dendritic spines and the performance of mice in the OiP task (Parihar et al., 2015). This study also demonstrated dose-dependent decreases in the total number of spines, total dendritic length, number of branch points, number of dendritic branches, and DI in both the NOR and OiP tasks when exposed to 0.05 and 0.3 Gy of 16O and 48Ti.

Changes in the levels of neurogenesis also exhibit dose-concordance with cognitive deficits. Whoolery et al. (2017) identified a 0.8- and 0.4-fold decrease in the number of Ki-67+ cells in male mice exposed to 0.2 and 1 Gy of 28Si particles, respectively. The mice also demonstrated a decrease in percentage freezing by 0.4-fold in contextual FC, indicative of impaired learning and memory, when exposed to 0.2 Gy, although no significant changes in learning and memory were seen at 1 Gy. Another study identified dose-dependent decreases in the numbers of Ki67+ and BrdU+ cells in three age groups (postnatal day (PD) 21, PD 50, and PD 70) in rats exposed to 0.3, 3, and 10 Gy of ionizing radiation (Achanta, Fuss & Martinez, 2009). In PD 21 rats, dose-dependent decreases in freezing response were observed at doses of 0.3 and 10 Gy during the trace fear conditioning/testing. These results show that higher doses of irradiation lead to greater alterations in neuronal integrity, correlated with greater cognitive impairments. Many other studies showed decreased neurogenesis or increased neurodegeneration and impaired learning and memory at doses from 0.3 to 25 Gy, with stressors like X-rays, gamma rays, carbon ions and iron ions (Achanta, Fuss & Martinez, 2009; Akiyama et al., 2001; Hodges et al., 1998; Madsen et al., 2003; Miry et al., 2021; Raber et al., 2004; Rola et al., 2004; Sorokina et al., 2021; Winocur et al., 2006).

Neuron excitation has also been measured to assess neuron integrity. After 0.18 Gy of 252Cf neutron, with an activity of 1.6 GBq, excitatory signaling in CA1 neurons of mice was reduced and both NOR and OiP showed decreased learning and memory (Acharya et al., 2019).

Synaptic composition in hippocampal neurons was assessed as well to determine neuron integrity. GluR1, involved in hippocampal-dependent working memory, and recognition memory were both reduced after 0.5 Gy of simulated galactic cosmic radiation (GCR) (Krukowski et al., 2018).

## Time Concordance

Evidence supporting time concordance between abnormal neural remodeling and impaired learning and memory come from several studies. Whoolery et al. (2017) found that the number of Ki-67+ cells decreased 24 hours after 0.2 Gy of 28Si radiation of mice, while contextual FC 3 months after radiation showed impaired memory. Similarly, after 10 Gy X-rays, cell proliferation and the number of immature neurons decreased by 95% in mice after 3 months, and a Barnes maze showed impaired hippocampal-dependent spatial learning and memory measured after proliferation at 3 months as well (Raber et al., 2004). After mice were irradiated with 16O and 48Ti particles at 0.05 and 0.30 Gy, dendritic complexity and spine density were reduced and synaptic spine puncta were increased 15 weeks post-irradiation while learning and memory was impaired 15 and 24 weeks post-irradiation (Parihar et al., 2016). Neurogenesis was reduced immediately after 3 Gy 300 kVp X-ray radiation in rats and remained low 2 and 6 weeks later, and memory was impaired 1, 3 and non-significantly at 7 weeks post-irradiation (Madsen et al., 2003). Rats irradiated with gamma rays showed decreased neurogenesis and impaired performance during FC both 4 weeks post-irradiation (Winocur et al., 2006). In a longer-term study, neurogenesis was impaired 1 and 3 months after 5 Gy X-ray radiation in mice, while the Morris Water Maze (MWM) demonstrated impaired learning and memory also at 3 months (Rola et al., 2004). This trend continued after 9 months where both learning and memory through NOR testing and abnormal neural remodeling through mushroom and thin spine density were reduced after mice were irradiated with 0.05 Gy 16O particles (Howe et al., 2019). Hodges et al. (1998) showed abnormal neural remodeling at 41 weeks after 250 kVp X-ray irradiation and impaired learning and memory from 35 to 44 weeks post-irradiation.

## Incidence Concordance

No available data.

## Essentiality
No identified studies applied countermeasures to support the causal relationship between abnormal neural remodeling and impaired learning and memory.

## Uncertainties and Inconsistencies

* One study observed cognitive deficits in the one- and two-way avoidance, press-lever avoidance, and water maze tests, but through pathological examination, no abnormalities were seen in the brains (white matter and axons were normal with no inflammation or glial response) (Lamproglou et al., 1995). This is indicative of impaired cognition without any changes in neuron integrity.

* At 25 Gy of X-irradiation, Hodges et al. (1998) observed both abnormal neural remodeling and impaired learning and memory. However, at 20 Gy in the same study, abnormal neural remodeling was not observed.

* Whoolery et al. (2017) found that abnormal neural remodeling was high at 1 Gy and low and 0.2 Gy but found impaired learning and memory at 0.2 Gy and not at 1 Gy.

* Miry et al. (2021) found that 1 Gy of 56Fe particles led to increased learning and memory 20 months after exposure, although this is part of the compensatory or repair mechanisms following early suppressive changes.

## Known modulating factors

<table><thead><tr><td>Modulating factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Sex</td><td>Female mice</td><td>Female mice were protected from the GCR-induced deficits in learning and memory and did not show changes in synapse levels</td><td>Krukowski et al., 2018</td></tr><tr><td>Antioxidant</td><td>α-tocopherol</td><td>Improved global cognitive ability, memory and executive function</td><td>Hladik & Tapio, 2016</td></tr><tr><td>Stem cells</td><td>Human neural stem cell treatment</td><td>Increased neurogenesis in the brain after radiation and improved learning and memory</td><td>Hladik & Tapio, 2016</td></tr><tr><td>Drug</td><td>Ramipril (Angiotensin converting enzyme inhibitor)</td><td>Mitigated neurodegeneration and prevented cognitive impairment</td><td>Hladik & Tapio, 2016</td></tr><tr><td>Drug</td><td>Memantine (NMDA receptor antagonist)</td><td>Reduced rate of memory decline after radiotherapy</td><td>Bálentová & Adamkov, 2020</td></tr><tr><td>Hypoxia</td><td>Systemic hypoxia</td><td>Systemic hypoxia reversed the effects of radiation on learning and memory</td><td>Bálentová & Adamkov, 2020; Hladik & Tapio, 2016</td></tr></tbody></table>

## Quantitative Understanding of the Linkage

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Design</td><td>Summary</td></tr></thead><tbody><tr><td>Acharya et al., 2019</td><td>In vivo. Mice were irradiated with neutrons from a 252Cf source over 180 days at 1 mGy/day. Electrophysiological measurements were taken from CA1 pyramidal neurons to assess neuron integrity. NOR, OiP and FE were used to assess learning and memory.</td><td>Spontaneous excitatory postsynaptic current (sEPSC) frequency decreased with a mean difference of approximately -0.81 Hz. Field excitatory postsynaptic potential (fEPSP) slopes also decreased in the dorsal hippocampus and cortical layer. Avoidance behavior was increased by 20 sec, time spent interacting was decreased by 10 sec, and the discrimination index in both NOR and object in place OiP were decreased by 19.</td></tr><tr><td>Parihar et al., 2016</td><td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with charged particles (16O and 48Ti) at 600 MeV/amu (0.05 to 0.25 Gy/min). In the medial prefrontal cortex, neuron integrity was measured through spine density, dendritic complexity and postsynaptic density protein 95 synaptic puncta,</td><td>Dendritic complexity and spine density were both decreased about 0.7-fold, while synaptic puncta were increased about 1.5-fold after both types of radiation at 0.05 and 0.3 Gy. Impairment in cognitive ability was observed at both 0.05 and 0.3 Gy. The impairment was typically greater in 48Ti than 16O. The correlation between the OiP DI and number of synaptic puncta was significant at 0.05 Gy 48Ti and 0.3 Gy</td></tr></tbody></table>
<table>
  <tr>
    <td></td>
    <td>while NOR, OiP, TO and FE tests were done to assess learning and memory. Wistar rats were used in an attentional set-shifting (ATSET) test.</td>
    <td>48Ti and 16O. The correlation between the OiP DI and number of dendritic spines was significant at 0.05 Gy 48Ti and 16O and 0.3 Gy 48Ti.</td>
  </tr>
  <tr>
    <td>Parihar et al., 2015</td>
    <td>In vivo. Thy1-1 EGFP transgenic mice were irradiated with 600 MeV/amu 16O and 48Ti particles (0.5-1 Gy/min). In the medial prefrontal cortex, neuron integrity was measured through spine density, dendritic complexity and postsynaptic density protein 95 synaptic puncta, while learning and memory was assessed with NOR and OiP.</td>
    <td>Dendritic complexity was decreased 0.6-fold, spine density decreased 0.5-fold and synaptic puncta increased 1.4-fold for both radiation types at 0.05 and 0.3 Gy. The DI for NOR and OiP was reduced dose- and particle size-dependently, decreasing from a control DI of 30-40 to DI &lt; 0 after 0.3 Gy of 48Ti. The correlation between OiP DI and number of dendritic spines was significant with 0.3 Gy 48Ti.</td>
  </tr>
  <tr>
    <td>Krukowski et al., 2018</td>
    <td>In vivo. Male and female C57BL/6J mice were irradiated with GCR (60% 252 MeV/n protons, 20% 249.3 MeV/n helium and 20% 594.4 MeV/n oxygen) at various doses. Neuronal integrity was measured through synaptic composition and number of synaptosomes. Memory was assessed through NOR.</td>
    <td>No changes were observed in female mice. Male mice showed a 0.9-fold decrease in the number of synaptosomes after 0.5 Gy. GluR1 (associated with hippocampal-dependent working memory) levels were decreased 0.8-fold. No changes in synapsin 1 and postsynaptic density protein 95 were observed. Recognition memory was reduced after 0.15 and 0.5 Gy irradiation in male mice, as irradiated mice did not spend significantly more time with the novel object.</td>
  </tr>
  <tr>
    <td>Raber et al., 2004</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with 10 Gy of X-rays. Ki-67 positive (proliferating) and DCx positive (immature) cells were measured to assess neuron integrity. NOR, MWM and Barnes maze were used to assess learning and memory.</td>
    <td>Before cognitive testing, the number of Ki-67 and DCx positive cells each decreased by 95% after irradiation. In the Barnes maze, irradiated mice took a longer path to reach the escape tunnel and also made more errors than control mice. Irradiated mice showed no impairment during NOR and the MWM.</td>
  </tr>
  <tr>
    <td>Madsen et al., 2003</td>
    <td>In vivo. Adult male Wistar rats were irradiated with 3 Gy of 300 kVp X-rays (4.58 Gy/min). Neuron integrity was assessed using BrdU (thymidine analog) staining to show neurogenesis in the DG. Learning and memory was assessed using NOR, novel location recognition (NLR) and a water maze.</td>
    <td>At a dose of 3 Gy, BrdU-positive cells decreased from approximately 1750 to values too small to be observed when injected during the second block of irradiation. The place recognition test showed reduced time spent in the novel location to almost 50% in irradiated rats. NOR and the water maze did not show any changes in cognitive function.</td>
  </tr>
  <tr>
    <td>Akiyama et al., 2001</td>
    <td>In vivo. Male Fischer 344 rats were irradiated with 25 Gy of 10 MeV X-rays. Bodian and neurofilament (NF) staining were used to identify axons in the corpus callosum. MWM and a passive avoidance test were used to assess learning and memory.</td>
    <td>Rats irradiated with 25 Gy had fewer axons and took longer in each trial to reach the platform in the MWM, although not significantly. When the platform was removed, irradiated rats crossed the area where the platform was 0.6-fold fewer times than control rats. During passive avoidance, the retention time after a shock was also decreased 0.6-fold in irradiated rats.</td>
  </tr>
  <tr>
    <td>Hodges et al., 1998</td>
    <td>In vivo. Male Sprague-Dawley rats were irradiated with 250 kVp X-rays at 1.4 Gy/min. Histological analysis was performed to measure damage and necrosis in the fimbria-fornix, hippocampus and corpus callosum. Learning and memory were measured through a T-maze and water maze.</td>
    <td>At 20 Gy, there was no histological evidence of abnormal neural remodeling, while learning and memory were impaired. At 25 Gy, various necrotic areas were observed in the brain, while learning and memory were impaired. For example, irradiated rats at both 20 and 25 Gy showed 2-fold more errors in the T-maze.</td>
  </tr>
  <tr>
    <td>Rola et al., 2004</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with X-rays at 1.75 Gy/min. Ki-67 positive (proliferating) and DCx positive (immature) cells were measured along with BrdU staining to assess neuron integrity in the subgranular zone. NOR, NLR, MWM, Barnes maze and passive avoidance learning were used to assess learning and memory.</td>
    <td>The number of Ki-67-positive and DCx-positive cells decreased linearly at 0, 2 and 5 Gy, then leveled off at 10 Gy, reaching reductions of 0.1-fold for Ki-67 and 0.3-fold for DCx. The number of BrdU-positive cells decreased by 70% after 5 Gy. Also, after 5 Gy in the MWM, irradiated mice spent 30% less time in the target quadrant compared to control mice, indicative of reduced memory retention. No changes were observed in NOR, NLR, Barnes maze and passive avoidance tests.</td>
  </tr>
  <tr>
    <td>Whoolery et al., 2017</td>
    <td>In vivo. Nestin-GFP male and female mice (C57BL/6J background) were irradiated with 300 MeV/n 28Si particles (linear energy transfer = 67 keV/µm) at 1 Gy/min. Stereological immunopositive cell counts were determined for BrdU, Ki-67 and DCx in the DG granule cell layer. FC was used to assess learning and memory.</td>
    <td>Ki-67-positive cells decreased 0.8-fold at 0.2 Gy and 0.4-fold at 1 Gy, which occurred in both males and females. BrdU-positive cells decreased by 0.8- and 0.7-fold at 1 Gy in males and females, respectively. DCx-positive cells decreased by 0.5-fold at 1 Gy in both sex groups. Small and nonsignificant decreases in BrdU- and DCx-positive cells occurred at 0.2 Gy. Mice exhibited a decrease in percentage freezing of 0.4-fold at 0.2 Gy in contextual FC. No changes in learning and memory were observed at 1 Gy.</td>
  </tr>
  <tr>
    <td>Howe et al., 2019</td>
    <td>In vivo. C57BL/6 mice were irradiated with heavy ions 16O (600 MeV/n) at 0.05 Gy. NOR was used to assess non-spatial declarative memory. Y-maze assessed short-term spatial memory of mice. Sholl analysis was performed to quantify the neurons in the hippocampus. The morphology and density of dendritic spines from the DG and dorsal CA1 and CA3 pyramidal neurons. Behavior was evaluated 9 months after irradiation.</td>
    <td>Overall dendritic complexity in the DG decreased 0.59-fold, while overall dendrite density in the DG decreased 0.94-fold. Irradiated animals exhibited a 0.11-fold change in the discrimination ratio in NOR, as the irradiated group was unable to discriminate between the familiar and novel object, indicating cognitive impairment. Irradiated mice spent more time exploring the novel object than the familiar object in the Y-maze, indicating no impairment on short-term spatial memory.</td>
  </tr>
</table>
<table><tr><td>Achanta, Fuss & Martinez, 2009</td><td>In vivo. Male Sprague Dawley rats were irradiated with 0.3, 3 or 10 Gy of X-rays. BrdU staining was used to assess neuronal proliferation. Changes in cognitive behavior was evaluated by FC/testing. Fear testing was performed at 90 days post-irradiation.</td><td>At 3-months post-irradiation, total number of Ki67+ cells (proliferative marker for cells) decreased by 0.8-, 0.7-, and 0.3-fold in the PD 21 group; 0.8-, 0.7-, and 0.3-fold in the PD 50 group; and 0.9-, 0.083-, and 0.3-fold in the PD 70 group, at 0.3, 3, and 10 Gy, respectively. Total number of BrdU+ cells decreased by 0.7-, 0.4-, and 0.1-fold in the PD 21 group; 0.6-, 0.4-, and 0.03-fold in the PD 50 group; and 0.9-, 0.6-, and 0.04-fold in the PD 70 group, at 0.3, 3, and 10 Gy, respectively. % mean freezing in the PD 21 group in response to a conditioned stimulus (CS) decreased by 0.7- and 0.6-fold in the trace fear conditioning after 3 Gy and 10 Gy, respectively, and 0.7- and 0.6-fold with an intertrial interval (ITI) after 3 Gy and 10 Gy, respectively. % mean freezing in the PD 50 group in response to a CS decreased by 0.6-fold in trace fear conditioning after 10 Gy, and 0.6-fold with an ITI after both 0.3 and 10 Gy. % mean freezing in the PD 70 group in response to a CS decreased by 0.6-fold in trace fear conditioning and 0.6-fold with an ITI after 10 Gy.</td></tr><tr><td>Winocur et al., 2006</td><td>In vivo. Long-Evans rats were irradiated with 7.5 or 10 Gy of 60Co gamma rays. BrdU staining was used to label the DNA of proliferating cells. DCX as an immature neuron marker and NeuN for mature neuron staining were used. Cognitive impairment was identified by contextual FC in the studied rats 4 weeks after irradiation.</td><td>Numbers of BrdU+ cells decreased by 0.2- and 0.2-fold after 10 and 7.5 Gy, respectively. Total number of DCX+ cells decreased by 0.03- and 0.10-fold, for 10 and 7.5 Gy, respectively. Times spent freezing in the irradiated context-only group were decreased by 0.1- and 0.4-fold at the short and long delays, respectively.</td></tr><tr><td>Simmons et al., 2019</td><td>In vivo. C57BL/6J mice were irradiated with 30 Gy electrons (16 and 20 MeV). Spatial and non-spatial object recognition was studied by object location and NOR 10 weeks after irradiation. Spine density was measured by Golgi-stained hippocampal neuron tracing using Neurolucida neuron tracing software and Neurolucida Explorer software (MBF Bioscience).</td><td>Apical dendritic spine density decreased 0.8-fold in 30 Gy conventional irradiated (given over 240 s) mice. The discrimination index for NOR decreased from 23% to 8% after 30 Gy given conventionally. The discrimination index for NOL decreased from 12% to -8% after 30 Gy given conventionally. Conventionally irradiated mice spent significantly less time with the object in a new location, indicating impairment to hippocampus learning and memory. Irradiated mice spent less time exploring a novel object than control mice. No significant changes were observed when the 30 Gy was given over 0.1-0.16 s.</td></tr><tr><td>Sorokina et al., 2021</td><td>In vivo. Male mice were irradiated with accelerated carbon ions at 0.7 Gy (450 MeV/n). Spatial learning, short-term and long-term hippocampus-dependent memory were studied using Barnes Maze and NOR 2 months after irradiation. Nissl staining was performed 1 month after cognitive evaluations to quantify neuronal cells.</td><td>Neuronal quantification revealed a decrease in the number of cells in irradiated mice by 0.9-fold in the DG. The length of the CA3c field of the dorsal hippocampus decreased by 0.89-fold. In the Barnes maze, the latency to the goal box was 3.1-fold higher in the irradiated mice than control on the 9th day after learning, indicating long-term decreased learning. NOR did not show any changes to learning and memory, shown through the 0.7 Gy irradiated mice spending significantly longer with the novel object.</td></tr><tr><td>Miry et al., 2021</td><td>In vivo. C57BL/6J mice were irradiated with 0.1, 0.5 and 1 Gy 56Fe. Active avoidance was used to study spatial learning and discrimination. Immunohistological analysis of proliferating neural cells were performed using DCX, an immature neural marker. Other tests performed included the Barnes Maze for spatial learning. Mice were studied at multiple time-points (2, 6, 12, and 20 months) post-exposure.</td><td>2 months post-exposure, levels of DCX+ cells decreased by 0.7- (0.1 Gy), 0.4- (0.5 Gy) and 0.6-fold (1 Gy) in male mice, and 0.7- (0.1 Gy), 0.6- (0.5 Gy) and 0.4-fold (1 Gy) in female mice. 12 months post-exposure, levels of DCX+ cells increased by 2.6- (0.1 Gy), 2.4- (0.5 Gy), and 2.3-fold (1 Gy) in male mice, and 2.2- (0.1 Gy), 1.1- (0.5 Gy), and 2-fold (1 Gy) in female mice. In the active avoidance task, the normalized error entries significantly increased in both male and female 0.5 Gy irradiated mice compared to the 0 Gy group. In Barnes maze, male mice 20 months post-exposure to 1 Gy 56Fe showed a significant decrease in latency to escape. The most profound change was measured on day 2 with a 0.6-fold decrease in latency to escape, as the irradiated mice learned the location of the escape box faster than the control group over the 5-day training period.</td></tr></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Design</td><td>Summary</td></tr></thead><tbody><tr><td>Parihar et al., 2016</td><td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with 600 MeV/amu charged particles (16O and 48Ti) (0.05 to 0.25 Gy/min) at 0.05 and 0.3 Gy. In the medial prefrontal cortex, neuron integrity was measured through spine density, dendritic complexity and postsynaptic density protein 95 synaptic puncta, while NOR, OiP, TO</td><td>Dendritic complexity and spine density were reduced 0.7-fold 15 weeks post-irradiation, while synaptic puncta were increased 1.3-fold 15 weeks post-irradiation and 1.5-fold 27 weeks post-irradiation. Learning and memory were impaired 15 weeks post-irradiation, and even further impaired 24 weeks post-irradiation.</td></tr></tbody></table>
<table>
  <tr>
    <td></td>
    <td>and fear extinction (FE) tests were done to assess learning and memory. Wistar rats were used in an ATSET test.</td>
    <td></td>
  </tr>
  <tr>
    <td>Raber et al., 2004</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with 10 Gy of X-rays. Ki-67 positive (proliferating) and DCx positive (immature) cells were measured to assess neuron integrity. NOR, MWM and Barnes maze were used to assess learning and memory.</td>
    <td>Before cognitive testing (3 months post-irradiation), the number of Ki-67 and DCx positive cells each decreased by 95% after irradiation. In the Barnes maze, irradiated mice took a longer path to reach the escape tunnel and also made more errors than control mice 3 months after irradiation. Irradiated mice showed no impairment during NOR and the MWM.</td>
  </tr>
  <tr>
    <td>Madsen et al., 2003</td>
    <td>In vivo. Adult male Wistar rats were irradiated with 3 Gy of 300 kVp X-rays (4.58 Gy/min). Neuron integrity was assessed using BrdU (thymidine analog) staining to show neurogenesis in the DG. Learning and memory was assessed using NOR, NLR and a water maze.</td>
    <td>At a dose of 3 Gy, numbers of BrdU-positive cells (neurogenesis) decreased from approximately 1750 to values too low to be observed when injected during the second block of irradiation. BrdU-positive cells decreased from 1300 to 300 when injected 2 weeks after end of irradiation. BrdU-positive cells decreased from 1300 to 250 when injected 6 weeks after end of irradiation. The place recognition test showed impairments in irradiated animals as time in the new arm decreased from 72% to 62% at 1 week post-irradiation; and 67% to 54% at 3 weeks post-irradiation. After 7 weeks, rats showed impaired location memory but not significantly.</td>
  </tr>
  <tr>
    <td>Hodges et al., 1998</td>
    <td>In vivo. Male Sprague-Dawley rats were irradiated with 250 kVp X-rays at 1.4 Gy/min and 20 or 25 Gy. Histological analysis was performed to measure damage and necrosis in the fimbria-fornix, hippocampus and corpus callosum. Learning and memory were measured through a T-maze and water maze.</td>
    <td>Abnormal neural remodeling, only measured at 41 weeks after radiation, was observed. Impaired learning and memory were observed from 35 to 44 weeks after radiation.</td>
  </tr>
  <tr>
    <td>Rola et al., 2004</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with X-rays at 1.75 Gy/min. Ki-67 positive and DCx positive cells were measured along with BrdU staining to assess neuron integrity in the subgranular zone. NOR, NLR, MWM, Barnes maze and passive avoidance learning were used to assess learning and memory.</td>
    <td>The number of Ki-67-positive and DCx-positive cells decreased 0.1-fold and 0.3-fold, respectively, 48h after 5 Gy. The number of BrdU-positive cells decreased by 70% at both 1 and 3 months after 5 Gy. Also, after 3 months, in the MWM, irradiated mice spent 30% less time in the target quadrant after 5 Gy compared to control mice. No changes were observed in NOR, NLR, Barnes maze and passive avoidance tests.</td>
  </tr>
  <tr>
    <td>Whoolery et al., 2017</td>
    <td>In vivo. Nestin-GFP male and female mice (C57BL/6J background) were irradiated with 300 MeV/n 28Si particles (linear energy transfer = 67 keV/µm) at 1 Gy/min. Stereological immunopositive cell counts were determined for BrdU, Ki-67 and DCx in the DG granule cell layer. FC was used to assess learning and memory.</td>
    <td>24 h post-irradiation, BrdU-positive cells decreased by 0.75- and 0.65-fold at 1 Gy in the male and female groups, respectively, DCx-positive cells decreased by 0.5-fold at 1 Gy in both sex groups and Ki-67-positive cells decreased by 0.8-fold at 0.2 Gy and 0.4-fold at 1 Gy in both males and females. At 3 months post-irradiation, BrdU-positive cells decreased by 0.83- and 0.31-fold in male mice only at 0.2 and 1 Gy, respectively. Ki-67 was not significantly changed and DCx was reduced at 1 Gy but only when both sexes were combined. 3 months post-irradiation, male mice exhibited a decrease in percentage freezing of 0.43-fold at 0.2 Gy in contextual FC.</td>
  </tr>
  <tr>
    <td>Howe et al., 2019</td>
    <td>In vivo. C57BL/6 mice were irradiated with heavy ions 160 (600 MeV/n) at 0.05 Gy. NOR was used to assess non-spatial declarative memory. Y-maze assessed short-term spatial memory of mice. Sholl analysis was performed to quantify the neurons in the hippocampus. The morphology and density of dendritic spines from the DG and dorsal CA1 and CA3 pyramidal neurons. Behavior was evaluated 9 months after irradiation.</td>
    <td>9 months after irradiation, mice exhibited a 0.1-fold change in discrimination ratio in NOR indicating cognitive impairment. Dendritic spine density at 9 months post-irradiation was found to be reduced in the DG.</td>
  </tr>
  <tr>
    <td>Achanta, Fuss & Martinez, 2009</td>
    <td>In vivo. Male Sprague Dawley rats were irradiated with 0.3, 3 or 10 Gy. BrdU staining was used to assess neuronal proliferation. Changes in cognitive behavior were evaluated by FC/testing. Fear testing was performed at 90 days post-irradiation.</td>
    <td>Total number of BrdU+ cells and total number of Ki67+ cells (proliferative marker for cells) 3-months post-irradiation decreased in PD 21, PD50 and PD 70 groups. Fear conditioning/testing at 90 days post-irradiation showed a decrease in % mean freezing in response to a CS in PD21, PD50 and PD 70. As well, ITI decreased in the studied groups.</td>
  </tr>
  <tr>
    <td>Winocur et al., 2006</td>
    <td>In vivo. Long-Evans rats were irradiated with 7.5 or 10 Gy of 60Co gamma rays. BrdU staining was used to label the DNA of proliferating cells. DCx as an immature neuron marker and NeuN for mature neuron staining were used. Cognitive impairment was identified by contextual fear conditioning in the studied rats 4 weeks after irradiation.</td>
    <td>Numbers of BrdU+ cells decreased by 0.22- and 0.23-fold 4 weeks after irradiation. Fear conditioning took place 4 weeks post-irradiation. The time spent freezing in the irradiated context-only group was decreased by 0.1- and 0.4-fold at the short and long delays, respectively.</td>
  </tr>
  <tr>
    <td>Sorokina et al., 2021</td>
    <td>In vivo. Male mice were irradiated with accelerated carbon ions at 0.7 Gy (450 meV/n). Spatial learning, revealed a decrease in the number of cells by 0.9-fold in the DG.</td>
    <td>1 month after cognitive evaluations, neuronal quantification revealed a decrease in the number of cells by 0.9-fold in the DG.</td>
  </tr>
</table>
<table><tr><td></td><td>short-term and long-term hippocampus-dependent memory were studied using Barnes Maze and NOR 2 months after irradiation. Nissl staining was performed 1 month after cognitive evaluations to quantify neuronal cells.</td><td>The length of the CA3c field of the dorsal hippocampus decreased by 0.5-fold.<br/>In the Barnes maze 2 months after irradiation, the latency to the goal box was 3.1-fold higher in the irradiated mice than control on the 9th day after learning. Meanwhile no significant changes in latency to the goal box were observed on the second day of learning. NOR did not show any changes to memory.</td></tr><tr><td>Miry et al., 2021</td><td>In vivo. C57BL/6J mice were irradiated with 0.1, 0.5 and 1 Gy of 56Fe ions. Active avoidance was used to study spatial learning and discrimination. Immunohistological analysis of proliferating neural cells were performed using DCX, an immature neural marker. Other tests performed included the Barnes maze for spatial learning. Mice were studied at multiple time-points (2, 6, 12, and 20 months) post-exposure.</td><td>2 months post-exposure, levels of DCX+ cells decreased by 0.4- to 0.7-fold in both male and female mice. 12 months post-exposure, DCX+ cells were increased in irradiated mice compared to control mice and levels of DCX+ cells increased by 1.1- to 2.6-fold in male and female mice.<br/>20 months after exposure, learning was found to be improved.</td></tr></table>
References

Achanta, P., M. Fuss and J. L. Martinez. (2009), "Ionizing Radiation Impairs the Formation of Trace Fear Memories and Reduces Hippocampal Neurogenesis", Behavioral Neuroscience, Vol. 123/5, https://doi.org/10.1037/a0016870.

Acharya, M. M. et al. (2019), "New concerns for neurocognitive function during deep space exposures to chronic, low dose-rate, neutron radiation", eNeuro, Vol. 6/4, Society for Neuroscience, https://doi.org/10.1523/ENEURO.0094-19.2019.

Akiyama, K. et al. (2001), "Cognitive Dysfunction and Histological Findings in Adult Rats One Year After Whole Brain Irradiation.", Neurologia medico-chirurgica, Vol. 41/12, Japan Neurological Society, https://doi.org/10.2176/nmc.41.590.

Bálentová, S. and M. Adamkov. (2020), "Pathological changes in the central nervous system following exposure to ionizing radiation", Physiological Research, Czech Academy of Sciences, https://doi.org/10.33549/PHYSIOLRES.934309.

Burgess, N. (2002), "The hippocampus, space, and viewpoints in episodic memory", The Quarterly Journal of Experimental Psychology, Vol. 55/4, Experimental Psychology Society, https://doi.org/10.1080/02724980244000224.

Cucinotta, F. A. et al. (2014), "Space radiation risks to the central nervous system", Life Sciences in Space Research, Vol. 2, Elsevier Ltd, Amsterdam, https://doi.org/10.1016/j.lssr.2014.06.003.

Desai, R. I. et al. (2022), "Impact of spaceflight stressors on behavior and cognition: A molecular, neurochemical, and neurobiological perspective", Neuroscience & Biobehavioral Reviews, Vol. 138, Elsevier, Amsterdam, https://doi.org/10.1016/j.neubiorev.2022.104676.

D'Hooge, R. and P. P. De Deyn. (2001), "Applications of the Morris water maze in the study of learning and memory", Brain Research Reviews, Vol. 36/1, Elsevier B.V., https://doi.org/10.1016/S0165-0173(01)00067-4.

Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research - Reviews in Mutation Research, Vol. 770, Elsevier B.V., https://doi.org/10.1016/j.mrrev.2016.08.003.

Hodges, H. et al. (1998), "Late behavioural and neuropathological effects of local brain irradiation in the rat", Behavioural Brain Research, Vol. 91/1-2, Elsevier, https://doi.org/10.1016/S0166-4328(97)00108-3.

Howe, A. et al. (2019), "Long-term changes in cognition and physiology after low-dose 16 O irradiation", International Journal of Molecular Sciences, Vol. 20/1, MDPI AG, https://doi.org/10.3390/ijms20010188.

Kiffer, F., M. Boerma and A. Allen. (2019b), "Behavioral effects of space radiation: A comprehensive review of animal studies", Life Sciences in Space Research, Vol. 21, Elsevier, Amsterdam, https://doi.org/10.1016/j.lssr.2019.02.004.

Krukowski, K. et al. (2018), "Female mice are protected from space radiation-induced maladaptive responses", Brain, Behavior, and Immunity, Vol. 74, Academic Press Inc., https://doi.org/10.1016/j.bbi.2018.08.008.
Lamproglou, I. et al. (1995), "Radiation-induced cognitive dysfunction: An experimental model in the old rat", International Journal of Radiation Oncology, Biology, Physics, Vol. 31/1, Elsevier, https://doi.org/10.1016/0360-3016(94)00332-F.

Madsen, T. M. et al. (2003), "Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat", Neuroscience, Vol. 119/3, Elsevier Ltd, https://doi.org/10.1016/S0306-4522(03)00199-4.

Miry, O. et al. (2021), "Life-long brain compensatory responses to galactic cosmic radiation exposure", Scientific Reports 2021 11:1, Vol. 11/1, Nature Publishing Group, https://doi.org/10.1038/s41598-021-83447-y.

Monje, M. L. and T. Palmer. (2003), "Radiation injury and neurogenesis", Current Opinion in Neurology, Vol. 16/2, Ovid Technologies (Wolters Kluwer Health), https://doi.org/10.1097/01.wco.0000063772.81810.b7.

National Council on Radiation Protection and Measures (NCRP). (2016). Commentary No. 25 – Potential for central nervous system effects from radiation exposure during space activities phase I: Overview.

Parihar, V. K. et al. (2016), "Cosmic radiation exposure and persistent cognitive dysfunction", Scientific Reports, Vol. 6/1, Nature Publishing Group, https://doi.org/10.1038/srep34774.

Parihar, V. K. et al. (2015), "What happens to your brain on the way to Mars", Science Advances, Vol. 1/4, American Association for the Advancement of Science, https://doi.org/10.1126/SCIADV.1400256.

Raber, J. et al. (2004), "Radiation-induced cognitive impairments are associated with changes in indicators of hippocampal neurogenesis", Radiation Research, Vol. 162/1, Allen Press, https://doi.org/10.1667/RR3206.

Rola, R. et al. (2004), "Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice", Experimental Neurology, Vol. 188/2, Academic Press Inc., https://doi.org/10.1016/j.expneurol.2004.05.005.

Romanella, S. M. et al. (2020), "Noninvasive Brain Stimulation & Space Exploration: Opportunities and Challenges", Neuroscience & Biobehavioral Reviews, Vol. 119, https://doi.org/10.1016/j.neubiorev.2020.09.005.

Simmons, D. A. et al. (2019), "Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation", Radiotherapy and Oncology, Vol. 139, https://doi.org/10.1016/j.radonc.2019.06.006.

Sorokina, S. S. et al. (2021), "Low dose of carbon ion irradiation induces early delayed cognitive impairments in mice", Radiation and Environmental Biophysics, Vol. 60, Nature, https://doi.org/10.1007/s00411-020-00889-0.

Tomé, W. A. et al. (2015), "Hippocampal-dependent neurocognitive impairment following cranial irradiation observed in pre-clinical models: current knowledge and possible future directions", The British Journal of Radiobiology, Vol. 89/1057, British Institute of Radiology, https://doi.org/10.1259/bjr.20150762.

Vorhees, C. V. and M. T. Williams. (2014), "Assessing Spatial Learning and Memory in Rodents", ILAR Journal, Vol. 55/2, Oxford University Press, Oxford, https://doi.org/10.1093/ilar/ilu013.
Whoolery, C. W. et al. (2017), "Whole-body exposure to 28Si-radiation dose-dependently disrupts dentate gyrus neurogenesis and proliferation in the short term and new neuron survival and contextual fear conditioning in the long term", Radiation Research, Vol. 188/5, Radiation Research Society, https://doi.org/10.1667/RR14797.1.

Winocur, G. et al. (2006), "Inhibition of neurogenesis interferes with hippocampus-dependent memory function", Hippocampus, Vol. 16/3, https://doi.org/10.1002/HIPO.20163.
## AOPs Referencing Relationship

<table><thead><tr><th>AOP Name</th><th>Adjacency</th><th>Weight of Evidence</th><th>Quantitative Understanding</th></tr></thead><tbody><tr><td>AOP: 483 – Deposition of Energy Leading to Learning and Memory Impairment</td><td>adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

Evidence for this relationship comes from human-derived cells, rat, and mouse models, with most of the evidence in mice. There is in vivo evidence in both male and female animals, with more evidence in males. Animal age is occasionally not indicated in studies, but most evidence is in adolescent rodent models with a few studies using adult animals.

## Taxonomic Applicability

<table><thead><tr><th>Term</th><th>Scientific Term</th><th>Evidence</th><th>Link</th></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>Moderate</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Low</td><td>NCBI</td></tr></tbody></table>

## Life Stage Applicability

<table><thead><tr><th>Term</th><th>Evidence</th></tr></thead><tbody><tr><td>Juvenile</td><td>Moderate</td></tr><tr><td>Adult</td><td>Low</td></tr></tbody></table>

## Sex Applicability

<table><thead><tr><th>Sex</th><th>Evidence</th></tr></thead><tbody><tr><td>Male</td><td>Moderate</td></tr><tr><td>Female</td><td>Low</td></tr><tr><td>Unspecific</td><td>Moderate</td></tr></tbody></table>

## Key Event Relationship Description

Alterations in signaling pathways can trigger disruption to neuronal structures, which can lead to altered morphology, changes in neurogenesis, neurodegeneration, apoptotic activity and synaptic activity, collectively known as abnormal neural remodeling (Cekanaviciute et al., 2018; Chakraborti et al., 2012; Hladik & Tapio, 2016). These intracellular pathways are key processes to control various cell functions such as cell growth, death or communication. Within the neuron, multiple signaling pathways influence its structure and function. For example, the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) family pathways are involved in neuronal survival, proliferation, morphology, and synaptic plasticity (Davis and Laroche, 2006; Long et al., 2021; Mazzucchelli and Brambilla, 2000). The senescence pathway induces cell cycle arrest and can restrict neurogenesis (McHugh and Gil, 2018). The apoptotic pathway can be initiated within the mitochondria due to dysfunction within the respiratory chain and induces various signaling proteins such as p53, BAX, caspases and cytochrome C (Betlazar et al., 2016; Mielke and Herdegen, 2000; Wang et al., 2020). Apoptosis of neurons results in a reduction in neuron numbers, which can be a marker of abnormal neural remodeling. A few studies also measure high apoptosis levels over time, indicating sustained neuron loss contributing to reduced neural activity (Chow, Li, and Wong, 2000; Limoli et al., 2004; Pius-Sadowska et al., 2016). Additionally, the brain-derived neurotrophic factor (BDNF)-cAMP-calcium response element binding protein (CREB) pathway is involved in the regulation of excitatory transmission as CREB-dependent transcription allows for persistent pre- and post-synaptic neurotransmitter release at excitatory synapses (Ran et al., 2012).
**Biological Plausibility**

Abnormal neural remodeling can occur through multiple signaling pathways, including MAPK signaling, PI3K/Akt signaling, senescence signaling, and apoptotic signaling. These pathways are involved in the homeostatic regulation of neuron numbers, morphology, proliferation, differentiation, and synaptic activity.

Like many signaling pathways, MAPK pathways help maintain the biological functions in neurons, and changes to the expression or activity of signaling molecules in MAPK pathways can result in altered structure and function of neurons. The extracellular signal-regulated protein kinase (ERK)1/2 MAPK pathway is crucial for modulating synaptic function and alteration in expression of critical proteins in this pathway will result in long-term potentiation (LTP) deficits (Davis and Laroche, 2006; Mazzucchelli and Brambilla, 2000). Research shows that modulations in ERK1/2 expression and activity can decrease cell proliferation in the hippocampus (Betlazar et al., 2016). The p38 MAPK pathway is also involved in maintaining neuronal plasticity and synaptic function, by inducing metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) in hippocampal neurons (Falcicchia et al., 2020). However, p38 demonstrates variable effects in neuronal survival and proliferation. Although p38 signaling is required for the survival of developing neurons, p38 can also be involved in the induction of apoptosis, and subsequent inhibition of p38 promotes cell survival (Mielke and Herdegen, 2000; Nebreda and Porras, 2000). The role of p38 is often dependent on the cell type and stimulus and will determine whether p38 has a positive or negative role on neural cell proliferation (Nebreda and Porras, 2000). The c-Jun NH2-terminal kinase (JNK) MAPK pathway plays a similar role to the p38 pathway, and its function is also dependent on the cell type and context of the cellular environment. JNK can induce apoptosis as well as regulate proteins like tau and microtubule-associated protein (MAP)2 involved in altering cytoskeletal dynamics and cell morphology (Mielke and Herdegen, 2000; Sherrin, Blank, and Todorovic, 2011). JNK is also involved in both pre- and post-synaptic function through the phosphorylation of AMPA receptors and postsynaptic density protein (PSD)95 (Sherrin, Blank, and Todorovic, 2011).

The PI3K/Akt pathway is involved in many neural functions. Activation of the PI3K/Akt pathway promotes transcription of survival genes and inhibits death genes, while also regulating the activity of various death pathways (Long et al., 2021; Rai et al., 2019).

Alterations in Akt expression and activity can decrease cell proliferation in the hippocampus (Betlazar et al., 2016). The pathway can also regulate neuron morphology, as neurite outgrowth can be induced by activation of the pathway (Rodgers and Theibert, 2002). Synaptic plasticity and LTP, which is induced by the activation of NMDA receptors and the subsequent insertion of AMPA receptors to the membrane, can additionally be regulated by the PI3K/Akt pathway. It has been shown that the mammalian target of rapamycin (mTOR), downstream of Akt, increases the expression of LTP-related proteins while PI3K guides AMPA insertion on the membrane (Long et al., 2021). Therefore, maintaining the appropriate expression levels of signaling molecules is critical for proper neural development and function.

The signaling molecules p53/p21 and p16 as part of the cellular senescence pathway can induce cell cycle arrest. For example in neural stem cells (NSCs), reduced functionality and limited neurogenesis is associated with increased senescence markers (McHugh and Gil, 2018).

The apoptosis pathway, consisting of the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, caspases, cytochrome C, and Bcl-2-associated X protein (BAX) as well as the anti-apoptotic B-cell leukemia/lymphoma (Bcl)-2 protein, contributes to a reduction in neuron numbers through cell death when activated (Betlazar et al., 2016; Wang et al., 2020). This pathway may be induced by perturbations to other signaling pathways, including MAPK, PI3K/Akt, and senescent pathways (Hladik and Tapio, 2016; Mielke and Herdegen, 2000). For example, JNK
and p53 can both antagonize the anti-apoptotic Bcl-2, while JNK can stabilize p53 and p53 enhances BAX (Mielke and Herdegen, 2000).

Apoptotic signaling pathways activated not just in neurons, but also in astrocytes and microglia, can lead to maladaptive neural remodeling. For example, it was previously mentioned that p38 signaling in neurons contribute to hippocampal mGluR-dependent LTD. In astrocytes, p38 signaling is necessary for NMDA-dependent LTD during astrocyte-to-neuron communication (Falcicchia et al., 2020). In addition, BDNF signaling in both neurons and astrocytes prevents cell death in the respective cells through activation of the ERK and PI3K/Akt pathways. Neuronal death can be prevented by BDNF signaling in both cell types because astrocytes release factors that prevent neuronal death (Rai et al., 2019).

Synergistic and antagonistic interactions between signaling pathways can also occur, contributing to the complexity and context- dependence of neural activity in response to various signaling pathways. For example, nuclear factor of activated T-cells (NFATc) nuclear translocation and transcriptional activation can be encouraged by the PI3K/Akt pathway and the ERK pathway and inhibited by p38 and JNK (Macian, 2005; Mielke and Herdegen, 2000). Activation of NFATc promotes neuronal survival, synaptic plasticity, and neurite outgrowth through the transcription of multiple target genes (Zhang et al., 2018). BDNF activation of the ERK, PI3K/Akt, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways through tropomyosin receptor kinase (Trk) activation results in the activation of CREB transcriptional activity (Cunha, Brambilla, and Thomas, 2010). CREB is essential for the regulation of excitatory transmission, and exogenous stressors can induce hippocampal neuronal damage through the inhibition of this pathway (Hladik and Tapio, 2016; Wang et al., 2020).

## Empirical Evidence

The empirical evidence for this KER comes from in vivo mouse and rat models as well as in vitro rat-, mouse-, and human-derived cell models. Stressors used included gamma ray radiation (El-Missiry et al., 2018; Eom et al., 2015; Ivanov and Hei, 2014; Kanzawa et al., 2006; Pius-Sadowska et al., 2016; Suman et al., 2013), X-ray radiation (Chow, Li and Wong, 2000; Huang et al., 2021; Limoli et al., 2004; Silasi et al., 2004), electron radiation (Ji et al., 2014; Zhang et al., 2018), iron-56 ion radiation (Suman et al., 2013), simulated ischemic stroke (Tian et al., 2020), and pharmacological modulation of signaling molecules (Kumar et al., 2005). Abnormal neural remodeling can be determined through various endpoints, including neuronal apoptosis, morphology, proliferation, differentiation, and synaptic activity.

## Dose Concordance

Several studies demonstrate dose concordance for this relationship. Studies have observed that altered signaling can occur at the same stressor doses as neural remodeling. X-irradiation at doses ranging from 0.5 Gy to 10 Gy resulted in changes in protein levels and phosphorylated proteins in key signaling pathways, as well as increased apoptotic activity and decreased number of neurons. Silasi et al. (2004) irradiated male and female mice with either acute (single 0.5 Gy dose) or chronic (0.05 Gy/day for 10 days) X-ray radiation at 0.002 Gy/s and found that chronic radiation decreased the levels of phosphorylated Akt, ERK1/2, CREB and CaMKII in males. Female mice showed a 1.3-fold increase in ERK1/2. A decrease in DCx+ cells was also observed in both sexes. At 2 Gy, Chow, Li and Wong (2000) reported an increase in p53+ cells and apoptosis within the brain. 10 Gy of X-irradiation resulted in an increase in BAX/Bcl-2 ratio, p53 and cleaved caspase 3 levels, as well as apoptosis within hippocampal neurons (Huang et al., 2021). Another radiation source is electrons, and doses from 2 to 20 Gy also affect protein levels and phosphorylated proteins in key signaling pathways, as well as neuronal structure and number. Zhang et al. (2018) reported a dose-dependent decrease in dephosphorylated (active) NFATc4/3 after 2 and 8 Gy, as well as an increase in phosphorylated (inactive) NFATc4/3 at these doses within the brains of rats. Total neurite length and branching points dose-dependently decreased at 2 and 8 Gy, whereas total dendritic length decreased at only 2 Gy. Whole-brain irradiation of rats at 20 Gy decreased p-ERK1/2, p-Akt, BDNF, p-TrkB, p-CaMKII, and
p-CREB. Irradiation at 20 Gy also reduced the number of DCx+ cells and the number of BrdU+/NeuN+ cells (Ji et al., 2014).

Furthermore, studies using gamma irradiation as a stressor have also shown changes within this KER. Doses ranged from 2-10 Gy and resulted in altered levels of phosphorylated and dephosphorylated proteins in the MAPK family, apoptotic pathway, senescence pathway and BDNF-pCREB pathway (El-Missiry et al., 2018; Ivanov & Hei, 2014; Kanzawa et al., 2006; Pius-Sadowska et al., 2016; Suman et al., 2013). In another study, Kumar et al. (2005) utilized pharmacological inhibition of various signaling proteins such as those associated with the PI3K-Akt and MAPK family pathway. The results elicited changes in hippocampal neuron morphology as defined by a reduction in dendritic branch length, number of terminal tips, soma area, spine density and filopodia density.

## Time Concordance

Multiple studies demonstrate that signaling pathways are altered before abnormal neural remodeling is observed in a time-course. Altered stress response signaling are often found as early as hours post-irradiation, while abnormal neural remodeling is first measured after days. NSCs isolated from rats showed an increase in p-JNK as early as 1h post-irradiation (10 Gy of gamma rays), while apoptosis was measured 48h post-irradiation (Kanzawa et al., 2006). After 5 Gy X-ray irradiation of rat neural precursor cells, p53 and p21 signaling was increased after 2h, while the first significant increase in apoptosis was observed after 24h (Limoli et al., 2004). In mice exposed to gamma ray irradiation at 10 Gy, multiple signaling molecules were increased as early as 3h post-irradiation, while measurements of apoptosis and neuronal morphological changes occurred 48h post-irradiation (Pius-Sadowska et al., 2016).

Neuron levels were found decreased 24h post-irradiation in this study, although this was in an in vivo mouse model. Human NSCs irradiated with 5 Gy of gamma rays showed many signaling molecules were dysregulated after 6h, apoptosis was first increased after 24h, and the differentiation of NSCs was decreased after 10-12 days (Ivanov and Hei, 2014).

A few studies also demonstrated time concordance when altered signaling was measured after days or longer. Rat neurons irradiated with electrons at 2 Gy showed higher inactive and lower active levels of NFATc4/3 after both 1 and 3 days (Zhang et al., 2018). The number of branching points were subsequently decreased after only 3 days. However, total neurite length was decreased after 1 day. Furthermore, total dendritic length decreased after both 14 and 28 days, although this was in an in vivo rat model (Zhang et al., 2018). In a longer-term study using both gamma ray (2 Gy) and 56Fe ion (1.6 Gy) irradiation of mice, Suman et al. (2013) found changes in apoptotic and senescent signaling pathways at both 2 and 12 months post-irradiation, while increased apoptosis and decreased cortical thickness were only measured after 12 months.

## Incidence Concordance

Several studies reported an incident-concordant relationship between altered stress response signaling and abnormal neural remodeling. In the study conducted by Suman et al. (2013), female mice were irradiated with either gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) and at both doses, an increase in p16, p21, p53, BAX and apoptosis was observed. A decrease in Bcl-2 and cortical thickness was also seen after both gamma and 56Fe irradiation. Another study irradiated neural stem cells with 6 Gy of gamma rays and found increased p-Akt, p-p53, p-STAT3, and mGluR1, with decreased neural stem cells (Eom et al., 2015). At 5 Gy of X-irradiation, Limoli et al. (2004) also found increased levels of p53 and phosphorylated p53 with increased apoptosis and decreased DCx+ cells.

Through the use of middle cerebral artery occlusion, a technique to simulate an ischemic stroke, alteration in signaling pathways were shown. Following the administration of this procedure in male mice, the ratios of phosphorylated to total ERK1/2, p38 and JNK increased, as well as levels of BAX, cleaved caspase-3 and the percent of apoptosis. Anti-apoptotic marker Bcl-2 also decreased after surgery (Tian et al., 2020).

## Essentiality
Attenuation of altered signaling consistently results in a reduction in endpoint measurements of abnormal neural remodeling. Multiple studies showed this using genetic knockout of signaling molecules. For example, genetic knockout of Src, an upstream activator of multiple signaling pathways, or a combination of p38 and ERK1/2 in mice with a simulated ischemic stroke led to the inhibition of signaling in the MAPK pathways, decreased apoptosis, and increased neuron levels compared to wild-type mice (Tian et al., 2020). In accordance, activation of Src led to increased MAPK signaling and apoptosis compared to wild-type mice after simulated ischemic stroke (Tian et al., 2020). Two studies showing that neurons decreased following 1-5 Gy irradiation of mice found that knockout of p53 decreased apoptosis and slightly restored neuron numbers (Chow, Li and Wong, 2000; Limoli et al., 2004).

Inhibition of various signaling molecules after irradiation also led to reduced abnormal neural remodeling. The mGluR1 inhibitor LY367385 restored NSC numbers after irradiation of neural-like stem cells with 6 Gy of gamma rays (Eom et al., 2015). Similarly, the JNK inhibitor SP600125 restored neuronal differentiation after 2 Gy gamma ray irradiation of NSCs (Kanzawa et al., 2006). Zhang et al. (2018) found that activation of NFATc4/3 nuclear translocation with BDNF was able to restore neurite length and the number of total branching points in rats and rat-derived neurons after 2 Gy electron irradiation, while inactivation of NFATc4/3 nuclear translocation with CsA produced the opposite effect in rat-derived neurons after irradiation.

## Uncertainties and Inconsistencies

* The changes to a signaling pathway may provide inconsistent outcomes in markers of abnormal neural remodeling. For example, the p38 pathway is involved in many, often opposing, biological processes (Nebreda and Porras, 2000). Different cell types and exposures can be associated with the expression of different receptors of the p38 pathway, resulting in different biological changes. In addition, signaling pathways that synergize or antagonize with each other may be influenced at the same time resulting in cumulative effects across different pathways (Nebreda and Porras, 2000).

* Eom et al., 2015: Irradiation of C17.2 mouse neural stem-like cells with 6 Gy of gamma rays resulted in an increase in β-III tubulin expression, indicating a rise in neurons post-irradiation. However, all other studies observed a decrease in neuron numbers post-irradiation.

## Known modulating factors

<table><thead><tr><th>Modulating factor</th><th>Details</th><th>Effects on the KER</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Genetic</td><td>Src (regulates the activation of MAPK pathways) knockout</td><td>Src knockout in mice inactivated MAPK and apoptotic signaling and reduced apoptosis in the brain after middle cerebral artery occlusion.</td><td>Tian et al., 2020</td></tr><tr><td>miR-137 (silences Src) knockout</td><td>miR-137 knockout in mice increased MAPK and apoptotic signaling and further increased apoptosis after middle cerebral artery occlusion.</td><td>Tian et al., 2020</td></tr><tr><td>p38 and ERK1/2 knockout</td><td>p38 and ERK1/2 knockout in mice inactivated MAPK and apoptotic signaling and reduced apoptosis in the brain after middle cerebral artery occlusion.</td><td>Tian et al., 2020</td></tr><tr><td>p53 knockout</td><td>Irradiation (1-5 Gy) of p53 knockout mice led to a higher number of neurons and decreased apoptosis compared to irradiation of wild-type mice.</td><td>Chow, Li and Wong, 2000; Limoli et al., 2004</td></tr><tr><td rowspan="3">Drug</td><td>LY367385 (mGluR1 inhibitor). mGluR1 is involved in neuronal differentiation.</td><td>LY367385 (25 M) increased the number of NSCs after 6 Gy radiation of C17.2 neural stem-like cells.</td><td>Eom et al., 2015</td></tr><tr><td>SP600125 (JNK inhibitor)</td><td>SP600125 (5 µM) restored neuronal differentiation after it was reduced by 2 Gy radiation of rat NSCs.</td><td>Kanzawa et al., 2006</td></tr><tr><td>Cyclosporin (CsA, prevents NFATc4/3 nuclear translocation)</td><td>CsA (1 µg/mL) further reduced the levels of dephosphorylated NFATc4/3 as well as total neurite length and branching points after both 2 and 8 Gy irradiation of rat neurons.</td><td>Zhang et al., 2018</td></tr></tbody></table>
<table><tr><td></td><td>BDNF (induces NFATc4/3 nuclear translocation)</td><td>BDNF (100 ng/mL in vitro, 0.75 µg/1.5 µL in vivo) slightly restored the levels of dephosphorylated NFATc4/3 after 2 Gy irradiation and completely restored neurite length and total branching points both in vitro and in vivo.</td><td>Zhang et al., 2018</td></tr><tr><td>Sex</td><td>Female mice</td><td>Male mice showed many changes in Akt and ERK1/2 activity following acute and chronic irradiation at 0.5 Gy. However, female mice showed only few changes. In addition, male mice showed a trend of fewer immature neurons after 0.5 Gy radiation.</td><td>Silasi et al., 2004</td></tr><tr><td>Exercise</td><td>Forced running in 30-minute intervals twice per day, 5 times per week for 3 weeks.</td><td>Forced running after irradiation completely restored the levels of the signaling molecules in the BDNF-pCREB pathway and slightly restored neurogenesis.</td><td>Ji et al., 2014</td></tr></table>

## Quantitative Understanding of the Linkage

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Kumar et al., 2005</td><td>In vitro. Hippocampal cornu ammonis (CA)3/CA1 pyramidal neurons extracted from rats were subjected to pharmacological inhibition of various signaling molecules. The level of various signaling molecules was determined with western blot. Confocal microscopy was used to assess hippocampal neuron morphology, including total dendritic branch length, number of terminal tips, soma area, spine density, and filopodia density.</td><td>The PI3K inhibitor LY294002, at a dose of 50 µM, resulted in a 0.4-fold decrease in p-Akt (activated Akt) and a decrease in p-S6 (activated S6, downstream in the PI3K/Akt pathway) to less than 0.1-fold. The mTOR inhibitor rapamycin, at a dose of 1 µM, resulted in a decrease in p-S6 to less than 0.1-fold. The MAPK/ERK kinase (MEK) (kinase upstream of ERK) inhibitor U0126, at a dose of 10 µM, resulted in a decrease in p-ERK to less than 0.1-fold. LY294002 decreased total dendritic branch length 0.7-fold, the number of terminal tips 0.7-fold, soma area 0.6-fold, spine density 0.8-fold, and filopodia density 0.7-fold. Rapamycin decreased total dendritic branch length 0.5-fold, the number of terminal tips 0.6-fold, soma area 0.6-fold, spine density 0.7-fold, and filopodia density 0.5-fold. U0126 alone did not show any changes to any markers of abnormal neural remodeling, but U0126 with overexpression of RasL61 (activates the signaling pathway) decreased the total dendritic branch length 0.8-fold, decreased the number of terminal tips 0.4-fold, increased spine density 1.4-fold, and decreased filopodia density 0.5-fold compared to just RasL61 alone.</td></tr><tr><td>Kanzawa et al., 2006</td><td>In vitro. NSCs isolated from the frontal cortex of embryonic Fisher 344 rats were irradiated with a maximum of 10 Gy 137Cs gamma rays at 3.4 Gy/min. Signaling was determined by western blot. Apoptotic morphology of cells was determined with Hoechst 33258 staining. Apoptosis of just neurons was measured by a TUNEL assay.</td><td>p-JNK (activated) increased a maximum of 60% after 10 Gy. No changes in p38 or ERK1/2 activation were observed. Cytochrome C and BAX were increased, and Bcl-2 was decreased after 10 Gy. The percent of apoptotic cells increased from 40 to 65% after 10 Gy. The percent of apoptotic neurons increased from 15 to 42% after 10 Gy. The percent of TUNEL+ cells that were p-JNK+ increased from 33 to 82% after 10 Gy as well.</td></tr><tr><td>Silasi et al., 2004</td><td>In vivo. Male and female C57/BL6 mice were whole-body irradiated with either acute (single 0.5 Gy dose) or chronic/fractionated (0.05 Gy/day for 10 days) X-ray radiation, both at 0.002 Gy/s. Western blot was used to assess the levels of proteins in various signaling pathways in the hippocampus. DCx staining (immature neurons) was performed to determine hippocampal neurogenesis.</td><td>After chronic radiation, male mice showed a 1.2-fold increase in Akt, a 0.95-fold decrease in p-Akt, a 0.6-fold decrease in p-ERK1/2, a 0.8-fold decrease in CaMKII, and a 0.9-fold decrease in p-CREB. Female mice showed a 1.3-fold increase in ERK1/2. After chronic radiation, DCx+ cells decreased 0.5-fold in both males and females. After acute radiation, a 0.6-fold decrease in p-ERK1/2 and a 0.7-fold decrease in CaMKII in males was observed, and DCx+ cells decreased 0.8-fold in males (non-significant) and 0.9-fold in females (non-significant).</td></tr><tr><td>Ivanov & Hei, 2014</td><td>In vitro. Human NSCs or neuroblastoma SK-N-SH cells were irradiated with 2.5, 5 or 10 Gy of 137Cs gamma rays (0.82 Gy/min). Western blot analysis was used to assess the levels of various signaling proteins. The percentage of hypodiploid nuclei was analyzed using flow cytometry to quantify apoptotic cells. Survival of differentiated cells</td><td>NSCs: p53 and TRAIL were increased at 2.5 and 5 Gy. Akt, p-Akt, p-p38, p-JNK, and pro-caspase-8 and -3 (inactive) were decreased at 5 Gy. The percent of NSCs that were apoptotic increased at 2.5 and 5 Gy, with a 5-fold increase at 10 Gy. At 5 Gy, just 11% of NSCs survived after differentiation compared to the unirradiated control.</td></tr></tbody></table>
<table><tr><td></td><td>was assessed with staining for Nestin (NSCs) and DCx (immature neurons).</td><td>Neuroblastoma cells: p53, BAX, and p-ERK1/2 were increased at 2.5, 5, and 10 Gy. p-p38 was increased at 5 and 10 Gy. p-JNK2 and p-Akt were decreased at 10 Gy. Differentiation of NSCs cultured with non-irradiated SK-N-SH cells led to a survival rate of 19%, while NSCs cultured with 5 Gy irradiated SK-N-SH cells had a survival rate of 5%.</td></tr><tr><td>Zhang et al., 2018</td><td>In vivo and in vitro. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 2 Gy. Primary cultured hippocampal neurons from 18-day-old Sprague-Dawley rat embryos were irradiated with electrons at 2 or 8 Gy. Western blot was used to assess phosphorylated (inactive) and dephosphorylated (active) NFATc4/3 levels in vitro. Neurite growth in vitro was determined by immunofluorescence of β-tubulin+ neurons. In vivo dendritic growth was determined in the dentate gyrus with a retrovirus labeling newborn neurons with green fluorescent protein.</td><td>In vitro: Dephosphorylated NFATc4/3 decreased by 20% at 2 Gy and 30% at 8 Gy. p-NFATc4/3 increased by 60% at 2 Gy and 90% at 8 Gy. Total neurite length decreased after 3 days by 24% at 2 Gy and 32% at 8 Gy. Branching points decreased by 29% at 2 Gy and 36% at 8 Gy after 3 days.<br/>In vivo: Total dendritic length in the dentate gyrus decreased about 30% after 2 Gy.</td></tr><tr><td>Chow, Li and Wong, 2000</td><td>In vivo. Female C57BL6/J mice (57 to 123 days old) were irradiated with X-rays to the entire brain (2 Gy). p53 levels were determined through immunohistochemistry. Apoptosis levels were quantified through morphological assessment after hematoxylin and eosin staining.</td><td>In the subependymal region of the brain, 2 Gy resulted in the identification of many p53+ cells while none were found in the control. Specifically in glial cells, 2 Gy in the subependyma increased the p53+ cells from 0.23 to 15.7%. Apoptosis in the subependyma increased from 0.33 to 11.5% at 2 Gy.</td></tr><tr><td>Huang et al., 2021</td><td>In vitro. HT22 hippocampal neuronal cells were irradiated with 10 Gy of X-rays (6 Gy/min). Levels of proteins in the PI3K/Akt, p53, and apoptotic signaling pathways were determined by western blot. Apoptosis was measured by flow cytometry with annexin V (marker for apoptotic cells) and propidium iodide staining.</td><td>The ratio of p-PI3K/PI3K increased 5-fold (non-significant), the ratio of p-Akt/Akt increased 2-fold (non-significant), the ratio of BAX/Bcl-2 increased 5-fold, p53 increased 2-fold, cleaved caspase-3 increased 2.7-fold, and apoptosis increased 9-fold all at 10 Gy.</td></tr><tr><td>El-Missry et al., 2018</td><td>In vivo. Adult male albino Wistar rats were whole-body irradiated with 137Cs gamma rays at 4 Gy (0.695 cGy/s). Levels of signaling proteins in the hippocampus were determined with respective assay kits. Hematoxylin and eosin staining in the hippocampal dentate gyrus was used for histopathological analysis. Apoptosis levels in the hippocampus were determined by flow cytometry with annexin V (marker for apoptotic cells) and propidium iodide staining. Radiation was delivered to the animal's entire body.</td><td>Radiation at 4 Gy resulted in 2- to 4-fold increases in p53, cytochrome C, BAX, and caspase-3, -8, and -9 levels. Radiation at 4 Gy also resulted in a 0.2-fold decrease in Bcl-2. The percent of live cells decreased 0.6-fold, the frequency of apoptosis increased 3- to 4-fold, and the frequency of necrosis increased 7-fold. In addition, 4 Gy resulted in extensive damage to the dentate gyrus.</td></tr><tr><td>Pius-Sadowska et al., 2016</td><td>In vivo. Female 6- to 8-week-old BALB/c mice were whole-brain irradiated with 60Co gamma rays at 10 Gy. After whole-brain irradiation, the levels of various signaling molecules were assessed with western blot in the brain. Apoptosis was measured with a TUNEL assay in the hippocampus. Nissl staining was used to assess neuron morphology in the hippocampus.</td><td>Irradiation at 10 Gy resulted in a maximum 1.8-fold increase in caspase-3, a 1.7-fold increase in BDNF, a 1.8-fold increase in TrkA, a 3.8-fold increase in TrkB, a 1.7-fold increase in TrkC, a 4.1-fold increase in p-ERK1/2, and a 2.9-fold increase in p-Akt. Without irradiation, no TUNEL+ cells were found in the brain, while 10 Gy resulted in the detection of apoptotic nuclei in the dentate gyrus. Also, degenerative morphological changes were observed in the hippocampus after 10 Gy.</td></tr><tr><td>Ji et al., 2014</td><td>In vivo. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 20 Gy. Western blot was performed to assess the levels and activity of proteins in the BDNF-pCREB pathway in the hippocampus. Neurogenesis in the dentate gyrus was assessed using DCx (immature neurons) or BrdU/NeuN (new neurons) staining.</td><td>Irradiation at 20 Gy decreased p-ERK1/2 by 15%, p-Akt by 34% BDNF by 38%, p-TrkB by 54%, p-CaMKII by 30%, and p-CREB by 29%. Irradiation at 20 Gy also reduced the number of DCx+ cells by 92% and the number of BrdU+/NeuN+ cells by 82%.</td></tr></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Kanzawa et al., 2006</td><td>In vitro. NSCs isolated from the frontal cortex of embryonic Fisher 344 rats were irradiated with 137Cs gamma rays at 10 Gy (3.4 Gy/min). Signaling was determined by western blot. Apoptotic morphology of cells was determined with Hoechst 33258 staining. Apoptosis of just neurons was measured by a TUNEL assay.</td><td>p-JNK increased 30% after 1h and 60% after 2h post-irradiation. Starting 24h post-irradiation, BAX and cytochrome C were increased, and Bcl-2 was decreased. Apoptosis increased 48h post-irradiation.</td></tr><tr><td>Suman et al., 2013</td><td>In vivo. Female 6- to 8-week-old C57BL/6J mice were irradiated with either 137Cs gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) both at 1 Gy/min. p16, p21, p53, BAX, and Bcl-2 levels were determined in the cerebral cortex with immunoblotting. Hematoxylin and eosin staining was used to measure</td><td>Altered signaling through p16, p21, p53, BAX, and Bcl-2 protein levels was observed as early as 2 months post-irradiation, while increased</td></tr></tbody></table>
<table><tr><td>Limoli et al., 2004</td><td>In vivo and in vitro. Male 2-month-old C57BL/J6 mice (1.75 Gy/min) and neural precursor cells (4.5 Gy/min) isolated from rats were irradiated with X-rays at 5 Gy. Mice were irradiated cranially. Western blot analysis was done in vitro to measure levels of p53 and p21 proteins. An antibody against DCx was used to detect immature neurons in the dentate gyrus in vivo. FACS analysis of propidium iodide fluorescence was used to assess apoptosis in vitro.</td><td>Apoptosis and decreased cortical thickness were only shown at 12 months.<br/>In vitro: At 2h post-irradiation, p53 protein levels increased 2-fold compared to unirradiated controls. At 6h post-irradiation, p53 protein levels increased 4-fold compared to unirradiated controls. At the same timepoints increases in p21 and p-p53 were observed. Apoptosis showed a maximum increase 12h post-irradiation.<br/>In vivo: DCx+ cells were decreased at 24h post-irradiation.</td></tr><tr><td>Ivanov & Hei, 2014</td><td>In vitro. Human NSCs or neuroblastoma SK-N-SH cells were irradiated with 5 Gy of 137Cs gamma rays (0.82 Gy/min). Western blot analysis was used to assess the levels of various signaling proteins. The percentage of hypodiploid nuclei was analyzed using flow cytometry to quantify apoptotic cells. Survival of differentiated cells was assessed with staining for Nestin (neuroprogenitors) and DCx (immature neurons).</td><td>Altered signaling in both cell types was observed 6h post-irradiation. Apoptosis of NSCs was observed only 24h post-irradiation. NSC differentiation was reduced 10-12 days post-irradiation.</td></tr><tr><td>Zhang et al., 2018</td><td>In vivo and in vitro. Male 1-month-old Sprague-Dawley rats were whole-brain irradiated with electrons (4 MeV) at 2 Gy. Primary cultured hippocampal neurons from 18-day-old Sprague-Dawley rat embryos were irradiated with electrons at 2 or 8 Gy. Western blot was used to assess phosphorylated (inactive) and dephosphorylated (active) NFATc4/3 levels in vitro. Neurite growth in vitro was determined by immunofluorescence of β-tubulin+ neurons. In vivo dendritic growth was determined with a retrovirus labeling newborn neurons with green fluorescent protein.</td><td>In vitro: Dephosphorylated NFATc4/3 was decreased and p-NFATc4/3 was increased at both 1- and 3-days post-irradiation. Although total neurite length was decreased at both 1 and 3 days as well, the number of branching points was only decreased at 3 days post-irradiation.<br/>In vivo: Total dendritic length decreased at both 14- and 28-days post-irradiation.</td></tr><tr><td>Pius-Sadowska et al., 2016</td><td>In vivo. Female 6- to 8-week-old BALB/c mice were whole-brain irradiated with 60Co gamma rays at 10 Gy. The levels of various signaling molecules were assessed with western blot in the brain. Apoptosis was measured with a TUNEL assay in the hippocampus. Nissl staining was used to assess neuron morphology in the hippocampus.</td><td>Following 10 Gy, the various signaling molecules were increased as early as 3h, while apoptosis and morphological changes were found 48h post-irradiation.</td></tr></table>

Incidence Concordance

<table><thead><tr><td>Reference</td><td>Experimental Description</td><td>Results</td></tr></thead><tbody><tr><td>Tian et al., 2020</td><td>In vivo. Male 8- to 10-week-old C57BL/6J mice were subjected to middle cerebral artery occlusion to simulate an ischemic stroke. MAPK signaling molecules and BAX/Bcl-2 apoptotic markers were measured with western blotting in ischemic brain tissues. Apoptosis was determined using a TUNEL assay in the ischemic cerebral cortex. Endpoints were measured 7 days after surgery.</td><td>After surgery, ERK1/2, p38 and JNK mRNA increased 2- to 3-fold. The ratios of phosphorylated to total ERK1/2, p38 and JNK increased 2- to 3-fold as well. BAX increased 2.5-fold, Bcl-2 decreased 0.15-fold, and cleaved caspase-3 increased 1.5-fold. The % of TUNEL+ cells increased 2-fold.</td></tr><tr><td>Eom et al., 2015</td><td>In vitro. C17.2 mouse neural stem-like cells were irradiated with 6 Gy of 137Cs gamma rays at 0.95 Gy/min. Protein levels in signaling pathways were determined by western blot. The number of cells expressing nestin (NSC marker) were quantified with immunocytochemistry. Endpoints were measured 72h post-irradiation.</td><td>Radiation at 6 Gy led to increased p-Akt, p-p53, p-STAT3, and mGluR1 at least 2-fold. NSCs decreased 0.4-fold.</td></tr><tr><td>Suman et al., 2013</td><td>In vivo. Female 6- to 8-week-old C57BL/6J mice were irradiated with either 137Cs gamma rays (2 Gy) or 1 GeV/n 56Fe ions (1.6 Gy) both at 1 Gy/min. p16, p21, p53, BAX, and Bcl-2 levels were determined in the cerebral cortex with immunoblotting. Hematoxylin and eosin staining was used to measure cerebral cortex thickness, and a TUNEL assay was used to measure apoptosis.</td><td>p16 increased a maximum of 3.4-fold after gamma rays and 5-fold after 56Fe radiation. p21 increased a maximum of 1.5-fold after gamma rays and 3-fold after 56Fe radiation. p53 increased a maximum of 8.4-fold after gamma rays and 9-fold after 56Fe radiation. BAX increased a maximum of 2.3-fold after gamma rays and 6.7-fold after 56Fe radiation. Bcl-2 decreased a maximum of 0.6-fold after gamma rays and 0.4-fold after 56Fe radiation. Gamma rays increased apoptosis 1.8-fold and 56Fe ions increased apoptosis 3.6-fold. Gamma rays decreased cortical thickness 0.9-fold and 56Fe ions decreased cortical thickness 0.7-fold.</td></tr><tr><td>Limoli et al., 2004</td><td>In vivo and in vitro. Male 2-month-old C57BL/J6 mice (1.75 Gy/min) and neural precursor cells (4.5 Gy/min) isolated from rats were irradiated with X-rays. Mice were irradiated cranially. Western blot analysis was done in vitro to measure levels of p53 and p21 proteins. An antibody against DCx was</td><td>In vitro: At 5 Gy, p53 levels increased a maximum of 4-fold, while p-p53 and p21 were also increased at this dose. Apoptosis was increased a maximum of 1.4-fold after 5 Gy.<br/>In vivo: The number of DCx+ cells decreased 0.4-fold after 5 Gy.</td></tr></tbody></table>
<table><tr><td>used to detect immature neurons in the dentate gyrus in vivo. FACS analysis of propidium iodide fluorescence was used to assess apoptosis in vitro.</td></tr></table>
References

Betlazar, C. et al. (2016), "The impact of high and low dose ionising radiation on the central nervous system", Redox Biology, Vol. 9, Elsevier, Amsterdam, https://doi.org/10.1016/j.redox.2016.08.002.

Cekanaviciute, E., S. Rosi and S. V. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI), Basel, https://doi.org/10.3390/IJMS19113669.

Chakraborti, A. et al. (2012), "Cranial Irradiation Alters Dendritic Spine Density and Morphology in the Hippocampus", PLOS ONE, Vol. 7/7, Public Library of Science, San Francisco, https://doi.org/10.1371/JOURNAL.PONE.0040844.

Chow, B. M., Y.-Q. Li and C. S. Wong. (2000), "Radiation-induced apoptosis in the adult central nervous system is p53-dependent", Cell Death & Differentiation, Vol. 7/8, Springer Nature, https://doi.org/10.1038/sj.cdd.4400704.

Cunha, C., R. Brambilla and K. L. Thomas. (2010), "A simple role for BDNF in learning and memory?", Frontiers in Molecular Neuroscience, Vol. 3/1, Frontiers, https://doi.org/10.3389/neuro.02.001.2010.

Davis, S. and S. Laroche. (2006), "Mitogen-activated protein kinase/extracellular regulated kinase signalling and memory stabilization: a review", Genes, Brain and Behavior, Vol. 5, Wiley, https://doi.org/10.1111/j.1601-183X.2006.00230.x.

El-Missiry, M. A. et al. (2018), "Neuroprotective effect of epigallocatechin-3-gallate (EGCG) on radiation-induced damage and apoptosis in the rat hippocampus", International Journal of Radiation Biology, Vol. 94/9, Informa, London, https://doi.org/10.1080/09553002.2018.1492755.

Eom, H. S. et al. (2015), "Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways", International Journal of Radiation Biology, Vol. 91/7, Informa, London, https://doi.org/10.3109/09553002.2015.1029595.

Falcicchia, C. et al. (2020), "Involvement of p38 MAPK in Synaptic Function and Dysfunction", International Journal of Molecular Sciences, Vol. 21/16, MDPI, Basel, https://doi.org/10.3390/ijms21165624.

Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research - Reviews in Mutation Research, Vol. 770, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003.

Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", Dose-Response, Vol. 19/1, SAGE publications, https://doi.org/10.1177/1559325820984944.

Ivanov, V. N. and T. K. Hei. (2014), "A role for TRAIL/TRAIL-R2 in radiation-induced apoptosis and radiation-induced bystander response of human neural stem cells", Apoptosis, Vol. 19/3, Springer Nature, https://doi.org/10.1007/s10495-013-0925-4.

Ji, J. et al. (2014), "Forced running exercise attenuates hippocampal neurogenesis impairment and the neurocognitive deficits induced by whole-brain irradiation via the BDNF-mediated pathway", Biochemical and Biophysical Research Communications, Vol. 443/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbrc.2013.12.031.
Kanzawa, T. et al. (2006), "Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway", Oncogene, Vol. 25/26, Springer Nature, https://doi.org/10.1038/sj.onc.1209414.

Kumar, V. et al. (2005), "Regulation of Dendritic Morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK Signaling Pathways", Journal of Neuroscience, Vol. 25/49, Society for Neuroscience, https://doi.org/10.1523/JNEUROSCI.2284-05.2005.

Limoli, C. L. et al. (2004), "Radiation Response of Neural Precursor Cells: Linking Cellular Sensitivity to Cell Cycle Checkpoints, Apoptosis and Oxidative Stress", Radiation Research, Vol. 161/1, BioOne, https://doi.org/10.1667/RR3112.

Long, H.-Z. et al. (2021), "PI3K/AKT Signal Pathway: A Target of Natural Products in the Prevention and Treatment of Alzheimer's Disease and Parkinson's Disease", Frontiers in Pharmacology, Vol. 12, Frontiers, https://doi.org/10.3389/fphar.2021.648636.

Macian, F. (2005), "NFAT proteins: key regulators of T-cell development and function", Nature Reviews Immunology, Vol. 5/6, Springer Nature, https://doi.org/10.1038/nri1632.

Mazzucchelli, C. and R. Brambilla. (2000), "Ras-related and MAPK signalling in neuronal plasticity and memory formation", Cellular and Molecular Life Sciences, Vol. 57/4, Springer Nature, https://doi.org/10.1007/PL00000722.

McHugh, D. and J. Gil. (2018), "Senescence and aging: Causes, consequences, and therapeutic avenues", Journal of Cell Biology, Vol. 217/1, Rockefeller University Press, New York, https://doi.org/10.1083/jcb.201708092.

Mielke, K. and T. Herdegen. (2000), "JNK and p38 stress kinases — degenerative effectors of signal-transduction-cascades in the nervous system", Progress in Neurobiology, Vol. 61/1, Elsevier, Amsterdam, https://doi.org/10.1016/S0301-0082(99)00042-8.

Nebreda, A. R. and A. Porras. (2000), "p38 MAP kinases: beyond the stress response", Trends in Biochemical Sciences, Vol. 25/6, Elsevier, Amsterdam, https://doi.org/10.1016/S0968-0004(00)01595-4.

Pius-Sadowska, E. et al. (2016), "Alteration of Selected Neurotrophic Factors and their Receptor Expression in Mouse Brain Response to Whole-Brain Irradiation", Radiation Research, Vol. 186/5, BioOne, https://doi.org/10.1667/RR14457.1.

Rai, S. N. et al. (2019), "The Role of PI3K/Akt and ERK in Neurodegenerative Disorders", Neurotoxicity Research, Vol. 35/3, Elsevier, Amsterdam, https://doi.org/10.1007/s12640-019-0003-y.

Ran, I., I. Laplante and J.-C. Lacaille. (2012), "CREB-Dependent Transcriptional Control and Quantal Changes in Persistent Long-Term Potentiation in Hippocampal Interneurons", Journal of Neuroscience, Vol. 32/18, Society for Neuroscience, https://doi.org/10.1523/JNEUROSCI.5463-11.2012.

Rodgers, E. E. and A. B. Theibert. (2002), "Functions of PI 3-kinase in development of the nervous system", International Journal of Developmental Neuroscience, Vol. 20/3–5, Wiley, https://doi.org/10.1016/S0736-5748(02)00047-3.

Sherrin, T., T. Blank and C. Todorovic. (2011), "c-Jun N-terminal kinases in memory and synaptic plasticity", Reviews in the Neurosciences, Vol. 22/4, De Gruyter, https://doi.org/10.1515/rns.2011.032.
Silasi, G. et al. (2004), "Selective brain responses to acute and chronic low-dose X-ray irradiation in males and females", Biochemical and Biophysical Research Communications, Vol. 325/4, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbrc.2004.10.166.

Suman, S. et al. (2013), "Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production", Aging, Vol. 5/8, https://doi.org/10.18632/aging.100587.

Tian, R. et al. (2020), "miR-137 prevents inflammatory response, oxidative stress, neuronal injury and cognitive impairment via blockade of Src-mediated MAPK signaling pathway in ischemic stroke", Aging, Vol. 12/11, https://doi.org/10.18632/aging.103301.

Wang, Q. et al. (2020), "Radioprotective Effect of Flavonoids on Ionizing Radiation-Induced Brain Damage", Molecules, Vol. 25/23, MDPI, Basel, https://doi.org/10.3390/molecules25235719.

Zhang, Q. et al. (2018), "The effect of brain-derived neurotrophic factor on radiation-induced neuron architecture impairment is associated with the NFATc4/3 pathway", Brain Research, Vol. 1681, Elsevier, Amsterdam, https://doi.org/10.1016/j.brainres.2017.12.032.
## AOPS Referencing Relationship

<table><thead><tr><th>AOP Name</th><th>Adjacency</th><th>Weight of Evidence</th><th>Quantitative Understanding</th></tr></thead><tbody><tr><td>AOP: 483 – Deposition of Energy Leading to Learning and Memory Impairment</td><td>adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

Evidence for this relationship is derived from studies that use human-derived cells and mouse models, with most of the evidence in mice. There is in vivo evidence in male animals. Most evidence is from adult models.

## Taxonomic Applicability

<table><thead><tr><th>Term</th><th>Scientific Term</th><th>Evidence</th><th>Link</th></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>Moderate</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Low</td><td>NCBI</td></tr></tbody></table>

## Life Stage Applicability

<table><thead><tr><th>Term</th><th>Evidence</th></tr></thead><tbody><tr><td>Juvenile</td><td>Low</td></tr><tr><td>Adult</td><td>Moderate</td></tr></tbody></table>

## Sex Applicability

<table><thead><tr><th>Sex</th><th>Evidence</th></tr></thead><tbody><tr><td>Male</td><td>Moderate</td></tr><tr><td>Female</td><td>Low</td></tr></tbody></table>

## Key Event Relationship Description

DNA single strand breaks (SSBs) and double strand breaks (DSBs) can lead to cell cycle arrest and apoptosis (Madabhushi, Pan and Tsai, 2014; Michaelidesova et al., 2019). In proliferative cells like neural stem/progenitor cells this will reduce neurogenesis within the brain (Alt and Schwer, 2018; Lee and McKinnon, 2007; Michaelidesova et al., 2019). Although the role of DSBs is less well-characterized in mature neurons (Lee and McKinnon, 2007; Thadathil fsylet al., 2019), some evidence suggests that unrepaired DNA strand breaks could also have deleterious effects in these neurons (Wang et al., 2017). Furthermore, there is evidence that DNA strand breaks can induce changes to neural plasticity and synaptic activity through changes in gene expression (Konopka and Atkin, 2022; Thadathil et al., 2019). This can occur via changes in N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor activity or changes in the expression of early response genes (ERGs) that encode transcription factors controlling processes like neurite outgrowth, synapse development and maturation and the balance between excitatory and inhibitory synapses (Konopka and Atkin, 2022).

## Evidence Supporting this KER

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.

Overall Weight of Evidence: Moderate

## Biological Plausibility
The biological plausibility between DNA strand breaks leading to abnormal neural remodeling is supported by literature.

Abnormal neural remodeling due to DNA strand breaks can occur through apoptosis (Abner and McKinnon, 2004; Desai et al., 2022; Madabhushi, Pan and Tsai, 2014; Michaelidesova et al., 2019; Wang et al., 2017; Zhu et al., 2019). Newly post-mitotic neurons with DSBs may undergo checkpoint mediated apoptosis as a mechanism to prevent their incorporation into the nervous system as mature neurons (Alt and Schwer, 2018; Lee and McKinnon, 2007). In response to DSBs, developing neural progenitor cells and a trace number of neural stem cells will undergo cell cycle arrest at critical stages. In the mammalian genome, DNA strand breaks can regulate checkpoint activation through the activation of phosphoinositide 3-kinase (PI3K)-related family of serine/threonine kinases (PIKK), ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR), that can phosphorylate many downstream proteins (Wang et al., 2017). Specifically, DSBs can activate ATM which phosphorylates p53, which can then act on apoptosis factors, p53-upregulated modulator of apoptosis (PUMA), CD95 (Fas/APO1) and apoptotic peptidase activating factor 1 (Apaf1) (Zhu et al., 2019). Activation of this pathway in proliferating cells like neuronal precursors can reduce neurogenesis (Wang et al., 2017; Michaelidesova et al., 2019).

DNA strand breaks can also lead to changes in synaptic activity, neural plasticity, proliferation, and differentiation. Neurons communicate electrically and chemically through synaptic contacts. Neural plasticity refers to the ability of the nervous system to modify its structure, function or connections in response to stimuli. DNA damage can modulate the activity and expression of glutamate receptors, including NMDA/AMPA, which are involved in synaptic activity, plasticity and neuronal activation in the central nervous system. The changes in receptor activity and expression modulate neural gene expression and lead to changes in plasticity (Konopka and Atkin, 2022). Additionally, changes in ERG expression following DNA damage can lead to certain abnormal neural remodeling changes, such as neurite outgrowth, synapse development and maturation (Konopka and Atkin, 2022). As well, inhibition of the cell cycle by DNA strand breaks can impair neurogenesis through decreased differentiation and proliferation of neural stem cells (NSCs) (Michaelidesova et al., 2019).

## Empirical Evidence

The empirical evidence for this KER comes from in vivo mouse models as well as in vitro mouse- and human-derived cell models. Stressors used included X-ray radiation (Barazzuol, Ju and Jeggo, 2017; Barazzuol et al., 2015; Huang et al., 2021; Zhang et al., 2017), 60Co gamma ray radiation (Zanni et al., 2015), 137Cs gamma rays (Acharya et al., 2010), and 6 MV photon radiation (Schmal et al., 2019). Abnormal neural remodeling can be determined through various endpoints, including neuronal apoptosis, morphology, proliferation, differentiation and altered synaptic activity. Markers of DNA strand breaks in this KER include p53 binding protein 1 (53BP1), phosphorylation of H2AX (γ-H2AX), and phosphorylation of ATM (p-ATM).

## Dose Concordance

Several studies demonstrate dose concordance for the relationship between DNA strand breaks leading to abnormal neural remodeling. Adult mice irradiated with X-rays showed increased 53BP1 foci in the lateral ventricle at doses as low as 0.1 Gy, while neuronal apoptosis and impaired neurogenesis were observed as low as 1 Gy (Barazzuol, Ju and Jeggo, 2017). Juvenile and adult male mice irradiated with 0.5 to 2 Gy of 6 MV photons showed increased DSBs in neurons at the same doses as impaired neurogenesis (Schmal et al., 2019). X-ray irradiation of mice aged 2 to 4 months with 50 mGy, 100 mGy and 200 mGy showed increased DSBs in the cerebellum, as well as apoptosis in the subventricular zone (SVZ) (Barazzuol et al., 2015).

## Time Concordance

Many studies demonstrate that DNA strand breaks occur prior to abnormal neural remodeling. Studies frequently measure increased DNA DSBs through γ-H2AX foci or 53BP1 foci within 1 h post-irradiation and increased neuronal apoptosis 4 to 6 h post-irradiation (Acharya et al., 2010; Barazzuol,
<table><tr><td></td><td>determining-region-Y) box 2 (SOX2)+ stem/progenitor cells in the subgranular zone (SGZ) of the hippocampal dentate gyrus.</td><td>cells did not change at 72 h post-irradiation, but decreased 0.6-fold in juvenile mice and 0.8-fold in adult mice at 2 Gy after 1 month.</td></tr><tr><td>Barazzuol et al., 2015</td><td>In vivo. C57BL/6 mice aged 2-4 months were irradiated with 50 mGy, 100 mGy and 200 mGy of X-rays. Apoptosis in the SVZ was determined with a TUNEL assay. DSBs in the cerebellum were quantified with 53BP1 immunofluorescence.</td><td>53BP1 foci increased linearly from 0.05 foci/cell at 0 Gy to 1.3 foci per cell at 200 mGy. The number of TUNEL+ cells increased linearly from 5 cells/section at 0 Gy to about 50 cells/section at 200 mGy.</td></tr><tr><td>Barazzuol, Ju and Jeggo, 2017</td><td>In vivo. C57BL/6 mice were irradiated with various doses of X-rays (0.5 Gy/min). Immunofluorescence was used to detect TUNEL+ cells (apoptotic), Ki67+ cells (proliferating) and DCX+ cells (neuron progenitors). 53BP1 was also detected by immunofluorescence.</td><td>53BP1 foci increased over 10-fold at 0.1 Gy and about 80-fold at 2 Gy in the lateral ventricle. At 1, 2, and 3 Gy, Ki67+ cells in the lateral ventricle decreased 0.2-fold, and TUNEL+ cells were increased in the lateral ventricle. At 2 Gy, DCX+ cells decreased to less than 0.1-fold.</td></tr></table>

## Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Acharya et al., 2010</td><td>In vitro. hNSCs were irradiated with 5 Gy of 137Cs gamma rays (2.2 Gy/min. γ-H2AX foci for DSBs were quantified with immunofluorescence. Apoptosis of hNSCs was measured using fluorescence-activated cell sorting (FACS) for poly (ADP-ribose) polymerase (PARP) cleavage (early marker) and annexin V binding (late marker). Differentiation of hNSCs was measured by β-III-Tubulin staining and cell numbers were measured by SYBR green fluorescence.</td><td>γ-H2AX foci were increased from 5% to 95% as early as 0.3 h post-irradiation. PARP+ cells were increased 3-fold at 6 h post-irradiation, while annexin V+ cells were increased 2-fold 48 h post-irradiation. hNSC differentiation was decreased 0.5-fold 2 days post-irradiation. hNSC cell numbers were decreased 0.3-fold 3 days post-irradiation.</td></tr><tr><td>Barazzuol et al., 2015</td><td>In vivo. C57BL/6 mice aged 2-4 months were irradiated with 50 mGy, 100 mGy and 200 mGy of X-rays. Apoptosis in the SVZ was determined with a TUNEL assay. DSBs in the cerebellum were quantified with 53BP1 immunofluorescence.</td><td>The earliest increase in 53BP1 foci was observed at 0.25 h post-irradiation. The earliest increase in TUNEL+ cells was observed at 6 h post-irradiation.</td></tr><tr><td>Zhang et al., 2017</td><td>In vitro. Cells from the HT22 mouse hippocampal neuronal cell line were irradiated with 12 Gy of X-rays (4 Gy/min). γ-H2AX and p-ATM protein expression was determined with western blot. Apoptosis was determined with flow cytometry using annexin V and propidium iodide staining.</td><td>At 30 min post-irradiation, γ-H2AX increased 3.2-fold and the ratio of p-ATM/ATM increased 4.4-fold. Apoptosis increased over 10-fold at 48 h post-irradiation.</td></tr><tr><td>Barazzuol, Ju and Jeggo, 2017</td><td>In vivo. C57BL/6 mice were irradiated with various doses of X-rays (0.5 Gy/min). Immunofluorescence was used to detect TUNEL+ cells (apoptotic), Ki67+ cells (proliferating) and DCX+ cells (neuron progenitors). 53BP1 was also detected by immunofluorescence.</td><td>A peak in 53BP1 foci occurred at 0.5 h post-irradiation. Changes to TUNEL+ cells, Ki67+ cells and DCX+ cells were observed at 6 h post-irradiation.</td></tr></tbody></table>

## Incidence Concordance

<table><thead><tr><td>Reference</td><td>Experimental Description</td><td>Results</td></tr></thead><tbody><tr><td>Acharya et al., 2010</td><td>In vitro. hNSCs were irradiated with 5 Gy of 137Cs gamma rays (2.2 Gy/min. γ-H2AX foci for DSBs were quantified with immunofluorescence. Apoptosis of hNSCs was measured using FACS for PARP cleavage (early marker) and annexin V binding (late marker). Differentiation of hNSCs was measured by β-III-Tubulin staining and cell numbers were measured by SYBR green fluorescence.</td><td>γ-H2AX foci were increased about 20-fold. PARP+ cells were increased 3-fold and annexin V+ cells were increased 2-fold. hNSC differentiation was decreased by 0.5-fold. hNSC cell numbers were decreased 0.3-fold.</td></tr><tr><td>Barazzuol et al., 2015</td><td>In vivo. C57BL/6 mice aged 2-4 months were irradiated with 50 mGy, 100 mGy and 200 mGy of X-rays. Apoptosis in the SVZ was determined with a TUNEL assay. DSBs in the cerebellum were quantified with 53BP1 immunofluorescence.</td><td>53BP1 foci increased almost 30-fold between 0 Gy and 200 mGy. The number of TUNEL+ cells increased about 10-fold between 0 Gy and 200 mGy.</td></tr></tbody></table>

## Known Feedforward/Feedback loops influencing this KER

Some studies suggest that neuron activity can generate DNA DSBs. Specifically, it has been shown that γ-H2AX foci can be formed by the activation of NMDA a https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/epstein-barr-virus.html and AMPA glutamate receptors (reviewed by Konopka and Atkin, 2022). Activity induced DSBs in mature neurons subsequently influence gene expression and neuronal activity (Alt and Schwer, 2018).
Abner, C. W. and P. J. McKinnon. (2004), "The DNA double-strand break response in the nervous system", DNA Repair, Vol. 3/8–9, Elsevier, Amsterdam, https://doi.org/10.1016/j.dnarep.2004.03.009.

Acharya, M. M. et al. (2010), "Consequences of ionizing radiation-induced damage in human neural stem cells", Free Radical Biology and Medicine, Vol. 49/12, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2010.08.021.

Alt, F. W. and B. Schwer. (2018), "DNA double-strand breaks as drivers of neural genomic change, function, and disease", DNA Repair, Vol. 71, Elsevier, Amsterdam, https://doi.org/10.1016/j.dnarep.2018.08.019.

Barazzuol, L., L. Ju, and P. A. Jeggo. (2017), "A coordinated DNA damage response promotes adult quiescent neural stem cell activation", PLoS biology, 15(5), PLOS, San Francisco, https://doi.org/10.1371/journal.pbio.2001264

Barazzuol, L. et al. (2015), "Endogenous and X-ray-induced DNA double strand breaks sensitively activate apoptosis in adult neural stem cells", Journal of Cell Science, Vol. 128/19, The Company of Biologists, Cambridge, https://doi.org/10.1242/jcs.171223.

Desai, R. I. et al. (2022), "Impact of spaceflight stressors on behavior and cognition: A molecular, neurochemical, and neurobiological perspective", Neuroscience & Biobehavioral Reviews, Vol. 138, Elsevier, Amsterdam, https://doi.org/10.1016/j.neubiorev.2022.104676.

Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", Dose-Response, Vol. 19/1, SAGE Publications https://doi.org/10.1177/1559325820984944.

Konopka, A. and J. D. Atkin. (2022), "The Role of DNA Damage in Neural Plasticity in Physiology and Neurodegeneration", Frontiers in Cellular Neuroscience, Vol. 16, Frontiers, https://doi.org/10.3389/fncel.2022.836885.

Lee, Y. and P. J. McKinnon. (2007), "Responding to DNA double strand breaks in the nervous system", Neuroscience, Vol. 145/4, Elsevier, Amsterdam, https://doi.org/10.1016/j.neuroscience.2006.07.026.

Madabhushi, R., L. Pan and L.-H. Tsai. (2014), "DNA Damage and Its Links to Neurodegeneration", Neuron, Vol. 83/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.neuron.2014.06.034.

Michaelidesova, A. et al. (2019), "Effects of Radiation Therapy on Neural Stem Cells", Genes, Vol. 10/9, MDPI, Basel, https://doi.org/10.3390/genes10090640.

Schmal, Z. et al. (2019), "DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis", Radiotherapy and Oncology, Vol. 137, Elsevier, Amsterdam, https://doi.org/10.1016/j.radonc.2019.04.021.

Thadathil, N. et al. (2019), "DNA double-strand breaks: a potential therapeutic target for neurodegenerative diseases", Chromosome Research, Vol. 27/4, Springer Nature, https://doi.org/10.1007/s10577-019-09617-x.

Wang, H. et al. (2017), "Chronic oxidative damage together with genome repair deficiency in the neurons is a double whammy for neurodegeneration: Is damage response signaling a potential therapeutic target?", Mechanisms of Ageing and Development, Vol. 161, Elsevier, Amsterdam, https://doi.org/10.1016/j.mad.2016.09.005.
Zanni, G. et al. (2015), "Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro", *Oncotarget*, Vol. 6/35, https://doi.org/10.18632/oncotarget.5191.

Zhang, L. et al. (2017), "The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy", *Scientific Reports*, Vol. 7/1, Springer Nature, https://doi.org/10.1038/s41598-017-16693-8.

Zhu, L.-S. et al. (2019), "Emerging Perspectives on DNA Double-strand Breaks in Neurodegenerative Diseases", *Current Neuropharmacology*, Vol. 17/12, Bentham Science Publishers, https://doi.org/10.2174/1570159X17666190726115623.
## AOPs Referencing Relationship

<table><thead><tr><td>AOP Name</td><td>Adjacency</td><td>Weight of Evidence</td><td>Quantitative Understanding</td></tr></thead><tbody><tr><td>AOP: 478 - Deposition of energy leading to occurrence of cataracts</td><td>adjacent</td><td>Moderate</td><td>Low</td></tr><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>adjacent</td><td>Moderate</td><td>Moderate</td></tr><tr><td>AOP: 470 - Deposition of energy leads to abnormal vascular remodeling</td><td>adjacent</td><td>High</td><td>Moderate</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

This KER is plausible in all life stages, sexes, and organisms with DNA. The evidence is from human, rodent, rabbit and bovine in vitro studies that do not specify the sex, as well as an adult rat in vivo study.

## Taxonomic Applicability

<table><thead><tr><td>Term</td><td>Scientific Term</td><td>Evidence</td><td>Link</td></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Low</td><td>NCBI</td></tr><tr><td>rabbit</td><td>Oryctolagus cuniculus</td><td>Low</td><td>NCBI</td></tr><tr><td>bovine</td><td>Bos taurus</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>Low</td><td>NCBI</td></tr></tbody></table>

## Life Stage Applicability

<table><thead><tr><td>Term</td><td>Evidence</td></tr></thead><tbody><tr><td>Adult</td><td>Low</td></tr><tr><td>Not Otherwise Specified</td><td>Low</td></tr></tbody></table>

## Sex Applicability

<table><thead><tr><td>Sex</td><td>Evidence</td></tr></thead><tbody><tr><td>Unspecific</td><td>Low</td></tr><tr><td>Male</td><td>Low</td></tr></tbody></table>

## Key Event Relationship Description

Oxidative stress is an event that involves both a reduction in free radical scavengers and enzymes, and an increase in free radicals (Brennan et al., 2012). Oxidative stress needs to be maintained within an organism to avoid an excess of damage to biological structures, such as DNA. A redox homeostasis between the radicals and the scavengers is necessary. Between reactive oxygen species (ROS) and reactive nitrogen species (RNS), collectively known as RONS, ROS is particularly significant to oxidative damage and disease states. Radicals such as singlet oxygen and hydroxyl radical are highly unstable and will react with molecules near their generation point, while radicals such as H2O2 are more stable and membrane permeable, meaning they can travel further to find electrons (Spector, 1990). Since DNA is mainly found in nucleus, ROS needs to reach the nucleus to induce breaks. Hydroxyl radicals, in addition to being highly reactive, are capable of causing DNA damage (Halliwell et al., 2021; Engwa et al., 2020). The regulation of these radicals is achieved by the antioxidant defense response (ADR), which includes enzymatic and non-enzymatic processes. The ADR is recruited to manage RONS levels, with antioxidants such as superoxide dismutase (SOD) functioning as the first line of defense (Engwa et al., 2020). These antioxidants act as scavengers to oxidants, reacting with them before reaching other structures within the cell such as DNA strands (Cabrera et al., 2011; Engwa et al., 2020). The backbone of DNA can fragment upon sustained exposure to ROS (Uwineza et al., 2019; Cannan et al., 2016). Due to low oxidation potentials, adenine and guanine are the DNA bases more prone to oxidation, with oxidation potentials (normal hydrogen electrode) at pH 7 of 1.3 eV and 1.42 eV compared to the 1.6 eV and 1.7 eV of cytosine and thymine (Fong, 2016; Halliwell et al.,
2021; Poetsch, 2020). In fact, certain radicals even target guanine in a selective fashion, including carbonate anion radical (CO3•-) and singlet oxygen (1O2) (Halliwell et al., 2021).

### Evidence Supporting this KER

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.

Overall Weight of Evidence: Moderate

### Biological Plausibility

The biological plausibility of the relationship between increased oxidative stress leading to increased DNA double strand breaks (DSBs) is highly supported by the literature. Evidence was collected from studies conducted using in vitro lens epithelial cell models and derived from humans, bovine and germ line cells (Spector, 1990; Stohs, 1995; Aitken et al., 2001; Spector, 1995). As this evidence is derived from studies using a human cell model it limits the ability to compare between different taxonomies (Ahmadi et al., 2022; Cencer et al., 2018; Liu et al., 2013; Meng et al., 2021; Smith et al., 2015; Zhou et al., 2016). Other evidence comes from human-derived and rodent models of neuronal and endothelial cells (Cervelli et al., 2014; El-Missiry et al., 2018; Huang et al., 2021; Sakai et al., 2017; Ungvari et al., 2013; Zhang et al., 2017).

ROS that are generated specifically as a result of radiation are highly localized, increasing the likelihood of clustered regions of damage. Naturally generated ROS are more widespread and as a result less capable of generating clusters of damage. ROS will act on DNA bases to oxidize or delete them from the sequence, which create nicks on the strand (Cannan et al., 2016). This damage can occur to any DNA base but bases such as guanine and adenine are most vulnerable due to their low oxidation potentials (Fong, 2016). The mechanism through which the strand break occurs is a result of base excision repair (BER) happening at multiple sites that are too close together, resulting in the spontaneous conversion to DSBs prior to completion of repair. ROS damage to bases clustered together means that multiple sites of BER are happening very close together and while the strand may be able to support the damaged area for one repair, concurrent repairs make surrounding areas more fragile and the strand breaks at the nick sites are under added strain (Cannan et al., 2016). Endogenous damage to DNA as a result of radicals appears over time and mainly as isolated lesions, a pattern understood to be due to the diffusion of the radicals resulting in homogenous distribution patterns. This differs from the specific situations where radiation acts as the stressor to increase oxidative stress, as the radiation track will be highly localized and form radicals within that hit space. This leads to non-homologous lesions and clustered damage to the DNA (Ward et al., 1985).

### Empirical Evidence

This relationship is well supported through empirical evidence from studies using stressors such as H2O2, photons, γ- and X-ray, which cause an increase in markers of oxidative stress such as ROS-generating enzymes (lactate dehydrogenase, LDH), and a decrease in free radical scavengers, resulting in DNA strand fragmentation. These studies include both in vivo and in vitro human lens epithelial cells (LECs), mouse, rat and rabbit models, including neuronal cells lines and endothelial cells (Ahmadi et al., 2022; Cencer et al., 2018; Cervelli et al., 2014; El-Missiry et al., 2018; Huang et al., 2021; Liu et al., 2013; Meng et al., 2021; Spector et al., 1997; Ungvari et al., 2013; Zhang et al., 2017; Zhou et al., 2016; Sakai et al., 2017).

### Dose/Incidence Concordance

There is high evidence to support a dose concordance between oxidative stress and DNA strand breaks. One in vitro study demonstrated that when ROS levels in LECs are 10% above control following 0.5 Gy gamma ray exposure, DNA strand breaks increased 15-20% above control (Ahmadi et al., 2021). Another study with ultraviolet (UV)B radiation demonstrated higher ROS levels after exposure to 0.14 J/cm2 on in vitro LECs as compared to a lower dose exposure (0.014 J/cm2) for the same time. This
corresponded to DNA strand break levels also increasing following high dose rate exposure, but not
with the low dose exposure (Cencer et al., 2018).

A 30 µM of H2O2 treatment of in vitro LECs is associated with a 1.4x increase in lactate dehydrogenase (LDH) and 55% more DNA strand breaks (Liu et al., 2013; Smith et al., 2015). Following exposure of in vitro LECs to 50 µM H2O2, increased ROS levels, 4x for LDH, and decreased antioxidant levels, 2x control for GSH-Px and SOD, are associated with a 3x increase in γ-H2AX, a marker of DNA strand breaks (Meng et al., 2021). SOD and GSH decreased by 2-fold following 100 µM H2O2 exposure on LECs with an in vitro model (Zhou et al., 2016). At 125 µM H2O2 intact DNA can be reduced to near 1% of pre-treatment levels for in vitro LECs (Spector et al., 1997). Following 400 µM H2O2 LDH increased to 1200% of control in neuroblastoma cells (Feng et al., 2016) and DNA strand breaks increased to over 150% of control in in vitro LECs (Li et al., 1998).

Exposure of in vitro mouse hippocampal neuronal cells (HT22 cell line) to 10 Gy of X-irradiation resulted in a 5x increase in ROS generation and 3x increase in γ-H2AX (Huang et al., 2021). Another study exposed the same cell line to 8 and 12 Gy of X-irradiation and found a ~2x increase in ROS at 8 Gy and a 4.4x and 3.2x increase in phosphorylation of ataxia telangiectasia mutated (ATM) and γ-H2AX, respectively, 30 minutes after 12 Gy (Zhang et al., 2017). A separate study exposed adult male rats to 4 Gy of γ-irradiation and found 2x increase in 4-hydroxy-2-nonenal (4-HNE) (lipid peroxidation marker) and 3x increase in protein carbonylation. Glutathione reductase decreased by approximately 5x, whereas glutathione and glutathione peroxidase levels decreased by approximately 3x each. Tail DNA %, tail length and tail moment (DNA strand break parameters) increased by approximately 2x, 3x and 6x, respectively (El-Missiry et al., 2018).

Endothelial cells exposed to irradiation also demonstrated the relation between oxidative stress and DNA strand breaks. Rat cerebromicrovascular endothelial cells (CMVECs) exposed to 8 Gy 137Cs gamma rays showed increased cellular peroxide production and mitochondrial oxidative stress. Tail DNA content indicating DNA damage was also increased from 0 to 45% (Ungvari et al., 2013). Human umbilical vein endothelial cells (HUVECs) were irradiated with single (0.125, 0.25, 0.5 Gy), or fractionated (2 × 0.125 Gy, 2 × 0.250 Gy) doses of X-rays. Intracellular ROS production increased in a dose-dependent manner following 0.125, 0.25, 0.5 Gy, and γ-H2AX foci positive cells were observed at all doses (Cervelli et al., 2014). Human aortic endothelial cells (HAECs) exposed to 100μM H2O2 showed 3.7-fold increase in intracellular ROS and a 3.4- and 4.7-fold increase in γ-H2AX and p-ATM, respectively (Sakai et al., 2017).

## Time Concordance

There is low evidence to support a time concordance between oxidative stress to strand breaks on DNA. Non-protein-thiol levels, an antioxidant, in in vitro LECs decreased to near zero by 30 minutes post-exposure to 300 µM H2O2, before recovering to 70% of control by 120 minutes. At 60 minutes post-exposure to 125 µM H2O2 there was a start to a divergence from control level DNA fragmentation, one that increased logarithmically, with the treated group having a 14~18% reduction in intact DNA by 9 h post-exposure (Yang et al., 1998). Time response information is difficult to monitor for DNA strand breaks because repair will occur, reducing the number of breaks over time. At 0 minutes post in vitro exposure to 40 µM H2O2 LECs had ~145% of control level DNA strand breaks but that number dropped to ~105% by 30 minutes post-exposure (Li et al., 1998).

## Essentiality

Oxidative stress has been found to increase levels of DNA strand breaks above background levels (Li et al., 1998; Liu et al., 2013; Cencer et al., 2018; Ahmadi et al., 2022; El-Missiry et al., 2018; Huang et al., 2021; Cervelli et al., 2017; Sakai et al., 2017). It has been shown that inhibition of oxidative stress leads to a reduction in DNA strand breaks. Sulforaphane (SFN) is an isothiocyanate, which provides chemical protection against ROS by activating the release of enzymatic scavengers. When SFN was added to in vitro LECs exposed to 30 µM H2O2, LDH decreased to near unexposed cell levels from the 1.4x control level without SFN. This LDH drop was associated with reducing the levels of DNA strand breaks induced by oxidative stress almost 3-fold as compared to cells without SFN
(Liu et al., 2013). In another study, intact DNA levels were returned to control when treated with μPx-11 (peroxidase that breaks down H2O2), following exposure to 125 μM H2O2. This was a near 100% recovery compared to the drop seen in LECs that did not contain μPx-11 (Spector et al., 1997).

Within the brain of Wistar rats, epigallocatechin-3-gallate (EGCG) ameliorated radiation-induced increases in lipid peroxidation and protein carbonylation, as well as decreases in glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) and reverted the levels back to those similar to controls. DNA strand break parameters also returned to those similar to controls after treatment with EGCG (El-Missiry et al., 2018). Similar effects were also shown in another study using treatment mesenchymal stem cell-conditioned medium in mouse hippocampal cells exposed to 10 Gy of X-irradiation (Huang et al., 2021).

HUVECs pretreated with the antioxidant mixture RiduROS blunted ROS generation in a concentration-dependent manner by 65% ± 5.6% and 98% ± 2%, at 0.1 and 1 μg/mL, respectively, compared with cells irradiated without pretreatment. Low-dose irradiation also increased DSB-induced γ-H2AX foci compared with control cells and 24 h of RiduROS pretreatment reduced the γ-H2AX foci number by 41% (Cervelli et al., 2017). Additionally, HAECs treated with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found significantly reduced intracellular ROS at 100μM, as well as reduced γ-H2AX foci formation by 47% and 48% following EPA and DHA treatment respectively. (Sakai et al., 2017).

## Known modulating factors

There is limited evidence demonstrating this relationship across different life stages/ages or sexes (Cencer et al., 2018; Li et al., 1998).

<table><thead><tr><td>Modulating Factors</td><td>MF Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Age</td><td>Reduced antioxidant capacities have been linked to aged lenses (in humans >30 years old). The development of a chemical barrier between the cortex and the nucleus is partially responsible, as it prevents GSH from protecting aged lens cells from ROS.</td><td>Prevention of RONS-mediated damage is primarily achieved by antioxidants, so a lowered capacity would likely lead to reduced damage mitigation abilities. 78% of lens over 30 had a low level of GSH in the center compared to 14% of lens under 30. Lens epithelial cells have an associated 3-fold increase in γ-H2AX (marker of DNA damage) when GSH-PX decreases by 2-fold.</td><td>Taylor & Davies, 1987; Cabrera & Chihuailaf, 2011; Quinlan & Hogg, 2018; Sweeney & Truscott, 1998; Meng & Fang, 2021</td></tr><tr><td>Free radical scavengers</td><td>ROS-scavengers are essential components of the body's natural defense against oxidative damage. Increased ROS production leads to increased incidence of electron donation by scavengers, thus reducing the overall level of free radical scavengers available to deal with ROS.</td><td>Isothiocyanates, such as sulforaphane (SFN), activate the release of more enzymatic scavengers. When SFN was added to in vitro LECs, LDH decreased to near unexposed cell levels and was associated with 3.3x less DNA strand breaks compared to the non-SFN cells following stressor exposure. Epigallocatechin-3-gallate (EGCG) also has antioxidant properties and was shown to alleviate radiation-induced increases in oxidative stress and DNA strand breaks within rat hippocampi.</td><td>Taylor et al., 1987; Cabrera et al., 2011; Liu et al., 2013; El-Missiry et al., 2018</td></tr><tr><td>Media</td><td>Mesenchymal stem cell-conditioned medium (MSC-CM), which has self-renewal, differential and proliferation capacities.</td><td>MSC-CM treatment has also been shown to improve ROS levels and decrease radiation-induced DNA strand breaks within mouse hippocampal neuronal cells.</td><td>Huang et al., 2021</td></tr></tbody></table>

## Quantitative Understanding of the Linkage

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

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Cencer et al., 2018</td><td>In vitro, human LECs exposed to UVB and tested for 120 min post exposure with fluorescent probes to detect ROS production and mitochondrial superoxide, and tetramethylrhodamine-dUTP (TMR) red assay to detect strand breaks.</td><td>Both ROS and DNA strand breaks were increased by both 0.014 J/cm2 and 0.14 J/cm2 UVB radiation. At 0.014 J/cm2, cellular ROS increased a maximum of 15 fluorescence units above the control at 5 min post-UVB, while DNA strand breaks increased about 115 fluorescence units above the control at</td></tr></tbody></table>
<table><tr><td>Ahmadi et al., 2021</td><td>In vitro, human LECs exposed to 0.065-0.3 Gy/min gamma radiation, with dihydroethidium (DHE) fluorescent probes to measure ROS levels and comet assay to measure strand breaks.</td><td>Human LECs exposed in vitro to 0.1-0.5 Gy gamma rays showed a gradual increase in ROS levels and a corresponding gradual increase in DNA in the tail from the comet assay (indicative of increased DNA strand breaks) with the maximum dose displaying a 10% increase in ROS levels and a 17% increase in DNA strand damage.</td></tr><tr><td>Li et al., 1998</td><td>In vitro, bovine LECs were exposed to 40 and 400 µM H2O2 with an alkaline unwinding assay to determine strand break levels.</td><td>Immediately after LECs were exposed to 40 µM and 400 µM H2O2, there were ~145% and ~150% DNA strand breaks compared to the unexposed control level, respectively. The amounts of DNA strand breaks in cells exposed to both concentrations were reduced to ~105% of the unexposed control level after 30 min. After 400 µM H2O2, oxidative stress as measured by LDH was 1200% of control in neuroblastoma cells.</td></tr><tr><td>Spector et al., 1997</td><td>In vitro, rat LECs exposed to 100 and 125 µM H2O2 with alkaline elution assay to determine single strand break level.</td><td>Exposure to 125 µM of H2O2 to lens epithelial cells resulted in reduction of intact DNA to near 1% by 9 hr post-exposure. Exposure to 100 µM H2O2 reduced SOD and GSH levels by 2-fold.</td></tr><tr><td>El-Missiry et al., 2018</td><td>In vivo, albino Wistar rats were exposed to 4 Gy of γ radiation (137Cs source) at 0.695 rad/s. Kits were used to measure 4-HNE (secondary product of lipid peroxidation) and protein carbonyl group levels as markers of oxidative stress. Antioxidants including GSH, GPx and GR were also assessed. The comet assay was used to analyze DNA strand breaks by visualizing DNA tail %, tail length and tail moment.</td><td>4-HNE and protein carbonyl levels increased by approximately 2- and 3-fold after radiation exposure. GSH and GPx levels decreased by approximately 3-fold each, whereas GR levels decreased by approximately 5-fold. Tail DNA %, tail length and tail moment increased by approximately 2-, 3- and 6-fold after exposure to 4 Gy.</td></tr><tr><td>Ungvari et al., 2013</td><td>In vitro, CMVECs and rat hippocampal neurons were irradiated with 2-8 Gy 137Cs gamma rays. 5(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) staining, and flow cytometry were used to measure ROS production. DNA damage was quantified by measuring the tail DNA content (as a percentage of total DNA) using the Comet Assay-IV software.</td><td>Day 1 post-irradiation showed increased cellular peroxide production and increased mitochondrial oxidative stress in CMVECs in a dose-dependent manner, increasing a maximum of ~3-fold at 8 Gy. Tail DNA content also increased in a dose-dependent manner with an approximate increase from 0 to 45% at 8 Gy.</td></tr><tr><td>Huang et al., 2021</td><td>In vitro, HT22 cells (mouse hippocampal neuronal cell line) were exposed to 10 Gy of X-irradiation at 6 Gy/min. ROS levels were measured using H2-DCFDA staining and fluorescence microscope analysis, whereas western blotting was used to detect γ-H2AX.</td><td>At 10 Gy, intracellular ROS generation increased by 5-fold and γ-H2AX increased by 3-fold.</td></tr><tr><td>Zhang et al., 2017</td><td>In vitro, HT22 cells were exposed to 8 and 12 Gy X-rays. Relative intracellular ROS levels were determined by DCFDA. p-ATM, γ-H2AX were measured with Western blot.</td><td>Following 8 Gy irradiation, intracellular ROS levels increased ~1.8-fold. Phosphorylation of ATM and γ-H2AX were increased 4.4-fold and 3.2-fold, respectively, 30 min after 12 Gy.</td></tr><tr><td>Cervelli et al., 2014</td><td>In vitro, HUVECs were irradiated with single doses (0.125, 0.25, 0.5 Gy), or fractionated doses (2 × 0.125 Gy, 2 × 0.250 Gy) of X-rays. Intracellular ROS generation was measured with a fluorescent dye, C-DCFDA, using a spectrofluorometer. Immunofluorescence microscopy was used to measure γ-H2AX foci.</td><td>Intracellular ROS production was significantly increased in a dose-dependent manner (1.6-, 2- and 2.8-fold at 0.125, 0.25, 0.5 Gy, respectively). When HUVECs were exposed to fractionated doses, no increase in ROS generation was observed, compared with respective single doses. 24h post-irradiation the percentage of foci-positive cells exposed to 0.125 Gy, 2 × 0.125 Gy, 0.250 Gy, 2 × 0.250 Gy and 0.5 Gy, was 1.68, 1.48, 3.53, 2.59, 8.74-fold over the control, respectively.</td></tr><tr><td>Sakai et al., 2017</td><td>In vitro, HAECs were exposed to 100µM H2O2. Intracellular ROS was measured by CM-H2DCFDA. DNA DSBs were detected by immunofluorescent analysis with γ-H2AX as a marker.</td><td>Intracellular ROS increased by ~3.7-fold p-ATM increased by ~4.7-fold. γ-H2AX increased by ~3.4-fold.</td></tr></table>

Incidence Concordance
<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Meng et al., 2021</td><td>In vitro, human LECs exposed to 50 µM H2O2 with DCFH-DA fluorescent probe to detect ROS levels and immunofluorescence and western blot assay to detect γ-H2AX.</td><td>50 µM H2O2 exposure to lens epithelial cells increased oxidative stress, with ROS measured by LDH, by 4-fold and decreased the level of antioxidants by 2-fold as measured by SOD and GSH-PX. This resulted in 3-fold increase in γ-H2AX.</td></tr><tr><td>Smith et al., 2015</td><td>In vitro, human LECs exposed to 30 µM H2O2 with alkaline comet assay to determine amount of strand breaks.</td><td>Treatment of lens epithelial cells to 30 µM H2O2 induced DNA strand breaks by 55% at 0.5 hr after exposure and increased the level of LDH by ~1.4 fold at 24 hr post-exposure.</td></tr><tr><td>Liu et al., 2013</td><td>In vitro, human LECs exposed to 30 µM H2O2 with alkaline comet assay determination of strand breaks.</td><td>LDH increased by ~1.4 fold at 24 hr post-exposure, with a 5x increase from control levels in DNA strand breaks.</td></tr></tbody></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Yang et al., 1998</td><td>In vitro, rabbit LECs exposed to H2O2 with TCA addition and thiol assay to determine non-protein thiol (NP-SH) level and alkaline elusion assay to determine strand breaks.</td><td>In rabbit LECs exposed in vitro to 125 µM H2O2, non-protein thiol levels decreased to <5% control (indicates oxidative stress) 30 min post-irradiation, and % DNA retained using alkaline elution decreased by 1.6 log (indicates increased DNA fragmentation) within the next 8.5 h.</td></tr></tbody></table>
Ahmadi, M. et al. (2021), “Early Responses to Low-Dose Ionizing Radiation in Cellular Lens Epithelial Models”, Radiation Research, Vol.197, Radiation Research Society, Indianapolis, https://doi.org/10.1667/RADE-20-00284.1.

Aitken, R.J. and C. Krausz. (2001), “Oxidative stress, DNA damage and the Y chromosome”, Reproduction, Vol.122/2001, Bioscientifica, Bristol, https://doi.org/10.1530/rep.0.1220497.

Annesley, S.J. and P.R. Fisher. (2019), “Mitochondria in Health and Disease”, Cells, Vol.8/7, MDPI, Basel, https://doi.org/10.3390/cells8070680.

Brennan, L., R. McGreal and M. Kantorow. (2012), “Oxidative stress defense and repair systems of the ocular lens”, Frontiers in Bioscience – Elite, Vol.4/E(1), Frontiers in Bioscience, Singapore, https://doi.org/10.2741/365.

Britton, S. et al. (2020), “ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks”, Nucleic Acids Research, Vol.48/17, Oxford University Press, Oxford, https://doi.org/10.1093/nar/gkaa723.

Cabrera, M. and R. Chihuailaf. (2011), “Antioxidants and the integrity of ocular tissues”, Veterinary Medicine International, Vol.2011, Hindawi Limited, London, https://doi.org/10.4061/2011/905153.

Cannan, W. and D. Pederson. (2016), “Mechanisms and consequences of double-strand DNA break formation in chromatin”, Journal of Cell Physiology, Vol.231/1, Wiley, Hoboken, https://doi.org/10.1002/jcp.25048.

Cencer, C. et al. (2018), “PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light”, Photochemistry and Photobiology, Vol.94/1, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/php.12814.

Cervelli, T. et al. (2014), “Effects of single and fractionated low-dose irradiation on vascular endothelial cells”, Atherosclerosis, Vol.235/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.atherosclerosis.2014.05.932.

Cervelli, T. et al. (2017), “A New Natural Antioxidant Mixture Protects against Oxidative and DNA Damage in Endothelial Cell Exposed to Low-Dose Irradiation”, Oxidative medicine and cellular longevity, Vol. 2017, Hindawi, London, https://doi.org/10.1155/2017/9085947.

Climent, M. et al. (2020), “MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases”, International Journal of Molecular Science, Vol.21/12, MDPI, Basel, https://doi.org/10.3390/ijms21124370.

Dahm-Daphie, J., C. Sass, and W. Alberti. (2000), “Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells”, International Journal Radiation Biology, Vol.76/1, Informa, London, https://doi.org/10.1080/095530000139023.

El-Missiry, M. A. et al. (2018), "Neuroprotective effect of epigallocatechin-3-gallate (EGCG) on radiation-induced damage and apoptosis in the rat hippocampus", International Journal of Radiation Biology, Vol. 94/9, https://doi.org/10.1080/09553002.2018.1492755.

Engwa, G.A., F.N. Nweke and B.N. Nkeh-Chungag. (2020), “Free Radicals, Oxidative Stress-Related Diseases and Antioxidant Supplementation”, Alternative therapies in health and medicine, Vol.28/1, InnoVision Health Media, Eagan, pp.114-128.
Feng, C. et al. (2016), "Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways", Molecular Medicine Reports, Vol.13/5, Spandidos Publications, Athens, https://doi.org/10.3892/mmr.2016.5056.

Fong, C.W. (2016), "Platinum anti-cancer drugs: Free radical mechanism of Pt-DNA adduct formation and anti-neoplastic effect", Free Radical Biology and Medicine, Vol.95/June 2016, Elsevier, Amsterdam, https://doi.org/10.1016/j.freeradbiomed.2016.03.006.

Halliwell, B. et al. (2021), "Hydroxyl radical is a significant player in oxidative DNA damage in vivo", Chemical Society Reviews, Vol.50, Royal Society of Chemistry, London, https://doi.org/10.1039/d1cs00044f.

Huang, Y. et al. (2021), "Mesenchymal Stem Cell-Conditioned Medium Protects Hippocampal Neurons From Radiation Damage by Suppressing Oxidative Stress and Apoptosis", Dose-Response, Vol. 19/1, https://doi.org/10.1177/1559325820984944.

Kay, J. et al. (2019), "Inflammation-induced DNA damage, mutations and cancer", DNA Repair, Vol.83, Elsevier, Amsterdam, https://doi.org/10.1016/j.dnarep.2019.102673.

Jeggo, P.A., V. Geuting and M. Löbrich. (2011), "The role of homologous recombination in radiation-induced double-strand break repair", Radiotherapy and Oncology, Vol.101/1, Elsevier, Amsterdam, https://doi.org/10.1016/j.radonc.2011.06.019.

Kurutas E. B. (2016), "The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state", Nutrition journal, Vol.15/1, Biomed Central, London, https://doi.org/10.1186/s12937-016-0186-5.

Kruk, J., K. Kubasik-Kladna and H. Aboul-Enein. (2016), "The Role Oxidative Stress in the Pathogenesis of Eye Diseases: Current Status and a Dual Role of Physical Activity", Mini-Review in Medicinal Chemistry, Vol.16/3, Bentham Science Publishers, Sharjah, https://doi.org/10.2174/1389557516666151120114605.

Li, Y. et al. (1998), "Response of lens epithelial cells to hydrogen peroxide stress and the protective effect of caloric restriction", Experimental Cell Research, Vol.239/2, Elsevier, Amsterdam, https://doi.org/10.1006/excr.1997.3870.

Liu, H. et al. (2013), "Sulforaphane can protect lens cells against oxidative stress: Implications for cataract prevention", Investigative Ophthalmology and Visual Science, Vol.54/8, Association for Research in Vision and Ophthalmology, Rockville, https://doi.org/10.1167/iovs.13-11664.

Meng, K. and C. Fang. (2021), "Knockdown of Tripartite motif-containing 22 (TRIM22) relieved the apoptosis of lens epithelial cells by suppressing the expression of TNF receptor-associated factor 6 (TRAF6)", Bioengineered, Vol.12/1, Taylor & Francis, Oxfordshire, https://doi.org/10.1080/21655979.2021.1980645.

Nishida, M. et al. (2005), "Ga12/13- and Reactive Oxygen Species-dependent Activation of c-Jun NH2-terminal Kinase and p38 Mitogen-activated Protein Kinase by Angiotensin Receptor Stimulation in Rat Neonatal Cardiomyocytes", Journal of Biological Chemistry, Vol.280/18, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1074/jbc.M409710200.

Poetsch, A.R. (2020), "The genomics of oxidative DNA damage, repair, and resulting mutagenesis", Computational and Structural Biotechnology Journal, Vol.18, Elsevier, Amsterdam, https://doi.org/10.1016/j.csbj.2019.12.013.
Quinlan, R.A., and P.J. Hogg. (2018), "γ-Crystallin redox-detox in the lens", Journal of Biological Chemistry, Vol.293/46, American Society for Biochemistry and Molecular Biology, Rockville, https://doi.org/10.1074/jbc.H118.006240.

Sakai, C. et al. (2017), "Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells", PloS one, Vol.12/11, https://doi.org/10.1371/journal.pone.0187934.

Scully, R. and A. Xie. (2013), "Double strand break repair functions of histone H2AX", Mutation Research, Vol.750/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrfmmm.2013.07.007.

Smith, A. et al. (2015), "Ku80 counters oxidative stress-induced DNA damage and cataract formation in the human lens", Investigative Ophthalmology and Visual Science, Vol.56/13, Association for Research in Vision and Ophthalmology, Rockville, https://doi.org/10.1167/iovs.15-18309.

Spector, A. et al. (1997), "Microperoxidases catalytically degrade reactive oxygen species and may be anti-cataract agents", Experimental Eye Research, Vol.65/4, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1997.0336.

Spector, A. et al. (1996), "Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress", Experimental Eye Research, Vol.62/5, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1996.0063.

Spector, A. (1995), "Oxidative stress-induced cataract: mechanism of action", The FASEB Journal, Vol.9/12, Federation of American Societies for Experimental Biology, Bethesda, https://doi.org/10.1096/fasebj.9.12.7672510.

Spector, A. (1990), "Oxidation and Aspects of Ocular Pathology", CLAO Journal, Vol.16/1, Lippincott, Williams and Wilkins Ltd, Philadelphia, S8-S10.

Stohs, S. (1995), "The role of free radicals in toxicity and disease", Journal of Basic Clinical Physiology and Pharmacology, Vol.6/3-4, Walter de Gruyter GmbH, Berlin, https://doi.org/10.1515/jbcpp.1995.6.3-4.205.

Sweeney, M.H.J. and R.J.W. Truscott. (1998), "An Impediment to Glutathione Diffusion in Older Normal Human Lenses: a Possible Precondition for Nuclear Cataract", Experimental Eye Research, Vol.67, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0549.

Taylor, A. and K. J. A. Davies (1987), "Protein oxidation and loss of protease activity may lead to cataract formation in the aged lens", Free Radical Biology & Medicine, Vol. 3, Pergamon Journals Ltd, United States of America, pp. 371-377

Ungvari, Z. et al. (2013), "Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosens", The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 68/12, https://doi.org/10.1093/gerona/glt057.

Uwineza, A. et al. (2019), "Cataractogenic load – A concept to study the contribution of ionizing radiation to accelerated aging in the eye lens", Mutation Research - Reviews in Mutation Research, Vol.779, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrrev.2019.02.004.
Ward, J.F., W.F. Blakely & E.I. Joner. (1985), "Mammalian Cells Are Not Killed by DNA Single-Strand Breaks Caused by Hydroxyl Radicals from Hydrogen Peroxide", Radiation Research, Vol.103/3, Radiation Research Society, Indianapolis, https://doi.org/10.2307/3576760.

Wu, H. et al. (2021), “Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli”, Signal Transduction and Targeted Therapy, Vol.6/1, Nature Portfolio, Berlin, https://doi.org/10.1038/s41392-021-00595-3.

Yang, Y. et al. (1998), “The effect of catalase amplification on immortal lens epithelial cell lines”, Experimental Eye Research, Vol.67/6, Academic Press Inc, Cambridge, https://doi.org/10.1006/exer.1998.0560.

Yuan, J., R. Adamski and J. Chen. (2010), “Focus on histone variant H2AX: To be or not to be”, FEBS Letters, Vol.584/17, Wiley, Hoboken, https://doi.org/10.1016/j.febslet.2010.05.021.

Zhang, L. et al. (2017), "The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy", Scientific Reports, Vol. 7/1, https://doi.org/10.1038/s41598-017-16693-8.

Zhou, Y. et al. (2016), “Protective Effect of Rutin Against H2O2-Induced Oxidative Stress and Apoptosis in Human Lens Epithelial Cells”, Current Eye Research, Vol.41/7, Taylor & Francis, Oxfordshire, https://doi.org/10.3109/02713683.2015.1082186.
## AOPs Referencing Relationship

<table><thead><tr><td>AOP Name</td><td>Adjacency</td><td>Weight of Evidence</td><td>Quantitative Understanding</td></tr></thead><tbody><tr><td>AOP: 272 - Deposition of energy leading to lung cancer</td><td>adjacent</td><td>High</td><td>High</td></tr><tr><td>AOP: 216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</td><td>adjacent</td><td>High</td><td></td></tr><tr><td>AOP: 238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</td><td>adjacent</td><td></td><td></td></tr><tr><td>AOP: 478 - Deposition of energy leading to occurrence of cataracts</td><td>adjacent</td><td>High</td><td>High</td></tr><tr><td>AOP: 470 - Deposition of energy leads to abnormal vascular remodeling</td><td>adjacent</td><td>High</td><td>High</td></tr><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>adjacent</td><td>High</td><td>High</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

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.

## Taxonomic Applicability

<table><thead><tr><td>Term</td><td>Scientific Term</td><td>Evidence</td><td>Link</td></tr></thead><tbody><tr><td>mouse</td><td>Mus musculus</td><td>High</td><td>NCBI</td></tr><tr><td>human</td><td>Homo sapiens</td><td>High</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>High</td><td>NCBI</td></tr><tr><td>bovine</td><td>Bos taurus</td><td>Low</td><td>NCBI</td></tr><tr><td>rabbit</td><td>Oryctolagus cuniculus</td><td>Low</td><td>NCBI</td></tr><tr><td>Pig</td><td>Pig</td><td>Low</td><td>NCBI</td></tr></tbody></table>

## Life Stage Applicability

<table><thead><tr><td>Term</td><td>Evidence</td></tr></thead><tbody><tr><td>All life stages</td><td>High</td></tr></tbody></table>

## Sex Applicability

<table><thead><tr><td>Sex</td><td>Evidence</td></tr></thead><tbody><tr><td>Unspecific</td><td>High</td></tr></tbody></table>

## Key Event Relationship Description

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 & 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). Thus, 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 Supporting this KER

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.

Overall Weight of Evidence: High

## Biological Plausibility

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; Valentin, 1998; UNSCEAR, 2000; Terato & Ide, 2005; Goodhead, 2006; Kim & Lee, 2007; Asaithamby et al., 2008; Hada & Georgakilas, 2008; Jeggo, 2009; Stewart, 2012; Okayasu, 2012b; M. E. Lomax et al., 2013; EPRI, 2014; Hamada, 2014; Moore et al., 2014; Desouky et al., 2015; Rothkamm 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 γ-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

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><img src="image_6.png" /><figcaption>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.</figcaption></figure>
<figure><img src="image_7.png" /></figure>

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 & 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).
## 210 | 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 & Lo, 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

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

<table><thead><tr><td>Modulating Factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Nitroxides</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>DeGraff et al., 1992; Citrin & Mitchel, 2014</td></tr><tr><td>5-fluorouracil</td><td>Increased concentration</td><td>Increased DNA strand breaks.</td><td>De Angelis et al., 2006; Citrin & Mitchel, 2014</td></tr><tr><td>Thiols</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>Milligan et al., 1995; Citrin & Mitchel, 2014</td></tr><tr><td>Cisplatin</td><td>Increased concentration</td><td>Decreased DNA break repair.</td><td>Sears & Turchi; Citrin & Mitchel, 2014</td></tr></tbody></table>

### Quantitative Understanding of the Linkage

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.

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Ward, 1988</td><td>In vitro. Cells containing approximately 6 pg of DNA were exposed to 1 Gy.</td><td>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.</td></tr><tr><td>Charlton, 1989</td><td>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.</td><td>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.</td></tr><tr><td>Sutherland, 2000</td><td>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.</td><td>Using isolated bacteriophage T7 DNA and 0-100 Gy of γ rays, observed a response of 2.4 DSBs per megabase pair per Gy.</td></tr><tr><td>Rogakou et al., 1999</td><td>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.</td><td>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).</td></tr><tr><td>Kuhne et al., 2005</td><td>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 CK X-rays (0.14 Gy/min). The number of DSBs were determined with pulsed-field gel electrophoresis.</td><td>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. CK X-rays: (12.1 ± 1.9) x 10-9 DSBs per base pair per Gy.</td></tr><tr><td>Rothkamm, 2003</td><td>In vitro. Primary human fibroblast cell lines MRC-5 (lung), HSF1 and HSF2 (skin), and 180BR (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.</td><td>X-ray irradiation of primary human fibroblasts (MRC-5) in the range 1 mGy - 100 Gy, 35 DSBs per cell per Gy.</td></tr><tr><td>Grudzenski et al., 2010</td><td>In vitro. Primary human fibroblasts (HSF1) and C57BL/6NcrI 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.</td><td>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/6NcrI 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.</td></tr><tr><td>de Lara, 2001</td><td>In vitro. Chinese hamster cells (V79-4) were exposed to 0 – 20 Gy of 60Co 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.</td><td>V79-4 cells irradiated with γ-rays and ultrasoft X-rays (carbon K-shell, copper L-shell, aluminium K-shell and titanium 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.</td></tr><tr><td>Rübe et al., 2008</td><td>In vivo. Brain, lung, heart and small intestine tissue from adult SCID, A-T, BALB/c and C57BL/6NcrI mice; Whole blood and isolated lymphocytes from BALB/c and C57BL/6NcrI 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.</td><td>Linear dose-dependent increase in DSBs in the brain, small intestine, lung and heart of C57BL/6NcrI 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).</td></tr><tr><td>Antonelli et al., 2015</td><td>In vitro. Primary human foreskin fibroblasts (AG01522) were exposed to 0 – 1 Gy of 137Cs γ-rays (1 Gy/min), protons (0.84 MeV, 28.5 keV/μm), carbon ions (58 MeV/u, 39.4 keV/μm), and alpha particles (americium-241, 0.75 MeV/u, 0.08 Gy/min, 125.2 keV/μm). γ-H2AX foci were determined with immunochemistry to measure DSBs.</td><td>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.</td></tr><tr><td>Barnard et al., 2019</td><td>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.</td><td>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.</td></tr><tr><td>Ahmadi et al., 2021</td><td>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</td><td>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</td></tr></tbody></table>
<table><tr><td></td><td>and 0.3 Gy/min. DNA strand breaks were measured using the comet assay.</td><td>followed a similar pattern with a lower amount of strand breaks.</td></tr><tr><td>Hamada et al., 2006</td><td>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 γ-H2AX foci/cell, which represented DNA strand breaks, was determined 6 – 7 min after irradiation through fluorescence microscopy.</td><td>Cells displayed a linear increase in the number of γ-H2AX foci/cell, with the maximum dose displaying a 125x increase compared to control (32 foci/Gy).</td></tr><tr><td>Schmal et al., 2019</td><td>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.</td><td>Only low 53BP1-foci levels (~0.03 foci/cell) were observed in non-irradiated controls. However, 0.1 h post-irradiation, directly after single dose exposure to 0.1 Gy, Approx 1 focus/cell was induced. Following fractioned low dose ionizing radiation, (20 × 0.1 Gy, 72 h post-IR) the number of persisting foci was higher in hippocampal neurons compared to non-irradiated wild-type (WT) mice.</td></tr><tr><td>Barazzuol et al., 2017</td><td>In vivo. C57BL/6 mice were exposed to 0.1 or 2 Gy of X-rays (250 kV) at a dose rate of 0.5 Gy/min. 53BP1 foci were quantified with immunofluorescence in neural stem cells and neuron progenitors in the lateral ventricle.</td><td>Both 0.1 and 2 Gy resulted in increased 53BP1 foci with a 10-fold increase at 0.1 Gy and an 80-fold increase at 2 Gy.</td></tr><tr><td>Sabirzhanov et al., 2020</td><td>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.</td><td>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.</td></tr><tr><td>Geisel et al., 2012</td><td>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.</td><td>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.</td></tr><tr><td>Ungvari et al., 2013</td><td>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.</td><td>DNA damage increased at all doses (2-10 Gy). In the control, less than 5% of DNA damage was in the tail, while by 6 Gy, 35% of the DNA damage was in the tail in cerebromicrovascular endothelial cells and 25% was in the tail in neurons.</td></tr><tr><td>Rombouts et al., 2013</td><td>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.</td><td>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.</td></tr><tr><td>Baselet et al., 2017</td><td>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.</td><td>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.</td></tr></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Rogakou et al., 1999</td><td>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 min. DNA breaks were visualized using immunofluorescence.</td><td>DSBs were present at 3 min and persisted from 15 - 60 min.</td></tr><tr><td>Hamada, 2017b</td><td>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.</td><td>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.</td></tr><tr><td>Hamada et al., 2006</td><td>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.</td><td>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.</td></tr><tr><td>Acharya et al., 2010</td><td>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.</td><td>The number of cells positive for nuclear γ-H2AX foci peaked at 20 min post-irradiation. After 1h, this level quickly declined.</td></tr></tbody></table>
<table><tr><td>Schmal et al., 2019</td><td>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.</td><td>To assess possible accumulation of persisting 53BP1-foci during fractionated radiation, juvenile and adult mice were examined 72 h after exposure to 5x, 10x, 15x, or 20x 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 min post-IR).</td></tr><tr><td>Dong et al., 2015</td><td>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.</td><td>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.</td></tr><tr><td>Barazzuol et al., 2017</td><td>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.</td><td>At both 0.5 and 6 h post-irradiation, increased 53BP1 foci were observed, with the highest level at 0.5 h.</td></tr><tr><td>Sabirzhanov et al., 2020</td><td>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.</td><td>In rat cortical neurons, γ-H2AX, p-ATM and p-ATR all increased at 30 min post-irradiation, with a sustained increase until 6 h.</td></tr><tr><td>Zhang et al., 2017</td><td>In vitro. HT22 hippocampal neuronal cells 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.</td><td>At 8 Gy, the comet assay showed an increased tail moment at both 30 min and 24 h post-irradiation. At 12 Gy, p-ATM was increased over 4-fold at both 30 min and 1 h post-irradiation. γ-H2AX was increased over 3-fold at 30 min post-irradiation and almost 2-fold at 1 and 24 h.</td></tr><tr><td>Geisel et al., 2012</td><td>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.</td><td>DSBs were increased at 1 h post-irradiation and returned to pre-irradiation levels by 24 h.</td></tr><tr><td>Park et al., 2022</td><td>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.</td><td>γ-H2AX, p-ATM, and 53BP1 increased at 1 h post-irradiation and slightly decreased for the rest of the 6 h but remained elevated above the control.</td></tr><tr><td>Kim et al., 2014</td><td>In vitro. Human umbilical vein endothelial cells were irradiated with 4 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence.</td><td>γ-H2AX foci greatly increased at 1 and 6 h post-irradiation, with the greatest increase at 1 h.</td></tr><tr><td>Dong et al., 2014</td><td>In vitro. Human umbilical vein endothelial cells were irradiated with 2 Gy of 137Cs gamma rays. γ-H2AX levels were determined with immunofluorescence.</td><td>γ-H2AX foci increased 8-fold at 3 h, 7-fold at 6 h, and 2-fold at 12 and 24 h post-irradiation.</td></tr><tr><td>Rombouts et al., 2013</td><td>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.</td><td>The greatest increase in γ-H2AX foci was observed 30 min post-irradiation, while levels were still slightly elevated at 24 h.</td></tr><tr><td>Nübel et al., 2006</td><td>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.</td><td>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.</td></tr><tr><td>Baselet et al., 2017</td><td>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.</td><td>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.</td></tr><tr><td>Gionchiglia et al., 2021</td><td>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.</td><td>In the forebrain, cerebral cortex, hippocampus and subventricular zone (SVZ)/ rostral migratory stream (RMS)/ olfactory bulb (OB), γ-H2AX and p53BP1 positive cells increased at both 15 and 30 mins post-irradiation, with the greatest increase at 30 min.</td></tr></table>

## Response-response Relationship

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

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 & 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).
References

Agrawala, P.K. et al. (2008), "Induction and repairability of DNA damage caused by ultrasoft X-rays: Role of core events.", Int. J. Radiat. Biol., 84(12):1093-1103. doi:10.1080/09553000802478083.

Ahmadi, M. et al. (2021), "Early responses to low-dose ionizing radiation in cellular lens epithelial models", Radiation research, Vol.197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00284.1

Ainsbury, E. A. et al. (2016), "Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research", Mutation research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.07.010

Alexander, J. L. and Orr-Weaver, T. L. (2016), "Replication fork instability and the consequences of fork collisions from replication", Genes & Development, Vol. 30/20, Cold Spring Harbor Laboratory Press, https://doi.org/10.1101/gad.288142.116

Allen, C. H. et al. (2018), "Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation", Physics in medicine & biology, Vol. 63/2, IOP Publishing, Bristol, https://doi.org/10.1088/1361-6560/aaa176

Antonelli, A.F. et al. (2015), "Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death", Radiat. Res. 183(4):417-31, doi:10.1667/RR13855.1.

Acharya, M. et al. (2010), "Consequences of ionizing radiation-induced damage in human neural stem cells", Free Radical Biology and Medicine. 49(12):1846-1855, doi:10.1016/j.freeradbiomed.2010.08.021.

Asaithamby, A. et al. (2008), "Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts.", Radiat Res. 169(4):437-446. doi:10.1667/RR11651.1.

Aufderheide, E. et al. (1987), "Heavy ion effects on cellular DNA: Strand break induction and repair in cultured diploid lens epithelial cells", International journal of radiation biology and related studies in physics, chemistry and medicine, Vol. 51/5, Taylor & Francis, London, https://doi.org/10.1080/09553008714551071

Bannik, K. et al. (2013), "Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?", Radiation and environmental biophysics, Vol. 52/2, Springer-Verlag, Berlin/Heidelberg, https://doi.org/10.1007/s00411-012-0451-8

Bains, S. K. et al. (2019), "Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells", International journal of radiation biology, Vol. 95/1, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1466066

Barazzuol, L et al. (2017), "A coordinated DNA damage response promotes adult quiescent neural stem cell activation. PLOS Biology, 15(5). https://doi.org/10.1371/journal.pbio.2001264

Barnard, S. G. R. et al. (2018), "Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage", International journal of radiation biology, Vol. 94/12, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1532609
Barnard, S. G. R. et al. (2019), "Inverse dose-rate effect of ionising radiation on residual 53BP1 foci in the eye lens", Scientific Reports, Vol. 9/1, Nature Publishing Group, England, https://doi.org/10.1038/s41598-019-46893-3

Barnard, S. G. R. et al. (2022), "Radiation-induced DNA damage and repair in lens epithelial cells of both Ptch1 (+/-) and Ercc2 (+/-) mutated mice", Radiation Research, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00264.1

Baselet, B. et al. (2019), “Pathological effects of ionizing radiation: endothelial activation and dysfunction”, Cellular and molecular life sciences, Vol. 76/4, Springer Nature, https://doi.org/10.1007/s00018-018-2956-z

Baselet, B. et al. (2017), “Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose”, Frontiers in pharmacology, Vol. 8, Frontiers, https://doi.org/10.3389/fphar.2017.00213

Baumstark-Khan, C., J. Heilmann, and H. Rink (2003), ‘Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation”, Advances in space research, Vol. 31/6, Elsevier Ltd, England, https://doi.org/10.1016/S0273-1177(03)00095-4

Beels, L. et al. (2009), "g-H2AX Foci as a Biomarker for Patient X-Ray Exposure in Pediatric Cardiac Catheterization", Are We Underestimating Radiation Risks?":1903–1909. doi:10.1161/CIRCULATIONAHA.109.880385.

Belli M, Cherunbini R, Vecchia MD, Dini V, Moschini G, Signoretti C, Simon G, Tabocchini MA, Tiveron P. 2000. DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study. Int J Radiat Biol. 76(8):1095-1104.

Brooks, A.L., D.G. Hoel & R.J. Preston (2016), "The role of dose rate in radiation cancer risk: evaluating the effect of dose rate at the molecular, cellular and tissue levels using key events in critical pathways following exposure to low LET radiation.", Int. J. Radiat. Biol. 92(8):405-426. doi:10.1080/09553002.2016.1186301.

Bucolo, C. et al. (1994), “The effect of ganglioside on oxidation-induced permeability changes in lens and in epithelial cells of lens and retina”, Experimental eye research, Vol. 58/6, Elsevier Ltd, London, https://doi.org/10.1006/exer.1994.1067

Cabrera et al. (2011), “Antioxidants and the integrity of ocular tissues”, in Veterinary medicine international, SAGE-Hindawi Access to Research, United States. DOI: 10.4061/2011/905153

Cadet, J. et al. (2012), “Oxidatively generated complex DNA damage: tandem and clustered lesions”, Cancer letters, Vol. 327/1, Elsevier Ireland Ltd, Ireland. https://doi.org/10.1016/j.canlet.2012.04.005

Cannan, W.J. & D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", J. Cell Physiol. 231(1):3–14. doi:10.1002/jcp.25048.

Cencer, C. S. et al. (2018), “PARP-1/PAR activity in cultured human lens epithelial cells exposed to two levels of UVB light”, Photochemistry and photobiology, Vol. 94/1, Wiley, Hoboken, https://doi.org/10.1111/php.12814

Chadwick, K.H., (2017), Towards a new dose and dose-rate effectiveness factor (DDREF)? Some comments., J Radiol Prot., 37:422-433. doi: 10.1088/1361-6498/aa6722.
Charlton, D.E., H. Nikjoo & J.L. Humm (1989), "Calculation of initial yields of single- and double-strand breaks in cell nuclei from electrons, protons and alpha particles.", Int. J. Rad. Biol., 53(3):353-365, DOI: 10.1080/09553008814552501

Christensen, D.M. (2014), "Management of Ionizing Radiation Injuries and Illnesses, Part 3: Radiobiology and Health Effects of Ionizing Radiation.", 114(7):556-565. doi:10.7556/jaoa.2014.109.

Citrin, D.E. & J.B. Mitchel (2014), "Public Access NIH Public Access.", 71(2):233-236. doi:10.1038/mp.2011.182.doi.

Dalke, C. et al. (2018), "Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk", Radiation and environmental biophysics, Vol. 57/2, Springer Berlin Heidelberg, Berling/Heidelberg, https://doi.org/10.1007/s00411-017-0728-z

Day, T.K. et al. (2007), "Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose.", Radiat Res. 167(6):682-692. doi:10.1667/rr0764.1.

De Angelis, P. M. et al. (2006), "Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery", Molecular Cancer, Vol. 5/20, BioMed Central, https://doi.org/10.1186/1476-4598-5-20

DeGraff, W. G. et al. (1992), "Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage", Free Radical Biology and Medicine, Vol. 13/5, Elsevier, https://doi.org/10.1016/0891-5849(92)90142-4

Desouky, O., N. Ding & G. Zhou (2015), "ScienceDirect Targeted and non-targeted effects of ionizing radiation.", J. Radiat. Res. Appl. Sci. 8(2):247-254. doi:10.1016/j.jrras.2015.03.003.

Dong et al. (2015), "Relationship between irradiation-induced neuro-inflammatory environments and impaired cognitive function in the developing brain of Mice. International Journal of Radiation Biology, 91(3):224-239. https://doi.org/10.3109/09553002.2014.988895

Dong, X. et al. (2014), "NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-κB signal pathway", Radiation Research, Vol. 183/1, BioOne, https://doi.org/10.1667/RR13682.1

Dubrova, Y.E. & M.A. Plumb (2002), "Ionising radiation and mutation induction at mouse minisatellite loci The story of the two generations", Mutat. Res. 499(2):143-150.

EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California

Falk, M., E. Lukášová & S. Kozubek (2008), "Chromatin structure influences the sensitivity of DNA to γ-radiation.", Biochim. Biophys. Acta. - Mol. Cell. Res. 1783(12):2398-2414. doi:10.1016/j.bbamcr.2008.07.010.

Feinendegen, L.E. (2005), "UKRC 2004 debate Evidence for beneficial low level radiation effects and radiation hormesis. Radiology.", 78:3-7. doi:10.1259/bjr/63353075.

Feinendegen, L.E., M. Pollycove & R.D. Neumann (2007), "Whole-body responses to low-level radiation exposure: New concepts in mammalian radiobiology.", Exp. Hematol. 35(4 SUPPL.):37-46. doi:10.1016/j.exphem.2007.01.011.
Flegal, M. et al. (2015), "Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and γ-Radiation.", (July):1–9. doi:10.3791/52912.

Foray, N., M. Bourguignon and N. Hamada (2016), “Individual response to ionizing radiation”, Mutation research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.09.001

Franken NAP, Hovingh S, Cate RT, Krawczyk P, Stap J, Hoebe R, Aten J, Barendsen GW. 2012. Relative biological effectiveness of high linear energy transfer alpha-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells. Oncol reports. 27:769-774.

Frankenberg D, Brede HJ, Schrewe UJ, Steinmetz C, Frankenberg-Scwager M, Kasten G, Pralle E. 1999. Induction of DNA Double-Strand Breaks by 1H and 4He Ions in Primary Human Skin Fibroblasts in the LET range of 8 to 124 keV/µm. Radiat Res. 151:540-549.

Geisel, D. et al. (2012), “DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study”, European Radiology, Vol. 22/8, Springer Nature, https://doi.org/10.1007/s00330-012-2426-1

Gionchiglia, N. et al. (2021), “Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice,” Biomedicines. 9(9):1166. doi: 10.3390/biomedicines9091166.

Goodhead, D.T. (2006), "Energy deposition stochastics and track structure: What about the target?", Radiat. Prot. Dosimetry. 122(1-4):3-15. doi:10.1093/rpd/ncl498.

Grudzenski, S. et al. (2010), "Inducible response required for repair of low-dose radiation damage in human fibroblasts.", Proc. Natl. Acad. Sci. USA. 107(32): 14205-14210, doi:10.1073/pnas.1002213107.

Hada, M. & A.G. Georgakilas (2008), "Formation of Clustered DNA Damage after High-LET Irradiation: A Review.", J. Radiat. Res., 49(3):203-210. doi:10.1269/jrr.07123.

Hamada, N. et al. (2006), “Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair”, Radiation Research, Vol. 166/1, Radiation Research Society, United States, https://doi.org/10.1667/RR3577.1

Hamada, N. (2014), “What are the intracellular targets and intratissue target cells for radiation effects?”, Radiation research, Vol. 181/1, The Radiation Research Society, Lawrence, https://doi.org/10.1667/RR13505.1

Hamada, N. and T. Sato (2016), “Cataractogenesis following high-LET radiation exposure”, Mutation Research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.005

Hamada, N. (2017a), “Ionizing radiation sensitivity of the ocular lens and its dose rate dependence”, International journal of radiation biology, Vol. 93/10, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2016.1266407

Hamada, N. (2017b), “Ionizing radiation response of primary normal human lens epithelial cells”, PloS ONE, Vol. 12/7, Public Library Science, San Francisco, https://doi.org/10.1371/journal.pone.0181530
Havas, M. (2017), "When theory and observation collide: Can non-ionizing radiation cause cancer?", Environmental pollution, Vol. 221, Elsevier Ltd, England.
https://doi.org/10.1016/j.envpol.2016.10.018

Hightower, K. R. (1995), "The role of the lens epithelium in development of UV cataract", Current eye research, Vol. 14/1, Informal UK Ltd, Philadelphia, https://doi.org/10.3109/02713689508999916

Iliakis, G., T. Murmann & A. Soni (2015), "Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.", Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 793:166-175. doi:10.1016/j.mrgentox.2015.07.001.

Joiner, M. (2009), "Basic Clinical Radiobiology", Edited by. [1] P.J. Sadler, Next-Generation Met Anticancer Complexes Multitargeting via Redox Modul Inorg Chem 52 21.:375. doi:10.1201/b13224.

Jorge, S.-G. et al. (2012), "Evidence of DNA double strand breaks formation in Escherichia coli bacteria exposed to alpha particles of different LET assessed by the SOS response.", Appl. Radiat. Isot. 71(SUPPL.):66-70. doi:10.1016/j.apradiso.2012.05.007.

Kadhim, M.A., M.A. Hill & S.R. Moore, (2006), "Genomic instability and the role of radiation quality.", Radiat. Prot. Dosimetry. 122(1-4):221-227. doi:10.1093/rpd/ncl445.

Khanna, K.K. & S.P. Jackson (2001), "DNA double-strand breaks: signaling, repair and the cancer connection.", Nature Genetics. 27(3):247-54. doi:10.1038/85798.

Kim, K. S. et al. (2014), "Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells", International Journal of Radiation Biology, Vol. 90/1, Informa, London, https://doi.org/10.3109/09553002.2014.859763

Kim, J. N. and B. M. Lee (2007), "Risk factors, health risks, and risk management for aircraft personnel and frequent flyers", Journal of toxicology and environmental health. Part B, Critical reviews, Vol. 10/3, Taylor & Francis Group, Philadelphia, https://doi.org/10.1080/10937400600882103

Kleiman, N. J., R. Wang and A. Spector (1990), "Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells", Current eye research, Vol. 9/12, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689009003475

Kuefner, M.A. et al. (2009), "DNA Double-Strand Breaks and Their Repair in Blood Lymphocytes of Patients Undergoing Angiographic Procedures.", Investigative radiology. 44(8):440-6. doi:10.1097/RLI.0b013e3181a654a5.

Kuefner, M.A. et al. (2015), "Chemoprevention of Radiation-Induced DNA Double-Strand Breaks with Antioxidants.", Curr Radiol Rep (2015) 3: 81. https://doi.org/10.1007/s40134-014-0081-9

Kuhne, M., G. Urban & M. Lo, (2005), "DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and Co γ-Rays.", Radiation Research. 164(5):669-676. doi:10.1667/RR3461.1.

de Lara, C.M. et al. (2001), "Dependence of the Yield of DNA Double-Strand Breaks in Chinese Hamster V79-4 Cells on the Photon Energy of Ultrasoft X Rays.", Radiation Research. 155(3):440-8. doi:10.1667/0033-7587(2001)155[0440:DOTYOD]2.0.CO;2.
Liao, J. et al. (2011), "Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: An implications for anti-cataract agent", International journal of molecular sciences, Vol. 12/10, MDPI, Basel. https://doi.org/10.3390/ijms12107059

Lipman, R. M., B. J. Tripathi, R. C. Tripathi (1998), "Cataracts induced by microwave and ionizing radiation", Survey of ophthalmology, Vol. 33/3, Elsevier Inc, United States, https://doi.org/10.1016/0039-6257(88)90088-4

Lomax, M.E., L.K. Folkes & P.O. Neill (2013), "Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy", Statement of Search Strategies Used and Sources of Information Why Radiation Damage is More Effective than Endogenous Damage at Killing Cells Ionising Radiation-induced Do. 25:578-585. doi:10.1016/j.clon.2013.06.007.

Long, A. C., C. M. H. Colitz, and J. A. Bomser (2001), "Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death", Experimental biology and medicine, SAGE Publications, London, https://doi.org/10.1177/153537020422901012

Lorat, Y. et al. (2015), "Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair.", DNA Repair (Amst). 28:93-106. doi:10.1016/j.dnarep.2015.01.007.

Maier, P. et al. (2016), "Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.", Int. J. Mol. Sci., 14;17(1), pii:E102, doi:10.3390/ijms17010102.

Markiewicz, E. et al. (2015), "Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape", Open biology, Vol. 5/4, Royal society, London, https://doi.org/10.1098/rsob.150011

Milligan, J. R. et al. (1995), « DNA repair by thiols in air shows two radicals make a double-strand break », Radiation Research, Vol 143/3, pp. 273-280

Moore, S., F.K.T. Stanley & A.A. Goodarzi (2014), "The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task.", DNA repair (Amst), 17:64-73. doi: 10.1016/j.dnarep.2014.01.014.

Mosconi, M., U. Giesen & F. Langner (2011), "53BP1 and MDC1 foci formation in HT-1080 cells for low- and high-LET microbeam irradiations.", Radiat. Envrion. Biophys. 50(3):345-352. doi:10.1007/s00411-011-0366-9.

Nagane, M. et al. (2021), "DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular disease", Journal of Radiation Research, Vol. 62/4, https://doi.org/10.1093/jrr/rrab032

Nenoi, M., B. Wang & G. Vares (2015), "In vivo radioadaptive response: A review of studies relevant to radiation-induced cancer risk.", Hum. Exp. Toxicol. 34(3):272-283. doi:10.1177/0960327114537537.

Nikitaki, Z. 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 Radiac. Res. 50(sup1):S64-S78, doi:10.1080/10715762.2016.1232484.

Nikjoo, H. et al. (2001), "Computational approach for determining the spectrum of DNA damage induced by ionizing radiation.", Radiat. Res. 156(5 Pt 2):577-83.
Nübel, T. et al. (2006), "Lovastatin protects human endothelial cells from killing by ionizing radiation without impairing induction and repair of DNA double-strand breaks", Clinical Cancer Research, Vol. 12/3, American Association for Cancer Research, https://doi.org/10.1158/1078-0432.CCR-05-1903

Ojima, M., N. Ban, and M. Kai (2008), "DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects", Radiation Research, Vol. 170/3, Radiation Research Society, United States, https://doi.org/10.1667/RR1255.1

Okayasu, R. (2012a), "Repair of DNA damage induced by accelerated heavy ions-A mini review.", Int. J. Cancer. 130(5):991-1000. doi:10.1002/ijc.26445.

Okayasu, R. (2012b), "Heavy ions — a mini review.", 1000:991–1000. doi:10.1002/ijc.26445.

Park, J. W. et al. (2022), "Metformin alleviates ionizing radiation-induced senescence by restoring BARD1-mediated DNA repair in human aortic endothelial cells", Experimental Gerontology, Vol. 160, Elsevier, Amsterdam, https://doi.org/10.1016/j.exger.2022.111706

Parris, C.N. et al. (2015), "Enhanced γ-H2AX DNA damage foci detection using multimagnification and extended depth of field in imaging flow cytometry.", Cytom. Part A. 87(8):717-723. doi:10.1002/cyto.a.22697.

Radulescu I., K. Elmroth & B. Stenerlöw (2006), "Chromatin Organization Contributes to Non-randomly Distributed Double-Strand Breaks after Exposure to High-LET Radiation.", Radiat. Res. 161(1):1-8. doi:10.1667/rr3094.

Rastogi, R. P. et al. (2010), "Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair", Journal of nucleic acids, Hindawi Ltd, United States. https://doi.org/10.4061/2010/592980

Reddy, V. N. et al. (1998), "The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium", Investigative ophthalmology & visual science, Vol. 39/2, Arvo, pp. 344-350

Rogakou, E.P. et al. (1999), "Megabase Chromatin Domains Involved in DNA Double-Strand Breaks In Vivo.", J. Cell Biol, 146(5):905-16. doi: 10.1083/jcb.146.5.905.

Rogers, C. S. et al. (2004), "The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells", Experimental eye research, Vol. 78/5, Elsevier Ltd, London, https://doi.org/10.1016/j.exer.2003.12.011

Rombouts, C. et al. (2013), "Differential response to acute low dose radiation in primary and immortalized endothelial cells", International Journal of Radiation Biology, Vol. 89/10, Informa, London, https://doi.org/10.3109/09553002.2013.806831

Rothkamm, K. et al. (2015), "Review DNA Damage Foci: Meaning and Significance.", Environ. Mol. Mutagen., 56(6):491-504, doi: 10.1002/em.21944.

Rothkamm, K. & M. Lo (2003), "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.", PNAS, 100(9):5057-62. doi:10.1073/pnas.0830918100.

Rübe, C.E. et al. (2008), "Cancer Therapy: Preclinical DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing.", Clin. Cancer Res., 14(20):6546-6556. doi:10.1158/1078-0432.CCR-07-5147.
Russo, A. et al. (2015), "Review Article Genomic Instability: Crossing Pathways at the Origin of Structural and Numerical Chromosome Changes.", Envrion. Mol. Mutagen. 56(7):563-580. doi:10.1002/em.

Rydberg B, Heilbronn L, Holley WR, Lobrich M, Zeitlin C et al. 2002. Spatial Distribution and Yield of DNA Double-Strand Breaks Induced by 3-7 MeV Helium Ions in Human Fibroblasts. Radiat Res. 158(1):32-42.

Sabirzhanov, et al. (2020), "Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.", Int J Mol Sci. 21(15):5239. doi: 10.3390/ijms21155239.

Sage, E. & N. Shikazono (2017), "Free Radical Biology and Medicine Radiation-induced clustered DNA lesions: Repair and mutagenesis.", Free Radic. Biol. Med. 107(December 2016):125-135. doi:10.1016/j.freeradbiomed.2016.12.008.

Sasaki, H. et al. (1998), "TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit", Investigative ophthalmology & visual sciences, Vol. 39/3, Arvo, Rockville, pp. 544-552

Schmal, Z. et al. (2019), “DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis”, Radiotherapy and Oncology. 137:45-54.
doi:10.1016/j.radonc.2019.04.021.

Sears, C. R. and J. J. Turchi (2012), “Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways”, Journal of biological chemistry, Vol 287/29, The American Society for Biochemistry and Molecular Biology, Inc, USA, https://doi.org/10.1074/jbc.M112.344911

Shah, D.J., R.K. Sachs & D.J. Wilson (2012), "Radiation-induced cancer: A modern view." Br. J. Radiol. 85(1020):1166-1173. doi:10.1259/bjr/25026140.

Shelke, S. & B. Das (2015), "Dose response and adaptive response of non-homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to γ radiation.", (December 2014):365-379. doi:10.1093/mutage/geu081.

Sidjanin, D., S. Zigman and J. Reddan (1993), “DNA damage and repair in rabbitlens epithelial cells following UVA radiation”, Current eye research, Vol. 12/9, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689309020382

Smith, J. et al. (2003), "Impact of DNA ligase IV on the delity of end joining in human cells.", Nucleic Acids Research. 31(8):2157-2167. doi:10.1093/nar/gkg317.

Smith, T.A. et al. (2017), "Radioprotective agents to prevent cellular damage due to ionizing radiation." Journal of Translational Medicine.15(1).doi:10.1186/s12967-017-1338-x.

Stewart, F. A. et al. (2012), “ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs – threshold doses for tissue reactions in a radiation protection context”, Annals of the ICRP, Vol, 41/1-2, Elsevier Ltd, London, https://doi.org/10.1016/j.icrp.2012.02.001

Sudprasert, W., P. Navasumrit & M. Ruchirawat (2006), "Effects of low-dose γ radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells.", Int. J. Hyg. Envrion. Health, 209:503-511. doi:10.1016/j.ijheh.2006.06.004.