Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitoc
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MOLECULAR TOXICOLOGY
Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals Wanda van der Stel1 · Giada Carta2 · Julie Eakins3 · Salihanur Darici1 · Johannes Delp4 · Anna Forsby5 · Susanne Hougaard Bennekou6 · Iain Gardner7 · Marcel Leist4 · Erik H. J. Danen1 · Paul Walker3 · Bob van de Water1 · Paul Jennings2 Received: 24 February 2020 / Accepted: 20 May 2020 © The Author(s) 2020
Abstract 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. Keywords Mitochondria · Seahorse · ETC · ECAR · MMP · RPTEC/TERT1 · HepG2
Introduction There is accumulating evidence that chemical-induced organ toxicity involves disruption of mitochondrial function more frequently than previously considered (Dykens and Will 2007; Will et al. 2019; Dreier et al. 2019). Mitochondrial perturbations can have major effects on tissues and organs Wanda van der Stel and Giada Carta contributed equally. * Bob van de Water [email protected] * Paul Jennings [email protected] Extended author information available on the last page of the article
due to their key role in fatty acid metabolism, energy production and generatio
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