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Toxicol In Vitro. 2019 Apr;56:93-100. doi: 10.1016/j.tiv.2019.01.002. Epub 2019 Jan 6.

Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides.

Author information

1
Division for Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
2
Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD, USA.
3
National Center for Computational Toxicology, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, NC, USA.
4
Division for Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA. Electronic address: mxia@mail.nih.gov.

Abstract

The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.

KEYWORDS:

Acetylcholinesterase (AChE); Liver microsomes; Metabolic activation; Organophosphorus; Quantitative high-throughput screening (qHTS)

PMID:
30625376
DOI:
10.1016/j.tiv.2019.01.002
[Indexed for MEDLINE]
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