Intestinal microbiota affect cell responses to ionizing radiation at the molecular level and can be linked to the development of the immune system, controlled cell death or apoptosis. We have developed a microbiota mouse model and report here that high-linear energy transfer (LET) radiation induced the repair of chromosomal DNA lesions more efficiently in conventional than in restricted intestinal microbiota mice. Based on different phylotype densities after whole-body irradiation, bacterial indicator phylotypes were found to be more abundant in restricted in microbiota than in conventional microbiota. Genotoxic phenotypes of irradiated restricted and conventional microbiota mice were compared with ataxia telangiectasia-deficient restricted and conventional microbiota mice, respectively. Those indicator phylotypes, including Bacteroides (Gram-negative bacterium cTPY-13), Barnesiella intestinihominis and others, which were identified in nonirradiated restricted microbiota mice, increase in radiation-exposed conventional microbiota along with a reduction of persistent DNA double-strand breaks in blood lymphocytes. The dynamic change of phylotype abundances elucidated a feedback mechanism and effect of intestinal microbiota composition on the adaptive response to high-LET radiation. Several other bacterial phylotypes (Helicobacter hepaticus, Helicobacter spp and others) were found to be more abundant in conventional than restricted microbiota. In this commentary, mouse models used in cancer research and radiotherapy for the study on the effects of intestinal microbiota composition on normal tissue radiation response are characterized and discussed. Highlights of this commentary: 1. Restricted microbiota phylotypes were correlated with persistent DNA double-strand breaks (DSBs) and were found to orchestrate onco-protective controlled cell death after radiation; 2. Restricted microbiota composition reduced proinflammatory extracellular-stimulated immune responses, but specifically increased anti-neoplastic cytolytic memory CD8 T cells by low taxonomic diversity and 3. DNA damage repair efficiency induced by a model of conventional microbiota most likely initiates an adaptive response to radiation through microbiota-induced intestinal sub-symptomatic inflammation.

Hypoxia is a major cause of radiation resistance, which may predispose to local recurrence after radiation therapy. While hypoxia increases tumor cell survival after radiation exposure because there is less oxygen to oxidize damaged DNA, it remains unclear whether signaling pathways triggered by hypoxia contribute to radiation resistance. For example, intratumoral hypoxia can increase hypoxia inducible factor 1 alpha (HIF-1α), which may regulate pathways that contribute to radiation sensitization or radiation resistance. To clarify the role of HIF-1α in regulating tumor response to radiation, we generated a novel genetically engineered mouse model of soft tissue sarcoma with an intact or deleted HIF-1α. Deletion of HIF-1α sensitized primary sarcomas to radiation exposure in vivo. Moreover, cell lines derived from primary sarcomas lacking HIF-1α, or in which HIF-1α was knocked down, had decreased clonogenic survival in vitro, demonstrating that HIF-1α can promote radiation resistance in a cell autonomous manner. In HIF-1α-intact and -deleted sarcoma cells, radiation-induced reactive oxygen species, DNA damage repair and activation of autophagy were similar. However, sarcoma cells lacking HIF-1α had impaired mitochondrial biogenesis and metabolic response after irradiation, which might contribute to radiation resistance. These results show that HIF-1α promotes radiation resistance in a cell autonomous manner.

Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway.

Nuclear workers worldwide have been studied for decades to estimate associations between their exposure to ionizing radiation and cancer. The low-level exposure of these workers requires pooling of large cohorts studied over many years to obtain risk estimates with appropriate latency and good precision. We assembled a pooled cohort of 119,195 U.S. nuclear workers at four Department of Energy nuclear weapons facilities (Hanford site, Idaho National Laboratory, Oak Ridge National Laboratory and Savannah River site) and at the Portsmouth Naval Shipyard. The cohort was followed at the start of the workers beginning their radiation work (at earliest, between 1944 and 1952) through 2005, and we compared its mortality to that of the U.S. population. We also conducted regression-modeling analysis to evaluate dose-response associations for external radiation exposure and outcomes: all cancers, smoking- and nonsmoking-related cancers, all lymphatic and hematopoietic cancers, leukemia (excluding chronic lymphocytic), multiple myeloma, cardiovascular disease and others. The mean dose observed among the cohort was 20 mSv. For most outcomes, mortality was below expectation compared to the general population, but mesothelioma and pleura cancers were highly elevated. We found an excess relative risk (ERR) per 10 mSv of 0.14% [95% confidence interval (CI): −0.17%, 0.48%] for all cancers excluding leukemia. Estimates were higher for nonsmoking-related cancers (0.70%, 95% CI: 0.058%, 1.5%) and lower for smoking-related cancers (−0.079%, 95% CI: −0.43%, 0.32%). The ERR per 10 mSv was 1.7% (95% CI: −0.22%, 4.7%) for leukemia, which was similar to the estimate of 1.8% (95% CI: 0.027%, 4.4%) for all lymphatic and hematopoietic cancers. The ERR per 10 mSv for multiple myeloma was 3.9% (95% CI: 0.60%, 9.5%). The ERR per 10 mSv for cardiovascular disease was 0.026% (−0.25%, 0.32%). Little evidence of heterogeneity was seen by facility, birth cohort or sex for most outcomes. The estimates observed here are similar to those found in previous large pooled nuclear worker studies and also (with the exception of multiple myeloma) to those conducted in the Life Span Study of Japanese atomic bomb survivors. The tendency of observed risks to persist many years after exposure for most outcomes illustrates the importance of continued follow-up of nuclear worker cohorts.

In the framework of the International Nuclear Workers Study conducted in France, the UK and the U.S. (INWORKS), updated and expanded methods were developed to convert recorded doses of ionizing radiation to estimates of organ doses or individual personal dose equivalent [H_p(10)] for a total number of 308,297 workers, including 40,035 women. This approach accounts for differences in dosimeter response to predominant workplace energy and geometry of exposure and for the recently published ICRP report on dose coefficients for men and women separately. The overall mean annual individual personal dose equivalent, including zero doses, is 1.73 mSv [median = 0.42; interquartile range (IQR): 0.07, 1.59]. Associated individual organ doses were estimated. INWORKS includes workers who had potential for exposure to neutrons. Therefore, we analyzed neutron dosimetry data to identify workers potentially exposed to neutrons. We created a time-varying indicator for each worker, classifying them according to whether they had a positive recorded neutron dose and if so, whether their neutron dose ever exceeded 10% of their total external penetrating radiation dose. The number of workers flagged as being exposed to neutrons was 13% for the full cohort, with 15% of the cohort in France, 12% of the cohort in the UK and 14% in the U.S. We also used available information on in vivo and bioassay monitoring to identify workers with known depositions or suspected internal contaminations. As a result of this work, information is now available that will allow various types of sensitivity analyses.

Leukocyte growth factors (LGF), such as filgrastim, pegfilgrastim and sargramostim, have been used to mitigate the hematologic symptoms of acute radiation syndrome (ARS) after radiation accidents. Although these pharmaceuticals are currently approved for treatment of chemotherapy-induced myelosuppression, such approval has not been granted for myelosuppression resulting from acute radiation exposure. Regulatory approval of drugs used to treat radiological or nuclear exposure injuries requires their development and testing in accordance with the Animal Efficacy Rule, set forth by the U.S. Food and Drug Administration. To date, filgrastim is the only LGF that has undergone efficacy assessment conducted under the Animal Efficacy Rule. To confirm the efficacy of another LGF with a shorter dosing regimen compared to filgrastim, we evaluated the use of pegfilgrastim (Neulasta®) in a lethal nonhuman primate (NHP) model of hematopoietic acute radiation syndrome (H-ARS). Rhesus macaques were exposed to 7.50 Gy total-body irradiation (the LD_50/60), delivered at 0.80 Gy/min using linear accelerator 6 MV photons. Pegfilgrastim (300 μg/kg, n = 23) or 5% dextrose in water (n = 23) was administered on day 1 and 8 postirradiation and all animals received medical management. Hematologic and physiologic parameters were evaluated for 60 days postirradiation. The primary, clinically relevant end point was survival to day 60; secondary end points included hematologic-related parameters. Pegfilgrastim significantly (P = 0.0014) increased 60 day survival to 91.3% (21/23) from 47.8% (11/23) in the control. Relative to the controls, pegfilgrastim also significantly: 1. decreased the median duration of neutropenia and thrombocytopenia; 2. improved the median time to recovery of absolute neutrophil count (ANC) ≥500/μL, ANC ≥1,000/μL and platelet (PLT) count ≥20,000/μL; 3. increased the mean ANC at nadir; and 4. decreased the incidence of Gram-negative bacteremia. These data demonstrate that pegfilgrastim is an additional medical countermeasure capable of increasing survival and neutrophil-related parameters when administered in an abbreviated schedule to a NHP model of lethal H-ARS.

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

Internal emitters such as Strontium-90 (⁹⁰Sr) pose a substantial health risk during and immediately after a nuclear disaster or detonation of an improvised device. The environmental persistency and potency of ⁹⁰Sr calls for urgent development of high-throughput tests to establish levels of exposure and to help triage potentially exposed individuals who were in the immediate area of the disaster. In response to these concerns, our team focused on developing a robust metabolomic profile for ⁹⁰Sr exposure in urine using a mouse model. The sensitivity of modern time-of-flight mass spectrometry (TOFMS) combined with the separation power of ultra performance liquid chromatography (UPLC) was used to determine perturbations in the urinary metabolome of mice exposed to ⁹⁰Sr. The recently developed statistical suite, MetaboLyzer, was used to explore the mass spectrometry data. The results indicated a significant change in the urinary abundances of metabolites pertaining to butanoate metabolism, vitamin B metabolism, glutamate and fatty acid oxidation. All of these pathways are either directly or indirectly connected to the central energy production pathway, the tricarboxylic acid (TCA) cycle. To our knowledge, this is the first in vivo metabolomics to evaluate the effects of exposure to ⁹⁰Sr using the easily accessible biofluid, urine.

The secondary radiation-induced radicals in lithium formate monohydrate were studied using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques complemented with periodic density functional theory (DFT) calculations. Single crystals of lithium formate monohydrate were X irradiated at 77 K and at room temperature. The main radicals present after irradiation at 77 K are the CO₂^•– radical (R1), the recently identified protonated electron-gain product, HCOOH^•– (R2) (Krivokapić et al., Radiat Res 2014: 181:503-11), and a different geometrical conformation of this latter radical, a species that, up until now, has remained unidentified (R3). The successful quantum chemical modeling of R3 confirmed its structure and also provided a possible mechanism for its formation. After irradiation at 295 K, the crystals were investigated both shortly after irradiation and after storage for eight months at room temperature in ambient environments. After long-term storage the CO₂^•– radical had significantly decayed and the EPR spectra were dominated by two minority radicals. Both of these radicals are most likely formate-centered π-radicals, and based on the observed EPR parameters (g- and hyperfine coupling tensors) tentative candidates are the CO^•– radical and the dimer formed by the CO₂^•– radical and a neighboring formate molecule yielding the radical ^–O₂C·O·^•CH·O^–.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (−25% to −95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

Radiotherapy for malignant tumors of the head and neck commonly leads to radiation-induced sialadenitis as a result of radiation-induced salivary gland dysfunction. We demonstrated previously that phenylephrine could protect the irradiated submandibular gland against apoptosis, although the mechanism is unclear. In this study, we investigated the influence of phenylephrine pretreatment on the expressions of aquaporin 5 (AQP5) and c-Jun N-terminal kinase (JNK) that were presumed to have a role in radiation-induced salivary gland dysfunction. Rats pretreated with phenylephrine (5 mg/kg) were locally irradiated (20 Gy) in the head and neck region. The submandibular glands were removed on day 7 after irradiation. The expression of AQP5 and activation of JNK were measured by immunohistochemistry and Western blot. The localization of AQP5 at the apical and lateral plasma membrane of acinar cells was significantly reduced by irradiation, but markedly enhanced with phenylephrine pretreatment. The protein expression of AQP5 was decreased by 84.91% in irradiated glands, whereas it was fully recovered to the control level in phenylephrine-pretreated glands. Moreover, many acinar, ductal and granular convoluted tubular cells in the irradiated glands exhibited intense immunoreactivity for p-JNK, while in the phenylephrine-pretreated irradiated glands, only a few acinar cells exhibited very faint immunoreactivity for p-JNK. The protein expression level of p-JNK was increased by 41.65% in the irradiated alone glands, but was significantly decreased in the phenylephrine-pretreated irradiated glands. These results suggest that the protective mechanism of phenylephrine might be related to the improved expression of AQP5 and decreased activation of JNK. Pretreatment with phenylephrine in patients undergoing radiotherapy may provide a helpful strategy for suppression of radiation-induced sialadenitis.

Although there has been extensive research done on the biological response to doses of ionizing radiation relevant to radiodiagnostic procedures, very few studies have examined radiation schemes similar to those frequently utilized in CT exams. Instead of a single exposure, CT exams are often made up of a series of scans separated on the order of minutes. DNA damage dose-response kinetics after radiation doses and schemes similar to CT protocols were established in both cultured (ESW-WT3) and whole blood lymphocytes and compared to higher dose exposures. Both the kinetics and extent of H2AX phosphorylation were found to be dose dependent. Damage induction and detection showed a clear dose response, albeit different, at all time points and differences in the DNA repair kinetics of ESW-WT3 and whole blood lymphocytes were characterized. Moreover, using a modified split-dose in vitro experiment, we show that phosphorylation of H2AX is significantly reduced after exposure to CT doses fractionated over a few minutes compared to the same total dose delivered as a single exposure. Because the split-dose exposures investigated here are more similar to those experienced during a CT examination, it is essential to understand why and how these differences occur. This work provides compelling evidence supporting differential biological responses not only between high and low doses, but also between single and multiple exposures to low doses of ionizing radiation.

The space radiation environment consists of multiple species of charged particles, including ²⁸Si, ⁴⁸Ti and protons that may impact cognition, but their damaging effects have been poorly defined. In mouse studies, C57Bl6/J homozygous wild-type mice and genetic mutant mice on a C57Bl6/J background have typically been used for assessing effects of space radiation on cognition. In contrast, little is known about the radiation response of mice on a heterozygous background. Therefore, in the current study we tested the effects of ²⁸Si, ⁴⁸Ti and proton radiation on hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory in C57Bl6/J × DBA2/J F1 (B6D2F1) mice three months after irradiation. Contextual fear memory was impaired at a 1.6 Gy dose of ²⁸Si radiation, but not cued fear memory. ⁴⁸Ti or proton irradiation did not affect either type of memory. Based on earlier space radiation cognitive data in C57Bl6/J mice, these data highlight the importance of including different genetic backgrounds in studies aimed at assessing cognitive changes after exposure to space radiation.

The purpose of this study was to characterize changes in tumor vascular parameters hours after a single radiation exposure in an orthotopic brain tumor model. U-251 human brain tumors were established intracerebrally in rat brains, and tumor blood flow, forward volume transfer constant (K^trans) and interstitial volume fraction (v_e) were measured using magnetic resonance imaging (MRI). Tumors were exposure to a single stereotactic radiation treatment of 20 Gy. Vascular parameters were assessed one additional time between 2 and 24 h after irradiation. After the second MRI session, brain tissue histology was examined for gross changes and apoptosis. In separate studies, cerebral blood flow was measured in nonimplanted controls before radiation exposure and 2 and 24 h after 20 Gy irradiation, and in implanted rats before radiation exposure and at 2 and 24 h after 6 Gy irradiation. Significant changes were observed in tumor-bearing rat brains in the hours after 20 Gy irradiation. Two hours after 20 Gy irradiation, tumor blood flow decreased nearly 80% and v_e decreased by 30%. At 4 h, the K^trans increased by 30% over preirradiation values. Extensive vacuolization and an increase in apoptosis were evident histologically in rats imaged 2 h after irradiation. Between 8 and 12 h after irradiation, all vascular parameters including blood flow returned to near preirradiation values. One day after irradiation, tumor blood flow was elevated 40% over preirradiation values, and other vascular parameters, including K^trans and v_e, were 20–40% below preirradiation values. In contrast, changes in vascular parameters observed in the normal brain 2 or 24 h after 20 Gy irradiation were not significantly different from preirradiation values. Also, tumor blood flow appeared to be unchanged at 2 h after 6 Gy irradiation, with a small increase observed at 24 h, unlike the tumor blood flow changes after 20 Gy irradiation. Large and significant changes in vascular parameters were observed hours after 20 Gy irradiation using noninvasive MRI techniques. It is hypothesized that cellular swelling hours after a high dose of radiation, coinciding with vacuolization, led to a decrease in tumor blood flow and v_e. Four hours after radiation exposure, K^trans increased in concert with an increase in tumor blood flow. Vascular permeability normalized, 24 h after 20 Gy irradiation, as characterized by a decrease in K^trans. Vascular parameters did not change significantly in the normal brain after 20 Gy irradiation or in the tumor-bearing brain after 6 Gy irradiation.