Research Report Effects of N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12 Suman Penugonda a , Suneetha Mare a , Glenn Goldstein b , William A. Banks c , Nuran Ercal a, * a Department of Chemistry, University of Missouri-Rolla, 1870 Miner Circle, 142 Schrenk Hall, Rolla, MO 65409, USA b Department of Surgery, New York University, New York, NY 10010, USA c Geriatric Research Education and Clinical Center (GRECC), VA Medical Center, St. Louis, MO 63106, USA Accepted 13 July 2005 Available online 24 August 2005 Abstract Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions such as cerebral ischemia and Parkinson’s disease. Elevated levels of glutamate are thought to be responsible for CNS disorders through various mechanisms causing oxidative stress induced by a nonreceptor-mediated oxidative pathway which blocks cystine uptake and results in depletion of intracellular glutathione (GSH). The newly designed amide form of N-acetylcysteine (NAC), N-acetylcysteine amide (NACA), was assessed for its ability to protect PC12 cells against oxidative toxicity induced by glutamate. NACA was shown to protect PC12 cells from glutamate (Glu) toxicity, as evaluated by LDH and MTS assays. NACA prevented glutamate-induced intracellular GSH loss. In addition, NACA restored GSH synthesis in a Glu (10 mM) plus buthionine – sulfoximine (BSO) (0.2 mM)-treated group, indicating that the intracellular GSH increase is independent of g-GSC (g-glutamylcysteinyl synthetase). The increase in levels of reactive oxygen species (ROS) induced by glutamate was significantly decreased by NACA. Measurement of malondialdehyde (MDA) showed that NACA reduced glutamate-induced elevations in levels of lipid peroxidation by-products. These results demonstrate that NACA can protect PC12 cells against glutamate cytotoxicity by inhibiting lipid peroxidation, and scavenging ROS, thus preserving intracellular GSH. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Oxidative stress; NACA; GSH; Glutamate; Neurodegeneration; PC12 cell 1. Introduction Glutamate, an excitatory amino acid, is one of the major neurotransmitters in the central nervous system (CNS). Elevated levels of extracellular glutamate can acutely damage neurons, have been postulated to mediate CNS damage from conditions such as hyperglycemia, ischemia, hypoxia [8], and may be involved in CNS disorders such as Huntington’s, Alzheimer’s, and Parkinson’s diseases [19,10]. Two mechanisms have been proposed for gluta- mate toxicity. The first mechanism explains the excitotox- icity of glutamate as being mediated through three types of excitatory amino acid receptors [20]. Besides receptor- mediated glutamate excitotoxicity, it has also been proposed that elevated levels of extracellular glutamate inhibits cystine uptake, which leads to a marked decrease in cellular GSH levels, resulting in the induction of oxidative stress [21]. Cysteine is the critical component for intracellular GSH synthesis. Because of redox instability, almost all of the extracellular cysteine is present primarily in its oxidized state, cystine, which is taken up by cells via a cystine/ glutamate transporter, the X c  system [3]. Studies indicate 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.07.032 * Corresponding author. Fax: +1 573 341 6033. E-mail address: nercal@umr.edu (N. Ercal). Brain Research 1056 (2005) 132 – 138 www.elsevier.com/locate/brainres