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High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells
Citation:
Karmaus, A., C. Toole, D. Filer, K. Lewis, AND M. Martin. High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 150(2):323-332, (2016).
Impact/Purpose:
Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. An in vitro high-throughput assay using H295R human adrenocortical carcinoma cells was optimized to evaluate the concentraiton-dependent effects of 2,060 chemical samples on steroidogenesis via quantification of 10 hormones.
Description:
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 2,060 chemical samples on steroidogenesis via HPLC-MS/MS quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a three 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 while the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were pre-stimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2,060 chemical samples evaluated, 524 samples were selected for six-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 five 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.
