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R A Prough

Publications and source records attributed to R A Prough.

At least 19 recordsLinked to original sources
PubMed1991

Characteristics of cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of rodent liver.

A second cholesterol-derived metabolite in addition to 7 alpha-hydroxycholesterol was observed to be produced from endogenous microsomal cholesterol in the presence of hamster liver microsomal fractions and NADPH, when analyzed by HPLC using the method of Ogishima and Okuda (Anal Biochem 158: 228-232, 1986). However, only 7 alpha-hydroxycholesterol was produced in the presence of rat hepatic microsomal protein fractions and NADPH. The second metabolite was facilely produced when endogenous 7 alpha-hydroxycholesterol was incubated with hamster liver microsomes and NADPH, but not with rat liver microsomes. The second metabolite derived from either endogenous cholesterol or exogenous 7 alpha-hydroxycholesterol contained three hydroxyl groups as shown by mass spectrometric analysis. After oxidation of the 3 beta-ol group by cholesterol oxidase, the metabolite comigrated with 7 beta-hydroxycholest-3-one on normal phase HPLC, but was resolved from both 7 alpha- and 7 beta-hydroxycholest-3-one on reverse phase HPLC. The data indicate that the second metabolite is a hydroxylated product of 7 alpha-hydroxycholesterol, possibly cholest-5-ene-3 beta,7 alpha, 12 alpha-triol. Cholestyramine feeding increased production of both 7 alpha-hydroxycholesterol and its metabolite from endogenous cholesterol by 3-fold in hamster liver microsomes in vitro. However, the direct conversion of 7 alpha-hydroxycholesterol to the metabolite by hamster liver microsomes was not increased appreciably after cholestyramine feeding (20-30%). The hydroxylation of 7 alpha-hydroxycholesterol was similar in characteristics to cholesterol 7 alpha-hydroxylase activity in that it was dependent on NADPH, was inhibited by several known P450 inhibitors, and was affected by an inhibitory autobody elicited against rat hepatic NADPH: cytochrome P450 oxidoreductase. 5,6- and 7,8-Benzoflavone were poor inhibitors (IC50 approximately 1 mM) of cholesterol 7 alpha-hydroxylase activity in liver microsomes from cholestyramine-fed rats, but caused a striking enhancement of the 7 alpha-hydroxylase activity of liver microsomes from untreated rats in vitro. In contrast, 7,8-benzoflavone inhibited cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of microsomes from normal and cholestyramine-fed hamsters. However, 5,6-benzoflavone stimulated cholesterol 7 alpha-hydroxylase activity in liver microsomes from normal and cholestyramine-fed hamsters, but inhibited 7 alpha-hydroxycholesterol hydroxylase activity by approximately 50%. These results suggest that hepatic cholesterol 7 alpha-hydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities apparently involve multiple forms of cytochrome P450 in untreated and cholestyramine-treated hamsters.

Animals↗
PubMed1991

Inhibition of carbamoyl phosphate synthetase-I by dietary dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), administered per os, serves to prevent or retard the development of a variety of genetic and induced disorders in mice and rats. This treatment also results in the development of hepatomegaly, a change of liver color from pink to mahogany, peroxisome proliferation in hepatocytes and alterations in hepatocyte mitochondria morphology and respiration. We used one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to identify changes in the relative levels of liver proteins produced by DHEA treatment of rodents. In mouse liver, there were apparent increases in the levels of 26 proteins and decreases in the levels of 7 proteins. Of the induced proteins the most prominent had Mr approximately 72 K; this protein was identified in a previous study as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Another protein of Mr approximately 28 K, of unknown nature, also was induced markedly by DHEA treatment of mice and rats. A protein of Mr approximately 160 K, which was identified as carbamoyl phosphate synthetase-I (CPS-I), was decreased markedly by DHEA action. This enzyme, which comprises approx. 15-20% of mitochondrial matrix protein, is involved in the entry and rate-limiting step of the urea cycle. The specific activity of CPS-I also was significantly decreased by DHEA, but serum urea levels were normal. To determine whether steroids other than DHEA also induced similar changes, mice were treated with various steroids for 14 days and, thereafter, liver proteins were evaluated by SDS-PAGE: estradiol-17 beta and isoandrosterone induced both the approximately 72 and approximately 28 kDa proteins, testosterone and androsterone induced the 28 kDa protein only, but etiocholanolone, pregnenolone and progesterone were without effect. The findings of this study serve to demonstrate that: (i) hepatic protein levels are affected by DHEA treatment of mice and rats; (ii) liver CPS-I activity is decreased significantly by DHEA treatment, but serum urea levels remain within the normal range; and (iii) sex steroids and some of their precursors, when administered per os, also alter liver protein levels.

Animals↗
PubMed1990

Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P450cam) by the chemopreventive agent 3-t-butyl-4-hydroxyanisole.

The efficacy of 2(3)-t-butyl-4-hydroxyanisole (BHA) and other chemicals as chemopreventive agents against chemically induced cancer or toxicity may involve direct modulation of cytochrome P450 activity. Direct interaction of BHA with cytochrome P450 was investigated using substrate-bound, oxyferrous cytochrome P450CIA1 either in a reconstituted system containing cytochrome P450CIA1, putidaredoxin, and putidaredoxin reductase with NADH as electron donor or in the absence of physiological electron donors. In the reconstituted system, BHA caused a concentration-dependent decrease in the production of 5-exo-hydroxycamphor and a substoichiometric increase in hydrogen peroxide production. However, BHA did not appreciably inhibit either NADH oxidation or oxygen utilization under conditions optimal for accumulation of oxyferrous cytochrome P450CIA1 during steady-state metabolism of camphor. In the absence of electron donor, BHA enhanced decomposition of the ternary oxyferrous substrate complex of cytochrome P450CIA1 without the formation of any apparent spectral intermediate(s). The rate of decomposition of the oxyferrous complex was pseudo-first order and was dependent upon the concentration of BHA present. Enhanced decomposition of the complex was not attributable to catalytic turnover of cytochrome P450CIA1 (i.e., acquisition of a second electron from an indeterminate source) since no appreciable metabolism of either camphor or BHA was observed. The enhanced decomposition was accompanied by a substoichiometric increase in hydrogen peroxide production, suggesting that BHA may facilitate four-electron reduction of molecular oxygen to water. These results indicate that BHA inhibits cytochrome P450 function, presumably by enhancing autoxidation of the substrate-bound oxyferrous complex.

Animals↗
PubMed1990

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding.

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1990

The effect of the tert-butylquinone metabolite of butylated hydroxyanisole on cytochrome P-450 monooxygenase activity.

1. The t-butylquinone metabolite of BHA was shown to redox cycle with NADPH-cytochrome P-450 reductase leading to enhanced NADPH-oxidase activity for both the purified and liver microsome-bound flavoprotein. Likewise, addition of t-butylquinone (20-100 microM) strikingly inhibited electron transfer from the flavoprotein reductase to cytochrome P-450 of liver microsomes from phenobarbital-treated rats. 2. When the effect of t-butylquinone on metabolism of biphenyl was evaluated with liver microsomal fractions or isolated hepatocytes, t-butylquinone was less effective as an inhibitor then BHA alone or vitamin K3 (menadione). Addition of dicoumarol had little or no effect on the inhibitory potency of either t-butylquinone or vitamin K3 in isolated hepatocytes. 3. t-Butylquinone was not an effective reductant for exogenous oxidants, such as cytochrome c, in the presence of purified, cytosolic NAD(P)H-quinone oxidoreductase (DT-diaphorase). This property is most probably due to the lower rate of reoxidation of t-butylquinone by molecular oxygen, relative to vitamin K3 (menadione).

Animals↗
PubMed1990

Dehydroepiandrosterone feeding and protein phosphorylation, phosphatases, and lipogenic enzymes in mouse liver.

Dehydroepiandrosterone (DHEA) treatment is effective in preventing or delaying the onset of various genetic and induced disorders of mice and rats. Associated with the beneficial therapeutic effects exerted by action of this steroid is the development of hepatomegaly. To determine whether the changes associated with hepatomegaly also involve alterations in activities of tissue enzymes, we evaluated the effects of DHEA (0.45% in food, w/w) on hepatic protein kinases, phosphatases, and lipogenic enzymes in mice of various strains. The rates of fatty acid and cholesterol syntheses also were evaluated. DHEA administration resulted in profound changes in the sodium dodecylsulfate-polyacrylamide gel electrophoresis patterns of endogenous radiophosphorylated proteins obtained by incubation of liver homogenates with (gamma-32P]ATP. These changes were dependent upon the medium used for homogenization. Thus, when homogenates of liver tissue of DHEA-treated mice were prepared in Tris buffer containing sucrose (0.25 M) there was a marked decrease in phosphorylation of the proteins of relative molecular weight approximately 116,000 (Mr approximately 116,000), approximately 82,000, approximately 80,000, approximately 58,000, approximately 56,000, approximately 48,000, approximately 34,000, and approximately 31,000 compared with controls. With liver homogenates of DHEA-treated mice prepared in Tris buffer alone, there was a marked increase in phosphorylation of the proteins of Mr approximately 70,000, approximately 49,000, approximately 34,000, approximately 31,000, and 28,000 compared with controls. Moreover, the specific activity of kinases for endogenous protein acceptors in liver of control mice was higher than that in liver of DHEA-treated animals. The specific activities of casein kinase, cAMP-dependent protein kinase, and cGMP-dependent protein kinase remained unchanged with DHEA treatment, but the specific activity of histone kinase was increased approximately 30%. Long-term administration of DHEA also was associated with increases in the specific activities of liver AMPase and GTPase (approximately two times), but not of other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. The activity of hepatic NADP-linked malic enzyme was increased significantly (two to three times) by DHEA treatment of female mice of three different strains, but was unchanged in male C57BL/6 mice. The specific activities of hepatic glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, and ATP-citrate lyase were not affected significantly by DHEA treatment of mice. The rate of hepatic lipogenesis, determined by incorporation of tritium from 3H2O into fatty acids, was decreased approximately 70% in DHEA-treated mice, while the rate of cholesterol synthesis was increased approximately 44% compared with controls.

ATP Citrate (pro-S)-Lyase↗
PubMed1990

Cytochrome P-450s as toxicogenic catalysts: the influence of dehydroepiandrosterone.

The cytochrome P-450s catalyze the oxidative transformation of a large number of endogenous and exogenous chemicals in plants, insects, and mammals. One consequence of this type of reaction is the generation of highly reactive electrophilic metabolites that can react with intracellular macromolecules. It has been postulated that the initiation reaction for chemical carcinogenesis and/or cellular toxicity involves the metabolism of xenobiotics by P-450s. The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of rodents, has been reported to have anti-carcinogenic and other chemoprotective activities. A change in the inventory of liver P-450s occurs during treatment of rodents with DHEA, the most pronounced being that involved in the omega-hydroxylation of medium-chain length fatty acids. In addition, changes in the activities of other liver P-450s, viz., P-450IIB1, P-450IIC11, and P-450IIIA, occur as shown by in vitro experiments to assess the P-450-dependent formation of hydroxylated metabolites of testosterone and androstenedione. The effect of feeding rodents a diet supplemented with DHEA mimics some of the changes seen when animals are treated with hypolipidemic drugs (such as ciprofibrate, and other chemicals which are known to be peroxisome proliferators). Studies comparing the enzymatic functions of the heterologous expressed recombinant forms of P-45017 alpha, responsible for the two step conversions of progesterone and pregnenolone to the C19-steroids, androstenedione, and DHEA, respectively, illustrated the marked differences in enzymatic properties between the human and the rat orthologues. The findings serve to demonstrate that DHEA is an obligatory intermediate for the synthesis of androgens in the human, but not in the rodent. The biochemical changes responsible for the anti-carcinogenic properties of DHEA remain to be identified. The studies presented here suggest that DHEA, when administered as a dietary supplement, functions like a xenobiotic, and that its effects may result from alterations in the inventory of cellular P-450s, thereby influencing the balance of metabolic activities associated with the initiation phase of chemical carcinogenesis and/or toxicity.

Animals↗
PubMed1990

Glucocorticoid regulation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferases, and NAD(P)H:quinone oxidoreductase in cultured fetal rat hepatocytes.

The regulation of polycyclic aromatic hydrocarbon-inducible enzymes, cytochrome P450IA1, NAD(P)H:quinone oxidoreductase, and glutathione S-transferases, by glucocorticoids was investigated using primary fetal rat hepatocyte culture. Treatment of cells in culture with 1,2-benzanthracene (100 microM, 72 hr) resulted in 60-, 2-, and 6-fold increases in cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone reductase activities, respectively. The inductive effect of 1,2-benzanthracene on cytochrome P450IA1 and glutathione S-transferase (1-chloro-2,4-dinitrobenzene conjugation) activities was potentiated approximately 3- and 2- to 3-fold, respectively, when dexamethasone (0.01-1 microM) was included in the culture medium. In contrast, 1 microM dexamethasone was found not to potentiate the induction of NAD(P)H:quinone oxidoreductase activity by 1,2-benzanthracene. Treatment of cultured hepatocytes with dexamethasone alone, at concentrations of up to 100 microM, resulted in a 2- to 4-fold increase in glutathione S-transferase and NAD(P)H:quinone oxidoreductase activity. Both the induction of glutathione S-transferase activity by high concentrations of dexamethasone alone and the potentiation of 1,2-benzanthracene induction by lower concentrations of dexamethasone were observed for other steroids of the glucocorticoid class in conjunction with a variety of polycyclic aromatic hydrocarbons. Western immunoblot analyses indicated that low concentrations of dexamethasone (0.1-1 microM) potentiated 1,2-benzanthracene-dependent induction of cytochrome P450IA1, glutathione S-transferase Ya/Yc subunit and NAD(P)H:quinone oxidoreductase content. Additionally, increased glutathione S-transferase activity in response to concentrations of dexamethasone exceeding 1 microM was associated with concomitant increases in Ya/Yc and Yb subunit content. Potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase protein content by low concentrations of glucocorticoids and induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase by high concentrations of glucocorticoids alone indicates the importance of these endogenous compounds in the regulation of some hepatic enzymes involved in xenobiotic metabolism.

Animals↗
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

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↗
PubMed1979

The metabolic activation of benzo(a)pyrene and 9-hydroxybenzo(a)pyrene by liver microsomal fractions.

A rat liver microsome-mediated bacterial mutagenicity test showed 9-hyroxybenzo(a)pyrene to be significantly more effective as a pre-mutagen than benzo(a)pyrene. Experiments measuring the ability of these compounds to be metabolically activated to moieties that alkylate exogenous DNA demonstrated that 9-hydroxybenzo(a)pyrene was almost six times more effective than benzo(a)pyrene itself. Addition of trichloropropene-2,3-oxide to the reaction mixture enhanced the mutagenicity and DNA alkylation by benzo(a)pyrene but had little or no effect on the 9-hydroxybenzo(a)pyrene-mediated mutagenicity and alkylation. On the other hand, 7,8-benzoflavone inhibited the microsome-mediated mutagenicity and DNA alkylating activity of both hydrocarbons.

Alkylation↗