Mercedes Lacort

Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microsomes.

In the present study, the antioxidant effects of estradiol (E2) and 2-hydroxyestradiol (2-OHE2) on microsomal lipid peroxidation induced by Fe3+/ADP/NADPH and Fe2+/ascorbate are described. The extent of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) detection, low-level chemiluminescence, and oxygen consumption. 2-OHE2 had a potent antioxidant activity, which in all cases was higher than that of E2. In the Fe2+/ascorbate model, 2-OHE2 showed a similar pattern of inhibition, irrespective of the presence of NADPH or the functionality of microsomes. However, E2 produced only a slight inhibition when either denatured microsomes or native microsomes without NADPH were used, whereas its protective effect increased considerably when microsomal E2 metabolism was favored. During enzymic Fe3+/ADP/NADPH-induced lipid peroxidation, both E2 and 2-OHE2 were found to provide good protection. Results underline the importance of the chemical structure of these compounds and the role of estradiol metabolism in its antioxidant effects.

PROTECTIVE EFFECT OF ESTROGENS AND CATECHOLESTROGENS AGAINST PEROXIDATIVE MEMBRANE DAMAGE IN VITRO

The antioxidant effects of natural estrogens (estrone E1; 17β-estradiol), synthetic estrogens (17α-ethynylestradiol, EE2; mestranol, MES; diethylstilbestrol, DES) and catechle-strogens (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)-OHE2 and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsómes, and (ii) the antioxidant activities of E1, EE2 and MES were dependent on the assay conditions with the activity being markedley 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.

tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals

tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A(2) (PLA(2)) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA(2)-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA(2) activation, this process being important in TBHP-induced hepatocyte injury.

Effects of estrogens on the redox chemistry of iron : a possible mechanism of the antioxidant action of estrogens

The preventive effects of estrogens on FeSO4-induced lipid peroxidation are closely correlated with their inhibition of Fe(II) oxidation during peroxidation. Estrogens affected the redox status of iron in aqueous solution with varying degrees of effectiveness. 2-Hydroxyestradiol substantially decreased the oxidation of Fe(II) and was the most potent Fe(III) reductant. Diethylstilbestrol and 4-hydroxyestradiol also exhibited reduction properties, whereas the phenolic estrogens 17 beta-estradiol, estrone, and 17 alpha-ethynylestradiol displayed slighter or no effects. Present results demonstrate that catecholestrogens and diethylstilbestrol directly alter the iron redox chemistry, this fact probably being involved in the antioxidant effects of these molecules.

Cytoprotective actions of estrogens against tert-butyl hydroperoxide-induced toxicity in hepatocytes

Estrogens are effective antioxidants in diverse biological systems. Despite their antioxidant activities, it is not known yet whether estrogens prevent or alleviate liver toxicity induced by oxidative stress. In the present work, we studied this possibility by examining in vitro the protective potential of different estrogen compounds (17beta-estradiol, 2-hydroxyestradiol, and diethylstilbestrol) against tert-butyl hydroperoxide-induced hepatocyte damage. Various parameters such as cell viability, lipid peroxidation, adenine nucleotide content, and thiol status were measured as an index of cytotoxicity. The protective effects of estrogens were compared to those of the iron chelator deferoxamine. The molecules tested prevented oxidant-induced cell death differently, showing variable degrees of protection. Deferoxamine was the most potent agent, followed by diethylstilbestrol and 2-hydroxyestradiol, 17beta-estradiol being the least efficient. The inhibitory effects on lipid and thiol oxidations paralleled the effects on cell viability. The molecules also reduced the oxidant-induced ATP depletion, except for 17beta-estradiol which had no effect on the decreased ATP levels. Our results suggest that the mechanisms of the preventive actions of estrogens may be related not only to their antioxidant activity against free radicals, but also and to a lesser extent to the maintenance of the normal redox status of the cell, which partially recovers the intracellular GSH levels.

Cholesteryl ester hydrolysis in rat liver cytosol. Modulation by female sex hormones

The regulation of cholesterol ester hydrolase activity by female sex hormones was studied in cytosolic preparations from female rat liver. The investigation was undertaken in order to determine whether a reduction in the enzyme activity might be responsible for the increased content of esterified cholesterol found in rat liver after estradiol or progesterone treatments. The single injection of estradiol (0.75 mg/100 g) or progesterone (1.50mg/100g) produced respectively significant decreases and increases in sterol hydrolase activity. Both opposite effects were noted after a similar lag period of 3–4 hr and were of short duration. No alterations were observed in rats receiving short-term treatments. When hormones were added to the incubation medium, the activity of cholesterol ester hydrolase decreased progressively with increasing concentrations of hormones. Kinetic studies demonstrate that both estradiol and progesterone compete with the substrate (cholesteryl oleate) for the active center. The findings of the present paper exclude a direct relationship between hepatic hydrolytic activity and lipid deposition. However, they provide evidences that female sex hormones act as modulatory agents of the hydrolysis of cholesteryl esters in rat liver cytosol and suggest that other factors besides competitive inhibition are involved in such regulatory effects.

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.

Effect of estradiol and progesterone on cholesterol 7α-hydroxylase activity in rats subjected to different feeding conditions

The regulation of cholesterol 7 alpha-hydroxylase activity by estradiol and progesterone was investigated in liver microsomes isolated from rats fed standard diet, either ad libitum or fasted for 24 h, and diet containing the bile acid sequesterant cholestyramine. Differential effects were observed when the direct action of estradiol and progesterone on microsome preparations was examined. Cholesterol 7 alpha-hydroxylase activity was inhibited by progesterone in a dose-dependent way to almost complete abolition; similar patterns of declines were found in the three feeding groups under study. In contrast, the addition of 5 microM estradiol induced small and selective 7 alpha-hydroxylase increases in fasting and cholestryamine-fed animals, then activity declined to control values and consistent decreases were found from 20 microM. The administration of estradiol (50 micrograms) or progesterone (100 micrograms) for 21 days resulted in depressed cholesterol 7 alpha-hydroxylase activity in rats with high bile acid synthesis basal rate due to cholestyramine feeding. In rats receiving a standard diet, either ad libitum or after 24 h fasting, the hormonal effects did not reach significance. Declines in the content of free cholesterol were provoked by progesterone, not by estradiol, in liver microsomes prepared from all feeding groups. No changes in cholesterol 7 alpha-hydroxylase activity and microsomal free cholesterol were observed after administration of the sex hormones for 3 days. Rapid and transient inhibitions in 7 alpha-hydroxylase activity were found after the single injection of progesterone to fed animals. Estradiol, on the contrary, was unable to alter rapidly the hepatic 7 alpha-hydroxylase capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal variations of rat liver enzymes catalyzing cholesterol ester hydrolysis

Cholesterol ester hydrolase activity was determined at 3 h time intervals over 24 h in lysosomes, cytosol and microsomes from ad libitum-fed and 24 h food-deprived female rat liver. Diurnal rhythms were identified for the acid and neutral esterases, which were strikingly changed by fasting. In fed animals, lysosomal esterase specific activity exhibited a peak at noon and a sustained medium rate at early darkness, whereas total esterase was maximal at midnight. The circadian patterns of the cytosolic and the microsomal esterases paralleled each other, though the amplitude of rhythms differed, showing higher activities around midnight. After fasting, cholesterol esterase activity from all cell fractions reached a maximum near dark onset. These results are the first to indicate that cholesteryl ester hydrolysis may play a role in generating the diurnal rhythm of hepatic cholesterol.

Topological studies on rat liver microsomal cholesterol ester hydrolase

Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal asymmetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles.