Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2)

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Human CYP1A1 allelic variants: baculovirus expression and purification,hydrodynamic, spectral,and catalytical properties and their potency in the formation of all-trans-retinoic acid

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.     

Amino acid 305 determines catalytic center accessibility in CYP3A4

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.     

Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1

Benzene is an occupational and environmental toxicant. The main human health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome p450 enzyme system. The cytochrome p450 enzymes CYP2E1 and CYP2F2 are the major contributors to the bioactivation of benzene in rats and mice. Although benzene metabolism has been shown to occur with mouse and human lung microsomal preparations, little is known about the ability of human CYP2F to metabolize benzene or the lung cell types that might activate this toxicant. Our studies compared bronchiolar derived (BEAS-2B) and alveolar derived (A549) human cell lines for benzene metabolizing ability by evaluating the roles of CYP2E1 and CYP2F1. BEAS-2B cells that overexpressed CYP2F1 and recombinant CYP2F1 were also evaluated. BEAS-2B cells overexpressing the enzyme CYP2F1 produced 47.4 +/- 14.7 pmols hydroxylated metabolite/10(6) cells/45 min. The use of the CYP2E1-selective inhibitor diethyldithiocarbamate and the CYP2F2-selective inhibitor 5-phenyl-1-pentyne demonstrated that both CYP2E1 and CYP2F1 are important in benzene metabolism in the BEAS-2B and A549 human lung cell lines. The recombinant expressed human CYP2F1 enzyme had a K(m) value of 3.83 microM and a V(max) value of 0.01 pmol/pmol p450 enzyme/min demonstrating a reasonably efficient catalysis of benzene metabolism (V(max)/K(m) = 2.6). Thus, these studies have demonstrated in human lung cell lines that benzene is bioactivated by two lung-expressed p450 enzymes.     

Leukotriene B4 omega-side chain hydroxylation by CYP4F5 and CYP4F6

Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.     

Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.     

Alpha-tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic alpha-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic alpha-tocopherol metabolism and excretion, rats received daily subcutaneous alpha-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver alpha-tocopherol concentrations increased from 12 +/- 1 nmol/g (mean +/- SE) to 819 +/- 74 (Day 3), decreased at Day 9 (486 +/- 67), and continued to decrease through Day 18 (338 +/- 37). alpha-Tocopherol metabolites and their intermediates increased and decreased similarly to alpha-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic alpha-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver alpha-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic alpha-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.     

Purification of recombinant wheat cytochrome P450 monooxygenase expressed in yeast and its properties

To investigate the properties of wheat cytochrome P450 and the characteristics of herbicide metabolism by cytochrome P450 in vitro, deeply understand the mechanisms of herbicide selectivity, recombinant wheat cytochrome P450 monooxygenase (CYP71Cv1) heterologously expressed in yeast was purified by DE-52 cellulose chromatography and fast protein liquid chromatography (FPLC) with Mono-Q column. The degree of purification was 1366-fold. The specific activity of purified cytochrome P450 reached to 512 nmol min-1 mg-1 protein with herbicide chlorsulfuron as substrate. The purified cytochrome P450 exhibited one band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular mass was 52.5 kDa. Kinetic parameter was determined in vitro. The Km values for chlorsulfuron and triasulfuron were 57 (+/-15) and 38 (+/-16) microM, respectively; and Vmax for chlorsulfuron and triasulfuron were 4.1 (+/-0.7) and 2.7 (+/-0.5) nmol min-1 mg-1protein in vitro, respectively.     

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

Catalytic turnover of pyrene by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.     

Localization of CYP4B1 in the rat nasal cavity and analysis of CYPs as secreted proteins

CYP4B1 is highly expressed in rat nasal respiratory mucosa, and to a lesser extent in olfactory mucosa. Examination of high-power photomicrographs suggests that CYP4B1 may be a secreted protein, based on the fact that immunoreactivity appears to be present in the lumens of ducts of Bowman's glands (rather than intracellular localization, as we observed with an antibody recognizing CYP2F4) and in secretory granules in respiratory mucosa. Furthermore, anti-CYP4B1 immunoreactivity is present on the surface of both respiratory and olfactory mucosa. We used SignalP 3.0 analysis to ascertain the likelihood that rat CYP4B1 is a secreted protein. While this analysis does not suggest that rat CYP4B1 is a secreted protein, several other cytochrome P450 enzymes were predicted to be secreted proteins. The observation that multiple human cytochrome P450s appear to be secreted proteins helps to explain the appearance of anti-cytochrome P450 antigens in cases of human autoimmune liver diseases.     

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.     

Short term treatment with St. John's wort, hypericin or hyperforin fails to induce CYP450 isoforms in the Swiss Webster mouse

This investigation was designed to determine whether St. John's wort (SJW)(435 mg/kg/d), a readily available antidepressant, or its purported active constituents hypericin (1 mg/kg/d) and hyperforin (10 mg/kg/d) were able to induce various hepatic cytochrome P450 (CYP450) isoforms. SJW, hypericin and hyperforin were administered to male Swiss Webster mice for four consecutive days and hepatic microsomes were prepared on day 5. None of the three treatments resulted in a statistical change in total hepatic CYP450 (SJW treated 0.95 +/- 0.09 nmol/mg vs control 1.09 +/- 0.14 nmol/mg). Furthermore, the catalytic activities of CYP1A2. CYP2E1 and CYP3A were unchanged from control following all three treatments as determined by ethoxyresorufin O-deethylation, p-nitrophenol hydroxylation and erythromycin N-demethylation respectively. Additionally, western immunoblotting demonstrated that there was no significant change in the polypeptide levels of any of the three isoforms. These results indicate that four days of treatment with moderate to high doses of SJW, hyperforin or hypericin fails to induce these CYP450 isoforms in the male Swiss Webster mouse.     

Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.