National Institute on Alcoholism, Kurihama National Hospital, 5-3-1 Nobi, Yokosuka, Kanagawa 239-0841,
1 Department of Surgery, Kawasaki Municipal Hospital, Kawasaki, Kanagawa 210-0013,
2 Department of Neuropsychiatry and
3 Department of Internal Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-0016 and
4 Department of Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
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Abstract |
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Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; OR, odds ratio.
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Introduction |
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The mutant allele encoding an inactive subunit of aldehyde dehydrogenase-2 (ALDH2*2) is prevalent in Asians [e.g. approximately 50% of Japanese (4)], but has not been found in Caucasians or AfricanAmericans (5). Its failure to promptly metabolize the ethanol metabolite acetaldehyde leads to excessive acetaldehyde accumulation after drinking (6,7). Although the inactive ALDH2 inhibits Asians from heavy drinking and the development of alcoholism by acetaldehydemia and alcohol flushing responses (8), the preventive effect of heterozygous ALDH2*1/2*2 is incomplete. Approximately 10% of Japanese alcoholics have ALDH2*1/2*2 (4).
Studies of various Japanese drinking populations have shown that the inactive form of ALDH2 encoded by the gene ALDH2*1/2*2 is a risk factor for oropharyngolaryngeal (9,10) and esophageal cancers (9,1113). Synchronous (14) and metachronous (15) multiple esophageal cancers have been found more frequently in Japanese alcoholics with the inactive ALDH2 than in those without it. In Japanese head and neck cancer patients, inactive ALDH2 was a risk indicator for the presence of multiple esophageal iodine-unstained lesions, which consist mainly of squamous epithelial dysplasia (16). Because acetaldehyde is an established animal carcinogen (17,18), all this evidence points to a role for acetaldehyde in the development of human cancers.
Our screening of Japanese male alcoholics, using upper gastrointestinal endoscopy combined with oropharyngolaryngeal inspection and esophageal iodine staining, detected aerodigestive tract cancer at extremely high rates (19,20). Synchronous and metachronous multiple esophageal cancers have been also frequently found in this population (14,15). The high rate of detected esophageal cancer is primarily due to the improved method of detection (esophageal iodine staining), but enhanced susceptibility by persons with inactive ALDH2 also explains the extremely high rate in Japanese alcoholics.
A genetic polymorphism of alcohol dehydrogenase-2 (ADH2) also has striking racial differences. The mutant ADH2*2 allele, which is highly prevalent among Asians [e.g. 93% of Japanese (4)], encodes a superactive subunit of ADH2. The homodimer of ADH2 encoded by ADH2*1/2*1 has only 1/100 and 1/200 of the ethanol oxidation capacity of the isozymes encoded by ADH2*1/2*2 and ADH2*2/2*2, respectively (21). The ADH2*2 allele has been found in only about 10% of Caucasians (5).
Inactive ALDH2 is responsible for most of the intense flushing responses in Asians, but the ADH2*2 allele also contributes to alcohol flushing (2224) and prevents people from developing alcoholism (4,25). Recently, the ADH2*1/2*1 genotype has been reported to be more strongly associated with the development of esophageal cancer than the ADH2*1/2*2 or ADH2*2/2*2 genotypes in both the alcoholic (24) and general populations (12) of Japan. The aim of this study was to define the individual and combined roles of the ADH2 and ALDH2 gene polymorphisms in the risk for cancer development and multicentric field cancerization of the oropharyngolaryngeal sites, esophagus and stomach in Japanese alcoholics.
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Materials and methods |
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For reference, we used data from 526 Japanese male alcoholics aged 40 years or older without cancer who were consecutively admitted to the Institute between January 1996 and May 1998. None of these patients had a history of cancer, and all were diagnosed as cancer-free by systematic examination using chest X-rays plus upper gastrointestinal endoscopy combined with oropharyngolaryngeal inspection and esophageal iodine staining, immunochemical fecal occult blood tests with subsequent double-contrast barium enema and/or colonoscopy and abdominal ultrasound.
The proposed study was reviewed and approved by the ethics committee of the Institute, and informed consent was obtained from each participating patient.
Drinking and smoking habits
Information on the drinking and smoking habits of all 159 alcoholic cancer patients and the 526 alcoholic patients without cancer was obtained from patients themselves and, when available, their partners. All of these alcoholics met the criteria for alcohol dependence of the Diagnostic and Statistical Manual of Mental Disorders [3rd Edn, revised (26)]. Daily alcohol consumption during the year preceding cancer diagnosis or admission was expressed in grams of ethanol per day, using a standard conversion for alcohol beverages. Beer was considered to be 5% ethanol (v/v); wine, 12%; sake, 16%; shochu, 25%; and whiskey, 40%.
ADH2 and ALDH2 genotyping
The methods of Xu et al. (27) and slight modifications of the methods of Harada and Zhang (28) were used for polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) for ADH2 and ALDH2 genotyping of lymphocyte DNA samples from the patients' blood. The modifications for ALDH2 genotyping were as follows: briefly, 100200 ng genomic DNA was mixed with 5 pmol each primer (5'-CAAATTACAGGGTCAACTGCT-3', sense; 5'-CCACACTCACAGTTTTCTCTT-3', antisense) in a total volume of 50 µl containing 50 µM each dNTP, 1.5 mM MgCl2 and 1 U Taq DNA polymerase (Promega, Madison, WI). Thirty-five cycles of PCR (denaturation at 94°C for 15 s, annealing at 58°C for 1.5 min and polymerization at 72°C for 30 s) were performed in a Perkin Elmer Cetus GeneAmp PCR System 9600. After purification, each PCR product was digested with MboII, electrophoresed on a 20% polyacrylamide gel, stained with ethidium bromide and viewed.
Statistical analysis
Data were expressed as mean ± standard deviation (SD), and the unpaired Student's t-test was used in comparing group statistics. The association between ADH2 and ALDH2 genotypes and cancer risk was expressed as the odds ratio (OR), adjusted for the effects of several possible confounders using a multiple logistic regression model. To assess the impact of ADH2 and ALDH2 gene polymorphisms on the occurrence of cancers at the alcoholic population level, we estimated the population attributable risk, using an approach based on unconditional logistic regression (29). By combining adjusted OR estimates and the observed prevalence of the risk factors under study, this approach yields a point estimate of adjusted population attributable risk for the cases. The following formula was used to calculate the adjusted population attributable risk:
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Results |
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Discussion |
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This gene-based cancer susceptibility adds a new dimension to the alcohol-cancer enigma. In individuals with inactive ALDH2 encoded by ALDH2*1/2*2, the blood acetaldehyde level after drinking is ~6-fold the concentration in those with active ALDH2 (7). There is sufficient evidence of acetaldehyde's carcinogenicity in experimental animals (17,18). In addition, its ability to form DNA adducts in humans may represent a critical initiating event in the process of carcinogenesis. The levels of acetaldehyde adducts in leukocyte DNA from alcoholics are much higher than those in healthy controls (30), and chromosome alterations have been observed more frequently in lymphocytes from drinkers with inactive ALDH2 (31). Together, this evidence of ALDH2-associated susceptibility suggests that acetaldehyde plays a pivotal role in carcinogenesis in alcoholics.
Although investigators have failed to establish any correlation between ADH2 genotype and peak levels of blood acetaldehyde after drinking (7), the high-risk ADH2 encoded by ADH2*1/2*1 has only a small fraction of the activity of the isozymes encoded by the ADH2*2 allele (21) and individuals with ADH2*1/2*1 might be exposed to lower doses of acetaldehyde following heavy alcohol drinking. That would argue against the important role of acetaldehyde in carcinogenesis of alcoholics. ADH2*1/2*1 might, under certain conditions, increase the risks for oropharyngolaryngeal and esophageal cancer via other mechanisms of the interaction between genes and drinking behavior.
The ADH2*1/2*1 and ALDH2*1/2*2 genotypes, each of which encodes the inactive form of its isozyme, have opposing effects on drinking behavior (4,25). ALDH2*1/2*2 generally serves as a strong protective factor against alcoholism, as exemplified by our earlier study, in which 12% of Japanese alcoholics had inactive ALDH2, compared with 42% of healthy controls (4). In addition, the ADH2*2 allele also protects many Japanese from alcoholism: 69% of our Japanese alcoholics had this allele, compared with 93% of healthy controls (4). This evidence suggests that cancer risks for ADH2 and ALDH2 genotypes should be adjusted by alcohol consumption. Another study, carried out in the surgical department of a general hospital, showed high frequencies of ADH2*1/2*1 and ALDH2*1/2*2 genotypes (22 and 75%, respectively) among the esophageal cancer patients, 93% of whom were drinkers or smokers (12). The findings are consistent with those of the present study. However, in that study (12) the investigators calculated the patients' risk in comparison with that of only healthy control subjects without adjustment for alcohol consumption. Moreover, they overestimated the risk associated with ADH2*1/2*1 and underestimated that for ALDH2*1/2*2.
The present study conducted in an alcoholic population showed cancer risks at only the highest level of alcohol exposure. At that level, both ADH2*1/2*1 and ALDH2*1/2*2 increased the risks considerably, but the role of ALDH2*1/2*2 predominated; the odds ratios for ALDH2*1/2*2 were 3- to 5-fold those for ADH2*1/2*1 with respect to the oropharyngolaryngeal and esophageal cancers.
Our previous estimate of ALDH2*1/2*2-associated risk for non-alcoholic everyday drinkers with esophageal cancer (11), whose average daily alcohol consumption was approximately half that of the alcoholics, is close to the odds ratio for ALDH2*1/2*2 found in this study. From the public health point of view, gene-based susceptibility to the carcinogenic action of alcohol may have more important implications at low levels of exposure. Among populations of European origin, a beneficial effect of light drinking has been demonstrated (32). Regardless of whether this is the case for Asians, carcinogenic interactions between the genotypes of alcohol-metabolizing enzymes and light alcohol consumption should be investigated.
There is a paradox in Japanese esophageal cancer statistics: per capita alcohol consumption by Japanese males is lower than that by European and American males (due to the high prevalence of ADH2*2 and ALDH2*2 alleles in Japanese); however, the death rates for esophageal cancer rank higher for Japanese males than for men from Western nations (33). Although some differences in other risk factors such as smoking and diet influence esophageal cancer mortality [e.g. higher prevalence of smoking among Japanese males than among Western males (34)], this paradox might be explained, in part, by the carcinogenic role of ALDH2*1/2*2 in Japanese, which predominates over the influence of ADH2*1/2*1 prevailing in Europeans and Americans.
We cannot adequately explain why inactive ALDH2 and severe acetaldehydemia selectively influence the development of cancers on the oropharyngolaryngeal sites and esophagus. One possible explanation is related to topical acetaldehyde exposure and accumulation in these organs. In addition to systemic acetaldehyde exposure, these organs are exposed to acetaldehyde derived from salivary and mucosal production. An alcohol-challenge test showed that the salivary acetaldehyde levels in Asians with ALDH2*1/2*2 were 9-fold higher than their blood acetaldehyde levels and 23-fold higher than the salivary acetaldehyde levels in those with ALDH2*1/2*1 (35). The gingiva, tongue and esophagus strongly express high Km ADH7, which would be very active in producing acetaldehyde upon exposure to a locally higher ethanol dose than most other organs, including the liver. Moreover, these organs lack ALDH2 activity (36,37), suggesting that after exposure to acetaldehyde derived from systemic, salivary or mucosal production, these organs' inefficient degradation of acetaldehyde enhances the chances for local acetaldehyde-associated carcinogenesis.
Our results show that ADH2*1/2*1 combines with ALDH2*1/2*2 to enhance the risks for oropharyngolaryngeal and esophageal cancers in a multiplicative fashion. Recently, using simple questions to learn about current or former facial flushing after drinking a glass of beer, we showed that, among alcoholics with ALDH2*1/2*2 who had esophageal cancer, 52% of those who also had ADH2*1/2*1 had never experienced alcohol flushing. Of those with both ALDH2*1/2*2 and ADH2*2 alleles, 92% reported current or former flushing (24). Accordingly, individuals with the combination of ADH2*1/2*1 and ALDH2*1/2*2 should be at high risk for acetaldehyde-related cancers because, as a result of their lack of protective flushing, they are more likely to drink to experience accelerated development of alcoholism, and thus to be exposed to higher levels of acetaldehyde earlier in life. However, because ADH2*1/2*1 increased the risk of oropharyngolaryngeal and esophageal cancers, even among the never-flushing alcoholics who have ALDH2*1/2*1, the ADH2 effects cannot be fully explained by the influence of alcohol flushing. Some unknown differences in the alcohol metabolism or drinking behaviors of alcoholics with and without ADH2*1/2*1 might influence the risk for cancer. Our recent examination of the temporal profile of disease development among alcoholics showed that those with ADH2*1/2*1 experienced each event in the history of alcoholism (e.g. binge drinking and withdrawal syndrome) earlier in life than those without this genotype (38,39), suggesting that ADH2*1/2*1-mediated acceleration in the occurrence of alcohol-related events contributes to the enhancement of cancer risk. During binge drinking, episodic alcohol consumption is generally very high, compared with average daily consumption; however, in the present study, we adjusted cancer risks for the subjects' drinking habits, using only the reported daily alcohol consumption during the year preceding cancer diagnosis or admission. Thus we cannot rule out the possibility that we underestimated the confounding effects of drinking behaviors relative to ADH2*1/2*1 genotype.
Epidemiologic studies have shown that the risks for cancer from drinking alcohol differ across the subsites of the oropharyngolarynx. An IARC international case-control study of southern European populations showed alcohol's similar distinct influence on the risks for epilaryngeal and hypopharyngeal cancers and its weak influence on endolaryngeal cancer risk (40); approximately equal numbers were found to have epilaryngeal/hypopharyngeal and endolaryngeal cancer among the cases classified in the highest category of alcohol consumption (121 g/day). In the present study, the cancer risks from ADH2*1/2*1 and ALDH2*1/2*2 were similarly enhanced for the oral cavity/oropharynx and epilarynx/hypopharynx; however, we found only two patients with endolaryngeal cancer. The disproportionate rarity of endolaryngeal cancer compared with epilaryngeal/hypopharyngeal cancer in Japanese alcoholics might, at least in part, be explained by the enhanced carcinogenic effect of alcohol on oropharyngolaryngeal subsites excluding the endolarynx, where the effect of tobacco seems to be more important. Of two Japanese case-control studies recently investigating the association between the ALDH2 genotype and the risk for oral cancer, one showed increased risk in the presence of ALDH2*1/2*2 in moderate drinkers (10), although the magnitude of increased risk (OR = 2.9) was considerably lower than the risk in alcoholics. The other study showed neither alcohol consumption nor ALDH2 genotype to be associated with cancer risk (41). In some non-alcoholic populations, alcohol actions and ALDH2 effects on oral carcinogenesis may be less important than other risk factors such as smoking, poor dentition and inadequate oral hygiene.
We have previously reported frequently finding multiple intra-esophageal cancers and multiple esophageal cancers with oropharyngolaryngeal and/or stomach cancer in Japanese alcoholics, especially those with ALDH2*1/2*2 (14,15,20). The present findings show that the role of ALDH2*1/2*1 predominates for multiple field cancerization, but that the ADH2 genotype is not associated with this multiplicity. Our observations indicated that these multiple cancers were strongly associated with severe acetaldehyde exposure, and suggested that the role of ADH2*1/2*1 in carcinogenesis might differ from the acetaldehyde scenario. The slightly higher odds ratios of oropharyngolaryngeal cancer for ALDH2*1/2*2 (ORs = 18.5228.92) compared with esophageal cancer (OR = 13.50) could be explained by different frequencies of multiple cancers in these organs between oropharyngolaryngeal cancer patients (45.5%) and esophageal cancer patients (19.6%).
Stomach cancer is one of the leading cancers in Japan, where its prevalence is near the highest in the world. There is little epidemiologic evidence that alcoholic beverages play a causal role in stomach cancer (1), but our endoscopic screening has yielded a higher rate of detection of stomach cancer in Japanese alcoholics than in the Japanese general population (20). Molecular epidemiology in the present study showed that in fact stomach cancer has a relatively weak but significant association with the ALDH2*1/2*2 genotype, but not with the ADH2*1/2*1 genotype. This positive association with ALDH2*1/2*2 was stronger for stomach cancer patients who also had oropharyngolaryngeal and/or esophageal cancer, but it was not observed for those with stomach cancer alone. These findings support the hypothesis that the pathogenesis of stomach cancer concurrent with oropharyngolaryngeal and/or esophageal cancer is associated with acetaldehyde exposure and distinct from other stomach cancer. The lack of association between stomach cancer and the ADH2 genotype further suggests that, unlike that of ADH2*1/2*1-associated cancers, the high prevalence of stomach cancer might not be associated with alcohol drinking per se but rather with other aspects of risk (e.g. diet, nutrition, smoking) in the alcoholics' lifestyle.
Regardless of the underlying mechanisms that link ADH2 and ALDH2 genotypes and alcohol-related aerodigestive tract cancers, the identification of flushing responses, which indicate inactive ALDH2 and associated risks could serve as a powerful means of preventing alcohol-related cancer in Japanese. The ethanol patch test (42) and flushing questionnaires (43,44) can reliably identify ALDH2 phenotypes in individuals who do not have ADH2*1/2*1. Unfortunately, however, these screening tools frequently yield false-negative results for persons with the combination of ADH2*1/2*1 and ALDH2*1/2*2 (22,24), who are at highest risk for cancer. Thus, we recommend testing the usefulness of combined ADH2 and ALDH2 genotyping as a method of screening for aerodigestive tract cancer among alcoholics in Japan.
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Notes |
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Acknowledgments |
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References |
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