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Biomedical subjects

R A Prough

Publications and source records attributed to R A Prough.

At least 73 records · Page 4Linked to original sources
PubMed1984

Cytosol-mediated reduction of resorufin: a method for measuring quinone oxidoreductase.

The reduction of resorufin (7-hydroxyphenoxazone) fluorescence was catalyzed by enzymes present in the hepatic cytosol of rats and hamsters. This reaction was mediated by either NADH or NADPH, was completely inhibited by 10 microM dicumarol, and was not affected by anaerobic conditions (purging the reaction cuvette with nitrogen). The enzyme-mediated decrease in resorufin fluorescence was also associated with the loss of the primary absorbance maximum at 570 nm as well as the shoulders at 530 and 600 nm. Similar spectral changes were observed after resorufin was nonenzymatically reduced by sodium dithionite. The enzymatic activity was induced 20- to 40-fold by animal pretreatment with Aroclor-1254 or methylcholanthrene, but only minimally by phenobarbital. A 2.5-fold increase in the rate of the reaction was noted when the pH of the reaction mixture was lowered from pH 7.5 to 6.0. This pH optimum was not a result of slower rates of reoxidation of the reduced resorufin at lower pH, but was due to increased rates of reduction of the compound. Several of the characteristics of the reaction were congruent with the involvement of DT-diaphorase (quinone oxidoreductase, EC 1.6.99.2), and this newly developed fluorimetric assay would appear to be a rapid, sensitive, and direct method for measurement of DT-diaphorase activity.

Animals↗
PubMed1984

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450.

The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethylhydrazine elicited no response, either in observable spectral changes or loss of CO-reactive cytochrome P-450. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.

Animals↗
PubMed1984

Evaluation of free radical effects and catecholamine alterations in adriamycin cardiotoxicity.

With the goal of evaluating the potential roles of free radicals and catecholamines in the pathogenesis of acute and chronic Adriamycin (ADR) cardiotoxicity, evidence was sought for myocardial free radical toxicity and alterations in myocardial catecholamine levels after acute and chronic administration of ADR to rabbits. In acute studies, male New Zealand white rabbits received intravenous ADR, 1.1 or 5 mg/kg per day for 1 or 3 days or 10 mg/kg for 1 day and were sacrificed 3-72 hours later. Because the glutathione-glutathione peroxidase system is a major pathway for free radical detoxification, glutathione levels and glutathione peroxidase activity were measured. In the acute studies, ADR-treated rabbits exhibited significantly increased levels (up to 50%) of total and reduced glutathione, unchanged levels of oxidized glutathione, and a slight decrease in the percentage of oxidized glutathione. Major effects of dose and sacrifice interval were not observed. However, in animals receiving three injections of 5 mg/kg or one injection of 10 mg/kg ADR, myocytes exhibited fine vacuolization, due to lipid accumulation and dilatation of the sarcoplasmic reticulum, without evidence of coagulation necrosis. In the chronic study, rabbits received 1.1 mg/kg ADR twice weekly for up to 10 weeks. Levels of total and reduced glutathione were increased significantly by 23-36% after 9-12 and 16-20 injections without change in the percentage of oxidized glutathione. The mean percentage of myocytes with vacuolar-myofibrillar degeneration, the characteristic lesion of chronic ADR cardiotoxicity, was 0 after 5-7 injections, 3.3 after 9-12 injection, and 17.2 after 16-20 injections. Glutathione peroxidase activity was not reduced significantly in any group of acute or chronic ADR-treated animals. Tests for lipid peroxidation (malondialdehyde and ethane production) were negative in acute studies. Myocardial catecholamine levels were unchanged in acute and chronic ADR animals. Thus, the cardiac glutathione-glutathione peroxidase system is activated with ADR treatment at the onset of cellular damage, and cellular damage progresses without further alteration of this system, loss of glutathione peroxidase activity, or reduction in myocardial catecholamines in rabbit models of ADR cardiotoxicity. These findings suggest that free radical generation in the heart may contribute to ADR cardiotoxicity, but that other factors probably play a more important role in the pathogenesis of the myocardial damage.

Animals↗
PubMed1984

Effect of cytosolic components on the metabolism of the hydrazide iproniazid.

The effects of thiols, such as glutathione (GSH), and the cytosolic glutathione S-transferases on the microsomal metabolism of the hydrazide iproniazid to hydrocarbon products were investigated. Thiol compounds stimulated propane production and depressed propylene production. Addition of preparations of cytosolic proteins to the microsomal reaction mixtures in the presence of GSH depressed production of propane by more than 80% and propylene by 50% compared to the GSH-mediated reaction. The purified glutathione S-transferases A and B were most potent in eliciting this effect; isozymes AA, C, and E had little or no effect on hydrocarbon production. Further, a mixture of these purified isozymes in the concentrations known to exist in cytosol affected hydrocarbon production in a manner similar to cytosol. Experiments performed with isolated hepatocytes and an inhibitor of these cytosolic enzymes further supported the involvement of these enzymes in altered hydrocarbon production. These isozymes were subsequently shown to catalyze the formation of a GSH conjugate, S-(2-propyl)glutathione. The decreases in hydrocarbon production by microsomes in the presence of the glutathione S-transferases and GSH were accompanied by production of slightly larger amounts of conjugate. These data indicate that the cytosolic glutathione S-transferases interact with an oxidative microsomal metabolite of iproniazid to enzymatically produce an S-(2-propyl)glutathione conjugate and thus prevent formation of a reactive species which would otherwise chemically decompose to yield hydrocarbons or to covalently bind to cellular macromolecules.

Animals↗
PubMed1984

Major isozymes of rat liver microsomal cytochrome P-450 involved in the N-oxidation of N-isopropyl-alpha-(2-methylazo)-p-toluamide, the azo derivative of procarbazine.

Seven isozymes of cytochrome P-450 were tested to establish whether they could N-oxidize azoprocarbazine to form the two isomeric azoxy metabolites after optimizing the reconstitution of various purified isozymes with regard to substrate concentration, exogenous lipid, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c (P-450) reductase concentration. Two isozymes, cytochromes P-450PB-C (an isozyme present in untreated rats or in rats treated with phenobarbital or beta-naphthoflavone) and P-450 beta NF-B (the major beta-naphthoflavone-induced isozyme), had appreciable turnover numbers for the N-oxidation reaction. The product ratio [N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide formation relative to N-isopropyl-alpha-(methyl-NNO-azoxy)-p-toluamide formation] obtained with cytochrome P-450PB-C was nearly identical to those values obtained with liver microsomes from untreated and phenobarbital-treated rats. In addition, cytochrome P-450 beta NF-B and liver microsomes from beta-naphthoflavone-treated rats had nearly identical product ratios. Specific inhibitory antibodies to four purified isozymes were used to titrate the N-oxidase activity of liver microsomes from untreated, phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats. Anti-cytochrome P-450PB-C globulin inhibited more than 70 to 90% of the formation of N-isopropyl-alpha-(methyl-ONN-azoxy)-p-toluamide in microsomes from untreated, phenobarbital-, and pregnenolone-16 alpha-carbonitrile-treated rats, respectively, but only 20 to 50% of N-isopropyl-alpha-(methyl-NNO-azoxy)-p-toluamide formation. A small amount (25 to 30%) of inhibition was observed with anti-cytochrome P-450PB/PCN-E globulin. Anti-cytochrome P-450 beta NF-B globulin inhibited more than 85% of the synthesis of either azoxy isomer catalyzed by liver microsomes from beta-naphthoflavone-treated rats. These results demonstrate that two isozymes are responsible for the oxidative metabolism of azoprocarbazine and can account for the major portion of this N-oxidase activity in liver microsomes from untreated and phenobarbital-, pregnenolone-16 alpha-carbonitrile-, or beta-naphthoflavone-treated rats.

Animals↗
PubMed1983

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions.

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.

Animals↗
PubMed1983

Studies on the pathway of methane formation from procarbazine, a 2-methylbenzylhydrazine derivative, by rat liver microsomes.

The oxidative metabolism of procarbazine, its azo, hydrazone, and two azoxy derivatives, and methylhydrazine by hepatic microsomes from phenobarbital-pretreated rats was investigated to elucidate the pathway of metabolism that resulted in methane formation from procarbazine. When incubated with microsomal reaction mixtures fortified with NADPH, all of the compounds, except the azoxy isomers, were metabolized to yield methane. A lag phase in methane formation was noted for procarbazine, but not for the other compounds. Kinetic and inhibition studies utilizing methimazole and ethylhydrazine precluded methylhydrazine as an intermediate in methane formation from procarbazine. When the azo derivative was oxidatively metabolized in the presence of liver microsomes, no hydrazone tautomer was detected. Upon monitoring the production of the azo and hydrazone metabolites formed during microsomal metabolism of procarbazine, the azo derivative was formed in sufficient quantities to account for the majority of the methane produced. In addition, small amounts of hydrazone were also detected. It was concluded that both the azo and hydrazone metabolites of procarbazine contribute to methane formation from the terminal methyl group of the hydrazine with the azo derivative being the predominant source and the hydrazone derivative being a minor source of methane. Consideration of the chemical and enzymatic pathways of procarbazine oxidation and the implication of a methyl radical intermediate in methane formation are discussed.

Animals↗
PubMed1982

The reaction of phenylhydrazine with microsomal cytochrome P-450. Catalysis of heme modification.

Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typical of many nitrogenous compounds. The phenylhydrazine-induced difference spectrum observed with oxidized microsomal cytochrome P-450 was converted, in a time-dependent process, to yield a new spectral intermediate with an absorbance maximum around 480 nm. The time required to form this new phenylhydrazine-induced spectral intermediate was decreased from hours to minutes when either NADPH or NADH was added to the reaction mixture. Phenyldiazene generated by addition of the decarboxylation product of methyl phenyldiazenecarboxylate or by addition of potassium ferricyanide and phenylhydrazine (2:1 molar equivalents) instantly formed the new spectral intermediate. This suggests that phenyldiazene is formed during the NADPH-dependent reaction. The appearance of the new spectral intermediate occurred concomitant with the loss of CO-reactive cytochrome P-450 (less than 90%) and loss of absorbance at 418 nm. The interpretation of the optical spectral changes was supported by a loss of the low spin signals characteristic of oxidized cytochrome P-450 as determined by electron paramagnetic resonance spectroscopy. The loss of CO-reactive cytochrome P-450 apparently resulted from the formation of a binary complex of phenyldiazene and the heme of oxidized cytochrome P-450 giving rise to the 480 nm spectral intermediate. In addition, the diazene-bound heme of cytochrome P-450 apparently was modified irreversibly in the presence of oxygen. The effects observed with phenylhydrazine could be produced to a lesser degree by other hydrazine derivatives. The possible role of phenylhydrazine as a new type of suicide substrate is discussed.

Aerobiosis↗
PubMed1982

Cytochrome P-450-dependent oxygenation of arachidonic acid to hydroxyicosatetraenoic acids.

Arachidonic acid is oxidized by a NADPH-dependent oxygenase of rat liver microsomes to a number of oxygen-containing products, which can be resolved by HPLC. Several of these products have been purified and characterized. They exhibit an absorbance in the UV region of the spectrum that has a maximum at approximately 235 nm, indicative of the presence of a conjugated diene function. Mass spectral analysis of the trimethylsilyl ether derivatives of the methyl esters of the hydrogenated and nonhydrogenated metabolites shows that they are the 9-, 11-, 12-, and 15-monohydroxy derivatives of arachidonic acid, the hydroxyicosatetraenoic acids (HETEs). Their UV absorbance and chromatographic properties suggest that these products possess cis,transdiene geometry characteristic of HETEs isolated from other mammalian sources. THe isolation of these isomeric HETEs suggests that cytochrome P-450 may play a role in the oxidative metabolism of arachidonic acid to physiologically and pharmacologically important hydroxylated unsaturated fatty acids.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1982

Monooxygenase activities of human liver, lung, and kidney microsomes - a study of 42 post mortem cases.

The cytochrome P-450-dependent monooxygenase system was examined in microsomal fractions prepared from 42 post mortem human livers and 9 lungs and kidneys. Electron microscopy studies indicated that the human liver samples were relatively free of mitochondrial and plasma membrane contamination, but samples of kidney and lung were less pure. The microsomal fractions from all organs were judged to be relatively free of haemoglobin and methaemoglobin. The specific enzyme activities for several drug substrates for the monooxygenase, NADPH-cytochrome c reductase activity and the content of the microsomal cytochromes were measured. The values of the biochemical parameters studied were found to be quite variable and the values for the human liver were appreciably lower than those obtained with liver microsomes from laboratory rodents. The enzyme activities of the human kidney and lung microsomal fractions were 1-10% of those seen for human liver samples, except for NADPH-cytochrome c(P-450) reductase activity. In order to evaluate any post mortem changes in human liver, correlations between drug metabolism activities and either cytochrome P-450 or NADPH-cytochrome c (P-450) reductase content were examined. Strong correlations (r greater than 0.91) were seen only between aminopyrine or ethylmorphine demethylase activity and cytochrome P-450 content in samples obtained within 4 hours of death. Longer post mortem times gave poorer correlation between activity and cytochrome content. These studies document several conditions required in order to obtain human microsomal fractions representative of the activities in fresh, viable tissue.

Adult↗
PubMed1981

The microsomal metabolism of the organometallic derivatives of the group-IV elements, germanium, tin and lead.

The NADPH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetra-ethyl-substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product. These reactions were shown to be catalysed by the liver microsomal cytochrome P-450-dependent mono-oxygenase. Since formation of ethane and ethylene was differentially inhibited by anaerobiosis, the results suggest that at least a large portion of the ethane produced may be derived by a reductive mechanism. Triethyltin bromide in both the absence and presence of NADPH was shown to convert cytochrome P-450 into cytochrome P-420 and to affect the function of the mono-oxygenase in vitro. Tetraethyltin caused the NADPH- and time-dependent formation of cytochrome P-420, suggesting that tetraethyltin is converted into triethyltin salts in significant concentrations. The order of potency in formation of cytochrome P-420 was closely paralleled by the ability of the tin derivatives to induce microsomal lipid peroxidation in vitro.

Animals↗
PubMed1981

Liver microsomal cytochrome P-450 and the oxidative metabolism of arachidonic acid.

Arachidonic acid is oxidatively metabolized by rat liver microsomes at a rate of approximately 5 nmol per min per mg of protein at 25 degrees C. This reaction is dependent on the presence of NADPH and oxygen. Studies with various inhibitors indicate a role for membrane-bound cytochrome P-450 in the transformation of arachidonic acid to a mixture of hydroxy acid derivatives. The stoichiometry of the reaction conforms to that of a monooxygenase reaction--i.e., one mole of NADPH is oxidized per mole of oxygen utilized--suggesting a reaction mechanism different from that proposed for lipid peroxidation reactions. No evidence for the formation of prostaglandin-like metabolites was obtained. The diene character of some of the metabolites formed suggests another role for cytochrome P-450--i.e., participation in hydrogen abstraction reactions for the activation of various substrates.

Animals↗