Li., L., Steinauer, K. L., Dirks, A. J., Husbeck, B., Gibbs, I. and Knox, S. J. Radiation-Induced Cyclooxygenase 2 Up-Regulation is Dependent on Redox Status in Prostate Cancer Cells. Radiat. Res. 160, 617–621 (2003).

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE₂. In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (γGCS), and the antioxidants α-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas α-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H₂O₂ had similar effects on COX2 up-regulation and PGE₂ production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE₂ production induced by radiation and H₂O₂ were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with α-lipoic acid or NAC for 24 h, both radiation- and H₂O₂-induced COX2 up-regulation and PGE₂ production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Wan, X. S., Zhou, Z. and Kennedy, A. R. Adaptation of the Dichlorofluorescein Assay for Detection of Radiation-Induced Oxidative Stress in Cultured Cells. Radiat. Res. 160, 622–630, (2003).

The oxidation of 2′7′-dichlorofluorescin (DCFH) to 2′7′-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to γ rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with γ rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.

Lestaevel, P., Clarençon, D., Gharib, A., Peinnequin, A., Cespuglio, R., Gourmelon, P., Alonso, A., Laval, J. D. and Multon, E. Nitric Oxide Voltammetric Measurements in the Rat Brain after Gamma Irradiation. Radiat. Res. 160, 631–636 (2003).

The effects of a lethal γ irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude ( 120%) 24 h later. A dose–effect relationship from 2 to 15 Gy for γ-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca -independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after γ-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.

Van der Meeren, A., Vandamme, M., Squiban, C., Gaugler, M-H. and Mouthon, M-A. Inflammatory Reaction and Changes in Expression of Coagulation Proteins on Lung Endothelial Cells after Total-Body Irradiation in Mice. Radiat. Res. 160, 637–646 (2003).

Inflammatory reaction is a classical feature of radiation exposure, and pneumonitis is a dose-limiting complication in the handling of hematological disorders treated with total-body irradiation. In the present study, we first evaluated the inflammatory response in C57BL6/J mice exposed to lethal doses of γ rays treated with antibiotics or not. Both interleukin 6 and KC (also known as Gro1) were increased in the plasma 10 to 18 days after radiation exposure, independent of bacterial infection, whereas fibrinogen release was linked to a bacterial infection. Furthermore, both Il6 and KC were increased in the lungs of irradiated mice. Our second objective was to characterize the endothelial cell changes in the lungs of total-body-irradiated mice. For this purpose, a quantitative RT-PCR was used to determine the expression of genes involved in inflammatory and coagulation processes. We found that the adhesion molecules P-selectin and platelet endothelial cell adhesion molecule 1 were up-regulated, whereas E-selectin remained unchanged. Tissue factor expression was up-regulated as well, and thrombomodulin gene expression was down-regulated. The investigation by immunohistochemistry of adhesion molecules confirmed the increase in the basal expression of both P-selectin and platelet endothelial cell adhesion molecule 1 on pulmonary endothelial cells. All together, our results suggest the involvement of endothelial cells in the development of radiation-induced inflammatory and thrombotic processes.

Bergner, A., Stief, J., Holdenrieder, S., Stieber, P., Haimerl, W., Passlick, B., Waldschmidt, A., Kummermehr, J., Gamarra, F. and Huber, R. M. Effects of Single-Dose Irradiation on Bronchial Epithelium: A Comparison of BEAS 2B Cell Monolayers, Human Organ Cultures, and Goettinger Minipigs. Radiat. Res. 160, 647–654 (2003).

To assess the effects of radiation on bronchial epithelium, BEAS 2B cells cultured as monolayers and human bronchial epithelium cultured as organ cultures were exposed to single doses of 0, 10 and 30 Gy. The lactate dehydrogenase in the supernatant of the BEAS 2B cells increased markedly 24 h after irradiation, whereas in the organ cultures only a minor increase was found after 48 h. The nucleosomes in the supernatant of the BEAS 2B cells showed a massive increase in response to irradiation, whereas in the organ cultures no change could be seen. The number of BEAS 2B cells was dramatically diminished after 96 h, whereas in the organ cultures a smaller decrease was observed no earlier than 21 days after irradiation. To assess the effects of brachytherapy in bronchial epithelium in vivo, brachytherapy with 30 Gy was performed in Goettinger minipigs, and histological sections of the bronchi were analyzed for morphological alterations and cell numbers. After 2 weeks, only slight cell damage was observable, and after 3 weeks, moderate morphological changes and decreased cell numbers were found. However, after 8 weeks, the epithelium had nearly regained its normal structure. We conclude that the bronchial epithelium has a remarkably high radioresistance and that organ cultures, but not monolayers of BEAS 2B cells, reflect the effects of radiation in vivo.

Satyamitra, M. M., Uma Devi, P., Murase, H. and Kagiya, V. T. In Vivo Postirradiation Protection by a Vitamin E Analog, α-TMG. Radiat. Res. 160, 655–661 (2003).

The water-soluble vitamin E derivative α-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with α-TMG against a range of whole-body lethal (8.5–12 Gy) and sublethal (1–5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.

Kim, J. H., Lew, Y. S., Kolozsvary, A., Ryu, S. and Brown, S. L. Arsenic Trioxide Enhances Radiation Response of 9L Glioma in the Rat Brain. Radiat. Res. 160, 662–666 (2003).

Arsenic trioxide (ATO) at low doses induces leukemia cells to undergo apoptosis and at higher doses causes blood flow to solid tumors to shut down. To determine whether a potential synergistic interaction exists between ATO at the non-toxic dose level in the rat and radiation, the present study was carried out with orthotopic 9L malignant gliomas growing in the brains of rats. Animals died within 50 days of treatment when 12-day-old 9L gliomas growing in the brain of Fischer rats were treated with either the drug alone (8 mg/kg) or radiation alone (25 Gy). In contrast, the overall tumor cure rate exceeded 50% at a follow-up time of 120 days after the combined treatment with radiation and ATO. Long-term surviving animals showed no clinical or disproportionately enhanced histopathological changes in the brain parenchyma. Early changes in tumor physiology showed that the vascular leakage of FITC-dextran conjugates was apparent within 8 h of drug administration. Last, the use of diffusion magnetic resonance imaging as an early surrogate marker of therapeutic efficacy corroborated the effects of drug with and without radiation on brain histology and animal survival.

Bench, G., Grant, P. G., Ueda, D. L., Autry-Conwell, S. A., Hou, Y. and Boggan, J. E. Assessment of Proton Microbeam Analysis of ¹¹B for Quantitative Microdistribution Analysis of Boronated Neutron Capture Agents in Biological Tissues. Radiat. Res. 160, 667–676 (2003).

The ¹¹B(p,α)⁸Be* nuclear reaction was assessed for its ability to quantitatively map the in vivo subcellular distribution of boron within gliosarcomas treated with a boronated neutron capture therapy agent. Intracranial 9L gliosarcomas were produced in Fischer 344 rats. Fourteen days later, the majority of the rats were treated with f-boronophenylalanine and killed humanely 30 or 180 min after intravenous injection. Freeze-dried tumor cryosections were imaged using the ¹¹B(p,α)⁸Be* nuclear reaction and proton microbeams obtained from the nuclear microprobe at Lawrence Livermore National Laboratory. The ¹¹B distributions within cells could be imaged quantitatively with spatial resolutions down to 1.5 μm, minimum detection limits of 0.8 mg/kg, and acquisition times of several hours. These capabilities offer advantages over α-particle track autoradiography, electron energy loss spectroscopy, and secondary ion mass spectrometry (SIMS) for quantification of ¹¹B in tissues. However, the spatial resolution, multi-isotope capability, and analysis times achieved with SIMS are superior to those achieved with ¹¹B(p,α)⁸Be* analysis. When accuracy in quantification is crucial, the ¹¹B(p,α)⁸Be* reaction is well suited for assessing the microdistribution of ¹¹B. Otherwise, SIMS may well be better suited to image the microdistribution of boron associated with neutron capture therapy agents in biological tissues.

Wojcik, A., Stephan, G., Sommer, S., Buraczewska, I., Kuszewski, T., Wieczorek, A. and Gózdz, S. Chromosomal Aberrations and Micronuclei In Lymphocytes of Breast Cancer Patients after an Accident during Radiotherapy with 8 MeV Electrons. Radiat. Res.160, 677–683 (2003).

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.

Hadjidekova, V. B., Bulanova, M., Bonassi, S. and Neri, M. Micronucleus Frequency is Increased in Peripheral Blood Lymphocytes of Nuclear Power Plant Workers. Radiat. Res. 160, 684–690 (2003).

Nuclear power plant workers are exposed to ionizing radiation at relatively low doses and for prolonged periods of time. To investigate the extent of genetic damage in these workers, a group of 133 nuclear power plant workers and 39 healthy controls were compared using the cytokinesis-block micronucleus assay. The frequency of micronuclei was significantly increased in peripheral lymphocytes of nuclear power plant workers (20.5 ± 9.7% compared to 13.7 ± 5.9%). A significant dose–response relationship was observed between micronucleus (MN) frequency and both the accumulated dose and the duration of employment (P < 0.01 for both variables after adjusting for age, gender and cigarette smoking) with an evident leveling off for exposures over 200 mSv. Accumulated dose and duration of employment were significantly correlated but exerted independent effects on MN frequency. For non-occupational parameters, age was significantly associated with the frequency of micronuclei, while gender was not. Smoking habit showed no overall effect, whereas increased chromosome damage was evident in smokers of more than 20 cigarettes per day. In conclusion, a dose-related association between MN frequency and exposure to ionizing radiation was evident in nuclear power plant workers, encouraging the application of the cytokinesis-block micronucleus assay in biomonitoring studies of human populations with prolonged exposure to ionizing radiation.

Travis, L. B., Hauptmann, M., Gaul, L. K., Storm, H. H., Goldman, M. B., Nyberg, U., Berger, E., Janower, M. L., Hall, P., Monson, R. R., Holm, L-E., Land, C. E., Schottenfeld, D., Boice, J. D., Jr. and Andersson, M. Site-Specific Cancer Incidence and Mortality after Cerebral Angiography with Radioactive Thorotrast. Radiat. Res. 160, 691–706 (2003).

Few opportunities exist to evaluate the carcinogenic effects of long-term internal exposure to α-particle-emitting radionuclides. Patients injected with Thorotrast (thorium-232) during radiographic procedures, beginning in the 1930s, provide one such valuable opportunity. We evaluated site-specific cancer incidence and mortality among an international cohort of 3,042 patients injected during cerebral angiography with either Thorotrast (n = 1,650) or a nonradioactive agent (n = 1,392) and who survived 2 or more years. Standardized incidence ratios (SIR) for Thorotrast and comparison patients (Denmark and Sweden) were estimated and relative risks (RR), adjusted for population, age and sex, were generated with multivariate statistical modeling. For U.S. patients, comparable procedures were used to estimate standardized mortality ratios (SMR) and RR, representing the first evaluation of long-term, site-specific cancer mortality in this group. Compared with nonexposed patients, significantly increased risks in Thorotrast patients were observed for all incident cancers combined (RR = 3.4, 95% CI 2.9–4.1, n = 480, Denmark and Sweden) and for cancer mortality (RR = 4.0, 95% CI 2.5–6.7, n = 114, U.S.). Approximately 335 incident cancers were above expectation, with large excesses seen for cancers of the liver, bile ducts and gallbladder (55% or 185 excess cancers) and leukemias other than CLL (8% or 26 excess cancers). The RR of all incident cancers increased with time since angiography (P < 0.001) and was threefold at 40 or more years; significant excesses (SIR = 4.0) persisted for 50 years. Increasing cumulative dose of radiation was associated with an increasing risk of all incident cancers taken together and with cancers of the liver, gallbladder, and peritoneum and other digestive sites; similar findings were observed for U.S. cancer mortality. A marginally significant dose response was observed for the incidence of pancreas cancer (P = 0.05) but not for lung cancer. Our study confirms the relationship between Thorotrast and increased cancer incidence at sites of Thorotrast deposition and suggests a possible association with pancreas cancer. After injection with >20 ml Thorotrast, the cumulative excess risk of cancer incidence remained elevated for up to 50 years and approached 97%. Caution is needed in interpreting the excess risks observed for site-specific cancers, however, because of the potential bias associated with the selection of cohort participants, noncomparability with respect to the internal or external comparison groups, and confounding by indication. Nonetheless, the substantial risks associated with liver cancer and leukemia indicate that unique and prolonged exposure to α-particle-emitting Thorotrast increased carcinogenic risks.

Land, C. E., Tokunaga, M., Koyama, K., Soda, M., Preston, D. L., Nishimori, I. and Tokuoka, S. Incidence of Female Breast Cancer among Atomic Bomb Survivors, Hiroshima and Nagasaki, 1950–1990. Radiat. Res. 160, 707–717 (2003).

An incidence survey of the Life Span Study (LSS) population found 1093 breast cancers among 1059 breast cancer cases diagnosed during 1950–1990. As in earlier breast cancer surveys of this population, a linear and statistically highly significant radiation dose response was found. In the analysis, particular attention was paid to modification of radiation dose response by age at exposure (e) and attained age (a). Dose-specific excess relative risk (ERR_1Sv) decreased with increasing values of e and a. A linear dose–response model analysis, with e and a as exponential age modifiers, did not conclusively discriminate between the two variables as modifiers of dose response. A modified isotonic regression approach, requiring only that ERR_1Sv be monotonic in age, provides a fresh perspective indicating that both e and a are important modifiers of dose response. Exposure before age 20 was associated with higher ERR_1Sv compared to exposure at older ages, with no evidence of consistent variation by exposure age for ages under 20. ERR_1Sv was observed to decline with increasing attained age, with by far the largest drop around age 35. Possible explanations for these observations are discussed, along with research approaches that might provide more information.

Pierce, D. A. Mechanistic Models for Radiation Carcinogenesis and the Atomic Bomb Survivor Data. Radiat. Res. 160, 718–723 (2003).

Recently, Heidenreich et al. (Radiat. Res., 158, 607–617, 2002) suggested that the Radiation Effects Research Foundation (RERF) A-bomb survivor cohort study is not large enough to discriminate between various possible carcinogenic mechanisms. At least with the current follow-up, this is true to some extent, but I think the specific issues are rather different than they suggest. In particular, I do not think it is true—as they further indicate—that various models fit the data about equally well while estimating very different patterns of excess risk, which would imply that these patterns cannot be reasonably well characterized. I will point to specific criticisms of their approach to the data and offer some more general comments on mechanistic modeling approaches. Although there are important distinctions, I suggest on a very optimistic note that the two major approaches may be converging, and soon the main differences may not be in the assumptions made but in the aims of the modeling.

Katz, R. The Parameter-Free Track Structure Model of Scholz and Kraft for Heavy-Ion Cross Sections. Radiat. Res. 160, 724–728 (2003).

The “parameter-free”, “local effects” theory of Scholz and Kraft is an extension to mammalian cells of the theory of RBE for dry enzymes and viruses of Butts and Katz. Its claim for parameter freedom has been challenged elsewhere. Here we examine its conceptual base and find errors in its use of the physical concept of cross section and its neglect of the radiobiological relationship between target size and radiosensitivity in evaluating the radiation damage to “point targets”.

Coleman, C. N., Stone, H. B., Alexander, G. A., Barcellos-Hoff, M. H., Bedford, J. S., Bristow, R. G., Dynlacht, J. R., Fuks, Z., Gorelic, L. S., Hill, R. P., Joiner, M. C., Liu, F-F., McBride, W. H., McKenna, W. G., Powell, S. N., Robbins, M. E. C., Rockwell, S., Schiff, P. B., Shaw, E. G., Siemann, D. W., Travis, E. L., Wallner, P. E., Wong, R. S. L. and Zeman, E. M. Education and Training for Radiation Scientists: Radiation Research Program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland, May 12–14, 2003. Radiat. Res. 160, 729–737 (2003).

Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204–223, 2002; Radiat. Res. 159, 812–834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences.