Tissue culture based systems to validate the enzymatic detoxification of organophosphorus neurotoxicants

Abstract

This study was conducted to develop cell culture based systems to test the efficacy of enzyme prophylaxsis for the detoxification of organophosphorus (OP) neurotoxicants. The assays utilized for detecting OP neurotoxicity and its amelioration by organophosphate hydrolase (OPH) were-. 1) inhibition of acetylcholine esterase (AChE), 2) inhibition of neurotoxic esterase (NTE), and 3) alterations in ionophore-induced Ca 21 fluxes in transformed neuronal cell lines SY5Y (human neuroblastoma) and PC12 (rat pheochromocytoma). It was observed that OPH suppressed the ability of paraoxon to inhibit ACHE, restoring activity from 33% to lOO% in PC12 cells and from 2% to 86% in SY5Y cells. OPH did not suppress NTE inhibition by mipafox within 5 minutes (59.5% inhibition by mipafox and 56.5% by hydrolyzed mipafox) suggesting that mipafox hydrolysis proceeds at a slow rate. Microfluoroscopy with the fluorescent calcium probe fluo-3 suggested that, contrary to expectations based on effects of other OP compounds, paraoxon had no effect of Ca'+ fluxes in cells exposed to ionomycin. Cells treated with hydrolyzed paraoxon showed a higher final fluorescence level after recovery from ionomycin (1 195 fluorescence units) than cells treated with paraoxon (874 fluorescence units), p-nitrophenol (951 fluorescence units) and OPH alone (871 fluorescence units) suggesting a potential role for paraoxon breakdown product diethyl phosphonic acid. Also cells treated with hydrolyzed paraoxon and p-nitrophenol showed significantly different recovery rates. The results of this study suggest that though enzymatic detoxification of OPs may selectively attenuate inhibition of enzyme systems, such detoxification may augment effects on [Ca 2+] i due to the production of organic phosphonic acids. Thus a battery of assays addressing different aspects of OP toxicity is indicated for validating enzymatic detoxification of OPs.