During the last few years, it has been documented that alcoholism is increasing among young persons (1). Alcohol is metabolized by the liver cells (hepatocytes) via 3 pathways: the alcohol dehydrogenase pathway in the cytosol, the microsomal ethanol oxidizing system in the endoplasmic reticulum and catalase in the peroxisomes. All 3 pathways result in the production of acetaldehyde, which is then metabolized by aldehyde dehydrogenase (ALDH) into acetate, a metabolite more toxic than alcohol itself (2–4).

Cytochrome P450 2E1 (CYP2E1) is the main enzyme in the microsomal ethanol oxidation system (5), and it is a member of the cytochrome P450 (CYP) superfamily, a membrane-bound protein primarily associated with endoplasmic reticulum membranes. CYP2E1 has also been shown to be involved in the metabolism of more than 80 low-molecular hydrophobic toxicologically dangerous compounds and contributes to the activation of a number of pro-carcinogens and drugs into highly reactive metabolites (6,7). Specifically, CYP2E1 activates N-nitrosamines contained in tobacco smoke and foodstuffs (8–10) and several industrial (11) and endogenous carcinogens (12,13); moreover, it is capable of reducing molecular oxygen to highly reactive compounds, such as superoxide anion radical (O₂⁻), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^·) leading to DNA damage and carcinogenesis (14,15).

The human CYP2E1 gene is located on chromosome 10q26.3 and consists of 9 exons and 8 introns. CYP2E1 gene expression may be regulated at the transcription and translation level, as well as the mRNA stabilization and protein degradation levels (16,17). Similar to other CYP genes, CYP2E1 has more than 10 different polymorphic sites, in the 5′-flanking region, in introns and transcribed gene regions. In particular, a polymorphic variable number tandem repeat (VNTR) sequence has been described ~2.0 kb pair upstream of the transcription start site (18).

There is a high variability among different ethnic groups in the activity of the CYP enzyme due to both environmental and genetic factors, such as polymorphisms. Polymorphic CYP genes are capable of creating differences in the ability to metabolize, detoxify or activate a number of substances. This ability, as is well known, provides one of the first lines of defence against xenobiotic chemicals; therefore, we hypthesized that an alteration in the activity of these enzymes may result in a greater susceptibility to certain diseases or cancer, although existing evidence on the matter is controversial (19). A number of studies have clarified that certain polymorphic genes, in association with alcohol consumption, are involved in the development of certain cancers (20–22). Despite the fact that alcohol is not itself a carcinogen, it acts as a co-carcinogen by enhancing the effects of other chemicals activated by certain enzymes, i.e. CYP2E1. Certain studies have demonstrated that CYP2E1 is highly expressed in both the liver and pancreas after ethanol administration (23). Acetaldehyde produced during ethanol oxidation may be the direct cause of cell damage via the production of reactive oxygen species (ROS). Moreover, it is currently well known that certain tumours are heritable (24,25) and that environmental hazards associated with particular habits may increase the risk of tumour insurgence. In this study, we investigated CYP2E1 VNTR polymorphism distribution among healthy subjects by analyzing whether particular CYP2E1 VNTR polymorphisms were associated with certain health-risk behaviours, such as alcohol consumption and/or cigarette smoking. Finally we analyzed patients with pancreatic adenocarcinoma (PA) and hepatocellular carcinoma (HCC) in order to find evidence of a possible correlation between certain CYP2E1 VNTR polymorphisms, risky habits and predisposition to cancer.

It is known that alcohol consumption and cigarette smoking are, unfortunately, increasing among young persons (1). Moreover, it is well known that these dangerous injuries exert an influence on susceptibility to tumours, such as PA (26) and HCC. These social and clinical problems drove us to explore whether an association exists between particular genetic factors, health-risk behaviours, as well as drinking- and/or smoking-related neoplastic diseases. We focused our attention on CYP genes and in particular, on CYP2E1, which is a polymorphic gene involved not only in the ethanol oxidative metabolism, but also in the transformation of many exogenous compounds, including N-nitrosamines, which may be activated into highly reactive metabolites. Polymorphisms in this gene can create differences in the ability to metabolize, detoxify or activate a number of substances. This ability can generate certain reactive substances which may consequently increase the risk of developing serious diseases, such as tumours (7,8,14,27).

The VNTR polymorphism in the 5′-flanking region of the CYP2E1 gene has previously been described (18). Moreover, to date, only a few studies have focused on this polymorphism and its asociation with drinking and/or smoking and cancer development (16). We formed the hypothesis that the VNTR polymorphism may be responsible (together with others) for the different susceptibility to serious diseases which are caused, directly or indirectly, by drinking and/or smoking. In this study, we report for the first time, the results of VNTR CYP2E1 genotyping, carried out in 166 young healthy individuals and 126 patients (60 PA and 66 HCC), residents of southern Italy.

In order to genotype the VNTR polymorphisms, we developed a simple and precise technique in our laboratory, suitable as a hospital diagnostic routine, consisting of PCR followed by enzyme digestion to improve sensitivity and resolution.

We found 7 out of 10 expected genotypes: A2/A2 was the most frequent and A1/A2, A3/A3 were the least frequent both in the healthy subjects and patients. The genotypes, A1/A3, A1/A4 and A3/A4, were not detected. As A2/A2 was the modal genotype in all the studied subjects, we believe that this allele provides a generic selective advantage in humans.

We considered the results regarding the other no-modal genotypes more interesting. In fact, in all the association analyses between the health-risk behaviours and VNTR genotypes, we found that individuals with the A1/A1 genotype had a lower inclination either to drink or smoke (the lowest OR values shown in Tables III and IV), whereas individuals with the A4/A4 genotype had an inclination to drink and those with the A2/A3 genotype a propensity to smoke. Consequently, individuals with the A1/A1 genotype are exposed neither to potentially dangerous molecules inhaled by smoking and transformed by the CYP2E1 enzymes, nor to the ROS produced by the CYP over-activity induced by alcohol ingestion. This genotype may determine a lower susceptibility to drinking- and/or smoking-related neoplastic diseases, hence it may be considered a protective genetic order. Otherwise, A4/A4 subjects are exposed to the continual alcohol induction of the CYP2E1 gene; therefore, they are promptly able to transfrom certain incidentally absorbed pro-carcinogens into active toxic molecules and to increase CYP activity-derived ROS production. This genotype, therefore, may be associated with the susceptibility to develop serious drinking-related diseases.

These results led us to investigate whether an association between the A4/A4 genotype and tumour susceptibility exists; therefore, we genotyped patients with drinking- and/or smoking-related tumours (i.e. PA and HCC). In confirmation of the results obtained from the healthy subjects, the A1/A1 genotype was found neither among PA nor HCC patients and A4/A4 PA cases were significant (~3-fold) with respect to the controls (OR=3.25; 95% CI 1.21–7.50) (Table V).

Nowadays, little is known about the risk factors for pancreatic cancer (20) and in particular, there are controversial data about alcohol consumption (28) as a susceptibility factor. Our results show that one of the VNTR CYP2E1 genotypes could be a susceptibility factor for PA, either directly, by conferring a higher expression of the CYP2E1 gene, or indirectly, by being associated with drinking habits. It has previously been reported that transcriptional activity is increased in subjects with the A4 allele (16) and the inductive effect of ethanol on CYP2E1 gene expression (12) and on ROS production is well known (29).

We hypothesized that the A4/A4 genotype, having a double dose of the A4 allele, may be associated with a greater susceptibility to PA, confirming the suspicions reported previously regarding other cancers (16), especially for subjects which have an inclination to alcohol consumption.

We consider that further studies are required to further confirm this hypothesis. In particular, it may be of interest to study the real contribution of VNTR alleles in CYP2E1 gene expression, as well as their regulatory elements contained in the 5′-flanking region, either genetic, or epigenetic.