Use of a High-Throughput Screening Approach Coupled with <i>In Vivo</i> Zebrafish Embryo Screening To Develop Hazard Ranking for Engineered Nanomaterials

TLDR

Safety concerns about engineered nanomaterials drive the need for high‑throughput screening and in‑silico tools to accelerate in‑vitro hazard ranking. The study aims to develop a multiparametric, automated screening assay and compare its in‑vitro results with zebrafish embryo responses to rank engineered nanomaterial hazards. The assay uses automated epifluorescence microscopy to measure ROS, calcium flux, mitochondrial depolarization, and membrane permeability, z‑scoring the data to generate heat maps and self‑organizing maps for hazard ranking, and applies the same nanoparticles to zebrafish embryos to assess mortality, hatching, cardiac rate, and morphology. The assay identified QD1 and nano‑ZnO as the most lethal, while Pt, Ag, SiO.

Abstract

Because of concerns about the safety of a growing number of engineered nanomaterials (ENM), it is necessary to develop high-throughput screening and in silico data transformation tools that can speed up in vitro hazard ranking. Here, we report the use of a multiparametric, automated screening assay that incorporates sublethal and lethal cellular injury responses to perform high-throughput analysis of a batch of commercial metal/metal oxide nanoparticles (NP) with the inclusion of a quantum dot (QD1). The responses chosen for tracking cellular injury through automated epifluorescence microscopy included ROS production, intracellular calcium flux, mitochondrial depolarization, and plasma membrane permeability. The z-score transformed high volume data set was used to construct heat maps for in vitro hazard ranking as well as showing the similarity patterns of NPs and response parameters through the use of self-organizing maps (SOM). Among the materials analyzed, QD1 and nano-ZnO showed the most prominent lethality, while Pt, Ag, SiO2, Al2O3, and Au triggered sublethal effects but without cytotoxicity. In order to compare the in vitro with the in vivo response outcomes in zebrafish embryos, NPs were used to assess their impact on mortality rate, hatching rate, cardiac rate, and morphological defects. While QDs, ZnO, and Ag induced morphological abnormalities or interfered in embryo hatching, Pt and Ag exerted inhibitory effects on cardiac rate. Ag toxicity in zebrafish differed from the in vitro results, which is congruent with this material's designation as extremely dangerous in the environment. Interestingly, while toxicity in the initially selected QD formulation was due to a solvent (toluene), supplementary testing of additional QDs selections yielded in vitro hazard profiling that reflect the release of chalcogenides. In conclusion, the use of a high-throughput screening, in silico data handling and zebrafish testing may constitute a paradigm for rapid and integrated ENM toxicological screening.