Association of Genetic Polymorphism of Glutathione S-transferase M1,T1, P1 and Susceptibility to Bladder Cancer

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1 European Urology European Urology 48 (2005) Translational ResearchöFrom Lab to Clinic: Oncology Association of Genetic Polymorphism of Glutathione S-transferase M1,T1, P1 and Susceptibility to Bladder Cancer Daya Shankar Lal Srivastava a, **, Dhruva Kumar Mishra a, Anil Mandhani a, Balraj Mittal b, Anant Kumar a, Rama Devi Mittal a, * a Department of Urology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow , Uttar Pradesh, India b Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow , Uttar Pradesh, India Accepted 9 February 2005 Available online 16 March 2005 Abstract Objective: Glutathione-S-transferases (GSTs) are active in the detoxification of wide variety of endogenous or exogenous carcinogens. We examined the association of the GST gene polymorphism with sporadic bladder cancer patients in Northern India. Material and methods: The study constituted of 106 bladder cancer cases and 370 age-matched controls. The GSTT1 and GSTM1 null genotypes were identified by multiplex PCR and GSTP1313 A/G by Polymerase Chain Reaction/Restriction Fragment Length Polymorphism method (PCR/RFLP). Results: We observed non-significant association in null alleles of the GSTM1 (p = 0.611, OR = 1.12, 95% CI = and GSTT1 (p = 0.135, OR = 1.45, 95% CI = ) with risk of bladder cancer. However, the G/G genotype of the GSTP1 gene polymorphism was highly significant when compared to controls (p = 0.000, OR = 7.12, 95% CI = ). The combined analysis of the three risk genotypes demonstrated further increase in the risk of bladder cancer (p = 0.000, OR = % CI = ). Conclusion: Our study demonstrated that GSTP1313 G/G polymorphism is a strong predisposing risk factor for bladder cancer. Combination of three GST genotypes association exhibiting gene-gene interaction further substantiates the increased risk of bladder cancer. # 2005 Elsevier B.V. All rights reserved. Keywords: Glutathione S-transferase; Genetic polymorphism; Detoxification; Gene-gene interaction; Bladder cancer 1. Introduction A great majority of chemical carcinogen incapable of producing DNA damage requires activation by enzyme system (such as Cytochrome P450) in order to become active carcinogen, for tumor initiation [1]. Conversely phase II metabolizing enzyme such as GST, NAT, epoxide hydroxylase and sulfotransferase are involved in * Corresponding author. Tel x2116; Fax: addresses: dshankarpgi@yahoo.com (D.S.L. Srivastava), rmittal@sgpgi.ac.in, ramamittal@yahoo.com (R.D. Mittal). ** Co-corresponding author: Tel ; Fax: detoxification of chemical carcinogen and subsequently their role is expected to be protective [2]. Exposure to tobacco smoke and other environmental and occupational chemicals have been described as a risk factor for bladder cancer such as 2-nepthylamine, benzidine and 4- aminobiphenyl[3]. Genetic difference in the metabolism of these chemicals has been recently suggested as modifiers of individual susceptibility to environmentally induced bladder cancer. Cytosolic glutathione S-transferases (GSTs) are a family of related isozymes that catalyze the conjugation of reduced glutathione to a wide range of electrophillic substrate [4]. Among them, most extensively studied are the GSTM1 null, the GSTP1313 A/G /$ see front matter # 2005 Elsevier B.V. All rights reserved. doi: /j.eururo

2 340 D.S.L. Srivastava et al. / European Urology 48 (2005) substitution and the GSTT1 null polymorphism. The GSTP1313 A/G polymorphism at the nucleotide level leads to an amino acid variation of isoleucine/ valine at codon 105 in the protein. Valine amino acid results in decreased enzymes activity [5]. Toruner et al (2001), reported that GSTP1313A/G or G/G genotype increases 1.75 fold of bladder cancer susceptibility [6]. Individuals with GSTT1 null genotype show reduced ability to detoxify metabolites of 1, 3 butadiene and ethylene oxide [7,8]. GSTM1 deficiency suspected to increased risk of bladder cancer was first reported by Bell et al, 1993 [9], a number of studies appeared with supporting finding [10,11]. In contrast to GSTM1, potential role of GSTT1 genotype in individual susceptibility to bladder cancer have been reported with inconsistent results. A few studies exhibited non-significant decreased risk of bladder cancer with GSTT1 null genotype [12,13] however; some other studies exhibited increased risk of bladder cancer with GSTT1 null genotype [14,15]. Inconsistent results have also emerged from recent studies exploring the combined effect of GSTM1 and GSTT1 genotype in the development of malignancy [14,16]. Although promising data from these studies are accumulating at a remarkable pace, they are still too sparse to support a role for a specific gene in bladder cancer risk. Recent study suggests that the frequencies of some polymorphisms in certain genes differ among different racial and ethnic groups [17]. Whether these genetic variants can help explain part of the large differences in bladder cancer risk between populations await further clarification. In the present study, we determined the genotypic frequency of the GSTM1 null, GSTT1 null polymorphism and GSTP1313 A/G in bladder cancer patients to substantiate its association with bladder cancer in the north Indian population DNA extraction and genotyping Five ml of blood was collected in EDTA vials from cases and controls. DNA was extracted from blood lymphocytes using the Proteinase K and phenol chloroform extraction procedure [18]. Analysis for GSTM1 and GSTT1 gene polymorphism was done by multiplex PCR as described by Rehman et al. [19]. Isolated DNA ( ng) was amplified in a total volume of 25 ml reaction mixture containing 20 pmol of each of the following primers. GSTM1: Forward, 5 0 -GAACTCCCTGAAAAGCTAAAGC-3 0 and Reverse, 5 0 -GTTGGGCTCAAATATACGGTGG-3 0 ; GSTT1: Forward, 5 0 -TTCCTTACTGGTCCTCACATCTC-3 0 ; Reverse, 5 0 -TCACGGGATCATGGCCAG CA-3 0. Exon 7 of CYP1A1 genes were co-amplified and used as an internal control using following primers: Forward, 5 0 -GAACTGCCACTTCAGCTGTCT-3 0 and Reverse, 5 0 CAGCTGCATTTGGAAGTGCTC-3 0. Each set of reaction included both positive and negative controls. The multiplex PCR method was used to detect the presence or absence of the GSTT1 and GSTM1 genes in the genomic DNA samples, simultaneously in the same tube. The reaction mixture was subjected to initial denaturation at 94 8C for 2 min, followed by 35 cycles of 94 8C for 2 min, 59 8C for 1 min and 72 8C for 1 min. The final extension was done at 72 8C for 10 min. The PCR products were eletrophoresed in 2% agarose gels, and visualized by ethidium bromide staining. DNA from samples positive for GSTM1 and GSTT1 genotypes yielded bands of 215 bp and 480 bp respectively while the internal positive control (CYP1A1) PCR product corresponded to 312 bp. GSTP1A313G polymorphism was analyzed using a previously described PCR-RFLP method [20]. Briefly, amplification was conceded using primers: Forward, 5 0 -ACCCCAGGGCTCTATGGGAA-3 0 and Reverse, 5 0 -TGAGGGCACAAGAAGCCCCT Materials and methods 2.1. Patients The study group consisted of 106 bladder cancer patients (all were transitional cell carcinoma; out of them 60% were stage I tumors, 28% were stage II, 3% were stage III and 9% were stage IV tumors) with mean age and 370 normal healthy individuals (controls) with mean age of Ethnic origin of cases and controls were same. Criteria for the patient selection were based on a questionnaire covering medical, pathological, and histo-pathological records from the outpatient department of Sanjay Gandhi postgraduate Institute of Medical Science, Lucknow. The inclusion criteria for the controls were absence of prior history of cancer or pre-cancerous lesions. The 176-bp amplified product was digested with Alw261 and eletrophoresed on 3% Agarose gel. Presence of restriction site resulting in only two fragments (91 bp and 85 bp) indicated mutant allele (G/G), and if A/G polymorphism incurred then it resulted in three fragments of 176 bp, 91 bp and 85 bp Statistical analysis Statistical analysis was done with SPSS software 11.5 version. Binary Logistic Regression Model (BLRM) assessed difference in genotype prevalence and association between case and control group. Multivariate analysis, correlation coefficient, odds ratio (OR) and its 95% confidence interval (CI) were used to describe the strength of association. p-value <0.05 was considered as statistically significant for the study.

3 D.S.L. Srivastava et al. / European Urology 48 (2005) Table 1 Distribution of GST genotypes among bladder cancer patients and control Genotype Controls n = 370 Patients (Ca-B) n = 106 p-value OR (95% CI) GSTM1 Present 230 (62.2%) 63 (59.4%) 1.0 (Ref.) Null 140 (37.8%) 43 (40.6%) ( ) GSTT1 Present 291 (78.6%) 78 (73.6%) 1.0 (Ref.) Null 79 (21.4%) 28 (26.4%) ( ) GSTP1 Ile/Ile 191 (51.6%) 33 (31.1%) 1.0 (Ref.) Ile/Val 166 (44.9%) 58 (54.7%) ( ) Val/Val 13 (3.5%) 15 (14.2%) ( ) 3. Results Table 1: (see also Figs. 1 and 2) presents the frequencies of GSTM1, GSTT1 and GSTP1 alleles and genotypes by case-control status and the association of GSTs genotypes with bladder cancer risk. In the control samples, frequency of GSTM1 null and GSTT1 null was 36.9 and 20.7 respectively. In GSTP1 polymorphism A allele (51.6%) while G allele was 3.5%; homozygous and the remaining 44.9% were heterozygous. Genotype distributions in controls were in agreement with Hardy Weinberg Equilibrium. We observed non-significant association with null genotype of GSTM1 (OR = 1.12, 95% CI = , p = 0.611) and GSTT1 (OR = 1.45, 95% CI = , p = 0.135). However, the A/G genotype (OR = 1.99, 95% CI = , p = 0.005) and G/ G genotype (OR = 7.12, 95% CI = , p = 0.000) of the GSTP1 gene polymorphism were highly significant as compared to the controls (Table 1). Combination of the two high-risk genotypes GSTM1 null and GSTT1 null or G allele of GSTP1 genotype revealed that the risk increased up to 2.63 times (OR = 2.63, 95% CI: ; p = 0.003) for GSTP1and GSTM1 and 1.9 times (OR = 1.90, 95% CI: ; p = 0.059) for GSTM1 and GSTT1 genotype. However, risk increased more than 2.91 folds (OR = 2.91, 95% CI: ; p = 0.004) for GSTT1 and GSTP1 genotype when compared with non-risk genotypes (Table 2). We further investigated the risk associated with all the three high-risk GST genotypes compared to nonrisk genotypes (positive genotypes of GSTM1 and GSTT1 and 313 A/A genotype of GSTP1 were designated as the reference group). The OR for the three high-risk genotypes verses non-risk genotypes was 7.29 folds higher (p = 0.000, OR = % CI = ) (Table 3, Figs. 1 and 2). The clinical stage of tumor was categorized into four groups: T1 (Ta + T1), T2, T3 and T4.We observed nonsignificant association (P > 0.05) for the GSTs genotypes with stage of bladder tumor (Fig. 3). Table 2 Distribution of doubles GST genotypes among bladder cancer patients and controls Genotypes Controls n = 370 Patients (Ca-B) n = 106 p-value OR (95% CI) GSTM1and GSTT1 Both Present 182 (49.2%) 51 (48.1%) 1.09 (Ref.) Either Null 156 (42.2%) 38 (35.9%) ( ) Both Null 32 (8.6%) 17 (16.0%) ( ) GSTM1 and GSTP1 M1 (+/+)&P1 (Ile/Ile) 123 (33.2%) 20 (18.8%) 1.0 (Ref.) M1 (+/+)&P1 (Ile/Val/or Val/Val) 109 (29.5%) 43 (40.6%) ( ) M1 ( / )&P1 (Ile/Ile) 68 (18.4%) 13 (12.3%) ( ) M1 ( / )&P1 (Ile/Val/or Val/Val) 70 (18.9%) 30 (28.3%) ( ) GSTT1 and GSTP1 T1 (+/+)&P1 (Ile/Ile) 151 (40.7%) 21 (19.8%) 1.0 (Ref.) T1 (+/+)&P1 (Ile/Val/or Val/Val) 139 (37.6%) 56 (52.8%) ( ) T1 ( / )&P1 (Ile/Ile) 38 (10.3%) 12 (11.3%) ( ) T1 ( / )&P1 (Ile/Val/or Val/Val) 42 (11.4%) 17 (16.1%) ( )

4 342 D.S.L. Srivastava et al. / European Urology 48 (2005) Table 3 Distribution of triple GST genotypes among bladder cancer patients and controls Genotypes (GSTM1, GSTT1 and GSTP1) Controls n = 370 Patients (Ca-B) n = 106 p-value OR (95% CI) M1& T1 (+/+)&P1 (Ile/Ile) 101 (27.3%) 12 (11.3%) 1.0 M1& T1 (+/+)&P1 (Ile/Val/or Val/Val) 82 (22.2%) 40 (37.7%) ( ) M1 ( / ), T1 (+/+)&P1 (Ile/Ile) 52 (14.1%) 9 (8.5%) ( ) M1 ( / ), T1 (+/+)&P1 (Ile/Val/or Val/Val) 56 (15.1%) 16 (15.1%) ( ) M1 (+/+), T1 ( / )&P1 (Ile/Ile) 21 (5.7%) 8 (7.5%) ( ) M1 (+/+), T1 ( / )&P1 (Ile/Val/or Val/Val) 26 (7.0%) 4 (3.8%) ( ) M1 ( / ), T1 ( / )&P1 (Ile/Ile) 17 (4.6%) 4 (3.8%) ( ) M1 ( / ), T1 ( / ) &P1 (Ile/Val/or Val/Val) 15 (4.5%) 13 (12.3%) ( ) Fig. 4. G1 = grade1, G2 = grade 2 and G3 = grade 3 of bladder tumor. Fig. 1. Lane M: 100 bp ladder; lane 1: negative controls, lane 7 depicts M1 and T1 null alleles; lane 2, 3, 9 signify M1 and T1 positive, lane 5 is M1 positive and lanes 4, 6, 8 are T1 positive. We also classified pathological findings of grade into three groups: well differentiated (Grade1), moderately differentiated (Grade2) and poorly differentiated (Grade3). Correlations of GSTs genotypes with tumor grade of the disease were also insignificant for the risk of bladder cancer (Fig. 4). 4. Discussion Fig. 2. Lane M: 100 bp ladder; lane 1, 3 wild type (Ile/Ile); lanes 2, 4, 5, 6 heterozygous (Ile/Val) and lane 7 indicate the mutant type (Val/Val) genotypes. Fig. 3. S1 = stage 1 (Ta and T1), S2 = stage2 and S3 S4 = stage 3 and stage 4 of bladder tumor. We observed an increased risk for bladder cancer associated with the GSTP1 A/G or G/G polymorphism. Our observation is in accord with findings in Turkish [6] and British population [20], where they observed significant increased risk of bladder cancer for GSTP1 A/G or G/G polymorphism. However, Steinhoff et al. [21] in German and Katoh et al. [22] in Japanese population observed no association for GSTP1 A/G or G/G genotype with susceptibility to bladder cancer. The present study indicates that polymorphic GSTP1 locus may be an important factor for genetic susceptibility to bladder cancer. Mechanism behind this association suggests that person having G allele of GSTP1 undergo lower activity towards detoxification of carcinogenic compounds due to inactive form of enzymes. In the present study, a tendency of increased risk was observed with GSTT1 null genotypes, although this difference was not statistically significant for bladder cancer when compared with the controls. Our findings

5 D.S.L. Srivastava et al. / European Urology 48 (2005) concur with previous studies [21,23]; however, in studies conducted in Slovakian and Egyptian [14,19] population, statistically significant risk of bladder cancer for GSTT1 null genotype was observed. Since GSTT1 is stalwartly involved in the metabolism of lower molecular weight electrophilic molecules [24], the decreased frequency of a genotype encoding active GSTT1 might impinge on role of carcinogenesis in bladder cancer. A non-significant risk was also observed for the GSTM1 null genotype. Our results were comparable to the observations made by Kim et al. in Korean population [17]. However, several studies reported significant association of GSTM1 in Bladder cancer. [14,21]. We also tried to evaluate an association by taking two risk genotypes (GSTM1 null and GSTT1 null or GSTP1313 A/G genotype) simultaneously (Table 2). When GSTT1 null and GSTP1313 A/G genotype were present then risk increased by 2.91 folds in comparison to non-risk genotype. This is suggestive of a synergistic function between GSTT1 and GSTP1313 A/G genotype in the etiology of bladder cancer. Interestingly, when we combined the three-risk genotypes then risk increased more than seven folds. Combined analyses of GSTM1/GSTT1 and GSTP1 gene loci and the significant association have been reported by Toruner et al. in Turkish population [6]. The individuals possessing null allele of GSTM1 and GSTT1 and Val allele of GSTP1 have higher risk of bladder cancer and more so in tobacco users due to reduced detoxification of carcinogens (6, 21, and 22). The present study indicated escalation of risk with multiple risk alleles of GSTs, and suggests that gene-gene interaction may contribute towards causative propensity for development of bladder cancer in north Indian population. We also attempted to correlate clinical stage and/or pathological grade with GSTs genotypes for the risk of bladder cancer, but no association was observed (Figs. 3 and 4). Our findings concur with previous studies that also showed non-significant association of GSTM1/GSTT1 genotypes with grade/or stage of the disease [16]. Bladder cancer is a malignancy in which, in addition to genetic status of the individuals, gene environment interactions are thought to play an important role. Interindividual differences in cancer susceptibility may be mediated in part through polymorphic variability in the bio-activation and detoxification of carcinogens. Glutathione S-transferases are active in the detoxification of wide variety of potentially toxic and carcinogenic electrophiles by conjugating with glutathione, and so their role is mainly detoxification. Moreover GSTs also modulate the induction of other enzymes and proteins important for cellular functions such as DNA repair. Hence, GSTs are important for maintaining cellular genomic integrity and as a result play an important role in cancer susceptibility [24]. Besides this they are also involved in the deactivation of oxidative metabolites of exogenous or endogenous carcinogenic agents (industrial chemicals, dietary compound, tobacco products, drugs and environmental carcinogens etc.) that are probably associated with bladder cancer risk [3,8,25]. Due to presence of inactive form of enzymes (null genotypes of GSTM1 or GSTT1 and G allele of GSTP1) detoxification of activated carcinogen is reduced, leading to progression of cancer. To the best of our knowledge, this is perhaps, the first genetic study on the association of GSTs with bladder cancer in the north Indian population. In conclusion, our study demonstrated that GSTP1313 G/G polymorphism is strong predisposing risk factor for bladder cancer. However, combination of three GST genotypes association demonstrated gene-gene interaction that could further contribute to the increased risk of bladder cancer. Acknowledgements One of the authors (D.S.L.Srivastava) is thankful to Council of Scientific and Industrial Research, New Delhi, for awarding senior research fellowship. References [1] Perera FP. Environment and cancer: who are susceptible? Science 1997;278: [2] Weber WW. Human drug metabolizing enzyme variants. In: Roberts Frazare JA, Carter CO, editors. Pharmacogenetics. London: Oxford University Press; p [3] Silverman DT, Rothman N, Devesa SS. Epidemiology of bladder cancer. In: Syrigos KN, Skinner DG, editors. Bladder cancer. Biology, Diagnosis, Management. New York: Oxford University Press Inc; p [4] Mannervik B. The isozymes of glutathione S-transferases. Adv Enzymol 1985;57: [5] Ali-Osman F, Akande N, Mao J. Molecular cloning, characterization, and expression in Escherichia coli of full-length cdna of three human glutathione S-transferase pi gene variants. Evidence for differential

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