Comparison of Proteomic Expression Profiles after Radiation Exposure across Four Different Species.

There is a need to identify biomarkers of radiation exposure for use in development of circulating biodosimeters for radiation exposure and for clinical use as markers of radiation injury. Most research approaches for biomarker discovery rely on a single animal model. The current study sought to take advantage of a novel aptamer-based proteomic assay which has been validated for use in many species to characterize changes to the blood proteome after total-body irradiation (TBI) across four different mammalian species including humans. Plasma was collected from C57BL6 mice, Sinclair minipigs, and Rhesus non-human primates (NHPs) receiving a single dose of TBI at a range of 3.3 Gy to 4.22 Gy at 24 h postirradiation. NHP and minipig models were irradiated using a 60Co source at a dose rate of 0.6 Gy/min, the C57BL6 mouse model using an X-ray source at a dose rate of 2.28 Gy/min and clinical samples from a photon source at 10 cGy/min. Plasma was collected from human patients receiving a single dose of 2 Gy TBI collected 6 h postirradiation. Plasma was screened using the aptamer-based SomaLogic SomaScan® proteomic assay technology to evaluate changes in the expression of 1,310 protein analytes. Confirmatory analysis of protein expression of biomarker HIST1H1C, was completed using plasma from C57BL6 mice receiving a 2, 3.5 or 8 Gy TBI collected at days 1, 3, and 7 postirradiation by singleplex ELISA. Summary of key pathways with altered expression after radiation exposure across all four mammalian species was determined using Ingenuity Pathway Analysis (IPA). Detectable values were obtained for all 1,310 proteins in all samples included in the SomaScan assay. A subset panel of protein biomarkers which demonstrated significant (p < 0.05) changes in expression of at least 1.3-fold after radiation exposure were characterized for each species. IPA of significantly altered proteins yielded a variety of top disease and biofunction pathways across species with the organismal injury and abnormalities pathway held in common for all four species. The HIST1H1C protein was shown to be radiation responsive within the human, NHP and murine species within the SomaScan dataset and was shown to demonstrate dose dependent upregulation at 2, 3.5 and 8 Gy at 24 h postirradiation in a separate murine cohort by ELISA. The SomaScan proteomics platform is a useful screening tool to evaluate changes in biomarker expression across multiple mammalian species. In our study, we were able to identify a novel biomarker of radiation exposure, HIST1H1C, and characterize panels of radiation responsive proteins and functional proteomic pathways altered by radiation exposure across murine, minipig, NHP and human species. Our study demonstrates the efficacy of using a multispecies approach for biomarker discovery.

Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs.

Gottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1-7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

Investigating the Multifaceted Nature of Radiation-Induced Coagulopathies in a Göttingen Minipig Model of Hematopoietic Acute Radiation Syndrome.

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.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Impairment of IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity and Mortality.

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling pathway is associated with radiosensitivity, we investigated the link between systemic or tissue-specific IGF-1 signaling and radiosensitivity. Adult male C3H/HeN and CD2F1 mice were irradiated using gamma photons at Lethal Dose-70/30 (LD70/30), 7.8 and 9.35 Gy doses, respectively. Those mice that survived up to 30 days post-irradiation, were termed the survivors. Mice that were euthanized prior to 30 days post-irradiation due to deteriorated health were termed decedents. The analysis of non-irradiated and irradiated survivor and decedent mice showed that inter-strain radiosensitivity and post-irradiation survival outcomes are associated with activation status of tissue and systemic IGF-1 signaling, nuclear factor erythroid 2-related factor 2 (Nrf2) activation, and the gene expression profile of cardiac mitochondrial energy metabolism pathways. Our findings link radiosensitivity with dysregulation of IGF-1 signaling, and highlight the role of antioxidant gene response and mitochondrial function in radiation sensitivity.

Gene Expression Profiles from Heart, Lung and Liver Samples of Total-Body-Irradiated Minipigs: Implications for Predicting Radiation-Induced Tissue Toxicity.

In the event of a major accidental or intentional radiation exposure incident, the affected population could suffer from total- or partial-body exposures to ionizing radiation with acute exposure to organs that would produce life-threatening injury. Therefore, it is necessary to identify markers capable of predicting organ-specific damage so that appropriate directed or encompassing therapies can be applied. In the current work, gene expression changes in response to total-body irradiation (TBI) were identified in heart, lungs and liver tissue of Göttingen minipigs. Animals received 1.7, 1.9, 2.1 or 2.3 Gy TBI and were followed for 45 days. Organ samples were collected at the end of day 45 or sooner if the animal displayed morbidity necessitating euthanasia. Our findings indicate that different organs respond to TBI in a very specific and distinct manner. We also found that the liver was the most affected organ in terms of gene expression changes, and that lipid metabolic pathways were the most deregulated in the liver samples of non-survivors (survival time <45 days). We identified organ-specific gene expression signatures that accurately differentiated non-survivors from survivors and control animals, irrespective of dose and time postirradiation. At what point did these radiation-induced injury markers manifest and how this information could be used for applying intervention therapies are under investigation.

Dysregulated Cardiac IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity.

Acute exposure to ionizing radiation leads to Hematopoietic Acute Radiation Syndrome (H-ARS). To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, adult males of two strains of minipig, one with higher radiosensitivity, the Gottingen minipig (GMP), and another strain with comparatively lower radiosensitivity, the Sinclair minipig (SMP), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling is associated with radiation sensitivity and regulation of cardiovascular homeostasis, we investigated the link between dysregulation of cardiac IGF-1 signaling and radiosensitivity. The adult male GMP; n = 48, and SMP; n = 24, were irradiated using gamma photons at 1.7-2.3 Gy doses. The animals that survived to day 45 after irradiation were euthanized and termed the survivors. Those animals that were euthanized prior to day 45 post-irradiation due to severe illness or health deterioration were termed the decedents. Cardiac tissue analysis of unirradiated and irradiated animals showed that inter-strain radiosensitivity and survival outcomes in H-ARS are associated with activation status of the cardiac IGF-1 signaling and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant gene expression. Our data link H-ARS with dysregulation of cardiac IGF-1 signaling, and highlight the role of oxidative stress and cardiac antioxidant response in radiation sensitivity.

Morphological and functional impairment in the gut in a partial body irradiation minipig model of GI-ARS.

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.

NBS1 interacts with HP1 to ensure genome integrity.

Heterochromatin Protein 1 (HP1) and the Mre11-Rad50-Nbs1 (MRN) complex are conserved factors that play crucial role in genome stability and integrity. Despite their involvement in overlapping cellular functions, ranging from chromatin organization, telomere maintenance to DNA replication and repair, a tight functional relationship between HP1 and the MRN complex has never been elucidated. Here we show that the Drosophila HP1a protein binds to the MRN complex through its chromoshadow domain (CSD). In addition, loss of any of the MRN members reduces HP1a levels indicating that the MRN complex acts as regulator of HP1a stability. Moreover, overexpression of HP1a in nbs (but not in rad50 or mre11) mutant cells drastically reduces DNA damage associated with the loss of Nbs suggesting that HP1a and Nbs work in concert to maintain chromosome integrity in flies. We have also found that human HP1α and NBS1 interact with each other and that, similarly to Drosophila, siRNA-mediated inhibition of NBS1 reduces HP1α levels in human cultured cells. Surprisingly, fibroblasts from Nijmegen Breakage Syndrome (NBS) patients, carrying the 657del5 hypomorphic mutation in NBS1 and expressing the p26 and p70 NBS1 fragments, accumulate HP1α indicating that, differently from NBS1 knockout cells, the presence of truncated NBS1 extends HP1α turnover and/or promotes its stability. Remarkably, an siRNA-mediated reduction of HP1α in NBS fibroblasts decreases the hypersensitivity to irradiation, a characteristic of the NBS syndrome. Overall, our data provide an unanticipated evidence of a close interaction between HP1 and NBS1 that is essential for genome stability and point up HP1α as a potential target to counteract chromosome instability in NBS patient cells.

Quantification of Self-renewal in Murine Mammosphere Cultures.

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value.

Loss of p53 function is invariably associated with cancer. Its role in tumor growth was recently linked to its effects on cancer stem cells (CSCs), although the underlying molecular mechanisms remain unknown. Here, we show that c-myc is a transcriptional target of p53 in mammary stem cells (MaSCs) and is activated in breast tumors as a consequence of p53 loss. Constitutive Myc expression in normal mammary cells leads to increased frequency of MaSC symmetric divisions, extended MaSC replicative-potential, and MaSC-reprogramming of progenitors, whereas Myc activation in breast cancer is necessary and sufficient to maintain the expanding pool of CSCs. Concomitant p53 loss and Myc activation trigger the expression of 189 mitotic genes, which identify patients at high risk of mortality and relapse, independently of other risk factors. Altogether, deregulation of the p53:Myc axis in mammary tumors increases CSC content and plasticity and is a critical determinant of tumor growth and clinical aggressiveness.

Neulasta Regimen for the Hematopoietic Acute Radiation Syndrome: Effects Beyond Neutrophil Recovery.

PURPOSE: Understanding the physiopathology underlying the acute radiation syndrome (ARS) and the mechanism of action of drugs known to ameliorate ARS is expected to help identify novel countermeasure candidates and improve the outcome for victims exposed to radiation. Granulocyte colony-stimulating factor (G-CSF) has been approved by the US Food and Drug Administration for treatment of hematopoietic ARS (H-ARS) because of its ability to alleviate myelosuppression. Besides its role in hematopoiesis, G-CSF is known to protect the cardiovascular and neurologic systems, to attenuate vascular injury and cardiac toxicity, to preserve gap junction function, and to modulate inflammation and oxidative stress. Here, we characterized the protective effects of G-CSF beyond neutrophil recovery in minipigs exposed to H-ARS doses. METHODS AND MATERIALS: Twenty male Göttingen minipigs were exposed to total body, acute ionizing radiation. Animals received either pegylated G-CSF (Neulasta) or dextrose at days 1 and 8 after irradiation. Survival was monitored over a 45-day period. RESULTS: Neulasta decreased mortality compared with the control, reduced nadir and duration of neutropenia, and lowered prevalence of organ hemorrhage and frank bleeding episodes. Neulasta also increased plasma concentration of IGF-1 hormone, activated the cardiovascular protective IGF-1R/PI3K/Akt/eNOS/NO pathway, and enhanced membrane expression of VE-cadherin in the heart, improving vascular tone and barrier function. Expression of the acute phase protein CRP, a mediator of cardiovascular diseases and a negative regulator of the IGF-1 pathway, was also induced but at much lower extent compared with IGF-1. Activity of catalase and superoxide dismutase (SOD-1) was only marginally affected, whereas activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was downregulated. CONCLUSIONS: In addition to a neutrophilic effect, amelioration of endothelial homeostasis and barrier function and reduction in NADPH oxidase contribute to the beneficial effects of Neulasta for the treatment of H-ARS.

Selective Insulin-like Growth Factor Resistance Associated with Heart Hemorrhages and Poor Prognosis in a Novel Preclinical Model of the Hematopoietic Acute Radiation Syndrome.

Although bone marrow aplasia has been considered for the past decades as the major contributor of radiation-induced blood disorders, cytopenias alone are insufficient to explain differences in the prevalence of bleeding. In this study, the minipig was used as a novel preclinical model of hematopoietic acute radiation syndrome to assess if factors other than platelet counts correlated with bleeding and survival. We sought to determine whether radiation affected the insulin-like growth factor-1 (IGF-1) pathway, a growth hormone with cardiovascular and radioprotective features. Gottingen and Sinclair minipigs were exposed to ionizing radiation at hematopoietic doses. The smaller Gottingen minipig strain was more sensitive to radiation; differences in IGF-1 levels were minimal, suggesting that increased sensitivity could depend on weak response to the hormone. Radiation caused IGF-1 selective resistance by inhibiting the anti-inflammatory anti-oxidative stress IRS/PI3K/Akt but not the pro-inflammatory MAPK kinase pathway, shifting IGF-1 signaling towards a pro-oxidant, pro-inflammatory environment. Selective IGF-1 resistance associated with hemorrhages in the heart, poor prognosis, increase in C-reactive protein and NADPH oxidase 2, uncoupling of endothelial nitric oxide synthase, inhibition of nitric oxide (NO) synthesis and imbalance between the vasodilator NO and the vasoconstrictor endothelin-1 molecules. Selective IGF-1 resistance is a novel mechanism of radiation injury, associated with a vicious cycle amplifying reactive oxygen species-induced damage, inflammation and endothelial dysfunction. In the presence of thrombocytopenia, selective inhibition of IGF-1 cardioprotective function may contribute to the development of hemostatic disorders. This finding may be particularly relevant for individuals with low IGF-1 activity, such as the elderly or those with cardiometabolic dysfunctions.

MODELING H-ARS USING HEMATOLOGICAL PARAMETERS: A COMPARISON BETWEEN THE NON-HUMAN PRIMATE AND MINIPIG.

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) 60Co 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.

Significance of Bioindicators for Early Predictions on Diagnosis and Therapy of Irradiated Minipigs.

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.

Gastrointestinal acute radiation syndrome in Göttingen minipigs (Sus scrofa domestica).

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; (60)Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.

Circulating interleukin-18 as a biomarker of total-body radiation exposure in mice, minipigs, and nonhuman primates (NHP).

We aim to develop a rapid, easy-to-use, inexpensive and accurate radiation dose-assessment assay that tests easily obtained samples (e.g., blood) to triage and track radiological casualties, and to evaluate the radioprotective and therapeutic effects of radiation countermeasures. In the present study, we evaluated the interleukin (IL)-1 family of cytokines, IL-1ß, IL-18 and IL-33, as well as their secondary cytokines' expression and secretion in CD2F1 mouse bone marrow (BM), spleen, thymus and serum in response to γ-radiation from sublethal to lethal doses (5, 7, 8, 9, 10, or 12 Gy) at different time points using the enzyme-linked immune sorbent assay (ELISA), immunoblotting, and cytokine antibody array. Our data identified increases of IL-1ß, IL-18, and/or IL-33 in mouse thymus, spleen and BM cells after total-body irradiation (TBI). However, levels of these cytokines varied in different tissues. Interestingly, IL-18 but not IL-1ß or IL-33 increased significantly (2.5-24 fold) and stably in mouse serum from day 1 after TBI up to 13 days in a radiation dose-dependent manner. We further confirmed our finding in total-body γ-irradiated nonhuman primates (NHPs) and minipigs, and demonstrated that radiation significantly enhanced IL-18 in serum from NHPs 2-4 days post-irradiation and in minipig plasma 1-3 days post-irradiation. Finally, we compared circulating IL-18 with the well known hematological radiation biomarkers lymphocyte and neutrophil counts in blood of mouse, minipigs and NHPs and demonstrated close correlations between these biomarkers in response to radiation. Our results suggest that the elevated levels of circulating IL-18 after radiation proportionally reflect radiation dose and severity of radiation injury and may be used both as a potential biomarker for triage and also to track casualties after radiological accidents as well as for therapeutic radiation exposure.

Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species.

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.

Significance of bioindicators to predict survival in irradiated minipigs.

The minipig is emerging as a potential alternative non-rodent animal model. Several biological markers (e.g., blood counts, laboratory parameters, and clinical signs) have been proposed for rapid triage of radiation victims. Here, the authors focus on the significance of bio-indicators for prediction of survivors after irradiation and compare it with human data; the relationship between these biomarkers and radiation dose is not part of this study. Male Göttingen minipigs (age 4-5 mo, weight 9-10 kg) were irradiated (or sham-irradiated) bilaterally with gamma-photons (6°Co, 0.5-0.6 Gy min⁻¹) in the dose range of 1.6-12 Gy. Peripheral blood cell counts, laboratory parameters, and clinical symptoms were collected up to 10 d after irradiation and analyzed using logistic regression analysis and calculating ROC curves. In moribund pigs, parameters such as decreased lymphocyte/granulocyte counts, increased C-reactive protein, alkaline phosphatase values, as well as increased citrulline values and body temperature, significantly (p < 0.002 up to p < 0.0001) discriminated non-survivors from survivors with high precision (ROC > 0.8). However, most predictive within the first 3 d after exposure was a combination of decreased lymphocyte counts and increased body temperature observed as early as 3 h after radiation exposure (ROC: 0.93-0.96, p < 0.0001). Sham-irradiated animals (corresponding to "worried wells") could be easily discriminated from dying pigs, thus pointing to the diagnostic significance of this analysis. These data corroborate with 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.

Accelerated hematopoietic syndrome after radiation doses bridging hematopoietic (H-ARS) and gastrointestinal (GI-ARS) acute radiation syndrome: early hematological changes and systemic inflammatory response syndrome in minipig.

PURPOSE: To characterize acute radiation syndrome (ARS) sequelae at doses intermediate between the bone marrow (H-ARS) and full gastrointestinal (GI-ARS) syndrome. METHODS: Male minipigs, approximately 5 months old, 9-12 kg in weight, were irradiated with Cobalt-60 (total body, bilateral gamma irradiation, 0.6 Gy/min). Endpoints were 10-day survival, gastrointestinal histology, plasma citrulline, bacterial translocation, vomiting, diarrhea, vital signs, systemic inflammatory response syndrome (SIRS), febrile neutropenia (FN). RESULTS: We exposed animals to doses (2.2-5.0 Gy) above those causing H-ARS (1.6-2.0 Gy), and evaluated development of ARS. Compared to what was observed during H-ARS (historical data: Moroni et al. 2011a , 2011c ), doses above 2 Gy produced signs of increasingly severe pulmonary damage, faster deterioration of clinical conditions, and faster increases in levels of C-reactive protein (CRP). In the range of 4.6-5.0 Gy, animals died by day 9-10; signs of the classic GI syndrome, as measured by diarrhea, vomiting and bacterial translocation, did not occur. At doses above 2 Gy we observed transient reduction in circulating citrulline levels, and animals exhibited earlier depletion of blood elements and faster onset of SIRS and FN. CONCLUSIONS: An accelerated hematopoietic subsyndrome (AH-ARS) is observed at radiation doses between those producing H-ARS and GI-ARS. It is characterized by early onset of SIRS and FN, and greater lung damage, compared to H-ARS.