Risk assessment of parabens in a transcriptomics-based in vitro test.

Parabens have been used for decades as preservatives in food, drugs and cosmetics. The majority however, were banned in 2009 and 2014 leaving only methyl-, ethyl-, propyl-, and butyl-derivates available for subsequent use. Methyl- and propylparaben have been extensively tested in vivo, with no resulting evidence for developmental and reproductive toxicity (DART). In contrast, ethylparaben has not yet been tested for DART in animal experiments, and it is currently debated if additional animal studies are warranted. In order to perform a comparison of the four currently approved parabens, we used a previously established in vitro test based on human induced pluripotent stem cells (iPSC) that are exposed to test substances during their differentiation to neuroectodermal cells. EC50 values for cytotoxicity were 906 µM, 698 µM, 216 µM and 63 µM for methyl-, ethyl-, propyl- and butylparaben, respectively, demonstrating that cytotoxicity increases with increasing alkyl chain length. Genome-wide analysis demonstrated that FDR-adjusted significant gene expression changes occurred only at cytotoxic or close to cytotoxic concentrations, for example 1720 differentially expressed genes (DEG) at 1000 µM ethylparaben, 1 DEG at 316 µM, and no DEG at 100 µM or lower concentrations. The highest concentration of ethylparaben that did not induce any cytotoxicity nor DEG was 1670-fold above the highest concentration reported in biomonitoring studies (60 nM ethylparaben in cord blood). In conclusion, cytotoxicity and gene expression alterations of ethylparaben occurred at concentrations of approximately three orders of magnitude above human blood concentrations; moreover, the substance fitted well into a scenario where toxicity increases with the alkyl chain length, and gene expression changes only occur at cytotoxic or close to cytotoxic concentrations. Therefore, no evidence was obtained suggesting that in vivo DART with ethylparaben would lead to different results as the methyl- or propyl derivates.

Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells.

Animal studies for embryotoxicity evaluation of potential therapeutics and environmental factors are complex, costly, and time-consuming. Often, studies are not of human relevance because of species differences. In the present study, we recapitulated the process of cardiomyogenesis in human induced pluripotent stem cells (hiPSCs) by modulation of the Wnt signaling pathway to identify a key cardiomyogenesis gene signature that can be applied to identify compounds and/or stress factors compromising the cardiomyogenesis process. Among the 23 tested teratogens and 16 non-teratogens, we identified three retinoids including 13-cis-retinoic acid that completely block the process of cardiomyogenesis in hiPSCs. Moreover, we have identified an early gene signature consisting of 31 genes and associated biological processes that are severely affected by the retinoids. To predict the inhibitory potential of teratogens and non-teratogens in the process of cardiomyogenesis we established the "Developmental Cardiotoxicity Index" (CDI31g) that accurately differentiates teratogens and non-teratogens to do or do not affect the differentiation of hiPSCs to functional cardiomyocytes.

Epigenetic mechanisms of Strip2 in differentiation of pluripotent stem cells.

Significant evidence points to Strip2 being a key regulator of the differentiation processes of pluripotent embryonic stem cells. However, Strip2 mediated epigenetic regulation of embryonic differentiation and development is quite unknown. Here, we identified several interaction partners of Strip2, importantly the co-repressor molecular protein complex nucleosome remodeling deacetylase/Tripartite motif-containing 28/Histone deacetylases/Histone-lysine N-methyltransferase SETDB1 (NuRD/TRIM28/HDACs/SETDB1) histone methyltransferase, which is primarily involved in regulation of the pluripotency of embryonic stem cells and its differentiation. The complex is normally activated by binding of Krueppel-associated box zinc-finger proteins (KRAB-ZFPs) to specific DNA motifs, causing methylation of H3 to Lysin-9 residues (H3K9). Our data showed that Strip2 binds to a DNA motif (20 base pairs), like the KRAB-ZFPs. We establish that Strip2 is an epigenetic regulator of pluripotency and differentiation by modulating DNA KRAB-ZFPs as well as the NuRD/TRIM28/HDACs/SETDB1 histone methyltransferase complex.

High Accuracy Classification of Developmental Toxicants by In Vitro Tests of Human Neuroepithelial and Cardiomyoblast Differentiation.

Human-relevant tests to predict developmental toxicity are urgently needed. A currently intensively studied approach makes use of differentiating human stem cells to measure chemically-induced deviations of the normal developmental program, as in a recent study based on cardiac differentiation (UKK2). Here, we (i) tested the performance of an assay modeling neuroepithelial differentiation (UKN1), and (ii) explored the benefit of combining assays (UKN1 and UKK2) that model different germ layers. Substance-induced cytotoxicity and genome-wide expression profiles of 23 teratogens and 16 non-teratogens at human-relevant concentrations were generated and used for statistical classification, resulting in accuracies of the UKN1 assay of 87-90%. A comparison to the UKK2 assay (accuracies of 90-92%) showed, in general, a high congruence in compound classification that may be explained by the fact that there was a high overlap of signaling pathways. Finally, the combination of both assays improved the prediction compared to each test alone, and reached accuracies of 92-95%. Although some compounds were misclassified by the individual tests, we conclude that UKN1 and UKK2 can be used for a reliable detection of teratogens in vitro, and that a combined analysis of tests that differentiate hiPSCs into different germ layers and cell types can even further improve the prediction of developmental toxicants.

Classification of Developmental Toxicants in a Human iPSC Transcriptomics-Based Test.

Despite the progress made in developmental toxicology, there is a great need for in vitro tests that identify developmental toxicants in relation to human oral doses and blood concentrations. In the present study, we established the hiPSC-based UKK2 in vitro test and analyzed genome-wide expression profiles of 23 known teratogens and 16 non-teratogens. Compounds were analyzed at the maximal plasma concentration (Cmax) and at 20-fold Cmax for a 24 h incubation period in three independent experiments. Based on the 1000 probe sets with the highest variance and including information on cytotoxicity, penalized logistic regression with leave-one-out cross-validation was used to classify the compounds as test-positive or test-negative, reaching an area under the curve (AUC), accuracy, sensitivity, and specificity of 0.96, 0.92, 0.96, and 0.88, respectively. Omitting the cytotoxicity information reduced the test performance to an AUC of 0.94, an accuracy of 0.79, and a sensitivity of 0.74. A second method, which used the number of significantly deregulated probe sets to classify the compounds, resulted in a specificity of 1; however, the AUC (0.90), accuracy (0.90), and sensitivity (0.83) were inferior compared to those of the logistic regression-based procedure. Finally, no increased performance was achieved when the high test concentrations (20-fold Cmax) were used, in comparison to testing within the realistic clinical range (1-fold Cmax). In conclusion, although further optimization is required, for example, by including additional readouts and cell systems that model different developmental processes, the UKK2-test in its present form can support the early discovery-phase detection of human developmental toxicants.

Road Map for Development of Stem Cell-Based Alternative Test Methods.

Much progress has been made in establishing strategies for differentiation of induced human pluripotent stem cells (hiPSCs). However, differentiated hiPSCs are not yet routinely used for prediction of toxicity. Here, limiting factors are summarised and possibilities for improvement are discussed, with a focus on hepatocytes, cardiomyocytes, tubular epithelial cells, and developmental toxicity. Moreover, we make recommendations for further fine-tuning of differentiation protocols for hiPSCs to hepatocyte-like cells by comparing individual steps of currently available protocols to the mechanisms occurring during embryonic development. A road map is proposed to facilitate test system development, including a description of the most useful performance metrics.

Effects of bisphenol A on the microtubule arrays in root meristematic cells of Pisum sativum L.

Bisphenol A (BPA), a widely used chemical in the plastics industry that displays weak oestrogenic properties, is an emerging environmental pollutant, potentially harmful to living organisms. The presumed cytotoxicity of BPA to plant cells has been poorly studied. To understand how BPA might influence plant cell division and affect the underlying cytoskeleton, the effects of BPA on the microtubule (MT) arrays of meristematic root-tip cells of Pisum sativum L. were investigated. Root tips of young seedlings were exposed to 20, 50 and 100mg/L BPA for 1, 3, 6, 12 and 24h. The effects of each treatment were determined by means of confocal laser scanning microscopy after immunolabelling of tubulin and counterstaining of DNA, and by use of light and transmission electron microscopy. It was found that BPA affected normal chromosome segregation, hampered the completion of cytokinesis and deranged interphase and mitotic MT arrays. BPA effects were dependent on the stage of each cell at the time of BPA entrance. Moreover, BPA induced the formation of macrotubules with a mean diameter of 32 ± 0.14 nm, compared with 23 ± 0.70 nm for the MT arrays in untreated cells. Finally, all MT arrays and macrotubules were depolymerised upon longer treatment. Taken together, the data suggest that BPA exerts acute anti-mitotic effects on meristematic root-tip cells of P. sativum, MT arrays constitute a primary sub-cellular target of BPA toxicity, and the manifested chromosomal abnormalities could be attributed to the disruption of the MT cytoskeleton.