Frankenberg-Schwager, M., Gebauer, A., Koppe, C., Wolf, H., Pralle, E. and Frankenberg, D. Single-Strand Annealing, Conservative Homologous Recombination, Nonhomologous DNA End Joining, and the Cell Cycle-Dependent Repair of DNA Double-Strand Breaks Induced by Sparsely or Densely Ionizing Radiation. Radiat. Res. 171, 265–273 (2009).

The cell cycle-dependent relative contributions of error-prone single-strand annealing (SSA), error-free conservative homologous recombination (HR), and potentially error-prone nonhomologous DNA end joining (NHEJ) to repair simple (induced by 200 kV X rays) or complex (induced by ²⁴¹Am α particles) DNA double-strand breaks (DSBs) in Chinese hamster ovary cells are reported for the first time. Cells of the parental cell line AA8 and its derivatives UV41 (SSA-deficient), irs1SF (HR-deficient) and V3 (NHEJ-deficient) were synchronized in G₁ or in S phase, and survival responses after exposure to either type of radiation were measured. It is demonstrated for the first time that in G₁-phase SSA is negligible for the repair of DSBs of various complexities. HR-deficient cells exposed to X rays or α particles in G₁ phase show enhanced radiosensitivity, but this does not necessarily mean that HR is important in G₁ phase. NHEJ appears to be the most important (if not the only) mechanism in G₁ phase acting efficiently on simple DSBs, but complex DSBs are a less preferred target. In contrast to X rays, NHEJ-deficient cells show no cell cycle-dependent variation in sensitivity to α particles. Surprisingly, when these cells are exposed to X rays in G₁ phase, they are even more sensitive compared to α particles. It is also shown for the first time that in S phase all three mechanisms play a role in the repair of simple and complex DSBs. A defect in SSA confers radiosensitivity to cells in S phase, suggesting that the error-prone SSA mechanism is important for the repair of specific simple and complex DSBs that are not a substrate for HR or NHEJ. The most important mechanism in S phase for the repair of simple and complex DSBs is HR. This is also emphasized by the finding that irs1SF cells, after complementation of their HR defect by human XRCC3 cDNA, show a greater radioresistance than parental cells, whereas resistance to mitomycin C is only partially restored. Complementation confers a greater resistance to α particles than X rays, suggesting an important role of HR, especially for the repair of complex DSBs. In S phase, NHEJ is more important than SSA for the repair of simple DSBs, but SSA is more important than NHEJ for the repair of complex DSBs.

Reiter, R., Deutschle, T., Wiegel, T., Riechelmann, H. and Bartkowiak, D. Absence of Inflammatory Response from Upper Airway Epithelial Cells after X Irradiation. Radiat. Res. 171, 274–282 (2009).

Radiotherapy of head and neck tumors causes adverse reactions in normal tissue, especially mucositis. The dose- and time-dependent response of upper airway cells to X radiation should be analyzed in terms of the pro-inflammatory potential. Immortalized BEAS-2B lung epithelial cells were treated with 2, 5 and 8 Gy. Out of 1232 genes, those that were transcribed differentially after 2, 6 and 24 h were assigned to biological themes according to the Gene Ontology Consortium. Enrichment of differentially regulated gene clusters was determined with GOTree ( http://bioinfo.vanderbilt.edu/gotm). Eleven cytokines were measured in culture supernatants. The cell cycle response up to 24 h and induction of apoptosis up to 4 days after exposure were determined by flow cytometry. A significant dose- and time-dependent gene activation was observed for the categories response to DNA damage, oxidative stress, cell cycle arrest and cell death/apoptosis but not for immune/inflammatory response. This correlated with functional G₂ arrest and apoptosis. Pro-inflammatory cytokines accumulated in supernatants of control cells but not of X-irradiated cells. The complex gene expression pattern of X-irradiated airway epithelial cells is accompanied by cell cycle arrest and induction of apoptosis. In vivo, this may impair the epithelial barrier. mRNA and protein expression suggest at most an indirect contribution of epithelial cells to early radiogenic mucositis.

Kondo, H., Searby, N. D., Mojarrab, R., Phillips, J., Alwood, J., Yumoto, K., Almeida, E. A. C., Limoli, C. L. and Globus, R. K. Total-Body Irradiation of Postpubertal Mice with ¹³⁷Cs Acutely Compromises the Microarchitecture of Cancellous Bone and Increases Osteoclasts. Radiat. Res. 171, 283–289 (2009).

Ionizing radiation can cause substantial tissue degeneration, which may threaten the long-term health of astronauts and radiotherapy patients. To determine whether a single dose of radiation acutely compromises structural integrity in the postpubertal skeleton, 18-week-old male mice were exposed to ¹³⁷Cs γ radiation (1 or 2 Gy). The structure of high-turnover, cancellous bone was analyzed by microcomputed tomography (microCT) 3 or 10 days after irradiation and in basal controls (tissues harvested at the time of irradiation) and age-matched controls. Irradiation (2 Gy) caused a 20% decline in tibial cancellous bone volume fraction (BV/TV) within 3 days and a 43% decline within 10 days, while 1 Gy caused a 28% reduction 10 days later. The BV/TV decrement was due to increased spacing and decreased thickness of trabeculae. Radiation also increased (∼150%) cancellous surfaces lined with tartrate-resistant, acid phosphatase-positive osteoclasts, an index of increased bone resorption. Radiation decreased lumbar vertebral BV/TV 1 month after irradiation, showing the persistence of cancellous bone loss, although mechanical properties in compression were unaffected. In sum, a single dose of γ radiation rapidly increased osteoclast surface in cancellous tissue and compromised cancellous microarchitecture in the remodeling appendicular and axial skeleton of postpubertal mice.

Tanaka, K., Kohda, A., Satoh, K., Toyokawa, T., Ichinohe, K., Ohtaki, M. and Oghiso, Y. Dose-Rate Effectiveness for Unstable-Type Chromosome Aberrations Detected in Mice after Continuous Irradiation with Low-Dose-Rate γ Rays. Radiat. Res. 171, 290–301 (2009).

Chronological changes in the chromosome aberration rates of splenocytes from specific-pathogen-free (SPF) mice after continuous and long-term exposure to low-dose-rate γ rays were studied. Incidences of dicentrics plus centric rings (Dic Rc), detected by conventional Giemsa staining, and dicentric chromosomes, detected by fluorescence in situ hybridization (Dic by FISH) using a centromere probe, showed an essentially linear increase up to a total accumulated dose of 8000 mGy after irradiation for about 400 days at a low dose rate of 20 mGy/day. For comparison, acute high-dose-rate and medium-dose-rate irradiation were performed. The values of the α coefficients in the linear regression lines for these unstable-type aberrations decreased as the dose rates were lowered from medium dose rates (200 and 400 mGy/day) to low dose rates (1 and 20 mGy/day). The dose and dose-rate effectiveness factor (DDREF), estimated by the ratio of calculated incidences using the best-fit regression lines at a high dose rate (890 mGy/min) and low dose rate (20 mGy/day), was 4.5 for Dic by FISH and 5.2 for Dic Rc, respectively, at the same dose of 100 mGy, while different DDREFs were obtained for different accumulated doses. This is the first study to provide information regarding the effects of long-term exposure to low-dose-rate radiation on chromosomes.

Suslova, K. G., Sokolova, A. B., Krahenbuhl, M. P. and Miller, S. C. The Effects of Smoking and Lung Health on the Organ Retention of Different Plutonium Compounds in the Mayak PA Workers. Radiat. Res. 171, 302–309 (2009).

The purpose of this study was to determine the effects of smoking and lung health on the pulmonary and extrapulmonary retention after inhalation of different chemical forms of plutonium with different solubilities in workers from the Mayak Production Association (Ozersk, Russia). Samples of lung, pulmonary lymph nodes, liver and skeleton were obtained from 800 workers who died between 1962–2000. The chemical form of plutonium aerosols, smoking history and presence of lung disease were determined. In workers with normal lung status, all plutonium chemical classes were about equally distributed between the lung parenchyma and pulmonary lymph nodes. The more insoluble chemical forms of plutonium had a greater retention in pulmonary than systemic tissues regardless of smoking history or lung health status. A history of smoking did, however, result in a significantly greater retention of less soluble chemical forms of plutonium in pulmonary tissues of workers with no lung disease. In workers with lung disease, smoking did not significantly influence the terminal organ retention of the different chemical forms of plutonium. These initial data can be used to modify dosimetry and biokinetics models used for estimating radiation risks from plutonium in humans.

Demidenko, E., Williams, B. B. and Swartz, H. M. Radiation Dose Prediction Using Data on Time to Emesis in the Case of Nuclear Terrorism. Radiat. Res. 171, 310–319 (2009).

A rigorous statistical analysis of the retrospective estimation of radiation dose received using time to emesis and its uncertainty is provided based on 108 observations associated with accidents with significant exposures to ionizing radiation in the period 1956–2001. The standard error, confidence interval, specificity and sensitivity, and Receiver Operating Characteristic (ROC) curve are used to characterize the uncertainty of the dose prediction. The relative error of the dose prediction using time to emesis data is about 200%. Consequently, if D is the dose assessment, the 95% confidence interval is approximately (D/4, 4D). Our assessment of the precision is applied to computation of the probabilities in triage medical management in the case of a nuclear terrorism event. We also note several factors that indicate that there are additional problems in the use of time to emesis for triage, including a lack of consideration of individuals that do not vomit, differences between the conditions under which the data were obtained and the conditions under which they are likely to be used, and the potential for the incidence of vomiting to be altered by factors unrelated to radiation exposure such as psychogenic factors and the use of emetic agents. In summary, while time to emesis is a rapid and inexpensive method for estimating the radiation dose, it should be used with caution because it is imprecise and may lead to a very high false positive rate. More reliable methods for after-the-fact assessment of radiation dose are needed to complement the use of time to emesis.

Fakir, H., Hofmann, W., Tan, W. Y. and Sachs, R. K. Triggering-Response Model for Radiation-Induced Bystander Effects. Radiat. Res. 171, 320–331 (2009).

We propose a mechanistic model for radiation cell killing and carcinogenesis-related end points that combines direct and bystander responses. The model describes the bystander component as a sequence of two distinct processes: triggering of signal emission from irradiated cells and response of nonirradiated recipient cells; in principle it can incorporate microdosimetric information as well as the random aspects of signal triggering and recipient response. Late effects are modeled using a one-stage model based on the concepts of inactivation and initiation, which allows for the proliferation of normal and initiated cells; proliferation of initiated cells is analyzed using a stochastic, birth-death approach. The model emphasizes the dependence of bystander effects on dose, which is important for the assessment of low-dose cancer induction by extrapolations of risk from high-dose exposures. The results obtained show adequate agreement with different in vitro bystander experiments involving ultrasoft X rays and α particles and correctly reflect the main features observed for several end points. Our results suggest signal transmission through the medium rather than gap junctions. We suggest that for many such experiments, a moderate increase in medium volume should have about the same effect as a moderate decrease in the fraction of irradiated cells.

Schreiber, E. C. and Chang, S. X. Monte Carlo Simulation of an X-Ray Pixel Beam Microirradiation System. Radiat. Res. 171, 332–341 (2009).

Monte Carlo simulations are used in the development of a nanotechnology-based multi-pixel beam array small animal microirradiation system. The microirradiation system uses carbon nanotube field emission technology to generate arrays of individually controllable X-ray pixel beams that electronically form irregular irradiation fields having intensity and temporal modulation without any mechanical motion. The microirradiation system, once developed, will be incorporated with the micro-CT system already developed that is based on the same nanotechnology to form an integrated image-guided and intensity-modulated microirradiation system for high-temporal-resolution small animal research. Prospective microirradiation designs were evaluated based on dosimetry calculated using EGSnrc-based Monte Carlo simulations. Design aspects studied included X-ray anode design, collimator design, and dosimetric considerations such as beam energy, dose rate, inhomogeneity correction, and the microirradiation treatment planning strategies. The dosimetric properties of beam energies between 80–400 kVp with varying filtration were studied, producing a pixel beam dose rate per current of 0.35–13 Gy per min per mA at the microirradiation isocenter. Using opposing multi-pixel-beam array pairs reduces the dose inhomogeneity between adjacent pixel beams to negligible levels near the isocenter and 20% near the mouse surface.

Roginskaya, M. and Razskazovskiy, Y. Selective Radiation-Induced Generation of 2-Deoxyribonolactone Lesions in DNA Mediated by Aromatic Iodonium Derivatives. Radiat. Res. 171, 342–348 (2009).

2-Deoxyribonolactone lesions were identified as major products of radiation damage to DNA mediated by o,o′-diphenyleneiodonium cations in a hydroxyl radical-scavenging environment. The highest selectivity toward deoxyribonolactone formation (up to 86% of all sugar-phosphate damages) and the overall reaction efficiency (up to 40% of all radiation-generated intermediates converted into products) was displayed by derivatives with positively charged (2-aminoethylthio)acetylamino and (2-aminoethylamino)acetylamino side chains. The reaction can be useful for random single-step incorporation of deoxyribonolactone lesions into single- and double-stranded oligonucleotides and highly polymerized DNA directly in commonly used buffers (PBS, phosphate, Tris-HCl, etc.) at room temperature. In combination with HPLC separation, this technique can serve as a source of short (<6 mer) sequences containing deoxyribonolactone lesions at known positions.

Marrale, M., Brai, M., Barbon, A. and Brustolon, M. Analysis of the Spatial Distribution of Free Radicals in Ammonium Tartrate by Pulse EPR Techniques. Radiat. Res. 171, 349–359 (2009).

Using pulse electron paramagnetic resonance (EPR) on a series of l( )-ammonium tartrate (AT) dosimeters exposed to radiations with different linear energy transfer (LET), we assessed the ability of pulse EPR spectroscopy to discriminate the quality of various radiation beams such as ⁶⁰Co γ-ray photons, protons and thermal neutrons at various doses by analyzing the local radical distributions produced by the different beams. We performed two types of pulse EPR investigations: two-pulse electron spin echo decay obtained by varying the microwave power, and a double electron-electron resonance (DEER) study. Both methods provide information about the dipolar interactions among the free radicals and about their spatial distributions. The first method provided information on the instantaneous diffusion and hence the microscopic concentration of the radicals that is compared with the macroscopic one obtained by CW-EPR. The DEER spectra yielded the distributions of distances between pairs of radicals two to five crystal cells apart produced by the same radiation event, a result reported here for the first time. The inter-radical distributions given by the DEER results have been simulated by modeling the radical distributions according to the details of the matter-radiation interactions for the various beams. The results of both types of pulse experiments are strongly dependent on the radiation quality. This was also observed for samples giving indistinguishable CW-EPR spectral profiles. We conclude that the pulse EPR measurements can be valuable tools for distinguishing the LET of the radiation beams, an important parameter for radiobiological considerations.

Hou, Y-N., Lavaf, A., Huang, D., Peters, S., Huq, R., Friedrich, V., Rosenstein, B. S. and Kao, J. Development of an Automated γ-H2AX Immunocytochemistry Assay. Radiat. Res. 171, 360–367 (2009).

γ-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify γ-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for γ-H2AX focus formation by immunofluorescence analysis. The γ-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and γ-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r² correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when γ-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of γ-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify γ-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.

Sieber, F., Muir, S. A., Cohen, E. P., North, P. E., Fish, B. L., Irving, A. A., Mäder, M. and Moulder, J. E. High-Dose Selenium for the Mitigation of Radiation Injury: A Pilot Study in a Rat Model. Radiat. Res. 171, 368–373 (2009).

The purpose of this study was to evaluate in an animal model the safety and efficacy of dietary supplementation with high doses of selenium for the mitigation of the type of radiation injury that might be sustained during a nuclear accident or an act of radiological terrorism. Age-matched male rats were exposed to 10 Gy (single dose) of total-body irradiation (TBI) followed by a syngeneic bone marrow transplant, then randomized to standard drinking water or drinking water supplemented with sodium selenite or seleno-l-methionine. At 21 weeks after TBI, most rats on standard drinking water had severe renal failure with a mean blood urea nitrogen (BUN) level of 124 ± 29 mg/dl (geometric mean ± SE) whereas rats on selenium-supplemented drinking water (100 μg/day) had a mean BUN level of 67 ± 12 mg/dl. The mitigating effect of selenium was confirmed by histopathological analyses. None of the animals on high-dose selenium showed signs of selenium toxicity. Our results suggest that dietary supplementation with high-dose selenium may provide a safe, effective and practical way to mitigate radiation injury to kidneys.

Leonard, B. E. The Range of the Bystander Effect Signal in three-Dimensional Tissue and Estimation of the Range in Human Lung Tissue at Low Radon Levels. Radiat. Res. 171, 374–378 (2009).

It is shown in the measurements of Belyakov et al. (Proc. Natl. Acad. Sci. USA 102, 14203–14208, 2005) that the distribution of microbeam-irradiated cells represents a finite line source distribution for the emission of bystander effect signals. By assuming a plane source configuration for the propagation of the bystander effect deleterious signal, in the modeling analysis of Shuryak et al. (Radiat. Res. 168, 741–749, 2007) there is an overestimate of the range to effectiveness. A correction is provided to this analysis to estimate the bystander signal propagation distance in the case of low domestic radon levels where single isolated cells are “hit” by α particles and emit the bystander signals as a “point” source to neighboring bystander effect receptor cells. The result is an estimate, but it shows that with the spatial correction the deleterious propagation range would be of the order of 210 μm, a factor of about 5 less than prior predictions.

Meesungnoen, J. and Jay-Gerin, J-P. High-LET Ion Radiolysis of Water: Oxygen Production in Tracks. Radiat. Res. 171, 379–386 (2009).

It is known that molecular oxygen is a product of the radiolysis of water with high-linear energy transfer (LET) radiation, a result that is of particular significance in radiobiology and of practical relevance in radiotherapy. In fact, it has been suggested that the radiolytic formation of an oxygenated microenvironment around the tracks of high-LET heavy ions is an important factor in their enhanced biological efficiency in the sense that this may be due to an “oxygen effect” by O₂ produced by these ions in situ. Using Monte Carlo track simulations of pure, deaerated water radiolysis by 4.8 MeV ⁴He² (LET ∼ 94 keV/μm) and 24 MeV ¹²C⁶ (LET ∼ 490 keV/μm) ions, including the mechanism of multiple ionization of water, we have calculated the yields and concentrations of O₂ in the tracks of these irradiating ions as a function of time between ∼10⁻¹² and 10⁻⁵ s at 25 and 37°C. The track oxygen concentrations obtained compare very well with O₂ concentrations estimated from the “effective” amounts of oxygen that are needed to produce the observed reduction in oxygen enhancement ratio (OER) with LET (assuming this decrease is attributable to the sole radiolytic formation of O₂ in the tracks). For example, for 24 MeV ¹²C⁶ ions, the initial track concentration of O₂ is estimated to be more than three orders of magnitude higher than the oxygen levels present in normally oxygenated and hypoxic tumor regions as well as in normal human cells. Such results, which largely plead in favor of the “oxygen in the heavy-ion track” hypothesis, could explain at least in part the greater efficiency of high-LET radiation for cell inactivation (at equal radiation dose).