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Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.

Mounting evidence supports a contribution of endogenous alcohol metabolism in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is not known whether the expression of alcohol metabolism genes is altered in the livers of simple steatosis. There is also a current debate on whether fatty acids induce CYP2E1 in fatty livers. In this study, expression of alcohol metabolizing genes in the liver biopsies of simple steatosis patients was examined by quantitative real-time PCR (qRT-PCR), in comparison to biopsies of NASH livers and normal controls. Induction of alcohol metabolizing genes was also examined in cultured HepG2 cells treated with ethanol or oleic acid, by qRT-PCR and Western blots. We found that the mRNA expression of alcohol metabolizing genes including ADH1C, ADH4, ADH6, catalase and CYP2E1 were elevated in the livers of simple steatosis, to similar levels found in NASH livers. In cultured HepG2 cells, ethanol induced the expression of CYP2E1 mRNA and protein, but not ADH4 or ADH6; oleic acid did not induce any of these genes. These results suggest that elevated alcohol metabolism may contribute to the pathogenesis of NAFLD at the stage of simple steatosis as well as more severe stages. Our in vitro data support that CYP2E1 is induced by endogenous alcohol but not by fatty acids.

The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 (“stomach” isoenzyme) are substantially larger (10–100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD + and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD + and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 A resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

Human alcohol dehydrogenases (ADH1 and ADH4) actively use retinoids oxidized at the cyclohexenyl ring (4-oxo-, 4-hydroxy-, and 3,4-didehydro-retinoids), which are functional compounds in several cells and tissues (i.e., in human skin). Remarkably, activities with 4-oxo-retinal and 4-hydroxy-retinol (kcat = 2050 min−1 for ADH4) are the highest among retinoids, similar to those of the best aliphatic alcohols. Thus, ADH1 and ADH4 provide a metabolic pathway for the synthesis of the corresponding retinoic acids. Tween 80, a widely used detergent in the retinoid activity assay, behaves as a competitive inhibitor. The Km values for all-trans-retinol (2–3 μM), estimated in the absence of detergent, are 10-fold lower than those obtained at the usual 0.02% Tween 80. This suggests a contribution of ADH to retinoid metabolism more relevant than previously expected. However, Tween 80 stabilizes retinoids in water solution and provides a reliable and reproducible assay, suitable for comparing different ADHs and different retinoid substrates.

Human alcohol dehydrogenase (ADH) constitutes a complex family. Class IV ADH (ADH4) is characteristic in its epithelial expression in the aerodigestive tract and high V(max) and K(m) for oxidation of ethanol. ADH4 exhibits the highest catalytic efficiency for retinol oxidation in human ADH family. Initial velocity, product inhibition, and dead-end inhibition studies indicate that ADH4, when functioning as ethanol dehydrogenase, conforms to an ordered sequential mechanism with coenzyme binding first and releasing last in catalytic cycle. When functioning as retinol dehydrogenase, the mechanism of ADH4 deduced from steady-state kinetic and equilibrium-binding studies is best described as a rapid equilibrium random mechanism with two dead-end ternary complex for retinol oxidation and a rapid equilibrium ordered mechanism with one dead-end ternary complex for retinal reduction, a unique mechanistic form for zinc-containing ADHs in the medium chain dehydrogenase/reductase superfamily. Kinetic and genetic studies support the proposal that ADH4 may play two important physiological roles, i.e., as a major contributor to first-pass metabolism of ethanol in stomach as well as involvement in the synthesis of retinoic acid, a hormonal ligand controlling a nuclear receptor signaling pathway that regulates growth, development, and epithelial maintenance. Quantitative simulation studies indicate that retinol metabolism through ADH pathway can be inhibited to a significant extent during alcohol consumption. The perturbation of retinoic acid synthesis by ethanol may underlie the pathogenesis of fetal alcohol syndrome and alcohol-related upper digestive tract cancer.

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD(50) studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only approximately 5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems — the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) — have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.

A full-length 1966-base pair clone of the human class IV alcohol dehydrogenase (sigma-ADH) was isolated from a human stomach cDNA library. The 373-amino acid sigma-ADH encoded by this cDNA was expressed in Escherichia coli. The specific activity of the recombinant enzyme for ethanol oxidation at pH 7.5 and 25 degrees C, calculated from active-site titration of NADH binding, was 92 +/- 9 units/mg. Kinetic analysis of the catalytic efficiency (kcat/KM) of recombinant sigma-ADH for oxidation of primary alcohols indicated broad substrate specificity. Recombinant human sigma-ADH exhibited high catalytic efficiency for oxidation of all-trans-retinol to all-trans-retinal. This pathway is important in the synthesis of the transcriptional regulator all-trans-retinoic acid. Secondary alcohols and 3 beta-hydroxysteroids were inactive with sigma-ADH or were oxidized with very low efficiency. The KM of sigma-ADH for ethanol was 25 mM, and the KM for primary straight chain alcohols decreased substantially as chain length increased. There are important amino acid differences in the alcohol-binding site between the human class IV (sigma) and human class I (beta) alcohol dehydrogenases that appear to explain the high catalytic efficiency for all-trans-retinol, the high kcat for ethanol, and the low catalytic efficiency for secondary alcohols of sigma-ADH relative to beta 1-ADH. For example, modeling the binding of all-trans-retinol in the human beta 1-ADH structure suggested that coordination of retinol to the active-site zinc is hindered by a loop from residues 114 to 120 that is at the entrance to the alcohol-binding site. The deletion of Gly-117 in human sigma-ADH and a substitution of Leu for the bulky Tyr-110 appear to facilitate retinol access to the active-site zinc.

Alcohol dehydrogenase classes exhibit important differences in both substrate specificity and tissue distribution which suggest distinct physiological functions. We have studied the kinetic constants at pH 7.5 of the rat alcohol dehydrogenase classes, purified from liver (classes I and III) and from stomach (class IV), with three groups of relevant physiological compounds: cytotoxic aldehydes generated in lipid peroxidation, omega-hydroxyfatty acids, and retinoids. Classes I and IV actively reduce 4-hydroxynonenal, 2-hexenal, and hexanal, which are toxic compounds known to be produced in significant amounts during lipid peroxidation. Class III shows poor activity with these aldehydes. Class IV exhibits the best kcat/Km values (2150 mM-1 x min-1 for 4-hydroxynonenal), which suggest a role for this enzyme in the elimination of the cytotoxic aldehydes in tissues that are susceptible to lipid peroxidation, such as skin, cornea, and mucosa of the respiratory and digestive tracts, where class IV is localized. The three classes are very active with omega-hydroxyfatty acids, suggesting that all of them are involved in the physiological oxidation of these compounds in the rat tissues. The kinetic constants support that oxidation of omega-hydroxyfatty acids is a physiological function for class III, in addition to its role as formaldehyde dehydrogenase. Finally, classes I and IV are active in retinol oxidation and retinal reduction. Class IV may play a crucial role in the generation of retinoic acid in epithelia, where this compound is involved in development and cell differentiation. In conclusion, alcohol dehydrogenase is an enzyme with multiple metabolic roles, and the different substrate specificity and tissue localization for each class provide organs and tissues with distinct physiological functions.

Alcoholic abuse is of great interest in forensic medicine, for the evaluation of driving and working ability, for permanent invalidity to alcohol-related pathologies and for the high incidence of alcohol-related deaths. It is a quantitative disorder, with a combined incidence of environmental and genetic factors. Since it was observed that variations in ADH4 gene are associated with alcoholism, SNPs mapping in different regions of ADH4 gene were selected. A new approach was applied in this study: mutations that could modify the splicing sites were selected using "Splice site prediction" available at http://www.fruitfly.orgseq_tools/sp/lice.html; in addition, since the correct splicing is finely regulated by RNA binding proteins, SNPs that could affect splicing, destroying or creating protein target sequences and changing RNA secondary structure, were predicted by a tool available at www.introni.it. 206 unrelated volunteers, diagnosed for alcohol dependence (DSM-IV), were included in this study. SNPs typing was performed setting up 2 multiplex PCRs and minisequencing reactions. 12 SNPs were selected from literature data and 26 SNPs, among 233 polymorphisms screened, were chosen by computational analyses.

The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh 1 gene was determined. The butanol dehydrogenase gene is referred to as the adh 1 gene since it was shown to have activity using butanol and ethanol as substrates. The adh 1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an M r of 43274. The adh 1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp. No promoter consensus sequences were identified in the intergenic upstream region and the adh 1 gene did not appear to be expressed off its own promoter in Escherichia coli . Three separate types of ADH have been recognized. The ADH1 from C. acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae , but showed little or no homology with the other characterised types of ADH.

A method has been developed for simultaneous analysis of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzymes in small (2.5 mg) liver biopsy cores by starch gel electrophoresis. All the currently recognized hepatic isoenzymes coded by ADH1, ADH2, ADH3 and ADH4 can be detected as can the five ALDH isoenzymes. Using this technique we have investigated the isoenzyme composition of liver samples from English and Chinese subjects and a group of chronic alcoholics. Pronounced racial differences in frequency of ADH2 and ALDH phenotypes were found--only 2 (4%) of English controls had the "atypical" ADH2 variant whereas this was present in 42 (84%) of Chinese subjects, and whereas all the English subjects had the rapidly migrating mitochondrial isoenzyme of ALDH, this was absent in 27 (54%) of Chinese. No differences in ADH or ALDH phenotype were seen in the chronic alcoholics, all of whom were of English origin, compared with the English controls, but there was a reduction in overall ALDH activity and particularly in the mitochondrial isoenzyme in those with cirrhosis. The reduction in ALDH activity is probably acquired; by limiting acetaldehyde oxidation it could be responsible for the rapid deterioration in liver function in patients who continue drinking excessively.