Polymorphisms of Glutathione- S -Transferase Genes and the Risk of Aerodigestive Tract Cancers in the Northeast Indian Population

Abstract

Background: Widespread use of tobacco and betel quid consumption and a high incidence of tobacco-associated aerodigestive tract cancers have been reported in different ethnic groups from several regions of Northeast (NE) India. This study was done to explore the possibility of phase II metabolic enzymes being responsible for the high prevalence of cancers in this region of India. Methods: Samples from 370 cases with oral, gastric, and lung cancers and 270 controls were analyzed for polymorphism of glutathione-S-transferase (GST) genes using polymerase chain reaction-restriction fragment length polymorphism-based methods. Results and Conclusions: Tobacco smoking and betel quid chewing were found to be high risk factors for oral and lung cancers but not for gastric cancer, whereas tobacco chewing was found to be a risk factor for oral cancer but not for gastric or lung cancer. The variant genotypes of GSTP1 were not associated with any of the aerodigestive tract cancers. GSTT1 and GSTM1 null genotypes appeared to play a protective role for lung cancer (odds ratio [OR] = 0.47, 95% confidence interval [95% CI]: 0.24-0.93, p = 0.03) and (OR = 0.52, 95% CI: 0.28-0.96, p = 0.04), but they were not associated with oral and gastric cancers. However, when data was analyzed in different geographic regions the GSTT1 null genotype was found to be a significant risk factor for oral (OR = 2.58, 95% CI 1.01-6.61, p = 0.05) as well as gastric cancer (OR = 3.08, 95% CI 1.32-7.19, p = 0.009) in samples obtained from the Assam region of NE India. This is the first study on the association of GST polymorphisms and aerodigestive tract cancers in the high-risk region of NE India.

Introduction

Northeastern states in India have reported a very high prevalence of aerodigestive tract cancers when compared with other regions of India (Phukan et al., 2004; Bhattacharjee et al., 2006). Prevalence of oral cancer is highest in the Kamrup district of Assam (age-adjusted rate [AAR]: 8.7 in males and 8.3 in females), whereas the prevalence of gastric cancer (AAR: 57.3 in males and 33.6 in females) and lung cancer (AAR: 39.3 in males and 42.2 in females) are highest in the Aizawl district of Mizoram (ICMR, 2006). The use of tobacco is very high in the Aizawl district, where locally prepared tobacco products such as Tuibur, a unique tobacco smoke-infused water, and Mieziol, a local cigarette made from vaihlo, are widely used. The habit of chewing betel quid, containing fresh betel nut and slaked lime wrapped in betel leaf, is also widespread in this region (Phukan et al., 2005). In the Assam region there is widespread chewing habit of tobacco with peculiarly fermented betel nut. Betel nut contains arecoline, which can produce 3-methyl nitrosamine propionitrile, a potent carcinogen, and safrole-like DNA adducts that have been shown to be genotoxic and mutagenic. In addition, when betel nut is fermented, it is contaminated by fungi, leading to production of carcinogenic aflatoxins (Chattopadhyay et al., 2007), which further adds to the risk for these cancers in this region.

The quantitative absorption, distribution, metabolism, and excretion of carcinogenic tobacco constituents depend on the activity and efficiency of metabolic and enzymatic detoxification pathways. The enzymatic detoxification process is mainly divided into three phases. Phase I involves activation of toxic compounds predominantly by oxidation into more reactive intermediates that are neutralized and conjugated by phase II family of enzymes such as glutathione-S-transferase (GST) (Guengerich, 1990; Sheehan et al., 2001). The resultant water-soluble and less-toxic conjugated product can easily be eliminated from the cell by phase III transport mechanisms for the elimination of glutathione conjugates.

The detoxification efficiency of GST enzymes is determined by the presence, amount, and nature of the isoenzymes coded by GSTT1, GSTM1, and GSTP1 genes. The allelic polymorphism of GSTT1 and GSTM1 are characterized by the deletion of a part of the gene. GSTP1 polymorphism is a single base pair substitution where adenine is replaced by guanine, resulting in an amino acid change in which isoleucine (I105) is replaced by valine (V105) (Watson et al., 1998; Coles and Kadlubar, 2003). Electrophilic compounds are reported to be detoxified less efficiently in individuals with null genotypes of GSTT1 and GSTM1 or variant genotypes of GSTP1 (Ile/Val and Val/Val) when compared with those with wild-type genotype (Bolt and Thier, 2006). The presence of GSTT1 and GSTM1 null genotypes have been reported to be associated with increased risk for several cancers including skin, lung, bladder, prostate, colorectal, and oral cancers (Gao et al., 2002; Jain et al., 2006). However, several other reports have failed to confirm this association (Buch et al., 2002; Sobti et al., 2008). In fact, GSTT1 null genotype had been reported to be a protective factor for oral cancer in a central Indian population (Anantharaman et al., 2007). Polymorphic variants of GSTP1 have also been reported to increase the risk of various cancers (Rebbeck, 1997; Hirvonen, 1999). Previous studies of gene polymorphisms and risk for tobacco-associated cancers have suggested that the polymorphisms in GSTT1, GSTM1, and GSTP1 increase cancer risk in tobacco consumers (Soya et al., 2007; Singh et al., 2008).

The prevalence of tobacco and betel quid chewing habits as well as the occurrence of tobacco-associated cancers is high in the northeast (NE) region of India. In a recent study, we have reported a higher prevalence of GSTT1 and GSTM1 null genotypes in this region when compared with other regions of India (Thoudam et al., 2010). GSTT1 null genotypes have also been reported to be associated with premalignant lesions of oral leukoplakia in the Assam region (Chatterjee et al., 2009). However, the prevalence of polymorphism in GST genes in tobacco-associated cancer patients from this region is not well known. The individual difference in susceptibility to chemically induced carcinomas may possibly be attributed to the genetic differences in the activation or detoxification of carcinogens due to polymorphic variants of GST genes. In the present study, the association of tobacco, betel quid habits, and polymorphism of GSTT1, GSTM1, and GSTP1 genes with aerodigestive tract cancers was evaluated to find out if this could explain the unusually high prevalence of cancers in the NE region of India.

Materials and Methods

Selection of cases

The present study was done on samples obtained from 370 histopathologically confirmed cases with upper aerodigestive cancers (oral squamous cell carcinoma [136], gastric adenocarcinoma [133], and squamous cell carcinoma of lung [100]). The patients were diagnosed at three different tertiary health facilities of NE India, including Dr. Bhubneshwar Borooah Cancer Institute, Guwahati, Assam; Sir T.N.M. Hospital, Gangtok, Sikkim; and Civil Hospital, Aizawl, Mizoram, between 2006 and 2008. Questionnaires containing information on age, sex, region of origin, occupation, duration, and type of tobacco and betel quid consumption habits were recorded by interviewing all participating individuals.

Selection of controls

Samples obtained from unrelated voluntary healthy individuals who were accompanying the patients to the hospital were included as controls. Each case of oral, gastric, and lung cancers was matched individually for age, sex, and ethnicity with the control samples (270 controls for oral and gastric cancers and 221 for lung cancer). Questionnaires containing information on age, sex, region of origin, occupation, duration, and type of tobacco and betel quid consumption habits were recorded by interviewing all participating individuals. Institutional ethical clearance was obtained as per the guidelines. An informed consent was signed and obtained from all subjects.

Collection and processing of samples

Two to 3 mL of peripheral blood samples were collected in tubes containing ethylenediaminetetraacetic acid, stored in a −20°C freezer, and transported in dry ice to the Institute of Pathology. Genomic DNA was extracted and purified using proteinase K phenol-chloroform extraction procedure (Sambrook and Russell, 2001).

Genotyping of GSTT1 and GSTM1

A multiplex polymerase chain reaction (PCR) method was used to detect the presence or absence of the GSTT1 and GSTM1 genes in the genomic DNA samples of patients and controls. Twenty-five microliters of PCR mixture was prepared by mixing 2.5 μL of 10 × Taq buffer, 1 μL of 25 mM MgCl₂, 0.5 μL of 10 mM dNTP mix, 0.5 μL of each forward and reverse primers (10 pM), 50-100 ng of template DNA, and 1 unit of Taq polymerase (M/s Fermentas, Vilnius, Lithuania). The primers were synthesized by M/s Microsynth, Lindau, Germany. The primer pairs were 5′-TTCCTTACTGGTCCTCACATCTC-3′ and 5′-TCACCGGATCATGGCCAGCA-3′ for GSTT1, 5′-GAACTCCCTGAAAAGCTAAAGC-3′ and 5′-GTTGGGCTCAAATATACGGTGG-3′ for GSTM1, and 5′-CAACTTCATCCACGTTCACC-3′ and 5′-GAAGAGCCAAGGACAGGTAC-3′ for β-globin. β-Globin (268-bp fragment) was used as an internal control to ensure PCR amplification if the samples had null genotypes of GSTM1 and GSTT1. To test for contamination, negative controls (without template) were included in every PCR run. PCR was carried out as follows: denaturation at 94°C for 4 min; followed by 20 cycles of denaturation at 93°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min; then additional 15 cycles of denaturation at 93°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were electrophoresed in 2% agarose gels containing ethidium bromide in 0.6× tris-borate ethylenediaminetetraacetic acid (TBE) buffer. The absence of 459 bp band indicated GSTT1 null genotype and the absence of 219 bp indicated GSTM1 null genotype. Approximately 10% of samples were randomly selected and repeated for genotyping.

Genotyping of GSTP1

Polymorphic variants of GSTP1 were detected by PCR-restriction fragment length polymorphism. Twenty-five microliters of PCR mixture was prepared by mixing 2.5 μL of 10 × Taq buffer, 2 μL of 25 mM MgCl₂, 1.25 μL of 10 mM dNTP mix, 1.25 μL of each forward (5′-CCAGTGACTGTGTGTTGATC-3′) and reverse (5′-CAACCCTGGTGCAGATGCTC-3′) primers (10 pM) for GSTP1, 50-100 ng of template DNA, and 1 unit of Taq polymerase. Cycling conditions were as follows: initial denaturation at 94°C for 3 min; followed by 35 cycles of 94°C for 1 min, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. The PCR product of GSTP1 was 189 bp in size. After testing for the amplification of PCR products in 2% agarose gel, 10 μL of PCR product was digested using BsmA1 restriction enzyme (M/s Fermentas) in a reaction volume of 30 μL by overnight incubation at 37°C. The products were separated by electrophoresis in 4% agarose gel in 0.6 × TBE. On the basis of the band patterns, three genotypic variants were identified. The wild-type genotype [Ile/Ile (A/A)], completely undigested, was represented by a single band at 189 bp. The genotypic variant [Val/Val (G/G)] was completely digested, yielding two bands of 148 and 41 bp with absence of a 189-bp fragment. The digested product that yielded all the three bands represented the heterozygous genotype [Ile/Val (A/G)]. A positive known control sample that had earlier been identified as Val/Val (G/G) variant of GSTP1 was included in all experiments. Approximately 10% of samples were randomly selected and genotyping was repeated. Genotyping procedures were validated by sequencing of representative samples.

Statistical analysis

Stata 8.0 version software was used for statistical analysis. Hardy-Weinberg equilibrium test was done to compare the difference between the observed and expected frequencies for GSTP1 genotype. The association for the considered covariates including tobacco use (no/yes), chewing (no/yes), smoking (no/yes), and the polymorphisms GSTT1 (present/null), GSTM1 (present/null), and GSTP1 (wild type/variant) with three cancer groups (oral, gastric, and lung) were assessed by applying the conditional logistic regression analysis (age and sex matched). The conditional logistic regression analysis was performed to get risk estimates for various cancers associated with these phenotypic and genotypic variables and these results were interpreted in terms of adjusted odds ratios (ORs) along with their corresponding 95% confidence intervals (95% CIs). To get the estimates of regression coefficient, standard error, and statistical significance for each of the variables, enter method was used during conditional logistic regression analysis. Table 1 exhibits the results of the conditional logistic regression analysis for each cancer with the phenotypic and genotypic variables. The adjusted estimates for the specific phenotypic variables (tobacco chewing, tobacco smoking, and betel quid chewing) and genotypic variables (GSTT1, GSTM1, and GSTP1) were adjusted for all other phenotypic and genotypic variables under consideration. Table 2 illustrates the region-specific estimates for various cancers with the adjusted estimates in accordance to as explained for Table 1 above.

Table 1.

Distribution and Association of Tobacco and Betel Quid Consumption Habits and Glutathione-S-Transferase Genotypes Among Cancer Cases and Controls

	Oral cancer				Gastric cancer				Lung cancer
	Cases n = 136	Controls n = 270	Adjusted ^a		Cases n = 133	Controls n = 270	Adjusted ^a		Cases n = 101	Controls n = 221	Adjusted ^a
Risk factors	n (%)	n (%)	OR (95% CI)	p	n (%)	n (%)	OR (95% CI)	p	n (%)	n (%)	OR (95% CI)	p
Tobacco chewing^a	103 (76)	152 (56)	2.44 (1.47-4.05)	0.001	86 (65)	152 (56)	1.44 (0.88-2.32)	0.14	55 (54)	123 (56)	0.73 (0.42-1.27)	0.27
Tobacco smoking^a	81 (60)	133 (49)	1.72 (1.08-2.73)	0.02	53 (40)	133 (49)	0.72 (0.47-1.11)	0.14	81 (80)	104 (47)	4.23 (2.24-7.96)	<0.001
Betel quid chewing^a	106 (78)	169 (63)	2.20 (1.29-3.76)	0.004	95 (71)	169 (63)	1.41 (0.88-2.28)	0.15	79 (78)	130 (59)	2.16 (1.05-4.43)	0.04
GSTT1
Present	94 (69)	185 (69)	1.00		83 (62)	185 (69)	1.00		82 (81)	152 (69)	1.00
Null^a	42 (31)	85 (31)	1.02 (0.61-1.71)	0.93	50 (38)	85 (31)	1.24 (0.76-2.03)	0.39	19 (19)	69 (31)	0.47 (0.24-0.93)	0.03
GSTM1
Present	70 (51)	150 (56)	1.00		84 (63)	150 (56)	1.00		68 (67)	118 (53)	1.00
Null^a	66 (49)	120 (44)	1.18 (0.74-1.88)	0.48	49 (37)	120 (44)	0.74 (0.46-1.17)	0.19	33 (33)	103 (47)	0.52 (0.28-0.96)	0.04
GSTP1
Ile/Ile	78 (57)	173 (64)	1.00		75 (56)	173 (64)	1.00		54 (54)	132 (60)	1.00
Ile/Val or Val/Val^a	58 (43)	97 (36)	1.35 (0.86-2.13)	0.19	58 (44)	97 (36)	1.25 (0.77-2.03)	0.37	47 (46)	89 (40)	1.50 (0.84-2.68)	0.17

A p-value of <0.05 is considered statistically significant.

Adjusted with all other risk variables under consideration.

OR, odds ratio; 95% CI, 95% confidence interval.

Table 2.

Region-Specific Demographic Characteristics and Distribution of Glutathione-S-Transferase Genotypes Among Cancer Cases and Controls

		Oral cancer				Gastric cancer				Lung cancer
State	Variables/genotypes	Cases n/N (%)	Controls n/N (%)	Adjusted ^a OR (95% CI)	p	Cases n/N (%)	Controls n/N (%)	Adjusted ^a OR (95% CI)	p	Cases n/N (%)	Controls n/N (%)	Adjusted ^a OR (95% CI)	p
Assam	Age (years ± SEM)	53 ± 11	49 ± 10			52 ± 12	49 ± 10			53 ± 10	49 ± 10
	Sex (M/F)	56/22	82/26			50/18	82/26			60/16	82/26
	Tobacco chewing^a	65/78 (83)	53/108 (49)	4.47 (1.93-10.32)	<0.001	38/68 (56)	53/108 (49)	1.24 (0.62-2.48)	0.55	42/76 (55)	53/108 (49)	1.44 (0.69-2.98)	0.32
	Tobacco smoking^a	43/78 (55)	64/108 (59)	0.72 (0.31-1.66)	0.48	28/68 (41)	64/108 (59)	0.38 (0.19-0.78)	0.008	60/76 (79)	64/108 (59)	3.12 (1.39-6.97)	0.006
	Betel quid chewing^a	67/78 (86)	90/108 (83)	0.77 (0.38-1.58)	0.60	63/68 (93)	90/108 (83)	4.12 (1.13-15.08)	0.03	62/76 (82)	90/108 (83)	0.74 (0.29-1.89)	0.53
	GSTT1 null^a	20/78 (26)	13/108 (12)	2.58 (1.01-6.61)	0.05	18/68 (26)	13/108 (12)	3.08 (1.32-7.19)	0.009	12/76 (16)	13/108 (12)	0.96 (0.36-2.54)	0.93
	GSTM1 null^a	35/78 (45)	36/108 (33)	1.50 (0.67-3.34)	0.32	20/68 (29)	36/108 (33)	1.01 (0.46-2.19)	0.99	26/76 (34)	36/108 (33)	0.87 (0.46-1.66)	0.67
	GSTP1 (Ile/Val or Val/Val)^a	37/78 (47)	39/108 (36)	1.36 (0.65-2.83)	0.41	34/68 (50)	39/108 (36)	1.56 (0.79-3.07)	0.19	37/76 (49)	39/108 (36)	1.60 (0.79-3.24)	0.19
Mizoram	Age (years ± SEM)	53 ± 12	54 ± 12			54 ± 12	54 ± 12			63 ± 10	63 ± 10
	Sex (M/F)	16/9	60/29			33/15	60/29			5/3	19/12
	Tobacco chewing^a	12/25 (48)	72/89 (81)	0.08 (0.013-0.48)	0.006	40/48 (83)	72/89 (81)	1.19 (0.43-3.27)	0.73	4/8 (50)	22/31 (71)	Not analyzed because of inadequate number of cases
	Tobacco smoking^a	17/25 (68)	30/89 (34)	3.74 (1.15-12.08)	0.03	17/48 (35)	30/89 (34)	1.11 (0.50-2.45)	0.80	7/8 (87)	11/31 (35)
	Betel quid chewing^a	16/25 (64)	54/89 (61)	1.04 (0.35-3.10)	0.94	31/48 (65)	54/89 (61)	1.13 (0.58-2.94)	0.51	7/8 (87)	17/31 (55)
	GSTT1 null^a	14/25 (56)	45/89 (51)	0.87 (0.28-2.67)	0.80	24/48 (50)	45/89 (51)	0.85 (0.42-1.71)	0.65	1/8 (12)	15/31 (48)
	GSTM1 null^a	17/25 (68)	47/89 (53)	2.20 (0.76-8.38)	0.15	18/48 (37)	47/89 (53)	0.55 (0.28-1.09)	0.09	3/8 (37)	15/31 (48)
	GSTP1 (Ile/Val or Val/Val)^a	10/25 (40)	30/89 (34)	2.30 (0.57-7.63)	0.27	19/48 (40)	30/89 (34)	1.41 (0.62-3.18)	0.41	3/8 (37)	17/31 (55)
Sikkim	Age (years ± SEM)	59 ± 14	62 ± 10			60 ± 11	62 ± 10			59 ± 11	62 ± 10
	Sex (M/F)	23/10	50/23			11/6	50/23			12/5	50/23
	Tobacco chewing^a	26/33 (79)	27/73 (37)	27.6 (3.07-248.3)	0.003	8/17 (47)	27/73 (37)	1.01 (0.25-4.06)	0.99	9/17 (53)	27/73 (37)	1.57 (0.45-5.54)	0.48
	Tobacco smoking^a	21/33 (64)	39/73 (53)	0.83 (0.27-2.53)	0.74	8/17 (47)	39/73 (53)	1.17 (0.32-4.31)	0.81	14/17 (82)	39/73 (53)	2.83 (0.65-12.33)	0.17
	Betel quid chewing^a	23/33 (70)	25/73 (34)	3.99 (1.20-13.21)	0.02	1/17 (6)	25/73 (34)	0.12 (0.01-1.13)	0.06	10/17 (59)	25/73 (34)	3.26 (0.75-14.25)	0.12
	GSTT1 null^a	8/33 (24)	27/73 (37)	0.20 (0.03-1.41)	0.10	8/17 (47)	27/73 (37)	1.77 (0.47-6.73)	0.40	6/17 (35)	27/73 (37)	0.73 (0.21-2.58)	0.63
	GSTM1 null^a	14/33 (42)	37/73 (51)	0.67 (0.13-3.36)	0.62	11/17 (65)	37/73 (51)	1.35 (0.36-5.08)	0.65	4/17 (23)	37/73 (51)	0.43 (0.12-1.59)	0.21
	GSTP1 (Ile/Val or Val/Val)^a	11/33 (33)	28/73 (38)	0.76 (0.20-2.91)	0.69	5/17 (29)	28/73 (38)	0.91 (0.25-3.31)	0.89	7/17 (41)	28/73 (38)	0.99 (0.27-3.57)	0.99

Adjusted with all other risk variables of the same region under consideration.

SEM, standard error of the mean.

Results

Oral cancer

One hundred thirty-six patients with oral cancer (78 from Assam, 33 from Sikkim, and 25 from Mizoram) and 270 healthy controls (108 from Assam, 73 from Sikkim, and 89 from Mizoram) were included in the study. The mean age (in years) of patients with oral cancer and normal healthy controls was 53 ± 11 and 49 ± 10 in Assam, 59 ± 14 and 62 ± 10 in Sikkim, and 53 ± 12 and 54 ± 12 in Mizoram, respectively. One hundred three (76%) cases with oral cancer and 152 (56%) controls were tobacco chewers, 81 (60%) cases and 133 (49%) controls were tobacco smokers, and 106 (78%) cases and 169 (63%) controls were betel quid chewers when we considered the NE population as one group. The conditional logistic regression analysis further revealed that the risk of developing oral cancer significantly increased in tobacco chewers (OR = 2.44, 95% CI: 1.47-4.05, p = 0.001), tobacco smokers (OR = 1.72, 95% CI: 1.08-2.73, p = 0.02), and betel quid chewers (OR = 2.20, 95% CI: 1.29-3.76, p = 0.004) (Table 1).

The frequency of GSTT1 and GSTM1 null genotype was 31% and 49% in samples obtained from patients with oral cancer and 31% and 44% in controls, respectively. When adjusted for other variables under consideration, no significant association was found for GSTM1 and GSTT1 null genotype independently or in combination with oral cancer risk. The frequency of the variant genotypes of GSTP1 (heterozygous Ile/Val and homozygous Val/Val) was higher in samples of patients with oral cancer (43%) when compared with those with controls (36%); however, this difference was not statistically significant (OR = 1.35, 95% CI: 0.86-2.13, p = 0.19) (Table 1).

Although no significant independent association of oral cancer with null genotypes of GSTT1, GSTM1, and the variant alleles of GSTP1 was found, individuals with both GSTM1 null genotype and variant alleles of GSTP1 were found to have marginal increased risk for developing oral cancer (OR = 1.84, 95% CI: 0.91-3.72, p = 0.08) (data not shown). When data were analyzed for each geographical region, GSTT1 null genotype was found to be significantly higher (OR = 2.58, 95% CI: 1.01-6.61, p = 0.05) in oral cancer cases (26%) when compared with controls (12%) for the Assam region (Table 2).

Gastric cancer

One hundred thirty-three patients with gastric cancer (68 from Assam, 17 from Sikkim, and 48 from Mizoram) and 270 normal healthy controls (108 from Assam, 85 from Sikkim, and 73 from Mizoram) were included in the study. The mean age of patients with gastric cancer and normal healthy controls was 52 ± 12 and 49 ± 10 in Assam, 60 ± 11 and 62 ± 10 in Sikkim, and 54 ± 12 and 54 ± 12 in Mizoram, respectively. Of these, 86 (65%) cases with gastric cancer and 152 (56%) controls were tobacco chewers, 53 (40%) cases and 133 (49%) controls were tobacco smokers, and 95 (71%) cases and 169 (63%) controls were betel quid chewers.

Betel quid chewing (71%) and tobacco chewing (65%) habits were higher in gastric cancer cases when compared with a control population (63% and 56%, respectively), but this difference was statistically insignificant (OR = 1.41, 95% CI: 0.88-2.28, p = 0.15 and OR = 1.44, 95% CI: 0.88-2.32, p = 0.14, respectively) (Table 1).

The frequency of GSTT1 and GSTM1 null genotype was 38% and 37% in samples obtained from patients with gastric cancer and 31% and 44% in controls, respectively. Variant genotypes (Ile/Val and Val/Val) of GSTP1 were found more frequently in samples from patients with gastric cancer (44%) when compared with controls (36%), but the difference was not statistically significant (OR = 1.25, 95% CI: 0.77-2.03, p = 0.37). When adjusted for tobacco and betel quid consumption habits, no significance of GSTT1, GSTM1, or GSTP1 polymorphism, either alone (Table 1) or in combination (data not shown), was found with gastric cancer. When data were analyzed for each geographical region, the prevalence of GSTT1 null genotype in Assam was found to be significantly higher (OR = 3.08, 95% CI: 1.32-7.19, p = 0.009) in gastric cancer cases (26%) when compared with controls (12%) (Table 2).

Lung cancer

One hundred one patients with lung cancer (76 from Assam, 17 from Sikkim, and 8 from Mizoram) and 221 normal healthy controls (77 from Assam, 70 from Sikkim, and 74 from Mizoram) were included in the study when NE was considered as single group for comparison. This matching was done by best possible matching of age and sex without looking at any other risk factors. However, the number of best matching controls from each region changed when matching was done geographically (108 from Assam, 73 from Sikkim, and 31 from Mizoram). The mean age of patients with lung cancer and the corresponding controls was 53 ± 10 and 49 ± 10 in Assam, 59 ± 11 and 62 ± 10 in Sikkim, and 63 ± 10 and 63 ± 10 in Mizoram, respectively. Of these, 81 (80%) of lung cancer cases and 104 (47%) controls were tobacco smokers, 55 (54%) cases and 123 (56%) controls were tobacco chewers, and 79 (78%) cases and 130 (59%) controls were betel quid chewers (Table 1).

Lung cancer was significantly associated with tobacco smoking (OR = 4.23 95% CI: 2.24-7.96, p < 0.001) and betel quid chewing (OR = 2.16, 95% CI: 1.05-4.43, p = 0.04) (Table 1). The frequency of GSTT1 and GSTM1 null genotype was 19% and 33% in lung cancer cases and 31% and 47% in controls, respectively. Variant genotypes of GSTP1 (Ile/Val and Val/Val) were found more frequently in lung cancer cases (46%) when compared with controls (40%), but the difference was not statistically significant (OR = 1.50, 95% CI: 0.84-2.68, p = 0.17). GSTT1 null (OR = 0.47, 95% CI: 0.24-0.93, p = 0.03) and GSTM1 null (OR = 0.52, 95% CI: 0.28-0.96, p = 0.04) genotypes appeared to be protective factors for lung cancer (Table 1).

The geographical and cancer-specific estimates obtained by conditional logistic regression analysis revealed that the chances of developing lung cancer were significantly higher for smoking individuals in Assam (OR = 3.12, 95% CI: 1.39-6.97, p = 0.006) (Table 2).

Discussion

Lack of GSTT1 and GSTM1 isoenzymes activity or differences in the activity and distribution of allelic variants of GSTP1 have been earlier implicated in increased cancer risk following exposure to environmental carcinogens. Of these, GSTT1 is responsible for the biotransformation of the constituents of tobacco smoke, such as alkyl halides, and its derivatives, such as monohaloethanes, ethylene oxide, benzo(a)pyrene diol epoxide, and acrolien (Pemble et al., 1994; Rebbeck, 1997). GSTM1 subfamily metabolizes lipid peroxidation products, DNA hydroperoxides, and polyaromatic hydrocarbons such as benzo [alpha] pyrene (Lear et al., 2000; Ye et al., 2004; Jain et al., 2006). The GSTP1 enzyme is widely expressed in tumor cells and is responsible for the detoxification of benzo(a)pyrene diol epoxide and acrolein present in cigarette smoke. The GSTP1 isoform is also known to metabolize tobacco-related carcinogens with elimination of the oxidative products of thymidine or uracil propenal (Matthias et al., 1998). Polymorphism of the GSTT1 and GSTM1 genes, which are located on chromosome 22q11.2 and 1p13.3, respectively, results in deletion of their loci with subsequent loss of specific enzymatic functional activity and reduced ability to detoxify potentially toxic substances. Polymorphism of GSTP1 gene, which is located on chromosome region 11q13, shows a single base pair substitution where adenine is replaced by guanine, resulting in amino acid isoleucine (I105) being replaced by valine (V105) (Watson et al., 1998; Coles and Kadlubar, 2003). As GST genes are involved in the detoxification of tobacco constituents, there is a possibility that the genetic polymorphisms of these enzymes may be a high risk factor for the widespread occurrence of tobacco-associated aerodigestive malignancies in NE Indians.

The association of oral and lung cancers with tobacco consumption have been well documented (Cho et al., 2006). In our study, tobacco and betel quid chewing habits were found to be significant risk factors for oral cancer, whereas tobacco smoking and betel quid chewing were found to be significant risk factors for lung cancer. However, no significant association of tobacco consumption in any form was found to be associated with gastric cancer. This was in contrast to earlier reports where tobacco consumption was found to be significantly associated with gastric cancer (Phukan et al., 2005; Cho et al., 2006). Helicobacter pylori is also a major cause of chronic gastritis and a firmly established carcinogen for gastric adenocarcinoma (Chiurillo et al., 2010). In an earlier study, we have found IgG antibodies to H. pylori in serum samples of almost 60% patients with gastric cancer (data not shown).

Earlier studies from different regions of the world have reported a higher risk for the occurrence of several cancers in patients with GSTT1 and GSTM1 null genotypes. However, many other studies have reported conflicting results. GSTM1 null genotype has been reported as a risk factor for oral cancer (Gattas et al., 2006; Duarte et al., 2008), gastric cancer (Lai et al., 2005; Martinez et al., 2006; Tripathi et al., 2008), and lung cancer (Lan et al., 2000; Chen et al., 2006). This is in contrast to other reports where no significant association of GSTM1 null genotype was found with risk of oral cancer (Losi-Guembarovski et al., 2008), gastric cancer (Wideroff et al., 2007), and lung cancer (Sorensen et al., 2007; Honma et al., 2008). In fact, there are reports that have shown GSTM1 null genotype as a protective factor for some cancers such as oral (Hatagima et al., 2008), breast (Roodi et al., 2004), and skin cancers (Heagerty et al., 1994; Ramachandran et al., 1999). GSTT1 null genotype has been reported as a risk factor for oral cancer (Bartsch et al., 1999; Jourenkova-Mironova et al., 1999; Duarte et al., 2008), gastric cancer (Lan et al., 2001; Boccia et al., 2007), and lung cancer (Chen et al., 2006), whereas no significant association of GSTT1 null genotype had been reported with oral cancer (Kietthubthew et al., 2001; Hatagima et al., 2008; Losi-Guembarovski et al., 2008), gastric cancer (Wideroff et al., 2007), and lung cancer (Sorensen et al., 2007; Honma et al., 2008) in other studies. As reported for GSTM1 null genotype, GSTT1 null genotype has been also reported as a protective factor for some cancers such as head and neck cancer (Evans et al., 2004), bladder cancer (Kim et al., 2002), and breast cancer (Garcia-Closas et al., 1999).

A review of studies done on these cancers in India also showed conflicting results of association with GST polymorphism. For example, GSTM1 null genotypes were reported as a significant risk for oral cancer in the western Indian population (Buch et al., 2002; Anantharaman et al., 2007), gastric cancer in the Kashmir valley population (Malik et al., 2009), and lung cancer in the Punjab state of North India (Sobti et al., 2004), whereas no risk was reported for lung cancer in a South Indian population (Sreeja et al., 2005) and for oral cancer in a North Indian population (Sharma et al., 2006). GSTT1 null genotype was reported to be a risk factor for oral cancer in a North Indian population (Sharma et al., 2006; Singh et al., 2008), gastric cancer in Kashmir valley (Malik et al., 2009), and lung cancer in a North Indian population (Sobti et al., 2004). However, no significant risk was reported in other studies for oral cancer in a western Indian population (Buch et al., 2002) and for lung cancer in the Punjab region of North India (Sobti et al., 2008) and in Trivandrum of South India (Sreeja et al., 2008). GSTP1 variants have been reported as a risk factor for gastric cancer in the Lucknow region of North India (Tripathi et al., 2008), for lung cancer in the South Indian population (Sreeja et al., 2008), and for oral cancer in an East Indian population (Sikdar et al., 2004), whereas no association has been found for gastric cancer in the Kashmir valley (Malik et al., 2009) and for lung cancer in a North Indian population (Kumar et al., 2009). Moreover, data from different geographical regions of India show large variation in different ethnic groups in a healthy population (Thoudam et al., 2010).

In the present study, GSTT1 null genotype was not found to be associated with risk of oral and gastric cancers when the NE population was taken as one group. However, analysis of GST polymorphisms in different geographic regions of NE India showed GSTT1 genotype to be a significant risk factor for oral and gastric cancers in the Assam region of NE India. Further, GSTT1 and GSTM1 null genotypes were more prevalent in the control samples when compared with samples from patients with lung cancer, suggesting that GSTT1 and GSTM1 null genotypes may in fact have a protective role in this cancer. In the present study, the variant GSTP1 Ile/Val and Val/Val genotypes were not significantly associated with oral, gastric, or lung cancer when the NE population was taken as one group or when analyzed for different geographical regions. Several earlier studies have also reported conflicting results for GSTP1 polymorphism, with both risk factor (Miller et al., 2003; Sreeja et al., 2008) and no association (Reszka et al., 2003; Sobti et al., 2008) having been reported. In addition, epigenetic factors such as hypermethylation of the promoter region of GSTP1 gene may lead to downregulated gene expression and reduced activity of the enzyme. Methylation of the GSTP1 promoter region has been earlier found to be associated with some cancers, particularly prostate cancers, where it has been used for its early diagnosis and prognosis (Duffy et al., 2009). However, no such significant association has been so far reported for aerodigestive tract cancers. Other epigenetic phenomena such as polymorphisms and mutations in mitochondrial DNA have also been reported in a variety of human cancers including prostate (Petros et al., 2005), gastric (Maximo et al., 2001), and esophageal (Hibi et al., 2001) cancers.

Exposure to the type and amount of environmental toxins is variable not only in different geographic regions, but also in different ethnic groups within the same geographic region because of variations in their dietary, social, and cultural habits. Although the samples included in our study belonged to a common geographical region of India, the inhabitants of this region are of different ethnic origins. As the ethnically different population inhabiting this region of India has been presumably exposed to shared environmental factors such as pesticide exposure and high level of tobacco and betel quid consumption, we have analyzed the data of different racial composition separately as well as a combined group from NE India. The inconsistency in results of association of GST polymorphism with various cancers may be due to different ethnicity or interaction between different environmental and genetic factors. For example, individuals who inherit the GSTT1 enzyme can produce a mutagenic and carcinogenic metabolite of the industrial chemical dichloromethane following conjugation with glutathione (Pemble et al., 1994). Further, although the GSTs are enzymes that are synthesized mainly in the liver (Jefferies et al., 2003), the localization and concentration of different classes of GSTs in the cytosol of different organs are variable. Moreover, some other properties of GST enzymes, such as affinity toward substrate and isoelectric focusing, are also variable in different tissues and organs (Awasthi et al., 1994). This may explain why the toxicological effect of carcinogens varies in different tissues and organs. For example, the significance of the GSTM1 null genotype may be lower for lung cancer when compared with some other cancers because the expression of GSTM1 is reported to be low in lung tissue (Yang et al., 2007). In contrast, GSTP1 is found in abundant concentration in the lung (Anttila et al., 1993). The distribution of GST enzymes in different organs may also vary with the age and sex of different individuals. These factors may lead to variation in the carcinogenic concentration of toxins in different tissues and to a variable role of GST genotypes in different populations exposed to different environmental carcinogens as has been found in our study. To the best of our knowledge, this is the first study on these detoxifying genes involving aerodigestive tract cancers in a high-risk region of India where the local population has peculiar betel quid and tobacco consumption habits. Further studies on large number of samples and investigation on other detoxifying genes may substantially enrich our knowledge on the reason for the unusually high prevalence of tobacco-associated cancers in the NE region of India.

Footnotes

Acknowledgments

The study was funded by a grant from the Indian Council of Medical Research, New Delhi, India (Ref. No. 49/3/RMRC/NE/2005-NCD-II dated March 28, 2005). One of the authors (D.S.Y.) thanks the University Grants Commission, India, for providing research fellowship. The authors are thankful to the clinical investigators involved in this study—Dr. K. Ahmed, Dr. B.K. Das, Dr. J. Purkaystha, Dr. C. Bhuyan, Dr. B.J. Saikia, Dr. N. Kalita, Dr. K. Lalbiakzuala, and Dr. Thomas Zomuana.

Disclosure Statement

No competing financial interests exist.

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