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Event: 1635

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Increase, DNA strand breaks

Short name

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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
DNA Strand Break	Deoxyribonucleic acid	increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Oxidative DNA damage, chromosomal aberrations and mutations	KeyEvent	Carole Yauk (send email)	Open for comment. Do not cite	WPHA/WNT Endorsed
Deposition of energy leading to lung cancer	KeyEvent	Vinita Chauhan (send email)	Open for citation & comment	WPHA/WNT Endorsed
Alkylation of DNA leading to reduced sperm count	KeyEvent	Carole Yauk (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to population decline via DSB and follicular atresia	KeyEvent	Knut Erik Tollefsen (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to population decline via DSB and apoptosis	KeyEvent	Knut Erik Tollefsen (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to cataracts	KeyEvent	Vinita Chauhan (send email)	Open for citation & comment
Deposition of Energy Leading to Learning and Memory Impairment	KeyEvent	Vinita Chauhan (send email)	Open for citation & comment
Deposition of energy leads to vascular remodeling	KeyEvent	Vinita Chauhan (send email)	Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

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

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Life stage	Evidence
All life stages	High

Sex Applicability

An indication of the the relevant sex for this KE. More help

Term	Evidence
Unspecific	High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

DNA strand breaks are a type of damage resulting from the hydrolysis of phosphodiester groups in the backbone of DNA molecules (Gates, 2009) and can occur on a single strand (single strand breaks; SSBs) or both strands (double strand breaks; DSBs). SSBs arise when the sugar phosphate backbones connecting adjacent nucleotides in DNA are simultaneously hydrolyzed such that the hydrogen bonds between complementary bases are not able to hold the two strands together. 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), as well as other normal cellular processes where DSBs act as genetic shufflers to generate genetic diversity for V(D)J recombination in lymphoid cells, and chromatin remodeling in both somatic cells and germ cells, and meiotic recombination in gametes. 

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. The 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 oxidized 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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.

Method of Measurement	References	Description	OECD Approved Method?
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
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	No
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	No
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	No
γ-H2AX Foci Quantification - ELISA	Ji et al., 2017	Measurement of γ-H2AX in cells by ELISA, normalized to total levels of H2AX	No
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	No
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	No
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	No
PCR assay	Figueroa‑González & Pérez‑Plasencia, 2017	Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification	No
Sucrose density gradient centrifuge	Raschke et al. 2009	Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion	No
Alkaline Elution Assay	Kohn, 1991	Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA	No
Unwinding Assay	Nacci et al. 1992	DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding	Yes
STRIDE assay	Zilio and Ulrich, 2021	STRIDE (SensiTive Recognition of Individual DNA Ends) combines in situ nick translation with the proximity ligation assay (PLA) to detect single-strand breaks (sSTRIDE) or double-strand breaks (dSTRIDE). In this process, lesions labeled through nick translation with biotinylated nucleotides are identified by a PLA signal, which arises from the interaction of two anti-biotin antibodies from different species.	No
sBLISS	Bouwmann et al. 2020	sBLISS (in-suspension breaks labeling in situ and sequencing) labels double-strand breaks (DSBs) in cells immobilized on glass coverslips, using double-stranded oligonucleotide adaptors that facilitate selective linear amplification through T7-mediated in vitro transcription (IVT), followed by next-generation sequencing (NGS) library preparation	No

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Taxonomic applicability: DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan & Pederson, 2016).

Life stage applicability: This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White & Vijg, 2016).

Sex applicability: This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012).

Evidence for perturbation by a stressor: There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing & non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan & Pederson, 2016; Yang et al., 1998).

References

List of the literature that was cited for this KE description. More help

Ager, D. D., et al. (1990). Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis. Radiation research, 122/(2), 181–187.

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

Bouwman, B. et al. (2020), “Genome-wide detection of DNA double-strand breaks by in-suspension BLISS”, Nature protocols,.15/12, Springer Nature, London, https://doi.org/10.1038/s41596-020-0397-2

Bryce, S. et al. (2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.”, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996.

Burma, S. et al. (2001), “ATM phosphorylates histone H2AX in response to DNA double-strand breaks.”, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200

Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231(/1), Wiley, New York, https://doi.org/10.1002/jcp.25048.

Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.(94/1), Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814.

Charlton, E. D. et al. (1989), “Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.”, Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141.

Collins, R. A. (2004), “The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.”, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249

EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California.

Figueroa‑González, G. and C. Pérez‑Plasencia. (2017), “Strategies for the evaluation of DNA damage and repair mechanisms in cancer”, Oncology Letters, Vol.133(/6), Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002.

Garcia-Canton, C. et al. (2013), “Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.”, Mutat Res. 757:158-166. Doi: 10.1016/j.mrgentox.2013.08.002

Gardiner, K. et al. (1986), “Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.”, Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665.

Garm, C. et al. (2012), “Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells”, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019.

Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Indianapolis, https://doi.org/10.1667/RR13505.1.

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.

Ji, J. et al. (2017), “Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.”, PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582

Kawashima, Y.(2017), “Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.”, Genes Cells 22:84-93. Doi: 10.1111/gtc.12457.

Khoury, L. et al. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.”, Environ Mol Mutagen, 54:737-746. Doi: 10.1002/em.21817.

Khoury, L. et al. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.”, Mutagenesis, 31:83-96. Doi: 10.1093/mutage/gev058.

Kohn, K.W. (1991), “Principles and practice of DNA filter elution”, Pharmacology & Therapeutics, Vol.49(/1), Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E.

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.

Mah, L. J. et al. (2010), “Quantification of gammaH2AX foci in response to ionising radiation.”, J Vis Exp(38). doi:10.3791/1957.

Nacci, D. et al. (1992), “Application of the DNA alkaline unwinding assay to detect DNA strand breaks in marine bivalves”, Marine Environmental Research, Vol.33(/2), Elsevier BV, Amsterdam, https://doi.org/10.1016/0141-1136(92)90134-8.

Nikolova, T., F. et al. (2017), “Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.”, Mutat Res 822:10-18. Doi: 10.1016/j.mrgentox.2017.07.004.

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 .

Olive, P. L., & Banáth, J. P. (2006), “The comet assay: a method to measure DNA damage in individual cells.”, Nature Protocols. 1(1): 23-29. doi:10.1038/nprot.2006.5.

Platel A. et al. (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. Doi: 10.1016/j.mrgentox.2011.09.003.

Raschke, S., J. Guan and G. Iliakis. (2009), “Application of alkaline sucrose gradient centrifugation in the analysis of DNA replication after DNA damage”, Methods in Molecular Biology, Vol.521, Humana Press, Totowa, https://doi.org/10.1007/978-1-60327-815-7_18.

Redon, C. et al. (2010), “The use of gamma-H2AX as a biodosimeter for total-body radiation exposure in non-human primates.”, PLoS One. 5(11): e15544. doi:10.1371/journal.pone.0015544

Revet, I. et al. (2011), “Functional relevance of the histone γH2Ax in the response to DNA damaging agents.” Proc Natl Acad Sci USA.108:8663-8667. Doi: 10.1073/pnas.1105866108

Rogakou, E.P. et al. (1998), “DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139.” , J Biol Chem, 273:5858-5868. Doi: 10.1074/jbc.273.10.5858

Rothkamm, K. & Horn, S. (2009), “γ-H2AX as protein biomarker for radiation exposure.”, Ann Ist Super Sanità, 45(3): 265-71.

White, R.R. and J. Vijg. (2016), “Do DNA Double-Strand Breaks Drive Aging?”, Molecular Cell, Vol.63, Elsevier, Amsterdam, http://doi.org/10.1016/j.molcel.2016.08.004.

Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67(/6), Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560. 

Zilio, N. and H. D. Ulrich (2021), “Exploring the SSBreakome: genome-wide mapping of DNA single-strand breaks by next-generation sequencing”, The FEBS journal, 288(13), Wiley, Hoboken, https://doi.org/10.1111/febs.15568