A RELATIVE STUDY TO FIND OUT THE RISK POPULATION IN VINDHYAN REGION BY CARCINOGEN ACTIVATING AND DEACTIVATING THE GSTM1 POLYMORPHISM

The Glutathione S-transferase are a family of phase II isoenzymes, believed to protect cells from reactive chemical intermediates and oxidative stress, resulting from a wide range of electrophilic xenobiotics (Example-PAH) and endogenous intermediates. Inheritance of null (gene deletion) alleles of the GSTM1 (chromosome 1p 13.3) genes is common in the population varies by ethnicity and is associated with the loss of enzymatic activity and cytogenetic damage. The studies have linked the gene deletion of GSTM1 to susceptibility to various cancers, including lung, bladder, Head, Neck, Colon and basal cell carcinoma. Variation in metabolism of carcinogens could increase or decrease exposure of cells to carcinogens. Ethnic variation in cancer incidence and mortality may be due in part because of differences in the distribution of polymorphisms, as well as differences in environmental and dietary exposures.


INTRODUCTION
Polymorphism is a process in which more than one allele occupies that gene's locus within a population. The Glutathione S-transferase are a family of phase II isoenzymes, believed to protect cells from reactive chemical intermediates and oxidative stress, resulting from a wide range of electrophilic xenobiotics (Example-PAH) and endogenous intermediates (Example- reactive oxygen species) ( 1,2,3,4,5,6,7) . Inheritance of null (gene deletion) alleles of the GSTM1 (chromosome 1p 13.3) genes is common in the population varies by ethnicity and is associated with the loss of enzymatic activity and cytogenetic damage. The studies have linked the gene deletion of GSTM1 to susceptibility to various cancers, including lung, bladder, Head, Neck, Colon and basal cell carcinoma (8) . Variation in metabolism of carcinogens could increase or decrease exposure of cells to carcinogens, and through this pathway affects susceptibility to cancer. These polymorphisms are often common, and that their prevalence differs between populations. Ethnic variation in cancer incidence and mortality may be due in part because of differences in the distribution of polymorphisms, as well as differences in environmental and dietary exposures.

MATERIAL & METHODS
Study population: In our study we have selected the vindhyan region, the heart land of India. This is constructed by four district of Madhya Pradesh and is the oldest habitat of mankind. The Kol, Gond, Bhil and Baiga are the native tribe of this region. Modern population is dominated by Hindu and Mohammedan religions. The study population consisted of 409 unrelated subjects comprising between 311 hindus, 25 muslims and 73 tribals.
Blood sample collection: Blood samples were drawn by the intra-venous injection and 3-5ml. blood were collected in the EDTA containing bowls and were stored in the -20 0 C till the used.
DNA isolation: Genomic DNA was extracted from whole blood by the modification of salting out procedure described by Miller. Thermal profile used for the amplification of desired segment of gene was as follows: Initial denaturation at 95 0 C for 5 min and 30 cycles of denaturation at 94 0 C for 45 Sec, annealing at 60 0 C for 45 sec and extension at 72 0 C for 1 min, followed by final extension at 72 0 C for 07 min. PCR products were separated on 2% agarose gel (2%w/v, SRL) using a 100 bp molecular weight (MW) marker to confirm the PCR product size of 215 bp.

Hardy -Weinberg equilibrium
The genotype frequencies of each study group were tested to be in accordance with hardy -Weinberg equilibrium using chi square test for independence. All the calculated values were compared with tabulated values and found that all the frequencies were in Hardy -Weinberg equilibrium (Table 4.1). The standard tabulated values was 3.8 at 1 degree of freedom and P = 0.05 level of significance.  figure 2).The associations of allele frequencies between groups were tested by Fisher's exact test and values obtained were P = 0.6797 between Hindu -Muslim, P = 0.6436 between Muslim -Tribal and P = 0.7949 between Hindu -Tribal population. All the values were statically Non significant between all the groups tested ( Table 2).

DISCUSSION
In past three decades the information about carcinogen metabolism has been improved. Due to this information the interest has grown about study of polymorphism among carcinogen activating and de-activating genes. In this respect we have conducted a broad polymorphism screening study in population of Vindhyan Region. The GSTM1 is important carcinogen metabolizing gene, as it is responsible for the metabolism of highly ubiquitous environmental carcinogen like Polycyclic Aromatic Hydrocarbon (PAH) which is also the most lethal constituent of cigarette smoke.
The allelic frequencies of Polycyclic aromatic Hydrocarbon (PAH) metabolizing gene was determined in a sample of typical population, which was stratified into three groups (Hindu, Muslim and Tribal) based on their ethnicity. The total 409 individuals were screened for the GSTM I Null/Present polymorphism, in which 311 individuals were Hindu, 25 individuals were Muslim and 73 individuals were Tribal.
The allele Frequency of GSTM1 were compared between the three groups of our study population and we found that the frequency distribution of Present and null allele were not statically different between these three groups, However the increase of Null allele frequency seen in Muslim than other groups. Where the frequency distribution of Present and Null allele were Present = 49.52% and Null = 50.48% in Hindu, Present = 44% and null = 56% in Muslim, and in tribal were Present = 52.25% and Null = 47.95%.
A slight difference were seen when result from our study compared with the finding of Kyoung-Mu Lee et al (186) in Asian population (present = 44.5 and Null = 55.6) and finding of Valder R. Anuda et al (187) in Caucasian population (present = 45% and null = 55%) which were corresponding to frequency in Muslim group of our population but different from Hindu and Tribal. These differences could be due to differences in mating pattern.