O2-04 Cigarette smoking, genetic polymorphisms and colorectal cancer - - PDF document

O2-04

Cigarette smoking, genetic polymorphisms and colorectal cancer risk

Hoirun Nisa, 銀光、 古野純典（ 九州大学・ 大学院医学研究院・ 予防医学分野) Background: It is uncertain whether smoking is related to colorectal cancer risk whereas smoking has consistently associated with increased risk of colorectal adenomas. Different types of tobacco carcinogens are activated and detoxified through different metabolic

• pathways. Cytochrome P-450 (CYP)1A1, glutathione-S-transferase (GST), quinone
• xidoreductase (NQO1) and epoxide hydrolase (EPHX1) are important enzymes in the

metabolism of tobacco carcinogens, and functional genetic polymorphisms are known of these enzymes. We investigated the relation of cigarette smoking and related genetic polymorphisms to colorectal cancer risk, with special reference to the interaction between smoking and genetic polymorphism. Methods: The subjects were 685 cases of colorectal cancer and 778 community controls who gave informed consent to genetic analysis in the Fukuoka Colorectal Cancer Study, a population-based case control study. Lifestyle factors were ascertained by interview, and DNA was extracted from buffy coat. Genotyping was done for CYP1A1 6235T>A (MspI) and 4889A>G (Ile462Val), GSTM1, GSTT1, NQO1 609C>T (Pro187Ser) and EPHX1 Ex4+52A>G (His139Arg). Logistic regression analysis was used to control for sex, age, area, and other factors. Likelihood ratio test was used to assess the interaction. Results: Cigarette smoking was not associated with an increased risk of colorectal cancer. None of the individual polymorphisms under study was related to colorectal cancer risk and showed a measurable effect modification on the relation between smoking and colorectal

• cancer. Of the gene-gene interactions studied, GSTT1 and CYP1A1 Ile462Val

polymorphisms showed a statistically significant interaction (P = 0.02). As compared with those having Ile/Ile genotype and GSTT1 non-null genotype, individuals with CYP1A1 462Val variant allele showed an odds ratio (OR) of 1.23 (95% CI: 0.90–1.69) when they had GSTT1 null genotype, and an OR of 0.76 (95% CI 0.56–1.01) when they had GSTT1 non-null genotype. The composite genotypes of these two polymorphisms, however, showed no measurable interaction with cigarette smoking in relation to colorectal cancer risk. Conclusion: The present study showed no clear association of cigarette smoking and genetic polymorphisms related to the metabolism of tobacco carcinogens with colorectal cancer risk. The observed interaction between GSTT1 and CYP1A1 Ile462Val polymorphisms may be a chance finding and needs further confirmation.

Introduction Both environmental and genetic factors are thought to play an important role in colorectal carcinogenesis [1]. The role of high-penetrance single genes in the etiology of colorectal cancer is estimated to be less than 10% [2,3] and the combination of low penetrance genes and environmental factors seem to contribute substantially to the

• ccurrence of colorectal cancer.

It has been a matter of interest whether smoking is related to increase risk of CRC [4-11]. A large number of studies have consistently found that cigarette smoking is associated with increased risk of colorectal adenoma, as reviewed extensively elsewhere [12, 13]. The results on smoking and CRC are, however, inconsistent [4-11]. Several studies suggested a modest increase in the risk of colorectal cancer associated with smoking. Even in the former studies, the findings were disparate with respect to sex or site-specific colorectal cancer. These inconsistent findings may be due to differences in study design and genetic susceptibility in study populations. Tobacco smoke contains various types of carcinogens such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines, aromatic amines, and N-nitrosamines which require metabolic activation and detoxification by different enzymatic pathways. PAHs are metabolized through complex phase I and phase II metabolic enzymes such as cytochrome P-450 (CYP), glutathione-S- transferases (GSTs), quinone oxidoreductase (NQO1) and epoxide hydrolase (EPHX1). Cytochrome P-450 CYP1A1 is a phase I, predominantly extrahepatic, microsomal enzyme involved in the bioactivation of PAHs including benzo(a)pyrene. Two genetic polymorphisms in CYP1A1, the T3801C substitution creating a Msp I restriction site in the 3’-flanking region and the A2445G substitution resulting in an amino acid change in the heme-binding region of exon 7 at codon 462 (Ile462Val), appear to be associated with higher

levels of biomarkers for PAH exposure [14, 15]. An increased risk of in situ colorectal cancer associated with CYP1A1 T3801C was reported in a case-control study in Hawaii, but no association between CYP1A1 T3801C and colorectal cancer was observed in subsequent

• studies. CYP1A1 Ile462Val was found to be unrelated to colorectal cancer risk those studies.

GSTs are a superfamily of detoxification enzymes that facilitate the inactivation of chemical carcinogens and environmental toxic compounds. GSTs consist of several classes of genes and the relation of genetic polymorphisms for GSTM1 and GSTT1 to cancer risk has been rather extensively studied. The “null” alleles of these polymorphisms result in a complete loss

• f enzyme function and thus may be at increased risk of tobacco related cancers. Results on

GSTM1 and GSTT1 in relation to colorectal cancer are inconsistent as reviewed elsewhere. The NQO1 primarily involved in detoxification through their two electron reduction to hydroquinones, thereby inhibiting the DNA. The functional C609T polymorphism (rs1800566) causing amino acid change (Pro187Ser) resulting in loss of NQO1 activity and therefore may increase susceptibility to the risk of cancer especially of tobacco-related cancers [25]. The effect of the NQO1 polymorphism seems to be more evident among smoking-related cancers as common environmental exposure to quinone arises from the

• xidative metabolite of benzo(a) pyrene [benzo(a)pyrene 3,6-quinones] present in the tobacco

smoke [24]. Hamajima et al. have demonstrated that the NQO1 Pro187Ser polymorphism had an association with the risk of lung cancer, and may modify the effect of smoking for cancers

• f the lung and esophagus [26]. Two studies have also reported a positive relation of the

NQO1 Pro187Ser polymorphism to the risk of bladder cancer among smokers [27-28]. Results are inconsistent regarding the NQO1 Pro187Ser polymorphism and colorectal neoplasia [21,26,29-32]. Mitrou et al. showed an evidence for the association between NQO1 Pro187Ser polymorphism and colorectal adenoma [29], and Hou et al. reported only a marginal association [21]. The role of the NQO1 Pro187Ser polymorphism in relation to an

increase risk of CRC has been positively observed in two studies [30,31], but there is no relation found in one study [32]. EPHX1 also plays an important role in determining the susceptibility of an individual to cancer. In exon 4 of the EPHX1 gene, the functional A415G polymorphism resulting a histidine to arginine change at codon 139 (His139Arg) and this change enhances enzyme activity by 25% through an increase in EPHX1 protein stability [33]. None of the previous studies examining the relationship between EPHX1 His139Arg polymorphism and risk of either colorectal adenoma [29,34-36] or carcinoma [37-38] have showed significant

• association. However, some studies have shown that an association between EPHX1

His139Arg polymorphism and risks for colorectal adenoma [29,34,36] and for CRC [37] tended to be highest among smokers. Recently many studies have focused on the relationship between genetic polymorphisms and risks of CRC. However, none of the studies have considered the cumulative effect of polymorphisms in five metabolic genes, CYP1A1, GSTM1, GSTT1, NQO1 and EPHX1, on the pathway to tobacco smoke metabolism in CRC. In light of this, we conducted a case control study to determine whether functionally characterized variants of the polymorphisms in CYP1A1, GSTM1, GSTT1, NQO1 and EPHX1 are associated with the risk of CRC. In addition, we assessed the interaction between smoking, genotype, the possibly combined genotypes and CRC risk. Materials and Methods The Fukuoka Colorectal Cancer Study is a case-control study of incident cases and community controls, with Fukuoka city and three adjacent areas as the study area. Details of methodological issues have been described elsewhere [39]. The study protocol was approved by the ethical committees of the Faculty of Medical Sciences, Kyushu University, but two of the participating hospitals which had no ethical committees at the time of survey obtained

permission from the director of each hospitals.

• Subjects. Cases were a consecutive series of patients with histologically confirmed

incident colorectal adenocarcinomas who were admitted to two university hospitals or six affiliated hospitals for surgical treatment during the period October 2000 to December 2003. By design, patients with prior history of partial or total removal of the colorectum, familial adenomatous polyposis or inflammatory bowel disease were excluded from the study. Controls were selected from community by two stage random sampling and were matched to each case on sex and age (within 10 years). Overall, 80% (840/1,053) of the eligible cases and 60% (833/1382) of the eligible controls were interviewed. A total of 685 cases and 778 controls (or 87% of the eligible interviewed subjects) gave an informed consent to genotyping.

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

physical activity, smoking, alcohol intake, and other factors using a uniform questionnaire. Detailed information on smoking history was collected from subjects with the first question was whether they had ever smoked cigarettes everyday for one year or longer. Then they were asked about the current status (before the symptom or screening in the cases). Age of starting smoking and that of quitting smoking (for past smokers) were ascertained, along with years of smoking and numbers of cigarettes smoked per day for each decade of age from the second to eighth decade. Cumulative exposure to cigarette smoking was expressed by cigarette-years, the number of cigarettes smoked per day multiplied by years of smoking, and classified into 0, 1-399, 400-799 and > 800 cigarette years.

• Genotyping. DNA was extracted from the buffy coat by using a commercial kit

(Qiagen GmbH, Hilden, Germany) and genotyping was performed using the PCR-RFLP

• method. The PCR was performed in a reaction mixture of 10 µL containing approximately

50-150 ng/µL.

Statistical analysis. The association of CYP2E1 polymorphisms with colorectal cancer was examined in terms of odds ratio (OR) and 95% confidence interval (CI), which were obtained from a logistic regression analysis. The 95% CI was derived from the standard error of the logistic regression coefficient. Statistical adjustment was made for 5-year age class (starting with the lowest class of < 45 years), sex, residency 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,

• r ≥ 2.0 units/day), type of job (sedentary work and no job, work with walking, or labor

work), leisure-time physical activity (0, 1–15.9, or ≥ 16 MET-hours/week), and parental history of colorectal cancer. Gene-gene and gene- environment (smoking) interactions were statistically evaluated based on the likelihood test, comparing the model including an interaction term and the model without. Interactions between genotype and lifestyle variables were statistically tested using the likelihood ratio test, comparing a model including interaction terms with one that

• nly included the main effects. Deviation from the Hardy-Weinberg equilibrium was

evaluated by chi-square test with 1 degree of freedom. Statistical significance was declared if a two-sided P-value was less than 0.05 or if the 95% CI did not include unity. Statistical analyses were carried out using SAS version 8.2 (SAS Institute, Cary, NC). Results Table 1 shows the association between cigarette smoking and colorectal cancer risk. Adjustment for the covariates did not change the results. As compared with lifelong nonsmokers, individuals with a light exposure to cigarette smoking (1-399 cigarette-years) had a statistically significant decrease in the OR of colorectal caner, and those with higher

categories showed only small increases in the OR. This pattern was also observed in both men and women. Table 1. Risk of colorectal cancer according to cigarette smoking Cigarette-years Number (%) OR (95% CI)* OR (95% CI) † Cases Controls Both sexes 299 (43.7) 326 (41.9) 1.00 (referent) 1.00 (referent) 1‒ 399 117 (17.1) 201 (25.8) 0.68 (0.50-0.94) 0.65 (0.47-0.89) 400‒ 799 195 (28.5) 180 (23.1) 1.21 (0.88-1.68) 1.16 (0.83-1.62) ≥800 74 (10.8) 71 (9.1) 1.21 (0.79-1.86) 1.14 (0.73-1.77) Men 80 (18.8) 92 (18.8) 1.00 (referent) 1.00 (referent) 1‒ 399 94 (22.1) 158 (32.2) 0.55 (0.32-0.95) 0.48 (0.27-0.85) 400‒ 799 182 (42.7) 171 (34.9) 1.44 (0.60-3.48) 1.47 (0.59-3.69) ≥800 70 (16.4) 69 (14.1) 2.29 (0.40-13.0) 1.87 (0.31-11.4) Women 219 (84.6) 234 (81.3) 1.00 (referent) 1.00 (referent) 1‒ 399 23 (8.9) 43 (14.9) 0.73 (0.49-1.09) 0.69 (0.45-1.03) ≥400 17 (6.5) 11 (3.8) 1.22 (0.85-1.75) 1.10 (0.76-1.61) * Adjusted for sex, age and residence area. † Adjusted for sex, age, residence area, alcohol consumption, body mass index, type of job, leisure-time physical activity and parental colorectal cancer. The genotype distribution was almost identical between cases and controls with regard to all genotypes status (Table 2).

Table 2. Risk of colorectal cancer by genotype status Cases, n = 685 Controls, n = 778 OR (95% CI)a n % n % CYP1A1 T6235C TT TC CC 283 307 95 41.31 44.82 13.87 305 368 105 39.20 47.30 13.50 1.00 (Referent) 0.90 (0.72 – 1.13) 0.97 (0.70 – 1.35) CYP1A1 Ile462Val Ile/Ile Ile/Val Val/Val 418 231 36 61.02 33.72 5.26 461 276 41 59.25 35.48 5.27 1.00 (Referent) 0.94 (0.75 – 1.17) 1.00 (0.62 – 1.62) GSTT1 Non-null Null 347 338 50.66 49.34 435 343 55.91 44.09 1.00 (Referent) 1.20 (0.97 – 1.48) GSTM1 Non-null Null 328 357 47.88 52.12 356 422 45.76 54.24 1.00 (Referent) 0.90 (0.73 – 1.11) GSTT1*GSTM1 Non-null Null 502 183 73.28 26.72 589 189 75.71 24.29 1.00 (Referent) 1.09 (0.86 – 1.39) NQO1 Pro187Serb Pro187/Pro187 Pro187/Ser Ser/Ser 259 336 89 37.81 49.05 12.99 282 392 103 36.25 50.39 13.24 1.00 (Referent) 0.94 (0.75 – 1.18) 0.97 (0.69 – 1.36) EPHX His139Arg His139/His139 His139/Arg Arg/Arg 485 182 18 70.80 26.57 2.63 525 224 29 67.48 28.79 3.73 1.00 (Referent) 0.88 (0.70 – 1.12) 0.65 (0.35 – 1.21)

aAdjusted for sex, age, city, smoking, alcohol, BMI, job related physical activity, MET,

family history. bOne case and one control were excluded because of undetermined genotype. OR, odds ratio. CI, confidence interval The risks of CRC, in terms of the OR, were not significant at the five percent level. Table 3 presents the ORs of CRC according to the combination of cigarette smoking and metabolic enzyme genotypes. In this analysis, individuals heterozygous for the CYP1A1, NQO1 Pro187Ser or EPHX1 His139Arg polymorphism were each combined with those homozygous for the variant allele. Compared with smokers with an exposure of < 400 cigarette-years and having null genotype of CYP1A1 6235T>C MspI (TT) or CYP1A1 Ile462Val (Ile/Ile), smokers who exposed to > 400 cigarette-years and had the common

genotype of CYP1A1 6235T>C MspI (TT) or CYP1A1 Ile462Val (Ile/Ile) were positively associated with CRC risk (OR 1.76; 95% CI 1.19 – 2.60 and OR 1.58; 95% CI 1.14 – 2.20, respectively). The elevated risk for CRC was also found significantly in those who had EPHX1 His139Arg common genotype (His/His) and who had an exposure of > 400 cigarette-years (OR 1.52; 95% CI 1.11 – 2.07). The association existed between the GST variants and CRC among smokers with > 400 cigarette-years but it only appeared to be confined to subjects who had GSTT1 non-null genotype or the combined GSTM1 and GSTT1 non-null genotype (OR 1.51; 95% CI 1.08 – 2.13 and OR 1.54; 95% CI 1.14 – 2.07, respectively). However, when tested for interaction, there was no evidence of synergistic effects among those polymorphisms and smoking. Table 3. Risk of colorectal cancer by cigarette-years and CYP1A1, GST, NQO1, EPHX polymorphismsa < 400 > 400 p interaction n+ OR (95% CI)b n+ OR (95% CI)b CYP1A1 T6235C TT TC + CC 161/207 255/320 1.00 (Referent) 1.04 (0.79 – 1.36) 122/98 147/153 1.76 (1.19 – 2.60) 1.30 (0.91 – 1.87) 0.14 CYP1A1 Ile462Val Ile/Ile Ile/Val + Val/Val 243/309 173/218 1.00 (Referent) 1.03 (0.79 – 1.34) 175/152 94/99 1.58 (1.14 – 2.20) 1.28 (0.88 – 1.86) 0.30 GSTT1 Non-null Null 211/301 205/226 1.00 (Referent) 1.24 (0.95 – 1.61) 136/134 133/117 1.51 (1.08 – 2.13) 1.67 (1.18 – 2.37) 0.60 GSTM1 Non-null Null 207/245 209/282 1.00 (Referent) 0.88 (0.68 – 1.14) 121/111 148/140 1.38 (0.96 – 1.97) 1.33 (0.94 – 1.87) 0.68 GSTT1*GSTM1 Non-null Null 304/407 112/120 1.00 (Referent) 1.20 (0.89 – 1.63) 198/182 71/69 1.54 (1.14 – 2.07) 1.41 (0.94 – 2.11) 0.29 NQO1 Pro187Ser Pro/Pro Pro/Ser + Ser/Ser 157/189 259/338 1.00 (Referent) 0.94 (0.72 – 1.23) 102/93 166/157 1.41 (0.95 – 2.09) 1.37 (0.96 – 1.94) 0.11 EPHX His139Arg His/His His/Arg + Arg/Arg 284/352 132/175 1.00 (Referent) 0.93 (0.70 – 1.23) 201/173 68/78 1.52 (1.11 – 2.07) 1.15 (0.77 – 1.72) 0.40

aAdjusted for sex, age, city, alcohol, BMI, job related physical activity, MET, family history.

n+, number of cases/controls.

All possible combinations of two genotypes for CYP1A1, GSTM1, GSTT1 and NQO1 were examined and shown in Table 4. There was no evidence of interaction for all of the gene-gene combinations except for the combination of GSTT1 and CYP1A1 Ile462Val genotype (p 0.02). The OR for those who had the combined GSTT1 null genotype and CYP1A1 Ile462Val variant allele (Ile/Val + Val/Val) genotype was 1.23 fold (95% CI: 0.90 – 1.69) greater than those who had the combined GSTT1 non-null genotype and CYP1A1 Ile462Val (Ile/Ile), but the OR was not significant at the five percent level. Evaluation of the combined gene-gene and smoking to CRC risk, however, showed that none of the tests for interaction was significant (Data not shown). Discussion Our results indicate that smoking had no association with an increased risk of CRC. This is conflicting with findings of previous Japanese and Dutch studies [5,9], but is in agreement with other findings [6-10]. The previous study of Japanese population found that smoking was associated significantly with CRC in men and revealed that 22% of CRC is attributable to currently and formerly smoking [5]. The latter study among Women’s Health Initiative participants observed statistically significant positive association between most measures of cigarette smoking and CRC, and that current smokers had only increased risk for rectal cancer compared to never smokers [4]. On the contrary, Terry et al. [11] in a prospective cohort study conducted in Sweden reported that smoking status was not clearly associated with the risk of CRC in women, but up to 30 years or more of smoking duration was associated with an increased risk of rectal cancer. Results from a population-based case control study in Germany also observed none of the smoking status was associated with an increased risk of CRC [7], but that smoking for a long duration at a high cumulative dose increased the risk for CRC. Most of the studies investigating the relation between smoking

and CRC have later revealed that smoking requires a long induction period to cause CRC, as has been discussed in a recent review [12]. Table 4. Gene-gene interactions for combinations of CYP1A1 and GST genotypes in CRC

n OR (95% CI)a p interaction Cases Controls GSTM1 Non-null Non-null Null Null CYP1A1 T6235C TT TC + CC TT TC + CC 122 206 161 196 135 221 170 252 1.00 (Referent) 1.02 (0.74 – 1.40) 1.01 (0.72 – 1.42) 0.84 (0.61 – 1.15) 0.34 GSTT1 Non-null Non-null Null Null CYP1A1 T6235C TT TC + CC TT TC + CC 147 200 136 202 158 277 147 196 1.00 (Referent) 0.75 (0.56 – 1.01) 0.93 (0.67 – 1.30) 1.07 (0.79 – 1.45) 0.06 GSTT1*GSTM1 Non-null Non-null Null Null CYP1A1 T6235C TT TC + CC TT TC + CC 203 299 80 103 222 367 83 106 1.00 (Referent) 0.88 (0.69 – 1.13) 0.99 (0.68 – 1.44) 1.03 (0.73 – 1.44) 0.51 GSTM1 Non-null Non-null Null Null CYP1A1 Ile462Val Ile/Ile Ile/Val + Val/Val Ile/Ile Ile/Val + Val/Val 187 141 231 126 207 149 254 168 1.00 (Referent) 1.06 (0.78 – 1.45) 0.98 (0.75 – 1.29) 0.83 (0.61 – 1.13) 0.29 GSTT1 Non-null Non-null Null Null CYP1A1 Ile462Val Ile/Ile Ile/Val + Val/Val Ile/Ile Ile/Val + Val/Val 214 133 204 134 239 196 222 121 1.00 (Referent) 0.76 (0.56 – 1.01) 0.98 (0.75 – 1.29) 1.23 (0.90 – 1.69) 0.02 GSTT1*GSTM1 Non-null Non-null Null Null CYP1A1 Ile462Val Ile/Ile Ile/Val + Val/Val Ile/Ile Ile/Val + Val/Val 300 202 118 65 337 252 124 65 1.00 (Referent) 0.90 (0.70 – 1.15) 1.00 (0.74 – 1.36) 1.13 (0.77 – 1.66) 0.39 NQO1 Pro187Ser Pro/Pro Pro/Pro Pro/Ser+Ser/Ser Pro/Ser + Ser/Ser CYP1A1 T6235C TT TC + CC TT TC + CC 126 133 157 268 118 164 187 308 1.00 (Referent) 0.78 (0.55 – 1.10) 0.81 (0.58 – 1.14) 0.83 (0.61 – 1.13) 0.30 NQO1 Pro187Ser Pro/Pro Pro/Pro Pro/Ser+Ser/Ser Pro/Ser + Ser/Ser CYP1A1 Ile462Val Ile/Ile Ile/Val + Val/Val Ile/Ile Ile/Val + Val/Val 162 97 256 169 176 106 285 210 1.00 (Referent) 1.04 (0.73 – 1.32) 1.00 (0.76 – 1.48) 0.90 (0.67 – 1.23) 0.14

aAdjusted for sex, age, city, smoking, alcohol, BMI, job related physical activity, MET,

family history The inconsistence findings may be affected by the complexity tobacco smoke constituents, variation in metabolics of cigarette smoke, differences in study design, and different distribution of genetic polymorphisms on smoking-related enzymes across

• population. Another explanation, moreover, may be due to different measures of cigarette

• smoking. The present study assessed an exposure to cigarette smoking as the product of the

number of cigarettes consumed per day within ten years of smoking. Thus, it is likely to fail an observed association since the duration of smoking is shorter than other studies that confirmed such an effect. The prevalence of smoking in our controls (56.35%) was somewhat higher than in Japanese population (38.7%) [40]. To interpret our findings, therefore, should be viewed with caution since smokers may differ from non smokers more so than in general

• population. It was unexpected in this study, however, that subjects who had an exposure of

1-399 cigarette years were inversely related to CRC risk. This unique finding might only have arisen by chance. To the best of our knowledge, this is the first study examined the effect of numerous

• f smoking related genetic polymorphisms and CRC risk, and the effect of their combination

with cigarette smoking. Our results do not show an association of CYP1A1 polymorphisms and CRC risk. Previous studies investigating these associations have been inconsistent [16-20,41]. In accordance with present study, results from a prospective study of Caucasian men concluded none of the MspI and Ile462Val polymorphisms had significant association with CRC risk [18]. A meta-analysis also failed to find any association with CRC risk [17]. In contrast with the findings showing no association, Sivaraman et al. [16] formerly conducted a small case-control study (n = 43 cases and 47 controls) and observed an eightfold increased risk of CRC with CYP1A1 Msp1 variant polymorphism only among individuals of Asian descent living in Hawaii. The effect of smoking on genetic susceptibility to colorectal cancer has been extensively studied, and results on the interaction between smoking and CYP1A1 polymorphisms have been conflicting. Two previous studies of CYP1A1 polymorphisms among smokers examined the effect of smoking dose on lung cancer, and noted an increased risk associated with 6235T>C MspI and Ile462Val homozygous variant genotypes with low

dose level of cigarette smoking (42,43). Our results were in disagreement with two lung cancer studies. In this study, we found an exposure of > 400 cigarette-years in subjects having 6235T>C MspI (TT) or Ile/Ile genotypes were at greater risk to develop CRC risk. Analysis

• f CYP1A1 polymorphisms and smoking, however, only revealed suggestive significant
• interactions. Slattery et al. [20] in a large case control study reported similar findings but

solely only for colon cancer and among men. An evidence of significant interaction with smoking for CYP1A1 6235T>C (MspI) and for CYP1A1 Ile462Val genotype have only been found in colorectal adenoma study [21] and in a recent study of colorectal polyps [41]. We observed no association of either GSTM1 or GSTT1 null genotypes with CRC risk and neither did with the combined null genotype of GSTM1 and GSTT1. In agreement with this study, a non significant association between the GSTM1 genotypes and CRC was also reported in three studies [44-46], but not in two studies [38, 47]. The risk of CRC with GSTT1 null genotype has been confirmed in English Caucasians study [47], but neither of the studies has found the association between the combined non-null genotype of GSTM1 and GSTT1 and CRC [44,46]. Because GSTs have an important role in the detoxification of smoking-related carcinogens, there are many cancer studies have been addressed and resulted more consistently associated with cigarette smoking, such as lung cancer. However, there are not frequent colorectal cancer studies assessed on interaction of cigarette smoking and GSTM1 or GSTT1 polymorphisms. Four studies and a huge review explored interactions between cigarette smoking and GSTM1

• r GSTT1

polymorphisms and CRC risk [44,45,48-50] have reported no interaction. The present study, although not statistically significant, showed possible interaction between GSTT1 genotypes and cigarette smoking and the only study showed for the combined non-null genotype of GSTM1 and GSTT1. Similar to

• ur findings but based on site-specific CRC, up till now there is only one study reported a

suggestive interaction between GSTT1 null genotype and cigarette smoking for colon cancer

among whites [48]. Another study found a borderline significant interaction between GSTM1 null genotype and cigarette smoking for rectal cancer in men [22]. The prevalence of GSTT1 null genotype (44.09%) in our controls group was similar to that seen in an earlier Japanese study (44.40%) [46]. In contrast to other reports, the frequency of GSTT1 null genotype was varied among or within racial groups, such as it is 36.10% in an English Caucasian study [47], 58.62% within the prospective Physicians’ Health Study [44], is ranged 15-26% in African and 10-21% in Europe [50]. These data of the frequency variations of GSTT1 null genotype revealed ethnic differences most probably may influence susceptibility to chemicals carcinogens detoxified by GSTT1, and susceptibility to specific exposure-induced diseases among these populations, therefore, may be unequal. Previous NQO1 studies reported inconsistent findings on association of colorectal cancer with NQO1 Pro187Ser. A recent meta analysis on whites suggested that, though with less confident because the heterozygous genotype have higher risk compare to the homozygous variant genotype, the presence of NQO1 Pro187Ser variant genotype was associated with increased risk of CRC [24]. Two studies previously have found the findings

• f a meta analysis study [30,31]. Lafuente et al [30] reported that ten fold increased risk of

CRC was associated with NQO1 Pro187Ser genotype for tumors containing K-ras codon 12

• mutations. On the other hand, one Japanese study did not show any effect of NQO1

Pro187Ser genotype with CRC risk [26]. In another study among Caucasians and the only study that has looked at the interactions between NQO1 Pro187Ser genotype and smoking in relation to CRC, no such association was found nor influence of NQO1 Pro187Ser genotype was observed among smokers [32], and these findings have repeated in the present study. In agreement with two previous studies [37,38], no significant association between EPHX1 His139Arg and CRC was found. However, Robien et al. reported that colon cancer risk tended to be increased among smokers with 139His genotype [37]. Three previous

studies have also supported possible effect modification of cigarette smoking on the association between EPHX1 His139Arg and colorectal adenoma [34-36]. Tranah et al. reported that a significant interaction was found between His139Arg and cigarette smoking among men with the highest risk was for subjects who had > 25 pack-years smoking and were homozygous for the slow genotype of His139Arg [36]. However, the risks for colorectal adenoma tended to be highest among smokers with the combined of two EPHX1 variants genotypes (113Tyr and 139Arg) [34,35]. Here, we failed to observe a significant interaction with smoking, but subjects who exposed to > 400 cigarette-years with 139His genotype were at increased risk for CRC. Few studies have examined the association between gene-gene interactions and the risk of CRC, and we then referred to their genotype combinations and to each putative function of polymorphisms in selecting gene-gene interactions in this study. A case-control study conducted by Sachse et al. [38] on 490 CRC patients and 593 controls failed to show significant interactions between GST and CYP1A1. Fan et al. [51] repeated evaluation on possible interactions between GST and CYP1A1 polymorphisms through a case-only study on 207 individuals with CRC in China, but no such interaction was reported. In contrast, we found a statistically significant interaction with CRC for combination of GSTT1 and CYP1A1 Ile462Val polymorphisms in our study population. For the interactions of the combined genotypes of these two polymorphisms and cigarette smoking, though non-significant, an increased risk of CRC was appeared greater among subjects who had an exposure of > 400 cigarette-years compared to subjects who had an exposure < 400 cigarette-years and the combined of GSTT1 non-null and Ile/Ile genotypes. The present study has some merits including the use of data from a large population-based case-control study, thus no issue of study size in investigating the role of rare genotypes in the gene-environment or gene-gene interaction has developed. The

participation rate in terms of genotyping was high in either cases (82%) or controls (93%). Further, the study subjects consist of an ethnically homogenous population of Japanese, and the concern over population stratification would be negligible. In conclusion, the present study showed no clear association of cigarette smoking and genetic polymorphisms related to the metabolism of tobacco carcinogens with CRC risk. We can not exclude the possibility that the observed interaction between GSTT1 and CYP1A1 Ile462Val polymorphism may represent chance finding, therefore further investigation is warranted. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports and Technology, Japan (1814022), and from a Grant-in-Aid in the year 2005, Fukuoka Cancer Society, Japan. The authors acknowledge support from Emeritus Professor Keizo Sugimachi; Professor Seiyo Ikeda, Takayuki Shirakusa, and Sumitaka Arima; and Drs. Motonori Saku, Yoichi Ikeda, Soichiro Maekawa, Kazuo Tanoue, Kinjiro Sumiyoshi, and Shoichiro Saito in conducting the survey of cases. The following physicians kindly supervised the survey of the controls at their clinics: Drs. Hideaki Baba, Tomonori Endo, Hiroshi Hara, Yoichiro Hirokata, Motohisa Ikeda, Masayoshi Ishibashi, Fumiaki Itoh, Yasuhiro Iwanaga, Hideki Kaku, Shoshi Kaku, Minori Kanazawa, Akira Kobayashi, Ryunosuke Kumashiro, Shinichi Matsumoto, Soukei Mioka, Umeji Miyakoda, Osamu Nakagaki, Nobuyoshi Nogawa, Nobuyoki Ogami, Toyoaki Okabayashi, Hironao Okabe, Nishiku Saku, Masafumi Tanaka, Masahiro Ueda, Bunichi Ushio, and

• KoheishoYasunaga. The authors are grateful to research nurses: Ms. Nobuko Taguchi, Yuriko

Moroe, Yuko Noda, Ryoko Tanaka, Hisako Nakagawa, and Yoko Mikasa; and research clerk

• Ms. Hiroko Mizuta; and the assistance of Ms. Masumi Koga, Ms. Akiko Koga, for their

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