Do smoking and polymorphisms in xenobiotic metabolizing enzymes affect the histological stage and grade of bladder tumors?

ARTICLE

Auteur(s) : S Ouerhani¹, K Rouissi¹, R Marrakchi¹, M Riadh Ben Slama², M Sfaxi², M Ayed², M Chebil², AB Elgaaied¹

¹Laboratoire de génétique, d’immunologie et de pathologies humaines, faculté des sciences de Tunis El-Mannar-I, 2092 Tunis, Tunisie
²Service d’urologie, hôpital Charles-Nicole, Tunis, Tunisie

Article reçu le 7 Novembre 2008, accepté le 19 Janvier 2009

Introduction

Cigarette smoking is the most important risk factor for bladder cancer, accounting for 50% of cases in men and 35% in women [3]. A meta-analysis reported that cigarette smokers have a risk of 2.57 (95% confidence intervals [CI]: [2.20-3.00]) compared to non-smokers [3]. Tobacco components, such as 4-aminobiphenyl (4-ABP), increase bladder cancer risk by inducing local somatic mutations. Indeed, the study of Feng et al. [4] has reported that cigarette smoke generates a substantial amount of 4-ABP and metabolically activated 4-ABP preferentially binds to codons 280 and 285 of the p53 gene. The p53 alterations occur predominantly in invasive and high-grade superficial tumors [5]. In fact, Thompson et al. [6] have suggested that bladder tumors in patients who smoke tend to be large, multifocal and demonstrate high-histological grade and stage.

Hypothetically, the mutational spectra of somatically altered genes (such as p53) in environmentally associated cancers such as bladder cancer may be influenced by the properties of relevant phase I and phase II xenobiotic metabolizing enzymes, as well as by the efficiency of the DNA repair system and other cellular host factors. The xenobiotic-metabolizing machinery includes oxidative enzymes (phase I), which may generally activate compounds to become carcinogenic and phase II conjugating enzymes, considered rather protective since they detoxify a number of reactive chemical carcinogens [7]. The conjugating process (phase II) is mainly controlled by the superfamilies of glutathione-S-transferases (GST) and N-acetyl-transferases (NAT) enzymes. Polymorphisms in NAT and GST genes alter the ability of these enzymes to metabolize carcinogens. Indeed, epidemiological studies have shown that some NAT2 polymorphisms have been correlated with decreased NAT2 enzyme activities (“slow” alleles). The slow NAT2 acetylation genotype compromises its detoxification ability, and studies have consistently observed an association between the slow NAT2 genotype and increased bladder cancer risk among smokers [8-10]. Moreover, deletion of the genes and lack of the encoded enzyme have been identified in both GSTM1 and GSTT1 loci. Subjects lacking GSTM1 are at increased risk of developing environmentally related cancers such as lung and bladder cancers [11, 12]. In some studies, the GSTT1 null genotype has been suggested to be associated with an increased risk of developing bladder [13] and lung cancers [14], whereas other studies have reported that the risk of cancer was increased only among those with the GSTT1 wild-type genotype [15, 16]. The identified polymorphisms in GST and NAT2 genes may influence, by modulation of mutagenic DNA adduct levels, the occurrence and type of critical mutations in oncogenes and in tumor suppressor genes and thereby affect the individual susceptibility to cancer.

Many previous case-control studies have analyzed the association between risk factors (tobacco or genetic polymorphisms in xenobiotic metabolizing enzyme) and bladder cancer development. However, the relationship between these risk factors, the stage and the grade of bladder tumors has not been evaluated. We therefore examined in this study the combined effect of smoking and polymorphisms in xenobiotic metabolizing enzymes (slow NAT2, GSTM1 and GSTT1 null genotypes) in different histological subgroups of bladder tumors aiming at clarifying whether smoking and genetic variations represent risk factors for the development of tumors with high stage and grade. Efficient study design utilizing these biomarkers should accelerate the development of optimum bladder cancer prevention strategies.

Patients and methods

Patients

Tumors were staged and graded according to the criteria of the tumor-node-metastasis classification (TNM) and the WHO-International Society of Urological Pathology as follows: 25 pTa GI, 11 pTa GII, 2 pTa GIII, 25 pT1 GII, 12 pT1 GIII and 22 invasive tumors (≥ pT2). After giving informed consent, peripheral blood samples were collected from all patients into tubes with EDTA at pH 8.

DNA preparation and genotyping

GSTM1 and GSTT1 null genotypes were identified using a multiplex-polymerase chain reaction (PCR)-based method as described by Arand et al. [18]. For NAT2, a PCR was carried out as described by Hsieh et al. [19]. The whole intronless NAT2 gene was resulted in a 1093-base pair fragment that was then digested with 5 U of KpnI, 10 U of BamHI, 5 U of TaqI and 10 U of AluI, to reveal NAT2*5, NAT2*6, NAT2*7 and NAT2*14 alleles, respectively. Digestions were performed at 37 °C overnight for KpnI, BamHI and AluI and at 65 °C for TaqI. The obtained fragments were separated on a 2% agarose gel.

Individuals with two wild-type alleles (NAT2*4/*4) were classified as rapid acetylators, the others were classified as intermediate acetylators when they had one mutant allele and as slow acetylators when they had two mutant alleles including NAT2*5, NAT2*6, NAT2*7 or NAT2*14 [20, 21].

Statistical analysis

Results

Among superficial tumors, 81.3% were with low-grade (GI or GII) and 18.7% (14/75) were with high-grade (GIII). More than 90% of patients with superficial low-grade tumors were smokers. This percentage was statistically different from that reported for patients with high-grade tumors (table 3). The high frequency of smokers in patients with superficial low-grade tumors (used as a reference group) compared to those with high-grade led to a RR < 1 (RR = 0.48; 95% CI: [0.26-0.88]). The comparison of GSTM1 and GSTT1 null genotype frequencies in patients with superficial low-grade and those with superficial high-grade tumors does not show a significant statistical difference (table 3). For these genotypes, significant statistical differences were only obtained, when we compared smokers to non-smoker patients (table 4). For NAT2 gene, a significant statistical difference in genotypic distribution between patients with superficial low-grade tumors and those with high-grade tumors was detected for the intermediate NAT2 genotype (P = 0.04). This genotype presented a 1.33-fold increased risk of developing high-grade bladder tumors compared to reference group (RR = 1.33; 95% CI: [1.03-1.72]). This risk increases to 3.67 in non-smoker patients carrying altered NAT2 genotypes compared to non-smoker patients carrying rapid NAT2 genotype (P = 0.02; RR = 3.67; 95% CI: [1.40-9.62]).
Table 1 The distribution of patients according to tumor stage; tobacco status and xenobiotic metabolizing enzyme genotypes.

Table 2 Stratification of GST and NAT2 genotypes in superficial and invasive tumors according to smoking status.

Table 3 Distribution of patients with superficial bladder tumors according to tumor grade, smoking status and xenobiotic metabolizing enzyme genotypes.

Table 4 Stratification of GST and NAT2 genotypes in superficial low- and high-grade tumors according to smoking status.

Discussion

Our data show that 81.45% (79/97) of patients were current smokers and 18.55% were non-tobacco consumers. It was found that 88.60% (70/79) of smokers were heavy smokers, and 53.16% (42/79) have smoked more than 40 PY. These results suggested the important role of tobacco in bladder cancer development in the Tunisian population. Indeed, other studies have reported that smokers are two to three times more likely to develop UCC, and 50% of all bladder tumors are directly attributable to cigarette smoking [23, 24]. The distribution of patients according to smoking status and histological tumors and stage has suggested that the frequency of smokers in the superficial tumor group was not different from that reported in the invasive tumor group (P > 0.05). Conversely, the percentage of smokers in the superficial low-grade tumor group was higher than that found in the superficial high-grade tumor group. We conclude that in Tunisian patients, tobacco does not appear to be a factor affecting the tumor stage, but was essentially associated with the development of superficial low-grade tumors. This finding was in contradiction with several other studies, which have suggested that bladder tumors in patients who smoke tend to be large, multifocal and demonstrate high-histological grade and stage [6].

The distribution of patients according to genotypic frequencies of GSTM1 and GSTT1 genotypes and histological tumors stage did not show a significant statistical difference. This result suggests that genetic polymorphisms in GST enzymes do not appear as a factor affecting the histological tumor stage. Similarly, the comparison of GSTM1 and GSTT1 genotype frequencies between patients with superficial low-grade, and those with superficial high-grade tumors did not show a significant statistical difference. The association between GSTM1 null genotype and the GSTT1 wild-type genotype and superficial low-grade tumors was only obtained when we compared smoking patients to non-smokers. This association was essentially attributed to the direct effect of tobacco carcinogens. Our hypothesis suggests that in superficial low-grade tumors, tobacco carcinogens in interaction with GST enzymes induce somatic mutations in FGFR3 oncogene (fibroblast growth factor receptor 3). This oncogene has been shown to be the most frequently altered gene in low-grade tumors [25]. Until now, only one study has studied the effect of tobacco carcinogens on the FGFR3 mutation spectrum and did not report any correlation [26].

The distribution of patients according to genotypic frequencies of NAT2 and histological tumor grade has suggested that non-smoker patients carrying an altered NAT2 genotypes presented a 3.67-fold increased risk of developing high-grade bladder tumors compared to non-smokers patients carrying a rapid NAT2 (P = 0.02; RR = 3.67; 95% CI: [1.40-9.62]). Our hypothesis suggests that in the absence of tobacco, which was the most important environmental risk factor, the role of genetic factors will be essential in determining the initiation of pathology. The altered NAT2 alleles increase the risk of bladder cancer development by increasing somatic mutation in tumors suppressor genes. Indeed, previous studies suggest that the NAT2 slow acetylator genotypes may be associated with an impaired metabolism of carcinogens that predispose individuals to p53 gene mutations, which were associated with the development of superficial high-grade bladder tumors [27].

Although some of the results presented here are novel, this study has some limitations. Firstly, the sample size is small, limiting the precision of the statistical analyses. Secondly, we have not information regarding somatic altered genes such as FGFR3 and p53, which were respectively, associated with superficial low- and high-grade bladder tumors. Besides that in the future, enlargement of sample sizes in the Tunisian population and analysis of somatic altered genes (which is already ongoing) will be essential to assess the role that environmental factors together with the genetic factors play as predictors of differential susceptibility to the presentation of malignancy.

Conclusion

Conflict of interest: Authors confirm that they do not have any disclosure to make at submission, and none of them has any potential financial conflict of interest related do this manuscript.

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