BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
BASF Metabolome Solution GmbH, Berlin, Germany.
Arch Toxicol. 2023 Nov;97(11):2903-2917. doi: 10.1007/s00204-023-03572-7. Epub 2023 Sep 4.
Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC-MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.
组学技术已逐渐被认为是下一代风险评估的有前途的工具。靶向代谢组学提供了提供关于受扰生物途径的易于解释的机制信息的优势。在这项研究中，应用了一种基于高通量 LC-MS/MS 的广泛靶向代谢组学系统来研究硝呋太尔随时间和浓度的代谢动态，并提供用于推导起始点 (PoD) 的基于机制的方法。在五个浓度（7.5 µM、15 µM、20 µM、30 µM 和 120 µM）和四个时间点（3、6、24 和 48 h）下暴露于硝呋太尔后，评估了 HepG2 细胞的细胞内代谢组。总共测量、注释和分配了 13 种不同代谢物类别的 256 种独特鉴定的代谢物。主成分分析 (PCA) 和单变量统计分析显示出基于代谢组的时间和浓度的明显影响。机制信息证明了细胞途径的差异激活，表明早期适应性和肝毒性反应。在低浓度下，主要影响能量和脂质代谢，在中浓度范围内，通过增加谷胱甘肽 (GSH) 和从头合成 GSH 途径的代谢物水平来证明抗氧化细胞反应的激活。在最高浓度下，GSH 的耗竭以及反映线粒体损伤的改变表明肝毒性反应。最后，使用整个测量代谢物集通过多元 PCA 衍生了基于代谢组学的 PoD。这种方法允许使用整个数据集并推导可以与已建立的关键事件相关联的 PoD。我们的研究结果表明高通量靶向代谢组学适用于研究肝毒性的机制，并衍生可以与现有的不良结局途径相关联并有助于开发新途径的起始点。
