Abstract
Currently, in the context of radiology, irradiation-induced and other genotoxic effects are determined by visualizing DSB-induced DNA repair through γ-H2AX immunofluorescence and direct counting of the foci by epifluorescence microscopy. This procedure, however, neglects the 3D nature of the nucleus. The aim of our study was to use confocal microscopy and 3D reconstructed images to improve documentation and analysis of γ-H2AX fluorescence signals after diagnostic examinations. Confluent, non-dividing MRC-5 lung fibroblasts were irradiated in vitro with a Cs-137 source and exposed to radiation doses up to 1000 mGy before fixation and staining with an antibody recognizing the phosphorylated histone variant γ-H2AX. The 3D distribution of γ-H2AX foci was visualized using confocal laser scanning microscopy. 3D reconstruction of the optical slices and γ-H2AX foci counting were performed using Imaris Image Analysis software. In parallel, γ-H2AX foci were counted visually by epifluorescence microscopy. In addition, whole blood was exposed ex vivo to the radiation doses from 200 to 1600 mGy. White blood cells (WBCs) were isolated and stained for γ-H2AX. In fibroblasts, epifluorescence microscopy alone visualized the entirety of fluorescence signals as integral, without correct demarcation of single foci, and at 1000 mGy yielded on average 11.1 foci by manual counting of 2D images in comparison to 36.1 foci with confocal microscopy and 3D reconstruction (p < 0.001). The procedure can also be applied for studies on WBCs. In contrast to epifluorescence microscopy, confocal microscopy and 3D reconstruction enables an improved identification of DSB-induced γ-H2AX foci, allowing for an unbiased, ameliorated quantification.
Highlights
Maintaining the genome integrity and stability is one central function of all cells
We investigated the induction of double strand breaks (DSBs) in confluent cultures of primary MRC-5 fibroblasts exposed to increasing radiation doses
Genotoxicity is a wide field ranging from desired DNA DSB in malignant cells to kill them to unwanted effects of chemical compounds including drugs and physical influences such as irradiation and heat for example
Summary
In higher organisms, mutations on the basis of DNA double strand breaks (DSBs) may be harmful, because they could be the origin of cancer (Berrington de Gonzalez and Darby 2004; Lobrich and Jeggo 2007). DNA damage includes a wide range of DNA base modifications, single-strand breaks (SSBs) and DSBs, which are a threat to genome stability (Tounekti et al 2001). There are several cellular repair mechanisms operating (Symington and Gautier 2011). If DSBs occur due to exogenous agents, such as ionizing radiation or during programmed recombination events, one of the first observed cellular responses is the rapid phosphorylation of the histone protein H2AX at serine 139 (Burma et al 2001; Rogakou et al 1998; Chen et al 2000). Phosphorylation of H2AX involves the phosphatidylinositol 3-kinase family consisting of ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase, ataxia telangiectasia and
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