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The antioxidant effects of natural estrogens (estrone, E1; 17 beta-estradiol), synthetic estrogens (17 alpha-ethynylestradiol, EE2; mestranol, MES; diethylstilbestrol, DES) and catecholestrogens (2-hydroxyestradiol; 4-hydroxyestradiol, 4-OHE2) on lipid peroxidation induced by different means in rat liver microsomes were investigated. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances. Prooxidants included Fe3+/ADP/reduced NADPH, Fe2+/ascorbate, tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane) (AAPH). Estrogens and catecholestrogens decreased lipid peroxidation in all four systems tested. In the iron/ascorbate model it was shown that (i) 4-OHE2 and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsomes, and (ii) the antioxidant activities of E1, EE2 and MES were dependent on the assay conditions with the activity being markedly higher when estrogen metabolism was favored. When peroxidation was initiated by the peroxyl radical generator AAPH, the inhibitory effects observed were least pronounced. Our data also showed that, in each of the systems, all inhibitors displayed the same order of inhibitory potency with DES and catecholestrogens being the most potent antioxidants under all experimental conditions used. The present results confirm earlier findings and point toward a link between estrogen metabolism and estrogen antioxidant activity. The data also indicate that estrogens and catecholestrogens interact with the peroxidative process at different levels with their interactions with iron or the metal-derived species being the most important modes of inhibition.

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Reactive oxygen species are important mediators of cellular injury via destruction of membranes or alteration of enzyme activities. The polyunsaturated fatty acids of the membranes are particularly susceptible to free radical attack, ultimately forming lipid hydroperoxides. Iron catalyzes lipid peroxidation because of its ability to react with oxygen to form species capable of initiating peroxidative events. Both Fe(I1) and Fe(II1) are required for the catalysis of lipid peroxidation [ 11.Estrogens and catecholestrogens have been shown to inhibit microsomal lipid peroxidation stimulated by iron and NADPH [2,3], which involves the enzymatic reduction of Fe(II1) by NADPH-cytochrome P450 reductase. Both the reductase system and the cytochrome P450 itself participate also in the metabolic transformations (hydroxylations) of estrogens into catecholestrogens [4].The present work was aimed to investigate if the antioxidant action of estrogens is caused by the inhibition of the reduction of the Fe(III)/ADP complex and/or by interactions of estrogens with the cytochrome P450 activity.We have used rat liver microsomes, prepared as described previously [5], as the lipid source. The microsomes (0.3 mg/ml) were resuspended in Tris/HCl buffer, pH 7.4, and preincubated with the agents dissolved in DMSO (1 %) at 37"C, under air. Reactions were initiated by the addition of Fe3CVADP (25 pM/2.5 mM) and NADPH (200pM). Reduction of Fe(II1) was monitored by estimating the formation rate of the Fe(II)/I , 10-phenantroline complex. NADPHcytochrome P450 reductase activity was estimated according to Lake [6] by following the reduction of cytochrome c at 550 nm for 2 min. The activity of 7-ethoxycoumarin 0-deethylase was measured fluorimetrically [7].As can be seen in Figure 1, both El and E2 inhibited the Fe(II1) reduction in a dose-dependent manner, probably by diverting reduction equivalents from the peroxidation process to its own metabolization. DES similarly affected the Fe(II1) reduction. By contrast, 2-OHE2, the most potent antioxidant [8], increased markedly the formation of Fe(I1) (Fig. I), suggesting a different inhibitory mechanism.Abbreviations used: El, estrone; E2, 17I3-estradiol; DES, diethylstilbestrol; 2-OHE2, 2-hydroxyestradiol. 2-OHE2 E l E 2 DES T Fig. 1. Effects of estrogens on the microsomal Incubations contained FeC13IADPRVADPH (25 pMI2.5 mM/200 pM) without (0) or with the indicated estrogens at the concentrations of 0 1, 5, 10, 15, 25, and 50 pM. Samples were taken at 10 min. Values are means f SD of 3 independent samples.We also studied the effects of these agents on the NADPH-cytochrome P450 reductase activity. 2-Hydroxyestradiol itself reduced cytochrome c and could not be assayed for the reductase activity. The rest of the molecules (El, E2, and DES) had no effects on transfer of electrons from NADPH to oxidized cytochrome c (control = 0.15 lfO.O1 AAsso/min/mg protein). However, the estrogenic compounds inhibited the NADPH-supported monooxygenase activity represented by 7-ethoxycoumarin 0-deethylase. At the con...