Identification of phthalate mixture exposure targets in the human and mouse ovary in vitro

Chemical health risk assessment is based on single chemicals, but humans and wildlife are exposed to extensive mixtures of industrial substances and pharmaceuticals. Such exposures are life-long and correlate with multiple morbidities, including infertility. How combinatorial effects of chemicals should be handled in hazard characterization and risk assessment are open questions. Further, test systems are missing for several relevant health outcomes including reproductive health and fertility in women. Here, our aim was to screen multiple ovarian cell models for phthalate induced effects to identify biomarkers of exposure. We used an epidemiological cohort study to define different phthalate mixtures for in vitro testing. The mixtures were then tested in five cell models representing ovarian granulosa or stromal cells, namely COV434, KGN, primary human granulosa cells, primary mouse granulosa cells, and primary human ovarian stromal cells. Exposures at epidemiologically relevant exposure level did not markedly elicit cytotoxicity or affect steroidogenesis in short 24-hour exposure. However, significant effects on gene expression were discovered by RNA-sequencing. Altogether, the exposures changed the average expression of 124 genes (9-479 genes per exposure) in human cell models, without obvious concentration or mixture-dependent effects on gene numbers. The mixtures stimulated distinct changes in different cell models. Despite differences, our analyses suggest commonalities in responses towards phthalates, which forms a starting point for follow-up studies on identification and validation of candidate biomarkers that could be developed to novel assays for regulatory testing or even into clinical tests. Samples from five different cell models (COV434 (three passages), KGN (three passages), primary human granulosa cells (three patients), primary human stromal cells (three patients) and primary mouse granulosa cells (four animals)), with nine different treatments in total (DMSO, WQS-m 1x, WQS-m 100x, EPIDEM 1x, EPIDEM 100x, EQUI 1x, EQUI 100x, MEHHP 1x and MEHHP 100x)