Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation
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Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays
Published on: March 7, 2025
Wanda van der Stel1, Huan Yang1, Nanette G Vrijenhoek1
1Division of Drug Discovery and Safety, Leiden Academic Centre of Drug Research, Leiden University, Leiden, The Netherlands.
Abstract:
Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.
Insights
Chemicals inhibiting electron transport chain (ETC) complexes I and III disrupt mitochondrial function and cell viability. A specific gene set aids adaptation, while amino acid response pathways are activated during mitochondrial stress.
Area of Science:
- Toxicology
- Cellular Biology
- Mitochondrial Research
Background:
- Mitochondrial dysfunction is central to chemical-induced organ toxicities.
- The precise mechanisms of electron transport chain (ETC) inhibitor toxicity are not fully understood.
- Understanding cellular responses to ETC inhibition is crucial for predicting and mitigating chemical toxicity.
Purpose of the Study:
- To investigate the impact of ETC complex I, II, and III (CI, CII, CIII) inhibitors on mitochondrial function, cellular stress responses, and viability in HepG2 cells.
- To identify specific gene expression patterns and cellular pathways involved in the response to ETC inhibition.
- To explore the role of identified genes and stress responses in hepatocyte toxicity.
Main Methods:
- Utilized high-content imaging and TempO-Seq for comprehensive analysis of HepG2 cells.
- Assessed mitochondrial membrane potential (MMP), ATP levels, and cell viability.
- Performed targeted TempO-Seq for mRNA expression profiling and identified responsive gene sets.
- Employed GFP reporter panels to monitor dynamic stress pathway activation.
- Conducted pathway and gene network analysis, including in silico validation in primary human hepatocytes.
Main Results:
- CI and CIII inhibitors, but not CII inhibitors, impaired MMP and ATP levels in a dose- and time-dependent manner.
- Inhibition of glycolysis exacerbated CI/CIII inhibitor-induced cell death.
- A specific gene set responsive to ETC inhibition was identified, with NOS3 silencing worsening toxicity.
- Amino acid response (AAR) and endoplasmic reticulum (ER) stress were identified as downstream events of ETC inhibition.
- Similar AAR was observed in primary human hepatocytes exposed to mitochondrial toxicants.
Conclusions:
- CI and CIII inhibitors induce significant mitochondrial perturbation and cellular stress, impacting cell viability.
- A distinct set of genes is involved in cellular adaptation to ETC inhibition.
- ER stress and AAR are key accompanying responses to ETC inhibition in hepatocytes, relevant to chemical toxicity.
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