In vitro screening of cell bioenergetics to assess mitochondrial dysfunction in drug development

Tilmant, Karen; Gerets, Helga; Ron, Pierrette De; Hanon, Étienne; Bento‐Pereira, Claudia; Atienzar, Franck

doi:10.1016/j.tiv.2018.07.012

Cited by 11 publications

(13 citation statements)

References 71 publications

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“…Altered glycolytic status can be quantified by assessing extracellular acidification rates (ECAR), or biochemically by measuring supernatant lactate (Limonciel et al 2011 ). The Seahorse bioanalyser coupled with the mitostress assay is becoming an industry standard for the quantification of OCR and ECAR (Divakaruni et al 2014 ; Eakins et al 2016 ; Tilmant et al 2018 ). Other methods to assess mitochondrial function include live cell dyes, which under optimised conditions can be related to MMP or comparing toxicity in the presence and absence of glucose as an energy source.…”

Section: Discussionmentioning

confidence: 99%

“…The mitostress test was performed as described (Eakins et al 2016 ; Tilmant et al 2018 ). Immediately before the assay, cell culture medium was replaced with 180 µL of Seahorse XF Base Medium without phenol red (Agilent, 1003335-100), supplemented with 10 mM d -glucose (Merck, G7021), 5 mM HEPES (Merck, H4034), 2 mM sodium pyruvate (Merck, P5281) and 1 mM l -glutamine (Merck, G8540).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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“…Considering the difficulties of detecting mitochondrial toxicity (mitotoxicity) in rodents, it is not surprising that many drugs that had to be withdrawn from the market (e.g., troglitazone and cerivastatin), had later been linked to mitotoxicity (Blomme and Will 2016;Tirmenstein et al 2002;Westwood et al 2005). Indeed, a sizable fraction of compounds causing, e.g., drug-induced liver injury are mitotoxicants (Aleo et al 2014;Begriche et al 2011;Pessayre et al 2012;Rana et al 2018;Tilmant et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

et al. 2019

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Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito-and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/ complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants. Keywords Neurotoxicity • Mitotoxicity • Metabolic reprogramming • High-throughput toxicity screening • High content imaging • Mechanistic safety assessment Abbreviations ADP Adenosine triphosphate AOP Adverse outcome pathway Asp l-Aspartate ATP Adenosine diphosphate cAMP N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate cI-V MRC complex I-V CNS Central nervous system Cyt c Cytochrome c FAD(H 2) Flavin adenine dinucleotide (FAD: oxidized, FADH2: reduced) FCCP Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone G6P Glucose-6-phosphate Gal1P Galactose-1-phosphate GALK Galactokinase GALT Galactose-1-phosphate uridylyltransferase GDNF Glial derived neurotrophic factor Glc1P Glucose-1-phosphate Hex Hexose, in this study either glucose or galactose HK Hexokinase Lac Lactate Mal Malate MoA Mode of action MPP 1-Methyl-4-phenylpyridinium Electronic supplementary material The online version of this article (

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“…Drugs targeting the mitochondrial respiratory chain (MRC) are relatively well tolerated in normal tissues (Blecha et al 2017). Many in vitro studies may also be insensitive to mitochondrial toxicants as cells are kept in supra-physiological (e.g., 3-5 times higher than normal plasma levels) glucose concentrations (Blomme and Will 2016;Broom et al 2016;Perron et al 2013;Tilmant et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

et al. 2021

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Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10–260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

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In vitro screening of cell bioenergetics to assess mitochondrial dysfunction in drug development

Cited by 11 publications

References 71 publications

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

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

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors

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