Partial-body Irradiation Research Articles

IEPA, a novel radiation countermeasure, alleviates acute radiation syndrome in rodents

Repurposing therapeutic agents with existing clinical data is a common strategy for developing radiation countermeasures. IEPA (imidazolyl ethanamide pentandioic acid) is an orally bioavailable small molecule pseudopeptide with myeloprotective properties, a good clinical safety profile, and stable chemical characteristics facilitating stockpiling. Here, we evaluated IEPA’s radiomitigative efficacy in the hematopoietic subsyndrome of acute radiation syndrome (H-ARS) using total-body irradiation (TBI) models in C57BL/6J mice and WAG/RijCmcr rats, applying various posology schemes and introducing syringe feeding of the IEPA formulation in the pudding. Additionally, we assessed IEPA in the delayed effects of acute radiation exposure (DEARE) model after partial-body irradiation (PBI) in WAG/RijCmcr rats. Endpoints included survival, body weight, hematology, and pulmonary parameters, depending on the model. Results from mouse and rat TBI models demonstrated survival improvements with repeated IEPA dosing at 10 mg/kg, with the largest benefits observed in the bi-daily (BID) treatment over the 30-day ARS phase in female rats. Survival across PBI-DEARE subsyndromes was comparable between IEPA and vehicle groups, though IEPA improved pulmonary parameters in female rats during the lung-DEARE phase. Sex-related differences in response to irradiation and IEPA were noted, with females showing a survival advantage. IEPA treatment is compatible with Neulasta® (Pegfilgrastim; PEG-G-CSF); adequately powered studies are needed to confirm the trend toward improved survival over standard care alone. IEPA is a promising development candidate as a medical countermeasure against the effects of acute radiation syndrome. Further confirmatory studies in small and large animal models should validate the robustness and translatability of preliminary rodent data on IEPA’s radiomitigative efficacy.

Selecting the Most Relevant Mouse Strains for Evaluating Radiation-Induced Multiple Tissue Injury after Leg-Shielded Partial-Body Gamma Irradiation.

Animal studies are needed that best simulate a large-scale, inhomogeneous body exposure after a radiological or nuclear incident and that provides a platform for future development of medical countermeasures. A partial-body irradiation (PBI) model using 137Cs gamma rays with hind limb (tibia) shielding was developed and assessed for the sequalae of radiation injuries to gastrointestinal tract, bone marrow (BM) and lung and among different genetic mouse strains (C57BL/6J, C57L/J, CBA/J and FVB/NJ). In this case, a marginal level of BM shielding (∼2%) provided adequate protection against lethality from infection and hemorrhage and enabled escalation of radiation doses with evaluation of both acute and delayed radiation syndromes. A steep radiation dose-dependent body weight loss was observed over the first 5 days attributed to enteritis with C57BL/6J mice appearing to be the most sensitive strain. Peripheral blood cell analysis revealed significant depression and recovery of leukocytes and platelets over the first month after PBI and were comparable among the four different mouse strains. Latent pulmonary injury was observed on micro-CT imaging at 4 months in C57L/J mice and confirmed histologically as severe pneumonitis that was lethal at 12 Gy. The lethality and radiological densitometry (HUs) dose responses were comparable to previous studies on C57L/J mice after total-body irradiation (TBI) and BM transplant rescue as well as after localized whole-thorax irradiation (WTI). Indeed, the lethal radiation doses and latency appeared similar for pneumonitis appearing in rhesus macaques after WTI or PBI as well as predicted for patients given systemic radiotherapy. In contrast, PBI treatment of C57BL/6 mice at a higher dose of 14 Gy had far longer survival times and developed extreme and debilitating pIeural effusions; an anomaly as similarly reported in previous thorax irradiation studies on this mouse strain. In summary, a radiation exposure model that delivers PBI to unanesthetized mice in a device that provides consistent shielding of the hind limb BM was developed for 137Cs gamma rays with physical characteristics and relevance to relatively high photon energies expected from the detonation of a nuclear device or accidental release of ionizing radiation. Standard strains such as C57BL/6J mice may be used reliably for early GI or hematological radiation syndromes while the C57L/J mouse strain stands out as the most appropriate for evaluating the delayed pulmonary effects of acute radiation exposure and recapitulating this disease in humans.

Impact of Partial Body Shielding from Very High Dose Rates on Untargeted Metabolomics in Biodosimetry.

A realistic exposure to ionizing radiation (IR) from an improvised nuclear device will likely include individuals who are partially shielded from the initial blast delivered at a very high dose rate (VHDR). As different tissues have varying levels of radiosensitivity, e.g., hematopoietic vs gastrointestinal tissues, the effects of shielding on radiation biomarkers need to be addressed. Here, we explore how biofluid (urine and serum) metabolite signatures from male and female C57BL/6 mice exposed to VHDR (5-10 Gy/s) total body irradiation (TBI, 0, 4, and 8 Gy) compare to individuals exposed to partial body irradiation (PBI) (lower body irradiated [LBI] or upper body irradiated [UBI] at an 8 Gy dose) using a data-independent acquisition untargeted metabolomics approach. Although sex differences were observed in the spatial groupings of urine signatures from TBI and PBI mice, a metabolite signature (N6,N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, taurine, and creatine) previously developed from variable dose rate experiments was able to identify individuals with high sensitivity and specificity, irrespective of radiation shielding. A panel of serum metabolites composed from previous untargeted studies on nonhuman primates had excellent performance for separating irradiated cohorts; however, a multiomic approach to complement the metabolome could increase dose estimation confidence intervals. Overall, these results support the inclusion of small-molecule markers in biodosimetry assays without substantial interference from the upper or lower body shielding.

Consequences of ionizing radiation exposure to the cardiovascular system.

Ionizing radiation is widely used in various industrial and medical applications, resulting in increased exposure for certain populations. Lessons from radiation accidents and occupational exposure have highlighted the cardiovascular and cerebrovascular risks associated with radiation exposure. In addition, radiation therapy for cancer has been linked to numerous cardiovascular complications, depending on the distribution of the dose by volume in the heart and other relevant target tissues in the circulatory system. The manifestation of symptoms is influenced by numerous factors, and distinct cardiac complications have previously been observed in different groups of patients with cancer undergoing radiation therapy. However, in contemporary radiation therapy, advances in treatment planning with conformal radiation delivery have markedly reduced the mean heart dose and volume of exposure, and these variables are therefore no longer sole surrogates for predicting the risk of specific types of heart disease. Nevertheless, certain cardiac substructures remain vulnerable to radiation exposure, necessitating close monitoring. In this Review, we provide a comprehensive overview of the consequences of radiation exposure on the cardiovascular system, drawing insights from various cohorts exposed to uniform, whole-body radiation or to partial-body irradiation, and identify potential risk modifiers in the development of radiation-associated cardiovascular disease.

Irradiation and Alterations in Hippocampal DNA Methylation.

The response of the brain to radiation is important for cancer patients receiving whole or partial brain irradiation or total body irradiation, those exposed to irradiation as part of a nuclear accident or a nuclear war or terrorism event, and for astronauts during and following space missions. The mechanisms mediating the effects of irradiation on the hippocampus might be associated with alterations in hippocampal DNA methylation. Changes in cytosine methylation involving the addition of a methyl group to cytosine (5 mC) and especially those involving the addition of a hydroxy group to 5 mC (hydroxymethylcytosine or 5 hmC) play a key role in regulating the expression of genes required for hippocampal function. In this review article, we will discuss the effects of radiation on hippocampal DNA methylation and whether these effects are associated with hippocampus-dependent cognitive measures and molecular measures in the hippocampus involved in cognitive measures. We will also discuss whether the radiation-induced changes in hippocampal DNA methylation show an overlap across different doses of heavy ion irradiation and across irradiation with different ions. We will also discuss whether the DNA methylation changes show a tissue-dependent response.

Serum sSelectin-L is an early specific indicator of radiation injury

ObjectiveIt's crucial to identify an easily detectable biomarker that is specific to radiation injury in order to effectively classify injured individuals in the early stage in large-scale nuclear accidents. MethodsC57BL/6J mice were subjected to whole-body and partial-body γ irradiation, as well as whole-body X-ray irradiation to explore the response of serum sSelectin-L to radiation injury. Then, it was compared with its response to lipopolysaccharide-induced acute infection and doxorubicin-induced DNA damage to study the specificity of sSelectin-L response to radiation. Furthermore, it was further evaluated in serum samples from nasopharyngeal carcinoma patients before and after radiotherapy. Simulated rescue experiments using Amifostine or bone marrow transplantation were conducted in mice with acute radiation syndrome to determine the potential for establishing sSelectin-L as a prognostic marker. The levels of sSelectin-L were dynamically measured using the ELISA method. ResultsSelectin-L is mainly expressed in hematopoietic tissues and lymphatic tissues. Mouse sSelectin-L showed a dose-dependent decrease from 1 day after irradiation and exhibited a positive correlation with lymphocyte counts. Furthermore, the level of sSelectin-L reflected the degree of radiation injury in partial-body irradiation mice and in nasopharyngeal carcinoma patients. sSelectin-L was closely related to the total dose of γ or X ray. There was no significant change in the sSelectin-L levels in mice intraperitoneal injected with lipopolysaccharide or doxorubicin. The sSelectin-L was decreased slower and recovered faster than lymphocyte count in acute radiation syndrome mice treated with Amifostine or bone marrow transplantation. ConclusionsOur study shows that sSelectin-L has the potential to be an early biomarker to classify injured individuals after radiation accidents, and to be a prognostic indicator of successful rescue of radiation victims.

Effects of Partial-Body, Continuous/Pulse Irradiation at Dose Rates from FLASH to Conventional Rates on the Level of Surviving Blood Lymphocytes: Modeling Approach. I. Continuous Irradiation.

A mathematical model developed by Cucinotta and Smirnova is extended to describe effects of continuous, partial-body irradiation at high doses D and at dose rates N from FLASH to conventional rates on the level of surviving blood lymphocytes in humans and small laboratory animals (mice). Specifically, whereas the applicability of the model is limited to the exposure times shorter than a single cardiac cycle T0, the extended model is capable of describing such effects for the aforementioned and longer exposure times. The extended model is implemented as the algebraic equations. It predicts that the level of surviving blood lymphocytes in humans and mice increases with increasing the dose rate from N= D/T0 to FLASH rates and approaches the upper limiting level of 1-vR, where vR is the fraction of blood volume in the irradiated part of the blood circulatory system. Levels of surviving blood lymphocytes computed at doses from 10 Gy to 40 Gy and at dose rates N, which equal or exceed 40 Gy/s for humans and 400 Gy/s for mice, are nearly indistinguishable from the upper limiting level. In turn, the level of surviving blood lymphocytes in humans and mice decreases with decreasing the dose rate from N= D/T0 to conventional rates and approaches a lower limiting level. This level strongly depends on the dose D (it is smaller at larger values of D) with a slight dependence on the dose rate N. The model with the parameters specified for mice (together with the model of the dynamics of lymphopoietic system in mice after partial-body irradiation) reproduce, on a quantitative level, the experimental data, according to which the concentration of blood lymphocytes measured in mice in one day after continuous, partial-body irradiation at a high dose and conventional dose rate is smaller at the larger value of vR. Additionally, the model predicts at the same high dose (>10 Gy) a faster restoration of the blood lymphocyte population in humans exposed to continuous, partial-body irradiation at a FLASH dose rate compared to a conventional dose rate.

Delayed effects of radiation exposure in a C57L/J mouse model of partial body irradiation with ~2.5% bone marrow shielding.

Mouse models of radiation injury are critical to the development of medical countermeasures (MCMs) against radiation. Now that MCMs against hematopoietic acute radiation syndrome (H-ARS) have achieved regulatory approval, attention is shifting to develop MCMs against the adverse effects of gastrointestinal acute radiation syndrome (GI-ARS) and delayed effects of acute radiation exposure (DEARE). The C57L/J mouse model of partial body irradiation (PBI) with 2.5% bone marrow shielding (BM2.5) is being leveraged to examine both GI-ARS and DEARE effects. Within days of PBI, mice may develop H- and GI-ARS followed several months later by DEARE as a multi-organ injury, which typically involves the lung and kidney (L- and K-DEARE, respectively). The objective of this manuscript is to describe the dose response relationship and progression of radiation injury in the C57L/J mouse and to evaluate its suitability for use in DEARE MCM testing. In two separate studies conducted over 2 years, male and female C57L/J mice were exposed to PBI BM2.5 with one hindlimb shielded from radiation, representing ~2.5% bone marrow shielding/sparing. Mice were X-ray irradiated at doses ranging from 9 to 13 Gy at 10 to 12 weeks of age for the purposes of assessing ARS survival at 30 days and DEARE survival at 182 days post-irradiation. Clinical indicators of ARS and DEARE were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging (MRI) of lung, and histopathology of selected tissues. C57L/J mice developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in dose dependent mortality at doses ≥11 Gy between 1- and 15-days post-irradiation. In animals that survived ARS, DEARE associated mortality occurred in dose dependent fashion at ≥9 Gy for both sexes between 60- and 159-days post-irradiation with histopathology examinations indicating lung injury as the primary cause of death in moribund animals. The PBI BM2.5 C57L/J mouse model reliably produced known H- and GI-ARS effects at doses greater than those resulting in DEARE effects. Because of this, the C57L/J mouse can be used to test MCMs against L-DEARE injury, while avoiding ARS associated mortality.

Histopathological studies of nonhuman primates exposed to supralethal doses of total- or partial-body radiation: influence of a medical countermeasure, gamma-tocotrienol

Despite remarkable scientific progress over the past six decades within the medical arts and in radiobiology in general, limited radiation medical countermeasures (MCMs) have been approved by the United States Food and Drug Administration for the acute radiation syndrome (ARS). Additional effort is needed to develop large animal models for improving the prediction of clinical safety and effectiveness of MCMs for acute and delayed effects of radiation in humans. Nonhuman primates (NHPs) are considered the animal models that reproduce the most appropriate representation of human disease and are considered the gold standard for drug development and regulatory approval. The clinical and histopathological effects of supralethal, total- or partial-body irradiations (12 Gy) of NHPs were assessed, along with possible protective actions of a promising radiation MCM, gamma-tocotrienol (GT3). Results show that these supralethal radiation exposures induce severe injuries that manifest both clinically as well as pathologically, as evidenced by the noted functionally crippling lesions within various major organ systems of experimental NHPs. The MCM, GT3, has limited radioprotective efficacy against such supralethal radiation doses.

Development of a Radiation-induced Pulmonary Fibrosis Partial Body Irradiation Model in C57BL/6 Mice.

With the current volatile geopolitical climate, the threat of nuclear assault is high. Exposure to ionizing radiation from either nuclear incidents or radiological accidents often lead to major harmful consequences to human health. Depending on the absorbed dose, the symptoms of the acute radiation syndrome and delayed effects of acute radiation exposure (DEARE) can appear within hours, weeks to months. The lung is a relatively radiosensitive organ with manifestation of radiation pneumonitis as an acute effect, followed by apparent fibrosis in weeks or even months. A recently developed, first-of-its-kind murine model for partial-body irradiation (PBI) injury, which can be used to test potential countermeasures against multi-organ damage such as gastrointestinal (GI) tract and lungs was used for irradiation, with 2.5% bone marrow spared (BM2.5-PBI) from radiation exposure. Long-term damage to lungs from radiation was evaluated using µ-CT scans, pulmonary function testing, histopathological parameters and molecular biomarkers. Pulmonary fibrosis was detected by ground glass opacity observed in µ-CT scans of male and female C57BL/6J mice 6-7 months after BM2.5-PBI. Lung mechanics assessments pertaining to peripheral airways suggested fibrotic lungs with stiffer parenchymal lung tissue and reduced inspiratory capacity in irradiated animals 6-7 months after BM2.5-PBI. Histopathological evaluation of the irradiated lungs revealed presence of focal and diffuse pleural, and parenchymal inflammatory and fibrotic lesions. Fibrosis was confirmed by elevated levels of collagen when compared to lungs of age-matched naïve mice. These findings were validated by findings of elevated levels of pro-fibrotic biomarkers and reduction in anti-inflammatory proteins. In conclusion, a long-term model for radiation-induced pulmonary fibrosis was established, and countermeasures could be screened in this model for survival and protection/mitigation or recovery from radiation-induced pulmonary damage.

Natural-history Characterization of a Murine Partial-body Irradiation Model System: Establishment of a Multiple-Parameter Based GI-ARS Severity-Scoring System.

The purpose of this investigation was to characterize the natural history of a murine total-abdominal-irradiation exposure model to measure gastrointestinal acute radiation injury. Male CD2F1 mice at 12 to 15 weeks old received total-abdominal irradiation using 4-MV linear accelerator X-rays doses of 0, 11, 13.5, 15, 15.75 and 16.5 Gy (2.75 Gy/min). Daily cage-side (i.e., in the animal housing room) observations of clinical signs and symptoms including body weights on all animals were measured up to 10 days after exposure. Jejunum tissues from cohorts of mice were collected at 1, 3, 7 and 10 days after exposure and radiation injury was assessed by histopathological analyses. Results showed time- and dose-dependent loss of body weight [for example at 7 days: 0.66 (±0.80) % loss for 0 Gy, 6.40 (±0.76) % loss at 11 Gy, 9.43 (±2.06) % loss at 13.5 Gy, 23.53 (± 1.91) % loss at 15 Gy, 29.97 (±1.16) % loss at 15.75 Gy, and 31.79 (±0.76) % loss at 16.5 Gy]. Negligible clinical signs and symptoms, except body weight changes, of radiation injury were observed up to 10 days after irradiation with doses of 11 to 15 Gy. Progressive increases in the severity of clinical signs and symptoms were found after irradiation with doses >15 Gy. Jejunum histology showed a progressive dose-dependent increase in injury. For example, at 7 days postirradiation, the percent of crypts, compared to controls, decreased to 82.3 (±9.5), 69.2 (±12.3), 45.4 (±11.9), 18.0 (±3.4), and 11.5 (± 1.8) with increases in doses from 11 to 16.5 Gy. A mucosal injury scoring system was used that mainly focused on changes in villus morphology damage (i.e., subepithelial spaces near the tips of the villi with capillary congestion, significant epithelial lifting along the length of the villi with a few denuded villus tips). Peak levels of total-abdominal irradiation induced effects on the mucosal injury score were seen 7 days after irradiation for doses ≥15 Gy, with a trend to show a decline after 7 days. A murine multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system was established based on clinical signs and symptoms that included measures of appearance (i.e., hunched and/or fluffed fur), respiratory rate, general (i.e., decreased mobility) and provoked behavior (i.e., subdued response to stimulation), weight loss, and feces/diarrhea score combined with jejunum mucosal-injury grade score. In summary, the natural-history radio-response for murine partial-body irradiation exposures is important for establishing a well-characterized radiation model system; here we established a multiple-parameter gastrointestinal acute-radiation syndrome severity-scoring system that provides a radiation injury gastrointestinal tissue-based assessment utility.

High variability in short and long-term recovery kinetic of blood cell count and blood chemistry in a partial body irradiation mouse model

Purpose In the aftermath of a nuclear disaster or accident, survivors will suffer from radiation-induced normal tissue damage. Recovery after radiation exposure is dictated by several factors, one of which is degree of shielding at time of exposure. This study aims to characterize the short and late term changes in kinetics and magnitude of pancytopenia and blood chemistry in a model of heterogeneous radiation exposure, or partial body irradiation (PBI), compared to whole body irradiation (WBI). Materials and methods Male C57BL/6 mice, 8-10 weeks of age, were WBI at 6 different doses (6, 6.1. 6.15, 6.2, 6.5, and 7.5 Gy) to establish the LD50. To determine the effect of shielding on blood cell counts and chemistry, animals were either WBI at 6 Gy (LD2230) or 6 Gy PBI with one leg shielding (LD030). Complete blood counts and chemistry were measured at 1, 5-, 10-, 20-, 30- and 120-days post-irradiation. Results and conclusions Irradiated animals had significant depletion of white blood cells, red blood cells and platelets up to 10 days post-irradiation. Separation between PBI and WBI were observed at 10- and 20-days post-irradiation at which point PBI animals showed sign of recovery while overall cell count remains depleted in WBI animals up to 30 days post-irradiation. In addition, significant changes were found in parameters indicative of hematopoietic injury including hemoglobin count, hematocrit count and white blood cell population. Significant changes were observed in kidney function with changes to blood urea nitrogen and calcium concentration at 5-days post-irradiation. At 10-days post-irradiation. liver function changes differentiated WBI from PBI animals. Long-term, irradiated animal’s chemistry values and many blood counts were not significantly different from Sham. In conclusion, partial shielding ensured complete survival and demonstrated a different recovery kinetics of blood and chemistry parameters after irradiation compared to survivors of whole body irradiation and no single hemopoietic parameter was able to consistently differentiate irradiated from Sham animals. This seems to indicate that there is no single robust hemopoietic parameter to differentiate those exposed from those who were not due to the inherent variability in individual responses. Furthermore, there were no significant long-term effects on these blood parameters between survivors of WBI and PBI except that shielding accelerated recovery.

Metabolomic Profiles in Tissues of Nonhuman Primates Exposed to Either Total- or Partial-Body Radiation.

A complex cascade of systemic and tissue-specific responses induced by exposure to ionizing radiation can lead to functional impairment over time in the surviving population. Current methods for management of survivors of unintentional radiation exposure episodes rely on monitoring individuals over time for the development of adverse clinical symptoms due to the lack of predictive biomarkers for tissue injury. In this study, we report on changes in metabolomic and lipidomic profiles in multiple tissues of nonhuman primates (NHPs) that received either 4.0 Gy or 5.8 Gy total-body irradiation (TBI) of 60Co gamma rays, and 4.0 or 5.8 Gy partial-body irradiation (PBI) from LINAC-derived photons and were treated with a promising radiation countermeasure, gamma-tocotrienol (GT3). These include small molecule alterations that correlate with radiation effects in the jejunum, lung, kidney, and spleen of animals that either survived or succumbed to radiation toxicities over a 30-day period. Radiation-induced metabolic changes in tissues were observed in animals exposed to both doses and types of radiation, but were partially alleviated in GT3-treated and irradiated animals, with lung and spleen being most responsive. The majority of the pathways protected by GT3 treatment in these tissues were related to glucose metabolism, inflammation, and aldarate metabolism, suggesting GT3 may exert radioprotective effects in part by sparing these pathways from radiation-induced dysregulation. Taken together, the results of our study demonstrate that the prophylactic administration of GT3 results in metabolic and lipidomic shifts that likely provide an overall advantage against radiation injury. This investigation is among the first to highlight the use of a molecular phenotyping approach in a highly translatable NHP model of partial- and total-body irradiation to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

The Roles of IL-18 in a Realistic Partial Body Irradiation with 5% Bone Marrow Sparing (PBI/BM5) Model.

IL-18 has been shown to play important roles in response to total body irradiation. However, homogenous total body irradiation is not a realistic model to reflect the radiation exposure in a real nuclear event. To further study the roles of IL-18 in a real nuclear scenario, we developed a mouse partial body irradiation with 5% bone marrow sparing (PBI/BM5) model to mimic the inhomogeneous radiation exposure. We established the dose response curves of PBI/BM5 using different radiation doses ranging from 12 to 16 Gy. Using the PBI/BM5 model, we showed that IL-18 knockout mice were significantly more radiation resistant than the wild-type mice at 14.73 Gy. We further studied the hematopoietic changes using a complete blood count, bone marrow colony-forming assays, and serum cytokine assays on the mice exposed to PBI/BM5 with IL-18BP treatment and wild-type/IL-18 knockout mice. In conclusion, our data suggest that IL-18 plays important roles in mouse survival in a realistic nuclear exposure model, potentially through the IL-18/IFNγ pathway.

Biodosimetry Based on Gamma-H2AX Quantification in Human Peripheral Blood Lymphocytes after Partial-body Irradiation.

Quantification of gamma-H2AX foci can estimate exposure to ionizing radiation. Most nuclear and radiation accidents are partial-body irradiation, and the doses estimated using the total-body irradiation dose estimation formula are often lower than the actual dose. To evaluate the dose-response relation of gamma-H2AX foci in human peripheral blood lymphocytes after partial-body irradiation and establish a simple and high throughput model to estimate partial-body irradiation dose, we collected human peripheral blood and irradiated with 0-, 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, and 8-Gy gamma rays to simulate total-body irradiation in vitro. Gamma-H2AX foci were quantitated by flow cytometry at 1 h after irradiation, and a dose-response curve was established for total-body irradiation dose estimation. Then, a partial-body irradiation dose-response calibration curve was established by adding calibration coefficients based on the Dolphin method. To reflect the data distribution of all doses more realistically, the partial-body irradiation dose-response calibration curve was divided into two sections. In addition, partial-body irradiation was simulated in vitro, and the PBI data were substituted into curves to verify the accuracy of the two partial-body irradiation calibration curves. Results showed that the dose estimation variations were all less than 30% except the 25% partial-body irradiation group at 1 Gy, and the partial-body irradiation calibration dose-response curves were YF 1 = - 3.444 x 2 + 18.532 x + 3.109, R 2 = 0.92 (YF ≤ 27.95); YF 2 = - 2.704 x 2 + 37.97 x - 56.45, R 2 = 0.86 (YF > 27.95). Results also suggested that the partial-body irradiation dose-response calibration curve based on the gamma-H2AX foci quantification in human peripheral blood lymphocytes is a simple and high throughput model to assess partial-body irradiation dose.

Human cell-expressed tag-free rhMFG-E8 as an effective radiation mitigator

Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and the possibility of antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 will have suitable structural and functional properties to be developed as a safe and effective novel biologic to treat inflammatory diseases including radiation injury. We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing an experimental rodent model of radiation injury, i.e., partial body irradiation (PBI). HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE with the standard human MFG-E8 loaded as control and, mass spectrometry followed by analysis using MASCOT for peptide mass fingerprint. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with a terminal elimination half-life in circulation of at least 1.45 h. In the 15 Gy PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 calculated using probit analysis was 1.058. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI suggesting it as a potential therapeutic candidate for a medical countermeasure for radiation injury. Our new human cell-expressed tag-free rhMFG-E8 has proper structural and functional properties to be further developed as a safe and effective therapy to treat victims of severe acute radiation injury.

Modulation of Hematopoietic Injury by a Promising Radioprotector, Gamma-Tocotrienol, in Rhesus Macaques Exposed to Partial-Body Radiation.

Currently, no radioprotectors have been approved to mitigate hematopoietic injury after exposure to ionizing radiation. Acute ionizing radiation results in damage to both hematopoietic and immune system cells. Pre-exposure prophylactic agents are needed for first responders and military personnel. In this study, the ability of gamma-tocotrienol (GT3), a promising radioprotector and antioxidant, to ameliorate partial-body radiation-induced damage to the hematopoietic compartment was evaluated in a nonhuman primate (NHP) model. A total of 15 rhesus NHPs were divided into two groups, and were administered either GT3 or vehicle 24 h prior to 4 or 5.8 Gy partial-body irradiation (PBI), with 5% bone marrow (BM) sparing. Each group consisted of four NHPs, apart from the vehicle-treated group exposed to 5.8 Gy, which had only three NHPs. BM samples were collected 8 days prior to irradiation in addition to 2, 7, 14, and 30 days postirradiation. To assess the clonogenic ability of hematopoietic stem and progenitor cells (HSPCs), colony forming unit (CFU) assays were performed, and lymphoid cells were immunophenotyped using flow cytometry. As a result of GT3 treatment, an increase in HSPC function was evident by an increased recovery of CFU-granulocyte macrophages (CFU-GM). Additionally, GT3 treatment was shown to increase the percentage of CD34+ cells, including T and NK-cell subsets. Our data further affirm GT3's role in hematopoietic recovery and suggest the need for its further development as a prophylactic radiation medical countermeasure.

Development of a Multi-Organ Radiation Injury Model with Precise Dosimetry with Focus on GI-ARS.

The goal of this study was to establish a model of partial-body irradiation (PBI) sparing 2.5% of the bone marrow (BM2.5-PBI) that accurately recapitulates radiological/nuclear exposure scenarios. Here we have reported a model which produces gastrointestinal (GI) damage utilizing a clinical linear accelerator (LINAC) with precise dosimetry, which can be used to develop medical countermeasures (MCM) for GI acute radiation syndrome (ARS) under the FDA animal rule. The PBI model (1 hind leg spared) was developed in male and female C57BL/6 mice that received radiation doses ranging from 12-17 Gy with no supportive care. GI pathophysiology was assessed by crypt cell loss and correlated with peak lethality between days 4 and 10 after PBI. The radiation dose resulting in 50% mortality in 30 days (LD50/30) was determined by probit analysis. Differential blood cell counts in peripheral blood, colony forming units (CFU) in bone marrow, and sternal megakaryocytes were analyzed between days 1-30, to assess the extent of hematopoietic ARS (H-ARS) injury. Radiation-induced GI damage was also assessed by measuring: 1. bacterial load (16S rRNA) by RT-PCR on days 4 and 7 after PBI in liver, spleen and jejunum, 2. liposaccharide binding protein (LBP) levels in liver, and 3. fluorescein isothiocyanate (FITC)-dextran, E-selectin, sP-selectin, VEGF, FGF-2, MMP-9, citrulline, and serum amyloid A (SAA) levels in serum. The LD50/30 of male mice was 14.3 Gy (95% confidence interval 14.1-14.7 Gy) and of female mice was 14.5 Gy (95% confidence interval 14.3-14.7 Gy). Secondary endpoints included loss of viable crypts, higher bacterial loads in spleen and liver, higher LBP in liver, increased FITC-dextran and SAA levels, and decreased levels of citrulline and endothelial biomarkers in serum. The BM2.5-PBI model, developed for the first time with precise dosimetry, showed acute radiation-induced GI damage that is correlated with lethality, as well as a response to various markers of inflammation and vascular damage. Sex-specific differences were observed with respect to radiation dose response. Currently, no MCM is available as a mitigator for GI-ARS. This BM2.5-PBI mouse model can be regarded as the first high-throughput PBI model with precise dosimetry for developing MCMs for GI-ARS under the FDA animal rule.

A New Method for Estimating Glomerular Filtration Rate in Rhesus Macaques (Macaca mulatta).

Late effects of total- or partial-body irradiation include chronic kidney injury (CKI), which increases morbidity and mortality. Glomerular filtration rate (GFR) is the gold standard measure of kidney function. Renal function markers, such as blood urea nitrogen (BUN) and serum creatinine (Cr), may not be higher than reference ranges until 50% or more of nephrons are affected. Currently available methods to measure GFR are difficult and expensive, requiring multiple blood draws or timed urine collections, but their use can provide a framework for the development of simpler GFR estimates. The measurement of iohexol clearance is a validated tool used to determine GFR in veterinary patients. In this study, we aimed to determine if the Schwartz formula as used in human pediatric medicine can estimate GFR in rhesus macaques. We hypothesized that iohexol-GFR would correlate with the Schwartz formula-estimated GFR (eGFR) in irradiated and non-irradiated rhesus macaques. Twelve rhesus macaques [age 5-14 years (mean 7 years); 5 females, 7 males] with a range of BUN levels were selected for comparison to 4 non-irradiated controls (2 females, 2 males). Irradiated animals were divided by BUN into 3 groups: BUN ≤20 mg/dL (n = 4), BUN >20-24 mg/dL (n = 4), and BUN ≥25 mg/dL (n = 4). Baseline serum chemistry and urinalysis were used to assess renal function. For measurement of GFR, macaques were maintained under general anesthesia and received an intravenous injection of iohexol (2 mL/kg, 300 mg I/mL). Whole blood was collected at 10, 30, 60 and 90 min post-iohexol injection. Plasma iohexol concentrations were determined by mass spectrometry. GFR was calculated from the peak iohexol concentration and trapezoidal area under the curve (tAUC). The iohexol-GFR significantly correlated with the Schwartz formula-eGFR. In macaques with renal irradiation doses below 6 Gy, GFR was higher for males than females. GFR was lower in macaques with renal irradiation doses greater than 6 Gy compared to macaques with renal doses less than 6 Gy. We conclude that use of the Schwartz formula can provide a rapid, non-invasive, cost-effective, and accurate estimation of GFR to aid in the clinical assessment of renal function in irradiated rhesus macaques.

Identification of the Kupffer cell-derived circulating IGFBP-3 as a universal radiation biomarker for heavy ion, proton, and X-ray exposure

The minimally invasive biomarkers that can facilitate a rapid dose assessment are valuable for the early medical treatment when accidental or occupational radiation exposure happens. Our previous proteomic research identified one kind of circulating protein, Insulin-like Growth Factor Binding Protein 3 (IGFBP-3), which showed a significant increase after total body exposure of mice to carbon ions and X-rays. However, several critical issues such as the responses to diverse radiation, the origin and underlying mechanism in radiation response obstruct the utilization of circulating IGFBP-3 as a reliable radiation biomarker. In this study, mice were subjected to total or partial body irradiation with carbon ions, protons or X-rays, or treated with chloroform as a comparison. The level of IGFBP-3 in serum and different organs were measured via Enzyme Linked Immunosorbent Assay (ELISA), Western blot (WB) and Immunohistochemistry (IHC). A significant increase of IGFBP-3 was discovered in serum and liver tissue post-irradiation with three kinds of radiation, but absent when challenged with chloroform. Likewise, a similar response was also observed in blood samples from patients receiving radiotherapy. Moreover, the effect of radiation on three main hepatic cells was investigated, the findings indicated that IGFBP-3 could be detected in the culture medium of Kupffer cells (MKC) alone and was elevated in cells and cultured medium of MKC post-irradiation. Additionally, we observed a co-expression effect between P53 and IGFBP-3 in liver tissues and MKC post-irradiation. Along with down-regulation of Trp53 by siRNA, the response of IGFBP-3 to radiation was attenuated. The present study demonstrated that circulating IGFBP-3 could be a promising universal biomarker for complex environmental radiation exposure, and the upregulation of IGFBP-3 is attributed to the MKC in a P53-dependent manner. Circulating IGFBP-3 assays would offer rapid, convenient and effective dose and toxicity assessment methods in occupational exposure or radiation disaster management.