Individual Cytochrome P450 Isozymes Research Articles

CYPlebrity: Machine learning models for the prediction of inhibitors of cytochrome P450 enzymes

The vast majority of approved drugs are metabolized by the five major cytochrome P450 (CYP) isozymes, 1A2, 2C9, 2C19, 2D6 and 3A4. Inhibition of CYP isozymes can cause drug-drug interactions with severe pharmacological and toxicological consequences. Computational methods for the fast and reliable prediction of the inhibition of CYP isozymes by small molecules are therefore of high interest and relevance to pharmaceutical companies and a host of other industries, including the cosmetics and agrochemical industries. Today, a large number of machine learning models for predicting the inhibition of the major CYP isozymes by small molecules are available. With this work we aim to go beyond the coverage of existing models, by combining data from several major public and proprietary sources. More specifically, we used up to 18815 compounds with measured bioactivities to train random forest classification models for the individual CYP isozymes. A major advantage of the new data collection over existing ones is the better representation of the minority class, the CYP inhibitors. With the new data collection we achieved inhibitor-to-non-inhibitor ratios in the order of 1:1 (CYP1A2) to 1:3 (CYP2D6). We show that our models reach competitive performance on external data, with Matthews correlation coefficients (MCCs) ranging from 0.62 (CYP2C19) to 0.70 (CYP2D6), and areas under the receiver operating characteristic curve (AUCs) between 0.89 (CYP2C19) and 0.92 (CYPs 2D6 and 3A4). Importantly, the models show a high level of robustness, reflected in a good predictivity also for compounds that are structurally dissimilar to the compounds represented in the training data. The best models presented in this work are freely accessible for academic research via a web service.

Development of a multitask deep learning QSAR model using data from individual cytochrome P450 isozymes

Development of a multitask deep learning QSAR model using data from individual cytochrome P450 isozymes

Cytochrome P450-inhibitory activity of parabens and phthalates used in consumer products.

The in vitro cytochrome P450 (CYP)-inhibitory effects of 11 parabens and 7 phthalates used in consumer products, as well as their hydrolytic metabolites, were investigated, using rat liver microsomes as an enzyme source. The effects on individual CYP isozymes were evaluated by assaying inhibition of activities towards specific substrates, i.e., ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), pentoxyresorufin O-dealkylase (PROD), 7-benzyloxy-4-trifluoromethylcoumarin dealkylase (BFCD), 7-methoxy-4-trifluoromethylcoumarin dealkylase (MFCD) and 7-ethoxy-4-trifluoromethylcoumarin dealkylase (EFCD) activities. These activities were dose-dependently inhibited, most potently by medium-side-chain parabens (C6-9) and phthalates (C4-6), and less potently by shorter- and longer-side-chain esters. The hydrolytic product of parabens, 4-hydroxybenzoic acid, was not inhibitory, while those of phthalates, phthalic acid monoesters, showed lower inhibitory activity than the parent phthalates. Parabens showed relatively potent inhibition of MFCD activity, considered to be mainly due to CYP2C, and phthalates showed relatively potent inhibition of PROD activity, considered to be mainly due to CYP2B.

Effect of biliary obstruction and internal biliary drainage on hepatic cytochrome P450 isozymes in rats

To investigate the total cytochrome P450 (CYP) content, microsomal mixed-function oxidase (MFO) activity, and expression of mRNAs for various CYP isozymes in a simple rat model of reversible obstructive jaundice. Obstructive jaundice was created in male rats by causing bile duct obstruction with polyester tape. In another group of rats, bile duct obstruction was followed by internal biliary drainage after releasing the tape. The expression of various CYP isozyme mRNAs was semi-quantitatively assessed by competitive RT-PCR. The total CYP content and microsomal MFO activity showed a significant decrease after biliary obstruction, but returned to respective control levels after biliary drainage. A marked reduction in the expression of CYP1A2, 2B1/2, 2C11, 2E1, 3A1, and 3A2 mRNA was detected during biliary obstruction, while expression increased significantly toward the control level after biliary drainage. Although expression of CYP4A1 mRNA showed no reduction during biliary obstruction, it still increased significantly after biliary drainage. These results suggest that not only obstructive jaundice, but also the subsequent internal biliary drainage may affect regulatory medications of the synthesis of individual CYP isozymes differently.

Identification and hepatic expression profiles of cytochrome P450 1–4 isozymes in common minke whales ( Balaenoptera acutorostrata)

Full-length cDNA sequences of cytochrome P450 (CYP) 2C78, 2E1, 3A72, 4A35 and 4V6 isozymes were isolated from a hepatic cDNA library of common minke whale ( Balaenoptera acutorostrata). The deduced amino acid sequences of minke whale CYP2C78, 2E1, 3A72, 4A35 and 4V6 showed high identities with cattle CYP2C86 (83%), pig CYP2E1 (85%), sheep CYP3A24 (82%), pig CYP4A21 (80%), and human CYP4V2 (76%), respectively. To investigate whether or not these CYP expression levels are altered by contamination of organochlorine contaminants (OCs), mRNA levels of these CYPs in the liver of common minke whale were measured using a quantitative real-time RT-PCR method, and the quantified mRNA levels were employed for the statistical analysis with the residue levels of OCs including PCBs, DDTs ( p,p′-DDT, p,p′-DDD and p,p′-DDE), chlordanes ( cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane), HCHs (α-, β- and γ-isomers) and hexachlorobenzene that have already been reported elsewhere. Spearman's rank correlation analyses showed no significant correlation between CYP expression levels and each OC level in the common minke whale liver, implying that these environmental chemicals have no potential to alter the expression levels of these CYPs or the residue levels encountered in the whale livers may not reach their transcriptional regulation levels. This suggests that the expression of individual CYPs in the whale liver may be at basal level. Relationships among hepatic mRNA expression levels of these CYP2–4 isozymes together with CYP1A1 and CYP1A2 were also examined. Significant positive correlations were detected among mRNA expression levels of individual CYP isozymes in most cases. These associations indicate that the transcriptional regulation of these CYPs examined in this study may be reciprocally related. CYP1A1 levels showed a positive correlation with CYP1A2 levels ( r = 0.64, p < 0.01) indicating that both CYP isozymes were regulated by aryl hydrocarbon receptor activated by endogenous ligands. A strong positive correlation between CYP2C78 and 3A72 ( r = 0.90, p < 0.001) suggests that expression of these CYP isozymes may be under a regulation mechanism of cross-talk in which specific nuclear receptors such as constitutive androstane receptor and pregnane X receptor are involved. The present study indicates that minke whale from the North Pacific may be a model species to investigate the mechanism of basal regulation of these CYPs.

Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine.

The psychotropic beta-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for the O-demethylations mediated by each isozyme and by pooled HLMs. K(cat) (min(-1)) and K(m) Awake M) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline.

The effects of α,β-unsaturated aldehydes on hepatic thiols and thiol-containing enzymes

The effects of series of α,β-unsaturated aldehydes on hepatic glutathione, cytochrome P450, and NADPH-cytochrome c reductase activity were compared with time. Male F-344 rats were dosed with muconaldehyde (36 μmol/kg), acrolein (89 μmol/kg), crotonaldehyde (450 μmol/kg), or the saturated aldehyde propionaldehyde (89 μmol/kg) and terminated 0.5, 4, or 24 hr later. Acrolein or muconaldehyde reduced glutathione to 51 and 75% of controls, respectively, at 4 hr; glutathione returned to control values at 24 hr. Only at 24 hr, acrolein, muconaldehyde, or crotonaldehyde decreased cytochrome P450 to 61, 71, and 67% of control values, respectively; ethylmorphine N-demethylation was decreased to a greater extent, i.e., to 35, 60, and 23% of controls. The reductase activity was unchanged at any time following the treatment with reactive aldehydes which were not hepatotoxic (as shown by glucose 6-phosphatase activity, histological changes, or serum enzymes). Propionaldehyde changed none of these activities. Acrolein (44.5 μmol/kg) given 4 hr prior to phenobarbital (50 mg/kg) for two consecutive days decreased the phenobarbital induction of cytochrome P450 to 45% of phenobarbital alone. This treatment also decreased the 2α, 2β, 6β, 16α, and 16β hydroxylation of testosterone as well as androstenedione formation showing effects on individual cytochrome P450 isozymes. NADPH-cytochrome c reductase induction was not decreased by this treatment, thus indicating that in vivo these changes are due to a mechanism other than generalized inhibition of protein synthesis.

The Effects of α,β-Unsaturated Aldehydes on Hepatic Thiols and Thiol-Containing Enzymes

The effects of series of α,β-unsaturated aldehydes on hepatic giutathione, cytochrome P450, and NADPH-cytochrome c reductase activity were compared with time. Male F-344 rats were dosed with muconaldehyde (36 μmol/kg), acrolein (89 μmol/ kg), crotonaldehyde (450 μmol/kg), or the saturated aldehyde propionaldehyde (89 μmol/kg) and terminated 0.5, 4, or 24 hr later. Acroiein or muconaldehyde reduced glutathione to 51 and 75% of controls, respectively, at 4 hr; glutathione returned control values at 24 hr. Only at 24 hr, acrolein, muconaldehyde, or crotonaldehyde decreased cytochrome P450 to 61, 71, and 67% of control values, respectively; ethylmorphine N-demethylation was decreased to a greater extent, i.e., to 35, 60, and 23% of controls. The reductase activity was unchanged at any time following the treatment with reactive aldehydes which were not hepatotoxic (as shown by glucose 6-phosphatase activity, histological changes, or serum enzymes). Propionaidehyde changed none of these activities. Acroiein (44.5 µmol/kg) given 4 hr prior to phenobarbital (50 mg/kg) for two consecutive days decreased the phenobarbital induction of cytochrome P450 45% of phenobarbital alone. This treatment also decreased the 2α, 2β, 6β, l6α, and 16β hydroxylation of testosterone as well as androstenedione formation showing effects on individual cytochrome P450 isozymes. NADPH-cytochrome c reductase induction was not decreased by this treatment, thus indicating that in vivo these changes are due to a mechanism other than generalized inhibition of protein synthesis.

Induction of cytochrome P-450 isozymes during long-term nifedipine administration

The wide substrate specificity of the microsomal monooxygenase system, catalyzing oxidation of many xenobiotics and endogenous compounds, is determined by the existence of multiple forms of cytochrome P-450 [13]. A modern approach to the characterization and identification of the individual cytochrome P-450 isozymes is to study specific reactions catalyzed by these hemoproteins. There are substrates whose metabolism is more specific for a particular cytochrome P-450 isozyme. For example, the O-de-ethylation of 7-ethoxyresorufin is catalyzed by cytochrome P-450 c [3] . The steroid hormones testosterone, androstenedione (AD), and progesterone are utilized as specific substrates by the microsomal monooxygenase of the liver, with well-marked regional and stereospecificity of metabolism [16]. The rate of formation of hydroxylated metabolites gives information on activity of different forms of cytochrome P-450. For example, the formation of 16a-OH-AD is catalyzed by cytochrome P-450h, which is characterized only of male rats. The formation of 16~-OH-AD is catalyzed by P-45%, that of 7a-OH-AD by cytochrome P-450a, and that of 6fl-OH-AD by cytochrome P-450p [15]. Thus the specificity of the reaction of metabolism of certain substrates can serve as an important criterion for the identification of many forms of cytochrome P-450. On the other hand, the immunochemical method using monospecific antibodies against individual cytochrome P-450 isozymes makes it quantitative and qualitative determination in microsomes possible. This combined approach can be used in pharmacologic investigations, especially those in which the drug influences the microsomal monooxygenase system. The calcium antagonist nifedipine, widely used in cardiologic practice, is metabolized by cytochrome P-450p in rat liver microsomes [6] or by P-450NF in human liver microsomes [14], which are induced by the synthetic steroid pregnenolone-16carbonitrile and steroids of the glucocorticoid class, such as dexamethasone and macrolide antibiotics [12]. Previously the writers described induction of a cytochrome P-450-dependent monooxygenase system after long-term administration of nifedipine to rats [7]. The aim of the present investigation was to discover what forms of cytochrome P-450 can be induced under these conditions.

Distribution of cytochromes P-450, cytochrome b5, and nadph-cytochrome P-450 reductase in an entire human liver

In rat liver there appear to be significant differences between lobes in the concentration of individual cytochrome P-450 isozymes (Sumner and Lodola, Biochem Pharmacol 36: 391–393, 1987). Because studies in patients often rely on small pieces of liver obtained from diverse anatomical locations, it seemed important to determine if the cytochromes P-450 were also heterogeneously distributed in human liver. Accordingly, tissue was obtained from ten different locations in a single human liver including those most commonly biopsied by percutaneous needles, and by surgeons during laparotomy. The differences observed between locations in the microsomal concentrations of carbon monoxide-binding protein (total cytochrome P-450), cytochrome b 5, and NADPH-cytochrome P-450 reductase appeared to be small and were not statistically significant. Likewise, no significant differences were observed between locations in the specific content of HLp, HLp3, HLj, HLx or P450MP. However, the specific concentrations of HLd varied almost 2-fold between the microsomes and this was statistically significant in some cases (P < 0.05). Our results suggest that, in human livers, regional differences in the content of cytochromes P-450 are generally small but may be significant for some isozymes. With the exception of HLd, tissue obtained by percutaneous or surgical liver biopsies is probably representative of the entire organ with regard to the enzymes assayed.

Rat but not human interferons suppress hepatic oxidative drug metabolism in rats

An animal model suitable for in vivo studies of interferon-mediated suppression of hepatic oxidative drug metabolism has been developed. Rats were injected with either recombinant human interferon αA, recombinant human interferon γ, recombinant rat interferon γ, or vehicle and experiments were performed 24 h later. In some animals theophylline elimination was determined twice (10 days apart), once after interferon and once after vehicle. Theophylline clearance was also determined in the isolated perfused rat liver after pretreatment of animals with interferon or vehicle. Pretreatment of animals with rat interferon γ significantly reduced theophylline clearance in the intact rat but neither human interferon αA nor human interferon γ altered theophylline elimination in vivo. Similar results were observed in the isolated perfused rat liver. We then examined whether the effects of interferon on hepatic drug metabolism were generalized or confined to individual cytochrome P450 isozymes; androstenedione hydroxylation pathways were used as catalytic probes for individual cytochrome P450 isozymes. Rat interferon γ (but not human interferon αA) decreased levels of total hepatic microsomal P450 and reduced androstenedione 16β-hydroxylation. The formation of three other hydroxylated androstenedione metabolites appeared reduced to a similar extent, although these changes were not significant. It is concluded that autologous but not heterologous interferons impair oxidative drug metabolism in the rat. The reduction of hepatic P450 produced by interferon may result from the suppression of multiple isozymes.

Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: Simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography

Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-l-phenyl-3-pyrazoline-5-one (AM-OH), and one unidentified metabolite. MAA was the major metabolite generated by the microsomes; its formation was induced by phenobarbital but not by 3-methylcholanthrene. Female rats had lower N-demethylation activity of aminopyrine than male rats. The production of AA by microsomes was low. The formation of AM-OH was strongly induced by phenobarbital, but treatment with 3-methylcholanthrene reduced its formation. These differences in the microsomal aminopyrine monooxygenase activity are dependent on the relative amounts of the individual cytochrome P-450 isozymes. Therefore, we examined aminopyrine metabolism in a reconstituted system with purified cytochrome P-450s. P-450 UT-2 ( P-450h) had high aminopyrine N-demethylation and hydroxylation activities, but P-450 F-2 ( P-450i) had low N-demethylation activity and no hydroxylation activity. P-450 PB-4 ( P-450b) and P-450 PB-5 ( P-450e) had high aminopyrine hydroxylation activity and their N-demethylation activity also was high. The 3-methylcholanthrene-inducible forms P-450 MC-1 ( P-450d) and MC-5 ( P-450c) had aminopyrine N-demethylation activity but no hydroxylation activity. P-450 UT-4 (RLM2) is a unique form that produced a large amount of the unknown metabolite. P-450 UT-7 had the highest N-demethylation activity. Addition of cytochrome b 5 to the reconstituted system enhanced the aminopyrine hydroxylation activities of P-450s UT-1, UT-2, PB-2, and PB-5. Also, the N-demethylation activities of P-450s UT-1, PB-1, PB-2, and MC-1 were increased by cytochrome b 5. Metyrapone inhibited the catalytic activities of P-450s PB-4, PB-5, MC-1, and MC-5, and especially those of P-450s UT-4, and UT-7. The kinetics of the four major cytochrome P-450s ( P-450 UT-2, UT-4, PB-4, and MC-5) for aminopyrine N-demethylation and hydroxylation activities were studied. P-450s PB-4 and UT-2 had similar K m values (0.50 and 0.62 m m, respectively) in aminopyrine N-demethylation activity. These K m values were similar to the K m of aminopyrine N-demethylase in microsomes from phenobarbital-treated rats. P-450 UT-4 had a higher K m (3.23 m m) than P-450 UT-2 by an order of magnitude. P-450 MC-5 gave the highest K m value for aminopyrine N-demethylation (8.55 m m).

Nutrition-related alterations in liver microsomal testosterone hydroxylases.

The oxidation products of testosterone formed by liver microsomes from normal-fed and protein-energy malnourished male rats have been analysed by HPLC. Microsomes from normal-fed rats oxidized testosterone at a rate of 4.52 nmol/min/mg protein. The major products formed were: 6 beta-, 7 alpha- and 16-alpha-hydroxytestosterone; these three metabolites represented 65% of the total testosterone metabolism. Microsomes from protein-energy malnourished rats oxidized testosterone at a reduced rate of 2.03 nmol/min/mg protein. The major product formed was 7 alpha-hydroxytestosterone, which accounted for 43% of total testosterone oxidation. Microsomes from protein-energy malnourished rats showed a CO-reduced cytochrome P-450 spectra with a maxima at 452 nm, and a 38% decrease in the total content of cytochrome P-450. Some testosterone hydroxylases were drastically affected by protein-energy malnutrition but others, such as 7 alpha-hydroxylase, remained unchanged. The present results suggest that nutritional status can modify the relative amounts of individual cytochrome P-450 isozymes, thus explaining the observed changes in several testosterone hydroxylases. Protein-energy malnutrition seems to be an excellent tool with which to obtain a microsomal fraction containing predominantly P-450 isozymes, which are probably involved in key mono-oxygenations of physiological substrates.

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16β- and 6β-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7α-hydroxylase activity was decreased to 0.8-fold of control and 16α-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite- P-450 complex revealed an even greater induction of 16β- and 6β-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16α-hydroxylase activity increased 2-fold after ferricyanide but 7α-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT- A decreased androst-4-ene-3,17-dione 16α-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16α-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT- A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB- B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16α-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT- A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT- F, which is active in steroid 7α-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.

Drug metabolism in cirrhosis: Selective changes in cytochrome P-450 isozymes in the choline-deficient rat model

The effect of a choline-deficient diet on microsomal cytochome P-450 and mixed-function oxidase (MFO) activity was investigated in relation to the development of nutritional cirrhosis. In rats that received the choline-deficient diet for 28 weeks cirrhosis was evident macroscopically and histologically; control rats that received an identical diet supplemented with choline had normal livers. Microsomal cytochrome P-450 and cytochrome b 5 were reduced in cirrhotic liver to 50% of control levels. Three MFO activities (ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase) were also reduced to 40–70% of control levels. However, the turnover number for the O-deethylation of 7-ethoxycoumarin was not reduced in cirrhotic liver. This finding suggested that certain drug oxidations may be selectively depressed in nutritional cirrhosis. To examine the possibility that selective changes in MFO activity may reflect the suppression of certain cytochrome P-450 isozymes, partially purified fractions of the cytochrome were prepared after solubilisation and hydrophobic affinity chromatography (on n-octylamino-Sepharose 4B) of cirrhotic and control liver microsomes. Analysis of these fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and laser densitometry indicated that a protein band of apparent minimum molecular weight 50.5 kD was primarily affected in cirrhotic rat liver microsomes. Levels of two other bands (apparent minimum molecular weight 48 and 52.5 kD) appeared essentially unaltered. Additional electrophoretic studies, conducted under non-reduced conditions, indicated the haemoprotein nature of protein bands in the 48–55 kD region. These data strongly suggest that cirrhosis produced in rats by a choline-deficient diet is associated with selective decreases in oxidative drug metabolism and individual cytochrome P-450 isozymes.

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 μmol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (~25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (~45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450 b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4′-penta- and 2,3,4,5,3′,4′-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2′,4′,5′-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.