Coupling the H295R with ERα and AR U2OS CALUX assays enables simultaneous testing for estrogenic, anti-androgenic and steroidogenic modalities

Endocrine active substances, including steroidogenesis modulators, have received increased attention. The in vitro H295R steroidogenesis assay (OECD TG 456) is commonly used to test for this modality. However, current detection methods often fail to capture alterations to estrogen biosynthesis. The present study explored the potential of ERα and AR CALUX bioassays to serve as a detection system for the original H295R assay, as they can quantify lower hormone concentrations and can simultaneously provide information about estrogen- and androgen-receptor activities. Using substances from the original OECD validation study, we obtained lowest observed effect concentrations for steroidogenesis mostly equivalent to those previously reported and sometimes lower for estrogen biosynthesis. However, categorization of many of these substances as receptor (ant)agonists or disruptors of steroidogenesis was difficult because often substances had both modalities, including some where the receptor-mediated activities were identified at concentrations below those triggering steroidogenic effects. When the leading activity was not accounted for, H295R-CALUX assay sensitivity in comparison to the OECD validation study was 0.50 for androgen and 0.78 for estrogen biosynthesis. However, upon reinterpretation of the combined assay results to identify endocrine activities without regard to the modality or direction of effects, assay sensitivity was equal to 1.00. These proof-of-concept study findings indicate the high relevance of this assay for the identification of endocrine active substances with additional valuable mode-of-action information and the capacity to detect smaller changes in estrogen biosynthesis, suggesting that the coupled H295R-CALUX assay has promise for the analysis of samples in a decision-making context.