Thierry-Chef, I., Simon, S. L., Land, C. E. and Miller, D. L. Radiation Dose to the Brain and Subsequent Risk of Developing Brain Tumors in Pediatric Patients Undergoing Interventional Neuroradiology Procedures. Radiat. Res. 170, 553– 565 (2008).

Radiation dose to the brain and subsequent lifetime risk of diagnosis of radiation-related brain tumors were estimated for pediatric patients undergoing intracranial embolization. Average dose to the whole brain was calculated using dosimetric data from the Radiation Doses in Interventional Radiology Study for 49 pediatric patients who underwent neuroradiological procedures, and lifetime risk of developing radiation-related brain tumors was estimated using published algorithms based on A-bomb survivor data. The distribution of absorbed dose within the brain can vary significantly depending on field size and movement during procedures. Depending on the exposure conditions and age of the patient, organ-averaged brain dose was estimated to vary from 6 to 1600 mGy. The lifetime risk of brain tumor diagnosis was estimated to be increased over the normal background rates (57 cases per 10,000) by 3 to 40% depending on the dose received, age at exposure, and gender. While significant uncertainties are associated with these estimates, we have quantified the range of possible dose and propagated the uncertainty to derive a credible range of estimated lifetime risk for each subject. Collimation and limiting fluoroscopy time and dose rate are the most effective means to minimize dose and risk of future induction of radiation-related tumors.

Hofer, M., Pospíšil, M., Holá, J., Vacek, A., Štreitová, D. and Znojil, V. Inhibition of Cyclooxygenase 2 in Mice Increases Production of G-CSF and Induces Radioprotection. Radiat. Res. 170, 566–571 (2008).

Meloxicam, a selective inhibitor of cyclooxygenase 2, was tested to determine its ability to modulate hematopoiesis and to influence survival of mid-lethally γ-irradiated mice. A single dose of meloxicam (20 mg/kg) administered to mice intraperitoneally 1 h before irradiation was shown to enhance serum levels of granulocyte colony-stimulating factor (G-CSF) during the first 24 h after irradiation, to elevate numbers of granulocytic precursor cells in bone marrow and granulocyte counts in peripheral blood on day 10 after irradiation, and to increase 30-day survival of these mice. The results provide new evidence for the protective ability of meloxicam administration to mice irradiated with mid-lethal doses and contribute to the understanding of the mechanisms of this meloxicam action by drawing attention to the possible role of increased endogenous G-CSF production.

Acevedo, S. F., Tittle, S., Merry, D. E. and Raber, J. Transgenic Expression of Androgen Receptors Improves Spatial Memory Retention in both Sham-Irradiated and ¹³⁷Cs γ-Irradiated Female Mice. Radiat. Res. 170, 572–578 (2008).

Using a water maze, it has been shown that both wild-type and apoE4-expressing female mice are at greater risk of developing age-related hippocampal-dependent impairments in spatial learning and memory than age-matched male mice of the same genotype. In addition, apoE4-expressing female mice were more sensitive to ¹³⁷Cs γ-radiation-induced impairment in spatial learning and memory than age-matched male mice of the same genotype. These findings imply that androgen receptors (ARs) contribute to spatial learning and memory, posing the question as to whether transgenic expression of AR in female mice might modulate hippocampal-dependent learning and memory under baseline conditions and after local brain irradiation. Hippocampal-dependent novel location recognition was comparable in wild-type and AR-Tg female mice. This function was impaired after irradiation in AR-Tg but not wild-type mice. In contrast, sham-irradiated wild-type and AR-Tg female mice showed hippocampal-independent novel location recognition, and this was not affected by radiation. After the second day of hidden platform training, in a water maze probe trial, sham-irradiated and irradiated AR-Tg female mice showed spatial memory retention but irradiated wild-type mice did not. After the third day of hidden platform training, only irradiated wild-type female mice did not show spatial memory retention in the water maze probe trial. Both sham-irradiated and irradiated wild-type and AR-Tg female mice showed passive avoidance learning and memory. These data support an important role for AR in spatial memory retention in water maze probe trials in female mice under baseline conditions and after cranial irradiation.

Lee, H-J., Lee, Y-J., Kang, C-M., Bae, S., Jeoung, D., Jang, J-J., Lee, S-S., Cho, C-K. and Lee, Y-S. Differential Gene Signatures in Rat Mammary Tumors Induced by DMBA and Those Induced by Fractionated γ Radiation. Radiat. Res. 170, 579–590 (2008).

The aim of this work was to identify specific genes involved in rat mammary tumors induced by dimethylbenz(a)anthracene (DMBA) or radiation. More TUNEL- and PCNA-positive cells were present in mammary tumors induced by radiation than in tumors induced by DMBA, whereas DNA damage responses like p53 accumulation and histone H2AX phosphorylation were higher in DMBA-induced tumors, even though the pathology was similar in both types of tumors. cDNA microarray and real-time RT-PCR analysis of radiation- or DMBA-induced tumor tissues, revealed that stanniocalcin 2 (Stc2), interferon regulatory factor 1 (Irf1), interleukin 18 binding protein (Il18bp), and chloride channel calcium activated 3 (Clca3) were expressed in both, and that arachidonate 5-lipoxygenase activating protein 1 (Alox5ap) and cathepsin S (Ctss) were expressed only in radiation-induced tumors. No DMBA-specific gene signatures were found. Soft agar growth assays were carried out to identify the carcinogenic features of these specific genes. Cells stably transfected with Alox5ap, Ctss, Stc2, Irf1, Il18bp and Clca3 showed morphological changes compared to controls. These findings indicate different gene alterations in carcinogen- or radiation-induced mammary tumors with similar pathological stages.

Van der Meeren, A., Tourdes, F., Grémy, O., Grillon, G., Abram, M-C., Poncy, J-L. and Griffiths, N. Activation of Alveolar Macrophages after Plutonium Oxide Inhalation in Rats: Involvement in the Early Inflammatory Response. Radiat. Res. 170, 591––603 (2008).

Alveolar macrophages play an important role in the distribution, clearance and inflammatory reactions after particle inhalation, which may influence long-term events such as fibrosis and tumorigenesis. The objectives of the present study were to investigate the early inflammatory events after plutonium oxide inhalation in rats and involvement of alveolar macrophages. Lung changes were studied from 3 days to 3 months after inhalation of PuO₂ of different isotopic compositions (70% or 97% ²³⁹Pu) and initial lung deposits (range 2.1 to 43.4 kBq/rat). Analyses of bronchoalveolar lavages showed early increases in the numbers of granulocytes, lymphocytes and multinucleated macrophages. The activation of macrophages was evaluated ex vivo by measurement of inflammatory mediator levels in culture supernatants. TNF-α and chemokine MCP-1, MIP-2 and CINC-1 production was elevated from 7 days after inhalation and remained so up to 3 months. In contrast, IL-1β, IL-6 and IL-10 production was unchanged. At 6 weeks, pulmonary macrophage numbers and activation state were increased as observed from an immunohistochemistry study of lung sections with anti-ED1. Similarly, histological analyses of lung sections also showed evidence of inflammatory responses. In conclusion, our results indicate early inflammatory changes in the lungs of PuO₂-contaminated animals and the involvement of macrophages in this process. A dose–effect relationship was observed between the amount of radionuclide inhaled or retained at the time of analysis and inflammatory mediator production by alveolar macrophages 14 days after exposure. For similar initial lung deposits, the inflammatory manifestation appears higher for 97% ²³⁹Pu than for 70% ²³⁹Pu.

Mazier, S., Villette, S., Goffinont, S., Renouard, S., Maurizot, J. C., Genest, D. and Spotheim-Maurizot, M. Radiation Damage to a DNA-Binding Protein. Combined Circular Dichroism and Molecular Dynamics Simulation Analysis. Radiat. Res. 170, 604–612 (2008).

The E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site.

Heidenreich, W. F. and Paretzke, H. G. Promotion of Initiated Cells by Radiation-Induced Cell Inactivation. Radiat. Res. 170, 613–617 (2008).

Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

Song, S., McCann, K. E. and Brown, J. M. Radiosensitization of Yeast Cells by Inhibition of Histone H4 Acetylation. Radiat. Res. 170, 618–627 (2008).

Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (γ-H2A) induction after irradiation but a reduced rate of loss of γ-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G₁ and G₂ checkpoints after exposure to ionizing radiation, suggesting that G₁ and G₂ checkpoint activation is independent of histone H4 acetylation.

Ross, C. D. and Fox, M. H. Multiparameter Analysis of CHO A_L Mutant Populations Sorted on CD59 Expression after Gamma Irradiation. Radiat. Res. 170, 628–637 (2008).

The flow cytometry mutation assay is based on detecting mutations in the CD59 gene on human chromosome 11 in CHO A_L cells with flow cytometry, but the kinetics of mutant expression and the histogram region for mutant selection have not been studied in detail. CHO A_L cells were analyzed by flow cytometry for CD59 expression at various times after irradiation. The mutant fraction increased to a maximum at day 6 but decreased to near background levels by day 20. Cells were sorted from six different regions on the CD59 histograms after irradiation. The growth rate was similar for cells from all regions, and the surviving fraction was 50% of that for control cells. By 14 days the CD59 expression of cells from regions 2–5 was reduced to that of region 1. Cells were also analyzed for simultaneous expression of CD59, CD44 and CD90 (all on chromosome 11) to roughly characterize the size of the mutations. Triple mutants from the sorted populations were reduced from 41% on day 6 to 8% on day 24. We conclude that the mutant region should be increased to include cells with intermediate CD59 expression; also, the loss of CD59 mutant expression over time could be explained in part by the loss of triple mutants from the population.

Gulliksrud, K., Vestvik, I. K., Galappathi, K., Mathiesen, B. and Rofstad, E. K. Detection of Different Hypoxic Cell Subpopulations in Human Melanoma Xenografts by Pimonidazole Immunohistochemistry. Radiat. Res. 170, 638–650 (2008).

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.

Baumann, R. P., Penketh, P. G., Seow, H. A., Shyam, K. and Sartorelli, A. C. Generation of Oxygen Deficiency in Cell Culture Using a Two-Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells. Radiat. Res. 170, 651–660 (2008).

The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to prodrugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.

Vrijheid, M., Cardis, E., Ashmore, P., Auvinen, A., Gilbert, E., Habib, R. R., Malker, H., Muirhead, C. R., Richardson, D. B., Rogel, A., Schubauer-Berigan, M., Tardy, H. and Telle-Lamberton, M., for the 15-Country Study Group. Ionizing Radiation and Risk of Chronic Lymphocytic Leukemia in the 15-Country Study of Nuclear Industry Workers. Radiat. Res. 170, 661–665 (2008).

In contrast to other types of leukemia, chronic lymphocytic leukemia (CLL) has long been regarded as non-radiogenic, i.e. not caused by ionizing radiation. However, the justification for this view has been challenged. We therefore report on the relationship between CLL mortality and external ionizing radiation dose within the 15-country nuclear workers cohort study. The analyses included, in seven countries with CLL deaths, a total of 295,963 workers with more than 4.5 million person-years of follow-up and an average cumulative bone marrow dose of 15 mSv; there were 65 CLL deaths in this cohort. The relative risk (RR) at an occupational dose of 100 mSv compared to 0 mSv was 0.84 (95% CI 0.39, 1.48) under the assumption of a 10-year exposure lag. Analyses of longer lag periods showed little variation in the RR, but they included very small numbers of cases with relatively high doses. In conclusion, the largest nuclear workers cohort study to date finds little evidence for an association between low doses of external ionizing radiation and CLL mortality. This study had little power due to low doses, short follow-up periods, and uncertainties in CLL ascertainment from death certificates; an extended follow-up of the cohorts is merited and would ideally include incident cancer cases.