Jacob, P., Bogdanova, T. I., Buglova, E., Chepurniy, M., Demidchik, Y., Gavrilin, Y., Kenigsberg, J., Meckbach, R., Schotola, C., Shinkarev, S., Tronko, M. D., Ulanovsky, A., Vavilov, S. and Walsh, L. Thyroid Cancer Risk in Areas of Ukraine and Belarus Affected by the Chernobyl Accident. Radiat. Res. 165, 1–8 (2006).

The purpose of the present study was to analyze the thyroid cancer incidence risk after the Chernobyl accident and its degree of dependence on time and age. Data were analyzed for 1034 settlements in Ukraine and Belarus, in which more than 10 measurements of the ¹³¹I content in human thyroids had been performed in May/June 1986. Thyroid doses due to the Chernobyl accident were assessed for the birth years 1968– 1985 and related to thyroid cancers that were surgically removed during the period 1990–2001. The central estimate for the linear coefficient of the EAR dose response was 2.66 (95% CI: 2.19; 3.13) cases per 10⁴ PY-Gy; for the quadratic coefficient, it was −0.145 (95% CI: −0.171; −0.119) cases per 10⁴ PY-Gy². The EAR was found to be higher for females than for males by a factor of 1.4. It decreased with age at exposure and increased with age attained. The central estimate for the linear coefficient of the ERR dose response was 18.9 (95% CI: 11.1; 26.7) Gy⁻¹; for the quadratic coefficient, it was −1.03 (95% CI: −1.46; −0.60) Gy⁻². The ERR was found to be smaller for females than for males by a factor of 3.8 and decreased strongly with age at exposure. Both EAR and ERR were higher in the Belarusian settlements than in the Ukrainian settlements. In contrast to ERR, EAR increases with time after exposure. At the end of the observation period, excess risk estimates were found to be close to those observed in a major pooled analysis of seven studies of childhood thyroid cancer after external exposures.

Dynlacht, J. R., Tyree, C., Valluri, S., DesRosiers, C., Caperell-Grant, A., Mendonca, M. S., Timmerman, R. and Bigsby, R. M. Effect of Estrogen on Radiation-Induced Cataractogenesis. Radiat. Res. 165, 9–15 (2006).

Cataractogenesis is a widely reported late effect that is observed in patients receiving total-body irradiation (TBI) prior to bone marrow transplantation or radiotherapy for ocular or head and neck cancers. Recent studies indicate that estrogens may protect against age-related and drug-induced cataracts. Moreover, other reports suggest that estrogen possesses antioxidant properties. Since the effect of estrogen on radiation cataractogenesis is unknown, we wished to determine whether estrogen modulates radiation-induced opacification of the lens. Intact or ovariectomized Sprague-Dawley rats were treated with either 17-β-estradiol or an empty silastic capsule. The right orbit was then irradiated with either 10 or 15 Gy of ⁶⁰Co γ rays using a Leksell Gamma Knife, and lenses were examined at various times postirradiation with a slit lamp or evaluated for light transmission. We found that for ovariectomized rats irradiated with 15 Gy, the lens opacity and the incidence of cataract formation in the estradiol-treated group were significantly increased compared to the control group at the end of the 25-week period of observation. Cataract incidence was also high in irradiated eyes of ovary-intact animals at 25 weeks postirradiation but was greatly reduced in the ovariectomized control group, with less than half of irradiated eyes showing evidence of cataractogenesis. Thus, after irradiation with 15 Gy of γ rays, estrogen increased the incidence of cataract formation. We also observed that although the incidence of cataract formation in rats irradiated with 10 Gy and receiving continuous estrogen treatment was not altered compared to rats in the control group that did not receive estrogen, the latent period for posterior subcapsular cataract formation decreased and the severity of the anterior cataract increased. Taken together, our data suggest that estrogen accelerates progression of radiation-induced opacification.

Brurberg, K. G., Thuen, M., Ruud, E. B. M. and Rofstad, E. K. Fluctuations in pO₂ in Irradiated Human Melanoma Xenografts. Radiat. Res. 165, 16–25 (2006).

Several studies have demonstrated that untreated tumors may show significant fluctuations in tissue oxygen tension (pO₂). Radiation treatment may induce changes in the tumor microenvironment that alter the pO₂ fluctuation pattern. The purpose of the present study was to investigate whether pO₂ fluctuations may also occur in irradiated tumors. A-07 human melanoma xenografts were irradiated with single doses of 0, 5 or 10 Gy. Fluctuations in pO₂ were recorded with OxyLite probes prior to irradiation and 24 and 72 h after the radiation exposure. Radiation-induced changes in the tumor microenvironment (i.e. blood perfusion and extracellular volume fraction) were assessed by dynamic contrast-enhanced magnetic resonance imaging. Seventy-two hours after 10 Gy, tumor blood perfusion had decreased to ∼40% of that prior to irradiation, whereas the extracellular volume fraction had increased by ∼25%. Fluctuations in pO₂ were seen in most tumors, irrespective of radiation dose and time after irradiation. The mean pO₂, the number of fluctuations around the mean pO₂, the number of fluctuations around threshold pO₂ values of 1, 2, 3, 5, 7 and 10 mmHg, and the amplitude of the fluctuations were determined for each pO₂ trace. No significant differences were detected between irradiated and unirradiated tumors. The results showed that pO₂ fluctuations may occur in irradiated tumors and that the pO₂ fluctuation pattern in A-07 tumors exposed to 5 or 10 Gy is similar to that in untreated tumors. Consequently, these doses did not induce changes in the tumor microenvironment that were sufficient to cause detectable alterations in the pO₂ fluctuation pattern.

Mothersill, C., Seymour, R. J. and Seymour, C. B. Increased Radiosensitivity in Cells of Two Human Cell Lines Treated with Bystander Medium from Irradiated Repair-Deficient Cells. Radiat. Res. 165, 26–34 (2006).

Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy γ rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of ⁶⁰Co γ rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.

Podtcheko, A., Ohtsuru, A., Namba, H., Saenko, V., Starenki, D., Palona, I., Sedliarou, I., Rogounovitch, T. and Yamashita, S. Inhibition of ABL Tyrosine Kinase Potentiates Radiation-Induced Terminal Growth Arrest in Anaplastic Thyroid Cancer Cells. Radiat. Res. 165, 35–42 (2006).

Gleevec®, a selective tyrosine kinase inhibitor, retarded the growth of anaplastic thyroid cancer cell lines in vitro and in vivo through selective inhibition of ABL tyrosine kinase activity. In the present study, we investigated the ability of Gleevec® to modulate the in vitro and in vivo radiation response of anaplastic thyroid cancer cells. Cell growth assays, colony formation assays and xenograft models were used to quantify the radiosensitizing effect of Gleevec® in cells of the anaplastic thyroid cancer cell lines ARO and FRO. FACS, Western blotting and histochemical techniques were employed to study the mechanisms of radiation response after exposure to Gleevec®. Gleevec® (7.0 μM) increased the anti-proliferative effect of radiation on the growth ARO and FRO cells in vitro. Clonogenic analysis demonstrated that Gleevec® reduced cell survival after irradiation. Gleevec® combined with radiation produced an increase in tumor growth inhibition compared to treatment with either modality alone in mice bearing anaplastic thyroid cancer xenografts. The drug suppressed radiation-induced ABL activation and promoted CDKN1A (p21^cip1) accumulation in irradiated anaplastic thyroid cancer cells. Gleevec® had an additional effect on radiation-induced apoptosis in cells of both cell lines and potentiated the induction of terminal growth arrest accompanied by the expression of senescence-associated β-galactosidase. The antitumor effect of Gleevec® is potentiated in adjunctive therapy with radiation not only due to inhibition of proliferative cell growth with transient cell cycle arrest and apoptosis, but also due to the terminal growth arrest associated with senescence, suggesting that tumor cell senescence is a mechanism for tumor targeting therapy in combination with ionizing radiation.

Kumar, P. R. V., Mohankumar, M. N., Hamza, V. Z. and Jeevanram, R. K. Dose-Rate Effect on the Induction of HPRT Mutants in Human G₀ Lymphocytes Exposed In Vitro to Gamma Radiation. Radiat. Res. 165, 43–50 (2006).

The influence of dose rate on expression time, cell survival and mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was evaluated in human G₀ peripheral blood lymphocytes exposed in vitro to γ rays at low (0.0014 Gy/min) and high (0.85 Gy/min) dose rates. A cloning assay performed on different days of postirradiation incubation indicated an 8-day maximum expression period for the induction of HPRT mutants at both high and low dose rates. Cell survival increased markedly with decreasing dose rate, yielding D₀ values of 3.04 Gy and 1.3 Gy at low and high dose rates, respectively. The D₀ of 3.04 Gy obtained at low dose rate could be attributed to the repair of sublethal DNA damage taking place during prolonged exposure to low-LET radiation. Regression analysis of the mutant frequency yielded slopes of 12.35 × 10⁻⁶ and 3.66 × 10⁻⁶ mutants per gray at high and low dose rate, respectively. A dose and dose-rate effectiveness factor of 3.4 indicated a marked dose-rate effect on the induced HPRT mutant frequency. The results indicate that information obtained from in vitro measurements of dose-rate effects in human G₀ lymphocytes may be a useful parameter for risk estimation in radiation protection.

Durante, M., George, K. and Cucinotta, F. A. Chromosomes Lacking Telomeres are Present in the Progeny of Human Lymphocytes Exposed to Heavy Ions. Radiat. Res. 165, 51–58 (2006).

High-charge and energy (HZE) nuclei represent one of the main health risks for human space exploration, yet little is known about the mechanisms responsible for the high biological effectiveness of these particles. We have used in situ hybridization probes for cross-species multicolor banding (RxFISH) in combination with telomere detection to compare yields of different types of chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to either high-energy iron ions or γ rays. Terminal deletions showed the greatest relative variation, with many more of these types of aberrations induced after exposure to accelerated iron ions (energy 1 GeV/nucleon) compared with the same dose of γ rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles after exposure and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. On the other hand, the fraction of cells carrying stable, transmissible chromosomal aberrations was similar in the progeny of cells exposed to the same dose of densely or sparsely ionizing radiation. The results demonstrate that unrejoined chromosome breaks are an important component of aberration spectra produced by the exposure to HZE nuclei. This finding may well be related to the ability of such energetic particles to produce untoward late effects in irradiated organisms.

Okayasu, R., Okada, M., Okabe, A., Noguchi, M., Takakura, K. and Takahashi, S. Repair of DNA Damage Induced by Accelerated Heavy Ions in Mammalian Cells Proficient and Deficient in the Non-homologous End-Joining Pathway. Radiat. Res. 165, 59–67 (2006).

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/μm carbon ions, and 200 keV/μm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = ∼1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G₁ premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.

Pecaut, M. J., Dutta-Roy, R., Smith, A. L., Jones, T. A., Nelson, G. A. and Gridley, D. S. Acute Effects of Iron-Particle Radiation on Immunity. Part I: Population Distributions. Radiat. Res. 165, 68–77 (2006).

Health risks due to exposure to high-linear energy transfer (LET) charged particles remain unclear. The major goal of this study was to confirm and further characterize the acute effects of high-LET radiation (⁵⁶Fe²⁶) on erythrocyte, thrombocyte and leukocyte populations in three body compartments after total-body exposure. Adult female C57BL/6 mice were irradiated with total doses of 0, 0.5, 2 and 3 Gy and killed humanely 4 days later. Body and organ masses were determined and blood, spleen and bone marrow leukocytes were evaluated using a hematology analyzer and flow cytometry. Spleen and thymus (but not body, liver and lung) masses were significantly decreased in a dose-dependent manner. In general, red blood cell (RBC) counts and most other RBC parameters were depressed with increasing dose (P < 0.05); the major exception was an increase in cell size at 0.5 Gy. Platelet numbers and volume, total white blood cell counts, and all three major types of leukocytes also decreased (P < 0.05). Lymphocyte populations in blood and spleen exhibited variable degrees of susceptibility to ⁵⁶Fe-particle radiation (B > T > NK and T cytotoxic > T helper cells). In the bone marrow, leukocytes with granulocytic, lymphocytic (“dim” and “bright”), and monocytic characteristics exhibited proportional variations at the higher radiation doses in the expression of CD34 and/or Ly-6A/E. The data are discussed in relation to our previous investigations with iron ions, other forms of radiation, and space flight in this same animal model.

Gridley, D. S., Dutta-Roy, R., Andres, M. L., Nelson, G. A. and Pecaut, M. J. Acute Effects of Iron-Particle Radiation on Immunity. Part II: Leukocyte Activation, Cytokines and Adhesion. Radiat. Res. 165, 78–87 (2006).

The effects of high-linear energy transfer (LET) radiation on immune function have not been clearly established. The major goal of this study was to evaluate leukocyte responses after whole-body exposure to high-LET radiation. C57BL/6 mice were exposed to 0, 0.5, 2 and 3 Gy ⁵⁶Fe²⁶ particles (1055 MeV/nucleon, 148.2 keV/μm) and killed humanely 4 days after exposure. Spontaneous synthesis of DNA in blood and spleen cells was increased significantly in groups receiving either 2 or 3 Gy (P < 0.001). In contrast, a significant depression in the response of T lymphocytes to phytohemagglutinin (PHA) and concanavalin A (ConA) was noted (P < 0.005); the response to lipopolysaccharide (LPS), a B-cell mitogen, was similar among groups. A cytometric bead array assay revealed that the level of tumor necrosis factor α (Tnfa) secreted by splenocytes increased significantly with increasing ⁵⁶Fe-particle dose (P < 0.05); interferon γ, interleukin2 (Il2), Il4 and Il5 were unaffected. Flow cytometry analysis showed that 2 and 3 Gy markedly reduced splenic mononuclear cells expressing the activation markers CD25 and CD71, both with and without the T-cell marker CD3 (P < 0.05); proportions also varied significantly. Similar patterns were noted in mononuclear and granular cells with adhesion markers CD11b and, to a lesser extent, CD54 (P < 0.05). The results show that a single, acute exposure to high-LET radiation induced changes that can profoundly alter leukocyte functions. The implications of the data are discussed in relation to low-LET radiation, altered gravity, and space flight.

Lantow, M., Schuderer, J., Hartwig, C. and Simkó, M. Free Radical Release and HSP70 Expression in Two Human Immune-Relevant Cell Lines after Exposure to 1800 MHz Radiofrequency Radiation. Radiat. Res. 165, 88–94 (2006).

The goal of this study was to investigate whether radiofrequency (RF) electromagnetic-field (EMF) exposure at 1800 MHz causes production of free radicals and/or expression of heat-shock proteins (HSP70) in human immune-relevant cell systems. Human Mono Mac 6 and K562 cells were used to examine free radical release after exposure to incubator control, sham, RF EMFs, PMA, LPS, heat (40°C) or co-exposure conditions. Several signals were used: continuous-wave, several typical modulations of the Global System for Mobile Communications (GSM): GSM-non DTX (speaking only), GSM-DTX (hearing only), GSM-Talk (34% speaking and 66% hearing) at specific absorption rates (SARs) of 0.5, 1.0, 1.5 and 2.0 W/kg. Heat and PMA treatment induced a significant increase in superoxide radical anions and in ROS production in the Mono Mac 6 cells when compared to sham and/ or incubator conditions. No significant differences in free radical production were detected after RF EMF exposure or in the respective controls, and no additional effects on superoxide radical anion production were detected after co-exposure to RF EMFs PMA or RF EMFs LPS. The GSM-DTX signal at 2 W/kg produced a significant difference in free radical production when the data were compared to sham because of the decreasing sham value. This difference disappeared when data were compared to the incubator controls. To determine the involvement of heat-shock proteins as a possible inhibitor of free radical production, we investigated the HSP70 expression level after different RF EMF exposures; no significant effects were detected.

Alfieri, R. R., Bonelli, M. A., Pedrazzi, G., Desenzani, S., Ghillani, M., Fumarola, C., Ghibelli, L., Borghetti, A. F. and Petronini, P. G. Increased Levels of Inducible HSP70 in Cells Exposed to Electromagnetic Fields. Radiat. Res. 165, 95–104 (2006).

Because reports in the literature on the effects of electromagnetic fields (EMFs) on expression of the 70-kDa heat-shock protein (HSP70) are somewhat contradictory, we studied the influence of low-frequency EMFs on the accumulation of inducible HSP70 in several cell models. Some of the cell types tested showed increased levels of HSP70 protein when exposed for 24 h to 50 Hz, 680 μT EMFs. In endothelial cells, EMFs alone induced only a poor and transient activation of the heat-shock transcription factor 1 (HSF1); however, neither the level of HSP70 mRNA nor the synthesis of HSP70 appeared to be altered significantly. Accordingly, transfection experiments involving HSP70 promoter showed that gene transcription was not affected. We also noted a marked reduction in proteasome activities in cell extracts exposed to EMFs. Interestingly, the heat-shock-induced levels of HSP70 mRNA and protein were increased by a concomitant weak stressor like EMFs. Taken together, our results indicate that in EMF-exposed endothelial cells, HSP70 gene transcription and translation are unaffected; however, EMFs alone promoted accumulation of the inducible HSP70 protein, probably by increasing its stability, and it enhanced accumulation and translation of the heat-induced HSP70 mRNA when applied in concert with heat shock.

Faraone, A., Luengas, W., Chebrolu, S., Ballen, M., Bit-Babik, G., Gessner, A. V., Kanda, M. Y., Babij, T., Swicord, M. L. and Chou, C-K. Radiofrequency Dosimetry for the Ferris-Wheel Mouse Exposure System. Radiat. Res. 165, 105–112 (2006).

Numerical and experimental methods were employed to assess the individual and collective dosimetry of mice used in a bioassay on the exposure to pulsed radiofrequency energy at 900 MHz in the Ferris-wheel exposure system (Utteridge et al., Radiat. Res. 158, 357–364, 2002). Twin-well calorimetry was employed to measure the whole-body specific absorption rate (SAR) of mice for three body masses (23 g, 32 g and 36 g) to determine the lifetime exposure history of the mice used in the bioassay. Calorimetric measurements showed about 95% exposure efficiency and lifetime average whole-body SARs of 0.21, 0.86, 1.7 and 3.4 W kg⁻¹ for the four exposure groups. A larger statistical variation in SAR was observed in the smallest mice because they had the largest variation in posture inside the plastic restrainers. Infrared thermography provided SAR distributions over the sagittal plane of mouse cadavers. Thermograms typically showed SAR peaks in the abdomen, neck and head. The peak local SAR at these locations, determined by thermometric measurements, showed peak-to-average SAR ratios below 6:1, with typical values around 3:1. Results indicate that the Ferris wheel fulfills the requirement of providing a robust exposure setup, allowing uniform collective lifetime exposure of mice.

Böcker, W. and Iliakis, G. Computational Methods for Analysis of Foci: Validation for Radiation-Induced γ-H2AX Foci in Human Cells. Radiat. Res. 165, 113–124 (2006).

Observation and counting of γ-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced γ-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and γ-H2AX foci. The computational methods presented extend to images generated by digital cameras.