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Genetic polymorphisms of alcohol metabolizing enzymes
Pathol Biol 2001 ; 49 : 703-9 2001 Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0369-8114(01)00242-5/FLA
Original article
Genetic polymorphisms of alcohol metabolizing enzymes D.P. Agarwal ∗ Institute of human genetics, university of Hamburg, Butenfeld 42, 22529 Hamburg, Germany (Received 10 July 2000; accepted 2 November 2000)
Summary Alcohol metabolism is one of the biological determinants that can significantly influence drinking behavior and the development of alcoholism and alcohol-induced organ damage. Most ethanol elimination occurs by oxidation to acetaldehyde and acetate, catalyzed principally by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Other ethanol oxidation pathways, including catalase and microsomal ethanol-oxidizing system (MEOS/CYP2E1), as well as the nonoxidative pathway (FAEES), which forms fatty acid ethyl esters, appear to play a minor role. The major alcohol metabolizing enzymes exhibit genetic polymorphism and ethnic variation. In this review recent advances in the understanding of the functional polymorphisms of ADH, ALDH and CYP2E1 and their metabolic, physiologic and clinical correlations are presented. 2001 Éditions scientifiques et médicales Elsevier SAS ADH / alcohol metabolism / ALDH / CYP2E1 / genetic polymorphism
Résumé – Les polymorphismes génétiques des enzymes du métabolisme de l’alcool. Le métabolisme de l’alcool est l’un des déterminants biologiques qui peut significativement avoir une influence sur le comportement d’alcoolisation, sur le développement de l’alcoolisme et sur les dommages de l’alcool au niveau des organes. L’éthanol est en majeure partie éliminé grâce à son oxydation en acétaldéhyde puis en acétate, ces réactions étant catalysées principalement par l’alcool deshydrogénase (ADH) et l’aldéhyde deshydrogénase (ALDH). D’autres voies d’oxydation de l’éthanol, telles que la catalase et le système microsomal d’oxydation de l’éthanol (MEOS/CYP2E1), de même que la voie non oxydative (FAEES) qui produit des esters éthyliques d’acide gras, semblent jouer un rôle mineur. La plupart des enzymes du métabolisme de l’alcool présentent un polymorphisme génétique et une variation ethnique. Cette revue présente les avancées récentes dans la compréhension des polymorphismes fonctionnels de l’ADH, de l’ALDH et du CYP2E1 ainsi que de leurs corrélations métaboliques, physiologiques et cliniques. 2001 Éditions scientifiques et médicales Elsevier SAS ADH / ALDH / CYP2E1 / métabolisme de l’alcool / polymorphisme génétique
Recent molecular genetic research into the causes of alcoholism has drawn attention to the potential important role of alcohol and acetaldehyde metabolizing enzymes. Functional polymorphisms have been observed at various genes encoding these enzyme proteins which all act to alter the rate of synthesis of the toxic metabolite acetaldehyde, or decrease its further oxidation. A positive selection of such genetic polymorphisms in some popula-
∗ Correspondence and reprints.
E-mail address: [email protected] (D.P. Agarwal).
tions might act as a protective factor against alcohol abuse and alcohol-related disease outcomes. Thus, elucidation of the molecular mechanisms that control and influence elimination and metabolism of alcohol is important in understanding the biochemical basis of alcohol toxicity and alcohol abuse-related pharmacological and addictive consequences in humans. The enzymatic pathways responsible for ethanol metabolism and their genetic as
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well as environmental determinants have been the focus of detailed investigations in the past decades. Identification of structural gene variants and the characterization of putative alcoholism vulnerability genes may help in improving preventive and treatment approaches. In humans, more than 90% of the ingested ethanol is degraded in the liver by oxidative and non-oxidative metabolic pathways. The major enzymes involved in the metabolism of ethanol are alcohol dehydrogenases (ADH), aldehyde dehydrogenase (ALDH) and cytochrome P450 (CYP2E1). In addition, catalase and fatty acid ethyl ester synthase (FAEES) are also considered to be involved in ethanol degradation. This short review presents current knowledge about the genetic polymorphisms of alcohol metabolizing enzymes and their functional role.
ALCOHOL DEHYDROGENASES (ADH) The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Human ADH is a dimeric protein consisting of two subunits with a molecular weight of 40 kD each. At least seven different genetic loci code for human ADH arising from the association of different types of subunits. Over 20 ADH isoenzymes are known which vary in their pharmacokinetic properties: – the types of alcohols they preferentially oxidize; – the amount of alcohol that has to accumulate before appreciable degradation occurs; – the maximal rate at which they oxidize alcohol. As presented in table I, various human ADH forms can be divided into five major classes or distinct groups (I–V) according to their subunit and isoenzyme composition as well as their physicochemical properties [1].
Class I ADH The class I ADH isoenzymes are formed by random dimeric association of any of the three types of polypeptide subunits, α, β and γ controlled by three separate gene loci, ADH1, ADH2, and ADH3, respectively. These isoenzymes belong to the low Km (< 4 mM) forms, and are considered to play a major role in ethanol metabolism. The molecular characterization of ADH2*1 allele shows that 9 exons are stretched over 15 kilobases (kb) in length. The complete nucleotide sequence of all 9 exons of an ADH2*2 allele, which encodes for the β2β2 isoenzyme has been determined using clones from a human genomic DNA library. The nucleotide sequence data indicate that the CGC/CAC substitution, responsible for the arginine/histidine exchange, is the only nucleotide polymorphism detected between the coding regions of ADH2*1 and ADH2*2 alleles.
Class II ADH Class II ADH, encoded by the ADH4 gene, is composed of π subunits. It exhibits a high catalytic efficiency for oxidation of long-chain aliphatic and aromatic alcohols (Km = 18–34 mM), migrates more anodically than the class I isoenzymes on starch gel electrophoresis, and is far less sensitive to pyrazole inhibition. Hence it is called π (pi, pyrazole insensitive) ADH.
Class III ADH Several ADH activity bands migrating anodically in starch gel electrophoresis and showing enzyme-specific staining activity only with medium chain alcohols (> 4 carbons) as substrate have been characterized. This class of ADH enzyme (Chi or χ -ADH) is encoded by the ADH5 gene. It has a very high Km for ethanol (> 3 M), but exhibits high activity for oxidation of long-chain alcohols. Chi-ADH is a zinc-containing dimeric enzyme responsible for the oxidation of long-chain alcohols and omega-hydroxyfatty acids. Recent evidence suggests that class-III ADH and formaldehyde dehydrogenase (FDH) are the same enzyme. ADH5 is composed of nine exons and eight introns.
Class IV ADH Human stomach mucosa contains an ADH isoenzyme (sigma, σ σ subunits) which is moderately active with ethanol and exhibits kinetic properties, electrophoretic mobility, isoelectric point, and structural characteristics different from those previously described for other ADHs [2]. This form is now recognized as a class IV enzyme encoded by ADH7 gene. The ADH7 gene was cloned from a Caucasian genomic DNA library and characterized [3]. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of sigma ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs.
Class V ADH A recently cloned ADH6 gene was found in liver and stomach, and an active enzyme has been expressed in Escherichia coli [4]. However, its expressed protein product has not yet been identified in human tissues.
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Table I. Human ADH genes. Class
Gene
Subunit
Dimeric isozymes
Chromosomal location
ADH1
ADH1* 1
α
αα, αβ1, αβ2, αγ1, αγ2
4q22
ADH2
ADH2* 1
β1
β1β1, β1β2, β1γ1, β1γ2
4q22
ADH* 2
β2
β2β2, β2γ1, β2γ2
ADH2* 3
β3
β3β3
ADH3* 1
γ1
γ1γ1, γ1γ2
ADH3* 2
γ2
γ2γ2, γ1γ2
ADH4
π
ππ
4q21-25
ADH5
χ
χχ
4q21-25
ADH7
σ
σσ
4q23-24
ADH6
?*
?*
4q21-25
Class I ADH
ADH3
4q22
Class II ADH ADH4 Class III ADH ADH5 Class IV ADH ADH7 Class V ADH ADH6
* Subunit composition not known.
MOLECULAR GENETICS OF ADH All ADH subunits consist of 374 amino acid residues and have about 10% total amino acid exchanges [4]. The β subunit differs from the α subunit at 24 amino acids and the α subunit differs from the γ at 20 residues. For class I ADH, only at three sequence positions, 143, 319 and 327, do all the three subunits differ from each other. The degrees of exchanges in the α, β and γ subunits are very similar suggesting separate but comparatively recent duplicatory diversions. Amino acid sequence data show that the “atypical” subunit (ADH2 locus) from Caucasian and Oriental livers is identical, but differs from the “typical” β1 subunit by a single amino acid exchange, Arg to His at 47th position due to a G/C to A/T base transition in exon 3. This arginine/histidine-47 mutational difference has been found to be responsible for the altered catalytic and functional properties including both a lower pH optimum and a higher turnover number of the atypical enzyme. The substitution in the β3 isoenzyme consists of a single amino acid exchange at position 369, Arg being replaced by Cys generated by C/G to T/A base transition in exon 9. Amino acid sequence analysis of the γ1 and γ2 subunits showed that both have the same sequence, Cys-Arg-Ser at their positions 46–48 but have two replacements at positions 271 (Arg/Gln) and 349 (Ile/Val).
Genetic polymorphisms Any one particular class I ADH isoenzyme may be composed of homodimeric subunits, consisting of two identical polypeptides (e.g., αα, ββ, γγ) coded by a specific allele at one of the loci, or heterodimeric subunits consisting of two non-identical polypeptides (e.g. αβ, αγ) coded by alleles at separate loci, or heterodimeric subunits coded by different alleles at the same locus (e.g., β1β2, γ1γ2). So far no allelic polymorphism has been reported in human populations for the α subunit of ADH1 (class I ADH), π-ADH (class II ADH) and χ ADH (class III ADH). While about 5–10% of the English, 9–14% of the German, and 20% of the Swiss population have been found to possess an “atypical” phenotype of ADH2 (allelic variant at ADH2 gene locus containing β2 subunit), this variant form occurs in at least 85% of the Japanese, Chinese, and other Oriental populations [5]. The frequency of the variant forms of ADH3 locus is relatively higher in Caucasians than in Oriental and African populations. In table II, the molecular basis of polymorphic changes in various ADH alleles are summarized.
CHROMOSOME MAPPING OF THE ADH GENE LOCI Analysis of DNA from hybrids containing fragments of human chromosome 4 has provided evidence that all
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Table II. Polymorphism of ADH genes. Allele
Subunit
ADH1* 1
α
Nucleotide change
Wild type
Effect on protein
Exon
ADH2* 1
β1
Wild type
ADH* 2
β2
47G > A
Arg > His
3
ADH2* 3
β3
369C > T
Arg > Cys
9
ADH3* 1
γ1
ADH3* 2
γ2
271C > T; 349G > A
Arg > Gln; Ile > Val
8
ADH4
π
−192; −159; −75
Reduced activity
Promoter
Wild type
ADH5
χ
Wild type
ADH6
?*
?*
ADH7
σ
Wild type
?*
* Gene product not exactly known.
classes of ADH genes are located on the long arm of human chromosome 4 between 4q21 and 4q25.
Physiological role The role of ADH in alcoholism and alcohol-related disorders has not been fully understood so far. Altered structures of the genes encoding for various ADH isoenzymes could potentially be the cause of observed genetic differences in alcoholics and nonalcoholics. Although the rate of ethanol metabolism has been found to be similar in alcoholics and nonalcoholics, elevated serum ADH activity has been noted in alcoholics with liver damage and other gastrointestinal disorders [5]. Variants at both ADH2 and ADH3 genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls [6]. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) they found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2.
MICROSOMAL ETHANOL OXIDIZING SYSTEM (MEOS) A small but significant part of the ingested ethanol (10% or less) is metabolized by alternative pathways. The microsomal metabolism of ethanol accounts for the major non-ADH pathway of alcohol oxidation in the liver [7]. The cytochrome P450 isoform, P4502E1 (CYP2E1), represents the major alternative system which metabolizes alcohol in the liver. After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases, with an associated rise in cytochromes
P-450, especially CYP2E1. Enhanced ethanol oxidation is associated with cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, there is increased conversion of known hepatotoxic agents (such as CCl4 ) to toxic metabolites, which may explain the enhanced susceptibility of alcoholics to the adverse effects of industrial solvents. CYP2E1 also has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis (e.g., from dimethylnitrosamine). Both ethanol-induced and enhanced transcriptional activity of the polymorphic form of CYP2E1 (c2/c2) might play a role in the development of severe alcoholic liver disease [8].
POLYMORPHIC ALLELES OF CYP2E1 Polymorphisms in various CYP2E1 genes are summarised in table III. A RsaI restriction fragment length polymorphism (RFLP) has been reported in the 5 -flanking region of the CYP2E1 gene. The rare mutant allele (c2 allele) that lacks the RsaI restriction site has been found to be associated with higher transcriptional activity, protein levels and enzyme activity than the common wild-type c1 allele. Moreover, the frequency of RsaI c2 allele varies in different populations [9]. The highest frequency has been observed in the Taiwanese (0.28) and Japanese (0.19–0.27), while the frequency is much lower (ranging between 0.01 and 0.05) in African-Americans, European Americans, and Scandinavians.
Catalase A third system with the potential to oxidize alcohols in human body is the enzyme catalase which appears to act only at high concentrations of alcohol. However, its overall role in ethanol elimination is unclear.
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Table III. Polymorphisms of CYP2E1 gene. Allele
Protein
Nucleotide changes
CYP2E1*1A
CYP2E1.1
None
CYP2E1*1B
CYP2E1.1
9893C > G
Effect
RFLP
TaqI-
CYP2E1*1C
CYP2E1.1
6 repeats in the 5 flanking region
CYP2E1*1D
CYP2E1.1
8 repeats in the 5 flanking region
CYP2E1*2
CYP2E1.2
1132G > A
R76H
CYP2E1*3
CYP2E1.3
10023G > A
V389I
CYP2E1*4
CYP2E1.4
4768G > A
V179I
DraI−+XbaI
CYP2E1*5A
CYP2E1.1
−1293G > C; −1053C > T; 7632T > A
PstI+RsaI− DraI−
CYP2E1*5B
CYP2E1.1
−1293G > C; −1053C > T
PstI+RsaI−
CYP2E1*6
CYP2E1.1
7632T > A
DraI−
CYP2E1*7A
CYP2E1.1
261T > A
CYP2E1*7B
CYP2E1.1
−71G > T; 261T > A
CYP2E1*7C
CYP2E1.1
261T > A; 280G > A
Fatty acid ethyl ester synthases (FAEES) Ethanol can condense with free fatty acids in a reaction catalyzed by FAEES to form abnormal compounds such as fatty acid ethyl esters. Three different synthases have been detected and these are designated as synthase I, II, III and IV. Each of these enzymes has been purified and characterized and several of them have been purified to homogeneity from the human myocardium [10]. The observation that FAEES are synthesized at high rates in the heart, and other organs that lack oxidative ethanol metabolism, provides a plausible link between the observed tissue damage, the ingestion of alcohol, and the subsequent development of alcohol-induced heart muscle disease [10].
ALDEHYDE DEHYDROGENASES Acetaldehyde is the first metabolic product of enzymatic ethanol oxidation in human liver, and is far more toxic than the parent compound ethanol. The major oxidation of acetaldehyde in the liver and other organs is catalyzed by the NAD+ -dependent aldehyde dehydrogenase (ALDH, aldehyde: NAD+ oxidoreductase, EC 1.2.1.3). A number of isoenzymes of ALDH coded by different gene loci have been detected in human organs and tissues which differ in their electrophoretic mobility, kinetic properties, as well as in their cellular and tissue distribution. Human ALDH are divided into nine major gene families (table IV). Members of the family 1 are cytoplasmic ALDHs (ALDH1), members of the family 2 are mitochondrial ALDHs (ALDH2), and members of the family 3 are the major constitutive and inducible high Km ALDH forms (ALDH3) found in rat and mouse tissue such as stomach,
Table IV. Human ALDH genes* . Gene
Trivial name
Chromosomal localization
ALDH1A1
ALDH1
9q21
ALDH1A6
ALDH6
15q26
ALDH1B1
ALDH5
9p13
ALDH2
ALDH2
12q24.2
ALDH3A1
ALDH3
17p11.2
ALDH3A2
ALDH10
17p11.2
ALDH3B1
ALDH7
11q13
ALDH3B2
ALDH8
11q13
ALDH4A1
ALDH4
1
ALDH5A1
SSDH
6p22
ALDH6A1
MMSDH
?
ALDH7A1
ATQ1
5q31
ALDH8A1
ALDH12
6q241.1-25
ALDH9A1
ALDH9
1q22-q23
* Adapted from [12].
SSDH: succinic semialdehyde dehydrogenase; MMSDH: methylmalonic acid semialdehyde dehydrogenase; ATQ1: antiquitin.
lung, liver, cornea as well as in human stomach, saliva, and hepatocarcinoma. Various ALDH isoenzymes show a broad range of substrate specificity for aliphatic and aromatic aldehydes. Whereas ALDH1 and ALDH2 both show Km values in the micromolar range with acetaldehyde and propionaldehyde, the Michaelis constants for ALDH3 and ALDH4 are in the millimolar range for these substrates. NAD+ is the preferred coenzyme for the low Km isoenzymes (ALDH1 and ALDH2), whereas the high Km isoenzymes
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D.P. Agarwal
(ALDH3 and ALDH4) can use either NAD+ or NADP+ . The major human liver ALDH1 and ALDH2 isoenzymes are homotetramers consisting of equal but isoenzymespecific subunits with a MW of about 54 kD each.
Additional ALDH isoenzymes As compiled in table IV, in addition to the three nonallelic genes, cytosolic ALDH1A1 (ALDH1), mitochondrial ALDH2, and the cytosolic stomach-specific ALDH3A1 (ALDH3), a number of additional genes have been cloned and characterized in humans [11]: – ALDH1B (ALDH5): a new functional ALDH gene has been identified and has been expressed in variuos tissues including liver, brain, adrenal gland, testis, stomach, and parotid gland; – ALDH1A6 (ALDH6): this isoenzyme is primarily expressed in the salivary gland, stomach, and kidney. The cDNA encodes 512 amino acid residues. A 70% sequence homology was observed with ALDH1. The gene spans about 37 kilobase pairs (kbp) in length and consists of 13 exons. The gene was assigned to chromosome 15q26; – ALDH3B1 (ALDH7): the gene codes for the isoenzyme expressed mainly in the kidneys and lungs, and the cDNA encodes 468 residues. There is a 52% positional identity with ALDH3. It consists of ten exons spanning 20 kbp. The ALDH7 gene was assigned to chromosome 11q13; – ALDH3B2 (ALDH8): the deduced amino acid sequence of the gene product is very similar to that of the ALDH7 (85% homology). The gene spans about 30 kbp and consists of ten exons. It has been assigned to chromosome 11q13; – ALDH9A1 (ALDH9): the cDNA and the gene for a human aldehyde dehydrogenase isozyme, which has a high activity for oxidation of gamma-aminobutyraldehyde and other amino aldehydes have been cloned and characterized. The gene is about 45 kb and consists of ten coding exons interrupted by nine introns. The gene was assigned to chromosome 1q22-q23, using fluorescence in situ hybridization. The gene is polymorphic, i.e., C or T at nt 327 and C or G at nt 344.
Polymorphisms of ALDH genes Genetic polymorphisms have beeen reported in a number of ALDH genes. In table V, nucleotide changes and effect on encoded proteins are compiled. Naming of the human alleles is based on the new nomenclature [12].
Polymorphism of ALDH2 The single genetic factor most strongly correlated with reduced alcohol consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The
Table V. Polymorphism of ALDH genes* . Allele symbol
Nucleotide change
ALDH1B1*1
Effect on protein
Exon
Wild type
2
ALDH1B1*2
257C > T
V69A
2
ALDH1B1*3
320T > G
R90L
2
ALDH1B1*4
183C > T
Silent
2
ALDH2*1
Wild type
ALDH2*2
1510G > A
E487K
12
ALDH2*3
1486G > A
E479K
12
ALDH2*4
1464G > A
Silent
12
ALDH3A1*1
Wild type 985C > G
P329A
ALDH3A2*2
521delT
Frameshift
4
ALDH3A2*3
808delG
Frameshift
6
ALDH3A2*4
941del3,
A314G, P315A,
7
94lins21
AKSTVG313ins
ALDH3A2*5
641G > A
G214Y
4
ALDH3A2*6
1297delGA
Frameshift
9
ALDH3A2*7
1311linsACAAA
Frameshift
9
ALDH3A2*8
1297delGA
Frameshift
9
ALDH3A1*2 ALDH3A2*1
9
Wild type
ALDH4A1*1
Wild type
ALDH4A1*2
21delG
ALDH4A1*3
1055C > T
S352L
§
ALDH4A1*4
47C > T
P16L
§
ALDH4A1*5
1563insT
Frameshift
§
ALDH9A1*1
Frameshift
§
Wild type
ALDH9A1*2
344G > C
C115S
2
ALDH9A1*3
327C > T
Silent
2
§ Gene structure not known yet. * Adapted from [12].
E487K variant of ALDH2 is found in approximately 50% of the Asian population, and is associated with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes [5, 13]. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. Functionally, a single base mutation at this position, resulting in loss of the catalytic activity, is compatible with the proximity in the primary structure between this region and the segment that contains cysteine residues.
Population distribution of ALDH2 variants Oriental populations of Mongoloid origin widely show the presence of the inactive ALDH2 isoenzyme phenotype whereas none of the Caucasian or Negroid
Genetic polymorphisms of alcohol metabolizing enzymes
populations have this isoenzyme abnormality [14]. Among native Indians, about 40% of the South American Indian tribes (Mapuche, Atacamen’s, Shuara) showed the presence of the deficient type of ALDH2. However, this variant isoenzyme was detected only in a very small percentage of the North American Indians (Sioux, Navajo and Mestizo). More recent genotyping data hint to a considerable genetic heterogeneity in the distribution of ALDH2*2 gene in American Indian and Central Asian populations [14, 15].
Polymorphism of ALDH3 The stomach ALDH3 isoenzyme band, focusing at about pH 6.0, displays several activity bands [2]. Three polymorphisms of ALDH3 (variants I, II, and III) were also detected in human saliva and hair root extracts.
Physiological role The physiological significance of ALDH relates mainly to its role in the detoxification of acetaldehyde and other aldehydes which show a variety of toxic effects in human organs and tissues. Many biogenic amines are converted to their corresponding aldehydes via monoamine and diamine oxidase systems. Moreover, in vivo biotransformation of many drugs and xenobiotics that are not aldehydes gives rise to aldehyde metabolites. For example, cyclophosphamide, which is pharmacologically inactive, needs to be biotransformed to its cytotoxic metabolite phosphoramide mustard via an intermediate metabolite 4-hydroxycyclophosphamide. The latter compound exists in equilibrium with aldophosphamide which can get converted to a non-cytotoxic compound (carboxyphosphamide) through irreversible oxidation of the aldehyde group catalyzed by one or more forms of aldehyde dehydrogenases. This enzymatic pathway leads to the detoxification of cyclophosphamide affecting its therapeutic efficiency [16]. More recent studies have focused on the putative role of alcohol metabolizing enzymes in alcohol elimination rate, acute reactions to alcohol, alcohol drinking habits and alcoholism across various ethnic groups [17]. For example, Orientals exhibit intense facial flushing after a mild dose of alcohol as compared to Caucasians affecting their alcohol drinking habits. Quantity-frequency-variability distribution indicates that the percentage of heavy and moderate drinkers is higher among Caucasians, while the percentage of abstainers and infrequent drinkers is higher among the Chinese, Japanese, and other Orientals [18 – 21]. The alcoholism rate as well as alcohol-related end-organ damage is also found to be lower among the Japanese, Chinese, and other Orientals as compared to Caucasian populations living in the Western society [6, 8, 20, 21]. A significantly fewer number of patients with alcoholism and alcoholic liver disease have been found to posssess the inactive ALDH2*2 gene.
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Original article
Genetic polymorphisms of alcohol metabolizing enzymes D.P. Agarwal ∗ Institute of human genetics, university of Hamburg, Butenfeld 42, 22529 Hamburg, Germany (Received 10 July 2000; accepted 2 November 2000)
Summary Alcohol metabolism is one of the biological determinants that can significantly influence drinking behavior and the development of alcoholism and alcohol-induced organ damage. Most ethanol elimination occurs by oxidation to acetaldehyde and acetate, catalyzed principally by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Other ethanol oxidation pathways, including catalase and microsomal ethanol-oxidizing system (MEOS/CYP2E1), as well as the nonoxidative pathway (FAEES), which forms fatty acid ethyl esters, appear to play a minor role. The major alcohol metabolizing enzymes exhibit genetic polymorphism and ethnic variation. In this review recent advances in the understanding of the functional polymorphisms of ADH, ALDH and CYP2E1 and their metabolic, physiologic and clinical correlations are presented. 2001 Éditions scientifiques et médicales Elsevier SAS ADH / alcohol metabolism / ALDH / CYP2E1 / genetic polymorphism
Résumé – Les polymorphismes génétiques des enzymes du métabolisme de l’alcool. Le métabolisme de l’alcool est l’un des déterminants biologiques qui peut significativement avoir une influence sur le comportement d’alcoolisation, sur le développement de l’alcoolisme et sur les dommages de l’alcool au niveau des organes. L’éthanol est en majeure partie éliminé grâce à son oxydation en acétaldéhyde puis en acétate, ces réactions étant catalysées principalement par l’alcool deshydrogénase (ADH) et l’aldéhyde deshydrogénase (ALDH). D’autres voies d’oxydation de l’éthanol, telles que la catalase et le système microsomal d’oxydation de l’éthanol (MEOS/CYP2E1), de même que la voie non oxydative (FAEES) qui produit des esters éthyliques d’acide gras, semblent jouer un rôle mineur. La plupart des enzymes du métabolisme de l’alcool présentent un polymorphisme génétique et une variation ethnique. Cette revue présente les avancées récentes dans la compréhension des polymorphismes fonctionnels de l’ADH, de l’ALDH et du CYP2E1 ainsi que de leurs corrélations métaboliques, physiologiques et cliniques. 2001 Éditions scientifiques et médicales Elsevier SAS ADH / ALDH / CYP2E1 / métabolisme de l’alcool / polymorphisme génétique
Recent molecular genetic research into the causes of alcoholism has drawn attention to the potential important role of alcohol and acetaldehyde metabolizing enzymes. Functional polymorphisms have been observed at various genes encoding these enzyme proteins which all act to alter the rate of synthesis of the toxic metabolite acetaldehyde, or decrease its further oxidation. A positive selection of such genetic polymorphisms in some popula-
∗ Correspondence and reprints.
E-mail address: [email protected] (D.P. Agarwal).
tions might act as a protective factor against alcohol abuse and alcohol-related disease outcomes. Thus, elucidation of the molecular mechanisms that control and influence elimination and metabolism of alcohol is important in understanding the biochemical basis of alcohol toxicity and alcohol abuse-related pharmacological and addictive consequences in humans. The enzymatic pathways responsible for ethanol metabolism and their genetic as
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well as environmental determinants have been the focus of detailed investigations in the past decades. Identification of structural gene variants and the characterization of putative alcoholism vulnerability genes may help in improving preventive and treatment approaches. In humans, more than 90% of the ingested ethanol is degraded in the liver by oxidative and non-oxidative metabolic pathways. The major enzymes involved in the metabolism of ethanol are alcohol dehydrogenases (ADH), aldehyde dehydrogenase (ALDH) and cytochrome P450 (CYP2E1). In addition, catalase and fatty acid ethyl ester synthase (FAEES) are also considered to be involved in ethanol degradation. This short review presents current knowledge about the genetic polymorphisms of alcohol metabolizing enzymes and their functional role.
ALCOHOL DEHYDROGENASES (ADH) The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Human ADH is a dimeric protein consisting of two subunits with a molecular weight of 40 kD each. At least seven different genetic loci code for human ADH arising from the association of different types of subunits. Over 20 ADH isoenzymes are known which vary in their pharmacokinetic properties: – the types of alcohols they preferentially oxidize; – the amount of alcohol that has to accumulate before appreciable degradation occurs; – the maximal rate at which they oxidize alcohol. As presented in table I, various human ADH forms can be divided into five major classes or distinct groups (I–V) according to their subunit and isoenzyme composition as well as their physicochemical properties [1].
Class I ADH The class I ADH isoenzymes are formed by random dimeric association of any of the three types of polypeptide subunits, α, β and γ controlled by three separate gene loci, ADH1, ADH2, and ADH3, respectively. These isoenzymes belong to the low Km (< 4 mM) forms, and are considered to play a major role in ethanol metabolism. The molecular characterization of ADH2*1 allele shows that 9 exons are stretched over 15 kilobases (kb) in length. The complete nucleotide sequence of all 9 exons of an ADH2*2 allele, which encodes for the β2β2 isoenzyme has been determined using clones from a human genomic DNA library. The nucleotide sequence data indicate that the CGC/CAC substitution, responsible for the arginine/histidine exchange, is the only nucleotide polymorphism detected between the coding regions of ADH2*1 and ADH2*2 alleles.
Class II ADH Class II ADH, encoded by the ADH4 gene, is composed of π subunits. It exhibits a high catalytic efficiency for oxidation of long-chain aliphatic and aromatic alcohols (Km = 18–34 mM), migrates more anodically than the class I isoenzymes on starch gel electrophoresis, and is far less sensitive to pyrazole inhibition. Hence it is called π (pi, pyrazole insensitive) ADH.
Class III ADH Several ADH activity bands migrating anodically in starch gel electrophoresis and showing enzyme-specific staining activity only with medium chain alcohols (> 4 carbons) as substrate have been characterized. This class of ADH enzyme (Chi or χ -ADH) is encoded by the ADH5 gene. It has a very high Km for ethanol (> 3 M), but exhibits high activity for oxidation of long-chain alcohols. Chi-ADH is a zinc-containing dimeric enzyme responsible for the oxidation of long-chain alcohols and omega-hydroxyfatty acids. Recent evidence suggests that class-III ADH and formaldehyde dehydrogenase (FDH) are the same enzyme. ADH5 is composed of nine exons and eight introns.
Class IV ADH Human stomach mucosa contains an ADH isoenzyme (sigma, σ σ subunits) which is moderately active with ethanol and exhibits kinetic properties, electrophoretic mobility, isoelectric point, and structural characteristics different from those previously described for other ADHs [2]. This form is now recognized as a class IV enzyme encoded by ADH7 gene. The ADH7 gene was cloned from a Caucasian genomic DNA library and characterized [3]. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of sigma ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs.
Class V ADH A recently cloned ADH6 gene was found in liver and stomach, and an active enzyme has been expressed in Escherichia coli [4]. However, its expressed protein product has not yet been identified in human tissues.
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Table I. Human ADH genes. Class
Gene
Subunit
Dimeric isozymes
Chromosomal location
ADH1
ADH1* 1
α
αα, αβ1, αβ2, αγ1, αγ2
4q22
ADH2
ADH2* 1
β1
β1β1, β1β2, β1γ1, β1γ2
4q22
ADH* 2
β2
β2β2, β2γ1, β2γ2
ADH2* 3
β3
β3β3
ADH3* 1
γ1
γ1γ1, γ1γ2
ADH3* 2
γ2
γ2γ2, γ1γ2
ADH4
π
ππ
4q21-25
ADH5
χ
χχ
4q21-25
ADH7
σ
σσ
4q23-24
ADH6
?*
?*
4q21-25
Class I ADH
ADH3
4q22
Class II ADH ADH4 Class III ADH ADH5 Class IV ADH ADH7 Class V ADH ADH6
* Subunit composition not known.
MOLECULAR GENETICS OF ADH All ADH subunits consist of 374 amino acid residues and have about 10% total amino acid exchanges [4]. The β subunit differs from the α subunit at 24 amino acids and the α subunit differs from the γ at 20 residues. For class I ADH, only at three sequence positions, 143, 319 and 327, do all the three subunits differ from each other. The degrees of exchanges in the α, β and γ subunits are very similar suggesting separate but comparatively recent duplicatory diversions. Amino acid sequence data show that the “atypical” subunit (ADH2 locus) from Caucasian and Oriental livers is identical, but differs from the “typical” β1 subunit by a single amino acid exchange, Arg to His at 47th position due to a G/C to A/T base transition in exon 3. This arginine/histidine-47 mutational difference has been found to be responsible for the altered catalytic and functional properties including both a lower pH optimum and a higher turnover number of the atypical enzyme. The substitution in the β3 isoenzyme consists of a single amino acid exchange at position 369, Arg being replaced by Cys generated by C/G to T/A base transition in exon 9. Amino acid sequence analysis of the γ1 and γ2 subunits showed that both have the same sequence, Cys-Arg-Ser at their positions 46–48 but have two replacements at positions 271 (Arg/Gln) and 349 (Ile/Val).
Genetic polymorphisms Any one particular class I ADH isoenzyme may be composed of homodimeric subunits, consisting of two identical polypeptides (e.g., αα, ββ, γγ) coded by a specific allele at one of the loci, or heterodimeric subunits consisting of two non-identical polypeptides (e.g. αβ, αγ) coded by alleles at separate loci, or heterodimeric subunits coded by different alleles at the same locus (e.g., β1β2, γ1γ2). So far no allelic polymorphism has been reported in human populations for the α subunit of ADH1 (class I ADH), π-ADH (class II ADH) and χ ADH (class III ADH). While about 5–10% of the English, 9–14% of the German, and 20% of the Swiss population have been found to possess an “atypical” phenotype of ADH2 (allelic variant at ADH2 gene locus containing β2 subunit), this variant form occurs in at least 85% of the Japanese, Chinese, and other Oriental populations [5]. The frequency of the variant forms of ADH3 locus is relatively higher in Caucasians than in Oriental and African populations. In table II, the molecular basis of polymorphic changes in various ADH alleles are summarized.
CHROMOSOME MAPPING OF THE ADH GENE LOCI Analysis of DNA from hybrids containing fragments of human chromosome 4 has provided evidence that all
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Table II. Polymorphism of ADH genes. Allele
Subunit
ADH1* 1
α
Nucleotide change
Wild type
Effect on protein
Exon
ADH2* 1
β1
Wild type
ADH* 2
β2
47G > A
Arg > His
3
ADH2* 3
β3
369C > T
Arg > Cys
9
ADH3* 1
γ1
ADH3* 2
γ2
271C > T; 349G > A
Arg > Gln; Ile > Val
8
ADH4
π
−192; −159; −75
Reduced activity
Promoter
Wild type
ADH5
χ
Wild type
ADH6
?*
?*
ADH7
σ
Wild type
?*
* Gene product not exactly known.
classes of ADH genes are located on the long arm of human chromosome 4 between 4q21 and 4q25.
Physiological role The role of ADH in alcoholism and alcohol-related disorders has not been fully understood so far. Altered structures of the genes encoding for various ADH isoenzymes could potentially be the cause of observed genetic differences in alcoholics and nonalcoholics. Although the rate of ethanol metabolism has been found to be similar in alcoholics and nonalcoholics, elevated serum ADH activity has been noted in alcoholics with liver damage and other gastrointestinal disorders [5]. Variants at both ADH2 and ADH3 genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls [6]. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) they found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2.
MICROSOMAL ETHANOL OXIDIZING SYSTEM (MEOS) A small but significant part of the ingested ethanol (10% or less) is metabolized by alternative pathways. The microsomal metabolism of ethanol accounts for the major non-ADH pathway of alcohol oxidation in the liver [7]. The cytochrome P450 isoform, P4502E1 (CYP2E1), represents the major alternative system which metabolizes alcohol in the liver. After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases, with an associated rise in cytochromes
P-450, especially CYP2E1. Enhanced ethanol oxidation is associated with cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, there is increased conversion of known hepatotoxic agents (such as CCl4 ) to toxic metabolites, which may explain the enhanced susceptibility of alcoholics to the adverse effects of industrial solvents. CYP2E1 also has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis (e.g., from dimethylnitrosamine). Both ethanol-induced and enhanced transcriptional activity of the polymorphic form of CYP2E1 (c2/c2) might play a role in the development of severe alcoholic liver disease [8].
POLYMORPHIC ALLELES OF CYP2E1 Polymorphisms in various CYP2E1 genes are summarised in table III. A RsaI restriction fragment length polymorphism (RFLP) has been reported in the 5 -flanking region of the CYP2E1 gene. The rare mutant allele (c2 allele) that lacks the RsaI restriction site has been found to be associated with higher transcriptional activity, protein levels and enzyme activity than the common wild-type c1 allele. Moreover, the frequency of RsaI c2 allele varies in different populations [9]. The highest frequency has been observed in the Taiwanese (0.28) and Japanese (0.19–0.27), while the frequency is much lower (ranging between 0.01 and 0.05) in African-Americans, European Americans, and Scandinavians.
Catalase A third system with the potential to oxidize alcohols in human body is the enzyme catalase which appears to act only at high concentrations of alcohol. However, its overall role in ethanol elimination is unclear.
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Table III. Polymorphisms of CYP2E1 gene. Allele
Protein
Nucleotide changes
CYP2E1*1A
CYP2E1.1
None
CYP2E1*1B
CYP2E1.1
9893C > G
Effect
RFLP
TaqI-
CYP2E1*1C
CYP2E1.1
6 repeats in the 5 flanking region
CYP2E1*1D
CYP2E1.1
8 repeats in the 5 flanking region
CYP2E1*2
CYP2E1.2
1132G > A
R76H
CYP2E1*3
CYP2E1.3
10023G > A
V389I
CYP2E1*4
CYP2E1.4
4768G > A
V179I
DraI−+XbaI
CYP2E1*5A
CYP2E1.1
−1293G > C; −1053C > T; 7632T > A
PstI+RsaI− DraI−
CYP2E1*5B
CYP2E1.1
−1293G > C; −1053C > T
PstI+RsaI−
CYP2E1*6
CYP2E1.1
7632T > A
DraI−
CYP2E1*7A
CYP2E1.1
261T > A
CYP2E1*7B
CYP2E1.1
−71G > T; 261T > A
CYP2E1*7C
CYP2E1.1
261T > A; 280G > A
Fatty acid ethyl ester synthases (FAEES) Ethanol can condense with free fatty acids in a reaction catalyzed by FAEES to form abnormal compounds such as fatty acid ethyl esters. Three different synthases have been detected and these are designated as synthase I, II, III and IV. Each of these enzymes has been purified and characterized and several of them have been purified to homogeneity from the human myocardium [10]. The observation that FAEES are synthesized at high rates in the heart, and other organs that lack oxidative ethanol metabolism, provides a plausible link between the observed tissue damage, the ingestion of alcohol, and the subsequent development of alcohol-induced heart muscle disease [10].
ALDEHYDE DEHYDROGENASES Acetaldehyde is the first metabolic product of enzymatic ethanol oxidation in human liver, and is far more toxic than the parent compound ethanol. The major oxidation of acetaldehyde in the liver and other organs is catalyzed by the NAD+ -dependent aldehyde dehydrogenase (ALDH, aldehyde: NAD+ oxidoreductase, EC 1.2.1.3). A number of isoenzymes of ALDH coded by different gene loci have been detected in human organs and tissues which differ in their electrophoretic mobility, kinetic properties, as well as in their cellular and tissue distribution. Human ALDH are divided into nine major gene families (table IV). Members of the family 1 are cytoplasmic ALDHs (ALDH1), members of the family 2 are mitochondrial ALDHs (ALDH2), and members of the family 3 are the major constitutive and inducible high Km ALDH forms (ALDH3) found in rat and mouse tissue such as stomach,
Table IV. Human ALDH genes* . Gene
Trivial name
Chromosomal localization
ALDH1A1
ALDH1
9q21
ALDH1A6
ALDH6
15q26
ALDH1B1
ALDH5
9p13
ALDH2
ALDH2
12q24.2
ALDH3A1
ALDH3
17p11.2
ALDH3A2
ALDH10
17p11.2
ALDH3B1
ALDH7
11q13
ALDH3B2
ALDH8
11q13
ALDH4A1
ALDH4
1
ALDH5A1
SSDH
6p22
ALDH6A1
MMSDH
?
ALDH7A1
ATQ1
5q31
ALDH8A1
ALDH12
6q241.1-25
ALDH9A1
ALDH9
1q22-q23
* Adapted from [12].
SSDH: succinic semialdehyde dehydrogenase; MMSDH: methylmalonic acid semialdehyde dehydrogenase; ATQ1: antiquitin.
lung, liver, cornea as well as in human stomach, saliva, and hepatocarcinoma. Various ALDH isoenzymes show a broad range of substrate specificity for aliphatic and aromatic aldehydes. Whereas ALDH1 and ALDH2 both show Km values in the micromolar range with acetaldehyde and propionaldehyde, the Michaelis constants for ALDH3 and ALDH4 are in the millimolar range for these substrates. NAD+ is the preferred coenzyme for the low Km isoenzymes (ALDH1 and ALDH2), whereas the high Km isoenzymes
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D.P. Agarwal
(ALDH3 and ALDH4) can use either NAD+ or NADP+ . The major human liver ALDH1 and ALDH2 isoenzymes are homotetramers consisting of equal but isoenzymespecific subunits with a MW of about 54 kD each.
Additional ALDH isoenzymes As compiled in table IV, in addition to the three nonallelic genes, cytosolic ALDH1A1 (ALDH1), mitochondrial ALDH2, and the cytosolic stomach-specific ALDH3A1 (ALDH3), a number of additional genes have been cloned and characterized in humans [11]: – ALDH1B (ALDH5): a new functional ALDH gene has been identified and has been expressed in variuos tissues including liver, brain, adrenal gland, testis, stomach, and parotid gland; – ALDH1A6 (ALDH6): this isoenzyme is primarily expressed in the salivary gland, stomach, and kidney. The cDNA encodes 512 amino acid residues. A 70% sequence homology was observed with ALDH1. The gene spans about 37 kilobase pairs (kbp) in length and consists of 13 exons. The gene was assigned to chromosome 15q26; – ALDH3B1 (ALDH7): the gene codes for the isoenzyme expressed mainly in the kidneys and lungs, and the cDNA encodes 468 residues. There is a 52% positional identity with ALDH3. It consists of ten exons spanning 20 kbp. The ALDH7 gene was assigned to chromosome 11q13; – ALDH3B2 (ALDH8): the deduced amino acid sequence of the gene product is very similar to that of the ALDH7 (85% homology). The gene spans about 30 kbp and consists of ten exons. It has been assigned to chromosome 11q13; – ALDH9A1 (ALDH9): the cDNA and the gene for a human aldehyde dehydrogenase isozyme, which has a high activity for oxidation of gamma-aminobutyraldehyde and other amino aldehydes have been cloned and characterized. The gene is about 45 kb and consists of ten coding exons interrupted by nine introns. The gene was assigned to chromosome 1q22-q23, using fluorescence in situ hybridization. The gene is polymorphic, i.e., C or T at nt 327 and C or G at nt 344.
Polymorphisms of ALDH genes Genetic polymorphisms have beeen reported in a number of ALDH genes. In table V, nucleotide changes and effect on encoded proteins are compiled. Naming of the human alleles is based on the new nomenclature [12].
Polymorphism of ALDH2 The single genetic factor most strongly correlated with reduced alcohol consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The
Table V. Polymorphism of ALDH genes* . Allele symbol
Nucleotide change
ALDH1B1*1
Effect on protein
Exon
Wild type
2
ALDH1B1*2
257C > T
V69A
2
ALDH1B1*3
320T > G
R90L
2
ALDH1B1*4
183C > T
Silent
2
ALDH2*1
Wild type
ALDH2*2
1510G > A
E487K
12
ALDH2*3
1486G > A
E479K
12
ALDH2*4
1464G > A
Silent
12
ALDH3A1*1
Wild type 985C > G
P329A
ALDH3A2*2
521delT
Frameshift
4
ALDH3A2*3
808delG
Frameshift
6
ALDH3A2*4
941del3,
A314G, P315A,
7
94lins21
AKSTVG313ins
ALDH3A2*5
641G > A
G214Y
4
ALDH3A2*6
1297delGA
Frameshift
9
ALDH3A2*7
1311linsACAAA
Frameshift
9
ALDH3A2*8
1297delGA
Frameshift
9
ALDH3A1*2 ALDH3A2*1
9
Wild type
ALDH4A1*1
Wild type
ALDH4A1*2
21delG
ALDH4A1*3
1055C > T
S352L
§
ALDH4A1*4
47C > T
P16L
§
ALDH4A1*5
1563insT
Frameshift
§
ALDH9A1*1
Frameshift
§
Wild type
ALDH9A1*2
344G > C
C115S
2
ALDH9A1*3
327C > T
Silent
2
§ Gene structure not known yet. * Adapted from [12].
E487K variant of ALDH2 is found in approximately 50% of the Asian population, and is associated with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes [5, 13]. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. Functionally, a single base mutation at this position, resulting in loss of the catalytic activity, is compatible with the proximity in the primary structure between this region and the segment that contains cysteine residues.
Population distribution of ALDH2 variants Oriental populations of Mongoloid origin widely show the presence of the inactive ALDH2 isoenzyme phenotype whereas none of the Caucasian or Negroid
Genetic polymorphisms of alcohol metabolizing enzymes
populations have this isoenzyme abnormality [14]. Among native Indians, about 40% of the South American Indian tribes (Mapuche, Atacamen’s, Shuara) showed the presence of the deficient type of ALDH2. However, this variant isoenzyme was detected only in a very small percentage of the North American Indians (Sioux, Navajo and Mestizo). More recent genotyping data hint to a considerable genetic heterogeneity in the distribution of ALDH2*2 gene in American Indian and Central Asian populations [14, 15].
Polymorphism of ALDH3 The stomach ALDH3 isoenzyme band, focusing at about pH 6.0, displays several activity bands [2]. Three polymorphisms of ALDH3 (variants I, II, and III) were also detected in human saliva and hair root extracts.
Physiological role The physiological significance of ALDH relates mainly to its role in the detoxification of acetaldehyde and other aldehydes which show a variety of toxic effects in human organs and tissues. Many biogenic amines are converted to their corresponding aldehydes via monoamine and diamine oxidase systems. Moreover, in vivo biotransformation of many drugs and xenobiotics that are not aldehydes gives rise to aldehyde metabolites. For example, cyclophosphamide, which is pharmacologically inactive, needs to be biotransformed to its cytotoxic metabolite phosphoramide mustard via an intermediate metabolite 4-hydroxycyclophosphamide. The latter compound exists in equilibrium with aldophosphamide which can get converted to a non-cytotoxic compound (carboxyphosphamide) through irreversible oxidation of the aldehyde group catalyzed by one or more forms of aldehyde dehydrogenases. This enzymatic pathway leads to the detoxification of cyclophosphamide affecting its therapeutic efficiency [16]. More recent studies have focused on the putative role of alcohol metabolizing enzymes in alcohol elimination rate, acute reactions to alcohol, alcohol drinking habits and alcoholism across various ethnic groups [17]. For example, Orientals exhibit intense facial flushing after a mild dose of alcohol as compared to Caucasians affecting their alcohol drinking habits. Quantity-frequency-variability distribution indicates that the percentage of heavy and moderate drinkers is higher among Caucasians, while the percentage of abstainers and infrequent drinkers is higher among the Chinese, Japanese, and other Orientals [18 – 21]. The alcoholism rate as well as alcohol-related end-organ damage is also found to be lower among the Japanese, Chinese, and other Orientals as compared to Caucasian populations living in the Western society [6, 8, 20, 21]. A significantly fewer number of patients with alcoholism and alcoholic liver disease have been found to posssess the inactive ALDH2*2 gene.
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