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药物和化学物毒性反应的遗传基础

Genetic basis of toxic reactions to drugs and chemicals

Ingolf Cascorbi  

Institute of Pharmacology, University Hospital Schleswig-Holstein, Hospitalstr. 4, D-24105 Kiel, Germany

Abstract

Inter-individual drug effects are subject to substantial variability. There are multiple reasons based on pathophysiological factors and environmental interactions, but also genetic characteristics. Groundbreaking successes have been achieved in the field of pharmacogenomics and toxicogenomics. In particular, the identification of hereditary polymorphisms in genes of the cytochrome P450 system and phase II-enzymes such as TMPT contributed considerably to the explanation of the individually varying pharmacokinetics of a number of drugs. Furthermore, hereditary variations in genes of membrane drug transporters were recently discovered. Along with these factors, which could influence pharmacokinetics, strong efforts have been undertaken to clarify the role of genetic polymorphisms in receptors or signal transduction proteins modulating drug efficacy. Particularly for malignant diseases such as bladder or lung cancer, polymorphic foreign compound metabolizing enzymes have been identified as susceptibility factors, modulating an individual‘s cancer risk dependent on the extent of environmental exposure.

This review focuses on the role of the polymorphic phase I enzymes cytochrome P450 1A1, 1A2, 1B1, 2C9, 2C19, 2D6, 3A5 and myeloperoxidase as well as on the phase II-enzymes arylamine N-acetyltransferases 1 and 2, glutathione S-transferases M1 and T1, and thiopurine S-methyltranferases as detoxifying but also toxifying factors, modulating pharmacokinetics and disease susceptibility.

Keywords: Drug metabolism; Idiosyncratic reactions; Toxification; Pharmacogenetics; Cytochrome P450

1. Introduction

The inter-individual effects of drugs and xenobiotics are based on pathophysiological factors and environmental interactions, but also genetic characteristics. In many cases, toxicity depends on the concentration at the side of action but may also be subject of idiosyncrathic reactions. Despite iatrogenic or patients failures, over-dosage may be the result of impaired elimination due to organ failures, drug interactions or hereditary consequences. On the other side, the same metabolizing enzymes may contribute to toxification of environmental compounds or bio-activation of pro-drugs. Thus induction of such pathways, e.g. Ah-receptor mediated enhancement of cytochrome P4501A1 activity may lead to elevated formation of reactive intermediates. These pathways, however, have been also shown to be subject of genetic variability. Aside the polymorphic drug metabolism, there is increasing evidence that genetic differences in membrane transporters may contribute to the explanation of inter-individual variability of susceptibility of adverse drug reactions or susceptibility to xenobiotic-related diseases.

strong efforts have been undertaken to clarify the role of genetic polymorphisms in receptors or signal transduction proteins modulating drug efficacy, as well as in factors involved in cancer etiology such as factors controlling cell cycle, apoptosis and DNA repair.

The broad field of research within pharmacogenomics is trying to elucidate the complex interaction of these polymorphic genes in order to explain and to develop improved therapies particularly for common illnesses such as cardiovascular und malignant diseases and to reduce the number of adverse drug events (Evans and Relling, 2004). This is both reasonable and necessary for several rationales; e.g. a prospective study in the USA points to the fact that 6.7% of hospitalisations can be traced to adverse drug side effects (Lazarou et al., 1998). The resulting costs in the USA have been estimated to amount yearly between 30 and 100 million US$ (White et al., 1999). Pharmacogenomics offers the opportunity, on one hand, to identify new drug targets, and, on the other hand, to adjust individually the required dosage of drugs available on the market (Kirchheiner et al., 2005).

This review focuses on the role of polymorphic drug metabolizing enzymes in the toxicity of drugs and other chemicals.

2. Adverse effects and toxicity due to impaired detoxification pathways

2.1. Cytochrome P450s

The phase I-enzymes catalyze oxidative and reductive reactions of the foreign compound metabolism, but also of transformation of certain lipids and steroids. Obviously, cytochrome P450s are most important and the CYP3A-family contributes to approximately 50% of the total cytochrome P450 activity of the adult human liver. It metabolizes about 60% of all usually prescribed drugs. However, nearly 30% of all drugs are polymorphically metabolized in the human liver particularly by cytochromes P450 (CYP) 2C9, 2C19, 2D6 (Table 1). There are many other polymorphisms known in P450s enzymes such as 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2E1, 3A4, 3A5, 3A7, 5A1, and 8A1, but the functional significance towards drug metabolism is discussed controversial (Ingelman-Sundberg, 2001a).

Table 1. Substrates of polymorphic cytochrome P450 enzymes, functional important alleles and frequencies in Caucasians^a

^a Data according to (Cascorbi et al., 1996, Ingelman-Sundberg, 2001b, Lang et al., 2001, Rylander-Rudqvist et al., 2003 and Sachse et al., 1997).
^b Among Caucasians

2.1.1. Cytochrome P4592C9

There is increasing evidence that the bleeding risk of patients, treated with the vitamin K-antagonist warfarin for thrombosis prophylaxis is increased among carriers of a low active variant of cytochrome P4502C9 (CYP2C9) (Aithal et al., 1999). Particularly the effective S-enantiomer of warfarin is significantly decreased in these individuals, resulting in augmented intermediate plasma concentrations. Consequently, the synthesis of the vitamin K-dependent coagulation factor is strongly inhibited, corresponding to elevated INR values and increased tendency to bleeding episodes (Daly and King, 2003).

Cytochrome P450 2C9 belongs to a close gene cluster on chromosome 10, comprising CYP2C8, 2C9, 2C18, and 2C19. The CYP2C9 deficiency is determined to a major extent by two missense point mutations that lead to the exchange of the amino acids Arg144Cys (CYP2C9*2) and Ile359Leu (CYP2C9*3) (Goldstein and de Morais, 1994). The allele frequencies in the Caucasian population are approximately 11% or 7%, respectively. Homozygous carriers who account for the phenotype of poor metabolizes have a prevalence of about 3–4%. Interestingly in Asians, an alternative Ile359Thr amino acid replacement was observed, termed CYP2C9*4 (Imai et al., 2000). Further polymorphisms were identified in African-Americans (Dickmann et al., 2001 and Kidd et al., 2001). However, the phenomenon of vitamin K-antagonists resistance is also due to genetic polymorphisms. Recently it was shown that subjects, providing variants in the gene of the vitamin K epoxide reductase complex, subunit 1 (VKORC1) require significantly higher warfarin doses than usually recommended (D’Andrea et al., 2005 and Yuan et al., 2005).

Beside warfarin (but to a much lower extent phenprocoumon), many non-steroidal antiphlogistics such as diclofenac, ibuprofen, and meloxicam, oral antidiabetics like tolbutamide and glimenclamide, the angiotensin receptor antagonists irbesartan and losartan as well as some other medications such as phenytoin are metabolized by CYP2C9 (Kirchheiner and Brockmoller, 2005). Indeed, cutaneous adverse reactions during phenytoin therapy account in part to the CYP2C9 genotype (Lee et al., 2004). Moreover, there is increasing evidence that the clearance of oral antidiabetics is directly dependent on the CYP2C9 genotype. The consequences are not always clear. After glubyride or glibenclamide intake, the area under the curve increased nearly three-fold, but glucose levels of heterozygous carriers of CYP2C9 variants did not differ (Niemi et al., 2002). In homozygous CYP2C9*3-carriers, these effects are more pronounced and are mirrored in accelerated insulin response to glucose stimulation (Kidd et al., 1999 and Kirchheiner et al., 2002).

2.1.2. Cytochrom P450 2C19

CYP2C19 (mephenytoine hydroxylase) catalyzes the hydroxylation particularly of proton pump inhibitors like omeprazole and lanzoprazole, but not rabeprazole. Beside many other variants, a splice-site mutation leads to total lack of any activity in 3–5% of Caucasians (De Morais et al., 1994). Interestingly, there is increasing evidence that poor metabolizers profit much stronger from helicobacter pylori eradication and gastroesophageal reflux therapy than extensive metabolizers (Tanigawara et al., 1999, Kawamura et al., 2003, Schwab et al., 2004 and Furuta et al., 2005). On the other hand, the elimination of the alkylating cytostatic cyclophosphamide is significantly impaired in CYP2C19 poor metabolizers (Timm et al., 2005). The clinical consequences remain currently open. Moreover, certain antidepressants are metabolized at least in part by CYP2C19. Many novel SNPs have been detected recently and currently there are 19 different alleles annotated, but most of the genetic variants identified have a very low prevalence. Aside the G681A splice site mutation, G636A (CYP2C19*3) should be considered genotyping Blacks and Orientals. However, this premature stop codon is rare in Caucasians.

2.1.3. Cytochrome P450 2D6

CYP2D6 is one of the best characterized cytochrome P450 enzymes (for review see Bertilsson et al., 2002). Approximately 7–10% of the European populations are CYP2D6-poor metabolizers who show reduced metabolism of numerous drugs like antiarrhythmics, antidepressents, neuroleptics, and some betablockers or opiates. Among this subgroup, clinically relevant drug side effects are more likely compared to extensive metabolizers. The occasionally observed absence of the desired effect accounts in some cases to the phenomenon of gene duplications, occurring with 1–3% in Middle-Europeans.

CYP2D6 belongs to a gene cluster on chromosome 22q13.1 of the highly homologues inactive pseudogenes CYP2D7 containing a single reading frame-disrupting insertion in its first exon and the real pseudogene CYP2D8 (Eichelbaum et al., 1987, Gough et al., 1993 and Kimura et al., 1989). The polymorphisms are well characterized and extensively described by Sachse et al. (1997). Currently more than 90 different CYP2D6 haplotypes are recorded by the human cytochrome P450 (CYP) allele nomenclature committee (http://www.imm.ki.se/CYPalleles). The alleles may be classified into functional, non-functional and reduced function groups. One of the major primary gene defect at the cytochrome P450 CYP2D locus is a 1846G > A splice site mutation (CYP2D6*4) (Gough et al., 1990) with a frequency of 20.7% in Caucasians (Sachse et al., 1997). In 4–6% of Caucasian and other ethnic populations, the entire coding region is deleted (Gaedigk et al., 1991). Hence, allele *5 is believed to have an ancient origin. Further relevant variants are a 2549A deletion in *3 (2.0%), and a 1707T deletion in *6 (0.9%), generating frame shifts. In contrast to these fatal polymorphisms, a triple-base-pair deletions in allele *9 (1.8%) does not significantly alter enzyme activity (Broly and Meyer, 1993) and a proline to serine exchange in codon 34 is associated with lower enzyme activity and particularly decreased stability of CYP2D6.10 (Nakamura et al., 2002). This variant occurs with 1–2% in Caucasians, but is the major cause of low CYP2D6 activity in Orientals (Bertilsson, 1995). In African-Blacks, *17 is one of the major reasons for low CYP2D6 activity.

The intermediate phenotype is also due to diminished expression rates of CYP2D6, e.g. there is convincing evidence that homozygous carriers of a C/G polymorphism 1584 bp upstream of the start codon (CYP2D6*41) exhibited only 50% of protein compared to carriers of the ?1584G variant (Zanger et al., 2001).

Extremely high CYP2D6 activities in 1–2% of Caucasians were identified to be due to gene duplications of the wild-type and allele CYP2D6*2 but possibly also of others. In Northern Europe the prevalence is below 2% (Dahl et al., 1995), but in some regions of Spain, frequencies of more than 7% were observed (Agundez et al., 1995). In Arabian countries (McLellan et al., 1997) as well is in the North-East-African Ethiopia, a prevalence of ultra rapid metabolizers of up to 29% is reported (Aklillu et al., 1996). In a few cases, there were families with up to thirteen gene copies identified. In contrast, in China, CYP2D6 gene duplications are absolutely rare, but the mean metabolic ratio of debrisoquine/4-hydroxydebrisoquine is increased compared to Caucasians (Johansson et al., 1994). This is due to the high prevalence of the low active (intermediate) CYP2D6*10 variant (Garcia-Barcelo et al., 2000). In Blacks, however, large heterogeneity seems to exist (Griese et al., 1999 and Masimirembwa et al., 1996).

The ultrarapid metabolizer phenotype of CYP2D6 has been well established as a relevant cause of non-response to antidepressant drug therapy. Clearance of such drugs like nortriptyline, desipramine, and to some extent imipramine and amitriptyline (Brosen et al., 1991, Ghahramani et al., 1997 and Venkatakrishnan et al., 1999) evidently depend on the CYP2D6 polymorphism. Specific serotonine reuptake inhibitors like fluoxetine, citalopram or paroxetine were shown to be inhibitors of CYP2D6 (Alfaro et al., 1999 and Crewe et al., 1992). The effects of the CYP2D6 polymorphism on antipsychotic therapy appear to be more pronounced in neuroleptics. Compounds like perphenazine, zuclopenthixol, thioridazine, haloperidol and risperidone are metabolized to a significant extent by CYP2D6. Poor metabolizers appeared to posses an elevated risk to suffer from side effects like extrapyramidal symptoms (Bertilsson et al., 2002, Brockmoller et al., 2002 and Scordo et al., 2000). Moreover, the antipsychotic efficacy seems to be influenced by the number of active copies of CYP2D6 genes (Brockmoller et al., 2002). The findings give rise to perform genotyping before treatment with polymorphically metabolized antipsychotics (Dahl, 2002).

Betablockers like metoprolol, and to some extent carvedilol are also CYP2D6 substrates (Oldham and Clarke, 1997, Pepper et al., 1991 and Rau et al., 2002). However, currently there is no data available if the clinical outcome of betablocker therapy is influenced by genetic polymorphisms of CYP2D6.

The beneficial use of CYP2D6 genotyping for drug therapy was demonstrated recently on the treatment of nausea and vomiting in cancer chemotherapy with the antiemetic 5-HT₃ receptor antagonist tropisetron (Kaiser et al., 2002). In this case, CYP2D6 poor metabolizer took advantage of antiemetic therapy compared to extensive metabolizers.

2.1.4. Cytochrome P450 3A

The CYP3A-family consists of the well known, numerous drugs metabolizing CYP3A4, of CYP3A5, of the fetal CYP3A7, as well as of the recently identified CYP3A43. The interindividual variability of the total CYP3A-activity accounts in part to the presence or absence of active CYP3A5. Only less than a third of Caucasians and 66% of African-Blacks exhibit CYP3A5 expression, caused by a G/A-DNA base exchange within intron 3. This single nucleotide polymorphism leads to alternative splicing and generation of a premature stop codon in exon 3B, resulting in the inactive CYP3A5*3 (Hustert et al., 2001a and Kuehl et al., 2001). This polymorphism explains in part the bidomal distribution of midazolam kinetics. Additionally among American-Blacks, a rare G/A-SNP was identified in exon 7, associated with reduced CYP3A5 activity (CYP3A5*6) (Kuehl et al., 2001).

Aside CYP3A5, CYP3A4 contributes to the clearance of midazolam. Recently, a functional significant polymorphism of CYP3A4 (L373F) was discovered. It reduces affinity of midazolam to CYP3A4 from a K_M of 8.7 μmol/l to a K_M of 36.4 μM. Further novel identified SNPs lead in part to lowered or even lack of expression. However, due to the rarity of these polymorphisms, they do not contribute to the explanation of individual variability of CYP3A4 activity (Eiselt et al., 2001).

CYP3A can be strongly induced by drugs like rifampicin, carbamazepine, phenobarbital and others. They interact with the nuclear pregnane X receptor (PXR) followed by dimerization with the 9-cis-retinoic acid receptor α (RXRα) and binding to the respective promoter responsive elements. Therefore genetic variants of the PXR gene could contribute to the variability of the CYP3A activity. Indeed, PXR exhibits numerous variants, at least six of them generate amino acid exchanges. The variant 163G leads to a 15-fold induction by rifampicin compared with a five-fold induction by 163D. Other variants such as V140M and A370T exhibit effects of minor significance (Hustert et al., 2001b). These discoveries show the great importance of gene–environmental interactions, since the PXR polymorphisms do not affect the basal CYP3A activity, but the CYP3A induction. Likewise CYP3A4 polymorphisms, the prevalence of PXR SNPs is rather low, hence the observed CYP3A activity can be described only to a small part by this variables.

2.2. Phase II-enzymes

Major enzymes of conjugation in Phase II are the UDP glucuronosyltransferase (UGT), sulfotransferases (SULT), arylamine N-acetyltransferases (NAT), glutathione S-transferases (GST), thiopurine S-methyltransferases (TPMT), catecholamine-O- methyltransferases (COMT) and others. The polymorphic character of NAT and GST and its role towards particularly towards malignancies is extensively investigated and the role of TPMT for azathioprine toxicity is well established. The role of polymorphisms of sulfotransferases is much more difficulty to consider, since the different isoforms of sulfotransferases are involved in detoxification, but also toxification pathways, leading to partly contradictory results (excellently reviewed by Glatt and Meinl (2004)). Also COMT is polymorphic and was related to neuro-psychiatric disorders (Glatt et al., 2003 and Redden et al., 2005) or malignancies (Mitrunen and Hirvonen, 2003), the associations are, however, weak or need confirmation. Also the UGT family provides certain polymorphic traits, hereditary defects may lead to mild or severe hyper-bilirubinemia, and there is increasing evidence that genetic variants may have an important pharmacological impact on e.g. anti-cancer therapy with irinotecan (Ando et al., 2000). For review see (Burchell, 2003).

2.3. Arylamine N-acetyltransferases

Arylamine N-acetyltransferases are responsible for the conjugation of drugs like isoniazid, dapsone, procainamide and many others. The slow NAT2 acetylator is supposed to be at higher risk for drug side effects such as peripheral neuropathia after isoniazid treatment (Yamamoto et al., 1996) or certain disorders such as drug-induced lupus erythematosus and Stevens–Johnsons syndrome (Wolkenstein et al., 1995). This severe diseases may be caused, in susceptible individuals, by a large number of drugs involved in acetylation metabolism, the there is no association to the idiopathic form of LE (Zschieschang et al., 2002). Peripheral neuropathy provoked by isoniazid over-dosage may be a major problem in ethnicities with a high frequency of slow acetylators as in Northern Africa. Low drug efficacy may be expected in rapid acetylators. NAT2 is expressed preferably in the human liver, whereas the sister gene NAT1 can be determined in a wide variety of different tissues.

Phenotyping studies in the last four decades disclosed distinct ethnic differences of slow acetylator frequencies (Evans, 1992). Extremes can be found between North Africa with a frequency of alleles coding for slow acetylation of 95% and the Far East Pacific region with a frequency of only 11%. The expected partition of slow acetylators in these populations spans 90–1.2% (Cascorbi et al., 1995, Ilett et al., 1993 and Lin et al., 1993). In African Blacks, there is an additional frequent 191G > A SNP, and a large diversity of haplotypes can be observed (Cascorbi et al., 1999).

The implication of both, oxidative metabolization by cytochrome P450s and conjugation with acetyl-CoA also plays a major role in an individual‘s risk to suffer from certain diseases, which are related to environmental toxicants, particular cancer.

Earlier phenotyping studies provided some evidence that slow acetylators are at increased risk for bladder cancer (Cartwright et al., 1982, Lower et al., 1979 and Vineis and Ronco, 1992). Later studies performed using genotyping methodologies confirmed these early findings (Brockm?ller et al., 1996 and Risch et al., 1995). It is hypothesized that in rapid acetylators arylamines, as contained in aniline dyes or cigarette smoke (e.g. 4-aminobiphenyl), are detoxified by N-acetylation in the liver and excreted in the urine. In contrast, low N-acetylation activity leads to increased formation of N-hydroxylated products. These hydroxylamines may undergo further O-acetylation in the urinary bladder preferentially by arylamine N-acetyltransferase 1 (NAT1), which was found to be expressed in the urinary epithelium (Kloth et al., 1994). The product, arylamine acetoxyesters are unstable in the acid environment and disintegrate spontaneously to arynitrenium ions. These highly reactive radicals may well interact with proteins and DNA of bladder epithelial cells forming adducts (Hein et al., 1993).

A number of in vitro studies gave evidence for this theory. Most carcinogenic compounds activated by acetyltransferases are heterocyclic aromatic amines like 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) which lead to dose-dependent effects in mutagenicity tests (Wild et al., 1995).

A meta-analysis of 22 published case-control phenotyping and genotyping studies conducted in a total of 2496 cases and 3340 controls in different populations revealed that slow acetylators had a 40% increased risk compared to rapid acetylators (odds ratio 1.4, 95% confidence interval 1.2–1.6) (Marcus et al., 2000). In particular, the largest genotyping studies clearly showed a gene-environment interaction (Vineis et al., 2001).

Aside from the unequivocal role of NAT2, the discovery of the polymorphic nature of NAT1 raised the question whether different NAT1 genotypes may additionally modulate bladder cancer susceptibility. Indeed, (Bell et al., 1995) reported two important facts: NAT1*10 was found to provide enhanced activity in bladder tissue compared to NAT1*4 and moreover, the frequency of NAT1*10 was increased among bladder cancer patients. However, these results are conflicting (Okkels et al., 1997) and recently we were able to show that NAT1*10 does not alter enzyme activity towards ex vivo formation of N-acetyl p-amino benzoic acid (Bruhn et al., 1999). We found a significant decrement of NAT1*10 genotypes among 425 bladder cancer patients; the adjusted odds ratio was 0.65 (95%-C.I. 0.46–0.91; P = 0.013) (Cascorbi et al., 2001). Considering the NAT2 genotype, a clear under-representation of NAT1*10 genotypes among rapid NAT2 genotypes in the cases studied (odds ratio 0.39; 95%-C.I. 0.22–0.68; P = 0.001), and a gene–gene–environment interaction was observed. NAT2*slow/NAT1*4 genotype combinations with a history of occupational exposure were six times more frequent in cancer cases than in controls without risk occupation (P < 0.0001).

Similar as in the bladder, one of the first steps of colon cancer may be initiated by DNA adducts, formed by heterocyclic arylamines, which had been activated by cytochromes P450s and N-acetyltransferases. It is well established that NAT1 as well as NAT2 are expressed in colon tissue (Hickman et al., 1998). Enhanced O-acetylation in the colon mucosa could therefore contribute to the formation of adducts (Nerurkar et al., 1995). Indeed, some studies suggested the rapid acetylators phenotype as a hereditary trait for predisposition of colorectal carcinomas particularly when patients had a history of smoking or red meat intake. (Chen et al., 1998, Roberts-Thomson et al., 1996 and Welfare et al., 1997). Independently of confounders, extended risk was observed rapid acetylators (Agundez et al., 2000 and Gil and Lechner, 1998). However, the findings on NAT2 as a susceptibility factor of colon cancer are not consistent less pronounced in several other large molecular epidemiological studies (Slattery et al., 1998).

2.3.1. Thiopurine-S-methyltransferase (TPMT)

A rare but serious side effect in treatment with the antimetabolites 6-mercatopurine and azathioprine is a severe bone marrow depression, which may result in lethal side effects (Krynetski and Evans, 2000). Detoxification takes place by TPMT, a phase-II enzyme, which however, is homozygously deficient in one of 300 individuals (Weinshilboum, 1992). Among these patients, metabolism takes place by an alternative pathway to the 6-thioguanin-nucleotide (6-TGN). The plasma concentration of 6-TGN correlates with the severity of the medication‘s side effects. However, a problem arises, since TPMT exhibits a high number of mutations, which allows a genotyping only to a limited degree. So far, eight mutations that determine an amino acid exchange and a splice site mutation are known. Apparently even more, though very rare, SNPs exist which determine a TPMT-poor metabolizer. Many clinics still routinely prefer ex-vivo phenotyping procedures to date, but increasing knowledge about rare variations and application of techniques like DHPLC will allow a reliable prediction of TMPT status by means of genotyping (Schaeffeler et al., 2001). The clinical importance was shown in a large study in pediatric acute lymphoblastic leukemia patients, showing that TPMT-poor metabolizers are at clear risk of severe azathioprine side effects, making a significant dose reduction necessary (Stanulla et al., 2005).

2.3.2. Glutathione S-transferases

Glutathione S-transferases of classes GSTA, -M, -P, -T, and -Z are conjugating a variety of exogenous and endogenous compounds including several cytostatics. Since glutathione-conjugation represents a detoxification pathway, it becomes rapidly clear that the total absence of GSTM1 activity (genotype GSTM1*0/*0) may be linked to increased drug toxicity or cancer susceptibility. Indeed, GSTM1 deficiency was shown by several studies to be a risk factor for lung (Houlston, 1999), laryngeal (Hashibe et al., 2003) and urinary bladder cancer (Brockmoller et al., 1994 and Engel et al., 2002), comprehensively reviewed by Parl (2005). Howewer, there is a clear lack of association between GSTM1, GSTT1, and GSTP1 and breast cancer (Vogl et al., 2004).

These findings may be partly explained due to the fact that glutathione S-transferase deficiency prevents from detoxification of dioepoxide from e.g. benzo(a)pyrene. Thus several studies could show elevated levels of DNA and protein adducts in carriers of GSTM1 and GSTT1 (Alexandrov et al., 2002 and Bartsch et al., 1999).

Interestingly GSTM1 deficiency was also reportedly associated with other smoking-related diseases such as atherosclerosis (Olshan et al., 2003) or lung emphysema.

3. Toxicity due to elevated toxification

This chapter deals with the role of polymorphic metabolizing enzymes in the activation of parent compounds to highly active intermediates, hence less active phenotypes would lower the risk of e.g. DNA aduct formation, whereas polymorphisms leading to elevated activity would be potentially associated with an increased risk of e.g. tobacco-smoke related cancers.

3.1. Cytochrome P450s

Bioactivation of xenobiotics accounts to some extend to the inducible cytochrome P450s 1A1, 1A2, and 1B1. CYP1A1 is a key enzyme in carcinogen metabolism and was proved as a promising genetic biomarker for susceptibility to certain malignancies, particularly lung cancer (Vineis et al., 2004). It metabolizes polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), a prominent and highly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in tobacco smoke, into benzopyrene diol epoxide (BPED) which reacts with DNA predominantly at the N²-position of guanine to produce primarily N²-guanine lesions, e.g., BPDE-N^2?dG adduct (Osborne, 1990). CYP1A1 is highly polymorphic, but most data is available on a T to C-transition 1194 bp downstream of exon 7, creating a new MspI-cleavage site at position 3801T > C (CYP1A1*2A). A meta-analysis by Vineis et al. (2003) revealed that smokers, homozygous for 3801C were at significantly elevated risk of lung cancer (odds ratio 2.36). However, an Ile462Val exchange is in strong linkage disequilibrium and the haplotype CYP1A1*2B was shown to be associated to lung cancer even in non-smokers (Raimondi et al., 2005). The particular role of these polymorphisms considering also the GSTM1 genotype, was shown for significantly elevated DNA adduct levels in lymphocytes and lung cancer tissue (Rojas et al., 2000 and Rojas et al., 2004). A further adjacent Thr461Asp amino acid replacement revealed no evidence of an association to lung cancer (Cascorbi et al., 1996), but this SNP modulates the substrate affinity particularly of 17beta-estradiol and estrone, giving rise to a possible involvement in hormone-related cancers (Kisselev et al., 2005 and Schwarz et al., 2001).

The role of the polymorphic CYP1B1 seems to be less important as cancer susceptibility factor. Although CYP1B1 is involved in estrogen metabolism, there is lack of evidence for an association to breast cancer in a recent large meta-analysis (Wen et al., 2005).

3.2. Myeloperoxidase

Myeloperoxidase (MPO) has been shown to transform environmental precarcinogens such as benzo(a)pyrene and aromatic amines to highly reactive intermediates (Kadlubar et al., 1992 and Mallet et al., 1991). MPO is a lysosomal hemoprotein expressed in polymorphonuclear leukocytes and monocytes that actually catalyzes the production of hypochlorous acid in physiologic situations which leads to microbicidal activity against a wide range of organisms (Foster et al., 1998). Strkingly, we found an association of MPO genotypes and clozapin-induced agranulocatosis, a severe and treatment-limiting side effect of treatment with this atypical neuroleptic drug (Mosyagin et al., 2004). On the other hand, the highly active ?463G allele is strongly associated to lung cancer (Cascorbi et al., 2000 and Schabath et al., 2002), indicating elevated formation of reactive intermediates, leading to DNA adducts, as could be demonstrated in bronchoalveolar lavage fluid; cytology (Van Schooten et al., 2004).

4. Conclusion