In Vitro Immunotoxicity Testing of Pesticides using Human Cytokine Promoter Based Reporter Cell Lines

Abstract

Pesticides including fungicides, herbicides and insecticides are the class of chemicals deliberately released into the environment because of their utility. Worldwide, an estimated, 3 million cases of pesticide exposure are reported per year. It has been established that pesticide chemicals can alter immune function and causes immunotoxicity. We have developed a model for immunotoxicity testing of chemicals in vitro by analyzing cytokine gene expression. The changes in cytokine geneexpression observed in vitro was used as an endpoint for studying perturbation of the immune system in vivo. This system employs cell lines that have been transformed using constructs that have promoter region of Th1, Th2 cytokines and pro-inflammatory cytokines. These promoter regions regulate the expression of areporter gene, Green Fluorescent Protein (GFP), and the activity of promoter to the external stimuli was compared with the expressionof endogenous cytokine genes. CyclosporinA, FK506 (tacrolimus) and pentoxifylline, have been tested on this model and they showed specific pattern of suppression of reporter gene expression treated with PMA/Ionomycin and LPS. These immunosuppressants also suppressed the PMA/Ionomycin and LPS induced endogenous gene expression of these cytokines. Thus our model has potential for application as a predictive screening test system for immunomodulatory activities of chemicals. The validation of the model was carried out using three pesticides chlorpyriphos, cypermethrin and captan that were known to have immunosuppressive activity on the expression of Th1 and Th2 cytokines. Following exposure to above pesticides, we also observed decrease in Th1 and Th2 cytokines promoter activity based GFP expression in our transformed cells line. Thus our model efficiently predicts the immunosuppressant nature of pesticides and therefore may serve as a tool for preliminary high throughput immunotoxicity screening of test agents such as pesticides and other environmental pollutants.