Human alcohol dehydrogenases and serotonin metabolism

Human liver alcohol dehydrogenases (ADH) may participate in serotonin (5-hydroxytryptamine) metabolism. Class I and II isozymes catalyze the oxidation of 5-hydroxytryptophol (5-HTOL) with kcat/Km values ranging from 10 to 100 mM-1 min-1 compared to 4-66 mM-1 min-1 for that of ethanol at pH 7.40, 25 degrees C. The product, 5-hydroxyindoleacetaldehyde, was purified as its semicarbazone and identified by mass spectrometry. Ethanol competitively inhibits 5-HTOL oxidation by beta 1 gamma 2 ADH with a Ki of 440 microM, a value similar to the Km of ethanol, 210 microM. The inhibition constants for 1,10-phenanthroline and 4-methylpyrazole are 20 microM and 80 nM respectively, essentially identical to those obtained with ethanol as substrate, 22 microM and 70 nM, respectively. The competition between ethanol and 5-HTOL for ADH can explain observations of ethanol induced changes in serotonin metabolism in vivo.     

Human class I alcohol dehydrogenases catalyze the interconversion of alcohols and aldehydes in the metabolism of dopamine

The class I human liver alcohol dehydrogenases (ADHs) catalyze the interconversion of the intermediary alcohols and aldehydes of dopamine metabolism in vitro, whereas those of the class II and class III do not. The individual, homogeneous class I isozymes oxidize (3,4-dihydroxyphenyl)ethanol and (4-hydroxy-3-methoxyphenyl)ethanol (HMPE) and ethanol with kcat/Km values in the range from 16 to 240 mM-1 min-1 and from 16 to 66 mM-1 min-1, respectively. They reduce the corresponding dopamine aldehydes (3,4-dihydroxyphenyl)acetaldehyde and (4-hydroxy-3-methoxyphenyl)acetaldehyde (HMPAL) with kcat/Km values varying from 7800 to 190,000 mM-1 min-1, considerably more efficient than the reduction of acetaldehyde with kcat/Km values from 780 to 4900 mM-1 min-1. For beta 1 gamma 2 ADH, ethanol competes with HMPE oxidation with a Ki of 23 microM. In addition, 1,10-phenanthroline inhibits HMPE oxidation and HMPAL reduction with Ki values of 20 microM and 12 microM, respectively, both quite similar to that for ethanol, Ki = 22 microM. Thus, both ethanol/acetaldehyde and the dopamine intermediates compete for the same site of ADH, a basis for the ethanol-induced in vivo alterations of dopamine metabolism.     

Hydrophobic anion activation of human liver chi chi alcohol dehydrogenase

Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substrates and hence the activity of class III ADH up to 30-fold. Thus, in the presence of 1 mM octanoate, ethanol displays Michaelis-Menten kinetics. The degree of activation depends on the size both of the substrate and of the activator; generally, longer, negatively charged activators result in greater activation. At pH 10, the activator binds to the E-NAD+ form of the enzyme to potentiate substrate binding. Pentanoate activates methylcrotyl alcohol oxidation and methylcrotyl aldehyde reduction 14- and 30-fold, respectively. Such enhancements of both oxidation and reduction are specific for class III ADH; neither class I nor class II shows this effect. The implications as to the nature of the physiological substrate(s) of class III ADH are discussed in light of the recent finding that this ADH and glutathione-dependent formaldehyde dehydrogenase are identical. A new rapid purification procedure for chi chi-ADH is presented.     

Regulation of human class I alcohol dehydrogenases by bile acids

Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.     

Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification

Humans are polymorphic at two of the alcohol dehydrogenase (ADH) loci important in ethanol metabolism, ADH2 and ADH3. Although the coding regions of these genes are 94% identical, they produce subunits that differ greatly in kinetic properties in vitro. These differences are likely to be reflected in the pharmacokinetics of alcohol metabolism, but studies have been hampered by the need to use liver biopsy specimens to determine the ADH phenotype. This problem has now been overcome by determining the genotype at these loci using DNA that has been amplified in vitro by the polymerase chain reaction. We report here the identification of all three of the ADH2 alleles and both of the ADH3 alleles. Any pair of ADH2 or ADH3 alleles can be distinguished using allele-specific oligonucleotide probes directed at their single base pair difference. In addition, ADH2(2) can be distinguished from ADH2(1) and ADH2(3) by detecting a new MaeIII site created in the third exon by the single base pair alteration in ADH2(2).     

The microsomal ethanol oxidizing system mediates metabolic tolerance to ethanol in deermice lacking alcohol dehydrogenase

Metabolic tolerance to ethanol has been attributed to enhanced mitochondrial reoxidation of reducing equivalents produced in the alcohol dehydrogenase (ADH) pathway or to non-ADH mechanisms. To resolve this issue, deermice lacking low Km hepatic ADH were fed for 2 weeks a liquid diet containing ethanol or isocaloric carbohydrate and hepatocytes were isolated. Ethanol (50 mM) oxidation increased (9.8 vs 4.5 nmol/min/10(6) cells in controls). To differentiate which of two non-ADH pathways (the microsomal ethanol oxidizing system (MEOS) or catalase) was responsible for the induction, four approaches were used. First, MEOS was assayed in hepatic microsomes and found to be increased (24.4 vs 6.8 nmol/min/mg protein in controls). Second, hepatocyte ethanol metabolism was measured after addition of the catalase inhibitor azide (0.1 mM) and found to be unchanged. By contrast, the competitive MEOS inhibitor, 1-butanol, depressed metabolism in a concentration-dependent manner. A third approach relied on measurement of isotope effects known to be different for MEOS and catalase. From the isotope effect values, MEOS was calculated to contribute 85% or more of total ethanol oxidation by cells from both ethanol-fed and control animals. A fourth approach involved in vivo pretreatment with pyrazole (300 mg/kg/day for 2 days), which reduced peroxidation by catalase to 13% of control values in liver homogenates while inducing MEOS activity to 152% of controls. Hepatocytes from pyrazole-treated deermice showed a 47% increase in ethanol metabolism, paralleling the MEOS induction and contrasting with the catalase suppression. These results indicate that since metabolic tolerance occurs in the absence of ADH, it is not necessarily ADH mediated, and further, that MEOS rather than catalase accounts for basal ethanol metabolism and its increase after chronic ethanol treatment.     

Purification and characterization of a new alcohol dehydrogenase from human stomach

Starch gel electrophoresis of homogenates from human stomach mucosa resolves three alcohol dehydrogenase (ADH) forms: the anodic chi-ADH (class III), the cathodic gamma-ADH (class I), and a new form of slow cathodic mobility that has not been previously characterized. In this work, we describe the purification in three chromatographic steps and the physical and kinetic characterization of this new human alcohol dehydrogenase, which we have named sigma-ADH. The enzyme exhibits the general physicochemical features (Mr, zinc content, subunit Mr, cofactor preference) of all mammalian alcohol dehydrogenases. The kinetic studies show a high Km value (41 mM) and a high kcat value (280 min-1) for ethanol at pH 7.5. The Km decreases as the alcohol increases its chain length. The aldehydes are better substrates than the corresponding alcohols, with m-nitrobenzaldehyde being the best substrate examined. sigma-ADH is strongly inhibited by 4-methylpyrazole, but with a Ki (10 microM) still higher than that for a class I isoenzyme. These properties suggest that sigma-ADH is a class II isoenzyme, different from pi-ADH and similar to that previously described by us in rat stomach. At the high ethanol concentrations in stomach after drinking, sigma-ADH is probably the ADH form with the largest contribution to human gastric ethanol metabolism.     

Organ specific alcohol metabolism: placental chi-ADH

Human placenta contains a single detectable isozyme of alcohol dehydrogenase that has been isolated and characterized. It migrates toward the anode on starch gel electrophoresis and can be stained with pentanol but not ethanol as substrate. Its kinetic and molecular characteristics are identical with those of the recently discovered chi-ADH (Class III) isozyme from human liver. Placental ADH is present in the cytosol of this organ in small amounts, 6 mg/kg fresh tissue. It oxidizes ethanol very slowly--even at ethanol concentrations that would reflect intoxication when found in serum. Thus, placental alcohol dehydrogenase cannot play a significant role in the ethanol metabolism of pregnant women.     

Characterization of three isoenzymes of rat alcohol dehydrogenase. Tissue distribution and physical and enzymatic properties

Rat tissues contain three different isoenzymes of alcohol dehydrogenase (ADH) that we have named ADH-1, ADH-2 and ADH-3, ADH-1 is an anodic isoenzyme present in high amounts in the ocular tissues, stomach and lung. ADH-2 is also anodic and has been found in all the rat organs examined. ADH-3 is the group of cathodic ADH forms, mainly present in liver, that has been the subject of the majority of the previous studies on rat ADH. The three isoenzymes have been purified to homogeneity and characterized. All of them have similar physical characteristics: Mr 80,000, with two subunits of Mr 40,000; they contain four atoms of Zn per molecule, and prefer NAD+ as cofactor. Isoelectric points are, however, different: 5.1 for ADH-1, 5.95-6.3 for ADH-2 and 8.25-8.4 for ADH-3. ADH-3 exhibits a Km for ethanol of 1.4 mM, a broad substrate specificity and is strongly inhibited by pyrazole (Ki = 0.4 microM). ADH-2 shows substrate specificity toward long-chain alcohols and aldehydes, cannot be saturated by ethanol and is practically insensitive to pyrazole (Ki = 78.4 mM). ADH-1 has intermediate properties, with a Km for ethanol of 340 mM, a broad substrate specificity and Ki for pyrazole of 0.56 mM. Rat ADH-1, ADH-2 and ADH-3 exhibit many analogies with human ADH classes II, III and I respectively. The specific localization and kinetic properties of rat ADH isoenzymes suggest that ADH-1 and ADH-3 may act as metabolic barriers to external alcohols and aldehydes whereas ADH-2 may have a function in the metabolism of the endogenous long-chain alcohols and aldehydes.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene.

Five non-allelic genes which encode five types of alcohol dehydrogenase subunits have been identified in humans. An additional gene (ADH6) and cDNA, whose coding sequences were not highly analogous to any of the known alcohol dehydrogenase subunits, were recently cloned (Yasunami et al., Proc. Natl. Acad. Sci. USA 88, 7610-7614, 1991). The full-length ADH6 cDNA was expressed in the E.coli expression system and in the in vitro translation system of rabbit reticulocytes. The protein produced had its isoelectric point at pH 8.6, optimum pH at pH 10, and a lower Km for benzylalcohol than for ethanol and propanol. These characteristics are compatible to the properties of mu- or sigma-alcohol dehydrogenase isozyme existing in human stomach, indicating that ADH6 gene encodes the mu- or sigma-alcohol dehydrogenase subunit.     

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio)

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.     

Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels.     

The effect of ethanol on 5'-nucleotidase of rat liver plasma membranes

The activity of 5'-nucleotidase of rat liver plasma membranes has been investigated in normal and acutely ethanol-intoxicated rats (7 g ethanol/Kg body wt). Ethanol was also added to the incubation mixture for 5'-nucleotidase assay. The alcohol modified the Km of the enzyme when added to plasma membranes of normal rats; moreover, it increased the activation energy of the reaction. The treatment with the alcohol in vivo lowered the Vmax, but no modifications of Km could be detected in this case, upon further addition of the toxic in vitro. It is concluded that ethanol is able to act by itself on 5'-nucleotidase activity of rat liver plasma membranes; however, ethanol produces other effects in vivo, probably due to its metabolism.     

Organ-specific human alcohol dehydrogenase: isolation and characterization of isozymes from testis

Class III alcohol dehydrogenase (ADH) predominates in human testis. The two isozymes of this class were isolated jointly by affinity and conventional ion exchange chromatography. They display anodic electrophoretic mobility at pH 8.2, are completely insensitive to 4-methylpyrazole inhibition and oxidize ethanol and other short-chain primary alcohols very poorly. Thus, their kinetic and inhibition characteristics are identical to human liver class III ADH. In contrast, class I ADH is a barely detectable component of testicular alcohol dehydrogenase. The physicochemical characteristics of class III ADH are virtually identical to those of alcohol dehydrogenases found in other organs.     

Purification and molecular properties of a Class II alcohol dehydrogenase (ADH-C2) from horse liver

An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. The enzyme (designated ADH-C2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. The enzyme exhibited similar kinetic properties to human pi-ADH and mouse ADH-C2, previously classified as class II ADHs [Vallee and Bazzone (1983) Isozymes, Vol. 8, pp. 219-244; Algar et al. (1983) Eur. J. Biochem. 137, 139-147] but differed in most respects from the extensively investigated horse Class I ADHs; EE, ES and SS. Horse ADH-C2 exhibited a Km value for ethanol of 42 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.     

The inhibition of alcohol dehydrogenase in vitro and in isolated hepatocytes by 4-substituted pyrazoles

As a means of comparing the functional properties of an enzyme in dilute solution in vitro with those for the same enzyme acting in its normal cellular environment, a study was conducted with 4-substituted pyrazoles as inhibitors of rat liver alcohol dehydrogenase in vitro and ethanol oxidation in isolated rat hepatocytes. Inhibitor constants (Ki's) for the same set of pyrazole derivatives were also determined for human liver alcohol dehydrogenase. The best-fitting equations were derived to relate the Ki's to the chemical nature of substituents. These quantitative structure-activity relationships show that pyrazoles with stronger electron-withdrawing substituents are weaker inhibitors both for the enzyme in vitro and, to an equal extent, for ethanol oxidation by intact cells. Inhibitor effectiveness is also dependent on substituent hydrophobicity, but, while increasing hydrophobicity makes stronger inhibitors of the enzyme in vitro, it can diminish the effectiveness in vivo by decreasing permeability through the cell membrane. A structure-activity analysis of published Ki's for pyrazoles acting against human pi-ADH indicates that its active site differs from those in other alcohol dehydrogenases.     

Stomach and duodenal alcohol and aldehyde dehydrogenase isozymes in Chinese

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozyme phenotypes were determined in surgical and endoscopic biopsies of the stomach and duodenum by agarose isoelectric focusing. gamma-ADH was found to be the predominant form in the mucosal layer whereas beta-ADH was predominant in the muscular layer. Low-Km ALDH1 and ALDH2 were found in the stomach and duodenum. High-Km ALDH3 isozymes occurred only in the stomach but not in the duodenum. The isozyme patterns of gastric mucosal ALDH2 and ALDH3 remained unchanged in the fundus, corpus, and antrum. The stomach ALDH3 isozymes exhibited a Km value for acetaldehyde of 75 mM, and an optimum for acetaldehyde oxidation at pH 8.5. Since the Km value was high, ALDH3 contributed very little, if any, to gastric ethanol metabolism. The activities of ALDH in the gastric mucosa deficient in ALDH2 were 60-70% of that of the ALDH2-active phenotypes. These results indicate that Chinese lacking ALDH2 activity may have a lower acetaldehyde oxidation rate in the stomach during alcohol consumption.