Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells

Redox Biol. 2015 Dec:6:607-616. doi: 10.1016/j.redox.2015.09.003. Epub 2015 Oct 23.


Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability. Integrated analysis of cells stably expressing cytosolic- or mitochondria-targeted variants of the reporter molecules HyPer (H2O2-sensitive and pH-sensitive) and SypHer (H2O2-insensitive and pH-sensitive), revealed that CI- and CIII inhibition increased cytosolic but not mitochondrial H2O2 levels. Total and mitochondria-specific lipid peroxidation was not increased in the inhibited cells as reported by the C11-BODIPY(581/591) and MitoPerOx biosensors. Also expression of the superoxide-detoxifying enzymes CuZnSOD (cytosolic) and MnSOD (mitochondrial) was not affected. Oxyblot analysis revealed that protein carbonylation was not stimulated by CI and CIII inhibition. Our findings suggest that chronic inhibition of CI and CIII: (i) increases the levels of HEt-oxidizing ROS and (ii) specifically elevates cytosolic but not mitochondrial H2O2 levels, (iii) does not induce oxidative stress or substantial cell death. We conclude that the increased ROS levels are below the stress-inducing level and might play a role in redox signaling.

Keywords: HyPer; Hydroethidine; Oxyblot; SypHer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimycin A / pharmacology
  • Cell Survival
  • Cytosol / metabolism
  • Electron Transport Complex I / antagonists & inhibitors*
  • Electron Transport Complex I / metabolism
  • Electron Transport Complex II / antagonists & inhibitors*
  • Electron Transport Complex II / metabolism
  • HEK293 Cells
  • Humans
  • Hydrogen Peroxide / metabolism*
  • Lipid Peroxidation
  • NADP / metabolism
  • Oxidation-Reduction
  • Oxidative Stress*
  • Phenanthridines / metabolism
  • Protein Carbonylation
  • Rotenone / pharmacology
  • Superoxide Dismutase / metabolism


  • Phenanthridines
  • Rotenone
  • hydroethidine
  • NADP
  • Antimycin A
  • Hydrogen Peroxide
  • Superoxide Dismutase
  • Electron Transport Complex II
  • Electron Transport Complex I