Partial-body Irradiation Research Articles

Mitigative and anti-inflammatory effects of Trichostatin A against radiation-induced gastrointestinal toxicity and gut microbiota alteration in mice

Purpose Radiation-induced gastrointestinal injury (RIGI) is a serious side effect of abdominal and pelvic radiotherapy, which often limits the treatment of gastrointestinal and gynaecological cancers. RIGI is also observed during accidental radiological or nuclear scenarios with no approved agents available till date to prevent or mitigate RIGI in humans. Trichostatin A (TSA), an epigenetic modulator, has been currently in clinical trials for cancer treatment and is also well known for its antibiotic and antifungal properties. Methods In this study, partial body (abdominal) irradiation mice model was used to investigate the mitigative effect of TSA against gastrointestinal toxicity caused by gamma radiation. Mice were checked for alterations in mean body weight, diarrheal incidence, disease activity index and survival against 15 Gy radiation. Structural abnormalities in intestine and changes in microbiota composition were studied by histopathology and 16S rRNA sequencing of fecal samples respectively. Immunoblotting and biochemical assays were performed to check protein nitrosylation, expression of inflammatory mediators, infiltration of inflammatory cells and changes in pro-inflammatory cytokine. Results TSA administration to C57Bl/6 mice improved radiation induced mean body weight loss, maintained better health score, reduced disease activity index and promoted survival. The 16S rRNA sequencing of fecal DNA demonstrated that TSA influenced the fecal microbiota dynamics with significant alterations in the Firmicutes/Bacteriodetes ratio. TSA effectively mitigated intestinal injury, down-regulated NF-κB, Cox-2, iNOS expression, inhibited PGE2 and protein nitrosylation levels in irradiated intestine. The upregulation of NLRP3-inflammasome complex and infiltrations of inflammatory cells in the inflamed intestine were also prevented by TSA. Subsequently, the myeloperoxidase activity in intestine alongwith serum IL-18 levels was found reduced. Conclusion These findings provide evidence that TSA inhibits inflammatory mediators, alleviates gut dysbiosis, and promotes structural restoration of the irradiated intestine. TSA, therefore, can be considered as a potential agent for mitigation of RIGI in humans.

Treatment of Radiation Lesions with Mesenchymal Stem Cells

Acute radiation syndrome (ARS) is an acute illness caused by exposure to a high dose of ionizing radiation. ARS is the deterministic effect of radiation exposure of the whole body or a significant body volume (partial body irradiation) above a threshold dose of about 1 Gy (gray). Radiation accidents, such as those in Chernobyl (1986) and Fukushima (2011), or the possible use of nuclear weapons during the hostilities or terrorist attacks, can lead to the massive development of ARS in humans. The aim of the work is to introduce a new method of post-radiation treatment – the use of allogeneic mesenchymal stem cells (MSCs). Materials and methods. The information contained in specialized scientific journals that are freely available and accessible through the global Internet was studied. Discussion of the results. In the scenario of mass exposure of the population, when from several tens (hundreds) to millions of people can be irradiated, the transfusion of hematopoietic stem cells traditionally used in such cases would be impossible. MSCs can possibly differentiate into specialized cells, that is, turn into cells of various organs and tissues or induce such kind of regeneration. For practical use, there are two main sources of their isolation and reproduction ex vivo – bone marrow and adipose tissue. To date, it has been shown that MSCs derived from adipose tissue can be effective in mitigating the effects of acute radiation illness. Intravenously applied MSCs are migrating mainly to the bone marrow and are partially restoring its function. Deep anatomical structures are also involved in local radiation injuries: bone, muscles, nerves, blood and lymphatic vessels and skin. There is a strong body of evidence suggesting the «repair effect» of MSCs when used to treat such lesions. This is because MSCs can induce the repair and regeneration of the anatomical structures which they are locally applied, possibly by the paracrine effect. The main advantage of allogeneic MSCs over autologous ones is their logistical accessibility. They can be produced in advance in quantities and stored frozen. After thawing, the cells must be cultured for at least 48 hours in humidified incubators with the addition of 5 % CO2. Findings. Treatment of MSCs should be started as soon as possible after radiation exposure. Rescue of damaged hematopoiesis in the bone marrow can be achieved by multiple intravenous administration of up to 1 million (106) freshly prepared allogeneic MSCs/kg body weight. Locally (around and in the irradiation area), the dose of MSCs may be lower – 20 million cells. Repeated topical application should be carried out at intervals of two to four weeks. Subsequent surgical reconstruction should be performed by an experienced surgeon and in a specialized center with concomitant topicalapplication of MSCs.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13Gy partial body irradiation at 42- and 90days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Sex as a Factor in Murine Radiation Research: Implications for Countermeasure Development

There is an increased threat of exposure to ionizing radiation; in the event of such exposure, the availability of medical countermeasures will be vital to ensure the protection of the population. Effective countermeasures should be efficacious across a varied population and most importantly amongst both males and females. Radiation research must be conducted in animal models which act as a surrogate for the human response. Here, we identify differences in survival in male and female C57BL/6 in both a total body irradiation (TBI) model using the Armed Forces Radiobiology Research Institute (AFRRI) 60Co source and a partial body irradiation (PBI) model using the AFRRI Linear Accelerator (LINAC) with 4 MV photons and 2.5% bone marrow shielding. In both models, we observed a higher degree of radioresistance in female animals and a corresponding radiosensitivity in males. One striking difference in male and female rodents is body size/weight and we investigated the role of pre-irradiation body weight on survivability for animals irradiated at the same dose of irradiation (8 Gy TBI, 14 Gy PBI). We found that weight does not influence survival in the TBI model and that heavier males but lighter females have increased survival in the PBI model. This incongruence in survival amongst the sexes should be taken into consideration in the course of developing radiation countermeasures for response to a mass casualty incident.

Transcriptome profile changes in the jejunum of nonhuman primates exposed to supralethal dose of total- or partial-body radiation

The risk of exposure of the general public or military personnel to high levels of ionizing radiation from nuclear weapons or radiological accidents is a dire national security matter. The development of advanced molecular biodosimetry methods, those that measure biological response, such as transcriptomics, to screen large populations of radiation-exposed victims is key to improving survival outcomes during radiological mass casualty scenarios. In this study, nonhuman primates were exposed to either 12.0 Gy cobalt-60 gamma (total-body irradiation, TBI) or X-ray (partial-body irradiation, PBI) 24 h after administration of a potential radiation medical countermeasure, gamma-tocotrienol (GT3). Changes in the jejunal transcriptomic profiles in GT3-treated and irradiated animals were compared to healthy controls to assess the extent of radiation damage. No major effect of GT3 on radiation-induced transcriptome at this radiation dose was identified. About 80% of the pathways with a known activation or repression state were commonly observed between both exposures. Several common pathways activated due to irradiation include FAK signaling, CREB signaling in the neurons, phagosome formation, and G-protein coupled signaling pathway. Sex-specific differences associated with excessive mortality among irradiated females were identified in this study, including Estrogen receptor signaling. Differential pathway activation was also identified across PBI and TBI, pointing towards altered molecular response for different degrees of bone marrow sparing and radiation doses. This study provides insight into radiation-induced changes in jejunal transcriptional profiles, supporting the investigation for the identification of biomarkers for radiation injury and countermeasure efficacy.

Biological sex differences in renin angiotensin system enzymes ACE and ACE2 regulate normal tissue response to radiation injury.

Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats. Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response. Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors. Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

TLR4-A Pertinent Player in Radiation-Induced Heart Disease?

The heart is one of the organs that is sensitive to developing delayed adverse effects of ionizing radiation (IR) exposure. Radiation-induced heart disease (RIHD) occurs in cancer patients and cancer survivors, as a side effect of radiation therapy of the chest, with manifestation several years post-radiotherapy. Moreover, the continued threat of nuclear bombs or terrorist attacks puts deployed military service members at risk of exposure to total or partial body irradiation. Individuals who survive acute injury from IR will experience delayed adverse effects that include fibrosis and chronic dysfunction of organ systems such as the heart within months to years after radiation exposure. Toll-like receptor 4 (TLR4) is an innate immune receptor that is implicated in several cardiovascular diseases. Studies in preclinical models have established the role of TLR4 as a driver of inflammation and associated cardiac fibrosis and dysfunction using transgenic models. This review explores the relevance of the TLR4 signaling pathway in radiation-induced inflammation and oxidative stress in acute as well as late effects on the heart tissue and the potential for the development of TLR4 inhibitors as a therapeutic target to treat or alleviate RIHD.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

The progression of radiation injury in a Wistar rat model of partial body irradiation with ∼5% bone marrow shielding

Purpose To describe the dose response relationship and natural history of radiation injury in the Wistar rat and its suitability for use in medical countermeasures (MCM) testing. Materials & Methods In two separate studies, male and female rats were exposed to partial body irradiation (PBI) with 5% bone marrow sparing. Animals were X-ray irradiated from 7 to 12 Gy at 7–10 weeks of age. Acute radiation syndrome (ARS) survival at 30 days and delayed effects of acute radiation exposure (DEARE) survival at 182 days were assessed. Radiation effects were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging of lung, whole-body plethysmography, and histopathology. Results Rats developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in mortality at doses ≥8Gy in males and ≥8.5 Gy in females. DEARE mortality occurred at doses ≥8Gy for both sexes. Findings indicate lung, kidney, and/or liver injury, and persistent hematological dysregulation, revealing multi-organ injury as a DEARE. Conclusion The Wistar rat PBI model is suitable for testing MCMs against hematopoietic and gastrointestinal ARS. DEARE multi-organ injury occurred in both sexes irradiated with 8–9Gy, also suggesting suitability for polypharmacy studies addressing the combination of ARS and DEARE injury.

IPW-5371 mitigates the delayed effects of acute radiation exposure in WAG/RijCmcr rats when started 15 days after PBI with bone marrow sparing

Purpose To test IPW-5371 for the mitigation of the delayed effects of acute radiation exposure (DEARE). Survivors of acute radiation exposure are at risk for developing delayed multi-organ toxicities; however, there are no FDA-approved medical countermeasures (MCM) to mitigate DEARE. Methods WAG/RijCmcr female rat model of partial-body irradiation (PBI), by shielding part of one hind leg, was used to test IPW-5371 (7 and 20 mg kg−1 d−1) for mitigation of lung and kidney DEARE when started 15 d after PBI. Rats were fed known amounts of IPW-5371 using a syringe, instead of delivery by daily oral gavage, sparing exacerbation of esophageal injury by radiation. The primary endpoint, all-cause morbidity was assessed over 215 d. Secondary endpoints: body weight, breathing rate and blood urea nitrogen were also assessed. Results IPW-5371 enhanced survival (primary endpoint) as well as attenuated secondary endpoints of lung and kidney injuries by radiation. Conclusion To provide a window for dosimetry and triage, as well as avoid oral delivery during the acute radiation syndrome (ARS), the drug regimen was started at 15 d after 13.5 Gy PBI. The experimental design to test mitigation of DEARE was customized for translation in humans, using an animal model of radiation that was designed to simulate a radiologic attack or accident. The results support advanced development of IPW-5371 to mitigate lethal lung and kidney injuries after irradiation of multiple organs.

Effects of Flash Radiotherapy on Blood Lymphocytes in Humans and Small Laboratory Animals.

A mathematical model, which describes the level of surviving lymphocytes in the blood after ultra-high (FLASH) and lower dose rates of partial-body irradiation, is developed. The model is represented by simple analytic formulae that involve a few parameters, namely, physiologic parameters (characteristics of the blood flow through the blood circulatory system and its irradiated part), a biophysical parameter (a characteristic of the blood lymphocytes radiosensitivity), and the physical parameters (characteristics of irradiation). The model predicts that the level of surviving blood lymphocytes increases as the dose rate increases and approaches the limiting level of (1 - vR), where vR is the fraction of the blood volume in the irradiated part of the blood circulatory system. The model also predicts that the level of surviving blood lymphocytes after the same exposure is higher for lower vR. It is found that FLASH irradiation in humans with doses of 10 to 40 Gy and with exposure times significantly less (<1 s) than the blood circulation time (∼60 s) leads to the maximal blood lymphocyte sparing. Simple formula, which determines effective dose rates for optimal blood lymphocyte sparing, is derived in the framework of the developed model. For the dose range specified above, the obtained modeling prediction of the range of effective dose rates for optimal blood lymphocyte sparing in humans (namely, N ≥40 Gy/s) coincides with the dose rate range in FLASH radiation therapy. It is revealed that the respective effective dose rates for mice are higher than those for humans (for the same dose range) due to the shorter blood circulation time in mice than in humans. Proceeding from the findings obtained in this paper, a hypothesis elucidating the mechanisms of the abscopal effect of FLASH radiation therapy (namely, an antitumor response on metastases located outside of irradiated part of a body) is proposed.

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0–2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844–1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.

Cytogenetic Effects in Patients after Computed Tomography Examination.

Millions of people around the world are exposed to low doses of ionizing radiation from diagnostic computed tomography (CT) scans. Currently available data on the potential cancer risk after CT scans are contradictory and therefore demand further investigations. The aim of the current study was to obtain estimations of genome damage after CT scans in 42 non-cancer patients and to conduct a comparison of the results with 22 control subjects. The frequency of dicentric ring chromosomes and chromosome breaks was significantly increased in irradiated patients compared to the controls. The distribution of dicentrics among the cells demonstrated non-Poisson distribution that reflected non-uniform and partial-body radiation exposure. A fraction of patients followed Poisson distribution, which is typical for uniform whole-body exposures. Some patients demonstrated a level of dicentrics similar to the control subjects. The individual variations in the frequency and dicentric distribution suggested complex mechanisms of chromosome aberration induction and elimination that could be associated with individual radiosensitivity, as well as previous diagnostics that used ionizing radiation or the redistribution of small fractions of irradiated lymphocytes within the circulatory pull. In conclusion, CT scans may cause genome damage and possible increases in cancer risk. The introduction of a specific follow-up of such patients, especially in the case of repeated CT scans, is suggested.

Profiling mRNA, miRNA and lncRNA expression changes in endothelial cells in response to increasing doses of ionizing radiation

Recent and past research have highlighted the importance of the endothelium in the manifestation of radiation injury. Our primary focus is on medical triage and management following whole body or partial-body irradiation. Here we investigated the usability of endothelial cells’ radiation response for biodosimetry applications. We profiled the transcriptome in cultured human endothelial cells treated with increasing doses of X-rays. mRNA expression changes were useful 24 h and 72 h post-radiation, microRNA and lncRNA expression changes were useful 72 h after radiation. More mRNA expressions were repressed than induced while more miRNA and lncRNA expressions were induced than repressed. These novel observations imply distinct radiation responsive regulatory mechanisms for coding and non-coding transcripts. It also follows how different RNA species should be explored as biomarkers for different time-points. Radiation-responsive markers which could classify no radiation (i.e., ‘0 Gy’) and dose-differentiating markers were also predicted. IPA analysis showed growth arrest-related processes at 24 h but immune response coordination at the 72 h post-radiation. Collectively, these observations suggest that endothelial cells have a precise dose and time-dependent response to radiation. Further studies in the laboratory are examining if these differences could be captured in the extracellular vesicles released by irradiated endothelial cells.

Determination of Lethality Curve for Cobalt-60 Gamma-Radiation Source in Rhesus Macaques Using Subject-Based Supportive Care.

Well-characterized and validated animal models are required for the development of medical countermeasures (MCMs) for acute radiation syndrome to mitigate injury due to high doses of total- or partial-body irradiation. Animal models used in MCM development must reflect a radiation dose- and time-dependent relationship, clinical presentation, and pathogenesis of organ injuries in humans. The objective of the current study was to develop the lethality curve for the Armed Forces Radiobiology Research Institute high level cobalt-60 gamma-radiation source in nonhuman primates (NHPs) after total-body irradiation. A dose-response relationship was determined using NHPs (rhesus macaques, N = 36, N = 6/radiation dose) irradiated with 6 doses in the range of 6.0 to 8.5 Gy, with 0.5 Gy increments at a dose rate of 0.6 Gy/min. Animals were provided subject-based supportive care including blood transfusions and were monitored for 60 days postirradiation. Survival was the primary endpoint of the study and the secondary endpoint included hematopoietic recovery. The lethality curve suggested LD30/60, LD50/60, and LD70/60 values as 5.71, 6.78, and 7.84 Gy, respectively. The results of this study will be valuable to provide specific doses for various lethalities of 60Co-gamma radiation to test radiation countermeasures in rhesus macaques using subject-based supportive care including blood transfusion.

Effects of Gamma-Tocotrienol on Partial-Body Irradiation-Induced Intestinal Injury in a Nonhuman Primate Model.

Exposure to high doses of radiation, accidental or therapeutic, often results in gastrointestinal (GI) injury. To date, there are no therapies available to mitigate GI injury after radiation exposure. Gamma-tocotrienol (GT3) is a promising radioprotector under investigation in nonhuman primates (NHP). We have shown that GT3 has radioprotective function in intestinal epithelial and crypt cells in NHPs exposed to 12 Gy total-body irradiation (TBI). Here, we determined GT3 potential in accelerating the GI recovery in partial-body irradiated (PBI) NHPs using X-rays, sparing 5% bone marrow. Sixteen rhesus macaques were treated with either vehicle or GT3 24 h prior to 12 Gy PBI. Structural injuries and crypt survival were examined in proximal jejunum on days 4 and 7. Plasma citrulline was assessed using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Crypt cell proliferation and apoptotic cell death were evaluated using Ki-67 and TUNEL staining. PBI significantly decreased mucosal surface area and reduced villous height. Interestingly, GT3 increased crypt survival and enhanced stem cell proliferation at day 4; however, the effects seemed to be minimized by day 7. GT3 did not ameliorate a radiation-induced decrease in citrulline levels. These data suggest that X-rays induce severe intestinal injury post-PBI and that GT3 has minimal radioprotective effect in this novel model.

Prediction of Total-Body and Partial-Body Exposures to Radiation Using Plasma Proteomic Expression Profiles.

There is a need to identify new biomarkers of radiation exposure for not only systemic total-body irradiation (TBI) but also to characterize partial-body irradiation and organ specific radiation injury. In the current study, we sought to develop novel biodosimetry models of radiation exposure using TBI and organ specific partial-body irradiation to only the brain, lung or gut using a multivariate proteomics approach. Subset panels of significantly altered proteins were selected to build predictive models of radiation exposure in a variety of sample cohort configurations relevant to practical field application of biodosimetry diagnostics during future radiological or nuclear event scenarios. Female C57BL/6 mice, 8-15 weeks old, received a single total-body or partial-body dose of 2 or 8 Gy TBI or 2 or 8 Gy to only the lung or gut, or 2, 8 or 16 Gy to only the brain using a Pantak X-ray source. Plasma was collected by cardiac puncture at days 1, 3 and 7 postirradiation for total-body exposures and only the lung and brain exposures, and at days 3, 7 and 14 postirradiation for gut exposures. Plasma was then screened using the aptamer-based SOMAscan proteomic assay technology, for changes in expression of 1,310 protein analytes. A subset panel of protein biomarkers which demonstrated significant changes (P < 0.01) in expression after irradiation were used to build predictive models of radiation exposure using different sample cohorts. Model 1 compared controls vs. all pooled irradiated samples, which included TBI and all organ specific partial irradiation. Model 2 compared controls vs. TBI vs. partial irradiation (with all organ specific partial exposure pooled within the partial-irradiated group), and model 3 compared controls vs. each individual organ specific partial-body exposure separately (brain, gut and lung). Detectable values were obtained for all 1,310 proteins included in the SOMAscan assay for all samples. Each model algorithm built using a unique sample cohort was validated with a training set of samples and tested with a separate new sample series. Overall predictive accuracies of 89%, 78% and 55% resulted for models 1-3, respectively, representing novel predictive panels of radiation responsive proteomic biomarkers. Though relatively high overall predictive accuracies were achieved for models 1 and 2, all three models showed limited accuracy at differentiating between the controls and partial-irradiated body samples. In our study we were able to identify novel panels of radiation responsive proteins useful for predicting radiation exposure and to create predictive models of partial-body exposure including organ specific radiation exposures. This proof-of-concept study also illustrates the inherent physiological limitations of distinguishing between small-body exposures and the unirradiated using proteomic biomarkers of radiation exposure. As use of biodosimetry diagnostics in future mass casualty settings will be complicated by the heterogeneity of partial-body exposure received in the field, further work remains in adapting these diagnostic tools for practical use.

Haptoglobin is an early indicator of survival after radiation-induced severe injury and bone marrow transplantation in mice

BackgroundHematopoietic stem cell transplantation (HSCT) is the main treatment for acute radiation sickness, especially after fatal radiation. The determination of HSCT for radiation patients is mainly based on radiation dose, hemogram and bone marrow injury severity. This study aims to explore a better biomarker of acute radiation injury from the perspective of systemic immune response.MethodsC57BL/6J female mice were exposed to total body irradiation (TBI) and partial body irradiation (PBI). Changes in haptoglobin (Hp) level in plasma were shown at different doses and time points after the exposure and treatment with amifostine or bone marrow transplantation. Student’s t-test/two tailed test were used in two groups. To decide the Hp levels as a predictor of the radiation dose in TBI and PBI, multiple linear regression analysis were performed. The ability of biomarkers to identify two groups of different samples was determined by the receiver operating characteristic (ROC) curve. The results were expressed as mean ± standard deviation (SD). Significance was set at P value < 0.05, and P value < 0.01 was set as highly significant. Survival distribution was determined by log-rank test.ResultsIn this study, we found that Hp was elevated dose-dependently in plasma in the early post-irradiation period and decreased on the second day, which can be used as a molecular indicator for early dose assessment. Moreover, we detected the second increase of Hp on the 3rd and 5th days after the lethal irradiation at 10 Gy, which was eliminated by amifostine, a radiation protection drug, while protected mice from death. Most importantly, bone marrow transplantation (BMT) on the 3rd and 5th day after 10 Gy radiation improved the 30-days survival rate, and effectively accelerated the regression of secondary increased Hp level.ConclusionsOur study suggests that Hp can be used not only as an early molecule marker of radiation injury, but also as an important indicator of bone marrow transplantation therapy for radiation injury, bringing new scientific discoveries in the diagnosis and treatment of acute radiation injury from the perspective of systemic immunity.

Lung transcriptome of nonhuman primates exposed to total- and partial-body irradiation

The focus of radiation biodosimetry has changed recently, and a paradigm shift for using molecular technologies of omic platforms in addition to cytogenetic techniques has been observed. In our study, we have used a nonhuman primate model to investigate the impact of a supralethal dose of 12 Gy radiation on alterations in the lung transcriptome. We used 6 healthy and 32 irradiated animal samples to delineate radiation-induced changes. We also used a medical countermeasure, γ-tocotrienol (GT3), to observe any changes. We demonstrate significant radiation-induced changes in the lung transcriptome for total-body irradiation (TBI) and partial-body irradiation (PBI). However, no major influence of GT3 on radiation was noted in either comparison. Several common signaling pathways, including PI3K/AKT, GADD45, and p53, were upregulated in both exposures. TBI activated DNA-damage-related pathways in the lungs, whereas PTEN signaling was activated after PBI. Our study highlights the various transcriptional profiles associated with γ- and X-ray exposures, and the associated pathways include LXR/RXR activation in TBI, whereas pulmonary wound-healing and pulmonary fibrosis signaling was repressed in PBI. Our study provides important insights into the molecular pathways associated with irradiation that can be further investigated for biomarker discovery.