Fournier, C.

Large, M.

Reichert, S.

Rödel, C.

Rödel, F.

GSI Repository

A discontinuous detection of phospho-histone H2AX in endothelial cellsfollowing low-dose irradiation is mediated by reactive oxygen species (ROS)∗S. Reichert1, M. Large1, C. Fournier2, C. Ro¨del1, and F. Ro¨del11Goethe-University Frankfurt am Main, Germany; 2GSI, Darmstadt, GermanyIntroductionSince decades an anti-inflammatory efficacy of radiationtherapy with single doses < 1 Gy is clinically well estab-lished in the treatment of benign and chronic degenerativediseases [1]. In line with that, recent experimental data in-dicate an involvement of a variety of cellular and molecularimmune-modulatory mechanisms in these effects. Amongthem, an impact of low-dose irradiation on endothelial cells(EC) and mononuclear/polymorphnuclear leukocyte activ-ity has been proven to comprise key elements in the mod-ulation of inflammatory cascades [2]. Moreover, a non-linear dose response relationship, shared with non (DNA)-targeted properties of ionizing radiation [3], is a majorcharacteristic of the mechanisms explored so far [4]. Re-cently, a putative interrelationship between DNA damagerepair, induction of reactive oxygen species (ROS) and fac-tors implicated in the regulation of antioxidant responsepathways have been proposed to contribute to these dis-continuous responses [5].Material and MethodsHUVEC derived immortalized EA.hy926 cells werestimulated in a pro-inflammatory manner by tumor necrosisfactor-α (20 ng/ml) 4 hours before irradiation with dosesranging from 0.3 to 1 Gy. To analyse DNA repair capacity,γ-H2AX foci were assayed at 1 hour, 4 hours and 24 hoursafter irradiation by immunofluorescence. ROS productionand superoxide dismutase (SOD) activity were analysedby fluorometric 2’,7’-dichlorodihydrofluorescein-diacetate(H2DCFDA) and colorimetric assays, respectively.Results and ConclusionIrrespective of stimulation by TNF-α, we observed inEA.hy926 EC a linear dose-response characteristic of γ-H2AX foci detection at early times (1 hours and 4 hours)after irradiation. On the contrary, at 24 hours the num-ber of residual γ-H2AX foci was significantly (p ≤ 0.05)elevated after a 0.5 Gy exposure (Figure 1a). To furtheranalyse molecular mechanisms implicated in the biphasicinduction of γ-H2AX foci the impact of ROS expressionand enzymatic activity of SOD, reported to be involved inanti-oxidant defence [6] were investigated. As a result, abiphasic induction of ROS became evident at 24 hours af-ter irradiation concomitant with a significant decrease in∗This research was funded by BMBF (GREWIS, 02NUK017F) andEU (DoReMi, FP7-249689)SOD activity, most pronounced at a dose of 0.5 Gy (Figure1b).Figure 1: Dose kinetics of γ-H2AX foci detection (a) andrelative SOD enzymatic activity (b) as measured by a col-orimetric assay in stimulated (TNF-α, 20 ng/ml) EA.hy926EC at 24 h following irradiation with the doses indicated.Data represent means ± SD from at least three indepen-dent experiments. Asterisks indicate significant differences(p < 0.05) vs. 0.3 Gy and 0.7 Gy irradiated ECs.In conclusion, these data implicate a non-linear regula-tion of SOD activity and ROS production in EA.hy926 ECfollowing irradiation with doses < 1 Gy that contributesto a discontinuous dose response relationship of γ-H2AXdetection.References[1] Seegenschmiedt et al. Int J Radiat Oncol Biol Phys2000;47:195–202.[2] Ro¨del et al. Front Oncol 2012;2:120.[3] Mothersill. Health Phys 2011;100:302.[4] Ro¨del et al. Curr Med Chem 2012;19:1741–1750.[5] Beels et al. Int J Radiat Biol 2010;86:760–768.[6] Fukai. Antioxid Redox Signal 2011;15:1583–1606.GSI SCIENTIFIC REPORT 2013 BIOPHYSICS-05doi:10.15120/GR-2014-1-BIOPHYSICS-05 207

A discontinuous detection of phospho-histone H2AX in endothelial cells following low-dose irradiation is mediated by reactive oxygen species (ROS)

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A discontinuous detection of phospho-histone H2AX in endothelial cells following low-dose irradiation is mediated by reactive oxygen species (ROS)

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