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Multi-tiered Approach to Development of Increased Throughput Assay Models to Assess Endocrine-Disrupting Activity of Chemicals
Citation:
Paul, K., J. Hedge, P. Hartig, E. Gray, K. Crofton, AND Steve Simmons. Multi-tiered Approach to Development of Increased Throughput Assay Models to Assess Endocrine-Disrupting Activity of Chemicals. Presented at Society for Environmental Toxicology and Chemistry (SETAC) 33rd Annual Meeting, November 11 - 15, 2012.
Impact/Purpose:
This abstract is for presentation at the Society for Environmental Toxicology and Chemistry (SETAC) meeting November 11-15, 2012, Long Beach, CA
Description:
Screening for endocrine-disrupting chemicals (EDCs) requires sensitive, scalable assays. Current high-throughput screening (HTPS) approaches for estrogenic and androgenic activity yield rapid results, but many are not sensitive to physiological hormone concentrations, suggesting that less potent EDCs may not be identified. And although thyroid hormone (TH) receptor assays are available, environmental thyroid-disrupting chemicals act primarily via other mechanisms, including inhibition of thyroid hormone synthesis; yet, no HTPS assays exist for TH synthesis targets. We used the following multi-tiered approach to improve assays available for EDC screening: (1) re-optimization of sensitive estrogen and androgen transcriptional activation assays (ER-TA, AR-TA) for HTPS; (2) re-development of an ex vivo rat thyroperoxidase (TPO) inhibition assay; and (3) de novo development of an in vitro model system for human TPO inhibition. We tested the hypothesis that T47D-kbluc ER-TA and MDA-kb2 AR-TA assays could be miniaturized to a 384-well plate, and used controls, dynamic range, variability, and Z´ to evaluate this effort. Results demonstrated that the AR-TA assay can be miniaturized without loss of dynamic range (10-fold), increased variability (< 10%), or loss of sensitivity, but that the ER-TA assay could only be automated in 96-well plates due to loss of sensitivity in the 384-well format. Cross-validation of these assays with other ER and AR assays is ongoing with a 90-chemical reference set. Further, we hypothesized that use of a peroxidase substrate (Amplex UltraRed, AUR, LifeTech) and automated reagent delivery could increase the throughput of an ex vivo rat TPO assay. The potency of methimazole (MMI)-induced TPO inhibition was similar with AUR compared to the existing guaiacol assay (IC50 reduced from 5 µM to ~1 µM), and the AUR signal was stable > 30 min after reaction initiation. Finally, preliminary work demonstrated that lentiviral-transduction of a human cell line (HEK293T) with recombinant TPO could be used to test for human TPO inhibition. Cell fractions showed responsivity to MMI using AUR as a substrate (IC100 = 100 µM). The results of this step-wise approach to improving and developing assays for EDC screening indicate that systematic evaluation of assay sensitivity and specificity is critical for adaptation of assays to HTPS for EDCs. This abstract does not necessarily reflect the policy of the US EPA.