Embryotoxicity and mixture effects of legacy PFAS in a human iPSC-based 3D model

Humans are continuously exposed to a wide array of exogenous chemicals via dietary intake, environmental sources, and the use of personal care products. This includes per- and polyfluoroalkyl substances (PFAS), a class of highly persistent compounds that have been associated with developmental effects in humans. This study assessed the effects of four legacy PFAS, namely PFOS, PFOA, PFNA and PFHxS, and mixtures thereof in the PluriLum assay, a 3D human induced pluripotent stem cell (hiPSC)-based model for embryotoxicity testing. We established the individual embryotoxic potencies of PFAS, with PFNA exhibiting the highest potency, followed by PFOS, PFOA and PFHxS. The four PFAS were evaluated in three reconstituted mixtures, prepared either to reflect identical potencies ("equipotent mixture") or the average serum concentrations reported for the European adult or child population ("real-life mixtures"). Comparing observed versus predicted mixture responses demonstrated concentration additivity throughout the entire range of tested concentrations. Studies on uptake in 3D embryoid bodies revealed the highest bioaccumulation of PFOS, followed by PFNA, PFOA, and PFHxS. Moreover, less than 2% of the nominally added PFAS could be recovered in the embryoid bodies. RNA sequencing showed that relatively few genes were affected by PFOS, PFNA and PFOA, however expression of genes related to focal adhesion and functional pathways associated with cardiac, cardiomyocyte and muscle tissue development was significantly changed. Notably, PFOS affected the greatest number of embryonic development pathways. In conclusion, the four tested PFAS significantly impaired cardiomyocyte differentiation, indicating embryotoxicity. The combined responses were consistent with the concentration addition principle, supported by shared functional pathways and indicative of common sites of molecular action.