AOP ID and Title:

Short Title: Deposition of energy leads to vascular remodeling

Authors

Tatiana Kozbenko^1,2, Nadine Adam, Veronica Grybas¹, Benjamin Smith¹, Dalya Alomar¹, Robyn Hocking¹, Janna Abdelaziz³, Amanda Pace³, Carole Yauk², Ruth Wilkins¹, Vinita Chauhan¹

(1) Health Canada, Ottawa, Ontario, K1A 0K9, Canada  

(2) University of Ottawa, Ottawa, Ontario K1N 6N5, Canada 

(3) Carelton University, Ottawa, Ontario K1S 5B6, Canada 

Consultants

Marjan Boerma¹, Omid Azimzadeh², Steve Blattnig³, Nobuyuki Hamada⁴

(1) University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

(2) Federal Office for Radiation Protection (BfS), Section Radiation Biology, 85764 Neuherberg, Germany

(3) NASA Langley Research Center Hampton, VA  23681, USA

(4) Biology and Environmental Chemistry Division, Sustainable System Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Tokyo, Japan

Status

Abstract

The present qualitative AOP (AOP#470) summarizes the evidence for a progression beginning with the deposition of energy from exposure to radiation to vascular remodeling. The pathway is initiated by ionization events from the deposition of energy (MIE: Event #1686). The resultant water radiolysis creates an environment of reactive oxygen species (ROS), if this occurs at a rate that outpaces the antioxidant defense system, oxidative stress ensues (KE: Event #1392). Deposition of energy can concurrently induce DNA strand breaks (KE: Event #1635) either directly or through damage from ROS. Excessive ROS damages cellular compartments, therebyaltering signaling pathways (KE: Event #2066), and increasing levels of pro-inflammatory mediators (KE: Event #1493). Within the vascular wall, activation of certain signaling molecules can alter nitric oxide (NO) levels (KE: Event #2067). All the upstream KEs of the pathway then converge to cause endothelial dysfunction (KE: Event #2068). Modified levels of NO can alter the blood flow within the endothelium resulting in subsequent compensatory vascular remodeling (AO: Event #2069). Vascular remodeling is an important precursor for many diverse cardiovascular pathologies and serves as an important marker for cardiovascular disease. Vascular remodeling includes many structural changes such as increased vessel stiffness, vessel wall thickening, and decreased capillary density. Studies informing this AOP include clinical follow-up studies of radiotherapy patients, epidemiological cohort studies of atomic bomb survivors and nuclear plant workers as well as biological studies using mouse and rat models. Knowledge gaps in the weight of evidence include inconsistencies in NO evaluation, and relatively few studies exploring chronic and low dose exposures including the lack of studies focusing on female biology. 

Background

Cardiovascular disease (CVD) includes any health condition affecting the heart and blood vessels. CVD is one of the leading causes of death worldwide, accounting for millions of deaths yearly and surpassed in some countries by only cancer (Bray et al., 2021; Tsao et al., 2022). This class of diseases includes congenital defects, as well as CVDs that can develop throughout life such as peripheral artery disease (PAD), atherosclerosis, coronary artery disease and myocardial infarction. While the progression to a CVD outcome can take significant time, many CVDs are often preceded by much earlier changes to vascular structure. Vascular remodeling entails various structural changes of existing vasculature arising from cell death, cell migration and changes to the endothelial cell membrane. It is important to note that changes to vascular structure are not inherently detrimental, and the cardiovascular system undergoes continuous adaptation to protect vascular health (Pries et al., 2001; Santamaría et al., 2020; Zakrzewicz et al., 2002). However, certain remodeling can also serve as an important marker and risk factor for future adverse cardiovascular events (Cohn et al., 2004; Van Varik et al., 2012). Changes to vascular structure can be triggered through various perturbations such oxidative stress, inflammation, and alterations to various cellular signaling pathways. Adverse remodeling of the vasculature encompasses structural and functional changes to vessel wall intima-media, elevated stiffness, and decreased lumen diameter which are all predictive of the development of and mortality and morbidity from CVD (Heald et al., 2006; Hodis et al., 1998; Polak et al., 2011; Zieman et al., 2005).  

The risk of CVD increases with several factors such as age. There is significant evidence suggesting that environmental factors such as radiation can contribute to the risk of development (Belzile-Dugas & Eisenberg, 2021; Boerma et al., 2016; Francula-Zaninovic & Nola, 2018; Wang et al., 2019). The deposition of energy from radiation exposure is a stochastic event and any part of the human body can be affected, with adverse effects emerging years or decades after the exposure (Boerma et al., 2016; Dörr, 2015; EPRI, 2020; Menezes et al., 2018). The effects of high-dose radiation on the cardiovascular system have been well-characterized while the effects of low-dose exposure are more contended. However, growing evidence suggests that lower doses than previously thought are linked to subsequent adverse cardiovascular outcomes (Boerma et al., 2016; EPRI, 2020; Little et al., 2021; UNSCEAR, 2008). Much of the high-dose data come from follow up studies in radiotherapy patient cohorts who are shown to be at elevated risk for adverse cardiovascular events (Zou et al., 2019). In addition to clinical exposure scenarios, epidemiological studies of occupational exposures and Japanese atomic bomb survivors have also been conducted. Cohort studies of atomic bomb survivors demonstrate various ways in which CVD risk is affected by exposure parameters, including factors such as age at exposure and estimated dose received (Ozasa et al., 2012; Preston et al., 2003; Shimizu et al., 2010; Takahashi et al., 2017). Long-term follow up of individuals exposed in the Chernobyl disaster region also identified statistically significant elevation in CVD risk (Ivanov et al., 2006; Kashcheev et al., 2017). Occupational exposure studies have also been conducted in various countries in an effort to understand the relationship between low-dose chronic exposure and cardiovascular health of nuclear workers (Azizova et al., 2018; Gillies et al., 2017; Zielinski et al., 2009). Occupational exposure studies suggest positive associations between received dose and excessive relative risk of circulatory diseases (Zielinski et al., 2009), CVD mortality (Gillies et al., 2017) and occurrence of ischemic and cerebrovascular disease involving the blood vessels (Azizova et al., 2018).  

Beyond earth, space travel presents an additional radiation exposure scenario. With future missions planned beyond low Earth orbit and the protective shield of the magnetosphere, understanding the unique challenges of space radiation is crucial for protection of travelers. In space, radiation is present in the form of high linear energy transfer (LET) particles and high mass, high energy ions (HZE) which indiscriminately impacts the whole body at a low fluence rate (Baker et al., 2011; Durante & Cucinotta, 2008; Norbury et al., 2016). While the present AOP includes an MIE focused on deposition of energy following radiation exposure, it is important to note that the space exposome contains multiple stressors to which space travelers will be exposed simultaneously. Particularly important, in the case of the cardiovascular system, is microgravity. The cardiovascular system is gravity sensitive, with the endothelial layer being responsive to changes in shear stress and blood pressure (Hughson et al., 2018; Maier et al., 2015; Versari et al., 2013). Variation to the pressure gradient throughout the body can also trigger regional adaptations to vascular structure (L. F. Zhang, 2013). While determining a mechanism for an MIE of microgravity has proven challenging, microgravity exposure has been shown to contribute to other KEs in the pathway and evidence from microgravity studies has been included in the weight of evidence (WOE).

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Overall Assessment of the AOP

Summary of evidence (KE & KER Relationships and evidence)  

Vascular remodeling was selected as the AO due to its implications in CVD. The AOP is supported by high biological plausibility and moderate empirical evidence. Research, primarily laboratory studies, has shown dose- and temporal-response relationships for each KER.  

Biological Plausibility  

Described below is the well-established understanding of the mechanisms underlying this AOP with supporting literature. More detailed examples of the empirical data can be found in the individual entries for each KER. 

It is well accepted that when energy is deposited in the cell from ionizing radiation (IR), direct damage to cellular structures can occur (Desouky et al., 2015). When traveling through a cell, IR can induce the radiolysis of water forming reactive oxygen species (ROS). Deposition of energy can also induce feedback loops of ROS production where structures and molecules damaged by ROS including the mitochondria and NADPH oxidase (NOX) further produce ROS (Mittal et al., 2014; Soloviev & Kizub, 2019). Additionally, deposited energy can directly upregulate enzymes involved in ROS and reactive nitrogen species (RNS) (collectively RONS) production (de Jager, Cockrell and Du Plessis, 2017). If reactive nitrogen species RONS production outpaces the antioxidant defense, a state of oxidative stress occurs (Fletcher et al., 2010; Slezak et al., 2017; Tahimic & Globus, 2017; Wang et al., 2019). Damage to macromolecules can occur due to oxidative stress, including strand breaks to DNA, oxidation of amino acid residues in proteins and peroxidation of lipids (Ping et al., 2020). Lipid peroxidation can induce further damage to cellular structures as a chain reaction is created by the lipid peroxidation radicals, attacking other lipids, proteins and nucleic acids (Ping et al., 2020). Consequently, oxidative stress can directly lead to multiple downstream KEs including altered signaling pathways, increased DNA strand breaks, increased pro-inflammatory mediators and altered nitric oxide (NO) levels. 

DNA strand breaks in endothelial cells can be induced either directly through energy deposition or indirectly through oxidative stress. DNA strand breaks can recruit and activate the protein kinases ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR) (Nagane et al., 2021). Downstream signaling pathways involved in cell death and senescence like the p53/p21 pathway can be activated by ATM/ATR. Furthermore, DNA strand breaks induced by radiation directly or through oxidative stress can cause mutations or changes in transcription of proteins in signaling pathways (Ping et al., 2020; Schmidt-Ullrich et al., 2000). Therefore, DNA strand breaks will induce death and senescence of endothelial cells through altered signaling, resulting in endothelial dysfunction. 

Oxidative stress can also induce altered signaling pathways. The effects of oxidative stress on signaling pathways occur through protein oxidation of signaling components (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Oxidation of cysteine and methionine residues, which are particularly sensitive to oxidation, can result in structural and functional detriments to the protein (Ping et al., 2020). RONS can influence various pathways including the Akt/PI3K/mTOR pathway, where impaired cell survival signaling can induce cellular senescence (Hassan et al., 2013; Ping et al., 2020). Additionally, inhibition of tyrosine phosphatases by ROS can increase the phosphorylation of mitogen-activated protein kinase (MAPK) pathways, resulting in various downstream effects (Schmidt-Ullrich et al., 2000; Valerie et al., 2007). A phosphorylated p53 induced by oxidative DNA damage can also activate MAPK signaling and initiates a cascade ending in apoptosis (Ashcroft et al., 1999; Gen, 2004). Through affecting cell signaling pathways, damage caused by elevated RONS affects cells beyond those that have been directly irradiated (Ramadan et al., 2021). 

Excessive RONS produced by IR disrupt cellular balance and can increase pro-inflammatory mediators (Lumniczky et al., 2021; Schaue et al., 2015). Similar to activation of the immune system by damage from a pathogen, activation by oxidative stress promotes many repair mechanisms, some of which involve rapid release of pro-inflammatory cytokines (Stanojković et al., 2020). The cytokines released vary based on tissue type and radiation parameters (Di Maggio et al., 2015), but tumor necrosis factor (TNF)-α and interleukin (IL)-1 can trigger a cytokine cascade that initiates an inflammatory response (Slezak et al., 2017; Srinivasan et al., 2017). A prolonged state of inflammation in endothelial cells can lead to endothelial dysfunction (Baran et al., 2021). 

Both oxidative stress and altered signaling can directly result in altered NO levels. NO is synthesized from L-arginine by the three nitric oxide synthase (NOS) enzymes, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). ROS can directly reduce NO levels by reacting with NO to produce the RNS peroxynitrite (Deanfield et al., 2007). Furthermore, the cofactor of NOS enzymes, tetrahydrobiopterin (BH4), can be oxidized by RONS leading to inhibition of NOS dimerization, also called NOS uncoupling (Deanfield et al., 2007). Uncoupled NOS will produce superoxide instead of NO, leading to a positive feedback loop of ROS production and reduced NO (Förstermann, 2010; Förstermann & Münzel, 2006; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019). 

Modulation of NO through altered signaling pathways occurs through changing the activity of NOS enzymes. Phosphorylation of eNOS at Ser1177 will activate the enzyme while phosphorylation at Thr495 inhibits it (Förstermann, 2010; Nagane et al., 2021). Protein kinase B (Akt), part of the phosphoinositide 3-kinase (PI3K)/Akt pathway, can activate eNOS through phosphorylation at Ser1177 to increase NO production (Karar & Maity, 2011). In contrast, activation of the RhoA/Rho kinase (ROCK) pathway will inhibit NO production by destabilizing eNOS mRNA and preventing Ser1177 phosphorylation by Akt (Yao et al., 2010). Angiotensin II (AngII), the end product of the renin-angiotensin-aldosterone system (RAAS), is involved in both downregulating Ser1177 phosphorylation to prevent NO creation (Ding et al., 2020) and activating eNOS as a corrective measure (Millatt et al., 1999). Alterations to these pathways due to IR will result in changes in NO levels. 

Each of the components of the pathway described above converge at endothelial dysfunction. Endothelial cells lining the blood vessels throughout the body are an important component for maintaining vascular homeostasis (Bonetti et al., 2003; Deanfield et al., 2007). Endothelial cells are quiescent with high levels of NO most of the time (Carmeliet & Jain, 2011). Endothelial dysfunction can occur due to prolonged activation of the endothelium, characterized by the prolonged lack of bioavailable NO, lack of endothelium-dependent vasodilation and chronic pro-thrombotic and inflammatory state (Baran et al., 2021; Bonetti et al., 2003; Deanfield et al., 2007; Krüger-Genge et al., 2019). A prolonged reduction in NO will decrease vasodilation, increase leukocyte adhesion and increase fibrous plaque formation contributing to the pro-thrombotic dysfunctional environment (Schiffrin, 2008; Senoner & Dichtl, 2019; Venkatesulu et al., 2018). Furthermore, signaling in pathways like p53/p21 or PI3K/Akt/mammalian target of rapamycin (mTOR) can induce apoptosis or premature senescence of endothelial cells as part of endothelial dysfunction due to DNA damage or oxidative stress (Borghini et al., 2013; Hughson et al., 2018; Schiffrin, 2008; Senoner & Dichtl, 2019; Soloviev & Kizub, 2019). Senescent cells have decreased levels of NO production and a pro-inflammatory secretory phenotype, which feed back to further promote endothelial dysfunction (Ungvari et al., 2013; Wang et al., 2016). 

Endothelial dysfunction subsequently leads to vascular remodeling, which encompasses multiple structural changes to the vasculature. Chronic inflammation combined with impaired healing and lack of endothelium-dependent vasodilation during endothelial dysfunction increases vulnerability to damage from non-laminar flow and maladaptive repair (Sylvester et al., 2018). As compensation, vessel walls can thicken and atherosclerotic risk can increase (Hughson et al., 2018; Slezak et al., 2017; Sylvester et al., 2018). In cases of maladaptive repair of vessels, vascular remodeling can be exhibited through an increase in fibrosis (Hsu et al., 2019). The pro-thrombotic environment with increased lymphocyte adhesion induced by endothelial cell senescence can increase the likelihood of vessel occlusion, decreasing vascular density such that the corresponding increase in vascular resistance will induce remodeling as a compensatory measure (Slezak et al., 2017). Thus, increased leukocyte adhesion during endothelial dysfunction occurs early in the development of atherosclerosis (Senoner & Dichtl, 2019). Increased arterial stiffness can also occur in response to endothelial dysfunction (Boerma et al., 2015, 2016; Patel et al., 2020), with increased collagen and smooth muscle content paired with decreased elastin and degradation of the extracellular matrix (Zieman et al., 2005). The changes to the vascular structure in response to the deposition of energy are similar to a form of accelerated age-related atherosclerosis (Boerma et al., 2016; Sylvester et al., 2018; Vernice et al., 2020). 

Temporal, Dose, and Incidence Concordance  

Evidence for time, dose, and incidence concordance in this AOP is moderate. It has been repeatedly shown using many study designs and systems that deposition of energy occurs immediately following irradiation, and downstream events occur at a later timepoint. Endpoints indicating oxidative stress have been observed within minutes following irradiation (Wortel et al., 2019). Studies show that oxidative stress, increased DNA strand breaks, increased pro-inflammatory mediators, and altered signaling may occur over a similar time period; however, alteration in signaling pathways, increased DNA strand breaks, and increased pro-inflammatory mediators can be observed following oxidative stress (Ramadan et al., 2020; Baselet et al., 2017; Sakata et al., 2015; Yang et al., 1998). Increases in NO levels occur in hours to weeks after irradiation (Azimzadeh et al., 2017; Sonveaux et al., 2003; Sakata et al., 2015). Then, from weeks to months following irradiation both endothelial dysfunction and vascular remodeling occur, though concordance between these events is difficult to determine, possibly due to inter-study differences in experimental design and markers (Yentrepalli et al., 2017; Soucy et al., 2007; Yu et al., 2011; Shen et al., 2018). 

Overall, the majority of studies demonstrate that upstream KEs occur at the same or lower doses and earlier or the same time as downstream KEs. For example, endothelial cells show a dose-dependent increase in oxidative stress to X-ray irradiation at 0.1 and 5 Gy, while 0.1 Gy induced few changes in pro-inflammatory mediators with significant increases only observed at 5 Gy (Ramadan et al., 2020). Some studies also show that the upstream and downstream KEs can be observed at the same doses of radiation. For example, X-ray irradiation of mice resulted in oxidative stress, altered signaling and reduced NO levels at both 8 and 16 Gy (Azimzadeh et al., 2015). Dose concordance is not consistent across studies, but this may be due to differences in models, timepoints, and radiation types used. Many studies only measure the KEs at one dose, and thus cannot be used to evaluate dose concordance.

A limited number of studies support incidence concordance. In these, the upstream KE demonstrates a greater change than the downstream KE following exposure to a stressor. For example, mice exposed to 18 Gy of X-rays showed a roughly 2-fold increases in both oxidative stress and pro-inflammatory markers. A 1.3-fold increase in markers for endothelial dysfunction was observed (Shen et al., 2018).

Uncertainties and inconsistencies  

The collection of WOE identifies several important uncertainties in the literature. These include lack of quantitative understanding, low-dose or chronic-exposure studies, data from female models and consistency in measurement of NO levels.  

The WOE contained data from a wide variety of interdisciplinary fields; consequently, experimental design was equally varied. Overall, studies did not use consistent doses, radiation types, time-points, or evaluation of endpoints. Since dose and type of radiation can affect biological responses, quantitative understanding of relationships could not be determined and was low overall. Additionally, most studies used single or select doses, with limited studies exploring relatively low doses (<0.5 Gy (EPRI, 2020)). Harmonized experiments evaluating changes to adjacent endpoints across a wide range of doses or time-points with consistency of radiation type would greatly benefit quantitative understanding for this AOP.  

Similarly, the WOE is lacking in evidence using female models. Sex is an important modulating factor in cardiovascular changes and studies suggest vascular remodeling responses of astronauts can vary by sex (Hughson et al., 2016). The consequence of the general bias in clinical research (Rios et al., 2020; Yakerson, 2019) from which the current WOE draws, is the very large knowledge gaps in mechanistic data for the female body. Filling these knowledge gaps at all levels of biological organization will be an important step in solidifying the AOP.   

Evaluation of NO levels was inconsistent between studies. According to the biological plausibility, deposition of energy and subsequent oxidative stress would lead to a decrease in NO that then contributes to impaired vascular relaxation as part of endothelial dysfunction. However, primary research concludes that NO can either increase (Abdel-Magied & Shedid, 2020; Hirakawa et al., 2002; Sakata et al., 2015; Sonveaux et al., 2003) or decrease (Baker et al., 2009; Fuji et al., 2016) following irradiation. Proxy measures used to detect NO, like NOS enzyme activities or nitrite/nitrate levels, may not directly correspond to changes in NO levels. Further standardization in NO measurement and interpretation could refine this KE to become the depletion of NO. 

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The empirical evidence supports that this AOP is relevant to human (Hong et al., 2013; Siamwala et al., 2010; Jiang et al., 2020; Lee, et al., 2020; Ramadan et al., 2020), rat (Hatoum et al., 2006; Soucy et al., 2010; Hong et al., 2013; Abdel-Magied & Shedid, 2019: Hasan et al., 2020), mouse (Yu et al., 2011; Coleman et al., 2015; Sofronova et al., 2015; Shen et al., 2018; Hamada et al., 2020), and rabbit (Soloviev et al., 2003, Hong et al., 2013) models. Biological plausibility suggests that events in this AOP are not sex specific; however, more studies used male models. Similarly, while biological plausibility suggests the pathway is not age-specific, most studies used adult models. 

Essentiality of the Key Events

The essentiality of the MIE to a downstream KE is supported by a non-irradiated control. The comparison of irradiated and non-irradiated groups has shown that the effects of downstream events are enhanced or accelerated by the deposition of energy.  

The essentiality of other KEs can be determined by the impact of the manipulation of the upstream KE on the resulting downstream effects. For example, the essentiality of oxidative stress is frequently assessed through antioxidant treatments, which can decrease oxidative stress markers through decreased ROS production or strengthened antioxidant defense activity. SOD administration decreased free radicals, superoxide and peroxide, and improved endothelium-dependent vasodilation, a downstream KE, which had been previously decreased due to radiation exposure (Hatoum et al., 2006). Additionally, oxypurinol treatment inhibited xanthine oxidase (XO) enzyme, which limited the enzyme’s contribution to cardiac ROS and improved endothelium-dependent vasodilation and the recovery of vascular stiffness to control levels (Soucy et al., 2007, 2010, 2011). 

The essentiality of DNA strand breaks was not assessed often. One study used mesenchymal stem cell conditioned media (MSC-CM) to reduce the level of ROS-mediated DNA double-stranded breaks and found decreases in signaling molecules including p53, Bax and cleaved caspase 3 (Huang et al., 2021). 

The essentiality for altered signaling pathways KE was evaluated by studies using pathway inhibitors or conditioned media. Signaling pathways were shown to be suppressed by inhibitors such as ROCK inhibitor Y27632 and acid sphingomyelinase (ASM) inhibitor desipramine (dpm), which have demonstrated decreased apoptosis and recovered endothelium-dependent vasodilation (Soloviev & Kizub, 2019; Venkatesulu et al., 2018; Wang et al., 2016). Incubation of endothelial cells in MSC-CM was shown to increase cell signaling components, Akt and p-Akt, and decrease apoptosis (Chang et al., 2017). PI3K inhibitors, such as LY294002 and wortmannin, and angiotensin-converting enzyme inhibitor bradykinin-potentiating factor (BPF) were studied for their impact on NO levels. The increase in p-Akt and subsequently eNOS, p-eNOS and NO levels were reversed following PI3K inhibition (Shi et al., 2012; Siamwala et al., 2010). AngII and iNOS levels were returned to control following BPF treatment of irradiated groups (Hasan et al., 2020). Further studies are required for a better understanding of the changes in NO levels and endothelial dysfunction due to altered signaling pathways. Overall, the flexibility of signaling pathways makes it difficult to assess essentiality. 

The essentiality for pro-inflammatory mediators was assessed by studies that suppress their expression. The decrease in pro-inflammatory mediators was observed following the use of TAT-Gap19 to block connexin43 hemichannels. This decrease was associated with a decrease in radiation-induced endothelial cell senescence (Ramadan et al., 2020). Additionally, MSC-CM incubation resulted in decreased pro-inflammatory cytokines, IL-1α, IL-6 and TNF-α and decreased endothelial apoptosis (Chang et al., 2017). 

Changes in vascular remodeling were evaluated through vascular structure, among other endpoints. Following hindlimb unloading, ASM inhibition in the small mesenteric artery was found to reverse the changes in apoptosis and intima-media thickness (IMT) (Su et al., 2020). Comparisons between irradiated and sham or non-irradiated control groups of various studies using animal and human models have demonstrated differences in vascular structures (Hamada et al., 2020, 2021; Sárközy et al., 2019; Shen et al., 2018; Sridharan et al., 2020; Yu et al., 2011).

Essentiality of the key events 

	Defining Question	High	Moderate	Low
Support for Essentiality of KEs	Are downstream KEs and/or the AO prevented if an upstream KE is blocked?	Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs	Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE	No or contradictory experimental evidence of the essentiality of any of the KEs
MIE: KE #1689 Deposition of energy	Evidence for Essentiality of KE: High  This event is difficult to test for essentiality as deposition of energy is a physical stressor and cannot be blocked/decreased using chemicals. However, studies show that control or sham-irradiated groups do not show the occurrence of downstream KEs.
KE #1392 Oxidative stress	Evidence for Essentiality of KE: High  Essentiality was well supported within the literature. Antioxidant treatments led to recovery of antioxidant enzyme activity, decreases in DNA strand breaks, and decreases in pro-inflammatory mediators, while ZNO-NP also restored NO levels. Oxypurinol (Oxp) treatment was found to aid in the acetylcholine (ACh) vasodilation response and restore NO levels as it decreased xanthine oxidase (XO) activity and reactive oxygen species (ROS).
KE #1635  Increase, DNA strand breaks	Evidence for Essentiality of KE: Low  Few studies use countermeasures to reduce the number of DNA strand breaks in cells. A few studies show that reducing DNA strand breaks induced by radiation restores signaling pathways and reduces endothelial dysfunction.
KE #1493 Increase, pro-inflammatory mediators	Evidence for Essentiality of KE: Low  Essentiality of this event can be determined with countermeasures that limit the increase of pro-inflammatory mediators. Limited research does show essentiality, evidenced by a decrease in apoptosis of endothelial cells following treatment with MSC-CM, which contains angiogenic cytokines that have therapeutic potential for microvascular injury, and a decrease in endothelial cell senescence following treatment with TAT-Gap19, a connexin hemichannel blocker.
KE #2066  Altered signaling pathways	Evidence for Essentiality of KE: Moderate  Essentiality of this relationship can be determined with the use of signaling molecule inhibitors. Signaling molecule inhibitors reduced downstream changes in eNOS, NO, p-Akt, angiotensin II (AngII) and aldosterone following stressors such as irradiation and altered gravity.  Inhibitors also prevented impaired contractile response and decreased apoptosis in the arterial endothelium.
KE #2067 Altered, NO levels	Evidence for Essentiality of KE: Moderate  The evidence for essentiality of this KE can be determined by using countermeasures that limit changes in NO levels, such as Oxp, L-NA (NOS inhibitor), AG (iNOS inhibitor), DAHP (Gch1 inhibitor) and losartan (AT1 receptor antagonist). Use of these countermeasures reduced NOS levels and decreased the ratio of couple-to-uncoupled eNOS. Endothelial relaxation increased after Oxp and losartan treatment after microgravity exposure, while relaxation decreased in the presence of DAHP, L-NA and AG. When treated with these countermeasures following radiation or microgravity, changes to NO were limited or restored and as a result, endothelial dysfunction was limited.
KE #2068  Increase, endothelial dysfunction	Evidence for Essentiality of KE: Moderate  The essentiality of endothelial dysfunction leading to vascular remodeling is moderately supported within literature. Oxp treatment, an XO inhibitor, restored vasodilator response and reduced vascular stiffness following irradiation. Both dpm and DOX decreased apoptosis and reduced Caspase-3 protein expression. Ceramide treatment following microgravity was found to return proliferation to control levels and increase apoptosis.

Weight of Evidence Summary

	Defining Question	High	Moderate	Low
Review of Biological Plausibility for the KER	Is there a mechanistic (structural or functional) relationship between the upstream KE and downstream KE consistent with established biological knowledge	The relationship is well understood based on extensive previous documentation and has an established mechanistic basis and broad acceptance	The KER is plausible based on an analogy to accepted biological relationships but scientific understanding is not completely established	There is empirical support for a statistical association between KEs but structural or functional relationship between them is not understood
Deposition of energy (MIE: KE #1686) leads to oxidative stress (KE #1392)	Evidence for Biological Plausibility of KER: High   Deposition of energy onto the water and biological components of a cell creates ROS, and as ROS production outpaces the cell’s antioxidant defense system, oxidative stress is induced. Both ROS production and subsequent oxidative stress have been extensively studied and the mechanisms are well described in numerous review articles across many biological systems.
Deposition of energy (MIE: KE #1686) leads to increase, DNA strand breaks (KE #1635)	Evidence for Biological Plausibility of KER: High  The deposition of energy onto the DNA molecule will directly cause single- or double-strand breaks in the DNA. Deposited energy can induce chemical modifications to the phosphodiester backbone of both strands of the DNA, possibly resulting in breaks in one or both strands.
Oxidative stress (KE #1392) leads to increase, DNA strand breaks (KE #1635)	Evidence for Biological Plausibility of KER: High  Increased ROS during oxidative stress can result in the oxidation of bases on the DNA strand, triggering base excision repair, which removes the oxidized bases. When multiple bases in close proximity are removed, the repair efforts cause strain which can lead to strand breaks.
Increase, DNA strand breaks (KE #1635) leads to altered signaling pathways (KE #2066)	Evidence for Biological Plausibility of KER: High  Strand breaks induce the recruitment of the kinases ataxia-telangiectasia mutated (ATM) and ATM/RAD3-related (ATR). ATM and ATR can subsequently phosphorylate multiple downstream signaling molecules. High levels of DNA strand breaks can increase the recruitment of ATM and ATR, leading to greater activation of pathways like the p53/p21 pathway and subsequently greater downstream effects.
Oxidative stress (KE #1392) leads to increase, pro-inflammatory mediators (KE #1493)	Evidence for Biological Plausibility of KER: High  Excess ROS during oxidative stress damages cellular structures and thus activates the immune system and repair mechanisms, many of which involve release of pro-inflammatory mediators. Cells involved with host-defense can themselves also produce ROS, further exacerbating the state of oxidative stress. The biological plausibility of the linkage between oxidative stress and increases in pro-inflammatory mediators is highly supported in literature.
Oxidative stress (KE #1392) leads to altered signaling pathways (KE #2066)	Evidence for Biological Plausibility of KER: High  Oxidative stress can alter signaling pathways both directly and indirectly. Directly, oxidative stress conditions can lead to oxidation of amino acid residues. This can cause conformational changes, protein expansion, and protein degradation, leading to changes in the activity and level of signaling proteins. Oxidation of key functional amino acids can also alter the activity of signaling proteins, resulting in downstream alterations in signaling pathways. Indirectly, oxidative stress can damage DNA causing changes in the expression of signaling proteins as well as the activation of DNA damage response signaling. The mechanisms of this relationship are widely accepted.
Oxidative stress (KE #1392) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Biological Plausibility of KER: High  ROS can interact with NO, taking a vasodilator crucial for endothelial function and turning it into peroxynitrite, a RNS that further contributes to oxidative stress. Furthermore, cellular senescence, inhibition of vasodilation, induced inflammatory environments and cellular apoptosis are all part of endothelial dysfunction that can be indirectly caused by oxidative stress.
Increase, pro-inflammatory mediators (KE #1493) leads to increase,  endothelial dysfunction (KE #2068)	Evidence for Biological Plausibility of KER: High  Inflammation provides a protective effect to the endothelium but prolonged or repeated exposure to a stressor can exhaust this, leading to senescence or apoptosis in endothelial cells and subsequent leading to endothelial dysfunction. This endothelial dysfunction can also manifest as a dysregulation of vasodilation. Prolonged inflammation is a widely accepted component in the development of endothelial dysfunction.
Altered signaling pathways (KE #2066) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Biological Plausibility of KER: High  Signaling pathways including the PI3K/Akt/mTOR, RhoA-Rho-kinase, ASM/cer pathway, and the p53-p21 pathway have downstream effects on endothelial apoptosis, premature endothelial cell senescence and cytoskeletal proteins to impair contraction, indicators of endothelial dysfunction.
Increase, endothelial dysfunction (KE #2068) leads to occurrence, vascular remodeling (AO: KE #2069)	Evidence for Biological Plausibility of KER: High  Key components of endothelial dysfunction include deficiency in bioavailable NO, impaired vasodilation, inflamed endothelium and prothrombotic environment. These events can ultimately lead to vascular remodeling to compensate for decreased capillary and vascular density and increased vascular resistance. Regional pressure changes in vessels due to microgravity can also result in regional changes to vascular structure.
Deposition of energy (MIE: KE #1686) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Biological Plausibility of KER: High  Irradiation can cause cellular and tissue level markers of endothelial dysfunction. Following prolonged exposure to radiation, the protective effect of the endothelium can become exhausted and lead to endothelial dysfunction. Consequently, endothelial cells may lose their integrity and become senescent or apoptotic via alterations to signaling pathways, leading to endothelial dysfunction evidenced by dysregulation of vasodilation. Endothelial dysfunction is commonly considered a hallmark for the development of various cardiovascular pathologies.
Deposition of energy (MIE: KE #1686) leads to occurrence, vascular remodeling (AO: KE #2069)	Evidence for Biological Plausibility of KER: High  Radiation can accelerate the natural processes of vascular remodeling related to aging. An increase in ROS, produced by IR, can reduce NO bioavailability, leading to endothelial dysfunction and vascular stiffness. In addition, the low level of inflammation during early stages of radiation leads to inhibition of tissue and vessel recovery, and later results in intimal thickening and vascular remodeling. Changes in vessel composition, such as collagen content, may also occur from energy deposition and affect vascular remodeling.
Deposition of energy (MIE: KE#1686) leads to altered, NO levels (KE #2067)	Evidence for Biological Plausibility of KER: High  NO is produced by NOS enzymes or by the reduction of nitrite to NO. Deposition of energy can interfere with this process in several ways. Radiolysis of water forms ROS that interacts with NO to produce peroxynitrite which reduces NO bioavailability. ROS can also cause NOS uncoupling, which can reduce NO levels. In contrast, NO can also increase as a result of IR through activation of iNOS during oxidative stress. IR can also influence various signaling pathways that control NO levels, causing radiation to indirectly affect NO levels.
Oxidative stress (KE #1392) leads to altered, NO levels (KE #2067)	Evidence for Biological Plausibility of KER: High  It is thought that excessive ROS production can lead to altered NO bioavailability both through direct interaction and indirectly through decreasing its production. Elevated O2- can interact with NO converting it to peroxynitrite leading to decreased bioavailability. ROS can also oxidize the eNOS cofactor BH4, causing eNOS uncoupling inhibiting NO production. Electron leakage in uncoupled eNOS produces additional ROS, exacerbating the state of oxidative stress.
Altered signaling pathways (KE #2066) leads to altered, NO levels (KE #2067)	Evidence for Biological Plausibility of KER: High  Various pathways are well known to influence NO levels. Some well-studied examples include the PI3K/Akt pathway, the RhoA/ROCK pathway, the RAAS pathway and the acidic sphingomyelinase/ceramide pathway. The PI3K/Akt, RhoA/ROCK and RAAS pathways and their components are involved in the phosphorylation of various eNOS residues affecting the enzymes activation. The activation or deactivation of eNOS affects the levels of NO production. In contrast, the acidic sphingomyelinase/ceramide pathway can activate NADPH oxidase (NOX), leading to the production of ROS that goes on to scavenge NO decreasing its bioavailability.
Altered, NO levels (KE #2067) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Biological Plausibility of KER: High  Lack of bioavailable NO is considered one of the key drivers of endothelial dysfunction. Under normal conditions NO binds with soluble guanylyl cyclase (sGC) creating cGMP and cAMP to activate cellular kinase cascades and Ca2+-dependent vasodilation through smooth-muscle relaxation. Lack of bioavailable NO interrupts this process, reducing the relaxation of smooth muscle cells and dilation of the blood vessels. In contrast, an increase in NO combined with simultaneous excessive ROS can drive cellular senescence through increased peroxynitrite formation. Prolonged impaired vasodilation and elevated premature endothelial cell senescence are important characteristics of endothelial dysfunction.

Quantitative Consideration

Despite biological plausibility and empirical evidence demonstrating the qualitative linkages within the AOP, quantitative understanding is low. As described above, the lack of quantitative understanding of the KERs is due to the diversity in experimental design, including doses tested and radiation types used. The evidence is primarily from laboratory studies that show dose and time response relationships for KEs; however, the strength of the response can vary with factors such as dose-rate, type of radiation, and cell type. Particularly relevant are the relative lack of low-dose studies and exposure scenarios relevant to space radiation. Future work could use the present qualitative AOP to guide experimental design and strengthen quantitative understanding. Standardized studies simultaneously measuring endpoints across several KEs, and across a range of doses and timepoints would be beneficial in filling important gaps in the quantitative understanding.

Deposition of energy (MIE: KE #1686) leads oxidative stress (KE #1392)	Evidence for Quantitative Understanding of KER: High  There is a large amount of evidence supporting how much of a change in the deposition of energy is needed to produce a change in the level of oxidative stress. Several different endpoints representing oxidative stress have been used, including changes in the levels or activity of catalase, GSH, superoxide dismutase, GSH-Px, MDA, and ROS. Measurements have also been made over a large range of doses and dose rates, and changes to oxidative stress levels have been shown to depend on the nature, dose and dose rate of energy deposition.
Deposition of energy (MIE: KE #1686) leads to increase, DNA strand breaks (KE #1635)	Evidence for Quantitative Understanding of KER: High  Studies examining energy deposition leading to strand breaks suggest a positive, linear relationship between these two events. The exact number of strand breaks is difficult to predict from the deposition of energy. The relationship depends on the biological model, the type of radiation, and the dose.
Oxidative stress (KE #1392) leads to increase, DNA strand breaks (KE #1635)	Evidence for Quantitative Understanding of KER: Moderate  There is a considerable amount of evidence showing increased DNA strand breaks following exposure to oxidative stress. However, no model has emerged that predicts the number of DNA strand breaks following oxidative stress. Measurements of oxidative stress vary across studies.
Increase, DNA strand breaks (KE #1635) leads to altered signaling pathways (KE #2066)	Evidence for Quantitative Understanding of KER: Moderate  There is much evidence showing changes in the expression or activity of signaling pathways following increased DNA strand breaks. However, no model has been developed to accurately predict the changes to signaling pathways due to increased DNA strand breaks. Furthermore, the changes to signaling pathways are very context- and cell type-dependent.
Oxidative stress (KE #1392) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Quantitative Understanding of KER: Low  Although studies quantitatively measure both oxidative stress and endothelial dysfunction following a stressor, it is difficult to compare results and identify a quantitative relationship as studies use different models, stressors, doses and time scales. In addition, many factors and pathways can contribute to endothelial dysfunction. Thus, no model has been established to predict the extent of changes in endothelial dysfunction after oxidative stress.
Oxidative stress (KE #1392) leads to increase, pro-inflammatory mediators (KE #1493)	Evidence for Quantitative Understanding of KER: Moderate  Current primary research shows that an increase in oxidative stress will be followed by a more significant increase in pro-inflammatory mediators. A quantitative association between the two KEs is difficult to determine, as multiple positive feedback mechanisms exist between oxidative stress and inflammation.
Increase, pro-inflammatory mediators (KE #1493) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Quantitative Understanding of KER: Low  Although studies reveal increases in markers for endothelial dysfunction in response to increased pro-inflammatory mediators, no quantitative understanding has been established to predict the changes in endothelial dysfunction markers. There are various pro-inflammatory mediators that may contribute to various markers of endothelial dysfunction such as apoptosis and cellular senescence. Studies investigate changes in the levels of different pro-inflammatory mediators and different measures of endothelial dysfunction; therefore, it is difficult to compare the results and identify trends.
Altered signaling pathways (KE #2066) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Quantitative Understanding of KER: Low  Although studies show increases in markers for endothelial dysfunction in response to altered signaling pathways, no quantitative understanding has been established to predict the changes in endothelial dysfunction markers. There are various signaling pathways that may contribute to endothelial dysfunction, including the Akt/PI3K/mTOR pathway, the RhoA-Rho-kinase pathway, and the ASM/cer pathway. Studies investigate changes to the levels of different signaling pathway molecules; therefore, it is difficult to compare the results and identify trends.
Increase, endothelial dysfunction (KE #2068) leads to occurrence, vascular remodeling (AO: KE #2069)	Evidence for Quantitative Understanding of KER: Low  Vascular remodeling is consistently shown with endothelial dysfunction. However, it is difficult to compare results and identify a quantitative relationship as various models, stressors, doses and endpoint measures were used. Thus, no model has been established to accurately predict the changes in vascular remodeling.
Deposition of energy (MIE: KE #1686) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Quantitative Understanding of KER: Low  Studies revealed consistent increases in levels of indicators of endothelial dysfunction such as apoptosis, premature endothelial cell senescence and diminished relaxation response. There is consistent evidence that shows that as the dose increases, the maximum relaxation response decreases. However, more studies are required to quantify this association to show how this relates to levels of cellular markers of apoptosis and senescence.
Deposition of energy (MIE: KE #1686) leads to occurrence, vascular remodeling (AO: KE #2069)	Evidence for Quantitative Understanding of KER: Low  Deposition of energy from IR is consistently demonstrated to drive vascular remodeling. However, it is difficult to compare results and quantify relationships as each study uses different models, stressors, doses and time scales. In addition, many factors and pathways contribute to the components of vascular remodeling. Thus, no model has been established to predict the changes in vascular remodeling after deposition of energy.
Deposition of energy (MIE: KE #1686) leads to altered, NO levels (KE #2067)	Evidence for Quantitative Understanding of KER: Low  Altered nitric oxide levels occur consistently with deposition of energy. However, it is difficult to compare results and determine a quantitative relationship as each study uses different models, stressors, doses and endpoint measures of NO. As well, cancerous cells and normal cells can show different production of NO. Thus, no model has been established to predict the changes in nitric oxide levels at a given dose of IR.
Oxidative stress (KE #1392) leads to altered, NO levels (KE #2067)	Evidence for Quantitative Understanding of KER: Low  Alterations in NO levels cannot be predicted from relevant measures of oxidative stress changes, such as increased ROS production and antioxidant enzyme activity. Nevertheless, a general decrease in NO is observed following ROS production.
Altered signaling pathways (KE #2066) leads to altered, NO levels (KE #2067)	Evidence for Quantitative Understanding of KER: Low  Altered NO, iNOS and eNOS levels occur in response to altered signaling pathways; however, a model has not been established to predict the changes in NO levels. Different models, stressors, time scales, doses and dose rates make trends difficult to identify. The studies investigated the levels of different altered signaling pathway molecules and their effects on NO levels, making it difficult to compare and identify quantitative relationships across the results.
Altered, NO levels (KE #2067) leads to increase, endothelial dysfunction (KE #2068)	Evidence for Quantitative Understanding of KER: Low  Increased vascular tension occurs consistently with decreased NO levels. Although many studies quantitatively measure a change in endothelial function after changes in NO levels, no model has been established. Each study cited used different models, stressors, time scales, doses and dose rates, which makes it difficult to determine if response levels are consistent between studies.

Considerations for Potential Applications of the AOP (optional)

The present AOP serves as a platform to promote broader collaborative efforts to understand non-cancer health risks from radiation exposures. It will be a foundational AOP of regulatory interest to researchers seeking areas of knowledge gaps to prioritize research in understanding mechanisms of CVD. The AOP is also relevant to space agencies and clinicians working to improve the guidance on health risks from long-term spaceflight and radiotherapy treatments, respectively. The WOE for this AOP may also inform parameters in biologically based risk models and can serve to develop countermeasures to protect the cardiovascular systems of future space travelers on deep space missions. The present qualitative AOP can be used to guide the design of experiments that will provide quantitative understanding for the KERs to support risk-model development and inform additional guidelines for radiation protection; additionally, the identified research gaps could help prioritize research needs for funding strategies.

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Appendix 1

List of MIEs in this AOP

Event: 1686: Deposition of Energy

Short Name: Energy Deposition

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

It is well documented that ionizing radiation( (eg. X-rays, gamma, photons, alpha, beta, neutrons, heavy ions) leads to energy deposition on the atoms and molecules of the substrate. Many studies, have demonstrated that the type of radiation and distance from source has an impact on the pattern of energy deposition (Alloni, et al. 2014). High linear energy transfer (LET) radiation has been associated with higher-energy deposits (Liamsuwan et al., 2014) that are more densely-packed and cause more complex effects within the particle track (Hada and Georgakilas, 2008; Okayasu, 2012ab; Lorat et al., 2015; Nikitaki et al., 2016) in comparison to low LET radiation. Parameters such as mean lineal energy, dose mean lineal energy, frequency mean specific energy and dose mean specific energy can impact track structure of the traversed energy into a medium (Friedland et al., 2017). The detection of energy deposition by ionizing radiation can be demonstrated with the use of fluorescent nuclear track detectors (FNTDs). FNTDs used in conjunction with fluorescent microscopy, are able to visualize radiation tracks produced by ionizing radiation (Niklas et al., 2013; Kodaira et al., 2015; Sawakuchi and Akselrod, 2016). In addition, these FNTD chips can quantify the LET of primary and secondary radiation tracks up to 0.47 keV/um (Sawakuchi and Akselrod, 2016). This co-visualization of the radiation tracks and the cell markers enable the mapping of the radiation trajectory to specific cellular compartments, and the identification of accrued damage (Niklas et al., 2013; Kodaira et al., 2015). There are no known chemical initiators or prototypes that can mimic the MIE.

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Energy can be deposited into any substrate, both living and non-living; it is independent of age, taxa, sex, or life-stage.

Taxonomic applicability: This MIE is not taxonomically specific.  

Life stage applicability: This MIE is not life stage specific. 

Sex applicability: This MIE is not sex specific. 

Key Event Description

(Adapted from AOP 272, KE: 1686- in blue)

Deposition of energy refers to events where subatomic particles or electromagnetic radiation deposit energy in the media through which they transverse. The energy may either be sufficient (e.g. ionizing radiation) or insufficient (e.g. non-ionizing radiation) to ionize atoms or molecules (Beir, et al. 1999).

Ionizing radiation can cause the ejection of electrons from atoms and molecules, thereby resulting in their ionization and the breakage of chemical bonds. The energy of these subatomic particles or electromagnetic waves ranges from 124 KeV to 5.4 MeV, and is dependent on the source and type of radiation (Zyla et al., 2020). Not all electromagnetic radiation is ionizing; as the incident radiation must have sufficient energy to free electrons from the atom or molecule’s electron orbitals. The energy can induce direct and indirect ionization events and can be via internal (injections, inhalation, injection) or external exposure. Direct ionization is the principal path where charged particles interact with DNA to cause a biological damage. Photons, which are electromagnetic waves can also cause direct ionization. Indirect ionization produces free radicals of other molecules, specifically water, which can transform to damage critical targets such as DNA (Beir, et al. 1999; Balagamwala et al., 2013). Given the fundamental nature of energy deposition by nuclei, nucleons or elementary particles in material, this process is universal to all biological contexts.

Energy deposition is influenced by the linear energy transfer (LET) (Hall and Giaccia, 2018 UNSCEAR, 2020). High LET refers to energy above 10 keV μm-1 which produces more complex, dense structural damage than low LET radiation (below 10 keV μm-1). Low-LET particles produce sparse ionization events such as photons (X- and gamma rays), as well as high-energy protons. Low LET radiation travels farther into tissue but deposits smaller amounts of energy, whereas high LET radiation, which includes heavy ions, alpha particles and high-energy neutrons, does not travel as far but deposits larger amounts of energy into tissue at the same absorbed dose. The biological effect of the deposition of energy can be modulated by varying dose and dose rate of exposure, such as acute, chronic, or fractionated exposures (Hall and Giaccia, 2018). 

Non-ionizing radiation is a type of electromagnetic radiation that lacks the energy to ionize atoms or molecules. Examples of non-ionizing radiation include radio waves (wavelength: 100 km-1m), microwaves (wavelength: 1m-1mm), infrared radiation (wavelength: 1mm- 1 um), visible light (wavelengths: 400-700 nm), and ultraviolet radiation of longer wavelengths such as UVB (wavelengths: 315-400nm) and UVA (wavelengths: 280-315 nm). UVC radiation (X-X nm) is, in contrast to UVB and UVA, considered to be a type of ionizing radiation. Exposure to non-ionizing radiation occurs either from natural or anthropogenic sources, and include radio waves used for communication (broadcasting and cell phones), microwaves used in cooking food and in radar systems, infrared radiation emitted by warm objects or used in remote controls, thermal imaging and medical treatments. Visible light is the range of electromagnetic radiation that we can see and that is commonly used in photosynthesis in primary producers. UV radiation has key functions in melanisation (tanning) of a number of species, and exhibit key signalling roles in navigation and communication (e.g insects, aquatic invertebrates and fish), locomotory and predatory behavior (e.g. reptiles, birds and crustaceans) and growth and development (e.g. plants). UV radiation is also used in some medical treatments such as skin diseases (e.g. psoriasis, eczema, vitiligo and skin cancers).  

How it is Measured or Detected

Radiation type	Assay Name	References	Description	OECD Approved Assay
Ionizing radiation	Monte Carlo Simulations (Geant4)	Douglass et al., 2013; Douglass et al. 2012; Zyla et al., 2020	Monte Carlo simulations are based on a computational algorithm that mathematically models the deposition of energy into materials.	No
Ionizing radiation	Fluorescent Nuclear Track Detector (FNTD)	Sawakuchi, 2016; Niklas, 2013; Koaira & Konishi, 2015	FNTDs are biocompatible chips with crystals of aluminium oxide doped with carbon and magnesium; used in conjuction with fluorescent microscopy, these FNTDs allow for the visualization and the linear energy transfer (LET) quantification of tracks produced by the deposition of energy into a material.	No
Ionizing radiation	Tissue equivalent proportional counter (TEPC)	Straume et al, 2015	Measure the LET spectrum and calculate the dose equivalent.	No
Ionizing radiation	alanine dosimeters/NanoDots	Lind et al. 2019; Xie et al., 2022		No
Non-ionizing radiation	UV meters or radiameters	Xie et at., 2020	UVA/UVB (irradiance intensity), UV dosimeters (accumulated irradiance over time), Spectrophoto meter (absorption of UV by a substance or material)	No

 

References

Balagamwala, E. H. et al. (2013), “Introduction to radiotherapy and standard teletherapy techniques”, Dev Ophthalmol, Vol. 52, Karger, Basel, https://doi.org/10.1159/000351045 

Beir, V. et al. (1999), “The Mechanistic Basis of Radon-Induced Lung Cancer”, in Health Risks of Exposure to Radon: BEIR VI, National Academy Press, Washington, D.C., https://doi.org/10.17226/5499 

Douglass, M. et al. (2013), “Monte Carlo investigation of the increased radiation deposition due to gold nanoparticles using kilovoltage and megavoltage photons in a 3D randomized cell model”, Medical Physics, Vol. 40/7, American Institute of Physics, College Park, https://doi.org/10.1118/1.4808150 

Douglass, M. et al. (2012), “Development of a randomized 3D cell model for Monte Carlo microdosimetry simulations.”, Medical Physics, Vol. 39/6, American Institute of Physics, College Park, https://doi.org/10.1118/1.4719963 

Hall, E. J. and Giaccia, A.J. (2018), Radiobiology for the Radiologist, 8th edition, Wolters Kluwer, Philadelphia.  

Kodaira, S. and Konishi, T. (2015), “Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector.”, Journal of Radiation Research, Vol. 56/2, Oxford University Press, Oxford, https://doi.org/10.1093/jrr/rru091 

Lind, O.C., D.H. Oughton and Salbu B. (2019), "The NMBU FIGARO low dose irradiation facility", International Journal of Radiation Biology, Vol. 95/1, Taylor & Francis, London, https://doi.org/10.1080/09553002.2018.1516906.

Sawakuchi, G.O. and Akselrod, M.S. (2016), “Nanoscale measurements of proton tracks using fluorescent nuclear track detectors.”, Medical Physics, Vol. 43/5, American Institute of Physics, College Park, https://doi.org/10.1118/1.4947128 

Straume, T. et al. (2015), “Compact Tissue-equivalent Proportional Counter for Deep Space Human Missions.”, Health physics, Vol. 109/4, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1097/HP.0000000000000334 

Niklas, M. et al. (2013), “Engineering cell-fluorescent ion track hybrid detectors.”, Radiation Oncology, Vol. 8/104, BioMed Central, London, https://doi.org/10.1186/1748-717X-8-141 

UNSCEAR (2020), Sources, effects and risks of ionizing radiation, United Nations. 

Xie, Li. et al. (2022), "Ultraviolet B Modulates Gamma Radiation-Induced Stress Responses in Lemna Minor at Multiple Levels of Biological Organisation", SSRN, Elsevier, Amsterdam, http://dx.doi.org/10.2139/ssrn.4081705 .

Zyla, P.A. et al. (2020), Review of particle physics: Progress of Theoretical and Experimental Physics, 2020 Edition, Oxford University Press, Oxford. 

 

 

List of Key Events in the AOP

Event: 1392: Oxidative Stress

Short Name: Oxidative Stress

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Platinum

Kruidering et al. (1997) examined the effect of platinum on pig kidneys and found that it was able to induce significant dose-dependant ROS formation within 20 minutes of treatment administration.

Aluminum

In a study of the effects of aluminum treatment on rat kidneys, Al Dera (2016) found that renal GSH, SOD, and GPx levels were significantly lower in the treated groups, while lipid peroxidation levels were significantly increased.

Cadmium

Belyaeva et al. (2012) investigated the effect of cadmium treatment on human kidney cells. They found that cadmium was the most toxic when the sample was treated with 500 μM for 3 hours (Belyaeva et al., 2012). As this study also looked at mercury, it is worth noting that mercury was more toxic than cadmium in both 30-minute and 3-hour exposures at low concentrations (10-100 μM) (Belyaeva et al., 2012).

Wang et al. (2009) conducted a study evaluating the effects of cadmium treatment on rats and found that the treated group showed a significant increase in lipid peroxidation. They also assessed the effects of lead in this study, and found that cadmium can achieve a very similar level of lipid peroxidation at a much lower concentration than lead can, implying that cadmium is a much more toxic metal to the kidney mitochondria than lead is (Wang et al., 2009). They also found that when lead and cadmium were applied together they had an additive effect in increasing lipid peroxidation content in the renal cortex of rats (Wang et al., 2009).

Jozefczak et al. (2015) treated Arabidopsis thaliana wildtype, cad2-1 mutant, and vtc1-1 mutant plants with cadmium to determine the effects of heavy metal exposure to plant mitochondria in the roots and leaves. They found that total GSH/GSG ratios were significantly increased after cadmium exposure in the leaves of all sample varieties and that GSH content was most significantly decreased for the wildtype plant roots (Jozefczak et al., 2015).

Andjelkovic et al. (2019) also found that renal lipid peroxidation was significantly increased in rats treated with 30 mg/kg of cadmium.

Mercury

Belyaeva et al. (2012) conducted a study which looked at the effects of mercury on human kidney cells, they found that mercury was the most toxic when the sample was treated with 100 μM for 30 minutes.

Buelna-Chontal et al. (2017) investigated the effects of mercury on rat kidneys and found that treated rats had higher lipid peroxidation content and reduced cytochrome c content in their kidneys.

Uranium

In Shaki et al.’s article (2012), they found rat kidney mitochondria treated with uranyl acetate caused increased formation of ROS, increased lipid peroxidation, and decreased GSH content when exposed to 100 μM or more for an hour.

Hao et al. (2014), found that human kidney proximal tubular cells (HK-2 cells) treated with uranyl nitrate for 24 hours with 500 μM showed a 3.5 times increase in ROS production compared to the control. They also found that GSH content was decreased by 50% of the control when the cells were treated with uranyl nitrate (Hao et al., 2014).

Arsenic

Bhadauria and Flora (2007) studied the effects of arsenic treatment on rat kidneys. They found that lipid peroxidation levels were increased by 1.5 times and the GSH/GSSG ratio was decreased significantly (Bhadauria and Flora, 2007).

Kharroubi et al. (2014) also investigated the effect of arsenic treatment on rat kidneys and found that lipid peroxidation was significantly increased, while GSH content was significantly decreased.

In their study of the effects of arsenic treatment on rat kidneys, Turk et al. (2019) found that lipid peroxidation was significantly increased while GSH and GPx renal content were decreased.

Silver

Miyayama et al. (2013) investigated the effects of silver treatment on human bronchial epithelial cells and found that intracellular ROS generation was increased significantly in a dose-dependant manner when treated with 0.01 to 1.0 μM of silver nitrate.

Manganese

Chtourou et al. (2012) investigated the effects of manganese treatment on rat kidneys. They found that manganese treatment caused significant increases in ROS production, lipid peroxidation, urinary H₂O₂ levels, and PCO production. They also found that intracellular GSH content was depleted in the treated group (Chtourou et al., 2012).

Nickel

Tyagi et al. (2011) conducted a study of the effects of nickel treatment on rat kidneys. They found that the treated rats showed a significant increase in kidney lipid peroxidation and a significant decrease in GSH content in the kidney tissue (Tyagi et al., 2011).

Zinc

Yeh et al. (2011) investigated the effects of zinc treatment on rat kidneys and found that treatment with 150 μM or more for 2 weeks or more caused a time- and dose-dependant increase in lipid peroxidation. They also found that renal GSH content was decreased in the rats treated with 150 μM or more for 8 weeks (Yeh et al., 2011).

It should be noted that Hao et al. (2014) found that rat kidneys exposed to lower concentrations of zinc (such as 100 μM) for short time periods (such as 1 day), showed a protective effect against toxicity induced by other heavy metals, including uranium. Soussi, Gargouri, and El Feki (2018) also found that pre-treatment with a low concentration of zinc (10 mg/kg treatment for 15 days) protected the renal cells of rats were from changes in varying oxidative stress markers, such as lipid peroxidation, protein carbonyl, and GPx levels.

nanoparticles

Huerta-García et al. (2014) conducted a study of the effects of titanium nanoparticles on human and rat brain cells. They found that both the human and rat cells showed time-dependant increases in ROS when treated with titanium nanoparticles for 2 to 6 hours (Huerta-García et al., 2014). They also found elevated lipid peroxidation that was induced by the titanium nanoparticle treatment of human and rat cell lines in a time-dependant manner (Huerta-García et al., 2014).

Liu et al. (2010) also investigated the effects of titanium nanoparticles, however they conducted their trials on rat kidney cells. They found that ROS production was significantly increased in a dose dependant manner when treated with 10 to 100 μg/mL of titanium nanoparticles (Liu et al., 2010).

Pan et al. (2009) treated human cervix carcinoma cells with gold nanoparticles (Au1.4MS) and found that intracellular ROS content in the treated cells increased in a time-dependant manner when treated with 100 μM for 6 to 48 hours. They also compared the treatment with Au1.4MS gold nanoparticles to treatment with Au15MS treatment, which are another size of gold nanoparticle (Pan et al., 2009). The Au15MS nanoparticles were much less toxic than the Au1.4MS gold nanoparticles, even when the Au15MS nanoparticles were applied at a concentration of 1000 μM (Pan et al., 2009). When investigating further markers of oxidative stress, Pan et al. (2009) found that GSH content was greatly decreased in cells treated with gold nanoparticles.

Ferreira et al. (2015) also studied the effects of gold nanoparticles. They exposed rat kidneys to GNPs-10 (10 nm particles) and GNPs-30 (30 nm particles), and found that lipid peroxidation and protein carbonyl content in the rat kidneys treated with GNPs-30 and GNPs-10, respectively, were significantly elevated.

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Occurrence of oxidative stress is not species specific.  

Life stage applicability: Occurrence of oxidative stress is not life stage specific. 

Sex applicability: Occurrence of oxidative stress is not sex specific. 

Evidence for perturbation by prototypic stressor: There is evidence of the increase of oxidative stress following perturbation from a variety of stressors including exposure to ionizing radiation and altered gravity (Bai et al., 2020; Ungvari et al., 2013; Zhang et al., 2009).  

Key Event Description

Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell.  As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.

In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).

ROS also undermine the mitochondrial defense system from oxidative damage. The antioxidant systems consist of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, as well as antioxidants such as α-tocopherol and ubiquinol, or antioxidant vitamins and minerals including vitamin E, C, carotene, lutein, zeaxanthin, selenium, and zinc (Fletcher, 2010). The enzymes, vitamins and minerals catalyze the conversion of ROS to non-toxic molecules such as water and O₂. However, these antioxidant systems are not perfect and endogenous metabolic processes and/or exogenous oxidative influences can trigger cumulative oxidative injuries to the mitochondria, causing a decline in their functionality and efficiency, which further promotes cellular oxidative stress (Balasubramanian, 2000; Ganea & Harding, 2006; Guo et al., 2013; Karimi et al., 2017).

However, an emerging viewpoint suggests that ROS-induced modifications may not be as detrimental as previously thought, but rather contribute to signaling processes (Foyer et al., 2017). 

Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).

Sources of ROS Production

Direct Sources: Direct sources involve the deposition of energy onto water molecules, breaking them into active radical species. When ionizing radiation hits water, it breaks it into hydrogen (H*) and hydroxyl (OH*) radicals by destroying its bonds. The hydrogen will create hydroxyperoxyl free radicals (HO₂*) if oxygen is available, which can then react with another of itself to form hydrogen peroxide (H₂O₂) and more O₂ (Elgazzar and Kazem, 2015). Antioxidant mechanisms are also affected by radiation, with catalase (CAT) and peroxidase (POD) levels rising as a result of exposure (Seen et al. 2018; Ahmad et al. 2021).

Indirect Sources: An indirect source of ROS is the mitochondria, which is one of the primary producers in eukaryotic cells (Powers et al., 2008).  As much as 2% of the electrons that should be going through the electron transport chain in the mitochondria escape, allowing them an opportunity to interact with surrounding structures. Electron-oxygen reactions result in free radical production, including the formation of hydrogen peroxide (H₂O₂) (Zhao et al., 2019). The electron transport chain, which also creates ROS, is activated by free adenosine diphosphate (ADP), O₂, and inorganic phosphate (P_i) (Hargreaves et al. 2020; Raimondi et al. 2020; Vargas-Mendoza et al. 2021). The first and third complexes of the transport chain are the most relevant to mammalian ROS production (Raimondi et al., 2020). The mitochondria have its own set of DNA and it is a prime target of oxidative damage (Guo et al., 2013). ROS are also produced through nicotinamide adenine dinucleotide phosphate oxidase (NOX) stimulation, an event commenced by angiotensin II, a product/effector of the renin-angiotensin system (Nguyen Dinh Cat et al. 2013; Forrester et al. 2018). Other ROS producers include xanthine oxidase, immune cells (macrophage, neutrophils, monocytes, and eosinophils), phospholipase A₂ (PLA₂), monoamine oxidase (MAO), and carbon-based nanomaterials (Powers et al. 2008; Jacobsen et al. 2008; Vargas-Mendoza et al. 2021).

How it is Measured or Detected

Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage. Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed

• Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)
• Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.
• Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). 
• TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. 
• 8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or  HPLC, described in Chepelev et al. (Chepelev, et al. 2015).

Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:

• Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus
• Western blot for increased Nrf2 protein levels
• Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus
• qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)
• Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014)
• OECD TG422D describes an ARE-Nrf2 Luciferase test method
• In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation

 

Assay Type & Measured Content	Description	Dose Range Studied	Assay Characteristics (Length / Ease of use/Accuracy)
ROS Formation in the Mitochondria assay (Shaki et al., 2012)	“The mitochondrial ROS measurement was performed flow cytometry using DCFH-DA. Briefly, isolated kidney mitochondria were incubated with UA (0, 50, 100 and 200 μM) in respiration buffer containing (0.32 mM sucrose, 10 mM Tris, 20 mM Mops, 50 μM EGTA, 0.5 mM MgCl2, 0.1 mM KH2PO4 and 5 mM sodium succinate) [32]. In the interval times of 5, 30 and 60 min following the UA addition, a sample was taken and DCFH-DA was added (final concentration, 10 μM) to mitochondria and was then incubated for 10 min. Uranyl acetate-induced ROS generation in isolated kidney mitochondria were determined through the flow cytometry (Partec, Deutschland) equipped with a 488-nm argon ion laser and supplied with the Flomax software and the signals were obtained using a 530-nm bandpass filter (FL-1 channel). Each determination is based on the mean fluorescence intensity of 15,000 counts.”	0, 50, 100 and 200 μM of Uranyl Acetate	Long/ Easy High accuracy
Mitochondrial Antioxidant Content Assay Measuring GSH content (Shaki et al., 2012)	“GSH content was determined using DTNB as the indicator and spectrophotometer method for the isolated mitochondria. The mitochondrial fractions (0.5 mg protein/ml) were incubated with various concentrations of uranyl acetate for 1 h at 30 °C and then 0.1 ml of mitochondrial fractions was added into 0.1 mol/l of phosphate buffers and 0.04% DTNB in a total volume of 3.0 ml (pH 7.4). The developed yellow color was read at 412 nm on a spectrophotometer (UV-1601 PC, Shimadzu, Japan). GSH content was expressed as μg/mg protein.”	0, 50, 100, or 200 μM Uranyl Acetate
H2O2 Production Assay Measuring H2O2 Production in isolated mitochondria (Heyno et al., 2008)	“Effect of CdCl2 and antimycin A (AA) on H2O2 production in isolated mitochondria from potato. H2O2 production was measured as scopoletin oxidation. Mitochondria were incubated for 30 min in the measuring buffer (see the Materials and Methods) containing 0.5 mM succinate as an electron donor and 0.2 µM mesoxalonitrile 3‐chlorophenylhydrazone (CCCP) as an uncoupler, 10 U horseradish peroxidase and 5 µM scopoletin.” (	0, 10, 30  μM Cd2+ 2  μM antimycin A
Flow Cytometry ROS & Cell Viability (Kruiderig et al., 1997)	“For determination of ROS, samples taken at the indicated time points were directly transferred to FACScan tubes. Dih123 (10 mM, final concentration) was added and cells were incubated at 37°C in a humidified atmosphere (95% air/5% CO2) for 10 min. At t 5 9, propidium iodide (10 mM, final concentration) was added, and cells were analyzed by flow cytometry at 60 ml/min. Nonfluorescent Dih123 is cleaved by ROS to fluorescent R123 and detected by the FL1 detector as described above for Dc (Van de Water 1995)”		Strong/easy medium
DCFH-DA Assay Detection of hydrogen peroxide production (Yuan et al., 2016)	Intracellular ROS production was measured using DCFH-DA as a probe. Hydrogen peroxide oxidizes DCFH to DCF. The probe is hydrolyzed intracellularly to DCFH carboxylate anion. No direct reaction with H2O2 to form fluorescent production.	0-400 µM	Long/ Easy High accuracy
H2-DCF-DA Assay Detection of superoxide production (Thiebault et al., 2007)	This dye is a stable nonpolar compound which diffuses readily into the cells and yields H2-DCF. Intracellular OH or ONOO- react with H2-DCF when cells contain peroxides, to form the highly fluorescent compound DCF, which effluxes the cell. Fluorescence intensity of DCF is measured using a fluorescence spectrophotometer.	0–600 µM	Long/ Easy High accuracy
CM-H2DCFDA Assay	**Come back and explain the flow cytometry determination of oxidative stress from Pan et al. (2009)**

Direct Methods of Measurement

Method of Measurement	References	Description	OECD-Approved Assay
Chemiluminescence	(Lu, C. et al., 2006;  Griendling, K. K., et al., 2016)	ROS can induce electron transitions in molecules, leading to electronically excited products. When the electrons transition back to ground state, chemiluminescence is emitted and can be measured. Reagents such as uminol and lucigenin are commonly used to amplify the signal.	No
Spectrophotometry	(Griendling, K. K., et al., 2016)	NO has a short half-life. However, if it has been reduced to nitrite (NO2-), stable azocompounds can be formed via the Griess Reaction, and further measured by spectrophotometry.	No
Direct or Spin Trapping-Based Electron Paramagnetic Resonance (EPR) Spectroscopy	(Griendling, K. K., et al., 2016)	The unpaired electrons (free radicals) found in ROS can be detected with EPR, and is known as electron paramagnetic resonance. A variety of spin traps can be used.	No
Nitroblue Tetrazolium Assay	(Griendling, K. K., et al., 2016)	The Nitroblue Tetrazolium assay is used to measure O2•– levels. O2•– reduces nitroblue tetrazolium (a yellow dye) to formazan (a blue dye), and can be measured at 620 nm.	No
Fluorescence analysis of dihydroethidium (DHE) or Hydrocyans	(Griendling, K. K., et al., 2016)	Fluorescence analysis of DHE is used to measure O2•– levels. O2•–  is reduced to O2 as DHE is oxidized to 2-hydroxyethidium, and this reaction can be measured by fluorescence. Similarly, hydrocyans can be oxidized by any ROS, and measured via fluorescence.	No
Amplex Red Assay	(Griendling, K. K., et al., 2016)	Fluorescence analysis to measure extramitochondrial or extracellular H2O2 levels. In the presence of horseradish peroxidase and H2O2, Amplex Red is oxidized to resorufin, a fluorescent molecule measurable by plate reader.	No
Dichlorodihydrofluorescein Diacetate (DCFH-DA)	(Griendling, K. K., et al., 2016)	An indirect fluorescence analysis to measure intracellular H2O2 levels. H2O2 interacts with peroxidase or heme proteins, which further react with DCFH, oxidizing it to dichlorofluorescein (DCF), a fluorescent product.	No
HyPer Probe	(Griendling, K. K., et al., 2016)	Fluorescent measurement of intracellular H2O2 levels. HyPer is a genetically encoded fluorescent sensor that can be used for in vivo and in situ imaging.	No
Cytochrome c Reduction Assay	(Griendling, K. K., et al., 2016)	The cytochrome c reduction assay is used to measure O2•– levels. O2•–  is reduced to O2 as ferricytochrome c is oxidized to ferrocytochrome c, and this reaction can be measured by an absorbance increase at 550 nm.	No
Proton-electron double-resonance imagine (PEDRI)	(Griendling, K. K., et al., 2016)	The redox state of tissue is detected through nuclear magnetic resonance/magnetic resonance imaging, with the use of a nitroxide spin probe or biradical molecule.	No
Glutathione (GSH) depletion	(Biesemann, N. et al., 2018)	A downstream target of the Nrf2 pathway is involved in GSH synthesis. As an indication of oxidation status, GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html).	No
Thiobarbituric acid reactive substances (TBARS)	(Griendling, K. K., et al., 2016)	Oxidative damage to lipids can be measured by assaying for lipid peroxidation with TBARS using a commercially available kit.	No
Protein oxidation (carbonylation)	(Azimzadeh et al., 2017; Azimzadeh etal., 2015; Ping et al., 2020)	Can be determined with enzyme-linked immunosorbent assay (ELISA) or a commercial assay kit. Protein oxidation can indicate the level of oxidative stress.	No
Seahorse XFp Analyzer	Leung et al. 2018	The Seahorse XFp Analyzer provides information on mitochondrial function, oxidative stress, and metabolic dysfunction of viable cells by measuring respiration (oxygen consumption rate; OCR) and extracellular pH (extracellular acidification rate; ECAR).	No

Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assays for Nrf2 activity include: 

Method of Measurement	References	Description	OECD-Approved Assay
Immunohistochemistry	(Amsen, D., de Visser, K. E., and Town, T., 2009)	Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus	No
Quantitative polymerase chain reaction (qPCR)	(Forlenza et al., 2012)	qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences)	No
Whole transcriptome profiling via microarray or via RNA-seq followed by a pathway analysis	(Jackson, A. F. et al., 2014)	Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway	No

References

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Antelmann, H. and J. D. Helmann (2011), “Thiol-based redox switches and gene regulation.”, Antioxidants & Redox Signaling, Vol. 14/6, Mary Ann Leibert Inc., Larchmont, https://doi.org/10.1089/ars.2010.3400

Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, https://doi.org/10.1007/978-1-59745-447-6_5 

Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, https://doi.org/10.1021/pr501141b

Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, https://doi.org/10.1080/09553002.2017.1339332

Azzam, E. I. et al. (2012), “Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury”, Cancer Letters, Vol. 327/1-2, Elsevier, Ireland, https://doi.org/10.1016/j.canlet.2011.12.012 

Bai, J. et al. (2020), “Irradiation-induced senescence of bone marrow mesenchymal stem cells aggravates osteogenic differentiation dysfunction via paracrine signaling”, American Journal of Physiology - Cell Physiology, Vol. 318/5, American Physiological Society, Rockville, https://doi.org/10.1152/ajpcell.00520.2019.

Balasubramanian, D (2000), “Ultraviolet radiation and cataract”, Journal of ocular pharmacology and therapeutics, Vol. 16/3, Mary Ann Liebert Inc., Larchmont, https://doi.org/10.1089/jop.2000.16.285. 

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Elgazzar, A. and N. Kazem. (2015), “Chapter 23: Biological effects of ionizing radiation” in The Pathophysiologic Basis of Nuclear Medicine, Springer, New York, pp. 540-548

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Forrester, S.J. et al. (2018), “Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology”, Physiological Reviews, Vol. 98/3, American Physiological Society, Rockville, https://doi.org/10.1152/physrev.00038.201

Foyer, C. H., A. V. Ruban, and G. Noctor (2017), “Viewing oxidative stress through the lens of oxidative signalling rather than damage”, Biochemical Journal, Vol. 474/6, Portland Press, England, https://doi.org/10.1042/BCJ20160814 

Ganea, E. and J. J. Harding (2006), “Glutathione-related enzymes and the eye”, Current eye research, Vol. 31/1, Informa, London, https://doi.org/10.1080/02713680500477347. 

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Event: 1635: Increase, DNA strand breaks

Short Name: Increase, DNA strand breaks

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: DNA strand breaks are relevant to all species, including vertebrates such as humans, that contain DNA (Cannan & Pederson, 2016).  

Life stage applicability: This key event is not life stage specific as all life stages display strand breaks. However, there is an increase in baseline levels of DNA strand breaks seen in older individuals though it is unknown whether this change due to increased break induction or a greater retention of breaks due to poor repair (White & Vijg, 2016). 

Sex applicability: This key event is not sex specific as both sexes display evidence of strand breaks. In some cell types, such as peripheral blood mononuclear cells, males show higher levels of single strand breaks than females (Garm et al., 2012). 

Evidence for perturbation by a stressor: There are studies demonstrating that increased DNA strand breaks can result from exposure to multiple stressor types including ionizing & non-ionizing radiation, chemical agents, and oxidizing agents (EPRI, 2014; Hamada, 2014; Cencer et al., 2018; Cannan & Pederson, 2016; Yang et al., 1998).  

Key Event Description

DNA strand breaks can occur on a single strand (SSB) or both strands (double strand breaks; DSB). SSBs arise when the phosphate backbone connecting adjacent nucleotides in DNA is broken on one strand. DSBs are generated when both strands are simultaneously broken at sites that are sufficiently close to one another that base-pairing and chromatin structure are insufficient to keep the two DNA ends juxtaposed. As a consequence, the two DNA ends generated by a DSB can physically dissociate from one another, becoming difficult to repair and increasing the chance of inappropriate recombination with other sites in the genome (Jackson, 2002). SSB can turn into DSB if the replication fork stalls at the lesion leading to fork collapse.

Strand breaks are intermediates in various biological events, including DNA repair (e.g., excision repair), V(D)J recombination in developing lymphoid cells and chromatin remodeling in both somatic cells and germ cells. The spectrum of damage  can be complex, particularily if the stressor is from large amounts of deposited energy which can result in complex lesions and clustered damage defined as two or more oxidized bases, abasic sites or starnd breaks on opposing DNA strands within a few helical turns. These lesions are more difficult to repair and have been studied in many types of models  (Barbieri et al., 2019 and Asaithamby et al., 2011). DSBs and complex lesions  are of particular concern, as they are considered the most lethal and deleterious type of DNA lesion. If misrepaired or left unrepaired, DSBs may drive the cell towards genomic instability, apoptosis or tumorigenesis (Beir, 1999).

 

How it is Measured or Detected

Please refer to the table below for details regarding these and other methodologies for detecting DNA DSBs.

 

Assay Name	References	Description	OECD Approved Assay
Comet Assay (Single Cell Gel Eletrophoresis - Alkaline)	Collins, 2004; Olive and Banath, 2006; Platel et al., 2011; Nikolova et al., 2017	To detect SSBs or DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at an alkaline pH (pH >13); DNA fragments are forced to move, forming a "comet"-like appearance	Yes (No. 489)
Comet Assay (Single Cell Gel Eltrophoresis - Neutral)	Collins, 2014; Olive and Banath, 2006; Anderson and Laubenthal, 2013; Nikolova et al., 2017	To detect DSBs, single cells are encapsulated in agarose on a slide, lysed, and subjected to gel electrophoresis at a neutral pH; DNA fragments, which are not denatured at the neutral pH, are forced to move, forming a "comet"-like appearance	N/A
γ-H2AX Foci Quantification - Flow Cytometry	Rothkamm and Horn, 2009; Bryce et al., 2016	Measurement of γ-H2AX immunostaining in cells by flow cytometry, normalized to total levels of H2AX	N/A
γ-H2AX Foci Quantification - Western Blot	Burma et al., 2001; Revet et al., 2011	Measurement of γ-H2AX immunostaining in cells by Western blotting, normalized to total levels of H2AX	N/A
γ-H2AX Foci Quantification - Microscopy	Redon et al., 2010; Mah et al., 2010; Garcia-Canton et al., 2013	Quantification of γ-H2AX immunostaining by counting γ- H2AX foci visualized with a microscope	N/A
γ-H2AX Foci Detection - ELISA and flow cytometry	Ji et al., 2017; Bryce et al., 2016	Detection of γ-H2AX in cells by ELISA, normalized to total levels of H2AX; γH2AX foci detection can be high-throughput and automated using flow cytometry-based immunodetection.	N/A
Pulsed Field Gel Electrophoresis (PFGE)	Ager et al., 1990; Gardiner et al., 1985; Herschleb et al., 2007; Kawashima et al., 2017	To detect DSBs, cells are embedded and lysed in agarose, and the released DNA undergoes gel electrophoresis in which the direction of the voltage is periodically alternated; Large DNA fragments are thus able to be separated by size	N/A
The TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Assay	Loo, 2011	To detect strand breaks, dUTPs added to the 3’OH end of a strand break by the DNA polymerase terminal deoxynucleotidyl transferase (TdT) are tagged with a fluorescent dye or a reporter enzyme to allow visualization (We note that this method is typically used to measure apoptosis)	N/A
In Vitro DNA Cleavage Assays using Topoisomerase	Nitiss, 2012	Cleavage of DNA can be achieved using purified topoisomerase; DNA strand breaks can then be separated and quantified using gel electrophoresis	N/A
PCR assay	Figueroa‑González & Pérez‑Plasencia, 2017	Assay of strand breaks through the observation of DNA amplification prevention. Breaks block Taq polymerase, reducing the number of DNA templates, preventing amplification	N/A
Sucrose density gradient centrifuge	Raschke et al. 2009	Division of DNA pieces by density, increased fractionation leads to lower density pieces, with the use of a sucrose cushion	N/A
Alkaline Elution Assay	Kohn, 1991	Cells lysed with detergent-solution, filtered through membrane to remove all but intact DNA	N/A
Unwinding Assay	Nacci et al. 1992	DNA is stored in alkaline solutions with DNA-specific dye and allowed to unwind following removal from tissue, increased strand damage associated with increased unwinding	N/A

References

Ager, D. D. et al. (1990). “Measurement of Radiation- Induced DNA Double-Strand Breaks by Pulsed-Field Gel Electrophoresis.” Radiat Res. 122(2), 181-7.

Anderson, D. & Laubenthal J. (2013), “Analysis of DNA Damage via Single-Cell Electrophoresis. In: Makovets S, editor. DNA Electrophoresis. Totowa.”, NJ: Humana Press. p 209-218.

Asaithamby, A., B. Hu and D.J. Chen. (2011) Unrepaired clustered DNA lesions induce chromosome breakage in human cells. Proc Natl Acad Sci U S A 108(20): 8293-8298 .

Barbieri, S., G. Babini, J. Morini et a l (2019). . Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures. Scientific Reports 9(1): 14019

Bryce, S. et al. (2016), “Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.”, Environ Mol Mutagen. 57:171-189. Doi: 10.1002/em.21996.

Burma, S. et al. (2001), “ATM phosphorylates histone H2AX in response to DNA double-strand breaks.”, J Biol Chem, 276(45): 42462-42467. doi:10.1074/jbc.C100466200

Cannan, W.J. and D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", Journal of Cellular Physiology, Vol.231/1, Wiley, New York, https://doi.org/10.1002/jcp.25048.  

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

Charlton, E. D. et al. (1989), “Calculation of Initial Yields of Single and Double Stranded Breaks in Cell Nuclei from Electrons, Protons, and Alpha Particles.”,  Int. J. Radiat. Biol. 56(1): 1-19. doi: 10.1080/09553008914551141.

Collins, R. A. (2004), “The Comet Assay for DNA Damage and Repair. Molecular Biotechnology.”, Mol Biotechnol. 26(3): 249-61. doi:10.1385/MB:26:3:249

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

Figueroa‑González, G. and C. Pérez‑Plasencia. (2017), “Strategies for the evaluation of DNA damage and repair mechanisms in cancer”, Oncology Letters, Vol.13/6, Spandidos Publications, Athens, https://doi.org/10.3892/ol.2017.6002. 

Garcia-Canton, C. et al. (2013), “Assessment of the in vitro p-H2AX assay by High Content Screening asa novel genotoxicity test.”, Mutat Res. 757:158-166.  Doi:  10.1016/j.mrgentox.2013.08.002

Gardiner, K. et al. (1986), “Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed-Field Gradient Gel Electrophoresis.”,  Somatic Cell and Molecular Genetics. 12(2): 185-95.Doi: 10.1007/bf01560665.

Garm, C. et al. (2012), “Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells”, Aging Cell, Vol.12/1, Blackwell Publishing Ltd, Oxford, https://doi.org/10.1111/acel.12019. 

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

Herschleb, J. et al. (2007), “Pulsed-field gel electrophoresis.”,  Nat Protoc. 2(3): 677-684. doi:10.1038/nprot.2007.94

Iliakis, G. et al. (2015), “Alternative End-Joining Repair Pathways Are the Ultimate Backup for Abrogated Classical Non-Homologous End-Joining and Homologous Recombination Repair: Implications for the Formation of Chromosome Translocations.”,  Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2(3): 677-84. doi: 10.1038/nprot.2007.94

Jackson, S. (2002). “Sensing and repairing DNA double-strand breaks.”,  Carcinogenesis. 23:687-696. Doi:10.1093/carcin/23.5.687.

Ji, J. et al. (2017), “Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.”,  PLoS One. 12(2): e0171582. doi:10.1371/journal.pone.0171582

Kawashima, Y.(2017), “Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.”,  Genes Cells 22:84-93. Doi: 10.1111/gtc.12457.

Khoury, L. et al. (2013), “Validation of high-throughput genotoxicity assay screening using cH2AX in-cell Western assay on HepG2 cells.”, Environ Mol Mutagen, 54:737-746. Doi:  10.1002/em.21817.

Khoury, L. et al. (2016), “Evaluation of four human cell lines with distinct biotransformation properties for genotoxic screening.”, Mutagenesis, 31:83-96. Doi: 10.1093/mutage/gev058.

Kohn, K.W. (1991), “Principles and practice of DNA filter elution”, Pharmacology & Therapeutics, Vol.49/1, Elsevier, Amsterdam, https://doi.org/10.1016/0163-7258(91)90022-E. 

Loo, DT. (2011), “In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. In: Didenko V, editor. DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Totowa.”, NJ: Humana Press. p 3-13.doi: 10.1007/978-1-60327-409-8_1.

Mah, L. J. et al. (2010), “Quantification of gammaH2AX foci in response to ionising radiation.”,  J Vis Exp(38). doi:10.3791/1957.

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Nikolova, T., F. et al. (2017), “Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.”,  Mutat Res 822:10-18. Doi: 10.1016/j.mrgentox.2017.07.004.

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Platel A. et al. (2011), “Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: Determination of No-Observed-Genotoxic-Effect-Levels.”,  Mutat Res 726:151-159. Doi: 10.1016/j.mrgentox.2011.09.003.

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Event: 1493: Increased Pro-inflammatory mediators

Short Name: Increased pro-inflammatory mediators

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

LIVER:

Human [Santibañez et al., 2011]

Rat [Luckey and Petersen, 2001]

Mouse [Nan et al., 2013]

BRAIN:

Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014

 

Taxonomic applicability: The inflammatory response and increase of the pro-inflammatory mediators has been observed across species from simple invertebrates such as Daphnia to higher order vertebrates (Weavers & Martin, 2020).

Life stage applicability: This key event is not life stage specific (Kalm et al., 2013; Veeraraghan et al., 2011; Hladik & Tapio, 2016).  

Sex applicability:  Most studies conducted were on male models, although sex-dependent differences in pro-inflammatory markers have been previously reported (Cekanaviciute et al., 2018; Parihar et al., 2020).  

Evidence for perturbation by a prototypic stressor: There is evidence of the increase of pro-inflammatory mediators following perturbation from a variety of stressors including exposure to ionizing radiation. (Abdel-Magied et al., 2019; Cho et al., 2017; Gaber et al., 2003; Ismail et al., 2016; Kim et al. 2002; Lee et al., 2010; Parihar et al., 2018).

Key Event Description

Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites. They can be divided into exogenous and endogenous mediators.

Exogenous mediators of inflammation are bacterial products or toxins like endotoxin or LPS. Endogenous mediators of inflammation are produced from within the (innate and adaptive) immune system itself, as well as other systems. They can be derived from molecules that are normally present in the plasma in an inactive form, such as peptide fragments of some components of complement, coagulation, and kinin systems. Or they can be released at the site of injury by a number of cell types that either contain them as preformed molecules within storage granules, e.g. histamine, or which can rapidly switch on the machinery required to synthesize the mediators.

Table1: a non-exhaustive list of examples for pro-inflammatory mediators

Classes of inflammatory mediators	Examples
Pro-inflammatory cytokines	TNF-a, Interleukins (IL-1, IL-6, IL-8), Interferons  (IFN-g), chemokines (CXCL, CCL, GRO-α, MCP-1), GM-CSF
Prostaglandins	PGE2
Bradykinin
Vasoactive amines	histamine, serotonin
Reactive oxygen species (ROS)	O2-, H2O2
Reactive nitrogen species (RNS)	NO, iNOS

The increased production of pro-inflammatory mediators can have negative consequences on the parenchymal cells leading even to cell death, as described for TNF-a or peroxynitrite on neurons (Chao et al., 1995; Brown and Bal-Price, 2003). Along with TNF-α, IL-1β and IL-6 have been shown to exhibit negative consequences on neurogenesis and neuronal precursor cell proliferation when overexpressed. IFN-γ is also associated with neuronal damage, although it is not as extensively studied compared to TNF-α, IL-1β and IL-6. In addition, via a feedback loop, they can act on the reactive resident cells thus maintaining or exacerbating their reactive state; and by modifying elements of their signalling pathways, they can favour the M1 phenotypic polarization and the chronicity of the inflammatory process (Taetzsch et al., 2015).

Basically, this event occurs equally in various tissues and does not require tissue-specific descriptions. Nevertheless, there are some specificities such as the release of glutamate by brain reactive glial cells (Brown and Bal-Price, 2003; Vesce et al., 2007).The differences may rather reside in the type of insult favouring the increased expression and/or release of a specific class of inflammatory mediators, as well the time after the insult reflecting different stages of the inflammatory process. For these reasons, the analyses of the changes of a battery of inflammatory mediators rather than of a single one is a more adequate measurement of this KE.

Regulatory examples using the KE

CD54 and CD 86 as well as IL-8 expression is used to assess skin sensitization potential (OECD TG 442E). IL-2 expression is used to assess immunotoxicity (and will become an OECD test guideline); for the latter see also doi: 10.1007/s00204-018-2199-7.

 

LIVER:

When activated, resident macrophages (Kupffer cells) release inflammatory mediators including cytokines, chemokines, lysosomal, and proteolytic enzymes and are a main source of TGF-β1 - the most potent pro-fibrogenic cytokine. Following the role of TGF-β is described in more detail.

Transforming growth factor β (TGF-β) is a pleiotropic cytokine with potent regulatory and

inflammatory activity [Sanjabi et al., 2009; Li and Flavell, 2008a;2008b]. The multi-faceted effects of TGF-β on numerous immune functions are cellular and environmental context dependent [Li et al., 2006]. TGF-β binds to TGF-β receptor II (TGF-βRII) triggering the kinase activity of the cytoplasmic domain that in turn activates TGF-βRI. The activated receptor complex leads to nuclear translocation of Smad molecules,

and transcription of target genes [Li et al., 2006a]. The role of TGF-β as an immune modulator of T cell activity is best exemplified by the similarities between TGF-β1 knockout and T cell specific

TGF-β receptor II knockout mice [Li et al., 2006b; Marie et al., 2006;Shull et al., 1992]. The animals in both of these models develop severe multi-organ autoimmunity and succumb to death within a few weeks after birth [Li et al., 2006b; Marie et al., 2006; Shull et al., 1992]. In addition, in mice where TGF-β signaling is blocked specifically in T cells, the development of natural killer T (NKT) cells, natural regulatory T (nTreg) cells, and CD8+ T cells was shown to be dependent on TGF-β signaling in the thymus [Li et al., 2006b; Marie et al., 2006].

TGF-β plays a major role under inflammatory conditions. TGF-β in the presence of IL-6 drives the differentiation of T helper 17 (Th17) cells, which can promote further inflammation and augment autoimmune conditions [Korn et al., 2009]. TGF-β orchestrates the differentiation of both Treg and Th17 cells in a concentration-dependent manner [Korn et al., 2008]. In addition, TGF-β in combination with IL-4, promotes the differentiation of IL-9- and IL-10-producing T cells, which lack

suppressive function and also promote tissue inflammation [Dardalhon  et al., 2008; Veldhoen et al., 2008]. The biological effects of TGF-β under inflammatory conditions on effector and memory CD8+ T cells are much less understood. In a recent study, it was shown that TGF-β has a drastically opposing role on naïve compared to antigen-experienced/memory CD8+ T cells [Filippi et al., 2008]. When cultured in vitro, TGF-β suppressed naïve CD8+ T cell activation and IFN-γ production, whereas TGF-β enhanced survival of memory CD8+ T cells and increased the production of IL-17 and IFN-γ [Filippi et al., 2008]. TGF-β also plays an important role in suppressing the cells of the innate immune system.

The transforming growth factor beta (TGF-β) family of cytokines are ubiquitous, multifunctional, and essential to survival. They play important roles in growth and development, inflammation and repair, and host immunity. The mammalian TGF-β isoforms (TGF-β1, β2 and β3) are secreted as latent precursors and have multiple cell surface receptors of which at least two mediate signal transduction. Autocrine and paracrine effects of TGF-βs can be modified by extracellular matrix, neighbouring cells and other cytokines. The vital role of the TGF-β family is illustrated by the fact that approximately 50% of TGF-1 gene knockout mice die in utero and the remainder succumb to uncontrolled inflammation after birth. The role of TGF-β in homeostatic and pathogenic processes suggests numerous applications in the diagnosis and treatment of various diseases characterised by inflammation and fibrosis. [Clark and Coker, 1998; Santibañez et al., 2011; Pohlers et al., 2009] Abnormal TGF-β regulation and function are implicated in a growing number of fibrotic and inflammatory pathologies, including pulmonary fibrosis, liver cirrhosis, glomerulonephritis and diabetic nephropathy, congestive heart failure, rheumatoid arthritis, Marfan syndrome, hypertrophic scars, systemic sclerosis, myocarditis, and Crohn’s disease. [Gordon and Globe,2008] TGF-β1 is a polypeptide member of the TGF-β superfamily of cytokines. TGF-β is synthesized as a non-active pro-form, forms a complex with two latent associated proteins latency-associated protein (LAP) and latent TGF- β binding protein (LTBP) and undergoes protolithic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Among the TGF-βs, six distinct isoforms have been discovered although only the TGF-β1, TGF-β2 and TGF-β3 isoforms are expressed in mammals, and their human genes are located on chromosomes 19q13, 1q41 and 14q24, respectively. Out of the three TGF-β isoforms (β1, β2 and β3) only TGF-β1 was linked to fibrogenesis and is the most potent fibrogenic factor for hepatic stellate cells. [Roberts, 1998; Govinden and Bhoola, 2003]. During fibrogenesis, tissue and blood levels of active TGF-β are elevated and overexpression of TGF-β1 in transgenic mice can induce fibrosis. Additionally, experimental fibrosis can be inhibited by anti-TGF-β treatments with neutralizing antibodies or soluble TGF-β receptors [Qi et al.; 1999; Shek and Benyon , 2004; De Gouville et al., 2005; Chen et al., 2009]. TGF-β1 induces its own mRNA to sustain high levels in local sites of injury. The effects of TGF-β1 are classically mediated by intracellular signalling via Smad proteins. Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory. [Parsons et al., 2013; Friedman, 2008; Kubiczkova et al., 2012] Smad1/5/8, MAP kinase (mitogen-activated protein) and PI3 kinase are further signalling pathways in different cell types for TGF-β1 effects.

TGF-β is found in all tissues, but is particularly abundant in bone, lung, kidney and placental tissue. TGF-β is produced by many, but not all parenchymal cell types, and is also produced or released by infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, all these cells are potential sources of TGF-β. In general, the release and activation of TGF-β stimulates the production of various extracellular matrix proteins and inhibits the degradation of these matrix proteins. [Branton and Kopp, 1999]

TGF-β 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. [Letterio and Roberts; 1998]

In the liver TGF-β1 is released by activated Kupffer cells, liver sinusoidal endothelial cells, and platelets; in the further course of events also activated hepatic stellate cells express TGF-β1. Hepatocytes do not produce TGF-β1 but are implicated in intracellular activation of latent TGF-β1. [Roth et al., 1998; Kisseleva and Brenner, 2007; Kisseleva and Brenner, 2008; Poli, 2000; Liu et al., 2006]

TGF-β1 is the most established mediator and regulator of epithelial-mesenchymal-transition (EMT) which further contributes to the production of extracellular matrix. It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4. [Kolios et al., 2006; Bataller and Brenner, 2005; Guo and Friedman,2007; Brenner,2009; Kaimori et al., 2007; Gressner et al., 2002; Kershenobich Stalnikowitz and Weisssbrod, 2003; Li et al., 2008; Matsuoka and Tsukamoto, 1990; Kisseleva and Brenner, 2008; Poli, 200; Parsons et al., 2007; Friedman 2008; Liu et al., 2006]

TGF-β1 induces apoptosis and angiogenesis in vitro and in vivo through the activation of vascular endothelial growth factor (VEGF) High levels of VEGF and TGF-β1 are present in many tumors. Crosstalk between the signalling pathways activated by these growth factors controls endothelial cell apoptosis and angiogenesis. [Clark and Coker; 1998]

 

How it is Measured or Detected

The specific type of measurement(s) might vary with tissue, environment and context and will need to be described for different tissue contexts  as used within different AOP descriptions.

In general, quantification of inflammatory markers can be done by:

• qRT-PCR (mRNA expression)
• ELISA
• Immunocytochemistry
• Immunoblotting

For descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014
 

LIVER:

There are several assays for TGB-β1 measurement available.

e.g. Human TGF-β1 ELISA Kit. The Human TGF-β 1 ELISA (Enzyme –Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human TGF-β1 in serum, plasma, cell culture supernatants, and urine. This assay employs an antibody specific for human TGF-β1 coated on a 96-well plate. Standards and samples are pipetted into the wells and TGF-β1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human TGF-β1 antibody is added. After washing away unbound biotinylated antibody, HRP- conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and colour develops in proportion to the amount of TGF-β1 bound. The StopSolution changes the colour from blue to yellow, and the intensity of the colour is measured at 450 nm [Mazzieri et al., 2000]

Listed below are common methods for detecting the KE, however there may be other comparable methods that are not listed.

Assay	Reference	Description	OECD Approved Assay
RT-qPCR  Q-PCR	(Veremeyko et al., 2012; Alwine et al, 1977; Forlenza et al., 2012)	Measures mRNA expression of cytokines, chemokines and inflammatory markers	No
Immunoblotting (western blotting)	(Lee et al., 2008)	Uses antibodies specific to proteins of interest, can used to detect presence of pro-inflammatory mediators in samples of cell or tissue lysate	No
Whole blood stimulation assay	(Thurm & Halsey, 2005)	Detects inflammatory cytokines in blood	No
Imaging tests	(Rollins & Miskolci, 2014)	A qualitative technique using a cytokine specific antibodies and fluorophores can be used to visualize expression patterns, subcellular location of the target and protein-protein interactions.   Common examples include double immunofluorescence confocal microscopy or other molecular imaging modalities.	No
Flow-cytometry	(Karanikas et al., 2000)	Detects the intracellular cytokines with stimulation.	No
Immunoassays (ex. enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot), radioimmunoassay)	(Amsen et al., 2009; Engvall & Perlmann, 1972; Ji & Forsthuber, 2016; Goldsmith, 1975)	Plate based assay technique using antibodies to detect presence of a protein in a liquid sample.   Can be used to identify presence of an inflammatory cytokine of interest especially when in low concentrations.	No
Inflammatory cytokine arrays	(Amsen et al., 2009)	Similar to the ELISA, except using a membrane-based rather than plate-based approach. Can be used to measure multiple cytokine targets concurrently.	No
Immunohistochemistry (IHC)	(Amsen et al., 2009; Coons et al., 1942)	Immobilized tissue or cell cultures are stained using antibodies for specificity of ligands of interest. Versions of the assays can be used to visualize localization of inflammatory cytokines.	No

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• Veldhoen M, Uyttenhove C, van Snick J, Helmby H, Westendorf A, Buer J, Martin B, Wilhelm C, Stockinger B. Transforming growth factor-beta 'reprograms' the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat Immunol 2008;9:1341–1346.

  

Abdel-Magied, N., S. M., Shedid and Ahmed, A. G. (2019), “Mitigating effect of biotin against irradiation-induced cerebral cortical and hippocampal damage in the rat brain tissue”, Environmental Science and Pollution Research, Vol. 26/13, Springer, London, https://doi.org/10.1007/S11356-019-04806-X.   

Alwine, J. C., D. J. Kemp and G. R. Stark (1977), “Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 74/12, United States National Academy of Sciences, Washington, D.C., https://doi.org/10.1073/pnas.74.12.5350 

Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, https://doi.org/10.1007/978-1-59745-447-6_5

Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel,  https://doi.org/10.3390/ijms19113669.  

Cho, H. J. et al. (2017), “Role of NADPH Oxidase in Radiation-induced Pro-oxidative and Pro-inflammatory Pathways in Mouse Brain”, International Journal of Radiation Biology, Vol. 93/11, Informa, London, https://doi.org/10.1080/09553002.2017.1377360.   

Coons, A. H. et al. (1942), “The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody”, The Journal of Immunology, Vol. 45/3, American Association of Immunologists, Minneapolis, pp. 159-169 

Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, The Journal of Immunology, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135 

Fan, L. W. and Y. Pang. (2017), "Dysregulation of neurogenesis by neuroinflammation: Key differences in neurodevelopmental and neurological disorders", Neural Regeneration Research, Vol. 12/3, Wolters Kluwer, Alphen aan den Rijn, https://doi.org/10.4103/1673-5374.202926.  

Forlenza, M. et al. (2012), “The use of real-time quantitative PCR for the analysis of cytokine mRNA levels” in Cytokine Protocols, Springer, New York, https://doi.org/10.1007/978-1-61779-439-1_2  

Gaber, M. W. et al. (2003), “Differences in ICAM-1 and TNF-alpha expression between large single fraction and fractionated irradiation in mouse brain”, International Journal of Radiation Biology, Vol. 79/5, Informa, London, https://doi.org/10.1080/0955300031000114738.   

Goldsmith, S. J. (1975), "Radioimmunoassay: Review of basic principles", Seminars in Nuclear Medicine, Vol. 5/2, https://doi.org/10.1016/S0001-2998(75)80028-6. 

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

Ismail, A. F. M., A.A.M. Salem and M.M.T. Eassawy (2016), “Modulation of gamma-irradiation and carbon tetrachloride induced oxidative stress in the brain of female rats by flaxseed oil”, Journal of Photochemistry and Photobiology B: Biology, Vol. 161, Elsevier, Amsterdam, https://doi.org/10.1016/J.JPHOTOBIOL.2016.04.031. 

Ji, N. and T. G. Forsthuber. (2014), "ELISPOT Techniques" (pp. 63–71), https://doi.org/10.1007/7651_2014_111. 

Kalm, M., K. Roughton and K. Blomgren. (2013), "Lipopolysaccharide sensitized male and female juvenile brains to ionizing radiation", Cell Death & Disease, Vol. 4/12, Nature Publishing Group, Berlin, https://doi.org/10.1038/cddis.2013.482. 

Karanikas, V. et al. (2000), “Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy”, Clinical Cancer Research, Vol. 6/3, American Association for Cancer Research, Philadelphia, pp. 829–837  

Kim, S. H. et al. (2002), “Expression of TNF-alpha and TGF-beta 1 in the rat brain after a single high-dose irradiation”, Journal of Korean Medical Science, Vol. 17/2, Korean Medical Association, Seoul, https://doi.org/10.3346/JKMS.2002.17.2.242.   

Lee, J. W. et al. (2008), “Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation”, Journal of Neuroinflammation, Vol. 5/1, BioMed Central, London, https://doi.org/10.1186/1742-2094-5-37 

Lee, W. H. et al. (2010), “Irradiation induces regionally specific alterations in pro-inflammatory environments in rat brain”, International Journal of Radiation Biology, Vol. 86/2, Informa, London, https://doi.org/10.3109/09553000903419346. 

Parihar, V. K. et al. (2018), “Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice”, Experimental Neurology, Vol. 305, Elsevier, Amsterdam, https://doi.org/10.1016/J.EXPNEUROL.2018.03.009. 

Parihar, V. K. et al. (2020), "Sex-Specific Cognitive Deficits Following Space Radiation Exposure", Frontiers in Behavioral Neuroscience, Vol. 14, https://doi.org/10.3389/fnbeh.2020.535885. 

Rollins, J. and V. Miskolci (2014), “Immunofluorescence and subsequent confocal microscopy of intracellular TNF in human neutrophils” in Cytokines Bioassays, Springer, London, https://doi.org/10.1007/978-1-4939-0928-5_24

Thurm, C. W. and J. F. Halsey (2005), “Measurement of Cytokine Production Using Whole Blood”, in Current Protocols in Immunology, John Wiley & Sons, Inc., Hoboken, https://doi.org/10.1002/0471142735.im0718bs66 

Veeraraghavan, J. et al. (2011), "Low-dose γ-radiation-induced oxidative stress response in mouse brain and gut: Regulation by NFκB–MnSOD cross-signaling", Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol. 718/1–2, Elsevier, Amsterdam, https://doi.org/10.1016/j.mrgentox.2010.10.006. 

Veremeyko, T. et al. (2012), “Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation”, Journal of Visualized Experiments: JoVE, Vol. 65, MyJove Corporation, Cambridge, https://doi.org/10.3791/4097

Weavers, H. and P. Martin (2020), “The cell biology of inflammation: From common traits to remarkable immunological adaptations”, Journal of Cell Biology, Vol. 219, Rockefeller University Press, New York, https://doi.org/10.1083/jcb.202004003 

Event: 2066: Altered Signaling Pathways

Short Name: Altered Signaling

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Altered signaling is applicable to all animals as cell signaling occurs among animal cells. This includes vertebrates such as humans, mice and rats (Nair et al., 2019).

Life stage applicability: This key event is not life stage specific.

Sex applicability: This key event is not sex specific.

Evidence for perturbation by a stressor: Multiple studies show that signaling pathways can be disrupted by many types of stressors including ionizing radiation and altered gravity (Cheng et al., 2020; Coleman et al., 2021; Su et al., 2020; Yentrapalli et al., 2013).

Key Event Description

Cells receive, process, and transmit signals to respond to their environment via signaling pathways. Signaling pathways are groups of molecules that work together in a cell to control physiological and metabolic processes. Kinases, for example, are important signaling molecules that can phosphorylate other proteins (Svoboda & Reenstra, 2002). Initiation of signaling pathways is an important component of cellular homeostasis including normal cell development and the response to cellular damage from exposure to external stressors (Esbenshade & Duzic, 2006). Signaling pathways are themselves activated by signals and the same signal can lead to different responses depending on the tissue type (Hamada, et al. 2011; Svoboda & Reenstra, 2002). Examples of signals include the activation of receptors to activate transcriptional targets, induction of receptor-ligand interactions and the initiation of cell-cell contact, or cell-extracellular matrix contact (Hunter, 2000). Many signalling pathways are crucial to intercellular communication via membrane receptors that transduce signals into the cell, while others are activated in an intracellular manner (Svoboda & Reenstra, 2002). Altered signalling (i.e., increase/decrease) can lead to different physiological outcomes, meaning that the directionality of the signaling response, determines the end outcome. For example, increase of the PI3K/Akt/mTOR pathway, which under physiological conditions is responsible for regulating the cell cycle, can lead to increased proliferation and decreased apoptosis. However, a decrease expression of this pathway can lead to an increase in apoptosis and decreased proliferation (Porta et al., 2014; Venkatesulu et al., 2018).

How it is Measured or Detected

Method of Measurement	Reference	Description	OECD Approved Assay
Kinase assays	(Svoboda & Reenstra, 2002)	Block kinase with inhibitors to monitor the activity of a kinase of interest.	No
Cell behaviour assays	(Svoboda & Reenstra, 2002)	Signal transduction events of cells are monitored. Cells are exposed to varying levels of signaling proteins and the resulting actions of a cell are observed (changes in structure, cell shape, matrix binding etc.).	No
Ratiometric or single-wavelength dyes	(Svoboda & Reenstra, 2002)	Detects alterations in signal-transduction activities via monitoring changes in detectable wavelengths.	No
Fluorescence microscopy/spectroscopy	(Oksvold et al., 2002)	Measures cell localization, protein interactions, signal propagation, amplification, and integration in the cell in real-time, or upon stimulation.	Yes
Green Fluorescent Protein (GFP)	(Zaccolo and Pozzan, 2000)	GFP assays act as fluorescent reporters but also as a marker of intracellular signalling events i.e. second messengers Ca2+ and cAMP, or for pH in different various cell compartments	No
Fluorescence Resonance Energy Transfer (FRET)	(Bunt and Wouters, 2017)	Assay helps illuminate the interactions between biological molecules	No
Fluorescence recovery after photobleaching (FRAP)	(Svoboda & Reenstra, 2002)	Determines mobility and diffusion of small molecules.	No
Immunoprecipitation	(Svoboda & Reenstra, 2002)	Involves isolating and concentrating a particular protein from mixed samples to detect changes in signalling molecule activity.	Chromatin immunoprecipitation approved for analyzing histone modifications
Immunohistochemistry	(Kurien et al., 2011; Svoboda & Reenstra, 2002)	Northern, western and southern blotting techniques can be used to visualize signal transduction events. For example, antibodies with recognition epitopes can be used to locate active configurations or phosphorylated proteins within a cell or cell lysate.	No
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)	(Veremeyko et al., 2012; Alwine et al, 1977)	Measures mRNA expression of the gene of interest.	No
Enzyme-linked immunosorbent assay (ELISA)	(Amsen et al., 2009; Engvall & Perlmann, 1972)	Plate-based assay technique using antibodies to detect presence of a protein in a liquid sample. Can be used to identify presence of a protein of interest especially in when in low concentrations	No

References

Alwine, J. C., D. J. Kemp and G. R. Stark (1977), “Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 74/12, United States National Academy of Sciences, Washington, D.C., https://doi.org/10.1073/pnas.74.12.5350

Amsen, D., de Visser, K. E., and Town, T. (2009), “Approaches to determine expression of inflammatory cytokines”, in Inflammation and Cancer, Humana Press, Totowa, https://doi.org/10.1007/978-1-59745-447-6_5

Bunt, G., and F. S. Wouters (2017), “FRET from single to multiplexed signaling events”, Biophysical reviews, Vol. 9, Springer, London, https://doi.org/10.1007/s12551-017-0252-z

Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, Pflugers Archiv European Journal of Physiology, Vol. 469, Springer, New York, https://doi.org/10.1007/s00424-017-1969-z

Coleman, M. A. et al. (2015), “Low-dose radiation affects cardiac physiology: gene networks and molecular signaling in cardiomyocytes”,  American Journal of Physiology - Heart and Circulatory Physiology, Vol. 309/11, American Physiological Society, Rockville, https://doi.org/10.1152/ajpheart.00050.2015

Engvall, E., and P. Perlmann (1972), “Enzyme-Linked Immunosorbent Assay, Elisa”, The Journal of Immunology, Vol. 109/1, American Association of Immunologists, Minneapolis, pp. 129-135

Esbenshade, T. A., and E. Duzic (2006), “Overview of signal transduction”, Current Protocols in Pharmacology, Vol. 31/1, John Wiley & Sons, Ltd., Hoboken, https://doi.org/10.1002/0471141755.ph0201s31

Hamada, N. et al. (2011), “Signaling pathways underpinning the manifestations of ionizing radiation-induced bystander effects”, Current Molecular Pharmacology, Vol. 4/2, Bentham Science Publishers, Sharjah UAE, https://doi.org/10.2174/1874467211104020079

Hunter, T. (2000), “Signaling - 2000 and beyond”, Cell, Vol. 100/1, Cell Press, Cambridge, https://doi.org/10.1016/s0092-8674(00)81688-8

Kurien, B. T. et al. (2011), “An overview of Western blotting for determining antibody specificities for immunohistochemistry”, in Signal Transduction Immunohistochemistry Methods and Protocols, Springer, London, https://doi.org/10.1007/978-1-61779-024-9_3

Nair, A. et al. (2019), “Conceptual Evolution of Cell Signaling”, International journal of molecular sciences, Vol. 20/13, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/ijms20133292

Oksvold, M. P. et al. (2002), “Fluorescent histochemical techniques for analysis of intracellular signaling”, The Journal of Histochemistry and Cytochemistry, Vol. 50/3, SAGE Publications, Thousand Oaks, https://doi.org/10.1177/002215540205000301

Porta, C., C. Paglino and A. Mosca (2014), “Targeting PI3K/Akt/mTOR Signaling in Cancer”, Frontiers in Oncology, Vol. 4, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fonc.2014.00064

Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, Life Sciences, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253

Svoboda, K. K. and W. R. Reenstra (2002), “Approaches to studying cellular signaling: a primer for morphologists”, The Anatomical record, Vol. 269/2, John Wiley & Sons, Ltd., Hoboken, https://doi.org/10.1002/ar.10074

Venkatesulu, B. P. et al. (2018), “Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms”, JACC: Basic to Translational Science, Vol. 3/4, Elsevier, Amsterdam, https://doi.org/10.1016/j.jacbts.2018.01.014

Veremeyko, T. et al. (2012), “Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation”, Journal of Visualized Experiments: JoVE, Vol. 65, MyJove Corporation, Cambridge, https://doi.org/10.3791/4097

Yentrapalli, R. et al. (2013), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, PloS one, Vol. 8/8, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0070024

Zaccolo, M. and T. Pozzan (2000), “Imaging signal transduction in living cells with GFP-based probes”, IUBMB life, Vol. 49/5, John Wiley & Sons, Ltd., Hoboken, https://doi.org/10.1080/152165400410218

Event: 2067: Altered, Nitric Oxide Levels

Short Name: Altered, Nitric Oxide Levels

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Altered nitric oxide is applicable to vertebrates only, as endothelial NO synthase (eNOS) is required for the formation of NO from the amino acid, L-arginine, and only vertebrates have a true endothelial lining (Yano et al., 2007).

Life stage applicability: This key event is not life stage specific.

Sex applicability: This key event is not sex specific (Soucy et al., 2011; Takeda et al., 2003).

Evidence for perturbation by a stressor: Current literature provides ample evidence of external stressors, including ionizing radiation exposure and altered gravity, inducing significant changes to levels of nitric oxide, nitrate, and NO synthase (Soucy et al., 2011; Zhang et al., 2009).

Key Event Description

Nitric oxide (NO) is a diffusible molecule produced by many cell types, including endothelial cells, and is responsible for vasodilation (Schulz, Gori & Münzel, 2011; Soloviev & Kizub, 2019). The source of endogenous NO is L-arginine (Burov et al., 2022). Production of NO in the body can occur through nitric oxide synthase (NOS), an enzyme that degrades L-arginine in the presence of oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) (Luiking, Engelen & Deutz, 2010). Tetrahydrobiopterin (BH4) is an important cofactor of NOS, allowing the enzymatic production of NO. A non-enzymatic method to produce NO includes the reduction of nitrite (Luiking, Engelen & Deutz, 2010). NO is constitutively produced by endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS), and can be increased by inducible NOS (iNOS) (Powers & Jackson, 2008). Changes in the expression or activity of NOS enzymes can cause changes in NO levels. For example, iNOS is mainly regulated through transcription and its upregulation can result in increased production of NO (Farah, Michel & Balligand, 2018). Also, eNOS can be regulated by Ca2+ concentrations and blood flow shear stress through phosphorylation at Ser1177 (activating) and Thr495 (inhibiting) (Förstermann, 2010).

How it is Measured or Detected

Without measuring NO levels directly, NOS levels can be used as a proxy to measure NO production. eNOS and iNOS are common points for assessing NO levels indirectly. Decreased NOS protein expression often corresponds to a decrease in NO. However, it is important to note that NOS levels do not perfectly correlate with NO levels. Increased NOS can also decrease NO if paired with a simultaneous increase in ROS, which, through oxidizing the enzyme’s cofactor BH4, causes NOS uncoupling (Forstermann, 2010; Zhang et al., 2009). Uncoupled NOS produces additional ROS that react with NO and reduce its overall abundance. Therefore, in this case, higher levels of NOS correlate to increased quantity of uncoupled NOS and a subsequent drop in NO bioavailability (Soloviev & Kizub, 2019). 

Assay	Reference	Description	OECD Approved Assay
Western blotting/immunoblotting	(Hong et al., 2013; Baker et al., 2009; Yan et al., 2020; Zhang et al., 2009; Zhang et al., 2008; Shi et al., 2012; Azimzadeh et al., 2017; Azimzadeh et al., 2015)	Western blotting/immunoblotting is used to determine levels of inducible and endothelial NOS (NO synthesizing enzyme) in its phosphorylated and unphosphorylated forms, as well as nitrotyrosine (an indicator of NO). NOS and nitrotyrosine are detected by antibodies of each protein, visualized using chemiluminescence, and quantified using densitometry.	No
Nitric oxide/nitrate/nitrate (NOx) assay kit (Griess assay)	(Azimzadeh et al., 2017; Adbel-Magied & Shedid, 2019; Yan et al., 2020; Cervelli et al., 2017; Siamwala et al., 2010)	Levels of nitrite/nitrate (NOx) are determined using the NO assay kit. Nitrate reductase is used to convert nitrate into nitrite and the Griess reagent is then used to quantify levels of nitrite.	No
Immunohistochemical staining	(Fuji et al., 2016)	Uses an antibody to detect and measure levels of eNOS.	No
Immunofluorescence	(Hamada et al., 2019)	Uses fluorescent dye-labeled eNOS antibodies to visualize and determine eNOS levels.	No
ELISA kit	(Hasan et al., 2020; Azimzadeh et al., 2015)	Used to determine levels of NO and iNOS in serum by immobilizing the target antigen and binding it to associated antibodies linked to reporter enzymes. The activity of the reporter enzymes is then measured to determine levels of NO and iNOS.
4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescent probe	(Soucy et al., 2011; Soucy et al., 2010)	Used to detect low concentrations of NO by reacting with it to become a fluorescent benzotriazole that can then be visualized and measured.	No

References

Abdel-Magied, N. and S. M. Shedid (2019), “Impact of zinc oxide nanoparticles on thioredoxin-interacting protein and asymmetric dimethylarginine as biochemical indicators of cardiovascular disorders in gamma-irradiated rats”, Environmental Toxicology, Vol. 35/4, John Wiley & Sons, Inc., Hoboken,  https://doi.org/10.1002/tox.22879

Azimzadeh, O. et al. (2015), “Integrative Proteomics and Targeted Transcriptomics Analyses in Cardiac Endothelial Cells Unravel Mechanisms of Long-Term Radiation-Induced Vascular Dysfunction”, Journal of Proteome Research, Vol. 14/2, American Chemical Society, Washington, https://doi.org/10.1021/pr501141b

Azimzadeh, O. et al. (2017), “Proteome analysis of irradiated endothelial cells reveals persistent alteration in protein degradation and the RhoGDI and NO signalling pathways”, International Journal of Radiation Biology, Vol. 93/9, Informa, London, https://doi.org/10.1080/09553002.2017.1339332

Baker, J. E. et al. (2009), “10 Gy total body irradiation increases risk of coronary sclerosis, degeneration of heart structure and function in a rat model”, International Journal of Radiation Biology, Vol. 85/12, Informa, London, https://doi.org/10.3109/09553000903264473

Burov, O. N. et al. (2022), “Mechanisms of nitric oxide generation in living systems”, Nitric Oxide, Vol. 118, Elsevier, Amsterdam, https://doi.org/10.1016/j.niox.2021.10.003.

Cervelli, T. et al. (2017), “A new natural antioxidant mixture protects against oxidative and DNA damage in endothelial cell exposed to low-dose irradiation”, Oxidative Medicine and Cellular Longevity, Vol. 2017, Hindawi, London, https://doi.org/10.1155/2017/9085947

Farah, C., L. Y. M. Michel and J.-L. Balligand. (2018), "Nitric oxide signalling in cardiovascular health and disease", Nature Reviews Cardiology, Vol. 15/5, Springer Nature, London, https://doi.org/10.1038/nrcardio.2017.224.

Förstermann, U. (2010), "Nitric oxide and oxidative stress in vascular disease", Pflügers Archiv - European Journal of Physiology, Vol. 459, Springer Nature, London, https://doi.org/10.1007/S00424-010-0808-2.

Fuji, S. et al. (2016), “Association between endothelial function and micro-vascular remodeling measured by synchrotron radiation pulmonary micro-angiography in pulmonary arterial hypertension”, General Thoracic and Cardiovascular Surgery, Vol. 64/10, Springer, London, https://doi.org/10.1007/s11748-016-0684-6

Hamada, N. et al. (2020), “Ionizing Irradiation Induces Vascular Damage in the Aorta of Wild-Type Mice”, Cancers, Vol. 12/10, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/CANCERS12103030

Hamada, N. et al. (2022), “Temporal Changes in Sparing and Enhancing Dose Protraction Effects of Ionizing Irradiation for Aortic Damage in Wild-Type Mice”, Cancers, Vol. 14/14, Multidisciplinary Digital Publishing Institute, Basel, https://doi.org/10.3390/cancers1414331

Hasan, H. F., R. R. Radwan and S. M. Galal (2020), “Bradykinin‐potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway”, Clinical and Experimental Pharmacology and Physiology, Vol. 47/2, Wiley-Blackwell, Hoboken, https://doi.org/10.1111/1440-1681.13202

Hong, C. W. et al. (2013), “Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage”, Journal of Radiation Research, Vol. 54/6, Oxford University Press, Oxford, https://doi.org/10.1093/JRR/RRT066

Powers, S. K. and M. J. Jackson. (2008), "Exercise-Induced Oxidative Stress: Cellular Mechanisms and Impact on Muscle Force Production", Physiological Reviews, Vol. 88/4, The American Physiological Society, Rockville, https://doi.org/10.1152/physrev.00031.2007.

Schulz, E., T. Gori and T. Münzel. (2011), "Oxidative stress and endothelial dysfunction in hypertension", Hypertension Research, Vol. 34/6, Nature Portfolio, London, https://doi.org/10.1038/hr.2011.39.

Shi, F. et al. (2012), “Effects of Simulated Microgravity on Human Umbilical Vein Endothelial Cell Angiogenesis and Role of the PI3K-Akt-eNOS Signal Pathway”, PLoS ONE, Vol. 7/7, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0040365

Siamwala, J. H. et al. (2010), “Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells”, Protoplasma, Vol. 242/1, Springer, London, https://doi.org/10.1007/S00709-010-0114-Z

Soloviev, A. I. and I. V. Kizub. (2019), "Mechanisms of vascular dysfunction evoked by ionizing radiation and possible targets for its pharmacological correction", Biochemical Pharmacology, Vol. 159, Elsevier, Amsterdam, https://doi.org/10.1016/J.BCP.2018.11.019

Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, Journal of Applied Physiology, Vol. 108/5, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00946.2009.

Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, Radiation Research, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1.

Yan, T., et al. (2020), “Ionizing radiation induces BH4 deficiency by downregulating GTP-cyclohydrolase 1, a novel target for preventing and treating radiation enteritis”, Biochemical Pharmacology, Vol. 180, Elsevier, Amsterdam, https://doi.org/10.1016/J.BCP.2020.114102

Takeda, I., et al. (2013), “Possible Role of Nitric Oxide in Radiation-Induced Salivary Gland Dysfunction”, Radiation Research, Vol. 159/4, BioOne, https://doi.org/10.1667/0033-7587(2003)159[0465:PRONOI]2.0.CO;2

Yano, K., et al. (2007), “Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium”, Blood, Vol. 109/2, American Society of Hematology, Washington, D.C., https://doi.org/10.1182/blood-2006-05-026401

Yao, L. et al. (2010), "The role of RhoA/Rho kinase pathway in endothelial dysfunction", Journal of Cardiovascular Disease Research, Vol. 1/4, Elsevier, Amsterdam, https://doi.org/10.4103/0975-3583.74258

Zhang, R. et al. (2009), “Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats”, Journal of Applied Physiology, Vol. 106, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.01278.2007

Event: 2068: Increase, Endothelial Dysfunction

Short Name: Increase, Endothelial Dysfunction

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Endothelial dysfunction is applicable to vertebrates as only vertebrates have a true endothelial lining (Yano et al., 2007).

Life stage applicability: Although endothelial dysfunction may occur due to aging (Hererra et al., 2010), this key event can occur at any life stage (Chang et al., 2017; Lee et al., 2020).

Sex applicability: This key event is not sex specific (Hughson et al., 2018; Lee et al., 2020).

Evidence for perturbation by a stressor: Multiple studies show that endothelial dysfunction can be triggered by many types of stressors including ionizing radiation and altered gravity (Cheng et al., 2017; Soucy et al., 2011; Su et al., 2020; Yentrapalli et al., 2013).

Key Event Description

The endothelium is the innermost lining of blood vessels consisting of a single layer of endothelial cells. As the layer separating blood and vessel walls, the endothelium controls the flow of molecules, fluid, and circulating blood cells between the two. However, the specific functions and even the structure of endothelial cells vary greatly depending on the organ (Ricard et al., 2021). Dysfunction to the vascular endothelium can age arteries and is the result of increased proliferation and apoptotic behaviour of cells including an increased response to endothelial constrictors. It is also represented by an imbalance between vasodilators and vasoconstrictors which are produced by the endothelium. The dysfunction can encompass vasospasm, thrombosis, penetration of immune cells (i.e macrophage) and an increase in cyclooxygenase. These processes can activate the endothelium and a prolonged state of activation is problematic and is referred to as endothelial dysfunction (Sitia et al., 2010; Deanfield et al., 2005; Konukoglu & Uzun, 2017; Korpela & Liu, 2014). Other factors leading to endothelial dysfunction are loss in endothelial function leading to cell senescence and a low proliferative capacity of endothelial progenitor cells.

How it is Measured or Detected

Endothelial cell senescence 

Assay	Reference	Description	OECD Approved Assay
Senescence-associated beta-galactosidase staining (SA-beta-gal)	(Farhat et al., 2008; González-Gualda et al., 2021; Hooten et al., 2017)	Can be used to measure senescence-associated β-galactosidase activity, a marker for senescent cells.	No
Bromodeoxyuridine (BrdU) detected with staining incorporation	(González-Gualda et al., 2021)	Reduced BrdU incorporation can indicate a lack of DNA synthesis.	No
Immunohistochemistry to detect senescence markers.	(González-Gualda et al., 2021)	Markers include Ki67 and Lamin B1. Reduced Ki67 can indicate reduced proliferation. Reduced Lamin B1 indicates impaired structural integrity of the nucleus.	No
Cell morphology and size measured with light microscopy or flow cytometry.	(González-Gualda et al., 2021)	Senescent cells exhibit an enlarged and flattened morphology.	No

 

Cell death: 

See the increase, cell death KE for methods to measure endothelial cell death. 

 

Impaired vasomotion

Assay	Reference	Description	OECD Approved Assay
Concentration-response curves to vasodilators/vasoconstrictors	(Deanfield et al., 2005; Verma et al., 2003)	Measurement of endothelial relaxation/contraction of blood vessels can give insight into endothelial dysfunction. This can be induced by endothelium-independent stimuli to stimulate vasodilation or vasoconstriction. A decreased stimuli response can be indicative of endothelial dysfunction.	No
Detection of contractile factors (eg. endothelin) using enzyme-linked immunosorbent assay (ELISA).	(Abdel-Sayed et al., 2003)	Endothelin is an endothelium-derived vasoconstrictor.	No

References

Abdel-Sayed, S. et al. (2003), “Measurement of plasma endothelin-1 in experimental hypertension and in healthy subjects”, American Journal of Hypertension, Vol. 16/7, Oxford University Press, Oxford, https://doi.org/10.1016/S0895-7061(03)00903-8

Chang, P. Y. et al. (2017), “MSC-derived cytokines repair radiation-induced intra-villi microvascular injury”, Oncotarget, Vol. 8/50, Impact Journals, Orchard Park, https://doi.org/10.18632/oncotarget.21236

Cheng, Y. P. et al. (2017), “Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats”, Pflugers Archiv European Journal of Physiology, Vol. 469, Springer, New York, https://doi.org/10.1007/s00424-017-1969-z

Deanfield, J. et al. (2005), “Endothelial function and dysfunction”, Journal of hypertension, Vol. 23/1, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1097/00004872-200501000-00004

Farhat, N. et al. (2008), “Stress-induced senescence predominates in endothelial cells isolated from atherosclerotic chronic smokers”, Canadian Journal of Physiology and Pharmacology, Vol. 86/11, Canadian Science Publishing, Ottawa, https://doi.org/10.1139/Y08-082

González-Gualda, E. et al. (2021), “A guide to assessing cellular senescence in vitro and in vivo”, The FEBS Journal, Vol. 288, FEBS press, https://doi.org/10.1111/febs.15570 

Herrera, M. D. et al. (2010), “Endothelial dysfunction and aging: An update”, Ageing Research Reviews, Vol 9/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.arr.2009.07.002

Hooten, N. N. and M. K. Evans (2017), “Techniques to Induce and Quantify Cellular Senescence”, Journal of Visualized Experiments: JoVE, Vol. 123, MyJove Corporation, Cambridge,  https://doi.org/10.3791/55533

Hughson, R. L., A. Helm and M. Durante (2018), “Heart in space: effect of the extraterrestrial environment on the cardiovascular system”, Nature Reviews Cardiology, Vol. 15/3, Nature Portfolio, London, https://doi.org/10.1038/nrcardio.2017.157

Konukoglu, D., and H. Uzun (2017), “Endothelial Dysfunction and Hypertension”, in Hypertension: from basic research to clinical practice, Springer, London, https://doi.org/10.1007/5584_2016_90

Korpela, E., and S. K. Liu (2014), “Endothelial perturbations and therapeutic strategies in normal tissue radiation damage”, Radiation Oncology, Vol. 9, BioMed Central, London, https://doi.org/10.1186/s13014-014-0266-7

Lee, S. et al.  (2020), “Arterial structure and function during and after long-duration spaceflight”, Journal of Applied Physiology, Vol. 129/1, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00550.2019

Ricard, N. et al. (2021), “The quiescent endothelium: signalling pathways regulating organ-specific endothelial normalcy”, Nature reviews cardiology, Vol. 18/8, Springer Nature, https://doi.org/10.1038/s41569-021-00517-4 

Sitia, S. et al. (2010), “From endothelial dysfunction to atherosclerosis”, Autoimmunity Reviews, Vol. 9/12, Elsevier, Amsterdam, https://doi.org/10.1016/j.autrev.2010.07.016

Soucy, K. G. et al. (2011), “HZE ⁵⁶Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, Radiation Research, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1

Su, Y. T. et al. (2020), “Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats”, Life Sciences, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253

Verma, S., M. R. Buchanan and T. J. Anderson (2003), “Endothelial function testing as a biomarker of vascular disease”, Circulation, Vol. 108/17, Lippincott Williams & Wilkins, Philadelphia, https://doi.org/10.1161/01.CIR.0000089191.72957.ED

Yano, K. et al. (2007), “Phenotypic heterogeneity is an evolutionarily conserved feature of the endothelium”, Blood, Vol. 109/2, American Society of Hematology, Washington, D.C., https://doi.org/10.1182/blood-2006-05-026401

Yentrapalli, R. et al. (2013), “The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation”, PloS one, Vol. 8/8, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0070024

List of Adverse Outcomes in this AOP

Event: 2069: Occurrence, Vascular Remodeling

Short Name: Occurrence, Vascular Remodeling

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Taxonomic applicability: Vascular remodelling is applicable to all species with a closed circulatory system where blood is transported throughout the body via blood vessels with corresponding vessel walls (Renna, Heras & Miatello, 2013). Closed circulatory systems are present in most vertebrates and some invertebrates.

Life stage applicability: This key event is not life stage specific. However, advancing age is a risk factor for vascular remodeling (Harvey, Montezano & Touyz, 2015).

Sex applicability: This key event is not sex specific. However, men are shown to develop vascular remodeling younger than women (Kessler et al., 2019).

Evidence for perturbation by a stressor: Current literature provides ample evidence of vascular remodelling being induced by stressors including ionizing radiation exposure and altered gravity (Shen et al. 2018; Su et al., 2020; Delp et al., 2000, Cheng et al., 2017, Yu et al., 2011; Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011).

Key Event Description

The vascular wall is composed of endothelial, smooth muscle and fibroblast cell interactions (Gibbons & Dzau, 1994; Renna, Heras & Miatello, 2013). The vasculature is capable of detecting changes in its surroundings and maintaining homeostasis (Gibbons & Dzau, 1994; Renna, Heras & Miatello, 2013). The functionality of blood vessels is highly dependent on their structure, with changes in arterial morphology being associated with downstream impacts (Gibbons & Dzau, 1994). Vascular remodeling is a term for many histological changes, including increased vascular stiffness, wall shear stress, intima-media thickening (IMT), increased intima-media section area, and increased vessel diameter (Herity et al., 1999). As blood vessels stiffen, this impacts systolic and diastolic pressure and pulse which can be indicators of vascular remodeling. Cellular level changes characterized by processes of growth, death, migration and production or degradation of the extracellular matrix (ECM) result in inflammation (increase in VCAM, ICAM, cytokines, chemokines) and calcification (changes in ratios of collagen and elastin) (Gibbons & Dzau, 1994). Initial tissue injury and resulting remodeling can also lead to turbulent blood flow causing further structural changes like increased vessel fibrosis.  Increased vascular remodelling is often associated with a build-up of plaque in the arteries (known as atherosclerosis) due to impaired healing, which forces the vessel walls to attempt to remodel to maintain blood flow (Sylvester et al., 2018).

How it is Measured or Detected

Assay	Reference	Description	OECD Approved Assay
Pulse wave velocity (PWV)	(Soucy et al., 2007; Soucy et al., 2010; Soucy et al., 2011)	Used to measure blood vessel stiffness. Calculated using measurements from a Doppler probe and electrocardiogram (ECG).	No
NIS-Elements image analysis software (Nikon)	(Soucy et al., 2011)	Used to measure intraluminal perimeter (which in turn is used to calculate circular luminal diameter) and vessel wall thickness.	No
Hematoxylin-eosin (HE) staining	(Shen et al., 2018; Su et al., 2020; Delp et al., 2000, Cheng et al., 2017, Yu et al., 2011)	Used to measure aortic wall thickness, intima-media wall thickness (IMT), wall shear stress, outer media perimeter, and media cross section area (CSA).	No
Wire myography	(Tarasova et al., 2020)	Blood vessels are mounted in wire myograph systems and the relaxed inner diameter is estimated from the passive length-tension relationship between each artery.	No
Verhoeff-van Gieson staining	(Sofronova et al., 2015)	Measures elastin-collagen content in blood vessels, with Verhoeff stain highlighting elastin and van Gieson highlighting collagen. The higher the ratio of elastin to collagen, the greater the distensibility of the vessel. A higher collagen ratio is associated with increased vascular stiffness.	No
Sonography	(Lee et al., 2020; Sarkozy et al., 2019; Sridharan et al., 2020)	Uses ultrasound waves to measure IMT and intima-media area, both of which are markers of vascular structure and are used to calculate vascular stiffness.	No

References

Cheng, Y. P. et al. (2017), "Acid sphingomyelinase/ceramide regulates carotid intima-media thickness in simulated weightless rats", Pflugers Archiv European Journal of Physiology, Vol. 469, Springer Nature, London, https://doi.org/10.1007/s00424-017-1969-z

Delp, M.D. et al. (2000), “Structural and functional remodeling of skeletal muscle microvasculature is induced by simulated microgravity”, American Journal of Physiology - Heart and Circulatory Physiology, Vol. 278, American Physiological Society, Rockville, https://doi.org/10.1152/ajpheart.2000.278.6.h1866

Gibbons, G. H., and V. J. Dzau (1994), “The Emerging Concept of Vascular Remodeling”, New England Journal of Medicine, Vol. 330/20, Massachusetts Medical Society, Waltham, https://doi.org/10.1056/NEJM199405193302008

Harvey, A., A. C. Montezano, & R. M. Touyz. (2015), “Vascular biology of ageing-Implications in hypertension”, Journal of molecular and cellular cardiology, Vol. 83, Elsevier, Amsterdam, https://doi.org/10.1016/j.yjmcc.2015.04.011

Herity, N.A. et al. (1999), “Review: Clinical Aspects of Vascular Remodeling”, Journal of Cardiovascular Electrophysiology, Vol.10/7, Wiley, https://doi.org/10.1111/j.1540-8167.1999.tb01273.x

Kessler, E. L. et al. (2019), “Sex-specific influence on cardiac structural remodeling and therapy in cardiovascular disease”, Biology of Sex Differences, Vol. 10, Springer Nature, https://doi.org/10.1186/s13293-019-0223-0

Lee, S. M. C. et al. (2020), “Arterial structure and function during and after long-duration spaceflight”, Journal of Applied Physiology, Vol. 129, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00550.2019

Patel, S. (2020), "The effects of microgravity and space radiation on cardiovascular health: From low-Earth orbit and beyond", IJC Heart and Vasculature, Vol. 30, Elsevier, Amsterdam, https://doi.org/10.1016/j.ijcha.2020.100595.

Renna, N. F., N. Heras, R. M. Miatello (2013), “Pathophysiology of Vascular Remodeling in Hypertension”, International Journal of Hypertension, Vol. 2013, Hindawi, London, http://doi.org/10.1155/2013/808353

Sárközy, M. et al. (2019), "Selective heart irradiation induces cardiac overexpression of the pro-hypertrophic miR-212", Frontiers in Oncology, Vol. 9, Frontiers Media S.A., Lausanne, https://doi.org/10.3389/FONC.2019.00598/FULL

Shen, Y. et al. (2018), “Transplantation of bone marrow mesenchymal stem cells prevents radiation-induced artery injury by suppressing oxidative stress and inflammation”, Oxidative Medicine and Cellular Longevity, Vol. 2018, Hindawi, London, https://doi.org/10.1155/2018/5942916.

Sofronova, S. I. et al. (2015), “Spaceflight on the Bion-M1 biosatellite alters cerebral artery vasomotor and mechanical properties in mice”, Journal of Applied Physiology, Vol. 118/7, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00976.2014.

Soucy, K. G. et al. (2011), “HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: Role of xanthine oxidase”, Radiation Research, Vol. 176/4, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2598.1.

Soucy, K. G. et al. (2010), “Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta”, Journal of Applied Physiology, Vol. 108/5, American Physiological Society, Rockville, https://doi.org/10.1152/japplphysiol.00946.2009.

Soucy, K. G. et al. (2007), “Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta”, Radiation and Environmental Biophysics, Vol. 46, Springer, New York, https://doi.org/10.1007/s00411-006-0090-z.

Sridharan, V. et al. (2020), "Effects of single-dose protons or oxygen ions on function and structure of the cardiovascular system in male Long Evans rats", Life Sciences in Space Research, Vol. 26, Elsevier, Amsterdam, https://doi.org/10.1016/j.lssr.2020.04.002.

Su, Y. T. et al. (2020), "Acid sphingomyelinase/ceramide mediates structural remodeling of cerebral artery and small mesenteric artery in simulated weightless rats", Life Sciences, Vol. 243, Elsevier, Amsterdam, https://doi.org/10.1016/j.lfs.2019.117253.

Sylvester, C. B. et al. (2018), "Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer", Frontiers in Cardiovascular Medicine, Vol. 5, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fcvm.2018.00005.

Tarasova, O. S. et al. (2020), "Simulated Microgravity Induces Regionally Distinct Neurovascular and Structural Remodeling of Skeletal Muscle and Cutaneous Arteries in the Rat", Frontiers in Physiology, Vol. 1, Frontiers Media SA, Lausanne, https://doi.org/10.3389/fphys.2020.00675.

Yu, T. et al. (2011), "Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-Deficient mice", Radiation Research, Vol. 175/6, Radiation Research Society, Bozeman, https://doi.org/10.1667/RR2482.1.

Appendix 2

List of Key Event Relationships in the AOP