Polymorphism in CYP1A1 and CYP2E1 genes and susceptibility to leukoplakia in Indian tobacco users

Abstract

Inter-individual genetic differences may contribute to differences in susceptibility to human diseases triggered by environmental exposures. In this study, we investigated polymorphisms at two sites in the CYP1A1 and three sites in the CYP2E1 genes in 99 leukoplakia patients and 227 controls from one Indian population. The frequencies of genotypes at these polymorphic sites (MspI and Ileu/Val) in the CYP1A1 and (PstI, RsaI and DraI) in the CYP2E1 genes, were similar in patient and control groups. But the combined rare and heterozygous genotypes (CC+CD) at the DraI site in the CYP2E1 gene were over-represented among patients compared with controls (age-adjusted odds ratio (OR)=2.02, 95% confidence interval (CI)=1.21–3.35). Light tobacco smokers (i.e. <21 pack-year) and light tobacco chewers (i.e. <104 chewing-year) with a “rare” C allele at the DraI site had high risk of leukoplakia (OR=2.88, 95% CI=1.16–7.22; OR=2.94, 95% CI=1.15–7.65, respectively). The “mixed tobacco” users with “rare” C allele are more susceptible to the disease than “exclusive” tobacco smokers and chewers. The results indicate that the “rare” C allele at the DraI polymorphic site in CYP2E1 gene may enhance susceptibility to leukoplakia among tobacco users in this population. But the low sample size limited the power to precisely estimate the tobacco–genotype interactions.

Introduction

Oral leukoplakia, a common pre-malignant lesion among smokers, is defined as “a chronic white mucosal macule which cannot be characterized clinically or pathologically as any other disease and is not associated with any physical or chemical causative agent except the use of tobacco” [1]. The different types of leukoplakia are classified as homogeneous, ulcerated, nodular and verrucous in order of their increasing severity. The annual incidence of oral leukoplakia among subjects over 15 years of age was reported as 0.2–11.7% in different populations of India [2]. Tobacco chewing and smoking has been reported as major risk factors for oral leukoplakia [2], [3], [4]. A study in India found that keeping chewed tobacco in the cheek overnight increases the risk of oral leukoplakia [5]. Tobacco smoking and alcohol consumption have been identified as main risk factors for oral cancer in Western population [6]. Polycyclic aromatic hydrocarbons (PAHs), aldehydes and nitrosamines are thought to be carcinogenic components present in tobacco smoking. But chewing of tobacco with betel quid increases the concentrations of carcinogenic tobacco-specific nitrosamines and reactive oxygen species in mouth [7]. As an early sign of damage to oral mucosa, tobacco smokers and chewers often develop precancerous lesions such as leukoplakia and submucosal fibrosis. These lesions are easily accessible to diagnosis and can be considered as indicators of oral cancer risk. About 2–12% of these lesions becomes malignant within several years [4]. Molecular epidemiological studies have now provided evidence that an individual's susceptibility to cancer is mediated by both genetic and environmental factors. Inherited differences in the effectiveness of the activation/detoxification of carcinogens play a crucial role in host susceptibility. Thus, there is an urgent need to know host genetic markers, which could predispose an individual to leukoplakia and ultimately to cancer.

Most procarcinogens require metabolic activation by Phase I enzymes (e.g. cytochrome P450 oxidases like CYP1A1 and CYP2E1) to act as carcinogens. But detoxification of it by Phase II metabolic enzymes (e.g. glutathione S-transferase (GST) M1, T1) is also maintained by body to protect itself against the ill effects of carcinogens. CYP1A1 and CYP2E1 genes are considered to play important roles in the activation of PAHs and nitrosamines, respectively [8]. The polymorphic MspI restriction enzyme site in CYP1A1 gene, at the 264th base downstream from additional poly (A) signal in the 3′-flanking region, could modulate expression of gene and has been shown to be associated with susceptibility to lung and oral squamous cell carcinoma (SCC) [9], [10], [11]. Another polymorphic site (Ileu/Val) in exon 7 of same gene has been reported to increase risk of oral cancer among tobacco users in different ethnic populations [12], [13], [14].

Another CYP enzyme, CYP2E1 is responsible for the metabolism of various xenobiotics [15], [16], [17]. CYP2E1 is expressed in cultured human oral epithelial cells [18], [19]. In addition to metabolizing potentially important carcinogens such as benzene, butadiene, carbon tetrachloride, vinyl chloride and low molecular weight nitrosamines [20], CYP2E1 is also involved in the activation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [21]. Two polymorphisms in this gene, at nucleotides −1259 and −1019 bp upstream of the CYP2E1 transcriptional start site, are detectable by PstI and RsaI restriction enzyme digestions, respectively [22]. Based upon the presence [+] or absence [−] of RsaI and PstI recognition sequences at these polymorphic sites, the “wild-type” haplotype (RsaI[+].PstI[−]) and one of the variant haplotypes (RsaI[−].PstI[+]) were designated as the “c1” and “c2” alleles, respectively. The PstI/RsaI polymorphic sites are present in a putative HNF-1 binding site and may play important roles in the regulation of CYP2E1 transcription and subsequent protein expression [23]. The DraI polymorphism in intron 6 of the CYP2E1 gene leads to a wild-type D allele (presence of DraI restriction site) and a variant C allele (absence of DraI restriction site) [24]. Several studies have reported that the variant “c2” and C alleles (of the RsaI/PstI and DraI sites, respectively) are associated with enhanced enzyme activity [25]. Results from other studies also suggested that there was no difference in CYP2E1 activity in human liver with different CYP2E1 genotypes [26], [27]. But the reasons for this discrepancy are not clear yet. Other CYP2E1 variant alleles identified so far are very rare in many populations and hence may lack functional significance in an association study [28], [29]. The hypothesis that polymorphisms in the CYP2E1 gene influence susceptibility to tobacco-related oral cancer has attracted attention in the recent past [30], [31], [32].

It is reported that homozygous deletions in GSTM1 and GSTT1 genes increase risk of leukoplakia in Indian tobacco user [33]. But the association study relating polymorphisms in CYP1A1 and CYP2E1 genes and risk of leukoplakia in tobacco users has not been reported yet. In this study, we examined whether the polymorphisms in these two genes could modify the risk of oral leukoplakia in individuals with tobacco smoking and/or chewing habits.