Carcinogenetic impact of ADH1B and ALDH2 genes on squamous cell carcinoma risk of the esophagus with regard to the consumption of alcohol, tobacco and betel quid
Article first published online: 21 NOV 2007
Copyright © 2007 Wiley-Liss, Inc.
International Journal of Cancer
Volume 122, Issue 6, pages 1347–1356, 15 March 2008
How to Cite
Lee, C.-H., Lee, J.-M., Wu, D.-C., Goan, Y.-G., Chou, S.-H., Wu, I.-C., Kao, E.-L., Chan, T.-F., Huang, M.-C., Chen, P.-S., Lee, C.-Y., Huang, C.-T., Huang, H.-L., Hu, C.-Y., Hung, Y.-H. and Wu, M.-T. (2008), Carcinogenetic impact of ADH1B and ALDH2 genes on squamous cell carcinoma risk of the esophagus with regard to the consumption of alcohol, tobacco and betel quid. Int. J. Cancer, 122: 1347–1356. doi: 10.1002/ijc.23264
- Issue published online: 21 JAN 2008
- Article first published online: 21 NOV 2007
- Manuscript Accepted: 25 SEP 2007
- Manuscript Received: 28 JUL 2007
- Taiwan National Science Council. Grant Numbers: NSC 95-2314-B-037-081, 95-2314-B-039-042-MY3
- Taiwan National Health Research Institutes. Grant Numbers: NHRI-CN-IN-9007P, NHRI-EX94-9428PI
- alcohol drinking;
- esophageal neoplasms;
- genetic poly-morphism;
The consumption of alcohol, tobacco and betel quid has been found to be an important contributor to esophageal squamous cell carcinoma (ESCC) in Taiwan. The genotoxic effect of the ADH1B and ALDH2 genes modulating an individual's alcohol-metabolizing capacity on ESCC may be linked to drinking behavior, intake pattern and other exogenous factors. To investigate the interplay of these genetic and environmental factors in determining the risk of ESCC, a multicenter case-control study was conducted. Here, 406 patients with pathology-proven ESCC, as well as 656 gender, age and study hospital matched controls were recruited. Genetic polymorphisms of ADH1B and ALDH2 appeared to correlate with the abstinence of alcohol, though not with tobacco and betel quid. Within the same levels of alcohol consumption, carcinoma risks increased along with an increase in the numbers of ADH1B*1 and ALDH2*2 alleles. The inactive ALDH2*1/*2 genotype was found to multiplicatively interact with a low-to-moderate (0.1–30 g/day) and a heavy (>30 g/day) ethanol intake to increase the ESCC risk (the joint aOR = 14.5 and 102.6, respectively). Among low-to-moderate drinkers, a smoking-dependent carcinogenetic effect for the ADH1B*1/*1 and ALDH2*1/*2+*2/*2 genotypes was recognized, with significant risks found in smokers, but not in nonsmokers. Further, a supra-multiplicative combined risk of ESCC for alcohol and tobacco use was identified among carriers of the ADH1B*1/*1 genotype (p for interaction = 0.042). In conclusion, the interplay of the ADH1B and ALDH2 genotypes, in conjunction with a behaved drinking habit and a practiced drinking pattern, along with continued tobacco consumption, plays an important pathogenic role in modulating ESCC risk. © 2007 Wiley-Liss, Inc.
Alcohol consumption has been causally linked to the cancerization of the esophagus in humans.1 Epidemiological surveys offered ample evidence that esophageal cancer risk due to this agent is not homogeneous across gender and populations, or even socioeconomic classes.1 Although discrepancies in the characteristics of alcohol intake have clarified parts of this issue,2 interindividual dissimilarities in regard to the capabilities of alcohol metabolism may be crucial in accounting for the diverse effect of alcohol in populations, since acetaldehyde, the primary intermediate metabolite of ethanol, has been well recognized as a carcinogen in animal models.3
Though the variant allele of the alcohol dehydrogenase-1B gene (ADH1B*2) that confers the “fast” metabolism of ethanol to acetaldehyde, and the mutant allele of the aldehyde dehydrogenase-2 gene (ALDH2*2) that results in a catalytically inactive subunit to oxidize acetaldehyde to acetate, are prevalent in Orientals, they are uncommon in Caucasians.4 Alcohol flushing syndrome, characterized by a facial flushing accompanied by palpitation, drowsiness, breathlessness or nausea is a condition related to a toxic accumulation of acetaldehyde in the human body.5 In Asians, such a reaction is found to be clustered in drinkers carrying the ALDH2*2 genetic variant and, to a certain extent, the ADH1B*2 homozygote was also observed to enhance this syndrome. While the effect of the fast-metabolizing ADH1B allele has not been evidently established,6, 7 earlier investigations reported that the ALDH2*2 allele significantly prevents individuals from further drinking due to unpleasant responses.7, 8 Alternatively, research in various populations have found that esophageal cancer risk in regard to alcohol intake is closely correlated to polymorphisms of the ADH1B and ALDH2 genes.6, 9–11 Although esophageal neoplasm is believed to develop under both genetic and environmental influences, information about how the influences of genetic susceptibility and drinking behavior, as well as the interplay between genetic variants and alcohol consumption exert, is still limited.
The relationship between the two alcohol-related metabolizing genes and esophageal cancer risk has been suggested to be ethanol-amount dependent.6, 12, 13 As seen in a meta-analysis, light-to-moderate alcohol consumption (<24 g/day) may not be harmful to certain diseases, for example, in regard to total and ischemic stroke.14 Whether the genetic vulnerability for esophageal cancer expands to this level of ethanol intake or its effect is also related to other exogenous factors, remains uncertain. Since there are a substantial proportion of people who consume such quantities of alcohol, this issue raises a major concern.
The ALDH2 activity in the mucosa of the upper aerodigestive tract (UADT) has been experimentally reported to be particularly weak.15, 16 Accordingly, the biomedical mechanism that increases UADT cancer risk among individuals with inactive ALDH2 heterozygote has been hypothesized to be connected to some unidentified systemic effects in regard to increased blood acetaldehyde levels.17 Recently, since a 2- to 3-fold elevated salivary acetaldehyde level was detected in ALDH2-deficient healthy Asians after a moderate alcohol ingestion, the local carcinogenic role of acetaldehyde that is associated with the variant ALDH2 allele in humans has been supported.18 In contrast, the possible pathogenic mechanisms behind ADH1B polymorphisms and the increased risk of esophageal cancer have remained indistinct. Experimental research on Japanese alcoholic patients indicated that the high levels of salivary acetaldehyde observed as a result of prolonged ethanol exposure and oral microorganism overgrowth are related to the less-active ADH1B genotype.19 If the locally carcinogenetic activity of acetaldehyde in the esophagus is substantial, the joint tumor-promoting effect of the inactive ALDH2 allele and the slow-metabolizing ADH1B*1/*1 homozygote on this neoplasm should be properly understood.
In addition to alcohol drinking, the consumption of tobacco and betel quid has been found to be an important contributor to the development of esophageal cancer in India and Taiwan.20–23 Previous investigations revealed that alcohol may interact with tobacco and areca nut in determining the genesis of this cancer.24, 25 However, researchers know very little about how/if the synergistic way that alcohol, perhaps acting as a solvent,1 exerts its carcinogenetic effect with these two substances, is linked to the rate of alcohol-elimination determined by the ADH1B genotypes. On the other hand, substantial concentrations of acetaldehyde derived from tobacco smoke have been found to dissolve into the saliva during active smoking.26 Such findings, together with the importance of the less-active ADH1B allele in the salivary acetaldehyde levels, make it seem prudent to take ADH1B polymorphisms into account when examining the synergistic effect of alcohol consumption and tobacco smoking on esophageal cancer.
The uses of alcohol, tobacco and betel quid have been shown to account for about 84% of the attributable fraction of esophageal cancer in Taiwan,24 and the ADH1B*2 and ALDH2*2 variants occurred in about 73% and 29% of the population, respectively.12 We therefore conducted a large-scale study to investigate the carcinogenetic impact of the ADH1B and ALDH2 genes on the cancerization risk of the esophagus. This was performed via the examination of the possible interplay between genetic polymorphisms with drinking behavior and one's alcohol intake pattern, as well as the other exogenous factors.
Material and methods
From 1996 to 2005, a multicenter case-control design for the research of esophageal cancer was conducted in 3 medical centers, National Taiwan University Hospital (NTUH, Taipei), Kaohsiung Medical University Hospital (KMUH, Kaohsiung) and Kaohsiung Veterans General Hospital (KVGH, Kaohsiung). These hospitals provide comprehensive medical services to patients of various socioeconomic levels in the greater Taipei and Kaohsiung metropolitan areas. The institutional review boards at NTUH and KMUH reviewed and approved this study, and all participants signed written sheets of informed consent.
The detailed study scheme for this research was described earlier.27 In brief, a survey network for speedy case recognition and verification was instituted among the aforementioned 3 hospitals so that incident esophageal cancer cases could be identified and enrolled into this study as soon as the respective diagnosis was confirmed. The study cases were those of newly diagnosed patients with esophageal squamous cell carcinoma (ESCC) that were recruited from the Department of Chest Surgery and the Department of Gastroenterology at the 3 medical centers. Although patient enrollment was initiated in 1996, the collection of blood specimens was launched from 2000. Therefore, the overall study period for this investigation was from 2000 to 2005. Of the 492 cases that have been histologically confirmed to have ESCC by the endoscopists, surgeons or pathologists, 414 patients took part in this study and provided blood samples. Although DNA genotyping for 8 patients was unsuccessful, 406 cases with complete polymorphism information remained available for analysis for our purposes. All of these study patients were interviewed within 1 week of their diagnosis.
The control subjects were recruited from the same 3 hospitals. Community residents aged 25 years and older who were hospitalized for 1 day in the Department of Preventive Medicine for routine health checkups were identified as potential controls after their first visit there. From a list of all potential controls, 1–4 controls (rarely, 3–4 controls) were selected to match each cancer patient with regard to gender, age (within a 4-year age difference) and hospitalization (within 4 weeks after each case was identified). Of the 694 matched controls, 660 subjects agreed to be interviewed and offered blood specimens. Genetic polymorphisms were successfully analyzed for 656 of these controls.
Detailed information in regard to demographic characteristics, substance use and daily diet was collected via an interview with participants utilizing a standardized questionnaire. Alcohol drinkers, tobacco smokers, and betel quid chewers were defined, respectively, as subjects who had consumed any alcoholic beverage ≥1 time per week, those who had smoked ≥10 cigarettes per week and individuals that had chewed ≥1 betel nut (measured as quid) per day for at least 6 months. The information about substance use was collected with regard to the age of respective commencement, their daily consumption, the duration of the use, and the type of substance consumed. To assess the risk of the total alcohol intake, data on the types of alcoholic beverage ingested (beer, wine, whisky, brandy, Taiwanese rice wine, Chu Yeh Ching liquor, Kaoliang Spirit, and so forth), the amount of ethanol from each type of beverage (according to the ethanol content of each beverage and the total amount consumed), the number of drinks per day and the average frequency per week was collected and converted to the total consumption of alcohol in grams per day.27 Since beer is the most popular alcoholic beverage in Taiwan, 1 drink was defined as having consumed 15.75 g of pure alcohol, which equates to one 350 ml of beer containing 4.5% ethanol. To examine the effect of cumulative lifetime exposure, the number of “drink × years” and “pack × years” was calculated by multiplying the amount of the substances consumed, 15.75 g-alcohol drinks per day drunk, 20-cigarette packs per day smoked or 10-betel quid packs per day chewed, by the corresponding years of the substance used. Daily dietary patterns were assessed by investigating the consumption of 20 food groups during the patients' 3 life stages (<20, 20–40, and >40 years of age). Information on the frequency and quantity of food intake was collected for each life stage. Only the consumption pattern of the latest stage for each patient was used for the data analyses.
All lymphocyte DNA was extracted from whole blood specimens. Laboratory analyses were performed by laboratory technicians who were blinded to the case-control status. The ADH1B genotype at codon 48 and ALDH2 polymorphism at codon 487 were determined by use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The detailed processes were described in earlier papers.24 In short, the genomic DNA sample was amplified with 2 primers: 5′-AAT CTT TTC TGA ATC TGA ACA G-3′ (forward) and 5′-GAA GGG GGG TCA CCA GGT TGC-3′ (reverse) for ADH1B polymorphisms, and 5′-CAA ATT ACA GGG TCA ACT GCT-3′ (forward) and 5′-CCA CAC TCA CAG TTT TCT CTT-3′ (reverse) for ALDH2 polymorphisms, respectively. The final PCR reaction products were treated with restriction enzymes, MaeIII for ADH1B, and MboII for ALDH2. The DNA digested samples for the 2 genes were then electrophorezed and examined, separately, in 3% agarose and 12% polyacrylamide gels. The representative figures as to the genetic analyses for the 2 genes are displayed in Figure 1.
To be assured of good quality control, DNA samples from cases and controls with the homozygous wild-type, heterozygous and homozygous variant genotypes for ADH1B and ALDH2 were initially sequenced to confirm genotype calling before the complete scale of genotyping was conducted. One positive control and 1 negative control without DNA templates were included in each batch of samples (∼10 samples). The positive control sample was included in each run of genotyping, to assure the complete digestion of the PCR product by a restriction enzyme. The negative control for detection of contamination was treated with the same reagents as those used with actual samples. To evaluate the reliability of the genotyping assays, about 8% of the study subjects (including both cases and controls) were randomly selected, and genetic polymorphism was reanalyzed. Results indicated that all of the ADH1B and ALDH2 genes were in 100% concordance.
The Hardy–Weinberg equilibrium test was used to assess the discrepancies between the observed and the expected genotype frequencies based on the allele frequencies that were observed. Unconditional multiple logistic regression models with maximum likelihood fitting were employed for all analyses.28 Odds ratios (OR), along with their 95% confidence intervals (CI), were computed so as to investigate the association between habitual alcohol drinking, tobacco smoking, betel quid chewing and the polymorphisms of ADH1B and ALDH2 genes. Tests for trends in the ORs across ordered exposure strata were performed by treating the categorical variable as a continuous predictor in the logistic regression model. The potential effect modification between the ADH1B and ALDH2 genes, or between the genes and substances used, was evaluated by fitting a multiplicative model with cross-product terms representing the interaction between the 2 variables studied. To test the significance of interaction, likelihood ratio tests for cross-product terms were applied.
The joint risks, in regard to developing ESCC, were calculated for variables that were coded for combinations of categorized factors explored using a common reference group. The OR ratio computed by exponentiating the appropriate linear combinations of coefficients in the fitted regression models was used to evaluate the capacity of different levels of exposure to distinguish the risk of contracting esophageal cancer. As covariates, gender, age (as a continuous variable), the study hospital (NTUH, KMUH, and KVGH) and one's ethnicity (Fukienese, Aborigine, Hakka and others) were included in the regression models. In addition, potential confounding factors, including the level of education (<7, 7–12, >12 years of schooling), cumulative tobacco smoking (Nonsmoker, 1–30, >30 pack × years), lifetime betel quid chewing (Nonchewer, 1–20, >20 pack × years), the consumption of vegetables and of fruits (<7, 7–14, >14 times per week), and, where appropriate, the ADH1B and ALDH2 polymorphisms, were adjusted in the multivariate models. The proportion of ESCC cases (population attributable risk percent; PAR%) attributable to one-specific level of alcohol consumption among carriers of different genotypes was calculated using the method described by Bruzzi et al.,29 which allows for an adjusted PAR% to be estimated on the data coming from the case-control studies.
As shown in Table I for selected demographic characteristics, the distributions of age, gender and ethnicity are reasonably similar for the cases and controls. The consumption of alcohol, tobacco and betel quid was all associated with a higher risk of contracting esophageal cancer, and a dose–response relationship was clearly observed here (p < 0.001).
|Factor/Category||Cases No. (%)||Controls No. (%)||aOR1 (95% CI)|
|<41||22 (5.4)||25 (3.8)|
|41–50||95 (23.4)||167 (25.5)|
|51–60||97 (23.9)||172 (26.2)|
|61–70||108 (26.6)||180 (27.4)|
|>70||84 (20.7)||112 (17.1)|
|Female||10 (2.5)||9 (1.4)|
|Male||396 (97.5)||647 (98.6)|
|Fukienese||306 (75.4)||479 (73.0)|
|Aborigines||71 (17.5)||111 (16.9)|
|Hakka||13 (3.2)||26 (4.0)|
|Others||16 (3.9)||40 (6.1)|
|Alcohol consumption (drink × years)2|
|Nondrinker||63 (15.5)||471 (71.8)||1.0|
|1–20||72 (17.7)||89 (13.6)||4.0 (2.4–6.5)|
|21–40||50 (12.3)||37 (5.6)||5.6 (3.1–10.0)|
|>40||221 (54.4)||59 (9.0)||13.7 (8.6–21.8)|
|p for trend||<0.001|
|Cigarette smoking (pack × years)2|
|Nonsmoker||40 (9.9)||388 (59.2)||1.0|
|1–30||167 (41.1)||152 (23.2)||5.8 (3.5–9.6)|
|>30||199 (49.0)||116 (17.7)||5.9 (3.6–9.9)|
|p for trend||<0.001|
|Betel quid chewing (pack × years)2|
|Nonchewer||221 (54.4)||621 (94.7)||1.0|
|1–20||42 (10.3)||11 (1.7)||5.9(2.7–12.9)|
|>20||143 (35.2)||24 (3.7)||6.2 (3.6–10.7)|
|p for trend||<0.001|
Table II presents the impact of ADH1B and ALDH2 genetic polymorphisms on the behaviors of alcohol drinking, tobacco smoking and betel quid chewing among both the controls and the cases. Taking the effects of ALDH2 polymorphisms and other covariates into account, control subjects who carried the slow-metabolizing ADH1B*1/*1 genotype were found to have a 2.2-fold higher risk of being a drinker than those who carried the fast-metabolizing genotype (ADH1B*2/*2). Alcohol abstinence was strongly associated with the inactive ALDH2 allele. Compared with the active ALDH2*1/*1 genotype, control subjects with the heterozygote and homozygote for the inactive ALDH2 variant allele were 0.3- and 0.03-fold less likely to be a drinker, respectively, with a significantly decreased trend in risk (p < 0.001). While an overwhelming majority of cancer patients were drinkers, a similar influence in regard to the 2 genes on drinking behavior was observed among the cases. Alternatively, the effect modification between the ADH1B and ALDH2 genotypes on drinking status was not significant in the controls (p for interaction = 0.404). No notable association was identified for the 2 alcohol-related metabolizing genes with respect to the habitual use of tobacco and betel quid. The allele frequency for the ADH1B and ALDH2 genes among the controls conformed well to the Hardy–Weinberg equilibrium.
|Genes/polymorphisms||p for H-W1||Total no.||Alcohol drinking||Tobacco smoking||Betel quid chewing|
|No. (%)||No. (%)||aOR2 (95% CI)||No. (%)||No. (%)||aOR2 (95% CI)||No. (%)||No. (%)||aOR2 (95% CI)|
|*2/*2 (fast)||335||94 (50.8)||241 (51.2)||1.0||136 (50.8)||199 (51.3)||1.0||17 (48.6)||318 (51.2)||1.0|
|*1/*2||275||72 (38.9)||203 (43.1)||1.0 (0.7–1.4)||111 (41.4)||164 (42.3)||1.0 (0.7–1.4)||13 (37.1)||262 (42.2)||0.9 (0.4–2.0)|
|*1/*1 (slow)||46||19 (10.3)||27 (5.7)||2.2 (1.1–4.5)||21 (7.8)||25 (6.4)||1.5 (0.8–2.8)||5 (14.3)||41 (6.6)||2.6 (0.9–7.8)|
|p for trend||0.139||0.463||0.314|
|*1/*1 (active)||325||134 (72.4)||191 (40.6)||1.0||135 (50.4)||190 (49.0)||1.0||19 (54.3)||306 (49.3)||1.0|
|*1/*2||286||50 (27.0)||236 (50.1)||0.3 (0.2–0.5)||112 (41.8)||174 (44.9)||0.9 (0.7–1.3)||13 (37.1)||273 (44.0)||0.8 (0.4–1.8)|
|*2/*2 (inactive)||45||1 (0.5)||44 (9.3)||0.03 (0.01–0.24)||21 (7.8)||24 (6.2)||1.3 (0.7–2.5)||3 (8.6)||42 (6.8)||1.2 (0.3–4.5)|
|p for trend||<0.001||0.776||0.947|
|p for interaction3||0.404||0.196||0.946|
|*2/*2 (fast)||140||111 (32.4)||29 (46.0)||1.0||129 (35.3)||11 (27.5)||1.0||58 (31.4)||82 (37.1)||1.0|
|*1/*2||149||123 (35.9)||26 (41.3)||1.5 (0.8–3.1)||130 (35.5)||19 (47.5)||0.7 (0.3–1.8)||72 (38.9)||77 (34.8)||1.0 (0.6–1.8)|
|*1/*1 (slow)||117||109 (31.8)||8 (12.7)||5.6 (2.0–16.0)||107 (29.2)||10 (25.0)||1.0 (0.4–3.0)||55 (29.7)||62 (28.1)||0.9 (0.5–1.6)|
|p for trend||0.001||0.859||0.472|
|*1/*1 (active)||111||94 (27.4)||17 (27.0)||1.0||100 (27.3)||11 (27.5)||1.0||34 (18.4)||77 (34.8)||1.0|
|*1/*2||281||243 (70.9)||38 (60.3)||0.7 (0.4–1.5)||255 (69.7)||26 (65.0)||1.0 (0.4–2.4)||145 (78.4)||136 (61.5)||1.7 (0.9–2.9)|
|*2/*2 (inactive)||14||6 (1.8)||8 (12.7)||0.07 (0.02–0.31)||11 (3.0)||3 (7.5)||0.3 (0.1–1.5)||6 (3.2)||8 (3.6)||2.2 (0.6–8.5)|
|p for trend||0.021||0.436||0.073|
|p for interaction3||NA4||NA4||0.874|
The joint ESCC risk and the combined adjusted PAR% for alcohol and aldehyde-metabolizing genes and alcohol consumption are displayed in Table III. Compared with nondrinkers possessing homozygous ADH1B*2, the cancer risk increased monotonically with the increase in numbers of the less-active ADH1B allele within strata of the same alcohol intake. Comparable findings were observed for the ALDH2 gene. Compared to the low-to-moderate drinkers (0.1–30 g/day) who carried the same genotypes, heavy drinkers (>30 g/day) were found to have a heterogeneously greater risk of contracting esophageal cancer (OR ratios = 3.2–7.1). Further, significant interactions between ALDH2*1/*2 heterozygote and the 2 levels of alcohol intake were identified based on a multiplicative model (p < 0.001). Heavy drinkers with inactive ALDH2*1/*2 genotype had the highest partial PAR% (31.5%) for esophageal cancer. Such a pattern of alcohol abuse, together with low-to-moderate drinking, accounted for 57.6% of the etiological fraction of this neoplasm among carriers of ALDH2*1/*2 polymorphism.
|Genes/polymorphisms||Nondrinkers||0.1–30 g/day of drinking||>30 g/day of drinking||>30 vs. 0.1–30 g/day||Nondrinkers||0.1–30 g/day||>30 g/day||Subtotal PAR%2|
|Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1 (95% CI)||OR ratio1 (95% CI)||Adj. PAR%1||Adj. PAR%1||Adj. PAR%1|
|*2/*2 (fast)||29/241||1.0 (Ref)||58/76||3.5 (1.9–6.5)||53/18||11.1 (5.0–24.4)||3.2 (1.5–6.8)||Ref||10.2||11.9||22.1|
|*1/*2||26/203||0.8 (0.4–1.6)||59/55||4.2 (2.2–7.9)||64/17||14.2 (6.6–30.6)||3.4 (1.6–7.3)||NA3||11.1||14.7||25.7|
|*1/*1 (slow)||8/27||1.2 (0.4–3.6)||45/15||10.6 (4.7–23.7)||64/4||71.9 (22.6–228.5)||6.8 (1.9–23.8)||NA3||10.0||15.5||25.6|
|p for interaction4||0.327||0.085|
|*1/*1 (active)||17/191||1.0 (Ref)||45/103||2.2 (1.1–4.5)||49/31||7.2 (3.3–15.9)||3.3 (1.6–6.5)||Ref||6.1||10.4||16.5|
|*1/*2||38/236||1.1 (0.6–2.3)||114/42||14.5 (7.1–29.6)||129/8||102.6 (38.3–274.8)||7.1 (2.9–17.2)||NA3||26.1||31.5||57.6|
|*2/*2 (inactive)||8/44||1.2 (0.4–3.4)||3/1||17.3 (1.4–213.7)||3/0||NA3||NA3||NA3||0.7||NA3||0.7|
|p for interaction4||<0.001||<0.001|
To obtain more robust estimates for the combined risks of ADH1B and ALDH2 genotypes associated with the quantities of alcohol consumed, nondrinkers with any genotypes were regarded as being a reference group (Table IV). This procedure is based on the results of a meta-analysis13 and our data, in those the cancer risk for nondrinkers with different polymorphisms of the 2 genes was nondiscernible. Controlling for the influence of ADH1B gene (within the same ADH1B polymorphisms), inactive ALDH2 genotypes (*1/*2 + *2/*2) had a higher ESCC risk than those with the ALDH2*1/*1 genotype, and a greater risk-difference was found to occur among heavy drinkers (OR ratios = 12.1–18.1) than among low-to-moderate drinkers (OR ratios = 6.5–6.8). Analogous effects were identified for the less-active ADH1B*1/*1 genotype relative to ADH1B variant genotypes (*2/*2 + *1/*2). However, significantly heterogeneous risk-difference was limited to drinkers with ALDH2*1/*1 polymorphism (OR ratios = 3.5–5.9). Although ingesters of alcohol who carried the slow-metabolizing ADH1B*1/*1 genotype and the inactive ALDH2*2 allele had the highest, though relatively imprecise, increased risk (aOR = 37.5 and 382.3 for 0.1–30 and >30 g/day of drinking, respectively), no noteworthy interaction between the 2 genes was discovered at each level of alcohol consumption. As expected, the effect of ALDH2 gene (OR ratios: 6.5–18.1) on cancer risk was stronger than that of ADH1B gene (OR ratios: 3.3–5.9).
|Genes/polymorphisms||0.1–30 g/day of drinking||>30 g/day of drinking|
|Nondrinkers (all genotypes)||ADH1B*2/*2+*1/*2||ADH1B *1/*1||*1/*1vs. *2/*2+*1/*2||ADH1B *2/*2+*1/*2||ADH1B*1/*1||*1/*1vsi. *2/*2+*1/*2|
|Cases/controls||aOR1||Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1 (95% CI)||OR ratio1 (95% CI)||Cases/controls||OR ratio1(95% CI)||Cases/controls||aOR1 (95% CI)||OR ratio1 (95% CI)|
|*1/*1||33/91||1.7 (0.9–3.0)||12/12||5.8 (2.1–15.6)||3.5 (1.2–9.9)||34/28||5.4 (2.7–10.8)||15/3||31.5 (7.4–133.9)||5.9(1.3–26.7)|
|*1/*2+*2/*2||84/40||11.2 (6.4–19.8)||33/3||37.5 (10.4–134.7)||3.3 (0.9–12.3)||83/7||97.3 (33.8–280.0)||49/1||382.3 (47.4–3084.9)||3.9(0.4–34.6)|
|*1/*2+*2/*2 vs.*1/*1 OR ratio1 (95% CI)||6.8 (3.5–13.2)||6.5 (1.4–30.2)||18.1 (5.9–55.8)||12.1 (1.1–139.2)|
|p for interaction3||0.955||0.949|
The pathogenic impact of alcohol-related metabolizing genes and alcohol intake in the esophagus with regard to tobacco consumption is assessed in Table V. Among smokers, drinking patients of all genotypes consistently had an increased risk of contracting ESCC at each level of alcohol consumption (aOR = 2.3–199.6). In contrast, among nonsmokers, an elevated risk was only observed in heavy drinkers who carried the ADH1B*2 allele, ADH1B*1/*1 genotype and ALDH2*2 variants (aOR = 9.3–82.2). Compared with the ADH1B*2/*2+*1/*2 and ALDH2*1/*1 genotypes, respectively, smoking patients with ADH1B*1/*1 and ALDH2*1/*2+*2/*2 genotypes correspondingly experienced an appreciably higher cancer risk (OR ratios = 2.9–6.8 for ADH1B*1/*1 and 7.2–10.1 for ALDH2*2 allele), with risk-enhanced effect here being identified as ingesting a higher amount of alcohol (OR ratios = 6.8–10.1). However, except for the ALDH2*2 variants, no comparable effect was found among nonsmokers.
|Nondrinkers||0.1–30 g/day of drinking||>30 g/day of drinking||Non-drinkers||0.1–30 g/day of drinking||>30 g/day of drinking|
|Cases/controls||aOR1||Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1||Cases/controls||aOR1 (95% CI)||Cases/controls||aOR1 (95% CI)|
|All genotypes||26/317||1.02 (Ref)||37/154||1.02 (Ref)|
|*2/*2+*1/*2*||4/54||1.6 (0.5–5.4)||5/11||9.3 (2.5–35.0)||113/77||9.0 (4.9–16.4)||112/24||29.3 (13.9–61.8)|
|*1/*1||1/4||6.7 (0.5–92.2)||4/2||19.2 (1.8–207.5)||44/11||25.9 (10.4–64.7)||60/2||199.6 (42.9–927.7)|
|*1/*1 vs. *2/*2 +*1/*2 OR ratio1(95% CI)||4.1 (0.3–65.0)||2.1 (0.2–23.7)||2.9 (1.3–6.6)||6.8 (1.5–31.6)|
|All genotypes||26/317||1.02 (Ref)||37/154||1.02 (Ref)|
|*1/*1||1/41||0.5 (0.1–3.9)||1/11||0.9 (0.1–8.1)||44/62||2.3 (1.2–4.2)||48/20||7.8 (3.7–16.3)|
|*1/*2+*2/*2||4/17||3.6 (0.9–13.5)||8/2||82.2 (10.8–628.4)||113/26||16.5 (8.5–32.0)||124/6||79.3 (28.8–218.1)|
|*1/*2+*2/*2 vs.*1/*1 OR ratio1(95% CI)||7.5 (0.7–78.9)||90.8 (5.4–1537.6)||7.2 (3.7–14.1)||10.1 (3.5–29.3)|
Because the less-active ADH1B*1 homodimers has an ∼40-fold lower Vmax for alcohol-elimination than the supper-active ADH1B*2 homodimers,30 the multiplicative synergy of alcohol with tobacco and betel quid on ESCC risk was evaluated by the 2 genotypes (Table VI). The combined risk (aOR = 45.0) for the ADH1B*1/*1 carriers who drank and smoked appeared to significantly surpass the expected level of risk (aOR = 4.5) predicted from the product of the risk for each factor acting separately (p for interaction = 0.042). While this effect is not significant, the joint risk of alcohol intake with tobacco smoking and betel quid chewing was, separately, 1.6- and 2.0-fold higher among carriers with the ADH1B*1/*1 genotype than among those with the ADH1B*2/*2 genotype.
|*2/*2 (fast)||*1/*1 (slow)||*1/*1 vs.*2/*2|
|Cases/controls||aOR (95% CI)||Cases/controls||aOR (95% CI)||OR ratio (95% CI)|
|Alcohol consumption/tobacco smoking1|
|No/Yes||22/80||3.3 (1.3–8.6)||3/8||1.2 (0.2–7.7)||0.4 (0.1–3.0)|
|Yes/No||4/38||3.1 (0.8–12.2)||5/6||3.7 (0.5–27.1)||1.2 (0.1–13.4)|
|Yes/Yes||107/56||28.6 (11.4–71.1)||104/13||45.0 (12.0–168.3)||1.6 (0.3–7.5)|
|p for interaction1,3||0.142||0.042|
|Alcohol consumption/betel quid chewing4|
|No/Yes||3/4||4.1 (0.7–25.0)||1/1||4.2 (0.2–87.6)||1.0 (0.1–34.8)|
|Yes/No||56/81||7.3 (3.8–14.0)||55/15||15.7 (4.5–54.9)||2.2 (0.6–8.1)|
|Yes/Yes||55/13||30.1 (12.2–74.0)||54/4||60.9 (13.7–271.1)||2.0 (0.4–10.4)|
|p for interaction3,4||0.969||0.905|
This study demonstrates that the carcinogenetic impact of the ADH1B and ALDH2 genotypes on ESCC not only correlates with the development of drinking behavior, but also with the ethanol amounts consumed, as well as the tobacco use. The genetic susceptibility that is associated with alcohol removal as determined by the ADH1B gene may be involved in the synergy action of alcohol with tobacco.
The genetic effects of the ADH1B and ALDH2 variants, which lead to a propensity to decline one's drinking due to acetaldehydemia with flushing syndrome, were detected in this study. While the ADH1B fast-metabolizing allele in the controls had a weaker impact (regarding ADH1B*1/*1 as the reference group, p for trend = 0.139) than that of the ALDH2 inactive allele (p for trend < 0.001) on drinking behavior, its influence remained, even after accounting for the strong effect of the ALDH2*2 allele. Similar research findings were observed in several Eastern populations,7, 8 but not in 5 countries in Central and Eastern Europe,6 in which no association between habitual drinking and 3 genes related to ethanol metabolism was identified. Since drinking characteristics and the frequency of ADH1B and ALDH2 variant alleles were dissimilar across these populations,6, 7, 8 these could explain part of the discrepancy in the results. Alternatively, although a comparable level of genetic protection against drinking conferred by the 2 genes was also found in the cancer cases, there was still an overwhelming majority of cancer patients who drank regularly. This phenomenon, as pointed out by Hampton,31 implies that there must be some important environmental factors, such as lifestyle and social factors, that encourage esophageal cancer patients with these genetic polymorphisms to overcome the physiological protection that they have inherited. The discovery of the acquired tolerance to severe acetaldehydemia among male ALDH2*1/*2 ESCC patients in Japan32, 33 provided further evidence that, through the increase in ethanol tolerance, chronic and heavy drinking may prevail over genetic traits to raise an individual's predisposition to alcohol-induced cancers.
Since substantially heterogeneous cancer risks between heavy and low-to-moderate alcohol intake among patients with the same genotypes were detected (OR ratio = 3.2–6.8 for ADH1B and 3.3–7.1 for ALDH2), although genetic susceptibility has an essential influence on ESCC, the dose-risk effect from alcohol consumption remains crucial. The findings that the OR ratio increases along with the increased number of slow-metabolizing allele in the 2 genes illustrated that such an effect is linked to ADH1B/ALDH2 genetic vulnerability. In accord with one study conducted in East Asia,9 our research exhibited that low-to-moderate alcohol drinkers who carried inactive ALDH2*1/*2 (aOR = 14.5) and ALDH2*2/*2 (aOR = 17.3) genotypes, respectively, had a higher cancer risk than heavy drinkers who carried the active ALDH2*1/*1 genotype (aOR = 7.2). This fact suggests that the carcinogenetic effect of alcohol on this neoplasm is more dependent on the polymorphisms of ALDH2 gene than on the ethanol amount that is consumed. Further, as has also been shown in earlier investigations,6, 34 a significant gene–environment interaction between the ALDH2*1/*2 genotype and alcohol intake was recognized in this study. Because acetaldehyde has been demonstrated to be carcinogenic in numerous cell cultures and animal models,35 and since ALDH2 is the key oxidation enzyme that determines the level of an individual's acetaldehyde exposure upon alcohol consumption,36 these findings give added support to the report evaluated by the International Agency for Research on Cancer indicating that “acetaldehyde derived from the metabolism of ethanol in alcoholic beverages contributes to causing malignant esophageal tumors.”37
Because of the fact that a noteworthy interaction between the ALDH2*1/*2 heterozygote and alcohol consumption has been reported,6, 34 the assessment of esophageal cancer risk for ALDH2 genotypes must be carefully carried out. Statistically multivariate manipulation performed by simply adjusting for the main effect of alcohol intake, assuming homogeneous risks between nondrinkers and drinkers with various intake levels, may conceal the real effect of inactive ALDH2 polymorphisms. In this research, the drinking-main-effect adjusted OR (4.4-fold) for the ALDH2*1/*2 genotype was found to be, respectively, moderately and sharply lower than that of the risk for this genotype within low-to-moderate (aOR = 6.5) and heavy (aOR = 14.2) alcohol drinking estimated from the models allowing for the modifying effects of alcohol use (data not shown). Alternatively, while the ALDH2*2/*2 genotype in one meta-analysis was observed in association with a reduced esophageal cancer risk, the drinking status was not incorporated into the risk evaluation there.13 A possible explanation of this result may be that a genetic predisposition that is susceptible to flushing syndrome appears to help protect one against becoming a drinker, thus indicating a lower cancer risk. However, ALDH2*2/*2 homozygote carriers who were unaffected by this protection and drank frequently were at an increased risk of contracting esophageal cancer, as shown in our findings.
Understanding the impact of gene–environment interplay on population etiologic fraction will lead us to new approaches of disease detection and prevention. In the studied population, although no meaningful PAR% associated with esophageal cancer was identified among nondrinkers, a greater partial PAR% for heavy drinkers (10.4–31.5%) than for low-to-moderate drinkers (6.1–26.1%) with the same ADH1B/ALDH2 genotypes was observed. Relative to drinkers carrying the active ALDH2*1/*1 genotype (16.5%), a remarkably higher proportion of etiological fraction is attributable to drinkers who carried the inactive ALDH2*1/*2 genotype (57.6%). Alcohol consumers possessing this polymorphism of ALDH2 are certainly expected to be an initiative target for disease prevention for this site of neoplasm.
Consistent with reports from East Asians,9, 10, 32 our study showed that the less-active ADH1B*1/*1 polymorphism is associated with an increased ESCC risk. As demonstrated in this research, the lack of a genetic effect from the ADH1B*2 allele on alcohol abstinence via aversion to alcohol explains, at least in part, why such a result was identified. In one Jewish study investigating the functional role of the ADH1B*2 allele using a clamping technique, the alcohol-elimination rate among the ADH1B*1 homozygote was observed to be significantly lower than that among the ADH1B*2 carriers.38 This implies that alcohol lingers in the body during the slow ethanol oxidation process that is modulated by the less-active ADH1B*1/*1 genotype. One Japanese experimental study indicated that alcoholic men of the ADH1B*1 homozygote have higher levels of ethanol and acetaldehyde lingering in the saliva and blood than those of other genotypes.19 Further, as also observed in an alcohol-challenge test study,39 strikingly greater acetaldehyde concentrations were detected in the saliva than in the blood.19 Because of a significantly positive correlation between salivary acetaldehyde production and the number of microorganisms in the oral cavity,19 high levels of salivary acetaldehyde were reported to be partly attributable to the oral microbial formation of acetaldehyde from the ethanol.19, 39 Extended exposure to carcinogenic salivary acetaldehyde for the ADH1B*1/*1 drinkers could be a powerful explanation for the higher ESCC risk for this genotype.
Our study illustrated that within the same levels of ethanol intake, the genotoxic effect of the ALDH2*2 allele on the esophagus is strengthened in a multiplicative way among carriers of the ADH1B*1 homozygote. As discussed previously, because high levels of carcinogenic salivary acetaldehyde associated with the less-active ADH1B*1/*1 genotype were observed in Asian male alcoholics,19 multiplicatively enhanced cancer susceptibility for such ADH1B*1/*1 and ALDH2*1/*2 polymorphisms of drinkers may be partially mediated by the increasing local accumulation of acetaldehyde due to inefficient elimination of the acetaldehyde by the inactive ALDH2.
The activity related to the β1 subunit encoded by the ADH1B*1 allele is highly capacity-limited after a large amount of alcohol ingestion.4, 30 Chronic alcohol intake has been manifested to such an extent as to bring about the induction of the microsomal cytochrome P4502E1, by which parts of the ethanol is metabolized to acetaldehyde.4, 40 It has been established that alcohol-induced P4502E1 is not only present in the liver, but is also in the alimentary tract, including the esophagus.41 Though in this study we did not have such information to verify, the elevated P4502E1 activity observed in the esophagus may also be involved in the enhanced risk of ESCC for chronic ALDH2*1/*2 drinkers with the ADH1B*1 homozygote.
Unlike heavy alcohol consumption, the particular effect of light-to-moderate alcohol intake on the genesis of esophageal cancer in earlier findings was inconsistent. Epidemiological studies in South India,21 the U.S.,42 Northern Italy43 and Hong Kong44 have consistently reported that an alcohol consumption level of about <140 g/week is associated with a minor increase, or even decrease, in the risk of esophageal cancer. However, a meta-analysis concluded that a daily intake of 25 g of alcohol conveyed a 50% excessive esophageal cancer risk.45 This present study, taking genetic susceptibility and tobacco smoking into account, recognized a smoking-dependent increase in cancer risk for a 0.1–30 g/day of alcohol intake, with significant risks found in smokers (aOR = 2.3–25.9, p < 0.05), but not in nonsmokers (aOR = 0.5–6.7, p > 0.05) among patients with the same genotypes. Further, in smokers, the ADH1B*1/*1 genotype and variant ALDH2*2 allele were found to confer an appreciably elevated risk for 0.1–30 g/day drinkers (OR ratio = 2.9–7.2). The risk of contracting this site of neoplasm for light-to-moderate alcohol consumers is closely linked to the tobacco use and genetic vulnerability of alcohol-related metabolizing genes.
Alcohol and tobacco abuse, individually and jointly, are 2 of the principle determinants of carcinoma of the esophagus.1, 46 As clearly evidenced in an in vivo study, active smoking was associated with a 7-fold higher salivary acetaldehyde.26 Such increased levels of acetaldehyde, together with those locally formed in the oral cavity by alcohol dehydrogenases and oral microorganism after alcohol consumption, may form an influential explanation for the synergistic and multiplicative ESCC risk stemming from the 2 agents. In this investigation, a supra-multiplicative cancer effect for consumption of alcohol and tobacco was found among patients carrying the ADH1B*1/*1 polymorphism; this suggests that alcohol-metabolizing genetic susceptibility may also be involved in the multiplicatively carcinogenetic action on the esophageal mucosa. On the other hand, our study identified a higher combined risk, though not a significant one, for alcohol use that occurs in conjunction with betel quid (OR ratio = 2.0) in ADH1B*1/*1 carriers, as compared to that in ADH1B*2/*2 carriers. The ADH1B*1 homozygote, which leads alcohol to linger in the esophagus due to “slow” ethanol oxidation, may be related to the tumorgenesis for the betel quid-derived carcinogens on this site of malignancy.
In this research, due to having observed an analogous pattern for the influences of the ADH1B and ALDH2 genes on drinking habits, as well identifying such in regard to smoking and chewing behaviors between the cases and controls, it is improbable that the 2 group participants were recruited with regard to specific genotypes. Moreover, since both the prevalence of drinking and smoking among the control subjects was comparable to those presented in 2 large-scale studies in Taiwan,20, 47 the under-representation of drinkers and smokers in this study is unlikely. With the social acceptability of alcohol and tobacco use in Taiwan, and as the study data was collected by well-trained interviewers, the extent to which recall biases have arisen in this investigation should be limited. On the other hand, poor oral health and hygiene, which are related to the overgrowth of oral microorganisms, have been reported to be important risk factors in the etiology of head and neck cancer.48 However, the present study has no available information that might serve to clarify this issue.
In summary, the interplay of the ADH1B and ALDH2 genotypes, in conjunction with a behaved drinking habit and a practiced drinking pattern, along with continued tobacco consumption, were found to have an important pathogenic role in modulating the risk of ESCC. It appears likely that exposure to high concentrations of salivary acetaldehyde, with regard to the functional polymorphism of ADH1B*1/*1, leads the carcinogenetic effect of the inactive ALDH2*2 allele to be strengthened in a multiplicative way by the ADH1B*1 homozygote.
- 1International Agency for Research on Cancer. Alcohol drinking, IARC monographs on the evaluation of carcinogenic risks of chemicals to humans.Alcohol drinking, vol. 44. Lyon: IARC, 1988.
- 11Chinese alcoholic patients with esophageal cancer are genetically different from alcoholics with acute pancreatitis and liver cirrhosis. Am J Gastroenterol 2000; 95: 2958–64., , , , , .Direct Link:
- 35International Agency for Research on Cancer. Acetaldehyde, inIARC monographs on the evaluation of carcinogenic risks of chemicals to humans: re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide, vol. 71, Part 2. Lyon: IARC, 1999. 319–35.
- 46International Agency for Research on Cancer. Tobacco smoke and involuntary smoking. IARC Monogr Eval Carcinog Risks Hum 2004; 83: 1–1438.