Effect of conazoles on steroidogenesis mapped by extended H295R-CALUX® assay and LC-MS/MS profiling

The H295R assay (OECD TG456) is used to screen chemicals for interference with steroidogenesis, measuring testosterone and estradiol levels as endpoints. Focusing on these two hormones, however, may overlook informative changes in other steroid classes, particularly progestogens and glucocorticoids. Here, we extended the effect-based H295R-CALUX® read-out to include progestogens and glucocorticoids using PR- and GR CALUX® assays. First, we evaluated whether PR- and GR-CALUX® could be used as H295R read-outs by determining whether control samples produced adequate hormone levels and whether responses to control compounds forskolin and prochloraz met predefined criteria. Progestogenic and glucocorticoid activity was consistently quantifiable, forskolin induced both modalities above the predefined threshold, and prochloraz inhibited glucocorticoid activity while increasing progestogenic activity, consistent with upstream pathway inhibition. The extended H295R-CALUX® read-out was then applied to analyse a set of conazoles. Each conazole was first pre-screened for ERα/AR/PR/GR agonism or antagonism and for cytotoxicity to avoid interference from direct receptor activation and cytotoxicity in the H295R read-out. In the H295R assay, all conazoles except 1,2,4-triazole, affected at least one hormone modality, with a common pattern of reduced androgenic, estrogenic and glucocorticoid activity and compound-specific increases or decreases in progestogenic activity. Further, targeted LC-MS/MS steroid profiling mirrored the effect-based results obtained with the H295R-CALUX® read-out and supported the hypothesis of CYP17A1-related perturbations for some conazoles. Overall, the addition of progestogen and glucocorticoid read-outs, alongside androgen and estrogens, enhances the resolution of the H295R assay, hence improving its relevance for endocrine safety assessment.