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Biomedical subjects

R A Prough

Publications and source records attributed to R A Prough.

At least 55 records · Page 3Linked to original sources
PubMed1989

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.

Actins↗
PubMed1989

Cytotoxicity and DNA damage caused by the azoxy metabolites of procarbazine in L1210 tumor cells.

Procarbazine, a chemotherapeutic hydrazine, is thought to be metabolized to an alkylating species similar to methyl carbonium ion by multistep reactions involving cytochrome P-450, monoamine oxidase, and cytosolic enzymes. The DNA-damaging and cytotoxic potential of procarbazine and its metabolites in murine L1210 leukemia tumor cells in vitro was determined using alkaline elution techniques and extrapolation of growth curves. Neither procarbazine nor any of the chemical degradation products (except for the aldehyde derivative at high concentrations) caused significant amounts of DNA strand breakage. The primary enzymatic oxidation product, azo-procarbazine, did not produce strand breakage. However, exposure of the cells to either of the two isomers of azoxy-procarbazine led to significant DNA damage and cytotoxicity. DNA damage included both single-strand breaks and alkali-labile sites. At equimolar concentrations, the azoxy 2 isomer of procarbazine caused 14 to 20 times more DNA damage than did the azoxy 1 metabolite. When cell growth is expressed as percentage survival of L1210 cells, the azoxy 2 isomer was approximately 7-fold more toxic than the azoxy 1 metabolite. The other metabolites tested showed little or no cytotoxicity. L1210 cells were shown to contain little or no cytochrome P-450 or monoamine oxidase activity, which may account for the lack of toxicity of the parent drug or the primary oxidative metabolite, azo-PCZ, to these cells. The conversion of procarbazine to the azoxy-procarbazine isomers in vivo must occur in cells which contain these enzymes, such as liver. However, the azoxy isomers of procarbazine were metabolized in L1210 cells, presumably leading to the DNA or cytotoxic damage observed.

Animals↗
PubMed1989

Potentiation of 3-methylcholanthrene induction of rat hepatic cytochrome P450IA1 by dexamethasone in vivo.

The potentiation of the expression of polycyclic aromatic hydrocarbon-inducible cytochromes P450IA1 and P450IA2, and phenobarbital-inducible cytochrome P450IIB1 and NADPH-cytochrome P-450 reductase by dexamethasone in vivo was investigated using adrenalectomized and sham-operated rats. Hepatic cytochrome P450IA1 activity (determined by ethoxyresorufin O-deethylase activity) was induced maximally (30-fold) in both sham-operated and adrenalectomized rats 24 hr after a single injection of 3-methylcholanthrene (10 mg/kg). Dexamethasone (10 mg/kg) increased hepatic tyrosine aminotransferase activity 3- to 4-fold, but had little or no effect on ethoxyresorufin O-deethylase activity. However, dexamethasone potentiated the 3-methylcholanthrene-dependent induction of hepatic P450IA1 activity 2- to 3-fold in adrenalectomized rats and 1.5- to 2-fold in sham-operated rats when administered concomitantly with 3-methylcholanthrene (P less than .05). The dose of dexamethasone required to potentiate 3-methylcholanthrene induction of P450IA1 activity (greater than 1 mg/kg) correlated well with the dose required to induce hepatic tyrosine aminotransferase activity; a marker of glucocorticoid action. Potentiation of 3-methylcholanthrene induction of P450IA1 activity in the rat appears to be tissue specific in that dexamethasone was found not to potentiate 3-methylcholanthrene induction of P450IA1 activity of rat lung or kidney. Cytochrome P450IA1 content induced by 3-methylcholanthrene in the liver was also potentiated 2- to 3-fold by dexamethasone. In contrast, potentiation of 3-methylcholanthrene induction of cytochrome P450IA2 content was not observed. Western blot analyses indicate that the increase in monooxygenase activity induced by 3-methylcholanthrene or 3-methylcholanthrene plus dexamethasone corresponds well only with the induction of cytochrome P450IA1 protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1988

Rat lung antioxidant enzyme activities and their specific proteins during hyperoxia.

The hyperoxia-induced increases in the activity of lung glucose-6-phosphate dehydrogenase (G-6-P) and glutathione reductase (GR) after exposure of rats to greater than 97% O2 for 6 days were accompanied by equivalent increases in the amount of the respective immunoreactive proteins. Hyperoxia also increased lung glutathione (GSH) + oxidized glutathione (GSSG) content and the magnitude of this hyperoxic response of increased GSH + GSSG, G-6-P, and GR (maximal 1.3- to 1.8-fold) declined as a function of age during the first 3 wk of life. Fetal rat lung explants cultured 4 days in 95% O2 showed increased G-6-P and GR activity and increased levels of the specific proteins 1.5-fold those of explants at 2 days of culture. We conclude that the hyperoxic response of increased rat lung G-6-P and GR activity in vivo and in vitro involves not just alteration of enzyme activity but also specific increases in the proteins catalyzing the reactions.

Aging↗
PubMed1988

Effects of induction on the metabolism and cytochrome P-450 binding of harman and other beta-carbolines.

1. The metabolism of harman by liver microsomes from non-induced, phenobarbitone (PB)-induced and 3-methylcholanthrene (MC)-induced mice was investigated. Initial reaction rates for harman disappearance were measured and showed a 4-fold induction by PB and a 10.6-fold induction by MC. 2. The major metabolite formed with each microsomal preparation was identified as 6-hydroxyharman. 3. Microsomal cytochrome P-450 binding was measured for harman and other beta-carbolines and both Type I and Type II binding spectra were observed, being dependent upon the mode of induction.

Alkaloids↗
PubMed1986

Synergistic induction of monooxygenase activity by glucocorticoids and polycyclic aromatic hydrocarbons in human fetal hepatocytes in primary monolayer culture.

The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus.

Aryl Hydrocarbon Hydroxylases↗
PubMed1986

Oxidative metabolism of some hydrazine derivatives by rat liver and lung tissue fractions.

The enzyme systems in rat liver and lung responsible for the oxidative metabolism of hydrazine derivatives were studied to determine whether these enzymes, cytochrome P-450 and monoamine oxidase, were responsible for metabolically activating hydrazines to carcinogenic/toxic metabolites. Cytochrome P-450 preferentially oxidized the nitrogen to nitrogen bond of 1,2-disubstituted hydrazines and hydrazides, while monoamine oxidase oxidized the nitrogen to nitrogen bond of all the classes of hydrazine derivatives that were tested. Oxidation of the nitrogen to nitrogen bond led to the formation of stable azo intermediates in the case of 1,2-disubstituted hydrazines and to unstable monoazo (diazene) metabolites in the case of monosubstituted hydrazines and hydrazides. In addition, cytochrome P-450 preferentially oxidized the carbon to nitrogen bond of monoalkylhydrazines; this reaction resulted in the formation of aldehyde metabolites (via hydrazone intermediates). Monosubstituted hydrazines were shown to be potent, irreversible inhibitors of mitochondrial monoamine oxidase. In contrast, the 1,2-disubstituted hydrazines appeared to be good substrates for the monoamine oxidase and served as competitive inhibitors at high concentrations. There did not appear to be any monoamine oxidase isozyme (form A or B) specificity in the metabolism of either the 1,2-disubstituted hydrazines or the monoalkylhydrazines, ethyl- and n-propylhydrazine.

1,2-Dimethylhydrazine↗
PubMed1986

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes.

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.

Animals↗
PubMed1985

Propane and propylene formation during the microsomal metabolism of iproniazid and isopropylhydrazine.

Both iproniazid and isopropylhydrazine were metabolized to the hydrocarbon products, propane and propylene, with nearly identical Michaelis constants and rates. This reaction appeared to be catalyzed by microsomal cytochrome P-450. Isonicotinic acid, a product of iproniazid hydrolysis by various amidases, was produced in only very small quantities, suggesting that the other amidase product, isopropylhydrazine, may not be an obligatory intermediate in the pathway of hydrocarbon formation from iproniazid. Hydrocarbon formation from iproniazid was more sensitive to inhibition in vitro by bis-p-nitrophenylphosphate (used in vivo as an amidase inhibitor) than was isopropylhydrazine. Iproniazid must be directly metabolized by cytochrome P-450 to yield propane and propylene, presumably via an azo ester intermediate which could give rise to an isopropyl radical, the chemical species presumed to be responsible for the hepatoxicity apparent after administration of large doses of iproniazid in vivo.

Alkenes↗
PubMed1985

Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs).

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.

8,11,14-Eicosatrienoic Acid↗
PubMed1985

Metabolic activation of the terminal N-methyl group of N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine).

The NADPH-dependent microsomal metabolism of [14C]procarbazine, labeled on the terminal N-methyl group, resulted in the covalent binding of the drug to exogenously added DNA; this reaction was inhibited by metyrapone. Procarbazine metabolism was also shown to result in covalent binding of the methyl group of the drug to microsomal protein upon metabolism, but the extent of protein binding was at least an order of magnitude smaller than that seen with its primary oxidative metabolite. N-isopropyl-alpha-(2-methylazo)-p-toluamide. The characteristics of the reactions leading to the covalent binding of the N-methyl group of the azo derivative to microsomal protein and its metabolism to form the hydrocarbon, methane, possessed a number of similarities in the apparent kinetic parameters (Km and Vmax), induction, and inhibition patterns indicating a common pathway of metabolism to form a reactive intermediate and the involvement of cytochrome P-450. Reduced glutathione stimulated methane formation and inhibited covalent binding to protein. One azoxy derivative, N-isopropyl-alpha-(2-methyl-ONN-azoxy)-p-toluamide, was chemically unstable and its decomposition was shown to lead to covalent binding to microsomal protein. A diazene intermediate and a methyl radical are proposed to be intermediates in the formation of methane during the oxidative metabolism of the azo derivative of procarbazine and a common intermediate in the activation of procarbazine may result in both covalent binding to cellular macromolecules and methane production. In addition, chemical decomposition of the azoxy metabolites may also contribute to a small portion of the covalent binding, but not to methane formation.

Animals↗
PubMed1985

The responses of glutathione and antioxidant enzymes to hyperoxia in developing lung.

Total glutathione levels and the activity of enzymes associated with antioxidant protection in neonatal lung are increased in response to hyperoxia. Glutathione levels in developing rat lung decreased from 24 nmol/mg protein on day 19 of gestation to approximately 12 nmol/mg protein at birth. The initial decrease in glutathione may be due to emergence of other antioxidant systems. Newborn rats placed in 100% oxygen showed a rapid and sustained increase in total glutathione levels which was primarily due to an increase in reduced glutathione. Explants obtained from 16-wk gestation human fetal lung or from 17- to 18-day fetal rat lung also showed increased total and reduced glutathione when cultured in 95% oxygen, 5% CO2 as compared with explants cultured in room air. Type II cells isolated from neonatal rats maintained in oxygen for 6 days also showed glutathione levels twice those found in cells isolated from animals in room air. The activity of antioxidant enzymes (glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase) was increased in lungs of newborn rats exposed to 100% oxygen either at birth or 2 days of age. Antioxidant enzyme activity of lung explants cultured in 95% oxygen, 5% CO2 was also higher than in explants maintained in room air. These results suggest that the increases in glutathione and of antioxidant enzymes in vivo and in vitro are a direct effect of oxygen exposure in lung and that the increase of both glutathione and antioxidant enzyme activity is intrinsic to the lung cell itself. It is likely that increases in glutathione in lung represent an important protective mechanism against oxidant injury.

Animals↗
PubMed1985

Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling.

A significant population of humans (5 to 10%) are phenotypic poor metabolizers of debrisoquine. We have isolated the cytochrome P-450 isozyme from rat liver responsible for this activity and have shown that antibodies raised against the protein are able to inhibit this catalytic activity in human liver microsomes (Distlerath, L. M., and Guengerich, F. P., Proc. Natl. Acad. Sci. USA, 81: 7348-7352, 1984). These antibodies were utilized to determine which metabolic transformations are linked to debrisoquine 4-hydroxylation in human liver microsomes using techniques of immunochemical inhibition. The antibodies almost completely inhibited debrisoquine 4-hydroxylation and bufuralol 1'-hydroxylation in microsomes prepared from several different human livers. The oxidation of the pyrrolizidine alkaloids lasiocarpine and monocrotaline were inhibited by roughly one-third. The antibodies did not inhibit N,N-dimethylnitrosamine N-demethylation, oxidation of vinylidene chloride to 2,2-chloroacetaldehyde, oxidation of trichloroethylene to chloral, N-oxidation of azoprocarbazine, morphine N-demethylation, diazepam N-demethylation, oxidation of benzo(a)pyrene to alkali-soluble metabolites, oxidation of benzo(a)pyrene 7,8-dihydrodiol to products covalently bound to DNA, the N- and ring-oxidation of 1- and 2-naphthylamine and 2-aminofluorene, or the conversion of aflatoxin B1 to DNA adducts or aflatoxin Q1. Studies with space-filling models of the drugs the metabolism of which is associated with debrisoquine 4-hydroxylase in the literature indicated that all can be fitted to a general structure in which a basic nitrogen is about 5 A away from the site of carbon hydroxylation and a hydrophobic domain is near the site of hydroxylation. These results may be useful in predicting which chemicals may or may not be metabolized in an atypical manner by a segment of the human population.

Aflatoxin B1↗
PubMed1985

Metabolism of 3-tert-butyl-4-hydroxyanisole by microsomal fractions and isolated rat hepatocytes.

3-tert-Butyl-4-hydroxyanisole is oxidatively metabolized in the presence of rat liver microsomes, reduced nicotinamide adenine dinucleotide phosphate, and oxygen to yield tert-butylhydroquinone, tert-butylquinone, and a polar metabolite(s). In the presence of human and rat liver microsomes or eight purified cytochrome P-450 isozymes reconstituted with NADPH-cytochrome P-450 reductase, this phenolic antioxidant is converted to the oxidoreduction-active metabolite, tert-butylquinone, that can stimulate the NADPH oxidase activities of these preparations by 2- to 7-fold. The rate of formation of each of the metabolites of 3-tert-butyl-4-hydroxyanisole was increased by pretreatment of rats with either 5,6-benzoflavone or phenobarbital. In addition the tert-butylhydroquinone and tert-butylquinone concentrations in solution reached apparent steady-state levels during metabolism; the steady-state concentrations were also increased by various animal pretreatment regimens. Furthermore it was shown that the metabolism of 3-tert-butyl-4-hydroxyanisole yielded material which was covalently bound to protein. In the presence of glutathione the rates of formation of the polar metabolite(s) were enhanced 3- to 4-fold, while covalently bound products were nearly stoichiometrically decreased. The increase in the amount of polar metabolite was due to the formation of a 3-tert-butyl-4-hydroxyanisole-glutathione conjugate. 3-tert-Butyl-4-hydroxyanisole was also oxidatively metabolized by rat lung microsomes to yield the polar metabolite(s) and tert-butylhydroquinone. The polar metabolite(s), tert-butylquinone, and tert-butylhydroquinone were also shown to be formed in isolated hepatocyte suspensions. They could be found as either the free hydroquinone, the sulfate conjugate, the glucuronide conjugate, and polar metabolites, presumedly the 3-tert-butyl-4-hydroxyanisole-glutathione conjugate. The total tert-butylhydroquinone concentration attained a steady-state level in a manner similar to that seen with the microsomal suspensions. In addition 3-tert-butyl-4-hydroxyanisole itself formed sulfate and glucuronide conjugates, the glucuronide being the major product.

Animals↗