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Carcinogenesis Advance Access originally published online on November 1, 2006
Carcinogenesis 2007 28(4):875-882; doi:10.1093/carcin/bgl194
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Role of NQO1C609T and EPHX1 gene polymorphisms in the association of smoking and alcohol with sporadic distal colorectal adenomas: results from the UKFSS Study

Panagiota N. Mitrou1, Mark A. Watson2, Alexandre S. Loktionov1, Christopher Cardwell3, Marc J. Gunter1, Wendy S. Atkin3, Christopher P. Macklin4, Tom Cecil5, D.Timothy Bishop4, John Primrose5 and Sheila A. Bingham1

1 Dunn Human Nutrition Unit, MRC/Wellcome Trust Building Hills Road, Cambridge CB2 2XY, UK
2 Department of General Surgery, Norfolk and Norwich Health Care NHS Trust, Norwich NR4 7U4, UK and Dorset County Hospital, Williams Avenue Dorchester, Dorset DT3 5NR, UK
3 Cancer Research UK, St Mark's Hospital Northwick Park, Harrow HA1 3UJ, UK
4 Cancer Research UK Genetic Epidemiology Section, St James's University Hospital Leeds LS9 7TF, UK
5 Cancer Sciences Division, University of Southampton Southampton SO16 6YD, UK

*To whom correspondence should be addressed. Tel: +44 1223 252760; Fax: +44 1223 252765; Email: sab{at}mrc-dunn.cam.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
NADP(H):quinone oxidoreductase 1 (NQO1) and microsomal epoxide hydrolase (EPHX1, also mEH) are attractive candidate enzymes for association with colorectal neoplasia because they metabolize a number of compounds including polycyclic aromatic hydrocarbons (PAHs) that have been linked with colorectal carcinogenesis. We examined the relationship between NQO1C609T, mEH3, mEH4 and risk of sporadic distal colorectal adenomas in one of the largest case–control studies of 946 polyp-free controls and 894 cases, all participants of the UK Flexible Sigmoidoscopy Screening (UKFSS) Trial. The polymorphisms were examined as independent risk factors and evidence for interaction with smoking and alcoholic drinks was sought. The NQO1 609*T allele was positively associated with high-risk adenoma in this population [odds ratio (OR), 1.36; 95% confidence interval (CI), 1.02–1.83]. Elevated risk estimates were seen in smokers independently of the genotype but the association was stronger among current smokers with the heterozygous variant genotype (OR, 4.24; 95% CI, 2.54–7.09). It was reported for the first time that the association between alcohol and colorectal adenoma was modified by NQO1C609T genotype, such that the relation between alcohol and colorectal adenoma was stronger among those with the common C/C genotype (OR, 1.49; 95% CI, 1.11–2.02; P-interaction = 0.024). There was no association between mEH3 and mEH4 variants and colorectal adenoma risk and no effect modification by alcohol and smoking. These findings provide evidence for an important role of the NQO1C609T polymorphism in susceptibility of colorectal adenomas. Alcohol increases risk of colorectal adenoma in carriers of the high-activity genotype possibly through enhanced activation of alcohol-related procarcinogens.

Abbreviations: CI, confidence interval; NQO1, NADP(H):quinine oxidoreductase 1; OR, odds ratio; PAHs, polycyclic aromatic hydrocarbons; UKFSS, UK Flexible Sigmoidoscopy Screening


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Enzymes involved in xenobiotic metabolism are thought to play a role in colorectal neoplasia (1). NADP(H):quinone oxidoreductase 1 (NQO1) and microsomal epoxide hydrolase (EPHX1, also mEH) are attractive candidate enzymes for association with colorectal neoplasia because they metabolize a number of compounds that have been linked with colorectal carcinogenesis. NQO1 is primarily involved in the detoxification of potentially mutagenic and carcinogenic quinones (derived from tobacco smoke or diet), through their two electron reduction to hydroquinones (2). However, depending on the stability of hydroquinone generated following reduction, NQO1 may produce a more active product that can result in the formation of reactive oxygen species (ROS) (3). NQO1 also protects cells from oxidative stress by maintaining antioxidant forms of ubiquinone and vitamin E (2). The most actively studied NQO1 polymorphism is a non-synonymous single nucleotide polymorphism, C609T, which gives rise to a proline to serine amino acid change in the encoded protein (4). Phenotyping studies of both human cell lines and primary human tissues demonstrated that the variant T genotype is associated with trace amounts of NQO1 protein but no NQO1 activity (5). In contrast, the more common (C/C) or heterozygous (C/T) genotype showed variable but detectable NQO1 levels. The NQO1C609T polymorphism exhibits ethnic variation (4–22%) with the highest prevalence of the T allele occurring in Asian populations (19% Korean Asians; 22% Chinese Asians) and the lowest in Caucasians (4%) (6,7). Several more SNPs have been identified in the NQO1 gene but most of them are rare variants with allele frequencies of <1% (8).

Studies on NQO1C609T polymorphism and colorectal neoplasia are limited. To date, four studies have examined the role of NQO1C609T polymorphism in relation to colorectal cancer susceptibility (9–12) and there is only one study on colorectal adenoma (13). The epidemiological data on NQO1 and colorectal cancer suggest that the NQO1C609T variant polymorphism might be a risk factor for the disease, whereas normal enzyme activity may confer the opposite effect. It might also be hypothesized that NQO1 activity might influence colorectal neoplasia risk in individuals exposed to NQO1 metabolizing compounds. Tobaccosmoke is a source of polycyclic aromatic hydrocarbons (PAHs), which are substrates of NQO1 and can be detoxified by its action. In light of the antioxidant function of NQO1, the significance of the NQO1C609T polymorphism may extend to a wide variety of agents that induce oxidative stress such as alcohol. None of these putative NQO1-alcohol/smoking interactions have been explored so far.

EPHX1 (mEH) has also a critical role in xenobiotic metabolism and the EPHX1 gene is highly polymorphic (14). Polymorphisms in exon 3 (referred to as mEH3) and exon 4 (referred to as mEH4) of the gene have been associated with decreased and increased enzyme activity, respectively (15). As EPHX1 is also involved in both activation and detoxification of PAHs (carcinogens found in cooked meat and tobacco smoke), mEH3 and mEH4 polymorphic variants taken separately or in combination, may influence the rate of PAH metabolism and subsequently modulate adenoma risk. It has been recently concluded that mEH3 and mEH4 genotypes may play a role in the earlier stages of colon carcinogenesis upon exposure to specific environmental carcinogens (16). In this, one of the largest studies to date, involving 1840 people from the UK Flexible Sigmoidoscopy Screening (UKFSS) Trial, the NQO1C609T, mEH3 and mEH4 polymorphisms were examined as independent risk factor of sporadic distal colorectal adenomas. In addition, we investigated the interaction between adenoma risk, genotype, smoking and alcoholic drink consumption which contain components suspected to interact with the mEH and NQO1 enzymes.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study population recruitment
This study was part of the UKFSS Trial, a multicentre randomized controlled trial in 14 centres throughout the UK designed to assess whether a single FS screening can lower the incidence and mortality of bowel cancer (17,18). Of 170 432 individuals aged 55–64 years who did not have a history of colorectal cancer, adenoma, inflammatory bowel disease or a severe or terminal disease with life expectancy of <5 years or who had had a sigmoidoscopy or colonoscopy within the past 3 years or who were incapability of providing informed consent. A total of 57 254 were randomized to be invited for screening and 40 674 attended (19). From baseline questionnaires, 1% or less were non-Caucasians. Full description of the study and recruitment of subjects are reported in detail elsewhere (19). After screening, cases were further classified into high- and low-risk cases according to the size and morphology of the polyps (high-risk criteria included any of the following: diameter of 1 cm or larger; three or more adenomas; tubulovillous or villous histology; severe dysplasia or malignancy).

In 3 of the 14 centres, (Norwich Leeds and Portsmouth) the baseline protocol was extended to include collection of blood samples at the time of screening for a case–control analysis of gene variants in relation to adenoma risk in 1997–98. In these 3 centres, 96% (8896) of those approached agreed to screening and 1134 adenomas were detected. Eighty-eight percent (1002) agreed to donate a blood sample for genotyping at the screening visit. There were 6148 polyp-free participants and for each polyp case, an age- and sex-matched polyp-free participant was asked by the registrar at the same screening visit to donate a blood sample for genotyping. A total of 1119 of these controls agreed to this request. The proportion of controls refusing a blood specimen was not recorded but was very low. Due to the low proportion of non-Caucasians in the study, information on ethnicity for individuals who attended the screening was also not recorded.

Alcohol and smoking consumption
At the time of the sigmoidoscopy, participants were asked whether they were never, former or current smokers, which was coded as never (one), former (two), current smoking (three). Smoking consumption was categorized as never, <20 to ≥20 cigarettes per day. Duration of smoking was classified as never, <30, ≥30 years. Smoking dose was assessed by the pack-years of smoking variable (number of packets of cigarettes smoked multiplied by years of smoking) and divided into three groups such as 0 pack-years, <20 pack-years and ≥20 pack-years. Participants were excluded from analyses if their responses did not comply with the cleaning data criteria used at the UKFSS centre in London. To clean the data prior to data release for analyses, if the ‘age stopped smoking’ was less than or equal to the age started smoking, then it was assumed that the number of years stopped smoking was actually the ‘age’ stopped smoking. An answer of ≥30 years was interpreted by assuming that the subject meant to record ‘age stopped smoking’. Data were also excluded if any of the following features were present: 1, if the age started smoking was between 1 and 6 years; 2, if the age started smoking corresponded to the actual age when the patient attended screening. On the basis of these criteria, out of the 1840 cases and controls, the number of individuals with missing smoking data were as follows: 158 for smoking status, 189 for smoking duration, 221 for smoking consumption and 245 for pack-years of smoking. These numbers were evenly distributed between the centres. In brief, only ~1% of data were missing after cleaning as outlined above, which would have had little impact on power calculations.

Participants were also asked to estimate alcohol consumption [wine, beer, spirits and port (which included sherry, vermouth and liqueurs)] in portions per month (p.p.m) using a previously validated questionnaire (also used in EPIC UK) (20). Alcohol variables were categorized in three groups (tertiles T1–T3) of almost equal size based on the combined distribution in cases and controls, using cut-off points of 0–6 p.p.m., 7–26 p.p.m., 27+ p.p.m.

Ethics
The study was approved by the Norwich District Ethics Committee, Portsmouth and South East Hampshire Health Authority Research Ethics Committee, United Leeds Teaching Hospitals Trust Research Ethics Committee of the participating centres.

DNA extraction and genotyping
Blood samples were collected into EDTA-containing tubes from all participants in the study at each centre on the day of the appointment for FS by the UKFSS endoscopists, and were stored at –70°C for DNA extraction. Genomic DNA was extracted using either a standard phenol–chloroform method or the Qiagen Extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR-based restriction fragment length polymorphism (RFLP) analysis was used to assess the NQO1 C609T (rs1800566), EPHX1 exon 3 (rs1051740) and EPHX1 exon 4 (rs2292566) polymorphisms. All analyses were performed blinded with regard to case–control status. There were at least three attempts to genotype samples of low DNA quality that resulted in poor amplification. Samples that did not show the same pattern when repeated or when the genotype was not easy to interpret were excluded. All genotyping results were checked by two individual researchers to confirm the genotype classification. To assess genotyping reproducibility, 94 randomly selected samples were regenotyped for each polymorphism and yielded concordant results.

NQO1 genotyping
Primers specific to exon 6 of the NQO1 gene were designed and the target DNA was amplified by PCR. The primer sequences used were the following: 5'-TCCTCAGAGTGGCATTCTGC-3' (sense) and 5'-CCTTCTTTGCGGACCTCTTAT-3' (antisense). Subjects with the more common (C/C) genotype were resistant to restriction with the HinfI restriction enzyme, displaying a single 131 bp fragment. Heterozygotes (C/T) were partially subjected to HinfI digestion, and resulted in fragments of 131, 88 and 44 bp, whereas subjects homozygous for the T allele were subjected to complete digestion and displayed fragments of 88 and 44 bp. The amplification of DNA from 10 cases and 5 controls was unsuccessful for NQO1 C609T genotyping.

EPHX1 genotyping
The polymorphism at amino acid 113 of EPHX1 was analysed by PCR–RFLP using the restriction enzyme EcoR V as described by Smith and Harrison (1997) (21). The primer sequences used were the following: sense primer, 5'-GATCGATAAGTTCCGTTTCAC-3' and antisense primer, 5'-ATCCTTAGTCTTGAAGTGAGG-3'. Since the mEH3 polymorphism is in close proximity and in high linkage disequilibrium with the codon 119G>A polymorphism (rs2292566), the mEH3 variant genotypes were combined to avoid any potential misclassification of the heterozygous mutant genotype (22,23). The polymorphism at amino acid 139 of EPHX1 was analysed by PCR–RFLP using the restriction enzyme RsaI as described by Hasset et al. (1994). The primer sequences were as follows: 5'-ATGAAGGGGCGGCGGGGGCACTAAGGG-3' and antisense primer 5'-CTTGGCGAGGACGGGGCAGTTATGGAA-3'. Samples from 82 cases and 131 controls were not successfully genotyped for the mEH3 polymorphism, whereas samples from 50 cases and 75 controls were not successfully genotyped for the mEH4 polymorphism.

Statistical analyses
Sample size calculations for the case and control group of the study were undertaken, based on control genotype frequencies from previous studies of Caucasian subjects (9,24). Calculations assumed 5% level of significance, 80% power and a genotypic risk of 2.0. The UKFSS Study had 80% power with five level of significance to detect a risk of 2.0 associated with homozygosity or carriage for the NQO1 rare allele, and the same risk for the main effect comparing the more common mEH3 or mEH4 genotype with the mEH3 or mEH4 variant genotypes.

Distributions of genotype frequencies were calculated for the two main study groups (controls and adenomas) and for those with high-risk and low-risk adenomas separately. These calculations were also done separately for men and women. Differences in genotype distributions between cases and controls were tested using the {chi}2 test. Tests for Hardy–Weinberg equilibrium (HWE) were conducted by comparing observed and expected genotype distributions by the {chi}2 goodness of fit test. The P-value applied to a {chi}2 statistic with 1 d.f. Statistical significance for departure of a genotype frequency from its expected frequency under the HWE model was set at P ≤ 0.05.

The data were analysed as an unmatched case–control series using logistic regression and associations were calculated as odds ratios (ORs) with 95% confidence intervals (CIs) with age, gender and sigmoidoscopy centre included in the models as covariates. The NQO1C609T and mEH4 genotypes were analysed under two separate models with homozygosity for the common allele as the reference group in all cases: a three level model (homozygosity for the common allele, heterozygosity, homozygosity for the variant allele) and a two level, or dominant model (homozygous for the common allele, at least one copy of the variant allele). The mEH3 variant genotypes were combined to avoid any potential misclassification of the heterozygous mutant genotype as mentioned above, and were compared with the homozygous common allele.

To investigate the effect of genotype, NQO1C609T, mEH3 and mEH4 were analysed separately with age at FS, centre and sex included as covariates. Alcohol was investigated in a separate model, stratified by the three level genotype variable, with age at FS, centre, smoking and sex included as covariates with low intake as the referent group. Smoking-related analysis was performed based on smoking status and on pack-years. Those with wild-type genotype and the ‘never smokers’ or ‘0 pack-years’ group were the referent group. Significance levels across tertiles by genotype were calculated. For the assessment of gene-smoking associations, smoking status was added to the model (including age, sex and centre) as a covariate. Potential two-way interactions between genotypes and alcohol were assessed by comparing models with and without the interaction term using the likelihood ratio test. All analyses were considered significant at the 5% level. STATA 7.0, Statistics/Data Analysis package for PC (Stata Corporation, College Station, TX) was used for all the analyses.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The characteristics of the study population are shown in Table I. The cases were further classified into high- and low-risk adenomas according to the morphology of the polyps, with a higher number of low-risk adenomas. There were more male subjects in the study and compared with females, males were over-represented among cases, especially among low-risk adenomas, as compared to controls (P = 0.02). Overall, controls were more likely to be never-regular smokers compared to cases (47.1 versus 31.9%, respectively), whereas there were more current smokers among cases (24.8 versus 10.9%) (P < 0.001), including high-risk (29.9 versus 10.9%) and low-risk adenomas (22.9 versus 10.9%). Moreover, cases had significantly higher total alcoholic drink consumption than controls (P < 0.001). This was also evident among high- and low-risk adenomas compared to controls (P < 0.0001). There were no significant differences in any of these characteristics between adenoma subgroups.


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  		Table I Selected characteristics of the UKFSS study population

 
Table II shows associations between NQO1C609T, mEH3 and mEH4 genotypes and colorectal adenoma risk including risk for high- and low-risk adenomas. The distribution of the NQO1C609T genotypes was consistent with HWE (P = 0.58). The low activity allele frequency of 0.17 in controls found in this case–control study is consistent with previous studies and the rare distribution of 2.5% for the homozygous mutant T/T genotype is within the range reported for other Caucasian populations (2,10). There were no overall associations between the NQO1C609T genotypes and colorectal adenoma (Table II). However, a positive association between the NQO1609 C/T genotype and high-risk colorectal adenoma was evident: compared to participants homozygous for the NQO1609*C allele, carriage of the C/T genotype conferred a 1.47-fold increased odds of disease (95% CI, 1.10–1.97; P = 0.19). In contrast, the OR of 0.18 for high-risk adenomas among subjects with the T/T genotype might be a chance finding due to the small number of individuals with that genotype (1 case and 24 controls). Assuming a dominant model of inheritance, the NQO1609 C/T and T/T genotype categories were collapsed to create a ‘T-carrier’ category. Compared to homozygosity for the common C-allele, presence of a T allele conferred a 1.36-fold increased risk of high-risk adenoma (95% CI, 1.02–1.83; P = 0.04). Subgroup analysis by sex and risk status strata showed some evidence of a positive effect of the heterozygous C/T genotype among men with high-risk adenomas (OR, 1.52; 95% CI, 1.10–2.20). The ORs for females were lower and close to unity but did not reach statistical significance (data not shown).


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  		Table II Associations between NQO1C609T, mEH3 and mEH4 genotypes and colorectal adenoma risk

 
The EPHX1 minor allele frequencies observed among the controls in this study (0.33 for the mEH3 His allele and 0.20 for the mEH4 Arg allele) were within the range of those reported in previous studies (Table II) (22,25–29). The genotype distributions for the mEH4 polymorphism were consistent with HWE (P > 0.05), whereas the genotype distributions for the mEH3 polymorphism did not conform to HWE. This has been reported in other investigations (11,15,25,28,30,31) and it has been proposed that an adjacent codon 119G>A polymorphism may interfere with mEH3 genotyping (22) (see Materials and methods). No statistically significant risks for colorectal adenoma were observed with any of the mEH3 and mEH4 variant genotypes. Subgroup analyses revealed no effect of sex (data not shown). However, a statistical significant increased risk of low-risk adenoma among carriers of the heterozygous or the homozygous variant mEH4 genotype was shown. The mEH4 homozygous mutant genotype (Arg/Arg) was associated with a significantly increased risk in the low-risk adenoma group (OR, 1.72; 95% CI, 1.04–2.86; P-trend = 0.02). A less pronounced effect was seen for the combined variant genotypes (OR, 1.24; 95% CI, 1.0–1.54; P = 0.05).

Table III presents risks of colorectal adenoma for the combined effects of NQO1C609T genotypes and smoking status. Smokers (former or current) of the NQO1 common genotype (C/C) or heterozygous mutant (C/T) genotypes were positively associated with colorectal adenoma risk. The presence of the homozygous mutant (T/T) genotype in smokers was not associated with colorectal adenoma risk but this might be due to the small number of individuals with this gene-smoking combination. To increase the power, the heterozygous and the homozygous variant genotypes were combined, and revealed statistically significant elevated risks for former smokers (OR, 1.67; 95% CI, 1.20–2.31) and for current smokers (OR, 4.0; 95% CI, 2.44–6.55). Similarly, statistically significant elevated risks of colorectal adenoma were associated with the C/T or T/T genotypes as pack-years of smoking increased (the trend in all cases was highly significant, P-trend = 0.0001) with the highest OR estimate seen in carriers of the C/T genotype with ≥20 pack-years of smoking (OR, 3.14; 95% CI, 2.10–4.70). However, when tested for interactions, no synergistic effects were found.


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  		Table III Smoking and NQO1C609T genotypes: adjusted ORsa (95%CI) for colorectal adenomas versus controls

 
A statistically significant interaction between alcohol consumption, NQO1 genotype and colorectal adenoma was evident (P for interaction: for three genotype levels, 0.024; for two genotype levels, 0.013). The association between high alcoholic drink consumption and colorectal adenoma was stronger among carriers of the C/C genotype versus the other genotypes (OR, 1.49; 95% CI, 1.11–2.02; P-trend = 0.005) (Table IV).


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  		Table IV Consumption of total alcoholic drink and NQO1 genotypes: adjusted ORsa (95% CI) for colorectal adenomas versus controls

 
There were no differences in risk between mEH3 and mEH4 genotypes with regard to smoking and alcoholic drink consumption (Table V).


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  		Table V Risk (OR and 95% CI)a of colorectal adenoma associated with EPHX1 genotypes and effect modification by alcohol drinks and smoking in cases and controls

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the largest study of NQO1 C609, mEH3 and mEH4 polymorphisms in relation to risk of distal adenomatous polyps. The NQO1 609 T allele was positively associated with high-risk adenoma in this population. In addition, we report for the first time that the association between alcohol consumption and colorectal adenoma was modified by NQO1C609T genotype, such that the relation between alcohol and colorectal adenoma was stronger among those with the more common C/C genotype. There was no overall association between mEH3 and mEH4 variants and colorectal adenoma risk and no effect modification by alcohol and smoking.

The positive associations between the variant NQO1C609T genotypes and adenoma risk were confined to the high-risk adenoma group; individuals with the heterozygous C/T genotype were at ~50% increased risk of developing high-risk adenomas compared to those with the more common genotype and a similar effect was seen for the combined variant genotypes. These results suggest that NQO1C609T polymorphism may increase susceptibility to high risk adenomas, which are thought to have a high malignant potential (32). Recently, Hou et al. (2005) (13) in a study of 725 distal advanced colorectal adenoma cases and 729 controls reported a marginally positive association between NQO1C609T variant genotypes and adenoma risk. To the best of our knowledge this is the only study of adenomas and NQO1C609T polymorphism in the literature and further supports our findings.

The finding in this study of an enhanced adenoma risk for the presence of the NQO1C609T mutant allele among current smokers and among individuals with high cumulative dose in pack-years is comparable with results obtained in two previous studies (9,13). However, when tested for interactions, no statistically significant results were revealed. All three studies, including our study, suggest a detoxification role for NQO1 under smoking exposures and hence the greater risk observed among NQO1609 C/T or T/T carriers may be attributed to reduced detoxification capacity of tobacco derived carcinogens associated with the reduced enzyme activity due to the NQO1C609T polymorphism. One potential mechanism that could explain the increased risk of high-risk adenomas or the increased risk of colorectal adenoma in presence of smoking among individuals with the low activity genotype as seen in this study, is through the induction of K-ras base pair substitutions. Lafuente and coleaugues (10) showed an association between NQO1C609T genotype and the risk for a specific subtype of colorectal cancer (K-ras codon 12 mutant) indicating that genetic variation affecting NQO1 locus may increase colorectal neoplasia in presence of NQO1 metabolizing agents that are capable of inducing K-ras mutations.

Alcohol may increase the risk of colorectal neoplasia through a number of mechanisms including free radical damage to DNA of colon cells (33). The oxidation of ethanol by CYP2E1 is believed to produce free radicals that may be directly genotoxic or that initiate lipid peroxidation and consequently carcinogenesis (34). It is known that the two and four electron reductions catalysed by NQO1 are beneficial to the cell by preventing redox cycling which leads to the generation of free radicals (8,35).

In this study, stratification by NQO1C609T genotype showed increased ORs for the C/C genotype and the trends across tertiles of alcoholic drink consumption were significant. There was some evidence of opposing effects with the mutant NQO1C609T genotypes but the trends across levels of intake were not significant. This difference in the effect of alcoholic drink consumption depending on NQO1 status was supported by the fact that the likelihood ratio test for an interaction between alcoholic drink consumption and NQO1C609T genotype was statistically significant (P interaction, 0.024 for three genotype levels and 0.013 for two genotype levels). It is known that NQO1 reduces quinones to hydroquinones in a single 2-electron step. However, not all hydroquinones are redox-stable, and in some cases metabolism by NQO1 yields a more active product that can react with molecular oxygen to form semiquinones and generate reactive oxygen species (36). In addition, the reduction of the quinone moiety can produce a compound that is capable of alkylating nucleophilic sites including DNA (2). Therefore, the positive effect seen in this study with the more common genotype might well mean that potential bioactive and procarcinogenic compounds such as nitrosamines found in alcoholic drinks interact with NQO1 and hence individuals with higher NQO1 activity may be more predisposed to adenoma formation because they are capable of further activating these compounds.

This is the largest study to date to examine epoxide hydrolase polymorphisms and their interaction with alcohol and smoking on modifying colorectal adenoma risk. Although there was no overall association between mEH3 and mEH4 polymorphisms, the latter was specifically associated with low-risk adenomas. However, this finding should be interpreted with caution given the lack of association in the main group. Previous studies have investigated the mEH3, mEH4 polymorphisms in relation to colorectal adenoma development and some but not all support the hypothesis that exposure to PAHs, i.e. through smoking may increase risk of adenomas, especially in individuals genetically more proficient (carriers of the high mEH activity genotype) in activating these compounds (29,37,38). The findings reported here do not support the hypothesis that EPHX1 genotype influences the relation between smoking and colorectal adenoma.

We also examined the association between intake of total alcoholic drinks and mEH3 and mEH4 genotypes based on the rationale that ethanol consumption is involved in benzo({alpha})pyrene (metabolite of PAH) biotransformation (39). However, there appeared to be no synergistic effect between alcoholic drink consumption and EPHX1 genotypes in this study of adenomas.

Despite the large size of the study, the sample size was still limited within certain genotype and exposure strata, which underscores the need for even larger epidemiological studies. The failure to identify any interactive effects, especially between NQO1C609T, mEH4 genotypes and smoking despite the statistically significant associations within specific strata, might also reflect that other genes and/or environmental factors may be relevant and may in a complex way be related to tobacco or diet carcinogen exposures, which underlines the necessity for multigene-smoking and -diet analysis. Furthermore, as with any measurement of exposure in epidemiological studies, there was the potential for misclassification regarding assessment of smoking habits and diet. The questions included in the FFQ were selected from the EPIC FFQ questionnaire which was fully validated and the correlation coefficient between alcohol consumption using this method with other more accurate measures such as 16 day weighed food records was r = 0.90. (20). Smoking information for the adenoma and control groups was relatively complete and participants provided information on smoking history, duration of smoking and smoking consumption. All smoking data were ‘cleaned’ before analysis to limit any obvious reporting errors; therefore, misclassification of smoking behaviour is thought to be relatively small. Furthermore, one of the strengths of this study is that the control group undertook FS, and therefore, was free of distal lesions. However, some degree of misclassification may have occurred as adenomas in the proximal colon cannot be detected during sigmoidoscopy.

In conclusion, we found no overall association between mEH3 and mEH4 variants and colorectal adenoma risk and no evidence for an interaction between alcohol, smoking and mEH3 and mEH4 genotypes. However, the results of this study provide evidence for an important role of the NQO1C609T polymorphism in susceptibility of adenomatous polyps, the outcome of which depends on the particular exposure. NQO1 has a dual role in xenobiotic metabolism, involving both an activation and a detoxification role, and therefore, the fact that the polymorphic variant allele was positively associated with adenoma risk in this study, especially in presence of tobacco smoke suggests a detoxification role for the enzyme following its induction by chemicals in tobacco smoke, i.e. PAHs. On the other hand, in presence of high alcoholic drink consumption, NQO1 further activates procarcinogens found in alcohol and this action is reflected into an increased risk among carriers of the high activity NQO1 genotype. Controlled feeding studies that examine genotype alongside dietary exposure are likely to determine whether the results found in this study are chance findings or have etiological implications. It might therefore be of particular interest to determine the phenotype–genotype relationship in individuals with different NQO1 genotypes and monitor their enzyme expression in response to alcoholic drink intake.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 6, 2006; revised September 19, 2006; accepted October 3, 2006.


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