# AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment

Ahmad Sleiman, Kathleen Miller, Danicia Flores, Jaqueline Kuan, Kaitlyn

Altwasser, Benjamin J. Smith, Tatiana Kozbenko, Robyn Hocking, Scott

Wood, Janice Huff, et al.

► To cite this version:

Ahmad Sleiman, Kathleen Miller, Danicia Flores, Jaqueline Kuan, Kaitlyn Altwasser, et al. AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment. Environmental and Molecular Mutagenesis, 2024, 65 (S3), pp.57-84. 10.1002/em.22622. hal-04886703

HAL Id: hal-04886703

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Submitted on 14 Jan 2025

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# AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment

Ahmad Sleiman<fnref n="1" /> | Kathleen B. Miller<fnref n="2" /> | Danicia Flores<fnref n="3" /> | Jaqueline Kuan<fnref n="3" /> |
Kaitlyn Altwasser<fnref n="3" /> | Benjamin J. Smith<fnref n="3" /> | Tatiana Kozbenko<fnref n="3" /> | Robyn Hocking<fnref n="3" /> |
Scott J. Wood<fnref n="4" /> | Janice Huff<fnref n="5" /> | Christelle Adam-Guillermin<fnref n="1" /> |
Nobuyuki Hamada<fnref n="6" /> | Carole Yauk<fnref n="7" /> | Ruth Wilkins<fnref n="3" /> | Vinita Chauhan<fnref n="3" />

## Correspondence

Vinita Chauhan, Consumer and Clinical
Radiation Protection Bureau, Environmental
and Radiation Health Sciences Directorate,
Health Canada, 775 Brookfield Road, Ottawa,
Ontario, Canada.

Email: vinita.chauhan@hc-sc.gc.ca

## Funding information

Solutions Fund; Genomics Research and
Development Initiative; Canadian Space
Agency; NASA Langley Research Center
Cooperative Agreement, Grant/Award
Number: 80LARC17C0004

Accepted by: J. OBrien

## Abstract

Understanding radiation-induced non-cancer effects on the central nervous system (CNS) is essential for the risk assessment of medical (e.g., radiotherapy) and occupational (e.g., nuclear workers and astronauts) exposures. Herein, the adverse outcome pathway (AOP) approach was used to consolidate relevant studies in the area of cognitive decline for identification of research gaps, countermeasure development, and for eventual use in risk assessments. AOPs are an analytical construct describing critical events to an adverse outcome (AO) in a simplified form beginning with a molecular initiating event (MIE). An AOP was constructed utilizing mechanistic information to build empirical support for the key event relationships (KERs) between the MIE of deposition of energy to the AO of learning and memory impairment through multiple key events (KEs). The evidence for the AOP was acquired through a documented scoping review of the literature. In this AOP, the MIE is connected to the AO via six KEs: increased oxidative stress, increased deoxyribonucleic acid (DNA) strand breaks, altered stress response signaling, tissue resident cell activation, increased pro-inflammatory mediators, and abnormal neural remodeling that encompasses atypical structural and functional alterations of neural cells and surrounding environment. Deposition of energy directly leads to oxidative stress, increased DNA strand breaks, an increase of pro-inflammatory mediators and tissue resident cell activation. These KEs, which are themselves interconnected, can lead to abnormal neural remodeling impacting learning and memory processes. Identified knowledge gaps include improving quantitative understanding of the AOP across several KERs and additional testing of proposed modulating factors through experimental work. Broadly, it is envisioned that the outcome of these efforts could be extended to other cognitive disorders and complement ongoing work by international radiation governing bodies in their review of the system of radiological protection.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.

© 2024 The Author(s). *Environmental and Molecular Mutagenesis* published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.

<footnote n="1">¹Institut de Radioprotection et de Süreté
Nucléaire, St. Paul Lez Durance, Provence,
France</footnote>

<footnote n="2">²Department of Health and Exercise Science,
Morrison College Family of Health, University
of St. Thomas, Saint Paul, Minnesota, USA</footnote>

<footnote n="3">³Consumer and Clinical Radiation Protection
Bureau, Environmental and Radiation Health
Sciences Directorate, Health Canada, Ottawa,
Ontario, Canada</footnote>

<footnote n="4">⁴NASA Johnson Space Center, Houston,
Texas, USA</footnote>

<footnote n="5">⁵NASA Langley Research Center, Hampton,
Virginia, USA</footnote>

<footnote n="6">⁶Biology and Environmental Chemistry
Division, Sustainable System Research
Laboratory, Central Research Institute of
Electric Power Industry (CRIEPI), Chiba, Japan</footnote>

<footnote n="7">⁷Department of Biology, University of Ottawa,
Ottawa, Ontario, Canada</footnote>
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### KEYWORDS

AOP, cognitive deficits, radiation, risk assessment, space exploration

# 1 | INTRODUCTION

The mechanistic underpinnings of cognitive injury and other adverse health effects following exposure to ionizing radiation (IR) on the central nervous system (CNS) are unclear. IR exposure may occur from natural sources, such as cosmic rays, or may be human made, with sources ranging from nuclear power generation to medical devices. Today, the most common human-made sources of IR are diagnostic or therapeutic medical exposures (ICRP, 2007; ICRP, 2012, p. 118).

Given that there are many contexts in which humans are exposed to IR, there is a need to better understand the impact of IR on the CNS. The space environment presents particular concerns due to the multitude of stressors that could be encountered. The three main sources of IR in space include solar particle radiation, trapped particles in the Earth's magnetosphere, and galactic cosmic rays. Of note, galactic cosmic rays include heavy ions, high-energy protons and secondary particles that have a high probability of inducing biological damage (Cucinotta et al., 2014; Simonsen et al., 2020). Although an increased incidence of cataracts is the only effect for which elevated radiation risk has been reported in astronauts (Hamada & Sato, 2016), other potential effects of space radiation exposure on humans include cancer and various non-cancer effects (cardiovascular diseases, neurological disorders, immune dysfunction, and bone loss). The present study scopes the impacts of space radiation on cognitive function, specifically learning and memory, as optimal cognitive function is essential for crewmember safety, mission success, and post-mission quality of life (Strangman et al., 2014).

The general approach to define health risks associated with space radiation exposure arises from extrapolation of experimental results from preclinical models, computational models, and terrestrial exposures within human cohorts (Cucinotta et al., 2013; Huff et al., 2022; Simonsen & Slaba, 2021). However, data is currently lacking to utilize this approach to estimate risks to the CNS (Miller et al., 2022; Nelson et al., 2016). The existing animal and cellular data suggest significant adverse effects of space-relevant charged particles at low (0.05 Gy) and high (4 Gy) doses on cognitive function, which may be relevant for a Mars mission or other long duration deep space missions (Cekanaviciute et al., 2018). Development of comprehensive frameworks that describe mechanistic pathways to impairment in cognitive function is an important goal for space radiation risk assessment and other exposures from a medical, occupational and environmental setting.

To identify knowledge gaps, an adverse outcome pathway (AOP) that describes the causal linkages and impact of IR on the CNS has been developed. An AOP is a conceptual construction that takes into account the existing knowledge of biological interactions between a molecular initiating event (MIE), and the resulting cascade of key events (KEs) up to the final adverse outcome (AO). The AOP

framework was adopted in 2012 by the Organisation for Economic Co-operation and Development (OECD) to improve efficiency for chemical safety assessment (https://aopwiki.org/) and is now being applied to radiation stressors. Herein, an AOP from deposition of energy to an AO of learning and memory impairment prioritizing space-relevant radiation stressors is described.

# 2 | BACKGROUND

Learning and memory are essential cognitive functions that are interconnected through common neural networks, synaptic plasticity, neuromodulators and the interactions between brain regions (Toricelli et al., 2021). Although mechanisms leading to cognitive impairment are incompletely understood, there are some critical events that are well understood and accepted. Impaired learning can be seen as diminished ability to acquire information and create new associative or non-associative relationships, whereas impaired memory consists of reduced ability to retrieve sensory, short-term, or long-term learned information (Desai et al., 2022; Kiffer et al., 2019b). Although they are two independent cognitive outcomes, the processes of learning and memory are intimately connected through shared mechanisms (Toricelli et al., 2021). There are multiple brain areas involved in learning and memory including: the hippocampal region, imperative for declarative or episodic memory and the process of long term potentiation; the amygdala, which can process emotional components to memory; the parietal lobe, which is involved in spatial memory; the prefrontal cortex, involved in regulating emotional behaviors, thoughts and actions; and the basal ganglia, which may be important for stimulus response associations. These areas do not act independently as multiple brain areas may be involved at any given time depending on the task or stimulus (Berryhill & Olson, 2008; Cucinotta et al., 2014; Desai et al., 2022; NCRP Commentary, 2016; NCRP Report, 2019; Phelps, 2004).

Deposition of energy from IR could lead to reduced cognitive function. The process begins at the macromolecular level through the generation of oxidative stress, deoxyribonucleic acid (DNA) strand breaks, alteration of signaling pathways and promotion of neuroinflammatory environments. The altered cellular environment could impact the functions of both neurons and glial cells. The alterations in neurons and glial cells include persistent DNA strand breaks and pro-inflammatory response, reduced neurogenesis and inhibition of neuronal connectivity and synaptic activity (Alt & Schwer, 2018; Cekanaviciute et al., 2018; Hladik & Tapio, 2016). Without the ability of neurons to communicate with each other effectively and the ability of glial cells to regulate the immune responses, normal CNS functions such as learning and memory may be impaired.
<figure><img src="image_3.png" /></figure>

It has been shown that patients undergoing high dose, fraction-
ated cranial radiotherapy (>15 Gy) are at a higher risk for developing
long-term impairment in learning and memory (Lehrer et al., 2022;
Pazzaglia et al., 2020). Long-term impairment in learning and memory
after radiation exposure have been reported in adult and pediatric
populations (Chen et al., 2015; De Ruiter et al., 2013; Mulhern
et al., 2004). However, the large number of assessment tools and
scales available to evaluate neurocognitive disorders make it difficult
to consolidate findings and estimate specific radiation related risks,
especially at low-to-moderate dose ranges (Pasqual et al., 2021). Nev-
ertheless, there have been multiple studies regarding the impact of IR
at low-to-moderate doses on humans that had accidental, occupa-
tional or wartime radiation exposures. The Life Span Study of the
Japanese atomic bomb survivors reported no increased radiation risk
for mortality from dementia (Yamada et al., 2004, 2009, 2016),
although another cohort study of French nuclear workers reported an
excess radiation risk of Alzheimer's disease (Laurent et al., 2022). In
addition, a recent systematic review and meta-analysis of cohorts
including nuclear workers and uranium miners, nuclear weapon tests
participants, and medical workers found no increase in standardized
mortality ratio from diseases of the nervous system compared with
the general population (Lopes et al., 2022). However, the meta-
analysis found an increased radiation risk for Parkinson's disease
(Lopes et al., 2022), which was first reported in a cohort of
22,377 Mayak workers exposed to chronic IR, with the maximum
gamma rays absorbed in the brain being 8.01 Gy in men and 6.14 Gy
in women (95th percentile 1.55 Gy for both and mean cumulated
dose of 0.46 ± 0.67 Gy in men and 0.36 ± 0.56 Gy in women)
(Azizova et al., 2020). Moreover, multiple cohorts of clean-up workers
involved in the Chernobyl catastrophe demonstrate cognitive dys-
function (Bazyka et al., 2015; Loganovsky et al., 2015, 2018, 2020).
Overall, impairment in learning and memory have mostly been
reported from radiation exposures on Earth at high doses. At low-
to-moderate doses, the impact on learning and memory impairment is
less clear, though multiple cohorts report cognitive complaints after
radiation exposure and meta-analysis results suggest an increased
radiation risk for dementia and Parkinson's disease (Azizova
et al., 2020; Lopes et al., 2022; Srivastava et al., 2023). Importantly,
existing studies on human cohorts have not evaluated exposure to
heavy ions, such as what would be present in the space environment,
although multiple studies in animal models report impairment in learn-
ing and memory functions after heavy ion exposure even at low-
to-moderate doses (Cekanaviciute et al., 2018; Kiffer et al., 2019b).
Therefore, it is critical to understand the underlying mechanisms that
connect IR exposure to impairment in learning and memory, especially
in the context of the spaceflight environment, which includes
heavy ions.

Although the translation of data from animal experiments to
humans is challenging, experimental studies in rodents can help detect
cognitive effects that may occur after IR exposure (doses ≤1 Gy). It
has been suggested that low-dose IR exposure could increase the inci-
dence of certain neurodegenerative diseases, such as Parkinson's dis-
ease (Azizova et al., 2020) and Alzheimer's disease (Laurent

et al., 2022) in human populations. Changes in gene expression related to DNA damage response, ion channel regulation, long-term potentiation/depression, and vascular damage have been observed in the brain tissues of irradiated mice (Lowe et al., 2009). Moreover, DNA damage has been induced at 0.1 Gy (X-rays, 250 kV) with 10-fold increase of p53 binding protein 1 (53BP1) foci, representing sites of DNA double strand break (DSB) in neurons of mice (Barazzuol et al., 2017). Also, doses below 0.5 Gy led to the activation of nuclear factor kappa B (NF-κB) target genes in the brain and increased superoxide dismutase 2 (SOD2) expression (Veeraraghavan et al., 2011). It was also reported that the damage induced by 0.05 Gy of ¹⁶O-particle radiation resulted in neuronal defects and increased the expression of pre- and post-synaptic markers significantly, which may cause impairment in cognition and learning (Howe et al., 2019). In fact, multiple studies have demonstrated structural and functional deficits in CNS tissues, and behavioral deficits from low-to-moderate doses of heavy ion IR in rodent models (Cekanaviciute et al., 2018; Kiffer et al., 2019b). These experimental data suggest that low-to-moderate doses of IR, similar to high dose irradiation, could impact neurocognitive functions in rodents under certain conditions. Although disparate responses to low-to-moderate and high doses of IR have been reported, highlighting the need to investigate further the potential effects of IR exposure, particularly in humans (Betlazar et al., 2016). Together, the mechanistic and epidemiological studies provide relevant information to construct an AOP with the MIE of deposition of energy and an AO of impairment in learning and memory.

3 | ADVERSE OUTCOME PATHWAY (AOP #483)—BRIEF SUMMARY

The AOP described (AOP #483 in Wiki; URL: https://aopwiki.org/aops/483) was developed as part of a broader network to three other AOs of vascular remodeling, bone loss, and cataracts (Figure 1). The KEs that constitute the AOP and available methods for measuring each KE are summarized in Table 1. The main endpoints that were commonly used to measure each KE are summarized in Figure 2. Detailed descriptions of each KE and KER are available online in the AOP-Wiki (AOP #483 in Wiki; URL: https://aopwiki.org/aops/483). The AOP captures the KEs that occur after the deposition of energy, the MIE, leading to learning and memory impairment, the AO, which is a neurotoxicity marker belonging to the OECD regulatory toolbox. The linkages between KEs are described in Tables 2 and 3. The quantity of evidence supporting each key event relationship (KER) is summarized in Figure 3. The empirical evidence is drawn from studies using various stressors (Figure 4), but primarily IR. However, it is important to note that not all types of stressors support each KER and the AOP is not stressor or exposure parameter specific; for details on individual KERs refer to the AOP-Wiki. Most data are reported from adult and adolescent rodents (Figure 5). The findings subdivided by evidence stream are summarized in Figure 6. This AOP is not designed to be a complete representation of the complex biological processes leading to learning and memory impairment, but rather provides a
<figure><img src="image_4.png" /><figcaption>**FIGURE 1** Flow chart of AOP #483 from deposition of energy to impaired learning and memory. The molecular initiating event (MIE) is the first interaction between a stressor and a biomolecule within an organism. Subsequent key events (KEs) leading to the adverse outcome (AO) were identified. Both adjacent key event relationships (KERs) (solid lines) and non-adjacent KERs (dashed lines) are included in this pathway. Adjacent KERs demonstrate the causal relationship between two neighboring KEs. Non-adjacent KERs could be used to support the weight of evidence (WOE) of the whole AOP by bypassing KEs with less evidence.</figcaption></figure>

structured and simplified way of organizing essential and measurable KEs through process of expert consultation in a manner that enhances its utility for decision support and risk assessment. Note that the scope of some KEs (altered stress response signaling pathways and abnormal neural remodeling) is broad as multiple measurable endpoints were used to support the empirical relationship, in order to represent better the current state of knowledge and meet stringent Bradford Hill criteria.

The six proposed KEs are associated with several AOPs and have been extensively discussed within the AOP-Wiki. Briefly, radiation causes a deposition of energy [MIE, KE #1686 in the AOP-Wiki] that interacts directly with the DNA double helix and produces damage in the form of strand breaks [KE #1635 in the AOP-Wiki]. It could also interact with water molecules and generate reactive oxygen species (ROS), as well as reduce antioxidant enzymes, elevating oxidative stress [KE #1392 in the AOP-Wiki]. In addition, deposition of energy could cause tissue resident cell activation, particularly in microglial cells and astrocytes [KE #1492 in the AOP-Wiki] and could increase pro-inflammatory mediators (interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon gamma (IFN-γ)) [KE #1493 in the AOP-Wiki], in the CNS. Oxidative stress represents a cornerstone at the molecular level in this AOP. In addition to its ability to activate the tissue resident cell and to induce DNA strand breaks in neurons, oxidative stress may cause alterations in signaling pathways related to synaptic function and cell senescence [KE #2244 in the AOP-Wiki]. The signaling of these pathways are also altered by DNA strand breaks through the DNA damage response. Altogether, alterations in stress response signaling pathways,

increased pro-inflammatory mediators and DNA strand breaks may lead to abnormal neural remodeling [KE #2098 in the AOP-Wiki]. Deposition of energy also has a non-adjacent connection to abnormal neural remodeling. Abnormal neural remodeling includes changes in the morphological properties of neural cells as well as altered functional properties such as impaired neurogenesis and neurodegeneration occurring in the hippocampus. Although neural remodeling is typically a beneficial and ongoing process that enables the brain to adapt, certain stressors such as the space environment may lead to maladaptation's, potentially resulting in cognitive deficits despite the brain's continued efforts to adjust. Abnormal neural remodeling has an adjacent connection to impaired learning and memory [KE #341 in the AOP-Wiki] whereas both deposition of energy and increased pro-inflammatory mediators have non-adjacent connections to impaired learning and memory.

## 4 | AOP DEVELOPMENT STRATEGY

The AOP was developed in accordance with guidelines proposed in the OECD AOP Users' Handbook (OECD, 2018). Alongside expert consultation, a scoping review (SR) was performed in the year 2021. A protocol was developed a priori that defined the criteria used to include and exclude sources of evidence and to identify relevant data based on a population, exposure, outcome and endpoint (PEO/E) statement. The protocol also included how the data would be extracted and presented (Kozbenko et al., 2022). Sciome Workbench for Interactive computer-Facilitated Text-mining (SWIFT)-Review [https://www.sciome.com/swift-review/) was used to pre-filter
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<table><caption>**TABLE 1** Summary of KEs in the learning and memory AOP and methods of measurements. Frequently used measurement methods are described. For details of all the measurement methods identified for each KE refer to the AOP-Wiki (www.aopwiki.org) and Figure 2.</caption><thead><tr><th>Type</th><th>Event title</th><th>Description</th><th>How to measure</th></tr></thead><tbody><tr><td>Molecular initiating event (MIE)</td><td>Deposition of energy Existing KE in the AOP-Wiki, KE #1686</td><td>This KE includes:<ul><li>Subatomic particles or electromagnetic waves (ionizing radiation) deposit sufficient energy to directly ionize the target</li><li>Direct ionization of charged particles with DNA</li><li>Indirect ionization, production of free radicals, which could lead to DNA damage</li></ul></td><td>Ionizing radiation<ul><li>Monte Carlo simulations (e.g., Geant4) (Douglass et al., 2012, 2013)</li><li>Fluorescent nuclear track detector (Kodaira et al., 2015; Niklas et al., 2013; Sawakuchi et al., 2016)</li><li>Tissue equivalent proportional counter (Straume et al., 2015)</li><li>Alanine dosimeters and nanoDots (Lind et al., 2019; Xie et al., 2022)</li></ul>Non-ionizing radiation<ul><li>UV meters or radiometers (Xie et al., 2022)</li></ul></td></tr><tr><td>Key event (KE)</td><td>Oxidative stress Several versions of this KE exist in the AOP-Wiki. Additions are to the KE titled: KE #1392: Oxidative stress</td><td>This KE includes:<ul><li>Imbalance in ROS and RNS (collectively RONS) production and antioxidant defense</li><li>Damage within the electron transport chain and within the mitochondria caused by RONS</li><li>Indirect damage of RONS through molecular changes during oxidative stress</li><li>Defense mechanisms includes antioxidants, vitamins and minerals</li><li>Direct sources of ROS are radical species formed from broken water molecules</li><li>Indirect sources of ROS include producers in the electron transport chain</li></ul></td><td>Direct ROS measurements:<ul><li>Fluorescence analysis of dihydroethidium or hydrocyanics (Griendling et al., 2016)</li><li>Dichlorodihydrofluorescein diacetate or amplex red assay (Griendling et al., 2016)</li><li>Chemiluminescence (Griendling et al., 2016)</li><li>Electron spin resonance microscopy (Griendling et al., 2016)</li></ul>Indirect ROS measurements by ROS-dependent cellular damage:<ul><li>Protein carbonylation (Griendling et al., 2016)</li><li>Lipid peroxidation assay with thiobarbituric acid reactive substances (Griendling et al., 2016)</li></ul>Antioxidant measurements:<ul><li>Antioxidant enzyme depletion, including superoxide dismutase, catalase, and glutathione peroxidase (Gille & Sigler, 1995)</li><li>GSH depletion (Biesemann et al., 2018)</li></ul></td></tr><tr><td>Key event (KE)</td><td>Increase, DNA strand breaks Existing KE in the AOP-Wiki, KE #1635</td><td>This KE includes:<ul><li>Nicks in the DNA strand could result in SSBs, or DSBs when two SSBs are close together in time and space</li><li>Complex lesions and clustered damage</li><li>Clustered oxidative DNA lesions</li></ul></td><td><ul><li>Comet assay to detect SSBs or DSBs through gel electrophoresis (Nikolova et al., 2017)</li><li>γ-H2AX quantification assays—flow cytometry, western blot, microscopy, ELISA (Bryce et al., 2016; Garcia-Canton et al., 2013; Ji et al., 2017; Revet et al., 2011)</li><li>Pulsed field gel electrophoresis (PFGE) to detect DSBs (Kawashima et al., 2017)</li><li>Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect strand breaks using reporter enzymes or fluorescent dyes (Loo, 2011)</li><li>Unwinding assay, where increased DNA unwinding is correlated with increased DNA breaks (Nacci et al., 1992)</li></ul></td></tr><tr><td>Key event (KE)</td><td>Increase pro-inflammatory mediators Existing KE in the AOP-Wiki, KE #1493</td><td>This KE includes:<ul><li>Increased production of pro-inflammatory mediators leading to cell death</li><li>Pro-inflammatory cytokines include TNF-α, interleukins (IL-1, IL-6, and IL-8), interferons (IFN-γ), chemokines (CXCL, CCL, and MCP-1), and GM-CSF</li></ul></td><td><ul><li>Localization of inflammatory cytokines by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) (Amsen et al., 2009; Coons et al., 1942; Goldsmith, 1975; Ji & Forsthuber, 2016)</li><li>mRNA levels of genes that express cytokines, chemokines, and inflammatory markers (e.g., RT-qPCR Q-PCR) (Alwine et al., 1977; Forlenza et al., 2012; Veremeyko et al., 2012)</li></ul></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Type</th><th>Event title</th><th>Description</th><th>How to measure</th></tr></thead><tbody><tr><td>Key Event (KE)</td><td>Tissue resident cell activation Existing KE in the AOP-Wiki, KE #1492</td><td>This KE includes:<ul><li>Adoption of hypertrophic morphology and a proliferate state, exhibiting rounding of the cell body and retraction of cell processes</li><li>Acute inflammation</li><li>Damage-associated molecular pattern molecules (DAMPs) which are danger signals or alarmins</li><li>Common markers of tissue activation (CD11b, Iba1, GFAP, CD68, CD86, Mac-1, NF-κB, AP-1, JNK, and P38/MAPK)</li></ul></td><td><ul><li>Immunoblotting (western blotting) to analyze the presence of pro-inflammatory mediators (Lee et al., 2008)</li><li>Flow cytometry for intracellular cytokines (Karanikas et al., 2000)</li><li>Whole blood stimulation assay for inflammatory cytokines in blood (Thurm & Halsey, 2005)</li></ul></td></tr><tr><td>Key event (KE)</td><td>Altered, stress response signaling Existing KE in the AOP-Wiki, KE #2244</td><td>This KE includes:<ul><li>Changes in signals from both external and internal stressors to coordinate protective responses, thereby improving the cell's ability to manage adverse conditions</li><li>The focus is on alterations towards a non-homeostatic state leading to downstream adverse effects</li><li>Key stress response pathways: AMP-PKA, MAPK, PI3K-Akt, NF-κB, JAK-STAT, and p53.</li></ul></td><td><ul><li>Neuroimaging techniques to visualize activation (e.g., magnetic resonance imaging and positron emission tomography) (Ho et al., 2013; Tronel et al., 2017)</li><li>Protein identification using various methods to detect evidence of cell activation (e.g., ELISA, western blotting, and immunohistochemistry) (Amsen et al., 2009; Coons et al., 1942; Falsig et al., 2003; Lund et al., 2006; Monnet-Tschudi et al., 2011; Yang & Mahmood, 2012)</li><li>mRNA levels of genes that express cell activation peptides, cytokines and chemokines (e.g., RT-qPCR, Q-PCR) (Alvarez & Doné, 2014; Falsig et al., 2003; Lund et al., 2006; Monnet-Tschudi et al., 2011)</li><li>Flow cytometry for cytokines (Karanikas et al., 2000)</li></ul></td></tr><tr><td>Key event (KE)</td><td>Increase, abnormal neural remodeling KE #2098</td><td>This KE includes:<ul><li>Abnormal alterations in structure and function of neurons</li><li>Changes in dendritic complexity</li><li>Changes to the levels of protein synthesis affecting neuronal communication</li><li>Changes in synaptic plasticity and action potential, ultimately leading to the disruption of neuronal signaling</li><li>Changes in electrophysiological and neurophysiological activity</li></ul></td><td><ul><li>ELISA (Kaur & Kaur, 2013)</li><li>Electrophoretic mobility shift assay (EMSA) (Broughton & Burfoot, 2001; Jiao et al., 2003)</li><li>cAMP-Glo™ Assay (Hu et al., 2019)</li><li>Chromatin immunoprecipitation (ChIP) (Vousden & Prives, 2009; Wei et al., 2024)</li><li>Western Blot (Kennedy et al., 2015; Kaur & Kaur, 2013; Sita et al., 2023; Thakur et al., 2019; Zheng et al., 2019)</li><li>RT-PCR or qRT-PCR (Kennedy et al., 2015; Zheng et al., 2019)</li><li>Immunofluorescence (Thakur et al., 2019; Zheng et al., 2019)</li></ul></td></tr><tr><td></td><td></td><td></td><td><ul><li>Detection of neurogenesis and degeneration (e.g., MRI scan, fluoro-Jade stain, bromodeoxyuridine labeling, and MTT assay) (Jiang et al., 2014; Riss et al., 2004; Schmued & Hopkins, 2000; Vallières et al., 2002)</li><li>Quantification of target molecules such as PSD-95, synapsin 1, and drebrin (e.g., western blot, ELISA, and immunoassay) (Vallières et al., 2002; Yang & Mahmood, 2012)</li><li>Golgi-Cox method to visualize neural morphology in vivo (Zaqout & Kaindl, 2016)</li><li>Synaptic proximity ligation assay (SYNPLA) to determine learning induced synaptic plasticity (Dore et al., 2020)</li><li>Electroencephalogram (EEG) nonlinear features (Cheng et al., 2019)</li><li>Immunochemistry (Cheng et al., 2019)</li></ul></td></tr></tbody></table>
<figure><img src="image_6.png" /></figure>

<table><caption>TABLE 1 (Continued)</caption><thead><tr><td>Type</td><td>Event title</td><td>Description</td><td>How to measure</td></tr></thead><tbody><tr><td>Adverse outcome (AO)</td><td>Impairment, learning and memory Existing KE in the AOP-Wiki, KE #341</td><td>This KE includes:<ul><li>Weakened ability to develop sensory, short-term or long-term memories and new associative or non-associative relationships</li></ul></td><td><ul><li>Spatial tasks to assess learning impairment (e.g., radial arm maze (RAM), Barnes maze, and Morris water maze (MWM)) (Vorhees & Williams, 2014)</li><li>Various tests to assess memory functions such as novel object recognition (NOR) and Mini Cog to test recognition memory and learning (Cohen & Stackman Jr., 2015; Gonzalez & Tadi, 2022)</li><li>Fear conditioning to elicit a freezing response (e.g., contextual fear conditioning and trace fear conditioning) (Curzon et al., 2009; Shors et al., 2001)</li></ul></td></tr></tbody></table>

Abbreviations: cAMP, cyclic adenosine monophosphate; CCL, chemokine ligand; CXCL, chemokine (C-X-C motif) ligand 1; DNA, deoxyribonucleic acid; DSB, double strand break; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; GSH, glutathione; IF, interferon; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; mRNA, messenger ribonucleic acid; MRI, magnetic resonance imaging; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction; MWM, Morris water maze; NOR, novel object recognition; PFGE, pulsed field gel electrophoresis; pH, potential hydrogen; PSD-95, postsynaptic density protein 95; Q-PCR, quantitative polymerase chain reaction; RAM, radial arm maze; ROS, reactive oxygen species; RT, reverse transcription; SSB, single strand break; SYNPLA, synaptic proximity ligation assay; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

<figure><img src="image_7.png" /><figcaption>**FIGURE 2** Description of the endpoints commonly used in studies to measure each key event (KE). KEs are ordered by level of biological organization. Green arrows indicate the endpoint increases in the KE, and red arrows indicate the endpoint decreases in the KE. No endpoints are identified for deposition of energy because this KE is measured by the dose of radiation in gray (Gy). 4-HNE, 4-hydroxynonenal; 53BP1, p53-binding protein 1; Akt, protein kinase B; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CAT, catalase; CCL, chemokine ligand; CD68, cluster of differentiation 68; COX, cyclooxygenase; DSB, DNA double strand break; ED1, ectodysplasin A; ERK, extracellular signal-regulated protein kinase; γ-H2AX, phosphorylated histone H2AX; GFAP, glial fibrillary acidic protein; GSH, reduced glutathione; Iba-1, ionized calcium binding adaptor molecule 1; IFN, interferon; IL, interleukin; JNK, c-Jun NH₂-terminal kinase; Mac-1, macrophage 1; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; PI3K, phosphatidyl-inositol-3-kinase; PSD-95, postsynaptic density protein 95; RONS, reactive oxygen and nitrogen species; SOD, superoxide dismutase; SSB, DNA single strand break; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.</figcaption></figure>
<table><caption>TABLE 2 Summary of adjacent KERs in AOP #483 and examples of empirical evidence.</caption><thead><tr><th>Key event relationship (KER) title</th><th>Description</th><th>Example of empirical evidence</th></tr></thead><tbody><tr><td>Deposition of energy leads to increases in oxidative stress KER #2769</td><td>Energy ionizes water and free radicals are produced that combine to create more stable ROS, such as hydrogen peroxide, hydroxide, superoxide, and hydroxyl. Ionizing radiation could also interact with other molecules, such as NO, to produce other less common free radicals, including RNS. Free radicals are then able to diffuse freely through the cell and damage vital cellular components, such as proteins, lipids, and DNA, as well as dysregulate cellular processes, such as cell signaling.</td><td>Empirical evidence: high<br>Dose concordance<br><ul><li>Mice exposed to gamma irradiation resulted in approximately 25% increase in SOD2 activity after 0.1 Gy and approximately 60% increase in SOD2 activity after 0.5 Gy (Veeraraghavan et al., 2011).</li></ul>Time concordance:<ul><li>Neural cells irradiated at 0.1 Gy demonstrated elevated reactive oxygen and nitrogen species (RONS) levels, particularly approximately 2-fold from 1 to 4 weeks, approximately 3-fold from 4 to 6 weeks and approximately 2-fold from 6 to 8 weeks (Tseng et al., 2014).</li></ul></td></tr><tr><td>Deposition of energy leads to increases in DNA strand breaks KER #1977</td><td>The deposition of energy could directly interact with the orbital electrons of DNA, inducing electron excitation and ionization, potentially leading to SSBs or DSBs. The number of breaks produced, and the complexity of the breaks is highly dependent on the amount of energy deposited on and absorbed by the cell.</td><td>Empirical evidence: high<br>Dose concordance:<ul><li>Both 0.1 and 2 Gy resulted in increased 53BP1 foci (marker for DNA strand break) within the brain of mice with a 10-fold increase at 0.1 Gy and an 80-fold increase at 2 Gy (Barazzuol et al., 2017).</li></ul>Time concordance:<ul><li>Mouse hippocampal neuronal cells demonstrated an increase in tail moment (DNA strand break marker) at both 30 min and 24 h after 8 Gy. At 12 Gy, γ-H2AX was increased over 3-fold at 30 min post-irradiation and almost 2-fold at 1 and 24 h (Zhang et al., 2017a).</li></ul></td></tr><tr><td>Deposition of energy leads to tissue resident cell activation KER #2832</td><td>Deposition of energy refers to radiation that has sufficient energy to penetrate biological tissue leading to ionization events that break water molecules resulting in free radical formation. Cells respond to damage by releasing danger signals such as alarmins or damage-associated molecular pattern molecules (DAMPs), either passively, when the ionization event is severe enough to induce cell death, or actively by cells undergoing life threatening stress. Resident immune cells, such as macrophages and dendritic cells, use pattern recognition receptors on their surfaces to detect alarmins and DAMPs which initiate their activation and proliferation.</td><td>Empirical evidence: moderate<br>Dose concordance<br><ul><li>After mice were irradiated with either 1.6 Gy <sup>56</sup>Fe or 2 Gy gamma rays, glial fibrillary acidic protein (GFAP) levels increased by 2.2-fold in the gamma group and 4.3-fold in the <sup>56</sup>Fe irradiated group (Suman et al., 2013).</li></ul>Time concordance:<ul><li>Immediate deposition of energy at varying doses of <sup>16</sup>O particles led to increases in GFAP levels in rats. After 36 h, GFAP was increased 1.2-fold after 1 Gy. After 75 days, GFAP increased 1.2-fold after 5 and 50 cGy and 1.3-fold after 1 Gy (Poulose et al., 2011).</li></ul></td></tr><tr><td>Oxidative stress leads to increases in DNA strand breaks KER #2811</td><td>Oxidative stress is an event that involves both a reduction in free radical scavengers and enzymes, and an increase in free radicals. Between reactive oxygen species (ROS) and reactive nitrogen species (RNS), collectively known as RONS, ROS is particularly significant to oxidative damage and disease states. Since DNA is mainly found in the nucleus, ROS needs to reach the nucleus to induce breaks. Hydroxyl radicals, in addition to being highly reactive, are capable of causing DNA damage, and the backbone of DNA could fragment upon sustained exposure to ROS.</td><td>Empirical evidence: moderate<br>Dose concordance<br><ul><li>Mouse hippocampal neuronal cells irradiated with 10 Gy resulted in a 5-fold increase in intracellular ROS generation and a 3-fold increase in γ-H2AX levels (Huang et al., 2021).</li></ul></td></tr><tr><td>Oxidative stress leads to tissue resident cell activation Existing KER in the AOP-Wiki, KER #1906</td><td>Oxidative stress encompasses an increase in the production of free radicals (e.g., superoxide, hydrogen peroxide, and hydroxyl radicals) and a loss of antioxidant mechanisms. The imbalance of high free radicals and compromised antioxidant mechanisms could lead to damaging byproducts that could activate tissue resident cells. Within the brain, oxidative stress will specifically activate microglial cells and astrocytes. Microglial cells are macrophages in the brain that respond to tissue</td><td>Empirical evidence: moderate<br>Incidence and dose concordance<br><ul><li>BV-2 microglia irradiated with gamma rays demonstrated approximately 7-fold increase in ROS, as well as a 2.5-fold increase in NF-κB and 2-fold increase in AP-1 DNA binding, indicating activated glial cells, at the same dose of 10 Gy (Schnegg et al., 2012).</li></ul><ul><li>Treatment with 4 µg/µL of the neurotoxin 6-hydroxydopamine caused decreased antioxidant</li></ul></td></tr></tbody></table>
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th>Key event relationship (KER) title</th><th>Description</th><th>Example of empirical evidence</th></tr></thead><tbody><tr><td></td><td>injury, provide surveillance to neurons, and maintain synaptic homeostasis.</td><td>levels (0.6-fold decrease in SOD and 0.3-fold decrease in GSH-Px), as well as a 1.7-fold increase in GFAP levels in rats (Wang et al., 2017a, 2017b).<br>Time concordance<ul><li>The immediate exposure of human glioblastoma astrocytes with 50 µM H<sub>2</sub>O<sub>2</sub> (a reactive oxygen species) resulted in slight increases of GFAP at 2, 6, and 12 h following the treatment (Daverey & Agrawal, 2016).</li></ul></td></tr><tr><td>Oxidative stress leads to increase, altered stress response signaling KER #3316</td><td>Many signaling pathways control and maintain physiological balance within a living organism, and these could be impacted by oxidative stress. Excessive RONS during oxidative stress could modify biological molecules and directly cause DNA damage, which could lead to altered signal transduction pathways. Some examples relevant to brain function include the PI3K/Akt pathway, cAMP response element-binding protein (CREB) pathway, caspase pathway, the p53/p21 pathway, as well as the MAPK family pathways, including JNK, ERK, and p38.</td><td>Empirical evidence: moderate<br>Dose/concentration concordance<ul><li>Neuronal cells exposed to 10 µM and 30 µM H<sub>2</sub>O<sub>2</sub> demonstrated 34% and 58% increases in ROS production, respectively. A 2-fold, 10-fold, and 9-fold increase in caspase-3 enzyme activity was also observed at 3, 10, and 30 µM, respectively (Carvour et al., 2008).</li></ul>Time concordance<ul><li>Mice experienced cold stress at various lengths and malondialdehyde (MDA) levels increased by approximately 3-fold at 1 week, approximately 4-fold at 2 weeks, and approximately 5-fold at 3 weeks. Phosphorylated JNK increased by approximately 10% (1 week) and approximately 30% at 2 and 3 weeks compared to room temperature control. Phosphorylated ERK and p38 also increased in a time-concordant manner (Xu et al., 2019).</li></ul></td></tr><tr><td>DNA strand breaks leads to increase, altered stress response signaling KER #3315</td><td>DNA strand breaks could lead to altered signaling of various pathways through the DNA damage response. Following DNA strand breaks, DNA damage response cellular signaling could phosphorylate downstream proteins and activate several transcription factors and pathways including ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR), two phosphoinositide 3-kinase (PI3K)-related serine/threonine kinases (PIKKs), p53/p21 pathway and caspase pathway.</td><td>Empirical evidence: moderate<br>Dose concordance<ul><li>Irradiated primary cortical neurons demonstrated increased γ-H2AX by 30-fold at both 8 and 32 Gy but not at 2 Gy. p-ATM was increased at all doses, increasing about 15-fold at 8 and 32 Gy. Signaling molecules including p-p53, p21, and cleaved caspase 3 were increased at all doses (Sabirzhanov et al., 2020).</li></ul>Time concordance<ul><li>Mouse hippocampal neuronal cells irradiated with 12 Gy resulted in a 4.4-fold and 3.2-fold increase in p-ATM and γ-H2AX, respectively, 30 min post-irradiation. p53 was increased 4.6-fold at 1 h post-irradiation and cleaved caspase 3 increase by 9-fold 48 h post-irradiation (Zhang et al., 2017a).</li></ul></td></tr><tr><td>Tissue resident cell activation leads to increase, pro-inflammatory mediators KER #2834</td><td>Activated tissue-resident cells demonstrate increased levels of transcription factor nuclear factor κB (NF-κB) and activated protein 1 (AP-1) DNA binding due to increased oxidative stress or DNA damage. Through the activity of NF-κB, AP-1 and other signaling pathways, activated immune cells could together produce/secrete tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, cyclooxygenase (Cox)-2, lipoxygenase (LOX) enzymes, intercellular adhesion molecule (ICAM)-1, and monocyte chemoattractant protein (MCP)-1.</td><td>Empirical evidence: moderate<br>Incidence and dose concordance<ul><li>Microglial activation increased after mice were irradiated with 0.05 and 0.3 Gy <sup>4</sup>He particles by 3.5-fold and 3.8-fold, respectively. Chemokine CCL-3 was increased by 2.2-fold after 0.3 Gy (Parihar et al., 2018).</li><li>Rats with 10 Gy gamma ray irradiation demonstrated a 3.6-fold and 2.8-fold increase in NF-κB and AP-1 DNA binding, respectively, which are indicators of activated microglia. At the same dose, pro-inflammatory mediators TNF-α, IL-1β, and MCP-1 also increased by 23-fold, 10-fold, and 1.6-fold, respectively (Lee et al., 2010).</li></ul>Time concordance<ul><li>After mouse cranial X-ray irradiation, the number of F4/80 positive cells/mm<sup>2</sup> (microglial activation marker) was significantly increased from 3 h to</li></ul></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 2 (Continued)</caption><thead><tr><th>Key event relationship (KER) title</th><th>Description</th><th>Example of empirical evidence</th></tr></thead><tbody><tr><td>Increase, pro-inflammatory mediators lead to abnormal neural remodeling KER #2835</td><td>Cytokines mediate immune response through ligand binding to cell surface receptors, which activate signaling cascades such as the JAK-STAT or MAPK pathways to produce or recruit more cytokines. Once organs initiate inflammatory reactions, the cytokines are capable of impairing neuronal function through direct effects on neurons or by indirect mechanisms mediated by microglia, astrocytes or vascular endothelial cells. Overexpression of pro-inflammatory mediators could disrupt the integrity of neurons through increased necrosis and demyelination, decreased neurogenesis, neural stem cell proliferation and synaptic complexity.</td><td>2 weeks with a maximum after 24 h, while TNF-α and IL-1β both demonstrated significantly increased levels and expression at many times from 3 h to 6 weeks post-irradiation, with maximum levels occurring after 24 h (Dong et al., 2015).<br>Empirical evidence: moderate<br>Incidence and dose concordance<br>• No available data<br>Time concordance<br>• X ray-induced increase in IL-1β and TNF-α at 3 and 6 h resulted in a time-dependent decrease in DCX<sup>+</sup> cells, a marker for neurogenesis, which persisted for 2 weeks (Dong et al., 2015).</td></tr><tr><td>Increase, altered stress response signaling leads to increase, abnormal neural remodeling KER #3321</td><td>Abnormal neural remodeling could be facilitated by changes in 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.</td><td>Empirical evidence: moderate<br>Dose and incidence concordance<br>• Neural stem-like cells exposed to 6 Gy of gamma irradiation demonstrated increased p-Akt, p-p53, p-STAT3, and mGluR1 at least 2-fold. Neural stem cells decreased 0.4-fold (Eom et al., 2015).<br>• Mice exposed to 2 Gy of gamma irradiation showed increases of pro-apoptotic markers p53 and BAX by 8.4-fold and 2.3-fold, respectively. A 0.6-fold decrease in Bcl-2 (anti-apoptotic) was also observed. Gamma rays decreased cortical thickness 0.9-fold (Suman et al., 2013).<br>Time concordance<br>• Neural stem cells irradiated with 10 Gy gamma rays demonstrated an increase in p-JNK of 30% at 1 h and 60% at 2 h post-irradiation. Apoptosis increased 48 h post-irradiation (Kanzawa et al., 2006).</td></tr><tr><td>Increase, abnormal neural remodeling leads to learning and memory, impairment KER #2836</td><td>Abnormal neural remodeling encompasses changes in neuronal structure and function including alterations in synaptic activity, necrosis, demyelination, neurogenesis, neurodegeneration, and dendrite morphology. When modified by stressors, these neuronal components could be disrupted and lead to an impairment of learning and memory.</td><td>Empirical evidence: moderate<br>Dose/concentration concordance<br>• Mice exposed to 0.05 and 0.3 Gy of <sup>48</sup>Ti demonstrated dose-dependent decreases in total dendritic length and number of dendritic branch points. When subjected to cognitive testing, mice exposed to 0.3 Gy demonstrated greater impairment in the NOR and OiP tests compared to 0.05 Gy exposed mice (Parihar et al., 2016).<br>Time concordance<br>• After mice were irradiated with <sup>16</sup>O and <sup>48</sup>Ti particles at 0.05 and 0.30 Gy, dendritic complexity and spine density were reduced 15 weeks post-irradiation. Synaptic spine puncta increased 15 and 24 weeks post-irradiation, while learning and memory were impaired 15 and 24 weeks post-irradiation (Parihar et al., 2016).</td></tr></tbody></table>

Abbreviations: AP-1, activator protein 1; ATM, ataxia telangiectasia mutated; ATR, ATM/RAD3-related; BAX, Bcl-2 associated X protein; CCL, chemokine ligand; COX, cyclooxygenase; CREB, cAMP response element-binding protein; DAMP, damage-associated molecular pattern molecule; DCX, doublecortin; DNA, deoxyribonucleic acid; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; GSH, glutathione; GSH-Px, glutathione peroxidase; ICAM, intercellular adhesion molecule; IL, interleukin; JAK-STAT, Janus kinase-signal transducer and activator of transcription; JNK, c-Jun NH<sub>2</sub>-terminal kinase; LOX, lipoxygenase; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; MDA, malondialdehyde; mGluR1, metabotropic glutamate receptor 1; NF-κB, nuclear factor kappa B; PI3K/Akt, phosphatidylinositol 3-kinase/protein kinase B; PIKK, phosphoinositide 3-kinase (PI3K)-related serine/threonine kinase; NO, nitric oxide; NOR, novel object recognition; OiP, object in place; RNS, reactive nitrogen species; ROS, reactive oxygen species; RONS, reactive oxygen and nitrogen species; SOD, superoxide dismutase; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor.
<figure><img src="image_8.png" /></figure>

relevant studies with desired stressors (both ionizing and non ionizing), models (brain relevant cell types, unless it is a shared KER with other AOs), and doses. Priority was given to in vivo/human relevant biological models and IR exposure at low-to-moderate dose relevant to space radiation exposures. Although the AOP is mostly supported by IR studies, a few studies relate to non-ionizing exposures, specifically macromolecular-level KERs. The collective impact of various stressors, including ultraviolet exposure, can lead to the generation of free radicals and then initiate downstream pro-inflammatory mediators. Since AOPs are stressor-agnostic, this collective burden (i.e., multiple stressor exposure) may overwhelm protective mechanisms, thereby triggering further KEs along the AOP.

In the case of insufficient articles, studies were included from in vitro models, high dose IR and/or non-IR studies. Of note, any articles published following completion of the SWIFT-Review would not have been identified. The filtered articles were then screened using DistillerSR [https://www.distillersr.com/] software based on the PEO/E criteria and relevant data were extracted using the modified Bradford Hill criteria (Becker et al., 2015). For all extracted studies information was tracked on exposure parameters, domain of applicability and how the study met the Bradford Hill criteria. This extracted data was used to generate visual graphics that provide a summary of dose range, stressor types, domain of applicability, and evidence stream used to support the AOP. The articles that failed to meet these requirements were excluded from further consideration. A number of

<table><caption>TABLE 3 Summary of non-adjacent KERs in AOP #483 and examples of empirical evidence.</caption><thead><tr><th>Key event relationship (KER) title</th><th>Description</th><th>Example of empirical evidence</th></tr></thead><tbody><tr><td>Deposition of energy leads to increase, abnormal neural remodeling<br>KER #2837</td><td>Deposited energy disrupts neuronal signaling through the induction of alterations to the neuronal architecture and synaptic activity. The energy induced damage includes increased necrosis and demyelination, and decreased neurogenesis and synaptic complexity; these together are important to maintain the integrity of the neurons.</td><td>Empirical evidence: moderate<br>Dose concordance<br><ul><li>Dose-dependent increases in neuronal apoptosis/ necrosis and decreases in F-Actin, drebrin and synapsin I protein clusters are observed at X-ray irradiation of 0.5, 4, and 10 Gy (Okamoto et al., 2009).</li><li>Time concordance<br><ul><li>A long-term study at 30 months (15 Gy of X-rays) revealed a reduction in computed tomography (CT) density, indicative of edema, demyelination, axonal swelling, and/or necrosis relative to earlier timepoints (Fike et al., 1984).</li></ul></li></ul></td></tr><tr><td>Deposition of energy to learning and memory, impairment<br>KER #2838</td><td>Deposition of energy could lead to reduced cognitive functions and the process begins at the macromolecular level, through the generation of oxidative stress and promotion of neuroinflammatory environments. In a chronic state, the functions of neurons and glial cells could deviate into a more detrimental role. Consequences include the persistence of a pro-inflammatory response, reduced neurogenesis and inhibition of neuronal connectivity and synaptic activity. Without the ability of neurons to communicate with each other effectively and glial cells to regulate the immune responses, normal CNS functions such as learning and memory are impaired.</td><td>Empirical evidence: moderate<br>Dose concordance<br><ul><li>Novel object recognition (NOR) task was used to assess recognition in mice. At 0.15 and 0.5 Gy galactic cosmic rays (GCR) (composed of protons, <sup>4</sup>He and <sup>16</sup>O), a concordant reduction in memory was observed (Krukowski et al., 2018).</li><li>Time concordance<br><ul><li>At 6 weeks after <sup>48</sup>Ti (0.05 and 0.30 Gy) and <sup>16</sup>O (0.30 Gy) irradiation, the discrimination index (DI) for NOR decreased significantly in mice (Parihar et al., 2015b).</li></ul></li></ul></td></tr><tr><td>Increase in DNA strand breaks leads to abnormal neural remodeling<br>KER #2841</td><td>DNA single strand breaks and double strand breaks could lead to cell cycle arrest and apoptosis. In proliferative cells like neural stem/progenitor cells this will reduce neurogenesis within the brain. In mature neurons, some evidence suggests that unrepaired DNA strand breaks could have deleterious effects. There is also evidence that DNA strand breaks could induce changes to neural plasticity and synaptic activity through changes in gene expression.</td><td>Empirical evidence: moderate<br>Dose and incidence concordance<br><ul><li>53BP1 foci increased over 10-fold in mice at 0.1 Gy and about 80-fold at 2 Gy in the lateral ventricle. At 1, 2, and 3 Gy, proliferating cells in the lateral ventricle decreased 0.2-fold (Barazzuol et al., 2017).</li><li>γ-H2AX foci in human neural stem cells were increased about 20-fold after 5 Gy. PARP+ (early apoptotic marker) cells were increased 3-fold and annexin V+ (late apoptotic marker) cells were increased 2-fold. Neural stem cell differentiation decreased 0.5-fold (Acharya et al., 2010).</li></ul><li>Time concordance<br><ul><li>30 min after 12 Gy irradiation, γ-H2AX increased 3.2-fold in mouse hippocampal neuronal cells. Apoptosis increased over 10-fold at 48 h post-irradiation (Zhang et al., 2017a).</li></ul></li></ul></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 3 (Continued)</caption><thead><tr><th>Key event relationship (KER) title</th><th>Description</th><th>Example of empirical evidence</th></tr></thead><tbody><tr><td>Increase in pro-inflammatory mediators leads to learning and memory, impairment KER #2839</td><td>Cytokines mediate immune response through ligand binding to cell surface receptors, which activate signaling cascades such as the JAK-STAT or MAPK pathways to produce or recruit more cytokines. Once organs initiate inflammatory reactions, the cytokines could modulate different metabolic and molecular pathways that have direct effects on neurons or indirect effects mediated by microglia, astrocytes or vascular endothelial cells. Modulation of these pathways ultimately affect crucial neuronal networks such as within the hippocampus, which is the main brain region responsible for learning and memory.</td><td>Empirical evidence: moderate<br>Incidence and dose concordance<ul><li>Mice injected with 100 µg of lipopolysaccharide (LPS) demonstrated approximately 27-fold increase in IL-1β and TNF-α increased by approximately 72-fold. Performance in the water maze was impaired as assessed by the distance swam, latency and swim speed after LPS injection, with a maximum 2-fold increased swim distance (Sparkman et al., 2006).</li><li>Mice irradiated with 10 Gy X-ray demonstrated a 3-fold increase in IL-6 secretion, as well as significant decreases in the discrimination index for the novel object recognition and object in place tasks (Bhat et al., 2020).</li><li>Rats irradiated with gamma rays demonstrated increased inflammation after 2 months, while memory was impaired after 6 months (Jenrow et al., 2013).</li></ul>Time concordance<ul><li>Rats irradiated with gamma rays demonstrated increased inflammation after 2 months, while memory was impaired after 6 months (Jenrow et al., 2013).</li></ul></td></tr></tbody></table>

Abbreviations: CNS, central nervous system; CT, computed tomography; DI, discrimination index; DSB, double strand break; GCR, galactic cosmic rays; IL, interleukin; JAK-STAT, Janus kinase-signal transducer and activator of transcription; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NOR, novel object recognition; PARP, poly (ADP-ribose) polymerase; SSB, single strand break; TNF, tumor necrosis factor.

<figure><img src="image_9.png" /></figure>

MIE1686: Deposition of energy

KE1392: Oxidative stress

KE1635: Increase, DNA strand breaks

KE2244: Altered stress response signaling

KE1492: Tissue resident cell activation

KE2097: Increased, pro-inflammatory mediators

KE2098: Increased, abnormal neural remodeling

AO341: Impaired learning and memory

<caption>**FIGURE 3** Systematic evidence map depicting the quantity of evidence supporting each key event relationship (KER). Key events (KEs) are represented as circles, and KERs as arrows. The size of each arrow represents the weight of evidence as determined by the number of articles supporting the relationship in the form of biological plausibility, empirical evidence and essentiality studies. The articles could be used to support multiple KERs.</caption>

KEs identified in DistillerSR were considered but are absent in the final pathway due to the lack of empirical evidence to support connectivity to another KE or the lack of a well-defined measurement. The three KEs, increased oxidative stress, increased pro-inflammatory mediators and tissue resident cell activation already exist within the AOP-Wiki and were not significantly modified.

KERs were all supported by the modified Bradford Hill considerations (Becker et al., 2015) that include (1) biological plausibility, (2) empirical support (dose-response, temporality, and incidence), and (3) essentiality of KEs to the relationships. In AOP #483, two sets of KERs connect the KEs in the network—adjacent (directly upstream

and downstream of each other in the pathway) and non-adjacent (between two KEs that are separated by one or more intermediate KEs) (Figure 1).

The adjacent and non-adjacent KERs within the AOP network are summarized in Tables 2 and 3, respectively. In Tables 2 and 3, each of the KERs are described and examples of empirical evidence supporting the relationships are provided. Extended sets of empirical evidence for each KER are available in the AOP-Wiki, and collectively, the empirical evidence for both types of KERs provides weight of evidence (WOE) for the overall AOP (OECD, 2018).
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**FIGURE 4** Summary of the Bradford Hill criteria (upper panel; plausibility, incidence concordance, dose concordance, time concordance, and essentiality) and the stressor types supporting the AOP (lower panel). The number of studies with each evidence stream and the number of studies using each stressor to support the AOP were determined. Note essentiality may be overrepresented relative to the other evidence streams due to the multiple KERs directly linked to the MIE that demonstrated essentiality through control and treatment groups (an area with an abundance of studies). Not all of these stressors support each KER, details can be found in the AOP-Wiki. UV, ultraviolet.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**FIGURE 5** Summary of the domain of applicability and doses used. Studies were broken down by animal life stage, taxonomy, and the dose of radiation used. Animal life stage was defined in Sengupta (2013) for rats, in Dutta and Sengupta (2018) for rabbits, and in Flurkey (2007) for mice. Not all of these doses support each KER, details can be found in the AOP-Wiki. Under taxonomic applicability, "other" refers to pigs (1%), dogs (1%), bovines (1%), and guinea pigs (0.5%). *Studies derived from in vitro models, as well as some in vivo models, do not specify life stage.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**FIGURE 6** Summary of findings. Visual representation of evidence supporting the AOP [AOP #483, subdivided based on evidence stream, taxonomic applicability, linear energy transfer (LET), and dose range]. The AOP is predominantly supported by ionizing radiation stressors and the breakdown of the doses is as follows: Low doses were defined as <0.1 Gy, moderate doses were in the range of 0.1–1 Gy, and high doses were >1 Gy. High LET radiation includes neutrons and heavy ions, and low LET radiation includes protons, X-rays, and gamma rays. Not all stressors and dose ranges support each KER in the AOP, details can be found in the AOP-Wiki. The size of each boxed section is representative of the relative number of articles supporting that category, although sections with two or less articles supporting it were removed. H/R/M refers to human, rat, and mouse species. Additionally, the number of articles was adjusted to make each label visible, but the figure is still representative of the summary of findings.</figcaption></figure>

# 5 | SUMMARY OF SCIENTIFIC EVIDENCE

## 5.1 | Description and evaluation of biological plausibility and empirical evidence

Biological plausibility is the highest form of evidence and identifies the known structural and functional aspect of each KER in the AOP. It is the fundamental biological understanding and the ability to infer the biological consequence of the perturbation from the exposure/stressor. The biological rationale linking deposition of energy to impaired learning and memory is strongly

supported in the literature, as reported by several studies in vitro and in vivo.

There is high biological plausibility that when energy is deposited onto a cell it excites/ionizes abundant molecules or directly impacts enzymes (i.e., catalase) to produce both acute and chronic elevation in ROS as approximately 80% of tissues are composed of water (Baluchamy et al., 2012; Baulch et al., 2015; de Jager et al., 2017; Giedzinski et al., 2005; Rehman et al., 2016; Tahimic & Globus, 2017; Wang et al., 2019). Deposited energy can also damage DNA through single or double strand breaks, base damage or DNA crosslinking. It is worth noting that double strand breaks are the most detrimental type
<figure><img src="image_14.png" /></figure>

of DNA damage (Christensen et al., 2014; Joiner & van der Kogel, 2009; Sage & Shikazono, 2017; Smith et al., 2003). In addition, ROS production from energy deposition can result in decreased levels of antioxidants, thus disrupting the antioxidant defense system, or upregulate these antioxidant enzymes such as superoxide dismutase (SOD) or catalase to combat the elevated ROS (Acharya et al., 2010; Baluchamy et al., 2012; Baulch et al., 2015; Veeraraghavan et al., 2011). Oxidative stress may subsequently lead to abnormal neural remodeling through three pathways: (1) by oxidizing DNA bases, which create nicks on the strand and leads to DNA strand breaks (Cannan & Pederson, 2016; Fong, 2016) which if persistent can induce neuronal apoptosis disrupting normal neuronal function (Abner & McKinnon, 2004; Desai et al., 2022; Madabhushi et al., 2014; Michaelidesová et al., 2019; Wang et al., 2017a, 2017b; Zhu et al., 2019); (2) by activation of tissue resident cells in the brain such as astrocytes and microglial cells which lead to increased pro-inflammatory mediators downstream and (3) by inducing changes in multiple signaling pathways, including mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling, senescence signaling, and apoptotic signaling (Hladik & Tapio, 2016; Simpson & Oliver, 2020). These signaling pathways are involved in regulating the structural and functional properties of neurons (NCRP, 2016), which are also directly altered by DNA strand breaks. Following these breaks, damage response cellular signaling could lead to phosphorylation of downstream proteins and activate several transcription factors and pathways, including p53/p21 (Abner & McKinnon, 2004; Baselet et al., 2019), caspase (Abner & McKinnon, 2004; Baselet et al., 2019) and MAPK family pathways (Ghahremani et al., 2002; Nagane et al., 2021). However, even if these pathways are activated as a normal response to facilitate DNA repair, uncontrolled DNA damage will overly activate these pathways, resulting in subsequent harmful downstream effects.

There is moderate evidence to suggest that microglial cells and astrocytes can change from resting to reactive states in response to stressors including IR and excess ROS (Chiang et al., 1993; Cucinotta et al., 2014; Hladik & Tapio, 2016; Lee et al., 2021). Reactive microglia and astrocytes release pro-inflammatory mediators and promote antioxidant defenses through toll-like receptors (TLRs) (Heidari et al., 2022; Simpson & Oliver, 2020). Activation of various pathways including the NF-κB transcription factor pathway, the MAPK-AP-1 signaling pathway, and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway could lead to production of pro-inflammatory mediators (Chen et al., 2018; Vezzani & Viviani, 2015). Under normal circumstances, the release of proinflammatory mediators are part of the immune response to maintain CNS homeostasis and protect against pathogens. However, dysregulation or chronic activation of glia cells can lead to an excessive release of proinflammatory mediators, contributing to neuroinflammation. This sustained inflammation in the nervous system is associated with various neurodegenerative diseases (Simpson & Oliver, 2020). Therefore, the biological plausibility of tissue resident cell activation to increase in pro-inflammatory mediators is also high.

There is moderate evidence to support biological plausibility for the link between increases in pro-inflammatory mediators and abnormal neural remodeling. Inflammation in CNS can decrease neuron production (Chen & Palmer, 2013), modulate neurogenesis (Cameron & Gould, 1994; Gould et al., 1992; Turnbull & Rivier, 1999), and contribute to the overall stability of neural networks collectively referred to as abnormal neural remodeling. Although cytokines and their receptors are constitutively expressed by CNS overexpression of pro-inflammatory, or T-helper type 1 (Th1) cytokines are implicated in the pathogenesis of neurotoxicity. Once bound to their receptors, these cytokines lead to the activation of intracellular signaling pathways and subsequent changes in cell structure and function (Mousa & Bakhiet, 2013). These changes include modifications in physical and electrophysical properties in neurons from cell culture lines, specific brain regions such as the hippocampus or dentate gyrus (DG) or even whole-brain samples (Fan & Pang, 2017; Jenrow et al., 2013; Wong et al., 2004). Various studies have also reported a reduction in the proliferation and differentiation of progenitor and neural stem cells, as well as a decrease in neurogenesis due to increased pro-inflammatory mediators such as IL-1β, TNF-α, and IL-6 (Tang et al., 2017; Wong et al., 2004; Zonis et al., 2015). For example, IL-1β could induce the expression of the nuclear receptor TLX, an orphan nuclear receptor required to maintain the neural precursor cell pool in neurogenic brain regions, and consequently cell proliferation (Ryan et al., 2013). TNF-α affects neuronal fate through tumor necrosis factor receptor 1 (TNFR1) mediated signaling, which could suppress NSC proliferation (Chen & Palmer, 2013). IL-6 has numerous mechanisms of action that could alter neurogenesis, one of which is the stimulation of the hypothalamic-pituitary-adrenal axis. This activation increases circulating glucocorticoids that can impede proliferation and cell growth in the DG (Cameron & Gould, 1994; Gould et al., 1992; Turnbull & Rivier, 1999).

In addition to activating tissue resident cells, oxidative stress through RONS has also been reported to induce alterations to various signaling pathways. There is high evidence surrounding the biological plausibility of increased oxidative stress to altered stress response signaling. Oxidative stress may directly impact proteins that are in pathways involved in synaptic function, morphology and proliferation, cellular senescence, and apoptosis. Specifically in the CNS, oxidative stress could impact the PI3K/Akt pathway, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) pathway, the p53/p21 pathway, as well as the MAPK family pathways, including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38 (Lehtinen & Bonni, 2006; Ramalingam & Kim, 2012). As these pathways are essential in many processes in neurons, including cell survival, morphology, proliferation, differentiation, and synaptic activity, disruptions to components of these pathways will lead to abnormal neural remodeling (Carvour et al., 2008; Lehtinen & Bonni, 2006). Oxidative stress may also impact signaling pathways within the mitochondria, such as apoptotic neuronal death signaling pathways (Niizuma et al., 2010). These intracellular pathways control various cell functions such as cell growth, death or communication. However, the complexity of the abnormal neural remodeling
<figure><img src="image_15.png" /></figure>

response to these pathways can be due to the synergistic and antagonistic interactions between signaling pathways. Therefore, the biological plausibility surrounding altered stress response signaling to abnormal neural remodeling is moderate.

Finally, there is moderate biological plausibility that abnormal neural remodeling leads to impairment of learning and memory. Abnormal neural remodeling refers to alterations through changes to neurogenesis, neurodegeneration, neuronal excitability and synaptic plasticity, demyelination and dendritic spine density (Hladik & Tapio, 2016; Makale et al., 2017). One of the structures in the brain with evidence of de novo adult neurogenesis is the hippocampus, specifically in the subgranular zone (SGZ) of the DG (Monje & Palmer, 2003; Tomé et al., 2015). Accordingly, reduced neurogenesis in the SGZ is associated with impaired learning and memory (Bálentová & Adamkov, 2020; Monje & Palmer, 2003). Similarly, apoptosis and neurodegeneration have been reported to impair cognitive function (Bálentová & Adamkov, 2020; Hladik & Tapio, 2016). Additional evidence suggests that impairment in hippocampal excitability and long-term potentiation, processes essential for learning and memory formation, are disrupted from radiation exposure and associated with learning and memory impairment (Acharya et al., 2019). Stressors such as IR could also cause decrements to CNS structures that are associated with impairment in learning and memory. For example, radiation induced changes in white matter, specifically demyelination and white matter necrosis, are associated with impaired learning and memory (Bálentová & Adamkov, 2020). In addition, morphometric changes in gray matter structures, such as reduced dendritic complexity and spine density, are associated with impaired learning and memory (Bálentová & Adamkov, 2020; Parihar et al., 2015a; Romanella et al., 2020).

There are three non-adjacent KERs. There is moderate biological plausibility describing the relationship between deposition of energy and increase, abnormal neural remodeling. Several reviews have described changes in the physical and electrophysiological properties of neurons in response to ionizing radiation exposure (Cekanaviciute et al., 2018; Hladik & Tapio, 2016; Makale et al., 2017). There is high biological plausibility describing the relationship between deposition of energy and impaired learning and memory, and this relationship has also been described in multiple review articles and reports (Cekanaviciute et al., 2018; Kiffer et al., 2019b; NCRP, 2016; Pasqual et al., 2021). Finally, there is moderate biological plausibility describing the relationship between pro-inflammatory mediators and impaired learning and memory. Although mechanisms remain unclear due to the complexity of inflammatory signaling, sufficient evidence shows that these inflammatory markers are involved in changes to neural circuits that regulate learning and memory processes (Bourgognon & Cavanagh, 2020; Pugh et al., 2001). For example, as IL-1β and its receptor are greatly expressed in the hippocampus, multiple studies have demonstrated that IL-1β plays an important role in the formation of hippocampal-dependent memory. Experimentally, the reported results include impaired performance in behavioral paradigms such as spatial memory, contextual learning, and passive avoidance tasks (Donzis & Tronson, 2014; Pugh et al., 2001; Yirmiya & Goshen, 2011).

Regarding the empirical evidence supporting this AOP, overall, many studies demonstrated that KE<sub>upstream</sub> occurred at lower or the same doses and at earlier or the same times as KE<sub>downstream</sub>. There were some inconsistencies where the KEs were only measured at one dose or time point. The relationship between energy deposition and both oxidative stress and DNA strand breaks is strongly supported by primary research on the effects of IR on ROS and antioxidant levels. Of the studies that examined the effects of IR on the CNS, the doses ranged from 1 cGy to 10 Gy from different radiation sources (⁵⁶Fe, X-rays, ¹³⁷Cs gamma rays, protons, ¹⁶O, ²⁸Si, and ⁴⁸Ti) being applied to primarily rodent models. Generally, these studies found a dose-dependent increase of oxidative stress markers in the brain after exposure to IR, along with some studies showing changes in antioxidant levels. Also, quantitative understanding of linkage between energy deposition and DNA strand breaks suggests that DSBs could be predicted upon exposure to ionizing radiation. The quantitative relationship is dependent on the biological model, the type of radiation and the radiation dose. In general, 1 Gy of radiation are thought to result in 3000 damaged bases (Maier et al., 2016), 1000 single-strand breaks, and 40 DSBs (Foray et al., 2016; Maier et al., 2016; Ward, 1988). In addition, the increase in DNA strand breaks is well supported after oxidative stress through empirical evidence from studies using photons, γ- and X-rays, both in vivo using rat models and in vitro in neuronal cells lines (El-Missiry et al., 2018; Huang et al., 2021; Zhang et al., 2017b). In the CNS, data obtained moderately supports the link of increased oxidative stress resulting in increased tissue resident cell activation and alteration of signaling pathways. Tissue resident cell activation in astrocytes was commonly measured by expression of glial fibrillary acidic protein (GFAP), activation in microglia commonly measured by CD86, while NF-κB and activator protein 1 (AP-1) DNA binding could assure activation of both microglia and astrocytes. Also, the relationship between tissue-resident cell activation leading to increased pro-inflammatory mediators was moderately supported by empirical evidence gathered from studies using human, in vitro cell cultures as well as rat and mouse in vivo models. The stressors used were gamma rays, X-rays, ⁴He ions, nominal photon energy, and lipopolysaccharide (LPS) treatment. In addition, the empirical evidence collected from both in vitro and in vivo studies with heavy ion or X-ray irradiation supports a causal relationship between the increase of pro-inflammatory mediators and abnormal neural remodeling. These studies provide evidence to support a causal association with time concordance between an increase in pro-inflammatory mediators and decreased neurogenesis. Furthermore, sufficient empirical evidence from in vivo mouse and rat models as well as in vitro rat-, mouse-, and human-derived cell models using different type of ionizing radiation supported the KE of abnormal neural remodeling following alteration of stress response signaling pathways (Chow et al., 2000; El-Missiry et al., 2018; Kumar et al., 2005; Suman et al., 2013) and increased DNA strand breaks (Barazzuol et al., 2015, 2017; Zanni et al., 2015). Finally, ample evidence supported the connection between the KEs for impairment of learning and memory. The evidence was gathered from studies using in vivo rodent models and human studies. The applied stressors ranged from
<figure><img src="image_16.png" /></figure>

0.5 cGy to 40 Gy and included heavy ions (i.e., ¹⁶O, ²⁸Si, ⁴⁸Ti, and ⁵⁶Fe), X-rays, gamma rays, protons, and α particles. Impairment in learning and memory were typically determined using tasks to identify changes in cognitive behavior. These tests include novel object recognition (NOR), object in place (Oip), light–dark box (LDB), forced swim test (FST), fear extinction (FE), Morris water maze, temporal order (TO), Barnes maze, passive avoidance task, T-maze, Y-maze, and fear conditioning (FC). These tests assessed various cognitive aspects such as short-term memory, long-term memory, recognition memory, spatial memory, working memory, declarative memory, associative learning, discrimination, and reversal learning.

# 6 | ESSENTIALITY OF KEY EVENTS

## 6.1 | KEs linked to the MIE

The essentiality of the MIE and the KE<sub>downstream</sub> was evaluated to be moderate overall. Since deposition of energy is a physical stressor, it could be shielded, but chemicals cannot effectively block or decrease it. Negative (non-irradiated) controls in experimental studies is used to support the essentiality of energy deposition for the KE<sub>downstream</sub>. Radiation exposure has been reported to increase levels of DNA strand breaks above the background levels in several studies. However, while the deposition of energy increases oxidative stress, other factors may contribute to oxidative stress even without energy deposition. Potential other factors contributing to oxidative stress include mitochondrial DNA damage, and presence of ozone, aromatic amines, organochlorines, and certain xenobiotics. Likewise, the absence of a radiation stressor, as exhibited in the controls of the empirical studies, demonstrates that tissue resident cell activation is not being initiated. These studies suggest that the presence of radiation stress is essential to the recruitment and activation of tissue resident cells. Also, several experiments demonstrate no changes in neuron structure and function in the absence of radiation exposure. Furthermore, studies have reported that energy deposition from different doses of radiation from X-rays, gamma rays, protons, and heavy ions leads to impaired learning and memory, while impaired learning and memory is not observed in non-irradiated young wild-type animals.

## 6.2 | KEs not linked to the MIE

Essentiality was demonstrated through treatment with chemical inhibitors or knockout genes. There is moderate evidence for the essentiality of the increase in oxidative stress to activation of tissue resident cells and DNA strand breaks. Various studies used antioxidants to inhibit oxidative stress and found a reduction in DNA strand breaks (Huang et al., 2021; Liu et al., 2013; Spector et al., 1997), as well as a reduction in tissue resident cell activation (Daverey & Agrawal, 2016; Schnegg et al., 2012; Wang et al., 2017a, 2017b; Zhang et al., 2017b). In addition, a SOD knockout model demonstrated increased tissue resident cell activation (Fishman et al., 2009). When activated,

microglia and astrocytes are sources of pro-inflammatory cytokines in the CNS through various pathways (i.e., NF-κB and MAPK-AP-1 signaling pathway). Likewise, tamoxifen (Liu et al., 2010), retinoic acid (Van Neerven et al., 2010) and the JNK inhibitor SP-600125 (Ramanan et al., 2008) were able to reduce the activation of tissue resident cells, suggesting moderate essentiality of tissue resident cell activation to produce the pro-inflammatory cytokines. Multiple studies demonstrate that a reduction in DNA strand breaks could ameliorate abnormal neural remodeling and signaling pathways alterations. Treatment of irradiated HT22 cells (10 Gy, X-rays) with mesenchymal stem cell conditioned media reversed abnormal neural remodeling by reducing the expression of phosphorylated H2AX (γ-H2AX) and reducing apoptosis, and reversed alterations of signaling pathways by decreasing the levels of p53, Bcl-2-associated protein X (Bax), cleaved caspase 3 and increasing the levels of B-cell leukemia/lymphoma-2 (Bcl-2) (Huang et al., 2021). Several studies provide evidence to support a causal association between an increase in pro-inflammatory mediators and decreased neurogenesis, but also provide evidence of reduced neuronal signaling, NSC and progenitor cell proliferation, as well as a change in overall neuronal shape. Their essentiality was assessed as moderate, as multiple studies use cytokine receptor antagonists or knockout key receptors to block the effects of IL-1β, TNF-α, and chemokine ligand 2 (CCL2), which have been reported to preserve neuron survival (Chen & Palmer, 2013; Green et al., 2012; Ryan et al., 2013; Wu et al., 2017). Additionally, it has been demonstrated that inflammatory mediators could influence the normal behavior of neuronal cells and their functional connection through decreased neurogenesis, synaptic complexity and increased necrosis and demyelination, ultimately impairing learning and memory. There is moderate evidence supporting that an alteration in KE<sub>upstream</sub> attenuates the detrimental effects seen in cognitive function. Treatments with anti-inflammatory drugs (MW-151), ethyl eicosapentaenoic acid (E-EPA), 1-[(4-nitrophenyl) sulfonyl]-4-phenylpiperazine (NSPP) (Bhat et al., 2020; Jenrow et al., 2013; Taepavarapruk & Song, 2010), or hormones (alpha-melanocyte-stimulating hormone (α-MSH)) (Gonzalez et al., 2009) have influenced the effects of pro-inflammatory mediators and rescued the impairment seen in learning and memory.

# 7 | MODULATING FACTORS

The relationship between energy deposition and learning and memory impairment could be influenced by several factors. One of the most studied are antioxidants, which catalyze the conversion of RONS into non-toxic molecules, and an imbalance in RONS and antioxidant levels could lead to oxidative stress. Increased antioxidants reduce oxidative stress, the subsequent DNA strand breaks (El-Missiry et al., 2018; Liu et al., 2013), alterations to glial cell activation and signaling pathways, and thus decrease impairment to learning and memory (Demir et al., 2020; Ismail & El-Sonbaty, 2016; Manda et al., 2007a, 2007b, 2008). Age and sex are also modulators of this AOP as older, aged models have lower levels of antioxidants, greater tissue resident cell activation, increased sensitivity to immune signals and inflammation,
<figure><img src="image_17.png" /></figure>

as well as greater decrements to radiation-related impairment in learning and memory (Barrientos et al., 2009, 2012; Casciati et al., 2016; Hanslik et al., 2021; Liguori et al., 2018; Patterson, 2015). In terms of sex, male animals demonstrated greater increases in tissue resident cell activation and learning and memory impairment, whereas female animals were not impacted, although not all studies reported similar findings (Krukowski et al., 2018; Parihar et al., 2020; Raber et al., 2019). Drugs are another modulator as several drugs could reduce the activation of tissue-resident cells and levels of pro-inflammatory mediators, resulting in the rescue of cognitive impairment (Bhat et al., 2020; Gonzalez et al., 2009; Jenrow et al., 2013; Komatsu et al., 2017; Kyrkanides et al., 2002; Liu et al., 2010; Ramanan et al., 2008; Taepavarapruk & Song, 2010; Tan et al., 2014; Van Neerven et al., 2010). Lastly, genetics plays a role in the development to the AO. For example, a polymorphism that increases the expression of the apolipoprotein E4 (APOE4) gene increases the risk of developing Alzheimer's disease, which generally consists of a decline in memory, executive function, and language abilities (Hunsberger et al., 2019; Zhu et al., 2012). A lifestyle of chronic psychological stress related to perceived risk of radiation exposure can also impact learning and memory (Collett et al., 2020).

## 8 | UNCERTAINTIES/INCONSISTENCIES AND KNOWLEDGE GAPS

There are several notable uncertainties/inconsistencies and knowledge gaps (summarized in Table 4) related to this AOP as described in the following section:

1. The AOP is intended for use in humans. However, the following KERs lack empirical support from human studies: energy deposition leading to abnormal neural remodeling, oxidative stress leading to altered stress response signaling, increased pro-inflammatory mediators leading to abnormal neural remodeling, and abnormal neural remodeling leading to impairment of learning and memory. The neocortex in mammals is involved in higher cognitive functions, and the principles of learning and memory are similar in all mammals, and thus our understanding of these aspects of brain development comes largely from animal models. However, genome-wide approaches have identified a small number of human-specific genes important for learning and memory such as Kallikrein-related peptidase 8 (KLK8) that is expressed only in humans (not in non-human primates) and produces human-specific type II KLK8 (Mella et al., 2020). Other differences between human and animal models related to the neuron number include the emergence of the outer subventricular zone in primates which may also drive differences in brain size and function compared with humans (Dehay et al., 2015). Therefore, despite the importance of findings in animal models, there is still a knowledge gap on how radiation exposure alters the human brain, leading to impaired learning and memory (Desai et al., 2022).

2. Empirical evidence supporting tissue resident cell activation following an increase of oxidative stress is inferred exclusively from gamma radiation studies. A knowledge gap exists regarding the impact of other forms of exposure.

3. Some data revealed that increased pro-inflammatory mediators had contradictory effects on abnormal neural remodeling (Borsini et al., 2015). For example, hyper-IL-6, a fusion of IL-6 and IL-6 receptor, was found to increase neurogenesis through the activation of the MAPK/CREB cascade (Islam et al., 2009), while in most studies, an increase in pro-inflammatory mediators has been associated with decreased neurogenesis.

4. The KE of abnormal neural remodeling has a wide range of endpoints. Abnormal neural remodeling encompasses changes to the physical and/or electrophysiological properties of neurons. Several endpoints are usually measured/analyzed for the KE such as demyelination, neurodegeneration, levels of neurogenesis, synapse formation/remodeling, synaptic activity, and dendritic complexity (spine number and density). Variations between protocols in different studies are the main source of inconsistency. The scope of abnormal neural remodeling could be refined and more precisely delineated in terms of specific endpoints once a definitive mechanism is identified.

5. In rodents, a variety of tests have been used to probe learning and memory. Under similar exposure conditions, tests may report contradictory results. Belarbi et al. (2013) did not find any changes in NOR performance after exposure to 10 Gy gamma rays. Forbes et al. (2014) reported that 40 Gy leads to impaired ability during NOR tests, but not during NOL tests. Kiffer et al. (2019a) reported impaired ability during NOR tests, but not during Y-maze tests after 0.1 and 0.25 Gy ¹⁶O particles. Furthermore, quantitative translation of experiments that test learning and memory in animal models to humans has not been defined.

6. A large amount of uncertainty surrounds the impact of low-dose IR on the occurrence of KEs, especially tissue resident cell activation and oxidative stress. Several studies found unexpected effects of low-dose radiation on oxidative stress. For example, low-dose neutron radiation increased the activity of antioxidant enzymes glutathione (GSH) and SOD, and the concentration of malondialdehyde, a product of oxidative stress, decreased (Chen et al., 2021). More evidence is required to determine the relationship between IR at doses <1 Gy and tissue-resident cell activation.

7. Higher levels of RONS within cells can deplete antioxidants, therefore some studies that use high doses of radiation observed declined antioxidants, while lower doses of radiation show the increased expression of antioxidant processes to eliminate the RONS.

8. There are many studies that investigate the MIE of deposition of energy. However, there are no studies that simultaneously include all of the KEs in the same analysis. The supporting evidence from this AOP comes from information collected from multiple different models, time points and doses.
<table><caption>TABLE 4 Identified knowledge gaps and proposed future directions.</caption><thead><tr><th>Research-lines</th><th>Knowledge gap</th><th>Details</th><th>Future directions</th></tr></thead><tbody><tr><td>Endpoint assessment</td><td>Defining the scope of endpoints related to KE#2098: increase, abnormal neural remodeling.<br>Studies that examine all KEs proposed in the AOP.</td><td>Identifying clear criteria for assessment of neural synaptic formation/activity, dendritic spine number and complexity, demyelination, neurodegeneration and levels of neurogenesis.<br>Most studies supporting the AOP assess a maximum of two to three endpoints (Figure 2).</td><td>Establish standardized test methods for assessment of abnormal neural remodeling endpoints.<br>Simulation/computer-based modeling and experimental studies in relevant models assessing all KEs.</td></tr><tr><td>Additional KEs</td><td>Strengthening KEs that lacked empirical evidence.</td><td>KEs considered but not included in the final AOP:<ul><li>Altered blood-brain barrier permeability</li><li>Inadequate repair of DNA</li><li>Oxidative damage to DNA</li></ul></td><td>Design experiments to assess causality of the additional KEs in context of the AOP.</td></tr><tr><td rowspan="3">Improvements in data consistency</td><td>Confirmation on the directionality (increase/ decrease) of certain KEs to AO.</td><td>Many reported inconsistencies in pro-inflammatory mediators leading to neurogenesis. Primary studies report both increase and decrease in pro-inflammatory mediators leading to the AO.</td><td>More research could be directed to understanding the role of proinflammatory mediators in neurogenesis and the relationship between pro-inflammatory mediators and the AO.</td></tr><tr><td>Lack of consistent results from tests used to assess learning and memory.</td><td>Under similar exposure conditions, tests may report contradictory results. Studies report KEs leading to both impaired learning and memory as well as no changes.</td><td>Design statistically relevant studies that systematically examine variables related to learning and memory under controlled conditions.</td></tr><tr><td>Harmonized data reporting</td><td>Inconsistency in data reporting and evaluation between studies.</td><td><ul><li>Co-ordination of experiments that are more informative</li><li>Development of standardized guidelines for reporting, evaluating and presenting data</li></ul></td></tr><tr><td rowspan="2">Understanding modulators</td><td>Age differences</td><td>Most studies researched adolescent and adult rodents.</td><td>Design studies using preadolescent models.</td></tr><tr><td>Sex differences</td><td>Most studies used male models.</td><td>Design studies to examine female models.</td></tr><tr><td>Stressors</td><td>Lack of combined exposures relevant to space.</td><td>For specific KERs, there was limited data from protons and electrons, alpha particles and heavy ions (Figure 4):<ul><li>KER #2841</li><li>KER #3315</li><li>KER #2835</li><li>KER #2833</li></ul>Limited data from altered gravity (Figure 4):<ul><li>KER #2835</li><li>KER #2833</li></ul></td><td>Exposures relevant to the space environment that examine multiple stressors strengthen the evidence for KERs.</td></tr><tr><td rowspan="2">Strengthening Bradford Hill criteria</td><td>Dose concordance</td><td>Lack of studies that performed a thorough dose-response analysis for certain KERs:<ul><li>KER #2835</li><li>KER #2833</li></ul></td><td>Studies designed to examine dose concordance across multiple endpoints proposed in the AOP.</td></tr><tr><td>Time concordance</td><td>Low number of studies that investigate a range of timepoints across certain KERs:<ul><li>KER #2833</li><li>KER #2811</li><li>KER #3316</li></ul></td><td>Studies designed to assess a multitude of time points across KERs in the AOP.</td></tr><tr><td></td><td>Incidence concordance</td><td>Improve the understanding of incidence concordance:<ul><li>KER #2835</li><li>KER #2833</li></ul></td><td>Explore all endpoints of the KER to strengthen incidence concordance.</td></tr><tr><td></td><td>Essentiality</td><td>Strengthen the evidence for essentiality:<ul><li>KER #2836</li><li>KER #3315</li></ul></td><td>Further research supporting the causal relationship between KERs.</td></tr><tr><td>Taxonomic applicability</td><td>Lack of empirical evidence using human models</td><td>Enrich KERs which lack data from human studies:<ul><li>KER #2837</li></ul></td><td>Design new experiments around KERs that lack human data.</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 4 (Continued)</caption><thead><tr><th>Research-lines</th><th>Knowledge gap</th><th>Details</th><th>Future directions</th></tr></thead><tbody><tr><td>Biological levels of organization</td><td>Validated test methods to understand connectivity of KEs from cell to organ level.</td><td>Most studies examine macromolecular-level events, and endpoints were only assessed at the cell-level, thus presenting a difficulty to demonstrate connectivity from cell to organ (Figures 2 and 3).</td><td><ul><li>Future studies should consider organoid models or lab-on-a-chip approaches.</li><li>Quantitative models could be developed that aid in translation of learning and memory tests from animal models to relevant human endpoints.</li></ul></td></tr><tr><td>Strengthening KERs</td><td>Improve quantitative understanding of KERs.</td><td>KERs with low quantitative understanding (Figure 3):<ul><li>KER #3315</li><li>KER #3321</li><li>KER #2835</li><li>KER #2841</li></ul></td><td>Explore patterns in relationships between KEs.</td></tr></tbody></table>

Abbreviations: AO, adverse outcome; AOP, adverse outcome pathway; KE, key event; KER, key event relationship; LET, linear energy transfer; NT2, teratocarcinoma cell line; PC12, derived from a rat adrenal medullary tumor (pheochromocytoma); SK-N-MC, human neuroblastoma cell line; KERs, KER #2811: increase oxidative stress to increase DNA strand breaks; KER #2833, increase oxidative stress to tissue resident cell activation; KER #2835, increase pro-inflammatory mediators to increase abnormal neural remodeling; KER #2836, increase abnormal neural remodeling to impairment, learning and memory; KER #2837, energy deposition to increase abnormal neural remodeling; KER #3321, altered stress response signaling to increase abnormal neural remodeling; KER #2841, increase DNA strand breaks to increase, abnormal neural remodeling; KER #3315 DNA strand breaks to altered stress response signaling; KER #3316, increase oxidative stress to altered stress response signaling.

9. Some evidence was generated from cells from other physiological systems that may respond differently compared to the nervous system. Neurons and other CNS cells have tissue-specific effects, and not every cell type is equally susceptible to radiation effects.

10. Most of the studies lack an extensive dose-response analysis. A lack of dose-response analysis leads to difficulties in describing concordance across multiple doses and deriving threshold values necessary for an effect if cognitive effects are classified as tissue reactions (formerly called deterministic effects) (Hamada & Fujimichi, 2014).

11. Improving the accuracy of quantifying/measuring energy deposition.

12. Inflammatory markers can have both anti-inflammatory and pro-inflammatory roles. Factors such as concentration, timing, and the specific microenvironment can influence whether a mediator acts as pro-inflammatory or anti-inflammatory (Lawrence & Gilroy, 2007; Nathan & Ding, 2010; Opal & DePalo, 2000).

13. The utilization of diverse assays to assess KEs may result in variations in the quantitative interpretation of observations across studies.

14. Limited data is available to support an understanding of oxidative stress and pro-inflammatory mediators at low doses <0.1 Gy.

# 9 | OVERALL ASSESSMENT

## 9.1 | Biological plausibility: high

*Rationale:* Considering the current literature and mechanistic understanding of effect of IR, the oxidative stress, and the induction of the AO, the biological plausibility of the AOP network is strong. There is high biological plausibility that when energy reaches a cell it reacts with water and

organic materials to produce ROS and induce DNA strand breaks. Many studies have reported a strong positive correlation between the level of ROS production and the increase in DNA strand breaks, the production of pro-inflammatory mediators and alteration of stress response signaling pathways. Research reveals that the three KEs can lead to abnormal neural remodeling, which in turn is strongly linked to impaired learning and memory. Sufficient data show changes in neurogenesis, neurodegeneration, neuronal excitability and synaptic plasticity, demyelination, and dendritic spine density culminating in impairment of learning and memory.

## 9.2 | Essentiality of the KEs: high

The essentiality of the MIE and the KE<sub>downstream</sub> was evaluated to be high overall through studies that attenuated the dose of the stressor to block effects. Several experiments demonstrate no changes in oxidative stress, inflammatory response and neural function or structure in the absence of radiation exposure. For KEs not linked to the MIE, essentiality was demonstrated through treatment with chemical inhibitors or knockout genes. Several studies provided evidence to support a causal association between the KE<sub>downstream</sub> and KE<sub>upstream</sub> (Bhat et al., 2020; Daverey & Agrawal, 2016; Gonzalez et al., 2009; Jenrow et al., 2013; Schnegg et al., 2012; Taepavarapruk & Song, 2010).

## 9.3 | Empirical evidence supporting the KERs: moderate

The empirical evidence supporting the KERs in this AOP was rated as moderate. Overall, many studies demonstrated that KE<sub>upstream</sub>
<figure><img src="image_18.png" /></figure>

occurred at lower or the same doses and at earlier or the same times as KE<sub>downstream</sub>. Among the KERs, the KER deposition of energy to oxidative stress [KER #2769 in the AOP-Wiki], had the highest empirical support. The evidence collected to support KERs was gathered from various studies using in vitro and in vivo rat, mice, rabbit, squirrel, bovine, and human models. Various stressors were applied, including ultraviolet (UV), UVB, UVA, gamma rays, X-rays, protons, neutrons, <sup>56</sup>Fe, and <sup>12</sup>C heavy ion irradiation. Studies using a wide range of doses were also collected. Doses as low as 5, 30, and 50 cGy were found sufficient to elicit a response and to establish a KER. Overall, dose and time concordance are unclear as there is limited data that describes KE<sub>upstream</sub> occurring at lower doses or before KE<sub>downstream</sub>.

## 9.4 | Quantitative understanding: low

Quantitative understanding describes whether the magnitude or probability of a KE<sub>downstream</sub> could be predicted by the magnitude or probability of an upstream KE (OECD, 2021). In general, quantitative understanding for direct KERs is difficult to discern, as studies normally do not examine two endpoints concurrently. Three KERs have a moderate amount of evidence surrounding a quantitative understanding (deposition of energy (MIE) → oxidative stress (KE #1392), deposition of energy (MIE) → increased DNA strand breaks (KE #1635), and deposition of energy (MIE) → tissue resident cell activation (KE #1493)); however, the current AOP has low quantitative evidence supporting the KERs overall. It was difficult to compare results and identify a trend as each study uses different models, stressors, doses and time scales. No trend or mathematical model has been established between the KEs. Only a qualitative or semi-quantitative prediction of the change in KE<sub>downstream</sub> could have been determined from a measure of KE<sub>upstream</sub>. Completing experiments with additional levels of dose- and time-concordance across multiple endpoints will improve the quantitative understanding of the relationships, which could support the development of risk models and tools for mitigating risk.

## 10 | POTENTIAL APPLICATIONS

The AOP has been developed specifically to integrate data, in particular those provided by high-throughput screening, exposure data, human data and those from alternative methods, in vitro and in silico. The AOP includes studies from multiple species at multiple life stages and IR exposures at different doses, dose rates, and radiation qualities. The information from this AOP could then be used for expansion to other cognitive disorders and diseases from an MIE of deposition of energy. Importantly, the AOP could be updated with the emergence of new evidence.

The strongest point of this AOP is the systematic collection and evaluation of evidence, especially with the high support of biological plausibility and empirical evidence to the KERs. Although a transparent and documented approach was used to identify evidence, risk-

of-bias evaluation was not undertaken and not a requirement of the OECD guidance document. This is predominantly due to the broad scope of data across levels of biological organization. However, this could be a consideration for future AOPs.

There are multiple considerations for potential applications of the AOP. The CNS could be exposed to IR in multiple events, including occupational exposures, accidental exposures, radiotherapy, and space travel; therefore, understanding its impact on CNS structure and function is essential. The current AOP identified a knowledge gap in quantitative evidence supporting the KERs, information that could be used to plan experiments to strengthen to the dose concordance data across KERs in the AOP. Furthermore, the proposed modulating factors, particularly those relevant to KERs at the macromolecular level, could be tested further as potential countermeasures for learning and memory impairment. Broadly, the AOP presented could serve as an integral component in the consideration of reclassifying health outcomes attributed to radiation exposure. Through evidence-based understanding of the associated risks and outcomes, the AOP structured framework could aid regulatory bodies and international governing bodies in re-evaluating and potentially refining the classification of health effects related to radiation exposures.

### AUTHOR CONTRIBUTIONS

V. C. conceptualized and managed the project. D. F. and J. K. conducted the literature review and development of the first draft of the AOP package. A. S. and K. M. led writing of the report. R. H., S. J. W., J. H., C. A., N. H., C. Y., and R. W. provided expert opinions during AOP development. All authors reviewed and finalized the report.

### ACKNOWLEDGMENTS

The authors are grateful to Drs. Sami Qutob and Chunsheng Li for their critical review of the manuscript. This work was supported by the Health Canada Solutions Fund, the Canadian Space Agency, and the Genomics Research and Development Initiative. This work was also supported by the NASA Langley Research Center Cooperative Agreement 80LARC17C0004 (KBM). Open Access funding provided by the Health Canada library.

### CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

### ORCID

Kathleen B. Miller https://orcid.org/0000-0003-2878-3978

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Sleiman, A., Miller, K.B., Flores, D., Kuan, J., Altwasser, K., Smith, B.J. et al. (2024) AOP report: Development of an adverse outcome pathway for deposition of energy leading to learning and memory impairment. *Environmental and Molecular Mutagenesis*, 65(Suppl. 3), 57–84.
Available from: https://doi.org/10.1002/em.22622