Oxidase and Oxygenase Function of the Microsomal Cytochrome P450 Monooxygenase System

Abstract

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of 0; radicals by microsomes, a stoichiometry of 0; to Hz02 close to 2 : 1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2Oz formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of HzO2.The rates of 0; as well as HzO2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H202 formation dropped to 55 "/,.It was concluded that most of the 0; originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H202 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H202 formation was only slightly decreased at 20 pM dioxygen suggesting that H202 formation via cytochrome P450 should also occur in vivo.After the early observation by Gillette et al. [I] it was confirmed repeatedly that liver microsomes generate hydrogen peroxide during the oxidation of NADPH [2-41. The reaction seemed to be linked to the monooxygenation system for xenobiotics, since pretreatment of the animals with phenobarbital or pregnenolone-l6a-carbonitrile increased the rate of H202 formation [3], and inhibitors of cytochrome P450 affected the production of H202 and the N-demethylation of ethylmorphine in a similar fashion [4]. However, the underlying mechanisms of hydrogen peroxide formation are not well understood [5]. It was found that some substrates of the monooxygenase system stimulate Hz02 formation whereas others are either not effective or are effective only in animals pretreated with phenobarbital but not with pregnenolone16a-carbonitrile [3,4]. A correlation of H202 formation with the spectral binding and hydroxylation of hexobarbital was observed by Heinemeyer et al. [6] from which a hypothetical peroxide complex of cytochrome P450 was postulated as a common intermediate for Hz02 formation and substrate hydroxylation.If this hypothesis is correct, an increased H202 formation should be observable upon addition of pseudo-substrates [7,8] of the microsomal cytochrome P450 monooxygenase system. This was reinvestigated in the present study.As a second possible pathway of NADPH-dependent formation of H202 in liver microsomes the release of superoxide anion radicals by autoxidation of microsomal redox systems, and the subsequent disproportionation of these Saarland, Homburg-Saar, Federal Republic of German...

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