Biphasic effect of Endosulfan on human PBMC proliferation and pro- and anti-inflammatory cytokines.

• ¹ University of Guadalajara, Mexico

Introduction. Endosulfan is a DDT-era organochlorine pesticide. Currently banned or being phased out in many parts of the world (Europe, USA), endosulfan is still in use in the developing world, including Mexico and many countries of Latin America. Bioaccumulation of past pesticide use, as well as environmental effects of current use indicate that the investigation of the effects of endosulfan exposure is of current importance. Endosulfan exposure has been strongly correlated with neural and endocrine system damage. The effects of this pesticide on the immune system are not fully characterized. We have previously shown the effects of endosulfan on non specific activation, proliferation, apoptosis and senescence in immune cells from Nile tilapia. Methods. PBMC from healthy donors were extracted via density separation and cultured for 16- 72 hours in the presence of 0.1-50 µM endosulfan. For mitogen induced proliferation, cells were pre exposed with endosulfan 4 hours prior to incubation with PHA. Proliferation was measured using WST-1; cytokines were quantified using the Bioplex Pro (BioRad) assay. Results. We observed a slight but significant increase in proliferation (p=0.03) when PBMCs were treated with 0.1µM endosulfan (72h). Endosulfan was found to have a significant inhibitory effect on 24-72 hour cell growth at concentrations of 17µM and above. PBMCs exposed to endosulfan and PHA mitogen demonstrated similar results, with endosulfan at 0.1µM increasing mitogen induced proliferation and endosulfan at 17µM inhibiting mitogen induced proliferation at 24, 48 and 72 hours. Cytokine analysis of supernatants from proliferating cells incubated with endosulfan at 17µM (from 9 different donors) demonstrated significantly increased pro inflammatory cytokines: TNF-alpha (up to 9 fold increase) and IL-6 (4-10 fold increase). Other cytokines were also increased: IL-4 (10%-300%), IFN-gamma (up to 50 fold increase) and GMCSF (50-150%). In contrast, endosulfan at 17µM had a significant inhibitory effect on the anti-inflammatory cytokine IL-10 (up to 90 fold decrease). Endosulfan at 0.1µM did not significantly increase or decrease pro inflammatory cytokine production, with approximately half the samples showing a slight increase and the other half a slight decrease. When stimulated with PHA, cytokine production was universally increased. Inclusion of endosulfan 17µM with PHA led to significantly lower cytokine production, with the exception of IL-6 (slight increase) and TNF-alpha (30-200% increase). Conclusion: At both high and low doses endosulfan provoked immunologically significant in vitro effects. Of note is the inability of cells treated with high dose endosulfan to produce cytokines when stimulated with PHA, suggesting that the initial 17µM endosulfan-induced increase in production of pro-inflammatory cytokines leads to exhaustion or an anergic/ senescent state. The increase of TNF-alpha and IL-6 in both unstimulated and PHA stimulated cells suggests that exposure to endosulfan might continue to provoke these pro-inflammatory cytokines in persons with chronic inflammation or autoimmunity. These results will be of interest in understanding the consequences of chronic and acute exposure of this pesticide on the human immune system and its possible implications in auto immune diseases.