Immunotoxic Effects of Mixtures of Endosulfan and Permethrin Via Caspase Dependent Thymocyte Apoptosis

Altered immune responses have been observed following occupational, inadvertent, or therapeutic exposure to xenobiotics. Many pesticides are known to cause immunotoxicity. Exposure to mixtures of pesticides, either concurrently or sequentially, may result in potentiating this effect partly because one can effect the metabolism of the other. The objective of this study was to determine the effect of the insecticides endosulfan, permethrin and their mixtures on C57/BL6 male mice thymocytes in vitro and to ascertain the mechanism by which these effects take place. Permethrin, a broadspectrum synthetic pyrethroid, is a widely used insecticide in agriculture and public health. Endosulfan is a highly toxic chlorinated hydrocarbon insecticide used worldwide. We examined the immunotoxic potential of these pesticides using a flow cytometric technique in combination with 7-Amino Actinomycin D (7AAD) to distinguish live, early apoptotic, and late apoptotic/necrotic cells. DNA ladder assay, a hallmark of apoptosis, was also used to determine the occurrence of apoptosis. Both endosulfan and permethrin were found to cause significant apoptotic death of thymocytes in a doseand timedependent manner. Thus, permethrin at 50, 100 or 300 μM was found to cause 5.5, 11.5 and 26.1% increases in early apoptotic cell death relative to control, respectively. Endosulfan at 25, 50 or 250 μM was found to cause 11.9, 15.7 and 68.0% early apoptotic cell death, respectively. For the mixture study, concentrations of 100 μM permethrin and 50 μM endosulfan were selected and found to cause 27.1% apoptosis. Thus, these pesticides in mixture have an additive immunotoxic effect. Increases in lateapoptotic/necrotic cells were found at these concentrations for either pesticide when exposed for 12 hours. DNA ladder assay confirmed the presence of DNA fragments and therefore the presence of significant apoptotic cell death. Apoptosis is a morphologically distinct form of cell death that can be mediated by a variety of pathological and physiological stimuli. Because permethrin and endosulfan were found to induce apoptosis in C57/BL6 mice thymocytes in vitro, the objective of the second half of this study was to elucidate the potential mechanism by which these pesticides regulate apoptosis in immune cells. Caspases are a family of cystinedependent, aspartate-directed proteases that have an integral role in apoptotic cell death. Caspases, which are normally inactive in healthy cells, are activated during apoptosis and form an irreversible cascade. There are two subsets of caspases, initiator caspases (i.e. caspase 8 and 9) and effector caspases (i.e. caspases 3 and 6). Caspase 3, a downstream effector of apoptosis, is activated by many different pathways and is an apoptotic marker in cells. Caspase 8 is the apical caspase in the extrinsic pathway. Caspase 9 is the apical caspase in the intrinsic pathway, therefore we investigated mechanisms of pesticide induced apoptosis involving the thymocyte caspase system. Thymocytes from C57/BL6 mice were incubated with varying concentrations of pesticides for varying amounts of time. Active caspase 3 was then measured using EnzCheck Caspase 3 Assay Kit. Relative fluorescence for permethrin exposed cells after 12 hours incubation in the presence of pesticides at 150, 100, and 50 μM and 40 minutes in the presence of AFC-