Epoxide hydrolase polymorphisms and risk of colorectal cancer: the Fukuoka Colorectal Cancer Study Hoirun Nisa 1,2* , Suminori Kono 1 1 Department 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. 2 Faculty 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 or 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, or 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 of 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/m 2 ) 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
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