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.

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.

Pollution effects on retinal health: A review on current methodologies and findings.

In our daily life, we are exposed to numerous industrial chemicals that may be harmful to the retina, which is a delicate and sensitive part of our eyes. This could lead to irreversible changes and cause retinal diseases or blindness. Current retinal environmental health studies primarily utilize animal models, isolated mammalian retinas, animal- or human-derived retinal cells, and retinal organoids, to address both pre- and postnatal exposure. However, as there is limited toxicological information available for specific populations, human induced pluripotent stem cell (hiPSC)-induced models could be effective tools to supplement such data. In order to obtain more comprehensive and reliable toxicological information, we need more appropriate models, novel evaluation methods, and computational technologies to develop portable equipment. This review mainly focused on current toxicology models with particular emphasis on retinal organoids, and it looks forward to future models, analytical methods, and equipment that can efficiently and accurately evaluate retinal toxicity.

Development of a simplified human embryonic stem cell-based retinal pre-organoid model for toxicity evaluations of common pollutants.

OBJECTIVE: To explore the retinal toxicity of pharmaceuticals and personal care products (PPCPs), flame retardants, bisphenols, phthalates, and polycyclic aromatic hydrocarbons (PAHs) on human retinal progenitor cells (RPCs) and retinal pigment epithelial (RPE) cells, which are the primary cell types at the early stages of retinal development, vital for subsequent functional cell type differentiation, and closely related to retinal diseases. MATERIALS AND METHODS: After 23 days of differentiation, human embryonic stem cell (hESC)-based retinal pre-organoids, containing RPCs and RPE cells, were exposed to 10, 100, and 1000 nM pesticides (butachlor, terbutryn, imidacloprid, deltamethrin, pendimethalin, and carbaryl), flame retardants (PFOS, TBBPA, DBDPE, and TDCIPP), PPCPs (climbazole and BHT), and other typical pollutants (phenanthrene, DCHP, and BPA) for seven days. Then, mRNA expression changes were monitored and compared. RESULTS: (1) The selected pollutants did not show strong effects at environmental and human-relevant concentrations, although the effects of flame retardants were more potent than those of other categories of chemicals. Surprisingly, some pollutants with distinct structures showed similar adverse effects. (2) Exposure to pollutants induced different degrees of cell detachment, probably due to alterations in extracellular matrix and/or cell adhesion. CONCLUSIONS: In this study, we established a retinal pre-organoid model suitable for evaluating multiple pollutants' effects, and pointed out the potential retinal toxicity of flame retardants, among other pollutants. Nevertheless, the potential mechanisms of toxicity and the effects on cell detachment are still unclear and deserve further exploration. Additionally, this model holds promise for screening interventions aimed at mitigating the detrimental effects of these pollutants.

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.

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.

Tetrabromobisphenol A (TBBPA): A controversial environmental pollutant.

Tetrabromobisphenol A (TBBPA) is one of the most widely used brominated flame retardants and is extensively used in electronic equipment, furniture, plastics, and textiles. It is frequently detected in water, soil, air, and organisms, including humans, and has raised concerns in the scientific community regarding its potential adverse health effects. Human exposure to TBBPA is mainly via diet, respiration, and skin contact. Various in vivo and in vitro studies based on animal and cell models have demonstrated that TBBPA can induce multifaceted effects in cells and animals, and potentially exert hepatic, renal, neural, cardiac, and reproductive toxicities. Nevertheless, other reports have claimed that TBBPA might be a safe chemical. In this review, we re-evaluated most of the published TBBPA toxicological assessments with the goal of reaching a conclusion about its potential toxicity. We concluded that, although low TBBPA exposure levels and rapid metabolism in humans may signify that TBBPA is a safe chemical for the general population, particular attention should be paid to the potential effects of TBBPA on early developmental stages.

The short-chain perfluorinated compounds PFBS, PFHxS, PFBA and PFHxA, disrupt human mesenchymal stem cell self-renewal and adipogenic differentiation.

Per- and polyfluorinated alkyl substances (PFASs) are commonly used in industrial processes and daily life products. Because they are persistent, they accumulate in the environment, wildlife and humans. Although many studies have focused on two of the most representative PFASs, PFOS and PFOA, the potential toxicity of short-chain PFASs has not yet been given sufficient attention. We used a battery of assays to evaluate the toxicity of several four-carbon and six-carbon perfluorinated sulfonates and carboxyl acids (PFBS, PFHxS, PFBA and PFHxA), with a human mesenchymal stem cell (hMSC) system. Our results demonstrate significant cyto- and potential developmental toxicity for all the compounds analyzed, with shared but also distinct mechanisms of toxicity. Moreover, the effects of PFBS and PFHxS were stronger than those of PFBA and PFHxA, but occurred at higher doses compared to PFOS or PFOA.

A human embryonic stem cell-based in vitro model revealed that ultrafine carbon particles may cause skin inflammation and psoriasis.

Air pollution has been linked to many health issues, including skin conditions, especially in children. Among all the atmospheric pollutants, ultrafine particles have been deemed very dangerous since they can readily penetrate the lungs and skin, and be absorbed into the bloodstream. Here, we employed a human embryonic stem cell (hESC)-based differentiation system towards keratinocytes, to test the effects of ultrafine carbon particles, which mimic ambient ultrafine particles, at environment related concentrations. We found that 10 ng/mL to 10 µg/mL ultrafine carbon particles down-regulated the expression of the pluripotency marker SOX2 in hESCs. Moreover, 1 µg/mL to 10 µg/mL carbon particle treatments disrupted the keratinocyte differentiation, and up-regulated inflammation- and psoriasis-related genes, such as IL-1ß, IL-6, CXCL1, CXCL2, CXCL3, CCL20, CXCL8, and S100A7 and S100A9, respectively. Overall, our results provide a new insight into the potential developmental toxicity of atmospheric ultrafine particles.

Toxicogenomic analyses of the effects of BDE-47/209, TBBPA/S and TCBPA on early neural development with a human embryonic stem cell in vitro differentiation system.

Flame retardants are detected in the environment worldwide, and thus pose great risks to human health. The potential effects of these chemicals on the development of the central nervous system, have recently raised public concern. In this study, to explore the toxicity of these chemicals during the early developmental stages of the human central nervous system, we induced human embryonic stem cells to differentiate into neural ectoderm in the presence of five halogenated flame retardants, BDE-47, BDE-209, TBBPA, TBBPS and TCBPA, individually or in combination. We identified a set of neural development-related biological processes that responded to these chemicals, by analyzing the whole transcriptional changes. We confirmed the RNA-seq results by qRT-PCR and found that transcription factors crucial for neural development, such as ZIC1, ZIC3, HES3, IGFBP3 and DLX5, were dysregulated by those chemicals. In addition, the five flame retardants might also influence axon growth/guidance and neuron transmission-related processes, by dysregulating genes including CNTN2, SLIT1, LRRC4C, RELN, CBLN1, CHRNB4 and GDF7. Furthermore, the chemical treatments seemed to interfere with the WNT and AHR signaling pathways. Overall, our findings revealed that BDE-209 had similar toxicity as BDE-47, whereas TBBPS and TCBPA might not be safe alternatives to TBBPA. Interestingly, we observed no obvious synergistic effects when we mixed those five flame retardants together.

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.

Typical halogenated flame retardants affect human neural stem cell gene expression during proliferation and differentiation via glycogen synthase kinase 3 beta and T3 signaling.

2',2',4,4'-tetrabromo diphenyl ether (BDE-47), one of the most abundant congeners of commercial pentaBDE utilized as flame retardants, has been phased out of production due to its potential neural toxicity and endocrine disrupting activities, and yet still present in the environment. Several alternatives to BDE-47, including tetrabromobisphenol A (TBBPA), tetrabromobisphenol S (TBBPS), tetrachlorobisphenol A (TCBPA) and decabromodiphenyl ether (BDE-209), are presently employed without restrictions and their potential toxic effects on human neural development are still unclear. In this study, we utilized a human neural stem cell (hNSC)-based system to evaluate the potential developmental neurotoxic effects of the above-mentioned five chemicals, at environment and human exposure relevant concentrations. We found that those compounds slightly altered the expression of hNSC identity markers (SOX2, SOX3 and NES), without impairing cell viability or proliferation, in part by either modulating glycogen synthase kinase 3 beta (GSK3ß) signaling (TBBPS, TCBPA and BDE-47), and slightly disturbing the NOTCH pathway (TBBPA, TBBPS and TCBPA). Moreover, the five chemicals seemed to alter hNSC differentiation by perturbing triiodothyronine (T3) cellular signaling. Thus, our findings suggest that the five compounds, especially TBBPS, TCBPA, and BDE-47, may affect hNSC self-renewal and differentiation abilities and potentially elicit neural developmental toxicity.

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.

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.

Bisphenol F Disrupts Thyroid Hormone Signaling and Postembryonic Development in Xenopus laevis.

The safety of bisphenol A (BPA) alternatives has attracted much attention due to their wide use. In this study, we investigated the effects of bisphenol F (BPF), an alternative to BPA, on thyroid hormone (TH) signaling and postembryonic development in vertebrates using T3-induced and spontaneous Xenopus metamorphosis as models. We found that in the T3-induced metamorphosis assay, higher concentrations of BPF (100-10000 nM) antagonized T3-induced TH-response gene transcription and morphological changes including intestinal remodeling in a concentration-dependent manner, whereas 10 nM BPF exerted stimulatory effects on T3-induced integral metamorphosis when inhibited T3-induced TH-response gene transcription, demonstrating TH signaling disrupting effects of BPF. In the spontaneous metamorphosis assay, correspondingly, BPF inhibited development at metamorphic climax (with high endogenous TH levels), but promoted pre- and pro-metamorphic development (with low endogenous TH levels), displaying a developmental stage-dependent manner. Importantly, we observed agonistic actions of BPF on Notch signaling in intestines, showing that BPF disrupts vertebrate development possibly via multi pathways besides TH signaling. Thus, we infer the biphasic concentration-response relationship between BPF exposure and T3-induced metamorphosis could result from the interactions of TH signaling with other signaling pathways such as Notch signaling. Our study highlights the adverse influences of BPF on vertebrate development.

TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways.

Tetrabromobisphenol A (TBBPA), as well as its alternatives Tetrabromobisphenol S (TBBPS) and Tetrachlorobisphenol A (TCBPA), are widely used halogenated flame retardants. Their high detection rates in human breast milk and umbilical cord serum have raised wide concerns about their adverse effects on human fetal development. In this study, we evaluated the cytotoxicity and neural developmental toxicity of TBBPA, TBBPS, and TCBPA with a mouse embryonic stem cell (mESC) system, at human body fluid and environmental relevant doses. All the three compounds showed similar trends in their cytotoxic effects. However, while TBBPA and TBBPS stimulated ESC neural differentiation, TCBPA significantly inhibited neurogenesis. Mechanistically, we demonstrated that, as far as the NOTCH (positive regulator) and WNT (negative regulator) pathways were concerned, TBBPA only partially and slightly disturbed them, whereas TBBPS significantly inhibited the WNT pathway, and TCBPA down-regulated the expression of NOTCH effectors but increased the WNT signaling, actions which both inhibited neural specification. In conclusion, our findings suggest that TBBPS and TCBPA may not be safe alternatives to TBBPA, and their toxicity need to be comprehensively evaluated.

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.

Vitamin E attenuates silver nanoparticle-induced effects on body weight and neurotoxicity in rats.

Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials; however, it remains unclear whether AgNPs induce neurotoxicity. Here, we investigated the potential neurological effects of AgNPs and the neuroprotective effect of vitamin E (VE). We found that intranasal instillation of AgNPs in neonatal Sprague-Dawley rats caused significant body weight loss. Moreover, histological examinations revealed activation of neuroglial cells with concomitant destruction of the granular layer of the cerebellum. Furthermore, western blot analyses showed an increase in the levels of the glial fibrillary acidic protein (GFAP), a marker of astrocyte activation. These observations suggest that AgNPs have significant neurotoxic effects on the rat cerebellum. Strikingly, oral administration of VE counterbalanced the toxic effects triggered by AgNPs. Taken together, our findings suggest that nasal administration of AgNPs may produce neurotoxicity in rats, and that VE supplementation attenuates these effects.

Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.