Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing.

To test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus of NKX2.5 of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established two NKX2.5 reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineered NKX2.5 reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25-300 µM) and thalidomide (0.1-36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

Perfluorooctanesulfonic acid (PFOS) disrupts cadherin-16 in the developing rat thyroid gland.

Perfluorooctanesulfonic acid (PFOS) can disrupt the thyroid hormone (TH) system in rodents, potentially affecting perinatal growth and neurodevelopment. Some studies also suggest that gestational exposure to PFOS can lead to lower TH levels throughout life, indicating that PFOS may compromise thyroid gland development. To address this question, we utilized a rat thyroid gland ex vivo culture system to study direct effects of PFOS on the developing thyroid. No significant changes to follicular structure or size were observed with 1 µM or 10 µM PFOS exposure. However, the transcription factor Foxe1, together with Tpo and Lrp2, were upregulated, whereas the key transcription factor Pax8 and its downstream target gene Cdh16 were significantly downregulated at the transcript level, observed with both RT-qPCR and RNAscope. Notably, Cdh16 expression was not uniformly downregulated across Cdh16-postive cells, but instead displayed a patchy expression pattern across the thyroid gland. This is a significant change in expression pattern compared to control thyroids where Cdh16 is expressed relatively uniformly. The disrupted expression pattern was also seen at the protein level. This suggests that PFOS exposure can impact follicular growth and structure. Compromised follicle integrity, if irreversible, could help explain reduced TH synthesis postnatally. This view is supported by observed changes to Tpo and Lrp2 expression, two factors that play a role in TH synthesis.

Antibiotic induced restructuring of the gut microbiota does not affect oral uptake and accumulation of perfluorooctane sulfonic acid (PFOS) in rats.

Perfluorooctane sulfonic acid (PFOS) is a manmade legacy compound belonging to the group of persistent per- and polyfluorinated substances (PFAS). While many adverse health effects of PFOS have been identified, knowledge about its effect on the intestinal microbiota is scarce. The microbial community inhabiting the gut of mammals plays an important role in health, for instance by affecting the uptake, excretion, and bioavailability of some xenobiotic toxicants. Here, we investigated (i) the effect of vancomycin-mediated microbiota modulation on the uptake of PFOS in adult Sprague-Dawley rats, and (ii) the effects of PFOS exposure on the rat microbiota composition. Four groups of twelve rats were exposed daily for 7 days with either 3 mg/kg PFOS plus 8 mg/kg vancomycin, only PFOS, only vancomycin, or a corn oil control. Vancomycin-induced modulation of the gut microbiota composition did not affect uptake of branched and linear PFOS over a period of 7 days, measured in serum samples. 16S rRNA amplicon sequencing of faecal and intestinal samples revealed that vancomycin treatment lowered microbial alpha-diversity, while PFOS increased the microbial diversity in vancomycin-treated as well as in non-antibiotic treated animals, possibly because an observed decrease in the Enterobacteriaceae abundance allows other microbial species to propagate. Colonic short-chain fatty acids were significantly lower in vancomycin-treated animals but remained unaffected by PFOS. Our results suggest that PFOS exposure may disturb the intestinal microbiota, but that antibiotic-induced modulation of the intestinal ecosystem does not affect systemic uptake of PFOS in rats.

PFOS disrupts key developmental pathways during hiPSC-derived cardiomyocyte differentiation in vitro.

Exposure to perfluorooctanesulfonic acid (PFOS) has been associated with congenital heart disease (CHD) and decreased birth weight. PFOS exposure can disrupt signaling pathways relevant for cardiac development in stem cell-derived cardiomyocyte assays, such as the PluriBeat assay, where spheroids of human induced pluripotent stem cells (hiPSCs) differentiate into contracting cardiomyocytes. Notably, cell line origin can also affect how the assay responds to chemical exposure. Herein, we examined the effect of PFOS on cardiomyocyte differentiation by transcriptomics profiling of two different hiPSC lines to see if they exhibit a common pattern of disruption. Two stages of differentiation were investigated: the cardiac progenitor stage and the cardiomyocyte stage. Many differentially expressed genes (DEGs) were observed between cell lines independent of exposure. However, 135 DEGs were identified as common between the two cell lines. Of these, 10 DEGs were associated with GO-terms related to the heart. PFOS exposure disrupted multiple signaling pathways relevant to cardiac development, including WNT, TGF, HH, and EGF. Of these pathways, genes related to the non-canonical WNTCa2+ signaling was particularly affected. PFOS thus has the capacity to disrupt pathways important for cardiac development and function.

PFOS-induced thyroid hormone system disrupted rats display organ-specific changes in their transcriptomes.

Perfluorooctanesulfonic acid (PFOS) is a persistent anthropogenic chemical that can affect the thyroid hormone system in humans and animals. In adults, thyroid hormones (THs) are regulated by the hypothalamic-pituitary-thyroid (HPT) axis, but also by organs such as the liver and potentially the gut microbiota. PFOS and other xenobiotics can therefore disrupt the TH system at various locations and through different mechanisms. To start addressing this, we exposed adult male rats to 3 mg PFOS/kg/day for 7 days and analysed effects on multiple organs and pathways simultaneously by transcriptomics. This included four primary organs involved in TH regulation, namely hypothalamus, pituitary, thyroid, and liver. To investigate a potential role of the gut microbiota in thyroid hormone regulation, two additional groups of animals were dosed with the antibiotic vancomycin (8 mg/kg/day), either with or without PFOS. PFOS exposure decreased thyroxine (T4) and triiodothyronine (T3) without affecting thyroid stimulating hormone (TSH), resembling a state of hypothyroxinemia. PFOS exposure resulted in 50 differentially expressed genes (DEGs) in the hypothalamus, 68 DEGs in the pituitary, 71 DEGs in the thyroid, and 181 DEGs in the liver. A concomitant compromised gut microbiota did not significantly change effects of PFOS exposure. Organ-specific DEGs did not align with TH regulating genes; however, genes associated with vesicle transport and neuronal signaling were affected in the hypothalamus, and phase I and phase II metabolism in the liver. This suggests that a decrease in systemic TH levels may activate the expression of factors altering trafficking, metabolism and excretion of TH. At the transcriptional level, little evidence suggests that the pituitary or thyroid gland is involved in PFOS-induced TH system disruption.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

Endocrine disrupting chemicals (EDCs) are a matter of great concern. They are ubiquitous in the environment, are considered harmful to humans and wildlife, yet remain challenging to identify based on current international test guidelines and regulatory frameworks. For a compound to be identified as an EDC within the EU regulatory system, a plausible link between an endocrine mode-of-action and an adverse effect outcome in an intact organism must be established. This requires in-depth knowledge about molecular pathways regulating normal development and function in animals and humans in order to elucidate causes for disease. Although our knowledge about the role of the endocrine system in animal development and function is substantial, it remains challenging to predict endocrine-related disease outcomes in intact animals based on non-animal test data. A main reason for this is that our knowledge about mechanism-of-action are still lacking for essential causal components, coupled with the sizeable challenge of mimicking the complex multi-organ endocrine system by methodological reductionism. Herein, we highlight this challenge by drawing examples from male reproductive toxicity, which is an area that has been at the forefront of EDC research since its inception. We discuss the importance of increased focus on characterizing mechanism-of-action for EDC-induced adverse health effects. This is so we can design more robust and reliable testing strategies using non-animal test methods for predictive toxicology; both to improve chemical risk assessment in general, but also to allow for considerable reduction and replacement of animal experiments in chemicals testing of the 21st Century.

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

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.

Exposure to human relevant mixtures of halogenated persistent organic pollutants (POPs) alters neurodevelopmental processes in human neural stem cells undergoing differentiation.

Halogenated persistent organic pollutants (POPs) like perfluorinated alkylated substances (PFASs), brominated flame retardants (BFRs), organochlorine pesticides and polychlorinated biphenyls (PCBs) are known to cause cancer, immunotoxicity, neurotoxicity and interfere with reproduction and development. Concerns have been raised about the impact of POPs upon brain development and possibly neurodevelopmental disorders. The developing brain is a particularly vulnerable organ due to dynamic and complex neurodevelopmental processes occurring early in life. However, very few studies have reported on the effects of POP mixtures at human relevant exposures, and their impact on key neurodevelopmental processes using human in vitro test systems. Aiming to reduce this knowledge gap, we exposed mixed neuronal/glial cultures differentiated from neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) to reconstructed mixtures of 29 different POPs using concentrations comparable to Scandinavian human blood levels. Effects of the POP mixtures on neuronal proliferation, differentiation and synaptogenesis were evaluated using in vitro assays anchored to common key events identified in the existing developmental neurotoxicity (DNT) adverse outcome pathways (AOPs). The present study showed that mixtures of POPs (in particular brominated and chlorinated compounds) at human relevant concentrations increased proliferation of NSCs and decreased synapse number. Based on a mathematical modelling, synaptogenesis and neurite outgrowth seem to be the most sensitive DNT in vitro endpoints. Our results indicate that prenatal exposure to POPs may affect human brain development, potentially contributing to recently observed learning and memory deficits in children.