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Real-Time Growth Kinetics Measuring Hormone Mimicry for ToxCast Chemicals in T‑47D Human Ductal Carcinoma Cells
Citation:
ROTROFF, D., D. J. DIX, K. A. HOUCK, R. J. KAVLOCK, T. B. KNUDSEN, M. T. MARTIN, D. REIF, A. M. RICHARD, N. SIPES, Y. A. ABASSI, C. JIN, M. STAMPFL, AND R. JUDSON. Real-Time Growth Kinetics Measuring Hormone Mimicry for ToxCast Chemicals in T‑47D Human Ductal Carcinoma Cells . CHEMICAL RESEARCH IN TOXICOLOGY. American Chemical Society, Washington, DC, 26(7):1097-1107, (2013).
Impact/Purpose:
Overall, the T-47D cell growth assay can provide important information regarding a chemical’s potential to interact with the ER signaling pathway. Our approach is to combine multiple ER assays (including the current one), plus physicochemical and chemical structure information to help classify chemicals as to their estrogenic potential. These data will then be combined with exposure information to develop a prioritization scheme for EDSP testing. Further work is needed to include a more comprehensive panel of assays, covering other key molecular initiating events in the ER pathway. Currently, efforts within the ToxCast program are underway to include assays that measure ER signaling before and after treatment of the test chemical with a liver S9 fraction to determine effects of xenobiotic metabolism and to include assays that measure receptor dimerization and nuclear translocation for both ERα and ERβ. The present analysis of T-47D cell growth provides significant information relating duration of exposure, potency, and efficacy to effects on cell growth or cell loss. In addition, the data-driven characterization of ER in vitro assays provides an important step toward developing a more comprehensive and integrated approach for testing chemicals for their potential modulation of ER signaling.
Description:
High-throughput screening (HTS) assays capable of profiling thousands of environmentally relevant chemicals for in vitro biological activity provide useful information on the potential for disrupting endocrine pathways. Disruption of the estrogen signaling pathway has been implicated in a variety of adverse health effects including impaired development, reproduction, and carcinogenesis. The estrogen-responsive human mammary ductal carcinoma cell line T-47D was exposed to 1815 ToxCast chemicals comprising pesticides, industrial chemicals, pharmaceuticals, personal care products, cosmetics, food ingredients, and other chemicals with known or suspected human exposure potential. Cell growth kinetics were evaluated using real-time cell electronic sensing. T-47D cells were exposed to eight concentrations (0.006−100 μM), and measurements of cellular impedance were repeatedly recorded for 105 h. Chemical effects were evaluated based on potency (concentration at which response occurs) and efficacy (extent of response). A linear growth response was observed in response to prototypical estrogen receptor agonists (17β-estradiol, genistein, bisphenol A, nonylphenol, and 4-tert-octylphenol). Several compounds, including bisphenol A and genistein, induced cell growth comparable in efficacy to that of 17β-estradiol, but with decreased potency. Progestins, androgens, and corticosteroids invoked a biphasic growth response indicative of changes in cell number or cell morphology. Results from this cell growth assay were compared with results from additional estrogen receptor (ER) binding and transactivation assays. Chemicals detected as active in both the cell growth and ER receptor binding assays demonstrated potencies highly correlated with two ER transactivation assays (r = 0.72; r = 0.70). While ER binding assays detected chemicals that were highly potent or efficacious in the T-47D cell growth and transactivation assays, the binding assays lacked sensitivity in detecting weakly active compounds. In conclusion, this cell-based assay rapidly detects chemical effects on T-47D growth and shows potential, in combination with other HTS assays, to detect environmentally relevant chemicals with potential estrogenic activity. [The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.]
