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.     

Inhibition of azoreductase activity by antibodies against cytochromes P-450 and P-448

Hepatic microsomal azoreductase activity in mice was induced with phenobarbital (PB) and 3-methylcholanthrene (3-MC). Antibodies against cytochrome P-450 inhibited azoreductase activity of PB-treated animals while antibodies against cytochrome P-448 inhibited liver azoreductase activity of 3-MC-treated animals, each by about 90%. These antibodies also inhibited microsomal 7-ethoxycoumarin-O-deethylase activity to the same extent. It is concluded that hepatic microsomal azoreductase activity is almost totally dependent on cytochromes P-450 and P-448 and the contribution, if any, of other microsomal components is negligible.     

Monoclonal antibodies against a high spin form of rat cytochrome P-448

Ten monoclonal antibodies reactive with a high spin form of rat cytochrome P-448 (P-448-H) were obtained from hybridoma clones established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells of a BALB/c mouse hyperimmunized with the cytochrome. One monoclonal antibody recognized an epitope characteristic for P-448-H. Five monoclonal antibodies were cross-reactive with a low spin form of rat cytochrome P-448, but not with cytochrome P-450. Reactivity of these monoclonal antibodies with microsomes of rats pretreated with drug metabolizing inducers and Western blots of the microsomal cytochrome P-450 components are also demonstrated.     

Organ selective induction of cytochrome P-448 isozymes in the rat by 2-methoxy-4-aminoazobenzene and 3-methylcholanthrene

Male Sprague Dawley rats were injected intraperitoneally with 2-methoxy-4-amino-azobenzene (2-MeO-AAB) or 3-methylcholanthrene (MC), and then the expression of microsomal cytochrome P-450 isozymes in liver and extrahepatic tissues was investigated by means of immunological methods and a bacterial mutation test. The results of protein A-enzyme-linked immunosorbent assaying and immunoblotting using anti-rat cytochrome P-448 monoclonal antibodies showed that MC induced at least two microsomal cytochrome P-448 isozymes, a high spin form (cytochrome P-448H) and a low spin form (cytochrome P-448L), in liver, but that it induced only cytochrome P-448L in extrahepatic tissues such as lung, kidney, small intestine, and colon. The results also indicated that, in contrast to MC, 2-MeO-AAB selectively induced microsomal cytochrome P-448H in liver but did not induce any cytochrome P-448 isozymes in extrahepatic tissues. The activities of 9,000 X g supernatants from the individual organs, as to the mutagenic conversion of 3 aromatic amines (3-amino-1-methyl-5H-pyrido(4,3-b)indole, 2-amino-6-methyldipyrido(1,2-a: 3',2'-d)-imidazole and 3-methoxy-4-aminoazobenzene), toward Salmonella typhimurium TA 98 bacteria were dependent upon the quantity and/or quality of the microsomal cytochrome P-448 isozymes in the organs.     

Studies on the mechanism of hepatic microsomal N-oxide formation. N-oxidation of NN-dimethylaniline by a reconstituted rabbit liver microsomal cytochrome P-448 enzyme system.

The N-oxidation of NN-dimethylaniline was studied by using a reconstituted rabbit liver microsomal enzyme system consisting of highly purified cytochrome P-448, NADPH-cytochrome c reductase and lipid factor. Both cytochrome P-448 and NADPH-cytochrome c reductase were required for optimum N-oxygenating activity; the catalytic capacity of the reductase fraction for supporting N-oxide formation varied with the isolation procedure applied. Addition of microsomal lipids to the assay media stimulated N-oxidation of the arylamine. N-Oxide formation appeared to be not generally controlled by electron transfer from cytochrome b5 to cytochrome P-448. The present work confirms that cytochrome P-448 can mediate about 44% of the rabbit liver microsomal N-oxidation of NN-dimethylaniline, thus reinforcing the existence of at least two distinct tertiary amine N-oxidases, i.e. haemoprotein and flavoprotein oxidase, in liver microsomal fractions.     

Benzpyrene hydroxylase activity in hepatic microsomal and solubilized systems containing rabbit or rat cytochrome P-448 or P-450

Treatment of adult, male rabbits and rats with 3-methylcholanthrene results in the formation of hepatic microsomal cytochrome P-448. In the rat, this occurs coincidently with an increase in hepatic microsomal benzpyrene hydroxylase activity. In the rabbit, benzpyrene hydroxylase activity is decreased following treatment with 3-methylcholanthrene. Benzpyrene hydroxylase activity in solubilized, reconstituted mixed-function oxidase systems containing rat cytochrome P-448 is about seven times higher than in systems containing rabbit cytochrome P-448. Evidence obtained by spectral analysis suggests that rabbit P-448 is combined with a type I compound. Residual ¹⁴C-3-methylcholanthrene does not appear to be responsible for the differences observed between rat and rabbit cytochrome P-448.     

Purification and partial characterization of hepatic microsomal cytochrome P-450s from phenobarbital- and 3-methylcholanthrene-treated rats.

Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.     

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.     

Induction of cytochrome P-448 isozyme(s) in primary cultured rat hepatocytes by drugs which induce different isozymes in vivo

Effect of 2-methoxy-4-aminoazobenzene (2-MeO-AAB) and 3-methylcholanthrene (MC) on the induction of microsomal cytochrome P-448 isozymes in primary cultured rat hepatocytes was examined by means of immunochemical methods such as protein A-enzyme-linked immonosorbent assay and immuno-blots using anti-rat cytochrome P-448 monoclonal antibodies and by means of bacterial mutation tests. Although 2-MeO-AAB selectively induced cytochrome P-448H and MC induced both cytochrome P-448H and a low spin form of cytochrome P-448 (P-448L) in the liver of rats, addition of these chemicals to primary cultured rat hepatocytes resulted in selective induction of cytochrome P-448L, as determined by the immunological methods. This was substantiated by the bacterial mutation test using Salmonella typhimurium TA 98 bacteria and two aromatic amine substrates with different specificities to the cytochrome P-448 isozymes. These results suggest that the responses of rat hepatocytes to cytochrome P-450 inducers are different in in vivo and in vitro.     

Liver microsomal cytochromes P-450 and azoreductase activity.

Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.     

A simple method for assessment of rat cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogenic chemicals

Cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes were prepared from the livers of Sprague-Dawley rats treated with 3-methylcholanthrene (MC) and with a combination of MC and carbon tetrachloride, respectively, and their activities for mediating mutagenic activation of 9 carcinogenic aromatic amines and benzo[a]pyrene, which are found to be different from cyt. P-450 isozymes as to mutagenic activation, were compared on the basis of microsomal cytochrome P-450 content using Salmonella typhimurium TA98 as a tester bacterium. With regard to the substrate-specificity of cytochrome P-448 isozymes, the present results reflected the reported results with use of a cytochrome P-450-reconstituted system. These findings indicate that the mutation test with cytochrome P-448H/L-enriched and cytochrome P-448L-enriched microsomes could be used as a simple method for the determination of the cytochrome P-448 isozymes responsible for the mutagenic activation of carcinogens and mutagens without the use of a cytochrome P-450-reconstituted system.     

Correlation between cytochrome P-448 content and benzopyrene hydroxylase activity during the induction of microsomal monooxygenases using methylcholanthrene-type xenobiotics

Using antibodies against electrophoretically homogeneous cytochrome P-448 from rat liver microsomes induced by 3-methylcholanthrene, the changes in the immunologic identity and contents by cytochrome P-448 induced by 3-methylcholanthrene, 3.4-benzpyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were studied. No cytochrome P-448 was detected in the liver microsomes of control or phenobarbital-induced rats. This form of the cytochrome makes up to about 35% of the total content of the CO-binding hemoprotein during TCDD induction and up to 90% during 3-methylcholanthrene and 3,4-benzpyrene induction. On the other hand, 3-methylcholanthrene, 3,4-benzpyrene and TCDD significantly and equally activates the cytochrome P-448-dependent benzpyrene hydroxylase, since the antibodies against cytochrome P-448 inhibit benzpyrene metabolism in the microsomes by 85-90%. The possible reasons for the TCDD-induced increase in the catalytic activity of cytochrome P-448 as compared to the immunologically identical cytochrome P-448 induced by 3-methylcholanthrene and 3,4-benzpyrene, are discussed.     

The effect of Sudan III on drug metabolizing enzymes

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.     

Purification and characterization of two forms of chicken liver cytochrome P-450 induced by 3,4,5,3',4'-pentachlorobiphenyl

3,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16 alpha-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16 alpha-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.     

Perfluorocarbons do not induce cytochrome P-448 in the rat liver

Cytochrome P-450 forms appearing in the liver after injection of methylcholanthrene, polychlorinated biphenyls and perfluorochemical emulsion to rats were studied. Activities of marker enzymes, benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, as well as the interaction of liver microsomal membranes with antibodies against different cytochrome forms were investigated. It was shown that fluorocarbon emulsion containing perfluorodecalin did not induce cytochrome P-448 in the rat liver.     

Drug-oxidizing mono-oxygenase system in liver microsomes of goldfish (Carassius auratus)

The fractionation of the liver of goldfish (Carassius auratus) was studied, and the properties of the microsomal fraction were examined. The microsomal fraction contained cytochrome P-450 and catalyzed the oxidation of aminopyrine, aniline, 7-ethoxycoumarin and benzo(a)pyrene. The oxidation activities were significantly lower than those of rat liver microsomes. The titration of cytochrome P-450 by potassium cyanide indicated the presence of multiple forms of cytochrome P-450 in goldfish liver microsomes. Feeding of goldfish with 3-methylcholanthrene-containing food greatly induced benzo(a)pyrene hydroxylation activity of the liver microsomes. The Soret peak of the carbon monoxide compound of cytochrome P-450 was shifted from 450 to 448 nm.     

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.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.