Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to in fection in animals. Often this manifestation is accompanied by atrophy of m ajor lymphoid organs. It has been suggested that increased apoptotic cell d eath leading to altered T-B cell ratios, and loss of regulatory cells in cr itical numbers leads to perturbations in immune function. The major objecti ve of our study was to define the mechanism by which endosulfan, an organoc hlorinated pesticide, induces human T-cell death using Jurkat, a human T-ce ll leukemic cell line, as an in vitro model. We exposed Jurkat cells to var ying concentrations of endosulfan for 0-48 h and analyzed biochemical and m olecular features characteristic of T-cell apoptosis. Endosulfan lowered ce ll viability and inhibited cell growth in a dose- and time-dependent manner . DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 mu M induced a significant percentage of cells to unde rgo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 mu M of endosulfan. We confirmed these observations using both DNA fragm entation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (Delta Psi(m)) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptos is. Within 30 min of chemical exposure, a significant percentage of cells e xhibited a decreased incorporation of DiOC(6)(3), a cationic lipophilic dye into mitochondria indicating the disruption of Delta Psi(m). This drop in Delta Psi(m) was both dose- and time-dependent and correlated well with oth er parameters of apoptosis. We also examined whether this occurred by the d own regulation of bcl-2 protein expression that is likely to increase the s usceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the in tracellular expression of bcl-2 protein was elevated in a dose dependent ma nner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathwa y. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by en dosulfan: uncoupling of oxidative phosphorylation --> excess ROS production --> GSH depletion --> oxidative stress --> disruption of Delta Psi(m) --> release of cytochrome C and other apoptosis related proteins to cytosol --> apoptosis. This study reports for the first time that endosulfan can induc e apoptosis in a human T-cell leukemic cell line which may have direct rele vance to loss of T cells and thymocytes in vivo. Furthermore, our data stro ngly support a role of mitochondrial dysfunction and oxidative stress in en dosulfan toxicity.