J Pineal Res 1996; zyxwvutsrqponmlk 20:226-231 Printed in the United States zyxwvutsrqp of America--all rights reserved. Copyrighf zyxw 0 Munksgaard, 1996 ISSN 0742-3098 zyx lournal zyxw of Pineal Research zy In zyxw vitro and in vivo protection against kainate- induced excitotoxicity by melatonin Giusti P, Franceschini D, Petrone M, Manev H, Floreani M. In vitro and in vivo protection against kainate-induced excitotoxicity by melatonin. J. Pineal Res. 1996; 20:226-23 1 . 0 Munksgaard, Copenhagen Abstract: In this study, the protective effect of melatonin against kainate (KA)-induced neurotoxicity was evaluated in vitro and in vivo. In rat brain synaptosomes, KA-induced oxidative stress was measured as shown by significant increases in both the basal generation of reactive oxygen species (ROS), assessed by a fluorescent method, and lipid peroxidation, evaluated as malondialdehyde (MDA) levels. Melatonin decreased, in a concentration-dependent manner, KA-induced lipid peroxidation. The intrinsic fluorescence of melatonin molecule hindered the evaluation of its protective effect against KA-induced ROS generation. However, melatonin was able to reduce FeSOJascorbate-induced ROS generation. The melatonin protective effect was confirmed by in vivo experiments: 73% of rats injected with KA (10 mg/kg i.p.) died within 5 days; melatonin administration i.p. significantly reduced mortality of the animals. The present results suggest that melatonin might be considered a pharmacological agent for the treatment of neurodegenerative pathologies. Introduction Intracerebral or systemic administration of kainate (kainic acid) (KA) causes in rats marked neuronal damage in selective brain areas [Kohler, 19841. KA binds to and activates a subtype of ionotropic recep- tor for the brain neurotransmitter glutamate [Nakanishi, 19921. In addition to inducing lesions directly, KA also triggers epileptiform activity and secondary brain injury that can be reduced by anti- convulsant drugs [Ben-Ari et al., 19791. A central role in the mechanisms of KA-induced neuronal death has been ascribed to an excessive production of reactive oxygen species (ROS) [Puttfarchen, 19931, which increases the release of glutamate [Pellegrini-Giampietro et al., 19901. A vicious cycle takes place, since glutamate per se increases ROS formation [Dickens, 19871. Therefore, it creates a feedback loop that produces progressive neuronal damage. ROS may cause a rapid proliferation of free radical reactions, which are involved in damage of many cellular elements, such as lipids, nucleic ac- ids, and proteins. This effect is particularly evident in the brain, owing to its low content of protective antioxidant compounds [Philips, 19941. In this light, Pietro Giusti,' Davide Franceschini,' Marcella Petrone,' Hari Manev,' and Maura Floreani' zy 'Department of Pharmacology, University of Padova, Padova, Italy; 'Departments of Psychiatry and Pharmacology, Medical College of Pennsylvania and Hahnemann University, Allegheny Campus, Pittsburgh, PA, U.S.A. Key words: melatonin - kainate - ROS - lipid peroxidation - rats - mortality Address reprint requests to Prof. Pietro Giusti, Department of Pharmacology, University of Padova, Largo Meneghetti, 235131 Padova, Italy. Received January 8, 1996; accepted April 2, 1996. it has been found that a significant attenuation in the ability of KA to induce neural damage can be obtained with various lipophilic antioxidants [Puttfarchen, 19931. Direct evidence of ROS involvement in KA neurotoxicity has been obtained in in vitro experi- ments by Bondy and Lee [1993], who reported that KA induced an increase in ROS generation. Recently, a protection effect against KA neuro- toxicity was observed in primary neuronal cultures by melatonin [Giusti et al., 19951. This effect was not associated with the action of melatonin on KA- sensitive glutamate receptor or GABA, receptors. Moreover, the concentration of melatonin was higher than that required for activation of specific melatonin receptors. Thus, it was hypothesized that the neuroprotective effect of melatonin was due to the property of the melatonin molecule itself [Giusti et al., 19953. The antioxidant properties of melatonin have been documented in vitro and in vivo [Reiter, 1995; Reiter et al., 19951. In vitro, melatonin is able to scavenge hydroxyl radicals [Tan et al., 19931 and to reduce the damaging biological action of singlet oxygen [Cagnoli et al., 19951. Furthermore, mela- tonin appears to be a potent peroxyl radical scav- 226