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Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microsomes
Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microsomes M a Begofia Ruiz-Larrea,*t Ana M a Leal,* Mariana Liza,* Mereedes Lacort,* and Herbert de Groott * Departamento de Fisiologia, Facultad de Medicina, Universidad del Pais Vasco, Bilbao, Spain; t Klinische Forschergruppe Leberschiidi#un#, Institut fiir Physiolo#ische Chemie L Heinrich-Heine-Universitiit Diisseldorf, Diisseldorf, Germany
In the present study, the antioxidant effects of estradiol (E2) and 2-hydroxyestradiol (2-OHE2) on microsomal lipid peroxidation induced by Fe 3 +/ A D P / N . 4 D P H and Fe 2 +/ascorbate are described. The extent of lipid peroxidation was measured by thiobarbituric acid reactive substances ( TBARS ) detection, low-level chemiluminescence, and oxygen consumption. 2-OHE 2 had a potent antioxidant activity, which in all cases was higher than that of E 2. In the Fe 2 +/ascorbate model, 2-OHE2 showed a similar pattern of inhibition, irrespective of the presence of N A D P H or the functionality of microsomes. However, E2 produced only a slight inhibition when either denatured microsomes or native microsomes without N A D P H were used, whereas its protective effect increased considerably when microsomal E 2 metabolism was favored During enzymic Fe 3+/ADP/NADPH-induced lipid peroxidation, both E 2 and 2- OHE: werefound 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. (Steroids 59:383-388, 1994)
Keywords:steroids;estradiol;2-hydroxyestradiol;rnicrosomallipid peroxidation;antioxidantaction
Introduction Estrogen treatments produce cytotoxic effects on liver and other tissues which have been associated with the oxidative metabolism of these compounds. 1-4 In the endoplasmic reticulum, estrogens are mainly converted into 2- and 4-hydroxyestrogens (catechol estrogens) by an NADPH-dependent cytochrome P450-1inked monooxygenase system) Oxidation of catechols generates, in turn, chemically reactive species (quinones, semiquinones, and superoxide anions), capable of consuming intracellular antioxidant defenses, in particular the reduced glutathione (GSH). 6-9 When hepatotoxic agents utilize GSH at a rate exceeding the hepatic ability to regenerate or synthesize it, the GSH depletion leads to an oxidative stress. This situation results in metabolic alterations and changes in membrane fluidity and/or in intracellular calcium homeostasis, which may account for early stages of cytotoxicity. Addressreprintrequeststo Dr. M"BegofiaRuiz-Larrea,Departamento de Fisiologia,Facultadde Medicina,Universidaddel Pais Vasco,P.O. Box 699, Bilbao,Spain. ReceivedAugust31, 1993; accepted December21, 1993
© 1994 Butterworth-Heinemann
In a recent work, we showed that 17fl-estradiol (E2) added to hepatocyte suspensions brought about a sharp dose-dependent decrease of GSH levels, thus underlining the importance of this detoxifying pathway for the elimination of reactive metabolites. 1° E 2 w a s also found to modify the cytosolic redox state and activate NADPH-regenerating systems in intact liver cells, tl't2 as has been described for a number of other toxic molecules. 13-i 5 Lipid peroxidation is an important process initiated by free radicals, which is involved in the events leading to cell death. Its rate has been reported to be higher in chicken erythrocytes ~6 and hamster kidney 17 after E 2 treatments. However, in other studies, a protective role has been claimed for estrogens and catechol estrogens as free radical scavengers. 18'19 According to these reports, we previously demonstrated antioxidant properties of E 2 against endogenous lipid peroxidation in isolated rat hepatocytes. 1° At present, we have investigated the effects of E2 and 2-hydroxyestradiol (2-OHE2) on iron-dependent lipid peroxidation in microsomes of rat liver, so as to characterize better the role of estrogens and catechol estrogens in the peroxidation process. The extent of lipid peroxidation was measured using
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Papers t h i o b a r b i t u r i c acid reactive substances (TBARS) detection, low-level chemiluminescence, a n d oxygen uptake.
Experimental
increase during the peroxidation phase. The lag time %, which precedes the onset of the peroxidation phase, was determined by extrapolation of the linear phase of chemiluminescence increase during the peroxidation phase to the baseline level, 23
Preparation of microsomes
Oxygen consumption
Male Sprague-Dawley rats weighing 180 g were given food and water ad libitum. For the preparation of hepatic microsomes, rats were anesthetized and the livers were perfused with saline and homogenized in 250 mM sucrose. Liver homogenates were centrifuged at 100 x 9 for 10 rain at 0-4 C, to eliminate nuclear debris. The supernatant was centrifuged at 22,000 x 9 for 20 rain and again at 105,000 × 9 for 60 rain. The microsomal pellet was washed once in 150 mM Tris/HC1 buffer, pH 8, and finally resuspended in 150mM Tris/HCl buffer, pH 7.4. Denaturation of microsomes was performed by a heat treatment (30rain at 80C). The microsomal protein was determined according to Bradford, using bovine serum albumin as standard. 2°
The rate of oxygen uptake was measured using a Clark-type oxygen electrode. 24 The volume of the reaction mixture was 1.4 mL and oxygen uptake was continuously recorded. The peroxidation induction system was added to the chamber by means of a microsyringe.
Incubation systems Liver microsomes (400-600#g of protein per mL) were preincubated in 150 mM Tris/HCl buffer, pH 7.4, with the chemicals dissolved in 5/~L dimethylsulfoxide (DMSO) (1%) in the absence or the presence of 200/~M NADPH, The same volume of D M S O alone was added to control incubates. After 10 rain preincubation, lipid peroxidation was initiated by the addition of either a) 25 IbM FeSO4, 100 pM ascorbate, and 10 mM KH2PO 4, or b) 2 #M FeCI3/200/~M ADP (previously complexed) and 200/~M NADPH. All additions are expressed as final concentrations in the incubation system, total volume 500 #L. Incubations were carried out at 37 C under air in a bath shaker. Other assay details are specified in the legends to the figures.
Assay for lipid peroxidation Lipid peroxidation was monitored by the formation of TBARS. 2~ Reactions were stopped at selected incubation times by adding 5%TCA. After centrifugation, 350/~L of the supernatant was added to 700 #L of 0.67% thiobarbituric acid (TBA), and this solution was heated in a boiling water bath for 15 rain. The mixture was allowed to cool and absorbance was measured at 535nm. Microsomal lipid peroxidation is expressed as nanomole of malondialdehyde (MDA) per milligram of protein. MDA values were calculated using a molar extinction coefficient of 156 m M - l c m - ~ at 535 nm. All values are the means of three determinations. In some experiments incubations were halted with TCA containing 0.02% butylated hydroxytoluene (BHT). The addition of BHT had no effect on the amount of MDA measured. Neither E 2 nor 2-OHE 2 interfered with the TBA assay. When the molecules were added to the reaction mixture at the end of the incubation time before the addition of TBA, no protection of lipid peroxidation was observed.
Results P e r o x i d a t i o n of m i c r o s o m a l lipids was s t i m u l a t e d by either one of the following systems: i) F e 3 + / A D P / NADPH enzymic system a n d it) F e 2 + / a s c o r b a t e n o n - e n z y m i c d e p e n d e n t system. In each case, previous e x p e r i m e n t s were d o n e in o r d e r to define the o p t i m a l c o n d i t i o n s of reaction.
TBA RS formation The effect of different c o n c e n t r a t i o n s of E z on the time course of a c c u m u l a t i o n of T B A R S initiated by F e 3 + / A D P / N A D P H was studied ( F i g u r e 1). M i c r o s o m e s were p r e i n c u b a t e d for 10 min with 200 # M N A D P H a n d E 2 ( 0 . 1 - 5 0 p M ) in D M S O . R e a c t i o n was initiated by a d d i t i o n of F e 3 + / A D P c o m p l e x (2/200llM). E z prevented the a c c u m u l a t i o n of T B A R S in a d o s e - d e p e n d e n t m a n n e r . At 0.1/~M c o n c e n t r a t i o n the h o r m o n e had no effect, whereas at 10 /~M the h o r m o n e c o m p l e t e l y suppressed lipid p e r o x i d a t i o n from the first 5 m i n i n c u b a t i o n , the half m a x i m a l c o n c e n t r a t i o n being 5/~M. D M S O itself h a d no effect on the reaction. W h e n N A D P H was a d d e d s i m u l t a n e o u s l y with the i n i t i a t o r system (zero time) no differences in p e r o x i d a t i o n values were observed. W e then studied the efficiency of E z in p r e v e n t i n g lipid p e r o x i d a t i o n using a n o n - e n z y m i c o o.1 5 1o 25 50
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Low-level chemiluminescence Low-level chemiluminescence was measured with a singlephoton counting-system equipped with a red-sensitive photomultiplier (EMI 9658 AM) and cooled to - 2 2 C by a thermoelectric cooler. 12 Measurements were carried out in a glass cuvette and the reaction mixture was maintained at 37 C under constant stirring during the measuring period. During the induction phase of lipid peroxidation, photoemission remained at about background level, followed by a stronger
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F i g u r e 1 Effect of E2 on time course of TBARS accumulation initiated by Fe3+/ADP/NADPH. Microsomes were preincubated for 10 rain with E2. At zero time, peroxidation was initiated by addition of Fea+/ADP/NADPH. Other experimental conditions were as described in the text. The M D A values are the means of 3 independent experiments with mean errors less than 10%,
Antioxidant action of estradiol: Ruiz-Larrea et al.
iron-dependent system. Microsomes were preincubated with the estrogen and reaction was started by 25/100/~M Fe 2 +/ascorbate. Figure 2 shows that E 2 (10 #M) blocked markedly peroxidation only when the reduced nicotinamide coenzyme was present in the incubation medium, whereas in its absence the steroid caused only a partial inhibition of lipid peroxidation during the first min of incubation (maximal 47% inhibition after 5min). Additional experiments were performed with denatured microsomes. Total amounts of TBARS in control tubes were similar to those in native microsomes peroxidized by the same system (Figure 3). Analysis of kinetics of MDA accumulation in the presence of Ez revealed that, irrespective of NADPH, the hormone had an effect
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F i g u r e 4 Antioxidant effects of 2-OHE 2 on microsoma[ lipid peroxidation induced by Fe3+/ADP/NADPH or Fe2+/ascorbate. Microsomes were preincubated for 1 0 m i n with 2-OHE 2 at the indicated final concentrations. At zero time the peroxidative system was added, and after 60 rain incubation M D A measured. Details of experiments as described in the text. Values are means +SE of 3 independent experiments.
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F i g u r e 2 Effect of E2 on time course of TBARS accumulation initiated by Fe2+/ascorbate. Microsomes were preincubated for 10 min with 10/~M E2 dissolved in 5 #L DMSO either in the absence or the presence of NADPH. Controls contained 5/~L DMSO. At zero time Fe2+/ascorbate was added and M D A measured at the indicated times. Details of experiments as described in the text. Curves are representative of 3 independent experiments.
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Figure 5 Antioxidant action of 2-OHE2 on Fe2+/ascorbate induced lipid peroxidation from native or denatured microsomes. Native and denatured microsomes were incubated with 2-OHE2 (10 #M) and Fe2+/ascorbate as described in the text. Values are means of three determinations with a single batch of microsomes. The individual values did not differ by more than 5% from the mean value.
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tion in denatured microsomes. Heat denatured microsomes were assayed under the experimental conditions described in the legend to Figure 2. The values at each point are the means of three determinations with a single batch of microsomes. The individual values did not differ by more than 5% from the mean value.
similar to that observed with native microsomes in the absence of N A D P H (54% inhibition after 5 min). We studied the effects of 2-OHE2 on microsomal lipid peroxidation. Figure 4 shows the concentration dependencies of the inhibiting effects of 2-OHE 2 during Fe 2 +/ascorbate and Fe 3 +/ADP/NADPH induced lipid peroxidation. The catechol estrogen did not require NADPH to exert its antioxidant effects in the Fe 2 +/ascorbate system. Moreover, the same results were obtained when denatured microsomes were used as the lipid source (Figure 5).
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Papers non-enzymic system, where the lag doubling concentrations of E 2 and 2-OHE~ were 5.4 and 0.21~M, respectively, compared to 1.5 and 0.7pM, in the Fe 3+/ADP/NADPH system.
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The initial step of lipid peroxidation was monitored by oxygen consumption. Control incubations presented a constant oxygen consumption rate of 5.2 nmol/min. The addition of FeZ+/ascorbate caused a strong oxygen consumption, which increased linearly with incubation time after a short lag phase. Both E 2 and 2-OHE 2 inhibited in a dose-dependent manner the oxygen consumption initiated by the non-enzymic peroxidative system. However, the mode of inhibition was different: E z did not modify the lag phase, but changed the rate of oxygen consumption, whereas 2-OHE2 prolonged the lag time before oxygen consumption, slightly affecting the maximal rate (Figure 7).
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Figure 6 Effects of E2 and 2-OHE 2 on the lag time (~0) of low-level chemiluminescence during lipid peroxidation induced by a) Fe3+/ADP/NADPH and b) Fe2+/ascorbate. Microsomes were incubated with the steroids and low-level chemiluminescence was monitored continuously. Ratio of lag time in the presence of antioxidant to the lag time in control (~/~0) is plotted versus concentration of antioxidant. Values are means of 2-4 independent experiments with mean errors less than 10%.
Chemiluminescence study Figure 6 shows the efficiencies of E 2 and 2-OHE2 in preventing iron-induced lipid peroxidation measured by low-level chemiluminescence. Experimental conditions were similar to those in the above experiments. Microsome samples were preincubated with the steroid for 10 rain prior to addition of the peroxidative system (Fe2+/ascorbate or Fe3+/ADP/NADPH). The cuvette was maintained at 37 C with constant stirring during incubation. The lag time was determined by extrapolation of the linear level according to Cadenas et al. 23 In control microsomes, the lag time ranged from 1 to 20 min, possibly depending on the initial concentration of endogenous antioxidants after microsome isolation. We, thus, represented the ratio of lag time in presence of antioxidant (~) to the lag time in its absence (%) versus the concentration of steroid. The antioxidant capacity was expressed as concentration of compound required to double lag time of control. In both iron-induced peroxidative systems E z and 2-OHE2 exhibited protection in a concentration-dependent manner. However, 2-OHE/ presented an inhibitory efficiency greater than E 2. This difference was more pronounced in the
386
Steroids, 1994, vol. 59, June
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Figure 7 Time-course of oxygen uptake during F e 2 + / a s c o r b a t e induced lipid peroxidation in rat liver microsomes incubated with a) E2 and b) 2-OHE2. Oxygen consumption was measured in an incubation system containing liver microsomes (400 #g/mL) and Fe2+/ascorbate in the absence and the presence of a range of a) E2 (2-10/~M) and b) 2-OHE 2 (0.1-0.5/~M) concentrations. Other incubation conditions as described in the text. Dotted lines represent the oxygen consumption in control incubations without peroxidation induction system. Curves are representative of 3 independent experiments.
Antioxidant action of estradiol: Ruiz-Larrea et al.
Discussion This study describes the antioxidant effects of E2 and 2-OHE2 on microsomal iron-induced lipid peroxidation. 2-OHE2 had a potent antioxidant activity, which in all cases was higher than that of E2. In the Fe z +/ascorbate model, the catechol estrogen showed a similar pattern of inhibition irrespective of either the presence of NADPH or the functionality of microsomes. In contrast, E 2 behaved differently depending on both the presence of NADPH and the integrity of microsomes. In studies with either denatured microsomes or native microsomes without NADPH (Figure 3) E 2 produced only a slight inhibition. At present, we cannot explain the mechanisms underlying this effect, but we believe that it is related to the chemical structure of the estrogen. Lipid peroxidation is a complex process, which can be considered as a sequence of events initiated by a hydrogen atom abstraction, followed by reaction of oxygen with the subsequently formed radical, and by further free radical chain reactions. Iron ions play a critical role, since they catalyze the first step of peroxidation, as well as accelerate the decomposition of lipid hydroperoxides that usually contaminate microsomal fractions. Data obtained from the experiments of oxygen uptake (Figure 7) suggested an inhibition in the early stages of lipid peroxidation (suppression of oxygen uptake). A variety of antioxidants with phenolic structure are known to act by their iron chelating ability, thus blocking at this level the peroxidation process. 25"26 Furthermore, estradiol, like other protective molecules, may act by regenerating endogenous antioxidants present in membranes, and so delaying the appearance of lipid peroxides. In this respect, certain estrogens have been described to have the ability to regenerate tocopheroxyl radical to tocopherol in benzene solutions. 27 On the other hand, phenolic compounds have free radical chain-breaking properties: they efficiently would donate a hydrogen atom from their phenolic hydroxyl group to a peroxyl or alkoxyl radical, so interfering with the propagation of lipid peroxidation. 2a,29 In conditions where microsomal metabolism was favored, the protective effect of E 2 in the Fe 2 +/ascorbate system was considerably increased (Figure 2). The oxidative transformation of E2 involves specific P450 monooxygenases and produces 2- and 4-hydroxyestradiol as major metabolites. Our results indicated that the additional hydroxylation in the A ring of the steroid would be the cause for these increased effects. The higher antioxidant properties of catechol estrogens are probably due to a superior stability of radical derived from catechol compared to that of phenoxyl radical. The strong inhibition exhibited by 2-OHE 2 makes it difficult to know whether the metabolism of 2-OHE2 is involved in its effects. E2 was found to provide good protection against the enzymic iron-dependent lipid peroxidation. During Fe3+/ADP/NADPH-induced lipid peroxidation, cytochrome P450 reductase as well as reducing cytochrome
P450, have been thought to act by donating electrons to some Fe(III)-complexes and so generating Fe(II), which stimulates peroxidation. 2s This reductase system also catalyzes the estrogen metabolization, so that E2 may protect lipid peroxidation by inhibiting iron reduction. This competition was dearly observed in experiments of low-level chemiluminescence, where E 2 produced a striking concentration-dependent decrease of the chemiluminescence slope (data not shown). In conclusion, EE and 2-OHE2 showed antioxidant activities on iron-induced lipid peroxidation in liver microsomes. Results strongly suggest that these effects are due in great part to their own chemical structure. Since these molecules may interfere at different stages in the peroxidation process, further studies are needed to clarify the underlying inhibitory mechanism.
Acknowledgments This work was supported by Scientific Research Grants (UPV 081.327-E070/91 and UPV 081.327-EA101/92) from the University of the Basque Country and by the Deutsche Forschungsgemeinschaft (Str 92/4-3). Ana M. Leal has been awarded a fellowship from the Spanish Education Ministry. We are grateful to David Hallet for reading the manuscript.
References 1.
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Haaf H, Li SA, Li JJ (1987). Covalent binding of estrogen metabolites to hamster liver microsomal proteins: inhibition by ascorbic acid and catechol-O-methyl tranferase. Carcinogenesis 8:209-215. Lamartiniere CA, Pardo GA (1988). Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment. J Biochem Toxicol 3:87-103. Morgan P, Maggs JL, Bulman-Page PC, Hussain F, Park BK (1992). The metabolism of 2- and 4-fluoro-173-oestradiol in the rat and its implications for oestrogen carcinogenesis. Biochem Pharmacol 43:985-994. Lax ER (1987). Mechanisms of physiological pharmacological sex hormone action on the mammalian liver. J Steroid Biochem 27:1119-1128. Jellink PH, Michnovicz JJ, Bradlow HL (1991). Influence of indole-3-carbinol on the hepatic microsomal formation of catechol estrogens. Steroids 56:446-450. Kalyanaraman B, Scaly RC, Liehr JG (1989). Characterization of semiquinone free radicals formed from stilbene catechol estrogens. An ESR spin stabilization and spin trapping study. J Biol Chem 264:11014-11019. Liehr JG, Roy D (1990). Free radical generation by redox cycling of estrogens Free Radic Biol Med 8:415-423. Roy D, Liehr JG (1988). Temporary decrease in renal quinone reductase activity induced by chronic administration of estradiol to male Syrian hamsters. Increased superoxide formation by redox cycling of estrogen. J Biol Chem 263:3646--3651. Roy D, Strobel HW, Liehr JG (1991). Cytochrome b5-mediated redox cycling of estrogen. Arch Biochem Biophys 285:331-338. Ruiz-Larrea MB, Garrido MJ, Lacort M (1993). Estradiolinduced effects on glutathione metabolism in rat hepatocytes. J Biochem 113: 563-567. Astiazar;in AM, Ochoa B, Lacort M (1988). Estradiolproduced modifications on the response to glucose of hepatocytes from fasted rats. Biochem Med Metab Biol 40:197-203.
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In the present study, the antioxidant effects of estradiol (E2) and 2-hydroxyestradiol (2-OHE2) on microsomal lipid peroxidation induced by Fe 3 +/ A D P / N . 4 D P H and Fe 2 +/ascorbate are described. The extent of lipid peroxidation was measured by thiobarbituric acid reactive substances ( TBARS ) detection, low-level chemiluminescence, and oxygen consumption. 2-OHE 2 had a potent antioxidant activity, which in all cases was higher than that of E 2. In the Fe 2 +/ascorbate model, 2-OHE2 showed a similar pattern of inhibition, irrespective of the presence of N A D P H or the functionality of microsomes. However, E2 produced only a slight inhibition when either denatured microsomes or native microsomes without N A D P H were used, whereas its protective effect increased considerably when microsomal E 2 metabolism was favored During enzymic Fe 3+/ADP/NADPH-induced lipid peroxidation, both E 2 and 2- OHE: werefound 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. (Steroids 59:383-388, 1994)
Keywords:steroids;estradiol;2-hydroxyestradiol;rnicrosomallipid peroxidation;antioxidantaction
Introduction Estrogen treatments produce cytotoxic effects on liver and other tissues which have been associated with the oxidative metabolism of these compounds. 1-4 In the endoplasmic reticulum, estrogens are mainly converted into 2- and 4-hydroxyestrogens (catechol estrogens) by an NADPH-dependent cytochrome P450-1inked monooxygenase system) Oxidation of catechols generates, in turn, chemically reactive species (quinones, semiquinones, and superoxide anions), capable of consuming intracellular antioxidant defenses, in particular the reduced glutathione (GSH). 6-9 When hepatotoxic agents utilize GSH at a rate exceeding the hepatic ability to regenerate or synthesize it, the GSH depletion leads to an oxidative stress. This situation results in metabolic alterations and changes in membrane fluidity and/or in intracellular calcium homeostasis, which may account for early stages of cytotoxicity. Addressreprintrequeststo Dr. M"BegofiaRuiz-Larrea,Departamento de Fisiologia,Facultadde Medicina,Universidaddel Pais Vasco,P.O. Box 699, Bilbao,Spain. ReceivedAugust31, 1993; accepted December21, 1993
© 1994 Butterworth-Heinemann
In a recent work, we showed that 17fl-estradiol (E2) added to hepatocyte suspensions brought about a sharp dose-dependent decrease of GSH levels, thus underlining the importance of this detoxifying pathway for the elimination of reactive metabolites. 1° E 2 w a s also found to modify the cytosolic redox state and activate NADPH-regenerating systems in intact liver cells, tl't2 as has been described for a number of other toxic molecules. 13-i 5 Lipid peroxidation is an important process initiated by free radicals, which is involved in the events leading to cell death. Its rate has been reported to be higher in chicken erythrocytes ~6 and hamster kidney 17 after E 2 treatments. However, in other studies, a protective role has been claimed for estrogens and catechol estrogens as free radical scavengers. 18'19 According to these reports, we previously demonstrated antioxidant properties of E 2 against endogenous lipid peroxidation in isolated rat hepatocytes. 1° At present, we have investigated the effects of E2 and 2-hydroxyestradiol (2-OHE2) on iron-dependent lipid peroxidation in microsomes of rat liver, so as to characterize better the role of estrogens and catechol estrogens in the peroxidation process. The extent of lipid peroxidation was measured using
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Papers t h i o b a r b i t u r i c acid reactive substances (TBARS) detection, low-level chemiluminescence, a n d oxygen uptake.
Experimental
increase during the peroxidation phase. The lag time %, which precedes the onset of the peroxidation phase, was determined by extrapolation of the linear phase of chemiluminescence increase during the peroxidation phase to the baseline level, 23
Preparation of microsomes
Oxygen consumption
Male Sprague-Dawley rats weighing 180 g were given food and water ad libitum. For the preparation of hepatic microsomes, rats were anesthetized and the livers were perfused with saline and homogenized in 250 mM sucrose. Liver homogenates were centrifuged at 100 x 9 for 10 rain at 0-4 C, to eliminate nuclear debris. The supernatant was centrifuged at 22,000 x 9 for 20 rain and again at 105,000 × 9 for 60 rain. The microsomal pellet was washed once in 150 mM Tris/HC1 buffer, pH 8, and finally resuspended in 150mM Tris/HCl buffer, pH 7.4. Denaturation of microsomes was performed by a heat treatment (30rain at 80C). The microsomal protein was determined according to Bradford, using bovine serum albumin as standard. 2°
The rate of oxygen uptake was measured using a Clark-type oxygen electrode. 24 The volume of the reaction mixture was 1.4 mL and oxygen uptake was continuously recorded. The peroxidation induction system was added to the chamber by means of a microsyringe.
Incubation systems Liver microsomes (400-600#g of protein per mL) were preincubated in 150 mM Tris/HCl buffer, pH 7.4, with the chemicals dissolved in 5/~L dimethylsulfoxide (DMSO) (1%) in the absence or the presence of 200/~M NADPH, The same volume of D M S O alone was added to control incubates. After 10 rain preincubation, lipid peroxidation was initiated by the addition of either a) 25 IbM FeSO4, 100 pM ascorbate, and 10 mM KH2PO 4, or b) 2 #M FeCI3/200/~M ADP (previously complexed) and 200/~M NADPH. All additions are expressed as final concentrations in the incubation system, total volume 500 #L. Incubations were carried out at 37 C under air in a bath shaker. Other assay details are specified in the legends to the figures.
Assay for lipid peroxidation Lipid peroxidation was monitored by the formation of TBARS. 2~ Reactions were stopped at selected incubation times by adding 5%TCA. After centrifugation, 350/~L of the supernatant was added to 700 #L of 0.67% thiobarbituric acid (TBA), and this solution was heated in a boiling water bath for 15 rain. The mixture was allowed to cool and absorbance was measured at 535nm. Microsomal lipid peroxidation is expressed as nanomole of malondialdehyde (MDA) per milligram of protein. MDA values were calculated using a molar extinction coefficient of 156 m M - l c m - ~ at 535 nm. All values are the means of three determinations. In some experiments incubations were halted with TCA containing 0.02% butylated hydroxytoluene (BHT). The addition of BHT had no effect on the amount of MDA measured. Neither E 2 nor 2-OHE 2 interfered with the TBA assay. When the molecules were added to the reaction mixture at the end of the incubation time before the addition of TBA, no protection of lipid peroxidation was observed.
Results P e r o x i d a t i o n of m i c r o s o m a l lipids was s t i m u l a t e d by either one of the following systems: i) F e 3 + / A D P / NADPH enzymic system a n d it) F e 2 + / a s c o r b a t e n o n - e n z y m i c d e p e n d e n t system. In each case, previous e x p e r i m e n t s were d o n e in o r d e r to define the o p t i m a l c o n d i t i o n s of reaction.
TBA RS formation The effect of different c o n c e n t r a t i o n s of E z on the time course of a c c u m u l a t i o n of T B A R S initiated by F e 3 + / A D P / N A D P H was studied ( F i g u r e 1). M i c r o s o m e s were p r e i n c u b a t e d for 10 min with 200 # M N A D P H a n d E 2 ( 0 . 1 - 5 0 p M ) in D M S O . R e a c t i o n was initiated by a d d i t i o n of F e 3 + / A D P c o m p l e x (2/200llM). E z prevented the a c c u m u l a t i o n of T B A R S in a d o s e - d e p e n d e n t m a n n e r . At 0.1/~M c o n c e n t r a t i o n the h o r m o n e had no effect, whereas at 10 /~M the h o r m o n e c o m p l e t e l y suppressed lipid p e r o x i d a t i o n from the first 5 m i n i n c u b a t i o n , the half m a x i m a l c o n c e n t r a t i o n being 5/~M. D M S O itself h a d no effect on the reaction. W h e n N A D P H was a d d e d s i m u l t a n e o u s l y with the i n i t i a t o r system (zero time) no differences in p e r o x i d a t i o n values were observed. W e then studied the efficiency of E z in p r e v e n t i n g lipid p e r o x i d a t i o n using a n o n - e n z y m i c o o.1 5 1o 25 50
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Low-level chemiluminescence Low-level chemiluminescence was measured with a singlephoton counting-system equipped with a red-sensitive photomultiplier (EMI 9658 AM) and cooled to - 2 2 C by a thermoelectric cooler. 12 Measurements were carried out in a glass cuvette and the reaction mixture was maintained at 37 C under constant stirring during the measuring period. During the induction phase of lipid peroxidation, photoemission remained at about background level, followed by a stronger
384
S t e r o i d s , 1 9 9 4 , v o l , 59, J u n e
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F i g u r e 1 Effect of E2 on time course of TBARS accumulation initiated by Fe3+/ADP/NADPH. Microsomes were preincubated for 10 rain with E2. At zero time, peroxidation was initiated by addition of Fea+/ADP/NADPH. Other experimental conditions were as described in the text. The M D A values are the means of 3 independent experiments with mean errors less than 10%,
Antioxidant action of estradiol: Ruiz-Larrea et al.
iron-dependent system. Microsomes were preincubated with the estrogen and reaction was started by 25/100/~M Fe 2 +/ascorbate. Figure 2 shows that E 2 (10 #M) blocked markedly peroxidation only when the reduced nicotinamide coenzyme was present in the incubation medium, whereas in its absence the steroid caused only a partial inhibition of lipid peroxidation during the first min of incubation (maximal 47% inhibition after 5min). Additional experiments were performed with denatured microsomes. Total amounts of TBARS in control tubes were similar to those in native microsomes peroxidized by the same system (Figure 3). Analysis of kinetics of MDA accumulation in the presence of Ez revealed that, irrespective of NADPH, the hormone had an effect
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F i g u r e 2 Effect of E2 on time course of TBARS accumulation initiated by Fe2+/ascorbate. Microsomes were preincubated for 10 min with 10/~M E2 dissolved in 5 #L DMSO either in the absence or the presence of NADPH. Controls contained 5/~L DMSO. At zero time Fe2+/ascorbate was added and M D A measured at the indicated times. Details of experiments as described in the text. Curves are representative of 3 independent experiments.
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Figure 5 Antioxidant action of 2-OHE2 on Fe2+/ascorbate induced lipid peroxidation from native or denatured microsomes. Native and denatured microsomes were incubated with 2-OHE2 (10 #M) and Fe2+/ascorbate as described in the text. Values are means of three determinations with a single batch of microsomes. The individual values did not differ by more than 5% from the mean value.
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tion in denatured microsomes. Heat denatured microsomes were assayed under the experimental conditions described in the legend to Figure 2. The values at each point are the means of three determinations with a single batch of microsomes. The individual values did not differ by more than 5% from the mean value.
similar to that observed with native microsomes in the absence of N A D P H (54% inhibition after 5 min). We studied the effects of 2-OHE2 on microsomal lipid peroxidation. Figure 4 shows the concentration dependencies of the inhibiting effects of 2-OHE 2 during Fe 2 +/ascorbate and Fe 3 +/ADP/NADPH induced lipid peroxidation. The catechol estrogen did not require NADPH to exert its antioxidant effects in the Fe 2 +/ascorbate system. Moreover, the same results were obtained when denatured microsomes were used as the lipid source (Figure 5).
Steroids, 1994, vol. 59, June
385
Papers non-enzymic system, where the lag doubling concentrations of E 2 and 2-OHE~ were 5.4 and 0.21~M, respectively, compared to 1.5 and 0.7pM, in the Fe 3+/ADP/NADPH system.
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The initial step of lipid peroxidation was monitored by oxygen consumption. Control incubations presented a constant oxygen consumption rate of 5.2 nmol/min. The addition of FeZ+/ascorbate caused a strong oxygen consumption, which increased linearly with incubation time after a short lag phase. Both E 2 and 2-OHE 2 inhibited in a dose-dependent manner the oxygen consumption initiated by the non-enzymic peroxidative system. However, the mode of inhibition was different: E z did not modify the lag phase, but changed the rate of oxygen consumption, whereas 2-OHE2 prolonged the lag time before oxygen consumption, slightly affecting the maximal rate (Figure 7).
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Figure 6 Effects of E2 and 2-OHE 2 on the lag time (~0) of low-level chemiluminescence during lipid peroxidation induced by a) Fe3+/ADP/NADPH and b) Fe2+/ascorbate. Microsomes were incubated with the steroids and low-level chemiluminescence was monitored continuously. Ratio of lag time in the presence of antioxidant to the lag time in control (~/~0) is plotted versus concentration of antioxidant. Values are means of 2-4 independent experiments with mean errors less than 10%.
Chemiluminescence study Figure 6 shows the efficiencies of E 2 and 2-OHE2 in preventing iron-induced lipid peroxidation measured by low-level chemiluminescence. Experimental conditions were similar to those in the above experiments. Microsome samples were preincubated with the steroid for 10 rain prior to addition of the peroxidative system (Fe2+/ascorbate or Fe3+/ADP/NADPH). The cuvette was maintained at 37 C with constant stirring during incubation. The lag time was determined by extrapolation of the linear level according to Cadenas et al. 23 In control microsomes, the lag time ranged from 1 to 20 min, possibly depending on the initial concentration of endogenous antioxidants after microsome isolation. We, thus, represented the ratio of lag time in presence of antioxidant (~) to the lag time in its absence (%) versus the concentration of steroid. The antioxidant capacity was expressed as concentration of compound required to double lag time of control. In both iron-induced peroxidative systems E z and 2-OHE2 exhibited protection in a concentration-dependent manner. However, 2-OHE/ presented an inhibitory efficiency greater than E 2. This difference was more pronounced in the
386
Steroids, 1994, vol. 59, June
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Figure 7 Time-course of oxygen uptake during F e 2 + / a s c o r b a t e induced lipid peroxidation in rat liver microsomes incubated with a) E2 and b) 2-OHE2. Oxygen consumption was measured in an incubation system containing liver microsomes (400 #g/mL) and Fe2+/ascorbate in the absence and the presence of a range of a) E2 (2-10/~M) and b) 2-OHE 2 (0.1-0.5/~M) concentrations. Other incubation conditions as described in the text. Dotted lines represent the oxygen consumption in control incubations without peroxidation induction system. Curves are representative of 3 independent experiments.
Antioxidant action of estradiol: Ruiz-Larrea et al.
Discussion This study describes the antioxidant effects of E2 and 2-OHE2 on microsomal iron-induced lipid peroxidation. 2-OHE2 had a potent antioxidant activity, which in all cases was higher than that of E2. In the Fe z +/ascorbate model, the catechol estrogen showed a similar pattern of inhibition irrespective of either the presence of NADPH or the functionality of microsomes. In contrast, E 2 behaved differently depending on both the presence of NADPH and the integrity of microsomes. In studies with either denatured microsomes or native microsomes without NADPH (Figure 3) E 2 produced only a slight inhibition. At present, we cannot explain the mechanisms underlying this effect, but we believe that it is related to the chemical structure of the estrogen. Lipid peroxidation is a complex process, which can be considered as a sequence of events initiated by a hydrogen atom abstraction, followed by reaction of oxygen with the subsequently formed radical, and by further free radical chain reactions. Iron ions play a critical role, since they catalyze the first step of peroxidation, as well as accelerate the decomposition of lipid hydroperoxides that usually contaminate microsomal fractions. Data obtained from the experiments of oxygen uptake (Figure 7) suggested an inhibition in the early stages of lipid peroxidation (suppression of oxygen uptake). A variety of antioxidants with phenolic structure are known to act by their iron chelating ability, thus blocking at this level the peroxidation process. 25"26 Furthermore, estradiol, like other protective molecules, may act by regenerating endogenous antioxidants present in membranes, and so delaying the appearance of lipid peroxides. In this respect, certain estrogens have been described to have the ability to regenerate tocopheroxyl radical to tocopherol in benzene solutions. 27 On the other hand, phenolic compounds have free radical chain-breaking properties: they efficiently would donate a hydrogen atom from their phenolic hydroxyl group to a peroxyl or alkoxyl radical, so interfering with the propagation of lipid peroxidation. 2a,29 In conditions where microsomal metabolism was favored, the protective effect of E 2 in the Fe 2 +/ascorbate system was considerably increased (Figure 2). The oxidative transformation of E2 involves specific P450 monooxygenases and produces 2- and 4-hydroxyestradiol as major metabolites. Our results indicated that the additional hydroxylation in the A ring of the steroid would be the cause for these increased effects. The higher antioxidant properties of catechol estrogens are probably due to a superior stability of radical derived from catechol compared to that of phenoxyl radical. The strong inhibition exhibited by 2-OHE 2 makes it difficult to know whether the metabolism of 2-OHE2 is involved in its effects. E2 was found to provide good protection against the enzymic iron-dependent lipid peroxidation. During Fe3+/ADP/NADPH-induced lipid peroxidation, cytochrome P450 reductase as well as reducing cytochrome
P450, have been thought to act by donating electrons to some Fe(III)-complexes and so generating Fe(II), which stimulates peroxidation. 2s This reductase system also catalyzes the estrogen metabolization, so that E2 may protect lipid peroxidation by inhibiting iron reduction. This competition was dearly observed in experiments of low-level chemiluminescence, where E 2 produced a striking concentration-dependent decrease of the chemiluminescence slope (data not shown). In conclusion, EE and 2-OHE2 showed antioxidant activities on iron-induced lipid peroxidation in liver microsomes. Results strongly suggest that these effects are due in great part to their own chemical structure. Since these molecules may interfere at different stages in the peroxidation process, further studies are needed to clarify the underlying inhibitory mechanism.
Acknowledgments This work was supported by Scientific Research Grants (UPV 081.327-E070/91 and UPV 081.327-EA101/92) from the University of the Basque Country and by the Deutsche Forschungsgemeinschaft (Str 92/4-3). Ana M. Leal has been awarded a fellowship from the Spanish Education Ministry. We are grateful to David Hallet for reading the manuscript.
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