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Relationship: 1977

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

Energy Deposition leads to Increase, DNA strand breaks

Upstream event

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

Energy Deposition

Downstream event

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

Increase, DNA strand breaks

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
Deposition of energy leading to lung cancer	adjacent	High	High	Brendan Ferreri-Hanberry (send email)	Open for citation & comment	WPHA/WNT Endorsed
Deposition of energy leading to population decline via DNA strand breaks and follicular atresia	adjacent	High		Evgeniia Kazymova (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis	adjacent			Agnes Aggy (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to occurrence of cataracts	adjacent	High	High	Arthur Author (send email)	Open for citation & comment
Deposition of energy leads to vascular remodeling	adjacent	High	High	Cataia Ives (send email)	Open for citation & comment
Deposition of Energy Leading to Learning and Memory Impairment	adjacent	High	High	Brendan Ferreri-Hanberry (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
mouse	Mus musculus	High	NCBI
human	Homo sapiens	High	NCBI
rat	Rattus norvegicus	High	NCBI
bovine	Bos taurus	Low	NCBI
rabbit	Oryctolagus cuniculus	Low	NCBI
Pig	Pig	Low	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

Direct deposition of ionizing energy refers to imparted energy interacting directly with the DNA double helix and producing randomized damage. This can be in the form of double strand breaks (DSBs), single-strand breaks, base damage, or the crosslinking of DNA to other molecules (Smith et al., 2003; Joiner, 2009; Christensen, 2014; Sage and Shikazono, 2017). Among these, the most detrimental type of DNA damage to a cell is DSBs. They are caused by the breaking of the sugar-phosphate backbone on both strands of the DNA double helix molecule, either directly across from each other or several nucleotides apart (Ward, 1988; Iliakis et al., 2015). This occurs when high-energy subatomic particles interact with the orbital electrons of the DNA causing ionization (where electrons are ejected from atoms) and excitation (where electrons are raised to higher energy levels) (Joiner, 2009). The number of DSBs produced and the complexity of the breaks is highly dependent on the amount of energy deposited on and absorbed by the cell. This can vary as a function of the dose-rate (Brooks et al., 2016) and the radiation quality which is a function of its linear energy transfer (LET) (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012). LET describes the amount of energy that an ionizing particle transfers to media per unit distance (Smith et al., 2003; Okayasu, 2012a; Christensen et al., 2014). High LET radiation, such as alpha particles, heavy ion particles, and neutrons can deposit larger quantities of energy within a single track than low LET radiation, such as γ-rays, X-rays, electrons, and protons (Kadhim et al., 2006; Franken et al., 2012; Frankenberg et al., 1999; Rydberg et al., 2002; Belli et al., 2000; Antonelli et al., 2015). As such, radiation with higher LETs tends to produce more complex, dense structural damage, particularly in the form of clustered damage, in comparison to lower LET radiation (Nikjoo et al., 2001; Terato and Ide, 2005; Hada and Georgakilas, 2008; Okayasu, 2012a; Lorat et al., 2015; Nikitaki et al., 2016). Some data reports that low dose and low LER radiation can lead to complex lesions, which can cause unrepairable DNA damage. However, determining the actual frequency of the complexity of these lesions has proven challenging (Wilkinson et al., 2023). The complexity and yield of clustered DNA damage increases with ionizing density (Ward, 1988; Goodhead, 2006). However, clustered damage can also be induced even by a single radiation track through a cell.

While the amount of DSBs produced depends on the radiation dose (see dose concordance), it also depends on several other factors. As the LET increases, the complexity of DNA damage increases, decreasing the repair rate, and increasing toxicity (Franken et al., 2012; Antonelli et al., 2015).

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 on radiation stressors 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 Supporting this KER

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

Overall Weight of Evidence for this KER: High

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The biological rationale linking the direct deposition of energy on DNA with an increase in DSB formation is strongly supported by numerous literature reviews that are available on this topic (J .F. Ward, 1988; Lipman, 1988; Hightower, 1995; Terato & Ide, 2005; Goodhead, 2006; Kim & Lee, 2007; Asaithamby et al., 2008; Hada & Georgakilas, 2008; Jeggo, 2009; Clement, 2012; Okayasu, 2012b; Stewart, 2012; M. E. Lomax et al., 2013; EPRI, 2014; Hamada, 2014; Moore et al., 2014; Desouky et al., 2015; Ainsbury, 2016; Foray et al., 2016; Hamada & Sato, 2016; Hamada, 2017a; Sage & Shikazono, 2017; Chadwick, 2017; Wang et al., 2021; Nagane et al., 2021; Sylvester et al., 2018; Baselet et al., 2019). Ionizing radiation can be in the form of high energy particles (such as alpha particles, beta particles, or charged ions) or high energy photons (such as gamma-rays or X-rays). Ionizing radiation can break the DNA within chromosomes both directly and indirectly, as shown through using velocity sedimentation of DNA through neutral and alkaline sucrose gradients. The most direct path entails a collision between a high-energy particle or photon and a strand of DNA.

Additionally, excitation of secondary electrons in the DNA allows for a cascade of ionization events to occur, which can lead to the formation of multiple damage sites (Joiner, 2009). As an example, high-energy electrons will traverse a DNA molecule in a mammalian cell within 10^-18 s and 10^-14 s, resulting in 100,000 ionizing events per 1 Gy dose in a 10 μm cell (Joiner, 2009). The amount of damage can be influenced by factors such as the cell cycle stage and chromatin structure. It has been shown that in more condensed, packed chromatin structures such as those present in intact cells and heterochromatin, it is more difficult for the DNA to be damaged (Radulescu et al., 2006; Agrawala et al., 2008; Falk et al., 2008; Venkatesh et al., 2016). In contrast, DNA damage is more easily induced in lightly-packed chromatin such as euchromatin and nucleoids, (Radulescu et al., 2006; Falk et al., 2008; Venkatesh et al., 2016).

Of the possible radiation-induced DNA damage types, DSB is considered to be the most harmful to the cell, as there may be severe consequences if this damage is not adequately repaired (Khanna & Jackson, 2001; Smith et al., 2003; Okayasu, 2012a; M. E. Lomax et al., 2013; Rothkamm et al., 2015).

A considerable fraction of DSBs can also be formed in cells through indirect mechanisms. In this case, deposited energy can split water molecules near DNA, which can generate a significant quantity of reactive oxygen species in the form of hydroxyl free radicals (Ward, 1988; Wolf, 2008; Desouky et al., 2015; Maier et al., 2016, Cencer et al., 2018; Bains, 2019; Ahmadi et al., 2021). Estimates using models and experimental results suggest that hydroxyl radicals may be present within nanoseconds of energy deposition by radiation (Yamaguchi et al., 2005). These short-lived but highly reactive hydroxyl radicals may react with nearby DNA. This will produce DNA damage, including single-strand breaks and DSBs (Ward, 1988; Sasaki, 1998; Desouky et al., 2015; Maier et al., 2016). DNA breaks are especially likely to be produced if the sugar moiety is damaged, and DSBs occur when two single-strand breaks are in close proximity to each other (Ward, 1988).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Empirical data strongly supports this KER. The evidence presented below is summarized in table 1. The types of DNA damage produced by ionizing radiation and the associated mechanisms, including the induction of DSBs, are reviewed by Lomax et al. (2013) and documents produced by international radiation governing frameworks (Valentin, 1998; UNSCEAR, 2000). Other reviews also highlight the relationship between the deposition of energy by radiation and DSB induction, and discuss the various methods available to detect these DSBs (Terato & Ide, 2005; Rothkamm et al., 2015; Sage & Shikazono, 2017). A visual representation of the time frames and dose ranges probed by the dedicated studies discussed here is shown in Figures 1 & 2 below.

Figure 1: Plot of example studies (y-axis) against equivalent dose (Sv) used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom.

Figure 2: Plot of example studies (y-axis) against time scales used to determine the empirical link between direct deposition of energy and DSBs. The z-axis denotes the equivalent dose rate used in each study. The y-axis is ordered from low LET to high LET from top to bottom.

Dose Concordance

There is evidence in the literature suggesting a dose concordance between the direct deposition of energy by ionizing radiation and the incidence (Grudzenski et al., 2010) of DNA DSBs. Results from in vitro (Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Frankenberg et al., 1999; Rogakou et al., 1999; Belli et al., 2000; Sutherland et al., 2000; Lara et al., 2001; Rydberg et al., 2002; Baumstark-Kham et al., 2003; Rothkamm and Lo, 2003; Long, 2004; Kuhne et al., 2005; Sudprasert et al., 2006; Beels et al., 2009; Grudzenski et al., 2010; Liao, 2011; Franken et al., 2012; Bannik et al., 2013; Shelke & Das, 2015; Antonelli et al., 2015; Markiewicz et al., 2015; Allen, 2018; Dalke, 2018; Bains, 2019; Ahmadi et al., 2021; Sabirzhanov et al., 2020; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017), in vivo (Reddy, 1998; Sutherland et al., 2000; Rube et al., 2008; Beels et al., 2009; Grudzenski et al., 2010; Markiewicz et al., 2015; Barnard, 2018; Barnard, 2019; Barnard, 2022; Schmal et al., 2019; Barazzuol et al., 2017; Geisel et al., 2012), ex vivo (Rube et al., 2008; Flegal et al., 2015) and simulation studies (Charlton et al., 1989) suggest that there is a positive, linear, dose-dependent increase in DSBs with increasing deposition of energy across a wide range of radiation types (iron ions, X-rays, ultrasoft X-rays, gamma-rays, photons, UV light, and alpha particles) and radiation doses (1 mGy - 100 Gy) (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al., 2000; de Lara et al., 2001; Baumstark-Khan et al., 2003; Rothkamm & Lo, 2003; Kuhne et al., 2005; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Shelke & Das, 2015; Antonelli et al., 2015; Dalke, 2018; Barazzuol et al., 2017; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017; Geisel et al., 2012). DSBs have been predicted to occur at energy deposition levels as low as 75 eV (Charlton et al., 1989).

Time Concordance

There is evidence suggesting a time concordance between the direct deposition of energy and the incidence of DSBs. A number of different models and experiments have provided evidence of ionizing radiation-induced foci (IRIF), which can be used to infer DSB formation seconds (Mosconi et al., 2011) or minutes after radiation exposure (Rogakou et al., 1999; Rothkamm and Löbrich, 2003; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Sabirzhanov et al., 2020; Rombouts et al., 2013; Nübel et al., 2006; Baselet et al., 2017; Zhang et al., 2017).

Essentiality

Deposition of energy is essential for DNA strand breaks. They can also be caused through other routes, such as oxidative stress (Cadet et al., 2012), but under normal physiological conditions deposition of energy is necessary. This was tested through many studies using various indicators such as 53BP1 foci/cell, γH2AX foci/cell, DNA migration, and the amount of DNA in tails for the comet assay. Various organisms such as humans, mice, rabbits, guinea pigs, and cattle were used. They showed that without the deposition of energy, there was only a negligible amount of DNA strand breaks (Aufderheide et al., 1987; Sidjanin, 1993; Bucolo, 1994; Reddy, 1998; Rogers, 2004; Bannik et al., 2013; Dalke, 2018; Bains, 2019; Barnard, 2019; Barnard, 2021).

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:

Studies have shown that dose-rates (Brooks et al., 2016) and radiation quality (Sutherland et al., 2000; Nikjoo et al., 2001; Jorge et al., 2012) are factors that can influence the dose-response relationship.
Low-dose radiation has been observed to have beneficial effects and may even invoke protection against spontaneous genomic damage (Feinendegen, 2005; Day et al., 2007; Feinendegen et al., 2007; Shah et al., 2012; Nenoi et al., 2015; Dalke, 2018). This protective effect has been documented in in vivo and in vitro, as reviewed by ICRP (2007) and UNSCEAR (2008) and can vary depending on the cell type, the tissue, the organ, or the entire organism (Brooks et al., 2016).
Depositing ionizing energy is a stochastic event; as such this can influence the location, degree and type of DNA damage imparted on a cell. As an example, studies have shown that mitochondrial DNA may also be an important target for genotoxic effects of ionizing radiation (Wu et al., 1999).

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
Nitroxides	Increased concentration	Decreased DNA strand breaks.	DeGraff et al., 1992; Citrin & Mitchel, 2014
5-fluorouracil	Increased concentration	Increased DNA strand breaks.	De Angelis et al., 2006; Citrin & Mitchel, 2014
Thiols	Increased concentration	Decreased DNA strand breaks.	Milligan et al., 1995; Citrin & Mitchel, 2014
Cisplatin	Increased concentration	Decreased DNA break repair.	Sears & Turchi; Citrin & Mitchel, 2014

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 DSBs can be predicted upon exposure to ionizing radiation. This is dependent on the biological model, the type of radiation and the radiation dose. In general, 1 Gy of radiation is thought to result in 3000 damaged bases (Maier et al., 2016), 1000 single-strand breaks, and 40 DSBs (Ward, 1988; Foray et al., 2016; Maier et al., 2016) . The table below provides representative examples of the calculated DNA damage rates across different model systems, most of which are examining DNA DSBs.

Dose Concordance

The following tables provide representative examples of the relationship, unless otherwise indicated, all data is significantly significant.

Reference	Experiment Description	Result
Ward, 1988	In vitro. Cells containing approximately 6 pg of DNA were exposed to 1 Gy.	Under the assumption of 6 pg of DNA per cell. 60 eV of energy deposited per event over a total of 1 Gy. Deoxyribose (2.3 pg/cell): 14,000 eV deposited, 235 events. Bases (2.4 pg/cell): 14.7 keV deposited, 245 events. Phosphate (1.2 pg/cell): 7,300 eV deposited, 120 events. Bound water (3.1 pg/cell): 19 keV deposited, 315 events. Inner hydration shell (4.2 pg/cell): 25,000 eV deposited 415 events.
Charlton, 1989	In-silico. A computer simulation/model was used to test various types of radiation with doses from 0 to 400 eV (energy deposited) on the amount of DNA damage produced.	Simulated dose-concordance prediction of increase in number of DSBs/54 nucleotide pairs as direct deposition of energy increases in the range 75-400 eV. In the range 100 - 150 eV: 0.38 DSBs/54 nucleotide pairs and at 400 eV: ~0.80 DSBs per 64 nucleotide pairs.
Sutherland, 2000	In vitro. Human cells were exposed to ¹³⁷Cs γ-rays (0 – 100 Gy, 0.16 – 1.6 Gy/min). The frequency of DSBs was determined using gel electrophoresis.	Using isolated bacteriophage T7 DNA and 0-100 Gy of γ radiations, observed a response of 2.4 DSBs per megabase pair per Gy.
Rogakou et al., 1999	In vitro. Normal human fibroblasts (IMR90) and human breast cancer cells (MCF7 were exposed to 0.6 and 2 Gy ¹³⁷Cs γ-rays delivered at 15.7 Gy/min. The number of DSBs were determined by immunoblotting for γ-H2AX.	Radiation doses of 0.6 Gy & 2 Gy to normal human fibroblasts (IMR90) and MCF7 cells resulted in 10.1 & 12.2 DSBs per nucleus on average (0.6 Gy), respectively; increasing to 24 & 27.1 DSBs per nucleus (2 Gy).
Kuhne et al., 2005	In vitro. Primary human skin fibroblasts (HSF2) were exposed to 0 – 70 Gy ⁶⁰Co γ-rays (0.33 Gy/min), X-rays (29 kVp, 1.13 Gy/min), and CKX-rays (0.14 Gy/min). The number of DSBs were determined with pulsed-field gel electrophoresis.	γ-ray and X-ray irradiation of primary human skin fibroblasts (HSF2) at 0 - 70 Gy. γ-rays: (6.1 ± 0.2) x 10-9 DSBs per base pair per Gy, X-rays: (7.0 ± 0.2) x 10-9 DSBs per base pair per Gy. CKX -rays: (12.1 ± 1.9) x 10-9 DSBs per base pair per Gy.
Rothkamm, 2003	In vitro. Primary human fibroblast cell lines MRC-5 (lung), HSF1 and HSF2 (skin), and180BR (deficient in DNA ligase IV) were exposed to 1 mGy – 100 Gy X-rays (90 kV). Low doses were delivered at 6 – 60 mGy/min and high doses were delivered at 2 Gy/min. The number of DSBs were determined with pulsed-field gel electrophoresis.	X-ray irradiation of primary human fibroblasts (MRC-5) in the range 1 mGy - 100 Gy, 35 DSBs per cell per Gy.
Grudzenski et al, 2010	In vitro. Primary human fibroblasts (HSF1) and C57BL/6NCrl adult mice were exposed to X-rays (2.5 – 200 mGy, 70 mGy/min), and photons (10 mGy – 1 Gy, 2 Gy/min (100 mGy and 1 Gy), and 0.35 Gy/min (10 mGy)). γ-H2AX immunofluorescence was observed to determine DSBs.	X-rays irradiating primary human fibroblasts (HSF1) in the range 2.5 - 100 mGy yielded a response of 21 foci per Gy. When irradiating adult C57BL/6NCrl mice with photons a response of 0.07 foci per cell at 10 mGy was found. At 100 mGy the response was 0.6 foci per cell and finally, at 1 Gy; 8 foci per cell.
de Lara, 2001	In vitro. Chinese hamster cells (V79-4) were exposed to 0 – 20 Gy of⁶⁰Co γ-rays (2 Gy/min), and ultrasoft X-rays (0.7 – 35 Gy/min): carbon-K shell (0.28 keV), copper L-shell (0.96 keV), aluminum K-shell (1.49 keV), and titanium K-shell (4.55 keV). The number of DSBs were determined with pulsed-field gel electrophoresis.	V79-4 cells irradiated with γ-rays and ultrasoft X-rays (carbon K-shell, copper L-shell, aluminium K-shell and titanum K-shell) in the range 0 - 20 Gy. Response (DSBs per Gy per cell): γ-rays: 41, carbon K-shell: 112, copper L-shell: 94, aluminum K-shell: 77, titanium K-shell: 56.
Rübe et al., 2008	In vivo. Brain, lung, heart and small intestine tissue from adult SCID, A-T, BALB/c and C57BL/6NCrl mice; Whole blood and isolated lymphocytes from BALB/c and C57BL/6NCrl mice were exposed to 0.1 – 2 Gy of photons (whole body irradiation, 6 MV, 2 Gy/min) and X-rays (whole body irradiation, 90 kV, 2 Gy/min). γ-H2AX foci were determined with immunochemistry to measure DSBs.	Linear dose-dependent increase in DSBs in the brain, small intestine, lung and heart of C57BL/6CNrl mice after whole-body irradiation with 0.1 - 1.0 Gy of radiation. 0.8 foci per cell (0.1 Gy) and 8 foci per cell (1 Gy).
Antonelli et al., 2015	In vitro. Primary human foreskin fibroblasts (AG01522) were exposed to 0 – 1 Gy of ¹³⁶Cs γ-rays (1 Gy/min), protons (0.84 MeV, 28.5 keV/um), carbon ions (58 MeV/u, 39.4 keV/um), and alpha particles (americium-241, 0.75 MeV/u, 0.08 Gy/min, 125.2 keV/um). γ-H2AX foci were determined with immunochemistry to measure DSBs.	Linear dose-dependent increase in the number of DSBs from 0 - 1 Gy for γ-rays and alpha particles as follows: γ-rays: 24.1 foci per Gy per cell nucleus, alpha particles: 8.8 foci per Gy per cell nucleus.
Barnard et al., 2019	In vivo. 10-week-old female C57BL/6 mice were whole-body exposed to 0.5, 1, and 2 Gy of 60Co γ-rays at 0.3, 0.063, and 0.014 Gy/min. p53 binding protein 1 (53BP1) foci were determined via immunofluorescence.	Central LECs showed a linear increase in mean 53BP1 foci/cell with the maximum dose and dose-rate displaying a 78x increase compared to control. Peripheral LECs and lower dose rates displayed similar results, with slightly fewer foci. Although an increase in dose-response was observed, an inverse-dose rate response was reported, with higher 53BP1 foci persisting at lower dose rates.
Ahmadi et al., 2021	In vitro. Human LEC cells were exposed to 137Cs γ-rays at doses of 0, 0.1, 0.25, and 0.5 Gy and dose rates of 0.065 and 0.3 Gy/min. DNA strand breaks were measured using the comet assay.	Human LECs showed a gradual increase in the tail from the comet assay with the maximum dose and dose-rate displaying a 3.7x increase compared to control. Lower dose-rates followed a similar pattern with a lower amount of strand breaks.
Hamada et al., 2006	In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy X-rays at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined 6 – 7 minutes after irradiation through fluorescence microscopy.	Cells displayed a linear increase in the number of H2AX foci/cell, with the maximum dose displaying a 125x increase compared to control.
Dubrova & Plumb, 2002		At 1 Gy observe 70 DSBs, 1000 single-strange breaks and 2000 damaged DNA bases per cell per Gy.
Sabirzhanov et al., 2020	In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure γ-H2AX, p-ataxia telangiectasia mutated (ATM) and p- ATM/RAD3-related (ATR) levels.	In rat cortical neurons, p-ATM increased at 2, 8, and 32 Gy, with a 15-fold increase at 8 and 32 Gy. γ-H2AX levels increased at 8 and 32 Gy.
Geisel et al., 2012	In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by γ-H2AX fluorescence.	There was a correlation between effective dose (in mSv) and DSBs. For both conventional coronary angiography and computed tomography, a dose of 10 mSv produced about 2-fold more DNA DSBs than a dose of 5 mSv.
Ungvari et al., 2013	In vitro. Rat cerebromicrovascular endothelial cells and hippocampal neurons were irradiated with 2-10 Gy of ¹³⁷Cs gamma rays. DNA strand breaks were assessed with the comet assay.	DNA damage increased at all doses (2-10 Gy). In the control, less than 5% of DNA was in the tail, while by 6 Gy, 35% of the DNA was in the tail in cerebromicrovascular endothelial cells and 25% was in the tail in neurons.
Rombouts et al., 2013	In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with various doses of X-rays (0.25 Gy/min). γ-H2AX foci were assessed with immunofluorescence.	More γ-H2AX foci were observed at higher doses in both cell types. In human umbilical vein endothelial cells, few foci/nucleus were observed at 0.05 Gy, with about 23 at 2 Gy. In EA.hy926 cells, few foci/nucleus were observed at 0.05 Gy, with about 37 at 2 Gy.
Baselet et al., 2017	In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for γ-H2AX and 53BP1 foci.	Doses of 0.05 and 0.1 Gy did not increase the number of γ-H2AX foci, but 0.5 Gy increased foci number by 5-fold and 2 Gy by 15-fold. A dose of 0.05 Gy did not increase the number of 53BP1 foci, but 0.1 Gy, 0.5 Gy and 2 Gy increased levels by 3-fold, 7-fold and 8-fold, respectively.

Time Concordance

Reference	Experiment Description	Result
Rogakou et al., 1999	In vitro. Normal human fibroblasts (IMR90), human breast cancer cells (MCF7), human astrocytoma cells (SF268), Indian muntjac Muntiacus muntjak normal skin fibroblasts, Xenopus laevisA6 normal kidney cells, Drosophila melanogaster epithelial cells, and Saccharomyces cerevisiae were exposed to 0.6, 2, 20, 22, 100, and 200 Gy 137Cs γ-rays. Doses below 20 Gy were delivered at 15.7 Gy/min and other doses were delivered in 1 minute. DNA breaks were visualized using γ-H2AX antibodies and microscopy.	DSBs were present at 3 min and persisted from 15 - 60 min.
Hamada & Woloschak, 2017	In vitro. human LECs were exposed to 0.025 Gy X-rays at 0.42 – 0.45 Gy/min. 53BP1 foci were measured via indirect immunofluorescence.	In cells immediately exposed to 0.025 Gy, the level of 53BP1 foci/cell increased to 3.3x relative to control 0.5 h post-irradiation.
Hamada et al., 2006	In vitro. Primary normal human diploid fibroblast (HE49) cells were exposed to 0.1, 0.5, and 4 Gy (deposition of energy) at 240 kV with a dose rate of 0.5 Gy/min. The number of H2AX foci/cell, which represented DNA strand breaks, was determined through fluorescence microscopy.	In cells immediately exposed to 0.5 Gy, 11% of cells had 18 foci six min post-irradiation, compared to 90% of controls having 0 foci.
Acharya et al., 2010	In vitro. Human neural stem cells were exposed to 1, 2 and 5 Gy of γ-rays at a dose rate of 2.2 Gy/min. The levels of γ-H2AX phosphorylation post irradiation were assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) analysis and γ-H2AX foci enumeration.	The number of cells positive for nuclear γ-H2AX foci peaked at 20 min post-irradiation. After 1h, this level quickly declined.
Schmal et al., 2019	In vivo. Juvenile and adult C57BL/6 mice were exposed to whole body 6-MV photons at 2 Gy/min. Irradiations were done in 5x, 10x, 15x and 20x fractions of 0.1 Gy. Double staining for NeuN and 53BP1 was used to quantify DNA damage foci and the possible accumulation in the hippocampal dentate gyrus.	To assess possible accumulation of persisting 53BP1-foci during fractionated radiation, juvenile and adult mice were examined 72 h after exposure to 5×, 10×, 15×, or 20× fractions of 0.1 Gy, compared to controls. The number of persisting 53BP1-foci increased significantly in both juvenile and adult mice during fractionated irradiation (maximum at 1 m post-IR).
Dong et al., 2015	In vivo. C57BL/6J mice were exposed to 2 Gy of X-rays at 2 Gy/min using a 6 MV source. γ-H2AX foci were assessed with immunofluorescence in the brain.	At 0.5 h, about 14 γ-H2AX foci/cell were present. This decreased linearly to about 2 foci/cell at 24 h, with no foci/cell from 48 h to 6 weeks.
Barazzuol et al., 2017	In vivo. C57BL/6 mice were exposed to 0.1 or 2 Gy of X-rays (250 kV) at a rate of 0.5 Gy/min. 53BP1 foci were quantified with immunofluorescence in neural stem cells and neuron progenitors in the lateral ventricle.	At both 0.5 and 6 h post-irradiation, increased 53BP1 foci were observed, with the highest level at 0.5 h.
Sabirzhanov et al., 2020	In vitro. Rat cortical neurons were exposed to 2, 8 or 32 Gy of X rays (320 kV) at a dose rate of 1.25 Gy/min. Western blot was used to measure γ-H2AX, p-ATM and p-ATR levels.	In rat cortical neurons, γ-H2AX, p-ATM and p-ATR all increased at 30 minutes post-irradiation, with a sustained increase until 6 h.
Zhang et al., 2017	In vitro. HT22 hippocampal neuronal cellsT were irradiated with X-rays (320 kVp) at 8 or 12 Gy at a dose rate of 4 Gy/min. The comet assay was preformed to assess the DNA double strand breaks in HT22 cells. Western blot was used to measure γ-H2AX and p-ATM.	At 8 Gy, the comet assay showed an increased tail moment at both 30 minutes and 24 h post-irradiation. At 12 Gy, p-ATM was increased over 4-fold at both 30 minutes and 1 h post-irradiation. γ-H2AX was increased over 3-fold at 30 minutes post-irradiation and almost 2-fold at 1 and 24 h.
Geisel et al., 2012	In vivo. Patients with suspected coronary artery disease receiving X-rays from computed tomography or conventional coronary angiography had levels of DSBs assessed in blood lymphocytes by γ-H2AX fluorescence.	DSBs were increased at 1 h post-irradiation and returned to pre-irradiation levels by 24 h.
Park et al., 2022	In vitro. Human aortic endothelial cells were irradiated with 137Cs gamma rays at 4 Gy (3.5 Gy/min). γ-H2AX was measured with western blot. p-ATM and 53BP1 were determined with immunofluorescence.	γ-H2AX, p-ATM, and 53BP1 were shown increased at 1 h post-irradiation and slightly decreased for the rest of the 6 h but remained elevated above the control.
Kim et al., 2014	In vitro. Human umbilical vein endothelial cells were irradiated with 4 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence.	γ-H2AX foci greatly increased at 1 and 6 h post-irradiation, with the greatest increase at 1 h.
Dong et al., 2014	In vitro. Human umbilical vein endothelial cells were irradiated with 2 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence.	γ-H2AX foci increased 8-fold at 3 h, 7-fold at 6 h, and 2-fold at 12 and 24 h post-irradiation.
Rombouts et al., 2013	In vitro. EA.hy926 cells and human umbilical vein endothelial cells were irradiated with X-rays (0.25 Gy/min). γ-H2AX foci were assessed with immunofluorescence.	The greatest increase in γ-H2AX foci was observed 30 minutes post-irradiation, while levels were still slightly elevated at 24 h.
Nübel et al., 2006	In vitro. Human umbilical vein endothelial cells were irradiated with gamma rays at 20 Gy. DNA strand breaks were assessed with the comet assay and western blot for γ-H2AX.	The olive tail moment increased 5-fold immediately after irradiation and returned to control levels by 4 h. A large increase in γ-H2AX was observed at 0.5 h post-irradiation, with lower levels at 4 h but still above the control.
Baselet et al., 2017	In vitro. Human telomerase-immortalized coronary artery endothelial cells were irradiated with various doses of X-rays (0.5 Gy/min). Immunocytochemical staining was performed for γ-H2AX and 53BP1 foci.	Increased γ-H2AX and 53BP1 foci were observed at 0.5 h post-irradiation, remaining elevated at 4 h but returning to control levels at 24 h.
Gionchiglia et al., 2021	In vivo. Male CD1 and B6/129 mice were irradiated with X-rays at 10 Gy. Brain sections were single or double-stained with antibodies against γ-H2AX and p53BP1.	In the forebrain, cerebral cortex, hippocampus and subventricular zone (SVZ)/ rostral migratory stream (RMS)/ olfactory bulb (OB), γH2AX and p53BP1 positive cells increased at both 15 and 30 minutes post-irradiation, with the greatest increase at 30 minutes.

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 between the deposition of energy and the frequency of DSBs. In studies encompassing a variety of biological models, radiation types and radiation doses, a positive, linear relationship was found between the radiation dose and the number of DSBs (Aufderheide et al., 1987; Sidjanin, 1993; Frankenberg et al., 1999; Sutherland et al., 2000; de Lara et al., 2001; Baumstark-Khan et al., 2003; Rothkamm & Lo, 2003; Kuhne et al., 2005; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Shelke & Das, 2015; Antonelli et al., 2015; Hamada, 2017b; Dalke, 2018; Barazzuol et al., 2017; Geisel et al., 2012; Ungvari et al., 2013; Rombouts et al., 2013; Baselet et al., 2017). There were, however, at least four exceptions reported. When human blood lymphocytes were irradiated with X-rays in vitro, a linear relationship was only found for doses ranging from 6 - 500 mGy; at low doses from 0 - 6 mGy, there was a quadratic relationship reported (Beels et al., 2009). Secondly, simulation studies predicted that there would be a non-linear increase in DSBs as energy deposition increased, with a saturation point at higher LETs (Charlton et al., 1989). Furthermore, primary normal human fibroblasts exposed to 1.2 – 5 mGy X-rays at 5.67 mGy/min showed a supralinear relationship, indicating at low doses, the DSBs are mostly due to radiation-induced bystander effects. Doses above 10 mGy showed a positive linear relationship (Ojima et al., 2008). Finally, in the human lens epithelial cell line SRA01/04, DNA strand breaks appeared immediately after exposure to UVB (0.14 J/cm2) and were repaired after 30 minutes. They then reappeared after 60 and 90 minutes. Both were once again repaired within 30 minutes. However, the two subsequent stages of DNA strand breaks did not occur when exposed to a lower dose of UVB (0.014 J/cm2) (Cencer et al., 2018).

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 DSBs likely occur within seconds to minutes of energy deposition by ionizing radiation. In a variety of biological models, the presence of DSBs has been well documented within 10 - 30 minutes of radiation exposure (Rogakou et al., 1999; Rube et al., 2008; Beels et al., 2009; Kuefner et al., 2009; Grudzenski et al., 2010; Antonelli et al., 2015; Acharya et al., 2010; Dong et al., 2015; Barazzuol et al., 2017; Sabirzhanov et al., 2020; Rombouts et al., 2013; Nübel et al., 2006; Baselet et al., 2017; Zhang et al., 2017; Gionchiglia et al., 2021); there is also evidence that DSBs may actually be present within 3 - 5 minutes of irradiation (Kleiman, 1990; Rogakou et al., 1999; Rothkamm & Lo, 2003; Rube et al., 2008; Grudzenski et al., 2010; Cencer et al., 2018). Interestingly, one study that focussed on monitoring the cells before, during and after irradiation by taking photos every 5, 10 or 15 seconds found that foci indicative of DSBs were present 25 and 40 seconds after collision of the alpha particles and protons with the cell, respectively. The number of foci were found to increase over time until plateauing at approximately 200 seconds after alpha particle exposure and 800 seconds after proton exposure (Mosconi et al., 2011).

After the 30 minute mark, DSBs have been shown to rapidly decline in number. By 24 hours post-irradiation, DSB numbers had declined substantially in systems exposed to radiation doses between 40 mGy and 80 Gy (Aufderheide et al., 1987; Baumstark-Khan et al., 2003; Rothkamm & Lo, 2003; Rube et al., 2008; Grudzenski et al., 2010; Bannik et al., 2013; Markiewicz et al., 2015; Russo et al., 2015; Antonelli et al., 2015; Dalke, 2018; Bains, 2019; Barnard, 2019; Ahmadi et al., 2021; Dong et al., 2015; Dong et al., 2014; Sabirzhanov et al., 2020; Rombouts et al., 2013; Baselet et al., 2017; Gionchiglia et al., 2021), with the sharpest decrease documented within the first 5 h (Kleiman, 1990; Sidjanin, 1993; Rogakou et al., 1999; Rube et al., 2008; Kuefner et al., 2009; Grudzenski et al., 2010; Bannik, 2013; Markiewicz et al., 2015; Shelke and Das, 2015; Cencer et al., 2018; Acharya et al., 2010; Park et al., 2022; Kim et al., 2014; Nübel et al., 2006). Interestingly, DSBs were found to be more persistent when they were induced by higher LET radiation (Aufderheide et al., 1987, Baumstark-Khan et al., 2003; Antonelli et al., 2015).

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 and human In vitro models that do not specify the sex.

References

List of the literature that was cited for this KER description. More help

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