Introduction

The glutathione S-transferase (GSTs) are one of the important xenobiotics metabolizing gene family; it plays a vital role in the metabolic detoxification of oxidative stress stimulating, electrophilic compounds, carcinogens, and environmental toxins [13, 16, 37]. By their origin, GSTs are classified as cytoplasmic, mitochondrial, and membrane-associated proteins (MAPEG). Seven classes of cytoplasmic GSTs are categorized as Alpha (GSTA), Mu (GSTM), Omega (GSTO), Pi (GSTP), Sigma (GSTS), Theta (GSTT), and Zeta (GSTZ) [38].

GSTs have the ability to detoxify toxic metabolic products, like reactive nitrogen and oxygen species via glutathione peroxidase activity [9, 26]. Other than detoxification, GSTs perform a variety of biological functions including regulation of S-glutathionylation cycle, and kinase-mediated signal transduction [18, 25, 26, 41].

Previous studies have shown that deletion variations associated with GSTM1 and GSTT1 genes are located at chromosome 1 (1p13.3) and (22q11.23), respectively. Individuals having deletion variants (null/null) of GSTM1 or GSTT1 genes demonstrate a complete lack of enzymatic activity for corresponding protein [26, 28, 40]. A loss of function in the GSTs classes (M1 and T1) due to a structural deletion (Null mutation) impacts an individual’s ability to detoxify genotoxic compounds [36, 37]. GSTM1 catalyzes and detoxifies polycyclic aromatic hydrocarbon diol epoxides, such as DNA hydroperoxides, alkyl halide (component of cigarette smoke), while GSTT1 catalyzes and detoxifies benzo (a) pyrene diol epoxide, and acrolein [17, 36]. GSTM1 and GSTT1 deficiencies, either alone or in combination, are thought to have reduced detoxifying characteristics; henceforth, contribute significantly toward increased susceptibility to various types of cancer [26, 42].

In the last few decades, GSTM1 and GSTT1 null genotypes have been investigated extensively in a variety of ethnic groups, and their widespread presence has been well established [8, 11, 32, 37]. For example, the prevalence of the GSTM1 null genotype was observed 47–57% in Caucasians, 42–54% in Asians, and 16–36% in Africans, respectively, while the GSTT1 null genotype was uncommon in Caucasians (13–26%), and shown higher prevalence among Asians (35–52%) [10, 37]. These variability of GSTM1 and GSTT1 null genotypes across the worldwide population might be linked to illness susceptibilities that are unique to each population. India’s ethnic communities are diverse, both physiologically and culturally [22, 37], and northern India’s ethnic groups are particularly diverse, because of various tribal groups of Indo-European ancestry that have lived here over several waves of migration. Therefore, we have been prompted to evaluate the GSTM1, GSTT1 genotype distribution in the Northern state (Himachal Pradesh) population and to compare it with various Indian states and global populations. This is one of the first research of its type to be conducted among the people of Himachal Pradesh.

Materials and methods

Studied population and sample collection

The study was carried out among 297 healthy unrelated Himachal Pradesh residents (232 males and 65 females) with ages, ranging from 20 to 62 years. The study’s goals were well explained to all the participants and were asked to fill out a questionnaire to collect personal details as well as information related to their socioeconomic status. The research was duly approved by Institutional Ethical Committee, Maharaja Agrasen University, Baddi (HP) India.

For DNA extraction, a blood sample (5 mL) was drawn from each of the participants and collected in a vacutainer tube containing K2EDTA. All samples were transported to the laboratory in an insulated ice bucket for further processing.

Genotyping of GSTM1 and GSTT1

Genomic DNA was extracted from 200 µL of whole blood using ReliaPrep™ Blood gDNA Miniprep kit (Promega, USA). The presence or absence of the GSTM1 and GSTT1 genes was determined by multiplex-PCR. As an internal control, a portion of exon 7 of the housekeeping gene such as CYP1A1 was also co-amplified. The primer sequences [1, 20] and product size of the GSTM1 and GSTT1 genes are listed in Table 1. Both genes were genotyped using a 25-μL reaction mixture containing 1 μL of genomic DNA template (100 ng/μL), 1 μL of each primer (20 pmol/mL), 0.5 μL of dNTPs (200 μM), 2.5 μL of PCR buffer with 15 mM/L MgCl2, and 0.5 μL of Taq polymerase (3 U/μL) and 19.5 μL sterile nuclease free water. In total, 30 thermal cycles were performed. PCR reaction cycle included, initial denaturation (at 94 °C for 10 min); denaturation (at 94 °C for 60 s); annealing (at 59 °C for 45 s); and extension (at 72 °C for 60 s). The final extension was carried out at 72 °C 10 min and the amplification results were examined on 2% agarose gel.

Table 1 GSTM1 and GSTT1 gene’s primer sequences
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Statistical analysis

Allelic frequencies of GSTM1 and GSTM1 were determined by direct counting and by ensuring presence or absence of DNA bands on agarose gel. Fisher’s exact test (SPSS 20; IBM, New York, USA) was applied for gender-wise distribution of both GSTs genotypes in the studied population.

Results

The outcomes of GSTM1 and GSTT1 genotyping, as well as the general frequency distributions of GST polymorphisms in the studied population, are shown in Fig. 1 and Table 2. In our study, the frequency distribution of the homozygous null genotypes of GSTM1, GSTT1 individually as well as combined was found as 33.3%, 32% and 9%, respectively. Upon gender-wise comparison, a non-significant distribution (p > 0.05) for null genotypes of GSTM1 (32.8% and 35.4%, OR-0.77, 95% CI 0.42–1.41), GSTT1 (33.2% and 27.7%, OR-1.12, 95% CI 0.63–2.0) individually and combined GSTM1 and GSTT1 (10.8% and 3.7%, OR-0.31, 95% CI 0.07–1.42) were observed in studied population (Table 2). Allelic distribution of GSTM1 and GSTT1 among various ethnic groups is shown in Table 3.

Fig. 1
figure 1

2% agarose gel is showing M lane is 100 pb marker, in lane 1, 3 and 7 are showing GSTT1 non-null and GSTM1 homozygous null genotyping. Lane 2 and 4 are showing GSTM1 non-null and GSTT1 homozygous null genotyping. Lane 5 is showing both GSTM1 and GSTT1 non-null genotyping and Lane 6 is showing both GSTM1 non-null and GSTT1 homozygous null genotyping

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Table 2 Overall distributions of GSTT1 and GSTM1 genotypes according to gender in the Himachal Population
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Table 3 GSTM1 and GSTT1 null genotype frequency distribution among the different ethnic groups
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Discussion

GSTM1 and GSTT1 are the most important phase II xenobiotic metabolizing or detoxifying enzymes that catalyze glutathione conjugation, play a key role in metabolism of different types of reactive species. GST enzymes protect nucleic acids such as DNA from endogenous oxidants and save the cells by detoxifying the carcinogens and environmental contaminants. GSTs family members are major candidates for cancer disease risk, because they have the potential to regulate a person’s ability to metabolize a carcinogen. It has been reported that carries with homozygous deletions lack GST-m and GST-θ enzyme activity. The presence of deletion variations (null type) in GSTM1 and GSTT1 genes, could damage DNA and result in different types of cancer. The results of our investigation suggest that the frequencies of GSTM1 and GSTT1 null genotypes in the Doon population of Himachal Pradesh (North Indian region of Asian continent) are comparatively higher.

The allelic distribution of GSTM1 and GSTT1 among various ethnic groups has been studied (Table 3). The highest percentage of null alleles of GSTM1 (66.3%) and GSTT1 (42.5%) was observed in Asian and American populations, respectively [4, 6], while the lowest percentage of GSTM1 (11.2%) and GSTT1 (10.4%) null alleles were found in African and Asian populations, respectively [8, 27].

In our study, 33.3% of the Doon population of Himachal Pradesh had GSTM1 homozygous null or deletion genotype (Tables 2 and 3). Our results were in agreement with those of Asian, who had GSTM1 null frequencies ranging from 36.4 to 37.8% [34, 39]. Furthermore, we observed 32% of Doon population of were homozygous null or deletion for GSTT1 gene. The prevalence of homozygous null genotype of GSTT1 in in Doon population (Table 2) shows similar trend to other northern Indian populations (Table 3), [20, 31]. When compared to other Asian populations, the frequency of GSTT1 null genotype varies 10.4–27.3% which was less than and contradicting our findings [14, 27] in Asian populations. However, few studies also have shown higher percentage (35.8–40.1) of GSTT1 null genotype among different ethnic groups [8, 39]. GSTM1 and GSTT1 null frequency among Europeans varied from 45.2 to 53.5% and 16.5 to 21.3%, respectively (Table 3). GSTM1 null variants in African people varied from 11.2 to 49.5%, while GSTT1 null frequencies ranged from 22 to 35.8% (Table 3). Genotyping studies of both GSTM1 null and GSTT1 null in Asian population varied from 23.0 to 66% and 10.4 to 40.1%, respectively (Table 3). In the American, the genotype percentage of GSTM1 and GSTT1 alleles varied from 26.9 to 44.8 and 12.2 to 42.5% (Table 3). In our study, we also found non-significant differences (p > 0.05) in homozygous null genotypes of GSTM1, GSTT1 gene, individually as well as in combination among males and females in the studied population (Table 2). Upon gender-wise comparison, the non-significant differences of GSTs null genotypes might be due to the unequal number of male and female participants in the study. However, our results were in agreement with findings of a few Asians studies [10]. The difference in the gene frequency of GSTM1 and GSTT1 among the various ethnic populations is largely due to their different evolutionary histories and method selection. Our research would be useful to the Doon population of Himachal Pradesh, and globally, it will allow us to find out risk factors for disease like cancer within the susceptible individuals.

Conclusions

GSTs are involved in the detoxification of various xenobiotic compounds; therefore, knowing the prevalence of various GST alleles in a population might help in determining a risk due to susceptibility. The current study reveals the allelic distribution of GSTM1 and GSTT1 genes in the Himachal Pradesh population of the Northern region of India. Our findings have further strengthened the heterogeneity of GSTT1 and GSTM1 gene polymorphisms in the global population and also highlighted its role as a biomarker of susceptibility. From future perspectives, our findings might lay down a pathway for future bio-monitoring studies involving the risk of xenobiotic exposure in Himachal Pradesh. Moreover, our findings might add to a better understanding of the relationship between ethnicity and illness prevalence. It also might be helpful in explaining the significance of gene behavior in epidemiological and clinical research.