Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--II. Evidence for a co-operative interaction between NADPH and NADH.

There was a significant reduction in apparent K_m (NADPH) values for both aminopyrine and ethylmorphine demethylases when the kinetic constants for NADPH were determined in the presence of constant NADH concentrations. NADH was also shown to significantly stimulate apparent V for NADPH cytochrome P450-reductase (in the presence of aminopyrine) without changing the apparent K_m (NADPH) value. Further, NADH stimulated the initial rapid phase of the biphasic reduction kinetics of NADPH cytochrome P450-reductase in the presence of aminopyrine. These findings suggest that in the presence of both pyridine nucleotides, there has been a change in the rate limiting step which, with NADPH alone, is generally accepted to be the reduction of the cytochrome P450-substrate complex. It has been necessary to make certain modifications to a previously proposed mechanism to explain the results obtained in the present study.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--IV. Inhibition of aminopyrine and ethylmorphine-N-demethylases by 2.6-dihydroxy-acetophenone.

Pyridine nucleotide kinetics for both aminopyrine and ethylmorphine-N-demethylases were evaluated in the presence of 2.6-dihydroxyacetophenone. (DiHAP) which inhibits certain microsomal mixed function oxidase reactions. The nature of the inhibition in the presence of varying NADPH concentrations was shown to be slope-linear, intercept-linear non-competitive. DiHAP was shown to combine preferentially with the ferric cytochrome P₄₅₀-substrate complex in the presence of aminopyrine, but had a greater affinity for the ferrous cytochrome P₄₅₀-substrate complex when ethylmorphine was present.There were changes in the values of K_ii and K_is without changes in the type of inhibition of aminopyrine-N-demethylase in the presence of both NADH and DiHAP, whereas with ethylmorphine-N-demethylase, the nature of the inhibition kinetics changed to slope-hyperbolic, intercept-hyperbolic non-competitive inhibition in the presence of both NADH and DiHAP.These results are consistent with the proposal that NADH interacts with the ferric cytochrome P₄₅₀-substrate complex prior to the reduction of this complex by an electron from NADPH.     

Pyridine nucleotide cofactor requirements of indicine N-oxide reduction by hepatic microsomal cytochrome P-450

Indicine N-oxide is reduced to indicine under anaerobic conditions by rat hepatic microsomal fraction in the presence of either NADH or NADPH. CO completely inhibits reduction, indicating the involvement of cytochrome P-450. In contrast to cytochrome P-450-catalyzed oxidations, for which NADH is about 15 per cent as effective as NADPH, NADH is 80 per cent as effective as NADPH in supporting indicine N-oxide reduction. In the presence of 3 mM NADH, the K_m for indicine N-oxide is 0.37 mM, and the V_max is 2.65 nmoles indicine formed/min/mg; with 3 mM NADPH the K_m is 0.51 mM, and the V_max is 3.00 nmoles/min/mg. NADH- and NADPH-dependent indicine N-oxide reduction appear to involve different pathways. NADH-supported reduction is inhibited 48 per cent by 0.5 mM KCN and 45 per cent by 0.8 M acetone, while NADPH-supported reduction is inhibited 3 per cent by 0.5 mM KCN and stimulated 28 per cent by 0.8 M acetone. The ability of NADH and NADPH at saturating concentrations to support indicine N-oxide reduction is additive, although this effect is not seen with phenobarbital- or 3-methylcholanthrene-pretreated animals. Phenobarbital pretreatment produces a selective increase in the V_max for NADPH-dependent reduction, to 5.75 nmoles/min/mg, but has no significant effect upon K_tm with NADPH or upon either the K_m for the f_max for NADH-supported indicine N-oxide reduction. Pretreatment with 3-methylcholanthrene has no significant effect upon the K_m or V_max for NADPH- or NADH-supported reduction. A possible explanation for the observations is a form of cytochrome P-450 which can accept electrons from NADH and catalyze indicine N-oxide reduction but which does not contribute to oxidative microsomal drug metabolism     

Propylene glycol as a drug solvent in the study of hepatic microsomal enzyme metabolism in the rat

Propylene glycol administered ip to rats at a dose of 4 ml/kg twice a day for 3 days caused a significant elevation of the in vitro hepatic microsomal metabolism of aniline and p-nitroanisole, but at the same time caused a significant decrease in aminopyrine demethylation with no significant change in p-nitrobenzoic acid metabolism. There was no change in cytochrome P-450 concentrations with this treatment, but the response, which was dose-dependent, could not be repeated by the in vitro addition of propylene glycol to incubating systems. In vivo inhibition of drug metabolism was demonstrated by increased hexobarbital sleeping times and zoxazolamine paralysis times after propylene glycol treatment. When administered concurrently with 75 mg of phenobarbital/kg for 3 days. an additive response was obtained with aniline and p-nitroanisole metabolism. Phenobarbital appeared to abolish the depressant effects observed in aminopyrine metabolism and the slight changes observed in p-nitrobenzoic acid reduction. Kinetic studies with microsomes from treated rats showed a reduced K_m and V_max for aminopyrine demethylation, while for aniline hydroxylation there was an increase in V_max but an unchanged K_m value.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--I. Factors that influence NADPH kinetic estimations during mixed function oxidase reactions.

The localisation of significant amounts of nucleotide pyrophosphatase activity in the rat hepatic microsomal fraction results in erroneous values of apparent K_m (NADPH) for aminopyrine-Ndemethylase. This was demonstrated by the inclusion of 20 mM pyrophosphate, which inhibits nucleotide pyrophosphatase activity and reduces the apparent K_m (NADPH) value from 27.9 μM to 7.92 μM. The apparent K_m (NADPH) values determined in the presence of three Type I substrates were not statistically different from each other, but the value in the presence of aniline was lower. The kinetic constants of NADPH for NADPH cytochrome P450-reductase in the presence of either aminopyrine, ethylmorphine or aniline, are also reported.     

The effect of dietary lipids and vitamin E on lipid peroxide formation, cytochrome P-450 and oxidative demethylation in the endoplasmic reticulum.

The effects of varying the lipid components of the diet have been studied on the cytochrome P-450 content and the rate of oxidative demethylation of aminopyrine in the liver endoplasmic reticulum. The cytochrome P-450 content and rate of oxidative demethylation (V_max) were lowest when a fat-free diet was fed, increased by addition of 10% lard (containing mainly saturated and mono-unsaturated fatty acids but 6% linoleic acid) and much more by addition of 10% corn oil (containing 50% linoleic acid). Following induction with phenobarbitone the rates of oxidative demethylation and cytochrome P-450 were also greatest in animals fed the corn oil diet and least in animals fed the fat-free diet. Addition of vitamin E (120 mg/kg diet) to the lard diet caused a significant increase in the rate of oxidative demethylation but the synthetic antioxidant 2,6,di-tert-butyl-p-cresol (BHT) was ineffective. The lipid peroxide content of the endoplasmic reticulum and the rate of NADPH stimulated peroxidation were much greater if the corn oil diet was fed than if the fat free diet was fed. Addition of vitamin E reduced the lipid peroxide in the endoplasmic reticulum when a lard diet was fed but BHT was ineffective. It is concluded that polyunsaturated fatty acids, primarily linoleic acid and vitamin E are essential in the diet for the content of cytochrome P-450 and the rate of oxidative demethylation to be a maximum in the endoplasmic reticulum.     

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

The development of sex differences in the demethylation of ethylmorphine and in its interaction with components of the hepatic microsomal cytochrome P450 system in mice.

The development of sex differences in ethylmorphine N-demethylation and several components of the reaction chain were studied in hepatic microsomes from mice of the CPB-SE strain between 3–11 weeks of age. Sex-specific changes were observed in demethylation rate, type 1 spectral interaction, cytochrome P450 content, and ethylmorphine-induced stimulation of NADPH-cytochrome P450 reductase activity. These changes occurred mainly between weeks 3–7 and were confined to females. It is concluded that the development of the cytochrome P450 system is repressed by androgen during sexual maturation. The kinetic constants of demethylation developed differently from ethylmorphine binding constants. Changes in demethylase were mainly restricted to K_m, whereas the changes in type 1 binding only involved the maximum spectral change. In combination with differences observed between the developmental patterns of demethylation rate and cytochrome P450 reductase activities, this demonstrated that the reduction of cytochrome P450-substrate complex is not rate-limiting in ethylmorphine demethylation. The type 1 spectral change was correlated with the amount of cytochrome P450 only when a large portion of the cytochrome was considered inactive in ethylmorphine binding. It is suggested that immature animals possess a low basal level of ethylmorphine binding type 1 sites, which is elevated selectively in females during sexual maturation.     

Kinetic parameters of drug-metabolizing enzymes in Ca 2+ -sedimented microsomes from rat liver

Kinetic parameters of five substrates of the mixed function oxidase system were determined to ensure that the mixed function oxidases were unaltered by the Ca²⁺-sedimented microsomes. Using either of the microsomal preparation procedures, no differences were noted in the V_max and K_m for aminopyrine, ethylmorphine and p-nitroanisole demethylation, aniline hydroxylation and hexobarbital oxidase. Also, no differences were seen in the NADPH cytochrome P-450 reductase activities. The Ca²⁺-sedimentation procedure was further simplified to allow microsomal preparation within 1 hr.     

Carbon tetrachloride-induced lipid peroxidation of rat liver microsomes in vitro.

Characteristics of carbon tetrachloride-induced lipid peroxidation of rat liver microsomes and effect on microsomal enzymes were studies in vitro. Microsomes isolated from well-perfused livers and washed with EDTA-containing medium exhibited low endogenous lipid peroxidation when incubated in a phosphate buffer (> 0.1 M) in the presence of NADPH, whereas carbon tetrachloride stimulated to a great extent the peroxidation under these conditions. The stimulation was dependent on the concentration of NADPH, neither NADH nor ascorbic acid being replaced. The stimulatory action by bromotrichloromethane was more marked than that by carbon tetrachloride, however chloroform had no stimulatory action. N,N-Diphenyl-p-phenylene diamine, diethyldithiocarbamate and disulfiram inhibited carbon tetrachloride-induced lipid peroxidation in low concentrations. Inhibitions by thiol compounds and EDTA were weaker. Ferricyanide, cytochrome c and vitamine K₃ inhibited the stimulation by carbon tetrachloride while no inhibition was seen with carbon monoxide. An increase in the degree of carbon tetrachloride-induced lipid peroxidation resulted in a coincidental decrease in microsomal cytochrome P-450 content accompanying a parallel loss in aminopyrine demethylase activity, while NADH-ferricyanide dehydrogenase and NAD(P)H-eytochrome c reductase activities, and cytochrome b₅ content remained unaffected. Similar results were obtained when microsomes were peroxidized with NADPH in combination with ferric chloride and pyrophosphate. Regarding the mechanism of hepatotoxic action of carbon tetrachloride, these results support the hypothesis of lipid peroxidation.     

Hepatic drug metabolism in normal and vitamin E-deficient female Merino sheep.

The basal values for hepatic microsomal enzyme activity in Merino ewes were higher per milligram of microsomal protein than those in female Wistar rats for para-nitroreductase, aminopyrine, and para-nitroanisole demethylases, but slightly lower for aniline para-hydroxylase. Vitamin E deficiency did not alter these parameters significantly, although there was a slight increase in NADPH oxidase activity. Pretreatment with phenobarbital gave significant increases in cytochrome P-450 and the other enzymes associated with microsomal drug metabolism. The exceptions were a significant reduction in the content of cytochrome b₅ and a slight reduction in NADPH cytochrome c reductase activity. The addition of 10^?3m flavine mononucleotide (FMN) to the incubation media elevated the otherwise low levels of nitroreductase activity approximately 11-fold.     

Mestranol-induced hypertension: characterization of cytochrome P-450 dependent catechol estrogen formation in brain microsomes

An animal model of estrogen-induced hypertension was used to study the effects of chronic administration of the synthetic estrogen mestranol on cytochrome P-450 content and catechol estrogen formation in brain microsomes. Cytochrome P-450 content of brain microsomes from untreated female rats in estrus was 0.034 nmole/mg protein and the dithionite-reduced carbon monoxide absorbance peak (γ_max) was 452 nm. Catechol estrogen formation in brain microsomes was optimal in the presence of both NADPH and NADH cofactors with an apparent K_m value of 71 μM for 17β-estradiol substrate. Brain microsomes from animals in estrus and diestrus were compared, and no significant differences were observed in cytochrome P-450 content, or in the apparent K_m and V_max values of catechol estrogen formation. Administration of mestranol, 15 μg biweekly, for 3–4 weeks resulted in a significant increase in systolic blood pressure in unanesthetized female rats. Mestranol treatment was not associated with a change in microsomal cytochrome P-450 content or the spectral γ_max. At 10 μM substrate concentration, catechol estrogen formation in microsomes from mestranol-treated animals was increased 2- to 3-fold, with enzyme activity being expressed either per mg protein or per nmole cytochrome P-450. In contrast, no difference was observed between groups when enzyme activity was measured at 100 μM substrate concentration. These data suggest that chronic administration of a synthetic estrogen can regulate the enzyme system involved in formation of brain catechol estrogen metabolites, a mechanism which may alter the biological impact of the parent steroid.     

Modification of carbon tetrachloride hepatotoxicity by chemicals.

Cobaltous chloride (60 mg/kg, sc, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CCl₄-induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against CCl₄ hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P-450 reduction by NADPH. Further studies revealed that CCl₄-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5-hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCl₄ as the activation reaction for CCl₄ but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for CCl₄ activation.     

Studies on the interaction of safrole with rat hepatic microsomes

(1) Similar to previous results with methylenedioxyphenyl compounds microsomes from safrole pretreated rats showed, on reduction with NADH, NADPH or Na₂S₂O₄, characteristic absorption maxima at 427 and 455 nm. The same spectrum can be obtained after incubation in vitro of control microsomes with safrole, NADPH and oxygen. (2) Subsequent addition of carbon monoxide to microsomes of safrole pretreated rats causes an absorption maximum at 448 nm, characteristic of the 3-methylcholanthrene type of induction of microsomal hydroxylase protein. (3) The suspected cytochrome P-450-safrole metabolite complex, which can be visualized only in the reduced state of cytochrome P-450, is very stable as witnessed by its preservation through the preparation procedure for microsomes or after dialysis or detergent treatment. However, when safrole or ethylbenzene is added, both absorption maxima decrease in a time dependent manner. This can be measured for each time point after complete reduction of the microsomal preparation by adding Na₂S₂O₄. (4) From this it is concluded that the carcinogen safrole leads to the biosynthesis of a 3-methylcholanthrene type cytochrome P-450 and formation of a safrole metabolite-cytochrome P-450 complex which in turn can be cleaved in vitro by safrole or other lipophilic compounds.     

In vitro hepatic drug metabolism and microsomal enzyme induction in genetically obese rats

Genetically obese Zucker-strain rats were used to determine if obesity produced alterations in in vitro drug metabolism in hepatic microsomes or cytosol. Adult homozygous obese rats of both sexes had significantly less active drug-metabolizing enzymes in vitro than homozygous lean Zucker rats or Sprague-Dawley rats. Specific activities of aniline hydroxylase, aminopyrine demethylase and arylhydrocarbon hydroxylase (AHH) from male obese rats were only 25–30 percent of control activities, while glutathione S-aryltransferase, biphenyl 4-hydroxylase, and cytochrome P-450 activities were 50–70 percent of control activities. K_m and V_max values for aminopyrine demethylation were significantly less in obese rats than in lean rats. Enzyme activities in weanling obese rats were nearly equivalent to activities in weanling lean rats. Pretreatment of obese and lean rats with 3-methylcholanthrene produced expected increases in activities of biphenyl 4-hydroxylase, AHH and cytochrome P-450. Phenobarbital (PB), however killed half of the PB-treated obese rats at 60 mg/kg, but killed no lean rats. At lower doses, PB pretreatment produced only marginal increases in specific activities of aniline hydroxylase, aminopyrine demethylase and cytochrome P-450 in obese rats, while producing larger increases in activities in lean controls. When genetically obese rats were pair-fed to body weights that were equal to lean rats, the deficiency in in vitro drug metabolism between lean and fat rats was qualitatively as great as in ad lib. fed rats. Testosterone treatment of obese rats produced no significant increase in in vitro drug metabolism. Heterozygous lean Zucker rats responded to all treatments in a manner similar to that of homozygous lean Zucker rats. The explanation for these results is complex and they cannot be accounted for by a simple increase in body weight.     

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.     

Cytochrome P450 2C9 is responsible for hydroxylation of the naphthoquinone antimalarial drug 58C80 in human liver

2-(4-t-Butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone (58C80) is an experimental naphthoquinone antimalarial drug which undergoes extensive alky hydroxylation in man. By means of purification, N-terminal amino acid sequencing and inhibition by antibodies and sulfaphenazole, we have identified the form of cytochrome P450 primarily responsible for 58C80 hydroxylation in human liver, P450hB_20–27, to be a member of the P450 2C9 subfamily. P450hB_20–27 is a low-spin haemoprotein with molecular mass 54 kDa. 58C80 hydroxylation in human liver microsomes was dependent on either NADPH or NADH, with the activity supported by NADH being 35% of that supported by NADPH. With purified P450hB_20–27 cytochrome b₅ stimulated the NADH-dependent activity 8-fold but inhibited the NADPH-dependent activity by 30%. 58C80 is a novel substrate structure for human P450 2C and these results significantly broaden the range of drugs which have been directly shown (i.e. using a purified enzyme as opposed to expressed cDNA) to be metabolized by human P450 2C forms that are incontrovertibly expressed in human liver in vivo.     

The reductive metabolism of halogenated alkanes by liver microsomal cytochrome P450

Under anaerobic conditions various polyhalogenated alkanes (CCl₃-CCl₃, HCl₂C-CCl₃, CF₃-CCl₃, CCl₄, CF₃-CHClBr) stimulate the oxidation of NADPH by liver microsomal fractions. The participation of cytochrome P450 in the NADPH oxidation was shown by inducers and inhibitors of the monooxygenase system. The products of the reductive pathway of hexachloroethane were tetrachloroethene (99.5%) and pentachloroethane (0.5%). From pentachloroethane as substrate trichloroethene (96%) and tetrachloroethane (4%) were produced. The stoichiometry of NADPH oxidation and product formation was close to 1:1. There was a synergistic effect in the presence of NADPH and NADH for both hexa- and pentachloroethane. The influence of dioxygen and radical traps (RSH) on the formation of products from hexachloroethane with reduced cytochrome P450 has been investigated. The results indicate the possibility of a reductive in vivo metabolism of polyhalogenated alkanes even at physiological dioxygen concentrations. For the reductive dehalogenation of polyhalogenated alkanes by microsomal cytochrome P450 a reaction scheme is proposed: the reduction proceeds by two subsequent one electron reductions forming first a radical and then a carbanion. The carbanion can form an alkene via β-elimination of chloride.     

Comparison of the drug metabolising ability of rat intestinal mucosal microsomes with that of liver.

A study of the drug metabolizing capabilities of rat intestinal microsomes was undertaken and a direct comparison made with analogous activity in hepatic microsomes. The cofactor requirements and K_m values of the cytochrome P-450 mediated 7-ethoxycoumarin O-de-ethylation in the epithelial cell microsomes was similar to that in hepatic microsomes, whereas the V_max values were 5–6 fold lower in the intestinal preparation. Greater differences were seen in other cytochrome P-450 dependent reactions. The K_m value for biphenyl-4-hydroxylation was an order of magnitude lower in the intestinal microsomes, whereas the 4-hydroxylation of aniline and acetanilide and the N-demethylation of ethylmorphine and aminopyrine, although very active in hepatic microsomes, were barely detectable in intestinal microsomes. The O-de-ethylation inhibition characteristics of metyrapone and 7,8-benzoflavone but not SKF 525 A, were similar in the liver and intestinal preparations.These findings and the alterations in drug oxidation activity after PB and 3MC pretreatment suggest that there are both quantitative and qualitative differences in the cytochrome P-450's between liver and intestine. This suggestion is further supported by the difference in the safrole adduct binding spectrum with NADPH or with cumene hydroperoxide between the two tissues from control, phenobarbitone and 3-MC pretreated rats.O-Deacetylation of 4-nitrophenyl acetate and indoxyl acetate in intestinal and liver preparations was very comparable while acetanilide N-deacetylation was three times more active in the liver. Glucuronidation of 1-naphthol by the intestinal microsomes showed a similar K_m but a lower V_max than the liver microsomes. No change was observed in any of these enzymes following pretreatment of rats with PB or with 3-MC.     

Induction of Hepatic Cytochrome P450 by Phenobarbitone in Rhesus Monkey (Macaca Mulatta)

ABSTRACT

An intraperitoneal administration of PB at a daily dose of 50 mg Kg^?1body wt for 4 days increased the specific content of hepatic microsomal heme, cytochrome P450 and the activity of aminopyrine N-demethylase by 1.8, 2.8 and 3.5 fold respectively. These results were substantiated by the intensification of the 52.5 KDa polypeptide in the electrophoretogram of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the hepatic microsomes obtained from PB -pretreated versus control macaques. PB did not affect the hepatic content of cytochrome b₅, and the activity of NADPH cytochrome c reductase, whereas it decreased the activity of NADH cytochrome c reductase in the rhesus monkeys. to the best of our knowledge this is a first report on the induction of hepatic cytochrome P450 and related enzymes by PB in rhesus monkeys.