Elsevier

Toxicology in Vitro

Volume 16, Issue 1, February 2002, Pages 1-10
Toxicology in Vitro

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation

https://doi.org/10.1016/S0887-2333(01)00094-7Get rights and content

Abstract

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe2+, which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as α-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe2+-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Introduction

During the past decade, along with the studies on hormonal blood pressure regulation, an intensive study of vascular tone regulation by a new class of neurotransmitters such as nitrogen and carbon monoxides has been developed (Moncada et al., 1991). While the action of nitrogen monoxide was investigated in some detail, the role of carbon monoxide, the sources of its formation and the mechanism of its action, have not yet been fully clarified (Verma et al., 1993).

According to the current literature the predominant source of CO formation is heme enzymatic oxidation to biliverdin (Docherty et al., 1984). Another source of its formation was revealed by Guengerich (1978) in a study devoted to the identification of heme degradation products under the action of hydrogen peroxide. The third possible source of CO formation—lipid peroxidation—has been suggested by Archakov et al. (1975) in studying Fe2+ and NADPH-dependent cytochrome b5 reduction in microsomes. These authors have shown that reduction of cytochrome b5 is accompanied by emergence of the absorption peak at 450 nm, indicating the occurrence of endogenous CO formation during lipid peroxidation (LPO). However, this reaction was not studied in sufficient detail. Recently it was shown that CO production is associated with lipid peroxidation in subcellular fractions containing the membranes of brain, kidney, lung, spleen and blood of untreated adult male Wistar rats, while no CO formation was detected in the liver and heart (Vreman et al., 1998). At the same time, CO generation was reported to occur in the course of hepatic microsomal oxidative metabolism of cannabidiol (Usami et al., 1995); pretreatment of mice with phenobarbital (but not with 3-methylcholanthrene) increased the CO formation (Watanabe et al., 1988, Usami et al., 1995).

In the present study, the feasibility of CO formation during NADPH-dependent, Fe2+-stimulated lipid peroxidation was examined in phenobarbital-induced rabbit liver microsomes, the cytochrome P450 2B4 content of which accounts for up to 60% of all the cytochromes P450 in this tissue (Archakov and Bachmanova, 1990). The aim of this work was to clarify the role of the cytochrome P450 during iron-dependent lipid peroxidation.

Section snippets

Preparation of the rabbit phenobarbital-induced liver microsomal fraction

The preparation of the rabbit phenobarbital-induced liver microsomal fraction was carried out as described earlier (Karuzina et al., 1999).

Determination of the endogenous CO formation rate

The incubation mixture contained: 1 mg/ml of microsomal protein; 200 mm Tris–HCl, pH 7.5; 0.4 mm NaPP (Na4P2O7); 200 μm NADPH or the NADPH-generating system. The reaction was started by addition of 24 μm Fe2+ (FeSO4(NH4) 2SO4×6H2O).

The NADPH-generating system contained: 0.3 mm NÀDP+; 8 mm MgCl2; 9 mm glucose-6-phosphate sodium salt; 50 mm nicotinamide; 140

Study of the mechanism of CO formation in microsomes

In order to study the feasibility of CO formation during NADPH, Fe2+-dependent lipid peroxidation, the carbon monoxide formation rate in rabbit liver, phenobarbital-induced microsomes was measured. As seen from Fig. 1, the emergence and sharp increase of the absorption peak at 450 nm as a function of time points to the formation of endogenous CO in the presence of 200 μm NADPH and Fe2+ ions and its subsequent complexation with cytochrome P450.

The amounts of CO formed with 200 μm NADPH or with

Acknowledgements

This Work Was Supported By RFBR Grants N 00-15-97926 and N 99-04-48081.

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