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Event: 2067

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Altered, Nitric Oxide Levels

Short name

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Biological Context

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Level of Biological Organization
Cellular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
nitric oxide homeostasis	endothelium	functional change

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Deposition of energy leads to vascular remodeling	KeyEvent	Vinita Chauhan (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 KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	High	NCBI
rat	Rattus norvegicus	High	NCBI
mouse	Mus musculus	Moderate	NCBI
rabbit	Oryctolagus cuniculus	Moderate	NCBI

Life Stages

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

Life stage	Evidence
Adult	Moderate
Not Otherwise Specified	Moderate

Sex Applicability

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

Term	Evidence
Male	High
Female	Low
Unspecific	Moderate

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

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

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

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).

References

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

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