Abstract
Tobacco smoking and environmental exposures are the main known risk factors for bladder cancer (BC) via exposure to chemical carcinogens. Genetic differences in the metabolism of chemicals have been suggested to be associated with individual susceptibility to BC. Polymorphisms in genes coding to metabolising enzymes, resulting in variation of carcinogen detoxification efficiency, may therefore change the response of individuals to chemical carcinogens and be associated with an increased BC risk. Patients and Methods: The aim of the study was to investigate the association between functional polymorphisms in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 genes and BC risk, through a hospital-based case-control study. The genotyping of 11 Single Nucleotide Polymorphisms (SNPs) was carried out on DNA of 51 bladder cancer male patients and 45 male controls. The technique of MGB (Minor Groove Binder) probes that utilize allelic discrimination with the Taqman® method was used. Results: Individuals with NAT2 slow acetylator genotypes had a significant increase in risk of BC compared to individuals with NAT2 rapid acetylators (OR adjusted for smoking status=2.70; 95% CI, 1.10-6.61). GSTP1 Ile105Val variants (deletion of one - Ile/Val- and two -Val/Val-, null genotype- copies) showed a marginal increased risk of BC with OR adjusted for smoking status of 2.27 (95% CI, 0.97-5.31) compared to individuals carrying wild-type genotype (Ile/Ile). No statistically significant effects on BC risk with CYP1A1, CYP11B1 and COMT genotypes were observed. Conclusion: The results are consistent with previous literature among Caucasian populations.
- Bladder cancer
- susceptibility
- metabolising gene
- CYP1A1
- CYP1B1
- COMT
- GSTP1
- NAT2
- genetic polymorphism
- single nucleotide polymorphism
Bladder cancer (BC) is one of the most common types of neoplasm in men in Western Europe (1). Cigarette smoke, containing several potent chemical carcinogens, is the predominant risk factor. Occupational or environmental exposure to carcinogens, particularly to some aromatic amines and to some polycyclic aromatic hydrocarbons may also be a risk factor (2, 3). Even if prevalence of BC in populations exposed to these risk factors is higher than that observed in non-exposed populations, not all exposed individuals develop BC. This leads one to speculate that factors other than exposure to chemical carcinogens such as genetic susceptibility may play a part in the occurrence of BC.
Interindividual variations in the functional activity of several metabolizing enzymes involved in the metabolism of chemicals could be an influence. It is hypothesized that the genetic variations in the genes coding to metabolising enzymes might contribute to these inter-individual variations, and thus to the individual susceptibility to BC (4).
Indeed, in humans, polymorphisms exist with regard to genes encoding for metabolizing enzymes, leading to variations in the enzyme activities. Thus, depending on gene polymorphisms, an enzyme may contribute to activation of chemicals into reactive metabolites for an individual, and then to carcinogenicity of these chemicals. In contrast gene polymorphisms may contribute to detoxification of the same chemicals for another individual, and then to protection against carcinogenic effects of these chemicals.
Previous epidemiological studies investigated the relationship between genetic polymorphisms of genes involved in xenobiotic metabolism and risk of BC, but to the best of the author's knowledge, no studies on this topic have been published on a French population.
Primers and probes used in the characterization of polymorphic sites in the studied genes.
Therefore, a hospital-based case control study on French Caucasian men was performed and 11 single nucleotide polymorphisms (SNP) were analyzed to investigate the association between polymorphisms of 5 enzymes involved in different xenobiotic metabolism pathways: Cytochrome P450 1A1 (CYP1A1), Cytochrome P450 1B1 (CYP1B1), Catechol-O-Methyl Transferase (COMT), Glutathione-Sulfo-Transferase P1 (GSTP1) and N-acetyltransferase 2 (NAT2), with BC risk.
Patients and Methods
Study subjects. The cases were newly diagnosed BC patients in the urology department of the University Hospital of Clermont-Ferrand with histological confirmation of the disease.
The controls were patients, having no history of cancer, admitted to the same urology department, during the same period of time, with a urological non-neoplasic disease: prostatic adenoma. The cases and controls were all Caucasian males. The study was approved by the local Ethical Committee. All study subjects received counselling and provided written consent for the study.
Blood collection, DNA extraction and genotyping. Blood samples from patients and controls were collected on FTA® cards (Whatman, France) which can immediately lyse cells and release genomic DNA within fibrous matrix. The cards were then archived. The elution of genomic DNA was performed at room temperature according to Whatman FTA procedure.
The following SNPs (Single Nucleotide Polymorphisms) were analyzed: CYP1A1 (Ex 7, 462Ile>Val, rs 1048943) CYP1B1 (Ex 3, 432Val>Leu, rs 1056836), COMT (158Val>Met, rs 4680), GSTP1 (Ex 5, 105Ile>Val, rs 1695) genes. In NAT2 gene the following SNPs relevant to Caucasians: T341C (rs 1801280), C481T (rs 1799929), A803G (rs 1208), C282T (rs 1041983), G590A (rs 1799930) and G857A (rs 1799931) were genotyped to determine NAT2*4, NAT2*4G, NAT2*5A, NAT2*5B, NAT2*5D, NAT2*5G, NAT2*6A, NAT2*6B, NAT2*6C, NAT2*12B and NAT2*13 alleles.
The corresponding probes were obtained from Applied Biosystems, FOSTER City, CA, USA. Sixteen ng of DNA were amplified by AmpliTaq Gold DNA polymerase included in Taqman Universal Master Mix (Applied Biosystems). The detection of alleles was based on fluorogenic Taqman MGB probes using the ABI PRISM 7700 Sequence Detection Systems (Applied Biosystems). Allele specific Taqman MGB probes were synthesised by Applied Biosystems, Chekshire, UK. Sequences of primers and probes are described in Table I. The PCR reaction consisted of one step of 10 minutes at 95°C followed by 40 cycles of two-step PCR with denaturation at 92°C for 15 s and annealing and extention at 60°C for 1 min. Ten percents of all samples were genotyped again for quality control.
Statistical analysis. For CYP1A1, CYP1B1, COMT and GSTP1 genes, individuals were classified according to their genotypes. Moreover, for analysis purposes, individuals with heterozygous variants were combined with individuals with homozygous variants.
For consistency with previous studies, individuals were dichotomized as slow NAT2 acetylators and rapid acetylors. Individuals were classified as slow NAT2 acetylator phenotype if they were homozygous for any combination of NAT2*4G, NAT2*5A, NAT2*5B, NAT2*5D, NAT2*5G, NAT2*6A, NAT2*6B, NAT2*6C alleles (5). The rest of the individuals were classified as rapid acetylators for NAT2.
The effects of genetic polymorphisms on the risk of BC were estimated with odds ratio (OR) and its 95% confidence interval (95% CI), using logistic regression models, adjusting for smoking status. Statistical analyses were performed using the software SEM (Centre Jean Perrin, Clermont-Ferrand, France) (6).
Polymorphisms studied in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 genes.
Results
The study population included 51 BC cases and 45 controls and all were genotyped for genes of interest.
The mean ages were not statistically different between cases and controls (67.6±11.4 and 67.8±11 years, respectively). As expected, the prevalence of smokers or previous smokers was significantly higher in the case group than in the control group (48/51 in cases, 32/45 in controls).
Table II presents the frequencies of studied genotypes by case-control status and the effect of studied genotypes on BC risk.
Compared to individuals with NAT2 rapid acetylators, individuals with NAT2 slow acetylators genotypes had a significant increased risk of BC (OR=2.70; 95% CI, 1.10-6.61) (Table III).
GSTP1 Ile105Val variants (deletion of one - Ile/Val- and two -Val/Val, null genotype-copies) showed a marginal increase in risk of BC with an OR of 2.27 (95% CI, 0.97-5.31), compared to individuals carrying wild-type genotype (Ile/Ile), used as reference category (Table III).
Finally, no statistically significant effect was observed by CYP1A1, CYP1B1 and COMT genotypes on BC risk (Table III).
Discussion
Although this study was limited due to the sample size, a significant increased BC risk of cases homozygous for NAT2 slow acetylator alleles, classified as slow-acetylator phenotype, was found, with an OR of 2.7 (95% CI=1,10-6,61). The NAT2 gene, encoding NAT2 enzyme, one of the major phase II enzymes, is subject to extensive polymorphisms (7). The lack of two functional NAT2 alleles leads to a slow acetylation phenotype that compromises NAT2 detoxification ability and may modify individual susceptibility to the adverse effects of bladder carcinogens, such as aromatic amines (8).
Genotype and allele frequencies of CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 polymorphisms among cases and controls and their association with bladder cancer.
Several epidemiological studies have assessed the association between BC risk and NAT2 polymorphisms. Many of them have consistently observed an association between the slow NAT2 genotype and increased bladder cancer risk (9-11). The pooled analysis, restricted to Caucasians, conducted by Vineis et al., including studies conducted in European countries, generated an overall OR estimated of 1.4 [1.1-1.8] associated with the slow acetylator genotype in Caucasians (12). García-Closas et al. combined data from 22 case-control studies of Caucasians and reported a statistically significant OR for BC in NAT2 slow acetylators (OR=1.4, 95% CI 1.3-1.5) (13).
Recently, Sanderson et al. in a literature-based systematic HuGE review and meta-analysis also found an increased risk of BC in NAT2 slow acetylators, as compared with rapid acetylators, with a similar OR, usually found in Caucasians (OR=1.46, 95% CI: 1.26, 1.68) (14). Thus, the present results are similar to those published. This finding, to some extent, validates the technique of genotyping. However, the OR of 2.7 is higher than OR previously observed in pooled and meta-analyses of studies in Europeans or Caucasians, producing a summary OR of 1.4-1.5. The possible explanation is the statistical limitation.
In the present study, a tendency of increased risk was observed with GSTP1 Ile/Val and Val/Val genotypes compared with GSTP1 Ile/Ile, although this difference was not statistically significant for BC when compared with the controls. The GSTP1 family consists of a single gene (GSTP1). The substitution of an Isoleucine by a Valine at codon 105 deteriorates GSTP1 activity. Several studies have been performed to explore the associations between the various GSTP1 polymorphisms with predisposition to BC; however, the results have been inconsistent. Significant increased risk of BC for some GSTP1 polymorphisms was described in Turkish, British, American and Indian populations (15-18).
The most notable risk was stated by Harries et al. (16). They observed an approximately threefold increase in risk between individuals with the GSTP1 (Val/ Val) allele and those with GSTP1 (Ile/Ile) variant for BC (16). Contrary to this, other studies observed no association between GSTP1 polymorphisms with susceptibility to BC (10, 13, 19, 20). Recently, Kellen et al. undertook a meta- and pooled analysis to examine the association between GSTP1 isoleucine (Ile) 105 valine (Val) and BC (21). In the meta-analysis (16 studies, 4,273 cases and 5,081 controls), the unadjusted summary ORs for GSTP1 Ile/Val and Val/Val compared with GSTP1 Ile/Ile were 1.44 (95% CI: 1.04, 1.57). The association appeared to be the strongest in Asian countries. When the analysis was limited to European descendents (9 studies), the summary OR decreased (OR=1.23, 95% CI: 1.00, 1.52). They concluded that GSTP1 Ile105Val appears to be associated with a modest increase in the risk of BC. No significant association between studied CYP1A1, CYP1B1 and COMT polymorphisms and BC risk was observed in the presented study. Many commonly occurring SNPs are reported in CYP1A1 in various populations that include, isoleucine to valine substitution at codon 462 in heme binding region in exon 7. Epidemiological studies regarding the association between CYP1A1 polymorphism and cancer risk have shown inconsistent results among various populations. CYP1A1 polymorphisms investigations in BC risk have been few.
Brockmoller et al. showed non-significant association with CYP1A1 gene polymorphism for BC risk (22), whereas Srivastava et al. observed a trend of association with CYP1A1 (2A*) for risk of BC (OR=1.56, statistically non-significant) (23).
CYP1B1 is a phase I metabolising enzyme. A G to A polymorphism resulting in an amino acid change from valine to leucine at codon 432 has been identified (24), and presents a lower catalytic efficiency for the 4-hydroxylation than the wild-type (25). This suggests a possible reduced risk of cancer.
The presented results are in agreement with a study by Hung et al. that did not observe an effect of Val432Leu polymorphisms on BC risk (10).
COMT catalyzes the methylation of various endobiotic and xenobiotic substances. One major polymorphism has been identified and consists of a G to A substitution resulting in the change from a methionine to a valine in codon 108 in the soluble form and in codon 158 in the membrane protein form. Polymorphisms of COMT have been investigated in various cancers, but, so far, there are only limited data on BC. To date, there is only one epidemiological study published. Hung et al. conducted a hospital-based case-control study among men in Northern Italy. They failed to observe an effect of COMT Val108Met polymorphism on BC risk (4).
In conclusion, this study is the first French epidemiologic study to investigate the association between metabolising gene polymorphisms and BC risk. Despite the limitations, our results indicate that the NAT2 polymorphisms may modulate the individual susceptibility to BC in a French Caucasian male population. This finding is consistent with previous literature for Caucasians. A significant association between the other studied metabolising gene polymorphisms (CYP1A1, CYP1B1, COMT and GSTP1) with BC risk was not found. These genes have not yet been widely investigated and therefore further examinations are required to assess their association with BC risk.
Acknowledgements
This study was supported by “La Ligue Nationale Française de Lutte Contre le Cancer” (Puy-de-Dôme, Allier, Cantal). Laetitia Delort is recipient of a grant from the «Association pour la Recherche sur le Cancer» and Nadège Rabiau is recipient of a grant “CIFRE” from Soluscience S.A., Clermont-Ferrand, France. We thank T. H. Gunnels for assisting with the English translation of this study.
- Received November 20, 2008.
- Revision received December 18, 2008.
- Accepted January 19, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
