Analysis of the interactions of cytochrome b5 with flavocytochrome P450 BM3 and its domains

Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b(5)-like proteins in the control of activities of certain prokaryotic P450s.     

Non-enzymatic reduction of aliphatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450

1. The mechanism of reduction of aliphatic tertiary amine N-oxides to tertiary amines in liver microsomes was examined and a novel type of reduction by cytochrome P450 was found. 2. Rat liver microsomes exhibited a significant N-oxide reductase activity toward brucine N-oxide and imipramine N-oxide in the presence of both NAD(P)H and FAD under anaerobic conditions. These N-oxide reductase activities were inhibited by carbon monoxide or air. However, the activities were not abolished by boiling the microsomes; indeed, in the case of brucine N-oxide, the activity was enhanced. 3. The activity toward brucine N-oxide was also observed after the conversion of cytochrome P450 to cytochrome P420. Cytochrome P4502B1 alone exhibited the reductase activity in the presence of both NAD(P)H and FAD. After the removal of haem from cytochrome P4502B1, the activity was observed in the haem moiety, but not in the cytochrome P450 apoprotein. 4. Photochemically reduced FAD was effective in the reduction in place of NAD(P)H and FAD. 5. The N-oxide reduction appears to proceed non-enzymatically, catalysed by the haem group of cytochrome P450 in the presence of a reduced flavin.     

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b₅ as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b₅ (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b₅ and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b₅ and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40±0.15 g/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.Part of this work was presented at the EUROTOX 2001, Istanbul, Turkey, 2001     

Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy

Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.     

The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (k_cat=4120 min⁻¹, K_m=77 μM for MTT and k_cat=6580 min⁻¹, K_m=51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.     

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine,Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17α-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17α-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17α-hydroxylated P4 and P5 (17α-OH P4 and 17α-OH P5) followed by the C17,20-lyase reaction for the conversion of these C₂₁-17α-hydroxylated steroids to C₁₉-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b₅ (b₅)-stimulated C17,20-lyase activity that converts 17αOH-P4 to androstenedione (AD) but also converts 17α-OHP5 to dehydroepiandrosterone (DHEA); (b) guinea pig P450c17 possesses a b₅-stimulated C17,20-lyase activity that converts 17α-OH P4 to AD but does not convert 17α-OH P5 to DHEA., and (c) bovine P450c17 possesses a b₅-stimulated C17,20-lyase activity that converts 17α-OH P5 to DHEA but does not convert 17α-OH P4 to AD. Thus, the P450c17 of each species differs in its ability to catalyze in vitro the conversion of C₂₁-steroids to C₁₉-steroids. In addition, each P450c17 is capable of catalyzing additional hydroxylation reactions leading to low levels of 2α-, 6β-, 16- and 21-hydroxy-metabolites. Porcine P450c17 also catalyzes the b₅-dependent synthesis of andien-β (androsta-5,16-dien-3β-ol) from P5.

When the amino acid sequences of the three P450c17s were aligned there was an approximate 50% variation in the alignment identity (227 differences in the sequences of 509 amino acids). Alignment did not permit the assignment of specific amino acids or domains to the observed differences in enzymatic activities.     

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: The effect of the expression of cytochrome b5 with recombinant P450 enzymes

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL_int) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b₅ were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b₅. The following prediction methods were compared to determine the CL_int value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V_max value; and (3) RAF estimated from the CL_int value. Estimating RAF from CL_int proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b₅ might be suitable for the prediction of the CL_int value.     

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Terfenadine metabolism of human cytochrome P450 2J2 containing genetic variations (G312R,P351L and P115L)

The human cytochrome P450 2J2 is involved in several metabolic reactions, including the oxidation of important therapeutics and epoxidation of endogenous arachidonic acid. At least ten genetic variations of P450 2J2 have been identified, but their effects on enzymatic activity have not been clearly characterized. Here, we evaluated the functional effects of three genetic variations of P450 2J2 (G312R, P351L, and P115L). Recombinant enzymes of wild-type and three variant P450 2J2 were heterologously expressed in Escherichia coli and purified. P450 expression levels in the wild-type and two variants (P351L and P115L) were 142–231 nmol per liter culture, while the G312R variant showed no holoenzyme peak in the CO-binding spectra. Substrate binding titrations to terfenadine showed that the wild-type and two variants displayed K_d values of 0.90–2.2 μM, indicating tight substrate binding affinities. Steady-state kinetic analysis for t-butyl methyl hydroxylation of terfenadine indicated that two variant enzymes had similar k_cat and K_m values to wild-type P450 2J2. The locations of mutations in three-dimensional structural models indicated that the G312R is located in the I-helix region near the formal active site in P450 2J2 and its amino acid change affected the structural stability of the P450 heme environment.     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The K _m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The V _max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA. Received: 26 January 1998 / Accepted: 26 February 1998     

Induction of CYP1A1, 2B, 2E1 and 3A in rat liver by organochlorine pesticide dicofol

The present study has determined the ability of dicofol, an organochlorine pesticide, to induce cytochrome P450 using rats treated with 1, 10, and 25 mg/kg dicofol intraperitoneally for 4 days. Treatments with 10 and 25 mg/kg dicofol produced dose-related increases of cytochrome P450 and cytochrome b₅ contents and NADPH-cytochrome c reductase, 7-ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. The treatments also increased glutathione S-transferase and superoxide dismutase activities in liver cytosol. Dicofol at 1 mg/kg produced a general trend towards increases of the aforementioned enzyme levels. The results of immunoblot analyses showed that 10 and 25 mg/kg dicofol increased protein levels of CYP1A1, CYP2B, CYP2E1, and 3A in liver. RT-PCR data indicated that dicofol induced mRNA expression of liver CYP1A1, CYP2B, and CYP3A. Pretreatments of rats with 10 and 25 mg/kg dicofol decreased phenobarbital-induced sleeping time by 34% and 39%, respectively. Dicofol pretreatment at 25 mg/kg increased CCl₄-induced serum alanine aminotransferase activity by 4.3-fold and aspartate aminotransferase activity by 4.1-fold. The present study demonstrates that dicofol has the ability to induce CYP1A1, CYP2B, CYP2E1, and CYP3A in the liver and increase phenobarbital metabolism and CCl₄ toxicity in rats.     

Inhibitory effects of memantine on human cytochrome P450 activities: prediction of in vivo drug interactions

Objective: The aim of the present study was to predict the drug interaction potential of memantine by elucidation of its inhibitory effects on cytochrome P450 enzymes using pooled human liver microsomes (HLM) and recombinant P450s. Methods: The inhibitory potency of memantine on CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 activities was examined with specific probe drugs in HLM and recombinant P450s. The in vivo drug interactions of memantine were predicted in vitro using the [I ]/([I ] + K_I) values. Results: In HLM, memantine inhibited CYP2B6 and CYP2D6 activities, with K_I (IC₅₀) values of 76.7 (279.7) and 94.9 (368.7) M, respectively. Both inhibitions were competitive. In addition, cDNA-expressed P450s were used to confirm these results. Memantine strongly inhibited recombinant CYP2B6 activity with IC₅₀ (K_I) value of 1.12 (0.51) M and activity of recombinant CYP2D6 with IC₅₀ (K_I) value of 242.4 (84.4) M. With concentrations up to 1,000 M, memantine showed no appreciable effect on CYP1A2, CYP2E1, CYP2C9, or CYP3A4 activities and a slight decrease of CYP2A6 and CYP2C19 activities. Based on [I ]/([I ] + K_I) values calculated using peak total plasma concentration (or enzyme-available concentration in the liver) of memantine and the K_I obtained in HLM, 1.3 (13.5), and 1.0% (11.2%), inhibition of the clearance of CYP2B6 and CYP2D6 substrates could be expected, respectively. Nevertheless, when considering K_I values obtained from cDNA-expressed CYP2B6, as generally recommended, even 66.2% (95.9%) decrease in metabolism of coadministered CYP2B6 substrates could be anticipated. Conclusion: Memantine exerts selective inhibition of CYP2B6 activity at clinically relevant concentrations, suggesting the potential for clinically significant drug interactions. Inhibition of other CYPs during memantine therapy is unlikely. Moreover, memantine represents a new, potent, selective inhibitor of recombinant CYP2B6, which may prove useful for screening purposes during early phases of in vitro drug metabolism studies with new chemical entities.     

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2α-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67–22%. CYP3A2-dependent monooxygenase, testosterone 6β-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2α-hydroxylase and testosterone 6β-hydroxylase activities. The Cl _int (V _max/K _m) value for testosterone 2α-hydroxylase was significantly decreased by OP at all doses, whereas the Cl _int value for testosterone 6β-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3- dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP. Received: 29 September 1999 / Accepted: 18 October 1999     

Cytochrome P450: Molecular Architecture,Mechanism, and Prospects for Rational Inhibitor Design

Cytochromes P450 catalyze the insertion of one O₂-derived oxygen atom into an aliphatic or aromatic molecule. P450s are best known for the metabolism of xenobiotic molecules, where hydroxylation renders insoluble hydrocarbons more soluble for easier elimination. In addition to this important catabolic function, P450s catalyze key steps in steroid and plant growth regulator metabolism. A variety of therapeutic, fungicidal, and agochemical agents that perturb these metabolic pathways very likely operate by binding in the lipophilic P450 active site and coordinating with the heme iron atom. Recent determination of a bacterial P450 crystal structure, P450cam from Pseudomonas putida, in addition to the crystal structure of four inhibited complexes, has provided some insight into the potential use of P450 as a model system for the rational design of therapeutic agents. The crystal structure has also shed light on the P450 catalytic mechanism. P450cam operates differently from peroxidase or catalase in cleaving the O–O bond, since unlike these other enzymes, P450 contains no acid–base catalytic groups near the oxygen binding site. Instead, the O₂ pocket is lined with aliphatic and aromatic residues. This strongly suggests that the catalytic push required to cleave the O–O bond resides with the ability of the Cys heme ligand to donate electron density to the heme–oxy system. A comparison of the substrate-free and -bound P450cam crystal structures has revealed some interesting aspects regarding the dynamics of substrate binding. The structures of both forms of P450cam are the same except that in the substrate-free enzyme, the active-site pocket fills with a network of water molecules, one of which coordinates with the iron atom. Despite this lack of any significant conformational rearrangement of protein groups, a careful analysis of crystallographic temperature factors shows dynamical differences. Segments of the protein that are separated in the sequence but that lie close to one another in the structure and that define a small entrance to the substrate pocket undergo significantly higher thermal motion in the substrate-free enzyme. This suggests that dynamical fluctuations at the molecular surface play an important role in controlling substrate binding.     

Predicting the hepatic clearance of xenobiotics in humans from in vitro data

Values for V_max and K_m determined during the in vitro metabolism of a xenobiotic to a known metabolite by a specific human isozyme of cytochrome P450 (P450) were used to predict the hepatic clearance (CL_H) of the xenobiotic to that metabolite. The calculated CL_H values were then compared to literature values of clearance (CL) to the same metabolite obtained during pharmacokinetic studies in humans. For the 6-hydroxylation of chlorzoxazone (P450 2E1) the predicted and actual clearances were 110±77 mL min^?1 and 110 mL min^?1, respectively. For the 6β-hydroxylation of cortisol, the deethylation of lidocaine (two studies), and the oxidation of nifedipine (all P450 3A3/4) the values were 13±15 mL min^?1 and 13 mL min^?1; 758±282 or 829±283 mL min^?1 and 875 mL min^?1; and 284±176 mL min^?1 and 294 mL min^?1, respectively. An increase to 72±25 mL min^?1 in the CL_H of cortisol to 6β-hydroxycortisol was calculated following rifampicin treatment. Finally, the polymorphic nature of the metabolism (P450 2D6) of mexiletine was confirmed. The usefulness of the method and its limitations are discussed.     

Synthesis and Evaluation of Azole-Substituted Tetrahydronaphthalenes as Inhibitors of P450 arom,P450 17, and P450 TxA2

In search of potential drugs for the treatment of estrogen- and androgen-dependent cancer as well as the prophylaxis of metastases, tetralones, tetralins, and dihydronaphthalenes bearing a OCH₃ substituent at the benzene nucleus and an imidazol-4-yl, imidazol-1-yl, or 1,2,4-triazol-1-yl substituent in 2-position were synthesized with and without C₁-spacer between the rings (compounds 2 – 26 ). The compounds were tested in vitro for inhibition of the three target enzymes P450 arom (human placental microsomes), P450 17 (rat testicular microsomes), and P450 TxA₂ (citrated human whole blood). To examine selectivity, some compounds were further tested in vitro for inhibition of P450 18 (bovine adrenal mitochondria), P450 see (bovine adrenal mitochondria) and corticoid formation (aldosterone, corticosterone; ACTH stimulated rat adrenal tissue). In vivo, selected compounds were examined in Sprague Dawley rats regarding P450 TxA₂ inhibition, reduction of plasma testosterone concentration, antiuterotrophic activity (inhibition of the uterotrophic activity of androstenedione), reduction of plasma estradiol concentration (pregnant mares' serum gonadotropin-primed rats), and mammary tumor inhibiting activity (dimethylbenzanthracene-induced tumor; pre- and postmenopausal model). In the series of imidazol-4-yl compounds, which represent a novelty in the field of azole inhibitors of steroidogenic P450 enzymes, strong inhibitors of P450 arom and/or P450 17 were found: 7-OCH₃-2-(imidazol-4-ylmethylene)-1-tetralone ( 4 ) and 7-OCH₃-2-(imidazol-4-ylmethyl)-tetralin ( 12 ) are among the most potent inhibitors of P450 arom in vitro known so far. Compound 4 is a selective inhibitor, whereas 12 shows in addition strong inhibition of P450 17. In contrast to 12 , the 6-OCH₃ derivative (compound 11 ) is a selective inhibitor of P450 17, being 50 times more potent than ketoconazole. Some imidazol-1-yl compounds show a marked inhibition of P450 TxA₂: 2-(imidazol-1-ylmethyl)-1-tetralone ( 13 ) is a selective inhibitor of P450 TxA₂, whereas 7-OCH₃-2-(imidazol-1-ylmethyl)-tetralin ( 17 ) as well as 2-(imidazol-1-ylmethyl)-tetralin ( 16 ) and 7-OCH₃-2-imidazol-1-yl-3,4-dihydronaphthalene ( 25 ) additionally show strong inhibition of P450 arom and P450 17. Regarding the other steroidogenic P450 enzymes as well as corticosterone formation, the compounds show only little inhibitory activity. Aldosterone formation, however, is inhibited at low concentrations. Nevertheless, 4 and 12 are more selective, i.e. inhibit aldosterone synthesis less than the well known inhibitor of P450 arom fadrozole. The compounds show activity in the aforementioned in vivo tests.     

Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy

Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b₅ alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine–DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b₅ enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine–DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine–DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b₅ might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b₅ in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b₅ and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.     

Functional properties of CYP2D6 1 (wild-type) and CYP2D6 7 (His324Pro) expressed by recombinant baculovirus in insect cells

The debrisoquine/sparteine or CYP2D6 genetic polymorphism of drug oxidation is a common cause for interindividual variability in drug response. We recently identified a mutant allele, designated CYP2D6-E or CYP2D6*7, which is associated with the poor metabolizer phenotype and occurs in Caucasian populations with a frequency of about 1%. In contrast to other loss-of-function alleles, a full length protein with a single amino acid substitution, His₃₂₄Pro, is encoded by the CYP2D6*7 allele. To functionally analyze this mutant protein form of CYP2D6, recombinant baculoviruses were constructed to express the CYP2D6 cDNA. Up to 0.33 nmol of spectrally detected P450/mg of cell protein were produced in Spodoptera frugiperda cells, whereas Trichoplusia ni 5B1-4 cells reproducibly produced 0.8 nmol/mg (4% of total cell protein). Insect cell membranes were functionally characterized with cumene hydroperoxide or after reconstitution with purified rat NADPH:cytochrome P450 reductase. K_m values for the substrates bufuralol and sparteine and other enzymatic properties were almost identical to those of human liver microsomes. The H₃₂₄P mutation was introduced into the cDNA by site-directed mutagenesis and recombinant baculovirus was obtained. Expression under a variety of conditions demonstrated that mutant protein amounts comparable to the wild-type enzyme were produced. However, no spectrally detectable P450 was formed and no catalytic activity was detected. Furthermore, in contrast to the wild-type protein, the mutant protein was almost exclusively located in a detergent-insoluble insect cell fraction. These results demonstrate that the H₃₂₄P mutation is responsible for the in vivo poor metabolizer phenotype associated with the CYP2D6*7 allele by preventing normal protein folding and heme incorporation. Received: 30 July 1996 / Accepted: 3 December 1996