Roles of defective ALDH2 polymorphism on liver protection and cancer development

Because serum transaminases elevate alcohol dose dependently as a consequence of liver injury, they serve as useful biological markers of excessive drinking. However, these markers are inadequate in individuals with a defective allele of the aldehyde dehydrogenase 2 gene, ALDH2*2, because they show a different correlation with the amount of ethanol. For example, the serum alanine aminotransferase (ALT) level could become even lower than the baseline after alcohol intake in ALDH2*2 carriers. In fact, multiple studies suggest that ALDH2*2 is a hepato-protective factor in healthy individuals. Importantly, excessive drinking is particularly dangerous in carriers of ALDH2*2 because the risk of alcohol-related cancer is much higher than that for ALDH2*1/*1 carriers. Without recognizing the genotype interaction on serum transaminase, the opportunity to warn people about potential cancer risks is missed owing to incorrect interpretation. This is particularly important in East Asian countries where approximately half of the population carries the ALDH2*2 allele. To date, the mechanism of liver protection from ethanol load in individuals with ALDH2*2 has not been fully elucidated. However, some reasonable mechanisms have been suggested by experimental studies, including remodelling of detoxifying systems. Further studies to uncover the whole mechanism are anticipated.

We herein describe the function of ALDH2 and the pathophysiological impact of its defective polymorphism, ALDH2*2, with particular focus on its effects on cancer development and serum transaminase levels associated with ethanol intake. We also discuss the significance of ALDH2*2 on the value of serum transaminases as a biological marker of excessive drinking.

ALDH2
ALDHs catalyse the oxidation of many chemically diverse aldehydes to their corresponding acid metabolites. The ALDH superfamily plays an important role in the biotransformation of a variety of exogenous and endogenous aldehydes [18]. The human ALDH gene superfamily comprises 19 genes in 11 families and 4 subfamilies [19,20].
ALDH2 has the highest affinity for acetaldehyde, an important intermediary product of ethanol metabolism. Although acetaldehyde is also detoxified by other ALDH isozymes, including cytosolic ALDH1A1 and mitochondrial ALDH1B1, ALDH2 is the primary ALDH responsible for the clearance of ethanol-derived acetaldehyde [3,18,25].

ALDH2 polymorphism
Based on the National Center for Biotechnology Information database, nearly 300 single nucleotide polymorphisms (SNPs) have been reported for ALDH2. Of these, approximately 180 mutations are associated with amino acid replacement. However, r671 is the only SNP resulting in a powerful phenotypic change due to compromised protein function. The mutation site is located at exon 12, nucleotide position 42421 (transition of G to A), resulting in a Glu504 substitution to Lys. Glu504Lys in the precursor protein is also described as Glu487Lys in the mature protein form. The allele with this mutation is called ALDH2*2, in contrast to the wild type ALDH2*1.
The Glu487Lys mutation severely decreases ALDH enzymatic activity with respect to all three of its enzymatic functions (as a dehydrogenase, esterase, and reductase) [22,26]. It is reported that this alteration in ALDH2 increases the Km value (the concentration at which the reaction rate is half the maximum rate) for NAD? (the cofactor of ALDH2) by 150-fold compared to the wild type enzyme [27]. In fact, an ethanol challenge of Chinese volunteers was shown to elicit a 20-fold higher blood acetaldehyde level in the ALDH2*1/*2 subjects than in ALDH2*1/*1 subjects [28].

Effect of ALDH2*2 on drinking behaviour
The ALDH2*2 polymorphism strongly inhibits drinking behaviour [10,29]. According to Peng et al. [28], ethanol administration caused a 50 % increase in heart rate and 'terrible feelings' in ALDH2*1/*2 subjects, commonly referred to as ''flushing syndrome'', whereas no change was observed in ALDH2*1/*1 subjects. Given such vulnerability to drinking alcohol, it is not surprising that individuals with the ALDH2*2 allele tend not to be habitual drinkers.
It is well established that DNA adduct formation has an aetiological role in cancer [48]. We previously reported that acetaldehyde-derived DNA adducts are elevated to a greater extent in the liver and stomach of Aldh2 knockout mice following ethanol administration than in the liver and stomach of wild type mice [49,50]. Similarly, Ishikawa and colleagues [51] showed that subjects carrying the ALDH2*2 allele who consumed ethanol more than three times per week had greater DNA damage, as reflected by an increased micronuclei frequency. Furthermore, cytochrome P450 (CYP2E1), which is known to form reactive oxygen species [52] and enhance pro-carcinogen activation [53], is upregulated in Aldh2 knockout mice, and is further induced by ethanol administration [54]. As such, ALDH2*2 has the potential to promote the risk of cancers induced by alcohol consumption (Fig. 1).

Effect of ALDH2*2 on indicators of excessive drinking
Serum alanine transaminase (ALT), aspartate aminotransaminase (AST), and gamma-glutamyltransferase (GGT) levels are generally used as biological markers of excessive drinking, which elevate dose dependently as a result of alcoholic liver injury [55,56]. These markers have been presumed to be particularly high in drinkers that carry ALDH2*2, because this allele is expected to promote alcoholic liver injury. However, in reality, the opposite results have been obtained in multiple epidemiological and experimental studies.
Takeshita and colleagues [57] studied Japanese male workers who were free from hepatic viruses and from high transaminase activities (\100 IU/L), and showed that subjects with ALDH2*2 consuming moderate to high levels of alcohol had lower serum AST, ALT, and GGT levels (e.g. 25, 27, and 64 IU/L, respectively, in subjects with ALDH2*1/*1 versus 22, 17, and 42 IU/L, respectively, in subjects with ALDH2*1/*2) (Fig. 1b). Another study on Japanese male workers suggested that ALDH2*2 is an independent protective factor against elevation of the serum ALT level (odds ratio of 0.16) from multivariate logistic regression analysis, including body mass index and drinking habits in the model [58]. This finding was confirmed in a subsequent large-scale epidemiological study [10].
Furthermore, animal experiments using Aldh2 knockout mice showed the same tendency; serum ALT decreased after 5 weeks of simple ethanol loading (without any additional treatments to induce liver injury) in Aldh2 Fig. 1 Interaction between ALDH2 polymorphism and alcohol intake on oesophageal cancer risk and indicators of excessive drinking. a Estimated odds ratios (OR) for oesophageal cancer with adjustment for cumulative smoking level reported by Koyanagi and colleagues (a Japanese case-control study) [47]. The error bars represent 95 % confidence intervals. b Indicators of excessive drinking reported by Takeshita and colleagues (population-based study for Japanese workers) [57]. Data represent mean value. AST aspartate transaminase, ALT alanine transaminase Environ Health Prev Med (2016) 21:395-402 397 knockout mice, while no change was observed in wild type mice [59]. In a subsequent study, the decrease of ALT level in Aldh2 knockout mice was reported to be sustained for as long as the ethanol loading was administered, whereas wild type mice showed elevation of serum ALT [60]. Moreover, a liver injury model induced with a high-fat diet and carbon tetrachloride also showed lower serum ALT values in Aldh2 knockout mice than in wild type mice [61].

Mechanism of liver damage amelioration induced by congenital lack of ALDH2
Some mechanisms have been suggested to explain these observed interactions between the ALDH2 genotype and drinking on serum transaminase levels (Fig. 2). For example, the effects of ALDH2*2 on curbing acetaldehyde metabolism could produce benefits. Cyanamide, an ALDH inhibitor, diminished the ethanol-elicited increase of oxidative stress by inhibition of reduced nicotinamide adenine dinucleotide (NADH) formation in the mitochondria during the metabolism of acetaldehyde to acetate [62,63]. This theory suggests that the ALDH2*2 allele can ameliorate the sharp increase of NADH that causes mitochondrial dysfunction, resulting in oxidative stress and hepatocyte damage from alcohol consumption. Supporting this hypothesis, Aldh2 knockout mice showed lower MDA and higher glutathione (GSH) levels after a single-dose administration of ethanol [64]. Ethanol loading to people with ALDH2*2 might decrease tumour necrosis factor-alpha (TNFa) through inhibition of the associated transcription pathways (nuclear factor-kappa B pathway and mitogen-activated protein kinase pathway, which are considered to be important pathways for alcoholic liver injury [65][66][67]) [59,68]. An in vivo experiment using Aldh2 knockout mice showed decreased serum ALT, TNFa, and extracellular signalregulated kinase 2 levels after 5 weeks of ethanol administration, and significant correlations were observed among these three factors [59].
A different mechanism was suggested using liver injury models, in which interleukin-6-induced signal transducer and activator of transcription 3 relieve hepatic steatosis and result in lower serum ALT levels [61]. Supporting this finding, Yokoyama and colleagues [69] performed an epidemiological study on Japanese alcoholic men and reported that the serum triglyceride concentration was lower in ALDH2*1/*2 carriers than in ALDH2*1/*1 carriers (the least squares means adjusted for age and drinking amount were 103 and 113 mg/dL, respectively).
Another convincing explanation for these associations involves remodelling of stress response systems that are induced in a compensatory manner to mitigate the cytotoxicity of endogenous aldehydes. This is because individuals lacking ALDH2 will likely be exposed to higher levels of endogenous aldehydes from birth, such as 4HNE and MDA [23,24], thereby requiring an alternate defence system. In fact, Aldh2 knockout mice had higher levels of heme oxidase 1, an essential defensive factor against cellular oxidative stress, without any pre-treatment [54]. Furthermore, it was reported that a series of GSH-producing enzymes was upregulated in ALDH2*2 targeting mice [70]. A higher GSH level in individuals with ALDH2*2 has been confirmed in humans according to a case-control study of children in China [71]. In this study, better clinical outcomes were reported for ALDH2*2 carriers, such as lower serum troponin level and shorter stay period in intensive care unit [71]. The compensatory protection system may protect ALDH2*2 carriers not only from alcoholic liver damage Fig. 2 Proposed mechanism of amelioration of liver damage and aggravation of cancer development by a combination of alcohol intake and possession of a defective polymorphism of ALDH2 (ALDH2*2). ALDH2 aldehyde dehydrogenase 2, TNFa tumour necrosis factoralpha, STAT3 signal transducer and activator of transcription 3 but also from other pathologies, including cancerous diseases. To the best of our knowledge, ALDH2*2 has not been reported as a cancerous risk factor for non-drinkers, in spite of the decreased metabolic ability for 4HNE, which is a well-known cancer initiator [72]. Interestingly, a recent epidemiological study in Taiwan demonstrated that ALDH2*2 was a protective factor of hepatocellular carcinoma (HCC) for patients with hepatitis B-positive cirrhosis [73]. In this study, HCC did not appear to be induced by alcohol; only 14 % of the subjects had a drinking habit and no association between drinking and HCC was found. The multivariate-adjusted hazard ratio of ALDH2*1/*2 for HCC development was calculated to be 0.35 when the ALDH2*1/*1 genotype was set as reference. This finding suggests that ALDH2*2 could be a protective factor for alcohol-independent HCC. Induction of anti-oxidative proteins might be one of the defence systems for HCC development. Another possible mechanism could be that possession of ALDH2*2 ameliorates PI3K/Akt pathway activation [74]. It is known that the PI3K/Akt pathway is hyper-activated in tumours, resulting in the promotion of malignant growth [75].
Such amelioration of liver damage conferred by ALDH2*2 may be limited to the type of injury. For example, the liver injury model mentioned above suggested protective effects such as inhibition of fatty degeneration; nevertheless, adverse effects were also reported, such as aggravation of the inflammatory response and fibrosis [61]. Simple ethanol administration does not cause such a marked discrepancy; however, hepatocyte ballooning was observed only in Aldh2 knockout mice in spite of their lower TNFa and serum ALT levels [59]. However, in practice, people who carry ALDH2*2 drink much less than the amount given to experimental animals (see ''Effect of ALDH2*2 on drinking behaviour''). This may explain why epidemiological studies have only observed the advantageous aspect of ALDH2*2 for liver tissue function.
Conclusion: significance of the interaction between ALDH2 polymorphism and alcohol intake Serum transaminase is a useful indicator of excessive drinking for self-monitoring by drinkers themselves and for their surrounding healthcare workers [55,56]. However, it is less useful in people with ALDH2*2, because it does not elevate dose dependently with ethanol in the same manner as observed in people with ALDH2*1*1 (see Effect of ALDH2*2 on indicators of excessive drinking). As a result, the opportunity to warn people with ALDH2*2 of excessive drinking for the prevention of alcohol-related diseases, particularly cancerous diseases, could be lost when relying on only serum transaminase levels as a marker. Furthermore, individuals with ALDH2*2 have higher risks of cancer development (see Effect of ALDH2*2 on cancerous diseases). In other words, ALDH2*2 carriers need to be more strict about limiting or refraining from drinking than ALDH2*1*1 carriers, and serum transaminase is an inadequate indicator of excessive drinking for carriers of ALDH2*2. Thus, effective risk assessment and disease prevention require screening for ALDH2 genotype.
The influence of the ALDH2*2 allele on the value of serum transaminases and GGT as markers of excessive drinking could be modified in individuals with additional pathological conditions such as metabolic disorders of lipids and sugars. It would be useful to investigate this issue further in the future. Moreover, the interaction of ALDH2 polymorphism and alcohol intake on other alcoholrelated diseases such as cardiovascular diseases and reproductive disorders should be well studied.