title: High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.
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.
authors.affiliation: *National Center for Computational Toxicology, US EPA, Research Triangle Park, North Carolina; martin.matt@epa.gov.
authors.forename: Agnes L
authors.initials: AL
authors.lastname: Karmaus
authors.affiliation: Cyprotex (Formerly CeeTox, Inc.), Kalamazoo, Michigan; and.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Toole
authors.affiliation: *National Center for Computational Toxicology, US EPA, Research Triangle Park, North Carolina;
authors.forename: Dayne L
authors.initials: DL
authors.lastname: Filer
authors.affiliation: OpAns, LLC, Durham, North Carolina.
authors.forename: Kenneth C
authors.initials: KC
authors.lastname: Lewis
authors.affiliation: *National Center for Computational Toxicology, US EPA, Research Triangle Park, North Carolina; martin.matt@epa.gov.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Martin
mesh_terms: D018268:Adrenocortical Carcinoma / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D052244:Endocrine Disruptors / Q000633:toxicity*; D057166:High-Throughput Screening Assays; D006728:Hormones / Q000096:biosynthesis*; D006801:Humans; D020714:Maximum Tolerated Dose; D013256:Steroids / Q000096:biosynthesis*; D053719:Tandem Mass Spectrometry
chemical_list: D052244:Endocrine Disruptors; D006728:Hormones; D013256:Steroids
keywords: H295R cells; ToxCast; high-throughput screening.; steroidogenesis