After a large-scale nuclear accident or an attack with an improvised nuclear device, rapid biodosimetry would be needed for triage. As a possible means to address this need, we previously defined a gene expression signature in human peripheral white blood cells irradiated ex vivo that predicts the level of radiation exposure with high accuracy. We now demonstrate this principle in vivo using blood from patients receiving total-body irradiation (TBI). Whole genome microarray analysis has identified genes responding significantly to in vivo radiation exposure in peripheral blood. A 3-nearest neighbor classifier built from the TBI patient data correctly predicted samples as exposed to 0, 1.25 or 3.75 Gy with 94% accuracy (P < 0.001) even when samples from healthy donor controls were included. The same samples were classified with 98% accuracy using a signature previously defined from ex vivo irradiation data. The samples could also be classified as exposed or not exposed with 100% accuracy. The demonstration that ex vivo irradiation is an appropriate model that can provide meaningful prediction of in vivo exposure levels, and that the signatures are robust across diverse disease states and independent sample sets, is an important advance in the application of gene expression for biodosimetry.

The structural maintenance of chromosome 1 (Smc1) protein is a member of the highly conserved cohesin complex and is involved in sister chromatid cohesion. In response to ionizing radiation, Smc1 is phosphorylated at two sites, Ser-957 and Ser-966, and these phosphorylation events are dependent on the ATM protein kinase. In this study, we describe the generation of two novel ELISAs for quantifying phospho-Smc1^Ser-957 and phospho-Smc1^Ser-966. Using these novel assays, we quantify the kinetic and biodosimetric responses of human cells of hematological origin, including immortalized cells, as well as both quiescent and cycling primary human PBMC. Additionally, we demonstrate a robust in vivo response for phospho-Smc1^Ser-957 and phospho-Smc1^Ser-966 in lymphocytes of human patients after therapeutic exposure to ionizing radiation, including total-body irradiation, partial-body irradiation, and internal exposure to ¹³¹I. These assays are useful for quantifying the DNA damage response in experimental systems and potentially for the identification of individuals exposed to radiation after a radiological incident.

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

The parotid gland is an important organ at risk of complications of radiotherapy for head and neck cancer. In this study, we examined the potential of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for assessment of radiation injury to the parotid glands. DCE-MRI was performed before and 3 months after radiotherapy in patients treated for head and neck cancer. DCE-MRI was analyzed using the pharmacokinetic model proposed by Tofts and Kermode to produce three DCE parameters: k_trans, v_e and v_p. These parameters were correlated with the dose of radiation delivered to the parotid glands and the degree of radiation-induced parotid atrophy. The mean radiation dose received by the parotid glands was 47.1 ± 6.6 Gy. All patients received concurrent chemotherapy. There was a significant rise in all three parameters after therapy (P < 0.0001). Baseline v_e and v_p and the post-treatment rise in v_e correlated with parotid gland atrophy (P  =  0.0008, 0.0003 and 0.0022, respectively). DCE-MRI has the potential to be used as a non-invasive technique for predicting and assessing radiation injury in the parotid glands.

The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3 PTX before being exposed to 8.5–13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3 PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3 PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3 PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3 PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3 PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

In the present study, novel preventive effects of repeated low-dose radiation exposure on diabetes-induced cardiac inflammation and cardiac damage were investigated. C57BL/6J mice were given multiple low doses of streptozotocin (STZ, 60 mg/kg × 6) to generate type 1 diabetes. A week after the last STZ injection, hyperglycemic mice were diagnosed and treated with and without whole-body low-dose radiation exposure (25 mGy X rays) once every 2 days for 2, 4, 8, 12 and 16 weeks. Diabetes caused significant increases in cardiac inflammation, shown by time-dependent increases in mRNA and protein expressions of interleukin 18 (IL-18), tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule 1 (ICAM-1), plasminogen activator inhibitor 1 (PAI-1), and monocyte chemoattractant protein 1 (MCP-1). Repeated exposure of control mice to low-dose radiation caused mild increases in these inflammatory factors, except for ICAM-1. Repeated exposure of diabetic mice to low-dose radiation significantly reduced diabetes-increased cardiac expression of IL-18, TNF-α, MCP-1 and PAI-1 at both the mRNA and protein levels. Furthermore, cardiac histopathological abnormalities, oxidative damage and fibrosis were significant in diabetic mice but to a lesser extent in diabetic mice with repeated low-dose radiation exposure. These results suggest that although low-dose radiation contributes to mild cardiac inflammation in control mice, it can significantly reduce diabetes-induced cardiac inflammation and associated pathological changes. Therefore, low-dose radiation may potentially become a novel approach to the prevention of diabetic cardiovascular complications.

Exposure to high doses of ionizing radiation unequivocally produces adverse health effects including malignancy. At low doses the situation is much less clear, because effects are generally too small to be estimated directly by epidemiology, and extrapolation of risk and establishment of international rules and standards rely on the linear no-threshold (LNT) concept. Claims that low doses are more damaging than would be expected from LNT have been made on the basis of in vitro studies of nontargeted bystander effects and genomic instability, but relevant investigations of primary cells and tissues are limited. Here we show that after low-dose low-LET in vivo radiation exposures in the 0–100-mGy range of murine bone marrow there is no evidence of a bystander effect, assessed by p53 pathway signaling, nor is there any evidence for longer-term chromosomal instability in the bone marrow at doses below 1000 mGy. The data are not consistent with speculations based on in vitro nontargeted effects that low-dose X radiation is more damaging than would be expected from linear extrapolation.

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1–40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.

Cranial radiotherapy during the treatment of pediatric malignancies may cause adverse late effects. It is important to find methods to assess the functional effects of ionizing radiation in animal models and to evaluate the possible ameliorating effects of preventive or reparative treatment strategies. We investigated the long-term effects of a single 8-Gy radiation dose to the brains of 14-day-old mice. Activity and learning were evaluated in adulthood using open field and trace fear conditioning (TFC). These established methods were compared with the novel IntelliCage platform, which enables unbiased analysis of both activity and learning over time in a home cage environment. Neither activity nor learning was changed after irradiation, as judged by the open field and TFC analyses. The IntelliCage, however, revealed both altered activity and learning impairment after irradiation. Place learning and reversal learning were both impaired in the IntelliCage 3 months after irradiation. These results indicate that activity and learning should be assessed using multiple methods and that unbiased analysis over time in a home cage environment may offer advantages in the detection of subtle radiation-induced effects on the young brain.

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

Ultraviolet B (UVB) radiation induces inflammation in human skin. Extracellular nucleotides are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. In this study, we investigated the involvement of extracellular nucleotides and P2 receptors in UVB-radiation-induced inflammation using human keratinocyte-derived HaCaT cells. UVB radiation induced rapid ATP release from HaCaT cells; this was inhibited by pretreatment with anion transporter blockers or maxi-anion channel blockers. In addition, the radiation-induced activation of p38 MAPK was significantly blocked by pretreatment with ecto-nucleotidase (apyrase) or P2Y6 receptor antagonist (MRS2578). Expression of COX-2, mediated by activation of p38 MAPK, was also induced by UVB radiation. Both pretreatment with MRS2578 and knockdown of the P2Y6 receptor by siRNA transfection attenuated the induction of COX-2 in HaCaT cells exposed to UVB radiation. Our results indicate that UVB radiation evokes ATP release from human keratinocytes and also that activation of P2Y6 receptor mediates the UVB-radiation-induced activation of p38 MAPK and expression of COX-2. Thus P2Y6 receptor is a mediator of UVB-radiation-induced inflammatory responses in keratinocytes.

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

Plaques containing fibrillar amyloid-beta (Abeta) are a characteristic finding in Alzheimer's disease. Although plaque counts correlate poorly with the extent of cognitive deficits in this disorder, fibrillar Abeta can promote neuronal damage through a variety of mechanisms. External beam radiotherapy has been reported to be an effective treatment for tracheobronchial amyloidosis, in which amyloid is deposited as submucosal plaques and tumor-like masses in the trachea and/or bronchi. Radiotherapy's effectiveness in this disorder is thought to be due to its toxicity to plasma cells, but direct effects of radiotherapy on amyloid may also be involved. On this basis, whole-brain radiotherapy has been suggested as a treatment for Alzheimer's disease. The objective of this study was to determine the effects of external beam radiation on preformed Abeta1-42 fibrils and on the formation of these fibrils. Using the Thioflavin-T assay, no effects of radiation were found on either of these parameters. Our results in this in vitro study suggest that whole-brain irradiation is unlikely to directly reduce plaque counts in the Alzheimer's disease brain. This treatment might still lower plaque counts indirectly, but any potential benefits would need to be weighed against its possible neurotoxic effects, which could induce further cognitive deficits.

Combined radiation-burn injury can occur in people exposed to nuclear explosions, nuclear accidents or radiological terrorist attacks. Using different combined radiation-burn injury animal models, the pathological mechanisms underlying combined radiation-burn injury and effective medical countermeasures have been explored for several years in China, mainly at our institute. Targeting key features of combined radiation-burn injury, several countermeasures have been developed. Fluid transfusion and the calcium antagonist verapamil can prevent early shock and improve myocardial function after combined radiation-burn injury. Recombinant human interleukin 4 ( rhIL-4) is able to effectively reduce bacterial infection and increase intestinal immunological ability. Chitosan-wrapped human defensin 5 (HD5) and glucagon-like peptide 2 (GLP-2) nanoparticles can increase the average survival time of animals with severe combined radiation-burn injury. After treatment by cervical sympathetic ganglia block (SB), hematopoietic function is promoted and the release of inflammatory cytokines is suppressed. The optimal time for escharectomy and allo-skin grafting is 24 h after injury. Transfusion of irradiated (20 Gy) or stored (4°C, 7 days) blood improves the survival of allo-skin grafting and allo-bone marrow cells. In conclusion, as our understanding of the mechanisms of combined radiation-burn injury has progressed, new countermeasures have been developed for its treatment. Because of the complexity of its pathology and the difficulty in clinical management, further efforts are needed to improve the treatment of this kind of injury.

The objectives of this study were to examine the association between contact current exposure and the risk of childhood leukemia and to investigate the relationship between residential contact currents and magnetic fields. Indoor and outdoor contact voltage and magnetic-field measurements were collected for the diagnosis residence of 245 cases and 269 controls recruited in the Northern California Childhood Leukemia Study (2000–2007). Logistic regression techniques produced odds ratios (OR) adjusted for age, sex, Hispanic ethnicity, mother's race and household income. No statistically significant associations were seen between childhood leukemia and indoor contact voltage level [exposure ≥90th percentile (10.5 mV): OR  =  0.83, 95% confidence interval (CI): 0.45, 1.54], outdoor contact voltage level [exposure ≥90th percentile (291.2 mV): OR  = 0.89, 95% CI: 0.48, 1.63], or indoor magnetic-field levels (>0.20 µT: OR  =  0.76, 95% CI: 0.30, 1.93). Contact voltage was weakly correlated with magnetic field; correlation coefficients were r  =  0.10 (P  =  0.02) for indoor contact voltage and r  =  0.15 (P  =  0.001) for outdoor contact voltage. In conclusion, in this California population, there was no evidence of an association between childhood leukemia and exposure to contact currents or magnetic fields and a weak correlation between measures of contact current and magnetic fields.

Biological dosimetry is an essential tool for estimating radiation dose. The dicentric chromosome assay (DCA) is currently the tool of choice. Because the assay is labor-intensive and time-consuming, strategies are needed to increase throughput for use in radiation mass casualty incidents. One such strategy is to truncate metaphase spread analysis for triage dose estimates by scoring 50 or fewer metaphases, compared to a routine analysis of 500 to 1000 metaphases, and to increase throughput using a large group of scorers in a biodosimetry network. Previously, the National Institutes for Allergies and Infectious Diseases (NIAID) and the Armed Forces Radiobiology Research Institute (AFRRI) sponsored a double-blinded interlaboratory comparison among five established international cytogenetic biodosimetry laboratories to determine the variability in calibration curves and in dose measurements in unknown, irradiated samples. In the present study, we further analyzed the published data from this previous study to investigate how the number of metaphase spreads influences dose prediction accuracy and how this information could be of value in the triage and management of people at risk for the acute radiation syndrome (ARS). Although, as expected, accuracy decreased with lower numbers of metaphase spreads analyzed, predicted doses by the laboratories were in good agreement and were judged to be adequate to guide diagnosis and treatment of ARS. These results demonstrate that for rapid triage, a network of cytogenetic biodosimetry laboratories can accurately assess doses even with a lower number of scored metaphases.