Differentiation of cytochrome P-450 inducers on the basis of 7-alkoxycoumarin O-dealkylase activities.

1. The high-affinity and total activities of 7-methoxycoumarin O-demethylase and of 7-ethoxycoumarin O-deethylase were measured in liver microsomes isolated from rats treated with prototypic inducers belonging to the main classes of microsomal enzyme inducers currently recognized. 2. Each inducer (with the exception of clofibrate which was ineffective) produced a unique induction profile, indicating the possibility of using these two enzymes, each measured at two substrate concentrations, for characterization of the inducing potential of novel xenobiotics. 3. Differences between p,p'-DDT and phenobarbitone in the induction profiles indicate that DDT may not be a pure 'phenobarbitone-type' inducer as has been proposed hitherto.     

Influence of medium composition on 7-alkoxycoumarin O-dealkylase activities of rat hepatocytes in primary maintenance culture

1. The activities of 7-methoxycoumarin (7-MCOD), 7-ethoxycoumarin (7-ECOD) and 7-propoxycoumarin (7-PCOD) O-dealkylases decreased by 75-90% during culture of rat hepatocytes for 72 h. 2. The addition of dexamethasone (D) produced a stabilization or modest enhancement of 7-ECOD and 7-PCOD activities depending on the medium used; D was without effect on 7-MCOD activity. 3. The addition of nicotinamide (N) produced some stabilization of 7-ECOD and 7-PCOD activities but not of 7-MCOD activity. 4. The addition of D + N was associated with large increases in 7-ECOD and 7-PCOD activities, again with little effect on 7-MCOD activity. 5. Ethoxyresorufin O-dealkylase activity correlated with 7-ECOD/7-PCOD activities, whereas pentoxyresorufin O-dealkylase activity correlated with 7-MCOD activity. 6. It is concluded that D exerts a selective effect on certain forms of cytochrome P-450, but that more than one mechanism is probably implicated in this effect. 7. The magnitudes of induction of 7-ECOD activity by phenobarbitone and beta-naphthoflavone were blunted when cells were treated in culture when compared to treatment in vivo; the presence of D in the culture medium did not modify this phenomenon.     

Daily fluctuation of 7-alkoxycoumarin O-dealkylase activities in the liver of male F344 rats under ad libitum-feeding or fasting condition.

It has been reported that drug metabolizing enzyme activities in the liver fluctuate daily, though the daily fluctuation of 7-alkoxycoumarin O-dealkylase (ACD) activities catalyzed by P450 has not been examined. The ACD activities are known to be useful to clarify the extensive induction of P450-dependent oxygenases. In the present study, the hepatic ACD activities, as well as P450 content, were investigated periodically in male F344 rats under ad libitum-feeding or fasting condition. The ACD activities in ad libitum-fed rats were found to follow obvious daily fluctuations with high values during dark periods and with low values during light periods. This agreed with the other previous results of drug metabolizing enzyme activities measured with other substrates. Because the daily fluctuation was also observed in fasted rats, the fluctuation in ACD activities was clearly not affected by feeding, an external factor. P450 content, however, showed different fluctuation patterns from the ACD activities. This suggests that the fluctuation of ACD activities might not be related to the quantitative variation of P450 proteins.     

Biotransformation of coumarin derivatives. (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phenobarbital- or beta-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.     

Influence of monooxygenase inducers on the metabolic profile of phenanthrene in rat liver microsomes

The oxidation of phenanthrene by rat liver microsomes significantly depends on the pretreatment of the animals as shown by means of gas chromatography/mass spectrometry. Whereas untreated animals convert phenanthrene exclusively into the 9,10-dihydrodiol (K-region), pretreatment with various polycyclic aromatic hydrocarbons and related compounds resulted in different rates of additional oxidation at the 1,2- and 3,4-position. Moreover, secondary metabolism to dihydrodiol epoxides, detected as triols, was observed. Despite considerable concentration of the proximate carcinogen of phenanthrene (1,2-dihydrodiol) only low concentrations of the ultimate carcinogen were detected which may explain the carcinogenic inefficiency of this hydrocarbon.     

Effects of various inducers on the activities of cytochrome P-450-mixed function oxidases and aflatoxin B1 activation in microsomes of hamster livers

The effects of various inducers on the activities of drug-metabolizing enzymes including aflatoxin B1 activation were studied in Syrian golden hamsters. Activity for aflatoxin B1 was determined by aflatoxin B1-DNA adducts formation. The treatments of hamsters with 3-methylcholanthrene, alpha-naphthoflavone and benzo(a)pyrene elevated markedly the activity for aflatoxin B1 by 2460, 1380 and 450%, respectively. Phenobarbital induced slightly and isosafrole and ethanol did not induce the activity for aflatoxin B1. Pregnenolone-16 alpha-carbonitrile decreased aflatoxin B1 activation to 51% of that of the non-treated animals. These results were in good accordance with the induction rate of a form of cytochrome P-450 (P-450AFB) which has potent activity to aflatoxin B1. Characteristics in the induction of mixed function oxidases of hamsters by these inducers, especially in respect to benzo(a) pyrene metabolizing enzyme, seemed to differ from those of rats. These results suggest that the activity for aflatoxin B1 in hamster is inducible by 3-methylcholanthrene-type inducers and that hamster is a suitable animals model to study the mechanism of aflatoxin B1-hepatocarcinogenesis.     

Cytochrome P-450-dependent monooxygenase activities in prenatal and postnatal human livers: comparison of human liver 7-alkoxycoumarin O-dealkylases with rat liver enzymes

Human liver samples from 17 embryos, 5 fetuses, 5 infants and 4 adults were used to investigate human liver cytochrome P-450-dependent 7-alkoxycoumarin O-dealkylase activities, and their drug-metabolizing activities were compared to those of rat livers. The O-dealkylase activities in human embryos and fetuses were very low, although detectable, similar to those in fetal rats. Both male and female rats showed a postnatal increase of hepatic O-dealkylase activities with a maximum at about 30-40 days after birth and then a decline in the activities which was marked in female rats. Adult female rats showed a marked decrease in the hepatic enzyme activity observed in the O-depropylation reaction rather than the O-demethylation and O-deethylation reactions. During the developmental period of human infants, the O-demethylase activity, but not O-depropylase activity, increased gradually. Enzymes in adult human livers metabolize the O-methyl derivative of 7-hydroxycoumarin in preference to the O-ethyl and O-propyl derivatives. The metabolic activities of human adult enzymes for 7-alkoxycoumarin resembled those in adult female rats and were quite different from those in male rats. The study demonstrated that caution must be exercised in extrapolating pharmacological results from animal to man in the field of drug metabolism.     

Biphenyl metabolism by rat liver microsomes: regioselective effects of inducers, inhibitors, and solvents

Examination of the regioselective metabolism of biphenyl was explored as a means of characterizing different forms of cytochrome P-450 in microsomal and purified mono-oxygenase systems. In the present study the effects of the inducers phenobarbital and 3-methylcholanthrene, the inhibitors 7,8-benzoflavone and 1-benzylimidazole, and the solvents methanol, acetone, and dimethyl sulfoxide on the 2-, 3-, and 4-hydroxylation of biphenyl and the O-deethylation of 7-ethoxycoumarin by rat liver microsomes were examined. Phenobarbital pretreatment primarily induced 2- and 3-hydroxylation, the latter most dramatically. 3-Methylcholanthrene pretreatment induced 2- and 3-hydroxylation to similar extents. The inhibitors and solvents had regioselective effects on biphenyl metabolism that were characteristic of the uninduced, phenobarbital-induced, and 3-methylcholanthrene-induced microsomes. The presence of multiple forms of cytochrome P-450 in uninduced microsomes is indicated by the regioselective effects of the solvents and the inhibitors. The 3-methylcholanthrene-dependent increases in 2- and 3-hydroxylation appear due to induction of a single form of cytochrome P-450, as indicated by similar dose-response relationships and similar changes in sensitivity to the inhibitors. The phenobarbital-dependent increases in 2- and 3-hydroxylation appear due to the induction of two forms of cytochrome P-450, as indicated by different changes in sensitivity to the effects of dimethyl sulfoxide and 7,8-benzoflavone. The results indicate that examination of the regioselectivity of biphenyl metabolism is a useful approach for characterizing microsomal mono-oxygenases, and they suggest that the approach may also be useful in the characterization of purified mono-oxygenase systems.     

Comparison of monooxygenase activities and cytochrome P-450 isozyme concentrations in human liver microsomes

Concentrations of three human liver microsomal cytochrome P-450 isozymes and 20 different monooxygenase activities were determined in human liver microsomal preparations. The results of correlation analysis suggest that: there are important variations in the amounts of the three cytochrome P-450 isozymes measured, particularly P-450(8) and P-450(9); aldrin epoxidase, d-benzphetamine N-demethylase, and S-warfarin 4-hydroxylase activities are linked to cytochrome P-450(5); aryl hydrocarbon (benzo(a)pyrene) hydroxylase and 4-nitroanisole-O-demethylase activities are linked to P-450(8); hydroxylations at the 4'-, 6-, 7-, and 8-positions of R-warfarin are closely linked to each other but are not correlated with other measured monooxygenase activities or P-450 isozyme levels; and P-450(9) is not related to any of the catalytic activities tested. Thus, certain monooxygenase activities can be attributed to specific cytochrome P-450 isozymes. This approach should be useful in suggesting the roles of different cytochromes P-450 in drug metabolism in man which can be further examined using in vitro and in vivo methods.     

Studies of UDP-glucuronyltransferase activities in human liver microsomes

Human liver samples from a "liver bank" which have been previously characterized by the ability to catalyze cytochrome P-450 dependent reactions were analyzed for various UDP-glucuronyltransferase activities. When stored at -80 degrees C, UDP-glucuronyltransferase activities and latency characteristics of the enzyme appeared to be stable for up to 2 years. The correlation of UDP-glucuronyltransferase activity (4-methylumbelliferone as substrate) with enzyme activities towards 4-nitrophenol and 1-naphthol was much higher (r greater than 0.8) than that (r less than 0.3) observed with other enzyme activities (4-hydroxybiphenyl, morphine, and chloramphenicol as substrates), suggesting the presence of multiple enzyme forms in human liver. Livers of three patients treated with phenytoin or pentobarbital showed significantly higher UDP-glucuronyltransferase activities towards 1-naphthol, 4-methylumbelliferone, and bilirubin than those from five patients who did not receive these inducing agents.     

Formation of carcinogenic and inactive chrysene metabolites by rat liver microsomes of various monooxygenase activities

Microsomal oxidation of chrysene in rat liver occurs at various positions (1,2-; 3,4-; 5,6-). This has been verified by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. After various rat pretreatments with inducers of the monooxygenase system the oxidation at the 3,4-position predominated in isolated microsomes. The formation of the ultimate carcinogen of chrysene—1, 2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene — was not detectable in untreated rats. However, it was observed as 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene-TMS-ether formed under workup and derivatisation conditions after pretreating the rats with phenobarbital, polychlorinated biphenyl, benzoflavone, or various polycyclic aromatic hydrocarbons. Polychlorinated biphenyls and benzoflavone were the most potent inducers for the formation of this metabolite.Abbreviations BjF Benzo(j)fluoranthene - BNF 5,6-benzoflavone - GC gas-liquid chromatography - HPLC high-pressure liquid chromatography - MS mass spectrometry - PAH polycyclic aromatic hydrocarbons - PCB polychlorinated biphenyl - TCBP 3,3,4,4-tetrachlorobiphenyl - TMS trimethylsilyl Assessment of environmental compounds by carcinogenspecific tests, part 1     

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.     

Regioselective and stereoselective metabolisms of pyrene and 1-bromopyrene by rat liver microsomes and effects of enzyme inducers

Due to the symmetrical property of pyrene (Py), trans-dihydrodiols formed at 4,5- and 9,10-positions are identical, as are the monohydroxylated products (phenols) formed at C1, C3, C6, and C8 positions. With a bromo substituent at C1 position of Py, 1-bromopyrene (1-BrPy) trans-4,5-dihydrodiol and 1-BrPy trans-9,10-dihydrodiol are distinctly different products, as are the phenolic products formed at C3, C6, and C8 positions. Products formed in the oxidative metabolism of 1-BrPy by rat liver microsomes were characterized by retention times on reversed-phase high performance liquid chromatography (HPLC), and by ultraviolet-visible absorption and mass spectral analyses. We have compared regioselective and stereoselective metabolisms at the K- and non-K-regions of Py and 1-BrPy by liver microsomes from untreated (control), phenobarbital (PB)-treated, 3-methylcholanthrene (MC)-treated, and polychlorinated biphenyls (PCB, Aroclor 1254)-treated rats. The effects of inducers on the relative amounts of non-K-region phenols formed in the metabolisms of Py and 1-BrPy by rat liver microsomes were: MC greater than PCB greater than PB greater than control. The relative order was PB greater than PCB greater than MC greater than control for the formation of both 1-BrPy trans-4,5-dihydrodiol and 1-BrPy trans-9,10-dihydrodiol in the metabolism of 1-BrPy. The ratios between metabolically formed 1-BrPy trans-4,5-dihydrodiol to Py trans-4,5-dihydrodiol, using 0.5 mg of microsomal protein per ml of incubation mixture, were between 0.4 and 0.6 in the presence of liver microsomes from untreated, PB-treated, and PCB-treated rats. However, the ratio was approximately 1.5 using liver microsomes from MC-treated rats. The ratios between the sum of 1-BrPy trans-9,10-dihydrodiol and 1-BrPy 9,10-epoxide to Py trans-9,10-dihydrodiol, and to 1-BrPy trans-4,5-dihydrodiol were in the range of 0.1 to 0.5 using four rat liver microsomal preparations. These data revealed the effects of a bromo substituent at C1 of Py on the regioselectivity of various rat liver microsomal enzymes toward the oxidative metabolism at various positions of 1-BrPy. The enantiomeric compositions of K-region dihydrodiols formed by four rat liver microsomal preparations were determined by chiral stationary phase HPLC and circular dichroism spectral analyses; the percentage of R,R-enantiomers were: Py trans-4,5-dihydrodiol, 78-79%; 1-BrPy trans-4,5-dihydrodiol, 74-77%; 1-BrPy trans-9,10-dihydrodiol, 86-97%.     

Comparison of the effects of various flavonoids on ethoxycoumarin deethylase activity of rat intestinal and hepatic microsomes

Fifteen flavonoids were examined for their effects on the activity of 7-ethoxycoumarin O-deethylase in rat hepatic and intestinal microsomes. The effect depended on both the chemical structure of the flavonoid and the origin of the microsomes. Polyhydroxylated flavonoids with a C2-C3 double bond (flavones and flavonols) were more effective inhibitors of the enzyme in both hepatic and intestinal microsomes than were the reduced homologues (flavanonols, flavanones and flavan-3-ols). In contrast, flavones lacking hydroxyl substituents (e.g. 5,6-benzoflavone, 7,8-benzoflavone and flavone) increased ethoxycoumarin deethylase activity in liver microsomes although they had an inhibitory effect in intestinal microsomes.     

Influence of chronic ethanol consumption on hamster liver microsomal O-dealkylase activities and cytochrome b5 content

The effects of two different methods of administering ethanol to hamsters on liver microsomal cytochrome levels and the activities of ethoxyresorufin O-deethylase and p-nitroanisole O-demethylase have been examined. Administration of ethanol in liquid diets resulted in enhanced levels of cytochrome P-450, NADPH-supported aniline hydroxylase (Form I), and both NADPH- and NADH-supported p-nitroanisole O-demethylase. NADH-ferricyanide reductase was also increased. No change in NADPH-cytochrome c reductase or in the NADPH-supported rate of ethoxyresorufin O-deethylase was observed. In contrast, both NADH-supported ethoxyresorufin O-deethylase and cytochrome b5 levels were decreased. Administration of ethanol in the drinking water to chow-fed animals had no effect on total cytochrome P-450 levels; however, the rates of NADPH-supported aniline hydroxylase (Form I) and p-nitroanisole O-demethylase activity were increased. No changes in NADPH-cytochrome c reductase, NADH-ferricyanide reductase, or NADH-supported p-nitroanisole O-demethylase activity were noted. Cytochrome b5 levels were decreased as were both the NADPH- and NADH-supported rates of ethoxyresorufin O-deethylase. These data suggest that chronic consumption of ethanol by hamsters either in liquid diet form or as ethanol-water solutions to chow-fed animals lowers cytochrome b5 levels. When cytochrome b5 levels are lowered and total chromosome P-450 levels remain unchanged, the NADPH-supported rate of microsomal O-dealkylation of ethoxyresorufin is decreased. These data suggest that cytochrome b5 participates in the NADPH-supported microsomal O-dealkylation of ethoxyresorufin.     

外消旋普罗帕酮在经诱导的大鼠肝微粒体中N-去丙基化的立体选择性

采用对照及β-萘黄酮(β-NF)或地塞米松(Dex)诱导的大鼠肝微粒体,应用GITC柱前衍生化,反相高效液相色谱法研究了消旋普罗帕酮〔(R/S)-PPF〕体外代谢的立体选择性. 实验结果表明,在Dex,β-NF诱导的微粒体中有N-去丙基普罗帕酮生成。在β-NF,Dex预处理组,(R/S)-PPF低浓度时的经肝微粒体代谢具有立体选择性,R(-)对映体的清除大于S(+)对映体,高浓度时的代谢无立体选择性. R(-)对映体的K_m值显著低于S(+)对映体,而V_max值无显著性差异. 在Dex预处理组中的立体选择性大于β-NF组. 在对照组中代谢无立体选择性,且K_m,V_max值均小于β-NF,Dex预 处理组。结果提示,CYP1A,CYP3A4亚族对普罗帕酮(PPF)的N-去丙基化有贡献. (R/S)-PPF的N-去丙基化具有浓度依赖性的立体选择性.     

Comparison of the effects of inducers of cytochrome P450 on Mongolian gerbil and rat hepatic microsomal monooxygenase activities

1. Basal cytochrome P450 content (nmol/mg protein) was higher in gerbil (1.10 ± 0.01) than in rat (0.81 ± 0.05) hepatic microsomes. Pretreatment of gerbils with phenobarbitone and β-naphthoflavone increased P450 contents by 200% and 60% respectively.

2. 7-Ethoxycoumarin O-deethylase, coumarin 7-hydrozylase and 4-nitrophenol hydroxylase activities were generally higher in gerbil liver microsomes, whereas erythromycin N-demethylase, and 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were higher in rat microsomes. Microsomal benzphetamine N-demethylase activities were similar in both species.

3. Induction of specific cytochrome P450 isozymes increased similar monooxygenase activities of rat and gerbil microsomes. Phenobarbitone, β-naphthoflavone, isoniazid and pregnenolone 16α-carbonitrile principally increased benzphetamine N-demethylase, 7-ethoxyresorufin O-deethylase, 4-nitrophenol hydroxylase and erythromycin N-demethylase activities respectively.

4. Constitutive 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were markedly lower in gerbil microsomes compared with rat microsomes, and pretreatment of gerbils with cytochrome P450 inducers did not significantly increase these activities.

5. Hepatic microsomal coumarin 7-hydroxylase activities were approximately 30–200 times greater (depending on the inducer) in the gerbil than in rat. The gerbil, due to is high coumarin 7-hydroxylase activity, would appear to be a more appropriate species than rat for investigations of coumarin metabolism and toxicity relevant to humans.     

Differential induction of human liver UDP-glucuronosyltransferase activities by phenobarbital-type inducers

(1) UDP-glucuronosyltransferase (UDP-GT) activities and their inducibility were investigated in human liver microsomes from a "liver bank". (2) UDP-GT activities were differentially induced in liver microsomes from patients treated with the phenobarbital-type inducers phenytoin or pentobarbital. UDP-GT activity towards bilirubin was induced 3-fold. Enzyme activities towards paracetamol, benzo(a)pyrene-3,6-quinol, 4-methylumbelliferone and 1-naphthol were moderately induced and to similar extents (2-fold). In contrast, morphine and 4-hydroxybiphenyl glucuronidation were not significantly affected. Cytochrome P-450 dependent 7-ethoxycoumarin O-deethylase was increased 5-fold. (3) A human hepatoma cell line (Hep G2) was studied to obtain information on the inducibility of human UDP-GT activities by 3-methylcholanthrene-type inducers. UDP-GT activities towards benzo(a)pyrene-3,6-quinol and 1-naphthol were moderately but significantly induced by 3-methylcholanthrene-treatment of the cells (2-fold), whereas 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase were increased over 100- and 10-fold, respectively. (4) The results suggest the existence of differentially inducible UDP-GT isoenzymes in human liver. The findings may be useful as a guide to characterize human liver UDP-GT isoenzymes.     

Effect of probucol on cytochrome P450 activities in human liver microsomes

The effects of probucol, a cholesterol-lowering agent, on several cytochrome P450 (CYP) isoform-specific reactions in human liver microsomes were investigated to predict drug interactions with probucol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. Probucol had neither stimulatory nor inhibitory effects on CYP1Al/2, 2A6, 2B6, 2C8/9, 2C19, 2D6, 2E1, and 3A4 activities at concentrations up to 300 microM, indicating that probucol, at the expected therapeutic concentrations, would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.