Assessment and Comparison of Early Developmental Toxicity of Six Per- and Polyfluoroalkyl Substances with Human Embryonic Stem Cell Models.

Per- and polyfluoroalkyl substances (PFAS) are extensively utilized in varieties of products and tend to accumulate in the human body including umbilical cord blood and embryos/fetuses. In this study, we conducted an assessment and comparison of the potential early developmental toxicity of perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid, perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate, and perfluorobutyric acid at noncytotoxic concentrations relevant to human exposure using models based on human embryonic stem cells in both three-dimensional embryoid body (EB) and monolayer differentiation configurations. All six compounds influenced the determination of cell fate by disrupting the expression of associated markers in both models and, in some instances, even led to alterations in the formation of cystic EBs. The expression of cilia-related gene IFT122 was significantly inhibited. Additionally, PFOS and PFOA inhibited ciliogenesis, while PFOA specifically reduced the cilia length. Transcriptome analysis revealed that PFOS altered 1054 genes and disrupted crucial signaling pathways such as WNT and TGF-ß, which play integral roles in cilia transduction and are critical for early embryonic development. These results provide precise and comprehensive insights into the potential adverse health effects of these six PFAS compounds directly concerning early human embryonic development.

Assessing developmental neurotoxicity of emerging environmental chemicals using multiple in vitro models: A comparative analysis.

Newly synthesized chemicals are being introduced into the environment without undergoing proper toxicological evaluation, particularly in terms of their effects on the vulnerable neurodevelopment. Thus, it is important to carefully assess the developmental neurotoxicity of these novel environmental contaminants using methods that are closely relevant to human physiology. This study comparatively evaluated the potential developmental neurotoxicity of 19 prevalent environmental chemicals including neonicotinoids (NEOs), organophosphate esters (OPEs), and synthetic phenolic antioxidants (SPAs) at environment-relevant doses (100 nM and 1 µM), using three commonly employed in vitro neurotoxicity models: human neural stem cells (NSCs), as well as the SK-N-SH and PC12 cell lines. Our results showed that NSCs were more sensitive than SK-N-SH and PC12 cell lines. Among all the chemicals tested, the two NEOs imidaclothiz (IMZ) and cycloxaprid (CYC), as well as the OPE tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), generated the most noticeable perturbation by impairing NSC maintenance and neuronal differentiation, as well as promoting the epithelial-mesenchymal transition process, likely via activating NF-κB signaling. Our data indicate that novel NEOs and OPEs, particularly IMZ, CYC, and TDCIPP, may not be safe alternatives as they can affect NSC maintenance and differentiation, potentially leading to neural tube defects and neuronal differentiation dysplasia in fetuses.

Understanding the effects of per- and polyfluoroalkyl substances on early skin development: Role of ciliogenesis inhibition and altered microtubule dynamics.

Per- and polyfluoroalkyl substances (PFAS) are a class of highly stable chemicals, widely used in everyday products, and widespread in the environment, even in pregnant women. While epidemiological studies have linked prenatal exposure to PFAS with atopic dermatitis in children, little is known about their toxic effects on skin development, especially during the embryonic stage. In this study, we utilized human embryonic stem cells to generate non-neural ectoderm (NNE) cells and exposed them to six PFAS (perfluorooctanoic acid (PFOA), undecafluorohexanoic acid (PFHxA), heptafluorobutyric acid (PFBA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS) and perfluorobutyric acid (PFBS)) during the differentiation process to assess their toxicity to early skin development. Our results showed that PFOS altered the spindle-like morphology of NNE cells to a pebble-like morphology, and disrupted several NNE markers, including KRT16, SMYD1, and WISP1. The six PFAS had a high potential to cause hypohidrotic ectodermal dysplasia (HED) by disrupting the expression levels of HED-relevant genes. Transcriptomic analysis revealed that PFOS treatment produced the highest number (1156) of differentially expressed genes (DEGs) among the six PFAS, including the keratinocyte-related genes KRT6A, KRT17, KRT18, KRT24, KRT40, and KRT81. Additionally, we found that PFOS treatment disturbed several signaling pathways that are involved in regulating skin cell fate decisions and differentiation, including TGF-ß, NOTCH, Hedgehog, and Hippo signaling pathways. Interestingly, we discovered that PFOS inhibited, by partially interfering with the expression of cytoskeleton-related genes, the ciliogenesis of NNE cells, which is crucial for the intercellular transduction of the above-mentioned signaling pathways. Overall, our study suggests that PFAS can inhibit ciliogenesis and hamper the transduction of important signaling pathways, leading potential congenital skin diseases. It sheds light on the underlying mechanisms of early embryonic skin developmental toxicity and provides an explanation for the epidemiological data on PFAS. ENVIRONMENTAL IMPLICATION: We employed a model based on human embryonic stem cells to demonstrate that PFOS has the potential to elevate the risk of hypohidrotic ectodermal dysplasia. This is achieved by targeting cilia, inhibiting ciliogenesis, and subsequently disrupting crucial signaling pathways like TGF-ß, NOTCH, Hedgehog, and Hippo, during the early phases of embryonic skin development. Our study highlights the dangers and potential impacts of six PFAS pollutants on human skin development. Additionally, we emphasize the importance of closely considering PFHxA, PFBA, PFHxS, and PFBS, as they have shown the capacity to modify gene expression levels, albeit to a lesser degree.

Transcriptomic Integration Analyses Uncover Common Bisphenol A Effects Across Species and Tissues Primarily Mediated by Disruption of JUN/FOS, EGFR, ER, PPARG, and P53 Pathways.

Bisphenol A (BPA) is a common endocrine disruptor widely used in the production of electronic, sports, and medical equipment, as well as consumer products like milk bottles, dental sealants, and thermal paper. Despite its widespread use, current assessments of BPA exposure risks remain limited due to the lack of comprehensive cross-species comparative analyses. To address this gap, we conducted a study aimed at identifying genes and fundamental molecular processes consistently affected by BPA in various species and tissues, employing an effective data integration method and bioinformatic analyses. Our findings revealed that exposure to BPA led to significant changes in processes like lipid metabolism, proliferation, and apoptosis in the tissues/cells of mammals, fish, and nematodes. These processes were found to be commonly affected in adipose, liver, mammary, uterus, testes, and ovary tissues. Additionally, through an in-depth analysis of signaling pathways influenced by BPA in different species and tissues, we observed that the JUN/FOS, EGFR, ER, PPARG, and P53 pathways, along with their downstream key transcription factors and kinases, were all impacted by BPA. Our study provides compelling evidence that BPA indeed induces similar toxic effects across different species and tissues. Furthermore, our investigation sheds light on the underlying molecular mechanisms responsible for these toxic effects. By uncovering these mechanisms, we gain valuable insights into the potential health implications associated with BPA exposure, highlighting the importance of comprehensive assessments and awareness of this widespread endocrine disruptor.

Tox21-Based Comparative Analyses for the Identification of Potential Toxic Effects of Environmental Pollutants.

Chemical pollution has become a prominent environmental problem. In recent years, quantitative high-throughput screening (qHTS) assays have been developed for the fast assessment of chemicals' toxic effects. Toxicology in the 21st Century (Tox21) is a well-known and continuously developing qHTS project. Recent reports utilizing Tox21 data have mainly focused on setting up mathematical models for in vivo toxicity predictions, with less attention to intuitive qHTS data visualization. In this study, we attempted to reveal and summarize the toxic effects of environmental pollutants by analyzing and visualizing Tox21 qHTS data. Via PubMed text mining, toxicity/structure clustering, and manual classification, we detected a total of 158 chemicals of environmental concern (COECs) from the Tox21 library that we classified into 13 COEC groups based on structure and activity similarities. By visualizing these COEC groups' bioactivities, we demonstrated that COECs frequently displayed androgen and progesterone antagonistic effects, xenobiotic receptor agonistic roles, and mitochondrial toxicity. We also revealed many other potential targets of the 13 COEC groups, which were not well illustrated yet, and that current Tox21 assays may not correctly classify known teratogens. In conclusion, we provide a feasible method to intuitively understand qHTS data.

In vivo and in vitro transcriptomics meta-analyses reveal that BPA may affect TGF-beta signaling regardless of the toxicology system employed.

Bisphenol A (BPA) is a high-production-volume monomer for the manufacture of a wide variety of polycarbonate plastics and resins. Evidence suggests BPA can induce carcinogenesis, reproductive toxicity, abnormal inflammatory or immune response, and developmental disorders of the brain or nervous system. However, whether BPA affects the very same basic molecular processes in all the in vivo and in vitro systems employed to exert its molecular mechanisms of toxicity remains to be clarified. In this study, we collected multi-source global transcriptomics datasets for BPA-exposed organisms and cells, and evaluated the adverse effects of BPA by using data integration and gene functional enrichment analyses. We found that BPA may affect basic cellular processes, such as cell growth, survival, proliferation, differentiation, and apoptosis, independent of species and specific in vivo or in vitro systems. Mechanistically, BPA could regulate cell-extra cellular matrix interactions via challenging TGF-beta signaling pathways. Furthermore, we compared our in vitro BPA-dependent mouse embryoid body (EB) global differentiation transcriptomics with all the other datasets. We verified the EB-based toxicological system could recapitulate several in vivo and other in vitro findings very efficiently, and in a less time- and resource-consuming fashion. Taken together, this study emphasizes the utility of meta-analyses to understand common molecular mechanisms of toxicity of synthetic chemicals.

Development of Human Lung Induction Models for Air Pollutants' Toxicity Assessment.

There is an urgent need for reliable and effective models to study air pollution health effects on human lungs. Here, we report the utilization of human pluripotent stem cell (hPSC) induction models for human lung progenitor cells (hLPs) and alveolar type 2 epithelial cell-like cells (ATLs) for the toxicity assessment of benzo(a)pyrene, nano-carbon black, and nano-SiO2, as common air pollutants. We induced hPSCs to generate ATLs, which recapitulated key features of human lung type 2 alveolar epithelial cells, and tested the induction models for cellular uptake of nanoparticles and toxicity evaluations. Our findings reveal internalization of nano-carbon black, dose-dependent uptake of nano-SiO2, and interference with surfactant secretion in ATLs exposed to benzo(a)pyrene/nano-SiO2. Thus, hLP and ATL induction models could facilitate the evaluation of environmental pollutants potentially affecting the lungs. In conclusion, this is one of the first studies that managed to adopt hPSC pulmonary induction models in toxicology studies.

New insights into mechanism of bisphenol analogue neurotoxicity: implications of inhibition of O-GlcNAcase activity in PC12 cells.

Bisphenol analogues including bisphenol A and its derivatives are ubiquitous environmental contaminants and have been linked to adverse neurodevelopment effects on animals and humans. Most toxicological research focused on estrogen receptor mediated pathways and did not comprehensively clarify the observed toxicity. O-GlcNAcase (OGA), the highest level in brain, plays a critical role in controlling neuronal functions at multi-levels from molecule to animal behaviors. In this work, we intend to investigate the underlying molecular mechanisms for the neurotoxicity of bisphenol analogues by identifying their cellular targets and the resultant effects. The inhibitory actions of seven bisphenol analogues on the OGA activity at molecular level were investigated by our developed electrochemical biosensor. We found that their potency varied with substituent groups, in which tetrabromo bisphenol A (TBBPA) was the strongest. The seven bisphenol analogues (0-100 µM exposure) significantly inhibited OGA activity and up-regulated protein O-GlcNAcylation level in PC12 cells. Inhibition of OGA by bisphenol analogues further induced intracellular calcium, ROS, inflammation, repressed proliferation, interfered with cell cycle, induced apoptosis. And especially, 10 µM tetrabromo bisphenol A (TBBPA) exposure could impair the growth and development of neurite in human neural stem cells (hNSCs). Molecular docking for OGA/bisphenol analogue complexes revealed the hydrophobicity-dominated inhibition potency. OGA, as a new cellular target of bisphenol analogues, would illuminate the molecular mechanism of bisphenol analogues neurotoxicity.

Assessment of the carcinogenic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin using mouse embryonic stem cells to form teratoma in vivo.

As the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has gained lots of concerns, due to its diverse deleterious effects. However, the knowledge on carcinogenic risk of TCDD during early stage of development remains scarce. The in vivo teratoma formation model based on the transplantation of embryonic stem cells (ESCs) in immunodeficient mice is appealing for studying pluripotency and tumorigenicity in developmental biology, and also shows promise in environmental toxicology, especially in carcinogenesis researches. In this study, the malignant transformation of mouse embryonic stem cells (mESCs) pretreated with TCDD was investigated during their in vivo differentiation using teratoma formation model. Based on characterization of the pluripotency and differentiation capabilities of mESCs, evil changes in teratomas derived from TCDD-exposed mESCs were systematically studied. The results showed that TCDD significantly up-regulated CYP1A1 transcriptional levels in mESCs, elevated the incidence of malignant change in mESC-derived teratomas, and caused indefinite proliferation capabilities in sequential cultures of tumor tissues. The findings suggested that TCDD could exert carcinogenic effect on mESCs during their differentiation into teratoma in vivo, and more attention should be paid to the adverse health effects of this chemical during gestation or early developmental period.

Embryonic stem cell- and transcriptomics-based in vitro analyses reveal that bisphenols A, F and S have similar and very complex potential developmental toxicities.

Bisphenol A (BPA) is a very versatile industrial chemical. Many reports have associated BPA with several health effects. Some bisphenol alternatives have been introduced to replace BPA in its many applications. However, comprehensive toxicological evaluations for these replacements are still lacking. In this study, we examined the potential effects of BPA, bisphenol F (BPF) and bisphenol S (BPS), on embryonic development with an in vitro stem cell toxicology system and transcriptomics analyses. Mouse embryonic stem cells (mESCs) were differentiated via embryoid body formation, either globally towards the three primary germ layers and their lineages, or specifically into neuroectoderm/neural progenitor cells. During the differentiation, cells were treated with BPA, BPF, BPS, or DMSO control. Samples were collected at different time points, for qRT-PCR and RNA-seq analyses. BPA, BPF and BPS disrupted many processes, during mESC global and neural differentiations, in very similar manners. In fact, at each time point the three chemicals differentially regulated analogous gene categories, particularly the ones involved in cell-matrix and cell-cell adhesion, signal transduction pathways, and medical conditions such as cardiovascular diseases and cancer. Our findings demonstrate once more then BPA substitutes may not be very safe. They potentially have a very complex developmental toxicity, similarly to BPA, and seem more toxic than BPA itself. In addition, our results reveal that stem cell-based developmental toxicity assays can be very comprehensive.

Establishment of a human embryonic stem cell-based liver differentiation model for hepatotoxicity evaluations.

The liver is one of the major targets of hormones, including thyroid hormones (THs), and many industrial chemicals, such as endocrine-disrupting chemicals. Those compounds may permeate the placenta barrier and pose a risk for embryonic development. Therefore, it is necessary to assess the toxic effects of those kind of industrial chemicals during liver development. In this study, to mimic liver specification in vitro, we differentiated human embryonic stem cells (ESCs) into functional hepatocyte-like cells. We performed this differentiation process in presence of two THs, triiodothyronine (T3) and thyroxine (T4), with the purpose of identifying biomarkers for toxicity screening. TH exposure (3, 30 and 300 nM) yielded to hepatocytes with impaired glycogen storage ability and abnormal lipid droplets' accumulation. Global gene expression analysis by RNA-seq identified a number of genes responsible for hepatic differentiation and function which were affected by 30 nM T3 and T4. Those differentially expressed genes were used to assess the potential developmental liver toxicity of two famous environmental pollutants, 2, 2, 4, 4-tetrabromodiphenyl ether (BDE-47) and decabromodiphenyl ether (BDE-209), at 10 nM to 1 µM treatments. Our findings demonstrate that BDE-47 and BDE-209, dysregulated pathways such as "chemical carcinogenesis", "steroid hormone biosynthesis" and "drug metabolism-cytochrome P450". Moreover, we were able to identify a set of 17 biomarkers, very useful to predict the potential developmental hepatotoxicity of industrial chemicals.

Environmental and human relevant PFOS and PFOA doses alter human mesenchymal stem cell self-renewal, adipogenesis and osteogenesis.

PFOS and PFOA are two of the most abundant perfluorinated compounds (PFCs) in the environment. Previous studies have reported they have a long half-life (up to five years) once they enter into the human body. Moreover, they can potentially promote the adipogenic process by activating PPARγ. However, little is known about PFOS and PFOA chronic health impacts on humans. In this study, we employed primary human mesenchymal stem cells (hMSCs) and demonstrated that PFOS and PFOA exerted acute cytotoxicity and affected adipogenesis and osteogenesis at environmental and human relevant doses. In fact, PFOS and PFOA impaired the proper expression of CD90 (a surface antigen highly enriched in undifferentiated hMSCs) and promoted adipogenesis, presumably via their interaction with PPARγ. Moreover, PFOA partly disturbed osteogenesis. Thus, our findings not only validated the health risks of PFOS and PFOA, but also revealed new potential long-term PFOS/PFOA impacts on humans.

Non-cytotoxic nanomolar concentrations of bisphenol A induce human mesenchymal stem cell adipogenesis and osteogenesis.

Bisphenol A (BPA) is a typical endocrine disrupting chemical with extensive applications, and has been correlated with various hazardous health effects, including obesity and other metabolic-related diseases. Human mesenchymal stem cells (hMSCs), due to their abilities to differentiate into adipocytes and osteoblasts, can be a good in vitro model to assess chemical-dependent toxicity on adipogenesis or osteogenesis. Here, we employed hMSCs as an evaluation system to assess BPA-related effects on cell viability, oxidative stress induction, self-renewal, and differentiation. Our results revealed that low concentrations (1 and 10 nM) of BPA did not impair cell proliferation nor self-renewal capacity, but stimulated adipogenesis and osteogenesis. Our findings support the concern of BPA contributing to the epidemic of obesity, and also reveal its underlying toxicity on osteogenesis.

The ex vivo and in vivo biological performances of graphene oxide and the impact of surfactant on graphene oxide's biocompatibility.

Graphene oxide (GO) displays promising properties for biomedical applications including drug delivery and cancer therapeutics. However, GO exposure also raises safety concerns such as potential side effects on health. Here, the biological effects of GO suspended in phosphate buffered saline (PBS) with or without 1% nonionic surfactant Tween 80 were investigated. Based on the ex vivo experiments, Tween 80 significantly affected the interaction between GO and peripheral blood from mice. GO suspension in PBS tended to provoke the aggregation of diluted blood cells, which could be prevented by the addition of Tween 80. After intravenous administration, GO suspension with or without 1% Tween 80 was quickly eliminated by the mononuclear phagocyte system. Nevertheless, GO suspension without Tween 80 showed greater accumulation in lungs than that containing 1% Tween 80. In contrast, less GO was found in livers for GO suspension compared to Tween 80 assisted GO suspension. Organs including hearts, livers, lungs, spleens, kidneys, brains, and testes did not reveal histological alterations. The indexes of peripheral blood showed no change upon GO exposure. Our results together demonstrated that Tween 80 could greatly alter GO's biological performance and determine the pattern of its biodistribution in mice.

Graphene oxide induces toll-like receptor 4 (TLR4)-dependent necrosis in macrophages.

Graphene and graphene-based nanomaterials display novel and beneficial chemical, electrical, mechanical, and optical characteristics, which endow these nanomaterials with promising applications in a wide spectrum of areas such as electronics and biomedicine. However, its toxicity on health remains unknown and is of great concern. In the present study, we demonstrated that graphene oxide (GO) induced necrotic cell death to macrophages. This toxicity is mediated by activation of toll-like receptor 4 (TLR4) signaling and subsequently in part via autocrine TNF-α production. Inhibition of TLR4 signaling with a selective inhibitor prevented cell death nearly completely. Furthermore, TLR4-deficient bone marrow-derived macrophages were resistant to GO-triggered necrosis. Similarly, GO did not induce necrosis of HEK293T/TLR4-null cells. Macrophagic cell death upon GO treatment was partially attributed to RIP1-RIP3 complex-mediated programmed necrosis downstream of TNF-α induction. Additionally, upon uptake into macrophages, GO accumulated primarily in cytoplasm causing dramatic morphologic alterations and a significant reduction of the macrophagic ability in phagocytosis. However, macrophagic uptake of GO may not be required for induction of necrosis. GO exposure also caused a large increase of intracellular reactive oxygen species (ROS), which contributed to the cause of cell death. The combined data reveal that interaction of GO with TLR4 is the predominant molecular mechanism underlying GO-induced macrophagic necrosis; also, cytoskeletal damage and oxidative stress contribute to decreased viability and function of macrophages upon GO treatment.

Effects of polycyclic musks HHCB and AHTN on steroidogenesis in H295R cells.

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(γ)-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) are widely used in personal care products. Previous studies showed that HHCB and AHTN can be found in various environmental matrices and have potential endocrine disrupting effects. However, the effects on adrenocortical function of HHCB and AHTN are not fully understood. This study evaluated the influences of HHCB and AHTN on seven steroid hormones (progesterone, aldosterone, cortisol, 17α-OH-progesterone, androstenedione, 17ß-estradiol, and testosterone) and 10 genes involved in steroidogenic pathways (HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP21, CYP11B1, CYP11B2, 17ßHSD, and CYP19) using the H295R cell line in the absence and presence of 8-Br-cAMP. MC2R transcription on the cell membrane was also examined to further investigate the effects of HHCB and AHTN on adrenal steroidogenesis. The results demonstrated that HHCB and AHTN could inhibit progesterone and cortisol production mainly by the suppression of 3ßHSD2 and CYP21. Meanwhile, high concentrations of AHTN can affect the sensitivity of H295R cells to ACTH by disrupting MC2R transcription. Overall, the results indicate that high concentrations of HHCB and AHTN can affect steroidogenesis in vitro using the H295R cell line.

Estrogen-like response of perfluorooctyl iodide in male medaka (Oryzias latipes) based on hepatic vitellogenin induction.

Using the in vivo male medaka model, we investigated the estrogen-like response of perfluorooctyl iodide (PFOI) which is a potential source of perfluorinated carboxylic acids. Using real-time quantitative polymerase chain reaction, the expression levels of related estrogenic genes including estrogenic receptor α (ERα), ERß, vitellogenin I (VTG I), and VTG II in the livers of male medaka exposed to PFOI were analyzed. The results showed that PFOI upregulated the expression levels of the tested genes in a dose-dependent manner. VTG protein levels increased in both dose- and time-dependent manners due to PFOI exposure. The results suggested that PFOI is a potential estrogenic compound.