Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds

Abstract

A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results.

High‐Throughput Label‐Free Enzymatic Assays Using Desorption Electrospray‐Ionization Mass Spectrometry

“…Chlorpyrifos‐oxon was found to be the strongest inhibitor (IC 50 =49 n m ), followed by paraoxon (140 n m ) and dichlorovos (890 n m ); all of these CWA simulants are stronger than the compounds screened earlier in this study. These trends agree with previous reports [61, 62] …”

High‐Throughput Label‐Free Enzymatic Assays Using Desorption Electrospray‐Ionization Mass Spectrometry

“…These trends agree with previous reports. [61,62] Dose-response curves were also obtained for the reactivation of the enzyme using 2-pralidoxime ( Figure S7). The oxime was added after 20 minutes of incubation of the enzyme with the CWA simulant, and it was incubated with the mixture for additional 25 minutes before the addition of the substrate and a final 5-minute reaction incubation.…”