Rolf Teschke

Increased Carbon Tetrachloride Hepatotoxicity, and its Mechanism, After Chronic Ethanol Consumption

Clinical observations suggest that alcoholism predisposes to CCl4 hepatotoxicity. To study whether hepatic changes secondary to chronic ethanol consumption play a role, female rats were pair-fed, for 4 to 5 weeks, a nutritionally adequate liquid diet containing either ethanol (36% of total calories) or isocaloric carbohydrate (controls). CCl4 (0.5 ml per kg) was given intragastrically 15 hr after ethanol withdrawal. Within 24 hr, serum ornithine carbamyl transferase activity as well as liver lipid increased, whereas hepatic cytochrome P-450 content and the activities of hepatic aminopyrine N-demethylase and glucose-6-phosphatase decreased. All of these CCl4-induced changes were significantly greater in rats fed ethanol chronically than in their pair-fed controls. Moreover, in the ethanol-CCl4 treated animals, the activities of serum ornithine carbamyl transferase and glutamic pyruvic transaminase, and the concentration of serum bilirubin and liver lipid were significantly higher, whereas the content of hepatic cytochrome P-450, as well as the activities of hepatic aminopyrine N-demethylase and of glucose-6-phosphatase, were significantly lower than the values obtained in rats treated with CCl4 alone. These findings indicate the in vivo potentiation of CCl4 hepatotoxicity by chronic ethanol consumption, even in the absence of ethanol at the time of CCl4 administration. To study the mechanism of this ethanol-CCl4 synergism, liver microsomes were incubated with CCl4 and a reduced nicotinamide adenine dinucleotide phosphate-generating system. Destruction of cytochrome P-450 in vitro caused by CCl4 was much greater in microsomes of ethanol-treated rats than in their controls. In addition, chronic ethanol consumption increased the covalent binding of 14CCl4 metabolites to microsomal protein in vitro. Furthermore, the metabolism of 14CCl4 to 14CO2 in vitro was also found to be enhanced in the ethanol-fed rats. These results suggest that the increase of the CCl4 hepatotoxicity in chronic ethanol-fed rats is due to an enhanced microsomal activation and biotransformation of CCl4.

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

Abstract 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 m m , insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 m m ) 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 μ m ), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H 2 O 2 -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.

Microsomal ethanol-oxidizing system (MEOS): Purification and properties of a rat liver system free of catalase and alcohol dehydrogenase

Abstract A separation of the microsomal ethanol-oxidizing system (MEOS) from alcohol dehydrogenase (ADH) and catalase in rat liver microsomes is described. After solubilization of microsomes, ADH and catalase were eluted by DEAE-cellulose chromatography with the hemoglobin containing fraction, whereas MEOS activity was recovered separately. The oxidation of ethanol to acetaldehyde required O2 and NADPH, and was partially inhibited by CO. The fractions exhibiting MEOS activity contained cytochrome P-450, NADPH-cytochrome c reductase and phospholipids. These three components also increased in total microsomes after chronic ethanol consumption and may play a role in the associated enhancement of MEOS activity.

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

Abstract 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 m m . Pyrazole (2 m m ) 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 m m ). 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-H 2 O 2 , 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-H 2 O 2 accounts for most if not all of the alcohol dehydrogenase-independent pathway.

[37] The Microsomal ethanol oxidizing systems (MEOS)

Publisher Summary This chapter describes procedures for assaying the activities of microsomal ethanol oxidizing system (MEOS) in total liver tissue, in microsomes, and in partially purified microsomal fractions. It is assumed that ethanol metabolism proceeds exclusively via alcohol dehydrogenase (ADH), an enzyme of the cell sap of the hepatocyte. Indeed, this concept is satisfactory at low ethanol concentrations because the oxidation of ethanol is almost completely abolished under these conditions by pyrazole, a potent inhibitor of alcohol dehydrogenase activity. Recent studies have shown that, in addition to ADH, ethanol can also be metabolized by the microsomal fraction of the hepatocyte, which comprises the endoplasmic reticulum. MEOS is also differentiated from ADH, in contrast to ADH the microsomal ethanol oxidation is more active with NADPH than with NAD, has a neutral pH optimum, and shows a relative insensitivity to the ADH inhibitors pyrazole and 4- methylpyrazole.

Acceleration of Ethanol Metabolism by High Ethanol Concentrations and Chronic Ethanol Consumption: Role of the Microsomal Ethanol Oxidizing System (MEOS)

Chronic ethanol consumption has been shown to be associated with an acceleration of ethanol metabolism in man (1–3) and rats (4–6). This phenomenon could not be explained by the activity of alcohol dehydrogenase itself, as reviewed elsewhere (7). Some other possible mechanisms have been suggested, including the adaptive increase of the microsomal ethanol oxidizing system (MEOS) (5, 8, 9), increased mitochondrial reoxidation of NADH (10–12) and enhanced catalase-H2O2 mediated peroxidation (13, 14).