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Genetic polymorphisms and chromosome damage
International Journal of Hygiene and Environmental Health
Int. J. Hyg. Environ. Health 204, 31 ± 38 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Genetic polymorphisms and chromosome damage Hannu Norppa Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland
Abstract Genetic polymorphisms that affect xenobiotic metabolism or cellular response to DNA damage can modulate individual sensitivity to genotoxins. Information on the effects of such polymorphisms on the level of chromosome damage may facilitate the identification of risk groups and increase the sensitivity of cytogenetic endpoints as biomarkers of genotoxic exposure and effect. Glutathione S-transferase M1 (GSTM1) is an important detoxification enzyme which, due to a homozygous gene deletion (null genotype), is lacking from about 50% of Caucasians. A higher level of DNA adducts and chromosome damage has been detected in lymphocytes of tobacco smokers and bus drivers who lack the GSTM1 gene. Other polymorphic glutathione S-transferases include GSTM3, GSTP1, and GSTT1. The GSTT1 null genotype (10 ± 20% of Caucasians) has been associated with an increased ªbaselineº level of sister chromatid exchanges (SCEs) in lymphocytes. N-acetyltransferase 2 (NAT2), metabolizing xenobiotics with primary aromatic amine and hydrazine structures, is another important polymorphic phase II enzyme. Subjects having the NAT2 slow acetylator genotype appear to show an increased baseline frequency of lymphocyte CAs in the absence of identified environmental exposure. Besides human biomonitoring studies, genetic polymorphisms may be important in explaining individual variation in genotoxic response observed in genetic toxicology tests with human cells. Several studies have suggested that blood cultures from GSTT1 null and GSTM1 null individuals have increased in vitro sensitivity to various genotoxins. The best-known example is probably the diepoxybutane sensitivity of GSTT1 null donors. Recently discovered polymorphisms affecting DNA repair may be expected to be of special importance in modulating genotoxic effects; the first available studies have suggested that the exon 10 Arg399Gln polymorphism of XRCC1 gene (X-ray repair cross-complementing group 1) could affect individual genotoxic response. In conclusion, the genetic polymorphism of GSTM1 influences the frequency of chromosome damage in exposed humans, while that of GSTT1 and NAT2 affect the ªbaselineº level of such damage. Both GSTM1 and GSTT1 genotypes may shape the in vitro genotoxic response of human lymphocytes. The significance of DNA repair polymorphisms is presently unclear. Key words: biomarker ± DNA repair ± chromosome damage ± genotoxicity ± polymorphism ± xenobiotic metabolism
Introduction Susceptibility to genotoxic exposure varies among individuals, due to acquired or inherited characteristics. During the last few years, increasing attention has been focused on genetic polymorphisms that could modulate human response to genotoxic insult
(see (Norppa, 1997, 2000; Au et al., 1999; Pavanello and Clonfero, 2000; Norppa and Hirvonen, 2000)). At the same time, chromosomal aberrations (CAs) in peripheral lymphocytes have received special interest, as international collaborative studies have shown that a high frequency of CAs is predictive of increased risk of cancer (see (Hagmar et al. 1998,
Corresponding author: Dr. Hannu Norppa, Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland, Phone: 358 9 4747 2336, Fax: 358 9 4747 2110, E-mail: [email protected] 1438-4639/01/204-31 $ 15.00/0
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2000)). Recent findings have indicated that the cancer predictivity of CAs is not explained by tobacco smoking or occupational exposure to carcinogens, but that high CA rates are indicative of an increased cancer risk also in ªunexposedº nonsmokers (Bonassi et al., 2000; Hagmar et al., 2000). This suggests a general role for CAs as a cancer risk biomarker and underlines the importance of individual susceptibility factors. Several studies have implied that genetic polymorphisms can influence the level of chromosome damage associated with some genotoxic exposures, but may also affect the apparent baseline level of cytogenetic alterations. Thus, genetic polymorphisms might partly explain the association between CA levels and cancer risk. In principle, any polymorphisms that affect xenobiotic metabolism or cellular response to DNA damage could alter individual sensitivity to genotoxins. Many studies have addressed the question whether a particular genotype of a xenobioticmetabolizing enzyme is over-represented among cancer patients (see (Hirvonen, 1999a; Strange et al., 1999; Wormhoudt et al., 1999)). Such investigations require a high number of cases and controls, and relationship to past exposure is often difficult to establish due to insufficient information and the long time that has elapsed between the exposure and the disease. Another approach is to examine the influence of genetic polymorphism on biomarkers of genotoxic effect, comparing biomarker levels in different genotype groups. The association between exposure and biomarker response is then not obscured by a long latency period, and reasonable information on exposure levels can be obtained. This may facilitate the identification of risk groups and increase the sensitivity of the biomarkers in biomonitoring. The present paper shortly reviews some recent examples of genotype effects on chromosome damage.
GSTM1, tobacco smoke, and epoxides The polymorphism of glutathione S-transferase M1 (GSTM1) has offered a good model to study geneenvironment interactions. Along with other members of the glutathione S-transferase (GST) superfamily, GSTM1 is primarily involved in the metabolic detoxification of various reactive chemicals (see (Strange et al., 2000)). GSTM1 activity is present in individuals who have at least one copy of either GSTM1*A or GSTM1*B allele, the alleles differing from each other by a base substitution which seems to have little effect on enzyme activity.
GSTM1 activity is, however, totally lacking in GSTM1 null individuals who are homozygous for GSTM1 gene deletion (see (Brockmöller et al., 1992; Strange et al., 2000)). The GSTM1 null genotype is very common among Caucasians, being found in roughly 50% of the population (see (Hirvonen, 1999a)). Thus, the prerequisites to study the effects of GSTM1 polymorphism on, e. g., biomarkers of genotoxicity has been ideal, because there is an extreme and undisputed difference between the two genotypes in enzyme activity (GSTM1 positive having activity, GSTM1 null lacking it) both genotypes being distributed about evenly in the population. There are, however, ethnic differences in the frequency of these genotypes. As the GSTM1 null genotype is expected to increase individual sensitivity to various reactive compounds such as PAH diol epoxides, its role as a risk factor of various types of human cancer has extensively been studied. Although the results have been somewhat conflicting, the homozygous GSTM1 deletion has been observed to increase the risk of lung and bladder cancer in tobacco smokers (see (Brockmöller et al., 1994; McWilliams et al., 1995; Hirvonen, 1999a; Strange et al., 1999)), and has also been suggested to play a role in lung cancer among nonsmokers exposed to environmental tobacco smoke (Bennett et al., 1999). Results on genotoxicity biomarkers have supported the idea that GSTM1 null individuals have reduced capacity to detoxify genotoxins in tobacco smoke. In comparison with GSTM1 positive (or GSTM1 proficient) smokers, GSTM1 null (or GSTM1 deficient) smokers showed an elevated level of DNA adducts (Butkiewicz et al., 1998, 2000; Rojas et al., 1998, 2000; Viezzer et al., 1999) (see (Pavanello and Clonfero, 2000)), CAs (Scarpato et al., 1997), and sister chromatid exchanges (SCEs; van Poppel et al., 1992) in their leukocytes. In addition to PAHs, these findings may also be related to other constituents of tobacco smoke; lymphocytes from GSTM1 null donors were more sensitive to CA (tandem probe assay) and SCE induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine (Salama et al., 1999). The GSTM1 null genotype may predispose its carriers to the genotoxic effects of PAH-containing air pollutants in general, since GSTM1 null bus drivers (all nonsmokers), who are exposed to polluted city air containing, e. g., diesel exhausts, showed a higher frequency of lymphocyte CAs than GSTM1 positive drivers (Knudsen et al., 1999). An effect of GSTM1 genotype on DNA adducts associated with tobacco smoking or exposure to
Genetic polymorphisms and chromosome damage
PAH has not been observed in all studies (see (Pavanello and Clonfero, 2000)). Many things may contribute to this discrepancy, but it has also to be remembered that there may be great qualitative and quantitative differences in exposure among different studies (and certainly among individuals) and also the statistical power of the studies differ. One cannot just list the positive and negative studies, but should consider the overall picture. The question of GSTM1 genotype and SCEs in smokers may provide an example of the importance of including enough subjects in the study. The fact that several studies have not seen a connection between GSTM1 genotype and SCEs may reflect the fact that the effect of smoking itself on the mean number of SCEs per cell, although reproducible and proven, is usually small (only about a 10% increase). In such a situation, the demonstration of an interaction between smoking and GSTM1 phenotype or genotype would require either that the smoking effect is exclusively seen in the deficient subjects, or that there are enough subjects in the study to allow a small difference to be detected. Van Poppel et al. (1992) who originally described the association between GSTM1 phenotype and SCE frequency in smokers, saw a 5.4% increase in mean SCEs/cell among 78 heavy smokers, studying altogether 154 smokers. Almost identical SCE increase was also observed by Scarpato et al. (1996) and Wu et al. (2000), but as there was less individuals (and no separate analysis of heavy smokers), no statistical significance was reached. Results of one study have suggested that GSTM1 polymorphism could influence SCE levels also in nonsmokers (Cheng et al., 1995; Duell et al., 2000). One of the reasons that GSTM1 genotype appears to have an effect on various genotoxicity biomarkers measured from leukocytes is probably the fact that GSTM1 is expressed in these cells in GSTM1 proficient individuals (Brockmöller et al. 1992). Therefore, the GSTM1 genotype of the blood donor may likewise influence the results of genotoxicity tests performed with human lymphocytes, as discussed above in connection with NNK. Cultured lymphocytes of GSTM1 deficient subjects were also shown to have increased sensitivity to in vitro SCE induction by trans-stilbene oxide, an epoxide and a model substrate for GSTM1 (Wiencke et al., 1990). Accordingly, subjects with the GSTM1 genotype were observed to show higher SCE induction by trans-stilbene oxide than GSTM1 positive individuals (Bernardini et al., 2001). The GSTM1 genotype was also found to affect SCE induction by another epoxide, 1,2-epoxy-3-butene (Uusküla et al., 1995; Sasiadek et al., 1999).
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GSTT1, epoxides, and baseline level of chromosome damage A homozygous deletion of the GSTT1 gene (null genotype) is found in 10 ± 20% of Caucasians, making them unable to perform GSTT1-mediated detoxification reactions. Interestingly, the GSTT1 null genotype has been associated with an increased ªbaselineº level of SCEs in lymphocytes, with no association to smoking or other known exposures (Schröder et al., 1995; Wiencke et al., 1995). The finding possibly reflects an interaction between the genotype and some common endogenous or exogenous exposure; ethylene oxide generated from endogenous ethylene was proposed as a possible explanation (Schröder et al., 1995). The GSTT1 genotype effect was roughly the size of the smoking effect, i. e., quite small, which may explain why it has not been detected in all studies (see (Norppa, 2000)). Also, the low frequency of the GSTT1 null genotype in Caucasians has complicated the evaluation of GSTT1 influence in many studies. Two papers have suggested that the GSTT1 null genotype is also associated with an increase in ªspontaneousº CAs in lymphocytes (Landi et al., 1998; SÏraÂm et al. 1998). The best-known example of genotype-dependent response to in vitro chemical treatment is probably the 1,2 : 3,4-diepoxybutane (DEB) sensitivity of GSTT1 null donors. DEB, an epoxide metabolite of 1,3-butadiene, is an efficient inducer of SCEs, and DEB sensitivity was originally described using the human lymphocyte SCE test (Wiencke et al., 1991). Subsequently, DEB sensitivity was observed to be explained by GSTT1 null genotype (Norppa et al, 1995; Wiencke et al., 1995; Landi et al., 1996, 1998; Kligerman et al., 1999; Hayes et al., 2000; Schlade-Bartusiak et al., 2000). Removal of erythrocytes from whole-blood lymphocyte cultures abolished the genotype difference, reflecting the fact most of blood GSTT1 activity is found in erythrocytes (Pelin et al., 1996). Whole-blood cultures of GSST1 null subjects were also shown to be hypersensitive to the induction of micronuclei by DEB (Vlachodimitropoulos et al., 1998). Also for CA induction by DEB in vitro, GSTT1 genotype was an important factor, although it explained smaller part of the variation than in the case of SCEs; for unknown reason, some GSTT1 null subjects were only sensitive to induction of CAs but not of SCEs by DEB (Landi et al., 1998). One study could not find a GSTT1 dependence for CA induction by DEB (Schlade-Bartusiak et al., 2000). Whole-blood cultures of donors with the GSTT1 null genotype were also observed to be more sensitive than GSTT1
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positive subjects to SCE induction by 1,2-epoxy-4butene (another metabolite of 1,3-butadiene), and styrene-7,8-oxide (Bernardini et al., 1998; Ollikainen et al., 1998). Erythrocytic GSTactivity (GSTT1) was found to be protective for SCE induction in human lymphocytes by methyl bromide, ethylene oxide, and dichloromethane (Hallier et al., 1993). As the in vitro genotoxicity of the epoxide metabolites of 1,3-butadiene depended on GSTT1 or GSTM1 genotypes of the cell donors, these genotypes might also affect the genotoxic effects in workers occupationally exposed to 1,3-butadiene. However, the results of the available investigations have been conflicting, and further studies are needed (Sorsa et al., 1996; Kelsey et al., 1995; SÏraÂm et al. 1998; Hayes et al., 2000) (see (Norppa, 2000)).
NAT2 and baseline level of chromosomal aberrations N-acetyltransferase 2 (NAT2), metabolizing xenobiotics with primary aromatic amine and hydrazine structures, is another important polymorphic phase II enzyme. Seven different nucleotide transitions have been shown to lead to amino acid changes in the NAT2 enzyme (see (Hirvonen et al., 1999b)). The various genotypes result in either fast or slow acetylation capacity. Rapid acetylators have inherited at least one NAT2*4 allele, while slow acetylators have two slow-acetylation associated alleles. Slow acetylators have been described to have an elevated risk of bladder cancer, while rapid acetylators have been observed to be over-represented among colon cancer patients (see (Hirvonen et al., 1999b)). The frequency of the NAT2 slow acetylator genotype was 50 ± 63% among Caucasians, but Japanese or Chinese were mostly (92% or 80%, respectively) rapid acetylators. Subjects having the NAT2 slow acetylator genotype showed an increased baseline frequency of lymphocyte CAs, with no association to known exposures (Knudsen et al., 1999). This effect seems to be reproducible, as we have seen it in two other studies as well (Norppa et al., 2000, 2001). It might be explained by exposure to commonly found environmental genotoxins, such as heterocyclic amines in heated food; N-acetylation could function as a detoxification route in peripheral blood cells where the activities of cytochrome P-450 monooxygenases capable of metabolically activating heterocyclic amines are low (Hukkanen et al., 1997; Raucy et al., 1999). Alternatively, NAT2 may detoxify important (as yet unknown) endogenously formed
genotoxins(s). Acetyltransferases are involved in the acetylation of histones (Grunstein, 1997) and interconversion of polyamines (Seiler et al., 1985), but there is no evidence for the involvement of NAT2 in such reactions. Another polymorphic acetyltransferase, NAT1, appears to function in the catabolism of folate (Minchin, 1995), but, again, NAT2 is not known to be involved.
CYP2E1 and chromosome damage The cytochrome P-450 (CYP) superfamily of monooxygenases is involved in the metabolic activation of various precarcinogens. Studies on the influence of polymorphisms affecting various CYP species with genotoxicity are complicated by, e. g., unclear associations between CYP genotype and phenotype (see (Hirvonen 1999a; Norppa, 2000)). Several types of CYPs are found in lymphocytes, although at low levels (Raucy et al., 1999), CYP2E1 being probably among the most important ones (Hukkanen et al., 1997). Thus, CYP2E1 polymorphisms might affect the outcome of cytogenetic biomonitoring studies and genotoxicity tests with CYP2E1 substrates in human lymphocytes. A base substitution in the 5' flanking region (C1017T) of CYP2E1 gene, expected to result in CYP2E1 over-expression, has been associated with a (non-significantly) increased SCE frequency in vinylchloride-exposed workers (Wong et al., 1998) and with an elevated in vitro CA induction (tandemprobe assay) by NNK (Abdel-Rahman et al., 2000a).
XRCC1 and chromosome damage It is obvious that genetic polymorphisms of proteins involved in DNA repair (Shen and Jones, 1998) are expected to affect the level of chromosome damage. At present, however, only a few studies exist on this topic, and the actual influence of many of the recently discovered polymorphisms on DNA repair capacity remains unclear. XRCC1 protein (X-ray repair cross-complementing group 1) is considered to be involved in the repair of DNA single-strand breaks after base excision repair of damage produced by ionizing radiations, alkylating agents, and reactive oxygen species. The Arg399Gln substitution at exon 10 has been suggested to be associated with reduced DNA repair efficiency. Homozygous carriers of this variant allele were
Genetic polymorphisms and chromosome damage
observed to show elevated aflatoxin B1 DNA adducts in placenta (Lunn et al., 1999) and an agedependent increase in polyphenol DNA adducts in mononuclear leukocytes (Duell et al., 2000). Smokers with this genotype also showed increased frequencies of glycophorin A variant erythrocytes and lymphocyte SCEs, although the findings were based on only four (Lunn et al., 1999) and three (Duell et al., 2000) Gln/Gln smokers. Lymphocytes from subjects homozygous or heterozygous for the 399Gln allele were found to be more sensitive than lymphocytes from Arg/Arg subjects to SCE induction by the tobacco-specific nitrosamine NNK (Abdel-Rahman and El-Zein, 2000b).
Conclusions Some genetic polymorphisms of xenobiotic-metabolizing enzymes have been observed to influence the level of genotoxic damage in humans. The genetic polymorphisms potentially important for a particular endpoint largely depend on the exposing agent, biological material examined, and ethnicity of the population under study. As there is individual variation also in the extent and nature of the exposure, correct identification of individual exposure levels is essential. The GSTM1 null genotype appears to increase the frequency of chromosome damage in smokers, while the glutathione GSTT1 and NAT2 slow acetylator genotype seem to affect the ªbaselineº level of such damage. Besides human biomonitoring studies, genetic polymorphisms may be important in explaining individual variation in genotoxic response observed in genetic toxicology tests with human cells. Several studies have suggested that blood cultures from GSTT1 null and GSTM1 null individuals have increased in vitro sensitivity to various genotoxins. New information is emerging on polymorphisms affecting DNA repair, which may be of special importance in modulating genotoxic effects. Understanding the influence of genetic polymorphisms on genotoxic response is expected to improve the applicability of genotoxicity assays in biomonitoring and testing.
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frequency in DNA repair genes in healthy humans. Cancer Res 58: 604 ± 608, 1998. Sorsa M, Osterman-Golkar S, Peltonen K, Saarikoski ST, SÏraÂm R. Assessment of exposure to butadiene in the process industry. Toxicology 113: 77 ± 83, 1996. SÏraÂm R, Rössner P, Peltonen K, Podrazilova K, MracÏkova G, Demopoulos NA, Stephanou G, Vlachodimitropoulos D, Darroudi F, Tates AD. Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1,3-butadiene-exposed workers. Mutat Res 419: 145 ± 154, 1998. Strange RC, Fryer AA. Chapter 19. The glutathione Stransferases: influence of polymorphism on cancer susceptibility. IARC Sci Publ 148: 231 ± 249, 1999. Strange RC, Jones PW, Fryer AA. Glutathione S-transferase: genetics and role in toxicology. Toxicol Lett 112 ± 113: 357 ± 363, 2000. Uusküla M, Järventaus H, Hirvonen A, Sorsa M, Norppa H. Influence of GSTM1 genotype on sister chromatid exchange induction by styrene-7,8-oxide and 1,2-epoxy-3-butene in cultured human lymphocytes. Carcinogensis 16: 947 ± 950, 1995. van Poppel G, de Vogel N, van Bladeren PJ, Kok FJ. Increased cytogenetic damage in smokers deficient in glutathione S-transferase isozyme m. Carcinogenesis 13: 303 ± 305, 1992. Viezzer C, Norppa H, Clonfero E, Gabbani G, Mastrangelo G, Hirvonen A, Celotti L. Influence of GSTM1, GSTT1, GSTP1 and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers. Mutat Res 431: 259 ± 269, 1999. Vlachodimitroloulos D, Norppa H, Autio K, CatalaÂn J, Hirvonen A, Tasa G, Uusküla M, Demopoulos NA, Sorsa M. GSTT1-dependent induction of centromerenegative and -positive micronuclei by diepoxybutane in cultured human lymphocytes. Mutagenesis 12: 397 ± 403, 1997. Wiencke JK, Kelsey KT, Lamela RA, Toscano WA Jr. Human glutathione S-transferase as a marker of susceptibility to epoxide-induced cytogenetic damage. Cancer Res 50: 1585 ± 1590, 1990. Wiencke JK, Christiani DC, Kelsey KT. Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations. Mutat Res 248: 17 ± 26, 1991. Wiencke JK, Pemble S, Ketterer B, Kelsey KT. Gene deletion of glutathione S-transferase q: correlation with induced genetic damage and potential role in endogenous mutagenesis. Cancer Epidemiol Biom Prev 4: 253 ± 259, 1995. Wong R-H, Wang J-D, Hsieh L-L, Du C-L, Cheng T-J. Effect on sister chromatid exchange frequency of acetaldehyde dehydrogenase 2 genotype and smoking in vinyl chloride workers, Mutat Res 420: 99 ± 107, 1998. Wormhoudt LW, Commandeur JN, Vermeulen NP. Genetic polymorphisms of human N-acetyltransfer-
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H. Norppa ase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit Rev Toxicol 29: 59 ± 124, 1999.
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Int. J. Hyg. Environ. Health 204, 31 ± 38 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/intjhyg
Genetic polymorphisms and chromosome damage Hannu Norppa Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland
Abstract Genetic polymorphisms that affect xenobiotic metabolism or cellular response to DNA damage can modulate individual sensitivity to genotoxins. Information on the effects of such polymorphisms on the level of chromosome damage may facilitate the identification of risk groups and increase the sensitivity of cytogenetic endpoints as biomarkers of genotoxic exposure and effect. Glutathione S-transferase M1 (GSTM1) is an important detoxification enzyme which, due to a homozygous gene deletion (null genotype), is lacking from about 50% of Caucasians. A higher level of DNA adducts and chromosome damage has been detected in lymphocytes of tobacco smokers and bus drivers who lack the GSTM1 gene. Other polymorphic glutathione S-transferases include GSTM3, GSTP1, and GSTT1. The GSTT1 null genotype (10 ± 20% of Caucasians) has been associated with an increased ªbaselineº level of sister chromatid exchanges (SCEs) in lymphocytes. N-acetyltransferase 2 (NAT2), metabolizing xenobiotics with primary aromatic amine and hydrazine structures, is another important polymorphic phase II enzyme. Subjects having the NAT2 slow acetylator genotype appear to show an increased baseline frequency of lymphocyte CAs in the absence of identified environmental exposure. Besides human biomonitoring studies, genetic polymorphisms may be important in explaining individual variation in genotoxic response observed in genetic toxicology tests with human cells. Several studies have suggested that blood cultures from GSTT1 null and GSTM1 null individuals have increased in vitro sensitivity to various genotoxins. The best-known example is probably the diepoxybutane sensitivity of GSTT1 null donors. Recently discovered polymorphisms affecting DNA repair may be expected to be of special importance in modulating genotoxic effects; the first available studies have suggested that the exon 10 Arg399Gln polymorphism of XRCC1 gene (X-ray repair cross-complementing group 1) could affect individual genotoxic response. In conclusion, the genetic polymorphism of GSTM1 influences the frequency of chromosome damage in exposed humans, while that of GSTT1 and NAT2 affect the ªbaselineº level of such damage. Both GSTM1 and GSTT1 genotypes may shape the in vitro genotoxic response of human lymphocytes. The significance of DNA repair polymorphisms is presently unclear. Key words: biomarker ± DNA repair ± chromosome damage ± genotoxicity ± polymorphism ± xenobiotic metabolism
Introduction Susceptibility to genotoxic exposure varies among individuals, due to acquired or inherited characteristics. During the last few years, increasing attention has been focused on genetic polymorphisms that could modulate human response to genotoxic insult
(see (Norppa, 1997, 2000; Au et al., 1999; Pavanello and Clonfero, 2000; Norppa and Hirvonen, 2000)). At the same time, chromosomal aberrations (CAs) in peripheral lymphocytes have received special interest, as international collaborative studies have shown that a high frequency of CAs is predictive of increased risk of cancer (see (Hagmar et al. 1998,
Corresponding author: Dr. Hannu Norppa, Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250 Helsinki, Finland, Phone: 358 9 4747 2336, Fax: 358 9 4747 2110, E-mail: [email protected] 1438-4639/01/204-31 $ 15.00/0
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2000)). Recent findings have indicated that the cancer predictivity of CAs is not explained by tobacco smoking or occupational exposure to carcinogens, but that high CA rates are indicative of an increased cancer risk also in ªunexposedº nonsmokers (Bonassi et al., 2000; Hagmar et al., 2000). This suggests a general role for CAs as a cancer risk biomarker and underlines the importance of individual susceptibility factors. Several studies have implied that genetic polymorphisms can influence the level of chromosome damage associated with some genotoxic exposures, but may also affect the apparent baseline level of cytogenetic alterations. Thus, genetic polymorphisms might partly explain the association between CA levels and cancer risk. In principle, any polymorphisms that affect xenobiotic metabolism or cellular response to DNA damage could alter individual sensitivity to genotoxins. Many studies have addressed the question whether a particular genotype of a xenobioticmetabolizing enzyme is over-represented among cancer patients (see (Hirvonen, 1999a; Strange et al., 1999; Wormhoudt et al., 1999)). Such investigations require a high number of cases and controls, and relationship to past exposure is often difficult to establish due to insufficient information and the long time that has elapsed between the exposure and the disease. Another approach is to examine the influence of genetic polymorphism on biomarkers of genotoxic effect, comparing biomarker levels in different genotype groups. The association between exposure and biomarker response is then not obscured by a long latency period, and reasonable information on exposure levels can be obtained. This may facilitate the identification of risk groups and increase the sensitivity of the biomarkers in biomonitoring. The present paper shortly reviews some recent examples of genotype effects on chromosome damage.
GSTM1, tobacco smoke, and epoxides The polymorphism of glutathione S-transferase M1 (GSTM1) has offered a good model to study geneenvironment interactions. Along with other members of the glutathione S-transferase (GST) superfamily, GSTM1 is primarily involved in the metabolic detoxification of various reactive chemicals (see (Strange et al., 2000)). GSTM1 activity is present in individuals who have at least one copy of either GSTM1*A or GSTM1*B allele, the alleles differing from each other by a base substitution which seems to have little effect on enzyme activity.
GSTM1 activity is, however, totally lacking in GSTM1 null individuals who are homozygous for GSTM1 gene deletion (see (Brockmöller et al., 1992; Strange et al., 2000)). The GSTM1 null genotype is very common among Caucasians, being found in roughly 50% of the population (see (Hirvonen, 1999a)). Thus, the prerequisites to study the effects of GSTM1 polymorphism on, e. g., biomarkers of genotoxicity has been ideal, because there is an extreme and undisputed difference between the two genotypes in enzyme activity (GSTM1 positive having activity, GSTM1 null lacking it) both genotypes being distributed about evenly in the population. There are, however, ethnic differences in the frequency of these genotypes. As the GSTM1 null genotype is expected to increase individual sensitivity to various reactive compounds such as PAH diol epoxides, its role as a risk factor of various types of human cancer has extensively been studied. Although the results have been somewhat conflicting, the homozygous GSTM1 deletion has been observed to increase the risk of lung and bladder cancer in tobacco smokers (see (Brockmöller et al., 1994; McWilliams et al., 1995; Hirvonen, 1999a; Strange et al., 1999)), and has also been suggested to play a role in lung cancer among nonsmokers exposed to environmental tobacco smoke (Bennett et al., 1999). Results on genotoxicity biomarkers have supported the idea that GSTM1 null individuals have reduced capacity to detoxify genotoxins in tobacco smoke. In comparison with GSTM1 positive (or GSTM1 proficient) smokers, GSTM1 null (or GSTM1 deficient) smokers showed an elevated level of DNA adducts (Butkiewicz et al., 1998, 2000; Rojas et al., 1998, 2000; Viezzer et al., 1999) (see (Pavanello and Clonfero, 2000)), CAs (Scarpato et al., 1997), and sister chromatid exchanges (SCEs; van Poppel et al., 1992) in their leukocytes. In addition to PAHs, these findings may also be related to other constituents of tobacco smoke; lymphocytes from GSTM1 null donors were more sensitive to CA (tandem probe assay) and SCE induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine (Salama et al., 1999). The GSTM1 null genotype may predispose its carriers to the genotoxic effects of PAH-containing air pollutants in general, since GSTM1 null bus drivers (all nonsmokers), who are exposed to polluted city air containing, e. g., diesel exhausts, showed a higher frequency of lymphocyte CAs than GSTM1 positive drivers (Knudsen et al., 1999). An effect of GSTM1 genotype on DNA adducts associated with tobacco smoking or exposure to
Genetic polymorphisms and chromosome damage
PAH has not been observed in all studies (see (Pavanello and Clonfero, 2000)). Many things may contribute to this discrepancy, but it has also to be remembered that there may be great qualitative and quantitative differences in exposure among different studies (and certainly among individuals) and also the statistical power of the studies differ. One cannot just list the positive and negative studies, but should consider the overall picture. The question of GSTM1 genotype and SCEs in smokers may provide an example of the importance of including enough subjects in the study. The fact that several studies have not seen a connection between GSTM1 genotype and SCEs may reflect the fact that the effect of smoking itself on the mean number of SCEs per cell, although reproducible and proven, is usually small (only about a 10% increase). In such a situation, the demonstration of an interaction between smoking and GSTM1 phenotype or genotype would require either that the smoking effect is exclusively seen in the deficient subjects, or that there are enough subjects in the study to allow a small difference to be detected. Van Poppel et al. (1992) who originally described the association between GSTM1 phenotype and SCE frequency in smokers, saw a 5.4% increase in mean SCEs/cell among 78 heavy smokers, studying altogether 154 smokers. Almost identical SCE increase was also observed by Scarpato et al. (1996) and Wu et al. (2000), but as there was less individuals (and no separate analysis of heavy smokers), no statistical significance was reached. Results of one study have suggested that GSTM1 polymorphism could influence SCE levels also in nonsmokers (Cheng et al., 1995; Duell et al., 2000). One of the reasons that GSTM1 genotype appears to have an effect on various genotoxicity biomarkers measured from leukocytes is probably the fact that GSTM1 is expressed in these cells in GSTM1 proficient individuals (Brockmöller et al. 1992). Therefore, the GSTM1 genotype of the blood donor may likewise influence the results of genotoxicity tests performed with human lymphocytes, as discussed above in connection with NNK. Cultured lymphocytes of GSTM1 deficient subjects were also shown to have increased sensitivity to in vitro SCE induction by trans-stilbene oxide, an epoxide and a model substrate for GSTM1 (Wiencke et al., 1990). Accordingly, subjects with the GSTM1 genotype were observed to show higher SCE induction by trans-stilbene oxide than GSTM1 positive individuals (Bernardini et al., 2001). The GSTM1 genotype was also found to affect SCE induction by another epoxide, 1,2-epoxy-3-butene (Uusküla et al., 1995; Sasiadek et al., 1999).
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GSTT1, epoxides, and baseline level of chromosome damage A homozygous deletion of the GSTT1 gene (null genotype) is found in 10 ± 20% of Caucasians, making them unable to perform GSTT1-mediated detoxification reactions. Interestingly, the GSTT1 null genotype has been associated with an increased ªbaselineº level of SCEs in lymphocytes, with no association to smoking or other known exposures (Schröder et al., 1995; Wiencke et al., 1995). The finding possibly reflects an interaction between the genotype and some common endogenous or exogenous exposure; ethylene oxide generated from endogenous ethylene was proposed as a possible explanation (Schröder et al., 1995). The GSTT1 genotype effect was roughly the size of the smoking effect, i. e., quite small, which may explain why it has not been detected in all studies (see (Norppa, 2000)). Also, the low frequency of the GSTT1 null genotype in Caucasians has complicated the evaluation of GSTT1 influence in many studies. Two papers have suggested that the GSTT1 null genotype is also associated with an increase in ªspontaneousº CAs in lymphocytes (Landi et al., 1998; SÏraÂm et al. 1998). The best-known example of genotype-dependent response to in vitro chemical treatment is probably the 1,2 : 3,4-diepoxybutane (DEB) sensitivity of GSTT1 null donors. DEB, an epoxide metabolite of 1,3-butadiene, is an efficient inducer of SCEs, and DEB sensitivity was originally described using the human lymphocyte SCE test (Wiencke et al., 1991). Subsequently, DEB sensitivity was observed to be explained by GSTT1 null genotype (Norppa et al, 1995; Wiencke et al., 1995; Landi et al., 1996, 1998; Kligerman et al., 1999; Hayes et al., 2000; Schlade-Bartusiak et al., 2000). Removal of erythrocytes from whole-blood lymphocyte cultures abolished the genotype difference, reflecting the fact most of blood GSTT1 activity is found in erythrocytes (Pelin et al., 1996). Whole-blood cultures of GSST1 null subjects were also shown to be hypersensitive to the induction of micronuclei by DEB (Vlachodimitropoulos et al., 1998). Also for CA induction by DEB in vitro, GSTT1 genotype was an important factor, although it explained smaller part of the variation than in the case of SCEs; for unknown reason, some GSTT1 null subjects were only sensitive to induction of CAs but not of SCEs by DEB (Landi et al., 1998). One study could not find a GSTT1 dependence for CA induction by DEB (Schlade-Bartusiak et al., 2000). Whole-blood cultures of donors with the GSTT1 null genotype were also observed to be more sensitive than GSTT1
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positive subjects to SCE induction by 1,2-epoxy-4butene (another metabolite of 1,3-butadiene), and styrene-7,8-oxide (Bernardini et al., 1998; Ollikainen et al., 1998). Erythrocytic GSTactivity (GSTT1) was found to be protective for SCE induction in human lymphocytes by methyl bromide, ethylene oxide, and dichloromethane (Hallier et al., 1993). As the in vitro genotoxicity of the epoxide metabolites of 1,3-butadiene depended on GSTT1 or GSTM1 genotypes of the cell donors, these genotypes might also affect the genotoxic effects in workers occupationally exposed to 1,3-butadiene. However, the results of the available investigations have been conflicting, and further studies are needed (Sorsa et al., 1996; Kelsey et al., 1995; SÏraÂm et al. 1998; Hayes et al., 2000) (see (Norppa, 2000)).
NAT2 and baseline level of chromosomal aberrations N-acetyltransferase 2 (NAT2), metabolizing xenobiotics with primary aromatic amine and hydrazine structures, is another important polymorphic phase II enzyme. Seven different nucleotide transitions have been shown to lead to amino acid changes in the NAT2 enzyme (see (Hirvonen et al., 1999b)). The various genotypes result in either fast or slow acetylation capacity. Rapid acetylators have inherited at least one NAT2*4 allele, while slow acetylators have two slow-acetylation associated alleles. Slow acetylators have been described to have an elevated risk of bladder cancer, while rapid acetylators have been observed to be over-represented among colon cancer patients (see (Hirvonen et al., 1999b)). The frequency of the NAT2 slow acetylator genotype was 50 ± 63% among Caucasians, but Japanese or Chinese were mostly (92% or 80%, respectively) rapid acetylators. Subjects having the NAT2 slow acetylator genotype showed an increased baseline frequency of lymphocyte CAs, with no association to known exposures (Knudsen et al., 1999). This effect seems to be reproducible, as we have seen it in two other studies as well (Norppa et al., 2000, 2001). It might be explained by exposure to commonly found environmental genotoxins, such as heterocyclic amines in heated food; N-acetylation could function as a detoxification route in peripheral blood cells where the activities of cytochrome P-450 monooxygenases capable of metabolically activating heterocyclic amines are low (Hukkanen et al., 1997; Raucy et al., 1999). Alternatively, NAT2 may detoxify important (as yet unknown) endogenously formed
genotoxins(s). Acetyltransferases are involved in the acetylation of histones (Grunstein, 1997) and interconversion of polyamines (Seiler et al., 1985), but there is no evidence for the involvement of NAT2 in such reactions. Another polymorphic acetyltransferase, NAT1, appears to function in the catabolism of folate (Minchin, 1995), but, again, NAT2 is not known to be involved.
CYP2E1 and chromosome damage The cytochrome P-450 (CYP) superfamily of monooxygenases is involved in the metabolic activation of various precarcinogens. Studies on the influence of polymorphisms affecting various CYP species with genotoxicity are complicated by, e. g., unclear associations between CYP genotype and phenotype (see (Hirvonen 1999a; Norppa, 2000)). Several types of CYPs are found in lymphocytes, although at low levels (Raucy et al., 1999), CYP2E1 being probably among the most important ones (Hukkanen et al., 1997). Thus, CYP2E1 polymorphisms might affect the outcome of cytogenetic biomonitoring studies and genotoxicity tests with CYP2E1 substrates in human lymphocytes. A base substitution in the 5' flanking region (C1017T) of CYP2E1 gene, expected to result in CYP2E1 over-expression, has been associated with a (non-significantly) increased SCE frequency in vinylchloride-exposed workers (Wong et al., 1998) and with an elevated in vitro CA induction (tandemprobe assay) by NNK (Abdel-Rahman et al., 2000a).
XRCC1 and chromosome damage It is obvious that genetic polymorphisms of proteins involved in DNA repair (Shen and Jones, 1998) are expected to affect the level of chromosome damage. At present, however, only a few studies exist on this topic, and the actual influence of many of the recently discovered polymorphisms on DNA repair capacity remains unclear. XRCC1 protein (X-ray repair cross-complementing group 1) is considered to be involved in the repair of DNA single-strand breaks after base excision repair of damage produced by ionizing radiations, alkylating agents, and reactive oxygen species. The Arg399Gln substitution at exon 10 has been suggested to be associated with reduced DNA repair efficiency. Homozygous carriers of this variant allele were
Genetic polymorphisms and chromosome damage
observed to show elevated aflatoxin B1 DNA adducts in placenta (Lunn et al., 1999) and an agedependent increase in polyphenol DNA adducts in mononuclear leukocytes (Duell et al., 2000). Smokers with this genotype also showed increased frequencies of glycophorin A variant erythrocytes and lymphocyte SCEs, although the findings were based on only four (Lunn et al., 1999) and three (Duell et al., 2000) Gln/Gln smokers. Lymphocytes from subjects homozygous or heterozygous for the 399Gln allele were found to be more sensitive than lymphocytes from Arg/Arg subjects to SCE induction by the tobacco-specific nitrosamine NNK (Abdel-Rahman and El-Zein, 2000b).
Conclusions Some genetic polymorphisms of xenobiotic-metabolizing enzymes have been observed to influence the level of genotoxic damage in humans. The genetic polymorphisms potentially important for a particular endpoint largely depend on the exposing agent, biological material examined, and ethnicity of the population under study. As there is individual variation also in the extent and nature of the exposure, correct identification of individual exposure levels is essential. The GSTM1 null genotype appears to increase the frequency of chromosome damage in smokers, while the glutathione GSTT1 and NAT2 slow acetylator genotype seem to affect the ªbaselineº level of such damage. Besides human biomonitoring studies, genetic polymorphisms may be important in explaining individual variation in genotoxic response observed in genetic toxicology tests with human cells. Several studies have suggested that blood cultures from GSTT1 null and GSTM1 null individuals have increased in vitro sensitivity to various genotoxins. New information is emerging on polymorphisms affecting DNA repair, which may be of special importance in modulating genotoxic effects. Understanding the influence of genetic polymorphisms on genotoxic response is expected to improve the applicability of genotoxicity assays in biomonitoring and testing.
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