Byrd Gabrielle, Goldstein-Plesser Alice, Nyffeler Jo, Willis Clinton M, Fisher Anna, Boyes William K, Harrill Joshua A
Center for Computational Toxicology and Exposure, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA; Oak Ridge Associated Universities (ORAU), Oak Ridge, TN, USA; Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN, USA.
Oak Ridge Associated Universities (ORAU), Oak Ridge, TN, USA; Center for Public Health & Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA.
Toxicol Appl Pharmacol. 2025 Sep;502:117444. doi: 10.1016/j.taap.2025.117444. Epub 2025 Jun 16.
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.
银纳米颗粒（AgNPs）商业化程度的不断提高，增加了人类接触的机会，且超出了依赖体内试验的传统风险评估方法的能力范围。因此，需要高效的方法来评估AgNPs的潜在危害。以往的研究确定了粒径和表面电荷是AgNP毒性的决定因素，这主要归因于活性氧的产生。这些研究通常集中在细胞毒性或靶向细胞效应上，可能忽略了关键的亚细胞毒性效应。在这里，使用细胞绘画分析的高通量表型分析（HTPP）来表征12种不同的AgNPs（≤30μg/mL）对人视网膜色素上皮细胞（ARPE-19）细胞器形态的影响。选择了三种具有不同表面电荷的涂层（支化聚乙烯亚胺、聚乙烯吡咯烷酮、柠檬酸盐），并在4种不同尺寸（40、60、80、100nm）下进行测试，以确定这些特性对毒性的影响。HTPP与一项涉及所有涂层类型60nm颗粒的转录组分析实验同时进行。这有助于评估使用HTPP观察到的形态学效应的作用机制。同时还进行了细胞活力和凋亡分析，并用于从表型和转录组分析中排除细胞毒性处理。HTPP揭示了亚细胞毒性表型谱的浓度依赖性出现，并按涂层类型聚类，表明在确定AgNP毒性方面，表面电荷比粒径更具影响力。转录组数据突出了氧化应激和细胞周期改变是AgNPs毒性的基本潜在机制。
