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

• Published in: Toxicology in vitro 2019-04, Vol.56, p.93-100
• Main Authors: Li, Shuaizhang, Zhao, Jinghua, Huang, Ruili, Santillo, Michael F., Houck, Keith A., Xia, Menghang
• Format: Article
• Language: English
• Subjects: Acetylcholinesterase; Acetylcholinesterase (AChE); Acetylcholinesterase - metabolism; Animals; Assaying; Biotransformation; Chemicals; Cholinesterase Inhibitors - toxicity; Colorimetry; Enzymes; Fluorescence; High-throughput screening; High-Throughput Screening Assays; Humans; In vivo methods and tests; Inhibition; Inhibitors; Liver; Liver microsomes; Measurement methods; Metabolic activation; Metabolic rate; Metabolism; Metabolites; Microsomes; Microsomes, Liver - drug effects; Microsomes, Liver - metabolism; Neurotoxicity; Organic chemistry; Organophosphorus; Organophosphorus Compounds - toxicity; Organophosphorus pesticides; Pesticides; Pesticides - toxicity; Quantitative high-throughput screening (qHTS); Rats; Reproducibility; Xenobiotics - toxicity
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2019.01.002

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. •Many OPs require metabolic activation by P450 isoenzymes to inhibit AChE activity for their pesticidal activity•In vitro AChE assays with xenobiotic metabolism capability were developed and validated in a qHTS platform.•Potency differences between the parent compounds and their metabolites were observed in assays with metabolic activation.•The screening platform can be used to evaluate compounds for potential neurotoxicity from AChE inhibition.