Drug metabolism polymorphisms as modulators of cancer susceptibility

Mutation Research 436 Ž1999. 227–261 www.elsevier.comrlocaterreviewsmr Community address: www.elsevier.comrlocatermutres

Drug metabolism polymorphisms as modulators of cancer susceptibility Maurizio Taningher a , Davide Malacarne a , Alberto Izzotti b, Donatella Ugolini a , Silvio Parodi a,) a

National Cancer Institute (IST) r Department of Oncology, Biology and Genetics, UniÕersity of Genoa, Largo R. Benzi No. 10, I-16132 Genoa, Italy b Health Sciences Department, Faculty of Medicine, UniÕersity of Genoa, Via A. Pastore No. 1, I-16132 Genoa, Italy Received 13 July 1998; received in revised form 19 February 1999; accepted 19 February 1999

Abstract Recently, several molecular genetic bases of polymorphic enzyme activities involved in drug activation and detoxification have been elucidated. Many molecular epidemiology studies based on these premises have sought to gather information on the association of genetically determined metabolic variants with different risks of environmentally induced cancer. While rare alterations of tumor suppressor genes dramatically raise cancer risk for the single affected subjects, far more common and less dramatic differences in genes encoding for drug metabolism enzymes can be responsible for a relatively small, but rather frequent increase of cancer risk at the population level. This increase could be especially important in specific cases of occupational, pharmacological or environmental exposure. Examination of the current literature reveals that the most extensively investigated metabolic polymorphisms are those of P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases ŽGSTs; M1 and, to a lesser extent, M3, P1 and T1. and N-acetyltransferases ŽNATs; NAT1 and NAT2.. Making reference to these enzymes, we have assayed the current knowledge on the relations among polymorphisms of human xenobiotic-metabolizing enzymes and cancer susceptibilities. We have found intriguing models of susceptibility toward different types of cancer. We have reviewed and commented these models on light of the complex balance among different enzyme activities that, in each individual, determines the degree of each cancer susceptibility. Moreover, we have found techniques of molecular genetic analysis, more suitable than previous ones on phenotypic expression, now allowing better means to detect individuals at risk of cancer. According to the models presently available, a systematic screening of individuals at risk seems to make sense only in situations of well defined carcinogenic exposures and when performed by the polymorphism analysis of coordinated enzyme activities concurring to the metabolism of the carcinogenŽs. in question.

AbbreÕiations: AHH, aryl hydrocarbon hydroxylase; AHR, Ah receptor; ARNT, Ah receptor nuclear translocator; BCC, basal cell carcinoma; CYP, cytochrome P450; EM, extensive metabolizer; GSH, reduced glutathione; GST, glutathione S-transferase; IM, intermediate metabolizer; NAC, N-acetylcysteine; NAT, N-acetyltransferase; NNK, 4-Žmethylnitrosamino.-1-Ž3-pyridyl.-1-butanone; PAH, polycyclic aromatic hydrocarbon; PCR, polymerase chain reaction; PM, poor metabolizer; RFLP, restriction fragment length polymorphism; RR, relative risk; TCDD, tetrachlorodibenzo-p-dioxin; UM, ultrarapid metabolizer ) Corresponding author. Tel.: q39-10-5600211r2r3; Fax: q39-10-5600210; E-mail: [email protected] 1383-5742r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 0 5 - 8

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Genetic polymorphism analysis can allow for the detection of patients more prone to some types of specific cancers, or to the adverse effects of specific pharmaceutical agents. Considering the increasingly confirmed double-edged sword nature of metabolism polymorphism Žboth wild-type and variant alleles can predispose to cancer, albeit in different situations of exposure., individual susceptibility to cancer should be monitored as a function of the nature, and mechanism of action, of the carcinogenŽs. to which the individual under study is known to be exposed, and with reference to the main target organ of the considered type of exposure. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chemical carcinogenesis; Drug metabolism; Genetic polymorphism; Cancer risk

1. Introduction Ten to twenty years ago, cancer susceptibility was mainly attributed to the level of exposure to carcinogenic agents, e.g., heavy smoking for a longer time interval increases cancer risk more than light smoking for a short time interval. Similarly, exposure to heavy air pollution increases the risk of lung cancer and other related cancers with an effect whose intensity is proportional to the concentration of pollutants and the length of exposure. While these relationships still hold true for the average population, in recent years we have learned that the long process that leads from exposure to a carcinogenic agent to cancer, 20 to 30 years later, is subject to individual modulations that we are only now able to appreciate. Rare individuals Ž1–2% of the population. are affected by alterations of one allele of a tumor suppressor gene w1x. Therefore, in some of their target organs, the number of steps required to complete the process of carcinogenesis is Ž n y 1. instead of n. These individuals display dramatic increases of cancer susceptibility for both the associated parameters of incidence and age of incidence. In addition to these very strong but rare cancer predispositions, there are milder ones concerning different stages of the carcinogenic process that are by far less dramatic, but that can affect large portions of the human population. In recent years, we have learned to measure with great sensitivity the level of carcinogen– DNA adducts w2x in different individuals apparently faced with similar levels of exposure. We have witnessed large interindividual variations, suggesting that metabolism of carcinogens and capability of DNA repair are subject to different modulations in different individuals. On the other hand, molecular epidemiology has recently elucidated several genetic

bases of polymorphic enzyme activities which modulate drug metabolism. To have an objective, yet synthetic, picture of the present knowledge on the relationship existing between drug metabolism polymorphisms and cancer susceptibility, and likewise of the possible role in cancer prevention played by the detection of predisposed individuals, we have examined papers of the last 5 years of literature. We have found that the most relevant metabolic enzymes studied for their polymorphism were P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases ŽGSTs; M1 and, to a lesser extent, M3, P1 and T1 enzymes., and N-acetyltransferases ŽNAT; NAT1 and NAT2.. Based on this survey, we have extracted and commented on studies pertaining to the polymorphisms of the genes encoding these enzymes. We have divided these studies into four sections, namely Cytochrome P450 1A1 Ž CYP1A1., Cytochrome P450 2D6 Ž CYP2D6 ., GSTs and NATs. To facilitate the reading, we have introduced each section with a short description of the features of the enzyme under analysis and of the genetic variants leading to its polymorphism. Moreover, to acquaint the reader with the historical background from which present knowledge has evolved, we have also reviewed some earlier studies cited in the ones collected in our update. Finally, to make our review concise, we have considered only those cancer susceptibilities that are most widely documented as being dependent on metabolic polymorphisms. These susceptibilities pertain to the cancer of lung and upper respiratory tract, of colorectal tract, of urinary bladder and of breast, and to cutaneous basal cell carcinoma ŽBCC.. Admittedly, the criteria we adopted for collecting and reviewing data could be considered simplistic, especially in view of the growing number of rela-

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tions among different metabolic enzymes found to be involved in cancer outcome. However, they seemed to be the only viable ones to obtain a schematic and objective overview of the present field, whose mere vastness demands—but likewise hinders—an ongoing encyclopedic review of all its elements. Given that exhaustive reviews on the polymorphisms of specific classes of enzymes are already present in the literature Žas also shown by our cited references., our purpose was not to review previous reviews, but to outline the overall current knowledge on the polymorphisms of drug metabolism enzymes from which the factors modulating the carcinogenic effects of these polymorphisms could be evinced. The understanding of these factors is not only useful to apprehend the apparently contradictory results that, as we will review, are recurrently found in this field of study, but also necessary to define the possible applications of enzyme polymorphism monitoring in cancer prevention. In this view, we have extensively discussed the individual susceptibility to a given type of cancer as the result of a complex balance among different metabolic enzyme activities. As we will see, this balance reflects not only interindividual quantitative differences of the enzyme activities as determined by genetic polymorphisms, but also qualitative differences, similarities or complementarities among these activities that modulate the carcinogenic effects of a given chemical exposure in the different target organs. We also provide examples highlighting the importance of such qualitative and quantitative parameters even in the induction of different tumor types within the same target organ. In keeping with our purpose, the conclusions we draw are addressed to these issues rather than to a systematic re-examination of the polymorphisms previously reviewed in the single sections.

2. CYP1A1 2.1. Background CYPs Žcytochrome P450s. catalyze the insertion of an atom from molecular oxygen into substrate, i.e., a typical activating Žor Phase I. reaction which

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converts procarcinogens to DNA-reactive electrophilic forms w3x. They are encoded by genes of the CYP superfamily w4x. In particular, CYP1A1 gene encodes for CYP1A1 Žpreviously named cytochrome P450c or P1-450 and, more recently, cytochrome P450 1A1., a P450 isozyme associated with the aryl hydrocarbon hydroxylase ŽAHH. activity w5x. A relevant feature of this cytochrome is its ability to catalyze the first step in the metabolism of polycyclic aromatic hydrocarbons ŽPAHs, for instance present in tobacco smoke. leading to electrophilic, carcinogenic molecules. The observation of a trimodal pattern of AHH inducibility suggests that CYP1A1 is genetically regulated w6,7x. As described by Nebert w8,9x, CYP1A1 polymorphism implies both differences in CYP1A1 regulation and differences in the CYP1A1 structural gene itself. The gene regulation starts with the binding of the inducing agent Žfor instance, a xenobiotic substrate to be metabolized. with the intracellular Ah receptor, where the occurrence of a high-affinity receptor determines a high CYP1A1 inducibility. The ligand binding leads to the dimerization of the Ah receptor with a protein, the Ah receptor nuclear translocator ŽARNT. protein. As described by Hoffman et al. w10x, the ARNT protein is not the ligand-binding subunit of the receptor, but a factor required for its translocation from the cytosol to the nucleus after ligand binding. Once reaching the nucleus, the ligand–receptor complex binds to specific DNA sequences Ži.e., the xenobiotic responsive elements. upstream of the gene to be transcribed Žin this case CYP1A1., and stimulates its transcription. The Ah receptor, whose endogenous ligand is unknown, can bind PAHs and many other aromatic compounds, for instance tetrachlorodibenzo-p-dioxin ŽTCDD. w11x Žthe hypothesis that the Ah receptor was developed during evolution, mainly to deal with xenobiotic agents cannot be discarded.. It has been shown that the TCDDP Ah receptor complex induces the expression of both CYP1A1 and CYP1A2 Žanother member of the CYP1A subfamily, providing arylamine oxidation. and the genes encoding Phase II enzymes, namely NADŽP.H:menadione oxidoreductase, aldehyde dehydrogenase, UDP-glucuronosyltransferase and GST w9x. As reviewed by Lucier w11x, the TCDDP Ah receptor complex can also interact with the activity of a variety of components of the endocrine system

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Žsuch as the tumor necrosis factor a , the epidermal growth factor receptor, the glucocorticoid and the estrogen receptors.. The properties found for the murine Ah receptor can probably be transposed to the human receptor Žnow named AHR w12x., as has been suggested by Nebert w8x and Nebert et al. w13x. Differences in the alleles of the AHR locus, which encodes AHR, might account for interindividual differences in the susceptibility to certain forms of cancer w13x, though the AHR polymorphism has yet to be fully identified at the DNA level. Interindividual differences of the AHR phenotype have been observed in humans, with about one-tenth of the population having the high-affinity AHR phenotype concomitant with CYP1A1 high-inducibility, as reviewed by Nebert et al. w13x. Passing to the structural gene polymorphisms, which are the subject of our review, an MspI restriction fragment length polymorphism ŽRFLP. has been identified in the 3X-flanking Žnon-coding. region of the CYP1A1 gene, stemming from a T ™ C transition at position 6235 Ž250 bp downstream from the polyadenylation site. w14,15x. This mutation determines three genotypes, namely m1rm1 Žor genotype A., which is the wild-type lacking the MspI cleavage site, m1rm2 and m2rm2 Žor genotypes B and C ., which are heterozygous and homozygous, respectively, for the mutant allele with the MspI site. As reviewed by Nebert et al. w13x, large ethnic differences in the allelic frequencies of this polymorphism have been observed, the frequency of the mutant allele being 31% and 12% among Japanese and Caucasian populations, respectively. The grouping of individuals who are either homozygous or heterozygous for this mutant, hyperactive, allele seems to be the most appropriate approach for interethnic comparisons, given the low frequency of mutant homozygotes occurring in Caucasian and African ethnic groups w16x and assuming that both homozygous and heterozygous states are determinant of an excess of function. A second point mutation, an A ™ G transition at position 4889 in exon 7, has been described and linked with MspI. This mutation leads to an isoleucine to valine substitution at amino acid residue 462 involving the heme binding region of the protein w17x, and is responsible for the so called Ile–Val polymorphism Žor exon 7 polymorphism.. The various degrees of Ile–Val polymorphism are

classified as IlerIle Žcorresponding to the wild-type., IlerVal and ValrVal Žcorresponding to the heterozygous and homozygous genotypes for the mutant allele, respectively.. The functional significance of these two mutations in the CYP1A1 structural gene is not yet fully defined. A functional impact could depend on either the linkage between MspI and Ile–Val polymorphisms or a linkage between Ile–Val and other gene polymorphisms which might affect the levels of CYP1A1 transcription, such as the polymorphisms of promoter, AHR or other metabolic genes, as suggested by Crofts et al. w18x. For instance, the mutation in exon 7 has been associated with both increased inducibility and increased activity of the CYP1A1 enzyme w18x. However, a recent work by Persson et al. w19x argues that the same mutation does not significantly modify the kinetic properties of CYP1A1, thus excluding the notion that CYP1A1 Ile–Val polymorphism is the basis for alterations in enzyme properties. As Rannug et al. w20x have reviewed, also MspI polymorphism was associated with CYP1A1 inducibility. However, subsequent evidence showed that CYP1A1 inducibility occurred more frequently than the m2 mutant allele, thus leading the authors to conclude that while the existence of an association was possible it appeared unlikely w20x. A third polymorphism, without linkage with either of the two polymorphisms described above, has been found in the African–American population ŽAA polymorphism.. It stems from a single A ™ G transition in the 3X Žnon-coding. region of the gene, ; 300 bp upstream from the polyadenylation site, and its functional consequences remain to be defined w21x. The importance of the existence of three major polymorphisms in the same gene is highlighted in a paper by Garte et al. w22x. As described by the authors, the varied combinations of these polymorphisms allow the occurrence of eight different haplotypes, some of which are characteristic of specific ethnic groups. Labeling the genotypes of the individuals according to these haplotypes rather than to a single polymorphism should prevent biases due to linkages among the CYP1A1 gene polymorphisms in future studies on the relationships between CYP1A1 genotype and cancer susceptibility w22x. Interestingly, the paper by Garte et al. w22x shows that the genotype formerly termed ‘wild-type’ Žcontaining none of the three CYP1A1 gene polymor-

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phisms in question. occurs in fewer than half of non-Caucasian ŽAsian and African. individuals, and that it should by no means be considered the ‘normal’ genotype. A further polymorphism, consisting of a C ™ A transversion at position 4887 in exon 7, has been described by Cascorbi et al. w23x. Its frequency was found to be 10-fold higher in Caucasians than in African–Americans Ž4.0% vs. 0.4%, respectively. w24x. The functional effect of this polymorphism Žwhich is located adjacent to the codon of the Ile–Val polymorphism. has not yet been reported. 2.2. Cancer susceptibilities related to CYP1A1 polymorphism A correlation of either MspI or Ile–Val polymorphisms with lung cancer risk was observed in Japanese w15,17,25x, but not in Caucasian or African–American populations w26–28x. This discrepancy might reflect the different frequencies of the CYP1A1 mutant alleles existing among ethnic groups w16x. A more recent genotyping study by Xu et al. w29x, in which heterozygous and homozygous MspI variants were pooled and considered as a whole, demonstrated a significant association between CYP1A1 polymorphism and lung cancer risk in the North American population, this association remaining significant even when non-Caucasian individuals were specifically excluded from the analysis. According to these authors, an increased risk of lung cancer stemming from CYP1A1 MspI variant exists in both Caucasian and Japanese individuals, and to a similar extent w29x. Many of the works on cancer risk arising from CYP1A1 polymorphism have studied the effects of modified CYP1A1 when combined with a deficient GST activity. As we will describe in a following section, GST activities detoxify a multitude of activated carcinogens that are produced during Phase I metabolism. In particular, class m GST enzymes ŽGSTM1 among them. are elective inactivators of carcinogenic PAH diol-epoxides resulting from the CYP1A1-mediated activation of PAHs w30x. In this view, an overefficient CYP1A1 combined with a deficient GST activity seems to be the most appropriate premise for lung cancer risk after exposure to PAHs. This assumption has been confirmed in a

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work by Nakachi et al. w31x studying the incidence of squamous cell carcinoma in Japanese smokers, where an increased risk was shown for individuals harboring the CYP1A1 Ž m2rm2 . or Ž ValrVal . genotype together with the lack of a m class GST. Notably, moreover, given that the risk of lung cancer was in any case higher at higher smoking-dose exposures, the relative risk ŽRR. of these individuals was found to be higher for subjects smoking a lower number of cigarettes w31x. This finding is in keeping with a previous one by the same authors that the RR of subjects harboring CYP1A1 Ž m2rm2 . was remarkably high at a low dose level of cigarette smoking, and that the risk ratio between CYP1A1 susceptible and normal genotypes was reduced at high dose levels w25x. More recently, the risk of squamous and small cell carcinoma of the lung modulated by the combination of CYP1A1 and GSTM1 polymorphisms has been investigated by Kihara et al. w32x, again in a Japanese population. Among male smoker cancer patients the frequency of subjects harboring the CYP1A1 Ž m2rm2 . or Ž ValrVal . genotype was substantially the same as that among male smoker controls. However, when subjects were categorized by both CYP1A1 and GSTM1 genotypes, the frequency of subjects harboring the CYP1A1 Ž m2rm2 . genotype and the GSTM1 null genotype Ži.e., lacking both alleles for GSTM1. became significantly higher among patients than among controls. Interestingly, subjects harboring CYP1A1 Ž m2rm2 . but with the GSTM1 positive genotype were associated with the lowest risk of developing tumors, thus suggesting that CYP1A1 Ž m2rm2 . might protect against tobacco-related lung cancer when combined with a proficient GSTM1 expression w32x. These results confirm that the combined CYP1A1 and GSTM1 genotype can be a potential predictor of smoking-related lung cancer risk in populations such as Japanese, where CYP1A1 mutant alleles are common w32x. Another interesting finding, again on the combined effects of CYP1A1 and GSTM1 polymorphisms on the susceptibility to lung squamous cell carcinoma, has been obtained by Alexandrie et al. w33x studying a Swedish population. As reported by these authors, the presence of the m2 allele in the CYP1A1 genotype combined with the GSTM1 null genotype was significantly increased in cancer patients diagnosed before 66 years of age. Due to the very low fre-

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quency of the m2 allele in Caucasian populations, Alexandrie et al. w33x claimed the need for a larger study to confirm their finding. Focusing on the relationship of the CYP1A1 gene polymorphism per se with lung cancer susceptibility in Caucasian individuals, we recall a review paper already mentioned by us by some of the above authors w20x. From the reviewed data, this paper concluded that the carriers of genotypes associated with increased CYP1A1 inducibility were at increased risk of cancer. However, at least for the Caucasian population, the recognized CYP1A1 mutations probably identified only a fraction of the individuals susceptible to cancer, while a polymorphic mutation in the ARNT gene w34x, or other still unidentified mutations interfering with the regulation of CYP1A1 expression, could be significant in determining these individuals w20x. A recurrent issue in the studies on CYP1A1 polymorphism and lung cancer risk is the association of this polymorphism with types or grades of differentiation of the tumors. As mentioned, the CYP1A1 Ž m2rm2 . and Ž ValrVal . genotypes have been found to be risk factors for both squamous cell carcinoma and Žto a lesser extent. adenocarcinoma of the lung in Japanese populations w25,31,32,35x. The study by Nakachi et al. w35x on the occurrence of lung adenocarcinoma showed that, contrary to what was observed for squamous cell carcinoma, the presence of these genotypes increased the risk only for poorly differentiated tumors and that the risk did not increase at a low dose level of cigarette smoking w35x. On the other hand, as commented by the authors, while lung squamous cell carcinoma has been known to be closely associated with cigarette smoking, only a weak or non-existent association was shown between this etiological factor and lung adenocarcinoma w35x. The effects of CYP1A1 inducibility Žas detected in specimens by immunochemical means. and of the GSTM1 null genotype on the histological type and location of lung tumors were studied by Anttila et al. w36x in non-Japanese current smoker patients. Patients with inducible CYP1A1, which is located in the peripheral lung, developed mainly peripheral adenocarcinoma, while the non-inducible CYP1A1 phenotype was associated with the occurrence of bronchial tumors alone Žmainly squamous cell carcinoma.. In patients with inducible CYP1A1 phenotype, the expressing GSTM1 showed

a protective effect against contracting bronchial, but not against peripheral cancer w36x. These results highlight the predominant role of CYP1A1 inducibility in determining type and location of lung tumors, and are in keeping with a minor functional role, if present, of CYP1A1 enzyme activity linked to the Ile– Val polymorphism w36x, as also suggested by the work of Persson et al. w19x. It may be added that, more recently, El-Zein et al. w37x have found a tissue-targeted effect in lung cancer onset also for a polymorphism causing CYP2E1 overexpression w38x. CYP2E1 is another cytochrome implied with smoking-related lung cancer Žw39,40x, see also Section 3.2.. As observed by El-Zein et al. w37x in a casecontrol study among lung cancer patients, those harboring at least one CYP2E1 high-activity mutated allele developed adenocarcinoma only, while neither GSTM1 polymorphism showed preferential tendency toward developing squamous cell carcinoma or adenocarcinoma. The results of both Anttila et al. w36x and El-Zein et al. w37x are in agreement with the hypothesis that localized chemical activation of tobacco-smoke carcinogens is dependent on the CYP polymorphism. These studies also show that the inheritance of polymorphic genes may not only predispose individuals to developing lung cancer, but may also contribute to the development of specific types of cancer. The relationship between CYP1A1 and GSTM1 polymorphisms and cancer development was studied by Goto et al. w41x for the prognostic significance of these polymorphisms in patients with non-small cell lung cancer. Both MspI genotypes B and C were associated with various clinical parameters Žsuch as histological type and extent of the primary tumors. responsible for a poorer prognosis. The GSTM1 null genotype also was associated with parameters typical of a malignant phenotype such as the extent of invasion of regional lymph nodes and distant metastases. The presence of both CYP1A1 and GSTM1 ‘susceptible’ genotypes further worsened the prognosis w41x. The association of colorectal cancer with both smoking and the consumption of foods containing PAHs is well recognized w42,43x. Thus, a case-control study Ž43 cases vs. 123 controls. was conducted by Sivaraman et al. w44x to see whether CYP1A1 polymorphisms could pose a risk of colorectal cancer in subjects of different ethnic groups. The MspI

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genotype C was significantly associated with cancer risk in Japanese and Hawaiianrpart-Hawaiian populations, whereas the study seemed to lack the power to detect a similar association in Caucasians. In the Japanese population, some excess of risk Žyet not significant. was also observed for the ValrVal genotype w44x. Although they evinced an important role of PAHs in the etiology of colorectal cancer in populations with frequently mutated CYP1A1, these results highlight the need for very large samples to study populations in which this gene is rarely mutated. Based on the notion that estrogen metabolism is in part determined by CYP1A1, Taioli et al. w45x studied the role of CYP1A1 polymorphisms in breast cancer susceptibility in Caucasian and African– American women Ž51 cases vs. 269 controls.. The Ile–Val and AA polymorphisms were not associated with breast cancer cases in either ethnic group. In Caucasian women, no association was observed for the MspI polymorphism. However, the MspI genotype C was significantly associated with cancer cases in African–American women, and the association between this genotype and cancer outcome was not modified by smoking status w45x. As commented by Taioli et al. w45x, the mechanism of the observed association was unclear and could be explained with a possible linkage between the MspI polymorphism and other polymorphisms related to breast cancer risk in the African–American population. More recently, however, this association was not confirmed by Bailey et al. w24x in a study with a larger population. These authors, if anything, found in the African–American population a trend Ž p s 0.08. for a protective role of the MspI polymorphism against breast cancer risk w24x. More generally, none of the known CYP1A1 polymorphisms was found to increase breast cancer risk, in either Caucasian or African–American populations w24x. The possible role of PAHs in the etiology of breast cancer was considered by Ambrosone et al. w46x. Indeed, PAHs can be metabolized by mammary epithelial cells and, being lipophilic, can be stored in the breast adipose tissue. Both CYP1A1 and GSTM1 polymorphisms were analyzed in postmenopausal Caucasian women and studied for their possible correlation with tobacco smoking w46x. A slightly, but not significantly, increased risk was associated with the CYP1A1 Ile–Val polymorphism and smoking.

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Moreover, no increased breast cancer risk could be associated with the GSTM1 null genotype, but some evidence indicated the association of this genotype with risk among the youngest postmenopausal women w46x. In this study, the statistical power to detect an effect might be limited by the small sample sizes Ž216 cases vs. 282 controls., as acknowledged by the same authors w46x. The research by Ambrosone et al. w46x has recently been taken up by Ishibe et al. w47x and by Bailey et al. w24x. The study by Ishibe et al. w47x did not find an overall increase in breast cancer risk arising from CYP1A1 Ž MspI and Ile–Val. polymorphisms in a population of predominantly Caucasian women. However, a suggestive increase in the risk was observed among women who began smoking before the age of 18 and who had the CYP1A1 MspI variant genotype compared to nonsmokers who were homozygous wild-type for this polymorphism. A similar, but nonsignificant, association was observed also for the CYP1A1 Ile–Val polymorphism w47x. The study by Bailey et al. w24x found no significant correlations with increased breast cancer risk not only for any of the known CYP1A1 polymorphisms Žas we mentioned in the above paragraph., but also for the occurrence of GSTM1 and GSTT1 null genotypes ŽGSTT1 is another GST enzyme, as described in a following section.. Moreover, these authors w24x did not find any correlations even when the genotype variants they analyzed were considered in combination, or when factors such as smoking history, age and stage of disease were taken into consideration. Notably, Fontana et al. w48x have recently corroborated the suggestion by Ambrosone et al. w46x that GSTM1 null genotype is related to breast cancer risk specifically in the youngest postmenopausal women. As observed by these authors w48x, in patients with primary breast cancer, while the overall frequency of the GSTM1 null genotype was 50% Ži.e., a value similar to those recurrent in the literature., the frequency of this genotype differed significantly between patients below 55 years and older ones Ž56% vs. 45%; p - 0.05.. The difference in frequency was even more significant when subjects with the GSTM1 null genotype and subjects lacking only one GSTM1 proficient allele were pooled together Ž65% vs. 52%; p - 0.01. w48x. The findings on breast cancer susceptibility mentioned above, together with others on lung cancer

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susceptibility w29,33x, suggest that the use of appropriate samples and grouping of the samples allow for the detection of cancer susceptibilities which may have escaped previous, less discerning, investigations. In this context, the identification by genotyping of the so called silent carriers Ži.e., subjects heterozygous for the mutant alleles that escaped usual phenotyping assays. may allow defining subtle, but perhaps meaningful, differences in cancer predisposition. More generally, we would remark that the described lung cancer predisposition in Japanese smokers, as determined by an overly efficient CYP1A1 combined with a deficiency of class m GST enzymes, seems to be one of the most straightforward models presently available on cancer susceptibility due to metabolic polymorphism. Of course, this is a model into which many metabolic pathways, specific for genotoxic and nongenotoxic compounds and for free-radical agents, must be inserted. In this model, the convergence of AHR and ARNT gene polymorphisms to CYP1A1 inducibility provides for a complex regulation of the interactions occurring between genotoxic and nongenotoxic mechanisms of carcinogenesis. For instance, an increased function of AHR could lead to increased levels of activated DNA-reactive carcinogens due to increased CYP1A1 induction. However, given that functionally active AHR can modulate not only CYP1A1 inducibility but also the activity of components of the endocrine system w11x, an increased function of AHR could also strengthen processes of hormonal carcinogenesis. Intriguingly, AHR activation can be induced by both typical DNA-reactive carcinogens Že.g., PAHs. and by hormone-like acting carcinogens Že.g., dioxins.. In addition, AHR-inducible CYP1A1 Žand CYP1A2. can play a role not only in the production of electrophiles but also in the metabolism of hormones Že.g., estrogens w45x.. Thus, even though straightforward in essence, this model is probably one among those depending most on individual parameters, such as hormonal and nutritional status and exposure to antioxidant micronutrients. The reviewed data show that the effects of CYP1A1 polymorphisms may be combined among themselves, according to the occurrence of different haplotypes w22x, as well as with the effects of the polymorphisms of other enzymes, particularly GSTM1 w31–33,41x. Moreover, they highlight the

relevance of AHR and ARNT protein polymorphisms in modulating CYP1A1 activity w13,18,20x. Finally, they point out the complex interaction of CYP1A1 with carcinogenic agents of different nature. Thus, the adoption of single CYP1A1 gene polymorphisms as individual markers of cancer susceptibility is likely too simplistic an approach. 3. CYP2D6 3.1. Background CYP2D6 gene belongs to the CYP2 gene family w4x. More than 80 clinically important drugs have been reported to be substrates for its encoded cytochrome, including drugs acting on the cardiovascular and central nervous systems w49x. The lack of its normal activity can produce severe clinical effects and, in extreme cases, death. Among representative substrates of CYP2D6 are the hypotensive drug debrisoquine and the rodent pulmonary carcinogen 4-Žmethylnitrosamino.-1-Ž3-pyridyl.-1-butanone ŽNNK., a component of tobacco smoke w50x. The majority of individuals have at least one intact allele of the CYP2D6 gene, and are functionally classified as extensive metabolizers ŽEMs.. Poor metabolizers ŽPMs., that are determined by the lack of the gene function or even of the gene itself, are in the minority. The frequency of PMs is around 5–10% in Caucasians and 2% in African–Americans, and less than 1% in Orientals, according to the literature w13,51,52x. PMs lack CYP2D6 protein in their liver and, hence, insufficiently activate carcinogens to electrophilic DNA-reactive moieties. Therefore, in exposures in which metabolites rather than the parent compounds are the effective cause of cancer, PMs should be less prone to developing tumors than subjects proficient for CYP2D6 activity. Differences in drug metabolism Žup to 1000-fold rate. have been observed among individuals endowed with CYP2D6 activity, thus determining the existence of subpopulations of intermediate ŽIM. and ultrarapid metabolizers ŽUM. at the boundaries of the EM phenotype, which is related to the presence of the wild-type Ž CYP2D6U 1. allele. While the IM phenotype is ascribed to mutations of the wild-type gene, the UM phenotype is explained by the amplification of either the wild-type gene or of mutated, yet proficient,

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forms of the gene w52x. The existence of UMs ensued from clinical evidence of patients requiring unusually high doses of medication to attain therapeutic plasma concentrations w52x. As reviewed in a recent paper by Sachse et al. w53x, while the alleles possible for the CYP2D6 gene consist mainly of point mutations, gene conversions Žresulting in ‘hybrid’ alleles. and gene duplications Žor higher order amplifications. have also been observed, in addition to the complete deletion of the gene. Thus, scale of phenotypes ranging from overefficient ones to those completely lacking the function is possible for CYP2D6 activity, while different molecular events ranging from single base changes to chromosome rearrangements may account for the different CYP2D6 alleles. An updated example of the complexity of CYP2D6 polymorphisms can be taken from the above mentioned study by Sachse et al. w53x on a German population. According to the authors, the frequency of the wild-type Ž CYP2D6U 1. allele was 36.4% and the frequencies of the alleles encoding for slightly Ž CYP2D6U 2 . or moderately ŽU 9 and U 10 . reduced activity were 32.4%, 1.8% and 1.5%, respectively. The frequencies of the alleles that encoded for the lack of enzyme activity were 20.7% Žfor CYP2D6U 4 ., 2.0% Žfor U 3 and U 5 ., 0.9% Žfor U 6 . and 0.1% Žfor U 7, U 15 and U 16 .. Other known defective alleles Ži.e., CYP2D6U 8, U 11, U 12, U 13 and U 14 . were not found. Moreover, using polymerase chain reaction ŽPCR.-based tests, three CYP2D6 gene duplication alleles were found. They were the alleles CYP2D6U 1 = 2, U 2 = 2 and U 4 = 2, with frequencies of 0.5%, 1.3% and 0.1%, respectively. To complete the picture of a such complex pattern, we should mention that the alleles CYP2D6U 2, U 4, U 7, U 8, U 10, U 11, U 12 and U 14 consist of single base changes; the alleles CYP2D6U 3 and U 6 consist of single base deletions and the allele U 15 of a single base insertion Ždeletions or insertions of a number of bases not multiple of three imply a potentially more severe disturbance of protein function than single base changes.; the alleles CYP2D6U 5 and U 9 are determined by the entire and partial deletion of the gene, respectively, while CYP2D6U 13 and U 16 are ‘hybrid’ alleles. In such an apparently boundless situation, however, reasonable criteria for the detection of individuals distant from the commonest phenotypeŽs. are definable. Again with reference to the

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paper by Sachse et al. w53x, all the PMs in their population sample Ž41 out of 589 individuals. could be ascribed to only five mutations Ždetermining the alleles CYP2D6U 3, U 4, U 5, U 6 and the rare CYP2D6U 15 allele. which were detectable by means of only four RFLP-PCR tests. According to the authors w53x, the four mutation tests, together with a test of gene duplication, could suffice for the clinical prediction of CYP2D6 capacity. Another rapid method for CYP2D6 genotype detection, presented in Stuven et al. w54x, states that using long distanceand multiplex-PCR allows the detection of the CYP2D6U 3, U 4, U 6, U 7 and U 8 mutant alleles in the same DNA fragment. As mentioned by the authors, these five alleles, together with the CYP2D6U 5 deletion allele Ždetectable in a separate PCR assay., are responsible for the PM phenotype in approximately 99% of Caucasians w54x. Given the nature of the chemicals metabolized by CYP2D6, the monitoring of PMs seems to be more suitable to detect subjects whose response to a given medication is anomalous than to predict subjects at increased risk of cancer. As we will describe below, an increased risk of cancer due to PM status could be ascribed to toxic–necrogenic effects of unmetabolized chemicals, i.e., effects too generic to be meaningfully attributed to some kind of exposure. A monitoring of PMs applied to clinical outcomes was reported in a paper by Chen et al. w55x, whose objective was to examine cost, reliability and value of determining the principal CYP2D6 alleles that were presumably associated with the adverse responses to drug treatments of some patients suffering from depression. According to the authors w55x, many of the drugs that are substrates for CYP2D6 are used in psychiatric practice. The study, conducted on a North American population, determined the frequency of the A, B, D, E and T alleles Žcorresponding to the systematic names of CYP2D6U 3, U 4, U 5, U 7 and CYP2D6U 6A. that, as described, cover nearly all PMs occurring in Caucasians. The observed frequency of alleles related to a deficient CYP2D6 expression was significantly higher in patients that experienced adverse effects Ž44%. than in other patients Ž21%. or in a random group from the general population Ž20%; p - 0.05. w55x. As discussed by the authors, the results showed that determining CYP2D6 expression may be valuable as a means to

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detect metabolic-based therapeutic problems, thus contributing to the individualization of dosing regimens, the swift attainment of therapeutic drug levels and the reduction of therapeutic costs w55x. Concerning cost and feasibility, the authors calculated that their monitoring could be done in 13 h at a cost Žin 1996. of US$84 per sample w55x. 3.2. Cancer susceptibilities related to CYP2D6 polymorphism A scoring of the earliest literature does not evince a clear distinction between the roles of EM and PM phenotypes in determining cancer susceptibility. For instance, the risks of lung cancer and urinary bladder cancer, two recurrent issues among studies on metabolic polymorphism, have been related from the earliest studies to the EM phenotype w56,57x; on the other hand, the PM phenotype was related to risk of other types of cancer, including leukemia w58x. This may be explained by the fact that PMs, though less exposed than EMs to genotoxic–carcinogenic drug metabolites, must be exposed for an extended time period to the toxic effects of unmetabolized drugs and of numerous dietary factors that at present have not been identified. As is known, toxic effects could contribute to carcinogenesis, for instance through a necrogenic response followed by compensatory increased cell division w59,60x. Obviously, the induction of significant levels of cell death requires significant levels of exposure to toxic effects. An ambivalent trend of EM and PM phenotypes, even within a same type of target organ, is confirmed by the most recent literature. Coherent with the existence of a CYP2D6-mediated activation of tobacco carcinogens was the finding by Bouchardy et al. w61x that the effect of tobacco on lung cancer risk rose significantly with increasing CYP2D6 activity, as determined by phenotyping in lung cancer patients and controls. Notably, however, high CYP2D6 activity was found to be a significant risk factor only among heavy smokers. As a countercheck, no higher risk could be observed when the tobacco exposure was measured as either duration of smoking or age at beginning of smoking, instead of daily amount. As claimed by the authors, this last finding might explain previous discordant studies which ignored the fact that the level of smoking can

modify CYP2D6 activity. The effect observed by Bouchardy et al. w61x for CYP2D6 activity is opposite to that observed by Nakachi et al. w25,31x for CYP1A1 reported in Section 2.2. As discussed by the authors w61x, an overall picture might be defined on the relationships between tobacco smoke exposure and lung cancer risk. While low smoking exposure was a risk factor linked to increased activities of CYP1A1 w31x and CYP2E1 w39x, high smoking exposure was a risk factor linked to increased activity of CYP2D6 w61x and to deficiency of GSTM1 Žw62x, see also Section 4.2.. In this picture, obviously, the risk of lung cancer was in general higher for higher smoking exposures, but the contribution to this risk given by increased CYP1A1 and CYP2E1 activities was more relevant for lower exposures than for higher exposures, while the opposite trend occurred for CYP2D6 increased activity and GSTM1 deficiency. Again, coherent with a CYP2D6-mediated activation of tobacco carcinogens was the finding by Kato et al. w63x that DNA adducts of the lung were increased as a function of CYP2D6 activity. As reported by the authors, the relative increase of DNA adducts was found to be higher among subjects with lower serum cotinine levels, thus indicating that lower exposures were associated with higher RR. According to the authors, this finding suggested that low smoking andror environmental pollution were the exposure levels more responsive to increased CYP2D6 activity Ža similar situation was ascertained by the authors for CYP2E1 activity as well w63x.. Moreover, coherent with the notion that CYP2D6 and CYP2E1 metabolized tobacco N-nitrosamines w50,51x, the DNA adducts measured by Kato et al. w63x were of the 7-alkyl-deoxyguanosine type. We cannot say to what extent these adducts can determine carcinogenesis in the lung. Nevertheless, the effect of the smoking dose on lung cancer susceptibility appears to be contradictory when comparing the findings of Kato et al. w63x and of Bouchardy et al. w61x. A further discrepancy with the results of Bouchardy et al. w61x was seen with a more recent study by London et al. w64x which, if anything, found a slightly stronger association between CYP2D6 activity and lung cancer incidence specifically for the lightest smokers. What is reported here is only an example of the many discrepancies traceable to the role of CYP2D6 in cancer susceptibility. We can say

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that the current state of research likely reports several contradictory studies for every one giving an association. Contrary to their expectations, Stucker et al. w65x ¨ could not observe a reduced lung cancer frequency in PM subjects in a genotyping study on a French population. However, they found a slightly Žthough not significantly. lower frequency of PMs among patients with squamous and small cell carcinoma, but an excess of PMs Žborderline to significance. among those with adenocarcinoma w65x. After a meta-analysis that pooled their results with those of 11 previous studies Žstarting from the first one by Ayesh et al. w56x., the authors concluded that the hypothesis of a protective role of PM against overall lung cancer was only partially supported Ži.e., when different histologic types were not taken into account.. Moreover, they observed that the strongest evidence for a PM protective effect was essentially associated with the early phenotyping assays. This observation suggested a possible underestimation of the PM frequency by the more recent genotyping assays, given that the mutations detected by these assays are only a part of those contributing to the PM phenotype w65x. However, considering the predominant role played by the genotyped mutations in determining the PM phenotype, the authors concluded that the discrepancies they found depended most likely on the weakness of any relationship existing between CYP2D6 and lung cancer. Nevertheless, they did not entirely exclude the existence of a relationship above a certain threshold level of tobacco smoking w65x. Similar conclusions have been drawn more recently by London et al. w64x, after examining a total of 16 studies Žincluding their own.. As discussed by these authors, the association between lung cancer risk and PM phenotype observed in earlier works may reflect parameters of biologic relevance that are not fully captured by the genotype analysis w64x. For example, the CYP2D6 phenotype could be modified by some other gene important in controlling expression or the CYP2D6 gene could be in linkage disequilibrium with another polymorphic gene important in the carcinogenic process Žas quoted from their paper w64x.. Interestingly, the authors also examined the possible role of CYP2D6 gene duplication in determining lung cancer risk. They found that subjects with more than two functional copies of the CYP2D6 gene and

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without inactivating alleles may be at increased risk of lung cancer, particularly for adenocarcinoma w64x. Given the low frequency of the gene duplication studied by these authors, this new finding obviously needs further confirmation. Another recent meta-analysis of the association between CYP2D6 polymorphism and lung cancer risk was that by Christensen et al. w66x in which 13 studies were identified. Although the heterogeneity of the studies for factors such as age, gender, race, smoking history and disease status prevented their pooling, a trend could be observed. Whereas studies on smaller samples suggested the existence of an association, studies on larger samples did not. This trend led the authors to conclude that no association existed between CYP2D6 polymorphism and lung cancer risk when sample size bias was taken into account w66x. As the literature shows, the involvement of CYP2D6 polymorphism in lung cancer susceptibility is still an issue which is difficult to make out, especially when smoking habits are not considered appropriately. As we described above, while EMs are particularly exposed to the genotoxic effects of DNA-reactive metabolites, PMs are particularly exposed to the possible toxic effects of unmetabolized chemicals, and both genotoxic and prolonged toxic effects may be causes of cancer. Thus, the dose and duration of the smoking exposure should have different carcinogenic effects, which should be mostly evident in EMs and PMs, respectively. Other factors determining heterogeneity of effects should entail the overlapping activity of different cytochromes toward some Žbut not all. substrates, and in some Žbut not all. tissues, such that a given individual pattern of metabolic enzyme activities would favor different types of carcinogenic effects depending on the type of individual carcinogenic exposure. These effects should be more or less marked, given that the activity rate of cytochromes can be individually determined by genetic polymorphism. An example of how CYP2D6 polymorphism could influence the intensity and the type of carcinogenic effect follows: in a given tissue, a low CYP2D6 activity could allow the accumulation of toxic chemicals that lead to tissue necrosis and could, simultaneously, allow CYP1A1mediated activation of genotoxic carcinogens such as PAHs to prevail, thus favoring the occurrence of two

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synergistic causes of cancer, namely, cell proliferation and DNA mutation, induced respectively by tissue necrosis and activated PAHs. Conversely, an overexpressed CYP2D6 activity could cause the activation of nitrosamines Žsuch as NNK. to prevail over that of PAHs. The preferential activation of a specific carcinogen by a specific type of cells could be linked to the prevalence of a specific histologic type of tumor. We must remember that carcinogenic potentials vary among different genotoxins w67x and that, in the lung, NNK and PAHs might preferentially act on different target cells w68x. On the whole, considering the variety of carcinogens present in tobacco smoke w69x plus the variety of micronutrients and environmental pollutants to which smokers are exposed, an algebraic sum of the CYP2D6-dependent effects, leading to the status quo, seems to be a quite realistic possibility, at least when evaluating lung cancer risk at the population level. Adapting a sentence by London et al. w64x, we conclude that ‘‘data suggest that the CYP2D6 genetic polymorphism is not the strong risk factor for lung cancer suggested by some studies of phenotype, but that it may play a minor role’’. It has to be proven that an assessment of function at the level of genotype Ževen if technologically more sophisticated. is really more significant than an assessment of function at the level of phenotype. Finally, to give an example in which the PM phenotype is associated only with increased cancer risk, we mention a study by Elexpuru-Camiruaga et al. w70x concerning the influence of CYP2D6 and GSTT1 allelisms on the susceptibility to astrocytoma and meningioma. As reported, either the null genotype for GSTT1 or the genotype determining CYP2D6 PM were significant risk factors for both astrocytoma and meningioma. This finding confirms once more the ambivalent nature of CYP2D6 in determining cancer onset, even without the concurrence of other types of genetic polymorphisms.

4. GSTs 4.1. Background GSTs constitute a family of multifunctional dimeric proteins which catalyze the conjugation be-

tween electrophiles and the nucleophile reduced glutathione ŽGSH.. Their primary function is to detoxify electrophiles capable of DNA binding, a typical reaction of Phase II metabolism w71x. Some exceptions are the halogenated hydrocarbons that, once conjugated with GSH, can be subsequently activated to alkylating reactive forms w71x. GSTs also catalyze a number of other reactions involving GSH, including organic hydroperoxide reduction w72x, thus playing an important role in protecting tissues from oxidative stress. Focusing on the human cytosolic enzymes, we can distinguish the following four classes: a ŽGSTA., m ŽGSTM., p ŽGSTP. and u ŽGSTT. w73x. Among them, GSTM1 and GSTT1 Žbelonging to m and u classes, respectively. are studied recurrently. Both GSTM1 and GSTT1 detoxify either the products of many CYP-catalyzed reactions or the lipid and DNA peroxides stemming from the oxidative stress w30,71,74,75x, thus suggesting their coordinated activity. Both GSTM1 and GSTT1 are subject to the complete deletion of their encoding genes w76,77x. The proficient GSTM1 gene occurs in two variants, GSTM1UA and GSTM1U B, which differ only for a single base in exon 7, and which encode monomers that can generate homodimeric ŽGSTM1a–1a or GSTM1b–1b. and heterodimeric ŽGSTM1a–1b. enzymes of very similar catalytic effectiveness in vitro w73,78x. Both the homozygous and the heterozygous deletion of the GSTM1 gene have been associated with increased susceptibility to cancer due to deficient detoxification. The homozygous genotype for the null allele, GSTM1 Žyry ., is named GSTM1 null genotype. The frequency of the individuals harboring the GSTM1 null genotype is ; 50% in the Caucasian population w76x, and ranges between ; 30% and ; 90% in different ethnic groups w79,80x. The polymorphism of GSTT1 is another common deletion polymorphism w77,80,81x, whose interaction with GSTM1 is currently under study. As is logical to assume, individuals null at both GSTM1 and GSTT1 loci are thought to be particularly prone to chemical carcinogenesis. Thus, new methods for genotyping GSTM1 and GSTT1 together have been designed. Applying one of them to Caucasian and African–American populations, Chen et al. w82x found that the prevalence of the GSTM1 null genotype was higher in Caucasians Ž53.5% vs. 27.6%; p - 0.001., while that of the GSTT1 null genotype

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Ži.e., the GSTT1 Žyry .., was higher in African– Americans Ž24.1% vs. 15.0%; p s 0.019.. Instead, no significant ethnic differences were observed for the occurrence of the double null genotype for both GSTM1 and GSTT1 w82x. Abdel Rahman et al. w83x, instead, found similar distribution patterns between North American and Egyptian populations, where the prevalence of the GSTM1 null genotype was 51% and 44%, that of the GSTT1 null genotype was 15% and 14.7%, and that of the double null genotype was 6.3% and 8.8%, respectively Žno information was available on the ethnic composition of the North American population examined by the authors.. Among the GST polymorphic enzymes drawing increasing interest are surely GSTM3 and GSTP1. The GSTM3 gene has been found to occur in two different alleles, GSTM3UA and GSTM3U B w84x, the latter of which is thought to be typical of more efficient phenotypes, being expressed at higher levels w85,86x. The expression of GSTM3 has been suggested to be coordinated with that of GSTM1 in a way such that subjects homozygous for the GSTM1 null allele express on average less GSTM3 than do subjects with other GSTM1 genotypes w85,86x. The polymorphisms of the GSTP1 gene were first reported by Board et al. w87x. They consist in an A ™ G transition at nucleotide 313 in exon 5 and in a C ™ T transition at nucleotide 341 in exon 6, which lead to an Ile ™ Val and to an Ala ™ Val substitution, respectively, in the substrate-binding active site of the enzyme w88x. Remarkably, the isoleucine to valine substitution has been associated with reduced conjugating activity of the enzyme w89x, and a recent work by Watson et al. w90x has shown proportionally lowered GST activities in lung tissues from individuals carrying one or both alleles leading to this amino acid substitution. The presence of GSTP1 in many different tissues, combined with its ability to inactivate important carcinogens Žsuch as benzow axpyrene diol-epoxides., suggests that this enzyme plays a key role in detoxifying pathways and, as recently demonstrated by Harries et al. w91x, that GSTP1 polymorphism is implicated with different cancer susceptibilities Žsee below.. Moreover, the high enzyme expression that has been found in tumoral tissues and associated with tumor drug resistance and poor patient survival suggests that GSTP1 polymorphism could play an important role in cancer

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etiology and therapy Žsee Watson et al. w90x and references therein.. 4.2. Cancer susceptibilities related to GST polymorphisms As mentioned in the previous sections, the deficiency of class m GSTs can be a risk factor for lung cancer. In 1986 Seidegard ˚ et al. w92x already showed that smokers deficient of GST m expression are at increased risk for lung cancer. In addition, the presence of the intact GSTM1 gene was shown to be protective against both chemically-induced cytogenetic damage w93,94x and DNA adducts in the lung w95,96x. In particular, Kato et al. associated the GSTM1 null genotype with increased levels of PAH-deoxyguanosine adducts in an already mentioned paper w63x. Moreover, in smokers that were carriers of the GSTM1 null genotype, Grinberg-Funes et al. w97x found an inverse relationship between PAH–DNA adducts and serum levels of vitamin E and vitamin C Žwith significance levels of p F 0.04 and p s 0.06, respectively., thus suggesting the importance of antioxidant micronutrients in the prevention of DNA-adduct formation Žeither from smoke or other environmental exposures. in subjects lacking GSTM1. Finally, Ryberg et al. w98x compared the levels of aromaticrhydrophobic DNA adducts between male and female cancer patients in relation to GSTM1 activity and smoking exposure. They found an excess of GSTM1-deficient male patients with high adduct levels, but higher adduct levels among females than in males when adjusted for smoking dose w98x. This finding may indicate that females are at higher risk than males for smoke-related lung cancer, but also indicate that sex may be an additional confounding factor in detecting smoke-related lung cancer susceptibilities. A more recent paper, again by Ryberg et al. w99x, showed that the level of hydrophobic DNA adducts in the lung of male smokers was influenced more by GSTP1 polymorphism than by GSTM1 polymorphism. According to these authors, smokers carrying at least one GSTP1 mutant allele Žputatively associated with decreased enzyme activity. showed significantly increased DNA-adduct levels with respect to controls, while smokers carrying the GSTM1 null genotype showed only non-

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significantly increased levels. The trend observed for DNA-adduct onset was concordant with that of the degree of lung cancer susceptibility w99x. In addition, when the combined GST M1 and P1 genotypes were examined, patients with the combination GSTM1 null genotype and at least one GSTP1 mutant allele had significantly higher adduct levels than all other genotype combinations w99x. As commented by the authors, their data showed that GSTM1 null genotype had less penetrance than GSTP1 genotype as a risk factor for lung cancer. This observed minor role of GSTM1 agreed with a meta-analysis by McWilliams et al. w100x, where GSTM1 deficiency was concluded to be only a moderate risk factor. Corroborated by the results of Ryberg et al. w99x are the results of To-Figueras et al. w101x, which showed that the GSTM1 null genotype was Žonly. slightly overrepresented in lung cancer cases in the Caucasian population they studied. In addition, the further subdivision of the patients with one or two copies of the GSTM1 gene according to a GSTM1UA, GSTM1U B or GSTM1UArGSTM1U B genotype did not show significant differences between cases and controls w101x. These results differed from what was observed by others for either urinary bladder or laryngeal and cutaneous tumors, for which a protective role of either GSTM1UA w102,103x or GSTM1UArGSTM1U B w104,105x was proposed Žsee the following.. As claimed by To-Figueras et al. w101x, while the initial phenotyping studies showed a clear association between class m GST deficiency and lung cancer, later genotyping studies yielded discordant results, thus suggesting that a model based on the hypothesis that GSTM1U 0 alone confers increased risk may be too simplistic, and that the role of the different alleles of GSTM1 and of other genes of the same family Žsuch as GSTM3 . should be taken into account w101x. Finally, a significant correlation between GSTM1 null genotype and increased lung cancer risk was found specifically in small cell carcinoma patients diagnosed before 66 years of age, in the already mentioned study by Alexandrie et al. w33x. From the literature we reviewed, the monitoring for GSTM1 genotype, as for CYP1A1, also seems useful only as a weak indicator of a predisposing condition, rather than as a marker of an enzyme deficiency strongly affecting the frequency of lung

cancer. As recently reviewed by Rebbeck w80x, the studies providing the most consistent evidence of a relationship between GSTM1 polymorphism and cancer susceptibility are those concerning lung cancer. However, this relationship becomes to be particularly evident when studying the effects of specific genotype–environment or genotype–genotype interactions, such are the interactions of the GSTM1 genotype with smoking andror CYP1A1 genotype. To conclude with respect to lung cancer susceptibility, we point out that the lung as a target organ may have two routes of exposure to environmental carcinogens: the direct absorption of carcinogens by inhalation Žprobably the prevalent route. and the intake of inhaled Žor otherwise assimilated. carcinogens via the circulation system, after their hepatic metabolism. On these bases, since GSTM1 is expressed more in liver than in lung tissue w74x, the presence of the GSTM1 null genotype could allow activated carcinogens, coming from the liver after escaping GSTM1-detoxification, to modify the balance among local metabolic pathways. More generally, given the different degrees of expression between liver and lung of the different GST enzymes, different substrate-carcinogens should determine different metabolic balances between the two organs. This should hold true for different types of carcinogens but, in some cases, for different amounts, too. For instance, high smoking doses might shift a prevalent lung metabolism of inhaled carcinogens to a prevalent hepatic one, given the partial overlapping of substrates for the different GSTs and assuming that these carcinogens are metabolized in the liver when exceeding a given threshold in the lung. This would agree with the theory that lung cancer RR posed by GSTM1 null genotype is highest in heaviest smokers w62x. Besides its putative relationship to lung cancer, GSTM1 deficiency has been associated to the risk of other environment-related cancers, such as those of head and neck, urinary bladder and colon w102,104,106x. An update on this issue is reported herein. An increased risk of head and neck cancer stemming from GST m ŽGSTM1. deficiency has been reported by different authors w107–109x, though recently not confirmed w110x. In a study by Kihara et al. w109x on a Japanese population, an increased frequency of the GSTM1 null genotype was particu-

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larly evident in smoker patients with head and neck non-larynx cancer Žessentially squamous cell carcinoma.. Moreover, among the smoker patients with larynx cancer, an increased frequency of the GSTM1 null genotype was markedly evident in subjects aged below 60 years. Taken together, these results suggest that GSTM1 polymorphism potentially modifies the risk of head and neck cancer depending on smoking history, age and region of cancer onset w109x. On the other hand, in a study by Jahnke et al. w104x on a German Caucasian population, the specific susceptibility to laryngeal squamous cell carcinoma was shown to depend on the null genotype for GSTT1. The study by Jahnke et al. w104x also showed a protective role of both the heterozygote GSTM1 ArB and the homozygote GSTM3 BrB Žaccounting for a high GSTM3 expression. specifically against the larynx tumors, but not against head and neck squamous cell carcinoma located at sites other than the larynx. Finally, a recent study by Jourenkova et al. w111x showed that in smokers the occurrence of the GSTM1 null genotype was significantly associated with increased larynx cancer risk. As regards to urinary bladder cancer, Anwar et al. w112x found in an Egyptian population that, while the frequency of GSTM1 null subjects was significantly increased among cancer patients, that of CYP2D6 EMs was increased only to a nonsignificant extent. Among cancer patients, however, EMs for CYP2D6 harboring the GSTM1 null genotype were significantly more frequent than EMs harboring the proficient GSTM1 genotype, thus suggesting that CYP2D6 EM could play a role in cancer susceptibility if associated with the GSTM1 null genotype w112x. A significantly increased frequency of the GSTM1 null genotype was found also by Katoh et al. w113x in Japanese patients with urothelial Žbladder, renal pelvis and ureter. cancer. Papers concerning cancers of the digestive tract include another work by Katoh et al. w114x in which the GSTM1 null genotype was significantly associated with the susceptibility to gastric adenocarcinoma but not colorectal adenocarcinoma. However, the GSTM1 null genotype became significantly associated with colorectal adenocarcinoma when referring specifically to tumors of the colorectal distal portion w114x. Though the results were valid independently of smoking exposure, the subgroup analysis of

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the gastric cancer patients showed the low-smoking group to have the highest RR for GSTM1 null genotype when compared to the high-smoking group and the non-smoker group w114x. As claimed by the authors, the associations observed were relatively weak, thus requiring further confirmation. Perhaps in this work, as possibly also in others, statistical fluctuations cannot be excluded. Interestingly, the higher predisposing effect to gastric cancer of the GSTM1 null genotype observed here in light smokers compared to heavy smokers, is diametrically opposed to what was observed for lung cancer by Kihara et al. w62x. This contrasting observation could be an example in which the same etiological agent Žtobacco smoke. can activate different metabolic balances depending on the type of target organ. Furthermore, the picture of the relationships between the null genotype for GSTT1 and the risk of gastrointestinal cancer is still fragmentary. As discussed by Katoh et al. w114x, the lack of association between GSTT1 null genotype and colorectal and gastric cancer they observed corroborated the results by Chenevix-Trench et al. w115x, according to which the frequency of this genotype was similar in both colorectal cancer patients and controls. However, it partially contrasted the observation by Deakin et al. w116x that there was a statistically higher frequency of GSTT1 null genotype in colorectal cancer patients, but not in gastric cancer patients. Again concerning colorectal cancer and GSTM1 polymorphism, no overall correlation could be found by Lin et al. w117x among null genotype, cancer risk and smoking habit. Nevertheless, current smoking increased the incidence of colorectal adenomas among both subjects proficient and deficient of GSTM1 w117x. To conclude our overview of colorectal cancer, we report a phenotyping study by Szarka et al. w118x in which the overall GST activities were studied in subjects at risk for colorectal cancer. The risks of these subjects were determined by factors such as personal or family history of colon cancer or of colon polyps. While the overall GST activity in blood lymphocytes of subjects at risk Žcases. was significantly lower than that of controls, no association could be observed between the frequency of the GST m phenotype and the risk for colorectal cancer. As concluded by the authors, the specific contribution of the GST m deficiency to the risk for colorectal cancer remains

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equivocally different from what occurs in lung, bladder and larynx cancer w118x. As reported in a previous section on CYP1A1, no evidence exists that GSTM1 deficiency could pose a risk for breast cancer among smokers, even when combined with CYP1A1 polymorphism w24x. Here we would mention that, in addition to typical smoke-related PAH diol-epoxides, GSTM1 can more generally detoxify other electrophilic moieties stemming from the activation of disparate environmental carcinogens. Thus, we would note that the finding by Fontana et al. w48x of a correlation between early age at tumor diagnosis and GSTM1 null genotype assimilates the risk of breast cancer to that of environmentally induced cancers, as commented by the same authors w48x. It could be added that, specifically for susceptibility to breast cancer, the role of both CYP and GST polymorphisms should prove to be even more difficult to study than in other cancers, due to the involvement of P450 cytochromes and GSTs in the processing of hormones or hormone-like molecules, which are more tissue-specific and difficult to characterize w119x than environmental genotoxins. As is reasonable to assume, the metabolic balance among endogenous hormones and xenobiotic molecules involved in so called receptor-mediated carcinogenesis w11x should be particularly important in breast cell transformation. Finally, concerning the importance of GSTP1 polymorphism in cancer susceptibility, we cite the papers by Ryberg et al. w99x and by Harries et al. w91x. The former authors found that the GSTP1 genotype homozygous for the low-activity allele was highly associated with the incidence of primary non-small cell lung cancer Žwhile only a slight, nonsignificant association was found for the GSTM1 null genotype. w99x. The latter authors found that homozygosity for the low-activity allele was significantly associated with the incidence of urinary bladder and testicular cancer and slightly Žbut not significantly. with that of lung cancer w91x. This association was not found for colon, breast and prostate cancer w91x. Specifically among prostate cancer patients, the authors found a marked Žthough statistically not significant. reduction in the frequency of individuals homozygous for the low-activity allele; however, they also found a highly significant reduction in individuals homozygous for the high-activity allele

and a significant increase in the proportion of heterozygotes w91x. Very recently, GSTP1 polymorphism has also been associated with increased risk of oralr pharynx squamous cell carcinoma w120x. Though the whole of the results indicates that we are faced once more with a complex situation that involves tumors of different origin and different inducibility, it suggests a pivotal role, unsuspected until recently, of GSTP1 polymorphism in cancer outcome. 4.3. Susceptibility to cutaneous BCC According to the notion that GSTM1 and GSTT1 can protect against free-radical damage, Heagerty et al. w105,121x studied GSTM1 and GSTT1 polymorphisms as possible risk factors for cutaneous tumors induced by the oxidative stress following UV exposure or exposure to chemical agents. The individuals expressing the GSTM1 ArB phenotype seemed to be protected from multiple BCC, while those harboring the GSTM1 null genotype were found to have a risk similar to the background risk for developing BCC. Since GSTM1 A and GSTM1 B phenotypes are generally composed of GSTM1U 0 Ži.e., GSTM1 null allele. heterozygotes, this result suggests that the expression of two alleles confers better protection against BCC than the expression of one allele alone w121x. Whether homozygosity for GSTM1UA or GSTM1U B confers protection tantamount to that of GSTM1UArGSTM1U B remained to be determined, since the rarity of the homozygous genotypes hindered their study w121x. On the other hand, a protective effect stemming from the combination of GSTM1UA and GSTM1U B was difficult to explain, due to the similar catalytic properties observed in vitro for their encoded proteins w73,78x. In any case, a sort of gene–dose effect in GSTM1 ArB individuals was observed also by Brockmoller ¨ et al. w102,103x and by Jahnke et al. w104x, who found these individuals to be more protected than individuals carrying one GSTM1U 0 allele against urinary bladder and laryngeal cancer, respectively. GSTM3 polymorphism, too, has been related with the susceptibility to developing BCC. A protective role of GSTM3 BrB against the risk of this cancer was found by Yengy et al. w86x. The results by Yengy et al. w86x, combined with those by Heagerty

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et al. w121x on GSTM1 ArB, are analogous to the findings by Jahnke et al. w104x reported before, and indicate that both GSTM1 ArB and GSTM3 BrB are protective factors against both cutaneous BCC and squamous cell carcinoma of the larynx. This sort of agreement appears to be quite surprising if we consider that these tumors are thought to be induced by different etiological agents Ži.e., by UV radiation the former w86,121x, by alcohol consumption and tobacco smoke the latter w104x.. By contrast, for squamous cell carcinomas of the upper aerodigestive tract, with cellular origin, route of exposure and etiological agents presumably similar to those of the larynx, a protective role of either GSTM1 ArB or GSTM3 BrB could not be demonstrated w104x. Though statistically significant differences in the observed behaviors could be due simply to random fluctuations Žsee Section 6., now that we are able to discern different GST m genotypes we can probably distinguish slightly different enzyme activities that lead to different responses toward the same exposure even in similar cells. Again concerning cutaneous BCC, the influence of different metabolic polymorphisms on the number of tumors and accrual of further lesions was investigated by Lear et al. w122x. The metabolic activities studied concerned P450 cytochromes ŽCYP1A1, CYP2D6. and GSTs ŽGSTM1, GSTT1.. Accrual was evaluated by the number of tumorsryear appearing from first presentation. Both CYP1A1 Žm1rm1. Žcorresponding to the MspI homozygous ‘normal’ genotype. and CYP2D6 EM were factors that were significantly associated with increased numbers of primary BCC. The GSTT1 null genotype and the CYP2D6 EM status were found to be significant determinants of tumor accrual. Moreover, CYP1A1 ŽIlerIle. was associated with slower accrual than CYP1A1 ŽIlerVal. and CYP1A1 ŽValrVal. w122x. While a link between a low GST-mediated detoxification of products of the UV-induced oxidative stress and increased cutaneous BCC is easily surmiseable, not as presumable are the implications found for P450 cytochromes. As discussed by the authors w122x, data showing UV-oxidized tryptophan binds to the Ah receptor and that UV induces CYP1A1 expression in skin suggest a role of CYP1A1 in skin carcinogenesis w123x. Perhaps, a role of CYP1A1 could also imply increased activation of xenobiotics

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Že.g., PAHs. contaminating the skin. In a more recent paper, again by Lear et al. w124x, the polymorphisms of P450 cytochromes and GSTs were investigated for their influence on the time elapsing between first primary BCC presentation and presentation of the next new primary lesion. In particular, the GSTT1 null genotype was found to be significantly associated with a decreased time from first primary tumor to next tumor presentation. Moreover, both GSTM1 null genotype and CYP2D6 EM, while not significant per se, were found to be significant factors in patients whose first tumor presentation was at a truncal site w124x. As discussed by the authors w124x, the role of CYP2D6 EM in cutaneous BCC outcome could likely be explained by a CYP2D6mediated hepatic metabolism of unknown carcinogens, given that CYP2D6 was never detected in the skin w20,123x. Again according to authors, the varying importance of different polymorphisms found in their studies on BCC development indicates that factors influencing one parameter could be different from those influencing another w124x.

5. NATs 5.1. Background NATs transfer an acetyl group to the nitrogen atoms of aromatic amines and hydrazines or to the oxygen atoms of hydroxylated arylamine metabolites. In this way, they can catalyze either generally considered detoxifying reactions, such as N-acetylations, or intermediate reactions leading to DNA reactive metabolites, such as O-acetylations of N-hydroxyarylamines stemming from previous P450 metabolism w125x. NAT-dependent susceptibility to cancer of different tissues seems to depend on different metabolic pathways. In this context, human susceptibility to tumors after exposure to aromatic and heterocyclic amines, a fundamental topic in the cancer prevention field, is believed to depend on a balance among the actions of NATs and of activating, conjugating and deconjugating enzymes; the most important of which are CYP1A2, UDPglucuronosyltransferases and b-glucuronidases, respectively w126x. This susceptibility entails: Ža. the

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nature of the substrate to metabolize and its relative rates of N-acetylation by NATs and cytochromemediated N-hydroxylation occurring in the liver; Žb. the route of excretion of its metabolites and the consequent type of targeted tissues; Žc. the rates of NAT-mediated activation and hydrolysis of its conjugated metabolites in the target tissues w126x. In humans there are two loci encoding functional NAT enzymes, designated NAT1 and NAT2, which are located on the short arm of chromosome 8 and are ; 85% identical at the nucleotide level w127x. Though NAT1 and NAT2 isozymes have overlapping specificities toward some arylamines, they also have unique activities. Low- and high-activity alleles of the human NAT1 gene have been shown w128,129x and, since the gene is expressed in a wide range of tissues, this polymorphism may be as important as the longer known NAT2 polymorphism. Four common alleles of the NAT1 gene have been reported, which differ by changes in the 3X region in and around the putative mRNA polyadenylation signal w130,131x. In particular, NAT1U 4 is the most common and is presumed to be the wild-type allele, while NAT1U 10, which is present in ; 30% of populations with European ancestry w132x, has been associated with an increased NAT1 activity w132– 134x. The gene sequence corresponding to the NAT1U 10 mutated allele contains a C ™ A change at nucleotide 1095 and a T ™ A change at nucleotide 1088, the latter resulting in a shift in the mRNA polyadenylation signal w130–134x. It has been speculated that this new polyadenylation signal could influence NAT1 enzyme levels by producing a more stable mRNA w133x. On this subject, NAT1 activity in individuals heterozygous for the NAT1U 10 allele has been found two-fold higher than in individuals homozygous for the NAT1U 4 allele w134x Žsee the following.. A novel NAT1 polymorphism accounting for low enzyme activity has very recently been found by Payton and Sim w135x. Of the two NAT activities, NAT2 has been the longest known to characterize rapid and slow acetylators w9,52,136x. Slow acetylators, which can occur with frequencies between 10% and 90% in various ethnic groups w136x, are thought to produce enzymes that are either poorly expressed or unstable, or that have partially reduced catalytic activity w137–139x. In spite of the increasing number of mutant alleles

identified for the NAT2 gene Žmore than 25 to our knowledge., those accounting for the majority of the slow acetylator phenotypes are relatively few. As recently reviewed by Meyer and Zanger w52x, the following mutant alleles, namely NAT2U 5A, B, C, NAT2U 6A, NAT2U 7B and NAT2U 13, account for more than 99% of slow acetylators in Caucasian populations. In Asian populations, such as Japanese, Chinese, Korean and Thai, the frequencies of slow acetylators range between 10% and 30%, while most populations of Europe and North America have frequencies between 40% and 70% w52x. Slow acetylators are determined by the homozygous presence of slow-activity alleles, while rapid acetylators include both homozygous and heterozygous carriers of the NAT2U 4 wild-type allele, the former having significantly higher acetylation rates than the latter w140x. Rare mutant alleles associated with rapid acetylator phenotype have also been described w52x. 5.2. Cancer susceptibilities related to NAT polymorphisms The increased susceptibility to cancer stemming from NAT polymorphism has been ascribed to exposure to aromatic amines. As mentioned above, the implications of NATs in cancer susceptibility may be different in different tissues, depending on the type of carcinogenic exposure and on the complex interaction between NATs and other metabolizing enzymes. For instance, both NAT1 and NAT2 rapid acetylators have been predicted to be at increased risk for colorectal cancer. This assumption is based on the existence of an in situ NAT-mediated activation of heterocyclic amines ingested with food Žas is known, heterocyclic amines may derive from the cooking of meat and fish w141x.. On the other hand, only NAT1 rapid acetylators have been predicted to be at increased risk for urinary bladder cancer, while NAT2 rapid acetylators have been predicted to be at lower risk. This assumption is based on the fact that inhaled environmental arylamines are metabolized not only in the urinary bladder but also in the liver, and that in the liver arylamines may undergo NAT2-mediated N-acetylation. This reaction, which competes with the CYP1A2-mediated activation to N-hydroxyarylamines w125,126x, is considered to be a detoxifying step for urinary bladder carcinogenesis

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w134x. Hence, a particularly slow NAT2 acetylation would allow an increase in production of N-hydroxyarylamines which, once reaching the bladder epithelium via the circulation, are converted into N-acetoxy esters through N,O- and O-acetylations, essentially mediated by NAT1 activity w134x. As is known, N-acetoxy esters of arylamines can be DNA-reactive and may lead to mutations w142x. The effect of the combined role of NAT2 and NAT1 on the susceptibility to urinary bladder cancer was provided in a work by Badawi et al. w134x, in which the occurrence of N-acetyltransferase and Oacetyltransferase reactions mediated by NATs and the induction of DNA adducts were studied. Only NAT1 activity was detectable in bladder samples, whereas NAT2 expression was undetectable, thus upholding the notion that NAT2 activity was prevalently expressed in the liver. Moreover, NAT1 activity correlated with the levels of arylamine–DNA adducts found in the urinary bladder samples Žtwofold higher levels of adducts could be associated with the rapid acetylator phenotype.. Finally, the sequencing of the NAT1 gene showed that a polyadenylation polymorphism, NAT1U 10, accounted for the increased NAT1 activity of rapid acetylators w134x. Given that hepatic NAT2 is a key detoxifying step for urinary bladder carcinogenesis while urothelial NAT1 is a major activating one, these results suggest that subjects who are slow NAT2 and rapid NAT1 acetylators are at highest risk of urinary bladder cancer w134x. The influence of NAT2 slow acetylation on the risk of urinary bladder cancer induced by chemicals was first considered in the early eighties w143–145x, and still represents a recurrent issue in studies on genetic polymorphism and cancer susceptibility. A recent paper by Vineis et al. w146x investigated the relationship between NAT2 polymorphism and urinary bladder cancer after low-level exposure to carcinogens. In that paper, the measured levels of 4aminobiphenyl-hemoglobin adducts and of DNA adducts in exfoliated bladder cells were studied as a function of the NAT2 phenotype, the corresponding genotype, and the degree of exposure to tobacco smoke Žas measured by the nicotine–cotinine urinary level.. The authors observed that the hemoglobin adducts in slow acetylators were higher than in rapid ones at low or no detectable nicotine–cotinine levels,

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while the difference between slow and rapid acetylators was less evident at increasing nicotine–cotinine levels w146x. This result showed that the polymorphism of NAT2 may be especially relevant at low exposure levels, as seen above with CYP1A1 and CYP2E1 w31,40x. The early observed correlation between NAT2 slow acetylator phenotype and bladder cancer risk w144x was reinvestigated by Risch et al. w147x using genotyping means. The mutant alleles searched were NAT2U 5A, B, C, NAT2U 6A and NAT2U 7B that, as mentioned by the authors, are responsible for the slow acetylator phenotype in more than 95% of Caucasians w148x. The obtained results confirmed the early findings w144x that the frequency of slow acetylators was significantly increased among both occupationally exposed and smoking cancer patients with respect to controls w147x. Moreover, an increased frequency of slow NAT2 acetylators was also found in those cancer patients without identified exposure to arylamines, thus suggesting that slow NAT2 acetylators are particularly prone to environmentally induced urinary bladder cancer Žthus in line with Vineis et al. w146x.. Furthermore, no single slow NAT2 allele seemed to implicate cancer development more strongly than other alleles w147x. Again on the risk of urinary bladder cancer, the influence of smoking on NAT1 and NAT2 polymorphisms has recently been reinvestigated at the genetic level by Okkels et al. w149x. Indeed, as noted by these authors, very few genotyping studies existed on this subject, contrary to the many early phenotyping studies. Although no overall correlations could be found, a small yet significant association between NAT2 slow acetylator genotype and bladder cancer risk was detectable when the authors restricted their analysis specifically to smokers. In particular, an overrepresentation of the slow acetylator NAT2U 5 was found in the allele frequencies of the case group. As noted by the authors w149x, these findings confirmed that in genotyping studies an influence of NAT2 genotype will be revealed only in analyses comparing exposed individuals w150x. Moreover, the combination of the normal NAT1 and NAT2 genotypes Žrespectively leading to NAT1 slow and NAT2 rapid acetylation. was deduced to be potentially protective against bladder cancer with respect to other genotype combinations w149x. To conclude on the subject of the urinary

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bladder, we again cite the paper by Brockmoller et ¨ al. w103x, where a combined analysis was performed of NAT2 polymorphism together with polymorphisms of GSTs ŽM1 and T1., of microsomal epoxide hydrolase and of cytochrome P450s ŽCYP1A1, 2C19, 2D6 and 2E1., as modulators of the risk of urinary bladder cancer. The analysis revealed that slow NAT2 acetylation was a significant risk factor in heavy smokers, while GSTM1 deficiency was a risk factor independent of smoking and occupation. Moreover, deficiencies of both NAT2 and GSTM1 failed to show significant synergistic Žor antagonistic. interactions. No significant correlations, or no correlations at all, were found for the other polymorphisms investigated. In conclusion, these results illustrated an increased risk for subjects deficient of GSTM1 and confirmed the increased risk for NAT2-slow acetylator smokers w103x. We could note at this point that while all the papers we reviewed give essentially concordant results on the linkage between NAT polymorphisms and urinary bladder cancer, some discrepancies emerge on the effects of low smoking andror environmental exposure to arylamines. A clear vision on the involvement of NATs in colorectal cancer is also lacking, though much has been done and many reference points established after early reports showed that this cancer was related to rapid acetylation w151,152x. In this case, as in others described above, the first to be associated with increased risk was NAT2 polymorphism, and subsequent genotyping studies w153–155x gave contradictory results. Results of difficult interpretation have also be obtained in the more recent studies on NAT1 polymorphism. As in other instances, here again the incomplete overlapping of the genotypes mapped with the phenotypes observed might lead to inconsistent experimental data. However, specifically for colorectal cancer, the type of tumors considered Žadenomas or late-stage tumors., the type of aromatic amines acting as etiological factors Žaryl- or heterocyclic amines. and their route of exposure Žinhalation or ingestion., the type of diet consumed and, finally, smoking seem to be very important factors in evaluating susceptibility, thus explaining apparently discordant reports recurring in the literature. Results of the papers reviewed below provide the basis for this claim.

Concerning NAT1 polymorphism, a study by Bell et al. w132x on a British population found a significant association between colorectal cancer risk and the rapid acetylator NAT1U 10 allele. Among cancer patients, the authors found that subjects harboring NAT1U 10 were more likely to have an advanced stage tumor. As Bell et al. w132x remarked, this last finding suggests that NAT1 has its greatest effect in modulating the carcinogenic process at the level of tumor progression, where activation of environmental carcinogens could be particularly important for the addition of genetic damage to preneoplasticrbenign lesions. Moreover, their finding of a significant correlation, even without grouping cases and controls by different kinds of risk factors, suggests a very strong implication of NAT1 rapid acetylator status in colorectal cancer risk w132x. A subsequent work by Probst-Hensch et al. w156x studied the correlation between NAT1 polymorphism and the occurrence of colorectal adenomas Žprecursors to colorectal cancer. in a Southern California population. The authors observed neither an increased prevalence of adenomas among subjects homozygous or heterozygous for the NAT1U 10 allele, nor an interaction between NAT1 and NAT2 genes. These results, while in keeping with previous ones by the same authors on colorectal adenomas and NAT2 polymorphism w155x, are apparently in contrast with those by Bell et al. w132x. However, these results can be explained by stating again that the effect of the NAT1U 10 allele and aromatic amines was found only at late stages in colorectal tumorigenesis, as described above w132x. The unequivocal but scarce results available led the authors to conclude that further alleles of the NAT1 gene must be investigated for more conclusive understanding of the relevance of NAT1 in colorectal cancer w156x. As mentioned above, more numerous Žbut no less conflicting. than those concerning NAT1, are the notions on the involvement of NAT2 polymorphism in colorectal cancer. A recent study by Welfare et al. w157x is enlightening for an understanding of previous, apparently discordant results. The study considered 174 incident cases of colorectal cancer and 174 matched controls of a British population. As reported by the authors, no difference in the frequency of the rapid NAT2 acetylator genotype was observed between cases and controls, and the analysis by sex,

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age and site also revealed no difference in acetylator genotype w157x. However, the analysis for risk factors showed that recent smoking Žand to a lesser extent heavy alcohol consumption, as well. was more recurrent in the slow NAT2 acetylator cases than in controls, while frequent fried meat intake was more recurrent in the rapid NAT2 acetylator cases than in controls w157x. These results indicate that there might be different environmental risk factors for colorectal cancer in rapid and slow acetylators, the most important of which would be fried meat consumption and smoking use, respectively. In such a situation, the multiplicity of possible combinations may result in an overall neutral effect of the acetylator status, as was observed by the authors studying the population as a whole w157x. Moreover, these results suggest that some previously found discrepancies between colorectal cancer risk and acetylator status can be simply explained by different environmental exposures of the subjects under study. Notably, they stress that also slow acetylation Žspecifically dependent on NAT2 polymorphism. may be involved in the carcinogenic effects of these exposures. Thus, arylamines inhaled with tobacco smoke Žand transported to the colon via circulation. would increase their carcinogenic potential in subjects with a low NAT2-mediated detoxification occurring in the liver. This cascade of events is concurrent with what was observed for bladder cancer susceptibility w103,149x. On the other hand, heterocyclic amines ingested with food Žand directly reaching the colon. would increase their carcinogenic potential in subjects particularly proficient in N,O- and O-acetylations, occurring locally, which convert oxidized metabolites into DNA-reactive N-acetoxy esters. This would be consistent with the greater risk of colorectal cancer observed in subjects with the rapid acetylator NAT1 mutated allele w132x. Hence, these observations uphold our previous statement that both type of aromatic amine and route of its intake are critical for susceptible subjects. To conclude our review on the relationships between colorectal cancer risk and heterocyclic amine ingestion, we report the results of a recent genotyping study performed by Gil and Lechner w158x on a Portuguese population. Colorectal cancer patients showed a higher frequency of subjects homozygous for the NAT2 wild-type allele Žaccounting for the

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rapid acetylator genotype. than the control population. Moreover, the observed difference was particularly marked among males, thus pointing to a sexspecific effect of the NAT2 rapid acetylator genotype. Considering the importance of broiled and fried fish in the diet of the Portuguese population, the authors w158x commented that the lack of association between NAT2 rapid acetylator genotype and colorectal cancer risk previously observed in other populations, such as the Japanese w153x and Scottish w154x ones, could be ascribed to differences of dietary habits. This comment agrees with what has been commented above. The association of cigarette smoking and NAT2 slow acetylator status was shown by Ambrosone et al. w159x to be a significant risk factor for breast cancer, also. As reported, while neither smoking nor NAT2 status was independently associated with breast cancer risk, smoking did increase the risk about four-fold among postmenopausal slow-acetylators. As commented by the authors, the different results they obtained between postmenopausal and premenopausal women could reflect different etiologic pathways or intrinsic differences in the disease w159x. Although statistically significant, the results obtained were considered by the authors as preliminary. As a matter of fact, the possible relationships between metabolic polymorphism and cancer susceptibility presently found are prevailingly viewed with caution, after decades of exciting, yet often conflicting, results. The need for caution, especially when results are obtained with small numbers of subjects, is highlighted in an editorial by Lin w160x in which the paper by Ambrosone et al. w159x is also discussed. 6. Conclusions The carcinogenic process is characterized by the sequential mutations of protooncogenes and tumor suppressor genes that lead to qualitative andror quantitative alterations of their encoded proteins, thus determining the transformation of a normal cell into a neoplastic one w161x. In this context, there are genes whose products are critical for protecting protooncogenesrtumor suppressor genes against mutations, which therefore themselves may be involved in cancer outcome. These genes include, among

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others, mitotic checkpoint genes protecting from chromosomal instability w162x, genes encoding for enzymes devoted to DNA replication fidelity w163x, DNA repair genes w164x, and, the subject of the present paper, genes related to metabolism of potentially DNA-damaging chemicals. Inherited alterations in carcinogen metabolism, while less dramatic than inherited alterations in oncogenesrtumor suppressor genes at the individual subject level, can have a stronger impact in the population at-large, as we have reviewed here, because they can affect large portions of an entire population. The first impression given by the literature reviewed is that, excluding cases of populations in which an allelic variant is markedly frequent, the statistical significance of the association between a genetic polymorphism and susceptibility to a given malignancy is difficult to establish. Apart from obvious confounding factors, such as differences between cases and controls in environmental exposures or in the intake of antioxidant micronutrients Žas exemplified by Ref. w97x., the detection of an association between a polymorphism and a given cancer susceptibility may be hidden by a series of different factors. These may include the simultaneous need for other polymorphisms to be expressed Žthe case of CYP1A1 polymorphism is classic. or, conversely, the overlapping of substrate affinities of several enzymes, such that deficiency of a given enzyme may be deputized by the presence of another Žas discussed before, partially overlapping activities are common to either CYP or GST or NAT enzymes.. In particular, the association between CYP1A1 activity and susceptibility to cancer appears to be correlated both with the expression of different types of polymorphism located in the same, CYP1A1, gene w22x and with genotype–genotype interactions, such as those entailing CYP1A1 and GSTM1 gene polymorphisms w31– 33,41x. Not only that, the polymorphism of CYP1A1 gene has been hypothesized to be linked with that of other metabolic genes and, also, of cancer associated genes Žsuch as oncogenes and tumor suppressor genes. falling into the same chromosomal region in which CYP1A1 is located w18,20x. Moreover, the level of CYP1A1 enzyme activity seems not only to depend on CYP1A1 gene polymorphism, but also on the polymorphism of AHR and ARNT genes, which encode for proteins regulating CYP1A1 transcription

w13,18,20x. It could be added that the reported abilities of the activated Ah receptor to interfere with components of the endocrine system w11x, and of the AHR-induced CYP1A1 and CYP1A2 enzymes to process hormones w45x, suggest the existence of further confounding factors, e.g., those linked with the hormonal status and sex of the subjects in which CYP1A1 polymorphism effects are studied. Other considerations could arise from the reported AHR ability to induce the synthesis not only of CYP1A1 and CYP1A2, but also of Phase II enzymes w9x, such that an altered efficiency of AHR Ždetermined by AHR and ARNT gene polymorphisms. could lead to either an increased activation or an increased detoxification of chemical carcinogens, depending on the type of carcinogens to be metabolized. For instance, an increased efficiency of AHR could lead to increased activation of carcinogens that are specifically activated by AHR-induced cytochromes, but that can be detoxified by different types of Phase II enzymes Žsuch that an increase of AHR-induced Phase II enzymes would not significantly increase the overall detoxifying metabolism of these carcinogens.. Also, a decreased AHR efficiency could lead to increased activation, in this case of carcinogens that are specifically detoxified by AHR-induced Phase II enzymes, but that can be activated by different types of cytochromes Žsuch that constitutive cytochrome activities could deputize the decreased activity of AHR-induced cytochromes.. Increased carcinogen detoxification could occur in the opposite cases. Another confounding factor is that even the allelic variants of a same gene that yields opposite effects Ži.e., higher or lower enzyme activity. may both be causes of increased cancer susceptibility, although by different means. A classic example in this regard is given by CYP2D6 gene polymorphism that, as previously discussed, can favor either the activation of genotoxins to DNA-reactive moieties or the storing of unmetabolized drugs, possibly leading to tissue necrosis. Both mutations induced by DNA damage, and cell proliferation induced by necrosis, are events entailed in the carcinogenesis process w2,3,59,60x. The above examples show some of the causes for difficulty in interpreting the relationships between enzyme polymorphisms Že.g., CYP polymorphisms. and cancer susceptibility. However, apart from the difficulties posed by the intrinsic nature of the inter-

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actions between chemical carcinogens and metabolic enzymes in biological targets, the increasingly available protocols for genotype analysis are promising as tools to overcome interpretation difficulties at the genetic level Žsuch as that posed by the role of AHR and ARNT genes in CYP1A1 induction.. The ability of the genotyping approach to detect heterozygous mutant genotypes already allows verifying cancer susceptibilities even among ‘silent carriers’ or, at any rate, among populations that were hard to categorize using old phenotyping techniques. On this subject, the association between the occurrence of at least a single CYP1A1 m2 allele and lung cancer risk, as observed in Caucasians by Xu et al. w29x and Alexandrie et al. w33x, or that between at least a single GSTM1 null allele and breast cancer risk, as observed by Fontana et al. w48x, were more revealing than similar phenotyping studies. Obviously, the usefulness of this approach should be based on a sort of gene–dose effect according to whether the gene can accurately predict the translated enzyme activity. On this basis, heterozygous genotypes of a hyperactive variant would be subject to excessive drug activation or, in the case of a hypoactive Phase II enzyme variant, to a reduced detoxification in tissues that are susceptible to carcinogenesis compared to their respective wild genotypes. On the other hand, our review seems to evince a difference in enzymatic capacity depending on the presence of one or two proficient alleles. The higher acetylation capacity ascribed to the homozygous carriers of the NAT2 high activity allele compared to the heterozygous ones w140x, or the protective gene–dose effect against some types of cancer found for proficient GSTM1 alleles w102–104,121x, are reviewed examples on this topic Žanother example of gene–dose effect wconcerning CYP1A1 and GSTM1 genotypesx has been reviewed by Rebbeck w80x, as discussed herein.. In addition, the genotype analysis allows categorizing specific haplotypes that are more or less prone to a given cancer risk Žan example is the paper of Garte et al. w22x. and, more generally, allows studying linkages among gene polymorphisms. As a matter of fact, the first observation we made after scoring most recent works is that many of their findings are conditioned by the word ‘however’, because of the many linkages increasingly found among different, polymorphic genes. As seen, the complexity of these

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findings may reflect both genetic linkages Žproximities on the same chromosome of different metabolic susceptibility enzymes. and interactions among the expression of different genotypes. These factors are probably the cause of the recurrent discrepancies found between genotyping and phenotyping results w64,65,101,153–155x, where the latter presumably reflect an overall balance among different metabolic activities, in turn reflecting a balance among different Žthough correlated. gene expressions. On one hand, this may show that the knowledge at the genotype level is far from exhaustive; on the other, it may indicate the possibility to know fragments of metabolic pathways linked to several polymorphisms which, assayed together, could be applied to detect individuals who are particularly cancer prone. In fact, given the low penetrance of the single metabolic genes in determining individual cancer susceptibility, only the screening for polymorphism of coordinated enzyme activities could be useful for the monitoring of each individual. It has to be underlined that the final role of a given Žactivating or detoxifying. enzyme function is played at the level of the pool of proteins of the same family that is globally involved in that catalytic function. Moreover, in view of the many possible interactions between carcinogens and target tissues, this monitoring should be applied only to types of exposure whose carcinogenic effects are presumed to exceed those of background environmental exposures. Thus, a suitable application of the genetic polymorphism analysis should consider not only genotype–genotype interactions, but also interactions among genotypes and specific conditions of exposure to the carcinogenic agent that are determinants for the cancer risk under evaluation. Examples of the associations existing between exposure condition and type of induced cancer are given by the carcinogenic effects of tobacco smoke. As reviewed, these effects occur in different target organs and may be amplified by both GSTM1 w92,109,111,114x and NAT2 w147,149,157,159x polymorphisms, and also by CYP1A1 and GSTM1 polymorphisms combined together w31,32x. The importance of genotype–genotype and genotype–environment interactions in determining cancer susceptibility is thoroughly discussed in the already cited paper by Rebbeck w80x, on GSTM1 and GSTT1 polymorphisms. As regards GSTM1 polymorphism,

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Rebbeck observed that the risk of cancer determined by the GSTM1 null genotype is small in magnitude Žcorresponding to an odds ratio of less than 2.. However, this risk grows when the interaction of the GSTM1 genotype with other factors, such as CYP1A1 genotype or tobacco smoking, is considered Žfor lung cancer, the odds ratio increases up to values of around 6.. In this model, the risk of lung cancer due to the interaction between GSTM1 genotype and environment increases with increasing smoking doses. On the other hand, the risk due to the interaction between GSTM1 and CYP1A1 genotypes increases with the increasing the number of ‘unfavorable’ allelic variants present, according to a gene–dose effect involving both genotypes Žthe individuals with the highest RR are carriers of the combined CYP1A1 ValrVal and GSTM1 null genotypes, while those with the lowest RR are carriers of the combined GSTM1 Žqrq . and CYP1A1 IlerVal genotypes, with GSTM1 Žqrq . : CYP1A1 IlerIle as the reference group.. According to this model, the cancer risk increases greatly when all the three predisposing factors Ži.e., GSTM1 and CYP1A1 polymorphisms and smoke exposure. are combined together Žfor lung cancer, the odds ratio increases up to 21.9 w32x.. Analogous considerations, though less complete, are made for the GSTT1 polymorphism w80x. As discussed by Rebbeck, these facts have implications for future applications of GSTM1 and GSTT1 polymorphisms as biomarkers in cancer prevention or control strategies. The common occurrence of GSTM1 and GSTT1 null genotypes implies that the proportion of cancers attributable to them may be large in the general population; at the same time, their relatively small contribution to the absolute risk of cancer suggests that they may be less suited for individual cancer risk assessment w80x. Further problems in establishing relationships between genetic polymorphisms and cancer susceptibility may stem from factors of enzyme location. While carcinogenesis as a process is basically similar in different tissues Žinvolving both alterations in protooncogenesrtumor suppressor genes and clonal expansion of preneoplastic cells., the chemicals involved in the process Žinducing oncogene alterations and sometimes favoring cell growth. may differ from one tissue to another. Apart from more obvious factors of distribution and absorption peculiar for

each tissue, this varying activity of chemical carcinogens also depends on the different expression of drug-metabolizing enzymes in different cell types, such that each chemical carcinogen is prevalently Žor exclusively. active in specific target tissues. The varying metabolizing behavior among different cells of the upper respiratory tract toward presumably identical environmental factors is representative of specific target tissues, as suggested by the work of Jahnke et al. w104x. Further, the intracellular balance among metabolic pathways may also depend on exposure levels, given that the interaction of several metabolizing enzymes with different substrates may vary depending on the concentration of substrate itself Žsee Refs. w31,39,61,62,146x and, for a review, Ref. w165x.. Finally, the varying activity of chemical carcinogens in different tissues may depend on different routes of chemical exposure to which tissues are subject. A classic example in this regard is the NAT-dependent carcinogenic effects following arylamine exposure Žas discussed herein.. To conclude, though probably not exhaustive, the papers we reviewed suffice to understand the many factors of variation of metabolic polymorphism effects. These factors have been summarized in Table 1. They may be genetic factors ŽNos. 1–5., or individual factors, linked to the hormonal status and lifestyle ŽNos. 6–9.. Moreover, they may be exposure factors, linked to the typerdosage of the carcinogens and the typermetabolic properties of the target tissues involved ŽNos. 9–15.. As we have reviewed, factors of a given type may both be interlaced among them and with factors of other types, thus generating a myriad of combinatorial possibilities. A different type of problem is posed by the statistical significance of the reported associations between metabolic polymorphisms and cancer susceptibilities in specific target tissues, given that in a complex system with many degrees of freedom Žas a biological organism consisting of multiple target tissues is. some association should be expected to be significant by chance, due to statistical fluctuation. Excessively general findings relying on a single positive epidemiologic study, even if based on statistically significant results, could induce to false conclusions. On the other hand, the weight attributable to negative studies should also be evaluated, taking into

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Table 1 Factors of variation of metabolic polymorphism effectsa Ž1. Linkage of the polymorphism of a drug metabolism gene with the function or the polymorphism of other genes not directly related with it Žamong which oncogenes. w18,20x Ž2. Linkage of the polymorphism of a drug metabolism gene with the polymorphism of other drug metabolism genes Žgenotype–genotype interactions. w31–33,41,80x Ž3. Linkage of the polymorphism of a drug metabolism gene with other polymorphisms of the same gene Žpossibility of different haplotypes. w22x Ž4. Linkage of the polymorphism of a drug metabolism gene with the polymorphisms of other genes regulating the expression of that gene Že.g., linkage of CYP1A1 gene with AH and ARNT genes. w13,18,20,34x Ž5. Gene–dose effects Žthe presence of two functional alleles may be more predisposing torprotective against cancer outcome than the presence of a single one. w80,102–104,121,140x Ž6. Sex w98,158x Ž7. Age at the outset of the carcinogenic exposure Žpossible relationships with the endocrine function and hormonal status. w47x Ž8. Linkage of the polymorphism of a drug metabolism gene with the endocrine function Že.g., by connections with hormone metabolism, or by affecting responses to hormones. w11,45x Ž9. Lifestyle Že.g., intake of antioxidant micronutrients, broiledrfried meats andror alcohol consumption, tobacco smoking. w43,44,47,97,109,111,157,159x Ž10. Type of carcinogenic agent Žgenotype–environment interactions. w80x Ž11. Type of exposure to the carcinogenic agent Že.g., lowrhigh dose exposure, shortrlong time exposure duration, lowrhigh cumulative dose. a Ž12. Type of slope of the dose–response curve Žthe substrate interaction of a drug metabolizing enzyme may vary as a function of the concentration of the substrate itself. w31,39,61,62,146,165x Ž13. Type of exposure to carcinogens of the target tissue Že.g, direct exposure to environmental arylamines, exposure to their metabolites by urine or through circulation. w134,157x Ž14. Type of metabolizing enzymes available to the target tissue w36,37x. Their possible overlapping of substrate specificities w51x Ž15. Double-edged sword nature of metabolic polymorphism Žwhat might be a susceptible genotype in one situation may be protective in another. w9,58x a

See the text for details.

account the power of the statistical tests used Ži.e., the probability of yielding a significant result in the presence of a ‘non-negligible’ effect.. In this context, particular caution is a must when studying diseases with high incidence and mortality, where even a modest increase of RR can have strong impact on public health policies. For these reasons, the practicability of a combined analysis Ži.e., a meta-analysis. that incorporates all the methodologically acceptable case-controlrcohort studies available in the literature, independent of their positive or negative results, must be considered. To decide on a possible association between a given polymorphism and a given type of tumor, analogous studies testing the same polymorphism vs. either other tumor histologic types or other target tissues should also be included in the analysis. Such a procedure should obviate, at least partially, the problem of downgrading the level of protection of the statistical test that occurs whenever multiple comparisons are independently tested for their significance Žmultiple testing

bias.. Moreover, both positive and negative studies should be evaluated for their validity Ži.e., the extent to which their design and conduct are likely to prevent systematic errors and biases. and precision Ždeducible by the size of the confidence interval surrounding the estimate.. It must be added that studies with negative results are more likely to remain unpublished because investigators Žor peer reviewers and editors. could be not inclined to publish ‘negative’ information Žpublication bias.. Finally, we recall that, most likely, results could be definitely accepted when ‘mechanistically explainable’, that is, when placeable in a scheme in which both the mechanism of action of the chemical agents considered and the metabolic capacities of the target tissues involved concur to explain findings. Considering the many limitations affecting any relationship between metabolic polymorphisms and cancer susceptibility, an analysis of these polymorphisms as possible markers for individual susceptibility must be limited to specific cancer risks follow-

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Table 2 Relationships traceable between polymorphic expression of drug-metabolizing enzymes and susceptibility to environmentrelated cancersa Smoke-related lung cancer CYP1A1 high inducibility CYP1A1 MspI mutant CYP1A1 MspI mutantq GSTM1 null CYP1A1 MspI mutantq GSTM1 positive GSTM1 null

GSTM1 null q GSTP1 low activity GSTP1 low activity CYP2D6 high activity

Head and neck cancer GSTM1 null GSTM1 ArB or GSTM3 BrB GSTT1 null Urinary bladder cancer GSTM1 null GSTP1 low activity GSTM1 A NAT2 slow

NAT1 rapid NAT1 slow q NAT2 rapid Gastrointestinal cancer CYP1A1 MspI homozygous mutant GSTM1 null

NAT2 slow NAT2 rapid

NAT1 rapid

Increased risk of adenocarcinoma in non-Japanese individuals w36x Increased cancer risk in both Japanese w17,25x and Caucasian individuals w29x. Worse prognosis w41x. Higher cancer RR at lower smoking exposure w31x Increased risk of squamous cell carcinoma in both Japanese w31,32x and Caucasian individuals w33x Protection against cancer outcome w32x Increased induction of PAH–DNA adducts in lung in Japanese individuals w63x. Slightly increased risk of adenocarcinomarsmall cell carcinoma w33x and of squamous cell carcinoma w101x in Caucasian individuals. Higher cancer RR at higher smoking exposure w62x Increased induction of aromaticrhydrophobic DNA adducts in lung in Caucasian individuals w99x Increased risk of squamous cell carcinoma w99x Increased induction of 7MeG–DNA adducts in lung, with higher increases with respect to control values at lower smoking exposures w63x. Increased risk of squamous and small cell primary lung cancer w61x. Higher cancer RR at higher smoking exposure w61x. ŽIncreased cancer risk, particularly of adenocarcinoma, was associated also with the presence of additional copies of proficient CYP2D6 gene w64x.. ŽNote: the systematic analysis of the literature shows that recent genotyping results are contradictory and do not support the former hypothesis that high CYP2D6 activity predisposes to overall lung cancer outcome.

Increased risk of larynx w111x and non-larynx w109x cancer in smokers Protection against laryngeal squamous cell carcinoma w104x Increased risk of laryngeal squamous cell carcinoma w104x

Increased cancer risk w112,113x. ŽA synergistic interaction with this polymorphism was suggested for CYP2D6 EM w112x. Increased cancer risk w91x Protection against cancer outcome w102,103x Increased induction of arylamine–hemoglobin adducts, with higher increases with respect to control values at lower arylamine exposure w146x. Increased cancer risk in both smokers w103,149x and non-smokers w147x. ŽNo synergistic or antagonistic interaction with GSTM1 was found for this polymorphism w103x. Increased induction of arylamine–DNA adducts in the bladder w134x. Increased cancer risk w134x Protection against cancer outcome w149x

Increased risk of colorectal cancer specifically in Japanese and Hawaiian individuals w44x Increased risk of gastric adenocarcinoma and distal colon adenocarcinoma w114x, but not of overall colorectal adenocarcinoma and adenoma w114,117x. ŽContradictory results were found on the association between GSTT1 null and colorectal cancer w114–116x. Increased risk of colorectal cancer in smokers w157x Increased risk of colorectal cancer if associated with fried meat consumption w157x. ŽHomozygotes for the NAT2 rapid acetylation allele were found to be at higher cancer risk than heterozygotes, particularly among males w158x. Occurrence of late-stage w132x, but not early stage w156x, colorectal cancer

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Table 2 Žcontinued. Relationships traceable between polymorphic expression of drug-metabolizing enzymes and susceptibility to environmentrelated cancersa Breast cancer CYP1A1 MspI mutant

GSTM1 null Žfor either one or both alleles. NAT2 slow

Increased cancer risk specifically in women smokers who began smoking before the age of 18 w47x. ŽContradictory results were found on the association of this polymorphism with increased cancer risk in African–American women w24,45x. Increased cancer risk in the youngest postmenopausal women w48x Increased cancer risk in postmenopausal women if associated with smoking w159x

Cutaneous BCC CYP1A1 MspI wild CYP1A1 IlerIle CYP2D6 EM GSTT1 null GSTM3 BrB or GSTM1 ArB

Increased numbers of primary tumors w122x Decreased tumor accrualb w122x Increased numbers and accrual of tumors w122x Increased tumor accrual w122x. Decreased time to presentation of further lesions w124x Protection against cancer outcome w86,121x

a

The different types of polymorphic enzyme expression are listed in the first column, while their related effects on cancer susceptibility are listed in the second one Žsee the text for details.. b Accrual is defined as the number of tumorsryear occurring from first tumor presentation.

ing specific types of exposure. In this context, some telling associations are now emerging in the literature. We have summarized them in Table 2, taking the most significant andror less controversial results from the papers we reviewed. The most consistent models of cancer susceptibity that are presently available regard the lungs Žand upper respiratory tract., the colon and the urinary bladder, i.e., target organs which may be directly exposed to environmental carcinogens. The urinary bladder can be directly exposed since conjugated metabolites present in the urine can be restored to carcinogens within this organ w126x. A local exposure Žto UV radiation. is reasonably implicated with cutaneous BCC susceptibility w122x, though a local and a systemic hepatic metabolism of xenobiotics can be also hypothesized. Somewhat less expected could be the classification of breast cancer as environment-related, arguable by its proposed relationship with cigarette smoking w47,159x. Among the metabolic pathways subject to genetic polymorphism, those mediated by NAT enzymes toward aromatic amines seem to be relatively easier to delineate. The possibility to refer to specific types of etiological agents of well-known chemical structure and mechanism of action allows tracing meaningful mechanistic schemes. Both rapid and slow acetylation have been associated with increased risk

of specific types of cancer, depending on one of two distinct biochemical pathways of amine processing: one occurs locally, in the organ liable to cancer; the other occurs systemically, through the liver. The first of these pathways leads to the activation of carcinogens in target tissues directly in contact with the environment; the second provides for the detoxification Žand facilitated urinary excretion. of carcinogens absorbed from the environment and then passed into circulation. Hence, we find that colon w132,157x is coupled with urinary bladder w134x in the risk of cancer stemming from a locally increased NAT activity, and that colon w157x, urinary bladder w103,149x and breast w159x are associated for the risk of smoking-related cancer, stemming from a low, NATmediated hepatic detoxification. From the literature, a model implicating both the local and remote metabolism of carcinogens also seems to emerge for lung cancer susceptibility, after exposure to PAHs and arylamines of tobacco smoke w62,85,166x. Among metabolic variants, those concerning detoxifying enzymes should be the most suitable to be tested as general biomarkers of cancer susceptibility. As seen before, the association of NAT activities to cancer susceptibility is two-fold, depending on the exposure route of carcinogens to the target organs in question. On the other hand, products of P450-mediated activation may be involved with the increase of

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Phase II detoxifying enzymes, or may have dual carcinogenic activities Žeither genotoxic metabolites or high levels of unmetabolized toxic substrates may be carcinogenic.. Therefore, while a polymorphism leading to decreased detoxification should only predispose individuals to cancer, polymorphisms leading to increased P450 or NAT activities may not always be expected to have such an effect. This seems to be confirmed by the data of Table 2, where the GSTM1 null genotype and, more generally, genotypes leading to decreased GSTs are always associated with an increased risk for all types of cancer considered. Pressed to hazard a hypothesis, the GSTM1 null genotype appears to be one of the most likely individual biomarkers of overall susceptibility to cancer. Even though the complex implications of GSTM1 in chemical carcinogenesis limit its penetrance as an individual risk factor for each single cancer type, the generalized presence of the GSTM1 null genotype among the factors determining many different types of cancer suggests that the monitoring for this genotype as a marker of overall cancer susceptibility could be a feasible hypothesis. Thus, considering that in Caucasians the GSTM1 null genotype occurs with a frequency of ; 50% among healthy subjects, a large-scale monitoring for the frequency of this genotype among overall cancer patients could be informative for prevention purposes. At the level of cancer prevention, given the possible associations of the GSTM1 gene with other genes w85,86x, and the possible interactions of the GSTM1 genotype with other genotypes and with the environment w31,32,80x, the individual detection of the GSTM1 null genotype could serve as the basis for further genotype analyses tailored to individual situations of cancer risk. A strategy of this kind would be also strengthened by the fact that, differently from most gene polymorphisms, that of GSTM1 depends only on the deletion of the entire gene, thus giving rise to a nearly absolute response that leaves little room for doubt. Obviously, this strategy should be implemented as a simple prescreening of individuals that are putatively at increased cancer risk. A more circumstantial application of GSTM1 genotyping should also consider both the risk posed by the heterozygous genotype for the null allele and the possibly different protective role against cancer exerted by GSTM1UA and GSTM1U B Žsee Ref. w80x

and references therein for further speculations.. Similar considerations could be valid for the GSTT1 null genotype, as well. Considering that the detoxifying role of GSTs stems from their ability to increase the rate of a reaction, i.e., substrate conjugation with GSH, which already occurs spontaneously w71,167x, individuals found to be deficient of GSTs could be helped to prevent cancer by increasing their GSH supply. In this view, treatment strategies with Nacetylcysteine ŽNAC. could prove useful, given the absence of significant negative side effects following its chronic administration. It has been shown that administration of NAC increases the intracellular level of cysteine and, subsequently, also the level of GSH w168x. Higher levels of GSH imply that its consumption during the process of conjugation with the genotoxic agent Žcatalyzed by different GSTs. will take place only at higher levels of exposure to the genotoxic xenobiotic. Another, more direct, preventive measure could be the intake of antioxidant micronutrients as dietary supplements Žas Ref. w97x suggests.. Presently, feasible applications of metabolic polymorphism analysis have been reviewed above. These allow detecting subjects more prone to specific types of cancer in case-control studies. The papers by Goto et al. w41x and by Lear et al. w122,124x on the prognostic significance of some CYP and GST polymorphisms for lung cancer and cutaneous BCC are exemplary. Moreover, they entail the prediction of susceptible subjects to adverse drug responses Žwhere the number of genes involved is expectably lower than in carcinogenesis., as exemplified by the paper of Chen et al. w55x. In conclusion, our review shows that the factors of variation of metabolic polymorphism effects are so numerous and disparate that what might be a susceptible genotype in one type of exposure may be protective in another. In addition, there are a multiplicity of metabolic enzymes in supergene families which have overlapping substrate specificities. Furthermore, these enzymes may play a non-negligible endogenous role. These facts demand that the definition of individuals as ‘susceptible to cancer’ be strictly circumstantial, and likewise warn against the potential abuse of labeling individuals as such. Given these premises, the following considerations can be made. Techniques of molecular genetic analysis now

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enable us to find more diverse metabolic polymorphisms, and to propose new or to define current pathways of cancer susceptibility. At present, genetic polymorphism monitoring exists for the detection of patients more prone to specific types of cancer or to the adverse effects of specific pharmaceutical agents. A systematic population screening of genetic polymorphisms to detect individuals predisposed to cancer seems to be an achievable goal only in a relatively distant future, once present uncertainties of metabolic pathways are resolved and techniques allowing the genotyping of large sets of genes are widely adopted Žthus encouraging a profitable costreffectiveness ratio.. At present, possible exceptions could be cases of high occupational exposure to specific chemical agents, or of high racial predisposition Žanalysis results should remain confidential, to avoid risks of individual discriminations.. Another exception could be the exposure to tobacco smoke Žthough, in this case, anti-smoking campaigns might still prove to be the most effective preventive measures.. Finally, given the double-edged sword nature of metabolism polymorphism, namely that both wild and mutant alleles can predispose individuals to cancer following different exposures, individual susceptibility should be monitored as a function of the nature and route of intake of the carcinogen to which the individual under study is known to be exposed. The new chip technologies that have emerged over the last few years w169,170x will allow for the genotyping not only of one or at most two or three metabolic susceptibility genes, but, systematically, for the genotyping of relatively large sets of all the major families of Phase I and Phase II metabolism genes. It will be possible to systematically explore not only DNA sequences, but also mRNA levels in different target tissues. The obvious advantages of these new technological possibilities will likely be accompanied by equally obvious problems: the groups of individuals with the same genotyping Žand similar mRNA levels. will become extremely small. In addition, the multiplicity of sets to be compared will become so large that it will be possible to generate even more easily than today apparently significant differences that are in fact only statistical fluctuations. Finally, given that the crucial events of activation and detoxification take place not at the DNA or mRNA level, but rather at the protein

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