Compiled and edited by
Ari Hirvonen
State of the art of genotype
vs. phenotype studies
potential of diet to prevent cancer, and of the ways by which heredity can affect individual suscepti-bility to carcinogens, with the ultimate aim of reducing the cancer burden in Europe.
ECNIS is coordinated by Prof. Konrad Rydzyński, The Nofer Institute of Occupational Medicine, św. Teresy 8, 91‑348 Łódź, Poland
This review has been prepared as part of ECNIS Work Package 7: Development and Validation of Biomarkers of Individual Susceptibility.
© ECNIS 2008
All rights reserved. No part of this book may be reproduced in any form without permission of the publisher.
Compiled and edited by Ari Hirvonen Finnish Institute of Occupational Health Topeliuksenkatu 41 a A FI-00250 Helsinki FINLAND Tel: +359 30 474 2204 Fax: +358 30 474 2110 ISBN 978‑83‑60818‑13‑8 ISSN 1899‑2692
Technical editor: Katarzyna Rogowska Cover design, layout: Beata Grabska
Computer typesetting: pery plate studio – Monika Popielata
Published by The Nofer Institute of Occupational Medicine Św. Teresy 8, 91‑348 Łódź, Poland
Tel.: +48 (0) 42 631 45 04 Fax: +48 (0) 42 656 83 31 E-mail: ecnis@ecnis.pl Website: http://www.ecnis.org
Executive summary . . . 5
1. Introduction Ari Hirvonen, Roger Godschalk, Micheline Kirsch-Volders . . . 7
2. Phase I enzyme genotypes and their toxicologically relevant phenotypes Roger Godschalk, Frederik Jan van Schooten . . . 11
2.1. Alcohol and aldehyde dehydrogenases . . . 11
2.2. Aryl hydrocarbon receptor . . . 13
2.3. Cytochrome P450’s . . . 14 2.3.1. CYP1A1 . . . 15 2.3.2. CYP1A2 . . . 17 2.3.3. CYP1B1 . . . 19 2.3.4. CYP2A6 . . . 21 2.3.5. CYP2C . . . 23 2.3.6. CYP2D6 . . . 24 2.3.7. CYP2E1 . . . 25 2.3.8. CYP3A4 . . . 27 2.4. Epoxide hydrolase . . . 28 2.5. Myeloperoxidase . . . 30 2.6. NADPH‑quinone oxidoreductase . . . 31
3. Phase II enzyme genotypes and their toxicologically relevant phenotypes Ari Hirvonen . . . 47 3.1. Catechol O-methyltransferase . . . 48 3.2. Glutathione S-transferases . . . 49 3.2.1. GSTM1 . . . 49 3.2.2. GSTM3 . . . 50 3.2.3. GSTP1 . . . 50 3.2.4. GSTT1 . . . 51
3.3.2. NAT2 . . . 54
3.4. Manganese superoxide dismutase . . . 55
3.5. Sulfotransferases . . . 57
4. DNA repair enzyme genotypes and their toxicologically relevant phenotypes Peter Aka, Raluca Mateuca, Micheline Kirsch-Volders . . . 71
4.1. 8‑oxoguanine‑DNA glycosylase . . . 71
4.2. X‑ray repair cross‑complementing group 1 . . . 74
4.3. X‑ray repair cross‑complementing group 3 . . . 76
4.4. Excision repair cross‑complementing group 2 . . . 79
5. Mendelian randomization Paul Brennan, Rayjean Hung . . . 91
6. Conclusions Ari Hirvonen . . . 95
The purpose of this review is to summarise the current situation regarding the genotype‑pheno-type studies of the phase I and phase II xenobiotic metabolizing enzymes and DNA repair enzymes of potential relevance in individual susceptibility to environmentally-induced cancer.
Altered function of low penetrance genes due to single nucleotide polymorphisms may affect the gene-environment and gene-gene interaction, thereby increasing the risk of cancer development. The balance between phase I and II enzymes, e.g., is an important determinant of whether expo-sure to carcinogens will result in toxicity or increased levels of genetic damage. Phase I enzymes are generally involved in the metabolism of xenobiotic compounds to facilitate their excretion via urine or faeces. However, phase I metabolism may also result in the metabolic activation of carcinogenic compounds, and as a result reactive derivatives are formed that can covalently interact with cellular macromolecules, like DNA. If these so‑called DNA adducts are not properly or timely repaired by the DNA repair enzymes, they may be converted into DNA mutations that are thought to initiate the process of carcinogenesis.
It is believed that large scale genotyping of samples from cancer patients compared to normal, healthy individuals with similar exposures, will lead to important breakthroughs in understanding gene-environment and gene-gene interactions as mechanistic basis for the common polygenic sporadic cancers. This has led to a huge amount of work, in attempt to identify individuals with increased sus-ceptibility to develop cancer on basis of their genetic background. However, outcomes of many of the studies have been disappointing; no clear associations between certain genotypes and disease have been seen. There are many possible reasons for this lack of associations. One of the most probable explanations is that often it is not clear whether a genetic variant actually had a phenotypic effect. Since it is the gene that eventually dictates how the enzyme is expressed, even under the most com-plicated influence of endogenous and exogenous factors, it should always be possible to understand the relationship between the genotype and the phenotype. For this, however, a complete understanding of the gene regulation is necessary. Based on this review it seems clear that except for few cases we are at the moment still far from this goal.
With the sequencing of the human genome, it is evident that about 99.9% of the DNA is
identical in every human genome [1–3]. The 0.1% difference is responsible for the inter‑
individual variation and the unique phenotype of each individual. These minor genetic
variations, seen as single base changes in the genome are known as single nucleotide
poly-morphisms (SNPs). SNPs play an important role in promoting susceptibility to diseases
as well as in the individual response to various drugs and environmental carcinogens [4].
Based on a systematic survey of SNPs in the coding regions of human genes, about 50%
of the SNPs result in a change in the amino acid sequence [5].
Altered function of low penetrance genes due to SNPs may affect the gene-environment
and gene‑gene interaction, thereby increasing the risk of cancer development. The balance
between phase I and II enzymes, e.g., is an important determinant of whether exposure
to carcinogens will result in toxicity or increased levels of genetic damage. Phase I enzymes
are generally involved in the metabolism of xenobiotic compounds to facilitate their
excre-tion via urine or faeces. However, phase I metabolism may also result in the metabolic
acti-vation of carcinogenic compounds, and as a result reactive derivatives are formed that can
covalently interact with cellular macromolecules, like DNA. If these so‑called DNA adducts
are not properly or timely repaired by the DNA repair enzymes, they may be converted into
DNA mutations that are thought to initiate the process of carcinogenesis.
It is believed that large scale genotyping of samples from cancer patients compared to
normal, healthy individuals with similar exposures, will lead to important breakthroughs
in understanding gene-environment and gene-gene interactions as mechanistic basis for the
common polygenic sporadic cancers. There is a large volume of literature available where
polymorphisms in low penetrance genes and environmental factors, especially life styles,
have been associated with increased risk of different cancers [6–8]. Subtle changes in the
phenotype, derived from SNPs in the respective genes are increasingly becoming considered
as cancer susceptibility factors [9].
Current molecular biological techniques enable relatively easy discovery and
characteriza-tion of new variant alleles and genetic changes. It’s also much easier to conduct the
straight-forward genetic epidemiological studies, than the more cumbersome studies addressing the
basic question as to how the genotype is determining the phenotype and, whether there is
any biologically plausible link to be expected between the genotypic differences and cancer
susceptibility. Eventually, however, knowledge of the complete sequence of events, from
the gene to the outcome, is anticipated to be needed to confidently see the implications
and possible preventive and treatment strategies to be employed in those cases, where clear
association between low penetrance genes and cancer susceptibility has been uncovered.
Ari Hirvonen
1, Roger Godschalk
2, and Micheline Kirsch-Volders
3 1 Finnish Institute of Occupational Health, Helsinki, Finland2 Maastricht University, Maastricht, The Netherlands
The discovery of many SNPs in genes involved in the metabolic (de)activation of
carcin-ogens has led to a huge amount of work, in attempt to identify individuals with increased
susceptibility to develop cancer on basis of their genetic background. However, outcomes
of many of the studies have been disappointing; no clear associations between certain
genotypes and disease have been seen. There are many possible reasons for this lack of
as-sociations. One of the most probable explanations is that often it is not clear whether a
ge-netic variant actually had a phenotypic effect. Theoretically, phenotypic assays would give
a better indication of the inter-individual differences in enzyme activity. Nonetheless,
geno-typing is often applied because it is quick, easy to perform and does not require probe drugs
with sampling of urine or blood at defined times (Table 1.1). Therefore, it is of importance
to further investigate the genotype-phenotype relationships to increase our knowledge on
the genetic basis of increased susceptibility to genotoxic compounds.
Phenotypic assays Genotyping
Advantages Gives indication of actual enzyme activity Simple and quick assays Historically well studied High throughput analysis is possible
May also identify heterozygous subjects
Most suitable to population and field studies
Disadvantages Requires probe drugs and collection of samples Requires genetic material with ethical complications at defined times Assumes genetic variants have functional effects Larger measurement error Ethnic differences in variant frequencies and types Effect-cause bias; disease may alter phenotype will complicate interpretation
Modulating factors, such as diet and smoking
behaviour
One of the most challenging problems in the characterization of the human xenobiotic
metabolism phenotype stems from the fact that most of the xenobiotic metabolizing
en-zymes (XMEs) are expressed in a tissue specific manner leading to great differences between
tissues in the activation and inactivation of carcinogens. Therefore the phenotype of interest
should, in principal, be determined in the tissue of interest. Because of the limited
availabil-ity of human samples, this may, however, be possible only in rare cases, e.g., occasionally
in relation to surgery, and it is thus difficult to design systematic studies on this topic.
The above mentioned problems have lead to various attempts in trying to estimate
the enzymatic activities in tissues of interest otherwise. One approach is to use indirect
measurements, such as surrogate tissues or probe drugs in vivo to extrapolate the activity
in the tissue of interest. Ideally, a surrogate tissue should represent the target tissue in such
a way that the behaviour of a carcinogen, which is of importance for the final outcome,
i.e., manifest cancer, is faithfully reproduced in (or is in a meaningful correlation with) the
surrogate tissue. Unfortunately, in most instances, this prerequisite for the use of surrogate
Table 1.1. Advantages and disadvantages of phenotypic and genotypic assays to predict inter- individual differences in enzyme activity
tissue cannot be addressed, simply because we often have only limited information about
the expression of relevant enzymes in the target tissue.
It is thus obvious that ideal target tissues are difficult to obtain. Nevertheless, because
of the difficulties in obtaining human material, surrogate tissues have been chosen for many
studies in a more or less opportunistic way by using “whichever is available”. Therefore for
example blood lymphocytes, hair follicles, surface epithelia from skin or buccal mucosa
or “surplus” tissue from surgery have been used. When using these tissues one should
always keep in mind that they may not properly reflect the activities in the target tissue
and, e.g., in ex vivo regulation studies their inducibility may be different from the target
tissue. In any case the human tissue samples are extremely valuable for basic studies when
one wants to obtain information on the tissue specificity, catalytic properties and mode
of regulation of human XMEs.
The purpose of this review is to summarise the current situation regarding the genotype‑
phenotype studies of the phase I and phase II XMEs, and DNA repair enzymes of potential
relevance in individual susceptibility to environmentally-induced cancer. Functional effects
of certain genotypes can of course be studied directly by assessing the actual enzyme
activ-ity of the variants. However, indirect assessment of phenotypic effects can also be obtained
by studying their effects on later end‑points like DNA damage and cancer risk. These
end-points are therefore also discussed when appropriate.
References
1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001;409:860–921.
2. Marth G, Yeh R, Minton M, Donaldson R, Li Q, Duan S, et al. Single‑nucleotide polymorphisms in the public domain: how useful are they? Nat Genet 2001;27:371–2.
3. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the hu-man genome. Science 2001;291:1304–51.
4. Hemminki K, Shields PG. Skilled use of DNA polymorphisms as a tool for polygenic cancers. Carcinogenesis 2002;23:379–80.
5. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, et al. Characterization of single‑ nucleotide polymorphisms in coding regions of human genes. Nat Genet 1999;22:231–8. 6. Fearon ER. Human cancer syndromes: clues to the origin and nature of cancer. Science
1997;278:1043–50.
7. Potter JD. Colorectal cancer: molecules and populations. J Natl Cancer Inst 1999;91:916–32. 8. Goode EL, Ulrich CM, Potter JD. Polymorphisms in DNA repair genes and associations with
cancer risk. Cancer Epidemiol Biomarkers Prev 2002;11:1513–30.
9. Evans MD, Cooke MS. Factors contributing to the outcome of oxidative damage to nucleic acids. Bioessays 2004;26:533–42.
and their toxicologically relevant
phenotypes
Roger Godschalk, and Frederik Jan van Schooten
Maastricht University, Maastricht, The Netherlands
In this chapter, the phenotype-genotype relationships for the following enzymes will
be discussed (in alphabetical order), selected on basis of their relevance for the metabolism
of xenobiotics:
1. Alcohol and aldehyde dehydrogenases (ADH2, ADH3 and ALDH2).
2. Aryl hydrocarbon receptor (AHR).
3. Cytochrome P450 isoenzymes (CYP1A1, 1A2, 1B1, 2A6, 2C, 2D6, 2E1 and 3A4).
4. Microsomal epoxide hydrolase (EPHX1).
5. Myeloperoxidase (MPO).
6. NADPH‑quinone oxidoreductase (NQO1).
It should be noted that the relationships between polymorphisms in individual phase I
enzymes and cancer incidence at various sites are far from being clearly established, and
firm conclusions are difficult to reach in view of the large number of contradictory
pub-lications. These contradictions can at least partly be explained by the use of genotyping
instead of phenotyping.
2.1. Alcohol and aldehyde dehydrogenases
Alcohol dehydrogenases (ADHs) form a group of dehydrogenase enzymes that facilitate the
conversion of alcohols to aldehydes or ketones, and they are predominantly located in the
lining of the stomach and in the liver [1]. After the oxidation of ethanol to acetaldehyde
(note that acetaldehyde is even more toxic than ethanol), acetaldehyde is quickly converted
to acetic acid and other less toxic compounds by, e.g., cytochrome P450 2E1 (CYP2E1) and
aldehyde dehydrogenase (ALDH). In fact, the first gene shown to affect the biological
ac-tions of alcohol was ALDH2 [1]. Deficiency of the ALDH2 enzyme, common in Asian
populations, results in a strong phenotype; the inhibited acetaldehyde metabolism results
in elevated blood acetaldehyde levels, making alcohol drinking less pleasant, which will
protect the individual from high consumption and alcoholism. As a result, virtually no
cas-es of alcoholism have been reported in individuals who are homozygous for the ALDH2*2
allele (about 5% to 10% of the entire Asian population). The heterozygous individuals (30%
to 40% of the entire Asian population) can drink but still develop a number of less intense
side effects. These individuals consume less alcohol than the carriers of the ALDH2*1/*1
genotype, but they may still develop alcoholism [1].
In humans, seven ADH genes have been mapped to chromosome 4, but relevant
poly-morphisms have been found only for ADH2 and ADH3 [2]. The effect on kinetic
param-eters was much stronger for ADH2 isoenzymes than for the ADH3 isoenzymes. For
instance, the maximum reaction rate (V
max) of ADH2*2 homodimers is around 40 times
that of ADH2*1 homodimers, whereas the V
maxof ADH3*1 homodimers is double that
of ADH3*2 homodimers. The role of the ADH polymorphisms in the adverse actions
of alcohol was first suggested by Stamatoyannopoulus et al. [3]. Subsequently, Shibuya
et al. [4] indeed observed a positive association between flushing and the prevalence
of the ADH2*2 allele. Later, similar associations have been reported [5,6]. In addition,
a number of studies have found that the ADH2*2 allele protects against alcohol abuse
and alcoholism in both Asian [7–10] and white populations where the prevalence is much
lower [11–13].
The ADH3*1 allele, which encodes for a more active enzyme, has also been associated
with reduced risk for alcohol dependence in Asians [9,10,14]. However, these rather weak
associations have been explained by linkage disequilibrium between ADH2 and ADH3
[8,11,15].
DNA damage and other biomarkers
Breakdown of ethanol produces acetaldehyde, which is able to induce chromosomal
aberrations (CAs),
micronuclei (MN) and sister chromatid exchanges (SCEs) in cultured
mammalian
cells [16]. Additionally, acetaldehyde can covalently interact with DNA to
form
DNA adducts, which may initiate the
multistage process of carcinogenesis. The
formation
of N
2‑ethyl‑2’‑deoxyguanosine, one major stable acetaldehyde–DNA
adduct
[17], was detected in DNA from the liver of ethanol‑treated
mice [18]. Levels of
acetal-dehyde DNA adducts in white blood cells
were much higher in alcohol abusers than the
corresponding levels in healthy
control individuals [19]. Although some studies show
that alcohol abuse may lead to increased levels of DNA damage, little work has been
done on the impact of ADH and ALDH gene polymorphisms on these levels, and more
work should be done. In vitro experiments have already shown that
lymphocytes from
habitual drinkers with the inactive form of
ALDH2, which cannot efficiently detoxify
acetaldehyde, have
higher SCE frequencies than lymphocytes
from individuals with
ac-tive ALDH2 [20].
Cancer risk
Alcoholic beverages are causally related to cancer of the oral cavity, pharynx, larynx and
esophagus. The responsible compound is probably acetaldehyde (formed by ADH and
re-moved by ALDH), which is an established carcinogen in experimental animals and can
interact with DNA. Case‑control studies of the effects of ALDH2, ADH2, and ADH3
genotypes consistently showed positive associations between the inactive heterozygous
in Asian heavy drinkers; this genetic vulnerability may also be present in light-to-moderate
drinkers [1,21]. Some studies suggest similar associations with the risk for head and neck
cancer [1]. This is somewhat surprising; one would expect that the highest risk should
be observed in populations with high activity associated ADH2 genotypes, because they
would form more acetaldehyde out of the ethanol. However, the explanation for this
discrepancy may be found in the differences in drinking behaviour between subjects with
the ADH2*2 allele (more adverse effects of alcohol drinking) as compared to those carrying
the ADH2*1 alleles.
Whether the ADH3 genotype influences head and neck cancer risk is still controversial.
Associations are stronger in Asian populations than in Caucasians, which can partly be
explained by differences in allele frequencies [1,21].
Conclusions
Overall, it can be concluded that variant alleles of ALDH2 and ADH2 have substantial
effects on enzyme activity, and may affect cancer risks, especially in alcohol abusers. Their
role in the modulation of DNA damage (as early cancer end‑point) is less well studied and
deserves further attention. The phenotypic effect of ADH3 is weak and could be due to
linkage disequilibrium with ADH2.
2.2. Aryl hydrocarbon receptor
The aryl hydrocarbon receptor (AHR) mediates many of the toxic responses induced by
polyhalogenated (dioxins) and polycyclic aromatic hydrocarbons (PAHs), which are
ubiq-uitous environmental contaminants. The AHR is a ligand‑activated transcription factor
involved in the regulation of several genes, including those encoding for XMEs
belong-ing to CYP1A and 1B families (see next section) [22]. Some variation in the induction
of CYP’s in humans is likely to be due to genetically based variations in the AHR
struc-ture. The functional effect of these genetic polymorphisms has been described in a
com-prehensive review by Harper et al. [22]. Human AHR polymorphisms include variants
at −459G>A in the 5’‑untranslated region [23], at codons 44 (132T>C), 517 (1549C>T)
[24], 554 (1661G>A) [25], and 570 (1708G>A) [24,26]. In rodent models, genetic
varia-tion in the AHR had strong phenotypic effects on responses to dioxins and PAH’s [27,28],
whereas in humans, the phenotypic effect of known genetic variants is less clear, also
because of linkage disequilibrium.
The most widely used method to assess AHR‑related phenotypes in human subjects and
populations has been to prepare cultures of peripheral blood lymphocytes, then test
induc-tion of CYP1A1 after treating the cells with AHR‑ligands such as benz[a]anthracene. An
alternative phenotyping approach involves comparing AHR-mediated responses in groups
of people who have different AHR genotypes. In circumstances where human tissues can
be harvested, e.g., lung from surgical resections [29], the amounts of AHR‑regulated gene
products have been measured at the level of mRNA, immunoreactive protein and CYP1A1
catalytic activity. Outcomes of such population studies are complex to interpret since many
factors that might influence the level of expression of AHR-target genes other than the
AHR will differ between the study populations.
The −459G>A variant in the 5’‑untranslated region of the AHR gene was found to be
unrelated to CYP1A1 induction in human lymphocyte cultures [23]. Similarly, the
poly-morphism at codon 44 was not found to affect the CYP1A1 expression in lungs of smokers
[29]. The polymorphism at codon 517 occurred only in combination with variant alleles
at codons 554 and 570 [24]. These alleles appear confined to persons of African descent
and occur with very low frequency. When the combination of 554 and 570 variant
al-leles or a 554, 570 and 517 variant allele haplotype were expressed in an artificial model
of Hepa‑1 Group B mutant cells, each failed to support TCDD‑induced CYP1A1 induction
[24]. There was no apparent effect of the codon 570 polymorphism by itself on CYP1A1
expression [26]. Moreover, the phenotypic impact of the codon 554 polymorphism
is not yet clear. Although the 3‑methylcholanthrene (3‑MC) induced CYP1A1 activity
was increased in peripheral blood lymphocytes [26], other studies showed no
associa-tion for i) 3‑MC induced aryl hydrocarbon hydroxylase activity [25], ii) CYP1A1 mRNA
expression, protein concentration and enzymatic activity in lung tissue (8), and iii)
benz[a]anthracene induced CYP1A1 activity in peripheral blood lymphocytes [23].
Conclusions
Overall, it can be concluded that human AHR polymorphisms have no substantial effects
on cellular responses to AHR-ligands and none of the polymorphisms have been found to
play a key role in the CYP1A1 inducibility or in the susceptibility to develop lung cancer.
2.3. Cytochrome P450’s
The CYPs are a large group of monooxygenase enzymes responsible for the metabolism
of toxic hydrocarbons; NADPH is required as a coenzyme and O
2is used as a
sub-strate. The CYP’s are located in the endoplasmic reticulum and are highly concentrated
in the liver. They are also found in the mitochondrial membrane. CYPs encompass
a highly diverse “superfamily” of hemoproteins, and one of their most relevant functions
is that of metabolizing drugs in humans. Over 60 key forms are known, with hundreds
of genetic variations, producing a wide variety of susceptibility to specific toxins.
In the present report, we will focus on the CYP‑isoforms mentioned in Table 2.1.
It needs to be noted that although the CYP‑phenotype is also highly dependent on
factors other than the CYP‑genotype, which may influence the actual CYP‑activities
(for instance smoking, diet or use of medication), these factors are often not taken into
account and may attenuate the actual genotype-phenotype relationships. It is also
im-portant to note, that historically CYPs have been associated with the metabolic
acti-vation of pre‑carcinogens. However, more recent work with cytochrome P450 knock‑
out mice have indicated that CYPs could as well be involved in the detoxification
of carcinogens [30], which may further complicate the interpretation of data on genetic
polymorphisms in CYPs. Furthermore, some CYPs have been reported to contribute
to the metabolism of some compounds in vitro, but they have a relatively high K
mvalue
for that particular compound, and are therefore unlikely to be relevant as principal
catalysts to the usual levels of carcinogen exposure.
Table 2.1. CYP’s in human liver, their variability and important examples of substrates
1A1 1A2 1B1 2A6 2C 2D6 2E1 3A CYP-isoform Relative presence in liver (%) Extent of variability in enzyme activity Factors influencing expression levels and activity Examples of substrates relevant for ECNIS
Presence in other tissues than liver < 1 8–15 < 1 5–12 ~ 18 2 7–11 30–40 Inducible Inducible Inducible 30–100 fold 25–100 fold > 1 000 fold 20 fold 20 fold Nutrition Smoking Drugs/carcinogens Nutrition Smoking Drugs/carcinogens Nutrition Smoking Drugs/ carcinogens Drugs/carcinogens Drugs/carcinogens Drugs/carcinogens Nutrition Alcohol Carcinogen exposures Nutrition Smoking Drugs/carcinogens PAH Aromatic amines Aflatoxins PAH Aromatic amines Nicotine Nitrosamines PAH Clinical drugs Alcohol Acrylamide Nitrosamines Broad substrate spectrum, incl. PAH and aflatoxins Lung, Kidney GI tract, Skin, Placenta Mainly located in liver Skin, Kidney, Prostate, Mammary gland Lung, Nasal membrane GI tract, Larynx, Lung Intestine, Kidney, Brain Lung, placenta GI-tract, Lung, Placenta, Foetus, Uterus, Kidney
2.3.1. CYP1A1
CYP1A1 is associated with the activation of procarcinogens such as PAHs. The CYP1A1
activity can be assessed by measuring the O‑deethylation of ethoxyresorufin (EROD‑
activity). Studies in which benz[a]anthracene‑induced EROD activity was measured in
hu-man lymphocytes, indicated that the data are compatible with a bimodal distribution [31].
This could hypothetically be due to genetic variation of AHR. However, since phenotypic
effects of the known AHR-polymorphisms are small, it is likely that genetic polymorphisms
in CYP1A1 gene itself play a more crucial role in this context.
To date several polymorphisms in CYP1A1 gene have been described the most studied
ones being the 6235T→C (in CYP1A1*2A allele) and 4889A→G (in CYP1A1*2B allele) SNPs.
The other CYP1A1 variants including the 5639T→C (in CYP1A1*3 allele) and 4887C→A
(in CYP1A1*4 allele) SNPs have been much less studied [32]. The CYP1A1*2A and *2B alleles
are in linkage disequilibrium, but are not strictly linked in all subjects.
Functional effects
Already in 1990, the high‑CYP1A1‑inducibility phenotype was shown to segregate with
the CYP1A1*2A allele. This allele was also experimentally related with increased catalytic
activity and a higher inducibility by 2,3,7,8‑tetrachlorodibenzo‑para‑dioxin (TCDD) [33].
However, the CYP1A1*2A allele did not affect the mRNA expression and more recent
studies have not found any phenotypic consequences for it. Moreover, no apparent
molecular mechanism exist for the effect of the homozygous CYP1A1*2A/*2A genotype
on gene expression, since the SNP lies outside the promoter or other regulatory regions
of the gene [32,34].
The CYP1A1 gene inducibility was found to be higher for the CYP1A1*2B allele as
com-pared to the wild‑type allele [35]. Moreover, the 4889A→G SNP is located in the
heme-binding region and is expected to result in a changed enzyme activity. However, this
poly-morphism did not consistently affect enzyme activity in in vitro studies; both increased
activity [36,37] and no effect [38,39] have been reported. Thus, the functional effect of
the CYP1A1*2B variant allele is still not completely clear.
Because of the position of the African American‑specific polymorphism, 5639T→C
(in CYP1A1*3 allele) upstream of the poly‑adenylation site, it was hypothesized to
have an adverse effect on mRNA stability, leading to a decreased steady-state level
of CYP1A1 mRNA. However, no evidence for such an effect has been reported [34].
DNA damage and other biomarkers
Both the CYP1A1*2A and *2B variant alleles were associated with higher levels
of benzo[a]pyrene (B[a]P) related DNA adducts in human white blood cells [40].
Moreover, the CYP1A1*2A variant was in some studies found to affect DNA adduct
levels in human lung, cord blood and placenta [40,41] However, lack of effect on DNA
adduct levels in lung has also been reported [42]. Furthermore, higher levels of 1‑OH‑pyrene
excretion were observed in subjects carrying the CYP1A1*2A allele, predominantly in light
smokers [43]. Finally, among smoking lung cancer patients homozygous carriers of variant
CYP1A1 alleles (*2A or *2B) have been shown to exhibit a higher frequency of mutations
in the p53 or Ki-Ras gene; these frequencies were even higher in subjects concurrently
lacking the GSTM1 gene ([44]; see Chapter 3). These data further suggest that cells
of subjects carrying mutant CYP1A1 alleles have a higher level of bio-activation and
in-creased DNA damage by cigarette smoke related compounds.
Cancer risk
Increased lung cancer risks have been reported for the carriers of the CYP1A1*2A variant
allele in Asians. Probably due to significant ethnic differences in the variant allele
frequen-cy it has been difficult to detect such an association in Caucasian populations.
Nonethe-less, a recent pooled analysis verified an effect of CYP1A1*2A allele on lung cancer risk
in Caucasians [45]. Probably for the same reason as for CYP1A1*A, it appeared difficult to find
an effect of the CYP1A1*2B allele on lung cancer risk in Caucasians. However, a recent
pooled analysis suggested that also the CYP1A1*2B allele may confer an increased risk
of lung cancer in Caucasians [46].
Conclusions
Overall, it can be concluded that the functional effects of both the CYP1A1*2A and the
CYP1A1*2B alleles are still not clear due to discrepancies in the literature. However, taken
together with data on DNA damage and (lung) cancer risk, both polymorphisms seem
to result in a higher inducibility. More research on the actual functional effects of the
polymorphisms is required.
2.3.2. CYP1A2
CYP1A2 is involved in the metabolic activation of several carcinogens, including
aromat-ic and heterocyclaromat-ic amines (AAs and HAs), nitroaromataromat-ic compounds, and mycotoxins.
CYP1A2 gene has 72% sequence identity with CYP1A1, but has been detected in liver only,
where it seems to be regulated by at least two mechanisms; one controlling constitutive
levels of expression and another regulating inducibility [47]. Large inter‑individual dif‑
ferences were observed, which may be due to factors such as gender, exposure to inducers,
race, and genetic polymorphisms [47].
The CYP1A2 activity can be assessed in a non‑invasive manner by the so‑called caffeine
test, in which metabolites of caffeine after coffee consumption are measured in urine as an
index of CYP1A2 activity. Variable results have been obtained with caffeine‑based
meth-ods; some caffeine metabolite ratios have given bimodal or trimodal distributions while
others have suggested normal or unimodal distributions (for a review, see [47]). Overall,
slow and intermediate CYP1A2 metabolizers represent about 50% of healthy Caucasians,
while their frequency in Japanese subjects seems to be much lower.
To date several polymorphisms have been desribed in the CYP1A2 gene, most of which
are located in the 5’‑flanking regulatory region [48]. One SNP was found in the first
in-tron of the gene [49]. Since the CYP1A2 polymorphisms are in linkage disequilibrium, only
the –163A→C (in CYP1A2*1F allele) and –2464T→delT (in CYP1A2*1D allele) need to be
analysed in the routine CYP1A2 genotype assessment [49].
Functional effects
Some genetic variants have been identified to be associated with CYP1A2 inducibility and
a polymorphism in intron 1 of the CYP1A2 gene has been linked to a higher catalytic
activity of the enzyme [49,50]. Of the polymorphisms located in the 5’‑flanking regulatory
region, the CYP1A2 –163C → A base change (in CYP1A2*1F allele) was associated with
in-creased inducibility in Caucasian smokers but not in non‑smokers [50]. Subsequently [49],
another polymorphism, CYP1A2 −2467T → delT (in CYP1A2*1D allele) was found to be the
most frequent together with −163C → A in Caucasians; this polymorphism was also linked
to increased enzyme activity.
The CYP1A2 −2467T → delT was identified as a common polymorphism to be analysed
in routine assessment of CYP1A2 SNPs in Caucasians. However, no association between
this genotype and caffeine phenotype was found, probably due to the small sample
popu-lation (n = 14) [49]. The CYP1A2 −3860G → A base change (in CYP1A2*1C allele), was
reported to decrease enzyme activity in Japanese smokers. Since the CYP1A2*1C variant
allele is rare in Caucasians, larger study populations are needed to assess the potential
im-pact of this polymorphism on CYP1A2 inducibility [32].
The SNPs cannot completely explain the large inter‑individual differences in the CYP1A2
related metabolic capacity, since many different factors can affect the induction or in-
ducibility of CYP1A2 activity. These factors include cigarette smoking, dietary factors, several
drugs, chronic hepatitis, ethnic/racial group, gender and exposure to polybrominated
biphe-nyls and 2,3,7,8‑tetrachlorodibenzo‑p-dioxin. Furthermore, gene-gene interactions have been
shown to play a role in this context. For instance, a higher inducibility was found in subjects
that were GSTM1-null (see Chapter 3) or carried the CYP1A1*2B allele [52]. As a result, there
is no overall detectable association between CYP1A2 genotype and caffeine phenotype.
DNA damage and other biomarkers
Extensive CYP1A2 activity resulted in increased levels of some biomarkers of genotoxic risk
such as urinary mutagenicity [53
–
56] and DNA and protein adducts [57
–
59] during
expo-sure to carcinogens from diet, cigarette smoke, and environment. However, more studies
are needed to investigate whether these differences can also be linked to genetic
polymor-phisms. Recently, urinary mutagenicity was shown to be significantly increased in heavy
smokers carrying the CYP1A2 *1D and *F alleles [48].
Cancer risk
Several epidemiological studies have been conducted on the relationship between the
can-cer risk and the CYP1A2 activity (either alone or in combination with other CYP’s) and
phase II XMEs, and cancer risk. For instance, risk of colorectal cancer, which has been
as-sociated with exposure to HAs in cooked foods, is strongly elevated in individuals with
the combined rapid phenotypes for both CYP1A2 and N‑acetyltransferase 2 (NAT2)
[60]. However, the link with CYP1A2 polymorphisms needs more attention. Only one
case‑control study has shown that patients with the intron 1 polymorphism may be
at increased risk for bladder cancer if they are smokers [32].
Conclusion
The mechanism of CYP1A2 induction is complex and still not fully understood. The
CYP1A2 promoter polymorphisms might be relevant only in smokers, in whom they
influence enzyme activity by affecting the mechanism of gene induction. The CYP1A2
phenotype and inducibility has been extensively studied by the so-called caffeine test,
but evidence for a possible link with the genotype is weak and needs further attention.
Additional factors that can affect the induction or inducibility of CYP1A2 activity need to
be addressed in these studies.
2.3.3. CYP1B1
CYP1B1 has catalytic activities overlapping with CYP1A1 and CYP1A2 with respect to
the oxidation of drugs and model CYP‑substrates. It is involved in
the metabolic activation
of PAHs and
in the hydroxylation of estradiol to 4‑hydroxyestradiol,
a potentially
genoto-xic metabolite that is suggested to play
a role in carcinogenesis (reviewed in [61]). CYP1B1
is expressed in many tissues, and it is also highly expressed in some tumour tissues. It is
regulated
through the AHR–mediated pathway,
which can be induced by several
environ-mental chemicals, including
PAHs and persistent organochlorine
pollutants such as
poly-chlorinated biphenyls (PCBs).
The CYP1B1 gene is highly polymorphic; to date nine common SNPs, five of which
cause amino acid substitutions, and seven CYP1B1 variant
alleles have been identified
(see [61]). It has been anticipated
that these polymorphisms might cause an altered function
of
the enzyme thereby determining inter-individual differences in
susceptibility to
carcino-genesis.
Functional effects
With regard to the metabolic conversion of B[a]P; the CYP1B1*7 allele has been shown
to encode for an enzyme with a significantly decreased capacity for the formation
of B[a]P‑7,8‑dihydrodiol from B[a]P as indicated by a lower V
max/K(m) [62]. A somewhat
decreased clearance was also observed for the isoform encoded by the CYP1B1*4 allele,
whereas no significant differences in kinetic properties among the remaining variants were
observed as compared with the wild‑type CYP1B1.
As for the metabolic conversion of estradiol [61], the transcrips of CYP1B1*6
and CYP1B1*7 alleles exhibited altered kinetics with significantly increased K
mand
lowered V
maxvalues for both the 2‑ and 4‑hydroxylation of 17 beta‑estradiol after
heterologous expression of the corresponding cDNAs in S. cerevisiae (i.e., impaired
es-tradiol clearance). On the other hand, the proteins encoded by the other constructs
(CYP1B1*2 through *5) were indistinguishable from the wild‑type enzyme. These
re-sults emphasize the necessity of a complete haplotype analysis of enzyme variants for
evaluation of functional consequences in vivo and for analyses of genetic polymorphisms
in relation to, e.g., cancer incidence.
DNA damage and other biomarkers
There is evidence of an association between the frequency of tobacco‑induced
p53
muta-tions and CYP1B1 genotypes in patients with head
and neck squamous cell cancer [63];
smokers carrying the 4326G variant were 20 times more likely to show p53 mutations
than
those with CYP1B1 wild type allele. The 4326C→G SNP, located in the locus encoding for
the heme binding domain of CYP1B1, is present in CYP1B1*3, *5, *6 and *7 alleles.
We recently observed an inverse relationship between the CYP1B1 4326C→G base
change and 4‑aminobiphenyl‑hemoglobin adduct levels in smokers (unpublished data),
in-dicating that CYP1B1 might also be involved in the metabolism of 4‑aminobiphenyl.
Cancer risk
Association of CYP1B1 polymorphism with increased risk of malignancies like ovarian
can-cer [64], endometrial cancan-cer [65], renal cancan-cer [66], smoking‑related
head and neck
can-cer [63,67], lung cancan-cer [68] and prostate
cancer [69–71] have been reported. An
associa-tion of CYP1B1 polymorphism with increased
risk of breast cancer has also been reported
in Asians [68,72], but no association has been
found in Caucasians [73,74].
Interesting-ly, the genotypes that are related to a decreased enzyme activity are related to increased
cancer risks, suggesting that CYP1B1 may predominantly be involved in the inactivation
of carcinogens.
Conclusion
CYP1B1*6 and *7 alleles (containing three and four SNPs, respectively) seem to have most
impact on CYP1B1 activity. Many studies on cancer risks focus on the 4326C→G base
change only. However, in addition to the CYP1B1*3 allele, this variation is also represented
in the CYP1B1*6 and *7 alleles. It thus seems necessary to analyze haplotypes (combination
of genotypes) instead of separate SNP’s to fully characterize the functional effects of the
CYP1B1 variants. The complexity of the haplotype distribution in study populations may
complicate the interpretation of the results.
Table 2.2. Common variant alleles of the CYP1B1 gene
Allele Nucleotide change
CYP1B1*1 None (wild-type) CYP1B1*2 142C
→
G; 355G→
T CYP1B1*3 4326C→
G CYP1B1*4 4390A→
G CYP1B1*5 142C→
G; 4326C→
G CYP1B1*6 142C→
G; 355G→
T; 4326C→
G CYP1B1*7 142C→
G; 355G→
T; 4326C→
G; 4360C→
G2.3.4. CYP2A6
CYP2A6 is involved in the metabolism of several carcinogens, including aflatoxin B1
(AFB1) and nitrosamines, and is also involved in the formation of cotinine from nicotine.
CYP2A6 also catalyzes the hydroxylation of coumarin. Substantial variation in CYP2A6
activity and protein levels exists. In vivo studies using coumarin [75–77] and nicotine
[78–80] as substrates indicate large inter‑individual variability in CYP2A6 activity. In
ad-dition, in vitro kinetic studies using human liver microsomes revealed inter-individual
vari-ability in cotinine‑to‑nicotine metabolic ratios [81,82]. Considerable interethnic
variabil-ity in coumarin [83]
and nicotine [84] metabolism has also been described. The presence
of environmental or therapeutic inducers and liver abnormality may contribute to these
differences [85]. However, this variability has been largely attributed to genetic
polymor-phisms in the CYP2A6 gene [86]. Presently, there are 26 known variants of the CYP2A6
gene. The frequencies of CYP2A6 alleles vary among ethnicities. Generally, variant
alleles resulting in reduced enzyme activity are more common among Asian populations
(Chinese, Japanese, and Korean) compared with Caucasian and African North American
populations. In addition, certain alleles vary substantially among Asian populations. For
instance, the CYP2A6*4 allele frequency is significantly higher among Japanese compared
with Chinese [84,87].
Functional effects
Table 2.3. (modified from Malaiyandi et al. [88]) summarizes the CYP2A6 variant alleles for
which functional consequences have been characterized in vitro or in vivo.
Table 2.3. Description of CYP2A6 alleles with known in vivo or in vitro functional conse-quences
Allele Nucleotide and structural changes Functional consequences CYP2A6*1A CYP2A6*8 CYP2A6*1x2 CYP2A6*2 CYP2A6*4A CYP2A6*4B CYP2A6*4D CYP2A6*5 CYP2A6*6 – 6600G>T (R485L) Reciprocal of *4A or *4D (gene duplication) 1799T>A (L160H) Unequal crossover (3’-UTR) with CYP2A7 (gene deletion) Unequal crossover (distal 3’-UTR) with CYP2A7 (gene deletion) Unequal crossover (intron 8) with CYP2A7 (gene deletion) 6582G>T (G479L) 1703G>A (R128Q) Normal activity Normal in vivo activity Increased nicotine clearance in vivo No in vivo or in vitro activity No in vivo activity No in vivo activity No in vivo activity No in vivo or in vitro activity Reduced in vitro activity
DNA damage and other biomarker
In experimental systems the cells over‑expressing CYP2A6 were 2‑fold more susceptible
towards AFB1 as compared to control cells [89], whereas the induction of p53 mutations
was not increased. In this context it has to be noted that in the 2A family, 2A5 seems to
have the highest affinity for AFB1.
A major pathway of nicotine metabolism is C-oxidation to cotinine, which is
cata-lyzed by CYP2A6. There are substantial data suggesting that the large inter‑individual
differences in cotinine formation are indeed associated with CYP2A6 gene
polymor-phism. Since the CYP2A6 genotype has a major impact on nicotine clearance, its
rela-tionships with smoking behaviour and the subsequent risk of lung cancer has also been
suggested [88].
Cancer risk
Case control studies have suggested that the CYP2A6*4 allele encoding for an inactive
en-zyme may be related to lower lung cancer risk [90–92]. However, contrasting results have
also been reported [93–95].
Taken together, smokers with CYP2A6 slow activity associated genotypes may be at
a lower risk for lung cancer due to the reduced bio-activation of tobacco smoke related
pro-carcinogens. In addition, these individuals may consume less cigarettes and smoke for fewer
years [87]. This will additionally reduce their overall exposure to tobacco smoke derived
procarcinogens [96,97].
Table 2.3. Description of CYP2A6 alleles with known in vivo or in vitro functional conse-quences — cont.
Allele Nucleotide and structural changes Functional consequences CYP2A6*7 CYP2A6*10 CYP2A6*11 CYP2A6*12 CYP2A6*9 6558T>C (I471T) 6558T>C +6600G>T (I471T+R485L) 3391T>C (S224P) Hybrid gene:CYP2A7:5’ regulatory to exon 2 CYP2A6:exon 3-3’-UTR 48T>G (TATA>TAGA box)
Reduced in vivo and in vitro activity toward nicotine but not coumarin
Absent or reduced in vivo activity
Reduced in vivo and in vitro activity toward tegafur, an antineoplastic agent
Reduced in vivo and in vitro activity toward coumarin
Decreased enzyme expression resulting in reduced activity in vivo and in vitro
A number of alleles with SNPs, both in regulatory regions (CYP2A6*1B, CYP2A6*1C, CYP2A6*1D, CYP2A6*1E, CYP2A6*1H, and CYP2A6*1J) and in coding regions (CYP2A6*1F, CYP2A6*1G, and CYP2A6*13-*16) have also been identified; however, little is known about their prevalence or functional effects.
Conclusions
Overall, it can be concluded that CYP2A6 has many functional polymorphisms,
predomi-nantly leading to enzymes with impaired/no activity. Although an effect of these
polymor-phisms on nicotine clearance is obvious, their role in the formation of DNA damage and
cancer risk is less clear and deserves to be studied further.
2.3.5. CYP2C
More than 100 currently used drugs have been identified as substrates of CYP2C isozymes
by biochemical analysis, corresponding to about 10% to 20% of commonly prescribed drugs
(predominantly subtrates for CYP2C9). The CYP2C isozymes were also found to
metabo-lize environmental and dietary carcinogens, such as PAHs [32].
Both CYP2C9 and CYP2C19 genes are located in a highly conserved gene cluster in
chro-mosome 10. The structure and function of CYP2C9 and CYP2C19, as well as their genetic
variants, have been extensively studied by means of enzymology, clinical pharmacokinetic
studies, observational clinical studies measuring drug responses and side effects, and, more
recently, protein crystallography [32,98–100].
Genetic polymorphisms affecting the CYP2C9 and 2C19 enzymes have been
extensive-ly studied during recent years. In this report we will summarize the most important
func-tional aspects; further information can be found in [98–100]. Since the CYP2C‑isoforms
have distinct substrate specificities, it is clear that the consequences of polymorphism need
to be considered separately.
Functional effects
In addition to the wild-type allele, at least five other CYP2C9 alleles are known to occur.
With the exception of one of these (CYP2C9*6), each of these alleles is associated with
a single nonsynonymous base change [100].
The two most common variant alleles are CYP2C9*2 (144Arginine→Cysteine) and
CYP2C9*3 (359 Isoleucine→Leucine); in northern Europeans, over 30% of the population
carry one or two of these alleles, with the overall allele frequencies of 0.10 and 0.08,
respec-tively [32,98–100]. The frequencies of CYP2C9*2 and CYP2C9*3 alleles are much lower
in other ethnic groups, including Orientals and African‑Americans.The other variant alleles
(CYP2C9*4 to CYP2C9*6) are all seen at frequencies of approximately 1%.
Each of the CYP2C variant alleles with coding region SNPs appear to be associated with
significantly impaired enzyme activity. In a study in Japanese subjects several upstream
CYP2C9 polymorphisms were found which may affect the gene expression. There is also
some evidence for the existence of additional polymorphisms in exons 4 and 7 [100].
Fur-ther studies are needed to assess the relevance of these polymorphisms to drug and
carcino-gen metabolism.
The CYP2C19 gene encodes for S-mephenytoin hydroxylase, which has been known
to be deficient in some individuals who are so-called slow metabolizers of the clinical
drug mephenytoin. This deficiency affects approximately 20% of Orientals and 3%
of Europeans [100]. Two relatively common variant alleles of CYP2C19 together with at least
five other rarer alleles have been identified and were associated with absence of the respective
enzyme activity. The two common alleles are associated with production of truncated
pro-teins, whereas absence of activity can also arise as a result of amino acid substitutions.
DNA damage and other biomarkers
The levels of smoking related DNA adducts in larynx were correlated with the presence
of CYP2C protein, suggesting a role of CYP2C9/CYP2C19 isoforms [101]. However,
although it was found that CYP2C9 can be involved in the metabolic activation of PAHs,
the CYP2C9 variant genotypes exhibited no statistically significant effect on PAH‑DNA
adduct levels in bronchial tissues of smokers [102]. Nonetheless, in that study, smokers
car-rying the CYP2C9*3/*3 genotype were found to have relatively high DNA adduct levels.
Cancer risk
The high activity associated CYP2C9 genotype has been shown to increase the risk
of colorectal cancer [103], while the low activity genotype posed an increased risk of
ad-enoma, especially in the presence of tobacco smoke [104]. Especially men seem to have
a reduced risk of colorectal cancer if they carry at least one CYP2C9 variant allele.
Consist-ent with the role of CYP2C9 in activation of carcinogens, the variant alleles appeared to
impair the metabolic activation of tobacco smoke derived carcinogens [105]. On the other
hand, Chan et al. [104] suggested that the CYP2C9 may also play a prominent role in
de-toxification because variant alleles appear to increase the risk of adenoma. Only one of two
large studies in African-Americans and Caucasians have revealed an association between
the CYP2C9 *2 allele and increased lung cancer risk.
Subjects with low activity CYP2C19 genotypes may have higher incidences of
esopha-gus cancer, adult acute leukaemia, stomach cancer and lung cancer. Conversely, they may
have a lower incidence of bladder cancer [32,100].
Conclusions
Overall, it can be concluded that the phenotypic effects of both CYP2C9 and CYP2C19
polymorphisms are clear and may result in differences in the metabolic
activation/detoxi-fication of carcinogens (variants strongly reduce enzyme activity). As a result, they may
also affect the individual cancer risks. Further studies are needed to assess the relevance
of certain novel polymorphisms to drug and carcinogen metabolism.
2.3.6. CYP2D6
CYP2D6 catalyzes hydroxylation or demethylation of more than 20% of drugs metabolized
in the human liver, such as neuroleptics, antidepressants, some beta-blockers and many
oth-ers like codeine [106]. Since many clinical drugs are metabolized by CYP2D6, the phenotypic
effects of known genetic polymorphisms in CYP2D6 are very well documented. Individual
responses to the drug therapy could also be well linked to the CYP2D6 genotypes. Therefore
a lot of attention has been paid to the CYP2D6 genotype-phenotype relationship from a
clini-cal point of view. Unlike other CYP enzymes, CYP2D6 is considered to be non‑inducible, but
was found to be inhibited by some clinical drugs, such as bupropion. This CYP2D6 inhibition
can be so strong that up to 80% of extensive metabolizers are ‘converted’ to poor
metabo-lizers, which markedly reduced their ability to metabolize CYP2D6 substrates [107]. Since
many good reviews on this topic have recently been published [106,108,109], the phenotypic
aspects of the CYP2D6 genotype will be discussed only briefly here.
Functional effects
Polymorphisms within the CYP2D6 cluster are well documented, including point
muta-tions, frame shift mutamuta-tions, deletions and splicing defects. These changes result in either
no, increased, or decreased activities against several clinical drugs. Individuals can thus be
characterised as either extensive (EM) or poor (PM) CYP2D6 metabolisers. Several silent
mutations have been detected among the EMs. The major PM‑phenotype associated alleles
are CYP2D6*3, *4 and *5 in Caucasians (*3 and *4 are rare in Asian populations).
An allele (CYP2D6*2) has also been described containing SNPs that lead to 296Arg→Cys
and 486Ser→Thr amino acid changes. This allele has been associated with ultra‑rapid
metab-olism of debrisoquine in case individuals carry multiple copies (also called CYP2D6*2XN);
a result of a gene amplification event. Such individuals are categorised as ultra-rapid
me-tabolisers (UMs). Only about 1–3% of Middle‑Europeans, but up to 29% of Ethiopians
displays such gene duplications.
Cancer risk
Although the potential role of CYP2D6 polymorphisms in the aetiology of human tobacco
smoke‑related cancers (lung, head and neck, and urinary tract) has been extensively studied
the results have remained largely inconclusive [32].
Conclusion
The clear genotype‑phenotype relationship of CYP2D6 genetic variants is very well
studied, because of its clinical relevance. There is now a good understanding of the
ge-netic background of poor, intermediate, extensive and ultrarapid metabolizers, enabling
a more precise DNA genotyping‑based prediction of drug plasma levels. However, more work
is needed regarding its involvement in the metabolism of human carcinogens.
2.3.7. CYP2E1
The CYP2E subfamily has only been described in mammals and only one CYP2E gene,
CYP2E1, has been identified in humans (see Ronis et al. [110]). Ethanol inducible CYP2E1
is expressed in the liver, and the control over its expression is very complex and involves
mechanisms at the transcriptional, mRNA, translational and post-translational level.
Fur-thermore, the intracellular transport of CYP2E1 (for example to the mitochondria) is
regu-lated in a complex manner, and can determine the fate of the enzyme. CYP2E1 has very
broad substrate specificity; more than 70 different chemicals with diverse structures have
been identified to be metabolised by CYP2E1 [110]. In general, most CYP2E1 substrates are
small and hydrophobic in character. Among the substrates are alcohols/ketones/aldehydes,
aromatic compounds, halogenated alkanes or alkanes, anaesthetics, drugs and carcinogens
(nitrosamines and azo carcinogens). Many of the CYP2E1 substrates also induce the enzyme.
Functional effects
Although in Caucasians no clear relationship was found between in vivo activity of CYP2E1
and genotypes, the presence of a variant allele (called c2) resulted in a significant reduction
of enzyme activity in Japanese subjects. SNPs exist in the 5’‑flanking region (‑1053C>T
detected by restriction enzyme RsaI) and in
intron 6 (7632T>A detected by restriction
en-zyme DraI) of the CYP2E1 gene [111]. Assessment of the relationships between these
(par-tially linked) variant alleles
and the CYP2E1 phenotype were performed in vitro [112,113]
and in vivo [114–116] in Caucasians. Although the rarity of the variant alleles severely
re-duces the power of the studies in this ethnic group, there
is suggestive evidence that the
variant c2 allele may be associated with reduced CYP2E1 activity [112–114,116]. It has to be
noted, however, that in vitro this allele
was found to increase expression of a reporter gene
construct [117,118].
To the best of our knowledge, the functional and biochemical characterization of DraI
variant alleles has not been fully conducted yet. There is no evidence whether or not this
rare allele affects CYP2E1 enzyme activity and contributes to individual susceptibility.
Furthermore, a polymorphic 100 bp insertion was found in the 5’‑flanking region of the
CYP2E1 gene in some individuals, which was associated with increased chlorzoxazone
hy-droxylation in a relatively small number of subjects which were obese and recently
con-sumed alcohol. It is unlikely that this polymorphism is of significance with respect to
altered levels of expression of CYP2E1 [119].
DNA damage and other biomarkers
Individuals that were exposed to safrole and carried at least one mutant CYP2E1 c2
al-lele had a significant higher frequency of safrole‑DNA adducts than those with
the CYP2E1 c1/c1 genotype [120]. Also the formation of 1‑styrene oxide‑adenine adducts
was found to be affected by CYP2E1 polymorphisms [121]. In smoking subjects, CYP2E1
genotypes were associated with higher 7‑methyl‑dGMP levels [122]. On the other hand,
other studies found no effect of CYP2E1 polymorphisms and further research seems
necessary [185]. Interestingly, the c2/c2 genotype was found to correlate positively with
the presence of p53 mutations in squamous cell carcinoma of the lung [123].
Cancer risk
Several studies observed significant associations between CYP2E1 polymorphisms and the
incidences of human cancer, e.g., in esophageal cancer, lung cancer, nasopharyngeal
carci-noma and colorectal cancer [124–128]. In contrast, other studies showed no such
associa-tions [129–131].
Conclusion
There are contradictory results as to whether CYP2E1 polymorphisms correlate to the
transcrip-tional rate and enzyme activity. The incidence of these polymorphisms has been investigated
in relation to DNA damage and different types of diseases (cancer, alcoholic liver disease and
alcoholism). However, the results are controversial and no firm conclusions can be drawn.
2.3.8. CYP3A4
The CYP3A gene cluster contains four genes including CYP3A4 located on the long arm
of chromosome 7. CYP3A4 has a large range of substrates and can activate procarcinogens,
including PAHs and nitrosamines. The CYP3A genes are expressed within the liver and in
some individuals make up to 60% of the livers total CYP‑content, and approximately 3%
of total liver protein [32,61]. Due to the high levels of expression with respect to other
mem-bers of the CYPs, variation in these genes is anticipated to be an important susceptibility
factor. However, it is not yet known whether the wide variation in 3A4 activity is caused by
genetic factors or is it solely attributable to environmental factors. Many different CYP3A4
variant alleles have been found, including missense mutations
(http://www.imm.ki.se/CYP-alleles/cyp3a4.htm) and alleles with mutations in the 5’‑upstream regulatory region.
Functional effects
Only a few of the genetic variants may result in altered properties of the enzyme. In
bac-teria, CYP3A4*2 (resulting in 222Ser→Pro amino acid change) and CYP3A4*12 (resulting
in 373Leu→Phe amino acid change) alleles yielded an enzyme with slightly altered substrate
specificity. The CYP3A4*8 (resulting in 130Arg→Gln amino acid change) and CYP3A4*13
(re-sulting in 416Pro→Leu amino acid change) alleles cause no expression of the CYP holoprotein
and CYP3A4*11 (resulting in 363Thr→Met amino acid change) is expressed at lower levels.
Other allelic variants did not cause any altered function in vitro of the enzyme [132]. The
vari-ants that were found to change the enzyme activity were, however, very rare; a recent
screen-ing for the presence of these variants has revealed that in a population of 500 Caucasians
only the CYP3A4*3 allele was found at a significant frequency (1%) whereas all other forms
were essentially absent [133]. Thus, the enzyme is extremely well preserved.
Cancer risk
To date, no extensive studies on CYP3A4 polymorphisms and cancer risk have been
reported.
Conclusion
At present the interindividual variation in CYP3A4 activity cannot be explained by genetic
polymorphisms. Thus, in order to find any possible genetic basis for the very high
interin-dividual variability in CYP3A4, studies are needed that focus on genetic variations in genes
that regulatie CYP3A4 transcription, (for instance PXR) or genes that control the post‑
translational regulation of the protein.
To better understand genotype‑phenotype relationships in CYPs, it may be necessary
in the future studies to assess the in vivo phenotype by reactions that are specific for certain
CYP iso‑enzymes in humans or cells in vitro. Table 2.4. lists the suggested substrates and
reactions to perform such studies.
2.4. Epoxide hydrolase
Microsomal epoxide hydrolase (EPHX1) is a critical biotransformation enzyme that
cata-lyzes the conversion of xenobiotic epoxides to the more polar diols. To date, several
poly-morphisms in the EPHX1 gene, localized at chromosome 6, have been described. These
polymorphisms include the exon 3 variation leading to Tyr113His amino acid change and
the exon 4 SNP leading to His139Arg amino acid change [135]. In a study among
Cauca-sian subjects [100], 36% of the study subjects were homozygous for the 113Tyr allele, 56%
were heterozygous for this allele, and 8% were homozygous for the 113His variant allele.
Table 2.4. Potential substrates to study the phenotypic effects of CYP genotypes in vitro and in vivo [134]
CYP Substrate Reaction Technique Intact cells In vivo
1A1 1A2 1B1 2A6 2C9 2C19 2D6 2E1 3A4 Ethoxyresorufin Methoxyresorufin Caffeine Phenacetin 17b-Estradiol Dimethylbenzanthracene (DMBA) Coumarin Tolbutamide Phenytoin Diclofenac (S)-mephenytoin Debrisoquine Bufuralol Dextromethorphan Chlorzoxazone 4-Nitrophenol N-Nitrosodimethylamine Aniline Nifedipine Erythromycin Testosterone Midazolam O-deethylation O-demethylation N-Demethylation O-deethylation 4-hydroxylation Oxidations 7-hydroxylation Methyl hydroxylation 4’-hydroxylation 4’-hydroxylation 4’-hydroxylation 4-hydroxylation 1-hydroxylation O-demethylation 6-hydroxylation 3-hydroxylation N-demethylation 4-hydroxylation Oxidations N-demethylation 6-hydroxylation 1-hydroxylation Fluorimetry Fluorimetry HPLC HPLC HPLC HPLC Fluorimetry HPLC HPLC HPLC HPLC HPLC HPLC HPLC HPLC Spectrometry Spectrometry Spectrometry HPLC Spectrometry HPLC HPLC – – Yes – – Yes Yes Yes Yes Yes – Yes – – – – Yes Yes – Yes Yes Yes Yes Yes – Yes Yes Yes -Yes Yes – Yes Yes Yes – – – Yes – Yes Yes
