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Relationship: 2716

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 Increase, Cell death

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

Increase, Cell death

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
Calcium overload in dopaminergic neurons of the substantia nigra leading to parkinsonian motor deficits	adjacent	Not Specified	Not Specified	Cataia Ives (send email)	Under development: Not open for comment. Do not cite
Deposition of energy leading to occurrence of bone loss	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	Moderate
Female	Moderate

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

Oxidative stress can cause cellular damage and activate signalling cascades that result in programmed cell death, including apoptosis and autophagy. Increased production of free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), collectively RONS, and a weakened antioxidant defense system can be detrimental. When free radicals overwhelm antioxidants, the resulting oxidative stress can cause damage to DNA, including base damage; strand breaks; and mutation, as well as damage to vital cellular components, such as lipid peroxidation within the cellular and mitochondrial membranes. Sufficient oxidative damage to the cell can result in programmed cell death (Pacheco and Stock, 2013; Tian et al., 2017). Overwhelming DNA damage from oxidative stress can result in cell damage and death.

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

When MC3T3-E1 murine preosteoblast cells underwent microgravity conditions in a 3D clinostat, CAT expression increased by ~1.25-fold. This response was the opposite of the other antioxidants that were measured and is contrary to the decrease in antioxidant expression normally seen after microgravity exposure (Yoo, Han & Kim, 2016).
Kondo et al. (2010) did not observe any significant effects to MDA+4-HNE levels or apoptosis after subjecting their C57BL/6J mice to hindlimb unloading.

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 SOD activity, reduced autophagy, reduced osteocyte cell death, and reduced the rate of apoptosis in hBMMSCs.	Liu et al., 2018; Li et al., 2018
Drug	Sema3a	Treatment with 50 ng/mL partially reduced ROS levels and promoted Raw264.7 cell apoptosis after irradiation.	Huang et al., 2018
Drug	AMI	Treatment with 30 mg/kg reversed the radiation-induced effects on ROS levels and reduced the percentage of apoptotic cells and DNA damage.	Huang et al., 2019
Nanoparticle	CeO2	Cerium oxide acts can switch between a fully reduced and fully oxidized state, allowing it to mimic antioxidants to mediate oxidative stress. Treatment with 100nM significantly attenuated IR-induced increases to ROS production and extracellular hydrogen peroxide, as well as causing cell viability to significantly recover.	Wang et al., 2016
Drug	Melatonin (antioxidant)	Treatment with 200nM melatonin reversed the effect of microgravity on Bcl-2, Bax, Cu/Zn-SOD and Mn-SOD to control levels.	Yoo, Han & Kim, 2016

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
Liu et al., 2018	In vitro. hBMMSCs were irradiated with 8 Gy of X-rays at a rate of 1.24 Gy/min. SOD activity and MnSOD protein expression levels were measured to assess oxidative stress. hBMMSCs were stained for Annexin V to determine cell death.	SOD activity decreased by ~0.5-fold compared to the non-irradiated control at 24 hours post-irradiation. MnSOD protein expression decreased by ~0.4-fold. This decrease in antioxidant defense resulted in a ~3-fold increase in the rate of apoptosis.
Huang et al., 2018	In vitro. Murine RAW264.7 macrophage cells were irradiated with 2 Gy of gamma rays at a rate of 0.83 Gy/min. ROS levels were measured to assess oxidative stress. Levels of Annexin binding was measured to determine cell death.	ROS levels had a maximum increase of ~2.5-fold compared to the non-irradiated control at 2 hours post-irradiation. This increase in oxidative stress was accompanied by a 5.3-fold increase in apoptotic cells (from 1.9% to 9.8%) at 24 hours post-irradiation.
Liu et al., 2019	In vivo. 8-10-week-old, female, SPF BALB/c mice underwent whole-body irradiation with 2 Gy of carbon ions (LET=31.6 KeV/µm in water) at a rate of 1 Gy/min. Femoral bone marrow mononuclear cells were then extracted and ROS levels were measured to assess oxidative stress, while Annexin binding was used to measure the number of apoptotic cells.	ROS levels increased by ~2.2-fold, compared to the non-irradiated control. This increase in oxidative stress was accompanied by a ~5.4-fold increase in early apoptosis and a ~4.2-fold increase in late apoptosis/necrosis.
Huang et al., 2019	Ex vivo. A single 2 Gy dose of ⁶⁰Co gamma radiation was administered to bmMSCs of Sprague Dawley rats at a rate of 0.83 Gy/min. ROS production was measured to assess oxidative stress and apoptosis was determined by Annexin V staining.	ROS production increased by ~2-fold compared to the non-irradiated control. This increase in oxidative stress was accompanied by a ~4-fold increase in osteoblast apoptosis.
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 gamma rays or sham-irradiated. Intracellular ROS and apoptotic cell numbers in the bone marrow cells of the right femora were assessed to determine oxidative stress and cell death, respectively. To assess oxidative damage MDA and 4-HNE were measured.	Following irradiation under normal loading, ROS production increased by ~1.3-fold at 1 Gy by day 3 post-irradiation and a ~1.2-fold at 2 Gy by day 3. The cumulative levels of MDA and 4-HNE increased by ~2-fold under exposure to both 1 and 2 Gy by day 10. This increase in oxidative stress was associated with a ~1.6-fold increase in bone marrow cell apoptosis at 2 Gy by day 3.
Wang et al., 2016	In vitro. Murine MC3T3-E1 osteoblast-like cells were irradiated with 6 Gy of X-rays. Intracellular ROS production and extracellular hydrogen peroxide levels were measured to assess oxidative stress and cell viability was measured to assess cell death.	Intracellular ROS production and extracellular hydrogen peroxide levels increased by ~1.75-fold at 24 hours post-irradiation and ~1.5-fold at 3 hours post-irradiation, respectively, compared to the non-irradiated control. This increase in oxidative stress was accompanied by a significant ~0.3-fold decrease in cell viability at 4 days post-irradiation (no significant decrease at 1 day).
Bai et al., 2020	Ex vivo. bmMSCs were taken from 4-week-old, male Sprague-Dawley rats. After extraction, cells were then irradiated with 2, 5, and 10 Gy of ¹³⁷Cs gamma rays. Intracellular ROS levels and relative mRNA expression of the antioxidants, SOD1, SOD2, and CAT2, were measured to assess the extent of oxidative stress induced by IR. Cell death was measured by a viability assay.	Cellular ROS levels increased significantly in a dose-dependent manner from 0-10 Gy. Compared to sham-irradiated controls, ROS levels increased by ~15%, ~55%, and ~105% after exposure to 2, 5, and 10 Gy, respectively. Antioxidant mRNA expression decreased in a dose-dependent manner from 0-10 Gy, with significant decreases seen at doses as low as 2 Gy for SOD1 and CAT2 and 5 Gy for SOD2. Compared to sham-irradiated controls, SOD1 expression decreased by ~9%, ~18%, and ~27% after exposure to 2, 5, and 10 Gy, respectively. SOD2 expression decreased by ~31% and ~41% after exposure to 5 and 10 Gy, respectively. CAT2 expression decreased by ~15%, ~33%, and ~58% after exposure to 2, 5, and 10 Gy, respectively. This increase in oxidative stress was associated with decreases in cell viability of ~33% and ~44% after 1 day post-exposure to 5 and 10 Gy, respectively, and ~3%, ~45%, and ~65% after 3 days post-exposure to 2, 5, and 10 Gy.
Li et al., 2020	In vitro. hBMMSCs were exposed to 8 Gy of X-ray radiation at a rate of 2.75 Gy/min. To assess IR-induced oxidative stress, ROS levels were measured. hBMMSC apoptosis was then measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and an Annexin V-FITC/PI apoptosis detection kit to assess cell subsequent cell death.	At 24 hours post-irradiation, ROS levels increased by ~3.3-fold and ~1.5-fold when measured with fluorescent microscopy and flow cytometry, respectively. At 24 hours post-irradiation, cell apoptosis increased by ~1.8-fold. TUNEL-positive cells experienced a maximum increase of ~1.75-fold compared to the non-irradiated control at 7 days post-irradiation.
Li et al., 2018	In vivo. The mandibles of Sprague-Dawley rats were exposed to a cumulative dose of 35 Gy of X-ray radiation fractionated into 7 Gy daily for 5 days. ROS activity was measured along with SOD activity to assess oxidative stress and empty lacunae were measured to assess cell death among osteocytes.	ROS activity increased significantly at days 1, 14, and 28, with a maximum increase of ~5-fold at day 28. SOD activity decreased significantly at days 1 and 14, with a maximum decrease of ~0.66-fold at day 1. The % of empty lacunae increased ~1.8-fold compared to the non-irradiated control at 4 months-post irradiation.
Yoo, Han & Kim, 2016	In vitro. MC3T3-E1 murine pre-osteoblast cells underwent microgravity conditions in a 3D clinostat. The expression of the antioxidants, Cu/Zn-SOD; Mn-SOD; and CAT, were measured to assess oxidative stress. The expression of the apoptosis/autophagy regulators, Bax and Bcl-2, were measured along with the autophagy marker, LC3 II, to assess IR-induced cell death	After 72 hours, expression of Cu/Zn-SOD and Mn-SOD decreased by ~0.25-fold and ~0.6-fold, respectively, while CAT expression increased by ~1.25-fold. LC3 II levels increased by ~2.25-fold compared to the normally loaded control. Bax levels increased by ~2.4-fold, while Bcl-2 levels decreased by ~0.6-fold.

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. Murine RAW264.7 macrophage cells were irradiated with 2 Gy of gamma rays (⁶⁰Co isotope) at a rate of 0.83 Gy/min. ROS levels were measured to assess oxidative stress. Levels of Annexin binding was measured to determine the effects of IR on cell death.	ROS levels increased by ~2.5-fold at 2 hours post-irradiation and ~2-fold at 8 hours. The increase in oxidative stress was followed by a ~5.26-fold increase in apoptotic cells (from 1.86% to 9.78%) at 24 hours post-irradiation.
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 gamma rays or sham-irradiated. Intracellular ROS and apoptotic cell numbers in the bone marrow cells of the right femora were assess to determine oxidative stress and cell death, respectively. To assess oxidative damage, MDA and 4-HNE levels were measured.	Following irradiation under normal loading, ROS production increased by ~1.3-fold at 1 Gy by day 3 post-irradiation and a ~1.2-fold at 2 Gy by day 10. The cumulative levels of MDA and 4-HNE increased by ~2-fold under exposure to both 1 and 2 Gy by day 10. This increase in oxidative stress was associated with a ~1.6-fold increase in bone marrow cell apoptosis at 2 Gy by day 3.
Li et al., 2020	In vitro. hBMMSCs were exposed to 8 Gy of radiation. To assess IR-induced oxidative stress, ROS levels were measured. hBMMSC apoptosis was then measured using TUNEL staining and Annexin V-FITC/PI staining to assess cell subsequent cell death.	ROS levels increased significantly at 24 hours post-irradiation. Cell apoptosis also increased significantly at 24 hours post-irradiation. IR-induced changes to the % of TUNEL-positive cells decreased over time, with increases of ~1.75-fold compared to the non-irradiated control at 7 days post-irradiation, ~1.35-fold at 14 days, and ~1.33-fold at 28 days.
Li et al., 2018	In vivo. The mandibles of Sprague-Dawley rats were exposed to a cumulative dose of 35 Gy of radiation fractionated into 7 Gy daily for 5 days. Empty lacunae were measured to assess cell death among osteocytes and ROS activity was measured along with SOD activity to assess oxidative stress.	ROS activity increased by ~4.9-fold compared to the non-irradiated control at day 1 post-irradiation, ~3.7-fold at day 14, and ~5-fold at day 28. SOD activity experienced a maximum decrease of ~0.66-fold at day 1 and recovered over time with a ~0.78-fold decrease at day 14, and a non-significant increase at day 28. The % of empty lacunae increased significantly compared to the non-irradiated control at 4 months-post irradiation.
Wang et al., 2016	In vitro. Murine MC3T3-E1 osteoblast-like cells were irradiated with 6 Gy of X-rays. Intracellular ROS production and extracellular hydrogen peroxide levels were measured to assess oxidative stress and cell viability was measured to assess cell death.	ROS production increased by ~1.75-fold at 24 hours post-irradiation and hydrogen peroxide levels increased by ~1.5-fold at 3 hours-post irradiation, while cell viability did not decrease significantly until 4 days post-exposure (~0.3-fold).

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

None 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 and in vitro animal-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.