Identification of candidate reference chemicals for in vitro steroidogenesis assays

Abstract

The Endocrine Disruptor Screening Program (EDSP) is transitioning from traditional testing methods to integrating ToxCast/Tox21 in vitro high-throughput screening assays for identifying chemicals with endocrine bioactivity. The ToxCast high-throughput H295R steroidogenesis assay may potentially replace the low-throughput assays currently used in the EDSP Tier 1 battery to detect chemicals that alter the synthesis of androgens and estrogens. Herein, we describe an approach for identifying in vitro candidate reference chemicals that affect the production of androgens and estrogens in models of steroidogenesis. Candidate reference chemicals were identified from a review of H295R and gonad-derived in vitro assays used in methods validation and published in the scientific literature. A total of 29 chemicals affecting androgen and estrogen levels satisfied all criteria for positive reference chemicals, while an additional set of 21 and 15 chemicals partially fulfilled criteria for positive reference chemicals for androgens and estrogens, respectively. The identified chemicals included pesticides, pharmaceuticals, industrial and naturally-occurring chemicals with the capability to increase or decrease the levels of the sex hormones in vitro. Additionally, 14 and 15 compounds were identified as potential negative reference chemicals for effects on androgens and estrogens, respectively. These candidate reference chemicals will be informative for performance-based validation of in vitro steroidogenesis models.

Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors

“…The Organisation for Economic Co-operation and Development's (OECD) H295R in vitro steroidogenesis assay, an internationally validated assay for regulatory contexts, has been used to study a chemical's impact on the steroidogenic pathway by measuring hormone concentrations following exposure to human H295R adrenocortical carcinoma cells, and similar approaches have been used in research settings (Caron-Beaudoin et al 2016Fan et al 2007;Hecker et al 2011;Pinto et al 2018;Strajhar et al 2017). Although initially only validated to measure the effects on E2 and testosterone, the H295R assay was subsequently modified to run in high-throughput (HT) format as part of the U.S. Environmental Protection Agency (EPA) ToxCast chemical screening program and to measure effects on 13 hormones involved in the steroidogenic pathway, including progestagens, corticosteroids, androgens, and estrogens (Haggard et al 2018;Karmaus et al 2016).…”

Cheminformatics analysis of chemicals that increase estrogen and progesterone synthesis for a breast cancer hazard assessment

“…This cluster included chemicals with a dihydroxy phosphanedithione substructure. Notably, this cluster included numerous insecticides as Sulprofos (15), Phosmet (16) or Malathion (17). Many insecticides in this group also have both E2up and P4up activity (Fig.…”

Primary Cultures and Cell Lines for <i>In Vitro</i> Modeling of the Human Adrenal Cortex

“…The human adrenal H295R line has recently been used to develop a high-throughput testing model to ascertain chemical effects on steroid synthesis (HT-H295R) (Karmaus et al 2016). The utility of the system results from the broad steroid metabolome secreted by H295R cells, which includes mineralocorticoids, glucocorticoids, progestins, androgens and estrogens (Botteri Principato et al 2018;Pinto et al 2018;Haggard et al 2018Haggard et al , 2019. This allows the cells to act as a "Tier 1" steroidogeneses screen that has been validated using a set of recommended international test guidelines.…”