API

Event: 1635

Key Event Title

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Increase, DNA strand breaks

Short name

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Increase, DNA strand breaks

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Cell term
eukaryotic cell


Organ term

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Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Oxidative DNA damage, chromosomal aberrations and mutations KeyEvent

Stressors

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Taxonomic Applicability

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Term Scientific Term Evidence Link
human and other cells in culture human and other cells in culture NCBI

Life Stages

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Life stage Evidence
All life stages

Sex Applicability

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Term Evidence
Unspecific Not Specified

Key Event Description

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DNA strand breaks can occur on a single strand (SSB) or both strands (double strand breaks; DSB). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002). SSB can turn into DSB if the replication fork stalls at the lesion leading to fork collapse.

Strand breaks are intermediates in various biological events, including DNA repair (e.g., excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells.


How It Is Measured or Detected

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  • Comet Assay (Single cell gel electrophoresis) 
    • There are two variations of the comet assay for measuring DNA strand breaks
    • Alkaline comet assay (pH >13) (Platel et al., 2011; Nikolova et al., 2017)
      • OECD test guideline for in vivo mammalian alkaline comet assay (#489) is available (OECD, 2014)
      • Detects SSB and DSB resulting from direct-acting genotoxicants, alkali labile sites, or strand breaks that are intermediates of DNA excision repair (OECD, 2014)
    • Neutral comet assay (Anderson and Laubenthal, 2013; Nikolova et al., 2017)
      • Electrophoresis is performed in neutral pH and DNA is not denatured – mostly detects DSB
  • γH2AX foci detection (Detects DSB)

Phosphorylation of histone H2AX (γH2AX) at serine 139 is an early response to DSB; it causes chromatin decondensation and plays a critical role in recruiting repair machineries to the site of damage (Rogakou et al., 1998). γH2AX foci can be detected by immunostaining on several platforms:

  • Flow cytometry (Bryce et al., 2016); γH2AX foci counting can be high-throughput and automated using flow cytometry-based immunodetection. 
  • Fluorescent microscopy (Garcia-Canton et al., 2013; Khoury et al., 2013); γH2AX foci can be counted in fluorescent microscope images. Image acquisition and foci count can be automated to increase the assay throughput
    • In-Cell Western technique (Khoury et al., 2013; Khoury et al., 2016) combines the principles of Western blotting (e.g., "blocking" to prevent non-specific antibody binding) and fluorescent microscopy for immunodetection of γH2AX foci.
  • Western blotting (Revet et al., 2011); this method does not provide a quantitative measurement of γH2AX foci and is no longer commonly applied in screening for γH2AX induction.
  • Pulsed field gel electrophoresis (detects DSB) (Kawashima et al., 2017)
    • Cells are embedded and lysed in agarose and fractionated by electrophoresis
    • The length of fragments can be determined by running a DNA ladder in the adjacent lane
  • The TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay
    • Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that adds deoxynucleotides to the 3’OH end of DNA strand breaks without the need for a template strand. The dUTPs incorporated at the sites of strand breaks are tagged with a fluorescent dye or a reporter enzyme to allow visualization (Loo, 2011).
    • We note that this method is typically used to measure apoptosis.

When measuring these events, it is important to distinguish between breaks that may lead to mutation or chromosomal aberrations, and those that are associated with cell death processes.


Domain of Applicability

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DNA strand breaks can occur in any eukaryotic or prokaryotic cell.


Evidence for Perturbation by Stressor



References

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Anderson D, Laubenthal J. (2013). Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa, NJ: Humana Press. p 209-218.

Bryce S, Bernacki D, Bemis J, Dertinger S. (2016). Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ Mol Mutagen 57:171-189.

Garcia-Canton C, Anadon A, Meredith C. (2013). Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test. Mutat Res 757:158-166.

Kawashima Y, Yamaguchi N, Teshima R, Narahara H, Yamaoka Y, Anai H, Nishida Y, Hanada K. (2017). Detection of DNA double-strand breaks by pulsed-field gel electrophoresis. Genes Cells 22:84-93.

Khoury, L., Zalko, D., Audebert, M. (2013), Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells, Environ Mol Mutagen, 54:737-746.

Khoury, L., Zalko, D., Audebert, M. (2016), Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening, Mutagenesis, 31:83-96.

Jackson S. (2002). Sensing and repairing DNA double-strand breaks. Carcinogenesis 23:687-696.

Loo DT. (2011). In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa, NJ: Humana Press. p 3-13.

Nikolova T, Marini F, Kaina B. (2017). Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays. Mutat Res 822:10-18.

OECD. (2014). Test No. 489: In vivo mammalian alkaline comet assay. OECD Guideline for the Testing of Chemicals, Section 4 .

Platel A, Nesslany F, Gervais V, Claude N, Marzin D. (2011). Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels. Mutat Res 726:151-159.

Revet I, Feeney L, Bruguera S, Wilson W, Dong T, Oh D, Dankort D, Cleaver J. (2011). Functional relevance of the histone γH2Ax in the response to DNA damaging agents  . Proc Natl Acad Sci USA 108:8663-8667.

Rogakou, E.P., Pilch, D., Orr, A., Ivanova, V., Bonner, W.M. (1998), DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139, J Biol Chem, 273:5858-5868.