Comparative effects of antithrombitic and antimycotic N-substituted imidazoles on rat hepatic microsomal steroid and xenobiotic hydroxylases in vitro

N-Substituted imidazoles have been shown to be potent inhibitors of microsomal mixed-function oxidase activities in vitro and in vivo. In the present study the effects of two antithrombitic (dazmegrel and dazoxiben) and four antimycotic (ketoconazole, econazole, miconazole and clotrimazole) imidazoles on microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases were studied in vitro. Despite the presence of the N-substituted imidazole moiety, the antithrombitic agents were essentially non-potent as inhibitors of all of the oxidase activities evaluated. In contrast, the antimycotic drugs were potent inhibitory compounds. Binding studies revealed that all six imidazoles elicited type II optical difference spectra and exhibited relatively high affinity for ferricytochrome P-450 in microsomal suspensions (Ks range 0.26-0.73 microM for the antimycotic agents and 6.5 microM and 21 microM for dazmegrel and dazoxiben, respectively). The structural feature that the antithrombitic compounds share is a carboxylate function so that, at physiological pH, less than 1% of the drug would be present in the unionised form. This functionality is absent from the structures of the antimycotic agents which possess much greater hydrophobic character. Even though the antithrombitic imidazoles elicit type II binding interactions of quite high affinity it would appear from this study that significant inhibition potency does not necessarily follow. The present findings also suggest that interesting differences exist between the active site binding regions in the cytochrome P-450 that catalyse thromboxane synthetase activity and those involved in microsomal drug oxidation. Inhibitor hydrophobicity is clearly an important factor in the inhibition of microsomal cytochromes P-450 whereas effective thromboxane synthetase inhibitors may be quite hydrophilic at physiological pH.     

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.     

Induction of drug metabolism. VI. Effects of phenobarbital and 3-methylcholanthrene administration on N-demethylating enzyme systems of rough and smooth hepatic microsomes

The induction of the microsomal hemoproteins, cytochromes P-450 and Pi-450, and of N-demethylase activities in hepatic microsomal subfractions from rats were studied at various times after administration of phenobarbital or 3-methylcholanthrene. After a single dose of phenobarbital, N-demethylase activity and cytochrome P-450 levels increased initially only in rough microsomes (RER) whereas a single dose of 3-methylcholanthrene caused almost simultaneous increases of the two enzymes in both RER and smooth microsomes (SER). The increases in N-demethylase activities during this early period of induction by 3-methylcholanthrene were paralleled by a change in P-450 hemoprotein from cytochrome P-450 to cytochrome P₁-450 in both microsomal subfractions, but the total amount of P-450 hemoprotein remained essentially unchanged. These results add to existing evidence that phenobarbital and 3-methylcholanthrene produce their inductive effects by different mechanisms and raise the possibility that cytochrome P₁-450 may be synthesized in both RER and SER.     

Induction and inhibition of rat hepatic drug metabolism by N-substituted imidazole drugs

Three daily administrations of N-substituted imidazole antimycotics, clotrimazole (CloTZ, 75 mg/kg/day), miconazole (MCZ, 150 mg/kg/day), or tioconazole (TCZ, 150 mg/kg/day), but not the 4,5-disubstituted imidazole cimetidine (350 mg/kg/day) or imidazole (200 mg/kg/day for 4 days), induced rat hepatic cytochrome P-450 and other drug-metabolizing enzymes. These findings paralleled in vitro observations where CloTZ, MCZ, and TCZ were several orders of magnitude more potent as inhibitors of p-nitroanisole O-demethylase activity in control male rat liver microsomes than cimetidine or imidazole. Although no marked difference in inhibitory potency was evident among the N-substituted imidazoles, there were qualitative and quantitative differences in the profiles and extents of induction of various cytochrome P-450-dependent monooxygenases and Phase II conjugation enzymes. Cytochrome P-450 was elevated dramatically by CloTZ (3-4 times the control) and to a lesser extent by MCZ and TCZ (congruent to 1.5 times the control). For all agents, there was an increase in metyrapone binding approximately equivalent to the additional (i.e. above control) cytochrome P-450. Despite the large difference in cytochrome P-450 induction by CloTZ, MCZ, and TCZ, these agents elevated p-nitroanisole demethylase and aniline hydroxylase to similar extents (3-5 X and 1-2 X control, respectively). All agents induced erythromycin and ethylmorphine demethylation in proportion to cytochrome P-450. Ethoxyresorufin O-de-ethylation was not substantially affected by any agent. Large differences in the extent and specificity of induction of microsomal glucuronide conjugations were also evident.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-benzylimidazole, a high magnitude inducer of rat hepatic cytochrome P-450 exhibiting both polycyclic aromatic hydrocarbon- and phenobarbital-type induction of phase I and phase II drug-metabolizing enzymes

The effects of N-benzylimidazole on hepatic microsomal and cytosolic drug-metabolizing enzymes were compared to the effects produced by phenobarbital, beta-naphthoflavone, a polycyclic aromatic hydrocarbon, and Aroclor 1254, a polychlorinated biphenyl mixture. N-Benzylimidazole was a "high magnitude" inducer of male rat hepatic cytochrome P-450, inducing cytochrome P-450 over 3 times above control. N-Benzylimidazole exhibited mixed type induction of cytochrome P-450, producing both polycyclic aromatic hydrocarbon- and phenobarbital-type induction. There was no evidence of imidazole (isoniazid) type induction characteristics. Microsomes from rats treated with either Aroclor 1254 or N-benzylimidazole showed a common pattern of induction of the cytochrome P-450-dependent properties and glucuronosyltransferase activities, and the electrophoretic profiles of proteins were also similar. Cytosolic glutathione transferase activity was also induced similarly after treatment with the two agents.     

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant (K_s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding (ΔA_max) was increased only following phenobarbital induction. The K_s values (ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant (K_m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V_max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO₂ and H₂O₂, but not NaIO₄, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b₅ or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.     

Altered induction response of hepatic cytochrome P-450 to phenobarbital, 3-methylcholanthrene, and beta-naphthoflavone in organotin-treated animals

The effects of tricyclohexyltin hydroxide on the induction of cytochrome P-450 in liver by phenobarbital, 3-methylcholanthrene and beta-naphthoflavone were studied. A single dose of the organotin (15 mg/kg body wt) prevented the full extent of phenobarbital induction of cytochrome P-450 from occurring; this was the case whether tricyclohexyltin was given 48 hr preceeding a single injection of phenobarbital, or administered simultaneously with the first of three daily doses of the drug. Elevation of hepatic heme oxygenase (EC 1.14.99.3) activity accompanied these changes in cytochrome P-450, but the induction of this enzyme was not affected by phenobarbital treatment. The induction of cytochrome P-448 by 3-methylcholanthrene and beta-naphthoflavone was not affected to the same extent by a single injection of tricyclohexyltin, while heme oxygenase induction was less pronounced when these cytochrome P-448 inducers were given together with the organotin. The changes in cytochrome P-450 content and in its functional activity resulting from the various treatments were further examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the microsomal fractions. The electrophoretic profiles illustrate clearly that the apoprotein moieties of the various cytochrome P-450 subspecies are affected to a considerable extent by treatment with tricyclohexyltin hydroxide alone, and staining in these bands was noticeably reduced even when phenobarbital was administered together with the organotin. In contrast, tricyclohexyltin failed to decreased the 3-methylcholanthrene- or beta-naphthoflavone-induced cytochrome P-450 subspecies. These data suggest that significant metabolic interactions can occur from exposure to a combination of environmental chemicals and drugs resulting in an altered metabolism of heme and cytochrome P-450.     

Comparative study of the inductive effect of two psychomoderators--tetrabamate and complexe 1656--and of phenobarbital on liver microsomal enzymes in rats

Tetrabamate (Atrium) and Complexe 1656, two psychosedative drugs, are complexes formed by phenobarbital with its N-substituted derivatives. We have studied the induction effect of all these compounds on the microsomal liver enzymes in male rats. Tetrabamate and Complexe 1656 were found to be equally effective in increasing the amount of microsomal proteins, cytochrome P-450 and the in vitro aniline p-hydroxylation and amino-pyrine N-demethylation activity. These effects were similar to those obtained after a treatment with an equivalent dose of phenobarbital. Febarbamate and difebarbamate had no significanteffect on microsomal enzymes. The inductive effect of tetrabamate and of its analogue seems essentially due to the phenobarbital content of these complexes.     

Induction of hepatic and extrahepatic cytochrome P-450 and monooxygenase activities by N-substituted imidazoles

1. Seven N-substituted imidazoles, with abilities to induce rat hepatic cytochrome P-450 from 1.5- to 4-fold after 3 days of treatment (75 mg/kg daily), were investigated for their concurrent inductive effect in kidney, intestine and lung. 2. The ability of a compound to induce cytochrome P-450 in the liver did not correlate with the ability to induce in extrahepatic tissues, the highest magnitude hepatic inducer (clotrimazole) having little inductive effect in other organs. 3. Induction of cytochrome P-450 concentration was greater in kidney and intestine than in lung but, with the exception of the two imidazoles bearing either a benzyl or a 2-naphthylmethyl substituent, the degree of induction in the extrahepatic organs did not approach that seen in liver. 4. Different monooxygenase activities were preferentially induced by the individual N-substituted imidazoles in a single tissue, and activities induced by a compound in one tissue were not uniformly induced by that compound in other tissues. Induction of activities did not always correlate with an increase in cytochrome P-450 concentration.     

Kinetic alterations in rat liver microsomal cholecalciferol 25-hydroxylase associated with phenobarbital administration

The kinetics of cholecalciferol 25-hydroxylase in vitamin D-depleted rat liver microsomes, before and after phenobarbital induction, were studied. Three days of pretreatment with phenobarbital altered significantly both the apparent K_m and the V_max of the hydroxylase. Untreated vitamin D-repleted rats had lower cytochrome P-450 content and aminopyrine demethylase activity than the vitamin D-depleted animals. Phenobarbital administration reversed this nutritional effect on aminopyrine demethylase but not on cytochrome P-450 content. Furthermore, vitamin D deficiency potentiated the phenobarbital inductive effect upon microsomal protein. No inhibition of aminopyrine demethylase could be elicited in the presence of cholecalciferol or 25-hydroxycholecalciferol either prior to or after phenobarbital treatment, suggesting that these two oxidases are different entities.     

Ketoconazole: a potent inhibitor of cytochrome P-450-dependent drug metabolism in rat liver

Ketoconazole, an orally active imidazole antimycotic agent, is shown to be a potent inhibitor of drug N-demethylase activities of liver microsomes from rats pretreated with phenobarbital or pregnenolone-16 alpha-carbonitrile, and an inhibitor of 7-ethoxyresorufin O-deethylase activity of liver microsomes from rats pretreated with 5,6-benzoflavone. Spectrophotometric studies reveal that the imidazole compound binds to the cytochrome P-450 component of the monooxygenase complex, and has little effect on NADPH-cytochrome c (P-450) reductase activity. These results are strongly suggestive that cytochrome P-450 is the site of action of this potent inhibitor of drug metabolism in liver microsomes.     

Induction of hepatic mono-oxygenase systems of pregnant rats with phenobarbital and 3-methylcholanthrene.

When sodium phenobarbital was given to pregnant and non-pregnant female rats (40 mg/kg for 4 days), ethylmorphine N-demethylase, a cytochrome P-450-dependent system, was induced about 4-fold in non-pregnant females, but only 2-fold in pregnant females. The induction of microsomal cytochrome P-450 was also lower in pregnant animals. This impairment of phenobarbital induction occurred within 3 days of conception and disappeared after parturition within 5 days. 3-Methylcholan-threne induction of hepatic benzo[a]pyrene hydroxylase, a cytochrome P₁-450-dependent mono-oxy-genase system not inducible by phenobarbital, was not impaired during pregnancy. The depressed response of the maternal liver to phenobarbital induction can be partially reversed by the coadministra-tion of 3-methylcholanthrene. The administration of a higher dose of sodium phenobarbital (80 mg/kg day for 4 days) overcame the pregnancy-related lowered response to phenobarbital induction observed with the smaller dose of the barbiturate. The similarity in responses of the maternal and fetal livers to inducing agents suggests that a common regulatory mechanism operates in both the fetus and the pregnant female.     

Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.     

Modulation of the induction of rat hepatic cytochromes P-450 by selenium deficiency

The induction by phenobarbital of liver microsomal cytochrome P-450 has been demonstrated to be impaired in rats fed a selenium-deficient diet. Cytochrome P-450 isozyme specific immunologic and molecular techniques were used in the present study to better define the role of selenium in the induction of cytochrome P-450 by phenobarbital. Phenobarbital treatment of the selenium-deficient rats resulted in an increase in the level of total cytochrome P-450 50% of that observed with control rats and in a 10-fold increase in microsomal heme oxygenase. Quantitative immunoblot analyses demonstrated that the levels of cytochromes P-450b + e and P-450p in the phenobarbital-treated selenium-deficient rats were approximately 50% of those found in the phenobarbital-treated control rats. Finally, RNA hybridization studies using cDNA probes to cytochromes P-450b + e or P-450p demonstrated that the accumulations of the RNAs encoding these cytochromes P-450 were unaffected by the selenium status of the rats. These studies suggest that the impaired phenobarbital induction of the cytochromes P-450 in the selenium-deficient rats is the result of an increase in the degradation of the cytochromes P-450 or a decrease in the translation of the mRNAs coding for them.     

In vitro effects of an imidazole antifungal, prochloraz, on spectral and catalytic properties of microsomal cytochromes P-450

1. The imidazole antifungal agent, prochloraz, elicited type II spectral interactions with microsomal cytochromes P-450 from rats pretreated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and dexamethasone (DEX). 2. Prochloraz interacts strongly with type I binding sites of both PB- and DEX-induced cytochromes P-450 and to a lesser extent with cytochromes P-450 from MC-induced rats. 3. The antifungal derivative was a more potent inhibitor of the troleandomycin-nitrosoalkyl-cytochrome P-450 complex formation in DEX-induced microsomes than of the isosafrole-carbene-cytochrome P-450 complex formation in MC-pretreated rats. 4. Prochloraz is a strong inhibitor of the cytochrome P-450-dependent mixed-function oxidases in rat liver microsomal preparations.     

The effects of differential induction of cytochrome P-450, carboxylesterase and glutathione S-transferase activities on malathion toxicity in mice

The organophosphorous pesticide malathion is metabolized by three hepatic enzyme systems: the microsomal cytochrome P-450-dependent monooxygenase system, the microsomal carboxylesterases, and the cytosolic glutathione S-transferases. We produced differential induction of these three enzyme systems in mice with phenobarbital and 2(3)-tert-butyl-4-hydroxyanisole (BHA) and examined the effects of the induction on the inhibition of acetylcholinesterases by malathion. Phenobarbital not only significantly induced hepatic microsomal cytochrome P-450 (p less than 0.05) but also increased microsomal carboxylesterase activity (p less than 0.05). BHA not only increased the activity of microsomal carboxylesterases (p less than 0.05) but also substantially increased cytosolic glutathione S-transferase activity (p less than 0.05). Despite the differential effects of phenobarbital and BHA on the three enzyme systems, neither agent protected the mice against malathion toxicity.     

In vitro effects of an imidazole antifungal,prochloraz, on spectral and catalytic properties of microsomal cytochromes P-450

1. The imidazole antifungal agent, prochloraz, elicited type II spectral interactions with microsomal cytochromes P-450 from rats pretreated with phenobarbital (PB), 3-methyl-cholanthrene (MC) and dexamethasone (DEX).

2. Prochloraz interacts strongly with type I binding sites of both PB-and DEX-induced cytochromes P-450 and to a lesser extent with cytochromes P-450 from MC-induced rats.

3. The antifungal derivative was a more potent inhibitor of the troleandomycin-nitrosoalkyl-cytochrome P-450 complex formation in DEX-induced microsomes than of the isosafrole-carbene-cytochrome P-450 complex formation in MC-pretreated rats.

4. Prochloraz is a strong inhibitor of the cytochrome P-450-dependent mixed-function oxidases in rat liver microsomal preparations.     

Effect of riboflavin deficiency on phenobarbital and 3-methylcholanthrene induction of microsomal drug-metabolizing enzymes of the rat

The effect of riboflavin deficiency on the induction of hepatic microsomal enzymes by phenobarbital and 3-methylcholanthrene has been investigated. A decrease in microsomal flavin levels of 56 per cent was associated with a decrease in NADPH cytochrome c reductase (52 per cent), azoreductase (71 per cent) and benzpyrene hydroxylase (74 per cent). Microsomal cytochrome P-450 content and aminopyrine demethylase were not significantly affected by flavin deficiency. Phenobarbital or 3-methylcholanthrene pretreatment did not affect hepatic microsomal flavin levels in normal or deficient animals. In flavin-deficient animals, phenobarbital pretreatment significantly increased cytochrome c reductase, cytochrome P-450 content, aminopyrine demethylase and azoreductase. Thus the carbon monoxide-sensitive pathway (cytochrome P-450 mediated) of azoreductase was essentially unaffected by flavin deficiency. In deficient animals, the carbon monoxide-insensitive microsomal azoreductase pathway (non-cytochrome P-450 mediated) normally induced by 3-methylcholanthrene was unaffected. Thus, induction of azoreductase by 3-methylcholanthrene was found to be flavin dependent. However, 3-methylcholanthrene did increase cytochrome P-450 content and benzpyrene hydroxylase in flavin-deficient animals. The induction of benzpyrene hydroxylase by 3-methylcholanthrene increased with increasing microsomal flavin content. Part of the mechanism of azoreductase induction by 3-methylcholanthrene was due to an induced change in the structure or composition of microsomal flavoprotein. This interpretation is supported by the findings that: (1) induction by 3-methylcholanthrene in riboflavin-deficient rats required a minimal flavin level, (2) increased enzyme activity was not compensated by an increase in microsomal flavin and (3) induction by 3-methylcholanthrene augmented FMN-stimulation of microsomal azoreductase in vitro.     

Effect of low-dose phenobarbital on hepatic microsomal UDP-glucuronyl transferase activity

To determine whether hepatic microsomal enzyme induction occurs in rats following administration of phenobarbital at doses similar to those used in humans (0.5 to 7.5 mg/kg), UDP-glucuronyl transferase (UDPGT) and cytochrome P-450 activities were measured in liver homogenate and microsomal preparations from control rats and rats treated for 6 days with phenobarbital at 1 and 3 mg per kg per day. While no significant increases in liver weight and protein content of homogenate and microsomal preparations were observed with either dose of the drug, both UDPGT and P-450 activities were enhanced significantly following administration of phenobarbital at 3 mg per kg per day. The activity of P-450 was increased by approximately 30% and that of UDPGT by 15-24 and 45-66%, respectively, employing bilirubin and p-nitrophenol as the acceptor substrate. The extent of induction of bilirubin or p-nitrophenol UDPGT was similar when measured with "native" enzyme or with enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate. These data suggest that the discordant effects of phenobarbital on UDPGT and cytochrome P-450 previously reported in humans and rats may not be attributable solely to differences in the drug doses employed.     

Ethanol-a direct inducer of drug metabolism.

The inductive effects of chronic phenobarbital and ethanol ingestion on hepatic microsomal drug metabolism were examined in rats consuming different proportions of dietary fat. Eight groups of rats were fed for 1 week on matched amounts of nutritionally adequate liquid diets providing 2, 5, 10 or 41% of calories as fat (safflower oil), two groups at each level. One group at each level received phenobarbital (80 mg/kg daily by intubation), the other getting water. In a parallel experiment, one group at each fat level received 36% of calories as ethanol, the other as sucrose, and these regimens were continued for 1 month. Dietary fat content per se had no significant effect on body weight, liver weight, microsomal protein, cytochrome P-450 content, or hepatic triglycerides (TG) during the 1-week experiment, but significantly increased all these measures in the 1-month experiment. Both phenobarbital and ethanol treatment increased all these measures except body weight, but phenobarbital had a greater effect on liver weight, microsomal protein and cytochrome P-450, while ethanol had a greater effect on hepatic TG and a significant interaction with dietary fat content in relation to TG levels. In contrast, microsomal metabolism of aminopyrine, aniline and meprobamate in vitro was increased approximately equally by phenobarbital and ethanol, while dietary fat content had no effect alone, and no significant interaction with phenobarbital or ethanol. Ethanol, like phenobarbital, therefore, appears to induce microsomal drug metabolism independently of its effects on TG metabolism.