This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Relationship: 2846

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

Oxidative Stress leads to Altered Bone Cell Homeostasis

Upstream event

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

Oxidative Stress

Downstream event

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

Altered Bone Cell Homeostasis

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	non-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	High	NCBI
rat	Rattus norvegicus	Moderate	NCBI

Sex Applicability

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

Sex	Evidence
Male	Moderate
Female	Low

Life Stage Applicability

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

Term	Evidence
Adult	Moderate
Juvenile	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

The tight regulation of differentiation pathways leading to bone-forming osteoblasts (osteoblastogenesis) and bone-resorbing osteoclasts (osteoclastogenesis) is essential for the maintenance of osteogenic balance, i.e., the deposition and resorption of bone matrix. As such, perturbations by the overproduction of reactive oxygen species (ROS) during oxidative stress can have devastating effects on the delicate balance of bone cell (i.e., osteocyte, osteoclast, and osteoblast) differentiation and function.

Oxidative stress disrupts the homeostatic balance of osteoblastic bone deposition and osteoclastic bone resorption by altering the osteoblastogenic/osteoclastogenic differentiation pathways through the overproduction of ROS (Tian et al., 2017). Briefly, ROS produced in pre-osteoblasts and pre-osteoclasts will affect the activities of different signaling molecules in the respective cell types. In osteoblasts, ROS naturally upregulate expression of the transcription factor forkhead box O (FoxO) which enhances cell antioxidant status. FoxO requires ß-catenin binding, which sequesters ß-catenin from the main osteoblast differentiation pathway, the Wnt/ß-catenin pathway, ultimately downregulating osteoblastogenesis and the expression of alkaline phosphatase (ALP) and osteocalcin (OCN) (Manolagas and Almeida, 2007; Tian et al., 2017). Further, ROS upregulate the receptor activator of nuclear factor kappa B ligand (RANK-L), which is the main regulator of osteoclastogenesis. By increasing RANK-L production, ROS inhibits osteoclast apoptosis and promotes osteoclastogenesis and the expression of tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CTSK), and HCl (Tian et al., 2017).

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

One study suggests X-ray radiation results in a dose-dependent increase in oxidative stress and bone resorption parameters only at doses above 2 Gy (Kook et al., 2015). This, however, is inconsistent with other studies performed at doses of 1-2 Gy, which indicate a significant effect of radiation on ROS production, TRAP expression, and ALP activity at lower doses (≤2 Gy) (Huang et al., 2018; Huang et al., 2019; Kondo et al., 2010; Zhang et al., 2020). Further research is needed to elucidate the effects of low doses, as well as the dose-dependent effect of increasing doses of ionizing radiation (IR).

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	α2M	Treatment reversed the radiation-induced effects on ALP and SOD activity	Liu et al., 2018
Drug	N-acetyl cysteine	2.5 and 5 mM reversed the effects of 8 Gy radiation on ROS levels and ALP activity	Kook et al., 2015
Drug	AMI	Treatment with 30 mg/kg reversed the radiation-induced effects on ROS levels, ALP activity and TRAP-5b levels	Huang et al., 2019; Zhang et al., 2020
Drug	CeO₂	Treatment with 100 nM lowered dihydroethidium (DHE) and H₂O₂ levels and partially restored Alizarin red optical density	Wang et al., 2016
Drug	Sema3a	Treatment with 50 ng/mL partially reduced ROS levels and reversed TRAP stain to below controls	Huang et al., 2018
Drug	Curcumin (antioxidant)	Fully reversed all oxidative stress and altered bone cell homeostasis	Xin et al., 2015
Drug	Hydrogen water	Reversed microgravity-induced effects on oxidative stress and altered bone cell homeostasis	Sun et al., 2013
Drug	Polyphenol S3	Fully reversed microgravity-induced oxidative stress, osteoblastogenesis and osteoclastogenesis	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 following are a few examples of quantitative understanding of the relationship. All data 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
Huang et al., 2018	In vitro. A single dose of 2 Gy ⁶⁰Co gamma radiation by linear accelerator was administered to murine RAW264.7 osteoclast-like cells at a rate of 0.83 Gy/min. ROS production was measured to assess oxidative stress and TRAP staining was used to measure subsequent osteoclastogenic changes.	2-fold increase in ROS production accompanied by a ~2-fold increase in the number of TRAP-positive cells in cells exposed to 2 Gy ⁶⁰Co gamma radiation relative to controls.
Huang et al., 2019	Ex vivo. A single dose of 2 Gy ⁶⁰Co gamma radiation was administered to bone marrow stromal stem cells of Sprague-Dawley rats at a rate of 0.83 Gy/min. ROS production was measured to assess oxidative stress and ALP activity was measured to determine subsequent imbalances in osteoblastogenesis.	~2-fold increase in ROS production with a 0.33-fold decrease in ALP activity in cells exposed to 2 Gy ⁶⁰Co gamma rays relative to unirradiated controls.
Zhang et al., 2020	In vitro. RAW264.7 cells were irradiated with 2 Gy of ⁶⁰Co gamma radiation at a rate of 0.83 Gy/min was administered. ROS production was measured to assess oxidative stress and TRAP staining was used to measure subsequent changes to osteoclastogenesis.	2-fold increase in ROS production in RAW264.7 cells and a 2-fold increase in the number of TRAP-positive osteoclasts when exposed to 2 Gy gamma radiation.
Kondo et al., 2010	In vivo. Male C57BL/6J mice at 17 weeks of age were hindlimb unloaded or normally loaded, 4 days later they were exposed to 1 or 2 Gy of ¹³⁷Cs or sham irradiated. Oxidative stress markers including, ROS production, MDA, and 4-hydroxynonenal (4-HNE) were measured along with tibial osteoclast surface.	In normally loaded mice, there was a ~1.3-fold increase in ROS at 1 Gy by day 3 and a ~1.2-fold increase in ROS at 2 Gy by day 10. There was a 2-fold increase in MDA and 4-HNE under exposure to either 1 or 2 Gy gamma radiation relative to control in normally loaded models by day 10. There was a 46%, 47% and 64% increase in tibiae osteoclast surface as a result of 2 Gy irradiation, hindlimb unloading and the combination of irradiation and hindlimb unloading, respectively.
Kook et al., 2015	In vitro. MC3T3-E1 cells were exposed to various doses of X-ray irradiation (0–8 Gy) at a rate of 1.5 Gy/min. Levels of ROS, superoxide dismutase (SOD), and glutathione (GSH) were measured to assess oxidative stress and ALP activity was measured to assess subsequent changes in osteoblastogenesis.	Roughly linear dose-dependent increase from 0-8 Gy (significant increases at 4 and 8 Gy) in intracellular ROS accumulation up to 1.39-fold of the control at 8 Gy. Dose dependent decrease from 2-8 Gy (significant decreases at 4 Gy and 8 Gy) in SOD and GSH activity to half at 8 Gy. Following 8 Gy of IR, OCN mRNA expression decreased 48% compared to the non-irradiated control. Irradiation at 4 Gy showed similar decrease in OCN mRNA expression. Mouse bone marrow stromal cell ALP activity saw a significant, 0.62-fold decrease following 8 Gy irradiation.
Liu et al., 2018	In vitro. Human bone marrow-derived mesenchymal cells (hBMMSCs) were irradiated with X-rays at a dose of 2, 4, 8 and 12 Gy and a dose rate of 1.24 Gy/min. SOD levels were measured to assess oxidative stress. ALP activity, calcium deposition and hBMMSCs proliferation were determined.	Following irradiation at 8 Gy, there was a ~0.5-fold decrease in osteoblast SOD activity. There was a dose-dependent decrease in hBMMSC proliferation following irradiation with 2, 4, 8, and 12 Gy, compared to the non-irradiated control. Changes in cell proliferation became significant at doses ≥8 Gy, with a maximum decrease of ~0.60-fold at 1 week-post irradiation with 12 Gy. 8 Gy of IR resulted in a 0.46 decrease in both ALP activity and calcium deposition compared to non-irradiated controls.
Wang et al., 2016	In vitro. MC3T3-E1 cells were exposed to X-ray irradiation at a dose of 6 Gy. Levels of ROS and H2O2 were measured along with ALP activity and calcium deposition.	1.5-fold increase in H2O2 accumulation and 1.75-fold increase in ROS staining intensity under 6 Gy X-rays. Measured at 1-week post-irradiation, following 6 Gy of IR, there was a 0.54-fold decrease in ALP activity compared to the non-irradiated controls. Measured at 3 weeks post-irradiation, Alizarin Red staining revealed a ~0.1-fold decrease in calcium deposition following exposure to 6 Gy of IR.
Xin et al., 2015	In vivo and in vitro. Sprague-Dawley rats (8 weeks old) were hindlimb suspended and after six weeks, oxidative stress markers and altered bone cell homeostasis were measured. MC3T3-E1 cells and RAW264.7 cells were exposed to modeled microgravity in the NASA rotating wall vessel bioreactor (RWVB). Intracellular ROS and ALP and TRAP levels were measured.	Rat femur MDA increased by ~1.4-fold. Rat femurs showed a ~2.5-fold increase in TRAP mRNA and a ~0.5-decrease in OCN mRNA. MC3T3-E1 cells found a ~1.3-fold increase in ROS formation and a ~0.75-fold decrease in ALP activity. RAW264.7 found a 2-fold increase in intracellular ROS and a 2-fold increase in TRAP positive osteoclasts.
Sun et al., 2013	In vivo and in vitro. Male Sprague–Dawley rats were subjected to hindlimb suspension for 6 weeks. RAW264.7 and MC3T3-E1 cells were exposed to modeled microgravity by RWVB (0.01xg). Femoral peroxynitrite (OONO-), MDA, and intracellular ROS were measured to assess oxidative stress and deoxypyridinoline (DPy), ALP levels, and TRAP-positive cells were subsequently measured to assess bone cell function.	Rats exposed to microgravity via unloading of hindlimbs showed a 2.5-fold increase in femoral peroxynitrite (OONO-) and a 1.3-fold increase in femoral MDA. This was accompanied by a roughly 1.8-fold increase in DPy excretion (biomarker of bone resorption) and 0.4-fold decrease in femoral ALP expression. Exposure to modeled microgravity in MC3T3-E1 (osteoblast cell line) led to a ~1.4-fold increase in intracellular ROS and a 0.75-fold decrease in osteoblast ALP activity. RAW264.7 (preosteoclast cell line) found a ~2-fold increase in ROS and a ~5-fold increase in TRAP mRNA expression.
Diao et al., 2018	In vivo. 50 Male Sprague-Dawley rats (6 weeks) were hindlimb suspended for 72 hours. SOD, catalase (CAT), and MDA were measured to assess oxidative stress and TRAP-5b, OCN and N-terminal type 1 collagen telopeptide (NTX) were measured to assess subsequent bone cell function.	Rats under hindlimb suspension showed a ~0.4-fold decrease in SOD, ~0.3-fold decrease in CAT activity, and a ~1.5-fold increase in MDA relative to unloaded controls in rat femur. This was accompanied by ~1.14-fold increase in serum TRAP-5b and ~1.3-fold increase in NTX. Relative mRNA OCN levels and mRNA collagen I alpha 1 in rat femur decreased significantly.

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
Huang et al., 2018	In vitro. A single dose of 2Gy ⁶⁰Co gamma radiation was administered to murine RAW264.7 osteoclast-like cells at a rate of 0.83 Gy/min. ROS production was measured to assess oxidative stress and TRAP staining was used to measure subsequent changes in osteoclastogenesis.	2-fold increase in ROS production after 2h accompanied by a ~2-fold increase in the number of TRAP-positive cells after 7 days in cells exposed to 2 Gy gamma radiation relative to controls.
Kondo et al., 2010	In vivo. Male C57BL/6J at 17 weeks of age were hindlimb unloaded or normally loaded, 4 days later they were exposed to 1 or 2 Gy of ¹³⁷Cs or sham irradiated. Oxidative stress markers including, ROS production, MDA, and 4-HNE were measured along with tibial osteoclast surface.	In normally loaded mice, at day 3, ROS in the 1 Gy group increased significantly. By day 10, however, ROS in the 1 Gy group had dropped relative to day 3, while ROS in the 2 Gy group reached significant levels compared to the control. Also, by day 10, MDA and 4-HNE increased ~2-fold in normally loaded mice. This was accompanied by a 46% increase in tibiae osteoclast surface due to irradiation at day 3.
Kook et al., 2015	In vitro. MC3T3-E1 cells were exposed to various doses of X-ray radiation (0–8 Gy) at a rate of 1.5 Gy/min. Levels of ROS, SOD, and GSH were measured to assess oxidative stress and ALP activity was measured to assess subsequent changes in osteoblastogenesis.	Roughly linear dose-dependent increase (after 2 Gy X-ray radiation) in intracellular ROS accumulation up to 1.39-fold of the control at 8 Gy after 1 day. Dose dependent decrease (after 2 Gy) in SOD and GSH activity to half at 8 Gy after 1 day. Markers for altered bone cell homeostasis were measured at 7 days post-irradiation. Following 8 Gy of IR, OCN mRNA expression decreased 48% compared to the non-irradiated control. Irradiation at 4 Gy showed similar decrease in OCN mRNA expression. Mouse bone marrow stromal cell ALP activity saw a significant, 0.62-fold decrease following 8 Gy irradiation.
Liu et al., 2018	In vitro. hBMMSCs were irradiated with X-rays at dose of 2, 4, 8 and 12 Gy, and a dose rate of 1.24 Gy/min. SOD levels were measured to assess oxidative stress. ALP activity, calcium deposition and hBMMSCs proliferation were determined.	0.5-fold decrease in osteoblast SOD activity after 1 day with 0.46-fold decrease in ALP activity after 1 week.

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 majority of the evidence is from in vitro human-derived cells. The relationship is supported by mice and rat models using male and female animals. The relationship is plausible at any life stage. However, most studies have used adolescent and adult animal models