Impact of Tobacco on Glutathione S Transferase Gene Loci of Indian Ethnics

Tobacco contains different carcinogens as DNA and protein adducts in its different form of usage. Polycyclic aromatic hydrocarbons (PAHs), volatile aldehydes, nitrosoamines, nitrosamino acids, phenols, volatile hydrocarbons, nitro compounds, organic and other inorganic compounds were found as carcinogenic agents in tobacco. Several compounds in tobacco like PAH, benzo (a) pyrene diol epoxide, 7-methylguanine, hydroxypyridyloxobutyl , 8-Oxo-deoxyguanosine, lipidperoxidation derived DNA adducts were few among the tobacco-carcinogen biomarkers (Kriek et al., 1998; Tang et al., 2001; Phillips, 2002; Boysen et al., 2003; Stephen, 2003). Drug/Xenobiotic compounds metabolism is essential for biotransforming hazardous pollutants or potent carcinogenic intermediates to their non toxic forms. The continuous exposure of various DNA adducts during the life time of an individual lead to possible mutations and could end in altered or loss of function of a gene that might regulate the cell cycle, cell division, cell death or other


Introduction
Tobacco contains different carcinogens as DNA and protein adducts in its different form of usage.Polycyclic aromatic hydrocarbons (PAHs), volatile aldehydes, nitrosoamines, nitrosamino acids, phenols, volatile hydrocarbons, nitro compounds, organic and other inorganic compounds were found as carcinogenic agents in tobacco.Several compounds in tobacco like PAH, benzo (a) pyrene diol epoxide, 7-methylguanine, hydroxypyridyloxobutyl , 8-Oxo-deoxyguanosine, lipidperoxidation derived DNA adducts were few among the tobacco-carcinogen biomarkers (Kriek et al., 1998;Tang et al., 2001;Phillips, 2002;Boysen et al., 2003;Stephen, 2003).Drug/Xenobiotic compounds metabolism is essential for biotransforming hazardous pollutants or potent carcinogenic intermediates to their non toxic forms.The continuous exposure of various DNA adducts during the life time of an individual lead to possible mutations and could end in altered or loss of function of a gene that might regulate the cell cycle, cell division, cell death or other

Impact of Tobacco on Glutathione S Transferase Gene Loci of Indian Ethnics
KP Senthilkumar 1,2 , Ramasamy Thirumurugan 1,3 * survival factors and cause various cancers (Hecht, 1999;Lacko et al., 2009;Zhuo et al., 2009).Drug/Xenobiotic compound metabolizing enzymes are capable of regulating the removal of this exogenous and endogenous DNA adducts/carcinogens from the cell (Hecht, 2003;Lacko et al., 2009).The mutation observed in GSTM1 or T1 was a deletion mutation of null genotype and completely lost its functions to become susceptible to various cancers or diseases and further the inheritance of the same (Evans et al., 2004;Anantharaman et al., 2007;Mo et al., 2009;Sam et al., 2009;Amer et al., 2011;Chuang et al., 2011;Kumar et al., 2011).In this study, we investigated the impact of tobacco usage on drug/Xenobiotic compound metabolizing GST gene loci mutation.The age old customary habituate of high tobacco usage among Gujarat population was considered as the suitable subject for the study among Indian ethnics reported earlier (Rani et al., 2003).Further we compared the frequency rate of GST polymorphism among the subjects with the low tobacco using other populations within India, reported by various investigators (Roy et al., 1998;Buch et al., 2001;Mishra 5038 et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010).

Subjects
The investigation comprised of 504 healthy (336 male and 168 female) unrelated volunteers of western India from Gujarat origin.The age of individuals ranged from 40-80 with the mean age of 60 years.Two ml of blood samples were collected from volunteers and informed consent was obtained.The institutional ethical committee of Sh.NP Cancer Institute, Rajkot Cancer Society; India, approved the study.

DNA isolation and Genotyping
Genomic DNA was isolated from whole blood by salting out method (Lahiri and Nurnberger, 1991).Multiplex polymerase chain reaction was performed for identification of GSTM1 and T1 null types with albumin gene as internal control (Huang et al., 2006).Primers used were GSTM1-F: 5' GAA CTC CCT GAA AAG CTA AAG C 3', GSTM1-R: 5' GTT GGG CTC AAA TAT ACG GTG G 3', GSTT1-F: 5' TTC CTT ACT GGT CCT CAC ATC TC 3', GSTT1-R: 5' TCA CCG GAT CAT GGC CAG CA 3', Albumin F: 5' GCC CTC TGC TAA CAA GTC CTA 3' and Albumin R: 5' GCC CTA AAA AGA AAA TCG CCA ATC 3'.The amplified 215 bp, 480 bp, and 350 bp were analyzed for GSTM1, GSTT1, and albumin presence respectively.Homozygous null types were identified by the absence of respective bands and the efficiency of the reaction was confirmed by the presence of the albumin band in all the samples.Amplification was done in a 25 µl reaction with 10 µl of 1:10 diluted DNA sample, 200 µM of all dNTPs, 5 pM of the forward and reverse primers for GSTM1, T1 and albumin, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 and 2.5 U of Taq DNA polymerase.

Stratification of Tobacco Usage among the subjects
The life time tobacco usage as smokeless and smoking form was calculated using the smoking Index and chewing index, Chewing Index (CI)=Frequency of chewing events per day×duration in years, Smoking Index (SI)=Number of cigarettes/20×duration in years or Number of bidis/5×duration in years.Both smoking Index and chewing index are stratified to six levels as per the usage is concerned from no habit to ≥301, with an increment of 75 SI and CI respectively.

Statistics
Null Allele frequency of GSTM1 and GSTT1 was analyzed using Statistical Package for Social Studies (SPSS 16) software for Windows.Hardy-Weinberg Equilibrium (HWE) Testing for GST was done by using online software of Rodriguez et al. 2009.Two sided Fisher's exact test was done with statistical significance set at p<0.05 to compare frequency variation of null alleles among other low tobacco using ethnics reported by various investigators within India, in reference to high tobacco using ethnics of Gujarat.

Genotype frequency of GST among the subjects
Among the 504 samples investigated, GSTM1 and T1 null genotype was 20% and 35.5% respectively and the stratified analysis for polymorphism based on sex revealed that there was significant difference (P>0.05) in the null allele frequency for GSTT1 and not for GSTM1 as shown in Table 1.The GST allele frequency distribution in the Gujarat population was not in Hardy Weinberg equilibrium according to chi square test for GSTM1 and T1 (Data not shown).

Tobacco usage among the subjects
Gender based stratified analysis for CI and SI sub population was done to identify the frequency rate for chewing and smoking respectively as shown in Table 2.No chewing was category observed in male, female and among the subjects in common with 27.1%, 33.9% and 29.4% respectively.Low chewing habit with CI of 1-75 was observed high in females (33.9%) than males (6.5%) and among the subjects in common (15.7%).High chewing habit with CI of 226-300 was observed more in males (37.5%) than females (14.3%) and among the subjects in general (29.8%).No smoking habit was observed high in females (91.1%) than to males (15.2%) and among the subjects in general (40.5%).Smoking habit of females was observed only with SI of 1-75 (8.9%); while in males it was observed in all categories.Frequency of GSTT1, M1 Genotypes with Tobacco usage among subjects Genotypes of homozygous wild type (+/+), heterozygous (+/-), homozygous null type (-/-) and either homozygous or heterozygous (+/+ or +/-) were categorized among the subjects for GSTM1 and GSTT1 gene loci.Distribution pattern of GSTM1 and GSTT1 frequency among the subjects was stratified by chewing index and smoking index to calculate the significant influence of tobacco usage among the subjects (Table 3).The GSTT1 null frequency was significant to the usage of tobacco in both chewing and smoking forms, while GSTM1 null frequency was found significant to smoking alone among the subjects.Distribution pattern of GSTT1  and M1 Genotypes with Tobacco usage among male and female subjects was observed separately to differentiate the level of tobacco influence in mutation (Table 4 and 5 respectively).In male the distribution pattern was found almost similar to the observation seen previously among the subjects (Table 3).But the distribution pattern for GSTT1 and M1 frequency with respect to stratified analysis for CI and SI in female could not predict the significant influence of tobacco usage in null frequencies, as more than one zero values were observed in the frequencies among female subjects (Table 5).

Null type frequency of GSTM1 and GSTT1 among Indian populations
The frequency of GSTT1 and M1 null type of various populations were analyzed and compared to the subjects for their tobacco usage (Rani et al., 2003).GSTM1 null allele frequency of Gujaratians (20%) was not significantly different to other Indian ethnic's frequency of 23% to 33% but GSTT1 null frequency (35.5%) was significantly different (Table 6) from 13% to 19.1% of various Indian populations reported earlier (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010).

Discussion
India is one of the top ten polluted nations in world and the study subjects were recruited from one of the most polluted states of India alongside high tobacco habituated populations (Rani et al., 2003;Singh and Kohli, 2012).Among the study population, higher null types of GST were observed in high tobacco using individuals of male than female as shown in Table 1.The distribution pattern of GST genotypes with stratified tobacco usage as CI and SI among the subjects was found with an interaction between the dose of tobacco use and null type frequency as illustrated in Table 2. GSTT1 null genotype frequency was significant to tobacco use in both the forms, while GSTM1, significant to smoking tobacco and not for chewing.The distribution patterns for GSTT1 and M1 genotypes were observed to be similar in general and male subjects as in Table 3 and 4, but not for female subjects as shown in Table 5.This difference in female subject's observation was possibly due to less and non frequent tobacco usage.The frequency of GSTT1 null genotype in Gujaratians was high among the other population with low tobacco usage (Rani et al., 2003).And it might be due to high tobacco usage in both chewing and smoking forms among the subjects than the other populations in India (Roy et al., 1998;Buch et al., 2001;Mishra et al., 2004;Naveen et al., 2004;Vetriselvi et al., 2006;Konwar et al., 2010).GSTM1 null frequency among different population in India was not significantly different to Gujaratians, as the influence was moderate due to smoking tobacco form alone and not by chewing form.
Both GSTT1 and M1 genotypes were deviating from HWE among the subjects.This might be due to the existence of recent somatic mutation, which has not attained equilibrium or null alleles (Liu, 2012;Xu, 2012), non-random mating selection (Lauren, 2012), overrepresentation of a certain allele in long time survivors (Nordfors, 2012).We claim this non existence of HWE in GSTT1 and M1 was due to all of the above mentioned reasons among the subjects.We claim that in Gujarat, the high tobacco usage and its related DNA adducts mediated the mutation in GSTT1 and M1 null alleles.The population of Gujarat were monogamy and marriages were done within the same community as non-random mating (Gadgil and Malhoera, 1983) and therefore the overrepresentation of null alleles among the population.In addition, under certain environmental conditions, natural selection of certain phenotypes allow for increased fitness among the populations (Barreiro, 2008;Lordelo, 2012).
In this study a probable gene-environment interaction was observed between high tobacco using subjects and their null type frequency in GST gene loci and in particular GSTT1 than GSTM1.This hypothesizes the impact of tobacco dependent mutation in GST gene loci and provides background for the tobacco dependent DNA adducts effect on null type frequency existence among the subjects.This observation of null type mutation in GSTT1 might have susceptible status or protective role to the individual for various diseases related to tobacco or their respective drug substrates.GSTT1 null type mutation leading to susceptibility for various cancers and diseases among the high frequency populations, reported earlier by various investigators (Lacko et al., 2009;Mo et al., 2009;Sam et al., 2009;Chuang et al., 2011;Kumar et al., 2011) were not yet uniformly demonstrated.In our observation, the DNA adducts of tobacco were causing mutation in GSTT1 gene loci and nullifying its detrimental function in carcinogenic compound biotransformation ability.In due course, the null type frequency was recorded high among the individuals with more habituate of tobacco pronounced in Gujarat.We claim that the tobacco dependent DNA adducts impact on GSTT1 null type mutation frequency among the population is a matter of concern in clinical aspects for therapeutic purposes.
In conclusion, GSTT1 null type frequency observed high among the habituates of more tobacco usage and this might be due to the possible gene-environment interactions.These null type mutations could be caused by DNA adducts of tobacco, as the allelic frequency was DOI:http://dx.doi.org/10.7314/APJCP.2012.13.10.5037Impact of Tobacco in Glutathione S Transferase Gene Loci of Indians high with respect to more habitual of tobacco.The GSTT1 null type mutation might influence the susceptibility nature of the Gujarat population for various diseased status and cancerous conditions as mentioned by various investigators (Evans et al., 2004;Anantharaman et al., 2007;Mo et al., 2009;Sam et al., 2009;Amer et al., 2011;Chuang et al., 2011;Kumar et al., 2011).In addition these populations were not able to utilize the GSTT1 dependent drugs for their treatment.Further molecular studies are needed in this aspect of tobacco related mutation in drug/ xenobiotic compound metabolizing genes to understand the multifaceted pathways involved in induction of various disorders or unhealthy conditions.The data reported in this study would add another vital reason for existing difficulties to cure the cancer patients with drugs that were not accustomed to the gene-environment interactions like habits of tobacco and GSTT1 metabolism.The study also reports the more habitual nature of Gujaratians for tobacco and their susceptibility to GSTT1 null type allele related disorders, which is a matter of concern to design drugs that could overcome the difficulties of these disease prone statuses of the ethnics too.