The 399Gln polymorphism in the DNA repair gene XRCC1 modulates the genotoxic response induced in human lymphocytes by the tobacco-specific nitrosamine NNK.

DNA repair plays a critical role in protecting the genome of the cell from the insults of cancer-causing agents such as those found in tobacco smoke. Reduced DNA repair capacity would, therefore, constitute a significant risk factor for smoking-related cancers. Recently, a number of polymorphisms in several DNA repair genes have been discovered, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate cancer susceptibility in exposed populations. In the current study, we explored the relationship between two polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codons 194 and 399) and the genotoxic response induced by the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The sister chromatid exchange (SCE) assay was used as a marker of genetic damage. Our results, using whole blood cultures from 47 volunteers, indicated that treatment of cells with 0.24, 0.72 and 1.44 mM of NNK induced a concentration-dependent increase in the mean number of SCE (P<0.001). There was a significant difference (P<0.05) in response to NNK treatment between cells from individuals with the 399Gln allele (either homozygous or heterozygous) and cells from individuals with the homozygous 399 Arg/Arg genotype. Treatment of cells that have the 399Gln allele with 0.24, 0.72 and 1.44 mM NNK resulted in 22.8, 35.8 and 52.8% increases in NNK-induced SCE, respectively. Treatment of cells with the 399 Arg/Arg genotype using the same NNK concentrations resulted in 16.0, 15.5 and 32.6% increases in NNK-induced SCE, respectively. In contrast, no significant difference in NNK-induced SCE was observed between cells with the codon 194 Arg/Arg genotype and cells with the codon 194 Arg/Trp genotype at all concentrations of NNK tested. These data suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair. Our study underscores the important role of polymorphisms in DNA repair genes in influencing the genotoxic responses to environmental mutagens, and justifies additional studies to investigate their potential role in susceptibility to cancer.

Association of the NAT1*10 genotype with increased chromosome aberrations and higher lung cancer risk in cigarette smokers.

The NAT1 gene exhibits polymorphisms in the non-coding polyadenylation region with a number of alleles. Of these alleles, NAT1*10 is responsible for increased NAT1 enzyme levels and is reported to be associated with increased risk for colorectal and bladder cancers. In view of the possible role of the NAT1 gene product in the metabolism of a number of cigarette smoke carcinogens, we tested the possibility that genetic variation in the NAT1 gene might also be associated with increased risk for lung cancer. Allelic variances of the NAT1 gene were analyzed in 45 lung cancer patients and 47 controls who were matched with respect to age, race and gender using restriction fragment length polymorphism (RFLP) analysis and allele-specific (AS)-PCR. Our results indicate that individuals who inherited the NAT1*10 allele had a 3.7-fold increased relative risk for lung cancer (95% CL = 1.2-16.0, p < 0.02). There was a 6.8-fold increase in relative risk for lung cancer associated with the inheritance of the NAT1*10 allele in younger individuals (< 60 years of age) compared to 2.2-fold increase in older individuals (> 60 years old) (OR = 6.8; 95% CL = 1.1-40.7, p < 0.01 and OR = 2.2; 95% CL = 0.5-11.1, p = 0.2, respectively). We have also applied the sensitive fluorescence in situ hybridization (FISH) tandem probe assay to elucidate the frequency of chromosome breakage among a subgroup of the studied individuals harboring the NAT1*10 allele (17 lung cancer patients, 17 smoking controls and 7 non-smoking controls). Our results indicate a significant increase (p < 0.001) in the frequency of chromosome breaks in lung cancer patients (mean +/- SE per 100 cells = 1.45 +/- 0.11) and in smoking controls (1.30 +/- 0.13) compared to non-smoking controls (0.47 +/- 0.07). Regression analysis indicated a highly significant positive correlation between the duration of smoking in years and the frequency of chromosome breaks in lung cancer patients (r = 0.62, p = 0.008), but not in smoking controls (r = 0.02; p = 0.91). These findings suggest that NAT1 polymorphism may be an important genetic determinant of lung cancer risk. In addition, these data provide a mechanistic link between the inheritance of the NAT1*10 allele and smoking-induced lung cancer. Given that the NAT1 enzyme can mediate activation and detoxication pathways for numerous carcinogens and given that this polymorphism is prevalent in the general population (20-50% frequency), it may play a significant role in influencing the outcome of a variety of environmental cancers.

Polymorphism of metabolizing genes and lung cancer histology: prevalence of CYP2E1 in adenocarcinoma.

The relationship between genetic predisposition and development of specific cancers has not been adequately elucidated. In this study, the involvement of three polymorphic genes (CYP2E1, GSTM1, and GSTT1) in the development of different histological types of lung cancer was investigated. DNA was extracted from peripheral blood lymphocytes of lung cancer patients who have been long-term cigarette smokers (n = 52). Allelic variants of CYP2E1 were detected using PCR followed by PstI restriction enzyme digest and RFLP analysis, which detects a specific mutation causing over-expression of the gene. GSTM1 and GSTT1 genotypes were detected using two separate differential PCR methods. Our results indicate a 13.5% allele frequency for the CYP2E1 rare PstI site among the lung cancer patients which represents a 3.4-fold increase over the normal controls (OR = 3.5, 95% CL = 0.65-25.8). A novel observation is that all the patients with this polymorphism had adenocarcinomas only, resulting in a significant association between them (OR = 16.17, 95% CL = 0.95-73, P = 0.02). The frequency of the null GSTM1 gene was 42.3% among the lung cancer patients with no preferential tendency towards developing squamous cell carcinoma versus adenocarcinoma (OR = 1.10, 95% CL = 0.3-4.14, P = 0.5). The GSTT1 gene was absent in 21.1% of the patients with a non-significant tendency towards developing squamous cell carcinoma (OR = 1.23, 95% CL = 0.25-6.1, P = 0.5). Another important observation is the significant predominance of the three predisposing polymorphic alleles among the adenocarcinoma patients (OR = 3.4, 95% CL = 0.78-16.1, P = 0.05) compared with the squamous cell carcinoma patients. The results of this study indicate that the inheritance of several polymorphic metabolizing genes, particularly the CYP2E1 gene, contributes not only to the development of lung cancer but also to the development of specific types of cancer.

A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies.

A deletion polymorphism in glutathione S-transferase theta (GSTT1) gene was recently discovered in humans. Similar to the GSTM1 gene, GSTT1 is also recognized as a risk modifier in exposed populations. To evaluate the role of genetic polymorphism in health effects, the combined genetic polymorphism of different genes should be taken into consideration. In the present study, we have developed a multiplex PCR approach for simultaneous replication of both genes for molecular analysis. The multiplex PCR protocol was validated using donor DNA with different polymorphic combinations for both genes from two different ethnic populations (North Americans and Egyptians). The prevalence of the GSTM1 null genotype was 51% among North Americans and 44% among Egyptians. The prevalence of the GSTT1 null genotype was 15% among North Americans and 14.7% among Egyptians. Combined polymorphism analysis of both genes revealed that 6.3% of North Americans harbor the deleted genotype of both genes compared to 8.8% of the Egyptians. The data indicate that there is no major difference in allelic distribution of both genes between the ethnic populations. The multiplex PCR assay used in this study has the advantage of reducing the time, effort and cost required to carry out such analysis. It will also significantly enhance the ability to use genetic screening techniques as a potential tool for early detection of health outcomes in exposed populations.

Genetic polymorphism of GSTM1, CYP2E1 and CYP2D6 in Egyptian bladder cancer patients.

Polymorphic changes in the GSTM1, CYP2E1 and the CYP2D6 genes have been reported to be individually associated with increased susceptibility to certain cancers. In the present study, the relationship between genetic polymorphism for these genes and development of urinary bladder cancer among Egyptian patients was investigated. Our results indicate that the frequency of bladder cancer patients with the GSTM1 null genotype is significantly higher than that of the normal controls (86.3 and 47.6%, respectively) with an odds ratio (OR) of 6.97 (95% CL -1.59-30.57, Fisher's exact P = 0.008). In contrast, our investigation failed to demonstrate any difference in the distribution of CYP2E1 polymorphism between bladder cancer patients and controls as detected by PstI restriction fragment length polymorphism (RFLP) analysis. RFLP analysis of the CYP2D6 gene revealed a non-significant increase in the number of extensive metabolizers (EM) among the patients compared to the controls (68 versus 48%). However, the EM genotypes enhances the risk further for individuals harboring the GSTM1 null genotype as individuals harboring both the EM and the GSTM1 null genotypes have an odds ratio of 14.0 (95% CL = 1.3- 151.4, Fisher's exact P = 0.02) compared to individuals harboring the EM and the GSTM1 +/+ genotypes. In conclusion, our results indicate that genetic polymorphism, especially in GSTM1 and CYP2D6 could play an important role as host risk factors for development of urinary bladder cancer among Egyptians.