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Evaluating perturbation of in vitro steroidogenesis using a high-throughput H295R assay
Citation:
Haggard, D., A. Karmaus, M. Martin, R. Judson, Woodrow Setzer, AND K. Paul-Friedman. Evaluating perturbation of in vitro steroidogenesis using a high-throughput H295R assay. Presented at Society of Toxicology Annual Meeting, San Antonio, Texas, March 11 - 15, 2018. https://doi.org/10.23645/epacomptox.6827660
Impact/Purpose:
The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program and the Organization for Economic Co-operation and Development (OECD) use a human adrenocarcinoma (H295R) cell-based assay to predict changes in testosterone (T) and 17-estradiol (E2) production. The ToxCast high-throughput H295R (HT-H295R) assay measures 11 hormones, including progestagens, glucocorticoids, androgens, and estrogens. To date, 2012 chemicals have been screened at a single concentration; 656 of these were screened in concentration-response. The objectives of this work were to: 1) develop an integrated statistical analysis of chemical-mediated effects on steroidogenesis in the HT-H295R assay; 2) evaluate whether the assay predicts T and E2 production via comparison with the OECD-validated H295R assay; and, 3) develop a pathway-based kinetic model of steroidogenesis in HT-H295R.
Description:
The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program and the Organization for Economic Co-operation and Development (OECD) use a human adrenocarcinoma (H295R) cell-based assay to predict changes in testosterone (T) and 17-estradiol (E2) production. The ToxCast high-throughput H295R (HT-H295R) assay measures 11 hormones, including progestagens, glucocorticoids, androgens, and estrogens. To date, 2012 chemicals have been screened at a single concentration; 656 of these were screened in concentration-response. The objectives of this work were to: 1) develop an integrated statistical analysis of chemical-mediated effects on steroidogenesis in the HT-H295R assay; 2) evaluate whether the assay predicts T and E2 production via comparison with the OECD-validated H295R assay; and, 3) develop a pathway-based kinetic model of steroidogenesis in HT-H295R. To support application of HT-H295R data to prioritization, a single value based on Mahalanobis distance was computed for 654 chemicals to indicate the magnitude of effects on 11 hormones. The maximum mean Mahalanobis distance (maxmMd) values were high for strong modulators (prochloraz, mifepristone) and lower for moderate modulators (atrazine, molinate). Twenty-five of 28 reference chemicals used for OECD validation were screened in HT-H295R, and produced qualitatively similar results, with accuracies of 0.90/0.75 and 0.81/0.91 for increased/decreased T and E2 production, respectively. To further mechanistic understanding of HT-H295R results, preliminary work to adapt a kinetic model of steroidogenesis included time-course experiments and addition of isozyme-specific kinetics. The HT-H295R assay provides robust information regarding production of T and E2, as well as additional steroid hormones. The statistical analysis presented here provides a data-driven approach to prioritizing chemical lists for putative effects on steroidogenesis, and development of a kinetic model may inform mechanistic hypotheses about chemical activity in the HT-H295R assay. This abstract does not necessarily reflect U.S. EPA policy.
URLs/Downloads:
DOI: Evaluating perturbation of in vitro steroidogenesis using a high-throughput H295R assay
HAGGARD_SOT_2018_POSTER_FINAL_030518.PDF (PDF, NA pp, 790.683 KB, about PDF)