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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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Explore in a Third Party Tool

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help

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

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	Brendan Ferreri-Hanberry (send email)	Open for comment. Do not cite	EAGMST Approved
Deposition of energy leading to lung cancer	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite	EAGMST Approved
Alkylation of DNA leading to reduced sperm count	KeyEvent	Brendan Ferreri-Hanberry (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to population decline via DSB and follicular atresia	KeyEvent	Evgeniia Kazymova (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to population decline via DSB and apoptosis	KeyEvent	Agnes Aggy (send email)	Under development: Not open for comment. Do not cite

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 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

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.

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 Detection - ELISA and flow cytometry	Ji et al., 2017; Bryce et al., 2016	Detection of γ-H2AX in cells by ELISA, normalized to total levels of H2AX; γH2AX foci detection can be high-throughput and automated using flow cytometry-based immunodetection.	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 (We note that this method is typically used to measure apoptosis)	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

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

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

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.” 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

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

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

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.

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.

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.

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.

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.