Bouffler, S. D., Bridges, B. A., Cooper, D. N., Dubrova, Y., McMillan, T. J., Thacker, J., Wright, E. G. and Waters, R. Assessing Radiation-Associated Mutational Risk to the Germline: Repetitive DNA Sequences as Mutational Targets and Biomarkers. Radiat. Res. 165, 249–268 (2006).

This review assesses recent data on mutational risk to the germline after radiation exposure obtained by molecular analysis of tandemly repeated DNA loci (TRDLs): minisatellites in humans and expanded simple tandem repeats in mice. Some studies, particularly those including exposure to internal emitters, indicate that TRDL mutation can be used as a marker of human radiation exposure; most human studies, however, are negative. Although mouse studies have suggested that TRDL mutation analysis may be more widely applicable in biomonitoring, there are important differences between the structure of mouse and human TRDLs. Mutational mechanisms probably differ between the two species, and so care should be taken in predicting effects in humans from mouse data. In mice and humans, TRDL mutations are largely untargeted with only limited evidence of dose dependence. Transgenerational mutation has been observed in mice but not in humans, but the mechanisms driving such mutation transmission are unknown. Some minisatellite variants are associated with human diseases and may affect gene transcription, but causal relationships have not yet been established. It is concluded that at present the TRDL mutation data do not warrant a dramatic revision of germline or cancer risk estimates for radiation.

Suzuki, M., Suzuki, K., Kodama, S. and Watanabe M. Phosphorylated Histone H2AX Foci Persist on Rejoined Mitotic Chromosomes in Normal Human Diploid Cells Exposed to Ionizing Radiation. Radiat. Res. 165, 269–276 (2006).

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that “residual” foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.

Nakamura, H., Yasui, Y., Saito, N., Tachibana, A., Komatsu, K. and Ishizaki, K. DNA Repair Defect in AT Cells and their Hypersensitivity to Low-Dose-Rate Radiation. Radiat. Res. 165, 277–282 (2006).

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of γ-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few γ-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of γ-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.

Han, J., Hendzel, M. J. and Allalunis-Turner, J. Quantitative Analysis Reveals Asynchronous and more than DSB-Associated Histone H2AX Phosphorylation after Exposure to Ionizing Radiation. Radiat. Res. 165, 283–292 (2006).

Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called γ-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual γ-H2AX foci. In addition, there are hundreds of smaller γ-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated γ-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each γ-H2AX focus as well as to discriminate DSB-related γ-H2AX foci from DSB-unrelated γ-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

Manti, L., Bowen, I. D., Stevens, D. L. and Court, J. C. Probing Lethal Damage Expression in Cytochalasin B-Induced Polykaryons by Radiation Quality. Radiat. Res. 165, 293–298 (2006).

The polykaryon-forming unit (PFU) cell survival assay is based on the postirradiation flow cytometric analysis of the DNA content accumulated in high-ploidy cells (polykaryons) induced by the cytokinesis inhibitor cytochalasin B and can provide a meaningful measure of cell radiosensitivity. In this assay, cell survival is defined as the ability to form a polykaryon of a given ploidy after irradiation. The slope of the polykaryon dose response has been shown to be highly correlated with the initial slope of the clonogenic survival curves after γ irradiation, which implies a common subset of lethal lesions. We reported previously on an apoptotic mode of cell death in the polykaryon system and on the heritability of small variations in polykaryon radioresponse. We now show that exposure of PFUs to a given dose of α particles results in a greater reduction in the proportion of cells able to reach at least 16C when compared to the same dose of low-LET radiation. This reduction is less than that observed in the low-dose (α term) region of the clonogenic curve. On the basis of published LET-dependent spectra of radiation-induced DNA damage, we suggest that this behavior reflects a differential expression of lethal damage that can be probed by varying the LET of the radiation and that base damages contributing additional complexity to clustered DNA lesions may be more deleterious in PFUs than in clonogens.

Kinoshita, M. and Hynynen, K. Mechanism of Porphyrin-Induced Sonodynamic Effect: Possible Role of Hyperthermia. Radiat. Res. 165, 299–306 (2006).

The biological effects of ultrasound have been investigated vigorously for various applications including the thermal coagulation of tissues, the opening of tight junctions, and localized gene or drug introduction. The synergistic cell killing effect of ultrasound and porphyrin derivatives, the so-called sonodynamic effect, holds promise for cancer treatment. Although several models to explain the sonodynamic effect have been proposed, its exact mechanism, especially in vivo, remains unknown. We examined the effect of a porphyrin derivative, protoporphyrin IX, on ultrasound-induced killing of HeLa cells. In some experiments, the intracellular protoporphyrin IX concentration was increased by 5-aminolevulinic acid treatment of the cells. Although extracellular protoporphyrin IX showed an enhanced cell killing effect by microbubble-enhanced ultrasound, intracellular protoporphyrin IX did not. On the other hand, intracellular protoporphyrin IX enhanced the cell killing effect of hyperthermia, which can be produced by ultrasound exposure, in a moderately acidic environment (pH 6.6). Because porphyrin derivatives are generally imported into the intracellular component in vivo, our results suggest that hyperthermia caused by ultrasound may play an important role in the sonodynamic effect induced by porphyrin derivatives.

Biaglow, J. E., Ayene, I. S., Tuttle, S. W., Koch, C. J., Donahue, J. and Mieyal, J. J. Role of Vicinal Protein Thiols in Radiation and Cytotoxic Responses. Radiat. Res. 165, 307–317 (2006).

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, β-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD (K1) and G6PD⁻ (E89) cells treated with l-buthionine sulfoximine (l-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of l-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.

Christiansen, H., Batusic, D., Saile, B., Hermann, R. M., Dudas, J., Rave-Frank, M., Hess, C. F., Schmidberger, H. and Ramadori, G. Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis. Radiat. Res. 165, 318–325 (2006).

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.

Ljungkvist, A. S. E., Bussink, J., Kaanders, J. H. A. M., Wiedenmann, N. E., Vlasman, R. and van der Kogel, A. J. Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model. Radiat. Res. 165, 326– 336 (2006).

Proliferation and hypoxia affect the efficacy of radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine adenocarcinoma tumor line (C38). The hypoxia marker pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second hypoxia marker, CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated caspase 3 staining. After immunohistochemical staining, the tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated tumors to 8% at both 8 h and 28 h after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated tumors and 90% after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in tumor oxygenation after irradiation. In addition, a decreased tumor cell density without a significant change in tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.

Takahashi, M. and Kojima, S. Suppression of Atopic Dermatitis and Tumor Metastasis in Mice by Small Amounts of Radon. Radiat. Res. 165, 337–342 (2006).

We examined the effect of radon in two experimental disease models in mice by administering radon dissolved in water at 68–203 Bq/liter. Administration of radon in drinking water to NC/Nga mice significantly delayed the progression of atopic dermatitis-like skin lesions induced by picrylchloride when administered prior to the induction of disease signs. The number of pulmonary metastatic foci in C57BL/6 mice inoculated with B16 melanoma cells was also reduced significantly by administration of radon in drinking water when the number of tumor cells was small and the radon treatment was started prior to tumor inoculation. The ratio of Ifng to Il4 produced by splenocytes from BALB/c mice immunized with DNP-Ascaris was significantly increased by administration of radon in drinking water. From these results, a modulation of immunity by radon was suggested.

Sommer, A. M. and Lerchl, A. 50 Hz Magnetic Fields of 1 mT do not Promote Lymphoma Development in AKR/J Mice. Radiat. Res. 165, 343–349 (2006).

Some epidemiological studies suggest that exposure to power-frequency magnetic fields increases the risk of leukemia, especially in children with high residential exposures. In contrast, most animal studies did not find a correlation between magnetic-field exposure and hematopoietic diseases. The present study was performed to investigate whether chronic, high-level (1 mT) magnetic-field exposure had an influence on lymphoma development in a mouse strain that is genetically predisposed to thymic lymphoblastic lymphoma. Three groups of 160 unrestrained female AKR/J mice were sham-exposed or exposed to sinusoidal 50 Hz magnetic fields beginning at the age of 12 weeks for 32 weeks, 7 days per week, either for 24 h per day or only during nighttime (12 h). Exposure was carried out in a blind design. Exposure did not affect survival time, body weight, lymphoma development or hematological parameters. The resulting data do not support the hypothesis that exposure to sinusoidal 50 Hz magnetic fields is a significant risk factor for hematopoietic diseases, even at this relatively high exposure level.

Damron, T. A., Horton, J. A., Naqvi, A., Loomis, R. M., Margulies, B. S., Strauss, J. A., Farnum, C. E. and Spadaro, J. A. Combination Radioprotectors Maintain Proliferation Better than Single Agents by Decreasing Early Parathyroid Hormone-Related Protein Changes after Growth Plate Irradiation. Radiat. Res. 165, 350–358 (2006).

Our hypothesis was that combinations of radioprotectors would be more effective than individual agents in minimizing the effects of radiation on the growth plate after single-fraction hind-limb irradiation of Sprague-Dawley rats. At 2 days postirradiation, the decrease in parathyroid hormone-related protein and parathyroid hormone receptor 1 expression in the irradiated growth plate transitional and hypertrophic zones was reversed in both of the combination groups but persisted in the groups treated with the individual drugs. By 2 weeks, positive findings unique to the combination-treatment animals included greater mean proliferation in the irradiated growth plate than on the contralateral side, smaller limb length discrepancies, reversal of the increased overall matrix area fraction, and reversal of the usual deficiency in Indian hedgehog staining in the irradiated hypertrophic zone. While all treatments had a positive effect in reversing the decrease in B-cell leukemia 2 protein and coincident increase in Bax previously observed 2 weeks postirradiation, the two combination groups had a more robust effect. Combinations of radioprotectors may achieve their beneficial additive effects in the growth plate by decreasing the usual early drop in parathyroid hormone-related protein and parathyroid hormone receptor 1 after irradiation, resulting in a cascade of parathyroid hormone-related protein-mediated events.

Nakamura, N., Cullings, H. M., Kodama, Y., Wada, T., Miyazawa, C., Lee, K. and Awa, A. A. A Method to Differentiate between the Levels of ESR Signals Induced by Sunlight and by Ionizing Radiation in Teeth from Atomic Bomb Survivors. Radiat. Res. 165, 359–364 (2006).

Electron spin resonance (ESR, or electron paramagnetic resonance, EPR) analysis of tooth enamel is an effective method for the retrospective estimation of individual radiation doses. One problem with this technique is that the observed ESR signal may include a contribution from ultraviolet (UV) light exposure from sunlight, especially in front teeth. Thus there has been a need to find ways to estimate the UV-light effect in the total signal so that the net ESR dose from ionizing radiation can be determined. To examine this issue, we measured 96 teeth of various types, but with buccal and lingual parts measured separately, from a control group of atomic bomb survivors (estimated dose <5 mGy). We found that, except for molars, the mean ESR-estimated dose for the buccal halves was, on average, nearly twice that from the lingual side, which indicates that the UV-light-induced lingual dose equals the difference between the two halves. Using these corrections for UV-light exposure to front teeth that had been exposed to both ionizing radiation and UV light, it was found that the estimated radiation doses closely approximated the previously estimated ESR dose to molars from the same donors or the estimated dose arrived at with cytogenetic methods. We concluded that, when using ESR to estimate radiation dose, measuring molars is the first choice, but if only front teeth are available, separate measurements to the buccal and lingual parts can provide an estimation of the mean UV-light contribution to the ESR-determined dose.

Cai, Z., Cloutier, P., Hunting, D. and Sanche, L. Enhanced DNA Damage Induced by Secondary Electron Emission from a Tantalum Surface Exposed to Soft X Rays. Radiat. Res. 165, 365–371 (2006).

Both thick and thin films of pGEM®-3Zf(-) plasmid DNA deposited on a tantalum foil were exposed to soft X rays (effective energy of 14.8 keV) for various times in air under a relative humidity of 45% (Γ ≈ 6, where Γ is the number of water molecules per nucleotide) and 84% (Γ ≈ 21), respectively. For a thick film, the DNA damage was induced chiefly by X-ray photons. For a thin film of DNA, X-ray-induced secondary electrons emitted from the tantalum result in a substantial increase in DNA damages. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis and laser scanning. The exposure curves for the formation of nicked circular (single-strand break, SSB), linear (double-strand break, DSB), and interduplex crosslink forms 1 and 2 were obtained for both thick and thin films of DNA. The secondary electron enhancement factor for SSBs, DSBs and crosslinks of the thin film of DNA were derived to be 3.8 ± 0.5, 2.9 ± 0.7 and 7 ± 3 at Γ ≈ 6 and 6.0 ± 0.8, 7 ± 1 and 3.9 ± 0.9 at Γ ≈ 21, respectively. This study provides a molecular basis for understanding the enhanced biological effects at interfaces during diagnostic X-ray examination and radiotherapy.