Jean-Gil Joly

Ethanol-induced cytochrome P-450: Catalytic activity after partial purification☆

Abstract Cytochrome P-450 was partially purified from liver microsomes obtained from control, ethanol, phenobarbital, and 3-methylcholanthrene-treated rats. Benzphetamine demethylation, benzpyrene hydroxylation and aniline hydroxylation activities were assayed in a reconstituted system using fixed amounts of reductase and lipids, and increasing amounts of cytochrome P-450 from each source. Cytochrome P-450 from ethanol-fed rats showed substrate specificity differing from cytochrome P-450 obtained from control, phenobarbital and 3-methylcholanthrene-treated rats.

Effects of chronic ethanol administration in the rat: Relative dependency on dietary lipids—I.: Induction of hepatic drug-metabolizing enzymes in vitro

Abstract Female Sprague-Dawley rats were fed nutritionally adequate liquid diets with or without ethanol, at two ethanol concentrations, 5 and 6% (w/v). In other animals, various degrees of caloric deficiency were obtained by replacing ethanol by water in one animal of a pair. Ethanol given as a 5% (w/v) solution with high amounts of dietary fat increased cytochrome P-450, the activities of NADPH-cytochrome P-450 reductase, benzphetamine demethylation, aniline hydroxylation and microsomal ethanol-oxidizing system (MEOS). When ethanol was given with a low fat diet as a 5% (w/v) solution, the increase in cytochrome P-450 and P-450 reductase was much less than with a high fat diet; the other enzyme activities, however, were enhanced to a level comparable to that achieved with the high fat diet. When ethanol was administered as a 6% (w/v) solution in presence of a low fat diet, caloric deficiency was observed and no significant induction of any parameter except aniline hydroxylation could be found. When it was given with a high fat diet, in spite of caloric deficiency and lower ethanol ingestion, cytochrome P-450 and P-450 reductase activities were enhanced while that of MEOS was not. Ingestion of ethanol as a 6% (w/v) solution with a high fat diet resulted in a negligible weight gain. Higher basal levels of cytochrome P-450, P-450 reductase and benzphetamine demethylation activities were found in animals rendered caloric-deficient. Ethanol is associated with a greater induction of drug-metabolizing enzyme activities in the high fat model compared to the low fat model. Induction of drug-metabolizing enzymes by ethanol is partly dependent on dietary lipids as well as on the amounts of ethanol ingested and on the caloric status of the animal.

Effect of chronic ethanol administration on bromobenzene liver toxicity in the rat

Female Sprague-Dawley rats were pair-fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 3 weeks. In vitro studies showed that chronic ethanol pretreatment preferentially increased the liver microsomal biotransformation of bromobenzene to p-bromophenol (via the toxic 3,4-epoxide) rather than to o-bromophenol (via the nontoxic 2,3-epoxide) and could thus potentiate bromobenzene hepatotoxicity. Bromobenzene administration (500 mg/kg body weight, ip), after an overnight fast, was associated in ethanol-pretreated rats with greater and accelerated liver glutathione depletion and subsequent decrease in liver cytochrome P-450 content than in controls. As assessed histologically and by determination of the rise in activities of serum enzyme markers of liver necrosis, chronic ethanol pretreatment, however, mainly resulted in earlier onset and resolution of bromobenzene-induced liver necrosis, with only a mild increase in the maximal severity of liver lesions. These results suggest that the twofold increase in liver microsomal bromobenzene 3,4-epoxidation by ethanol, being much less than that seen after phenobarbital pretreatment in our animal model and in that of others, is apparently not sufficient to markedly affect the severity of bromobenzene-induced liver toxicity.

Differences in the duration of the enhancement of liver mixed-function oxidase activities in ethanol-fed rats after withdrawal

Liver microsomal mixed-function oxidase activities were determined in female Sprague-Dawley rats after 3 weeks of ethanol feeding and for up to 10 days after withdrawal. Ethanol (36% of total calories) was administered in a high fat liquid diet and was replaced isocalorically by carbohydrates in controls. Chronic ethanol feeding similarly enhanced both microsomal cytochrome P-450 content and benzphetamine N-demethylase activity, per mg of protein, and resulted in a disproportionate increase in both aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities. A 6- to 7-day withdrawal period was apparently necessary for the overall disappearance of these effects of ethanol. Marked differences, however, were seen in the time courses of return of these variables to control levels, as also indicated by changes, during this period and specially during the first 24 hr after withdrawal, in the apparent molar activity of the microsomal fraction with the three substrates tested. The results were interpreted as indicating that the distinct ethanol-inducible cytochrome P-450 isozyme, with a high specific activity toward aniline, undergoes a very rapid turnover in liver microsomes. Induction of another form of cytochrome P-450, differing from the former by its slower turnover rate, would explain the induction by ethanol of 7-ethoxycoumarin O-deethylase activity. The withdrawal of ethanol was followed by a rapid but transient increase in benzphetamine N-demethylase activity above the ethanol-induced level, at a time when other activities were rapidly declining. This could suggest that the microsomal content of other cytochrome P-450 isozyme(s), with high specific activity toward this substrate, would also be temporarily altered during ethanol withdrawal. Important alterations in microsomal cytochrome P-450-dependent mixed-function oxidase activities occurred during the initial 24-hr period of withdrawal, even in the absence of a change in microsomal cytochrome P-450 content, indicating that the effects of chronic ethanol ingestion on hepatic drug-metabolizing enzyme activities may also be highly dependent on the proximity of ethanol intake.

CATECHOLAMINE LEVELS IN PORTAL, HEPATIC, AND SYSTEMIC VENOUS BLOOI IN PORTAL HYPERTENSION

Abstract Combined hepatic and umbilico-portal venous catheterisation was used to sample blood in the vena cava, the hepatic vein, and the portal vein in eleven patients with hypertension and five patients without portal hypertension. Catecholamine levels were detremined by means of a fluorometric method. No difference in adrenaline and noradrenaline plasma levels was found in the two groups. Mean portal venous plasma levels of noradrenaline were higher than mean hepathic venous plasma levels, suggesting that the liver metabolises circulating noradrenaline. It is concluded that cate- cholamine plasma levels have no hearing on portal hypertension.

Mechanism of induction of hepatic drug-metabolizing enzymes by ethanol—I: Limited role of microsomal phospholipids☆

Abstract Two groups of female Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing 36 per cent of total calories as ethanol or additional carbohydrate (controls). One group was fed a high fat diet (35 per cent of total calories as fat); the other group was fed a low fat diet (2 per cent of total calories as linoleate as the only source of fat). When given with the high fat diet, ethanol increased cytochrome P-450 and microsomal phospholipid content per g of liver. When given with a low fat diet, it increased cytochrome P-450 to a lesser extent and did not alter the microsomal phospholipid content when expressed per g of liver or per 100 g of body weight. Phosphatidylcholine accounted for the greater porportion of phospholipids and was increased by ethanol only with the high fat diet. L -Methionine-methyl[ 3 H] and [ 14 C]choline incorporation into phosphatidylcholine was unaltered by ethanol in either model. The fatty acid composition of phosphatidylcholine was altered by ethanol more significantly with the high fat diet. Since ethanol similarly enhances the activity of benzphetamine demethylation in both dietary models, quantitative and qualitative differences in microsomal phospholipids produced by ethanol are probably not responsible for the induction by ethanol of this drug-metabolizing enzyme activity. Further evidence to that effect was obtained from the measurement of benzphetamine demethylation in vitro by partially purified cytochrome P-450, reductase and lipid fractions of ethanol-fed and control rats fed a high fat diet. The stimulation of benzphetamine demethylation by the lipid fraction was identical whether using lipids from microsomes of ethanol-fed animals or control animals. Dietary fat plays a role in the induction by ethanol of cytochrome P-450 and NADPH-cytochrome P-450 reductase and on microsomal phospholipid content and composition. The effects of ethanol on microsomal phospholipids are probably not related to the induction of benzphetamine demethylation activity.

Effects of chronic acetone administration on ethanol-inducible monooxygenase activities in the rat

Liver microsomal monooxygenase activities known to be ethanol-inducible were determined in female Sprague-Dawley rats after 2-week treatment with 1% (v/v) acetone. Daily acetone intake was in the order of 1.2 g/kg. The final body weight, liver weight and microsomal protein content of acetone-treated rats were identical to those of untreated controls. Microsomal NADPH-cytochrome c reductase activity was also unaffected, while cytochrome P-450 content was only increased 12-18%. Ethanol-inducible p-nitrophenol hydroxylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation activities were enhanced 5.3-, 4.4- and 2.6-fold, respectively, by chronic acetone treatment. The sex-dependent inducing effect of ethanol on benzphetamine N-demethylation activity in female rats was not observed however, after acetone. Addition of acetone in vitro had a stimulatory effect on aniline hydroxylation by microsomes from control and acetone-induced rats. Acetone, however, was found to be a competitive inhibitor of p-nitrophenol hydroxylation activity (apparent Ki = 1.8 mM), an observation suggesting that p-nitrophenol is a more selective substrate than aniline for rat liver ethanol- and acetone-inducible cytochrome P-450j. Interruption of the chronic acetone treatment for 24 hr resulted in the almost complete disappearance of its inducing affects, this treatment apparently reproducing only the rapidly reversible preferential inducing effects of chronic ethanol administration. This experimental model of induction by acetone in the rat, when compared to chronic ethanol administration, would thus permit a more selective look at the consequences of these common inducing effects in particular, with respect to drug metabolism and toxicity in vivo, and this, in the absence of the hepatotoxic effects of ethanol itself.

Effects of chronic ethanol administration in the rat: relative dependency on dietary lipids--III. In vivo tolerance to hexobarbital.

Abstract Chronic administration of ethanol with either a high-fat (35% cal., including 2% cal. as linoleate) or a low-fat (2% cal. as linoleate) diet reduces similarly the hexobarbital sleeping times in the rat. Ethanol decreased the plasma and total body half-lives of hexobarbital in both dietary models, but they were decreased significantly more with the high-fat diet. A good correlation between hexobarbital plasma half-life and sleeping time was found only with the high-fat dietary model; the sleeping time was not a good index of hexobarbital metabolism in the low-fat model. Ethanol-fed rats awaken with significantly higher hexobarbital concentrations in brain and other tissues; this phenomenon is significantly more important in the high-fat model. Ethanol, administered chronically in nutritionally adequate liquid diets, increases tolerance to hexobarbital by increasing drug disposition and by decreasing central nervous system sensitivity. Both factors in tolerance are altered by modifications of the dietary lipid intake.

MECHANISM OF INDUCTION BY ETHANOL OF HEPATIC MICROSOMAL DRUG METABOLIZING ENZYMES

It is widely accepted that alcoholics when sober are tolerant to a number of drugs. Although some effects of chronic ethanol administration are due to alterations of the sensitivity of the target organ to drugs (1), it has also been shown that ethanol accelerates the metabolism of xenobiotics (2). It is now accepted that microsomal hepatic drug biotransformation is stimulated by chronic ethanol administration (3,4).