Toxicology 204 (2004) 175–185 Methylmercury cytotoxicity in PC12 cells is mediated by primary glutathione depletion independent of excess reactive oxygen species generation Rita Gatti a,∗,1 , Silvana Belletti a,1 , Jacopo Uggeri a , Maria Vittoria Vettori b , Antonio Mutti c , Renato Scandroglio a , Guido Orlandini a a Department of Experimental Medicine, via Volturno 39, Universit` a degli Studi di Parma, 43100 Parma, Italy b ISPESL Research Centre, via Gramsci 14, Universit` a degli Studi di Parma, 43100 Parma, Italy c Department of Clinical Medicine, Nephrology and Health Sciences, via Gramsci 14, Universit` a degli Studi di Parma, 43100 Parma, Italy Received 28 May 2004; received in revised form 21 June 2004; accepted 21 June 2004 Available online 28 July 2004 Abstract Low doses, chronic exposure to mercurial organic compounds is a worldwide health concern and could be pathogenetically relevant as co-factor in several neurodegenerative diseases. In this in vitro study we wanted to further improve our knowledge on the mechanisms of toxicity of methylmercury hydroxide (MeHgOH) in the unprimed PC12 cell line. Cell viability, mitochondrial function, redox state, and cell morphology were recorded at different time points to sequence the events leading to cell death. The lowest cytotoxic concentration and EC 50 were 0.3 and 1.3 M, respectively. 5 M MeHgOH was fatal for 80% of the cell population after 24 h; within 1 h it caused glutathione (GSH) depletion and a partial dissipation of ψ m . At this concentration, reactive oxygen species (ROS) generation was only slight and delayed. After 6h more than 50% of ATP was available and caspase 3 was active. Time-lapse confocal microscopy showed that only a fraction of the cells completed apoptosis while others turned toward necrosis (necrapoptosis). Pre-incubation with N-acetylcysteine (NAC) and GSH but not Cyclosporin A rescued over 80% of the cells. Abbreviations: ANOVA, analysis of variance; ψ m , mitochondrial membrane potential; AFC, 7-amino-4-trifluoromethyl coumarin; BCA, bicinchoninic acid; calcein-AM, calcein-acetoxymethyester; CLSM, confocal laser scanning microscopy; CM-H 2 DCFDA, chloromethyl- dihydrodichlorofluorescein diacetate; FCCP, carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone; GSH, glutathione; MeHg, methylmer- cury; MeHgOH, methylmercury hydroxide; MPT, mithocondrial permeability transition; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra- zolium bromide; NAC, N-acetyl-cysteine; PBS, phosphate buffered saline; ROS, reactive oxygen species; TMRM, tetramethylrhodamine methyl ester ∗ Corresponding author. Tel.: +39 052 1903919; fax: +39 052 1903912. E-mail address: rita.gatti@unipr.it (R. Gatti). 1 Contributed equally to this work. 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.06.023