P450 Monooxygenase System Research Articles

Oxidase and oxygenase function of the microsomal cytochrome P450 monooxygenase system.

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

Is thiobenzamide a specific substrate for the microsomal FAD-containing monooxygenase?

While the hepatotoxicant thiobenzamide is S-oxidized primarily by the microsomal FAD-containing monooxygenase, a significant part of the oxidation in mouse liver is due to the cytochrome P-450-dependent monooxygenase system. The oxidation of thiobenzamide by both the FAD-containing monooxygenase and the cytochrome P-450 monooxygenase system was demonstrated using purified enzymes from mouse liver. An inhibitor of the cytochrome P-450 monooxygenase system reduced the overall oxidation of thiobenzamide by approximately 30 percent in microsomes from uninduced mouse liver. One form of cytochrome P-450 purified from rabbit lung was also active in the oxidation of thiobenzamide.

Sequential administrations of polyriboinosinic acid and polyribocytidylic acid induce interferon and depress the hepatic cytochrome P-450-dependent monooxygenase system

Double-stranded polyriboinosinic acid·polyribocytidylic acid is a potent interferon inducing agent and depressant of hepatic cytochrome P-450 monooxygenase systems. Single-stranded polyriboinosinic acid or polyribocytidylic acid are not. However, it is known that interferon is induced in mice when the administration of polyriboinosinic acid is followed shortly thereafter by the administration of polyribocytidylic acid. The current study demonstrates that this sequential administration of single-stranded polynucleotides induces serum and hepatic interferon and depresses the cytochrome P-450 systems. Neither of these effects were seen when the order of administration of these polynucleotides was reversed.

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in [formula omitted

When grown in high concentrations of glucose, the yeast Saccharomyces cerevisiae produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes P-448 P-450 in yeast are briefly discussed.

Heterogeneous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes.

Enzyme distribution in rat liver lobes was investigated using liver homogenate as the enzyme source. When the content or the activity was expressed on the basis of unit (gram) wet weight of liver tissue, the protein concentration was almost the same throughout the liver, but several enzymes were distributed heterogeneously within the liver. Cytochrome P-450 monooxygenase activity was higher in the median and right lobes of livers, while the left lobe contained a higher concentration of mitochondrial enzymes. Phenobarbital induced cytochrome P-450 monooxygenase, and the activity was still higher in the median and right lobes even after the pretreatment of rats with phenobarbital. On the other hand, administration of beta-naphthoflavone resulted in a uniform increase of cytochrome P-450 (P-448) content throughout the liver to almost the same concentration. Increase of cytochrome P-448-dependent O-dealkylation activity of 7-alkoxycoumarin by the administration of beta-naphthoflavone was much greater in the left lobe compared to that in the median and the right lobes. Heterogeneous distribution of the enzymes in various liver lobes was also determined employing liver samples obtained from various physiological states of rats (fasting, glucose refeeding after fasting and hyperthyroidism), although the cytochrome P-450 content and drug-metabolizing activity were altered markedly depending upon the physiological states of the rats.

Metabolism of chlorpromazine by pulmonary microsomal enzymes in the rat and rabbit

Pulmonary metabolism of chlorpromazine (CPZ) was compared using isolated microsomes of rat and rabbit lungs. CPZ-metabolizing activity of the rat lung was found to be 10-fold higher than that of the rabbit lung. The principal metabolic pathways were N-oxidation in the rat lung and N-demethylation in the rabbit lung. Kinetic analyses revealed that, although the values for apparent K m were roughly similar for both pathways, V max for N-oxidation by the rat lung was approximately ten times greater than that for N-demethylation by the rabbit lung. N-Oxidation by the rat lung had a broad range of pH optimum of 7–8, whereas N-demethylation by the rabbit lung had a pH optimum 8–9. SKF525-A, piperonyl butoxide, n-octylamine and CO did not inhibit N-oxidation by the rat lung, but inhibited N-demethylation by the rabbit lung. SKF525-A and n-octylamine stimulated the CPZ- N-oxidation by the rat lung. Hg 2+ and Mg 2+ inhibited N-oxidation by the rat lung. These results indicate that pulmonary metabolism of CPZ in the rat is catalyzed by a microsomal flavoprotein monooxygenase, while pulmonary metabolism in the rabbit is catalyzed by a cytochrome P-450 monooxygenase system, and that a marked species variation exists with respect to pulmonary metabolism of CPZ.

Caffeine metabolism in liver slices during postnatal development in the rat

The metabolism of caffeine was investigated in liver slices of young and adult rats. Liver slices from adult male rats metabolized caffeine at an initial rate of 48.31 ± 3.71 nmoles · (g liver) −1 · hr −1 to four main metabolite fractions. By a combination of thin-layer radiochromatography and high performance liquid chromatography, theophylline, paraxanthine and 1, 3, 7-trimethyldihydrouric acid were identified as caffeine metabolites. Apparent V max of the overall reaction was 83.30 nmoles caffeine metabolites formed · (g liver) −1 · hr −1. Theophylline competitively inhibited caffeine metabolism [the apparent K m was 19.20, μM in the absence of theophylline, the apparent k i was 36.50 μM in the presence of theophylline (100 μM)]. SKF 525-A inhibited caffeine metabolism; the formation of all of the metabolite fractions was inhibited to a similar extent. Allopurinol (100 μM) had no effect. The specific activity of the enzyme system was extremely low when liver slices of 2-day-old-rats were used [1.46 ± 0.08 nmoles caffeine metabolites formed · (g liver) −1 · hr −1]; the reaction velocity increased gradually with increasing age and reached a peak [52.26 ± 1.41 nmoles caffeine metabolites formed · (g liver) −1 · hr −1]at 30 days of age. Changes in the formation of the four metabolite fractions with age followed the pattern of the overall caffeine metabolism. These results demonstrate that the liver of the newborn rat has an extremely limited capacity to metabolize caffeine in vitro and are consistent with the proposed involvement of the liver microsomal cytochromes P-450 monooxygenase system in the metabolism of caffeine. N-Demethylation is the main pathway of in vitro caffeine metabolism in the rat liver at all ages.

Metabolic activation and cytotoxicity of cyclophosphamide in primary cultures of postnatal rat hepatocytes

We have developed a model of primary cultures of postnatal rat hepatocytes to characterize the metabolic activation of xenobiotics to toxic intermediates and to study the mechanism(s) by which these chemicals produce cellular injury. This model was employed to investigate the cytochrome P-450 mediated biotransformation of cyclophosphamide (CP) to cytotoxic metabolites that nonspecifically alkylate DNA and cellular proteins. The parenchymal cells were isolated by an in situ collagenase perfusion technique and cultured for 24 hr prior to drug treatment. The cultures were then exposed to CP concentrations ranging from 1 × 10 −4 M to 1 × 10 −3 M for 24 hr. Initial studies indicated minimal toxicity to non-replicating parenchymal hepatocytes maintained in ornithine-supplemented, arginine-deficient medium. The addition of arginine permitted the overgrowth of fibroblasts in the same culture system. These fibroblasts then became the target of alkylating CP metabolites produced by the par-enchymal cells. By day 3 after CP administration, cell number and total protein per dish decreased by over 40 percent. The morphology of the cultures changed dramatically because of fibroblast destruction. The cytotoxicity to dividing fibroblasts was eliminated by administering 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF 525-A), an inhibitor of the cytochrome P-450 monooxygenase system, to the co-cultures treated with CP. The alkylating metabolites of CP produced by the parenchymal cells and released into the culture medium were quantitated by reacting aliquots of medium from CP-treated cells with 4-( p-nitrobenzyl)pyridine. These results provide both direct and indirect evidence of drug metabolism in cultured cells and suggest that this co-culture system can be utilized to evaluate the metabolic activation of xenobiotics.

Abstracts of papers presented at the Twenty-first Annual Meeting of the Japanese Teratology Society. Kumamoto, Japan, July 10-11, 1981.

Cyclophosphamide must be enzymatically activated to be either mutagenic or teratogenic. This activation is thought to be catalyzed by the cytochrome P-450 monooxygenase system. To study the relationship between the mutagenic and teratogenic metabolites of cyclophosphamide, the mutagenicity and teratogenicity of this drug were compared after activation by rats pretreated with chemicals (phenobarbital and beta-naphthoflavone) inducing different cytochromes P-450. Activation of cyclophosphamide to mutagenic metabolites by enzyme fractions from rats on day 13 of gestation was measured with the Ames test using Salmonella typhimurium TA1535. Teratogenicity was assessed in vivo by treatment of rats with cyclophosphamide on day 13 of gestation. Cyclophosphamide was activated to mutagenic metabolites to the same extent (on a tissue wet weight basis) by enzyme fractions from maternal liver, kidney and placenta, despite differences in cytochrome P-450 content. Fetal homogenates did not activate cyclophosphamide. Phenobarbital pretreatment increased the activation of cyclophosphamide to mutagenic metabolites by maternal liver microsomes 10-fold and liver cytochrome P-450 content 1.8 fold; however, this drug did not alter the activation of cyclophosphamide by maternal kidney, by placenta or by the fetus. Phenobarbital pretreatment increased the teratogenicity of cyclophosphamide in rats on day 13 of gestation (increased incidence of malformed embryos, decreased fetal weight). Pretreatment with beta-naphthoflavone did not induce liver cytochrome P-450 in the pregnant rat and did not change the activation of cyclophosphamide to mutagenic metabolites by liver, kidney, placenta or the fetus. Pretreatment with this polycyclic aromatic hydrocarbon had no effect or decreased the teratogenicity of cyclophosphamide. Thus, these experiments suggest that the mother, rather than the fetus, is the site of activation of cyclophosphamide; after phenobarbital pretreatment the predominant site of cyclophosphamide activation is the maternal liver. There appears to be a correlation between the teratogenicity and mutagenicity of cyclophosphamide after induction of the cytochromes P-450. We can speculate that the "proximate teratogen" of cyclophosphamide may also be the "proximate mutagen".

Effects of in vivo carbon disulfide administration on the hepatic monooxygenase systems of microsomes from rats

In vivo administration of carbon disulfide to fasted rats leads to the disappearance of 35% of liver microsomal P-450 hemoproteins measured by the CO-binding spectrum of dithionite-reduced microsomes. P-450 hemoprotein disappearance is not accompanied by any change in the other components of P-450 monooxygenase systems (i.e., cytochrome P-420, cytochrome b 5 and NAD(P)H reductases). The spectrophotometrically remaining P-450 hemoproteins after carbon disulfide administration have increased peroxidase activity, increased apparent K S and A max for hexobarbital and aniline bindings, and decreased aniline hydroxylase activity. The lower wavelength peak of pyridine binding is increased whereas the higher wavelength peak of the binding is decreased at all pH values used. The mechanism by which CS 2 leads to these effects is suggested to be an alteration of a CS 2-sensitive population of liver P-450 hemoproteins. This, however, remains to be demonstrated.

Hydroxylation of 9-hydroxystearate by a soluble cytochrome P-450-dependent fatty acid hydroxylase from [formula omitted

A soluble cytochrome P-450 monooxygenase system from Bacillus megaterium ATCC 14581, previously shown to catalyze the monohydroxylation of longchain unsubstituted fatty acids, has now been found to convert 9-D-hydroxystearate to a mixture of ω-1, ω-2, ω-3 and ω-4 dihydroxystearate isomers. 9-D-Hydroxystearate has a significantly higher affinity than stearate for the enzyme and is a strong competitive inhibitor of palmitate hydroxylation. These results suggest that the enzyme surface has a non-hydrophobic, sterically-permissive binding region between the methyl-group and carboxyl-group binding sites that interacts with polar substituents near the middle of the substrate chain.

Modulation of 25-hydroxyvitamin D 3-24-hydroxylase by aminophylline: A cytochrome P-450 monooxygenase system

The chick kidney mitochondrial 25-hydroxyvitamin D 3-24-hydroxylase is a cytochrome P-450 catalyzed monooxygenase system. The administration of aminophylline [3,7-Dihydro-1,3-dimethyl-1H-purine-2,6-dione compounded with 1,2-ethanediamine (2:1)], a cyclic nucleotide phosphodiesterase inhibitor, to vitamin D 3-sufficient chicks at a dose of 78 mg/kg body weight increased the 24-hydroxylase activity by 10-fold without a corresponding increase in the kidney mitochondrial cytochrome P-450 concentration. The product of the reaction comigrated with authentic 24,25-dihydroxyvitamin D 3 in high pressure liquid chromatography and was quantitatively sensitive to periodate oxidation cleavage. The increase in the enzyme activity occurred while serum calcium concentration fell from 14 to 9 mg%. Serum phosphate concentration remained unchanged. These results support the idea that the regulation of kidney 24-hydroxylase activity may be mediated by tissue levels of cyclic nucleotides.

Distribution of polycyclic hydrocarbon metabolism-linked enzymes in specialized regions of the endoplasmic reticulum

Microsomal enzymes were partially separated by centrifugation in continuous sucrose gradients containing a low concentration of detergent into four bands in which the following enzymes were enriched: (I) glucose-6-phosphatase ( d-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9); (II) cytochrome P-450, NADPH-cytochrome c reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4), monooxygenase ( 7 -ethoxycoumarin-O -deethylase) ; (III) cytochrome b 5 , NADH cytochrome c reductase (NADH:ferricytochrome oxidoreductase, EC 1.6.2.2), epoxide hydratase (glycol hydro-lyase (epoxide-forming), EC 4.2.1.63), and UDPglucuronyltransferase (UDPglucoronate β-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17); (IV) glutathione-S -transferases (RX:glutathione R-transferase, EC 2.5.1.18). The distribution of epoxide hydratase in the active fractions was identical whether determined with styrene oxide or benzo(a)pyrene-4,5-oxide as substrate giving further support to the conclusion that both substrates are hydrated by the same enzyme. The fact that epoxide hydratase, UDPglucuronyltransferase and microsomal glutathione-S- transferase activities were not enriched in the same fractions as the cytochrome P-450 monooxygenase system argues against a firm spatial association of the bulk of any of the former enzymes with the bulk of the monooxygenases system, although they catalyze consecutive reactions. Inclusion of antibodies to epoxide hydratase in the sucrose gradients inhibited the epoxide hydratase activity but did not affect monooxygenase activity which again suggests spatial independence of these enzymes.

Stimulation of caffeine metabolism in the rat by 3-methylcholanthrene

Groups of adult male rats treated with 3-methylcholanthrene, phenobarbital or vehicles alone, were administered caffeine either orally or intravenously. Serum caffeine concentrations were measured by radioimmunoassay. In vehicle and phenobarbital pretreated animals, caffeine elimination kinetics were non-linear. In control animals, the in vivo apparent Km was 8 μg·ml −1 (40 μM) and the apparent Vmax was 0.1 μg·ml −1·min −1 (0.5 μM·min −1). Phenobarbital pretreatment did not change the apparent Km but slightly increased the apparent Vmax. 3-Methylcholanthrene pretreatment dramatically altered the elimination kinetics of caffeine, whether caffeine was given orally or intravenously. The elimination of caffeine from serum of 3-methylcholanthrene pretreated rats was first order with a t 1 2 of approximately 14 minutes. Our results are consistent with the proposed involvement of the cytochromes P-450 monooxygenase system in the elimination of caffeine. In addition, our results suggest that caffeine is a moderately poor substrate for the cytochromes P-450 present in control and phenobarbital-pretreated rats, but a particularly good substrate for the form(s) induced by 3-methylcholanthrene.

Determination of aminocresol isomers by high-speed liquid chromatography

Aminocresol isomers (4-hydroxy-m-toluidine [II], 3-hydroxy-p-toluidine [II], 2-hydroxy-p-toluidine [III]) and p-aminophenol have been separated and determined by a high-speed liquid Chromatographie method. Since this method is applicable in aqueous media, it was used to investigate the suitability of a haemin-cysteine system as a model for the cytochrome P-450 mono-oxygenase system, by determination of the [I], [II], [III] and p-aminophenol formed.

Developmental aspects of the hepatic cytochrome P450 monooxygenase system.

Developmental aspects of the hepatic cytochrome P450 monooxygenase system.

Metabolism of prostaglandin A1 by hepatic microsomal monooxygenase P-450 system in the guinea pig and rat.

Abstract This study demonstrates the metabolic transformation of prostaglandin A 1 (PGA 1 ) by guinea pig and rat liver microsomes. The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products. Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones. It was demonstrated that PGA 1 metabolism in the guinea pig and the rat was inhibited by the addition of SKF-525A, metyrapone, carbon monoxide and cytochrome C ; nicotinamide (10 mM) inhibited only the guinea pig system. These findings indicate that the enzymatic activity responsible for PGA 1 metabolism is composed of a typical cytochrome P-450 monooxygenase system.

Reversal of the inhibitory effect of lipid peroxides on the hepatic cytochrome P-450 monooxygenase system by a soluble factor from liver and a commercial isocitric dehydrogenase preparation from hog heart

Summary A factor (SF) contained in the 105,000 × g supernatant fraction of rat liver homogenate, previously described as a requirement for the maximal activity of the cytochrome P-450-dependent monooxygenase system of hepatic microsomes, was found to function by reversing the inhibitory effect of TPNH-supported microsomal lipid peroxidation. Commercial hog heart isocitric dehydrogenase preparations contain a large amount of SF or a substance like SF.

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGAI, PGA2, PGE 1 , PGE 2 , PGF 1α and PGF 2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K s ) were highest with PGA 1 and PGA 2 . With liver microsomes from control or 3-methylcholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a weak spectrum, designated here as type “II” was at times observed. With microsomes from phenobarbital (Pb)-treated rats only PGA 1 and PGA 2 yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA 1 and PGA 2 to liver microsomes from Pb-treated rats inhibited the microsomal mediated hydroxylation of hexobarbital. The inhibition by PGA 1 was competitive; the K i = 8.2 × 10 −4 M was found to be similar in magnitude to the K s = 7.3 × 10 −4 M of PGA 1 observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGAI, PGA2, PGE 1, PGE 2, PGF 1α and PGF 2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K s) were highest with PGA 1 and PGA 2. With liver microsomes from control or 3-methylcholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a weak spectrum, designated here as type “II” was at times observed. With microsomes from phenobarbital (Pb)-treated rats only PGA 1 and PGA 2 yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA 1 and PGA 2 to liver microsomes from Pb-treated rats inhibited the microsomal mediated hydroxylation of hexobarbital. The inhibition by PGA 1 was competitive; the K i = 8.2 × 10 −4 M was found to be similar in magnitude to the K s = 7.3 × 10 −4 M of PGA 1 observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.