Di-n-butyltin dichloride (DBTC) and
tri-n-butyltin chloride (TBTC) cause thymus
atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these
organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min
after treatment, was followed by a dose- and time-dependent generation of
reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of
cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of
cytochrome c were reduced by
BAPTA, an intracellular Ca(2+)
chelator, or
rotenone, an inhibitor of the electron entry from complex I to
ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that
rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both
BAPTA and
Z-DEVD FMK (mainly a
caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by
organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and
cytochrome c from mitochondria, activation of
caspases, and finally results in DNA fragmentation.