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Department of Preventive Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan [H. I., C. K., T. M., S. S., S. K.]; Department of Internal Medicine, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan [E. T., K. H.]; Fukuoka Women’s University, Fukuoka 813-8529, Japan [H. Hay.]; Self Defense Forces Fukuoka Hospital, Fukuoka 816-0824, Japan [K. O., H. Ham.]; and Self Defense Forces Kumamoto Hospital, Kumamoto 862-0901, Japan [H. K.]
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ABSTRACT |
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INTRODUCTION |
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Genetic polymorphisms are known for enzymes involved in the activation of tobacco-related carcinogens such as PAHs,3 nitrosamines, and arylamines. PAHs are activated by CYP1A1, a phase I drug-metabolizing enzyme, into reactive forms that produce DNA adducts, and the reactive metabolites of PAHs are detoxified by glutathione S-transferases, a phase II enzyme. A rare mutation at the MspI site in the CYP1A1 gene has been reported to be associated with an increased risk of lung cancer, particularly of squamous cell carcinoma (7, 8) . There is an ethnic difference in the frequency of the CYP1A1 MspI mutation allele, and the homozygous MspI mutation is relatively common in the Japanese population (9) . Interestingly, a case-control study of Japanese men in Hawaii reported an 8-fold increase in the risk of colorectal in situ carcinoma associated with MspI homozygous mutation (10) . The relation between GSTM1 polymorphism and cancer risk has also drawn much interest recently (11) . At least six studies have addressed the relation between the GSTM1-null genotype and colorectal cancer or adenomas. Whereas two of these studies suggested an increased risk among those with the GSTM1-null genotype (12, 13) , others found no difference in the risk between the GSTM1-null genotype and the non-null genotype (14–17) . None of these studies examined the combined effect of CYP1A1 MspI and GSTM1 genotypes. We investigated the relation of the CYP1A1 MspI variant genotype and the GSTM1-null genotype to the risk of colorectal adenomas with special reference to interaction with cigarette smoking in middle-aged Japanese men.
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MATERIALS AND METHODS |
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Of 803 men in the consecutive series, 778 underwent total or partial colonoscopy. Excluded from the study were 41 men with a prior history of colorectomy (n = 3), colorectal polypectomy (n = 36), or malignant neoplasms (n = 4). The macroscopic findings of the colonoscopy among 737 men were as follows: (a) normal, 368 men; (b) polyps, 325 men; (c) carcinoma, 2 men; and (d) other lesions, 42 men. A total of 209 of 325 men with colorectal polyps were found to have histologically confirmed colorectal adenomas without in situ or invasive carcinoma; the histological findings of the remaining 116 men were no biopsy (n = 16), normal or inflammatory tissue (n = 63), hyperplastic polyp without adenoma (n = 34), carcinoid (n = 1), and carcinoma (n = 2). Of the 368 men with normal study results, 230 men who had received total colonoscopy were used as controls; the colorectum of the remaining 138 men was not completely examined. Blood samples for DNA analysis were not available for 4 cases of adenomas and 10 controls, and the present study finally used 205 cases of colorectal adenomas and 220 controls with normal total colonoscopy.
Adenomas located more than 60 cm from the anus were defined as proximal adenomas, and the others were defined as distal adenomas. Cases with proximal adenomas only, distal adenomas only, and both proximal and distal adenomas numbered 69, 110, and 26, respectively. Those with both proximal and distal adenomas were treated as proximal adenoma cases. Cases with an adenoma of <5 mm, 5+ mm, and unknown size (diameter of the largest adenoma in multiple adenoma cases) numbered 129, 71, and 5, respectively.
Smoking and Alcohol Use.
Information on smoking habits, alcohol use, and other lifestyle factors
was obtained before colonoscopy by use of a self-administered
questionnaire, with a supplementary interview for unanswered questions.
Subjects were asked whether they were lifelong nonsmokers, former
smokers, or current smokers, and former and current smokers reported
the average number of cigarettes smoked per day and the number of years
of smoking. Cumulative exposure to cigarette smoking was expressed by
cigarette-years, the number of cigarettes smoked per day multiplied by
years of smoking. Alcohol drinkers were defined as those having ever
drunk alcohol at least once a week over a period of 1 year or longer.
Former alcohol drinkers were separated from lifelong nondrinkers. For
current alcohol drinkers, alcohol consumption was estimated from
reported frequencies and amounts of consumption of five types of
alcoholic beverages (sake, shochu, beer, spirits, and wine) on average
in the past year.
Laboratory Analysis.
Genomic DNA was extracted from buffy coat stored at -80°C using the
QIAamp blood kit (QIAGEN, Santa Clarita, CA). The genotype of the
CYP1A1 MspI polymorphism was determined by the PCR-RFLP
method as described by Hayashi et al. (19)
. The
PCR products of a 340-base region containing the MspI
restriction site were digested with MspI (Nippon Gene,
Toyama, Japan) and subjected to electrophoresis to identify wild-type
homozygotes (genotype A), heterozygotes (genotype B), and variant
homozygotes (genotype C). The GSTM1-null genotype was
determined by PCR analysis using the method described by Zhong et
al. (12)
. All PCR assays were done by one of the
authors (H. I.) without knowledge of adenoma status.
Statistical Analysis.
Logistic regression analysis was used to examine the relation of
cigarette smoking and genetic polymorphism to colorectal adenomas.
Statistical adjustment was made for cigarette smoking, alcohol use,
body mass index, hospital, and rank in the SDF. Age was not taken into
account because age was in a limited range (47–55 years). Cigarette
smoking was categorized into 0, 1–399, 400–799, and 800+
cigarette-years; subjects were also classified into lifelong
nonsmokers, past smokers, and current smokers consuming <25 or 25+
cigarettes/day. Alcohol use was categorized into never, past, and
current use with a consumption of <30, 30–59, or 60+ ml of alcohol
per day; and rank in the SDF was categorized into low, middle, and high
ranks. Body mass index was used as a continuous variable. Indicator
variables were created for the categories of the covariates. ORs and
95% CIs were calculated from logistic regression coefficients and SEs
for the corresponding indicator variables. The trend for association
between cigarette-years and adenoma risk was tested by the Wald
statistic in logistic regression analysis with ordinal scores of 0, 1,
2, and 3 assigned to the four levels of cigarette smoking. Interaction
between the genotypes and cigarette smoking on the risk of colorectal
adenomas was assessed by the likelihood ratio test using an ordinal
variable for cigarette-years (which were scored from 0–3). Two-sided
P values less than 0.05 were regarded as statistically
significant. All statistical computations were done by using PC-SAS
version 6.12 software (SAS Institute, Inc., Cary, NC).
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RESULTS |
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). Whereas the OR for past smokers compared with lifelong nonsmokers did
not differ much from unity, current smokers showed a nearly 4-fold
increase in the ORs. When the association with smoking was examined in
terms of cigarette-years, the ORs increased progressively with
increasing levels of cumulative exposure.
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). Thus, the risk of colorectal adenomas, in terms of the OR, did not
vary with either of the two genetic polymorphisms, regardless of
whether the covariates were adjusted for or not. The associations did
not change when adjustment was made for current smoking status (never,
past, <25 or 25+ cigarettes/day) instead of cigarette-years (data not
shown).
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). CYP1A1 MspI genotype C was associated with a statistically
nonsignificant decrease in the risk of proximal adenomas but not of
distal adenomas. GSTM1 genotype was not related to either
proximal or distal adenomas. No apparent difference was noted between
small and large adenomas with regard to the association with either
CYP1A1 MspI or GSTM1 genotype (data not shown).
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presents the ORs of colorectal adenomas according to the combination of
smoking levels and CYP1A1 MspI genotypes. As compared with
lifelong nonsmokers with genotype A, nonsmokers with genotype C showed
a statistically significant decrease in adenoma risk. The OR for the
highest level of cigarette smoking was approximately 2-fold greater in
those with genotype A than in those with genotypes B or C, but the 95%
CIs overlapped well. Increasing trends of ORs with increasing levels of
cigarette smoking were statistically significant within each group of
genotypes and did not differ significantly according to the genotype
(interaction P = 0.31).
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). Although the association with cigarette smoking tended to be slightly
stronger in those with the GSTM1 non-null genotype than in
those with the null genotype, the interaction between the
GSTM1 genotype and smoking was not statistically significant
(P = 0.35).
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). Results from the analysis of current smokers only were almost the
same as those for former and current smokers combined (data not shown).
There was no variation in the risk of colorectal adenomas according to
the categories of the two genotypes. Because of the limited number of
subjects, analysis of these two genetic polymorphisms in
combination was not possible for nonsmokers.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Homozygous mutant allele (genotype C) at CYP1A1 MspI is rare in Caucasians but is relatively common in the Japanese. Thus, the Japanese are a suitable population for study of the role of CYP1A1 MspI polymorphism in human carcinogenesis. The prevalence of genotype C in the control subjects of the present study was comparable to those reported among healthy subjects in Japan (9) . The present study showed no association between CYP1A1 MspI genotype and colorectal adenomas as a whole. The risk of proximal but not distal adenomas tended to be lower among men with genotype C as compared with those with genotype A. Unexpectedly, genotype C was associated with a decreased risk of colorectal adenomas among nonsmokers. The findings are in disagreement with the observation regarding in situ colorectal cancer in a Japanese population in Hawaii (10) . In that study, an approximately 8-fold increased risk was observed in association with genotype C. Genotype C accounted for only 4% of the control subjects in the Hawaiian Japanese study; this figure is much lower than that observed in the present study and elsewhere in Japan (9) . The observed protective association between genotype C and colorectal adenomas among nonsmokers may be due to chance because the number of men with genotype C was still small. Although much larger studies are needed to clarify the role of the CYP1A1 MspI genotype in colorectal carcinogenesis, the present study provides no evidence that CYP1A1 MspI genotype C confers increased susceptibility to colorectal adenomas in conjunction with exposure to cigarette smoking. An accentuated effect of smoking among those with genotype C has also been observed for squamous cell carcinoma of the lung (8) . Increased inducibility of aryl hydrocarbon hydroxylase, an enzyme activating PAHs, was recently observed among subjects with genotype C (20) . Compounds other than PAHs in tobacco smoke may also be responsible for the occurrence of colorectal adenomas.
The frequency of the GSTM1-null genotype has been reported to be approximately 50% in Japanese as well as in Caucasian populations (11) . In the present study, half of both cases and controls were classified as having the GSTM1-null genotype. GSTM1 polymorphism did not materially modify the relation between cigarette smoking and colorectal adenomas. Two case control studies in the United Kingdom (12) and Japan (13) reported an increased risk of colorectal cancer among those with the GSTM1-null genotype as compared with those with the non-null genotype. However, two other case-control studies and one prospective study failed to find an increased susceptibility to colorectal cancer among individuals with the GSTM1-null genotype (14–16) . One of these studies was based on nearly 2000 cases of colorectal cancer and observed no interaction with various indices of smoking (16) . A study of colorectal adenomas also found neither an overall association with the GSTM1-null genotype nor an interaction between the null genotype and smoking habits (17) . Our findings corroborated the lack of effect of the GSTM1-null genotype on colorectal adenomas.
GSTs have four classes of isoenzymes, i.e., 
, µ, 
,
and theta (21)
. GSTM1 genotype is only one of
several genetic determinants of glutathione S-transferase
activity. Although GSTM1 is considered to be a critical enzyme
detoxifying reactive metabolites of PAHs, other isoenzymes, especially
glutathione S-transferase T, may also function in the
detoxification of tobacco-related carcinogens (21)
. Thus,
the lack of an association between the GSTM1-null genotype
and colorectal adenomas does not necessarily imply that different
activities in the detoxification of PAH metabolites are irrelevant to
colorectal adenomas. We expected that the risk of colorectal adenomas
would be higher in smokers with CYP1A1 MspI genotype C and
the GSTM1-null genotype than in smokers having another
combination of these two genetic polymorphisms. However, the present
results were not in line with such a hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid from the
Ministry of Education, Science, Sports and Culture and from the
Ministry of Health and Welfare, Japan. 
2 To whom requests for reprints should be
addressed, at the Department of Preventive Medicine, Graduate School of
Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku,
Fukuoka 812-8582, Japan.
3 The abbreviations used are: PAH, polycyclic
aromatic hydrocarbon; SDF, Self Defense Forces; OR, odds ratio; CI,
confidence interval.
Received 10/ 4/99. Accepted 5/15/99.
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REFERENCES |
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