Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

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.     

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.     

Detection of a system of microsomal monooxygenases in the rodent malaria parasite Plasmodium berghei

A microsomal fraction from the cells of the malaria parasite of rodent Plasmodium berghei was obtained. The spectral properties of microsomal preparations suggest that P. berghei microsomes contain cytochromes b5 and P-420. Electrophoretic separation of microsomal proteins revealed the presence of proteins whose molecular mass corresponds to NADPH-cytochrome c reductase, cytochrome P-450 and epoxide hydratase. The activities of NADPH-cytochrome c reductase and benzpyrene hydroxylase were determined. The spectral parameters, electrophoretic data and enzymatic activities of microsomal proteins indicate that P. berghei cells contain a cytochrome P-450 monooxygenase system. The interrelationship between the activity of the microsomal monooxygenase system and the resistance of P. berghei cells to the antimalaria preparation chloroquine is discussed.     

Activity of NADPH-dependent monooxygenase hydroxylating system of rat liver under the effect of tetramethylthiuramdisulfide

Changes in the activity of a NADPH-dependent monooxygenase system of the rat liver are studied under the effect of tetramethylthiuramdisulphide. Under these conditions aniline hydroxylation is shown to be inhibited to a higher extent than amidopyrine demethylation. Besides a decrease in the level of cytochrome P-450, the central component of the microsomal system of hydroxylation, there appears cytochrome P-420--an inactivated form of cytochrome P-450.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole

Inhibition studies were used to investigate the identity of the microsomal enzyme(s) responsible for the NADPH-dependent N-hydroxylation of 2-amino-6-nitrotoluene. The N-hydroxylation reaction was inhibited by several cytochrome P-450 inhibitors as well as by methimazole, a substrate for flavin-containing monooxygenase. Heat inactivation of flavin-containing monooxygenase had no effect on the rate of the reaction but abolished the inhibition by methimazole. These results indicate that the flavin-containing monooxygenase-mediated metabolism of methimazole produced an inhibitor of the cytochrome P-450-catalyzed N-hydroxylation reaction. When glutathione was included in the incubation the inhibition by methimazole was abolished, presumably due to the reduction of oxygenated metabolites of methimazole. These results show that methimazole inhibition does not necessarily implicate flavin-containing monooxygenase in microsomal N-hydroxylation reactions.     

Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro effects of trans-4-hydroxy-2-alkenals on mouse liver cytochrome P-450

Under in vitro conditions, trans-4-hydroxy-2-hexenal (t-4HH), trans-4-hydroxy-2-nonenal (t-4-HN) and trans-2-hexenal (t-2H) significantly reduced the levels of mouse liver microsomal cytochrome P-450. Incubation of trans-4-hydroxy-alkenals, under anaerobic conditions in the absence of an NADPH-generating system indicated that these compounds were converting cytochrome P-450 to cytochrome P-420. Prior activation by the mixed function oxidase system was not required for trans-4-hydroxy-alkenals to alter cytochrome P-450 concentrations. trans-4-Hydroxy-alkenals and non-hydroxylated alpha,beta-unsaturated aldehydes may be exerting their effects on cytochrome P-450 by binding to sulfhydryl groups in a similar manner as reported for sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and p-chloromercuribenzoate.     

Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.     

An immunochemical analysis of the induction of cytochrome P-450 isoforms in the liver of fresh-water fishes by 3-methylcholanthrene, beta-naphthoflavone and aroclor 1254

The cytochrome P-450 isoforms have been studied in liver microsomes of some fish species from Lake Baikal. Using the inhibitory analysis of microsomal monooxygenase activities carried out by the specific polyclonal antibodies it has been shown that 3-methylcholanthrene, beta-naphthoflavone and arochlor 1254 induce isoforms immunologically related to cytochrome P-488c but not to the rat cytochrome P-450b in fish liver microsomes. The immunologic identity in isoforms of fish and rat cytochromes induced by methylcholanthrene has not been revealed. A possibility to use the method of the inhibitory analysis of fish microsomal activities by specific antibodies to the rat cytochrome P-450 isoforms for biomonitoring and biotesting of polycyclic hydrocarbons and polychlorinated biphenyls in aquatic systems is discussed.     

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.     

Effect of inducers of DT-diaphorase on the haemolytic activity and nephrotoxicity of 2-amino-1,4-naphthoquinone in rats

Reduction of naphthoquinones by DT-diaphorase is often described as a detoxification reaction. This is true for some naphthoquinone derivatives, such as alkyl and di-alkyl naphthoquinones, but the situation with other substances, such as 2-hydroxy-1,4-naphthoquinone, is more complex. In the present study, the effect of several substances that are known to increase tissue activities of DT-diaphorase on the toxicity of 2-amino-1,4-naphthoquinone has been investigated. Like 2-hydroxy-1,4-naphthoquinone, the 2-amino-derivative was found to cause both haemolytic anaemia and renal tubular necrosis in rats. Again like 2-hydroxy-1,4-naphthoquinone, the severity of the haemolysis induced by the 2-amino derivative was increased in animals pre-treated with inducers of DT-diaphorase, but the degree of nephrotoxicity was decreased. With these substances, therefore, DT-diaphorase both activates and detoxifies the quinone, depending on the target organ. It is not possible to generalise with regard to the effects of modulation of tissue levels of DT-diaphorase on naphthoquinone toxicity in vivo, since this may change not only the severity of the toxic effects, but also the target organ specificity. In evaluating the possible therapeutic applications of such compounds, the possibility of toxic effects upon the blood and kidney must be borne in mind. In man, renal damage by compounds such as 2-hydroxy- and 2-amino-1,4-naphthoquinone may be a particular problem, because of the low level of DT-diaphorase in human liver.     

Enzymes metabolizing xenobiotics in spontaneous tumors in mice

The microsomal monooxygenase activity in spontaneous mouse hepatomas has been studied. The cytochrome P-450 level in hepatomas was shown to be 2 times as low as that in the liver. The reduction of the cytochrome P-450 content in the tumour was accompanied by a decrease in the activity of benz(a)pyrene hydroxylase, amino-pyrene-N-demethylase and p-nitroanisole-O-demethylase. However, 7-ethoxycoumarin-O-deethylase activity in hepatomas was much higher than in the liver both estimated as mg of the microsomal protein and nmol of cytochrome P-450. The cytochrome b5 content in the hepatomas was comparable with its level in the liver. A more elevated content of NADPH-cytochrome c reductase and microsomal epoxide hydrolase activity was found in the hepatomas. The results obtained provide evidence of different oxidation effects regarding some substrates in the liver and hepatomas. The ratio of cytochrome P-450 isoforms is likely to change in the hepatomas in contrast with that in the liver.     

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism. Hepatic NADPH-dependent metabolism of pNA and reduction of cytochrome c are inhibited to the same extent with varying amounts of antibodies to NADPH cytochrome P-450 reductase. The same relationship between inhibition of monooxygenase and reductase activities is observed for the hepatic and pulmonary metabolism of benzphetamine and 7-ethoxycoumarin. In contrast, the relationship between inhibition of the pulmonary NADPH-dependent metabolism of pNA and reductase activity is biphasic; at 75% inhibition of reductase activity, metabolism of pNA is inhibited by less than 25%. For NADH-dependent metabolism of pNA, our results indicate that both electrons are transferred to cytochrome P-450 from cytochrome b5.