Selective inhibitory interactions of alkoxymethylenedioxybenzenes towards mono-oxygenase activity in rat-hepatic microsomes

A series of eight 4-n-alkoxymethylenedioxybenzene (AMDB) derivatives were evaluated for their inhibitory effects on several mono-oxygenase reactions and their capacity to form metabolite complexes with cytochrome P-450 in vitro in hepatic microsomes from phenobarbital (PB)-and Beta-naphthoflavone (Beta NF)-induced rats. Ethoxyresorufin O-deethylase in Beta NF-induced microsomes and aminopyrine N-demethylase in PB-induced microsomes were most susceptible to inhibition by the test compounds. In contrast, aldrin epoxidation and arylhydrocarbon hydroxylase in PB-and Beta NF-induced microsomes, respectively, were not inhibited by derivatives of AMDB. All AMDB derivatives elicited spectral complexes with cytochrome P-450, the characteristics of which were influenced by the microsomes employed and by the length of the AMDB alkoxy side-chain. Derivatives containing short-chain alkoxy substituents (C1 to C3) formed unstable metabolite complexes and generated substantial quantities of carbon monoxide (CO), those with intermediate length alkoxy groups (C4 to C6) generated little CO and rapidly formed intense spectral complexes (large delta A max), and those with the largest alkoxy groups (C7 and C8) formed no CO and elicited complexes of high stability. Quantitative structure-activity analyses showed that the biological data could be described by parabolic equations in II, the hydrophobic constant of the alkoxy substituent, and suggested the importance to AMDB interactions of a lipophilic-binding region at the active centre of the cytochrome P-450. The alkoxy chain length for optimal mono-oxygenase inhibition and complex formation with cytochrome P-450 appeared to be about five or six carbon atoms. The data suggest that the capacity of AMDB compounds to form stable inhibitory complexes with cytochrome P-450 may not always be associated with their ability to inhibit mono-oxygenase activity.     

Effects of a series of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on the major inducible cytochrome P-450 isozymes of rat liver

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) by destroying the heme prosthetic group. We have examined the isozyme selectivity of representative DDC analogues with respect to the major inducible P-450 isozymes of rat liver. Hepatic microsomes from untreated, phenobarbital (PB)-treated, beta-naphthoflavone (beta NF)-treated, and dexamethasone (DEX)-treated rats were incubated with a DDC analogue and NADPH and were subsequently analyzed for P-450 and heme content, P-450 isozyme immunoreactivity, and enzyme activity. Compared with the uninduced state, 4-isopropyl-DDC caused slightly less P-450 destruction following beta NF induction and much greater destruction following DEX pretreatment. Also, 4-hexyl-DDC was found to cause less P-450 destruction following PB or DEX pretreatment, compared with results obtained with untreated rats. These results suggest that DDC analogues possess different isozyme selectivity profiles. Monoclonal antibodies (MAbs) directed against the major inducible isozymes of P-450 were used to probe Western blots of microsomal protein following DDC analogue treatment. The formation of lower molecular mass (45-55 kDa) immunoreactive proteins in microsomes from beta NF-treated rats following DDC analogue treatment was revealed by two MAbs (1-31-2 and 1-36-1), suggesting that the apoprotein of the major beta NF-inducible isozyme, P-450c, is subject to alteration by DDC analogues. In microsomes from DEX-treated rats, DDC analogues caused the formation of higher molecular mass (80, 94, and 115 kDa) proteins showing immunoreactivity with MAb 2-13-1, directed against a major DEX-inducible isozyme belonging to the P-450p family. These immunochemical findings are supported by the demonstration that DDC analogues also caused mechanism-based inhibition of the catalytic activity of P-450c (7-ethoxyresorufin O-deethylase) and P-450p (erythromycin N-demethylase) but not that of the major PB-inducible isozyme, P-450b (7-pentoxyresorufin O-dealkylase). The combined immunochemical and enzymic studies indicate that rat liver P-450 c and p are targets for mechanism-based inactivation by DDC analogues.     

Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species

Effects of SKF 525A (0.1 mM), metyrapone (0.1 mM), alpha-naphthoflavone (ANF) (0.5 mM) and pyrazole (1.0 mM) on N-nitrosodimethylamine (NDMA), N-nitrosomethylbutylamine (NMBuA) and N-nitrosomethylbenzylamine (NMBeA) metabolism by hepatic microsomes from rats pretreated with inducers were investigated. NDMA demethylation was weakly increased by phenobarbital (PB) treatment. The demethylation was inhibited by SKF 525A and enhanced by metyrapone in non-treated and PB-treated microsomes, and weakly inhibited by ANF in 3-methylcholanthrene(MC)-treated microsomes. NMBuA demethylation was increased by PB treatment and inhibited by SKF 525A in all microsomes. Metyrapone inhibited the demethylation in PB-treated microsomes. NMBuA debutylation was increased by PB and MC treatments, and inhibited by metyrapone in all microsomes. The strongest inhibition by metyrapone was observed in PB-treated microsomes. The debutylation was inhibited by SKF 525A in non-treated and PB-treated microsomes and by ANF in MC-treated microsomes. NMBeA demethylation was decreased by MC treatment and weakly inhibited by SKF 525A in all microsomes. The effects of the inducers and inhibitors on NMBeA debenzylation were almost the same as those on NMBuA debutylation except that the increasing effect of MC was small. Pyrazole was a relatively selective inhibitor of NDMA demethylation. These results suggest the following: NDMA demethylation is catalyzed by PB-induced cytochrome P-450 species (P450-PB) and MC-induced cytochrome P-450 species (P448-MC). But their specific activity is low and the other cytochrome P-450 species demethylate NDMA. NMBuA demethylation is catalyzed by P450-PB. But the specific activity is not high and the other cytochrome P-450 species also demethylate NMBuA. NMBuA debutylation is catalyzed by P450-PB and P448-MC. Almost all of NMBeA demethylation is catalyzed by cytochrome P-450 species other than P450-PB and P448-MC. NMBeA debenzylation is catalyzed by P450-PB and P448-MC, but the specific activity of P448-MC is not high.     

Qualitative and quantitative differences in the induction and inhibition of hepatic benzo[a]pyrene metabolism in the rat and hamster

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 5 micrograms/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., X 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., X 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., X 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10- and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.     

Selective inactivation by chloramphenicol of the major phenobarbital-inducible isozyme of dog liver cytochrome P-450

Chloramphenicol (CAP) is a potent and effective mechanism-based inactivator of the major phenobarbital (PB)-inducible isozyme of dog liver cytochrome P-450 (PBD-2) in vitro. In a reconstituted system containing PBD-2, CAP causes a time- and NADPH-dependent irreversible loss of 7-ethoxycoumarin deethylase activity, with no loss of spectrally detectable cytochrome P-450. Inactivation is enhanced by cytochrome b5, and, in the presence of cytochrome b5, the concentration of CAP at which the rate constant for inactivation is half-maximal (Kl) and the maximal rate constant for inactivation (Kinact) are 5 microM and 1.2 min-1, respectively. CAP binds covalently to PBD-2 with a stoichiometry of 1 nmol of [14C]CAP bound/nmol of cytochrome P-450 inactivated. In addition, CAP is a selective inactivator of PBD-2. In intact liver microsomes from PB-treated dogs, CAP irreversibly inhibits androstenedione 16 alpha and 16 beta, but not 6 beta hydroxylation. Covalent binding of [14C]CAP to dog liver microsomes in vitro is increased 5.5 times by PB induction. This increase correlates well with the increased levels of immunochemically determined PBD-2 (5.8-fold) and 16 alpha and 16 beta hydroxylation of androstenedione (5.7- and 5.8-fold) in microsomes from PB-treated compared to control animals. Anti-PBD-2 IgG specifically inhibits by greater than 80% the covalent binding of [14C]CAP to microsomes from control and PB-treated dogs. Finally, in liver microsomes from PB-treated and control dogs, CAP appears to bind covalently to a single protein with the same molecular weight as PBD-2 as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.     

Studies of the mechanisms of metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by rabbit liver microsomes

Based on the protein content of microsomes, the administration of 3-methylcholanthrene (3-MC) and phenobarbital (PB) to adult rabbits leads to an increased rate of metabolism of parathion (diethyl 4-nitrophenyl phosphorothionate) by rough-surfaced and whole microsomes but not by smooth-surfaced microsomes. Although prior administration of both PB and 3-MC increased the cytochrome P-450 content of the microsomes, when the rate of metabolism of parathion was calculated on the basis of the concentration of cytochrome P-450 in these microsomes, there is no difference in the rate of metabolism of parathion by rough-surfaced and smooth-surfaced microsomes from the untreated, 3-MC-treated and PB-treated animals. However, based on the cytochrome P-450 concentration, the rate of metabolism of parathion by whole microsomes from 3-MC and PB-treated animals is less than the rate with whole microsomes from untreated animals. Further studies have shown there is no correlation between the concentration of high spin or low spin cytochrome P-450 in any of the microsomal fractions or subfractions and the rate of metabolism of parathion to paraoxon or diethyl phosphorothionate.     

Ontogeny of the chicken cytochrome P-450 enzyme system. Expression and development of responsiveness to phenobarbital induction

The sensitivity of the developing embryo to toxins and drugs is highly dependent on the state of development of the cytochrome P-450 system. Previous work in this laboratory has demonstrated the genotoxicity of aflatoxin B1 (AFB1) to the chicken embryo at 3 days of incubation (DI) and induction of AFB1 genotoxicity by phenobarbital at 7 DI. In this study, the basal and 24-hr phenobarbital (PB) induced levels of aminopyrine-N-demethylase (AMPD) and cytochrome P-450 were assayed in hepatic microsomes from 7 DI to 36 days posthatching (PH) and in microsomes from whole embryos at 5 DI. A dose-response for induction by PB was observed in embryonic hepatic microsomes as early as 7 DI, whereas a low level of cytochrome P-450 was detected in control 7 DI microsomes using the reduced CO vs oxidized CO difference spectrum. Basal levels of AMPD and cytochrome P-450 in hepatic microsomes increased steadily throughout development as did the responsiveness of the embryonic liver to induction with PB. Hepatic microsomes from control and PB-induced chickens had the highest AMPD activities posthatching particularly from 1 to 3 days PH. Maximal induced levels, which were 2- to 3-fold over control throughout development, ranged from 1.22 at 7 DI to 12.72 nmol HCHO/mg protein/min at 2 days PH. The potency of PB as an inducer increased about 1000-fold between 7 DI and hatching. PB induction did not increase the specific activity of AMPD at any period of development. The specific activity of AMPD posthatching increased about 3-fold above embryonic levels, indicating the development of a cytochrome P-450 complex more active toward aminopyrine in the neonatal period.     

Monoclonal antibodies to phenobarbital-induced rat liver cytochrome P-450

Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450_LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.     

Effects of enzyme induction on microsomal benzene metabolism

The effect of induction by phenobarbital (PB), beta-naphthoflavone (BNF), and benzene on benzene metabolism was studied in hepatic microsomes from male Sprague-Dawley rats. Two distinct forms of mixed-function oxidase activity appeared to metabolize benzene. One form was active at all substrate concentrations in microsomes from control, benzene-treated, and BNF-treated animals, and at benzene concentrations of 0.8 mM and below in microsomes from PB-treated animals. It was saturated at benzene concentrations above 0.4 mM, had a pH optimum of approximately 6.6, and was stimulated by fluoride. Pretreatment with benzene, but not BNF, increased benzene metabolism in these preparations. Benzene metabolism in microsomes from PB-induced rats was less active than in controls at benzene concentrations below 0.8 mM, but increased rapidly at higher benzene concentrations. Further characteristics of the PB-induced enzyme activity were that saturation was not observed at benzene concentrations as high as 4 mM, the pH optimum for benzene metabolism in these preparations was 7.1, metabolism was not stimulated by fluoride, and metabolism was inhibited by metyrapone. Both phenol and an unidentified polar component were formed from benzene in all microsomal preparations. Formation of the polar component was increased by PB pretreatment and inhibited by metyrapone, suggesting that formation of the polar component involves a step requiring cytochrome P-450.     

Oxidation of butylated hydroxytoluene to toxic metabolites. Factors influencing hydroxylation and quinone methide formation by hepatic and pulmonary microsomes.

The effects of inducing agents and inhibitors on the cytochrome P-450-catalyzed oxidations of butylated hydroxytoluene (BHT) to form three metabolites were investigated with liver and lung microsomes from rats and mice. These compounds, the quinone methide (QM) formed by two-electron oxidation of BHT, the hydroxy-tert-butyl analog of BHT (BHT-OH) resulting from aliphatic hydroxylation, and the hydroxy-quinone methide (QM-OH) derived from BHT-OH, have been implicated previously as intermediates or products involved in BHT bioactivation and toxicity. Although there was little or no increase in BHT metabolism in pulmonary microsomes from either species following phenobarbital (PB) administration, 6- to 37-fold enhancements occurred in the transformation of BHT to QM, and the conversion of BHT-OH to QM-OH with hepatic microsomes from both species. The first step in QM-OH formation, hydroxylation of BHT to BHT-OH, is a minor pathway with hepatic microsomes from treated or untreated rats, thereby explaining the lack of QM-OH formation from BHT by that species. The two-step oxidation of BHT to QM-OH, however, is a relatively important metabolic pathway with hepatic microsomes from PB-treated mice, due to an unusually large (111-fold) PB-induced increase in the tert-butyl hydroxylation step. These results demonstrate that pulmonary microsomes from mice, but not rats, have relatively high constitutive P-450 activity for the formation of QM-OH from BHT, supporting the proposal that this metabolite is involved in BHT-induced pneumotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of isozymes of cytochrome P-450 in the metabolism of N,N-dimethyl-4-aminoazobenzene in the rat

The metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) was investigated in vitro by use of hepatic 10,000g supernatant fraction, microsomes, and purified cytochromes P-450 prepared from rats. Position-selective metabolism was studied in response to induction by 3-methylcholanthrene (MC), phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN) as well as inhibition by SKF 525-A, metyrapone, alpha-naphthoflavone, and piperonyl butoxide. The principal phase I pathways are demethylation of the tertiary (DAB) and secondary (MAB) amines and ring hydroxylation. When metabolism was measured with 10,000g supernatant fractions, each pathway responded differently and often independently to the inducers and inhibitors, suggesting that they are catalyzed preferentially by different isozymes of cytochrome P-450. Microsomes from PB-treated animals demethylated and hydroxylated DAB at the same rate as did control microsomes, based on cytochrome P-450 content, whereas microsomes from BNF- or MC-treated animals demethylated more rapidly and hydroxylated more slowly. Microsomes from PB-treated animals demethylated the secondary amine, MAB, more rapidly than the tertiary amine, DAB. Purified cytochrome P-448 from MC-treated animals catalyzed DAB demethylation very readily but hydroxylation very poorly. The turnover number was 10 times that seen in microsomes from MC-treated animals. Only one of the four cytochrome P-450 fractions isolated from PB-treated animals had significant activity with DAB and the turnover number of one of these (fraction B) was approximately that seen in microsomes. This study supports the concept of selectivity of various isozymes of cytochrome P-450 for the different steps in phase I metabolism of DAB. Furthermore, it is apparent that the association of certain inhibitors with specific isozymes of cytochrome P-450 is a generalization that requires qualification in terms of the substrates(s) involved.     

Immunochemical study on the contribution of hypolipidaemic-induced cytochrome P-452 to the metabolism of lauric acid and arachidonic acid

The influence of four hypolipidaemic drugs (clofibrate, WY-14,643, clobuzarit and bezafibrate) on hepatic cytochrome P-450 and fatty acid metabolism in male rat liver microsomes has been investigated. All of the hypolipidaemic drugs tested significantly induced the hydroxylation of lauric acid and, furthermore, this was accompanied by a concomitant 3-fold induction of a specific isoenzyme of cytochrome P-450 (termed cytochrome P-452) as determined by a single radial immunodiffusion technique. In addition, immunochemical quantitation of cytochrome P-452 in control, uninduced rat liver microsomes revealed that this particular isoenzyme constituted 22% of the total carbon monoxide-discernible cytochrome P-450 population. This has led us to the conclusion that cytochrome P-452 is a constitutive cytochrome P-450 isoenzyme and therefore that hypolipidaemic agents function as inducers of constitutive haemoprotein isoenzymes. Cytochrome P-452 plays a significant role in the hydroxylation of lauric acid as evidenced by inhibition of hydroxylase activity in the presence of an anti-P-452 IgG fraction. In addition, this antibody preferentially inhibits the 12-hydroxylation of lauric acid in rat liver microsomes by comparison to the 11-hydroxylase activity. Our studies have also shown that arachidonic acid serves as an excellent substrate for hypolipidaemic-induced cytochrome P-452, resulting in the formation of several metabolites that have been separated by reverse phase HPLC. Furthermore, a specific metabolite (or group of metabolites) of arachidonic acid is induced by clofibrate pretreatment and that the formation of this metabolite(s) is inhibited by an antibody to cytochrome P-452. By comparison, other metabolites of arachidonic acid remain refractory to induction by clofibrate and are not inhibited by the presence of anti-P-452 IgG. In addition, a reconstituted enzyme system containing highly purified cytochrome P-452 actively catalyses the above specific oxidation of arachidonic acid, a reaction that is significantly stimulated by the presence of cytochrome b5. Taken collectively, our data provide compelling evidence that hypolipidaemic agents induce a specific isoenzyme of hepatic microsomal P-450 that readily oxidizes fatty acids and that arachidonic acid may serve as an excellent endogenous substrate for this novel haemoprotein.     

Testosterone metabolite profiles reveal differences in the spectrum of cytochrome P-450 isozymes induced by phenobarbitone, 2-acetylaminofluorene and 3-methylcholanthrene in the chick embryo liver

The regiospecificity and stereoselectivity of testosterone hydroxylation by hepatic microsomes prepared from control, PB, 3MC and 2AAF treated chick embryos has been analysed. Microsomes prepared from control animals hydroxylate testosterone at the 16 alpha and 6 beta positions exclusively: 3MC treatment only causes comparatively minor alterations in the rates of these conversions. PB and 2AAF treatment induced 16 beta-hydroxylation, whilst only 2AAF caused a substantial induction of 6 beta hydroxylation. This data suggests that in the chick, 2AAF not only induces P-450 subforms which are also induced by PB but additional subforms which are not markedly induced by either PB or 3MC.     

Effect of cytochrome P-450 and flavin-containing monooxygenase modifying factors on the in vitro metabolism of amiodarone by rat and rabbit

Experiments were conducted to affirm hepatic cytochrome P-450 involvement in the biotransformation of the class III antiarrhythmic agent, amiodarone (Am; Cordarone X) to its major metabolite, desethylamiodarone (DEA). Male Sprague-Dawley rats and male New Zealand white rabbits were treated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) (to induce cytochrome P-450 (PB-inducible cytochrome(s) P-450) or P-448 (MC-inducible cytochrome P-450). In vivo decreases in rat hepatic microsomal cytochrome P-450 were achieved either by a single ip dose of CCl4 or by a 2-day treatment with CoCl2. In vitro biotransformation of Am by hepatic microsomes from PB-induced and 3-MC-induced rats and PB-induced rabbits was significantly greater than that from noninduced animals. Conversely, in vitro DEA production was significantly decreased with hepatic microsomes from CCl4- and CoCl2-pretreated rats. The classic P-450 inhibitors, piperonyl butoxide, SKF 525A, n-octylamine, and CO provided a significant reduction in the in vitro formation of DEA by microsomes from induced animals. In vitro DEA formation by hepatic microsomes from PB- and 3-MC-induced rats was significantly decreased by 0.5 mM chloroquine (specific inhibitors of PB-inducible cytochrome(s) P-450) and 0.3 mM quinacrine (specific inhibitor of MC-inducible cytochrome(s) P-450), respectively. Further evidence for involvement of gut microsomal flavin-containing monooxygenase was provided by the inhibition of gut microsomal-mediated in vitro DEA formation in the presence of methimazole. Methimazole had no effect on hepatic microsomal DEA production in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

omega-Hydroxylation of 15-hydroxyeicosatetraenoic acid by lung microsomes from pregnant rabbits

15-Hydroxyeicosatetraenoic acid (15-HETE) was converted by lung microsomes from pregnant rabbits to a polar metabolite that was identified by mass spectrometry as the 15,20-dihydroxyeicosatetraenoic acid. The formation of the 20- or omega-hydroxylated product was NADPH dependent, with a specific activity of 1.87 +/- 0.53 nmol/min/mg of microsomal protein. Other hydroxylated derivatives of eicosatetraenoic acid that possessed hydroxy groups at the 5- and 12-carbon atoms were not metabolized by the lung microsomes. This hydroxylation of 15-HETE was observed in lung microsomes of pregnant rabbits and only minor amounts were formed by nonpregnant rabbits. The specific activity for 15-HETE omega-hydroxylation was similar to the value obtained for prostaglandin E1 (1.48 +/- 0.33 nmol/min/mg). It is known that rabbit lungs possess a cytochrome P-450 that is induced during pregnancy and catalyzes the 20-hydroxylation of prostaglandins. The addition of the antibody to cytochrome P-450 prostaglandin omega-hydroxylase or prostaglandin E1, a substrate of this enzyme, resulted in potent inhibition of 15-HETE omega-hydroxylation, providing strong evidence that a common cytochrome P-450 catalyzes the omega-hydroxylation of both prostaglandins and 15-HETE.     

Identification of the cyanopregnenolone-inducible form of hepatic cytochrome P-450 as a catalyst of aldrin epoxidation

In light of recent suggestions that hepatic microsomal aldrin expoxidation activity selectively reflects the phenobarbital (PB)-inducible form(s) of cytochrome P-450 (P-450PB), we tested the effect of pregnenolone-16 alpha-carbonitrile (PCN), a synthetic steroid that induces P-450PCN, a form of the cytochrome biochemically and immunochemically distinguishable from P-450PB. In hepatic microsomes prepared from rats receiving PB, 3-methylcholanthrene (3-MC), or PCN, the latter compound produced a greater increase in aldrin epoxidation activity relative to control than did PB, whereas 3-MC decreased enzyme activity. Moreover, the aldrin epoxidation activity in microsomes prepared from PCN- or PB-pretreated rats was selectively inhibited by form-specific antibodies directed against P-450PCN or P-450PB, respectively, whereas anti-P-450MC antibodies gave no inhibition with microsomes prepared from induced or control animals. We conclude that P-450PCN, P-450PB, and probably other cytochromes P-450 catalyze aldrin epoxidation, precluding use of this enzyme as a specific marker of a single form of the cytochrome.     

Induction of cytochrome P-450 isozymes in liver microsomes of rats treated with the cardiotonic agent sulmazole

Pretreatment of adult male rats with sulmazole (AR-L 115 BS) results in a 2-6-fold increase of the liver microsomal scoparone O-demethylation and 7-ethoxycoumarin O-deethylation activity. The change in the ratio of the demethylation products scopoletin to isoscopoletin from 1 : 1.8 +/- 0.1 in control microsomes to 1: 2.5 +/- 0.1 in microsomes from sulmazole pretreated rats is statistically significant. Sulmazole produces a modified type II difference spectrum when added to microsomes of control or sulmazole-pretreated rats. Immunoquantitation of seven cytochromes P-450 showed that two forms, namely P-450 beta NF/ISF-G and P-450 beta NF-B, are increased 3-4-fold in the microsomes of sulmazole-pretreated rats.     

Evidence for the stability and cytochrome P450 specificity of the phenobarbital-induced reductive halothane-cytochrome P450 complex formed in rat hepatic microsomes

The hypothesis that the reduced spectral halothane-cytochrome P450 complex formed in rat hepatic microsomes is a stable cytochrome P450 specific species was examined. Comparisons of the cytochrome P450 inducers, phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN) and beta-naphthoflavone (beta-NF) showed that PB was the most effective inducer of the halothane-cytochrome P450 complex and the cytochrome P450 which liberates the halothane metabolites, 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). However, the ratio of CDE produced to quantity of complex was found to be reduced 70-77% in these microsomes. A large portion of total microsomal cytochrome P450 was destroyed upon halothane reduction (up to 39%), yet the complexed cytochrome P450, particularly in microsomes from PB-treated animals, was resistant to the irreversible inactivation mechanisms of halothane reduction. The effects of reductive halothane metabolism on subsequent warfarin metabolism showed that 7-hydroxywarfarin formation from either (R)- or (S)-warfarin in microsomes from PCN-treated, PB-treated or untreated rats was highly susceptible to irreversible inhibition. In microsomes from PB-treated, but not PCN or untreated rats, the formation of one warfarin metabolite, 4'-hydroxywarfarin from (R)-warfarin, could be shown to be increased when complex was eliminated by photodissociation. These results suggest that PB-B is preferentially bound as complex and resistant to inactivation because of complex stability, and that halothane reduction readily destroys the cytochrome P450 form, PB-C.     

Differential metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate by rat and chicken hepatic microsomes

The in vitro hepatic metabolism of O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) was investigated in the hen (a species that is sensitive to EPN delayed neurotoxicity) and the rat (an insensitive species). EPN, which produced a Type I binding spectrum on incubation with cytochrome P-450, was converted by liver microsomes from both species to its oxygen analog, O-ethyl O-4-nitrophenyl phenylphosphonate (EPNO), and to p-nitrophenol (PNP). The formation of EPNO and PNP was dependent on the presence of NADPH in the reaction mixture and could be inhibited by either SKF-525A or by anaerobic conditions. The rates of EPNO and PNP formation by rat liver microsomes were, however, 3- and 20-fold higher, respectively, than the rates of formation by chicken liver microsomes. There was also a 4-fold difference in the cytochrome P-450 contents of the liver microsomes. The EPNO-hydrolyzing activity of rat liver microsomes was much greater than that of chicken liver microsomes. EPNO metabolism, in contrast to EPN metabolism, did not require NAPDH nor was it inhibited by SKF-525A or by anaerobic conditions. Prior exposure of rats to phenobarbital (PB) or Arochlor 1254 resulted in an increase in hepatic microsomal EPN metabolism and cytochrome P-450 content. On the other hand, 3-methylcholanthrene (3-MC) treatment elevated microsomal cytochrome P-450 but did not increase EPNO or PNP formation. Pretreatment with EPN did not alter either microsomal EPN metabolism or cytochrome P-450 levels. In chickens, prior exposure to PB, 3-MC or 100 mg/kg EPN increased EPNO and PNP formation by liver microsomes as well as cytochrome P-450 levels; prior exposure of chickens to 15 mg/kg EPN did not alter these variables. The λ_max Soret bands of the reduced hepatic cytochrome P-450 complexes from these animals differed as follows (rat then chicken): untreated, 450 vs 452 nm; PB-treated, 450 vs 451 nm; and 3-MC-treated, 448 vs 449 nm. None of the above treatments had an effect on EPNO metabolism by liver microsomes.     

Differential induction of cytochromes P450 and cytochrome P450-dependent arachidonic acid metabolism by 3,4,5,3',4'-pentachlorobiphenyl in the rat and the guinea pig

Differential induction of hepatic cytochromes P450 by 3,4,5,3',4'-pentachlorobiphenyl (PENCB) has been observed in the rat and the guinea pig: (1) in rat and guinea pig, treatment with the chosen dose levels resulted in significant induction of total, carbon monoxide-discernible cytochrome P450 content; the absorption maximum of the CO-adduct of the dithionite-reduced microsomes from PENCB-induced rat liver was shifted from 450 to 448 nm, whereas its counterpart in the guinea pig did not; (2) PENCB treatment significantly increased EROD activity in rat liver microsomes (up to 60-fold), but the increase in the guinea pig was less than fivefold; (3) PENCB-induced rat liver microsomes significantly induced the omega-1 hydroxylation of arachidonic acid (AA); however, omega-1 hydroxylation of AA was hardly affected by PENCB treatment in the guinea pig. Instead, omega-hydroxylation was significantly increased in this latter species. In addition to omega-1 hydroxylation in the rat or omega-hydroxylation in the guinea pig, an additional AA metabolite (designated peak III) was significantly induced by PENCB in both rat and guinea pig; (4) Western blot and ELISA analyses with polyclonal anti-P450 IA1/IA2 and IVA1 antibodies demonstrated that P450 IA1 was significantly induced in the rat but only slightly induced in the guinea pig, whereas P450 IVA1 was significantly suppressed in the rat but significantly induced in the guinea pig by PENCB treatment. The induction of the third arachidonic acid metabolite peak, Peak III, in both rat and guinea pig, particularly in the guinea pig, is obviously neither mediated by P450 IA1 nor by P450 IV A1. At present, it is still unclear which form(s) of cytochrome P450 isoenzymes is responsible for this latter hydroxylation of arachidonic acid.