Emanuel Rubin

Hepatic Microsomal Enzymes in Man and Rat: Induction and Inhibition by Ethanol

The feeding of ethanol increased significantly the activities of hepatic pentobarbital and benzpyrene hydroxylases in rats, and, in human volunteers, doubled pentobarbital hydroxylase activity. In vitro ethanol inhibited aniline, pentobarbital, and benzpyrene hydroxylases. These data may explain, at least in part, the increased tolerance of alcoholics to sedatives when sober, and the enhanced sensitivity to sedatives when inebriated.

Ethanol Increases Hepatic Smooth Endoplasmic Reticulum and Drug-Metabolizing Enzymes

Rats were fed ethanol for 2 weeks along with diets either adequate or deficient in protein and choline, the latter intake being similar to that of many alcoholics. Hepatic lipids, smooth endoplasmic reticulum, and the activities of drug-metabolizing enzymes (aniline hydroxylase and nitroreductase) were increased with the adequate diet but more so with the deficient one. These results may explain the increased tolerance by alcoholics of drugs such as sedatives.

Effects of chronic ethanol treatment on mitochondrial functions damage to coupling site I

Abstract Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg 2+ - and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD + -dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD + -dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the P O ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD + -dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca 2+ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding. Concentrations of acetaldehyde (1–3 m m ) which inhibited phosphorylation associated with the oxidation of NAD + -dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro .

Ethanol Produces Muscle Damage in Human Volunteers

Repeated administration of ethanol (42 percent of total calories) for 28 days increased serum creatine phosphokinase activity and produced ultrastructural changes in skeletal muscle of human volunteers. The data suggest that alcoholic myopathy results from ethanol toxicity, rather than from nutritional or other factors.

The effect of acetaldehyde on mitochondrial function

Abstract The oxidation of ethanol by the liver produces acetaldehyde, which is a highly reactive compound. Low concentrations of acetaldehyde inhibited mitochondrial respiration with glutamate, β-hydroxybutyrate, or α-ketoglutarate as substrates, but not with succinate or ascorbate. High concentrations led to respiratory inhibition with all substrates. Inhibition of succinate- and ascorbate-linked oxidation by acetaldehyde correlates with the inhibition of the activities of succinic dehydrogenase and cytochrome oxidase. A site more sensitive to acetaldehyde appears to be localized prior to the NADH-ubiquinone oxidoreductase segment of the respiratory chain. Acetaldehyde inhibits energy production by the mitochondria, as evidenced by its inhibition of respiratory control, oxidative phosphorylation, the rate of phosphorylation, and the ATP- 32 P exchange reaction. Energy utilization is also inhibited, in view of the decrease in both substrate- and ATP-supported Ca 2+ uptake, and the reduction in Ca 2+ -stimulated oxygen uptake and ATPase activity. The malate-aspartate, α-glycerophosphate, and fatty acid shuttles for the transfer of reducing equivalents, and oxidation by mitochondria, were highly sensitive to acetaldehyde. Acetaldehyde also inhibited the uptake of anions which participate in the shuttles. The inhibition of the shuttles is apparently caused by interference with NAD + -dependent state 3 respiration and anion entry and efflux. Ethanol (6–80 m m ) had no significant effect on oxygen consumption, anion uptake, or mitochondrial energy production and utilization. The data suggest that acetaldehyde may be implicated in some of the toxic effects caused by chronic ethanol consumption.

Interaction of calcium with microsomes: a modified method for the rapid isolation of rat liver microsomes.

The addition of 8 mM Ca++ to a dilute post-mitochondrial supernatant of rat liver allows the isolation of the microsomal fraction by centrifugation at 30 xg for 10 minutes. The yield of microsomal protein and phospholipids is comparable to that obtained by centrifuging the untreated supernatant at 105,000 xg for one hour. The composition and functional activities of the membranes isolated by both methods are also similar. This method circumvents the need for an ultracentrifuge and results in a saving of time, particularly when multiple washes are necessary.

The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes

Abstract The NADPH-dependent oxidation of ethanol by rat liver microsome preparations was studied in the presence of azide to inhibit the peroxidatic activity of catalase. Dimethylsulfoxide, benzoate, mannitol and thiourea, four compounds that react rapidly with hydroxyl radicals, each inhibited the oxidation rate of ethanol. Inhibition was competitive with respect to ethanol. In contrast, urea, a compound that reacts poorly with hydroxyl radicals, was essentially without effect. Dimethylsulfoxide at concentrations that inhibited the oxidation of ethanol had no effect on the xanthine oxidase-mediated oxidation of ethanol or on aniline hydroxylase or aminopyrine demethylase activity of microsomes. These results suggest that ethanol oxidation by microsomes can be dissociated from drug metabolism and that the mechanism of ethanol oxidation may involve, in part, the interaction of ethanol with hydroxyl radicals that are generated by microsomes during the oxidation of NADPH.

Effect of ethanol and acetaldehyde on the (Na+ + K+)-activated adenosine triphosphatase activity of cardiac plasma membranes

Abstract Ethanol and acetaldehyde inhibited the (Na + + K + )-activated ATPase activity of plasma membranes prepared from the guinea-pig heart. The degree of inhibition was dose-dependent and antagonized by the K + concentration in the reaction mixture. The inhibition is not attributable to increase in osmolality. The presence of ethanol or acetaldehyde in the reaction mixture was necessary for the inhibitory effect. Plasma membranes treated with ethanol or acetaldehyde and subsequently washed showed no impairment of (Na + + K + )-activated ATPase activity. Prolonged exposure of the plasma membranes to a low concentration of ethanol was ineffective in increasing the inhibition of ATPase activity.

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria.

Abstract The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.

Effects of ethanol on calcium binding and calcium uptake by cardiac microsomes

Abstract Calcium uptake and Ca binding by cardiac microsomes enriched in fragmented sarcoplasmic reticulum were inhibited by ethanol. After 5 min of incubation, half-maximal inhibition of the former was seen at approximately 1.3 M ethanol, that of the latter was seen at approximately 1.9 M ethanol. Neither Ca uptake nor Ca binding was stimulated at lower concentrations of ethanol; Prolonged exposure of the microsomes to ethanol increased the extent of inhibition of both Ca uptake and Ca binding. These inhibitory effects were almost completely reversed when microsomes were washed after exposure to ethanol. Inclusion of 0.12 M Na+ instead of 0.12 M K+ increased the inhibitory effect of ethanol on Ca uptake, but did not significantly change the sensitivity of Ca binding to ethanol. These inhibitory effects of ethanol were seen at concentrations that generally stabilize membrane structures and are associated with an anesthetic action. Even though the concentrations of ethanol needed to inhibit Ca uptake and Ca binding exceeded those found in the blood of chronic alcoholic patients, the time dependence of these effects raises the possibility that prolonged exposure to concentrations of ethanol might contribute to the myocardial weakness of chronic alcoholics by interfering with the retention of Ca2+ within the cardiac sarcoplasmic reticulum.