Słonina, D., Biesaga, B., Urbański, K. and Kojs, Z. Low-Dose Radiation Response of Primary Keratinocytes and Fibroblasts from Patients with Cervix Cancer. Radiat. Res. 167, 251–259 (2007).

The aim of the present study was to examine, using the micronucleus (MN) assay, the low-dose radiation response of normal skin cells from cancer patients and to determine whether the hyper-radiosensitivity (HRS)-like phenomenon occurs in cells of these patients. Primary skin fibroblasts and keratinocytes derived from 40 patients with cervix cancer were studied. After in vitro γ irradiation with single doses ranging from 0.05 to 4 Gy, MN induction was assessed. For each patient, the linear-quadratic (LQ) model and the induced repair (IR) model were fitted over the whole data set. In fits of the IR model, an HRS-like response after low doses (seen as the deviation over the LQ curve) was demonstrated for the fibroblasts of two patients and for the keratinocytes of four other patients. The α_s/α_r ratio for the six patients ranged from 2.7 to 15.4, whereas the values of the parameter d_c ranged from 0.13 to 0.36 Gy. No relationship was observed between chromosomal radiosensitivity of fibroblasts and keratinocytes derived from the same donor in the low-dose (0.1–0.25 Gy) region. In conclusion, the fact that low-dose chromosomal hypersensitivity was observed for cells of only six of the patients studied suggests that it is not a common finding in human normal cells and can represent an individual characteristic.

Krueger, S. A., Joiner, M. C., Weinfeld, M., Piasentin, E. and Marples, B. Role of Apoptosis in Low-Dose Hyper-radiosensitivity. Radiat. Res. 167, 260–267 (2007).

Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557–566, 2004). We also show that enriching populations of MR4 and V79 cells with G₁-phase cells, to minimize the numbers of G₂-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G₂-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G₂-phase cells harboring unrepaired DNA breaks entering mitosis.

Rosi, A., Grande, S., Luciani, A. M., Palma, A., Giovannini, C., Guidoni, L., Sapora, O. and Viti, V. Role of Glutathione in Apoptosis Induced by Radiation as Determined by ¹H MR Spectra of Cultured Tumor Cells. Radiat. Res. 167, 268–282 (2007).

The relationship between apoptosis induced by γ radiation and glutathione in cells of two human cancer cell lines, HeLa from cervix carcinoma and MCF-7 from mammary carcinoma, was examined. MCF-7 cells appeared to be more radioresistant than HeLa cells, and radiation-induced apoptosis, which was monitored by assessing phosphatidylserine externalization, was observed in HeLa cells but not in MCF-7 cells. Glutathione levels monitored by ¹H MRS were higher in MCF-7 cells than in HeLa cells, while the opposite was true for the free glu signals. MCF-7 cells became more radiosensitive when treated with 0.1 mM buthionine sulfoximine, which inhibits GSH synthesis through inactivation of γ-glutamylcysteine synthetase, with the concomitant appearance of radiation-induced apoptosis. We can thus reasonably associate, at least in part, the resistance of MCF-7 cells to apoptosis with a high level of glutathione and probably with a high activity of γ-glutamylcysteine synthetase. A late decrease in glutathione concentration after irradiation was observed in MCF-7 cells, but not in HeLa cells and to a lesser degree in buthionine sulfoximine-treated MCF-7 cells. This would indicate that the radiation-induced decrease in glutathione concentration is not related to the onset of apoptosis, but it is more likely related to glutathione consumption as a result of detoxification reactions.

Shao, C., Furusawa, Y., Matsumoto, Y., Pan, Y., Xu, P. and Chen, H. Effect of Gap Junctional Intercellular Communication on Radiation Responses in Neoplastic Human Cells. Radiat. Res. 167, 283–288 (2007).

Gap junctional intercellular communication (GJIC) is an important function of metazoan cells and is believed to have beneficial effects in anti-tumor therapy. In this study, we found that, when neoplastic human salivary gland (HSG) cells were irradiated with a 100 keV/μm carbon-ion beam, micronuclei, G₂/M-phase arrest, and cell killing were induced and that their induction increased with dose. Treatment of confluent HSG cells with 8-Br-cAMP increased GJIC between cells. After release from this treatment, the cell cycle progress and the formation of binucleated cells were still similar to those of untreated cells. However, radiation-induced cellular damage, including micronucleus (MN) formation and G₂/M-phase arrest of that cAMP-treated population, was less than that of the untreated population and that the surviving fraction was slightly enhanced by cAMP treatment, suggesting that increased GJIC protects HSG cells from lethal radiation damage. Moreover, when confluent HSG cells were treated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of nitric oxide (NO) free radical, MN induction and cell killing in the irradiated population were increased. Our results indicate that NO may be involved in GJIC-mediated radioprotection of HSG cells, which may have implications for radiotherapy.

Epperly, M. W., Wegner, R., Kanai, A. J., Kagan, V., Greenberger, E. E., Nie, S. and Greenberger, J. S. Effects of MnSOD-Plasmid Liposome Gene Therapy on Antioxidant Levels in Irradiated Murine Oral Cavity Orthotopic Tumors. Radiat. Res. 167, 289–297 (2007).

Intraoral manganese superoxide dismutase (SOD2)-plasmid liposome (PL) radioprotective gene therapy prolongs the survival of mice with orthotopic oral cavity tumors within the irradiated field. To determine whether the mechanism involved effects in antioxidant pool, C57BL/6J mice bearing orthotopic oral cavity squamous cell carcinoma SCC-VII tumors received intraoral or intravenous MnSOD-PL gene therapy 24 h prior to 18 Gy irradiation to the head and neck region. Glutathione (GSH) levels and levels of radiation-generated nitric oxide and peroxynitrite were measured in orthotopic tumors and in adjacent oral mucosa. MnSOD-PL transfection of the SCC-VII tumor cells, but not normal embryo fibroblasts, produced acute radiosensitization. Furthermore, SCC-VII tumor cells demonstrated increased relative hydrogen peroxide (the product of MnSOD superoxide dismutation)-induced apoptosis in vitro. Radiation decreased levels of GSH and increased GPX in both tumor and normal cells in vitro, effects that were blunted by MnSOD-PL treatment. In vivo irradiation decreased GSH and GPX more effectively in tumors, and the decrease was not reversed by MnSOD-PL therapy. Intravenous but not intraoral administration of epitope-tagged hemagglutinin MnSOD-PL resulted in significant uptake in orthotopic tumors and decreased the levels of radiation-induced nitric oxide and peroxynitrite. Thus normal tissue radioprotective MnSOD-PL gene therapy radiosensitizes tumor cell lines in vitro and has a therapeutic effect on orthotopic tumors in part through its effects on tumor antioxidants.

Yang, G., Wu, L. J., Chen, L. Y., Pei, B., Wang, Y. G., Zhan F. R., Wu, Y. J. and Yu, Z. L. Targeted Irradiation of Shoot Apical Meristem of Arabidopsis Embryos Induces Long-Distance Bystander/Abscopal Effects. Radiat. Res. 167, 298– 305 (2007).

Bystander effects induced by low-dose ionizing radiation have been shown to occur widely in many cell types and may have a significant impact on radiation risk assessment. Although the region of radiation damage is known to be much greater than the initial target volume irradiated, it remains to be seen whether this response is limited to the specific organ irradiated, spans a limited region of the body, or even covers the whole body of the target. To determine whether long-distance bystander/abscopal effects exist in whole organisms and to clarify the problem of intercellular communication, in the present study a specific cell group, the shoot apical meristem in Arabidopsis embryo, was irradiated with a defined number of protons and examined for root development postirradiation. The results showed that after direct damage to the shoot apical meristem from ion traversals, root hair differentiation, primary root elongation and lateral root initiation were all inhibited significantly in postembryonic development, suggesting that radiation-induced long-distance bystander/abscopal responses might exist in the whole organism. To further scrutinize the mechanism(s) underlying these inhibitory effects, a DR5-GUS transgenic Arabidopsis was used. The results showed that accumulation of the reporter GUS gene transcript in irradiated shoot apical meristem embryos decreased in the postembryonic development. Treatment with either 2,4-dichlorophenoxyacetic acid, a synthetic plant auxin, or DMSO, a effective reactive oxygen species (ROS) scavenger, could rescue the reporter GUS enzyme accumulation and the length of primary root in irradiated shoot apical meristem embryos, indicating that ROS or probably the ROS related auxin and auxin-dependent transcription process may be involved in radiation-induced long-distance bystander/abscopal effects.

Zeni, O., Di Pietro, R., d'Ambrosio, G., Massa, R., Capri, M., Naarala, J., Juutilainen, J. and Scarfì, M. R. Formation of Reactive Oxygen Species in L929 Cells after Exposure to 900 MHz RF Radiation with and without Co-exposure to 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone. Radiat. Res. 167, 306–311 (2007).

The aim of this study was to investigate the induction of reactive oxygen species in murine L929 fibrosarcoma cells exposed to radiofrequency (RF) radiation at 900 MHz, with or without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent environmental carcinogen produced during chlorination of drinking water. Both continuous-wave and GSM mobile phone signals were applied for 10 or 30 min at specific absorption rates of 0.3 and 1 W/kg. Simultaneous sham exposures were performed for each exposure condition. MX treatment was performed at a subtoxic level of 500 μM, and the RF-field exposure was carried out during the first 10 or 30 min of the chemical treatment. The formation of reactive oxygen species was followed soon after the exposure and at different harvesting times until 1 h after RF-field treatment. The studied provided no indication that 900 MHz RF-field exposure, either alone or in combination with MX, induced formation of reactive oxygen species under any of the experimental conditions investigated. In contrast, exposure to MX resulted in a statistically significant increase in the formation of reactive oxygen species for all the treatment durations investigated, confirming that MX is an inductor of oxidative stress in L929 cells.

Claesson, A. K., Stenerlöw, B., Jacobsson, L. and Elmroth, K. Relative Biological Effectiveness of the α-Particle Emitter ²¹¹At for Double-Strand Break Induction in Human Fibroblasts. Radiat. Res. 167, 312–318 (2007).

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the α-particle emitter ²¹¹At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with α particles from ²¹¹At, ⁶⁰Co γ rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp–5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with α particles from ²¹¹At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for ⁶⁰Co γ rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with ²¹¹At α particles or ⁶⁰Co γ rays at 2°C or 37°C and intact cells were irradiated with ²¹¹At α particles at the same temperatures. The dose-modifying factor (DMF_temp) for irradiation of nuclear monolayers at 37°C compared with 2°C was 1.7 for ²¹¹At α particles and 1.6 for ⁶⁰Co γ rays. No temperature effect was observed for intact cells irradiated with ²¹¹At. In conclusion, irradiation with α particles from ²¹¹At induced two to three times more DSB than γ rays and X rays.

Elsässer, T. and Scholz, M. Cluster Effects within the Local Effect Model. Radiat. Res. 167, 319–329 (2007).

The local effect model predicts the relative biological effectiveness (RBE) for different ions and cell lines starting from the corresponding experimental photon data and an amorphous track structure model. Here we present an extension of the model that takes cluster effects of single-strand breaks (SSBs) at the nanometer scale into account. In line with the main idea of the local effect model, we take the yields of SSBs and double-strand breaks (DSBs) from experimental photon data and use a Monte Carlo method to distribute them onto the DNA. We score clusters of SSBs where individual SSBs are separated by less than 25 bp as additional DSBs. Assuming that the number of DSBs is a measure of cell lethality, we derive a modified cell survival curve for photons that takes these cluster effects into account. In combination with an improved radial dose distribution, we find that the extended local effect model including cluster effects reproduces most experimental data better than the original local effect model and thus enhances the accuracy of the local effect model.

Lin, Z.W. and Adams, J. H., Jr. Effects of Nuclear Cross Sections at Different Energies on the Radiation Hazard from Galactic Cosmic Rays. Radiat. Res. 167, 330–337 (2007).

The radiation hazard for astronauts from galactic cosmic rays (GCR) is a major obstacle to long-duration human space exploration. Space radiation transport codes have been developed to calculate the radiation environment on missions to the Moon, Mars and beyond. We have studied how uncertainties in fragmentation cross sections at different energies affect the accuracy of predictions from such radiation transport calculations. We find that, in deep space, cross sections at energies between 0.3 and 0.85 GeV/nucleon usually have the largest effect on the dose equivalent behind shielding in solar minimum GCR environments and that cross sections at energies between 0.85 and 1.2 GeV/nucleon have the largest effect in solar maximum GCR environments. At the International Space Station, cross sections at higher energies have the largest effect due to the geomagnetic cutoff.