XRCC1 Polymorphisms, Cooking Oil Fume And Lung Cancer In Chinese Women Nonsmokers

IntroductionLung cancer incidence has increased rapidly in China, especially in females. Although cigarette smoking remains the predominant cause of lung cancer, it can not fully explain epidemiologic characteristics of lung cancer in nonsmoker. The latter constitute a special subset of patients which include a higher proportion of women, presence of adenocarcinoma, as well as an earlier age at diagnosis Recent genetic association studies on cancer risk have focused on assessing effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly studied because of their pivotal role in maintaining genome integrity. Sequence variants in DNA repair genes are thought to modulate DNA repair capacity and consequently are suggested to be associated with altered cancer risk.The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in two interconnected DNA repair pathways: base excision repair (BER) and single-strand break repair (SSBR)The most extensively studied single nucleotide polymorphisms in XRCC1 gene are Arg194Trp on exon 6, Arg280His on exon 9, and Arg399Gln on exon 10. The Argl94Trp variant has been shown to be associated with lower bleomycin and benzo (α) pyrene diolepoxide sensitivity in vitro. The Arg280His is located in the proliferating cell nuclear antigen binding region. The Arg280His variant protein is defective in its efficient localization of a damaged site in the chromosome, thereby reducing the cellular BER/SSBR efficiency. The 399Gln allele is situated within the BRCT-1 region harboring the ADPRT binding domain that is likely to affect functional interaction between XRCC1 and ADPRT. An altered DNA repair activity has been suggested to be associated with this polymorphism. 5'UTR -77T>C is a novel polymorphism identified in the XRCC1 gene located in the 5' untranslated region. It was reported that functional SNP -77T>C decreased transcriptional activity of C-allele-containing promoter with higher affinity to Sp1 binding.ObjectiveIn this study including a relatively large number of women nonsmokers, we investigated the possible association of four polymorphisms with susceptibility to lung cancer. We also evaluated other possible etiological factors for lung cancer in women nonsmokers.MethodThis was a hospital-based case-control study in the northeast of China, comprising 350 primary lung cancer cases and 350 cancer-free hospital controls. All subjects were unrelated ethnic Han Chinese. Cases were recruited from January 2002 to January 2006 at the First Affiliated Hospital of the China Medical University and the Liaoning Cancer Hospital & Institute. They were all newly diagnosed with histopathologically confirmed primary lung cancer and surgically treated, before any radiotherapy and chemotherapy. Cancer-free controls were selected from the same hospital during the same period as the cases. The selection criteria included no previous or present history of malignant disease and they were frequency matched to case subjects by age (±5 years).Genomic DNA samples from cases were isolated from surgically resected normal tissues adjacent to the tumors of lung cancer patients using Trizol Reagent. In controls, we collected approximately 5-ml venous blood and extracted genomic DNA by the Guanidine Hydrochloride (Gu·HCl) Method. XRCC1 genotypes were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods as described previously. The PCR primers (Takara Biotechnology Dalian Co. Ltd, China) for amplifying DNA fragment containing the -77T>C (rs3213245), Argl94Trp (rsl799782), Arg280His (rs25489), or Arg399Gln (rs25487) site were 77 F5'GAG GAA ACG CTC GTT GCT AAG 3', R 5'TCC TCA TTA ATT CCC TCA CGT C 3'; 194 F5' GCC AGG GCC CCT CCT TCA A 3', R 5'TAC CCT CAG ACC CAC GAG T 3'; 280F5'CCA GTG GTG CTA ACC TAA TC 3', R5'CCTA CAT GAG GTG CGT GCT GT 3'; 399 F5'TTG TGC TTT CTC TGT GTC CA 3', R 5'TCC TCC AGC CTT TTC TGA TA 3' respectively. The PCR products were digested with restriction enzyme BsrBI (for -77T>C), PvuII (for Argl94Trp), RsaI (for Arg280His), or MspI (for Arg399Gln) to determine the genotypes. The chi-square test was used to examine differences in demographic variables and distribution of genotypes, alleles and haplotypes between cases and controls. The associations between genotype and risk of lung cancer were estimated by calculating crude and adjusted odd ratio (OR) and 95% confidence interval (95% CI) with unconditional logistic models. The Hardy-Weinberg equilibrium for each SNP was tested with Pearson's x~2 test. On the basis of the observed frequencies of the selected SNPs, we calculated linkage disequilibrium index (D' and r~2) and inferred haplotype frequencies by using the SHEsis analysis platform. To evaluate gene-environment interaction, the risks were estimated by logistic regression model. Data were analyzed with Statistical Product and Service Solutions (SPSS) software, Version 10.0 if not otherwise specified.Results1. Subject characteristicsThe mean ages of cases and controls (mean±S.D.) being almost identical (55.5±11.3 and 57.5±9.4 years, respectively). Cases included 203 adenocarcinomas, 78 squamous cell carcinomas and 69 other tumors with a variety of different pathologies (including large-cell and small-cell carcinomas, carcinoids and mixed types). No significant differences were found between cases and controls regarding passive smoking (P=0.2), family history of cancer (P=0.08) and fuel smoke exposure (P=0.08). However, cases have a higher prevalence of cooking oil fume exposure compared with controls (crude OR = 2.60, 95% CI [1.87-3.61], P<0.001).2. SNPs frequencies and association with lung cancerIn the controls, frequencies of the variant alleles were as follows: XRCC1-77C = 0.09; XRCCl-194Trp = 0.25; XRCCl-280His = 0.11; XRCCl-399Gln = 0.25. All allele distributions were consistent with Hardy-Weinberg equilibrium. Among these SNPs, homozygous carriers of the XRCC1 399Gln/Gln variant genotype had a 1.73-fold risk of lung cancer compared with the homozygous wild genotype (95%CI [1.01-2.97], P=0.01). Individuals carrying the XRCC1-77TC heterozygote genotype had a 1.51 fold increased risk of lung cancer compared with the wild genotype (OR = 1.51, 95% CI [1.01-2.24], P=0.04) and those carrying the -77CC homozygote genotype had a 3.14-fold (non-significant) increased risk (95% CI [0.96-10.30], P=0.091). 3. Stratified analysisIn the adenocarcinoma (AC) subgroup, we found that the impact of 399 Gln and -77C variant alleles was more evident than the analysis including all cases. Other SNPs including Arg194Trp and Arg280His were not associated with lung cancer in this study. Because the -77CC genotype was rare in this population, it was combined with the -77TC genotype for subsequent estimation of lung cancer risk. As a result, the combined -77TC/CC genotypes were associated with an increased risk of lung cancer with an adjusted OR of 1.61 for overall cases and 1.81 for AC. In the subgroup of squamous cell carcinoma (SCC) and other types, the distribution of genotypes was very similar among cases and controls for all polymorphisms.4. Multivariate analysisWe first eliminated the statistically non-significant SNPs (P>0.1) observed at individual SNP analyses. Thus only 2 SNPs were selected in the logistic regression model in which two nongenetic covariates (age and cooking oil expose) remained. the environmental factor cooking oil fume was the main risk factor in this model (OR = 2.51 for overall cases and 2.75 for AC), and in the presence of cooking oil fume exposure in the same model, genetic factors appeared to have a relatively moderate effect on risk of female nonsmoker lung cancer. An increased risk of lung cancer was found for 399Gln/Gln genotype carriers (Gln/Gln versus Arg/Arg, OR = 1.75, 95% CI [1.02-3.01]) and -77C variant allele carriers (TT+TC versus TT: OR = 1.66, 95% CI [1.13-2.42]). The analysis of the histological subtype AC revealed slightly higher differences between cases and controls than the analysis including all cancer cases with an OR for the 399Arg/Gln genotype of 1.63 (95% CI [1.11-2.40], P=0.014).5. Association with HaplotypesWe estimated haplotype frequencies for cases and controls by the SHEsis analysis platform. Our analysis showed that the four SNPs are in linkage disequilibrium in this study population, the minimum D' between any pair of SNPs analyzed being 0.72 (all P<0.0001). We combined all haplotypes that had an allele frequency of less than 0.03. Table 4 summarizes the association between the haplotypes and risk of lung cancer in women nonsmokers. Six possible haplotypes represented 86.0% of the chromosomes for the cases and 93.9% for the controls. Comparisons of overall haplotype distribution profiles revealed a statistically significant difference between cases and controls (global test: p<0.0001). The T-Trp-Arg-Arg and T-Arg-His-Arg haplotypes were associated with (non-significantly) reduced risks of lung cancer (OR = 0.67 and 0.75, respectively), compared with the T-Arg-Arg-Arg haplotype, whereas T-Trp-Arg-Gln was associated with a significantly increased risk (OR = 2.26). 6. Analysis of gene-environment interactionsWe analyzed the interaction of XRCC1 Arg399Gln or XRCC1 T-77C with cooking oil fume on lung cancer among women nonsmokers in a logistic regression model. We combined the heterozygous and homozygous variant genotypes for analysis. Increased lung cancer risk for XRCC1 399Gln/Gln or Arg/Gln carriers (OR = 3.46, 95% CI [0.77-15.56]) or for XRCC1-77TC or CC carriers (OR = 4.35, 95% CI [0.82-23.0]) among cooking oil fume exposure was observed, however, as these findings were not statistically significant, a further histological subgroup analysis was not performed in this paper.ConclusionIn conclusion, our study revealed that XRCC1 Arg399Gln and T-77C polymorphisms may alter the risk of lung cancer in women nonsmokers in China. Moreover, because genetic polymorphisms often vary between ethnic groups, further studies are needed to clarify the association between XRCC1 polymorphisms and lung cancer in diverse ethnic women nonsmokers.