Purification and characterization of three forms of microsomal cytochrome P-450 in liver from 3-methylcholanthrene-treated guinea pigs

One molecular form of cytochrome P-450IIA from liver microsomes of guinea pigs treated with 3-methylcholanthrene was purified to a specific content of 17.4 nmoles/mg of protein. The difference spectrum of reduced hemoprotein-carbon monoxide complex of this cytochrome exhibits an absorption maximum at 448 nm. The absolute absorption spectrum of the oxidized form of this hemoprotein suggests a high-spin state of heme iron. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein shows a single band of polypeptide stained with Coomassie brilliant blue at the position corresponding to Mr 54,000. On the other hand, the other two forms of cytochrome P-450, cytochrome P-450I and P-450IIB, were also separated and purified to specific contents of 8.7 and 5.2 nmoles/mg of protein, respectively. Both cytochrome P-450I and P-450IIB exhibit absorption maxima at 450 nm in the difference spectrum of reduced hemoprotein-carbon monoxide complex, and a low-spin state of ferric iron in the heme. The spectrophotometrical property of cytochrome P-450I and P-450IIB was clearly different from that of cytochrome P-450IIA. Molecular activities of a reconstituted aryl hydrocarbon hydroxylase (EC 1.14.14.1) containing, respectively, cytochrome P-450I, P-450IIA, and P-450IIB were 0.224, 0.250, and 0.395 (moles per minute per mole of cytochrome P-450), and were estimated to be one-tenth that of cytochrome P-448 induced in rat liver by 3-methylcholanthrene, indicating the presence of the low inducibility by 3-methylcholanthrene of aryl hydrocarbon hydroxylase in liver microsomes of guinea pigs.     

Immunohistochemical localization of cytochrome P-450a in liver sections from untreated rats and rats treated with phenobarbital or 3-methylcholanthrene

Antibodies against cytochrome P-450a purified from hepatic microsomes of Long-Evans rats were used for indirect immunofluorescent localization of cytochrome P-450a within the livers of untreated, phenobarbital-treated, and 3-methylcholanthrene-treated mature and immature male and female rats. Immunohistochemical localization of cytochrome P-450a in liver sections from untreated mature male rats revealed fluorescence primarily in the central and midzonal regions, and moderate fluorescence in the periportal region. This pattern of fluorescence was not significantly altered in liver sections from mature male rats treated with phenobarbital or 3-methylcholanthrene. Liver sections from untreated mature female and immature male and female rats were primarily fluorescent in the central region with weak fluorescence in the midzonal and periportal regions of the lobule. Phenobarbital or 3-methylcholanthrene treatment markedly enhanced the intensity and lobular distribution of the fluorescence in liver sections from mature female and immature male and female rats. The apparent induction of cytochrome P-450a in the mature female and immature male and female rats, but not in mature males, is consistent with our previous immunochemical studies on the quantity of cytochrome P-450a in hepatic microsomes from untreated and treated rats. In addition, this study demonstrates that the lobular distribution of cytochrome P-450a in untreated mature male rats is different from the distribution of cytochrome P-450a in untreated female or immature rats.     

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.     

Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram

Microsomes from male rats treated with picloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16 alpha-hydroxylase activity when incubated with (4-14C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P-450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450 in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to picloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbital-inducible forms of cytochrome P-450. Third, hepatic microsomes from picloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98 revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from picloram-treated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Induction of drug metabolism. VI. Effects of phenobarbital and 3-methylcholanthrene administration on N-demethylating enzyme systems of rough and smooth hepatic microsomes

The induction of the microsomal hemoproteins, cytochromes P-450 and Pi-450, and of N-demethylase activities in hepatic microsomal subfractions from rats were studied at various times after administration of phenobarbital or 3-methylcholanthrene. After a single dose of phenobarbital, N-demethylase activity and cytochrome P-450 levels increased initially only in rough microsomes (RER) whereas a single dose of 3-methylcholanthrene caused almost simultaneous increases of the two enzymes in both RER and smooth microsomes (SER). The increases in N-demethylase activities during this early period of induction by 3-methylcholanthrene were paralleled by a change in P-450 hemoprotein from cytochrome P-450 to cytochrome P₁-450 in both microsomal subfractions, but the total amount of P-450 hemoprotein remained essentially unchanged. These results add to existing evidence that phenobarbital and 3-methylcholanthrene produce their inductive effects by different mechanisms and raise the possibility that cytochrome P₁-450 may be synthesized in both RER and SER.     

Characterization of hepatic and pulmonary cytochromes P-450 in 3-methylcholanthrene-treated hamsters

Two major forms of hepatic cytochrome P-450 (hepatic P-450_MCI and P-450_MCII) were purified approximately 5-fold from liver microsomes in Syrian golden hamsters treated with 3-methylcholanthrene (MC). The purified preparations of hepatic P-450_MCI and P-450_MCII contained 9.6 and 8.3 nmol cytochrome P-450 (P-450) per mg protein, respectively, and were essentially free from NADPH-cytochrome c (P-450) reductase (fpT), NADH-cytochrome b₅ reductase and cytochrome b₅. By sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of hepatic P-450_MCI and P-450 _mMCII were estimated to be 56 000 and 53 500. Further, a major form of pulmonary P-450 (P-450_MC) were purified from lung microsomes of MC-treated hamster, and contained 14.2 nmol P-450 per mg protein, and estimated to be 56 000 in monomeric molecular weight, indicating the similar molecular weight to hepatic P-450_MCI in the hamster. From the absorption spectra the oxidized forms of hepatic P-450_MCI and P-450_MCII were high- and low-spin ferric hemoproteins, respectively, and pulmonary P-450_MC was similar to hepatic P-450_MCII in their hemoprotein spin state. No difference, however, was observed in the CO-reduced forms among hepatic P-450_MCI, P-450_MCII and pulmonary P-450_MC, all exhibiting 446.5 nm Soret bands. In a reconstituted system containing fpT and dilauroylphos-phatidylcholine (DLPC), pulmonary P-450_MC efficiently catalyzed benzo[a]pyrene (BP) hydroxylation at a rate of 11.4 mol formed per min per mol P-450, but hepatic P-450_MCI and P-450_MCII both exhibited lower levels, e. g., 0.49 and 0.54, respectively. These findings indicated a clear tissue difference in the activity of BP hydroxylation between lung and liver in MC-treated hamsters.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

Genotoxic and mutagenic activation of aflatoxin B1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B1 (AFB1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/S. typhimurium TA98 system by activated metabolites of AFB1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB1 in rat liver microsomes.     

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats. Effects of 3-methylcholanthrene and beta-naphthoflavone

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b5, NADH- and NADPH- dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-488) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and beta-naphthoflavone, increased the cytochrome content 3- to 10-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 micrometers alpha-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.     

Evaluation of monooxygenase induction as a means of enhancing the yield of the rat liver cytochrome P-450 isoenzyme with high affinity for cimetidine

The binding of cimetidine to liver microsomes prepared from untreated rats and rats pretreated with phenobarbitone or 3-methylcholanthrene has been investigated by difference spectroscopy and equilibrium partition methods. In M/15 phosphate buffer, pH 7.9, microsomes from each group of rats yielded markedly biphasic spectral binding curves, which have been interpreted in terms of two independent classes of cytochrome P-450 site with widely differing binding affinities for the drug. Support for such interpretation was provided by the finding that the spectral binding curve for a purified sample of the principal cytochrome P-450 isoenzyme from liver microsomes of phenobarbitone-pretreated rats could be described adequately by a single rectangular hyperbolic relationship, the spectral dissociation constant being indistinguishable experimentally from that for the weaker class of cytochrome P-450 binding site in the corresponding microsomes. The spectral dissociation constants were 2 μM and 80 μM for microsomes from untreated rats; 44 μM and 540 μM for those from phenobarbitone-pretreated rats; and 34 μM and 540 μM for microsomes from rats pretreated with 3-methylcholanthrene. On this basis, both classes of P-450 site in the microsomes from rats subjected to either pretreatment exhibited lower affinity for cimetidine than their counterparts in microsomes from untreated rats. Equilibrium partition studies of the higher-affinity class of microsomal binding site for cimetidine showed that the twofold increase in the cytochrome P-450 content of microsomes effected by 3-methylcholanthrene pretreatment was more than offset by a diminished proportion of the total cimetidine-binding capacity present as the higher-affinity, pharmacologically significant, receptor (18%, cf. 48% in control microsomes); and that phenobarbitone pretreatment resulted in replacement of the high-affinity receptor by one with a threefold weaker cimetidine-binding affinity. Thus the use of these monooxyginase inducers to enhance the cytochrome P-450 content of liver microsomes would seem to offer little potential in the isolation of the isoenzyme with high affinity for cimetidine.     

Effect of cytochrome P-450 and flavin-containing monooxygenase modifying factors on the in vitro metabolism of amiodarone by rat and rabbit

Experiments were conducted to affirm hepatic cytochrome P-450 involvement in the biotransformation of the class III antiarrhythmic agent, amiodarone (Am; Cordarone X) to its major metabolite, desethylamiodarone (DEA). Male Sprague-Dawley rats and male New Zealand white rabbits were treated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) (to induce cytochrome P-450 (PB-inducible cytochrome(s) P-450) or P-448 (MC-inducible cytochrome P-450). In vivo decreases in rat hepatic microsomal cytochrome P-450 were achieved either by a single ip dose of CCl4 or by a 2-day treatment with CoCl2. In vitro biotransformation of Am by hepatic microsomes from PB-induced and 3-MC-induced rats and PB-induced rabbits was significantly greater than that from noninduced animals. Conversely, in vitro DEA production was significantly decreased with hepatic microsomes from CCl4- and CoCl2-pretreated rats. The classic P-450 inhibitors, piperonyl butoxide, SKF 525A, n-octylamine, and CO provided a significant reduction in the in vitro formation of DEA by microsomes from induced animals. In vitro DEA formation by hepatic microsomes from PB- and 3-MC-induced rats was significantly decreased by 0.5 mM chloroquine (specific inhibitors of PB-inducible cytochrome(s) P-450) and 0.3 mM quinacrine (specific inhibitor of MC-inducible cytochrome(s) P-450), respectively. Further evidence for involvement of gut microsomal flavin-containing monooxygenase was provided by the inhibition of gut microsomal-mediated in vitro DEA formation in the presence of methimazole. Methimazole had no effect on hepatic microsomal DEA production in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the concentration of seven forms of cytochrome P-450 in primary cultures of adult rat hepatocytes

We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.     

Identification of the cyanopregnenolone-inducible form of hepatic cytochrome P-450 as a catalyst of aldrin epoxidation

In light of recent suggestions that hepatic microsomal aldrin expoxidation activity selectively reflects the phenobarbital (PB)-inducible form(s) of cytochrome P-450 (P-450PB), we tested the effect of pregnenolone-16 alpha-carbonitrile (PCN), a synthetic steroid that induces P-450PCN, a form of the cytochrome biochemically and immunochemically distinguishable from P-450PB. In hepatic microsomes prepared from rats receiving PB, 3-methylcholanthrene (3-MC), or PCN, the latter compound produced a greater increase in aldrin epoxidation activity relative to control than did PB, whereas 3-MC decreased enzyme activity. Moreover, the aldrin epoxidation activity in microsomes prepared from PCN- or PB-pretreated rats was selectively inhibited by form-specific antibodies directed against P-450PCN or P-450PB, respectively, whereas anti-P-450MC antibodies gave no inhibition with microsomes prepared from induced or control animals. We conclude that P-450PCN, P-450PB, and probably other cytochromes P-450 catalyze aldrin epoxidation, precluding use of this enzyme as a specific marker of a single form of the cytochrome.     

Participation of cytochrome P-450 isozymes in N-demethylation, N-hydroxylation and aromatic hydroxylation of methamphetamine

1. Five isozymes of cytochrome P-450 were purified from liver microsomes of phenobarbital-pretreated (P-450-SD-I and -II), 3-methylcholanthrene-pretreated (P-450-SD-III) and untreated rats (P-450-SD-IV and -V) to determine their catalytic activities in metabolic reactions of methamphetamine. 2. All the isozymes except P-450-SD-III showed considerably high N-hydroxylating activity of methamphetamine. The cytochromes P-450 initiate N-demethylation of this drug by two metabolic pathways, C-hydroxylation and N-hydroxylation. 3. Both N-demethylation and N-hydroxylation of methamphetamine were efficiently catalysed by the phenobarbital-inducible forms P-450-SD-I and -II and constitutive forms P-450-SD-IV and -V. 4. The constitutive forms P-450-SD-IV and -V revealed high catalytic activities of p-hydroxylation of methamphetamine, but phenobarbital- and 3-methylcholanthrene-inducible isozymes showed only low activities. 5. The present results indicate that the different extents of the metabolic intermediate complex formation with cytochrome P-450 (455 nm complex) in the microsomes from phenobarbital-, 3-methylcholanthrene-pretreated, and untreated rats is not attributable to the activities of the respective isozymes of cytochrome P-450 to form the precursor of the complex, N-hydroxymethamphetamine.     

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

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

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant (K_s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding (ΔA_max) was increased only following phenobarbital induction. The K_s values (ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant (K_m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V_max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO₂ and H₂O₂, but not NaIO₄, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b₅ or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.     

A Comparative Study on Drug Hydroxylation and Glucuronidation in Liver Microsomes of Phenobarbital and 3-Methylcholanthrene Treated Rats

The treatment of hepatic microsomes with phospholipase A or phospholipase C decreased the drug hydroxylase activity. The exception was the aryl hydrocarbon hydroxylase in hepatic microsomes from 3-methylcholanthrene pre-treated rats. Its specific activity remained unchanged after phospholipase A digestion. In hepatic microsomes from phenobarbital pretreated rats the aryl hydrocarbon hydroxylase was not significantly increased and after phospholipase A or phospholipase C digestion, it decreased in both treated and control rats. The phenobarbital or 3-methylcholanthrene induced p-nitroanisole 0-demethylase activity in liver microsomes decreased in a manner corresponding to the decrease in cytochrome P-450 (P-448) concentrations. The treatment of hepatic microsomes with phospholipase A or phospholipase C activated the membrane-bound UDP glucuronosyltransferase both in control and drug-treated rats. Phospholipase A increased the measurable UDP glucuronosyltransferase activity particularly in hepatic microsomes from 3-methylcholanthrene pretreated rats. However, digestion with trypsin was necessary in order to detect the induction in UDP glucuronosyltransferase after phenobarbital pretreatment of rats. Other agents studied, such as phospholipase A or C, digitonin, cetylpyridinium chloride, or NaSCN, were unable to do this. Digestion with trypsin released considerable amounts of UDP glucuronosyltransferase activity into 27,000 g supernatant, especially from phenobarbital microsomes, whereas phospholipase A was more active in releasing UDP glucuronosyltransferase into 27,000 g supernatant from hepatic microsomes of 3-methylcholanthrene pretreated rats. Membrane perturbating agents seem to differ in their action towards the hepatic microsomal membranes obtained from rats treated with phenobarbital or 3-methylcholanthrene.     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

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

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

Binding of reactive metabolites of CCl4 to specific microsomal proteins

The covalent binding of [14C]carbon tetrachloride to microsomal proteins in rat liver microsomes under anaerobic conditions was investigated by SDS-polyacrylamide slab gel electrophoresis and fluorography. Most of the labeled proteins were observed in the molecular weight range of 52-61 kDa, indicating that cytochrome P-450 forms (EC 1.14.14.1) were labeled. Protein bands at the position of the NADPH-cytochrome P-450 reductase (78 kDa) (EC 1.6.2.4) and NADH-cytochrome b5 reductase (33 kDa) (EC 1.6.2.2) also showed radioactivity. The fluorographic pattern of the protein labeling was cytochrome P-450-dependent, as was demonstrated by CO and metyrapone inhibition as well as by pretreatment of rats with inducing drugs such as 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Aroclor 1254. Immuno-precipitation with a purified anti-P-450 immunoglobulin against cytochrome P-450 PB-B (52 kDa) of rat liver indicated that this protein contained about 10-20% of the total bound radioactivity in an average ratio of 0.8 mol [14C]CCl4-metabolite/mol cytochrome P-450 PB-B.