Mechanisms of hydroxylation by cytochrome P-450: Exchange of iron-oxygen intermediates with water

Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from ¹⁸O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H₂¹⁸O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from ¹⁸O₂ into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O₂ plus NADPH.     

High activity of cytochrome P-450-linked aminopyrine N-demethylase in mouse brain microsomes, and associated sex-related difference.

The presence of cytochrome P-450 and associated mono-oxygenase activities was examined in brain microsomes from male and female mice. Although the cytochrome P-450 level in male mouse brain was very low as compared with mouse liver, the aminopyrine N-demethylase and morphine N-demethylase specific activities in male mouse brain were much higher than those observed in mouse liver. Ethoxycoumarin O-de-ethylase and aniline hydroxylase activities were, however, not detected in mouse brain. Sex-related differences were observed in both the cytochrome P-450 levels and aminopyrine N-demethylase activity in mouse brain, the levels of both being higher in male mouse brain as compared with female mouse brain. Aminopyrine N-demethylase activity in mouse brain microsomes was dependent on the presence of oxygen and NADPH and could be inhibited by piperonyl butoxide, N-octyl imidazole and carbon monoxide. Antiserum raised to the phenobarbital-inducible form of rat liver cytochrome P-450 [P-450(b+e)] inhibited mouse brain aminopyrine N-demethylase activity by around 80+ mouse brain microsomal protein exhibited cross-reactivity against this antiserum when examined by Ouchterlony double diffusion and immunoblotting. The present results indicate the presence of a phenobarbital-inducible form of cytochrome P-450 (or a form of cytochrome P-450 that is similar immunologically) in mouse brain microsomes, which is associated with a sex-related difference.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

Selenium and hepatic microsomal hemoproteins

The microsomal share of liver homogenate ⁷⁵Se after injection of a tracer dose of ⁷⁵SeO₃^2? was three times greater in rats fed a selenium-deficient diet than in rats fed a selenium-adequate diet. Basal levels of microsomal cytochromes P-450 and b₅ were unaffected by selenium deficiency. However, induction of these cytochromes by phenobarbital was markedly inpaired in selenium-deficient rats, whereas liver weight increase and NADPH cytochrome $\text{c}$ reductase induction were not impaired. These data indicate that selenium is essential for phenobarbital induction of microsomal hemoproteins.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

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.     

Specificity of antisera directed against rat liver cytochrome P-448

A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b₅, NADH-cytochrome b₅ reductase, NADPH-cytochrome c reductase or epoxide hydrase.     

Aniline is hydroxylated by the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism

Hydroxylation of aniline, catalyzed by rabbit liver microsomal cytochromes P-450 in reconstituted systems, was inhibited by catalase, superoxide dismutase, catechol, mannitol, hydroquinone, dimethylsulfoxide and benzoate, whereas the cytochrome P-450-catalyzed O-demethylation of paranitroanisole, measured under the same conditions, was unaffected by these agents. A similar inhibition profile of the hydroxylation reaction was observed in reconstituted systems where cytochrome P-450 had been replaced by hemoglobin. The results indicate that aniline hydroxylation is mediated by hydroxyl radicals generated in an iron-catalyzed Haber-Weiss reaction between O₂^? and H₂O₂ and may explain some of the special properties of this reaction previously described.     

Highly purified microsomal P-450: the oxyferro intermediate stabilized at low temperature.

The oxyferro intermediate of highly purified microsomal P-450 from rabbit liver was formed and stabilized at ?30°C in a mixture of aqueous buffer and glycerol ($\text{1}\text{1}$). Absolute and difference (Fe^2·+O₂-Fe³⁺) spectra of this intermediate appear to be very similar to those obtained under either steady state kinetics or stopped flow conditions on the same cytochrome as well as on bacterial P-450_cam. (Absolute and difference spectra present maxima at 420 and 557–558 nm and a broad maximum at 442 nm respectively). As temperature increases the oxyferro intermediate autoxidizes and ferric cytochrome P-450 is restored. This reaction appears to follow biphasic first order kinetics. The rate constant of both phases decreases with temperature and increases with protons concentrations.     

Suicidal inactivation of microsomal cytochrome P-450 by halothane under hypoxic conditions

Under anaerobic conditions the addition of halothane to NADPH-reduced liver microsomes from phenobarbital-pretreated male rats resulted in a pronounced inactivation of microsomal cytochrome P-450, presumably produced by covalent binding of reactive halothane metabolites such as the CF₃CHCl-radical. Compared with microsomes from phenobarbital-pretreated rats, the loss of active cytochrome P-450 was markedly decreased in microsomes from both 3-methylcholanthrene-pretreated and untreated rats. Increasing the O₂-partial pressure decreased the amount of cytochrome P-450 inactivated by halothane metabolites. At an O₂-partial pressure of approximately 40 mm Hg the inactivation was virtually eliminated.     

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

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

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

Carbon tetrachloride activation in liver microsomes from rats induced with 3-methylcholantrene

The irreversible binding of¹⁴C from¹⁴CCl₄ to microsomal lipids is decreased in animals treated with 3-methylcholantrene (3-MC), while it is increased in animals induced with phenobarbital (PB). CCl₄-induced lipid peroxidation in 3-MC treated rats is as intense as in controls. Destruction of glucose 6-phosphatase (G6P-ase) by CCl₄ is smaller in 3-MC treated rats than in controls. Destruction of total cytochrome P-450 (P-450 + P₁-450) by CCl₄ is smaller in 3-MC treated than in PB treated rats but similar to that obtained in controls. Results would indicate that P-450 would participate in CCl₄ activation much more effectively than P₁-450.     

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

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

The degradation of different forms of cytochrome P-450 in vivo by fluroxene and allyl-iso-propylacetamide.

Treatment of uninduced, phenobarbital and 3-methylcholanthrene induced rats with fluroxene and allyl-$\text{iso}$-propylacetamide decreased hepatic microsomal cytochrome P-450 and equivalently decreased microsomal heme, aniline binding and $\text{p}$-nitroanisole demethylase. In contrast, ethylmorpnine demethylase, benzpyrene-3-hydroxylase and ethoxyresofurin deethylase were not in all cases decreased in proportion to the loss of cytochrome P-450. After phenobarbital induction fluroxene and allyl-$\text{iso}$-propylacetamide degrade multiple forms of cytochrome P-450, but degrade in the greatest amounts the form(s) of cytochrome P-450 inducible by phenobarbital. After 3-methylcholanthrene induction fluroxene preferentially degrades cytochrome P-448, while allyl-$\text{iso}$-propylacetamide is relatively specific for the form(s) of cytochrome P-450 inducible by phenobarbital.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

Effect of a single dose of inducers and inhibitors on the rate of synthesis of cytochromes and reductases in liver organelles

Summary Measurement of the effect of drugs on the in vivo rates of synthesis of rabbit liver organelle bound proteins were measured following individual treatments with the inducers phenobarbital, 3-methylcholanthrene and PCB (a mixture of polychlorinated biphenyls) and the inhibitors, cycloheximide, aflatoxin 13, chloramphenicol and actinomycin D. Following their isolation from a homogenate containing the combined livers of ¹⁴C-leucine injected experimental animals and 3H-leucine injected control animals, purified fractions of the following proteins were prepared: microsomal cytochrome b₅, cytochrome P-450, NADH-cytochrome b₅ reductase, NADPH-cytochrome P-450 reductase and proteolipids, outer mitochrondrial membrane cytochrome b5, NADH-cytochrome b₅ reductase and proteolipids, inner mitochrondrial membrane cytochrome c, NADH dehydrogenase and proteolipids, intermitochrondrial membrane cytochrome b₅ and circulating serum albumin.This research was supported by grants from the PSC-BHE Research Award Program of The City University of New York, US Public Health Service Grant SO 7 PRO7132-07 and the Alma Toorock Fund for Cancer Research.