Search

The identification and validation of radiation biomarkers is critical for assessing the radiation dose received in exposed individuals and for developing radiation medical countermeasures that can be used to treat acute radiation syndrome (ARS). Additionally, a fundamental understanding of the effects of radiation injury could further aid in the identification and development of therapeutic targets for mitigating radiation damage. In this study, blood samples were collected from fourteen male nonhuman primates (NHPs) that were exposed to 7.2 Gy ionizing radiation at various time points (seven days prior to irradiation; 1, 13, and 25 days post-irradiation; and immediately prior to the euthanasia of moribund (preterminal) animals). Plasma was isolated from these samples and was analyzed using a liquid chromatography tandem mass spectrometry approach in an effort to determine the effects of radiation on plasma proteomic profiles. The primary objective was to determine if the radiation-induced expression of specific proteins could serve as an early predictor for health decline leading to a preterminal phenotype. Our results suggest that radiation induced a complex temporal response in which some features exhibit upregulation while others trend downward. These statistically significantly altered features varied from pre-irradiation levels by as much as tenfold. Specifically, we found the expression of integrin alpha and thrombospondin correlated in peripheral blood with the preterminal stage. The differential expression of these proteins implicates dysregulation of biological processes such as hemostasis, inflammation, and immune response that could be leveraged for mitigating radiation-induced adverse effects., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

In times of war, radiological/nuclear emergency scenarios have become a reemphasized threat. However, there are challenges in transferring whole-blood samples to laboratories for specialized diagnostics using RNA. This project aims to miniaturize the process of unwieldy conventional RNA extraction with its stationed technical equipment using a microfluidic-based slide (MBS) for point-of-care diagnostics. The MBS is thought to be a preliminary step toward the development of a so-called lab-on-a-chip microfluidic device. A MBS would enable early and fast field care combined with gene expression (GE) analysis for the prediction of hematologic acute radiation syndrome (HARS) severity or identification of RNA microbes. Whole blood samples from ten healthy donors were irradiated with 0, 0.5 and 4 Gy, simulating different ARS severity degrees. RNA quality and quantity of a preliminary MBS was compared with a conventional column-based (CB) RNA extraction method. GE of four HARS severity-predicting radiation-induced genes (FDXR, DDB2, POU2AF1 and WNT3) was examined employing qRT-PCR. Compared to the CB method, twice as much total RNA from whole blood could be extracted using the MBS (6.6 ± 3.2 µg vs. 12.0 ± 5.8 µg) in half of the extraction time, and all MBS RNA extracts appeared DNA-free in contrast to the CB method (30% were contaminated with DNA). Using MBS, RNA quality [RNA integrity number equivalent (RINe)] values decreased about threefold (3.3 ± 0.8 vs. 9.0 ± 0.4), indicating severe RNA degradation, while expected high-quality RINe ≥ 8 were found using column-based method. However, normalized cycle threshold (Ct) values, as well as radiation-induced GE fold-changes appeared comparable for all genes utilizing both methods, indicating that no RNA degradation took place. In summary, the preliminary MBS showed promising features such as: 1. halving the RNA extraction time without the burden of heavy technical equipment (e.g., a centrifuge); 2. absence of DNA contamination in contrast to CB RNA extraction; 3. reduction in blood required, because of twice the biological output of RNA; and 4. equal GE performance compared to CB, thus, increasing its appeal for later semi-automatic parallel field applications., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8-7.2 Gy (LD29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. At the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at -80°C for internal experimental analyses. Leftover tubes from these already ongoing studies were kindly provided to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and for further gene expression (GE) studies quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

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., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Coagulopathies are well documented after acute radiation exposure at hematopoietic doses, and radiation-induced bleeding is notably one of the two main causes of mortality in the hematopoietic acute radiation syndrome. Despite this, understanding of the mechanisms by which radiation alters hemostasis and induces bleeding is still lacking. Here, male Göttingen minipigs received hematopoietic doses of 60Co gamma irradiation (total body) and coagulopathies were characterized by assessing bleeding, blood cytopenia, fibrin deposition, changes in hemostatic properties, coagulant/anticoagulant enzyme levels, and markers of inflammation, endothelial dysfunction, and barrier integrity to understand if a relationship exists between bleeding, hemostatic defects, bone marrow aplasia, inflammation, endothelial dysfunction and loss of barrier integrity. Acute radiation exposure induced coagulopathies in the Göttingen minipig model of hematopoietic acute radiation syndrome; instances of bleeding were not dependent upon thrombocytopenia. Neutropenia, alterations in hemostatic parameters and damage to the glycocalyx occurred in all animals irrespective of occurrence of bleeding. Radiation-induced bleeding was concurrent with simultaneous thrombocytopenia, anemia, neutropenia, inflammation, increased heart rate, decreased nitric oxide bioavailability and endothelial dysfunction; bleeding was not observed with the sole occurrence of a single aforementioned parameter in the absence of the others. Alteration of barrier function or clotting proteins was not observed in all cases of bleeding. Additionally, fibrin deposition was observed in the heart and lungs of decedent animals but no evidence of DIC was noted, suggesting a unique pathophysiology of radiation-induced coagulopathies. These findings suggest radiation-induced coagulopathies are the result of simultaneous damage to several key organs and biological functions, including the immune system, the inflammatory response, the bone marrow and the cardiovasculature., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Purpose: In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes., Materials and Methods: EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood., Results: The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude., Conclusions: T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3 . This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.

High-dose radiation exposure results in organ-specific sequelae that occurs in a time- and dose-dependent manner. The partial body irradiation with minimal bone marrow sparing model was developed to mimic intentional or accidental radiation exposures in humans where bone marrow sparing is likely and permits the concurrent analysis of coincident short- and long-term damage to organ systems. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the plasma proteome of non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing with 6 MV LINAC-derived photons at 0.80 Gy min over a time period of 3 wk. The plasma proteome was analyzed by liquid chromatography-tandem mass spectrometry. A number of trends were identified in the proteomic data including pronounced protein changes as well as protein changes that were consistently upregulated or downregulated at all time points and dose levels interrogated. Pathway and gene ontology analysis were performed; bioinformatic analysis revealed significant pathway and biological process perturbations post high-dose irradiation and shed light on underlying mechanisms of radiation damage. Additionally, proteins were identified that had the greatest potential to serve as biomarkers for radiation exposure.

Exposure to total- and partial-body irradiation following a nuclear or radiological incident result in the potentially lethal acute radiation syndromes of the gastrointestinal and hematopoietic systems in a dose- and time-dependent manner. Radiation-induced damage to the gastrointestinal tract is observed within days to weeks post-irradiation. Our objective in this study was to evaluate plasma biomarker utility for the gastrointestinal acute radiation syndrome in non-human primates after partial body irradiation with minimal bone marrow sparing through correlation with tissue and histological analyses. Plasma and jejunum samples from non-human primates exposed to partial body irradiation of 12 Gy with bone marrow sparing of 2.5% were evaluated at various time points from day 0 to day 21 as part of a natural history study. Additionally, longitudinal plasma samples from non-human primates exposed to 10 Gy partial body irradiation with 2.5% bone marrow sparing were evaluated at timepoints out to 180 d post-irradiation. Plasma and jejunum metabolites were quantified via liquid chromatography-tandem mass spectrometry and histological analysis consisted of corrected crypt number, an established metric to assess radiation-induced gastrointestinal damage. A positive correlation of metabolite levels in jejunum and plasma was observed for citrulline, serotonin, acylcarnitine, and multiple species of phosphatidylcholines. Citrulline levels also correlated with injury and regeneration of crypts in the small intestine. These results expand the characterization of the natural history of gastrointestinal acute radiation syndrome in non-human primates exposed to partial body irradiation with minimal bone marrow sparing and also provide additional data toward the correlation of citrulline with histological endpoints.

Purpose: Göttingen minipig (G-MP) displays classic gastrointestinal acute radiation syndrome (GI-ARS) following total body irradiation (TBI) at GI doses which are lethal by 10-14 days. In collaboration with BARDA, we are developing a hemi-body/partial body irradiation (PBI) model by exposing only the abdomen and lower extremities to study GI structure/function impairment, natural history of injury and recovery, as well as correlative biomarkers out to 30 days. Materials and methods: Twenty-four G-MP were exposed to either 12 or 16 Gy (LINAC Elekta); head, forelimbs, and thorax were outside the irradiation field, sparing ∼50% of the bone marrow. Animals were followed for 30 days with euthanasia scheduled at pre-set intervals to study the time course of GI injury and recovery. Hematological profiles, clinical symptoms, gross- and histo-pathology including markers of proliferation and apoptosis in the small intestines, gut function parameters (food tolerance, digestion, absorption, citrulline production), and levels of two biomarkers, CRP and IGF-1, were evaluated. Results: PBI at 16 Gy yielded higher lethality than 12 Gy. Unlike TBI, PBI did not cause severe pancytopenia or external hemorrhage, as expected, and allowed to focus the injury on GI organs while sparing the radiation sensitive heart and lung. Compromised animals showed inactivity, anorexia, vomiting, diarrhea, and weight loss. Histology revealed that in 12 Gy irradiated animals, lesions recovered overtime. In 16 Gy irradiated animals, lesions were more pronounced and persistent. BrdU and Ki67 labelling demonstrated dose-dependent loss of crypts and subsequent mucosal ulceration which recovered over time. Minimal apoptosis was observed at both doses. Reductions in food tolerance, digestion, absorption, and citrulline production were time and dose-dependent. Loss of citrulline reached a nadir between 6-12 days and then recovered partially. CRP and IGF-1 were upregulated following PBI at GI doses. Conclusions: This lower hemi-body irradiation model allowed for extended survival at GI-specific ARS doses and development of a well-controlled GI syndrome with minimal hematopoietic injury or confounding mortality from cardiopulmonary damage. A dose-dependent impairment in the intestinal structure resulted in overall decreased gut functionality followed by a partial recovery. However, while the structure appeared to be recovered, not all functionality was attained. PBI induced systemic inflammation and altered the IGF-1 hormone indicating that these can be used as biomarkers in the minipig even under partial body conditions. This PBI model aligns with other minipig models under BARDA's large animal consortium to test medical countermeasure efficacy against a less complex GI-specific ARS injury.

Purpose: Characterization of a novel partial-body irradiation (PBI) shielding strategy in nonhuman primates (NHP; rhesus macaques), aimed at protecting the oral cavity, with respect to various gastrointestinal acute radiation syndrome (GI-ARS) syndrome parameters as well as buccal ulceration development. Materials and methods: NHPs were irradiated using a Cobalt-60 gamma source, in a single uniform dose, ranging from 9-13 Gy and delivered at 0.60-0.80 Gy min -1 . Animals were either partially shielded via oral cavity shielding (PBI OS ) or underwent total-body irradiation (TBI). Results: Clinical manifestations of GI-ARS, and also radiation-induced hematology and clinical chemistry changes, following PBI OS were comparable to the PBI NHP GI-ARS model utilizing shielding of the distal pelvic limbs and were significantly milder than TBI at similar radiation doses. Nadir citrulline levels were comparable between PBI OS and TBI but signs of recovery appeared earlier in PBI OS -treated animals. The PBI OS model prevented oral mucositis, whereas the TBI model presented buccal ulcerations at all tested radiation dose levels. Conclusions: Taken together, these results suggest that the PBI OS model is a suitable alternative to traditional PBI. For GI-ARS investigations requiring orally administered medical countermeasures, PBI OS confers added value due to the prevention of oral mucositis over traditional PBI.

The group of radiation victims who had received radiation injures similar to those of Chernobyl accident victims was evaluated in terms of retrospective cytogenetic biodosimetry in the long term period of from 17 y up to 50 y after irradiation. Based on the existing results of the long-term cytogenetic examination of the victims injured after the Chernobyl accident, an original method was developed. This method of retrospective dose recovery was based on the use of a special computer program, the time elapsed after irradiation and the frequency of atypical chromosomes. Both patient groups were examined using conventional cytogenetic analysis. The new method of a retrospective biodosimetry was tested on the non-Chernobyl group. As a result the multiple regression equations which included frequency atypical chromosomes produced better results because the majority of the estimates of the retrospective doses fell into the 95%-prediction intervals for the reference group of the Chernobyl victims., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Results from archived (1986 and 1996) experiments were used to establish a baboon radiation-quality dose-response database with haematology biomarker time-course data following exposure to mixed-fields (i.e. neutron to gamma ratio: 5.5; dose: 0-8 Gy) and 60Co gamma-ray exposures (0-15 Gy). Time-course (i.e. 0-40 d) haematology changes for relevant blood-cell types for both mixed-field (neutron to gamma ratio = 5.5) and gamma ray alone were compared and models developed that showed significant differences using the maximum likehood ratio test. A consensus METREPOL-like haematology ARS (H-ARS) severity scoring system for baboons was established using these results. The data for mixed-field and the gamma only cohorts appeared similar, and so the cohorts were pooled into a single consensus H-ARS severity scoring system. These findings provide proof-of-concept for the use of a METREPOL H-ARS severity scoring system following mixed-field and gamma exposures., (Published by Oxford University Press 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Acute radiation syndrome (ARS) occurs as a result of partial- or whole-body, high-dose exposure to radiation in a very short period of time. Survival is dependent on the severity of the hematopoietic sub-syndrome of ARS. In this study, we investigated the mitigating effects of a lipid molecule, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), on the kinetics of hematopoietic cells, including absolute neutrophil count (ANC), red blood cells (RBCs) and platelet counts, in mice after gamma-ray total-body irradiation (TBI). Male and female BALB/c mice (11 weeks old) received a LD 70/30 dose of TBI. PLAG significantly and dose-dependently attenuated radiation-induced mortality ( P = 0.0041 for PLAG 50 mg/kg; P < 0.0001 for PLAG 250 mg/kg) and body weight loss ( P < 0.0001 for PLAG 50 and 250 mg/kg) in mice. Single-fraction TBI sharply reduced ANC within 3 days postirradiation and maintained the neutropenic state (ANC < 500 cells/µl) by approximately 26.8 ± 0.8 days. However, administration of PLAG attenuated radiation-induced severe neutropenia (ANC < 100 cells/µl) by effectively delaying the mean day of its onset and decreasing its duration. PLAG also significantly mitigated radiation-induced thrombocytopenia ( P < 0.0001 for PLAG 250 mg/kg) and anemia ( P = 0.0023 for PLAG 250 mg/kg) by increasing mean platelet and RBC counts, as well as hemoglobin levels, in peripheral blood. Moreover, delayed administration of PLAG, even at 48 and 72 h after gamma-ray irradiation, significantly attenuated radiation-induced mortality in a time-dependent manner. When compared to olive oil and palmitic linoleic hydroxyl (PLH), only PLAG effectively attenuated radiation-induced mortality, indicating that it has a distinctive mechanism of action. Based on these preclinical observations, we concluded that PLAG has high potential as a radiation countermeasure for the improvement of survivability and the treatment of hematopoietic injury in gamma-ray-induced ARS.

Prostaglandins and inhibitors of their synthesis (cyclooxygenase (COX) inhibitors, non-steroidal anti-inflammatory drugs) were shown to play a significant role in the regulation of hematopoiesis. Partly due to their hematopoiesis-modulating effects, both prostaglandins and COX inhibitors were reported to act positively in radiation-exposed mammalian organisms at various pre- and post-irradiation therapeutical settings. Experimental efforts were targeted at finding pharmacological procedures leading to optimization of therapeutical outcomes by minimizing undesirable side effects of the treatments. Progress in these efforts was obtained after discovery of selective inhibitors of inducible selective cyclooxygenase-2 (COX-2) inhibitors. Recent studies have been able to suggest the possibility to find combined therapeutical approaches utilizing joint administration of prostaglandins and inhibitors of their synthesis at optimized timing and dosing of the drugs which could be incorporated into the therapy of patients with acute radiation syndrome., Competing Interests: The authors declare no conflict of interest.

Radiological exposure scenarios involving large numbers of people require a rapid and high-throughput method to identify the unexposed, and those exposed to low- and high-dose radiation. Those with high-dose exposure, e.g., >2 Gy and depending on host characteristics, may develop severe hematological acute radiation syndrome (HARS), requiring hospitalization and treatment. Previously, we identified a set of genes that discriminated these clinically relevant groups. In the current work, we examined the utility of gene expression changes to classify 1,000 split blood samples into HARS severity scores of H0, H1 and H2-4, with the latter indicating likely hospitalization. In several previous radiation dose experiments, we determined that these HARS categories corresponded, respectively, to doses of 0 Gy (unexposed), 0.5 Gy and 5 Gy. The main purpose of this work was to assess the rapidity of blood sample processing using targeted next-generation sequencing (NGS). Peripheral blood samples from two healthy donors were X-ray irradiated in vitro and incubated at 37°C for 24 h. A total of 1,000 samples were evaluated by laboratory personnel blinded to the radiation dose. Changes in gene expression of FDXR , DDB2 , POU2AF1 and WNT3 were examined with qRT-PCR as positive controls. Targeted NGS (TREX) was used on all samples for the same four genes. Agreement using both methods was almost 78%. Using NGS, all 1,000 samples were processed within 30 h. Classification of the HARS severity categories corresponding to radiation dose had an overall agreement ranging between 90-97%. Depending on the end point, either a combination of all genes or FDXR alone (H0 HARS or unexposed) provided the best classification. Using this optimized automated methodology, we assessed 100× more samples approximately three times faster compared to standard cytogenetic studies. We showed that a small set of genes, rather than a complex constellation of genes, provided robust positive (97%) and negative (97%) predictive values for HARS categories and radiation doses of 0, 0.5 and 5 Gy. The findings of this study support the potential utility of early radiation-induced gene expression changes for high-throughput biodosimetry and rapid identification of irradiated persons in need of hospitalization.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid β oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Detonation of a radiological or nuclear device in a major urban area will result in heterogenous radiation exposure, given to the significant shielding of the exposed population due to surrounding structures. Development of biodosimetry assays for triage and treatment requires knowledge of the radiation dose-volume effect for the bone marrow (BM). This proof-of-concept study was designed to quantify BM damage in the non-human primate (NHP) after exposure to one of four radiation patterns likely to occur in a radiological/nuclear attack with varying levels of BM sparing. Rhesus macaques (11 males, 12 females; 5.30-8.50 kg) were randomized by weight to one of four arms: 1. bilateral total-body irradiation (TBI); 2. unilateral TBI; 3. bilateral upper half-body irradiation (UHBI); and 4. bilateral lower half-body irradiation (LHBI). The match-point for UHBI vs. LHBI was set at 1 cm above the iliac crest. Animals were exposed to 4 Gy of 6 MV X rays. Peripheral blood samples were drawn 14 days preirradiation and at days 1, 3, 5, 7 and 14 postirradiation. Dosimetric measurements after irradiation indicated that dose to the mid-depth xiphoid was within 6% of the prescribed dose. No high-grade fever, weight loss >10%, dehydration or respiratory distress was observed. Animals in the bilateral- and unilateral TBI arms presented with hematologic changes [e.g., absolute neutrophil count (ANC) <500/ll; platelets <50,000/ll] and clinical signs/symptoms (e.g., petechiae, ecchymosis) characteristic of the acute radiation syndrome. Animals in the bilateral UHBI arm presented with myelosuppression; however, none of the animals developed severe neutropenia or thrombocytopenia (ANC remained >500/µl; platelets >50,000/µl during 14-day follow-up). In contrast, animals in the LHBI arm (1 cm above the ilieac crest to the toes) were protected against BM toxicity with no marked changes in hematological parameters and only minor gross pathology [petechiae (1/5), splenomegaly (1/5) and mild pulmonary hemorrhage (1/5)]. The model performed as expected with respect to the dose-volume effect of total versus partial-BM irradiation, e.g., increased shielding resulted in reduced BM toxicity. Shielding of the major blood-forming organs (e.g., skull, ribs, sternum, thoracic and lumbar spine) spared animals from bone marrow toxicity. These data suggest that the biological consequences of the absorbed dose are dependent on the total volume and pattern of radiation exposure.

Sex is an important confounding variable in biomarker development that must be incorporated into biomarker discovery and validation. Additionally, understanding of sex as a biological variable is essential for effective translation of biomarkers in animal models to human populations. Toward these ends, we conducted high-throughput targeted metabolomics using liquid chromatography tandem mass spectrometry and multiplexed immunoassay analyses using a Luminex-based system in both male and female mice in a model of total-body irradiation at a radiation dose consistent with the hematopoietic acute radiation syndrome. Metabolomic and immunoassay analyses identified metabolites and cytokines that were significantly different in plasma from naive and irradiated C57BL/6 mice consisting of equal numbers of female and male mice at 3 d after 8.0 or 8.72 Gy, an approximate LD60-70/30 dose of total-body irradiation. An additional number of metabolites and cytokines had sex-specific responses after radiation. Analyses of sham-irradiated mice illustrate the presence of stress-related changes in several cytokines due simply to undergoing the irradiation procedure, absent actual radiation exposure. Basal differences in metabolite levels between female and male were also identified as well as time-dependent changes in cytokines up to 9 d postexposure. These studies provide data toward defining the influence of sex on plasma-based biomarker candidates in a well-defined mouse model of acute radiation syndrome.

The increasing potential for accidental radiation exposure from either nuclear accidents or terrorist activities has escalated the need for radiation countermeasure development. We previously showed that a 30-day course of high-dose captopril, an ACE inhibitor, initiated 1-4 h after total body irradiation (TBI), improved Hematopoietic Acute Radiation Syndrome (H-ARS) and increased survival in mice. However, because of the time likely required for the deployment of a stockpiled radiation countermeasure to a radiation mass casualty site, there is a need for therapies that can be administered 24-48 hours after initial exposure. Using C57BL/6 mice exposed to an LD 50-80/30 of 60 Co TBI (7.75-7.9 Gy, 0.615 Gy/min), we show that low-dose captopril administration, initiated as late as 48 h post-TBI and continued for 14 days, significantly enhanced overall survival similarly to high-dose, rapid administration. Captopril treatment did not affect radiation-induced cell cycle arrest genes or the immediate loss of hematopoietic precursors. Reduced mortality was associated with the recovery of bone marrow cellularity and mature blood cell recovery at 21-30 days post-irradiation. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA in the plasma. Finally, delayed captopril administration mitigated brain micro-hemorrhage at 21 days post-irradiation. These data indicate that low dose captopril administered as late as 48 h post-TBI for only two weeks improves survival that is associated with hematopoietic recovery and reduced inflammatory response. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure.

Threats of nuclear terrorism coupled with potential unintentional ionizing radiation exposures have necessitated the need for large-scale response efforts of such events, including high-throughput biodosimetry for medical triage. Global metabolomics utilizing mass spectrometry (MS) platforms has proven an ideal tool for generating large compound databases with relative quantification and structural information in a short amount of time. Determining metabolite panels for biodosimetry requires experimentation to evaluate the many factors associated with compound concentrations in biofluids after radiation exposures, including temporal changes, pre-existing conditions, dietary intake, partial- vs. total-body irradiation (TBI), among others. Here, we utilize a nonhuman primate (NHP) model and identify metabolites perturbed in serum after 7.2 Gy TBI without supportive care [LD 70/60 , hematologic (hematopoietic) acute radiation syndrome (HARS) level H3] at 24, 36, 48 and 96 h compared to preirradiation samples with an ultra-performance liquid chromatography quadrupole time-of-flight (UPLC-QTOF) MS platform. Additionally, we document changes in cytokine levels. Temporal changes observed in serum carnitine, acylcarnitines, amino acids, lipids, deaminated purines and increases in pro-inflammatory cytokines indicate clear metabolic dysfunction after radiation exposure. Multivariate data analysis shows distinct separation from preirradiation groups and receiver operator characteristic curve analysis indicates high specificity and sensitivity based on area under the curve at all time points after 7.2 Gy irradiation. Finally, a comparison to a 6.5 Gy (LD 50/60 , HARS level H2) cohort after 24 h postirradiation revealed distinctly increased separations from the 7.2 Gy cohort based on multivariate data models and higher compound fold changes. These results highlight the utility of MS platforms to differentiate time and absorbed dose after a potential radiation exposure that may aid in assigning specific medical interventions and contribute as additional biodosimetry tools.

The search for and development of radiation countermeasures to treat acute lethal radiation injury has been underway for the past six decades, resulting in the identification of multiple classes of radiation countermeasures. However, to date only granulocyte colony-stimulating factor (Neupogen) and PEGylated granulocyte colony-stimulating factor (Neulasta) have been approved by the U.S. Food and Drug Administration for the treatment of hematopoietic acute radiation syndrome. Gamma-tocotrienol has demonstrated radioprotective efficacy in murine and nonhuman primate models. Currently, this agent is under advanced development as a radioprotector, and the authors are trying to identify its efficacy biomarkers. In this study, global metabolomic changes were analyzed using ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry. The pilot study using 16 nonhuman primates (8 nonhuman primates each in gamma-tocotrienol- and vehicle-treated groups), with samples obtained from gamma-tocotrienol-treated and irradiated nonhuman primates, demonstrates several metabolites that are altered after irradiation, including compounds involved in fatty acid beta-oxidation, purine catabolism, and amino acid metabolism. The machine-learning algorithm, Random Forest, separated control, irradiated gamma-tocotrienol-treated, and irradiated vehicle-treated nonhuman primates at 12 h and 24 h as evident in a multidimensional scaling plot. Primary metabolites validated included carnitine/acylcarnitines, amino acids, creatine, and xanthine. Overall, gamma-tocotrienol administration reduced high fluctuations in serum metabolite levels, suggesting an overall beneficial effect on animals exposed to radiation. This initial assessment also highlights the utility of metabolomics in determining underlying physiological mechanisms responsible for the radioprotective efficacy of gamma-tocotrienol.

Use of plasma proteomic and hematological biomarkers represents a promising approach to provide useful diagnostic information for assessment of the severity of hematopoietic acute radiation syndrome. Eighteen baboons were evaluated in a radiation model that underwent total-body and partial-body irradiations at doses of Co gamma rays from 2.5 to 15 Gy at dose rates of 6.25 cGy min and 32 cGy min. Hematopoietic acute radiation syndrome severity levels determined by an analysis of blood count changes measured up to 60 d after irradiation were used to gauge overall hematopoietic acute radiation syndrome severity classifications. A panel of protein biomarkers was measured on plasma samples collected at 0 to 28 d after exposure using electrochemiluminescence-detection technology. The database was split into two distinct groups (i.e., "calibration," n = 11; "validation," n = 7). The calibration database was used in an initial stepwise regression multivariate model-fitting approach followed by down selection of biomarkers for identification of subpanels of hematopoietic acute radiation syndrome-responsive biomarkers for three time windows (i.e., 0-2 d, 2-7 d, 7-28 d). Model 1 (0-2 d) includes log C-reactive protein (p < 0.0001), log interleukin-13 (p < 0.0054), and procalcitonin (p < 0.0316) biomarkers; model 2 (2-7 d) includes log CD27 (p < 0.0001), log FMS-related tyrosine kinase 3 ligand (p < 0.0001), log serum amyloid A (p < 0.0007), and log interleukin-6 (p < 0.0002); and model 3 (7-28 d) includes log CD27 (p < 0.0012), log serum amyloid A (p < 0.0002), log erythropoietin (p < 0.0001), and log CD177 (p < 0.0001). The predicted risk of radiation injury categorization values, representing the hematopoietic acute radiation syndrome severity outcome for the three models, produced least squares multiple regression fit confidences of R = 0.73, 0.82, and 0.75, respectively. The resultant algorithms support the proof of concept that plasma proteomic biomarkers can supplement clinical signs and symptoms to assess hematopoietic acute radiation syndrome risk severity.

There is still a need for better protection against or mitigation of the effects of ionizing radiation following conventional radiotherapy or accidental exposure. The objective of our current study was to investigate the possible roles of matrix metalloproteinase inhibitor, ilomastat, in the protection of mice from total body radiation (TBI), and the underlying protective mechanisms. Ilomastat treatment increased the survival of mice after TBI. Ilomastat pretreatment promoted recovery of hematological and immunological cells in mice after 6 Gy γ-ray TBI. Our findings suggest the potential of ilomastat to protect against or mitigate the effects of radiation., (Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

In presented article, by means of a comparative analysis of the relationship between the dose-dependent alterations in the organism's redox status, measured by the innovative method developed by us and the standard methods used for assessing catalase and superoxide dismutase activity, and an end radiobiological effect, was attempted the preliminary assessment of the possibility to apply the parameter of blood plasma total antioxidant activity (TAA) as marker of dose and effect of radiation exposure. The experiments were carried out on white mice randomly divided into groups of irradiated and sham irradiated animals. The mice were exposed to a whole body gamma irradiation by source Cesium-137 (137Cs) at doses of 5 and 7 Gr, a dose rate of 1.1 Gr / min. After 5 days of beginning of observation in animals' blood measurements of the activity of antioxidant enzymes (superoxide dismutase - SOD and catalase - CAT) and total antioxidant activity by spectrophotometric method were evaluated. Parallel monitoring of animal survival was conducted. At the given stage of the study, applicability of OAA as a marker of dose-dependent alterations in antioxidant status was assessed by the criteria of sensitivity and linearity, and as a marker of the effect the strength of the relation between the antioxidant status indicators and the final radiobiological effect, measured by animal life span in post- radiation period (time-effect) For comparative analysis of the effects of radiation on the levels of antioxidant status indicators (SOD, catalase, OAA), ANOVA methods were used, the nature of the causal relationship between levels of antioxidant status and the life span of laboratory animals was analyzed on the basis of the Cox proportional intensity model with time covariates, preliminary processing of data, basic calculations and visualization of the results were carried out using a mathematical package that " STATISTIC 12". The received results testify to the significantly high sensitivity of the total redox-status indicator (OAA) to the dose of irradiation, which makes it possible with full justification to consider it as a promising candidate of the biological exposure dose marker. In addition, the indicator of the total antioxidant status (OAA) of the body allows predicting the dose dependence of the survival of laboratory mice more accurately than the indices of the activity of individual antioxidant enzymes (catalase and SOD), which makes it possible to consider it as a promising candidate for the biomarker of the dose and the effect of radiation exposure.

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Exposure to ionizing radiation alone or combined with traumatic tissue injury is a crucial life-threatening factor in nuclear and radiological incidents. Radiation injuries occur at the molecular, cellular, tissue and systemic levels; their mechanisms, however, remain largely unclear. Exposure to radiation combined with skin wounding, bacterial infection or burns results in greater mortality than radiation exposure alone in dogs, pigs, rats, guinea pigs and mice. In the current study we observed that B6D2F1/J female mice exposed to 60 Co gamma-photon radiation followed by 15% total-body-surface-area skin wounds experienced an increment of 25% higher mortality over a 30-day observation period compared to those subjected to radiation alone. Radiation exposure delayed wound healing by approximately 14 days. On day 30 post-injury, bone marrow and ileum in animals from both groups (radiation alone or combined injury) still displayed low cellularity and structural damage. White blood cell counts, e.g., neutrophils, lymphocytes, monocytes, eosinophils, basophils and platelets, still remained very low in surviving irradiated alone animals, whereas only the lymphocyte count was low in surviving combined injury animals. Likewise, in surviving animals from radiation alone and combined injury groups, the RBCs, hemoglobin, hematocrit and platelets remained low. We observed, that animals treated with both pegylated G-CSF (a cytokine for neutrophil maturation and mobilization) and Alxn4100TPO (a thrombopoietin receptor agonist) at 4 h postirradiation, a 95% survival (vehicle: 60%) over the 30-day period, along with mitigated body-weight loss and significantly reduced acute radiation syndrome. In animals that received combined treatment of radiation and injury that received pegylated G-CSF and Alxn4100TPO, survival was increased from 35% to 55%, but did not accelerate wound healing. Hematopoiesis and ileum showed significant improvement in animals from both groups (irradiation alone and combined injury) when treated with pegylated G-CSF and Alxn4100TPO. Treatment with pegylated G-CSF alone increased survival after irradiation alone and combined injury by 33% and 15%, respectively, and further delayed wound healing, but increased WBC, RBC and platelet counts after irradiation alone, and only RBCs and platelets after combined injury. Treatment with Alxn4100TPO alone increased survival after both irradiation alone and combined injury by 4 and 23%, respectively, and delayed wound healing after combined injury, but increased RBCs, hemoglobin concentrations, hematocrit values and platelets after irradiation alone and only platelets after combined injury. Taken together, the results suggest that combined treatment with pegylated G-CSF and Alxn4100TPO is effective for mitigating effects of both radiation alone and in combination with injury.

Rapid clinical triage of radiation injury patients is essential for determining appropriate diagnostic and therapeutic interventions. We examined the utility of blood cell counts (BCCs) in the first three days postirradiation to predict clinical outcome, specifically for hematologic acute radiation syndrome (HARS). We analyzed BCC test samples from radiation accident victims (n = 135) along with their clinical outcome HARS severity scores (H1-4) using the System for Evaluation and Archiving of Radiation Accidents based on Case Histories (SEARCH) database. Data from nonirradiated individuals (H0, n = 132) were collected from an outpatient facility. We created binary categories for severity scores, i.e., 1 (H0 vs. H1-4), 2 (H0-1 vs. H2-4) and 3 (H0-2 vs. H3-4), to assess the discrimination ability of BCCs using unconditional logistic regression analysis. The test sample contained 454 BCCs from 267 individuals. We validated the discrimination ability on a second independent group comprised of 275 BCCs from 252 individuals originating from SEARCH (HARS 1-4), an outpatient facility (H0) and hospitals (e.g., leukemia patients, H4). Individuals with a score of H0 were easily separated from exposed individuals based on developing lymphopenia and granulocytosis. The separation of H0 and H1-4 became more prominent with increasing hematologic severity scores and time. On day 1, lymphocyte counts were most predictive for discriminating binary categories, followed by granulocytes and thrombocytes. For days 2 and 3, an almost complete separation was achieved when BCCs from different days were combined, supporting the measurement of sequential BCC. We found an almost complete discrimination of H0 vs. irradiated individuals during model validation (negative predictive value, NPV > 94%) for all three days, while the correct prediction of exposed individuals increased from day 1 (positive predictive value, PPV 78-89%) to day 3 (PPV > 90%). The models were unable to provide predictions for 10.9% of the test samples, because the PPVs or NPVs did not reach a 95% likelihood defined as the lower limit for a prediction. We developed a prediction model spreadsheet to provide early and prompt diagnostic predictions and therapeutic recommendations including identification of the worried well, requirement of hospitalization or development of severe hematopoietic syndrome. These results improve the provisional classification of HARS. For the final diagnosis, further procedures (sequential diagnosis, retrospective dosimetry, clinical follow-up, etc.) must be taken into account. Clinical outcome of radiation injury patients can be rapidly predicted within the first three days postirradiation using peripheral BCC.

Multiple hematological biomarkers (i.e. complete blood counts and serum chemistry parameters) were used in a multivariate linear-regression fit to create predictive algorithms for estimating the severity of hematopoietic acute radiation syndrome (H-ARS) using two different species (i.e. Göttingen Minipig and non-human primate (NHP) (Macacca mulatta)). Biomarker data were analyzed prior to irradiation and between 1-60 days (minipig) and 1-30 days (NHP) after irradiation exposures of 1.6-3.5 Gy (minipig) and 6.5 Gy (NHP) 60 Co gamma ray doses at 0.5-0.6 Gy min -1 and 0.4 Gy min -1 , respectively. Fitted radiation risk and injury categorization (RRIC) values and RRIC prediction percent accuracies were compared between the two models. Both models estimated H-ARS severity with over 80% overall predictive power and with receiver operating characteristic curve area values of 0.884 and 0.825. These results based on two animal radiation models support the concept for the use of a hematopoietic-based algorithm for predicting the risk of H-ARS in humans., (Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2-4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2-3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI)., Methods and Materials: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison., Results: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies., Conclusions: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted proteins were identified in urinary and serum exosomes. Together, these data showed the feasibility of defining biomarkers that could elucidate tissue-associated and systemic response caused by high-dose ionizing radiation. This is the first report using an exosome proteomics approach to identify radiation signatures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In radiation accidents, determining the radiation dose the victim received is a key step for medical decision making and patient prognosis. To reconstruct and evaluate the absorbed dose, researchers have developed many physical devices and biological techniques during the last decades. However, using the physical parameter "absorbed dose" alone is not sufficient to predict the clinical development of the various organs injured in an individual patient. In operational situations for radiation accidents, medical responders need more urgently to classify the severity of the radiation injury based on the signs and symptoms of the patient. In this work, the author uses a unified hematopoietic model to describe dose-dependent dynamics of granulocytes, lymphocytes, and platelets, and the corresponding clinical grading of hematopoietic acute radiation syndrome. This approach not only visualizes the time course of the patient's probable outcome in the form of graphs but also indirectly gives information of the remaining stem and progenitor cells, which are responsible for the autologous recovery of the hematopoietic system. Because critical information on the patient's clinical evolution can be provided within a short time after exposure and only peripheral cell counts are required for the simulation, these modeling tools will be useful to assess radiation exposure and injury in human-involved radiation accident/incident scenarios.

Decisions on whether to start a therapeutic intervention for management of the Acute Radiation Syndrome (ARS) should be made early after exposure, and it should be based on readily available clinical signs and laboratory parameters. Here, the authors use the minipig to assess if early prediction of the later developing clinical outcome and necessity of therapeutic interventions can be determined within the first 3 d after exposure and whether it is comparable to human data. Retrospective analysis of data accumulated in the period 2009-2012 was used. Male Göttingen minipigs (age 4-5 mo, weight 9-10 kg) were irradiated (or sham-irradiated) bilaterally with gamma-photons (Co, 0.5-0.6 Gy min) in the dose range of 1.6-12 Gy. Complete blood counts, serum chemistry, and clinical symptoms were collected up to 60 d after irradiation in untreated minipigs. Changes in these early parameters (up to 3 d after exposure) were correlated with later occurrence (10-60 d after irradiation) of (1) hematological severity scores, (2) severe thrombocytopenia, (3) severe neutropenia, as well as need for (4) therapeutic intervention, (5) administration of cytokines/antibiotics, or (6) thrombocyte transfusions. Binary endpoints were analyzed using logistic regression analysis and calculating receiver operating characteristic (ROC) curves. Most predictive were decreased lymphocyte counts and increases in body temperature at 3 h after irradiation. These data corroborate earlier findings performed on human radiation victims suffering from severe hematological syndrome and provide further evidence for the suitability of the minipig model as a potential alternative non-rodent animal model.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13-17 Gy and 8-16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72-13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 μM and 63.1 ± 2.1 μM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 μM, very similar to that of humans (∼40 μM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

The search for treatments to counter potentially lethal radiation-induced injury over the past several decades has led to the development of multiple classes of radiation countermeasures. However, to date only granulocyte colony-stimulating factor (G-CSF; filgrastim, Neupogen)and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved by the United States Food and Drug Administration (FDA) for the treatment of hematopoietic acute radiation syndrome (ARS). Gamma-tocotrienol (GT3) has demonstrated strong radioprotective efficacy in the mouse model, indicating the need for further evaluation in a large animal model. In this study, we evaluated GT3 pharmacokinetics (PK) and efficacy at different doses of cobalt-60 gamma radiation (0.6 Gy/min) using the nonhuman primate (NHP) model. The PK results demonstrated increased area under the curve with increasing drug dose and half-life of GT3. GT3 treatment resulted in reduced group mean neutropenia by 3-5 days and thrombocytopenia by 1-5 days. At 5.8 and 6.5 Gy total-body irradiation, GT3 treatment completely prevented thrombocytopenia. The capability of GT3 to reduce severity and duration of neutropenia and thrombocytopenia was dose dependent; 75 mg/kg treatment was more effective than 37.5 mg/kg treatment after a 5.8 Gy dose. However, the higher GT3 dose (75 mg/kg) was associated with higher frequency of adverse skin effects (small abscess) at the injection site. GT3 treatment of irradiated NHPs caused no significant difference in animal survival at 60 days postirradiation, however, low mortality was observed in irradiated, vehicle-treated groups as well. The data from this pilot study further elucidate the role and pharmacokinetics of GT3 in hematopoietic recovery after irradiation in a NHP model, and demonstrate the potential of GT3 as a promising radioprotector.

Acute radiation syndrome results from radiation exposure, such as in accidental nuclear disasters. Safe and effective radioprotectants, mitigators, and treatment drugs must be developed as medical countermeasures against radiation exposure. Here, the authors evaluated CCM-Ami, a novel polyethylene glycol micelle encapsulated with amifostine, for its radioprotective properties after total-body irradiation from a 60Co source. Male C57BL/6 mice (6-8 wk old) were intravenously injected with 45 mg kg(-1) of CCM-Ami 90 min before exposure to 7.2 and 8.5 Gy irradiation at a dose rate of 0.04 Gy min(-1). Both survival benefit and hematopoietic protection were observed after prophylactic CCM-Ami administration when compared with the effects measured in excipient control and amifostine groups. Pharmacokinetic results showed that after the intravenous injection, the plasma concentration of WR-1065, the active form of amifostine, was higher in CCM-Ami-treated mice than in amifostine-treated mice. These findings suggest that CCM-Ami-mediated hematopoietic protection plays a key role in enhancing survival of mice exposed to radiation toxicity and thus indicate that CCM-Ami is a radioprotectant that can be used safely and effectively in nuclear disasters.

The U.S. Food and Drug Administration (FDA) recently approved Neupogen(®) (filgrastim) for the treatment of patients with radiation-induced myelosuppression following a radiological/nuclear incident. It is the first medical countermeasure currently approved by the FDA for this indication under the criteria of the FDA "animal rule". This article summarizes the consequences of high-dose radiation exposure, a description of the hematopoietic acute radiation syndrome (H-ARS), the use of hematopoietic growth factors in radiation accident victims and current available treatments for H-ARS with an emphasis on the use of Neupogen in this scenario., (Copyright 2015 Prous Science, S.A.U. or its licensors. All rights reserved.)

Biologically motivated mathematical models of the dynamics of the major hematopoietic lineages (the thrombopoietic, erythropoietic, granulopoietic, and lymphopoietic systems) in mammals (rodents) exposed to nonuniform acute irradiation are developed and thoroughly investigated. These models are based on earlier developed mathematical models, which are capable of predicting the dynamics of these systems in mammals (rodents) exposed to uniform acute/chronic irradiation. The developed models are the systems of nonlinear ordinary differential equations whose variables and constant parameters have clear biological meaning. It is found that these models are capable of reproducing a lesser depletion of the major hematopoietic lineages and faster recovery in rodents (rats, mice) after nonuniform acute irradiation than those after uniform acute irradiation at equal whole-body doses of such exposures. The nature of this phenomenon is elucidated in the framework of the models. Thorough comparative analysis of effects of nonuniform (partial) and uniform acute irradiation on the major hematopoietic lineages is performed. It is revealed that the lymphopoietic system is the most susceptible major hematopoietic lineage both to uniform and nonuniform (partial) acute irradiation. It is argued that the first-day level of the concentration of functional blood cells in this system (blood lymphocytes) after nonuniform (partial) acute irradiation can serve for early assessment of the risk of acute radiation syndrome. It is also shown that the modeling results on the first-day levels of the blood lymphocyte concentration after various partial acute exposures are in a very good agreement with the relevant experimental data. This agreement testifies to the applicability of the developed model of the lymphopoietic system to be used in the prognostic aims; in particular, for predicting the development of lymphocytopenia after partial acute irradiation. The modeling results imply that the prognostic use of a whole-body dose of nonuniform acute irradiation is not effective. The obtained results testify to the efficiency of the proposed dynamic modeling approach in the study of the effects of nonuniform acute irradiation on the major hematopoietic lineages and the validity of using developed models of the major hematopoietic lineages in the prediction of the effects of nonuniform acute irradiation on these systems in rodents and, after appropriate modification of the models, in humans.

Accidental exposure to uranium is a matter of concern, as U(VI) is nephrotoxic in both human and animal models, and its toxicity is associated to chemical toxicity instead of radioactivity. We synthesized different PAMAM G4 and G5 derivatives in order to prove their interaction with uranium and their effect on the viability of red blood cells in vitro. Furthermore, we prove the effectiveness of the selected dendrimers in an animal model of acute uranium intoxication. The dendrimer PAMAM G4-Lys-Fmoc-Cbz demonstrated the ability to chelate the uranyl ion in vivo, improving the biochemical and histopathologic features caused by acute intoxication with uranium.

Purpose: The effects of radiation exposure are long-lasting. Long-term monitoring is imperative to diagnose late effects and improve our far-sightedness about possible events in the future. A radiation accident occurred in Istanbul in 1998 that resulted in mild to moderate acute radiation syndrome (ARS). In this study we aimed to investigate the changes in hematological parameters at the long-term follow-up of ARS patients., Methods: Ten adults were hospitalized after exposure to a 60Co source. Seven were diagnosed as having ARS and had severe and symptomatic pancytopenia. All of the exposed people recovered following intensive treatment. Treatment was supportive with transfusion, granulocyte-colony stimulating factor, and anti- infective management covering antifungal agents. Patients were closely monitored. Nine years after the accident, the initial and follow-up complete blood count examinations and peripheral blood smears (PBS) were comparatively evaluated by an experienced hematologist. The hematological laboratory values of the patients on admission, after treatment, and nine years after the accident were documented and compared., Results: Biodosimetric analysis revealed that whole-body doses ranged from 1-1.9 Gy. All subjects have shown complete recovery of the hematological laboratory values after treatment. All but one of the subjects showed complete blood cell recovery. The improvement of the blood cell count of the excepted patient stalled at a mildly reduced level and his bone marrow was still hypocellular nine years after the accident; however, no malignant changes were detected. Values at admission were significantly different compared with post treatment and present values of all patients. Post treatment and follow-up values were similar. One of the patients died of lung cancer. None of the patients developed hematological malignancy., Conclusions: In this study, the recovery from ARS was complete after treatment. The small population, short follow-up period, and the relatively small doses resulted in no long-term adverse effects, as would be predicted.

The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.

The effects of fibroblast growth factors and their potential as broad-spectrum agents to treat and mitigate radiation injury have been studied extensively over the past two decades. This report shows that a peptide mimetic of basic fibroblast growth factor (FGF-P) protects and mitigates against acute radiation syndromes. FGF-P attenuates both sepsis and bleeding in a radiation-induced bone marrow syndrome model and reduces the severity of gastrointestinal and cutaneous syndromes; it should also mitigate combined injuries. FGF-2 and FGF-P induce little or no deleterious inflammation or vascular leakage, which distinguishes them from most other growth factors, angiogenic factors, and cytokines. Although recombinant FGFs have proven safe in several ongoing clinical trials, they are expensive to synthesize, can only be produced in limited quantity, and have limited shelf life. FGF-P mimics the advantageous features of FGF-2 without these disadvantages. This paper shows that FGF-P not only has the potential to be a potent yet safe broad-spectrum medical countermeasure that mitigates acute radiotoxicity but also holds promise for thermal burns, ischemic wound healing, tissue engineering, and stem-cell regeneration.

In an effort to expand the worldwide pool of available medical countermeasures (MCM) against radiation, the PEGylated G-CSF (PEG-G-CSF) molecules Neulasta and Maxy-G34, a novel PEG-G-CSF designed for increased half-life and enhanced activity compared to Neulasta, were examined in a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS), along with the lead MCM for licensure and stockpiling, G-CSF. Both PEG-G-CSFs were shown to retain significant survival efficacy when administered as a single dose 24 h post-exposure, compared to the 16 daily doses of G-CSF required for survival efficacy. Furthermore, 0.1 mg kg of either PEG-G-CSF affected survival of lethally-irradiated mice that was similar to a 10-fold higher dose. The one dose/low dose administration schedules are attractive attributes of radiation MCM given the logistical challenges of medical care in a mass casualty event. Maxy-G34-treated mice that survived H-ARS were examined for residual bone marrow damage (RBMD) up to 9 mo post-exposure. Despite differences in Sca-1 expression and cell cycle position in some hematopoietic progenitor phenotypes, Maxy-G34-treated mice exhibited the same degree of hematopoietic stem cell (HSC) insufficiency as vehicle-treated H-ARS survivors in competitive transplantation assays of 150 purified Sca-1+cKit+lin-CD150+cells. These data suggest that Maxy-G34, at the dose, schedule, and time frame examined, did not mitigate RBMD but significantly increased survival from H-ARS at one-tenth the dose previously tested, providing strong support for advanced development of Maxy-G34, as well as Neulasta, as MCM against radiation.

Multivariate radiation injury estimation algorithms were formulated for estimating severe hematopoietic acute radiation syndrome (H-ARS) injury (i.e., response category three or RC3) in a rhesus monkey total-body irradiation (TBI) model. Classical CBC and serum chemistry blood parameters were examined prior to irradiation (d 0) and on d 7, 10, 14, 21, and 25 after irradiation involving 24 nonhuman primates (NHP) (Macaca mulatta) given 6.5-Gy (60)Co Υ-rays (0.4 Gy min(-1)) TBI. A correlation matrix was formulated with the RC3 severity level designated as the "dependent variable" and independent variables down selected based on their radioresponsiveness and relatively low multicollinearity using stepwise-linear regression analyses. Final candidate independent variables included CBC counts (absolute number of neutrophils, lymphocytes, and platelets) in formulating the "CBC" RC3 estimation algorithm. Additionally, the formulation of a diagnostic CBC and serum chemistry "CBC-SCHEM" RC3 algorithm expanded upon the CBC algorithm model with the addition of hematocrit and the serum enzyme levels of aspartate aminotransferase, creatine kinase, and lactate dehydrogenase. Both algorithms estimated RC3 with over 90% predictive power. Only the CBC-SCHEM RC3 algorithm, however, met the critical three assumptions of linear least squares demonstrating slightly greater precision for radiation injury estimation, but with significantly decreased prediction error indicating increased statistical robustness.

Purpose: We are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the acute radiation syndrome (ARS) to enhance the discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematologic parameters and dynamics of cell loss/recovery after irradiation provide a convenient means to compare the efficacy of drugs known to affect bone marrow cellularity and hematopoiesis., Methods and Materials: Male Gottingen minipigs, 4 to 5 months old and weighing 9 to 11 kg, were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen, 10 μg/kg/day for 17 days), at the estimated LD70/30 total-body γ-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation., Results: The results indicated that granulocyte colony stimulating factor (G-CSF) enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, the administration of G-CSF resulted in maturation of monocytes/macrophages., Conclusions: These results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and they suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing the numbers of circulating granulocytes., (Published by Elsevier Inc.)

Acute radiation sickness (ARS) is expected to occur in astronauts during large solar particle events (SPEs). One parameter associated with ARS is the hematopoietic syndrome, which can result from decreased numbers of circulating blood cells in those exposed to radiation. The peripheral blood cells are critical for an adequate immune response, and low blood cell counts can result in an increased susceptibility to infection. In this study, Yucatan minipigs were exposed to proton radiation within a range of skin dose levels expected for an SPE (estimated from previous SPEs). The proton-radiation exposure resulted in significant decreases in total white blood cell count (WBC) within 1 day of exposure, 60% below baseline control value or preirradiation values. At the lowest level of the blood cell counts, lymphocytes, neutrophils, monocytes and eosinophils were decreased up to 89.5%, 60.4%, 73.2% and 75.5%, respectively, from the preirradiation values. Monocytes and lymphocytes were decreased by an average of 70% (compared to preirradiation values) as early as 4 h after radiation exposure. Skin doses greater than 5 Gy resulted in decreased blood cell counts up to 90 days after exposure. The results reported here are similar to studies of ARS using the nonhuman primate model, supporting the use of the Yucatan minipig as an alternative. In addition, the high prevalence of hematologic abnormalities resulting from exposure to acute, whole-body SPE-like proton radiation warrants the development of appropriate countermeasures to prevent or treat ARS occurring in astronauts during space travel., (© 2013 by Radiation Research Society)

Background/aim: To evaluate the impact of an antibiotic, minocycline, on several immune parameters in response to radiation in a mouse model., Materials and Methods: C57BL/6 mice were treated with minocycline (i.p.) for 5 days, beginning immediately before radiation with 1-3 Gy (60)Co γ-rays. Spleen and blood were collected on day 4 post-irradiation. Cell populations were determined in the blood and spleen. Splenocytes were activated with anti-CD3 antibody for 48 h and cytokines were quantified., Results: Minocycline increased the counts and/or percentages of splenic macrophages, granulocytes, natural killer, T- and CD8(+) T-cells (p<0.05 versus radiation alone). Minocycline significantly increased the expression of interleukin-1α and β, which are radioprotective, as well as the ones of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, which accelerate neutrophil recovery (p<0.05 versus radiation alone), while suppressing cytokines that could prevent hematopoiesis, e.g. macrophage inflammatory protein-1α, tumor necrosis factor-α and interferon-γ., Conclusion: These data indicate that minocycline should be further tested for use in restoration of the hematopoietic system after radiation exposure.

Background: Alexander Litvinenko died on November 23, 2006, from acute radiation sickness syndrome caused by ingestion of polonium-210 (²¹⁰Po)., Objective: The objective was to assess the prevalence of and risk factors for internal contamination with ²¹⁰Po in healthcare workers (HCWs) caring for the contaminated patient., Setting: Hospital., Participants: HCWs who had direct contact with the patient., Methods: We interviewed 43 HCWs and enquired about their activities and use of personal protective equipment (PPE). Internal contamination was defined as urinary ²¹⁰Po excretion above 20 mBq within 24 hours. We obtained risk ratios (RRs) for internal contamination using Poisson regression., Results: Thirty-seven HCWs (86%) responded, and 8 (22%) showed evidence of internal contamination, all at very low levels that were unlikely to cause adverse health outcomes. Daily care of the patient (washing and toileting the patient) was the main risk factor (RR, 3.6 [95% confidence interval (CI), 1.1-11.6]). In contrast, planned invasive procedures were not associated with a higher risk. There was some evidence of a higher risk associated with handling blood samples (RR, 3.5 [95% CI, 0.8-15.6]) and changing urine bags and/or collecting urine samples (RR, 2.7 [95% CI, 0.8-9.5]). There was also some evidence that those who reported not always using standard PPE were at higher risk than were others (RR, 2.5 [95% CI, 0.8-8.1])., Conclusions: The sensitive quantitative measurement enabled us to identify factors associated with contamination, which by analogy to other conditions with similar transmission mechanisms may help improve protection and preparedness in staff dealing with an ill patient who experiences an unknown illness.

Immunological parameters in different periods of acute radiation syndrome (ARS) of experimental animals and Chernobyl reactor accident-injured patients have been studied. 148 patients and experimental animals (123 dogs and 198 monkeys) were observed after radiation exposure of different levels (from a sub-lethal dose to the minimal absolute lethal dose). We have found the increase in the C-reactive protein, fluctuation of normal antibody titers and the complement in blood serum, as well as the growing number of skin microbes after exposures to lethal doses. Experimental results match clinical data in terms of ARS progress phases but differ from the latter in terms of the time of clinical manifestations. The highest rate of clinical manifestations is observed on the 7-14 days for experimental animals (rats, dogs and monkeys) and on the 20-30 days for patients after radiation exposure. Regenerative processes in animals run faster than those in humans.

Additional large animal models for the acute radiation syndrome (ARS) would facilitate countermeasure development. We demonstrate here that Gottingen minipigs develop hematopoietic ARS symptoms similar to those observed in canines, non-human primates (NHPs) and humans. Dosimetry for whole-body γ irradiation (0.6 Gy/min) was performed using electronic paramagnetic resonance (EPR) with alanine; National Institute of Standards and Technology (NIST)-calibrated alanine pellets and water-filled Plexiglas phantoms were used. After irradiations of 1.6-2.0 Gy, blood pancytopenia was observed for several weeks, accompanied by the characteristic ARS stages: prodromal symptoms, latent period, illness and recovery or morbidity. Morbidity occurred between days 14 and 27, with a preliminary LD(50/30) estimate between 1.7 and 1.9 Gy. The criterion of whether platelet counts were <200 × 10(3)/µl 7 days postirradiation predicted whether animals would survive in 18 out of 20 cases. The degree of granulocytosis 3 h postirradiation was inversely correlated with survival. Animals euthanized based on preset morbidity criteria displayed signs of multi-organ dysfunction, including widespread internal hemorrhage and alterations in organ function reflected in blood chemistry. Circulating C-reactive protein (CRP), a marker for inflammation, became elevated within hours after irradiation, subsided after several days, and increased again after 14 days. The results support further development of the Gottingen minipig as a model for ARS.