Wilson, P. F., Nagasawa, H., Warner, C. L., Fitzek, M. M., Little, J. B. and Bedford, J. S. Radiation Sensitivity of Primary Fibroblasts from Hereditary Retinoblastoma Family Members and Some Apparently Normal Controls: Colony Formation Ability during Continuous Low-Dose-Rate Gamma Irradiation. Radiat. Res. 169, 483–494 (2008).

We previously described an enhanced sensitivity for cell killing and G₁-phase cell cycle arrest after acute γ irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1 ^/− probands and unaffected RB1 ^/ parents) as well as distinctive gene expression profiles in unirradiated cultures by microarray analyses. In the present study, we measured the colony formation ability of these cells after exposure to continuous low-dose-rate (0.5–8.4 cGy/h) ¹³⁷Cs γ radiation for a 2-week growth period. Fibroblasts from all RB family members (irrespective of RB1 genotype) and from 5 of 18 apparently normal Coriell cell bank controls were significantly more radiosensitive than the remaining apparently normal controls. The average dose rates required to reduce relative survival to 10% and 1% were ∼3.1 and 4.7 cGy/h for the Coriell control strains with normal radiosensitivity and ∼1.4 and 2.5 cGy/h for the radiosensitive RB family member and remaining apparently normal Coriell control strains. The finding that a significant proportion of fibroblast strains derived from apparently normal individuals are sensitive to chronic low-dose-rate irradiation indicates such individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes that may likewise predispose them or their children to cancer.

Murley, J. S., Nantajit, D., Baker, K. L., Kataoka, Y., Li, J. J. and Grdina, D. J. Maintenance of Manganese Superoxide Dismutase (SOD2)-Mediated Delayed Radioprotection Induced by Repeated Administration of the Free Thiol Form of Amifostine. Radiat. Res. 169, 495–505 (2008).

Thiol-containing drugs such as WR1065, the free thiol form of amifostine, have been shown to induce a delayed radioprotective effect in both malignant and non-malignant cells. In mammalian cells exposed to a dose as low as 40 μM WR1065, the redox-sensitive nuclear transcription factor κB (NFκB) is activated, leading to an elevation in the expression of the antioxidant gene manganese superoxide dismutase (SOD2) and a concomitant increase in active SOD2 enzyme levels that peaks 24 to 32 h later. Exposure of cells to ionizing radiation during the period of elevated SOD2 enzymatic activity results in an enhanced radiation resistance. This is seen as an increase in surviving fraction as determined by standard colony formation assays. To determine whether this delayed radioprotection can be maintained over a prolonged period in cells of either malignant or non-malignant origin, both human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were grown to confluence and exposed to 40 μM WR1065 using three administration protocols: (1) daily drug exposure for 10 days followed each day by irradiation with 2 Gy; (2) drug exposure once every 48 h followed by irradiation with 2 Gy 48 h later for 14 days; and (3) drug exposure every 72 h followed by irradiation with 2 Gy 72 h later for 12 days. As a function of each experimental condition, cell numbers and associated SOD2 enzymatic activities were measured at the time of each irradiation. None of the treatment conditions were toxic to either HMEC or SA-NH cells. SOD2 activity was elevated 5.3- and 1.8-fold over background on average for HMEC exposed to 40 μM WR1065 every 24 or 48 h, respectively. Likewise, SOD2 activity was elevated in SA-NH mouse sarcoma cells 7.8- and 4.9-fold after daily exposure to WR1065 or exposure to WR1065 once every 48 h, respectively. Both HMEC and SA-NH cells exhibited enhanced radiation resistance that correlated with the increase in SOD2 activity. The average respective increases in cell survival were 1.33 ± 0.01 (SEM), 1.23 ± 0.01 and 1.04 ± 0.01 for HMEC exposed to WR1065 every 24, 48 and 72 h, respectively, and 1.27 ± 0.01, 1.18 ± 0.02 and 1.02 ± 0.02 for SA-NH cells exposed to WR1065 every 24, 48 and 72 h, respectively. Both the elevation in WR1065-induced SOD2 enzymatic activity and the corresponding increase in radiation resistance were completely inhibited in HMEC and SA-NH cells transfected with human or mouse SOD2 siRNA oligomers and irradiated 24 h later. These data demonstrate that a delayed radioprotective effect can be induced and maintained over a prolonged period in both non-malignant and malignant cells exposed to thiol-containing drugs such as WR1065. For non-malignant cells this represents a novel paradigm for radiation protection. The ability of WR1065 to induce a persistent elevated radiation resistance in malignant cells, however, suggests a new potential concern regarding the issue of tumor protection in patients exposed to thiol-containing drugs.

Karlsson, K. H., Radulescu, I., Rydberg, B. and Stenerlöw, B. Repair of Radiation-Induced Heat-Labile Sites is Independent of DNA-PKcs, XRCC1 and PARP. Radiat. Res. 169, 506– 512 (2008).

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after γ irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t_1/2 = 7–8 min) and slow (t_1/2 = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60–90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.

Li, H., Liu, N., Rajendran, G. K., Gernon, T. J., Rockhill, J. K., Schwartz, J. L. and Gu, Y. A Role for Endogenous and Radiation-Induced DNA Double-Strand Breaks in p53-Dependent Apoptosis during Cortical Neurogenesis. Radiat. Res. 169, 513–522 (2008).

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.

Vlkolinský, R., Krucker, T., Nelson, G. A. and Obenaus, A. ⁵⁶Fe-Particle Radiation Reduces Neuronal Output and Attenuates Lipopolysaccharide-Induced Inhibition of Long-Term Potentiation in the Mouse Hippocampus. Radiat. Res. 169, 523–530 (2008).

Exposure to space radiation consisting of high-energy charged ⁵⁶Fe particles represents a significant health risk for astronauts. ⁵⁶Fe-particle radiation affects the synaptic plasticity of the hippocampus and alters its response to the experimental immunological stressor lipopolysaccharide (LPS). We previously showed in mice that 1 month after exposure to ⁵⁶Fe-particle radiation, the LPS-induced inhibition of hippocampal long-term potentiation (LTP) was significantly attenuated, resulting in seemingly normal LTP. In the current study, we investigated this phenomenon further at longer times postirradiation. We exposed mice to accelerated iron particles (⁵⁶Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only), and 1, 3, 6 or 12 months postirradiation we administered LPS. Four hours after the intraperitoneal LPS injection, we prepared hippocampal slices to measure synaptic excitability and plasticity between CA3–CA1 neurons. In unexposed mice, we confirmed that LPS inhibited LTP at all times. However, in mice exposed to 2 Gy, the LPS-induced LTP inhibition was attenuated and reversed to control values. Such reversal was evident at 1 and 3 months but not 6 and 12 months postirradiation. In addition, at 6 and 12 months postirradiation, we observed inhibition of population spike (PS) amplitudes at 4 Gy that correlated with decrements in dendritic potentials, suggesting synaptic damage. Our data show that ⁵⁶Fe-particle radiation affects the response of the hippocampus to an immunological stressor and that the alterations progress over time.

Haase, M. G., Klawitter, A., Bierhaus, A., Yokoyama, K. K., Kasper, M., Geyer, P., Baumann, M. and Baretton, G. B. Inactivation of AP1 Proteins by a Nuclear Serine Protease Precedes the Onset of Radiation-Induced Fibrosing Alveolitis. Radiat. Res. 169, 531–542 (2008).

Radiation-induced lung damage comprises inflammation (alveolitis) as well as disturbed regulation of cell differentiation and proliferation (fibrosis). The transcriptional regulation of this process is poorly understood. One key transcription factor involved in the regulation of proliferation and differentiation is AP1 (activator protein 1). The present study examined changes in the DNA-binding activity of AP1 after irradiation and defined the underlying molecular mechanisms in an animal model. The right lungs of Fischer rats received a single radiation dose of 20 Gy. Lung tissue was tested for AP1 DNA-binding activity, AP1 mRNA, and levels of AP1 proteins as well as for c-Jun specific proteolytic activity. After an initial increase, the AP1 DNA-binding activity was completely lost starting at 5.5 weeks after irradiation, which is 2.5 weeks before the onset of fibrosing alveolitis. This was not caused by reduction of mRNA levels or size. Instead, a selective nuclear cleavage of c-Jun by a serine protease caused the loss of AP1 activity. Considering the central role of AP1 in cell proliferation and differentiation and the strict timely correlation to the onset of the disease, the complete loss of AP1 function is likely to play a critical role in radiation-induced fibrosing alveolitis.

Bertho, J. M., Roy, L., Souidi, M., Benderitter, M., Gueguen, Y., Lataillade, J. J., Prat, M., Fagot, T., De Revel, T. and Gourmelon, P. New Biological Indicators to Evaluate and Monitor Radiation-Induced Damage: An Accident Case Report. Radiat. Res. 169, 543–550 (2008).

The aim of this work was to use several new biological indicators to evaluate damage to the main physiological systems in a victim exposed accidentally to ionizing radiation. Blood samples were used for biological dosimetry and for measurement of the plasma concentrations of several molecules: Flt3 ligand to assess the hematopoietic system, citrulline as an indicator of the digestive tract, and several oxysterols as lipid metabolism and vascular markers. The cytogenetic evaluation estimated the dose to the victim to be between 4.2 and 4.8 Gy, depending on the methodology used. Monitoring the Flt3 ligand demonstrated the severity of bone marrow aplasia. In contrast, the citrulline concentration showed the absence of gastrointestinal damage. Variations in oxysterol concentrations suggested radiation-induced damage to the liver and the cardiovascular system. These results were correlated with those from classic biochemical markers, which demonstrated severe damage to the hematopoietic system and suggested the appearance of subclinical damage to the liver and cardiovascular system. These results demonstrate for the first time the importance of a multiparameter biological approach in the evaluation of radiation damage after accidental irradiation.

Wilkins, R. C., Romm, H., Kao, T-C., Awa, A. A., Yoshida, M. A., Livingston, G. K., Jenkins, M. S., Oestreicher, U., Pellmar, T. C. and Prasanna, P. G. S. Interlaboratory Comparison of the Dicentric Chromosome Assay for Radiation Biodosimetry in Mass Casualty Events. Radiat. Res. 169, 551–560 (2008).

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose–effect calibration curve for the yield of dicentrics for ⁶⁰Co γ rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c αD βD². For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.

Vijayalaxmi and Prihoda, T. J. Genetic Damage in Mammalian Somatic Cells Exposed to Radiofrequency Radiation: A Meta-analysis of Data from 63 Publications (1990–2005). Radiat. Res. 169, 561–574 (2008).

During the last several decades, numerous researchers have examined the potential of in vitro and/or in vivo exposure of radiofrequency (RF) radiation to damage the genetic material in mammalian somatic cells. A meta-analysis of reported data was conducted to obtain a quantitative estimate (with 95% confidence intervals) of genotoxicity in RF-radiation-exposed cells compared with sham-exposed/unexposed control cells. The extent of genotoxicity was assessed for various end points, including single- and double-strand breaks in the DNA, incidence of chromosomal aberrations, micronuclei and sister chromatid exchanges. Among the several variables in the experimental protocols used in individual investigations, the influence of three specific variables related to RF-radiation exposure characteristics was examined in the meta-analysis: frequency, specific absorption rate, and exposure as continuous-wave, pulsed-wave and occupationally exposed/cell phone users. The overall data indicated that (1) the difference between RF-radiation-exposed and sham-/unexposed controls as well as the effect size or standardized mean difference due to RF-radiation exposure was small with very few exceptions; (2) at certain RF radiation exposure conditions, there were statistically significant increases in genotoxicity for some end points; and (3) the mean indices for chromosomal aberrations and micronuclei in RF-radiation-exposed and sham-/unexposed controls were within the spontaneous levels reported in the historical database. Considerable evidence for publication bias was found in the meta-analysis.

Manti, L., Braselmann, H., Calabrese, M. L., Massa, R., Pugliese, M., Scampoli, P., Sicignano, G. and Grossi, G. Effects of Modulated Microwave Radiation at Cellular Telephone Frequency (1.95 GHz) on X-Ray-Induced Chromosome Aberrations in Human Lymphocytes In Vitro. Radiat. Res. 169, 575–583 (2008).

The case for a DNA-damaging action produced by radiofrequency (RF) signals remains controversial despite extensive research. With the advent of the Universal Mobile Telecommunication System (UMTS) the number of RF-radiation-exposed individuals is likely to escalate. Since the epigenetic effects of RF radiation are poorly understood and since the potential modifications of repair efficiency after exposure to known cytotoxic agents such as ionizing radiation have been investigated infrequently thus far, we studied the influence of UMTS exposure on the yield of chromosome aberrations induced by X rays. Human peripheral blood lymphocytes were exposed in vitro to a UMTS signal (frequency carrier of 1.95 GHz) for 24 h at 0.5 and 2.0 W/kg specific absorption rate (SAR) using a previously characterized waveguide system. The frequency of chromosome aberrations was measured on metaphase spreads from cells given 4 Gy of X rays immediately before RF radiation or sham exposures by fluorescence in situ hybridization. Unirradiated controls were RF-radiation- or sham-exposed. No significant variations due to the UMTS exposure were found in the fraction of aberrant cells. However, the frequency of exchanges per cell was affected by the SAR, showing a small but statistically significant increase of 0.11 exchange per cell compared to 0 W/kg SAR. We conclude that, although the 1.95 GHz signal (UMTS modulated) does not exacerbate the yield of aberrant cells caused by ionizing radiation, the overall burden of X-ray-induced chromosomal damage per cell in first-mitosis lymphocytes may be enhanced at 2.0 W/kg SAR. Hence the SAR may either influence the repair of X-ray-induced DNA breaks or alter the cell death pathways of the damage response.

Dingfelder, M., Ritchie, R. H., Turner, J. E., Friedland, W., Paretzke, H. G. and Hamm, R. N. Comparisons of Calculations with PARTRAC and NOREC: Transport of Electrons in Liquid Water. Radiat. Res. 169, 584–594 (2008).

Monte Carlo computer models that simulate the detailed, event-by-event transport of electrons in liquid water are valuable for the interpretation and understanding of findings in radiation chemistry and radiation biology. Because of the paucity of experimental data, such efforts must rely on theoretical principles and considerable judgment in their development. Experimental verification of numerical input is possible to only a limited extent. Indirect support for model validity can be gained from a comparison of details between two independently developed computer codes as well as the observable results calculated with them. In this study, we compare the transport properties of electrons in liquid water using two such models, PARTRAC and NOREC. Both use interaction cross sections based on plane-wave Born approximations and a numerical parameterization of the complex dielectric response function for the liquid. The models are described and compared, and their similarities and differences are highlighted. Recent developments in the field are discussed and taken into account. The calculated stopping powers, W values, and slab penetration characteristics are in good agreement with one another and with other independent sources.

Brown, E. T., Robinson-Benion, C. and Holt, J. T. Radiation Enhances Caspase 3 Cleavage of Rad51 in BRCA2-Defective Cells. Radiat. Res. 169, 595–601 (2008).

After DNA damage, caspases cleave and activate proteins involved in cell death by apoptosis but also cleave and inactivate proteins implicated in DNA repair. Here we report a rapid onset of Rad51 cleavage by caspase 3 in BRCA2-defective mouse and human cells. This rapid cleavage was reduced markedly by transfer of full-length human BRCA2 into BRCA2-defective mouse or human cells, which also blocked the association of caspase 3 and Rad51 proteins. Overall caspase 3 activity was increased in BRCA2-defective cells, but the time course was much slower than that for Rad51 cleavage. We further showed that caspase 3 cleavage of Rad51 resulted in a functional decrease in Rad51 strand exchange activity and that inhibition of caspase 3 activity increased Rad51 protein levels and Rad51 foci. These findings indicate that BRCA2 inhibits Rad51 cleavage and subsequent apoptosis.