Interaction of XRCC1 and XPD Gene Polymorphisms with Lifestyle and Environmental Factors Regarding Susceptibility to Lung Cancer in a High Incidence Population in North East India

Exposure to tobacco smoke, fumes and airborne particulates in the indoor environment and ionizing radiations are regarded as triggering factors for DNA damages (Sterpone and Cozzi, 2010; Tang et al., 2010). Converging lines of evidence suggest that cancer can be initiated by DNA damage, which if not repaired, can cause errors during DNA synthesis (Maynard et al., 2009). Humans are routinely exposed to mutagenic and carcinogenic aromatic amines via smoking, cooking of food and other sources (Zheng and Lee, 2009). DNA sodamaged is typically repaired by certain DNA-repairing enzymes. These enzymes are fundamental for the maintenance of genomic integrity in case of replication errors. Therefore individuals with impairment in DNA repair capability are often at an elevated risk of cancer development (Berwick and Vineis, 2000). In humans more than 100 proteins are involved in DNA repair system (Lopez-Cima et al., 2007). These proteins are implicated


Introduction
Exposure to tobacco smoke, fumes and airborne particulates in the indoor environment and ionizing radiations are regarded as triggering factors for DNA damages (Sterpone and Cozzi, 2010;Tang et al., 2010).Converging lines of evidence suggest that cancer can be initiated by DNA damage, which if not repaired, can cause errors during DNA synthesis (Maynard et al., 2009).Humans are routinely exposed to mutagenic and carcinogenic aromatic amines via smoking, cooking of food and other sources (Zheng and Lee, 2009).DNA sodamaged is typically repaired by certain DNA-repairing enzymes.These enzymes are fundamental for the maintenance of genomic integrity in case of replication errors.Therefore individuals with impairment in DNA RESEARCH ARTICLE

Interaction of XRCC1 and XPD Gene Polymorphisms with Lifestyle and Environmental Factors Regarding Susceptibility to Lung Cancer in a High Incidence Population in North East India
Bhaskar Jyoti Saikia, Rup Kumar Phukan*, Santanu Kumar Sharma, Gaganpreet Singh Sekhon, Jagadish Mahanta in various DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) (Li et al., 2011).The X-ray repair cross-complementing group 1 (XRCC1) gene plays an important role in the development of lung cancer (Wang et al., 2014).XRCC1 protein plays a central role in base excision repair (BER) pathway by interacting with other DNA repair proteins (Yin et al., 2009).XRCC1 interacts with polynucleotide kinase enzyme, DNA pol-β, PARP1 and DNA ligase IIIα (Pramanik et al., 2011;Mutairi et al., 2013).Three coding polymorphisms in the XRCC1 gene are at codons 399 (Arg to Gln), 280 (Arg to His) and 194 (Arg to Trp) (Shen et al., 1998).In particular, 399 Gln/Gln allele is found to be significantly associated with higher level of DNA adducts, somatic mutations, sister chromatid exchanges and chromosomal damages (Lunn et al., 1999).Xerodermapigmentosum group D (XPD) encodes an evolutionary conserved ATP dependent helicase, a subunit of transcription factor II H (TFIIH) which is essential for transcription and NER (Coin et al., 1999;Li et al., 2013).
Mutation of XPD codons 312 and 751 increases the risk of lung cancer (Zhou et al., 2012).XPD 751Gln/Gln has been demonstrated to have suboptimal DNA-repair capacity to remove UV photoproducts when compared to the XPD 751 Lys/Lys and Lys/Gln genotypes (Qiao et al., 2002).
Lung cancer (LC) is leading cause of cancer death worldwide with an annual mortality of 18.2 % cancer death (Ferlay et al., 2010a).India contributes 6.2% cases of LC with approximately 58,000 incidence cases reported in 2008 (Ferlay et al., 2010b).North Eastern (NE) parts of India represent a unique, strategic geographic location with a demographic diverse population.Manipur and Mizoram are two states from NE parts of India.LC is mostly predominant in NE parts of India, with highest ageadjusted rate (AAR) in Mizoram (28.3 per 10 5 in male and 28.7 per 10 5 in female).Manipur also contributes a very high incidence of LC (with AAR of 14.1 per 10 5 in males and 11.9 per 10 5 in females) (NCRP, 2013).These areas are also reported for a unique consumption of tobacco, betel quids and cooking habits that are different from other places (Phukan et al., 2001(Phukan et al., , 2005(Phukan et al., , 2006)).
High risk of LC in the study population may be an outcome of genetic and environmental risk factors or a complex interaction of both.Studies have also reported association of XRCC1 and XPD allelic polymorphisms for LC (De-Ruyck et al., 2007;Li et al., 2013;Natukula et al., 2013).Lack of data on XRCC1 and XPD polymorphisms and high incidence of LC in NE parts of India prompted us to explore and evaluate any relevance of these polymorphisms in the study population.We also wished to explore the interaction of XRCC1 and XPD gene with smoking, betel quid chewing, alcohol consumption, exposure of wood combustion during cooking and cooking oil fumes (COF).

Study design
Present study was an age (±5 years), sex and ethnicity matched population based case-control study.The study duration was from June 2010-May 2013.The work was carried out at Regional Medical Research Centre (RMRC) NE Region, Indian Council of Medical Research (ICMR); India in collaboration with Population Based Cancer Registry (PBCR), Imphal, Manipur and Aizawl, Mizoram, India.Incident cases and control subjects willing to participate in the study were indigenous people of Manipur and Mizoram.Histopathologically or cytologically confirmed LC cases with no evidence of pulmonary inflammation or benign lung tumors were included in the study.Cases too old to be interviewed elaborately and who refused to be interviewed were excluded from this study.Cancer free control subjects with age (±5 years), sex and ethnicity matched were selected from healthy population of the states.None of the controls subjects had consanguinity with the cases or had any non-communicable diseases.Information of smoking, betel quid chewing, consumption of alcohol, exposure  doi.org/10.7314/APJCP.2014.15.5.1993 XRCC1 andXPD SNPs andOther Factors Interact Regarding Susceptibility to Lung Cancer in North East India.to household combustion and COF were recorded in a structured pre-designed questionnaire.The time period set for exposure of wood combustion during cooking and COF was 25 years, the participants were asked whether they have been exposed to the aforesaid time period or not.Those who were found to be exposed were taken as 'yes' for exposure of wood combustion and COF where as those who were not exposed for the set time period of 25 years were taken as 'no'.Written informed consent was taken from all subjects for participation in a protocol approved by the Institutional ethical committee of RMRC, NE Region (Indian Council of Medical Research).Thus a total of confirmed 272 LC cases and 544 controls matched for age (±5 years), sex and ethnicity were enrolled in the study.

DNA extraction
Four ml. of blood was collected from all subjects in EDTA vials.DNA was extracted by standard phenol chloroform method (Landi et al., 2006) and stored at -80º C till further analysis.

Genotyping of XRCC1 and XPD gene
Genotyping of XRCC1 and XPD were done by polymerase chain reaction based restriction fragment length polymorphism.All of the PCR reactions were carried out by a Master cycler gradient thermo cycler (Bio-Rad, United States) in a final volume of 25 ul containing 200 ng of each primer (Sigma, United States), 50 ng genomic DNA, 1.0mM MgCl 2 (Roche, Germany), 200 ul dNTPs (Roche, Germany) and 2.0 unit of Taq DNA Polymerase (Roche).The PCR product was visualized in 2% agarose gel electrophoresis by gel documentation system (Cell Biosciences).XRCC1 PCR products were amplified with primers 5'-GCCCCGTCCCAGGTAAG-3' (sense) and 5'-AGCCCCAAGACCCTTTCACT-3'(antisense) (Park et al., 2002) followed by MspI (Promega) restriction digestion.The homozygous Gln allele was determined by presence of an uncut 615-bp band (indicative of absence of MspI cutting site), homozygous Arg allele was determined by presence of two bands at 377 and 238 bp while the heterozygous Arg/Gln allele was characterized by presence of three bands at 615, 377 and 238 bp (Figure 1).XPD PCR products were amplified with the primers 5'-CCTCTCCCTTTCCTCTGTTC-3' (sense) and 5'-CAGGTGAGGGGGACATCT-3' (antisense) (Vettriselvi et al., 2007) and digested with PstI (New England BioLabs, Inc.).The homozygous Lys/Lys allele was characterized by an undigested band of 734bp, homozygous Gln/Gln allele determined by 646bp and 88bp, while heterozygous Lys/Gln genotype had three bands of 734bp, 646bp and 88bp (Figure 2).10% of

Statistical analysis
Difference in demographic characteristics, tobacco smoking, betel quid chewing and genotype frequencies between cases and controls were evaluated using Chi Square (χ 2 ) test.Estimates of LC risk, imparted by genotypes were determined by deriving odds ratio (OR) and its corresponding 95% confidence interval (95% CI) using multivariable conditional logistic regression after adjusting for potential confounders.For all tests, a two sided p≤0.05 was considered statistically significant.All statistical analysis were done using SPSS version 17.0.Tests for Hardy-Weinberg equilibrium amongst cases and control were conducted using observed genotype frequencies and a chi-square test featuring one degree of freedom.

Results
The details of demographic characteristics among cases and controls enrolled in this study are shown in Table 1.There was no statistically significant difference in term of mean age of cases (61.96±11.91years, range 21-88) and controls (61.79±12.21years, range 21-89) (p=0.711) of study subjects.58.8% of cases were of non small squamous cell carcinoma, 23.9% were of non small adenocarcinoma and 9.6% were of small cell carcinoma and 7.7% others.Significant risk was observed for smoking (OR=1.62,CI=1.17-2.24;p=0.004).Risk was observed for betel-quid chewing (OR=1.36,CI=0.99-1.87;p=0.056), exposure of wood combustion (OR=1.32,CI=0.96-1.81;p=0.088) and COF (OR=1.27,CI=0.93-1.75;p=0.133) but results are not statistically significant.Tests for Hardy-Weinberg equilibrium amongst cases and controls were conducted using observed genotype frequencies and a chi-square test featuring one degree of freedom.The distribution of genotypes for both XRCC1 and XPD genes among cases and controls were in Hardy-Weinberg equilibrium (Table 1).

Discussion
In this study, we examined whether association of XRCC1 and XPD genes polymorphisms and their interaction with indoor household exposure during cooking, tobacco smoking and betel quid chewing are implicated in development of LC in population with high incidence of lung cancer from North East India.Observation on association of XRCC1 and XPD on LC was inconsistent in different ethnic and geographical region with varying allele frequency (Lopez-Cima et al., 2007;Improta et al., 2008;Karkucak et al., 2012).In present study, no significant independent association of XRCC1 and XPD polymorphisms for LC was observed.These findings are concordant with some of the previous reports over different ethnic population (David-Beabes et al., 2001;Huang et al., 2008;Sun et al., 2013).However present study reveals significant association when XRCC1 Gln/Gln genotype interact with exposure of wood combustion, exposure of COF and tobacco smoking, while XPD Gln/Gln genotype with betel quid chewing and tobacco smoking.Combined effect of XRCC1 (Arg399Gln) and XPD (Lys751Gln) were also analysed.Result suggested that individuals with both XRCC1 Gln/Gln and XPD Gln/Gln genotype seemed to have synergistically increased risk for LC compared with those of either of them.Environmental exposure primarily tobacco smoke and other household exposure contain complex mixture of certain substances, it is plausible that repair of DNA damage intrigued by these mixed substances either by BER pathway or NER pathway.The failure or diminished on either side may cause LC risk.As expected, our study also confirmed the well established association between tobacco smoking and LC.Because of the traditional culture of study population, responsibility of cooking lies mostly with women; they are more exposed to COF and other household exposure.Interestingly in the present study significantly higher risk was observed in women than in males for LC in terms of exposure of wood combustion (OR=1.58,CI=1.01-2.48;p=0.046) and COF (OR=1.61,CI=1.02-2.53;p=0.039).Study conducted in Shenyang also observed a positive association between COF and LC risk among women (Li et al., 2008).Another study conducted by Hung et al. (2007) reported that COF is capable of causing cellular destruction of genetic material.
Wood combustion, cooking oil emission, tobacco, betel quids primarily constitute large number of polycyclic aromatic hydrocarbon (PAH), alkaloids and other phenolic compounds which are considered as a prime risk factors of LC (Seow et al., 2000;Pfeifer et al., 2002;Li et al., 2008;Hosgood et al., 2010;Mandal et al., 2013).Individuals differ widely in their capacity to repair DNA damage from both exogenous agents, such as wood and tobacco smoke, exposure to COF as well as endogenous reactions.Present study reports for XRCC1 and XPD gene polymorphisms, their interaction with exposure of COF, wood combustion, betel quid chewing, tobacco smoking and alcohol consumption and its association with LC in a high risk area from NE parts of India.Though no significant association for XRCC1 and XPD genotype on LC was observed, the Gln/Gln allele of XRCC1 seems to contribute significant risk modifiers for exposure of wood combustion, exposure of COF and tobacco smoking while the Gln/Gln allele of XPD with betel quid chewing and tobacco smoking.Studies conducted by Lunn et al. (1999) reveals that Gln allele is associated with higher DNA adduct level or lower DNA repair efficiency.PAH-induced bulky DNA adducts, such as benzo[a]pyrenediol epoxide-DNA adducts, which are the most potent premutagenic adducts are mainly repaired by NER.A variety of reactive oxygen species, such as hydroxyl radical and hydrogen peroxide are generated during enzymatic oxidation of PAH (Park et al., 2002).These reactive oxygen species can lead to DNA damages which may be quantitatively a predominant PAH-induced DNA damage.Oxidative DNA damages are primarily removed via BER, including XRCC1.
Our study has several strengths and findings.It was a population based case-control study with a high participation rates.Our cases were incident; therefore possibility of observer or recall bias can be nullified.Also case-control matching was done in reference to age (±5 years), sex and ethnicity, thereby controlling for any confounding effect on account of these variables.
Present study indicates that there is no significant relationship between XRCC1, XPD polymorphisms in study population.Significant risk was observed for interaction of these genes with some environmental factors.However a validation of these results will require its replication in a larger sample size.Taking into account other factors such as susceptibility differences of familial aggregation studies, epigenetic mechanism and infection (Human papilloma virus) will gives us more probable factors for increase risk of LC in NE parts of India.

Table 2 . Genetic Interaction and Distributions of XRCC1 Arg399Gln and XPD Lys751Gln Genotypes and Risk of Lung Cancer
* Significant; † Adjusted OR were estimated by adjusting exposure of wood combustion, cooking oil fumes, betel-quid chewing and tobacco smokingin conditional multiple logistic regression model; ‡Hardy-Weinberg equilibrium test is calculated for 1 (one) degree of freedom and values rounded to two decimals randomly selected samples were randomly sequenced to verify genotyping results and 100% concordance was found.

Table 4 . Interaction of XRCC1 Arg399Gln and XPD Lys751Gln Genotypes with Exposure of Wood Combustion, Cooking Oil Fumes, Betel-Quid Chewing and Tobacco Smoking
Significant; †Exposure of cooking oil fumes, betel-quid chewing and tobacco smokingwere adjusted to estimate adjusted OR in each model; ‡Exposure of wood combustion, betel-quid chewingand tobacco smokingwere adjusted to estimate adjusted OR in each model; §Exposure of wood combustion, exposure of cooking oil fumesand tobacco smokingwere adjusted to estimate adjusted OR in each model; ¶Exposure of wood combustion, exposure of cooking oil fumes and betel-quid chewing were adjusted to estimate adjusted OR in each model; #Adjusted OR were estimated through conditional multiple logistic regression model *