Genetic Polymorphism of Alcohol Metabolyzing Enzymes and Its Implication to Human Ecology. Institute of Community Medicine, University of Tsukuba

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1 REVIEW ARTICLE Genetic Polymorphism of Alcohol Metabolyzing Enzymes and Its Implication to Human Ecology Shoji HARADA Institute of Community Medicine, University of Tsukuba Abstract Individual and racial differences in alcohol metabolism and their implications in acute and chronic intoxication of alcohol intake were reviewed in the relationships of genetic polymorphism of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The recent studies revealed that the catalytic deficiency of ALDH2 isozyme is responsible to the flushing symptom as well as other vasomotor symptoms caused by a higher acetaldehyde level after alcohol consumption. Subsequently the deficiency of ALDH2 has been prevalently found among only the peoples of Mongoloid origin and the individuals deficient in ALDH2 refrain from excessive drinking of alcohol due to averse reaction leading to protection against alcoholism. Moreover, many studies based on DNA analysis have confirmed these findings. As ALDH2 polymorphism was found only in Mongoloid, population studies on the ALDH2 mutant will provide the important information to estimate the dispersal of the ethnic groups in Asia and Oceania and also to prevent alcohol abuse in the developing countries. Keywords ADH, ALDH, Genetic polymorphism, Alcohol sensitivity, Alcoholism, Racial difference Introduction One of the main components in alcohol beverages is ethanol, which is readily absorbed from the digestive tracts such as stomach and intestine after oral intake. In the major pathway for the ethanol metabolism, the first metabolite of ethanol by alcohol dehydrogenase (ADH, EC: ) is acetaldehyde which is catalyzed by aldehyde dehydrogenase (ALDH, EC: ) into acetic acid. A number of studies in the past decades have indicated that individual and ethnic differences have been found in the alcohol metabolyzing enzymes as well as the symptoms after alcohol intake. The typical symptoms such as facial flushing, tachicardia, headache, peripheral vasodilation after drinking a low dose of alcohol beverage have been observed at high percentages in Oriental population, whereas those symptoms have not been noted in European and American Whites (WOLFF,1972, 1973; EWING et al., 1974; ZEINER et al., 1979; Article No Received February 6, 1991

2 124 S. HARADA REED et al., 1976; MIZOI et al., 1979). BY the discovery of the multiple forms of ADH isozymes (VON WARTBURG et al., 1965; SMITH et al., 1971), STAMATOYANNOPOULAS et al. (1975) have hypothesized that the ADH polymorphism could lead to individual difference on alcohol sensitivity and metabolism. Subsequently, the discovery of the multiple forms of ALDH isozymes and the polymorphism (HARADA et al., 1980a, 1980b, 1981) and the observed correlation between the ALDH polymorphism as well as pharmacogenetic differences in such symptoms as flushing due to acetaldehyde accumulation have shown doubt on the role of ADH (HARADA et al., 1982a). Human ALDH isozymes consist of four different components (ALDH1,2,3,4) as revealed by using electrophoresis. Among these isozymes, ALDH2 mainly contributes to the oxidation of acetaldehyde. Japanese and other Mongoloid peoples show genetic polymorphism in the isozyme of ALDH2 and the inactive type of ALDH2 is responsible for higher acetaldehyde levels and the flushing symptoms as well as other vasomotor symptoms after alcohol intake (HARADA et al., 1982a, b). Subsequent studies indicate that the polymorphism of ALDH2 isozyme is widely prevalent among the Mongoloid peoples (GOEDDE and AGARWAL, 1986; GOEDDE et al., 1986, 1990; HARADA, 1989). Recent studies based on molecular biology revealed that the cause for the isozyme deficiency is a structural mutation leading to the synthesis of an enzymatically inactive protein (IMPRAIM et al., 1982; Hsu et al., 1987). Epidemiological relationship between ALDH2 polymorphism and alcoholism has been reported among Japanese (HARADA et al., 1982b). The frequency of the ALDH2 isozyme deficiency in alcoholics was significantly lower than that of healthy controls. This finding indicates that individuals showing the deficiency of ALDH2 isozyme may refrain from excessive drinking due to adverse reactions caused by raised blood acetaldehyde level and this may explain why alcoholism in Japanese and other Mongoloid groups has been less common than in the Caucasian groups. In this paper, a review on the implication of genetic polymorphism of ADH and ALDH isozymes in racial differences of alcohol sensitivity are presented. Also, the pharmacogenetic importance of ALDH mutant gene is discussed. Population surveys of ALDH2 polymorphism is considered on the significance in dispersal of Mongoloid population groups. Isozyme Variation Enzymatic pathways for ethanol metabolism can be lead by two main enzymes ALDH. - ADH and Both enzymes can react with ethanol and acetaldehyde which is the metabolite of ethanol, as follows: C2H5OH+NAD+*ADH*CH3CHO+NADH+H+ CH3CHO+NAD+*ALDG*CH3COOH+NADH+H+ Ten percent or less of the ingested ethanol is metabolized by two different nonadh ethanol oxidizing enzymes; microsomal ethanol oxidizing system (MEOS) linked to the cytochrome P-450 oxygenases (LIEBER and DECARLI, 1972; TESCHKE et al., 1977; LIEBER, 1977) and catalase (MIWA et al., 1978). However, the contribution of these two enzymes for ethanol oxidation is thought to be minor in moderate drinking. 1) ADH Multiple molecular forms of human ADH are divided into three classes according to electrophoretic migration and biochemical properties. The class I isozymes are products formed by a

3 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 125 random association of *, * and * polypeptide subunits, which are controlled by three separate gene loci, ADHI, ADH2 and ADH3 (SMITH et al., 1971, 1972, 1973). Because all classes of ADH are dimeric enzyme, class I polypeptide subunits form homodimeric isozymes (*, * and *) and heterodimeric isozymes (*, *, *). However, no heterodimers are formed between class I, II and III enzymes. Class II ADH was commonly designated as *-ADH (BOSRON et al., 1977, 1980; HOOD et a1.,1987) encoded by the ADH4 locus. The *-ADH is insensitive to pyrazol and is immunologically no cross-reactive with class I ADH. Class III ADH is designated as *-ADH and possesses different kinetic and structural properties as compared to other ADH classes (PARES and VALLEE, 1981). Genetic variations of ADH isozymes have been so far reported only at the ADH2 and ADH3 loci. As class I ADH isozymes can form heterodimers through the random combination among each subunits, the isozyme patterns obtained by starch gel electrophoresis are complicated. Isozyme bands and schematic patterns are shown in Figs. 1 a and l b. Two different polypeptide subunits designated as *1 and *2 have been identified by using electrophoresis and biochemical analysis (VON WARTBURG and SCHURCH,1968; SMITH et al.,1971). The homoand heterodimers containing *2 subunit are commonly called as atypical type (A) and exhibit higher activity at ph 8.8. On the contrary, usual type (U) contains only * subunit and exhibits optimum activity at ph Beside of the two alleles, later on, a new allele (*3) was found in Black Americans and has been biochemically characterized (BOSRON and LI, 1987). Isozyme containing *3 exhibits a faster electrophoretic migration toward the cathode and possesses a dual ph optimum (7.0 and 10.0). The polymorphic products at ADH3 locus are *1 and *2 subunits and each subunit forms homo- and heterodimers among all subunits at class I ADH loci. So far, the incidence of three alleles at the ADH2 locus and two alleles at the ADH3 locus have been reported by a number of authors (VON WARTBURG and SCHURCH,1968; SMITH et al., 1971,1972; SCHULZ et al., 1976; HARADA et al., 1978b, 1980a, b; FUKUI and WAKASUGI, 1972; TENG et al., 1979; AZEVEDO et al., 1975; REX et al., 1985; BOSRON et al., 1983). In Table 1, allele frequencies in different population are presented. The frequency of ADH2*2 (*2) is variable among different racial population. Japanese and Chinese possesses 65-70%, while that occurs in the frequencies of 5-10% among American and European Whites as well as Blacks. However, the frequency of ADH3*2 was found to be 40-50% in Whites, whereas the frequencies in Japanese and Chinese were to be 5-10% and Blacks exhibit about 15%. The gene frequency of ADH3*2 among American Indians in New Mexico was higher (56&) than in the other Mongoloid populations (REx et al., 1985). 2) ALDH NAD-dependent ALDH is the main enzyme involved in the oxidation of acetaldehyde, which is the primary product of alcohol metabolism in humans. During the last ten years or more, multiple forms and gene loci of ALDH have been found and characterized in human tissues (GREENFIELD and PIETRUSZKO, 1977; PIETRUSZKO et al., 1977; HARADA et al., 1978a, 1980a, 1981; FORTE-MCROBBIE and P IETRUSZKO, 1985; DULEY et al., 1985, REX et al., 1985). Human ALDH isozymes consist of four different components (ALDH1,2,3,4) as revealed by electrophoretic and kinetic properties as well as their cellular and tissue distribution. As shown in Fig. 2, a total of four ALDH bands

4 126 S. HARADA Fig. 1. a) Polymorphism of ADH2 and ADH3 isozymes detected by starch gel electrophoresis. b) Schematic patterns of the different isozymes formed by ADH class I subunits. with different electrophoretic mobilities were detectable in various tissue extracts using starch gel electrophoresis. The two fast migrating isozymes (ALDHI and 2) were found mainly in liver and kidney. In stomach and lung, a slower migrating isozyme (ALDH3) was detectable. The fourth isozyme (ALDH4) occurred mainly in liver and kidney. Biochemical properties and tissue distribution of these isozymes have been investigated using purified enzymes (HARADA et al., 1980a, b). Tables 2 indicates that ALDH2 has a lower Km

5 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 127 Table 1. Gene frequencies of ADH2 and ADH3 alleles in different racial populations Fig. 2. Distribution of four different ALDH isozymes in various tissues. The arrows indicate ALDH2, ALDH1, ALDH3 and ALDH4 enzymes from the top. value for acetaldehyde than that of ALDH1, while ALDH3 and ALDH4 have much higher Km values than the others. The low Km isozyme (ALDH2) is predominantly localized in the mitochondria and the other high Km isozymes are detectable in the cytosol (KOIVULA, 1975; PIETRUSZKO et al., 1978; JENKINS and PETERS, 1983; MEIER-TACKMAN et al., 1988). Using starch gel electrophoresis or polyacrylamide gel isoelectric focusing technique, two different types of ALDH2 were detected in Japanese autopsy liver extracts. As shown in Fig. 3, 48% of the liver specimens exhibited ALDH2 isozyme band and 52% of the specimens had no activity (deficiency) in Japanese. Because of difficulty in obtaining biopsy liver samples from normal individuals, hair root samples were analyzed using polyacrylamide gel isoelectric focusing. Thus, the isozyme variation of ALDH2 was demonstrated in hair root lysates in Fig. 4 (GOEDDE et al., 1980; HARADA et al., 1982a). The immunological properties of ALDH1 and 2 using monospecific antisera against each isozyme. As examined by double immunodiffusion, the antiserum against ALDH1 reacted only with ALDH1 and antiserum against ALDH2 reacted only with ALDH2. Moreover, the liver extract showing an ALDH2 deficiency reacted

6 128 S. HARADA Table 2. Kinetic properties of ALDH isozymes and their cellular and tissue distribution Fig. 3. Polymorphism of ALDH2 isozyme. 1, 2, 5 indicate catalytic deficiency of ALDH2, and 3, 4 indicate normal. with the antiserum against ALDH2. This indicates that the ALDH2 deficiency possesses enzymatically inactive but immunologically crossreactive material (IMPRAIM et al., 1982; YOSHIDA et al., 1984; AGARWAL et al., 1984) and the amino acid substitution between the active and inactive ALDH2 isozymes is glutamic acid to a lysine (HEMPEL et al., 1984, 1985b; HSU et al., 1985). Molecular Genetics of the Isozyme Variations Recent studies based on the molecular biology have revealed that the cause for the isozyme variation of ADH and ALDH is a structural mutation leading to the synthesis of polypeptide subunits exhibiting different kinetic and

7 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 129 Fig. 4. Polymorphism of ALDH2 isozymes in hair root lysates (2*8) by isoelectric focusing. l: Liver homogenate (normal). 3: Deficiency of ALDH2. The other numbers (2, 4*8) indicate normal. biochemical properties. Moreover, the determination of the genotypes of the each locus in the both enzymes is now possible using the advanced techniques for DNA analysis. 1) ADH Complete amino acid sequencies have been reported on the subunits of the class I ADH (JORNVALL et al., 1984, 1987). All subunits consist of 374 residues and have about 10% total amino acid exchanges. In addition the degree of exchanges in the *, * and * subunits are quite similar. The amino acid data on *1 and *2 subunits indicated that a single amino acid substitution at position Arg-47 in the site of *1 to His-47 occurs in the *2 subunit (JORNVALL et al., 1987; BUHLER et al., 1984; DUESTER et al., 1986). This mutation in the active site has lead to the change of kinetic and functional properties such as different ph optimum and Vmax of the atypical type of ADH. Amino acid analysis of *1, *2 and,*3 subunits indicated that the substitution of Arg of *1 and *2 at the 47th residue occurs and *3 has Cys at the 369th position (BURNELL et al., 1987). Amino acid sequences of the *1 and *2 subunits have the same ones except to replacements at position 271(Arg/Gln) and 349 (IIe/Val) (BUHLER et al., 1984; HEMPEL et al., 1985a). Also, the analysis on cdnas of class I ADH has confirmed the same amino acid sequences of each subunit. Table 3 shows the amino acid and DNA sequences corresponding to different * and * subunits. All genes encoding ADHJ, 2 and 3 located in tandem on chromosome 4 (SMITH et al., 1986). 2) ALDH Although the evidence of ALDH isozymes encoded in at least four separated loci have been described according to the electrophoretic and biochemical characterization (HARADA et al., 1980b; PIETRUSZKO et al., 1977; PIETRUSZKO, 1980; DULEY et al., 1985; SANTISTEBAN et al.,

8 130 S. HARADA Table 3. Differences of amino acid sequences among human ADH subunit ALDH indicate that ALDHI, 2 and 3 localize at chromosome 9, 12 and 17, respectively (HSU et al., 1986; SANTISTEBAN et al., 1985). 3) Genotyping by Polymerase Chain Reaction Now, determination of the genotypes of ADH and ALDH have been widely carried out for the population studies based on the information of DNA sequences around point mutation areas (HSU et al., 1987; XU et al., 1988; GOEDDE et al., 1989; HARADA, 1990). DNA amplification technique by polymerase chain reaction (PCR) 1985), so far, only the subunits of ALDH 1 and 2 have been reported on the primary structure of amino acid and DNA sequences. The both of subunits have 500 residues (JORNvALL et al., 1987) and the identity of two isozymes are approximately 68% (HEMPEL et al., 1985b). The structural difference of the two alleles of ALDH2 isozymes showing polymorphism have been determined on amino acid as well as DNA sequence (HEMPEL et al., 1985b; HSU et al., 1985, 1988). According to the information, the catalytic deficiency is caused by a structural point mutation at amino acid position 487 of the polypeptide subunit of ALDH2 normal. At this position a substitution of Glu to Lys resulting from a transition of G(C) to A(T) at DNA level has occured. Recently, direct detection of genotypes of the both alleles of ALDH2 has become possible using the genomic DNA in blood samples (HSU et al., 1987; GOEDDE et al., 1989; SINGH et al., 1989; HARADA et al., 1990). Several studies on chromosome localization of (SAIKI et al.,1985,1988) have enabled to detect genetic polymorphism responsible for a point mutation of nucleotide using small amount of blood and tissue samples. Genotyping using DNA amplification and Southern blot techniques involves generally the following five procedures: i) Amplification by PCR. ii) Transfer to nylon membrane and fixation of the amplified oligonucleotides. iii) Hybridization of allele specific oligonucleotide probes labelled with 32P ATP. iv) Stringent washing of the membrane. v) Autoradiography. Genotypes of ADH2 and ALDH2 using DNAs extracted from blood samples could be determined using PCR and slot-blot hybridization with synthesized oligonucleotide probes. In Fig. 5, genotypes of ADH2 and ALDH2 loci were demonstrated. Pharmacogenetic Basis of Alcohol Sensitivity It has been hypothesized previously that individual and racial differences in isozyme variation of ADH2 could lead to pharmacogenetic differences after intake of alcohol (von WART- BURG et al., 1965; STAMATOYANNOPOULAS et al., 1975). These authors proposed that the individuals possessing *2 subunit show a higher acetaldehyde level after alcohol consumption due to strong catalytic property at physiological ph.

9 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 131 Fig. 5. Genotypes of two alleles at ADH2 and ALDH2 loci obtained by southern hybridization and autoradiography. *1: ADH2*1 probe, 2: ADH2*2 probe. N: ALDH2*1 probe, D: ALDH2*2 probe. * Numbers (1 to 8) indicate subjects as follows. 1: ADH2*l/ADH2*l, ALDH2*1/ALDH2*2. 2: ADH2*2/ADH2*2, ALDH2*1/ALDH2*1. 3: ADH2*2/ADH2*2, ALDH2*1/ALDH2*2. 4: ADH2*2/ADH2*2, ALDH2*1/ALDH2*1. 5: ADH2*2/ADH2*2, ALDH2*1/ALDH2*2. 6: ADH2*l/ADH2*2, ALDH2*1/ALDH2*2. 7: ADH2*1/ADH2*2, ALDH2*2/ALDH2*2. 8: ADH2*1/ADH2*2, ALDH2*1/ALDH2*1. However, the subsequent studies have not supported this hypothesis. No significant difference in the rate of alcohol metabolism was found between the individuals possessing ADH2 usual and atypical types (EDWARDS and EVANS, 1967), and also no difference in the elimination rate of alcohol was observed between flusher and non-flusher (MIZOI et al., 1979). On the basis of ALDH2 polymorphism detected in liver extracts using starch gel electrophoresis, a new hypothesis was first proposed (HARADA et al., 1980). The catalytic deficiency of ALDH2 isozyme is responsible for the higher acetaldehyde level and flushing symptom after alcohol intake. In earlier studies (HARADA et al., 1981, Mizoi et al., 1983, 1985), hair roots lysates were used for the determination of ALDH2 phenotypes using isoelectric focusing. The mean peak blood acetaldehyde level after alcohol intake

10 132 S. HARADA are significantly higher (35.4*12.8*mol/l) in the group of individuals of ALDH2 deficiency than the group (2.1*1.7*mol/1) possessing the catalytic isozyme. However, the peak blood ethanol levels are similar in both groups. Therefore, it can be concluded that the delayed oxidation of acetaldehyde may be responsible for the marked sensitivity to ethanol as observed in the Oriental populations. Recently, the determination of the genotypes of ALDH2 locus has become possible using the advanced techniques for DNA analysis. Based on the technology, the genotypes of ALDH2 locus was determined in healthy Japanese and their acetaldehyde metabolism after oral moderate alcohol injection was investigated using gas chromatography (HARADA et al., 1990). Genetic study on the relationship of enzymatic activity and ALDH2 phenotypes formed with the subunits of ALDH2* 1 and ALDH2*2 have revealed that only the homozygous genotype (ALDH2* 1 /ALDH2* 1) shows an catalytic activity (HSU et al., 1987). The blood acetaldehyde concentration after alcohol intake was compared among the groups of three different genotypes formed by combining of ALDH2*1 and ALDH2*2 genes in healthy controls. As shown in Fig. 6, homozygous genotype of ALDH2*1(NN) showed the lowest concentration (4.9*1.7*M) and the subjects showing heterozygous genotype of ALDH2*1/ALDH2*2 (ND) had lower concentration (24.6*8.4*M) than those of homozygous genotype of ALDH2*2 (DD) (95.0*2.6*M) (HARADA, 1990). Consequently, the individuals possessing the hetero- or homozygous genotype of ALDH2*2 gene show the sensitivity to alcohol due to a higher formation of blood acetaldehyde after alcohol intake. Epidemiological Implication of ALDH Polymorphism Fig. 6. Blood acetaldehyde concentration after alcohol intake (0.4ml ethanol/kg body weight) in the groups of three genotypes. MN: ALDH2*1/ALDH2*1, ND: ALDH2*1/ALDH2*2, DD: ALDH2*2/ALDH2*2. As the prevalence of ALDH2 deficiency may produce an aversion to alcohol consumption and affect the incidence of alcohol-related problems, genetic polymorphism of ALDH2 locus observed in Mongoloid involves a significant importance from the aspect of drinking behavior as well as chronic alcohol intoxication. Epidemiological studies have indicated that a positive correlation exists between flushing response and alcohol drinking pattern (WILSON et al., 1978; REED, 1978). Genetic investigation concerning the alcohol consumption per capita and the frequency of ALDH2 deficiency have certified a high correlation among them (HARADA et al., 1985). As shown in Table 4, in Gifu a higher percentage of liver ALDH2 deficiency was found and the alcohol consumption was relatively low. In Sendai, the situation is reversed. Later on, investigation of a large number of Japanese con-

11 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 133 Table 4. Alcohol consumption and frequency of ALDH2 isozyme deficiency in two different districts in Japan firmed this observation (SUWAKI and OHARA, 1985). The frequency of alcoholics has been reported to be much lower among Japanese, Chinese and other Mongoloid populations (REED, 1978). Population study to confirm the observation has been made in alcoholics, schizophrenics, drug dependents and normal subjects from Japanese (HARADA et al., 1985). This observations indicate that individuals possessing ALDH2*2 gene may refrain from excessive drinking due to adverse reactions such as flushing, increase of heart rates, headache etc, caused by a raise of blood acetaldehyde level. This may explain why alcoholism in Japanese and other Mongoloid groups has been less common than in the Caucasian. Protective reaction to alcohol abuse do not work among Caucasians due to the absence of aversion caused by ALDH2 deficiency. Preliminary studies on the relationship between ALDH2 deficiency and alcoholics has indicated that a low percentage of ALDH2 deficiency was found in alcoholics (2.3%) compared with healthy controls (41.0%). The frequencies of other patients were also similar to that of healthy controls (HARADA et al., 1982b). Furthermore, the gene frequencies of ADH2 and Table 5. Comparisons of the allelic frequencies at ADH2 and ALDH2 loci between healthy controls and alcoholics Controls: ADH2*1=0.38, ADH2*2=0.62 Alcoholics: ADH2*1=0.39, ADH2*2=0.61 Controls: ALDH2*1=0.756, ALDH2*2=0.244 Alcoholics: ALDH2*1=0.98, ALDH2*2=0.02

12 134 S. HARADA ALDH2 locus were compared between alcoholics and healthy control groups (HARADA, 1990). Although the genotypic distributions of ADH2*1 and ADH2*2 were found to be not significantly different among the both groups, the gene frequency of ALDH2*2 in alcoholics was significantly lower than that of healthy controls as shown in Table 5. Dispersal of ALDH2*2 Gene in Mongoloid A number of genetic markers have been used for estimating the genetic distance among different racial and ethnic populations. As genetic polymorphism of ALDH2 was observed only in Mongoloid, population surveys of ALDH2 mutant (ALDH2*2) have been made using hair root samples as well as DNA samples extracted from blood. Distribution of genotypes and gene frequency of ALDH2 locus among Mongoloid population groups are summarized in Tables 6a, b, c (GOEDDE and AGARWAL, 1986; GOEDDE et al., 1983,1984a, 1984b, 1985, 1986, 1990; HARADA et al., 1980a, 1986b, 1989, 1990; SINGH et al., 1989; O'DOWD et al., 1990). ALDH2*2 gene was found only among individuals belonging to the Mongoloid race. However, such a mutant gene was not detected in Caucasoid and Negroid groups. The gene frequency of ALDH2*2 was found to be higher Table 6. Frequencies of genotypes of ALDH2 locus in Mongoloids (a), Caucasoids (b) and Negroids (c) Mongoloids (a)

13 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 135 Caucasoids (b) Negroids (c) in the areas of China and Japan than in Thailand, Philippines and Malaysia. On the contrary the mutant gene was very rare in American Indian, Papua New Guinea and Australian Aborigines. The mutant gene might have accumulated for a long time among new Mongoloid populations for some causes such as gene-environmental interaction and genetic drift. During the time the gene has dispersed to the neighbouring population groups through emigration and exogamy. If the hypothesis might be accepted, we could estimate on the dispersal of Mongoloid by the gene marker. Furthermore, studies on population genetics as to the natives in Asia, Oceania and America will be necessary to elucidate the questions.

14 136 S. HARADA Acknowledgements The author is grateful to Prof. Yasuhiko MIZOI, Osaka Medical College for reading this paper critically and he wishes to express thanks to Prof. Morihiko OKADA, University of Tsukuba for giving him a chance to present this paper. References AGARWAL, D.P., R. ECKEY, S. HARADA and H.W. GOEDDE, 1984: Basis of aldehyde dehydrogenase deficiency in Orientals: Immunochemical studies. Alcohol, 1: AZEVEDO, E.S., O.M.C., DA-SILVA and J. TAVARES- NETO, 1975: Human alcohol dehydrogenase ADH1, ADH2 and ADH3 loci in a mixed population of Bahia, Brazil. Ann. Hum. Genet., 39: BOSRON, W.F. and T-K LI, 1987: Catalytic properties of human liver alcohol dehydrogenase isoenzymes. Enzyme, 37: BOSRON, W.F., T-K LI, L.G. LANGE, W.P. DAFAL- DECKER and B.L. VALLEE, 1977: Isolation and characterization of an anodic form of human liver alcohol dehydrogenase. Biochem. Biophys. Res. Comm., 74: BOSRON, W.F., T-K LI and B.L. VALLEE, 1980: New molecular forms of liver alcohol dehydrogenase: Isolation and characterization of ADH Indianapolis. Proc. Natl. Acad. Sci. USA, 77: BOSRON, W.F., L.J. MAGNES and T-K. LI, 1983: Human liver alcohol dehydrogenase: ADH Indianapolis results from the genetic polymorphism at the ADH2 gene locus. Biochem. Genet., 21: BUHLER, R., J. HEMPEL, J.P. von WARTBURG and H. JORNVALL, 1984: Human liver alcohol dehydrogenase: The unique properties of the "atypical" isoenzyme *2/* 2-Bern can be explained by a single base mutation. Alcohol, 2: BURNELL, J.C., L.G. CARR, F.E. DWULET, H.J. EDENBERG, T-K. LI and W.F. BOSRON, 1987: The human *3 alcohol dehydrogenase subunit differs from *1 by a cys for arg-369 substitution which decreased NAD(H) binding. Biochem. Biophys. Res. Commun., 146: DUESTER, G., M. SMITH, V. BLANCHIONE and G.W. HATFIELD, 1986: Molecular analysis of the human class I alcohol dehydrogenase gene family and nucleotide sequence of the gene encoding the subunit. J. Biol. Chem., 261: DULEY, J.A., O. HARRIS and R.S. HOLMES, 1985: Analysis of human alcohol- and aldehyde metabolizing isozymes by electrophoresis and isoelectric focusing. Alcoholism: Clin. Exp. Res., 9: EDWARDS, J.A. and D.A.P., EVANS, 1967: Ethanol metabolism in subjects possessing typical and atypical liver alcohol dehydrogenase. Clin. Pharmacol. Therapeut., 8: SWING, J.A., B.A. ROUSE and E.D. PELLIZZARI, 1974: Alcohol sensitivity and ethnic background. Am. J. Psychiatry, 131: FORTE-MCROBBIE, C.M, and R. PIETRUSZKO, 1985: Aldehyde dehydrogenase content and composition of human liver. Alcohol, 2: FUKUI, M. and C. WAKASUGI, 1972: Liver alcohol dehydrogenase in Japanese population. Jap. J. Legal Med., 26: GOEDDE, H.W., D.P. AGARWAL and S. HARADA, 1980: Genetic studies on alcohol metabolizing enzymes: Detection of isozymes in human hair roots.

15 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 137 Enzyme, 25: GOEDDE, H.W., D.P. AGARWAL, S. HARADA, D. MEIER-TACKMANN, D. RUOFU, U. BIENZLE, A. KROEGER and L. HUSSEIN, 1983: Population genetic studies on aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity in four different Chinese populations. Hum. Hered., 34: GOEDDE, H.W., HG. BENKMANN, L. KRIESE, P. BOGDANSKI, D.P. AGARWAL, D. RUOFU, C. LIANGZHONG, C. MEIYING, Y. YIDA, X. JIUSIN, L. SHIZHE and W. YONGFA, 1984a: Aldehyde dehydrogenase isozyme deficiency and alcohol sensitivity in four different Chinese populations. Hum. Hered., 34: GOEDDE, H.W., F. ROTHHAMMER, H.G. BENKMANN and P. BOGDANSKI, 1984b: Ecogenetic studies in Atacameno Indians. Hum. Genet., 67: GOEDDE, H.W., D.P. AGARWAL, R. ECKEY and S. HARADA, 1985: Population genetic and family studies on aldehyde dehydrogenase deficiency and alcohol sensitivity. Alcohol, 2: GOEDDE, H.W. and D.P. AGARWAL, 1986: Aldehyde oxidation: Ethnic variation in metabolism and response. In: KALOW, W., H.W. GOEDDE and D.P. AGARWAL (eds.) Ethnic Differences in Reactions to Drugs and Xenobiotics. Alan R. Liss Inc., New York, pp GOEDDE, H.W., D.P. AGARWAL, S. HARADA, J.O. WHITTAKER, F. ROTHHAMMER and R. LISKER, 1986: Aldehyde dehydrogenase polymorphism in North American, South American and Mexican Indians. Am. J. Hum. Genet., 38: GOEDDE, H.W., G. FRITZE, S. SINGH, D.P. AGARWAL and D. MEIER-TACKMAN, 1990: Genotypes of ADH and ALDH. Population genetic and family studies in Mongoloids, Negroids and Caucasoids and relationship to alcohol metabolism. Alcoholism: Clin. Exp. Res., 14: 293. GOEDDE, H.W., S. SINGH, D.P. AGARWAL, G. FRITZE, K. STAPEL and Y.K. PAIK, 1989: Genotyping of mitochondrial aldehyde dehydrogenase in blood samples using allele-specific oligonucleotides: comparison with phenotyping in hair roots. Hum. Genet., 81: GREENFIELD, N.J. and R. PIETRUSZKO, 1977: Two aldehyde dehydrogenases from human liver: Isolation via affinity chromatography and characterization of the isozymes. Biochem. Biophys. Acta, 483: HARADA, S.,1989: Polymorphism of aldehyde dehydrogenase and its application to alcoholism. Electrophoresis, 10: HARADA, S., 1990: Genetic polymorphism of aldehyde dehydrogenase and its physiological significance to alcohol metabolism. In: OGGITA, Z. and L.C. MARKERT (eds.), Isozymes, Structure, Function and Use in Biology and Medicine. Wiley-Liss, New York, pp HARADA, S, D.P. AGARWAL and H.W. GOEDDE, 1978a: Human liver alcohol dehydrogenase isozyme variations. Improved separation methods using prolonged high voltage starch gel electrophoresis and sioelectric focusing. Hum. Genet., 40: HARADA, S., D.P. AGARWAL and H.W. GOEDDE, 1978b: Isozyme variations in acetaldehyde dehydrogenase (EC ) in human tissues. Hum. Genet., 44: HARADA, S., D.P. AGARWAL and H.W. GOEDDE, 1980a: Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues. Life Sci., 26: HARADA, S., S. MISAWA, D.P. AGARWAL and H.W. GOEDDE, 1980b: Liver alcohol and aldehyde dehydrogenase in the Japanese: Isozyme variation and its possible role in alcohol intoxication. Am. J. Hum. Genet., 32: HARADA, S., D.P. AGARWAL and H.W. GOEDDE, 1981: Aldehyde dehydrogenase deficiency as cause of facial flushing reaction to alcohol in Japanese. Lancet, ii: 982. HARADA, S., D.P. AGARWAL and H.W. GOEDDE, 1982a: Mechanism of alcohol sensitivity and desulfiram-ethanol reaction. Substance Alcohol Actions/Misuse, 3: HARADA, S., D.P. AGARWAL, H.W. GOEDDE, S. TAGKAKI and B. ISHIKAWA, 1982b: Possible protective role against alcoholism for aldehyde dehydrogenase isozyme deficiency in Japan. Lancet, ii: 827. HARADA, S., D.P. AGARWAL and H.W. GOEDDE, 1985: Aldehyde dehydrogenase polymorphism and alcohol metabolism in alcoholics. Alcohol, 2: HEMPEL, J., B. HOLMPUIST, L. FLEETWOOD, R. KAISER, J. BARROS-SODERLING, R. BUHLER, B. VALLE and H. JORNVALL, 1985a: Structural relationship among class I isozymes of human liver alcohol dehydrogenase. Biochemistry, 24: HEMPEL, J., R. KAISER and H. JORNVALL, 1984: Human liver mitochondrial aldehyde dehydrogenase: A C-terminal segment positions and defines the structure corresponding to the one reported to differ in the Oriental enzyme variant. FEBS Letter, 173: HEMPEL, J., R. KAISER and H. JORNVALL, 1985b: Mitochondrial aldehyde dehydrogenase from human liver: Primary structure, differences in relation to the cytosolic enzyme and functional correlation. Eur. J. Biochem., 153: HOOG, J-O., H. von BAHR-LINDSTROM and L-O. HEDEN, 1987: Structure of the class II enzyme of human liver alcohol dehydrogenase. Combined cdna

16 138 S. HARADA and protein sequence determination of the * subunit. Biochemistry, 26: HSU, L.C., RE. BENDEL and A. YOSHIDA, 1987: Direct detection of usual and atypical alleles on the human aldehyde dehydrogenase2 locus. Am. J. Hum. Genet., 41: HSU, L.C., R.E. BENDEL and A. YOSHIDA, 1988: Genomic structure of human alcohol mitochondrial aldehyde dehydrogenase gene. Genomics, 2: HSU, L.C., K. TANI, T. FUJIYOSHI, K. KARACHI and A. YOSHIDA, 1985: Cloning of cdnas for human aldehyde dehydrogenasesl and 2. Proc. Natl. Acad. Sci. USA, 82: HSU, L.C., A. YOSHIDA and T. MOHANDAS, 1986: Chromosomal assignment of the genes for human aldehyde dehydrogenase-1 and aldehyde dehydrogenase-2. Am. J. Hum. Genet., 38: IMPRAIM, C., G. WANG and A. YOSHIDA, 1982: Structural mutation in a major human aldehyde dehydrogenase gene results in loss of enzyme activity. Am. J. Hum. Genet., 34: JENKINS, W.J. and T.J. PETERS, 1983: Subcellular localization of acetaldehyde dehydrogenase in human liver. Cell. Biochem. Funct., 1: JORNVALL, H., J. HEMPEL and B. VALLEE, 1987: Structures of human alcohol and aldehyde dehydrogenases. Enzyme, 37: JORNVALL, H., J. HEMPEL, B.L., VALLEE, W.F. BOSRON and T-K. LI, 1984: Human liver alcohol dehydrogenase: amino acid substitution in the *2*2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme. Proc. Natl. Acad. Sci. USA, 81: KOIVULA, T., 1975: Subcellular distribution and characterization of human liver aldehyde dehydrogenase fraction. Life Sci., 16: LIEBER, C.S., 1977: Metabolism of ethanol, In: LIEBER, C.S. (ed.) Metabolic Aspects of Alcoholism. University Park Press, Baltimore, pp LIEBER, C.S. and L.M. DECARLI, 1972: The role of hepatic microsomal ethanol oxidizing system (MEOS) for ethanol metabolism in vivo. J. Pharmacol. Exp. Ther., 181: MEIER-TACKMANN, D., G.C. KORENKE, D.P. AGARWAL and H.W. GOEDDE, 1988: Aldehyde dehydrogenase isozymes: Subcellular distribution in livers from alcoholics and healthy subjects. Alcohol, 5: MIWA, G.T., W. LEVIN, P. THOMAS and A.Y.H. LU, 1978: The direct oxidation of ethanol by catalase- and alcohol dehydrogenase-free reconstituted system containing cytochrome p-450. Arch. Biochem. Biophys., 178: 464. MIZOI, Y., I. IJIRI, T. TATSUNO, T. KIJIMA, S. FUJIWARA and J. ADACHI, 1979: Relationship between facial flushing and blood acetaldehyde levels after alcohol intake. Pharmacol. Biochem. Behav., 16: MIZOI, Y., M. KOGAME, T. FUKUNAGA, Y. UENO, J. ADACHI and S. FUZIWARA,1985: Polymorphism of aldehyde dehydrogenase and ethanol elimination. Alcohol, 2: MIZOI, Y., Y. TATSUNO, I. ADACHI, M. KOGAME, T. FUKUNAGA, S. FUJIWARA, S. HICHIDA and I. IJIRI, 1983: Alcohol sensitivity related to polymorphism of alcohol-metabolizing enzymes in Japanese. Pharmacol. Biochem. Behav., 18: O'DOWD, B.F., F. ROTHHAMMER and Y. ISRAEL, 1990: Genotyping of mitochondrial aldehyde dehydrogenase in hair root samples of native American Indians. Alcoholism: Clin. Exp. Res., 14: 321. PARES, X. and B.L. VALLEE, 1981: New human liver alcohol dehydrogenase forms with unique kinetic characteristics. Biochem. Biophys. Res. Comm., 98: PEIETRUSZKO, R., 1980: Alcohol and aldehyde dehydrogenase isozymes from mammalian liver - their structural and functional differences. In: RATTAZZI, M.C., J.G. SCANDALIOS and G.S. WHITT (eds.) Isozymes: Current Topics in Biological and Medical Research. Vol. 4, Alan R. Liss Inc., New York, pp PIETRUSZKO, R., N.J. GREENFIELD and C.R. EDSON, 1977: Human liver aldehyde dehydrogenase. In: THURMAN, R., J.R. WILLIAMSON, H.R. DROTT and B. CHANCE (eds.) Alcohol and Aldehyde Metabolizing Systems. Vol II, Academic Press, New York, pp PIETRUSZKO, R., M.T. RYZLAK and C.M. FORTE- MCROBBIE, 1978: Multiplicity and identity of human aldehyde dehydrogenases. Alcohol & Alcoholism, Suppl., 1: REED, T.E., H. KALANT, R.J. GIBBINS and B.M. HANNA, 1976: Alcohol and acetaldehyde metabolism in Caucasians, Chinese and Amerinds. Can. Med. Assoc. J., 115: REX, D.K., W.F. BOSRON, J.E. SMIALEK and T.K. LI, 1985: Alcohol and aldehyde dehydrogenase isoenzymes in North American Indians. Alcoholism: Clin. Exp. Res., 9: SAIKI, R.K., S. SCHARF, F. FALOONA, K.B. MULLIS, G.T. HORN, A.H. ERLICH and N. ARNHEIM,1985: Enzymatic amplification of *-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230: SAIKI, R.K., D.H. GELFAND, S. STOFFEL, S.J. SCHARF, R. HIGUCHI, G.T. HORN, K.B. MULLIS and H.A. ERLICH, 1988: Primer directed enzymatic

17 Genetic Polymorphism of Alcohol Metabolyzing Enzymes 139 amplification of DNA with a thermostabile DNA polymerase. Science, 239: SANTISTEBAN, I., S. POVEY, L.F. WEST, J.M. PARRINGTON and D.A. HOPKINSON, 1985: Chromosome assignment, biochemical and immunological studies on a human aldehyde dehydrogenase, ALDH3. Ann. Hum. Genet., 49: SCHULZ, W., S. KREUZBERG, H.G. NEYMEYER, U. SCHWARZ and A. PACHALY, 1976: Uber die Haufigkeit der atypischen ADH in Leverbiopsiematerial und den Einflul* auf den Athanolumsatz in vivo. Krimialistik and forensische Wissenschaften, 26: SINGH, S., G. FRITZE, B. FANG, S. HARADA, Y.K. PARK, R. ECKEY, D.P. AGARWAL and H.W. GOEDDE, 1989: Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese, Japanese and South Korean families reveals dominance of the mutant allele. Hum. Genet., 83: SMITH, M., 1986: Genetics of human alcohol and aldehyde dehydrogenases. In: HARRIS, H. and K. HIRSHHORN (eds.) Advances in Human Genetics. vol. 15, Plenum Press, New York, pp SMITH, M., D.A. HOPKINSON and H. HARRIS, 1971: Developmental changes and polymorphisms in human alcohol dehydrogenase. Ann. Hum. Genet., 34: SMITH, M., D.A. HOPKINSON and H. HARRIS, 1972: Alcohol dehydrogenase isoenzymes in adult human stomach and liver: Evidence for activity of the ADH3 locus. Ann. Hum. Genet., 35: SMITH, M., D.A. HOPKINSON and H. HARRIS, 1973: Studies on the subunit structure and molecular size of the human alcohol dehydrogenase isozymes determined by the different loci ADH1, ADH2, ADH3. Ann. Hum. Genet., 36: STAMATOYANNOPOULAS, G., S.H. CHEN and F. FUKUI, 1975: Liver alcohol dehydrogenase in Japanese: High population frequency of atypical form and its possible role in alcohol sensitivity. Am. J. Hum. Genet., 27: SUWAKI, H. and H. OHARA, 1985: Alcohol-induced facial flushing and drinking behavior in Japanese men. J. Stud. Alcohol., 46: TENG, Y.S., S. JEHAN and L.E. LIE-INJO, 1979: Human alcohol dehydrogenase ADH2 and ADH3 polymorphism in ethnic Chinese and Indians of West Malaysia. Hum. Genet., 53: TESCHKE, R., S. MATSUZAKI, K. OHNISHI, L.M. DECARLL and C.S. LIEBER, 1977: Microsomal ethanol oxidizing system (MEOS): Current status of its characterization and its role. Alcoholism: Clin. Exp. Res., 1: VON WARTBURG, J.P., J. PAPENBERG and H. AEBI, 1965: An atypical human alcohol dehydrogenase. Canad. J. Biochem., 43: VON WARTBURG, J.P. and P.M. SCHURCH, 1968: Atypical human liver alcohol dehydrogenase. Ann. NY. Acad. Sci. USA, 151: WILSON, J.R., G.E. MCCLEAN and R.C. JOHNSON, 1978: Ethnic variation in the use and effects of alcohol. Drug Alcohol Depend., 3: WOLFF, P.H., 1972: Ethnic differences in alcohol sensitivity. Science, 175: WOLFF, P.H., 1973: Vasomotor sensitivity to alcohol in diverse Mongoloid populations. Am. J. Hum. Genet., 25: XU, Y., L.G. CARR, W.F. BOSRON, T.K. LI and H.J. EDENBERG, 1988: Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification. Genomics, 2: YOSHIDA, A., I-YIH. HUANG and M. IKAWA, 1984: Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals. Proc. Natl. Acad. Sci. USA, 81: ZEINER, A.R., A. PAREDES and D. CHRISTIANSEN, 1979: The role of acetaldehyde in mediating reactivity to an acute dose of ethanol among different racial groups. Alcoholism: Clin. Exp. Res., 3: Shoji HARADA Institute of Community Medicine, University of Tsukuba Tennodai 1-1-1, Tsukuba 305, Japan