Abstract
Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.
Highlights
ΓH2AX is a recognized marker for DNA double-strand breaks (DSBs) in radiation biology [1]
We found that plasma but not UVinduced γH2AX induction was dependent on apoptosis and caspase activation, making DNA damage marked via γH2AX rather a consequence than the cause for plasma-induced cell death
While catalase and glutathione (GSH) conferred protection, superoxide dismutase (SOD) did not. To confirm that this finding was related to protection from reactive oxygen and nitrogen species (ROS), cells were stained with CM-H2DCFDA, which after intracellular modifications fluoresces upon oxidation with, e.g., plasma treatment (Figure 1(g))
Summary
ΓH2AX is a recognized marker for DNA double-strand breaks (DSBs) in radiation biology [1]. Phosphorylation at serine139 of the histone 2AX occurs rapidly, and approximately 1% of all H2AX proteins are phosphorylated per gray irradiation via a molecular machinery [2]. Based on these findings in radiobiology, γH2AX has been used as direct surrogate and correlate of DNA DSBs in a variety of studies testing chemical and physical treatments, for example, in the field of oncology [3]. One novel physical treatment modality for the treatment of cancer is cold physical plasma [4]. Medical plasmas are multicomponent systems consisting of, e.g., electrons and ions, electric fields, and a multiplicity of different reactive oxygen and nitrogen species (ROS) [9]
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