Bioremediation of organophosphorus neurotoxicity and genotoxicity in vitro by organophosphorus hydrolase

The toxicity of OPs is primarily associated with cholinesterase inhibition in the exposed organism. Some OP compounds also produce a delayed neurotoxicity called organophosphate—induced delayed neuropathy (OPIDN). Furthermore, certain OP compounds produce genotoxic effects in vitro and in vivo. The overall purpose of the work described in this dissertation was to develop and partially characterize in vitro systems for assessing the remediation of OP toxicity by organophosphorus hydrolase (OPH). Two types of in vitro systems were examined: a Salmonella typhimurium system, in which mutagenic potential was assayed, and the human neuroblastoma cell line, SH-SY5Y, in which neurotoxic potential was assayed. Methyl parathion (MP) was confirmed to have a positive genotoxic response in S. typhimurium strain TA100. MP genotoxicity could be eliminated by treatment with genetically engineered intact E. coli cells expressing the OPH activity. The OPH-treated MP failed to induce a genotoxic response. These experiments indicate that OPH is capable of degrading MP and eliminating its genotoxic potential in liquid culture. In a subsequent study, molecular markers for OPIDN were identified in an in vitro system with the SY5Y cell line differentiated with nerve growth factor (NGF). Mipafox applied during late stage in cell differentiation increased NF200 protein expression in SY5Y cells in a dose dependent manner. Paraoxon applied continuously from an early stage in cell differentiation produced down-regulation of NF200 expression at and above 0.3 μM. Although NGF induced a significant increase in GAP43 protein expression, mipafox up to 30 μM did not alter GAP43 expression. Finally, biodegradation of model OP compounds and the efficiency of OPH to remove or reduce toxicity of the compounds were tested in the SY5Y cell line. OPs preincubated with the OPH showed reduced or remediated inhibition of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in exposed SY5Y cells. Mipafox (40 μM) produced significant modifications in expression of MAP2ab and NF200 proteins in SY5Y cells while equimolar paraoxon did not. Biodegradation of mipafox with OPH significantly reduced neurotoxicological stress on modulation of these neuroskeletal protein markers. These results support the conclusion that OPH can be used to biodegrade OPs and remediate their neurotoxic effects in vitro.