Whitehead, T. D., Moros, E. G., Brownstein, B. H. and Roti Roti, J. L. Gene Expression does not Change Significantly in C3H 10T ½ Cells after Exposure to 847.74 CDMA or 835.62 FDMA Radiofrequency Radiation. Radiat. Res. 165, 626–635 (2006).

In vitro experiments with C3H 10T½ mouse cells were performed to determine whether Frequency Division Multiple Access (FDMA) or Code Division Multiple Access (CDMA) modulated radiofrequency (RF) radiations induce changes in gene expression. After the cells were exposed to either modulation for 24 h at a specific absorption rate (SAR) of 5 W/ kg, RNA was extracted from both exposed and sham-exposed cells for gene expression analysis. As a positive control, cells were exposed to 0.68 Gy of X rays and gene expression was evaluated 4 h after exposure. Gene expression was evaluated using the Affymetrix U74Av2 GeneChip® to detect changes in mRNA levels. Each exposure condition was repeated three times. The GeneChip® data were analyzed using a two-tailed t test, and the expected number of false positives was estimated from t tests on 20 permutations of the six sham RF-field-exposed samples. For the X-ray-treated samples, there were more than 90 probe sets with expression changes greater than 1.3-fold beyond the number of expected false positives. Approximately one-third of these genes had previously been reported in the literature as being responsive to radiation. In contrast, for both CDMA and FDMA radiation, the number of probe sets with an expression change greater than 1.3-fold was less than or equal to the expected number of false positives. Thus the 24-h exposures to FDMA or CDMA RF radiation at 5 W/kg had no statistically significant effect on gene expression.

Qutob, S. S., Chauhan, V., Bellier, P. V., Yauk, C. L., Douglas, G. R., Berndt, L., Williams, A., Gajda, G. B., Lemay, E., Thansandote, A. and McNamee, J. P. Microarray Gene Expression Profiling of a Human Glioblastoma Cell Line Exposed In Vitro to a 1.9 GHz Pulse-Modulated Radiofrequency Field. Radiat. Res. 165, 636–644 (2006).

The widespread use of mobile phones has led to public concerns about the health effects associated with exposure to radiofrequency (RF) fields. The paramount concern of most persons relates to the potential of these fields to cause cancer. Unlike ionizing radiation, RF fields used for mobile telecommunications (800–1900 MHz) do not possess sufficient energy to directly damage DNA. Most rodent bioassay and in vitro genotoxicity/mutation studies have reported that RF fields at non-thermal levels have no direct mutagenic, genotoxic or carcinogenic effects. However, some evidence has suggested that RF fields may cause detectable postexposure changes in gene expression. Therefore, the purpose of this study was to assess the ability of exposure to a 1.9 GHz pulse-modulated RF field for 4 h at specific absorption rates (SARs) of 0.1, 1.0 and 10.0 W/kg to affect global gene expression in U87MG glioblastoma cells. We found no evidence that non-thermal RF fields can affect gene expression in cultured U87MG cells relative to the nonirradiated control groups, whereas exposure to heat shock at 43°C for 1 h up-regulated a number of typical stress-responsive genes in the positive control group. Future studies will assess the effect of RF fields on other cell lines and on gene expression in the mouse brain after in vivo exposure.

Natarajan, M., Nayak, B. K., Galindo, C., Mathur, S. P., Roldan, F. N. and Meltz, M. L. Nuclear Translocation and DNA-Binding Activity of NFKB (NF-κB) after Exposure of Human Monocytes to Pulsed Ultra-wideband Electromagnetic Fields (1 kV/cm) Fails to Transactivate κB-Dependent Gene Expression. Radiat. Res. 165, 645–654 (2006).

The objective of this study was to investigate whether exposure of human monocytes to a pulsed ultra-wideband electromagnetic field (EMF) of 1 kV/cm average peak power triggers a signaling pathway responsible for the transcriptional regulation of NFKB (NF-κB)-dependent gene expression. Human Mono Mac 6 (MM6) cells were exposed intermittently to EMF pulses for a total of 90 min. The pulse width was 0.79 ± 0.01 ns and the pulse repetition rate was 250 pps. The temperature of the medium was maintained at 37°C in both sham- and EMF-exposed flasks. Total NFKB DNA-binding activity was measured in the nuclear extracts by the electrophoretic mobility shift assay. Cells exposed to the EMFs and incubated for 24 h postexposure showed a 3.5 ± 0.2-fold increase in the NFKB DNA-binding activity. Since activation of NFKB was observed, the possibility of κB-dependent gene expression in response to exposure to the EMFs was investigated using NFKB signal-specific gene arrays. The results revealed no difference in the NFKB-dependent gene expression profiles at 8 or 24 h postexposure, indicating that activated NFKB does not lead to the differential expression of κB-dependent target genes. To determine whether the absence of the κB-dependent gene expression was due to compromised transcriptional regulation of NFKB, the functional activity of NFKB was examined in cells transiently transfected with Mercury Pathway™ constructs containing 4× NFKB binding sites associated either with the luciferase reporter system or a control vector. Pulsed EMF exposure did not induce NFKB-driven luciferase activity in these cells, indicating that the activation of NFKB at 24 h after the 1 kV/cm EMF exposure is functionally inactive. From these results, it is clear that the EMF-induced NFKB activation is only a transient response, with minimal or no downstream effect.

Scarfì, M. R., Fresegna, A. M., Villani, P., Pinto, R., Marino, C., Sarti, M., Altavista, P., Sannino, A. and Lovisolo, G. A. Exposure to Radiofrequency Radiation (900 MHz, GSM signal) does not Affect Micronucleus Frequency and Cell Proliferation in Human Peripheral Blood Lymphocytes: An Interlaboratory Study. Radiat. Res. 165, 655–663 (2006).

The objective of this study was to investigate whether 24 h exposure to radiofrequency electromagnetic fields similar to those emitted by mobile phones induces genotoxic effects and/or effects on cell cycle kinetics in cultured human peripheral blood lymphocytes. The effect of 900 MHz exposure (GSM signal) was evaluated at four specific absorption rates (SARs, 0, 1, 5 and 10 W/kg peak values). The exposures were carried out in wire patch cells under strictly controlled conditions of both temperature and dosimetry, and the induction of genotoxic effects was evaluated in lymphocyte cultures from 10 healthy donors by applying the cytokinesis-block micronucleus assay. Positive controls were provided by using mitomycin C. Two research groups were involved in the study, one at ENEA, Rome, and the other at CNR-IREA, Naples. Each laboratory tested five donors, and the resulting slides were scored by both laboratories. Following this experimental scheme, it was also possible to compare the results obtained by cross-scoring of slides. The results obtained provided no evidence for the existence of genotoxic or cytotoxic effects in the range of SARs investigated. These findings were confirmed in the two groups of five donors examined in the two laboratories and when the same slides were scored by two operators.

Nasta, F., Prisco, M. G., Pinto, R., Lovisolo, G. A., Marino, C. and Pioli, C. Effects of GSM-Modulated Radiofrequency Electromagnetic Fields on B-Cell Peripheral Differentiation and Antibody Production. Radiat. Res. 165, 664–670 (2006).

We examined the effects of in vivo exposure to a GSM-modulated 900 MHz RF field on B-cell peripheral differentiation and antibody production in mice. Our results show that exposure to a whole-body average specific absorption rate (SAR) of 2 W/kg, 2 h/day for 4 consecutive weeks does not affect the frequencies of differentiating transitional 1 (T1) and T2 B cells or those of mature follicular B and marginal zone B cells in the spleen. IgM and IgG serum levels are also not significantly different among exposed, sham-exposed and control mice. B cells from these mice, challenged in vitro with LPS, produce comparable amounts of IgM and IgG. Moreover, exposure of immunized mice to RF fields does not change the antigen-specific antibody serum level. Interestingly, not only the production of antigen-specific IgM but also that of IgG (which requires T-B-cell interaction) is not affected by RF-field exposure. This indicates that the exposure does not alter an ongoing in vivo antigen-specific immune response. In conclusion, our results do not indicate any effects of GSM-modulated RF radiation on the B-cell peripheral compartment and antibody production and thus provide no support for health-threatening effects.

Epperly, M. W., Goff, J. P., Zhang, X., Niu, Y., Shields, D. S., Wang, H., Shen, H., Franicola, D., Bahnson, A. B., Nie, S., Greenberger, E. E. and Greenberger, J. S. Increased Radioresistance, G₂/M Checkpoint Inhibition, and Impaired Migration of Bone Marrow Stromal Cell Lines Derived from Smad3^−/− Mice. Radiat. Res. 165, 671–677 (2006).

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3^−/− mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3^−/− mice and homozygous littermate ^/ mice. Smad3^−/− cells displayed a significant increase in radiation resistance with a D₀ = 2.25 ± 0.14 Gy compared to Smad3 ^/ cells with a D₀ = 1.75 ± 0.03 (P = 0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D₀ = 1.49 0.10 (P = 0.028) for Smad3^−/−(3) cells. More Smad3^−/− cells than Smad3 ^/ cells were in the G₂/M phase; Smad3^−/−(3) cells were similar to Smad3 ^/ cells. Smad3 ^/ cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3^−/− cells exposed to either dose. The movement of Smad3^−/− cells, measured in an automated cell tracking system, was slower than that of Smad3 ^/ cells. Smad3^−/−(3) cells resembled Smad3 ^/ cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G₂/M cells, and radioresistance. The decreased migratory capacity of Smad3^−/− cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.

Li, Z., Zhao, D., Gong, B., Xu, Y., Sun, H., Yang B. and Zhao, X. Decreased Saliva Secretion and Down-Regulation of AQP5 in Submandibular Gland in Irradiated Rats. Radiat. Res. 165, 678–687 (2006).

The molecular mechanisms of radiation-induced xerostomia remain unclear. The purpose of this study was to investigate the alterations of aquaporins (AQPs) and Na /K -ATPase in irradiated rat submandibular glands and to test the hypothesis that down-regulation of AQP5 expression in irradiated salivary glands is one of the mechanisms of radiation-induced xerostomia. Saliva from control and irradiated rat submandibular glands was analyzed. The mRNA level of AQP5 in the submandibular glands was assessed by semi-quantitative RT-PCR and in situ hybridization. The protein expression of AQP5, AQP1 and Na /K -ATPase was determined by Western blotting and immunohistochemistry. The body weight, submandibular gland weight, and saliva secretion of irradiated rats significantly decreased by 12, 24 and 32% on day 3 and 24, 16 and 38% on day 30 postirradiation, respectively. There was a significant increase in the protein concentration and osmolality of saliva in irradiated rats on days 3 and 30 postirradiation. However, there was no significant difference between irradiated and control rats in total saliva protein secretion. RT-PCR analysis showed that mRNA expression of AQP5 was significantly down-regulated by 37 and 51% in irradiated rats on days 3 and 30 postirradiation, respectively. Immunoblotting showed that the AQP5 protein level was decreased by 40 and 60% in irradiated glands, in contrast to the slight reductions of AQP1 and Na /K -ATPase proteins. Immunohistochemical analysis demonstrated that loss of AQP5 protein occurred throughout the irradiated glands, while no significant reduction was detected in AQP1 and Na / K -ATPase labeling density. These results suggest that the preferential down-regulation of AQP5 with minor effects on AQP1 and Na /K -ATPase may contribute to radiation-induced salivary dysfunction.

Kishikawa, H., Wang, K., Adelstein, S. J. and Kassis, A. I. Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123. Radiat. Res. 165, 688–694 (2006).

The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated ¹²⁵I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765–13770, 2002). We have repeated these studies using cells exposed to lethal doses of ¹²³I, an Auger electron emitter whose emission spectrum is identical to that of ¹²⁵I, and report herein that the decay of ¹²³I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects (¹²⁵I) and stimulatory bystander effects (¹²³I) are obtained in vitro. Moreover, supernatants from cultures with ¹²⁵I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with ¹²³I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose–response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all “calculated” dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.

Viola, A., Major, T. and Julow, J. Comparison of ¹²⁵I Stereotactic Brachytherapy and LINAC Radiosurgery Modalities based on Physical Dose Distribution and Radiobiological Efficacy. Radiat. Res. 165, 695–702 (2006).

The goal of this study was to make a comparison between stereotactic brachytherapy implants and linear accelerator-based radiosurgery of brain tumors with respect to physical dose distributions and radiobiological efficacy. Twenty-four treatment plans made for irradiation of brain tumors with low-dose-rate ¹²⁵I brachytherapy and multiple-arc LINAC-based radiosurgery were analyzed. Using the dose–volume histograms and the linear-quadratic model, the brachytherapy doses were compared to the brachytherapy-equivalent LINAC radiosurgery doses with respect to the predicted late effects of radiation on normal brain tissue. To characterize the conformity and homogeneity of dose distributions, the conformal index, external volume index, and relative homogeneity index were calculated for each dose plan and the mean values were compared. The average tumor volume was 5.6 cm³ (range: 0.1–19.3 cm³). At low doses, the calculated radiobiological late effect on normal tissue was equivalent for external-beam and brachytherapy dose delivery. For brachytherapy at doses greater than 30 Gy, the calculated equivalent dose to normal tissues was less than for external-beam radiosurgery. However, the dose-calculated homogeneity was better for the LINAC radiosurgery, with a mean relative homogeneity index of 0.62 compared to the calculated value of 0.19 for the brachytherapy (P = 0.0002). These results are only predictions based on calculations concerning normal tissue tolerance. More data and research are needed to understand the clinical relevance of these findings.

Ito, A., Nakano, H., Kusano, Y., Hirayama, R., Furusawa, Y., Murayama, C., Mori, T., Katsumura, Y. and Shinohara, K. Contribution of Indirect Action to Radiation-Induced Mammalian Cell Inactivation: Dependence on Photon Energy and Heavy-Ion LET. Radiat. Res. 165, 703–712 (2006).

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/μm to 440 keV/μm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/μm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.

Belli, M., Campa, A., Dini, V., Esposito, G., Furusawa, Y., Simone, G., Sorrentino, E. and Tabocchini, M. A. DNA Fragmentation Induced in Human Fibroblasts by Accelerated ⁵⁶Fe Ions of Differing Energies. Radiat. Res. 165, 713–720 (2006).

DNA fragmentation was studied in the fragment size range 0.023–5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with γ rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/μm, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after γ irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than γ rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.

Li, X., Sanche, L. and Sevilla, M.D Base Release in Nucleosides Induced by Low-Energy Electrons: A DFT Study. Radiat. Res. 165, 721–729 (2006).

Low-energy electrons are known to induce strand breaks and base damage in DNA and RNA through fragmentation of molecular bonding. Recently the glycosidic bond cleavage of nucleosides by low-energy electrons has been reported. These experimental results call for a theoretical investigation of the strength of the C₁′–N link in nucleosides (dA, dC and dT) between the base and deoxyribose before and after electron attachment. Through density functional theory (DFT) calculations, we compare the C₁′–N bond strength, i.e., the bond dissociation energy of the neutral and its anionic radical, and find that an excess electron effectively weakens the C₁′– N bond strength in nucleosides by 61–75 kcal/mol in the gas phase and 76–83 kcal/mol in the solvated environment. As a result, electron-induced fragmentation of the C₁′–N bond in the gas phase is exergonic for dA (ΔG = −14 kcal/mol) and for dT (ΔG = −6 kcal/mol) and is endergonic (ΔG = 1 kcal/ mol) only for dC. In the gas phase all the anionic nucleosides are found to be in valence states. Solvation is found to increase the exergonic nature by an additional 20 kcal, making the fragmentation both exothermic and exergonic for all nucleoside anion radicals. Thus C₁′–N bond breaking in nucleoside anion radicals is found to be thermodynamically favorable both in the gas phase and under solvation. The activation barrier for the C₁′–N bond breaking process was found to be about 20 kcal/mol in every case examined, suggesting that a 1 eV electron would induce spontaneous cleavage of the bond and that stabilized anion radicals on the DNA strand would undergo base release at only a modest rate at room temperature. These results suggest that base release from nucleosides and DNA is an expected consequence of low-energy electron-induced damage but that the high barrier would inhibit this process in the stable anion radicals.

Mokrini, R., Trouillas, P., Kaouadji, M., Champavier, Y., Houée-Lévin, C., Calliste, C. A. and Duroux, J. L. Radiolytic Transformation of 2,2′,4′-Trihydroxychalcone. Radiat. Res. 165, 730–740 (2006).

Radiolysis of 2,2′,4′-trihydroxychalcone, a natural antioxidant present in fruit and vegetables, was performed in ethanol in the absence or in the presence of dioxygen. The degradation process of chalcone was followed in deaerated solution by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC. Under anaerobic conditions, six new products (three couples of diastereoisomers) were identified. Four of them kept the chalcone skeleton with OCH₂CH₃, CH(OH)CH₃ and H substitutions on C_α and C_β. Thus the target was the α-β double bond on which ethanol radicals were added. The two other compounds were formed in a second stage and exhibited a cyclization between the substituent on C_β and the carbonyl group. In the presence of dioxygen, these reactions were prevented and chalcone was protected. This study was the first step toward understanding of the behavior chalcone in irradiated fruits and vegetables.

Bijwaard, H., Brugmans, M. J. P. and Schöllnberger, H. Can Promotion of Initiated Cells be Explained by Excess Replacement of Radiation-Inactivated Neighbor Cells? Radiat. Res. 165, 741–744 (2006).

Recently, the observed promotion in the clonal expansion of a two-stage cancer model was attributed to a small excess replacement probability for the initiated cells. The proposed mechanism of excess replacement was evaluated for single intermediate cells surrounded by normal cells. This paper investigates this mechanism further using the same biological parameters. If the formation of clones of intermediate cells is taken into account in a quantitative analysis of the proposed mechanism, it turns out that (1) for the initial strong increase of the promotional effect with exposure, a much larger and unlikely excess replacement probability is needed, and (2) the leveling of the promotional effect for high exposures cannot be explained by multiple normal neighbors of an intermediate cell being inactivated within one cell cycle, as it had been suggested. Perhaps these discrepancies could be partly resolved by a rescaling of the original parameters, but this should be investigated further.

Mayo, M. S., Gajewski, B. J. and Morris, J. S. Some Statistical Issues in Microarray Gene Expression Data. Radiat. Res. 165, 745–748 (2006).

In this paper we discuss some of the statistical issues that should be considered when conducting experiments involving microarray gene expression data. We discuss statistical issues related to preprocessing the data as well as the analysis of the data. Analysis of the data is discussed in three contexts: class comparison, class prediction and class discovery. We also review the methods used in two studies that are using microarray gene expression to assess the effect of exposure to radiofrequency (RF) fields on gene expression. Our intent is to provide a guide for radiation researchers when conducting studies involving microarray gene expression data.