Common Polymorphisms in GSTA1, GSTM1 and GSTT1 Are Associated with Susceptibility to Urinary Bladder Cancer in Individuals from Balkan Endemic Nephropathy Areas of Serbia

Abstract

Balkan endemic nephropathy (BEN) is a chronic familial form of interstitial nephritis that might eventually lead to end stage renal disease. This nephropathy affects individuals living along of the Danube River and its tributaries in Serbia, Bosnia, Croatia, Bulgaria and Romania. The increased incidence of urinary tract tumors in the BEN areas is well described, but its specific genetic predisposition is still unclear. Certain nephrocarcinogenic compounds, including those associated with BEN, are metabolized by glutathione S-transferase (GST) superfamily of phase II detoxication enzymes. Importantly, the GST-mediated detoxification may result in formation of more toxic compounds. We examined the association of common GST polymorphisms and bladder cancer (BC) risk in individuals from BEN areas in Serbia. A hospital-based case-control study included 201 BC cases (67 from BEN region) and 122 controls. Each polymorphism was identified by a PCR-based method. Individuals from BEN region with low-expression GSTA1 genotype (AB+BB) exhibited a 2.6-fold higher BC risk compared to those with GSTA1 (AA) genotype who were from non-BEN region (OR = 2.60, p = 0.015). In contrast, carriers of GSTM1-active genotype from BEN region had a 2.9-fold increased BC risk compared to those with GSTM1-active genotype from non-BEN region (OR = 2.90, p = 0.010). Likewise, carriers with GSTT1-active genotype from BEN region exhibited 2.1-fold higher BC risk compared to those from non-BEN region with GSTT1-active genotype (OR = 2.10, p = 0.027). Thus, common polymorphisms in GSTA1, GSTM1 and GSTT1 are associated with susceptibility to BC in individuals from BEN areas of Serbia.

Introduction

Balkan endemic nephropathy (BEN) is a chronic familial form of interstitial nephritis that might eventually lead to end stage renal disease (Stefanovic and Cosyns 2004). This nephropathy represents an endemic disease affecting individuals living along of the Danube River and its tributaries in Serbia, Bosnia, Croatia, Bulgaria and Romania (Jelakovic et al. 2014). Ever since the 1950s, when the disease was first described, its geographic distribution has remained the same. The most important epidemiologic characteristics of BEN are its focal appearance in certain villages and small towns in endemic region, in residents who have lived there for at least 15 years, as well as, familial character of the disease, although it is not inheritable (Stefanovic and Cosyns 2004). Moreover, BEN is associated with increased incidence of urinary tract transitional cell carcinoma (TCC), which is localized, either in renal pelvis and ureter, or in renal pelvis and urinary bladder (Stefanovic and Cosyns 2004).

Despite extensive research conducted over the past 60 years, the etiology of BEN still remains obscure and it seems that a polygenic susceptibility to the disease, in interaction with multiple environmental factors, exists (Toncheva et al. 1998; Batuman 2006). Although European Commission has adopted aristolochic acid as a cause of BEN (Cosyns 2003), still there is much evidence in favor of other environmental toxins, such as fungal mycotoxin ochratoxin A (OTA) (Castegnaro et al. 2006). However, it is still unclear whether there is a specific genetic predisposition leading to a highly increased TCC risk in patients with BEN.

Certain nephrocarcinogenic compounds, including those associated with BEN, such as OTA, are metabolized by cytosolic glutathione S-transferase (GST) family of enzymes (Tozlovanu et al. 2012; Reljic et al. 2014). Cytosolic GSTs belong to phase II detoxifying enzymes that protect normal cells by catalyzing conjugation of electrophylic compounds of exogenous and endogenous origin with glutathione (Hayes et al. 2005). They are also an important line of defense in the cell protection from oxidative damage (Hayes et al. 2005). Based on their primary structures, cytosolic GSTs have been divided into several classes, including GSTs classes alpha (GSTA), mu (GSTM), pi (GSTP) and theta (GSTT). Common polymorphisms occur in almost all members of GSTs and several types of allelic variations have been observed. Within GSTM1 and GSTT1 class deletion polymorphism (GSTM1-null and GSTT1-null) have been described. Thus, genotypes, referred as GSTM1-null and GSTT1-null, are homozygous for the GSTM1*0 and GSTT1*0 alleles. Individuals with these genotypes produce no GSTM1 and GSTT1 protein and consequently have complete lack of GSTM1 and GSTT1 enzymatic activity. It has been reported that approximately 50% and 20% of Caucasian population possess GSTM1-null and GSTT1-null genotype, respectively (Landi 2000; Eaton and Bammler 1999). GSTP1 enzyme activity may be altered due to single-nucleotide polymorphism (SNP), A to G transition, which results in substitution of amino acid isoleucine (Ile) with valine (Val) (Watson et al. 1998). The relative frequencies of GSTP1 Ile/Ile (A/A), Ile/Val (A/G) and Val/Val (G/G) genotype are 50%, 40% and 10% in white population, respectively (Watson et al. 1998). There are three linked SNPs (G-52A, C-69T and T-567G) in proximal promoter region of GSTA1 gene (Coles and Kadlubar 2005). This results in two GSTA1 promoter variants which are characterized by two alleles GSTA1*A (-52G, -69C, -567T) and GSTA1*B (-52A, -69T, -567G), with differential hepatic expression (Coles and Kadlubar 2005). Namely, the nucleotide change at position -52 prevents Sp1 transcription factor binding, leading to 4-5 fold lower transcriptional activation of variant GSTA1*B allele (Morel et al. 2002). In Caucasians the distribution of GSTA1AA, AB and BB genotypes is 38%, 48% and 14%, respectively (Coles and Kadlubar 2005).

It has been shown that polymorphic expression of GSTs is associated with the altered xenobiotics detoxification efficiency, thus influencing an individual’s susceptibility to carcinogens and various diseases (Di Pietro et al. 2010; Suvakov et al. 2013; Eslami and Sahebkar 2014; Kim et al. 2015). Still, there are no data regarding the association between GST genetic polymorphism and bladder cancer risk, neither in patients with BEN, nor in individuals who originate from BEN regions. There is evidence on the role of GSTM1 and GSTT1 genetic polymorphisms in the sensitivity of uroepithelial cells to genotoxic OTA effects in vitro (Lebrun et al. 2006). Furthermore, there are some data indicating that GSTA1 may catalyze the formation of OTA conjugates (Reljic et al. 2014).

Given the fact that GSTs are included in the metabolism of potential cause of BEN, it is very important to investigate to what extent the presence of GST genetic variations contributes to bladder cancer susceptibility in individuals originating from BEN region. Hence, we examined the associations between polymorphisms within the key GST genes and bladder cancer risk in a Serbian group of bladder cancer patients from BEN.

Materials and Methods

Study subjects

This case-control study included 201 histological confirmed TCC patients, among which 67 of them were from BEN region. A total of 122 individuals with nephrolithiasis (26 from BEN region) and no previous record of any malignant disease represented the control group. Both patients and controls were recruited from the Clinic of Urology and Nephrology, Clinical Centre of Serbia, Belgrade. All participants gave informed consent and were able to leave the study by wish. The ethical approval for study protocol was obtained by local Committee of Medical Faculty and was in compliance with the Helsinki Declaration.

Well-trained interviewers collected demographic characteristics, cigarette smoking and occupational exposure data from each participant by using a standard questionnaire, based on which two different exposure categories were defined: unexposed and exposed. The life-time occupational exposure history included all jobs (both full and part-time jobs) in period longer than 6 months. Job description consisted of the job title, the industry or type of business, employment dates and duration, company name and location.

DNA extraction and genotyping

DNA was isolated from whole blood using commercial extraction kit (Qiagen, Inc., Chatsworth, CA, U.S.A.). GSTM1 and GSTT1 polymorphism were identified by PCR, while GSTA1 and GSTP1 were identified by restriction fragment length polymorphism PCR (RFLP-PCR) method as previously described (Suvakov et al. 2013).

Statistical analysis

Hardy-Weinberg equilibrium was tested using χ² test. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) in order to assess the associations of GST genotypes and BEN region with bladder cancer risk. All calculations were adjusted to age, gender and smoking status as potential confounders.

Results

Demographic characteristics of TCC patients and controls were presented in Table 1. No statistical significance was obtained with respect to age, gender or educational level between patients and controls. The BEN region prevalence among patients was higher than in control group (33% and 21%, respectively). We found that individuals who were from BEN region had significantly higher TCC risk then individuals originating from non-BEN region (OR = 1.84, p = 0.022). Besides, the smoking prevalence among cases was higher (74%) than in controls (54%) and occupationally exposed individuals were more frequent among patients than controls (36% and 14%, respectively).

A significant association was observed between GSTA1, M1 and T1 polymorphisms, susceptibility to urinary bladder cancer and BEN region residence as presented in Table 2. Regarding GSTA1 polymorphism, the strongest association of GSTA1 genotype with the risk of urinary bladder cancer in individuals from BEN region residence was found in individuals with GSTA1/AB+BB genotype. These individuals, with lower GSTA1 expression, exhibited a 2.6 times higher bladder cancer risk compared to those with GSTA1/AA genotype who were from non-BEN region (OR = 2.60, p = 0.015, 95% CI = 1.20-5.50). Interestingly, similar combined effect for bladder cancer risk was observed for GSTM1 polymorphism in individuals from BEN region. As shown, individuals with GSTM1-active genotype from BEN region had a 2.9 times higher risk of developing bladder cancer compared to those with the same GSTM1 genotype from non-BEN region (OR = 2.90, p = 0.010, 95% CI = 1.30-6.60). In case of GSTT1 genotype the strongest effect for bladder cancer risk was found in carriers of GSTT1-active genotype from BEN region. They exhibited 2.1-fold higher bladder cancer risk in comparison with individuals from non-BEN region and GSTT1-active genotype (OR = 2.10, p = 0.027, 95% CI = 1.10-4.00). Analysis of GSTP1 polymorphism showed that carriers of GSTP1Ile/Val+ Val/Val genotype from BEN region had 1.9 times higher bladder cancer risk than individuals from non-BEN region with GSTP1Ile/Ile genotype. However, this association lacked statistical significance (OR = 1.90, p = 0.098, 95% CI = 0.90-4.20).

Table 1.

Selected characteristics of patients with bladder cancer and controls.

OR, odds ratio; CI, confidence interval; BEN, Balkan endemic nephropathy.

Table 2.

Combined effect of BEN region and GST genotype on risk of bladder cancer.

Adjusted for age, gender and smoking. ^aActive (present) if at least one active allele present. ^bInactive (null) if no active alleles present.

OR, odds ratio; CI, confidence interval; GSTA1, glutathione S-transferase A1; GSTM1, glutathione S-transferase M1; GSTP1, glutathione S-transferase P1; GSTT1, glutathione S-transferase T1.

Discussion

The results of this study showed that low-activity GSTA1/AB+BB, as well as, GSTM1 and GSTT1-active genotypes in combination with BEN residence contribute towards the risk of bladder cancer in Serbian TCC patients. Regarding GSTP1 polymorphism, no association was observed between susceptibility of bladder cancer development and BEN residence.

Recently, our group showed that individuals with one or both low-activity GSTA1*B alleles are at 1.6-fold higher risk of BEN than individuals carrying the wild type homozygous GSTA1*A/*A genotype (Reljic et al. 2014). The main result obtained from this study pointed out that TCC risk in individuals from BEN areas is modified by GSTA1 gene polymorphism. Specifically, individuals with at least one GSTA1*B allele who originated from BEN region had 2.6 times higher TCC risk compared to homozygous GSTA1*A/*A individuals who were not from this region. This is not surprising, since GSTA1 has overlapping specificity towards many different substrates (Honaker et al. 2013). Thus, GSTA1 enzyme is involved in both peroxidase and glutathione-conjugation reaction, due to which it is a key GST involved in detoxification of xenobiotics and antioxidant protection in liver. Homozygous carriers of GSTA1 variant allele are shown to have four- to five-fold lower expression of this enzyme in liver (Coles and Kadlubar 2005). Since extensive production of reactive oxygen species leading to oxidative stress has been implicated in tumor development, it may be suggested that liver, by its GSTA1 conjugating and peroxidase activity, plays an important role in protection against bladder carcinogens. Given a thought that GSTs catalyze some reactions of OTA metabolism, one of the possible causes of BEN, we hypothesized that GST polymorphisms might alter individual susceptibility to bladder cancer in individuals from BEN-region. Indeed, our previous results based on in silico simulation suggested that GSTA1 is involved in metabolizing OTA quinone derivatives (Reljic et al. 2014). As GSTA1 gene polymorphism results in lowering of enzyme activity, it is reasonable to assume that these individuals will be under both increased oxidative stress and risk of cancer development when exposed to higher concentration of OTA metabolites. However, to confirm this hypothesis, further studies conducted on larger study population are necessary.

Regarding GSTM1 polymorphism in patients with TCC originating from BEN region, our results demonstrated that individuals with GSTM1-active genotype are at 2.9-fold higher risk of bladder cancer compared to those from non-endemic areas. These results are in accordance with the study of Andonova et al. (2004), which reported that GSTM1-active genotype is more prevalent among BEN patients than in endemic control group. Similarly, Toncheva et al. (2004) have conducted another study in Bulgarian population on 95 BEN patients and 112 non-endemic controls and they have also found higher GSTM1-active genotype frequency in BEN patients compared to controls, but without statistical significance. Having in mind the role in detoxification, as well as, antioxidant role of GSTM1, our results are quite unexpected especially in the view of the fact that large meta-analysis unambiguously showed association between GSTM1-null genotype and TCC risk, independently or in association with smoking (Johns and Houlston 2000). One of the possible explanations for this result is that sometimes GST-mediated detoxification by glutathione conjugation may result in bioactivation and formation of even more toxic compounds (van Bladeren 2000). The data on potential role of GSTM1 in metabolism of OTA and aristolochic acid are scarce and inconsistent.

Interestingly, GSTM1 is not the only active form of GST associated with TCC risk in patients originating from BEN region. Our results also shown that BEN-area residents carriers of GSTT1-active genotype have two times higher risk to develop TCC of urinary bladder in comparison to non-BEN-area residents with the same genotype. These findings may be explained by the well-known role of GSTT1 in bioactivation of various cancerogenic compounds, such as trichloroethylene (Moore et al. 2010). Indeed, our group has showed that GSTT1-active genotype is associated with TCC risk in men exposed to pesticides (Matic et al. 2014). Regarding the potential role of GSTT1 in metabolism of OTA and aristolochic acid, the results of our recent investigation did not show involvement of this enzyme in OTA metabolism (Reljic et al. 2014), while data on aristolochic-acid metabolism and GSTT1 are lacking. Still, it is noteworthy mentioning that our results on association of GSTT1 polymorphism between risk of TCC bladder cancer and residence in BEN-area are in line with results of Lebrun et al. (2006) who pointed out the role of GSTT1 polymorphism in the extent of DNA damage induced by OTA. Namely, when uroepithelial cells from different human donors were treated with OTA, in cells with GSTT1-active genotype the DNA damage was more extensive than in those with GSTT1-null genotype.

In conclusion, the results obtained from this study may improve our understanding of the potential role of GST polymorphism on TCC risk in BEN region residents. However, there are certain limitations that should be considered. The first one is the relatively small sample size that is in favor of creating potential biases. The second one is our hospital-based control group that may not reflect the general population characteristics. Although we adjusted all results by age, gender and smoking status, there are probably more confounding factors that might influence the results. Therefore, further investigations with larger sample size and more rigorous designs are needed for further elucidation of the role of GST polymorphisms in relation to BEN and bladder cancer risk.

Acknowledgments

This study was supported by a Grant 175052 from Serbian Ministry of Education and Science.

Conflict of Interest

We declare no conflict of interest.

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