Genetic Polymorphism of Glutathione S-transferases M 1 and T 1 , Tobacco Habits and Risk of Stomach Cancer in Mizoram , India

Stomach cancer is the second leading cause of cancer death and forth most common malignancy in the world which accounts for 9.7% of total cancer deaths (Ferlay et al., 2010). In India, the incidence of stomach cancer is highest in the Aizawl district of the state of Mizoram with an age-adjusted rate (AAR) of 55.4 in male and 24.4 in female per 105 populations (NCRP, 2010). The ageadjusted rate (AAR) of stomach cancer for the entire state of Mizoram are 42.9 and 20.5 per 105 populations in male and female, respectively (NCRP, 2010). This incidence is quite high as compared to other regions of India where the incidence of stomach cancer per 105 varies from 1.0-12.3 in males and 0.6-11.0 in females. There are considerable regional differences in the incidence of stomach cancer worldwide and the highest incidence among men is reported from Changle, China (AAR=145.0) and among women in Yamagata, Japan (AAR=38.9) per 105 populations (Parkin et al., 2002). Numerous studies have shown that consumption of alcohol, tobacco and different food habits are important


Introduction
Stomach cancer is the second leading cause of cancer death and forth most common malignancy in the world which accounts for 9.7% of total cancer deaths (Ferlay et al., 2010).In India, the incidence of stomach cancer is highest in the Aizawl district of the state of Mizoram with an age-adjusted rate (AAR) of 55.4 in male and 24.4 in female per 10 5 populations (NCRP, 2010).The ageadjusted rate (AAR) of stomach cancer for the entire state of Mizoram are 42.9 and 20.5 per 10 5 populations in male and female, respectively (NCRP, 2010).This incidence is quite high as compared to other regions of India where the incidence of stomach cancer per 10 5 varies from 1.0-12.3 in males and 0.6-11.0 in females.
There are considerable regional differences in the incidence of stomach cancer worldwide and the highest incidence among men is reported from Changle, China (AAR=145.0)and among women in Yamagata, Japan (AAR=38.9) per 10 5 populations (Parkin et al., 2002).Numerous studies have shown that consumption of alcohol, tobacco and different food habits are important
Glutathione-s-transferases (GSTs), a supergene family of detoxification enzymes, provide protection against genotoxic and carcinogenic effects of numerous substances of both xenobiotic and endogenous origins (Pemble et al., 1994).The GSTM1 gene is classified into the mu class and the GSTT1 gene belongs to the theta class.Genes coding for GSTM1 and GSTT1 proteins are polymorphic in humans and GSTM1 is absent in 35-60% of individuals (Bell et al., 1993).Similarly, GSTT1 is absent in 10-65% of human populations (Chenevix-T et al., 1995).The phenotypic absence of GSTM1 and GSTT1 activity is due to homozygocity for an inherited deletion of these genes, termed the null genotype (Pemble et al., 1994).Individuals with the homozygous GSTM1 null genotype express no protein and expected to have reduced abilities to detoxification of hazardous compounds, particularly epoxides (Cai et al., 2001).Many epidemiological studies have reported differently about the association of GSTM1 and GSTT1 gene polymorphism with stomach cancer (Katoh et al., 1996;Setiawan et al., 2000;Cai et al., 2001;Tripathi et al., 2011;Zang et al., 2011;García-G et al., 2012).The present study was undertaken to examine the association and interaction of GSTM1 and GSTT1 gene polymorphism and tobacco consumption with stomach cancer for the first time in this high risk state of Mizoram in the north eastern part of India.

Materials and Methods
This study was a population based matched casecontrol study carried out from March, 2009-2012.All cases and controls were ethnic Mizos of the Mizoram state.All cases (n=102) were newly diagnosed and histopathologically confirmed stomach cancer patients from the Aizawl civil hospital and other private clinics of Aizawl, Mizoram.The patients with severe clinical symptoms, patients with recurrent cancer or too old to be interviewed elaborately and who refused to be interviewed were excluded from this study.Two age (±5 years) and sex-matched population based healthy neighborhood controls (n=204) were selected for each case.Information on socio-demographic and other risk factors like consumption pattern of tobacco intake, alcohol drinking, betel nut chewing was collected from cases and controls by face-to-face interviews and information was recorded in a pre-designed questionnaire.
5 to 10 ml of peripheral whole blood was collected from each of the study subject in EDTA-containing vials and stored at -80 0 C until analyzed.
The institutional ethical committee of the Regional Medical Research Centre, Dibrugarh approved this study.All participants were given an explanation of the nature of the study and informed and written consent were obtained from all cases and controls.

DNA extraction and PCR
Extraction and purification of high-molecularweight genomic DNA was carried out with Quiagen DNeasy (R) Blood and Tissue Kit.The PCR reactions were performed by using two previously described methods (Wiencke et al., 1995;Cai et al., 2001) with slight modifications.A reaction mixture of 50 µL volume was prepared containing Promega 2X master mix (GoTaq DNA polymerase, 2X GoTaq Reaction Buffer, pH 8.5, 400 µmol.L -1 of each dNTP and 3 mM MgCl 2 ), 0.14 µmol.L -1 of each primer, 200 ng of template DNA and 0.1 mgml -1 of BSA.Primer sequences for GSTM1 were 5'-GCTTCACGTGTTATGGAGGTTC-3' and 5'-GAGATGAAGTCCTCCAGATTT-3' which produced a 157 base-pair band.For GSTT1 the primers used were 5'-TTCCTTACTGGTCCTCACATCTC-3' and 5'-TCACCGGATCATGGCCAGCA-3' which produced a 480 base-pair band.β-globin gene was used as a positive internal control in all the PCR reactions.The primers used for β-globin gene amplification were 5'-CAACTTCATCCACGTTCACC-3' and 5'-GAAGAGCCAAGGACAGGTAC-3'.The PCR amplification was carried out in Applied Biosystems Thermal Cycler.The PCR cycles for GSTM1 and β-globin were 94°C for 8 min, followed by 35 cycles of 94°C for 30s, 60°C for 40s and 72°C for 1 min with a final extension at 72°C for 10 min.PCR cycles for GSTT1 were 94°C for 8 min, followed by 35 cycles of 94°C for 30s, 62°C for 40s and 72°C for 1 min with a final extension at 72°C for 10 min.The PCR products were run in 2-2.5% of agarose gel, stained in ethidium bromide and documented in Gel-Doc.

Detection of Helicobacter pylori infection
Indirect IgG ELISA (BIO-RAD) was used to detect antibodies against Helicobacter pylori in serum of both cases and controls.

Statistical analysis
Univariate and multiple logistic regressions were used for data analysis.The conditional maximum likelihood method (Breslow et al., 1980) was used to estimate the parameters of the regression model because of the matched study design and significance was taken at p≤0.05 (two tailed).Initially a univariate analysis was carried out.The crude measure of association between single putative risk factor and stomach cancer was expressed as odds ratio (OR) and its 95% confidence interval (95%CI) was calculated from the standard error (SE) of the regression   ,2,3,4,5,6,9,12 co-efficient.To control the confounding variable and other co-variates such as smoking, alcohol drinking, level of education taken, occupation, income etc, the data were analyzed by conditional multiple logistic regression to evaluate the extent of risk association.The statistical package used for the analysis were SPSS version 17.0 and Epi-Info version 3.5.4.

Results
The main characteristics of the cases and controls.The mean age of the cases and controls were 59.3 (SD±13.4)and 59.4 years (SD±13.8),respectively.The distribution of sex and age among cases and controls were not statistically significant (p>0.05) which suggest that the age and sex matching was effective.Majorities (52.9%) of the stomach cancer cases were in the age group of 31-60 years at the time of diagnosis and 76.5% were males.To control for confounding, level of education, income groups and occupation were also included in the analysis.
The data in Table 1 indicates the risk of stomach cancer associated with tobacco habits.Odds ratios (OR) were calculated using non-smokers as reference group.The matched univariate OR for both ex-smokers and current smokers was 3.32 (95%CI, 1.66-6.64)and 2.55 (95%CI, 1.38-4.73),respectively.Higher risk of stomach cancer among past and current smokers was seen even after controlling for confounding factors which indicates its independent effect on the development of stomach cancer.High risk (OR, 23.02; 95% CI, 5.02-105.63)was observed in individuals smoking both cigarettes and meiziol (prepared from locally available tobacco).However, among individuals smoking only one type of tobacco, higher risk was observed in meiziol smokers (OR,4.42;95%CI,) in the multivariate model in contrast to cigarette only smokers.A significant higher risk was observed among smokers who smoked more than 10 times per day (OR,5.38;95%CI,.Longer duration of smoking increased the risk of stomach cancer significantly.Risk of stomach cancer also tended to decline significantly among ex-smokers who have quit smoking more than 10 years ago. Tuibur (tobacco smoke infused water used in Mizoram) consumption and the risk of stomach cancer was also checked (Table 1) which revealed higher risk in individuals who takes tuibur for more than 15 years (OR, 2.04; 95%CI, 1.00-4.17) in the univariate analysis.In stratified analysis among tobacco smokers, tuibur user showed three times more risk (OR, 3.32; 95%CI, 1.13-9.74) of stomach cancer than tuibur non-users (Table 1).

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The data in Table 2 indicates the risk involved with different tobacco related habits either in single or in combination.Significant high risk was observed in persons who practice meiziol and tuibur together (OR, 3.84; 95%CI, 1.36-10.86)followed by individuals who take meiziol and betel nut together (OR, 3.28; 95%CI, 1.39-7.75).

GSTM1 and GSTT1 polymorphism
The frequency distribution of homozygous GSTM1 and GSTT1 null and non-null genotype in stomach cancer cases and healthy controls were shown in Table 3.The data reveals that GSTM1 and GSTT1 null genotype are distributed unevenly between stomach cancer cases and controls.The GSTM1 null genotype frequency was increased in stomach cancer cases (55.9%) as compared to controls (47.5%).Individuals with GSTM1 null genotype showed higher risk of stomach cancer (OR, 1.48; 95%CI, 0.90-2.43)than GSTM1 non-null genotype carrying people, while persons carrying GSTT1 null genotype demonstrated decreased risk of stomach cancer (OR, 0.69; 95%CI, 0.42-1.13)than the non-null genotype carriers of GSTT1, although these associations failed to attain statistical significance.Among patients of stomach cancer, 36.3% (37/102) were GSTT1 homozygous null genotype as compared to 45.6% (93/204) of controls.
The odds ratio of stomach cancer associated with combined genotypes of the polymorphisms of GSTM1 and GSTT1 genes are also shown in Table 3.No significant association was found between combined genotypes of GSTM1 and GSTT1 genotypes and stomach cancer.However, persons who carry the GSTM1 null and GSTT1 non-null genotype were found to have more risk involved (OR, 1.50; 95%CI, 0.78-2.86)than other combinations of GSTM1 and GSTT1 genotypes, though it could not attain statistical significance.

GSTM1 polymorphism, smoking, tuibur and alcohol habit
Data of Table 4 reveals that among non-smokers, persons carrying GSTM1 null genotype had significantly higher risk of stomach cancer than non-smokers carrying GSTM1 non-null genotype.Both ex-smokers as well as current smokers whether carrying GSTM1 non-null or null genotype had significantly higher risk of stomach cancer than non-smokers non-null genotype carriers.Persons who smoked more than 10 times per day had significantly higher risk of stomach cancer irrespective of their genotypic status (GSTM1 non-null or null genotype) as compared to non-smokers non-null GSTM1 genotype carriers.All persons who smoked for more than 15 years had a higher risk of stomach cancer irrespective of whether they belong to GSTM1 null or non-null genotype.Reduction in the risk of stomach cancer was seen in persons who left smoking for more than 10 years in both GSTM1 non-null and null genotyped groups.The effect of the type of smoking and risk of stomach cancer was also similar in both GSTM1 null and non-null genotype carriers.Meiziol smokers had significantly higher risk as compared to persons who smoked cigarette only.
The effect of GSTM1 polymorphism on the risk of stomach cancer in tuibur users revealed that persons having GSTM1 null genotypes using tuibur had 2.4 times (OR, 2.40; 95%CI, 1.03-5.59)higher risk of stomach cancer as compared to non-consumers of tuibur having non-null genotype (Table 4).Among alcohol drinkers, no significant effect of GSTM1 polymorphism on the risk of stomach cancer was found (data not shown).

GSTT1 polymorphism, smoking, tuibur and alcohol habit
Table 4 reveals that among non-smokers, there was no effect of GSTT1 polymorphism and risk of stomach cancer.Ex-smokers as well as current smokers had significantly higher risk of stomach cancer, if they carries GSTT1 nonnull genotype than non-smokers carrying GSTT1 non-null genotype.Similarly, GSTT1 non-null meiziol smokers had significantly higher risk of stomach cancer (OR, 4.05; 95%CI, 1.74-9.43)than GSTT1 non-null non-smokers.Persons who smoked more than 10 times per day had significantly higher risk of stomach cancer than GSTT1 non-null carriers of non-smokers.However, this risk was higher among persons who belong to GSTT1 non-null genotype (OR, 5.76; 95%CI, 2.41-13.77)than persons belonging to GSTT1 null genotype.Similarly, persons with GSTT1 non-null genotype and who smoked for more than 30 years had significantly higher risk of stomach cancer (OR, 4.04; 95%CI, 1.66-9.86)as compared to nonsmokers non-null genotype carriers.Persons who stopped smoking for last 10 years or more had a decreased risk of stomach cancer as compared to persons who stopped smoking less than 10 years ago.This decrease in risk of stomach cancer was similar in both GSTT1 null or nonnull genotypes.
Among tuibur consumers and alcohol drinkers, the effect of GSTT1 polymorphism on the risk of stomach cancer was also examined, however, no significant effect was observed (data not shown).

Association of Helicobacter pylori with stomach cancer
The frequency of Helicobacter pylori in matched healthy population control was found to be 69.4% (n=193) based on the detection of antibodies against H. pylori using IgG indirect ELISA.Among stomach cancer patients, H. pylori infection was slightly higher (73.7%, n=99) than

Discussion
The state of Mizoram is a high risk region of stomach cancer in India (Rao et al., 1998;Phukan et al., 2004).Few studies have been carried out earlier to detect the risk factors for stomach cancer in India.Our study is the first population based matched case-control study of stomach cancer in Mizoram state of India which also considers GSTM1 and GSTT1 genetic polymorphism.
Tobacco smoking was found to be a significant risk factor of stomach cancer in our study which is consistent with the work of other investigators and also our earlier study (Phukan et al., 2005).Both current and ex-smokers showed significantly higher risk in comparison to the nonsmokers.The meiziol smokers demonstrated high risk than the non-smokers and cigarette only smokers.Although cigarette smoking alone showed no significant association with stomach cancer, however synergistic interaction was found between cigarette and meiziol smokers, as persons who are exposed to the habits of cigarette smoking along with meiziol, showed nearly five times higher risk than those who smokes only meiziol.IARC monoghaph (IARC, 2002) and several cohort and case-control studies (Hansson et al., 1994;Tredaniel et al., 1997;Russo et al., 2001;García-G et al., 2012) reveal that tobacco smoking is a risk factor for stomach cancer.However, in Indian context, many studies reported variably about the involvement of the risk of tobacco-smoking for stomach cancer.Rao et al. (2002) and Yadav et al. (2010) did not get any relationship between tobacco use and stomach cancer.However, Gajalakshmi et al. (1996) and in one of our previous study (Phukan et al., 2005), we have reported tobacco-smoking as a significant risk factor for stomach cancer, which is consistent with this study also.
In our earlier study (Phukan et al., 2005), we have reported tuibur as a risk factor for stomach cancer in Mizoram which is consistent with this current study.No significant association between alcohol drinking and stomach cancer was seen like most of the previous studies (Hansson et al., 1994;Gajalakshmi et al., 1996;You et al., 2000;Rao et al., 2002); except a few studies (Boeing et al., 1991;Sumathi et al., 2009) reported alcohol drinking as risk factor for stomach cancer.
The present study is the first study to describe the GSTM1 and GSTT1 genetic polymorphism of stomach cancer in Mizoram.The GSTM1 null genotype was found to be high in stomach cancer cases (55.9%) as compared to healthy neighborhood controls (47.5%),although this difference was not statistically significant.Another three studies carried out in China (Cai et al., 2001;Jing et al., 2012) and Japan (Katoh et al., 1996) also reported high frequency of GSTM1 null genotype among stomach cancer cases than in controls.GSTT1 homozygous null genotype was 36.3% in cases and 45.6% in controls which difference was also not significant.No significant association of GSTM1 and GSTT1 gene polymorphism and stomach cancer was found.
In this study, relationship between risk of gastric cancer, GSTM1 polymorphism and smoking habit revealed that persons carrying GSTM1 null genotype among nonsmokers had significantly higher risk of stomach cancer than non-smokers carrying GSTM1 non-null genotype.Among tuibur users, people who carry GSTM1 null genotype have more than two fold higher risk of stomach cancer than tuibur non-users who carry GSTM1 non-null genotype.Earlier two studies reported association of GSTM1 null genotype with stomach cancer (Katoh et al., 1996;Saadat et al., 2001).The GSTT1 non-null genotype carriers among ex-smokers as well as current smokers, meiziol smokers, persons who smoked more than 10 times per day showed higher risk of stomach cancer than nonsmokers belonging to GSTT1 non-null genotype carriers.Another study in Taiwan reported protective role of GSTT1 null genotype for stomach cancer (Wang et al., 1998).A non significant increase of stomach cancer risk was observed among those persons, who carry GSTM1 null and GSTT1 non null genotype.One earlier study (Cai et al., 2001) showed similar increased risk among GSTM1 null and GSTT1 non null genotype carrying persons.Another study from India revealed protective role of GSTT1 null genotype in oral cancer (Anantharaman et al., 2007).
In this study, no association of H. pylori infection and stomach cancer in Mizoram was found.Although several meta-analyses revealed a strong relationship between H. pylori and gastric cancer (Eslick et al., 1999;Xue et al., 2001), it is still controversial in different Asian countries (Miwa et al., 2002;Jing et al., 2012).Various studies from India also failed to show an association between H. pylori infection and gastric cancer (Kate et al., 2000;Khanna et al., 2002;Singh et al., 2006).These disparate observations have created an enigmatic situation in Indian context and needs serious approach to solve this problem.
In conclusion, this study reveals that tobacco-smoking either in single or in combined mode of habit is an important risk factor for stomach cancer in Mizoram state of India.Meiziol is the important risk factor among different tobacco habits.Cigarette, tuibur and betel nut modulates the risk when taken with meiziol.Among tobacco smokers, tuibur users have three times more risk of stomach cancer than tuibur non-users.Frequency of GSTM1 null and GSTT1 null genotype in the control population of Mizoram were 47.5% and 45.6% respectively, whereas among cases they were 55.9% and 36.3%respectively.Polymorphism of GSTM1 and GSTT1 gene are not found to be directly associated with stomach cancer in Mizoram.However, GSTM1 and GSTT1 genes modify the effect of tobacco habits.Persons who smoke tobacco and / or take tuibur had increased risk of stomach cancer if they carry GSTM1 null genotype and GSTT1 non-null genotype.Tuibur users carrying GSTM1 null genotypes had 2.4 times higher risk of stomach cancer than non-consumers of tuibur having GSTM1 non-null genotype.No association of H. pylori infection and stomach cancer in Mizoram was found.Similar types of studies with large population in different geographical areas may through important light and will help to understand and to confirm these preliminary findings in coming period.

Table 1 . Tobacco use and Risk of Stomach Cancer
,13=GSTT1 non-null genotype with 480 bp band.

Table 2 . Different Tobacco and Betel Nut Related Behavior and Risk of Stomach Cancer
*Matched (cases and controls were matched for age, sex and ethnicity) univariate odds ratio estimated by conditional logistic regression analysis, **Adjusted odds ratios (adjusted for alcohol drinking, betel nut chewing, level of education, occupation and income group) obtained by matched conditional multiple logistic regression analysis using maximum likelihood approach.

Table 3 . Association of GSTM1 and GSTT1 and Stomach Cancer
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