Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates
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Methods are needed for rapid screening of environmental compounds for neurotoxicity, particularly ones that assess function. To demonstrate the utility of microelectrode array (MEA)-based approaches as a rapid neurotoxicity screening tool, 1055 chemicals from EPA’s phase II ToxCast library were evaluated for effects on neural function and cell health. Primary cortical networks were grown on multi-well microelectrode array (mwMEA) plates. On day in vitro 13, baseline activity (40 min) was recorded prior to exposure to each compound (40 µM). Changes in spontaneous network activity [mean firing rate (MFR)] and cell viability (lactate dehydrogenase and CellTiter Blue) were assessed within the same well following compound exposure. Following exposure, 326 compounds altered (increased or decreased) normalized MFR beyond hit thresholds based on 2× the median absolute deviation of DMSO-treated wells. Pharmaceuticals, pesticides, fungicides, chemical intermediates, and herbicides accounted for 86% of the hits. Further, changes in MFR occurred in the absence of cytotoxicity, as only eight compounds decreased cell viability. ToxPrint chemotype analysis identified several structural domains (e.g., biphenyls and alkyl phenols) significantly enriched with MEA actives relative to the total test set. The top 5 enriched ToxPrint chemotypes were represented in 26% of the MEA hits, whereas the top 11 ToxPrints were represented in 34% of MEA hits. These results demonstrate that large-scale functional screening using neural networks on MEAs can fill a critical gap in assessment of neurotoxicity potential in ToxCast assay results. Further, a data-mining approach identified ToxPrint chemotypes enriched in the MEA-hit subset, which define initial structure–activity relationship inferences, establish potential mechanistic associations to other ToxCast assay endpoints, and provide working hypotheses for future studies.
KeywordsNeurotoxicity screening ToxCast Microelectrode array
The authors wish to thank Ms Kathleen Wallace and Ms Theresa Freudenrich of the US Environmental Protection Agency for their outstanding technical contributions in support of the tissue cultures needed for this work. Further, the authors greatly appreciate the constructive comments from Drs William Mundy and Joshua Harrill from the US Environmental Protection Agency on a draft version of this manuscript.
Compliance with ethical standards
Conflict of interest
At the time these experiments were conducted, JDS was an employee of Axion Biosystems, which makes microelectrode array equipment and supplies. The other authors have no conflicts to report.
Preparation of this document has been funded by the U.S. Environmental Protection Agency. This document has been subjected to review by the National Health and Environmental Effects Research Laboratory (NHEERL) and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. This work was supported in part by CRADA 644-11 between the US EPA and Axion Biosystems.
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