In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca2+](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25 mu M of the fungal metabolite gliotoxin for 72 h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alpha II-spectrin breakdown fragments confirmed activity of the proteases, and that all-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca2+-mediated. (c) 2006 Elsevier Inc. All rights reserved.