Concentration dependence of the mechanisms of tributyltin-induced apoptosis.

Tributyltin chloride (TBT), an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide, and component of antifouling paints. In this study, we investigated the concentration dependence of the mechanisms of tributyltin cytotoxicity in PC12 cells. Exposure of PC12 cells to both 500 nM and 2 microM tributyltin increased the number of cells showing nuclear fragmentation, a typical apoptotic feature, and activated caspase-3. The peak Ca(2+) concentration in 2 microM tributyltin-treated cells was higher than that in 500 nM tributyltin-treated cells. The intracellular Ca(2+) increase induced by 2 microM tributyltin was mediated by Ca(2+) release from both inositol 1,4,5-trisphosphate receptor and ryanodine receptor, while the Ca(2+) increase induced by 500 nM tributyltin was mediated through the voltage-dependent calcium channel (VDCC). Next, we investigated whether the mechanisms leading to cell death after Ca(2+) increase were different. Reactive oxygen species (ROS) were involved only in 2 microM tributyltin-induced cell death, while c-jun N-terminal kinase (JNK) mediated only 500 nM tributyltin-induced toxicity. Thus, caspase-dependent apoptosis caused by 2 microM tributyltin was mediated by a large Ca(2+) increase via inositol 1,4,5-trisphosphate receptor and ryanodine receptor, followed by generation of ROS. Apoptosis caused by 500 nM tributyltin was mediated by a moderate Ca(2+) increase through the VDCC, followed by phosphorylation of JNK. These results suggest that apoptosis by TBT is induced via distinct pathways depending on the TBT concentration, and we showed a rare example that upstream mechanisms of apoptosis are distinct depending on strength of toxic insult.