Biomarkers for Radiation Biodosimetry and Injury Assessment after Mixed-field (Neutron and Gamma) Radiation in the Mouse Total-body Irradiation Model.

The risk of potential radiation exposure scenarios that include detonation of nuclear weapons, terrorist attacks on nuclear reactors, and the use of conventional explosives to disperse radioactive substances has increased in recent years. The majority of radiation biodosimetry and countermeasure studies have been performed using photon radiation even though many exposure scenarios predict mixed-field (neutron and photon) radiation. Hence, there is a need to evaluate biomarkers and accurately determine exposure levels of mixed-field combinations of neutrons and photons for an individual. These biomarkers will be critical for biodosimetry triage, treatment, and follow-up visits with such individuals. We evaluated the utility of multiple blood biomarkers for early response assessment of radiation exposure using a mouse (B6D2F1, males and females) total-body irradiation model exposed to a mixed-field (neutrons and gamma rays) using the Armed Forces Radiobiology Research Institute's Mark F nuclear research reactor. Total-body irradiation was given as a single exposure over a dose range from 1.5 to 6 Gy, dose rates of 0.6 and 1.9 Gy min, and different proportions of neutrons and gammas: either (67% neutrons + 33% gammas) or (30% neutrons + 70% gammas). Blood was collected 1, 2, 4, and 7 d after total-body irradiation. Radiation-responsive protein biomarkers were measured using the Meso Scale Diagnostics' high-throughput MULTI-ARRAY plate-format platform (QuickPlex 120 Imager) and enzyme-linked immunosorbent assay kits. Results demonstrate (1) dose- and time-dependent changes in fms-related tyrosine kinase 3 ligand, interleukins IL-5, IL-10, IL-12, and IL-18, granulocyte and granulocyte-macrophage colony-stimulating factors, thrombopoietin, erythropoietin, acute-phase proteins (serum amyloid A and lipopolysaccharide binding protein), surface plasma neutrophil (CD45) and lymphocyte (CD27) markers, ratio of CD45 to CD27, and procalcitonin; (2) dose- and time-dependent changes in blood cell counts (lymphocytes, neutrophils, platelets, red blood cells, and ratio of neutrophils to lymphocytes); (3) levels of IL-18, granulocyte and granulocyte-macrophage colony-stimulating factors, serum amyloid A, and procalcitonin were significantly higher in animals irradiated with 67% neutrons + 33% gammas compared to those irradiated with 30% neutrons + 70% gammas (p < 0.015), while no significant differences (p > 0.114) were observed in hematological biomarker counts; (4) exposure with 3-fold difference in dose rate (0.6 or 1.9 Gy min) revealed no significant differences in hematological and protein biomarker levels (p > 0.154); and (5) no significant differences in hematological and protein biomarker levels were observed in the sex-comparison study for any radiation dose at any time after exposure (p > 0.088). Results show that the dynamic changes in the levels of selected hematopoietic cytokines, organ-specific biomarkers, and acute-phase protein biomarkers reflect the time course and severity of acute radiation syndrome and may function as prognostic indicators of acute radiation syndrome outcome. These studies supplement an ongoing effort to deliver U.S. Federal Drug Administration-approved biodosimetry capabilities, which assess mixed-field radiation exposure.

Comparison of Biodosimetry Biomarkers for Radiation Dose and Injury Assessment After Mixed-Field (Neutron and Gamma) and Pure Gamma Radiation in the Mouse Total-Body Irradiation Model.

The detonation of a nuclear weapon and the occurrence of a nuclear accident represent possible mass-casualty events with significant exposure to mixed neutron and gamma radiation fields in the first few minutes after the event with the ensuing fallout, extending for miles from the epicenter, that would result primarily in photon (gamma- and/or x-ray) exposure. Circulating biomarkers represent a crucial source of information in a mass-casualty radiation exposure triage scenario. We evaluated multiple blood biodosimetry and organ-specific biomarkers for early-response assessment of radiation exposure using a mouse (B6D2F1, males and females) total-body irradiation model exposed to Co gamma rays over a broad dose range (3-12 Gy) and dose rates of either 0.6 or 1.9 Gy min and compared the results with those obtained after exposure of mice to a mixed field (neutrons and gamma rays) using the Armed Forces Radiobiology Research Institute Co gamma-ray source and TRIGA Mark F nuclear research reactor. The mixed-field studies were performed previously over a broad dose range (1.5-6 Gy), with dose rates of either 0.6 or 1.9 Gy min, and using different proportions of neutrons and gammas: either (67% neutrons + 33% gammas) or (30% neutrons + 70% gammas). Blood was collected 1, 2, 4, and 7 d after total-body irradiation. Results from Co gamma-ray studies demonstrate: (1) significant dose- and time-dependent reductions in circulating mature hematopoietic cells; (2) dose- and time-dependent changes in fms-related tyrosine kinase 3 ligand, interleukins IL-5, IL-10, IL-12, and IL-18, granulocyte colony-stimulating factors, thrombopoietin, erythropoietin, acute-phase proteins (serum amyloid A and lipopolysaccharide binding protein), surface plasma neutrophil (CD45) and lymphocyte (CD27) markers, ratio of CD45 to CD27, procalcitonin but not in intestinal fatty acid binding protein; (3) no significant differences were observed between dose-rate groups in hematological and protein profiles (fms-related tyrosine kinase 3 ligand, IL-5, IL-12, IL-18, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, CD27, CD45, and ratio of CD45 to CD27) for any radiation dose at any time after exposure (p > 0.148); (4) no significant differences were observed between sex groups in hematological and protein profiles (fms-related tyrosine kinase 3 ligand, IL-18, erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, serum amyloid A, CD45) for any radiation dose at any time after exposure (p > 0.114); and (5) PCT level significantly increased (p < 0.008) in mice irradiated with 12 Gy on day 7 post-total-body irradiation without significant differences between groups irradiated at dose rates of either 0.6 or 1.9 Gy min (p > 0.287). Radiation-quality comparison results demonstrate that: (1) equivalent doses of pure gamma rays and mixed-field radiation do not produce equivalent biological effects, and hematopoietic syndrome occurs at lower doses of mixed-field radiation; (2) ratios of hematological and protein biomarker means in the Co study compared to mixed-field studies using 2× Co doses vs. 1× TRIGA radiation doses (i.e., 3 Gy Co vs. 1.5 Gy TRIGA) ranged from roughly 0.2 to as high as 26.5 but 57% of all ratios fell within 0.7 and 1.3; and (3) in general, biomarker results are in agreement with the relative biological effectiveness = 1.95 (Dn/Dt = 0.67) reported earlier by Armed Forces Radiobiology Research Institute scientists in mouse survival countermeasure studies.

Urine Interleukin-18 (IL-18) as a Biomarker of Total-Body Irradiation: A Preliminary Study in Nonhuman Primates.

We have reported that circulating IL-18 can be used as a radiation biomarker in mice, minipigs and nonhuman primates (NHPs, Macaca mulatta). Here, we report the levels of IL-18 in individual NHP's urine before and at 6 h-7 days after 5.0, 6.5 and 8.5 Gy 60Co total-body irradiation (TBI) using enzyme linked immunosorbent assay (ELISA). Six animals (3.5-5.5 kg, 3-4 years old) per radiation dose were investigated. Correlation values between urine IL-18 and blood cell counts and serum chemistry parameters including lactate dehydrogenase (LDH), lipase, and serum total protein (TP), as well as between urine IL-18 and 60-day survival, were analyzed. Our data, to the best of our knowledge, for the first time, demonstrate that concentrations of urine IL-18 from irradiated NHPs were increased in a radiation dose-dependent manner compared to pre-TBI levels in samples from these animal (N = 18, 11.02 ± 1.3 pg/ml). A 5.0 Gy low dose of radiation (∼LD10/60) did not increase urine IL-18 levels. In contrast, high-dose TBI significantly increased urine IL-18 at day 1 to day 5 in a bell-shaped time course, reaching a peak of 5- to 10-fold of control levels on day 3 after 6.5 Gy (∼LD50/60) and 8.5 Gy (∼LD90/60), respectively. Statistical analysis using receiver operator characteristic (ROC) and MultiROC analysis indicated that white blood cell and platelet counts, serum LDH, lipase and TP, when combined with urine IL-18, provide discriminatory predictors of total-body radiation injury with a very high ROC area of 0.98. Urine IL-18 measurement, as an early prognostic indicator of survival, may facilitate rapid detection of lethal doses of radiation, based on the currently available data set.

Non-human Primate Total-body Irradiation Model with Limited and Full Medical Supportive Care Including Filgrastim for Biodosimetry and Injury Assessment.

An assessment of multiple biomarkers from radiation casualties undergoing limited- or full-supportive care including treatment with filgrastim is critical to develop rapid and effective diagnostic triage strategies. The efficacy of filgrastim with full-supportive care was compared with results with limited-supportive care by analyzing survival, necropsy, histopathology and serial blood samples for hematological, serum chemistry and protein profiles in a non-human primate (Macaca mulatta, male and female) model during 60-d post-monitoring period following sham- and total-body irradiation with 6.5 Gy 60Co gamma-rays at 0.6 Gy min-1 Filgrastim (10 µg kg-1) was administered beginning on Day 1 post-exposure and continued daily until neutrophil counts were ≥2,000 µL-1 for two consecutive days. Filgrastim and full-supportive care significantly decreased the pancytopenia duration and resulted in improved animal survival and recovery compared to animals with a limited-supportive care. These findings also identified and validated a multiparametric biomarker panel to support radiation diagnostic device development.

Acute Radiation Syndrome Severity Score System in Mouse Total-Body Irradiation Model.

Radiation accidents or terrorist attacks can result in serious consequences for the civilian population and for military personnel responding to such emergencies. The early medical management situation requires quantitative indications for early initiation of cytokine therapy in individuals exposed to life-threatening radiation doses and effective triage tools for first responders in mass-casualty radiological incidents. Previously established animal (Mus musculus, Macaca mulatta) total-body irradiation (γ-exposure) models have evaluated a panel of radiation-responsive proteins that, together with peripheral blood cell counts, create a multiparametic dose-predictive algorithm with a threshold for detection of ~1 Gy from 1 to 7 d after exposure as well as demonstrate the acute radiation syndrome severity score systems created similar to the Medical Treatment Protocols for Radiation Accident Victims developed by Fliedner and colleagues. The authors present a further demonstration of the acute radiation sickness severity score system in a mouse (CD2F1, males) TBI model (1-14 Gy, Co γ-rays at 0.6 Gy min) based on multiple biodosimetric endpoints. This includes the acute radiation sickness severity Observational Grading System, survival rate, weight changes, temperature, peripheral blood cell counts and radiation-responsive protein expression profile: Flt-3 ligand, interleukin 6, granulocyte-colony stimulating factor, thrombopoietin, erythropoietin, and serum amyloid A. Results show that use of the multiple-parameter severity score system facilitates identification of animals requiring enhanced monitoring after irradiation and that proteomics are a complementary approach to conventional biodosimetry for early assessment of radiation exposure, enhancing accuracy and discrimination index for acute radiation sickness response categories and early prediction of outcome.

Early-response biomarkers for assessment of radiation exposure in a mouse total-body irradiation model.

Nuclear accidents or terrorist attacks could expose large numbers of people to ionizing radiation. Early biomarkers of radiation injury will be critical for triage, treatment, and follow-up of such individuals. The authors evaluated the utility of multiple blood biomarkers for early-response assessment of radiation exposure using a murine (CD2F1, males) total-body irradiation (TBI) model exposed to 6°Co γ rays (0.6 Gy min⁻¹) over a broad dose range (0-14 Gy) and timepoints (4 h-5 d). Results demonstrate: 1) dose-dependent changes in hematopoietic cytokines: Flt-3 ligand (Flt3L), interleukin 6 (IL-6), granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), erythropoietin (EPO), and acute phase protein serum amyloid A (SAA); 2) dose-dependent changes in blood cell counts: lymphocytes, neutrophils, platelets, and ratio of neutrophils to lymphocytes; 3) protein results coupled with peripheral blood cell counts established very successful separation of groups irradiated to different doses; and 4) enhanced separation of dose was observed as the number of biomarkers increased. Results show that the dynamic changes in the levels of SAA, IL-6, G-CSF, and Flt3L reflect the time course and severity of acute radiation syndrome (ARS) and may function as prognostic indicators of ARS outcome. These results also demonstrate proof-in-concept that plasma proteins show promise as a complimentary approach to conventional biodosimetry for early assessment of radiation exposures and, coupled with peripheral blood cell counts, provide early diagnostic information to manage radiation casualty incidents effectively, closing a gap in capabilities to rapidly and effectively assess radiation exposure early, especially needed in case of a mass-casualty radiological incident.