Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants.
Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
Wang
Yuxia
Y
University of Nebraska Medical Center, Eppley Institute, Omaha, NE 68198-6805, USA.
Schopfer
Lawrence M
LM
Duysen
Ellen G
EG
Nachon
Florian
F
Masson
Patrick
P
Lockridge
Oksana
O
D000818:Animals; D002800:Cholinesterase Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D002802:Cholinesterases / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004351:Drug Resistance; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D009943:Organophosphorus Compounds / Q000494:pharmacology* / Q000633:toxicity*; D010641:Phenotype; D013053:Spectrophotometry
D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; D002802:Cholinesterases