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Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates
Citation:
Strickland, J., M. Martin, A. Richard, K. Houck, AND Tim Shafer. Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates. Archives of Toxicology. Springer, New York, NY, 92(1):487-500, (2018). https://doi.org/10.1007/s00204-017-2035-5
Impact/Purpose:
Thousands of chemicals in the environment have not been adequately characterized for their potential toxicities, including neurotoxicity. Because testing these compounds with conventional animal-based studies is too costly and time consuming, novel, high-throughput, low-cost methods are needed to screen and prioritize chemicals for toxicity testing, including for neurotoxicity. The studies conducted in the attached manuscript demonstrate an approach to screening chemicals for their ability to disrupt the function of interconnected neural networks in vitro. The work demonstrates that this approach is amenable to screening over 1000 compounds and detects broad classes of chemicals known to cause neurotoxicity. As such, this approach can be a useful addition to large-scale screening and predictive toxicity approaches such as those taken by the EPA’s ToxCast Program. The EPA’s ToxCast Program incorporates over 800 assays for a wide variety of cellular responses including ~300 signaling pathways. However, few of these assays provide direct measures of chemical effects on function of neurons, thus creating a potential gap in the biological space covered by ToxCast Assays. The attached manuscript evaluates effects of ToxCast phase I and II chemicals on function of neural networks grown on microelectrode arrays. This allows for determination of a chemical’s ability to alter the function of networks of interconnected neurons, and published work demonstrates that many chemicals that are neurotoxic in vivo alter network function in vitro. In this study, primary cultures of cortical neurons from neonatal rats were plated into each well of a 48 well microelectrode array plate; each well contained 16 microelectrodes. After allowing neural networks to form over at least 13 days in vitro, baseline recordings of network activity were made for 40 minutes followed by treatment with test chemical (concentration 0.3 to 30 µM) or vehicle and recording for an additional 40 minutes. Chemical induced changes in network function were determined based on their magnitude relative to the vehicle. These data demonstrate that measurement of chemical effects on neural network function using microelectrode arrays is a rapid and economical method to screen compounds for the potential to cause neurotoxicity.
Description:
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.
URLs/Downloads:
DOI: Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates