Embryonic Stem Cell Test Research Articles

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs.

Animal-based test systems have traditionally been used to screen for the potential teratogenic activity of drugs. Still, their deficits in predicting precise human-specific outcomes and ethical concerns have led to a need for alternative approaches. In vitro, teratogenicity testing using cell cultures or other in vitro systems is a potential alternative. Of the different in vitro platforms, the mouse embryonic stem cell test (mEST) is currently the most widely used and validated in vitro test for assessing the potential effects of teratogens on early embryonic development. The mEST involves exposing mouse embryonic stem cells to the test compound and monitoring their differentiation for several days.Nevertheless, its predictive ability was comparatively lower when distinguishing weak developmental toxicants from non-toxic substances. Since then, several modifications and adaptations of the mEST protocol have been developed. This chapter describes an alternative method based on molecular approaches to predict embryotoxicity. This method, originated from the mEST, analyzes the expression of differentiation genes involved in the development of mesoderm, endoderm, and stoderm and allows screening embryo-toxicants with different mechanisms of action. The hanging drops embryoid bodies used in the original mEST protocol have been replaced with monolayer culture, and thus the process has been shortened. In general, the method shows higher predictability compared with the traditional ones.

Evaluating the in vitro developmental toxicity potency of a series of petroleum substance extracts using new approach methodologies (NAMs)

The present study evaluates the in vitro developmental toxicity and the possible underlying mode of action of DMSO extracts of a series of highly complex petroleum substances in the mouse embryonic stem cell test (mEST), the zebrafish embryotoxicity test (ZET) and the aryl hydrocarbon receptor reporter gene assay (AhR CALUX assay). Results show that two out of sixteen samples tested, both being poorly refined products that may contain a substantial amount of 3- to 7-ring polycyclic aromatic compounds (PACs), induced sustained AhR activation in the AhR CALUX assay, and concentration-dependent developmental toxicity in both mEST and ZET. The other samples tested, representing highly refined petroleum substances and petroleum-derived waxes (containing typically a very low amount or no PACs at all), were negative in all assays applied, pointing to their inability to induce developmental toxicity in vitro. The refining processes applied during the production of highly refined petroleum products, such as solvent extraction and hydrotreatment which focus on the removal of undesired constituents, including 3- to 7-ring PACs, abolish the in vitro developmental toxicity. In conclusion, the obtained results support the hypothesis that 3- to 7-ring PACs are the primary inducers of the developmental toxicity induced by some (i.e., poorly refined) petroleum substances and that the observed effect is partially AhR-mediated.

Comparative assessment of embryotoxicity of 2,4,6-triiodophenol to mouse blastoid and pre-implantation embryo models

Embryonic developmental effects of disinfection by-products, which are generated during drinking water treatment and widely detected in environment, have gained more and more attention nowadays, calling for construction of in vitro research models which can mimic early embryonic development to evaluate the embryotoxicity. The embryonic stem cell test offers a promising assay to predict embryotoxicity of environmental pollutions. However, it is not appropriate for the toxicological study of preimplantation embryos. Here, we used mouse extended stem cells (mEPS) to reconstruct embryo-like structures (blastoid), furtherly attempting to evaluate the reliability of this model for the prediction of possible developmental toxicity of 2,4,6-triiodophenol (TIP, 5–50 μM), a novel halogenated disinfection byproduct widely detected in water and even drinking water, to mammalian preimplantation embryo. To verify this, we treated mouse embryo derived from in vitro fertilization (IVF-embryo) as reference. The results showed that mEPS-blastoid was like natural blastocyst in morphology, cell composition, and could recapitulate key developmental events happened during mouse preimplantation stage. When blastoid and IVF-embryo models were separately exposed to TIP, their final blastocyst formation rates were not impaired, according to morphological features, meanwhile that TIP exposure caused slight cell apoptosis. Besides, TIP induced an ICM cell bias in cell fate decision, resulting in cell proportion change, which implied abnormal developmental potential. Though we could not evaluate TIP’s embryotoxicity before 8-cell stage using blastoid model, its viability as a novel and high-throughput assessment platform for increasing environmental pollutants was still recognized.

In vitro tests to evaluate embryotoxicity and irritation of Chinese herbal medicine (Pentaherbs formulation) for atopic dermatitis

Ethnopharmacological relevanceAtopic dermatitis (AD) is a common chronic inflammatory skin disorder and its prevalence is increasing in the last few decades. No treatment can cure the condition. Pregnancy often worsens the clinical manifestation. There are considerable interests in Chinese Herbal Medicine (CHM) as an alternative treatment for AD. A well tolerated CHM formula (Pentaherbs formulation, PHF) has been proven efficacious in improving life quality and reducing topical corticosteroid use in children with moderate-to-severe AD. However, safety data of PHF are not available. Aim of the studyOur study aimed to evaluate the safety of PHF and its 5 individual herbal extracts, including embryotoxicity by Embryonic Stem Cell Test (EST) and irritation by Skin Irritation Test (SIT). Materials and methodsQuality of 5 herbal extracts of PHF was confirmed by chromatography. In EST, mouse embryonic stem cell line (D3) and mouse fibroblast cell line (3T3) were used to study potential embryotoxicity. Three endpoints were assessed by concentration-response curves after 10 days’ culture: 50% inhibition of D3 differentiation into beating cardiomyocytes (ID50D3), 50% cytotoxic effects on D3 (IC50D3) and on fibroblasts (IC503T3). A biostatistically based prediction model (PM) was applied to predict the embryotoxic potentials of each CHM. In SIT, epidermis equivalent commercially available kits (EpiDerm™) were used, and concentration-viability curves were obtained by MTT assay to detect skin irritations of each CHM. ResultsChemical authentication confirmed that 5 test herbal extracts contained their main active compounds. EST results indicated that the formula PHF and its individual CHMs were non-embryotoxic, except one CHM, Amur Corktree Bark (Huang Bai, Phellodendron chinense C.K.Schneid), was weakly embryotoxic. SIT results showed that cell viability was above 50% after treatment with different concentrations of all tested CHMs. ConclusionsOur in vitro tests provided preliminary evidence for safety of the formula PHF in embryonic stem cell test and skin irritation model, but PHF shall be cautiously used in pregnant women with AD. Further studies are needed to support its clinical application as an alternative treatment for AD, especially to the patients who plan for pregnancy or at lactation stages.

A preliminary cumulative risk assessment of Diethylhexyl phthalate and Dibutyl phthalate based on the inhibition of embryonic development via the PPARγ pathway

BackgroundDiethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are the two most widely used plasticizers (PAEs) that may act as endocrine disruptors and cause developmental toxicity. MethodsWe measured intrauterine exposure to DEHP and DBP which are the two most widely used phthalates (PAEs) in the cord blood of 50 postpartum women using ultra-HPLC-tandem mass spectrometry and solid-phase extraction. The embryotoxicity of DEHP and DBP was evaluated using the human embryonic stem cell test (hEST). Based on the intrauterine exposure concentration of DEHP and DBP in pregnant women and the reference point of toxic effects in hEST, we used the reference point index (RPI) method to assess the cumulative risk of DBP and DEHP. ResultsThe mean concentrations of DEHP and DBP were 99.9 μg/L and 24.7 μg/L, respectively. DEHP and DBP were weakly embryotoxic, and the benchmark dose lower confidence intervals were 29.99 and 0.99 μg/mL, respectively, as determined using hEST. Both DEHP and DBP inhibited embryonic development via PPAR/PTEN/Akt signal pathway. ConclusionThe findings suggest that the cumulative risk in pregnant women with a high level of exposure should receive more attention in the future.

Optimization of the TeraTox Assay for Preclinical Teratogenicity Assessment.

Current animal-free methods to assess teratogenicity of drugs under development still deliver high numbers of false negatives. To improve the sensitivity of human teratogenicity prediction, we characterized the TeraTox test, a newly developed multilineage differentiation assay using 3D human-induced pluripotent stem cells. TeraTox produces primary output concentration-dependent cytotoxicity and altered gene expression induced by each test compound. These data are fed into an interpretable machine-learning model to perform prediction, which relates to the concentration-dependent human teratogenicity potential of drug candidates. We applied TeraTox to profile 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Comparing TeraTox predictions with known human or animal toxicity, we report an accuracy of 69% (specificity: 53%, sensitivity: 79%). TeraTox performed better than 2 quantitative structure-activity relationship models and had a higher sensitivity than the murine embryonic stem cell test (accuracy: 58%, specificity: 76%, and sensitivity: 46%) run in the same laboratory. The overall prediction accuracy could be further improved by combining TeraTox and mouse embryonic stem cell test results. Furthermore, patterns of altered gene expression revealed by TeraTox may help grouping toxicologically similar compounds and possibly deducing common modes of action. The TeraTox assay and the dataset described here therefore represent a new tool and a valuable resource for drug teratogenicity assessment.

NH115a, anovel inhibitor of the La protein: Effect on expression of multiple RNAs in hepatitis B virus-infected hepatoma cells and embryotoxicity profile.

The La protein binds to RNA and protects replication of the hepatitis B virus (HBV). We recently developed the compound nH115a, an inhibitor of the La protein that has high stability and anti-HBV activity. However, the mechanism, by which this compound inhibits HBV infection and its safety to embryos, remains unclear. Our goal was to examine the molecular mechanism, by which nH115a inhibits HBV infection, and to characterize its embryotoxicity. Microarray experiments using HepG2.2.15 cells (established by transfecting an HBV plasmid into HepG2 hepatoma cells) and bioinformatics analyses were used to measure the effect of nH115a on the expression of lncRNAs, mRNAs, and circRNAs. The embryonic stem cell test was used to assess the embryotoxicity of nH115a. nH115a significantly altered the expression of 2402 lncRNAs, 338 mRNAs, and 559 circRNAs. Gene Ontology (GO) analysis indicated the differentially expressed transcripts functioned in interleukin-2 production, I-SMAD binding, RNA-induced silencing complex (RISC), NLRP3 inflammasome complex assembly, cytoplasmic sequestering of nuclear factor kappa-B (NF-κB), and death receptor binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the most enriched pathways included transforming growth factor-β (TGF-β) signaling, pathways in cancer, ubiquitin mediated proteolysis, p53 signaling, antigen processing and presentation, Fc gamma R-mediated phagocytosis, and B cell receptor signaling. The results of the embryonic stem cell test indicated that nH115a exhibited weak embryotoxicity. In conclusion, immune responses, TGF-β/SMAD signaling, and cancer-related pathways may function in the nH115a-mediated inhibition of HBV replication. Keywords: hepatitis B virus; La protein; inhibitor; nH115a.

Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration–response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).

Integrating in vitro chemical transplacental passage into a generic PBK model: A QIVIVE approach

With the increasing application of cell culture models as primary tools for predicting chemical safety, the quantitative extrapolation of the effective dose from in vitro to in vivo (QIVIVE) is of increasing importance. For developmental toxicity this requires scaling the in vitro observed dose-response characteristics to in vivo fetal exposure, while integrating maternal in vivo kinetics during pregnancy, in particular transplacental transfer. Here the transfer of substances across the placental barrier, has been studied using the in vitro BeWo cell assay and six embryotoxic compounds of different kinetic complexity. The BeWo assay results were incorporated in an existing generic Physiologically Based Kinetic (PBK) model which for this purpose was extended with rat pregnancy. Finally, as a “proof of principle”, the BeWo PBK model was used to perform a QIVIVE based on developmental toxicity as observed in various different in vitro toxicity assays. The BeWo results illustrated different transport profiles of the chemicals across the BeWo monolayer, allocating the substances into two distinct groups: the ‘quickly-transported’ and the ‘slowly-transported’. BeWo PBK exposure simulations during gestation were compared to experimentally measured maternal blood and fetal concentrations and a reverse dosimetry approach was applied to translate in vitro observed embryotoxicity into equivalent in vivo dose-response curves. This approach allowed for a direct comparison of the in vitro dose-response characteristics as observed in the Whole Embryo Culture (WEC), and the Embryonic Stem Cell test (cardiac:ESTc and neural:ESTn) with in vivo rat developmental toxicity data. Overall, the in vitro to in vivo comparisons suggest a promising future for the application of such QIVIVE methodologies for screening and prioritization purposes of developmental toxicants. Nevertheless, the clear need for further improvements is acknowledged for a wider application of the approach in chemical safety assessment.

Evaluation of developmental toxicity of ammonium dinitramide by micromass culture and embryonic stem cells models

Objective: To evaluated the potential developmental toxicity and teratogenicity of ammonium dinitroamide (ADN) by micromass test (MM Test) and embryonic stem cell test models. Methods: In September 2018, rat embryos were isolated and limb bud cells were collected. The limb bud cells were treated with different concentrations of ADN (0, 312.50, 625.00, 1250.00, 2500.00, 5000.00, 10000.00 μg/ml) . Half proliferation inhibitory concentration and half differentiation inhibitory concentration were calculated and the teratogenic effects were evaluated according to the criteria. For the embryonic stem cell test, the effects of different concentrations of ADN (0, 39.06, 78.13, 156.25, 312.50, 625.00, 1250.00, 2500.00 μg/ml) on the differentiation of mouse embryonic stem cells (mESCs) into myocardial cells and the cytotoxicity of mESCs and 3T3 cells were detected. The embryonic toxicity was evaluated according to the criteria. In this study, both 5-fluorouracil (5-FU) , a known strong embryonic toxic drug, and penicillin-G (P-G) , a non-embryonic toxic drug, were used to verify the effectiveness of the model, and the validated test model was applied to evaluate the embryonic toxicity of ADN. Results: In the MM Test, the inhibition rates of proliferation and differentiation of limb bud cells in ADN groups were higher than that in control group (P<0.05) . And the half proliferation inhibitory concentration and half differentiation inhibitory concentration of ADN on limb bud cells were 7480.32 and 4526.09 μg/ml, respectively. ADN was determined to be non-teratogenic by standard. In the embryonic stem cell test, the inhibition rates of mESCs proliferation in ADN groups were higher than that in control group, and the inhibition rates of 3T3 cells in 156.25, 312.50, 625.00, 1250.00, 2500.00 μg/ml ADN groups were higher than that in control group (P<0.05) . The half proliferation inhibitory concentration and half differentiation inhibitory concentration of ADN on mESCs were 1851.73 and 1796.39 μg/ml, respectively, and the half proliferation inhibitory concentration on 3T3 cells was 3334.35 μg/ml. ADN was determined to be non-embryotoxic by standard. Conclusion: After evaluation by MM Test and embryonic stem cell models, ADN has no embryo toxicity and is a non-teratogenic substance.

Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies

Concurrent with the ‘3R’ principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2–5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Cell differentiation in the cardiac embryonic stem cell test (ESTc) is influenced by the oxygen tension in its underlying embryonic stem cell culture

Oxygen (O2) levels in the mammalian embryo range between 2.4% and 8%. The cardiac embryonic stem cell test (ESTc) is a model for developmental toxicity predictions, which is usually performed under atmospheric O2 levels of 20%. We investigated the chemical sensitivity of the ESTc carried out under 20% O2, using embryonic stem cells (ESC) cultured under either 20% O2 or 5% O2. ESC viability was more sensitive to valproic acid (VPA) but less sensitive to flusilazole (FLU) when cultured under 5% versus 20% O2. For beating cardiomyocyte differentiation, lower ID50 values were found for FLU and VPA when the ESCs had been cultured under 5% versus 20% O2. At differentiation day 4, gene expression values were primarily driven by the level of O2 during ESC culture instead of exposure to FLU. In addition, using ESCs cultured under 5% O2 tension, VPA enhanced Nes (ectoderm) expression. Bmp4 (mesoderm) was enhanced by VPA when using ESCs cultured under 20% O2. At differentiation day 10, using ESCs cultured under 5% instead of 20% O2, Nkx2.5 and Myh6 (cardiomyocytes) were less affected after exposure to FLU or VPA. These results show that O2 tension in ESC culture influences chemical sensitivity in the ESTc. This enhances awareness of the standard culture conditions, which may impact the application of the ESTc in quantitative hazard assessment of chemicals.

The oxygen tension during embryonic stem cell culture preceding differentiation influences chemical sensitivity in the embryonic stem cell test (EST)

The oxygen tension during embryonic stem cell culture preceding differentiation influences chemical sensitivity in the embryonic stem cell test (EST)

Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro\u2013in silico approach

Developmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro–in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration–response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose–response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose–response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro–in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.

Establishment of a developmental neurotoxicity test by Sox1-GFP mouse embryonic stem cells

Developmental toxicity tests have been generated by applying the embryonic stem cell tests at the European Centre for the Validation of Alternative Methods, or by using the embryoid body test in our laboratory. This study was undertaken to explore novel developmental neurotoxicity (DNT) assay, using a Sox1-GFP cell line (mouse embryonic stem cells with an endogenous Sox1-GFP reporter). The expression of Sox1, a marker for neuroepithelial cells, is detected by green fluorescence, and the fluorescence intensity is a critical factor for achieving neuronal differentiation. Sox1-GFP cells cultured for 24 h were exposed to eleven neurotoxicants and four non-neurotoxicants. CCK-8 assays were performed to determine IC50 values after 48 h of chemical treatment. The fluorescence intensity of GFP was measured 4 days after treating the cells, and it was observed to decrease after exposure to neurotoxicants at higher concentrations, thereby indicating that the neuronal differentiation of Sox1-GFP cells is inhibited by the chemicals. Taken together, the results obtained in this study provide a model for DNT using embryonic stem cells, which may be applied to evaluate the toxicity of new chemicals or new drug candidates.

Individual and combined effects of BPA, BPS and BPAF on the cardiomyocyte differentiation of embryonic stem cells

Exposure to many kinds of bisphenols (BPs) is common, and the effects of BP mixtures may differ from those of individual BPs. Therefore, evaluating combined exposure effects is necessary. Our study evaluated the individual and combined exposure effects of bisphenol A (BPA), bisphenol S (BPS) and bisphenol AF (BPAF) on embryonic development using an embryonic stem cell test (EST) and a concentration additive (CA) model at relatively high doses to uncover the interaction model of the three BPs. Environmentally relevant concentrations were then used to evaluate the possible effects of the individual and combined BPs at actual human exposure levels. Exposure to relatively high-dose BPA, BPS and BPAF inhibited embryonic stem cell differentiation into cardiomyocytes and exhibited weak embryo toxicity. Individually, BPA, BPS and BPAF inhibited endoderm, mesoderm and ectoderm marker expression but enhanced pluripotency marker expression. Combined exposure to BPs had an additive effect on cardiomyocyte differentiation and embryonic stem cell proliferation based on the CA model. Environmentally relevant individual or combined BP doses (10 ng/ml individual BPA, BPS and BPAF doses or 1 ng/ml and 10 ng/ml BP mixture doses) failed to cause oxidative stress, DNA damage or apoptosis changes in stem cell differentiation. The cardiomyocyte differentiation ratio also did not change significantly. Individual and combined exposure to environmentally relevant BP doses led to a significant increase in collagen expression. BPAF and the combination of BPs increased the type 1 collagen level, while the combination also increased the type 3 collagen level, which may be related to p38 pathway activation. The p38 pathway inhibitor SB203580 inhibited the increase in collagen during cardiomyocyte differentiation caused by low-dose BPs. These results suggest that relatively high-dose BPs in combination have an additive effect on cardiomyocyte differentiation. Low-dose BPs individually and in combination may affect cardiomyocyte collagen through the p38 pathway.

Developmental toxicity testing of the fume condensate extracts of bitumen and oxidized asphalt in a series of in vitro alternative assays

The potential developmental toxicity and mode-of-action of fume condensate extracts of bitumen and oxidized asphalt were evaluated in the aryl hydrocarbon receptor (AhR) CALUX assay, the zebrafish embryotoxicity test (ZET), and the mouse embryonic stem cell test (mEST). In the AhR CALUX assay, both fume condensate extracts showed a concentration-dependent AhR induction following 6-h of exposure, but this activity was substantially reduced after 24-h, indicating a transient AhR activation. The main effect observed in the ZET was early embryonic lethality that occurred mostly in the 24 h-post-fertilization (hpf). This typically reflects non-specific toxicity rather than in vitro developmental toxicity of the fume condensate extracts tested since this effect was not seen as a result of the whole cumulative exposure period in the ZET (up to 96 hpf). No malformations were seen in any zebrafish embryo exposed to these fume condensate extracts, although some developed pericardial and/or yolk-sac edemas. Furthermore, both fume condensate extracts tested negative in the mEST. In conclusion, the results show that fume condensate extracts of bitumen and oxidized asphalt do not induce any in vitro developmental toxicity, which is in line with the results observed in the in vivo prenatal developmental toxicity studies performed with the same materials.

Fine particles in surgical smoke affect embryonic cardiomyocyte differentiation through oxidative stress and mitophagy

Surgical smoke is widespread in operating rooms, and fine particles are the main toxic components. However, the effect of fine particles in surgical smoke on embryonic development has not yet been studied. This study evaluated the effect of fine particles in surgical smoke on embryonic development and compared it with that of atmospheric fine particles. Afterwards, differentiated cardiomyocytes were purified, and the effect of exposure to fine particles in surgical smoke on cardiomyocyte differentiation was evaluated. Fine particles in surgical smoke exhibited weak embryotoxicity toward an embryonic stem cell test model, and their inhibitory effect on cardiomyocyte differentiation was slightly stronger than that of atmospheric fine particles. Fine particles in surgical smoke specifically inhibited the differentiation of the mesoderm lineage and promoted the differentiation of the ectoderm lineage. Furthermore, fine particles in surgical smoke reduced the beating rate of purified cardiomyocytes, promoted mitophagy, reduced ATP production and increased the reactive oxygen species (ROS) content. Antioxidants attenuated the inhibition of cardiomyocyte differentiation and the reduction in the cardiomyocyte beating rate caused by fine particles in surgical smoke and simultaneously restored mitophagy and other processes to the control levels. However, mitophagy inhibitors treatment blocked only the inhibition of cardiomyocyte differentiation caused by fine particles in surgical smoke; it had little effect on other changes caused by fine particles. Based on the results described above, we propose that fine particles in surgical smoke and atmospheric fine particles exhibit similar levels of toxicity toward embryonic development. Fine particles in surgical smoke potentially affect the beating of cardiomyocytes by damaging mitochondria and increasing oxidative stress.