Developmental Effects of Per- and Polyfluoroalkyl Substances (PFASs): From hiPSC-derived cardiomyocytes to thyroid hormone system disruption

As a group, per- and polyfluoroalkyl substances (PFASs) comprise more than 10 000 different chemicals. The unique properties of PFASs have made them useful in a number of industrial and household applications for multiple decades. As a result, they are commonly used in products such as non-stick pans, water-resistant clothing, food-contact materials, and firefighting foams. However, these characteristics also make them resistant to degradation and, as a result, prone to accumulate in the environment and in humans; PFASs have been found in blood samples collected worldwide. Exposure to PFASs has been associated with many adverse health effects, such as congenital heart disease (CHD), decrease in birthweight and disruption of the thyroid hormone (TH) system. This PhD thesis investigates the underlying molecular basis behind these effects, focusing on cardiac development and TH system disruption. The aim was to improve our understanding of how PFASs cause the aforementioned health effects, and possibly elucidate mechanisms that can contribute to the screening of novel PFASs. As such, the thesis covers two broad subject areas in relation to PFASs, namely heart development and the thyroid hormone system.

The heart is among the first organs to form during development in order to support the growing fetus. Hence disruption to this process may have implications for both fetal survival and growth. To investigate the potential of PFASs to disrupt cardiac development, I used a cardiomyocyte differentiation assay based on human induced pluripotent stem cells (hiPSCs). In this assay, named PluriBeat, hiPSCs are led to become cardiomyocytes through an 8-day differentiation protocol. This protocol is meant to take the cells through a series of developmental stages similar to those occurring in the embryo during early cardiogenesis. In the initial study three prominent PFASs were tested, perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (GenX) for their ability to disrupt cardiac differentiation. Both PFOS and PFOA disrupted cardiac differentiation in the cell line initially tested, PFOS showed the highest potency and efficacy. GenX did not affect differentiation in the cell line initially tested but disrupted cardiac differentiation when tested in a second cell line. This was followed up with a transcriptomics study to further investigate how PFASs disrupt cardiac differentiation. Here, two hiPS cell lines originating from two different donors were taken through the same differentiation protocol while being exposed to PFOS. The transcriptomic profiles of the two cell lines following exposure to PFOS were compared to find overlapping pathways and markers. This revealed that PFOS exposure changed the expression of genes related to WNT, TGF, Hh, EGF, and mTOR signaling pathways. Many of the identified genes were associated with CHD. Indicating that PFASs such as PFOS can disrupt cardiac development and contribute to the incidence of CHDs, and by extension possibly lower birth weight. However, due to the limitations of the assay, it is impossible to establish with certainty that these effects translate to the in vivo situation. Further studies are needed to establish the connection between the disruption of signaling pathways in vitro and cardiac development in vivo.

To understand how chemicals such as PFOS can affect the TH system, adult male rats were treated with PFOS for 7 days. PFOS treatment caused a hypothyroxinemia-like effect with decreased serum levels of circulating TH, without a compensatory increase in TSH. Hypothalami, pituitaries, thyroids and livers were excised for transcriptomic analysis. The transcriptomics showed that PFOS (1) induced genes related to hepatic enzymes in the liver, (2) changed expression of genes related to neuron function and vesicle transport in the hypothalami, and (3) did not change expression of genes directly related to the TH system in the pituitaries or thyroids. This indicated that PFOS affected the TH balance without disturbing the TH regulatory system. Additionally, the effects of PFOS exposure could not be disentangled from the effects of decreased TH levels, making the mode of action elusive. This indicates a complicated effect pattern, which reflects the intricate nature of the TH system. The ability of PFOS to disrupt the TH system is concerning as THs are essential for fetal growth and brain development, as well as maintaining metabolic homeostasis in adults. Hence, disruption of the TH system can have detrimental and long-lasting effects on the afflicted organism. Therefore, future studies need to focus on further elucidating the mode of action behind TH disruption and the downstream implications. Especially during development.

Building on the notion that PFOS disrupt TH balance in the adult rat, the effects of PFOS exposure on the developing thyroid was investigated. Thyroids excised from rat pups on gestational day 21 was cultured ex vivo and exposed to PFOS for 48h. PFOS disrupted the network of transcription factors regulating thyroid development, decreasing expression of Pax8 and increasing expression of Foxe1. Expression of Cdh16, a gene directly downstream of Pax8 was also decreased, indicating downregulation of folliculogenesis, a process crucial for the maturation of the thyroid and its ability to initiate TH synthesis. Additionally, genes related to TH synthesis and transport was also upregulated. This suggested that PFOS disrupted thyroid development and function, which can have implications for growth and development. However, more studies are needed to confirm these effects using protein measurements and histology.

Taken together, these studies highlight the many challenges that face modern chemical testing. Modern in vitro methods such as hiPSC-cardiomyocyte assays offer invaluable insight into the fundamental molecular mechanisms of chemical disruption and provide potential markers for screening purposes. However, they often provide a simplified image of the in vivo effects, which obscure extrapolation from these models. Similarly, it is challenging to devise reliable in vitro-based test strategies based on complex endocrine systems such as the TH system. This especially rings true when considering the enigmatic nature of PFOS disruption to the TH system. However, these are two sides of the same coin that bring to light challenges tied to the fundamental understanding of the underlying biological systems. Hence, an improved understanding of cardiac development and the TH system will aid interpretation of data generated from these type of studies as well as facilitate the development of future test assays. As such more studies are needed to help decode the effects of chemical disruption and aid translation between model systems. In turn, this will improve our capacity to test and screen novel chemicals for their disruptive capabilities and meet the future needs of chemical testing.