Liver ADH Activity Research Articles

Effects of the extracts and an active compound curcumenone isolated from Curcuma zedoaria rhizomes on alcohol-induced drunkenness in mice

The Curcuma zedoaria rhizome has been used traditionally to treat gastrointestinal diseases as an aromatic stomachic drug, and this is currently used to treat alcohol-induced loss of appetite and nausea in Japan. We examined the effects of various fractions and isolated compounds on alcohol-induced drunkenness and blood alcohol concentrations in mice. The 30% ethanol-extract (1000mg/kg) of C. zedoaria rhizome prevented drunkenness 60 and 120min after 40% alcohol administration. The n-hexane-soluble fraction (300mg/kg) and an isolated compound (3, 10 or 30mg/kg) prevented drunkenness at 30, 60 or 120min. The extract, n-hexane-soluble fraction and isolated compound reduced the elevation in blood alcohol concentrations 30 and 60min after 40% alcohol administration. The isolated compound (10 and 30mg/kg) enhanced liver ADH activity 30 and 60min after 40% alcohol administration. The compound was identified as curcumenone by a direct comparison of 1H- and 13C-NMR spectral data. In conclusion, the protective effect of the C. zedoaria extract on drunkenness might be due to an active substance, curcumenone, and decreases in the elevation of blood alcohol concentrations through increased liver alcohol dehydrogenase activity.

Reduction of Ethanol Consumption in Alcohol-Preferring Rats by Dual Expression Gene Transfer

To mimic, in an animal model of alcoholism, the protective phenotype against alcohol consumption observed in humans carrying a fast alcohol dehydrogenase (ADH1B*2) and an inactive aldehyde dehydrogenase (ALDH2*2). We developed a multiple expression cassette adenoviral vector (AdV-ADH/asALDH2) encoding both a fast rat ADH and an antisense RNA against rat ALDH2. A control adenoviral vector (AdV-C) containing intronic non-coding DNA was also developed. These adenoviral vectors were administered intravenously to rats bred as high alcohol-drinkers (University of Chile bibulous) that were previously rendered alcohol dependent by a 75-day period of voluntary 10% ethanol intake. Animals administered AdV-ADH/asALDH2 showed a 176% increase in liver ADH activity, whereas liver ALDH2 activity was reduced by 24%, and upon the administration of a dose of ethanol (1g/kg, i.p.), these showed arterial acetaldehyde levels that were 400% higher than those of animals administered AdV-C. Rats that received the AdV-ADH/asALDH2 vector reduced by 60% their voluntary ethanol intake versus controls. This study provides evidence that the simultaneous increase of liver ADH and a reduction of ALDH activity by gene transfer could constitute a potential therapeutic strategy for the treatment of alcoholism.

Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse.

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V max⁡/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3.

A new view of alcohol metabolism and alcoholism--role of the high-Km Class III alcohol dehydrogenase (ADH3).

The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol intake increases. Furthermore, ADH3 is induced in damaged cells that have greater hydrophobicity, whereas ADH1 activity is lower when there is severe liver disease. These data suggest that chronic binge drinking and the resulting liver disease shifts the key enzyme in alcohol metabolism from low-Km ADH1 to high-Km ADH3, thereby reducing the rate of alcohol metabolism. The interdependent increase in the ADH3/ADH1 activity ratio and AUC may be a factor in the development of alcoholic liver disease. However, the adaptive increase in ADH3 sustains alcohol metabolism, even in patients with alcoholic liver cirrhosis, which makes it possible for them to drink themselves to death. Thus, the regulation of ADH3 activity may be important in preventing alcoholism development.

Effect of catalase activators and inhibitors on ethanol pharmacokinetic parameteres and ethanol and aldehyde-metabolizing enzyme activities in the rat liver and brain

The effects of catalase regulators (aminotriazole, lead acetate, taurine, di-2-ethylhexylphthalate) on the preference for ethanol, its pharmacokinetics, and activities of rat liver and brain ethanol and acetaldehyde-metabolizing enzymes were studied. Lead acetate (100 mg/kg, i.p., 7 days), aminotriazole (1 g/kg, i.p., 7 days), and taurine (650 mg/kg, i.g., 14 days) decreased ethanol consumption under conditions of free choice (10% ethanol water), whereas di-2-ethylhexylphthalate (300 mg/kg, i.g., 7 days) did not exert any effect on this parameter. Taurine, lead acetate and di-2-ethylhexylphthalate significantly activated liver ADH, MEOS and catalase peroxidase activity. Aminotriazole also activated ADH and MEOS, but inhibited liver catalase. The activities of liver and brain A1DH as well as catalase were insignificantly changed by this treatment. The 7-day administration of lead acetate, di-2-ethylhexylphthalate and aminotriazole administrations significantly influenced the ethanol (2 g/kg., i.p.) pharmacokinetic parameters: the area under the pharmacokinetic curve and the elimination half-life time were significantly reduced, whereas the elimination constant and clearance were increased. This unequivocally indicates accelerated ethanol elimination. The 14-day ingestion of taurine insignificantly changed the parameters of ethanol pharmacokinetics in rats.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

ALCOHOL ELIMINATION AND THE REGULATION OF ALCOHOL CONSUMPTION IN AA AND ANA RATS

Previous studies have usually found that animals with either higher alcohol elimination rates or ADH (alcohol dehydrogenase, EC1.I.I.I) activities have higher voluntary intakes of alcohol than ones with lower elimination rates. This relationship has now been studied in the AA and ANA rat lines genetically developed, respectively, for high and low alcohol consumption. Female AA and ANA rats had their alcohol elimination rate measured before being given a free choice between 10% (v/v) alcohol and water for 3 weeks. The elimination rate was then measured again and liver ADH activity was determined. The alcohol elimination rate was significantly higher in AA than ANA rats before drinking and was increased by alcohol drinking in AA but not ANA rats. ADH activity was similar in both lines and unrelated to either alcohol drinking or elimination rates, suggesting that the enzyme activity is not a rate-limiting factor in the alcohol metabolism of these two lines. The present results support the conclusion that alcohol elimination and alcohol consumption are partially determined by genetics. Furthermore, although alcohol elimination itself probably does not have direct control over drinking, some factor related to the alcohol elimination rate appears to be among the mechanisms influencing the level of alcohol drinking.

Structure of the mouse Adh-1 gene and identification of a deletion in a long alternating purine-pyrimidine sequence in the first intron of strains expressing low alcohol dehydrogenase activity

The ethanol-active alcohol dehydrogenase (ADH-A 2) expressed at high levels in mouse liver is encoded by the Adh-1 gene. Inbred strains differ in the amount of ADH-A 2 expressed. We report here the cloning and sequencing of the Adh-1 genes from mouse strains that express high and low amounts of ADH-A 2 in liver (strains YBR/Ki and Balb/c respectively). The gene contains nine exons, and encodes an ADH-A subunit identical to that encoded by the cDNA isolated from DBA/2J, a strain with low liver ADH activity. This demonstrates that the difference between strains in liver ADH activity is not due to differences in the amino acid sequence of the ADH-A 2. The 5'-nontranslated region and at least the first 225 bp 5' to the transcriptional start point are identical in both strains. We have found restriction fragment length polymorphisms in the Adh1 gene that correlate with the level of expression of ADH-A 2 in different strains. One of these RFLPs is within a remarkably long (288 bp) strictly alternating purine-pyrimidine sequence located in the first intron. This region in YBR/Ki contains 25 copies of the sequence ATGT(A/G)T (four of them inverted), which closely resembles important elements in the SV40 enhancer region. Balb/c mice, which express Adh-1 at lower levels, have a deletion that removes 101 bp of this sequence and also have several transition mutations; the comparable region has nine fewer ATGT(A/G)T repeats. These results suggest that the difference in gene expression may be due to differences in these hexamers or in other portions of the alternating purine-pyrimidine sequences, rather than in cis-acting sequences in the proximal 5' (promoter) region.

The effects of starving and refeeding on the intra-acinar distribution pattern of alcohol-dehydrogenase activity in rat liver.

Microquantitative determinations of ADH activity were carried out on the livers of male and female rats. The animals were either starved for 84 h, or starved and then refed with a carbohydrate-rich diet for 6 nights. When the enzyme activity is expressed in mumoles/min/g dry weight, fasting does not appear to alter liver ADH activity, while in starved and subsequently refed rats it is diminished by 20%. Microquantitative measurements of ADH activity in 50-150 ng lyophilized tissue samples, microdissected the whole way along the sinusoidal length, made the computer-aided plotting of intra-acinar distribution patterns possible. The results showed that, under the feeding conditions selected, only minor changes in the ADH activity profiles occur in the liver acinus. These are within the range of the standard deviations of the normal mean values. From these results it can be deduced that fasting and refeeding do not lead to specific inhibition or induction of liver ADH activity. - The decrease of ADH activity of total liver (mumol/min) per total body weight in starved rats is obviously the result of a loss of protein which affects the liver cells of all acinar zones almost equally.

Genetic considerations in the effects of ethanol in mice. I. Genotype-dependent alterations in alcohol dehydrogenase activity.

Most genetic studies on individual and racial differences in sensitivity to alcohol intoxication have concentrated on genetic variations associated with structural genes for the enzymes involved in alcohol metabolism, including alcohol dehydrogenase (ADH; E.C. 1.1.1.1). We studied the ethanol-induced regulation of ADH following chronic administration of ethanol in mice. Newly weaned males from six inbred strains (BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, S.W., and 129/ReJ) were subjected to ethanol administration. Alterations in the level of liver ADH activity, relative to matched littermate controls, were evaluated. The change in ADH activity was found to be strain (genotype) specific, which may explain the contradictory results in the literature. Strains which showed induction of ADH activity, in general, reflected a strain-specific time-dependent profile. Strains which showed repression, however, were independent in the degree of repression to the duration of ethanol exposure. Such variable, ethanol-induced regulatory responses (induction/repression) in ADH activity of different genotypes may account for individual and population variations in response to alcohol. Additional work, however, is needed to establish the molecular bases of ADH inducibility and its specific role in relative susceptibility to alcohols.

Differences between the sexes in voluntary alcohol consumption and liver ADH-activity in inbred strains of mice

During a self-selection period of four weeks, tests were made of the voluntary alcohol consumption of 21 males and 20 females of an inbred CBA mouse strain, and 23 males and 19 females of a C57BL-strain. The difference in alcohol consumption between the strains proved to be highly significant. There was also apparent a highly significant difference in the alcohol consumption of different sexes of the C57BL-strain, a strain which evidences a high spontaneous alcohol preference. Statistically significant differences in the total fluid consumption and in the body weight were also discernible between the strains and the sexes. Strain differences in voluntary alcohol consumption explained 69.4 per cent of the total variance of the males, and 94.2 per cent of that of the females; this indicates that the genetic strain differences are more pronounced in females. The sex differences explained 53.3 per cent of the variance of the C57BL-strain, and 0 per cent of the variance of CBA. The liver ADH-activity was determined in respect of 20 subjects, representing equal numbers of strains and sexes. The difference in ADH-activity was in the same direction as voluntary alcohol consumption, although less in extent.

Ethanol inhibition of sorbitol oxidation in liver slices of propyl thiouracil-treated and control rats

Experiments have been made with a view to studying the effect of ethanol on sorbitol oxidation in liver slices from PTU-treated and normal rats. The liver ADH and SDH enzyme activities were measured, and compared with the oxidation rates of ethanol and sorbitol in slice experiments. The liver ADH activity of the group treated with PTU was significantly higher than that of the control group. This effect of the drug may have been mediated by the thyroid gland. No significant differences were discernible either in the SDH activity or the oxidation rate of sorbitol alone in liver slices from the groups. The addition of ethanol to the incubation medium significantly inhibited sorbitol oxidation, in the liver slices from both PTU-treated and control rats.

Genetic and biochemical correlates of alcohol preference in mice.

Certain strains of mice prefer alcohol to water in preference tests whereas others avoid alcohol. This is the behavior which we are trying to explain, and we have formulated several explanatory hypotheses: 1. It was hypothesized that animals of high preference strains metabolize alcohol more quickly than animals of low preference strains. This hypothesis was confirmed. 2. We have asked the question whether there are differences in the activity of liver ADH, the enzyme which catalyzes the rate limiting reaction in alcohol metabolism. We have found that the activity of this enzyme is significantly greater in high than in low preference strains. 3. We have asked whether there are differences in the rate of accumulation of acetaldehyde in high and low preference strains. Tests indicated that significantly greater amounts of blood acetaldehyde accumulate during alcohol metabolism in low than in high preference strains. 4. We have asked whether there are differences in aldehyde dehydrogenase which might account for this differential acetaldehyde accumulation in high and low preference strains. Only an indirect answer is available, but it was found that Antabuse, an inhibitor of aldehyde dehydrogenase, acts differently in high and low preference strains. 5. We have asked whether Antabuse will decrease voluntary consumption of alcohol in normally high or intermediate preference strains. Antabuse was found to decrease voluntary alcohol intake in all strains tested. 6. We have asked whether in mice which show a decrease in alcohol preference with age, liver ADH activity also decreases with time. The answer to this question was affirmative. 7. It was hypothesized that mice which are selectively bred for high and low alcohol preference show a correlated selection response in terms of liver ADH activity. The answer to this question was affirmative; mice selected for high alcohol preference have higher liver ADH activity than mice selected for low alcohol preference. A number of questions were asked about the metabolism of alcohol in different strains of mice in attempting to discover the underlying biochemical reasons for these differences in alcohol preference behavior. In every case the answers make it appear as if we are heading in the right direction. No one, of course, can say whether these differences which we have described are the real answer to why certain mice drink alcohol and others do not. What can be said, however, is that a good start in that direction has been made.