Epoxide hydrolase polymorphisms and risk of colorectal cancer: the - - PDF document

Epoxide hydrolase polymorphisms and risk of colorectal cancer: the Fukuoka Colorectal Cancer Study

Hoirun Nisa1,2*, Suminori Kono1

1Department of Preventive Medicine, Graduate School of Medical Sciences, Kyushu

University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan 812-8582. Phone: (092) 642-6113; Fax: (092) 642-6115.

2Faculty of Medicine and Health Sciences, State Islamic University Jakarta, Indonesia. *Corresponding author

Abstract We investigated the relation the relation of polymorphisms in exon 3 (Y113H) and exon 4 (H139R) of the Epoxide Hydrolase 1 (EPHX1) with the colorectal cancer risk, and the interaction between these polymorphisms and cigarette smoking on the risk of colorectal

• cancer. We used data from Fukuoka Colorectal Cancer Study, a population-based case-control

study, including 685 cases and 778 controls who gave informed consent to genetic analysis. Interview was conducted to assess lifestyle factors, and DNA was extracted from buffy coat. Neither of the two polymorphisms was associated with the risk of colorectal cancer. Odds ratios of colorectal cancer for predicted intermediate and high EPHX1 activities in comparison with predicted low EPHX1 activity were 1.03 (95% confidence interval, 0.81-1.30) and 0.92 (0.67-1.25), respectively. No interaction between EPHX1 polymorphisms

• r the combination of EPHX1 polymorphisms and cigarette smoking on colorectal cancer risk

was shown. These findings suggest that EPHX1 polymorphisms do not play an important role in colorectal carcinogenesis. Keywords: Cancer genetics, Colorectal cancer, Smoking

Background Colorectal cancer accounts for 10% of all cancers and is the third most common cancer in the world [1]. In Japan, the temporal trend showed a marked increase in the incidence of and mortality from colorectal cancer until 1990s [2], and the rates are currently among the highest in the world [1]. Risk for colorectal cancer is influenced by both environmental and genetic factors [3]. Several lifestyle factors such as obesity, physical inactivity, alcohol use, and high intake of red meat have been implicated as conferring increased risk of colorectal cancer [4]. It has been a recent interest whether smoking is related to increased risk of colorectal cancer [5]. While smoking has consistently been related to increased risk of colorectal adenomas [6], epidemiological studies on smoking and colorectal cancer are rather disparate in their findings. However, a recent meta-analysis reported a modest increase in the risk of colorectal cancer associated with a long-term smoking [7]. Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the metabolism of reactive epoxides including polycyclic aromatic hydrocarbons (PAH), carcinogens found in cigarette smoke [8]. The EPHX1 converts benzo(a)pyrene 7,8 epoxide to the less reactive and more water-soluble dihydrodiol, benzo[a]pyrene 7,8 diol [8]. Although this reaction is generally considered as a detoxification reaction, the less reactive dihydrodiol can be further activated into a highly reactive benzo(a)pyrene 7,8 dihydrodiol 9,10 epoxide [9]. Two functional polymorphisms are known in the EPHX1 gene; one is the Y113H in exon 3 (rs 1051740), and the other is the H139R in exon 4 (rs 2234922) [10]. In vitro, the EPHX1 113H allele (slow allele) is associated with a 40% decrease in enzyme activity, and the 139R allele (rapid allele) has increased activity about 25% [10]. Individuals homogyzous or heterozygous for the 113H were shown to have decreased risks of lung cancer [11-13] and upper aerodigestive cancer [14]. Furthermore, high-activity phenotype imputed from the combined

genotypes of the Y113H and H139R was associated with increased risk among those with a high exposure to cigarette smoking [11, 14]. These findings suggest that the EPHX1 polymorphisms may play a role in the development of tobacco-related cancers. The 113H allele was associated with an increased risk of bladder cancer [15], however. Several studies have also addressed the association of the EPHX1 polymorphisms with colorectal cancer [16-21] and adenomas [21-26], but reporting inconsistent findings. Individuals with the 113HH genotype had an increased risk of colorectal cancer in the earliest study [16] but a decreased risk in the subsequent study [17]. The other studies showed no measurable association of Y113H, H139R polymorphism or the imputed activity phenotype in relation to colorectal cancer risk [18-21]. On the other hand, high-activity phenotype was associated with an increased risk of colorectal adenomas [22, 23] among smokers, whereas individuals homozygous for the 113H allele and those with the composite genotype representing very slow activity showed an increased risk of colorectal adenomas when they had a high exposure to smoking [26]. In the present study, we examined the risk of colorectal cancer in relation to the EPHX1 Y113H and H139R polymorphisms and assessed the interaction between these polymorphisms and cigarette smoking in the Fukuoka Colorectal Cancer Study, a community-based case-control study in Japan. Materials and Methods The Fukuoka Colorectal Cancer Study aims to examine the relation of lifestyle factors and genetic susceptibility to the risk of colorectal cancer among residents living in Fukuoka City and three adjacent areas. The study protocol was approved by the ethics committee of the Kyushu University Faculty of Medical Sciences and all but two participating hospitals. These

two hospitals had no ethics committee at the time of the survey, and an approval was obtained from the director of each hospital. Details of methodological issues have been previously described [27].

• Subjects. Cases were incident cases of colorectal cancer who were admitted for

surgery to one of the participating hospitals (two university hospitals and six affiliated hospitals) during the period of September 2000 to December 2003. Eligible cases were those who were at the age of 20 to 74 years at the time of diagnosis, lived in the study area, and had no prior history of partial or total removal of the colorectum, familial adenomatous polyposis,

• r inflammatory bowel disease. They had to be mentally competent to give an informed

consent and to complete the interview. Of the 1,053 eligible cases, a total of 833 (80%) participated in the interview and 685 gave an informed consent for the genotyping. Controls were randomly selected from the study areas by frequency-matching to the expected distribution of incident cases with respect to gender and 10-year age class. Criteria for eligible controls were the same as described for cases except that they had no prior diagnosis

• f colorectal cancer. Of 1500 persons control candidates who were selected by a two-stage

random sampling, 833 participated in the survey and 778 gave an informed consent for

• genotyping. The participation rate for the interviews was calculated as 60% (833 of 1,382),

after excluding 118 persons for the following reasons: death (n = 7), migration from the study area (n = 22), undelivered mail (n = 44), mental incompetence (n = 19), history of partial or total removal of the colorectum (n = 21), and diagnosis of colorectal cancer after the survey (n = 5).

• Interview. Research nurses interviewed cases and controls in person regarding

smoking, alcohol intake, physical activity and other factors using a uniform questionnaire. Interviews for cases were conducted during admission, and those for controls were mostly

conducted at public community centers or collaborating clinics. The index date for cases was the date of the onset of symptoms or screening, and that for controls was the time of the

• interview. Height (cm), body weight (kg) currently and 10 years earlier were elicited. Body

mass index (kg/m2) 10 years earlier was used because the current body mass index was unrelated to the risk [28]. Body weight 10 years earlier was not ascertained from 2 cases and 10 controls and was substituted with the current body weight. Smoking history was ascertained by asking the individuals whether they had ever smoked cigarettes daily for one year or longer. Years of smoking and numbers of cigarettes smoked per day were ascertained for each decade of age. Cumulative exposure to cigarette smoking until the beginning of the previous decade of age was expressed by cigarette-years, the number of cigarettes smoked per day multiplied by the years of smoking, and classified into 0, 1–399, 400–799 and >800 cigarette-years. Alcohol consumption at the time of five years prior to the referent time was elicited. Questions on physical activities elicited type of job and non-occupational activities five years prior to the referent time. As previously described in detail [28], non-occupational physical activity was expressed as a sum of metabolic equivalents (MET) multiplied by hours of weekly participation in each activity. Genetic Data. DNA was extracted from the buffy coat by using a commercial kit (Qiagen GmbH, Hilden, Germany). The following genotyping procedures used 1 µL template DNA with a concentration of 10 ng/µL. Genotyping of the EPHX1 Y113H polymorphism was carried out by the TaqMan assay (Applied Biosystems, Inc., Foster City, CA), using the Stratagene Mx3000P Real-Time QPCR system (Agilent Technologies, USA). The EPHX1 H139R polymorphism was determined by the PCR-RFLP method as described elsewhere [29], using primers 5′-GGTGCCAGAGCCTGACCGTGC-3′ (sense) and

5′-ATGGAACCTCTAGCAGCCCCGTACC-3′ (anti-sense). The PCR product of 319 bp was digested with RsaI, resulting in fragments of 297 and 22 bp for the 139His allele and fragments of 177, 122 and 22 bp for the 139Arg allele. Statistical Methods. The EPHX1 activity phenotype was imputed on the basis of the number of putative high-activity alleles (113Y and 139R) in the combined genotype [11]. Associations of EPHX1 genotypes with colorectal cancer risk were examined in terms of

• dds ratio (OR) and 95% confidence interval (CI) which were obtained from logistic

regression analysis. Statistical adjustment was made for 5-year age class (starting with the lowest class of <50 years), sex, residence area (Fukuoka City or the adjacent areas), body mass index 10 years ago (<22.5, 22.5–24.9, 25.0–27.4, or ≥27.5 kg/m2), smoking (0, 1–399, 400–799, or ≥800 cigarettes-years), alcohol intake (0, 0.1–0.9, 1.0–1.9, or ≥2.0 units/day), type of job (sedentary, moderate, or hard), non-occupational physical activity (0, 1–15.9, or ≥16 MET-hours/week), and parental history of colorectal cancer. Trend of the association was assessed with scores 0, 1, and 2 assigned to the 3 genotype

• categories. Effect modification of smoking was tested by the Wald statistic for a product term
• f the ordinal variable for genotype and a dichotomous variable for smoking category.

Deviation from the Hardy-Weinberg equilibrium was evaluated by χ2 test with 1 degree of

• freedom. Statistical significance was declared if a two-sided P-value was less than 0.05.

Statistical analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA). Results Genotype distribution of the controls was in Hardy-Weinberg equilibrium for both the EPHX1 Y113H (p=0.349) and H139R (p=0.405). Frequencies of the EPHX1 113His allele were 0.42 in cases and 0.44 in controls, and frequencies of the EPHX1 139Arg allele were

0.16 in cases and 0.18 in controls. The OR of colorectal cancer associated with the EPHX1 Tyr113His and His139Arg polymorphisms and predicted EPHX1 activity are shown in Table 1. The EPHX1 113HH genotype was associated with a slightly decreased risk of colorectal cancer, and the EPHX1 139R allele was related to a decreased risk. These decreases were far from the statistical

• significance. The imputed phenotype activity was unrelated to colorectal cancer (Table 1).

Table 1. EPHX1 polymorphisms and colorectal cancer risk Number (%) Genotype Cases, n = 685 Controls, n = 778 OR (95% CI)* Tyr113His (Exon 3) 0 (His/His) 115 (16.8) 143 (18.4) 1.00 (Referent) 1 (His/Tyr) 342 (49.9) 396 (50.9) 1.09 (0.81-1.45) 2 (Tyr/Tyr) 228 (33.3) 239 (30.7) 1.22 (0.89-1.67) His139Arg (Exon 4) 0 (His/His) 485 (70.8) 525 (67.5) 1.00 (Referent) 1 (His/Arg) 182 (26.6) 224 (28.8) 0.88 (0.70-1.12) 2 (Arg/Arg) 18 (2.6) 29 (3.7) 0.65 (0.35-1.21) Predicted activity 0-1 (low) 367 (53.6) 414 (53.2) 1.00 (Referent) 2 (medium) 223 (32.6) 246 (31.6) 1.03 (0.81-1.30) 3-4 (high) 95 (13.9) 118 (15.2) 0.92 (0.67-1.25) *Adjusted for sex, age, residence area, cigarette smoking, alcohol consumption, body mass index, type of job, leisure-time physical activity and parental colorectal cancer.

In the analysis stratified by smoking (<400 and >400 cigarette-years), neither of the polymorphisms was related to colorectal cancer risk in each stratum. Nor was the imputed phenotype associated with colorectal cancer risk in either low or high smoking category. The results were the same when stratified with never-smoking and ever-smoking (Table 2). Table 2. Effect modification of cigarette smoking on colorectal cancer risk associated with EPHX1 polymorphisms

< 400 ≥ 400 P for interaction Genotype No.* OR (95% CI)† No.* OR (95% CI)† Tyr113His (Exon 3) His/His 59/89 1.00 (Referent) 56/54 1.54 (0.90-2.63) 0.82 His/Tyr or Tyr/Tyr 357/438 1.17 (0.81-1.68) 213/197 1.68 (1.09-2.58) His139Arg (Exon 4) His/His 284/352 1.00 (Referent) 201/173 1.52 (1.11-2.07) 0.40 His/Arg or Arg/Arg 132/175 0.93 (0.70-1.23) 68/78 1.15 (0.77-1.72) Predicted activity 0-1 (low) 209/278 1.00 (Referent) 158/136 1.62 (1.15-2.28) 0.64 2 (medium) 146/166 1.13 (0.84-1.51) 77/80 1.34 (0.89-2.02) 3-4 (high) 61/83 0.96 (0.65-1.41) 34/35 1.37 (0.80-2.36)

* Number of cases/controls.

† Adjusted for sex, age, residence area, cigarette smoking, alcohol consumption, body mass

index, type of job, leisure-time physical activity and parental colorectal cancer. Discussion In this study, neither of the EPHX1 Y113H and H139R polymorphisms nor the combination of these polymorphisms predicting EPHX1 activity was associated with the risk

• f colorectal cancer. Moreover, cigarette smoking showed no effect modification on the

association with the EPHX1 genotype. The present findings are consistent with the results from four [18-21]of the six previous studies [16-21]. Two British case-control studies reported contradictory findings on the association between the EPHX1 Y113H polymorphism and colorectal cancer. The EPHX1 113HH genotype was associated with a 3.8-fold increase in the risk of colorectal cancer in a small case-control study [16], but was associated with a 32% decrease in the risk in another larger study [17]. However, genotype distribution of the EPHX1 Y113H polymorphism differed substantially and was not in Hardy-Weinberg equilibrium in these two studies. The 113HH genotype accounted for 21.8% of the controls in the latter study [17] and 6.4% of the controls in the former [16]. Deviation from the Hardy-Weinberg equilibrium was highly significant in the latter (P < 0.001) and marginally significant in the former (P = 0.04). The PCR-RFLP was used in these two studies, and their results should be interpreted with caution. It is reported that the PCR-RFLP method may have caused an error in genotyping the Y113H polymorphism due to the presence of a synonymous polymorphism (AAG to AAA) at codon 119 [30]. The reverse primer used by Harrison and Smith [16] contains this polymorphism, and the heterozygous 113YH genotype may be misclassified as the homozygous 113HH genotype due to failure of the 113Y allele to be amplified. The 119AAA is present in linkage with the 113Y allele [30]. Approximately 40% of the heterozygotes were misclassified as 113H homozygotes among Caucasians [12, 31]. On the other hand, the Y113H polymorphisms was unrelated to colorectal cancer risk in the other four studies based on the allele-specific discrimination assay [18-21], in all of which the genotype distribution was in Hardy-Weinberg equilibrium. A somewhat decreased risk of colorectal cancer among those with the 139RR

genotype in the present study is consistent with the finding in the Physicians’ Health Study, in which the OR for 139HR and 139RR compared with 139HH were 0.68 (95% CI 0.47-0.97) and 0.94 (95% CI 0.38-2.32), respectively, the OR for 139HR and 139RR combined being 0.70 (75% CI 0.49-0.99). However, no such association was replicated in the Nurses’ Health Study [18] and the other three [16, 17, 19]. As regards the imputed phenotype of EPXH1 activity, the three-category classification was used in the present study, as used in many other studies [11, 13, 14, 17, 23, 31]. The imputed phenotype was classified into four categories of “rapid”, “normal”, “slow”, and “very slow” groups in the previous studies on colorectal cancer [18-20]. The two groups of “slow” and “very slow” in the four-category classification were combined as a single group in the three-category classification. The repeated analysis based on the four-group classification did not find a specific imputed phenotype associated with colorectal cancer risk. No interaction between cigarette smoking and the EPHX1 polymorphisms on colorectal cancer was found in the present study, and this finding was inconsistent with the previous study[18]. Most of the studies on colorectal carcinogenesis addressed an interaction between cigarette smoking and the EPHX1 polymorphisms in colorectal adenoma. Associations between cigarette smoking and either of the EPHX1 polymorphisms in colorectal adenoma risk differed by sex in a study of 932 cases and 1282 controls in the USA [25]. In the study from the USA, a marginally significantly interaction between EPHX1 ‘rapid’ 113Tyr allele and cigarette smoking was found in women, and a statistically significantly interaction between the EPHX1 ‘slow’ 139His allele and cigarette smoking was found in men, but interaction was not observed for the combination of the two polymorphisms in both sexes and in combination of sexes. The differences observed between

men and women in that study [25] were probably chance findings by statistical evaluation of subgroups with small numbers of individuals. On the other hand, a statistically significantly interaction between cigarette smoking and predicted EPHX1 activity in colorectal adenoma was reported for both sexes combined in a study of 772 cases and 777 controls [23]. In that study, an increased risk of colorectal adenoma associated with the increasing number of putative high activity alleles was most apparent among current and recent cigarette smokers. Two other results from the studies on colorectal adenomas reported only suggestive interactions between cigarette smoking and either of the EPHX1 polymorphisms [26] or predicted EPHX1 activity [22, 26]. The use of community controls and the large number of subjects were strengths of the present study. The other strength was that the study subjects consisted of an ethnically homogenous population of Japanese, and concern over population stratification would be

• negligible. The statistical power was fairly large for EPHX1 Tyr113His, but we had

insufficient power for EPHX1 His139Arg due to the small number of homozygotes of the rare allele in cases and controls. There were several limitations to be discussed. In the present study, the participation rate in terms of genotyping was rather low (65% in cases and 56% in controls). However, it is unlikely that participation had been affected by genetic polymorphisms under study. As reported previously [32], although older persons and women were less likely to give consent for the genotyping, there was no difference between those who gave consent and those who did not in terms of smoking, residence area, and alcohol use. In conclusion, the present study showed that neither of the EPHX1 polymorphisms nor the combination of the EPHX1 polymorphisms (predicted EPHX1 activity) was associated with colorectal cancer risk. No interaction between the EPHX1 polymorphisms or

the combination of the EPHX1 polymorphisms and cigarette smoking on colorectal cancer risk was found in this study. These findings suggest that EPHX1 polymorphisms do not play an important role in colorectal carcinogenesis References

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