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Relationship: 2774


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

Oxidative Stress leads to Altered, Nitric Oxide Levels

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

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

AOPs Referencing Relationship

AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Deposition of energy leads to vascular remodeling adjacent Moderate Low Cataia Ives (send email) Open for citation & comment

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help
Term Scientific Term Evidence Link
human Homo sapiens Low NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help
Sex Evidence
Male Moderate
Female Low
Unspecific Low

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help
Term Evidence
Adult Moderate
Juvenile Low

Key Event Relationship Description

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

The increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during oxidative stress can lead to altered nitric oxide (NO) levels, specifically a reduction in its bioavailability.  

Although RNS can also interfere with NO levels, most studies focus on ROS and not RNS (Nagane et al., 2021). Oxidative stress influences the production and activity of endothelial nitric oxide synthase (eNOS), thereby altering and reducing NO levels and its bioavailability. eNOS, otherwise known as NOS3, is an enzyme that catalyzes NO production from the amino acid L-arginine in vascular endothelial cells. NO mediates vascular tone and blood flow via the activation of soluble guanylate cyclase (sGC) within the vascular smooth muscle (Chen, Pittman and & Popel, 2008). A form of ROS known as superoxide anion (O2-) causes increased NO degradation (Incalza et al., 2018), converting NO into the RNS peroxynitrite. In addition, ROS can uncouple eNOS by oxidation of the enzyme’s cofactor, BH4 (Matsubara et al., 2015; Forstermann, 2010). Uncoupled eNOS produces ROS instead of NO, which can further convert existing NO into peroxynitrite (Forstermann, 2010). 

Evidence Collection Strategy

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

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

Evidence Supporting this KER

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

Overall weight of evidence: Moderate

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

The biological rationale for the relationship between increased oxidative stress and altered NO levels is well-supported by the literature, validated by many studies presented in this area of research. The biological mechanism of this relationship is well-known and widely accepted. The reaction between NO and free radicals leads to the creation of peroxynitrite, which results in reduced NO bioavailability (Incalza et al., 2018; Mitchell et al., 2019; Nagane et al., 2021; Soloviev & Kizub, 2019; Wang, Boerma & Zhou, 2016). In addition, in vascular tissues, increased levels of O2- or peroxynitrite can oxidize BH4 and lead to uncoupled eNOS (Forstermann, 2010; Matsubara et al., 2015; Soloviev & Kizub, 2019). When uncoupled, eNOS transfers electrons to O2- rather than L-arginine, causing O2- production instead of NO production, further reducing NO bioavailability. Although much of the biological plausibility indicates that NO decreases with oxidative stress, ROS have been associated with increased NO as well (Nagane et al., 2021; Soloviev & Kizub, 2019). This is likely due to the complexity of NO regulation in signaling pathways, upregulation of inducible nitric oxide synthase (iNOS), as well as variations in stressors, doses, dose rates, models, duration of study and diseases present in studies (Nagane et al., 2021).

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help
  • The directionality of changes to NO is inconsistent between studies, as some studies show increased NO levels and other studies show decreased NO levels. Improved methods are needed to assess NO levels directly, which may facilitate an understanding of the relationship (Cervelli et al., 2017, Hasan, Radwan & Galal, 2019). This, along with variation in experimental conditions, can account for the inconsistencies in NO changes between studies.

Known modulating factors

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

Modulating factor 


Effects on the KER 



BPF (ACE inhibitor) 

BPF treatment led to decreased iNOS, angiotensin II and aldosterone following irradiation. Oxidative stress indices returned closer to control levels. 

Hasan, Radwan & Galal, 2019 


OXP (XO inhibitor) 

OXP can increase NO levels and decrease ROS after irradiation. 

Soucy et al., 2010; Soucy et al., 2011 


RiduROS (A combination of antioxidants resveratrol, extramel, seleno-L-methionine, Curcuma longa, reduced L-glutathione, vitamin C) 

RiduROS led to decreased ROS and NO production after irradiation. 

Cervelli et al., 2017 


DAHP (Gch1 inhibitor which is involved in BH4 synthesis) 

DAHP can decrease ROS production and increase NO production after irradiation. 

Yan et al., 2020 


Losartan (AT1 receptor antagonist) 

Treatment with Losartan after microgravity decreased superoxide production and returned eNOS and iNOS to control levels. 

Zhang et al., 2009 


ZnO-NPs (Zinc oxide nanoparticles that act as antioxidants) 

Treatment with ZnO-NPs returned serum and cardiac NO levels and ROS indicators closer to control levels after irradiation. 

Abdel-Magied & Shedid, 2020 



Biotin (6mg) increased GSH content, SOD activity, and CAT activity closer to control levels following irradiation in hippocampus. NOx levels also returned to near control values. 

Abdel-Magied & Shedid, 2020 

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

Dose/Incidence Concordance


Experiment Description 


Cervelli et al., 2017 

In vitro. HUVECs were irradiated with 0.25 Gy X-rays. Levels of ROS as well as nitrite and nitrate (NO markers) were determined using a fluorescent probe and Griess assay, respectively. 

The irradiated samples had a 2.8-fold increase in ROS (not significant) and a 1.6-fold increase in nitrite/nitrate compared to controls. 

Yan et al., 2020 

In vitro and in vivo. Rat arteries were irradiated with 4 Gy abdominal X-ray radiation. HUVEC were irradiated by 10 Gy X-rays. Oxidative stress was indicated by superoxide anions. Nitrite and eNOS levels were measured by NO assay kit and SDS-PAGE. 

Following 4 Gy irradiation of rats, O2- levels increased by 2.1-fold. Nitrite (NO metabolite and marker) and eNOS ratio decreased by 0.6-fold.  

Following 10 Gy irradiation of HUVEC, O2- levels increased by 3.6-fold and nitrite along with eNOS decreased 0.5 and 0.6-fold respectively. 

Soucy et al., 2011 

In vivo. Male rats irradiated with 1 Gy 56Fe ions. ROS and NO levels in rat aorta were measured using fluorescence rates of dihydroethidium and diaminofluorescein, respectively. 

Iron ion irradiation at 1 Gy produced a 1.8-fold increase in ROS levels and 0.8-fold decrease in NO levels compared to controls. 

Soucy et al., 2010 

In vivo. 4-month-old rats were irradiated with 5 Gy 137Cs gamma radiation. ROS and NO levels in rat aorta were measured using fluorescence rates of dihydroethidium and diaminofluorescein, respectively. 

After 5 Gy, ROS increased 1.7-fold. NO production decreased 0.7-fold compared to controls. 

Hasan, Radwan & Galal, 2019 

In vivo. Rats were irradiated by 6 Gy 137Cs gamma rays and serum was collected. Oxidative damage biomarker MDA ROS-clearing enzyme reduced GSH and FRAP were measured using various assays. 

Irradiation with 6 Gy led to a 1.4-fold increase in MDA, a 0.5-fold decrease in GSH and a 0.4-fold decrease in FRAP. A 3.3-fold increase in iNOS levels was observed. 

Zhang et al., 2009 

In vivo. Hindlimb unweighted (HU) rats had superoxide and NOS levels measured in carotid arteries. Western blot was used to measure eNOS and iNOS levels, while dihydroethidium fluorescence was used to measure superoxide. 

In HU rats, eNOS levels increased 2-fold in carotid arteries. A 4.2- and 3.3-fold increase in iNOS in carotid and cerebral arteries, respectively, was found in HU rats. Superoxide levels were not quantitatively shown but increased greatly after altered gravity. 

Abdel-Magied & Shedid, 2020 

In vivo. Male rats were irradiated with 8 Gy 137Cs gamma irradiation at 0.4092 Gy/min. Oxidative damage biomarker MDA and ROS-clearing enzymes SOD, CAT and glutathione peroxidase (GPx) activities and GSH were measured using respective assay kits. Total nitrite/nitrate content was measured with nitrite/nitrite assay kit. The oxidative damage biomarker and ROS-clearing enzymes were measured in heart tissue and nitrite/nitrate was measured in heart tissue and blood serum. 

Irradiated cardiac tissue showed a 1.8-fold increase in MDA levels, 0.4-fold decrease in GSH levels, 0.4-fold decrease in CAT activity, 0.5-fold decrease in SOD activity and a 0.5-fold decrease in GPx activity compared to control. XO levels increased 2-fold. Irradiated heart tissue showed a 2-fold increase in nitrite/nitrate content, while irradiated blood serum showed a 1.8-fold increase in nitrite/nitrate content compared to the control. 

Sakata et al., 2015 

In vitro. HUVECs were irradiated with various doses of X-rays. eNOS, p-eNOS (Ser1177 & Thr495), iNOS, citrulline (produced by NOS with NO) and NOx levels were measured with western blots for proteins and various assay kits for molecules. ROS intensity was measured using fluorescence microscopy. 

At maximum after 10 Gy, ROS increased 15.5-fold, eNOS expression did not significantly change, p-eNOS (Ser1177) increased 1.8-fold, p-eNOS (Thr495) decreased 0.3-fold, iNOS showed a non-significant 1.4-fold increase and citrulline increased 1.3-fold. NOx increased consistently from 1-20 Gy with significant fold changes at 10 and 20 Gy and a maximum 10-fold increase after 10 Gy. 

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

Time Concordance 


Experiment Description 


Cervelli et al., 2017 

In vitro. HUVECs were irradiated with 0.25 Gy X-rays. Levels of ROS as well as nitrite and nitrate (NO markers) were determined using a fluorescent probe and Griess assay, respectively.

The irradiated samples had a 2.8-fold increase in ROS (not significant) measured after 45 minutes and a 1.6-fold increase in nitrite/nitrate measured after 24 hours. 

Hasan, Radwan & Galal, 2019 

In vivo. Rats were exposed to irradiation by 6 Gy 137Cs gamma rays and serum was collected. Oxidative damage biomarker MDA, ROS-clearing enzyme reduced GSH and FRAP were measured using various assays. 

Irradiation with 6 Gy led to a 1.4-fold increase in MDA, a 0.5-fold decrease in GSH and a 0.4-fold decrease in FRAP all measured after 4 weeks. A 3.3-fold increase in iNOS levels was observed after 4 weeks as well. 

Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Not Identified.

Domain of Applicability

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

The evidence for the taxonomic applicability to humans is low as evidence comes from in vitro human cell-derived models. Many studies use in vivo rat models, predominately males. Occasionally, animal age is not specified in studies; most studies indicate the animals are adult or adolescent. In addition, the relationship is also plausible in preadolescent animals.


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

Abdel-Magied, N. and S. M. Shedid (2020), “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, Wiley,

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,

Chen, K., R. N. Pittman and A. S. Popel (2008), “Nitric oxide in the vasculature: where does it come from and where does it go? A quantitative perspective”, Antioxidants & redox signaling, Vol. 10/7, Mary Ann Liebert, Inc., Larchmont,

Förstermann, U. (2010), “Nitric oxide and oxidative stress in vascular disease”, Pflugers Archiv : European journal of physiology, Vol. 459/6, Springer, Berlin,

Hasan, H. F., R. R. Radwan and S. M. Galal (2019), “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,

Incalza, M. A. et al. (2018), “Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases”, Vascular pharmacology, Vol. 100, Elsevier, Amsterdam,

Kojsová, S. et al. (2006), “The effect of different antioxidants on nitric oxide production in hypertensive rats”, Physiological research, Vol. 55, Czech Academy of Sciences, Prague,  

Kozbenko, T. et al. (2022), “Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example”, International Journal of Radiation Biology, Vol. 98/12.

Matsubara, K. et al. (2015), “Nitric oxide and reactive oxygen species in the pathogenesis of preeclampsia”, International journal of molecular sciences, Vol. 16/3, Multidisciplinary Digital Publishing Institute, Basel,

Mitchell, A. et al. (2019), “Cardiovascular effects of space radiation: implications for future human deep space exploration”, European Journal of Preventive Cardiology, Vol. 26/16, SAGE Publishing, Thousand Oaks,

Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular diseases”, Journal of Radiation Research, Vol. 62/4, Oxford University Press, Oxford,  

Sakata, K. et al. (2015). “Roles of ROS and PKC-βII in ionizing radiation-induced eNOS activation in human vascular endothelial cells”, Vascular Pharmacology, Vol. 70, Elsevier, Amsterdam,

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,

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,

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,

Wang, Y., M. Boerma and D. Zhou (2016), “Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases”, Radiation research, Vol. 186/2, Radiation Research Society, Bozeman,

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,

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. 106/1, American Physiological Society, Rockville,