Neurotoxicity Research, 2002 VOL. 4 (2). pp. 87-93 aylor&Francis health$clences Peroxynitrite-induced Cytotoxicity in Cultured Astrocytes is Associated with Morphological Changes and Increased Nitrotyrosine Immunoreactivity MARIANA PEHAR a'b, LAURA MARTINEZ-PALMA b, HUGO PELUFFOb, ANDR]~S KAMAIDb, PATRICIA CASSINAb and LUIS BARBEITO a'b'* aNeurobiologla Celulary Molecular, Instituto de Investigaciones Biol6gicas ClementeEstable, Montevideo, Uruguay; bDepartamentoHistologia y Embriologia, Facultad de Medicina, Universidadde la Repf~blica,Montevideo, Uruguay (Received 1 May 2001; Revised 10 May 2001) We have established a cell culture model of spinal cord astrocytes to study the cytotoxicity of peroxynitrite. Nitric oxide (NO) has been implicated as a key contributor to neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidant and nitrating agent with deleterious cytotoxic and pro-apoptotic effects. Perox- ynitrite and nitrotyrosine are formed in damaged motor neurons in amyotrophic lateral sclerosis (ALS), which are surrounded by reactive astrocytes. To determine the effects of extracellular addition of peroxynitrite, purified astrocytes monolayers prepared from neonatal rat spinal cords were exposed to peroxynitrite (0.25-0.75mM) for 5 min and further incubated in culture medium for 24- 72h. Peroxynitrite exposure did not result in apparent cell loss or damage of the monolayer. However, a substantial number of cells adopted reactive features, with long processes displaying intense immunoreactiv- ity to glial fibrillary acidic protein (GFAP). Western blot analysis performed 24h after peroxynitrite treatment showed that GFAP levels were not modified by the oxidant. There were no changes in cell viability parameters in astrocyte cultures after peroxynitrite, indicating that astrocytes are more resistant to the oxidant than other cell types. Peroxynitrite reacts with protein-bound tyrosine residues to form nitrotyrosine. We observed a modest to strong nitrotyrosine immunor- eactivity in astrocytes 24h following peroxynitrite exposure. There was a remarkable association between nitrotyrosine and high-intensity GFAP immunoreactivity in astrocytes bearing long processes. These results suggest that peroxynitrite induces a characteristic long- lasting reactive astrocytic phenotype and provide new insight into understanding the origin of reactive astrocytes occurring in ALS. Keywords: Amyotrophic lateral sclerosis (ALS); astrocyte; cyto toxicity; glial fibrillary acidic protein (GFAP); nitrotyrosine; peroxynitrite; spinal cord INTRODUCTION Oxidative stress is a major neurotoxic mechanism involved in the pathogenesis of neurological dis- orders such as trauma, ischemia and neurodegen- erative diseases (Wolozin and Behl, 2000). Among the reactive species overproduced in these con- ditions, nitric oxide (NO) has been implicated as a key contributor to neurotoxicity (Dawson et aL, 1993; Lipton et al., 1993). NO is a free radical with low toxicity by itself, but it is able to react with metal centers and other reactive oxygen species. In particular, NO reacts with superoxide by a diffusion limited reaction to generate peroxynitrite (Huie and Padmaja, 1993), a strong oxidant and nitrating agent with deleterious cytotoxic effects. The reaction between NO and superoxide is three *Corresponding author. Departamento de Neurobiologia Celular y Molecular, Insfituto Clemente Estable, Avenida Italia 3318, CP 11600, Montevideo, Uruguay. Tel.: +598-2-487-16-16 (ext 171). Fax: +598-2-487-55-48. E-mail: lbarb@iibce.edu.uy ISSN1029-8428 print/ISSN 1476-3524 online 2002Taylor & FrancisLtd DOh 10.1080/10298420290015818