and two proteins that influence the generation of IP6 are required for proper
and efficient export of mRNA from the nucleus to the cell. They identified
three genes in the yeast that are involved in the inositol signaling pathway:
PLC1 encoding phospholipase C (which converts PIP2 to IP3 and DAG), IPK1
encoding the inositol polyphosphate kinase Ipk1p (which converts IP5 to IP6),
and IPK2 encoding Ipk2p (which converts IP3 to IP4 and IP5). A mutation in
any of these genes blocks export of mRNA from the nucleus to the cytoplasm. IP6
being the end product of this metabolic pathway is therefore the most likely effec-
tor molecule controlling the mRNA export. The IPK2 gene is identical to the
yeast gene ARG8, which encodes Arg82p, a pleiotropic kinase that regulates
processes as diverse as response to stress, sporulation, and mating. Incidentally,
in yeast, stress increases IP6 level (66) causing increased export of certain
mRNAs that when translated into proteins would counteract the stressful
stimuli. Arg82 has a predicted molecular mass similar to the yeast IP3 – IP4
kinase activity. Along with Arg80, Arg81, and Mcm1, Arg82p is also an essential
component of ArgR – Mcm1 transcription complex essential for proper transcrip-
tional control that activates or represses genes involved in arginine metabolism.
Odom et al. (67) now show that this arginine production and breakdown is
dependent on the kinase activity of Ipk2p and the generated IP4 and IP5.
Chromatin Remodeling
The packaging of DNA into chromatin in eukaryotic cells limits its access to
DNA-binding proteins. Chromatin remodeling is therefore essential for efficient
transcription of eukaryotic genes, which use ATP-dependent chromatin remode-
ling complexes to regulate gene expression. The SWI2/SNF2 family of ATP-
dependent chromatin remodeling complexes is used to regulate DNA accessibility
for transcription. Four related classes of protein complexes use the energy of ATP
hydrolysis to alter nucleosome architecture. Shen et al. (68) and Steger et al. (69)
report that mutations in genes encoding inositol polyphosphate kinases that
produce IP4, IP5, and IP6 impair transcription in vivo, providing link between
inositol polyphosphates, chromatin remodeling, and gene expression.
NF-kB (Nuclear Transcription Factor-kB)

NF-kB proteins are gene regulatory proteins that also play an important role in
intercellular signaling during normal vertebrate development. There are five
NF-kB proteins in mammals (RelA, RelB, c-Rel, NF-kB1, and NF-kB2). They
form a variety of homodimers and heterodimers each of which activates different
sets of genes, the most abundant dimer is a p50/p65 heterodimer that binds to
DNA. In unstimulated cells, inhibitory proteins called IkB bind tightly to the
dimers and hold NF-kB in an inactive state within large protein complexes in
the cytoplasm preventing the latter (NF-kB) from localizing to the nucleus and
binding to the DNA. Inflammatory cytokines such as tumor necrosis factor a
(TNF-a) and interleukin-1 (IL-1) trigger a signaling pathway resulting in phos-
phorylation, ubiquitylation, and finally degradation of IkB by 26S proteasome
complex. This degradation of IkB exposes a nuclear localization signal on the
NF-kB proteins allowing them now to move into the nucleus and stimulate the
transcription of specific genes such as those involved in cell cycle regulation,
cell adhesion, and programmed cell death (apoptosis).
412 Shamsuddin
Since NF-kB is constitutively active in advanced and androgen-independent

human prostate cancer cell line DU145 cells, Agarwal et al. (70) assessed whether
IP6 inhibits this constitutively active NF-kB and associated upstream effectors.
They also investigated whether such an affect of IP6 has biological relevance in
causing inhibition of proliferation and induction of apoptosis in DU145 cells.
Treatment of cells with 1 and 2 mM IP6 resulted in a strong inhibition of
NF-kB activation. This finding was consistent with a decrease in nuclear levels
of NF-kB subunit proteins p65 and p50. IP6-treated cells also showed a strong
inhibition in phospho-IkBa protein levels concomitant with a significant increase
in total IkBa levels. This effect of IP6 also correlated with a significant inhibition
in IkBa kinase (IKKa) activity; and all of these correlating with a strong inhi-
bition in cell proliferation and induction of apoptosis in IP6-treated cells (70).
In a similar manner, in mouse epidermal JB6 cells, IP6 strongly blocked
UVB-AP-1 and NF-kB transcriptional activities in a dose-dependent fashion.
IP6 also suppressed UVB-induced AP-1 and NFkB –DNA binding activities
and inhibited UVB-induced phosphorylation of extracellular signal-regulated
protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs). IP6 also
blocked UVB-induced phosphorylation of IkB-alpha, which is known to result
in the inhibition of NF-kB transcriptional activity. Contrary to the epidermal
growth factor-induced PI 3-K activity (62), IP6 does not block UVB-induced
PI 3-K activity, suggesting that the inhibition of UVB-induced AP-1 and
NkF-B activities by IP6 is not mediated through PI-3 kinase (71).
Zinc-Finger Motif
A group of DNA-binding motifs consisting of an a-helix and a b-sheet held
together by one or more zinc atoms serves as an important regulator of transcrip-
tion. These are often found as a cluster with additional zinc fingers arranged one
after the other. This arrangement allows the a-helix of each zinc finger to come in
contact with the major groove of DNA for a considerable length along the groove.
Though there has not been any experimental demonstration of removal of the zinc
atoms from the zinc-finger motifs, it has nonetheless been speculated by some
that IP6 could in theory bind the zinc atoms and in turn affect the ability of
zinc-finger motifs (now without the zinc) to bind to the DNA. It has also been
speculated that IP6 by removing zinc could inhibit thymidine kinase, an
enzyme essential for DNA synthesis (72).
DNA Repair
There are other ways by which IP6 could influence the various activities within the
cells. For instance, repair of double-strand breaks in DNA is essential for maintain-
ing the stability of the genome; failure to repair may result in loss of genetic infor-
mation, chromosomal translocation, and even cell death. Two mechanisms for this
repair has been described—homologous recombination or non-homologous end-
joining, IP6 has been demonstrated to stimulate nonhomologous end-joining; it
has been proposed to be brought about by the binding of IP6 to the DNA-dependent
protein kinase DNA–PKcs (73). A more recent study reports that it is not DNA–
PKcs (a large protein of 3500 amino acids, Mw 465 kDa), but the DNA end
binding protein Ku (consists of Ku70—70 kDa, and Ku86—83 kDa) that binds to
IP6 (74). Be that as it may, these studies, in spite of their differences in their specific
findings clearly show a very important role of IP6 in DNA repair mechanism.
Once the assault on the cell has gone past the scope of DNA repair, the
otherwise heretofore normal cell is likely to transform to a malignant (cancer)
cell. Insofar as the transformation of cells from normal to malignancy is
concerned, there are various models and pathways, one of these pathways is
the activation of transcription factors activating protein-1 (AP-1) and nuclear
factor NF-kB via phosphatidylinositol 3-kinase (PI-3 kinase). Using tumor
promoter-induced cell transformation of human skin JB6 cells, Huang et al. (62)
have demonstrated that IP6 blocks epidermal growth factor-induced PI-3 kinase
and AP-1 activity. Zi et al. (36) reported similar results on DU145 human prostate
cancer cells along with concomitant inhibition of cell growth. Upstream of these
pathways lies the MAPK, which are serine/threonine kinases that are rapidly
activated upon extracellular stimulation. This family of kinases include extra-
cellular signal-regulated kinases (Erks), c-Jun N-terminal kinases (JNKs), and
p38 kinases. IP6 inhibited the activities of Erks and JNKs, but not of the p38
kinases in human skin, prostate, and breast cancer cells (36,62,75,76). Thus,
given the commonality shared by these three divergent cell types, the blocking
of this cellular to nuclear signaling pathway appears as an important mechanism
of anticancer action of IP6.
ROLE OF IP6 IN ENERGY TRANSDUCTION

Back in 1963, Morton and Raison (77) first suggested a link between IP6 and ATP
regeneration and its importance in seed development and germination. As usual,
there was considerable skepticism to such “outlandish” ideas. It was not till 1978
that the hypothesis appeared more plausible: an IP6 –ADP phosphotransferase
described by Biswas et al. (78) could use IP6 as a phosphate donor for the
conversion of ADP to ATP by transferring a phosphate group from 2-position
of IP6 to ADP in developing mung bean (Phaseolus aureus) seeds (78).
Additional support came from Phillippy et al. (79) who isolated a inositol
1,3,4,5,6-pentakisphosphate 2-kinase, which is involved in both formation of
IP6 in maturing seeds and catalyze the conversion of ATP from ADP in germinat-
ing seeds, in this case the soybean. It should not be an “outlandish” idea to think
that in mammalian cells too, IP6 may be crucial in providing the phosphates so
necessary in the conversion of ADP ! ATP.
ANTICANCER ACTION OF IP6: FROM THE LABORATORY

TO THE CLINIC
Since the first reports from my laboratory in the early 1987 (80 –82), we and sub-
sequently others have established the role IP6 as a broad-spectrum anticancer
414 Shamsuddin
agent, effective against cancers of different experimental models (43 – 45).

myo-Inositol itself was also demonstrated to have anticancer function, albeit
modest; however, the combination of inositol þ IP6 produced the best results,
being significantly better in different cancers than either one alone (43 – 46).
This was based on my working hypothesis that the anti-proliferative action of
IP6 is via the lower inositol phosphates and that adding inositol to IP6 would
increase the mass of these molecules through the activities of the kinases and
phosphatases as:
Inositol þ IP6 ! 2IP3 :
In addition, since the lower inositol phosphates (IP3 in particular) are the signal
transduction molecules (as you have gathered from the preceding discussions)
common to most forms of life, the anticancer effect would be seen in a wide
variety of tissues and organs. But that is where the ideal evolution of hypoth-
esis-driven scientific exercise ends as I only had a hypothesis and the burden
of proof was upon me. You will note from the bibliography that all the various
pathways of signal transduction have been described since my demonstration
of anticancer action in the later part of 1980s. In essence, my colleagues and I
were fortunate to have observed some fascinating phenomena in the form of strik-
ing anticancer action of IP6 and we are now beginning to understand some of the
molecular mechanisms. Science would like to have it the other way round: mech-
anisms first, demonstrate the effect (if any) later.
Perhaps (in part) owing to the lack of this knowledge as to the mechanism
of the function of IP6, there has not been the expected enthusiasm to translate
these rather amazing laboratory findings to the clinic. Belated though it may
be, following availability of IP6 þ inositol, since 1998, pilot clinical trials have
been started, which indeed confirm the broad-spectrum anticancer action in the
human (83,84). An enhanced anticancer activity without compromising the
patient’s quality of life was demonstrated in a pilot clinical trial of patients
with advanced colorectal cancer (Dukes C and D) with multiple liver and lung
metastasis (84). IP6 þ inositol was given as an adjuvant to chemotherapy accord-
ing to Mayo protocol. One patient with liver metastasis who refused chemother-
apy was treated only with IP6 þ inositol; her control ultrasound and abdominal
computed tomography scan after 14 months showed a significantly reduced
growth rate. A reduced tumor growth rate was noticed overall and in some
cases a regression of lesions was noted. Additionally, when IP6 þ inositol was
given in combination with chemotherapy, side effects of chemotherapy (drop
in leukocyte and platelet counts, nausea, vomiting, alopecia) were diminished
and patients were able to perform their daily activities (84). Notwithstanding
the politics of medical economics, it is hoped that reports such as this will
bring the benefit of IP6 þ inositol to the doors of cancer patients and to the popu-
lation at large for cancer prevention.
OTHER BIOLOGICAL EFFECTS OF IP6

IP6 is not only virtually safe and has nontoxic effects, but it also has many other
beneficial health effects such as inhibition of kidney stone formation and
reduction in risk of developing cardiovascular disease. IP6 was administered
orally either as the pure sodium salt or in a diet in order to reduce hypercalciuria
and to prevent formation of kidney stones, and no evidence of toxicity was
reported (85). A potential hypocholesterolemic effect of IP6 may be very signifi-
cant in the clinical management of hyperlipidemia and diabetes (86). IP6 inhibits
agonist-induced platelet aggregation (87) and efficiently protects myocardium
from ischemic damage and reperfusion injury (88), both of which are important
for the prevention and management of cardiovascular diseases.
CONCLUSION
I have attempted to give a somewhat comprehensive up-to-date review of the role
of IP6 and other inositol phosphates in cellular signal transduction. Beyond
reasonable doubt, IP6 plays a crucial role in cellular signaling. Likewise, its anti-
cancer actions and other health benefits are also unquestionable. In addition, this
is a compound that is present in physiological concentrations in various cells,
tissues, and in our body fluid, the level of which changes with intake and
deficiency (89,90). Hopefully, some day, it may join the list of compounds as
a vitamin!
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20
Modulation of Gene Expression
by Dietary Iron
Paul Sharp
King’s College, London, UK
Introduction 421
Dietary Iron Absorption 422
Iron-Dependent Regulation of Intestinal Iron Absorption 423
Iron Regulatory Proteins 424
Iron Responsive Elements 424
Regulation of DMT1 426
Regulation of IREG1 426
Regulation of Dcytb and Hephaestin 427
Hypoxia 427
The Role of Hepcidin in Regulating Body Iron Metabolism 428
Hemochromatosis 430
Iron: A Pro-oxidant 432
Conclusions 433
References 433
INTRODUCTION
Iron is an essential trace metal in our diet and plays a key role in a plethora of
biochemical processes in the body including the binding and release of oxygen
421
422 Sharp
in hemoglobin and electron transfer in mitochondrial oxidative phosphorylation

by the cytochromes. These processes can be compromised if dietary iron supply
is reduced with obvious consequences for human health. However, despite being
an essential nutrient, iron is a powerful pro-oxidant, and in excess can lead to
oxidative damage to cells and tissues through the formation of free radicals
(1). Consequently, body iron levels must be regulated within strictly defined
limits to avoid pathologies associated with iron deficiency and overload. The
major iron-related diseases are iron deficiency anemia, which affects up to 2
billion people and is regarded as the most prevalent nutritional deficiency dis-
order worldwide, and hereditary hemochromatosis, a genetic disease that predis-
poses 1 person in every 200 of northern European decent to body iron loading. To
maintain homeostatic control over iron nutriture, a number of regulatory mech-
anisms have evolved to match dietary iron absorption to the body’s physiological
requirements. This chapter will review the current knowledge regarding the
major iron-sensitive signals involved in the homeostatic regulation of body
iron metabolism.
DIETARY IRON ABSORPTION

Most western diets contain a mixture of heme (found exclusively in animal tissues)
and nonheme iron (not only found extensively in cereals and vegetables, but also
in meat). Heme accounts for 5– 10% of the daily iron intake in industrialized
countries (2); whereas in vegetarian diets and in developing countries, the
heme iron intake is negligible. The main form of iron in all diets is nonheme
iron. These different forms of iron are absorbed by totally independent transport
mechanisms that reside on the apical membrane of the duodenal enterocytes
(Fig. 20.1). Heme is removed from hemoglobin and myoglobin in the intestinal
lumen and is absorbed intact through an as yet uncharacterized heme transporter.
Inside the cell, iron is removed from the porphyrin shell by the action of heme
oxygenase 1 (3).
Nonheme iron in the diet is present largely in its ferric form, which is non-
bioavailable and must therefore be reduced to ferrous prior to uptake into the duo-
denal enterocytes. This is achieved by the action of a number of reducing agents
in the diet, including ascorbic acids and small peptides containing cysteinyl and
histidyl residues. In addition, the apical membrane of duodenal enterocytes con-
tains ferric reductase activity in the form of the recently characterized Dcytb
enzyme (Duodenal cytochrome b) (4). The relatively low pH of the duodenal
lumen together with the acid microclimate that covers the apical surface of enter-
ocyte (5,6) stabilizes iron in the ferrous form and provides the inwardly directed
proton gradient that drives iron uptake through the apical membrane iron trans-
porter DMT1 (7,8). Nonheme iron together with iron liberated from heme
enter a common pool inside the duodenal enterocytes and this iron can either
be stored in the form of ferritin inside the cells or can be transferred across the
basolateral membrane via IREG1 (9) before being re-oxidized back to Fe3þ by
Modulation of Gene Expression by Dietary Iron 423
Fe 3+
ferritin
ascorbate
etc.
Dcytb Fe3+
Fe 2+ DMT1 Fe 2+ Hp
Fe3+ Fe3+
LIP Fe2+ IREG1 Tf
Haem HT HO
apical basolateral
Figure 20.1 Duodenal iron transport. Nonheme iron is present in the diet largely in the
less bioavailable ferric or Fe3þ form. To facilitate absorption, iron must first be reduced to
the Fe2þ or ferrous state and this is achieved by the presence of dietary reducing agents,
such as ascorbate, in the duodenal lumen and by the endogenous ferric reductase Dcytb,
which resides on the apical membrane of enterocytes. Fe2þ is readily transported into
enterocytes through DMT1 via a proton-dependent uptake mechanism. Heme enters the
cell intact through an as yet uncharacterized transporter (HT) and the iron contained
within the porphyrin ring is excised under the action of heme oxygenase (HO). Iron
derived from both heme and nonheme sources enters a common pool inside the enterocytes
and can either be targeted for storage in ferritin, if body iron requirements are low, or can
enter a labile iron pool (LIP) from where it is processed for onward transport out of the
cell. Efflux across the basolateral membrane occurs through IREG1 as Fe2þ, though
iron is rapidly oxidized to Fe3þ in the presence of the ferrioxidase activity of hephaestin
(Hp) and picked up by transferrin (Tf ) for transport to its sites of utilization in the body.
hephaestin (10). Ferric iron leaving the cell is picked up by transferrin for onward
delivery to the iron stores in the liver and to the utilizing tissues.
IRON-DEPENDENT REGULATION OF INTESTINAL

IRON ABSORPTION
All of the key components of the intestinal iron transport machinery identified
earlier are subject to tight regulation by the dietary iron load, the state of the
body iron stores, and the iron requirements for erythrocyte production.
Regulation of these intestinal genes by iron utilizes several mechanisms.
All cells and tissues must maintain a homeostatic balance between receiv-
ing an adequate iron supply while avoiding the build up of free iron inside the cell
that might lead to permanent cellular damage through the generation of reactive
oxygen species (ROS). In most cells, iron supply is regulated by the number of
424 Sharp
transferrin receptors (TfR) expressed on the cell surface; whereas inside the cell,
excess iron is sequestered in ferritin. The expression of both TfR and ferritin is
directly related to intracellular iron levels through posttranscriptional mechan-
isms that involve interactions between cytosolic iron regulatory proteins (IRP)
and stem loop structures, known as iron responsive elements (IRE), that exist
in either the 50 or 30 untranslated region (UTR) of several target mRNA species.
Iron Regulatory Proteins

Two cytosolic IRP, IRP-1 and IRP-2, are known to exist in most cells. Both of
these proteins can bind to IRE structures within RNA sequences when cellular
iron levels are depressed. However, under iron replete conditions, RNA
binding is quickly inactivated by either posttranslational modification of IRP-1
(there is no decrease in total IRP-1 levels) or degradation of IRP-2. The under-
lying mechanism behind the iron-dependent posttranslational inactivation of
IRP-1 has been studied extensively. Structurally, IRP-1 is remarkably similar
in its amino acid sequence to the mitochondrial acontitase (11) that converts
citrate to isocitrate in the tricarboxylic acid cycle. Under iron replete conditions,
IRP-1 can act as a cytoplasmic aconitase and this dual function is controlled by
the presence or absence of an iron sulfur cluster (Fig. 20.2). When cellular iron is
high, a 4Fe– 4S cluster is inserted into IRP-1 and is held in place by three con-
served cysteine residues (also present in mitochondrial aconitase). Under these
conditions, IRP-1 has a closed conformation and cannot bind IREs. The fourth
position iron in the cluster is highly labile and is readily removed when cellular
iron levels are low, leading to disassembly of the Fe – S cluster which permits the
apoprotein to bind IRE sequences (12 – 14).
IRP-2 is less abundant in cells than IRP-1, and is subject to de novo syn-
thesis when cellular iron levels are low and proteosomal degradation when
iron levels are high (15). IRP-2 contributes significantly to the total IRP RNA
binding activity in several tissues but particularly in the brain (16) and intestine
(17). Both IRP-1 and IRP-2 bind successfully to the consensus IRE sequence;
however, recent evidence suggests that IRP-2 may be able to recognize exclu-
sively a specific subset of IRE sequences (18,19).
Iron Responsive Elements

The IRE is a stem loop structure within the UTRs of several mRNA species. A
single IRE was first identified in the 50 UTR of ferritin mRNA (20 –23), and
shortly afterwards five similar sequences were identified in the 30 UTR of the
TfR mRNA (24). The sequence of the hairpin loop 50 -CAGUGN-30 is highly con-
served and there is a further C six bases upstream of this central motif that is
unpaired and forms a bulge in the stem (Fig. 20.2). However, the sequence
within the stem is variable and can give rise to several different predicted
secondary structures.
G
A U
(a) C G (b)
N
N-N
N-N
N-N +Fe
N-N C C C C
C
N-N C C
N-N
N-N
N-N IRP activity aconitase activity
N-N
N-N
5’ – N-N – 3’
(c) (d)
5’ ORF
5’ 43S entry site ORF 3’

AAA - 3’
X
Figure 20.2 IRE/IRP interactions regulate the expression of a number of iron responsive
mRNA species. IREs are stem loop structures present within the UTR of a number of mam-
malian mRNA species (a). A number of variations in the structure of individual IREs have
been observed but they are characterized by an asymmetrical bulge within the RNA stem,
due to the presence of an unpaired C, and the consensus sequence 50 -CAGUGN-30 which
forms the loop. Under low cellular iron conditions, IRE interact with cytosolic IRP (b).
However, this binding is lost in iron replete cells due to the insertion of a cubic 4Fe–4S
cluster that is held in place by three conserved cysteine residues. In the case of IRP-1, the
presence of the iron–sulfur cluster converts the RNA binding protein into one that exhibits
cytoplasmic aconitase activity. IRP binding to IRE in the 50 UTR (e.g., in ferritin) prevents
the binding of the eIF4F complex to the 43S entry site that is required protein translation
(c)—hence under low cellular iron conditions, ferritin protein is decreased. TfR contains
five consensus IRE sequences in its 30 UTR (d). Binding of IRP in this scenario blocks endo-
nuclease-mediated mRNA degradation, thus increasing mRNA half-life.
Under iron replete conditions, ferritin mRNA is efficiently translated to

form of the cell’s major storage protein. However, when cellular iron levels
decrease, ferritin protein levels are also lowered. This decrease in ferritin is
directly attributable to the position of the IRE in the 50 UTR. The IRE in both
ferritin H and L chains (and other iron responsive with 50 IRE sequences such
as mitochondrial aconitase and erythroid 5-ALA-synthase) is less than 40
bases from the AUG site, and binding of IRP to these IRE sequences prevents
the binding of the eukaryotic initiation factor (eIF4F) complex to the 43S riboso-
mal subunit that is necessary for protein translation (25).
The regulation of the TfR by iron is directly opposite to the control of
ferritin. Under iron replete conditions, TfR protein levels are low; whereas,
426 Sharp
when cellular iron is low, the expression of TfR protein is elevated due to acti-
vated IRP binding to all or a number of the five IRE sequences present in the
30 UTR. It is believed that IRP/IRE binding does not alter the rate of TfR
protein translation but rather promotes mRNA stability by protecting it from
endonucleolytic degradation, thus increasing TfR mRNA half-life (24,26,27).
Regulation of DMT1
The overall regulation of DMT1 by iron is a complex issue. Analysis of the
DMT1 gene has revealed evidence that it encodes at least four variants through
two alternate splicing events—one at the 50 end of the gene leads to two separate
initiation sites in exon 1A or exon 1B, repectively, both of which are in frame
with exon 2 (28) and an alternate splice site in exon 16 that is in frame with
exon 16A or exon 17 (29). The exon 16A variant leads to the transcription of
mRNA containing a single IRE in the 30 UTR, whereas the exon 17 transcript
lacks this IRE. With regard to the exon 1A and exon 1B variants, it has been
suggested that DMT1A is predominantly found in epithelial tissues, whereas
DMT1B is ubiquitously expressed (28). The relative abundance of these four
possible variants (i.e., 1A/16A, 1A/17, 1B/16A, or 1B/17) and their function
in terms of iron uptake in various tissues are still unclear.
Currently, there is a great deal of confusion regarding the role of the 30 IRE
in DMT1 regulation by iron. Initial studies in animal models of iron deficiency
demonstrated that DMT1 mRNA was regulated appropriately by iron status for
mRNA containing an IRE in the 30 UTR, that is, an increase under iron-deficient
conditions (7). Furthermore, in vitro binding studies established that the 30 IRE
recognizes and binds cytosolic IRP (30). However, recent data suggest that
despite binding IRP, the DMT1 IRE might not be functionally involved in regu-
lating mRNA levels (31). This is an area that clearly needs to be resolved.
Mechanisms other than IRE/IRP interactions are also implicated in the
regulation of DMT1. Our own work and that of others (28) has shown that the
expression of non-IRE containing DMT1 transcripts is regulated by both iron
deficiency and iron loading. This suggests that the promoter region of the gene
may play an important role in the overall regulation of DMT1 expression. To
this end, several putative transcription factor binding sites have been identified
upstream of the exon 1B variants (29,32). Using a DMT1 construct in which
1.5 kb of the DMT1 1B promoter was cloned upstream of a luciferase reporter
gene, we have demonstrated that several transition metals (iron, zinc, and
copper) can induce luciferase activity (33). At this stage, the nature of the
metal-sensitive transcription factors activated in these studies remains elusive.
Regulation of IREG1
IREG1—also known as ferroportin 1 (34) and MTP1 (35)—mRNA also contains
an IRE sequence, though in contrast to DMT1 it resides in the 50 UTR. In general,
binding of IRP to a 50 IRE is thought to lead to a decrease in protein translation
(e.g., ferritin). However, in our studies using the intestinal Caco-2 cell line, we do
not see any change in IREG1 mRNA or protein expression or alterations in cel-
lular iron efflux following iron loading (36). Indeed, several studies have reported
that duodenal IREG1 expression is upregulated in iron-deficient human and
animals as well as in hemochromatotic patients (9,37). These findings, taken
together, question the role of the IRE in regulating IREG1 expression. Previous
studies have shown that IRE/IRP interactions that take place at a distance of
greater than 67 nucleotides from the transcription start site cannot repress protein
translation (38). In the duodenal IREG1 transcript, the distance from the tran-
scription start site to the base of the IRE is somewhat greater than 67 bases
(9,35) suggesting that the IRE may not be functional. Intriguingly, IRE-mediated
regulation of IREG1 does take place in hepatocytes (39,40) and in a number of
other tissues (41) suggesting that IREG1 mRNA processing is tissue-specific
and is designed to match the function of those tissues in body iron metabolism.
Regulation of Dcytb and Hephaestin

Efficient transepithelial transport of iron in the intestine relies on two key redox
reactions, reduction of ferric iron to ferrous to allow entry into the enterocyte
across the apical membrane and oxidation of ferrous iron leaving the cell to
ferric to permit loading onto transferrin for onward transport in the plasma.
These processes are governed by Dcytb, the recently characterized ferrireductase
(4), and hephaestin, a ceruloplasmin homolog that acts as a ferrioxidase in enter-
ocytes (10), respectively. Dcytb in particular is strongly iron-regulated,
expression is increased in iron deficiency and by hypoxia (4). However,
despite the crucial role played by hephaestin in iron efflux, highlighted by sla
mice that have a profound anemia due to a hephaestin mutation, there is still
some question as to whether it is regulated by iron. In rodent models, dietary
iron manipulation has no discernable effect on hephaestin expression (42),
whereas in patients with iron deficiency, hephaestin levels are upregulated
(43). Importantly, this latter study demonstrated that the expression of DMT1,
Dcytb, IREG1, and hephaestin were positively related to each other indepen-
dently of iron status, suggesting that a co-ordinated mechanism exists for the
transfer of dietary iron across the intestine and into the blood. However, unlike
the transporters themselves, Dcytb and hephaestin do not contain IRE sequences
in their UTRs indicating perhaps that iron status regulates these genes at the tran-
scriptional level.
HYPOXIA
The development of iron deficiency anemia is a multistage process, initiated by a
decrease in body iron stores (characterized by reduced serum ferritin levels),
progressing onto iron-deficient eryrthropoiesis (when the stores are empty) that,
if untreated, ultimately leads to the development of a microcytic hypochromic
428 Sharp
anemia. The decrease in hemoglobin production seriously compromises the

body’s ability to distribute oxygen to the tissues resulting in hypoxic stress.
Hypoxia itself has long been recognized as an important regulator of body iron
metabolism. Approximately 1% of the circulating red blood cell population is
removed and degraded by the reticuloendothelial macrophages every day and
replaced with new erythrocytes. The rate of erythropoiesis is controlled by the
hormone erythropoietin, produced in cells residing in the kidney and liver, and
the primary stimulus for its production is tissue hypoxia (44,45). The trigger
for induction of erythropoietin production by hypoxia is the transcription
factor HIF-1 (hypoxia inducible factor-1) (46,47), which is formed of two sub-
units (a and b) that combine and bind to hypoxia responsive elements—DNA
sequences in the flanking regions of a number of hypoxia-sensitive genes (48).
Hypoxia has been shown to upregulate duodenal iron absorption in rats (49)
and in humans (50) presumably to satisfy the increased requirement for iron in
erythropoiesis. The cellular mechanisms involved in this adaptation are emerging
and include an increased electrical driving force for iron uptake (51), an increased
expression of proteins involved in iron transport including Dcytb (4), IREG1 (9)
and a TfR (52). In TfR, the effect of hypoxia is mediated by the presence of a
hypoxia responsive element consensus sequence (52). Interestingly, analysis of
the DMT1 promoter identified two potential hypoxia responsive sequences
suggesting that it too may be a hypoxia-regulated gene (29). However, it is
unknown whether similar sequences exist in the flanking regions of other key
components of the iron transport machinery.
A further link between hypoxia and iron metabolism has been identified
recently. In mice subjected to hypobaric pressure and in hepatoma cell lines
maintained under low oxygen conditions, the expression of hepcidin, an import-
ant peptide involved in the regulation of body iron metabolism (see later), was
significantly decreased (53). This gives rise to the possibility that hypoxic
stimuli could also exert effects on genes that do not contain classical DNA
sequences recognizing the HIF-1 transcription factor.
THE ROLE OF HEPCIDIN IN REGULATING BODY

IRON METABOLISM
The nature of the signals generated by the body iron stores has been the subject of
much speculation. Recently, a candidate peptide that might explain the link
between iron requirements and the regulation of intestinal absorption has been
identified. Hepcidin is predominantly expressed in the liver and its mRNA
encodes a 25 amino acid peptide with significant antimicrobial activity (54,55).
In addition to this antimicrobial action, hepcidin expression is associated with
body iron status and is dramatically upregulated when liver iron is high and
downregulated when the stores are depleted (56). A role for hepcidin in iron
metabolism was subsequently established using knockout mice in which the
USF2 transcription factor had been deleted. These animals developed a severe
iron overload, strikingly similar to that found in human hemochromatosis and
in the Hfe 2/2 mouse (57). Subsequent examination of the Usf 2 2/2 mice
revealed that the hepcidin gene had also been disrupted (the two mouse genes
are only 1240 bp apart) (56). An alternative gene targeting methodology con-
firmed that it was the disruption of the hepcidin gene and not USF2 that resulted
in the iron overloading phenotype (58). Interestingly, recent studies have demon-
strated a link between hepcidin expression and the regulation of human iron
metabolism. Two families have been identified who have mutations in the hepci-
din gene that led to a novel variant of juvenile hemochromatosis, a particularly
severe form of the iron loading disease that typically affects people in their
late teens and early twenties (59).
Recently, transgenic mice over expressing hepcidin have been generated
(58). These animals have severe body iron deficiency and microcytic hypochro-
mic anemia suggesting a reciprocal relationship between hepcidin expression and
iron accumulation. Furthermore, studies in humans have demonstrated that inap-
propriate expression of hepcidin is associated with the anemia of chronic disease
(53,60). In one of these studies (60), two patients with severe iron deficiency were
identified who had large hepatic adenomas. Analysis of the tumors showed that
hepcidin mRNA was massively over expressed, a situation that would lead to
increased circulating hepcidin levels. Resection of the tumors reversed the clini-
cal anemia suggesting that changes in hepcidin expression provide the link for the
perturbations in iron metabolism observed in chronic disease. Further evidence
for this link is afforded by recent work demonstrating that hepcidin mRNA is
also increased in animal models of inflammation (53) and following exposure
of human hepatocytes to monokines and cytokines (61).
Hepcidin expression following administration of an iron-deficient diet is
inversely correlated with intestinal iron transporter expression (62). In this
model, it is proposed that changes in dietary iron levels affect the degree of trans-
ferrin saturation by iron. Consequently, the changes in transferrin saturation are
detected by the liver, and hepcidin expression is regulated accordingly—
decreased when transferrin saturation is low, increased when transferrin satur-
ation is high. However, to add a further level of intrigue to the role of hepcidin
in the regulation of body iron metabolism, recent studies have shown that
exposure of isolated hepatocytes (61) and hepatoma cells (63) to iron down
regulated hepcidin mRNA.
In current models regarding the regulation of intestinal iron absorption,
hepcidin is thought to be the major modulator involved. Elevated levels of hep-
cidin are believed to act directly on the intestinal epithelium and act as a repressor
of iron absorption. However, at this time the precise intestinal site of action of
hepcidin is unclear with the arguments focusing on whether it contributes to
the programing of the duodenal crypt cells or acts directly on the mature
enterocytes (Fig. 20.3).
430 Sharp
Dcytb enterocyte
Hp
IREG1
DMT1
hepcidin
? IRP Fe2+
gene
transcription
crypt
DMT
HEPATOCYTE cell
Fe2+
Tf
HFE
hepcidin
mRNA
?
Fe3+
Fe3+
HFE HFE
Tf
Tf
Fe3+
Fe3+
TfR hepcidin TfR
Figure 20.3 Regulation of duodenal iron transport: roles of HFE/TfR and hepcidin. Cir-
culating transferrin (Tf ) binds to transferrin receptors (TfR) located on the serosal surface
of the duodenal crypt cells. HFE is also localized to this membrane due to its interaction
with b2-microglobulin and binds to TfR regulating the rate of receptor recycling. This
level of control is lost in hemochromatosis. The HFE/Tf/TfR complex is endocytosed
and iron dissociates from Tf in the acidified environment of the endosome and is trans-
ported into the cytosol via DMT1. Changes in the cellular iron content influence the
binding of IRP to IRE in the UTR of a number of mRNA species (including DMT1,
TfR, IREG1, and ferritin) and alters protein translation accordingly. HFE/TfR interactions
also, in part, regulate iron accumulation by hepatocytes. Hepcidin, a 25 amino acid
peptide, is synthesized and released from the hepatocyte when body iron levels are high
and is thought to act directly on the intestinal epithelium as a negative regulator of
dietary iron absorption. The intestinal site of action of hepcidin is still unclear. It could
bind within the crypts and contribute to the overall programing of duodenal iron transport
by regulating the transcription of a number of genes involved in cellular iron metabolism.
Alternatively, hepcidin could act directly on the mature enterocytes to influence the cell
surface expression of the essential components of the iron transport machinery.
HEMOCHROMATOSIS
Hereditary hemochromatosis is a relatively common inborn error of iron metab-
olism (1:200 people mainly of northern European decent are affected) that is
characterized by excess iron accumulation and deposition within several tissues,
especially the liver, which can lead to cell and tissue damage (e.g., fibrosis and
cirrhosis in the liver). The most common form of hemochromatosis arises from an
autosomal recessive mutation that leads to the substitution of tyrosine for
cysteine at amino acid 282 of the HFE protein (64). Subsequent analysis of the
hfe gene has demonstrated that a number of other less pathogenic (in terms of
body iron status) mutations also exist in addition to C282Y (65). The involvement
of HFE in iron metabolism was confirmed following the production of a knockout
mouse, which subsequently developed iron overload and resembled the human
hereditary hemochromatosis phenotype (66).
The HFE protein is localized to a number of tissues that have major roles in
body iron metabolism including the duodenum (where it is found exclusively in
the crypts of Lieberkühn) (67), the liver (in Kupffer cells and hepatocytes)
(68,69), and in tissue macrophages and circulating monocytes (70). HFE is a
member of the MHC class 1 family of molecules, and not an iron transport
protein as first predicted, and as such it needs to be associated with b2-
microglobulin for normal intracellular processing and cell surface expression
(71 – 73). b2-Microglobulin itself is known to play an essential role in iron metab-
olism since deletion of this gene leads to a progressive iron overload similar to
that seen in hemochromatosis patients (74,75). In addition to its interaction
with b2-microglobulin, HFE also binds to TfR regulating the rate at which trans-
ferrin-bound iron can enter the cell (76,77). Normally, iron uptake by the duode-
num from the plasma is directly proportional to the plasma iron concentration.
However, in hfe knockout mice duodenal iron uptake from plasma transferrin
is significantly lower than in control mice, supporting the hypothesis that HFE
plays a crucial role in regulating the uptake of transferrin-bound iron (78).
The HFE protein is thought to be a major regulator of intestinal iron absorp-
tion (Fig. 20.3) through its interaction with TfR since this establishes the prevail-
ing cellular iron concentration within the duodenal crypt cells, which ultimately
determines the level of expression of the proteins involved in iron absorption
(i.e., DMT1 and Dcytb at the apical membrane and IREG1 plus hephaestin at
the basolateral surface) in enterocytes as they migrate along the crypt-villus
axis. Since only the mature enterocytes, located on the upper third of the
villus, participate in translocation of dietary iron from the intestinal lumen to
the blood, this essentially means that transporter protein levels are pre-programed
in the cells leaving the crypt and that re-programing in response to changes in
body iron status would take a further 2– 3 days to be established (i.e., the time
taken from crypt stem cell division to arrival of the mature enterocyte at the
villus tip). Given that the C282Y mutant protein does not interact with TfR, it
has been postulated that in hemochromatotic patients as well as knockout
mouse models of the disease, dietary iron absorption proceeds in a less regulated
fashion and is therefore inappropriately high in relation to the body iron stores.
Several pieces of evidence suggest that HFE/TfR interaction cannot be the
only regulatory pathway controlling intestinal iron absorption. First, in studies
employing hfe knockout mice, levels of the iron transporters and ferrireduc-
tase activity are not compromised compared with wild type littermates (79).
432 Sharp
Furthermore, these animals still respond appropriately to dietary iron deficiency

or loading even in the absence of functional HFE protein (80 – 83). In nonphlebo-
tomized patients with hemochromatosis, levels of DMT1 and IREG1 also were
unaltered compared with iron replete subjects (84). This apparent paradox can
perhaps be explained by the recent observation that HFE status can regulate
hepatic hepcidin production. In hemochromatotic patients, and in hfe knockout
mice, hepcidin mRNA levels are inappropriately low given the degree of iron
loading (85,86), which might in turn account for the relatively high intestinal
iron absorption that characterizes the disease. Furthermore, when hfe knockout
mice were crossed with transgenic mice that constitutively expressed hepcidin
intestinal, iron absorption was significantly reduced (87) adding credence to
the hypothesis that hepcidin and not HFE is the master controller of body iron
metabolism.
IRON: A PRO-OXIDANT
Despite its essential role in metabolism, iron is also a prospective pro-oxidant and
is therefore potentially harmful. Excess iron promotes lipid peroxidation and
tissue damage in vitro (1) raising the possibility that, via these pro-oxidant
effects, disturbances in iron metabolism may play a pathogenic role in a
number of diseases. Iron participates in the Fenton reaction with hydrogen per-
oxide (a by-product of cellular oxygen metabolism) to produce the highly dama-
ging hydroxyl radical.
Fe2þ þ H2 O2 ¼ Fe3þ þ OH þ OH†
Hydroxyl radicals once generated react with virtually all molecules in

living cells, resulting, for example, in the peroxidation of membrane lipids or
DNA strand breakages leading to cell aging and death. Iron chelated in low mol-
ecular weight complexes in particular has been shown to lead to the generation of
ROS that in turn impact on the modulation of cellular signal transduction path-
ways (e.g., those leading to NFkB activation) with downstream consequences
for gene transcription (88). Furthermore, iron-dependent generation of lipid
peroxidation products is associated with altered expression of a number of
genes (89).
The central nervous system in particular appears to be very sensitive to
damage by ROS and there is an increasing body of evidence that suggests that
ROS generated through metal-dependent processes might lead to neurodegenera-
tive diseases such as Alzheimer’s disease, Friedriech’s ataxia, and Parkinson’s
disease (90). Iron plays an essential role in neural function as it is a cofactor
for the production of the neurotransmitters dopamine, noradrenaline, and 5-HT
(91). An intriguing link between iron and Alzheimer’s disease has emerged
with the discovery that the amyloid precursor protein (APP) contains a type II
IRE in its 50 UTR (92) in a region containing mutations associated with familial
Alzheimer’s disease (93 – 95). Studies have shown that IRP binding to the APP 50
UTR is reduced after treatment of cells with the iron chelator desferrioxamine,
suggesting a significant role for iron in the metabolism of APP as well as
highlighting a possible therapeutic role for iron chelators in the treatment of
Alzheimer’s disease (92). The IRE/IRP system per se may be very important
in regulating brain iron metabolism. Studies in mice with a targeted disruption
of the IRP2 gene have revealed that the mutant mice have disregulated iron
metabolism in the CNS and ultimately develop movement disorders character-
ized by ataxia, bradykinesia, and tremor that are associated with iron accumu-
lation in the white matter tracts and nuclei (96).
In certain situations, excess cellular iron is associated with cancer, for
example, death from hepatocellular carcinoma is increased several hundred
fold in patients diagnosed with hereditary hemochromatosis (97). The link
between iron and cancer rests on the fact that proliferating cells have an absolute
requirement for iron to catalyze a number of key reactions involved in oxygen
sensing, energy metabolism, and DNA synthesis (in the absence of iron, cells
cannot proceed from G1 to S phase of the cell cycle). Indeed iron chelators
are being increasingly used as anticancer agents since they can exert anti-
proliferative effects on cancer cells (98).
CONCLUSIONS
The studies reviewed here highlight the numerous pathways by which iron can
influence gene expression and physiological function. These range from the
well-characterized interactions between cytosolic IRP and IRE sequences
present in the UTR of a number of genes through to the influences of novel pep-
tides (hepcidin) and ROS on the cellular signals that control the expression of key
components of the body’s iron regulatory system. The role of hepcidin in particu-
lar is an area attracting much current research interest since is appears to act as
the common pathway that relays the information from both the main site of
iron storage (the liver) and the iron utilization (the bone marrow) that is necessary
to match intestinal iron absorption in line with the body’s physiological require-
ments. Perturbations in body iron metabolism do not just lead to the development
of iron deficiency anemia or iron loading disorders but also impact on a number
of other multifactorial disease processes including neurodegeneration and cancer.
One thing that is clear from this survey is that many of the iron-sensitive mech-
anisms involved in the regulation of gene expression are incompletely understood
at present and this rapidly developing area of nutigenomics is ripe for further
extensive study.
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21
Dietary Selenium and Gene Expression
Alexandra Fischer
University of Oxford, Oxford, UK
Josef Pallauf
Justus-Liebig-University of Giessen, Giessen, Germany
Introduction 441
Selenium Metabolism 442
Symptoms of Dietary Selenium Deficiency 443
Regulation of Selenoprotein Expression 444
Dietary Selenium and Selenoprotein Expression 447
Dietary Selenium and Differential Gene Expression 449
References 452
INTRODUCTION
The element selenium (Se34) was discovered in 1817 by the Swedish chemist
Jacob J. Berzelius. Historically, selenium was regarded as a naturally occurring
toxic agent, but this perspective has undergone a radical transformation in the
past 50 years. In 1973, it was established that selenium is an essential micronu-
trient in mammals (1,2). It exerts a number of important health benefits including
protection against oxidative stress, cancer, AIDS, inflammatory diseases, and
male infertility. On the molecular level, selenium shows extraordinary behavior,
as it is incorporated into proteins as selenocysteine, for which the UGA codon is
441
442 Fischer and Pallauf
transformed from one that signals translation termination to one specific for
selenocysteine.
SELENIUM METABOLISM
The absorption of selenium from relevant organic and inorganic resources is
usually very high (70 – 95%). In contrast to many other micronutrients, the
absorption is not influenced by the selenium status of the organism (3), so that
selenium-homeostasis is believed to be regulated by excretion via the urine
(4). The amount and the actual transport through the intestinal border are depen-
dent on the selenium compound. For selenate a sodium cotransport, as well as an
anion exchange with hydroxylanion, has been found (5). Selenite very likely
reacts with membrane-bound thiols (6) to form aminoacid-like products, for
example, selenodicysteine. These products can be transported via aminoacid
transporters, which are also believed to be responsible for the transport of sele-
noaminoacids, such as selenomethionine and selenocysteine.
Throughout the intermediate metabolism of selenium, mammals synthesize
several different metabolites in the course of converting inorganic selenium to
organic forms and vice versa (Fig. 21.1). Hydrogen selenide (H2Se) is a key
metabolite, formed from inorganic sodium selenite (oxidation state þ4) via sele-
nodiglutathione (GSSeSG) through reduction by thiols and NADPH-dependent
nonspecific
selenate selenite selenocysteine selenomethionine proteins (e.g.
4 GSH
albumin)
GSSG
GSSeSG
NADPH Se±0
+
NADP + GSSG
specific
selenoproteins tRNASEC H2Se
(e.g. GPx,TrxR)
+CH3
CH3SeH
+CH3
(CH3)2Se
+CH3
(CH3)3Se+
Figure 21.1 Intermediate selenium metabolism, modified according to Combs (63).

Dietary Selenium and Gene Expression 443
reductases. Furthermore, H2Se can be released from selenocysteine by lyase

action (7). It provides selenium for the synthesis of selenoproteins after activation
to selenophosphate. The reduction from selenate to selenite is believed to be
analogous to the reduction of sulfur in the body (8). Nonspecific incorporation
of selenium into proteins occurs through substitution of selenomethionine for
methionine, especially under high selenomethionine intake (9).
Selenium can be excreted either via the lungs or via the urine after methyl-
ation from selenide to methylselenocysteine, dimethylselenide, or trimethylsele-
nonium. Almost all selenium found in the feces represents the nonabsorbed
selenium from the diet (10). The dose and status of selenium influence the
amount and form of excretion. In rats given orally 16 mg Se/kg body weight,
only 10% of the total selenium excreted in urine consisted of trimethylseleno-
nium, whereas in animals which received doses of 1500 mg Se/kg body
weight, it was the main excretion form (65%) in urine (11). Injections in
pharmacological doses (5 mg/kg body weight) led 24 h later to excretion of
selenium via the lungs as dimethlyselenide in rats (12).
SYMPTOMS OF DIETARY SELENIUM DEFICIENCY

Dietary selenium deficiency has been implicated as a factor in Keshan disease, a
cardiomyopathy that affects young women and children in certain regions of
China that have selenium-poor soil. This disease can be prevented by selenium
supplementation, as can Kashin-Beck disease, a deforming osteoarthritis also
found in China. Although these selenium deficiency diseases have been recog-
nized for sometime, evidence is mounting that less-overt deficiency can also
cause health problems and furthermore that supra-nutritional levels of selenium
may give additional protection from disease. Whereas deficiency has an adverse
effect on immunocompetence, selenium supplementation appears to enhance the
immune response. Selenium appears to be a key nutrient in counteracting certain
viral infections; thus, in a selenium-deficient host, the benign coxsackie
virus becomes virulent causing heart damage (13), the influenza virus causes
more serious lung pathology (14), and HIV infection progresses more rapidly
to AIDS (15). Furthermore, moderate selenium insufficiency may contribute
to male infertility (16) and aging (17). Findings have been equivocal in linking
selenium to cardiovascular disease risk, although other conditions involving
oxidative stress and inflammation have shown some association with selenium
status (18). There is growing evidence that higher selenium intakes are associated
with reduced cancer risk. The results of animal experiments, epidemiological
studies, and clinical intervention trials support the hypothesis that dietary
selenium reduces the risk of certain types of cancers (19).
These effects are at least partly due to the function of selenium as an essen-
tial factor in the selenoproteins, whose expression is reduced when selenium is
limited. It could be demonstrated that the function of one or more of these sele-
noproteins is essential for life, as a knockout of the selenium-specific tRNA gene
in mice resulted in early embryonic lethality (20). In these proteins, selenium is

present in the form of selenocysteine (Sec). The estimates of the actual number of
selenoproteins vary greatly. Recently, Kryukov et al. (21) have identified seleno-
protein genes in sequenced mammalian genomes by searching for selenocysteine
insertion RNA structures, the coding potential of UGA codons, and the presence
of cysteine-containing homologs. By this method, they have identified 25 seleno-
proteins in the human genome, but the functions of the encoded proteins are
largely unknown (Table 21.1).
Selenoproteins with known functions play critical roles in a variety of bio-
logical processes, and several of them are involved in antioxidant defense. Two
of the glutathione peroxidases (GPx), the cellular GPx and the plasmatic GPx,
protect cells against peroxidative damage by reducing hydrogen peroxide and
free fatty acid hydroperoxides (22). An antioxidant function has also been pro-
posed for the plasma protein, selenoprotein P (SelP) (23). Another GPx family
member, phosopholipid hydroperoxide glutathione peroxidase (PHGPx), reduces
phospholipids, cholesterol, and cholesteryl ester hydroperoxides, thereby protect-
ing cells against membrane lipid peroxidation (24). PHGPx also plays a structural
role in the mitochondrial capsule of mature spermatozoa where the protein
becomes oxidatively cross-linked and inactive (16). This noncatalytic function
of PHGPx may be responsible for the male infertility seen in selenium deficiency.
Further selenoproteins in mammals are three thioredoxin reductases, which func-
tion in cellular redox homeostasis by reducing thioredoxin and other substrates.
This could be the link to the protective role of selenium in inflammatory diseases
(25). Other oxido-reductases that contain Sec include the family of diodinases,
which are involved in thyroid hormone metabolism (26), and selenophosphate
synthetase 2 (SPS2), which synthesizes the selenium donor for Sec biosynthesis.
This enzyme is unique in that it is the only selenoprotein expressed in both
prokaryotes and eukaryotes (27).
Several selenoproteins have no known enzymatic activity, including SelW,
which is expressed in cardiac and skeletal muscles (28), and Sep15, which is
implicated in preventing prostate cancer (29).
In addition, recent studies have revealed that one of the new selenoproteins,
SelR, is a zinc-containing methionine sulfoxide reductase (30). With the discov-
ery of this enzyme, a possibility is raised that selenium is also involved in aging
as an enzyme catalyzing a complementary reaction (MsrA) has been implicated
in antioxidant defense and the life-span of mammals (31).
REGULATION OF SELENOPROTEIN EXPRESSION

Given that the nutritional requirement for selenium is partly related to its function
in the selenoproteins, the synthesis of this class of proteins is important. In pro-
karyotes, archaebacteria, and eukaryotes, Sec is encoded by a UGA codon. The
incorporation of Sec into protein requires a novel Sec-charged tRNA that
Table 21.1 Mammalian Selenocysteine-Containing Proteins, Modified According to

Refs. (21,40)
Sec location
in protein
Common (length
Selenoprotein abbreviations of protein) Function
Cytosolic GPx cGPx, GPx1 47 (201) Protection of oxidation

Gastrointestinal GI-GPx, GPx2 40 (190) Local redoxprotection?
GPx
Plasma GPx pGPx, GPx3 73 (226) Redoxbuffer?,
regulation of
prostanoidmetabolism?
Phospholipid PHGPx, GPx4 73 (197) Redoxregulation,
hydroperoxide sperm maturation
GPx GPx6 73 (221) Unknown
0 0
5 -Deiodinase, 5 DI-I 126 (249) T3-synthesis:
type I T4 þ 2e2 þ Hþ !
T3 þ I2; degradation
of T3 and T4
50 -Deiodinase, 50 DI-II 133 (265) T3-synthesis:
type II T4 þ 2e2 þ Hþ !
T3 þ I2
50 -Deiodinase, 50 DI-III 144 (278) Degradation of
type III T3 and T4
Thioredoxin TR1, TrxR 498 (499) Reduction of thioredoxin,
reductase DNA-synthesis,
thioldisulfide balance
Thioredoxin TR2 655 (656) Unknown
reductase 2
Mitochondrial TR3 522 (523) Unknown
thioredoxin
reductase
Selenophosphate SPS2 60 (448) Selenoprotein
synthetase-2 synthesis
15 kDa Sep15 93 (162) Unknown,
selenoprotein proteinfolding?
Selenoprotein H SelH 44 (122) Unknown
Selenoprotein I SelI 387 (397) Unknown
Selenoprotein K SelK 92 (94) Unknown
Selenoprotein M SelM 48 (145) Unknown
Selenoprotein N SelN 428 (556) Unknown
Selenoprotein O SelO 667 (669) Unknown
(continued )

Sec location
in protein
Common (length
Selenoprotein abbreviations of protein) Function
Selenoprotein P SelP 59, 300, 318, 330, Unknown,

345, 352, 367, 369, redoxprotection?
376, 378 (381)
Methionine SelR 95 (116) Antioxidant
sulfoxide defense?
reductase
Selenoprotein S SelS 188 (189) Unknown
Selenoprotein T SelT 36 (182) Unknown
Selenoprotein V SelV 273 (346) Unknown
Selenoprotein W SelW 13 (87) Unknown,
redoxprotection?
contains the anticodon UGA. As UGA normally signals the termination of

protein synthesis, how does the ribosome cope with a codon that encodes two
different functions?
The underlying mechanisms in eukaryotes are poorly understood and are
under intensive investigation, but some insights can be drawn from the genetic
and biochemical studies of selenoprotein synthesis in Escherichia coli carried
out by Böck and co-workers (32). Four genes (selA –selD) were identified as
being involved in this process. The gene products have been identified as a sele-
nocysteine synthase (SELA), a selenocysteine-specific elongation factor (SELB),
a selenocysteine-specific tRNA (tRNASec, selC gene product), and a seleno-
phosphate synthetase (SELD). tRNASec is first charged with serine by seryl-
tRNA synthetase. Selenocysteine synthase then catalyzes the conversion of
seryl-tRNASec to selenocysteyl-tRNASec using selenophosphate as the active
selenium donor (33). Selenophosphate synthetase catalyzes the synthesis of
selenophosphate from selenide and ATP. Recoding of UGA as a selenocysteine
codon requires a mRNA secondary structure called selenocysteine-insertion
sequence (SECIS), which is localized in eukaryotes in the 30 -nontranslated
region and thus decoding from an appreciable distance is necessary (33). For
the incorporation, at least two additional proteins are involved, the elongation
factor eEFsec (SELB homolog) and SBP2 (34). SECIS elements function by
recruiting SBP2 to form a tight SECIS– SBP2 complex. An RNA-binding
domain was identified in the C-terminal sequence of SBP2 and an additional
domain was identified that was required for Sec insertion. Besides binding to
SECIS elements, SBP2 binds eEFsec which in turn recruits tRNASec and
inserts Sec into nascent polypeptides in response to UGA codons (35).
This mechanism shows that Sec is dramatically different from any other of
the 20 protein aminoacids in the mode of its incorporation and basic biosynthetic
steps. It is the only aminoacid that directly requires a structural element in mRNA
in addition to the information specified by the genetic code. It is synthesized on
its own tRNA, whereas free Sec is not a substrate for selenoprotein synthesis. The
Sec biosynthetic machinery is strikingly different from that of other aminoacids
and employs additional Sec-specific components. These unique features of Sec
biosynthesis and insertion favor the view that Sec was added to the already exist-
ing genetic code to take advantage of the unique chemistry of selenium to coun-
teract environmental stress and/or evolve new functions (36). This is underlined
by the fact that most of the functions of selenoenzymes identified to date catalyze
oxido-reduction reactions in which the selenocysteine residue exists in the active
site. Substitution of the structurally related aminoacid, cysteine, for selenocys-
teine produced functional enzymes, but significantly decreased the Kcat , indicat-
ing that the presence of selenocysteine confers specific biochemical properties.
DIETARY SELENIUM AND SELENOPROTEIN EXPRESSION

The biosynthesis of selenoproteins is primarily dependent on the bioavailability
of selenium and consequently on the formation of tRNASec, which regulates
the level of all selenoproteins. In selenium deficiency, UGA is interpreted as a
stop-codon, which results in a premature chain termination at the UGA codon.
The decrease in selenoprotein synthesis is further accompanied by a fall in the
levels of the respective mRNAs. This is not due to a lower transcription rate
but due rather to a loss of stability, for example, enhanced degradation of the
mRNA (37). This occurs by the surveillance pathway, designated nonsense-
mediated decay, where the UGA Sec codon is recognized as nonsense (38,39).
For an optimum translation of a selenoprotein apart from selenium supply,
its mRNA appears to require an optimum UGA location, an optimum UGA
context, and the SECIS element to optimize selenocysteine incorporation. Sur-
prisingly, individual selenoproteins vary in their specific responses to selenium
deprivation, a phenomenon referred to as “the hierarchy of selenoproteins”
(40). Although some of the selenoproteins disappear rapidly in selenium depri-
vation and are slowly resynthesized when resupplemented, others decline gradu-
ally in the presence of a limited selenium supply and rebound immediately at
resupplementation. After 8– 10 weeks of selenium deficiency, cGPx activity in
the liver of pigs (41), rats (42), and rabbits (43) was reduced to 3– 10%
when compared with selenium supplemented control animals. In other organs
(e.g., heart and skeletal muscles), the extent of the loss of activity was consider-
ably less given an inherent generally lower cGPx activity in these organs (41,43).
The greater sensitivity of cGPx activity to selenium deficiency has been attribu-
ted largely to an increased turnover in mRNA (44). The position of the UGA Sec
codon relative to the sole, downstream intron in cGPx mRNA determines
whether the mRNA is subject to nonsense-mediated decay (45). However,
other selenoprotein mRNAs, such as 50 DI-I, PHGPx, and SelP, are not as sensi-
tive as cGPx during selenium deprivation despite the presence of introns down-
stream of their UGA codons (46). SBP2 has also been reported as preferentially
recognizing SECIS elements in specific selenoprotein mRNAs suggesting a
mechanism which accounts, at least in part, for selenoprotein expression hierar-
chy during selenium deficiency (47). In selenium-deficient rats and rabbits,
cGPx mRNA was found to be downregulated by 85– 90% after 8 –10 weeks of
selenium deficiency (42,48).
Irrespective of the mechanisms involved, the hierarchy in the biosynthesis
of selenoproteins is considered to reflect their biological importance. The follow-
ing ranking has been suggested:
GI-GPx . TrxR, SelP, 50 DI-I . cGPx
Furthermore, a characteristic of selenium deficiency in mammals is that a hierar-

chy exists with respect to the retention of selenium in different organs. During
selenium deprivation in the diets of rats and mice, the amounts of this element
were substantially reduced in the liver and kidney, whereas the brain and testes
retained most of their selenium (49 – 51). The same finding is reflected in seleno-
protein activities. In rats made selenium-deficient for two generations, total GPx
activity was below 5% in liver, kidney, lung, heart, adrenal gland, pancreas, and
muscle, below 10% in male thymus and testes, but only decreased to 50% in the
brain and to 25% in the ovaries and female thymus (52). Interestingly, transgenic
mice expressing i6-deficient Sec tRNA(Ser)Sec had reduced levels of selenium in
their tissues and a hierarchy of selenoprotein activities similar to that observed
with selenium-deficient mice (53).
From these results, it can be concluded that mechanisms must exist that guide
selenium to a specific enzyme within a particular tissue, probably via tissue-
specific regulation of mRNA stability. cGPx and PHGPx are preferentially retained
under selenium-limiting conditions in the brain, reproductive organs, and endocri-
nological tissues. Brigelius-Flohé (54) proposes the ranking order of tissue-specific
stabilities, which do not reflect absolute activities, for cGPx as follows:
brain thymus . thyroid . heart . liver, kidney, lung
For some selenoproteins, a hormone-dependent regulation could be observed.

Human TrxR is rapidly induced in monocytes through 1,25-dihydroxy vitamin
D3 and in fetal osteoblast-like cells through certain cytokines and growth factors
(55). The three deiodinase isoenzymes are regulated by thyroid hormones, reti-
noids, sexual hormones, gluco- and corticosteroids, and a series of growth
factors and cytokines (56). For PHGPx, a sexual hormone-dependent expression
has been suggested.
An induction of GPx genes by oxidative stress was often postulated, but has
never been convincingly demonstrated in vivo. The induction of cGPx through an
oxygen-responsive element has only been described in vitro (57).
DIETARY SELENIUM AND DIFFERENTIAL GENE EXPRESSION

Apart from the direct influence of dietary selenium intake and selenium status on
selenoprotein expression, selenium seems to have a major impact on overall gene
expression as well. With the introduction of array technology to measure diffe-
rential expression of thousands of genes simultaneously, new insights into
selenium-dependent gene expression in rodents have been revealed in recent
years. The results from these studies, which will be elucidated in more detail
in the following section, are summarized in Table 21.2.
Rao et al. (58) used Affymetrix high density oligonucleotide arrays (Gene
Chipw MU6500) representing 6347 murine genes to determine the transcriptional
profile associated with low selenium status in the intestines of C57BI/6J mice.
Mice were fed with either a torula yeast, selenium-deficient diet (,0.01 mg
Se/kg), or the same diet supplemented with a high level of dietary selenium
(1 mg Se/kg, as seleno-L -methionine) for 90 days. Total RNA for the expression
profiles was obtained from the middle segment of the small intestine correspond-
ing to the jejunum after 111 days of feeding. A comparison between mice fed
with the selenium-deficient diet or the high selenium diet revealed that selenium
status was associated with alterations in specific mRNA levels, which may reflect
changes in gene expression, mRNA stability, or both. Of the 6347 genes sur-
veyed, 48 displayed a greater than 2-fold decrease in expression in response to
low selenium status, whereas 84 displayed a greater than 2-fold increase in
expression. These genes were consistent with a state of DNA damage, genetic
instability, and oxidative stress. These included alterations in expression of the
cell cycle arrest/DNA damage inducible genes GADD34, GADD45ß,
GADDg, and XP-E, as well as the molecular chaperones hsp27 and hsp40.
Table 21.2 Global View of Transcriptional Changes Induced by Dietary Selenium

Deficiency in Rodents
#Selenoproteins
Reduced antioxidant capacity
"Apoptosis/cell cycle control/oncogenesis "Stress response/inflammation
Induction of: Induction of:
Protein phosphorylation Heat shock response
Signal transduction DNA damage-inducible genes
Angiogenesis Oxidative stress-inducible genes
Cell adhesion
Intestine specific changes Liver specific changes
#Xenobiotic metabolism "Xenobiotic metabolism
#Lipid metabolism
Tumorigenic stress specific changes
#Decreased apoptotic ability
Also induced were the mitogen- and stress-activated protein kinase AMPKg,
metallothionein-I, a free radical scavenger implicated in oxidative damage pro-
tection, and MDM2, an oncogene. Low selenium status was also associated
with changes in the expression of genes involved in cell proliferation, such as
M-phase inducer phosphatase 2, G2/mitotic-specific cyclin B2, cyclin-dependent
kinase 1 and PAC-1. Furthermore, of the 48 genes that decreased in expression in
mice of low selenium status, 15% were genes that participate in lipid metabolism,
especially in lipid transport, including apolipoprotein AI, AIV, CIII, APOBEC-1,
nonspecific lipid-transfer protein, and fatty acid binding protein. Apo-AI is the
major determinant of the capacity of HDL particles to promote cholesterol
efflux and is associated with the inhibition of atherosclerosis.
Zeng et al. (59) investigated the protective role for selenium enriched broc-
coli in tumorigenesis, thereby a gene profile in the liver of multiple intestinal neo-
plasi (Min) mice was conducted. Mice were fed with either 0.11 mg Se/kg control
diet or 2.1 mg Se/kg selenobroccoli diets for 10 weeks. Expression profiles were
obtained in mouse liver samples with mouse pathways finder-1 GEArray mem-
brane (Superarry, Bethesda, MD, USA), which contained 23 sequence-verified
known marker genes. Furthermore, the authors applied DNA mobility shift assay
to define the transcriptional response in Min mouse liver. When compared with
the low selenium control diet, selenium-enriched broccoli upregulated the
mRNA levels of ikBa, hsp86, and gadd45. In addition, the analysis of binding
of liver nuclear proteins to 32P-labeled probes demonstrated that selenium-
enriched broccoli enhanced the binding of the transcription factors p53, NF1B,
and AP-1 to their cis-acting elements. ikBa and NF1B are main mediators of
the cellular response to a variety of extracellular stress stimuli, such as initiating
apoptosis. Similarly, the expression of hsp86 and gadd45 genes correlated with
induction of apoptosis. These results suggest that dietary selenium intake can acti-
vate pro-apoptotic gene expression and enhance the DNA binding of pro-apoptotic
transcription factors in response to tumorigenic stress.
Christensen et al. (60) compared the expression in rat liver of genes for
transferrin, transferring receptor, ferritin light and heavy chains, and iron-
regulatory proteins in selenium adequacy and deficiency. Weanling male
Sprague– Dawley rats were fed with torula yeast selenium-deficient diets
supplemented with either 0 or 0.15 mg Se/kg diet as sodium selenite for
15 weeks. To examine differential gene expression, a multiplex relative reverse
transcriptase –polymerase reaction method was applied. Three of the six genes
examined showed modest, but consistent upregulation in selenium deficiency.
Transferrin mRNA was 30% more abundant in deficient liver than in
selenium-adequate liver. For the transferrin receptor, the difference was 32%,
and for iron regulatory protein 1, it was 63%. The authors concluded a possible
role for dietary selenium in moderating iron metabolism.
To examine the molecular events associated with selenium deficiency in
rats, we applied cDNA array technology to define the transcriptional response
in rat liver after 7 weeks on a selenium deficient torula yeast based diet
(,0.01 mg Se/kg), compared with rats fed the control diet (200 mg Se/kg as
sodium selenite) (42). AtlasTM Rat cDNA Toxicology Array II from Clontech
was used to monitor simultaneously the expression of 465 genes, whereby a
fold-change of two or more was considered as significant.
Besides a 13.9-fold downregulation of the cGPx gene, selenium deficiency
was accompanied by an increase in the expression of UDP-glucuronosyltrans-
ferase 1 and bilirubin UDP-glucuronosyltransferase isoenzyme 2. These two
enzymes are known to have an important function in the detoxification of
xenobiotics in liver. Likewise, rat liver cytochrome P450 4B1, also involved in
xenobiotic metabolism and inducible by glucocorticoids, was induced 2.3-fold.
The mRNA levels of arachidonate 12-lipoxygenase (ALOX 12) were 2.4-fold
higher in selenium deficient animals when compared with controls. It has been
shown that ALOX12 and PHGPx are opposite enzymes balancing the intracellu-
lar concentration of hydroperoxy lipids (61), whereby an inhibition of PHGPx
activity increases the enzymatic catalysis of ALOX 12 (62).
These results provide evidence that selenium deficiency has an impact on
selenoprotein expression and probably as a secondary effect on induction of a
stress response, for example, modulation of inflammatory and cell cycle dependent
genes. This response may be due to oxidative stress, DNA damage, or both, and
could be related to a reduction in the activity of selenoproteins and detoxification
enzymes. Because some of the selenium-dependent enzymes, such as GPx, thiore-
doxin reductase, and selenoprotein P, function as antioxidants, it seems plausible
that low selenium status is associated with some forms of oxidative stress.
However, differences can be seen between the different animal models
and/or the different tissues investigated. In the selenium-deficient mouse, no
upregulation in expression of any gene involved in drug detoxification could
be observed, whereas in the liver of selenium-deficient rats, genes encoding for
xenobiotica metabolizing proteins, such as P450, were significantly induced.
This induction may be liver specific, or may be mediated at the protein level
in the intestine as opposed to increases in mRNA abundance.
Taken as a whole, these results suggest that suboptimal intake of a single
trace mineral can induce multiple transcriptional pathways suggestive of oxi-
dative stress, DNA damage, and alterations in cell cycle progression and
thereby can have widespread effects on gene expression patterns, providing a fra-
mework for understanding the multiple roles of selenium in human health. The
overall screening for the mRNAs of genes with altered expression due to
dietary selenium status has yielded new avenues of investigation and confirmed
previous observations associated with dietary selenium status. Efforts to identify
nutrient-regulated genes will significantly enhance the value and usefulness of
the genome and EST databases by providing a physiological response that can
be associated with the new genes. In addition, functional genomic techniques,
such as array technology and two-dimensional protein gel electrophoresis, will
play an increasing role in linking physiological perturbations to the molecular
and cellular mechanisms.
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22
Modulation of Gene Expression
by Dietary Zinc
Raymond K. Blanchard and Robert J. Cousins

University of Florida, Gainesville, Florida, USA
Introduction 457
Zinc as an Antioxidant 458
Ionic Zinc 458
MT as an Antioxidant 458
Molecular Mechanisms of Zinc-Regulated Gene Expression 460
Expression Profiling of Zinc-Regulated Genes 464
References 468
INTRODUCTION
Zinc is defined in classical literature as a redox neutral atom. This is in contrast
to other nutrient trace elements, such as iron and copper, which change ionic
charge during biological functions. Functions of zinc have been categorized as
catalytic, structural, and regulatory (1). In each of these functional categories,
zinc plays a role more analogous to calcium, perhaps related to exchange
rates with a ligand center, in comparison with the redox metals such as
copper and iron (2). Consequently, the properties of zinc that are considered
as antioxidant in nature are indirect and do not involve direct interaction with
reactive oxidant species.
457
458 Blanchard and Cousins
Literature on the antioxidant properties of zinc is voluminous and involves

descriptions of the effects of cellular defense against oxidative and nitrosative
stresses (3 –5). Therefore, our comments will be focused on the nutritional geno-
mics of zinc as an antioxidant. Much of this available information was generated
with in vitro systems, where individual molecules interact depending on redox
potentials, or with isolated cells perturbed to induce a change in redox state
and/or initiate a stress response. Evidence with integrative systems occasionally
supports the participation of zinc in one or more antioxidant functions. The
purpose of this review is to discuss evidence on zinc-regulated genes and induc-
tion of zinc-responsive networks that have antioxidant properties.
ZINC AS AN ANTIOXIDANT
Ionic Zinc
Galvanizing, the industrial coating of iron-containing metal objects with zinc to
prevent oxidation (rust formation), has been in practice for over a century. Zinc is
a strong Lewis acid (electron acceptor) that binds strongly to thiolates and amines
(2). For most functions of zinc, it must undergo intracellular trafficking much like
calcium with concentrations regulated by transporters. These concepts were
initially developed by Williams and colleagues (2). Estimates of the free Zn2þ
concentration are placed at or below the nanomolar range (2), but may actually
be in the order of only a few atoms per cell (6). The trafficking rate of zinc
may be the most important factor so that, while only a few atoms may be
“free,” the flux rate through that “free” pool may be tremendous.
Quantitation of the “free” zinc pool size, though important, is less relevant
to the concept of zinc trafficking than is the Zn2þ exchange rate from ligands of
specific binding complexes. Exchange rates are more a function of secondary/
tertiary protein structure and folding energies than the binding constant of a
specific motif for zinc. Zinc exchange rates for metalloproteins range from
minutes, in the case of metallothionein (MT), to days (alkaline phosphatase)
(2). Relevant to antioxidant properties is that, when zinc is bound to a redox-
active species, for example, a protein thiol, oxidation can result in Zn2þ
release (4), which may involve a transition into the “free” zinc pool. That
release would be followed by rebinding of Zn2þ to another ligand or transport
into a vesicle.
MT as an Antioxidant
A number of important findings converge to strongly suggest MT, with its high
cysteine-thiol content and ability to bind seven atoms of Zn2þ, has a role in cel-
lular redox. Paramount among these is the inducibility of the MT gene by zinc
and specific hormones and cytokines [reviewed in Ref. (7)]. Other issues
include: (i) correlation of cytoprotection and MT induction; (ii) altered cellular
response to oxidative stress in MT-null mice; (iii) evidence for MT in protective
Modulation of Gene Expression by Dietary Zinc 459
mechanisms in both oxidative and nitrosative stress (5); (iv) indications that some
interactions between MT and specific redox-active molecules [e.g., nitric oxide
(NO)] may have signaling roles in normal physiology, including antimicrobial
activity [reviewed in Ref. (7)].
An antioxidant function for MT was first advanced in 1985 by Thornalley
and Vasak (8). Using in vitro radical-generating systems, they observed that MT
was particularly effective in quenching OH† radicals. Subsequent findings by
numerous laboratories have implicated MT in antioxidant roles for reactive
oxygen species [ROS (including OH† and superoxide)], carbon-centered radicals
(e.g., CCl3† ), and nitrogen species [e.g., nitric oxide (NO) and peroxynitrite]
(3 – 5,7,9,10). Confounding a definitive relationship has been the difficulty in
separating the effects of zinc from those produced by MT (11). This was particu-
larly an issue where zinc is capable of causing MT induction in cell and animal
models. Further complicating interpretations of zinc and MT antioxidant effects
are the concurrent effects of other nutrient antioxidants (e.g., vitamins C and E)
and cellular antioxidant systems, including the glutathione (GSH – GSSG)
couple, catalase, glutathione S-transferases, superoxide dismutase, and gluta-
thione peroxidases (12). In addition, effects of zinc nutritional status (both
deficient and excessive) in animal models, those of zinc treatment of cells, and
the MT induction in cells and animals by oxidants all present difficulties in deli-
neating unequivocal zinc/MT antioxidant effects. These problems are not neces-
sarily circumvented with the use of transgenic knockout or MT-overexpressing
mice or cells, as compensatory mechanisms are frequently induced, which can
influence interpretation of the results. As will be discussed subsequently, the
spectrum of zinc-regulated genes is such that many yet uncharacterized proteins
may influence zinc-related antioxidant effects.
A theoretical breakthrough to explain the antioxidant properties of MT was
produced by Maret and colleagues. Specifically, their data suggest that MT
should be viewed as a reduced species that, upon interaction with an oxidant,
results in sulfhydryl oxidation and Zn2þ release (4). Mobilization of Zn2þ
from metalloproteins by the oxidant hypochlorous acid (HOCl) had previously
been demonstrated. HOCl is produced by neutrophils to inactivate cellular pro-
teins during host defense. Zn2þ released by HOCl was advanced as a mechanism
to explain oxidant-induced tissue injury during inflammation (13).
In contrast, Pitt and coworkers proposed S-nitrosylation of MT sulfhydryls
by NO stress regulates the MT – thionein redox pair through Zn2þ release (9).
NMR evidence suggests NO selectively releases Zn2þ from the amino-terminal
(b domain) metal cluster of MT, which coordinates three zinc atoms (14). The
b domain of MT is believed to have a lower binding affinity for Zn2þ, suggesting
that it may function in homeostasis. NO appears to preferentially release
those zinc ions. It has been suggested that NO may interact with bacterial zinc
finger proteins involved in growth, causing Zn2þ release as well as inhibition
of bacterial DNA replication (15). Intracellular pathogens may be killed by
NO-producing phagocytic cells through such a mechanism. NO-induced
release of Zn2þ from cytoplasmic and nuclear pools was actually documented a
decade ago (16). Additionally, intracellular NO, produced by inducible NOS
(iNOS) in response to cytokines, causes an MT-dependent release of nuclear
Zn2þ in endothelial cells (17). Therefore, it is not surprising that exogenous
NO causes induction of MT gene expression through release of intracellular
Zn2þ in these cells. Of note in this context is that dietary zinc deficiency produces
an upregulation of intestinal iNOS mRNA stimulated by IL-1a and is associated
with intestinal injury that can lead to diarrhea in a rat model (18,19). Down-
regulation of intestinal iNOS expression upon zinc repletion in zinc-deficient
rats is rapid, suggestive of a direct link among Zn2þ, NO, and iNOS gene
expression (19).
A possible mechanism for the involvement of MT against carbon-centered
oxidative damage is less clear. Using carbon tetrachloride (CCl4) as a hepato-
toxin, isolated cultured rat hepatocytes were protected from damage when
cellular zinc and MT levels were elevated by zinc added to the medium or by
stimulation of MT expression with dexamethasone and IL-6 (10). A protective
effect of zinc against CCl4 in rat liver was first observed three decades ago
(20). Subsequently, this protective effect was ascribed to MT as a zinc-inducible
antioxidant [reviewed in Ref. (7)]. When such protective effects were tested in
MT-null and MT-overexpressing mice, results were less conclusive (21). Specifi-
cally, CCl4 clearly produced hepatic oxidative damage in the MT-null mice.
However, MT overexpression did not have a beneficial effect. In contrast to
these findings, ROS- and NO-induced damage to the pancreas was prevented
in MT-overexpressing mice (22). This protective action of MT was viewed as
potentially advantageous for pancreatic islet survival during early phases of
transplantation. The murine pancreas is very sensitive to dietary zinc status
(23), which suggests that this organ may be particularly susceptible to zinc-
related oxidative effects. In that regard, it must be kept in mind that the pancreas
is also a target of zinc toxicity [reviewed in Ref. (1)].
The cytoprotective role of zinc and the involvement of MT have not been
fully defined. On a molecular basis, MT is clearly a reductant that, when acted
upon by an oxidant, undergoes oxidative release of Zn2þ with disulfide formation
in the protein (Fig. 22.1). In this context, zinc or MT should be considered as an
“antioxidant.” The fate of Zn2þ could be one that activates the metal-responsive
transcription factor (MTF-1) and/or other transcription factors and thus signals a
cascade of modulated genes including those beneficial for health. Oxidative Zn2þ
release from MTF-1 or other transcription factors could similarly influence genes
that are beneficial or lead to cell death through apoptosis.
MOLECULAR MECHANISMS OF ZINC-REGULATED

GENE EXPRESSION
Zinc-modulated gene expression has been studied most thoroughly for the
MT family of genes. MT protein, with its variable abundance in many cell
O2
Cytokines
iNOS
O2• –
cNOS
NO
SOD
Cl – H2O2
MPO
ClO– OH•
Zn Zn
Zn Finger b a
Proteins Metallothionein
Zn2+
Release
ARE-TF
Zn 2+
MTF -1
Zn Zn Oxidative
Repressed Induced Stress
Genes Response
? MRE Genes ARE Genes
Antioxidant
Effects
Figure 22.1 A hypothetical phagocytic cell responding to activation by lipopolysac-

charide (LPS) and/or cytokines (e.g., IL1, IL6, or IFNg). Oxidative release of Zn2þ
from the b metal-binding domain of MT by ROS (e.g., hydroxyl radical, OH† , and hypo-
chlorite anion, ClO2) or reactive nitrogen species (e.g., nitric oxide, NO) is shown.
Zn2þ-stimulated activation of the MTF-1 and zinc-responsive gene activation and
decrease in activation of other genes by Zn2þ are also shown. Principal enzymes, nitric
oxide synthases (iNOS and cNOS), superoxide dismutase (SOD), and myeloperoxidase
(MPO) are indicated. ROS also interact with ARE transcription factors (ARE-TF ) to
induce oxidative stress response genes, including the MT gene.
types, unusually high cysteine content, and tremendous zinc-binding capacity,

quickly evoked questions about its cellular function as well as regulatory inter-
actions between the protein and the essential micronutrient. Radioisotopes of
zinc were used to examine the kinetics and physiological compartmentalization
of the metal, but the regulation of MT gene expression awaited the advent of
molecular biology. The earliest reports showing dietary zinc as a regulator of
MT gene expression were in 1975 when Richards and Cousins (24) demonstrated
that induction in both intestine and liver could be blocked by actinomycin D and
therefore required RNA synthesis. Later, Shapiro and Cousins (25) were able to
demonstrate similar results from isolated polyribosomes, indicating a direct
increase in the amount of mRNA encoding MT. As northern blotting and other
expression analysis tools have been developed, the MT genes have become the
prototypical zinc-responsive gene model due in part to the large magnitude
and rapid rate of change observed in both mRNA and protein.
After the cloning of the MT-I gene, Mayo et al. found that the gene retained
its metal responsiveness after transfection into cells (26,27). Thus began the
search for the cis-acting elements within the gene that are responsible for impart-
ing zinc, as well as cadmium, inducibility. Sequence analysis of the proximal
promoters for the human and mouse MT-I and -II genes identified several con-
served sequence elements including one that was implicated in metal regulation.
Deletion analysis using reporter gene constructs showed that multiple 12 bp
elements were involved in zinc responsiveness and these were named metal regu-
latory elements, or MREs (28). Each element could individually confer, with
varying degrees of magnitude, metal responsiveness to an unrelated, nonrespon-
sive promoter. Insertion of an MRE consensus element into the upstream proxi-
mal promoter of a gene was subsequently demonstrated to be necessary and
sufficient to confer zinc responsiveness. The human MT-I and -II genes have
seven and eight MREs, respectively, within 500 bp of the transcription start
site (29), designated MREa through MREh. They are found in both directional
orientations and, while the core element of the MRE is tightly conserved, the sur-
rounding bases confer differing magnitudes of response; therefore, all MREs are
not equivalent. Although MREs act synergistically, with more copies allowing
greater maximal induction, different consensus variants were identified and the
MT-I MREd was determined to be the most responsive (30).
Another development that has confounded the characterization of MREs is
the interaction of other promoter elements adjacent to or overlapping with MRE
sequences. This was not surprising for MT, which was known to respond to a
variety of stress stimuli in addition to toxic metals. Promoter element interactions
initially came to light during the detailed mapping of a mouse MRE where a
potential SP1 transcription factor binding site was found to be overlapping the
MRE (31). Subsequent reports have identified glucocorticoid response elements,
antioxidant response elements (AREs), as well as USF and NFI transcription
factor binding sites within promoters as active elements regulating MT
expression (32 –35). Additional examination of the SP1 element associated
with the hMT-II MREb indicates that the SP1 transcription factor is a negative
regulator of MT expression (36).
Zinc-regulated gene expression has long been linked to oxidative stress
responses. For example, induction of MT protein in isolated rat liver cells was
observed upon treatment with the free-radical-generating compounds, t-butyl
hydroperoxide and 3-methylindole (11). Additional linkage of MT promoter
MREs to antioxidant responses was demonstrated subsequently (35). While func-

tionally defining an ARE element in the proximal mouse MT-I promoter, Dalton
et al. reported that even with the ARE deleted there was still some MT induction
by H2O2 . Further analysis demonstrated that MREs by themselves were sufficient
for H2O2 induction of MT.
Having identified the promoter element responsible for zinc responsive-
ness, the search began for transcription factors that bind this sequence. A
variety of DNA-binding methodologies used to analyze nuclear protein extracts
have identified several candidates by their ability to specifically bind the MRE
consensus sequence [reviewed in Ref. (30)]. The zinc-inducible MRE-binding
MTF-1 was the first MRE-binding factor characterized and cloned (37,38).
The cDNA revealed that the N-terminal half of the 675 AA protein contains
six consecutive zinc finger motifs, whereas the C-terminal half has three distinct
transcriptional activation domains (39). Further characterization revealed signifi-
cant functional heterogeneity in the zinc fingers. Fingers 2 –4 constitute the
DNA/MRE-binding core, whereas the most N-terminal finger, #1, appears to
function as a unique zinc-binding sensor (40). Fingers 5 and 6 also have
characteristics that may function as parts of the zinc sensor. Thus, MTF-1 not
only functions as a transcriptional inducer but also as an intracellular zinc detec-
tor. In addition, one or more zinc-binding domains of MTF-1 may be susceptible
to oxidant-induced zinc release.
Since its discovery, several features of MTF-1 have been identified that
may play significant roles in further understanding its function. First, MTF-1
levels may be regulated by zinc. Moreover, immunolocalization studies have
demonstrated that MTF-1 is translocated from the cytoplasm to the nucleus
after treatment of the cells with zinc, cadmium, or other stressors such as heat
shock or H2O2 (41,42). Perhaps, most interesting is the fact that a transgenic
knockout mouse model for MTF-1 displays embryonic lethality for the
homozygous mutant by day 14 of gestation (43). This indicates an essential
role in cellular biology beyond MTF-1’s ability to induce MT, because the
homozygous MT-I and -II knockout mouse is viable and appears to develop
normally (44).
Other reports add to the evidence that MTF-1 serves a role beyond metal
regulation of gene expression. In one report, a search conducted to identify
additional targets for MTF-1 yielded a diverse panel of candidates including
a-feto protein, C/EBPa transcription factor, and tear lipocalin (45). Another
indicates that MTF-1 may partner with p53 to transcriptionally regulate the
synthesis of the ribosomal S25 protein (46). Additionally, MTF-1 appears to
be directly involved in responding to oxidative stress produced by H2O2 and
tert-butylhydroquinone, which activate MTF-1 DNA-binding activity (47).
Further evidence suggests that this activation occurs via an increase in the
level of intracellular zinc released from MT after oxidative damage (48). Thus,
it appears that the roles of zinc-regulated gene expression and cellular antioxidant
response are intricately linked at multiple levels.
EXPRESSION PROFILING OF ZINC-REGULATED GENES

By the early 1990s, the number of genes being reported in the literature as
zinc responsive in a variety of different metabolic pathways from different
tissues led to a growing interest in identifying as many of these genes as
possible. As interest in this functional role for zinc expanded, so did an inter-
est in relating expression of specific genes to the pathology of nutritional
zinc deficiency. Although essentiality of this micronutrient in animals, includ-
ing humans, has been known for decades, specific biochemical defects that
could explain the plethora of clinical findings, including tissue peroxidation,
have not emerged.
Prior to the development of genomics and the sequencing of the human
genome, few techniques were available to clone the cDNAs of unknown differ-
entially expressed genes. The first article to report the results of differential zinc
expression utilized differential hybridization cDNA library screening. Shay and
Cousins (49) identified, and confirmed by northern blot analysis, nine differen-
tially expressed mRNAs from rat small intestine after 16 days of dietary zinc
deficiency. Among the mRNAs identified in this report were intestinal fatty
acid-binding protein, aldolase B, calbindin D-28K, apolipoprotein A-I, ubiquitin,
and cytochrome c oxidase, which is the first screening evidence of zinc-
modulated genes involved in cellular oxidative status. These moderately to
highly abundant genes encompassed a broad spectrum of metabolic pathways,
a precedent that has been consistently seen in all subsequent analyses.
Though the field of genomics was getting started in the mid-1990s, there
was still a paucity of genes represented in the DNA sequence databases; there-
fore, the most robust methods for identifying differentially expressed mRNAs
relied on de novo cloning and screening of cDNAs. The next report of zinc-
regulated expression screening utilized the PCR-based method of mRNA differ-
ential display (DD). Using the same rat small intestinal model with an additional
zinc-supplemented dietary treatment group, we identified another 13 dietary zinc-
regulated mRNAs (50). However, as the amplification by PCR allowed for the
detection of much rarer mRNA species, and the genome databases were far less
complete than they are today, only three could be matched to genes of known
function and four to expressed sequence tags (ESTs), whereas six sequences
were completely novel. These genes, which were suppressed by excess dietary
zinc and induced by zinc deficiency, included two peptide hormones, cholecysto-
kinin and uroguanylin, and ubiquinone oxidoreductase, a mitochondrial electron
transport component. Interestingly, though both of the hormones serve very
different physiological functions, their prohormones are proteolytically activated
extracellularly by zinc metallopeptidases. Of particular interest is that each of
these dysregulated genes could be linked to clinical signs of zinc deficiency,
specifically, cholecystokinin (anorexia), uroguanylin (intestinal fluid secretion
and diarrhea), and mitochondrial oxidoreductase (apoptosis). Subsequent experi-
ments showed, at the proteome level, that upregulated uroguanylin mRNA levels
were concurrent with increased immunoreactive uroguanylin peptide levels in a

zinc-dependent fashion (19).
Given the human health issues associated with the immunodeficiency result-
ing from clinical zinc deficiency, the focus on identification of zinc-responsive
genes was expanded to an immune organ (the thymus). Moore et al. (51) used
DD to examine such genes in the mouse thymus after 3 weeks of dietary zinc
excess and deficiency. This was also the first zinc-based expression profiling
study to theoretically complete a survey of an entire transcriptome. Approximately
265 differentially displayed cDNA bands were identified out of 48,000 cDNA
bands examined and, of 42 selected for follow-up, all but one were identified
with known genes or ESTs in GenBank. Among this list, were those for four ribo-
somal subunit proteins and the mitochondrial 16S ribosomal RNA indicating a
significant effect of zinc status on the protein synthesis pathways within thymo-
cytes. In addition, four distinct heat shock proteins showed decreased expression
due to zinc deficiency. Of the immunologically relevant genes identified, the
T cell cytokine receptor, which was induced by zinc deficiency, and the H2-Aa
subunit of the MHC class II receptor, which was suppressed in deficiency, were
confirmed by quantitative reverse transcriptase real-time PCR (Q-PCR). Together,
these indicate a zinc status effect at the level of cytokine signaling pathways that
control T cell development. Those findings support a role for zinc in immune
cell development and function advanced by Fraker and Prasad (52,53).
The foundations of genomics had been well established by the late 1990s,
when the first applications to nutrition, or nutrigenomics, were reported using the
new resources available. The first two reports of zinc-regulated gene expression
used macroarrays containing 1200 unique cDNA probes. In the first, with zinc-
deficient rat small intestine, 32 differentially expressed genes were reported,
and represented a variety of different cellular systems (54). Six of these
expression changes were confirmed using Q-PCR. Perhaps most interesting, in
regard to zinc antioxidant capabilities, is the large proportion of glutathione
S-transferases and other redox-associated genes that were modulated by zinc
deficiency. Three different glutathione S-transferase mRNAs were depressed
by zinc deficiency, which suggests that thiol status as well as redox status is dis-
turbed. In addition, depression of ATP synthases and intracellular ATPases point
towards decreased overall cellular energy level, which correlates with stunted
growth clinically observed during zinc deficiency. The correlation between clini-
cal observations and types of genes affected by zinc deficiency is also seen in the
multiple immune-related genes with altered expression that were reported.
The other macroarray paper reported, and subsequently confirmed by
Q-PCR, four additional mouse thymus genes that were modulated by 3 weeks of
zinc deficiency (23). As expected of the thymus, three of these, the myeloid cell
leukemia sequence-1, the laminin receptor, and the lymphocyte-specific protein
tyrosine kinase (lck) are immune-related genes, whereas the fourth, RAD23B, is
a DNA damage repair and recombination protein. Lymphocyte-specific protein
kinase was of particular interest because of the documented use of a zinc ion to
interact with its companion protein, the CD4 receptor (55). This highlights an area
of zinc functionality that is not often considered, the role of zinc to mediate struc-
tural or regulatory interactions between two different proteins. Most of the struc-
tural aspects of zinc in protein biochemistry have focused on medium to high
affinity binding sites within a single protein molecule; yet, a growing number of
reports suggest that lower affinity binding of zinc may play a crucial role in a
variety of protein–protein interactions of regulatory significance.
It should be noted that, in these two studies, the difference in number of
genes detected is generally a reflection of tissue type. Intestinal RNA gave detect-
able signals for 30– 40% of the cDNAs on the array, whereas it was only 19%
for thymus RNA. This is due to a bias towards metabolic genes in the choice
cDNAs to be spotted on these limited content arrays rather than tissue-specific
differences in gene expression between the intestine and the thymus. As arrays
become more comprehensive in their gene content, the bias has decreased until
it is simply a reflection of biological difference between the number of genes
required for different physiological pathways.
A commercial cDNA macroarray of 1200 probes, as well as a glass oligo-
nucleotide microarray of 1353 probes, were used to examine expression changes
for genes in rat liver after a zinc-deficient diet was fed for 11 days (56). Of 1550
mRNAs detected, 31 were found to be upregulated and 35 downregulated by zinc
deficiency. A similar wide distribution of functional classes was shown and
included the previously identified genes, fatty acid-binding protein, and gluta-
thione S-transferases. Of particular note was the zinc-deficient suppression of
three cytochrome P450s and the NADPH-cytochrome P450 reductase, which
may result in altered hepatic metabolism of xenobiotics.
Recently, high density oligonucleotide microarrays such as Affymetrix
GeneChips were used to examine zinc-regulated human gene expression. Using
the human U133A GeneChipw composed of 22,000 gene elements, we surveyed
changes due to zinc deficiency as well as supplementation in the human monocytic
cell line, THP-1 (57). This system serves as an in vitro model for circulating
PBMCs, where Cao et al. (58) demonstrated similar zinc responses for MT and
the zinc transporter Zip2 between THP-1 cells and human PBMCs. Treatment
with the cell permeable zinc chelator TPEN at 10 mM for 4 h was used to create
a severe, acute zinc deficiency while supplementation was at 40 mM zinc for the
same time. By setting up zinc concentrations on both sides of normal culture con-
ditions, the genes that are directly zinc responsive should display a continuity of
regulation across the treatments, that is, the gene would either be downregulated
in deficiency and upregulated in supplementation or the reverse.
Of 19,000 genes detected on triplicate GeneChips for each treatment,
1045 were identified under high statistical significance (P , 0.0001) as
changed in at least one condition. Of these, only 283 were significantly different
from normal in both treatments, and K-means clustering was used to group them
according to similar responses. Of that group, 104 genes positively correlated
with zinc levels, whereas 86 inversely correlated with zinc levels across all
three treatments. After breaking down the members of each correlation group by
functional category, 30 – 40% of each group were uncharacterized gene products,
which was not entirely unexpected given that a major portion of the gene
elements on the array have no annotated function. Of those in the positive corre-
lation group, the next most highly represented functional group was nucleic acid-
binding proteins, followed equally by genes involved in metabolism and signal
transduction. In the inverse correlation group, metabolism genes were most
highly represented, followed by signal transduction and then genes that influence
immune/cytokine function. Interestingly, the prototypical zinc-responsive gene
family, MT gene, does not show up in either of these groups owing to a combi-
nation of small fold change in the downregulation in zinc deficiency and moder-
ate variance that did not meet the stringent statistical analysis used. Strikingly,
the mRNA for tristetraprolin (TTP) showed the greatest magnitude of change
with a 14-fold decrease in zinc deficiency and an almost two-fold increase in
supplementation. TTP is a zinc finger protein that binds to TNF-a mRNA and
accelerates its degradation (59). Alterations of TNF-a gene expression are
commonly transduced by the NFkB transcription factor; therefore, it was very
striking to find prominently positioned in the list of zinc-modulated genes both
inhibitory proteins of NFkB, IkB-a and IkB-b. These findings provide additional
mechanisms to support the correlation between zinc status and immune function,
especially in the TNF cytokine signal transduction pathway.
An additional report analyzes zinc-deficient changes in gene expression with
a stress and aging-specific low-density microarray of 204 probes. In diversifying
from previous reports, the human lung fibroblast cell line, IMR90, was chosen
because the lung is chronically exposed to oxidative conditions and, therefore,
potentially very sensitive to zinc depletion. These cells were evaluated for anti-
oxidant and DNA repair gene expression changes under two different conditions
of zinc deficiency (60). The first zinc-deficient treatment was accomplished by
growing cells for 5 days in medium prepared from zinc-depleted FBS, whereas
the second used 40 mM TPEN for 2 h. Given the differences in treatment times
and deficiency models, as well as a conservative two-fold change cut-off, out of
the 30 genes changed by TPEN treatment and the 32 changed by depleted media,
there were only three found to be in common. These mRNAs encoded the proteins
glutathione peroxidase, proteasome 26S subunit, and chromosome segregation 1.
Nonetheless, when functional grouping of modulated genes were examined, they
showed similar types of changes in both types of deficiency, which supports the
theory that zinc-deficient cells are more susceptible to oxidative DNA damage.
By way of comparison, array analysis in the context of micronutrients and
antioxidants can be found in two reports from the area of selenium nutrition.
Macroarrays were used to evaluate selenium deficiency and its interaction with
vitamin E in rat liver, whereas microarrays were used to evaluate low selenium
status in mouse intestine (61,62). Both found that selenium deficiency, like
zinc, affects a diverse population of genes and this includes a significant
number that are involved in cellular redox maintenance.
Though dietary zinc-modulated genes are found in a wide variety of cellu-

lar pathways, all of the expression profiling to date shows that the percentage of
altered genes is still a small fraction of the transcriptome population. As
expression profiling provides information about multiple genes in the same
experiment, one of the questions raised is whether observed changes are due to
direct effects of the nutrient upon a gene or whether changes are a response to
the altered expression of direct responding genes. Often, this cannot be answered
in the context of the experimental system being profiled, as it requires very
precise manipulation of the cellular environment, which is not possible to
control in whole animal models with their extensive homeostatic mechanisms.
Therefore, classical reporter gene experiments in a cell culture system or RNA
interference techniques are desirable alternatives to determine whether nutrients
are acting directly on gene promoter elements.
The various methods utilized for expression profiling have different advan-
tages and disadvantages and most have been brought to bear upon the question of
zinc-responsive genes. DD of mRNA and serial analysis of gene expression
(SAGE) have been available the longest and have the advantage of requiring
minimal genomic sequence information for the system being evaluated. And
although SAGE analysis has not yet been applied to zinc-regulated expression pro-
filing, the increasing availability of SAGE libraries may soon change that fact.
Indeed, one of the benefits to applying multiple methods of detection is that inherent
bias in each technique may prejudice the selection of genes identified. This is
especially the case for detecting low abundance genes and reflects part of the
growing conundrum that, while low or even transiently expressed gene products
perform physiologically relevant functions, they present a challenge to our current
analytical capabilities. In addition, different methods of detection often present dif-
fering magnitudes of change although they generally agree on the direction of the
change. In all reports to date, there have been appreciable inconsistencies among
changes detected by array analysis vs. those from northern analysis or Q-PCR.
The latter techniques tend to indicate a greater magnitude of change than observed
on arrays, but there is still a strong concordance regarding the direction of change.
The application of large-scale mRNA expression profiling has provided
new understanding as well as new questions regarding the antioxidant properties
of zinc. Therefore, though nutrigenomics has seen increased application within
the nutritional science community, it will need to be followed by the application
of proteomics to fully understand the ramifications of zinc as an antioxidant and
regulator of gene expression.
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Index
Adipocyte, fatty acid regulation of, Anticarcinogenic properties

189 – 190 soy isoflavones, 327 – 336
Adipocyte differentiation, a-lipoic acid, tocotrienols, 213
290 – 294 Antioxidant
Aging AP-1 mediated gene expression,
inflammatory stress effects on, 103 148 – 152
low-grade inflammation applications, 160 – 164
mechanisms, 105– 106 atherogenesis, DNA
oxidative stress effects on, 103 methylation, 158
Alanine (ALA), 16 caspase-3 proteins, 129
a-lipoic acid cellular viability, 125
adipocyte differentiation, chromatin structure, transcription,
290 – 294 157 – 160
cell signaling properties, differential gene expression, 5 – 6
283 – 295 endothelial cells, 141 –167
glucose metabolism, diabetes, immune function and, 110 – 112
type 2, 287– 288 inflammation, 106
insulin-signaling pathway, 288 molecular mechanism, 134 – 137
a-tocopherol, 208– 210 NF-kB mediated gene expression,
molecular targets, 206 endothelium, oxidants,
NO, platelet aggregation, 210– 211 143 – 148
protein kinase C, 206– 208 pro-inflammatory genes, 164 – 167
VE sensitive genes, 211– 213 vitamin E, 152 – 157
Animal physiology zinc, 458 – 460
b,b-caroteine-15,150 - Antioxidant defenses
mono-oxygenases, 226–227 immune function, 97 – 117
vitamin A, 223– 225 infection/injury, 102 – 103
VP14-homologs, 227– 229 Antioxidant properties
Animal studies, isoflavones, procyanidins, 379 – 390
308 – 309 VE, chemistry, 202 –204
473
474 Index
AP-1 mediated gene expression, Caenorhabditis elegans, 68

endothelium, 148– 152 DAF-16 transcription target,
Apoptosis 76 – 77
cell death, 409–410 insulin/IGF-1 signaling, 70 – 73
gene expression, 125– 131 stress resistance, 73 – 76
PI 3-kinase, 409– 410 lifespan regulation, 69 – 81
Arterial wall gene expression, mitochrondrial electron
fatty acid regulation, transport, 78 –81
190 –191 nervous system, 78
Arteriosclerosis, potential reproduction system, 77 –78
mechanisms, 308 Caloric restriction (CR), 68
Ascorbate modulation Cancer related genes,
cell differentiation, 267–268 procyanidins, 384 – 385
collagen formation, 265– 267 Cancer, GSTM1 biomarkers, 21
nitric oxide production, 271–272 Cancer, GSTT1 biomarkers, 24
Ascorbate recycling, Cancer, MnSOD biomarkers,17– 18
metabolism, 263– 265 Capase-3 proteins, 129
Ascorbate-induced modulation Cardiovascular effects,
DNA repair, 272 isoflavones, 308
molecular functions, 272– 273 Cardiovascular related genes,
Ascorbic acid procyanidins, 384
ascorbate recycling, 260–263 Carotenoids
cellular redox activity, 257– 273 b,b-caroteine-15,150 -mono-
collagen formation, 265– 267 oxygenases, 226 – 227
immune function, anti-oxidants, color function, 222 – 223
effects on, 111 Catalase (CAT), 27
molecular functions, 265– 273 GST genes, 27 – 28
cell differentiation, 267– 268 cDNA array techniques
DNA repair, 272 complementary DNA, 386
nitric oxide production, procyanidins, 386 –389
271 –272 Cell adhesion proteins, 208 – 209
transcription factor Cell cycle progression inhibition,
modulation, 268– 271 lycopene, 246 – 247
nutritional aspects, 259– 260 Cell cycle, IP6 ,
redox status, 260– 265 cell proliferation, 405 – 410
Asthma, GSTP1, Cell differentiation
health conditions, 23 ascorbic acid, 267 – 268
Atherogenesis IP6, 408 – 409
DNA methylation, 158 Cell functions, MAP kinase
pro-inflammatory genes, 164– 167 signaling, 360 – 361
Atherosclerosis, vitamin E, 153 Cell proliferation normalization
cell cycle, 405 – 410
IP6, 405 – 408
b,b-caroteine-15,150 -mono-oxygenases, ras proteins, 407 – 408
carotenoids, 226– 227 retinoblastoma protein, IP6,
Blood pressure 406 – 407
isoflavones, 310, 313 Cell regulatory activity, tocopherols/
Breast cancer, isoflavones, 329– 333 tocotrienols, 201 – 214
Index 475
Cell signaling properties DAF-16 transcription target, 76 – 77

a-lipoic acid, 283– 295 Data analysis, 47 – 60
IP6, 397 –415 class prediction methods, 59 – 60
Cell survival, apoptosis, 125– 131 cross-validation, 60
Cellular redox activity, ascorbic acid, expression data, fold changes,
257 – 273 48 – 50
Cellular signaling hierarchical trees, 53
inositol compounds, 399– 400 k-means clustering, 50 – 52
IP3 receptor, inositol compounds, principal component analysis, 53
399 – 400 self-organizing maps, 52 – 53
IP6, signal transduction, 401– 402 Diabetes
Chemokines, scavenger receptors, a-lipoic acid, 283 – 295
a-tropomyosin, alpha tocopherol, insulin-signaling pathway,
cell adhesion proteins, 208– 209 285 – 287
Chromatin remodeling, IP6, nuclear treatment strategies, 284 – 285
inositol signaling, 411 type 2, a-lipoic acid, 287 –288
Chromatin structure, antioxidants, Dietary iron, gene expression
transcription, 157–160 modulation, 421 – 433
Class discovery, microarray data analysis Dietary selenium
hierarchical trees, 53 gene expression, 441 – 451
k-means clustering, 50– 52 selenoprotein expression, 447 – 448
principal component analysis, 53 Dietary zinc, 457 – 468
self-organizing maps, 52– 53 Differential gene expression
Class prediction methods, dietary selenium, 449 – 451
microarray data analysis Ginkgo biloba extract Egb 761,
cross-validation, 60 341 – 349
supervised learning methods, 59– 60 neuronal cells, 344 – 346
Clustering, hierarchical trees, 53 oxidants, antioxidants, 5– 6
Collagen formation, DMT1 regulation, iron, intestinal
ascorbic acid, 265– 267 absorption, 426
Colon cancer, isoflavones, 334– 335 DNA methylation, atherogenesis, 158
Colonic metabolism, flavonoids in vivo, DNA repair
356 – 358 ascorbate-induced modulation, 272
Complementary DNA arrays (cDNA), nuclear inositol signaling, 411
386 DNA synthesis, cell
Copper, zinc superoxide dismutase proliferation, 406
(SOD3), 18– 19 Drosophila, lifespan regulation, 81 – 83
Coronary heart disease (CHD), 181
fatty acids, gene expression 181– 186 Egb 761 Ginkgo biloba
isoflavones diets, 310– 319 chemical composition, 341 – 344
Cross-validation, microarray, class differential gene expression
prediction methods, 60 brain 346 –348
Cyclic strain, 160 neuronal cells, 344 – 346
Cyclooxygenase, Endocytosis, IP6 interactions, 402 – 405
alpha tocopherol, 209– 210 Endothelial cells, antioxidants,
Cytokines 141 – 167
adverse effects, 101 Endothelial gene expression,
infection/injury, 99– 101 vitamin E, 152 – 157
476 Index
Endothelial nitric oxide synthase. flavonols, 362 – 365

(eNOS), 25 MAP kinase signaling, 360 –361
dietary interventions, 26–27 Fold changes, microarray
health conditions, 26 data analysis, 48 – 50
smokers, 26 Folic acid, immune function, 115
Endothelium oxidants
AP-1 mediated gene expression, Gene clusters, hierarchical trees, 56 – 57
148 –152 Gene expression modulation, 457 – 468
NF-kB mediated gene expression, cancer related genes, 384 – 385
143 –148 cardiovascular related genes, 384
Endothelium reactivity, platelet cDNA array techniques, 386 – 389
aggregation, 317– 318 dietary iron, 421 – 433
Estrogen receptor inflammation related genes,
isoflavones, 305 385 – 386
mechanisms of action, 306 procyanidins, 383 –389
Exocytosis/endocytosis, vesicle Gene expression
trafficking, 402– 405 adipocytes, 189 –190
Expression data, fold changes, 48– 50 apoptosis, 125 – 131
arterial wall gene expression,
Fatty acid regulation 190 – 191
adipocytes, 189– 190 coronary heart disease, 181 – 196
arterial wall gene expression, dietary selenium, 441 – 451
190 –191 endothelial cells, 141 –167
coronary heart disease, 181– 196 fatty acid regulation, 187 –191
lipoprotein metabolism, 195– 196 glucose insulin homeostasis,
PPAR ligands, 193 195 – 196
PUFA induction microarray data analysis, 43 – 62
hepatic lipogenesis, 187–189 neutrigenomics, 1 – 9
lipid oxidation, 189 NF-kB, 143
PUFA mechanisms, 191–192 oxidants/antioxidants, 3 – 4
transcription factors, 192– 194 oxidative stress, 68– 86
peroxisome proliferators-activator profiling, 464 – 468
receptors, 192– 193 PUFA induction, lipid oxidation, 189
Fatty acids, metabolism, 183– 187 PUFA mechanisms, fatty acid
absorption, 183 regulations, 191 – 192
lipoproteins, 184– 185 PUFA, hepatic lipogenesis, fatty acid
Fatty acid structure, regulation, 187 – 189
tissue sources, 182– 183 transcription factors
Flavanols, signaling cascade fatty acid regulations, 192 – 194
modulations, 362–365 peroxisome proliferators-activator
Flavonoids receptors, 192 – 193
bioactive forms, 355– 359 PPAR ligands, 193
colonic metabolism, 356– 358 zinc, 460 – 463
intracellular metabolism, 358– 359 GI tract, flavonoids metabolism, 356
metabolism, GI tract and liver, 356 Ginkgo biloba extract Egb 761
metabolites, 353– 367 chemical composition, 341 – 344
signaling cascade modulations, differential gene expression,
359 –367 341 – 349
Index 477
Glucose insulin homeostasis, gene Hierarchical trees (clustering), 53

expression, 195– 196 class discovery, 53
Glucose metabolism, experiment, gene clusters, 56 – 57
diabetes type 2, 287– 288 measurements, 54 – 55
Glutathione, anti-oxidants, QT clustering, 57 – 59
effects on, 111– 112 HMG-CoA reductase,
Glutathione peroxidase (GPX1), 29 lycopene, 249 – 250
polymorphisms, 29 Hormesis, lifespan regulation,
Glutathione-s-transferase M1, C. elegans, 69 – 70
(GSTM1), 20 Human stress varations
oxidative stress, 19– 20 glutathione-s-transferases, 19 – 20
Glutathione-s-transferase P1 manganese superoxide
(GSTP1), 22– 24 dismutase, 15 – 18
asthma, 23 MnSOD health conditions, 17 –18
ile allele associations, 23 MnSOD polymorphisms, 16 – 17
val allele associations, 23 oxidative stress, 13 – 32
Glutathione-s-transferase T1 SOD3, 18 – 19
(GSTTI), 24 GST genes, 20 – 24
biomarkers, 24 Hypoxia, iron, 427 – 428
cancer, 24
GSH, immune function, 112– 114 Ile allele associations, GSTP1, 23
GST genes Immune function
catalase, 27– 28 antioxidant defenses, 101 – 102
endothelial nitric oxide antioxidant effects on, 110 – 112
synthase, 25–27 ascorbic acid, 111
glutathione peroxidase, 29 glutathione, 111 – 112
glutathione-s transferase M1, 20 vitamin E, 110 – 111
gluthathione-s-transferase P1, 22– 24 antioxidant modulation, 97 – 117
gluthathione-s-transferase T1, 24 cytokines, infection, injury, 99 – 101
GSTM1 biomarkers, 21 folic acid, 115
myeloperoxidase, 30 GSH, 112 – 114
NADPH dehydrogenase, quinine 1, 31 infection injury, 98
NADPH oxidase, 28 oxidants, 106
oxidative stress, 20– 24 pro-inflammatory, adverse effects, 101
GSTM1 biomarkers taurine, 115 – 116
GST genes, 21, 23 vitamin B6, 114 – 115
glutathione-s-transferase M1, 20 Infection injury
cancer, 21 anti-oxidant defenses, 102 – 103
lung disease, 21– 22 cytokines, 99 –101
other disorders, 22 inflammatory agents, 98
Inflammation, immune function,
Hemocromatosis, iron, 430– 432 effects on, 106
Hepatic lipogenesis, PUFA, 187–189 Inflammation mechanisms,
Hepatic nuclear receptor-4(HNF-4), low-grade, 105 – 106
transcription factors, 194 Inflammation-related genes,
Hepcidin, iron, metabolism, 428– 429 procyanidins, 385 – 386
Hephaestin regulation, iron, intestinal Inflammatory agents,
absorption, 427 infection injury, 98
478 Index
Inflammatory genes, vitamin E, programmed cell death,

155 –156 apoptosis, 409 – 410
Inflammatory stress, aging, effect on, 103 signal transduction, cellular
Injury, infection signaling, IP3, 401 – 402
anti-oxidant defenses, 102– 103 IP6 interactions
immune function, cytokines, 99– 101 cellular signaling, 402 –405
Inositol compounds vesicle trafficking, exocytosis/
cellular signaling, 399– 400 endocytosis, 402 – 405
IP3 receptor, 399–400 IREG1 regulation, iron, intestinal
IP6 signal molecule, 400– 405 regulation, 426 – 427
protein macromolecules, 402– 405 Iron, dietary
Inositol hexaphosphate (IP6), 397– 415 absorption, 422 – 423
Insects, vitamin A, 229– 230 gene expression modulation, 421 – 433
Insulin/IGF-1 signaling Iron, hemocromatosis, 430 – 432
C. elegans, 70 – 73 Iron, hypoxia, 427 – 428
lifespan regulation, 73– 76 Iron, intestinal absorption, 423 –427
Insulin-like growth factor (IGF-1) Dcyth regulation, 427
signaling, lycopene, 247– 249 DMT1 regulation, 426
Insulin-signaling pathway hephaestin regulation, 427
a-lipoic acid, 288 IREG1 regulation, 426 – 427
diabetes, 285– 287 regulatory proteins, 424
Intracellular communication modulation, responsive elements, 424 – 426
lycopene, 245– 246 Iron, metabolism, hepcidin, 428 – 429
Intracellular metabolism Iron, pro-oxidant, 432 – 433
fatty acids, 186 Isoflavone, 329 – 336
flavonoids in vivo, 358– 359 absorption, metabolism, 303 – 305
Ionic zinc, 458 arteriosclerosis, 308
IP3 receptor, inositol blood pressure, 310, 313
compounds, 399– 400 breast cancer, 329 – 332
IP3/IP6, inositol compounds, 401– 402 cardiovascular effects, 308
IP5/IP6 interactions, 402 colon cancer, 334 – 335
IP6 diets, mechanisms of action,
anticancer agent, 413– 414 301 – 319
cell differentiation induction, 408– 409 estrogen receptor, mechanisms of
cell proliferation normalization, action, 305
405 –408 anti-oxidant activity, 306
cell cycle, 405– 410 inflammation, cell adhesion,
DNA synthesis, 406 313, 315 – 317
protein kinase C, 407 lipid metabolism, 308 – 310
ras proteins, 407– 408 animal studies, 308 –309
retinoblastoma protein, 406– 407 clinical studies, 309 – 310
energy transduction, 413 phytoestrogens, 302 –303
nuclear inositol signaling, 410– 413 platelet aggregation, endothelium
chromatin remodeling, 411 reactivity, 317 – 318
DNA repair, 411– 412 prostate cancer, 332 – 333
mRNA transport, 410– 411 soy
NF-kB, 411– 412 blood pressures, 314
zinc-finger motif, 411– 412 serum lipids, 311 – 312
Index 479
k-Means clustering, microarray data Manganese superoxide dismutase

analysis, 50– 52 (MnSOD), 15
oxidative stress, 15 – 18
Late-onset neurological disorders, 17 MAP kinase signaling, signaling
Lifespan, replicative, 85– 86 cascade modulations,
Lifespan regulation, C. elegans, 69 – 81 360 – 361
DAF-16 transcription target, 76– 77 Mechanisms of action
insulin/IGF-1 signaling, 70– 73 estrogen receptors, 306
stress resistance, 73– 76 isoflavones, 305
mitochrondrial electron Metabolism
transport, 78– 81 absorption, fatty acids, 183
nervous system, 78 ascorbate recyling, 263 – 265
reproduction system, 77– 78 fatty acids, 183 – 187
stress, hormesis, 69– 70 flavonoids in vivo, 356
Lifespan regulation, drosophila, 81 – 83 hepcidin, iron, 428 – 429
Lifespan regulation, mammals, 83– 85 intracellular metabolism,
Linear, microarray, normalization fatty acids, 186
methods, 45 isoflavones, 303 – 305
Lipid metabolism lipoproteins, 184 – 185
isoflavones, 308– 310 selenium 442 – 443
animal studies, 308– 309 Microarray, gene expression data
clinical studies, 309– 310 organization (MGED), 47
Lipid oxidation, PUFA induction, 189 Microarray, normalization
Lipoprotein metabolism methods, 44 – 47
fatty acids, 184– 185 linear, 46
glucose insulin homeostasis, per array, 46 – 47
195 – 196 per gene, 46 –47
Lung disease, GSTM1 specific samples, 46 – 47
biomarkers, 21–22 Microarray data, gene expression,
Lycopene 43 – 62
cell cycle progression Microarray data analysis, 47– 60
inhibition, 246– 247 class discovery, 50 – 53
HMG-CoA reductase, hierarchical trees, 53
inhibition of, 249– 250 k-means clustering, 50 – 52
insulin-like growth factor (IGF-1) principal component
signaling, inhibition of, analysis, 53
247 – 249 self-organizing maps, 52 – 53
intracellular communication class prediction methods, 59 – 60
modulation, 245 –246 cross-validation, 60
molecular mechanisms, health benefits, expression data, fold changes,
241 – 251 48 – 50
oxidative stress, 243– 245 software, 61 – 62
validation, 60 –61
Macromolecules, proteins, IP6 in silico, 60 – 61
interactions, 402– 405 laboratory based, 61
Mammalian cells, cellular signaling, Minimal information about
400 – 401 a microarray experiment
Mammals, lifespan regulation, 83– 85 (MIAME), 9, 47
480 Index
Mitochrondrial electron transport, Nitric oxide production, ascorbate

C. elegans, 78 – 81 modulation, 271 – 272
MnSOD health conditions NO, platelet aggregation, alpha
cancer, 17– 18 tocopherol, 210 – 211
neurological disorders, 17 Normalization methods,
oxidative stress, 17– 18 microarray, 44 – 47
Model organism, molecular linear, 45
analyses, 229 per array, 45 – 46
Molecular analyses per gene, 46 – 47
vertebrates, 231– 235 specific samples, per gene, 46 – 47
vitamin A biosynthetic Nuclear factor kappa B (NF-kB),
pathway, 229 transcription factors, 194
Molecular functions Nuclear inositol signaling
ascorbic acid, 265–273 chromatin remodeling, IP6, 411
DNA repair, 272 DNA repair, IP6, 411
nitric oxide production, 271– 272 IP6, 410 – 413
ascorbate-induced modulation, mRNA transports, IP6, 410 – 411
272 –273 Nutrigenomics
cellular redox activity, 257– 273 definition, 1 –2
transcription factor modulation, methods, applications, 4, 7 – 9
268 –271 oxidants, antioxidants, gene
Molecular mechanisms expression, 1 – 9
lycopene, 241– 251
zinc, gene expression, 460–463 Oxidants/antioxidants
MRNA transports, nuclear inositol differential gene expression, 3 – 6
signaling, 410– 411 nutrigenomics, 1 –9
Myeloperoxidase (MPO), 30 endothelium, 148 – 152
immune function, effects on, 106
NADPH NF-kB mediated gene expression,
health conditions, 28– 29 143 – 148
polymorphisms, 28 Oxidative stress
NADPH dehydrogenase quinine 1 aging, effect on, 103
(NQO1), 31 gene expression, 68 – 86
GST genes, 31 human stress variation, 13 – 32
polymorphisms, 31 manganese superoxide dismutase,
NADPH oxidase, GST genes, 28 15 – 18
Nervous system, C. elegans, 78 MnSOD health conditions, 17 – 18
Neuronal cells, differential gene MnSOD polymorphisms, 16 – 17
expression, 344–346 glutathione-s-transferases, 19 – 20
Neuroprotection, tocotrienols, GST genes, 20 – 24
213 –214 SOD3, 18 – 19
NF-kB mediated gene expression, 143 lycopene, 243 – 245
endothelium, oxidants, antioxidants, replicative lifespan, 85 – 86
143 –148
IP6, nuclear inositol signaling, 411 PCA (principal component analysis), 53
nuclear transcription factor-kB, Peroxisome proliferators-activated
411 –412 receptors (PPAR), 192
proteins regulated by, 145 transcription factors, 192 – 193
Index 481
Phytoestrogens, isoflavones, 302– 303 Ras proteins, cell proliferation

PI 3-kinase, apoptosis, 409– 410 normalization, 407 – 408
Platelet aggregation Reactive oxygen species (ROS), 68
alpha tocopherol, NO, 210– 211 Redox status
endothelium reactivity, isoflavones, ascorbate recycling, 260 – 263
317 – 318 ascorbic acid, 260 – 265
Polymorphisms Replicative lifespan, oxidative stress,
CAT, 27 85 – 86
GPX1, 29 Reproduction system, C. elegans,
MPO, 30 – 31 77 – 78
NADPH, 28 Retinoblastoma protein, cell proliferation
NQO1, 31 normalization, 406 – 407
SOD3, 19
Polyphenols, procyanidins, 380 Scavenger receptors,
Polyunsaturated fatty acids (PUFA), 203 chemokines, 208 – 209
epatic lipogenesis, 187– 189 Selenium
induction, 189 deficiency, symptoms, 443 – 444
mechanisms, 191– 192 gene expression, 441 – 451
polyunsaturated fatty acids, 203 metabolism, 442 – 443
PPAR ligands, transcription selenocysteine-containing
factors, 193 proteins, 445 – 446
Procyanidins selenoprotein expression, 447 – 448
antioxidant properties, 379–390 Selenocysteine-containing
gene expression modulation, proteins, 445 – 446
383 – 389 Self-organizing maps (SOM), 52
cancer related genes, 384– 385 class discovery, 52 – 53
cardiovascular related genes, 384 Serum lipids, soy isoflavones, 311 – 312
cDNA array techniques, 386– 389 Shear stress, 161 – 164
inflammation related genes, Signaling cascade modulations, 362 –365
385 – 386 flavonoids, 359 – 367
polyphenols, 380 flavonols, 365 –367
sources, bioavailability, 380–382 MAP kinase signaling, 360 – 361
Programmed cell death, IP6, 409– 410 Single nucleotide polymorphisms
Pro-inflammatory genes, antioxidants, (SNPS), 14
164 – 167 Smoking, eNOS health conditions, 26
Pro-inflammatory, cytokines, immune SOD2, MnSOD, 15
function, 101 SOD3
Pro-oxidant, iron, 432– 433 copper, zinc superoxide dismutase,
Prostate cancer, isoflavones, 332–333 18 – 19
Protein(s), macromolecules oxidative stress, humans stress
IP6 interactions, 402– 405 variations,18 –19
Protein kinase C polymorphisms, 19
alpha tocopherol, 206– 208 Software, microarray analysis, 61 – 62
IP6, cell proliferation Soy isoflavones
normalization, 407 anti-carcinogenic properties,
327 – 336
QT clustering, hierarchical trees, blood pressure, 314
57 – 59 serum lipids, 311 – 312
482 Index
Sterol regulatory element-binding in silico, 60 – 61

protein, transcription laboratory based, 61
factors, 194 Valine (VAL), 16
Stress hormesis, C. elegans, 69– 70 Vesicle trafficking,
Stress resistance, insulin/IGF-1 IP6 interactions, 402 – 405
signaling, 73– 76 Vitamin A
Stress-induced premature senescense biosynthetic pathway, molecular
(SIPS), 85 analyses
Supervised learning methods, 59– 60 model organism, 229
vertebrates, 231 – 235
Taurine, immune function, 115– 116 carotenoids
Tocopherol, alpha, molecular b,b-caroteine-15,150 -mono-
targets, 206 oxygenases, 226 – 227
Tocopherols/tocotrienols, cell regulatory color function, 222 – 223
activity, 201– 214 deficiency (VAD), 223
Tocotrienols functions, 223 –225
biological properties, 213– 214 insects, 229 – 230
anticarcinogenic properties, 213 molecular analysis, 221 – 236
neuroprotection, Vitamin A deficiency (VAD), 223
Src activity, 213 –214 Vitamin E (VE), 202
cell regulatory activity, tocopherols, absorption, transport,
201 –204 metabolism, 204 – 206
Transcription factors atherosclerosis, 153
ascorbic acid, molecular functions, chemistry, antioxidant
268 –271 properties, 202 – 204
fatty acid regulations, gene expression, endothelial gene expression,
192 –194 152 – 157
hepatic nuclear receptor-4(HNF-4), immune function, effects on,
194 110 – 111
nuclear factor kappa B (NF-kB), 194 inflammatory genes, 155 – 156
peroxisome proliferators-activator VP14-homologs, animal physiology,
receptors, 192– 193 227 – 229
PPAR ligands, 193
sterol regulatory element-binding Zinc
protein, 194 as antioxidant, 458 – 460
Transcriptomics, applications, ionic, 458
techniques, 7 MT, 458 – 460
Type 2 diabetes, a-Lipoic acid, glucose dietary, gene expression modulation,
metabolism, 287– 288 457 – 468
expression profiling, 464 –468
Val allele associations, molecular mechanisms, 460 – 463
health conditions, 23 Zinc-finger motif, IP6, nuclear inositol
Validation, microarray analysis, 60– 61 signaling, 411

Nutrigenomics (Oxidative Stress and Disease) (PDFDrive)

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Nutrigenomics

Boca Raton London New York Singapore

© 2005 by Taylor & Francis Group, LLC

Library of Congress Cataloging-in-Publication Data

Catalog record is available from the Library of Congress

Visit the Taylor & Francis Web site at

Oxygen is a dangerous friend. Through evolution, oxygen—itself a free radical—

GERALD RIMBACH is Professor of Food Science and Director of the Institute

JÜRGEN FUCHS is Professor, Department of Dermatology, Johann Wolfgang

LESTER PACKER is Adjunct Professor of Molecular Pharmacology and Toxi-

Max O. Bingham Food Microbial Sciences Unit, School of Food Biosciences,

Raymond K. Blanchard Nutritional Genomics Laboratory, Food Science and

Aedin Cassidy School of Medicine, Health Policy & Practice, University of

Rainer Cermak Institute of Animal Nutrition and Physiology, Christian

Kyung-Joo Cho Laboratory of Cell Biology, Korea Advanced Institute of

An-Sik Chung Department of Biological Sciences, Korea Advanced Institute

Robert J. Cousins Nutritional Genomics Laboratory, Food Science and

S. G. Cremers School of Animal and Microbial Sciences, University of

Sonia De Pascual-Teresa Department of Plant Food Science and Technology,

B. A. Ewins School of Food Biosciences, University of Reading, Reading, UK

Alexandra Fischer Department of Cardiovascular Medicine, Wellcome Trust

Glenn R. Gibson Food Microbial Sciences Unit, School of Food Biosciences,

R. D. Gill-Garrison Sciona, Ltd., Havant, UK

K. Grimaldi Sciona, Ltd., Havant, UK

Robert Francis Grimble Institute of Human Nutrition, Biomedical Sciences

O. Gulati Horphag Research Ltd., Geneva, Switzerland

Shuji Honda Aging Redox Regulation Research Group, Tokyo Metropolitan

Yoko Honda Aging Redox Regulation Research Group, Tokyo Metropolitan

John K. Lodge School of Biomedical and Molecular Sciences, University of

Anne M. Minihane Hugh Sinclair Unit of Human Nutrition, University of

Ken Mills Department of Haematology, Wales College of Medicine, Cardiff

Hadi Moini Department of Molecular Pharmacology and Toxicology, School

B. A. Nier School of Animal and Microbial Sciences, University of Reading,

Estibaliz Olano-Martin School of Food Biosciences, University of Reading,

Lester Packer Department of Molecular Pharmacology and Toxicology,

Series Introduction ....................................... iii

1. Application of Nutrigenomics Tools to Analyze the Role of

2. Oxidative Stress and Human Genetic Variation . . . . . . . . . . . . . . 13

3. Analysis of Microarray Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

4. Oxidative Stress, Gene Expression, and Lifespan . . . . . . . . . . . . . 67

5. Anti-Oxidant Modulation in Immune Function . . . . . . . . . . . . . . . 97

6. Concentration-Dependent Gene and Protein Expressions of

7. Effects of Antioxidants on Gene Expression in

8. Fatty Acids, Gene Expression, and

9. Cell Regulatory Activity of Tocopherols

10. Molecular Analysis of the Vitamin A

11. Molecular Mechanisms Underlaying the Health

12. Cellular Redox Activity and Molecular Functions

13. Cell Signaling Properties of a-Lipoic Acid:

14. Dietary Isoflavones and Coronary Artery

15. Anti-Carcinogenic Properties of Soy Isoflavones . . . . . . . . . . . . . . 327

16. Effect of Ginkgo biloba Extract EGb 761

17. Interactions of Flavonoids and Their Metabolites

18. Antioxidant and Gene Regulatory Properties

19. Cell Signaling Properties of Inositol

20. Modulation of Gene Expression by Dietary Iron . . . . . . . . . . . . . . 421

21. Dietary Selenium and Gene Expression . . . . . . . . . . . . . . . . . . . . 441

22. Modulation of Gene Expression by Dietary Zinc . . . . . . . . . . . . . 457

Figure 1.1 Nutrigenomics as a discipline at the interface between genetics, molecular

Nutrigenomics applies high-throughput molecular biology techniques inclu-

Figure 1.2 Genomics, transcriptomics, proteomics, and metabolomics as analytical

DO OXIDANTS AND ANTIOXIDANTS AFFECT GENE EXPRESSION?

METHODS AND APPLICATIONS IN NUTRIGENOMICS