Ronald W. Estabrook

The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with a cytosolic epoxide hydrolase

Epoxyeicosatrienoic acids, formed during the cytochrome P-450-catalyzed oxidation of arachidonic acid, react with a liver cytosolic epoxide hydrolase to form vicinal diols of eicosatrienoic acid. The role of this cytosolic enzyme, rather than a microsomal bound type, explains previous results illustrating the ability to accumulate epoxides during the in vitro aerobic steady state of oxidative metabolism of arachidonic acid by liver microsomes. The inability of the 5,6-epoxyeicosatrienoic acid to serve as a suitable substrate for this enzyme is discussed in light of recent studies concerning possible unique physiological functions for this metabolite.

Heterogeneity of liver microsomal cytochrome P-450: The spectral characterization of reactants with reduced cytochrome P-450☆

Abstract The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.

Possible higher valence states of cytochrome P-450 during oxidative reactions

Abstract The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.

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.

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).

Transformation of an arachidonic acid hydroperoxide into epoxyhydroxy and trihydroxy fatty acids by liver microsomal cytochrome P-450

In the absence of NADPH, the addition of an arachidonic acid hydroperoxide, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, to liver microsomes, prepared from phenobarbital-treated rats, resulted in the formation of two major metabolites and several minor products, some of which have been purified by reverse-phase high-performance liquid chromatography. We propose the structures of the two major products to be 13-hydroxy-14,15-epoxyeicosa-5,8,11-trienoic acid and 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid based on spectral characteristics and mass spectral analysis of derivatives of the compounds. A potential heterolytic cleavage product, 15-hydroxyeicosa-5,8,11,13-tetraenoic acid, was not a product of the reaction. Ferric cytochrome P-450 catalyzed the formation of these products as shown by the inability of boiled microsomes to support the reaction, the inhibition of epoxyhydroxy and trihydroxy fatty acid formation by imidazole derivatives which bind tightly to the ferric heme iron of cytochrome P-450, and the inability of carbon monoxide (which binds to ferrous P-450) and free iron chelators (EDTA and diethylenetriaminepentaacetic acid) to inhibit product formation. These results show that liver microsomal cytochrome P-450, in addition to its role in the NADPH-dependent metabolism of arachidonic acid, can utilize a hydroperoxide to produce an interesting series of potentially important arachidonic acid metabolites.

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 effect of extra bound cytochrome b5 on cytochrome P-450-dependent enzyme activities in liver microsomes

Abstract Binding of increasing amounts of detergent-purified cytochrome b 5 to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome b 5 in these two reactions functions as an intermediate electron carrier to cytochrome P-450.

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.

Stimulation of NADH oxidation during NADPH dependent microsomal electron transport reactions.

Abstract Initiation of NADPH oxidation by rat liver microsomes results in a marked stimulation of the rate of NADH oxidation. The amount of NADH rapidly oxidized depends on the ratio of NADPH NADH and the presence of various substrates for the function of cytochrome P-450. The present paper illustrates a simple direct method of quantitatively assessing these changes in NADH oxidation and the influence of the anaesthetic, halothane, or the drug, ethylmorphine, on the pattern of changes in NADH oxidation during microsomal electron transport reactions.