Effect of XPD/ERCC2 Polymorphisms on Chromosome Aberration Frequencies in Smokers and on Sensitivity to the Mutagenic Tobacco-Specific Nitrosamine NNK

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1 Environmental and Molecular Mutagenesis 44:65 73 (2004) Effect of XPD/ERCC2 Polymorphisms on Chromosome Aberration Frequencies in Smokers and on Sensitivity to the Mutagenic Tobacco-Specific Nitrosamine NNK Alessandra A. Affatato, Kevin J. Wolfe, Mirtha S. Lopez, Csilla Hallberg, Marinel M. Ammenheuser, and Sherif Z. Abdel-Rahman* Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas Polymorphisms in DNA-repair genes could contribute to the interindividual differences in cancer susceptibility in smokers. By reducing DNA-repair capacity, these polymorphisms may influence the net level of smoking-induced genetic damage significantly, a critical step in the cascade of events leading to cancer. In this biomonitoring study, we examined the relationship between polymorphisms in the DNA-repair gene XPD/ERCC2 and genetic damage. We tested the hypothesis that coding polymorphisms in XPD/ERCC2 limit DNA-repair efficiency in humans leading to increased frequencies of chromosome aberration (CA) in their lymphocytes. We also used the mutagen-sensitivity assay, with the tobacco-specific nitrosamine NNK as a model mutagen, to determine whether lymphocytes from individuals with the variant XPD alleles are more sensitive to this tobacco-specific carcinogen. We calculated odds ratios (ORs) as estimates of relative risk of increased frequencies of CA associated with two XPD polymorphisms (Asp312Asn in exon 10 and Lys751Gln in exon 23). We observed a 2.57-fold (95% confidence limit [CL] ; P 0.10) increase in risk of elevated in vivo frequencies of CA associated with the variant 312Asn allele in the total population. The relative risk was more pronounced in smokers (OR 4.67; 95% CL ; P 0.04) and in all subjects 48 years old (OR 7.33; 95% CL ; P 0.01). Similarly, elevations in NNK-induced aberrations were significantly associated with the 312Asn allele (OR 3.69; 95% CL ; P 0.02). The risk was higher in smokers (OR 4.62; 95% CL ; P 0.04) and in subjects 48 years old (OR 5.76; 95% CL ; P 0.03). No significant effect was observed with the 715Gln variant allele in relation to either in vivo or NNK-induced CA. These data suggest that the Asp312Asn polymorphism may alter the phenotype of the XPD protein, resulting in reduced DNA-repair capacity. Environ. Mol. Mutagen. 44: 65 73, Wiley-Liss, Inc. Key words: DNA-repair genes; chromosome aberration; NNK; biomarkers INTRODUCTION The fact that only 10 20% of tobacco smokers develop lung cancer suggests that certain individuals may be highly resistant to the adverse effects of tobacco carcinogens, while others may be highly sensitive. Consistent with this concept, and further indicating that there is considerable interindividual variation in sensitivity to tobacco carcinogens, a number of cytogenetic studies have demonstrated a significant increase in the frequency of chromosome aberration (CA) in lymphocytes of cigarette smokers [Obe et al., 1984; Galloway et al., 1986; Milillo et al., 1996], while other studies of different populations have shown no significant differences between smokers and nonsmokers [Anderson et al., 1988; Oesch et al., 1994; Kasuba et al., 1995]. The frequency of CA in peripheral blood lymphocytes (PBLs) is a relevant biomarker for cancer risk in humans, reflecting both early biological effects of exposure to genotoxic carcinogens and individual cancer susceptibility [Hagmar et al., 1994, 1998; Bonassi et al., 1995, 2000]. Since DNA repair plays a critical role in protecting the genome of the cell from carcinogen-induced DNA damage, reduced DNA-repair capacity should constitute a significant risk factor for lung cancer and other smoking-related malignancies. In fact, phenotypic DNA-repair assays using human lymphocytes have indicated that reduced DNA-re- Grant sponsor: NIEHS; Grant number: ES06676; Grant number: T ; Grant sponsor: Philip Morris; Grant sponsor: National Center for Research Resources, NIH, USPHS; Grant number: M01 RR Grant sponsor: Doris Duke Charitable Foundation. Grant number: ICRA *Correspondence to: Sherif Z. Abdel-Rahman, Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX sabdelra@utmb.edu Received 20 January 2004; provisionally accepted 4 March 2004; and in final form 18 March 2004 DOI /em Published online in Wiley InterScience ( com) Wiley-Liss, Inc.

2 66 Affatato et al. pair capacity is associated with increased risk of tobaccorelated cancers [Wei and Spitz, 1997; Shen et al., 2003]. For example, cells from patients with lung cancer, compared with cells from healthy controls, are five times more likely to have reduced ability to repair DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), a metabolite of one of the polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke [Wei et al., 1996]. Furthermore, it was recently reported that there is a significant dose-response association between decreased DNA-repair capacity and increased risk of lung cancer, particularly for non-small cell lung carcinoma [Shen et al., 2003]. The repair of tobacco smoke-induced genetic damage involves several DNA-repair pathways [Cloutier et al., 2001]. Several polymorphisms in DNA-repair genes, representing different DNA-repair pathways, have been reported, and a number of these polymorphisms result in amino acid substitutions [Shen et al., 1998], which may lead to altered structure and/or function of the corresponding DNA-repair enzymes. Consequently, this may result in a compromise in DNA-repair capacity. Polymorphisms in genes involved in both the base excision repair (BER) and nucleotide excision repair (NER) pathways were reported to be associated with increased levels of smoking-induced genetic damage [Lei et al., 2002] and with increased risk of tobacco-related cancers [Sturgis et al., 1999; Divine et al., 2001]. The NER gene XPD, also known as ERCC2, has at least 17 single nucleotide polymorphisms that have been identified [Shen et al., 1998]. Two occur at a relatively high frequency (allele frequency 0.30) in the general population and result in amino acid changes. These are the G3A transition in exon 10, codon 312, resulting in an amino acid change from aspartic acid to asparagine (Asp312Asn), and the A3 C transversion in exon 23, codon 751, that results in a lysine to glutamine amino acid change (Lys751Gln) [Shen et al., 1998; Benhamou and Sarasin, 2002]. The variant 312Asn and 751Gln alleles of the XPD gene have been associated with a higher risk of tobacco-related cancers [Spitz et al., 2001; Zhou et al., 2002; Liang et al., 2003; Sturgis et al., 2000]. One way to detect potential variations in susceptibility to the effects of specific mutagenic agents is through the mutagen-sensitivity assay. The mutagen-sensitivity assay is based on the quantitation of mutagen-induced CA in cultured PBLs obtained from study subjects. This assay is considered an integrated biomarker that reflects individual sensitivity to the mutagen used in the assay, as well as DNA-repair capacity in the study subject [Hsu et al., 1989]. Using the mutagen-sensitivity assay, several investigators have reported that patients with tobacco-related cancers, such as head and neck and lung cancers, express the mutagen-sensitive phenotype significantly more often than cancer-free control subjects [Shen et al., 2003; Schantz et al., 1990; Spitz et al., 1995; Wu et al., 2000]. Several groups, including our laboratory, are using the mutagen-sensitivity approach to elucidate the functional significance of genetic polymorphisms and the genotype/phenotype relationship in cells following exposure to specific environmental toxicants [Uuskala et al., 1995; Norppa et al., 1995; Abdel-Rahman et al., 1999; Abdel-Rahman and El-Zein, 2000]. In the current study, we tested the hypothesis that polymorphisms coding for amino acid changes in the NER gene XPD limit the efficiency of DNA repair in humans, leading to an increased accumulation of both in vivo baseline and in vitro mutagen-induced genetic damage in PBLs. For the mutagen-sensitivity assay, we selected the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as our model mutagen. NNK is one of the most potent carcinogens in tobacco [Spiegelhalder and Bartsch, 1996] and is metabolized through -hydroxylation of either the -methyl or -methylene carbon, resulting in the formation of several reactive intermediates that form adducts with DNA [Hecht, 1998]. The repair of NNK-induced genetic damage involves several DNA-repair pathways, including the BER and NER pathways [Cloutier et al., 2001]. Our working hypothesis is that lymphocytes from individuals who inherit the variant alleles of the XPD gene are more sensitive to the mutagenic effects of this tobacco carcinogen than are individuals who are homozygous for the wild-type alleles. The data from our in vivo biomonitoring study indicate that, in smokers, the inheritance of the Asp312Asn polymorphism in the XPD gene is associated with an increase in the frequency of CA in their circulating lymphocytes. Results with the mutagen-sensitivity assay also indicate that the same polymorphism in the XPD gene plays a significant role in modulating the genotoxic response to the carcinogenic tobacco-specific nitrosamine NNK. MATERIALS AND METHODS Study Subjects and Collection of Blood Samples This study involved 65 healthy volunteers who were recruited, regardless of age, sex, or ethnicity, from the staff and students attending the University of Texas Medical Branch (UTMB) in Galveston, Texas. These individuals had responded to posted notices and advertisements requesting volunteers for genetic studies of smokers and nonsmokers. Nonsmokers were defined as individuals who had smoked less than 100 cigarettes during their lifetime. Smokers were defined as current smokers who have been smoking at least 10 cigarettes per day for at least three years prior to enrollment in the study. All volunteers were residents of the Houston Galveston metropolitan area. The study followed a protocol approved by the UTMB Institutional Review Board. Participants were asked to sign a consent form and to fill out a questionnaire that provided background demographic, occupational, personal and medical information. The questionnaire also included detailed information on smoking-related behavior and tobacco usage (e.g., number of cigarettes smoked per day; brand; use of smokeless tobacco, pipes, or cigars; former tobacco use; and duration of smoking). Criteria for exclusion of volunteers included current or recent acute bacterial or viral infections or major chronic illnesses (e.g., present or previous cancer, or autoimmune diseases), recent blood transfusion, or medical treatment with potentially mutagenic agents (e.g., chemotherapeu-

3 XPD Polymorphisms and NNK-Induced Genetic Damage 67 tic drugs or radiotherapy). Exclusion criteria also included excessive consumption of alcoholic beverages, and employment (present or past) involving exposure to toxic or mutagenic chemicals (e.g., benzene, nitrosamines, PAHs) or radiation. After informed consent was obtained, a blood sample was collected from each volunteer. A portion of the blood was used for the cytogenetic cultures; an additional portion of blood was used to obtain DNA for genotype analysis. PBL Cultures and NNK Treatment Cytogenetic cultures were established according to standard procedures [Evans and O Riordan, 1975]. Briefly, aliquots of blood (0.5 ml each) were cultured in 4.5 ml RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 10% fetal bovine serum (FBS) and 2 mm L-glutamine (all from Gibco-Invitrogen, Carlsbad, CA). PBLs were stimulated to proliferate by adding 0.18 mg/ml phytohemagglutinin (PHA; Remel, Lenexa, KS). For each subject, two cultures were prepared: a nontreated culture to provide an in vivo baseline level of CA, and a second culture for the mutagen-sensitivity assay. For the mutagen-sensitivity assay, 46 hr after stimulation, cells were separated from the culture fluids by centrifugation, and the supernatant growth medium was reserved. The PBLs were resuspended in 5 ml RPMI 1640 medium, without serum supplement, containing freshly prepared 0.24 mm NNK and incubated at 37 C for 1 hr. NNK (CAS No ) was obtained as purified crystalline powder from the National Cancer Institute, Midwest Carcinogen Repository, Kansas City, MO (lot No CSL A). After NNK treatment, the cells were washed twice in serum-free RPMI 1640, then resuspended in the reserved growth medium and transferred into clean tubes until harvested at 48 hr with the untreated cultures. Cell Culture Harvest and Cytogenetic Analysis Prior to harvest, the cells were treated with colcemid (Gibco, Invitrogen; final concentration 0.1 g/ml) for 1 hr to arrest the cells at the metaphase stage of the cell cycle. After centrifugation, cells were resuspended in hypotonic solution (0.075 M KCl), fixed with Carnoy s fixative (3:1 methanol to acetic acid), and stored overnight at 4 C. Cytogenetic slides were prepared for the evaluation of CA. The slides were coded so that the slide readers were not aware of their origin. After staining with Giemsa, the slides were scored. For each study participant, 50 metaphase cells were evaluated for in vivo CA and 50 cells for NNK-induced CA using a Nikon 400 light microscope according to standard procedures [ISCN, 1985]. The recorded aberrations were either chromosome breaks or frank chromatid breaks. In the final analysis, each chromatid break was scored as one break, and each chromosome break was scored as two breaks. Gaps or attenuated regions were also recorded, but not included in computing aberration frequencies. Total aberrant cells, including chromatid- and chromosometype aberrations, were recorded and reported as percentage of aberrant cells (breaks/100 cells). Genotypic Analysis of the XPD/ERCC2 Gene For determination of the exon 10 polymorphism (Asp312Asn), the PCR-RFLP technique described by Spitz et al. [2001] was used with slight modifications to the PCR conditions. Briefly, after an initial denaturation at 95 C for 5 min, the DNA was amplified by 30 cycles of melting (94 C, 1 min), annealing (63 C, 30 sec) and extension (72 C, 1 min), followed by a final extension step of 5 min at 72 C. The amplicon was digested with StyI (New England Biolabs, Beverly, MA), and the three possible genotypes were identified by three different banding patterns, as previously reported by Spitz et al. [2001]. For the determination of the exon 23 polymorphism, the procedure described by Dybdahl et al. [1999] was used. A PCR product containing the polymorphic site was generated and then subjected to restriction digestion by PstI (New England Biolabs), which recognizes the variant allele. This procedure allows the identification of three possible genotypes according to the different banding patterns of the digest: homozygous wild-type (Lys/Lys) and the homozygous (Gln/Gln) and heterozygous (Lys/Gln) variant genotypes [Dybdhal et al., 1999]. Statistical Analysis The main task of the statistical analysis was to assess whether there is an association between the inheritance of the polymorphisms under study and increased frequencies of either the in vivo frequencies of CA or the mutagen-induced CA in the PBLs of the study subjects. For each of the two polymorphisms studied, the homozygous and heterozygous carriers of the variant alleles were combined in the statistical analysis, as was done in many previous studies in order to increase the statistical power [Duell et al., 2000; Sturgis et al., 2000; Hou et al., 2002]. Odds ratios (ORs) and 95% confidence limits (CLs) were calculated as estimates of relative risk and strength of the association. To calculate the ORs and 95% CLs, we used the median in vivo background CA (breaks/100 cells) in the total population as the cutoff value for baseline frequencies of CA. Similarly, we used the median NNK-induced frequencies of breaks/100 cells in the total population as the cutoff value for the mutagen-induced CA. Values greater than the median were considered high, and values below the median were considered low (within the normal range). Stratified analysis was then used to test for the association in smokers and in nonsmokers, and in older ( 48 years old) and younger ( 48 years old) subjects. The median age of the population (48 years) was used as the cutoff value for this analysis. The Fisher s exact test was used to assess the statistical significance of the association. A two-sided P-value of 0.05 was considered statistically significant. All analyses were performed with Statistical Analysis System Software (SAS) (version 6.12; SAS Institute, Cary, NC). RESULTS Demographic Characteristics of the Study Population The population of subjects who participated in this study consisted of 30 males (46%) and 35 females (54%). The majority (73%) of the recruited subjects were White non- Hispanics (n 47). There were 12 African Americans (18%), and the remaining 6 subjects (9%) were Hispanics. Among the 65 participants, 39 were smokers and 26 were nonsmokers. The smokers had smoked at least 10 cigarettes per day (range of cigarettes, mean SD ) for at least 3 years (mean SD ). The nonsmokers were subjects who had smoked less than 100 cigarettes during their lifetime. The mean age of smokers ( SD) was not significantly different from the mean age of nonsmokers ( years for smokers and years for nonsmokers). The volunteers were predominantly middle-aged individuals, with a few older subjects; the mean age ( SD) was years. The median age of the study population was 48 years. Based on this median age, subjects were classified into one of two categories, 48 or 48 years of age. In Vivo and NNK-Induced Chromosome Aberration Frequencies The mean values ( SE) of the in vivo and the NNKinduced CA frequencies are presented in Table I. There was

4 68 Affatato et al. TABLE I. In Vivo and NNK-Induced Chromosome Aberration (CA) Frequencies No. of subjects In vivo (baseline) CA frequency a (mean SE) NNK-induced CA frequency a (mean SE) All subjects (n 65) Smokers (n 39) Nonsmokers (n 26) years old (n 35) years old (n 30) a CAs are reported as breaks/100 cells and included chromatid-type and chromosome-type aberrations. See text for details. The means within the groups (smoker vs. nonsmoker and older vs. younger) and between the groups (smokers or nonsmokers vs all subjects and older or younger vs. all subjects) were not significantly different (the P-value was 0.05). no statistically significant difference in baseline in vivo CA frequencies between smokers vs. nonsmokers and between older ( 48 years) vs. younger ( 48 years) individuals. In addition, the difference in CA frequencies was not statistically significant when comparing the CA levels among each of the four groups and the levels observed in all the study subjects. In all cases, the P-value was The same observation was also true when lymphocytes were treated with NNK. The frequencies of CA observed among the various strata (i.e., smokers vs. non smokers and older vs. younger individuals) were not significantly different from each other and no significant difference was observed between the frequencies of breaks among these groups compared to the frequencies observed in all study subjects. In all cases, the P-value was XPD Exon 10 (Codon 312) and Exon 23 (Codon 751) Genotypes Among the Study Population The distribution of XPD exon 10 (codon 312) and exon 23 (codon 751) genotypes among the study population is presented in Table II. Overall, no significant difference was observed in the genotypic distribution among all the subjects and the subgroups with respect to both of the polymorphisms considered. The variant 312Asn allele frequency in exon 10 was 0.32 for all subjects. This frequency was similar in smokers (0.33) and in nonsmokers (0.31), as well as in older subjects, ( 48 years, 0.33) and in younger subjects ( 48 years, 0.32). With respect to the variant 751Gln allele in exon 23, the frequency of 0.34 observed in the total population studied was also similar in smokers (0.33) and nonsmokers (0.34). However, it was slightly under-represented in older individuals (0.26) and over-represented in younger individuals (0.43). The allele frequencies are consistent with those previously reported in studies conducted on healthy subjects [Spitz et al., 2001; Hemminki et al., 2001] indicating that our sample is representative of the general population. Effect of XPD Polymorphisms on In Vivo Chromosome Aberration Frequencies Evaluation of the in vivo CA levels in PBLs of all study subjects revealed a range of 0 to 6 breaks/100 cells with a median of 4 breaks/100 cells. We confirmed by bootstrap resampling techniques that the median number of breaks/ 100 cells was the optimal cutoff value to use for calculation of relative risk estimates. The estimates of the relative risks (ORs) of increased in vivo baseline levels of CA associated with the two XPD polymorphisms are summarized in Table III. A 2.57-fold increase in risk of elevated baseline frequencies of breaks was associated with the inheritance of the 312Asn allele (the exon 10 polymorphism) when the total population was considered (OR 2.57; 95% CL ; two-sided P 0.10). The risk associated with this polymorphism was statistically significant in smokers (OR 4.67; 95% CL ; P 0.04), but not in nonsmokers. In addition, in the total population of older individuals (smokers and nonsmokers 48 years old), there was a highly significant risk of increased levels of breaks associated with this polymorphism (OR 7.33; 95% CL ; P 0.01). There was no increase in relative risk with this polymorphism in younger ( 48 years old) subjects when smokers and nonsmokers were analyzed together. With regard to the effect of the 751Gln variant allele (the exon 23 polymorphism), there was no significant overall increase in the odds ratio. There appeared to be a slight increase in risk in smokers and in older individuals, but this was not statistically significant (Table III). Effect of XPD Polymorphisms on NNK-Induced Chromosome Aberration Frequencies Using the mutagen-sensitivity approach, we evaluated the response to treatment with NNK associated with the inheritance of the 312Asn and the 751Gln variant alleles of the XPD gene in lymphocytes from the study subjects (Table IV). Evaluation of NNK-induced CA levels in PBLs of all study subjects revealed a range of 0 to 18 breaks/100 cells with a median of 8 breaks/100 cells. This value was used as the cutoff for the calculation of the relative risk estimates (ORs). Individuals were considered sensitive to NNK if the frequency of breaks was equal to or greater than the cutoff value. As shown in Table IV, the sensitivity to NNK was significantly associated with the inheritance of the 312Asn allele. The estimate of the relative risk of this association was OR 3.69 (95% CL ; P 0.02) in the total population studied. When smoking was considered, the risk was significantly elevated in smokers (OR 4.62; 95%

5 XPD Polymorphisms and NNK-Induced Genetic Damage 69 TABLE II. XPD Exon 10 (Codon 312) and Exon 23 (Codon 751) Genotypes Among the Study Population Genotypes Exon 10 Exon 23 No. of subjects Asp/Asp Asp/Asn Asn/Asn Lys/Lys Lys/Gln Gln/Gln All subjects (n 65) 25 (38) 38 (59) 2 (3) 27 (42) 32 (49) 6 (9) Smokers (n 39) 14 (36) 24 (62) 1 (2) 16 (41) 20 (51) 3 (8) Nonsmokers (n 26) 11 (42) 14 (54) 1 (2) 11 (42) 12 (46) 3 (12) 48 years old (n 35) 14 (40) 19 (54) 2 (6) 19 (54) 14 (40) 2 (6) 48 years old (n 30) 11 (37) 19 (63) 0 (0) 8 (27) 18 (60) 4 (13) TABLE III. Association Between XPD Polymorphisms and In Vivo Frequencies of Chromosome Aberrations No. of subjects OR a 95% CL P b Effect of the XPD312 Asn polymorphism All subjects n Smokers n * Nonsmokers n years old n * years old n Effect of the XPD 751Gln polymorphism All subjects n Smokers n Nonsmokers n years old n years old n Chromosome aberrations (CAs) were reported as total number of breaks/100 cells. a Odds ratios (ORs) are estimates of the relative risk of increased CA associated with the variant allele compared with the homozygous wild-type genotype. b Two-sided P-value (Fisher s exact test). *Statistically significant (P 0.05). TABLE IV. Association Between XPD Polymorphisms and Frequencies of NNK-Induced Chromosome Aberrations No. of subjects OR a 95% CL P b Effect of the XPD 312Asn polymorphism All subjects n * Smokers n * Nonsmokers n years old n * years old n Effect of the XPD 751Gln polymorphism All subjects n Smokers n Nonsmokers n years old n years old n Chromosome aberrations (CAs) were reported as total number of breaks/100 cells. a Odds ratios (ORs) are estimates of the relative risk of increased CA associated with the variant allele compared with the homozygous wild-type genotype. b Two-sided P-value (Fisher s exact test). *Statistically significant (P 0.05). CL ; P 0.04) but not significantly elevated in nonsmokers (OR 2.62; 95% CL ; P 0.43). The results, as presented in Table IV, also indicate that this exon 10 polymorphism is associated with higher mutagen-sensitivity in older subjects than in younger subjects. A significant 5.76-fold increase in risk was observed in the total population of subjects older than 48 years of age (OR 5.76; 95% CL ; P 0.03) compared to a nonsignificant OR of only 2.40 (95% CL ; P 0.45) in younger individuals ( 48 years old). With regard to the mutagen-sensitivity associated with the 751Gln variant allele, no significant effect was observed in any group across the strata. DISCUSSION In population studies, the use of biological markers, such as CA, offers a convenient method for assessing genotoxic effects associated with human exposure to potentially hazardous agents. The biological markers facilitate the evaluation of adverse responses to exposures at the time of their

6 70 Affatato et al. occurrence. Since these biomarkers represent early events in the continuum leading from exposure to clinical disease they typically respond to effects from exposure in a much larger proportion of an exposed population than the fraction that eventually will develop disease. A biomonitoring study, such as ours, can be conducted with a much smaller group of subjects than would be required for traditional epidemiological studies based on cancer incidence or mortality as an outcome [De Caprio, 1997; Abdel-Rahman et al., 2000, 2001, 2003; Ammenheuser et al., 2001]. In this study, we investigated the relationship between polymorphisms in the XPD gene and the development of genetic damage using both biomarkers of susceptibility (polymorphisms in the XPD gene) and early biological effects (CA). Although researchers have investigated XPD polymorphisms as possible risk modifiers for smoking-related cancers, very few studies have attempted to address the genotype/phenotype relationship of these single nucleotide polymorphisms using CA as an effect biomarker. The results of our in vivo biomonitoring study show that the inheritance of the 312Asn variant allele in exon 10 is associated with a significant increase in CA frequencies in smokers. An increased frequency of CA in circulating lymphocytes is generally considered indicative of an increased risk of the development of cancer [Bonassi et al., 1995, 2000; Hagmar et al., 1994, 1998]. The fact that the effect of the 312Asn polymorphism in our study was significant in smokers, but not in nonsmokers, is not surprising, since the biological concept is that a reduced repair capacity would be more important if exposure has occurred. The effect observed in smokers suggests a possible gene-smoking interaction, and indicates that coding polymorphisms in the XPD gene could affect the repair of genetic damage induced by carcinogens in tobacco smoke. A plausible biological explanation for such an interaction is that, in smokers, continuous exposure to carcinogens and mutagens present in tobacco smoke could overwhelm the DNA-repair machinery, making the effect of a polymorphism that reduces repair capacity more pronounced. Thus, the inheritance of polymorphisms that result in even a slight decrease in DNA repair could lead to more noticeable genetic damage in such individuals compared to nonsmokers. Our data indicate that the apparent effect of the 312Asn polymorphism is more pronounced in older individuals than in younger individuals. This finding suggests that individuals with this polymorphism may have a reduced ability to repair the DNA damage that tends to accumulate over the years. An age-related decrease in cellular DNA-repair capacity is a well-recognized biological phenomenon associated with increased levels of DNA damage and consequent increases in the risk of developing cancers associated with environmental exposures [Wei et al., 1993; Wei, 1998; Moriwaki et al., 1996; Barnett and King, 1995]. In addition to the in vivo biomonitoring study, we used the mutagen-sensitivity approach to test the hypothesis that individuals who inherit variant alleles of the XPD gene are more sensitive to the mutagenic effects of tobacco carcinogens than individuals homozygous for the wild-type alleles. Several case-control studies have indicated that mutagen-sensitivity is an independent risk factor for tobaccorelated cancers [Cloos et al., 1996; Schantz et al., 1990; Spitz et al., 1995]. The tobacco-smoke carcinogen NNK was chosen as the mutagen in our study, since it represents an important class of carcinogens, the tobacco-specific nitrosamines, which are known to be associated with the development of lung cancer, particularly adenocarcinoma, which is still on the rise [Hecht, 1998]. Studies on the metabolism of NNK have shown that NNK activation leads to the formation of several reactive intermediates, which induce cross-links in DNA, interact with DNA forming different types of adducts, and increase the frequencies of CA [Weitberg and Corvese, 1993; Berwick and Vineis, 2000]. The NER pathway, in which XPD is involved, participates in the repair of cross-links, oxidative DNA damage, and DNA adducts induced by tobacco carcinogens [Berwick and Vineis, 2000; Sancar and Tang, 1993; Wilson and Thompson, 1997], including NNK [Cloutier et al., 2001]. Our data indicate that the inheritance of the 312Asn allele is associated with a significantly increased sensitivity to NNK-induced genetic damage. Our results support recent findings indicating that the inheritance of the 312Asn variant allele is also associated with a reduced repair capacity for benzo[a]pyrene-induced genetic damage [Spitz et al., 2001] and decreased repair of aromatic DNA adducts [Hou et al., 2002]. Again, the sensitivity to NNK observed in the current study was higher in smokers and in older subjects, thus confirming the in vivo CA findings, and further documenting that increased genomic instability, either spontaneous or mutagen-induced, can be considered a predisposing factor for neoplastic transformation. Our data may also provide at least a partial explanation for the apparent increase in the risk of smoking-induced lung cancer associated with the XPD exon 10 polymorphism, as reported in several case-control studies [Spitz et al., 2001; Liang et al., 2003]. In some studies, the association between the 312Asn polymorphism and increased lung cancer risk was observed in nonsmokers and light smokers, but not in heavy smokers [Hou et al., 2002; Zhou et al., 2002]. In our studies, the effect of the intensity of smoking and the role of XPD polymorphisms in actual cancer risk were not the main focus. Our focus was on understanding the relationship between polymorphisms in the XPD gene and the development of genetic damage (i.e., the genotype/phenotype relationship) using CA frequencies as a biomarker of early biological effect as an end point. The mechanism by which smoking alters cytogenetic responses following exposure to specific chemicals such as NNK is not fully understood. However, in studies reported from our laboratory and others, exacerbation of genetic

7 XPD Polymorphisms and NNK-Induced Genetic Damage 71 damage in smokers exposed to environmental mutagens or carcinogens was observed. For example, increased CA frequencies were observed in smokers exposed to pesticides [Rupa et al., 1989], and to bleomycin [Albrecht et al., 2004], and elevated sister chromatid exchange (SCE) were also observed in smokers following exposure to asbestos [Kelsey et al., 1986]. The precise mechanism by which polymorphisms in the XPD gene affect DNA-repair capacity is not totally clear. Our findings indicate that the 312Asn polymorphism, rather than the 751Gln polymorphism, is associated with reduced capacity to repair chromosomal damage. Theoretically, the consequences of the 751Gln polymorphism should have been more important, since it is located in the important domain for interaction between the XPD. protein and its helicase activator, the p44 protein, inside the TFIIH complex [Coin et al., 1998]. Alteration in this interaction between XPD and p44 could, consequently, result in decreased repair. However, our data suggest that the 751Gln polymorphism is not significantly associated with reduced repair of chromosomal damage. These findings are in agreement with the results recently reported by Duell et al. [2000]. Using SCEs as a biomarker, they failed to observe a correlation between the inheritance of the 751Gln polymorphism and the levels of polyphenol-dna adducts. Also, the same variant allele did not seem to have a significant effect on the repair of bulky DNA adducts in cells from bladder cancer patients [Matullo et al., 2001]. Our observation regarding the 751Gln polymorphism is also in agreement with that of Seker et al. [2001], who reported that the 751Gln polymorphism neither increased nor reduced the apoptotic response of cells exposed to UV or ionizing radiation. A higher apoptotic response can effectively eliminate mutation-prone cells, thus providing a protective mechanism against cancer [Seker et al., 2001]. It should be noted that the 312Asn variant results in the removal of the acidic moiety of aspartic acid [Benhamou and Sarasin, 2002], and it is possible that this can alter the folding of the resulting protein, thus affecting down-stream sites of protein interaction and function. Studies are currently in progress in our laboratory to clarify the functional significance of the 312Asn polymorphism in the XPD gene. Taken together, our investigation provides evidence that the inheritance of the 312Asn variant allele of the XPD gene is associated with increased frequencies of CA in the lymphocytes of smokers. Furthermore, our data indicate that this polymorphism plays a significant role in modulating the genotoxic response to the carcinogenic tobacco-specific nitrosamine NNK. Additional studies, currently in progress in our laboratory, will address important questions raised by the current investigation, such as the combined effect of age and smoking. In the current study, we investigated the effect of smoking as a dichotomized variable (yes or no). Future studies with increased numbers of participants will address the possibility of increased risk of genetic damage associated with the XPD polymorphisms as a function of smoking duration and intensity (e.g., number of pack/years smoked). This should further substantiate the possible gene-smoking interaction observed in the current study. In our study, the more pronounced mutagenic response in a subset of susceptible individuals who are smokers raises an obvious public health concern. This is related to the question of whether these susceptible individuals have an elevated risk of the development of cancer. Concerns about cancer risk on an individual basis, however, must be tempered because other types of personal exposure to environmental carcinogens, unaccounted for, or other life-style factors that were not considered in this study, may have contributed to the elevation in CAs detected in smokers with the XPD 312Asn polymorphism. Nevertheless, although individual results for a susceptible subject in a biomarker study such as this one should not be used to specifically predict his or her future disease risk, the results for a group of susceptible subjects may be indicative of risks to that subpopulation as a whole. ACKNOWLEDGMENTS The authors thank Lori Galbert for her technical assistance. 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