Chlorpyrifos oxon

The five CWNAs evaluated were tabun (GA; O-ethyl N,N-dimethyl phosphoramidocyanidate), sarin (GB; O-isopropyl methylphosphonofluoridate), soman (GD; O-pinacolyl methylphosphonofluoridate), cyclosarin (GF, cyclohexyl methylphosphonofluoridate), and VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate). They were obtained from the U.S. Army Edgewood Chemical Biological Center (Aberdeen Proving Ground, MD). The purity values of the CWNAs were >98.5 percent as determined by gas chromatography. Chlorpyrifos oxon (purity ≥98 percent) and paraoxon (purity ≥98 percent) were purchased from Chem Service, Inc, West Chester, PA. Phorate oxon (purity ≥97.1 percent) was synthesized at Battelle’s Analytical Chemistry Development Department (Columbus, OH). GB, GD, GF, and VX were diluted in 0.9 percent saline; GA and phorate oxon were diluted in multisol (a biocompatible solution of 48.5 percent water, 40 percent propylene glycol, 10 percent ethanol, and 1.5 percent benzyl alcohol, all v/v); and Chlorpyrifos oxon and paraoxon were diluted in ethanol (99.96 percent), with the dosing solution concentration of each pesticide being limited to that which would allow the total volume of ethanol injected to be no more than 0.06 percent (v/w) of the body mass.

Eagle’s Minimum Essential Medium and fetal bovine serum were purchased from Quality Biological Inc. Chlorpyrifos oxon (CPO, >97% purity) was purchased from Chem Service and kept desiccated under nitrogen at −70°C. The desiccator containing CPO was brought to room temperature under the fume hood before opening to minimize any CPO hydrolysis. Bradford reagent, 5-amino-2-methoxypyridine (AMP), ethylenediaminetetraacetic acid (EDTA), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), Trizma base, Trizma hydrochloride, Triton X-100, sodium phosphate, sodium chloride, and bovine serum albumin were all purchased from Sigma-Aldrich (St. Louis, MO). Octanoyl methoxypyridine and was a kind gift from Dr. Bruce Hammock, UC Davis. TaqMan gene expression assays, TaqMan Fast Advanced Master Mix No AMP Erase UNG, and the High Capacity cDNA Reverse Transcription Kit were all purchased from Applied Biosystems. Anandamide, oleoylethanolamide, and GW9662 were purchased from Cayman Chemical. DMSO was purchased from ATCC. Human recombinant insulin in zinc solution was obtained from Gibco. Phosphate buffered saline (PBS) was purchased from Corning. Trizol reagent was purchased from Ambion Life Sciences. 5-amino-2-methoxypyridine (AMP) as well as all chemicals required for the MTT assay were purchased from Sigma Aldrich.

Twenty-six AChE inhibitors (Table 1) were selected from the Tox21 10 K library. Amplite™ Colorimetric Acetylcholinesterase Assay kit (Ellman assay) and Amplite™ Fluorimetric Acetylcholinesterase Assay kit (Green Fluorescence) were purchased from AAT Bioquest, Inc. (Sunnyvale, CA, USA). Chlorpyrifos-oxon was purchased from Chem Service, Inc. (West Chester, PA, USA). Chlorpyrifos (CAS#: 2921–88-2), BW284c51, β-Nicotinamide adenine dinucleotide 2′-phosphate (NADPH), dimethyl sulfoxide (DMSO) and purified recombinant human AChE protein were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). InVitroCYP™ 150-D human liver microsomes (HLM), prepared from 150 donor human liver tissue fraction pools with mixed gender, were purchased from BIOIVT (Baltimore, MD, USA). Acroclor 1254-induced male Sprague-Dawley rat liver microsomes (RLM) were purchased from Molecular Toxicology Inc. (Boone, NC, USA).

Considering previous observations that fluorophosphonate probes hydrolyze when stored under neat conditions,^{29 (link)} probes were dissolved in anhydrous DMSO shortly after synthesis and characterization and divided into aliquots for longer term storage, avoiding freeze–thaw cycles.
Organophosphates were purchased from the following suppliers and used without further purification: paraoxon (Chem Cruz, sc-208151, Lot D2117) and chlorpyrifos oxon (98.8%, Chem Service, MET-114590, Lot 525430). Stock solutions of OPs were prepared in dry DMSO and stored at −70 °C, avoiding freeze–thaw cycles. 2-Pralidoxime chloride (2-PAM) was purchased from Sigma-Aldrich. Stock solutions of 2-PAM were prepared freshly on the day of the experiment in Milli-Q water and maintained on ice.
Electric eel acetylcholinesterase (Sigma-Aldrich) was prepared at 2 mg/mL concentration in 100 mM PBS, pH 7.4, with 1 mg/mL bovine serum albumin (BSA) and stored at 4 °C for up to 3 months.
Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Scientific Pierce). Colorimetric assays were performed using a Molecular Devices plate reader.
Synthetic procedures are described in the Supporting Information.