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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	Vinita Chauhan (send email)	Open for citation & comment	Under Review

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 signaling 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 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: High

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

The biological rationale for linking direct deposition of energy to altered bone cell homeostasis is strongly supported in the literature, as documented by several review articles published on the subject (Donaubauer et al., 2020; Pacheco and Stock, 2013; Smith, 2020; Willey et al., 2011). These articles are of particular relevance, as they discuss the effects of environmental perturbations in the form of deposition of energy on osteoblast and osteoclast differentiation pathways. Deposition of energy in the form of IR has been shown to have a wide range of effects on osteoclasts, ranging from increased to decreased number and activity. Irradiated bone has an increased amount and activity of osteoclasts when compared to osteoblasts. Recent research suggests that low-dose (<1Gy) radiation can cause osteoclastogenesis in the acute phase due to inflammatory cytokines that stimulate osteoclastogenesis in the surrounding irradiated tissue. Increased bone resorption and increased bone turnover occur from increased osteoclast and decreased osteoblast activity (Pacheco and Stock, 2013; Sakurai et al., 2007; Willey et al., 2011; Willey et al., 2010).

Deposition of energy into bone cells results in osteoclast activation by upregulating the differentiation of precursors and increasing bone resorption. Osteoclast precursors are recruited to bone remodeling units (BRUs) to differentiate into mature osteoclast by binding macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) secreted in the bone microenvironment by osteoblasts and osteocytes (Donaubauer et al., 2020; Smith, 2020). Upregulation of osteoclastogenesis signaling pathways downstream to RANKL and M-CSF by radiation significantly enhanced osteoclast activity. Deposition of energy can also induce osteocyte apoptosis, resulting in proinflammatory signaling that upregulates the recruitment of osteoclasts to the area. In vitro experiments on osteoblast/osteoclast activity have shown enhanced osteoclastogenesis under exposures to radiation, as the deposition of energy in osteoblasts and osteocytes decreased their secretion of osteoprotegerin (OPG), a RANKL inhibitor, ultimately enhancing osteoclast stimulation (Donaubauer et al., 2020; Smith, 2020). The RANKL/OPG ratio is necessary for normal osteoclast activity, as increasing the proportion of RANKL to its inhibitor, OPG, results in stimulation of osteoclastogenesis. In addition, deposition of energy in bone cells results in upregulation of osteoclast stimulatory molecules, such as interleukin (IL)-6, high mobility group box 1 (HMGB1), and TNF-a, leading to enhanced osteoclast formation. Enhanced osteoclast formation leads to enhanced bone resorption (Donaubauer et al., 2020; Pacheco and Stock, 2013; Smith, 2020; Willey et al., 2011).

Radiation-induced damage to osteoblasts and osteocytes within the bone microenvironment is considered a significant factor and an exemplary instance of the effect of deposition of energy on bone cell function. Both in vivo and in vitro data suggest that radiation reduces osteoblast proliferation and differentiation, causing cell cycle arrest, reducing collagen production, and increasing apoptotic sensitivity. Radiation-induced oxidative stress likely damages osteoblast precursors, reducing cell viability and differentiation. Under energy deposition, osteoblast numbers and activity remain relatively unchanged, while significant bone degradation occurs, therefore, suggesting enhanced osteoclast activity as part of the altered bone cell homeostasis observed (Willey et al., 2011). Directly irradiated bone shows reduced mesenchymal stem cell (MSC) numbers and reduced colony formation when directed to bone cell precursors, which delays the recovery of damaged osteoblasts (Willey et al., 2011). Osteoclasts can degrade the bone matrix through the release of amino acids such as hydroxyproline (HP), fragments of collagen type I, including C- and N-terminal telopeptides (CTX and NTX), pyridinoline (PYD) and deoxypyridinoline (DPD) as well as proteases, including Cathepsin K (CTSK) and matrix metalloproteinases (MMP9 and MMP14) (Smith, 2020; Stavnichuk et al., 2020). Bone morphogenic protein 2 (BMP-2), a transcription factor regulating osteoblast differentiation can indicate impaired osteoblast differentiation following irradiation (Sakurai et al., 2007).

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

The empirical data relevant to this KER provides strong support for the linkage between deposition of energy and altered bone cell homeostasis. The evidence supporting this link comes from literature on radiation exposure directly or indirectly increasing osteoclast activity and decreasing osteoblast activity in a dose and/or time concordant manner. Radiation-specific studies examined the effects of irradiation with doses ranging from 0-30 Gy of X-rays, gamma rays, and heavy ions irradiation (da Cruz Vegian et al., 2020; Huang et al., 2019; Kook et al., 2015; Li et al., 2020; Liu et al., 2018; Sakurai et al., 2007; Wang et al., 2016; Willey et al., 2008; Willey et al., 2010; Wright et al., 2015; Zhang et al., 2020).

Dose Concordance

Current literature on the effects of radiation on bone cell function provided strong evidence for a dose concordance relationship between deposition of energy and altered bone cell homeostasis. Once energy is deposited into matter at all doses, follow-on downstream events are immediately initiated. In humans after spaceflight, where the time of flight is used as proxy for the dose of radiation received, osteoclast markers increased hyperbolically with a t1/2 of 11 days and a plateau at 113% increased, while osteoblast markers increased linearly at 7% per month (Stavnichuk et al., 2020). In mice after spaceflight, compared to ground controls, an increase of osteoclasts numbers in the bone surface by 197% was observed. The surface of the bone covered by osteoclasts also increased by 154% (Blaber et al., 2013). Relevant primary research shows that indicators of osteoblastogenesis, including osteoblast number and surface area, MSC proliferation, alkaline phosphatase (ALP) and osteocalcin (OCN) expression, and levels of calcium deposition, all decrease in response to IR exposure (Huang et al., 2019; Kook et al., 2015; Li et al., 2020; Liu et al., 2018; Sakurai et al., 2007; Wang et al., 2016; Wright et al., 2015. Studies also show that indicators of osteoclastogenesis, including osteoclast number and surface area as well as tartrate-resistant acid phosphatase (TRAP) expression increase in response to IR exposure (da Cruz Vegian et al., 2020; Kondo et al., 2009; Li et al., 2020; Willey et al., 2008; Willey et al., 2010; Wright et al., 2015; Zhang et al., 2020). At an irradiation dose less than or equal to 2 Gy X-rays or gamma rays, various models consistently showed a significant increase in the number of osteoclasts/mm2 of bone surface, as well as the bone surface area covered by osteoclasts (Kondo et al., 2009; Willey et al., 2008; Willey et al., 2010; Wright et al., 2015; Zhang et al., 2020). A few studies also reported a decrease in osteoblasts following a 2 Gy irradiation dose (Sakurai et al., 2007; Wright et al., 2015). IR-induced changes in osteoclast histology appear to be dose-dependent, as Kondo et al. (2009) found that 3 days after irradiation with gamma rays there was a greater increase in the number of osteoclasts in samples exposed to 2 Gy of gamma rays than to 1 Gy. Li et al. (2020) observed that exposure to 8 Gy resulted in a greater increase in osteoclast number than that to lower dose. At higher doses, such as a total dose of 30 Gy, an even greater fold increase in TRAP was observed (Da Cruz Vegian et al., 2020). This dose-dependent relationship between IR and altered bone cell homeostasis is further supported by measurements of osteoblast markers across a range of radiation doses. Studies that analyzed ALP levels across multiple doses of X-rays, found that expression decreased in a dose-dependent manner (Kook et al., 2015; Sakurai et al., 2007). Kook et al. (2015) also observed a dose dependent decrease in the osteoblast marker, OCN, following exposure to 0-8 Gy of X-rays. Following irradiation with 2, 4, 8, and 12 Gy of X-rays, Liu et al. (2018) found that human bone marrow MSC (hBMMSC) proliferation experienced a dose-dependent decrease. Lastly, OCN and osteoblast differentiation decreased 2-fold with each increasing dose (15 and 20 Gy) relative to control (Jia et al., 2011).

Time Concordance

Moderate evidence exists in the current literature suggesting a time concordance relationship between the deposition of energy and altered bone cell homeostasis. When energy is deposited into biological models it immediately causes ionization events which directly lead to downstream events occurring at later time points. In general, data collected from experiments show that overall changes in osteoblast and osteoclast activity occur primarily in the first week post-exposure. At 3 days post-exposure, osteoblast surface was decreased (Willey et al., 2008), TRAP levels increased (Swift et al., 2015; Willey et al., 2008; Zhang et al., 2020) and osteoclast number and surface were increased (Willey et al., 2008). OCN levels decreased on day 3 after radiation exposure, indicating a decrease in osteoblast markers (Swift et al., 2015).

10 days post-exposure revealed decreased osteoblasts in calvarial bone that was not significantly shown at earlier timepoints (Wright et al., 2015), Increased osteoclast surface and number was observed at 3 days, 1 week, and 10 days after irradiation (Kondo et al., 2009; Willey et al., 2010; Willey et al., 2008), however no further changes occurred after 2 and 3 weeks (Willey et al., 2010).

Da Cruz Vegian et al. (2020) found that both TRAP and OCN levels increased at day 3 post-irradiation of rats with a total of 30 Gy gamma rays. Chen et al. (2014) found that OCN protein levels increased at day 10 and continued onto day 14 in the 0.5 Gy MC3T3-E1 cells irradiated group. MC3T3-E1 cells irradiated with 0.5 or 5 Gy X-rays revealed an increase in ALP level at day 7 and day 10, then decreased at day 14 (Chen et al., 2014). As well, the in vivo models showed decreased osteoclast number as early as day 7, increased OCN protein levels at day 14 in the 5 Gy group (Chen et al., 2014). Oest et al. (2015) observed increased osteoclast numbers correlated temporally with trabecular resorption, most pronounced 2 weeks post-irradiation (5 Gy and 4X5Gy of X-rays). Mice irradiated with 5 Gy of gamma radiation showed significant increase in osteoblast activity 10 days following irradiation when compared to the control (Green et al. 2021). A single 50 Gy dose of electron beam energy to the proximal tibia of rabbits showed late onset decrease in the number of viable osteocytes (52 weeks) (Sugimoto et al., 1993).

Essentiality

Studies examining the effects of different methods of prevention or treatment of bone resorption under IR suggest a strong relationship between deposition of energy and altered bone cell homeostasis. Altered bone cell homeostasis mainly occurs in the bone tissue directly receiving radiation. Contralateral bone tissue (bone tissue that was shielded from radiation but was removed from irradiated rats) was extracted and compared to bone tissue that was directly exposed to radiation in several studies. Analysis of the shielded bone tissue indicated changes in osteoblast and osteoclast markers such as ALP and TRAP5b were less significant in contralateral bone compared to irradiated bone tissue (Wright et al., 2015).

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).
Mice receiving 6 Gy of radiation showed a significant increase in the osteoblasts and osteoclast-lined bone perimeter, as opposed to a decrease in osteoblast after a high dose of radiation (Turner et al., 2013).
Cao et al. (2010) observed decreased osteoclast numbers in the distal femora four weeks following 4 Gy irradiation, which is contrary to the increase in osteoclast observances found in other studies.

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 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 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.
Blaber et al., 2013	In vivo. 16-week-old female mice were subjected to 15-days of spaceflight. The quantity of osteoclasts of the right proximal femur were measured with TRAP staining.	Following spaceflight, there was a 197% increase in osteoclast numbers in the bone surface compared to the ground controls. The bone surface covered by osteoclasts also increased by 154% in microgravity.
Jia et al., 2011	In vivo and Ex vivo. 10 to 12 –weeks-old male mice were exposed to 0, 15 and 20 Gy of X-ray. Serum levels of the bone formation marker osteocalcin (OCN) were analyzed with the Mouse Osteocalcin EIA Kit. Osteoblast forming cells of the left tibia and left femur were counted based on the formation of alkaline phosphatase (ALP)-positive osteoblastic colonies.

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.
Oest et al., 2015	In vivo. 12-week-old female mice were irradiated with a single 5 Gy of X-ray or 4 fractions of 5 Gy of X-ray. The number of osteoclasts were enumerated over the distal 5 mm of each femur and stained with TRAP. They were evaluated histologically at 1, 2, 4, 8, 12, and 26 weeks post-irradiation.	1 week after irradiation, a 2-fold increase for the 5 Gy total osteoclasts compared to the control and 4x5Gy groups was observed. 2 weeks post-irradiation, the number of osteoclasts increased more than a 2-fold for both doses of 5 Gy and 4x5Gy compared to the control.
Green et al., 2013	In vivo, eight-week old male C57BL/6 mice were irradiated with gamma irradiation at a total dose of 5 Gy. Osteoclast activity was measured by the serum concentration of tartrate-resistant acid phosphatase (TRAP5b) assay.	At 2 days post-irradiation, a significant increase in osteoblast activity by 43±35% when compared to the control and remained elevated in compared to control group for 10 days following irradiation.
Lima et al. 2017	In vivo, 4-month-old female BALB/cBYJ mice were administered 0,0.17,0.5, and 1 Gy of X-ray radiation. Osteoclast numbers were measured using a tartrate-resistant acid phosphatase (TRAP+) staining kit. Marrow aspirate was used to determine osteoblast colony-forming unit.	3 days following radiation exposure, there was a significant increase in osteoclasts when compared to the control group. Osteoblast colony numbers were significantly decreased in the 0.5 Gy and the 1 Gy irradiated groups when compared to the control group 3 days post exposure.
Sugimoto et al., 1993	In vivo. The proximal tibia of rabbits was exposed to a single dose of 50 Gy of a 14-MeV electron beam generated by a betatron. Osteocytes were quantified by counting silver grains on autoradiographs, using 3H-cytidine as a tracer.	The proximal tibia of rabbits showed late onset decrease in the number of viable osteocytes after 52 weeks after irradiation.

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

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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.

References

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

Blaber, E.A. et al. (2013), “Microgravity induces pelvic bone loss through osteoclastic activity, osteocytic osteolysis, and osteoblastic cell cycle inhibition by CDKN1a/p21”, PloS one, Vol. 8/4, e61372. https://doi.org/10.1371/journal.pone.0061372

Chen, M., et al. (2014), “Low-dose X-ray irradiation promotes osteoblast proliferation, differentiation and fracture healing”, PloS one, Vol. 9/8, PLOS, San Francisco, https://doi.org/10.1371/journal.pone.0104016

da Cruz Vegian, R. M. et al. (2020), "Systemic and local effects of radiotherapy: an experimental study on implants placed in rats", Clinical Oral Investigations, Vol. 24, Nature, https://doi.org/10.1007/s00784-019-02946-5.

Donaubauer, A. J. et al. (2020), "The influence of radiation on bone and bone cells—differential effects on osteoclasts and osteoblasts", International Journal of Molecular Sciences, Vol. 21/17, Nature, https://doi.org/10.3390/ijms21176377.

Fu, J. et al. (2021), “Bone health in spacefaring rodents and primates: systematic review and meta-analysis", npj Microgravity Vol. 7, Nature, https://doi.org/10.1038/s41526-021-00147-7

Green, D. E., Adler, B. J., Chan, M. E., Lennon, J. J., Acerbo, A. S., Miller, L. M., & Rubin, C. T. (2013). Altered composition of bone as triggered by irradiation facilitates the rapid erosion of the matrix by both cellular and physicochemical processes. PloS one, 8(5), e64952. https://doi.org/10.1371/journal.pone.0064952

Huang, B. et al. (2019), "Amifostine suppresses the side effects of radiation on BMSCs by promoting cell proliferation and reducing ROS production", Stem Cells International, Vol. 2019, Hindawi, https://doi.org/10.1155/2019/8749090.

Jia, D. et al. (2011), “Rapid loss of bone mass and strength in mice after abdominal irradiation”, Radiation research, Vol. 176/5, 624–635. https://doi.org/10.1667/rr2505.1

Kondo, H. et al. (2009), "Total-body irradiation of postpubertal mice with 137Cs acutely compromises the microarchitecture of cancellous bone and increases osteoclasts", Radiation Research, Vol. 171/3, BioOne, https://doi.org/10.1667/RR1463.1.

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, Nature, https://doi.org/10.1007/s11010-015-2559-z.

Li, J. et al. (2020), "Effect of α2-macroglobulin in the early stage of jaw osteoradionecrosis", International Journal of Oncology, Vol. 57/1, Spandidos Publications, https://doi.org/10.3892/IJO.2020.5051https://doi.org/10.3892/IJO.2020.5051.

Lima, F. et al. (2017). Exposure to Low-Dose X-Ray Radiation Alters Bone Progenitor Cells and Bone Microarchitecture. Radiation Research, 188(4.1), 433–442. http://www.jstor.org/stable/26428489
Liu, Y. et al. (2018), "Protective effects of α‑2‑macroglobulin on human bone marrow mesenchymal stem cells in radiation injury", Molecular medicine reports, Vol. 18/5, Spanditos Publications, https://doi.org/10.3892/MMR.2018.9449.
Oest, M. E. et al. (2015), “Long-term loss of osteoclasts and unopposed cortical mineral apposition following limited field irradiation”, Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Vol. 33/3, 334–342. https://doi.org/10.1002/jor.22761

Pacheco, R. and H. Stock. (2013), "Effects of Radiation on Bone", Current Osteoporosis Reports, Vol. 11, Nature, https://doi.org/10.1007/S11914-013-0174-Z.

Sakurai, T. et al. (2007), "Radiation-induced Reduction of Osteoblast Differentiation in C2C12 cells", Journal of Radiation Research, Vol. 48/6, Oxford University Press, Oxford, https://doi.org/10.1269/jrr.07012.

Smith, J. K. (2020), "Osteoclasts and microgravity", Life, Vol. 10/9, MDPI, Basel, https://doi.org/10.3390/life10090207.

Stavnichuk, M., et al. (2020), “A systematic review and meta-analysis of bone loss in space travelers”, npj microgravity, Vol. 6, Nature, https://doi.org/10.1038/s41526-020-0103-2

Sugimoto, M. et al. (1993), “Osteocyte viability after high-dose irradiation in the rabbit”, Clinical orthopaedics and related research, Vol. 297, 247–252.

Swift, J. M., et al. (2015), “Skin wound trauma, following high-dose radiation exposure, amplifies and prolongs skeletal tissue loss”, Bone, Vol. 81, Elsevier, https://doi.org/10.1016/j.bone.2015.08.022

Turner, R. T., Iwaniec, U. T., Wong, C. P., Lindenmaier, L. B., Wagner, L. A., Branscum, A. J., Menn, S. A., Taylor, J., Zhang, Y., Wu, H., & Sibonga, J. D. (2013). Acute exposure to high dose γ-radiation results in transient activation of bone lining cells. Bone, 57(1), 164–173. https://doi.org/10.1016/j.bone.2013.08.002

Wang, C. et al. (2016), "Protective Effects of Cerium Oxide Nanoparticles on MC3T3-E1 Osteoblastic Cells Exposed to X-Ray Irradiation", Cellular Physiology and Biochemistry, Vol. 38, Karger, Basel, https://doi.org/10.1159/000443092.

Willey, J. S. et al. (2011), "Ionizing Radiation and Bone Loss: Space Exploration and Clinical Therapy Applications", Clinical Reviews in Bone and Mineral Metabolism, Vol. 9, Nature, https://doi.org/10.1007/s12018-011-9092-8.

Willey, J. S. et al. (2010), "Risedronate prevents early radiation-induced osteoporosis in mice at multiple skeletal locations", Bone, Vol. 46/1, Elsevier, https://doi.org/10.1016/j.bone.2009.09.002.

Willey, J. S. et al. (2008), "Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays", Vol. 170/3, BioOne, https://doi.org/10.1667/RR1388.1

Wright, L. E. et al. (2015), "Single-Limb Irradiation Induces Local and Systemic Bone Loss in a Murine Model", Journal of Bone and Mineral Research, Vol. 108/2, Wiley, https://doi.org/10.1002/jbmr.2458

Zhang, L. et al. (2020), "Amifostine inhibited the differentiation of RAW264.7 cells into osteoclasts by reducing the production of ROS under 2 Gy radiation", Journal of Cellular Biochemistry, Vol. 121/1, Wiley, https://doi.org/10.1002/jcb.29247