Original Contribution PROGRESSIVE IRON ACCUMULATION INDUCES A BIPHASIC CHANGE IN THE GLUTATHIONE CONTENT OF NEUROBLASTOMA CELLS MARCO T. NU ´ N ˜ EZ,* ,y VIVIAN GALLARDO,* P ATRICIA MUN ˜ OZ, y VICTORIA TAPIA, y ANDRE ´ S ESPARZA, y JULIO SALAZAR,* and HERNA ´ N SPEISKY z *Department of Biology, Faculty of Sciences, and z Micronutrients Unit, Instituto de Nutricio ´n y Tecnologı ´a de los Alimentos, Universidad de Chile, Santiago, Chile; and y Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, Santiago, Chile Abstract—Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH- SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 AM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2  GSSG)) glutathione. Cell exposure to high iron concentrations (20 – 80 AM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 AM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson’s disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity. Keywords—SH-SY5Y cells, Oxidative stress, Redox potential, Viability, Parkinson’s Disease, Free radicals INTRODUCTION Brain cells have a highly active oxidative metabolism with elevated levels of ATP consumption, yet they contain only low to moderate superoxide dismutase, catalase, nd glutathione peroxidase (GPx) activities [1,2]. Thus, their antioxidant defenses rely mainly on cellular glutathione (GSH) levels [3–5]. GSH reacts nonenzymatically with free radicals, serves as electron donor for GPx-catalyzed reduction of peroxides, and interacts with sulfhydryl groups of proteins to keep them in the reduced state. These functions are funda- mental to maintain the intracellular thiol redox potential [6] and involve the oxidation of GSH into its disulfide form, GSSG. GSH is regenerated from GSSG in a reaction catalyzed by GSH reductase (GR), which transfers electrons from NADPH to GSSG. In addition, GSH is nonoxidatively consumed via generation of glutathione S-conjugates by glutathione S-trans- ferase (GT) [7]. GSH also reacts with nitric oxide and peroxynitrite [8]. Brain antioxidant defenses function properly during most of human life. However, a number of neurodegen- erative processes, which involve reactive oxygen (ROS) and nitrogen species, become evident with age [9]. In particular, changes in GSH levels occur in Alzheimer’s disease (AD) and Parkinson’s disease (PD). Elevated GSH levels in hippocampus and midbrain were reported in AD [10], whereas treatment of NT2 cells with Ah-amyloid Address correspondence to: Marco T. Nu ´n ˜ez, Departamento de Biologı ´a, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. Fax: +56 2 2712983; E-mail: mnunez@uchile.cl. 953