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.

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.

The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

The utilisation of genome-wide transcriptomics has played a pivotal role in advancing the field of toxicology, allowing the mapping of transcriptional signatures to chemical exposures. These activities have uncovered several transcriptionally regulated pathways that can be utilised for assessing the perturbation impact of a chemical and also the identification of toxic mode of action. However, current transcriptomic platforms are not very amenable to high-throughput workflows due to, high cost, complexities in sample preparation and relatively complex bioinformatic analysis. Thus, transcriptomic investigations are usually limited in dose and time dimensions and are, therefore, not optimal for implementation in risk assessment workflows. In this study, we investigated a new cost-effective, transcriptomic assay, TempO-Seq, which alleviates the aforementioned limitations. This technique was evaluated in a 6-compound screen, utilising differentiated kidney (RPTEC/TERT1) and liver (HepaRG) cells and compared to non-transcriptomic label-free sensitive endpoints of chemical-induced disturbances, namely phase contrast morphology, xCELLigence and glycolysis. Non-proliferating cell monolayers were exposed to six sub-lethal concentrations of each compound for 24 h. The results show that utilising a 2839 gene panel, it is possible to discriminate basal tissue-specific signatures, generate dose–response relationships and to discriminate compound-specific and cell type-specific responses. This study also reiterates previous findings that chemical-induced transcriptomic alterations occur prior to cytotoxicity and that transcriptomics provides in depth mechanistic information of the effects of chemicals on cellular transcriptional responses. TempO-Seq is a robust transcriptomic platform that is well suited for in vitro toxicity experiments.

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.