C70-09 Organophosphorus Pesticides That Cause Neuronally Mediated Airway Hyperreactivity Bind to Macrophage Carboxylesterase 1

Background Exposure to organophosphorus pesticides is a substantial risk factor for the development of asthma. We have shown that a single, low dose of an organophosphorus pesticide, like chlorpyrifos (CPF), causes neuronally mediated airway hyperreactivity in guinea pigs that is mediated by macrophage TNFα. Liver cytochrome P450s convert CPF to CPF oxon, which is the active, toxic isoform that inhibits acetylcholinesterase. However, the inactive, parent compound CPF, but not the CPF oxon, directly stimulates cultured macrophages to release TNFα. The mechanism by which CPF activates macrophages to produce TNFα is not known. Methods To identify the cellular target of CPF in macrophages, we synthesized a CPF-based photoaffinity probe containing an alkyne handle for biorthogonal tagging and a diazirine moiety for UV-induced photo-crosslinking. First, we demonstrated that the CPF probe similarly stimulated differentiated THP1 cells (a human macrophage cell line) to release TNFα. To identify the protein target, we incubated differentiated THP1 cells for 4h with 10 μM CPF probe, and UV-crosslinked the probe to its protein targets. Cell lysates were subjected to copper-catalyzed azide-alkyne cycloaddition with biotin azide, and probe-protein complexes were isolated using streptavidin-conjugated beads. Proteins were separated by SDS-PAGE and analyzed by mass spectrometry. Competitive binding experiments were conducted by co-incubating cells with increasing concentrations of free CPF (3-300 µM) to demonstrate specific, saturable binding. Results Click chemistry experiments identified a 60 kDa protein band that decreased as the concentration of free CPF increased, demonstrating specific saturable binding. Mass spectrometry identified several proteins of interest, but the 60 kDa protein was identified to be carboxylesterase 1 (CES1). CES1 is a serine hydrolase involved in xenobiotic metabolism, lipid metabolism, and inflammation. Loss of CES1 activity has been shown to potentiate dendritic cell activation and increase lung inflammation. Conclusions Our findings demonstrate that the organophosphorus pesticide CPF binds specifically to CES1 in THP1 macrophages. Binding of the inactive parent form of CPF rather than the toxic CPF oxon underscores our previous finding that cholinesterase inhibition is not the mechanism of CPF induced airway hyperreactivity. Determining whether eliminating CES1 prevents CPF-induced increase in TNFα and airway hyperreactivity will be important for understanding the importance of CES1 in CPF-mediated effects in the lung and other organs. This abstract is funded by: HL155623, HL171226, HL163087, HL164474