Tributyltin-induced cell death is mediated by calpain in PC12 cells

Tributyltin, an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated whether calpain is involved in tributyltin toxicity in undifferentiated PC12 cells. Tributyltin (2 microM) induced an increase of lactate dehydrogenase release, a marker of cytotoxicity, in PC12 cells in a time-dependent manner. It also induced calpain activation in a dose-dependent manner, and a calpain inhibitor, MDL28170 (40 microM), decreased the cellular toxicity, suggesting that calpain is involved in tributyltin toxicity in PC12 cells. Because calpain is a calcium-dependent protease, we examined the effect of EGTA, an extracellular Ca(2+) chelator and BAPTA-AM, an intracellular Ca(2+) chelator. Calpain activation induced by tributyltin was decreased by BAPTA-AM (50 microM), but not by EGTA (1 mM), suggesting that calpain activation is associated with calcium release from intracellular Ca(2+) stores. Further, we investigated the relationship between caspase-3 and calpain. Inhibition of caspase-3 reduced calpain activity induced by tributyltin. In conclusion, we have demonstrated that tributyltin induced cell death through calpain activation, and that intracellular Ca(2+) increase and caspase-3 activation are required for calpain activation by tributyltin.