Human Cytochrome P450
Enzymes
F. Peter Guengerich
9.1 History
The history of cytochrome P450 (P450) really
began with studies on the metabolism of drugs,
carcinogens, and steroids The early research in
these fields necessarily involved animal mod-
els, but the intent was always to understand the
human systems in the context of the enzymes
catalyzing the observed transformations
A number of in vivo experiments in the realm
of clinical pharmacology showed that drug me-
tabolism was inducible [1–3] and varied among
individuals [4] Such phenomena were attrib-
uted to P450 enzymes after the development of
research with experimental animals, but the mo-
lecular basis was unknown
Early in vitro studies with human tissues were
done but were difficult because of the limited
availability of samples It was possible to docu-
ment the variability of human drug metabolism
[5], although there were caveats about the quality
of samples, etc
The next phase of research was the purifica-
tion of human P450s from liver microsomes
Some early efforts in this area were in the labo-
ratories of Coon [6], Beaune [7], Kamataki [8],
and Guengerich [9] Highly purified P450s could
F P Guengerich ()
Department of Biochemistry and Center in Molecular
Toxicology, Vanderbilt University School of Medicine,
638 Robinson Research Building, 2200 Pierce Avenue,
Nashville, TN, 37232-0146, USA
e-mail: fguengerich@vanderbiltedu
P R Ortiz de Montellano et al (eds), Cytochrome P450, DOI 101007/978-3-319-12108-6_9
© Springer International Publishing Switzerland 2015
9
be obtained, but analysis of catalytic specificity
was generally limited to sets of a few typical sub-
strates used with rat and rabbit P450 enzymes
However, some studies with warfarin oxidation
were to provide insight, in that distinct activities
were noted [10] Clearly, multiple P450 existed
in humans, as already appreciated in rats and rab-
bits However, there was no clear indication how
many human P450s might exist or how many
would be involved in xenobiotic metabolism
The human studies of Smith and his associates
[11], along with others [12, 13], were very use-
ful in that they first showed that the metabolism
of an individual drug was genetically controlled
Monogenic control of the oxidation of a drug
suggested that a single P450 would be dominant
in its metabolism This information led to a dif-
ferent plan to study human P450s: Purification
was monitored with analysis of individual drug
oxidation activities, rather than simply purify-
ing the colored hemoproteins and then trying
to establish their activities The approach was,
however, technically challenging in that indi-
vidual fractions recovered from chromatogra-
phy needed to be depleted of detergent, recon-
stituted with nicotinamide adenine dinucleotide
phosphate-cyrochrome P450 (NADPH-P450)
reductase, and monitored for activity using gas or
liquid chromatography (LC) Nevertheless, with
debrisoquine 4-hydroxylation and phenacetin O-
deethylation, the approach yielded what are today
termed P450s 2D6 and 1A2 [14] Further work in
this laboratory led to the purification of what are
known today as P450s 2C8, 2C9 [15], 3A4 [16],
523
524 F. P. Guengerich

2A6 [17], and 1A1 [18] Work in other laborato-
ries also yielded these same P450s purified from
human liver [19–21] and P450s 2C19 [22] and
2E1 [23], plus P450 3A7 from fetal liver [24]
The purified P450s and their antibodies could
be utilized to define the roles of individual P450s
in the metabolism of individual drugs, carcino-
gens, and steroids Other approaches developed
during the 1980s included correlation of indi-
vidual catalytic activities (in liver microsomes
prepared from different individuals [25], or im-
munochemically determined levels of P450s [26,
27]) and the development/application of selective
chemical inhibitors [14, 28–31]
Despite all of this progress in enzymology,
there were still issues that could not be addressed
easily Some P450s were not expressed at levels
high enough to be purified this way (and affin-
ity chromatography methods were not effective)
The need for large amounts of P450s in the future
was a limitation The development of recombi-
nant DNA technology in the 1980s was yield-
ing complementary DNAs (cDNAs) for P450s,
but the only way to associate these with isolated
P450 proteins was by N-terminal amino acid se-
quence analysis, using Edman degradation
cDNA clones for many of the human P450s
were rapidly isolated and used to determine nu-
cleotide (and predictably amino acid) sequences,
following the elegant work of Fujii-Kuriyama
and his associates with rat P450 2B1 [32] Much
of the cDNA work was done by Gonzalez and his
laboratory [33] The cDNA work led to insight
into the basis of the debrisoquine polymorphism
described by Smith [11, 34]
After the success of cDNA cloning, practi-
cal heterologous expression of P450 enzymes
was achieved in cells being CV-1 in origin and
carrying the SV40 genetic material (COS) cells
[35] and yeast [36] and then, very importantly,
achieved in bacterial systems in the early 1990s
[37–39] The high-yield expression methods
were important for the crystallization of human
P450s, which was done primarily by Johnson
and his associates following their success with
a rabbit subfamily 2C P450 [40, 41] Today, the
three-dimensional structures of at least 21 human
P450s have been determined (Table 91)
Recombinant DNA technology allowed for
insight into the regulation of human P450 genes
and also for the analysis of single nucleotide
variations (SNVs), which could sometimes be as-
sociated with altered drug or steroid metabolism

Table 9.1   Classification of human P450s based on major substrate class

Sterols Xenobiotics Fatty acids Eicosanoids Vitamins Unknown
a a
1B1 1A1 2J2 4F2 2R1a 2A7
7A1a 1A2a 2U1 4F3 24A1c 2S1
7B1 2A6a 4A11 4F8 26A1 2W1
8B1 2A13a 4B1 5A1 26B1 4A22
11A1a 2B6a 4F11 8A1a 26C1 4F22
11B1 2C8 a 4F12 27B1 4X1
11B2* 2C9a 4V2 27C1 4Z1
17A1a 2C18 20A1
19A1a 2C19a
b
21A2 2D6a
27A1 2E1a
39A1 2F1
46A1a 3A4a
51A1a 3A5
3A7
3A43
a X-ray crystal structure(s) reported (for human enzyme)
b Bovine X-ray crystal structure reported [42]
c Rat X-ray crystal structure reported [43]
9  Human Cytochrome P450 Enzymes 525

Table 9.2   Human P450 locations and marker reactions

P450 Tissue sites Subcellular Typical reactionb
localizationa
1A1 Lung, several extrahepatic sites ER Benzo[a]pyrene 3-hydroxylation
1A2 Liver ER Caffeine N3-demethylation
1B1 Several extrahepatic sites ERc 17β-Estradiol 4-hydroxylation
2A6 Liver, lung, and several extrahepatic ER Coumarin 7-hydroxylation
sites
2A7 ER
2A13 Nasal tissue ER Activation of 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK)
2B6 Liver, lung ER ( S)-Mephenytoin N-demethylation
2C8 Liver ERc Taxol 6α-hydroxylation
2C9 Liver ER Tolbutamide methyl hydroxylation
2C18 Liver ER
2C19 Liver ER ( S)-Mephenytoin 4ʹ-hydroxylation
2D6 Liver ERc Debrisoquine 4-hydroxylation
2E1 Liver, lung, other tissues ERc Chlorzoxazone 6-hydroxylation
2F1 Lung ER 3-Methylindole activation
2J2 Lung ER Arachidonic acid oxidations
2R1 Liver ER Retinoic acid oxidation
2S1 Lung ER (several drug reductions)
2U1 Thymus, brain ER Arachidonic acid oxidation
2W1 Tumors ER
3A4 Liver, small intestine ERc Testosterone 6β-hydroxylation
3A5 Liver, lung ER Testosterone 6β-hydroxylation
3A7 Fetal liver ER Testosterone 6β-hydroxylation
3A43 Brain, liver ER
4A11 Liver, kidney ER Fatty acid ω-hydroxylation
4A22 Liver, kidney ER
4B1 Lung ER Lauric acid ω-hydroxylation
4F2 Liver ER Leukotriene B4 ω-hydroxylation
4F3 Neutrophils ER Leukotriene B4 ω-hydroxylation
4F8 Seminal vesicles ER Prostaglandin ω-2 hydroxylation
4F11 Liver ER Fatty acid ω-hydroxylation
4F12 Liver ER Arachidonic acid ω-,ω-1 hydroxylation
4F22 Liver ER Vitamin K ω-hydroxylation
4V2 Eye ER Fatty acid ω-hydroxylation
4X1 Liver, brain ER
4Z1 Breast cancer ER
5A1 Platelets ER Thromboxane A2 synthase reaction
7A1 Liver ER Cholesterol 7α-hydroxylation
7B1 Brain ER DHEA 7α-hydroxylation
8A1 Aorta, others ER Prostacyclin synthase reaction
8B1 Liver ER 7α-Hydroxycholesterol
12-hydroxylation
11A1 Adrenals, other steroidogenic tissues Mit Cholesterol side-chain cleavage
11B1 Adrenals Mit 11-Deoxycortisol 11-hydroxylation
11B2 Adrenals Mit Corticosterone 18-hydroxylation
17A1 Steroidogenic tissues ER Pregnenolone 17α-hydroxylation
19A1 Steroidogenic tissues, adipose ER Androgen aromatization
20A1 Liver, other tissues ER
526 F. P. Guengerich

Table 9.2  (continued)

P450 Tissue sites Subcellular Typical reactionb
localizationa
21A2 Steroidogenic tissues ER 17α-Hydroxyprogesterone
21-hydroxylation
24A1 Kidney Mit 25-Hydroxyvitamin D3 24-hydroxylation
26A1 Several ER Retinoic acid 4-hydroxylation
26B1 Brain ER Retinoic acid 4-hydroxylation
26C1 ER Retinoic acid 4-,18-hydroxylation
27A1 Liver Mit Sterol 27-hydroxylation
27B1 Kidney Mit Vitamin D3 1-hydoxylation
27C1 Liver Mit
39A1 Liver, other tissues ER 24-Hydroxycholesterol 7-hydroxylation
46A1 Brain ER Cholesterol 24-hydroxylation
51A1 Liver, testes ER Lanosterol 14α-demethylation
DHEA dehydroepiandrosterone
a
ER endoplasmic reticulum (microsomal), Mit mitochondria
b If known
c Mainly ER, some detected in mitochondria

(http://wwwcypalleleskise) (The term “varia- 9.2 Relevance of P450s in Drug

tions” will be used here, in that “polymorphism”
is usually defined as an occurrence at a ≥ 1 %
frequency [44], and many of the cases to be de-
scribed here are observed at lower frequencies)
Ultimately, the availability of the human genome
nucleotide sequence led to the discovery of more
P450 genes Most of the P450s listed in the
“unknown” substrate column in Table 91 were
found in this way, on the basis of the signature
sequence surrounding the Cys residue that serves
as the axial heme ligand Very importantly, the
number of P450 genes was set at 57 (Tables 91
and 92), thereby closing old debates on the sub-
ject [45, 46]
As mentioned earlier, the history of P450 re-
search can be traced to early studies on the me-
tabolism of drugs, carcinogens, and steroids
Application in these areas was remarkable in
the period 1985 to present, and each area will be
treated separately Overall, the P450 field can be
considered a model for how basic research can
lead to important developments for human medi-
cine Defects in several of the P450s have been
linked to serious human diseases (Table 93)
Metabolism
P450s are the major enzymes involved in human
drug metabolism (Fig 91) In looking at the
fraction of the number of (small molecule) drugs
processed by enzymes (Fig 91a), P450s account
for ~ 75 % Constructing a figure of this type can
be somewhat misleading in that the contribution
of each P450 is more difficult to evaluate in vivo
than in vitro (for an earlier tabulation, see [51])
The large contributions of P450s 3A(4) and 2C9
are driven to a large extent by the high levels of
expression of these two enzymes in human liver
(and small intestine) and to their broad substrate
specificity (Figs 92 and 93) The charts do not
necessarily reflect all drugs currently in devel-
opment A current tendency has been the devel-
opment of larger molecules as drug candidates,
in order to achieve target specificity and affin-
ity, and a general axiom is that these are more
readily accommodated by P450s 3A4 and 2C9
In recent years, pharmaceutical companies have
tried to avoid developing drug candidates that are
substrates (or inhibitors) for the highly variant
9  Human Cytochrome P450 Enzymes 527

Table 9.3   Some diseases associated with defects in CYP genes [47, 48]
Gene Disorder
CYP1B1 Primary congenital glaucoma (buphthalmos)
CYP2R1 Rickets
CYP4A Defects in salt metabolism, water balance leading to arterial hypertension
CYP4F22 Ichthyosis
CYP4V2 Bietti’s crystalline dystrophy
CYP5A1, 8A1 Defects leading to clotting and inflammatory disorders, coronary artery disease, and pulmo-
nary hypertension
CYP7A1 Hypercholesterolemia
CYP7B1 Severe hyperoxysterolemia and neonatal liver disease
CYP11A1 Lipoid adrenal hyperplasia; occasional congenital adrenal hyperplasia (CAH)
CYP11B1 Occasional CAH
CYP11B2 Corticosterone methyloxidase deficiency type I, or type II; occasional CAH
CYP11B1, 11B2 Chimeric enzymes causing glucocorticoid-remediable aldosteronism; occasional CAH
CYP17A1 Mineralocorticoid excess syndromes, glucocorticoid and sex hormone deficiencies; associa-
tion with increased risk of prostate cancer and benign prostatic hypertrophy; occasional CAH
CYP19A1 Loss of function: virilization of females, hypervirilization of males, occasional CAH; gain of
function: gynecomastia in young males
CYP21A2 > 90 % of all CAH
CYP24A1a Hypervitaminosis D
CYP27A1 Cerebrotendinous xanthomatosis
CYP27B1 Vitamin D-dependent rickets type I
CYP46A1a Learning disability
a Evidence of disease in animal models but not yet in clinical studies

P450s 2D6 and 2C19 With all of these caveats
in hand, the allocation of the P450s in the chart in
Fig 91b is probably a good estimate and will not
change considerably in the near future However,
a point to be made here is that the metabolism of
many drugs is a function not only of P450s but
also other enzymes and, as recognized more in
recent years, transporters that alter the concentra-
tions of drugs within cells A discussion of drug
transporters is outside the scope of this chapter,
and the reader is referred elsewhere [57–59]
The subjects of P450 regulation and variation
have already been mentioned and will be treated
again with individual P450s At this point, some
general practical considerations are discussed If
one considers the total concentration of P450 in
liver samples from different healthy individuals
(on a milligram protein basis), most individuals
fall within a range of ~ threefold [28] Howev-
er, when individual “drug-metabolizing” P450s
(eg, families 1, 2, 3) are considered, the varia-
tion is considerable, with five- to tenfold being
common and 40-fold not unusual, eg, P450 1A2
[60] With P450 1A2, a similar variability (40-
fold) is seen in in vivo caffeine pharmacokinetics
[61] With some enzymes, the variability in the
same in vivo pharmacokinetic parameters can be
104-fold (Fig 94)
Two examples of studies of the variability
among individuals are presented in Fig 95 (Cau-
casians) and Fig 96 (Caucasians and Japanese)
Gender has not been shown to have a major influ-
ence on levels of expression of the major xeno-
biotic-metabolizing P450s [64] (with a German
P450 3A4 study seemingly unusual [65]), and in-
ter-gender pharmacokinetic differences are proba-
bly due to other influences of bioavailability [66]
Racial differences exist due to allelic variations,
which may influence either levels of expression
or the inherent catalytic activity of the P450s (eg,
P450 2D6 [67]) Some apparent racial differences
are seen here (Fig 96) and have also been report-
ed in in vivo studies (eg, P450 3A4 [68], P450
2E1 [69]) Controlling diets is an issue in many
in vivo studies of this type, and in vitro studies
can also be affected In general, the differences in
activities of a given P450 between races are much
less than within a race (eg, Fig 96) Finally, the
528 F. P. Guengerich

NAT (1%)
FMO (1%)
MAO (1%)
Esterase (9%)

UGT (15%)

P450 (74%)

a
1A1 (2%) 1A2 (7%)
2B6 (2%)

2C9 (16%)

2C19 (13%)

2D6 (12%)

b 2E1 (3%)

Fig. 9.1   The enzymes of drug metabolism a Contribu- drug metabolism [49] (see also [50]) UGT UDG glucuro-
tions of different enzymes to drug metabolism b Con- nosyl transferase, FMO flavin-containing monoxygenase,
tributions of individual human P450 enzymes to (P450) NAT N-acetyltransferase, MAO monoamine oxidase
 9  Human Cytochrome P450 Enzymes

Fig. 9.2   Relative concentrations of P450 in human liver microsomes a P450s in 60 liver samples were estimated using immunochemical methods (electrophoresis/immunoblot-
ting) [52] Because of cross-reactivity, the individual P450s in subfamilies are not distinguished The “unknown” fraction is the difference between the sum of the immunochemi-
cally determined forms and the total amount, calculated from Fe2 + ·CO versus Fe2 +  difference spectroscopy [53] b–d Estimates were made using liquid chromatography–mass
spectrometry (LC–MS) proteomic analysis with heavy-atom peptides b Results of an analysis of 50 pooled human liver samples (XenoTech, HLM610 preparation) [54] c Re-
sults reported in the same reference as Part B [54] as means from analysis of ten individual human samples d Analysis of a pooled set of 23 human liver samples by another
laboratory [55]
529
530
Fig. 9.3   Relative concentrations of individual P450s in human intestine (determined immunochemically) [56] 3A
indicates all subfamily 3A P450s
point should be made that the levels of the P450s
involved in steroid hormone metabolism (eg,
first column of Table 91) vary considerably less
than do the xenobiotic-metabolizing P450s (fami-
lies 1, 2, 3), probably due to their well-defined
roles in regulation of physiological processes
Many chemicals are capable of inducing
P450s, as clearly demonstrated in animals and
with cell culture systems [70] In vivo induction
experiments with humans are not as readily done
as with animals, but ample evidence for P450 in-
duction is available, going back to the barbiturate
observations of Remmer in the 1950s [2] A short
list of some established P450 inducers is present-
ed in Table 94 This list is rather conservative
in that only information is included from stud-
ies in which in vivo evidence has been obtained
Much of the studies have involved pharmacoki-
netics, but some “moderately invasive” studies
have involved direct measurement of proteins,
messenger RNA (mRNA), or enzyme activi-
ties in peripheral blood cells or small intestinal
biopsies; liver biopsy data are rare Table 94
could probably be expanded considerably if all
information from in vitro studies were included,
eg, P450s 1B1 and 2S1 are probably inducible
by aryl hydrocarbon receptor (AhR) ligands [71,
F. P. Guengerich
72] The major problem in demonstrating human
P450 induction in vivo is the lack of diagnostic
pharmacokinetic parameters for many of P450s
The clinical influence of differences in P450
activity can be rationalized using the scheme of
Fig 97 A list of major drug substrates of each
human P450, from the Indiana University website
(http://medicineiupuiedu/clinpharm/ddis/main-
table/), is presented in Tables 95, 96, and 97
This is intended to be useful but not comprehen-
sive, and of course more drugs will continue to be
added Drug doses are generally developed with
the extensive metabolizers (EMs) as the general
population of major interest, or at least this was
the emphasis in the past The plasma concentra-
tion rises to a peak ( Cp,max) following the first
dose and then decreases to a lower level prior to
the next dose With subsequent doses, the plas-
ma concentration remains within this region and
yields the desired pharmacological effect With-
out prior knowledge about a problem with this
drug, the poor metabolizer (PM; lower panel of
Fig 97) would be administered the same doses
Very limited metabolism would occur between
doses, and the plasma concentration of the drug
(and presumably the concentration of the drug in
the target tissue) will rise to an unexpectedly high
9  Human Cytochrome P450 Enzymes
25
EM
20
15
Frequency
10
UM
5
0
-20 -15 -10 -5
log10 Metabolic Ratio
Fig. 9.4   Frequency distribution histogram of (in vivo)
debrisoquine 4-hydroxylation in a Caucasian population
[62] The metabolic ratio is the ratio of debrisoquine/4-
hydroxydebrisoquine in the urine of individuals who were
administered debrisoquine (10-mg free base) 8 h previ-
ously The groups are designated PM (poor metabolizers,
level, with an attendant increase in the area under
the curve (AUC) The simplest effect would be
an exaggerated (and probably undesirable) phar-
macological response Sometimes there is a situ-
ation in which metabolism is more rapid than ex-
pected in the typical patient (Fig 94), eg, due to
gene amplification or enzyme induction In this
case, Cp,max and AUC would be smaller than in
531
PM
0 5 10 15 20
) and EM (extensive metabolizers, ) The group labeled
UM (ultra-metabolizer) is from retrospective research
[63] and probably represents gene duplication (With kind
permission from Springer Science + Business Media:
[149], Fig 105)
the case of the EM (Fig 97, upper panel), and
decreased drug efficacy would be expected
Some practical situations follow and can be
addressed in the context of our current general
knowledge of substrates, inducers, and inhibitors
(Fig 98, Tables 95, 96, and 97) With regard
to polymorphisms and other variations, several
are known that can render some drugs impracti-
532
Fig. 9.5   Variation in levels of five P450s in 18 human
liver samples Individual P450s and catalytic activities are
indicated on each chart [2768] Sample number refers to
cal due to toxicity (eg, perhexiline, leading to
peripheral neuropathy due to lack of metabo-
lism by P450 2D6 [75]) or can alter the recom-
mended dose (eg, warfarin/P450 2C9 [76–78]
and omeprazole/P450 2C19 [79, 80]) Perhaps
surprisingly, no deaths have been documented
to date due to PM phenotypes (to the author’s
knowledge and in a discussion with Robert
Smith), although it is possible that these have
occurred but not recognized However, a death
of a nursing infant occurred because the mother
was an ultrarapid metabolizer (Fig 94) and the
codeine she used resulted in an overdose of the
P450 2D6 product morphine in her breast milk
[81]
Drug interactions are a serious problem, and
pharmacokinetic interactions have several mo-
lecular bases One is enzyme induction, which
usually results in decreased bioavailability The
decreased bioavailability of a drug can be the
result of induction by that same drug or by an-
other drug A classic example is the decreased
F. P. Guengerich
a code from this laboratory (With kind permission from
Springer Science + Business Media: [149], Fig 101)
bioavailability of the oral contraceptive 17α-
ethinylestradiol following treatment of individu-
als with rifampicin, barbiturates, or St John’s
wort, leading to P450 3A4 induction [26, 82,
83] Another aspect of drug–drug interactions in-
volves P450 inhibition The inhibition can be of a
competitive nature, ie, two substrates competing
for a limiting amount of a P450 or a bona fide in-
hibitor (no enzymatic transformation) competing
with substrates An example here is the antihista-
mine terfenadine, the metabolism of which is in-
hibited by the P450 3A4 inhibitors erythromycin
and ketoconazole Another major type of P450
inhibition is “mechanism-based” (or “suicide”)
inactivation, in which oxidation of a substrate
destroys the P450 [84, 85] An example here is
the inactivation of P450 3A4 by bergamottin and
other flavones found in grapefruit juice [86–89]
In the above cases, the effects have been
discussed only in terms of altered bioavail-
ability; ie, with increased clearance of 17α-
ethinylestradiol, unexpected menstrual bleeding
 9  Human Cytochrome P450 Enzymes

Fig. 9.6   Comparison of some immunochemically determined levels of individual P450s and catalytic activities in human liver microsomes Results from samples obtained from
Caucasian ( C) and Japanese ( J) males ( M), females ( F), and a single neonatal sample ( N) are shown [52] The vertical axis is nmol P450/mg protein in the Total P450 chart and
533

% of total P450 in all other cases (With kind permission from Springer Science + Business Media: [149], Fig 102)
534 F. P. Guengerich

Table 9.4   Some major inducers of human P450 enzymes

Class of inducers Some sources Example Subfamily P450s induceda
AhR ligands Tobacco, broiled meat, accidental Polychlorinated 1A1, 1A2
exposures to pollutants biphenyls
Barbiturates and similar Drugs, some polyhalogenated biphe- Diphenylhydantoin 2C, 3A
compounds nyls, DDT
PXR ligands Some steroids and antibiotics, other Rifampicin 3A
drugs
P450 2E1 inducers Ethanol, isoniazid Ethanol 2E1
AhR aryl hydrocarbon receptor, DDT dichlorodiphenyltrichloroethane, PXR pregnane X receptor
a
Based on in vivo responses

Extensive Metabolizer cause the accumulation of the parent (prodrug)

(EM, normal)
c mias [92, 95] Another possibility is that block-
p,max
Plasma AUC
level of
drug Poor Metabolizer
(PM)
Time (arrows show repeated doses)
Fig. 9.7   Significance of low metabolism of a drug by
P450s (or other enzymes) A “typical” pattern is seen
in the upper panel ( EM), where the plasma level of the
drug is maintained in a certain range when a particular
repetitive dose is prescribed Unusually, slow metabolism
(lower panel, PM) results in an elevated plasma level of
the drug Cp,max = maximum plasma concentration, AUC
area under the curve (Reproduced with kind permis-
sion from Springer Science + Business Media: [149],
Fig 108)
and pregnancies have resulted [83, 90, 91] Some
of the drug interaction problems can be more
complex, even when the analysis is restricted to
pharmacokinetic aspects For instance, in the ex-
ample mentioned above, terfenadine can be con-
sidered a prodrug [92]; in most individuals, the
P450 oxidation (followed by further oxidation)
yields fexofenadine, the circulating (and active)
form of the drug Low levels of P450 3A4 activ-
ity (due to inhibition or other reasons) [93, 94]
terfenadine to toxic levels that can cause arrhyth-
ing a primary route of metabolism of a drug may
favor secondary pathways that lead to toxicity,
eg, blocking phenacetin O-deethylation (P450
1A2) can lead to deacetylation, N-oxygenation,
and methemoglobinemia [96] Although a good
example is not available, it is possible that block-
ing the oxidation of one drug by a P450 could
cause it to accumulate and behave as an inhibitor
towards another A potential example would be
decreasing the P450 3A4-catalyzed oxidation of
quinidine and having the accumulated drug in-
hibit P450 2D6 [97] P450 induction could result
not only in decreased oral availability but also in
the enhanced bioactivation of chemicals This is
a general concern with potential carcinogens, as
discussed in the next section of this chapter, and
one of the reasons why regulatory agencies have
concern about P450 1A inducers
In the process of drug development, there are
several guiding principles to dealing with P450
metabolism, aside from details of each specific
case: (1) use of in vitro screening to eliminate
compounds that will have poor bioavailability
(ie, rapid in vitro oxidation); (2) use of in vitro
screens to avoid obvious problems of toxicity,
induction, and inhibition; (3) searching for drug
candidates in which the metabolism is the result
of several different enzymes and not dependent
upon a single one, particularly a highly variable
P450 (or other enzyme); and (4) use of in vivo
human studies to address in vitro predictions as
early as possible
Table 9.5   Human P450 drug interactions—substrates (Reproduced from http://medicineiupuiedu/clinpharm/ddis/main-table/ with permission from the Indiana University
Division of Clinical Pharmacology)
1A2 2B6 2C8 2C9 2C19 2D6 2E1 3A4,5,7
Clozapine Bupropion Paclitaxel NSAIDs PPIs Beta-blockers Anesthetics Macrolide antibiotics
cyclophosphamide Diclofenac Lansoprazole Enflurane
Cyclobenzaprine Torsemide
Duloxetine Amodiaquine Ibuprofen Omeprazole ( S)-metoprolol Halothane Clarithromycin
Fluvoxamine Efavirenz Cerivastatin Piroxicam Pantoprazole Propafenone Isoflurane Erythromycin
Haloperidol Ifosfamide Repaglinide Rabeprazole Timolol Methoxyflurane (not 3A4)
Imipramine Methadone Oral hypoglycemics Sevoflurane NOT
Mexiletine Antiepileptics Antidepressants Azithromycin
Nabumetone Tolbutamide Amitriptyline Others Telithromycin
9  Human Cytochrome P450 Enzymes

Naproxen Glipizide Diazepam Clomipramine Acetaminophen

Benzene
Olanzapine Angiotensin II blockers Phenytoin Desipramine Chlorzoxazone Anti-arrhythmics
Riluzole Phenobarbitone Imipramine Ethanol
Tacrine Losartin Paroxetine N,N-dimethyl-formamide quinidine→3-OH
Theophyline Irbesartan Others
Tizanidine Amitriptyline Antipsychotics Theophyline
Triamterene Others Clomipramine Haloperidol Benzodiazepines
Zileuton Celecoxib Clopidogrel Risperidone Alprazolam
Zolmitriptan Fluvastatin Cyclophosphamide Thioridazine Diazepam
Naproxen Midazolam
Phenytoin Progesterone Others Triazolam
Rosiglitazone Aripiprazole
Sulfamethoxazole Codeine Immune modulators
Tamoxifen Dextromethorphan
Tolbutamide Duloxetine Cyclosporine
Torsemide Flecainide Tacrolimus
Warfarin Mexiletine (FK506)
Ondansetron
Tamoxifen HIV antivirals
Tramadol Indinavir
Venlafaxine Ritonavir
Saquinavir
535
 Table 9.5  (continued)
536

1A2 2B6 2C8 2C9 2C19 2D6 2E1 3A4,5,7

Prokinetics
Cisapride
Antihistamines
Astemizole
Chlorpheniramine
Calcium channel blockers
Amiodipine
Diltiazem
Felodipine
Nifedipine
Nisoldipine
Nitrendipine
Verapamil
HMG-CoA reductase
inhibitors
Atorvastatin
Lovastatin
Simvastatin
Others
Aripiprazole
Boceprevir
Buspirone
Gleevec
Haloperidol
Methadone
Pimozide
Quinine
Sildenafil
Tamoxifen
Telaprevir
Trazodone
Vincristine
NSAID nonsteroidal anti-inflammatory drug, PPI proton pump inhibitor, HMG-CoA 3-hydroxy-3-methyl-glutaryl-coenzyme A
F. P. Guengerich
Table 9.6   Human P450 inhibitors (http://medicineiupuiedu/clinpharm/ddis/main-table/)
1A2 2B6 2C8 2C9 2C19 2D6 2E1 3A4,5,7
Cimetidine Thiotepa Gemfibrozil Fluconazole Esomeprazole Bupropion Disulfiram HIV antivirals
Fluoroquinolones Ticlopidine Montelukast Amiodarone Fluoxetine Fluoxetine
Fluvoxamine Isoniazid Fluvoxamine Paroxetine Indinavir
Ticlopidine Ketoconazole Quinidine Nelfinavir
Lansoprazole Duloxetine Ritonavir
Omeprazole Amiodarone
Ticlopidine Cimetidine Antimicrobials
Chlor-pheniramine
Clarithromycin
9  Human Cytochrome P450 Enzymes

Doxepin
Haloperidol Itraconazole
Methadone Ketoconazole
Mibefradil Nefazodone
Ritonavir
Others
Erythromycin
Grapefruit
Juice
Verapamil
Suboxone
Diltiazem
Cimetidine
Amiodarone
Fluvoxamine
Mibefradil
Troleandomycin
537
538 F. P. Guengerich

Table 9.7   Human P450 inducers (http://medicineiupuiedu/clinpharm/ddis/main-table/)

1A2 2B6 2C8 2C9 2C19 2D6 2E1 3A4,5,7
Smoking Phenobar- Rifampin Ethanol Carbamaze-
bital pine
Phenytoin Secobarbital Isoniazid Phenobar-
bital
Rifampin Phenytoin
Pioglitazone
Rifabutin
Rifampin
St John’s
Wort
Troglitazone

Fig. 9.8   A summary of major human P450s involved
in drug metabolism, including major substrates, inhibi-
tors, and inducers (adapted from [73, 74] The sizes of
the circles indicate the approximate mean percentages of
the total hepatic P450 attributed to each P450 (see also
Fig 92) The overlap of the circles is to make the point
that overlap of catalytic action is often observed, although
the overlap does not necessarily refer to the indicated sub-
strates (or inhibitors) (With kind permission from Spring-
er Science + Business Media: [149], Fig 104)
9  Human Cytochrome P450 Enzymes
Fig. 9.9   Carcinogen metabolism by human enzymes
[99] a Contributions of different (human) enzyme sys-
tems to carcinogen activation b Contributions of differ-
ent (human) enzyme systems to carcinogen detoxication
FMO flavin-containing monoxygenase, NAT N-acetyl-
Fig. 9.10   Contributions of individual human P450s to the P450 sector of carcinogen activation [99]
9.3 Relevance of P450s in Toxicology
and Cancer Risk
Historically, much of the attention given to P450s
has come from the interest in cancer, going back
to some of the first demonstrations of oxidation
and reduction reactions in the metabolism of
chemical carcinogens [98] and the inducibility
of P450s by carcinogens [1] (Figs 99 and 910)
539
transferase, SULT sulfotransferase, AKR aldo-keto reduc-
tase, COX cyclooxygenase/prostaglandin synthase, UGT
UDG glucuronosyl transferase , GST glutathione transfer-
ase, COMT catechol O-methyl transferase
The interest in P450s was also extended to chem-
ical toxicities other than cancer with the dem-
onstration of bioactivation of compounds such
as the drug acetaminophen [100] and the insec-
ticide parathion [101, 102] Many studies have
been done with P450 animal models, particularly
using P450 inducers and inhibitors and geneti-
cally modified mice, either naturally occurring or
transgenic These studies provide strong evidence
that alterations in the activities of P450s can
540
modify the sensitivity of mice to various chemi-
cals For instance, the Ah locus (which controls
P450s 1A1, 1A2, and 1B1 as well as some con-
jugating enzymes) can modify the sensitivity in
AhR-deficient mice, depending upon the chemi-
cal and the organ site [103] Effects of specific
P450 knockouts have been reported in transgenic
mice as well, eg, prevention of acetaminophen
toxicity by deleting P450s 2E1 and 1A2 [104,
105] and of 7,12-dimethylbenz[a]anthracene-
induced lymphomas by deleting P450 1B1 [106]
When the enzymes involved in the activation
of chemical carcinogens in humans are consid-
ered, two-thirds of the reactions are catalyzed
by P450 enzymes (Fig 99a) [99] Of the human
P450s, six account for 77 % of these reactions
(Fig 910) The three family 1 P450s (1A1, 1A2,
1B1) account for one half of the reactions [99]
Two other points should be made One is that the
reported distributions (Fig 910) are a function of
how many compounds in prominent classes have
been considered That is, P450s 1A2 and 1B1
activate many arylamines, P450s 1A1 and 1B1
activate many polycyclic hydrocarbons, P450
2A6 and 2E1 activate many N-nitrosamines, etc
Therefore, the pattern may change in the future
as other categories are studied more The other
point is that P450s are involved in detoxication
reactions About 14 % of the enzymatic detoxi-
cation reactions are done by P450s (Fig 99b),
including C-hydroxylations, reductions, and N-
oxidations [99]
Despite the strong evidence for effects of vari-
ability of P450 on chemical toxicity and cancer
risk in animals and the knowledge that human
P450 levels vary considerably (Figs 95, 96,
97, and 911), demonstrating relationships with
human disease has been difficult In the 1960s,
the demonstration of the inducibility of aryl hy-
drocarbon hydroxylase (P450 1A1 and possibly
P450 1B1) by Nebert and Gelboin [107] led to
more investigations with human samples, par-
ticularly peripheral blood cells The work of
Shaw and Kellerman [108, 109] suggested that
the inducibility of aryl hydrocarbon hydroxylase
(now recognized as P450 1A1 and 1B1 under
these conditions) is correlated with susceptibil-
F. P. Guengerich
ity of smokers to lung cancer In the early work,
this apparently genetic variability was trimodal
Subsequently, this phenomenon has proven dif-
ficult to study, in part due to technical difficulties
in the earlier phases of the work [110] Many of
the early problems have been circumvented with
the ability to measure mRNA expression and the
access to DNA sequences While evidence for
correlation of P450 1A1 mRNA expression with
lung cancer incidence has been obtained [111],
an unresolved issue is the nature of any genetic
variability In contrast to the situation seen in
mouse models [112], the allelic variations in the
human AhR (which has apparently considerably
lower affinity for many of the ligands of interest
than the mouse receptor [113]) do not appear to
account for interindividual levels of inducibility
of P450 1A1 [114, 115] Epidemiological evi-
dence has been presented for association of lung
cancer incidence with an MspI polymorphism of
P450 1A1 [116] However these results, obtained
in studies done with Japanese, have not been re-
produced in Caucasians [117–119] Further, the
heterologously expressed human P450 1A1 al-
lelic variant (V462I) showed only a relatively
small change in oxidation of the prototypic poly-
cyclic aromatic hydrocarbon (PAH) carcinogen
benzo[a]pyrene [120, 121] A possible explana-
tion to the quandary comes from the report that
P450 1B1, not P450 1A1, is the major P450 re-
sponsible for the aryl hydrocarbon hydroxylation
activity in lymphocytes and that it is P450 1B1
expression that shows the classic trimodality, not
P450 1A1 [122]
Today the search for roles of a particular P450
in human disease follows a route similar to that
just discussed for P450 1A1, ie, the identifica-
tion of SNVs (see earlier note about difference
between variations and polymorphisms, vide
supra) is a basis for epidemiological associations
with various maladies This approach is com-
monly applied to the possible roles of P450s
in cancers at various organ sites The positive
aspects of this strategy are that we have an ex-
tensive knowledge base of allelic variations of
P450s (eg, http://wwwimmkise/cypalleles/),
sophisticated and very sensitive biological tools,
 9  Human Cytochrome P450 Enzymes

Fig. 9.11   Correlation of catalytic activities with immunochemically determined levels of five P450s in human liver microsomes. The correlation coefficients ( r) were determined
using linear regression analysis [52] (With kind permission from Springer Science + Business Media: [149], Fig 107)
541
542
and the potential to noninvasively analyze large
populations, at least in the case of some diseases
and P450s On the negative side, the ability to
rapidly screen for associations without serious
consideration of present or past chemical expo-
sure levels has led to many studies with little or
only marginal biological plausibility Many as-
sociation studies have been difficult to repeat
An example in point is the reported association
of attenuated lung cancer risk (of smokers) with
the P450 2D6 PM phenotype Although the ini-
tial reports were quite exciting [123], subsequent
studies yielded variable results and meta-analysis
has not supported an association [124]; no real
experimental support for a biological association
was ever found [125] A review by Vineis [126]
concludes that the risks of cancer due to genetics
are considerably less than those associated with
smoking or some other environmental factors
What associations of P450 have been ad-
equately demonstrated? The list below is short
and not intended to necessarily be totally in-
clusive but emphasizes some of the more posi-
tive associations found to date (The absence of
several of the steroid-oxidizing P450s is known
to be debilitating, but these are not treated here
(Table 93); see the sections on individual P450s
and reference [47]) The possible association
between P450 1A1 and lung cancer has already
been discussed above; a confounding factor may
be expression of P450 1B1 Truncation of P450
1B1 is associated with glaucoma, for unknown
reasons [127]; this defect has also been seen in
P450 1B1-knockout mice, but the molecular
basis is not known [128] Allelic variants in P450
1B1 do not appear to have major effects in the
oxidation of carcinogens [129]; some differences
in cancer risk have been reported in the epide-
miology literature [130, 131] P450 1A2 activ-
ity has been reported to be associated with colon
cancer incidence, when the factors of N-acetyl-
transferase and well-done meat intake are consid-
ered [132]; an association has plausibility in the
activation of heterocyclic amines by P450 1A2
[31] One of the stronger associations reported to
date involves that of P450 2A6 with lung can-
cer; the association is driven by the data obtained
with individuals with the gene deletion [133] A
F. P. Guengerich
relationship has plausibility in the demonstrated
ability of P450 2A6 to activate N-nitrosamines
(Table 98) and possibly in the decreased smoke
intake of null-type individuals due to impaired
metabolism of nicotine [134] (see Sect 747,
vide infra) Although many epidemiological
studies have been done with SNVs of P450 2E1,
any putative changes in P450 2E1 phenotype
have not been validated with in vivo assays and
must be considered suspect [135]
In the process of drug development, the induc-
tion of P450 family 1 and P450 2B enzymes (in
animals or in human cell or reporter assays) has
often been considered an issue for potential tox-
icity [136, 137] The concern about induction is
that the rodents may be likely to develop liver
or other tumors in cancer bioassays with these
compounds, and any association between these
inductions and human cancer is not established;
eg, epileptics with long-term exposure to bar-
biturates and hydantoins have not been found to
have more cancer [138] Likewise, the induction
of subfamily 4A P450s is an indicator of peroxi-
somal proliferation, a phenomenon associated
with rodent liver tumors but probably not human
[139] Thus, induction of rodent P450s has been
shown to be a means of identifying types of po-
tential rodent toxicity [140], some of which may
be relevant to humans, but should not be used
as evidence for adverse roles of these agents in
humans Transgenic studies with “humanized”
mouse models have provided some insight into
more appropriate risk assessment [141, 142]
9.4 Relevance of P450s in
Endocrinology
Another area that has driven the P450 field is
steroid metabolism (Fig 912) As the structures
of the important steroids were elucidated in the
first half of the twentieth century, it became ap-
parent that the metabolic pathways linking these
were dominated by oxidation and reduction Sub-
sequently, roles of P450s were discovered in the
hydroxylations and even more complex oxida-
tions involving C–C bond scissions One of the
first P450 reactions demonstrated was the steroid
Table 9.8   Some human P450 enzymes involved in the activation of carcinogens [30, 99] (See also Table 910 and Fig 910)
P450 1A1, 1B1 P450 1A2, 1B1 P450 2A6, 2A13 P450 2E1 P450 3A4
Benzo[a]pyrene and other PhIP N,N-Dimethylnitrosamine Benzene Aflatoxin B1
polycyclic hydrocarbons 2-Amino-6-methyl-dipyrido[1,2- (DMN) Styrene Aflatoxin G1
2-Amino-1-methyl- a:3,2’-d]-imidazole N,N-Diethylnitrosamine (DEN) Acrylonitrile Sterigmatocystin
6-phenylimidazo- (Glu P-1) NNK Vinyl carbamate 7,8-Dihydroxy-7,8-dihydrobenzo[a]
4,5-b]pyridine 2-Aminodipyrido- 4-(Methylnitrosamino)- Vinyl chloride pyrene
(PhIP) [1,2-a:3,2’-d]imidazole 1-(3-pyridyl)-1-butanol Vinyl bromide and some other polycyclic hydrocarbons
(Glu P-2) (NNAL) Ethyl carbamate 17β-Estradiol
2-Amino-3-methylimidazo-[4,5-f] Nornitrosonicotine (NNN) Trichlorethylene 6-Aminochrysene
quinoline 4-(Methylnitrosamino)- Carbon tetrachloride Senecionine
(IQ) 1-(3-pyridyl)-1-butanone (NNK) Chloroform 4,4´-Methylene-bis
2-Amino-3,5-dimethyl-imidazo[4,5-f] DMN (2-chloroaniline)
9  Human Cytochrome P450 Enzymes

quinoline (MeIQ) DEN (MOCA)

2-Amino-3,8-dimethyl-imidazo[4,5-f] NNK tris(2,3-Dibromopropyl) phosphate
quinoline (MeIQx) NNAL
3-Amino-1-methyl-5H-pyrido[4,3-b] NNN
indole Butadiene
(Trp P-2)
4-Aminobiphenyl
2-Naphthylamine
2-Aminofluorene
2-Acetylaminofluorene
543
 544

Fig. 9.12   A view of the metabolic pathway of steroidogenesis and the major P450s involved [47] (With kind permission from Springer Science + Business Media: [149],
Fig 1013)
F. P. Guengerich
9  Human Cytochrome P450 Enzymes
aromatase reaction (conversion of androgens to
estrogens) [143] Incidentally, one of the first
(1952) prominent uses of (microbial) P450s was
in the practical synthesis of cortisone by the Up-
john Company [144]
The interest in P450 metabolism of steroids
has been driven by several factors One is that
many steroids are used as drugs, and this section
of the chapter is not independent of the one on
drug metabolism The other driving feature is
inborn errors of metabolism involving steroids
(Table 93) The subject of P450s in steroidogen-
esis and clinical features is treated in more depth
in another chapter in this book [145] and will not
be reiterated here However, the point is made
that genetic deficiencies in the steroid-metaboliz-
ing P450s usually result in obvious clinical phe-
notypes, as opposed to the polymorphisms in the
P450s that metabolize xenobiotics
One example of a genetic problem is P450
21A2, where about 1 in 15,000 births is affected
[145] More than 100 different gene variants have
been identified in individuals presenting at the
clinic The consequences can range considerably
With P450 17A1, ~ 50 different genetic variants
have been identified P450 19A1 insufficiency,
somewhat surprisingly, is fairly infrequent
Some general points should be made here Al-
though androgens and estrogens are often con-
sidered male and female steroids, respectively,
this is not really true Both genders have some
of each, and imbalances cause problems in both
genders Another point is that steroids are not re-
stricted only to a few organs Neurosteroids are
produced by P450s in the brain and other nervous
tissues Placental steroid metabolism is important
to both the mother and child [145]
Finally, some of the steroid-metabolizing
P450s are drug targets themselves, in that pro-
duction of androgens and estrogens is a driving
factor in some tumors Individual P450s will be
discussed below, but suffice it to say for now that
inhibition of estrogen production by P450 19A1
is an important aspect of many chemotherapies
for breast and endometrial cancers [146], and
abiraterone, an inhibitor of P450 17A1, is used
in treatment of androgen-stimulated prostate can-
cers [147]
545
9.5 Approaches to Defining Catalytic
Specificity of Human P450s
Knowledge of the roles of individual P450s in
specific reactions (Fig 98) is critical in the ap-
plication of P450 biochemistry to practical is-
sues in drug metabolism Originally some of the
P450s were purified on the basis of their catalytic
activities towards certain specific drugs [14–16,
21], but even with such a strategy there are the
issues of the extent of contribution of that form
and the involvement of that P450 in other reac-
tions, particularly with new substrates Identi-
fication of the individual P450s contributing to
the metabolism of a new drug candidate is rou-
tinely done in the pharmaceutical industry This
information is often requested by the US Food
and Drug Administration at the time of an IND
(“Investigational New Drug”) application Iden-
tifying P450s involved in oxidations is important
in predicting drug–drug interactions and the ex-
tent of variation in bioavailability In general, it
is desirable to develop drugs for which several
P450s have a contribution to metabolism Drug
candidates that are metabolized exclusively by a
highly variant P450 (eg, 2D6, 2C19) are often
dropped from further development
A combination of methods involving the use
of human tissues and recombinant human P450s
is usually used to identify P450s involved in a
particular reaction, using an approach outlined
earlier [30, 148, 149] A combination of the fol-
lowing methods is usually done, not necessarily
in a particular order Lu has also reviewed these
approaches [150]
9.5.1 Inhibitors
The reaction is demonstrated in NADPH-forti-
fied human liver microsomes (if the reaction of
interest is restricted to another tissue, then this
tissue would be used instead) The effects of se-
lective inhibitors on the reaction are examined A
list of some of the inhibitors that have been used
was presented previously and a revised one is in-
cluded elsewhere in this monograph by Correia
and Hollenberg [85, 151]
546
The choice of substrate concentration is im-
portant in this and some other approaches Ide-
ally the effect of the substrate concentration on
the rate of catalytic activity should be determined
in the absence of inhibitor to determine Vmax and
Km parameters If this information is available,
the inhibition experiments are best done with a
concentration of substrate at or below the Km,
in order to observe the effect of the inhibitor on
the ratio Vmax/Km, which is the parameter usu-
ally most relevant to human drug metabolism If
the Vmax and Km information is not available, an
alternative is to select a substrate concentration
near that expected for the in vivo plasma concen-
tration ( Cp,max or less) Modern mass spectrom-
etry methods have been very useful in pushing
the sensitivity limits
With regard to inhibitor concentration, ide-
ally a range of concentrations would be used
However, if a single concentration of the diag-
nostic inhibitor is used, it must be selected on
the basis of previous literature because nonselec-
tive effects are often observed For instance, α-
naphthoflavone ( α-NF) can inhibit P450s other
than P450 1A2 at high concentrations [152], and
azoles inhibit many P450s at higher concentra-
tions [85] Use of a titration approach (concentra-
tion dependence) has merit [150]
Another general issue is the selection of a pro-
tein concentration Microsomal proteins can bind
drugs in a nonselective manner and effectively
lower the free concentration of substrate or in-
hibitor [153, 154], which can influence the inter-
pretation of results Another point is that the con-
centration of the P450 of interest should be less
than that of the drug and the inhibitor, in order
for the basic assumptions about steady-state ki-
netics to apply (and for the reaction to remain
linear during the incubation time, although some
of the inhibitors are mechanism based and the
loss of activity will be time dependent, requiring
preincubation) A corollary of these latter points,
which also apply to the other approaches that fol-
low, is that having a very sensitive assay method
is very desirable Thus, methods such as high-
performance liquid chromatography–mass spec-
trometry (HPLC–MS) have gained popularity
Finally, the choice of an organic solvent (to
deliver the substrate) is an issue Most P450
F. P. Guengerich
substrates are hydrophobic Ideally the substrate
should be dissolved in H2O or very little organic
solvent, but this may not be possible with many
drugs Several examinations of the effects of
individual solvents on human P450s have been
published [155, 156] Some very hydrophobic
substrates (eg, cholesterol) should be delivered
in cyclodextrins [157]
In principle, the extent of inhibition of a re-
action by a P450-selective inhibitor indicates the
fraction of that reaction attributable to that P450
For instance, if a 1 µM concentration of quinidine
(a P450 2D6 inhibitor) inhibits 50 % of a reac-
tion, then 50 % of that reaction may be attributed
to P450 2D6 To obtain a more global view than
possible with a single liver sample, a pooled set
of microsomes (e.g., from ≥ 10 samples, balanced
on the basis of liver weight or protein) is gener-
ally used for the inhibition assays However, if
one desires to examine the differences among in-
dividuals in terms of the contribution of a P450,
then doing several experiments with individual
liver samples is the approach to use
9.5.2 Correlations
Another approach with a set of human tissue mi-
crosomal samples is to measure the new reaction
of interest in each and attempt correlation with
rates of marker activities (for individual P450s)
[25] Lists are also published in this monograph
by Correia and Hollenberg [85] and elsewhere
[158, 159]
Correlation can be done by plotting the specif-
ic activity for the new reaction versus the marker
reaction (Fig 911) In principle, the correlation
coefficient r2 estimates the fraction of the vari-
ance attributable to the relationship between the
two activities, ie, the fraction of the activity cat-
alyzed by the particular enzyme (assuming that
all of the marker activity is catalyzed by this en-
zyme) In some cases, excellent correlations have
been reported [26, 60] An alternative method of
analysis is a Spearman rank plot, which has some
deficiencies but avoids the overweighting of un-
usually high or low values [27]
Although the approach works well when high
correlation coefficients are generated, the method
9  Human Cytochrome P450 Enzymes
is less useful when several P450s contribute to a
reaction, ie, r2 < 04 The results should, in all
cases, be considered in the context of results ob-
tained with other approaches
9.5.3 Antibody Inhibition
The points raised in the above section, Inhibitors,
apply to antibodies as well Antibodies are used
to inhibit activities in human liver (or other tis-
sue) microsomes and are of several general types:
(1) polyclonal antibodies raised against purified
animal P450s, (2) polyclonal antibodies raised
against purified human P450s, (3) monoclonal
antibodies raised against purified human P450s,
(4) polyclonal antibodies raised against peptide
fragments of P450s, and (5) antibody phage dis-
play library antibodies selected for recognition of
individual P450s
At this time, almost all antibodies raised
against intact P450s have been generated using
recombinant P450s (or against peptides), in con-
trast to early work in the field with P450s iso-
lated from liver and other tissues Another point
to make is that not all antibodies inhibit catalytic
activity Further, specificity in one immunochem-
ical assay (eg, electrophoretic/immunoblotting)
does not necessarily implicate specificity in an-
other (immunoinhibition)
Three points should be made in designing im-
munoinhibition experiments (1) The concentra-
tion of antibody should be varied and increased
to the point where the extent of inhibition is
constant (2) A nonimmune antibody should be
used as a control, using the same concentrations
as with the antibody raised against the P450 (3)
The antibody should be shown not to inhibit reac-
tions known to be attributable to other P450s Im-
munoglobulin G fractions are generally preferred
in that they produce less nonspecific inhibition
than crude preparations such as sera Polyclonal
antibodies can vary in their specificity and titer
from one animal to another and from one bleed to
another, so constant properties cannot necessarily
be assumed In principle, monoclonal antibodies
and antibodies eluted from phage display librar-
ies should not vary (among individual prepara-
547
tions), although this has not always been the case
with monoclonals
In general, antibodies are often selective for
individual P450 families/subfamilies, eg, 1
versus 2A versus 2B versus 2C, etc, but cross-
reaction among families can be detected, and in
some cases the (P450) sites of cross-reactivity
have been identified [160] Achieving selectiv-
ity among individual P450 subfamily members
(eg, P450 3A4 versus 3A5 versus 3A7) is more
difficult With polyclonal antibodies, this can be
achieved by cross-absorption [161]; with mono-
clonals and phage display libraries, this can be
done by selection The point should be made that
any selectivity demonstrated among classes of
animal P450s (eg, rat P450 families) cannot be
assumed to carry over to human P450s
Anti-peptide antibodies have become popular
in recent years and have two major advantages:
(1) peptides can be synthesized and readily puri-
fied by HPLC, avoiding the need to express and
rigorously purify P450 proteins (although dem-
onstration of purity by HPLC, capillary electro-
phoresis, and mass spectrometry is still in order),
and (2) peptides can be selected for use as anti-
gens by sequence comparisons, favoring specific
regions
Phage display antibody libraries are relatively
new and have been used in a few P450 appli-
cations to date [162] These have a number of
advantages, including potential selectivity due
to the large number of potential antibodies in li-
braries, the ability to avoid animal protocols, the
immediate availability of libraries (as opposed
to waiting on animals to develop antibodies),
the consistency of reproduction of the proteins
propagated in bacterial systems, and the ability
to include a second “epitope tag” for recovery
9.5.4 Demonstration of Reaction with
Recombinant P450
In early work in this field, this point would have
been the demonstration of the reaction of inter-
est with an enzyme purified from tissue Today
P450 proteins are generally produced in recom-
binant systems and seldom purified from tissue
548
sources In routine practice in the pharmaceuti-
cal industry, new reactions are examined with a
battery of the major recombinant human (liver)
P450s, many of which are available from com-
mercial sources Systems used for expression
include bacteria, yeast, baculovirus (-infected
insect cells), and mammalian cells The P450
need not be purified for these comparisons but
must have suitable provision for NADPH-P450
reductase in a crude system (and cytochrome b5
in certain cases)
Usually activity results obtained with several
of the major P450s are compared to each other
and to those obtained with tissue microsomes,
in order to put the work in context Ideally as-
says are done at several substrate concentrations
and the parameters kcat ( Vmax) and kcat/Km are ob-
tained These values should be normalized on the
basis of P450 concentration, in that any values
based on mg protein for the expression system
cannot be used for comparisons with tissue mi-
crosomes In principle, the kcat (total P450 basis)
should be at least as high for the recombinant
reaction than the tissue microsomes A more re-
alistic way to make a comparison is to immuno-
quantify the amount of the particular P450 in the
tissue microsomes and then use this value in cor-
recting the microsomal kcat for comparison to the
recombinant system The matter of scaling these
parameters to generate predicted microsomal (or
in vivo) rates from in vitro experiments with re-
combinant enzymes is not trivial, but there has
been considerable progress in this area and there
is commercial software in wide use [163]
9.6 Interindividual Variation
9.6.1 Genetic
Variability in patterns of drug metabolism has
been recognized for some time, even before
the discovery of P450s For instance, the field
of pharmacogenetics had been identified by the
1950s [44, 164] and the early work of Remmer
[2] showed the influence of barbiturates upon
drug metabolism Further, a number of congeni-
tal defects in steroid metabolism were known and
F. P. Guengerich
some could be attributed to alterations in specific
hydroxylations [165] Much of the subsequent
work on inducibility has been done in experimen-
tal animal models [1] and, later, in cell culture
In the 1960s and 1970s, a number of accounts
appeared describing variations in rates of metabo-
lism of drugs in human liver biopsy samples [28]
The first characterization of a monogenic vari-
ability in a human drug-metabolizing P450 was
the work of Smith with debrisoquine [11], as well
as Tucker and Lennard [12], which was paral-
leled by the work of Dengler and Eichelbaum on
sparteine [13] This polymorphism was first de-
scribed in the context of EMs and PMs (Fig 94)
[62, 63] These polymorphisms were first studied
at the level of the phenotype, ie, pharmacoki-
netics and in some cases unusual responses to
drugs due to reduced metabolism [166] The area
of pharmacogenetics (now expanded to “pharma-
cogenomics”) was facilitated by the identifica-
tion of the P450 enzymes involved in the drug
metabolism phenotypes and particularly by the
development of molecular biology, which al-
lows the precise characterization of genetic dif-
ferences between individuals The majority of
the allelic differences are SNVs, or single base
changes As anticipated from previous knowl-
edge of pharmacoethnicity, many of these SNVs
and polymorphisms show racial linkage (Again,
a polymorphism is generally defined as a ≥ 1 %
frequency of an allelic variant in a population;
below this frequency, the term “rare allele” is
applied or, in the case of a very detrimental al-
lele, a mutant or “inborn error of metabolism”
Therefore, as mentioned earlier, the terms “vari-
ant” and “SNV” will be used to include both, not
distinguishing for frequency)
The debrisoquine polymorphism is now well
understood in terms of P450 2D6 and has been
a prototype for research in this area The char-
acterization of the gene [34] yielded a basic un-
derstanding of the PM phenotype The incidence
of the PM phenotype is ~ 7 % in most northern
European populations, with different phenotypic
incidence (and SNVs) in other racial groups [62,
67, 167, 168] More than 160 allelic variants are
now known, and 98 % of the PMs in northern Eu-
ropean populations can be accounted for by four
9  Human Cytochrome P450 Enzymes
variant alleles [67, 169] A nomenclature system
has been set up for P450 alleles (using the suffix-
es *1 (where *1 is the “wild type,” or most com-
mon gene), *2, *3…) and is maintained at http://
wwwcypalleleskise Reference to this database
will be made with most of the individual P450s
Several P450 2D6 allelic variants clearly lead
to the PM phenotype, for a variety of reasons A
relatively rare case is a gene deletion (*5) [170]
The most common (Caucasian) PM phenotype
is an SNV that leads to aberrant RNA splicing
(ie, in splice site) and no mRNA or protein (*4)
Other alleles involve deletions (eg, *5), frame-
shifts (eg, *3A), and coding for proteins with
either intrinsically low catalytic activity or insta-
bility (reduced half-life) These general patterns
have been seen in other P450s (and other genes)
In addition to the EM and PM phenotypes, there
is also a “very extensive metabolizer” (or “ultra-
metabolizer,” UM) phenotype (Fig 94), due to
gene duplication (*2XN) A Swedish family was
identified with 13 gene copies, in principle lead-
ing to 13 times more enzyme [63] The level of
hepatic P450 2D6 and a parameter of in vivo
debrisoquine metabolism (the urinary metabolic
ratio = urinary debrisoquine/4-hydroxydebriso-
quine) vary ~ 104-fold among people (Fig 94)
With P450 2D6 and several other P450s, the al-
leles describing the most commonly observed
high and low levels of metabolism have been de-
scribed, but the kinetic parameters for many of
the alleles have not been determined by heterolo-
gous expression and measurements This is still
the general case with most of the human P450s
P450 2D6 is regulated by a hepatic nuclear factor
(HNF) element [171] but is not considered to be
inducible by xenobiotics With many other P450s,
there is regulation and variability due to noncod-
ing region SNVs, levels of inducers consumed,
and interactions between P450s and transporters
such as P-glycoprotein [66, 172] may influence
the phenotype
Although the level of P450 2D6 may have a
dramatic effect on the metabolism of certain drugs
(Fig  94), no other striking biological changes
have been reported in PMs (but see some of the
epidemiology under Sect 7127) This appears to
be the general case for many of the hepatic P450s
549
primarily involved in the metabolism of xenobi-
otics, and few observable physiological effects
have been reported in transgenic mice in which
these genes have been deleted [128] As pointed
out earlier, however, deficiencies in some of the
steroid-hydroxylating P450s can be very debili-
tating or lethal [145, 165] In general, the varia-
tion in the levels of these “more critical” P450s
is limited in most of the population, compared to
the xenobiotic-metabolizing P450s in which an
order of magnitude variation is not unusual [52]
Another general point to make is that, in con-
trast to some animal models [173], human P450
expression shows little if any gender differences
[64]
9.6.2 Environmental Variation
Interindividual variability of P450 activity can be
due to genetics or to environmental factors, ie,
anything that is not genetic These factors also
give rise to intraindividual variations, which can
be equally important in predicting how an indi-
vidual will respond to a drug These variations
may be caused by drugs, food, tobacco, alcohol,
and other influences The three major issues here
are enzyme induction, downregulation, and inhi-
bition These topics are dealt with elsewhere in
the book and will only be discussed briefly, in-
sofar as they relate to human P450s One other
topic, enzyme stimulation, is also discussed
below
When developmental differences are seen in
humans, they tend to be relatively soon after birth
(eg, P450 3A4, 3A7 [174, 175]), and changes
in expression in the elderly have not been very
dramatic [176–178]
9.6.2.1 Induction
Induction is a process that is relatively common
among the P450s involved in the oxidation of xe-
nobiotic chemicals (second column of Table 91)
The overall process can be seen as an adaptive
one, at least in some cases, in which a person
responds to a chemical in the environment by
synthesizing an enzyme to metabolize that com-
pound or a set of similar ones
550
The general model is one of transcriptional
regulation, based on a paradigm developed for
the steroid nuclear receptor family (Fig 913),
which is considered in more detail in Chap 10
A ligand is bound to a cytosolic receptor, which
facilitates heterodimer formation with another
protein This complex then translocates to the
nucleus and binds to a specific nucleotide se-
quence (5ʹ) upstream of the P450 structural gene.
Coactivator proteins are often recruited to the
complex This process has the net effect of chro-
matin remodeling and opening the promoter site
to allow RNA polymerases binding and initiation
of transcription
Several major systems are known to be in-
volved with (human) P450s The AhR system
involves the AhR and AhR nuclear transporter
(ARNT) proteins, regulating P450s 1A1, 1A2,
1B1, and 2S1 The constitutive androgen recep-
Fig. 9.13   Generalized model for regulation of P450
genes by induction L ligand, R receptor, R′ partner pro-
tein for heterodimer of R, Coactiv coactivator, RNA pol
F. P. Guengerich
tor (CAR) dimerizes with the retinoid X receptor
(RXR), which is loaded with retinoic acid CAR
can bind a strong ligand (eg, 1,4-bis[2-(3,5-
dichloropyridyloxy)]benzene (TCBOPOP)) but
usually acts without a ligand Recent evidence
indicates that phenobarbital, the classic barbitu-
rate inducer, binds to the epidermal growth factor
receptor (EGFR), leading to a cascade of extra-
cellular signal-regulated kinase (ERK) phos-
phorylation, and then dephosphorylation of CAR
(at Thr-38) leads to transport of the CAR–RXRα
complex to the nucleus and gene activation
[179] This process induces subfamily 2C and
2B P450 genes, plus possibly some others The
pregnane X receptor (PXR) binds a number of
steroids, drug, and other ligands and, like CAR,
heterodimerizes with retinoid-activated RXRα,
moves to the nucleus, and activates the transcrip-
tion of P450 subfamily 3A genes, particularly
RNA polymerase (With kind permission from Springer
Science + Business Media: [149], Fig 106)
9  Human Cytochrome P450 Enzymes
P450 3A4 The peroxisomal proliferator-activat-
ing receptor α (PPARα) binds fatty acids and a
number of hydrophobic drugs, heterodimerizes
with retinoid-activated RXRα, and induces P450
4A11 and 4X1 [180]
Some of the steroid-oxidizing P450s are regu-
lated by adrenocorticotropic hormone (ACTH)
and cyclic adenosine monophosphate (AMP)
pathways [181]
Evidence has been presented that some P450s
are regulated at post-transcriptional levels, in-
cluding stabilization of mRNA or protein [182]
The regulation of P450 2E1 is extremely com-
plex, at least in animal models [183, 184] Sev-
eral reports of epigenetic regulation of P450s
have appeared, including gene methylation (eg,
P450s 2A13, 2E1, 2R1, 5A1, 8A1, 19A1, 24A1,
27A1, 27B1, 2W1 [185, 186]), microRNAs (eg,
P450s 1B1, 2E1, 3A4, 24A1) [187], and histone
acetylation (eg, P450s 2A13, 2E1, 46A1 [188])
9.6.2.2 Downregulation of P450s
It should be pointed out that several of the P450s
can be downregulated by cytokines, and the re-
sult has practical significance in the impairment
of drug metabolism in individuals with colds or
flu or who have received vaccinations [189]
Another phenomenon observed in rat models
is the downregulation of some constitutive P450s
by the same chemicals that induce others, eg,
phenobarbital and 3-methylcholanthrene [190]
The mechanism of this response is at the tran-
scriptional level [191] but beyond this mecha-
nism remains unknown Whether this phenom-
enon is operative in humans (in vivo) is also
unknown
9.6.2.3 Inhibition
The subject of P450 inhibition is also treated
separately in this book (Chap 5) [85], and this
section is brief, focused on human P450s A rela-
tively extensive set of P450 inhibitors is now
available, and many of these can be used in a
diagnostic way for “reaction phenotyping,” ie,
identifying which P450s catalyze a newly discov-
ered reaction in tissue microsomes (see Sect 5)
Inhibition of human P450s is an important
practical matter on drug discovery/development
551
and in clinical practice (Table 96) (medicine
iupuiedu/clipharm/ddis) P450 inhibition has the
same effect as a genetic deficiency (attenuation of
drug metabolism, leading to enhanced pharmaco-
logical response), but can be even more problem-
atic because of temporal changes For instance,
some drugs can produce a delayed response for
various reasons, and the pharmacokinetics of a
drug (substrate) may vary with time Another im-
portant point is that not all human P450 inhibitors
are drugs For instance, an inhibitor in grapefruit
(bergamottin) explains the interaction with P450
3A4 [88] A number of herbal medicines contain
P450 inhibitors that attenuate drug metabolism
[192]
9.6.2.3.1  Reversible Inhibitors
Competitive inhibitors are common They act by
binding in the active site, in competition with the
substrate For instance, two substrates of P450
2D6 would be expected to compete for access to
the area surrounding the iron atom This behavior
is described by the simple equation
[S ]
v = Vmax ⋅ + [ S ]
 [I ] 
K m 1 + 
 KI 
Noncompetitive inhibition is the result of an en-
zyme interaction of a ligand at a site other than
the substrate-binding site The equation
1 1  [ I ]  Km  [ I ]  1
= 1 + + 1 + 
v Vmax  K I  Vmax  K I  [ S ]
indicates that the Km will not change but the Vmax
( kcat) will
In uncompetitive inhibition, the inhibitor com-
bines with only the ES form of the enzyme, and
the inhibitor constant KI is based on the interac-
tion of the inhibitor with this complex,
so that
1 1  [ I ]  Km 1
= 1 + + ⋅ ,
v Vmax  K I  Vmax [ S ]
552
and in a classic Lineweaver–Burk double recip-
rocal plot (1/v versus 1/[S]), two parallel lines are
obtained, ie, both Vmax and KI change [193]
In practice, the most common type of re-
versible inhibition relevant to human P450s
and drug metabolism is the competitive mecha-
nism Uncompetitive inhibition is very rare; one
(non-P450) example is the inhibition of steroid
5α-reductase by the drug finasteride [194] An
example of noncompetitive inhibition is that of
cholesterol blocking the oxidation of nifedipine
and quinidine by P450 3A4, even though choles-
terol is also a substrate for the enzyme [157]
9. 6. 2. 3. 2  Irreversible Inhibition
For several reasons these mechanisms are com-
monly seen in P450 reactions In a sense, they are
more problematic than competitive inhibitors, in
that the inhibition is more persistent, ie, the en-
zyme is generally inactivated and activity will
not be restored until new synthesis is completed
Metabolite Intermediates
Metabolite intermediate complexes are formed
by the oxidation of amines to C-nitroso com-
pounds or from oxidation of methylene dioxy-
phenyl compounds to carbenes [195] These bind
extremely tightly to ferrous P450 iron Both of
the bound forms are characterized by their 455-
nm absorption bands, which can be produced in
in vitro experiments A classic example is seen
with troleandomycin (TAO) and P450 3A4 [196]
These complexes can be disrupted by K3Fe(CN)6
oxidation of the iron (in vitro)
Covalent Binding
Covalent binding, where σ chemical bonds are
formed, is the result of the generation of electro-
philic species in the course of P450 oxidation of
compounds The binding may occur to the heme,
the apoprotein, or both (ie, cross-linking, a rare
but documented event [197, 198]) A number of
chemical moieties are notorious for such mech-
anism-based inactivation, including acetylenes,
some terminal olefins, and cyclopropylamines
[199] The destruction of heme is probably due
to very transient species that are generated dur-
ing catalysis and do (usually) not leave the en-
F. P. Guengerich
zyme In many cases, the covalent binding to
protein is restricted to the P450 that activates the
compound, which is one of the marks of an en-
zyme intermediate However, in some cases there
may be reactions with the P450 and with other
proteins In this case, the reactive products are
long-lived and there is concern not only about the
(P450) enzyme inhibition but also potential tox-
icity due to modification of other proteins
Along with a chapter on P450 inhibition
(Chap 5) [85], inhibitors of each human P450
are discussed in the appropriate Sects (7X6) of
this chapter (X indicates each of the 57 P450s)
9.6.2.4 Stimulation
Enzyme stimulation is an increase in enzyme
activity resulting directly from the addition of a
chemical This is a somewhat unusual phenom-
enon in enzymology, usually relegated to classi-
cally allosteric systems [200] The concept is that
a chemical stimulates the catalytic activity of an
enzyme This cooperativity may be considered in
two aspects One is homotropic cooperativity, in
which a chemical stimulates its own biotransfor-
mation This is usually manifested in sigmoidal
(S-shaped) plots of v versus S Heterotropic co-
operativity is the stimulation of catalytic activity
by direct addition of a different compound
Both of these phenomena have been observed
with P450s in vitro Heterotropic stimulation was
reported with animal-derived P450 systems [201,
202] and then human systems [203] Homotropic
cooperativity was reported later, actually first
with human systems [204, 205] Homotropic co-
operativity can be shown in hepatocytes [206],
but it may be unrealistic to observe this phenom-
enon in vivo Evidence for in vivo cooperativity
comes from a number of studies with experimen-
tal animals [202, 207] Whether this phenomenon
presents itself clinically is unknown It would not
generally be desirable in that the effects on phar-
macokinetics would be rather unpredictable
At least four pieces of evidence suggest that
such behavior is possible: (1) homotropic coop-
erativity has been reported in hepatocyte cultures
[206]; (2) an early experiment with neonatal
mice (individual P450s unknown) by Conney’s
group indicated the immediate enhancement of
9  Human Cytochrome P450 Enzymes
an activity by flavones [202]; (3) the work of
Slattery and Nelson with rats shows interaction
between caffeine and acetaminophen that imply
such behavior [208]; and (4) quinidine enhanced
the in vivo oxidation of diclofenac in monkeys,
in a manner consistent with in vitro human work
[207, 209] The first example (hepatocytes) re-
lates to homotropic cooperativity, but this would
be very hard to demonstrate in vivo, except per-
haps in the interpretation of unusual nonlinear
pharmacokinetics, if induction can be ruled out
The other three (in vivo) are cases of heterotropic
cooperativity If stimulation does occur in vivo,
it is a phenomenon that has been very difficult
to predict (even in vitro), and in the case of P450
3A4 substrates, the situation would probably be
further complicated by issues involving P-glyco-
protein behavior (and P-glycoprotein also shows
cooperativity of its own [210])
The mechanistic basis of P450 stimulation has
been studied extensively Some aspects of P450
stimulation will be treated under the topic of
P450 3A4 (Sect 7204), with which much of the
work has been done An open question is whether
such behavior occurs in humans Many classic al-
losteric enzymes have distinct regulatory sites for
binding chemicals, but to date there has been no
clear evidence for this One of the early proposals
was that the second ligand fits into the canoni-
cal active site, near the substrate [205] This view
was advanced in a number of indirect studies that
supported the concept [211, 212] Although a
number of different (human) P450s have exhib-
ited cooperative behavior, much of the emphasis
has been on P450 3A4 This was the first human
P450 to show heterotropic cooperativity [204,
205] In addition, its broad substrate specificity
allows the examination of more chemicals, both
substrates and effector molecules Although there
had been many postulates of multiple ligand oc-
cupancy in P450 3A4, this was first demonstrat-
ed with X-ray diffraction, ie, two ketoconazole
molecules in P450 3A4 [213]
The physical presence of two ligands in an ac-
tive site can be readily linked to sigmoidal kinet-
ics if activity towards the substrate is dependent
upon the presence of two substrates [214]
553
Two X-ray structures of P450 3A4 have re-
ported a single steroid molecule bound at a pe-
ripheral site [215, 216], although the relationship
to function is unclear Evidence from this labo-
ratory [217, 218] and others [219] has provided
evidence that binding of at least some substrates
to P450 3A4 involves rapid binding to a periph-
eral site followed by a slower movement to the
heme area Evidence for a similar course of sub-
strate movement has been observed with P450s
1A2 and 19A1 [214, 220]
Two-ligand occupancy of a P450 active site,
with the ligands stacked together, has now been
observed with bacterial P450s 107 [221] and
158A2 [222] and human P450s 2C8 [223], 3A4
[213], and 21A2 [42] The case that coopera-
tivity is due to multiple-ligand occupancy now
has physical support, but the question arises as
to why cooperativity has not been seen in P450s
that do have two ligands, eg, P450 2C8 [223]
(With P450 21A2, there was no evidence for co-
operativity but there was for two affinities [42])
9.7 Individual Human P450 Enzymes
Each of the 57 human P450 genes/gene products
will be covered here Clearly much more infor-
mation is available about some than others Points
to be covered with each, when possible, include
sites of expression and relative abundance, regu-
lation, genetic variation, substrates and reactions,
structure, inhibitors, and clinical issues It must
be emphasized that this chapter is not intended to
be comprehensive, and the literature accumulates
rapidly; the reader is encouraged to do further lit-
erature searches for each P450 of interest
9.7.1 P450 1A1
9.7.1.1 Sites of Expression
The gene has seven exons, and the cDNA region
is ~ 70 % identical to that of the closest relative,
P450 1A2 P450 1A1 is expressed in fetal liver but
not at appreciable levels in adult liver [224–226]
P450 1A1 can be induced in primary human he-
554
patocyte cultures [227] The dominance of he-
patic P450 1A2 over 1A1 in vivo may be due to
preferential induction of P450 1A2 > 1A1 at low
doses of inducers (a phenomenon established in
rats [228]) or to the presence of factors in liver
that are not preserved in hepatocyte cultures
P450 1A1 is expressed in human lung and was
partially purified [18] One estimate of a median
level of P450 in human lung [229] was 60 pmol
P450 1A1 in nonsmokers’ lungs ( n = 7), 16 pmol/
mg in smokers ( n = 18), and 19  pmol lung pro-
tein in ex-smokers ( n = 7). The variation in levels
of P450 1A1 is very high (> 100-fold) [18, 229],
as suggested from earlier work in which only
benzo[a]pyrene hydroxylation was used as an
indicator [230]
P450 1A1 is also expressed in placenta [231]
and peripheral blood cells (lymphocytes, mono-
cytes) [232], and these tissues have been used in
many studies Expression (at least at the mRNA
level) has been reported in a number of other ex-
trahepatic tissues, including pancreas, thymus,
prostate, small intestine, colon, uterus, and mam-
mary gland [233]
Another aspect of P450 1A1 expression in-
volves mitochondria P450 1A1 has both endo-
plasmic reticulum and mitochondrial-targeting
domains [234] and distributes into both organ-
elles, utilizing adrenodoxin for functional elec-
tron transfer in the mitochondria
9.7.1.2 Regulation
The induction of P450 1A1 has been studied ex-
tensively and has been discussed elsewhere in this
series [235] Briefly, the AhR resides in the cyto-
sol and, when activated by binding of an appro-
priate agonist, loses the accessory protein Hsp90
and dimerizes with the ARNT protein, moving to
the nucleus and interacting with a xenobiotic-re-
sponsive element (XRE) to initiate transcription
(Fig 913, with R = Ah receptor, R1 = ARNT, and
L = 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
or other inducer) A number of details regarding
this scheme remain to be elucidated, eg, roles
of coactivators, whether an endogenous ligand
exists, and if so what it is The list of inducers re-
ported from in vitro studies includes TCDD and
is quite long The list of compounds for which in
F. P. Guengerich
vivo evidence of induction is more limited but
is generally accepted includes cigarette smoke,
heterocyclic amines, polychlorinated biphenyls
[236], and some drugs (eg, omeprazole [237])
At least six human AhR genetic variants have
been identified and found to vary in functional
activity but surprisingly (based on mouse work)
only ~ two-fold [238]
In Michigan Cancer Foundation-7 (MCF-
7) breast cancer cells, regulation of P450 1A1
(via AhR) is dependent on the Ca2 +/calmodulin/
CaMKIα pathway [239] Epidermal growth fac-
tor (EGF) has been reported to downregulate
AhR in human keratinocytes [240, 241] There
is also cross talk of AhR systems with the estro-
gen receptor (ER)α [240] CAR transcriptionally
activates both P450 1A1 and 1A2 genes through
a common 5ʹ-flanking region regulatory element
[242] Liver X receptor α (LXRα) also regulates
human P450 1A1 [243] In (human) HepG2 cells,
P450 1A1 gene regulation by ultraviolet (UV)
light (UVB) involves cross talk between AhR
and the nuclear factor NFκB [244], which also
has relevance to an inflammatory response [245]
and possibly humans
An unusual mechanism of regulation involves
inhibition of the clearance of an endogenous AhR
agonist, 6-formylindolo[3,2-b]carbazole (FICZ;
a tryptophan photodegradation product), as a
mechanism for activating AhR [246] Another
unusual regulatory mechanism, demonstrated
only in mice thus far, involves activation of
AhR by modified low-density lipoprotein (LDL)
[247] Finally, 1-nitropyrene has been reported
to stabilize mouse P450 1A1 mRNA via an Akt
pathway [248]
9.7.1.3 Genetic Variation
Currently at least 13 alleles are known, plus an-
other seven single nucleotide polymorphisms
(SNPs) in which the haplotype has not been de-
termined (http://wwwcypalleleskise)
As mentioned earlier, there is also informa-
tion available about genetic variation in the AhR,
which controls P450 1A1 transcription [238]
Polymorphism in the inducibility of benzo[a]-
pyrene hydroxylation activity has attracted con-
siderable interest following the early reports of
9  Human Cytochrome P450 Enzymes
Shaw and Kellerman [108, 109] that the induc-
tion in lymphocytes of smokers can be associated
with susceptibility to lung cancer The link to lung
cancer has been studied extensively, but few gen-
eral conclusions can be reached Smoking clearly
induces levels of lung P450 1A1 [111, 229, 249]
Some epidemiological investigations have linked
the *2A ( MspI) and *2B (I462V) polymorphisms
to lung cancer incidence in Japanese [116], but
this association has not been reproduced in other
studies with Caucasians [117, 118] These two
alleles are in linkage disequilibrium [119] Two
studies with recombinant human P450 1A1 have
not shown a major difference in any catalytic
activities due to the substitution at codon 462
[120, 121] Although there is a general consensus
that phenotypic variation in the inducibility of
P450 1A1 is observed, extensive searches have
not associated the inducibility with any known
polymorphisms in the P450 1A1, AhR, or ARNT
genes [250, 251]
9.7.1.4 Substrates and Reactions
This enzyme was first explored in the con-
text of an aryl hydrocarbon hydroxylase, using
fluorescence assays that measure primarily the
3-hydroxylation of benzo[a]pyrene [107] (It
should be noted that the fluorescence assay
also picks up other fluorescent products, eg,
9-hydroxybenzo[a]pyrene, and that other P450s
also catalyze the 3-hydroxylation reaction, eg,
P450 2C9 in human liver [252]) Another clas-
sic model reaction used for P450 1A1 is 7-eth-
oxyresorufin O-deethylation [253, 254], but a
number of other P450s also catalyze this reac-
tion Human P450 1A1 oxidizes benzo[a]pyrene
to a variety of products [255, 256] Many other
polycyclic hydrocarbons are substrates for P450
1A1 and have been studied extensively [257,
258] (Fig 910) Some heterocyclic and aro-
matic amines can also be activated by P450 1A1
(Fig  910) [259] P450 1A1 does not appear to
play a major (in vivo) role in the metabolism of
many drugs, possibly because of its locations of
expression
Human P450 1A1 activates aminomethyl-
phenylnorharman, a fusion product of norharman
[260], but not as well as P450 1A2 P450 1A1
555
is involved in the detoxication of the important
toxic natural product aristolochic acid [261]
The EGFR antagonist erlotinib is activated to a
reactive electrophile by human P450 1A1 [262]
Some substituted benzothiazole compounds can
be activated (quinones, N-hydroxylation) by
human P450 1A1 [263]
TCDD and other dioxins can be oxidized (al-
beit slowly) by P450 1A1 enzymes, but rat P450
1A1 is more active than human P450 [264] 1-Ni-
tropyrene is deactivated by P450 1A1, to a prod-
uct that does not induce the tumor suppressor p53
[265]
Cytochrome b5 has generally been considered
not to stimulate P450 1A1 [266], but some ex-
amples have been published [267]
9.7.1.5 Structure
Early work on pharmacophore models for rat
P450 1A1 was done by Jerina’s group [268]
Some homology modeling was done by Lewis
[269] The lack of effect of interchanging Val and
Leu at position 462 has already been mentioned
[120, 121]
An X-ray crystal structure of human P450
1A1 has been published by Scott and her associ-
ates [270] Because the structure contains α-NF,
it can be compared directly with the structures
of the related proteins P450 1A2 [271] and P450
1B1 [272] The planar region of α-NF is packed
flat against the I-helix, with the 2-phenyl substit-
uent oriented towards the iron atom of the heme
π–π stacking with Phe-224 was observed [270]
As in the case of P450 1A2 (Sect 725, vide
infra), α-NF has the site of oxidation (5,6-epoxi-
dation [256]) furthest away from the iron atom
and the observed P450 1A1 α-NF structure is
presumably not a catalytically productive com-
plex Docking studies could place α-NF in a jux-
taposition to explain the oxidation [270] (which
is known to be slow, but faster with P450 1A1
than P450 1A2 [256])
A combinatorial approach has been used to
“mix” human P450 1A1 and 1A2 to define resi-
dues that contribute to the “identity” of each of
these two P450s [273]
556
9.7.1.6 Inhibitors
Despite the long interest in this enzyme, the list
of inhibitors is relatively short, and many in-
hibitors show overlap with P450s 1A2 and 1B1
[274] For instance, α-NF is often used as in-
hibitor but is more effective against P450 1A2
[274, 275] Another inhibitor is ellipticine [159]
1-(1ʹ-Propynyl)pyrene and 2-(1ʹ-propynyl)phen-
anthrene were found to be selective P450 1A1
inhibitors when compared against human P450s
1A2 and 1B1 [274]
More efforts have been made to synthesize
new inhibitors of P450 1A1 [276] Several or-
ganoselenium compounds are inhibitors [277], as
well as flavonoid derivatives [278] The natural
product rhapontigenin is a low KI inhibitor of
P450 1A1 [279] The furanocoumarin chalepen-
sin is a mechanism-based inactivator of P450 1A1
[280] Finally, the endogenous (tryptophan pho-
tolysis product) AhR ligand FICZ ( vide supra)
is a high-affinity ligand/inhibitor of human P450
1A1 [246]
9.7.1.7 Clinical Issues
Because of a rather limited role of P450 1A1 in
drug metabolism, there are no real pharmacoki-
netic issues The issue with P450 1A1 is induc-
tion and a possible role in chemical carcinogen-
esis Work with animal models shows that P450
1A1 inducers can be cocarcinogens [70, 103]
Thus, regulatory agencies have tended to look
unfavorably at induction of P450 1A1 by poten-
tial drugs in animal models However, the point
should be made that there is presently little ex-
perimental or epidemiological evidence to sup-
port this hypothesis, and Ah inducers can afford
protection from cancer in some animal models
[103] (Figs 99 and 910)
Very little evidence has been obtained that the
common genetic variations in human P450 1A1
have functional consequences with carcinogen
metabolism, eg, Ile-462 versus Val-462 [120]
However, genetic variations have been exam-
ined for relationship to overall cancer [281] and
to breast [282], colorectal [283], lung [284], oral
[285], and endometrial [286] cancers The over-
all evidence for relationship in any case is still
very limited
F. P. Guengerich
9.7.2 P450 1A2
9.7.2.1 Sites of Expression
As mentioned earlier, human P450s 1A1 and
1A2 both have seven exons and 70 % sequence
identity in their coding regions These two genes
both show similar patterns of regulation by the
AhR system, but P450 1A2 is essentially only ex-
pressed in the liver [233], probably due to the in-
volvement of HNF in its regulation ( vide infra)
Several lines of evidence indicate that the level of
expression is substantial (Fig 92), ~ 6–13 % of
the total P450 on the average, with levels varying
~ 40-fold among individuals (Figs 95 and 911)
A similar fold variation is seen in the in vivo me-
tabolism of the marker drug caffeine [61]
One LC–MS proteomic analysis of human
liver microsomes yielded a mean of 29 pmol
P450 1A2/mg microsomal protein (range 29–
104) [55] while another yielded 11–18 pmol/mg
microsomal protein [54]
Occasional reports cite mRNA expression in
some extrahepatic tissues, eg, colon [287] Ex-
tensive searches have not found expression in
human lung [233]
9.7.2.2 Regulation
The variability and inducibility of P450 1A2
have been recognized for some time, indirectly,
going back to studies on phenacetin metabolism
by Conney and his associates [288] The char-
acterization of P450 1A2 (“P450PA”) as the low
Km phenacetin O-deethylase [14] led to some in-
terpretation of the earlier results P450 1A2 was
shown to be the caffeine N3-demethylase [60],
and the 40-fold variation in levels of liver P450
1A2 is reflected in the 40-fold variation in some
in vivo parameters of caffeine metabolism [61]
Some of Vesells’s earlier work on the metabolism
of antipyrine in twins suggests a role for genetic
polymorphism in P450 1A2 activity [4], and a
more recent twin study confirms the strong ge-
netic component of caffeine demethylation [289]
One complication with genetic polymor-
phism, as with P450 1A1 ( vide supra), is the in-
ducibility Because of the availability of markers
of hepatic P450 1A2 function (phenacetin is no
longer used, due to its carcinogenicity in animal
9  Human Cytochrome P450 Enzymes
tests, but caffeine and theophylline are), dem-
onstrating in vivo changes in P450 is relatively
easy to do and the effects are consistently seen,
at least quantitatively The mechanism of induc-
tion appears to be similar to that of P450 1A1
(Fig 913), with expression restricted to the liver
because of the need for HNFα [290] An inter-
esting observation made recently in mice is that
the inducer 3-methylcholanthrene causes a per-
sistent induction (of P450 1A1) in liver, lasting
beyond the time suggested by pharmacokinetic
expectations [291] One interpretation is that a
P450 1A2-generated metabolite is involved Fur-
ther details and any relevance to humans remain
to be established With animal P450 1A2, one
mechanism of induction involves protein stabili-
zation, eg, by isosafrole-derived products [292]
Whether or not this mechanism is relevant in
humans is unknown Reported inducers include
cigarette smoking, charbroiled food (presum-
ably polycyclic hydrocarbons and heterocyclic
amines), cruciferous vegetables, vigorous exer-
cise [293], and the drug omeprazole (actually a
metabolite) [294]
The nuclear receptor LXRα has been found
to be involved in the regulation of both human
P450 1A2 and 1A1 [295] Dehydroepiandros-
terone (DHEA) has been reported to downregu-
late human P450 1A2 through an unusual mecha-
nism, destabilizing the mRNA [296] P450 1A2
phosphorylation has also been reported in vivo
[297]
Table 9.9   Some drug substrates for human P450 1A2a
Druga Reference
Acetaminophen (3ʹ) [304]
Antipyrine (4,3-methyl) [305]
Bufuralol (1,4) [306]
Caffeine (3) [60]
Clozapine [67]
Olanzapine [67]
Ondansetron (7,8) [307]
Phenacetin [14]
Tacrine [308, 309]
Theophylline (1,3,8) [310]
a Site of oxidation indicated in some cases See also Ren-
dic [51]
557
9.7.2.3 Genetic Variation
Although many early studies in this field dis-
counted a genetic contribution to the variability
of P450 1A2 levels due to lack of sharp breaks
in frequency distribution plots [132, 298], the
gene has been shown to be rather polymorphic/
variable At least 41 alleles are known [168], and
five additional SNPs remain to be characterized
for haplotype (http://wwwcypalleleskise) Of
these, several have changes in the coding se-
quences that cause amino acid changes Recent
work in this laboratory with the expressed coding
region variants indicates that most do not differ
more than twofold in their kinetic parameters for
several assays (phenacetin O-deethylation and N-
hydroxylation of heterocyclic amines), although
one of the variants (R431W) did not express
holoprotein in Escherichia coli [299] In cases
where analysis has been done, the variations gen-
erally lead to lower activity (http://wwwcypal-
leleskise) An exception is CYP1A2*1F (− 163
C > A), which is associated with higher induc-
ibility P450 1A2 is now considered to be more
variable than previously thought, as evidenced by
additional sites identified in an Ethiopian study
[300]
Genome-wide association studies (GWAS)
have identified sites in the P450 1A2 and AhR
genes as being determinants for coffee consump-
tion and induction of P450 1A2 by coffee [301–
303]
9.7.2.4 Substrates and Reactions
The list of drug substrates is long [51], and only
a few of the more well-known reactions are listed
in Tables 95, 96, 97, and 99
The only major endogenous substrates are
17β-estradiol and estrone (2-hydroxylation, with
some 4- and 16α-hydroxylation) The physiolog-
ical relevance of this reaction is unknown, par-
ticularly because of the wide variation in levels
of P450 1A2 (this reaction is also catalyzed by
other P450s, eg, 3A4 [311]) Induction of P450
1A2 and 2-hydroxylation has been proposed as a
means of preventing oxidation of 17β-estradiol to
the potentially more reactive 4-hydroxy product
[312, 313]
558
P450 1A2 is prominent among the human
P450s involved in carcinogen bioactivation [99]
Many carcinogens are substrates, particularly
aromatic and heterocyclic amines (Table 98,
Figs 99 and 910) Other carcinogens shown to
be substrates include polycyclic hydrocarbons,
nitropolycyclic hydrocarbons, and some N-ni-
trosamines [314] One of the most relevant car-
cinogens is aristolochic acid, a causative agent in
human nephropathy and urothelial cancer [315]
Although P450 1A2 is not generally consid-
ered to be a P450 stimulated by cytochrome b5
[266], it has been reported that cytochrome b5
can shift the balance of ellipticine from detoxica-
tion to bioactivation [267]
Chemical mechanisms of P450 1A2 reactions
have been considered, particularly for N-oxy-
genation A classical view involves the so-called
compound I (FeO3 +) entity, acting via 1-electron
oxidation followed by oxygen rebound [316,
317] A deficiency of this model is that electron-
withdrawing groups did not perturb N-oxygen-
ation of a series of N,N-dimethylanilines [317],
in contrast to N-dealkylation (which showed
a negative ρ value in Hammett analysis) [317–
319] Other mechanisms have been proposed
[320], including a recent “anionic” intermediate
model based on theoretical studies [321]
9.7.2.5 Structure
In 2007, Johnson and his associates [271] re-
ported an X-ray crystal structure of human P450
1A2 complexed with α-NF That structure may
be compared with the subsequently published
structures of P450 1A1 [270] and 1B1 [323],
which also contain the same ligand P450 1A2
has a compact, closed active site that is appropri-
ate for relatively large plasma molecules In the
published structure, as with P450 1A1 [270], the
site of the α-NF that is oxidized (to the 5,6-ep-
oxide is furthest away from the heme iron [214,
256]) (However, the rate of oxidation is very
slow and may reflect the tendency to bind in an
unproductive conformation) The issue of co-
operativity will be discussed later under P450
3A4 Cooperativity has not been reported for the
human P450, but behavior of the rabbit ortholog
has been interpreted in the context of multiple,
F. P. Guengerich
overlapping binding sites [214, 324] Docking
studies suggest that two molecules of pyrene (or
other small ligands) can be accommodated in the
P450 1A2 site [214, 325] (Fig 914)
9.7.2.6 Inhibitors
Several human P450 1A2 inhibitors are known
from clinical work, including furafylline (mecha-
nism based) [326] and fluvoxamine α-NF is a
readily commercially available and strong inhibi-
tor of human P450 1A2 ( KI ~ 6  nM [274]) for in
vitro work A number of polycyclic acetylenes
are potent inhibitors of P450 1A2 [274] With
rat P450 1A2, TCDD and some polyhalogenated
biphenyls are strong inhibitors, but these stud-
ies have not been extended to human P450 1A2
[327]
The multikinase inhibitor axitinib is also a po-
tent inhibitor of human P450 1A2 (IC50 01 µM)
[328] Some 7-ethynylcoumarin inhibitors have
been synthesized that are selective inhibitors of
human P450 1A1 and 1A2 [329] Other ethinyl
derivatives and some natural products also selec-
tively inhibit the three human P450s in family 1
[274, 278]
9.7.2.7 Clinical Issues
Some drug interactions have been reported An
older example is that of low activity towards
phenacetin favoring a potentially toxic secondary
pathway, deacetylation followed by quinonei-
mine formation and methemoglobinemia [96]
Furafylline was a drug candidate but was never
developed because of its strong P450 1A2 inhi-
bition and interference with caffeine metabolism
[330] High levels of P450 1A2 activity have also
been associated with ineffectiveness of theophyl-
line therapy (for asthma) [331, 332]
The other concern about P450 1A2 is the same
discussed earlier for P450 1A1, the cocarcino-
genic effect In this regard, there is some epide-
miological evidence that high P450 1A2 activ-
ity (measured as in vivo caffeine metabolism) is
associated with enhanced risk of colon cancer,
although the effect was not seen in the absence
of high N-acetyltransferase activity and high con-
sumption of charbroiled meat [132]
9  Human Cytochrome P450 Enzymes
Fig. 9.14   Docking of two pyrene molecules into the active site of human P450 1A2 [214] Pyrene molecules are in
green, and the heme is at the bottom of the figure
Some evidence has been reported that P450
1A2 genetic variants can be correlated with lung
cancer incidence [333]
In addition to the caffeine metabolism method
of noninvasive phenotyping [61], a [13C]-meth-
acetin breath test has been reported [334]
9.7.3 P450 1B1
9.7.3.1 Sites of Expression
P450 1B1 was originally discovered in keratino-
cyte cultures in a search for new dioxin-inducible
genes [71] and in work on adrenals in animal
models [335] In contrast to P450 1A1 and 1A2
(seven exons), the P450 1B1 gene has only three
exons and is located on chromosome 2 instead of
15 [336] Although most of the detailed studies of
tissue-specific expression have been done at the
mRNA level and not protein, strong responses
559
are seen in fetal kidney, heart, and brain, in that
order [259] In adults (human), there is little de-
tectable expression in liver but expression in kid-
ney, spleen, thymus, prostate, lung, ovary, small
intestine, colon, uterus, and mammary gland
[259] Many of these tissues are of particular in-
terest because of the tumors that develop there
Immunochemical staining of P450 1B1 has been
reported in a variety of different malignant tu-
mors [337]
The level of expression (of the protein)
in human lung has been estimated to be at the
level of ~ 1 pmol/mg microsomal protein in non-
smokers and 2–4 pmol/mg microsomal protein
in smokers, levels an order of magnitude lower
than for P450 1A1 [229] These low values may
explain the lack of immunostaining in (non-
tumor) tissues reported by Murray et al [337]
Specific values for levels of expression in tissues
other than lung have not been published Traces
560
of P450 1B1 mRNA were found in human liver
using real-time polymerase chain reaction (PCR),
but the protein was undetectable within the limits
of sensitivity [338]
The eye is an important site relevant to the
glaucoma associated with loss of activity alleles
(Sect 737, vide infra) In the eye (human), P450
1B1 mRNA is present at a high level in the iris
and ciliary body and at lower levels in the cor-
nea, retinal pigment epithelium, and retina [127,
339] P450 1B1 protein is absent in the trabecu-
lar network but present in nonpigmented ciliary
epithelium, corneal epithelium and keratocytes,
both layers of the iris pigmented epithelium, and
retina [127, 339]
P450 1B1 expression (at the protein level)
has been detected in human lungs and is higher
(18 pmol/mg microsomal protein) in smokers
[229] The level was even higher (44 pmol/mg
microsomal protein) in ex-smokers
It has recently been demonstrated that pro-
cessing of P450 1B1 by a cytosolic serine pro-
tease activates a mitochondrial-targeting signal
of P450 1B1 and leads to mitochondrial localiza-
tion and activity, where functional activity results
from coupling with the adrenodoxin electron de-
livery system [340]
9.7.3.2 Regulation
In vitro experiments show the inducibility of
P450 1B1 in patterns expected for an Ah-respon-
sive gene, which is one way in which the gene
was found [71] Unlike P450 1A1 and particular-
ly P450 1A2 ( vide supra), there is limited direct
evidence for inducibility of human P450 1B1 in
vivo because of the low, extrahepatic expression
and the lack of a diagnostic probe drug Although
the expression of P450 1B1 is driven by the AhR
system, additional factors must be involved be-
cause of the known tissue and cell line selectivity
of expression For instance, major differences are
seen between HepG2, MCF-7, and ACHN cells
(of liver, breast, and kidney tumor origin, re-
spectively) [336] With the information available
today, one would expect the gene to be induced
(in extrahepatic tissue) by the compounds that in-
duce P450s 1A1 and 1A2
F. P. Guengerich
In addition to the AhR regulation, the human
P450 1B1 gene is also regulated by estrogens via
the ER [341] Human P450 1B1 is also regulated
by microRNA [342]
9.7.3.3 Genetic Variation
Levels of P450 1B1 in human lung vary by at
least one order of magnitude [229] An interest-
ing observation is that a termination variant of
P450 1B1 is strongly associated with glaucoma
[127, 343] Other polymorphisms of (human)
P450 1B1 are known and are predominantly in a
set of haplotypes involving four variations, Arg/
Gly-48, Ala/Ser-119, Val/Leu-432, and Asn/Ser-
453 Assays involving the metabolism of 17β-
estradiol and polycyclic hydrocarbons by these
recombinant P450 1B1 variants show some vari-
ations but have not been particularly dramatic
(reviewed by Shimada et al [129])
At this time, the http://wwwcypalleleskise
website shows 26 allelic variants of P450 1B1,
plus six additional ones where the haplotype
has not been determined The number of allelic
variants listed in http://wwwcypalleleskise is
an underestimate, in that many more have been
reported to be associated with glaucoma (at least
82) [339] The functional effects on some of the
coding sequence variants have been determined
[129, 344] but are not particularly strong ( vide
supra) There is considerable interest in genetic
variations of P450 1B1 in the context of cancer
and glaucoma (Sect 737, vide infra)
9.7.3.4 Substrates and Reactions
Human P450 1B1 has never been purified from
tissue, and all of our information has come from
the protein expressed in heterologous systems
7-Ethoxyresorufin O-deethylation can be used
as a model reaction [345] The catalytic activity
of P450 1B1 is intermediate between P450s 1A1
and 1A2 [274] Some other model reactions can
be used as well [345]
Much of the interest in P450 1B1 has been be-
cause of its ability to activate a very broad spec-
trum of chemical carcinogens, including polycy-
clic hydrocarbons and their oxygenated deriva-
tives, heterocyclic amines, aromatic amines, and
nitropolycyclic hydrocarbons [259] (Table 910,
9  Human Cytochrome P450 Enzymes 561

Table 9.10   Some carcinogens activated by human P450 1B1

Substrate Reference
Polycyclic aromatic hydrocarbons
Benzo[a]pyrene [274]
Benzo[a]pyrene-4,5-diol [259]
(+) Benzo[a]pyrene-7,8-diol [259]
(−) Benzo[a]pyrene-7,8-diol [259]
Dibenzo[a, l]pyrene [344]
Dibenzo[a, l]pyrene-11,12-diol [259]
Benz[a]anthracene [274]
Benz[a]anthracene-1,2-diol [259]
Benz[a]anthracene-cis-5,6-diol [259]
7,12-Dimethylbenz[a]anthracene [259]
7,12-Dimethylbenz[a]anthracene-3,4-diol [259]
Benzo[c]phenanthrene-3,4-diol [259]
Fluoranthene-2,3-diol [259]
Benzo[b]fluoranthene-9,10-diol [259]
Chrysene-1,2-diol [259]
5-Methylchrysene [344]
5-Methylchrysene-1,2-diol [259]
5,6-Dimethylchrysene-1,2-diol [259]
Benzo[g]chrysene-11,12-diol [259]
6-Aminochrysene-1,2-diol [259]
Heterocyclic amines
MeIQ [259]
MeIQx [259]
IQ [259]
Trp-P1 [259]
Trp-P2 [259]
PhIP [259]
Aromatic amines
2-Aminoanthracene [259]
2-Aminofluorene [259]
4-Aminobiphenyl [259]
3-Methoxy-4-aminoazobenzene [259]
o-Aminoazotoluene [259]
6-Aminochrysene [259]
Nitropolycyclic hydrocarbons
1-Nitropyrene [346]
2-Nitropyrene [259]
6-Nitrochrysene [259]
2-Nitrofluoranthene [346]
3-Nitrofluoranthene [346]
6-Nitrobenzo[a]pyrene [346]
1,8-Dinitropyrene [346]
1-Aminopyrene [346]
Estrogens
17β-Estradiol [347]
Estrone [348]
562
Fig 910) This broad specificity of human P450
1B1 in activating aryl and heterocyclic amines,
polycyclic hydrocarbons, and other carcinogens
has been reviewed elsewhere [99, 259, 349–351]
Of particular interest is the observation that
human P450 1B1 is at least as active as P450
1A1 in the conversion of the classic carcinogen
benzo[a]pyrene to the 7,8-dihydrodiol, the first
step in the formation of the (7,8) diol (9,10) ep-
oxide [352] In general, it would appear that the
rodent P450 1B1 enzymes have similar catalytic
specificity as human P450 towards carcinogens,
from the available information [353] If this is a
valid view, then the observation that P450 1B1-
knockout mice do not form tumors when admin-
istered 7,12-dimethylbenz[a]anthracene is of
particular importance [106]
One of the interesting findings with human
P450 1B1 is that this enzyme is an efficient cata-
lyst of 17β-estradiol hydroxylation and that the
pattern is for 4- > 2-hydroxylation [311, 347,
354] This pattern is the opposite seen for P450s
1A2 and 3A4 (2- > 4-hydroxylation) [311, 355]
and is of significance because 4-hydroxyestra-
diol is chemically more reactive with oxygen and
also more likely to oxidize (to an o-quinone) and
bind DNA [356] Thus, 4-hydroxyestrogens are
considered to be candidates for causing estrogen-
dependent tumors [357] However, mouse P450
1B1 preferentially catalyzes estrogen 2-hydrox-
ylation compared to 4-hydroxylation, in sharp
contrast to human P50 1B1 [353], providing a
potentially important difference with the human
enzyme This apparent lack of conservation of se-
lectivity has relevance in use of mouse (and rat)
models in some of the biology, eg, the human
glaucoma mentioned earlier [127, 343]
9.7.3.5 Structure
Johnson and his associates [272] reported an
X-ray crystal structure of human P450 1B1
bound to α-NF The structure can be compared
directly with that P450 1A2 [271] and with P450
1A1 [270] with the same ligand bound Both
P450s 1A2 and 1B1 have narrow active site cavi-
ties, explaining the preference for flat aromatic
substrates A distortion of helix F places the resi-
F. P. Guengerich
due Phe-231 in position for π–π stacking with α-
NF [272]
Nishida et al [358] reported that mutagenesis
of Val-395 of human P450 1B1 to Leu changed
the regioselectivity of 17β-estradiol hydroxyl-
ation from the C4 position to C2, demonstrating
the sensitive nature of the active site, at least with
regard to some reactions The effects of the al-
lelic variants are probably not strong enough to
be of much use in understanding the effects of
those residues [129]
9.7.3.6 Inhibitors
α-NF is a strong inhibitor, as in the case of P450
1A2 [274] Some acetylenes developed by Al-
worth’s group have been found to selectively in-
hibit P450 1B1 (at least relative to P450s 1A1
and 1A2), including 2-ethynylpyrene [274] A
potential drawback to these compounds is that
they are rapidly oxidized by P450 1B1
The polyphenol resveratrol is found in red
grapes and has been of interest in the context of
its potential to inhibit cancer [359] Resveratrol
is a noncompetitive inhibitor of P450 1B1, with a
KI value of 23 µM in model systems [360] (with
selectivity towards P450 1A1) Potter et al [361]
reported that P450 1B1 oxidizes resveratrol to the
known anticancer agent piceatannol, a tyrosine
kinase inhibitor A series of methoxy-substituted
trans-stilbene compounds of the resveratrol/rha-
pontigenin family were prepared and tested; of
these, 2,4,3ʹ,5ʹ-tetramethoxystilbene was found
to be a strong and selective competitive inhibitor
of P450 1B1 ( KI 3 nM) and resisted demethyl-
ation [279]
Because of the roles of P450 1B1 in the ac-
tivation of carcinogens [99] (Fig 910), there is
strategic interest in developing inhibitors of P450
1B1 [362, 363] Another tetramethoxystilbene
(2,2ʹ,4,6ʹ-) has been reported to be a strong inhib-
itor of human P450 1B1 [364], in addition to the
2,4,3ʹ,5ʹ-substituted stilbene [279] A number of
other compounds have been considered regard-
ing their inhibition of P450 1B1, including de-
rivatives of flavonoids, stilbenes, pyrenes, naph-
thalenes, phenanthrenes, and biphenyls [365]
9  Human Cytochrome P450 Enzymes
9.7.3.7 Clinical Issues
No issues regarding drug interactions have been
raised The two dominant clinical issues with
P450 1B1 are its potential roles in cancer and
glaucoma As with the subfamily P450 1A en-
zymes, an issue is that induction of P450 1B1
might increase the activation of procarcinogens
(Fig 910) This issue may be real, although pres-
ently there is no strong epidemiological evidence
to support such a relationship Although the cod-
ing region polymorphisms have only indicated a
limited potential for contribution to cancer ( vide
supra), the evidence for its trimodal expression
[122] is certainly of interest, particularly in light
of the number of carcinogens that P450 1B1 ac-
tivates (Table 910) The issue of oxidation of es-
trogens to reactive products is one worth consid-
ering, in light of the long-standing experimental
evidence for tumorigenicity of estrogens [366]
Another matter that has only begun to be ad-
dressed is the possible metabolism of the various
estrogens in postmenopausal hormone treatments
(eg, Premarin® by P450 1B1 (eg, see [356,
367] regarding DNA adducts formed by some of
these estrogens)
Because P450 1B1 has such a prominent role
in carcinogen activation in vitro (Fig 910) [99,
259], there is considerable interest in molecu-
lar epidemiology on the subject, as reviewed by
Roos and Bolt [368] Kamataki and his associ-
ates [122] found that the trimodal distribution
of inducibility of aryl hydrocarbon hydroxylase
activity (benzo[a]pyrene hydroxylation) is due to
the induction of P450 1B1, not P450 1A1 This
information is relevant to the earlier findings of
Shaw and Kellerman [108, 109] correlating the
inducibility with lung cancer risk in smokers
However, apparently no major progress has been
reported in this area following the report of Toide
et al [122] P450 genetic variations have been
considered in the epidemiology of breast [369],
head and neck [370], endometrial [371], pan-
creatic [372], colorectal [373], hormonal [374],
and prostate [375] cancers, although overall the
evidence is not strong In a mouse model, P450
1B1 is associated with smoking-induced bone
loss [376]
563
The other major clinical issue is glaucoma,
where P450 1B1 variants are clearly associated
with the disease [127, 339] The condition is re-
produced in a mouse CYP1B1 knockout, but the
mechanism is still elusive [106, 339] As men-
tioned previously, P450 1B1 has a broad catalytic
specificity, and estrogens, arachidonic acid, reti-
noids, and melatonin have all been considered as
possibly being involved [339]
Finally, P450 1B1 has been considered to have
a role in hypertension, possibly involving its role
in arachidonic acid ω-hydroxylation [377, 378]
9.7.4 P450 2A6
9.7.4.1 Sites of Expression
P450 2A6 (formerly termed IIA3 and 2A3 [379])
was purified from human liver microsomes [17]
and a cDNA was first isolated from a human liver
library [380] The protein is expressed at medium
levels in liver (Fig 92) In one study, the frac-
tion of total human liver P450 attributed to P450
2A6 ranged from < 02 to 13 % among individual
samples, with a mean of ~ 4 % [52] P450 was not
found in placenta (full term) [381] In a recent
LC–MS proteomic study, P450 2A6 was found
at a mean level of 63 pmol/mg liver microsomal
protein, almost as high as P450 3A4 (Fig 92d)
[55] However, LC–MS-determined levels were
not this high in other studies [54] (Fig 92b, c)
P450 2A6 is also expressed in other tissues,
particularly in the nasal–pharyngeal region Ex-
pression has been detected in nasal mucosa, tra-
chea, lung [382], and esophageal mucosa [383]
These sites of expression are of interest regarding
certain cancers In liver cancers, overexpression
of P450 2A6 protein was associated with chronic
inflammation and cirrhosis [384]
P450 2A6 was found to be overexpressed in
colorectal tumors [385] P450s 2A6 and 2A13
are very similar proteins (94 % identity) but dif-
fer in structure (Sect 745, vide infra) and some
activities, as well as localization Both P450 2A6
and 2A13 are expressed in epithelial cells of tra-
chea and bronchi, and only P450 2A6 (no 2A13)
was detected in bronchial epithelial cells of pe-
ripheral lungs [386]
564
9.7.4.2 Regulation
The regulation of P450 2A6 expression has been
studied in primary cultures of human hepato-
cytes Expression (mRNA and protein) is induc-
ible by rifampicin [387], phenobarbital [388],
and (to a lesser extent) clofibrate, cobalt, griseo-
fulvin, and pyrazole [388] The nuclear receptor
HNF-4 is involved in expression in cultured he-
patocytes [389]
P450 2A6 transcriptional regulation has been
reviewed by Pitarque et al [390] Induction has
been shown to involve the PXR, along with
PPARα [391] P450 2A6 is also induced by es-
trogen via the ER [392], which may be relevant
to a reported influence of the menstrual cycle on
P450 2A6 activity (and the cardiovascular effects
of nicotine) [393]
Other factors influencing P450 2A6 transcrip-
tion are NF-Y [394] and nuclear factor-erythroid
2-related factor 2 [395] In addition, heteroge-
neous nuclear ribonucleoprotein A1 has been re-
ported to be involved in post-transcriptional regu-
lation of P450 2A6 [396], and a polymorphism in
the 3ʹ-untranslated region affects mRNA stability
and enzyme expression [397] Finally, P450 2A6
phosphorylation has been detected in vivo [297],
although the effect is not known
9.7.4.3 Genetic Variation
At least 86 allelic variants are known, with eight
haplotypes yet to be determined (http://www
cypalleleskise) These include a splice variant
(*12) in which CYP2A7 exons are included and
the protein has lost catalytic activity [398, 399]
Some are deletions and the activities of some of
the coding region variants are known to be de-
creased [400] Another SNV (*2), recognized
earlier, is the L160H change which yields very
low catalytic activity [401] At least one poly-
morphism is important for promoter activity
[402] Also of interest is a gene deletion (*4)
The incidence of these variants is racially linked
[168]
In part, because of the extensive genetic varia-
tion and the metabolism of carcinogens, genetic
variations have been extensively considered re-
garding cancer (Sect 747, vide infra) P450
2A6 is involved in nicotine oxidation, and Tyn-
dale and her group reported that individuals with
F. P. Guengerich
low P450 2A6 activity smoke less and might have
lower cancer risk [134] This proposal seems
reasonable, but the findings have been ques-
tioned General agreement exists that defective
P450 2A6 genes cause reduced nicotine metabo-
lism (the presumed basis for reduced smoking)
[403–405] Several reports conclude that having
deficient P450 2A6 reduces smoking [406–409]
and also lung cancer [133, 410, 411] in smokers
The latter hypothesis has biological plausibility
because many carcinogens from tobacco are ac-
tivated by P450 2A6 (Table 98 and vide infra)
However, other studies have not revealed any
relationship between P450 2A6 genotype and
smoking; cancer is also somewhat controversial
[412–415] Some of the discrepancies may be ra-
cial [416], but even this is unclear [417] Some
problems are attributed to technical shortcomings
in genotype analyses [418], and a definite rela-
tionship is still lacking [418] in Caucasians but is
more likely in Asians [419], where the incidence
of gene deletion is higher
9.7.4.4 Substrates and Reactions
The most characteristic and specific reaction of
P450 2A6 is coumarin 7-hydroxylation [17, 380]
Coumarin 7-hydroxylation has also been used as
an in vivo diagnostic assay [420–422]
Soucek [423] demonstrated that a 1:1 ratio of
cytochrome b5 to P450 was required for optimal
coumarin 7-hydroxylation catalyzed by the pu-
rified recombinant enzyme The effect of cyto-
chrome b5 on catalytic selectivity has not been
evaluated in all reports on P450 2A6
Coumarin 7-hydroxylation can be used in
vivo with humans as a phenotypic assay An
alternative procedure is to administer caffeine
to individuals and determine the conversion of
1,7-dimethylxanthine to 1,7-dimethyluric acid, a
reaction catalyzed by P450 2A6 [424]
Some industrial chemicals are substrates for
oxidation by P450 2A6, including alkoxyethers
(used as fuel additives, eg, tert-butyl methyl
ether) [425] and the vinyl monomer 1,3-butadi-
ene, a cancer suspect [426]
Some drugs are also substrates, including
(+)cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-
4-one (SM-12502) [427, 428] and tegafur [429,
430], which is converted to 5-fluorouracil Halo-
9  Human Cytochrome P450 Enzymes
thane is reductively converted to a free radical by
P450 2A6, which can yield at least two products
and initiate lipid peroxidation [431]
Some of the catalytic selectivity of P450 2A6
overlaps with that of P450 2E1 ( vide infra) One
area in which the overlap has been noted is in the
oxidation of nitrosamines P450 2A6 preferen-
tially catalyzes the oxidation (and activation) of
N-nitrosodiethylamine, in contrast to P450 2E1,
which oxidizes N-nitrosodimethylamine [432,
433] P450 2A6 is also involved in the oxidation
of many tobacco-specific nitrosamines, including
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) [433–436] P450 2A6 appears to be the
major human P450 involved in the activation of
N-nitrosobenzylmethylamine [437], N-nitroso-
dipropylamine, N-nitrosobutylamine, N-nitroso-
phenylmethylamine, and N-nitrosonornicotine
(NNN) [438] Fujita and Kamataki [439] studied
the bacterial mutagenicity of a number of tobac-
co-specific N-nitrosamines and concluded that
P450 2A6 is the major human enzyme involved
in activation of all
P450 2A6 is also involved in the metabolism
of nicotine ( vide supra) P450 2A6 is the main
catalyst in the oxidation of nicotine to cotinine
[440–442] P450 2A6 is also involved in the
3ʹ-hydroxylation of cotinine [443] In addition,
P450 2A6 catalyzes 2ʹ-hydroxylation of nicotine,
yielding a precursor of a lung carcinogen [444]
P450 2A6 can also N-demethylate hexameth-
ylphosphoramide [445]
Several forms of human P450 catalyze the
3-hydroxylation of indole [446], and the product
dimerizes to the dye indigo P450 2A6 was the
most active human P450 identified for this activi-
ty and could also catalyze several oxidations of in-
dole [446] Mutants of P450 2A6 generated from
a randomized library were shown to catalyze the
oxidation of several substituted indoles to gener-
ate variously colored indigos and indirubins [447]
Other substrates of interest include 1,7-di-
methylxanthine, a major caffeine metabolite
[448] (this can be applied in phenotyping stud-
ies), pilocarpaine [449], bilirubin [450], and met-
ronidazole [451]
More recently, Shimada et al [351] have dem-
onstrated that P450 2A6 can catalyze the bioacti-
vation of a number of PAHs and arylamines
565
Yun et al [452] analyzed the kinetics of the
catalytic cycle of P450 2A6 with coumarins and
concluded that substrate binding, product release,
electron transfer, and oxygen binding were all
rapid steps and that C–H bond cleavage is prob-
ably mainly rate limiting
9.7.4.5 Structure
In 2005, Johnson and his associates reported
the X-ray crystal structure of P450 2A6 com-
plexed with coumarin and methoxysalen [453]
Subsequent structures with synthetic inhibitors
[454] and mutants [455] added to the knowledge
of this P450 It has one of the smallest active
sites (~ 260 Å3 volume) and is relatively rigid,
although some larger ligands can be accommo-
dated
The structure of P450 2A6 has been compared
with those of P450s 2A13 and 2E1 (with pilo-
carpine bound) [456] and inferences about im-
portant residues differing between these proteins
have been made [457]
Some mutations, developed in random muta-
genesis work [458], result in large change in the
active site volume of P450 2A6 [459] (Fig 915)
Lewis published several homology models of
P450 2A6 and also attempted to rationalize the
pattern of nicotine oxidation using molecular or-
bital calculations [460]
9.7.4.6 Inhibitors
Several selective inhibitors of P450 2A6 are
known Diethyldithiocarbamate appears to be a
mechanism-based inactivator, although the inac-
tivation has not been extensively characterized
[433] Diethyldithiocarbamate and its oxidized
form, disulfiram, also inhibit P450 2E1 [461] In
vivo single-dose treatment of people with disul-
firam inhibits P450 2E1 but not P450 2A6 [462]
A number of chemicals have been tested as
inhibitors of P450 2A6 in human liver micro-
somes [463] Of these, the most selective and po-
tent inhibitors appear to be 8-methoxypsoralen,
tranylcypromine, and tryptamine, with KI values
~ 1 µM [463–465] The inhibition by the natural
product 8-methoxypsoralen (present in many
foods) is mechanism based [466] 8-Methoxy-
psoralen (methoxysalen) inhibits P450 2A6 in
vivo [462] and has also been reported to decrease
566 F. P. Guengerich

Fig. 9.15   Active site of P450 2A6 a In the wild-type with the orange mesh (total 440 Å3) b Minimized energy
enzyme, Ile-300 and Asn-297 restrict the available space docking of the substrate 5-benzoylindole to the P450 2A6
to the area shown with the purple mesh (359 Å3) The N297Q/I300V mutant [459]
extra space made available in the I300V mutant is shown
9  Human Cytochrome P450 Enzymes
nicotine metabolism in smokers [467] Both the
inhibitors 8- and 5-methoxypsoralen were cova-
lently bound to P450 2A6 during incubation with
NADPH [468] Menthofuran, another natural
product, is also a mechanism-based inactivator of
P450 2A6 [469] Isoniazid has been reported to
be a weak mechanism-based inactivator of P450
2A6 [470]
A number of heterocyclic inhibitors of P450
2A6 have been synthesized [471], and the in-
teraction of some other new inhibitors has been
visualized in P450 2A6 crystal structures [454]
The selectivity of P450 2A6 “reaction pheno-
typing” inhibitors was reevaluated by Stephens
et al [472], who compared chemicals for inhibi-
tion of P450s 2A6 and 2A13 ( vide infra): tran-
ylcypromine and ( R)-(+)-menthofuran had > ten-
fold selectivity in favor of P450 2A6 > P450 2A13
and 8-methoxypsoralen had a sixfold lower KI
for P450 2A13 Khojasteh et al [473] concluded
that 3-(pyridine-3-yl)-1H-(pyrazol-5-yl)pyridine
was more selective than tranylcypromine
Another inhibitor of P450 2A6 is chalepensin
(mechanism based) [474] Heteroatom nicotine
derivatives have been identified as inhibitors
[475], and P450 2A6 is inactivated during the
oxidation of nicotine itself [476]
Finally, a variety of chemicals, including
PAHs, chlorinated biphenyls, and flavonoids
were demonstrated to interact with (spectral
binding) and to inhibit P450 2A6 (as well as
P450 2A13) [477] This inhibition has relevance
to potential use of some of these compounds as
therapeutic inhibitors as well as to interactions in
the activation of them by P450 2A6 (and 2A13)
[351]
Much of the enthusiasm about inhibitors of
P450 2A6 stems from the hope of cancer preven-
tion, in that 8-methoxpsoralen (despite the ca-
veats about human P450 2A13 selectivity, vide
supra) effectively deceased tumors in an NNK
treatment mouse model [478]
9.7.4.7 Clinical Issues
As indicated in Sect 742, the major issue re-
garding P450 2A6 polymorphisms is the effects
on lung and esophageal cancers and smoking
habits, for which there is epidemiological evi-
567
dence in Asians [410], but reports remain contro-
versial in Caucasians [416, 418, 419, 479, 480]
As pointed out above, some drugs are P450
substrates, although the relative contribution of
P450 2A6 is still so small (Fig 91b) that P450
2A6 reactions are generally not included in
screens
P450 2A6 expression has been reported to be
induced during infection by (carcinogenic) liver
flukes [481] and downregulated during infection
by hepatitis A virus [482]
Genetic variations in P450 2A6 have been ex-
tensively considered in regard to nicotine metab-
olism and smoking cessation therapy [483–486],
and genetic variations have been considered in
the direct context of smoking-related cancers
[487–490] P450 2A6 genetic variation has also
been considered in the context of hepatoxicity of
coumarin [491] and pancreatic cancer [492]
9.7.5 P450 2A7
The situation involving the CYP2A7 gene is
complex, and sometimes this has even been er-
roneously referred to as a pseudogene [168] Two
pseudogenes ( CYP2A7PTX and CYP2A7PCX)
are known The P450 2A7 mRNA transcript is
produced in human liver, at roughly the same
level as that for P450 2A6 [398, 493] Gonzalez’s
laboratory had isolated cDNA clones now recog-
nized as P450 2A6, the 2A6 variant L160H, and
2A7 and expressed all three in HepG2 cells [380]
Of the three, only the “wild-type” P450 2A6 in-
corporated heme Others have also attempted to
express P450 2A7 in heterologous systems but
not reported any evidence of a catalytically ac-
tive P450 2A7 holoprotein [398] Whether or not
a functional P450 2A7 is transcribed from the
mRNA in human tissues is still unclear, and noth-
ing can be said about catalytic activity
Gene conversion events between the CYP2A6
and CYP2A7 genes have been reported, yield-
ing chimeric proteins in humans [398, 399, 494]
These proteins have some of the coumarin 7-hy-
droxylation conferred by the 2A6 component
[399]
568
It has been reported that there are at least four
polymorphic P450 2A7 gene variants, and some
of these can be confounding when genotyping for
certain P450 2A6 alleles [495]
9.7.6 P450 2A13
9.7.6.1 Sites of Expression
P450 2A13 cDNA was first cloned from a human
nasal mucosa library [445] mRNA was detected
primarily in nasal mucosa, trachea, and lung,
with the level in liver being only ~ 1 % of that
in nasal mucosa [496] This is in sharp contrast
to P450 2A6, which is primarily a liver enzyme
(see Sect 751, vide supra) At the protein level,
immunochemical analysis has shown P450 2A13
in the epithelial cells of human bronchus and tra-
chea [386, 497] P450 2A13 has also been detect-
ed in human bladder [498], and there are reports
of some expression in brain, mammary gland,
prostate, testes, and uterus [496] and pancreatic
α-islet cells [499]
P450 2A13 mRNA was reported to be elevat-
ed in small-cell lung cancer tissue in one study
[500] but was not detected or was downregulated
in any lung cancers in two other studies [497,
501]
9.7.6.2 Regulation
P450 2A13 transcription involves CcATT/en-
hancer-binding protein (C/EBP) transcription
factors [502] This interaction is believed to be
responsible for olfactory mucosa-specific ex-
pression in humans In addition, there is evidence
for epigenetic regulation of P450 2A13 expres-
sion, at both the levels of DNA methylation and
histone acetylation [502, 503]
9.7.6.3 Genetic Variation
At least 21 different CYP2A13 gene variants
have been reported (http://wwwcypalleleskise)
There is evidence that some of those that produce
amino acid changes can alter catalytic properties
and that expression levels can change [504–508]
Genetic differences are racially linked [503, 509–
511]
F. P. Guengerich
9.7.6.4 Substrates and Reactions
Although P450 2A13, 94 % identical to P450
2A6, can oxidize some relatively common
subfamily 2A P450 substrates such as couma-
rin [512], the interest in P450 2A13 has been
driven by its ability to activate procarcinogens
[496] The catalytic efficiency in activating the
so-called tobacco-specific nitrosamines (NNK,
NNN) is considerably higher than P450 2A6
When coupled with the selective expression of
P450 2A13 in the respiratory tract, there is poten-
tial for understanding aspects of tobacco-induced
cancers of the lung and the rest of the respiratory
tract [496, 513]
The active site of P450 2A13 is larger than
that of P450 2A6 ( vide infra), and a number of
additional substrates of P450 2A13 have been
identified, including nicotine and cotinine [514],
the nicotinium Δ5 (1ʹ) iminium ion [515], afla-
toxin B1 [516, 517], phenacetin and theophylline
[518], 4-aminobiphenyl [498], and 5-methoxyp-
soralen [519] In addition, P450 2A13 was found
to activate a large variety of PAHs (and their di-
hydrodiol derivatives), arylamines, and heterocy-
clic amines to genotoxic products [351]
The relevance of the activation of all of these
procarcinogens can be addressed in a P450 2A13-
humanized mouse model [520]
9.7.6.5 Structure
Some early site-directed mutagenesis work im-
plicated roles of certain amino acids in the meta-
bolic activation of NNK [521] A structure of
P450 2A13 was reported by Scott’s laboratory
in 2007 [522] Although no substrate had been
added, the finished structure revealed the pres-
ence of indole, which is known to be a substrate
Like P450 2A6, the active site cavity is relatively
small and hydrophobic, with a cluster of Phe resi-
dues composing the roof The size of the active
site appears to be larger than that of P450 2A6
Residues at positions 117, 300, 301, and 208 help
define differences with P450 2A6 [522] Some
computational work has also appeared [523]
Another structure has been reported with pi-
locarpine (an imidazole) bound [456] As might
be expected from the imidazole ring and the type
9  Human Cytochrome P450 Enzymes
II binding spectrum, the imidazole ring was clos-
est to the heme (the Fe–N distance was 23 Å)
The pilocarpine-bound structure was compared
to that of P450 2A6 and 2E1 [456]
9.7.6.6 Inhibitors
In light of the activation of procarcinogens by
P450 2A13, there is interest in developing inhibi-
tors to prevent cancer, and some success has been
achieved [471, 524–526] One of the issues is se-
lective inhibition of P450 2A13 relative to P450
2A6 Some compounds, eg, 8-methoxypsoralen,
menthofuran, and β-tyramine, show an order of
magnitude selectivity for P450 2A13 > P450 2A6
[472, 527, 528] Nicotine is a mechanism-based
inactivator of P450 2A13 [476] Shimada et al
[477] examined 66 chemicals as inhibitors of
P450 2A13, including a variety of flavonoids and
polycyclic hydrocarbons Several selectively in-
hibited P450 2A13 (relative to P450 2A6), with
low- or sub-µM IC50 values One of the conclu-
sions, based upon spectral binding studies, was
that the active site of P450 2A13 is more spa-
cious than that of P450 2A6, consistent with the
X-ray crystal structure ( vide supra)
9.7.6.7 Clinical Issues
P450 2A13 can oxidize some drugs [518], but
there is no evidence that it makes a major con-
tribution to the clearance of any (Fig 91b) The
major issue is possible contribution to cancers of
the respiratory tract, particularly those caused by
smoking [496] Accordingly, a number of epide-
miological studies have been done, particularly
with regard to alleles associated with lower meta-
bolic activity [503, 505, 507, 529–533], with at
least some of the studies showing significant cor-
relations of lung cancer with risk in smokers as-
sociated with P450 2A13 genotypes [503, 505]
9.7.7 P450 2B6
9.7.7.1 Sites of Expression
P450 2B6 is expressed primarily in liver, and the
protein was partially purified [534] The protein
has also been detected in human lung [535]
569
Much of the early work with P450s in experi-
mental animals was focused on the phenobarbi-
tal-inducible enzymes now recognized to be in
the P450 2B subfamily [536, 537] and a general
expectation was that similar P450s would be
prominent in human liver (and further suggested
by immunochemical studies [9] and early cloning
work [538]) However, the major P450 in human
liver (and small intestine) proved to be P450 3A4
(Figs 92 and 93) The mean level of P450 2B6
in human liver has been somewhat controversial
One of the problems has been antibody speci-
ficity Antibodies raised against rat P450 2B1
have not been very specific [534]; unfortunately
many papers in this area show only limited sec-
tions of gels (or actually show major cross-reac-
tive material present) The results tend to fall into
two groups One set reports levels vary from low
to 80 pmol P450 2B6 per mg protein [539–541]
Another set of reports ranges from near-zero lev-
els to 28 pmol P450 2B6/mg microsomal protein
[534, 542–545] However, the mean values dif-
fer considerably in the former and latter groups
While some of the discrepancy may be attribut-
able to the differences in liver samples, the main
difference may be with the antibodies used and
cross-reactivity with other proteins, as well as
error inherent in other aspects of immunochemi-
cal methods Our own work is in line with the
lower set of estimates of expression levels (mean
1–2 % of total P450, with values rarely exceeding
5 % even in samples from individuals adminis-
tered inducers) [545] This level is an order of
magnitude less than for P450 3A4 (Fig 92)
Recently Achour et al [55] used an LC–MS
proteomic approach with human liver micro-
somes and reported a mean value of 39 pmol/
mg protein This value was ~ one half of that
found for P450 3A4 in the same set of samples
(Fig 92d) The concentration was much less in
the other samples (Fig 92a, b, c), with another
LC–MS study reporting only 05 and 7 pmol
P450 2B6/mg protein [54]
9.7.7.2 Regulation
Studies with HepG2 cells (derived from hepato-
cytes) have shown the role of CAR, a member
570
of the steroid receptor superfamily, and its in-
teraction with the phenobarbital-responsive en-
hancer module (PBREM) in the region between
− 1733 and − 1683  bp in the 5ʹ-flanking region
[546] Other work with HepG2 cells implicated
the liver-selective transcription factor C/EBPα
[547] Kliewer’s group [548] also demonstrated
the involvement of another previously orphan re-
ceptor, PXR, in binding to PBREM in primary
human hepatocytes to induce P450 2B6 PXR
is active only when ligand activated, but CAR
apparently acts without an added ligand; both
CAR and PXR heterodimerize with (liganded)
RXR [549] (Fig 913) “Cross talk” also exists
at the PBREM site with the vitamin D receptor
(VDR) as well as CAR and PXR [550, 551] The
levels of CAR and PXR mRNA in individual
human livers correlate with the level of P450
2B6 mRNA [552] The regulation of P450 2B6
has considerable similarity to those of the P450
2C and 3A subfamilies ( vide infra), with some
differences CAR does have ligand-activated ef-
fects and 6-(4-chlorophenyl)imidazo[2,1-b;1,3]
thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)
oxime has been identified as an agonist [553]
A novel distal enhancer regulated by PXR and
CAR was identified in the CYP2B6 gene [554]
The roles of nuclear receptors in P450 2B6 in-
duction have been reviewed by Wang and Negi-
shi [555] and Wang and LeCluyse [556] In pri-
mary human hepatocytes, P450 2B6 was induced
by clotrimazole, phenobarbital, rifampicin, rito-
navir, carbamazepine, and phenytoin, with all but
the latter two compounds apparently activating
via PXR [557]
Negishi’s group also reported a novel CAR-
mediated mechanism for synergistic activation of
two distinct elements within the P450 2B6 gene
[558] Neurosteroids and nicotine were identified
as PXR activators [559] Negishi and his asso-
ciates were able to classify P450 2B6 inducers
in terms of PXR versus CAR mechanisms [560]
They also showed that CAR was an early growth
response factor in activating the P450 2B6 gene
[561] Oltipraz, generally considered in the con-
text of Nrf2, also activates CAR [562] Metfor-
min represses P450 2D6 by modulating CAR
signaling [563]
F. P. Guengerich
P450 2B6 has been found to be phosphory-
lated in vivo, although the effect on activity is
unknown [297]
9.7.7.3 Genetic Variation
As mentioned in the previous edition of this
chapter [149], P450 2B6 is highly polymorphic
At least 63 allelic variants have been identified,
and at least six more variants are known in which
the haplotypes have not been determined yet
(http://wwwcypalleleskise) A number of these
are known to be associated with lower activity,
and a number of clinical consequences have been
reported (Sect 777, vide infra) A partial dele-
tion of the P450 2B6 gene has been attributed to
crossover with the pseudogene CYP2B7 [564]
9.7.7.4 Substrates and Reactions
The number of P450 2B6 substrates has grown
with time but is still not as extensive as for P450
3A4 (Sect 7204, vide infra) However, with the
availability of more knowledge about genetic
variants ( vide supra) and diagnostic marker sub-
strates, it has been possible to show the relevance
of P450 2B6 in vivo in several cases
The substrate specificity of P450 2B6 has
been reviewed [565–567] “Marker” fluorescent
substrates are available for some in vitro uses
[568]
One diagnostic substrate is efavirenz [569],
which also has clinical issues (Sect 777, vide
infra) Perhaps the most widely accepted refer-
ence substrate for P450 2B6 (in vitro) is bupro-
pion [570, 571] Efavirenz has been utilized as a
marker in vivo [572]
An important substrate for P450 2B6 is the
antimalarial drug artemisinin [573] Another
is methadone, used in treating heroin addiction
[574] P450 2B6 is also involved in the metabo-
lism of a number of environmental chemicals,
including the pesticide chlorpyrifos [575]
9.7.7.5 Structure
Relatively little site-directed mutagenesis has
been done with P450 2B6 Halpert’s laboratory
modified ten residues and measured some activi-
ties, although most of the changes were ≤ twofold
[576] Halpert and his associates have published
9  Human Cytochrome P450 Enzymes
several X-ray crystal structures of P450 2B6,
some with inhibitors [577–579] and one with a
substrate, amlodipine [580] (this is also a sub-
strate for P450 3A4) Several features are of note
The apparent size of the active site is large but not
as large as that of P450 3A4 or P450 2C9 Two
amlodipine molecules are bound in the enzyme
structure Finally, the protein is malleable and
residues move to accommodate different ligands
Several homology models of P450 2B6 have
been published [581, 582], including one using
molecular dynamics [583]
Yamazaki and his associates have published a
two-dimensional model for rationalizing and pre-
dicting substrates for P450 2B6 [584] In silico
approaches have been used for the prediction of
P450 2B6 substrates [585]
9.7.7.6 Inhibitors
A list of the reported inhibitors of P450 2B6 has
been compiled by Rendic [51] Orphenadrine
had been utilized in some work with microsomes
but does not appear to be particularly selective
[586, 587] 2-Isopropenyl-2-methyladamantane
and 3-isopropenyl-3-methyldiamantane have
been reported as selective inhibitors of P450 2B6
[588] Triethylenethiophosphoramide has also
been reported to be a selective inhibitor of P450
2B6 [589]
Khojasteh et al [473] reported that 2-phe-
nyl-2(1-piperidinyl)propane is the most selective
in vitro inhibitor for use in reaction phenotyping
New inhibitors have been considered based on
structure–activity relationships [590] The effect
of a K262R substitution on inhibition by several
drugs has been noted by Hollenberg and his as-
sociates [591], and Thr-302 has been implicated
(by the same group) in irreversible inactivation
by tert-butylacetylene [592]
The oral contraceptive 17α-ethinylestradiol
is a mechanism-based inactivator of P450 2B6
and modifies the (apo) protein [593, 601], but the
in vivo relevance of the inhibition has not been
established Inhibition by duloxetine has been
described as being both reversible and time de-
pendent [594] Other P450 2B6 inhibitors include
an acetylenic drug candidate [595], ticlopidine,
clopidogrel [596, 597], phencyclidine [598], di-
571
aziridines [599], peroxynitrite [600], methadone
[602], selegiline [603], sibutramine [604], and
ritonavir [605] Another clinically relevant inhi-
bition involves grapefruit juice [606]
9.7.7.7 Clinical Issues
Some of the clinical issues have been reviewed
recently by Zanger and Klein [607] The major
issues are interindividual variations due to induc-
tion and genetic variation as well as some inhibi-
tions The effects of genetic variation have been
reported for the drug efavirenz (used for HIV)
[608–611] Another drug in which genetic varia-
tions make an in vivo difference is bupropion,
used in smoking cessation therapy [612, 613]
Efavirenz–bupropion interactions have also been
reported [614]
Genetic variations have not been found to
effect nicotine metabolism (or plasma levels)
[615, 616] However, genetic variations in P450
2B6 have been associated with the outcome of
cyclophosphamide therapy [617–619] and the
doses of methadone used in addiction therapy
[620] Other drugs for which genetic variations
have been shown to be important are sibutramine
[621] and imatinib [622] Genetic variation has
also been reported to contribute to the metabo-
lism of the insecticide chloropyrifos [623]
The phenomenon of barbiturate-like enzyme
induction is still an issue in drug development,
however The point is not only drug interactions
but particularly the prospect of tumor promotion
in rodent cancer bioassays, which is probably un-
related to the P450 induction [138]
9.7.8 P450 2C8
The P450s in the 2C subfamily have been of in-
terest for some time Some of the first human
P450 preparations purified were probably P450
2C9, in retrospect [9, 10] A major impetus for re-
search in this field was the observed genetic poly-
morphism in ( S)-mephenytoin 4ʹ-hydroxylation
[624, 625], which led to efforts at purification
Purified proteins had some catalytic activity to-
wards mephenytoin [15], but subsequent in vivo
pharmacokinetic [626] and heterologous expres-
572
sion experiments [627] demonstrated a distinc-
tion between tolbutamide and ( S)-mephenytoin
hydroxylation Genomic analysis indicated the
complexity of the CYP2C gene subfamily [628]
Subsequently the subfamily was characterized in
terms of four P450s: 2C8, 2C9, 2C18, and 2C19
[629] P450 2C19 is the polymorphic ( S)-me-
phenytoin 4ʹ-hydroxylase [22, 630]; P450 2C9 is
involved in a considerable number of drug oxida-
tions (Fig 93) Two previous entries in the P450
nomenclature, 2C10 and 2C17, are considered al-
lelic variants of other genes or other artifacts and
have been deleted [631]
9.7.8.1 Sites of Expression
P450 2C8 was first purified from human liver
[15]; the enzyme is known to be expressed in
liver and kidney [632] The level of expression of
P450 2C8 has been estimated at 11–29 pmol/mg
in liver microsomes using LC–MS [54, 55] but
may be one of the more substantial P450s in the
kidney Other sites of P450 2C8 (mRNA) include
adrenal gland, brain, uterus, mammary gland,
ovary, and duodenum [633] Expression has also
been detected in cardiovascular tissue [634]
Proteomic analysis of human liver indicated
P450 2C8 was detected in all samples analyzed
[54, 55, 635] A lack of effect of gender, age, or
genotype on expression has also been reported
[636]
Kemper and his associates have presented evi-
dence that P450 2C8 exists as a dimer in membranes
[637] Avadhani and his associates have reported
that a significant fraction of P450 2C8 is localized
and functionally active in mitochondria [638]
9.7.8.2 Regulation
The level of P450 2C8 expression in human liver
varies at least 20-fold [54, 55, 639] Rifampicin
induces P450 2C8 in hepatocyte culture [387]
The enzyme appears to be inducible by barbitu-
rates [640] Transcriptional regulation involves
the nuclear receptors CAR, PXR, HNF-1α, and
the glucocorticoid receptor [641]
As mentioned earlier, P450 2C8 has reported
to be phosphorylated in vivo [297], but the effect
on catalytic activity is unknown Post-transcrip-
tional control of P450 2C8 by microRNAs 103
and 107 has been reported in human liver [642]
F. P. Guengerich
An interesting approach with the inhibi-
tor gemfibrozil has been used to estimate the
(human) in vivo half-life of P450 2C8 at 20 h
[643]
9.7.8.3 Genetic Variation
The http://wwwcypalleleskise website cur-
rently lists 16 allelic variants of P450 2C8 The
functional effects of eight of these have been re-
viewed by Totah and Rettie [644] For in vitro
studies on the functional effects of P450 2C8
variations, see [645, 646] Two coding region
polymorphisms involve the amino acid substitu-
tions I264M and K399R, with the latter appear-
ing in a haplotype with R139K [639] The rate of
oxidation of taxol (paclitaxel) is decreased with
the *3 allele (K399R/R139K), but the extent of
the decrease has been variable in different stud-
ies, ranging from 90 % [632] to 25 % [639, 647]
The *1C polymorphism appears to cause some
attenuation of the mean level of expression [639]
In vivo clinical effects of P450 2C8 variants have
been reviewed by Daily and Aquilante [648], and
the results are not always consistent with in vitro
studies
P450 2C8 variants show racial linkage [644,
649]
Some of the drugs considered for response (in
metabolism) in regard to genetic variation include
rosiglitazone [650, 651], amiodarone [652, 653],
and paclitaxel and 13-cis-retinoic acid [654]
9.7.8.4 Substrates and Reactions
P450 2C8 does not appear to have the general
significance of P450 2C9 or 2C19 in drug me-
tabolism (Fig 91b) An important substrate is
taxol (paclitaxel)(6α-hydroxylation) [152, 655]
Another substrate for P450 2C8 is all-trans-reti-
noic acid [656] P450 2C8 also contributes to the
oxidation of troglitazone [657] and verapamil,
rosiglitazone, cerivastatin, amiodarone, dapsone,
and amodiaquine [51, 639]
In general, P450 2C8 has relatively low cata-
lytic activity towards the known substrates of
P450s 2C9 and 2C19 However, Mansuy and his
associates have synthesized model substrates that
all of the human subfamily 2C P450s have activ-
ity towards [658, 659]
9  Human Cytochrome P450 Enzymes
The substrates of P450 2C8 have been re-
viewed by Totah and Rettie [644] and more re-
cently by Niwa and Yamazaki [660] P450 2C8 is
involved in the oxidation of pioglitazone [661],
repaglinide [662], montelukast (now consid-
ered a “classic” P450 2C8 ligand) [663, 664],
an endothelin ETA receptor antagonist (( H)-
(5S,6R,7R-2-isopropylamino-7-[4-methoxy-
2-[(2R)-3-methoxy-2-methylpropyl]]-5-(3,4-
methylenedioxyphenyl)cyclopenteno(1,2-b)pyri-
dine 6-carboxylic acid)) [665], imatinib [666],
and 4-hydroxyretinoic acid [667] P450 2C8 has
been assigned major roles in the metabolism of
amiodarone, amodiaquine, arachidonic acid,
cerivastatin, chloroquine, paclitaxel (taxol), re-
paglinide, retinoic acid, tazarotenic acid, and tro-
glitazone [644]
Molecular differences in genetic variants re-
garding several probe substrates have been con-
sidered in the context of binding affinity [668]
9.7.8.5 Structures
An X-ray crystal structure of P450 2C8 was
published by Johnson and his associates in 2004
[669] This structure is of interest in that two
molecules of palmitic acid, derived from the bac-
teria (used for heterologous expression), were
bound to the dimer interface Another series of
structures from the Johnson group [223] were
solved with montelukast, troglitazone, felodip-
ine, and 9-cis-retinoic acid present The size of
the active site is large (~1400 Å3), similar to that
of P450 3A4 ( vide infra), but more rigid, with
an “L-shape” In the case of 9-cis-retinoic acid,
a second molecule was located above the proxi-
mal ligand and is postulated to “push” the first
for more efficient oxygenation (although no evi-
dence for binding or catalytic cooperativity was
found) [223] There is flexibility in the active
site, and the ability of Arg-241 and other residues
to reorient was noted
A gating mechanism has been proposed for
P450 2C8 based on theoretical studies [670]
9.7.8.6 Inhibitors
In contrast to P450 2C9, sulfaphenazole is not a
strong inhibitor of P450 2C8 Mansuy’s group
synthesized several sulfaphenazole-based selec-
573
tive inhibitors of individual P450 2C enzymes,
including P450 2C8 [671, 672] Early work on
paclitaxel metabolism suggests that high con-
centrations of the natural flavonoids naringenin,
quercetin, and kaempferol and the synthetic α-NF
inhibit [152], but little in vivo inhibition would be
expected Walsky et al [673] have screened 204
drugs for P450 2C8 inhibition P450 2C8 inhibi-
tors have also been reviewed by Totah and Rettie
[644] and Niwa and Yamazaki [660] One of the
most useful diagnostic inhibitors is montelukast,
a leukotriene receptor antagonist that has also
been used in a crystal structure (Sect 785, vide
supra) [223] and has clinical significance [674]
Another selective inhibitor reported recently is
the tyrosine kinase inhibitor nilotinib [675]
Another selective inhibitor with clinical sig-
nificance is the fibrate gemfibrozil The mecha-
nism is unusual in that the glucuronide conjugate
is oxidized in the (large) active site of P450 2C8,
leading to irreversible inactivation due to heme
alkylation [676–678]
9.7.8.7 Clinical Issues
Some of the current issues have been reviewed
by Totah and Rettie [644] and Niwa and Yamaza-
ki [660]
Induction and inhibition of P450 2C8 are not
major issues at this point (Tables 96 and 97)
The epoxides formed from arachidonic acid (ep-
oxyeicosatrienoic acids or “EETs”) by P450 2C8
(and 2C9) have been considered in cardiovascu-
lar protection and in cancer therapy [679] How-
ever, no disease etiology with P450 2C8 has been
implicated at this point The most serious issue
is probably any impact on the disposition of the
cancer chemotherapeutic agent paclitaxel Poly-
morphisms may have some effect on in vivo 6α-
hydroxylation [632, 639], although any influence
may be modulated in part by the contribution of
P450 3A4 to other reactions [152]
One issue was the statin (3-hydroxy-3-meth-
yl-glutaryl-coenzyme A (HMG-CoA) reductase
inhibitor) cerivastatin, which was withdrawn
shortly after marketing due to rhabdomyolysis
issues This drug had several issues, but some
are related to it being a P450 2C8 substrate Two
problems were the interaction with the fibrate
574
gemfibrozil ( vide supra) [680] and the influence
of genetic variations of P450 2C8 [681–683]
Genetic variations in P450 2C8 have been
related to amodiaquine efficacy in malaria treat-
ment [684], response to paclitaxel treatment for
breast cancer [685], pioglitazone pharmacokinet-
ics [686], and celecoxib pharmacokinetics [687]
9.7.9 P450 2C9
In retrospect, many of the observations regarding
in vivo metabolism of barbiturates [2, 688] are
some of the first reports on what is now known as
P450 2C9 P450 2C9 is one of the major enzymes
involved in drug metabolism (Fig 91b) Some
of the first purified human liver P450s can now
be recognized as P450 2C9 [9, 10] A protein pu-
rified with some mephenytoin 4ʹ-hydroxylation
activity (P450MP-1) was also P450 2C9 [15], and
the cDNA corresponded to the N terminus de-
termined by Edman degradation [689] Proteins
now recognized as P450 2C9 were also purified
from liver on the basis of their oxidation of tol-
butamide [626] and hexobarbital [690, 691] The
human P450 2C subfamily is complex [628], and
characterization of individual members was not
achieved without heterologous expression and
careful analysis of catalytic activities [627, 692]
A transcript designated as P450 2C10 from this
laboratory had only two coding region changes
[628] This is now recognized as an allelic variant
of P450 2C9; the original assignment had been
based on the unexplained distinct 3ʹ noncoding
sequence [628]
9.7.9.1 Sites of Expression
P450 2C9 is primarily a liver P450 The hepatic
level of expression is probably the highest, on the
average, except for P450 3A4 (Figs 92 and 96)
[52]
All subfamily 2C P450 enzymes are expressed
at only low levels in fetal liver, including P450
2C9 [689], and levels rise quickly in the first
month after birth [693] Very low levels of P450
2C9 (1–2 % of adult values) were detected dur-
ing the first trimester in fetal livers with values
rising to ~ 30 % in the second and third trimes-
F. P. Guengerich
ters [694] Pharmacokinetic experiments with ac-
cepted P450 2C9 substrates indicate that the level
of hepatic P450 2C9 does not change with age, at
least to 68 years [695]
P450 2C9 is also expressed in the small intes-
tine (Fig 93) [696] P450 2C9 has also been de-
tected in aorta and coronary artery [634], which
may have relevance to hypertension and other
cardiovascular disease
In human (adult) liver microsomes, P450 2C9
is one of the most plentiful P450s, usually fol-
lowing only P450 3A4 One LC–MS proteomic
analysis gave a mean of 40 pmol P450 2C9/mg
microsomal protein (range 17–139) [55] An-
other analysis [54] reported 80 pmol P450 2C9/
mg microsomal protein for a pooled sample and a
mean of 28 pmol P450 2C9/mg microsomal pro-
tein (range 8–61) for another set (Fig 92)
9.7.9.2 Regulation
Early work with human hepatocytes showed in-
duction of P450 2C9 by barbiturates and rifam-
picin [697], consistent with earlier in vivo work
on the induction of barbiturate metabolism [688]
Subsequent studies have shown that P450 2C9
expression is induced by rifampicin, dexameth-
asone, and phenobarbital in hepatocytes [640,
698] The induction involves the glucocorticoid
receptor, CAR, and PXR, with CAR and PXR ap-
parently competing at the same site [699]
Recently evidence for action of CAR at an ad-
ditional site has been presented [700] It should be
emphasized that the action of CAR is somewhat
different than that of other receptors from the ste-
roid superfamily, in that it enhances transcription
in the absence of a bound ligand and some of the
control is at the level of nuclear translocation re-
lated to dephosphorylation of Thr-38 [179, 701]
Other factors involved are HNF-4 [702] and C/
EBPα [547], accounting at least in part for he-
patic localization
The P450 2C9 promoter contains several reg-
ulatory elements, including two HNF-4α sites, a
PXR site, a CAR site, and a glucocorticoid re-
sponse element [703, 704] In addition, GATA-4
[705] and ER α [706] regulation have been re-
ported
9  Human Cytochrome P450 Enzymes
9.7.9.3 Genetic Variation
The genetic polymorphism of P450 2C9 has
been studied extensively and has major clini-
cal significance, although P450 2C9 has not
been shown to have a critical function in normal
physiology Tolbutamide metabolism had been
reported to display polymorphism [707], which
was an impetus to purify the protein catalyzing
the hydroxylation [626] At least 65 alleles are
known, plus eight SNPs that have not been clas-
sified as to haplotype (http://wwwcypalleles
kise) Some of these are in the promoter region
and have functional consequences for drug thera-
py, eg, phenytoin [708] Two of the most studied
polymorphisms are *2 (R144C, rs1799853) and
*3 (I359 L, rs1057910) Both have much lower
frequencies in Asians and Africans [703] A six-
base deletion in the coding region lowered cata-
lytic activity in a recombinant enzyme [709] A
number of P450 2C9 SNVs have been identified
[710] and their racial linkage has been explored
[711] Of some interest, in addition to the *2 and
*3 alleles with generally lower catalytic activity,
is the *5 allele (of higher frequency in Africans)
with lower catalytic activity [712] Some of the
SNPs occur in the 5ʹ-flanking region and attenu-
ate the expression of P450 2C9 [713] Also of
interest is an unusual phenomenon in which the
CYP2C18 exon 1-like locus is fused with com-
binations of exons and introns from CYP2C9 to
yield chimeric RNA transcripts [714] Finally,
linkage between CYP2C8 and CYP2C9 genetic
polymorphisms has been reported [715]
The functional difference of 36 (protein) vari-
ants was analyzed in vitro and showed a 100-
fold variation in the catalytic efficiency towards
losartan ( kcat/Km) [716] Another study analyzed
the functional effects of 32 variants with warfarin
and tolbutamide, also reporting a variation of at
least two orders of magnitude [717]
It has long been known that the functional ef-
fect of a genetic variant in (the coding region)
of P450 2C9 is substrate dependent, which is not
surprising in light of our current understanding of
P450 function [718]
Some unusual variants are those involving
promoter variations [708], and a splice variant
with a ten-residue section substituted for the nor-
575
mal 18 residues near the N terminus gave a typi-
cal Fe2 + ·CO versus Fe2 + difference spectrum but
no catalytic activity [719]
The reason for the lower activities of the com-
mon *2 and *3 variants has been considered One
report has attributed the effect to changes in un-
coupling [720] Our own work, using arachidonic
acid as a ligand, indicates that the difference can
be explained simply by rates of reduction of P450
2C9, the step which appears to be rate limiting
[721]
9.7.9.4 Substrates and Reactions
P450 2C9 is one of the major P450s involved in
drug metabolism (Fig 91b) Some earlier aspects
of substrate specificity were reviewed by Miners
and Birkett [722] and by Rendic [51] One of the
early substrates examined was phenytoin, which
undergoes 4-hydroxylation [15] P450s 2C18 and
2C19 can also catalyze this reaction, but P450
2C9 is the major catalyst [723]
Mansuy’s group used the P450 2C9 inhibitor
sulfaphenazole to build a substrate common to all
four subfamily 2C P450 enzymes [658]
Some compounds normally in the body are
oxidized by P450 2C9, including arachidonic
and linoleic acids (epoxidation) [724] and vita-
min A (all-trans-retinoic acid, 4-hydroxylation)
[725], although the physiological significance is
unknown P450 2C9 oxidizes arachidonic acid to
several of the epoxides (EETs), which have im-
portant vascular and other properties [726–731]
Several reactions have been used as in vivo
probes, including tolbutamide, warfarin, flurbi-
profen, and losartan [732]
One substrate of recent interest is celocoxib,
a cyclooxygenase-2 inhibitor (Celebrex®) P450
2C9 is the major catalyst of oxidation, and vari-
ants affect the in vivo pharmacokinetic param-
eters [733, 734]
Several aspects of P450 2C9 reactions are of
concern regarding interpretation of results, at
least in in vitro research One issue is the effect
of solvents on catalytic activity [735] A concen-
tration of 1 % (v/v) CH3CN markedly inhibited
the catalytic activity of P450 2C9 [735] Another
issue is the enhancement of most reactions by
cytochrome b5 [266] Further work also showed
576
that apo-cytochrome b5 (devoid of heme) was as
effective as cytochrome b5 [736], arguing against
a need for electron transfer Other work showed
that even other P450s could enhance the rates
of some P450 2C9 reactions, even though those
P450s did not catalyze the reactions themselves
[266] These results are reminiscent of some of
the interactions of rabbit P450s 1A2 and 2B4 re-
ported by Backes [737]
Other work with P450 2C9 has provided
evidence for cooperativity in some reactions,
although the area has not been as developed as
for P450 3A4 ( vide infra) Dapsone and some
analogs enhance the binding and 4-hydroxylation
of diclofenac [738, 739] However, the activity
of P450 2C9 towards dapsone is unaffected by
diclofenac, in a situation similar to that of P450
3A4, aflatoxin B1, and α-NF [740] The inter-
pretation that P450 2C9 uses two binding sites
in these interactions is probably valid [739], al-
though (as with P450 3A4) the mechanism re-
mains to be elucidated (including the exact na-
ture of the binding)
The substrates for P450 2C9 have been re-
viewed by Niwa and Yamazaki [660] and com-
pared with the other three subfamily 2C P450s
Important drugs that are oxidized (mainly) by
P450 2C9 include irbesartan, losartan, phenyt-
oin, cyclophosphamide, tamoxifen, fluvastatin,
celocoxib, diclofenac, ibuprofen, lornoxicam,
meloxicam, naproxen, glibenclamide, glipizide,
tolbutamide, and warfarin [703] A list of the
drugs for which genetic variation in P450 2C9
has been an issue in clinical practice has also
been published (Table 95) [741] For in vitro
work, tolbutamide and diclofenac are considered
the most validated substrates [742] Tolbutamide,
recognized early as a substrate [626], is also used
for in vivo phenotyping [743]
P450 2C9 contributes to the 2-hydroxylation
of the oral contraceptive 17α-ethinylestradiol
[744] Another substrate is nabumetone [745]
Some compounds are activated to potentially
dangerous electrophilic products, including the
natural product safrole [746] and two drug-relat-
ed thiophenes [747]
F. P. Guengerich
9.7.9.5 Structure
Two important X-ray crystal structures have been
published, one with bound warfarin [41] and one
with flurbiprofen [748] The active site is rela-
tively large, allowing many drug substrates, and
Arg-108 is involved in binding to the carboxyl-
ates of some of the substrates [748] The structure
has been compared with those of P450 2C8 and
2C9 [749]
The importance of Arg-108 has been un-
derscored by site-directed mutagenesis stud-
ies [750], although the picture is more complex
than simple substrate charge pairing The roles of
other residues have also been studied by site-di-
rected mutagenesis, including Phe-114, Phe-476,
and Leu-208 [751] Movement of the helix B–C
loop and Arg-108 between the open and closed
(substrate bound) forms has been proposed [749]
Theoretical studies have been done on P450
2C9 protein dynamics and substrate binding [752,
753] Structures and other information have been
utilized to develop models for the prediction of
substrate binding and reactivity [754–758]
Changes in particular residues of P450 2C9
yield markedly different effects depending on the
substrate and reaction under consideration For
instance, the polymorphism *3 (I359 L), which
appears to be very conservative, changed catalyt-
ic efficiencies of different reactions by factors of
3- to 27-fold (in vitro) [759] Although the *2 and
*3 polymorphisms cause considerable changes
with some substrates, diclofenac metabolism
is not altered [760], consistent with the in vitro
findings
With the above caveats, the roles of a num-
ber of amino acids have been examined with
several reactions, although extrapolation to more
reactions requires caution Changes in Arg-97
and Arg-98 affected activity towards diclofenac
[761] Asp-293 has been shown to have a rela-
tively general structural role, possibly by bond-
ing to a partner amino acid or amide [762] Stud-
ies with coumarins suggested two sites, one for
π-stacking of aromatic rings and an ionic binding
site for organic anions [763]; many P450 2C9 li-
gands have an anionic charge [764, 765]
9  Human Cytochrome P450 Enzymes
P450 2C9 was converted into an enzyme with
( S)-mephenytoin 4ʹ-hydroxylation activity (i.e.,
P450 2C19-like) with a relatively small number
of changes (I99H, S220P, P221T, S286N, V292A,
F295 L) Conversely, P450 2C19 could be trans-
formed to an enzyme with warfarin hydroxyl-
ation activity similar to that of P450 2C9 (and
also sulfaphenazole binding) with the changes
N286S, I289N, and E241K [766] Mansuy’s lab-
oratory identified residues 476, 365, and 114 as
being important in diclofenac and sulfaphenazole
binding and in inactivation by tienilic acid [767]
Phe-114 is proposed to be involved in π-stacking
[767], perhaps serving the role proposed in the
coumarin studies mentioned earlier [763]
9.7.9.6 Inhibitors
Sulfaphenazole has been recognized as a highly
selective competitive inhibitor of P450 2C9 for
some time [768] and has relatively poor affin-
ity for other subfamily 2C P450 enzymes [671]
Mansuy’s group examined some other similar
compounds as ligands and inhibitors [764, 769]
Other inhibitors have been reported, although
some have relatively poor affinity [770, 771],
including several warfarin analogs [772] For an
early compilation of inhibitors, see Rendic [51]
Inhibitors of the subfamily 2C P450s have been
reviewed more recently by Niwa and Yamazaki
[660] See also Table 96 Hanatani et al [773]
reported no differences in the effects of inhibitors
on the *1 and *3 proteins (wild type and R144C),
although it seems likely that some coding region
variants may be found to differ
Tienilic acid is a mechanism-based inactiva-
tor of P450 2C9 [774] The mechanism involves
S-oxygenation, and the unstable product reacts
with P450 2C9 [775] Subsequently, autoim-
mune antibodies develop in some patients who
recognize unmodified P450 2C9 [774] Exactly
how (or if) this process is related to the hepatitis
seen in some individuals who used tienilic acid
is still unclear [776], but the phenomenon has
raised concerns about whether such processes
might be associated with other drugs that cova-
lently modify proteins and could lead to idiosyn-
cratic drug reaction in patients, one of the major
concerns today for safety assessment in drug de-
577
velopment Structure–activity relationships have
been reported on thiophenes other than tienilic
acid [765]
A series of type II (spectra) π-binding ligands
have been analyzed, in regard to their physical
parameters [777] Tienilic acid and (±) suprofen
are mechanism-based inhibitors [778, 779]
Finally, some hydroxylated products of warfa-
rin have been reported to be potent inhibitors of
P450 2C9 [780], although not the ones derived
from warfarin by P450 2C9
9.7.9.7 Clinical Issues
One of the major current clinical issues regard-
ing P450 2C9 is warfarin therapy (blood thinning
for strokes) The safety margin is narrow, and too
much warfarin can lead to internal hemorrhaging
There is a relationship between P450 2C9 geno-
type and warfarin dose [76, 781], and one issue is
whether genotyping is useful in management of
the drug [782] Both negative [783] and affirma-
tive [784–786] opinions have been expressed
Another interesting issue regarding P450 2C9
involves the drug tienilic acid The compound is
a substrate and a mechanism-based inactivator
of P450 2C9 [778] A product of tienilic acid be-
comes selectively covalently bound to P450 2C9
(Sect 796, vide supra) Some patients treated
with tienilic acid develop liver injury (hepati-
tis) Some patients treated with tienilic acid also
present with so-called liver–kidney microsomal
(LKM) antibodies in their blood These antibod-
ies react with unmodified P450 2C9 [774] Al-
though it could be proposed that the modified
P450 2C9 produces these autoantibodies and that
they are involved in the liver injury, a causal rela-
tionship has never been demonstrated
Genetic variations in P450 2C9 can lead to el-
evated levels of meloxicam [787] and celocoxib
[788] Polymorphisms have also been related to
the response to celocoxib in cancer prevention
[789]
The incidence of the *2 genotype has been
related to bosentan-induced liver injury [790,
791] The *2 genotype has also been reported
to increase the risk for hypoglycemia in diabetic
patients treated with sulfonylureas (eg, tolbuta-
mide) [792]
578
Finally, P450 2C9 genetic variation has been
reported to contribute to the incidence of stroke
[793] and to colorectal cancer [794]
9.7.10 P450 2C18
9.7.10.1 Sites of Expression
Relatively little has changed regarding P450
2C18 since the previous edition of this chapter
was published [149] Of the four human P450
subfamily 2C members, the level of hepatic ex-
pression is lowest for 2C18, at both the mRNA
[629, 795, 796] and protein levels [297, 796,
797] In intestine, P450 2C18 mRNA levels were
high, but no protein was detected [796] Expres-
sion in lung and skin has been reported to be sig-
nificant [382, 797–800]
9.7.10.2 Regulation
Relatively limited information is available about
regulation of P450 2C18 The levels of P450
2C18 mRNA in human liver and intestine were
both reported to vary 25-fold [796] At the pro-
tein level, expression in liver is reported to be
very low (< 25 pmol/mg protein) [55, 797]
Rae et al [387] reported that P450 2C18 was
not inducible by rifampicin in human hepato-
cytes, in contrast to P450s 2C8, 2C9, and 2C19
In a humanized transgenic mouse model,
P450 2C18 was expressed in liver and kidney in
a male-specific manner [801], but the relevance
to humans is unknown
9.7.10.3 Genetic Variation
Variations in the CYP2C18 gene have been re-
ported [802] but are not included on the website
http://wwwcypalleleskise Effects on expres-
sion and catalytic activities are not well charac-
terized One variant has an exon 5 deletion [803]
9.7.10.4 Substrates and Reactions
P450 2C18 has low catalytic activity in tolbu-
tamide methyl hydroxylation [803] P450 2C18
is active in phenytoin metabolism, having an
enzyme efficiency ( kcat/Km) for 4-hydroxylation
comparable to P450 2C9 and being more effi-
cient in bioactivation to a reactive product [800]
Catalytic activities have also been reported with
F. P. Guengerich
the substrates bisphenol A, diclofenac, the diclof-
enac derivative 2-[2(2,6-dichlorophylamino)]
phenylethanol, and verapamil [660, 795] Re-
cently, P450 2C18 has been reported to oxidize
5-hydroxythalidomide to a reactive product (but
does not catalyze the oxidation of thalidomide
itself) [804]
9.7.10.5 Structure
No crystal structures have been published In-
formation about the active site of P450 2C18
is relatively limited beyond comparisons of the
substrates mentioned above [795], the interaction
of other P450 2C proteins with general P450 sub-
family 2C substrates [659] and inhibitors [805],
and inferences from the crystal structures of the
other three P450 subfamily 2C crystal structures
(ie, 2C8, 2C9, 2C19) At least one homology
model has been published [806]
9.7.10.6 Inhibitors
P450 2C18 is appreciably inhibited by sulfa-
phenazole, a classical inhibitor of P450 2C9
Mansuy’s group has published on a set of sulfa-
phenazole derivatives that can be used in vitro
[671, 672]
9.7.10.7 Clinical Issues
The limited expression and repertoire of catalytic
activity for P450 2C18 still precludes consider-
ation of any clinical issues at this time
9.7.11 P450 2C19
Interest in P450 2C19 developed from the dis-
covery of the polymorphic metabolism of the
S-isomer of mephenytoin, the first major poly-
morphism to be studied following P450 2D6
[624, 625] Initial work led to the purifica-
tion of an enzyme with some ( S)-mephenytoin
4ʹ-hydroxylation activity [15] Exactly how this
and other gene products from the complex P450
2C subfamily [628, 689] were involved was un-
clear [807, 808] Although there were some in-
dications that the hexobarbital 3ʹ-hydroxylase
(P450 2C9) was the enzyme involved in mephe-
nytoin hydroxylation [691, 809], expression of
P450 2C9 cDNA [689] in yeast yielded a protein
9  Human Cytochrome P450 Enzymes
with activity towards tolbutamide but not ( S)-
mephenytoin [627, 692] P450 2C18 had also
been suggested to be the enzyme [629]
Wrighton [630] compared ( S)-mephenytoin
4ʹ-hydroxylation activity in different liver sam-
ples with a protein gel band recognized by anti-
rat P450 2B1 and correlated this with P450 2C19,
a sequence which had been reported earlier Sub-
sequently, Goldstein et al [22] expressed several
subfamily 2C P450 cDNAs in yeast and identi-
fied P450 2C19 has having the highest activity
with mephenytoin
9.7.11.1 Sites of Expression
Apparently, significant expression only occurs in
the liver As with other human P450s examined
to date, there appears to be no gender difference
[810] P450 2C19 has been detected in human
liver microsomes using LC–MS proteomics
methods [297] P450 2C19 is a relatively minor
P450 in its abundance, probably accounting
for < 5 % of total P450 even in EM liver samples
(Fig 92) [54]
Neither P450 2C19 nor ( S)-mephenytoin
4ʹ-hydroxylation activity was detected in fetal
liver samples [689]
9.7.11.2 Regulation
In vivo work had shown that the enzyme was
inducible by rifampicin [811] Thus, this P450
differed from P450 2D6 in that it was both poly-
morphic and inducible Analysis of the regula-
tory system has not been extensive, but studies
with human hepatocytes demonstrated induction
of P450 2C19 mRNA by rifampicin, dexametha-
sone, and phenobarbital [698]
The regulation of transcription of P450 2C19
has been reviewed elsewhere [812] P450 2C19
expression is downregulated by ER α [706] Reg-
ulatory variations (eg, *17) can increase rates
of transcription (~ twofold) [813], and this vari-
ant has been associated with peptic ulcer disease
[814]
9.7.11.3 Genetic Variation
The variation and polymorphisms are now rela-
tively well understood The incidence of the PM
phenotype in Caucasians is generally 2–3 %, but
579
the incidence in Asians (at least Japanese, Kore-
ans, Chinese) is ~ 20 % [167] On some Pacific
islands, the incidence has been reported to be as
high as 75 % [815, 816] In Thai, Burmese, and
Karen populations, the incidence of PMs is “in-
termediate,” ie, 8–11 % [817]
The major defect in Caucasians and Japanese
was first identified in an exon 5 mutation that
leads to an aberrant splice site and yields a trun-
cated protein [818] Other variants are collected
at the website http://wwwkise/cypalleles/ These
are rather diverse and include a mutation of the
initiation codon [819] and altered enzymatic
properties [815] At the time of the update of this
chapter, at least 48 allelic variants are known,
with an additional 20 SNVs for which haplotypes
have not been determined
9.7.11.4 Substrates and Reactions
( S)-Mephenytoin 4ʹ-hydroxylation is the classic
reaction attributed to P450 2C19 ( vide supra)
Early studies on the basis of the polymorphism
of tolbutamide hydroxylation suggested that the
same enzyme might be responsible for both ac-
tivities [626], but in vivo work [626] and heterol-
ogous expression studies [627] distinguished the
two activities Nevertheless, recombinant P450
2C19 has now been shown to have some tolbuta-
mide hydroxylation activity [820]
A list of P450 2C19 reactions has been pub-
lished by Rendic [51] Another list of P450 2C19
substrates has been compiled, and catalytic ef-
ficiencies are compared to the other subfam-
ily 2C P450s [660] The scope of P450 2C19 in
drug metabolism is rather significant (Fig 91b,
Tables 95, 96 and 97) One drug of particular
interest is the ulcer drug omeprazole (and re-
lated compounds), because individuals with low
enzyme activity show a better response to treat-
ment for ulcers [79, 80] Some of the early varia-
tions seen in warfarin metabolism [821] can be
explained by the finding that P450 2C19 cata-
lyzes the 8-hydroxylation of ( R)-warfarin [822]
18-Methoxycoronaridine is O-demethylated by
P450 2C19 [823] P450 2C19 is responsible for
the 5- and 5ʹ-hydroxylation of thalidomide, an
older drug notorious for teratogenic effects that
has been “rediscovered” [824] Whether the ge-
580
netic variation is related to the birth defects is
unclear
P450 2C19 can also catalyze steroid oxida-
tions, including progesterone 21-hydroxylation
and testosterone 17-hydroxylations [825] The
organophosphate insecticide diazinon is activat-
ed in human liver by P450 2C19 [826]
One of the more well-studied substrates is the
drug clopidogrel (Plavix®), which is converted
to its active form in two steps, both catalyzed (in
large part) by P450 2C19 [827] (see Sect 7117
regarding clinical issues) P450 2C19 is involved
in the N-oxidation of voriconazole [828], and
genotype is a major factor contributing to the
highly variable in vivo pharmacokinetics [829]
Another substrate is the drug clobazam [830],
and genetic variation in P450 2C19 affects the
efficacy of therapy [831] Other drug substrates
of interest are escitalopram [832], fenbendazole
[833], and thalidomide [834, 835]
As with other P450 2C subfamily enzymes,
P450 2C19 activities are usually stimulated by
cytochrome b5 [736] In this case, stimulation
is not dependent on the heme in the cytochrome
b5 and thus electron transfer cannot be involved
[736]
9.7.11.5 Structures
Johnson and his associates [749] have reported
an X-ray crystal structure of P450 2C19 con-
taining the inhibitor (2-methyl-1-benzofuran-
3-yl)-(4-(hydroxy-3,5-dimethylpentyl) metha-
none A comparison has been made with the
available structures of P450 2C8 and 2C9 ( vide
supra) The size of the active site is similar to
that of P450 2C9 and much smaller than that of
P450 2C8
Goldstein and her associates did chimera
analysis and then site-directed mutagenesis on
P450 2C9 to convert it to a protein with P450
2C19-characteristic omeprazole hydroxylation
activity [836] Only three changes were needed
to achieve the activity of wild-type P450 2C19—
I99H, S200P, and P221T However, at least
three different mutations were needed to convert
P450 2C9 to an enzyme with ( S)-mephenytoin
4ʹ-hydroxylation activity, even to a catalytic ef-
ficiency one third of wild-type P450 2C19 (*1)
F. P. Guengerich
[837] In an opposite experiment, P450 2C19
was converted to a P450 2C9-like warfarin hy-
droxylase with high sensitivity to sulfaphenazole
[766] Residues 286 and 289 appear to be im-
portant However, these residues may exert an
indirect influence by adjusting the active site or
substrate access channels [837]
9.7.11.6 Inhibitors
Niwa and Yamazaki [660] have compiled a list
of inhibitors of subfamily 2C P450s Two diag-
nostic inhibitors validated for P450 2C19 reac-
tion phenotyping (in liver microsomes) are (+)N-
3-benzylnirvanol [838] and (−)N-3-benzylpheno-
barbital [839] The point has been made that the
choice of “probe” substrates can influence in vitro
inhibition profiles [840], which is not surpris-
ing in light of experience with P450 3A4 ( vide
infra). As indicated in Sect. 7.11.5 ( vide supra),
(2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-
dimethyl)methanone was the inhibitor used to
obtain the P450 2C19 crystal structure [749]
Two interesting inhibitors of practical interest
are cannabidiol (marijuana constituent) [841] and
grapefruit juice (extensively studied with P450
3A4) [842]
9.7.11.7 Clinical Issues
The issue is the genetic variation, particularly
so for drugs marketed in Asian populations At
least eight alleles have been associated with the
PM phenotype [816] Desta et al [816] reviewed
some of the drugs for which the 2C19 phenotype
is a problem (Tables 95, 96 and 97) Most phar-
maceutical companies and regulatory agencies
discourage development of a P450 2C19 substrate
because of potential problems for PM individu-
als Mephenytoin itself is seldom used and is not
an issue Several studies indicate that PM patients
may have more effective therapy (for ulcers)
with omeprazole and related compounds [816,
843–846] The popular proton pump inhibitors
omeprazole, lansoprazole, pantoprazole, and ra-
beprazole are metabolized by P450 2C19 (but not
esomeprazole), and genetic variation is an issue in
use for acid-related intestinal disease [847]
Another major drug of interest is clopido-
grel (Plavix®), which is a P450 2C19 substrate
9  Human Cytochrome P450 Enzymes
(converting the drug to the active form in two
steps) [827] The question has been raised as to
whether the use of genotyping is useful in pre-
scribing (correct doses of) this drug [848] Both
positive [849–852] and negative [853] opinions
have been expressed An Australian study con-
cluded that genotyping for the use of clopidogrel
was economically justified but for ticagrelor was
not [854]
Other drug issues regarding P450 2C19 varia-
tion involve thalidomide therapy [835] and treat-
ment of small-cell lung cancer with tivantinib
and erlotinib [855]
As with many polymorphisms, epidemiology
studies have been done to explore risks to dis-
eases in the absence of information about etiol-
ogy, substrates, etc Some of the reports include
suggestion of more hepatocellular cancer in PMs
[856] and lack of association of leukemia with
polymorphism [857] Other possible relation-
ships have been explored, but evidence for any
associations is limited at this time [816] Genetic
variation in P450 2C19 has also been considered
in regard to cancers of the breast (decreased with
*17) [858], biliary tract [859], and digestive sys-
tem [860] Other diseases in which P450 2C19
genetic variation has been considered include en-
dometriosis [861], essential tremor [862], peptic
ulcers [814], and mortality following acute myo-
cardial infarction [863]
9.7.12 P450 2D6
P450 2D6 is one of the main enzymes involved
in drug metabolism (Fig 91b) It was the first
“xenobiotic-metabolizing” P450 recognized to
be under monogenic regulation [11]
9.7.12.1 Sites of Expression
P450 2D6 is expressed mainly in liver and was
first purified from liver microsomes [14, 19] In
the average person, P450 2D6 accounts for ~ 5 %
of total P450 (with wide variation) [52] Esti-
mates of the level of P450 2D6 vary in different
studies An older immunoblotting analysis of 60
samples (one-half Caucasian, one-half Japanese)
showed a mean of 5 pmol P450 2D6/mg micro-
581
somal protein [52] Similar levels were reported
in adolescents by Stevens et al [864] One LC–
MS analysis gave a mean of 30 pmol P450 2D6/
mg microsomal protein [865], but a more recent
LC–MS analysis gave a mean value of 12 pmol
P450 2D6/mg microsomal protein [55] Another
yields values of 4–12 pmol P450 2D6/mg mi-
crosomal protein [54] However, this enzyme is
involved in the oxidation of ~ 25 % of all drugs
oxidized by P450s (Fig 91b)
Developmental studies showed little P450
2D6 in early fetal liver and a rapid increase in
protein shortly after birth, yielding a peak accu-
mulation in newborns and decline in adulthood
[866] In another study, P450 2D6 levels increase
during development, being low in fetal liver, in-
creasing the third trimester and then somewhat
high postnatally, increasing during childhood and
adolescence [864]
P450 2D6 is also expressed at low levels in
lung (bronchial mucosa and lung parenchyma)
[867] Another site of P450 2D6 expression is
brain, with localization in large principal neurons
[868] Higher levels of brain expression have
been reported in alcoholics [869]
In the central nervous system, there is evi-
dence of several endogenous substrates and for
neurophysiological differences in different geno-
types ( vide infra) Recently, a transgenic mouse
line expressing human P450 2D6 has been devel-
oped and may provide insight [870]
P450 2D6 is generally considered a microsom-
al protein, but Avadhani and his associates have
shown that an N-terminal chimeric signal in the
protein (residues 23–33) also mediates targeting to
mitochondria [871] Naturally occurring variants
can affect the localization, and phosphorylation has
a role [872] In the mitochondria, P450 2D6 is ca-
pable of using electrons from adrenodoxin, and the
mitochondrial localization may be an issue in the
bioactivation of the neurotoxicant 1-methyl-4-phe-
nyl-1,2,3,6-tetrahydropyridine (MPTP) [873]
9.7.12.2 Regulation
All information available indicates that P450
2D6 is not inducible Some factors are known to
be involved in constitutive expression, including
C/EBPα [547] and HNF4α [171]
582
Phosphorylation of P450 2D6 (in vivo) has
also been reported [297]
9.7.12.3 Genetic Variation
The wide variability in the activity of P450 2D6
is attributed to genetic variation (Fig 94) Re-
duced ability to metabolize the drug debriso-
quine was first noted (personally) by Smith in a
drug trial Subsequent work led to the report of
polymorphic hydroxylation of debrisoquine [11],
including a phenotypic hypotensive response
[874] Racial differences were first noted in Afri-
cans [62] The phenomenon of polymorphic de-
brisoquine hydroxylation [875] was also reported
for sparteine oxidation [13, 876]
Today, P450 2D6 is considered to be a very
polymorphic P450 At least 165 genetic variants
are known (and 26 more not characterized for
haplotype) (http://wwwcypalleleskise) The ef-
fects of formation of some have been identified
[877] but not all (particularly the coding region
variants, where function may vary depending
upon the substrate and inhibitor) There is also
variation of activity in vivo within each genotype
[878], possibly due to differences in regulatory
factors (or possibly the existence of endogenous
or food-borne inhibitors)
The most significant decreases in activity for
P450 2D6 alleles, aside from mRNA splicing
problems and gene deletion [170], are considered
to result from less stable proteins [879], although
low-activity P450 2D6 variant proteins have also
been reported [880, 881] Some of the allelic dif-
ferences are present as haplotypes [882]
In addition to the “poor” and “intermediate”
metabolizer phenotypes, a “very extensive” or
“ultra-metabolizer” (UM) phenotype was iden-
tified in early work (Fig 94) Ingelman-Sund-
berg’s group identified the basis for this as a gene
duplication, with up to 13 copies present in some
individuals [63] The main form of this phenom-
enon is a haplotype resulting from gene duplica-
tion [63, 883] The amplification appears to result
from unequal segregation and extrachromosomal
replication of the acentric DNA [884] As many
as 7 % of Caucasians show some of this effect,
and the incidence is even higher in some Middle
Eastern populations [885]
F. P. Guengerich
9.7.12.4 Substrates and Reactions
Since the original work with debrisoquine [11],
many substrates and reactions have been re-
ported for P450 2D6 In some cases, the role of
P450 2D6 is very dominant in vivo and the clini-
cal manifestations of genetic polymorphism are
important and even deadly [874, 886] Lists of
P450 2D6 substrates have been published [51];
see Table 95
P450 2D6 catalyzes many of the basic kinds
of oxidative reactions of P450s, eg, aliphatic
and aromatic hydroxylations, heteroatom deal-
kylations, etc [887] In early work in this labora-
tory [888], the observation was made that many
of the substrates contained a basic nitrogen atom
situated ~ 5 Å away from the site of oxidation,
possibly due to a specific anionic charge in P450
2D6 Subsequently more detailed pharmaco-
phore models have been developed [889–892]
All of these are based on the premise that a basic
nitrogen atom in the molecule interacts (coulom-
bic bond) with an acidic amino acid in P450 2D6,
usually Asp-301 in most studies (More recent
work shows a role for Glu-216, however, vide
infra)
The use of these models requires some cave-
ats Although the pKa of the substrate has been
proposed to have a dominant influence [893],
work in this laboratory has shown that the in-
trinsic pKa of a substrate can be altered in the
active site of P450 2D6 [894] Another issue is
that some compounds with a single amine ni-
trogen undergo N-dealkylation, eg, deprenyl
[895], which cannot be easily rationalized with
an amine-oxidation site interatomic distance of
5–7 Å Some substrates devoid of basic nitrogen
(and any nitrogen) have been reported, including
steroids [896, 897] Spirosulfonamide and sever-
al analogs do not have a basic nitrogen but have
been shown to be good substrates and ligands for
P450 2D6 [898] (Fig 916)
A large fraction of the population is devoid of
functional P450 2D6 but appears to function well
This information may be interpreted to mean that
P450 2D6 has no “physiological” substrate Nev-
ertheless, some reactions may be catalyzed by
P450 2D6 and yield physiological responses that
yield less than obvious changes For instance,
9  Human Cytochrome P450 Enzymes 583

Fig. 9.16   Analogs of spirosulfonamide and other P450 2D6 ligands Kd values were estimated by spectral titration
[898] (With kind permission from Springer Science + Business Media: [149], Fig 1010)

overexpression of human P450 2D6 in trans-
genic mice produces a higher capability to adapt
to anxiety [870] Tryptamine has been proposed
as a physiological substrate in one study [899]
but discounted in another [900] Proposed physi-
ological reactions catalyzed by P450 2D6 are
the O-demethylation of 5-methoxytryptamine,
5-methoxy-N,N-dimethyltryptamine, and pino-
line (6-methoxy-1,2,3,4-tetrahydro-β-carboline)
[900, 901] Whether significant catalysis is seen
at the low concentrations seen in vivo and what
the effect is remains to be established
P450 2D6 catalyzes tamoxifen α-hydroxylation
and formation of α,4-dihydroxy tamoxifen [902]
P450 2D6 has been reported to be the major en-
zyme involved in the O-demethylation of the de-
signer drug p-methoxymethamphetamine [903]
MPTP, a breakdown product of a designer drug,
is oxidized by P450 2D6-catalyzed aromatic hy-
droxylation and N-demethylation [904] P450
2D6 can also convert MPTP to MPP+ (1-methyl-
4-phenylpyridine), as shown in mitochondria,
and contributes to neurotoxicity in the substantia
nigra [873]
584
Possible endogenous substrates have also
been considered, including 5-methoxyindole-
thylamine [900] Human P450 2D6 also cata-
lyzes some important steps in mammalian opioid
biosynthesis, including conversion of ( R)-retic-
uline to salutaridine, thebaine to oripavine, and
codeine to morphine (Fig 917) [905, 906]
Modi et al [907] reported differences in prod-
uct profiles of P450 2D6 reactions supported
with artificial oxygen surrogates and NADPH-
P450 reductase and interpreted these as evidence
for an allosteric influence of the reductase Sub-
sequent experiments in this laboratory did not
support this conclusion and are in accord with
some differences in the chemical mechanisms for
the oxygen surrogates [908]
Detailed experiments have been done on the
O-demethylation of 3- and 4-methoxyphenethyl-
amine by P450 2D6 [909] Analysis of kinetic
deuterium isotope effects, kinetic simulation, and
other experiments yields evidence that both late
steps in O2 activation and C–H bond breaking
contribute to kcat The exact meaning of Km is still
not defined with this and most P450 reactions
Some of the P450 2D6 allelic variants show no
changes in kcat for certain reactions but do show
Km differences [910]; these are probably more
complex than simple “affinity” for the substrate
9.7.12.5 Structure
The active site of P450 2D6 has been the subject
of considerable interest, probably because of the
relevance to issues in the pharmaceutical indus-
try Some residues have been identified as being
important, and early homology and pharmaco-
phore models have been published [889–892,
911–917]
The original clone reported by Gonzalez [34]
had Met at position 374, but this now appears to
be a gene variant or artifact; the correct residue is
Val [918, 919] This residue appears to be in the
active site and affects activity
Rowland et al [920] published an X-ray crys-
tal structure of a slightly modified P450 2D6
without a ligand Johnson and his associates
[323] subsequently published a structure of P450
2D6 with the ligand prinomastat bound The lat-
ter structure had the *1 Val-374 instead of Met-
F. P. Guengerich
374 and differed significantly from the earlier
P450 2D6 structure devoid of a ligand The dif-
ferences in the structure are attributed to the flex-
ibility of P450 2D6 and conformational changes
seen with binding [323] High-pressure experi-
ments indicate that P450 2D6 is a much more
rigid molecule when a substrate is bound [921]
The crystal structures indicated that both Asp-
301 and Glu-216 are in position to form ionic
bonds to charged amines [323] Although much
of the earlier literature was focused on Asp-301,
both Asp-301 and Glu-216 have anionic changes
that are used in binding positively charged sub-
strates [922, 923] Interestingly, site-directed mu-
tagenesis of a few residues of P450 2D6 allowed
oxidation of quinidine [924], which is only an
inhibitor of the wild-type enzyme [97] Previous
studies had shown that neutral molecules are li-
gands of P450 2D6 (Fig 917) [898], in contrast
to earlier views about the need for basic atoms in
ligands Even acidic (eg, pactimibe) molecules
can be ligands and substrates [925]
Newer predictive pharmacophore schemes
have been developed, some based in part on the
available crystal structure of P450 2D6 [926–
928]
9.7.12.6 Inhibitors
Many inhibitors of P450 2D6 have been reported
(Table 96) [51, 890] Inhibition of P450 2D6 is
an undesirable issue in drug development, and
most pharmaceutical companies have screening
programs in place As with some other P450s
(eg, P450 3A4, vide infra), inhibitor screening
results have been reported to be dependent upon
the test substrate used [929] Structure–activity
relationship studies have been done with quini-
dine analogs [930]
The most established inhibitor of P450 2D6
is quinidine [931] The KI is ~ 50 nM and inhibi-
tion is competitive Interestingly, quinidine is not
a substrate for P450 2D6 [97, 909]
Mechanism-based inactivation of P450 2D6
is known, eg, 5-fluoro-2-[4-[(2-phenyl-1H-im-
idazoyl-5-yl)methyl]-1-piperazinyl]pyrimidine
(SCH66712) [932] In the case of this compound,
covalent binding to protein was detected, but the
position of attachment has not been identified
 9  Human Cytochrome P450 Enzymes

Fig. 9.17   Steps in mammalian morphine synthesis [905, 906]. P450 3A4 and 3A5 catalyze the O6-demethylation of thebaine with 3A5 being ~ 10-fold more active than P450
3A4 [906]. The microsomal oxidation of oripavine is very slow and is considered not to contribute. The conversion of codeinone to codeine is catalyzed by a reductase. (Adapted
with kind permission from Springer Science + Business Media: [149])
585
586
Two related mechanism-based inactivators,
1-[2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-
4-[4-(trifluoromethyl)-2-pyrinyl]piperazine
[933] and 5-fluoro-2-[4-[(2-phenyl-1H-imidzo-
5-yl)methyl]-1–1piperazinyl]pyrimidine [934],
modify the apoprotein Several other mechanism-
based inactivators of P450 2D6 have been report-
ed [935], including methylenedioxymethamphet-
amine [936] but not metoclopramide [937]
Other reported P450 2D6 inhibitors include
sanguinarine [938] and cannabidiol, a marijuana
constituent [939]
9.7.12.7 Clinical Issues
The clinical issues regarding P450 2D6 are con-
siderable due to the large variation in the genetics
in the population (Figs 91b, 95, and 96) and
the contribution of P450 2D6 in the total scheme
of drug metabolism (Fig 91b) Individuals seem
to be rather tolerant of the wide variability in ex-
pression with many marketed drugs, probably
because of generally wide therapeutic windows
selected for in the basic process of drug develop-
ment However, P450 2D6 PMs can be at consid-
erable risk when they encounter certain drugs, as
first observed by Smith [11, 874] The problem is
seen with drugs having a relatively narrow thera-
peutic index, eg, debrisoquine [11], phenformin
[940], captopril [941] The effects of P450 2D6
deficiency are seen not only in short-term treat-
ments but also in long-term therapy [942] The
issue of ineffectiveness of drugs that are very
rapidly metabolized by “very extensive” (UM)
metabolizers is an issue (Fig 94) Modeling of
the variability is still an issue [943] and may be a
function of particular drugs The issue of whether
genotyping/phenotyping is economical has been
considered, particularly in the case of neuroac-
tive and antipsychotic drugs [944, 945] The
overlap between P450 2D6 substrates and neuro-
active drugs is also an issue in drug development,
largely due to the overlap of these two groups of
compounds [946]
Zhang et al [947] have commented on the role
of genetic polymorphisms in withdrawal of drugs
from the market Another concern, in the context
of drug–drug interaction, is “phenoconversion,”
making an individual a “PM” due to inhibition
F. P. Guengerich
(eg, prescribing quinidine to a P450 2D6 “EM”)
[948]
Although it seems very possible that individu-
als might die due to drug interactions related to
genetic variations, there is actually only very lim-
ited evidence that this has happened A study of
individuals who died due to drug toxicity did not
show any relationships to known genetic varia-
tions in P450s [949] There is a report of near-
fatal tramadol cardiotoxicity in a P450 2D6 ultra-
rapid metabolizer [950] In 2004, an infant died
due to codeine intoxication when nursing from
his mother, who was an ultrarapid (UM) P450
2D6 metabolizer (and generated an overdose of
morphine) [81]
One of the substrates of P450 2D6 is tamoxi-
fen [902], an ER antagonist used extensively in
breast cancer therapy There has been contro-
versy regarding application of genotyping to im-
prove therapy Recommendations in favor of ge-
notyping and against it [951] have appeared, and
several meta-analyses conclude that more study
is needed [952–954]
Another issue with P450 2D6 is the relevance
of the genetic variation to cancer risks In 1984,
Idle [123] reported an association of lower risk
of lung cancer (in smokers) with the P450 2D6
PM phenotype These epidemiological results
were repeated in some studies [955] but not oth-
ers [124] Attempts were made to resolve the
discrepancies on the basis of levels of smoking
[956] Although some expression of P450 2D6 is
detectable in lung [867], no clear role for P450
2D6 in carcinogen activation could be estab-
lished, even with crude tobacco smoke fractions
[125] The issue of whether lung cancer is associ-
ated with P450 2D6 was not resolved by chang-
ing analyses from phenotyping to genotyping
The generally accepted epidemiological conclu-
sion today is that P450 2D6 is not related to lung
cancer [124, 957–960]
Other epidemiology studies have suggested
relationships of P450 2D6 with other cancers
[961, 962], but these findings have not been scru-
tinized as rigorously as the lung cancer hypoth-
esis
Another disease in which P450 2D6 has been
proposed to play a role, on the basis of epidemiol-
9  Human Cytochrome P450 Enzymes
ogy, is Parkinson’s disease [963] Contradictory
findings have been reported [964, 965] Although
a hypothesis has been raised that induction of
P450 2D6 by smoking might explain some dis-
crepancies [966], this proposal lacks biological
plausibility in light of the known refractory re-
sponse of P450 2D6 to induction Some positive
evidence for risk of Parkinson’s disease with
“PM” P450 2D6 status has been published [953]
Autoantigens (LKM1) that recognize P450
2D6 have been known for some time [967, 968]
These antibodies are associated with some cases
of hepatitis The exact mechanism of how they
arise is still unclear, as is the relationship with
hepatitis The antibodies may arise by molecu-
lar mimicry [969] or they may result from P450
2D6 translocation to the outer plasma membrane
[970, 971] These LKM1 antibodies may serve as
diagnostic tools for particular types of hepatitis
[972, 973], but causal relationships have never
been demonstrated
P450 2D6 genetic variation has been consid-
ered, with some evidence, in explaining depres-
sion [974], suicide in relation to serotonin use
[975], and type A versus type B personality [976]
9.7.13 P450 2E1
The microsomal mixed-function oxidation of
ethanol was reported nearly 50 years ago [977]
The view that ethanol could be a P450 substrate
was not readily accepted because of the hydro-
philic nature of the molecule, but Lieber’s group
characterized the enzymatic reaction in rat liver
[978, 979] Collaborative work with Levin led
to the isolation of a P450 (“j”), which was also
found to be inducible by isoniazid [980] Human
P450 2E1 was purified by Wrighton et al [23],
and Gonzalez’s group characterized the human
gene [981]
9.7.13.1 Sites of Expression
The greatest concentration is in the liver, and P450
2E1 is a moderately abundant P450 (Fig 92)
Using LC–MS proteomics, Shrivas et al [635]
detected P450 2E1 in all human liver microsomal
samples analyzed Seibert et al [982] used LC–
587
MS and reported values of 88–200 pmol (P450
2E1)/mg microsomal protein in four samples,
which seems unusually high compared to other
P450s (Fig 92) [55] and an average total of
~ 500 pmol/mg protein [10] The interindividual
variation is an order of magnitude (Fig 96) [52,
983] A racial difference exists, with Japanese
samples having mean expression levels lower
than Caucasians (Fig 96) [69]
P450 2E1 was reported not to be present in
fetal liver but appears within a few hours after
birth, regardless of the gestational age [984]
However, P450 2E1 has also been reported to be
detectable as early as gestational day 93 in fetal
liver [985] The activity increases during the first
year of childhood, and transcriptional regulation
due to hypermethylation has been proposed
P450 2E1 is expressed in many extrahepatic
sites, including lung [535], esophagus, small
intestine [382], brain [986, 987], nasal mucosa
[988], and pancreas [989] (some of the evidence
is extrapolated from rat work and not necessarily
extendable to humans)
P450 2E1 is found mainly in the endoplasmic
reticulum With heterologous expression in bac-
teria (rabbit), P450 2E1 was membrane bound
and catalytically active even when amino acids
3–29 are deleted [38, 990] The same bacterial
localization was seen with human P450 2E1 from
which 21 N-terminal residues were deleted [991]
However, P450 2E1 can show some unusual lo-
calization in mammalian systems Ingelmann-
Sundberg’s group deleted residues 2–29 of rat
P450 2E1 and demonstrated the presence of a
fragment in the mitochondria of a mouse hepa-
toma cell line [992] Avadhani’s group found
P450 2E1 intact in rat liver mitochondria and re-
ported that it could couple with adrenodoxin and
adrenodoxin reductase with full catalytic activity
[993] Subsequent work demonstrated a cryptic
mitochondrial-targeting signal at positions 21–31
that was activated by cyclic AMP-dependent
phosphorylation of Ser-129 [994] Neve et al
[995] found that the charge of the N terminus of
(rat) P450 2E1 was such that part is directed to
either the lumen of the endoplasmic reticulum or
the outside of the plasma membrane Migration
of human P450 2E1 into mitochondria and the
588
relevance to oxidative stress have been published
[996, 997]
9.7.13.2 Regulation
Early work in experimental animals was focused
on the induction of P450 2E1 in rat liver [978]
Subsequently many other chemicals, includ-
ing isoniazid and some solvents, were shown to
induce P450 2E1 [998] It is also of interest to
note that some of the common polycyclic hydro-
carbons and other inducers of P450 1 family en-
zymes attenuated the level of P450 2E1 in rats
[998] The regulation of P450 2E1 has come to
be recognized to be relatively complex, involv-
ing transcriptional activation, mRNA stabiliza-
tion, increased mRNA translation efficiency, and
decreased protein degradation [999]
HNF-1 is believed to regulate CYP2E1 gene
transcription [183] Obesity and diabetes are
known to modulate P450 2E1 in rat models In rat
hepatocyte cell culture, insulin attenuated mRNA
levels and glucagon or dibutyryl cyclic AMP
elevated mRNA, with the latter effect down-
regulated by a protein kinase A inhibitor [1000]
mRNA levels are also selectively attenuated in
mice or cell culture (relative to other P450s) by
interleukin-6 [1001], interleukin-4 [1002], or
interleukin-1β or tumor necrosis factor (TNF)α
[1003] Multiple mechanisms have been invoked,
including kinase pathways, control of HNF-1α
function, and regulation of other transcription
factors
Evidence for control at the level of mRNA
stability and enhanced translation efficiency
has been presented by Novak [182, 1004] The
3ʹ-region of the gene appears to be important
in stability The relevance of this rat model to
human P450 2E1 is still unknown
Another mechanism, generally well accepted
although not completely understood, involves
protein stabilization by substrate Rat studies (in
vivo) showed that ~ half of P450 2E1 was lost in
1 h, and a ubiquitin-linked pathway was invoked
[1005] Similar findings were also reported for
human P450 2E1 in HepG2 cells [1006] An at-
tempt has been made to estimate the half-life of
P450 2E1 in humans in vivo using chlorzoxa-
zone pharmacokinetics and a P450 2E1 inhibitor
F. P. Guengerich
[1007] The half-life was estimated at 50 ± 19 h,
but this approach may not be sensitive enough to
detect a short-lived P450 2E1 pool
Some aspects of P450 2E1 regulation have
been reviewed by Gonzalez [184] P450 2E1 is
also regulated by miR-378 [1008] Daly has re-
viewed genetic variation involving P450 2E1
gene regulation [1009]
P450 2E1 phosphorylation has been detected
in vivo [297], although the relevance is not yet
clear
9.7.13.3 Genetic Variation
P450 2E1 is polymorphic At least 14 allelic vari-
ants of P450 2E1 are known, with four additional
variants for which the haplotype has not been de-
termined (http://wwwcypalleleskise) In some
cases, the functional effects of coding region
substitutions have been defined [1010] Because
of the nature of many of the substrates, many ef-
forts have been made to determine the relevance
of SNPs and other variations to disease and risk
of injury. A polymorphism in the 5ʹ-flanking re-
gion was suggested to be related to the binding of
a transcription factor and related to alcohol intake
[168, 1011] A number of other polymorphisms
have been identified [168, 1012, 1013] Howev-
er, the evidence to date indicates that these varia-
tions do not seem to have much significance in
terms of their effects on in vitro or in vivo activ-
ity of P450 2E1 [69, 1012, 1014–1016]
9.7.13.4 Substrates and Reactions
P450 2E1 was originally characterized as an etha-
nol-oxidizing enzyme P450 2E1 can oxidize some
compounds that are present in the body, including
acetone and possibly other ketones involved in
certain physiological syndromes (fasting, diabe-
tes) [1017] Transgenic P450 2E1-knockout mice
appear to be relatively normal, although the blood
acetone levels become much higher (than in wild-
type mice) after fasting [1018]
The role that P450 2E1 plays in ethanol
metabolism has been debated for many years
[1019] What seems to be the general consensus
is that alcohol dehydrogenase is the main en-
zyme involved in ethanol oxidation The overall
contribution of P450s to the oxidation of etha-
9  Human Cytochrome P450 Enzymes
nol is considered elsewhere, relative to alcohol
dehydrogenase and catalase [1020] The point is
made that even if an overall role of an enzyme
(P450) is low, there may be strong “local” ef-
fects Somewhat surprisingly, the experimental
survey of human P450 enzymes did not show a
strong role for P450 2E1 relative to other P450s
[1021] P450 2E1 may contribute at very high
ethanol concentrations or in individuals with low
levels of alcohol dehydrogenase activity P450
2E1-knockout mice have blood ethanol levels not
significantly different from wild-type animals
after administration of ethanol [1022] Acetal-
dehyde, the product of ethanol oxidation, is also
oxidized to acetic acid by rat and human P450
2E1 (Fig 919) [1023–1025]
The oxidation of 4-nitrophenol to 4-nitro-
catechol has been used as an in vitro marker of
human P450 2E1 [1026] Chlorzoxazone 6-hy-
droxylation was demonstrated to be a relatively
specific reaction catalyzed by human P450 2E1;
other enzymes (eg, P450 1A1) can catalyze the
reaction but with poor catalytic efficiency [1027,
1028] Chlorzoxazone is a relatively innocuous
muscle relaxant, and the assay can be used in
vivo to estimate hepatic P450 2E1 function non-
invasively [69, 1016]
One group of substrates of interest is N-
alkylnitrosamines, which are carcinogens at
many sites and can be formed by chemical re-
actions within the body (eg, stomach acid)
[1029] Early research on the activation of N-ni-
trosodimethylamine ( N,N-dimethylnitrosamine)
indicated biphasic kinetics of the activating
N-demethylation reaction in liver microsomes
and the possible contribution of multiple P450s
Fig. 9.18   Sequential oxidation of N,N-dimethylnitrosamine to formaldehyde and formic acid by P450 2E1 [1035]
589
and possibly other enzymes [1030, 1031] The
enzyme involved in the “low Km” reaction was
shown to be P450 2E1 in rat and human liver
[1032, 1033] An in vivo role of P450 2E1 has
been confirmed in rats [1034] However, P450
2A6 has a significant share of the role of activa-
tion of some more complex nitrosamines, even
N-nitrosodiethylamine [432, 433] The oxidation
of N-nitrosodimethylamine is actually a two-step
reaction leading to formic acid, which appears to
be relatively processive (Fig 918) [1035]
P450 2E1 has been shown to be a major
P450 involved in the oxidation of a number of
low molecular weight procarcinogens, includ-
ing not only nitrosamines but also benzene, sty-
rene, CCl4, CHCl3, CH2Cl2, CH3Cl, CH3CCl3,
1,2-dichloropropane, ethylene dichloride, eth-
ylene dibromide, vinyl chloride, vinyl bromide,
acrylonitrile, vinyl carbamate, ethyl carbamate,
and trichloroethylene [461] The oxidations by
P450 2E1 all have relevance to the activation
and detoxication of these compounds and their
risk assessment (Figs 99 and 910) [461, 1036]
Another substrate is the gasoline additive methyl
tert-butyl ether [1037] A role of P450 2E1 has
been shown in the activation of some of these
chemicals in knockout mice [1038, 1039]
Another substrate for human P450 2E1 is
lauric acid, which undergoes 11-hydroxylation
[1040, 1041] The physiological relevance of this
reaction is unknown Indole is oxidized by P450
2E1 (3-hydroxylation to indoxyl, generating in-
digo) as well as by other P450s, particularly P450
2A6 and 2C19 [446, 1042] The relevance of this
reaction to the urinary excretion of indigoids
[1043] is still unclear
590
Relatively few drugs are oxidized by P450
2E1 (Fig 91b) Chlorzoxazone is one [1027]
Halogenated anesthetics are often metabolized
by P450 2E1, including halothane [1044] and
isoflurane [1045] For more substrates, see Ren-
dic [51]
Another example of an N-oxygenation by
P450 2E1 has been reported, that of nicotinamide
[1046], to go with pyridine N-oxygenation
A detailed kinetic analysis of the human P450
2E1-catalyzed oxidation of ethanol showed that
the product acetaldehyde was converted to acetic
acid in a rather processive manner [1025, 1047]
Both reactions occur with burst kinetics, ie, a
rate-limiting step occurs after product formation,
and the actual rate of oxidation (formal C–H bond
cleavage) is very fast [1047] Similar phenomena
were observed with P450 2E1 oxidation of N-
nitrosomethylamine ( N,N-dimethylnitrosamine),
in terms of oxidation of the resulting formalde-
hyde to formic acid (and processive oxidation of
N-nitrosodiethylamine to acetaldehyde to acetic
acid) [1035] This processivity is rather unique to
P450 reactions, including steroid hydroxylations
[220] but has also been observed with P450 2A6
in nitrosamine oxidations [1048] These phenom-
ena are related to the expression of kinetic deu-
terium isotope effects in the Km parameter [1025,
1047] The intermolecular isotope effect is ex-
pressed in the Km parameter, which includes the
C–H bond-breaking step kcat is governed largely
by an enzyme physical step after oxidation of
the substrate In this system, the Km term con-
tains kcat as a variable [1025, 1047, 1049] The
reasons for the processivity in these reactions
are not clear yet, in that there does not appear
to be an intrinsic chemical affinity for the alde-
hyde products to P450 2E1 (or P450 2A6) [1025,
1047, 1048, 1050] One possibility, which can be
rationalized in kinetic models, is that a confor-
mational change occurs after the initial substrate
binding and that this stays “locked” after the al-
dehyde forms, leading to a favorable oxidation of
the aldehyde [1048]
One of the issues in P450 2E1 in vitro reac-
tions is the need for cytochrome b5, first demon-
strated with the rat enzyme [1032] and also the
human enzyme [1033, 1047]; the involvement
F. P. Guengerich
also exists in microsomes [1051] Cytochrome
b5 also augments P450 2E1 activity in bacterial
expression systems [736, 1052] In contrast to
several of the P450s, apo-cytochrome b5 (minus
heme) does not function, arguing for a “classic”
role of electron donation in enhancement of ca-
talysis [736, 1053]
Other unusual phenomena have been reported
in P450 2E1 reactions, including negative coop-
erativity and inhibition at high substrate concen-
trations [1054] These effects have been rational-
ized in terms of multiplicity of ligand binding,
although there has been no structural support for
this hypothesis yet (Sect 7135, vide infra)
Mathematical models have also been devel-
oped for rates of oxidation by P450 2E1 [1055,
1056] In essence, these are based on chemi-
cal reactivity at individual substrate atom sites
In both of the cited examples [1055, 1056], the
models were used for relatively small sets of re-
lated compounds and may have some utility An
inherent problem in more extended sets is the dif-
ficulty in interpretation of the parameters kcat and
Km Thus, the rate-limiting step may not be relat-
ed to hydrogen abstraction or a similar chemical
step involving the substrate ( vide supra)
9.7.13.5 Structure
In 2008, Scott and her associates reported X-ray
crystal structures of human P450 2E1 with imid-
azole and the inhibitor 4-methylpyrazole bound
[1057] Her group has also published structures
with imidazole-modified fatty acids [1058] and
pilocarpine [456] The structures reveal an extra
pocket near the binding site of a small molecule,
and with different ligands the size of the active
space available to the substrate can vary from
190 to 470 Å3 [456] Thus, P450 2E1 is some-
what flexible, and this behavior can explain the
range in the size of substrates from ethanol to
long-chain fatty acids (Sect 7134, vide supra)
A pharmacophore template for prediction of
oxidations by P450 2E1 has been published by
Yamazoe et al [1059]
The kinetics of CO binding to human P450
2E1 following flash photolysis [1060] appeared
to be monophasic and the rate was decreased in
the presence of (400 mM) ethanol One interpre-
9  Human Cytochrome P450 Enzymes
tation of the results is that binding of the substrate
makes P450 2E1 more rigid [1060]
9.7.13.6 Inhibitors
As mentioned earlier, many low molecular weight
solvents are substrates for P450 2E1 These are
also inhibitors of P450 2E1 [155, 156] Such in-
hibition is a problem in that historically many
insoluble P450 substrates are added to enzymes
with solvent concentrations of 1 % (v/v), which
is often ~ 100 mM, and thus care is needed in
analyses It is possible to dilute many of the P450
2E1 low molecular weight substrates directly in
water to add them to incubations, eg, methylene
chloride (normally considered immiscible) has a
solubility of ~ 100 mM in H2O [1061]
Some of the alcohol and aldehyde dehydroge-
nase inhibitors are also inhibitors of P450 2E1,
making interpretations of in vivo ethanol me-
tabolism studies difficult 4-Methylpyrazole is
an excellent inhibitor [314, 1062] and probably
the best one for in vitro experiments at this time
3-Amino-1,2,4-triazole [1063] and diethyldithio-
carbamate [461] are mechanism-based inactiva-
tors The latter is of interest in that the oxidized
form, disulfiram (Antabuse®), is an aldehyde
dehydrogenase inhibitor used with patients in
alcohol aversion therapy Many of the early ani-
mal and human studies on interactions of ethanol
and disulfiram with various chemicals can now
be rationalized in the context of P450 2E1 [1064,
1065]
A number of compounds of natural origin
have also been examined as P450 2E1 inhibi-
tors, many of which are derived from vegetables
such as onions, garlic, and cruciferous vegetables
[1066, 1067]
In addition, the characterization of mecha-
nism-based inhibition of P450 2E1 by diethyldi-
thiocarbamate [1068], 3-hydroxyacetanilide (the
“meta” isomer of acetaminophen) [1069], and
the chemopreventive agent phenethyl isothiocya-
nate [1070] have been reported The inhibition
by diethyldithiocarbamate has been proposed to
involve modification of one of the thiol groups of
P450 2E1 [1068]
591
9.7.13.7 Clinical Issues
Gonzalez has reviewed some of the clinical and
practical aspects of P450 2E1 [184], which in-
clude the role of P450 2E1 in the oxidation of
certain drugs, alcoholism, oxidative stress, and
risk from cancer
As pointed out earlier, the most generally ac-
cepted noninvasive human assay involves 6-hy-
droxylation of the muscle relaxant chlorzoxazone
[1016, 1027] Studies with humans show little
effect of diabetes [1016, 1071] but an effect of
body weight/obesity [1071, 1072] As mentioned
before, genotype has shown little impact on the
in vivo parameters to date [69, 1072]
Another issue is drug metabolism and toxicity
Acetaminophen (paracetamol) overdose remains
a major cause of liver failure in the USA and Eu-
rope Several P450s are involved in the oxidation
to the reactive iminoquinone [304] Studies with
P450 2E1-knockout mice indicate that P450 2E1
is probably a major determinant of acetamino-
phen toxicity in humans, because the toxicity was
considerably attenuated in P450 2E1-knockout
animals [104]
P450 2E1-null mice have the same blood
ethanol levels as wild-type animals after ethanol
dosing [1022], suggesting that P450 2E1 activ-
ity is not a major factor in ethanol metabolism,
at least in mice The situation regarding a role
for P450 2E1 in alcohol-induced liver injury in
other models is unclear, with some reports sug-
gesting a link [1073, 1074] and others not [1022,
1075] Autoantibodies against P450 2E1 have
been reported in alcoholics [1076] and attributed
to hydroxyethyl radicals [1077] (which may arise
from lipid peroxidation processes rather than as
intermediates in P450-catalyzed oxidation, vide
supra) P450 2E1 is also a major autoantigen as-
sociated with halothane hepatitis, a rather idio-
syncratic response [1078] As with other autoim-
munities involving P450s (2C9, 2D6, 21A2, vide
supra and vide infra), causal associations remain
to be demonstrated
Many studies have been reported on the rela-
tionship of CYP2E1 genetic variations to risk of
diseases Benzene poisoning in Chinese workers
592
showed some changes in risk with one genotype
but only in smokers With regard to cancers, the
results appear to be very mixed An early report
suggested a link of lung cancer with a polymor-
phism, but since then the results have been mixed
for cancers of the lung [1079–1084], oral cavity
[1085, 1086], and stomach [1087] In most of
these cases, it should be emphasized that there
is little information about exposure and the only
relevant etiology is probably tobacco-derived
nitrosamines In a study of workers exposed to
vinyl chloride (a P450 2E1 substrate [461]), some
association was found between a P450 2E1 poly-
morphism and p53 mutations [1088] However,
it should be emphasized again that the relevance
of CYP2E1 genetic variants to known P450 2E1
reactions is unclear, particularly in vivo [1072],
and it is difficult to define roles of these genetic
polymorphisms in cancer risk; overall, P450 2E1
expression due to environmental influences may
have a role but is more difficult to establish
Because of the role of P450 2E1 in the me-
tabolism of industrial chemicals, there is con-
siderable interest in the field of occupational
medicine [1089] Genetic variations of P450 2E1
in human population have been linked to vinyl
chloride-induced liver fibrosis [1090] and risk
assessment of volatile organic chemicals [1091]
Physiologically based pharmacokinetic models
have been developed to incorporate variation in
human population, using trichloroethylene as an
example [1092] Efforts have been made to relate
genetic variations in P450 2E1 to cancer of the
lung [1093], head and neck [1094], gastric tract
[1095, 1096], and colon/rectum [1097] and vari-
ous chemically induced cancers [1098]
Autoantibodies to P450 2E1 have also been
detected in cases of chronic hepatitis C infection
[1099, 1100]
There is an extensive literature relating P450
2E1 to generation of reactive oxygen species and
oxidative stress, eg, [1101–1104] Ingelman-
Sundberg reported that P450 2E1 contributed
~ 20 % of the NADPH-dependent lipid peroxida-
tion in rat liver microsomes (and 45 % in micro-
somes prepared from rats treated with acetone to
induce P450 2E1) [1105] Transfection of human
P450 2E1 into a rat hepatic stellate cell culture
F. P. Guengerich
system elevated the production of reactive spe-
cies [1106] Cederbaum [1107] has reviewed
studies on the relationship of oxidative stress to
P450 in liver cell models However, almost all of
the studies on P450 oxidative stress are in vitro
studies (including cell culture), and there have
been few in vivo studies Even in the in vivo work
that has been done, the biomarkers for oxidative
stress are not ideal [1108, 1109] Results from
this laboratory showed that F2 isoprostanes, con-
sidered the most reliable biomarkers of oxidative
stress [1110], were not altered in rats treated with
isoniazid to induce P450 2E1 [1111] The same
findings were observed (for liver, kidney, brain,
and urinary isoprostanes) in mice [1112] Further,
no differences in the levels of the isoprostanes
were seen between CYP2E1 + /+  and CYP2E1 −/−
mice Mice with an Nrf2 reporter transgene sys-
tem did not show increased activity when treated
with isoniazid to induce P450 2E1 and did not
show changes [1112], in marked contrast to in
vitro studies on P450 2E1 in HepG2 cell culture
[1102] Although “global” oxidative stress does
not appear to be associated with P450 2E1 in ro-
dent models, the production of local “pockets” of
reactive oxygen species, eg, in mitochondria (as
documented by isoprostane formation in in vitro
systems [997])
P450 2E1 may also be involved in nonalco-
holic fatty liver disease, although this area is also
controversial and genetic variations have not
been implicated [184, 1113]
9.7.14 P450 2F1
9.7.14.1 Sites of Expression
P450 2F1 was originally cloned from a human
lung library [1114] It is expressed in bronchial
epithelial cells This is considered a lung-specific
P450, although there have been some repeats of
protein expression in liver [635] and of mRNA at
some other sites, eg, nasal mucosa [1115] and
placenta [381]
9.7.14.2 Regulation
A lung-specific factor (LSF) protein has been re-
ported to bind in the -152 to -182 5ʹ-region of the
9  Human Cytochrome P450 Enzymes
gene to yield the preferential expression in lung
[1116] The factors Sp1 and Sp3 have also been
implicated in the expression of P450 2F1 [1117]
Metabolites of the P450 2F1 substrate 3-me-
thylindole have been reported to induce P450
2F1 by a non-AhR mechanism [1118]
9.7.14.3 Genetic Variation
Polymorphisms have been reported in the
CYP2F1 gene [1119, 1120] The http://www
cypalleleskise website currently shows eight al-
leles, with two frameshift variants and five cod-
ing region variants The most frequent (*2A) is a
frameshift and does not lead to a functional P450
2F1 [1119]
9.7.14.4 Substrates and Reactions
Several model fluorescent substrates have been
used with P450 2F1 [1121], but most of the inter-
est in P450 2F1 has been in regard to its ability to
activate several potential toxicants and carcino-
gens, including 4-ipomeanol [1121], 3-methylin-
dole [1122, 1123], styrene [1124], and naphtha-
lene [1125]
9.7.14.5 Structure
No crystal structures have been reported At least
one homology model has been published [1126]
9.7.14.6 Inhibitors
The substrate 3-methylindole has been also re-
ported to be a mechanism-based inactivator of
P450 2F1 [1127]
9.7.14.7 Clinical Issues
Clinical issues have not been considered Al-
though functional polymorphisms have been re-
ported [1120] and potential carcinogens can be
activated by P450 2F1 ( vide supra), epidemio-
logical reports have not appeared
9.7.15 P450 2J2
9.7.15.1 Sites of Expression
P450 2J2 is generally considered an extrahe-
patic P450 The highest level of expression is in
the heart, but expression is also seen in skeletal
593
muscle, placenta, small intestine, kidney, lung,
pancreas, seminal vesicles, leukocytes, and brain
[1128–1136] The protein has been detected in
human liver microsomes using LC–MS [635],
although at a low level in one study [55] High
levels of P450 2J2 are expressed in adult human
primary cardiomyocytes [1129] Varying levels
of P450 2J2 are expressed in human fetal tissues
[1137] P450 2J2 has also been reported to be ex-
pressed at higher levels in some tumors [833]
9.7.15.2 Regulation
The general consensus in the literature is that
P450 2J2 is not very inducible [833, 1129] To-
tah’s laboratory reported a twofold induction
of P450 2J2 mRNA by rosiglitazone in human
primary cardiomyocytes [1129] It has been re-
ported that some regulation of P450 2J2 occurs
through an AP-1 site and with microRNA let-7B
[833, 1138–1140]
9.7.15.3 Genetic Variation
At least ten genetic variants of the CYP2J2 gene
have been reported (http://wwwcypalleleskise)
Of the six alleles examined (other than wild
type), five resulted in lower activity [1141] Ra-
cial differences have been reported [1142]
Associations have been considered for a num-
ber of disease states, including diabetes [1143],
hypertension [1144, 1145], ischemia [1146],
and myocardial infarction [1147] Other disease
states have been considered with P450 2J2 in ani-
mal models
9.7.15.4 Substrates and Reactions
The major endogenous substrate known for P450
2J2 is arachidonic acid, which is converted to
all four epoxides (EETs) [1128] These epoxides
have a variety of biological activities and are a
considerable source of interest (see also P450
2C9, Sect 794)
P450 2J2 has also been found to be rather
proficient in the oxidation of a number of drugs,
including terfenadine [1129, 1148], ebastine
[1149], astemizole [1150, 1151], hydroxyebas-
tine and carebastine [1152], eperisone [1153],
vorapaxar [1154], amiodarone [1155], albenda-
zole and fenbendazole [833], thioridazine, me-
594
soridazine, danazol [1148], apixaban [1156], and
some model substrates [1157] With these drugs,
it is not clear how much the generally extrahe-
patic metabolism of these contributes to the over-
all clearance, but in some cases local metabolism
may be important
9.7.15.5 Structure
No crystal structures have been reported Some
homology models have been proposed [1157,
1158]
9.7.15.6 Inhibitors
Several inhibitors of P450 2J2 have been syn-
thesized, some with sub-µM KI values [1157,
1159, 1160] One of the goals is to inhibit P450
2J2 in tumors [1159] Of the available drugs, da-
nazol was the most selective and potent inhibitor
( KI 20 nM for inhibiting artemizole oxidation)
[1155]
To date, there appear to be no reports of issues
of drug–drug interactions due to inhibition
9.7.15.7 Clinical Issues
As indicated earlier, there have not been any is-
sues of drug–drug interaction with P450 2J2, and
exactly how much this P450 contributes to over-
all drug clearance is unknown
The major issue with P450 2J2 is its role in
endogenous metabolism (ie, arachidonic acid
oxidation) and the etiology of several diseases,
including hypoxia [1161], cardiotoxicity [1162,
1163], coronary artery disease [1164–1167],
myocardial infarction [1168–1170], atheroscle-
rosis [1171], hypertension [1172, 1173], asthma
[1174], stroke [1169, 1175], hyperhomocyste-
inemia [1176], diabetes [1177], preeclampsia
[1178], Crohn’s disease [1179], and others [1130,
1180–1182]
9.7.16 P450 2R1
9.7.16.1 Sites of Expression
In the last edition of this chapter [149], nothing
was known about P450 2R1 Today, this P450 is
recognized as a major contributor in vitamin D
metabolism and a three-dimensional structure is
available [1183–1185]
F. P. Guengerich
Russell and his associates [1183] first cloned
mouse P450 2R1 in a search for a liver micro-
somal vitamin D3 25-hydroxylase The mRNA
is abundant in liver and testis of mice and was
also identified (mice) in kidney, brain, epididy-
mis, skin, heart, muscle, and spleen [1183] In
humans, a similar mRNA profile was reported
[1186], with the highest levels in testis, followed
by pancreas, and then the tissues reported by
Cheng et al [1183], including liver Thus, P450
2R1 mRNA is expressed in many tissues Protein
detection has not been reported
9.7.16.2 Regulation
Almost all of the work on regulation comes from
cell culture systems DNA methylation levels
have been reported to predict variations in re-
sponse to vitamin D [1187] In a prostate can-
cer cell line (LNCaP cells) and skin fibroblasts,
calcitriol suppressed P450 2R1 mRNA levels
[1188] The drug efavirenz suppressed P450 2R1
in fibroblasts but not LNCaP cells
9.7.16.3 Genetic Variation
With the finding that P450 2R1 is a major vitamin
D 25-hydroxylase [1183], considerable effort has
been put into establishing the relationships of
genetic variations Shortly after the report that
P450 2R1 is a vitamin D 25-hydroxylase [1183],
Russell’s group also reported that a patient with
low circulating levels of 25-hydroxyvitamin D
had an L99P change, which was associated with
the defect [1189] Surprisingly, no other poly-
morphisms have been entered in the http://www
cypalleleskise site as of this writing A GWAS
of circulating vitamin D levels also identified an
SNV in CYP2R1 [1190]
However, a number of studies (not all cited
here) have been done, and not all associated
diseases under investigation are linked with the
variation (see Clinical Implications, vide infra)
9.7.16.4 Substrates and Reactions
The only reaction attributed to P450 2R1 is the
25-hydroxylation of both vitamin D2 and D3
[1191]
A number of animal and human P450s (at
least six) have been reported to catalyze vita-
min D 25-hydroxylation, including P450s 2R1,
9  Human Cytochrome P450 Enzymes
27A1, 3A4, 2J3, 2J2, 2D25, and 2C11 [1192]
In mice, a CYP2R1 knockout lowered the level
of 25-hydroxyvitamin D in serum by 50 % and a
CYP27A1 deletion had no further effect [1184]
At least in mice, there may be another as yet
unknown vitamin D 25-hydroxylase [1184] Of
the human P450 enzymes examined, P450 2R1
had > 20-fold higher catalytic efficiency than
any other P450 in vitamin D3 25-hydroxylation
[1191]
9.7.16.5 Structure
A crystal structure of P450 2R1 with bound vita-
min D3 has been reported [1185] Cyclodextrin
(used to solubilize the ligand vitamin) was pres-
ent near the F–G loop Vitamin D3 was bound at
a channel between the G- and I-helices and the
B1 helix/B–C loop, in an elongated conforma-
tion The C-25 carbon distance to the heme iron
was 65 Å, slightly longer than might be expect-
ed However, this distance might change with the
redox state or binding of P450 2R1 to accessory
enzymes
9.7.16.6 Inhibitors
Apparently, no inhibitors of P450 2R1 have been
reported
9.7.16.7 Clinical Issues
The major issue is vitamin D-dependent rickets,
a rare autosomal recessive disease associated
with low levels of activated vitamin D3 This is
the disease associated with the L99P variant by
Cheng et al [1189]
Since then a number of studies have been
done to associate P450 2R1 with other diseases,
including asthma [1193, 1194], diabetes [1195],
multiple sclerosis [1196], and cancers [1197,
1198]
9.7.17 P450 2S1
9.7.17.1 Sites of Expression
P450 2S1 was discovered by Ingelman-Sun-
dberg’s group [1199] in searching databases
mRNA and protein blotting work indicate high-
est levels of expression in trachea, lung (and
595
fetal lung), stomach, small intestine, and spleen
mRNA expression was also detected in colon,
appendix, liver [1200], kidney, thymus, brain
(substantia nigra), peripheral leukocytes, and
placenta [1199, 1201] Recently the protein was
detected in human liver [635]
9.7.17.2 Regulation
Rivera et al [72] reported that both mouse and
human P450 2S1 mRNA transcripts are inducible
by TCDD in cell culture, in a mechanism involv-
ing the AhR Interestingly, induction is not seen
in rats [1202] Downregulation by corticosteroids
in cell culture has been reported [1203]
9.7.17.3 Genetic Variation
Genetic variation appears to be extensive, with
at least 13 alleles reported [1204, 1205] (http://
wwwcypalleleskise) Most of these are outside
of the coding region, and in no case have any re-
sulting phenotypic changes been identified
9.7.17.4 Substrates and Reactions
The identification of substrates for human P450
2S1 has been somewhat controversial Reports of
two oxidations—retinoic acid and naphthalene
[1206, 1207]—have not been repeatable, at least
with an E. coli recombinant enzyme [350, 1208]
Bui et al [1209] reported that P450 2S1 could not
be reduced by NADPH-P450 reductase, but this
was disproven in a series of reduction reactions
[263, 1208, 1210]
Bui et al [1209] reported “peroxygenase”-
type reaction of P450 2S1 with hydroperoxides
Such reactions have long been known in the P450
field [1211, 1212], but their physiological rele-
vance has never been established In these perox-
ygenase reactions, a number of polycyclic hydro-
carbons and aflatoxin B1 were substrates [1209]
However, one point that should not be dismissed
is that these compounds were oxidized in cells in
which P450 2S1 was transfected [1213], regard-
less of the mechanism It is conceivable that the
N-terminal modification used to express P450
2S1 might alter its catalytic selectivity, but the
expressed form is definitely capable of accepting
electrons from NADPH-P450 reductase [1210]
Other substrates for P450 2S1 include some aryl-
596
hydroxylamines, which are reduced to arylamines
[263, 1214] (the corresponding arylamines were
not substrates for oxidation) Some N-oxides are
also reduced by P450 2S1 [1208, 1210]
Surprisingly, then, P450 2S1 is left without
catalyzing any typical mixed-function oxida-
tions, only reductions and peroxygenations It
seems highly unlikely it would only catalyze
reductions A metabolomic search of lungs from
CYP2S1(−/−) mice revealed the accumulation of
two molecules, taurocholic acid and tauro-β-
muricholic acid, but only in female mice (Xiao,
Y, Ding, X, and Guengerich, FP, unpublished)
Neither compound was found to be a substrate
for human P450 2S1 nor were any of the precur-
sors, so that a number of other explanations must
be considered Nevertheless, the relevance to any
particular catalytic selectivity is unknown
(Human) P450 2S1 was found not to appre-
ciably activate any of a battery of procarcinogens
tested [350]
9.7.17.5 Structure
No structure has been reported One homology
model has been published [1209]
9.7.17.6 Inhibitors
No inhibitors have been reported, in that defini-
tive oxidations have not been identified
9.7.17.7 Clinical Issues
The only clinical issue involves searches for as-
sociation of cancer and respiratory diseases with
genotype [1215, 1216]
9.7.18 P450 2U1
9.7.18.1 Sites of Expression
Essentially all of the expression reports have
been at the mRNA level P450 2U1 mRNA ex-
pression has been reported in brain and thymus
[1217, 1218] Some expression was also detected
in other tissues, including heart, kidney, liver,
lung, testes, and leukocytes [1217] In the brain,
the highest level of mRNA was in the cerebel-
lum, as well as limbic structures and cortex, plus
cerebellum, olfactory bulbs, and pons and medul-
F. P. Guengerich
la [1217]; see also [1219] Another site of expres-
sion is white adipose tissue [1220] P450 2U1 is
also expressed in skin [1221]
9.7.18.2 Regulation
Relatively little is known about regulation of
P450 2U1, other than what might be inferred
from aspects of tissue localization ( vide supra)
P450 2U1 mRNA was upregulated in leukocytes
following trauma, for unknown reasons [1222]
9.7.18.3 Genetic Variation
Genetic variation of P450 2U1 has been reported
in a French population, with four variants report-
ed [1223] All of these four variations are outside
of the protein coding region
9.7.18.4 Substrates and Reactions
Chuang et al expressed P450 2U1 in a baculo-
virus-based system and reported the ω- and ω-1
hydroxylation of arachidonic acid [1217] Other
long-chain fatty acids were oxidized (sites not
identified) but short-chain fatty acids were not
Substrates included arachidonic, palmitic, palmi-
toleic, stearic, and vaccenic acids, plus eicosa-
pentaenoic and docosahexaenoic acids No ki-
netic parameters were reported [1217]
A metabolomics-based search for P450 2U1
substrates revealed arachidonic acid and also N-
arachidonoylserotonin as substrates [1224] The
site of oxidation of N-arachidonoylserotonin was
identified as the C-2 of the indole ring [1224] N-
Arachidonoylserotonin, an inhibitor of fatty acid
amide hydrolase [1225], was shown to be pres-
ent in human brain, and the oxidation at the C-2
site attenuated its ability to inhibit the hydrolase
[1224]
9.7.18.5 Structure
No information is presently available
9.7.18.6 Inhibitors
No information about inhibitors is presently
available
9.7.18.7 Clinical Issues
At the present time, there are no clinical issues
regarding P450 2U1 The only clinical issues in-
9  Human Cytochrome P450 Enzymes
volve the potential of P450 2U1 as a tumor mark-
er [1226] A variant has been associated with
complicated forms of hereditary spastic parapa-
resis [1227]
9.7.19 P450 2W1
9.7.19.1 Sites of Expression
mRNA searches showed little expression in most
tissues [350, 1228] but expression in colorectal
tumors [1228] However, the protein has also
been detected in human liver [635]
9.7.19.2 Regulation
P450 2W1 has been shown to be regulated by
gene methylation [185] The protein has also
been reported to be glycosylated in human em-
bryonic kidney (HEK)-293 cells and to have in-
verted endoplasmic reticulum topology [185]
9.7.19.3 Genetic Variation
Several reports have appeared on the genetic
variation of P450 2W1 [1229–1232] The http://
wwwcypalleleskese website lists seven known
alleles, five of which lead to coding changes (ef-
fects are unknown) One of the issues is potential
relationship to colon cancer prognosis
9.7.19.4 Substrates and Reactions
Although P450 2W1 could probably still be con-
sidered an “orphan” P450 (Table 91), a number
of catalytic activities have now been ascribed to
it P450 2W1 activates a number of procarcino-
gens, including PAHs, aflatoxins, and aryl- and
heterocyclic amines [350, 1233] A cancer che-
motherapeutic agent, AQ4N, is reduced by P450
2W1 [1208] P450 2W1 also activates several
cancer chemotherapeutic agents by oxidation,
including aryl benzothiazoles [1214, 1234] and
duocarmycin analogs [1235]
A metabolomic search for endogenous sub-
strates for P450 2W1 revealed lysolecithins
[1236] Hydroxylation and epoxidation at the in-
ternal carbons of the fatty acids were observed,
and the reaction occurred with other monoacyl
(but not diacyl) glycerophospholipids [1236]
Other reported substrates are indole, 3-methylin-
597
dole, and chlorzoxazone [1237] Only very low
catalytic activity towards arachidonic acid is ob-
served [350, 1228, 1237]
9.7.19.5 Structure
A homology model of P450 2W1 has been pub-
lished [1238]
9.7.19.6 Inhibitors
No inhibitors of P450 2SW1 have been reported
9.7.19.7 Clinical Issues
The only clinical issues reported relevant to P450
2W1 relate to the possibility of P450 2W1 ex-
pression as a cancer marker [1230, 1239, 1240]
9.7.20 P450 3A4
P450 3A4 is the most abundant P450 in the
human body (eg, Figs 92 and 93) and has a
dominant role in drug metabolism (Fig 91b)
Some of the earliest preparations of human P450
[9, 10] were retrospectively found to be P450
3A4 Two approaches led to an extensive charac-
terization Watkins et al [1241] isolated a P450
from human liver using immunochemical cross-
reactivity with what is now recognized as a rat
subfamily 3A P450 This laboratory isolated an
enzyme from human livers that catalyzed the oxi-
dation of the hypotensive dihydropyridine drug
nifedipine [16] cDNA cloning yielded sequences
corresponding to CYP3A3 [1242] and CYP3A4
[1243] (The former differed from CYP3A4 at
14 sites and could be considered a rare allele, al-
though it has not been reported again [1244–1246]
and originally came from the same single-liver
cDNA library as the CYP3A4 clone; CYP3A3 has
accordingly been dropped from the nomenclature
and earlier references to this should probably be
considered to indicate P450 3A4)
Subsequently studies with microsomes, an-
tibodies, and purified P450 3A4 quickly indi-
cated that nifedipine was not the only substrate;
other substrates included other dihydropyridines
[1247], steroids [16, 1248], quinidine [97], the
oral contraceptive 17α-ethinylestradiol [26], and
the carcinogen aflatoxin B1 [29] With more stud-
598
ies and the application of recombinant systems,
the repertoire of substrates expanded rapidly
[1249]
9.7.20.1 Sites of Expression
P450 3A4 is the most abundant P450 in human
liver and in the small intestine The average frac-
tion of the total P450 in liver accounted for by
P450 3A4 has been estimated to be 25–30 % [52]
(Figs 92 and 96); in small intestine, the fraction
attributed to P450 3A4 is even higher (Fig 93)
A study with the selective inhibitor gestodene,
which destroys P450 3A4, indicated that P450
3A4 can constitute 60 % of the total hepatic P450
[1250] Several estimates have been made of the
absolute amount of P450 3A4 (Fig 92b, c, and
d) One estimate with a pool of Japanese samples
was 64 pmol P450 3A4/mg protein, but analysis
of nine individual samples in the same laboratory
yielded a mean of 9 (pmol P450 3A4/mg micro-
somal protein, range 1–28) [54] Another labora-
tory reported a mean of 68 (pmol P450 3A4/mg
microsomal protein, range 10–262) [55]
P450 3A4 is also expressed in some extra-
hepatic tissues, including lung [382, 1251],
stomach, colon [382], brain [1252], and adrenal
(weak) [1253] P450 3A4 has not been reported to
be expressed in kidney, prostate, testis, or thymus
but other subfamily 3A P450s are [1253] P450
3A4 expression has been reported in brain at both
the mRNA and protein levels, particularly in the
cortex, neurons, and blood–brain barrier endo-
thelial cells [1252, 1254, 1255] This location is
of relevance regarding not only drug metabolism
of neurochemical drugs but also metabolism of
endogenous chemicals there, eg, morphine
(Sect 7204, vide infra; Fig 917) The literature
is mixed on whether expression occurs in periph-
eral blood lymphocytes or not [1253, 1256]
P450 3A4 is expressed in some tumors, al-
though the literature is very mixed as to reports
of levels being lower or higher than the surround-
ing tissue [1257–1259]
A significant gender difference in P450 3A4
expression does not appear to occur [52, 64] (al-
though one report indicated a difference [65]),
and some apparent pharmacokinetic gender dif-
ferences may be attributable to P-glycoprotein
F. P. Guengerich
not P450 3A4 [66] In fetal liver, P450 3A7 is
the most abundant form and P450 3A4 expres-
sion is very low [174, 1260] P450 3A4 expres-
sion increases rapidly after birth and reaches
50 % of adult levels between 6 and 12 months of
age [1260] Although many general regulatory
concerns have been expressed about additional
safety margins for children with drugs and other
chemicals, the evidence in this case indicates that
P450 3A4 activity levels in infants are slightly
higher than in adults [1260] Other studies concur
that there is a marked development of P450 3A4
(switch from P450 3A7 expression fetal period)
following birth and increase during the first year
of life [1261], with relatively little change after
childhood [64]
9.7.20.2 Regulation
The CYP3A4 gene is at chromosome 7q221
[1262] Although 3A subfamily enzymes were
long known to be inducible in animals [1263]
and considerable literature existed on the in vivo
induction of many activities by barbiturates and
macrolide antibiotics (eg, rifampicin) [2], early
demonstrations of inducibility were indirect but
some progress was made [1241] A general corre-
lation between enzymes and mRNA levels could
be shown in human liver samples [1242, 1244]
Defining the mechanism of regulation was dif-
ficult [1264], to some extent because of the dif-
ficulty in finding appropriately responsive cells
to utilize the CYP3A4 gene and vector constructs
derived from it Guzelian’s laboratory reported
that the source of liver cells was a greater issue
than the CYP3A regulatory region in comparing
interspecies differences in CYP3A gene regula-
tion [1265], and this result can now be rational-
ized in the context of new knowledge about re-
ceptors ( vide infra)
Although most CYP3A subfamily genes are
inducible by dexamethasone, the classic gluco-
corticoid receptor was shown not to be involved
in rat liver [1266] In early 1998, Maurel and his
associates reported that the macrolide antibiotic
rifampicin acted as a nonsteroidal ligand and
agonist of the human glucocorticoid receptor,
providing a possible mechanism for regulation
and a difference with the rodent systems [1267]
9  Human Cytochrome P450 Enzymes
The interpretation of these conclusions was ques-
tioned by Ray et al [1268]
Shortly thereafter, Kliewer’s group character-
ized the human homologue of mouse PXR, which
bound steroids and interacted with CYP3A sub-
family genes in the manner expected for a major
regulatory influence [1269, 1270] (some litera-
ture also refers to the human PXR as “SXR”)
This member of the steroid receptor family “or-
phan” group (PXR) interacted with barbiturates,
steroids (including dexamethasone), statin drugs,
macrolide antibiotics, and some organochlorine
pesticides [1270, 1271]
Knowledge of PXR and its cognate binding
site has led to the development of PXR receptor
and reporter assays to screen for P450 3A4 in-
duction with new drug candidates [1272–1274]
The discovery of PXR suggested that alleles of
this receptor might be responsible for the vari-
able inducibility in different individuals How-
ever, the PXR SNVs found to date have not been
found to control P450 3A4 induction [1275] The
regulation of CYP3A4 expression is more com-
plicated than simple loading of activated PXR
(eg, Fig 913), as suggested by Kliewer’s early
work showing the roles of coactivators [1269,
1270] However, the glucocorticoid-mediated in-
duction of P450 3A4 is mediated by elements in
addition to the canonical PXR site [1276, 1277]
Some compounds (eg, ketoconazole) suppress
CYP3A4 gene expression, apparently via binding
to the PXR and interaction with “corepressors”
(NCoR, SMRT) [1278] CAR (see Sect 672)
appears to interact with the CYP3A4 gene at
the PXR site to cause induction [1279] Further,
there is evidence that 1α,25-dihydroxyvitamin
D3 (see Sect 653) also controls the transcrip-
tion of P450 3A4 [1280] This effect is mediated
through the VDR [551], which has similarity to
PXR and CAR in the steroid receptor superfami-
ly Further, kinases have been shown to modulate
the induction of P450 3A4 via VDR in Caco-2
cells [1281]
Other factors also contribute to P450 3A4 reg-
ulation Among these are C/EPPα, DBP [1282],
and HNF-4α [1283] Interleukin-6 has been re-
ported to downregulate P450 3A4 through trans-
lational induction of the repressive C/EBPβ-LIP
599
protein [1284] Thus, the transcriptional regula-
tion of P450 3A4 expression centers on PXR but
involves many other aspects A systematic tran-
scriptomic analysis of the regulation of human
P450s, including P450 3A4, has been published,
based on pathway analysis in human liver sam-
ples [64, 1285]
Regulation of P450 3A4 expression has been
reviewed by Schuetz [1286] The P450 3A4
gene is somewhat unique in having an upstream
proximal ER6 element, with xenobiotic response
enhancer module (XREM) and constitutive liver
enhancer module (CLEM) [1287] The novel
enhancer CLEM4 is important, and HNF-1α,
HNF-4α, upstream stimulatory factor (USF) 1,
and Ap-1 all interact with CLEM-4 [1288] It
is also polymorphic HNF-4α determines PXR-
and CAR-mediated induction of P450 3A4
[1289] Nuclear factors (eg, VDR) can compete
with PXR for binding to its cognate site [1290]
PPARα has also been reported to regulate P450
3A4 [1291]
Based on results obtained with endometrial
samples, it has been postulated that estrogens up-
regulate P450 3A4 [1292] In addition, Wolbold
et al [1293] reported twofold higher levels of
P450 3A4 in livers from women than men in a
collection of 94 samples However, this gender
dimorphism has not been observed in other stud-
ies except for Schirmer et al [65], which was
only seen when testosterone 6b-hydroxylation
activity was considered (but not when midazol-
am was the test substrate) and was not statisti-
cally significant [64]
Another aspect of P450 3A4 regulation in-
volves degradation TAO, erythromycin, and
some related amine macrolide antibiotics
form “metabolite complexes” (C-nitroso:iron
(R–N = O:Fe)) and inactive protein accumulates
[1294, 1295] These studies have relevance to in
vivo P450 3A4 inhibition by these drugs
Degradation of P450 3A4 appears to be de-
graded by a ubiquitin-linked pathway [336] Cor-
reia and her associates also reported that protein
kinase C-modified P450 3A4 at Thr-264 and
Ser-420; the relevance of these phosphorylations
to ubiquitin-linked degradation is yet unknown
[1296]
600
Phosphorylation of P450 3A4 has been re-
ported in liver samples [297] The effect on
catalytic activity is not known Phosphorylation
(Thr-264, Ser-420, Ser-478) is also important
in ubiquitin-dependent proteasomal degrada-
tion [1297], which involves gp78 and CHIP E3
ligases [1298] Conformational phosphodegrous
(negatively charged patches) have been consid-
ered for (ubiquitin) E2/E3 recognition [1299]
The NFκB pathway has also been considered to
interact with proteasomal degradation in regulat-
ing the stability of the P450 3A4 protein [1300]
Hughes et al [1301] reported that progester-
one receptor membrane component 1 (PGTMC1,
or Dop1) binds and regulates (human) P450 3A4,
based on work with a yeast model However, in
studies in mammalian cell culture, downregula-
tion of PGTMC1 did not affect expression or lo-
calization of P450 3A4 [1302] Transfection of
PGRMC1 along with P450 3A4 resulted in the
inhibition of P450 3A4, and this inhibition was
relieved by increased expression of NADPH-
P450 reductase
9.7.20.3 Genetic Variation
The issue of genetic variation is considered in
the context of attempts to explain the population
variability in P450 3A4 activity, which does not
show truly modal distribution [1303]
At least 43 alleles of P450 3A4 are known,
and an additional four SNVs have yet to be char-
acterized with regard to haplotype (http://www
cypalleleskise) The SNVs and other variants
identified have not yet shown much relationship
to catalytic activities [1304–1310]
Some of the variants have impaired func-
tion [1311, 1312] The *17 allele (coding for an
F189S change) had < 1 % of the normal catalytic
activity [1313] Polymorphisms in transcription
factors and other regulatory proteins can influ-
ence P450 3A4 expression [1314]
Klein and Zangar [1315] have reviewed the
contributions of various genetic components in
the context of the overall variation in P450 3A4
9.7.20.4 Substrates and Reactions
Analysis of the catalytic activity of P450 3A4
and other subfamily 3A P450 enzymes is not
F. P. Guengerich
always easy to assess because of nuances about
the effects of the membranes and other proteins
Wrighton examined P450s 3A4, 3A5, and 3A7
under identical conditions and concluded that
P450 3A4 is generally more catalytically active
than 3A4 or 3A7 towards all substrates examined
[1316]
9.7.20.4.1  Substrates
P450 3A4 contributes to the metabolism of
~ 50 % of the drugs on the market or under de-
velopment (Fig 91b) For lists, see Table 95
and Rendic [51] Many of these are important
drugs such as simvastatin (Zocor®) and some
other statins [1317], the prostate hypertrophy in-
hibitor finasteride (Proscar®/Propecia®) [1318],
the immune suppressant cyclosporin [20, 1319],
protease inhibitors such as indinavir [1320], and
sildenafil (Viagra®) [1321]
In the course of these reactions, P450 3A4
catalyzes some atypical reactions [887], includ-
ing desaturation [1317], oxidative carboxylic
acid ester cleavage [1322], and oxidation of a ni-
trile to an amide [1323] An unexpected reaction
encountered in this laboratory was the oxidation
of alkylphenyl ether nonionic detergents, which
have been commonly used in enzyme purifica-
tion [537] and also have some medical and in-
dustrial applications [1324] Methylene hydrox-
ylations yield hemiacetals, which break down to
shorten the chains [1324]
One of the classic (and fastest) reactions
catalyzed by P450 3A4 is testosterone 6β-
hydroxylation [16] However, the physiological
significance of this and several other steroid hy-
droxylations [1248] is unclear The significance
of P450 3A4 in physiology may be questioned,
given its variability (Fig 95) However, some
contributions are possible and may be suggest-
ed from recent work Cholesterol is oxidized by
P450 3A4 to 4β-hydroxycholesterol, a major cir-
culating oxysterol [1325, 1326] P450 3A4 also
catalyzes the 25-hydroxylation of 5β-cholestane-
3α,7α,12α-triol [1327, 1328] The product is a
potent PXR agonist, and this system might func-
tion as an autoregulatory pathway (ie, excess
triol activates PXR and P450 3A4, which reduces
the level of triol [1329])
9  Human Cytochrome P450 Enzymes
P450 3A4 also functions in the metabolism
of cancer chemotherapeutic drugs In addition,
attention has been given to activations of drugs
and chemical carcinogens P450 3A4 activates
the ER antagonist tamoxifen to produce DNA ad-
ducts (Fig 910) [1330] Another example of car-
cinogen activation involves aflatoxin B1, which
undergoes both a detoxicating 3α-hydroxylation
and formation of the highly mutagenic 8,9-exo-
epoxide [29, 1331, 1332] Some other carcinogen
substrates of P450 3A4 are listed in Table 98
One of the issues with P450 3A4 is which
reaction provides the most appropriate index
of activity, both in vitro and in vivo Histori-
cally nifedipine oxidation and testosterone 6β-
hydroxylation were among the first activities
identified [16] and are still used in vitro [158]
Midazolam 1ʹ-hydroxylation has also been used
extensively [158], in part because of its accep-
tance for in vivo assays
Some higher-throughput fluorescence assays
were also developed and gained commercial
appeal [1333, 1334] One issue regarding these
and also several other P450 3A4 reactions is that
they show variable effects of added chemicals,
ie, one compound may inhibit a certain P450
3A4 reaction but stimulate another Chauret et al
[1335] reported a fluorescence reaction that be-
haves in a very similar way to testosterone 6β-
hydroxylation Houston has examined the be-
havior of P450 3A4 probe substrates in vitro and
grouped them into two categories Although all of
these reactions are catalyzed by P450 3A4, they
have been categorized into two groups by their
behavior in the presence of other compounds,
as mentioned above [1336] One group includes
testosterone, cyclosporin, and erythromycin
The second includes midazolam, triazolam, dex-
tromethorphan, and diazepam Terfenadine fits
in either group, and nifedipine seemed to have
properties unique from both groups [1336]
The ambivalence about the variability of
probe drugs is even greater for in vivo human
experiments than in vitro, as one might expect
A number of reactions have been used, includ-
ing nifedipine oxidation [1337], erythromycin
N-demethylation [1338], lidocaine oxidation
[1339], dapsone N-hydroxylation [1340], mid-
601
azolam 1ʹ-hydroxylation [1341], and quinine
3ʹ-hydroxylation [1342] In most cases, the test
drug is administered orally for convenience,
except for some uses of erythromycin and mid-
azolam (iv) The ratio of (endogenous) urinary
6β-hydroxycortisol to cortisol has also been used
to assess P450 3A4 function [1343] Many of
the assays reflect the activity of P450 3A4 in the
small intestine, particularly with the drugs ad-
ministered orally The erythromycin breath test
(exhaled CO2 produced from the HCHO released
in the reaction) is generally used to estimate he-
patic P450 3A4 and has been used as an aid in
selecting cyclosporin doses for liver transplant
patients [1344] The lack of correlation of these
indicators is a problem in the practical analysis
of drug interactions [1345–1347] Some of the
discrepancies are probably inherent in the nature
of P450 3A4 itself (ie, see in vitro assays, vide
supra) Other issues involve the lack of coordi-
nate regulation of hepatic and intestinal P450
3A4 [1348] and the activity of P-glycoprotein
[1349] which shows some overlap in regulation
patterns with P450 3A4 [1350] and influences
the availability of substrates to P450 3A4 in both
small intestine and liver
The substrates of most interest with P450 3A4
are drugs, steroids, and carcinogens It is very
clear that P450 3A4 is a major factor in drug
metabolism (Figs 91b, 92, and 93) P450 3A4
catalyzes many steroid reactions, although the
physiological significance of these remains to
be established P450 3A4 is also able to activate
many procarcinogens (Fig 910) [99], although
the impact on human cancer remains to be estab-
lished
P450 3A4 is involved in the oxidation of
cholic acid to 3-dehydrocholic acid and of che-
nodeoxycholic acid (CDCA; 6α-hydroxylation)
[1351] P450 3A4 hydroxylates cholesterol at the
4β-position, and this product accumulates and
can be of use as a noninvasive marker of P450
3A4 function [1352–1354] Cholesterol is also
hydroxylated at the 25 position by P450 3A4
[1355, 1356]
P450 3A4 has also been demonstrated to cata-
lyze testosterone 1β-hydroxylation [1357] and
progesterone 21-hydroxylation [1358] P450
602
3A4 has long been known to catalyze estradiol
2- (and some 4-) hydroxylation [16]; more recent
studies with transgenic mice suggest that P450
3A4 may have an important physiological role in
catalyzing this reaction in vivo [1359]
In addition to the list of major drugs for which
P450 3A4 has a major role (Tables 95, 96, 97,
and Fig 91b), the enzyme has more recently
been shown to have roles in the oxidation of
thalidomide [1360, 1361], and tamoxifen ( α-
hydroxylation) [1362]
P450 has also been demonstrated to play im-
portant roles in the biosynthesis of endogenous
morphine in mammals, catalyzing both (1) the
cyclizations of ( R)-reticuline to salutaridine
[905] and (2) the elusive O6-demethylation of
thebaine involved in the latter stages of morphine
synthesis [906] (Fig 917) With this, a minimal
scheme can be proposed with P450 enzymes ca-
pable of all oxidative steps in the pathway
9.7.20.4.2  Catalytic Mechanism
The mechanism of P450 3A4 has been studied
extensively, and several aspects of it bear dis-
cussion (along with structure considerations,
Sect 7205) before considering the issue of co-
operativity The basic P450 catalytic scheme is
actually a rather minimal scheme Studies with
substrates and inhibitors provided evidence that
substrate binding is a multistep kinetic process
[217, 218], as corroborated by others [219] The
evidence for multiple occupancy of P450 3A4
(Sect 7205, vide infra), coupled with the mul-
tistep binding, makes the process difficult Sligar
and his associates have shown that the oxida-
tion–reduction potential of P450 3A4 is lowered
by at least some substrates [1363], and the pres-
Fig. 9.19   Stereoselective removal of 6β-hydrogen from testosterone by P450 3A4 [1372]
F. P. Guengerich
ence of substrate is known to facilitate rates of
reduction of ferric P450 3A4 [1364, 1365] The
FeO22 + complex has been observed (stabilized in
the presence of substrate) but is less stable than in
several other P450s and degrades rapidly [1366,
1367] Some, but not all, P450 3A4 reactions
are stimulated by the presence of cytochrome
b5 [1365] Two surfaces of cytochrome b5 have
major and minor roles in interactions with P450
3A4 [1368] Electron transfer is not required for
the stimulatory role of cytochrome b5 (with P450
3A4), in that apo-cytochrome b5 (without heme,
devoid of electron transfer capability) is also ef-
fective [1369, 1370]
One point that can be made here (but that ap-
plies to many P450s) is that they exist, in part,
in the ferrous state in the cell [1371] Thus, the
ferric state is not necessarily the starting point in
the catalytic cycle
Deuterated testosterone has been used to
probe the catalytic mechanism of P450 3A4
[1372] (Fig 919) Abstraction of the 6β hydro-
gen of testosterone is highly stereoselective, with
the oxygen rebound also going only to the β po-
sition The use of both 6-deuterated and 6-triti-
ated testosterone led to the conclusion that the
6β-hydroxylation step has a high intrinsic kinetic
deuterium isotope effect, which is considerably
attenuated in the steady state [1372] The conclu-
sion is that steps other than C–H bond breaking
limit rates of the steady-state reaction
More recently, P450 3A4 was also shown to
oxidize 4,5-dihydrotestosterone, a more potent
androgen that differs only from testosterone in
the pucker of the A-ring (Fig 920) The sites of
hydroxylation were the two axial methyls (C-18,
C-19; Fig 920), which is surprising on the basis
9  Human Cytochrome P450 Enzymes
Fig. 9.20   Regioselectivity of oxidation of testosterone
and dihydrotestosterone by P450 3A4 [1357, 1373]
of both chemical reactivity and modeling predic-
tions [1373]
9. 7.20.4.3  Cooperativity
At the outset, cooperativity of P450s was re-
garded as a curiosity, but today there is interest in
practical settings, as reviewed by Obach [1374]
Both heterotropic and homotropic cooperativ-
ity have been observed with several human (and
other P450s), although it has been most reported
with P450 3A4 over the past 20 years [1375]
For a review of the mechanistic issues with P450
3A4, see Sevrioukova and Poulos [1376]
Although there are older examples of in vivo
heterotropic cooperativity [202], it is difficult to
assign these to particular P450s The results of
Tang and Stearns with quinidine and warfarin in
animals are probably attributable to subfamily
3A P450s [207] Evidence for heterotropic oxi-
dation of thalidomide in a transgenic P450 3A4
mouse has been presented [1377] Also, mid-
azolam oxidation could be stimulated by a drug
candidate—5-(4-fluorobenzyl)-2-((3-fluorophe-
noxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]
pyrazine—that also had (in vivo) enhancing ef-
fects in rats [1378]
Suggestions of multiple occupancy of the ac-
tive site of P450 3A4 were made in the 1990s
[205, 211, 212] However, dual occupancy was
not definitively demonstrated until the X-ray
603
crystallography work of Ekroos and Sjogren
[213] appeared A number of kinetic and spec-
troscopic measurements were analyzed and esti-
mates of the number of ligands included in the ac-
tive site of 2–4 were made using various models
[1379–1384] Cytochrome b5 has been reported to
induce P450 3A4 substrate cooperativity [1385]
To summarize the cited literature (and much
more for which space was not available), the
evidence is in favor of cooperativity involving
multiple occupancy within the active site (of
P450 3A4 in this case), and there is little if any
evidence for a completely distinct allosteric site
on the protein Direct evidence (X-ray crystal-
lography) exists for multiple occupancy [213],
and Auclair and her associates have shown that
attaching a large molecule (theobromine) to sub-
strates not only allows catalysis but also changes
the regioselectivity of oxidation [1386] Mod-
els based on kinetic systems are very complex,
particularly in light of limited information about
what step(s) is rate limiting in most cases [1372]
and the demonstrated complexity of ligand bind-
ing [217, 218, 1387] Hill plot n values for coop-
erativity are low (< 15) in most cases (subject to
error), and artifactual sigmoidicity can be intro-
duced simply by running low substrate concen-
tration reactions beyond linearity The number of
variables often greatly exceeds the experimental
parameters used Another obstacle is finding a
satisfactory substrate and effector, in that the pat-
terns with different P450 3A4 ligands are rather
unpredictable
In many respects, equilibrium physical mea-
surements could be considered most valuable
Nuclear magnetic resonance (NMR) spectra (T1
paramagnetic relaxation) were used to probe co-
operativity of midazolam with testosterone and
α-NF [1388] Atkins and his associates [1389]
used a single-molecule fluorescent approach to
show “allosteric” effects of one ligand on the dis-
sociation rate of another substrate, Nile red Nile
red is an allosteric fluorescent substrate and has
utility for such studies [1390]; evidence could
also be obtained for a second binding site [1391]
Another aspect and possibly another solution
to the issue comes from work by Friedman using
flash photolysis kinetics (of CO rebinding after
604
photodissociation from ferrous P450 3A4) The
kinetics were multiphasic and were selectively
altered by the presence of different substrates
[1392] Heterotropic effects were observed with
benzo[a]pyrene and α-NF [1393] The interpreta-
tion of the results is that different substrates dif-
ferentially modulate these kinetics by (1) chang-
ing the P450 conformation to alter the rate, and/
or (2) steric effects (of ligands) that reduce rates
[1394] Both effects are possible, although the
enhancement of rates in some cases [1392] argue
against the generality of the latter explanation and
in favor of multiple conformations for P450 3A4
bound to various ligands The concept advanced
is that some ligands act as allosteric factors to
“switch” P450 3A4 conformations [1395] Some
possibly relevant work has been done by Anzen-
bacherová [1396], who did pressure studies on
P450 3A4 and found that the compressibility of
P450 3A4 was less than that of bacterial P450
102A1; the compressibility was modified by the
ligand TAO The concept of preexisting multiple
conformers of P450 3A4 is an explanation for the
flash photolysis work [1392–1395] and has sup-
port in newer nonclassical approaches to general
protein chemistry [1397–1399] This view dif-
fers from the more general static “lock-and-key”
view of enzyme/substrate complexes and the in-
duced-fit theory in which enzymes are shaped by
their substrates The basic concept is that protein
dynamics present an ensemble of structures of an
enzyme in solution and different ligands bind to
individual states depending upon their comple-
mentarity [1397–1399] Another consideration in
this discussion, somewhat related, is that there is
good evidence that P450 conformations change
during the course of the catalytic cycle [1400],
and evidence has already been presented that dif-
ferent forms of P450 3A4 can differ in their bind-
ing of a ligand (eg, ferric and ferrous) [211]
Where does all of the work to date leave us in
this area? A recent review by Atkins et al [1401]
summarizes much of the work in more detail and
presents a cogent analysis Summarizing and
expanding on this, there are several major pos-
sibilities to explain the observed cooperativity of
P450 (and the other P450s showing this behav-
ior), which are not necessarily exclusive: (1) a
F. P. Guengerich
“classic” allosteric model with binding of effec-
tors at a site that then regulates the conformation
for substrate binding; (2) a relatively rigid P450
with a large active site that can accommodate
two to three ligands, with the results depending
on the chemical interactions of the two ligands
with each other and with P450 residues; and (3) a
series of preexisting conformations of P450 3A4
that selectively interact with individual ligands
[1397–1399] A general concept of induced fit is
related to the third possibility, as in the phenom-
ena already mentioned that different protein con-
formations exist throughout the catalytic cycle,
can differ in affinities and substrate orientation,
and may not be in rapid equilibria Many steady-
state kinetic schemes have been proposed but,
in considering the possible origins [200, 1402],
cannot be considered unique and do not provide
mechanistic answers
To return to the questions raised by Sevri-
oukova and Poulos [1376], there are still many
unanswered questions about cooperativity, even
20 years after the first reports with P450 3A4
[204] and 45 years after the first general reports
of the phenomenon with P450s [201, 203, 1403],
explaining the mechanisms at a molecular level
is not yet within our grasp However, the bat-
tery of structural, spectroscopic, and other tools
available is promising There is evidence that the
phenomenon may contribute to drug–drug in-
teractions and human variability in response to
molecules
9.7.20.5 Structure
A number of site-directed mutagenesis studies
on the possible roles of individual residues have
been published Phe-304 [1404] and Ala-305
[1405], in the putative I-helix, are proposed to
control access to the catalytic center Phe-304
was also implicated in the partitioning of aflatox-
in B1 oxidation (between 3α-hydroxylation and
8,9-exo-epoxidation) [1406] A role for Asn-206
was also proposed in the work with aflatoxin B1
[1406] Leu-211 is also postulated to control the
size of the active site [1407]
A number of X-ray crystal structures of P450
3A4 are now available, including the protein
without a ligand [1408] and with bound metyra-
9  Human Cytochrome P450 Enzymes
pone, progesterone [215], erythromycin, keto-
conazole [213], and ritonavir and several ritona-
vir analogs [1387, 1409, 1410] The active site is
large (~ 1285 Å3), and in the case of ketoconazole
two molecules of the ligand are present, only one
of which is in a position to be oxidized [213]
Another interesting aspect is the binding of pro-
gesterone at a peripheral site, 17 Å from the iron,
in a position incompatible with catalysis [215]
Collectively these structures are very valuable
in understanding how this enzyme handles so
many reactions ( vide infra, Fig 91b) One gen-
eral conclusion from all of the structural work is
that P450 3A4 can use multiple conformations to
accommodate different ligands, ie, has “malle-
ability” [1410, 1411] Support for this malleabil-
ity of P450 3A4 comes from molecular dynamics
simulations, which show much more flexibility
for P450 3A4 than for P450 2A6 or 2C9 [1412]
Whether the site of progesterone binding in
the structure of Williams et al [215] is relevant
is an interesting question Subsequently evidence
has been presented that P450 3A4 can have ini-
tial binding to P450 3A4 prior to moving near
the heme iron [217], and the peripheral site might
represent this Davydov et al [1413] also report-
ed a peripheral binding site for a dye (fluorol-
7GA) using fluorescence energy transfer
Cross-linking studies and mass spectrometry
have been used to characterize a site of interac-
tion of P450 3A4 with cytochrome b5/apo-cyto-
chrome b5 [1414]
Numerous systems have been developed to
predict sites of oxidation by P450 3A4, eg,
[926]
9.7.20.6 Inhibitors
Inhibition of P450 3A4 is a major issue in the
pharmaceutical industry and the cause of a
number of important drug–drug interactions
(Table 96) A compendium of P450 3A4 inhibi-
tors has also been compiled by Rendic [51] Only
a few other specific examples of P450 inhibi-
tors will be mentioned here One example of a
problem leading to recall of a drug is that of terf-
enadine [92–95] Inhibition of P450 3A4 is a fre-
quent problem with drug candidates, particularly
unsuspected mechanism-based inactivation, and
605
strategies have been developed for minimizing it
[1415] or making in vitro assessment as to the
extent it may be an issue in vivo [1416]
The inhibition of P450 3A4 has been shown
to be altered by the presence of (coding region)
variations [1417]
Erythromycin and ketoconazole are two of the
most established inhibitors of P450 3A4, based
on clinical experience Ketoconazole, used at
~ 1 µM, is probably the best established P450
3A4 inhibitor for in vitro use [85] Another P450
inhibitor is TAO [1418], which also has clinical
implications TAO has been used as a diagnostic
in vitro inhibitor of P450 3A4, although its mode
of action (activation to a nitroso derivative that
complexes P450 iron) requires time for the inhi-
bition to occur
One issue is the inhibition of P450 3A4 by
grapefruit juice, first reported by Bailey [1419]
The effect was rather specific for grapefruit and
a few other citrus fruits (not orange), and warn-
ing labels now include this contraindication for
many drugs [1420] Naringenin has some effect
[1421], but the most active compounds appear
to be the furanocoumarins bergamottin and 6ʹ,7ʹ-
dihydroxybergamottin, which behave as mech-
anism-based inactivators to destroy intestinal
P450 3A4 [88, 89] The magnitude of the effect
of the interaction varies with drugs, with some
of the statins, buspirone, terfenadine, astemizole,
and amiodaraone reported to show the greatest
interactions [1420]
Many of the HIV protease inhibitors are also
potent inhibitors of P450 3A4 as well as sub-
strates in some cases [1422] Because of the vari-
ety of drugs that AIDS patients use, the potential
for interactions is considerable
The effects of some herbal medicines on P450
3A4 have already been mentioned In addition to
P450 3A4 induction (eg, St John’s wort), some
of these materials also contain inhibitors For in-
stance, kava kava extracts produce kavapyrones
that inhibit P450 3A4 [1423]
Oral contraceptives contain acetylenes and
can be mechanistic inactivators of P450 3A4
Inactivation has been demonstrated for 17α-
ethinylestradiol, the major estrogenic component
of oral contraceptives [26, 1424], and several of
606
the progestogenic components, particularly ges-
todene [1250] Because of the very low doses of
these contraceptives that are used today, the ef-
fects might be expected to be small [1425], al-
though some in vivo effects have been reported
[1426, 1427]
Some chemicals and also oxidants have been
shown to cause the covalent cross-linking of
heme to apo-P450 [197] Correia’s group has
characterized the products of the destruction of
P450 3A4 with cumene hydroperoxide; the infor-
mation is consistent with a dipyrrolic fragment
of heme bound to fragment of the protein [1428]
Among diagnostic inhibitors used for reac-
tion phenotyping, ketoconazole (at 1–2 µM) re-
mains a popular choice, although it will not dis-
tinguish among individual subfamily 3A P450s
[473] Azamulin has some advantages [1429],
and “CYP3cide” (PF-04981517; 1-methyl-3-
[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-
yl]-1H-pyrolo[3,4-d]pyrimidine) [1430] and
ML-368 [1431] are P450 3A4-specific Li et al
[1432] have described a P450 3A4-selective in-
hibitor (1-(4-imidazopyridinyl-7-phenyl)-3-(4ʹ-
cyanobiphenylurea (SR9186)) that can be uti-
lized for inhibiting only P450 3A4 and not 3A5
Ritonavir is one of the most potent inhibitors
of P450 3A4 [1416] A number of analogs have
been compared using spectral, kinetic, and struc-
tural (crystallography) approaches [1409, 1410,
1433]
P450 3A4 is involved in the bioactivation of a
number of chemical carcinogens (Fig 910) [99],
and one strategy for chemoprevention is to in-
hibit P450 3A4 A number of flavonoid inhibitors
have been characterized [365] cis-Terpenones
have been shown to block aflatoxin B1 cytotoxic-
ity in vitro [1434]
Other inhibitors reported for P450 3A4 are
4-ipomeanol [1435], raloxifene [1436], and ber-
gamottin, the active principle of grapefruit juice
( vide supra) [1437] In the latter two cases, the
site of attachment (in the P450) has been identi-
fied
9.7.20.7 Clinical Issues
The major clinical issues with P450 3A4 are rapid
clearance (of drugs), variable bioavailability, and
F. P. Guengerich
enzyme induction and inhibition [1438, 1439]
One of the concerns is intestinal first-pass me-
tabolism of drugs, which usually inactivates them
[1440] One strategy to improve predictability in
drug development is the use of transgenic “hu-
manized” mice expressing P450 3A4, which
have been developed using different approaches
[1441, 1442] High enzyme activity towards a
drug will reduce bioavailability, and variations
in levels of P450 3A4 can cause clinical prob-
lems when the therapeutic window is narrow For
instance, low cyclosporin levels will not prevent
organ rejection during transplant, but high levels
cause renal toxicity, so adjustment of the dose is
critical [1443] Terfenadine has a relatively wide
window for use, but a few serious problems were
encountered [95, 1444] Renwick has considered
population models of P450 3A4 variability and
concluded that there is more interindividual vari-
ability from the oral route than iv, which is not
surprising in light of the previous discussion of
the intestinal contribution to drug metabolism
A “default factor” for adults of 32-fold is pre-
sented, but a factor of 12(-fold) was calculated to
be needed to cover 99 % of the neonates as well
[1445]
The effect of disease on P450 3A4 has been
considered P450 3A4 expression appears to be
decreased as a result of liver cirrhosis or cancer
[983, 1257, 1446] P450 3A4 levels were also
decreased in celiac disease and reversed by a
change in diet [1447]
The interactions of herbal medicines with
P450 3A4 have already been mentioned and
are one of the worst problems with these mix-
tures [1448] One of the most studied issues is
St John’s wort, which induces P450 3A4 as an
agonist of the receptor PXR [1449, 1450] The
induction of P450 3A4 by St John’s wort has
been responsible for the loss of the effectiveness
of oral contraceptives [83, 1451] The resulting
pregnancies are the result of more rapid elimi-
nation of 17α-ethinylestradiol, a phenomenon
previously reported for P450 3A4 induction by
rifampicin and barbiturates [26, 82, 90]
P450 3A4 is also of interest regarding cancer,
regarding exogenous carcinogens (Fig 910),
drugs used to treat cancer, and metabolism of ste-
9  Human Cytochrome P450 Enzymes
roids or other compounds that may affect cancer
risk or response to chemotherapy Some chemical
carcinogens activated by P450 3A4 are shown
in Table 98 The activation and detoxication of
aflatoxin B1 have already been discussed in the
context of 3α-hydroxylation (to aflatoxin Q1) and
formation of the highly reactive exo-8,9-epoxide
[29, 1331] However, aflatoxin B1 is a hepatocar-
cinogen and must reach the liver to cause dam-
age In a rat model, induction of rat P450 led to
an increase in small intestinal DNA adducts, sug-
gesting that activation of aflatoxin B1  at this site
constitutes a detoxication process, in that these
cells are rapidly sloughed and do not progress to
tumors [1452]
P450 3A4 genotypes have been reported to
be related to leukemias caused by prior treat-
ment with epipodophyllotoxin [1453] P450 3A4
expression, measured at the mRNA level, has
shown an inverse correlation with response of
breast cancer patients to docetaxel, presumably
due to changes in bioavailability [1454] Howev-
er, no relationships were found for any P450 3A4
genotypes in therapy-related myeloid malignan-
cies [1455] One of the more controversial issues
involves whether P450 3A4 genotypes are linked
with prostate cancer, with reports for and against
an association [1456–1461] The point should be
made that strong evidence for a change in an ac-
cepted P450 3A4 phenotype has not been made
in many of these cases
9.7.21 P450 3A5
P450 3A5 has 85 % sequence identity with P450
3A4 and, although generally accepted to have
less importance than P450 3A4, is of interest be-
cause of its highly polymorphic and racial distri-
bution and possible relevance to clinical issues
with P450 3A subfamily reactions
9.7.21.1 Sites of Expression
P450 3A5 (“HLp3”) was first purified from
human adult liver and found to be polymorphi-
cally expressed [1462] Gonzalez used a liver
sample apparently expressing only P450 3A5 and
not 3A4 to clone a cDNA [1463]
607
P450 3A5 expression has been reported in
liver, small intestine, kidney, lung, prostate, ad-
renal gland, and pituitary [1253, 1464–1466]
In human brain, both P450 3A4 and 3A5 were
detected (by form-specific antibodies) in the mi-
crosomal fractions of cortex, hippocampus, basal
ganglia, amygdala, and cerebellum [1252] Both
(P450 3A4 and 3A5) were localized in the soma
and axonal hillock of neurons and varied accord-
ing to cell type and cell layer Some researchers
have reported expression of P450 3A5 in periph-
eral blood cells (and not P450 3A4) [1467], but
others have not [1253]
P450 3A5 is expressed in fetal liver, in con-
trast to P450 3A4, but in a polymorphic man-
ner [1468] The overall expression of P450 3A5
(mRNA) as a part of all subfamily 3A P450 tran-
scripts has been estimated at 2 % [1253] Howev-
er, only about 25 % of Caucasians express P450
3A5, and when it is present, the level is usually
less than that of P450 3A4 However, a few indi-
viduals have been identified in which P450 3A5
is the predominant P450 3A subfamily enzyme
The variability in expression levels has been
linked to a polymorphism ( vide infra)
Recently Achour et al [55] have used LC–MS
to quantitate P450 3A5 in human liver micro-
somes, with a mean of 14 pmol/mg microsomal
protein (and a 100-fold range)
9.7.21.2 Regulation
The regulation of the CYP3A5 gene seems to be
similar to that of CYP3A4, although P450 3A5
does not seem as inducible The fetal/adult selec-
tivity of P450 3A4/3A7 is not seen [1468]
Maurel [1469] reported genomic clones and
found a CATA box (not TATA) in the promoter
The responses to glucocorticoids are probably ex-
plained by the PXR system [1470] The general
conclusion has been reached that P450s 3A4 and
3A5 are coregulated in the liver and intestine, in
terms of transcriptional control [1471], although
other factors may alter the expression [1348]
9.7.21.3 Genetic Variation
At least 26 alleles have been identified, and six
more SNVs have not been classified regarding
haplotype yet (http://wwwcypalleleskise)
608
Most of the genetic variants are loss of func-
tion due to splicing, etc However, one with a
single coding change (*11, Y53C) results in a
protein with only ~ 20 % of the wild-type cata-
lytic activity towards nifedipine [1472] The *17
allele is also very deficient in activity [1313]
Individuals with the *1 allele express the func-
tional (wild-type) protein, but those with the *3
allele express low to undetectable levels (of P450
3A5) The allele frequencies vary considerably
with regard to race, with the frequency of the
*1 allele being 10–30 % in Caucasians, 30–40 %
in Asians, and 50–70 % in an African American
population [1310, 1473, 1474] In *1 homozy-
gotes, P450 3A5 levels as high on 50 % of the
total subfamily 3A P450 pool have been reported
[1310] If there is a difference in catalytic activity
between P450 3A4 and 3A5, the genotype may
be important (see Sect 7217, vide infra)
Other alleles are known, including changes in
the 5ʹ-regulatory region where transcription fac-
tors bind [1475]
The in vivo consequences of 3A5 polymor-
phism are not clear For instance, Huang found
no significant effect of the *3 polymorphism on
midazolam pharmacokinetics [1476]
9.7.21.4 Substrates and Reactions
Since the discovery of P450 3A5, the catalytic
selectivity has been known to be similar to that
of P450 3A5 [1462], and subsequent compari-
sons with P450 3A4 confirmed this view [1477]
However, a general problem with P450 subfam-
ily 3A P450 enzymes is that they are sensitive
to the membrane environment Many P450 3A4
and 3A5 reactions—but not all reactions—are
stimulated by the addition of cytochrome b5 [736,
1478] Lee and Goldstein [1479] reported similar
patterns of dependence for P450s 3A4 and 3A5
In a few cases, the selectivity of P450 3A5 for dif-
ferent oxidation sites appears to differ from that
of P450 3A4, eg, aflatoxin B1 3α-hydroxylation
versus 8,9-epoxidation [1406, 1478] Wrighton
reported an extensive comparison of many re-
actions by recombinant P450s 3A4, 3A5, 3A7
under identical reconstitution conditions and
concluded that P450 3A5 had equal or reduced
F. P. Guengerich
activity compared to P450 3A4 in all cases ex-
amined [1316]
One of the most important issues is to what
extent P450 3A5 participates in a reaction, rela-
tive to P450 3A4 If P450 3A5 plays a major role,
then P450 3A5 genetic variations (Sect 7213)
may become important in vivo Niwa et al [1375]
have catalogued a number of reactions and found
that the ratio of P450 3A4/3A5 activity varies
Amlodipine oxidation is catalyzed almost exclu-
sively by P450 3A4 [1480]
Interesting recent results indicate that P450
3A5 is more active than P450 3A4 in some reac-
tions One is the activation of lapatinib [1481]
Another is the O6-demethylation of thebaine
(Fig 917), a critical step in the synthesis of en-
dogenous morphine [906], where P450 3A5 is
> 10-fold more active than P450 3A4 [906] The
presence of P450 3A5 in brain (Sect 7211) may
have implications in the relevance of the path-
way
Another issue with P450 3A4 (and some other
P450s) is cooperativity (Sect 7204, vide supra)
Niwa et al [1375] have reviewed heterotropic
cooperativity in P450s, including human subfam-
ily P450 3A P450s Recently, P450 3A5 has been
shown to exhibit homotropic cooperativity in the
oxidation of thalidomide [1360, 1482]
9.7.21.5 Structure
Because of the similarity of reactions of P450s
3A4 and 3A5, homology models based on P450
3A4 structures are probably reasonably valid
for P450 3A5 Relatively little site-directed mu-
tagenesis of P450 3A5 has been done, but one
study of note is the effort by Correia and Halp-
ert to utilize the differences in reactions with af-
latoxin B1 [1331, 1478] to probe the effects of
changing residues in the active site [1406]
9.7.21.6 Inhibitors
In general, the P450 3A4 inhibitors also inhibit
P450 3A5 For instance, ketoconazole and fluco-
nazole inhibit both P450s 3A4 and 3A5 [1483]
The mechanism-based inactivator gestodene
[1250] also inhibits P450 3A5 [1477]
9  Human Cytochrome P450 Enzymes
In light of the importance of distinguish-
ing whether reactions are catalyzed by P450
3A4 or 3A5 ( vide supra), Li et al [1432]
have described a P450 3A4-selective inhibi-
tor (1-(4-imidazopyridinyl-7-phenyl)-3-(4ʹ-
cyanobiphenylurea (SR9186))) that can be uti-
lized for this purpose Another selective P450
3A4 inhibitor (not affecting P450 3A5) is CY-
P3cide (PF-4981517; 1-methyl-3-[1-methyl-5-
(4-methylphenyl)-1H-pyrazol-4yl]-4-[13S]-3-pi-
peridin-1-yl-pyrrolidin-1-yl]-1H-pyrazolo[3,4-d]
pyrimidine) [1430], a mechanism-based inacti-
vator Another is ML-368 [1431]
Cannabidiol, a major substituent of marijuana,
inhibits several human P450s and inhibited P450
3A5 with an IC50 tenfold lower than P450 3A4
[939]
9.7.21.7 Clinical Issues
At this point, the significance of the wide vari-
ability in P450 3A5 is still difficult to assess As
mentioned previously, Huang [1476] found no
significant effect of the *3 allele on midazolam
pharmacokinetics in Chinese individuals How-
ever, it is possible that the extrahepatic expres-
sion [1253] may influence the course of particu-
lar drugs and other chemicals
More recent studies have reported a lack of a
major contribution in the cases of oxidation of
amlodipine [1480], midazolam [1484], and ator-
vastatin [1485] There has been considerable in-
terest in whether P450 3A5 genetic testing is of
use in dosing of tacrolimus (FK-506), an immu-
nosuppressant widely used in organ transplanta-
tion, long known to involve P450 3A4 oxidation
[1486, 1487] P450 3A5 genotyping (with tacro-
limus use) has been concluded by some to be
beneficial [1488, 1489] but not by others [1490,
1491]
9.7.22 P450 3A7
Early work in the field of human P450 research
by Kamataki and his associates with fetal sam-
ples led to the purification of a P450 termed
HFLα, now known as P450 3A7 [8, 24] Early
609
research established that this is a major P450 in
fetal liver (not in adult liver) and that the enzyme
could catalyze several reactions [24]
9.7.22.1 Sites of Expression
Early work established that P450 3A7 is the
major P450 present in fetal liver [24] and is also
present in other fetal tissues, including kidney,
adrenal, and lung [1492] Further work by Ka-
mataki’s group showed the existence of some im-
munochemically detectable P450 3A7 in gyne-
cologic tumors and in human placenta, but inter-
estingly not in cynemologous monkey placenta
[1493] Guzelian’s group also reported P450 3A7
protein in human placenta and endometrium,
with elevation in the latter site during pregnancy
or during the secretory phase of the menstrual
cycle [1494] Subsequently Sarkar et al [1495]
reported tenfold greater expression of P450 3A7
in endometrium in the proliferative rather than
the secretory phase Hakkola et al [1496] re-
ported some expression of P450 3A7 mRNA in
some first trimester placenta but not in full-term
placenta [381] Juchau’s group found expression
of P450 3A7 in early fetal tissue (50–60 days)
[1497] Schuetz et al [1498] found P450 3A7
mRNA in all fetal liver samples analyzed and
also reported its presence in one half of adult
liver samples However, the issue may be the
level of expression, because Kamataki’s group
[174] had reported the fetal > adult selectivity de
Wildt et al [1260] also found fetal specificity and
only very low levels of P450 3A7 in adults P450
3A7 expression was high during embryonic and
fetal life and decreased rapidly during the first
week of life Similar findings were reported by
Hakkola et al [1468] Also, the variability of
P450 3A7 expression was fivefold in fetal tissue
(and 77-fold in mRNA) In another report [1499],
P450 3A7 also disappeared rapidly after infancy
More recently, Gonzalez and his associates
[1500] have developed a mouse model in which
P450 3A7 is expressed in the fetus and a decrease
is seen after birth In the model, the CYP3A7 is
regulated by glucocorticoids through the gluco-
corticoid receptor
610
9.7.22.2 Regulation
The regulation of this gene is complex, as one
might expect after considering the temporal pat-
terns of expression during development ( vide
supra) Kamataki’s group published the cDNA
[1501] and genomic [1502] sequences, which
are similar to those of P450 3A4 However, more
identity (~90 %) is seen in the coding region than
elsewhere [1469, 1502] Recent work by Koch
et al [1253] reestablished that P450 3A7 only
accounted for < 2 % of all P450 expression in
adult human liver; a bimodality of P450 3A7 ex-
pression was seen, however P450 3A4 and 3A7
constructs were expressed in various cell lines by
Ourlin et al [1282], who showed differential re-
sponses to C/EBPα and DBP As in the case with
P450 3A4, P450 3A7 was inducible by rifampi-
cin in cell culture [1503] P450 3A7 has a func-
tional PXR element [1504], as does P450 3A4
( vide supra), explaining the rifampicin response
Thus, one would expect fetal P450 3A7 induction
by the usual P450 3A4 inducers
Bertilsson et al [1505] reported a distal
XREM in the CYP3A7 gene. An NFκB-like ele-
ment in CYP3A7 is inactive in CYP3A4 [1506],
and this element has recently been shown to re-
spond to p53 CYP3A7 expression is regulated
by Sp1, Sp3, HNF-3β, and USF1 Far upstream
(~ 11 kb), there are HNF-1 and HNF-4 and USF1
elements, which differ from the CYP3A4 gene
Exactly how this and other sequence differences
are involved in the rapid onset of P450 3A4 and
decrease in P450 3A7 shortly after birth [175] is
unclear
The exact basis of the postpartum shift from
P450 3A7 to 3A4 expression is still not clear
Although P450 3A7 has a PXR element, Mat-
sunuga et al [1507] reported that P450 3A7 was
induced by dexamethasone but not rifampicin in
fetal human hepatocyte culture This finding is
consistent with the report of Pang et al [1500]
with the transgenic P450 3A7 mouse model, in
which induction with glucocorticoids suggests
control by the glucocorticoid receptor, not PXR,
is important [1473]
The CYP3A7*1C alleles is expressed in adult
liver because the G > 219T substitution creates a
binding site for HNF-3 and the associated A233C
change destroys an HNF-3 binding site, creating
F. P. Guengerich
a putative octamer identical to that found up-
stream for P450 3A4 [1473, 1508]
Phosphorylation of P450 3A7 has also been
detected in vivo [297]
9.7.22.3 Genetic Variation
At least seven alleles are known (http://www
cypalleleskise) One (*1C) has been mentioned
above regarding its expression in adults [1473,
1509] A null allele (frameshift) (*3) has been
identified [1510] The *1C allele was associat-
ed with a 50 % reduction in serum DHEA 16α-
sulfate (in adults) [1511] The effect of this in
fetal life is unknown Some interesting variants
of CYP3A7 genes have been reported An mRNA
species was found that contains exons 2 and 13 of
a nearby CYP3A pseudogene spliced at the three
end [1512] The CYP3A7*1C allele is unusual
in the sense that a part of the CYP3A4 promoter
replaces the corresponding region of CYP3A7
(ER6 motif) and thus confers high levels of ex-
pression to CYP3A7*1C [1513]
The overall variability of P450 3A7 mRNA in
fetal liver varied 630-fold [1514] This observed
variability could not be attributed to the *2 or
other known polymorphisms
9.7.22.4 Substrates and Reactions
Early studies with P450 3A7 purified from
fetal liver established that testosterone 6β-
hydroxylation is catalyzed by this enzyme [1515]
Another early study indicated 16α-hydroxylation
of DHEA 3-sulfate [1516] These activities were
later verified with the use of recombinant P450
3A7 [1517]
In general, P450 3A7 has catalytic activities
rather similar to P450 3A4 and 3A5 [1518, 1519]
Activation of aflatoxin B1 [1520–1522] and het-
erocyclic amines [1520] has been observed in
various recombinant and transgenic systems,
including transgenic mice [1523] Retinoic acid
4-hydroxylation by P450 3A7 has also been re-
ported [1524] Wrighton’s laboratory reported an
extensive comparison of catalytic activities and
concluded that rates for P450 3A7 are generally
considerably lower for P450 3A7 than for P450
3A4 or 3A5 under similar conditions [1316]
The consensus about generally similar but
quantitatively lower catalytic activities of P450
9  Human Cytochrome P450 Enzymes
3A7 relative to P450s 3A4 and 3A5 still appears
to hold [1473], although some new information
is available Lee et al [1525] reported that P450
3A7 uniquely had a similar level of estrone 16α-
hydroxylation activity compared to 2-hydroxyl-
ation, in contrast to P450 3A4 That was not the
case for 17β-estradiol The possibility exists that
P450 3A7 may be important in the local or sys-
temic formation of 16α-hydroxyestrone (which is
procarcinogenic in some animal models)
Two of the substrates of P450 3A7 that may
be most important are DHEA 3-sulfate (16α-
hydroxylation) and retinoic acid, in terms of
protection of the fetus However, the finding that
fetal levels of P450 3A7 can vary 630-fold [1514]
raises questions about how important such any
regulation of these steroid and retinoid levels by
P450 3A7 really is
9.7.22.5 Structure
Much less has been done with P450 3A7 than
with P450s 3A4 and 3A5 Because the catalytic
selectivity of P450 3A7 is similar to P450s 3A4
and 3A5, models are probably about as appli-
cable One point of interest is the work of Ka-
mataki’s group showing that the substitution
T485P improved holoprotein expression in E.
coli [1526]
9.7.22.6 Inhibitors
Inhibitors have not been studied extensively, but
presumably most general inhibitors of P450 3A4
are effective with P450 3A7, eg, ketoconazole,
TAO, etc
9.7.22.7 Clinical Issues
The general point has already been made that
P450 3A7 is the major human fetal P450 and
therefore makes a major contribution to drug
metabolism in the fetus Thus, many, if not most,
of the considerations regarding drug interactions
etc with P450 3A4 should be considered with re-
spect to the fetus during pregnancy At this time,
there is still no clear consensus that the level or
activity of P450 3A7 in the fetus will have a major
physiological effect due to altered metabolism of
endogenous compounds The best candidates,
if indeed these are candidates, are retinoids and
DHEA 3-sulfate What is probably of more con-
611
cern is the role of P450 3A7 in the (fetal) metabo-
lism of drugs Even in pediatric medicine, there is
limited information to guide dosing [1527, 1528]
and the knowledge base regarding in vivo fetal
drug metabolism is even more limited
Another potentially important aspect is a re-
port that P450 3A7 expression increases in hepa-
tocellular carcinoma [1529], possibly as a part of
dedifferentiation
9.7.23 P450 3A43
9.7.23.1 Sites of Expression
In 2001, three groups reported the characteriza-
tion of a fourth subfamily 3A P450 member, P450
3A43 [1530–1532] The sequence identity with
other P450 3A subfamily members is 71–76 %
Expression (mRNA) is seen in liver, kidney,
pancreas, and prostate, and testis [1473] More
recently, high levels of expression have been re-
ported in brain, as high or higher than in liver
[1254] The results are discordant, in that previ-
ously the 3A43/3A4 mRNA expression ratio was
1/103, but in the brain study the liver ratio was
one fifth [1254] Very low levels (< 1 pmol/mg
protein) have been detected in human liver mi-
crosomes using LC–MS [54, 55]
9.7.23.2 Regulation
As with other P450 3A subfamily members, ri-
fampicin induces P450 3A43 [1530], presumably
via the PXR system
9.7.23.3 Genetic Variation
Genetic polymorphism in the CYP3A43 gene has
been reported [1533], and the http://wwwcypal-
leleskise website currently lists five alleles Two
are frameshifts and should not yield functional
protein
9.7.23.4 Substrates and Reactions
The initial studies with heterologously expressed
P450 3A43 (in E. coli) showed only low testos-
terone 6β-hydroxylation activity (a marker for
other 3A subfamily members) [1531] Agarwal
et al [1254] reported different catalytic specific-
ity in alprazolam oxidation compared to P450
3A4 and a relatively high level of activity, con-
612
cluding that P450 3A43 was more important than
P450 3A4 in brain metabolism of this drug
9.7.23.5 Structure
No structures or homology models of P450 3A43
have been reported
9.7.23.6 Inhibitors
Specific P450 3A43 inhibitors have not been re-
ported, perhaps in part due to the low catalytic
activities Presumably, other P450 3A subfamily
inhibitors such as ketoconazole would be effec-
tive
9.7.23.7 Clinical Issues
A polymorphism in P450 3A43 has been used to
explain a racial difference in olanzapine clear-
ance [1534] The conclusion is surprising in that
another study reported that P450s 1A2 and 2D6
(and FMO) are more involved in olanzapine me-
tabolism [1535]
9.7.24 P450 4A11
9.7.24.1 Sites of Expression
P450 4A11 was first cloned from a kidney cDNA
library [1536] and later identified in human liver
microsomes [1537] The originally reported
P450 4A11 sequence was subsequently found to
be that of P450 4A22 [1538] and the correction
has been made P450 4A11 is expressed largely
in the liver and kidney A proteomic study found
P450 4A11 peptides in all human livers sampled
[635] The level of expression of P450 4A11 is
much higher than that of P450 4A22 [1539]
9.7.24.2 Regulation
P450 4A11 is induced in HepG2 cells by both
peroxisome proliferators (PPARα) and dexa-
methasone [1539] Presumably a PPARα site(s)
exists in the gene Clofibrate is also an inducer
[1540]
9.7.24.3 Genetic Variation
In addition to wild-type P450 4A11, at least nine
variants are known (http://wwwcypalleleskise)
[1541] As discussed later, there is considerable
F. P. Guengerich
interest in the variants in relationship to hyper-
tension and other cardiovascular diseases [1542]
9.7.24.4 Substrates and Reactions
P450 4A11 catalyzes the ω-hydroxylation of
fatty acids, with a small amount of ω-1 product
[1536, 1537, 1543–1545] 20-Hydroxyeicosatet-
raenoic acid (20-HETE), a primary product, is a
potent vasoactive and natriuretic eicosanoid in
human kidney, and there is considerable inter-
est in the P450 4A11-catalyzed conversion of
arachidonic acid to this product (Sect 7247,
Clinical Issues, vide infra) Some prostaglan-
dins (stable analogs) have also been reported
to be ω-hydroxylated by P450 4A11, including
9,11-diazo-15-deoxy-PGH2 (U51605), 9,11-ep-
oxymethano-PGH2 (U44069), and 11,9-epoxy-
methoPGH2 (U46619) Thus, P450 4A11 may
oxidize other long-chain alkyl molecules
Some other points should be made about these
reactions of P450 4A11 First, in P450 4A11
much of the heme is covalently linked to the apo-
protein [1546] However, this attachment is not
critical to catalytic activity [1546] Second, the
reaction is stimulated ~ twofold by cytochrome b5
[1547], and the stimulation does not occur with-
out the heme [1548], arguing for electron transfer
Kinetic analysis indicates that the “second” elec-
tron transfer (from cytochrome b5) and the C–H
bond-breaking step are both rate limiting [1548]
An apparently high intrinsic kinetic deuterium
isotope effect shows considerable attenuation Fi-
nally, several of the sulfhydryls (cysteines) in the
protein are readily oxidized (to a disulfide and to
sulfenic acids), and the reactions are enhanced by
reductants, eg, dithiothreitol, tris(2-carboxyeth-
yl)phosphine, glutathione The latter phenomenon
is observed in human liver microsomes, but its in
vivo relevance is still under investigation [1549]
9.7.24.5 Structure
No crystal structures of P450 4A11 have been
reported Some homology models have been
published [1550, 1551] As mentioned earlier,
two phenomena observed with P450 4A11 are
the dependence of catalytic activity on free thiols
[1549] and the autocatalytic covalent attachment
of heme [1546]
9  Human Cytochrome P450 Enzymes
9.7.24.6 Inhibitors
HET0016 is a strong competitive inhibitor of
P450 4A11 and can be used in vivo (in ani-
mals) [1552] Another inhibitor is “20-SOLA”
(2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hy-
droxyeicosa-6(Z),15(Z)-dienoate)
9.7.24.7 Clinical Issues
P450 4A11 does not appear to be involved in the
metabolism of any drugs, and the major issue is
the role of P450 4A11 in cardiovascular diseases,
particularly salt-sensitive hypertension [1542,
1553–1571]
The hypertension problem is complex An as-
sociation between the rs1126742 C allele (cod-
ing for an F434S variant) and hypertension was
reported in 2005 [1542] and has been rather re-
producible in other human studies, with some
exceptions [1572] The working hypothesis has
been that the 20-hydroxylation ( ω) of arachidonic
acid is involved, in that this is the only measured
physiologically relevant catalytic activity The
F434S variant had a catalytic efficiency ~ 40 %
lower than the WT (*1) enzyme [1542] Deleting
the Cyp4a10 or Cyp4a14 gene from mice renders
them hypertensive, but neither of these enzymes is
an effective arachidonic ω-hydroxylation catalyst
[1573, 1574] P450 4a12 is the major arachido-
nate ω-hydroxylase in mice [1575], but this gene
has not been deleted yet It is possible that the 20-
HETE produced by P450 4A11 induces P450 2C/c
subfamily enzymes that make protective epoxides,
and deletion of mouse Cyp2c44 also causes hy-
pertension [1576] However, transgenic mice ex-
pressing human P450 4A11 have higher levels of
plasma 20-HETE and have hypertension [1577]
Thus, it is not clear exactly what role P450 4A11
has in hypertension Unresolved issues are the
importance of the site of P450 4A11 expression
within the kidney, in that 20-HETE can act as a va-
soconstrictor or a vasodilator, and the effect of the
rs1126742 genotype on the stability and level of
expression of P450 4A11 in the kidney and liver
9.7.25 P450 4A22
Relatively little is known about P450 4A22 The
originally reported CYP4A11 gene [1578] was
613
subsequently shown to be CYP4A22 [1538], but
the cDNA and protein have not been reported
The similarity of the two genes is 96 %
Johnson’s laboratory [1539] has reported that
P450 4A22 is expressed at lower levels than
P450 4A11 in human liver, as well as kidney
[1538] There was no correlation of expression
levels of P450 4A11 and 4A22 in human liver
[1539] P450 4A22 expression could not be ob-
served in HepG2 cell or PPARα-overexpressing
cells [1539]
P450 4A22 protein has been detected in
human liver using LC–MS [635]
9.7.25.1 Regulation
Relatively little information is available Savas
et al [1539] reported that P450 4A22 was only
expressed at low levels in human hepatoma
HepG2 cells and was refractory to treatment with
the PPARα inducer Wyeth 14,643 or dexametha-
sone, in contrast to P450 4A11
9.7.25.2 Genetic Variation
The CYP4A22 gene is highly polymorphic [1551,
1579] At least 22 variants have been identified
(http://wwwcypalleleskise)
9.7.25.3 Substrates and Reactions
Presumably the catalytic activity of the enzyme
is fatty acid ω-hydroxylation However, a litera-
ture search did not reveal an actual assay with
P450 4A22, and it is not known if the protein has
ever been expressed
9.7.25.4 Structure
No structure is available, but at least two homol-
ogy models have been published [1551, 1580]
9.7.25.5 Inhibitors
No inhibitors have been reported, although
HET0016 might be expected to be an inhibitor in
light of its activity against P450 4A11
9.7.25.6 Clinical Issues
In contrast to P450 4A11, there are no clinical
issues with P450 4A22 due to the evidence for
lower levels of expression
614
9.7.26 P450 4B1
9.7.26.1 Sites of Expression
P450 4B1 was cloned by Nhamburo et al [1581]
from a human lung cDNA library P450 4B1 ex-
pression has been reported (in addition to lung)
in kidney, bladder [1582], breast [1583], and
prostate [1584] Expression has also been re-
ported in bladder and breast tumors [1582] and
lung tumors [1585] It should be emphasized that
the tissue-selective expression of P450 4B1 var-
ies considerably among species, as discussed by
Baer and Rettie [1586]
9.7.26.2 Regulation
As with the tissue-specific expression ( vide
supra), the regulation of P450 4B1 expression
varies considerably among species [1586], and
caution is advised in the extrapolation of results
from any animal species to humans Poch et al
[1587] utilized A549 lung carcinoma cells and
HepG2 human hepatocarcinoma cells to identify
a proximal positive regulatory element located
between − 118 and − 73, a liver-selective nega-
tive regulatory element located between − 457
and − 216, and a distal lung-selective positive el-
ement located between − 1052 and − 1008. Three
possible binding sites were found for the Sp/
XKLF family of transcription factors The Sp1
and Sp3 transcription factors regulate P450 4B1
through the proximal regulatory element, but the
transcription factors involved in the distal lung-
selective positive element could not be identified
9.7.26.3 Genetic Variation
Genetic variation in the CYP4B1 gene appears to
be extensive, with several of the variants leading
to loss of function [1588, 1589] The interest in
P450 4B1 variation has been linked to possible
roles in cancers [1589]
9.7.26.4 Substrates and Reactions
The literature contains a considerable amount
of information concerning substrates and reac-
tions of P450 4B1 enzymes However, most of
this involves animal systems, and Baer and Rettie
[1586] have pointed out the problems in extrapo-
lation to human P450 4B1 A number of technical
F. P. Guengerich
problems have plagued heterologous expression
studies with human P450 4B1 [1586], and ac-
cordingly the information is limited A transgenic
mouse model expressing human 4B1 in (mouse)
liver was developed sometime ago [1590] More
recently, a CYP4B1 knockout mouse has also
been developed [1591], and it should be possible
to combine these systems
Substrates and reactions have been summa-
rized by Baer and Rettie [1586] Proven sub-
strates are lauric acid and 2-aminofluorene, al-
though both of these must only be considered
models for related compounds of interest
9.7.26.5 Structure
No structure is available Heme is covalently
linked to the apoprotein (Glu-310) through an
ester linkage [1592] Apparently no homology
models have been published, and the issues about
species extrapolation etc. ( vide supra) would
suggest caution in such efforts
9.7.26.6 Inhibitors
No specific inhibitors have been identified
9.7.26.7 Clinical Issues
There are two clinical issues regarding P450 4B1
One is a possible epidemiological link to can-
cers [1582, 1589], largely driven by work with
P450 4B1 from animal models The other issue
is the potential use of P450 4B1 (endogenous or
instilled by gene therapy) in the bioactivation of
cancer prodrugs [1593]
9.7.27 P450 4F2
9.7.27.1 Sites of Expression
The Kusenose laboratory reported the cloning of
a human liver cDNA corresponding to the leukot-
riene B4 ω-hydroxylase [1594] The site of ex-
pression was distinct from P450 4F3, which is re-
stricted to polymorphonuclear leukocytes P450
4F2 is found not only in liver but also in several
extrahepatic tissues [1595], including kidney (S2
and S3 segments of proximal tubules, in cortex,
and outer medulla) The extent of variation of
P450 4F2 in human liver was ~ fivefold [1596]
9  Human Cytochrome P450 Enzymes
9.7.27.2 Regulation
Relatively limited information about regulation
is available about P450 4F2 Expression is con-
trolled by sterol regulatory element-binding pro-
teins (SREBPs) [1597] In human hepatocytes,
lovastatin induced P450 4F2 and this effect was
blocked by 25-hydroxycholesterol
9.7.27.3 Genetic Variation
At least two genetic variants are known (http://
wwwcypalleleskise), coding for the W12G
and V433M forms The latter has been studied
in detail with regard to its role in warfarin me-
tabolism, ie, association with increased warfarin
dosing [1598, 1599] The genotype controls the
level of protein expression [1600]
9.7.27.4 Substrates and Reactions
P450 4F2 catalyzes ω-hydroxylation of several
lipids, including leukotriene B4 [1596, 1601],
arachidonic and [1545], and 6-trans-leukotriene
B4, lipoxin A4, 8-hydroxyeicosatetraenoic acid,
12-hydroxyeicosatetraenoic acid, and 12-hy-
droxystearic acid [1602] The physiological rel-
evance of some of these reactions is of interest,
but the effects of variability of P450 4F2 have not
been demonstrated Part of the interest lies in the
fact that leukotriene B4 is a potent proinflamma-
tory agent [1595, 1596]
In addition, P450 4F2 can also catalyze ω-
hydroxylation of arachidonic acid [1598]
Several drugs are also oxidized by P450 4F2,
including DB289 (2,5-bis[4-amidinophenyl]
furan-bis-O-methylamidoxime) [1603] and fin-
golimod (FTY720) [1604] A polymorphism
(V433M) in P450 4F2 has been shown to affect
the clinical warfarin dose [1599], but the enzyme
does not oxidize warfarin Rettie and his associ-
ates showed that the reason was that P450 4F2
is a vitamin K oxidase, explaining the effect in
terms of a physiological reaction [1605] The
reaction involves ω-oxidation and further oxida-
tion to the carboxylic acid [1600] P450 4F2 is
also an ω-oxidase for vitamin E [1606]
9.7.27.5 Structure
No structural information is available yet for
P450 4F2
615
9.7.27.6 Inhibitors
No inhibitors have been reported In that this is
an ω-hydroxylase, HET0016 might be expected
to inhibit
9.7.27.7 Clinical Issues
The major clinical issue with P450 4F2 is the role
in warfarin dose adjustment [1607–1610] due to
its activity in vitamin K oxidation [1605] The
issue is not a change in the activity of the enzyme
(V433M) but the protein stability [1600]
9.7.28 P450 4F3
9.7.28.1 Sites of Expression
A P450 4F3 cDNA was first cloned from a
human leukocyte library in 1993 [1611] The
CYP4F gene family is clustered in the p13 region
of chromosome 19, and P450 4F3 expression re-
sults in the synthesis of two enzymes, P450 4F3A
and P450 4F3B, resulting from alternate splicing
of a single pre-mRNA precursor [1612] As a re-
sult of tissue control, P450 4F3A contains exon 4
(but not 3) and is expressed in neutrophils P450
4F3B contains exon 3 (but not 4) and is expressed
in fetal and adult liver and kidney, trachea, and
gastrointestinal tract [1613, 1614]
9.7.28.2 Regulation
The tissue-specific expression of P450 4F3A/B
has been mentioned ( vide supra) Induction of
transcription has been reported with prostaglan-
din A1 (4F3B) in a human hepatocyte-derived
cell line [1615] and with benzene metabolites (in
promyelocytic leukemia cell lines and in blood
neutrophils [1616, 1617]) P450 4F3B expres-
sion is associated with differentiation of HepaRG
human hepatocytes and unaffected by fatty acid
overload [1618] Statins have been reported to
increase P450 4F3 in human liver cells through
a PXR-dependent mechanism [1619] All-trans-
retinoic acid has been reported to induce P450
4F3A in HL-60 cells [1620]
9.7.28.3 Genetic Variation
Although the CYP4F3 gene is subject to alternate
splicing, few reports of genetic variation have yet
616
appeared An SNV has been related to celiac and
Crohn’s disease [1612, 1621]
9.7.28.4 Substrates and Reactions
The two proteins generated by alternate splicing,
P450 4F3A and 4F3B, have different catalytic
specificities P450 4F3 is an ω-hydroxylase, but
the 4F3A form is more active with leukotriene B4
and the 4F3B form is more efficient with arachi-
donic acid [1614]
P450 4F3B has reported to have some ability
to oxidize the drug fingolimod (FTY720) [1604]
9.7.28.5 Structure
Little is known about the active site, including
the features associated with the differential selec-
tivity of the P450 4F3A and 4F3B enzymes A
fraction of the heme was shown to be covalently
attached to the protein [1622]
9.7.28.6 Inhibitors
A search did not identify reports of inhibitors of
P450 4F3A/B It is possible that HET0016 might
be one, on the basis of its inhibition of other ω-
hydroxylases
9.7.28.7 Clinical Issues
As mentioned under Sect.  7.28.3 ( vide supra),
there has been some association of SNVs with
celiac and Crohn’s disease [1612, 1621]
9.7.29 P450 4F8
9.7.29.1 Sites of Expression
Bylund et al [1134] first isolated the cDNA from
a human seminal vesicle library Expression has
also been reported in human epidermis, hair folli-
cles, sweat glands, corneal epithelium, proximal
renal tubules, and epithelial linings of the gut and
urinary tract [1623]
9.7.29.2 Regulation
P450 4F8 expression is upregulated in psoriasis
[1623, 1624] The mechanism has not been elu-
cidated A possible relationship with fenofibrate
treatment has been reported [1625]
F. P. Guengerich
9.7.29.3 Genetic Variation
No reports on genetic variation were identified
in a search
9.7.29.4 Substrates and Reactions
P450 4F8 was shown to catalyze hydroxylation
of prostaglandin endoperoxides [1134, 1626]
The recombinant enzyme also catalyzed the
ω-2 hydroxylation of arachidonic acid and three
stable prostaglandin H2 analogs, but prosta-
glandins D2, E1, E2, and F2α and leukotriene B4
were poor substrates [1626] These findings are
of relevance in that 19-hydroxyprostaglandins
have several biological activities [1623] (19R)-
Hydroxy prostaglandins E1 and E2 are the main
prostaglandins of human seminal fluid Bylund
et al [1626] have proposed that ω-1 hydroxyl-
ation of prostaglandins H1 and H2 by P450 4F8
occurs in seminal vesicles and that isomerization
to (19R)-hydroxyprostaglandin E is the result of
the action of prostaglandin E synthase
P450 4F8 has also been reported to form ω-3
hydroxy products of arachidonyl epoxy alcohols
with a 11,12-epoxy-10-hydroxy configuration
[1627] The 8,9- and 11,12-epoxides are also sub-
strates for ω-3 hydroxylation
9.7.29.5 Structures
No information is available about the structure
or active site
9.7.29.6 Inhibitors
No inhibitors have been reported for P450 4F8
9.7.29.7 Clinical Issues
As mentioned ( vide supra), P450 4F8 expression
is associated with psoriasis [1623, 1624], but its
role in the etiology of the disease is unclear P450
4F8 has also been identified as a potential target
in prostate cancer [1628]
9.7.30 P450 4F11
9.7.30.1 Sites of Expression
P450 4F11 has been reported to be expressed (at
the mRNA level) mainly in liver, followed by
9  Human Cytochrome P450 Enzymes
kidney, heart, skeletal muscle, and brain [1629]
Expression of P450 4F11 in liver has been con-
firmed at the protein level [297, 635, 1600]
9.7.30.2 Regulation
The regulation of P450 4F11 has been studied
in cell culture In human keratinocyte (HaCaT)
cells, the proinflammatory cytokines TNFα and
interleukin-1β induce P450 4F11 transcription
The c-Jun N-terminal kinase (JNK) pathway is
involved [1630] An RXR agonist induced P450
4F11 transcription and a retinoic acid receptor
(RAR) agonist attenuated transcription [1630]
In HepG2 cells (human liver carcinoma line),
TNFα also stimulated P450 4F11 transcription
through the JNK pathway, and NFκB attenuated
transcription [1631]
9.7.30.3 Genetic Variation
Only limited genetic variation has been reported
in P450 4F11 The rs11553651 (15016G > T) vari-
ant was reported not to be associated with breast
cancer in a study of Mexican women [1230] A
D466N substitution is also known but did not in-
fluence vitamin K ω-hydroxylation [1600]
9.7.30.4 Substrates and Reactions
P450 4F11 catalyzes a number of reactions In
light of the fact that it has ~ 80 % sequence iden-
tity to other subfamily 4F P450s, known to be
ω-hydroxylases, it is not surprising that these
reactions occur with P450 4F11 [1632] Studies
with P450 4F11 expressed in yeast, insect cells,
and bacteria have all shown ω-hydroxylation ac-
tivities towards several long-chain fatty acids,
plus leukotriene B4, lipoxin A4, and 8-HETE (but
not 5- or 12-HETE) [1632–1634] Interestingly,
much higher catalytic activity was seen with β-
hydroxy fatty acids [1633, 1634] The activities
towards fatty acids are probably relevant in that
(1) antibodies blocked activity in liver micro-
somes [1633], and (2) screening of liver extracts
with recombinant P450 4F11 in a metabolomics
approach also yielded stearic, oleic, arachidonic,
and docosahexaenoic acids as substrates [1634]
In all of these cases, only ω-hydroxylation was
observed
617
Recently P450 4F11 has also been shown
to be a catalyst of ω-hydroxylation of MK4, a
menaquinone form of vitamin K [1600] Further
research has also shown a role in vitamin E oxi-
dation [1635]
Some drugs are oxidized (at rates of < 1 min−1)
by P450 4F11, including amitriptyline, benzphet-
amine, chlorpromazine, erythromycin, ethylmor-
phine, fluoxetine, imipramine, pirenzepine, the-
ophylline, and verapamil [1632, 1634]
9.7.30.5 Structure
No crystal structures are available A homology
model (based on P450s 2C5, 101A1, 102A1,
108A1, and 107A1) has been published [1632]
9.7.30.6 Inhibitors
No information on specific inhibitors of P450
4F11 has been published
9.7.30.7 Clinical Issues
The extent to which P450 4F11 contributes to
the oxidation of drug substrates is unknown The
same applies to vitamin K (MK4), and P450 4F2
is also a catalyst, with similar expression levels
and catalytic efficiency [1600]
9.7.31 P450 4F12
9.7.31.1 Sites of Expression
P450 4F12 was originally cloned from human
liver [1636] and small intestine [1637] cDNA
libraries Expression has been reported in liver,
kidney, colon, small intestine, and heart [1636,
1638] There are also reports of expression in
gastrointestinal and urogenital epithelia [1639]
9.7.31.2 Regulation
PXR has been reported to regulate P450 4F12 ex-
pression in hepatocytes [1640]
9.7.31.3 Genetic Variation
At least seven variants in the CYP4F12 gene
have been identified, some with loss of func-
tion [1533, 1641] Some of the activity changes
have been reported with coding region variations
[1642]
618
9.7.31.4 Substrates and Reactions
Reactions identified include ω-, ω-2, and ω-3
hydroxylation of arachidonic acid [1637] and the
ω-hydroxylation of leukotriene B4 [1636, 1637]
and some prostaglandin analogs [1636] Hydrox-
ylation of the antihistamine ebastine has also
been reported [1149]
9.7.31.5 Structure
No structures have been reported The effects of
variations at Tyr-125 have been reported [1642]
This enzyme has covalently bound heme, at-
tached via Glu-328 Mutation at that site shifted
the regioselectivity of oxidation of arachidonic
acid [1642]
9.7.31.6 Inhibitors
Inhibitors of P450 4F12 have not been reported
9.7.31.7 Clinical Issues
No clinical issues have been reported
9.7.32 P450 4F22
9.7.32.1 Sites of Expression
P450 4F22 is associated with a skin disease called
ichthyosis and accordingly is expressed in skin
[1643] Specifically, it is expressed at the onset of
keratinization during skin development [1644]
Interestingly, an extensive analysis of (any) other
sites of localization has not been reported
9.7.32.2 Regulation
Although P450 4F22 is expressed during skin ke-
ratinization, molecular mechanisms of regulation
have not been reported
9.7.32.3 Genetic Variation
The ichthyosis is an autosomal recessive congenital
disease, and several CYP4F22 variants have been
identified in individuals with the disease [1643,
1645–1647]: F59I, R243H, R372W, H456Y, and
H436D, plus a frameshift and a large deletion
9.7.32.4 Substrates and Reactions
Epoxy alcohols (HEETs) and epoxides (EETs) of
arachidonic acid appear to be important for the
F. P. Guengerich
water permeability barrier of skin (ie, keeping it
from drying out) P450 4F22 oxygenated arachi-
donic acid at the ω-2 position but did not oxidize
HEETs [1627] However, it has been pointed out
that the reported catalytic activity is one to two
orders of magnitude lower than that of P450 4F8,
so the significance is unclear [1643]
The suggestion has been made that hepoxil-
ins are more relevant lipids regarding this disease
[1648] The hepoxilins (or trioxilins) might be
oxidized and play a role in signaling, rather than
acting directly as barrier lipids [1643]
9.7.32.5 Structure
No structure is available but a homology model
has been published [1649]
9.7.32.6 Inhibitors
No inhibitors have been reported, which is not
surprising in terms of the limited evidence of a
relevant reaction
9.7.32.7 Clinical Issues
The clinical issue is ichthyosis, a serious disease
Variants ( vide supra) in CYP4A22 and several
other genes [1650] are clearly involved More re-
mains to be learned about the molecular basis of
the disease before intervention is possible
9.7.33 P450 4V2
9.7.33.1 Sites of Expression
P450 4V2 is expressed at the mRNA level in a
variety of tissues [1651] The protein has been
detected by LC–MS in (female) livers, although
whether or not a gender difference really exists
is unknown [635] Antibodies have been used to
detect P450 4V2 protein in the retina and cor-
neum (eye), which is the site of most relevance
[1652]
9.7.33.2 Regulation
The regulation of the P450 4V2 expression has
not been studied, probably because of the em-
phasis on genetic variation as the major factor
in P450 4V2 activity and disease relevance ( vide
infra)
9  Human Cytochrome P450 Enzymes
9.7.33.3 Genetic Variation
P450 4V2 first attracted attention because a ge-
netic defect was implicated in Bietti’s crystalline
dystrophy, a recessive degenerative eye disease
[1651] The information is not included on the
http://wwwcypalleleskise website, but > 80 %
of the mutant alleles related to the disease are
attributed to three variants—two splice site al-
terations and one missense mutation (992 C > A,
yielding the protein variant H331P) [1652] The
H331P was not expressed in HepG2 transfected
with the cDNA and is concluded to be unstable
[1652] An I111T mutation has also been report-
ed to cause the disease [1653]
9.7.33.4 Substrates and Reactions
P450 4V2 has been characterized as a fatty acid
ω-hydroxylase [1654] Subsequent work sug-
gests ω-3 polyunsaturated as the substrates most
relevant to Bietti’s crystalline dystrophy [1652]
A search with a battery of carcinogens [350]
indicated that none are substrates for bioactiva-
tion (Xiao, Y, and Guengerich, FP, unpublished
results)
9.7.33.5 Knowledge About Active Site
No definite information is available, although
at least one homology model has been reported
[1655]
9.7.33.6 Inhibitors
HET0016 (an inhibitor of ω-hydroxylation re-
actions of other subfamily 4A P450s) inhibited
P450 4V2-catalyzed lauric acid ω-hydroxylation
with an IC50 of 38 nM [1654]
9.7.33.7 Clinical Issues
The only clinical issue relevant to P450 4V2 is
Bietti’s crystalline dystrophy [1651] This is a
rare ocular disorder and a progressive disease that
leads to atrophy of the retinal epithelium, con-
striction of the visual field, and night blindness
The role of P450 4V2 has been corroborated in a
number of genetic studies [1651–1653] At this
time, the basis appears to be the accumulation of
619
the fatty acids that are normally cleared by P450
4V2 [1652]
9.7.34 P450 4X1
9.7.34.1 Sites of Expression
P450 4X1 mRNA is found in a number of tissues,
including liver, kidney, skeletal muscle, aorta,
trachea, breast, ovary, and uterus [180] Another
site is brain, with P450 4X1 being found in the
cerebellum, amygdala, and basal ganglia [1656]
Expression of the protein has also been detected
in human liver [635]
9.7.34.2 Regulation
The only major study on regulation is work by
Johnson and his associates [180] in human hepa-
toma HepG2 cells The gene is regulated by the
PPARα receptor, which regulates some other
subfamily 4A P450s
9.7.34.3 Genetic Variation
Apparently no work has been published on P450
4X1 polymorphism or other genetic variation
9.7.34.4 Substrates and Reactions
The only reported substrate for P450 4X1 is anan-
damide ( N-arachidonylethanolamine) [1656],
with the reaction yielding the 14,15-epoxide Ar-
achidonic acid was also slowly converted to its
8,9- and 14,15-epoxides A study with a battery
of carcinogens [350] yielded no positive results
for the activation of any carcinogen [350] by bac-
ulovirus-expressed P450 4X1 (Y Xiao, and F P
Guengerich, unpublished results)
9.7.34.5 Information About Active Site
Presently no information about the active site is
available
9.7.34.6 Inhibitors
No inhibitors of P450 4X1 have been reported
9.7.34.7 Clinical Issues
At this point, no clinical issues have been identi-
fied
620
9.7.35 P450 4Z1
9.7.35.1 Sites of Expression
Most of the reports of P450 4Z1 are focused on
the expression of P450 4Z1 in breast cancer cells
[1657, 1658] P450 4Z1 has also been considered
as a marker for prostate cancer [1659] and ovar-
ian cancer [1660] P450 4Z1 is also expressed in
normal breast tissue [180]
9.7.35.2 Regulation
Limited information is available Savas et al
[180] utilized T47-D and MCF-7 human mam-
mary carcinoma cells and found considerable
induction with dexamethasone or progester-
one These results implicate the glucocorticoid
and progesterone receptors, and mifepristone
(RU486), an inhibitor of both, blocked induction
9.7.35.3 Polymorphism and Genetic
Variation
No reports have appeared regarding polymor-
phism or other genetic variation at this time
9.7.35.4 Substrate and Reactions
The only reactions reported for P450 4Z1 are
ω-2, ω-3, ω-4, and ω-5 hydroxylations of lauric
and myristic acids [1661] The significance of
these reactions is unclear, in that these are not
very physiologically relevant in mammals, and
longer-chain fatty acids were not considered
Because of the possible relevance of P450
4Z1 to cancer, we expressed the enzyme (baculo-
virus system) in our own laboratory and screened
a battery of carcinogens [350] for activation, but
all were negative (Y Xiao and F P Guengerich,
unpublished results)
9.7.35.5 Structure
At this point, no information is available
9.7.35.6 Inhibitors
No inhibition studies have been reported
9.7.35.7 Clinical Issues
P450 4Z1 is not an issue in terms of its metabolic
capability The clinical interest in P450 4Z1 in-
F. P. Guengerich
volves the use of mRNA expression as a tumor
marker [180, 1226, 1657–1660]
9.7.36 P450 5A1
P450 5A1 is the classification of thrombox-
ane synthase, which converts prostaglandin H2
to thromboxane (Fig 921) Thromboxane, the
product, causes vasoconstriction and platelet ag-
gregation, which are of considerable interest
Search names include CYP5A1, P450 5A1,
and TBXAS1 for this enzyme, with the latter
dominating the literature
9.7.36.1 Sites of Expression
P450 5A1 is expressed in platelets and also
erythroleukemia cells [1663] The enzyme is also
found in human monocytes [1664], leukocytes
[1665], and kidney interstitial dendritic reticu-
lum cells surrounding the tubules [1666] Some
expression is also seen in lung and liver [1664]
9.7.36.2 Regulation
As one might expect from its physiological func-
tion, P450 5A1 is a highly regulated enzyme
Dexamethasone induces P450 5A1 in human
monocytes [1664] Phorbol esters also induce
P450 5A1 (eg, 12-O-tetradecanoyl-phorbol-
13-acetate) in human erythroleukemia cells
[1667] Patients with systemic sclerosis showed
sixfold enhanced levels of leukocyte P450 5A1
[1665]
Promoter analysis indicates a 39-bp core pro-
moter, containing TATA and initiator elements
that control transcription Binding of the tran-
scription factor NF-E2 is critical both for altera-
tion of the nucleosomal structure and for activa-
tion of the P450 5A1 promoter [1668]
Further, Nrf2 has been reported to regulate
P450 5A1 in human lung cells [1669] Reduced
methylation of the gene is correlated with in-
creased expression levels (of P450 5A1) and pre-
eclampsia [1670]
9.7.36.3 Genetic Variation
Chevalier et al [1671] identified 11 variants
in the CYP5A1 gene, including eight missense
9  Human Cytochrome P450 Enzymes 621

Fig. 9.21   Rearrangement of prostaglandin H2 to prostacyclin (PGI2) by P450 8A1 and thromboxane (TXA2) by P450
5A1 [1662] (With kind permission from Springer Science + Business Media: [149], Fig 1012)

changes in the coding region The effects of these
changes have not been reported yet The current
http://wwwcypalleleskise website shows 12 al-
lelic variants reported to date, mostly those of
Chevalier et al [1672] Racial differences have
been reported between Caucasian and African
American populations [1673] Some in vitro
functional characterization of variants has been
reported [1674] Polymorphisms have been asso-
ciated with cerebral infection [1675]
622
9.7.36.4 Substrates and Reactions
The thromboxane synthase reaction has been
known for many years but was shown to be a
P450 by Ullrich and his associates, first in spectral
studies [1676] and then by purification [1677]
With the purified enzyme or one expressed in a
baculovirus system [1678], prostaglandin H2 was
converted to thromboxane A2 and 12-hydroxy-
heptatrienoic acid (HHT) plus malondialdehyde,
in equimolar amounts [1679] (Fig 921) Prosta-
glandin G2 was transformed to malondialdehyde
and the corresponding 15- and 12-hydroperoxy
products Prostaglandin H1 was enzymatically
transformed into 12( L)-hydroxy-8,10-hep-
tadecadienoic acid, and prostaglandin H3 yielded
thromboxane B3 and 12( L)-hydroxy-5,8,10,14-
heptadecatetraenoic acid [1679] (Fig 921)
These are all rearrangement reactions, not
involving input of O2 or electrons from pyri-
dine nucleotides The reaction mechanism has
been reviewed [1680] The reaction of the “ox-
ygen-surrogate” iodosylbenzene with a P450
5A1-containing preparation and the stable pros-
taglandin H2 analog 15( S)-hydroxy-11α,9α-
epoxymethano-5( Z),13( E)-prostadienoic acid
(U46619) yielded three oxidation products (that
could also be formed in a similar system using
rat liver microsomes) [1681] These and other
studies led Hecker and Ullrich [1682] to propose
a mechanism involving homolytic cleavage of
the prostaglandin endoperoxide (with the FeIV
bonded to one oxygen and the other oxygen bear-
ing a radical), transfer of the radical to a carbon,
further electron transfer to generate FeIII plus a
carbocation, and collapse of the bis-ionic struc-
ture to yield thromboxane A2 (Fig 921) [1662,
1680] Fragmentation competes with the electron
transfer step to also yield malondialdehyde and
heptatrienoic acid [1662]
9.7.36.5 Structure
Although a more soluble form of P450 5A1 has
been engineered [1683], no reports of crystal
structures have appeared Several spectroscopic
[1684, 1685] and modeling [1686] studies have
been published One conclusion has been that
the active site is relatively large and hydrophobic
[1685] As indicated, the protein does not bind
NADPH-P450 reductase Presumably the active
F. P. Guengerich
site is rather specific, although iodosylbenzene
could be utilized as an oxygen surrogate [1681]
9.7.36.6 Inhibitors
Thromboxane synthase inhibitors have been a
matter of interest for many years because of their
potential use in preventing plugs of platelets, and
efforts at development preceded the characteriza-
tion of the enzyme as a P450 [1687–1689] Many
of these inhibitors have a basic nitrogen atom that
binds to the P450 5A1 heme [1690]
For a review of both P450 5A1 inhibitors and
thromboxane receptors, which have been used
together, see [1691] Quantitative structure–ac-
tivity relationships of both have been reviewed
[1692] Among the uses for P450 5A1 inhibi-
tors are platelet function [1693], atherosclero-
sis [1694], inflammatory bowel disease [1695],
lung cancer [1696], and production of hepatitis
C virus (in a humanized mouse model) [1697]
9.7.36.7 Clinical Issues
As indicated earlier, platelet aggregation due to
thromboxanes is important, but overproduction
can yield clots, so control of homeostasis is de-
sirable Much of the clinical interest is in inhib-
iting this enzyme Most of the issues are with
cardiovascular diseases related to platelet func-
tion Genetic variations have been considered in
relation to aspirin tolerance in asthmatics [1698]
and acute urticaria induced by nonsteroidal anti-
inflammatory drugs [1699] P450 5A1 signaling
relationships with cancer have also been consid-
ered [1700, 1701]
9.7.37 P450 7A1
P450 7A1 catalyzes cholesterol 7α-hydroxylation,
the rate-limiting step in bile acid synthesis The
enzyme was isolated from rabbit and rat liver
[1702, 1703] and partially purified from human
liver [1704]; the cDNA was cloned by several
groups in 1990 [1705–1707]
9.7.37.1 Sites of Expression
Apparently the only major site of P450 7A1
expression is the liver The CYP7A1 gene is on
chromosome 8q11–q12 and contains recognition
9  Human Cytochrome P450 Enzymes
sequences for a number of liver-specific tran-
scription factors ( vide infra) [1708–1710]
The level of the enzyme in liver appears to be
similar to some of the low-to-moderately abun-
dant xenobiotic-metabolizing enzymes in liver
9.7.37.2 Regulation
The regulation of the CYP7A1 gene is very com-
plex, as might be expected from the important
physiological role this enzyme plays
P450 7A1 activity has long been known to be
upregulated by dietary cholesterol in most animal
models [1706], although there are some excep-
tions [1711] Feeding rats the competitive in-
hibitor 7-oxocholesterol led to reduced bile acid
synthesis (due to inhibition) and a compensatory
increase in P450 7A1 synthesis [1712] Chiang
[1713] identified a bile acid-responsive element
in the CYP7A1 promoter
Studies with CYP7A1-knockout mice show
that this reaction (cholesterol 7α-hydroxylation)
is essential for proper absorption of dietary lipids
and fat-soluble vitamins in newborn mice but not
for maintenance of cholesterol and lipid levels
[1714] The mice exhibit a complex phenotype
with abnormal lipid excretion, skin pathologies,
and behavioral irregularities The cholesterol lev-
els were not altered Interestingly, vitamin D3 and
E levels were low to undetectable
A new era in the regulation of P450 7A1
began with reports of the involvement of some
of the orphan steroid receptors The proximal
promoter region interacts with LXRα The oxy-
sterols 24( S)-hydroxycholesterol and 24( S)-
epoxycholesterol activate LXRα (and LXRβ)
[1715] Further, mice devoid of LXRα fail to
induce CYP7A1 transcription [1716] Two other
proteins, farnesoid X receptor (FXR) and cleav-
age and polyadenylation factor (CPF), are also
involved [1717–1719] Chenodeoxycholate, a
bile acid derived from cholesterol, interacts with
FXR to suppress CYP7A1 transcription [1720]
However, the action of FXR has been reported
to be indirect [1720] PXR binds lithocholic acid
and downregulates CYP7A1 [1721] Thus, cho-
lesterol metabolites control their synthesis in the
liver through feedback suppression of CYP7A1
[1717] Hylemon [1722] concluded that the dom-
inant factor is LXRα CPF binds to the promoter
623
(as a monomer) and leads to CYP7A1 transcrip-
tion [1719]
Other studies have addressed the role of
PPARα in P450 7A1 downregulation [1723]
However, differences between human and mice
gene responses have been observed, with the
mouse gene showing an enhanced response to
ligands because of an additional binding site
[1724] (further, humans have much less PPARα
than rodents [1725]) Chiang [1726] analyzed the
PPARα response and provided evidence that the
downregulation by the PPARα-agonist complex
is due to competition with HNF-4 for the DR-1
sequence
The regulation of P450 7A1 by other factors
has been considered Downregulation by TNFα
has been interpreted in the context of MEKK1,
an upstream nitrogen-activated protein kinase,
affecting HNF-4 [1727] The same mechanism
may be involved in the repression by endotox-
in and interleukin-1 [1728] A novel CYP7A1
site appears to be involved in the repression of
CYP7A1 by thyroid hormone (T3) [1729] Stud-
ies with rats indicate differences in the regulation
of P450 7A1 and P450 27A1, a sterol 27-hydrox-
ylase [1730] Human CYP7A1 expression is also
repressed by insulin and phorbol esters [1731]
Estrogen (100 µg/kg/week) increased hepatic
cholesterol 7α-hydroxylation 27-fold in ovariec-
tomized baboons [1732]
In addition to the mouse CYP7A1 knockouts,
work has been done with overexpression in mice
[1733, 1734] The mice did not exhibit altered
cholesterol levels [1734] The lack of an LXR
element in a region (− 56 to − 49) of the human
promoter may dictate some of the differences
seen in mouse and human models With regard to
humans, one study of biopsy samples from gall-
stone patients led to the conclusion that there was
no correlation between levels of total bile acids
and P450 7A1 activity [1735] A correlation was
seen with levels of CDCA
A long-standing observation from rodent stud-
ies is the apparent circadian rhythm of P450 7A1
[1736] This phenomenon has been suggested to
be indicative of a short half-life of the enzyme
[1737, 1738] The phenomenon has also been re-
ported in nonhuman primates [1739] The circa-
dian rhythm can be demonstrated at the level of
624
actual P450 7A1 in rats [1740] The molecular
mechanism of the rhythm is still not clear One
aspect is the reported instability of P450 7A1 in
microsomes (in vitro), with a t1/2 of ~ 1–2 h in hu-
mans and rats [1741] Alternatively, the mRNA
has a short t1/2 and the circadian rhythm can be
seen at the mRNA level [1742] Another unre-
solved aspect of P450 7A1 research is the issue
of phosphorylation, postulated early in the field
[1743] In vitro experiments with microsomes
show some effects of various treatments [1744–
1746], although the in vivo significance is yet
unclear ( vide infra)
Since the last edition of this chapter was pub-
lished [149], the complexity of P450 7A1 regu-
lation has increased The hepatocyte growth fac-
tor signaling pathway has been shown to inhibit
P450 7A1 expression [1747] Fibrates inhibit
P450 7A1 expression in culture via the LXRα
and PPARα heterodimers [1748] The LXR re-
pression of P450 7A1 expression in human he-
patocytes contrasts with the stimulation seen in
rodent liver [1749] The species selectivity of
P450 7A1 gene regulation has also been noted by
others [1750] ( vide supra)
Glucose stimulates P450 7A1 gene tran-
scription in human hepatocytes [1751] Insulin
regulates P450 7A1 expression (in human he-
patocytes) via Forkhead box O1 and SREBP 1c
[1752] SREBP-1c is responsible for mediating
the functional interaction of HNF-4 and PPARγ
coactivator 1α [1753, 1754]
The coactivator PGC-1α also activates P450
7A1 expression [1755] Under-expression of
both PGC-1α and SRC1 impairs HNF-4α and
promotes dedifferentiation in human hepatoma
cells and downregulation of P450 7A1 [1756]
Retinoic acid represses P450 7A1 expression
in human hepatocytes and HepG2 cells via both
FXR/RXR-dependent and independent mecha-
nisms [1757] Glycosylation of fibroblast growth
factor receptor 4 (GRF4) was shown to down-
regulate P450 7A1 [1758] Ligand-dependent
regulation of the orphan nuclear receptor small
heterodimer partner (SHP) is involved in repres-
sion of P450 7A1 [1759] Further, HNF-4α and
liver receptor homolog-1 (LRH-1) cooperate in
the regulation of P450 7A1 [1760] Thyroid hor-
F. P. Guengerich
mone was reported to regulate human P450 7A1
in humanized mice [1761]
A possible role of microRNA in P450 7A1
regulation was reported [1747]
Another aspect of P450 7A1 regulation is
phosphorylation The topic has been reviewed
by Stroup [1762, 1763] Multiple sites of phos-
phorylation have been proposed [1762], although
a proteomic search did not reveal any phosphory-
lated P450 7A1 peptides [297]
9.7.37.3 Genetic Variation
Gentic variations in the coding and noncoding
regions of the CYP7A1 gene are known [1764]
Some have been associated with clinical changes
[1765] but others have not [1766]
A promoter variant has been considered with
plant sterols and shown to yield increased P450
7A1 transcriptional activity in (transfected)
HepG2 cells [1767] Genetic variants have also
been considered in regard to colorectal [1768]
and gallbladder [1769, 1770] cancers
9.7.37.4 Substrates and Reactions
The classic reaction of P450 7A1 is cholesterol
7α-hydroxylation [37], and esterified choles-
terol is not a substrate [1771] The enzyme also
catalyzes the 7α-hydroxylation of 24-hydroxy-
cholesterol, with preference for the ( S)-isomer
[1772] 7α-Hydroxylation (with recombinant
human P450 7A1) was observed with 20( S)-
hydroxycholesterol, 25-hydroxycholesterol, and
27-hydroxycholesterol [1773] The relevance of
the activity towards 25( S)-hydroxycholesterol is
unknown compared to P450 39A1 [1774]
The P450 7A1-catalyzed 7α-hydroxylation of
cholesterol appears to be among the fastest reac-
tions for a mammalian P450, with kcat ~ 190 min− 1
and kcat/Km of ~ 24 × 106 M− 1/s [1775] (P450
21A2 is also a very efficient enzyme, vide infra)
Pre-steady-state kinetic analysis and kinetic deu-
terium isotope effects were used to establish that
the reduction of ferric iron is the rate-limiting
step in the 7α-hydroxylation [1775]
In addition to cholesterol, several other sterols
bind to P450 7A1 and show some conversion to
(uncharacterized) oxidation products, ie, epi-
cholesterols, 5-androstene-3β-ol [1776]
9  Human Cytochrome P450 Enzymes
P450 7A1 has also been demonstrated to
convert lathosterol to 7-ketolathosterol (the im-
mediate precursor of cholesterol in the normal
pathway) to 7-ketocholesterol and a trace of
the 7,8-epoxide [1777]. The reaction with Δ7-
dehydrocholesterol is proposed to be responsible
for the high level of the oxysterol 7-ketocho-
lesterol in individuals with Smith–Lemli–Opitz
syndrome [1777], and the ketone is formed in
a “direct’ reaction (carbocationic intermediate,
with hydride transfer) rather than via rearrange-
ment of the epoxide [1777] The relevance of this
reaction has been demonstrated in Smith–Lemli–
Optiz syndrome and cerebrotendinous xantho-
matosis patients [1778]
9.7.37.5 Structure
The binding of several cholesterol analogs was
used to propose a homology model [1776, 1779]
The region 214–227 has been postulated to inter-
act with the membrane and to serve as a substrate
access channel [1780] Mutations in the regions
yielded some changes in kinetic parameters to-
wards cholesterol
X-ray crystal structures of human P450 7A1
are available, unliganded and with cholest-4-en-
3-one and 7-ketocholesterol (PDB 3DAX, 3SNS,
3V8D, http://wwwrscborg, Strushkevich et al,
online but not published in periodicals)
9.7.37.6 Inhibitors
Limited information about inhibitors is available
As indicated earlier, 7-ketocholesterol is a (com-
petitive) inhibitor [1712]
9.7.37.7 Clinical Issues
P450 7A1 has been a topic of considerable inter-
est in the areas of hepatology and gastroenterol-
ogy Efforts to use drugs to utilize P450 7A1 to
lower cholesterol have been reviewed [1781]
The hypersecretion of cholesterol in obe-
sity does not appear to be due to reduced 7α-
hydroxylation [1782] Coffee terpenes (eg, caf-
estol) inhibit P450 7A1 and also raise cholesterol
levels [1783], although it is not clear that the two
phenomena are linked The complex regulation
of P450 7A1 makes interpretation of experi-
ments difficult Overexpression of P450 7A1 in
625
HepG2 cells increased bile acid synthesis but led
to decreased HMG-CoA reductase activity (rate-
limiting step in cholesterol biosynthesis) [1784]
Alterations in P450 7A1 were not seen in
hypo- or hyperthyroidism [1785]
A 10-week-old child with a stop codon muta-
tion and lacking P450 7A1 presented with severe
cholestasis, cirrhosis, and liver synthetic failure
[1765] A frameshift leading to (homozygous)
lack of P450 7A1 was associated with high LDL
cholesterol but not total cholesterol [1786] Het-
erozygotes were also hyperlipidemic However,
Beigneux et al [1787] have discussed some of
the caveats associated with interpretation of re-
sults of family and experimental studies with
P450 7A1
Several studies have been published on the
effects of genetic variants on plasma lipid com-
position [1788–1790] and also on response to
a high-fat diet [1791, 1792] Genetic variations
have also been linked to responses to fibrates
[1793] and statins [1794–1796]
Genetic variations in P450 7A1 have also
been related to gallstone disease [1797], bile acid
synthesis rates following ileal resection [1798],
risk of neuromyelitus optica [1799], and hyper-
tension [1800]
9.7.38 P450 7B1
P450 7B1, a microsomal P450, was discovered
as an “alternative” 7α-hydroxylase that used oxy-
sterols as substrates [1801, 1802] The enzyme
is conserved in nature, even in a Japanese fire-
bellied newt and the fungus Aspergillus niger
[1802]
9.7.38.1 Sites of Expression
P450 7B1 mRNA is found not only in liver but
also in the steroidogenic tissues testes, ovary, and
prostate, in brain, and in colon, kidney, and small
intestine [1803, 1804] The tissue specificity of
expression varies among species Human mRNA
levels are highest in kidney and brain, but ex-
pression is also seen in tissues involved in steroid
biosynthesis (testes, ovary, prostate) and bile acid
synthesis (liver) and reabsorption (colon, small
626
intestine) [1805] As will be seen later, the clini-
cal issues are mainly associated with the lack of
the enzyme in liver and brain [1802] P450 7B1
is overexpressed in prostate during progression
of prostate adenocarcinoma [1806] Evidence
was presented for the existence of multiple sterol
7α-hydroxylases [1801, 1807], and a novel rat
brain gene was identified [1808, 1809] Although
much of the literature involves animal models, a
considerable amount of interest has been gener-
ated regarding human P450 7B1 because of its
role in multiple diseases [1802]
9.7.38.2 Regulation
In mice, a gender variation has been reported,
along with hormonal regulation, but whether any
of this applies to humans is unknown Expres-
sion is regulated by androgens and estrogens in
prostate cancer LNCaP cells [1810] and HEK293
cells [1810] A possible role for estrogenic regu-
lation of P450 7B1 controlling DHEA levels in
human tissues has been proposed [1810] HNF-
1α and Sp1 regulation has been reported [1811–
1813] In mice, the CYB7B1 gene is regulated
by RORα and LXR [1814], but this has not been
confirmed in a human-based system P450 7B1
expression was upregulated in (human) prostate
during prostatic adenocarcinoma [1806] Human
CYP7B1 gene expression is controlled by SREBP
[1754]
9.7.38.3 Genetic Variation
At least 17 different variants have been found in
> 20 unrelated families due to the significance of
diseases ( vide infra) [1765, 1802, 1815–1818]
Not surprisingly, there are ethnic differences
[1819] A number of variants have been identi-
fied in patients with hereditary spastic paraplegia
type 5 [1815, 1820–1826 and liver failure [1827]
Other variants have been identified but not nec-
essarily related to diseases [1819, 1828]
9.7.38.4 Substrates and Reactions
Human P450 7B1 has not been purified or char-
acterized in kinetic terms, and much of what is
concluded is based on inference from animal
models [1802] The oxidations are 7α- and 6α-
hydroxylation of several steroids and oxyster-
F. P. Guengerich
ols (eg, 25- and 27-hydroxycholesterol, 7α-
hydroxylation of pregnenolone, DHEA, 25-hy-
droxycholesterol, and 27-hydroxycholesterol and
6α-hydroxylation of 5α-androstane-3β, 17β-diol
[1802–1804] Other reported substrates include
testosterone and 17β-estradiol [1803, 1804]
DHEA is a “prohormone,” secreted by the adre-
nals, and undergoes tissue-specific metabolism
to yield multiple products that have a variety of
biological effects [1803, 1804], producing com-
pounds important in cognition, behavior, and im-
mune response [1808, 1829]
5α-Androstene-3β,17β-diol (“anediol”) un-
dergoes 6α-hydroxylation, and this reaction
occurs in prostate The rest of the reactions
are all 7α-hydroxylations In the liver, the 7α-
hydroxylations of 25- and 27-hydroxycholes-
terol are associated with bile acid synthesis In
the brain, 7α-hydroxylation of pregnenolone
and DHEA is part of steroid hormone metabo-
lism Metabolism of ER ligands involves 7α-
hydroxylation of DHEA in the prostate and
27-hydroxycholesterol in the vasculature (as well
as 6α-hydroxylation of 5α-androstene-3β,17β-
diol) Immunoglobulin production (in immune
cells) involves 7α-hydroxylation of 25-hydroxy-
cholesterol Another known reaction is the 7α-
hydroxylation of 5α-androstene-3β,17β-diol
(“enediol”), at least with the rat enzyme
9.7.38.5 Structure
No structures of P450 7B1 are available, in that
the enzyme has not been reported to be purified
yet At least two homology models have appeared
[1820, 1830]
9.7.38.6 Inhibitors
No specific inhibitors of P450 7B1 have been
reported A nonselective inhibitor, clotrimazole,
was used to inhibit the rat enzyme in prostate
fractions [1831] Schwarz et al [1809] note
that nafimidone has been reported to inhibit the
mouse enzyme but not the human
9.7.38.7 Clinical Issues
Stiles et al [1802] reviewed the two major issues,
both of which are related to genetic variations
One is liver failure in children and the other is
9  Human Cytochrome P450 Enzymes
neuropathy in adults These seemingly unrelated
diseases may be understood in the variety of P450
7B1 substrates and the diversity of biological ac-
tions of steroids The biological roles of P450
7B1 include hepatic bile salt synthesis (25- and
27-hydroxycholesterol being substrates), brain
steroid hormone metabolism (pregnenolone and
DHEA being substrates), prostate and vascula-
ture metabolism of ER ligands (5α-androstane-
3β,17β-diol, DHEA, and 27-hydroxycholesterol
being substrates), and immunoglobulin produc-
tion in immune cells (25-hydroxycholesterol
being substrate) Overall, there are two driving
issues, the production of appropriate steroid hor-
mones and the removal of deleterious oxysterols,
depending upon the site
The two major clinical issues are liver failure
in children (due to genetic insufficiency) [1802,
1827, 1832, 1833], and neuropathy (in adults),
particularly the autosomal recessive disorder
spastic paraplegia type 5 [1802, 1815–1818,
1824, 1825] Possible association with Alzheim-
er’s disease has also been reported [1834] An
association with rheumatoid arthritis has been
considered [1835] P450 7B1 has also been men-
tioned regarding (low activity) and the promotion
of cell-autonomous ER-positive breast cancer
[1836]
9.7.39 P450 8A1
Prostacyclin (prostaglandin I2) has strong vaso-
dilation and anti-aggregation effects on platelets,
and the imbalance of prostacyclin and thrombox-
ane A2 (product of P450 5A1) is a factor in sev-
eral diseases, eg, myocardial infarction, stroke,
atherosclerosis [1837, 1838] The reaction yield-
ing prostacyclin from prostaglandin H2 is another
“internal” oxygen transfer, without the input of
O2 and electrons from NADPH (Fig 921), and
the involvement of a P450 was not immediately
obvious Ullrich hypothesized P450 involvement
on the basis of spectral interaction studies [1839]
DeWitt and Smith [1840] used a monoclonal an-
tibody to purify catalytically active prostacyclin
synthase from bovine aorta and demonstrated a
P450 Fe2 + ·CO spectrum Subsequently P450
627
8A1 was cloned from bovine endothelial cells
[1841]
9.7.39.1 Sites of Expression
A human P450 8A1 cDNA was cloned from
aorta endothelial cells by the Tanabe laboratory
[1838] The mRNA is widely expressed in human
tissues, including ovary, heart, skeletal muscle,
lung, prostate [1838], and umbilical vein [1842]
There is also localization in the brain, including
neurons [1843, 1844] Another site of expression
is fallopian tubes, with expression in luminal epi-
thelia, tubal smooth muscle, vascular endothelial
cells, and vascular smooth muscle cells [1845]
9.7.39.2 Regulation
P450 8A1 is constitutively expressed in human
endothelial cells [1842] The human CYP8A1
gene (chromosome 20) has ten exons [1846–
1848] and has consensus sequences for Sp1, acti-
vating protein-2 (AP-2), an interferon-γ response
element, GATA NFκB, a CACCC box, gluco-
corticoid receptor, and a shear stress-responsive
element (GAGACC) [1846] Whether or not all
of these are functional and how they interact to
maintain constitutive expression is not well un-
derstood yet
Hypermethylation of the promoter has been
reported as a frequent event in colorectal cancer
[1849]
One posttranslational aspect of regulation is
redox control of P450 8A1 Peroxynitrite causes
nitration of Tyr-430 [1850], causing inactivation
due to steric hindrance of the active site [1851]
This nitration has been reported to be associated
with enhanced retinal cell apoptosis in diabetes
[1852]
9.7.39.3 Genetic Variation
Variants have been of interest because of disease
relevance At least 14 alleles have been reported,
yielding four different proteins (http://wwwcyp-
alleleskise) Haplotypes have been considered
in the context of essential and thromboembolic
preliminary hypertension [1853, 1854], myocar-
dial infarction [1855], left main coronary artery
disease [1856], and cardiovascular disease in
general [1857]
628
In the 5ʹ-region, these are variants involving a
variable number of tandem repeats (VNTR) that
affect transcription, as demonstrated in reporter
systems in vitro [1672] An association between
this VNTR polymorphism and cerebral infarction
has been reported [1858]
An SNV in exon 8 has been reported to be
linked to myocardial infarction, although no
amino acid change occurs [1859] However, the
VNTR variation does not appear to be related
to essential hypertension [1860], nor does the
5ʹ-flanking region SNV T192G [1861] However,
a novel splicing variation leading to skipping of
exon 9 has been linked to hypertension [1862]
9.7.39.4 Substrates and Reactions
P450 8A1 has a very limited catalytic specific-
ity, functioning only as the prostacyclin synthase
(Fig  921) Prostaglandins G2, H2, 13( S)-hy-
droxy H2, 15-keto H2, and H3 are isomerized to
the corresponding prostacyclins [1682] Spectral
binding studies with 9,11-epoxymethano pros-
taglandins F2 and F2α lead to the view that the
binding juxtaposition is the key determinant in
distinguishing the courses of catalysis by P450s
5A1 and 8A1 [1682] A mechanism consistent
with available data has been proposed (Fig 923)
[1662, 1682]
Yeh et al [1680] used 15-hydroperoxyeico-
satetraenoic acid (15-HPETE) as a substrate for
P450 8A1 and found both hemolytic (15-ketoe-
icosatetraenoic acid) and heterolytic (15-hy-
droxyeicosatetraenoic acid) products, with the
former reaction accounting for ~80 % of the total
9.7.39.5 Structure
A crystal structure of human P450 8A1 was re-
ported by Chiang et al [1863] in 2006 This struc-
ture did not include a substrate In 2008, another
structure was published by the same group, with
a substrate (U51605) analog and an inhibitor (mi-
noxidil) [1864] Relative to the unliganded mol-
ecule, conformational changes were observed at
the proximal side of and in the heme itself
Other work has been on membrane topology,
and antibody studies indicate that P450 8A1 is
mainly exposed on the cytoplasmic site of the
endoplasmic reticulum with a single transmem-
F. P. Guengerich
brane anchor [1865, 1866] The (unstable) sub-
strate, prostaglandin H2, is produced in the lumen
and apparently passes through the membrane to
reach P450 8A1
9.7.39.6 Inhibitors
Relatively little interest has been shown in devel-
opment of drugs that inhibit P450 8A1 because
inhibition is generally considered to be deleteri-
ous Phenylbutazone has been reported to inhibit
[1867]
The prostaglandin synthase inhibitor rofe-
coxib (Vioxx®, now withdrawn from the market)
was reported to inhibit P450 8A1 [1868]
P450 8A1 is slowly inactivated during the
normal reaction itself, apparently by one of
the reactive intermediates in the catalytic cycle
(Fig  923) [1869] A kinactivation of 006 s− 1 was
reported [1869]
Peroxynitrite is a powerful inhibitor of P450
8A1, with a reported KI of 50 nM [1870] Per-
oxynitrite is formed by the chemical reaction of
NO·and O2 − [1871] The mechanism is believed
to involve tyrosine nitration [1872], and recently
Tyr430 has been implicated as the site of nitra-
tion [1873]
9.7.39.7 Clinical Issues
As mentioned earlier, prostacyclin is a powerful
vasodilator and inhibits platelet adhesion and un-
desired cell growth Although this view may be
overly simplistic, prostacyclins are a counterbal-
ance to thromboxanes in a “yin and yang” rela-
tionship Thus, the action of P450 8A1 balances
that of P450 5A1 Several of the genetic vari-
ants (Sect 7393, vide supra) have been related
to diseases, particularly cardiovascular disease
[1874]
Decreased expression of P450 8A1 has been
reported in severe pulmonary hypertension
[1875] With regard to general cardiovascular
disease, a study of Japanese subjects associated
the VNTR variation with hypertension (odds
ratio 19) [1876] Individuals with three to four
repeats had less promoter activity and higher
risk In experimental studies, the overexpression
of P450 8A1 in transgenic mice protected against
the development of hypoxic pulmonary hyper-
9  Human Cytochrome P450 Enzymes
tension [1877] In another study, the expression
of human P450 8A1 in the carotid arteries of rats
after arterial balloon injury (using a virus) led
to increased synthesis of prostacyclin and to re-
duced neointimal formation [1878]
P450 8A1 also has relevance in cancer treat-
ment Transfection of colon adenocarcinoma
cells with P450 8A1 led to slower growth and
reduced vascular development following inocu-
lation into syngeneic mice [1879] P450 8A1 has
also been considered in the context of cancer as a
target in non-small cell lung cancer [1700]
Finally, antibodies in the sera of some patients
with hypersensitivity reactions to phenytoin and
carbamazepine recognize rat P450 3A1 but not
human P450 3A [1880] The antisera also recog-
nizes P450s 8A1 and 51A1, although relation-
ships of etiology and causality are unclear
9.7.40 P450 8B1
9.7.40.1 Sites of Expression
P450 8B1 is a sterol 12α-hydroxylase expressed
in the liver The human CYP8B1 gene was char-
acterized on the basis of the rabbit and mouse or-
thologs [1881] Of interest is the finding that this
gene is devoid of introns, unique for this gene
among the P450 family [1881]
9.7.40.2 Regulation
Regulation of the gene is of interest, in that P450
8B1 catalyzes the synthesis of cholic acid and
controls the ratio of cholic acid to CDCA in the
bile [1882] Much of what has been reported
in the literature is with animal models HNF4α
activates human CYP8B1 expression in HepG2
cells [1882] Bile acids and FXR downregulate
HNFα expression Inflammation in liver cells
causes increased synthesis of α1-antitrypsin, a
serum protease inhibitor, and in a derived pep-
tide (C-36) C-36 appears to interact with the α1-
fetoprotein transcription factor (FTF) site in the
human CYP8B1 promoter, inducing a conforma-
tional change to lower DNA binding ability, and
suppressing the transcription of the CYP8B1 (and
CYP7A1) genes [1883, 1884] HNFα could over-
come the inhibitory effects of FTF and bile acids
629
[1884] Thus, regulation of P450 8B1 is involved
in bile acid feedback inhibition
Ligand-dependent regulation of the orphan
nuclear receptor SHP has been reported to down-
regulate P450 8B1 expression is HepG2 cells
[1759] Phenobarbital regulated P450 8B1 in
HepaRG cells [1885] The corepressor GOS2 has
also been reported to regulate P450 8B1 [1886]
Soy isoflavones upregulated human P450 8B1
[1887] Based on animal models, cytokines and
liver factor HNF-1α regulate P450 8B1 [1888,
1889]
The in vivo phosphorylation of P450 8B1 has
been reported [297]
P450 8B1 has been reported to show circadian
rhythm [1803]
9.7.40.3 Genetic Variation
Limited reports on genetic variation have ap-
peared [1890, 1891]
9.7.40.4 Substrates and Reactions
P450 8B1 catalyzes the 12α-hydroxylation
of several oxysterols, including 4β- and 7α-
hydroxycholesterol and 7α, 24- and 7α,27-
dihydroxycholesterol, yielding (following P450
27A1 action) the primary bile acid cholic acid
[1803] P450 8B1 controls the balance between
cholic acid and CDCA, adjusting the hydropho-
bicity of the bile (cholic acid is more hydrophilic
than CDCA) However, variations in the cholic
acid to CDCA ratio do not seem to be controlled
by genetic variation in P450 8B1 [1803]
9.7.40.5 Structure
No structures have been reported, and a literature
search did not reveal any homology models
9.7.40.6 Inhibitors
No selective inhibitors have been published
CDCA has been reported to inhibit P450 8B1 A
limitation of inhibition of P450 8B1 activity is
that a decrease in the cholic acid to CDCA ratio
might cause hepatotoxicity, which was observed
in patients treated with CDCA for gallstones
[1803, 1892]
630
9.7.40.7 Clinical Issues
An SNV in the CYP8B1 gene has been associ-
ated with gallstone disease in a Han Chinese
population [1890] However, P450 8B1 has been
reported to have a smaller effect on bile acid syn-
thesis than P450 7A1 in human liver [1893]
9.7.41 P450 11A1
P450 11A1 is the enzyme involved in the initia-
tion of hormonal steroid synthesis (Fig 912) It
catalyzes the conversion of cholesterol to preg-
nenolone by side-chain cleavage and has been
referred to in the older literature as P450scc or
cholesterol desmolase The enzyme was first pu-
rified from bovine adrenal cortex mitochondria
[1894] The human gene was cloned by Omura
and Fujii-Kuriyama in 1987 [1895] and includes
nine exons Of historical significance is the fact
that this P450 only contains a single cysteine
and further establishes the position of the heme
thiolate peptide in P450s, extending the work on
the location from the original crystal structure of
bacterial P450 101A1 [1896]
9.7.41.1 Sites of Expression
P450 11A1 is found primarily in steroidogenic
tissues, including adrenal cortex and gonads, in-
cluding ovary (corpus luteum [1897, 1898] and
theca interna cells [1899] and others [1900]) Of
interest are reports of P450 11A1 in brain [1901–
1904] and pancreas [1905]
P450 11A1 is one of several P450s localized
in the mitochondria (Table 92, Fig 912) Studies
with the bovine enzyme demonstrated that P450
11A1, synthesized on ribosomes in the cytosol,
is imported into mitochondria without processing
of the amino terminal extension peptide [1906]
The protein moves to the mitochondrial inner
membrane and is then cleaved to yield the mature
form [1906] Alteration of the basic amino acid
residues of the N terminus resulted in less effi-
cient mitochondrial import [1907] Miller and his
associates constructed vectors that could be used
to direct P450 11A1 to the endoplasmic reticulum
and found that the enzyme was inactive [1908]
The membrane environment was concluded to be
F. P. Guengerich
more important in modulating catalytic function
than the nature of the electron transfer partners
9.7.41.2 Regulation
The regulation of P450 11A1 is relatively com-
plex, as might be expected for the initial step in
steroid formation [1900] Moreover, the system
must be able to respond to signals in many dif-
ferent tissues Much of our understanding of the
regulation of P450 11A1 expression is based on
studies with CYP11A1 genes of experimental ani-
mals and reinvestigated with human CYP11A1
P450 11A1 has long been known to be regu-
lated by ACTH and cyclic AMP In the bovine
CYP11A1 gene, two Sp1-binding sites mediate
cyclic AMP transcription through the protein
kinase A signaling pathway, utilizing the rather
ubiquitous transcription factor Sp1 [1909] Ste-
roidogenic factor-1 (SF-1) activates CYP11A1
transcription through interaction with protein
factors upstream [1900] An upstream cAMP re-
sponse element-binding protein (CREB)-binding
region and an AP-1 site are also involved in the
cyclic AMP response Sp3 can also be involved
[1910] The TATA box drives cell type-specific
cyclic AMP-dependent transcription [1911]
SF-1 also interacts with Sp1 [1912–1914] Thus,
the regulation of the human CYP11A1 gene in-
volves all the above factors plus an AdE element
[1900] Expression of the human gene has been
shown to involve the zinc finger protein TreP-
132, interacting with both CBP/p300 [1915] and
SF-1 [1916] Also, salt-inducible kinase (SIK)
represses cyclic AMP-dependent protein kinase-
mediated activation through the CREB basic leu-
cine zipper domain [1917] In human placenta,
AP-2 assumes the role of SF-1 by binding to an
overlapping promoter element [1918]
An analysis of the P450 11A1 promoter has
been reported [1919]
The orphan nuclear receptor LRF-1 regulates
P450 11A1 expression in human granulosa cells
[1920] The human transcription factor LBP-32
(also termed mammalian grainyhead, MGR, or
LBP-32/MGR) has been reported to be a repres-
sor of P450 11A1 [1921] Cyclic AMP has been
reported to stimulate SF-1-dependent expression
of P4540 11A1 through homeodomain-interact-
9  Human Cytochrome P450 Enzymes
ing protein kinase 3-mediated JNK and c-Jun
phosphorylation [1922]
Abnormal expression of uncoupling protein-2
has been correlated with altered P450 11A1 ex-
pression in polycystic ovary syndrome (PCOS), the
main cause of infertility in women [1923] Further,
studies in PCOS theca cells showed that basal and
forskolin-stimulated P450 11A1 mRNA levels and
promoter activity were increased [1924] The tran-
scription factor nuclear factor 1C2 regulated the
basal activity of the minimal P450 11A1 promoter
element The P450 11A1 mRNA t1/2 increased
> twofold in the PCOS cells compared to normal
ones. The 5ʹ-untranslated region of the P450 11A1
mRNA conferred the added stability [1924]
9.7.41.3 Genetic Variation
Variations in CYP11A1 can cause congenital
adrenal insufficiency Arg-353 was found to be
critical in a study with an afflicted patient [1925]
The relationship of PCOS to the P450 11A1
promoter variants was already mentioned in
Sect.  7.41.3. ( vide supra) This issue has been
considered in a large genetic study [1926] Other
genetic studies have been reported on P450 11A1
and PCOS [1927, 1928], including microsatellite
variants [497]
Disruption of the P450 11A1 gene has been
associated with premature birth, sex reversal, and
adrenal failure [1929] Genetic variations have
also been linked to adrenal and gonadal deficien-
cy [1930, 1931]
P450 11A1 variants have also been related to
breast [1932, 1933] and endometrial [1934] cancers
9.7.41.4 Substrates and Reactions
The P450 11A1 reaction proceeds in a three-step
sequence, with generation of (22R)-20α, 22-dihy-
droxycholesterol as an intermediate (Fig 922)
[1935] Oxidative cleavage of the diol to pregnen-
olone and 4-methylpentanal (isocaproic aldehyde)
completes the overall reaction The mechanism of
the last step is not completely clear, but some pro-
posals have been presented [1936–1938]
The rate of electron transfer from adrenodoxin
is important and appears to be the rate-limiting
step for the enzyme in human placenta [1939]
The redox potential of adrenodoxin can be varied
631
by site-directed mutagenesis but had little effect
on rates of electron transfer, consistent with the
view that other factors such as protein–protein
interactions are more important than the intrin-
sic thermodynamics [1940] When P450s 11A1
and 11B1 are expressed together in cells, they
can compete for reducing equivalents from ad-
renodoxin [1941]; exactly how important the
competition is in tissues is unclear Another re-
port indicates interaction of P450 11A1 with and
enhancement by cytochrome b5 [1942], although
the relevance is unclear because of the compart-
mental separation of P450 11A1 (mitochondria)
and cytochrome b5 (endoplasmic reticulum)
P450 11A1 has now been found to be less
specific than originally thought Vitamin D3 is
oxidized to a number of different products, on
the “side chain,” by P450 11A1, mainly 20-hy-
droxy- and 20,23-dihydroxyvitamin D3 [1943,
1944] In addition, 23-hydroxy-, 17α-hydroxy-,
17α,20-dihydroxy- [1944], and 20,22-dihy-
droxyvitamin D3 [1945] are produced [1946]
1α-Hydroxyvitamin D3 can yield 1α,20-
dihydroxyvitamin D3 [1947] Several of these
products have biological activities [1945, 1948]
and are formed in vivo (animal models) [1949]
7-Dehydrocholesterol is also a substrate for
P450 11A1 [1943], forming five 5,7-dienal
products, with mono- and dihydroxy substitu-
tion [1949] These include the 22-hydroxyl and
20,22-dihydroxy 7-dehydrocholesterol products
Human P450 11A1 also oxidizes ergosterol
(the vitamin D2 precursor) to two major and four
minor products [1950] The major products have
been characterized as 20-hydroxy-22,23-epoxy-
and 22-keto-23-hydroxyergosterol
Finally, rat and human P450 11A1 have
been implicated in the metabolism and bio-
activation of a drug candidate, BMS-A (( N-
(4-((1H-pyrrolo[2,3[b]pyridine-4-yl)oxy)-
3-flurophenyl)2-oxo-1,2-dihydropyridine-3-car-
boxamide) [1951] The bioactivation was impli-
cated in the vacuolar degeneration and necrosis
of the adrenal cortex of rats
In conclusion, the specificity of P450 11A1 is
not so stringent as originally thought [149] Thus,
in considering P450 11A1 in a classification such
as that in Table 91, it joins other steroid metabo-
 632

Fig. 9.22   Three multistep C–C steroid bond cleavage P450 reactions (P450s 11A1, 19A1, 51A1) Aromatization reactions catalyzed by P450 19A1: The three distinct steps are
shown with the substrate testosterone Other physiologically relevant substrates are androstenedione and 16α-hydroxytestosterone
F. P. Guengerich
9  Human Cytochrome P450 Enzymes
lism P450s such as P450s 1B1, 3A4, 24A1, and
46A1 in bridging among steroid, vitamin, and xe-
nobiotic substrates
9.7.41.5 Structure
In 2011, Pikuleva’s group [1952] reported a
structure of bovine P450 11A1 bound to 22-hy-
droxycholesterol, the first reaction product (from
cholesterol) The active site cavity can be de-
scribed as a long curved tube that extends from
the surface to the heme group (A linker was used
to tether adrenodoxin to P450 11A1) The [2Fe–
2S] iron cluster of adrenodoxin was positioned
17 Å away from the heme iron of P450 11A1
A crystal structure of a human P450 11A1–
adrenodoxin complex was also reported in the
same year [1953], in the presence of 22-hydroxy-
cholesterol A structure with 20,22-dihyroxycho-
lesterol has also been published [1953]
Limited proteolysis experiments done with
P450 11A1 in E. coli membranes identified
peptides from the putative F–G loop (residues
218–225) and the C-terminal portion of the G-
helix (residues 238–250) as being involved in
membrane binding [1954] (these assignments are
consistent with the crystal structures)
Studies with bovine P450 11A1 indicated
the significance of Lys-377 and Lys-381 in ad-
renodoxin binding [1955] As indicated earlier, a
mutation at Arg-353 was found to attenuate the
function of P450 11A1 in a patient [1925] Site-
directed mutagenesis of human P450 11A1 (in
E. coli) indicated that Ile-462 had some effect on
kinetic parameters [1956]
9.7.41.6 Inhibitors
A number of inhibitors of P450 11A1 have been
reported, although some were studied only with
the bovine enzyme [1957, 1958], including some
acetylenic mechanism-based inactivators [1959]
With regard to the human enzyme, there is some
potential for the use of inhibitors in treatment of
prostatic cancer, and prodrug forms of amino-
glutethimide have been examined [1960] Anti-
convulsants have been reported to inhibit P450
11A1, but the interaction is not strong [1961]
Pikuleva’s group has published a study of
the inhibition of P450 11A1 by a selected set of
633
drugs [1962] When tested at a concentration of
10 µM (cf 1 µM cholesterol as substrate), only
ketoconazole, carbenoxolone, and selegiline in-
hibited > 50 % No IC50 values were calculated,
but spectral analysis yielded Kd values of 15 and
10 µM for ketoconazole and posaconazole
9.7.41.7 Clinical Issues
Several issues are of interest P450 11A1 insuf-
ficiency and relationship to diseases in general
have been reviewed by Miller and Auchus [1963]
Because of the nature of P450 11A1 in initiating
steroidogenesis, genetic variation in P450 11A1
is related to adrenal insufficiency and to congeni-
tal adrenal hyperplasia [1931, 1964–1966] Rab-
bit and mouse models show the effects [1967,
1968] CYP11A1-null mice die shortly after birth
but can be rescued by steroid injection [1968]
ACTH levels become very high due to lack of
feedback regulation by glucocorticoids Male
null mice are feminized with female external
genitalia and underdeveloped male accessory sex
organs These manifestations resemble various
human steroid deficiency syndromes
Another issue is autoantibodies to P450 11A1
(and also P450 17A1) in patients with autoimmune
polyglandular syndrome types I and II and Addi-
son’s disease [1969–1971] As with other P450s
recognized by autoantibodies, causal relationships
between immunity and disease are unclear
The relationship of P450 11A1 genetic varia-
tion and PCOS has already been mentioned in
Sect.  7.41.3 ( vide supra), including premature
birth, sex reversal, and severe adrenal failure
[1926, 1929, 1972]
Variants have also been linked to reduced
P450 11A1 ovarian transcription during experi-
mental nephrotic syndrome [1973] Finally, P450
11A1 variants have been associated with breast
[1974] and prostate [1975] cancers
9.7.42 P450 11B1
P450s 11B1 and 11B2 differ in only 32 residues
P450 11B1 catalyzes the 11β-hydroxylation of
deoxycortisol to yield cortisol (Fig 923), the
main glucocorticoid in the body Deficiencies in
 634

Fig. 9.23   Reactions catalyzed by P450s 11B1 and 11B2

F. P. Guengerich
9  Human Cytochrome P450 Enzymes
the enzyme are known, causing congenital adre-
nal hyperplasia [47, 1976]
9.7.42.1 Sites of Expression
P450 11B1 is expressed in the adrenal cor-
tex, specifically the zona fasciculata/reticularis
[1976] In rats, some expression has been detect-
ed in brain, but the relevance is not clear
P450 11B1 is synthesized in the cytosol and
directed to the mitochondria with a 24-residue
N-terminal-targeting sequence (where this is lost
after entry) As with the other six (exclusively)
mitochondrial P450s (Table 92), P450 11B1
receives electrons from adrenodoxin instead of
NADPH-P450 reductase
The characterization of the CYP11B1 gene has
developed considerably in recent years Much of
the early research in this field was done with bo-
vine adrenal glands because of the need for large
amounts of material, but the bovine P450 11B1
protein has the function that P450 11B1 (11-hy-
droxylation) and P450 11B2 (11-hydroxylation,
18-hydroxylation, and oxidation of the 18-alco-
hol to an aldehyde) have in most other species,
including humans [1977] The two human genes
( CYP11B1, CYP11B2) were characterized and
clearly shown to both be essential [1978–1981]
P450 11B1 expression has also been deleted
in human fetal adrenal gland, particularly in the
“fetal zone” (as opposed to neocortex) [1982]
9.7.42.2 Regulation
Much of the background on regulation of P450
11B1 comes from studies with the bovine gene,
which responds to ACTH and has six cis-acting
regulatory elements [1983] The protein (Ad4BP)
that binds to one of these (Ad4) is a member of
the steroid hormone receptor superfamily [1984]
Other studies by Omura [1985] indicated the co-
operative nature of these elements in transcrip-
tion Work with the rat CYP11B1 gene showed
that ACTH stimulates transcription by changing
composition in AP-1 factors (Fos, Jun) [1986]
The human gene also has a cyclic AMP re-
sponse element (CRE) [1987] The Ad1 element
binds CRE-binding protein, activating transcrip-
tion factor-1 (ATF-1), and ATF-2 SF-1 interacted
at the Ad4 site (− 242/− 234) and is required for
635
transcription [1987, 1988], which contrasts with
the lack of response of CYP11B2
ACTH modulation of transcription factors in-
volved in regulation has been reviewed by Sewer
and Waterman [181]
The orphan nuclear receptors NURR1 and
NGF1B regulate P450 11B1 transcription in
human H295R adrenocortical cells, and transfec-
tion with SF-1 activated P450 11B1 expression
[1989]
P450 11B1 expression (mRNA and protein)
was significantly higher in patients with subclini-
cal Cushing’s syndrome [1990], although the mo-
lecular basis is not known
MicroRNA-24 was reported to regulate P450
11B1 expression in a human adrenocortical cell
line [1991] The human P450 11B1 promoter con-
tains two Alu elements embedded in a truncated
L1 element, breaking L1 into three individual
fragments [1139] The effect of Alu is blocked by
a second L1 element (CYP11B1-L12) inserted
between the first one and the conserved proximal
upstream region The CYP11B1-L12 element
can be transcribed from the core promoter in
an opposite direction (and a smaller magnitude)
compared to P450 11B1 Deletion of CYP11B1-
L12 greatly increased P450 11B1 promoter ac-
tivity and restored the effect of Alu [1139] The
Ad5 and SF-1 binding elements in the proximal
core promoter play a role in basal expression
A polychlorinated biphenyl (PCB126) has
been reported to upregulate P450 11B1 tran-
scription in human adrenocortical cells due to
enhancement of mRNA stability but not an AhR
mechanism [1992] The practical significance of
the results is unclear, in that only concentrations
≥ 10 µM were used.
9.7.42.3 Genetic Variation
Many variants are known because of the relation-
ship of the gene with congenital adrenal hyper-
plasia [1976] Genetic variants related to phe-
notype and to inborn errors of metabolism have
been reviewed [1993, 1994]
A large number of genetic variants in P450
11B1 have now been identified and related to
high 18-hydroxycortisol [1995], to low 11β-
hydroxylation [1996–2002], congenital adre-
636
nal hyperplasia [2003–2010], and hypertension
[2011] The variants include a five-base dupli-
cation [2012] and clusters of mutations in exons
6–8 [2013] The high similarity and proximity of
the CYP11B1 and CYP11B2 genes appear to lead
to variants generated by unequal crossover and
inactive chimeric products [2014–2017] Splice
donor site variants are also known [2018]
9.7.42.4 Substrates and Reactions
As indicated previously, the only reported sub-
strate for P450 11B1 is deoxycortisol, which
undergoes 11β-hydroxylation to yield cortisol
(Fig 912)
9.7.42.5 Structure
One of the concerns about studies on the func-
tion of particular residues in site-directed muta-
genesis is that expression in some cellular sys-
tems leads to competition between P450s 11A1
and 11B1 for (adrenodoxin) reducing equivalents
in cellular systems [1941] Another issue is that
human P450s 11B1 and 11B2 have been difficult
to express in bacteria, so that most experiments
have relied on mammalian cells ( Schizosac-
charomyces pombe has provided some success)
[1976]) Information about function has also
been obtained from patients’ samples [1976]
Although no crystal structures of P450 11B1
have been published, structures of the highly
similar P450 11B2 (one with substrate, one with
an inhibitor) have appeared [2019] and, at the
very least, should facilitate future modeling
The close similarity of P450s 11B1 and 11B2
(and their reactions) has also facilitated studies
Making the changes S288G and V320A yielded
an enzyme with both P450 11B1 and 11B2 activi-
ties [2020] Changes at positions 147 [2021, 2022]
and 301/355 [2023] have also had the same effect
Homology models of P450 11B1 have also been
published [1976, 2008, 2024–2026], although the
effects of all of the mutants known to alter func-
tion have not been systematically rationalized
9.7.42.6 Inhibitors
Compared with some of the other steroidogenic
P450s, there is some reason to develop P450
11B1 inhibitors High levels of cortisol are asso-
ciated with Cushing’s syndrome [1976] Cellular
F. P. Guengerich
expression systems have been set up to assay for
inhibitors, using measurements of concentrations
of steroids [2027, 2028]
18-Vinylprogesterone and 18-ethinylproges-
terone have been reported to be mechanism-
based inactivators of bovine P450 11B but ap-
parently have not been tested with human P450
11B1 [2029]
Since the previous edition of this book [149],
work on more P450 11B1 inhibitors has been
published [147, 2030–2036] Some of these have
been developed with the specific goals of treat-
ing prostate cancer [2037] and cardiovascular
disease [2038, 2039] One case report involves a
beneficial effect in the management of an elderly
patient with an androgen-producing inoperable
adrenal tumor [2040]
9.7.42.7 Clinical Issues
As indicated previously, the main issue with
P450 11B1 is the impaired synthesis of cortisol
and congenital adrenal hyperplasia, characterized
by hypertension and signs of androgen excess
[2041, 2042] The role of P450 11B1 insufficien-
cy in congenital adrenal hyperplasia has been re-
viewed [2043, 2044] The same condition is seen
in a knockout mouse model [2045] Overproduc-
tion of glucocorticoids, which could have any of
several causes, including overactive P450 11B1,
is associated with Cushing’s syndrome [1976]
A number of genetic variations associated with
disease are cited in Sect. 7.42.3 ( vide supra) A
chromosome inversion was also seen in a fam-
ily [2046] Testicular tumors in patients with
P450 11B1-related congenital adrenal hyperpla-
sia showed functional features of adrenocortical
tissues [2047] Hyperplasia of adrenal rest tissue
was implicated in causing a retroperitoneal mass
in a child with P450 11B1 deficiency [2048]
P450 11B1 deficiency has also been associ-
ated with hypertension [2049–2051] rhabdomy-
olysis [2052], virilization [2053], and prepubertal
gynecomastia [2054]
9.7.43 P450 11B2
P450 11B2 is highly related to P450 11B1 ( vide
supra) and has a somewhat similar function P450
9  Human Cytochrome P450 Enzymes
11B2 catalyzes the 11β-hydroxylation of 11-de-
oxycorticosterone followed by 18-hydroxylation
and 2-electron oxidation of the 18-alcohol to an
aldehyde (Figs 912 and 923) Changes in the
gene can lead to corticosterone methyloxidase
deficiency and hyperaldosteronism [47, 1980,
2055, 2056] In the older literature, this P450 is
sometimes termed “P450aldo”
9.7.43.1 Sites of Expression
P450 11B2 is expressed in the adrenal cortex
(zona glomerulosa) and involved in the synthe-
sis of aldosterone (the 11β-hydroxy, 19-alde-
hyde product) It is a mitochondrial P450, as are
the other family 11 P450s The cDNA was first
cloned from the adrenal tumor of a patient suffer-
ing from primary aldosteronism [2057] Another
early study showed higher levels of P450 11B2 in
aldosterone-secreting tumors [2058]
There is some evidence for the synthesis of
aldosterone outside of the adrenals, and Li et al
[2059] reported expression of P450 11B2 in he-
patic stellate cells of liver; the activation of these
cells is a key event in liver fibrogenesis
9.7.43.2 Regulation
Some of the research on regulation overlaps that
presented for the CYP11B1 gene ( vide supra) A
CRE/Ad1 element and ATF-1 (and ATF–2?) play
roles with both the CYP11B1 and CYP11B2 genes
[2060] However, SF-1 does not appear to regu-
late P450 11B2, in contrast with CYP11B1 [1988]
Many aspects of regulation remain to be further
investigated, including the mechanisms of the ob-
served Ca2 + and cyclic AMP signaling [2061] and
the effects of kinase inhibitors [2062, 2063]
Transforming growth factor (TGF) β1 inhibits
aldosterone production in human adrenocortical
cells by inhibiting P450 11B2 expression [2064]
P450 11B2 expression (in a human adrenocorti-
cal cell line) was increased by the orphan nuclear
receptors NURR1 and NGF1B [1989] Levels of
NURR1 and NGF1B were strongly induced by
angiotensin II, the major regulator of human P450
11B2 expression in vivo The NBRE-1, Ad5, and
Ad1/CRE cis elements were all concluded to be
involved in both basal and angiotensin-stimulat-
ed transcription of human P450 11B2 [1989]
637
The protein kinase C ligand 12-O-tetradec-
anoy-pharbol-13-acetate (TPA) has been re-
ported to inhibit angiotensin II-stimulated P450
11B2 gene expression (in a H295R human adre-
nocortical cell line) [2065] TPA was concluded
to inhibit the angiotensin II-dependent activation
of P450 11B2 transcription via the p44/42 mito-
gen-activated protein kinase (MAPK) signaling
pathway, leading to an increase in the level of
nuclear JunB [2065] In addition, protein kinase
C–E inhibits P450 11B2 gene expression through
the ERK/1 signaling pathway (and Jun B) [2066]
Calcineurin mediates angiotensin II-induced
upregulation of P450 11B2 transcription [2067]
Like P450 11B1, the P450 11B2 gene is regu-
lated by transposable elements and conserved cis
elements [2068] The promoter contains two Alu
elements imbedded in a truncated L1 element,
breaking up L1 into three fragments Alu func-
tions as an enhancer in P450 11B2, as in P450
11B1 ( vide supra) As mentioned earlier, Ad5
and SF-1 binding elements in the proximal core
promoter are important in transcription [2068]
Polychlorinated biphenyls have been reported
to upregulate P450 11B2 [2069, 2070], appar-
ently via increasing mRNA stability by an un-
known mechanism [1992] However, concentra-
tions < 10 µM were not used, and the relevance of
these findings to health is unclear
9.7.43.3 Genetic Variation
As in the case of the CYP11B1 gene, many CY-
P11B2 variants have now been defined from clin-
ical studies For review, see [2071] The many
variants [2072, 2073] have been related to a
number of diseases, including congenital hypoal-
dosterism [2074], salt-wasting syndrome [2075],
adenoma [2076, 2077], treatment for diabetic
nephropathy [2078], high-altitude pulmonary
edema [2079, 2080], metabolic syndrome [2081],
hypertension [2082–2095], stroke [2096], atrial
fibrillation [2097], and other cardiovascular risks
[2098]
The “crossovers” between P450s 11B1 and
11B2 yield inactive P450 11B2, as well as P450
11B1 [2016, 2017, 2099, 2100] Other variants
in CYP11B2 were associated with corticosterone
methyloxidase I and II deficiency [2055, 2056,
638
2101] Variants in CYP11B2 have also been
linked to idiopathic hyperaldosteronism, a con-
dition characterized by autonomous production
of aldosterone and arterial hypertension [2102]
A variant in the promoter region of CYP11B2
(− 344 TK) has been associated with predisposi-
tion to essential hypertension [2103]
9.7.43.4 Substrates and Reactions
P450 11B2 catalyzes the three-step conversion of
11-deoxycorticosterone to aldosterone, with 11β-
hydroxylation, 18-hydroxylation, and 2-elec-
tron oxidation of the 18-carbinol (Figs 912 and
923) No other substrates are known Informa-
tion about the processivity of the human enzyme
(ie, extent of release of intermediate products) is
not available at this time
Strushkevich et al [2019] have presented
evidence arguing the three-step oxidation of de-
oxycorticosterone to aldosterone (Figs 923b
and 924) is a processive one, in that 11β-
corticosterone was not oxidized to the product
However, the question has not been analyzed in
the usual ways of addressing these questions, eg,
with time course and pulse-chase experiments
Another recent development is the oxidation
of the nonclassical substrate methandienone by
P450 11B2 [2104] (Fig 925) The 11β- and 20β-
hydroxynorsteroids were formed Thus, the cata-
lytic selectivity of this steroid hydroxylase may
be more relaxed than previously assumed
9.7.43.5 Structure
In 2013, Strushkevich et al [2019] published
structures of human P450 11B2 with the substrate
deoxycorticosterone and an imidazole-based in-
hibitor, fadrozole The active site is lined with the
same residues as present in P450 11B1 (in that
region), and most of the divergent residues ap-
parently associated with the P450 11B2 catalyze
activity (18-hydroxylation) are located in the I-
helix and loops around the H-helix [2019]
Homology and pharmacophore models have
been published [2026, 2073, 2105]
9.7.43.6 Inhibitors
Progress towards clinically useful inhibitors of
P450 11B2 has been reviewed recently [2106,
F. P. Guengerich
2107] A number of inhibitors have been pro-
duced [2036, 2038, 2108–2114] These inhibi-
tors are intended for use in congestive heart
failure, myocardial fibrosis [2030, 2115–2117],
and prostate cancer [2039] Another intended
use is hypertension [2030], and one inhibitor has
reached a clinical trial (phase 2) [2118]
9.7.43.7 Clinical Issues
Although there is a rationale for developing in-
hibitors of P450 11B2 (Sect 7435, vide supra),
the major clinical issue is genetic disorders of
P450 11B2 insufficiency Genetic variants and
relationship to several diseases, particularly hy-
pertension, have been covered in Sect 7433
( vide supra) In addition, age-related associa-
tion of variants has been considered in relation to
breast cancer risk [2119]
The issues of congenital adrenal hyperplasia
and types I and II corticosterone methyloxidase
deficiency in individuals with attenuated P450
11B2 activity have already been mentioned The
other issue also mentioned is elevated aldoste-
rone Several studies have reported an associa-
tion between variants and essential hypertension,
although the measurements of aldosterone excre-
tion are still lacking in some studies [2120] Other
studies show association of the − 344C allele with
increased left ventricular size [2121–2123] The
hypertension association has been seen in several
studies [2082–2095, 2120, 2121, 2124, 2125] but
not in a Japanese study [2126]
9.7.44 P450 17A1
17α-Hydroxylation and the 17α,20-lyase reac-
tion (“desmolase”) are two important reactions in
steroid biosynthesis (Figs 924 and 926) Clon-
ing of a cDNA which, when expressed, yielded
both activities established the role of what is
now known as human P450 17A1 (previously
termed P45017α, etc) [2127] The gene [2128]
showed similarity to CYP21A1 The demonstra-
tion of both 17α-hydroxylation and 17α,20-lyase
catalytic activities in a single protein established
work previously done with purified hog protein
[2129] The two activities have long been known
 9  Human Cytochrome P450 Enzymes

Fig. 9.24   Multistep P450 reactions catalyzed by human P450s 11B2 and 17A1
639
 640

Fig. 9.25   Oxidation of methandienone by P450 11B2

F. P. Guengerich
 9  Human Cytochrome P450 Enzymes

Fig. 9.26   Oxidation of steroids by P450 17A1. P450 17A1 is designated E (enzyme). Pregnenolone (shown) and progesterone are oxidized to DHEA and androstenedione,
respectively, via 17α-hydroxy intermediates. The relative rates of the individual steps, especially the dissociation of the 17α-hydroxy product, determine the processivity of the
reaction. In teleost fish, P450 17A2 is also present and catalyzes only the first reaction [2176].
641
642
to be regulated by cytochrome b5 [2130, 2131],
and aspects of this duality of function still remain
unclear
9.7.44.1 Sites of Expression
P450 17A1 is a microsomal enzyme (Fig 924,
Table  92) Human P450 17A1 is expressed in
steroidogenic tissues, including adrenals and
gonads The enzyme has also been reported in
fetal kidney, thymus, and spleen [2132] The en-
zyme has also been found in human (adult) heart
[2133] and adipose tissue [2134] Recently P450
17A1 expression in the human fetal nervous sys-
tem has been reported [2135]
9.7.44.2 Regulation
As with the other steroidogenic P450s, the regula-
tion of the CYP17A1 gene is relatively complex
Induction of P450 17A1 has long been known to
be cyclic AMP mediated and the induction is sup-
pressed by testosterone (mouse model) [2136],
and a cyclic AMP response region was mapped
in porcine Leydig cells [2137]
Nuclear factor-1 was implicated in the up-
regulation of P450 17A1, acting on the promoter
in the cells isolated from patients with PCOS
[2138] Sphingosine was reported to regulate
P450 17A1 transcription by binding to SF-1
[2139] The regulatory protein SMAD3 was re-
ported to inhibit SF-1-dependent activation of the
P450 17A1 promoter in human H295R cell cul-
ture [2140] TGFβ inhibited P450 17A1 transcrip-
tion in the H295R cells via activin receptor-like
kinase 5 [2141] Phosphorylation of CtBP1 by
cyclic AMP-dependent protein kinase modulated
induction by stimulating partnering of CtBP1 and
2 [2142] Protein kinase C-induced activin A sup-
pressed P450 17A1 expression [2143]
The homeodomain protein Pbx1 was shown
to interact with protein kinase A in the cyclic
AMP-dependent regulation (at − 250/− 241) of
the human CYP17A1 gene [2144] Further analy-
sis showed interaction at a cyclic AMP-related
site (− 80/− 40) by SF-1 [2145] Further, interac-
tions were shown for Sp1 and Sp3 (− 227/− 184)
and NF-1C (− 107/− 85 and − 178/− 152) [2146]
SF-1 ( vide supra) also interacts with p54nrb,
NonO, and protein-associated splicing factor
F. P. Guengerich
[2147] The ACTH/cyclic AMP response is de-
pendent upon phosphatase activity, as well as
kinase activity [2148, 2149] The cyclic AMP-
dependent protein kinase enhances transcription
via MKP-1 activation, involving phosphorylation
of SF-1 [181]
The Miller laboratory has presented evidence
that P450 17A1 is phosphorylated and that this
has the effect of stimulating only the lyase ac-
tivity [2150–2152] In the most recent work, the
phosphorylation is attributed to the (Ser/Thr) ki-
nase p38α [2152] The increase in lyase activity
was ~ two-fold The site(s) of phosphorylation is
unknown, and no isolation of a phosphorylated
protein has been isolated from a tissue
9.7.44.3 Genetic Variation
At least 49 different variants have been identi-
fied in P450 17A1 from clinical studies [2153]
These will not be reiterated here; some references
to roles in individual diseases are presented in
Sect. 7.44.7 ( vide infra) See also Chap 10 [145]
9.7.44.4 Substrates and Reactions
The generally accepted reactions of P450
17A1 are the 17α-hydroxylation of pregneno-
lone to 17α-hydroxypregnenolone and of pro-
gesterone to 17α-hydroxyprogesterone 17α-
Hydroxypregnenolone is also oxidized to DHEA,
and 17α-hydroxyprogesterone is oxidized to
androstenedione in the 17,20-lyase reaction
(Figs  912, 924, and 926) [2154, 2155] The
mechanism of the lyase reaction is not complete-
ly established, but mechanisms have been pro-
posed using analogs [2156] Lieberman [2157]
proposed alternative reactions, although the sug-
gested pathway involves what would be a very
unstable diradical No other substrates are known
presently, other than pregnenolone and proges-
terone and possibly closely related analogues
Soucy et al [2158] have provided evidence that
human P450 17A1 also converts pregnenolone
into 5,16-androstadien-3β-ol, a “16-ene syn-
thase” reaction (without intermediate formation
of an alcohol)
The lyase reaction is more prominent in
adult adrenals with the Δ5 steroids (than Δ4;
ie, with 17α-hydroxypregnenolone than 17α-
9  Human Cytochrome P450 Enzymes
hydroxyprogesterone), and this also applies in
(human) fetal testis [2159] P450 17A1 also has
trace 21-hydroxylation activity [2160], and the
mutation A105 L yields a protein with some 16α-
hydroxylation activity [2160, 2161]
A kinetic deuterium isotope of ~ 4 was ob-
served for the 17α-hydroxylation reaction
[2160] The mechanism of this hydroxylation is
presumed to be relatively straightforward “com-
pound I”-type hydroxylation, with C–H bond
breaking being at least partially rate limiting
Rates of individual steps in the reaction have not
been reported
The second reaction, the 17,20-lyase reac-
tion, is more complex and difficult to rational-
ize with a classic compound I mechanism Work
from Akhtar’s laboratory led to the proposal that
the reaction involves a nucleophilic attack of the
ferric peroxide (anion; FeO2 +, or FeIIO2−) on
the C-20 carbonyl (of 17α-hydroxyprogesterone
or pregnenolone) [2156, 2162] One of the key
pieces of evidence was the result of 18O2 labeling
experiments [2156] The results of site-directed
mutagenesis studies on Thr-306 are also con-
sistent with the conclusions about FeIIO2 − in-
volvement [2156, 2163] Sligar’s group has also
presented resonance Raman spectra [2164] and
solvent deuterium kinetic isotope effect studies
[2165] in support of the involvement of this en-
tity
Further work on the differential effects of cy-
tochrome b5 on individual catalytic activities has
been reported [2166] The ratio of cytochrome
b5 to P450 is high in testis and this phenomenon
might regulate the two activities of P450 17A1
Miller’s group has proposed that phosphorylation
of Ser and Thr residues in P450 17A1 may alter-
natively influence the two activities [2152, 2167,
2168]
A second cytochrome b5 gene has been identi-
fied recently and this protein also has the same
stimulatory effect on lyase activity [2169] Au-
chus et al [2170] also demonstrated that the same
stimulatory effect of cytochrome b5 could be ob-
tained with apo-cytochrome b5, arguing against
the requirement for electron transfer P450 17A1
enzymes from other species vary in their ability
to catalyze the 17,20-lyase reaction, and compar-
643
isons of the rat and human enzymes also led to
the conclusion that selective enhancement of the
lyase reaction was not due to changes in electron
transfer [2171]
The concertedness of the P450 17A1 17,20-
lyase reaction has been examined, and two stud-
ies both reached the conclusion that much of
the 17α-hydroxypregenolone dissociates [2172,
2173] In one of the studies [2172], the authors
concluded that the off-rate was an important
factor in determining the balance between 17α-
hydroxypregnenolone and DHEA with the beef
enzyme Exactly how cytochrome b5 would con-
trol this rate, which was modeled to be rather
slow (26–29 min− 1), is unclear unless the effect
is on the protein conformation
Studies with human P450 17A1 in this labora-
tory show that the human P450 17A1 enzyme is
relatively distributive for the two reactions, 17α-
hydroxylation and the 17,20-lyase cleavage This
was shown using pulse-chase experiments with
14C progesterone or pregnenolone and then add-
ing varying amounts of unlabeled 17α-hydroxy
steroid, measuring the attenuation of radiolabel
incorporated into the final product However, the
reaction shows more processivity with pregneno-
lone than progesterone Further evidence for the
distributive nature of the enzyme comes from
studies with the inhibitor orteronel (TAK-700),
which preferentially inhibits the lyase (second
reaction) [2174] If the enzyme were totally pro-
cessive, this result would be impossible
Teleost fish P450 17A1 enzymes catalyze
both reactions, similar to human P450 17A1
[2175] That enzyme is also distributive, more
so with progesterone than pregnenolone [2176]
The related fish P450 17A2 catalyzes only the
17α-hydroxylation, with or without cytochrome
b5 [2175, 2176] Cytochrome b5 did not affect
the processivity of the fish P450 17A1 reactions
[2176]
Another issue already mentioned (Sect 7442)
is phosphorylation, which has been reported to
favor the second reaction (lyase) [2152] This re-
sult is a bit of an enigma in that cytochrome b5
is considered to have an anionic region (“patch”)
that binds to basic residues in P450 17A1, as
evidenced by site-directed mutagenesis [2177]
644
A phosphate group (in this region?) would add
a negative charge and tend to prevent interaction
with cytochrome b5
Another enigma about P450 17A1 catalysis is
raised from the results of Scott and her associ-
ates [2178], who presented NMR evidence that
cytochrome b5 and NADPH-P450 reductase bind
to the same section of P450 17A1 and therefore
compete for binding Strong evidence has been
presented that electron transfer is not involved in
the stimulation of P450 17A1 by cytochrome b5
[2170] Therefore, both of the electrons used in
the P450 17A1 reaction (regardless of whether it
is a compound I or ferric peroxide mechanism)
must come from the reductase Thus, the P450
must be reduced to the ferrous state, bind O2, ac-
cept another electron, and thus be in the FeO2 +
state before the reductase dissociates This must
happen rapidly, and the formal FeO2 + entity must
be stable enough to persist until the cytochrome
b5 is bound and apparently “allosterically” per-
turbs P450 17A1 FeO2 + to catalyze the lyase re-
action The reaction has a kcat of ~ 1 min− 1, so this
must happen in seconds during every catalytic
cycle (and the cytochrome b5 must leave again
for NADPH-P450 reductase to begin and reiniti-
ate catalysis) Exactly what occurs will require
further study
What step is rate limiting in the lyase reac-
tion is presently unknown That reaction seems
impervious to the use of kinetic isotope effects
to study the nature of C–C bond cleavage (unless
13C isotope effects could be used)
9.7.44.5 Structures
Much of the information about the significance of
active site residues comes from analysis of mu-
tations in patients presenting with diseases (see
Sect 7442, vide supra) The changes H373 L
and P409R [2179] led to a loss of heme incor-
poration Mutation at Thr-306, possibly involved
in protonation of Fe–OO− or O–O cleavage, im-
paired 17α-hydroxylation more than the lyase
reaction [2180] However, the change R346A
selectively abolished lyase activity [2181], as
did F417C [2182] Mutations at Lys-83, Arg-
347, Arg-358, and Arg-449 produced proteins
that were refractory to cytochrome b5 stimulation
F. P. Guengerich
and attenuated in lyase activity [2183–2185] Of
these, only R347H and R358Q have been found in
patients [2186] Some variants found in patients
do cause the loss of both 17α-hydroxylation and
the lyase reaction, however [2187, 2188]
Some animal P450 17A1 enzymes have dif-
ferent ratios of 17-hydroxylation/lyase activities,
and efforts have been made to use these proper-
ties to define more elements controlling the lat-
ter steps, although the results have been limited
[2189, 2190]
A number of additional homology models
have been published [2024, 2191–2197]
In 2012, DeVore and Scott [2153] published
an X-ray crystal structure of human P450 17A1
bound to the inhibitors abiraterone and TOK-001
(Sect 7446, vide infra) As might be anticipated,
the pyridine nitrogen is bound to the heme iron
The binding mode was considered to be different
than observed for a number of other P450s that
use steroids as substrates This structure may be
useful in rationalizing the variants seen in clini-
cal problems
As discussed in Sect. 7.44.4 ( vide supra), one
of the mechanistic curiosities is the interaction of
cytochrome b5 with P450 17A1, which (in part)
regulates the balance between the 17α-hydroxy
and 17,20-lyase products An NMR study with
cytochrome b5 led to the conclusion that the
protein occupies a position at a site on P450
17A1, including Arg-347, Arg-358, and Arg-449
[2178] The same site is believed to be occupied
by NADPH-P450 reductase
The dual nature of the P450 17A1 in catalyz-
ing sequential reactions can be addressed using
fish orthologs Teleost fish have two P450 17A
genes, 17A1 and 17A2 [2175] Fish P450 17A1
resembles mammalian P450 17A1 in catalyzing
both the 17α-hydroxylation and the lyase reac-
tions, but (fish) P450 17A2 only catalyzes the
17α-hydroxylation [2175] Fish or human cyto-
chrome b5 stimulates only the lyase activity This
laboratory, collaborating with Prof Martin Egli,
has crystallized both zebra fish P450 17A1 and
17A2, with abiraterone bound to each and pro-
gesterone bound to P450 17A2 [2176], as in the
human P450 17A1 structure [2153]
9  Human Cytochrome P450 Enzymes
9.7.44.6 Inhibitors
Inhibitors of P450 17A1 have been studied for
some time Interestingly, ketoconazole inhibits
lyase activity but not 17α-hydroxylation activ-
ity [2198] 7α-Thiospirolactone is a mechanism-
based inhibitor of (guinea pig) P450 17A1 [2199]
A number of steroidal inhibitors have been
studied, primarily with the goal of treating can-
cers [2200–2202] [2203, 2204] The enantiomer
of progesterone ( ent-progesterone) is reported
to be a competitive inhibitor of P450 17A1 ( KI
02 µM) [2205]
Nonsteroidal inhibitors have also been studied
[2206, 2207]
Molecular modeling (Sect 7445) has also
been applied to searches for inhibitors [2197,
2208] Other approaches utilize P450 17A1 ex-
pressed in E. coli to screen for P450 17A1 in-
hibition in medium- to high-throughput systems
[2209, 2210]
One interest in inhibition of P450 17A1 is
treating prostate cancer The concept is that pros-
tate cancer is stimulated by androgens, and the
goal is to block production of androstenedione
(from progesterone/17α-hydroxyprogesterone)
This is a particular issue in “castration-resistant”
prostate cancer
A number of inhibitors have been published
[2035, 2037, 2211–2215] For reviews see
[2216–2219] Abiraterone is a leading inhibitor,
currently approved for use for prostate cancer
[2220–2225] Another drug in clinical trials is
orteronel (TAK-700), which shows selective in-
hibition of the lyase reaction [2174, 2226] The
concept is to block androgen production (ie, an-
drostenedione formation) and maintain produc-
tion of other steroids for normal physiology
9.7.44.7 Clinical Issues
P450 17A1 has a central role in human steroid
metabolism because of its role in regulating ste-
roid flux (Fig 912) There are two dominant
clinical issues with P450 17A1 One is various
diseases associated with hormone imbalance
P450 17A1 is at a branch point and involved in
production of glucocorticoids and sex hormones
(androgens and estrogens), and therefore a vari-
645
ety of maladies can be associated with changes
The other issue, addressed under Sect 7446
( vide supra), is the use of P450 17A1 inhibitors
(especially lyase inhibitors) to treat androgen-
stimulated tumors The second point will not be
treated further here
The clinical issues for which research has
been done to implicate associations with P450
17A1 status (usually genetic) include endome-
trial cancer [2227], prostate cancer [2228 2003,
53455, 2229], breast cancer [852, 2230], endo-
metrial cancer [2231], non-Hodgkin’s lymphoma
[2232], infertility [2233], pregnancy loss [2234],
early embryonic lethality [2235], short menstrual
cycles/early contraceptive use/BRCA mutations
[2236], secondary amenorrhea [2237], PCOS
[2238], endometriosis [2239], and acne [2240]
Perturbations in P450 17A1 lead to problems
in adrenarche, aging, and PCOS [2155, 2241]
Some of the more serious variants have been
mentioned already Another variant is related to
a case of pseudohermaphroditism involving lack
of lyase activity [2242]
Some of the other possible disease conditions
or risks are being studied in relationship to less
serious variants In most of these cases, the rela-
tionships are more difficult to establish than in the
serious diseases A possible link of CYP17A1 de-
ficiency has been made with rheumatoid arthritis
[2243] Little influence of genetic variation was
seen on age of menarche [2244] However, a link
was made between a particular variant and the
prediction to use hormone replacement therapy
(ie, postmenopausal estrogen therapy) [2245]
No association was found with PCOS in a study
with an SNV at the regulatory Sp1 site [2246]
Much attention has been given to the possi-
bility of a link between CYP17A1 allelic SNVs
and breast cancer risk [2247] The epidemiology
results are mixed at best [2248–2251], and a con-
clusion in favor of a relationship cannot be made
at this time [2230, 2252, 2253]
As with some other P450s, circulating anti-
bodies to P450 17A1 are seen in some autoim-
mune diseases, eg, autoimmune polyglandular
syndrome and Addison’s disease [1969, 2254],
but no causal relationship has been demonstrated
646
9.7.45 P450 19A1
P450 19A1 is the classic “aromatase,” often
known by that name in endocrinology This en-
zyme oxidizes the androgens (eg, androstendi-
one and testosterone) to estrogens (estrone and
17β-estradiol, respectively) (Fig 922) This pro-
cess is very important in normal physiology and
also a target for inhibition in some tumors
9.7.45.1 Sites of Expression
Estrogens have a number of functions, not only
in feminization Although estrogens are often
considered “female” hormones, they are also
important in males (eg, see material regarding
brain, vide infra) P450 19A1 is even found in the
penis [2255] and is important in male reproduc-
tion [2255, 2256] Sites of (human) expression
include the ovaries and testes, placenta and fetal
(but not adult) liver, adipose tissue, chondrocytes
and osteoblasts of bone, vasculature smooth
muscle, and several sites in brain, including parts
of the hypothalamus, limbic system, and cere-
bral cortex [2257] As discussed later, regulatory
mechanisms differ considerably in these tissues
P450 19A1 is also expressed in some tumors,
particularly those derived from these tissues
Evidence for P450 19A1 in the brain has been
reported [2258], and a mouse CYP19A1 knock-
out provides evidence that estrogens are required
for brain development [2259] The actions of an-
drogens and estrogens in the gonadal tissues are
fairly well understood but less is known in the
brain Androgens and androgen-derived estrogens
regulate complementary and interacting genes in
many neural networks [2260] P450 19A1 expres-
sion in skeletal muscle has been reported [2261]
Although P450 19A1 is generally considered
an extrahepatic P450, there is evidence for some
expression in human liver [2262] P450 19A1
peptides have been detected in liver microsomes
(treated with trypsin) by LC–MS [635] Oxida-
tions of dihydrotestosterone attributable to P450
19A1 have been observed in human liver micro-
somes [1373]
Evidence has been presented that P450 19A1
dimers exist in membranes and that P450 19A1
does not dimerize with P450 17A1 [2263]
F. P. Guengerich
9.7.45.2 Regulation
The regulation of the CYP19A1 gene is quite
complex, primarily because of the use of four
tissue-selective promoters [2257, 2264] The
promoters have been reviewed [2265] Much of
the research has been in the area of cancer Either
the Il, I4, If, and I6 sequence is read as exon
I and spliced in to the mRNA, depending upon
the tissue However, exon I does not code for the
protein, so the P450 19A1 enzyme is always the
same
In preovulatory follicles and corpora lutea of
human ovary, the 5ʹ-untranslated region of P450
19A1 transcripts is encoded by exon IIa [2266]
The major operatives here are CRE and SF-1 ele-
ments [2257]
In adipose tissue, the promoter from exon
I4 is utilized [2257] The same exon is utilized
in bone and skin [2257] and in leiomyoma tis-
sue derived in myometrium [2267] This system
is regulated with Sp1, a glucocorticoid regula-
tory element, STAT3, and possibly PPARγ [2257,
2268] Preadipocytes also involve regulation
with LRH-1 [2269]
In placenta exon I1, an 89-kb upstream ele-
ment is utilized [2257] This is a strong promoter
and involves C/EBP-β [2257] A strong posi-
tive enhancer element between − 42 and − 501 is
present [2270] The possibility exists that VDR/
RXRα heterodimers and PPARγ may have effects
[2257]
Regulation in bone uses exon I6 [2257] The
study of regulation in bone is less extensive than
in other sites, and 1,25-dihydroxycholecalciferol,
interleukins, TNFα, and TGF-β1 have stimula-
tory activity
Regulation in brain uses exon If and has also
not been as extensively studied [2257] P450
19A1 does seem to be upregulated by androgens
Regulation in fetal liver involves exon I4, as
with adipose tissue [2257] The same pattern ap-
pears to apply in skin fibroblasts and intestine
In cancer cells, alternate regulatory pathways
are utilized [2257] EP2 and EP4 receptors regu-
late P450 19A1 expression in human adipocytes
and in breast cancer cells, involving BRCA1-
p300 exchange [2271] CCAAT/EBPβ upregu-
lated promoters I2/II in breast cancer epithelial
9  Human Cytochrome P450 Enzymes
cells [2272] P450 19A1 transcription is also en-
hanced by RXRα/RORα in breast cancer cells
[2273, 2274] In skeletal muscle, the P450 19A1
gene is a target of the factor Runx2 [2275] In
human ovarian granulosa cell-like KGN cells, ac-
tivin stimulates P450 19A1 gene expression via
the Smad2 signaling pathway [2276] In granu-
losa cell tumors, P450 19A1 is a direct target of
FOXL2 to C134W via a single highly conserved
binding site in the ovarian-specific promoter
[2277] In human placental syncytiotrophoblasts,
cortisol induces P450 19A1 expression through
the cyclic AMP/Sp1 pathway [2278]
A vitamin D analog inhibits P450 19A1 ex-
pression by dissociation of the comodulator Wil-
liams syndrome transcription factor (WSTF)
from the promoter [2279] PPARγ agonists down-
regulate P450 19A1, via BRCA1 and prostaglan-
din E2 [2280, 2281] TCDD has been reported to
induce P450 19A1 in human glioma cells [2282]
In the area of post-transcriptional regulation, the
alternative miscoding exons 1 are involved [2283]
Some evidence for control of genes by DNA meth-
ylation has also been reported [2284, 2285]
9.7.45.3 Genetic Variation
The cypalleles website (http://wwwcypalleles
kise) shows only five allelic variants of the
human CYP19A1 gene, which seems surpris-
ing compared to some of the other steroidogenic
P450s, eg, P450s 17A1 and 21A2 (Sects 744
and 746, respectively) These have been studied
with regard to breast cancer but without convinc-
ing relationships ( vide infra); also, there was no
relationship with breast density [2286]
Relatively few cases of aromatase deficiency
have been reported [2257, 2287], and some of the
clinical cases may be the result of NADPH-P450
reductase deficiency In vitro steady-state kinetic
analysis of one variant (T201M) has been found
it to be more active than the *1 (wild type) P450
19A1 [2288] See Sect 7457 for clinical issues
related to genetic abnormalities
9.7.45.4 Substrates and Reactions
The reaction involves three steps and has been
the subject of considerable mechanistic inter-
est (Fig 922) Androstenedione is converted
647
to estrone, testosterone to 17β-estradiol, and
16α-hydroxytestosterone to estriol The first
two steps are relatively straightforward, eg,
RCH3→RCH2OH→CHO (at C19). The third
step was difficult to rationalize with “classic”
FeO3 + chemistry, and there has been general ac-
ceptance of a FeO2 − based mechanism originally
developed by Akhtar [2289, 2290] and Robinson
[2291] and further studied in models by Coon
and Vaz [2292]
The possibility of utilization of DHEA as a
substrate for estrone synthesis has been proposed
but not addressed directly [2293]
P450 19A1 also catalyzes oxidation reactions
with related compounds, some of which may have
physiological relevance (Fig 927) 3-Deoxy an-
drogens are oxidized (19-methyl deformylation)
in a similar manner [2294] Recently this labo-
ratory has demonstrated that androstenedione
and testosterone are oxidized to the 19-formic
acid derivatives by 2-electron oxidation of the
19-aldehyde [2295] This product (previously
reported as an androstenedione derivative in por-
cine granulosa cells [2296]) apparently is stable
and is not oxidized to an estrogen (it is sensitive
to acid-catalyzed decarboxylation, which yields
19-nor-androgens) We also found that the three
porcine P450 19A1 enzymes all make as much of
the 19-formic acid product as estrogen, from ei-
ther testosterone or androstenedione The in vivo
relevance remains to be established The product
estrone is known to be further oxidized (slowly)
by 2-hydroxylation [2297, 2298]
Dihydrotestosterone, a more potent physiologi-
cal androgen, is also oxidized by P450 19A1 in the
same general way as the other androgens [1373]
(Fig  927) The products are deformylated and
one is further oxidized (2-hydroxylation), but es-
trogens are not formed [1373] Whether the 19-al-
dehyde forms the 19-carboxylic acid is not known
The three-step reaction has been shown to be
mainly distributive [220] The reaction can be initi-
ated with any of the intermediate steroids (Fig 922;
yielding the final estrogenic product) Pulse-chase
experiments show the distributive nature of the
products, and a reaction with a limited amount
of androstenedione shows a smooth progression
through the two intermediate products [220]
 648

Fig. 9.27   Known reactions of human P450 19A1 (in addition to aromatization of androgens) [1373, 2300]
F. P. Guengerich
9  Human Cytochrome P450 Enzymes
Exactly which catalytic step is rate limiting
within each of the three reaction steps is not
clear With placental microsomes as the enzyme
source, an intermolecular kinetic deuterium iso-
tope effect of 32 was reported for the first step
and no kinetic isotope effect for the second step
[2299] An even higher kinetic hydrogen isotope
effect was estimated by Osawa et al [2300]
Sligar and his associates reported spectroscopic
studies on the Fe2 + O2 form of the enzyme in the
presence of androstenedione, with a decomposi-
tion rate of 07 s− 1 (42 min− 1) at 37 °C [2301]
which is roughly equivalent to the rate constant in
a model for the first step [220] Sligar’s group has
also presented electron paramagnetic resonance
(EPR) spectral evidence for the formation of an
FeO2 + (FeIIO2 −) intermediate, formed by cryora-
diolysis (at 77 K), using EPR detection [2302]
The relevance to catalysis has not been investi-
gated (ie, product formation was not measured)
The first two oxidations in the conversion of
androgens to estrogens are relatively straightfor-
ward and can be readily rationalized with classic
compound I mechanisms, ie, hydroxylation of
a methyl group and the oxidation of a carbinol
to a gem-diol/aldehyde The third step has been
problematic and has invited a number of propos-
als over the years, including (A-ring) 1-hydrox-
ylation, 2-hydroxylation, 4,5-epoxidation, and
hydrogen abstraction from C-19 followed by
rearrangement [2289, 2291, 2299, 2303–2317]
Pathways to estrogens (and formic acid) can be
drawn in these cases, but they have been ruled
out for one reason or another, eg, 18O labeling
results for the 2-hydroxy mechanism [2315]
Currently the most widely accepted mechanism
is probably the ferric peroxide mechanism pro-
posed by Akhtar and supported by some 18O2
labeling results [2305] An alternate mechanism
proposed by Hackett et al [2308] involves com-
pound I hydrogen abstraction of H-1β [2303,
2304] followed by an electronic rearrangement
and a “concerted” C–C bond scission without
formal hydroxylation This is an adaptation of
a mechanism proposed by Covey et al earlier,
which begins with hydrogen atom abstraction
from the C-19 gem-diol [2312]
649
The mechanism has involved considerable
debate over the years [2289, 2304, 2305, 2315]
A number of approaches can be applied to the
mechanistic question, including studies with
simplified models [2291, 2318–2321], synthesis
of potential intermediates for testing with the en-
zyme [2303, 2304], application of theory [2308,
2309], spectroscopy [2301, 2302], and isotopic
labeling studies [2299, 2305]
Recent work in this laboratory bears on the
mechanism [2295] As mentioned earlier, puri-
fied recombinant P450 19A1 converts 19-alde-
hyde androgens (and 19-methyl androgens) to
the 19-formic acid derivatives (Fig 928) The
products appear to be relatively stable (forma-
tion of estrogens requires base-catalyzed release
of the carbon as CO2) These findings indicate
that P450 19A1 compound I is capable of being
formed and used in the last step
When either 19-deuterated 19-oxoandro-
stenedione or testosterone was incubated with
recombinant P450 19A1 and 18O2, 18O label
was not incorporated into the recovered formic
acid (Fig 928) These results differ from those
reported previously [2289, 2305]; the major
technical differences are the use of recombinant
purified P450 19A1, a more sensitive probe for
trapping and analyzing formic acid, and the use
of UPLC-coupled high-resolution mass spec-
trometry, which avoided issues inherent in analy-
sis of labeled formic acid [2295] The results are
not consistent with the proposed ferric peroxide
mechanism, in which an 18O atom is expected to
be recovered in formic acid
An issue with the ferric peroxide mechanism
is that the substrate is in the (hydrated) gem-diol
form following the second reaction (Fig 928)
However, the proposed ferric peroxide mecha-
nism involves a nucleophilic attack (Fe2 + O2 −) on
the carbonyl (aldehyde) Thus, the gem-diol must
be dehydrated before this step can run The rate
of dehydration has been estimated at > 05 s− 1 (in
the absence of P450 19A1 using 18O exchange
methods [2295], which is faster than the kcat
(8 min− 1) for going from 19-hydroxy androstene-
dione to estrone [220] The reaction could occur
with the aldehyde or the gem-diol, the latter of
650
Fig. 9.28   Unified mechanism of C–C cleavage in the
third oxidation step of androgen conversion to estro-
gens by P450 19A1, including the formation of androgen
which is more consistent with the proposals of
Covey et al [2312] and Hackett et al [2308]
It is of interest to note that hogs have three
P450 19A1 genes, and one of these converts tes-
tosterone to 1β-hydroxytestosterone [2322] Both
androstenedione and testosterone are converted
to some 1β- and 2β-hydroxy products by puri-
fied human P450 19A1 [2300], indicating that
the FeO3 + complex can position itself to abstract
an H-1 or H-2 atom from the androgen substrate
9.7.45.5 Structure
One of the historic problems in studying struc-
ture–function relationships in P450 19A1 has
F. P. Guengerich
19-carboxylic acids The mechanism is based on labeling
studies with 18O2 [2295]
been the availability of expression systems Re-
cently several E. coli systems have been devel-
oped [220, 2323–2325]
Some homology modeling studies have ap-
peared [2326, 2327] In 2009, Ghosh et al [2328]
published a crystal structure of P450 19A1 puri-
fied from human placental samples, without any
modification (even at the N terminus), the only
mammalian P450 to be crystallized in such a way
The structure contains a bound androstenedione
substrate, positioned in a manner to make oxida-
tion feasible The structure has been utilized in
the design of new inhibitors [2329]
9  Human Cytochrome P450 Enzymes
More recently, recombinant ( E. coli) P450
19A1 has been expressed and crystallized, along
with some active site mutants [2325]
9.7.45.6 Inhibitors
The literature on clinical use of aromatase inhibi-
tors for cancer treatment is immense, and much
has been published since the last version of this
chapter [149] The topic has been reviewed many
times [2330–2333], including some reviews by
A Brodie, a pioneer in this area [2334, 2335]
Breast cancer is probably the major target area
for P450 19A1 inhibition, but other cancers are
also under investigation [2336]
Today the process has reached the stage of
“third-generation” inhibitors [2337], moving
beyond early drugs such as aminoglutethimide
[2338] The newer inhibitors are more effective
in lowering the body load of estrogens [2339]
One example of a newer drug is exemestane, a
site-directed Michael acceptor (compared with
the ER antagonist tamoxifen) [2338–2341]
The leading P450 19A1 inhibitors in use today
are primarily (but not exclusively, Sect 7457,
vide infra) exemestane, anastrozole, and letro-
zole [2342, 2343] Although the potency of these
three inhibitors is excellent, efforts to develop
new inhibitors are continuing using other chemi-
cal approaches [2329, 2344–2349]
These inhibitors are not without toxicities
[2350], although most of the expected issues can
be anticipated due to generalized attenuation of
estrogen levels throughout the body Other in-
hibitory Michael agents have been prepared from
prostaglandin J2, but detailed characterization
has not been done [2351] Other nonsteroidal
inhibitors of P450 19A1 are also under consid-
eration [2352]
The point has been made by Simpson et al
[2257] that a future goal of P450 19A1 inhibi-
tion should be tissue selectivity The diverse role
of P450 19A1 in different tissues might indicate
that generalized inhibition of estrogen synthesis
may be less than desirable Targeted inhibition of
P450 19A1 could, in principle, be achieved by
(1) selective targeting of inhibitors of P450 19A1
catalysis to tumors/individual organs or (2) tar-
geted downregulation of P450 19A1 synthesis in
selected areas
651
Finally, CYP19A1 genetic variants have been
considered in relationship to breast cancer patient
response to inhibitors [2353]
9.7.45.7 Clinical Issues
The two major clinical issues with P450 19A1
are (1) disease states associated with genetic
variations and (2) use of aromatase inhibitors to
block estrogen-dependent diseases [2354] Seri-
ous cases of congenital aromatase deficiency in
adults appear to be relatively rare [2257, 2355,
2356] and have been treated with estrogen re-
placement therapy [2357] However, some chil-
dren are considered to have attenuated P450
19A1 activity [2358] Studies with P450 19a1-
knockout mice show expected reproductive and
sexual phenotypes and also adipose and bone
phenotypes [2355, 2359], as well as a socio-
sexual behavior phenotype [2360] There are
known gain-of-function variants, with some is-
sues [2361]
For a review of the significance in cancers, see
[2362] There is consideration of the use of inhib-
itors for breast cancer prevention in high-risk in-
dividuals P450 19A1 inhibitors have been used
extensively for breast cancer (see Sect 8456)
[2363], epilepsy [2364], children with short stat-
ure [2365], other pediatric disease [2366] en-
dometriosis [2367], male infertility [2368], and
induction of ovulation [2369] P450 19A1 inhibi-
tors have also been reported to cause arthralgia
[2370]
A number of studies have been made on the
relationship of CYP19A1 polymorphisms with
breast cancer, but the evidence has not shown a
change in risk [2286, 2371] No strong associa-
tion was seen for endometriosis [2372] Genetic
variation in P450 19A1 has been considered re-
garding breast cancer [2373–2376], prostate can-
cer [2377], lung cancer [2378], response to ther-
apy with aromatase inhibitors [2379], estrogen
levels and bone structure in older women [2380],
bone loss [2381], polycystic ovary disease
[2382], age at menarche [2383], essential hy-
pertension [2384, 2385], craving during alcohol
withdrawal [2386], obsessive compulsive behav-
ior and Parkinson’s disease [2386], testicular dis-
ease [2387], pubertal sagittal jaw growth [2388],
and reading, speech, and language [2389]
652
9.7.46 P450 20A1
9.7.46.1 Sites of Expression
P450 20A1 expression has been reported in
liver and brain In brain, expression was noted
in substantia nigra, hippocampus, and amygdala
[2390]
9.7.46.2 Regulation
To date, no reports on the regulation of P450
20A1 have appeared
9.7.46.3 Genetic Variation
No reports of polymorphism or other genetic
variation of P450 20A1 have appeared
9.7.46.4 Substrates and Reactions
Attempts to identify substrates with a recombi-
nant P450 20A1 expressed in E. coli have been
negative [2390] However, the expression level
was low and should be improved
It is of interest to note that P450 20A1 is un-
usual and an ortholog even appears in sponges It
is possible that 20A1 has an important physiolog-
ical function One could consider this to be the
“most orphan” of the orphan P450s (Table 91)
9.7.46.5 Structure
No information is yet available
9.7.46.6 Inhibitors
Obviously, since no catalytic activity has been
reported, there are no inhibitors
9.7.46.7 Clinical Issues
No clinical issues have been considered, in light
of the lack of information about function
9.7.47 P450 21A2
P450 21A2 is the enzyme involved in the 21-hy-
droxylation of progesterone and 17-hydroxy-
progesterone, yielding deoxycorticosterone
and 11-deoxycortisol from the two substrates,
respectively (Fig 912) The 21-hydroxylation
reaction is an important step in the synthesis of
glucocorticoids and mineralocorticoids, and de-
F. P. Guengerich
ficiencies lead to “salt-wasting syndrome,” if not
treated, and to congenital adrenal hyperplasia in
the worst cases
9.7.47.1 Sites of Expression
The major site of expression is the adrenal cor-
tex This reaction has been known for some time,
and many of the early biochemical studies were
done with beef adrenals because of the need for
large amounts of tissue [2391]
Low amounts of P450 21A2 have been re-
ported in human lymphocytes [2392] and brain
[2393] Any specific function in these tissues is
unknown at this time
9.7.47.2 Regulation
The regulation of P450 21A2 has some similar-
ity to that of P450 17A1, in that both are regu-
lated by ACTH The cyclic AMP-responsive
sequence in the 5ʹ-flanking region [2394] uses
adrenal-specific protein factor and an Ad4-like
sequence [2395] One issue in the regulation of
the CYP21A2 gene is the neighboring homolo-
gous but nonfunctional CYP21A1 pseudogene,
which can compete for transcription factors and
other regulatory proteins [2396] In other work,
protein kinases A and C and Ca2 + were found to
regulate CYP21A2 gene expression in a human
cortical cell line [2397]
Another interesting aspect of the regulation of
the CYP21A2 gene is that it is located very close
to the major histocompatibility locus, 23-kb
downstream from the C4 gene Transcriptional
regulatory elements for the CYP21A2 gene lie
within intron 35 kb of the C4 gene [2398]
Evidence for regulation by vitamin D has ap-
peared [2399]
9.7.47.3 Genetic Variation
Steroid 21-hydroxylase deficiency is the most
common cause of congenital adrenal hyperpla-
sia, and many variants are now known to be as-
sociated with the disease To date, more than 150
clinical variants have been reported, with > 97
consisting of missense variants [42, 2400] In ad-
dition to missense variants, deletions [2401] and
copy number variants [2402, 2403] have been
reported Ethnic links of the variations have also
9  Human Cytochrome P450 Enzymes
been reported, eg, [2404] Variations in the pro-
moter region are also known [2405]
The genetics of P450 21A2 variation have
been reviewed recently [2406, 2407] Many ge-
netic variants are the result of recombination
with the related pseudogene [2408, 2409] Some
are in the coding region [2410–2412] and the
5ʹ-flanking region [2413] The incidence of car-
riers of congenital adrenal hyperplasia is 1–2 %
in the population, and many deleterious variants
have now been identified [2414–2421]
9.7.47.4 Substrates and Reactions
The primary substrates are progesterone and 17α-
hydroxyprogesterone, which are hydroxylated
only at the 21-position (Fig 912) A minor activ-
ity seen with P450 21A2 is 16α-hydroxylation of
progesterone, better revealed by use of the (C-17)
deuterated substrate (due to “metabolic switch-
ing”) [2160] The rates of 21-hydroxylation of
progesterone and 17α-hydroxyprogesterone are
among the fastest of all mammalian P450s, with
catalytic efficiencies of 107 and 2 × 106 M− 1/s ob-
served in this laboratory for the wild-type human
enzyme [2769]
9.7.47.5 Structure
Homology models have been reported [2024,
2422–2426] The amount of site-directed mu-
tagenesis has been limited, but the disease has
yielded many locations for loss of function be-
cause the severity of the disease is (inversely)
correlated to the residual 21-hydroxylation ac-
tivity Many of the variants could be rational-
ized in the context of a homology model [2422],
although some associated with disease are more
subtle (eg, E380D) A structure of the human
P450 21A2 protein is not available, but a struc-
ture of bovine P450 21A2 is [42] More than 80 %
of the variants known to be adverse from clinical
studies can be rationalized in the bovine structure
(although more details of exactly why these are
debilitating will require more study) [42, 2400]
The published bovine P450 21A2 structure
includes the substrate 17α-hydroxyprogesterone
[42]; a human P450 21A2 structure with proges-
terone is also available [2769] An interesting
feature of the published bovine P450 21A2 struc-
653
ture is the presence of two molecules of the sub-
strate (17α-hydroxyprogesterone) [42] One is in
an appropriate position for 21-hydroxylation, but
the other is on the “other” side of the substrate
near the heme and not in a position for hydrox-
ylation Spectral binding and reduction experi-
ments are consistent with the occupancy by two
substrates, as well as the crystal structure [42]
Dual occupancy does not lead to cooperative in-
teraction [2769]
9.7.47.6 Inhibitors
Relatively little has been published about inhibi-
tors Detrimental effects of spironolactone have
been attributed to inhibition of 21-hydroxylation
[2427], although further details with this P450
are lacking Recently Auchus and his associates
[2205] reported that the enantiomeric form of
progesterone ( ent-progesterone) is a competitive
inhibitor of P450 21A2 (although not as effective
as with P450 17A1) Apparently no new inhibi-
tors of P450 21A2 have been published since the
last edition of this chapter was published [149]
9.7.47.7 Clinical Issues
As mentioned earlier, the incidence of defects is
relatively frequent and the ability to form corti-
sol is a problem Patients who cannot synthesize
sufficient aldosterone may lose sodium balance
and can develop a fatal “salt-wasting” syndrome
Treatment involves administration of mineralocor-
ticoids and glucocorticoids Females with severe,
classic P450 21A2 deficiency are exposed to ex-
cess androgens prenatally and born with virilized
external genitalia, but prenatal diagnosis permits
prenatal treatment of affected females [2414]
For a review of aspects of P450 21A2 diagno-
sis and management (in adolescents), see Lin-Su
et al [2428] In addition to adrenal hyperplasia,
P450 21A2 insufficiency has also been consid-
ered in relationship to bone density [2429], adre-
nal mass [2430], Cushing’s disease [2431], risk
of cardiovascular disease [2432], virilization of
female genitalia [2433], and female [2434] and
male [2435] infertility See also [2436, 2437] for
more on virilizing congenital adrenal hyperpla-
sia
654
Autoantibodies against P450 21A2 have been
detected in autoimmune Addison’s disease pa-
tients [1971]
9.7.48 P450 24A1
The next three P450s (24A1, 27A1, 27B1) are
involved in vitamin D metabolism (Fig 929)
All three are mitochondrial and receive electrons
from the iron–sulfur protein adrenodoxin (via the
flavoprotein NADPH-adrenodoxin reductase)
(Table 92)
9.7.48.1 Sites of Expression
The 24-hydroxylation of 25-hydroxyvitamin D3
has long been known to occur in the kidney mi-
tochondrial membrane [2438] Following the pu-
rification of a rat P450 with this activity [2439],
cDNA clones for chicken [2440] and human
[2441] homologs were obtained
The enzyme is expressed in both proximal and
distal kidney tubules [2442] but has also been
found in human non-small cell lung carcino-
mas [2443] This would appear to be a relatively
low abundance P450 Expression has also been
reported in human keratinocytes [2444, 2445],
Fig. 9.29   Overview of P450s involved in key steps of vitamin D activation [47] (With kind permission from Springer
Science + Business Media: [149], Fig 1016)
F. P. Guengerich
colon carcinoma cells [2446], and prostatic can-
cer cells [2447]
P450 24A1 has also been found expressed in
the male reproductive tract [2448], and expres-
sion at the annulus of human spermatozoa has
been considered as a marker of semen quality
[2449]
Peptides corresponding to P450 24A1 have
been identified in human liver tissue [635]
9.7.48.2 Regulation
The regulation of the CYP24A1 gene appears
to be complex, although some phenomena ob-
served in animal models have not been examined
in as much detail in humans The activity has
long been known to be inducible by vitamin D,
perhaps to relieve the cells of an overload, and
a VDR element has been found in the 5ʹ-region
of the CYP24A1 gene [2450, 2451] Parathyroid
hormone and cyclic AMP both enhance induction
by the VDR [2442]
In human keratinocytes, P450 24A1 mRNA
was also elevated by 1α,25-dihydroxyvitamin D3
[2444] Studies with rat systems indicate that this
response is also mediated by VDR response ele-
ments and that two of these (VDRE-1, VDRE-2)
operate synergistically [2452] A functional Ras-
9  Human Cytochrome P450 Enzymes
dependent Ets-binding site is located downstream
from the proximal vitamin D response element
(VDRE) site and was critical; the model indicates
transcriptional cooperation between Ras-activat-
ed Ets proteins and the VDR–RXR complex in
mediating 1α,25-dihydroxyvitamin D action
on the P450 24A1 promoter [2453] The YY1
transcription factor has been reported to repress
1α,25-dihydroxyvitamin D3-induced transcrip-
tion in cell culture [2454] The isoflavone genis-
tein was reported to block the transcription of the
CYP24A1 gene in human prostatic cancer cells,
and this block could be relieved with the histone
deacetylase inhibitor trichostatin A [2447] Fi-
nally, the earlier results with Ets proteins ( vide
supra) have been expanded to show distinct
roles of the MAP kinases ERK1/ERK2 and
ERK5 [2455] Induction of P450 24A1 by 1α,25-
dihydroxyvitamin D3 involves Ets-1 phosphory-
lation at Thr-38, but 1α,25-dihydroxyvitamin
D3 stimulation of ERK1/ERK2 required RXRα
phosphorylation on Ser-260 [2455]
1α,25-Dihydroxyvitamin D3 has been report-
ed to induce P450 24A1 (mRNA) expression in
colon cells [2456] The human P450 24A1 pro-
moter has been characterized, and a short vita-
min D stimulating element (VSE) found in rat is
absent [2457] Induction of human P450 24A1
by 1α,25-hydroxyvitamin D3 is dependent upon
a promoter region spanning nucleotides -470 to
-392 [2457] Both a proximal and a downstream
element VDR element bind the VDR/RXR het-
erodimer and somehow lead to induction [2458]
Coregulators are also involved and are respon-
sible for increased RNA polymerase II activity
and histone H4 acetylation
PXR (liganded) can activate the P450 24A1
gene by directly binding to and transactivating
vitamin D-responsive elements within the pro-
moter region [2459]
Vitamin D3 activates the P450 24A1 promoter
by dissociating the corepressor silencing me-
diator for retinoid and thyroid (SMRT) hormone
receptors from the VDR on those VDREs PXR
strongly represses vitamin D3 activation of the
P450 24A1 gene indirectly by binding to and
preventing vitamin D3-dependent dissociation of
SMRT from the P450 24A1 promoter The degree
655
of the PXR-mediated locking of SMRT depends
on the relative concentration of vitamin D3 to
the human PXR activator rifampicin; SMRT in-
creases dissociation as this ratio increases CAR
is also found to prevent dissociation of SMRT
from the CYP24A1 promoter [2459] An SNV in
the promoter blocks protein binding and gene ac-
tivation [2460] P450 24A1 is also regulated by
proinflammatory cytokines (in a cultured human
trophoblast system)
P450 24A1 also appears to be subject to epi-
genetic regulation Studies in human prostate
cancer cells show that repression of the expres-
sion of the gene is mediated by promoter DNA
methylation and repressive histone modifications
[2461] Placental-specific gene methylation has
also been reported [2462] Along with other evi-
dence for epigenetic control (in prostate cancer
cells) [2461], evidence for a change in gene copy
number has been reported [2463, 2464] The
DNA methylation levels have been reported to
predict vitamin D response variation [1187]
9.7.48.3 Genetic Variation
Genetic variations in P450 24A1 are known and
have been considered in the context of clinical
issues regarding vitamin D [2465–2468]
9.7.48.4 Substrates and Reactions
Both 25-hydroxyvitamin D3 and 1α,25-
dihydroxyvitamin D3 are substrates for 24-hy-
droxylation (Fig 929), with the latter being the
preferred substrate [2469] However, human
P450 24A1 can also catalyze other side-chain
reactions (Fig 930) [2470, 2471] Studies with
side chain-fluorinated vitamin D analogs also
provide evidence for some flexibility of this
side chain in allowing P450 24A1 to oxidize
different sites [2472, 2473] Rat P450 24A1 dif-
fers from the human ortholog in taking 1α,25-
dihydroxyvitamin D3 on to calcitroic acid instead
of the products shown in Fig 30 [2474–2476]
A number of additional studies with substrate
analogs have appeared since the last edition of
this book was published [149] These include the
metabolism of A-ring diastereomers of 1α,25-
dihydroxyvitamin D3 [2477] The 23-hydrox-
ylation occurs, and 25,26,27-trinor-23-ene vi-
 656

Fig. 9.30   C-23 hydroxylation pathway for 25-hydroxyvitamin D3 (25(OH)) oxidation catalyzed by P450 24A1 [2470]. (With kind permission from Springer Science + Business
Media: [149], Fig. 10.17)
F. P. Guengerich
9  Human Cytochrome P450 Enzymes
tamin D3 and 25,26, and 27-trinor-23-ene-1α
vitamin D3 are the respective products formed
from 25-hydoxy- and 1α,25-dihydroxyvitamin
D3, both oxidized by P450 24A1 [2478] Other
work showed that different 2α-substituted 1α,25-
dihydroxyvitamin D3 analogs were processed in
different ways [2479] 25-Hydroxy-19-nor- and
1α,25-dihydroxy-19-nor vitamin D3 are substrates
[2480] Several pathways of oxidation were
seen with 1α-propoxyl-1α,25-dihydroxyvitamin
D3 [2481] Urushino et al [2482] reported that
1α,25-dihydroxyvitamin D2 (differing from vita-
min D3 only in the presence of a double bond at
the 22, 23 position) is converted into at least ten
products by human P450 24A1 (but only to one
by rat P450 27A1)
A single (A326G) mutation has been shown
to convert human P450 24A1 from a 24- into a
23-hydroxylase [2483] Also, a V391 L mutation
of human P450 24A1 changed the site of hydrox-
ylation to C-25 [2484]
9.7.48.5 Structure
Several reports involved site-directed mutagen-
esis and homology modeling to gain insight in
the structure of P450 24A1 [2485–2487] How-
ever, given the diversity of products observed
with minor modifications of either the substrate
or protein (Sect 7484), it is difficult to make
definite conclusions from much of this work An
X-ray crystal structure of rat P450 24A1 has been
published [43], the first structure of a mitochon-
drial P450 The structure is an open form, without
a substrate, although a 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS)
detergent molecule is present in the structure The
substrate was docked into the structure
9.7.48.6 Inhibitors
As discussed with other enzymes involved in
vitamin A or D metabolism, there is interest in
developing inhibitors of vitamin D degradation
as opposed to administration of vitamin D itself,
to raise levels of active vitamin D metabolites
Schuster [2445, 2488, 2489] has identified some
inhibitors that differ in their selectivity between
P450 24A1 and P450 27B1 and have sub-µM IC50
values More inhibitors have been published in the
657
literature [2490–2492] Some of these are of inter-
est in specifically inhibiting P450 24A1 in cancer
therapies related to vitamin D [2493–2495]
9.7.48.7 Clinical Issues
The scheme presented in Fig 929 depicts P450
24A1 as an enzyme involved in deactivating the
activated form of vitamin D The possibility has
been considered that defects in P450 24A1 might
lead to hypervitaminosis D [47] An overactive
P450 24A1 could lead to vitamin D deficiency
Henry [2496] has reviewed the role of P450
24A1 and made comparisons to other “multistep”
P450 enzymes The possibility is raised that P450
24A1 could serve to generate products with their
own biological activities, with P450 24A1 thus
being involved in an anabolic pathway Trans-
genic rats overexpressing (rat) P450 24A1 were
found to have low plasma levels of 24,25-dihy-
droxyvitamin D3 [2497], which was unexpected
Further, the transgenic rats developed albumin-
uria and hyperlipidemia shortly after weaning
and later developed atherosclerotic lesions in the
aorta These results raise the possibility that P450
24A1 is involved in functions other than vitamin
D metabolism [2497]
P450 24A1 can be an issue in situations in-
volving changes in levels of active forms of vi-
tamin D Some aspects involve cancer; further,
P450 24A1 has been considered as a biomarker
for some cancers [2448, 2465, 2498–2500] P450
24A1 has also been considered in the context of
kidney disease [2501, 2502] Because of the rela-
tionship of vitamin D with bone, loss-of-function
P450 24A1 genetic variants are an issue in hyper-
calcemia [2503, 2504]
9.7.49 P450 26A1
9.7.49.1 Sites of Expression
P450 26A1 is expressed mainly in liver [2505–
2507] The level is not high, ie, highest value
28 pmol/mg microsomal protein [2508] Expres-
sion of P450 26A1 at the mRNA level is also
considerable in brain, lung, and artery, with the
highest brain levels being in the cerebral cortex,
hippocampus, and temporal lobe [2506] Ex-
658
pression is also seen in testis and uterus At the
protein level, the highest expression was in lung,
pancreas, and uterus [2506], with some in adi-
pose, intestine, skin, and spleen
P450 26A1 is present at an earlier stage of em-
bryonic development than P450 26B1 or 26C1
[2509]
9.7.49.2 Regulation
P450 26A1 is regulated by its substrate, retinoic
acid, via RAR Zhang et al [2510] analyzed a
22-kb 5-flanking region of the human CYP26A1
gene and identified three conserved hexamet-
ric direct repeat -5 elements for RAR binding
(RARE -1, -2, -3) and an RAR element half-site
The combined element was functional in HepG2
cells Their results suggest a cooperative model
in which the binding to multiple RAR elements
may account for the strong inducibility of P450
26A1 in liver, possibly involving looping of the
distal region to position it close to the transcrip-
tion start site [2510]
RARα is considered to be the major RAR form
responsible for the induction of P450 26A1 (and
RARβ) in HepG2 cells [2507] The PPARγ agonists
proglitazone and rosiglitazone upregulated P450
26A1 expression tenfold (in HepG2 cells) Further
work by Tay et al [2507] indicated differences in
the regulation of P450 26A1 and 26B1 The altera-
tion of P450 26A1 regulation by drugs may have
relevance to therapy and drug safety [2507]
A number of other chemicals have been re-
ported to regulate P450 26A1 in rodents and
other experimental models [2507], although the
relevance to humans has not been established
9.7.49.3 Genetic Variation
In adult human liver, the levels of P450 26A1 are
highly variable [2509], but this has been attribut-
ed to the variability of vitamin A intake At least
four alleles have been identified (http://www
cypalleleskise) Two have been linked to lower
activity [2511]
9.7.49.4 Substrates and Reactions
Only two substrates of P450 26A1 are known,
all-trans-retinoic acid and 9-cis-retinoic acid
The latter is a much poorer substrate for P450
F. P. Guengerich
26A1 (but not for P450 26C1 [2509]) All-trans-
retinoic acid forms 4( S)-hydroxyretinoic acid
[667]. Both ( R) (formed by some other P450s)
and ( S)-4-hydroxyretinoic acid are substrates for
P450 26A1, yielding 4-oxo-retinoic acid [667]
Other products are 4,18-dihydroxyretinoic acid
and 4-oxo-18-hydroxyretinoic acid (plus 18-hy-
droxyretinoic acid) [667, 2509]
Although all-trans-retinoic acid can be oxi-
dized by P450 3A subfamily enzymes and P450
2C8, P450 26A1 is the predominant enzyme in-
volved in retinoic acid oxidation [2508]
9.7.49.5 Structure
No crystal structures of P450 26A1 are available
A homology model based on P450 3A4 has been
published [667]
9.7.49.6 Inhibitors
There is interest in inhibition of endogenous reti-
noid degradation as an alternative to administra-
tion of retinoids [2512] A number of inhibitors
of P450 26A1 are known, the best ones contain-
ing imidazoles and triazoles, some having sub-
µM IC50 values One novel approach is use of a
vaccine targeting (mouse) P450 26A1 as an im-
munopreventive strategy to prevent breast carci-
noma Selectivity between P450s 26A1 and B1
(and C1) is an issue, although realistically inhib-
iting both (all three) is a part of the overall strat-
egy [2509, 2512–2520] Gudas has shown a role
of P450 26A1 in stem cell differentiation, and
blocking P450 26A1 may be an approach in cell/
differentiation therapy to treat neurodegenerative
diseases [2521]
9.7.49.7 Clinical Issues
P450 26A1 has considerable relevance in devel-
opmental biology, at least in model organisms,
because of the control of retinoid homeostasis
CYP26A1(−/−) mice show distinct malformations
and lethality [2507] P450 26A1 knockout em-
bryonic stem cells have been reported to exhibit
reduced differentiation and growth arrest when
retinoic acid is added [2522]
Malfunction of P450 26A1 in retinoid homeo-
stasis is the major clinical issue One study [2523]
suggests a role of P450 26A1 genetic variation in
9  Human Cytochrome P450 Enzymes
nonsyndromic bilateral and unilateral optic nerve
aplasia Genetic variation of P450 26A1 has been
suggested to be involved in spina bifida [2524]
and caudal regression syndrome [2525] P450
26A1 has been considered in the context of ab-
normalities of limb development [2526]
9.7.50 P450 26B1
9.7.50.1 Sites of Expression
P450 26B1 was originally identified as a “sec-
ond” P450 family 26 gene in zebra fish and hu-
mans [2527], with a prominent brain localization
[2528] In contrast to P450 26A1, P450 26B1 is
not expressed in human liver [2506] Expression
(at the mRNA level) is seen at the highest levels
in brain (cerebellum, cerebral cortex, hippocam-
pus, temporal lobe), vein, artery, adipose, blad-
der, kidney, testis, and skin [2506] At the protein
level, the highest expression was seen in uterus,
pancreas, lung, skin, and spleen, with some seen
in intestine, adipose, and kidney [2506]
During fetal development, levels in cephalic
tissues are tenfold higher at days 57–100 than in
later gestation (112–224 days)
9.7.50.2 Regulation
As in P450 26A1, P450 26B1 is also induced
by (all-trans)-retinoic acid [2505, 2509] The
transcription factors SOX9/SF-1 and FOXL2
have been reported to be antagonistic regula-
tors of P450 26B1 during gonadal development
(in mice) [2529] Expression of P450 26B1 in T
cells is inhibited by TGF-β [2530] As with P450
26A1, PPARγ agonists regulate P450 26B1 tran-
scription and a PPARγ antagonist (the latter ef-
fect in contrast to P450 26A1) [2507]
9.7.50.3 Genetic Variation
Genetic variation is recognized in the CYP26B1
gene Possible linkages to Crohn’s disease [2531]
and congenital limb deficiencies [2526] have
been proposed
9.7.50.4 Substrates and Reactions
The substrates and reactions are essentially the
same as for P450 26A1; ie, all-trans-retinoic
659
acid is oxidized to the 4- and 18-hydroxy prod-
ucts and the 4-alcohol is further oxidized to
4-oxo derivatives [2509] 9-cis-Retinol and other
retinoids are poor substrates
9.7.50.5 Structure
No crystal structure is available At least one ho-
mology model has been published [2532]
9.7.50.6 Inhibitors
As discussed under P450 26A1 (Sect 7496, vide
supra), there is a general concept of giving drugs
to block the metabolism of endogenous retinoids
rather than administering retinoids themselves
[2512] Most inhibitors of retinoid oxidation
have been developed as general inhibitors and do
not distinguish between P450s 26A1 and 26B1
[2512, 2515]
9.7.50.7 Clinical Issues
Animal studies show that P450 26B1, like P450
26A1, is important in development [2509] Some
possible outcomes of genetic variations are men-
tioned in Sect 7503 P450 26B1 plays a major
role in retinoid metabolism and signaling in
human aortic smooth muscle cells The potential
for inhibition has already been addressed
9.7.51 P450 26C1
9.7.51.1 Sites of Expression
In contrast to the other P450 family 26 P450s,
P450 26C1 appears to be expressed mainly dur-
ing embryonic development, at least in animal
models [2509] Sites of expression include hind-
brain, inner ear, first bronchial arch, tooth buds,
and equatorial retina (of mice) [2533, 2534]
However, low levels (of mRNA) can be detected
in adult adrenal gland, lung, spleen, testis, and
brain [2509, 2535] Expression is also seen in
keratinocyte cell lines treated with 9-cis- or all-
trans-retinoic acid [2509]
9.7.51.2 Regulation
Relatively little information is available, particu-
larly in humans As indicated above, in keratino-
660
cyte cultures transcription is induced by retinoic
acid [2509]
9.7.51.3 Genetic Variation
Some maladies have been suggested to be linked
to genetic variation in CYP26C1, including focal
facial dermal dysplasia type IV [2536] and non-
syndromic bilateral and unilateral optic nerve
aplasia [2523]
9.7.51.4 Substrates and Reactions
P450 26C1 oxidizes both 9-cis- and all-trans-ret-
inoic acid to the 4-hydroxy, 4-oxo-, and 18-hy-
droxy products (and combinations) [2509, 2537]
These are inactivated (with regard to retinoid
receptors) and are the same products formed by
P450s 26A1 and 26B1 [2509]
9.7.51.5 Structure
No information about the structure of P450 26C1
is available
9.7.51.6 Inhibitors
Blocking the metabolism of endogenous reti-
noids is considered an alternative to administra-
tion of retinoids Some azoles are in development
as inhibitors, but it is not clear if their selectivity
towards individual 26C family P450s (or others)
has been studied and reported yet [2509, 2538]
9.7.51.7 Clinical Issues
The potential clinical issues related to P450 26C1
have not been considered in detail but would in-
volve issues with retinoids, ie, lack of retinoid
metabolism would lead to an overload of ret-
inoid-induced problems As mentioned above
(Sect 7513), some possibilities exist with ge-
netic variations in focal facial dermal dysplasia
type VI and nonsyndromic bilateral and unilat-
eral optic nerve aplasia
9.7.52 P450 27A1
This is a mitochondrial enzyme that was charac-
terized on the basis of two rather divergent cata-
lytic activities, the 25-hydroxylation of vitamin
D3 (Fig 929) and the oxidation of cholesterol at
F. P. Guengerich
the C27 position (Fig 931) Thus, the enzyme
bridges between hormone (vitamin D) and oxy-
sterol pathways, and the clinical relevance of
P450 27A1 is considerable
9.7.52.1 Sites of Expression
The enzyme is localized in liver mitochondria
Some confusion existed in the early literature
because some animal species have liver micro-
somal vitamin D3 25-hydroxylases (eg, hog
liver and kidney P450 2D25 [2540, 2541]), but
not humans [2542] The rat and human liver mi-
tochondrial P450 27A1 recombinant enzymes
were clearly shown to catalyze both vitamin D3
25-hydroxylation and the 27-hydroxylation of
the side chains of cholesterol and several deriva-
tives [2543, 2544]
Expression, at least at the mRNA level, has
also been reported in leukocytes [2545], skin fi-
broblasts [2546], kidney [2547] (and fetal liver
and kidney [2547]), and the arterial wall [2548]
Other sites (in humans) include the male repro-
ductive tract (round and elongated spermatids,
vesicles within the caputepididymis, glandular
epithelium of canda epididymis, seminal vesi-
cles, prostate, and spermatozoa [2448] and reti-
na pigment epithelial cells [2549, 2550]) P450
27A1 has been detected in human liver using
LC–MS [635]
9.7.52.2 Regulation
Several aspects of regulation of the CYP27A1
gene have been studied In rats, the enzyme can
be induced by gonadotropin [2551] A hamster
model showed downregulation of the gene in
cholestatic liver [2539], although human P450
27A1 (used in HepG2 cells) was not subject to
negative feedback regulation [1730]
Since the previous edition of this chapter
[149], the literature has several additions, indi-
cating that regulation of expression is even more
complex Androgens upregulate expression in
HepG2 cells, utilizing a region upstream from
− 792 [2552] Estrogens downregulate expres-
sion via both ER α and β pathways [2552] Ex-
pression is also regulated by T Fβ1 [2553], RXR,
and PPARγ pathways [2554, 2555], and PXR
[2556] pathways There is some discrepancy as to
9  Human Cytochrome P450 Enzymes 661

Fig. 9.31   Bile acid synthesis from cholesterol [2539] The steps shown with dashed arrows are tentative (With kind
permission from Springer Science + Business Media: [149], Fig 1018)
662
the involvement of LXR [2554, 2555] in human
macrophages Phenobarbital induces P450 27A1
[2557], although it is unknown if this involves
PXR or CAR
One report involves epigenetic control, ie,
DNA methylation [1187]
9.7.52.3 Genetic Variation
In “normal” human population, the variation in
the steady-state P450 27A1 mRNA level was re-
ported to be ~ 25-fold, compared with 60-fold for
P450 7A1 in the same study [1730] However,
at least two polymorphisms (≥ 1 % incidence,
no dramatic effect) and 42 other genetic vari-
ants (rare alleles, usually debilitating) are known
[2546, 2558] Truncation mutations are known
[2545], as well as splice variants [2559] Defects
in the CYP27A1 gene are associated with a condi-
tion known as cerebrotendinous xanthomatosis, a
rare, autosomal recessive disorder characterized
by accumulation of cholestanol and cholesterol in
many tissues The clinical manifestations include
tendon xanthoma, premature cataracts, juvenile
atherosclerosis, and a progressive neurological
syndrome involving mental retardation, cerebel-
lar atoxia, pyramidal tract signs, myelopathy, and
peripheral neuropathy [47, 2558]
The known variants leading to cerebrotendi-
nous xanthomatosis have been reviewed [2560]
Mutation in the gene has been associated with
fatal cholestasis in infancy [2561] Variants have
also been linked to susceptibility to amyotrophic
lateral sclerosis [2562]
9.7.52.4 Substrates and Reactions
Expanding on the previous discussion, P450
27A1 catalyzes the 25-hydroxylation of vita-
min D3 (Fig 929), 1α-hydroxyvitamin D3, vi-
tamin D2, and 1α-hydroxyvitamin D2 and also
the 27-hydroxylation of cholesterol and several
derivatives (Fig 931) [2563, 2564] The choles-
terol alcohols are further oxidized by the enzyme
to aldehydes and then carboxylic acids [2565]
The available information suggests release of the
intermediates in the pathway [2565] The regi-
oselectivity of the enzyme is considered to be a
function of the distance of the hydroxylation site
to the end of the side chain [2566]
F. P. Guengerich
More detailed analysis of the vitamin D3 re-
action has been done with E. coli recombinant
P450 27A1, with evidence for the following
products (from vitamin D3): 25-hydroxy, 26-hy-
droxy, 27-hydroxy, 24R,25-dihydroxy, 1α,25-
dihydroxy, 25,26-dihydroxy, 25-,27-dihydroxy,
27-oxo, and an unidentified dehydrogenated
product [2544, 2567]
P450 27A1 occupies a place at the intersection
of metabolism of vitamin D (a secosteroid) and
sterol metabolism (Table 91), and perhaps be-
cause of this less than stringent substrate selectiv-
ity, the list of substrates and reactions continues to
grow 2α-Propoxy- and 2α(3-hydroxypropoxy)-
1α,25-dihydroxyvitamin D3 are substrates
Human P450 27A1 also converted 25-hy-
droxyvitamin D3 into 1α,25-dihydroxyvitamin
D3 and 25,27-dihydroxyvitamin D3, as well as
the conversion of 25-hydroxyvitamin D3 into
4β,25-dihydroxyvitamin D3 [2568] Thus, 4β-
hydroxylation was catalyzed
In the retina, P450 27A1 catalyzed the conver-
sion of the oxysterol 7-ketocholesterol to 27-hy-
droxy and 27-carboxy products [2549] Further
work by Pikuleva and her associates showed that
several other cholesterol precursors (in addition
to Δ7-dehydrocholesterol) are substrates (for
27-hydroxylation), including desmosterol, zymo-
sterol, and lanosterol [2569] The dehydrocholes-
terol products (25-hydroxy and 26/27-hydroxy)
modulate LXR activity [2570] Progesterone is a
substrate, undergoing reduction of the C-20 keto
group (to an alcohol) [2571]
A study of the selectivity of sterol analogs as
substrates indicates that more polar derivatives
(eg, cholesterol sulfate) are better substrates
[2572]. Sterols with a 3-oxo-Δ4 structure were
found to be hydroxylated at higher rates than
3-hydroxy-Δ5 analogs The very high activity
with the cholestanol precursor 4-cholesten-3-one
may be of importance in the accumulation of
cholestanol in patients with cerebrotendinous
xanthomatosis disease
9.7.52.5 Structure
Some information about the roles of amino acids
can be inferred from the knowledge of alleles in-
volved in cerebrotendinous xanthomatosis; many
9  Human Cytochrome P450 Enzymes
of these proteins were unstable when attempts
were made at heterologous expression [2573]
Other work by Pikuleva et al [2574] with the pu-
tative F and G helices has shown differences due
to substitution of Phe-207, Ile-211, Phe-215, Trp-
235, and Tyr-238 Interestingly, the I211K and
F215K mutations affected the regioselectivity and
enabled the enzyme to catalyze C–C bond cleav-
age Further work with mutants in this region led to
weaker association of P450 27A1 with the mem-
brane, and some of the nonconservative changes
yielded impaired catalytic activity [2575]
Human P450 27A1 can be contrasted with
porcine P450 2D25, which also catalyzes vitamin
D3 25-hydroxylation The only human subfam-
ily 2D P450 enzyme is P450 2D6, which does
not have activity towards vitamin D Further,
changing a set of residues of porcine P450 2D25
to their counterparts in (human) P450 2D6 abol-
ished the activity towards vitamin D3 [2576]
No structures have yet appeared but more
models have, some based on site-directed muta-
genesis work [2483, 2577, 2578]
9.7.52.6 Inhibitors
Apparently, little specific work has been done on
inhibition of this enzyme Inhibition of this en-
zyme by a drug would probably be undesirable
9.7.52.7 Clinical Issues
Low serum 25-hydroxyvitamin D3 concentrations
have been reported in a variety of other medical
conditions and are considered to be a potential
problem [2579] Although cerebrotendinous xan-
thomatosis is linked with defective P450 27A1
[47], there are a number of enigmas about the eti-
ology A heterozygote showed frontal lobe demen-
tia and abnormal cholesterol metabolism [2580]
Compound heterozygous mutations have also
been reported to cause a variation of cerebroten-
dinous xanthomatosis [2558] P450 7A1 may also
play a role in the etiology of the disease [1778]
Björkhem [2581] has recently reviewed the
issue of whether oxysterols (eg, hydroxycho-
lesterol) control cholesterol homeostasis Stud-
ies with rodents and cultured cells have not been
very clear to date For instance, disruption of the
mouse Cyp21a1 gene yielded reduced bile acid
663
synthesis but no change in levels of cholesterol or
1α,25-dihydroxyvitamin D3 [2582] P450 27A1
is constitutively expressed in the normal artery
wall and is substantially upregulated in athero-
sclerosis, and the possibility has been raised that
P450 27A1 may be a protective mechanism for
removing cholesterol [2548] Further, immune
complexes and IFN-γ decreased P450 27A1 ex-
pression in human aortic endothelial cells, pe-
ripheral blood mononuclear cells, monocytes-
derived macrophages, and a human monocytoid
cell line, suggesting downregulation of P450
27A1 to maintain cholesterol homeostasis in the
arterial wall [2583]
In Cyp27a1-/- mice, a dramatic increase in the
level of P450 3A enzymes was seen; some ste-
rols accumulate and induce via the mouse PXR
system [2584] P450 3A4 has some side-chain
hydroxylation activities towards cholesterol-
derived sterols [1328] However, elevated P450
3A4 activity was not increased in cerebrotendi-
nous xanthomatosis [1328], indicating a differ-
ence in the murine and human systems Recently
Escher et al [2585] have reported that cholester-
ol efflux in CHOP cells is enhanced by heterolo-
gous expression of human P450 27A1, and the
authors suggest this as part of a protective sys-
tem against atherosclerosis The basis is probably
the ability of 27-hydroxycholesterol to act as an
endogenous ligand for the LXR in cholesterol-
loaded cells [2586]
In considering the general question of whether
oxysterols (eg, 27-hydroxycholesterol) control
cholesterol homeostasis, the hypothesis is still
open and the rodent data are not totally clear
here Björkhem [2581] has made the point that
humans lacking P450 27A1 have normal circu-
lating levels of cholesterol
Reference has already been made to genetic
variants in Sect.  7.52.3 ( vide supra) A genetic
association with obesity traits has been consid-
ered [2587], as well as with coronary artery dis-
ease [2588] The area of cerebrotendinous xan-
thomatosis has been reviewed recently, including
P450 27A1 [2589]
The production of 27-hydroxycholesterol by
(P450 27A1) has been linked to breast cancer
pathophysiology, in that it serves as an ER and
664
LXR ligand and increases ER-dependent growth
and LXR-dependent metastasis in a mouse mod-
els of breast cancer [2590] Accordingly, lower-
ing circulating cholesterol levels and inhibiting
P450 27A1 have been proposed as mechanisms
to treat breast cancer
9.7.53 P450 27B1
As discussed earlier, vitamin D is an important
hormone A critical step in activation is 1α-
hydroxylation [2591] (Fig 929) Early work es-
tablished the P450 nature of the enzyme, localized
in kidney mitochondria [2592] Subsequent work
demonstrated that the 1α- and 24-hydroxylation
activities could be attributed to different enzymes
[2593, 2594] Some early work had suggested
that the 1α- and 25-hydroxylation activities were
associated with the same enzyme [2595], but
later work showed that these activities were due
to P450 27B1 and 27A1, respectively
9.7.53.1 Sites of Expression
The cloning of the human cDNA for what is
now known as P450 27B1 established kidney
mitochondrial P450 27B1 as the vitamin D3 1α-
hydroxylase [2596]
P450 27B1 is expressed in many parts of the
human kidney, including the distal convoluted
tubule, the cortical and medullary part of the col-
lecting ducts, and the papillary epithelia [2597]
Lower expression was observed along the thick
ascending limb of the loop of Henle and Bow-
man’s capsule Some, weaker expression was
observed in glomeruli or vascular structures In
normal humans, the distal nephron is the predom-
inant site of expression [2597]
P450 27B1 is also expressed in many extrare-
nal sites (human) where it is involved in vitamin
D-related activities, including skin (basal kerati-
nocytes, hair follicles), lymph nodes (granuloma-
ta), colon (epithelial cells and parasympathetic
ganglia), pancreas (islets), adrenal medulla, brain
(cerebellum and cerebral cortex) [2598], placenta
(decidual and trophoblastic cells) [2598–2600],
cervix [2601], and parathyroid glands [2602]
Thus, P450 27B1 may be an intracrine modula-
F. P. Guengerich
tor of vitamin D function in peripheral tissues
[2598] The expression of P450 27B1 was el-
evated in parathyroid adenomas but attenuated
in carcinomas, relative to normal parathyroid
tissue [2602] P450 27B1 has also been found
in (human) and endometrial tissue [2603] For
reviews on the significance of extrarenal P450
27B1, see [2604, 2605]
9.7.53.2 Regulation
Although the CYP27B1 gene is only 5 kb in size
[2606], the regulation is quite complex The pro-
moter is in the  − 85/+ 22 region and requires a
functional CCATT element Three consensus
AP-1 sites are upstream [2607] Enzyme activ-
ity has long been known to be stimulated by low
phosphorus diets (in animal models) [2608], and
more recently this phenomenon has been linked
to a growth hormone mechanism [2609, 2610];
its relevance in humans is not known
Complexity is seen in different models Para-
thyroid hormone-related protein and Ca2 + have
conflicting actions in a rude rat model of humoral
hypercalcemia of malignancy [2611] In differ-
entiated Caco cells, there is upregulation of P450
27B1 expression by 1α,25-dihydroxyvitamin D3
and EGF but downregulation in less differentiat-
ed Caco cell lines [2446] P450 27B1 is regulated
by proinflammatory cytokines in human tropho-
blasts [2612] Immune regulation of P450 27B1
has been reported in monocytes [2613], and ure-
mia downregulated P450 27B1 in monocytes
[2614] Gene amplification (and splice variants)
has been reported in gliobastoma cells [2615] A
number of growth factors have been reported to
regulate (mostly suppress) P450 27B1 expres-
sion, including growth factor independent-1
(GFI-1) [2616], TGFβ [2617], nuclear recep-
tor 4A2 and CIEBPβ [2618], thyroid hormone
[2619], and NFκB [2620]
Regulation of P450 27B1 expression by (the
product) 1α,25-dihydroxyvitamin D3 has been re-
viewed [2621] The product downregulates P450
27B1 in colon cells [2456, 2622] Part of the mech-
anism has been attributed to hypermethylation
[2623], although increased copy number (and not
hypomethylation) has been identified as the cause
of overexpression in colorectal cancer [2464]
9  Human Cytochrome P450 Enzymes
9.7.53.3 Genetic Variation
Another aspect of regulation of P450 27B1 is
genetic; P450 27B1 deficiency results in type I
vitamin D-dependent rickets [2624] The genet-
ics have been established in more than 20 vari-
ants [2625, 2626] At least 13 missense variants
have been observed, none of which encode an
active protein Some of the mutants are splicing
defects [2627] Some variants in CYP27B1 are
also involved in what is termed pseudovitamin
D-deficiency rickets [2628, 2629]
Since the last edition [149], the genetic in-
formation has greatly expanded The number of
variants has increased, and CYP27B1 associa-
tions have been considered with diabetes [2630–
2632], brain and skin cancers [2633], Graves’
disease, Addison’s disease, and Hashimoto’s thy-
roiditis [2631, 2634–2636], congestive heart fail-
ure [2637], and multiple sclerosis [2638–2640]
The biochemical effects of the variants are
reviewed in [2623, 2641] Perhaps the most bio-
logically plausible relationships of P450 27B1
variants are with rickets disease type I [2642] and
fracture risk in the elderly [2643]
9.7.53.4 Substrates and Reactions
P450 27B1 can catalyze the 1α-hydroxylation
of both 25-hydroxy and 24( R),25-dihydroxyvi-
tamin D3 [2475, 2644] (Fig 33) The intrinsic
activity (catalytic efficiency, kcat/Km) for the re-
combinant human enzyme is better for 24( R),25-
hydroxy vitamin D3, but this does not mean that
this is the favored substrate in the cell because of
the balance of vitamin D metabolites regulated
by P450s 24A1 and 27A1 [47] Apparently, the
25-hydroxy group is an obligatory requirement
[2470, 2644]
In addition, 19-nor vitamin D3 analogs are
substrates [2480] The products of the reactions
of P450 11A1 on vitamin D3 are also substrates
[2645, 2646]
9.7.53.5 Structure
No crystal structures have been published
Some information is available from the natural
mutants of P450 27B1, even if the basis for loss
of activity is not obvious Inouye’s group [2573]
has provided evidence that Arg-107, Gly-125,
665
and Pro-497 are not simply involved in binding
substrate but required for proper folding It was
also suggested that Arg-389 and Arg-453 are in-
volved in heme binding and that Asp-164 stabi-
lizes the bundle of the D, E, I, and J helices Thr-
321 is suggested to be involved in O2 activation
[2573] The natural mutants L343F and E189G
show partial activity and the individuals bearing
these have only marginal impairment [2647]
Several homology models have been proposed
[2476, 2648, 2649]
9.7.53.6 Inhibitors
Little has been done because impairment of this
enzyme is a clinical problem Some thiavitamin
D analogs have been evaluated in animal models
[2650]
9.7.53.7 Clinical Issues
The significance of the enzyme is due to the
pleiotropic actions of the active form of vitamin
D, 1α,25-dihydroxyvitamin D3, which include
regulation of calcium homeostasis, control of
bone cell differentiation, and modification of im-
mune responses [2651] The 1α-hydroxylation
reaction is rate limiting and hormonally con-
trolled The expression of the gene is usually
tightly regulated ( vide supra), but gene defects
are responsible for type I vitamin D-dependent
rickets [2652] At least 30 different variants are
known in patients [2624, 2653] Even the “mild”
phenotype of type I rickets is due to deficiency in
P450 27B1 [2654]
Cyp27b1-knockout mice have been char-
acterized and show a typical rickets phenotype
[2655] Another knockout mouse model showed
skeletal, reproductive, and immune dysfunction
[2656] Rickets was also observed in a condition-
al knockout model [2657]
Patients with severe renal insufficiency show
attenuated 1α-hydroxylation activity [2658]
Another aspect of P450 27B1 research in-
volves cancer Increased activity was reported in
parathyroid tumors [2659] Some splice variants
of the CYP27B1 gene (coding for truncated pro-
teins) were amplified in human (brain) gliomas
[2660] Reports have also appeared on the rela-
tionship of P450 27B1 expression to various bio-
666
logical processes in human non-small cell lung
carcinomas [2443], colon tumors [2661–2663],
and prostate cancers [2664, 2665], generally with
decreased expression in tumors
Finally, 1α,25-dihydroxyvitamin D3 is used
to treat psoriasis, and patients can develop re-
sistance An experimental model for therapy
involves enhancement of the endogenous pro-
duction of 1α 25-dihydroxyvitamin D3 by gene
therapy [2666]
The potential disease relevance of several
genetic variations has already been presented in
Sect. 7.53.3 ( vide supra) Since the previous edi-
tion of this chapter [149], vitamin D 25-hydroxy-
lase deficiency has been reviewed [2667, 2668]
Studies with CYP27B1-knockout mice have also
been reviewed [2669] Rickets (type I) still ap-
pears to be the most relevant issue [2649]
9.7.54 P450 27C1
9.7.54.1 Sites of Expression
P450 27C1 is expressed, at least at the mRNA
level, in liver and a number of other tissues, in-
cluding kidney, pancreas, lung, adrenal, salivary
gland, and more [2670] It is of interest to note
that rats and mice do not have this gene
The sequence identity to P450s 27A1 and
27B1 indicate that it should be a mitochondrial
P450, although direct evidence is not available
9.7.54.2 Regulation
No information is available about the regulation
of P450 27C1
9.7.54.3 Genetic Variation
No information has been published
9.7.54.4 Substrates and Reactions
The protein was expressed in E. coli using an
E. coli-optimized cDNA [2670] The purified
enzyme, reconstituted with recombinant adreno-
doxin and NADPH-adrenodoxin reductase, did
not catalyze the oxidation of cholesterol, vitamin
D3, 1α-hydroxyvitamin D3, or 25-hydroxyvita-
min D3 In other studies, none of a test set of pro-
carcinogens [350] was activated to a genotoxic
product
F. P. Guengerich
9.7.54.5 Structures
No structural information is available
9.7.54.6 Inhibitors
No inhibition results have been published
9.7.54.7 Clinical Issues
P450 27C1 was a high-frequency gene in an anal-
ysis of factors involved in avascular necrosis of
the femoral head [2671]
9.7.55 P450 39A1
9.7.55.1 Sites of Expression
Much of our knowledge of this enzyme is still
based on animal models Russell and his asso-
ciates used expression cloning to characterize a
cDNA coding for a 24-hydroxycholesterol 7α-
hydroxylase [2672] Expression in the liver ap-
pears constitutive and abundant Expression has
also been detected in the ciliary nonpigmented
epithelium of (bovine) eye [2673] Those studies
have not really been extended to humans [2674]
9.7.55.2 Regulation
Very little information is available One study
showed that carbamazepine, a barbiturate-like in-
ducer (using PXR and CAR), upregulated hepatic
P450 39A1 mRNA in patients [2675]
9.7.55.3 Genetic Variation
At least three variants have been report-
ed ( rs7761731 (N324K), rs93981468, and
rs953062) [2676, 2677]
9.7.55.4 Substrates and Reactions
All of our knowledge is still based on the
presumed similarity to the mouse enzymes
[2672] That expressed enzyme oxidizes (7α-
hydroxylation) 24S-hydroxycholesterol much
more efficiently than 25- or 27-hydroxycholes-
terol These results suggest that the enzyme is
highly selective for 24S-hydoxycholesterol (a
product of P450 39A1)
9.7.55.5 Structure
No structures or homology models have been re-
ported
9  Human Cytochrome P450 Enzymes
9.7.55.6 Inhibitors
No inhibitors have been reported
9.7.55.7 Clinical Issues
Interestingly, at least two reports associate CY-
P39A1 SNPs with changes in drug metabolism
[2676, 2677] However, in neither case was the
enzyme actually shown to be involved in the
metabolism, and one report [2676] has a caveat
about a possible artifact with a SNP for a trans-
porter It is possible that P450 39A1, like P450
46A1 ( vide infra), may oxidize drugs, but pres-
ently there is no other evidence for this
9.7.56 P450 46A1
9.7.56.1 Sites of Expression
P450 46A1 is characterized as a brain P450 It
was identified first (in mice) in a search for extra-
hepatic enzymes catalyzing the 24-hydroxylation
of cholesterol [2678, 2679] P450 46A1 is also
expressed in neurons of the neural retina [2680]
In humans, there is a lack of enzyme and prod-
uct (24-hydroxycholeseterol) in retina but not
in brain [2681] A heavy isotope (full protein)
method was utilized in quantitating P450 46A1
in human brain (temporal lobe) and retina (01–
04 pmol/mg tissue protein) [2682] None was
detected in retinal pigment epithelium
9.7.56.2 Regulation
One interesting aspect of P450 46A1 is the re-
ported learning disability in CYP46A1-knockout
mice [2679] Abnormal induction was reported in
glial cells of Alzheimer’s disease [2683] Tran-
scriptional regulation in brain involves Sp fac-
tors (Sp3, Sp4) [2684] Part of this process may
involve histone deacetylation [2685] Neuronal
differentiation alters the ratio of Sp transcription
factors required for the P450 46A1 promoter
Chromatin-modifying agents increase transcrip-
tion of P450 46A1, ie, the demethylating agent
5ʹ-aza-2ʹ-deoxycytidine induced P450 46A1,
acting synergistically with trichostatin A in ac-
tivating transcription Further work showed that
this reagent (azadeoxycytidine) induced gene
expression in a DNA methylation-independent
mechanism, decreasing Sp3/histone deacetylase
667
binding to the proximal promoter [2686] Oka-
daic acid has been reported to inhibit the tricho-
statin A-mediated expression of P450 46A1 in an
ERK1/2-Sp3-dependent pathway [2687]
9.7.56.3 Genetic Variation
Variations in the CYP46A1 gene have been of in-
terest in large part due to the possible relevance
to Alzheimer’s disease ( vide supra) [2388, 2683,
2688–2712] However, not all studies agree that a
relationship exists [2713–2716] A meta-analysis
has been published [2717] Genetic polymor-
phisms have been associated with age-related
macular degeneration [2718]
9.7.56.4 Substrates and Reactions
Cholesterol 24-hydroxylation is the main reac-
tion ascribed to P450 46A1 [2679] However,
several other sterols are also substrates, including
24( S)-hydroxycholesterol (25- and 27-hydroxyl-
ations), 7α-hydroxycholesterol, cholestanol, pro-
gesterone, and testosterone [2719] Some drugs
are also substrates [2719] 7-Dehydrocholesterol
has been reported not to be a substrate [2720], but
if not, then the source of 24-hydroxy-7-dehydro-
cholesterol is unclear Recently we have found
that recombinant human P450 46A1 catalyzes
the 24- and 25-hydroxylation of 7-dehydrocho-
lesterol and the 24S,25-epoxidation and 27-hy-
droxylation of desmosterol, with efficiencies
similar to cholesterol [2721]
Interestingly, P450 46A1 binds and oxidizes a
number of drugs [2719, 2722] This is an interest-
ing phenomenon in that most of the P450s that ap-
pear to be specialized for oxidation of endogenous
substrates do not use xenobiotics as substrates
The overall in vivo contribution of P450 46A1 to
the metabolism of these drugs, even in brain, is
unknown Further, P450 46A1 activity (towards
cholesterol) is stimulated by binding to some
drugs (eg, efavirenz, acetaminophen, mirtazap-
ine, galantamine), and the in vivo relevance of this
effect has been shown in a mouse model [2723]
9.7.56.5 Structure
X-ray crystal structures of P450 46A1 have been
reported in the absence and presence of the sub-
strate cholesterol 3-sulfate [2722, 2724] As with
668
many other P450s, there is a major conformation-
al change upon binding
Structures have also been reported with drugs
bound [2722, 2725, 2726] Some of these are
substrates
9.7.56.6 Inhibitors
Largely due to the studies that involved drug
binding to P450 46A1, a number of inhibitors
have been identified, including fluvoxamine
[1962, 2725, 2727] In a mouse model, in vivo
inhibition was reported [2727]
With P450 46A1, there appears to be no im-
petus to develop an inhibitor, in light of the is-
sues What is a more important issue is avoiding
inhibition, given the literature on the drugs that
do this Several azoles used in the clinic (eg,
posaconazole, voriconazole, clotrimazole) are
tightly bound [2726]
9.7.56.7 Clinical Issues
The major clinical issues are the possible genetic
links to Alzheimer’s disease ( vide supra) [2712,
2728–2730] and glaucoma [2708] Other issues
are related to brain injury [2731, 2732] P450
46A1 has also been considered in relation to dis-
ease manifestation of acute autoimmune enceph-
alomyelitis (actually the level of 24-hydroxycho-
lesterol) [2733]
9.7.57 P450 51A1
Lanosterol is an important intermediate in cho-
lesterol synthesis, and 14α-demethylation is es-
tablished as a step in the pathway Yoshida’s lab-
oratory had studied the yeast enzyme for many
years and then demonstrated the reaction in rat
liver microsomes in 1994 [2734] Subsequently
the reaction was also demonstrated in rat brain
microsomes [2735]
The literature associated with P450 51A1 is
largely devoted to the enzyme in parasites and
to developing approaches to inhibition to treat
diseases The information regarding the human
enzymes is more limited, although now there is
significant regulatory and structural information
F. P. Guengerich
9.7.57.1 Sites of Expression
Waterman and Rozman identified the human
CYP51A1 gene and two pseudogenes [2736]
mRNA blot analysis showed the highest levels in
testis, ovary, adrenal, prostate, liver, kidney, and
lung In mouse testis, P450 51A1 was localized in
both round and elongated spermatids [2737] The
enzyme is also found in (rodent) oocytes [2738]
9.7.57.2 Regulation
With regard to regulation of the human gene,
primer extension studies indicated predominant
transcription initiation sites in liver, lung, and
kidney, and placenta 250- and 249-bp upstream
from the translation start site and a second major
site at − 100  bp, with the absence of TATA and
CAAT patterns and a GC-rich sequence in the
promoter region [2736] Multiple (rat) testis-
specific transcripts arise from differential poly-
adenylation site usage [2739]
In human adrenocortical H295R cells (in cul-
ture), cholesterol deprivation led to a 26–38-
fold induction of P450 51A1 mRNA, which was
suppressed by the addition of 25-hydroxycho-
lesterol [2740] In the liver and other somatic
tissues, the P450 51A1 gene is regulated by a
sterol/SREBP-dependent pathway [2741] In tes-
tis, cAMP/cAMP-responsive element modulator
CREM1-dependent regulation predominates Sp1
functions to maximize the sterol regulatory path-
way of P450 51 [2742]
Insulin is an essential factor in “basal” ex-
pression of P450 51A1 in rat liver, with possible
involvement of SREBP-1c [2743] In a porcine
vascular endothelial cell model (and in arterial
wall), LDLs downregulated P450 51A1 through
an SREBP-2 mechanism [2744]
P450 51A1 has been identified as an early
response gene [2745] Hughes et al [1301] re-
ported that Dap1/PGRMC1 binds and regulates
mammalian P450 51A1 (and 3A4), based on
work in a yeast model This result was confirmed
for P450 51A1 in mammalian cell culture (HEK
293 and HepG2 cells) [1302]
9.7.57.3 Genetic Variation
The enzyme is also found in (rodent) oocytes
[2738]
9  Human Cytochrome P450 Enzymes
Rozman and her associates have analyzed ge-
netic variations and reported that the P450 51A1
gene contains fewer variants than any other
human P450 gene [2746, 2747] This may be re-
lated to the importance of this enzyme; ie, ab-
normalities might be lethal
9.7.57.4 Substrates and Reactions
Stimulation of human P450 51A1 activity by cy-
tochrome b5 in a reconstituted system has been
reported by Kelly’s laboratory [2748]
The normal mammalian substrate for P450 51A1
is lanosterol [2749], with the 14α-demethylation
process proceeding in what are assumed to be
three consecutive steps, as with some other P450s,
eg, 11A1, 17A1, 19A1 (Fig 922) Interestingly,
both human and yeast ( Candida albicans) P450
51A1 showed relatively little selectivity among a
closely related group of analogs [2749] It is also
interesting to note that even though this P450 has
a relativity defined role in a physiological process,
the kinetic parameters are relatively poor among
P450s ( kcat/Km 300 M− 1/s) [2749]
9.7.57.5 Structure
Three X-ray crystal structures of human P450
have been reported, ligand-free and with the anti-
fungal drugs ketoconazole and econazole [2750]
As observed with many other P450s, a substan-
tial conformational change in the enzyme occurs
on binding ligand In this case, the changes are in
B-helix and F–G loop regions Azole binding oc-
curs mainly through hydrophobic residues in the
active site Presumably, similar changes would
occur upon binding of the substrate lanosterol
9.7.57.6 Inhibitors
Most of the interest in inhibition has been with
fungal P450 51, as a target for antimycotic drugs
The goal is to select candidate drugs inhibitory to
fungal P450 51 but not human P450 51
Some work on the interaction of azoles with
human P450 51A1 has been published [2751]
Although human P450 51A1 has been suggested
as a target for cholesterol-lowering drugs, appar-
ently little has been done and potential toxicity
due to the steroidogenic and potential germ cell
side effects ( vide supra) could be an issue
669
A comparison of inhibitors of human and
Candida albicans P450 51A1 with a series of
azoles has been reported [2752], and some more
inhibitors have been considered regarding block-
ing cholesterol synthesis [2753]
9.7.57.7 Clinical Issues
Most of the work discussed here is from experi-
mental studies on the possible role of P450 51A1
in reproduction, and the translation of phenom-
ena from animal models to humans is still some-
what speculatory However, the very high level
of P450 51A1 expression in postmeiotic haploid
spermatids is striking The action of P450 51A1
is proposed to lead to the production of signaling
steroids in haploid germ cells [2754] Meiosis-
activating substances (MAS) are produced by
14-reduction of the products of the action of P450
51A1 on lanosterol [2754] Follicular fluid MAS
(FF-MAS) is formed from lanosterol in rat sper-
matids [2755] Yoshida’s group has reported go-
nadotropin-dependent expression of P450 51A1
in rat ovaries and the product of MAS [2756]
The reaction and possible physiological sig-
nificance of the system in reproduction have been
reviewed recently by Rozman [2757] Leidig
cells and acrosomes of spermatids have the high-
est P450 51A1 levels, and primary mouse oocytes
and granulose cells also contain P450 51A1
MAS may have a role in fertilization [2757]
As mentioned earlier, P450 51A1 deficiency
has been considered in the context of Antley–Bix-
ler syndrome [2758], and CYP51A1-knockout
mice show a resemblance to this syndrome [2759]
9.8 Conclusions and Future Issues
with Human P450
Having just celebrated the 50th anniversary of
the discovery of P450 [2760], one can look back
on the success in the area of P450s and medicine
The attack on the human P450s did not start in
earnest until 20 years after the original discov-
ery of P450 but has proven to be a remarkable
success story in the translation of discoveries in
basic science Today we are generally capable of
understanding most aspects of (oxidative) drug
670
metabolism and even predicting it based on in
vitro experiments Medicinal chemists have logi-
cal paths to improve human pharmacokinetics
Steroid metabolism can largely be understood at
a genetic level, even if the basis for loss of func-
tion in each case is not yet known
Pharmacists can avoid many adverse drug–
drug interactions based on knowledge—or elec-
tronic histories—of P450 selectivities
Having accomplished all of this, what does
the future hold for P450 research? Clearly, there
are still many basic questions, many of which
are general questions about P450 function For
instance, the role of cytochrome b5 in the P450
17A1 17,20-lyase reaction is not solved More
than half of the human P450s do not have crystal
structures, and some of these will be problematic
until more substrates are discovered Following
are four of the more translational areas in which
P450 research can be applied and is needed
9.8.1 Orphan P450 and Their
Reactions
As pointed out in Table 91 and the description
of individual P450s, relatively little information
is still available about this group of the human
P450s Some substrates are being found, both
endogenous and xenobiotic, but some of these
P450s still have no real substrate at all (eg,
20A1) There is no compelling evidence that
these P450s make major contributions to the me-
tabolism of many drugs (with some occasional
exceptions [1214]) Some interesting reactions
with endogenous compounds have been identi-
fied, but exactly what their physiological impor-
tance is remains unknown The approaches in this
area are difficult, but the annotation of functions
of genes is probably one of the most important
aspects of biochemistry and biology
9.8.2 Pharmacogenetics and Pharma-
cogenomics
Advances in recombinant DNA technology, in-
cluding the completion of the Human Genome
Project, have made it possible to rapidly map and
F. P. Guengerich
screen for SNVs in individuals Knowledge of
P450 SNVs was seen as a major aspect of “per-
sonalized medicine” [2761] However, the de-
velopment of this area has been somewhat slow
since 2000, when “personalized medicine” was
touted as “just around the corner” At this time,
there is still no drug on the market for which the
US Food and Drug Administration or similar
agencies in other countries require genetic tests
[782] More recently, some hospitals and medical
centers are doing some SNV analysis for a few
drugs As an example, at the author’s own institu-
tion (Vanderbilt), CYP2C19 analysis is used with
administration of clopidogrel, in order to predict
which patients will respond [782, 848–854]
More tests like this will probably follow in the
future (Of course, genotype analysis is already
extensively used in drug discovery and develop-
ment, mainly to avoid drug candidates that might
show highly variable pharmacokinetics)
The major issues in the implementation are
added costs (although one can argue that SNV
analysis is a cost-effective investment and could
reduce hospital stays) and the limited number
of proven successes The author’s own opinion
is that there will be more progress, particularly
with drugs used in oncology, but exactly how fast
the field develops is still a matter of speculation
9.8.3 P450s and Cancer Susceptibility
As mentioned previously, carcinogen metabo-
lism has been one of the drivers for the P450
field There is strong evidence that the P450 com-
position can strongly influence chemical carcino-
genesis in experimental animal models [2762],
reinforced with transgenic mouse models [1441]
Nevertheless, there is still relatively lim-
ited evidence that variations in P450 influence
cancer risk in humans As mentioned earlier
(Sect 727), high P450 1A2 levels can influence
colon cancer risk, but only when coupled with an
N-acetyltransferase polymorphism and high in-
take of well-done meat [132] Although a number
of P450–cancer relationships have been proposed
[108, 109, 123, 1079–1087, 2763, 2764], the evi-
dence is still limited The difficulty in establish-
ing relationships results from the lack of defined
9  Human Cytochrome P450 Enzymes
exposure data available in most cases and the
long time period needed to develop cancer At the
molecular level, many P450s not only activate
carcinogens but also detoxicate them as well,
eg, aflatoxin B1 [1331] (Figs 99 and 910)
One issue is that epidemiology studies are
often initiated in the absence of any information
regarding relevant substrates and reactions, and
(not surprisingly) weak associations are found
and are difficult to repeat Clearly, more innova-
tive approaches are needed to address the issues
9.8.4 P450 and Chronic Diseases
In addition to cancer (and endocrine and drug in-
teractions), there is evidence that human P450s
can influence chronic diseases (Table 93) Sev-
eral P450s, including those in the 2C, 2J, 4A,
and 4F subfamilies, have been proposed to be in-
volved in hypertension, as judged by both basic
models and epidemiological studies [2765] Epi-
demiological studies may suggest a role of P450
2D6 in Parkinson’s disease [953], although the
relationship probably has limited support
Although a degree of skepticism is necessary
when considering translational reports regard-
ing P450, what we know about the P450s does
argue that we still have important relationships to
discover Several of products of the sterol-metab-
olizing enzymes are generally powerful biologi-
cal mediators (eg, oxysterols that are ligands of
nuclear receptors [1715, 1716, 1749]) As in the
case of cancer, the differences in life span etc
may be subtle and difficult to detect Again, new
strategies are needed in this area
My discussion of research needs in the P450
field has been restricted here to the translational
aspects of human P450 research For more con-
sideration of the present state and future direction
of P450 research in general, see [2760]
9.9 Some Final Thoughts
Reviewing the progress in research on human
P450s in the past 10 years is exciting but also
humbling, in that even what I consider to be a pro-
ductive laboratory of my own contributed < 1 %
671
of that literature After dealing with all 57 human
P450 genes (yielding about the same number of
proteins, ie, see P450s 2A7, 4F3), a few major
“take-home” messages can be summarized:
We still have more to learn about some chemi-
cal mechanisms (eg, C-C cleavage), even some
that have been thought to be firmly established
The number of X-ray crystal structures of
human P450s is rather amazing (Table 91)
Nothing seems impossible for the next 10 years
There is a problem in that some P450s do not
have substrates yet, and the usefulness of an unli-
ganded P450 structure is limited
The regulation of many of the P450 genes
is quite complex Finding one regulatory factor
leads to another
The “orphan” P450s (those without estab-
lished function) (Table 91, see also [149]) are
falling into categories, at least in terms of some
of the reactions that they can do Some of those
were previously in the “orphans” category [149]
now have as much information as some already
in the xenobiotics or fatty acids categories
Regarding tissue distribution, the information
can be rather variable, even comparing mRNA
studies to each other and immunoblotting or
LC–MS results with each other The distributions
may not reflect where the most physiologically
important site(s) of expression are
Catalytic efficiency (ie, kcat/Km) is not a reli-
able guide to the biological importance of P450s
Even among the bacterial P450s, most are rela-
tively slow, and only few well-studied reactions
have high rates, eg, P450 101A1 and 102A1,
with camphor and fatty acid oxidations, respec-
tively Of these, the function of P450 102A1 is
still unclear Among the mammalian P450s using
redox partners (ie, excluding P450s 5A1 and
8A1), P450s 7A1 and 21A2 appear to be the most
efficient, kcat/Km ~25 × 106 M− 1/s [1775] and
~107 M− 1/s, respectively Several of the mamma-
lian P450s have much lower catalytic efficiencies
but are clearly shown to be important in genetic
studies For instance, P450 46A1 has a kcat/Km of
500 M− 1/s (with cholesterol as substrate [2721,
2766]), but Cyp46a1(− /− ) mice have a deficient
memory phenotype [2679] Effects of drugs on
drug-metabolizing P450s can yield serious drug–
drug interactions
672
Some of the P450s involved in the metabo-
lism of endogenous substrates are proving to be
less selective than originally thought, eg, P459s
7A1, 11A1, 46A1
Following up on point vii, we are seeing more
overlap between oxidations of the endogenous
and xenobiotic substrates (eg, see the Substrates
and Reactions section for P450s 1B1, 11A1,
and 46A1) Of course, drugs can be made to tar-
get P450s that use endogenous substrates (eg,
P450 19A1 and numerous others) There are is-
sues in that preclinical drug candidate decisions
may need to involve not only predictions of drug
interactions with the major drug-metabolizing
P450s (Fig 91b) but also the P450s involved in
oxidation of endogenous substrates, eg, see ex-
ample with P450 11A1 [1951]
Acknowledgments  P450 work in the author’s laboratory
is supported by US Public Health Service (National Insti-
tutes of Health) grants R37 CA090426, R01 GM103937,
and P01 DK038226 Thanks are extended to individuals
who provided unpublished results, to Profs M R Water-
man and E M J Gillam for comments, and to present and
past members of the author’s laboratory for their involve-
ment in some of the original research This chapter is
dedicated to my postdoctoral mentor, Prof M J Coon,
who introduced me to these remarkable enzymes more
than 40 years ago In all honesty, I had not anticipated
working on them for the rest of my career I also particu-
larly thank K Trisler for her assistance in the preparation
of this manuscript
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