Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Oxidative DNA damage, chromosomal aberrations and mutations||KeyEvent|
|Ionizing energy leading to lung cancer||KeyEvent|
|Alkylation of DNA leading to reduced sperm count||KeyEvent|
|human and other cells in culture||human and other cells in culture||NCBI|
|All life stages||High|
Key Event Description
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. Th spectrum of damage can be complex, particularily if the stressor is from large amounts of deposited energy which can result in complex lesions and clustered damage defined as two or more oxidzed bases, abasic sites or starnd breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models (Barbieri et al., 2019 and Asaithamby et al., 2011). DSBs and complex lesions are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).
How It Is Measured or Detected
- 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.
Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.
|Assay Name||References||Description||OECD Approved Assay|
|Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)||Collins, 2004; Olive and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017||To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH >13); DNA fragments are forced to move, forming a "comet"-like appearance||Yes (No. 489)|
|Comet Assay (Single Cell Gel Eltrophoresis - Neutral)||Collins, 2014; Olive and Banath, 2006; Anderson and Laubenthal, 2013; Nikolova et al., 2017||To detect DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at a neutral pH; DNA fragments, which are not denatured at the neutral pH, are forced to move, forming a "comet"-like appearance||N/A|
|γ-H2AX Foci Quantification - Flow Cytometry||
Rothkamm and Horn, 2009; Bryce et al., 2016
|Measurement of γ-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX||N/A|
|γ-H2AX Foci Quantification - Western Blot||
Burma et al., 2001; Revet et al., 2011
|Measurement of γ-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX||N/A|
|γ-H2AX Foci Quantification - Microscopy||
Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al., 2013
|Quantification of γ-H2AX immunostaining by counting γ-H2AX foci visualized with a microscope||N/A|
|γ-H2AX Foci Quantification - ELISA||Ji et al., 2017||Measurement of γ-H2AX in cells by ELISA, normalized to total levels of H2AX||N/A|
|Pulsed Field Gel Electrophoresis (PFGE)||
Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et al., 2017
|To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus able to be separated by size||N/A|
|The TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay||Loo, 2011||To detect strand breaks, dUTPs added to the 3’OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization||N/A|
|In Vitro DNA Cleavage Assays using Topoisomerase||Nitiss, 2012||Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis||N/A|
Domain of Applicability
DNA strand breaks can occur in any eukaryotic or prokaryotic cell.
Evidence for Perturbation by Stressor
Ager, D. D. et al. (1990). “Measurement of Radiation- Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis.” Radiat Res. 122(2), 181-7.
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.
Asaithamby, A., B. Hu and D.J. Chen. (2011) Unrepaired clustered DNA lesions induce chromosome breakage in human cells. Proc Natl Acad Sci U S A 108(20): 8293-8298 .
Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019
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Herschleb, J. et al. (2007), “Pulsed-field gel electrophoresis.”, Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94
Iliakis, G. et al. (2015), “Alternative End-Joining Repair Pathways Are the Ultimate Backup for Abrogated Classical Non-Homologous End-Joining and Homologous Recombination Repair: Implications for the Formation of Chromosome Translocations.”, Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2(3): 677-84. doi: 10.1038/nprot.2007.94
Jackson, S. (2002). “Sensing and repairing DNA double-strand breaks.”, Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687.
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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.doi: 10.1007/978-1-60327-409-8_1.
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Nitiss, J. L. et al. (2012), “Topoisomerase assays. ”, Curr Protoc Pharmacol. Chapter 3: Unit 3 3.
OECD. (2014). Test No. 489: “In vivo mammalian alkaline comet assay.” OECD Guideline for the Testing of Chemicals, Section 4 .
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