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The growing number of manufactured nanomaterials (MNMs) used in consumer products and industrial applications is leading to increased exposures whose consequences are not yet fully understood in terms of potential impacts on human and ecosystem health. Thus, there is an urgent need to further develop high throughput testing for hazard prediction in nanotoxicology. The zebrafish (Danio rerio) embryo has emerged as a vertebrate model organism for nanotoxicity studies, as it provides superior characteristics for microscopy-based screening and can describe the complex interactions of MNMs with a living organism. Automated microscopy coupled with image acquisition has facilitated hazard assessment of chemicals by quantification of various endpoints (e.g., mortality, malformations, hatching), an approach which we employed here to address adverse effects of a selected library of MNMs of different compositions and surface functionalities. We investigated metal and metal oxide MNMs with mean primary particle sizes of 5 to 50 nm, different chemical compositions (silica, ceria, titania, zinc oxide and silver) and various surface coatings / functionalisation, including OECD reference materials. In addition, we tested as proxies representatives for of nanoplastics polystyrene and polymethylmethacrylate nanoparticles which were surface modified by either amino- or carboxyl-groups. Our systematic analysis to establish quantitative property-activity relationships revealed a pronounced toxicity of silver and amino-modified polystyrene nanoparticles (NPs), evidenced by reduced survival and malformations. Interference with hatching, however, was the most sensitive endpoint, as zinc oxide as well as silver NPs reduced hatching already at lower concentrations (i.e., 0.5 and 1 µg/mL, respectively) and early time points (3 days post fertilization, dpf). Interestingly, carboxylated polystyrene NPs and carboxylated or amino-modified polymethylmethacrylate NPs were non-toxic, highlighting both surface modification and core chemistry of nanoplastics as key determinants of biocompatibility.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical–chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Growth factor withdrawal induces rapid apoptosis via mitochondrial outer membrane permeabilization. We had previously observed that cell death of IL-3-dependent Ba/F3 cells, induced by removal of the growth factor, required the activity of the kinase GSK-3. Employing CRISPR/Cas9-mediated gene knockout, we aimed to identify pro-apoptotic GSK-3 regulated factors in this process. Knockout of either Puma or Bim demonstrated that the induction of Puma, but not Bim, was crucial for apoptosis induced by IL-3 deprivation. Thus, we aimed at identifying the GSK-3-dependent PUMA regulator. Loss of FOXO3A reduced the induction of Puma, while additional loss of p53 completely repressed induction upon growth factor withdrawal. A constitutively active mutant of FOXO3A, which cannot be controlled by AKT directly, still required active GSK-3 for the full transcriptional induction of Puma and cell death upon IL-3 withdrawal. Thus, the suppression of GSK-3 is the key function of PI3K signaling in order to prevent the induction of Puma by FOXO3A and p53 and thereby apoptosis upon growth factor withdrawal.