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

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

doi:10.1007/s00204-020-02792-5

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.

Highlights

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 and metabolic parameters were measured upon exposure to a broad concentration range of in total 21 mitochondrial electron transport chain (ETC) CI, CII and CIII inhibitors
Cell viability decreased in a concentration-dependent manner upon exposure to 15 out of 21 complex inhibitors in the RPTEC/ TERT1 cell line, whereas only rotenone mildly affected the viability of HepG2 cells (Fig. 2)

Summary

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. There are several mechanisms of direct mitochondrial perturbation including electron transport chain (ETC) inhibition, mitochondrial DNA damage, ROS, cardiolipin binding, Krebs cycle inhibition, disturbances of fatty acid shuttling, beta oxidation inhibition and protonphoretic (uncoupling) activity (Boelsterli 2003). The subsequent dysfunction of these organelles can have several adverse effects, which is both dependent on the target tissue’s reliance on mitochondrial function and the type of mitochondrial perturbation. Several drugs have been withdrawn from the market due to organ toxicity, which has subsequently been proven or has strong evidence supporting a

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