Microsomal Monooxygenase System Research Articles

Detection of reactive metabolites in vitro

The ability of 27 compounds to mediate depletion of glutathione (GSH) in a fortified liver microsome incubation via production of reactive metabolites generated by microsomal mono-oxygenase (MMO) metabolism has been studied. The majority of compounds tested of this type were positive in this assay, with the exception of iproniazid and naphthalene. Thioacetamide, the reactive metabolite of which binds via lysine residues, was negative. Acrolein and hexachlorobutadiene produced depletion in the absence of prior activation, as did menadione but for which enzyme induction increased the magnitude of depletion. Allyl alcohol produced MMO-mediated depletion of GSH. Some depletion of GSH occurred in the presence of common substrates of the MMO system. It is suggested that this depletion assay may be a valid test for detection of reactive metabolites generated by MMO metabolism.

Developmental Changes in the Microsomal Monooxygenase System and the In Vivo Metabolism of Aldrin in Larvae of the Mexican Bean Beetle (Coleoptera: Coccinellidae)

In vivo metabolism of topically applied aldrin and the specific levels of whole body and abdominal microsomal cytochrome P-450, P-420, b5 and creductase were measured during development in the penultimate and last instar of the Mexican bean beetle, Epilachna varivestis Mulsant. There were no significant differences between molting larvae (from the penultimate to the last instar), last-instar feeding larvae and prepupae in these monooxygenase properties for whole-body microsomal preparations, and in respect to in vivo metabolism. Cytochrome P-450, b5, and creductase were higher in the abdominal microsomes of feeding-stage larvae than in whole-body microsomes from the same stage, and cytochrome P-450 and b5 were higher in the abdominal microsomes of feeding-stage larvae than in the abdominal microsomes from prepupae. The molar ratio of P-450 to P-420 was constant (approximately 1) for all microsomal preparations examined. The high tolerance of Mexican bean beetle larvae to topically applied aldrin could not be predicted based on poor penetration or the inability of aldrin to be epoxidized to dieldrin.

Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly ( Musca domestica)

Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica, Rutgers strain) were characterized. Cytochrome P-450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5. Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a]pyrene in microsomal preparations was high although neither O-dealkylation of 7-ethoxyresorufin nor N-dealkylation of p-chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p-nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P-450 fractions, purified house fly or rat liver NADPH-cytochrome P-450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P-450, phosphatidylethanolamine was more active than phosphatidylcholine.

Induction of cytochrome P-450 mRNA in rainbow trout: In vitro translation and immunodetection

The time course of induction of the raingow trout microsomal hepatic monooxygenase (MO) system was examined by determination of levels of mRNA and corresponding levels of catalytic activity. Animals were pretreated with β-naphthoflavone (β-NF, ip, 100 mg/kg) and terminated at 0, 2, 6, 18, and 48 hr postinjection. Levels of mRNA were determined by immunoprecipitation of in vitro translation products. Levels of mRNA coding for the cytochrome P-450 LM 4b isozyme were maximally increased (13-fold) at 18 hr and had decreased almost to pretreatment levels by 48 hr post-treatment. This was in contrast to the catalytic activity in which ethoxyresorufin- O-deethylase (EROD) and ethoxycoumarin- O-deethylase (ECOD) were significantly elevated at both 18 hr (25- and 5-fold, respectively) and 48 hr (46- and 8-fold, respectively). Pretreatment with β-NF (ip, 100 mg/kg) or 2,4,5,2′,4′,5′-hexachlorobiphenyl (6-CB, ip, 150 mg/kg) for 18 hr resulted in significant differences in levels of mRNA in only the β-NF-treated group. The LM 2 P-450 isozyme could not be detected by immunoprecipitation with anti-LM 2 IgG in trout treated with these same inducers. The results suggest a difference between the time course of induction of the mRNA for cytochrome P-450 LM 4b isozyme and the induction of catalytic activity. Under the detection system utilized, the results suggest that the phenobarbital-like inducer, 6-CB, does not induce cytochrome activity nor does it induce the mRNA for cytochrome P-450 LM 4b isozyme.

Cytochrome P450 induction by phenobarbital (PB) is inhibited by 12-O-tetradecanoylphorbol-13-acetate(TPA): Evidence that protein kinase C regulates induction

The hepatic microsomal monooxygenase system was studied in hypophysectomized male rats exposed for 24 or 48 h to PB and/or TPA, an activator of kinase C. TPA attenuated basal and PB-induced levels of P450, aniline hydroxylase (ANH), ethylmorphine demethylase (EDM) and cytochrome c reductase. Hence, PB may effect induction via the inhibition of kinase C. Supporting this is spectral evidence that PB and TPA do not bind and the fact that TPA did not decrease P450 when co-incubated with O2 and NADPH. Hemin failed to increase P450 levels previously depressed by TPA indicating that TPA acts by lowering apocytochrome levels. This is consistent with its attenuation of PB-effected increases in hepatic RNA. TPA effects were associated with increased hepatic RNA and were blocked by puromycin.

Quantitative structure-activity relationship of coumarin derivatives : Involvement of partition between aqueous and membrane phase

In order to understand the substrate behaviour of several 7-alkoxycoumarins and 7-alkoxy-4-alkylcoumarins towards the liver microsomal monooxygenase system, their lipophilic properties have been examined. As a model for the lipophilicity the reversed-phase liquid chromatographic retention parameter log k w has been used. In a system with methanol-water as the mobile and RP-18 (octadecylsilica) as the stationary phase, we found a quadratic relationship between the volume fraction of the organic solvent and the logarithm of the capacity factor (log k′). The extrapolation to a pure aqueous phase reveals a linear relationship of the theoretical capacity factor log k w with the chain length. This holds for 1–12 carbon atoms in the alkoxy chain and for zero to three carbon atoms in the alkyl chain. Moreover, the incremental effect of the methylene residues on the lipophilicity of the compounds (Δlog k w/ΔCH 2) is found to be 0.60 ± 0.01. If the coumarin derivatives are used as substrates for the liver microsomal monooxygenase system, no systematic dependence of the enzymic data (Michaelis-Menten constant K m) on the lipophilic data (log k w) can be demonstrated. The metabolism of these compounds by the microsomal monooxygenase system seems to be limited by the partition between the membrane and the aqueous phase. Whether other factors, e.g. the lateral diffusion of the substrates versus the membrane-bound enzyme system or enzyme active-site characteristics, govern the metabolism remains to be investigated.

Theophylline and antipyrine disposition in smoking and non-smoking epileptic subjects.

Theophylline and antipyrine disposition has been compared in smoking epileptic patients, non-smoking epileptic patients and non-smoking healthy volunteers. Although clear differences in drug clearance and half-life were evident as a result of anticonvulsant drug therapy, no effect of smoking was discernible. Thus, additive effects from induction of the hepatic microsomal monooxygenase system in man by anticonvulsant drugs and polycyclic aromatic hydrocarbons (in cigarette smoke) were not evident.

Genotoxic and mutagenic activation of aflatoxin B 1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B 1 (AFB 1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB 1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB 1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB 1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/ S. typhimurium TA98 system by activated metabolites of AFB 1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB 1 in rat liver microsomes.

Molecular, cellular and physiological effects of oil-derived hydrocarbons on molluscs and their use in impact assessment

The impact of pollutants on an organism is realized as perturbations at different levels of functional complexity. This presentation considers responses to oil-derived hydrocarbons at the molecular, subcellular, cellular and whole animal levels of organization, with particular emphasis on the use of marine molluscs as sentinel organisms for assessing pollutant effects. A number of biological effects measurements are described which have been used in the development of early-warning systems based upon reactions to hydrocarboninduced damage. These include those of the microsomal cytochrome P-450 dependent monooxygenase system involved in metabolism of organic xenobiotics, functional and structural responses of lysosomes to hydrocarbons, quantitative structural alterations in the cells of the digestive and reproductive systems and effects on physiological scope for growth and parallel correlations of this latter parameter with ecological parameters such as species diversity. Aspects of recovery processes are also considered. Examples of both laboratory and field studies are cited to illustrate both the application of these approaches and the functional integration of the responses at the various levels of biological organization. This ability to link the various parameters in a functional manner is believed to strengthen the rationale for their use in impact assessment.

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.

Inhibition of microsomal N-nitrosodimethylamine demethylase by diethyl ether and other anesthetics

The inhibitory actions of diethyl ether and several other anesthetics on the metabolism of N-nitrosodimethylamine (NDMA) and other substrates were studied with rat liver microsomes. Diethyl ether was an effective inhibitor of the low K m , NDMA demethylase, showing characteristics of a competitive inhibition. Inhibition of NDMA metabolism was also observed in the liver post-mitochondrial supernatant fraction prepared from ether-anesthetized rats. Selectivity in the inhibitory action of diethyl ether was demonstrated; the ether was most effective against NDMA demethylase, less potent against p-nitroanisole demethylase and N-nitrosomethylbenzylamine demethylase, and not effective against the metabolism of aminopyrine or benzphetamine. Other anesthetics such as chloroform, isoflurane, enflurane, and halothane also effectively inhibited NDMA demethylase. The work demonstrates that diethyl ether is an efficient inhibitor of NDMA metabolism by the microsomal mono-oxygenase systems.

Sex difference in responsiveness to aztreonam of monooxygenase system in liver microsomes from rats

Effect of successive administration of Aztreonam on microsomal monooxygenase system was investigated in male and female Sprague-Dawley rats. The activities of benzphetamine N-demethylase, aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes from male rats were decreased dose-dependently by Aztreonam. On the contrary, the activities in liver microsomes from female rats were slightly increased rather than decreased by the administration of Aztreonam. In addition, Aztreonam was found to decrease the specific content of microsomal cytochrome P-450 in male rats but not in female rats. The decreases in the activities observed in male rats were accompanied by a parallel decrease in the specific content of cytochrome P-450. Furthermore, the results of quantitation of P-450 (M-1), one of the male specific forms of cytochrome P-450, indicated that the administration of Aztreonam resulted in a dose-dependent decrease in the content of P-450 (M-1) in liver microsomes from male rats.

DIFFERENCE SPECTRAL CHARACTERIZATION OF MICROSOMAL CYTOCHROME P-450 FROM LARVAE OF MOSQUITOES(Culex pipiens pallens Coq.)

Cytochrome P-450 is widely distributed in different species of animals. As a terminal oxidase ot the microsomal monooxygenase system, cytochrome P-450 is the key element of the enzyme system. Because of its broad substrate specificity, it is involved in the biotransformation of many xenobiotic and endogenous compounds which are important in envitonmental protection, pharmacology and carcinogenicity. Microsomal P-450 is also a very

Methylglyoxal: genotoxicity studies and its effect in vivo on the hepatic microsomal mono-oxygenase system of the mouse.

The genotoxic potential of methylglyoxal (MG) was studied in Saccharomyces cerevisiae D7 and in Salmonella typhimurium TA97 and TA102 in the presence and in the absence of metabolic activation system (S9 fraction) prepared from mouse liver induced with beta-naphthoflavone (beta-NF) and sodium phenobarbital (PB). The in vivo effects on the hepatic microsomal mixed function mono-oxygenase system induced by MG were studied in untreated, beta-NF or PB pre-treated mice. MG was a direct-acting mutagen in S. typhimurium TA97 and TA102 when tested up to a maximum concentration of 0.47 mg/plate. Mitotic gene conversion was also induced by MG in the yeast S. cerevisiae D7. A weak but significant effect on reverse point mutation was also found in S. cerevisiae. Genetic activity was lower in the presence of S9 fraction in yeast test. In the in vivo studies, MG (at the total dose of 600 mg/kg) was shown to increase the aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities in uninduced mice. Cytochrome P-450 content (cyt P-450) and ethoxycoumarin O-deethylase activity (ECD) were also weakly enhanced by MG treatment. In contrast, no significant changes in mono-oxygenase activities were seen in beta-NF- or PB-treated mice after MG injection.

Effects of Aroclor 1254 on cytochrome P-450-dependent monooxygenase, glutathione S-transferase, and UDP-glucuronosyltransferase activities in channel catfish liver

Channel catfish ( Ictalurus punctatus) were treated with single intraperitoneal injections of Aroclor 1254, ranging from 1–100 mg Aroclor 1254/kg body wt, and effects of the Aroclor on several xenobiotic-metabolizing enzymes were evaluated. Hepatic microsomal monooxygenase (MO) activity toward 7-ethoxyresorufin, 7-ethoxycoumarin and benzo[ a]pyrene was increased in a dose-dependent manner. 7-Ethoxyresorufin- O-deethylase activity was increased by a dose of 1 mg Aroclor 1254/kg, whereas 7-ethoxycoumarin- O-deethylase and benzo[ a]pyrene hydroxylase activities were increased by doses of 10 mg/kg and higher. Maximal increases in MO activity ranged from three- to 15-fold, depending upon the substrate used. Treatment with 100 mg Aroclor 1254/kg increased hepatic cytosolic glutathione S-transferase (GST) activity toward 1,2-epoxy-3-( p-nitrophenoxy)propane about 20%; however, GST activity toward four other substrates was unaffected. Also, hepatic microsomal UDP-glucuronosyl-transferase (UDPGT) activity was increased about two-fold in catfish treated with 100 mg Aroclor 1254/kg. Treatment with doses of Aroclor 1254 lower than 100 mg/kg did not significantly increase either GST or UDPGT activities. Although MO, GST, and UDPGT activities all were increased by Aroclor 1254, the MO system was by far the most sensitive. Because these enzyme systems are functionally linked in the biotransformation of certain xenobiotics, differential effects, such as those produced by Aroclor 1254, could alter patterns of xenobiotic metabolism and toxicity.

Comparative metabolism of BHA, BHT and other phenolic antioxidants and its toxicological relevance

Following oral administration, butylated hydroxyanisole (BHA) is absorbed and rapidly excreted by the rat, rabbit and man, with little evidence of long-term tissue storage. The major metabolic pathways for BHA are conjugation (phase 2) reactions, oxidative metabolism ( O-demethylation) being relatively unimportant. In the dog, the extent of absorption and urinary excretion is less, and oxidative metabolism is more important than in other species. In contrast, butylated hydroxytoluene (BHT) is cleared less rapidly from most species, enterohepatic circulation being partly responsible for the delay. Tissue accumulation is also greater for BHT than for BHA. Oxidative metabolism (phase 1 reactions) mediated by the microsomal monooxygenase system is the major route for BHT degradation; oxidation of the ring methyl group predominates in the rat, rabbit and monkey, and oxidation of the tert-butyl groups in man. Gallates and 2- tert-butylhydroquinone are mainly metabolized by non-oxidative pathways (methylation or conjugation with sulphate and glucuronic acid). The different biological properties of these compounds may be related to the differences in their absorption and metabolic disposition. Thus, whereas BHT, which is metabolized by oxidation reactions, is an inducer of the microsomal mono-oxygenase system, the other phenolic antioxidants, including BHA, are only weak inducers.

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes.

Immobilization of pig liver microsomes

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1'-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate and kappa-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by the N-demethylation of aminopyrine, the O-demethylation of p-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30-40% and a 60-70% decrease in Vappmax for the O- and N-demethylations were respectively observed. The Vappmax values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4 degrees C, microsomes entrapped inside kappa-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped inside kappa-carrageenan or Ca-alginate can be used to follow up the continuous metabolization of p-nitroanisole for several hours in a stirred-batch reactor.

The effect of substrate on the expression of activity catalyzed by cytochrome P-450: Metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems

The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo- p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[ a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo- p-dioxin-treated rabbits. Although benzo[ a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo- p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When α-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.

Partial purification of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and ω-aminohexyl-agarose. The reductase was further purified by chromatography on 2′,5′-ADP-agarose to a specific activity of about 16 μmoles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after ω-aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.