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

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.     

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

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

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[³H] and [¹⁴C]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.     

Hypo activity of cytochrome P-450 after triacetyloleandomycin administration

In a preceding communication we reported that triacetyloleandomycin (TAO) induces its own transformation into a metabolite forming a stable complex with the iron (II) of reduced cytochrome P-450; the complex is unstable in the ferric state and disrupted by potassium ferricyanide. In this communication, we report the effects of TAO administration on various monooxygenase activities. Repeated administration of TAO, 1 mmole/kg i.p. daily for 4 days, markedly prolonged the hexobarbital sleeping time, decreased the in vivo disappearance rate of hexobarbital from the liver, and decreased the in vitro activity of hexobarbital hydroxylase, benzo(a)pyrene hydroxylase, ethylmorphine N-demethylase and the in vitro conversion of 17β-estradiol into water-soluble metabolites. Hypoactivity of musomal enzymes was not detected 1 hr after a single dose and was moderate 24 hr after a single dose of TAO, 1 mmole/kg i.p. In vitro, addition of 0.3 mM TAO to the incubation mixture decreased hexobarbital hydroxylase activity by only 6% in control musomes or musomes from rats killed 1 hr after a single dose of TAO, but decreased it by 30% in musomes from rats killed 24 hr after a single dose of TAO and by 60% in musomes from rats killed after repeated doses of TAO. Addition of 50 μM potassium ferricyanide to the musomes did not modify monooxygenase activities in control musomes but increased them in musomes from rats treated with repeated doses of TAO. It is concluded that the administration of TAO decreases several monooxygenase activities and that hypoactivity is at least partly related to the in vivo formation, in TAO-induced rats, of an inactive cytochrome P-450-TAO metabolite complex.     

Protective effect of 16,16-dimethyl prostaglandin E2 on the hepatotoxicity of bromobenzene in mice

It has been suggested that 16,16-dimethyl prostaglandin E2 may have a cytoprotective effect in the liver. To assess this hypothesis, we determined the effects of this prostaglandin on the metabolism and toxicity of bromobenzene in mice. Administration of 16,16-dimethyl prostaglandin E2 (50 micrograms/kg s.c., 30 min before, and every 6 hr after, the administration of bromobenzene) did not modify the disappearance curves of unchanged bromobenzene from plasma and liver, and did not modify the amount of bromobenzene metabolites covalently bound to hepatic proteins 1-24 hr after the administration of a toxic dose of bromobenzene (0.36 ml/kg i.p.). The prostaglandin, however, markedly reduced serum alanine aminotransferase activity, the extent of liver cell necrosis, the depletion of glutathione, and the disappearance of cytochrome P-450 after administration of this toxic dose of bromobenzene (0.36 ml/kg i.p.). It also markedly reduced mortality after administration of a lethal dose of bromobenzene (0.43 ml/kg i.p.). We conclude that 16,16-dimethyl prostaglandin E2 can prevent hepatic necrosis without decreasing the covalent binding of bromobenzene metabolites to hepatic proteins. The mechanism for this dissociation between covalent binding and toxicity remains unknown.     

Evidence against a physiological role of cAMP in choleresis in dogs and rats.

The relationship between liver cAMP concentration and bile flow was studied after administration of several compounds which increase the bile acid independent flow (BAIF). In the dog, the three drugs tested (glucagon, aminophylline and DB cAMP¹) increased bile flow whereas only two of them (glucagon and DB cAMP) increased liver cAMP concentrations. In the rat, none of the drugs tested (glucagon, aminophylline and MIX²) modified bile flow whereas two of them (glucagon and MIX) increased liver cAMP concentrations. Therefore, there were no parallel variations between the increase in liver cAMP concentrations and the increase in choleresis. Furthermore, (1) no relationship was found between the time course of accumulation of cAMP and the increase in bile flow and (2) no relationship was found between the magnitude of the cAMP accumulation and the increase in bile flow. These data do not support the view that cAMP has a physiological role in bile formation.     

Assessment of mercuric chloride-induced nephrotoxicity by p-aminohippuric acid uptake and the activity of four gluconeogenic enzymes in rat renal cortex.

p-Aminohippurate (PAH) uptake and the activities of four gluconeogenic enzymes were used in vitro as indicators of nephrotoxicity in rats. The purpose was to compare the relative sensitivities of the gluconeogenic enzymes (pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-diphosphatase, and glucose-6-phosphatase), measured in renal cortical homogenates, and PAH uptake, measured in renal cortical slices, following ip treatment with mercuric chloride (HgCl₂). The morphologic aspects of the nephrotoxicity produced by HgCl₂ were also evaluated. A time study (30 days) following administration of 2 mg/kg of HgCl₂ showed that PAH uptake was more sensitive to HgCl₂ than the activities of the four gluconeogenic enzymes. The greatest inhibitory effect was noted on Day 6 and an increase toward normal was seen from Day 7. A dose-response study undertaken 6 days after HgCl₂ showed that 1 mg/kg caused significant decreases in the activities of fructose-1,6-diphosphatase and pyruvate carboxylase. However, PAH uptake was significantly reduced at 0.5 mg/kg; with this dosage no morphologic renal alterations were observed. PAH uptake was found to be more sensitive for detecting HgCl₂-induced nephrotoxicity than were the activities of the four gluconeogenic enzymes, with fructose-1,6-diphosphatase being the most sensitive of the four enzymes.     

Effects of phenobarbital and diphenylhydantoin on acute vitamin D3 toxicity in the rat.

Methylene chloride is biotransformed in vivo to carbon monoxide (CO), and increased carboxyhemoglobin (COHb) concentrations occur in animals and man following exposure. Binding of CO to hemoglobin causes a shift to the left in the position of the oxyhemoglobin dissociation curve (ODC), theoretically resulting in a diminished ability to release oxygen in tissue. This study was designed to determine the effect of methylene chloride alone and in combination with CO on altering the ODC. Methylene chloride and CO, alone and in combination, were incubated with rat and human blood in vitro and the position of the ODC was measured by the P50 value. The results indicate clearly that methylene chloride alone had no effect on changing the P50 value of the ODC. It was concluded that any shift to the left of the ODC following exposure to methylene chloride is due solely to CO formed from the biotransformation of methylene chloride. Thus, speculation that methylene chloride increases the affinity of Hb for CO is not supported.     

Effects of biogenic amines on the concentration of adnosine 3',5'-monophosphate in bovine superior cervical ganglion.

The accumulation of cyclic AMP elicited in bovine superior cervical ganglia by several biogenic amines in the presence of theophylline was investigated in vitro. The response to dopamine (10^?4M), after 6 min of incubation, was found to exceed that induced by equimolar concentrations of norepinephrine. Experiments with an inhibitor of dopamine-beta-oxidase showed that prior conversion of dopamine to norepinephrine was not necessary for the activation of ganglionic adenylate cyclase. The effect of norepinephrine could be prevented by pre-incubation with a combination of alpha- and beta-adrenergic antagonists; the cyclic AMP increase caused by epinephrine was predominantly beta-sensitive. Tyramine, 5-hydroxytryptamine and histamine caused a moderate increase in the concentration of cyclic AMP. The effect of histamine, which could be reduced by pre-incubation with an antihistamine, burimamide, was found to be additive to that of dopamine and norepinephrine.     

Isomerically pure bromobiphenyl congeners and hepatic mono-oxygenase activities in the rat: influence of position and degree of bromination.

Isomerically pure di-, tri-, tetra-, penta-, hexa-, and octabromobiphenyls were injected ip into weanling rats at a dosage of 0.2 mmol/kg/day for 3 consecutive days and the animals were killed 96 hr after the last injection. The influence of position and degree of bromination of the biphenyl nucleus on hepatic function was assessed by measuring changes in activities of hepatic p-nitroanisole O-demethylase (OD) and aniline hydroxylase (AH), by examining morphological changes by electron microscopy, and by comparing the results with those of animals treated with equivalent doses of Firemaster BP6. Bromobiphenyl residues were extracted from liver and analyzed by gas-liquid chromatography. Hepatic O-demethylase activities were increased proportionally as the degree of bromination increased, observations which correlated well with the increased tissue residues and changes in morphology. Marked increases in aniline hydroxylase were observed following treatment with certain di- and tribromobiphenyl congeners but, as the degree of bromination increased above four atoms per molecule, aniline hydroxylase activity was reduced to levels equal to or below those of control, vehicle-treated animals. In contrast, increases in both mono-oxygenases were observed following treatment with Firemaster BP6, fivefold increases for OD and twofold for AH. In vitro experiments with highly brominated congeners suggested that these influenced the AH assay by interacting with microsomal cytochrome P-450.