Thiolic antioxidants protect from nitric oxide-induced toxicity in fetal midbrain cultures

Nitric oxide (NO) may act as a neuroprotector or neurotoxic agent in dopamine neurons, depending on cell redox status. We have investigated the effect of several thiolic antioxidants, glutathione (GSH), its cell permeable analog GSH ethyl ester (GSHEE), and the GSH synthesis precursor L-N-acetyl cysteine (L-NAC), as well as non-thiolic antioxidants like ascorbic acid (AA) and uric acid, on NO-induced toxicity in fetal midbrain cultures. The cultures were treated for 8-24 h with neurotoxic doses of the NO donor diethylamine/nitric oxide complex sodium DEA/NO (200-400 muM) and/or antioxidants. Thiolic antioxidants, at equimolar concentrations, added at the same time or previous to DEA/NO, protected from cell death, from tyrosine hydroxylase (TH) positive cell number decrease and from intracellular GSH depletion, induced by DEA/NO, without increasing intracellular GSH content. In these conditions, S-nitrosothiol compound formation was detected in the culture media. Protection disappeared when antioxidants were supplied 30 min after NO treatment. Nevertheless, non-thiolic antioxidants, AA and uric acid, with similar peroxynitrite scavenging activity to thiolic antioxidants, and free radical-scavenging enzymes as catalase and Cu/Zn-superoxide dismutase, which prevent extracellular peroxynitrite ion fort-nation, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), which prevents intracellular peroxynitrite ion formation, did not rescue cell cultures from neurotoxicity induced by NO. In addition, AA exacerbated DEA/NO-induced toxicity in a dose-dependent manner from 200 muM AA. The present results suggest that only antioxidants with thiol group exert neuroprotection from NO-induced toxicity in fetal midbrain cultures, probably by direct interaction of NO and thiol groups, resulting in NO blocking. On the other hand, some classical antioxidants, like AA, exacerbate neurotoxicity due to NO. (C) 2002 Elsevier Science Ltd. All rights reserved.