Degradation of Organophosphorus Neurotoxicity in SY5Y Neuroblastoma Cells by Organophosphorus Hydrolase (OPH)

Abstract

Numerous approaches have been studied to degrade organophosphorus (OP) compounds and ameliorate their toxicity. In the current study, the potential of genetically engineered organophosphorus hydrolase (OPH) enzymes to functionally biotransform OP neurotoxicants was examined by assessing effects of OPH-hydrolyzed OPs on acute and delayed indicators of neurotoxicity. SY5Y human neuroblastoma cells were used as a model test system, as these cells respond distinctly to mipafox, which produces OP-induced delayed neuropathy, and paraoxon, which does not. Short-term effects of four OPH-treated OPs on acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were measured in retinoic acid-differentiated or undifferentiated cells, and delayed effects of OPH-treated paraoxon or mipafox on levels of neuronal cytoskeletal proteins in nerve growth factor (NGF)-differentiated cells. The anti-AChE activity of paraoxon (maximum 3 μM) and anti-NTE activity of mipafox (250 μM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kD neurofilament protein NF200 were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, as this endpoint is insensitive to paraoxon. However, NF200 levels rose in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below which SY5Y cells can maintain their viability for compensating cellular damage due to mipafox in neurite elongation. These results indicate that OPH may be used to biodegrade OPs and remediate their neurotoxic effects in vitro and that AChE and NTE are suitable detectors for OPH amelioration. We are grateful to Dr. Ling-Yu He for her assistance in setting up gas chromatography and Dr. Janet K. Grimsley for her preparation of organophosphorus hydrolase (OPH) for this study. We thank Dr. Marion F. Ehrich for her kind advice on NTE assay. This study was supported by National Institutes of Health grants P30 ES09106 and T32 ES07273 and by ATSDR grant U61/ATU684505.