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Increasing the Throughput of a High-Content Imaging-Based Developmental Neurotoxicity Proliferation Assay
Citation:
Byrd, G., T. Freudenrich, S. Choo, M. Culbreth, T. Shafer, AND J. Harrill. Increasing the Throughput of a High-Content Imaging-Based Developmental Neurotoxicity Proliferation Assay. International Neurotoxicology Association (INA), Durham, NC, May 21 - 25, 2023. https://doi.org/10.23645/epacomptox.23148746
Impact/Purpose:
Poster presented to the International Neurotoxicology Association (INA) Meeting symposium May 2023
Description:
The traditional reliance of developmental neurotoxicity (DNT) hazard assessment on in vivo studies has limited the acquisition of DNT data for the ever-expanding number of chemicals in our environment. Financial and temporal limitations, as well as calls to reduce animal testing, have encouraged the development of DNT new approach methods (NAM) that allow for the efficient screening of larger quantities of chemicals while reducing overall cost. The EPA DNT NAMs battery includes multiple 96-well format high-content imaging-based assays. To further increase throughput and cost-efficiency, enhanced laboratory automation and imaging capabilities can be leveraged to optimize these assays to 384-well format. As a first step in achieving this goal, this study has adapted an existing 5-bromo-2’-deoxyuridine (BrdU) incorporation-based cell proliferation assay in hNP1 human neural progenitor cells to 384-well format. Nine chemicals were assessed to determine the relative success of the optimization. Four known antiproliferative chemicals (5-fluorouracil, aphidicolin, cytosine arabinoside, and hydroxyurea), two suspected antiproliferative chemicals (ketamine and caffeine), two negative controls (saccharin and sorbitol), and one positive cell viability control (staurosporine) were evaluated. AC50 and EC50 concentrations were determined based on curve fitting of vehicle control normalized percent response values for 5-fluorouracil, aphidicolin, cytosine arabinoside, hydroxyurea, and staurosporine. Excluding staurosporine, which has not been previously assessed for effects on proliferation, observations of activity and inactivity were consistent with previous work in the lower-throughput 96-well format for 7/8 chemicals. Hydroxyurea, interestingly, stimulated proliferation at high concentrations, though the calculated EC50 value was extrapolated above the highest tested concentration (100 µM). Future work will entail multiplexing BrdU incorporation with caspase activation to simultaneously assess cell proliferation and apoptosis. This abstract does not reflect U.S. EPA policies.
URLs/Downloads:
DOI: Increasing the Throughput of a High-Content Imaging-Based Developmental Neurotoxicity Proliferation Assay
INA_2023_POSTER_GB_V4.PDF (PDF, NA pp, 920.128 KB, about PDF)