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Anti and Androgenic Activities in MDA-KB2 Cells: A Comparison of Performance in 96 Well Versus HTS Assays
Citation:
Gray, E., N. Evans, AND V. Wilson. Anti and Androgenic Activities in MDA-KB2 Cells: A Comparison of Performance in 96 Well Versus HTS Assays. Presented at Society of Toxicology Conference, San Diego, CA, March 23 - 27, 2015.
Impact/Purpose:
This abstract will be presented at the Society of Toxicology Annual Meeting , March 23-27, 2015, San Diego, CA
Description:
We developed the MDA-kb2 cell line to screen androgen agonists/antagonists (Wilson et al., ToxSci 66:69, 2002). MDA-kb2 has been used to quantify anti- and androgenic activities of chemicals, mixtures, combustion by-products, oil dispersants and waste, source and drinking water samples. Recently, EPA released the iCSS Dashboard, including HTS data for 800+ assays including an MDA-kb2 assay. We downloaded the data (v. beta 0.5) and compared performance of the HTS assay to our 96 well plate assay. Data are being analyzed to determine EC50, goodness of fit (R2), and variability. EC50 values for the 10 agonists common to both data sets compared favorably. However, some other HTS data for “active” agonists were highly variable (e.g., tannic acid, bromoxynil) and should be rerun before any designation is assigned. E.g., simvastatin was designated an “active” agonist with an AC50 of 0.7 µM. However, in one run it was a full agonist whereas it was completely negative in the other run; simvastatin is not an agonist in our assay or in vivo. Data for HTS “active” agonists phenolphthalein, nitrophenol, and metolachlor also were unstable; no effect in one run and high activity declining as concentration increased in the other run. 18 antagonists run in our assay also were tested in HTS. Of these, 12 were “inactive” in HTS, including cyproterone acetate, vinclozolin, prochloraz, and linuron; all are active in vivo. In conclusion: 1) some HTS results are uninterpretable due to high variability; 2) run-to-run variability may be due in part to differences in chemical purity; 3) performance criteria are needed for HTS assays; 4) concentrations used in HTS should be adjusted down for potent and up for weak ligands; and 5) modification of the criteria used to classify chemicals as “active” or “inactive” is warranted. We understand that subsequent analyses of the HTS data will include modifications to address some of these issues. This abstract does not reflect EPA policy.