Alpha-lipoic acid prevents ethanol-induced protein oxidation in mouse hippocampal HT22 cells Matthias Pirlich a,b , Karoline Kiok b , Grit Sandig b , Herbert Lochs a , Tilman Grune b, * a Department of Gastroenterology and Hepatology, University Hospital Charite ´, Humboldt-University Berlin, Schumannstr. 20/21, 10098 Berlin, Germany b Neuroscience Research Center, University Hospital Charite ´, Humboldt-University Berlin, Schumannstr. 20/21, 10098 Berlin, Germany Received 28 January 2002; received in revised form 9 April 2002; accepted 15 April 2002 Abstract Oxidative stress is involved in a number of neurological disorders, including the neurotoxic effects of ethanol. Recent studies have described a neuroprotective potential of a-lipoic acid (LC) in several models of neuronal cell death related to oxidative stress. We tested the hypothesis that LC could be effective in preventing ethanol-induced neurotoxicity employing the clonal hippocampa cell line HT22. A 24 h incubation with ethanol 100–600 mM caused a dose-dependent loss of cell viability and a significant increase of the overall intracellular protein oxidation. Coincubation with LC 0.1 mM resulted in a significant decrease of ethanol-related neurotoxicity and a complete prevention of the ethanol-induced intracellular protein oxidation. These results indicate that the radical scavenging properties of LC are effective to amelio- rate ethanol-induced neurotoxicity. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: a-Lipoic acid; Ethanol; Neurotoxicity; Oxidative stress; Protein oxidation; HT22 Oxidative stress has been implicated in a number of neuro- logical disorders including neuronal degenerative diseases, ischemia-reperfusion injury and experimental models of neurotoxicity [5,9]. Recently the ethanol-induced toxicity in cultured neurons has also be found to be associated with oxidative stress [8,10], mediated by increased generation of reactive oxygen species and subsequently increased lipid peroxidation. Furthermore, increased protein oxidation and accumulation of oxidatively damaged and inactivated intra- cellular proteins has been suggested to mediate the ethanol- related toxicity in animal models of chronic liver disease [6]. It is tempting to speculate that protein oxidation could also contribute to ethanol-induced neurotoxicity. On the other hand, recent studies have described a neuro- protective potential of a-lipoic acid and its metabolite dihy- drolipoate in several in vitro and in vivo models of neuronal cell death [1,9,12]. a-Lipoic acid is a thiol-replenishing and redox modulating agent, and acts as an effective scavenger of hydroxyl, peroxyl and superoxide radicals in both the aqueous and lipid phases. Regarding ethanol-toxicity bene- ficial effects of a-lipoic acid have been demonstrated in animal models of ethanol induced gastric mucosa damage [7] as well as in cellular models of ethanol-associated toxi- city of acetaldehyde-protein adducts [17]. However, to our knowledge the potential protective effect of a-lipoic acid on ethanol-induced neurotoxicity has not yet been evaluated. In the present study we have investigated the potential protective capacity of a-lipoic acid in a cellular model of ethanol-induced oxidative stress and neurotoxicity employ- ing the clonal mouse hippocampal cell line HT22, which is frequently used to study neurotoxicity related to oxidative stress [2,15]. Cells were maintained in 75 cm 2 flasks using Dulbecco’s Modified Eagle Medium (DMEM) supplemen- ted with 10% of fetal calf serum and 1% glutamine under 5% of CO 2 at 378C. All media, sera and media supplements were from Seromed (Berlin, Germany). All substrates and antiox- idants were from Sigma-Aldrich (Deisenhofen, Germany). Cells were subcultivated before reaching confluence using a seeding density of 5 £ 10 4 cells/cm 2 . The medium was changed three times a week. Before (24 h) experiments were started cells were dissociated and seeded into 25 or 75 cm 2 flasks at a density of 1 £ 10 5 cells per cm 2 . Cell viability was determined by Trypan blue exclusion expressing the number of non-stained (i.e. viable cells) under treatment with ethanol and/or antioxidants as a Neuroscience Letters 328 (2002) 93–96 0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(02)00415-9 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 149-30-450-539041; fax: 149-30- 450-539931. E-mail address: tilman.grune@charite.de (T. Grune).