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Relationship: 954

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

Oxidation, Glutathione leads to S-Glutathionylation, eNOS

Upstream event

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

Oxidation, Glutathione

Downstream event

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

S-Glutathionylation, eNOS

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

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
Homo sapiens	Homo sapiens	High	NCBI
Bos taurus	Bos taurus	High	NCBI
Mus musculus	Mus musculus	High	NCBI
Rattus norvegicus	Rattus norvegicus	High	NCBI

Sex Applicability

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

Sex	Evidence
Unspecific	High

Life Stage Applicability

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

Term	Evidence
All life stages	High

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

Under oxidative stress, reduced glutathione (GSH) is oxidized to glutathione disulfide (GSSG), which then induces S-glutathionylation of eNOS at its cysteine residues, resulting in eNOS uncoupling (Chen et al., 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

Evidence Supporting this KER

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

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

In an in vitro system, GSSG induced S-glutathionylation of human eNOS in a dose-dependent manner, which was reversed by reducing agents 2-mercaptoethanol and dithiothreitol (Chen et al., 2010). Cysteine residues 689 and 908 were identified as the critical sites for S-glutathionylation of eNOS since their mutatation resisted glutathionylation. These results were confirmed in bovine aortic endothelial cells (BAECs) and in aortae of spontaneously hypertensive (SHR) rats. Treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, induced an increase in GSSG and eNOS S-glutathionylation as confirmed by mass spectrometry in which Cys 689 was shown to be more than 50% S-glutathionylated. Aortae of SHR rats showed S-glutathionylation via immunohistology whereas normotensive rats had little S-glutathionylation. In human aortic endothelial cells (HAECs) exposed to ultrafine particles (UFP), GSSG levels were increased in parallel with protein S-glutathionylation, which was confirmed by ELISA to be eNOS S-glutathionylation (Du et al., 2013). This was recapitulated in LDLR-null mice exposed to UFP. Additional evidence was observed in BAECs undergoing hypoxia and reoxygenation where eNOS S-glutathionylation increased by three-fold compared to control cells and GSSG levels were increased (De Pascali et al., 2014). These effects were reversed with treatment of N-acetyl-l-cysteine, which increased cellular concentration of GSH. These results support strong biological plausibility for this key event relationship.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Include consideration of temporal concordance here

Treatment with GSSG (0.5 mM, 1 mM, 2 mM) for one hour induced a dose-dependent increase in human eNOS S-glutathionylation in vitro whereas BCNU (25 μM, 80 μM) also resulted in increased eNOS S-glutathionylation in a dose-dependent manner in BAECs (Chen et al., 2010).

Exposure to UFP and hypoxia/reoxygenation demonstrated a response-response relationship between GSH oxidation and eNOS S-glutathionylation in HAECs and BAECs, respectively (Du et al., 2013; De Pascali et al., 2014). Exposure to 50 μg/mL UFP for 6 hours caused a reduction in GSH from 17.1±1.8 μM in controls to 12.0±2.4 μM in treated cells, which is indicative of increased GSSG, and increased eNOS S-glutathionylation from 0.008±0.001 OD450 (as measured by ELISA) in controls to 0.042±0.006 OD450 in treated cells (Du et al., 2013). Similarly, following hypoxia and reoxygenation in BAECs, decreased GSH levels (1.64 ± 0.03 to 0.32 ± 0.03 μmol/mg) and increased S-glutathionylation of eNOS (0.3 to 1) were observed (De Pascali et al., 2014).

Overall, there is moderate empirical support for glutathione oxidation leading to eNOS S-glutathionylation.

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

No uncertainties or inconsistencies were found for this KER.

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

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

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

Based on results by Chen et al.(2010), a concentration of 0.5 mM GSSG was able to induce a significant increase in eNOS S-glutathionylation; and increasing concentrations of GSSG affected eNOS S-glutathionylation in a dose-responsive manner in vitro. UFP and hypoxia/reoxygenation were demonstrated to be modulators of the response-response relationship between glutathione oxidation and S-glutathionylation of eNOS (Du et al., 2013; De Pascali et al., 2014). Additional experiments using other stressors or oxidants would be beneficial in further understanding of this relationship, specifically around the temporal aspect (i.e. does glutathione oxidation occur at an earlier time point than eNOS S-glutathionylation?).

Response-response Relationship

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

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

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

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 supporting this key event relationship are from BAECs, HAECs, LDLR-null mice, and SHR rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).

References

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

Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem. Biophys. Res. Commun. 436, 462–466.