Rat Hepatic Microsomal Cytochrome P-450 Research Articles

Metabolism of bifenthrin, β-cyfluthrin, λ-cyhalothrin, cyphenothrin and esfenvalerate by rat and human cytochrome P450 and carboxylesterase enzymes

The metabolism of bifenthrin (BIF), β-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes. The pyrethroids were also metabolised by human liver microsomes and cytosol. Some species differences were observed. Pyrethroid metabolism by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. CYFL, CYHA, CYPH and ESF were metabolised by rat plasma CES enzymes, whereas none of the pyrethroids were metabolised by human plasma. This study demonstrates that the ability of male rats to metabolise these pyrethroids by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. All pyrethroids were metabolised by some of the human expressed CYP enzymes studied and apart from BIF were also metabolised by CES enzymes.

Rat cytochromes P450 oxidize 3-aminobenzanthrone, a human metabolite of the carcinogenic environmental pollutant 3-nitrobenzanthrone

3-Aminobenzanthrone (3-ABA) is a human metabolite of carcinogenic 3-nitrobenzanthrone (3-NBA), which occurs in diesel exhaust and air pollution. Understanding which cytochrome P450 (CYP) enzymes are involved in metabolic activation and/or detoxication of this toxicant is important in the assessment of an individual's susceptibility to this substance. The aim of this study was to evaluate the efficiency of rat hepatic CYPs to oxidize 3-ABA and to examine the metabolites formed during such an oxidation. The metabolites formed by CYPs in rat hepatic microsomes were separated by high performance liquid chromatography (HPLC). 3-ABA is oxidized by these enzymes to three metabolites, which were separated by HPLC as distinguish product peaks. Using co-chromatography with synthetic standards, two of them were identified to be oxidative metabolites of 3-ABA, N-hydroxy-3-ABA and 3-NBA. The structure of another 3-ABA metabolite remains to be characterized. To define the role of rat hepatic CYP enzymes in metabolism of 3-ABA, we investigated the modulation of its oxidation using different inducers of CYPs for treatment of rats to enrich the liver microsomes with individual CYPs. Based on these studies, we attribute most of 3-ABA oxidation in rat hepatic microsomes to CYP2B, followed by CYP1A, although a role of other hepatic CYPs cannot be ruled out. Inhibition of 3-ABA oxidation by selective inhibitors of individual CYPs, supported this finding.

2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Atropisomers Interact Enantioselectively with Hepatic Microsomal Cytochrome P450 Enzymes

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)-, and (+/-)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with beta-naphthoflavone or 3-methylcholanthrene, phenobarbital, and dexamethasone (prototypical inducers of CYP1A, CYP2B, and CYP3A, respectively) were used to determine first, whether the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer and second, whether P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)-, or (+/-)-PCB 136 were added to hepatic microsomes, and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together, these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice.

ANTITUMOR DRUG ELLIPTICINE INHIBITS THE ACTIVITIES OF RAT HEPATIC CYTOCHROMES P450

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. Here, we study the effect of ellipticine on CYP enzymes in rat hepatic microsomes, studying its binding to the enzymes and its potential to inhibit the CYP activities measured with their selective substrates. Although ellipticine was reported to be a selective and strong inhibitor of CYP1A1/2, we found that its inhibitory potential is non-specific. Ellipticine is the most potent inhibitor for CYP3A-dependent 6beta-hydroxylation of progesterone, followed by CYP1A1/2-dependent ethoxyresorufin O-deethylation and CYP2B-mediated pentoxyresorufin O-depentylation. Lower inhibition was detected for 1'-hydroxylation of bufurarol, 21-hydroxylation of progesterone and 6-hydroxylation of chlorzoxazone catalyzed by CYP2D, CYP2C and CYP2E1, respectively. Ellipticine binds to several CYPs of rat hepatic microsomes. The binding titration of ellipticine typically give reverse type I spectrum with CYPs in rat hepatic microsomes. The results indicate that inhibition of CYPs by ellipticine cannot be explained only by its differential potency to bind to individual CYPs.

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (Km/Ki ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of Km/Ki ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (Km/Ki ratio of 8.0 versus 4.0).The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1 min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67 μM in microsomes from DEX-induced and control rats.In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (Ki = 0.37 μM; Km/Ki ratio about 73). Inhibition was not enhanced at parathion concentrations near the Ki by a preincubation step with NADPH.In control microsomes parathion elicited a type I binding interaction with oxidized CYP (Ks = 7.7 μM, ΔAmax = 2.2 × 10−2 a.u. nmol CYP−1; ΔAmax/Ks 2.86 × 103 a.u. nmol CYP−1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

Effect of 4-(4-chlorobenzyl)pyridine on rat hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in vivo and in vitro.

The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12), and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.

Climbazole is a new potent inducer of rat hepatic cytochrome P450.

We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver.

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2-dipyridyl amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in P450 content. 2,2-'Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.

Sex-related effect of hemin on cytochrome P450 and drug-metabolizing enzymes in rat liver.

We examined the effect of hemin on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (P450 2E1, 3A2, 2C11 and 2C12) under the induction of heme oxygenase activity in male and female rats. Hemin produced an inverse relationship between the induction of heme oxygenase activity and the decrease of P450 content in a dose-dependent manner. A time course study revealed that hemin produced a significant decrease in total P450 content in male rats to about 37% that of the controls at 24 hr after its administration. Western and Northern blot analyses revealed that the increase in both heme oxygenase-1 (HO-1) protein and HO-1 mRNA reached a maximum at 24 hr and returned to control levels within 120 hr in both sexes. With respect to P450-dependent monooxygenase activities, we found that there was a significant decrease of aniline p-hydroxylase activity to about 30% of the control animals, but not in erythromycin N-demethylase activity at various time intervals. Immunoblot analysis revealed that P450 2E1 in male rat liver was dramatically decreased at 24 hr to about 20% of the controls, but not P450 3A2. Parallel to the decrease of androstenedione 16 alpha-hydroxylase activity, there was a marked decrease of P450 2C11 or 2C12 in male or female rats, respectively, at 72 hr after the treatment; however, hemin did not decrease androstenedione 16 alpha-hydroxylase activity in phenobarbital-pretreated rat liver. These findings suggest that hemin predominantly affects constitutively expressed isozymes rather than inducible P450 species in rat liver.

Evaluation of octamethylcyclotetrasiloxane (D4) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: a 28-day inhalation study.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Amlodipine Inhibits Rat Microsomal Cytochrome P450-Mediated Drug Biotransformation

□ Calcium channel antagonists have been shown to inhibit cytochrome P-450-mediated metabolism both in vitro and in vivo. The purpose of the present study was to examine the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities to determine the potential for drug interactions. In this study, amlodipine (0.05 and 0.5mM) decreased CYP1A-mediated ethoxyresorufin O-deethylase activity in microsomes prepared from noninduced (56 and 73% inhibition) and pyridine-induced (30 and 51% inhibition) rats. Amlodipine reduced pentoxyresorufin O-deethylase activity (a marker for CYP2B) to 15% of control in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes. The para-nitrophenol hydroxylase, erythromycin N-demethylase, and lauric acid ω and ω–1 hydroxylase activities were significantly inhibited by 1mM amlodipine in both noninduced and induced microsomes. These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs.

Liver transplantation induces cytochrome P450 1A1 dependent monooxygenase activity in rat lung and kidney.

Although liver transplantation has been the subject of intensive investigation, comparatively little is known regarding the effects of this procedure on the metabolism of xenobiotics. The objective of the present study was to examine the effect of orthotopic liver transplantation on rat hepatic, pulmonary, and renal microsomal cytochrome P450 (P450) monooxygenase activity through the use of isozyme-selective substrates. Pulmonary microsomal P450 1A1 dependent 7-ethoxyresorufin O-deethylation (ERFD) activity increased over time in recipient rats, with maximal induction (750% of donor) observed after 21 days. Similarly, ERFD activity in renal microsomes was increased (200% of donor) after 21 days. Both pulmonary and renal microsomal P450 2B dependent 7-pentoxyresorufin O-depentylation (PRFD) activity was decreased (50 and 75% of donor) 1 day after transplantation but was essentially unchanged 3, 7, and 21 days after transplantation. Pulmonary and renal microsomal heme oxygenase activities were not significantly affected by liver transplantation. In contrast, total hepatic microsomal P450 concentrations were decreased maximally (to 45% of donor concentration) 7 days after transplantation and remained low (55% of donor) up to 21 days. Similarly, hepatic P450 1A dependent ERFD and P450 2B dependent PRFD activities were maximally depressed (20 and 25% of donor activities) after 7 days and remained low (75 and 30% of donor) up to 21 days after transplantation. The decreases in rates of hepatic P450 monooxygenation were accompanied by significant increases in microsomal heme oxygenase activity. The data presented in this study suggest the existence of generalized stress responses to inflammation that result in tissue- and isozyme-selective modulation of P450 monooxygenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Diminished drug metabolism due to hormonal effects of cobalt mesoporphyrin on flavoproteins

Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 μmol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by >50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 μmol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 μmol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 μmol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 μmol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.

Effect of immobilization on the activity of rat hepatic microsomal cytochrome P450 enzymes

Cytochrome P450 enzyme preparations were obtained from phenobarbital-treated rats. On immobilization in Romicon PM 10 polysulfone anisotropic hollow fibers, 20–25% of the original activity remained. The immobilization procedure did not significantly alter the K m of the preparations when 7-ethoxycoumarin was the substrate. Coimmobilization of the preparations with glucose-6-phosphate dehydrogenase allowed cofactor recycling to take place but resulted in an apparent drop in the value for the K m. The V max of immobilized preparations with or without cofactor recycling was about 25% of that observed with the nonimmobilized preparations. The flow rate through the reactors greatly affected the time at which product was detected in the effluent from the reactor and the amount of 7-hydroxycoumarin produced. It is suggested that this reflects alternative fates of the NADPH cofactor when residence times in the reactor are lengthened. Increasing the amounts of protein immobilized increased the K m and decreased the V max values in comparison to the free enzyme. Immobilization increased the stability of preparations with respect to ionic strength and storage. Little effect was noted with regard to changes in temperature stability or pH optima of immobilized preparations.

Interaction of monocrotophos and its novel thion analogues with microsomal cytochrome P-450: in vivo and in vitro studies in rat

The binding of monocrotophos (MCP) and its two thion analogues (coded as RPR-II and RPR-V) to rat hepatic microsomal cytochrome P-450 (HMC) were investigated in vitro by difference spectroscopy. These three organophosphorus insecticides were found to bind stoichiometrically to HMC with very high affinity ( Ks 34–50 μM). RPR-V showed the highest binding affinity followed by RPR-II and MCP. Association of these compounds with HMC occured within 2 min of addition in the cuvette and therefore, appeared to be tight binding ligands of cytochrome P-450. In vivo studies at equitoxic doses of the three compounds 24 h after oral treatment in rats revealed that they all caused reduction in MC content in liver, lung, kidney and brain, as against induction in cardiac and spleenic cytochrome P-450. These in vivo results suggest organ specificity in modulating the microsomal cytochrome P-450 (MC) content of hepatic and extra-hepatic tissues by the three compounds. Apparently, their binding affinity with HMC is strongly correlated with their LD 50 value and has a substantial co-relationship with the cytochrome P-450 level in the liver.

Inhibition and metabolite complexation of rat hepatic microsomal cytochrome p450 by tricyclic antidepressants

Administration of imipramine (IMIP) and other tricyclic antidepressants to humans and experimental animals has been associated with inhibition of hepatic cytochrome P450 (P450)-mediated drug oxidation. This study investigated the capacity of several structurally related tricyclic antidepressants to inhibit musomal P450 activity in vitro. It was found that IMIP, desipramine (DES), amitriptyline (AMIT) and nortriptyline (NOR) were poor inhibitors of P450 activity unless they were preincubated with musomes and NADPH prior to transfer to flasks containing substrate. Thus, subsequent experiments characterized the time-dependent intensification of inhibition produced by the drugs. Preincubation of the N-methylaminoalkyi agents DES and NOR (200 μM) with NADPH-supplemented musomes for 30 min led to an approximate 30% decrease in spectrally apparent P450 content; the N, N-dimethylaminoalkyl drugs IMIP and AMIT did not significantly decrease apparent P450 content. Analysis of optical difference spectra of musomes during NADPH-mediated metabolism of these drugs revealed a prominent increase in absorbance at 454 nm with DES and NOR but not IMIP or AMIT. Monospecific antibodies to the male-specific P450 2C11 and, to a lesser extent, P450 3A2 were effective in preventing the formation of the DES metabolite 454 nm-Soret peak. In addition, the 454 nm absorbance was not produced by the incubation of DES with NADPH-fortified hepatic musomes from adult female or immature male rats. Studies with the steroid substrate testosterone, which undergoes P450-specific positional hydroxylation, indicated that P450 2C11-mediated 2α- and 16α-hydroxylation were most susceptible to the time-dependent intensification of inhibition produced by DES (8.5 and 7.0 min preincubation required for loss of 50% activity, respectively) and NOR (4.0 and 4.0 min for loss of 50% of both activities). The 6β- (P450 3A2) and 7α-hydroxylase (P450 2A1) pathways were somewhat less susceptible to inhibition than 2α- and 16α-hydroxylation. These findings suggest that DES and NOR form a metabolite intermediate (MI)-complex, characterized by a Soret region absorbance maximum near 454 nm in the optical difference spectrum, with musomal P450 in male rat liver in vitro. Studies with the steroid substrate testosterone as well as immunoinhibition experiments are consistent with the proposition that this MI complex forms principally with the male-specific enzymes P450 2C11 and 3A2. Although a human orthologue of P450 2C11 has not yet been identified, P450s of the 3A subfamily are quantitatively important enzymes in human liver. MI complexation of such enzymes could be a feasible underlying mechanism for certain clinically important drug interactions involving tricyclic antidepressants.

Reductive metabolism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole derivative.

1. The metabolism of zonisamide in vitro was characterized through aerobic and anaerobic incubations with rat liver subcellular fractions and cultured gastrointestinal microflora. 2. Zonisamide reacted with rat hepatic microsomal cytochrome P-450 and exhibited a Type I binding spectrum. 3. Metabolism of zonisamide in vitro by hepatic subcellular fractions and cultured gastrointestinal flora produced a single metabolite, 2-(sulphamoylacetyl)-phenol (2-SMAP), by reductive cleavage of the 1,2-benzisoxazole ring. 4. The reductive metabolism of zonisamide was primarily mediated by microsomal cytochrome P-450. The soluble fraction enhanced reduction when combined with the microsomal fraction but itself possessed only weak reductive activity. 5. Reduction of zonisamide by the most enzymically active liver fractions required NADPH, was stimulated by FMN and SKF-525A, and was inhibited by CO or air, as well as by n-octylamine. 6. Unlike their involvement in the reduction of numerous nitro, azo, and N-oxide compounds, cultured aerobic and anaerobic intestinal flora were not principally involved in the reduction of zonisamide.

Interaction of benzanthrone with cytochrome p450: Altered patterns of hepatic xenobiotic metabolism in rats

Benzanthrone, an anthraquinone dye intermediate, is commonly used for the synthesis of a number of polycyclic vat and disperse dyes. Our prior studies have shown that benzanthrone can be metabolized by rat hepatic microsomal cytochrome P450 (P450) (Biochem. Int., 18, 1989, 1237). In this study, the interaction of benzanthrone with rat hepatic microsomal P-450 and its effect on xenobiotic metabolism have been investigated. Parenteral administration of benzanthrone (40 mg/kg body weight) for 3, 7, or 21 days caused no change in the relative body weight or organ weight of rats. The levels of P450 were found to be reduced (33%-50%) in all the benzanthrone-exposed animals at all the time periods. In vitro addition of benzanthrone caused a spectral change with oxidized P450 and concentration-dependent reduction in the carbon monoxide spectrum of dithionite-reduced P450. The addition of benzanthrone to hepatic microsomes prepared from phenobarbital-treated rats resulted in spectral changes characterized by an absorbance maximum at 397 nm indicative of type I binding. In vitro addition of benzanthrone showed a concentration-dependent inhibition of hepatic aminopyrine N-demethylase (APD) and ethoxyresorufin-O-deethylase (ERD) activities with respective I50 values of 9.5 x 10(-4) and 8.0 x 10(-5) M. However, the inhibition of aryl hydrocarbon hydroxylase (AHH) even at the highest concentration of benzanthrone (10(-2) M), was of the order of only 29%. In vivo administration of benzanthrone also led to the inhibition of APD, AHH, and ERD activities at all treatment times although the magnitude of inhibition was of a lower order.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 3-(2-phenylethyl)-4-methylsydnone and related sydnones on heme biosynthesis

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS) cause mechanism-based inactivation of rat hepatic microsomal cytochrome P-450 and the formation of N-alkylprotoporphyrins in rat liver. In the present study, we have shown that both TTMS and PEMS cause mechanism-based inactivation of chick embryo hepatic microsomal cytochrome P-450. TTMS also caused the inhibition of ferrochelatase activity, the accumulation of protoporphyrin IX, and an increase in the activity of δ-aminolevulinic acid synthase in chick embryo liver cell culture. PEMS was devoid of effect on ferrochelatase activity, porphyrin accumulation, and δ-aminolevulinic acid synthase activity. There are two possible explanations for the lack of effect of PEMS on heme biosynthesis: (1) the ring-A- and/or ring-B-substituted regioisomers of the N-phenylethyl-and N-phenylethenylprotoporphyrins which are produced during the mechanism-based inactivation of cytochrome P-450 by PEMS are too bulky to fit into the active site of ferrochelatase to inhibit its activity, in contrast to the N-vinylprotoporphyrin formed from TTMS; and (2) the N-alkylprotoporphyrins produced consist of the ring-C- and/or ring-D-substituted regioisomers, which are not inhibitors of ferrochelatase activity.

エチレングリコールの肝シトクロームP-450に及ぼす影響

The effect of ethylene glycol on rat hepatic microsomal cytochrome P-450 was studied in vitro and in vivo. The destruction of cytochrome P-450 was not seen in vitro. The addition of 1 mM NADPH also did not change. When ethylene glycol was added to drinking water at a concentration of 1.0% for 7 days, there was no change in the contents of microsomal protein, cytochrome P-450, b5 and heme. While NADPH-cytochrome C reductase activity of the exposed group did not change, NADH-ferricyanide reductase activity increased significantly.