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Toxicol Sci. 2016 Apr;150(2):323-32. doi: 10.1093/toxsci/kfw002. Epub 2016 Jan 18.

High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

Author information

1
*National Center for Computational Toxicology, US EPA, Research Triangle Park, North Carolina; martin.matt@epa.gov.
2
Cyprotex (Formerly CeeTox, Inc.), Kalamazoo, Michigan; and.
3
*National Center for Computational Toxicology, US EPA, Research Triangle Park, North Carolina;
4
OpAns, LLC, Durham, North Carolina.

Abstract

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis.

KEYWORDS:

H295R cells; ToxCast; high-throughput screening.; steroidogenesis

PMID:
26781511
PMCID:
PMC4809454
DOI:
10.1093/toxsci/kfw002
[Indexed for MEDLINE]
Free PMC Article

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