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.

线粒体功能障碍在包括代谢性疾病，加速衰老，神经退行性疾病以及某些异种生物诱导的器官毒性在内的几种病理中起着核心作用的证据越来越多。评估线粒体扰动并非无关紧要，此类研究的结果取决于所用细胞类型和所用测定法。在这里，我们系统地研究了两种人类细胞类型HepG2和RPTEC / TERT1中电子传输链（ETC）抑制剂对多个线粒体相关参数的影响。将细胞暴露于浓度范围广泛的20种抑制ETC的农药和辣椒素中，辣椒素由NADH脱氢酶（化合物I，CI），琥珀酸脱氢酶（化合物II，CII）和细胞色素bc1复合物（化合物III，CIII）的抑制剂组成。使用了一系列测试，包括活力测定，乳酸生成，线粒体膜电位（MMP）和海马生物分析仪，它们同时可测量细胞外酸化率[ECAR]和耗氧率[OCR]。在两种细胞类型中，CI抑制剂均导致OCR的有效降低，线粒体膜电位的降低，ECAR的增加和乳酸生成的增加。在葡萄糖存在下二十四小时暴露于CI抑制剂会降低RPTEC / TERT1细胞和3D球形培养的HepG2细胞的活力。CI抑制剂仅在不存在葡萄糖的情况下才降低2D HepG2的活力。CII抑制剂对高达10 µM的完整细胞没有显着影响。CIII抑制剂的作用与CI抑制剂相似。抗霉素A是最有效的CIII抑制剂，其活性在纳摩尔范围内。拟议的CIII抑制剂cyazofamid在两种细胞类型中均显示线粒体解偶联信号。该研究提供了线粒体评估工作流程的综合示例，并建立了可测量的ETC抑制关键事件。