Effect of isolation stress on ethanol pharmacodynamics in the blood of rats

Head space chromatography was used to study the rate of ethanol excretion as criterion of the activity of the ethanol-oxidizing enzymatic system. Isolational stress was shown to be one of the factors determining induction of the ethanol-oxidizing erzymatic systems. The isolation-induced activity of these systems was reduced by diazepam administration. This fact may evidence the existence of central regulation of the activity of the enzymatic systems that metabolize ethanol.     

Hepatic ethanol metabolism: Respective roles of alcohol dehydrogenase,the microsomal ethanol-oxidizing system,and catalase

The respective role of alcohol dehydrogenase, of the microsomal ethanol-oxidizing system, and of catalase in ethanol metabolism was assessed quantitatively in liver slices using various inhibitors and ethanol at a final concentration of 50 mm. Pyrazole (2 mm) virtually abolished cytosolic alcohol dehydrogenase activity but inhibited ethanol metabolism in liver slices by only 50–60%. The residual pyrazole-insensitive ethanol oxidation in liver slices remained unaffected by in vitro addition of the catalase inhibitor sodium azide (1 mm). At this concentration, sodium azide completely abolished catalatic activity of catalase in liver homogenate as well as peroxidatic activity of catalase in liver slices in the presence of dl-alanine. Similarly, in vivo administration of 3-amino-1,2,4-triazole, a compound which inhibits the activity of catalase but not that of the microsomal ethanol-oxidizing system, failed to decrease both the overall rates of ethanol oxidation and the activity of the pyrazole-insensitive pathway. Finally, butanol, a substrate and inhibitor of the microsomal ethanol-oxidizing system but not of catalase-H₂O₂, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H₂O₂ accounts for most if not all of the alcohol dehydrogenase-independent pathway.     

Pharmacological analysis of the participation of ethanol-oxidizing enzyme systems in ethanol metabolism at different stages of experimental alcoholism

Using head space chromatography, the pharmacological analysis of changes in the activity of ethanol-oxidizing enzymatic systems: alcohol dehydrogenase, catalase, microsomal ethanol-oxidizing system under the effect of pyrazole and aminotriazole, has been performed on the model of experimental alcoholism in rats. It was shown that the rate of ethanol elimination from the rats' blood at all stages of experimental alcoholism was determined by alcohol dehydrogenase, while catalase and microsomal ethanol-oxidizing system activities did not play an important role.     

Hepatic microsomal ethanol-oxidizing system: solubilization, isolation, and characterization

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD⁺ to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K_m for ethanol (7.2 mm, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 mm) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K_m for oxygen (8.3 μm), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H₂O₂-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.     

Ethanol-metabolizing enzymes from human testis

Testicular ethanol-metabolizing enzymes (alcohol dehydrogenase, microsomal ethanol-oxidizing system, catalase) were investigated. Alcohol dehydrogenase was purified to homogeneity and its main kinetic parameters were analyzed. It was shown that alcohol dehydrogenase corresponds to class III isozymes and does not participate in ethanol oxidation. The testicular microsomal ethanol-oxidizing activity does not exceed 0.02 nmol/min/mg of protein. The activity of catalase and its peroxidase component is far lower in the testes than in the liver. On the whole, testicular tissue is rather inactive in respect of ethanol oxidation.     

Ethanol metabolism in the yeasts Yarrowia and Torulopsis (a review)

Results of research into ethanol metabolism in yeast organisms with highly pronounced aerobic metabolism are reviewed. The low activity of NAD-dependent alcohol dehydrogenase (EC 1.1.1.1), observed under the conditions of aerobic yeast growth on ethanol, demonstrates that alternative enzyme systems--alcohol oxidase (EC 1.1.3.13), microsomal ethanol-oxidizing system (including cytochrome P-450), and catalase (EC 1.11.1.6)--may be involved in the alcohol oxidation. The role of these systems in alcohol oxidation and conditions favoring their operation in this processes are analyzed. It is concluded that iron ions are important regulators of ethanol metabolism the microorganisms of this group.     

Ethanol Metabolism in the Yeasts <Emphasis Type="Italic">Yarrowia</Emphasis> and <Emphasis Type="Italic">Torulopsis</Emphasis>: A Review

Results of research into ethanol metabolism in yeast organisms with highly pronounced aerobic metabolism are reviewed. The low activity of NAD-dependent alcohol dehydrogenase (EC 1.1.1.1), observed under conditions of aerobic yeast growth on ethanol, demonstrates that alternative enzyme systems—alcohol oxidase (EC 1.1.3.13), microsomal ethanol-oxidizing system (including cytochrome P-450), and catalase (EC 1.11.1.6)—may be involved in the alcohol oxidation. The role of these systems in alcohol oxidation and the conditions favoring their operation in this processes are analyzed. It is concluded that iron ions are important regulators of ethanol metabolism for the microorganisms of this group.     

Respective role of superoxide and hydroxyl radical in the activity of the reconstituted microsomal ethanol-oxidizing system.

Superoxide dismutase, a scavenger of O^?₂. does not affect the rate of ethanol oxidation in a reconstituted system containing purified cytochrome P-450, NADPH-cytochrome c reductase, and dilauroyl l-3-phosphatidyl choline. The same concentration of Superoxide dismutase (50 μg/ml) completely abolishes the oxidation of epinephrine in this reconstituted system and ethanol oxidation by the xanthine-xanthine oxidase. Ethanol is not oxidized by the reconstituted system when NADPH is replaced by H₂O₂ but the addition of H₂O₂ to this sytem containing NADPH accelerates ethanol oxidation. This increase is abolished by the addition of Superoxide dismutase. Hydroxyl radical scavengers (50 mm dimethylsulfoxide, 100 mm benzoate, 100 mm mannitol, 20 mm thiourea) diminish the oxidation of ethanol in the reconstituted system by 48 to 76%. Thus hydroxyl radical may participate in the activity of reconstituted ethanol-oxidizing system, whereas Superoxide is not involved.     

Activities of hepatic enzymes related to ethanol oxidation and effects of ethanol on hepatic metabolites in rats with chronic liver injury.

To study the effects of ethanol on liver chronically injured by CCl4, activities of hepatic enzymes related to ethanol oxidation, influences of ethanol on hepatic metabolites, and blood ethanol disappearance were observed. (1) Activities of alcohol dehydrogenase, low- and high-Km aldehyde dehydrogenase, microsomal ethanol-oxidizing system and drug-metabolizing enzyme were remarkably decreased in the injured liver. (2) Increases in lactate/pyruvate and beta-hydroxybutyrate/acetacetate ratios were shown in control liver 2 h after ethanol ingestion. Similar but less pronounced effects of ethanol on the 'redox state' were also seen in rats with chronic liver injury. (3) Delay in ethanol disappearance was not observed until 12 h after ethanol ingestion. The ethanol-induced changes in the redox state in the injured liver were similar to those in controls. Higher ethanol concentrations in blood from rats with chronic liver injury could be related to potentiate the injured liver.     

Effect of thyroid hormones on the activities of hepatic alcohol metabolizing enzymes.

Treatment with thyroxine or triiodothyronine for 7 days in order to simulate a hyperthyroid state results in an enhanced activity of the microsomal ethanol oxidizing system. Conversely, a decrease of hepatic alcohol dehydrogenase activity was observed under these experimental conditions, whereas hepatic catalase activity remained unchanged. These findings suggest that if chronic ethanol consumption simulates a “hyperthyroid hepatic state”, increased rates of ethanol metabolism observed following prolonged alcohol intake might therefore be attributed at least in part to an induction of microsomal ethanol oxidizing system activity in the liver.     

Absence of ethanol metabolism in "acatalatic" hepatic microsomes that oxidize drugs

When liver slices of Cs^a and Cs^b mice were incubated $\text{in}\text{vitro}$, they had similar catalase activities and equal rates of ethanol metabolism. While incubated liver homogenates and microsomes from Cs^a mice oxidized ethanol and retained catalase activity, preparations from Cs^b mice did not oxidize ethanol and lost all catalase activity. Addition of beef liver catalase restored ethanol oxidation by Cs^b microsomes. The oxidations of aniline and aminopyrine proceeded at the same rate in Cs^a and Cs^b microsomes and were inhibited by ethanol. It is evident that (a) the microsomal drug-metabolizing pathway is not involved in ethanol oxidation, and (b) the postulation of a unique microsomal ethanol-oxidizing system (“MEOS”) that is independent of microsomal catalase is unwarranted.     

The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles

The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and affinity elution with nucleotides. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers. ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented. Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.     

Effect of central adrenergic alpha receptor blockader IEM-611 on the activity of alcohol and aldehyde dehydrogenase in rat liver

The effect of IEM-611 (30 mg/kg) on alcohol consumption in rats under the conditions of voluntary choice between water and 15% ethanol was studied as that on alcohol dehydrogenase (ADH) in postmitochondrial supernatant and in NAD-dependent aldehyde dehydrogenases (A1DH) in liver mitochondria. Administration of IEM-611 during 6 or 12 days reduces ethanol consumption by 29 and 30%, respectively, activates ADH and appreciably decreases overall activity of NAD-dependent A1DH. At the same time the ADH/A1DH ratio increases. Activation of ADH and A1DH and the decreased ADH/A1DH ratio were disclosed in alcohol preferring rats as compared to water preferring animals. IEM-611 shifts enzymatic activity of ethanol metabolism towards the level characteristic for water preferring rats. It is suggested that variation of the ADH/A1DH ratio is one of the mechanisms responsible for the decreased ethanol consumption in rats.     

Effect of alcohol in combination with stress in the prenatal period on adult mice behaviour

The aim of the present study was to investigate the effects of the prenatal alcohol and stress on behaviour of adult CBA/LacJ male mice. Pregnant mice were given ethanol 11% from to 21 days of the gestation and were exposed to restraint stress for two hours daily from 15 to 21 days gestation. At 3 months of age, the offspring were tested for behaviour. Alcohol and stress-exposed animals buried more marbles in the marble-burying test, which models obsessive-compulsive disorders (OCD). In addition, the alcohol and stress-exposed males showed increased social activity. No significant effects of the prenatal alcohol and stress exposure on locomotor activity, anxiety, exploring activity of the adult male mice were revealed. Conclusion was made that exposure to the alcohol and stress combination in prenatal period produces predisposition to OCD.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

The microsomal ethanol oxidizing system: its role in ethanol and xenobiotic metabolism

After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases and contributes to ethanol tolerance, as most conclusively shown in alcohol-dehydrogenase-negative deermice. In man and animals, there is an associated rise in microsomal cytochrome P-450, including a specific form (P-450IIEI) with high affinity for ethanol and for the activation of some drugs (i.e. acetaminophen), carcinogens (i.e. N-nitrosodimethylamine) and hepatotoxic agents (i.e. CCl4), thereby contributing to the susceptibility of alcoholics to xenobiotics, including industrial solvents. In addition, a benzoflavone-inducible liver cytochrome P-450 isoenzyme distinct but catalytically similar to cytochrome P-450IIE1 was purified which may play a significant role in drinkers who also are heavy smokers. Cross-induction of other microsomal enzymes is associated with enhanced metabolism of various drugs, resulting in drug tolerance. Catabolism of retinol was also found to be accelerated, in part through activation of newly discovered vitamin A depletion and possibly toxicity. Thus, elucidation of the microsomal metabolism of ethanol explains a number of complications that develop in alcoholics.     

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.     

The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C_1a or C₂ domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C_1a or C₂ domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl₂, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC₅₀ were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.     

Circadian rhythms of alcohol dehydrogenase and MEOS in the rat

The circadian peak in alcohol dehydrogenase (ADH) activity fell near the time of maximal blood ethanol clearance rates both in groups of rats injected with a single ethanol dose (acute group) and in rats continuously exposed to ethanol for 22 weeks (chronic group). However, at all timepoints investigated ADH activity levels were lower and fluctuated less in the chronic group than in either the acute or control (ethanol naive) groups. In contrast, activity levels of the microsomal ethanol oxidizing system (MEOS) revealed a prominent rhythm that was 180 degrees out of phase with the ADH rhythm in the chronic group, while MEOS activity showed very low levels in the acute and control groups and did not vary over the circadian span.