Developmental effects of PFOS, PFOA and GenX in a 3D human induced pluripotent stem cell differentiation model

Nichlas Davidsen,Anna Kjerstine Rosenmai,Karin Lauschke,Terje Svingen,Anne Marie Vinggaard

doi:10.1016/j.chemosphere.2021.130624

Nichlas Davidsen, Anna Kjerstine Rosenmai + Show 3 more

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https://doi.org/10.1016/j.chemosphere.2021.130624

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Journal: Chemosphere	Publication Date: Apr 22, 2021
Citations: 15	License type: cc-by

Affiliation: Technical University of Denmark

Abstract

Polyfluoroalkyl substances (PFASs), including perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), are persistent pollutants routinely found in human blood. PFASs have been associated with health issues such as decreased birth weight and impaired vaccination response in children. Substitutes to these PFASs, such as ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX) have been introduced, although hazard information is limited. Human induced pluripotent stem cell (hiPSC) based models are valuable for studying these compounds, as they mimic human embryonic development. We used our recently developed PluriBeat assay to investigate PFOS, PFOA and GenX for effects on early embryonic development in vitro. In our assay hiPSCs go through the early stages of embryonic development in 3D cultures of embryoid bodies (EBs) that mimic the human blastocyst until they finally form beating cardiomyocytes. Both PFOS and PFOA had a strong effect on cardiomyocyte differentiation at non-cytotoxic concentrations, with PFOS being more potent than PFOA. Moreover, both compounds decreased EB size at the highest test concentrations. GenX induced a weak concentration-dependent effect on differentiation of one hiPSC line, but not of another. Transcriptional analysis of mRNA from the cardiomyocytes showed that PFOS increased expression of the early cardiac marker ISL1, whereas PFOA decreased expression of the cardiomyocyte marker MYH7. This suggest that PFOS and PFOA perturb cardiomyocyte differentiation by disrupting molecular pathways similar to those taking place in the developing embryo. Based on these findings, we conclude that our PluriBeat assay has the potential to become a valuable, sensitive model system for elucidating embryotoxic effects of PFASs in future.

Highlights

Neither perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA) nor GenX showed any statistically significant effects on viability of embryoid bodies (EBs) at concentrations up to 200 mM, a non-significant trend towards slight cytotoxicity was observed for GenX at the highest test concentrations (Fig. 1)
We proceeded using a maximum concentration of 200 mM for PFOS and PFOA, with test concentrations of 12.5, 25, 50, 100, and 200 mM in the PluriBeat assay, whereas 6.25, 12.5, 25, 50 and 100 mM GenX was used due to minor indication of cytotoxicity at 200 mM
Initial testing of PFOS and PFOA in the PluriBeat assay led to marked reductions of beat scores at all test concentrations, and test concentrations were adjusted to include data points at the linear part of the concentration-response curves

Summary

Introduction

The molecular mechanisms behind these effects remain largely unknown (Knutsen et al, 2018; Liew et al, 2018) These findings, in addition to other adverse effects, has led to the phase-out and restrictions of long-chain PFASs (UNEP, 2018, 2008), including PFOS and PFOA, and the emergence of substitute PFASs with shorter perfluorinated chains (Scheringer et al, 2014), such as ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (HFPO-DA, known as GenX) (Sun et al, 2016), for which an increasing number of studies are reporting adverse effects (Blake et al, 2020; Conley et al, 2021; Wen et al, 2020)

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