The Important Role Of PP2A And JNK In TBT-induced Apoptosis In Vivo And Vitro

Tributyltin (TBT), a highly toxic environmental contaminant, has been reported to cause serious health problems and has a significant immunologic, neurological, reproductive and hepatic toxicity. The induction of apoptosis is believed to play a pivotal role in TBT-induced toxicity. TBT has been shown to induce Bax/Bcl-2-evoked mitochondrial apoptosis pathway. However, the upstream signal transduction pathways involved in TBT-induced apoptosis are still not fully elucidated. In addition, although cultured cells are indispensable research tools to eludiate the mechanisam of TBT-induced apoptosis, the mechanisms derived from cell culture experiments require further validation through in vivo experiments to understand and predict the effects of toxicants in humans.The present study using the human amnion cells was focused on (a) protein phosphatase2A activity, the protein profile of PP2A composition subunits and the post-translational modification of PP2A-C,(b) microtubule organization and the co-localization of PP2A C and a-tubulin,(c) the activation of mitogen-activated protein kinases (MAPKs), including ERK cascades, JNK and p38as well as their downstream transcription factors, c-Jun and ATF-2, and the role of JNK and p38activation in TBT-induced apoptosis. Then the in vivo study using mouse was undertaken to further verify the results of the in vitro study by evaluating (d) PP2A activity and the level of reactive oxygen species (ROS),(e) the activation of MAPK including ERK, JNK and p38,(f) apoptosis inducing in livers. These results give a comprehensive and novel description of the mechanism of TBT-induced toxicity both in vitro and in vivo.Results:1. TBT induced a concentration-dependent inhibition of PP2A activity, and reduced the expression of composition subunits including A and B55a subunit, as well as the methylation of PP2A-C in apoptotic FL cells.2. TBT inhibited tubulin polymerization and triggers dissociation of PP2A from microtubules structure in apoptotic FL cells.3. TBT activated JNK and p38, as well as their downstream targets, c-Jun and ATF-2in apoptotic FL cells4. TBT inhibited ERK cascades by inactivating c-Raf, then dephosphorylating MEK1/2, ERK1/2, as well as c-myc in apoptotic FL cells5. The JNK inhibitor, but not p38inhibitor, significantly inhibited the TBT-mediated elevation of apoptotic rate.6. The JNK inhibitor significantly inhibited the TBT-mediated activity of caspase-3, however, had no effect on the expression and localization of Bax, as well as the expression and phosphorylation of the Bcl-2.7. TBT inhibited PP2A activity and elevated the ROS level in mouse livers.8. TBT activated ERK, JNK and p38MAPKs in mouse livers.9. TBT induced apoptosis characterized by DNA degradation, chromatin condensation, and the elevation of Bax to Bcl-2ratio as well as the activation of caspase-3.Conclusions:1. The study denmonstrated that the manner of TBT-induced apoptosis in vivo was different with in vitro. This in vitro study showed that TBT might induce a mitochondrial-independent activation of caspase-3and subsequent apoptosis in FL cells, whereas this in vivo study showed that TBT might induce Bax/Bcl-2-evoked mitochondrial apoptosis pathway in mouse livers. 2. The mechanism of TBT-induced apoptosis in vivo was different with in vitro study. It was concluded that TBT induced caspase-3activation dependent of the activation of JNK and c-Jun in FL cells, whereas TBT induced caspase-3activation dependent of Bax/Bcl-2-evoked apoptosome formation in mouse livers.3. The upstream pathway of TBT-induced apoptosis in vivo is similar to the in vitro results that TBT induces caspase-3-dependent apoptosis via an inhibition of PP2A, followed by JNK activation. Therefore, PP2A and JNK are critical targets of TBT-induced toxicity both in vitro and in vivo.