Investigating the applicability domain of the hiPSC-based PluriLum assay: an embryotoxicity assessment of chemicals and drugs.

To meet the growing demand for developmental toxicity assessment of chemicals, New Approach Methodologies (NAMs) are needed. Previously, we developed two 3D in vitro assays based on human-induced pluripotent stem cells (hiPSC) and cardiomyocyte differentiation: the PluriBeat assay, based on assessment of beating differentiated embryoid bodies, and the PluriLum assay, a reporter gene assay based on the expression of the early cardiac marker NKX2.5; both promising assays for predicting embryotoxic effects of chemicals and drugs. In this work, we aimed to further describe the predictive power of the PluriLum assay and compare its sensitivity with PluriBeat and similar human stem cell-based assays developed by others. For this purpose, we assessed the toxicity of a panel of ten chemicals from different chemical classes, consisting of the known developmental toxicants 5-fluorouracil, all-trans retinoic acid and valproic acid, as well as the negative control compounds ascorbic acid and folic acid. In addition, the fungicides epoxiconazole and prochloraz, and three perfluoroalkyl substances (PFAS), PFOS, PFOA and GenX were tested. Generally, the PluriLum assay displayed higher sensitivity when compared to the PluriBeat assay. For several compounds the luminescence readout of the PluriLum assay showed effects not detected by the PluriBeat assay, including two PFAS compounds and the two fungicides. Overall, we find that the PluriLum assay has the potential to provide a fast and objective detection of developmental toxicants and has a level of sensitivity that is comparable to or higher than other in vitro assays also based on human stem cells and cardiomyocyte differentiation for assessment of developmental toxicity.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

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

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

Protease Activity Profiling of Snake Venoms Using High-Throughput Peptide Screening.

Snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) are among the most abundant enzymes in many snake venoms, particularly among viperids. These proteinases are responsible for some of the clinical manifestations classically seen in viperid envenomings, including hemorrhage, necrosis, and coagulopathies. The objective of this study was to investigate the enzymatic activities of these proteins using a high-throughput peptide library to screen for the proteinase targets of the venoms of five viperid (Echis carinatus, Bothrops asper, Daboia russelii, Bitis arietans, Bitis gabonica) and one elapid (Naja nigricollis) species of high medical importance. The proteinase activities of these venoms were each tested against 360 peptide substrates, yielding 2160 activity profiles. A nonlinear regression model that accurately described the observed enzymatic activities was fitted to the experimental data, allowing for the comparison of cleavage rates across species. In this study, previously unknown protein targets of snake venom proteinases were identified, potentially implicating novel human and animal proteins that may be involved in the pathophysiology of viper envenomings. The functional relevance of these targets was further evaluated and discussed. These new findings may contribute to our understanding of the clinical manifestations and underlying biochemical mechanisms of snakebite envenoming by viperid species.