Kossenko, M. M., Thomas, T. L., Akleyev, A. V., Krestinina, L. Yu., Startsev, N. V., Zhidkova, C. M., Vyushkova, O. V., Hoffman, D. A., Preston, D. L., Davis, F. and Ron, E. The Techa River Cohort: Study Design and Follow-up Methods. Radiat. Res. 164, 591–601 (2005).

Residents living on the banks of the Techa River in the Southern Urals region of Russia were exposed to radioactive contamination from the Mayak plutonium production and separation facility that discharged liquid radioactive waste into this river. This paper describes the methods used to establish and follow the Extended Techa River Cohort (ETRC), which includes almost 30,000 people living along the Techa River who were exposed to a complex mixture of radionuclides, largely ⁹⁰Sr and ¹³⁷Cs. The system of regular follow-up allows ascertainment of vital status, cause of death and cancer incidence. With over 50 years of follow-up and over 50% deceased, the ETRC now provides a valuable opportunity to study a wide range of health effects, both early and late, associated with protracted internal and external radiation exposures. The wide range of doses allows analysis of the nature of the dose–response relationship based on internal comparisons. Other features of the cohort are the high proportion (40%) exposed under age 20, and the inclusion of both sexes. The limitations of the study include loss to follow-up due to difficulties in tracing some cohort members and migration and incomplete ascertainment of cause of death.

Krestinina, L. Y., Preston, D. L., Ostroumova, E. V., Degteva, M. O., Ron, E., Vyushkova, O. V., Startsev, N. V., Kossenko, M. M. and Akleyev, A. V. Protracted Radiation Exposure and Cancer Mortality in the Extended Techa River Cohort. Radiat. Res. 164, 602–611 (2005).

In the 1950s many thousands of people living in rural villages on the Techa River received protracted internal and external exposures to ionizing radiation from the release of radioactive material from the Mayak plutonium production complex. The Extended Techa River Cohort includes 29,873 people born before 1950 who lived near the river sometime between 1950 and 1960. Vital status and cause of death are known for most cohort members. Individualized dose estimates have been computed using the Techa River Dosimetry System 2000. The analyses provide strong evidence of long-term carcinogenic effects of protracted low-dose-rate exposures; however, the risk estimates must be interpreted with caution because of uncertainties in the dose estimates. We provide preliminary radiation risk estimates for cancer mortality based on 1,842 solid cancer deaths (excluding bone cancer) and 61 deaths from leukemia. The excess relative risk per gray for solid cancer is 0.92 (95% CI 0.2; 1.7), while those for leukemia, including and excluding chronic lymphocytic leukemia, are 4.2 (CI 95% 1.2; 13) and 6.5 (CI 95% 1.8; 24), respectively. It is estimated that about 2.5% of the solid cancer deaths and 63% of the leukemia deaths are associated with the radiation exposure.

Montoro, A., Rodríguez, P., Almonacid, M., Villaescusa, J. I., Verdú, G., Caballín, M. R., Barrios, L. and Barquinero, J. F. Biological Dosimetry in a Group of Radiologists by the Analysis of Dicentrics and Translocations. Radiat. Res. 164, 612–617 (2005).

The results of a cytogenetic study carried out in a group of nine radiologists are presented. Chromosome aberrations were detected by fluorescence plus Giemsa staining and fluorescence in situ hybridization. Dose estimates were obtained by extrapolating the yield of dicentrics and translocations to their respective dose–effect curves. In seven individuals, the 95% confidence limits of the doses estimated by dicentrics did not include 0 Gy. The 99 dicentrics observed in 17,626 cells gave a collective estimated dose of 115 mGy (95% confidence limits 73–171). For translocations, five individuals had estimated doses that were clearly higher than the total accumulated recorded dose. The 82 total apparently simple translocations observed in 9722 cells gave a collective estimated dose of 275 mGy (132–496). The mean genomic frequencies (×100 ± SE) of complete and total apparently simple translocations observed in the group of radiologists (1.91 ± 0.30 and 2.67 ± 0.34, respectively) were significantly higher than those observed in a matched control group (0.53 ± 0.10 and 0.87 ± 0.13, P < 0.01 in both cases) and in another occupationally exposed matched group (0.79 ± 0.12 and 1.14 ±0.14, P < 0.03 and P < 0.01, respectively). The discrepancies observed between the physically recorded doses and the biologically estimated doses indicate that the radiologists did not always wear their dosimeters or that the dosimeters were not always in the radiation field.

Kodama, Y., Ohtaki, K., Nakano, M., Hamasaki, K., Awa, A. A., Lagarde, F. and Nakamura, N. Clonally Expanded T-Cell Populations in Atomic Bomb Survivors Do Not Show Excess Levels of Chromosome Instability. Radiat. Res. 164, 618– 626 (2005).

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.

Howe, O., O'Malley, K., Lavin, M., Gardner, R. A., Seymour, C., Lyng, F., Mulvin, D., Quinlan, D. M. and Mothersill, C. Cell Death Mechanisms Associated with G₂ Radiosensitivity in Patients with Prostate Cancer and Benign Prostatic Hyperplasia. Radiat. Res. 164, 627–634 (2005).

Cells respond to genotoxic insults such as ionizing radiation by halting in the G₂ phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G₂, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G₂ chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G₂ chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G₂ chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G₂ assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G₂ compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G₂ checkpoint control in the prostate patients. There was no significant correlation of G₂ radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G₂ radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G₂ aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

Casado, J. A., Núñez, M. I., Segovia, J. C., Ruiz de Almodóvar, J. M. and Bueren, J. A. Non-homologous End-Joining Defect in Fanconi Anemia Hematopoietic Cells Exposed to Ionizing Radiation. Radiat. Res. 164, 635–641 (2005).

Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca^−/− hematopoietic progenitor cells was observed compared to Fanca ^/ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca^−/− mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.

Pauwels, B., Korst, A. E. C., Andriessen, V., Baay, M. F. D., Pattyn, G. G. O., Lambrechts, H. A. J., De Pooter, C. M. J., Lardon, F. and Vermorken, J. B. Unraveling the Mechanism of Radiosensitization by Gemcitabine: The Role of TP53. Radiat. Res. 164, 642–650 (2005).

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.

Springer, D. L., Ahram, M., Adkins, J. N., Kathmann, L. E. and Miller, J. H. Characterization of Medium Conditioned by Irradiated Cells Using Proteome-Wide, High-Throughput Mass Spectrometry. Radiat. Res. 164, 651–654 (2005).

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based on immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.

Zhu, A., Zhou, H., Leloup, C., Marino, S. A., Geard, C. R., Hei, T. K. and Lieberman, H. B. Differential Impact of Mouse Rad9 Deletion on Ionizing Radiation-Induced Bystander Effects. Radiat. Res. 164, 655–661 (2005).

The cellular response to ionizing radiation is not limited to cells irradiated directly but can be demonstrated in neighboring “bystander” populations. The ability of mouse embryonic stem (ES) cells to express a bystander effect and the role of the radioresistance gene Rad9 were tested. Mouse ES cells differing in Rad9 status were exposed to broad-beam 125 keV/ μm ³He α particles. All populations, when confluent, demonstrated a dose-independent bystander effect with respect to cell killing, and the Rad9^−/− genotype did not selectively alter that response or cell killing after direct exposure to this high-LET radiation. In contrast, relative to Rad9 ^/ cells, the homozygous mutant was sensitive to direct exposure to α particles when in log phase, providing evidence of a role for Rad9 in repair of potentially lethal damage. Direct exposure to α particles induced an increase in the frequency of apoptosis and micronucleus formation, regardless of Rad9 status, although the null mutant showed high spontaneous levels of both end points. All populations demonstrated α-particle-induced bystander apoptosis, but that effect was most prominent in Rad9^−/− cells. Minimal α-particle induction of micronuclei in bystander cells was observed, except for the Rad9^−/− mutant, where a significant increase above background was detected. Therefore, the Rad9 null mutation selectively sensitizes mouse ES cells to spontaneous and high-LET radiation-induced bystander apoptosis and micronucleus formation, but it has much less impact on cell killing by direct or bystander α-particle exposure. Results are presented in the context of defining the function of Rad9 in the cellular response to radiation and its differential effects on individual bystander end points.

Brown, W. R., Thore, C. R., Moody, D. M., Robbins, M. E. and Wheeler, K. T. Vascular Damage after Fractionated Whole-Brain Irradiation in Rats. Radiat. Res. 164, 662–668 (2005).

Whole-brain irradiation of animals and humans has been reported to lead to late delayed structural (vascular damage, demyelination, white matter necrosis) and functional (cognitive impairment) alterations. However, most of the experimental data on late delayed radiation-induced brain injury have been generated with large single doses or short fractionation schemes that may provide a less accurate indication of the events that occur after clinical whole-brain radiotherapy. The pilot study reported here investigates cerebral vascular pathology in male Fischer 344 rats after whole-brain irradiation with a fractionated total dose of ¹³⁷Cs γ rays that is expected to be biologically similar to that given to brain tumor patients. The brains of young adult rats (4 months old) were irradiated with a total dose of 40 Gy, given as eight 5-Gy fractions twice per week for 4 weeks. Brain capillary and arteriole pathology was studied using an alkaline phosphatase enzyme histochemistry method; vessel density and length were quantified using a stereology method with computerized image processing and analysis. Vessel density and length were unchanged 24 h after the last dose, but at 10 weeks postirradiation, both were substantially decreased. After 20 weeks, the rate of decline in the vessel density and length in irradiated rats was similar to that in unirradiated age-matched controls. No gross gliosis or demyelination was observed 12 months postirradiation using conventional histopathology techniques. We suggest that the early (10-week) and persistent vascular damage that occurs after a prolonged whole-brain irradiation fractionation scheme may play an important role in the development of late delayed radiation-induced brain injury.

Kühne, M., Urban, G., Frankenberg, D. and Löbrich, M. DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to C_K Characteristic X Rays, 29 kVp X Rays and ⁶⁰Co γ Rays. Radiat. Res. 164, 669–676 (2005).

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for C_K characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for ⁶⁰Co γ rays. DSB induction by C_K characteristic X rays was about twofold higher than for ⁶⁰Co γ rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with ⁶⁰Co γ rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.

Sowa, M. B., Kathmann, L. E., Holben, B. A., Thrall, B. D. and Kimmel, G. A. Low-LET Microbeam Investigation of the Track-End Dependence of Electron-Induced Damage in Normal Human Diploid Fibroblasts. Radiat. Res. 164, 677–679 (2005).

Using a pulsed electron beam, we investigated the dependence of micronucleus formation on the incident electron energy in AG01522 human diploid fibroblasts after nontargeted irradiations at 25 and 80 keV. Examining the dose response, we found that 25 keV electrons are more effective than 80 keV electrons at producing biological damage for a given dose. Our results demonstrating the induction of micronuclei as a function of incident electron energy offer direct support for the hypothesis that the electron track end is responsible for the biological damage occurring in the cell.

Bendel, P., Margalit, R., Koudinova, N. and Salomon, Y. Noninvasive Quantitative In Vivo Mapping and Metabolism of Boronophenylalanine (BPA) by Nuclear Magnetic Resonance (NMR) Spectroscopy and Imaging. Radiat. Res. 164, 680–687 (2005).

¹⁰B-enriched l-p-boronophenylalanine (BPA) is one of the compounds used in boron neutron capture therapy (BNCT). In this study, several variations of nuclear magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) were applied to investigate the uptake, clearance and metabolism of the BPA-fructose complex (BPA-F) in normal mouse kidneys, rat oligodendroglioma xenografts, and rat blood. Localized ¹H MRS was capable of following the uptake and clearance of BPA-F in mouse kidneys with temporal resolution of a few minutes, while ¹H MRSI was used to image the BPA distribution in the kidney with a spatial resolution of 9 mm³. The results also revealed significant dissociation of the BPA-F complex to free BPA. This finding was corroborated by ¹H and ¹¹B NMR spectroscopy of rat blood samples as well as of tumor samples excised from mice after i.v. injection of BPA-F. This investigation demonstrates the feasibility of using ¹H MRS and MRSI to follow the distribution of BPA in vivo, using NMR techniques specifically designed to optimize BPA detection. The implementation of such procedures could significantly improve the clinical efficacy of BNCT.

Meesungnoen, J. and Jay-Gerin, J-P. Effect of Multiple Ionization on the Yield of H₂O₂ Produced in the Radiolysis of Aqueous 0.4 M H₂SO₄ Solutions by High-LET ¹²C⁶ and ²⁰Ne⁹ Ions. Radiat. Res. 164, 688–694 (2005).

Monte Carlo track structure simulations were performed to investigate the effect of multiple ionization of water on the primary (or “escape”) (at ∼10⁻⁶ s) yield of hydrogen peroxide (G_H2O2) produced in the radiolysis of deaerated 0.4 M H₂SO₄ solutions by ¹²C⁶ and ²⁰Ne⁹ ions at high linear energy transfer (LET) up to ∼900 keV/μm. It was found that, upon incorporating the mechanisms of double, triple and quadruple ionizations of water in the calculations, a quantitative agreement between theory and experiment can be obtained. The curve for G_H2O2 as a function of LET reaches a well-defined maximum of ∼1.4 molecules/100 eV at ∼180–200 keV/μm, in very good accord with the available experimental data. Our results also show that, for the highest LET values considered in this study, the H₂O₂ escape yields obtained in 0.4 M sulfuric acid solutions are about 45% greater in magnitude than those found in neutral water. Contrary to a recent assumption suggesting that the limiting value of G_H2O2 at infinite LET should be ∼1 molecule/100 eV, somewhat similar for neutral and acidic water, our simulations show a clear decrease in the primary H₂O₂ yields with increasing LET at high LET, indicating that the question of the limiting value of G_H2O2 at very high LET for both neutral and acidic liquid water is still open.

Sowa, M. B., Murphy, M. K., Miller, J. H., McDonald, J. C., Strom, D. J. and Kimmel, G. A. A Variable-Energy Electron Microbeam: A Unique Modality for Targeted Low-LET Radiation. Radiat. Res. 164, 695–700 (2005).

We have designed and constructed a low-cost, variable-energy low-LET electron microbeam that uses energetic electrons to mimic radiation damage produced by γ and X rays. The microbeam can access lower regions of the LET spectrum, similar to conventional X-ray or ⁶⁰Co γ-ray sources. The device has two operating modes, as a conventional microbeam targeting single cells or subpopulations of cells or as a pseudo broad-beam source allowing for direct comparison with conventional sources. By varying the incident electron energy, the target cells can be selectively exposed to different parts of the energetic electron tracks, including the track ends.

Fink, C. A. and Bates, M. N. Melanoma and Ionizing Radiation: Is There a Causal Relationship? Radiat. Res. 164, 701–710 (2005).

This review was initiated in response to concerns that ionizing radiation could be a cause of melanoma. Studies presenting the relative risks for melanoma after external ionizing radiation exposure were in seven categories: (1) The Canadian Radiation Dose Registry, (2) nuclear industry workers, (3) subjects near nuclear test blasts, (4) survivors of the atomic bombings of Japan, (5) airline pilots and cabin attendants, (6) recipients of medical radiation, and (7) radiological technicians. Relative risks for leukemia in each of the studies were used to confirm the likelihood of exposure to ionizing radiation. When studies within a category were compatible, meta-analytic methods were used to obtain combined estimates of the relative risk, and a meta-regression analysis of melanoma relative risk compared to leukemia relative risk was used to examine consistency across exposure categories. Generally, exposure categories with elevated relative risks of leukemia had proportionately elevated relative risks of melanoma. This suggests that people exposed to ionizing radiation may be at increased risk of developing melanoma, although alternative explanations are possible. Future epidemiological studies of ionizing radiation effects should include melanoma as an outcome of interest.