Phase I enzyme genotypes and their toxicologically relevant phenotypes.

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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

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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_max of 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 mammaliancells [16]. Additionally, acetaldehyde can covalently interact with DNA to formDNA adducts, which may initiate themultistage process of carcinogenesis. The formationof N2_{‑ethyl‑2’‑deoxyguanosine, one major stable acetaldehyde–DNA}_adduct [17], was detected in DNA from the liver of ethanol‑treatedmice [18]. Levels of acetal-dehyde DNA adducts in white blood cellswere much higher in alcohol abusers than the corresponding levels in healthycontrol 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 thatlymphocytes from habitual drinkers with the inactive form ofALDH2, which cannot efficiently detoxify acetaldehyde, havehigher SCE frequencies than lymphocytesfrom 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 ALDH2 genotypes and the less-active ADH2 genotypes, and the risk for esophageal cancer

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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

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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 O₂ is 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

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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_m value 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.

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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].

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.

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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].

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

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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 inducibility 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.

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.

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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 inthe metabolic activation of PAHs andin the hydroxylation of estradiol to 4‑hydroxyestradiol,a potentially genoto-xic metabolite that is suggested to playa role in carcinogenesis (reviewed in [61]). CYP1B1 is expressed in many tissues, and it is also highly expressed in some tumour tissues. It is regulatedthrough the AHR–mediated pathway,which can be induced by several environ-mental chemicals, includingPAHs and persistent organochlorinepollutants 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 variantalleles have been identified (see [61]). It has been anticipatedthat these polymorphisms might cause an altered function ofthe enzyme thereby determining inter-individual differences insusceptibility to carcino-genesis.

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 Km and lowered V_max values 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.

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There is evidence of an association between the frequency of tobacco‑inducedp53 muta-tions and CYP1B1 genotypes in patients with headand neck squamous cell cancer [63]; smokers carrying the 4326G variant were 20 times more likely to show p53 mutationsthan 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‑relatedhead and neck can-cer [63,67], lung cancan-cer [68] and prostatecancer [69–71] have been reported. An associa-tion of CYP1B1 polymorphism with increasedrisk of breast cancer has also been reported in Asians [68,72], but no association has beenfound 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→G

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2.3.4. 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].

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

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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.

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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.

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%

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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.

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

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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.

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

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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.

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 inintron 6 (7632T>A detected by restriction en-zyme DraI) of the CYP2E1 gene [111]. Assessment of the relationships between these (par-tially linked) variant allelesand 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, thereis 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 allelewas 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].

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].

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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.

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.

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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

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The respective frequencies for the 139His homozygotes, 139His heterozygotes, and 139Arg homozygotes were 59%, 37%, and 4%.

The EPHX1 genotype-phenotype associations have been studied by using purified pro-teins or with microsomal preparations obtained from liver tissues that were derived from individuals with the homozygous EPHX1 genotypes. Studies with the purified enzymes showed that when 113His was replaced by 113Tyr, a 40% loss of EPHX1 activity was ob-served. Arg139His replacement, on the other hand, resulted in a 25% increase in the activity [135]. An intermediate activity level was observed when the cDNA contained both 113His and 139Arg alleles. However, the maximal velocities in human liver microsomal prepara-tions among the variant EPHX1 proteins were not different. These results indicate that the structural differences may have only modest impact on the enzyme’s specific activity in vivo. Moreover, the base substitutions at codons 113 or 139 had no effect on the gene transcription. It may thus well be that variation in EPHX1 protein expression is in part regulated at posttranscriptional level [136].

There is now evidence that EPHX1 could play a role in protecting human DNA from the genotoxic effects of aflatoxin B1 (AFB1). In subjects with the 113His allele (reduced activity), higher levels of serum AFB1‑albumin adducts were observed [137] However, the importance of this detoxification pathway, relative to other detoxification pathways like that catalyzed by GSTM1 (see Chapter 3), remains to be elucidated. Furthermore, the role of EPHX1 in the detoxification of AFB1 remains controversial [138]. Data on chromosom-al aberrations provide evidence that supports a role of EPHX1 chromosom-alleles in the development of lung cancer from cigarette smoking [139]. With regard to smoking induced DNA dam-age, our own data suggest that higher levels of DNA adducts can be detected in subjects carrying low EPHX1 activity genotypes [140].

Cancer risk

To investigate the role of EPHX1 genotypes in lung cancer, a meta-analysis of seven pub-lished studies and a pooled analysis of eight studies (four pubpub-lished and four unpubpub-lished at that time) were performed [141]. This study concluded that there is a protective effect of exon 3 variant allelels in modulating lung cancer, especially adenocarcinoma of the lung. This effect may vary among different populations, possibly because of interactions with genetic or environmental factors.

Conclusions

It can be concluded that there is evidence for functional effects of EPHX1 polymorphisms in vitro. However, their in vivo effects are less clear, because the EPHX1 activity seems to be influenced by other (yet unknown) factors as well. Nonetheless, a pooled analysis on lung cancer showed a preventive effect by the exon 3 variant. Further studies are still necessary.

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2.5. Myeloperoxidase

Myeloperoxidase (MPO) is a heme protein synthesized during myeloid differentiation that constitutes the major component of neutrophilic granulocytes. Upon activation, neu-trophils release MPO, which catalyses the conversion of H₂O₂ into HOCl; HOCl is the main oxidant responsible for the bactericidal action of neutrophils [142]. In addition to the direct damaging effects of the oxidants generated by MPO, MPO is able to activate PAHs, such as the conversion of B[a]P into its reactive metabolite B[a]P‑diolepoxide (BPDE) [143,144]. This is especially true for tissues and/or organs in which the levels of CYPs are relatively low such as lung.

A –463G→A SNP is located at 463 base pairs upstream of the gene, in the binding site of a SP1‑transcription factor. 2–10% of the Caucasian population is homozygous for the −463A variant allele, 31–43% is heterozygous for GA, and 49‑64% is homozygous for the wildtype G‑allele [145,146]. Recently, some new SNPs were discovered [147] in exons 2 (V53F), 6 (M251T), 7 (A332V), 11 (I642L) and 12 (I717V), with allelic frequencies of 6.0%, 1.8%, 1.8%, 0.2% and 1.8%, respectively. Also a new polymorphism located in the intron 11 3'‑splice site was found (0.4%).

Four new polymorphisms in the promoter region were also recently detected [147]. Linkage analysis showed a strong disequilibrium between the ‑463G>A polymorphism and exonic SNPs. However, too little is currently known about these new polymorphisms and further studies need to be performed. Another polymorphism in the promoter region of MPO is located 129 bp upstream of the gene (‑129G→A) [148].

The MPO 463G→A polymorphism is located in the binding site of the SP1‑transcription factor. Due to this transition the SP1‑transcription factor binds less efficient, and as a result gene‑expression is reduced. Indeed, lower levels of MPO mRNA were detected in subjects carrying the MPO‑A alleles [149]. We observed [150] a gene‑dose effect in MPO‑activity (corrected for the amount of neutrophils) in broncho‑alveolar lavage fluids of subjects with inflammatory lung diseases; MPO‑463AA <AG <GG. The base change at ‑463 bp up-stream of the MPO gene destroys a SP1‑binding site, but creates an estrogen binding site. Rutgers et al. [148] indeed found gender‑dependent differences in MPO activity in older age groups. In that same study, a reduction of MPO activity of neutrophils was found for the MPO‑129A variant in vitro, indicating lower levels of transcription as compared to carriers of the MPO‑129G allele.

The activation of PAHs by MPO may be quantitatively relevant during inflammatory re-sponses. As a result, reduced MPO activity in subjects carrying the variant MPO‑463AA or AG genotypes would decrease the formation of reactive metabolites upon exposure to PAHs; PAH‑DNA adduct levels are expected to be less in these individuals as compared to those with the wild‑type (‑463GG) genotype. In two studies, the results are in concordance

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with this hypothesis; DNA‑adduct levels in mutant carriers were lower as compared with the wild type carriers [150,151]. Opposite results were recently observed in a study inves-tigating aromatic‑DNA adduct levels in breast tissue obtained at surgery for breast cancer or reduction mammoplasty [152]. However, it is very likely that the absence of inflamma-tion may explain the contrasting results in breast tissue.

Cancer risk

A number of case control studies showed that the MPO ‑463G→A polymorphism is closely related with lung cancer risk; persons carrying the mutant AA genotype have a 40–70% reduced risk for lung cancer [145,146,153–159]. However, not all studies could confirm these results [160,161]. The protective effect was predominantly observed in Caucasians. Interest-ingly, in a recent study the protective effect was strongest for squamous cell lung cancer (SCLC), which is the type of lung cancer most strongly associated with cigarette smoking [146]. The study also indicated that the selection of the control group is essential to de-tect an effect of the MPO polymorphism on lung cancer risk, since a control group which included subjects with non-malignant inflammatory diseases attenuated the underlying relationship (personal communication). Furthermore, in some studies in which the MPO polymorphism did not modulate lung cancer risk, it did so when combined with genetic polymorphisms in other genes like CYP1A1 [162] and GSTM1 [163].

Conclusions

Overall, it can be concluded that the MPO ‑463G→A and MPO 129G→A polymorphisms are likely to be functional polymorphisms, which result in a reduced activity of MPO in carriers of the A-alleles. For the MPO ‑463G→A, this may subsequently lead to a reduc-tion in DNA damage and lung cancer risk. The recent finding that this ‑463‑polymorphism is linked to other exonic SNPs in the MPO gene deserves further attention, since these polymorphisms may (at least partly) account for the observed change in enzyme activity.

2.6. NADPH-quinone oxidoreductase

NADPH dependent quinone oxidoreductase (NQO1) is a cytosolic protein that catalyz-es the metabolic reduction of quinoncatalyz-es [164]. Its purpose is to protect cells against redox cycling and a subsequent increase of oxidative stress. Interestingly, not all hydroquinones are chemically stable and in some cases metabolism by NQO1 yields a more active product. Thus, NQO1 may be involved in detoxification as well as activation of pro‑carcinogens [164].

Two polymorphisms have been characterized in the NQO1 gene. An 609C→T base change (in NQO1*2 allele) leads to a proline to serine substitution in the human protein structure. The frequency of the NQO1*2/*2 genotype varies across ethnic group from 4% in Caucasians, 5% in African–Americans, 16% in Mexican Hispanics to 22% in Chi-nese populations [165]. The NQO1*2 allele frequencies were reported to be 0.16 in Cauca‑ sians, 0.4 in Native Indians, 0.46 in Inuits and 0.49 in Chinese [166]. The NQO1*3 allele

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contains a 465C→T base change that results in an arginine to tryptophan substitution in the respective protein. The frequency of this NQO1*3 allele is low and varies from 0 to 0.05 in different ethnic groups [166].

The NQO1*2 allele has been fully characterized and shows profound phenotypic conse-quences [167,168]. Genotype‑phenotype studies of the NQO1*2 allele have been performed using both cell systems and tissues. No detectable or only trace levels of mutant NQO1 protein could be observed in cell lines and in saliva, bone marrow or lung samples from individuals with the NQO1*2/*2 genotype [168,169]. The mutant NQO1 protein has only from 2 to 4% of the activity of the wild‑type protein [168] due to diminished ability to bind FAD [170]. However, the mechanism underlying the lack of NQO1 activity in NQO1*2/*2 individuals appears primarily to be due to a lack of protein [168], because of an accelerated degradation of the mutant NQO1 protein [171].

The NQO1*3 variant allele was studied by mitomycin C reduction, and the enzyme activity was 60% reduced as compared to the wild‑type [172,173].

Quinones have the possibility to undergo redox-cycling, by which large amounts of re-active oxygen species (ROS) can be formed. These ROS can give rise to oxidative forms of DNA damage such as 8‑oxo‑dG, DNA adducts by lipid peroxidation products and DNA strand breaks. NQO1 catalyzes the metabolic reduction of quinones to their hydroqui-nones, preventing redox‑cycling; the two‑electron reduction by NQO1 directly competes with cellular one‑electron reduction. Studies focusing on oxidative types of DNA dam-age in subjects exposed to benzene, showed an effect of NQO1. Benzene‑induced toxicity is indeed associated with benzoquinone formation. For instance, NQO1 variant alleles had a significant enhancing effect on single strand breaks in 158 Bulgarian petrochemical workers [174]. In another study, benzene exposure was characterized by urinary excretion of S‑phenylmercapturic acid (S‑PMA), and correlated with strand breaks, predominantly in subjects with NQO1*1/*2 and *2/*2 genotypes [175]. Furthermore, the NQO1 genotype was associated with increased frequencies of aneuploidy among the benzene‑exposed indi-viduals even after adjustment for age, alcohol consumption and smoking [176].

On the other hand, DNA adducts associated with oxidative stress, for instance etheno‑ adducts and 8‑oxo‑dG, were not found to be modulated by the NQO1 polymorphisms in pancreatic tissue of a limited set of samples, indicating that these adducts were not derived from NQO1‑mediated redox cycling [177] Furthermore, exposure to quinones was not well characterized.

Cancer risk

Because the homozygous NQO1*2/*2 genotype essentially results in a null phenotype [166–168], it is expected to have a detectable impact on cancer risks. Indeed, the NQO1*2 allele has been associated with an increased risk of urothelial tumors [178], therapy‑related

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acute myeloid leukemia [179], cutaneous basal cell carcinomas [180] and pediatric leukemi-as [181]. Furthermore, the NQO1*2 allele wleukemi-as a significant risk factor for the development of benzene‑induced hematotoxicity in exposed workers [182]. The NQO1*2 allele does not appear to be a risk factor for prostate cancer [183]. On the other hand, in lung can-cers a protective effect was observed; the NQO1*2 allele was under-representated in lung cancer cases as compared to controls [184,185]. This can be explained by the fact that NQO1 could also act as an activating enzyme. Probably due to the low frequency of the NQO1*3 allele, no information is available on the relationship between this polymorphism and cancer risk.

Conclusions

Overall, it can be concluded that both the NQO1*2 and NQO1*3 alleles result in functional def-icits of the enzyme (NQO1*2 >> NQO1*3). Nonetheless, little is known about the biological consequences of NQO1*3, because of its very low frequency in different ethnic groups (< 5%).

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