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Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides
Citation:
Li, S., J. Zhao, R. Huang, M. Santillo, K. Houck, AND M. Xia. Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides. TOXICOLOGY IN VITRO. Elsevier Science Ltd, New York, NY, 56:93-100, (2019). https://doi.org/10.1016/j.tiv.2019.01.002
Impact/Purpose:
Acetylcholinesterase (AChE), located at the neuromuscular junctions and cholinergic nerve synapses, terminates neurotransmission at cholinergic synapses by catalyzing the breakdown of acetylcholine (ACh) into choline [1]. Such activity is exploited for pesticidal activity against a wide range of insects as well as in human therapeutic applications and chemical warfare agents. . Inhibition of AChE activity leads to cholinergic poisoning including vomiting, diarrhea, muscle fasciculation and even flaccid paralysis [2]. Inhibition of AChE has significant consequences in human health, and many compounds including synthesized drug candidates, food additives, and industrial chemicals have not been thoroughly evaluated for effects on AChE activity. Moreover, some of the chemicals may need metabolic activation to show inhibitory effects. Therefore, there is a great need to develop in vitro high-throughput methods with xenobiotic metabolism capability for identifying AChE inhibitors, which can prioritize more compounds for subsequent toxicological study.
Description:
The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as neurotoxic effects. The metabolites of some chemicals including organophosphorus compounds become more potent AChE inhibitors compared to their parental compounds. In order to identify the AChE inhibitors that need xenobiotic metabolism to become active or more potent, we have developed and validated a high-throughput screening method with metabolic capability for identifying AChE inhibitors. In this study, an enzyme-based high-throughput assay was developed 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 assays with microsome were validated by testing a group of well-known AChE inhibitors including parent compounds and their metabolites. Large potency difference between the parent compounds and the metabolites were observed in the assay with microsome addition. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that need metabolic activation for inhibiting AChE activity.
URLs/Downloads:
DOI: Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides