J. Capdevila

Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets.

Metapyrone and eicosatetraynoic acid but not indomethacin are effective inhibitors of the secretory response of isolated rat pancreatic islets to arginine and glucose. Epoxyeicosatrienoic acids, products of the cytochrome P-450-NADPH dependent arachidonic acid epoxygenase activity, are potent and selective mediators for the in vitro release of either insulin or glucagon from preparations of isolated rat pancreatic islets.

Novel epoxides formed during the liver cytochrome P-450 oxidation of arachidonic acid

Abstract Arachidonic acid is enzymatically oxidized by the rat liver microsomal mixed-function oxidase system, in the presence of NADPH and oxygen, to a wide variety of products. We report here, the identification of the major organic-soluble metabolites. They are the 5,6-,8,9-,11,12-, and 14,15-epoxy-eicosatrienoic acid derivatives of arachidonic acid.

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450☆

Abstract The mechanism of NADPH- and cumene hydroperoxide-supported hydroxylation of benzo(a)pyrene as catalyzed by liver microsomes was studied using high pressure liquid chromatography, fluorescence, and spectrophotometric methods. Repetitive scan difference spectral analysis clearly demonstrated that during the steady state of these reactions, different products were formed. While the major products noted with NADPH were phenols, only low concentrations of phenols were observed in the presence of cumene hydroperoxide using all three analytical methods. With the organic hydroperoxide, the metabolite profile was shifted from the preponderate production of phenols and dihydrodiols to the production of the three quinone isomers of the hydrocarbon. At several concentrations of cumene hydroperoxide, epoxide hydrase activity and the stability of the arene oxide substrates tested were unaffected during a 2-min incubation period. The transient nature of benzo(a)pyrene phenol formation was investigated; the 3- and 6-phenols were easily oxidized to quinones by cumene hydroperoxide in a cytochrome P-450-dependent oxidation process which most likely involves the formation of free radicals by a one-electron process. These results indicate that the reaction mechanism operative in the presence of the organic hydroperoxide differs in several regards from that functional in the presence of NADPH and that a common oxidative mechanism may not exist.

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.

Influence of a fibric acid type of hypolipidemic agent on the oxidative metabolism of arachidonic acid by liver microsomal cytochrome P-450

The regiospecificity of arachidonic acid oxygenation, catalyzed by rat liver microsomal fractions in the presence of NADPH, can be altered by animal pretreatment with a fibric acid type of hypolipidemic drug, ciprofibrate. While microsomal fractions isolated from either control or phenobarbital-treated animals oxygenate arachidonic acid to mainly epoxyeicosatrienoic acids (EETs), animal pretreatment with ciprofibrate results in an eightfold stimulation of omega and omega-1 oxidation, concomitant with a net decrease in the formation of both HETEs and EETs. The isomeric composition of the EETs and of the omega and omega-1 oxidation products formed is also dependent on the type of animal pretreatment. Associated decreases in the amounts of HETEs and the rate of hydrogen peroxide formation suggests a modification of the uncoupler action of arachidonic acid during the function of different cytochromes P-450.

The existence of a benzo(a)pyrene-3,6-quinone reductase in rat liver microsomal fractions

Abstract Analysis of repetitive scan difference spectra of incubation mixtures containing liver microsomes from phenobarbital-pretreated rats, benzo(a)pyrene-3,6-quinone, and NADPH reveals the rapid reduction of the quinone to a steady-state level of hydroquinone and the subsequent reoxidation of the hydroquinone. This cyclic process results in NADPH oxidation coupled to oxygen reduction and hydrogen peroxide formation. The reduction of the benzo(a)pyrene-3,6-quinone is not supported by NADH. The initial rate of the NADPH-supported reaction is inhibited by NADP+, metyrapone, and antiNADPH-cytochrome P-450 ( c ) reductase globulin, but not by dicumarol, anaerobiosis, or a gas mixture of carbon monoxide and oxygen (4:1, v v ). These results suggest that cytochrome P-450 and its reductase are involved in this reaction. During the steady-state of metabolism of benzo(a)pyrene by liver microsomes, the 3,6-quinone produced must exist largely as the hydroquinone and may be suitable for disposition as a water-soluble conjugate(s).

Hydrogen peroxide-supported oxidation of benzo [a]pyrene by rat liver microsomal fractions

Abstract In the presence of liver microsomes from phenobarbital-pretreated rats, hydrogen peroxide oxidized benzo [a]pyrene to a number of biologically significant products at a rate that was approximately 20 per cent as fast as that seen by us and others with NADPH and oxygen. As with NADPH-dependent reactions [J. Capdevila, R. W. Estabrook, and R. A. Prough, Archs. Biochem. Biophys.200, 186 (1980)], the hydrogen peroxide-dependent reactions resulted in the production of relatively large quantities of dihydrodiols as metabolites. This was in marked contrast to the product distribution observed when cumene hydroperoxide was utilized as a cosubstrate (foregoing reference). The formation of the various organic-soluble metabolites was dependent on the presence of functional liver microsomal cytochrome P-450 in the reaction mixture. Approximately 48 per cent of the benzo[a]pyrene metabolites, however, was observed to be bound to microsomal protein, and inhibition of cytochrome P-450 function, by metyrapone or N-octylamine did not affect the extent of covalent binding of the hydrocarbon to the microsomal protein. The differences noted during benzo[a]pyrene metabolism using hydrogen peroxide strongly suggest that at least two distinct mechanisms exist to account for the oxidation of the hydrocarbon, i.e. epoxidation and one-electron oxidation reactions.

The microsomal metabolism of benzo(a) pyrene phenols.

Abstract Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region. Based on the chromatographic retention time, absorbance, and fluorescence spectra, the two major products of 9-hydroxybenzo(a)pyrene metabolism may be diphenols. The existence of spectral intermediates which resemble phenols rather than quinones during the steady-state metabolism of 3-hydroxybenzo(a)pyrene strongly indicates that either the major product is a diphenol which slowly oxidizes to yield 3,6-quinone and/or that an active quinone reductase exists in liver microsomes.

The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

Abstract The oxidative metabolism of benzo[ a ]pryrene (B[ a ]P) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-B[ a ]P is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-B[ a ]P. However, the distribution of products obtained with 9-hydroxy-B[ a ]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the 9-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P -450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P -450. These metabolites of the benzo[ a ]pyrene phenols are most likely related to the putative phenol-oxides of benzo[ a ]pyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[ a ]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.

Polycyclic hydrocarbon metabolism: A plethora of phenomena

Studies of benzo(a)pyrene metabolism by liver microsomes have revealed a complex pattern of products formed. The distribution of products generated depends on the types of cytochromes P-450 present and the mechanism of oxidation reactions catalyzed. It is proposed that cytochrome P-450 may function to catalyze the oxidation of benzo(a)pyrene not only by a NADPH-dependent monooxygenation type reaction, but also concomitantly by a peroxidatic type reaction. In particular, attention is directed to the role of the latter type of reaction for the formation of benzo(a)pyrene quinones. The pathways for the metabolic generation of quinones, as well as the reactivity of hydroquinones with oxygen, provide additional pathways for the formation of highly reactive, toxic products which may contribute to the alterations of cellular characteristics that result in either tumor growth or cell death.