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Event: 1194

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 damage

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 Damage

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

Level of Biological Organization
Molecular

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

Cell term
eukaryotic cell

Organ term

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

Process	Object	Action
	deoxyribonucleic acid	functional change

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
ER activation to breast cancer	KeyEvent	Molly M Morgan (send email)	Open for adoption
Increased DNA damage leading to breast cancer	MolecularInitiatingEvent	Jessica Helm (send email)	Under development: Not open for comment. Do not cite	Under Development
RONS leading to breast cancer	AdverseOutcome	Jessica Helm (send email)	Under development: Not open for comment. Do not cite	Under Development

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

Name
Ionizing Radiation
Estrogen

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

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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 nucleotide damage, single, and double strand breaks occur in the course of cellular operations such as DNA repair and replication and can be induced directly and in neighboring “bystander” cells by internal or external stressors like reactive oxygen species, chemicals, and radiation. Ionizing radiation and RONS such as hydroxyl radicals or peroxide can create a range of lesions (a change in molecular structure) in the base of the nucleotide, with guanine particularly vulnerable because of its low redox potential (David, O'Shea et al. 2007). The same stressors can also break the sugar (deoxyribose)-phosphate backbone creating a single strand break. Simultaneous proximal breaks in both strands of DNA form double strand breaks, which are considered to be more destructive and mutagenic than lesions or single strand breaks. Double strand breaks can generate chromosomal abnormalities including changes in chromosomal number, breaks and gaps, translocations, inversions, and deletions (Yang, Craise et al. 1992; Haag, Hsu et al. 1996; Ponnaiya, Cornforth et al. 1997; Yang, Georgy et al. 1997; Unger, Wienberg et al. 2010; Behjati, Gundem et al. 2016; Morishita, Muramatsu et al. 2016).

However, DNA lesions and single strand breaks can also be destructive and mutagenic. Lesions can lead to point mutations (David, O'Shea et al. 2007) or single strand breaks (Regulus, Duroux et al. 2007). Lesions and single strand breaks can also promote the formation of double strand breaks: replication fork collapse and double strand breaks sometimes occur during mitosis when the replisome encounters an unrepaired single strand break (Kuzminov 2001), and clustered lesions and closely opposed single strand breaks can also form double strand breaks (Chaudhry and Weinfeld 1997; Vispe and Satoh 2000; Shiraishi, Shikazono et al. 2017). Complex damage consists of any combination of closely opposed DNA lesions, abasic sites, crosslinks, single, or double strand breaks in proximity. While classically induced by ionizing radiation, there is also evidence that it can be induced by oxidative activity (Sharma, Collins et al. 2016) or even by a single oxidizing particle (Ravanat, Breton et al. 2014). Complex damage is more difficult to repair (Kuhne, Rothkamm et al. 2000; Stenerlow, Hoglund et al. 2000; Pinto, Prise et al. 2005; Rydberg, Cooper et al. 2005).

DNA damage and resulting repair activity can trigger a halt in the cell cycle, cell death (apoptosis), and cause permanent changes to DNA including deletions, translocations, and sequence changes. DNA damage is also associated with an increase in genomic instability - the new appearance of DNA damage including double strand breaks, mutations, and chromosomal damage following repair of initial damage in affected cells or in clonal descendants or neighbors of DNA damaged cells. The mechanism behind this long term DNA damage is not clear, but telomere erosion appears to play a major role (Murnane 2012; Sishc, Nelson et al. 2015). Genomic instability is more common and longer lasting following complex damage (Ponnaiya, Cornforth et al. 1997), and is influenced by multiple factors including variants in DNA repair genes (Ponnaiya, Cornforth et al. 1997; Yu, Okayasu et al. 2001; Yin, Menendez et al. 2012), RONS (Dayal, Martin et al. 2008), estrogen (Kutanzi and Kovalchuk 2013), caspases (Liu, He et al. 2015), and telomeres (Sishc, Nelson et al. 2015).

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). ?

DNA damage can be studied in isolated DNA, fixed cells, or living cells. Types of damage that can be detected include single and double strand breaks, nucleotide damage, complex damage, and chromosomal or telomere damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis. Finally, tests for mutations reveal past DNA damage that resulted in a heritable change, and these are described in the key event ‘Increase in Mutation’.

Many other (non-test guideline) techniques have been used to examine specific forms of DNA damage (Table 1). Double strand breaks are commonly reported because of the significant risk attributed to breaks and the relative ease of detecting and quantifying them. Historically, single and double strand breaks were measured using gel electrophoresis, but are now commonly visualized microscopically using fluorescent or other labeled probes for double and single strand break repair such as H2AX and XRCC2. Base lesions can also be detected using labeled probes for base excision repair enzymes, or by chemical methods such as mass spectroscopy. Refinements on these methods can be used to characterize complex or clustered damage, in which various forms of damage occur in close proximity on a DNA molecule (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).

Certain challenges are common to all methods of detecting DNA damage. In the time required to initiate the detection method, some DNA may already be repaired, leading to undercounting of damage. On the other hand, apoptotic DSBs may be incorrectly included in a measurement of direct (non-apoptotic) induction of DSB damage unless controlled. All methods have difficulty distinguishing individual components of clustered lesions, and microscopic methods may undercount disparate breaks that are processed together in repair centers (Barnard, Bouffler et al. 2013). Methods that use isolated DNA (gel electrophoresis, analytical chemistry) are vulnerable to artifacts and must ensure that the DNA sample is protected from oxidative damage during extraction (Pernot, Hall et al. 2012; Barnard, Bouffler et al. 2013; Ravanat, Breton et al. 2014).

Table 1. Common methods of detecting DNA damage

Target	Name	Method	Strengths/Weaknesses
Nucleotide damage	Single cell gel electrophoresis (comet assay) with restriction enzymes (Collins 2004)	Gel electrophoresis	A variant of the comet assay in which restriction enzymes allow the identification of different types of nucleotide damage. The comet assay is more sensitive than PFGE, detecting damage from 0.1 Gy ionizing radiation (Pernot, Hall et al. 2012). A reproducible high-throughput application of the assay is available (Ge, Prasongtanakij et al. 2014; Sykora, Witt et al. 2018), and the test requires only a small (single cell) sample. Requires destruction of the cell.
Nucleotide damage	Labeled probes including Biotrin OxyDNA and anti- 8-oxoguanine-DNA glycosylase (OGG1) for oxidative damage and AP endonuclease (APE1) for Base Excision Repair of less bulky lesions such as oxidative damage.	Microscopy, FACS	Most useful with FACS or other measures of average or relative intensity, as locations and numbers of damaged nucleotides can be difficult to distinguish using fluorescence microscopy. (Ogawa, Kobayashi et al. 2003; Nikitaki, Nikolov et al. 2016).
Nucleotide damage	High performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS)	Analytical chemistry	Capable of quantifying low levels of specific nucleotide lesions (Madugundu, Cadet et al. 2014; Ravanat, Breton et al. 2014). Requires destruction of the cell.
Nucleotide damage	Unscheduled DNA synthesis test OECD Test Guideline 486 (OECD 1997)	Autoradiography	Measures DNA damage that is repaired using Nucleotide Excision Repair - mostly bulky adducts (OECD (Organisation for Economic Co-operation and Development) 2016).
Non-specific DNA strand breaks	Single cell gel electrophoresis (comet assay), alkali conditions OECD Test Guideline 489 (OECD 2016)	Gel electrophoresis	When used in alkali conditions, the comet assay reveals single and double strand breaks and alkali-sensitive nucleotide lesions. See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments.
Single strand breaks	Labeled probe pXRCC1 (Lorat, Brunner et al. 2015)	Microscopy	Fluorescent probes can label single strand breaks in cells, while immunogold labeling is able to distinguish multiple single strand breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016).
Double strand breaks	Single cell gel electrophoresis (comet assay), neutral conditions	Gel electrophoresis	Neutral conditions help minimize the release of single strand breaks coiled DNA and alkali lesions, allowing the measurement of double strand breaks. Since single strand breaks can still appear, assay is not very sensitive or specific to double strand breaks (Pernot, Hall et al. 2012). See single cell gel electrophoresis (comet assay) with restriction enzymes above for further comments.
Double strand breaks	Pulsed field gel electrophoresis (PFGE)	Gel electrophoresis	Permits the quantitative measurement of double strand breaks, and can be combined with immunoblotting to detect DNA-associated proteins (Lobrich, Rydberg et al. 1995; Kawashima, Yamaguchi et al. 2017). Considered less sensitive than comet assay, but detected damage from 0.25 Gy ionizing radiation (Gradzka and Iwanenko 2005). Requires destruction of the cell.
Double strand breaks	Labeled probes including phosphorylated H2AX, 53BP1, Ku70, ATM (Lorat, Brunner et al. 2015)	Microscopy	Fluorescent probes can label individual double breaks in cells allowing for quantification, with immunogold labeling resolving breaks in clusters (Lorat, Timm et al. 2016; Nikitaki, Nikolov et al. 2016). Sensitive: detects damage from 0.001 Gy ionizing radiation (Rothkamm and Lobrich 2003; Ojima, Ban et al. 2008).
Chromosomal damage	Chromosomal aberrations and micronuclei OECD Test Guidelines 473, 475, 483, and 487 (OECD 2016; OECD 2016; OECD 2016; OECD 2016)	Microscopy	Detects major DNA damage resulting from large breaks and rearrangements, or mitotic failures. Damage does not appear until DNA undergoes mitosis, so slower and limited to damage in replicating cells. Insensitive tosmall deletions and substitutions.

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

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

Stressors include:

Ionizing radiation

Estrogen

Ionizing Radiation

When ionizing radiation enters a cell and interacts with cellular components including double stranded DNA, it releases energy that leads to DNA damage. This energy ejects electrons from atoms and molecules, and these electrons can produce more electrons, directly ionize DNA, or radiolyze water to form hydroxyl molecules which damage DNA (Hutchinson 1985; Ward 1988; Ravanat, Breton et al. 2014). DNA damage observed after IR includes oxidized base, sugar (deoxyribose), and phosphate lesions, single and double strand breaks, and cross-linking (Ward 1988; Roots, Holley et al. 1990; Haegele, Wolfe et al. 1998; Pouget, Frelon et al. 2002; Rothkamm and Lobrich 2003). DNA damage from IR can occur in a clustered pattern, even from a single particle or photon (Sutherland, Bennett et al. 2002). The type and amount of DNA damage depends on both the quality and dose of radiation. Higher LET radiation such as alpha particles generates more complex clusters of damage including more frequent double strand breaks (Ottolenghi, Merzagora et al. 1997; Rydberg, Heilbronn et al. 2002; Watanabe, Rahmanian et al. 2015; Nikitaki, Nikolov et al. 2016) and other chromosomal abnormalities (Yang, Georgy et al. 1997; Anderson, Stevens et al. 2002), while lower LET radiation (gamma rays, X-rays) generates more oxidized base damage and single strand breaks (Douki, Ravanat et al. 2006).

Damage is also observed in DNA in cells not directly in the path of ionizing radiation, or at a delay following exposure. Indirect or bystander effects are mediated by multiple factors including RONS (Yang, Asaad et al. 2005), TGF-β (Dickey, Baird et al. 2009), and other cytokines (Havaki, Kotsinas et al. 2015). DNA damage following ionizing radiation in directly and indirectly damaged cells is repaired over the first few hours or days (Nikitaki, Nikolov et al. 2016), but long term DNA damage can reoccur as genomic instability weeks, months, or even years after the initial exposure and persist in subsequent generations of cells in vivo (Pazhanisamy, Li et al. 2011; Datta, Suman et al. 2012; Mukherjee, Coates et al. 2012; Snijders, Marchetti et al. 2012) and in vitro (Moore, Marsden et al. 2005; Natarajan, Gibbons et al. 2007; Buonanno, de Toledo et al. 2011; Bensimon, Biard et al. 2016).

Double strand breaks occur linearly with dose between 0.001 Gy (the lowest dose at which an effect has been reliably observed) to over 80 Gy in irradiated cells (Rydberg, Heilbronn et al. 2002; Rothkamm and Lobrich 2003; Yang, Asaad et al. 2005; Asaithamby and Chen 2009). Some low dose studies find a steeper slope between 0.001 and 0.01 Gy for X-rays (although not gamma rays), possibly due to underassessment at higher doses or to a bystander effect superimposed on a linear response (Ojima, Ban et al. 2008; Beels, Werbrouck et al. 2010). Clustered DNA damage also occurs linearly from at least 0.05 Gy (the lowest dose tested) (Sutherland, Bennett et al. 2002), and single strand breaks and alkali sensitive lesions are linear with dose in isolated DNA (Roots, Holley et al. 1990). Chromosomal aberrations appear to be linear or supralinear with dose for low LET radiation (Yang, Georgy et al. 1997; Ryu, Kim et al. 2016) and linear with dose for high LET radiation (Yang, Georgy et al. 1997; Jones, Riggs et al. 2007) at doses examined as low as 0.01 Gy (Schiestl, Khogali et al. 1994; Iwasaki, Takashima et al. 2011). DNA damage measured in bystander cells 1 hour to 3 days after exposure is dose-dependent at low doses (0.001-0.005 Gy), but may approach a maximum between 0.005 and 0.1 Gy (Yang, Anzenberg et al. 2007; Ojima, Ban et al. 2008).

Anderson, R. M., D. L. Stevens, et al. (2002). "M-FISH analysis shows that complex chromosome aberrations induced by alpha -particle tracks are cumulative products of localized rearrangements." Proceedings of the National Academy of Sciences of the United States of America 99(19): 12167-12172.

Asaithamby, A. and D. J. Chen (2009). "Cellular responses to DNA double-strand breaks after low-dose gamma-irradiation." Nucleic acids research 37(12): 3912-3923.

Beels, L., J. Werbrouck, et al. (2010). "Dose response and repair kinetics of gamma-H2AX foci induced by in vitro irradiation of whole blood and T-lymphocytes with X- and gamma-radiation." International journal of radiation biology 86(9): 760-768.

Bensimon, J., D. Biard, et al. (2016). "Forced extinction of CD24 stem-like breast cancer marker alone promotes radiation resistance through the control of oxidative stress." Mol Carcinog 55(3): 245-254.

Buonanno, M., S. M. de Toledo, et al. (2011). "Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress." Radiation research 175(4): 405-415.

Datta, K., S. Suman, et al. (2012). "Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine." PLoS One 7(8): e42224.

Dickey, J. S., B. J. Baird, et al. (2009). "Intercellular communication of cellular stress monitored by gamma-H2AX induction." Carcinogenesis 30(10): 1686-1695.

Douki, T., J. L. Ravanat, et al. (2006). "Minor contribution of direct ionization to DNA base damage inducedby heavy ions." International journal of radiation biology 82(2): 119-127.

Haegele, A. D., P. Wolfe, et al. (1998). "X-radiation induces 8-hydroxy-2'-deoxyguanosine formation in vivo in rat mammary gland DNA." Carcinogenesis 19(7): 1319-1321.

Havaki, S., A. Kotsinas, et al. (2015). "The role of oxidative DNA damage in radiation induced bystander effect." Cancer Lett 356(1): 43-51.

Hutchinson, F. (1985). "Chemical changes induced in DNA by ionizing radiation." Progress in nucleic acid research and molecular biology 32: 115-154.

Iwasaki, T., Y. Takashima, et al. (2011). "The dose response of chromosome aberrations in human lymphocytes induced in vitro by very low-dose gamma rays." Radiation research 175(2): 208-213.

Jones, J. A., P. K. Riggs, et al. (2007). "Ionizing radiation-induced bioeffects in space and strategies to reduce cellular injury and carcinogenesis." Aviat Space Environ Med 78(4 Suppl): A67-78.

Moore, S. R., S. Marsden, et al. (2005). "Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells." Radiation research 163(2): 183-190.

Mukherjee, D., P. J. Coates, et al. (2012). "The in vivo expression of radiation-induced chromosomal instability has an inflammatory mechanism." Radiation research 177(1): 18-24.

Natarajan, M., C. F. Gibbons, et al. (2007). "Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells." Br J Radiol 80 Spec No 1: S13-22.

Nikitaki, Z., V. Nikolov, et al. (2016). "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)." Free radical research 50(sup1): S64-S78.

Ojima, M., N. Ban, et al. (2008). "DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects." Radiation research 170(3): 365-371.

Ottolenghi, A., M. Merzagora, et al. (1997). "DNA complex lesions induced by protons and alpha-particles: track structure characteristics determining linear energy transfer and particle type dependence." Radiation and environmental biophysics 36(2): 97-103.

Pazhanisamy, S. K., H. Li, et al. (2011). "NADPH oxidase inhibition attenuates total body irradiation-induced haematopoietic genomic instability." Mutagenesis 26(3): 431-435.

Pouget, J. P., S. Frelon, et al. (2002). "Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles." Radiation research 157(5): 589-595.

Ravanat, J. L., J. Breton, et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.

Roots, R., W. Holley, et al. (1990). "The formation of strand breaks in DNA after high-LET irradiation: a comparison of data from in vitro and cellular systems." International journal of radiation biology 58(1): 55-69.

Rothkamm, K. and M. Lobrich (2003). "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses." Proceedings of the National Academy of Sciences of the United States of America 100(9): 5057-5062.

Rydberg, B., L. Heilbronn, et al. (2002). "Spatial distribution and yield of DNA double-strand breaks induced by 3-7 MeV helium ions in human fibroblasts." Radiation research 158(1): 32-42.

Ryu, T. H., J. H. Kim, et al. (2016). "Chromosomal Aberrations in Human Peripheral Blood Lymphocytes after Exposure to Ionizing Radiation." Genome integrity 7: 5.

Schiestl, R. H., F. Khogali, et al. (1994). "Reversion of the mouse pink-eyed unstable mutation induced by low doses of x-rays." Science 266(5190): 1573-1576.

Snijders, A. M., F. Marchetti, et al. (2012). "Genetic differences in transcript responses to low-dose ionizing radiation identify tissue functions associated with breast cancer susceptibility." PLoS One 7(10): e45394.

Sutherland, B. M., P. V. Bennett, et al. (2002). "Clustered DNA damages induced by x rays in human cells." Radiation research 157(6): 611-616.

Ward, J. F. (1988). "DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability." Progress in nucleic acid research and molecular biology 35: 95-125.

Watanabe, R., S. Rahmanian, et al. (2015). "Spectrum of Radiation-Induced Clustered Non-DSB Damage - A Monte Carlo Track Structure Modeling and Calculations." Radiation research 183(5): 525-540.

Yang, H., V. Anzenberg, et al. (2007). "The time dependence of bystander responses induced by iron-ion radiation in normal human skin fibroblasts." Radiation research 168(3): 292-298.

Yang, H., N. Asaad, et al. (2005). "Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts." Oncogene 24(12): 2096-2103.

Yang, T. C., K. A. Georgy, et al. (1997). "Initiation of oncogenic transformation in human mammary epithelial cells by charged particles." Radiat Oncol Investig 5(3): 134-138.

Estrogen

Metabolites created through the oxidative metabolism of estrogens form pro-mutagenic adducts with guanine and adenine. These adducts rapidly undergo depurination leaving abasic sites that can contribute to point mutations or to double strand breaks and further errors if not correctly repaired prior to replication (Cavalieri, Chakravarti et al. 2006; Savage, Matchett et al. 2014; Yager 2015; Yasuda, Sakakibara et al. 2017). The metabolic cycling of the same estrogen metabolites contributes to the formation of ROS, which can oxidatively damage DNA. However, this does not appear to be the major mechanism of DNA damage by estrogen (Cavalieri, Chakravarti et al. 2006). The creation of estrogen metabolites depend on an imbalance in estrogen synthesis and metabolism. DNA damage and mutation is enhanced under conditions that promote estrogen synthesis or inhibit further metabolism including the inactivation of the DNA-damaging metabolites (Cavalieri, Chakravarti et al. 2006; Yager 2015).

Estrogen can also increase double strand breaks through a transcription and replication-dependent mechanism (Stork, Bocek et al. 2016). Estradiol increases double strand breaks and rearrangements at R-loops (RNA-DNA hybrids with an associated single-stranded DNA) formed at ERa-mediated transcription sites. This damage is dependent on Transcription-Coupled Nucleotide Excision Repair and occurs after a delay compared with the ER-independent breaks. This mechanism is a major contributor to overall double strand break formation after estrogen treatment (Stork, Bocek et al. 2016).

Estrogen also affects DNA damage less directly through effects on cell cycle checkpoint regulation and DNA repair mechanisms (Caldon 2014; Li, Chen et al. 2014; Schiewer and Knudsen 2016). It enhances some aspects of the cellular response to DNA damage including enhancing Rad51 recruitment of repair machinery but inhibits others aspects of the response including suppressing multiple regulators of cell cycle checkpoints and delaying complete repair of DSBs (Caldon 2014; Li, Chen et al. 2014). Estrogen also promotes relatively error-prone NHEJ repair mechanisms (Caldon 2014). The net effect promotes survival and replication at the expense of genomic integrity. This effect of estrogen on cell cycle and repair also serves to promote the more direct DNA damaging effects of estrogen, since several of the mechanisms by which estrogen damages DNA require replication before repair is complete (Savage, Matchett et al. 2014; Stork, Bocek et al. 2016). Interestingly, the protection against breast cancer afforded by early parity may at least partially be mediated by a change in the response to estrogen signaling to promote p53 activity and genomic integrity at the cost of proliferation (Jerry, Dunphy et al. 2010).

The effect of estrogen on cell cycle machinery is closely linked with the canonical proliferative effect of estrogen. Since replication can create DNA damage through collapse of replicative forks encountering unrepaired sites to form DSBs, the proliferative effect itself promotes DNA damage even in the absence of other mechanisms.

Caldon, C. E. (2014). "Estrogen signaling and the DNA damage response in hormone dependent breast cancers." Front Oncol 4: 106.

Cavalieri, E., D. Chakravarti, et al. (2006). "Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention." Biochim Biophys Acta 1766(1): 63-78.

Jerry, D. J., K. A. Dunphy, et al. (2010). "Estrogens, regulation of p53 and breast cancer risk: a balancing act." Cellular and molecular life sciences : CMLS 67(7): 1017-1023.

Li, Z., K. Chen, et al. (2014). "Cyclin D1 integrates estrogen-mediated DNA damage repair signaling." Cancer Res 74(14): 3959-3970.

Savage, K. I., K. B. Matchett, et al. (2014). "BRCA1 deficiency exacerbates estrogen-induced DNA damage and genomic instability." Cancer Res 74(10): 2773-2784.

Schiewer, M. J. and K. E. Knudsen (2016). "Linking DNA Damage and Hormone Signaling Pathways in Cancer." Trends Endocrinol Metab 27(4): 216-225.

Stork, C. T., M. Bocek, et al. (2016). "Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage." Elife 5.

Yager, J. D. (2015). "Mechanisms of estrogen carcinogenesis: The role of E2/E1-quinone metabolites suggests new approaches to preventive intervention--A review." Steroids 99(Pt A): 56-60.

Yasuda, M. T., H. Sakakibara, et al. (2017). "Estrogen- and stress-induced DNA damage in breast cancer and chemoprevention with dietary flavonoid." Genes Environ 39: 10

Regulatory Significance of the Adverse Outcome

An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP. For KEs that are designated as an AO, one additional field of information (regulatory significance of the AO) should be completed, to the extent feasible. If the KE is being described is not an AO, simply indicate “not an AO” in this section.A key criterion for defining an AO is its relevance for regulatory decision-making (i.e., it corresponds to an accepted protection goal or common apical endpoint in an established regulatory guideline study). For example, in humans this may constitute increased risk of disease-related pathology in a particular organ or organ system in an individual or in either the entire or a specified subset of the population. In wildlife, this will most often be an outcome of demographic significance that has meaning in terms of estimates of population sustainability. Given this consideration, in addition to describing the biological state associated with the AO, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to describe regulatory examples using this AO. More help

DNA damage increases the susceptibility to and probability of subsequent mutations, described in the key event ‘Increase in Mutation’. Mutations can impair the functional capacity of the cell and are an endpoint of regulator significance in their own right.

Multiple guideline toxicity tests exist for DNA damage. The OECD test guideline for DNA synthesis Test No. 486 (OECD 1997) detects nucleotide excision repair, so it will reflect the formation of bulky DNA adducts but not the majority of oxidative damage to nucleotides, which is typically repaired via the Base Excision Repair pathway. The OECD test guideline alkaline comet assay Test No. 489 (OECD 2016) detects single and double strand breaks, including those arising from repair as well as some (alkali sensitive) nucleotide lesions including some lesions from oxidative damage. OECD tests for chromosomal damage and micronuclei Test No. 473, 475, 483, and 487 measure longer term effects of DNA damage but these tests require the damaged cell to subsequently undergo replication (OECD 2016; OECD 2016; OECD 2016; OECD 2016). They can therefore reflect a wider range of sources of DNA damage including changes in mitosis.

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

Barnard, S., S. Bouffler, et al. (2013). "The shape of the radiation dose response for DNA double-strand break induction and repair." Genome integrity 4(1): 1.

Behjati, S., G. Gundem, et al. (2016). "Mutational signatures of ionizing radiation in second malignancies." Nat Commun 7: 12605.

Chaudhry, M. A. and M. Weinfeld (1997). "Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA." The Journal of biological chemistry 272(25): 15650-15655.

Collins, A. R. (2004). "The comet assay for DNA damage and repair: principles, applications, and limitations." Molecular biotechnology 26(3): 249-261.

David, S. S., V. L. O'Shea, et al. (2007). "Base-excision repair of oxidative DNA damage." Nature 447(7147): 941-950.

Dayal, D., S. M. Martin, et al. (2008). "Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells." Biochem J 413(1): 185-191.

Ge, J., S. Prasongtanakij, et al. (2014). "CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells." Journal of visualized experiments : JoVE(92): e50607.

Gradzka, I. and T. Iwanenko (2005). "A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells." DNA repair 4(10): 1129-1139.

Haag, J. D., L. C. Hsu, et al. (1996). "Allelic imbalance in mammary carcinomas induced by either 7,12-dimethylbenz[a]anthracene or ionizing radiation in rats carrying genes conferring differential susceptibilities to mammary carcinogenesis." Mol Carcinog 17(3): 134-143.

Kawashima, Y., N. Yamaguchi, et al. (2017). "Detection of DNA double-strand breaks by pulsed-field gel electrophoresis." Genes to cells : devoted to molecular & cellular mechanisms 22(1): 84-93.

Kuhne, M., K. Rothkamm, et al. (2000). "No dose-dependence of DNA double-strand break misrejoining following alpha-particle irradiation." International journal of radiation biology 76(7): 891-900.

Kutanzi, K. and O. Kovalchuk (2013). "Exposure to estrogen and ionizing radiation causes epigenetic dysregulation, activation of mitogen-activated protein kinase pathways, and genome instability in the mammary gland of ACI rats." Cancer Biol Ther 14(7): 564-573.