The Pseudo-Pelger HuËt Cell-A New Permanent Radiation Biomarker.

Using archival peripheral blood slides obtained from patients in the 1958 Y-12 criticality accident, the authors have recently described the pseudo-Pelger Huët anomaly (PHA) in neutrophils as a new radiation-induced biomarker. The current work provides additional evidence that PHA is also a permanent biomarker, potentially useful in retrospective dosimetry. In the Y-12 cohort, the high dose group (n = 5, 2.98-4.61 Gy-Eq) exhibited 13.0 ± 0.85 % Pelger Huët cells (mean ± SEM) in the neutrophil population compared to 6.8 ± 1.6 % in the low dose group (n = 3, 0.29-0.86 Gy-Eq; p = 0.008). An age and gender-matched control group (n = 8) exhibited 3.6 ± 0.9 % PH cells. Results of a one-way ANOVA show that the high dose group is statistically different from both the low dose group and the control group (p = 0.002). In the Y-12 cohort, PHA appears <12 h post-accident and is permanent for more than 16 y. Similar long-term persistence of the PHA mutation has been obtained from examination of peripheral blood slides from the 1971 Co accident at the Variable Dose Rate Irradiation Facility (VDRIF) in Oak Ridge, TN. In order to investigate the pseudo-PH cell as a biomarker in animal studies under well controlled dosimetry, peripheral blood slides were obtained from animals in a nonhuman primate (NHP) (Macaca mulatta) total-body irradiation (TBI) model (Co γ rays at 0.6 Gy min; dose range 1-8.5 Gy, LD50/60 6.44 Gy). In the NHP studies, the first measurement of PHA is taken at 5 h post-irradiation, then daily for days 1-5 and every 5-10 d thereafter. In the TBI model, the PH cell appears quickly (<5 h) post-irradiation, and the dose-dependent PH percentage is constant from 1 d over the 60-d monitoring period of the experiments. Using the average of data from 1-60 d, a linear dose response (PHA % slope = 0.49 ± 0.07 % Gy, r = 0.92) is obtained over the dose range 0-8.5 Gy. The authors conclude that ionizing radiation induces dose-dependent internuclear bridges in circulating neutrophils, and this morphological change can be used both as an acute phase biomarker and as a tool for retrospective dosimetry.

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.

Wound trauma alters ionizing radiation dose assessment.

BACKGROUND: Wounding following whole-body γ-irradiation (radiation combined injury, RCI) increases mortality. Wounding-induced increases in radiation mortality are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to bacterial infection. Among these factors, cytokines along with other biomarkers have been adopted for biodosimetric evaluation and assessment of radiation dose and injury. Therefore, wounding could complicate biodosimetric assessments. RESULTS: In this report, such confounding effects were addressed. Mice were given 60Co γ-photon radiation followed by skin wounding. Wound trauma exacerbated radiation-induced mortality, body-weight loss, and wound healing. Analyses of DNA damage in bone-marrow cells and peripheral blood mononuclear cells (PBMCs), changes in hematology and cytokine profiles, and fundamental clinical signs were evaluated. Early biomarkers (1 d after RCI) vs. irradiation alone included significant decreases in survivin expression in bone marrow cells, enhanced increases in γ-H2AX formation in Lin+ bone marrow cells, enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood, and concomitant decreases in γ-H2AX formation in PBMCs and decreases in numbers of splenocytes, lymphocytes, and neutrophils. Intermediate biomarkers (7 - 10 d after RCI) included continuously decreased γ-H2AX formation in PBMC and enhanced increases in IL-1ß, IL-6, IL-8, and G-CSF concentrations in blood. The clinical signs evaluated after RCI were increased water consumption, decreased body weight, and decreased wound healing rate and survival rate. Late clinical signs (30 d after RCI) included poor survival and wound healing. CONCLUSION: Results suggest that confounding factors such as wounding alters ionizing radiation dose assessment and agents inhibiting these responses may prove therapeutic for radiation combined injury and reduce related mortality.

Delta-tocotrienol protects mouse and human hematopoietic progenitors from gamma-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling.

BACKGROUND: Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. DESIGN AND METHODS: CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. RESULTS: δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells. CONCLUSIONS: Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.