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Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates
Neuroscience Letters 323 (2002) 55–59 www.elsevier.com/locate/neulet
Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates S. Ortega-Gutie´rrez a, J.J. Garcı´a a,b, E. Martı´nez-Balları´n a, R.J. Reiter b,*, S. Milla´n-Plano a, M. Robinson a, D. Acun˜a-Castroviejo c a Department of Pharmacology and Physiology, University of Zaragoza, Zaragoza, Spain Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA c Department of Physiology, University of Granada, Granada, Spain
b
Received 29 August 2001; received in revised form 28 November 2001; accepted 29 November 2001
Abstract Deferoxamine (DF) is an antioxidant molecule because of its ability to chelate iron. This study compared the ability of DF alone or in combination with melatonin, 5-methoxytryptophol or pinoline in preventing lipid peroxidation due to hydrogen peroxide (H2O2) in rat brain homogenates. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the homogenates were measured as indices of lipid peroxidation. Incubation of homogenates with DF reduced, in a dose-dependent manner, MDA 1 4-HDA formation due to H2O2. When melatonin, 5-methoxytryptophol or pinoline were added to the incubation medium, the efficacy of DF in preventing lipid peroxidation was enhanced. These cooperative effects between DF, melatonin, and related pineal products may be important in protecting tissues from the oxidative stress due to iron overload. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Melatonin; Deferoxamine; Hydrogen peroxide; Lipid peroxidation; Brain homogenates
Although iron is an essential element in the human, uncontrolled ferrous ions promote lipid peroxidation and oxidative DNA damage, since iron is involved in the formation of hydroxyl radicals (·OH). Deferoxamine (DF) is an iron chelator, isolated from Streptomyces pilosus, that is widely used in the treatment of iron overload [15]. Moreover, DF is proposed as a treatment in ischemia/reperfusion injury [32] and it is known to reduce lipid peroxidation [4]. The protective effects of DF may be explained, at least partially, by its ability to form complexes with iron (thereby reducing ·OH generation) and also because DF reacts directly with ·OH [14,16]. Melatonin is synthesized in the pineal gland of mammals but its production is not limited to this organ [6,9,17,18,27, 29,39,44]. Numerous studies have shown that melatonin is a powerful antioxidant [31,34,41]. In addition to its ability to
* Corresponding author. Department of Cellular and Structural Biology, Mail code 7762, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Tel.: 11-210-567-3860; fax: 11-210-567-6948. E-mail address: [email protected] (R.J. Reiter).
scavenge directly peroxyl radicals (LOO·), ·OH and other reactive oxygen species, melatonin also stimulates enzymes related to the antioxidative defense system [33,42]. The brain is especially susceptible to free radical injury because of its elevated metabolic rate and rich lipid composition. Also, several areas in the brain have a high iron content and it may produce H2O2 in vivo [1,37]. The aim of this study was to test the effect of the combination of DF and melatonin (also 5-methoxytryptophol and pinoline) in preventing lipid peroxidation due to H2O2 in rat brain homogenates. Thirty male Sprague–Dawley rats (225 ^ 25 g body weight) were purchased from Harlan (Barcelona, Spain). The procedures used in these studies were approved by the appropriate animal care committee. After being acclimated for 2 weeks under a light/dark cycle of 12/12 h (lights on at 07:00 h), they were anesthetized with sodium thiopental administered intraperitoneally (60 mg/kg body weight) and perfused through the heart with an ice-cold saline solution (0.9% NaCl) between 10:00 and 13:00 h. The brains were quickly removed, washed in saline and homogenized (1:10 w/v) in 20 mM Tris–HCl buffer (pH 7.4). Aliquots of total brain homogenates (5 mg protein/ml) were incubated
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at 37 8C in a water bath with shaking in the presence of H2O2 at a concentration of either 0.01, 0.1, 0.5, 1, 2.5, 5 or 10 mM for 60 min or at a concentration of 5 mM H2O2 for either 0, 10, 30, 60 or 120 min (Experiment 1). Brain homogenates also were incubated with 5 mM H2O2 for 60 min in the presence or absence of either DF (10, 50, 100, 200, 400, 600, 800 nM), 100 mM melatonin, 100 mM 5-methoxytryptophol or 10 mM pinoline (Experiment 2). Melatonin, 5methoxytryptophol and pinoline concentrations were chosen based on their antioxidative activity as described elsewhere [11]. Lipid peroxidation was stopped by placing the homogenates into ice-cold water for 10 min and the homogenates were then centrifuged at 3000 £ g for 10 min at 4 8C. Malondialdehyde and 4-hydroxyalkenals (MDA 1 4-HDA) concentrations, indices of lipid peroxidation, were measured in the supernatants using a colorimetric commercial assay kit from Calbiochem (San Diego, CA). Results are expressed in nmol MDA 1 4-HDA/mg protein. Protein concentrations were measured by the Bradford method using bovine serum albumin as a standard [3]. Student’s two-tailed t-test was used for comparison of the means. Incubation of the homogenates with H2O2 induced lipid peroxidation in a concentration- and time-dependent manner (Fig. 1). When DF was combined with either melatonin, 5methoxytryptophol or pinoline in the incubation medium, they reduced the stimulatory effect of H2O2 on MDA 1 4HDA levels (Fig. 2). The concentrations of DF in the presence or absence of 100 mM melatonin, 100 mM 5-methoxytryptophol or 10 mM pinoline, each of which reduced MDA 1 4-HDA concentrations by 50% (IC50), are presented in Table 1. Both indoleamines, melatonin and 5-methoxytryptophol, and the b-carboline, pinoline, enhanced the protective effect of DF against lipid peroxidation. A cooperative effect of DF and indoleamines in protecting against the oxidative breakdown of lipids is apparent; this may relate to the antioxidant properties of these agents. The present results are in agreement with previous observations which have documented the antioxidant activity of DF [15]. DF protects against oxidative stress due to reoxygenation after ischemia in lung, skin, kidney, gut and heart, and it also reduces infarct volume following coronary artery occlusion [15,32]. Moreover, DF prevents the toxicity of CCl4 and paraquat, free radical generating toxins [22,46]. DF is not only an iron chelator but it also scavenges directly the ·OH as well as the superoxide radical with rates constant around 10 10 and 10 2 M 21 s 21, respectively [5,15,16]. Melatonin is an endogenous molecule that also reduces oxidative injury due to many processes [8,10,45] including that caused by ischemia/reperfusion, just as DF, in brain, stomach, liver and heart [2,20,23,24,43]. Each molecule of melatonin directly reacts with two ·OH, which are produced from H2O2 in the presence of iron (via the Fenton reaction), yielding a tricyclic compound identified as cyclic 3-hydroxymelatonin [40]. Melatonin also scavenges H2O2 and neutralizes LOO·; the latter are produced as a consequence
Fig. 1. (A) The effect of increasing concentrations of H2O2 on malondialdehyde plus 4-hydroxyalkenals (MDA 1 4-HDA) formation in brain homogenates. The incubation time was 60 min. (B) Time-dependent changes in MDA 1 4-HDA accumulation in brain homogenates following incubation for either 0, 10, 30, 60 or 120 min in the presence (W) or absence (X) of 5 mM H2O2. Values are means ^ standard errors (n ¼ 4). *P , 0:05 versus no H2O2.
of the interaction of ·OH with the phospholipids in biological membranes [28]. Relative to membrane preservation, melatonin stabilizes hepatic microsomal membranes and reduces the rigidity caused by lipid peroxidation induced by FeCl3, ADP and NADPH [12,13]. Both 5-methoxytryptophol and pinoline also improved the antioxidant activity of DF. 5-Methoxytryptophol is an indoleamine derived from serotonin which has free radical scavenging activity as indicated by its ability to reduce the oxidation of a radical trapping reagent, 2,2 0 -azino-bis(3ethyl-benz-thiazoline-6-sulfonic acid) [29]. Pinoline is a b-carboline isolated from the pineal gland and formed by the condensation of indoleamines and aldehydes [4]. Previously, pinoline was shown to reduce nitric oxideinduced lipid peroxidation in retinal homogenates [38] and it protects DNA against oxidative damage induced by chromium [31]. Herein we report that pinoline prevents lipid
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Fig. 2. Protective effect of DF (A) on lipid peroxidation induced by H2O2 (5 mM) in rat brain homogenates. The addition of either 100 mM melatonin (B), 100 mM 5-methoxytryptophol (C) or 10 mM pinoline (D) improved the antioxidant activity of DF. The values represent the means ^ standard errors obtained in five to six independent experiments. *P , 0:05 versus only H2O2.
peroxidation due to H2O2 in brain homogenates [11]. The ability of 5-methoxytryptophol and melatonin to scavenge the ·OH may depend on both the indolic ring and the methoxy group [26]. Although iron chelation and antioxidant abilities of DF suggest its clinical use, high doses of DF result in growth retardation, ototoxicity and ocular toxicity, as well as bone deformities [30]. Moreover, some in vitro studies demonstrated that DF stimulates the formation of nitroxide free Table 1 IC50 concentrations of DF, in the presence or absence of melatonin, 5-methoxytryptophol or pinoline, in preventing lipid peroxidation in rat brain homogenates induced by hydrogen peroxide (5 mM) IC50 (nM) DF DF 1 100 mM melatonin DF 1 100 mM 5-methoxytryptophol DF 1 10 mM pinoline
526.2 472.8 474.4 411.8
radicals and promotes lipid peroxidation [21,35]. In reference to melatonin, no toxicity has been reported to date; also, it has not been possible to define the LD50 for this molecule [34]. Melatonin may allow the use of DF at lower doses, thereby reducing the risk of DF intoxication. DF penetrates slowly into most animal cells and when it is orally administered it is poorly absorbed from the gut [15]. Moreover, DF may interfere with physiological iron absorption from the diet. By contrast, when melatonin is orally administered it is readily absorbed [33]. Furthermore, melatonin easily crosses biological membranes [7] and enters all cells quickly. Many recent studies suggest that melatonin is safe even when given over long periods of time [18,19,36], but there may be some situations in which it may be counterindicated [25]. M.R. is a fellow from the Gobierno de Arago´ n (B036/ 2000), Spain. [1] Beard, J.L., Connor, J.R. and Jones, B.C., Iron in the brain, Nutr. Rev., 51 (1993) 157–170.
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Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates S. Ortega-Gutie´rrez a, J.J. Garcı´a a,b, E. Martı´nez-Balları´n a, R.J. Reiter b,*, S. Milla´n-Plano a, M. Robinson a, D. Acun˜a-Castroviejo c a Department of Pharmacology and Physiology, University of Zaragoza, Zaragoza, Spain Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA c Department of Physiology, University of Granada, Granada, Spain
b
Received 29 August 2001; received in revised form 28 November 2001; accepted 29 November 2001
Abstract Deferoxamine (DF) is an antioxidant molecule because of its ability to chelate iron. This study compared the ability of DF alone or in combination with melatonin, 5-methoxytryptophol or pinoline in preventing lipid peroxidation due to hydrogen peroxide (H2O2) in rat brain homogenates. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the homogenates were measured as indices of lipid peroxidation. Incubation of homogenates with DF reduced, in a dose-dependent manner, MDA 1 4-HDA formation due to H2O2. When melatonin, 5-methoxytryptophol or pinoline were added to the incubation medium, the efficacy of DF in preventing lipid peroxidation was enhanced. These cooperative effects between DF, melatonin, and related pineal products may be important in protecting tissues from the oxidative stress due to iron overload. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Melatonin; Deferoxamine; Hydrogen peroxide; Lipid peroxidation; Brain homogenates
Although iron is an essential element in the human, uncontrolled ferrous ions promote lipid peroxidation and oxidative DNA damage, since iron is involved in the formation of hydroxyl radicals (·OH). Deferoxamine (DF) is an iron chelator, isolated from Streptomyces pilosus, that is widely used in the treatment of iron overload [15]. Moreover, DF is proposed as a treatment in ischemia/reperfusion injury [32] and it is known to reduce lipid peroxidation [4]. The protective effects of DF may be explained, at least partially, by its ability to form complexes with iron (thereby reducing ·OH generation) and also because DF reacts directly with ·OH [14,16]. Melatonin is synthesized in the pineal gland of mammals but its production is not limited to this organ [6,9,17,18,27, 29,39,44]. Numerous studies have shown that melatonin is a powerful antioxidant [31,34,41]. In addition to its ability to
* Corresponding author. Department of Cellular and Structural Biology, Mail code 7762, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Tel.: 11-210-567-3860; fax: 11-210-567-6948. E-mail address: [email protected] (R.J. Reiter).
scavenge directly peroxyl radicals (LOO·), ·OH and other reactive oxygen species, melatonin also stimulates enzymes related to the antioxidative defense system [33,42]. The brain is especially susceptible to free radical injury because of its elevated metabolic rate and rich lipid composition. Also, several areas in the brain have a high iron content and it may produce H2O2 in vivo [1,37]. The aim of this study was to test the effect of the combination of DF and melatonin (also 5-methoxytryptophol and pinoline) in preventing lipid peroxidation due to H2O2 in rat brain homogenates. Thirty male Sprague–Dawley rats (225 ^ 25 g body weight) were purchased from Harlan (Barcelona, Spain). The procedures used in these studies were approved by the appropriate animal care committee. After being acclimated for 2 weeks under a light/dark cycle of 12/12 h (lights on at 07:00 h), they were anesthetized with sodium thiopental administered intraperitoneally (60 mg/kg body weight) and perfused through the heart with an ice-cold saline solution (0.9% NaCl) between 10:00 and 13:00 h. The brains were quickly removed, washed in saline and homogenized (1:10 w/v) in 20 mM Tris–HCl buffer (pH 7.4). Aliquots of total brain homogenates (5 mg protein/ml) were incubated
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 53 0- 7
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at 37 8C in a water bath with shaking in the presence of H2O2 at a concentration of either 0.01, 0.1, 0.5, 1, 2.5, 5 or 10 mM for 60 min or at a concentration of 5 mM H2O2 for either 0, 10, 30, 60 or 120 min (Experiment 1). Brain homogenates also were incubated with 5 mM H2O2 for 60 min in the presence or absence of either DF (10, 50, 100, 200, 400, 600, 800 nM), 100 mM melatonin, 100 mM 5-methoxytryptophol or 10 mM pinoline (Experiment 2). Melatonin, 5methoxytryptophol and pinoline concentrations were chosen based on their antioxidative activity as described elsewhere [11]. Lipid peroxidation was stopped by placing the homogenates into ice-cold water for 10 min and the homogenates were then centrifuged at 3000 £ g for 10 min at 4 8C. Malondialdehyde and 4-hydroxyalkenals (MDA 1 4-HDA) concentrations, indices of lipid peroxidation, were measured in the supernatants using a colorimetric commercial assay kit from Calbiochem (San Diego, CA). Results are expressed in nmol MDA 1 4-HDA/mg protein. Protein concentrations were measured by the Bradford method using bovine serum albumin as a standard [3]. Student’s two-tailed t-test was used for comparison of the means. Incubation of the homogenates with H2O2 induced lipid peroxidation in a concentration- and time-dependent manner (Fig. 1). When DF was combined with either melatonin, 5methoxytryptophol or pinoline in the incubation medium, they reduced the stimulatory effect of H2O2 on MDA 1 4HDA levels (Fig. 2). The concentrations of DF in the presence or absence of 100 mM melatonin, 100 mM 5-methoxytryptophol or 10 mM pinoline, each of which reduced MDA 1 4-HDA concentrations by 50% (IC50), are presented in Table 1. Both indoleamines, melatonin and 5-methoxytryptophol, and the b-carboline, pinoline, enhanced the protective effect of DF against lipid peroxidation. A cooperative effect of DF and indoleamines in protecting against the oxidative breakdown of lipids is apparent; this may relate to the antioxidant properties of these agents. The present results are in agreement with previous observations which have documented the antioxidant activity of DF [15]. DF protects against oxidative stress due to reoxygenation after ischemia in lung, skin, kidney, gut and heart, and it also reduces infarct volume following coronary artery occlusion [15,32]. Moreover, DF prevents the toxicity of CCl4 and paraquat, free radical generating toxins [22,46]. DF is not only an iron chelator but it also scavenges directly the ·OH as well as the superoxide radical with rates constant around 10 10 and 10 2 M 21 s 21, respectively [5,15,16]. Melatonin is an endogenous molecule that also reduces oxidative injury due to many processes [8,10,45] including that caused by ischemia/reperfusion, just as DF, in brain, stomach, liver and heart [2,20,23,24,43]. Each molecule of melatonin directly reacts with two ·OH, which are produced from H2O2 in the presence of iron (via the Fenton reaction), yielding a tricyclic compound identified as cyclic 3-hydroxymelatonin [40]. Melatonin also scavenges H2O2 and neutralizes LOO·; the latter are produced as a consequence
Fig. 1. (A) The effect of increasing concentrations of H2O2 on malondialdehyde plus 4-hydroxyalkenals (MDA 1 4-HDA) formation in brain homogenates. The incubation time was 60 min. (B) Time-dependent changes in MDA 1 4-HDA accumulation in brain homogenates following incubation for either 0, 10, 30, 60 or 120 min in the presence (W) or absence (X) of 5 mM H2O2. Values are means ^ standard errors (n ¼ 4). *P , 0:05 versus no H2O2.
of the interaction of ·OH with the phospholipids in biological membranes [28]. Relative to membrane preservation, melatonin stabilizes hepatic microsomal membranes and reduces the rigidity caused by lipid peroxidation induced by FeCl3, ADP and NADPH [12,13]. Both 5-methoxytryptophol and pinoline also improved the antioxidant activity of DF. 5-Methoxytryptophol is an indoleamine derived from serotonin which has free radical scavenging activity as indicated by its ability to reduce the oxidation of a radical trapping reagent, 2,2 0 -azino-bis(3ethyl-benz-thiazoline-6-sulfonic acid) [29]. Pinoline is a b-carboline isolated from the pineal gland and formed by the condensation of indoleamines and aldehydes [4]. Previously, pinoline was shown to reduce nitric oxideinduced lipid peroxidation in retinal homogenates [38] and it protects DNA against oxidative damage induced by chromium [31]. Herein we report that pinoline prevents lipid
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Fig. 2. Protective effect of DF (A) on lipid peroxidation induced by H2O2 (5 mM) in rat brain homogenates. The addition of either 100 mM melatonin (B), 100 mM 5-methoxytryptophol (C) or 10 mM pinoline (D) improved the antioxidant activity of DF. The values represent the means ^ standard errors obtained in five to six independent experiments. *P , 0:05 versus only H2O2.
peroxidation due to H2O2 in brain homogenates [11]. The ability of 5-methoxytryptophol and melatonin to scavenge the ·OH may depend on both the indolic ring and the methoxy group [26]. Although iron chelation and antioxidant abilities of DF suggest its clinical use, high doses of DF result in growth retardation, ototoxicity and ocular toxicity, as well as bone deformities [30]. Moreover, some in vitro studies demonstrated that DF stimulates the formation of nitroxide free Table 1 IC50 concentrations of DF, in the presence or absence of melatonin, 5-methoxytryptophol or pinoline, in preventing lipid peroxidation in rat brain homogenates induced by hydrogen peroxide (5 mM) IC50 (nM) DF DF 1 100 mM melatonin DF 1 100 mM 5-methoxytryptophol DF 1 10 mM pinoline
526.2 472.8 474.4 411.8
radicals and promotes lipid peroxidation [21,35]. In reference to melatonin, no toxicity has been reported to date; also, it has not been possible to define the LD50 for this molecule [34]. Melatonin may allow the use of DF at lower doses, thereby reducing the risk of DF intoxication. DF penetrates slowly into most animal cells and when it is orally administered it is poorly absorbed from the gut [15]. Moreover, DF may interfere with physiological iron absorption from the diet. By contrast, when melatonin is orally administered it is readily absorbed [33]. Furthermore, melatonin easily crosses biological membranes [7] and enters all cells quickly. Many recent studies suggest that melatonin is safe even when given over long periods of time [18,19,36], but there may be some situations in which it may be counterindicated [25]. M.R. is a fellow from the Gobierno de Arago´ n (B036/ 2000), Spain. [1] Beard, J.L., Connor, J.R. and Jones, B.C., Iron in the brain, Nutr. Rev., 51 (1993) 157–170.
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