Comparison of steroidogenic disruption in adrenal H295R and ovarian KGN and COV434 cell models and the implications to assess female reproductive toxicity

To investigate the need for an ovarian-specific steroidogenesis assay in regulatory testing, steroid hormone profiles from adrenal H295R and ovarian KGN and COV434 cell cultures were compared with primary human cultures using LC-MS/MS. Hierarchical clustering showed close similarity between steroid hormone profiles of H295R and human fetal adrenal and ovarian tissue cultures, while KGN and COV434-derived profiles closer resembled human adult ovarian profiles. COV434 cultures showed the lowest number of steroid hormones even after forskolin stimulation. Specifically, in vehicle-treated controls 17 out of 21 steroid hormones were quantified in H295R cultures, 10 in KGN cultures and 7 in COV434 cultures. These included three backdoor pathway steroids, which were detected in different levels in the three cell cultures. Reference chemicals for steroidogenic disruption showed different effects in the cell lines, with most striking results early in the steroidogenic pathway. Atrazine, ketoconazole and aminoglutethimide affected pregnenolone and progesterone concentrations in KGN cultures more potently than in H295R cultures. Ketoconazole and propylparaben increased progesterone levels in H295R, but decreased levels in KGN cultures. Despite the differences in responses, classification of the test chemicals to act as steroidogenic disruptors was similar in H295R and KGN cell lines, but only when the OECD criteria were applied to a broad steroidome. These data do not support the use of KGN cells as an ovarian-specific steroidogenesis assay in a regulatory context, but do support the inclusion of more steroid hormones in the current test guideline to enhance the information that can be retrieved from the H295R assay.