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Relationship: 3319

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

Increase, Oxidative Stress leads to Altered cell differentiation signaling

Upstream event

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

Increase, Oxidative Stress

Downstream event

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

Altered cell differentiation signaling

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 leading to occurrence of bone loss	adjacent	High	Low	Vinita Chauhan (send email)	Open for citation & comment	WPHA/WNT Endorsed

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	High	NCBI
rat	Rattus norvegicus	High	NCBI
Pig	Pig	Moderate	NCBI

Sex Applicability

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

Sex	Evidence
Male	High
Female	Low
Unspecific	Low

Life Stage Applicability

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

Term	Evidence
Juvenile	High
Adult	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

Oxidative stress arises when the generation of free radicals surpasses the ability of cellular antioxidant defenses to neutralize them (Cabrera & Chihuailaf, 2011). Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) are types of free radicals that can lead to oxidative stress (Ping et al., 2020); however, ROS are more frequently studied than RNS (Nagane et al., 2021). ROS can cause oxidative damage to biomacromolecules by reacting with DNA, proteins, and lipids, leading to functional alterations in these molecules (Ping et al., 2020). For instance, ROS interacting with lipids results in lipid peroxidation (Cabrera & Chihuailaf, 2011).

Cell differentiation pathways are the processes through which unspecialized cells, such as stem cells, develop into specialized cells with distinct functions (Soumelis and Liu, 2006). Disruptions in cell differentiation pathways can occur due to various mechanisms, including ROS production. Persistent activation or inhibition of these pathways can lead to aberrant cell fate decisions (Kharrazian, 2021). ROS plays a crucial role in inducing cell differentiation by modulating signaling pathways and influencing transcription, and excessively high or low amounts of ROS can hinder cell differentiation. For instance, the oxidation of specific cysteine residues in signaling proteins can alter their activity, leading to aberrant activation or inhibition of pathways such as Wnt, Notch, or TGF-β, which are essential for maintaining proper cell differentiation. The disrupted signaling can cause cells to deviate from their intended differentiation path, leading to abnormal cell states or even contributing to pathological conditions. For example, ROS plays crucial role in regulating bone remodeling and repair, impacting osteoblasts and osteoclast activities. Under healthy conditions, ROS generated by osteoclasts activate and balance bone resorption with bone reformation. Exogenous ROS, such as H2O2 and superoxide's initiate RANK signaling in macrophages and endogenous ROS activates TNF receptor associated factor 6, NOX1, and the transcription factors NFκB and nuclear factor of activated T-cells (Riegger et al., 2023). These disruptions can have significant consequences, contributing to various diseases (Wu et al., 2023). An increase of ROS can cause osteocyte apoptosis, leading to bone resorption through osteoclastogenic RANKL and additionally impair Wnt/β-catenin signaling (Riegger et al., 2023). The MAPK family pathway, crucial in regulating molecular processes such as cell differentiation, is activated in response to ROS production through calcium-induced phosphorylation of several kinases (Sinkala et al., 2021). ROS can activate the JAK/STAT pathway through oxidation of glutathione, a pathway which affects cell differentiation (Hu et al., 2023; Villalpando-Rodriguez & Gibson, 2021). Oxidative stress in bone cells can lead to increased expression of the receptor activator of nuclear factor kappa B ligand (RANKL) and Nrf2 activation (Tahimic & Globus, 2017; Tian et al., 2017). Following activation, Nrf2 then interferes with the activation of runt-related transcription factor 2 (Runx2), and depending on the level of oxidative stress, this may result in altered bone cell function (Kook et al., 2015).

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

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

Many reviews describe the role of oxidative stress in cell differentiation signaling. The mechanisms through which oxidative stress can contribute to cell differentiation signaling via changes in signaling pathways are well-described. For example, oxidative stress can directly alter various pathways through protein oxidation (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 cause conformational change, protein expansion, and degradation, leading to changes in the protein levels of pathways involved in cell differentiation (Ping et al., 2020). Furthermore, oxidation of key residues in signaling proteins can alter their function, resulting in cell differentiation signaling. For example, oxidation of methionine 281 and 282 in the Ca2+/calmodulin binding domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII) leads to constitutive activation of its kinase activity and subsequent downstream alterations in cell differentiation signaling. (Li et al., 2013; Ping et al., 2020). Similarly, during oxidative stress, tyrosine phosphatases can be inhibited by oxidation of a catalytic cysteine residue, resulting in increased phosphorylation of proteins in various cell differentiation signaling pathways (Schmidt-Ullrich et al., 2000; Valerie et al., 2007). Particularly relevant to this are the MAPK pathways. For example, the extracellular signal-regulated kinase (ERK) pathway is activated by upstream tyrosine kinases and relies on tyrosine phosphatases for deactivation (Lehtinen & Bonni, 2006; Valerie et al., 2007).

Furthermore, oxidative stress can indirectly influence cell differentiation signaling through oxidative DNA damage which can lead to mutations or changes in the gene expression of proteins in signaling pathways (Ping et al., 2020; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). DNA damage surveillance proteins like ataxia telangiectasia mutated (ATM) kinase and ATM/Rad3-related (ATR) protein kinase phosphorylate over 700 proteins, leading to changes in downstream signaling (Nagane et al., 2021; Schmidt-Ullrich et al., 2000; Valerie et al., 2007). For example, ATM, activated by oxidative DNA damage, phosphorylates many proteins in the ERK, p38, and Jun N-terminal kinase (JNK) MAPK pathways, leading to various downstream effects (Nagane et al., 2021; Schmidt-Ullrich et al., 2000).

The response of oxidative stress on cell differentiation signaling has been studied extensively in disease. Here presented are examples relevant to a few cell types related to bone loss. Many other pathways are plausible but available research has highlighted these to be critical to disease.

Oxidative stress can induce signaling changes in the Wnt/β-catenin pathway, the RANK/RANKL pathway, the Nrf2/HO-1 pathway, and the MAPK pathways (Domazetovic et al., 2017a; Manolagas & Almeida, 2007; Tian et al., 2017).

The mechanisms of oxidative stress leading to cell differentiation signaling may be different for each pathway. For example, ROS-induced MAPK activation can be initiated through Ras-dependent signaling. Firstly, oxygen radicals mediate the phosphorylation of upstream epidermal growth factor receptors (EGFRs) on tyrosine residues, resulting in increased binding of growth factor receptor-bound protein 2 (Grb2) and subsequent activation of Ras signaling (Lehtinen & Bonni, 2006). Direct inhibition of MAPK phosphatases with hydroxyl radicals also activates this pathway (Li et al., 2013). In another mechanism, ROS competitively inhibit the Wnt/β-catenin pathway through the activation of forkhead box O (FoxO), which are involved in the antioxidant response and require binding of β-catenin for transcriptional activity (Tian et al., 2017).

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

A few studies demonstrate greater changes to oxidative stress than to altered signaling. Microgravity exposure to preosteoblast cells showed a 0.24-fold decrease to the antioxidant Cu/Zn-superoxide dismutase (SOD) and a 0.36-fold decrease to p-Akt (Yoo et al., 2016). Bai et al. (2020) also demonstrated with multiple endpoints that ROS levels increased, and antioxidant enzyme levels decreased more than signaling pathways were altered.

Dose Concordance:

Many studies demonstrate dose concordance for this relationship, at the same doses. After simulated microgravity, changes to signaling pathways, increased ROS and MDA, and decreased antioxidants were found both in in vitro mouse-derived bone cells and in in vivo rat femurs. In Bone marrow mesenchymal stem cells (BM-MSCs) exposed to 0, 6 and 16 Gy of Co-60 gamma rays, ROS levels increased concurrently with decreased β-catenin expression levels when compared to controls (Zuo et al. 2019). Increased ROS levels and decreased antioxidants were found with changes in the RANK/RANKL pathway, Wnt/β-catenin pathway, Runx2, and MAPK pathways (Diao et al., 2018; Sun et al., 2013; Xin et al., 2015; Yoo et al., 2016). Osteocyte-like cells (MLO-Y4) treated with 20, 40 and 80 μM of ferric ammonium citrate (FAC) had increased ROS levels of 1.5-folds, 2-folds and 2.5-folds compared to the control respectively, and increased RANKL/β-actin expression levels (~1.8, 3.5 and 5.5-folds compared to the control respectively) (Ma et al. (2022).

A few studies also find that oxidative stress often occurs at lower doses than altered cell differentiation signaling. Bai et al. (2020) measured oxidative stress, shown by increased ROS and decreased antioxidant expression, at 2, 5, and 10 Gy of gamma rays. They also found Runx2 increased at the same doses, but the p53/p21 pathway was only significantly altered at 5 and 10 Gy (Bai et al., 2020). At similar doses, X-ray irradiated mouse osteoblast-like cell line MC3T3-E1 cells showed increased ROS and decreased antioxidants both 4 and 8 Gy (Kook et al., 2015). While HO-1 also increased at both 4 and 8 Gy, Nrf2 and Runx2 were measured altered at 8 Gy (Kook et al., 2015).

Time Concordance

Limited evidence shows that oxidative stress leads to altered signaling pathways in a time-concordance manner. When irradiated with X-rays and MCT3T3-E1 osteoblast-like cells show increase in ROS or levels of protein carbonylation, or a decrease in the levels of superoxide dismutase (SOD), catalase (CAT), GSTO1 or glutathione (GSH) at earlier timepoints than alterations in the signaling molecules p16, p21, Ceramide, Runx2, and HO-1 (Kook et al., 2015). The molecular-level changes occur quickly after irradiation. In human osteoblast-like SaOS-2 cells treated with various concentrations of butionine sulfoximine (BSO), GSH levels showed a significant decrease compared to the control 0, 3 and 6 days post treatment. The treated cells concurrently demonstrated a significant decrease of RANKL/OPG levels at 0,3 and 6 days post treatment (Romagnoli et al., 2013). In another study, starved MLO-Y4 osteocyte-like cells demonstrated that both H2O2 and RANKL significantly increased 24 hours post starvation treatment when compared to controls (Fontani et al. (2015).

Essentiality

Several studies have investigated the essentiality of the relationship, where the blocking or attenuation of the upstream KE causes a change in frequency of the downstream KE. Antioxidants (N-acetyl cysteine, curcumin) were shown to restore or reduce ROS levels closer to control levels following radiation or microgravity exposure, respectively. Signaling proteins in the RANKL/osteoprotegerin (OPG) ratio were decreased and brought closer to control levels (Kook et al., 2015; Xin et al., 2015). Hydrogen rich medium showed reduced ROS with restoration of RANKL signaling levels to controls (Sun et al., 2013). Polyphenol S3 (60 mg/kg/d) treatment was found to reverse the effect of microgravity on CAT, SOD and MDA, returning the levels to near control values. Meanwhile, Runx2 mRNA levels and β-catenin/β-actin levels increased following treatment (Diao et al., 2018). It was observed when treated with butionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, GSH levels were significantly decreased for all days compared to the control and RANKL/OPG were significantly increased for all days (0, 3, 6 days) relative to the control (Romagnoli et al. 2013).

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

MAPK pathways can exhibit varied responses after exposure to oxidative stress. The expected response is an increase in the activity of the ERK, JNK, and p38 pathways as protein phosphatases, involved in the inactivation of MAPK pathways, are deactivated by oxidative stress (Valerie et al., 2007). Although some studies show a decrease (Yoo et al., 2016).
The assays employed in studies to assess the KEs may lead to variations in the quantitative understanding of observations.

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	N-acetyl cysteine (antioxidant)	Treatment of osteoblast-like cells with 5 mM restored ROS levels, SOD activity, and the level of proteins in the Nrf2/HO-1 pathway.	Kook et al., 2015
Drug	Curcumin (antioxidant)	Treatment of osteoblast-like cells with 4 µM reduced ROS levels and the RANKL/OPG ratio. Treatment of rats with 40 mg/kg of body weight reduced oxidative stress and the RANKL/OPG ratio.	Xin et al., 2015
Media	Hydrogen-rich (antioxidant)	Osteoblasts in a medium consisting of 75% H2, 20% O2, and 5% CO2 (vol/vol/vol) showed a reduction in ROS production and restoration of normal signaling.	Sun et al., 2013
Drug	Melatonin (antioxidant)	Treatment with 200 nM melatonin reversed the effect of microgravity on Cu/Zn-SOD and Mn-SOD to control levels.	Yoo et al., 2016
Drug	Polyphenol S3	Polyphenol S3 treatment reverses the effect of microgravity on CAT, SOD and MDA, returning the levels to near control values when S3 is used at high dose (60mg/kg/d). β-catenin/β-actin levels increased following treatment and simulated microgravity.	Diao et al., 2018

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 table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All reported findings are statistically significant at various alpha levels as listed in the original sources

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
Kook et al., 2015	In vitro. MC3T3-E1 osteoblast-like cells were irradiated with 2, 4, and 8 Gy of X-rays (1.5 Gy/min). ROS were measured with a fluorescent probe, and SOD, CAT, and GSH antioxidant activities were determined with assay kits. Protein levels in the Nrf2/HO-1 signaling pathway were determined by either western blot or RT-PCR.	ROS increased linearly at 2 and 4 Gy up to 1.4-fold at 8 Gy (significant changes at 4 Gy and 8 Gy). GSH and SOD were decreased 0.7-fold at 4 Gy and 0.5-fold at 8 Gy (insignificant increases at 2 Gy). CAT was also decreased but not significantly. HO-1 increased 3.3-fold after 4 Gy and 4.9-fold after 8 Gy (insignificant increase at 2 Gy). Nrf2 increased 2.3-fold after 8 Gy. Runx2 mRNA was decreased 0.5-fold after 8 Gy.
Bai et al., 2020	In vitro. Rat-derived bone marrow-derived mesenchymal stem cells (bmMSCs) were irradiated with 2, 5, and 10 Gy of 137Cs gamma rays. Mitochondrial and cellular ROS levels were determined with fluorescent probes. RT-qPCR was performed to measure antioxidant enzyme expression. Protein expression in various signaling pathways was measured by western blot.	Mitochondrial ROS increased 1.6-fold at 2 Gy (non-significant), 2-fold at 5 Gy, and 2.3-fold at 10 Gy. Cellular ROS increased 1.2-fold at 2 Gy, 1.5-fold at 5 Gy, and 2.1-fold at 10 Gy. Antioxidants SOD1, SOD2, and CAT all decreased about 0.9-fold (ns for SOD2) after 2 Gy, 0.8- to 0.7-fold at 5 Gy, and 0.7- to 0.4-fold at 10 Gy. Runx2 decreased 0.9-fold at 2 and 5 Gy and 0.6-fold at 10 Gy. p21 increased 1.6-fold at 5 Gy and 2.5-fold at 10 Gy. p53 increased 1.6-fold at 5 Gy and 1.7-fold at 10 Gy. p16 remained unchanged.
Xin et al., 2015	In vitro and in vivo. In vitro. MC3T3-E1 osteoblast-like cells were exposed to microgravity for 96 hours. ROS were determined with a fluorescent probe and the RANK/RANKL pathway was measured using RANKL and OPG assay kits. In vivo. Male 8-week-old Sprague-Dawley rats were exposed to hind-limb suspension for 6 weeks. Femur and plasma MDA and femur sulfhydryl levels were measured with assay kits and the RANK/RANKL pathway was measured in the femur using RANKL and OPG assay kits.	In vitro. ROS increased 1.5-fold and the RANKL/OPG ratio increased 1.6-fold. In vivo. Serum and femur MDA increased 1.4-fold and femur sulfhydryl decreased 0.6-fold. The RANKL/OPG ratio increased 3.5-fold.
Sun et al., 2013	In vitro. MC3T3-E1 osteoblast-like cells were exposed to microgravity (0.01G) for 96 hours. ROS production was measured by a fluorescent probe. The RANKL/OPG ratio was determined by assay kit and Runx2 mRNA expression was determined by RT-qPCR	ROS increased 1.5-fold. The RANKL/OPG ratio increased 1.6-fold. Runx2 expression decreased 0.4-fold.
Yoo et al., 2016	In vitro. Preosteoblast MC3T3-E1 cells were exposed to microgravity conditions by a 3D clinostat at a speed from 1-10 rpm. Oxidative stress was measured by Cu/Zn-SOD, Mn-SOD and catalase activity. Signaling molecules, phosphorylation of the mechanistic target of rapamycin p-(mTOR), and p-ERK were measured by western blot.	Following microgravity exposure, Cu/Zn-SOD and Mn-SOD levels decreased by 0.24 and 0.65-fold, respectively. Signaling molecules p-mTOR and p-ERK decreased by 0.58-fold.
Diao et al., 2018	In vivo. The left femur of rats was studied after exposure to simulated microgravity. Oxidative stress was measured by MDA, SOD, and CAT levels. RANK/RANKL signaling pathway was measured in rat femur by enzyme-linked immunoassay detection of OPG/RANKL molecules. Signaling molecule, Runx2, mRNA levels were measured by quantitative real time PCR. The Wnt/β-catenin pathway was measured by western blot for β-catenin protein levels.	MDA increased by 1.4-fold. SOD and CAT levels decreased by 0.4-fold. OPG/RANKL decreased by 0.6-fold. Runx2 mRNA levels decreased 0.04-fold (Fig 8d). β-catenin decreased 0.6-fold.
Ma et al., 2022	In vitro. MLO-Y4 osteocyte-like cells were treated with ferric ammonium citrate (FAC) at concentration of 20, 40 and 80 μM. ROS levels were measured with flow cytometry. RANKL/β-actin levels were assessed with western blotting.	ROS levels increased 1.5-folds, 2-folds and 2.5-folds with each increasing dose of FAC (20, 40, 80 μM) compared to the control. RANKL/β-actin expression levels increased approximately 1.8-folds, 3.5-folds and 5.5-folds with each increasing dose of FAC (20, 40, 80 μM) compared to the control.
Zuo et al., 2019	Ex vivo. Bone marrow mesenchymal stem cells (BM-MSCs) were isolated from 3-month-old female rats exposed to 0, 6 and 16 Gy of Co-60 gamma rays at 0.56 Gy/min. Cellular ROS was measured with a 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) stain and examined by flow cytometry. β-catenin expression levels were measured with quantitative real-time PCR, western blot and immunochemistry.	ROS levels increased significantly after 6 Gy of radiation compared to the control. β-catenin expression levels decreased after 6 Gy of radiation compared to the control.

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
Kook et al., 2015	In vitro. MC3T3-E1 osteoblast-like cells were irradiated with X-rays (1.5 Gy/min). ROS were measured with a fluorescent probe, and SOD, CAT, and GSH antioxidant activities were determined with assay kits. Protein levels in the Nrf2/HO-1 signaling pathway were determined by either western blot or RT-PCR.	After 1 day and 8 Gy, ROS increased 1.4-fold, GSH decreased 0.5-fold, and SOD decreased 0.5-fold. CAT was also decreased but not significantly. After 1 day and 8 Gy, Nrf2 increased 2.3-fold. After 2 days and 8 Gy, HO-1 increased 4.9-fold. After 3 days and 8 Gy, Runx2 mRNA was decreased 0.5-fold.
Romagnoli et al., 2013	In vitro. Human osteoblast-like SaOS-2 cells were treated with concentrations butionine sulfoximine (BSO). Cellular GSH protein levels were measured using bicinchoninic acid. RANKL/OPG markers were assessed with Quantitative real-time PCR.	Cells treated with BSO showed significant progressive decrease of GSH levels over the course of 6 days compared to the control group. Concurrently, levels of RANKL/OPG significantly increased with increasing days (0, 3, 6 days) compared to the control.
Fontani et al., 2015	In vitro. MLO-Y4 osteocyte-like cells were starved. Intracellular H2O2 was measured by cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate, a chemically reduced form of fluorescein and analyzed by fluorescence spectrophotometric analysis. RANKL was measured using quantitative sandwich enzyme immunoassay kits.	H2O2 significantly increased 24 hours post starvation treatment when compared to controls. Concurrently, RANKL increased significantly 24 hours post treatment when compared to controls.

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

ROS can upregulate protein kinase C, which stimulates the production of ceramide from sphingomyelinase. Ceramide activates NADPH oxidase, which can then produce more ROS (Soloviev & Kizub, 2019). Lastly, the MAPK pathway also exhibits a feedback loop. ERK can regulate ROS levels indirectly through p22phox, which increases ROS and upregulates antioxidants by Nrf2 activation. JNK activation can lead to FoxO activation, thereby resulting in antioxidant production (Arfin et al., 2021; Essers et al., 2004).

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

Based on the prioritized studies presented here, the evidence of taxonomic applicability is low for humans despite there being strong plausibility as the evidence only includes in vitro human cell-derived models. The taxonomic applicability for mice and rats is considered high as there is much available data using in vivo rodent models that demonstrate the concordance of the relationship. In terms of sex applicability, all in vivo studies that indicated the sex of the animals used male animals, therefore, the evidence for males is high and females is considered to be low for this KER. The majority of studies used adolescent animals, with a few using adult animals. Preadolescent animals were not used to support the KER; however, the relationship in preadolescent animals is still plausible.

References

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

Arfin, S. et al. (2021), “Oxidative Stress in Cancer Cell Metabolism”, Antioxidants 2021, Vol. 10/5, MDPI, Basel, https://doi.org/10.3390/ANTIOX10050642

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, https://doi.org/10.1152/ajpcell.00520.2019

Cabrera, M. P. and R. H. Chihuailaf (2011), “Antioxidants and the integrity of ocular tissues”, Veterinary medicine international, Vol. 2011, https://doi.org/10.4061/2011/905153

Diao, Y. et al. (2018), "Polyphenols (S3) Isolated from Cone Scales of Pinus koraiensis Alleviate Decreased Bone Formation in Rat under Simulated Microgravity", Scientific Reports, Vol. 8/1, Nature, https://doi.org/10.1038/s41598-018-30992-8

Domazetovic, V. et al. (2017a), "Oxidative stress in bone remodeling: role of antioxidants", Clinical cases in mineral and bone metabolism, Vol. 14/2, https://doi.org/10.11138/ccmbm/2017.14.1.209

Fontani, F. et al. (2015), “Glutathione, N-acetylcysteine and lipoic acid down-regulate starvation-induced apoptosis, RANKL/OPG ratio and sclerostin in osteocytes: involvement of JNK and ERK1/2 signalling”, Calcified tissue international, Vol. 96/4, Springer, London, https://doi.org/10.1007/s00223-015-9961-0

Hu, Q. et al. (2023), “JAK/STAT pathway: Extracellular signals, diseases, immunity, and therapeutic regimens”, Frontiers in bioengineering and biotechnology, Vol. 11, Frontiers Media, Lausanne, https://doi.org/10.3389/fbioe.2023.1110765

Kharrazian D. (2021), “Exposure to Environmental Toxins and Autoimmune Conditions”, Integrative medicine (Encinitas, Calif.), Vol. 20/2.

Kook, S. H. et al. (2015), “Irradiation inhibits the maturation and mineralization of osteoblasts via the activation of Nrf2/HO-1 pathway”, Molecular and Cellular Biochemistry, Vol. 410/1–2, Springer Nature, London, https://doi.org/10.1007/s11010-015-2559-z

Lehtinen, M. and A. Bonni. (2006), "Modeling Oxidative Stress in the Central Nervous System", Current Molecular Medicine, Vol. 6/8, https://doi.org/10.2174/156652406779010786.

Li, J. et al. (2013), "Oxidative Stress and Neurodegenerative Disorders", International Journal of Molecular Sciences, Vol. 14/12, https://doi.org/10.3390/ijms141224438.

Ma, J. et al. (2022), “Iron overload induced osteocytes apoptosis and led to bone loss in Hepcidin-/- mice through increasing sclerostin and RANKL/OPG”, Bone, Vol. 164, Elsevier, Amsterdam, https://doi.org/10.1016/j.bone.2022.116511

Manolagas, S. C. and M. Almeida. (2007), "Gone with the Wnts: β-Catenin, T-Cell Factor, Forkhead Box O, and Oxidative Stress in Age-Dependent Diseases of Bone, Lipid, and Glucose Metabolism", Molecular Endocrinology, Vol. 21/11, Oxford University Press, Oxford, https://doi.org/10.1210/me.2007-0259

Essers, M. A. et al. (2004), “FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK”. The EMBO journal, Vol. 23/24, EMBO, https://doi.org/10.1038/sj.emboj.7600476

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, https://doi.org/10.1093/jrr/rrab032

Ping, Z. et al. (2020), "Oxidative Stress in Radiation-Induced Cardiotoxicity", Oxidative Medicine and Cellular Longevity, Vol. 2020, Hindawi, London, https://doi.org/10.1155/2020/3579143

Ramadan, R. et al. (2021), "The role of connexin proteins and their channels in radiation-induced atherosclerosis", Cellular and Molecular Life Sciences, Vol. 78, Nature, https://doi.org/10.1007/s00018-020-03716-3

Riegger, Jana et al. (2023) “Oxidative stress as a key modulator of cell fate decision in osteoarthritis and osteoporosis: a narrative review”, Cellular & molecular biology letters, Vol. 28/1 doi:10.1186/s11658-023-00489-y

Romagnoli, C. et al. (2013), “Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells”, The FEBS journal, Vol. 280/3, Wiley, Hoboken, https://doi.org/10.1111/febs.12075

Schmidt-Ullrich, R. K. et al. (2000), "Signal transduction and cellular radiation responses.", Radiation research, Vol. 153/3, BioOne, https://doi.org/10.1667/0033-7587(2000)153[0245:stacrr]2.0.co;2

Sinkala, M. et al. (2021), “Integrated molecular characterisation of the MAPK pathways in human cancers reveals pharmacologically vulnerable mutations and gene dependencies”, Communications biology, Vol. 4/1, Springer, London, https://doi.org/10.1038/s42003-020-01552-6

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

Soumelis, V. and Y. J. Liu (2006), “From plasmacytoid to dendritic cell: morphological and functional switches during plasmacytoid pre-dendritic cell differentiation”, European journal of immunology, Vol. 36/9, Wiley, Hoboken, https://doi.org/10.1002/eji.200636026

Sun, Y. et al. (2013), "Treatment of hydrogen molecule abates oxidative stress and alleviates bone loss induced by modeled microgravity in rats", Osteoporosis International, Vol. 24/3, Nature, https://doi.org/10.1007/s00198-012-2028-4

Tahimic, C. G. T. and R. K. Globus. (2017), “Redox Signaling and Its Impact on Skeletal and Vascular Responses to Spaceflight”, International Journal of Molecular Sciences, Vol. 18/10, MDPI, Basel, https://doi.org/10.3390/IJMS18102153

Tian, Y. et al. (2017), "The impact of oxidative stress on the bone system in response to the space special environment", International Journal of Molecular Sciences, Vol. 18/10, MDPI, Basel, https://doi.org/10.3390/ijms18102132

Valerie, K. et al. (2007), "Radiation-induced cell signaling: inside-out and outside-in", Molecular Cancer Therapeutics, Vol. 6/3, American Association for Cancer Research, https://doi.org/10.1158/1535-7163.MCT-06-0596

Villalpando-Rodriguez, G. E. and S. B. Gibson (2021), “Reactive Oxygen Species (ROS) Regulates Different Types of Cell Death by Acting as a Rheostat”, Oxidative medicine and cellular longevity, Vol. 2021, John Wiley & Sons, Hoboken, https://doi.org/10.1155/2021/9912436

Wu, H. et al., (2023), “Molecular mechanisms of environmental exposures and human disease. Nature reviews” Genetics, Vol. 24/5, Springer, London, https://doi.org/10.1038/s41576-022-00569-3

Xin, M. et al. (2015), "Attenuation of hind-limb suspension-induced bone loss by curcumin is associated with reduced oxidative stress and increased vitamin D receptor expression", Osteoporosis International, Vol. 26/11, Nature, https://doi.org/10.1007/s00198-015-3153-7

Yoo, Y. M., T. Y. Han and H. S. Kim. (2016), "Melatonin suppresses autophagy induced by clinostat in preosteoblast MC3T3-E1 cells", International Journal of Molecular Sciences, Vol. 17/4, MDPI, Basel, https://doi.org/10.3390/ijms17040526

Zuo, R. et al. (2019), “BM-MSC-derived exosomes alleviate radiation-induced bone loss by restoring the function of recipient BM-MSCs and activating Wnt/β-catenin signaling”, Stem cell research & therapy, Vol. 10/1, Spinger, London, https://doi.org/10.1186/s13287-018-1121-9