The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.     

Effects of conditions for reconstitution with cytochrome b5 on the formation of products in cytochrome P-450-catalyzed reactions

Evidence is presented that the method of reconstitution of the cytochrome P-450-containing liver microsomal enzyme system with cytochrome b5 (b5), including the order of addition of the components, the concentration of the b5, and the length of incubation prior to initiation of the reaction by NADPH, governs the steady state catalytic activity obtained. For example, the addition to cytochrome P-450 isozyme 2, NADPH-cytochrome P-450 reductase, and phosphatidylcholine of concentrated b5 (0.4 microM) results in extensive inhibition of benzphetamine demethylation and NADPH oxidation, whereas the addition of dilute b5 (0.02 microM) to the other components results in extensive stimulation of the demethylation reaction. The inhibition is partly relieved by prolonged incubation. The effects of pH and buffer concentration were determined, and the optimal molar ratio of b5 to cytochrome P-450 isozyme 2 was shown to be about 2.0 for stimulation of benzphetamine demethylation, dimethylaniline demethylation, and cyclohexanol oxidation to cyclohexanone. Cytochrome P-450 isozyme 4-catalyzed aminopyrine demethylation and aniline p-hydroxylation are not stimulated by b5, as predicted from a model based on stopped flow kinetic measurements [Pompon and Coon: J. Biol. Chem. 259, 15377 (1984)]. End-point stoichiometry measurements were carried out with cytochrome P-450 isozyme 2 in the absence of b5 or in the presence of b5 under optimal conditions. The results indicate that when b5 is reconstituted with the cytochrome P-450 isozyme 2 enzyme system under optimal conditions, substrate monooxygenation is enhanced, NADPH oxidation is unaffected, and hydrogen peroxide formation is decreased.     

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

Three N-aralkylated derivatives of 1-aminobenzotriazole as potent and isozyme-selective, mechanism-based inhibitors of guinea pig pulmonary cytochrome P-450 in vitro

The potency and cytochrome P-450 (P-450) isozyme selectivity of 1-aminobenzotriazole (ABT) and three of its N-aralkylated analogues, N-benzyl-1-aminobenzotriazole (BBT), N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB), and the newly synthesized N-alpha-ethylbenzyl-1-aminobenzotriazole (alpha EB), as mechanism-based inhibitors were compared in pulmonary microsomes of untreated and beta-naphthoflavone (beta-NF)-induced guinea pigs. All four compounds were suicide substrates for pulmonary P-450, resulting in the loss of spectrally assayed hemoprotein (up to 50%). Monooxygenase activities were measured with isozyme-selective/specific substrates; the O-dealkylation of 7-pentoxyresorufin (PRF) for the guinea pig ortholog of rabbit P-450IIB4, the O-deethylation of 7-ethoxyresorufin for P-450IA1, and the N-hydroxylation of the aromatic amine 4-aminobiphenyl for P-450IVB1, BBT, alpha MB, and alpha EB were selective for the suicidal inhibition of P-450IIB4; for example, 1 microM alpha MB inactivated 95% of P-450IIB4-, and approximately 10% of P-450IA1- and IVB1-catalyzed, activity in microsomes from beta-NF-induced lungs. Isozyme selectivity was approximately the same for alpha EB and slightly lower for BBT, which inactivated relatively more P-450IA1. At low concentrations, 1 and 10 microM, respectively, ABT preferentially inactivated P-450IVB1, consistent with the efficient N-hydroxylation of aromatic amines by this form of P-450. alpha EB also was shown to efficiently inactivate P-450IIB4-catalyzed PRF activity in microsomes prepared from liver of phenobarbital-induced guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphism of human cytochrome P-450

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of microsomal drug oxidation activities by incorporation into microsomes of purified NADPH-cytochrome c (P-450) reductase.

The effects of addition of purified NADPH-cytochrome c (P-450) reductase on microsomal activities of aniline hydroxylation, p-phenetidine O-deethylation and ethylmorphine and aminopyrine N-demethylations were investigated utilizing microsomes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats. The purified reductase was incorporated into microsomes. The drug oxidation activities were increased by the fortification of microsomes with the reductase while the extent of increase in the activities varied with the substrate and microsomes employed. The most pronounced enhancement was seen in p-phenetidine O-deethylation, followed by aniline hydroxylation and aminopyrine and ethylmorphine N-demethylations. The enhancement was more remarkable in microsomes from rats treated with 3-methylcholanthrene or phenobarbital. alpha-Naphthoflavone inhibited p-phenetidine O-deethylation activity markedly when the reductase was incorporated into microsomes, indicating that a larger amount of a species of cytochrome P-450 sensitive to the inhibitor was capable of participating in the oxidation of this substrate in the presence of the added reductase. One of the two Km values seen with higher concentrations of aniline or aminopyrine was altered by the fortification of microsomes with the purified NADPH cytochrome c (P-450) reductase. From these results, we propose that NADPH-cytochrome c (P-450) reductase transfers electrons to the selected one or two of multiple species of cytochrome P-450 more preferentially depending upon the substrate and the concentration of the substrate in microsomal membranes.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

A novel form of cytochrome P-450 in beagle dogs. P-450-D3 is a low spin form of cytochrome P-450 but with catalytic and structural properties similar to P-450d

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.     

Interactions of diethylphenylphosphine with purified, reconstituted mouse liver cytochrome P-450 monooxygenase systems

Purified mouse liver cytochrome P-450 reconstituted with purified NADPH-cytochrome P-450 reductase and phosphatidylcholine metabolized diethylphenylphosphine to diethylphenylphosphine oxide. NADPH was required for the reaction and the amount of oxide formed was time and cytochrome P-450 dependent. Purified phenobarbital-induced cytochrome P-450 produced more oxide per nmole enzyme than any of the purified uninduced cytochrome P-450s. the phosphine oxide was also formed in lesser amounts in incubation mixtures containing only NADPH-cytochrome P-450 reductase and NADPH. Diethylphenylphosphine bound to oxidized purified phenobarbital-induced cytochrome P-450 and uninduced cytochrome P-450 with Ks values of 16 microM and 11-18 microM respectively. Diethylphenylphosphine was also a competitive inhibitor of p-nitroanisole O-demethylation catalyzed by a reconstituted phenobarbital-induced cytochrome P-450-dependent monooxygenase system, with a Ki value of 5 microM. The phosphine oxide produced no observable optical difference spectrum with oxidized phenobarbital-induced cytochrome P-450 and caused no inhibition of p-nitroanisole O-demethylation.     

Significance of cytochrome P-450 (P-450 HFLa) of human fetal livers in the steroid and drug oxidations

The purpose of this study was to clarify the pharmacological and physiological significance of P-450 HFLa. Thus, correlations between cytochrome P-450 (P-450 HFLa) level and different monooxygenase activities were investigated in liver homogenates from human fetuses. Poor correlation was seen between P-450 HFLa level and the activity of benzphetamine N-demethylation or aniline hydroxylation. In contrast, the content of P-450 HFLa was highly correlated with the activity of benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation or testosterone 6 beta-hydroxylation. In microsomes from human adult livers, a moderate relationship was also observed between testosterone 6 beta-hydroxylation and P-450 HFLa level. Furthermore, antibodies to P-450 HFLa inhibited testosterone 6 beta-hydroxylase activity in fetal and adult livers to similar extents. We conclude that P-450 HFLa is a form of cytochrome P-450 which catalyzes testosterone 6 beta-hydroxylation and limited drug oxidations in human fetal and adult livers.     

Different effects of cyanide on the activities of 7-ethoxycoumarin O-deethylation catalyzed by two forms of cytochrome P-450 purified from 3-methylcholanthrene-treated rats

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.     

Betamethasone-mediated activation of biphenyl 2-hydroxylation in rat liver microsomes. Studies on possible mechanisms

The addition of the steroid betamethasone to intact or detergent-solubilized rat liver microsomes caused a concentration-dependent increase in the rate of biphenyl 2-hydroxylation. Betamethasone (100 microM) increased the apparent Vmax for 2-hydroxybiphenyl formation 2- to 4-fold but had no effect on the apparent Km when either the biphenyl or NADPH concentration was varied. Betamethasone had little or no effect on the apparent Vmax or apparent Km of the 3- and 4-hydroxylations of biphenyl. The steroid did not enhance biphenyl 2-hydroxylation through a peroxidative mechanism. Betamethasone had little or no effect on the rate of the NADPH-dependent reduction of cytochrome c or total microsomal cytochrome P-450. The addition of purified NADPH-cytochrome P-450 reductase to cholate-solubilized liver microsomes increased the rate of hydroxylation of biphenyl in positions 2 and 4. Betamethasone (100 microM) decreased the apparent Km for purified cytochrome P-450 reductase by 48% and increased the apparent Vmax of 2-hydroxybiphenyl formation by 2-fold when the concentration of cytochrome P-450 reductase was varied. The steroid did not alter the Km or Vmax values for the 4-hydroxylation of biphenyl. The data suggest that betamethasone enhances the interaction between the reductase and the form(s) of cytochrome P-450 responsible for the 2-hydroxylation of biphenyl.     

The effect of the tert-butylquinone metabolite of butylated hydroxyanisole on cytochrome P-450 monooxygenase activity

1. The t-butylquinone metabolite of BHA was shown to redox cycle with NADPH-cytochrome P-450 reductase leading to enhanced NADPH-oxidase activity for both the purified and liver microsome-bound flavoprotein. Likewise, addition of t-butylquinone (20-100 microM) strikingly inhibited electron transfer from the flavoprotein reductase to cytochrome P-450 of liver microsomes from phenobarbital-treated rats. 2. When the effect of t-butylquinone on metabolism of biphenyl was evaluated with liver microsomal fractions or isolated hepatocytes, t-butylquinone was less effective as an inhibitor then BHA alone or vitamin K3 (menadione). Addition of dicoumarol had little or no effect on the inhibitory potency of either t-butylquinone or vitamin K3 in isolated hepatocytes. 3. t-Butylquinone was not an effective reductant for exogenous oxidants, such as cytochrome c, in the presence of purified, cytosolic NAD(P)H-quinone oxidoreductase (DT-diaphorase). This property is most probably due to the lower rate of reoxidation of t-butylquinone by molecular oxygen, relative to vitamin K3 (menadione).     

EFFECTS OF MOTORCYCLE EXHAUST ON CYTOCHROME P-450-DEPENDENT MONOOXYGENASES AND GLUTATHIONE S-TRANSFERASE IN RAT TISSUES

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450) -dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/ kg markedly elevated benzo[a]pyrene hydroxylation and 7- ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/ kg/d for 4d resulted in significant increases of P-450 and cytochrome b contents and NADPH-cytochrome c reductase 5 activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S -transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Polyamines as modulators of drug oxidation reactions catalyzed by cytochrome P-450 from liver microsomes

The effect of polyamines on the activity of the mixed-function oxidase (MFO) system from human, rat and rabbit liver microsomes was investigated in detail. It was shown that polyamine (spermine) stimulates NADPH-dependent activity of the MFO system several-fold whatever the substrate (foreign drug or natural), not only with microsomes but also with the reconstituted system consisting of highly purified cytochrome P-450 (LM₂ isozyme), cytochrome P-450 NADPH reductase and dilauroylphosphorylcholine. Stimulation (extent and concentration dependence) appeared to be dependent on a number of parameters such as ionic strength, pH, animal species and treatment, nature of the substrate, and was stereospecific (different effect on 6β-and 16α-testosterone hydroxylation). Further, the spermine effect was evaluated on some elementary steps of the cytochrome P-450 reaction cycle, like substrate binding, P-450 reduction and second electron transfer. Finally, it was shown that the organic peroxide dependent activity was not stimulated by spermine with microsomes nor with the purified P-450 LM₂ isozyme.On the basis of this study, it was concluded that the locus of polyamine action is cytochrome P-450 and that stimulation could result either from increased stability of the oxyferrous intermediate of P-450 or from an increased rate of second electron transfer from reductase to P-450.     

One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.     

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds.

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.     

The mechanism of inhibition by 2,2'-pyridylisatogen tosylate of NADPH-linked enzyme activities in microsomes isolated from rat liver

Microsomal preparations isolated from rat liver were used to study the action of 2.2'-pyridylisatogen tosylate (PIT) on aniline hydroxylation, cytochrome c reduction and NADPH oxidation. PIT was found to inhibit both the NADPH-dependent (5-100 microM, PIT) and the NADPH-independent (0.05-2.5 mM, PIT) hydroxylation of aniline, but had no significant effect on either the NADPH-dependent oxidation of hexobarbital, or the NADPH-independent hydrolysis of glucose-6-phosphatase. PIT was also found to inhibit cytochrome c reductase competitively (Ki = 35 microM) and to stimulate NADPH oxidation (ED50 = 6.5 microM) PIT and aniline were both found to bind to the microsomal haemoprotein cytochrome P-450 and produce Type II spectral changes. It is proposed that PITs ability to bind to the haemoprotein and its ability to accept electrons from the microsomal NADPH-cytochrome c reductase system leads to the inhibition of aniline hydroxylase activity.     

Generation of low-level chemiluminescence during the metabolism of 1-naphthol by rat liver microsomes

The metabolism of 1-naphthol in rat liver microsomal fractions supplemented with NADPH is accompanied by low-level chemiluminescence which reflects the formation of molecular excited states. Photoemission consists of two phases which both are dependent on microsomal protein and 1-naphthol concentration. The involvement of cytochrome P-450 in the microsomal metabolism of 1-naphthol was indicated by an inhibition of chemiluminescence by aminopyrine or metyrapone. Oxygen is required for light emission. Whereas phase I is hardly influenced by superoxide dismutase, phase II is suppressed. Chemiluminescence was not associated with malondialdehyde accumulation, in contrast to NADPH-dependent lipid peroxidation in microsomal fractions in the absence of 1-naphthol. Phase I of chemiluminescence appears to directly reflect cytochrome P-450-dependent hydroxylation, and phase II is attributed to redox cycling of products arising from these reactions, e.g. the 1,4- and/or 1,2-naphthoquinones as oxidation products of the corresponding dihydroxynaphthalenes.