Immunotoxic and Oxidative Effects of Endosulfan and Permethrin on Murine SPlenocytes, in vitro

Indiscriminate use of pesticides appears to alter immune response in non-target organisms such as humans and other animals. Thus, immune modulation is considered as one of the potential risks and consequences following exposure to these chemicals. Because of the widespread usage, exposure to mixtures of pesticides during the lifetime of individuals is unavoidable and can result in potentiation of the toxic effects. Because immune cells are more susceptible to toxic insults at a lower dose than most other cell types, the effects of pesticides and their mixtures on murine splenocytes were evaluated. C57BL/6 male mouse splenocytes, in vitro, were exposed to permethrin and endosulfan, individually and in-combination (25-200 µM). The immunotoxic potential of these pesticides was monitored using a flow cytometric technique in combination with 7-Amino Actinomycin D (7-AAD) staining. Endosulfan exposures (25-150 µM) resulted in time- and dose-dependent increase in apoptotic and necrotic cell death in murine splenocytes, in vitro. Permethrin exposure (50-200 µM) resulted in neither a time-dependent/dose-dependent loss of splenocyte viability nor induction of apoptosis in splenocytes. With mixtures of permethrin and endosulfan, depressed viability and enhanced early apoptosis and late apoptosis/necrosis were observed. Exposure to mixtures of 50 µM endosulfan with 50 or 100 µM permethrin increased late apoptosis/necrosis compared to exposure to either chemical alone. DNA fragmentation, a hall mark of apoptosis was observed by DNA ladder technique, confirming the occurrence of apoptosis. Morphological observation using cytospun slides was also carried out to further confirm the presence of apoptosis and necrosis. These findings suggest that the immunotoxicity of endosulfan both individually and in mixtures with permethrin is associated with the occurrence of apoptotic and necrotic processes.

Further, the ability of these pesticides to alter the oxidative status of the cells, via reactive oxygen species (ROS) generation and modulation of intracellular antioxidant enzymes levels, was investigated. We monitored the generation of ROS such as hydrogen peroxide (H₂O₂) with 2´, 7´- dichlorofluorescin diacetate (DCFH-DA) assay and superoxide anion (O₂⁻) with hydroethidine (HE) assay in combination with flow cytometry. Spectrophotometric techniques were used to measure antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPX). Results of the analyses revealed that individual pesticides increased the production of H₂O₂ in a time and dose-dependent manner. Both time and dose-dependent increases in O₂⁻ production were caused by permethrin; whereas endosulfan exposure resulted in only a dose-dependent increase. However, exposure to mixtures of these pesticides had little or no effect on the generation of H₂O₂ and O₂⁻ radicals as compared to individual pesticides. The levels of SOD and GPX in pesticide-treated splenocytes were found to be not different from solvent control. An increase in GR and CAT levels in cells was noticed with permethrin (100 µM) exposure. These findings suggest that permethrin and endosulfan have the ability to affect the cellular oxidative status and can cause toxicity in immune cells, in vitro.