3-methoxybenzidine: A potent inhibitor of cholesterol side chain cleavage cytochrome P-450

The effect of 3-methoxybenzidine on the conversion of cholesterol to pregnenolone was investigated using a reconstituted enzyme system comprised of adrenodoxin, adrenodoxin reductase and cytochrome P-450scc purified from bovine adrenal cortex. Under conditions where the cytochrome P-450scc concentration was rate-limiting, 3-methoxybenzidine was found to be a potent inhibitor, causing 50% inhibition at 7 μM when using a cholesterol concentration of 70 μM. The parent compound, benzidine, was much less effective, exhibiting an Icn value of approximately 40 μM. No effect of 3-methoxybenzidine was observed on the adrenodoxin reductase and adrenodoxin-catalyzed reduction of cytochrome c by NADPH, and it is concluded that 3-methoxybenzidine acts on cytochrome P-450scc in inhibiting cholesterol side chain cleavage.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Oxidative cleavage of carboxylic esters by cytochrome P-450

Cytochrome P-450 was demonstrated to catalyze the oxidative cleavage of carboxylic acid esters to the corresponding carboxylic acids. 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester and related dialkyl esters were shown to serve as substrates in NADPH-fortified rat liver microsomes and reconstituted systems containing purified cytochrome P-450 enzymes. The ethyl group gave rise to acetaldehyde. The reactions proceed with large kinetic deuterium isotope effects, consistent with the view that P-450 abstracts a hydrogen atom in the mechanism. Oxygen rebound to the radical site is then postulated to complete the reaction and lead to a hemiacetal-like structure which collapses to give the products. Rate studies with differing alkyl substituents showed that the reaction was more rapid with removal of an ethyl than a methyl or isopropyl group, consistent with the view that the ethyl optimizes steric and inductive effects. Oxidative cleavage of carboxylic acid esters has little biochemical precedent, due to the difficult character of the reaction, and should be considered as an alternative to direct hydrolysis.     

Properties of an adrenal cytochrome P-450 (P-450scc) for the side chain cleavage of cholesterol

A highly purified (12 nmol of P-450-heme per milligram of protein) bovine adrenal cortex mitochondrial cytochrome P-450, termed P-450_sce, which cleaves the side chain of cholesterol to yield pregnenolone, is obtained in the substrate-bound ferric form with observed absorption maxima at 393 nm and 645 nm and a shoulder around 540 nm. The absorption spectra of the P-450_scc, whether in the substrate-bound ferric form or in the CO-complexed ferrous form, are subject to environmental perturbation. The addition of adrenal ferredoxin readily restores full ferric high spin type spectrum of the substrate-bound P-450_scc or, together with cholesterol and Tween 20, restores the CO-spectrum of the P-450_scc, exhibiting stable and typical spectra of cytochrome P-450. Tween 20, at concentration of 0.3%, remarkably increases the P-450_scc-catalyzed cholesterol side chain cleavage activity. Based on these findings, a highly reactive and reliable assay has been developed for the conversion of cholesterol to pregnenolone. The specific activity of the P-450_scc, thus determined in the presence of NADPH, NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1), adrenal ferredoxin, cholesterol, and molecular oxygen, is 16 mol of pregnenolone formed per minute per mole of P-450-heme and V of enzyme catalyzed reaction was 30 mol/min/mol of P-450-heme. Apparent K_m values are 120 μm for cholesterol and 1.5 μm for adrenal ferredoxin. The P-450_scc has a pH optimum at pH 7.2 and is most active at ionic strength of 0.1.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Hydroperoxidation by cytochrome P-450 oxygenase: metabolism of 9-methylfluorene

9-Methylfluorene was metabolized by rat liver microsomes to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene. The results were confirmed by using a reconstituted cytochrome P-450 oxygenase system purified from phenobarbital-induced rat liver which established its involvement. SKF-525A strongly inhibited the formation of both oxygenation products. Cytochrome P-450 alone brought about the conversion of the hydroperoxide to its alcohol. NADPH augmented the peroxidative reaction, but the presence of NADPH-cytochrome P-450 reductase was without effect. Certain microsomal preparations and reconstituted enzyme yielded little or no detectable amounts of hydroperoxide. This was due to a too rapid conversion of the hydroperoxide to its alcohol. The addition of metyrapone, a compound that inhibited such conversion, resulted in accumulation of 9-hydroperoxy-9-methylfluorene for positive identification. Incubation of 9-methylfluorene with microsomes and NADPH resulted in covalent binding of its metabolite to microsomal proteins. Incubation of 14C-labeled 9-hydroperoxy-9-methylfluorene caused covalent binding of label to proteins, RNA, and DNA.     

Immunochemical examinations of cytochrome P-450 in various tissues of human fetuses using antibodies to human fetal cytochrome P-450, P-450 HFLa

P-450 HFLa is a form of cytochrome P-450 purified from human fetal livers. The amounts of P-450 HFLa in several fetal tissues were determined immunochemically. Detectable amounts presented in livers, kidneys, adrenals, lungs and some other tissues of human fetuses. The amounts were the highest in livers. Activities of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase in livers but not in adrenals were inhibited by the anti-P-450 HFLa antibodies, probably suggesting that distinct forms of cytochrome P-450 are responsible for the oxidations in livers and adrenals.     

Substrate-binding region of cytochrome P-450scc (P-450 XIA1). Identification and primary structure of the cholesterol binding region in cytochrome P-450scc

Cytochrome P-450_scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [¹⁴C]methoxychlor. [¹⁴C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450_scc) and the inactivation was prevented in the presence of cholesterol. The binding of [¹⁴C]methoxychlor and cytochrome P-450_scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450_scc was similar with the methoxychlor-induced difference spectrum. [¹⁴C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450_scc modified with [¹⁴C]methoxychlor. Determination of the sequence of the amino-acid residues of a [¹⁴C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28.     

Leukotriene B4 metabolism by hepatic cytochrome P-450

Leukotriene B4 (LTB) was found to be metabolized by suspensions of rat liver microsomes in the presence of NADPH and oxygen. The rate of LTB metabolism was also measured in reconstituted systems of both micelles and phospholipid vesicles containing cytochrome P-450-LM2, NADPH cytochrome P-450 reductase, and cytochrome b5. A 1 microM concentration of LTB was metabolized by rat hepatic microsomes at a rate of 4 pmol LTB/min/nmole P-450, and by vesicle and micelle reconstituted systems at 3 pmole/min/nmole P-450-LM2. At this rate a 10 g rat liver exposed to 1 microM LTB can metabolize 30 micrograms per hour. In that the leukotrienes are pharmacologically active at nanomolar concentrations, hepatic metabolism may be an important pathway of leukotriene inactivation.     

Effect of ketoconazole, etomidate and other inhibitors of steroidogenesis on cytochrome P-450sccII-catalyzed reactions

The effects of a variety of certain inhibitors of adrenal steroidogenesis have been studied on the reconstituted C21-steroid 17 alpha-hydroxylase-17,20-lyase system, whose protein components, the enzyme 17 alpha-hydroxylase-17,20-lyase(P-450sccII) and its reductase, are extensively purified from pig testis microsomes. We found: (1) Ketoconazole (cis-1-acetyl-4-[4-((2-(2,4-dichlorophenyl)-2-(1H-imidazole-1- ylmethyl-1,3-dioxalan-4-ol)methoxy)phenyl] piperazine and Etomidate(R-(+)-ethyl-[1-(a-methyl-benzyl)-indol-5-carboxylatel), inhibited cleavage of 17 alpha-hydroxy progesterone at the 17,20-bond to give androstenedione in a dose-dependent fashion. (2) Some other inhibitors of steroidogenesis, Metyrapone (2-methyl-1.2di-3-pyridyl-1-propanone), Trilostane (4,5-epoxy-17-hydroxy-3-oxo androstane-2-carbonitrile),o,p'DDD (1-(O-chlorophenyl)-1-(p-chlorophenyl)2,2-dichloroethane) and Aminoglutethimide (p-(alpha-aminopheny)-alpha-ethylglutaramide) did not inhibit the same 17,20-lyase system. (3) All of the above listed inhibitors, over a wide variety of concentration ranges, had no significant effect on the 17 alpha-hydroxylation of 11 beta-hydroxyprogesterone, which had been shown to be catalyzed by the same P-450sccII. (4) NADPH:P-450 reductase was not inhibited by all of the above listed inhibitors.     

The role of cytochrome P-450 in cholesterol biogenesis and catabolism

1. Adjuvant-induced arthritis in rats is accompanied by a loss of activity of the drug-metabolizing enzyme system and a decrease in hepatic cytochrome P-450. 2. Arthritic rats have normal serum and liver cholesterol concentrations. 3. The rate of biogenesis of cholesterol in vivo and in vitro from either [(14)C]acetate or [(14)C]mevalonate in arthritic rats was the same as or greater than that found in control rats. 4. Treatment of rats with carbon disulphide (1ml/kg) resulted in a loss of drug-metabolizing-enzyme activity and increased cholesterol biogenesis. 5. The activity of cholesterol 7alpha-hydroxylase in adjuvant-induced arthritic rats did not differ significantly from that in control rats. 6. Rats fed with cholestyramine had an elevated hepatic cholesterol 7alpha-hydroxylase activity, but neither the concentration of cytochrome P-450 nor the activity of the drug-hydroxylating enzyme, aminopyrine demethylase, was affected. 7. The relationships between drug hydroxylation and cholesterol metabolism are discussed.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

The anticarcinogen 3,3′-diindolylmethane is an inhibitor of cytochrome P-450

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3′-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with K_i values in the low micromolar range. We now report a similar potent inhibition by 3,3′-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3′-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B₁. There was no inhibition of cytochrome c reductase. In addition, we found 3,3′-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3′-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans. © 1995 John Wiley & Sons, Inc.     

Purification and characterization of an anticonvulsant-induced human cytochrome P-450 catalysing cyclosporin metabolism.

A form of human hepatic microsomal cytochrome P-450 (P450hA7) with subunit Mr 50,400 has been purified from an epileptic who had been receiving long-term treatment with anticonvulsant drugs. P450hA7 metabolized the immunosuppressant drug cyclosporin A and the dihydropyridine calcium channel antagonist nifedipine, but did not metabolize a similar dihydropyridine drug, nicardipine, nor a series of alkoxyresorufin model substrates. The hepatic microsomal concentration of P450hA7 was higher in five individuals who had been receiving long-term anticonvulsant treatment than in any of 21 individuals who had not been similarly treated. The mean P450hA7 concentration in the treated individuals was 5-fold higher than the mean concentration in the untreated individuals. It is concluded that P450hA7 is a member of the cytochrome P450III family which is induced by anticonvulsant drugs in man.     

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.