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Relationship: 2773

Title

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

Altered Signaling leads to Altered, Nitric Oxide Levels

Upstream event

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

Altered Signaling

Downstream event

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

Altered, Nitric Oxide Levels

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
mouse	Mus musculus	Moderate	NCBI
rat	Rattus norvegicus	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	Low
Female	Low
Unspecific	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult	Low
Not Otherwise Specified	Moderate

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

Multiple signaling pathways can regulate nitric oxide (NO) levels. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can activate nitric oxide synthase (NOS), an enzyme that produces NO, through phosphorylation (Hemmings & Restuccia 2012; Nagane et al., 2021). The RhoA/Rho kinase (ROCK) pathway inhibits both the expression and phosphorylation of NOS (Yao et al., 2010). Furthermore, the renin-angiotensin-aldosterone system (RAAS) can both inhibit NOS to reduce vasodilation and activate NOS as a countermeasure for vasoconstriction (Millatt, Abdel-Rahman & Siragy, 1999). The extracellular signal-regulated protein kinase 5 (ERK5)/kruppel-like factor 2 (KLF2) pathway can increase transcription of endothelial NOS (eNOS), which results in increased NO levels (Paudel, Fusi & Schmidt, 2021). Lastly, the acidic sphingomyelinase/ceramide pathway can activate NADPH oxidase (NOX) production of reactive oxygen species (ROS) that react with NO, resulting in lower NO levels (Soloviev & Kizub, 2019). Alterations in these pathways will result in altered NO levels.

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 Map 2.0

ID	Experimental Design	Species	Upstream Observation	Downstream Observation	Citation (first author, year)	Notes

Evidence Map

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

Title	First Author	Biological Plausibility	Dose Concordance	Temporal Concordance	Incidence Concordance

Biological Plausibility

Dose Concordance Evidence

Temporal Concordance Evidence

Incidence Concordance Evidence

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

Due to the high reactivity of NO, it can be difficult to obtain its direct measures (Luiking, Engelen & Deutz, 2010). The inconsistencies in NO levels may be attributed to the challenges in measuring NO. Directionality of NO changes cannot be compared between studies due to a variety of experimental conditions like stressor type, dose, dose rate, model and time course of the experiment.

Known modulating factors

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

Modulating factor	Details	Effects on the KER	References
Drug	BPF (ACE inhibitor)	BPF treatment led to decreased iNOS, angiotensin II and aldosterone following irradiation.	Hasan, Radwan & Galal, 2019
Drug	LY294002 (PI3K inhibitor)	LY294002 treatment led to reduced phosphorylation of both Akt and eNOS.	Shi et al., 2012;
Drug	Wortmanin (PI3K inhibitor)	Wortmanin decreased NO production.	Siamwala et al., 2010
Drug	Fenofibrate (PPARα activator)	Treatment with PPARα prevented the decrease in eNOS levels after irradiation.	Azimzadeh et al., 2021
Drug	Atorvastatin (HMG-CoA reductase inhibitor)	Treatment with atorvastatin increased KLF2 and eNOS levels slightly.	Sadhukhan et al., 2020
Drug	Gamma tocotrienol (HMG-CoA reductase inhibitor)	Treatment with gamma tocotrienol prevented the radiation-induced decrease in KLF2 and eNOS levels.	Sadhukhan et al., 2020
Drug	Geranylgeranyltransferase I inhibitor 298	Treatment with Geranylgeranyltransferase I inhibitor 298 prevented the radiation-induced decrease in KLF2 and eNOS levels.	Sadhukhan et al., 2020

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The following are a few examples of quantitative understanding of the relationship. All data that is represented is statistically significant unless otherwise indicated.

Response-response Relationship

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

Dose/Incidence Concordance

Reference	Experiment Description	Result
Shi et al., 2012	In vitro. Microgravity was stimulated with a clinostat using rotation at a slow speed to negate centrifugal force and simulate weightlessness. eNOS expression and Akt phosphorylation in HUVEC-C were measured by western blot.	After clinorotation, eNOS and p-eNOS expression increased by 5.2-fold and 5.5-fold respectively. p-Akt increased by 2.9-fold, while there was no significant change in Akt after clinorotation in HUVEC-C.
Hasan, Radwan & Galal, 2019	In vivo. Rat heart serum were exposed to irradiation by 6 Gy Gamma rays. iNOS levels were measured by ELISA. RAAS indices, AngII and aldosterone, were also measured in serum by ELISA.	Irradiation with 6 Gy led to a 3.3-fold increase in iNOS expression, ~1.4-fold increase in AngII and aldosterone.
Azimzadeh et al., 2015	In vivo. Mice were exposed to heart X-ray irradiation at either 8 or 16 Gy. Levels of proteins in the insulin-dependent PI3K/Akt pathway with and without phosphorylation were determined along with levels of NO and eNOS. Protein levels in various signaling pathways were measured using immunoblotting, and NO was measured using an ELISA assay.	Many key proteins in each pathway showed significant changes in abundance and phosphorylation after 8 and 16 Gy irradiation. For example, phosphorylation of the insulin receptor IGFR1 decreased 0.4-fold after 8 Gy and 0.2-fold after 16 Gy. Similarly, p-Akt decreased 0.2-fold at 8 Gy and 0.1-fold at 16 Gy. p-eNOS decreased 0.6-fold after 8 Gy and 0.2-fold after 16 Gy. The ERK/MAPK pathway was found decreased 0.5-fold at 16 Gy and the p38/MAPK pathway was found increased 1.3-fold at 16 Gy. NO decreased 0.3-fold after 8 Gy and 0.2-fold after 16 Gy.
Azimzadeh et al., 2017	In vitro. HCAECs were irradiated with 0.5 Gy X-ray irradiation over 1 minute. Phosphorylated RhoGDI and eNOS levels were determined using immunoblotting, and NO levels were determined using ELISA assay.	p-RhoGDI decreased 0.7-fold, p-eNOS decreased 0.6-fold, HSP90 (positive regulator of eNOS) decreased 0.8-fold and NO decreased 0.8-fold after 0.5 Gy irradiation.
Sonveaux et al., 2003	In vitro. Following X-ray irradiation of BAECs and HUVECs, levels of eNOS, p-eNOS, Akt, and p-Akt were measured with immunoblotting at various doses (0.86 Gy/min).	Compared to control, 24 h after irradiation, the ratio of p-Akt/Akt increased 1.3-fold after 2 Gy (not significant) and 5.6-fold after 6 Gy. eNOS increased 1.3-fold after 2 Gy (not significant), 2.1-fold after 4 Gy (not significant), 3-fold after 6 and 8 Gy, 4.3-fold after 10 Gy and 3.4-fold after 20 Gy. Compared to control, 24 h after irradiation, p-eNOS increased 1.2-fold after 2 Gy and 1.7-fold after 6 Gy.
Azimzadeh et al., 2021	In vivo. Male C57BL/6J were irradiated with an acute dose of 16 Gy X-rays. Activation of the PI3K-Akt pathway was determined through p-PPARα (deactivated) levels from Ponceau S staining. eNOS activity and NO were measured using fluorometric assay and Griess assay, respectively. The level of proteins in MAPK pathways were determined by ELISA in heart tissue.	After 16 Gy, p-PPARα increased 1.3-fold. After 16 Gy, p-ERK increased 1.5-fold, and p-p38 increased 1.3-fold, eNOS was unchanged, p-eNOS decreased 0.8-fold and NO decreased to 65% of control levels.
Sadhukhan et al., 2020	In vitro. HUVECs were irradiated with various regimens and doses of gamma radiation. Signaling from the ERK5/KLF2 pathway was determined through KLF2 and p-ERK5 levels from western blot. eNOS levels were determined by western blot. Fractionated doses were separated by 24 h.	p-ERK5 decreased 0.6-fold after 5 doses of 2 Gy, increased 1.5-fold after acute 10 Gy, decreased 0.5-fold after 5 doses of 2.5 Gy, and increased 1.3-fold after acute 12.5 Gy. KLF2 and eNOS showed similar responses and were often slightly decreased after acute doses.

Time-scale

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

Time Concordance

Reference	Experiment Description	Result
Azimzadeh.et al., 2017	In vitro. AECs were irradiated with 0.5 Gy X-rays over 1 minute. Phosphorylated RhoGDI and eNOS levels were determined using immunoblotting, and NO levels were determined using ELISA assay. Measurements were taken after 1, 7 or 14 days.	p-RhoGDI significantly decreased after 1 day, while NO only significantly decreased after 7 days. However, p-eNOS significantly decreased after 1 day.
Sonveaux et al., 2003	In vitro. Levels of eNOS, p-eNOS, Akt, and p-Akt after X-ray irradiation of BAECs and HUVECs were measured with immunoblotting at various doses (0.86 Gy/min).	p-Akt was significantly increased 24 h after irradiation, while eNOS was significantly increased after 12, 24, and 48 h and p-eNOS was significantly increased after 24 h.
Sadhukhan et al., 2020	In vitro. HUVECs were irradiated with various regimens and doses of gamma radiation. Fractionated doses were separated by 24 h. Signaling from the ERK5/KLF2 pathway was determined through KLF2 and p-ERK5 levels from western blot. eNOS levels were determined by western blot. Measurements were taken 4 or 24 h post-irradiation.	After 4 h, p-ERK5 decreased 0.6-fold after 5 doses of 2 Gy, increased 1.5-fold after acute 10 Gy, decreased 0.5-fold after 5 doses of 2.5 Gy, and increased 1.3-fold after acute 12.5 Gy. Both KLF2 and eNOS decreased similar to p-ERK5 after 4 h and were slightly decreased after 24 h.

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 majority of the evidence for this KER is from rat and mouse models. Most evidence regarding sex and lifestage is unspecified with a small amount of evidence from adult models.