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 broad-spectrum 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 dose- and time- dependent 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 late-apoptotic/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 cystine-dependent, 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-substrate was found to be 387, 386, and 297, respectively. Relative fluorescence for endosulfan exposed cells at 150, 100 and 50 µM was 188, 177, and 294. Caspase 3 activity increased as permethrin concentrations increased and decreased as endosulfan concentrations were increased. Then the extrinsic and intrinsic pathways of apoptosis were further investigated. Active caspase 8 was measured using the ApoAlert Caspase Fluorescent Assay Kit. Relative fluorescence for permethrin exposed cells after 7 hours incubation in the presence of pesticides at 100, 150, and 200 µM was found to be 35.5, 10.5, and 0, respectively. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 32.8, 63.8, 69.5, and 55.5, respectively. A mixture study was then performed using endosulfan (50, 100, 150 µM) combined with permethrin (100 µM). All combinations were found to have more than an additive effect, therefore the extrinsic pathway seems to be involved. Caspase 9 activity was measured using Caspase 9/Mch6 Fluorometric Protease Assay Kit. Relative fluorescence for endosulfan exposed cells after 7 hours incubation at 25, 50, 100 and 150 µM was found to be 43, 73, 78.9, and 5.12, respectively. Relative Fluorescence for permethrin exposed cells at 100, 150 and 200 µM was found to be 34.5, 39, and 55.5, respectively. A mixture study was then performed using endosulfan (25, 50 µM) combined with permethrin (100 µM). Both combinations were found to have less than an additive effect. These results suggest that apoptosis caused by both endosulfan and permethrin exert their effects via the caspase pathway. The results also show that mixtures of pesticides have a less than additive effect on caspase 9 activation and more than an additive effect on caspase 8 activation, therefore the extrinsic pathway is predominantly involved in thymocyte apoptosis caused by mixtures of permethrin and endosulfan.