Epidemiologic studies have implicated exposure to tobacco smoke and high intakes of cooked, broiled, or well-done meats in the increased risk of colorectal cancers (1-4). Microsomal epoxide hydrolase (mEH) is a phase II biotransformation enzyme which detoxifies epoxides, including carcinogens such as polycyclic aromatic hydrocarbons found in cigarette smoke and cooked meats (5). A tyrosine to histidine substitution in exon 3 (Y113H) of the mEH gene decreases in vitro enzyme activity by 40%, whereas a histidine to arginine substitution in exon 4 (H139R) increases in vitro enzyme activity by 25% (6). Smaller epidemiologic studies evaluating the relationship between the mEH polymorphisms and risk of colorectal cancer and its precursors have been inconclusive (7-9). We evaluated the risk of colon cancer associated with both the mEH Y113H and H139R genotypes in a large case-control study.

Methods for selection of cases and controls and data collection have been described in detail elsewhere (10-12). Briefly, participants were subjects from the Kaiser Permanente Medical Care Program of Northern California, an eight-county area in Utah, and the metropolitan Twin Cities area of Minnesota. Eligibility criteria have been previously described (10). Controls who had never had a previous colorectal tumor were selected from Kaiser Permanente Medical Care Program membership lists in California; driver's license lists, random-digit-dialing, or Centers for Medicare and Medicaid Services lists for Utah; and driver's license or state identification lists in Minnesota.

mEH Genotyping

The mEH Y113H, H139R polymorphisms were detected using the 5′ nuclease assay on a 7900HT sequence detection system (Applied Biosystems, Foster City, CA). Primers and probes and PCR core reagents were purchased from Applied Biosystems. The assays were validated by genotyping 100 individuals by both 5′ nuclease assay and RFLP or sequencing. There were no discrepancies between the two assays. Genotyping was done in 20 μL reactions containing 1× Taqman PCR core reagents 4 mmol/L MgCl2, 200 nmol/L primers (Y113H: 5′CTGGAAGAAGCAGGTGGAGATT3′, 5′TGGCTGGCGTTTTGCAA3′; H139R: 5′TCCACCCTGACTGTGCTCTGT3′, 5′TGGGATGATCTTATAAAACTCGTAGAAA3′), 100 nmol/L probes (Y113: VIC-5′TCAACAGATACCCTCACT3′-NFQ; 113H: 6FAM-5′AACAGACACCCTCACT3′-NFQ; H139: VIC-5′CAGGCCATACCCCGA3′_NFQ; 139R: FAM-5′AGGCCGTACCCCGA3′-NFQ), and 3 ng DNA. Amplification cycles were 50°C for 5 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Positive controls for all the genotypes and reagent controls were included in each plate. Genotyping of 94 randomly selected samples was repeated for each polymorphism. There were no discrepancies.

Statistical Methods

Unconditional logistic regression models were used. Multivariate adjustment included age, sex, body mass index (kg/m2), vigorous physical activity index (13), regular use of aspirin or nonsteroidal anti-inflammatory drugs, and usual number of cigarettes smoked per day. This study had 90% power to detect an odds ratio (OR) of 1.4 for the main effect comparing homozygous wild-type mEH 113YY with homozygous variant mEH 113HH genotype, and 80% power to detect an OR of 1.4 for the equivalent comparison within the H139R genotype.

Of 4,403 eligible participants with valid data for the study, 3,553 participants with available DNA (1,593 cases and 1,960 controls) were genotyped for both the mEH Y113H and H139K polymorphisms. Characteristics of the study population have been described elsewhere (14). Genotype frequencies for both the Y113H and H139K polymorphic sites were in Hardy-Weinberg equilibrium among the controls and among the cases, and did not vary significantly between cases and controls. Frequencies for both the Y113H H allele (0.27 for cases; 0.32 for controls) and H139K K allele (0.17 for cases; 0.22 for controls) are consistent with previously reported genotype frequencies (7, 8, 15-19).

Risks of colon cancer associated with the Y113H and H139K genotypes, combined genotypes, and imputed phenotypes as proposed by Smith and Harrison (19) are summarized in Table 1. Greater usual number of cigarettes smoked increased risk of colon cancer overall, especially when restricted to microsatellite instability–positive colon cancers; however, tests for trend did not identify significant differences by mEH Y113H or H139R genotypes. The risk estimates did not vary significantly when stratified by total mutagen index (20), dietary intake of red or white meat, tumor site (proximal versus distal), or genotype for the NAT2, GSTM1, or CYP1A1 biotransformation enzymes (data not shown).

Table 1.

Risk of colon cancer associated with mEH genotypes

mEH genotypeMain effects*Usual number of cigarettes smoked per day*
Total mutagen index*,
None≤20>200-451452-831≥832
Y113H        
    Y/Y 1.0 (ref.) 1.0 (ref.) 1.2 (0.96-1.5) 1.6 (1.2-2.1) 1.0 (ref.) 1.2 (0.9-1.5) 1.1 (0.9-1.4) 
 789/991 330/473 305/380 154/138 223/315 284/334 282/342 
    Y/H 1.0 (0.9-1.2) 1.0 (0.8-1.3) 1.3 (1.0-1.6) 1.5 (1.1-2.1) 1.0 (0.8-1.3) 1.2 (0.9-1.6) 1.2 (0.9-1.5) 
 655/795 272/376 269/314 114/105 193/265 223/260 239/270 
    H/H 1.1 (0.8-1.4) 1.1 (0.7-1.6) 1.3 (0.90-1.9) 1.5 (0.9-2.7) 1.2 (0.8-1.8) 1.0 (0.6-1.5) 1.4 (0.9-2.1) 
 149/174 55/74 66/75 28/25 48/58 44/63 57/53 
H139R        
    H/H 1.0 (ref.) 1.0 (ref.) 1.3 (1.1-1.5) 1.5 (1.2-1.9) 1.0 (ref.) 1.2 (0.9-1.4) 1.2 (0.9-1.4) 
 1,051/1,267 436/610 425/481 190/176 304/415 371/433 376/419 
    H/R 1.0 (0.8-1.1) 1.0 (0.8-1.2) 1.1 (0.9-1.4) 1.6 (1.2-2.3) 1.0 (0.8-1.3) 1.1 (0.8-1.4) 1.0 (0.8-1.4) 
 479/605 194/276 190/248 95/81 146/192 154/191 179/222 
    R/R 0.8 (0.6-1.2) 1.0 (0.6-1.7) 0.9 (0.5-1.5) 1.4 (0.6-3.2) 0.6 (0.3-1.2) 1.0 (0.6-1.8) 1.2 (0.7-2.2) 
 63/88 27/37 25/40 11/11 4/31 26/33 23/24 
Imputed phenotypes        
    Rapid 1.0 (0.8-1.2) 1.0 (0.8-1.4) 1.1 (0.8-1.5) 1.7 (1.1-2.6) 1.1 (0.7-1.5) 1.3 (0.9-1.7) 1.1 (0.8-1.5) 
 282/351 109/151 113/148 60/52 81/107 103/116 98/128 
    Normal 1.0 (ref.) 1.0 (ref.) 1.2 (0.9-1.5) 1.6 (1.2-2.2) 1.0 (ref.) 1.2 (0.9-1.5) 1.2 (0.9-1.5) 
 694/882 306/441 261/329 127/112 197/288 236/290 261/304 
    Slow 1.1 (0.9-1.2) 1.0 (0.8-1.3) 1.4 (1.1-1.7) 1.4 (1.0-2.0) 1.1 (0.8-1.4) 1.3 (0.97-1.7) 1.2 (0.9-1.6) 
 468/553 187/257 200/217 81/79 138/185 168/188 162/180 
    Very slow 1.1 (0.8-1.4) 1.1 (0.7-1.6) 1.3 (0.9-1.9) 1.5 (0.9-2.7) 1.2 (0.8-1.8) 1.0 (0.7-1.6) 1.4 (0.9-2.2) 
 149/174 55/74 66/75 28/25 48/58 44/63 57/53 
Combined genotypes        
    113YY/139HH 1.0 (ref.)       
 507/640       
    113YY/139HR 1.0 (0.8-1.2)       
 247/310       
    113YH/139HH 1.1 (0.9-1.3)       
 444/513       
    113YH/139HR 1.0 (0.8-1.2)       
 187/242       
    113HH/139HH or 139HR 1.1 (0.8-1.4)       
 145/167       
    Any Y113H/139RR 0.9 (0.6-1.2)       
 63/88       
mEH genotypeMain effects*Usual number of cigarettes smoked per day*
Total mutagen index*,
None≤20>200-451452-831≥832
Y113H        
    Y/Y 1.0 (ref.) 1.0 (ref.) 1.2 (0.96-1.5) 1.6 (1.2-2.1) 1.0 (ref.) 1.2 (0.9-1.5) 1.1 (0.9-1.4) 
 789/991 330/473 305/380 154/138 223/315 284/334 282/342 
    Y/H 1.0 (0.9-1.2) 1.0 (0.8-1.3) 1.3 (1.0-1.6) 1.5 (1.1-2.1) 1.0 (0.8-1.3) 1.2 (0.9-1.6) 1.2 (0.9-1.5) 
 655/795 272/376 269/314 114/105 193/265 223/260 239/270 
    H/H 1.1 (0.8-1.4) 1.1 (0.7-1.6) 1.3 (0.90-1.9) 1.5 (0.9-2.7) 1.2 (0.8-1.8) 1.0 (0.6-1.5) 1.4 (0.9-2.1) 
 149/174 55/74 66/75 28/25 48/58 44/63 57/53 
H139R        
    H/H 1.0 (ref.) 1.0 (ref.) 1.3 (1.1-1.5) 1.5 (1.2-1.9) 1.0 (ref.) 1.2 (0.9-1.4) 1.2 (0.9-1.4) 
 1,051/1,267 436/610 425/481 190/176 304/415 371/433 376/419 
    H/R 1.0 (0.8-1.1) 1.0 (0.8-1.2) 1.1 (0.9-1.4) 1.6 (1.2-2.3) 1.0 (0.8-1.3) 1.1 (0.8-1.4) 1.0 (0.8-1.4) 
 479/605 194/276 190/248 95/81 146/192 154/191 179/222 
    R/R 0.8 (0.6-1.2) 1.0 (0.6-1.7) 0.9 (0.5-1.5) 1.4 (0.6-3.2) 0.6 (0.3-1.2) 1.0 (0.6-1.8) 1.2 (0.7-2.2) 
 63/88 27/37 25/40 11/11 4/31 26/33 23/24 
Imputed phenotypes        
    Rapid 1.0 (0.8-1.2) 1.0 (0.8-1.4) 1.1 (0.8-1.5) 1.7 (1.1-2.6) 1.1 (0.7-1.5) 1.3 (0.9-1.7) 1.1 (0.8-1.5) 
 282/351 109/151 113/148 60/52 81/107 103/116 98/128 
    Normal 1.0 (ref.) 1.0 (ref.) 1.2 (0.9-1.5) 1.6 (1.2-2.2) 1.0 (ref.) 1.2 (0.9-1.5) 1.2 (0.9-1.5) 
 694/882 306/441 261/329 127/112 197/288 236/290 261/304 
    Slow 1.1 (0.9-1.2) 1.0 (0.8-1.3) 1.4 (1.1-1.7) 1.4 (1.0-2.0) 1.1 (0.8-1.4) 1.3 (0.97-1.7) 1.2 (0.9-1.6) 
 468/553 187/257 200/217 81/79 138/185 168/188 162/180 
    Very slow 1.1 (0.8-1.4) 1.1 (0.7-1.6) 1.3 (0.9-1.9) 1.5 (0.9-2.7) 1.2 (0.8-1.8) 1.0 (0.7-1.6) 1.4 (0.9-2.2) 
 149/174 55/74 66/75 28/25 48/58 44/63 57/53 
Combined genotypes        
    113YY/139HH 1.0 (ref.)       
 507/640       
    113YY/139HR 1.0 (0.8-1.2)       
 247/310       
    113YH/139HH 1.1 (0.9-1.3)       
 444/513       
    113YH/139HR 1.0 (0.8-1.2)       
 187/242       
    113HH/139HH or 139HR 1.1 (0.8-1.4)       
 145/167       
    Any Y113H/139RR 0.9 (0.6-1.2)       
 63/88       

NOTE: All estimates are multivariate adjusted for age, sex, body mass index (kg/m2), vigorous physical activity index, regular use of aspirin or nonsteroidal anti-inflammatory drugs, and usual number of cigarettes smoked per day where appropriate.

*

Reported as OR (95% confidence interval), number of cases/number of controls.

Total mutagen index is calculated as the frequency of cooked red meat, poultry, and fish consumption plus the use of drippings multiplied by usual doneness of meat (1, rare; 2, medium-rare; 3, medium-well; 4, well done) and the microwave factor (1, never used; 0.75, sometimes used; 0.5, often used; 0.25, always used; ref. 20).

Imputed phenotype, as classified by Smith and Harrison (19): rapid, 113YY/139HR or 113YY/139RR; normal, 113YY/139HH or 113YH/139HR; slow, 113YH/139HH or 113YH/139RR; and very slow, 113HH/139HH.

Previous reports evaluating the association between mEH Y113H genotype and colorectal cancer have been contradictory. Harrison et al. (7) reported that the 113HH genotype was associated with an increased risk of colon cancer (OR, 3.8; 95% confidence interval, 1.8-8.0), whereas Sachse et al. (9) found the 113HH genotype to be associated with a decreased risk of colorectal cancer (OR, 0.7; 95% confidence interval, 0.5-0.95). Controls for the studies by Sachse et al. (9) and Harrison et al. (7) were not in Hardy-Weinberg equilibrium for the Y113H polymorphism, as has been frequently reported in studies using PCR-RFLP mEH Y113H genotyping methods (6, 9, 16). Neither group evaluated whether environmental factors such as smoking or well-cooked meat intake modified their findings.

With regard to the H139R polymorphism, analyses by Harrison et al. (7), Sachse et al. (9), and Mitrou et al. (8) all failed to find an association between genotype and risk of colorectal cancer. However, among smokers, Mitrou et al. reported an increased risk of colorectal and distal colon cancers with the 139RR genotype (OR, 2.9; 95% confidence interval, 1.3-6.0 and OR, 3.7; 95% confidence interval, 1.6-8.2, respectively).

Both Sachse et al. (9) and Harrison et al. (7) reported a significant difference in Y113H genotype frequencies between cases and controls, suggesting that Y113H genotype may be a susceptibility factor in the development of colorectal cancer. These findings of an altered risk were not confirmed in our study, and may be partially attributable to the fact that the previous studies included cases of rectal cancers, whereas rectal cancer was an exclusion criterion for our study.

It would be expected that mEH genotypes would alter risk of colon cancer under specific conditions such as exposure to cigarette smoke and dietary mutagens. However, most of the previous studies did not explore these interactions. This study, which found no association between mEH Y113H or H139R genotype and colon cancer risk, is the largest to date, and included evaluation of exposure to cigarette smoke and well-cooked meats. Although we did not observe any difference by mEH genotype, environmental exposure to cigarette smoke and dietary mutagens have predicted risk of colon cancer and adenomatous and hyperplastic polyps in our past research (15, 20-22).

Studies of adenomatous polyps, possible precursors of colorectal cancer, have similarly failed to find an association between mEH genotype and risk of polyps. However, some have observed interactions with smoking and cooked meat by mEH genotype (15, 16, 23, 24).

We conclude that the mEH Y113H and H139R genotypes do not affect risk of colon cancer, and may only be a factor in the development of adenomatous polyps (earlier stages of colon carcinogenesis) under conditions of elevated carcinogen exposure.

Grant support: NIH research grants CA48998, CA59045, and CA61757 and National Cancer Institute training grant R25 CA94880 (K. Robien).

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.

We thank Juanita Leija for technical assistance with the mEH genotype determinations.

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