Immunocytological and biochemical analysis of the mode of action of bis (tri-n-butyltin) tri-oxide (TBTO) in Jurkat cells

Bis (tri-n-butyltin) oxide (TBTO) is one of the organotin compounds known to induce immunosuppression. Previously, we examined the effect of TBTO on whole-genome mRNA expression in the human T lymphocyte cell line Jurkat, which led to the hypothesis that induction of endoplasmic reticulum (ER) stress is the first initiated event, which induces a rise of intracellular calcium levels, activation of NFkB and NFAT, T cell activation response and oxidative stress together finally resulting in apoptosis. The present study verified this hypothesis with biochemical and cytological experiments. The induction of ER stress was confirmed by the rapid raise in protein levels of ATF3 and DDIT3. Moreover, the impairment of cell viability by TBTO was moderated by the ER stress inhibitor phenyl butyric acid. Real-time fluorescence microscopy confirmed that TBTO increases intracellular calcium levels within 2 min of exposure. Furthermore, the involvement of increased calcium levels in the effects of TBTO was evident from the induction of three calcium-dependent events: (1) activation of the protease activity of M-calpain, (2) induction of NF-kB (p65) expression, and (3) activation of NFAT. The induction of oxidative stress was verified by detection of increased levels of reactive oxygen species and a decrease in amount of reduced glutathione. We also showed that TBTO induces cleavage of caspase-3, an event known to mediate apoptosis. Finally, comparative microarray data analysis showed that many of the processes observed in vitro also occur in vivo in thymuses of TBTO-treated mice.