Research in Translation The Alcohol Flushing Response: An Unrecognized Risk Factor for Esophageal Cancer from Alcohol Consumption Philip J. Brooks*, Mary-Anne Enoch, David Goldman, Ting-Kai Li, Akira Yokoyama*

pproximately 36% of East Summary Points DNA damage and has other cancer- Asians (Japanese, Chinese, and promoting effects [5–7]. Acetaldehyde sÈ !,$(ÈDEFICIENCYÈRESULTINGÈFROMÈTHEÈ Koreans) show a characteristic is subsequently metabolized to acetate, A ALDH2 Lys487 allele contributes to physiological response to drinking mainly by the enzyme ALDH2 [8]. In both the alcohol flushing response alcohol that includes facial flushing East Asian populations there are two and an elevated risk of squamous (see Figure 1), nausea, and tachycardia main variants of ALDH2, resulting cell esophageal cancer from alcohol [1] . This so-called alcohol flushing from the replacement of glutamate consumption. response (also known as “Asian flush” (Glu) at position 487 with lysine (Lys) or “Asian glow”) is predominantly sÈ +NOWLEDGEÈOFÈTHEÈFLUSHINGÈRESPONSEÈISÈ [9]. The Glu allele (also designated due to an inherited deficiency in the useful clinically, as it allows doctors to ALDH2*1) encodes a protein with enzyme aldehyde dehydrogenase 2 identify their ALDH2-deficient patients normal catalytic activity, whereas (ALDH2) [2]. Although clinicians in a simple, cost-effective, and non- the Lys allele (ALDH2*2) encodes and the East Asian public generally invasive manner. an inactive protein. As a result, Lys/ know about the alcohol flushing sÈ $OCTORSÈSHOULDÈCOUNSELÈTHEIRÈ!,$( Lys homozygotes have no detectable response (e.g., http://www.echeng. deficient patients to limit alcohol com/asianblush/), few are aware consumption and thereby reduce the Funding: The authors received no specific funding of the accumulating evidence that risk of developing esophageal cancer. for this article. ALDH2-deficient individuals are at sÈ )NÈVIEWÈOFÈTHEÈAPPROXIMATELYÈÈ Competing Interests: The authors have declared that no competing interests exist. much higher risk of esophageal cancer million ALDH2-deficient individuals in (specifically squamous cell carcinoma) the world, many of whom now live in Citation: BrooksPJ, Enoch M-A, Goldman D, Li T-K, from alcohol consumption than Western societies, even a small percent Yokoyama A (2009) The alcohol flushing response: individuals with fully active ALDH2. An unrecognized risk factor for esophageal cancer reduction in esophageal cancers due from alcohol consumption. PLoS Med 6(3): e1000050. This is particularly unfortunate as to a reduction in alcohol drinking doi:10.1371/journal.pmed.1000050 esophageal cancer is one of the would translate into a substantial This is an open-access article distributed under the deadliest cancers worldwide [3], with number of lives saved. terms of the Creative Commons Public Domain five-year survival rates of 15.6% in the declaration, which stipulates that, once placed in the United States, 12.3% in Europe, and public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or 31.6% in Japan [4]. reduce alcohol consumption, and otherwise used by anyone for any lawful purpose. Our goal in writing this article is high-risk patients can be assessed for to inform doctors firstly that their Abbreviations: ADH, alcohol dehydrogenase; endoscopic cancer screening. Based on ALDH2, aldehyde dehydrogenase 2; Glu, glutamate; ALDH2-deficient patients have an the sizes of the Japanese, Chinese, and Lys, lysine; OR, odds ratio; UADT, upper aerodigestive increased risk for esophageal cancer Korean populations and the expected tract if they drink moderate amounts of frequency of ALDH2-deficient Philip J. Brooks, Mary-Anne Enoch, and David alcohol, and secondly that the alcohol individuals in each [1], we estimate that Goldman are in the Laboratory of Neurogenetics, flushing response is a biomarker National Institute on Alcohol Abuse and Alcoholism, there are at least 540 million ALDH2- National Institutes of Health, Rockville, Maryland, for ALDH2 deficiency. Because of deficient individuals in the world, United States of America. Ting-Kai Li is the former the intensity of the symptoms, most representing approximately 8% of the Director of the National Institute on Alcohol Abuse people who have the alcohol flushing and Alcoholism, National Institutes of Health. He is population. In a population of this size, now a Professor at the Duke–National University of response are aware of it. Therefore even a small reduction in the incidence Singapore Graduate Medical School, Durham, North clinicians can determine ALDH2 of esophageal cancer could result in Carolina, United States of America. Akira Yokoyama is Director of Clinical Research Unit, National Hospital deficiency simply by asking about a substantial reduction in esophageal Organization Kurihama Alcoholism Center, Nobi, previous episodes of alcohol-induced cancer deaths worldwide. Yokosuka, Kanagawa, Japan. Philip J. Brooks and flushing. As a result, ALDH2-deficient Akira Yokoyama were participants in the 2007 International Agency for Research on Cancer Working patients can then be counseled to A Primer on the Genetics of Group on alcoholic beverage consumption. Alcohol Metabolism * To whom correspondence should be addressed. Ethanol is first metabolized primarily E-mail: [email protected]; a_yokoyama@ Research in Translation discusses health interventions by alcohol dehydrogenase (ADH) into kurihama1.hosp.go.jp in the context of translation from basic to clinical acetaldehyde (Figure 2), a mutagen research, or from clinical evidence to practice. Provenance: Not commissioned; externally peer and animal carcinogen that causes reviewed

PLoS Medicine | www.plosmedicine.org 0258 March 2009 | Volume 6 | Issue 3 | e1000050 has also confirmed an increased risk, even among moderate drinking heterozygotes [11]. In the Japanese and Taiwanese studies, a strikingly high proportion (58%–69%) of the excessive risk for esophageal cancer is attributable to drinking by low-activity ALDH2 heterozygous individuals [13,14]. Consistent with the results of case control studies, prospective studies in cancer-free alcoholics have also shown that the relative hazard for future upper aerodigestive tract (UADT) cancers in low-activity ALDH2 heterozygotes is approximately 12 times higher than in individuals with active ALDH2 [15]. (The UADT includes the oral cavity, pharynx, larynx, and doi:10.1371/journal.pmed.1000050.g001 esophagus.) In addition, alcohol consumption in low-activity ALDH2 Figure 1. The Alcohol Flushing Response heterozygotes has been associated Facial flushing in a 22-year-old ALDH2 heterozygote before (left) and after (right) drinking alcohol. with other cancer-related outcomes, The individual pictured in this figure has given written consent for publication of his picture using the PLoS consent form. including the presence of multiple areas of esophageal dysplasia (i.e., premalignant lesions) and multiple ALDH2 activity. Because the Lys allele paradoxically, it is the more common independent UADT cancers [13]. acts in a semi-dominant manner, low-activity ALDH2 heterozygous It is important to note that ALDH2 ALDH2 Lys/Glu heterozygotes have genotype that is associated with deficiency does not influence far less than half of the ALDH2 activity greatest risk of esophageal cancer from esophageal cancer risk in non-drinkers of Glu/Glu homozygotes; in fact, drinking alcohol. [11]. Furthermore, the magnitude the reduction in ALDH2 activity in of the ALDH2-associated esophageal heterozygotes is more than 100-fold [8]. Evidence That ALDH2 Deficiency cancer risk depends on the relative Alcohol consumed by ALDH2- Increases the Risk of Alcohol- importance of alcohol versus other risk deficient individuals is metabolized to Related Squamous Cell Esophageal factors in a given population. In rural acetaldehyde, which accumulates in Cancer areas of China, where there is a high the body due to absent ALDH2 activity Following the first study [12], which rate of esophageal cancer but alcohol and results in facial flushing (Figure was conducted in the Japanese drinking plays a less important role 1), nausea, and tachycardia [2]. These population, case control studies in than in Japan and Taiwan, there is a unpleasant effects are the result of Japan and Taiwan have consistently more modest positive association (ORs, diverse actions of acetaldehyde in the demonstrated a strong link between 1.7 to 3.1) between low-activity ALDH2 body, including histamine release the risk of esophageal squamous cell heterozygotes and esophageal cancer [10]. Because of the intensity of this carcinoma (Figure 3) and alcohol risk (e.g., [16]). unpleasant response, ALDH2 Lys/Lys consumption in low-activity ALDH2 homozygotes are unable to consume Acetaldehyde Is Responsible for heterozygotes, with odds ratios significant amounts of alcohol. As a Facial Flushing and Esophageal (ORs) ranging from 3.7 to 18.1 after result, they are protected against the adjustment for alcohol consumption. Cancer Risk in ALDH2-Deficient increased risk of esophageal cancer Moreover, most studies show ORs Individuals from alcohol consumption [11]. This of over 10 for increased risk in Acetaldehyde is responsible for the observation also provided evidence for a heterozygotes who are heavy drinkers facial flushing and other unpleasant causative role for ethanol in esophageal [13,14]. An independent meta-analysis effects that ALDH2-deficient cancer, and a key role for acetaldehyde in mediating this effect [11]. ALDH2 Lys/Glu heterozygotes experience a less severe manifestation of the flushing response due to residual but low ALDH2 enzyme activity in their cells. As a result, some are able to develop tolerance to acetaldehyde doi:10.1371/journal.pmed.1000050.g002 and the flushing response and become Figure 2. The Ethanol Metabolic Pathway and the Role of the ALDH2 Variants in habitual heavy drinkers, due in part to Acetaldehyde Accumulation the influence of societal and cultural It should be noted that ADH is also polymorphic, and genetic variants in ADH1B interact with the factors (see below). Therefore, ALDH2 variant to modify risk [13].

PLoS Medicine | www.plosmedicine.org 0259 March 2009 | Volume 6 | Issue 3 | e1000050 amounts of alcohol, providing a likely times higher than in blood, due to the mechanism for the increased cancer local formation of acetaldehyde by oral risk. A study in Japanese alcoholics [17] microorganisms [21]. Importantly, showed that the amount of mutagenic ALDH2 heterozygotes had two to three acetaldehyde-derived DNA adducts times the acetaldehyde levels in their (Figure 4) in white blood cells was saliva compared to fully active ALDH2 significantly higher in ALDH2-deficient individuals after a moderate dose of heterozygotes than in individuals with oral ethanol [22]. active ALDH2 (Table 1). In this study, while the two groups were matched Social and Cultural Factors for alcohol consumption, the ALDH2- Modulate Alcohol Drinking by deficient group consumed slightly less ALDH2 Heterozygotes doi:10.1371/journal.pmed.1000050.g003 alcohol on average than the controls. Alcohol consumption is a social Also, ALDH2 heterozygotes who drank activity, and as such can be strongly Figure 3. A Late-Stage Squamous Cell alcohol had higher levels of white influenced by cultural and social forces. Carcinoma of the Esophagus in a 51-Year- Old Male with a Known History of Flushing blood cells with chromosomal damage In Japan, where the risk of alcohol- and Alcohol Drinking than drinkers with active ALDH2 [18]. related esophageal cancer in ALDH2 Because of these as well as other data, heterozygotes has been most well individuals experience when they the 2007 International Agency for documented, going out drinking after drink alcohol [10]. Importantly, there Research on Cancer Working Group work with colleagues is an essential is now direct evidence that ALDH2- on alcohol and cancer specifically element of Japanese business society, deficient individuals experience noted the substantial mechanistic and the idea of group harmony is higher levels of acetaldehyde-related evidence supporting a causal role particularly powerful. The percentage DNA and chromosomal damage than for acetaldehyde in alcohol-related of heavy drinking men who are low- individuals with fully active ALDH2 esophageal cancer [19]. activity ALDH2 heterozygotes has risen when they consume equivalent While the UADT is exposed to substantially in the last few decades, acetaldehyde from alcoholic beverages in parallel with the proliferation of [20] and tobacco smoke, increasing business society in Japan and increases Five Key Papers in the Field evidence points to the metabolism in per capita alcohol consumption. of ethanol by microorganisms in Harada et al. [23] first reported that Harada et al., 1981 [2] The first the oral cavity as an important the frequency of inactive ALDH2 documentation of the relationship source of acetaldehyde in saliva was very low (only 2%) in Japanese between ALDH deficiency and the and, by extension, in the esophagus. alcoholics in 1982. In a later study flushing reaction. Acetaldehyde levels in saliva are 10–20 using archival DNA samples, Higuchi Yoshida et al., 1984 [9] Identification of the amino acid variant responsible for ALDH deficiency. Yokoyama et al., 1996 [12] The first evidence demonstrating that ALDH2- deficient individuals have a dramatically elevated risk of esophageal cancer when they drink alcohol. Yokoyama et al., 2003 [25] Demonstrates that an updated flushing questionnaire containing two simple questions is approximately 90% sensitive and specific for identifying ALDH2- deficient individuals. Baan et al., 2007 [19] Summary of the conclusions from the 2007 International Agency for Research on Cancer Working Group on the Consumption of Alcoholic Beverages. This is the first report to conclude that ethanol in alcoholic beverages is carcinogenic to humans. The report also adds the female breast and colorectum to the list of sites for alcohol-related carcinogenesis and notes doi:10.1371/journal.pmed.1000050.g004 substantial mechanistic evidence linking acetaldehyde to esophageal cancer risk Figure 4. Chemical Structures of Deoxyguanosine, the DNA Base That Is the Target for based on studies from ALDH2-deficient Acetaldehyde, As Well As the Acetaldehyde-Derived DNA Lesions Referred To in Table 1 individuals. The atoms in red represent the acetaldehyde-derived chemical modifications.

PLoS Medicine | www.plosmedicine.org 0260 March 2009 | Volume 6 | Issue 3 | e1000050 when administered to Japanese women Table 1. Acetaldehyde-Derived DNA Damage in ALDH2-Deficient Alcoholics [29]. ALDH2 Acetaldehyde-Derived DNA Adduct Genotype Once ALDH2-deficient patients ALDH2487 Glu/Glu ALDH2487 Glu/Lys p-Value have been identified, they should be (*1/*1) [n = 19] (*1/*2) [n = 25] informed about their elevated risk of N2-Et-dG 17.8 ± 15.9 130 ± 52 0.03 developing esophageal cancer risk α-S-Me-γ-OH-PdG 42.9 ± 6.0 92.4 ± 12.9 0.01 from drinking alcohol. As can be seen α-R-Me-γ-OH-PdG 61.3 ± 6.4 114 ± 15 0.02 from Figure 5, ALDH2 deficiency increases esophageal cancer risk Values are means ± standard error of the mean (fmol/µM dG); p-values were calculated by the Wilcoxon-Mann-Whitney test (N2-Et-dG) and t-test (α-Me-γ-OH-PdGs). at all three drinking levels, but the For illustrations of these adducts, see Figure 4. These data are from [17]. slope of the line relating alcohol α-R-Me-γ-OH-PdG, α-R-methyl-γ-hydroxy-1, N2-propanodeoxyguanosine; α-S-Me-γ-OH-PdG, α-S-methyl-γ-hydroxy-1, consumption to esophageal cancer N2-propanodeoxyguanosine; N2-Et-dG, N2-ethyl-deoxyguanosine. doi:10.1371/journal.pmed.1000050.t001 risk is steeper in ALDH2-deficient individuals. Clinicians might therefore et al. [24] determined that in 1979, 3% experienced the alcohol flushing use this graph to explain the increased of Japanese alcoholics were ALDH2 response. In the Japanese population, risk when counseling their ALDH2- heterozygotes, compared with 8% ALDH2 deficiency can be identified deficient patients to reduce alcohol in 1986 and 13% in 1992. In a more accurately based on the answers to consumption. recent study, approximately 26% of a flushing questionnaire consisting The ORs in Figure 5 are adjusted heavy drinking (consuming more of two questions (see Box 1) about for smoking. However, patients should than about 400 g of ethanol per week) previous episodes of facial flushing also be informed that smoking further men in Tokyo were ALDH2 Lys487 after drinking alcohol [25]. The two increases the esophageal cancer risk in heterozygotes [35]. In other East Asian questions can be easily included as part a synergistic manner with alcohol [30]. countries, estimates of the percentage of a standard clinical interview. In a As noted above, cigarette smoking of alcoholics who are low-activity Japanese male population, the flushing dramatically increases acetaldehyde ALDH2 heterozygotes range from 17% questionnaire had a 90% sensitivity levels in saliva, and ALDH2-deficient in Taiwan in 1999 [26] to 4% in Korea and 88% specificity [25] and a positive individuals have a reduced capacity to in 2007 [27]. Taken together, these predictive value of 87% (based on the clear salivary acetaldehyde. observations indicate that the inhibitory tabulated data in [25]). The flushing For patients at high risk of effect of heterozygous ALDH2- questionnaire gave a similarly high esophageal cancer, doctors should also deficiency on alcohol consumption can sensitivity (88%) and specificity (92%) consider endoscopy for early cancer be strongly influenced by local social and cultural factors which may change over time. There are many East Asians now living in Western societies, particularly at universities and in metropolitan areas. A sub-population of special concern is ALDH2-deficient university students who may face peer pressure for heavy drinking and binge drinking. Furthermore, anecdotal evidence indicates that some young people view the facial flushing response as a cosmetic problem and use antihistamines in an effort to blunt the flushing while continuing to drink alcohol [28]. This practice is expected to increase the likelihood of developing esophageal cancer. Education and Early Detection Can Reduce the Global Health Burden of Esophageal Cancer Clinicians who treat patients of East doi:10.1371/journal.pmed.1000050.g005 Asian descent need to be aware of the Figure 5. Odds Ratios for Esophageal Cancer at Different Amounts of Alcohol Consumption risk of esophageal cancer from alcohol In Relation To the Flushing Response consumption in their ALDH2-deficient Alcohol consumption amounts: low, 1–8.9 units/week; moderate, 9–17.9 units/week; high, ≥18 patients. Importantly, clinicians can units/week; where 1 unit = 22 g of ethanol. The referent (OR = 1) is never/rare drinkers (<1 unit/ determine whether an individual of week) of either genotype. Odds ratios were adjusted for age, frequency of drinking strong alcohol beverages, pack-years of smoking, and intake of fruit and green-yellow vegetables, based on a East Asian descent is ALDH2 deficient multiple logistic regression model. Error bars are 95% confidence intervals. The graph is based on simply by asking whether they have the data in [25].

PLoS Medicine | www.plosmedicine.org 0261 March 2009 | Volume 6 | Issue 3 | e1000050 detection. A health risk assessment tool Box 1. Clinical Tests To Assess Supporting Information to select candidates for endoscopic ALDH2 Deficiency Due To the Alternative Language Summary S1. cancer screening, including data on ALDH2 Lys487 Allele Translation of the article summary into alcohol flushing as well as alcohol Japanese by AY. 1. The Flushing Questionnaire consumption, smoking, and dietary Found at doi:10.1371/journal. habits, is currently being developed The flushing questionnaire consists of two pmed.1000050.sd001 (23 KB DOC). and validated [31]. Using a version of questions: (A) Do you have a tendency to develop facial flushing immediately after References the health risk assessment that includes 1. Eng MY, Luczak SE, Wall TL (2007) ALDH2, the flushing questionnaire as a major drinking a glass (about 180 ml) of beer?; ADH1B, and ADH1C genotypes in Asians: A component, it has been estimated (B) Did you have a tendency to develop literature review. Alcohol Res Health 30: 22- facial flushing immediately after drinking 27. that approximately 58% of esophageal 2. Harada S, Agarwal DP, Goedde HW (1981) cancers in the Japanese population a glass of beer in the first one or two Aldehyde dehydrogenase deficiency as cause of could be detected by screening only the years after you started drinking? For both facial flushing reaction to alcohol in Japanese. questions, the choice of answers are: yes, Lancet 2: 982. individuals with the top 10% risk scores 3. Umar SB, Fleischer DE (2008) Esophageal [31]. no, or unknown. cancer: Epidemiology, pathogenesis and If an individual answers yes to either prevention. 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