Binding of benzo[a]pyrene to DNA by cytochrome P-450 catalyzed one-electron oxidation in rat liver microsomes and nuclei

To investigate whether cytochrome P-450 catalyzes the covalent binding of substrates to DNA by one-electron oxidation, the ability of both uninduced and 3-methylcholanthrene (MC) induced rat liver microsomes and nuclei to catalyze covalent binding of benzo[a]pyrene (BP) to DNA and formation of the labile adduct 7-(benzo[a]pyren-6-yl)guanine (BP-N7Gua) was investigated. This adduct arises from the reaction of the BP radical cation at C-6 with the nucleophilic N-7 of the guanine moiety. In the various systems studied, 1-9 times more BP-N7Gua adduct was isolated than the total amount of stable BP adducts in the DNA. The specific cytochrome P-450 inhibitor 2-[(4,6-dichloro-o-biphenyl)oxy]ethylamine hydrobromide (DPEA) reduced or eliminated BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The effects of the antioxidants cysteine, glutathione, and p-methoxythiophenol were also investigated. Although cysteine had no effect on the microsome-catalyzed processes, glutathione and p-methoxythiophenol inhibited BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The decreased levels of binding of BP to DNA in the presence of glutathione or p-methoxythiophenol are matched by decreased amounts of BP-N7Gua adduct and of stable BP-DNA adducts detected by the 32P-postlabeling technique. This study represents the first demonstration of cytochrome P-450 mediating covalent binding of substrates to DNA via one-electron oxidation and suggests that this enzyme can catalyze peroxidase-type electron-transfer reactions.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Comparison of the formation of benzo[a]pyrene diolepoxide-DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2

The involvement of cytochrome P-450 isozymes in the activation of benzo[a]pyrene (BP) by human placental and liver microsomes was studied in vitro using monoclonal antibodies (Mab) toward the major 3-methylcholanthrene (MC)-inducible and phenobarbital-inductible rat liver P-450 isozymes (Mab 1-7-1 and Mab 2-66-3, respectively). Microsomes from human placenta and liver and rat liver were incubated with BP and DNA, and BP-diolepoxide-DNA (BPDE-DNA) adducts were measured by synchronous fluorescence spectrophotometry (SFS). The only BP metabolite giving the same fluorescence peak as chemically modified BPDE-DNA was BP-7,8-dihydrodiol. Five (smokers) out of 29 human placentas (smokers and nonsmokers), and five out of nine human livers were able to metabolically activate BP to BPDE-DNA adducts in this system. The Mab 1-7-1 totally inhibited the formation of BPDE-DNA adducts in placental microsomal incubations. Inhibition using rat or human liver microsomes was 50-60% and about 90%, respectively. The Mab 2-66-3 had no effect in any of the microsome types. Adduct formation was inhibited more strongly and at lower concentrations of Mab 1-7-1 compared with the inhibition of AHH activity. This study is a clear indication of the major role of P-450IA1 (P-450c) in human placenta and probably P-450IA2 (P-450d) in human liver in BP activation, while other isozymes also take part in the activation in rat liver. Furthermore, this clearly indicates that AHH activity and BP activation are not necessarily associated.     

Hydroperoxide-dependent epoxidation of 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene by ram seminal vesicle microsomes and by hematin

Addition of arachidonic acid to ram seminal vesicle microsomes oxidizes 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene (BA-3,4-diol) to five more polar products. Four of the products are identified by chromatographic and spectroscopic analysis as tetrahydrotetraols, which are solvolysis products of dihydrodiolepoxides. The fifth product is a 10-methyl ether formed by methanolysis of the anti-diolepoxide. Quantitation of the individual products indicates that anti-diolepoxides predominate over syn-diolepoxides by approximately 2:1. Identical product profiles are detected from the reaction of BA-3,4-diol with hematin and 13-hydroperoxy-octadecadienoic acid in the presence of Tween 20. No other products are detected in either system, which indicates that peroxyl radicals oxidize BA-3,4-diol exclusively by epoxidation of the 1,2-double bond. The stereochemical and regiochemical differences between oxidation of BA-3,4-diol by peroxyl radicals and cytochrome P-450 are dramatic and suggest that BA-3,4-diol is uniquely suited as a probe to quantitate peroxyl radical-dependent epoxidation in vitro and in vivo.     

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.     

Xenobiotic-metabolizing enzymes and benzo[a]pyrene metabolism in the benzo[a]pyrene-sensitive mutant strain of Drosophila simulans.

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364yv, which is sensitive to the toxic and mutagenic effects of benzo[a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364yv flies with a rare mutation in one of the P-450 regulating genes.     

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.     

The role of vitamin K3 in the hydroxylation of benz(a)pyrene in rat liver mitochondria after induction with 3-methylcholanthrene and phenobarbital

The "fast" phase reduction of microsomal cytochromes P-450 and P-448 and their benz(a)pyrene (BP) hydroxylase activity was investigated as a function of menadione concentrations. Within a narrow concentration range (1.5-3 microM) menadione activates cytochrome P-448 reduction and the BP hydroxylase activity. At higher concentrations menadione inhibits cytochromes P-450 and P-448 reduction and BP hydroxylation with participation of the both cytochromes. These data suggest that menadione molecules present in membrane lipids serve as an additional electron carrier to cytochrome P-448, the active site of which is embedded into lipids. The activating effect is unobserved is case of cytochrome P-450 with an active site localized in the aqueous phase. The number of different BP metabolites formed at low (3 microM) menadione concentrations in the microsomes of rats induced with 3-methylcholanthrene (MC) and phenobarbital (PB) was compared. In PB-induced microsomes the amount of 7,8-dihydrodiol rises whereas the total content of BP metabolites decreases. Contrariwise, in MC-induced microsomes the synthesis of all BP metabolites is augmented. Menadione has a very weak effect on the ratio of different BP metabolites in PB- and MC-microsomes, but strongly inhibits the formation of more polar metabolites. This results in a marked reduction of the number of "dangerous" BP diolepoxides.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

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.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

17R(18S)epoxyeicosatetraenoic acid, a cytochrome P-450 metabolite of 20:5n-3 in monkey seminal vesicles, is metabolized to novel prostaglandins

Eicosapentaenoic acid (20:5n-3) is metabolized by cytochrome P-450w3 of monkey seminal vesicles to 17R(18S)epoxy-5,8,11,14-eicosatetraenoic acid (17R(18S)EpETE). PGH synthase is abundant in this tissue. Racemic 17(18)EpETE was therefore investigated as a substrate of PGH synthase. The main products were identified as two diastereoisomers of 17(18)epoxyprostaglandin E2, which were formed in a 4:5 ratio. The structures were confirmed by authentic material. The natural epoxide enantiomer can thus be metabolized to novel 17R(18S)epoxyprostaglandins.     

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.     

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.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Nonadditive effects of combined in vivo hepatic microsomal enzyme inducers in the Wistar rat

Compounds that are known to increase the hepatic microsomal cytochrome P-450 dependent monooxygenases were administered to adult female rats, alone or in combination, to determine whether their effects on certain substrate oxidations were additive. 3-Methylcholanthrene (3-MC) and pregnenolone-16 alpha-carbonitrile (PCN), known to induce different forms of cytochrome P-450, when administered together increased benzo[a]pyrene oxidation to the same level as observed following 3-MC treatment alone. Phenobarbital (Pb) and PCN when administered concomitantly increased benzo[a]pyrene, amino-pyrine, and ethylmorphine metabolism to the same extent as seen following PCN administration alone. Both compounds are known to induce different forms of cytochrome P-450. Nonadditive effects were also observed with Pb and spironolactone, as well as with Pb and trans-stilbene oxide. Treatment of adult male rats with either PCN or 3-MC resulted in significantly smaller increases in benzo[a]pyrene oxidation than observed in adult female rats. These results suggest that oxidative metabolism in hepatic microsomes is not the sum of activities of a number of cytochrome P-450s, but may represent the activity of a single predominant hemeprotein. In addition, it appears that the oxidation of substrate by a particular cytochrome P-450, in intact microsomes, is greatly influenced by the presence of another form.     

A class of strong inhibitors of microsomal monooxygenases: the ellipticines.

Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.     

The specificity and multiplicity of human placental xenobiotic-metabolizing monooxygenase system studied by potential substrates, inhibitors and gel electrophoresis

The specificity of the placental monooxygenase system to metabolize foreign compounds was studied by using different potential substrates and inhibitors and by performing electrophoresis of placental microsomes. Placental preparations from smokers catalyzed benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation and 2,5-diphenyloxazole hydroxylation, but not biphenyl hydroxylation at 2-, 3- or 4-carbon, aldrin epoxidation to dieldrin or coumarin hydroxylation or aminopyrine N-demethylation. Enzyme activities were inhibited by alpha-naphthoflavone, but to a much lesser extent by SKF 525-A or metyrapone. Correlations between the metabolism of benzo(a)pyrene, 7-ethoxycoumarin and 2,5-diphenyloxazole were highly significant. There was a clear difference in Michaelis-Menten constant of 7-ethoxycoumarin O-deethylation between placentas from smokers and nonsmokers. Gel electrophoresis revealed that protein bands of placental microsomes in the region of cytochrome P-450 enzymes were less prominent than those of rat liver microsomes, a finding that accorded with the relative amounts of cytochrome P-450. There were no consistent differences in the electrophoretic pattern between placentas of variable benzo(a)pyrene hydroxylase activities. Results show that the human placental monooxygenase system is restricted in substrate specificity, that there may be a qualitative difference between smokers and nonsmokers and that the increase in several enzyme activities by cigarette smoking cannot be detected by the standard gel electrophoresis.     

Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and metabolism of benzo(a)pyrene

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.     

Positive effectors of the binding of an active site-directed amino steroid to rabbit cytochrome P-450 3c

The binding of the amino steroid, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), to rabbit liver cytochrome P-450 3c was studied using purified P-450 3c and liver microsomes prepared from rifampicin-treated B/J rabbits. 22-ABC binds to purified cytochrome P-450 3c producing a type II spectral change reflecting the coordination of the amine with the heme iron of the protein. In the absence of allosteric effectors, the binding is characterized by a Ks of 5 microM. In the presence of alpha-naphthoflavone or progesterone, the Ks decreases to 0.8 microM, indicating that these two compounds serve as positive effectors of the binding of 22-ABC to cytochrome P-450 3c. The antibiotic rifampicin induces cytochrome P-450 3c in rabbit liver microsomes, and the benzo(a)pyrene hydroxylase, estradiol 2-hydroxylase, and progesterone 6 beta-hydroxylase activities of these microsomes are stimulated by alpha-naphthoflavone. Moreover, the progesterone 6 beta-hydroxylase activity catalyzed by these microsomes exhibits a dependence on substrate concentration that is consistent with activation of the enzyme by the substrate, progesterone. The magnitude of the type II spectral change elicited by 22-ABC for microsomes prepared from rifampicin-treated B/J rabbits is greater than that observed for microsomes from untreated rabbits. For microsomes from rifampicin-treated rabbits, the apparent binding constant for 22-ABC was decreased 5-fold in the presence of alpha-naphthoflavone. We propose that the effects of alpha-naphthoflavone and progesterone on the binding of 22-ABC to cytochrome P-450 3c mimic the effects of the two positive effectors on the metabolism of substrates by increasing the affinity of the enzyme for substrate.