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. 2022 Jun 1;444:116032. doi: 10.1016/j.taap.2022.116032. Epub 2022 Apr 26.
The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.
美国环境保护署（EPA）提出了一种基于新方法（NAM）的化学危害评估分层测试策略。第一层次包括适用于许多（人类）细胞类型的体外分析方法，如高通量转录组学（HTTr）和高通量表型分析（HTPP）。本研究的目的是：（1）协调 U-2 OS 人骨肉瘤细胞在两种测定中的接种密度；（2）比较 11 种具有不同作用机制的化学物质的 HTTr-和 HTPP 衍生的效力估计值；（3）确定候选参考化学物质，用于监测未来筛选中的测定性能；（4）详细描述全反式视黄酸（ATRA）的转录和表型变化，作为一种已知对成骨细胞分化有不良影响的模型化合物。该评估的结果表明：（1）HTPP 在低（400 个细胞/孔）和高（3000 个细胞/孔）接种密度下进行时，可获得可比的效力估计值，并且对测试化学物质的表型图谱相似；（2）HTPP 和 HTTr 分别导致细胞形态和基因表达变化的可比效力估计值；（3）三种测试化学物质（依托泊苷、ATRA、地塞米松）对细胞形态和基因表达产生浓度依赖性影响，与已知的作用模式一致，表明它们适合用作监测测定性能的参考化学物质；（4）ATRA 产生的表型变化与其他视黄酸受体激动剂（AM580、阿罗汀酸）和一些视黄醇 X 受体激动剂（贝沙罗汀、美福酸）高度相似。这种表型与 RARB 基因的自身调节同时出现。两种作用均通过用各自受体的药理学拮抗剂预处理 U-2 OS 细胞而被阻止。因此，观察到的表型可被认为是 U-2 OS 细胞中视黄酸途径激活的特征。这些发现为使用 HTTr 和 HTPP 对化学物质进行组合筛选奠定了基础，为基于 NAM 的化学危害评估策略的第一层次生成互补信息。
