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Relationship: 2847

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

Energy Deposition leads to Altered Bone Cell Homeostasis

Upstream event

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

Energy Deposition

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	High	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	Moderate	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	Low

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

Energy deposition in the form of ionizing radiation (IR) exposure can result in a loss of homeostasis among the osteocyte, osteoclast, and osteoblast bone cells. The severity of the irradiation effects is influenced by dose, dose rate, and the level of linear energy transfer (LET) between IR and bone tissue. The energy deposited into cells causes ionization events that can lead to oxidative stress, which may induce cell death and alter signalling pathways in the bone microenvironment that regulate the differentiation and activity of bone remodeling cells (Willey et al., 2011). Bone cells can be dysregulated by deposited energy from a variety of IR types, including X-rays, gamma rays, and heavy ions, and has been observed at a wide range of doses from 0-30 Gy. IR-induced changes to bone cell homeostasis are defined by progenitor cell proliferation, markers for osteoblast and osteoclast activity, and the number and surface area of both cell types on a sample.

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

Not all radiation qualities and doses of radiation will alter bone cell homeostasis in the same way. Low doses (<1 Gy) of low LET electromagnetic radiation (X-rays and gamma rays) are shown to increase osteoblasts and decrease osteoclasts, while high doses do the opposite (Donaubauer et al., 2020). This is in contrast with particle irradiation, where osteoblasts are decreased and osteoclasts are increased at low and high doses (Donaubauer et al., 2020).
There are differences in the mechanisms of altered bone cell homeostasis between humans and animals during spaceflight. In humans, increased osteoclast activity is the main cause of bone loss, while in rats, resorption was unchanged (Fu et al., 2021; Stavnichuk et al., 2020). However, microgravity is also a stressor in this case and not just radiation, and there are differences in how this is measured between humans and animals.
At 3 days post-irradiation, da Cruz Vegian et al. (2020) found that, in addition to an IR-induced increase in TRAP levels (osteoclastogenesis marker), rats that underwent 30 Gy irradiation also experienced a significant, ~8-fold increase in levels of the osteoblastogenesis marker, OCN, compared to non-irradiated controls. In addition, TRAP levels experienced a time-dependent decrease. This is contrary to the increase in osteoclastogenesis and decrease in osteoblastogenesis generally seen post-irradiation.

Chen et al. (2014) showed increased OCN mRNA expression and protein activity after 0.5 or 5 Gy X-ray irradiation, which is contrary to the decrease in osteoblastogenesis following irradiation observed in other studies. This may be explained by the survival strategy of osteoblasts to retain cell division for DNA repair as opposed to undergoing programmed death (Chen et al., 2014).

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	Risedronate (osteoporosis drug that blocks osteoclast activity)	Returned TRAP5b levels to near baseline and reduced the osteoclast count after radiation	Willey et al., 2010
Drug	α-2-macroglobulin (α2M); a radio-protective macromolecule	Treatment at 0.25 and 0.5 mg/mL slightly restored ALP activity.	Liu et al., 2018
Age	Old age	Lower estrogen at old age is thought to increase osteoclast activity, compounding with the effects of radiation.	Pacheco and Stock, 2013

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 Concordance

Reference	Experiment Description	Result
Stavnichuk et al., 2020	In vivo. A meta-analysis that extracted biochemical markers in 124 astronauts from articles from 1971 to 2019. The longer the spaceflight, the higher dose of ionizing radiation the astronauts received, although ionizing radiation was not the only stressor that the astronauts would have received. Markers for osteoblast activity included serum ALP and C-terminal cleaved collagen type 1 propeptide (PICP). Markers for osteoclast activity included urine HP, NTX, CTX, and DPD.	Early increases in resorption markers and early decreases in formation markers were observed, with late increases in formation markers. Bone resorption markers increased hyperbolically with a t1/2 of 11 days and a plateau at 113%. Formation markers increased linearly at 7% per month. Resorption markers dropped to pre-flight levels after flight, while formation markers continued to increase at 84% per month for 3-5 months.
Kook et al., 2015	In vitro. Mouse bone marrow stromal cells and the MC3T3-E1 murine osteoblast cell line were both irradiated with 0-8 Gy of X-rays at a rate of 1.5 Gy/min. Levels of the osteoblast mineralization proteins, ALP and OCN, were measured 7 days post-irradiation to observe changes to osteoblast activity.	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.
da Cruz Vegian et al., 2020	In vivo. Sixty male Wistar rats were implanted with grade V titanium femur implants and were separated into four groups: (a) no-irradiation group (N-Ir); (b) early-irradiation group (E-Ir); (c) late-irradiation group (L-Ir); and (d) previous-irradiation group (P-Ir). The animals in the E-Ir, L-Ir, and P-Ir groups were irradiated in two fractional stages of 15 Gy of ⁶⁰Co gamma rays for a total of 30 Gy. Blood samples were collected at the time of euthanasia. Cells were measured for TRAP and OCN levels.	At 3-days post-irradiation, rats observed significant, ~8-fold increase in TRAP levels compared to the non-irradiated control.
Zhang et al., 2020	In vitro and in vivo. Male Sprague-Dawley rats and the RAW264.7 cell line were irradiated with 2 Gy of ⁶⁰Co gamma rays at a rate of 0.83 Gy/60 seconds. TRAP staining was used to determine changes to osteoclast numbers following IR exposure.	Following exposure to IR, there was a ~2-fold and ~2.7-fold increase in the number of TRAP-positive osteoclasts in RAW264.7 and rat femur samples, respectively, compared to the non-irradiated control.
Huang et al., 2019	In vitro. Bone marrow MSCs (bmMSCs) from the tibiae and femur of rats were irradiated with 2 Gy of ⁶⁰Co gamma rays at a rate of 0.83 Gy/min. bmMSCs were analyzed for changes in bone cell function through measuring levels of ALP, calcium deposition and proliferation of the bmMSCs.	Following IR exposure, there was a ~0.6-fold decrease in bmMSC proliferation compared to non-irradiated controls. Levels of ALP activity and calcium deposition saw a 0.33-fold 0.66-fold decrease, respectively, from 0 Gy to 2 Gy.
Liu et al., 2018	In vitro. hBMMSCs were irradiated with 2, 4, 8, and 12 Gy of X-rays at a rate of 1.24 Gy/min. Cells were analyzed for progenitor cell proliferation, ALP activity, and calcium deposition to determine the effect of IR on osteoblast function.	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.
Li et al., 2020	In vitro. hBMMSCs were exposed to 8 Gy of X-rays. To determine the effects of IR on bone cell function, TRAP staining was used to determine the number of osteoclasts/mm2 of bone surface and the CCK-8 assay was used to measure hBMMSC proliferation.	Following exposure to 8 Gy of IR, there was a ~3-fold increase in osteoclast number at 7 days post-irradiation, compared to the non-irradiated control. There was a 0.77-fold decrease in hBMMSC proliferation after 72 hours post-irradiation, compared to the non-irradiated control.
Wang et al., 2016	In vitro. The MC3T3-E1 osteoblast-like cell line was irradiated with 6 Gy of X-rays. Following irradiation, ALP activity and calcium deposition were measured to determine the effects of IR on osteoblast activity.	Measured at 1-week post-irradiation, 6 Gy of IR resulted in 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.
Wright et al., 2015	In vivo and ex vivo. the right hindlimbs of 20-week-old male C57BI/6 mice were irradiated with 2 Gy of X-rays at a rate of 1.6 Gy/min. In addition, the calvariae of 4-day-old Swiss White mice were extracted and irradiated with 2 and 10 Gy of X-rays at a rate of 0.244 Gy/min. The number of TRAP5b-positive osteoclasts and osteoblasts/ mm2 of bone surface were measured in models. In vitro. Osteocyte-like cells (MLO-Y4) and osteoblast cells (MC3T3) were irradiated with 0-20 Gy X-rays.	Following in vivo irradiation of the right hindlimb of C57BI/6 mice with 2 Gy of IR, there was a ~1.7-fold increase in osteoclast number over bone surface compared to the non-irradiated control. There was no significant difference in osteoblast number following irradiation. While 2 Gy of IR did not lead to a significant change in osteoblast number, exposure to 10 Gy eventually resulted in a significant, ~0.4-fold decrease in calvarial bone-derived osteoblasts at 10 days post-irradiation, compared to the non-irradiated control.
Willey et al., 2008	In vivo. Thirty-two 13-week-old C57BL/6 mice were either irradiated by 2 Gy X-rays or served as controls. Osteoclast surface, osteoblast surface, osteoclast number and TRAP-5b levels were measured after 3 days to determine the effects of IR on bone cell function.	The stained bone sections of the irradiated mice showed a 44% increase in the number of osteoclasts/mm2 of bone surface, a 14% increase in serum levels of TRAP5b, and a 213% increase in osteoclast-covered bone surface area compared to the control. The irradiated bone sections were also tested for changes in serum levels of OCN (osteoblast activity marker), showing a non-significant radiation-induced decrease.
Willey et al., 2010	In vivo. 20-week-old female C57BL/6 mice were irradiated with 2 Gy X-rays, and left/right hind limbs, along with the vertebral column trabecular bone was analyzed, in addition to blood samples taken for serum analysis. Osteoblast marker OCN and osteoclast marker TRAP-5b was measured with enzyme-linked immunosorbent assay (ELISA). Osteoblast and osteoclast surfaces were determined as well.	Osteoblast surface did not change, but osteoclast surface increased 1.6-fold. Analysis of blood serum samples showed a 21% increase in the serum levels of TRAP-5b at 1 week post-irradiation compared to the control group. Serum levels of OCN were also measured, but no significant differences were found at 1, 2, or 3 weeks post-irradiation. Osteoclast number relative to bone surface increased 218% in the irradiated group, compared to the non-irradiated group.
Kondo et al., 2009	In vivo. 17-week-old male mice were exposed to 1 and 2 Gy of ¹³⁷Cs gamma-rays and their trabecular bone tissue was analyzed at 3- and 10-days post-irradiation. The number of osteoclasts was measured with TRAP staining.	At 3 days post-irradiation, the number of osteoclasts/square mm of bone surface area was ~2-fold higher than the control (0 Gy) under 1 Gy of radiation and ~2.5-fold higher under 2 Gy of radiation. At 10 days post-irradiation, the number of osteoclasts was ~3-fold higher than the control under 1 Gy of radiation and ~2.5-fold higher under 2 Gy of radiation.
Sakurai et al., 2007	In vitro. To evaluate the effects of radiation on osteoblast differentiation, murine C2C12 myoblast cells (osteoblast-like cells) were irradiated in vitro with 2 and 4 Gy of X-rays, differentiation was induced with BMP-2 and heparin over the course of 3 days. Collagen type 1 and ALP were used as markers of osteoblast differentiation.	When exposed X-rays, ALP activity of the C2C12 cells showed a significant, dose-dependent response. C2C12 cells experienced a ~0.3-fold decrease in ALP activity from 0 Gy to 4 Gy, and a 0.5-fold decrease from 0 Gy to 2Gy. Collagen type I was significantly reduced at both doses.

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
da Cruz Vegian et al., 2020	In vivo. Sixty male Wistar rats were implanted with grade V titanium femur implants and were separated into four groups: (a) N-Ir; (b) E-Ir; (c) L-Ir; and (d) P-Ir. The animals in the E-Ir, L-Ir, and P-Ir groups were irradiated in two fractional stages of 15 Gy ⁶⁰Co gamma radiation for a total of 30 Gy. Blood samples were collected at the time of euthanasia. Cells were measured for TRAP and OCN levels.	OCN levels in the irradiated groups increased greater than non-irradiated levels at 3 days, By the second week, only P-Ir OCN levels were greater than the N-Ir group. TRAP was greater than N-Ir in all irradiated group at day 3. At week 2, L-Ir TRAP levels fell below control levels, followed by a slight increase in TRAP in all irradiated groups by week 7.
Zhang et al., 2020	In vivo and in vitro. 2 Gy of ⁶⁰Co gamma rays were given to male rats and the RAW264.7 cell line. To detect changes in osteoclast activity following IR exposure, the number of osteoclasts and levels of TRAP5b were measured 1, 3, 5, and 7 days after exposure.	Samples of blood from rat tail vein were obtained and TRAP5b levels in the serum were measured. In the 2 Gy irradiated group, TRAP5b levels in serum increased 1.7-fold after 3 days and 2.6-fold after 5 days, followed by a slight decrease to a 2.4-fold change at day 7 (Fig. 6).
Willey et al., 2008	In vivo. Thirty-two C57BL/6 mice were either irradiated by 2 Gy X-rays or served as controls. Osteoclast surface, osteoblast surface, osteoclast number and TRAP-5b levels were measured after 3 days to determine the effects of IR on bone cell function.	In the radiated group, osteoclast surface, osteoclast number, and TRAP-5b level increased after 3 days by 213%, 44%, and 14%, respectively, compared to the control group. Osteoblast surface was decreased by 3% after 3 days compared to the non-radiated group.
Chen et al., 2014	In vitro and in vivo. In vitro MC3T3-E1 cells were exposed to a single 0.5 Gy or 5 Gy dose of X-ray irradiation at a rate of 200 cGy/min. In vivo male Sprague-Dawley rats were exposed to 0.5 Gy or 5 Gy dose of X-ray irradiation. Rats were euthanized 7, 14, 21 and 28 days after irradiation. Osteoblast differentiation markers, such as OCN and ALP, were measured post-irradiation by western blot. TRAP positive cells were used to determine osteoclast counts.	In vitro. ALP levels in all three groups (control, 0.5 Gy, and 5 Gy) were roughly the same levels relative to each other at day 4 and day 14. Irradiation-induced increases in ALP occurred on day 7 and 10 post-irradiation in both irradiated groups. OCN protein level was increased at day 10 in both irradiated groups, with the increases in the 0.5 Gy group continuing onto day 14 post-irradiation. In vivo. TRAP staining indicated an increase in the number of osteoclasts in the 0.5 Gy irradiated group at day 14, followed by a decrease to below control levels on day 21. Meanwhile, in the 5 Gy irradiated group, number of osteoclasts were decreased as early as 7 days post-irradiation. ALP mRNA expression increased in both irradiated group at day 14 and remained above control levels at day 21 in the 5 Gy group. OCN mRNA expression was increased as early as day 14 and remained increased at day 21 and 28. OCN positive cells in calluses indicated that OCN protein levels increased at day 14 in the 0.5 and 5 Gy groups.
Swift et al., 2015	In vivo. Female, B6D2F1/J mice were divided into 4 groups: Sham (0 Gy), Wound (W; 15% total body surface area), Radiation Injury (RI, 8 Gy ⁶⁰Co gamma rays), or Combined Injury (CI; RI + W). Mice were euthanized after irradiation at days 3, 7 and 30. The radiation group received a single whole-body dose of 8 Gy gamma rays at a rate of 0.4 Gy/min. Osteoblast surface, osteoclast surface, and osteoclastogenesis markers such as TRAP-5b and OCN were measured post-irradiation to determine the effects of IR on bone cell function.	Irradiated mice showed an increase in TRAP-5b from 38% to 83% from days 3-30. OCN in serum was decreased from –35% to –83% compared to sham mice on day 3.
Kondo et al., 2009	In vivo. 18-week-old male mice were exposed to 1 and 2 Gy of ¹³⁷Cs gamma rays at a dose of 0.915 Gy/min and their trabecular bone tissue was analyzed at 3- and 10-days post-irradiation. The number of osteoclasts was measured with TRAP staining.	Exposure to 1 Gy led to a ~2-fold increase in osteoclast number at day 3, and ~3-fold increase by day 10 post-irradiation. ~2.5-fold increase in osteoclast number by day 3 which remained constant up to day 10 post-irradiation. A marked ~150% increase in osteoclast number and surface were observed at day 3 and day 10 and at doses 1 and 2 Gy.
Willey et al., 2010	In vivo. 20-week-old female mice were irradiated with 2 Gy of X-rays, and bone and blood samples were taken to analyze levels of markers for osteoclast and osteoblast activity. Osteoblast marker OCN and osteoclast marker TRAP5b were measured with ELISA. Osteoclast and osteoblast surfaces were measured as well.	Osteoblast surface did not change. Osteoclast surface increased 1.6-fold after 1 week, but no change was observed after 2 and 3 weeks. A 21% increase in the levels of TRAP5b was observed within the irradiated group compared to the control group at week 1, but no further differences were observed between the irradiated and non-irradiated groups at weeks 2 and 3. The serum level of OCN did not change. A 218% increase in osteoclast number over bone surface was found at 1-week post-irradiation.
Wright et al., 2015	In vivo and ex vivo. the right hindlimbs of 20-week-old male C57BI/6 mice were irradiated with 2 Gy of X-rays at a rate of 1.6 Gy/min. In addition, the calvariae of 4-day-old Swiss White mice were extracted and irradiated with 2 and 10 Gy of X-rays at a rate of 0.244 Gy/min. The number of TRAP5b-positive osteoclasts and osteoblasts/ mm2 of bone surface were measured in models. In vitro. Osteocyte-like cells (MLO-Y4) and osteoblast cells (MC3T3) were irradiated with 0-20 Gy X-rays.	Following irradiation, a significant ~0.4-fold decrease in calvarial bone-derived osteoblasts was found at 10 days post-irradiation compared to the non-irradiated control. Earlier time points, such as day 4 and day 7, showed non-significant decreases in osteoblasts.

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 moderate as majority of the evidence is from in vitro human-derived cells, but one study performed a meta-analysis of astronauts. The relationship is supported in vivo mainly by mouse models with a few studies looking at rat models. The relationship has been shown in both male and female animal models. The relationship is plausible at any life stage. However, majority of studies have used adult animal models.