Ming-Fang Wang

Interaction between the Functional Polymorphisms of the Alcohol- Metabolism Genes in Protection against Alcoholism

The genes that encode the major enzymes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), exhibit functional polymorphism. The variant alleles ADH2*2 and ADH3*1, which encode high-activity ADH isoforms, and the ALDH2*2 allele, which encodes the low-activity form of ALDH2, protect against alcoholism in East Asians. To investigate possible interactions among these protective genes, we genotyped 340 alcoholic and 545 control Han Chinese living in Taiwan at the ADH2, ADH3, and ALDH2 loci. After the influence of ALDH2*2 was controlled for, multiple logistic regression analysis indicated that allelic variation at ADH3 exerts no significant effect on the risk of alcoholism. This can be accounted for by linkage disequlibrium between ADH3*1 and ADH2*2 ALDH2*2 homozygosity, regardless of the ADH2 genotypes, was fully protective against alcoholism; no individual showing such homozygosity was found among the alcoholics. Logistic regression analyses of the remaining six combinatorial genotypes of the polymorphic ADH2 and ALDH2 loci indicated that individuals carrying one or two copies of ADH2*2 and a single copy of ALDH2*2 had the lowest risk (ORs 0.04-0.05) for alcoholism, as compared with the ADH2*1/*1 and ALDH2*1/*1 genotype. The disease risk associated with the ADH2*2/*2-ALDH2*1/*1 genotype appeared to be about half of that associated with the ADH2*1/*2-ALDH2*1/*1 genotype. The results suggest that protection afforded by the ADH2*2 allele may be independent of that afforded by ALDH2*2.

Polymorphism of ethanol-metabolism genes and alcoholism: correlation of allelic variations with the pharmacokinetic and pharmacodynamic consequences.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for metabolism of ethanol. Both ADH and ALDH exhibit genetic polymorphisms among racial populations. Functional variant alleles ADH1B*2 and ALDH2*2 have been consistently replicated to show protection against developing alcohol dependence. Multiple logistic regression analyses suggest that ADH1B*2 and ALDH2*2 may independently influence the risk for alcoholism. It has been well documented that homozygosity of ALDH2*2 almost fully protects against developing alcoholism and that the heterozygosity only affords a partial protection to varying degrees. Correlations of blood ethanol and acetaldehyde concentrations, cardiovascular hemodynamic responses, and subjective perceptions have been investigated in men with different combinatorial ADH1B and ALDH2 genotypes following challenge with ethanol for a period of 130 min. The pharmacokinetic and pharmacodynamic consequences indicate that acetaldehyde, rather than ethanol, is primarily responsible for the observed alcohol sensitivity reactions, suggesting that the full protection by ALDH2*2/*2 can be ascribed to the intense unpleasant physiological and psychological reactions caused by persistently elevated blood acetaldehyde after ingesting a small amount of alcohol and that the partial protection by ALDH2*1/*2 can be attributed to a faster elimination of acetaldehyde and the lower accumulation in circulation. ADH1B polymorphism does not significantly contribute to buildup of the blood acetaldehyde. Physiological tolerance or innate insensitivity to acetaldehyde may be crucial for development of alcohol dependence in alcoholics carrying ALDH2*2.

Pharmacokinetic and pharmacodynamic basis for partial protection against alcoholism in Asians, heterozygous for the variant ALDH2*2 gene allele.

Objectives It has been well documented that although homozygosity of the variant aldehyde dehydrogenase-2 (ALDH2) gene allele, ALDH2*2, in Asians almost fully protects against alcoholism, the heterozygosity only affords a partial protection to varying degrees. The full protection against alcoholism has been ascribed to the low-amount of alcohol hypersensitivity accompanied by a prolonged and large accumulation of acetaldehyde in blood (Peng et al. Pharmacogenetics 1999; 9:463−476). The physiological basis for the partial protection, however, remains unclear. Methods To address this question, we recruited a total of 32 adult Han Chinese men, matched by age, body-mass index, and nutritional state from a population base of 404 men. The subjects were divided into 3 combinatorial genotypic groups of alcohol dehydrogenase (ADH) and ALDH, that is, ALDH2*1/*1–ADH1B*1/*1−ADH1C*1/*1 (n=8), ALDH2*1/*1−ADH1B*2/*2−ADH1C*1/*1 (n=8), and ALDH2*1/*2−ADH1B*2/*2−ADH1C*1/*1 (n=16). Following a moderate dose of ethanol (0.5u2009g/kg body weight), blood ethanol/acetaldehyde/acetate concentrations, cardiac and extracranial/intracranial arterial hemodynamic parameters, as well as self-rated subjective sensations, were measured for 130u2009min. Results Heterozygotic ALDH2*1/*2 subjects were found to be strikingly responsive to the moderate amount of alcohol, as evidenced by the prominent cardiovascular effects as well as subjective perceptions of general discomfort for as long as 2u2009h following ingestion. The ADH1B polymorphisms did not appear to correlate with the pharmacokinetic and pharmacodynamic effects. Conclusions These results indicate that acetaldehyde, rather than ethanol or acetate, is primarily responsible for the observed alcohol sensitivity reactions and suggest that substantially lower accumulation of blood acetaldehyde in the heterozygotes significantly reduces the aversion reaction to low amounts of alcohol that permits other biological as well as environmental factors to facilitate drinking and the according risk for the disease.

Pharmacokinetic and pharmacodynamic basis for overcoming acetaldehyde-induced adverse reaction in Asian alcoholics, heterozygous for the variant ALDH2*2 gene allele

Objectives It has been well documented that although homozygosity of the variant aldehyde dehydrogenese-2 (ALDH2) gene allele, ALDH2*2, in Asians almost fully protects against alcoholism, the heterozygosity only affords a partial protection to varying degrees. The partial protection against alcoholism has been ascribed to the faster elimination of acetaldehyde by residual hepatic ALDH2 activity and the lower accumulation in circulation in nonalcoholic heterozygotes. The physiological basis for overcoming the protection in ALDH2*1/*2 alcoholics, however, remains unclear. Methods To address this question, we recruited a total of 27 Han Chinese alcohol-dependent men, matched by age and body mass index, controlled for normal liver and cardiovascular functions, from a population base of 221 alcoholics. The participants were divided into ALDH2*1/*1 homozygotes (nu2009=u200913) and ALDH2*1/*2 heterozygotes (nu2009=u200914). After a moderate dose of ethanol (0.5u2009g/kg body weight), blood ethanol/acetaldehyde/acetate concentrations, cardiac and extracranial/intracranial arterial hemodynamic parameters, as well as self-rated subjective sensations, were measured for 130u2009min. Results ALDH2*1/*2 alcoholics exhibited significantly higher blood acetaldehyde levels as well as prominent cardiovascular effects and the subjective perceptions, compared with the ALDH2*1/*1 alcoholics. Comparable profiles of blood acetaldehyde were found between heterozygotic alcoholics and the previously reported nonalcoholic heterozygotes intaking the same dose of ethanol. ALDH2*1/*2 alcoholics revealed, however, significantly lower intensities in both physiologic and psychologic responses than did the nonalcoholic heterozygotes. Conclusion These results indicate that acetaldehyde, rather than ethanol or acetate, is primarily responsible for the observed alcohol sensitivity reactions in heterozygotic alcoholics and suggest that physiological tolerance and/or innate low sensitivity may play a crucial role in overcoming the deterring response. A potential pharmacogenetic classification of acetaldehydism and alcoholism for alcoholics carrying the different ALDH2 genotypes is proposed.

Substrate specificity of human and yeast aldehyde dehydrogenases

Human aldehyde dehydrogenase (ALDH) family may contribute to metabolism of hydrocarbons, biogenic amines, retinoids, steroids, and lipid peroxidation. We previously reported kinetic properties of human cytosolic ALDH1 and mitochondrial ALDH2 towards oxidation of the straight-chain and branched-chain aliphatic aldehydes with various chain lengths [S.J. Yin, M.F. Wang, C.L. Han, S.L. Wang, Substrate binding pocket structure of human aldehyde dehydrogenases: a substrate specificity approach, Adv. Exp. Med. Biol. 372 (1995) 9-16]. We present here substrate specificities for aromatic and heterocyclic aldehydes with purified human liver ALDH1 and ALDH2, and also with yeast mitochondrial ALDH2 for comparison. Kinetic assay for human ALDHs was performed in 50mM HEPES, pH 7.5 and 25 degrees C, containing 0.5mM NAD(+), 1.7% (v/v) acetonitrile (as a solvent carrier for aldehydes) and varied concentrations of substrate, and for yeast ALDH2 the assay was determined in the same reaction mixture except containing 3mM NAD(+) and addition of 200 mM KCl. With respect to phenylacetaldehyde, 2-phenylpropionaldehyde, benzaldehyde, p-nitrobenzaldehyde, cinnamaldehyde, 2-furaldehyde and indole-3-acetaldehyde, human liver ALDH1 exhibited K(M) ranging from 0.25 to 4.8 microM, V(max) of 0.34-2.4U/mg, and catalytic efficiency, V(max)/K(M), 0.070-3.9U/(mg microM); human ALDH2 exhibited K(M) ranging from less than 0.15-0.74 microM, V(max) of 0.039-0.51 U/mg, and V(max)/K(M), 0.15-1.0U/(mg microM). Human ALDH1 and ALDH2 exhibited substate inhibition constants (K(i)) for phenylacetaldehyde, 95 and 430 microM, respectively. Yeast ALDH2 exhibited K(M) for straight-chain aliphatic aldehydes (C1-C10), 2.3-210 microM, and substrate inhibition constants (C2-C10), 79-2900 microM, with a trend of being smaller K(M) and K(i) for longer chain lengths; and K(M) for cinnamaldehyde, benzaldehyde, and 2-furaldehyde, 5.0, 79, and 1000 microM, respectively. Therefore human ALDH1/ALDH2 and yeast ALDH2 can contribute to detoxification or metabolism of various exogenous/endogenous aliphatic and aromatic aldehydes. The systematic changes in kinetic parameters for oxidation of structurally related aldehydes may reflect subtle functional topographic distinctions of substrate pocket for human and yeast ALDHs.

The role of ALDH2 and ADH1B polymorphism in alcohol consumption and stroke in Han Chinese

The genes encoding the enzymes for metabolising alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) -- exhibit genetic polymorphism and ethnic variations. Although the ALDH2*2 variant allele has been widely accepted as protecting against the development of alcoholism in Asians, the association of the ADH1B*2 variant allele with drinking behaviour remains inconclusive. The goal of this study was to determine whether the polymorphic ADH1B and ALDH2 genes are associated with stroke in male Han Chinese with high alcohol consumption. Sixty-five stroke patients with a history of heavy drinking (HDS) and 83 stroke patients without such a history (NHDS) were recruited for analysis of the ADH1B and ALDH2 genotypes from the stroke registry in the Tri-Service General Hospital, Taipei, Taiwan, between January 2000 and December 2001. The allelotypes of ADH1B and ALDH2 were determined using the polymerase chain reaction-restriction fragment length polymorphism method. The HDS patients (3 per cent) showed a significantly lower ALDH2*2 allele frequency than NHDS patients (27 per cent) (p < 0.001). After controlling for age, patients with HDS were associated with a significantly higher occurrence of cigarette smoking (p < 0.01) and liver dysfunction (p < 0.01). Multiple logistic regression analyses revealed that the ALDH2*2 variant allele was an independent variable exhibiting strong protection (odds ratio 0.072; 95 per cent confidence interval 0.02-0.26) against HDS after adjustment for hypertension, diabetes mellitus, smoking status and liver dysfunction. By contrast, allelic variations in ADH1B exerted no significant effect on HDS. The present study indicated that, unlike ALDH2*2, ADH1B*2 appears not to be a significant negative risk factor for high alcohol consumption in male Han Chinese with stroke.

Substrate Binding Pocket Structure of Human Aldehyde Dehydrogenases

Human aldehyde dehydrogenases (AIDH), catalyzing irreversible oxidation of various aliphatic and aromatic aldehydes to the corresponding carboxylic acids, constitute a complex enzyme family (Pietruszko, 1983; Hempel and Jornvall, 1989; Hempel and Lindahl, 1989; Yoshida et al., 1991). There are at least five AlDHs that have been purified and characterized from human liver or stomach: AlDH1 and A1DH2 (E1 and E2; Greenfield and Pietruszko, 1977), A1DH3 (Wang et al., 1990), A1DH4 (E4, glutamic g-semialdehyde dehydrogenase; Forte-McRobbie and Pietruszko, 1986), and g-aminobutyraldehyde dehydrogenase (E3, Kurys et al., 1989). Kinetically, the AlDHs can be classified into two groups according to their Km values for acetaldehyde. The low Km (mM range) forms comprise AlDH1, A1DH2, and g-aminobutyraldehyde dehydrogenase (Greenfield and Pietruszko, 1977; Kurys et al., 1989). A1DH3 and A1DH4 belong to the high Km (mM range) forms (Yin et al., 1989; Forte-McRobbie and Pietruszko, 1986). Structurally, AlDHl and A1DH2 show highest sequence homology (68%) among the above AlDHs (Hempel et al., 1984, 1985; Hsu et al., 1985). The degree of positional identity between AIDH1/2, A1DH3, and g-aminobutyraldehyde dehydrogenase is 30–40% (Hsu et al., 1985, 1992; Yin et al., 1991; Kurys et al., 1993), suggesting a distant relationship. A1DH4 has a larger subunit which shows no significant sequence homologies to all the known AlDHs (Forte-McRobbie and Pietruszko, 1986; Hempel et al., 1992), thus excluding it from the AlDH family. Recently, genes, i.e. AIDH5 through AIDH8, encoding four additional human AlDHs have been cloned and characterized (Hsu et al., 1991, 1994). The kinetic properties of these new AlDH forms remain to be elucidated.

ALDH2*2 but not ADH1B*2 is a causative variant gene allele for Asian alcohol flushing after a low-dose challenge: correlation of the pharmacokinetic and pharmacodynamic findings.

Objective It has been well documented that variant alleles of both ADH1B*2 of alcohol dehydrogenase (ADH) and ALDH2*2 of aldehyde dehydrogenase (ALDH) protect against the development of alcoholism in East Asians. However, it remains unclear whether ADH1B*2 contributes significantly toward the accumulation of systemic blood acetaldehyde and whether it plays a critical role in the alcohol flushing reaction. Participants and methods Sixty-one adult Han Chinese men were recruited and divided into six combinatorial genotypic groups: ALDH2*1/*1–ADH1B*1/*1 (12), ALDH2*1/*1–ADH1B*1/*2 (11), ALDH2*1/*1–ADH1B*2/*2 (11); ALDH2*1/*2–ADH1B*1/*1 (9), ALDH2*1/*2–ADH1B*1/*2 (9), and ALDH2*1/*2–ADH1B*2/*2 (9). After ingesting 0.3u2009g/kg of alcohol, blood ethanol, acetaldehyde, and acetate concentrations, as well as the facial skin blood flow (FSBF) and pulse rate were measured for 130u2009min. Results The ALDH2*1/*2 heterozygotes carrying three ADH1B allelotypes showed significantly higher peak levels and areas under the concentration curve (AUCs) of the blood acetaldehyde as well as significantly greater increases in the peak pulse rate and peak FSBF compared with the ALDH2*1/*1 homozygotes. However, no significant differences in peak levels and AUCs of blood ethanol, acetaldehyde or acetate, or the peak cardiovascular responses, were found between the ADH1B allelotypes carrying ALDH2*1/*1 or between those with ALDH2*1/*2. Partial correlation analyses showed that peak blood acetaldehyde, rather than the blood ethanol or acetate, was correlated significantly with the peak responses of pulse rate and FSBF. Conclusion Findings indicate that ALDH2*2, rather than ADH1B2*2, is a causal variant allele for the accumulation of blood acetaldehyde and the resultant facial flushing during low alcohol consumption.