Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals

Wanda van der Stel, Giada Carta, Julie Eakins, Salihanur Darici, Johannes Delp, Anna Forsby, Susanne Hougaard Bennekou, Iain Gardner, Marcel Leist, Erik H J Danen, Paul Walker, Bob van de Water, Paul Jennings*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)2707-2729
Number of pages23
JournalArchives of Toxicology
Issue number8
Early online date18 Jul 2020
Publication statusPublished - Aug 2020


This project was funded by the EU-ToxRisk project, grant agreement No 681002, funded by the European Union’s Horizon 2020 research and innovation programme. We would like to thank all our EUToxRisk colleagues for their fruitful discussions and inputs. We especially want to thank Thomas Cole at the JRC for procuring and distributing the all 21 ETC inhibitors.

FundersFunder number
Horizon 2020 Framework Programme681002


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