1 National Institute on Alcoholism, Kurihama National Hospital, Yokosuka, Kanagawa 239-0841,
2 Surgery Division, National Cancer Center Hospital and
11 Cancer Information and Epidemiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045,
3 Department of Technology Assessment and Biostatistics, National Institute of Public Health, Wako, Saitama 351-0104,
4 Department of Surgery, National Osaka Hospital, Osaka, Osaka 540-0006,
5 Internal Medicine Division, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577,
6 Department of Surgery, Kawasaki Municipal Hospital, Kawasaki, Kanagawa 210-0013,
7 Kamio Memorial Hospital, Chiyoda-ku, Tokyo 101-0063,
8 Kumagai Satellite Clinic, Shinjuku-ku, Tokyo 169-0074,
9 Mitsukoshi Health and Welfare Foundation, Shinjuku-ku, Tokyo 160-0023,
10 Department of Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan
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Abstract |
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Abbreviations: ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; CI, confidence interval. GSTM1, glutathione S-transferase M1; OR, odds ratio; PAR, population-attributable risk.
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Introduction |
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In persons with ALDH2*2, a mutant allele that is prevalent in east Asians, ALDH2 is inactive. Inactive ALDH2 generally inhibits East Asians from heavy drinking by causing acetaldehydemia and alcohol flushing responses, which include facial flushing, tachycardia and drowsiness (11). However, the preventive effect of heterozygous ALDH2*1/2*2 is incomplete and influenced by sociocultural factors. Recent decades have seen a dramatic increase in the proportion of heavy drinkers who have inactive heterozygous ALDH2. More specifically, 2.5% of Japanese alcoholics in 1979, 8.0% in 1986 and 13.0% in 1992 had the inactive heterozygous ALDH2 (12). The paradoxical combination of heavy drinking and the gene that makes individuals susceptible to alcohol is most hazardous with regard to esophageal cancer; thus, heavy drinking by ALDH2 heterozygotes should be taken more seriously.
Some attention has been focused on the mechanisms through which drinkers with inactive ALDH2 overcome acetaldehydemia and alcohol flushing. Drinking is important in Japanese business culture. Occasionally getting drunk is much more acceptable to Japanese men than to American men of Japanese ancestry (13). Under the influence of these drinking customs, the flushing response diminishes in intensity in males with long or heavy drinking histories. Among Japanese ALDH2 heterozygotes, 77.0% of all men over 50 years of age (14) and 71.5% of alcoholic males with esophageal cancer (15) reported alcohol flushing currently or during the first to second year after they started drinking. However, only 64.6% and 8.2% of these two groups reported `current alcohol flushing.' These negative changes in the flushing responses suggest that heavy drinkers develop tolerance to severe acetaldehydemia. That tolerance may enhance their vulnerability to heavy drinking and alcohol-induced cancers.
Like ALDH2, alcohol dehydrogenase (ADH)-2 is polymorphic, and its mutant ADH2*2 allele is highly prevalent among east Asians. Although the ALDH2 genotype, but not the ADH2 genotype, determines an individual's peak blood acetaldehyde concentration (16), the ADH2*2 allele encodes a superactive subunit of ADH2, and superactive ADH2*2 homodimers have about 40 times higher Vmax than the less active ADH2*1/2*1 form of ADH2 (8). ADH2*1/2*1 has been consistently demonstrated to enhance the risk for esophageal cancer in East Asian drinkers (2,5,7,15). Among alcoholics, the ALDH2*1/2*2 and ADH2*1/2*1 gene combination enhanced the risk for esophageal cancer in a multiplicative fashion (7,15).
In fact, 52% of alcoholic men with esophageal cancer who have both the ALDH2*1/2*2 and the ADH2*1/2*1 genotypes reported `never alcohol flushing' (15). The flushing response may be triggered by an initially steep rise in blood acetaldehyde after drinking. This dramatic change may not occur in persons with the much less active form of ADH2, in spite of the presence of inactive heterozygous ALDH2. Local enzyme activities in the skin might affect alcohol flushing, as suggested by cutaneous flushing induced by the ethanol patch test (17). Ethanol patch test results differ by ADH2 genotype (18). Regardless of the mechanism affecting alcohol flushing, individuals with this gene combination should be considered at the highest risk for esophageal cancer, because, as a result of their lack of protective alcohol flushing, they are likely to become heavy drinkers, and to be exposed to higher levels of acetaldehyde.
Among western populations, ADH3 gene polymorphism is the rate-limiting factor in acetaldehyde metabolism (8). Enzymes encoded by the ADH3*1 allele produce acetaldehyde twice as fast as those encoded by the ADH3*2 allele (8). Individuals possessing ADH3*1 are hypothesized to be at greater risk for alcohol-related cancer. Although published studies have not yielded a consistent pattern of association (1924), several have suggested that individuals who have ADH3*1/3*1 are at greater risk for upper aerodigestive tract cancer when there is very high, chronic alcohol exposure (19,20,24). It has been consistently reported that in East Asians the ADH3*2 allele enhances the risk of alcoholism through linkage with the ADH2*1 allele (2527).
Whether the enzyme-related vulnerability for esophageal cancer can be extended to light to moderate drinkers is a very important issue needing immediate attention because of its potential role in the prevention of cancer in the general Japanese population. Among populations of European origin, light to moderate drinking has demonstrated health benefits, i.e. protection against heart disease and stroke (28). Although there are a few reports showing a similar trend (29,30), it has yet to be evaluated whether this is the case for east Asians.
Proof that certain combinations of ALDH2, ADH2 and ADH3 genotypes markedly increase the risk for esophageal cancer among light- to moderate-drinking populations could change our views on the beneficial health effects of light to moderate drinking. Therefore, to shed more light on the overall risk for esophageal cancer, we designed a multi-institutional casecontrol study to determine the relative impact of combinations of alcohol-metabolizing enzymes, drinking behavior and other environmental factors, including smoking and a diet poor in vegetables and fruit. By broadening the concept of what constitutes high-risk drinking, we may adopt a new strategic approach aimed at the prevention of esophageal cancer.
Glutathione S-transferase M1 (GSTM1) is a multifunctional enzyme that detoxifies toxic and carcinogenic electrophiles in tobacco smoke (31). Individuals with the GSTM1 deletion genotype (0/0) may have an impaired ability to detoxify carcinogenic compounds and may be at increased risk for certain smoking-related cancers (31). The results of several Asian studies using middle-sized samples (164296) to test this hypothesis regarding esophageal cancer were mixed (2,3235). Inasmuch as the risk conferred to individuals with the GSTM1 deletion genotype appears to be small (31), study size is critical to reliable estimation of cancer risk. Therefore, in this larger study, we also examined the association between GSTM1 genotype and esophageal cancer susceptibility.
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Materials and methods |
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The ethics committee of each collaborating institution reviewed and approved the proposed study, and each of the participants gave informed consent.
Each participant independently completed a structured questionnaire concerning his drinking, smoking, and dietary habits; those with cancer were instructed to report on their habits before they got sick. Each was asked to classify himself as a never drinker, a current drinker, or an ex-drinker, and to report alcohol intake as the frequency of consumption and the usual amount(s) and type(s) of alcoholic beverage(s) consumed. The frequency of consumption was divided into five categories: never; 13 days/month; 12 days/week; 34 days/week, and 5 or more days/week. The weekly frequency for drinking was calculated according to a score assigned to each frequency category (0, 0.5, 1.5, 3.5, and 6, respectively). Weekly ethanol consumption was calculated using a standard conversion for alcoholic beverages in which 180 ml sake was considered to be 22 g ethanol; 180 ml shochu, 36 g; 633 ml beer, 25 g; 30 ml whiskey, 10 g; and 120 ml wine, 11 g. Weekly alcohol intake was then converted into the number of units per week by dividing the total ethanol consumption in grams by 22 g per unit (the ethanol content of one serving of sake). The subjects were classified as never/rare drinkers, ex-drinkers, or current drinkers who consumed 18.9 units/week (light drinkers), 917.9 units/week (moderate drinkers), or 18+ units/week (heavy drinkers). The questionnaire also asked about the frequency of drinking strong alcoholic beverages straight. In addition, the subjects reported on smoking and the frequency of their intake of green and yellow vegetables and fruit as well as their preference for high-temperature food. The intake frequency of green and yellow vegetables and fruit was respectively asked using five categories: seldom; 12 days/month; 12 days/week; 34 days/week, and almost every day.
Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) methods or a PCR method were performed on lymphocyte DNA samples from all participants, without knowledge of cancer status, to determine the genotypes for ALDH2 (5,36), ADH2 (37), ADH3 (37), and GSTM1 (38).
Fisher's exact test and the MantelHaenszel chi-square test were used in comparing group statistics. The associations between ALDH2, ADH2, ADH3, and GSTM1 genotypes and esophageal cancers were expressed in terms of the odds ratio (OR) and the population-attributable risk (PAR; (39) and adjusted for the effects of several possible confounders by the use of a multiple logistic regression model. These analyses were done with the SAS statistical package (version 8.2; SAS Institute, Cary, NC). The maximum likelihood estimates of linkage disequilibrium statistic D between ADH2 and ADH3 gene polymorphisms were obtained using the ASSOCIATE program version 2.36 provided by Dr J.Ott (http://linkage.rockefeller.edu/ott/linkutil.htm). The haplotype frequencies in the cancer patients and controls were calculated allowing for linkage disequilibrium by the use of EH program version 1.11 (http://linkage.rockefeller.edu/soft/).
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Results |
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Significant linkage disequilibrium was detected between the ADH2 and ADH3 gene polymorphisms, not only for the cancer patients (D = 0.044, 2 = 39.1, df = 1, P < 0.0001) but for the controls (D = 0.022,
2 = 38.6, df = 1, P < 0.0001). The haplotype including the ADH2*1 and ADH3*2 alleles was over represented among both the cancer patients and controls. The frequencies of estimated haplotypes were significantly different between the cancer patients and controls (Table IV
). The proportion of haplotypes that included ADH2*1 allele was higher in the cancer patients than in controls regardless of the accompanied ADH3 allele. By contrast, the proportion of haplotypes that included ADH3*2 allele was similar between the cancer patients and controls when the accompanied ADH2 allele was identical.
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Discussion |
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In contrast to the long-held belief that the risk for alcohol-related esophageal cancer increases in dose-dependent fashion (40,41), our new findings show the difference between risks for drinkers with and without inactive ALDH2. An alcohol challenge test showed that in ALDH2 heterozygotes the average peak blood acetaldehyde after drinking a small amount of ethanol (0.1 g/kg body wt) was five times the average peak blood acetaldehyde in active ALDH2 homozygotes after drinking a moderate amount of ethanol (0.8 g/kg body wt; 42). Thus, with regard to the ALDH2 genotype, the levels of acetaldehyde in blood may determine dose dependency after drinking. Although multivariate analysis showed a significantly increased risk for esophageal cancer in the presence of the ALDH2*2/2*2 genotype, the small number of cases with the ALDH2*2/2*2 genotype in this study prevents reaching any conclusion.
These findings may force changes in our long-held views of Japanese men's risky drinking, but confirmation of these associations is needed. One recent study (6) reported enhanced risk for esophageal cancer due to the inactive heterozygous ALDH2 in heavy drinkers (OR = 16.4), but the risk did not reach significance in non-heavy drinkers (OR = 1.7). That study's non-heavy-drinking cancer population consisted of 34 cases, of which 16 were female. Its small sample size and the inclusion of women might have hampered that study from obtaining significant results. Analysis of women's risk for esophageal cancer is another focus of ongoing research in our laboratories.
There is ample evidence of acetaldehyde's carcinogenicity in experimental animals (9,10). In addition, the levels of acetaldehydeDNA adducts are higher in leukocytes from alcoholics than in those from healthy controls (43), and chromosomal alterations are observed more frequently in lymphocytes from drinkers with inactive ALDH2 (44). It is reasonable to speculate, therefore, that the high risk for esophageal cancer in persons with inactive ALDH2 is the consequence of repeated exposure to acetaldehyde. Individuals with inactive ALDH2 are incapable of rapidly eliminating this carcinogenic compound after drinking ethanol.
The reason for the selective influence of acetaldehyde on the esophagus is still open to question, however. This influence may be related to the rate of ethanol metabolism by topical mucosal enzymes in the esophagus (45). High-Km ADH7 is strongly expressed in the esophagus, which is active in producing acetaldehyde upon exposure to a locally high dose of ethanol. Moreover, salivary acetaldehyde levels are high after a moderate dose of alcohol in individuals with the ALDH2*2 allele (46). Alcoholic beverages themselves contain high levels of acetaldehyde (47), and normal oral microflora also form acetaldehyde from ethanol and contribute to acetaldehyde levels in saliva (48).
The esophagus lacks ALDH2 activity or expresses it extremely weakly, if at all (45). After exposure to acetaldehyde derived from systemic, mucosal, salivary or bacterial production, and alcoholic beverages per se, inefficient degradation of the acetaldehyde in the esophagus may enhance the risk for cancer. Comparison between the acetaldehyde concentrations in the esophagus and other organs would provide information crucial to the elucidation of this issue.
Although overall trends suggest that all types of alcoholic beverages increase the risk for esophageal cancer, some epidemiologic studies have shown stronger associations between esophageal cancer and alcoholic beverages high in ethanol (40). The present multivariate analyses confirm our earlier reports that alcoholic Japanese men's risk for esophageal cancer increases significantly with the use of strong alcoholic beverages (49,50). Our results stress not only the importance of the local effect of ethanol and its solvent action on tobacco and other carcinogens penetrating the mucosa but also the role of higher levels of acetaldehyde as a carcinogenic ingredient in stronger alcoholic beverages (47).
Other intriguing results from this study are the interactions between the ALDH2 and ADH2 genotypes. On initial examination of this issue, we observed significantly enhanced risks for esophageal cancer among those with the ADH2*1/2*1 genotype throughout all but the ex-drinking category. In addition, the much less active enzyme encoded by ADH2*1/2*1 enhanced the carcinogenic effect of the inactive ALDH2 in a multiplicative fashion. Investigators have found no correlation between the ADH2 genotype and acetaldehydemia (16). Rather, the presence of the ADH2 isozyme encoded by ADH2*1/2*1 has been associated with slower production of acetaldehyde from ethanol in vitro (8). This finding refutes the ALDH2-associated hypothesis that acetaldehyde plays a critical role in enhancing esophageal carcinogenesis.
We had earlier reported that among Japanese alcoholic men, the much less active form of ADH2 tended to mask alcohol flushing in spite of the presence of inactive ALDH2 and severe acetaldehydemia (15), and that the less active form of ADH2 increased the carcinogenic effect of inactive ALDH2 on the esophagus (5,15). In the present study, we found that individuals (cases and controls) with both ALDH2*1/2*2 and ADH2*1/2*1 genotypes also tended not to experience alcohol flushing, and that those (controls) with the ALDH2*1/2*2 genotype who reported weaker alcohol flushing tended to be heavier drinkers (T.Yokoyama et al., in preparation). Because the ALDH2 genotype is the basis on which to determine the level of an individual's acetaldehyde exposure after drinking (16), it may be that the ADH2 genotype affects cancer susceptibility, in part, through its influence on alcohol flushing. If the much less active form of ADH2 diminishes alcohol flushing in ordinary drinkers with inactive ALDH2, the diminished intensity of the protective response may enhance the individual's vulnerability to drinking and his exposure to acetaldehyde. Thus, interactions between the flushing response, ALDH2 and ADH2 genotypes and cancer susceptibility are of great interest for future studies.
Because in the presence of ADH2*1/2*1 the risk for esophageal cancer was higher, even among individuals with ALDH2*1/2*1, the effects of ADH2 cannot be fully explained by the influence of alcohol flushing. We have reported a similarly increased risk for esophageal cancer associated with ADH2*1/2*1 in an alcoholic populations with ALDH2*1/2*1 (5,15). Alcoholics with ADH2*1/2*1 tend to have experienced binge drinking and withdrawal syndrome earlier in life than those with other genotypes (51). Thus, ADH2*1/2*1- mediated acceleration of the occurrence of alcohol-related events may contribute to the enhancement of cancer risk in alcoholics. Some yet unknown differences in esophageal alcohol metabolism or in the drinking behaviors of individuals with and without ADH2*1/2*1 might influence the risk for cancer in the present population. ADH2 is the main enzyme among low-Km class I ADHs expressed in the esophagus (45). In ADH2*1/2*1 homozygotes, low concentrations of ethanol may linger in the esophageal mucosa during the slow oxidation of low Km ADH2 and thus may be associated with esophageal carcinogenesis.
Increased cancer risks were apparent, though to a lesser extent, in groups of moderate to heavy drinkers with either of the less active enzymes encoded by ADH3*1/3*2 and ADH3*2/3*2. Because of the proximity of the ADH2 and ADH3 loci on the human genome, ADH2 gene polymorphism is in linkage disequilibrium with ADH3 gene polymorphism (25,26), such that the two less active alleles, ADH2*1 and ADH3*2, are linked. The difference in enzymatic activity between the ADH3*1 and ADH3*2 proteins is estimated to be much smaller than that between the ADH2*1 and ADH2*2 proteins (8). The ADH2*1 and ADH3*2 alleles have been associated with greater risks for alcoholism in east Asians (25,52), but when recent Chinese studies considered this linkage disequilibrium, they found no relationship between ADH3 and alcoholism (26,27). Therefore, it is reasonable to assume that the ADH3*2 effect on cancer risk also reflects the effects of ADH2*1 through the linkage. Our study also confirmed high linkage disequilibrium statistic D values in both the cancer patients and the controls. The haplotype analysis revealed that ADH2*1 allele was more prevalent in the cancer patients than in controls whether the accompanied ADH3 allele was ADH3*1 or ADH3*2, whereas the prevalence of ADH3*2 allele was similar between the cancer patients and controls when the accompanied ADH2 allele was identical. This clearly indicated that ADH2*1 but not ADH3*2 per se had an individual effect on the risk of cancer. Furthermore, after adjustment for the ALDH2 and ADH2 genotypes, ADH3 showed no significant effects on cancer risk, also suggesting that its linkage with the ADH2 genotype could explain the apparent increase in risk associated with the ADH3 genotype.
Our findings concerning ADH3 are in contrast with those of two European studies, which found that alcoholics possessing the ADH3*1 genotype were at greater risk for upper aerodigestive tract cancer (19,24). There is marked racial diversity in ALDH2, ADH2, and ADH3 gene polymorphisms (8). Europeans' low prevalence of the ALDH2*2 (0%) and ADH2*2 alleles (010%) exclude the interactions between ALDH2, ADH2, and ADH3 genotypes. `At-risk' interactions may also account, in part, for a paradox in Japanese esophageal cancer statistics: due to the high prevalence of the ALDH2*2 and ADH2*2 alleles, per capita alcohol consumption by Japanese is lower than that of Europeans and Americans; however, the death rates for esophageal cancer rank higher for Japanese men than for men from western nations (53).
There is substantial evidence that smoking (40,41) and poor intake of vegetables and fruit (54) enhance the risk for esophageal cancer, and our risk estimates were in accord with previous studies. In comparison with the estimated PARs for preference for strong alcoholic beverages (30.7%), smoking (53.6%), and lower intake of green and yellow vegetables (25.7%) and fruit (37.6%), an extraordinarily high proportion of the excessive risk for esophageal cancer in the Japanese men can be attributed to drinking (90.9%), particularly drinking by persons with inactive heterozygous ALDH2 (68.5%). Our moderate-to-heavy drinkers with inactive heterozygous ALDH2, who corresponded to 9.8% of the controls, yielded a PAR of 57.2%.
Our large sample size, which was sufficient for performing subgroup analyses among heavy smokers and non-heavy smokers, showed no significant association between the GSTM1 genotype and the risk for esophageal cancer. The GSTM1 genotype did not appear to play any role in the development of esophageal cancer in this population, a finding that is in accordance with two previous Japanese studies (2,32).
Our study has several potential limitations. With any casecontrol study of this type, there is always the risk of population stratification. The controls were recruited from Tokyo, while the cases were recruited from four sites in Japan: three in or near Tokyo (Tokyo, Kanagawa, and Chiba) and one distinct area (Osaka). However, all of the hospitals were located in the urban areas of central portion of Honshu Island. Studies have reported that the frequencies of the ALDH2 and ADH2 genotypes do not differ between the general populations of these areas (2,6,14,18,25). The drinking and smoking habits of the general populations are reportedly similar between Tokyo and Osaka (55). Comparison of drinking, smoking, dietary habits, and ALDH2/ADH2 genotypes of our case populations from the four hospitals showed no significant geographical heterogeneity.
An additional limitation is that the background characteristics might reflect better health among the controls attending the clinics for annual health checkups than actually exists among the general urban population. Estimation of PAR requires that controls be representative of the general population. According to National Nutrition Survey in Japan in 2000 (56), a nationwide population-based survey using representative samples, the age-adjusted prevalence of current smokers and the proportion of men who drink alcohol 3 days or more per week and consume at least 1 unit at a time are 37.5% and 55.8%, respectively, in Tokyo. Our study's finding of similar values (40.7% and 56.0%, respectively) for our control group implies that they represented the general population of Tokyo well, at least with regard to drinking and smoking habits.
Because the age distribution of our controls did not match that of the cases well, especially at ages 7079 years, there was concern that adjustment for age might have unexpected effects. Repeating our analyses and excluding men >70 years of age confirmed the results obtained in our overall analyses. Nevertheless, these older subgroups deserve further investigation.
Professional and public education about risky conditions connected with inactive ALDH2 is vitally important in a new strategic approach to the prevention of esophageal cancer. The development of screening tests for inactive ALDH2 based on alcohol flushing responses (14,17,57) is also important. Because the intensity of the flushing response is decreased in individuals with long or heavy drinking histories, we designed a questionnaire that can detect changes in flushing responses over time, by asking about both current and past flushing (14). This simple questionnaire has high sensitivity for detecting inactive ALDH2 in men >50 years old (96.1%; 14) and in patients with esophageal or oropharyngolaryngeal cancer (95.6%; 58). Knowledge of the present findings and use of this highly sensitive flushing questionnaire will benefit many people who can identify their own risks for esophageal cancer. It may also help clinicians diagnose esophageal cancer earlier, by screening the high-risk population.
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Notes |
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Acknowledgments |
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References |
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