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High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells
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).
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.
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.