Bandstra, E. R., Pecaut, M. J., Anderson, E. R., Willey, J. S., De Carlo, F., Stock, S. R., Gridley, D. S., Nelson, G. A., Levine, H. G. and Bateman, T. A. Long-Term Dose Response of Trabecular Bone in Mice to Proton Radiation. Radiat. Res. 169, 607–614 (2008).

Astronauts on exploratory missions will experience a complex environment, including microgravity and radiation. While the deleterious effects of unloading on bone are well established, fewer studies have focused on the effects of radiation. We previously demonstrated that 2 Gy of ionizing radiation has deleterious effects on trabecular bone in mice 4 months after exposure. The present study investigated the skeletal response after total doses of proton radiation that astronauts may be exposed to during a solar particle event. We exposed mice to 0.5, 1 or 2 Gy of whole-body proton radiation and killed them humanely 117 days later. Tibiae and femora were analyzed using microcomputed tomography, mechanical testing, mineral composition and quantitative histomorphometry. Relative to control mice, mice exposed to 2 Gy had significant differences in trabecular bone volume fraction (−20%), trabecular separation ( 11%), and trabecular volumetric bone mineral density (−19%). Exposure to 1 Gy radiation induced a nonsignificant trend in trabecular bone volume fraction (−13%), while exposure to 0.5 Gy resulted in no differences. No response was detected in cortical bone. Further analysis of the 1-Gy mice using synchrotron microCT revealed a significantly lower trabecular bone volume fraction (−13%) than in control mice. Trabecular bone loss 4 months after exposure to 1 Gy highlights the importance of further examination of how space radiation affects bone.

Kennedy, A. R., Davis, J. G., Carlton, W. and Ware, J. H. Effects of Dietary Antioxidant Supplementation on the Development of Malignant Lymphoma and Other Neoplastic Lesions in Mice Exposed to Proton or Iron-Ion Radiation. Radiat. Res. 169, 615–625 (2008).

Malignancy is considered to be a particular risk associated with exposure to the types of ionizing radiation encountered during extended space flight. In the present study, two dietary preparations were evaluated for their ability to prevent carcinogenesis in CBA mice exposed to different forms of space radiation: protons and highly energetic heavy particles (HZE particles). One preparation contained a mixture of antioxidant agents. The other contained the soybean-derived Bowman-Birk protease inhibitor (BBI), used in the form of BBI Concentrate (BBIC). The major finding was that there was a reduced risk of developing malignant lymphoma in animals exposed to space radiation and maintained on diets containing the antioxidant formulation or BBIC compared to the irradiated animals maintained on the control diet. In addition, the two different dietary countermeasures also reduced the yields of a variety of different rare tumor types observed in the animals exposed to space radiation. These results suggest that dietary supplements could be useful in the prevention of malignancies and other neoplastic lesions developing from exposure to space radiation.

Rola, R., Fishman, K., Baure, J., Rosi, S., Lamborn, K. R., Obenaus, A., Nelson, G. A. and Fike, J. R. Hippocampal Neurogenesis and Neuroinflammation after Cranial Irradiation with ⁵⁶Fe Particles. Radiat. Res. 169, 626–632 (2008).

Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. In the central nervous system (CNS), loss of critical cellular components may lead to performance decrements that could ultimately compromise mission goals and long-term quality of life. Hippocampal-dependent cognitive impairments occur after exposure to ionizing radiation, and while the pathogenesis of this effect is not yet clear, it may involve the production of newly born neurons (neurogenesis) in the hippocampal dentate gyrus. We irradiated mice with 0.5–4 Gy of ⁵⁶Fe ions and 2 months later quantified neurogenesis and numbers of activated microglia as a measure of neuroinflammation in the dentate gyrus. Results showed that there were few changes after 0.5 Gy, but that there was a dose-related decrease in hippocampal neurogenesis and a dose-related increase in numbers of newly born activated microglia from 0.5–4.0 Gy. While those findings were similar to what was reported after X irradiation, there were also some differences, particularly in the response of newly born glia. Overall, this study showed that hippocampal neurogenesis was sensitive to relatively low doses of ⁵⁶Fe particles, and that those effects were associated with neuroinflammation. Whether these changes will result in functional impairments or if/how they can be managed are topics for further investigation.

Carsten, R. E., Bachand, A. M., Bailey, S. M. and Ullrich, R. L. Resveratrol Reduces Radiation-Induced Chromosome Aberration Frequency in Mouse Bone Marrow Cells. Radiat. Res. 169, 633–638 (2008).

Resveratrol, a polyphenol compound with reported antioxidant and anticarcinogenic effects, a wide range of molecular targets, and toxicity only at extreme doses, has received considerable attention. We evaluated the radioprotective effect of orally administered resveratrol on the frequencies of chromosome aberrations in irradiated mouse bone marrow cells. CBA/CaJ mice were divided into four groups: (1) no treatment, (2) resveratrol only, (3) radiation only, and (4) resveratrol and radiation. Resveratrol treatment (100 mg/kg daily) was initiated 2 days prior to irradiation. Bone marrow was then harvested at 1 and 30 days after a single dose of 3 Gy whole-body γ radiation. A statistically significant (P < 0.05) reduction in the mean total chromosome aberration frequency per metaphase at both times postirradiation in the resveratrol and radiation group compared to the radiation-only group was observed. This study is the first to demonstrate that resveratrol has radioprotective effects in vivo. These results support the use of resveratrol as a radioprotector with the potential for widespread application.

Rothkamm, K., Gunasekara, K., Warda, S. A., Krempler, A. and Löbrich, M. Radiation-Induced HPRT Mutations Resulting from Misrejoined DNA Double-Strand Breaks. Radiat. Res. 169, 639–648 (2008).

DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and α-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.

Elsässer, T., Brons, S., Psonka, K., Scholz, M., Gudowska-Nowak, E. and Taucher-Scholz, G. Biophysical Modeling of Fragment Length Distributions of DNA Plasmids after X and Heavy-Ion Irradiation Analyzed by Atomic Force Microscopy. Radiat. Res. 169, 649–659 (2008).

The investigation of fragment length distributions of plasmid DNA gives insight into the influence of localized energy distribution on the induction of DNA damage, particularly the clustering of double-strand breaks. We present an approach that determines the fragment length distributions of plasmid DNA after heavy-ion irradiation by using the Local Effect Model. We find a good agreement of our simulations with experimental fragment distributions derived from atomic force microscopy (AFM) studies by including experimental constraints typical for AFM. Our calculations reveal that by comparing the fragmentation in terms of fluence, we can uniquely distinguish the effect of different radiation qualities. For very high-LET irradiation using nickel or uranium ions, no difference between their fragment distributions can be expected for the same dose level. However, for carbon ions with an intermediate LET, the fragmentation pattern differs from the distribution for very high-LET particles. The results of the model calculations can be used to determine the optimal experimental parameters for a demonstration of the influence of track structure on primary radiation damage. Additionally, we compare the results of our model for two different plasmid geometries.

Little, M. P., Hoel, D. G., Molitor, J., Boice, J. D., Jr., Wakeford, R. and Muirhead, C. R. New Models for Evaluation of Radiation-Induced Lifetime Cancer Risk and its Uncertainty Employed in the UNSCEAR 2006 Report. Radiat. Res. 169, 660–676 (2008).

Generalized relative and absolute risk models are fitted to the latest Japanese atomic bomb survivor solid cancer and leukemia mortality data (through 2000), with the latest (DS02) dosimetry, by classical (regression calibration) and Bayesian techniques, taking account of errors in dose estimates and other uncertainties. Linear-quadratic and linear-quadratic-exponential models are fitted and used to assess risks for contemporary populations of China, Japan, Puerto Rico, the U.S. and the UK. Many of these models are the same as or very similar to models used in the UNSCEAR 2006 report. For a test dose of 0.1 Sv, the solid cancer mortality for a UK population using the generalized linear-quadratic relative risk model is estimated as 5.4% Sv⁻¹ [90% Bayesian credible interval (BCI) 3.1, 8.0]. At 0.1 Sv, leukemia mortality for a UK population using the generalized linear-quadratic relative risk model is estimated as 0.50% Sv⁻¹ (90% BCI 0.11, 0.97). Risk estimates varied little between populations; at 0.1 Sv the central estimates ranged from 3.7 to 5.4% Sv⁻¹ for solid cancers and from 0.4 to 0.6% Sv⁻¹ for leukemia. Analyses using regression calibration techniques yield central estimates of risk very similar to those for the Bayesian approach. The central estimates of population risk were similar for the generalized absolute risk model and the relative risk model. Linear-quadratic-exponential models predict lower risks (at least at low test doses) and appear to fit as well, although for other (theoretical) reasons we favor the simpler linear-quadratic models.

Koch, C. J. Importance of Antibody Concentration in the Assessment of Cellular Hypoxia by Flow Cytometry: EF5 and Pimonidazole. Radiat. Res. 169, 677–688 (2008).

The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of ¹⁴C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO₂ from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO₂. The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] – pimonidazole ≪ EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO₂ and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO₂. The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO₂ dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO₂.

Egeland, T. A. M., Gaustad, J-V., Benjaminsen, I. C., Hedalen, K., Mathiesen, B. and Rofstad, E. K. Assessment of Fraction of Hypoxic Cells in Human Tumor Xenografts with Necrotic Regions by Dynamic Contrast-Enhanced MRI. Radiat. Res. 169, 689–699 (2008).

The potential usefulness of gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for assessing hypoxia in tumors with significant necrosis was investigated. Small (100–350 mm³) and large (500–1000 mm³) D-12 and U-25 tumors were subjected to DCE-MRI, measurement of the fraction of necrotic tissue, and measurement of the fraction of radiobiologically hypoxic cells. Images of E·F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) and λ (λ is proportional to extracellular volume fraction) were produced by subjecting the DCE-MRI data to Kety analysis. Necrotic tissue could be identified in λ images but not in E·F images of the tumors. Most voxels in viable tissue showed λ values of 0.15–0.70, whereas the λ values of most voxels in necrotic tissue were either <0.15 or >0.70. The E·F and λ frequency distributions of the viable tissue, but not the E·F and λ frequency distributions of the whole tissue, were consistent with the observation that the four groups of tumors showed similar fractions of radiobiologically hypoxic cells. E·F and λ images may thus provide useful information on the extent of hypoxia in tumors provided that voxels in necrotic tumor regions are identified and excluded from the images.

Miller, J. H., Jin, S., Morgan, W. F., Yang, A., Wan, Y., Aypar, U., Peters, J. S. and Springer D. L. Profiling Mitochondrial Proteins in Radiation-Induced Genome-Unstable Cell Lines with Persistent Oxidative Stress by Mass Spectrometry. Radiat. Res. 169, 700–706 (2008).

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.

Parashar, V., Frankel, S., Lurie, A. G. and Rogina, B. The Effects of Age on Radiation Resistance and Oxidative Stress in Adult Drosophila melanogaster. Radiat. Res. 169, 707–711 (2008).

Drosophila melanogaster (fruit fly) is a well-established model organism for genetic studies of development and aging. We examined the effects of lethal ionizing radiation on male and female adult Drosophila of different ages, using doses of radiation from 200 to 1500 Gy. Fifty percent lethality 2 days postirradiation (LD_50/2) in wild-type 1-day-old adult fruit flies was ∼1238 Gy for males and 1339 Gy for females. We observed a significant age-dependent decline in the radiation resistance of both males and females. Radiation damage is postulated to occur by the generation of oxygen radicals. An age-related decline in the ability of flies to resist an agent that induces oxygen radicals, paraquat, was observed when comparing 10- and 20-day adults. Female flies are more resistant to paraquat than male flies. Oxidative stress mediated by paraquat was additive with sublethal exposures to radiation in young adults. Therefore, the ability to repair the damage caused by oxygen radicals seems to decline with the age of the flies. Because Drosophila adults are largely post-mitotic, our data suggest that adult Drosophila melanogaster can serve as an excellent model to study the factors responsible for radiation resistance in post-mitotic tissue and age-dependent changes in this resistance.

DiCarlo, A. L., Hatchett, R. J., Kaminski, J. M., Ledney, G. D., Pellmar, T. C., Okunieff, P. and Ramakrishnan, N. Medical Countermeasures for Radiation Combined Injury: Radiation with Burn, Blast, Trauma and/or Sepsis. Report of an NIAID Workshop, March 26–27, 2007. Radiat. Res. 169, 712–721 (2008).

Non-clinical human radiation exposure events such as the Hiroshima and Nagasaki bombings or the Chernobyl accident are often coupled with other forms of injury, such as wounds, burns, blunt trauma, and infection. Radiation combined injury would also be expected after a radiological or nuclear attack. Few animal models of radiation combined injury exist, and mechanisms underlying the high mortality associated with complex radiation injuries are poorly understood. Medical countermeasures are currently available for management of the non-radiation components of radiation combined injury, but it is not known whether treatments for other insults will be effective when the injury is combined with radiation exposure. Further research is needed to elucidate mechanisms behind the synergistic lethality of radiation combined injury and to identify targets for medical countermeasures. To address these issues, the National Institute of Allergy and Infectious Diseases convened a workshop to make recommendations on the development of animal models of radiation combined injury, possible mechanisms of radiation combined injury, and future directions for countermeasure research, including target identification and end points to evaluate treatment efficacy.

Zou, Z., Sun, H., Su, Y., Cheng, T. and Luo, C. Progress in Research on Radiation Combined Injury in China. Radiat. Res. 169, 722–729 (2008).

The significant feature of radiation combined injury is the occurrence of a combined effect. For decades our institute has focused on studying the key complications of radiation-burn injury, including shock, suppression of hematopoiesis and immunity, gastrointestinal damage and local refractory wound healing. Here we summarize recent advancements in elucidating the mechanisms of and potential treatments for radiation combined injury. Concerning the suppression and regeneration of hematopoiesis in radiation combined injury, mechanisms of megakaryocyte damage have been elucidated and a new type of fusion protein stimulating thrombopoiesis has been developed and is being tested in animals. With regard to the damage and repair of intestinal epithelium, the important molecular mechanisms of radiation combined injury have been clarified, and new measures to prevent and treat gastrointestinal tract injury are proposed. With respect to the difficulties encountered in wound healing, the underlying causes of radiation combined injury have been proposed, and some potential methods to accelerate wound closure are under study. Systemic experiments have been done to determine the appropriate time for eschar excision and skin grafting, and the results provided significant insight into clinical treatment of the injury. In the search for early therapeutic regimens for severe burns and radiation combined injury to prevent deterioration of injuries and to improve survival, cervical sympathetic ganglion block was used for the treatment of animals with radiation combined injury and had significant benefits. These research advancements have potential for application in on-site emergency rescue and in-hospital treatment of radiation combined injury.