Ebselen as protection against ethanol-induced toxicity in rat stomach.

The mucosal protective effect of ebselen was examined in an ethanol-induced rat gastric lesion model. Examination of gastric tissue samples by light microscopy showed that i.g. exposure to 50% ethanol induced gastric injury, which was more prominent in female rats. Ethanol did not effect the gastric acid secretion examined by means of H(+)-K+ATPase, the increment of which might be harmful in the stomach. But ebselen with or without ethanol kept H(+)-K+ATPase below control levels. Gastric alcohol dehydrogenase (ADH) was mainly responsible for oxidation of ethanol in the stomach before it enters the bloodstream. I.g. ethanol exposure inhibited the ADH activity but ebselen eliminated the ethanol-induced inhibition of this enzyme. Therefore, ebselen exhibited a beneficial effect by increasing the gastric ethanol metabolism and by ameliorating the possible tissue toxicity of ethanol. Consistently, we also found that ebselen diminished the blood ethanol level. A gender difference in the blood ethanol levels existed following the same dose of ethanol but there was no difference in ADH activity. Histologically, mucosal injury following ebselen exposure together with ethanol was less severe compared with ethanol treatment alone. We concluded that the decrease in ethanol-induced mucosal injury following ebselen may have contributed to the inhibition of H(+)-K+ATPase and the activation of ADH by ebselen.     

Genetic considerations in the effects of ethanol in mice. II. A trans-acting inducibility regulator(s) affecting alcohol dehydrogenase (ADH) activity

The inducibility of alcohol dehydrogenase (ADH) has been recognized in different systems including maize, Drosophila, and mice. Our earlier results showed strain-specific ADH responses to chronic ethanol administration relative to matched littermate controls in mice. For this study we used two strains which showed induction (BALB/c and S.W.) and two strains which showed repression (C57BL/6J and 129/ReJ) to produce three sets of F₁ hybrids and their reciprocals and one set (BALB/c×C57BL/6J) of recombinant inbred (RI) lines. The ADH properties of the resulting genotypes were again evaluated following 15% ethanol treatment in drinking water (2 weeks) in relation to their littermate matched controls in replicated trials. Our F₁ results suggest complete dominance for induction over repression at the phenotypic level, and the two repressed strains showed complementation. No significant differences were observed in the reciprocal F₁'s and all pairs of a given genotype-treatment combination yielded consistent results. The 1:1 segregation of RI lines suggests a single gene difference for ADH inducibility between BALB/c and C57BL/6J. These findings suggest the presence of a trans-acting inducibility regulator(s) for ADH which may or may not represent a single locus. Variability for such regulatory elements may provide an explanation for the commonly observed individual differences in natural populations for response to alcohol including alcohol metabolism.     

Variability in the effect of alcohol on alcohol metabolizing enzymes may determine relative sensitivity to alcohols: a new hypothesis

Individual and racial differences in response to alcohol and with respect to alcoholism have strong genetic predispositions. Most studies on the actual genetic determinants have concentrated on the isozymes of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), the two enzymes of the primary pathway of alcohol metabolism. Although few "activity" variants (associated with mutations in the structural genes) of the two enzymes are known to exist in susceptible groups, these observations do not offer an adequate explanation for the observed variability in response to alcohols in the population. Some recent studies have reported alterations in the specific activity of the two enzymes following exposure to alcohol for different lengths of time in man, rat, and mice. The induction-repression so observed is hypothesized to be regulated by one or more inducibility genetic elements (IGE) associated with the structural loci of the two enzymes. Variability in IGE will permit a genotype (individual) specific response in ADH and ALDH specific activity when challenged with a given level of alcohol. Considering the relative toxicity of acetaldehyde, the primary metabolite of this pathway, the resistant individuals would be expected to show ALDH induction. Conversely, the susceptible individuals should respond to alcohol by ALDH repression. The ability of an individual to show induction or repression following alcohol ingestion will depend on his or her IGE genotype(s) associated with specific enzyme loci. Also, the degree of polymorphism at these loci would be expected to be extensive and yet population and race specific. Once experimentally established, this approach could have important implications in screening, counselling, prevention, and in novel approaches to treatment.     

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. α,α-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile defect in cardiac myocytes

Alcoholic cardiomyopathy is characterized by impaired ventricular function although its toxic mechanism is unclear. This study examined the impact of cardiac overexpression of alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde (ACA), on ethanol-induced cardiac contractile defect. Mechanical and intracellular Ca(2+) properties were evaluated in ventricular myocytes from ADH transgenic and wild-type (FVB) mice. ACA production was assessed by gas chromatography. ADH myocytes exhibited similar mechanical properties but a higher efficiency to convert ACA compared with FVB myocytes. Acute exposure to ethanol depressed cell shortening and intracellular Ca(2+) in the FVB group with maximal inhibitions of 23.3% and 23.4%, respectively. Strikingly, the ethanol-induced depression on cell shortening and intracellular Ca(2+) was significantly augmented in the ADH group, with maximal inhibitions of 43.7% and 40.6%, respectively. Pretreatment with the ADH inhibitor 4-methylpyrazole (4-MP) or the aldehyde dehydrogenase inhibitor cyanamide prevented or augmented the ethanol-induced inhibition, respectively, in the ADH but not the FVB group. The ADH transgene also substantiated the ethanol-induced inhibition of maximal velocity of shortening/relengthening and unmasked an ethanol-induced prolongation of the duration of shortening/relengthening, which was abolished by 4-MP. These data suggest that elevated cardiac ACA exposure due to enhanced ADH expression may play an important role in the development of alcoholic cardiomyopathy.     

Alcohol Dehydrogenase Accentuates Ethanol-Induced Myocardial Dysfunction and Mitochondrial Damage in Mice: Role of Mitochondrial Death Pathway

Objectives

Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH).

Methods

ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined.

Results

Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O₂ ^•−. Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-α, Fas receptor, Fas L and cytosolic AIF.

Conclusions

Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.     

Influence of gender on ethanol-induced ventricular myocyte contractile depression in transgenic mice with cardiac overexpression of alcohol dehydrogenase

Acute ethanol exposure depresses ventricular contractility and contributes to alcoholic cardiomyopathy in both men and women chronically consuming ethanol. However, a gender-related difference in the severity of myopathy exists with female being more sensitive to ethanol-induced tissue damage. Acetaldehyde (ACA), the major oxidized product of ethanol, has been implicated to play a role in the pathogenesis and gender-related difference of alcoholic cardiomyopathy, possibly due to its direct cardiac effect and interaction with estrogen. This study was designed to compare the effects of cardiac overexpression of alcohol dehydrogenase (ADH), which converts ethanol into ACA, on the cardiac contractile response to ethanol in ventricular myocytes isolated from age-matched adult male and female transgenic (ADH) and wild-type (FVB) mice. Mechanical properties were measured with an IonOptix SoftEdge system. ACA production was assessed by gas chromatography. The ADH myocytes from both genders exhibited similar mechanical properties but a higher efficacy to produce ACA compared to FVB myocytes. Exposure to ethanol (80-640 mg/dl) for 60 min elicited concentration-dependent decrease of cell shortening in both FVB and ADH groups. The ethanol-induced depression on cell shortening was significantly augmented in female but not male ADH group. ADH transgene did not exacerbate the ethanol-induced inhibition of maximal velocity of shortening/relengthening in either gender. In addition, neither ethanol nor ADH transgene affect the duration of shortening and relengthening in male or female mice. These data suggest that females may be more sensitive to ACA-induced cardiac contractile depression than male, which may attribute to the gender-related difference of alcoholic cardiomyopathy.     

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

Isolation ofCaenorhabditis elegans mutants lacking alcohol dehydrogenase activity

Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematodeCaenorhabiditis elegans. Extracts of wild-typeC. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols.C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression inC. elegans.     

Cardiac overexpression of catalase antagonizes ADH-associated contractile depression and stress signaling after acute ethanol exposure in murine myocytes.

Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanol-induced cardiac depression, although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca(2+) properties and reactive oxygen species generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca(2+) properties. ADH or ADH-CAT myocytes had higher acetaldehyde-producing ability. Ethanol (80-640 mg/dl) suppressed FVB cell shortening and intracellular Ca(2+) transients with maximal inhibitions of 43.5 and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca(2+) was augmented in ADH group with maximal inhibitions of 66.8 and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0 and 47.2% for cell shortening and intracellular Ca(2+), respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca(2+). ADH amplified ethanol-induced reactive oxygen species generation, which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation, but it cancelled ADH-induced effects. These data suggest that antioxidant CAT may effectively antagonize ADH-induced enhanced cardiac depression in response to ethanol.     

Alcohol dehydrogenase (ADH). Influence of homo- and heterologous ADH administration on albino rats craving for alcohol and on ADH isozyme activity in the liver.

The effects of homo- and heterologous alcohol dehydrogenase (ADH) administration into albino rats were investigated. It was found that homologous ADH increases and heterologous ADH decreases the craving for ethanol. The latter effect was accompanied by the appearance of anti-ADH-3 antibodies and by a decrease in ADH-3 activity in the liver. Craving for alcohol decreased after both active and passive immunization against ADH.     

Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective

Individuals who carry the most active alcohol dehydrogenase (ADH) isoforms are protected against alcoholism. This work addresses the mechanism by which a high ADH activity leads to low ethanol intake in animals. Male and female ethanol drinker rats (UChB) were allowed access to 10% ethanol for 1 h. Females showed 70% higher hepatic ADH activity and displayed 60% lower voluntary ethanol intake than males. Following ethanol administration (1 g/kg ip), females generated a transient blood acetaldehyde increase ("burst") with levels that were 2.5-fold greater than in males (P < 0.02). Castration of males led to 1) an increased ADH activity (+50%, P < 0.001), 2) the appearance of an acetaldehyde burst (3- to 4-fold vs. sham), and 3) a reduction of voluntary ethanol intake comparable with that of na?ve females. The ADH inhibitor 4-methylpyrazole blocked the appearance of arterial acetaldehyde and increased ethanol intake. Since the release of NADH from the ADH.NADH complex constitutes the rate-limiting step of ADH (but not of ALDH2) activity, endogenous NADH oxidizing substrates present at the time of ethanol intake may contribute to the acetaldehyde burst. Sodium pyruvate given at the time of ethanol administration led to an abrupt acetaldehyde burst and a greatly reduced voluntary ethanol intake. Overall, a transient surge of arterial acetaldehyde occurs upon ethanol administration due to 1) high ADH levels and 2) available metabolites that can oxidize hepatic NADH. The acetaldehyde burst is strongly associated with a marked reduction in ethanol intake.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

Expression, localization and potential physiological significance of alcohol dehydrogenase in the gastrointestinal tract.

ADH1 and ADH4 are the major alcohol dehydrogenases (ADH) in ethanol and retinol oxidation. ADH activity and protein expression were investigated in rat gastrointestinal tissue homogenates by enzymatic and Western blot analyses. In addition, sections of adult rat gastrointestinal tract were examined by in situ hybridization and immunohistochemistry. ADH1 and ADH4 were detected along the whole tract, changing their localization and relative content as a function of the area studied. While ADH4 was more abundant in the upper (esophagus and stomach) and lower (colorectal) regions, ADH1 was predominant in the intestine but also present in stomach. Both enzymes were detected in mucosa but, in general, ADH4 was found in outer cell layers, lining the lumen, while ADH1 was detected in the inner cell layers. Of interest were the sharp discontinuities in the expression found in the pyloric region (ADH1) and the gastroduodenal junction (ADH4), reflecting functional changes. The precise localization of ADH in the gut reveals the cell types where active alcohol oxidation occurs during ethanol ingestion, providing a molecular basis for the gastrointestinal alcohol pathology. Localization of ADH, acting as retinol dehydrogenase/retinal reductase, also indicates sites of active retinoid metabolism in the gut, essential for mucosa function and vitamin A absorption.     

Ethanol sensitivity of rice and oat coleoptiles

The ability to avoid the ethanol-induced injury was evaluated in rice ( Oryza sativa L.) and oat ( Avena sativa L.) coleoptiles. The growth of the rice and oat coleoptiles was inhibited by ethanol exogenously applied at concentrations greater than 200 and 30 m M , respectively. At 300 m M ethanol, oat coleoptiles were brown and flaccid but rice coleoptiles did not show any visible symptoms of toxicity. The acetaldehyde level in rice and oat coleoptiles was increased by exogenously applied ethanol and the increases were greater in oat than in rice coleoptiles under aerobic and anaerobic conditions. At 300 m M ethanol, the acetaldehyde concentrations in the rice and oat coleoptiles were 46 and 87 nmol g⁻¹ FW under aerobic conditions, respectively, and 52 and 124 nmol g⁻¹ FW under anaerobic conditions, respectively. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) in the direction of ethanol to acetaldehyde was greater in oat than in rice coleoptiles and ADH protein in oat coleoptiles was more induced by exogenously applied ethanol than that in rice coleoptiles. These results suggest that in vivo conversion rate of ethanol to acetaldehyde by ADH is lower in rice than oat coleoptiles, which may be one of the reasons that ethanol sensitivity of rice is much lower than that of oat coleoptiles. The great ability of rice to avoid the ethanol-induced injuries may contribute its anoxia tolerance when glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of energy under anaerobic conditions.