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Assessing the Effects of Silver Nanoparticles on ARPE-19 Cells via High-Throughput Phenotypic Profiling with the Cell Painting Assay.

ABSTRACT: Increasing commercialization of silver nanoparticles (AgNPs) has resulted in elevated opportunity for human exposure and outpaced traditional risk assessment approaches that rely on in vivo testing. Therefore, efficient methods are needed to evaluate potential hazards of AgNPs. Previous studies identified particle size and surface charge as determinants of AgNP toxicity, which has primarily been attributed to reactive oxygen species generation. Those studies have generally focused on cytotoxicity or targeted cellular effects, potentially missing critical sub-cytotoxic effects. Here, high throughput phenotypic profiling (HTPP) with the Cell Painting assay was used to characterize the effects of 12 distinct AgNPs (≤ 30 µg/mL) on organelle morphology in human retinal pigmented epithelial cells (ARPE-19). Three coatings (branched polyethyleneimine, polyvinylpyrrolidone, citrate) were selected for their distinct surface charges and tested at 4 different sizes (40, 60, 80, 100 nm) to determine the effects of these properties on toxicity. HTPP was conducted in conjunction with a transcriptomic profiling experiment involving 60 nm particles of all coating types. This facilitated evaluation of mechanisms of action underlying morphological effects observed using HTPP. A cell viability and apoptosis assay was also run in parallel and used to exclude cytotoxic treatments from phenotypic and transcriptomic analyses. HTPP revealed concentration-dependent emergence of sub-cytotoxic phenotypic profiles that clustered by coating type, suggesting surface charge to be more influential than particle size in the determination of AgNP toxicity. The transcriptomic data highlighted oxidative stress and cell cycle alterations as fundamental underlying toxicity mechanisms of AgNPs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE297342 | GEO | 2025/05/20

REPOSITORIES: GEO

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Project description:We examined the use of toxicogenomics data to 1) elucidate the underlying mechanisms of pulmonary responses following exposures to titanium dioxide (TiO2) nanoparticles (NPs) of different size and surface charge, and 2) determine if such responses are influenced by embedding particles in complex paint matrix. Adult C57BL/6 mice were exposed via single intratracheal instillation to three doses of free forms of NRCWE-030 (10.5 nm), NRCWE-025 (38 nm), NRCWE-001 (10 nm, neutral charge) NRCWE-002 (10 nm, positive charge) or to sanding dusts of paint consisting of NRCWE-030+NRCWE-025 or NRCWE-025 alone. Controls were exposed to dispersion medium without NPs. TiO2NPs were characterized for size, surface area, and agglomeration status in the exposure medium. Hyperspectral microscopy was performed to detect TiO2NPs in lungs and to determine the in vivo status of matrix-embedded TiO2NPs. Pulmonary gene expression profiles were generated using DNA microarrays on lung tissues collected at 24 h and 28 d post exposure time points. Bioinformatics tools were used to characterize size and surface property-pertinent effects. The data from 360 individual arrays were collapsed into 567 differentially expressed genes (False discovery rate adjusted P ≤ 0.05 and fold change ≥ 1.5 in any one condition). Unsupervised hierarchical clustering of the 567 genes revealed that TiO2NPs clustered mainly based on the post-exposure time points and then by the particle types. A meta analysis using pathway tools showed that the combination of smaller size and positive surface charge contributes to the potency of TiO2NPs. Although embedding in matrix dampens the overall toxicity of TiO2NPs, the smaller size positively influences the toxicity. Paint matrix itself did not induce any response. Finally, the observed differences in response reflected the differences in severity of the response and not the underlying mechanisms. Thus, transcriptional profiling is an effective tool to determine the important properties responsible for eliciting a response.

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Assessing the Effects of Silver Nanoparticles on ARPE-19 Cells via High-Throughput Phenotypic Profiling with the Cell Painting Assay.

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