Methamidophos, dichlorvos, O‐methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry

Abstract

Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O-methoate, and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20-fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC-purified peptides were acquired on a MALDI-TOF-TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O-methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono-thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.

A high‐throughput UHPLC–MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents

“…The average BChE nonapeptide concentration in human serum was found to be 40.0 ± 9.8 ng/mL with a range of 18.4-61.7 ng/mL, which was in agreement with results previously reported . A BChE nonapeptide concentration of 40.0 ng/mL in serum is representative of a BChE enzyme concentration of 50 nM which is consistent with previously reported concentrations of BChE in human serum (Lockridge, 2015;Noort, Benschop, & Black, 2002;Tacal and Lockridge, 2010 (Berry and Davies, 1966).…”

“…(Pantazides et al, 2014) A BChE nonapeptide concentration of 40.0 ng/mL in serum is representative of a BChE enzyme concentration of 50 nM which is consistent with previously reported concentrations of BChE in human serum. (Lockridge, 2015, Noort et al, 2002, Tacal and Lockridge, 2010) Only the unadducted BChE biomarker was found to be present in the convenience set; no aged OPNA-BChE adducts were detected.…”

Comparative effect of technical and commercial formulations of methamidophos on sperm quality and DNA integrity in mice

“…We reported that single doses of diazinon (commercial‐grade) phosphorylated nuclear sperm protamines at serine residues altering sperm chromatin structure in mice (Piña‐Guzmán et al,). Similarly, MET (technical‐grade) efficiently inhibited purified butyrylcholinesterase forming adducts with the enzyme by the addition of methoxy aminophosphate (Tacal and Lockridge,). Studies are in progress to evaluate METt phosphorylating ability in sperm cells.…”

Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms

“…), including serine, tyrosine, lysine and histidine in various proteins. Organophosphorylation at amino acid residues is dose‐ and site‐dependent, and most identified modifications occurred at tyrosine and lysine residues on protein surface regions in the vicinity of positively charged amino acids …”

“…Organophosphorylation at amino acid residues is dose-and site-dependent, and most identified modifications occurred at tyrosine and lysine residues on protein surface regions in the vicinity of positively charged amino acids. [24,25,28] Each OP molecule makes a particular added mass when adducted to an amino acid residue (see Table 1) and can be identified by MS, mostly via bottom-up approaches. Using MS, multiple covalent modifications have been identified in various proteins, and MS/MS has determined the modification sites for albumin, tubulin, actin, keratin, transferrin, ubiquitin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mostly at tyrosine, lysine and serine residues.…”