De Ruyck, K., Wilding, C. S., Van Eijkeren, M., Morthier, R., Tawn, E. J. and Thierens, H. Microsatellite Polymorphisms in DNA Repair Genes XRCC1, XRCC3 and XRCC5 in Patients with Gynecological Tumors: Association with Late Clinical Radiosensitivity and Cancer Incidence. Radiat. Res. 164, 237–244 (2005).

This study investigates the association of microsatellite polymorphisms in XRCC1, XRCC3 and XRCC5 with the development of late radiation-induced radiotherapy reactions and examines the correlation between these microsatellites and cancer incidence. Sixty-two women with cervical or endometrial cancer treated with radiotherapy were included in the study. According to the CTCAEv3.0 scale, 22 patients showed late adverse radiotherapy reactions (grade 2 or more). PCR on lymphocyte DNA followed by automated fragment analysis was performed to examine the number of tandem repeat units at each locus. No significant association was found between the repeat length at any of the microsatellites in XRCC1, XRCC3 or XRCC5 and the incidence of late radiotherapy complications. Since higher odds ratios (ORs) were found for the rare XRCC1 [AC]₁₁ and [AC]₂₁ repeats (OR = 2.65, P = 0.325 and OR = 8.67, P = 0.093, respectively), the possible involvement of these small and large repeats in clinical radiosensitivity cannot be completely ruled out. When specific numbers of repeats were examined, no significant correlation was found between the microsatellite repeat length in XRCC1 and XRCC5 and cancer incidence. A weak correlation between XRCC3 [AC]₁₆ homozygotes and cancer incidence was found (OR = 2.56, P = 0.055). A large-scale multicenter study of cancer patients with a high number of radiosensitive individuals is needed to clarify the value of rare polymorphic microsatellite repeats in XRCC1 and XRCC3 as a biomarker of clinical radiosensitivity or increased cancer risk.

van Laarhoven, H. W. M., Bussink, J., Lok, J., Verhagen, I., Punt, C. J. A., Heerschap, A., Kaanders, J. H. A. M. and van der Kogel, A. J. Modulation of Hypoxia in Murine Liver Metastases of Colon Carcinoma by Nicotinamide and Carbogen. Radiat. Res. 164, 245–249 (2005).

There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.

Zhao, H., Cai, Y., Santi, S., Lafrenie, R. and Lee, H. Chloroquine-Mediated Radiosensitization is due to the Destabilization of the Lysosomal Membrane and Subsequent Induction of Cell Death by Necrosis. Radiat. Res. 164, 250–257 (2005).

The anti-malarial drug chloroquine (CQ) is also thought to be a potential radiation sensitizer. To gain a better understanding of how the lysomotropic CQ can potentiate the effects of ionizing radiation, we investigated the effects of CQ on lysosomal and mitochondrial membrane stability, the subcellular localization of ceramide, plasma membrane permeability, and the mode of cell death in response to irradiation. We found that CQ accumulated in the lysosomes and thus lysosomal volumes increased. As a result, both the lysosomal and plasma membranes were destabilized. After 7 Gy irradiation, most ceramide was associated with the lysosomes in the cells treated with CQ but not in the CQ-untreated control. The elevated levels of ceramide in the lysosomes of the CQ-treated cells appeared to further destabilize the lysosomal and plasma membranes of the cell. Both CQ-treated and -untreated cells had approximately the same rate of cell death by apoptosis after 7 Gy irradiation (P > 0.05, ns). However, in contrast to the CQ-untreated control, the CQ-treated cells underwent massive cell death by necrosis at 24–48 h after irradiation (P < 0.05). Taken together, our data support the idea that the increase in cytotoxic effects by the combination of CQ and radiation is due to radiation-mediated apoptosis and CQ-mediated necrosis.

Franke, H., Streckert, J., Bitz, A., Goeke, J., Hansen, V., Ringelstein, E. B., Nattkämper, H., Galla, H-J. and Stögbauer, F. Effects of Universal Mobile Telecommunications System (UMTS) Electromagnetic Fields on the Blood-Brain Barrier In Vitro. Radiat. Res. 164, 258–269 (2005).

The extensive use of mobile phone communication has raised public concerns about adverse health effects of radiofrequency (RF) electromagnetic fields (EMFs) in recent years. A central issue in this discussion is the question whether EMFs enhance the permeability of the blood-brain barrier (BBB). Here we report an investigation on the influence of a generic UMTS (Universal Mobile Telecommunications System) signal on barrier tightness, transport processes and the morphology of porcine brain microvascular endothelial cell cultures (PBEC) serving as an in vitro model of the BBB. An exposure device with integrated online monitoring system was developed for simultaneous exposure and measuring of transendothelial electrical resistance (TEER) to determine the tightness of the BBB. PBEC were exposed continuously for up to 84 h at an average electric-field strength of 3.4–34 V/m (maximum 1.8 W/kg) ensuring athermal conditions. We did not find any evidence of RF-field-induced disturbance of the function of the BBB. After and during exposure, the tightness of the BBB quantified by ¹⁴C-sucrose and serum albumin permeation as well as by TEER remained unchanged compared to sham-exposed cultures. Permeation of transporter substrates at the BBB as well as the localization and integrity of the tight-junction proteins occludin and ZO1 were not affected either.

Scarfí, M. R., Sannino, A., Perrotta, A., Sarti, M., Mesirca, P. and Bersani, F. Evaluation of Genotoxic Effects in Human Fibroblasts after Intermittent Exposure to 50 Hz Electromagnetic Fields: A Confirmatory Study. Radiat. Res. 164, 270–276 (2005).

The aim of this investigation was to confirm the main results reported in recent studies on the induction of genotoxic effects in human fibroblasts exposed to 50 Hz intermittent (5 min field on/10 min field off) sinusoidal electromagnetic fields. For this purpose, the induction of DNA single-strand breaks was evaluated by applying the alkaline single-cell gel electrophoresis (SCGE)/comet assay. To extend the study and validate the results, in the same experimental conditions, the potential genotoxicity was also tested by exposing the cells to a 50 Hz powerline signal (50 Hz frequency plus its harmonics). The cytokinesis-block micronucleus assay was applied after 24 h intermittent exposure to both sinusoidal and powerline signals to obtain information on cell cycle kinetics. The experiments were carried out on human diploid fibroblasts (ES-1). For each experimental run, exposed and sham-exposed samples were set up; positive controls were also provided by treating cells with hydrogen peroxide or mitomycin C for the comet or micronucleus assay, respectively. No statistically significant difference was detected in exposed compared to sham-exposed samples in any of the experimental conditions tested (P > 0.05). In contrast, the positive controls showed a statistically significant increase in DNA damage in all cases, as expected. Accordingly, our findings do not confirm the results reported previously for either comet induction or an increase in micronucleus frequency.

Luceri, C., De Filippo, C., Giovannelli, L., Blangiardo, M., Cavalieri, D., Aglietti, F., Pampaloni, M., Andreuccetti, D., Pieri, L., Bambi, F., Biggeri, A. and Dolara, P. Extremely Low-Frequency Electromagnetic Fields do not Affect DNA Damage and Gene Expression Profiles of Yeast and Human Lymphocytes. Radiat. Res. 164, 277–285 (2005).

We studied the effects of extremely low-frequency (50 Hz) electromagnetic fields (EMFs) on peripheral human blood lymphocytes and DBY747 Saccharomyces cerevisiae. Graded exposure to 50 Hz magnetic flux density was obtained with a Helmholtz coil system set at 1, 10 or 100 μT for 18 h. The effects of EMFs on DNA damage were studied with the single-cell gel electrophoresis assay (comet assay) in lymphocytes. Gene expression profiles of EMF-exposed human and yeast cells were evaluated with DNA microarrays containing 13,971 and 6,212 oligonucleotides, respectively. After exposure to the EMF, we did not observe an increase in the amount of strand breaks or oxidated DNA bases relative to controls or a variation in gene expression profiles. The results suggest that extremely low-frequency EMFs do not induce DNA damage or affect gene expression in these two different eukaryotic cell systems.

Hu, B., Han, W., Wu, L., Feng, H., Liu, X., Zhang, L., Xu, A., Hei, T K. and Yu, Z. In Situ Visualization of DSBs to Assess the Extranuclear/Extracellular Effects Induced by Low-Dose α-Particle Irradiation. Radiat. Res. 164, 286–291 (2005).

Extranuclear/extracellular effects may have a significant effect on low-dose radiation risk assessment as well as on the shape of the dose–response relationship. Numerous studies using different end points such as sister chromatid exchanges, micronuclei and mutation have shown that this phenomenon exists in many cell types. However, these end points mostly reflect the late events after radiation damage, and little is known about the early response in this phenomenon. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using γ-H2AX immunofluorescence staining, and there is evidence that the number of γ-H2AX foci can be closely correlated with DSBs induced. Here we used γ-H2AX as a biomarker to assess the extranuclear/extracellular effects induced by low-dose α particles in situ. The results show that a greater fraction of positive cells with DSBs (48.6%) was observed than the number of cells whose nuclei were actually traversed by the 1-cGy dose of α particles (9.2%). The fraction of DSB-positive cells was greatly reduced after treatment with either lindane or DMSO. These results suggest that in situ visualization of DSBs can be used to assess radiation-induced extranuclear/extracellular effects soon after irradiation. Moreover, the in situ DSB assay may provide a means to evaluate the spatial effect on unirradiated cells that are located in the neighboring region of cells irradiated by α particles.

Short, S. C., Bourne, S., Martindale, C., Woodcock, M. and Jackson, S. P. DNA Damage Responses at Low Radiation Doses. Radiat. Res. 164, 292–302 (2005).

Increased cell killing after exposure to low acute doses of X rays (0–0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.

Svoboda, P. and Harms-Ringdahl, M. Influence of Chromatin Structure and Radical Scavengers on Yields of Radiation-Induced 8-oxo-dG and DNA Strand Breaks in Cellular Model Systems. Radiat. Res. 164, 303–311 (2005).

Radiation-induced formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) and DNA strand breaks was studied in cultured cells with normal or modified chromatin structure. Human fibroblasts were irradiated as cellular monolayers (intact cells), nuclear monolayers (permeabilized cells with intact chromatin structure), and nucleoid monolayers (permeabilized and salt-treated cells with histone-free DNA). 8-oxo-dG was assayed with reverse-phase HPLC coupled to an electrochemical detector and strand breaks with the alkali unwinding assay. Depletion of low-molecular-weight nuclear components increased the radiation-induced formation of 8-oxo-dG fivefold compared to twofold for the formation of strand breaks. Removal of both low-molecular-weight components and histones increased the yield of 8-oxo-dG 46-fold and the yield of strand breaks 43-fold. Removal of only the histones thus leads to a two times greater increase in the yield of strand breaks compared to 8-oxo-dG. Addition of radical scavengers to nuclear and nucleoid monolayers provided a significantly better protection against the formation of 8-oxo-dG relative to the formation of strand breaks. These results suggest that in intact cells, 8-oxo-dG is preferentially formed in histone-free structures of chromatin, indicating a larger role for the indirect effect of radiation in the formation of 8-oxo-dG than in the formation of strand breaks.

Burmeister, J., Kota, C. and Maughan, R. L. Measured Microdosimetric Spectra and Therapeutic Potential of Boron Neutron Capture Enhancement of ²⁵²Cf Brachytherapy. Radiat. Res. 164, 312–318 (2005).

Californium-252 is a neutron-emitting radioisotope used as a brachytherapy source for radioresistant tumors. Presented here are microdosimetric spectra measured as a function of simulated site diameter and distance from applicator tube ²⁵²Cf sources. These spectra were measured using miniature tissue-equivalent proportional counters (TEPCs). An investigation of the clinical potential of boron neutron capture (BNC) enhancement of ²⁵²Cf brachytherapy is also provided. The absorbed dose from the BNC reaction was measured using a boron-loaded miniature TEPC. Measured neutron, photon and BNC absorbed dose components are provided as a function of distance from the source. In general, the absorbed dose results show good agreement with results from other measurement techniques. A concomitant boost to ²⁵²Cf brachytherapy may be provided through the use of the BNC reaction. The potential magnitude of this BNC enhancement increases with increasing distance from the source and is capable of providing a therapeutic gain greater than 30% at a distance of 5 cm from the source, assuming currently achievable boron concentrations.

Rivard, M. J., Melhus, C. S., Zinkin, H. D., Stapleford, L. J., Evans, K. E., Wazer, D. E. and Odlozilíková, A. A Radiobiological Model for the Relative Biological Effectiveness of High-Dose-Rate ²⁵²Cf Brachytherapy. Radiat. Res. 164, 319– 323 (2005).

While there is significant clinical experience using both low- and high-dose-rate ²⁵²Cf brachytherapy, there are minimal data regarding values for the neutron relative biological effectiveness (RBE) with both modalities. The aim of this research was to derive a radiobiological model for ²⁵²Cf neutron RBE and to compare these results with neutron RBE values used clinically in Russia. The linear-quadratic (LQ) model was used as the basis to characterize cell survival after irradiation, with identical cell killing rates (S_N = S_γ) between ²⁵²Cf neutrons and photons used for derivation of RBE. Using this equality, a relationship among neutron dose and LQ radiobiological parameter (i.e., α_N, β_N, α_γ, β_γ) was obtained without the need to specify the photon dose. These results were used to derive the ²⁵²Cf neutron RBE, which was then compared with Russian neutron RBE values. The ²⁵²Cf neutron RBE was determined after incorporating the LQ radiobiological parameters obtained from cell survival studies with fast neutrons and teletherapy photons. For single-fraction high-dose-rate neutron doses of 0.5, 1.0, 1.5 and 2.0 Gy, the total biologically equivalent doses were 1.8, 3.4, 4.7 and 6.0 RBE Gy with ²⁵²Cf neutron RBE values of 3.2, 2.9, 2.7 and 2.5, respectively. Using clinical data for late-responding reactions from ²⁵²Cf, Russian investigators created an empirical model that predicted high-dose-rate ²⁵²Cf neutron RBE values ranging from 3.6 to 2.9 for similar doses and fractionation schemes and observed that ²⁵²Cf neutron RBE increases with the number of treatment fractions. Using these relationships, our results were in general concordance with high-dose-rate ²⁵²Cf RBE values obtained from Russian clinical experience.

Sachs, R. K., Chan, M., Hlatky, L. and Hahnfeldt, P. Modeling Intercellular Interactions during Carcinogenesis. Radiat. Res. 164, 324–331 (2005).

By modulating the microenvironment of malignant or premalignant cells, inhibitory or stimulatory signals from nearby cells can play a key role in carcinogenesis. However, current commonly used quantitative models for induction of cancers by ionizing radiation focus on single cells and their progeny. Intercellular interactions are neglected or assumed to be confined to unidirectional radiation bystander effect signals from cells of the same tissue type. We here formulate a parsimoniously parameterized two-stage logistic (TSL) carcinogenesis model that incorporates some effects of intercellular interactions during the growth of premalignant cells. We show that for baseline tumor rates, involving no radiation apart from background radiation, this TSL model gives acceptable fits to a number of data sets. Specifically, it gives the same baseline hazard function, using the same number of adjustable parameters, as does the commonly used two-stage clonal expansion (TSCE) model, so it is automatically applicable to the many data sets on baseline cancer that have been analyzed using the TSCE model. For perturbations of baseline rates due to radiation, the models differ. We argue from epidemiological and laboratory evidence, especially results for the atomic bomb survivors, that for radiation carcinogenesis the TSL model gives results at least as realistic as the TSCE or similar models, despite involving fewer adjustable parameters in many cases.

Yoshida, T., Tanabe, T., Okabe, Y., Sawasaki, T. and Chen, A. Decomposition of Carbon Dioxide by Metals during Gamma Irradiation. Radiat. Res. 164, 332–335 (2005).

We have proposed a technique to enhance the decomposition of carbon dioxide by γ irradiation. This is possible by putting metal components into CO₂ gas to promote the conversion of γ rays to lower-energy electrons through Compton, photoelectron and cascading electron knock-on events in metals. Numerical simulations using the EGS code indicated that the number of lower-energy electrons ejected from metals into CO₂ gas increases with increasing Z number and/or density of the metals; this was supported by the experimental results, i.e., the CO₂-containing metals with a higher Z number exhibited a greater efficiency for production of CO. In addition, production of CO could be enhanced by carefully controlling the volume and surface area of metal components as well as the proximity to adjacent metal components. These experimental results successfully demonstrated that modification of the kinds of metal components and metal structures can control the energy and the number of electrons ejected from the metals and can lead to enhancement of production of CO from CO₂.

Nakagawa, K. and Sato, Y., Investigation of Heavy-Ion-Induced Sucrose Radicals by Electron Paramagnetic Resonance. Radiat. Res. 164, 336–338 (2005).

The production of sucrose radicals with heavy-ion irradiation was investigated by an EPR (electron paramagnetic resonance) spectroscopic method. We examined the correlation between the production of sucrose radicals and the ion species, as well as LET (linear energy transfer). The spectral pattern obtained was the same for various ion species, including helium, carbon, neon, argon and iron ions. Quantitative EPR analyses showed that the production of sucrose radicals depended on both the ion species and the LET for the same dose of 50 Gy. The spin yield obtained showed a logarithmic correlation with the LET. In addition, the EPR response had a linear relationship with dose in the dose range of 5–60 Gy. Thus the present EPR results show that sucrose can be used to monitor the ionizing particle based on the radical yield.

Miller, R. W. and Zanzonico, P. B. Radioiodine Fallout and Breast-Feeding. Radiat. Res. 164, 339–340 (2005).

The rationale for suspension of breast-feeding after exposure to radioiodine in fallout is presented.