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 infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell 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 microM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 microM of endosulfan. We confirmed these observations using both DNA fragmentation 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 (deltapsim) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of deltapsim. This drop in deltapsim was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. 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 endosulfan: uncoupling of oxidative phosphorylation --> excess ROS production --> GSH depletion --> oxidative stress --> disruption of deltapsim --> release of cytochrome C and other apoptosis related proteins to cytosol --> apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.
Kannan
K
K
Feist-Weiller Cancer Center, Arthritis Center, LSU Health Sciences Center, Shreveport, LA 71130, USA.
Holcombe
R F
RF
Jain
S K
SK
Alvarez-Hernandez
X
X
Chervenak
R
R
Wolf
R E
RE
Glass
J
J
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D004396:Coloring Agents / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004726:Endosulfan / Q000737:chemistry / Q000494:pharmacology*; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D010078:Oxazines; D018384:Oxidative Stress; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000187:drug effects*; D013997:Time Factors; D014966:Xanthenes
D017304:Annexin A5; D004396:Coloring Agents; D010078:Oxazines; D019253:Proto-Oncogene Proteins c-bcl-2; D014966:Xanthenes; C005843:resazurin; D005978:Glutathione; D004726:Endosulfan