Functional RsaI/PstI Polymorphism in Cytochrome P450 2E1 Contributes to Bladder Cancer Susceptibility: Evidence from a Meta-analysis

Bladder cancer was the ninth most common malignancy and the thirteenth most common cancer-related cause of mortality in the world, which was a complex disorder with both environmental and genetic influences (Parkin, 2008). The major environmental factors included tobacco smoking and occupational exposures (Clapp et al., 2008; Strope and Montie, 2008), which could cause DNA damage, such as cross-links, bulky adducts and single or double strand breaks resulting in unregulated cell growth and even cancer (Johansson et al., 1990; Hoeijmakers, 2001; Yue et al., 2009). Nevertheless, only a small proportion of the individuals exposed to these environmental factors eventually developed bladder cancer, indicating that host genetic factors may play an important role in bladder carcinogenesis (Taioli and Raimondi, 2005). A few gene polymorphisms associated with bladder cancer risk have been identified. Metabolizing enzymes were involved in the bioactivation and detoxification of xenobiotics,


Introduction
Bladder cancer was the ninth most common malignancy and the thirteenth most common cancer-related cause of mortality in the world, which was a complex disorder with both environmental and genetic influences (Parkin, 2008). The major environmental factors included tobacco smoking and occupational exposures (Clapp et al., 2008;Strope and Montie, 2008), which could cause DNA damage, such as cross-links, bulky adducts and single or double strand breaks resulting in unregulated cell growth and even cancer (Johansson et al., 1990;Hoeijmakers, 2001;Yue et al., 2009). Nevertheless, only a small proportion of the individuals exposed to these environmental factors eventually developed bladder cancer, indicating that host genetic factors may play an important role in bladder carcinogenesis (Taioli and Raimondi, 2005). A few gene polymorphisms associated with bladder cancer risk have been identified. Metabolizing enzymes were involved in the bioactivation and detoxification of xenobiotics, particularly the cytochrome P450 2E1 (CYP2E1), and its polymorphisms might be associated with bladder cancer risk (Gonzalez, 2005).
The CYP2E1 gene, located on chromosome 10q26.3, is a member of the CYP450 superfamily, is constitutively expressed in various organs and tissues including urothelial cells (Sheweita et al., 2001). It is a key ethanol-inducible enzyme in the metabolic activation of many low-molecular-weight carcinogens, such as vinyl chloride, benzene, and tobacco-specific nitrosamines (Guengerich et al., 1991;Yamazaki et al., 1992). Of the many known CYP2E1 genetic polymorphisms, RsaI/PstI polymorphism in the 5'-flanking region were in close linkage disequilibrium and affected the transcriptional activation of the gene (Hayashi et al., 1991). The wild allele (C1) and/or the less mutant allele (C2) of CYP2E1 RsaI/PstI polymorphism have been reported as conferring higher risk for developing liver, esophageal and lung cancer by meta-analysis (Wang et al., 2009;Leng et al., 2012;Tian et al., 2012). Therefore, the CYP2E1 RsaI/PstI polymorphism was believed to be risk factors for bladder cancer.
The associations of the CYP2E1 RsaI/PstI polymorphism and bladder cancer susceptibility have been extensively studied (Brockmoller et al., 1996;Choi et al., 2003;Mittal et al., 2005;Shao et al., 2008;Cantor et al., 2010;Basma et al., 2013). However, these studies yielded contradictory results, some studies showing significant association (Choi et al., 2003;Cantor et al., 2010;Basma et al., 2013), while others did not show such association, even in the same population (Brockmoller et al., 1996;Mittal et al., 2005;Shao et al., 2008). The inconsistency results might be resulted from a single study and the relatively small sample size, which had lower statistical power to detect the overall effects. Therefore, a quantitative synthesis of the combined data from different studies was necessary to estimate the association between CYP2E1 RsaI/PstI polymorphism and bladder cancer risk. In our study, we performed a systematic review and meta-analysis of the currently available literatures of the literature to clarify the accurate relationship between CYP2E1 RsaI/PstI polymorphism and bladder cancer risk.

Identification and eligibility of relevant studies
We conducted a comprehensive search in the PubMed, Medline, Embase, and Web of Science databases for all literatures about the association between CYP2E1 RsaI/ PstI polymorphism and bladder cancer (updated on Feb, 2014). Search term combinations were as follows: (Cytochrome P4502E1 or CYP2E1), (polymorphisms or SNPs or mutation or variant or variation) and (bladder cancer or bladder neoplasm or bladder tumor). All reference lists from the main literatures and relevant reviews were hand searched for additional eligible studies. Only those studies assessing the association between the CYP2E1 RsaI/PstI polymorphism and bladder cancer risk were included in this meta-analysis: (1) Case-control studies (retrospective or nested case-control); (2) Only English language articles reporting human studies were considered; (3) Studies with available data for estimating odds ratios (ORs) and the 95% confidence interval (CI); (4) For duplicated publications, only the study with the largest sample numbers was included; (5) We did not define a minimum number of cases or controls in the meta-analysis.

Data extraction
Information was independently extracted from all eligible publications by two investigators (Deng XD and Qin Gao) according to the inclusion criteria. The original extraction data were checked by Ma Y, and in case of disagreement, an agreement was reached after a discussion. For each of the eligible case-control studies, the following data were recorded: first author's last name, year of publication, ethnicity, country, number of cases and controls, number of different genotypes in cases and controls, Hardy-Weinberg equilibrium (HWE), genotyping methods, matching criteria. The main data of eligible studies are presented in Table 1. Different ethnicity descents were categorized as Asian, Caucasian, and African.

Statistical analysis
The HWE was assessed by Fisher's exact test and P value less than 0.05 was considered significant. Summary ORs with corresponding 95%CIs were used to evaluate the strength of association between CYP2E1 RsaI/PstI polymorphism and bladder cancer risk under different genetic models, including heterozygote (C1C2 vs C1C1), homozygote (C2C2 vs C1C1), dominant (C2C2/C1C2 vs C1C1), and recessive (C2C2 vs C1C1/ C1C2) genetic model. The Q test and I 2 statistics were evaluated to test statistical heterogeneity among studies (Higgins and Thompson, 2002). Pooled ORs estimation of each study was calculated by the fixed effects model (Mantel and Haenszel, 1959) or the random effects model (DerSimonian and Laird, 1986) according to the heterogeneity. The fixed-effects model was adopted when the studies were found to be homogeneous (P Q >0.1 and I 2 <50%). Otherwise, the random-effects model was applied. Subgroup analysis was conducted by ethnicity.  Sensitivity analysis was carried out to assess the stability of the results. Publication bias among the literatures was assessed by Begg's funnel plot and Egger's regression asymmetry test. All statistical tests were performed by Stata software, version 11.0 (STATA Corp, College Station, TX).

Main results of meta-analysis
The main results of the meta-analysis and heterogeneity test are listed in  (Figure 2), but not in Asians.
All I 2 values of heterogeneity were less than 50% and P Q values were greater than 0.10 in overall, unfortunately, significant heterogeneity with subgroup analysis were found in Caucasians under dominant genetic models (dominant: I 2 =57.6%, P Q =0.094, P OR =0.077, OR=0.598, 95%CI=0.338-1.058) ( Table 2). Due to only 3 studies in Caucasians, we failed to explore the sources of heterogeneity.

Sensitivity analysis and Publication bias
Sensitivity analysis was carried out by sequential omission of individual studies. The significance of   summary ORs was not influenced excessively by omitting any single study under different genetic models, indicating that currently meta-analysis results were statistically reliable. The shape of funnel plots did not show any evidence of obvious asymmetry under all genetic model (Figure 3), and the result s of Egger's test did not reveal any evidence of publication bias (P>0.361).

Discussion
Bladder cancer is increasing common cancer which is likely to be caused by multi-factors, including environmental, genetic and their interactions factors (Parkin, 2008). Only environmental factor cannot explain the phenomenon completely, and then genetic factors may play an important role. In recent years, genetic susceptibility was used to evaluate the risk of bladder cancer, but the results were inconsistent. In our study, we conducted a systematic review and meta-analysis to confirm the accurate relationship between the CYP2E1 RsaI/PstI polymorphism and bladder cancer risk.
In current meta-analysis, we found that the CYP2E1 RsaI/PstI polymorphism were significantly associated with bladder cancer susceptibility including 6 casecontrol studies, especially in Caucasians (Table 2). It was indicated that the C2 carrier genotypes of CYP2E1 RsaI/ PstI polymorphism might be a protective factor which decreased the risk of bladder cancer. The bladder was prone to expose under carcinogens which were known to induce DNA strand breaks in the bladder epithelium cell due to being the urine collecting area (Johansson et al., 1990;Hoeijmakers, 2001;Yue et al., 2009). The CYP2E1 played an important role in the metabolic activation of low molecular weight compounds and pro-carcinogens such as benzene, N-nitrosamines, and halogenated hydrocarbons, which might be involved in bladder cancer development (Guengerich et al., 1991;Yamazaki et al., 1992). The population and molecular biological studies indicated that the C2 allele or the C2 carrier genotypes of the CYP2E1 RsaI/PstI polymorphism had a lower ethanol-induced enzyme activity and basal CYP2E1 activity because the CYP2E1 PstI and RsaI restriction sites located in the transcription-regulation region might affect transcriptional activity, and decrease/lose the inducibility to pro-carcinogen (Uematsu et al., 1991;Lucas et al., 1995;Carriere et al., 1996;Kim et al., 1996). Although studies showed that the C2/C2 genotype produced higher enzyme activity than the Cl/Cl genotype in vitro (Hayashi et al., 1991;Ladero et al., 1996). However, this finding could not be verified in several in vivo and in vitro phenotyping studies (Kim and O'Shea, 1995;Lucas et al., 1995;Carriere et al., 1996;Kim et al., 1996). Additionally, a number of studies have suggested that individuals with C2 allele have lower risk in developing cancers of the lung, liver, and esophagus (Persson et al., 1993;Yu et al., 1995;Le Marchand et al., 1998;Lin et al., 1998). Thus, in view of the role of CYP2E1 in the metabolic activation of pro-carcinogen and our results suggesting a protective effect of the C2/C2 genotype against bladder cancer, we consider that this genotype may result in poor CYP2E1 activity/inducibility toward bladder epithelial cells procarcinogens than the corresponding Cl/Cl genotype. Unfortunately, due to power limitations, ethnic difference and the fact that other contributors of CYP2E1 variability were not adjusted, other studies have not found such a relation (Hirvonen et al., 1993;London et al., 1996). Although the explanation for the discordancy is unknown, power limitations, ethnic difference and other contributors of sex, dietary, age and smoking, for example, may be provide a mechanistic explanation (Zgheib et al., 2010).
Subgroup analysis based on the ethnic showed that Caucasians with carried the C2 genotypes had a decreased risk of bladder cancer, but not in Asians. It was indicated that the genetic diversity and variants among different ethnicities or populations might contribute to cancer risk (Shahriary et al., 2012;Lakkakula et al., 2013). Although the underlying mechanisms were not clear, ethnic diversity might affect bladder cancer risk. The study showed that the C2 allele frequencies of Asians (~25-50%) were significantly higher than those of Caucasians (~5-10%) (Stephens et al., 1994). Of course, it might also exist in weak effect or some selection bias due to small sample size. Further investigations are needed to confirm the possible effects of CYP2E1 RsaI/PstI polymorphism on bladder cancer risk, such as gene-gene and gene-environment interaction from different genetic background and lifestyles, in which it may play a role.
Although this meta-analysis has been recognized as a more precise and systematic method to evaluate the effect of selected genetic polymorphisms on the risk of disease than single case-control study and cohort study (Munafo and Flint, 2004), some limitations should be acknowledged in this meta-analysis. Firstly, bladder cancer was considered to be a multi-factorial disease, interacted by environmental factors and many genetic factors. A major route of metabolism from the body for most drugs and chemical carcinogens is mainly constituted by drugmetabolizing enzymes (DMEs) with phase I oxidation enzymes and phase II enzymes system. Cytochrome P450 (CYP) is the most important phase I enzymes system, which is usually involved in the activation of carcinogens; and phase II enzymes, particularly N-acetyltransferase (NAT) and glutathione s-transferase (GSTs), which mostly detoxify the products to be excreted in the urine and possibly played an important role in cancer etiology (Steck and Hebert, 2009;Zgheib et al., 2010). Although studies suggested that CYP2E1 genetic polymorphisms have an impact on the incidence of cancer (Danko and Chaschin, 2005), epidemiological studies suggest that the NAT1, NAT2, GSTM1 and GSTP1 polymorphisms modify the risk of developing cancers of the urinary bladder Pandith et al., 2013;Zabost et al., 2013). Therefore, not only is CYP2E1 suspected to be involved with the development of bladder cancer, but also other DMEs genetic factors may be associated with bladder cancer. However, lacking the original data of gene-environment and gene-gene interactions limited a more precise analysis. Secondly, in the current metaanalysis, only six studies were collected, statistical power was limited to assess the effects well. Thus, the results should be interpreted with caution. Functional RsaI/PstI Polymorphism in Cytochrome P450 2E1 Contributes to Bladder Cancer Susceptibility In summary, the present meta-analysis suggested that CYP2E1 RsaI/PstI polymorphism might be associated with bladder cancer risk in Caucasians. However, further studies with larger sample sizes and well-designed randomized studies in various ethnicities are needed to verify this association comprehensively.