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Event: 1461

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

DNA double-strand break

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
DSB

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
topoisomerase II binding, infant leukaemia KeyEvent Agnes Aggy (send email) Open for comment. Do not cite EAGMST Under Review

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

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

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help

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 double-strand breaks (DSB) is formed as a consequence of the production of excision repair breaks opposite each other on the two strands of DNA, and by the production of an excision repair break opposite a DNA daughter-strand gap. DSB are considered to be critical primary lesions in the formation of chromosomal aberrations.

To repare this potentially lethal damage, eukaryotic cells have evolved a variety of repair pathways related to homologous and illegitimate recombination, also called non-homologous DNA end joining (NHEJ), which may induce small scale mutations and chromosomal aberration (Pfeiffer et al. 2000). Repair by NHEJ often leads to small deletions at the site of the DSB and is considered error prone. The second repair mechanism, the Homologous Recombination (HR) is directed by extensive homology in a partner DNA molecule. In mitotic cells NHEJ occurs throughout all phases of the cell cycle, whereas HR is largely restricted to the S and G2 phases when the sister chromatid is available to mediate the repair process (Reynard et al. 2017). Persistent or incorrectly repaired DSBs can result in chromosome loss, deletion, translocation, or fusion, which can lead to carcinogenesis through activation of oncogenes or inactivation of tumor-suppressor genes (Raynard et al.2017). The DSB repair pathways apper to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. (Shrivastav et al. 2008).

DSBs are induced by agents such as ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy (Shrivastav et al. 2008). DSBs also arise during DNA replication when the DNA-polymerase ensemble encounters obstacles such as DNA lesions or unusual DNA structures (Raynard et al 2017). Additional endogenous sources include reactive oxygen species, generated during cellular metabolism, collapsed replication forks and nucleases(Shrivastav et al. 2008) .

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). ?

 A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage. H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells resulting in discrete γ-H2AX (phosphorylated H2AX) foci at the DNA damage sites. H2AX phosphorylation also appearsto be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells and meiotic recombination in mice. Phosphorylation of H2A at serine 139 causes chromatin decondensation and appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage.  DNA DSB staining  based on the phosphorylation of the histone H2A.X at serine 139 in response to DNA damaging agents which cause double strand breaks in cells that are cultured in microtiter plates is a rapid metod for the identification and quantification of the damage (Sealunavov et al.2002).

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a DNA-binding dye provides a way of measuring DNA damage and of assessing heterogeinicity in DNA damage within a mixed cell population. (Olive P. et al. 1991).

Pulsed field gel electrophoresis (PFGE) is the main method used for measurement of DNA DSB in mammalian cells (Blocker D et al. 1989 and 1990, Stamato T et al 1990, Ager D et al 1990). Alternatively the DNA is size fractioned in the pulsed-field gel, and the weight fraction of DNA below a certain defined size is measured (Erixo K. et al. 1990, Stenelow B. et al. 1995). An additional method to measure prompt DSBs without including heat-labile sites is also reported (Stenerlow B. et al. 2003).

In vitro assays for topoisomerase II based on the decantation of double strand DNA are extensively reported in Nitiss et al. 2012.

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

DSB occurs in eukaryotic and procaryoytic cells. There is good evidence for conservativism of DSB processing pathways in human cells (Gravel et al. 2008).

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., W. C. Dewey, K. Gardiner, W. Harvey, R. T. Johnson, and C. A. Waldren. Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis. Radiat. Res 122:181–187.1990

Blöcher, D., M. Einspenner, and J. Zajackowski. CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks. Int. J. Radiat. Biol 56:437–448.1989.

Blöcher, D. In CHEF electrophoresis a linear induction of dsb correspond to a nonlinear fraction of extracted DNA with dose. Int. J. Radiat. Biol 57:7–12.1990

Blöcher, D. In CHEF electrophoresis a linear induction of dsb correspond to a nonlinear fraction of extracted DNA with dose. Int. J. Radiat. Biol 57:7–12.1990.

Erixon, K., B. Cedervall, and R. Lewensohn. Pulsed-field gel electrophoresis for measuring radiation-induced DNA double-strand breaks. Comparison to the method of neutral filter elution. In Ionizing Radiation Damage to DNA: Molecular Aspects (R. Painter and S. Wallace, Eds.), pp. 69–80. UCLA Symposium on Molecular and Cellular Biology, New Series, Vol. 136, Wiley-Liss, New York, 1990.

Gravel S., Chapman JR., magill C., and jackson SP. 2008. DNA helicases Sgs1 and BLM promote DNA double-strand break resectio. Genes & Dev. 22:2767-2772.

Nitiss JL, Soans E, Rogoljina A, Seth A, Mishina M. 2012 Topoisiomerase assays. Current Protoc Pharmacol. Chapter: Unit 3.3.

Olive. PL, Wlodek D., Banath JP. 1991. DNA double-strand break measured in individual cells subjected to gel electrophoresis. Cnancer research. 51, 4671-4676, September 1.

Pfeiffer P., Goedeke W. and Gunter Obe. 2000. mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis vol15 n 4 289-302.

Raynard S., Niu H. and Sung P. 2017. 2002. DNA double-strand break processing: the beginning of the end. Genes & Dev. 22: 2903-2907.

Shrivastav M, De Haro LP, Nickoloff JA. 2008. Regulation of DNA double-strand break repair pathway choice. Cell Research, 18: 134-147.

Seluanov A, Zhiyong Mao, and Vera Gorbunova. 2002. Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells. J Vis Exp. 2010; (43): 2002. Published online 2010 Sep 8. doi:  10.3791/2002 PMCID: PMC3157866

Stamato, T. D. and N. Denko. Asymmetric field inversion gel electrophoresis: A new method for detecting DNA double-strand breaks in mammalian cells. Radiat. Res 121:196–205.1990. 

Stenerlow, B., J. Carlsson, E. Blomquist, and K. Erixon. Clonogenic cell survival and rejoining of DNA double-strand breaks: Comparisons between three cell lines after photon or He ion irradiation. Int. J. Radiat. Biol 65:631–639.1994. 

Stenerlöw B., Karin H. KarlssonBrian CooperBjörn Rydberg 2003 Measurement of Prompt DNA Double-Strand Breaks in Mammalian Cells without Including Heat-Labile Sites: Results for Cells Deficient in Nonhomologous End JoiningRadiation Research 159(4):502-510. https://doi.org/10.1667/0033-7587(2003)159[0502:MOPDDS]2.0.CO;2

 

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