A method to determine precise benchmark doses for carbamate anticholinesterases.
In determining benchmark doses for risk assessment and regulation of carbamate anticholinesterase pesticides like formetanate, oxamyl, and methomyl, one needs to quantitate low levels of cholinesterase inhibition. For improved accuracy while using fewer subjects, we developed an assay based on the recognized ability of carbamates to protect cholinesterase from irreversible inactivation. This assay measures enzyme that survives diisopropylfluorophosphate exposure in vitro and then reactivates by decarbamylation after small molecules are removed with size-exclusion centrifugation. The 99% silencing of unprotected cholinesterase yields a low background. Comparisons of recovered activity with initial activity (representing carbamate-free enzyme) use each sample as its own control. As a result, carbamate-protection assays can demonstrate a statistically significant 2-3% inhibition of brain cholinesterase in a single experimental group of modest size. When applied to brain samples from formetanate-treated rats, such an assay predicted a benchmark dose of 0.19 mg/kg for 10% inhibition (BMD10), with a lower 95% confidence limit of 0.15 mg/kg (BMDL10). Protection assays should enable precise determinations of benchmark doses for other carbamates, as well as accurate assessment of in vivo inhibition half-lives under low-dose scenarios.
Lassiter
T Leon
TL
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA. lassiter.tommie@mayo.edu
Brimijoin
Stephen
S
D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002219:Carbamates / Q000737:chemistry / Q000633:toxicity*; D002498:Centrifugation / Q000379:methods; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000633:toxicity*; D002802:Cholinesterases / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D007531:Isoflurophate / Q000737:chemistry; D007700:Kinetics; D008297:Male; D008724:Methomyl / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D008956:Models, Chemical; D010575:Pesticides / Q000633:toxicity*; D051381:Rats; D020318:Rats, Long-Evans; D012016:Reference Values; D015203:Reproducibility of Results; D018570:Risk Assessment; D012680:Sensitivity and Specificity; D013997:Time Factors
D002219:Carbamates; D002800:Cholinesterase Inhibitors; D010575:Pesticides; D007531:Isoflurophate; D008724:Methomyl; C100163:formetanate; D002802:Cholinesterases; C011960:oxamyl