An Easy-to-use Genotoxicity Assay Using EGFP-MDC1-expressing Human Cells

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Author(s)

Abstract

Histone H2AX phosphorylated at Ser139 (γ-H2AX) is a useful biomarker for DNA double-strand breaks. However, γ-H2AX detection has methodological disadvantages such as the requirement of expensive anti-γ-H2AX antibody and time-consuming handling for its staining. Mediator of DNA damage checkpoint 1 (MDC1) is a central adaptor protein which recruits various DNA damage response proteins to γ-H2AX and thus forms nuclear foci in the same location as γ-H2AX in response to DNA damage. Here, we describe an easy-to-use genotoxicity assay which combines enhanced green fluorescence protein (EGFP)-fused MDC1-expressing cells with a free <i>R</i> program for image-processing and quantification of foci area/nucleus. The workflow of this assay is simple: mutagen treatment, imaging, and <i>R</i>-processing. This assay does not need antibodies or staining handling and it detected the genotoxicity of a range of mutagens, including camptothecin (topoisomerase I inhibitior), cisplatin (crosslinker), and 4-nitroquinoline 1-oxide and benzo[<i>a</i>]pyrene (bulky DNA-adduct forming compounds), as increased fluorescence of EGFP-MDC1 foci. Furthermore, cotreatment with arabinofuranosyl cytosine/hydroxyurea and mutagens sensitized EGFP-MDC1 foci formation to bulky DNA adduct-type mutagens. Additionally, the established cells can be monitored in real-time using live cell imaging to obtain detailed dynamics of MDC1 in response to mutagens. The simple handling of this assay is expected to enable its full automation, thus making it useful for high-throughput genotoxicity screening of chemicals and monitoring of environmental mutagens.<br>

Journal

  • Genes and Environment

    Genes and Environment 36(1), 17-28, 2014

    The Japanese Environmental Mutagen Society

Codes

  • NII Article ID (NAID)
    130004481211
  • NII NACSIS-CAT ID (NCID)
    AA1212552X
  • Text Lang
    ENG
  • ISSN
    1880-7046
  • NDL Article ID
    025316290
  • NDL Call No.
    Z78-A397
  • Data Source
    NDL  J-STAGE 
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