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Relationship: 1911

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Increase, DNA strand breaks leads to Inadequate DNA repair

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Increase, DNA strand breaks

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Inadequate DNA repair

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Oxidative DNA damage leading to chromosomal aberrations and mutations	adjacent	High	Low	Brendan Ferreri-Hanberry (send email)	Open for comment. Do not cite	WPHA/WNT Endorsed
Deposition of energy leading to lung cancer	adjacent	Moderate	Moderate	Brendan Ferreri-Hanberry (send email)	Open for citation & comment	EAGMST Approved
Deposition of energy leading to occurrence of cataracts	adjacent	Moderate	Moderate	Arthur Author (send email)	Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	High	NCBI
mouse	Mus musculus	High	NCBI
rat	Rattus norvegicus	High	NCBI

Sex Applicability

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

Sex	Evidence
Unspecific	High

Life Stage Applicability

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

Term	Evidence
All life stages	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

The maintenance of DNA integrity is essential for genomic stability; for this reason cells have multiple response mechanisms that enable the repair of damaged DNA. Thus when DNA double strand breaks (DSBs) occur, the most detrimental type of lesion, the cell will initiate repair machinery. These mechanisms are not foolproof, and emerging evidence suggests that closely spaced lesions can compromise the repair machinery. The two most common DSB repair mechanisms are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is initiated in G1 and early S phases of the cell cycle (Lieber et al., 2003) and is preferentially used to repair DSB damage (Godwint et al., 1994), as it is rapid and more efficient than HR (Lliakis, 1991; Jeggo, 1998; Mao et al., 2008). In higher-order eukaryotes such as humans, NHEJ is the favoured DNA repair mechanism because of the large non-coding regions within the genome. NHEJ can occur through one of two subtypes: canonical NHEJ (C‐NHEJ) or alternative non-homologous end joining (alt-NHEJ). C-NHEJ, as the name suggests, simply ligates the broken ends back together. In contrast, alt‐NHEJ occurs when one strand of the DNA on either side of the break is resected to repair the lesion (Bétermier et al., 2014). Both repair mechanisms are error‐prone, meaning insertions and deletions are sometimes formed due to the DSBs being repaired imperfectly (Thurtle-Schmidt and Lo, 2018). However, alt-NHEJ is considered more error-prone than C-NHEJ, as studies have shown that it more often leads to chromosomal aberrations (Zhu et al., 2002; Guirouilh-Barbat et al., 2007; Simsek & Jasin, 2010). HR is mostly operative during S and G2 phases because of the presence of the sister chromatid that can be used as template for repair (Van Gent et al 2001). Because of the reliance on the undamaged sister chromatid to repair the DSB, HR is less error-prone than NEHJ. Nevertheless, defects in HR are known to contribute to genomic instability and the formation of chromosomal aberrations (Deans et al 2000)

There is extensive evidence that DNA repair capacity can be overwhelmed or saturated in the presence of high numbers of strand breaks. This is demonstrated by decades of studies showing dose-related increases in chromosomal exchanges, chromosomal breaks and micronuclei following exposure to double-strand break inducers. Inadequate repair not only refers to overwhelming of DNA repair machinery, but also the use of repair mechanisms that are error-prone (i.e., misrepair is considered inadequate repair).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Evidence Map 2.0

ID	Experimental Design	Species	Upstream Observation	Downstream Observation	Citation (first author, year)	Notes

Evidence Map

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Title	First Author	Biological Plausibility	Dose Concordance	Temporal Concordance	Incidence Concordance

Biological Plausibility

Dose Concordance Evidence

Temporal Concordance Evidence

Incidence Concordance Evidence

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Uncertainties and inconsistencies in this KER are as follows:

There is controversy surrounding how error-prone NHEJ truly is. Recent studies suggest that the process may be quite accurate (reviewed in (Bétermier et al. 2014)). The accuracy of NHEJ may actually be dependent on the structure of the termini. Thus, the termini processing rather than the NHEJ mechanism itself is argued to be the error-prone process (Bétermier et al. 2014).
There may be different cellular responses associated with low-dose radiation exposure and high-dose radiation exposure; these differences may also be dependent on a DSB threshold being exceeded prior to initiation repair. It has been suggested that DNA repair may not be activated at low doses of radiation exposure in order to prevent the risk of mutations from error-prone repair mechanisms (Marples 2004).
DSB repair fidelity varies in terms of confounding factors and the genetic characteristics of individuals (Scott 2006). For example, individuals who smoke have a 50% reduction in the mean level of DSB repair capacity relative to the non-smokers; this is due to an increased methylation index in smokers. A higher methylation index indicates more inactivation of gene expression. It is thus possible that expression of DNA repair proteins in smokers is decreased due to increased methylation of the genes encoding for repair proteins. In terms of individual genetics, single nucleotide polymorphisms (SNPs) within the MRE11A, CHEK2, XRCC3, DNA-PKcs, and NBN repair genes have been highly associated with the methylation index (Leng et al. 2008). SNPs can critically affect the function of these core proteins, varying the fidelity of DNA repair from person to person.
Cells containing DNA damaged may be eliminated by apoptotic pathways, therefore not undergo repair, alternatively evidence has also shown that damaged cells can propagate due to lack of detection by repair machinery (Valentin 2005).
The focus of this KER was on DSBs because there is lack of data to support that SSBs lead to inadequate repair. Multiple SSBs can lead to DSBs. Thus, DSBs are the focus as they can drive the cell towards genomic instability, apoptosis or tumorigenesis. Further quantitative evidence to define the extent of SSBs leading to DSBs and the relationship with repair is necessary.
Ercc2+/- mice have a mutation in a gene involved in the nucleotide excision repair (NER) pathway, leading to DNA repair deficiency. However, when compared to wild type mice Ercc2+/- mice had fewer DNA strand breaks. This was true of both central and peripheral lens cells, as well as 4 and 24 h after irradiation (60Co γ-rays, 0.3, 0.063 Gy/min) (Barnard et al., 2021).
DNA damage repair times can vary depending on the stressors that instigate the DNA damage. For example, it has been found that some types of radiation i.e., high linear energy transfer (LET) increases the amount of time required to repair DNA breaks (Aufderheide, 1987; Frankenburg-Schwager et al., 1994; Rydberg et al., 1994; Baumstark-Khan et al., 2003; Tsao, 2007; Blakely, 2012), however Stenerlöw et al. (2000) found that repair half-times were independent of LET.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating Factor	Details	Effects on the KER	References
Linear energy transfer (LET)	Increased LET	As the LET of the stressor increases, the amount of misrepaired and unrejoined DSBs also increases. One possible explanation for this is that DSB free ends are closer together at higher LETs, making it easier for misrepair to occur. Furthermore, higher LET stressors produce more complex, clustered breaks which also increasing repair difficulty. At very high LET values (over 10 000 keV/um), no significant DNA repair is detected.	Aufderheide, 1987; Rydberg et al., 1994; Durante et al., 1998; Kuhne et al., 2000; Stenerlöw et al., 2000; Baumstark-Khan et al., 2003; Tsao, 2007; Mukherjee et al., 2008; Blakely, 2012; Hamada, 2017
Oxygen	Decreased oxygen levels	Cells in an anoxic environment will rejoin DNA breaks more quickly than those in an oxic environment because oxygen can attach to the broken ends of DNA, fixing the damage and making it unrepairable.	Frankenburg-Schwager et al., 1994

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Quantitative understanding of this linkage suggests that DSB repair can be predicted from the presence of DSBs. The following tables provide representative examples of the relationship, unless otherwise indicated, all data is significantly significant. In terms of DNA repair in response to radiation-induced DSBs, one study suggests that complete DNA DSB repair occurs starting at a threshold dose of 5 mGy (0.005 Gy), as measured by the presence of γ-H2AX (Lobrich et al., 2005) and presence of 53BPI foci (Asaithamby & Chen, 2009). After a 10 Gy dose of radiation, approximately 10 - 15% of DSBs were found to be misrepaired (Mcmahon et al., 2016); at a dose of 80 Gy, the relative percentage of DSBs incorrectly repaired was estimated at 50 - 60% (Kuhne et al., 2000; Lobrich et al., 2000; Mcmahon et al., 2016). Twenty-four hours post-irradiation, this rate increased to approximately 80% for alpha particle irradiation at 80 Gy, and remained constant until the end of the assay (10 days) (Kuhne et al., 2000).

Dose Concordance

Reference	Experiment Description	Result
Rydberg et al., 1994	In vitro. Human VA13 lung fibroblast and GM38A skin fibroblast cells were exposed to neon ions (425 MeV/u, 1 – 5 Gy/min, 80 Gy), iron ions (600 MeV/u, 1 – 5 Gy/min, 50 Gy), and X rays (425 MeV/u, 1 – 2 Gy/min, 80 Gy) to induce DNA strand breaks. Initial breaks after exposure were measured via the fraction of activity released (FAR) assay, with an increased FAR value indicating an increased number of breaks. Repair was measured using the FAR assay after a period of incubation.	Exposure to X-rays, neon, and iron ions led to a 90, 70, and 50% FAR increase relative to control respectively, indicating the highest level of breaks in samples exposed to X-rays. Four h later, 15, 20, and 73% of the DNA strand breaks had not been repaired.
Kuhne et al., 2000	In vitro. Human lung fibroblast cells were exposed to X-rays (23 Gy/min) at doses from 0 - 320 Gy. Following this, both correct (measured via hybridization assay), and total (measured via FAR assay) breaks remaining were measured. Therefore, allowing for calculation of the amount of misrepaired breaks.	Cells exposed to 0 - 320 Gy X-rays displayed an approximately linear increase in DSBs. This led to a gradual increase in the % DSBs misrejoined, which began to plateau after 80 Gy at a misrejoining frequency of 50%.
Baumstark-Khan et al., 2003	In vitro. Bovine LECs were exposed to X-rays (5 Gy/min, 0 to 50 Gy), 16O (3.4, 8.7 MeV/u, 230.5 to 642.9 Gy), 40Ar (2.7, 6.2, 10.5, 19.3 MeV/u, 0 to 190 Gy), 132Xe (5.4, 10.1, 16.5 MeV/u, 0 to 80 Gy), 208Pb (3.0, 6.8, 15.4 MeV/u, 0 to 50 Gy), 238U (1.5, 1.9, 2.6, 4.0 MeV/u, 0 to 150 Gy). This led to the induction of both SSBs and DSBs, whose repair was measured using a method similar to the hydroxyapatite chromatography of alkaline unwound DNA.	Irradiation below 10 000 keV/μm led to almost 100% rejoining of SSBs and DSBs. At LETs above 10 000 keV/μm the rejoining capacity varied depending on the original level of damage. After irradiation with 238U (LET ~ 20 000 keV/μm) rejoining capacity as t -> ∞ ranged from 50 to 100%. After irradiation with 208Pb (LET ~ 18 000 keV/μm) rejoining capacity as t -> ∞ ranged from 15 to 28%. 48Ti was an exception, with an LET of 1440 keV/μm that resulted in a rejoining capacity of only 65% rather than almost 100% as t -> ∞.
Aufderheide, 1987	In vitro. Bovine lens epithelial cells (LECs) were exposed to 238U (5, 10, 20 x 106 P/cm2), 132Xe (3, 5, 7, 12, 20 x 106 P/cm2), 84Kr (9, 21 x 106 P/cm2), 40Ar (24 x 106 P/cm2), 16O (80 x 106 P/cm2), and X-rays (20, 40, 200 Gy). The radiation exposure induced DNA breaks were measured using the DNA unwinding method described by Rydberg (1975). The DNA then underwent a period of repair incubation lasting between 5 to 40 h, after which any remaining DNA damage was measured using the same method as before.	Bovine LECs exposed to 21 x 106 P/cm2 84Kr displayed a 1.3x increase in DNA breaks and a 5% decrease in the level of breaks repaired compared to cells exposed to 9 x 106 P/cm2.
Stenerlöw et al., 2000	In vitro. Human skin fibroblast cells were exposed to 100 Gy of photons (60Co, < 0.5 keV/um), nitrogen ions (80, 125, 175, 225 keV/um), and helium ions (40 keV/um), resulting in the formation of DSBs. Their number was calculated by fragment analysis, based upon the fraction of DNA less than 5.7 Mbp, under the assumption that the breaks were evenly distributed. DNA repair was also measured via fragment analysis.	Exposure to increasing LET of radiation at 100 Gy led to increasing DSBs, in general, with about 600 DSBs/Gbp after γ-ray irradiation and about 700 DSBs/Gbp after 225 keV/um nitrogen ion irradiation. A dose of 100 Gy also led to decreased repair at increased LET. About 20-22 h after γ-ray irradiation, 4% of DSBs were unrepaired, while 20-22 h after 225 keV/um nitrogen ion irradiation, 12% of DSBs were unrepaired.
Coquerelle et al., 1987	In vitro. Human skin fibroblast cells were exposed to ⁶⁰Co (1.5, 0.35 Gy/min) and alpha particles (120 keV/µm). Alkaline elution assay was used to detect DNA strand breaks. Repair of breaks were determined over time. The % rejoined DNA was calculated from the mean values of the entire elution profile.	Exposure to 25 Gy gamma rays or alpha particles resulted in ~20% strand breaks. 80% of these breaks were repaired 30 mins after the exposure.

Incidence Concordance

No studies were found.

Time Concordance

Reference

Experiment Description

Result

Durante et al., 1998

In vitro. Human, male, lymphocyte cells were exposed to either iron ions (140 keV/μm, 2 Gy), or carbon ions (42 keV/μm, 5 Gy) to induce DNA strand breaks. Misrepair was measured by producing chromosome spreads and evaluating them using a microscope and the PAINT classification code.

Exposure to 2 Gy iron particles resulted in about 0.45 breaks/cell, of which 50% were repaired 10 h later. However, there were 0.1 translocations/cell, 0.08 incomplete exchanges/cell, 0.075 complex exchanges/cell, and 0.07 dicentrics/cell.

Exposure to 5 Gy carbon ions resulted in 1.15 breaks/cell, of which 25% were repaired 10 h later. However, there were 0.35 translocations/cell, 0.28 incomplete exchanges/cell, 0.43 complex exchanges/cell, and 0.29 dicentrics/cell.

Rydberg et al., 1994

In vitro. Human VA13 lung fibroblast and GM38A skin fibroblast cells were exposed to neon ions (425 MeV/u, 1 – 5 Gy/min, 80 Gy), iron ions (250, 400, 600 MeV/u, 1 – 5 Gy/min, 50 Gy), and X rays (425 MeV/u, 1 – 2 Gy/min, 80 Gy) to induce DNA breaks. Their repair was measured using pulsed-field gel electrophoresis and determining the amount of DNA released from the gel plug (fraction of activity released – FAR).

In GM38A cells, exposure to 80 Gy of all three radiation types led to DNA breaks. Repair was observed between 0.5 and 4 h after this.

The most breaks remained after exposure to iron ions (75% of breaks remained), 25 – 42% remained after neon exposure, and only 15 – 20% remained after X ray irradiation.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

There is evidence of a response-response relationship for DNA repair of radiation-induced DSBs. The frequency of DSBs has been shown to increase linearly with radiation dose (Löbrich et al., 2000; Rothkamm & Lo, 2003; Kuhne et al., 2005; Asaithamby & Chen, 2009). For DNA repair, increasing doses of a radiation stressor were found to cause a linear-quadratic relationship between the radiation dose and the number of misrejoined DSBs per cell (Kuhne et al., 2005). Interestingly, the relationships between radiation and DNA repair were found to vary depending on the type of radiation. There was a more linear response between radiation dose and the number of misrejoined DSBs for high LET particles relative to a more curvilinear relationship for lower LET particles (Rydberg et al., 2005). Additionally, a linear relationship was defined for low dose-rate radiation and the number of non-repaired DNA DSBs, but a linear-quadratic equation was described for high dose-rate radiation (Dikomey & Brammer, 2000).

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Data from temporal response studies suggests that DSB repair may occur within 15 - 30 minutes of a DSB-inducing radiation stressor (Paull et al., 2000; Rothkamm & Lo, 2003; Pinto & Prise, 2005; Dong et al., 2017), with foci documented as early as 3-5 minutes post-irradiation (Asaithamby & Chen, 2009). The majority of DSB repair has been reported to occur within the first 3 - 6 hours following DSB induction (Rothkamm & Lo, 2003; Pinto & Prise, 2005; Asaithamby & Chen, 2009; Dong et al., 2017), with complete or near-complete DSB repair within 24 hours of the radiation stressor (Dikomey & Brammer, 2000; Lobrich et al., 2000; Rothkamm & Lo, 2003; Asaithamby & Chen, 2009; Mcmahon et al., 2016). In one 48-hour time-course experiment for DSB repair using two different types of radiation, the following repair progression was found at 30 minutes, 1 hour, 3 hours, 24 hours and 48 hours, respectively: 40 - 55%, 55 - 70%, 85%, 97 - 98% and 98% repair for X-rays and 30%, 45 - 50%, 65 - 70%, 85 - 90% and 90 - 96% repair for alpha particles (Pinto & Prise, 2005). Twenty-four hours post-irradiation, the frequency of DSB misrejoining was found to remain constant at approximately 80% for the 10 days that the DSB repair was monitored (Kuhne et al., 2000).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Not identified.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

This KER is plausible in all life stages, sexes, and organisms with DNA. The majority of the evidence is from in vivo adult mice with no specification on sex, and in vitro human models that do not specify sex.