Glutathione-S-transferase M1 and T1 gene Polymorphism as Risk Factors of Oral Squamous Cell Carcinoma: A Preliminary Investigation

 

Shiv Kumar Patel*, Moumita Sinha, Mitashree Mitra

School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur-492010 (C.G.)

*Corresponding Author E-mail: shivpatel.anthro@gmail.com

ABSTRACT:

Tobacco is an established risk factor for oral cancer. Polymorphism of a great number of phase I and phase II XME genes have been widely elucidated over the last three decades, may play a role in determining an individual’s susceptibility to oral cancer in particular and in combination with specific environmental exposure such as smoking. Glutathione S-transferases (GSTM1 and GSTT1) involved in detoxify tobacco smoke constituents and polymorphisms within GSTM1 and GSTT1 genes can result in a complete lack of enzyme activity.

 

In this hospital based case-control study, 104 oral cancer patients (cases) and 104 controls were studied to determine the risk of the disease due to polymorphisms at GSTM1 and GSTT1 loci.

 

Overall, we observed no statistically significant associations between the polymorphisms and oral cancer genotypes on GSTT1 locus, but the simultaneous presence of two mutant genotypes, one of each of two loci (GSTM1 and GSTT1) increased about four fold risk of oral cancer (OR= 3.79, 95% CI- 0.32-3.54).

 

In conclusion, our preliminary results are consistent with earlier published study supporting associations between oral cancer and GSTM1 and GSTT1 homozygous null genotypes.

 

 

INTRODUCTION:

Cancer is one of the five main causes of death in all societies, its relative position varying with age and sex. Oral cancer is the most common cause of cancer related death, although many people are unaware of its existence (WHO, 2004). Oral cancer is the sixth most common cancer worldwide continues to be the most prevalent cancer related to the consumption of tobacco, alcohol and other carcinogenic products (Atkinson et al. 1982).The synthetic chemical compounds which are present in tobacco and tobacco related products are causally associated with oral cancer. Genetic polymorphisms of drug metabolizing enzymes involved in the detoxification pathways of carcinogenic substances may influence cancer risk. Xenobiotic metabolizing enzymes (XMEs) are responsible for the metabolism of many of these exogenous chemicals that are toxic, mutagenic and carcinogenic.

 

Glutathione S-transferases (GSTs), a multigene family of phase II metabolic enzymes, are active in the detoxification of a wide variety of potentially toxic and carcinogenic electrophiles by conjugating them to glutathione (Pemble et al.1986). The human cytosolic GSTs comprise four main classes, based on sequence homology and substrate specificity: α (GSTA), µ(GSTM), π(GSTP) and θ(GSTT). About 50% of the Caucasian population is homozygous for a deletion of the GSTM1 gene, they possess the null genotype. The prevalence of GSTT1 null individuals is 10% to 20% among Caucasians (Nelson et al. 1995).

 

The purpose of this study was to determine the effect of these metabolic gene (null-genotypes of GSTM1 and GSTT1) polymorphisms on the risk of oral squamous cell carcinoma (OSCC).

 

MATERIAL AND METHOD:

Unrelated patients diagnosed with squamous cell carcinoma in the oral cavity were recruited between 2008 and 2010 from Indira Gandhi Regional Cancer Center, Pt. J.N.M. Medical College, Raipur (C.G).  For all patients, the department of pathology from the same hospital did histopathologic conformation of the lesions. Unrelated controls who came for treatment of dental ailments, but without any previous and present lesions in the oral cavity, and patients were personally interviewed using a questionnaire cum-schedule by individual informed written consent. Information on age, sex, occupation, alcohol consumption, type, frequency and duration of tobacco habits, economic status, place of job, and food habit etc. were recorded. Findings pertaining to histopathological diagnosis and clinical staging were obtained from the pathological reports of the biopsy materials. About 5 ml of biological sample was collected by vein puncture from patients (cancer = 104) and healthy controls (n = 104) was provided to us and stored at -20°C for further analysis. Genomic DNA was isolated from whole blood by the salt precipitation method (Miller et al. 1988).

 

GSTM1 and GSTT1 null genotypes (homozygous) were determined using a polymerase chain reaction (PCR) procedure, both genes were amplified simultaneously with and an internal control, in GeneAmp PCR system 9700 (Applied Biosystems, USA). The primer sequences were similar as described by Nair et al. (1999). The PCR was performed in 10µl reaction buffer containing 200 µM dNTPs, 2 mM MgCl2, 10 pmol of each primer, about 1µg DNA and 2 units of thermostable Taq DNA polymerase using a programmable thermocycler. After 6 min of pretreatment at 96°C, 30 cycles of 30 second denaturation at 94°C, 30 second annealing at 55°C, and 1 min extention at 72°C were performed. Homozygous GSTM1 and GSTT1 null genotypes were determined by the absence of 215 bp and 480 bp fragments in the PCR products, respectively, along with the presence of an internal control band of 916 bp ALAD fragment. For evaluating the GSTM1 and GSTT1 polymorphism the amplification products were analyzed by gel electrophoresis (1.5% Agarose) and visualized under UV light in Gel Doc 200 Imaging System.

 

Genotype distributions were compared between groups using the χ2 test. The group attributable risk was estimated by standard methods (Odd Ratio). Statistical analyses were performed using SPSS 16.0 version and Microsoft Excel (Microsoft Office 2007).

 

 

 

RESULTS:

Fig. 1. Band pattern of GSTM1 and GSTT1 and ALAD (internal control) polymorphisms. ladder ΦX 174 DNA marker, GSTM1 (215 bp)GSTT1 (480bp) ALAD (916bp)

 

Table No. 1 Age and gender distribution of case and control subjects

Variable	Case	Control
No. of subject	104 (50.0%)	104 (50.0%)
Age (yrs) Mean ± SD* (range)	48.30 ± 10.58 (23-70)	46.96 ± 9.49 (23-72)
Gender
Male	77 (74.00%)	72 (69.20%)
Female	27 (26.00%)	32 (30.80%)

 

 

Table No. 2 Distribution of risk and non-risk genotypes in case and control subjects and risk to oral cancer

Genotypes	Case	Control	χ2 value	OR (95% CI)
GSTM1 (+/+)	43 (41.3)	60 (57.7)	χ2 = 5.558 p<0.05 (df 1)	1.00 (Reference
GSTM1 (-/-)	61 (58.7)	44 (42.3)		2.38 (0.67-8.49)
GSTT1 (+/+)	48 (46.2)	55 (52.9)	χ2 = 0.942 P> 0.05 (df 1)	1.00 (Reference)
GSTT1 (-)	56 (53.8)	49 (47.1)		1.79 (0.68-4.80)
GSTM1(+/+),GSTM1 (+/+)	35 (33.7)	47 (45.2)	χ2 = 6.493 p<0.05 (df 2)	1.00 (Reference)
GSTM1(+/-),GSTM1 (+/-)	27 (26.0)	32 (30.8)		1.67 (0.53 – 2.98)
GSTM1(-/-),GSTM1 (-/-)	42 (40.4)	25 (24.0)		3.79 (0.32 – 3.54)

 

DISCUSSIONS:

Table 1 shows the distribution of case and control subject by age and sex. Molecular epidemiologic studies have now provided evidence that individual susceptibility to cancer is mediated by both genetic and environmental factors. Most of the cancer genes are rare and highly penetrant (Hayashi et al. 1992). The individual distribution and the combined distribution of normal and mutant alleles of GSTM1 and GSTT1 genes in patient and control groups are shown in table 2. According to the presence of active or mutant GSTM1 and GSTT1 alleles, all the individuals examined were divided in three groups: (i) those with the GSTM1+/+ and GSTT1+/+; (ii) those with the GSTM1+/+ and GSTT1-/- or GSTM1-/- and GSTT1 +/+ and (iii) those with GSTM1-/- and GSTT1 -/- (mutant) genotype (fig.1). The “wild type” genotypes (GSTM1+/+ and GSTT1+/+) was found in 45.2% healthy control but in only 33.7% patients (χ2= 6.493, p<0.05, df 1). The proportion of patients with both mutant genotypes (GSTM1-/- and GSTT1 -/-) was 40.4% as oppose to only 24.0% of controls. The comparable Odds ratio in the presence of both mutant genotype was 3.79 (95% CI, 0.32-3.54).

 

CONCLUSION:

To the best of our knowledge, this is the first report on the effect of GSTM1 and GSTT1 polymorphisms and susceptibility to oral cancer in Chhattisgarh population.

 

Our findings show that frequency of the GSTM1 gene statistically differs in oral cancer patients and controls. The combination of mutant genotype in GSTM1 and GSTT1 is having high risk for susceptibility to oral cancer in Chhattisgarh population. The simultaneous presence of two mutant genotypes, one of each of two loci (GSTM1 and GSTT1) increased about four fold risk of oral cancer (OR= 3.79, 95% CI- 0.32-3.54).

 

In conclusion, our result is consistent with earlier published study from different region of India.

 

ACKNOWLEDGEMENTS:

The authors are grateful for the cooperation received from all patients who participated in the present study. We thank Dr. Vivek Choudhary, HOD, Department of Radiotherapy, Pt. J.N.M. Medical College, Raipur (C.G) for his support throughout the study. Technical help provided by the laboratory technicians of the same department is duly acknowledged.

 

REFERENCES:

1.       Atkinson B, Ernst CS, Herlyn M, Steplewski Z, Sears HF, Koprowski H. (1982) Gastrointestinal cancer associated antigen in immunoperoxidase assay. Cancer Res.; 42: 4820–2.

2.       Cotton S.C., Sharp L., Little J. and Brockton N., Glutathione S Transferase polymorphism and Colorectal Cancer, HuGE Review,1999, Page 1-19.

3.       Hayashi S, Watanabe J, Kawajiri K. High susceptibility tolung cancer analyzed in terms of combined genotypes of P4501A1 and Mu class glutathione S-transferase genes. Jpn J Cancer Res 1992;83:866–870.

4.       Miller S.A. Dykes D.D and H.F.Polesky (1988) A simple salting out procedure for extracting DNA from human nucleated cells, Nucleic Acids Research, Vol. 16 Number 3 1988

5.       Nair VJ, Nair J, Mathew B amd Bartsch H (1999): Glutathione Stransferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers.Carcinogenesis, 20: 743-8.

6.       Nelson HH, Wiencke JK, Christiani DC and et al. (1995): Ethnic differences in the prevalence of the homozygous deleted genotype of glutathione S transferase theta. Carcinogenesis, 16:1243-5.

7.       Pemble, S. E., Taylor, J. B., Craig, R. K. & Ketterer, B. (1986)Biochem. J. 238, 373-378

8.       World Health Organization. The World Health Report 2004: changing history. Geneva: WHO; 2004.

 

 

Accepted on 23.07.2012      © RJPT All right reserved