Center for Computational Toxicology & Exposure, Office of Research and Development, US Environmental Protection Agency, Durham, NC 27711, United States of America; Oak Ridge Institute for Science and Education (ORISE) Postdoctoral Fellow, Oak Ridge, TN 37831, United States of America.
Center for Computational Toxicology & Exposure, Office of Research and Development, US Environmental Protection Agency, Durham, NC 27711, United States of America.
Toxicol Appl Pharmacol. 2023 Jun 1;468:116513. doi: 10.1016/j.taap.2023.116513. Epub 2023 Apr 11.
'Cell Painting' is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.
“细胞染色”是一种基于成像的高通量表型分析（HTPP）方法，其中培养细胞被荧光标记，以可视化亚细胞结构（即核、核仁、内质网、细胞骨架、高尔基体/质膜和线粒体），并定量测量化学物质或其他干扰物引起的形态变化。HTPP 是一种高通量、具有成本效益的生物活性筛选方法，可非靶向地检测与许多不同分子机制相关的效应，从而能够快速进行体外危害评估数以千计的化学品。在这里，使用 HTPP 在人类 U-2 OS 细胞中以浓度反应的方式筛选了 ToxCast 文库中的 1201 种化学物质，最高可达约 100μM。对于在测试范围内有效的化学物质，估计了表型改变浓度 (PAC)。PAC 往往高于从广泛的靶向高通量测定中估计的下限效力值，但低于细胞毒性阈值。体外到体内外推（IVIVE）用于根据 PAC 估计基于 PAC 的给药等效剂量 (AED)，以与人类暴露预测进行比较。18/412 种化学物质的 AED 与预测的人类暴露重叠。还利用表型谱信息来识别潜在的作用机制和分组化学物质。在 58 种已知的核受体调节剂中，只有糖皮质激素和视黄酸产生特征谱；而这两种受体类型都在 U-2 OS 细胞中表达。在二次筛选中测试了 13 种与糖皮质激素谱相似的化学物质，其中一种化学物质芘被正交基因表达测定确认为新型潜在的 GR 调节化学物质。大多数活性化学物质表现出与已知作用机制无关的特征谱。然而，许多结构相关的化学物质产生了相似的谱，例外情况如烯菌唑，其谱与其他活性唑类不同。总的来说，本研究通过一系列示例和简要案例研究展示了如何在筛选水平的化学评估中应用 HTPP。
