Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450

Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB] and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and beta-naphthoflavone (beta NF)-treated rats. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456 nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456-458 nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428 nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[alpha]pyrene)hydrolase (AHH) activity. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDP and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from β-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation.

2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex.

3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c.

4. Both ethoxyresorufin and benzo[α]pyrene inhibited the development of the type 1 optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[α]pyrene was noncompetitive in nature.

5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type 1 optical difference spectrum non-competitively.

6. The results indicate that ethoxyresorufin and benzo[α]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Interactions of imidazole antifungal agents with purified cytochrome P-450 proteins

The imidazole N-substituted antifungal agents ketoconazole, miconazole and clotrimazole have been shown to be potent inhibitors of oxidative metabolism by both a phenobarbital-induced cytochrome P-450 (P-450b) and a 3-methylcholanthrene-induced cytochrome P-448-protein (P-450c) in reconstituted systems. All three compounds inhibited the cytochrome P-450b-dependent 7-pentoxyresorufin-O-dealkylase and the cytochrome P-450c-dependent 7-ethoxyresorufin-O-deethylase activities. When 7-benzyloxyresorufin and 7-ethoxycoumarin were employed as substrates with both cytochrome preparations, all three antifungal compounds exhibited selective inhibition of the cytochrome P-450b preparation; ketoconazole was always the weakest inhibitor. The three antifungal agents were also shown to elicit a type II difference spectral interaction with both isoenzymes, the magnitude of the spectral interaction being greater with the cytochrome P-450b preparation.     

Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent mono-oxygenases from pig liver microsomes

1. Two distinct microsomal pathways involved in the metabolism of albendazole (ABZ) to albendazole-sulphoxide (SO. ABZ) by pig liver microsomes have been identified and quantified.

2. The binding of ABZ to microsomal cytochrome P-450 (Type I spectrum. Ks = 25.5/μM), the decrease of the rate of sulphoxidation by antibody against NADPH cytochrome c reductase, and the use of purified cytochrome P-450 A demonstrated the contribution of a cytochrome P-450-dependent mono-oxygenase to the metabolism of ABZ.

3. The involvement of FAD-containing mono-oxygenase (FMO) was shown by thermal pretreatment of microsomes, n-octylamine activation of the reaction, and by using purified pig liver FMO.

4. From K_m and V_max values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.     

Interaction of 4-methylbenzaldehyde with rabbit pulmonary cytochrome P-450 in the intact animal, microsomes, and purified systems. Destructive and protective reactions.

About 50 per cent of rabbit pulmonary cytochrome P-450 is destroyed by treatment of the intact animal, microsomes, or systems reconstituted from purified pulmonary mono-oxygenase components with 4-methylbenzaldehyde. The loss of the cytochrome is accompanied by an equimolar loss of heme. The action of 4-methylbenzaldehyde requires the presence of NADPH and O₂ and appears to result from cytochrome P-450-catalyzed metabolism. Selective destruction of one of the known forms of rabbit pulmonary cytochrome P-450 does not account for the lack of complete destruction of pulmonary P-450 by 4-methylbenzaldehyde; loss of about 50 per cent of each form of the cytochrome occurs in vivo and in reconstituted systems. However, form II is affected to a greater extent than form I when microsomes are incubated with 4-methylbenzaldehyde. The portion of the cytochrome not degraded by 4-methylbenzaldehyde appears to be protected by some factor produced from 4-methylbenzaldehyde during the incubation. This factor also protects against complete destruction of the cytochrome by cumene hydroperoxide     

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives

1. Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats.

2. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[α]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes.

3. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450.

4. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.     

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents

1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats.

2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes.

3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome.

4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.     

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 ρ-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.     

Benzo[a]pyrene metabolism by purified cytochrome P-450 from 3-methylcholanthrene-treated rats

The metabolism of benzo[a]pyrene in reconstituted pulmonary mono-oxygenase systems has been studied. Metabolites formed by pulmonary cytochrome P450MC, a major form of pulmonary cytochrome P-450 isolated from 3-methylcholanthrene-treated rats, were analysed by h.p.l.c. The profiles of benzo[a]pyrene metabolites formed by the reconstituted P-450MC systems were different from that obtained with rat-lung microsomes, indicating the presence of several unknown metabolites in the reconstituted systems containing NADPH-cytochrome P-450 reductase and epoxide hydrolase. 3-Hydroxybenzo[a]pyrene was a major product formed by pulmonary cytochrome P-450MC, in the absence or presence of epoxide hydrolase. The addition of purified epoxide hydrolase to the reconstituted systems increased the formation of dihydrodihydroxy-benzo[a]pyrenes, particularly 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene. The 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major dihydrodiol formed by pulmonary cytochrome P-450MC. By the addition of epoxide hydrolase the total amount of phenols decreased in parallel with an increased production of dihydrodiol, but the amount of quinones was not changed. Similar results concerning the related production of phenols and dihydrodiols, in the absence and presence of epoxide hydrolase, were obtained in reconstituted systems of hepatic cytochrome P-450MC, the major form of hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats.     

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.     

The hepatic microsomal mixed-function oxygenase (MFO) system of Alligator mississippiensis: induction by 3-methylcholanthrene (MC)

1. Pretreatment of alligators i.p. with 3-methylcholanthrene (MC) resulted in a 1.6-fold increase (P<0.001) in cytochrome P-450 specific content and a bathochromic shift in the absorption maximum of reduced, CO-liganded microsomes (448?nm).

2. Control and MC microsomal cytochrome P-450 binding spectra with a number of type I and type II ligands were similar.

3. MC treatment of alligators resulted in a 12-fold increase in benzo[a]pyrene hydroxylase activity, which was inhibited 82% by 0.1 mM α-naphthoflavone. The turnover number (units/nmol P-450) of aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase were unaffected by MC treatment.

4. The O-dealkylation (OD) of a series of alkoxyresorufins (ethoxyresorufin (ER), methoxyresorufin (MR), benzyloxyresorufin (BR), and pentoxyresorufin (PR)) was investigated. MC treatment resulted in a significant (P<0.001) increase in turnover number of EROD, MROD, and BROD over control values. The turnover number of PROD was unaltered by MC treatment.

5. Western blots showed that control alligator microsomes contain a protein band of lower mol. wt. than either rat cytochrome P-450c (P450 IA1) or P-450d (P450 IA2), which was recognized by antibodies to both P-450c and P-450d but preferentially by that against P-450c. This protein band was induced 3-4-fold by MC. MC treatment induced a second protein band in alligator microsomes of the same mol. wt. as rat P-450d, recognized preferentially by antibodies to rat cytochrome P-450d.

6. These results illustrate that the alligator mixed-function oxidase (MFO) system responds to MC in a similar manner as described in mammals, i.e. induction in P-450 content, increases in specific MFO activities, and the apparent expression of different P-450 isoenzymes.     

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450

1. The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied.

2. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethyl-hydrazine elicited no response, cither in observable spectral changes or loss of CO-reactive cytochrome P-450.

3. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.     

Effects of arsenite on hepatic mixed-function oxidase activity in rats

1. Injection of arsenite (As³⁺) to control rats results in losses of total hepatic cytochrome P-450 and significant decreases of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) activities. However, As³⁺ appears to decrease the activity of these enzymes differentially, with EROD showing greater sensitivity than ECOD.

2. Injection of As³⁺ to rats treated with phenobarbital and isosafrole significantly decreases the total content of hepatic cytochrome P-450 and various mixed function oxidase (MFO) activities, with the exception of ECOD which appears to be insensitive to As³⁺.

3. 3-Methylcholanthrene administration apparently protects against the effects of As³⁺ on the cytochrome P-450 system, since total content of the cytochrome P-450 and various MFO activities were all insensitive to this treatment.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs.

2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc.

3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)).

4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, α-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra.

5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes.

6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH.

7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones.

8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

Induction of hepatic mono-oxygenase systems in fetal and neonatal rats with phenobarbital, polycylic hydrocarbons and other xenobiotics.

The induction of hepatic P-450 hemoprotein-dependent mono-oxygenase systems was studied in fetal and neonatal rats. The fetal liver was refractive to phenobarbital induction of aminopyrine and ethylmorphine N-demethylase. which are cytochrome P-450-dependent mono-oxygenases, but was not refractive to the 3-methylcholanthrene induction of benzo[a]pyrene hydroxylase. a cytochrome P₁-450-dependent mono-oxygenase. After parturition, all three enzyme activities were inducible. These and other observations suggest that a control mechanism operates in the fetal rat which selectively suppresses the induction of cytochrome P-450. but allows induction of cytochrome P₁-450. This selective suppression of phenobarbital induction in the fetus was reversed in part by the simultaneous administration of 3-methylcholanthrene. Several other inducing agents also partially reversed the suppression of phonobarbital induction in Fetal livers: dibenz-[a,c]anthracene, 2-diethylaminoethyl-2.2-diphenyl-valerate (SKF 525-A), and 3β-hydroxy-20-oxopregn-5-ene-16α-carbonitrile (PCN). Other inducing agents were inactive: α-naphthoflavone, β-naphthoflavone. 1,1-bis[p-chlorophenyl]2,2,2-trichloroethane (DDT). 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). dieldrin. chlordane and chlorpromazine.     

Role of lipid in the electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 from mammalian liver cells

1. The anaerobic NADPH-reduction of the isozymes cytochrome P-450 LM2 and LM4 was used as a functional tool to study the component interaction in reconstituted monooxygenase systems in dependence on different phospholipids.

2. The isozymes were shown to exhibit similar lipid interaction. The lipids generally favour a catalytically active 1 :1 complex formation between reductase and cytochrome P-450 as the rate-determining unit in electron transfer.

3. The cytochrome P-450 reduction proceeds in a biphasic reaction. In dilauroyl phosphatidylcholine (DLPC)-reconstituted systems the amount of the fast reduction ψ₁, is stoichiometrically limited by the reductase in deficit: ψ₁, corresponds to the 1:1 complex formation capability of the reductase.

4. In vesicle-reconstituted systems an ‘overstoichiometric’ reductase cycling is observed which gives rise to a significantly increased amount of fast reduction ψ₁, Reductase cycling is proposed to occur in protein clusters of cytochrome P-450 and reductase in deficit.

5. The dissociation constant K_RP of the functionally active reductase—cytochrome P-450 complex has been determined by means of the amount of ψ₁, (DPLC) and the rate constant K^app₁ (vesicles) of the fast reduction as a measure of the complex formation in dependence on the protein molar ratio. Taking into account the actual protein concentration in the vesicular lipid phase, K_RP in vesicles has been calculated to be about 3 orders of magnitude increased in comparison to DLPC-reconstituted systems.

6. V_max data reveal almost the same catalytic activity of both reconstitution modes, which justifies DLPC-reconstitution in model investigations. The vesicle-specific increased accumulation of reduced cytochrome P-450 in the steady state as originated by reductase cycling may offer the physiological advantage of an increased capacity of cytochrome P-450 for synergistic substrate conversion via cytochrome b₅.     

Role of hepatic microsomal and purified cytochrome P-450 in one-electron reduction of two quinone imines and concomitant reduction of molecular oxygen

The possible role of cytochrome P-450 in one-electron reduction of quinoid compounds as well as in the formation of reduced oxygen species was investigated in hepatic microsomal and reconstituted systems of purified cytochrome P-450 and purified NADPH-cytochrome P-450 reductase using electron spin resonance (ESR) methods. Two compounds were selected as model compounds: N-acetyl-parabenzoquinone imine (NAPQI) and 3,5-dimethyl-N-acetyl-para-benzoquinone imine (3,5-dimethyl-NAPQI). Both compounds could be reduced by oxyhaemoglobin, the semiquinones formed were detectable by ESR and did not reduce molecular oxygen. Both NAPQI and 3,5-dimethyl-NAPQI underwent one-electron reduction in microsomal systems and in fully reconstituted systems of cytochrome P-450 and NADPH-cytochrome P-450 reductase under anaerobic and aerobic conditions. In both incubation systems the semiquinone formation was diminished under aerobic circumstances and concomitant reduction of oxygen occurred, leading to the formation of hydrogen peroxide and hydroxyl free radicals. Both the reduction of the quinone imines and the reduction of oxygen were found to be cytochrome P-450 dependent. Both activities of cytochrome P-450 may also be involved in the bioactivation of other compounds with quinoid structural elements, like many chemotherapeutic agents.     

Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent mono-oxygenases from pig liver microsomes

1. Two distinct microsomal pathways involved in the metabolism of albendazole (ABZ) to albendazole-sulphoxide (SO.ABZ) by pig liver microsomes have been identified and quantified. 2. The binding of ABZ to microsomal cytochrome P-450 (Type I spectrum, Ks = 25.5 microM), the decrease of the rate of sulphoxidation by antibody against NADPH cytochrome c reductase, and the use of purified cytochrome P-450 A demonstrated the contribution of a cytochrome P-450-dependent mono-oxygenase to the metabolism of ABZ. 3. The involvement of FAD-containing mono-oxygenase (FMO) was shown by thermal pretreatment of microsomes, n-octylamine activation of the reaction, and by using purified pig liver FMO. 4. From Km and Vmax values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.     

Stereospecificity in the oxidation of phorate and phorate sulphoxide by purified FAD-containing mono-oxygenase and cytochrome P-450 isozymes

1. Both the cytochrome P-450-dependent mono-oxygenase system and the FAD-containing mono-oxygenase catalyse the sulphoxidation of thioether-containing organophosphate insecticides. Using purified FAD-containing mono-oxygenase and purified cytochrome P-450 isozymes isolated from mouse liver microsomes, the stereospecificity of the oxidation of phorate to (+)-and (-)-phorate sulphoxide and the further oxidations of the (+)-and (-)-phorate sulphoxides to the sulphone, the oxon sulphoxide and the oxon sulphone were examined. 2. The FAD-containing mono-oxygenase catalysed the formation of (-)-phorate sulphoxide, while two cytochrome P-450 isozymes (cytochrome P-450-B2, a constitutive form, and cytochrome P-450-PB, the principal form induced by phenobarbital) produced (+)-phorate sulphoxide. The other three constitutive cytochrome P-450 isozymes examined yielded racemic mixtures. 3. The FAD-containing mono-oxygenase had the lowest Km for the sulphoxidation reaction, 32 microM, while the Km values for the cytochrome P-450 isozymes ranged from 67 microM to 250 microM. No additional oxidation of phorate sulphoxide by the FAD-containing monooxygenase was detected using either (+)-phorate sulphoxide or (-)-phorate sulphoxide as substrates. 4. In contrast, all five cytochrome P-450 isozymes tested formed additional oxidation products; the (+)-phorate sulphoxide was the preferred substrate for all cytochrome P-450 forms. 5. The final oxidation product, phorate oxon sulphone, was derived by desulphuration of phorate sulphone, with the formation of the oxon sulphoxide being a terminal pathway.