Division of Drug Discovery and Safety, Leiden Academic Centre of Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, AIMMS, Vrije Universiteit Amsterdam, De Boelelaan, 1108, 1081 HZ, Amsterdam, The Netherlands.
Arch Toxicol. 2020 Aug;94(8):2707-2729. doi: 10.1007/s00204-020-02792-5. Epub 2020 Jul 18.
Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.
越来越多的证据表明，线粒体功能障碍在多种病理中起着核心作用，包括代谢疾病、加速衰老、神经退行性疾病以及某些外源物诱导的器官毒性。评估线粒体扰动并不简单，此类研究的结果取决于所使用的细胞类型和所采用的测定方法。在这里，我们系统地研究了电子传递链 (ETC) 抑制剂对两种人类细胞类型 HepG2 和 RPTEC/TERT1 中多个与线粒体相关参数的影响。细胞暴露于广泛浓度的 20 种抑制 ETC 的农用化学品和辣椒素中，这些抑制剂包括 NADH 脱氢酶 (复合物 I，CI)、琥珀酸脱氢酶 (复合物 II，CII) 和细胞色素 bc1 复合物 (复合物 III，CIII) 的抑制剂。我们利用了一系列测试，包括活力测定、乳酸生成、线粒体膜电位 (MMP) 和 Seahorse 生物分析器，该分析器可同时测量细胞外酸化率 [ECAR] 和耗氧量 [OCR]。CI 抑制剂可显著降低两种细胞类型的 OCR，降低线粒体膜电位，增加 ECAR 并增加乳酸生成。24 小时暴露于 CI 抑制剂会降低 RPTEC/TERT1 细胞的活力，并降低存在葡萄糖时 3D 球体培养的 HepG2 细胞的活力。CI 抑制剂仅在没有葡萄糖的情况下降低 2D HepG2 的活力。在完整细胞中，CII 抑制剂在高达 10 μM 的浓度下没有明显作用。CIII 抑制剂与 CI 抑制剂具有相似的作用。安密妥 A 是最有效的 CIII 抑制剂，其活性处于纳摩尔范围内。拟议的 CIII 抑制剂 cyazofamid 在两种细胞类型中均表现出线粒体解偶联信号。该研究提供了一个全面的线粒体评估工作流程示例，并确定了 ETC 抑制的可测量关键事件。
