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Event: 1635

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Increase, DNA strand breaks

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Increase, DNA strand breaks

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). 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 signalling 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. 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 Carole Yauk (send email) Open for comment. Do not cite EAGMST Under Review
Deposition of energy leading to lung cancer KeyEvent Vinita Chauhan (send email) Under development: Not open for comment. Do not cite EAGMST Under Review
Alkylation of DNA leading to reduced sperm count KeyEvent Carole Yauk (send email) Under development: Not open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. 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. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. 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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. 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).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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

Evidence for Perturbation by Stressor

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). 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.