Protective effects of selenium on acetaminophen-induced hepatotoxicity in the rat

Experiments were undertaken to examine the ability of selenium to protect against acetaminophen-induced hepatotoxicity and to examine possible mechanisms for this protective effect. Pretreatment of male, Sprague-Dawley rats with sodium selenite (12.5 mumol Se/kg, ip) 24 hr prior to acetaminophen administration produced a significant protection against the hepatotoxic effects of acetaminophen as assessed by a decrease in the plasma appearance of alanine aminotransferase and aspartate aminotransferase activities following acetaminophen. This was accompanied by an increase in the hepatic glutathione levels in selenium-treated animals and an inhibition in the decrease in hepatic glutathione content observed in animals receiving hepatotoxic doses of acetaminophen. Selenium pretreatment decreased the in vivo covalent binding of acetaminophen metabolites to hepatic protein, but did not alter hepatic microsomal cytochrome P-450 content or NADPH cytochrome c reductase activity, suggesting that selenium does not significantly alter the metabolism of acetaminophen to reactive electrophilic metabolites by the cytochrome P-450-dependent mixed-function oxidase enzyme system. Selenium produced an increase in the activity of gamma-glutamylcysteine synthetase which may account for the increased glutathione availability in selenium-treated animals and increased the activities of glutathione S-transferase and glucose-6-phosphate dehydrogenase. Examination of the urinary metabolite profile in selenium-treated animals revealed that the urinary excretion of acetaminophen and its metabolites was significantly increased over a 72-hr period. The increase occurred in the AAP-glucuronide metabolite while parent AAP and AAP-sulfate were actually decreased in selenium-treated rats. No change in recovery was observed in the AAP-glutathione or AAP-mercapturate urinary metabolites. While the glutathione conjugating system is enhanced by selenium treatment, amelioration of acetaminophen toxicity is most likely the result of enhanced glucuronidation which effectively diverts the amount of acetaminophen to be converted by the cytochrome P-450 system to the toxic metabolite.     

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.     

Cytochrome P-450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes.

The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence the hepatic metabolism of substrates associated with the P450 I, IIB, IIE, III and IV families of cytochromes. In contrast, cytosolic glutathione S-transferase activity was markedly reduced and glutathione levels were significantly lowered. These findings raise the possibility that patients suffering from this disease may be more susceptible to chemicals that rely on glutathione conjugation for their deactivation.     

Inhibition of coal fly ash polycyclic aromatic hydrocarbons and metals induced mixed-function oxidase activity in rat lung and liver by vitamin A and citrate

Administration of benzene-soluble fraction (FAE) and benzene-insoluble fraction (FAR) of fly ash for 3 consecutive days to rats significantly raised cytochrome P-450 levels, aryl hydrocarbon hydroxylase (AHH) activity, and glutathione S-transferase activity in liver. This treatment also significantly increased pulmonary AHH and glutathione S-transferase activity. Intratracheal administration of FAR (5 mg/100 g body weight) alone for 6 consecutive days also significantly increased hepatic cytochrome P-450 levels and the activity of glutathione S-transferase. Intragastric administration of retinyl palmitate (5000 IU/100 g body weight), along with intratracheal FAE and FAR administration, significantly reduced P-450 levels, activity of glutathione S-transferase in liver, and activity of AHH and glutathione S-transferase in lung of rats. Intraperitoneal administration of citrate (40 mg/100 g body weight) along with FAR significantly reduced FAR-induced increase in hepatic cytochrome P-450 levels and glutathione S-transferase activity. The activity of AHH was not affected by these treatments.     

Mixed-function oxidase system induction and propylene hepatotoxicity

Propylene is hepatotoxic to male Charles River COBS Sprague-Dawley rats pretreated with polychlorinated biphenyls (PCB: Aroclor 1254). Four-hour inhalation exposure to 50,000 ppm propylene increased liver weight/body weight ratios and elevated serum enzyme activities in PCB-pretreated animals. Hepatic microsomal cytochrome P-450 content of PCB-pretreated rats dropped profoundly during propylene exposure and remained depressed for at least 24 h. In addition, PCB-pretreated, propylene-exposed rats exhibited a decrease in the specific activity of hepatic microsomal aniline hydroxylase. However, there was no change in activities of either hepatic microsomal aminopyrine demethylase or glucose-6-phosphatase. Propylene exposure of rats pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), or a mixture of BNF and PB was not hepatotoxic. However, there was, in these animals, a substantial decline in hepatic microsomal cytochrome P-450 levels 24 h after the start of propylene exposure. Hence, the propylene-dependent process resulting in hepatic cytochrome P-450 destruction is qualitatively or quantitatively different from the process that causes acute hepatotoxicity. Preexposure fasting had no effect on the hepatotoxicity resulting from a 4-h exposure of PCB-pretreated rats to 50,000 ppm propylene. Administration of SKF-525A to PCB-pretreated rats immediately prior to propylene exposure completely prevented elevations in serum enzyme activities and liver weight/body weight ratios. In vitro incubation of hepatic microsomes prepared from either BNF-, PB-, or PCB-pretreated rats with an atmosphere of 20% propylene/80% air produced in NADPH-dependent decrease in cytochrome P-450 content. These results suggest that PCB pretreatment is a prerequisite for propylene hepatotoxicity in the rat. Cytochrome P-450-dependent bioactivation of propylene is associated with this hepatotoxicity, but further studies are needed to characterize the mechanism of the PCB-propylene interaction.     

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats

Halothane-induced hepatic microsomal lipid peroxidation in guinea pigs and rats was examined with respect to the mixed function oxidase system, anaerobic dehalogenation activity of halothane, and the antioxidant system. The levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase were significantly higher in guinea pigs than in rats. There was no difference between the two animals in anaerobic dehalogenation activity of halothane per cytochrome P-450 in microsomes. Microsomal alpha-tocopherol was significantly lower in guinea pigs than in rats, and was increased by multiple exposure to halothane in guinea pigs but remained lower than in rats. Microsomal alpha-tocopherol was decreased in rats by multiple exposure. The concentration of reduced glutathione and ascorbic acid was decreased significantly by multiple exposure to halothane in guinea pigs but not in rats. These results suggest that the higher level of halothane-induced hepatic microsomal lipid peroxidation in guinea pigs is due to the large production of radical metabolites resulting from the large amounts of cytochrome P-450, the high activity of NADPH-cytochrome P-450 reductase, and the low concentration of microsomal alpha-tocopherol.     

Aging and drug metabolism: alteration of liver drug metabolizing ability in male rats. Is it functional deterioration or feminization of the liver?]

The profiles of hepatic drug metabolism were obtained by using young and old male and female rats. The profile obtained from old male rats was completely different from that from young male rats, while it was almost identical to those of females of any ages. This was due to the selective decrease in male hepatic enzyme activities showing higher activities than females to the activity levels of females which did not alter much with aging. Castration of young adult male rats caused a decrease in enzyme activities but did not result in the feminization of the metabolic profile. Administration of testosterone to old male rats resulted in the recovery of the profile of young male rats, but the levels of activities were not as high as young male rats. Plasma testosterone levels were found to decrease in parallel with drug metabolizing activities of male rats during aging. These results suggest that sex hormones play important roles in the alteration of drug metabolizing activities in male rats with aging. The loss of male characteristics in profile of drug metabolism during aging was evaluated by use of antibody to the male specific cytochrome P-450, P-450 m1. Anti-P-450 ml strongly inhibited imipramine N-demethylase activity, which showed marked sex (male greater than female) and age (young greater than old) differences, while this did not inhibit imipramine 2-hydroxylase activity, which showed no sex or age differences. The portion of N-demethylase activity inhibited by this antibody decreased in old rats while the portion not inhibited did not decrease with age. These results indicate that the decrease of sex specific cytochrome P-450 is responsible for the age-associated decrease in at least one of the drug metabolizing enzyme activities in male rats. It is suggested that some processes of the control mechanism of the gene expression of male specific cytochrome P-450 may be altered with old age.     

Effects of piperonyl butoxide on halothane hepatotoxicity and metabolism in the hyperthyroid rat

A series of experiments were conducted to examine the potential role of phase I metabolism in halothane-induced liver injury in the hyperthyroid rat. The metabolism of halothane was determined in both hyperthyroid (triiodothyronine, 3 mg/kg per day, for 6 days) and euthyroid rats and in animals pre-treated with the cytochrome P-450 inhibitor piperonyl butoxide (75-100 mg/kg, i.p.). It was found that the hyperthyroid state, which is associated with a substantial increase in sensitivity to the hepatotoxic effects of halothane, decreases both oxidative and reductive routes of halothane metabolism in the rat. The production of trifluoroacetic acid (TFA), an oxidative metabolite, as well as that of chlorodifluoroethylene (CDF) and chlorotrifluoroethane (CTF), 2 reductive metabolites, was significantly reduced in hyperthyroid animals. Consistent with these findings serum and urinary bromide levels resulting from the formation of TFA, CDF or CTF were significantly reduced. The only route of halothane metabolism significantly increased by the hyperthyroid condition was the defluorination of halothane. Piperonyl butoxide administration did not render euthyroid animals sensitive to the halothane-induced hepatotoxicity and had no effect on the defluorination of halothane in euthyroid animals. However, piperonyl butoxide markedly increased the hepatotoxicity of halothane in hyperthyroid rats and, except for a modest increase in debromination reactions, decreased all measured indices of halothane metabolism including the defluorination of halothane. Thus, none of the observed changes in halothane metabolism produced by triiodothyronine or piperonyl butoxide treatment could be consistently correlated to the increases in hepatotoxicity linked to these 2 treatments. Based on these studies we suggest that the halothane hepatotoxicity induced in the hyperthyroid rat results from effects produced by either the parent compound or an as yet unidentified metabolite. In addition, these studies further demonstrate that considerable mechanistic differences exist for halothane-induced hepatotoxicity when comparing euthyroid and hyperthyroid animal models.     

Induction of the hepatic cytochrome P-450 dependent monooxygenase system by cis- and trans-5,10-dihydrogen mirex

The hepatic induction of cytochrome P-450-dependent monooxygenase components by cis- and trans-5,10-dihydrogen mirex was studied in male and female laboratory rats. There were sex-dependent differences between the two isomeric derivatives of mirex. The cis-isomer significantly increased aniline hydroxylase activity in the female, but not in the male. In contrast, aminopyrine N-demethylase was significantly increased by the cis-isomer in both sexes. The trans-isomer increased the hydroxylase and N-demethylase activities in both sexes. The cis-isomer induced NADPH-cytochrome c reductase in the female, and the trans-isomer did not. Both isomers induced hepatic reductase activity in the male rat. No sex-dependent differences in the hepatic induction of cytochrome P-450 were observed for either isomer.     

Effect of monensin on liver glutathione, glutathione S-transferase and monooxygenases in rats.

Monensin administered ip to male rats at a dosage of 2.5 mg/kg/d for 3 consecutive days did not change the liver levels of glutathione, but depressed significantly the amount of cytochrome P-450 and the activities of aniline hydroxylase and a cytosolic CDNB-specific glutathione S-transferase. There was a marked decrease in the aminopyrine N-demethylase activity and a significant increase in the pentobarbital sleeping time in rats treated with monensin. In contrast, no change in these parameters was found 2 h after a single ip dose (7.5 mg/kg) of monensin. The results suggest that monensin-induced inhibition of the liver cytosolic glutathione S-transferase and microsomal monooxygenases is non-specific.     

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.     

Induction of hepatic drug-metabolising enzymes following treatment of rats and mice with chlordimeform

We investigated the effects of the formamidine insecticide chlordimeform upon the activities of various hepatic drug metabolising enzymes in rats and mice. Chlordimeform treatment induced several enzyme activities. However, the extent of induction depended upon the activity studied, the sex of the animal and the species selected. Microsomal cytochrome P-450 content was elevated in both male and female rats and mice. Ethoxycoumarin O-deethylase activity was induced in male and female rats but not in mice, whilst ethylmorphine N-demethylase activity was elevated in mice, but not in rats. Benzo(a)pyrene hydroxylase activity was increased in female rats and mice, but not in males. UDP-glucuronyl transferase, glutathione S-transferase and microsomal epoxide hydrolase were induced in a dose-dependent manner in male rats, and female rats and mice, but not in male mice.     

Modulation of carbon tetrachloride hepatotoxicity and xenobiotic-metabolizing enzymes by corticosterone pretreatment, adrenalectomy and sham surgery

These investigations sought to determine the role of physiological concentrations of natural glucocorticoids in modulating chemical toxicity, and to ascertain if effects on toxicity may be due to alterations of chemical metabolizing enzymes by glucocorticoids. The hepatotoxic response to carbon tetrachloride (CCl4) in adrenalectomized or naive Long Evans rats treated with corticosterone was assessed. Alterations of hepatic cytochrome P-450 concentration, mono-oxygenase activities, NADPH-cytochrome (P-450)c reductase activity, and glutathione S-transferase activity were examined. Adrenalectomy and to a lesser extent sham surgery were protective, but corticosterone administration increased CCl4 hepatotoxicity. Corticosterone administration to adrenalectomized or sham-operated rats reduced the protective effect of these treatments. Correlating with the in vivo response, mono-oxygenase activities decreased after adrenalectomy and sham surgery, but increased with glucocorticoid administration. These studies suggest that basal, stress-associated, and pharmacological concentrations of a natural glucocorticoid can modify chemical toxicity and alter hepatic enzymes important to chemical metabolism.     

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.     

Effect of length of exposure to malathion on xenobiotic biotransformation in male rat liver

The effect of exposure to malathion on several parameters of hepatic xenobiotic biotransformation was studied in male Sprague-Dawley rats. Groups of rats dosed i.p. daily for 1 or 2 weeks with 40 or 200 mg/kg malathion showed an increase in epoxide hydrolase activity (1 week, 200 mg/kg) and glutathione S-transferase activity (1 week, 200 mg/kg; 2 weeks 40 and 200 mg/kg). Aldrin epoxidation was decreased after 1 week of exposure to 200 mg/kg and by both dosage regimens after 2 weeks. After 9 weeks exposure to 40 mg/kg malathion administered i.p. 3 times per week, however, no changes in hepatic xenobiotic biotransformation were noted. The results demonstrate that only continuous exposure to high doses of malathion results in an induction of epoxide hydrolase and glutathione S-transferase activities. Inductive effects on hepatic cytochrome P-450 monooxygenase activity were not observed irrespective of whether exposure was short- or medium-term.     

Effect of subchronic cholestasis on microsomal mixed-function oxidases and the glutathione-conjugating enzyme system in rat liver

Bile duct ligation in male rats for two weeks led to a marked increase in both serum sorbitol dehydrogenase activity and serum bile acid concentration indicating cholestatic liver injury. Furthermore, a rise in the hepatic hydroxyproline level indicating collagen accumulation was observed. As a consequence of these alterations, the hepatic microsomal mixed-function oxidase system was impaired as evidenced by a decrease in cytochrome P-450 content and in the activities of NADPH-cytochrome c reductase and aminopyrine-demethylase. While the hepatic glutathione content remained unaffected, the cytosolic GSH S-transferase activity was clearly suppressed due to subchronic cholestasis.     

Effects of model traumatic injury on hepatic drug metabolism in the rat. VI. Major detoxification/toxification pathways

A previously validated small mammal trauma model, hindlimb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on two of the major hepatic enzymes of detoxification, glutathione S-transferase and epoxide hydrolase. Hepatic cytosolic glutathione S-transferase activity toward a variety of substrates showed a 26-34% decrease at 24 hr after model injury. Hepatic microsomal epoxide hydrolase activity toward 1,2-epoxy-3-(p-nitrophenoxy)propane was diminished by 53% after model trauma. Both enzymatic activities toward styrene oxide were similarly depressed. The toxicological sequelae of these derangements were illustrated by administering a dose of styrene oxide (300 mg/kg, ip) which was below the threshold dose (350 mg/kg, ip) necessary to produce hepatotoxicity in control animals. Model trauma dramatically enhanced the hepatotoxic effects of the subthreshold dose, as well as the covalent binding of labeled styrene oxide to liver proteins. These findings indicate that traumatic injury renders the animal more susceptible to agents which are detoxified by glutathione S-transferase and epoxide hydrolase. Conversely, model trauma provided almost complete protection from the hepatotoxic effects of a standard dose (200 mg/kg, ip) of bromobenzene. This protection appeared to derive from a post-traumatic alteration of cytochrome P-450 subpopulations that decreased the formation of the potentially toxic 3,4-epoxide metabolite, despite an increase in the cytochrome P-448-mediated generation of the nontoxic 2,3-epoxide. For bromobenzene, the change in cytochrome P-450-mediated activation appeared quantitatively more significant in overall toxicity than the post-traumatic depression of detoxification pathways described above, leading to decreased toxicity in vivo. For other compounds, the combination of post-traumatic influences on cytochrome P-450/P-448 activity and epoxide hydrolase/glutathione S-transferase activities could lead to markedly enhanced toxicity.     

Effects of long-term choline deficiency on hepatic microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases in the female rat

Total cytochrome P-450 levels decreased to about 80% of control in hepatic microsomes from female rats maintained for 30 weeks on a choline-deficient diet. Livers from these rats were fibrotic and had extensive fatty infiltration but, unlike livers of male rats on the same regimen, were not cirrhotic. Steroid hydroxylase activities were assessed in microsomes of female rats that received the choline-deficient diet and it was noted that the activity of the cytochrome P-450 UT-F-mediated steroid 7 alpha-hydroxylase was decreased to about 50% of the activity present in choline-supplemented control rat microsomes. Similar decreases were observed for microsomal androstenedione 6 beta-hydroxylase and aniline 4-hydroxylase activities. In female rat hepatic microsomes these two activities are probably mediated by the isozyme cytochrome P-450 ISF-G. In contrast to these findings, the activities of four other xenobiotic metabolising enzymes, as well as rates of microsomal steroid 16 alpha- and 16 beta-hydroxylation, were unchanged from control. Thus, in hepatic microsomes from choline-deficient female rats, it appears likely that levels of the non-sexually differentiated cytochromes P-450 UT-F and ISF-G are decreased. Unlike the situation in male rats, long term choline deficiency does not appear to influence levels of sexually-differentiated P-450 enzymes in the female rat.     

Species and sex differences in the inhibitory action of the corticosteroid alclometasone dipropionate on the hepatic drug-metabolizing system

The effect of successive administration of the corticosteroid alclometasone dipropionate (ACM) on the hepatic drug-metabolizing system was examined using male and female rats. Although some pharmacological changes such as increases in plasma enzyme activity, lipid level and protein concentration appeared similarly in ACM-treated male and female rats, the activities of 7-alkoxycoumarin O-dealkylase, especially the O-depropylation activity, decreased dose-dependently by ACM administration only in male rats. ACM did not affect the hepatic drug-metabolizing activity in female rats and mice of both sexes. Also, ACM did not inhibit androgen-independent aniline hydroxylase activity even in male rats. The time course of changes of the drug-metabolizing system in male rats showed a rapid decrease in cytochrome P-450 content and O-depropylation activity following successive treatments with ACM, but there was a slow onset in the decreases of the O-demethylation and O-deethylation activities of 7-alkoxycoumarin. When ACM was withdrawn, the O-demethylation and O-deethylation activities rapidly returned to their control levels, while recovery of the O-depropylation activity was slow. These results suggested that ACM inhibits the hepatic drug-metabolizing enzyme activity associated with a specific form(s) of androgen-dependent cytochrome P-450 in male rats.     

Altered activity of hepatic mixed-function mono-oxygenase enzymes in streptozotocin-induced diabetic rats.

1. In streptozotocin-induced diabetic male rats, hepatic microsomal aminopyrine N-demethylase activity was depressed, whereas aniline hydroxylase activity and cytochrome P-450 content were increased over control values. 2. In diabetic female rats, hepatic microsomal aminopyrine N-demethylase activity, aniline hydroxylase activity, biphenyl 4-hydroxylase activity, and cytochrome P-450 content were increased over control values. 3. Insulin treatment of diabetic male and female rats antagonized all physical and biochemical abnormalities of the diabetic state; 4. Methyl analogues of streptozotocin did not produce a diabetic state when injected into female rats, and resulted in no changes in aminopyrine N-demethylase activity, aniline hydroxylase activity, or cytochrome P-450 content. 5. Insulin treatment of non-diabetic female rats resulted in slight decreases in aminopyrine N-demethylase and aniline hydroxylase activities, but no changes in cytochrome P-450 content. These observations suggest that insulin primarily influences drug metabolism of diabetic animals through correction of the insulin-deficient diabetic state.