Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10µM) on the uptake of [(3)H]-estradiol-17ß-d-glucuronide (1µM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15.

Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials.

Correct prediction of human pharmacokinetics (PK) and the safety and efficacy of novel compounds based on preclinical data, is essential but often fails. In the current study, we aimed to improve the predictive value of ApoE*3Leiden (E3L) transgenic mice regarding the cholesterol-lowering efficacy of various statins in humans by combining pharmacokinetic with efficacy data. The efficacy of five currently marketed statins (atorvastatin, simvastatin, lovastatin, pravastatin, and rosuvastatin) in hypercholesterolemic patients (low-density lipoprotein ≥ 160 mg/dl) was ranked based on meta-analysis of published human trials. Additionally, a preclinical combined PK efficacy data set for these five statins was established in E3L mice that were fed a high-cholesterol diet for 4 weeks, followed by 6 weeks of drug intervention in which statins were supplemented to the diet. Plasma and tissue levels of the statins were determined on administration of (radiolabeled) drugs (10 mg/kg p.o.). As expected, all statins reduced plasma cholesterol in the preclinical model, but a direct correlation between cholesterol lowering efficacy of the different statins in mice and in humans did not reach statistical significance (R(2) = 0.11, P < 0.57). It is noteworthy that, when murine data were corrected for effective liver uptake of the different statins, the correlation markedly increased (R(2) = 0.89, P < 0.05). Here we show for the first time that hepatic uptake of statins is related to their cholesterol-lowering efficacy and provide evidence that combined PK and efficacy studies can substantially improve the translational value of the E3L mouse model in the case of statin treatment. This strategy may also be applicable for other classes of drugs and other preclinical models.

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1.

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial beta-oxidation and the activity of catalase, glutathione S-transferase and glutathione peroxidase.

The effect of hypolipidaemic compounds on peroxisomal fatty acid beta-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial beta-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial beta-oxidation activities in intact cells. Overall beta-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate beta-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal beta-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate beta-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the effects of model inducers of cytochrome P450 on the biotransformation of scoparone in rat and hamster liver.

The hamster is known to display very high rates of monooxygenase-mediated biotransformation. In comparison with other species little knowledge has been gathered with respect to the nature of its cytochrome P450 enzymes and their respective inducibility. We studied the consequences of induction of P450 enzymes in rats and Syrian golden hamsters using the regioselective oxidative O-demethylation of the coumarin derivative scoparone. This metabolic conversion indicates differential effects of P450 inducers in the rat, in which various types of inducers cause different shifts in the isoscopoletin/scopoletin metabolite ratio (I/S-ratio). Liver microsomes from hamster not treated with P450 inducers oxidized scoparone much more efficiently than liver microsomes of untreated rats. In rat liver microsomes total demethylation rates of scoparone increased upon in vivo treatment with phenobarbital or beta-naphthoflavone. Phenobarbital reduced the I/S-ratio whereas beta-naphthoflavone caused an increase in this ratio. In hamster liver microsomes both phenobarbital and beta-naphthoflavone treatments resulted in a decrease in the I/S ratio. In this species the total scoparone demethylation rate was not much affected by phenobarbital, but beta-naphthoflavone caused a huge increase in over-all scoparone biotransformation. In both species, dexamethasone, isoniazid and clofibrate were much less effective. In contrast to the rat, in the hamster the scoparone biotransformation profile cannot be used to differentiate between phenobarbital- or beta-naphthoflavone-treated animals.

Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures.

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500 mg indole-3-carbinol (13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450 activities were already induced, but the isoenzyme pattern induced was different in the liver and the small intestine. Hepatic P4501A1, P4501A2 and P4502B1 apoprotein levels were dose-relatedly enhanced, whereas in the small intestine induced levels of P4502B1 and P4501A1 were detected but P4501A2 was not induced. Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as well as in the small intestine (8- and 13-fold, respectively, at 500 mg 13C/kg diet). Testosterone 16 alpha- and 16 beta-hydroxylation in the small intestine were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities were only slightly enhanced from day 7 onwards. Thus, the major forms induced in the liver appear to be P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A, whereas in the small intestine all of the effects that were found are associated with only one cytochrome P-450, P4502B1. After 2 days I3C (500 mg/kg) induced glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold). Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and 28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine (1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat hepatocytes with indole-3-acetonitrile and 3,3'-diindolylmethane, but not I3C and indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16 alpha- and 2 alpha-hydroxylation. All four indoles slightly induced glutathione S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that the in vivo effects of I3C have to be attributed to indole-condensation products, such as 3,3'-diindolylmethane, but not to I3C itself.

Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes.

The effects of three acid condensation products of indole-3-carbinol (I3C), i.e. 3,3'-diindolylmethane (DIM), 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI) and 2,3-bis[3-indolylmethyl]indole (BII), on cytochrome P450 and phase II enzymes were studied in primary cultures of rat and cynomolgus monkey liver cells. In rat hepatocytes all three indole derivatives dose-relatedly induced the ethoxyresorufin O-dealkylation (EROD) activity (to 24-fold) and 7 alpha-hydroxylation of testosterone (to 4-fold), whereas all three decreased the 16 alpha- and 2 alpha-testosterone hydroxylation (DIM to 60%, CTI and BII to a mere 5% of the control cells). Treatment of monkey hepatocytes with DIM and BII enhanced the EROD activity to 6- and 9-fold, respectively. Furthermore, BII decreased the 6 beta-hydroxylation of testosterone (to 60% of the untreated cultures) in monkey cells. Phase II enzymes were also affected. In rat hepatocytes DIM, CTI and BII enhanced DT-diaphorase (DTD) (= NAD(P)H-quinone reductase) activity, and DIM and BII the glucuronidation of 1-naphthol. In monkey cells BII only enhanced DTD, and no changes were observed in the glucuronidation of 1-naphthol after treatment with either DIM or BII. The indole derivatives did not affect glutathione S-transferase activity and sulfation of 1-naphthol in either rat or monkey hepatocytes. These results identify two novel acid condensation products of I3C, CTI and BII, as potent compounds in affecting biotransformation in rat as well as in monkey hepatocytes.

Effects of cooked brussels sprouts on cytochrome P-450 profile and phase II enzymes in liver and small intestinal mucosa of the rat.

Male Wistar rats were given semi-synthetic diets supplemented with 0, 2.5, 5 and 20% cooked Brussels sprouts for 2, 7, 14 or 28 days. The effects on several cytochrome P-450 enzymes and phase II enzymes (glutathione S-transferase (GST), glucuronyl transferases 1 and 2 (GT1 and GT2) and DT-diaphorase (DTD)) in the liver and small intestinal mucosa were investigated. From 2 days of exposure onwards Brussels sprouts induced P4501A2 and--to a lesser extent--P4501A1 apoprotein levels in the liver, whereas in the small intestine markedly enhanced P4502B apoprotein levels could be detected. No enhanced P4503A apoprotein levels were observed. The 5 and 20% sprouts diets increased the intestinal pentoxyresorufin depentylation (PROD, 4.5-9-fold), and the hydroxylation of testosterone at the 16 alpha- and 16 beta-site (2.6-4.2-fold) after 2 days of exposure. In addition, the 20% sprouts died also enhanced the intestinal ethoxyresorufin deethylation (EROD) activity (c. 5-fold), the hepatic EROD and PROD activities (c. 2-fold) and the formation of 6 beta-hydroxytestosterone (c. 1.6-fold); the formation of 2 alpha-hydroxytestosterone in the liver was decreased (to c. 70% of the control value). GST activity was induced both in the liver (5 and 20% diet) and intestine (20% diet only) throughout the experiment. The 20% sprouts diet enhanced the hepatic DTD and GT1 activities, whereas the GT2 activity was decreased. The induction of DTD in the small intestine after 2 days (2.5-3.2-fold with 5 and 20% sprouts diets, respectively) diminished during the experiment. These results indicate that dietary exposure to cooked Brussels sprouts for only 2 days can change the metabolic activities of several phase II enzymes and cytochrome P-450 enzymes, of which P4502B is the predominant form induced in the small intestine.

Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions.

Assay conditions and results of cytochrome P-450 dependent 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation (OD) by rat liver microsomes were compared by four laboratories in the Netherlands. Microsomal mixtures were prepared from control, 3-methylcholanthrene and phenobarbital pretreated animals, resulting in different levels of cytochrome P-450 isozymes. EROD and PROD activities were determined in each laboratory according to their own protocols. Considerable variability was found both between and within laboratories. Further studies demonstrated that protocol differences are important factors causing this interlaboratory variation. Main factors of influence were buffer type, batch of resorufin used for calibration, substrate solvent and substrate concentration. Based on the results obtained, standardized protocols for optimized measurement of microsomal EROD and PROD activities were developed. Additional experiments demonstrated that the use of these standardized protocols reduced intralaboratory variation in both the EROD and the PROD assay, whereas it also reduced the interlaboratory variability for the PROD determinations. The interlaboratory variation for measurement of microsomal EROD activities was only reduced for the laboratories using a Cobas-Bio analyzer. The results of the present study demonstrate clearly that data obtained with EROD and PROD activity measurements are highly sensitive to factors frequently varying from one laboratory to another. In addition, they demonstrate the necessity to be careful with absolute values presented in the literature for these activities, unless well characterized assay conditions are applied.

Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities.

The metabolic profile of seven subfamilies of cytochrome P450 (P450IA, IIA, IIB, IIC, IIE, IIIA, IVA) was studied in rat liver (in vivo) and in primary hepatocyte cultures (in vitro) after treatment with various inducers. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), aniline 4-hydroxylation and the regio- and stereoselective hydroxylation of testosterone were measured to characterize the isoenzyme pattern in intact hepatocytes and in liver microsomes. Occurrence of isoenzyme apoproteins was determined using Western blotting. Primary cultures of rat hepatocytes retain the capacity to respond to inducers of isoenzymes belonging to six different subfamilies (P450IA, IIA, IIB, IIC, IIIA and IVA). Treatment of cells with beta-naphthoflavone revealed a P450-activity profile similar to in vivo, namely a highly induced EROD (P450IA1), a small enhancement of testosterone 7 alpha-hydroxylation (P450IIA) and a marked reduction in 2 alpha- and 16 alpha-hydroxylation (P450IIC11). Exposure of cultured cells to phenobarbital resulted in a higher testosterone 16 beta-hydroxylation (reflecting P450IIB), though to a lesser extent than in vivo. The induction of P450IIIA due to both phenobarbital and dexamethasone, as mirrored by 6 beta- and 15 beta-hydroxylation of testosterone, was the same in cultured hepatocytes and in vivo. Treatment of cells with clofibric acid resulted in an induction profile similar to the one observed in liver microsomes from clofibrate-treated rats: the apoprotein P450IVA as well as the apoprotein P450IIB1/2 and its associated activities (PROD and testosterone 16 beta-hydroxylation) were induced. Isoniazid, a known in vivo inducer of P450IIE1 and aniline 4-hydroxylation, did not change any of the determined P450-dependent activities in vitro.

Structure elucidation of acid reaction products of indole-3-carbinol: detection in vivo and enzyme induction in vitro.

The potency of indole-3-carbinol (I3C) to form condensation products under acidic aqueous conditions was studied. After identifying a known dimer, 3,3'-diindolylmethane (DIM), we elucidated the structures of two trimers also found in acid reaction mixtures: 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI), and 2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers was shown to be pH dependent. The highest amounts of DIM and BII were formed in aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected at pH values above 4.5. In rats that received an oral dose of I3C we could detect DIM and BII in gastric contents, stomach tissue, small intestine and liver. No CTI could be detected in vivo after oral exposure to I3C. In in vitro experiments, using rat hepatocytes, the cytochrome P-450IA1 apoprotein level, 7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD) were markedly enhanced by DIM and CTI as well as BII.

The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers.

Changes in the isoenzyme pattern of cytochrome P450 during culture were investigated in primary cultures of rat hepatocytes, measuring specific enzyme activities in microsomes prepared from cultured cells as well as in intact monolayers. Assays of 7-ethoxyresorufin O-deethylation (EROD), 7-pentoxyresorufin O-depentylation (PROD), aniline 4-hydroxylation (AH) and the specific regioselective hydroxylation of testosterone were used as representatives of the activities of seven isoenzymes of cytochrome P450. The isoenzyme profile expressed as catalytic activities was qualitatively and quantitatively similar in microsomes obtained from freshly isolated hepatocytes in comparison with microsomes obtained from whole livers of untreated rats. There was a relatively high activity in EROD, AH and the oxidation of testosterone at the 7 alpha, 2 alpha, 6 beta, 16 alpha and 17 sites (androstenedione). During culture, these microsomal enzyme activities declined at a similar rate to ca. 50% of the activities of microsomes prepared from freshly isolated hepatocytes after 24 hr and to 15% after 96 hr. The overall decline of cytochrome P450-dependent activities during culture was not accompanied with gross changes in catalytic profile. Determining the same drug-metabolizing activities directly in intact hepatocyte monolayers revealed a much higher metabolic rate for all measured P450-dependent activities. The profile of the catalytic activities was essentially the same as measured in microsomes prepared from cultured hepatocytes. The relatively low activity towards the 7 alpha site of testosterone measured in intact hepatocytes, however, remained constant during culture. Determination of enzyme activities directly in intact hepatocytes is a convenient way of studying changes in monooxygenase activities of different P450 isoenzymes in vitro.

Effect of normobaric hyperoxia on antioxidant defenses of HeLa and CHO cells.

The effect of increased intracellular oxygen activation on cellular antioxidant defenses in CHO and HeLa cells was studied. In both cell types, hyperoxic exposure (up to 4 days, 600-700 mm Hg O2) and in CHO cells menadione (up to 3 days, 15 microM) failed to affect the enzymatic antioxidant defenses Mn-containing superoxide dismutase (Mn-SOD), CuZn-SOD, catalase and glutathione peroxidase. The markedly increased antioxidant enzyme activities observed in a recently obtained oxygen-tolerant CHO variant persisted under normoxia. These data suggest that the synthesis of antioxidant enzymes is constitutive. Glutathione levels of HeLa cells did not respond to hyperoxia whereas in CHO cells hyperoxia and menadione exposure resulted in a 2- and 7-fold increase in glutathione contents, respectively. However, considering the large variations in glutathione contents observed under normal culture conditions, it is uncertain whether this increase is to be considered as a true adaptive response.

Antioxidant status of Fanconi anemia fibroblasts.

Several observations in the recent literature have indicated that Fanconi anemia (FA) cells may be primarily deficient in the detoxification of activated oxygen species. To evaluate the antioxidant status of FA fibroblasts, we measured Mn-containing superoxide dismutase (Mn-SOD), CuZn-containing superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase activities, as well as cellular glutathione contents and total nonenzymatic antioxidant potential in FA and control fibroblasts at multiple time points during a single passage. All parameters exhibited a characteristic pattern of changes during a period of 19 days following trypsinization. Unlike FA erythrocytes, which are known to be deficient in CuZn-SOD, FA fibroblasts exhibited normal CuZn-SOD activities. Also, the nonenzymatic "antioxidant potential" as well as glutathione levels were similar in FA and control fibroblasts. However, Mn-SOD, catalase, and glutathione peroxidase activities were consistently higher in FA fibroblasts. We hypothesize that the elevation of these enzyme activities might reflect a cellular "prooxidant" state in FA resulting from an increased formation of endogenous oxidizing molecular species that trigger enhanced synthesis of certain enzymatic antioxidant defenses.

Induction and activity of several isoenzymes of cytochrome P-450 in primary cultures of rat hepatocytes, in comparison with in vivo data.

Changes in cytochrome P-450 isoenzymes were studied in rat liver and in primary cultures of rat hepatocytes after treatment with compounds belonging to various classes of inducers, including phenobarbital (PB), beta-naphthoflavone (BNF), and clofibrate/clofibric acid (CLOF/CLOFA). The enzyme activity toward specific substrates was measured, and the presence of apoprotein of several P-450 isoenzymes was determined semiquantitatively by Western blotting. In untreated cultures the P-450 content and activities of 7-ethoxyresorufin O-deethylation (EROD) and aniline 4-hydroxylation (AH) declined with time at different rates. In cultures treated with BNF, the protein levels of isoenzyme P-450IA1 and P-450IA2 were elevated, as in vivo. This induction was reflected in a markedly increased EROD activity. CLOFA enhanced the AH and EROD activity in primary cultures at the same level as in vivo. The monooxygenase activity pentoxyresorufin O-depentylation (PROD) was stimulated by PB and CLOF in vivo, which correlated with the enhanced protein level of P-450IIB1/2. In contrast, the PROD activity was not induced when cultures were treated with PB or CLOFA, although we could detect apoprotein of P-450IIB1/2 by immunoblotting.