Influence of the CYP1A1 T3801C Polymorphism on Tobacco and Alcohol-Associated Head and Neck Cancer Susceptibility in Northeast India

Background: Tobacco and alcohol contain or may generate carcinogenic compounds related to cancers. CYP1A1 enzymes act upon these carcinogens before elimination from the body. The aim of this study was to investigate whether CYP1A1 T3801C polymorphism modulates the relationship between tobacco and alcohol-associated head and neck cancer (HNC) susceptibility among the northeast Indian population. Materials and Methods: One hundred and seventy histologically confirmed HNC cases and 230 controls were included within the study. The CYP1A1 T3801C polymorphism was determined using PCR-RFLP, and the results were confirmed by DNA sequencing. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approaches were applied for statistical analysis. Results: The CYP1A1 CC genotype was significantly associated with HNC risk (P=0.045). A significantly increased risk of HNC (OR=6.09; P<0.0001) was observed in individuals with combined habits of smoking, alcohol drinking and tobacco-betel quid chewing. Further, gene-environment interactions revealed enhanced risks of HNC among smokers, alcohol drinkers and tobacco-betel quid chewers carrying CYP1A1 TC or CC genotypes. The highest risk of HNC was observed among smokers (OR=7.55; P=0.009) and chewers (OR=10.8; P<0.0001) carrying the CYP1A1 CC genotype. In MDR analysis, the best model for HNC risk was the three-factor model combination of smoking, tobacco-betel quid chewing and the CYP1A1 variant genotype (CVC=99/100; TBA=0.605; P<0.0001); whereas interaction entropy graphs showed synergistic interaction between tobacco habits and CYP1A1 . Conclusions: Our results confirm that the CYP1A1 T3801C polymorphism modifies the risk of HNC and further demonstrated importance of gene-environment interaction.


Introduction
for a variety of cancers (Olshan et al., 2000). Human cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) is one of the major phase I enzymes, involved in the activation of carcinogens related to head and neck cancers, such as polycyclic aromatic hydrocarbon (PAH), heterocyclic amines, N-nitrosamine, alkaloids, polyphenols and tannins present in tobacco smoke and betel quid (Wu et al., 2004;Sharan et al., 2012). Ethanol and acetaldehyde of alcoholic drinks are important chemical agents related to various cancers (Lachenmeier et al., 2012). Therefore, modulation of CYP1A1 activity may be important in the aetiology of HNC. Both environmental and genetic factors influence the activity of CYP1A1. Tobacco smoking and betel quid consumption increases CYP1A1 activity in humans. CYP1A1 activity is also modulated by specific polymorphisms in the CYP1A1 gene. One of its common polymorphisms CYP1A1 T3801C (CYP1A1-MspI or CYP1A1*2A or rs4646903) has been associated with higher induction of CYP1A1. The higher enzyme activity would result in increased levels of carcinogenic intermediates, leading to greater risk of cancer development. The associations of these CYP1A1 single nucleotide polymorphisms (SNPs) with cancers have been well documented Liu et al., 2014;Lu et al., 2014).
The aim of this study was to investigate the frequency of CYP1A1 T3801C polymorphism in patients with HNC and its role in modulating the relationship between tobacco and alcohol consumption, and HNC risk. Furthermore, multifactor dimensionality reduction (MDR) approach was used to investigate the high-degree gene-environmental interaction in NPC carcinogenesis.

Study population
The present study consists of 170 histologically confirmed, untreated HNC cases (diagnosed between December, 2009 andJuly, 2013) and 230 healthy controls (without family history of cancer). The oral swab and/ or peripheral blood of the participating subjects were collected upon written consent. Controls were individually matched to cases in sex, age, ethnicity and neighborhood. Further, a pre-designed questionnaire was used to collect demographic information including family history of cancer, habits of tobacco, and alcohol consumption as well as their frequencies. Tobacco habits in our study include smoking (cigarettes and beedi), tobacco chewing (khaini, zarda, gutkha), and betel quid chewing (betel leaf along with areca nut, lime with or without tobacco). The study was approved by the Institutional Ethics Committee, Assam University, Silchar. Precautions were taken to avoid contamination while collecting and processing the samples.

DNA extraction and genotyping
Genomic DNA was isolated from the collected blood or oral swabs by phenol/chloroform/ isoamylalcohol (Ghosh and Mondal, 2012) method, and then stored in T.E buffer (10Mm Tris-HCl, pH 0.8, 1 Mm EDTA) at -20ºC for further used. The polymorphisms of CYP1A1 T3801C gene was analysed by polymerase chain reaction restriction length polymorphism (PCR-RFLP) method (Ng et al., 2005). PCR amplification of a 343-base DNA fragment containing MspI restriction site was performed, using the primers 5/-TAGGAGTCTTGTCTCATGCCTT-3/ and 5/-CAGTGAAGAGGTGTAGCCGCT-3/. PCR programme was performed at 95°C for 5 min for the initial denaturation, following 30 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 45 s, extension at 72°C for 30 s and final extension at 72°C for 5 mins. For the CYP1A1 T3801C genotype analysis, the PCR product was digested with the MspI restriction enzyme (New England BioLabs, USA); a single 343 bp fragment represents the wild-type allele (TT), three fragments of 343, 200 and 143 bp indicates for the heterozygous (TC) and two fragments of 200 and 143 bp for the variant allele (CC). The RFLP results were confirmed by sequencing 10% of randomly selected samples from both cases and controls by Sanger sequencing using Genetic Analyzer 3500, Applied BioSystems (Molecular Medicine Lab, Department of Biotechnology, Assam University, Silchar, India).

Statistical analysis
Statistically significant differences between cases and controls for demographic characteristics were assessed by χ 2 test. The association between smoking, alcohol drinking and tobacco-betel quid chewing and CYP1A1 T3801C polymorphism in HNC risk were analyzed by calculating odds ratios (ORs), 95% confidence intervals (95% CI), and their corresponding P-values. A P-value of <0.05 were considered to indicate statistically significant. Departures from Hardy-Weinberg equilibrium for CYP1A1 T3801C genotype were evaluated by comparing the observed genotype frequencies with expected frequencies using χ 2 tests. Statistical analyses were performed using SPPS software, version 16 for Windows.

Multifactorial dimensionality reduction analysis
The MDR analysis is an advanced non-parametric approach (www. multifactordimensionalityreduction. org) used to detect the gene-environment interactions associated with disease risk (Hahn et al., 2003). The advantage of using MDR is it overcomes the sample size limitations often encountered by parametric approaches (example: logistic regression analysis) while studying high-level interaction. The best prediction model was selected on the basis of maximum training balance accuracy (TrBA), testing balance accuracy (TBA) and cross validation consistency (CVC). The best model with highest TrBA, TBA and CVC was tested by 1000 fold permutation testing and χ 2 test at 0.05 significance levels during MDR analysis.

Interaction entropy graphs
The interaction entropy graphs were constructed using MDR results to determine synergistic and nonsynergistic interactions among the variables (Lavender et al., 2009;Yu et al., 2014). The graphs comprise of nodes containing percentage entropy of each individual variables and connections joining them pairwise showing entropy of interaction between them. Positive entropy signifies

Polymorphism in CYP1A1 gene and association with HNC risk
The genotypes of CYP1A1 T3801C were determined by detecting the PCR-RFLP band pattern on 1.5% agarose gel. The PCR-RFLP result was confirmed by randomly sequencing 10% of the samples (Figure 1). The three genotypes of CYP1A1 T3801C viz. TT, TC and CC had frequency distributions of 45.3%, 41.2%, 13.5% and 54.3%, 36.1%, 9.6% in cases and controls, respectively. Logistic regression analysis show that CYP1A1 CC (OR=2.06, 95% CI: 1.01-4.17; P=0.045) genotype had significant risk association with HNC in the study population when compare to the TT genotype (Table 2). Combine TC and CC (TT + CC) genotypes also showed a risk (OR=1.54, 95% CI: 1.02-2.39, P=0.039) of HNC. The risk associated with each allele was also investigated. We observed significant risk of HNC in those individual carrying the C-allele as compared to T-allele (P=0.04). Further, the association between CYP1A1 T3801C genotype and different types of HNC was analysed. It was observed that CC (P=0.014) and combined TC+CC (P=0.049) genotypes were significantly associated with the risk of oral cancer (Table 2). However, CYP1A1 polymorphism was not associated with the risk of other types HNC in our study.
The combinations of smoking, alcohol drinking and tobacco-betel quid chewing habits and their interactions with CYP1A1 T3801C polymorphism in HNC risk were also considered (Table 3). A significantly elevated risk of

MDR analysis
MDR analysis was used to determine the best-model gene-environment interaction for HNC risk (Table 4). The analysis suggest that smoking was the best one-factor model with CVC 100/100 (TBA=0.59; P<0.001). While smoking and CYP1A1 variant genotype was the best two factors model with CVC of 94/100 (TBA=0.57; P<0.001).
The best four-factors model was the combinations of smoking, tobacco-betel quid chewing, alcohol drinking and CYP1A1 variant genotype with CVC of 100/100 and TBA (0.51) and P<0.0001. However, the best model of all the predictive models was the three-factor model combination of smoking, tobacco-betel quid chewing, and CYP1A1 variant genotype having CVC of 99/100 and highest TBA of 0.60 (P<0.0001).

Interaction entropy graphs
Interaction entropy graph was constructed using MDR results to determine synergistic or antagonistic interactions between the genetic and environmental factors in HNC risk (Figure 2). Entropy graph revealed smoking, and tobacco-betel quid chewing had highest independent effect with percentage entropy of 2.49% and 2.14%, respectively. Tobacco-betel quid chewing had a synergistic interaction with CYP1A1 variant (0.64%) by removing 0.49% of entropy. Alcohol consumption (0.83%) might also explain considerable entropy independently.

Discussion
In this study, we investigated the role of CYP1A1 T3801C polymorphism in modulating the relationship between smoking, alcohol drinking and tobacco-betel quid chewing and HNC risk among the northeast Indian population. Our findings suggested that the variant CYP1A1 T3801C genotype, by increasing the CYP1A1 activity, modified HNC risk by interacting with tobacco and alcohol.
Northeast India is reported to have a very high morbidity and mortality rates of HNC (Bhattacharjee et al., 2006). Epidemiological studies from the northeast India reported tobacco smoking as one of the strongest risk factor for HNC (Yadav et al., 2010;Talukdar et al., 2013;Choudhury and Ghosh, 2014;Ghosh et al., 2014a) . Moreover, it was seen that tobacco-betel quid chewing also had a very high impact on HNC risk (Sharan et al., 2012;Choudhury et al., 2014) . Tobacco in any form generates free radicals that deplete antioxidants and cause oxidative damage to DNA, proteins and lipids resulting in cancers. Similarly; alcohol consumption is linked to an increase in risk of HNC (Bektas-Kayhan et al., 2014;Ghosh et al., 2014a;Krishna et al., 2014). Recent studies have revealed mutagenic effects of alcohol on human chromosomes in vitro (Hsu et al., 1991). Our results also suggest for a strong risk of HNC with smoking, interaction and HNC risk. This graphical model explains the percent of the entropy in case-control removed by each factor (independent effect) and by each pair-wise combination of attributes (interaction effect). Positive percentage of entropy indicating synergistic interaction and negative values of entropy represent redundancy. The red colour indicating a high degree of synergistic interaction, orange a lesser degree whereas; gold represent midpoint; blue represents the highest level of redundancy followed by green. TBC-tobacco-betel quid chewing, TBS-tobacco smoking, and ALC-alcohol consumption; (B) Summary of the four-factor model (CYP1A1, alcohol, smoking and tobacco-betel quid chewing) in MDR analysis. The distribution of high risk (dark shading) and low risk (light shading) combinations associated with HNC risk. The percentage of patients having HNC was represented by left column in each box, whereas right column in each box indicated percentage of controls Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 6959 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.16.6953 CYP1A1 T3801C Polymorphism and Tobacco and Alcohol-Associated Head and Neck Cancer Susceptibility in Northeast India alcohol drinking and tobacco-betel quid chewing (Table  1).
In our study, significant associations between HNC risk and CYP1A1 T3801C variant genotypes were observed (Table 2). Our study suggests an increased risk of HNC in the CYP1A1 CC genotypes (2 fold) that could reflect the higher enzymatic activity. CYP1A1 belongs to a superfamily of phase I xenobiotics metabolizing enzymes. These enzymes play a vital role in resisting a large variety of chemical carcinogens and environmental toxicants that are probably associated with cancer risk. Altered forms of enzymes are known to enhance enzyme activities and therefore, have been linked with increasing incidence of cancers (Sharma et al., 2013;Shukla et al., 2013), most likely due to increased susceptibilities to environmental toxins and carcinogens. Moreover, they are involved in the induction of various enzymes and proteins important in cellular function, e.g., the modulation of DNA repair genes that in turn determines individual susceptibility to cancer risks (Guo et al., 2008).
In the present study, the confounding effect of smoking on the HNC association with CYP1A1 T3801C polymorphism shows that CYP1A1 CC genotypes were strongly associated with risk of HNC in smokers (Table  3). Recent studies also observed that the risk of cancers in smokers was modulated by CYP1A1 genetic variations (Guo et al., 2008;Sam et al., 2008;Liu et al., 2013;Jiang et al., 2014). However, these studies were conducted in a bi-modal manner (presence/absence) whereas our study was conducted on a dose-dependent manner. Tobacco smoke is a complex mixture of over 4,000 compounds; more than 60 of the compounds are potent carcinogens, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, N-nitroso compounds. CYP1A1 is known to metabolize PAHs generated from tobacco smoke such as benzo [α] purene in addition to a various other carcinogenic compounds (Guengerich and Shimada, 1998;Bartsch et al., 2000). The carcinogenic substrates on exposure to CYP1A1 may therefore increase the potential for genomic damage (Sam et al., 2008). Studies also revealed the expression of CYP1A1 in the buccal mucosa, suggesting the in situ activation of tobacco carcinogens (Vondracek et al., 2001). Moreover, cigarette smoke has also been shown to up regulate CYP1A1 under in vitro conditions as well as in smokers (Nagaraj et al., 2006;Chi et al., 2009). Previous studies reported genetic variability altered enzyme activity and subsequently affect carcinogens detoxification in tobacco smoke, thereby increasing susceptibility to HNC risk (Soya et al., 2007;Sabitha et al., 2010;Sam et al., 2010;Tai et al., 2010;Khlifi et al., 2014). However, few studies did not find a relationship between smoking and risk of HNC among cases with the CYP1A1 polymorphisms (Matthias et al., 1998;Oude Ophuis et al., 1998;Varela-Lema et al., 2008). In addition, CYP1A1 TC and CC genotypes appeared to be strongly associated with risk of HNC in tobacco-betel quid chewers. Betel-quid chewing along with tobacco results in the exposure to nitrosamines derived from tobacco and areca or betel nut alkaloids. Betel quid chewing generate a high amount of ROS, which has been implicated in multistage carcinogenesis (Soya et al., 2007). Recent studies reported a significant association between CYP1A1 polymorphism and HNC among tobacco-betel quid chewers (Anantharaman et al., 2007;Hernando-Rodriguez et al., 2012;Chaudhuri et al., 2013), whereas few studies did not found any relationship between CYP1A1 and chewing (Khlifi et al., 2014).
Furthermore, a strong risk modulation of HNC was observed among individual carrying the CYP1A1 CC genotype and alcohol drinking (Table 3). This is the first study to report an interaction of CYP1A1 T3801C polymorphism and environmental factors in the development of HNC in northeast Indian population. Similar type of result, have been reported in the previous other study in oral cancers (Chatterjee et al., 2009;Chatterjee et al., 2010). On the other hand, individual carrying the CYP1A1 TC and CC genotypes and taking alcohol either with smoking or tobacco-betel quid chewing has an overall higher risk for HNC risk. Alcohol in general is not a genotoxic substance; however, its degradation results in the formation of acetaldehyde, which is carcinogenic in nature. Besides alcohol drinking suppresses the removal of nitrosamine molecules of low molecular weight released by tobacco in the liver and inhibiting several isoforms of the cytochrome P450 superfamily (Cury et al., 2012). Furthermore, there was an increase of nitrosamines to post-hepatic tissues and an increase in DNA adducts formation, which can lead to cancer development (Warnakulasuriya et al., 2005;Hashibe et al., 2007).
We include MDR analysis to study the higher order gene-environmental interaction. MDR approach identifies the combination of tobacco habits and CYP1A1 T3801C polymorphism as the best model for HNC risk in our study population (Table 4). Interaction entropy graph was drawn to determine synergistic or antagonistic interactions using the MDR results ( Figure 2). Entropy graph, revealed tobacco habits had highest independent effect and had a synergistic interaction with CYP1A1 variant genotypes. These results indicated that besides tobacco exposure, individual detoxification capacity also plays an important role in the development of HNC. Our study might have certain drawback for predicting high-order interactions due to the relatively small sample size; however, MDR approach overcomes the limitation for low sample sizes by using cross validation and permutation testing strategy.
Our study indicates that smoking, alcohol drinking and tobacco-betel quid chewing are risk factors associated with HNC in the northeast Indian population. In addition, it is suggested that CYP1A1 T3801C polymorphism is a predisposing risk factor for HNC. The result also indicates that the risk of HNC associated with tobacco and alcohol is modulated by CYP1A1 T3801C polymorphism. Highest risks of HNC were observed among individual consuming tobacco and alcohol and carrying the CYP1A1 CC genotypes. These findings demonstrated importance of gene-environment interaction in prediction the susceptibility of HNC.