Delayed Effects Of Acute Radiation Exposure Research Articles

Genetic Upregulation of Activated Protein C Mitigates Delayed Effects of Acute Radiation Exposure in the Mouse Plasma.

Exposure to ionizing radiation, accidental or intentional, may lead to delayed effects of acute radiation exposure (DEARE) that manifest as injury to organ systems, including the kidney, heart, and brain. This study examines the role of activated protein C (APC), a known mitigator of radiation-induced early toxicity, in long-term plasma metabolite and lipid panels that may be associated with DEARE in APCHi mice. The APCHi mouse model used in the study was developed in a C57BL/6N background, expressing the D168F/N173K mouse analog of the hyper-activatable human D167F/D172K protein C variant. This modification enables increased circulating APC levels throughout the mouse's lifetime. Male and female cohorts of C57BL/6N wild-type and APCHi transgenic mice were exposed to 9.5 Gy γ-rays with their hind legs shielded to allow long-term survival that is necessary to monitor DEARE, and plasma was collected at 6 months for LC-MS-based metabolomics and lipidomics. We observed significant dyslipidemia, indicative of inflammatory phenotype, upon radiation exposure. Additionally, observance of several other metabolic dysregulations was suggestive of gut damage, perturbations in TriCarboxylic Acid (TCA) and urea cycles, and arginine metabolism. We also observed gender- and genotype-modulated metabolic perturbations post radiation exposure. The APCHi mice showed near-normal abundance for several lipids. Moreover, restoration of plasma levels of some metabolites, including amino acids, citric acid, and hypoxanthine, in APCHi mice is indicative of APC-mediated protection from radiation injuries. With the help of these findings, the role of APC in plasma molecular events after acute γ-radiation exposure in a gender-specific manner can be established for the first time.

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.

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.

Nicotinamide riboside intervention alleviates hematopoietic system injury of ionizing radiation-induced premature aging mice.

Radiotherapy destroys cancer cells and inevitably harms normal human tissues, causing delayed effects of acute radiation exposure (DEARE) and accelerating the aging process in most survivors. However, effective methods for preventing premature aging induced by ionizing radiation are lacking. In this study, the premature aging mice of DEARE model was established after 6 Gy total body irradiation (TBI). Then the therapeutic effects and mechanism of nicotinamide riboside on the premature aging mice were evaluated. The results showed that 6 Gy TBI induced premature aging of the hematopoietic system in mice. Nicotinamide riboside treatment reversed aging spleen phenotypes by inhibiting cellular senescence and ameliorated serum metabolism profiles. Further results demonstrated that nicotinamide riboside supplementation alleviated the myeloid bias of hematopoietic stem cells and temporarily restored the regenerative capacity of hematopoietic stem cells probably by mitigating the reactive oxygen species activated GCN2/eIF2α/ATF4 signaling pathway. The results of this study firstly indicate that nicotinamide riboside shows potential as a DEARE therapeutic agent for radiation-exposed populations and patients who received radiotherapy.

Further Characterization of Multi-Organ DEARE and Protection by 16,16 Dimethyl Prostaglandin E2 in a Mouse Model of the Hematopoietic Acute Radiation Syndrome.

Survivors of acute radiation exposure suffer from the delayed effects of acute radiation exposure (DEARE), a chronic condition affecting multiple organs, including lung, kidney, heart, gastrointestinal tract, eyes, and brain, and often causing cancer. While effective medical countermeasures (MCM) for the hematopoietic-acute radiation syndrome (H-ARS) have been identified and approved by the FDA, development of MCM for DEARE has not yet been successful. We previously documented residual bone marrow damage (RBMD) and progressive renal and cardiovascular DEARE in murine survivors of H-ARS, and significant survival efficacy of 16,16-dimethyl prostaglandin E2 (dmPGE2) given as a radioprotectant or radiomitigator for H-ARS. We now describe additional DEARE (physiological and neural function, progressive fur graying, ocular inflammation, and malignancy) developing after sub-threshold doses in our H-ARS model, and detailed analysis of the effects of dmPGE2 administered before (PGE-pre) or after (PGE-post) lethal total-body irradiation (TBI) on these DEARE. Administration of PGE-pre normalized the twofold reduction of white blood cells (WBC) and lymphocytes seen in vehicle-treated survivors (Veh), and increased the number of bone marrow (BM) cells, splenocytes, thymocytes, and phenotypically defined hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) to levels equivalent to those in non-irradiated age-matched controls. PGE-pre significantly protected HPC colony formation ex vivo by >twofold, long term-HSC in vivo engraftment potential up to ninefold, and significantly blunted TBI-induced myeloid skewing. Secondary transplantation documented continued production of LT-HSC with normal lineage differentiation. PGE-pre reduced development of DEARE cardiovascular pathologies and renal damage; prevented coronary artery rarefication, blunted progressive loss of coronary artery endothelia, reduced inflammation and coronary early senescence, and blunted radiation-induced increase in blood urea nitrogen (BUN). Ocular monocytes were significantly lower in PGE-pre mice, as was TBI-induced fur graying. Increased body weight and decreased frailty in male mice, and reduced incidence of thymic lymphoma were documented in PGE-pre mice. In assays measuring behavioral and cognitive functions, PGE-pre reduced anxiety in females, significantly blunted shock flinch response, and increased exploratory behavior in males. No effect of TBI was observed on memory in any group. PGE-post, despite significantly increasing 30-day survival in H-ARS and WBC and hematopoietic recovery, was not effective in reducing TBI-induced RBMD or any other DEARE. In summary, dmPGE2 administered as an H-ARS MCM before lethal TBI significantly increased 30-day survival and ameliorated RBMD and multi-organ and cognitive/behavioral DEARE to at least 12 months after TBI, whereas given after TBI, dmPGE2 enhances survival from H-ARS but has little impact on RBMD or other DEARE.

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.

Pharmacokinetic and metabolomic studies with a BIO 300 Oral Powder formulation in nonhuman primates

BIO 300, a pharmaceutical formulation of genistein, is being developed as a radiation countermeasure to treat hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE). Several studies have affirmed its safety and efficacy in alleviating the damaging effects of ionizing radiation. However, dose optimization of any drug has always been an important area of research because unnecessarily high drug doses may result in serious complications. In this study, we assessed the pharmacokinetics (PK) and metabolic profiles of two different doses of a novel solid-dosage formulation of BIO 300 (BIO 300 Oral Powder; 100 mg/kg and 200 mg/kg), when administered orally to nonhuman primates (NHPs). While the Tmax values of both doses remained the same, the area under the curve at 48 h (AUC0-48) was tripled by doubling the dose. Additionally, we monitored serum samples for global metabolomic/lipidomic changes using high resolution mass spectrometry followed by functional pathway analysis prior to and at various time points up to 48 h post drug administration. Interestingly, the metabolomic profiles of sera from NHPs that received the lower dose demonstrated a transient perturbation in numerous metabolites between the 4 and 12 h time points. Eventually, the metabolite abundance reverted to near-normal by 48 h. These study results are consistent with our previous studies focused on the PK and metabolomic analysis for parenteral and oral aqueous nanosuspension formulations of BIO 300. This study affirms that administration of a single dose of up to 200 mg/kg of BIO 300 Oral Powder is safe in NHPs and conferred no metabolomic-mediated safety features.

Mitigation of Multi-Organ Radiation Injury with ACE2 Agonist Diminazene Aceturate.

The renin-angiotensin system (RAS) is known to regulate the pathogenesis of radiation-induced injury as inhibitors of the RAS enzyme angiotensin converting enzyme (ACE) have established function as mitigators of multi-organ radiation injury. To further elucidate the role of RAS signaling during both the acute and delayed syndromes of radiation exposure, we have evaluated whether pharmacologic modulation of alternate RAS enzyme angiotensin converting enzyme 2 (ACE2) reduces the pathogenesis of multi-organ radiation-induced injuries. Here, we demonstrate pharmacologic ACE2 activation with the small molecule ACE2 agonist diminazene aceturate (DIZE) improves survival in rat models of both hematologic acute radiation syndrome (H-ARS) and multi-organ delayed effects of acute radiation exposure (DEARE). In the H-ARS model, DIZE treatment increased 30-day survival by 30% compared to vehicle control rats after a LD50/30 total-body irradiation (TBI) dose of 7.75 Gy. In the mitigation of DEARE, ACE2 agonism with DIZE increased median survival by 30 days, reduced breathing rate, and reduced blood urea nitrogen (BUN) levels compared to control rats after partial-body irradiation (PBI) of 13.5 Gy. DIZE treatment was observed to have systemic effects which may explain the multi-organ benefits observed including mobilization of hematopoietic progenitors to the circulation and a reduction in plasma TGF-beta levels. These data suggest the ACE2 enzyme plays a critical role in the RAS-mediated pathogenesis of radiation injury and may be a potential therapeutic target for the development of medical countermeasures for acute radiation exposure.

Impact of Age, Sex, and Genetic Diversity in Murine Models of the Hematopoietic Acute Radiation Syndrome (H-ARS) and the Delayed Effects of Acute Radiation Exposure (DEARE).

Malicious or accidental radiation exposure increases risk for the hematopoietic acute radiation syndrome (H-ARS) and the delayed effects of acute radiation exposure (DEARE). Radiation medical countermeasure (MCM) development relies on robust animal models reflective of all age groups and both sexes. This review details critical considerations in murine H-ARS and DEARE model development including divergent radiation responses dependent on age, sex, and genetic diversity. Radioresistance increases with murine age from pediatrics through geriatrics. Between sexes, radioresistance is higher in male weanlings, pubescent females, and aged males, corresponding with accelerated myelopoiesis. Jackson diversity outbred (JDO) mice resemble non-human primates in radiation response for modeling human diversity. Weanlings and JDO models exhibit less DEARE than other models. Highly characterized age-, sex- and diversity-conscious murine models of H-ARS and DEARE provide powerful and essential tools in MCM development for all radiation victims.

Interspecies Comparison and Radiation Effect on Pharmacokinetics of BIO 300, a Nanosuspension of Genistein, after Different Routes of Administration in Mice and Non-Human Primates.

BIO 300, a suspension of synthetic genistein nanoparticles, is being developed for mitigating the delayed effects of acute radiation exposure (DEARE). The purpose of the current study was to characterize the pharmacokinetic (PK) profile of BIO 300 administered as an oral or parenteral formulation 24 h after sham-irradiation, total-body irradiation (TBI) with 2.5-5.0% bone marrow sparing (TBI/BMx), or in nonirradiated sex-matched C57BL/6J mice and non-human primates (NHP). C57BL/6J mice were randomized to the following arms in two consecutive studies: sham-TBI [400 mg/kg, oral gavage (OG)], TBI/BM2.5 (400 mg/kg, OG), sham-TBI [200 mg/kg, subcutaneous (SC) injection], TBI/BM2.5 (200 mg/kg, SC), sham-TBI (100 mg/kg, SC), or nonirradiated [200 mg/kg, intramuscular (IM) injection]. The PK profile was also established in NHP exposed to TBI/BM5.0 (100 mg/kg, BID, OG). Genistein-aglycone serum concentrations were measured in all groups using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. The PK profile demonstrates 11% and 19% reductions in Cmax and AUC0-inf, respectively, among mice administered 400 mg/kg, OG, after TBI/BM2.5 compared to the sham-TBI control arm. Administration of 200 mg/kg SC in mice exposed to TBI/BM2.5 showed a 53% increase in AUC0-inf but a 28% reduction in Cmax compared to the sham-TBI mice. The relative bioavailability of the OG route compared to the SC and IM routes in mice was 9% and 7%, respectively. After the OG route, the dose-normalized AUC0-inf was 13.37 (ng.h/mL)/(mg/kg) in TBI/BM2.5 mice compared to 6.95 (ng.h/mL)/(mg/kg) in TBI/BM5.0 NHPs. Linear regression of apparent clearances and weights of mice and NHPs yielded an allometric coefficient of 1.06. Based on these data, the effect of TBI/BMx on BIO 300 PK is considered minimal. Future studies should use SC and IM routes to maximize drug exposure when administered postirradiation. The allometric coefficient is useful in predicting therapeutic drug dose regimens across species for drug approval under the FDA animal rule.

Rat Models of Partial-body Irradiation with Bone Marrow-sparing (Leg-out PBI) Designed for FDA Approval of Countermeasures for Mitigation of Acute and Delayed Injuries by Radiation.

The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI) to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5-14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to d 7 and diarrhea scoring to d 10. Differential blood counts were analyzed between d 1-42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between d 28-110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between d 90-180. The LD50/30, including both lethality from GI-ARS and H-ARS, for female and male rats are 14.0 Gy and 13.5 Gy, respectively, while the LD50/7 for only GI-ARS are 14.3 Gy and 13.6 Gy, respectively. The all-cause mortalities, including ARS and L-DEARE, through 120 d (LD50/120) are 13.5 Gy and 12.9 Gy, respectively. Secondary end points confirmed occurrence of four distinct sequelae representing GI, hematopoietic, lung, and kidney toxicities after leg-out PBI. Adult rat models of leg-out PBI showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.

Effect of Radiation on the Essential Nutrient Homeostasis and Signaling of Retinoids in a Non-human Primate Model with Minimal Bone Marrow Sparing.

High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

A novel oral formulation of BIO 300 confers prophylactic radioprotection from acute radiation syndrome in mice

Purpose Exposure to high doses of ionizing radiation can result in hematopoietic acute radiation syndrome (H-ARS) and delayed effects of acute radiation exposure (DEARE). There is no radiation medical countermeasure (MCM) approved by the U.S. Food and Drug Administration which can be used prior to radiation exposure to protect exposed individuals. Different formulations containing synthetic genistein (BIO 300) are being developed to counter the harmful effects of radiation exposure. Materials and methods We investigated the efficacy of a BIO 300 oral powder (OP) formulation as a prophylactic radiation MCM against a lethal dose of cobalt-60 gamma-radiation in CD2F1 male mice while comparing to other formulations of BIO 300 and Neulasta (PEGylated filgrastim), a standard of care drug for H-ARS. Results BIO 300 OP provided significant radioprotection against ionizing radiation in mice when administered twice per day for six days prior to total-body radiation exposure. Its radioprotective efficacy in the murine model was comparable to the efficacy of a single subcutaneous (sc) injection of Neulasta administered after total-body radiation exposure. Conclusions Our results demonstrate that BIO 300 OP, which can be administered orally, is a promising prophylactic radiation countermeasure for H-ARS.

Observation of Unique Circulating miRNA Signatures in Non-Human Primates Exposed to Total-Body vs. Whole Thorax Lung Irradiation.

A radiological/nuclear (RAD-NUC) incident, especially in an urban setting, results in diverse radiation-induced injuries due to heterogeneities in dose, the extent of partial-body shielding, human biodiversity and pre-existing health conditions. For example, acute radiation syndrome (ARS) can result in death within days to weeks of exposure to 0.7-10 Gy doses and is associated with destruction of the bone marrow, known as hematopoietic ARS (H-ARS). However, partial-body shielding that spares a portion of the bone marrow from exposure can significantly reduce the occurrence of H-ARS, but delayed effects of acute radiation exposure (DEARE) can still occur within months or years after exposure depending on the individual. In a mass casualty event, ideal triage must be able to pre-symptomatically identify individuals likely to develop radiation-induced injuries and provide an appropriate treatment plan. Today, while there are FDA approved treatments for hematopoietic ARS, there are no approved diagnosis for radiation injury and no approved treatments for the broad spectra of injuries associated with radiation. This has resulted in a major capability gap in the nations preparedness to a potentially catastrophic RAD-NUC event. Circulating microRNA (miRNA) are a promising class of biomarkers for this application because the molecules are accessible via a routine blood draw and are excreted by various tissues throughout the body. To test if miRNA can be used to predict distinct tissue-specific radiation-induced injuries, we compared the changes to the circulating miRNA profiles after total-body irradiation (TBI) and whole thorax lung irradiation (WTLI) in non-human primates at doses designed to induce ARS (day 2 postirradiation; 2-6.5 Gy) and DEARE (day 15 postirradiation; 9.8 or 10.7 Gy), respectively. In both models, miRNA sequences were identified that correlated with the onset of severe neutropenia (counts <500 µL-1; TBI) or survival (WTLI). This method identified panels of eleven miRNA for both model and assigned functional roles for the panel members using gene ontology enrichment analysis. A common signature of radiation-induced injury was observed in both models: apoptosis, DNA damage repair, p53 signaling, pro-inflammatory response, and growth factor/cytokine signaling pathways were predicted to be disrupted. In addition, injury-specific pathways were identified. In TBI, pathways associated with ubiquitination, specifically of histone H2A, were enriched, suggesting more impact to DNA damage repair mechanisms and apoptosis. In WTLI, pro-fibrotic pathways including transforming growth factor (TGF-β) and bone morphogenetic protein (BMP) signaling pathways were enriched, consistent with the onset of late lung injury. These results suggest that miRNA may indeed be able to predict the onset of distinct types of radiation-induced injuries.

Prostaglandin E2 Promotes Recovery of Thymus in Survivors of the Hematopoietic Acute Radiation Syndrome (H-ARS)

We have previously developed a murine model of the hematopoietic acute radiation syndrome [H-ARS, total body irradiated (TBI)] and delayed effects of acute radiation exposure (DEARE) for efficacy testing of potential medical countermeasures (MCM) against these two syndromes. Using this model, we have previously reported that prophylactic use of 16,16 dimethyl-prostaglandin E2 (dmPGE2), a long-acting formulation of PGE, before irradiation significantly increases 30-day survival rate. The current study investigated the effects of the prophylactic use of dmPGE2 with or without therapeutic use of dmPGE2 in survivors on long-term thymus recovery in H-ARS survivors at 6 and 12-months post-LD70/30 (872cGy, 137Cs, 95.2±4.8 cGy/min). Mice were irradiated at 12 weeks of age and then injected subcutaneously with dmPGE2 (1 dose of 35 ug dmPGE2/mouse) or vehicle (Veh; ethanol/PBS solution) at 30“ or -1hr pre-TBI. Survivors were injected monthly with dmPGE2 or Veh and then analyzed for evidence of thymic reconstitution at 6 and 12 months.post-TBI. The three test groups were: prophylactic dmPGE2 + monthly dmPGE2 (PP), prophylactic dmPGE2 + monthly Veh (PV), and prophylactic Veh + monthly Veh (VV), n = 3-6 mice/group. Thymus weight and cellularity of mice in the dmPGE groups, PV and PP, were increased compared to Veh both at 6-months and 12-months post-TBI (p <0.05). The difference was not related to body weight. No difference was observed between PP and PV group. The number of thymocyte subgroups (CD4 single positive, CD8 single positive, double positive and double negative) was also increased in PV and PP mice. Complete blood count (CBC) was also analyzed in the same mice. The VV group showed myeloid skewing, evidenced as an increase in neutrophils and decrease in lymphocytes. The PV and PP groups both exhibited reversal of myeloid skewing, and once again dmPGE maintenance did not provide extra benefit. Thymic progenitors in bone marrow, known as the lymphoid-primed multipotent progenitors (LMPP, Lin-Kit+Sca1+Flt3hiCD34+) and common lymphoid progenitor (CLP, Lin-IL7R+KitloSca1lo Flt3+) were also increased in PV and PP groups compared with VV group (p <0.05). No difference was observed in common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and MEP (megakaryocyte-erythroid progenitor) between PP and PV group. Competitive bone marrow transplantation showed more than 10 times higher donor-derived chimerism in the thymus of PP and PV mice compared with the VV group. Again, no difference was observed between PP and PV group. In conclusion, these data show that dmPGE2, given before irradiation, protects thymic settling progenitors in bone marrow, resulting in reversal of thymic involution, increased peripheral lymphocytes, and partial alleviation of the myeloid skewing observed in radiation survivors. dmPGE maintenance does not bring extra benefit. F unded by Department of the Army PR140433P1 and NIAID 1U01AI107340-01. DisclosuresNo relevant conflicts of interest to declare.

Sex-dependent effects of genetic upregulation of activated protein C on delayed effects of acute radiation exposure in the mouse heart, small intestine, and skin.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.

Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

There is a need for countermeasures to mitigate lethal acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE). In WAG/RijCmcr rats, ARS occurs by 30-days following total body irradiation (TBI), and manifests as potentially lethal gastrointestinal (GI) and hematopoietic (H-ARS) toxicities after >12.5 and >7 Gy, respectively. DEARE, which includes potentially lethal lung and kidney injuries, is observed after partial body irradiation >12.5 Gy, with one hind limb shielded (leg-out PBI). The goal of this study is to enhance survival from ARS and DEARE by polypharmacy, since no monotherapy has demonstrated efficacy to mitigate both sets of injuries. For mitigation of ARS following 7.5 Gy TBI, a combination of three hematopoietic growth factors (polyethylene glycol (PEG) human granulocyte colony-stimulating factor (hG-CSF), PEG murine granulocyte-macrophage-CSF (mGM-CSF), and PEG human Interleukin (hIL)-11), which have shown survival efficacy in murine models of H-ARS were tested. This triple combination (TC) enhanced survival by 30-days from ∼25% to >60%. The TC was then combined with proven medical countermeasures for GI-ARS and DEARE, namely enrofloxacin, saline and the angiotensin converting enzyme inhibitor, lisinopril. This combination of ARS and DEARE mitigators improved survival from GI-ARS, H-ARS, and DEARE after 7.5 Gy TBI or 13 Gy PBI. Circulating blood cell recovery as well as lung and kidney function were also improved by TC + lisinopril. Taken together these results demonstrate an efficacious polypharmacy to mitigate radiation-induced ARS and DEARE in rats.

Establishing Pediatric Mouse Models of the Hematopoietic Acute Radiation Syndrome and the Delayed Effects of Acute Radiation Exposure.

Medical countermeasures (MCMs) for hematopoietic acute radiation syndrome (H-ARS) should be evaluated in well-characterized animal models, with consideration of at-risk populations such as pediatrics. We have developed pediatric mouse models of H-ARS and delayed effects of acute radiation exposure (DEARE) for efficacy testing of MCMs against radiation. Male and female C57BL/6J mice aged 3, 4, 5, 6, 7 and 8 weeks old (±1 day) were characterized for baseline hematopoietic and gastrointestinal parameters, radiation response, efficacy of a known MCM, and DEARE at six and 12 months after total-body irradiation (TBI). Weanlings (age 3 weeks) were the most radiosensitive age group with an estimated LD50/30 of 712 cGy, while mice aged 4 to 8 weeks were more radioresistant with an estimated LD50/30 of 767-787 cGy. Female weanlings were more radiosensitive than males at 3 and 4 weeks old but became significantly more radioresistant after the pubertal age of 5 weeks. The most dramatic increase in body weight, RBC counts and intestinal circumference length occurred from 3 to 5 weeks of age. The established radiomitigator Neulasta® (pegfilgrastim) significantly increased 30-day survival in all age groups, validating these models for MCM efficacy testing. Analyses of DEARE among pediatric survivors revealed depressed weight gain in males six months post-TBI, and increased blood urea nitrogen at 12 months post-TBI which was more severe in females. Hematopoietic DEARE at six months post-TBI appeared to be less severe in survivors from the 3- and 4-week-old groups but was equally severe in all age groups by 12 months of age. Similar to our other acute radiation mouse models, there was no appreciable effect of Neulasta used as an H-ARS MCM on the severity of DEARE. In summary, these data characterize a pediatric mouse model useful for assessing the efficacy of MCMs against ARS and DEARE in children.

Comparative proteomic analysis of serum from nonhuman primates administered BIO 300: a promising radiation countermeasure

Hematopoietic acute radiation syndrome (H-ARS) and delayed effects of acute radiation exposure (DEARE) are detrimental health effects that occur after exposure to high doses of ionizing radiation. BIO 300, a synthetic genistein nanosuspension, was previously proven safe and effective against H-ARS when administered (via the oral (po) or intramuscular (im) route) prior to exposure to lethal doses of total-body radiation. In this study, we evaluated the proteomic changes in serum of nonhuman primates (NHP) after administering BIO 300 by different routes (po and im). We utilized nanoflow-ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (NanoUPLC-MS/MS) methods for comprehensive global profiling and quantification of serum proteins. The results corroborate previous findings that suggest a very similar metabolic profile following both routes of drug administration. Furthermore, we observed minor alterations in protein levels, 2 hours after drug administration, which relates to the Cmax of BIO 300 for both routes of administration. Taken together, this assessment may provide an insight into the mechanism of radioprotection of BIO 300 and a reasonable illustration of the pharmacodynamics of this radiation countermeasure.