title: A non-linear detection of phospho-histone H2AX in EA.hy926 endothelial cells following low-dose X-irradiation is modulated by reactive oxygen species.
abstract: BACKGROUND
  A discontinuous dose response relationship is a major characteristic of the anti-inflammatory effects of low-dose X-irradiation therapy. Although recent data indicate an involvement of a variety of molecular mechanisms in these characteristics, the impact of reactive oxygen species (ROS) production to give rise or contribute to these phenomena in endothelial cells (EC) remains elusive.
  
  
  METHODS
  HUVEC derived immortalized EA.hy926 cells were stimulated by tumor necrosis factor-α (TNF-α, 20 ng/ml) 4 h before irradiation with doses ranging from 0.3 to 1 Gy. To analyse DNA repair capacity, phospho-histone H2AX foci were assayed at 1 h, 4 h and 24 h after irradiation. ROS production and superoxide dismutase (SOD) activity were analysed by fluorometric 2',7'-dichlorodihydrofluorescein-diacetate (H2DCFDA) and colorimetric assays. A functional impact of ROS on γH2AX production was analysed by treatment with the scavenger N-acetyl-L-cysteine (NAC).
  
  
  RESULTS
  Irrespective of stimulation by TNF-α, EA.hy926 cells revealed a linear dose response characteristic of γH2AX foci detection at 1 h and 4 h after irradiation. By contrast, we observed a discontinuity in residual γH2AX foci detection at 24 h after irradiation with locally elevated values following a 0.5 Gy exposure that was abolished by inhibition of ROS by NAC. Moreover, SOD protein expression was significantly decreased at doses of 0.5 Gy and 0.7 Gy concomitant with a reduced SOD activity.
  
  
  CONCLUSIONS
  These data implicate a non-linear regulation of ROS production and SOD activity in EA.hy926 EC following irradiation with doses < 1 Gy that may contribute to a discontinuous dose-response relationship of residual γH2AX foci detection.
authors.forename: Martin
authors.initials: M
authors.lastname: Large
authors.forename: Sebastian
authors.initials: S
authors.lastname: Reichert
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hehlgans
authors.forename: Claudia
authors.initials: C
authors.lastname: Fournier
authors.forename: Claus
authors.initials: C
authors.lastname: Rödel
authors.affiliation: Department of Radiotherapy and Oncology, Goethe-University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. franz.roedel@kgu.de.
authors.forename: Franz
authors.initials: F
authors.lastname: Rödel
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002460:Cell Line; D004307:Dose-Response Relationship, Radiation; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D017711:Nonlinear Dynamics; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011829:Radiation Dosage; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology*; D014965:X-Rays
chemical_list: D016166:Free Radical Scavengers; C468783:H2AX protein, human; D006657:Histones; D017382:Reactive Oxygen Species; D000111:Acetylcysteine