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      Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

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          Abstract

          The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [ 3H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.

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          Most cited references34

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          Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells.

          Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.
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            The comet assay: topical issues.

            The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.
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              Use of the γ-H2AX assay to monitor DNA damage and repair in translational cancer research.

              Formation of γ-H2AX in response to DNA double stranded breaks (DSBs) provides the basis for a sensitive assay of DNA damage in human biopsies. The review focuses on the application of γ-H2AX-based methods to translational studies to monitor the clinical response to DNA targeted therapies such as some forms of chemotherapy, external beam radiotherapy, radionuclide therapy or combinations thereof. The escalating attention on radiation biodosimetry has also highlighted the potential of the assay including renewed efforts to assess the radiosensitivity of prospective radiotherapy patients. Finally the γ-H2AX response has been suggested as a basis for an in vivo imaging modality. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 July 2015
                2015
                : 10
                : 7
                : e0132702
                Affiliations
                [1 ]Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
                [2 ]Department of Biomedical Magnetic Resonance, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
                [3 ]Department of Radiology and Nuclear Medicine, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
                [4 ]Medipan GmbH, Dahlewitz/Berlin, Germany
                [5 ]Faculty of Natural Sciences, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany
                [6 ]Leibniz Institute for Neurobiology, Magdeburg, Germany
                [7 ]Center for Behavioral Brain Sciences, Magdeburg, Germany
                [8 ]German Center for Neurodegenerative Disease, Magdeburg, Germany
                National Research Council, ITALY
                Author notes

                Competing Interests: The corresponding authors has read the journal's policy and the authors of this manuscript have the following competing interests: Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH and Medipan GmbH, and is employed by Medipan GmbH. Both companies are diagnostic manufacturers. The remaining authors declare no conflict of interest. The Dept. of Biomedical Magnetic Resonance receives research support from Siemens Healthcare. This support, however, is not related to the subject of the current study. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AR MF BF JR D. Reinhold OS. Performed the experiments: AR MF BF KG RH FG. Analyzed the data: AR D. Roggenbuck D. Reinhold. Contributed reagents/materials/analysis tools: JR D. Roggenbuck D. Reinhold OS. Wrote the paper: AR MF BF.

                ‡ These authors also contributed equally to this work and shared senior authorship.

                Article
                PONE-D-15-12566
                10.1371/journal.pone.0132702
                4503586
                26176601
                e696cc8c-3efc-409b-9f62-216a71061a1d
                Copyright @ 2015

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 23 March 2015
                : 17 June 2015
                Page count
                Figures: 4, Tables: 0, Pages: 14
                Funding
                Mahsa Fatahi was supported by the HiMR (High Field Magnetic Resonance), an initial training network, funded by the FP7 Marie Curie Actions of the European Commission (FP7-PEOPLE-2012-ITN-316716). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. GA Generic Assays GmbH and Medipan GmbH provided support in the form of salaries for author DR, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.
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                All relevant data are within the paper and its Supporting Information files.

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