The present study adapted an existing high content imaging-based high-throughput phenotypic profiling (HTPP) assay known as "Cell Painting" for bioactivity screening of environmental chemicals. This assay uses a combination of fluorescent probes to label a variety of organelles and measures a large number of phenotypic features at the single cell level in order to detect chemical-induced changes in cell morphology. First, a small set of candidate phenotypic reference chemicals (n = 14) known to produce changes in the cellular morphology of U-2 OS cells were identified and screened at multiple time points in concentration-response format. Many of these chemicals produced distinct cellular phenotypes that were qualitatively similar to those previously described in the literature. A novel workflow for phenotypic feature extraction, concentration-response modeling and determination of in vitro thresholds for chemical bioactivity was developed. Subsequently, a set of 462 chemicals from the ToxCast library were screened in concentration-response mode. Bioactivity thresholds were calculated and converted to administered equivalent doses (AEDs) using reverse dosimetry. AEDs were then compared to effect values from mammalian toxicity studies. In many instances (68%), the HTPP-derived AEDs were either more conservative than or comparable to the in vivo effect values. Overall, we conclude that the HTPP assay can be used as an efficient, cost-effective and reproducible screening method for characterizing the biological activity and potency of environmental chemicals for potential use in in vitro-based safety assessments.

中文翻译：

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使用基于成像的高通量表型分析对环境化学品进行生物活性筛选。


本研究采用了现有的基于高内涵成像的高通量表型分析（HTPP）测定（称为“细胞绘画”），​​用于环境化学品的生物活性筛选。该测定使用荧光探针组合来标记各种细胞器，并在单细胞水平上测量大量表型特征，以检测化学诱导的细胞形态变化。首先，以浓度-反应形式在多个时间点鉴定和筛选一小组已知可引起 U-2 OS 细胞形态变化的候选表型参考化学品 (n = 14)。许多这些化学物质产生了独特的细胞表型，这些表型在质量上与先前文献中描述的相似。开发了一种用于表型特征提取、浓度响应建模和化学生物活性体外阈值确定的新工作流程。随后，以浓度响应模式筛选了 ToxCast 库中的一组 462 种化学物质。使用反向剂量测定法计算生物活性阈值并转换为施用当量剂量 (AED)。然后将 AED 与哺乳动物毒性研究的效果值进行比较。在许多情况下 (68%)，HTPP 衍生的 AED 要么比体内效应值更保守，要么与体内效应值相当。总体而言，我们得出的结论是，HTPP 测定可作为一种高效、经济且可重复的筛选方法，用于表征环境化学品的生物活性和效力，以用于体外安全评估。