Epoxide hydrolase polymorphisms, cigarette smoking and risk of colorectal adenoma in the Nurses' Health Study and the Health Professionals Follow-up Study

Gregory J. Tranah1,3, Edward Giovannucci1,2,4, Jing Ma4, Charles Fuchs4, Susan E. Hankinson1,4 and David J. Hunter1,3,4,5

1 Department of Epidemiology, 2 Department of Nutrition and 3 Harvard Center for Cancer Prevention, Harvard School of Public Health, Boston, MA 02115, USA and 4 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA

5 To whom correspondence should be addressed Email: nhdjh{at}channing.harvard.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Microsomal epoxide hydrolase (mEH) is involved in the bioactivation and detoxification of polycyclic aromatic hydrocarbons derived from tobacco smoke and charred meat intake. Two coding region mEH variants located in exon 3 (Tyr113His) and exon 4 (His139Arg) have been described and may affect the enzyme's specific activity. We investigated these polymorphisms and tested interactions with smoking and charred meat intake in relation to risk of colorectal adenoma in two case–control studies nested in the Nurses' Health Study (NHS) and Health Professionals Follow-up Study (HPFS) cohorts. mEH exon 3 and exon 4 polymorphisms were not associated with overall risk of adenoma among 556 incident cases and 557 controls from the NHS or 376 prevalent cases and 725 controls from the HPFS. A statistically significant interaction was found between the exon 4 polymorphism and smoking for men (P = 0.03) and a borderline significant interaction was found between the exon 3 polymorphism and smoking for women (P = 0.06). Women having the exon 3 ‘rapid’ Tyr/Tyr genotype were at increased risk when exposed to either ≥25 pack-years smoking [relative risk (RR) = 2.43, 95% confidence interval (CI) 1.47–4.01] or <25 pack-years of smoking (RR = 1.73, 95% CI 1.10–2.73) relative to non-smokers. Men with the exon 4 ‘slow’ His/His genotype were at increased risk when exposed to ≥25 pack-years smoking (RR = 2.21, 95% CI 1.43, 3.41) or <25 pack-years smoking (RR = 1.71, 95% CI 1.13–2.59) relative to non-smokers. Charred meat intake was not associated with adenoma risk and there was no significant interaction with either mEH polymorphism. Our results indicate that individuals exposed to ≥25 pack-years smoking were at increased risk for colorectal adenoma and that risk is related to dose of tobacco carcinogens and mEH activity level, but the results were not consistent between men and women.

Abbreviations: BMI, body mass index; CI, confidence interval; HPFS, Health Professionals Follow-up Study; mEH, microsomal epoxide hydrolase; NHS, Nurses' Health Study; PAHs, polycyclic aromatic hydrocarbons; PMH, post-menopausal hormone; RR, relative risk; SNP, single nucleotide polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exposure to tobacco smoke and charred red meat has been associated with the formation of colorectal adenomas and carcinogenic substances derived from these exposures, such as polycyclic aromatic hydrocarbons (PAHs) and nitrosamines, may contribute to the elevated risk of adenoma (14). PAHs are metabolized through complex enzymatic pathways involving phase I and phase II biotransformation reactions. The metabolism of PAHs is carried out by cytochrome P450 enzymes, glutathione S-transferases and epoxide hydrolase, and functional polymorphisms in these genes have been associated with adenoma risk in some studies (57). Microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of reactive epoxide intermediates in preparation for conjugation reactions prior to excretion (8). While mEH usually results in detoxification of xenobiotic substances, it is also involved in the metabolic activation of PAHs, triggering the formation of highly reactive metabolites that can damage DNA, RNA and protein components (4,9). Specifically, mEH catalyzes the metabolic conversion of benzo[a]pyrene (a component of cigarette smoke and charred meat) to the highly mutagenic (+)-anti-7,8-diol compound, which is converted by cytochrome P450 enzymes CYP1A1 and CYP3A4 into the (+)-anti-7,8-diol-9,10-epoxide. Evidence indicates that the (+)-anti-7,8-diol-9,10-epoxide intercalates into DNA by binding to the exocyclic 2-amino position of guanine (10) and is active in DNA adduct formation in human colon mucosa (11).

Two coding region mEH variants located in exon 3 (Tyr113His) and exon 4 (His139Arg) have been described by Hassett et al. (12). Early in vitro analyses of the resultant proteins showed a 40% decrease (Tyr113His) and a 25% increase (His139Arg) in expression, possibly caused by alterations in protein stability (12). More recent experiments have demonstrated that structural differences encoded by the variants may have only a modest effect on the enzyme's specific activity and that inter-individual differences in mEH expression range from 2- to 10-fold (1215).

In a case–control study of 530 cases and 649 controls Ulrich et al. (7) found that neither polymorphism was associated with an increased risk of colorectal adenoma. However, in the presence of smoking and high cooked meat intake, genotypes producing low activity (slow) phenotypes were associated with increased risk, although no statistically significant gene–environment interactions were observed (7). Cortessis et al. (5) found no association between either mEH polymorphism and risk of adenoma in a case–control study of 464 cases and 510 controls. In an analysis combining exon 3 and exon 4 genotypes into low (slow), medium and high (rapid) activity phenotypes, medium and high mEH activity was associated with adenoma among current smokers. An increased risk was also observed among high (rapid) mEH activity individuals that consumed charred meat; this interaction was statistically significant (5). Harrison et al. (16) found an association between the low activity (slow) genotype and increased risk of colorectal cancer.

Associations have been reported between mEH exon 3 high activity (rapid) genotype and increased risk of ovarian cancer (17), lung cancer (18) and cancer of the oral cavity (19), while the low activity (slow) genotype has been associated with increased risk of cervical cancer (20). The aim of this study was to investigate the Tyr113His and His139Arg mEH polymorphisms and their potential interaction with smoking and intake of charred meat in relation to risk of colorectal adenoma. We analyzed samples from two case–control studies nested in the Nurses' Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) cohorts.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
The NHS is an ongoing prospective study of 121 700 US female registered nurses. Details of the design and follow-up of this cohort have been described previously (21). Briefly, at enrollment in 1976, the participants, who were 30–55 years old, completed a mailed questionnaire providing information on risk factors for cancer and cardiovascular disease. Biennially, updated exposure and disease information was collected by mail, including reports of endoscopy and polyp diagnosis. Self-reported diagnoses of colorectal adenomas were confirmed through histopathological reports reviewed by a study investigator. From 1989 to 1990, blood samples were collected from 32 826 of the NHS participants. To be eligible for selection as a case or control for the colorectal adenoma analyses, women must have supplied a blood sample between 1989 and 1990, must have undergone sigmoidoscopy or colonoscopy after the date of return of the blood sample and not have had a cancer or adenoma diagnosis, excluding non-melanoma skin cancer, prior to the date of endoscopy. One control was matched to each case on year of birth, month of blood draw, fasting status, time period of endoscopy (within 2 years) and routine screening, gastrointestinal symptoms or family history of colorectal cancer as indication(s) for endoscopy. A total of 557 incident cases and 557 matched controls were identified. Subsequently, one colon polyp case was identified as hyperplastic and removed from the final analysis, which included 556 cases and 557 controls.

The HPFS is an ongoing prospective study of the causes of chronic diseases in men. The cohort began in 1986 when 51 529 US male dentists, optometrists, osteopaths, podiatrists, pharmacists and veterinarians, aged 40–75 years, responded to a mailed questionnaire (22). These men provided baseline information on age, marital status, height and weight, ancestry, medications, smoking history, medical history, physical activity and diet. Exposure and medical history information were updated every 2 years. When a participant reported a diagnosis of colorectal adenomas on the follow-up questionnaires, we asked for permission to acquire the relevant medical records. All cases in this analysis were confirmed through histopathological reports reviewed by a study investigator. Blood samples were collected between 1993 and 1994 from 18 025 participants, among whom 367 had been diagnosed with adenomatous polyps between 1986 and 1994 and were confirmed by medical records. To be eligible for selection as a case or control, a man must have completed a valid dietary questionnaire in 1986, supplied a blood sample in 1993–1994, have undergone sigmoidoscopy or colonoscopy after the date of return of the blood sample and not have had a cancer diagnosis, excluding non-melanoma skin cancer, prior to the date of endoscopy. Seven hundred and thirty-six men who were diagnosed polyp-free at the time of case assessment were selected as controls and two controls matched to each case on year of birth, year of endoscopy and whether they had had a previous endoscopy. Subsequently, nine controls were identified as cases, so the final analysis included 376 cases and 725 controls.

Sample collection
Venous blood samples were separated into plasma, buffy coat and red blood cells and stored in liquid nitrogen freezers. Genomic DNA was extracted from 50 µl of buffy coat diluted with 150 µl of phosphate-buffered saline and using the QIAmp (Qiagen Inc., Chatsworth, CA) 96-spin blood protocol according to the manufacturer's instructions. Genomic DNA concentrations were calculated in 96-well format using PicoGreen technology (Molecular Probes, Eugene, OR).

mEH genotypes
Genotyping of mEH Tyr113His (rs1051740) and His139Arg (rs2234922) was carried out using the TaqMan allelic discrimination system. PE Applied Biosystems synthesized TaqMan primers and probes (Table I). Following PCR amplification, end-point fluorescence was read with an Applied Biosystems 7900HT instrument and genotypes were assigned using Allelic Discrimination software (Applied Biosystems SDS Software v.1.7a). Ten percent quality control samples were included and each analysis included no DNA template controls. Laboratory personnel were blind to quality control and case–control status. A genotyping success rate of >95% was attained for each mEH Tyr113His and His139Arg.


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Table I. mEH TaqMan based genotyping analysis

 
Statistical analyses
Adenoma risk was considered in relation to mEH genotypes. Polymorphisms were categorized into Tyr/Tyr (rapid), Tyr/His and His/His (slow) for mEH exon 3 and His/His (slow), His/Arg and Arg/Arg (rapid) genotypes for exon 4, respectively. In addition, we examined four genotype combinations representing imputed phenotypes according to Smith and Harrison (9): rapid, exon 3 Tyr/Tyr and exon 4 Arg/Arg or His/Arg; normal, exon 3 Tyr/Tyr and exon 4 His/His or exon 3 Tyr/His and exon 4 His/Arg; slow, exon 3 Tyr/His and exon 4 His/His; very slow, exon 3 His/His and exon 4 His/His.

Genotypes were considered in combination with pack-years of smoking and intake of charred meat. Data on charred meat consumption were obtained from participant responses to the following question: ‘How often do you eat meat that was charred during cooking (e.g. during barbecuing or broiling)?’. The charred meat question had six possible responses ranging from ‘never’ to ‘two or more times/week’. Subjects were categorized into three groups for pack-years of smoking (0, <25 and ≥25 years) and two groups for charred meat intake (<2 and ≥2 times/month) based on information collected in 1986 for the HPFS. All NHS analyses were based on smoking information collected in 1990, 1992, 1994 and 1996 and charred meat intake information collected in 1990.

Both conditional and unconditional logistic regression were used to compute odds ratios to estimate relative risk (RR), 95% confidence interval (CI), tests for interaction and to control for potential confounding variables. Although the dataset was initially based on matched cases and controls, some of the stratified analyses required unconditional analysis to maximize power. Separate analyses of men and women using unconditional logistic regression were controlled for age, history of previous endoscopy, year of endoscopy, family history of colorectal cancer, smoking history, aspirin use, body mass index (BMI), post-menopausal hormone (PMH) use (NHS only), physical activity and intake of red meat, charred meat, folate and alcohol. Combined NHS and HPFS analyses using unconditional logistic regression were controlled for age, sex, family history of colorectal cancer, smoking history, aspirin use, BMI, physical activity and intake of folate, red meat and alcohol. The results from conditional and unconditional logistic regression were similar, so only results for unconditional logistic regression are presented here. Interactions between genotype and exposure to smoking and charred meat were tested by computing a cross-product term using the three-category and imputed four-category ordinal values of genotype with the three-category smoking and two-category charred meat variables. The P value for interaction was based on the Wald test for the cross-product term in a model containing the main effects of genotype and exposure variable. To test for interaction between genotype, smoking and sex, we compared cross-product (genotype x smoking) ß terms obtained separately from the NHS and HPFS using a {xi}2 test with 1 degree of freedom. All P values are based on two-sided tests. All statistical analyses were done using the SAS 6.0 statistical package (SAS Institute).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diet and lifestyle factors
We first examined whether the risk factors for colorectal adenoma were similar in the cases and controls that provided blood samples compared with previous observations for each cohort (Table II). For HPFS, the risk patterns observed for this sample were largely similar to that from the entire cohort for adenoma cases, as previously reported (2326). For colorectal adenoma, family history of colorectal cancer was associated with increased risk, smoking was associated with an increased risk relative to never smokers, alcohol intake of ≥30 g/day was associated with increased risk relative to infrequent drinkers and regular aspirin use was associated with a slightly lower risk relative to non-users (RR = 0.80, 95% CI 0.61–1.04). Physical activity, BMI, red meat and folate intake were not associated with risk of total adenoma in the HPFS sample. For the NHS, the risk patterns observed for this sample were largely similar to that from the entire cohort for adenoma cases, as previously reported (22,27). For total colorectal adenoma, smoking ≥25 pack-years was associated with an increased risk relative to never smokers, while regular aspirin use 1–6 and ≥7 times per week was associated with a slightly decreased risk (RR = 0.86, 95% CI 0.64–1.15; RR = 0.79, 95% CI 0.57–1.10, respectively) relative to non-users, while BMI>30 was associated with a slightly increased risk. Physical activity, PMH use and alcohol, red meat and folate intake were not significantly associated with risk of total adenoma in the NHS blood group. In previous analyses, BMI, physical activity and PMH were strongly related to large adenomas (2227).


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Table II. Relative risks for risk factors for colorectal adenoma in the Nurses' Health Study (NHS) and the Health Professionals Follow-up Study (HPFS)

 
mEH polymorphisms
Table III presents the main associations between mEH genotypes and adenoma risk. In the NHS and HPFS control populations the exon 4 genotype distribution was in Hardy–Weinberg equilibrium. For men and women the control genotype distributions for exon 3 were not in Hardy–Weinberg equilibrium (P < 0.001), which was also reported in Cortessis et al. (5) and Sachse et al. (28). Our study had 80% power at the two-sided P = 0.05 level to detect odds ratios of 1.30 for the variant 113His allele and 1.35 for the variant 139Arg allele in the combined NHS and HPFS groups. With regard to risk of adenoma, neither mEH genotype showed a statistically significant association, except for a slightly increased risk for the rapid exon 4 homozygous variants in both the NHS and HPFS cohorts and in the combined NHS + HPFS analysis. No clear patterns emerged when the imputed phenotypes were considered.


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Table III. Associations between mEH genotype and colorectal adenoma risk in the Nurses' Health Study and the Health Professionals Follow-up Study

 
We next examined each mEH polymorphism stratified by pack-years of smoking and intake of charred meat (Tables IV and V). For pack-years of smoking we found a significant interaction with the exon 4 polymorphism in the HPFS (P = 0.03) and a borderline significant interaction with the exon 3 polymorphism in the NHS (P = 0.06). Women having the exon 3 ‘rapid’ Tyr/Tyr genotype were at the highest risk of colorectal adenoma when exposed to either ≥25 pack-years smoking (RR = 2.43, 95% CI 1.47–4.01) or <25 pack-years of smoking (RR = 1.73, 95% CI 1.10–2.73) relative to non-smokers with the rapid Tyr/Tyr genotype. Women exposed to ≥25 pack-years smoking and carrying the Tyr/His genotype were also at increased risk (RR = 1.74, 95% CI 1.01–2.99) relative to non-smokers with the rapid Tyr/Tyr genotype. Among men, risk of colorectal adenoma increased with pack-years smoking across all levels of mEH exon 3 genotype. For the HPFS, a significant interaction with pack-years of smoking and the exon 4 polymorphism was present (P = 0.03). There was a significantly increased risk for colorectal adenoma among men having the ‘slow’ His/His genotype exposed to ≥25 pack-years smoking (RR = 2.21, 95% CI 1.43–3.41) and <25 pack-years smoking (RR = 1.71, 95% CI 1.13–2.59) relative to non-smokers with the rapid His/His genotype, while men homozygous for the variant genotype were at a non-significant increased risk regardless of exposure. Women were at increased risk if they had the exon 4 ‘slow’ His/His genotype and were exposed to ≥25 pack-years smoking (RR = 1.78, 95% CI 1.16–2.72) relative to non-smokers with the rapid His/His genotype. Due to the conflicting results obtained for women and men, we tested the three-way interaction between genotype, smoking and sex to determine if sex contributes to the genotype–smoking interaction. We compared the (genotype x smoking) ß terms obtained separately from the NHS and HPFS for each polymorphism to test for a sex interaction. Neither three-way interaction was significant (exon3, P = 0.29; exon 4, P = 0.38). Collapsing the two populations did not increase our power to detect genotype and smoking interactions (exon 3, P = 0.22; exon 4, P = 0.12).


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Table IV. Relationship of pack-years of smoking and intake of charred meat to colorectal adenoma risk stratified by mEH exon 3 genotype in the Nurses' Health Study and the Health Professionals Follow-up Study

 

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Table V. Relationship of pack-years of smoking and intake of charred meat to colorectal adenoma risk stratified by mEH exon 4 genotype in the Nurses' Health Study and the Health Professionals Follow-up Study

 
We then examined the imputed mEH genotypes in combination with pack-years smoking and intake of charred meat (Table VI). Unlike the results for individual mEH exon 3 and exon 4 genotypes, interactions between pack-years smoking and the four imputed genotypes were not apparent. The combined ‘slow’ phenotype was consistently associated with a significantly increased risk among individuals that smoked ≥25 years relative to non-smokers with the imputed ‘normal’ phenotype. Intake of charred meat was not appreciably related to risk of colorectal adenoma among men or women and showed no clear pattern with mEH genotype.


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Table VI. Relationship of pack-years of smoking and intake of charred meat to colorectal adenoma risk stratified by mEH exon 3 and exon 4 combined genotypes in the Nurses' Health Study and the Health Professionals Follow-up Study

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we observed no apparent association between mEH polymorphisms at exon 3 and exon 4 and risk of colorectal adenoma. These results for the main effects of mEH are compatible with previously reported findings for adenoma risk and mEH exon 3 and 4 polymorphisms (5,7). Two previous investigations reported that the mEH Tyr113His genotype distribution was not in Hardy–Weinberg equilibrium (5,28). Assay-introduced bias is unlikely since each study, including the current study, used different genotyping methods to detect the Tyr113His polymorphism.

Since exposure to carcinogenic substances (e.g. PAHs) derived from tobacco smoke and charred red meat is associated with the formation of colorectal adenomas (13) and mEH functions to regulate the oxidation status of a wide range of xenobiotic substrates like PAHs, we predicted the associations between mEH activity and adenoma risk to be modified by such substances. Significant interactions were found between mEH genotype and smoking for the exon 3 polymorphism in women and for the exon 4 polymorphism in men. Among women, the highest risk was for individuals who had accumulated either <25 or ≥25 pack-years smoking and were homozygous for the ‘rapid’ genotype at exon 3. Among men, smokers were at significantly increased risk if they were homozygous for the ‘slow’ genotype at exon 4. Risk was slightly increased for all men homozygous for the ‘rapid’ allele at exon 4. Inspection of the non-significant smoking and mEH interactions for men (exon 3) and women (exon 4) showed similar patterns, with individuals accumulating ≥25 pack-years smoking at increased risk regardless of genotype. We found no significant interactions between the imputed activity levels based on combined exon 3 and exon 4 genotypes and smoking. Risk increased as smoking exposure increased and was not affected by genotype, although for women and men combined the greatest risk was for individuals having the imputed ‘slow’ phenotype.

These results are in contrast to those reported in a case–control study by Ulrich et al. (7), who reported that individuals with a high level of smoking or intake of charred meat who carried ‘slow’ activity alleles at exon 3 and exon 4 were at highest risk of adenoma. They also found that individuals with the imputed ‘very slow’ phenotype had a very strong increase in risk associated with smoking and intake of charred meat (7). In contrast, Cortessis et al. (5) carried out an analysis on combined exon 3 and exon 4 genotypes with imputed slow, medium and rapid activity phenotypes. Their activity levels were based on the number of ‘stable’ (rapid) alleles present in the combined genotype (Tyr at exon 3, Arg at exon 4). The slow activity phenotype included no or one ‘stable’ alleles, medium activity included two ‘stable’ alleles and rapid activity included three or more ‘stable’ alleles. Risk of adenoma among current smokers versus never smokers increased with increasing mEH activity level and was highest for those with the combined ‘rapid’ activity genotype with no significant interaction. Cortessis et al. (5) also reported a significant interaction between combined genotype and consumption of charred meat, with the highest risk among individuals having the rapid activity genotype and a high intake of charred meat.

Our study differed from the clinic-based Ulrich et al. (7) and Cortessis et al. (5) studies in several ways. The participants in our study, like those in the Ulrich et al. (7) study, were mostly Caucasian (>95%), while the participants in the Cortessis et al. (5) study were 55% Caucasian with 45% of the subjects consisting of Hispanic, Asian and African-American individuals. Diagnosis of colorectal adenomas was based in colonoscopy in the Ulrich et al. study (7), while diagnosis in our study and in Cortessis et al. (5) was based on sigmoidoscopy and/or colonoscopy. Our exposure information on smoking and diet was collected prospectively and thus less prone to recall bias.

While we did not find an association with adenoma risk and intake of charred meat, our measure of intake may not account for total carcinogen intake from charred meats due to measurement error resulting from the simple questionnaire used during follow-up in this study (a more complex questionnaire on cooked meats was added to the studies later). Also, exposure to PAHs from cigarette smoking is higher and more continuous than the dose associated with sporadic consumption of charred meat, while the specific carcinogens in cigarette smoke may differ from those found in charred meat.

Overall, individuals exposed to ≥25 pack-years smoking were consistently at increased risk for colorectal adenoma at all mEH activity levels. In addition, the effect of tobacco smoking on risk is modified by mEH activity level, suggesting that metabolic activity alters dose of tobacco carcinogens in men and women. Although a hormonal effect could explain the differences observed between men and women in this study, the role of mEH in hormone function and metabolism remains unclear. In vitro studies indicate the involvement of mEH in ovarian estrogen production (29) and that mEH is one of two protein components of the heterodimeric anti-estrogen binding site complex (30). The differences observed between women and men, though, may suggest that the interactions between mEH genotype and smoking seen in this study are chance results. The inconsistencies among the current and the previous studies may be due to the complexities of benzo[a]pyrene metabolism and the role of mEH, cytochrome P450 enzymes and glutathione S-transferases in this process. Specifically, mEH lies on one of two bioactivator pathways responsible for the transformation of benzo[a]pyrene into adduct-forming compounds. Reactions involving mEH, CYP1A1 and CYP3A4 lead to the formation of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts. Chemical inhibition of mEH, though, leads to the preferential formation of 9-OH-benzo[a]pyrene adducts through the alternative adduct-forming pathway involving CYP1A1 and CYP2C9 (31). In addition to PAH metabolism, CYP1A1 and CYP2C9 are responsible for metabolizing a wide range of chemicals while mEH might be involved in mammalian development and physiological homeostasis (32), emphasizing the complex functions carried out by these metabolic enzymes. Ultimately, the metabolic processing of benzo[a]pyrene and other PAHs found in cigarette smoke and charred meats is carried out by cytochrome P450 enzymes, glutathione S-transferases and mEH through a complex set of chemical reactions. In order to better understand the role of PAHs in the etiology of colorectal adenoma, it is important to consider polymorphisms in the constituent enzymes responsible for the metabolism of these compounds (mEH, CYP1A1, CYP2C9, CYP3A4 and glutathione S-transferases) and how their interactions ultimately regulate the process of cellular detoxification.


    Notes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    Acknowledgments
 
We thank the participants of the Nurses' Health Study and the Health Professionals Follow-up Study for their cooperation and participation. The authors are grateful to Hardeep Ranu and Yan Liu for technical assistance. This work was supported by National Institutes of Health research grants CA70817, CA87969 and CA55075. G.J.T. is supported by training grant CA 09001-27 from the National Institutes of Health.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 20, 2003; revised January 22, 2004; accepted February 14, 2004.