title: Trolox-sensitive reactive oxygen species regulate mitochondrial morphology, oxidative phosphorylation and cytosolic calcium handling in healthy cells.
abstract: OBJECTIVE
  Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts.
  
  
  RESULTS
  Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O(2) consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca(2+) transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide.
  
  
  CONCLUSIONS
  Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Felix
authors.initials: F
authors.lastname: Distelmaier
authors.forename: Federica
authors.initials: F
authors.lastname: Valsecchi
authors.forename: Marleen
authors.initials: M
authors.lastname: Forkink
authors.forename: Sjenet
authors.initials: S
authors.lastname: van Emst-de Vries
authors.forename: Herman G
authors.initials: HG
authors.lastname: Swarts
authors.forename: Richard J T
authors.initials: RJ
authors.lastname: Rodenburg
authors.forename: Eugène T P
authors.initials: ET
authors.lastname: Verwiel
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D002839:Chromans; D017382:Reactive Oxygen Species; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D002118:Calcium