Polymorphisms in XRCC 1 Gene , Alcohol drinking , and Risk of Colorectal Cancer : a Case-control Study in Jiangsu Province of China

The x-ray cross-complementig group 1 (XRCC1) is a DNA repair gene. This gene code for protein involved in the repair of single-strand breaks (SSB) and in base excision repair (BER) of damaged DNA bases caused by endogenous and exogenous oxidants. The XRCC1 protein is a scaffolding protein directly associated with polymerase and functions in a complex to facilitate the BER and SSB processes. Genetic polymorphisms in XRCC1 gene may influence individual variation in DNA repair capacity, which may be associated with a higher risk of developing cancer. The polymorphisms occurring at conserved sequences in the XRCC1 gene were reported by Shen et al (1998).. These coding polymorphisms, resulting in amino acid substitutions, were detected at codons 194 (Arg-Trp, rs1799782), 399 (Arg-Gln, rs25487) and 280 (Arg-His). Many authors have analysed relationship between the XRCC1 polymorphisms and the risk of several cancers, including colorectal cancer, however study findings have been inconsistent (Abdel-Rahman et al., 2000; Yeh et al., 2005; Stern et al., 2007; Jin et al., 2007, 2008; Improta et al., 2008; Curtin et al., 2009; Wang et


Introduction
The x-ray cross-complementig group 1 (XRCC1) is a DNA repair gene.This gene code for protein involved in the repair of single-strand breaks (SSB) and in base excision repair (BER) of damaged DNA bases caused by endogenous and exogenous oxidants.The XRCC1 protein is a scaffolding protein directly associated with polymerase and functions in a complex to facilitate the BER and SSB processes.Genetic polymorphisms in XRCC1 gene may influence individual variation in DNA repair capacity, which may be associated with a higher risk of developing cancer.The polymorphisms occurring at conserved sequences in the XRCC1 gene were reported by Shen et al (1998)..These coding polymorphisms, resulting in amino acid substitutions, were detected at codons 194 (Arg-Trp, rs1799782), 399 (Arg-Gln, rs25487) and 280 (Arg-His).Many authors have analysed relationship between the XRCC1 polymorphisms and the risk of several cancers, including colorectal cancer, however study findings have been inconsistent (Abdel-Rahman et al., 2000;Yeh et al., 2005;Stern et al., 2007;Jin et al., 2007Jin et al., , 2008;;Improta et al., 2008;Curtin et al., 2009;Wang et Canbay et al., 2011;Gsur et al., 2011;Reeves et al., 2012;Jelonek et al., 2012;Karahalil et al., 2012).Hong et al. (2005) in South Korean and Yin et al. (2012) in Japanese observed an increased risk of colorectal cancer associated with the XRCC1 Arg399Gln polymorphism and high alcohol intake.Whereas in another study in Japanese, Kasahara et al. (2008) found XRCC1 Arg399Gln was not associated with a colorectal cancer risk.To evaluate possible relationship between the XRCC1 codons 194 and 399 polymorphisms and the risk of colorectal cancer in the Jiangsu Province of China, we conducted a populationbased case-control study.

Study Subjects
We recruited colorectal cancer cases using data of the Cancer Registries in Huian and Jintan Cities of the Jiangsu Province of China, and also recruited cases who visited Jiangsu Province Cancer Hospital from these cities from August 2000 to September 2002.All were histopathologically diagnosed as having a primary colorectal cancer, and have never been other cancers.Physicians at the hospital or families of patients asked eligible cases to participate in our study, and doctors or nurses interviewed the subjects and collected their blood samples from a peripheral vein after obtaining informed consent.Population-based controls were selected from healthy residents in eight villages or towns of Huian and Jintan Cities.They must be not any cancers.Doctors of the public health center randomly selected one or two controls for each case, after matching for ethnicity, sex and age within 2 years using the records of residents at the local governmental office, and then asked eligible residents for their participation, and conducted interviews and collected blood samples in the same manner.A total of 10 patients and 33 residents refused to participate in our study, but the response rates was 97% for cases and 93% for controls.
The ethics committee of the Jiangsu Province Institute of Cancer Research approved this study.

Background Data and environmental factors
All participants completed an in-person interview that used a structured questionnaire.The items of our questionnaire covered demographic background, height, body weight, alcohol drinking and smoking habits.In this study, smoking status was categorized as never and ever-smokers (including both current and former smokers) and drinking status as drinkers/non-drinkers (the latter including individuals whose average alcohol consumption <1 g/day).Alcohol consumption was calculated according to 40g/100g of hot wine, 3.5g/100 g of beer and 12g/100g of grape wine.The body mass index (BMI) was calculated based on body weights and heights, and stratified into three categories (<23, 23-29.9,≥30).

DNA extraction and genotyping
Whole blood was collected into EDTA-coated tubes and centrifuged for 15 min, and the buffy coat layer was isolated.Genomic DNA was extracted from 200 μL of buffy coat using a Qiagen QIAamp DNA Blood Mini Kit (QIAGEN Inc, Valencia, CA).

Statistical analysis
Associations between the XRCC1 codon 194 and codon 399 polymorphisms and colorectal cancer risk were estimated by odds ratio (OR), using the unconditional logistic regression model.We calculated adjusted ORs for age (continuous), sex, body mass index (BMI), smoking and drinking habits.The procedure LOGISTIC from the statistical package SAS was employed for the calculations.The probability of Hardy-Weinberg equilibrium was assessed by the χ 2 test.

Results
A total of 190 male and 125 female cases with colorectal cancer, and 223 male and 216 female controls, were included in this study.The background characteristics of cases and controls have been described in previous articles (Cao et al., 2008;Gao et al., 2010).The proportional distribution of females in controls was higher than that in colorectal cases.The mean age and BMI did not significantly differ between cases and controls.The proportional distributions of smokers and alcohol drinkers were higher in colorectal cancer cases than in controls (Table 1).
The genotype distributions of the XRCC1 codon 194 and 399 are presented Table 1.The proportional distributions of the XRCC1 codon 194 Arg/Arg, Arg/Trp and Trp/Trp genotypes were 50.8%, 39.6% and 9.6% in controls, 46.0%, 44.8% and 9.2% in colorectal cancer case, respectively.The genotype distributions were in Hardy-Weinberg Equilibrium in controls (P value > 0.05).It shows that subjects from population are representative.The proportional distributions of the XRCC1 codon 399 Arg/Arg, Arg/Gln and Gln/Gln genotypes were 50.1%, 42.5% and 7.4% in controls, and 48.9%, 42.5% and 8.6% in colorectal cancer cases, respectively.The genotype distributions of the XRCC1 codon 399 also were in Hardy-Weinberg Equilibrium in controls (P value > 0.05).No significant differences were observed among the studied groups with regard to the genotype distributions of the XRCC1 codons 194 and 399 (all P value > 0.05).
Data for associations between the XRCC1 codons 194 and 399 polymorphisms and the colorectal cancer risk are presented Table 2.The XRCC1 polymorphisms were not associated significantly with risk of the colorectal cancer.Similar results also were observed in subgroup analyses based on gender and cancer locations (Table 1, Table 2).The data were further analyzed to examine the combined effects of XRCC1 codon 194 and 399 genotypes on risk of the colorectal, colon and rectal cancers (Table 3), however, no significant combined effects were observed.Among controls with the 194Trp allele, the frequencies of the 399Arg and Gln alleles were 0.80 and 0.20, respectively, were not in Hardy-Weinberg Equilibrium (χ 2 =5.939,P value <0.02), suggesting that the XRCC1 194 and 399 polymorphisms were in linkage disequilibrium.
Table 4 shows the relationship of alcohol drinking and colorectal cancer risk.Alcohol drinkers showed increased ORs for colorectal, colon and rectal cancers.The ORs were increased along with an increase in alcohol intake, the trend being significant for colorectal (p<0.0001),colon (P=0.0020) and rectal (P<0.0001)cancers.
Table 5 summarizes the combined effects of the XRCC1 polymorphisms and alcohol consumption on colorectal cancer risk.As compared with non-drinkers (average alcohol consumption <1 g/day) who had the 194 Arg/Arg genotype, individuals who had high alcohol intake (average consumption >20 g/day) and with same genotype had an increased OR for colorectal cancer (2.40, 95%CI: 1.26-4.57),whereas individuals who had high alcohol intake and with the 194 Trp allele had a further increased OR for colorectal cancer (3.18, 95%CI: 1.68-5.99).As compared with nondrinkers who had the 399 Arg/Arg genotype, individuals who had high alcohol intake and with same genotype had an increased OR for colorectal cancer (1.93, 95%CI: 1.00-3.70),whereas individuals who had high alcohol intake and with the 399 Gln allele also had a further increased OR for colorectal cancer (2.17, 95%CI: 1.13-4.17).However, among the subjects with highest alcohol intake, the increased OR by 194Trp or 399Gln allele had not statistics significance.Similar combined effects also were observed in subgroup analyses based on cancer locations (colon, rectum, Table 5).
The combined effects of the XRCC1 polymorphisms and smoking on colorectal cancer risk also were examined, but no significant combined effect was observed (data not shown).

Discussion
In this study, we investigated the associations between genetic polymorphisms in the DNA repair gene XRCC1 and colorectal cancer risk.results showed that the XRCC1 codons 194 and 399 polymorphisms themselves no significant associate with the risk of colorectal cancer, and that no significant combined effect between XRCC1 codon 194 and 399 polymorphisms on colorectal cancer risk.
There have been a lot of papers on the relation between XRCC1 gene polymorphism and cancer risk, including colorectal cancer.However, results in these papers have been inconsistent.In a larger case-control study of Taiwan of China, Yeh et al. (2005) found the risk for colorectal cancer did not appear to differ significantly amongst individuals featuring the XRCC1 399Arg/Arg genotype.Stern et al. (2007) in the Singapore Chinese Health Study found that no significant association between the XRCC1 codon 194 and 399 polymorphisms and the colorectal cancer risk.Two studies in Han people of China (Jin et al., 2007;2008) and seven studies in various countries (Improta et al., 2008;Kasahara et al., 2008;Curtin et al., 2009;Canbay et al., 2011;Gsur et al., 2011;Muñiz-Mendoza et al., 2012;Reeves et al., 2012) also discovered that single XRCC1 gene polymorphisms have not correlations to the colorectal cancer susceptibility.Our present findings are consistent with these previous studies.
Although some studies suggested the XRCC1 399Gln/Gln genotype was associated with colorectal (Abdel-Rahman et al., 2000;Hong et al., 2005;Karahalil et al., 2012;Yin et al., 2012), colon (Jelonek et al., 2012) or rectal (Wang et al., 2010) cancer risk.Hsieh et al. (2003) reported linkage disequilibrium between Arg194Trp and Arg399Gln, by showing that all carrying the 194Trp allele also carried the 399Arg allele..2013.14.11.6613 XRCC1 Gene SNPs, Alcohol and Risk of Colorectal Cancer: a Case-control Study in Jiangsu, China Three studies (Hong et al., 2005;Jin et al., 2007;Yin et al., 2012) also observed a close linkage among Arg194Trp, Arg280His and Arg399Gln.In this study, we found that among controls with the 194Trp allele, frequencies of the 399Arg and Gln alleles were 0.80 and 0.20, respectively, were not in Hardy-Weinberg Equilibrium.Our results also suggesting that the XRCC1 194 and 399 polymorphisms were in linkage disequilibrium.
The environment factors are important in development of colorectal cancer.The gene-environment interactions also have been noted for colorectal cancer risk.In the present study, we found that high alcohol consumption is associated with increased risk of colorectal cancer.We also found that the effect of high alcohol intake in elevate the risk of colorectal cancer was more notable in carriers of the XRCC1 codons 194 Trp allele or 399 Gln allele.This finding is similar to the study results in Korea (Hong et al., 2005) and in Fukuoka of Japan (Yin et al., 2012).However, we also found the increased OR by 194Trp or 399Gln allele among the subjects with highest alcohol intake had not statistics significance.These results suggesting that XRCC1 polymorphisms themselves only has a little or no biological impact on colorectal cancer susceptibility.An interaction effect between XRCC1 polymorphisms and alcohol intake on colorectal cancer risk is biologically plausible.The XRCC1 is a gene of the DNA base-excision repair and radiation-induced damage repair.XRCC1 polymorphisms affect DNA repair capacity.High alcohol intake is an important risk factor for colorectal cancer, may induce carcinogenesis through DNA damage caused by the toxic effects of alcohol or its metabolites (Brooks, 1997;Blasiac, 2001).Alcohol is converted to acetaldehyde in the colonic lumen, which induces the formation of DNA adducts and produces oxidative DNA damage (Brooks et al., 2005).Alcohol intake also is associated with the production of reactive oxygen species, including oxygen free radicals, which may generate DNA base lesions (Hoek et al., 2002).
Smoking is an important environmental risk factor in human cancers, but in previous study (Gao et al., 2010), we found no relationship between cigarette smoking and colorectal cancer risk.In the present study, we also found no interaction between smoking and XRCC1 polymorphisms on the risk of colorectal cancer.This finding is consistent with the study results in Japanese population (Kasahara et al., 2008;Yin et al., 2012).
Finally, some limitations in this study require further discussion.In this study, we did not deal with some reported risk factors for colorectal cancer such as dietary habits, physical activity and family history.These risk factors could be potential confounder of XRCC1 polymorphisms or could interact with XRCC1 polymorphisms, therefore influence the effects.The sample size in this study was not sufficient for stratified subgroup analyses, with consequent reduction in the magnitude of statistical power and increase in the potential for random error.Another possible problem is selection bias for controls, these being recruited by local health staff, although from the general population with a high response rate.The proportional distribution of females in controls was higher than that in colorectal cases, which may have caused a lower prevalence of smokers and alcohol drinkers in the present controls, although we adjusted for sex, smoking and drinking in all statistical analyses.
In summary, this study showed that the XRCC1 codon 194 and 399 polymorphisms themselves were not associated with colorectal cancer risk, but there is a cooperative action between the 194Trp allele or the 399Gln allele and high alcohol intake on increasing colorectal cancer risk.These results support the fact that colorectal cancer susceptibility may be altered by interaction of gene-environment.However, the genetic susceptibility showed variable in different ethnicity, therefore, further large sample multicenter studies are needed to confirmation.

Table 2 . Polymorphisms in XRCC1 Gene and Risk of Colorectal Cancer
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.11.6613XRCC1 Gene SNPs, Alcohol and Risk of Colorectal Cancer: a Case-control Study in Jiangsu, China ORs are adjusted for age, sex, smoking, alcohol drinking and BMI

Table 3 . Combined Effects of XRCC1 194 and 399 Polymorphisms on Risk of Colorectal Cancer
ORs are adjusted for age, sex, smoking, alcohol drinking and BMI