Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

Hepatic cytochrome P450 2B induction by ethyl/ phenyl-substituted congeners of phenobarbital in the B6C3F1 mouse

The abilities of structural congeners of phenobarbital to induce immunoreactive hepatic cytochrome P450 2B (CYP2B) protein and associated catalytic activity (benzyloxyresorufin O-dealkylation) in the male B6C3F1 mouse were examined. Interspecies differences in inducing ability were examined through comparison of the results with induction data obtained previously with the male F344/NCr rat. The congeners were administered in the diet for 2 weeks at concentrations equimolar to 500 ppm of the prototype CYP2B inducer, phenobarbital. Of the series of compounds tested, phenobarbital was the most effective inducer of benzyloxyresorufin O,-dealkylation and immunoreactive CYP2B protein, with 2-ethyl-2-phenylsuccinimide, 5-ethyl-5-phenylhydantoin, primidone, and glutethimide being only 19–42% as effective. 5-Ethyl-5-phenyloxazolidinedione and the ring-opened and decarboxylated congeners, N-(2-ethyl-2-phenylacetyl)urea and 2-ethyl-2-phenyl-malonamide, displayed minimal induction of these catalytic activities. Dose-response experiments performed with 5-ethyl-5-phenylhydantoin indicated that the intrinsic CYP2B-inducing activity of this congener was as great as that of phenobarbital in the mouse, although a fourfold greater dietary concentration of this hydantoin (2000 ppm) was required to elicit a response equivalent to that caused by 500 ppm phenobarbital. When extent of induction was related to serum total xenobiotic concentration rather than to administered dietary concentration, the potencies of the two congeners were determined to be more similar (58 vs. ≥78 μ M for phenobarbital and 5-ethyl-5-phenylhydantoin, respectively).     

Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.     

Oxidation of indole by cytochrome P450 enzymes

Indole is a product of tryptophan catabolism by gut bacteria and is absorbed into the body in substantial amounts. The compound is known to be oxidized to indoxyl and excreted in urine as indoxyl (3-hydroxyindole) sulfate. Further oxidation and dimerization of indoxyl leads to the formation of indigoid pigments. We report the definitive identification of the pigments indigo and indirubin as products of human cytochrome P450 (P450)-catalyzed metabolism of indole by visible, (1)H NMR, and mass spectrometry. P450 2A6 was most active in the formation of these two pigments, followed by P450s 2C19 and 2E1. Additional products of indole metabolism were characterized by HPLC/UV and mass spectrometry. Indoxyl (3-hydroxyindole) was observed as a transient product of P450 2A6-mediated metabolism; isatin, 6-hydroxyindole, and dioxindole accumulated at low levels. Oxindole was the predominant product formed by P450s 2A6, 2E1, and 2C19 and was not transformed further. A stable end product was assigned the structure 6H-oxazolo[3,2-a:4, 5-b']diindole by UV, (1)H NMR, and mass spectrometry, and we conclude that P450s can catalyze the oxidative coupling of indoles to form this dimeric conjugate. On the basis of these results, we propose that the P450/NADPH-P450 reductase system can catalyze oxidation of indole to a variety of products.     

Characterization of human cytochrome P450 enzymes.

Many biochemical approaches have been applied to the human cytochrome P450 enzymes, and more than 20 different gene products have been characterized with regard to their properties and catalytic specificities. The complement of the various cytochrome P450 enzymes in a given individual varies markedly, and dramatic differences may be seen in drug metabolism, pharmacological response, and susceptibility to toxic effects. An understanding of the nature of the individual cytochrome P450 enzymes and their regulation should be useful in determining the most suitable animal models, ascertaining risk from chemicals, and in avoiding undesirable drug interactions.     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

Heterobifunctional photoaffinity probes for cytochrome P450 2B.

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed.     

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.     

Differential effect of phenobarbital and beta-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB-P450) by phenobarbital and its repression by β-naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH-cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β-naphthoflavone.     

Probing the world of cytochrome P450 enzymes

Variability in drug response can be attributed in part to variability in the activity of drug-metabolizing enzymes. One of the most important drugmetabolizing enzyme systems in humans is the cytochrome P450 (CYP) enzyme family, which is responsible for the oxidative metabolism of numerous endogenous compounds and xenobiotics. The clinical relevance of factors that influence CYP-mediated metabolism can be appreciated by estimating in vivo enzyme activity (i.e., the phenotype) through the use of "probe drugs," which are drugs predominately or exclusively metabolized by an individual CYP enzyme. Thus, the use of probe drugs alone or in combination (i.e., the cocktail approach) can provide an invaluable tool to explore the clinical relevance of genetic and nongenetic factors that affect CYP enzyme activity and thereby contribute substantially to variability in response to therapeutic drugs.     

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.     

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.     

Phosphorylation of cytochrome P450: regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.     

Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase

Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.     

New perspectives on the conformational equilibrium regulating multi-phasic reduction of cytochrome P450 2B4 by cytochrome P450 reductase

The pre-steady-state reduction of cytochrome P450 (P450) 2B4 by P450 reductase (reductase) was modeled by assuming that an equilibrium between three catalytic conformers of P450 regulates the multi-phasic reduction of the enzyme. This model was compared to a model of reduction involving a minimum number of phases. Based on several criteria, the former model seems to provide an improved fit to the reduction data. Substrates were divided into two groups based on their effects at different concentrations of reductase. Surprisingly, in the presence of some substrates (group 1) but not others (group 2), the rate of reduction was actually slower with an excess of reductase than with equimolar reductase and P450. Presumably, oxidized reductase binds differently to P450 than reduced reductase. A schematic model based on two sites of interaction between reductase and P450 2B4 is offered to explain the unusual reduction kinetics with the two different groups of substrates.     

Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.     

Differential effect of phenobarbital and β-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB-P450) by phenobarbital and its repression by β-naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH-cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β-naphthoflavone.