1 3 Arch Toxicol DOI 10.1007/s00204-013-1147-9 ORGAN TOXICITY AND MECHANISMS "Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites Daniel José Barbosa · João Paulo Capela · Renata Silva · Luísa Maria Ferreira · Paula Sério Branco · Eduarda Fernandes · Maria Lourdes Bastos · Félix Carvalho Received: 13 August 2013 / Accepted: 7 October 2013 © Springer-Verlag Berlin Heidelberg 2013 differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabo- lites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metab- olites was observed, which was potentiated by hyperther- mia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA- exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stabil- ity of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differ- ently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA’s neurotoxicity. Keywords 3,4-Methylenedioxymethamphetamine (MDMA; “ecstasy”) · MDMA metabolites · Hyperthermia · SH-SY5Y differentiated cells · Neurotoxicity Introduction 3,4-Methylenedioxymethamphetamine (MDMA; “ecstasy”) is a worldwide major drug of abuse known to elicit neu- rotoxic effects, which are well documented in laboratory animals (Bai et al. 2001; Granado et al. 2011), nonhuman Abstract 3,4-Methylenedioxymethamphetamine (MDMA; “ecstasy”) is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic for- mation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mecha- nisms involved are still unclear. Here, we studied the neu- rotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α- MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normother- mic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y D. J. Barbosa (*) · J. P. Capela · R. Silva · M. L. Bastos · F. Carvalho (*) REQUIMTE (Rede de Química e Tecnologia), Toxicology Laboratory, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal e-mail: daniel.barbosa@ff.up.pt F. Carvalho e-mail: felixdc@ff.up.pt J. P. Capela Faculty of Health Sciences, University Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal L. M. Ferreira · P. S. Branco REQUIMTE/CQFB (Centro de Química Fina e Biotecnologia), Department of Chemistry, Faculty of Science and Technology, University Nova de Lisboa, 2829-516 Caparica, Portugal E. Fernandes REQUIMTE, Laboratory of Applied Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal