CYTOCHROME P-450 ONDER CONDITIONS

or OXIDATIVE STRESS:

ROLE OF ANTIOXIDANT RECYCLING IN THE PROTECTION MECHANISMS E. Serbinova, S. Ivanova, A. Kirova, S. Kitanova, L. Packer, and V. Kagan Institute of Physiology, Bulgarian Academy of Sciences Sofia, 1113 Bulgaria University of California, Berkeley CA 94720, USA Institute of Medical and Biomedical Problems Moscow, USSR Free radicals initiate lipid peroxidation of microsomal membranes by rapidly forming lipid hydroperoxides (LOOH) from endogenous polyunsaturated fatty acid residues of phospholipids. Lipid hydroperoxides may in turn propagate lipid peroxidation via the cytochrome P-450-dependent mechanism. Indeed cytochrome P-450 catalyzes the oxidative cleavage of lipid hydroperoxides to a pool of a1koxyl- (LO) and peroxyl(LOO) radicals which induce an additional formation of lipid hydroperoxides. Propagation and termination of lipid peroxidat ion in the cell are accompanied by a concomitant accumulation of numerous lipid peroxidation products possessing different effects on biomembranes and membrane bound enzymes (Halliwell and Gutteridge, 1985; Kagan, 1988). However, these products originate from hydroperoxides, the primary molecular lipid peroxidation products, which can be reduced to corresponding hydroxy-compounds by peroxidases (Christophersen, 1969; Ursini et al., 1985) thus preventing the formation of various scission products.Lipid peroxidation is considered to be an efficient triggering mechanism of the disassembly of microsomal membranes and cytochrome P-450. An inverse correlation exists between the steady-state concentrations of lipid hydroperoxides and cytochrome P-450 content in liver endoplasmic reticulum membranes (Kagan et aI, 1974). This makes it reasonable to believe that lipid peroxidation activation triggers the chain of reactions leading in the end to disappearance of cytochrome P-450. Can it be concluded that lipid hydroperoxides themselves are responsible for cytochrome P-450 degradation? The first shade of doubt appears when one compares kinetic curves of cytochrome P-450 destruct ion in liver microsomal membranes with the curves of lipid

Oxygen Transport /0 TIJISIII! XIII, Ildited by T.K. Goldstick e/ al. PIe"..". Press, New York, 1992

223

hydroperoxides and secondary lipid peroxidation products (TBAreacting carbonyl compounds), (Fig.1). In this in vitro system' the time-course of lipid hydroperoxides passes through a' maximum, whereas cytochrome P-450 declines following monotonous kinetics as does the accumulation of TBA-reactive substances. Thus it seems unlikely that primary lipid peroxidation products, hydroperoxides, are directely involved in cytochrome P-450 disassembly.

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Fig. 1 Accumulation of lipid hydroperoxides. TSARS and degradation of cytochrome P-450 in rat liver microsomes incubated with Fe-ADP (20 IlMl20 Ill) + NADPH 500llM

This question will remain open until the effect of hydroperoxides only, (but not of the mixture of different lipid peroxidation products including hydroperoxides as is in the case of lipid peroxidation induction by Fe 2 + + NADPH) is clarified. Accumulation of lipid hydroperoxides as the only (or at least predominant) lipid peroxidation products can be achieved by the use of reticulocyte lipoxygenase (Schewe et al, 1981; Lankin et al, 1985). Typical UV-spectra of rat liver microsomes incubated in the presence of reticulocyte lipoxygenase are shown in Fig.2. Addition of increasing amounts of the enzyme led to the increased absorption at 234 nm, characteristic of lipid hydroperoxides with conjugated dienes (in the reference compartment lipoxygenase and microsomes were present in the different cuvettes), whereas no peaks in the region of 270-280 nm, characteristic of secondary lipid peroxidation products appeared. Under the conditions used, 224

addition of 70J.Ll of lipoxygenase resulted in conversion of approximately 15% membrane phospholipids into phospholipid hydroperoxides. However, this caused neither significant destruction of cytochrome P-450 nor its conversion into cytochrome P-420 (Fig.3). Thus it can be concluded that phospholipid hydroperoxides accumulating among the other lipid peroxidation products in the course of lipid peroxidation are not responsible for cytochrome P-450 destruction.

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In contrast to reactions (2) and (3) where chromanoxyl radicals are irreversibly lost interaction of chromanoxyl regenerates radicals with reductants [reaction (4)] 226

antioxidant molecules, which can be repeatedly used for formation of chromanoxyl radicals. Under the conditions used, alpha-tocopherol gave characteristic pentameric ESR spectra of chromanoxyl radicals with g-values of the components 2.0122, 2.0092, 2.0061, 2.0028 and 1.9993. In the presence of ascorbate ESR signal of ascorbyl radical was observed first, which decayed and was progressively substituted by the appearing and growing signal of the tocopheroxyl radical. After reaching the maximal magnitude, the signals of tocopheroxyl radical followed typical decay kinetics (Fig. 4B).

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Fig. 4 ESR spectra (B) and time-course of chromanoxyl radicals (A) generated from alpha-tocopherol by lipoxygenase+ linolenic acid oxidation system in the presence of rat liver microsomes and their recycling by NADPH and ascorbate. Conditions: microsomes 27 mg protein/mi. linolenic acid 14 mM, lipoxygenase 90 Utili. alpha-tocopherol 8 mM, NADPH 5 mM, ascorbyl palmitate 2.5 mM.

The addition of another reductant, NADPH, to the incubation medium caused a pronounced decrease in the magnitude of ESR signals of tocopheroxyl radical (Fig. 4A). The signals reappeared, but their magnitudes increased in the course of subsequent incubation and coincided with the decay curves obtained in the absence of NADPH (Fig. 4B). This transient NADPH-dependent disappearance of the ESR signal of phenoxyl radicals was found to occur not only with alpha-tocopherol but also with other phenoxyl radicals such as alpha-tocopherol homologues, butylated hydroxy-toluene and its homologues, (Kagan et al, 1990, 1990a) It is well known that microsomal cytochrome P-450 supported reactions are able to generate radical intermediates from their substrates in the course of monooxygenase reactions 227

(Utsumi et al.. 1990; Epe and Metzler, 1985). These radical intermediates can be both suicidal for cytochrome P-450 and damaging for other neighboring macromolecules (Rahimtula, 1983). Our data gives an evidence for the presence of a "selfdefense" mechanism in the monooxygenase system, which is capable of reducing radical species to nondangerous stable molecules. However, under harsh oxidative conditions, this free radical reductase protective mechanism is probably not efficient enough to maintain steady-state vitamin E concentration in membranes. We investigated the effects of different kinds of oxidative stress on cytochrome P-450. Oxidative stress was induced either by long-term exposure of rats to hypokinesia (45 days) or by short-term exposure to iron loading, exhausttive physical exersise, hyperoxia or a combination of these factors.The data in Table 1 shows that intramuscular injection of iron (Ferrum Hausmann, single dose, 500 mg iron/kg b.w.) to rats results in the development of oxidative stress, viz. a decrease in vitamin E content and increase in the amount of lipid peroxidation products. It is documented that accumulation of lipid peroxidation products in microsomal membranes results in destruction of cytochrome P-450 (Kitada et aI, 1989; Iba et Mannering, 1987) ,but in this study we found only a slight decrease in cytochrome P-450 content in iron-loaded rats (11% decrease of the control, p