Study of the anti-inflammatory effects of low-dose radiation: The contribution of biphasic regulation of the antioxidative system in endothelial cells.

BACKGROUND: We examined (a) the expression of the antioxidative factor glutathione peroxidase (GPx) and the transcription factor nuclear factor E2-related factor 2 (Nrf2) following low-dose X-irradiation in endothelial cells (ECs) and (b) the impact of reactive oxygen species (ROS) and Nrf2 on functional properties of ECs to gain further knowledge about the anti-inflammatory mode of action of low doses of ionizing radiation. MATERIAL AND METHODS: EA.hy926 ECs and primary human dermal microvascular ECs (HMVEC) were stimulated by tumor necrosis factor-α (TNF-α, 20 ng/ml) 4 h before irradiation with single doses ranging from 0.3 to 3 Gy. The expression and activity of GPx and Nrf2 were analyzed by flow cytometry, colorimetric assays, and real-time PCR. The impact of ROS and Nrf2 on peripheral blood mononuclear cell (PBMC) adhesion was assayed in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and Nrf2 activator AI-1. RESULTS: Following a low-dose exposure, we observed in EA.hy926 EC and HMVECs a discontinuous expression and enzymatic activity of GPx concomitant with a lowered expression and DNA binding activity of Nrf2 that was most pronounced at a dose of 0.5 Gy. Scavenging of ROS by NAC and activation of Nrf2 by AI-1 significantly diminished a lowered adhesion of PBMC to EC at a dose of 0.5 Gy. CONCLUSION: Low-dose irradiation resulted in a nonlinear expression and activity of major compounds of the antioxidative system that might contribute to anti-inflammatory effects in stimulated ECs.

A non-linear detection of phospho-histone H2AX in EA.hy926 endothelial cells following low-dose X-irradiation is modulated by reactive oxygen species.

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. MATERIAL AND 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. CONCLUSION: 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.