Small Molecule Discovery Center and Department of Pharmaceutical Chemistry, University of California, San Francisco, California.
Current Address: National Center for Advancing Translational Sciences, Rockville, Maryland.
Curr Protoc. 2021 Mar;1(3):e75. doi: 10.1002/cpz1.75.
The liver is the primary organ responsible for drug detoxification. Drug-induced liver injury (DILI) is a leading cause of attrition during drug development and is one of the main reasons that drugs are withdrawn from the market. Hence, the prevention of DILI plays a central role in the overall drug-discovery process. Most of the liver's energy supply comes in the form of adenosine triphosphate (ATP), which is largely generated by mitochondria. This article describes the evaluation of drug-induced mitochondrial dysfunction using the Seahorse Extracellular Flux Analyzer (Agilent). The described protocols detail the accurate measurement of ATP production rate in HepG2 cells after exposure to a panel of potentially toxic compounds. This assay measures changes in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indicators of glycolysis and mitochondrial respiration-the two major energy-generating pathways in a cell. This assay provides a useful model to predict mitochondrial dysfunction-mediated DILI. © 2021 Wiley Periodicals LLC. Basic Protocol: Measurement of cellular ECAR, OCR, and ATP production in live HepG2 cells Support Protocol 1: Culturing and maintaining of HepG2 cells Support Protocol 2: Determining optimal cell density per well.
肝脏是主要的解毒器官。药物性肝损伤（DILI）是药物开发过程中淘汰的主要原因之一，也是药物退出市场的主要原因之一。因此，预防 DILI 在整体药物发现过程中起着核心作用。肝脏的大部分能量供应来自三磷酸腺苷（ATP），而 ATP 主要由线粒体生成。本文描述了使用 Seahorse 细胞外通量分析仪（Agilent）评估药物诱导的线粒体功能障碍。所描述的方案详细说明了在 HepG2 细胞中暴露于一组潜在有毒化合物后，准确测量 ATP 产生速率的方法。该测定法可测量细胞外酸化率 (ECAR) 和耗氧量 (OCR) 的变化，作为糖酵解和线粒体呼吸的指标-这是细胞中两种主要的能量产生途径。该测定法提供了一种有用的模型，可预测线粒体功能障碍介导的 DILI。© 2021 Wiley Periodicals LLC. 基本方案：活 HepG2 细胞中细胞 ECAR、OCR 和 ATP 产生的测量 支持方案 1：HepG2 细胞的培养和维持 支持方案 2：确定每孔的最佳细胞密度。
