The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Oxidation of arylcyclopropanes by rabbit liver cytochrome P-450. Mechanistic implications of a multiple oxidation.

The arylcyclopropanes (cyclopropylarenes) cyclopropylbenzene and diphenylcyclopropane are oxidized by rabbit liver microsomal cytochrome P-450, both by the microsomal fraction and by the purified cytochrome in a reconstituted system. The products formed, principally benzoic acid, are due to an unusual triple oxidation of the substrate, which probably remains attached to the active site during the several steps of the oxidation. Both substrates were found to be inhibitors of the cytochrome P-450-dependent O-de-ethylation of 7-ethoxycoumarin. Model oxidation studies with cumene hydroperoxide as oxidizing agent and rabbit liver microsomal fraction as source of enzyme gave similar products to the microsomal and reconstituted systems. The significance of these results in the mechanism of oxidation catalysed by cytochrome P-450 is discussed.     

The direct oxidation of ethanol by a catalase- and alcohol dehydrogenase-free reconstituted system containing cytochrome P-4501.

Ethanol oxidation activity has been reconstituted in a system composed of NADPH-cytochrome c reductase, synthetic dilauroylglycerol-3-phosphorylcholine and cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats. This system is free of alcohol dehydrogenase and catalase activities. Furthermore, sodium azide (1 mm), a catalase inhibitor, is without effect on ethanol metabolism. There is a requirement for both NADPH-cytochrome c reductase and cytochrome P-450 and a partial requirement for phospholipid for ethanol oxidation by the reconstituted system. In addition, both NADPH and O₂ are required for catalysis. Under optimal reaction conditions, the rate of acetaldehyde formation if 25 to 50 nmol/min/nmol of cytochrome P-450. Cytochrome P-450 from other sources, including the homogeneous P-450LM₂ from phenobarbital-treated rabbits, have also been found to catalyze ethanol oxidation in reconstituted systems. Antibody prepared against cytochrome P-450 inhibits ethanol metabolism in the reconstituted system consistent with a cytochrome P-450-mediated reaction. Furthermore, cumene hydroperoxide can replace both NADPH and NADPH-cytochrome c reductase in ethanol oxidation and catalysis can be demonstrated in a system composed of only cytochrome P-450, lipid, ethanol, and cumene hydroperoxide. These data implicate cytochrome P-450 in the direct oxidation of ethanol by this system.     

Possible higher valence states of cytochrome P-450 during oxidative reactions

The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the function of cytochrome P-450

The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide. The stoichiometry of lipid peroxidation and the yields of 2-phenyl-2-propanol (a major product of the reaction) and acetophenone (a minor product) observed with liver microsomes prepared from untreated rats is greater than that seen with liver microsomes from ciprofibrate-treated rats which, in turn, is greater than that observed with liver microsomes from phenobarbital-treated rats. The Km's and Vmax's of oxygen uptake varied with the type of rat liver microsomes used. Cytochrome P-450 substrates and inhibitors decreased the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation. A mechanism is proposed involving the cytochrome P-450-catalyzed homolytic cleavage of the cumene hydroperoxide O-O bond to give the cumyloxyl radical. It is proposed that this oxygen-centered radical abstracts a hydrogen atom from an unsaturated fatty acid associated with a lipid (initiating lipid peroxidation) to give 2-phenyl-2-propanol or that the radical undergoes beta-scission to produce acetophenone and a methyl radical.     

Oxidation of ethanol by cumene hydroperoxide in the presence of cytochrome P-450 LM2 and hemoglobin

Ethanol oxidation by cumene hydroperoxide (CHP) with participation of cytochrome P-450 LM-2 (pH 7.4) and hemoglobin (pH 7.0) was studied at 37 degrees C in phosphate buffer. Both hemoproteins form complexes with CHP that are decomposed with the liberation of the RO2., RO. and HO. radicals, thus initiating the chain oxidation of ethanol. Ethanol oxidation catalyzed by cytochrome P-450 LM-2 and hemoglobin occurs only through a radical formation and is competitively inhibited by the radical scavenging agents, e.g., 1-naphthol, thiourea, mannitol and dimethylsulfoxide (DMSO). The values of effective inhibition constants were determined for all antioxidants whose activity decreases in the following order: 1-naphthol greater than thiourea greater than mannitol greater than DMSO. The non-inhibited oxidation of ethanol in "CHP-hemoproteins" systems is characterized by low ethanol conversion because of bimolecular termination of radicals and biocatalyst destruction.     

Ethanol oxidation by components of rat liver microsomes

A new method was employed for the purification of cytochrome P-450 from rat liver microsomes. The purified cytochrome was essentially free from possible contaminants and the recovery and degree of purification were high. Although 15% of the original P-450 was recovered through the purification procedure used, only 0.8% of the total original microsomal ethanol oxidation activity was associated with this fraction. Addition of this purified fraction to other fractions isolated did not further stimulate ethanol oxidation. The component of rat liver microsomes that was found most efficient in the oxidation of ethanol was the mixture of catalase and NADPH - cytochrome c - reductase. It is concluded that highly purified cytochrome P-450 by itself does not oxidize ethanol to any appreciable degree.     

Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus.

The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

Covalent binding to apoprotein is a major fate of heme in a variety of reactions in which cytochrome P-450 is destroyed

The heme in rat liver microsomal cytochrome P-450 was labeled with 14C or 3H and the microsomes were fractionated after in vitro incubations with a variety of agents known to destroy cytochrome P-450 heme. A major fraction of the heme label was irreversibly bound to apoprotein in all cases, including incubations with fluroxene, 1-octene, vinyl bromide, trichloroethylene, vinyl chloride, parathion, cumene hydroperoxide, NaN3, or iron-ADP complex. Label was also extensively bound to apoprotein when purified and reconstituted cytochrome P-450 was incubated with NADPH and vinyl chloride. This process appears to be widespread and involved to a significant extent in the cytochrome P-450 heme destruction observed with many compounds.     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Cytochrome P-450-mediated redox cycling of estrogens

The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro. Depending on the cofactor used, microsomal enzymes catalyzed reductions and/or oxidations of the estrogens: Phenobarbital-induced rat liver microsomes catalyzed the oxidation of DES to 4',4"-diethylstilbestrol quinone (DES quinone) with cumene hydroperoxide as cofactor. The quinone was unstable and spontaneously rearranged to (Z,Z)-dienestrol. DES quinone was reduced to a mixture of E- and Z-isomers of DES by NADPH catalyzed by purified cytochrome P-450 reductase. After rearrangement of the quinone to (Z,Z)-dienestrol, reduction reactions did not proceed. Rat liver microsomes and NADPH catalyzed the conversion of DES to (Z,Z)-dienestrol and (Z)-DES, but DES quinone could not be detected. The reactions described provide direct evidence for microsome-mediated redox cycling of estrogens. Although DES quinone could not be detected in the incubation of DES, microsomes, and NADPH as cofactor, the intermediacy of the quinone is demonstrated by the formation of (Z,Z)-dienestrol, the marker product for oxidation. The quinone could not be detected because it was rapidly reduced to DES and its Z-isomer. Microsome-mediated redox cycling between 2-hydroxyestradiol and the corresponding quinone was also demonstrated. Using cumene hydroperoxide as cofactor, the oxidation to the quinone was favored, while with NADPH as cofactor the reduction to 2-hydroxyestradiol was preferred. It is postulated that microsome-mediated redox cycling of estrogens plays a role in hormonal carcinogenesis.     

Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica

The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Role of cytochrome P-450IIE1 in N-nitroso-N-methylaniline induced hepatocyte cytotoxicity.

1. The cytotoxicity of N-nitrosomethylaniline (NMA) towards hepatocytes isolated from rats was prevented by acetone or ethanol (inhibitors for cytochrome P-450IIE1) but not by metyrapone or SKF525A (inhibitors for cytochrome P-450IIB1/2). Various alcohols, secondary ketones and isothiocyanates that induced cytochrome P-450IIE1 were also found to be protective. Various aromatic and chlorinated hydrocarbon solvents that are substrates or inducers of cytochrome P-450IIE1 also prevented NMA cytotoxicity. Nitrogen-containing heterocycles that induced cytochrome P-450IIE1 were less effective. Further evidence that cytochrome P-450IIE1 was responsible for the activation of NMA was the marked increase in hepatocyte susceptibility if hepatocytes from pyrazole-induced rats were used. 2. NMA was more cytotoxic to hepatocytes isolated from phenobarbital-pretreated rats than uninduced rats. However, metyrapone now prevented and SKF525A delayed the cytotoxicity whereas ethanol, acetone, allyl isocyanate, isoniazid or trichloroethylene had no effect on the susceptibility of phenobarbital-induced hepatocytes. Furthermore, microsomes isolated from phenobarbital-pretreated rats had higher NMA-N-demethylase activity which was more inhibited by metyrapone and SKF525A than that of uninduced microsomal activity. By contrast the N-demethylase activity of phenobarbital induced microsomes was more resistant to acetone, ethanol, hexanal, trichloroethylene and toluene than uninduced microsome. 3. The above results suggest that cytochrome P-450IIE1 catalyses the cytotoxic activation of NMA in normal or pyrazole-induced hepatocytes whereas cytochrome P-450IIB1/2 is responsible for cytotoxicity in phenobarbital-induced hepatocytes.     

Significant pathways of hepatic ethanol metabolism.

Rat liver microsomes oxidized ethanol two to three times faster than propanol when incubated with either an NADPH- or an H2O2-generating system. In addition, solubilized, purified microsomal subfractions were found to contain protein with an electrophoretic mobility identical to rat liver catalase on SDS polyacrylamide gels, suggesting that the separation of catalase from cytochrome P-450 and other microsomal components may not be feasible. These data support the postulate that catalase is responsible for NADPH-dependent microsomal ethanol oxidation. Direct read-out techniques for pyridine nucleotides, the catalase-H2O2 complex, and cytochrome P-450 were utilized to evaluate the specificity of inhibitors of alcohol dehydrogenase (4-methylpyrazole; 4 mM) and catalase (aminotriazole; 1.0 g/kg) qualitatively in perfused rat livers. 4-Methylpyrazole and aminotriazole are specific inhibitors for alcohol dehydrogenase and catalase, respectively, under these conditions. Neither inhibitor nor a combination of them altered the mixed function oxygen of p-nitroanisole to p-nitrophenol as observed by oxygen uptake and product formation. When ethanol utilization was measured over the concentration range 20-80 mM in perfused liver, a concentration dependence was observed. At low concentrations of ethanol, ethanol oxidation was almost totally abolished by 4-methylpyrazole; however, the contribution of 4-methylpyrazole-insensitive ethanol uptake increased as a function of ethanol concentration. At 80 mM ethanol, ethanol utilization was nearly 50% methylpyrazole-insensitive. This portion of ethanol oxidation, however, was abolished by aminotriazole. The data indicate that alcohol dehydrogenase and catalase-H2O2 are responsible for hepatic ethanol oxidation. At low ethanol concentrations (less than 20 mM), alcohol dehydrogenase is predominant; however, at higher ethanol concentrations (up to 80 mM), the contribution of catalase-H2O2 to overall ethanol utilization is significant. No evidence that the endoplasmic reticulum is involved in ethanol metabolism in the perfused liver emerged from these studies.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.