Potent protective effect of melatonin on in vivo paraquat-induced oxidative damage in rats

Life Sciences, Vol. 56, No. 2, pp. 83-89, 1995 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/95 $9_5O + .00

Pergamon 0024-3205(94)00417-X

POTENT PROTECTIVE EFFECT OF MELATONIN ON IN VIVO PARAQUAT-INDUCED OXIDATIVE DAMAGE IN RATS Daniela Melchiorri t'2, Russel J. Reiter t.3,Ahmed M. Attia ~, Masayuki Hara t, Alejandro Burgos3, Giuseppe Nistico 'z. tDepartment of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Cud Drive, San Antonio Texas 78284-7762, U.S.A. 2Department of Biology, University of Rome "Tot Vergata", Rome 00173, Italy. (Received in final form October 17, 1994) Summary The in vivo effect of melatonin on paraquat-induced oxidative damage in rat lung and liver was studied using two parameters: the concentration of malonaldehyde and 4hydroxyalkenals as indices of lipid peroxidation; changes in total and oxidized glutathione. Melatonin (10 mg/kg) or an equal volume of saline were administered intraperitoneally (ip) to rats 30 min prior to an ip injection of paraquat (20mg/kg or 70mg/kg). After paraquat treatment, the animals received melatonin or saline ip injections every six hours for 24 hours. Rats were killed 24 hours after paraquat injection. In lung, both the low and high dose of paraquat, when administered with saline, augmented lipid peroxidation (100% and 18%, respectivly) above levels found in control animals. Treatment with melatonin completely reversed this effect. In liver, paraquat (70 mg/kg) increased lipid peroxidation by 40% over the levels of control animals. The increase was completely abolished by treatment with melatonin. Paraquat at 20mg/kg did not induce any significant change in liver lipid peroxidation. Paraquat treatment resulted in a significant decrease of total glutathione concentration and increased oxidized glutathione in both lung and liver. These effects were abolished by treatment with melatonin. The results suggest that melatonin confers marked protection against paraquat-induced oxidative toxicity in both the lung and liver. Key Words: melatonin,paraquat, fipid peroxidation,lung, liver

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a commonly used herbicide which has caused many deaths either accidently or after its deliberate ingestion. Paraquat causes extensive damage of human and animal lungs, liver and kidneys (1,2). Hepatic injury consists of centrolobular necrosis and fatty metamorphosis (1). Pulmonary damage is manifested by edema, hemorrhage, interstial inflammation and proliferation of bronchial epithelial cells (3). Death is usually caused by pulmonary impairment. The great sensitivity of lung to paraquat is probably due to the presence of a sodium-independent uptake mechanism in epithelial cells which increases local concentrations of the compound (4).

3Russel J. Reiter, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio 78284-7762, U.S.A.

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In vitro studies have proposed the following mechanism for paraquat toxicity: the compound easily undergoes redox cycling by NADPH-cytochrome C reductase with subsequent production of oxygen free radicals and oxidative damage to membrane lipids (2,5). Lipid peroxidation is thus generally believed to be a biochemical event in paraquat toxicity (6). Several pharmacological strategies have been used to reduce paraquat induction of reactive oxygen species with the intent of lowering its toxicity.Antioxidants such as catalase, superoxide dismutase reduced glutathione, vitamin E and ascorbic acid have been tested in paraquat-exposed humans and animals (7-9). The results, however, have not been satisfactory. None of these agents greatly reduced the in vivo toxic actions of the herbicide, probably because of their inability to cross cell membrane barriers and/or their rapid clearance from cells. Recently the pineal hormone, melatonin (5-methoxy-N-acetyl tryptamine), was found to have potent free radical scavenging activity (10). Differently from other known low molecular weight antioxidants, melatonin, once oxidized, cannot be reduced or involved in regenerating processes that may cause autooxidative radical formation or toxic redox recycling (11). Moreover, owing to its highly lipophilic nature, melatonin can provide efficient on-site protection to potentially all biomolecules within every cellular compartment (12,13). In the present study we examined the in vivo protective effect of melatonin on paraquat-induced oxidative damage in rat lung and liver utilizing the measurement of two different parameters: concentration of malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) as indices of lipid peroxidation and changes in total and oxidized glutathione. Materials and Methods Chemicals, All reagents were of the highest quality available. Paraquat (purity = 98%) was purchased from Chem Service (West Chester, PA). Melatonin, saturated picric acid, NADPH tetrasodium salt, 5-5'-dithio-bis(2-nitrobenzoicacid)(DTNB), reduced glutathione (GSH) and glutathione reductase (GSSG-R) were obtained from Sigma (St. Louis, Mo). 2-vinyl-pyridin monomer was purchased from Fluka (Ronkonkoma, N.Y.) Methods. The experiment was conducted in May. Adult male Sprague-Dawley rats (body weight 125-135 g) were obtained from Harlan, Houston, and housed in Plexiglass cages with 3 per cage. The animal rooms were windowless with automatic temperature controls (22+2 °C) and lighting (light on 07.00h and off 21.00h; 14h light/10h dark). The rats received standard laboratory chow and water ad libitum. Paraquat was dissolved in saline and administered ip in two different doses: 20 mg/kg (LoPQ) and 70 mg/kg (HiPQ). Melatonin was dissolved in absolute ethanol (the alcohol concentration in the final solution was 4%) and administered ip in one single dose of 10 mg/kg. Animal treatment. After one week of acclimation, the animals were divided into 6 groups of 8 animals each. Treatment began 24 hours before sacrifice. Each group received 2 ip injections within a 30 min interval. Group 1 (control) received two injections of saline. Group 2 received an injection of saline followed by a LoPQ injection. Group 3 was injected with melatonin and received LoPQ. Group 4 was treated with saline and HiPQ. Group 5 received melatonin and HiPQ. Group 6 was injected with melatonin and saline. After paraquat treatment, the animals received melatonin or saline injections every 6 hours for 24 hours.

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Groups 1, 2 and 4 were injected with saline; groups 3, 5 and 6 received melatonin. The aim of this injection scheme was to keep constantly high melatonin levels following paraquat adminstration. When the injections occurred at night, they were performed under dim red-light; this wavelength and light intensity are not capable of influencing nighttime pineal melatonin synthesis. The animals were killed at night, 24 hours after treatment began. Tissue preparation and assays. Soon after decapitation, the animal's abdomen was opened and heart was perfused by injectiong ice-cold saline. Lungs and liver were removed and cooled on dry ice. Approximately 100 mg of lung and liver were homogenized with Wheaton sticcer in icecold 20raM Tris-HC1 buffer, pH 7.4, to produce a 1/10 homogenate. The homogenate was centrifuged at 2500 g for 30 min at 4°C. The supernatant was collected and immediately tested for lipid peroxidation. Malonaldehyde (MDA) and 4-hydroxyalkenals concentrations provide a convenient index of lipid peroxidation (14). The Bioxytech LPO-586 kit, purchased from Cayman Chemical (Ann Arbor, MI) was used. The kit takes advantage of a chromogenic reagent which reacts with MDA and 4-HDA at 45°C yielding a stable chromophore with maximal absorbance at the 586nm wavelength. The light wavelength and the low temperature of incubation used for these measurements eliminate interference and undesiderable artefacts. Total [reduced (GSH) and oxidized glutathione (GSSG)] and oxidized glutathione (GSSG) concentrations in lung and liver homogenates were determined as described by Griffith (15). Proteins were determined by the Lowry procedure, with albumin as standard (16). Statistical analysis. All data were analyzed by one-way analysis of variance (ANOVA). If the F values were significant, the Student-Newman-Keuls test was used to compare the treated and control groups. The level of significance was accepted at p <0.05. Results Lung and liver lioid peroxidation. At 70 mg/kg dose, paraquat, when followed by saline injections, augmented lipid peroxidation in the lungs by 100% above levels found in control animals (Fig. I, A). When rats were treated with melatonin, the increase induced by paraquat was prevented. The low paraquat dose (20 mg/kg) itself increased lipid peroxidation in the lung by 18% above levels found in control animals.The increase was abolished by treatment with melatonin. Melatonin alone did not change the level of lipid peroxidation in lungs compared to control animals (Fig. 1, A). In the liver, the HiPQ induced a 40% increase in lipid peroxidation compared to control animals. The effect was reversed by treatement with melatonin (Fig.l, B). The LoPQ did not produce a significant increase in lipid peroxidation (Fig.l, B). Total and oxidized ~lutathione. Lung total glutathione levels after 70mg/kg paraquat were significantly lower than those of control animals (Fig.2, A). The decrease was abolished by treatment with melatonin. Likewise, liver total glutathione levels were significantly reduced after the administration of HiPQ (Fig.2, B). All measured decreases in total glutathione were accompanied by a concomitant increase in GSSG concentrations (Fig. 3, A and B). These data suggest that glutathione was oxidized following paraquat treatment and the administration of melatonin provided protection against GSH oxidation.

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Two high dose paraquat-treated animals died 18 hours following the herbicide injection. Their death was probably due to pulmonary impairment as the animals showed acute respiratory distress. On the other hand, none of the high dose-paraquat and melatonin-treated animals died or showed acute respiratory deficiencies.

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Discussion The results of this study show that melatonin confers potent protection against paraquat-induced lung and liver oxidative damage. It is well known that exposure of biological membranes to oxidative stress results in progressive degeneration of membrane structure and loss of activity. The measurement of lipid peroxidation is thus a convenient method to monitor oxidative cell damage (17). Malonaldehyde and 4-hydroxyalkenals are important products of peroxides derived from polynsaturated fatty acids and related esters (14). Consequently, the increase of such aldehydes, caused by paraquat administration, represent lung and liver peroxidative damage. These observations are in agreement with other reports which indicate that lipid peroxidation is a mechanism by which paraquat produces toxicity in vivo (18-22). Furthermore, in vitro studies (5) have demonstrated that paraquat toxicity is due to its generation of superoxide radicals. Paraquat is converted to a paraquat radical in the presence of NADPH and its radical rapidly reacts with molecular oxygen to generate superoxide anions. These may serve as a source for hydrogen peroxide as well as reduce Cu(II) or Fe(III) to the corresponding cuprous and ferrous states. Cu(I) or Fe(II), thus formed, bind to biological macromolecules and may react with hydrogen peroxide in a site-specific Fenton reaction to yield hydroxyl radicals, potent initiators of lipid peroxidation. Also, the paraquat radical has been reported to be a more powerful Fe(III) reductant than the hydroxyl radical, effectively out-competing the latter for metal ion in most reaction systems (23). Recently, melatonin was reported to be a potent endogenous hydroxyl radical scavenger (10,12,13). Thus, we suggest that melatonin protection against paraquat toxicity may be due, at least in part, to its radical scavenging capacity. Furthermore, considering the high level of oxidative damage protection following melatonin administration, other mechanisms of action cannot be excluded.

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This interpretation of our results is consistent with the finding that melatonin completely protects against paraquat-induced oxidation of GSH which serves as a substrate in the GSH peroxidase/GSSG reductase system to detoxify lipid peroxides. The role of GSH in protecting cells from peroxidative damage induced by several xenobiotics has been, in fact, reported by many investigators (24). The higher levels of lipid peroxidation products detected in lung compared to liver are consistent with the energy-dependent accumulation of paraquat in lung (6) and with the particular structure and function of this organ. Lung oxygen content is in fact much greater than liver and paraquat toxicity is augmented by a high amount of oxygen (25). Also, lung lipid content is very high. As this paper was being returned to the jorunal for final consideration, another report appeared also suggesting melatonin is an important protector against lipid peroxidation. Thus, Pieri et al (26) provide evidence that melatonin is twice as potent as vitamin E in scavenging the peroxyl radical. Acknowledgments Supported by NSF grant IBN 91-21262. References

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