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

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


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adrenocortical Carcinoma / metabolism
  • Adrenocortical Carcinoma / pathology
  • Cell Culture Techniques
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Chromatography, High Pressure Liquid
  • Dose-Response Relationship, Drug
  • Endocrine Disruptors / toxicity*
  • High-Throughput Screening Assays
  • Hormones / biosynthesis*
  • Humans
  • Maximum Tolerated Dose
  • Steroids / biosynthesis*
  • Tandem Mass Spectrometry


  • Endocrine Disruptors
  • Hormones
  • Steroids