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High-throughput H295R steroidogenesis assay: utility as an alternative and a statistical approach to characterize effects on steroidogenesis
Citation:
Haggard, D. AND K. Paul-Friedman. High-throughput H295R steroidogenesis assay: utility as an alternative and a statistical approach to characterize effects on steroidogenesis. Presented at Comptox Communities of Practice, RTP, NC, December 14, 2017. https://doi.org/10.23645/epacomptox.6863771
Impact/Purpose:
The purpose of this presentation is to give an update on analysis of the high-throughput H295R data for use as an alternative to the H295R assay and for generation of a prioritization metric.
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 chemical perturbation ofchanges in testosterone (T) and 17-estradiol (E2) production. The ToxCast high-throughput H295R (HT-H295R) assay includes measuresement of 11 hormones, including progestagens, glucocorticoids, androgens, and estrogens. To date, 2012 chemicals have been screened at one 1 concentration; with 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 specifically 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 the synthesis of 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 the HT-H295R assay, 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 published 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 lists of chemicals lists for putative effects on steroidogenesis.
URLs/Downloads:
DOI: High-throughput H295R steroidogenesis assay: utility as an alternative and a statistical approach to characterize effects on steroidogenesis
2017_COP_DRAFT_12112017.PDF (PDF, NA pp, 2356.419 KB, about PDF)