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Using adverse outcome pathway analysis to guide development of high-throughput screening assays for thyroid-disruptors
Citation:
Paul, K., J. Hedge, D. Rotroff, K. Crofton, M. Hornung, AND Steve Simmons. Using adverse outcome pathway analysis to guide development of high-throughput screening assays for thyroid-disruptors. Presented at CCT/SOT MEETING, Chapel Hill, NC, January 16 - 17, 2014.
Impact/Purpose:
This work is an important contribution to the emerging field of thyroid-disruptor screening. This oral postal pesentation details the development of a new thyroperoxidase inhibition assay to meet this salient need. This will also describe preliminary screening work with the assay. This will be presented at the Contemporary Concepts in Toxicology (CCT)/Society of Toxicology (SOT) Meeting, January 16-17, 2014, Chapel Hill, NC
Description:
Using Adverse Outcome Pathway Analysis to Guide Development of High-Throughput Screening Assays for Thyroid-Disruptors Katie B. Paul1,2, Joan M. Hedge2, Daniel M. Rotroff4, Kevin M. Crofton4, Michael W. Hornung3, Steven O. Simmons2 1Oak Ridge Institute for Science Education Postdoctoral Fellow; 2Integrated Systems Toxicology Division; and, 3Mid-Continent Ecology Division, NHEERL; 4National Center for Computational Toxicology; ORD, U.S. EPA, RTP, NC and Duluth, MN Adverse outcome pathway analysis illustrates several molecular-initiating events (MIEs) for adverse thyroid–mediated toxicities that are not covered by high-throughput screening (HTS) assays. In response, we developed a HTS assay to detect thyroperoxidase (TPO) inhibitors. TPO, the catalyst for thyroid hormone synthesis, is inhibited by a number of environmental contaminants. We adapted the guaiacol oxidation assay for TPO inhibition to HTS by replacing guaiacol with a fluorescent peroxidase substrate in a rat thyroid-based assay. We tested the hypothesis that use of a peroxidase substrate (Amplex UltraRed, AUR, LifeTech) in automated 96- or 384-well plate formats would increase throughput and maintain predictivity. Methimazole (MMI), a classic TPO inhibitor, was active in this new assay with an IC50 value of 0.025 µM in the 384-well format, compared to 2.20 µM in the guaiacol-based 96-well format. The AUR signal was stable from 30-120 min after initiation, and the dynamic range and Z’ score with MMI were 11-fold and 0.93, respectively, for the 384-well AUR-TPO assay. The assay demonstrated a 100% true positive rate and a 0% false negative rate with a 21-chemical training set. Work is underway to screen the ToxCast Phase I & II libraries (1060 chemicals) with the AUR-TPO assay. This approach to developing HTS assays for thyroid-disruptor screening based on AOPs has enabled the largest screening for TPO inhibition to date, will drastically reduce animal tissue use, and fits into a prioritization testing scheme for thyroid-disrupting chemicals. This abstract does not necessarily reflect the policy of the US EPA.