Tomasek, L., Rogel, A., Tirmarche, M., Mitton, N. and Laurier, D. Lung Cancer in French and Czech Uranium Miners: Radon-Associated Risk at Low Exposure Rates and Modifying Effects of Time since Exposure and Age at Exposure. Radiat. Res. 169, 125–137 (2008).

Radon is recognized as a public health concern for indoor exposure. Precise quantification derived from occupational exposure in miners is still needed for estimating the risk and the factors that modify the dependence on cumulated exposure. The present paper reports on relationship between radon exposure and lung cancer risk in French and Czech cohorts of uranium miners (n = 10,100). Miners from these two cohorts are characterized by low levels of exposure (average cumulated exposure of less than 60 WLM) protracted over a long period (mean duration of exposure of 10 years) and by a good quality of individual exposure estimates (95% of annual exposures based on radon measurements). The modifying effect of the quality of exposure on the risk is analyzed. A total of 574 lung cancer deaths were observed, which is 187% higher than expected from the national statistics. This significantly elevated risk is strongly associated with cumulated radon exposure. The estimated overall excess relative risk per WLM is 0.027 (95% CI: 0.017–0.043, related to measured exposures). For age at exposure of 30 and 20 years since exposure, the ERR/WLM is 0.042, and this value decreases by approximately 50% for each 10-year increase in age at exposure and time since exposure. The present study emphasizes that the quality of exposure estimates is an important factor that may substantially influence results. Time since exposure and simultaneously age at exposure were the most important effect modifiers. No inverse exposure-rate effect below 4 WL was observed. The results are consistent with estimates of the BEIR VI report using the concentration model at an exposure rate below 0.5 WL.

Eidemüller, M., Ostroumova, E., Krestinina, L., Akleyev, A. and Jacob, P. Analysis of Solid Cancer Mortality in the Techa River Cohort Using the Two-Step Clonal Expansion Model. Radiat. Res. 169, 138–148 (2008).

In this study the solid cancer mortality data in the Techa River Cohort in the Southern Urals region of Russia was analyzed. The cohort received protracted exposure in the 1950s due to the releases of radioactive materials from the Mayak plutonium complex. The Extended Techa River Cohort includes 29,849 people who resided along the Techa River between 1950 and 1960 and were followed from January 1, 1950 through December 31, 1999. The analysis was done within the framework of the biologically based two-stage clonal expansion (TSCE) model. It was found that about 2.6% of the 1854 solid cancer deaths (excluding 18 bone cancer cases) could be related to radiation exposure. At age 63, which is the mean age for solid cancer deaths, the excess relative risk (ERR) and excess absolute risk (EAR) were found to be 0.76 Gy⁻¹ (95% CI 0.23; 1.29) and 33.0 (10⁴ PY Gy)⁻¹ (95% CI 9.8; 52.6), respectively. These risk estimates are consistent with earlier excess relative risk analyses for the same cohort. The change in the ERR with age was investigated in detail, and an increase in risk with attained age was observed. Furthermore, the data were tested for possible signs of genomic instability, and it was found that the data could be described equally well by a model incorporating effects of genomic instability. Results from the TSCE models indicated that radiation received at older ages might have stronger biological effects than exposure at younger ages.

Obenaus, A., Huang, L., Smith, A., Favre, C. J., Nelson, G. and Kendall, E. Magnetic Resonance Imaging and Spectroscopy of the Rat Hippocampus 1 Month after Exposure to ⁵⁶Fe-Particle Radiation. Radiat. Res. 169, 149–161 (2008).

The response of the central nervous system to space radiation is largely unknown. The hippocampus, which is known for its critical role in learning and memory, was evaluated for its response to heavy-ion radiation. At 1 month, animals exposed to brain-only ⁵⁶Fe-particle irradiation (0–4 Gy) were examined using contrast-enhanced T1 imaging (CET1), T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and ¹H-magnetic resonance spectroscopy (MRS). Correlative histology was performed after imaging. The T2WI, DWI and CET1 images revealed no overt anatomical changes after irradiation. Quantitative analysis demonstrated a significant increase in T2 at 2 Gy compared to 0 Gy. The apparent diffusion coefficient (ADC) revealed an inverse dose-dependent quantitative change in water mobility. Compared to 0 Gy, the ADC increased 122% at 1 Gy and declined to 44% above control levels at 4 Gy. MRS showed a significant increase in the N-acetylaspartate/choline ratio at 4 Gy and a lactate peak. Histology demonstrated no overt pathological changes in neuronal and astrocyte populations. However, a significant inverse dose-dependent morphological change in the microglial population was detected in irradiated animals. Our results suggest that early tissue matrix modifications induced by ⁵⁶Fe-particle radiation can be detected by MRI in the absence of evident histopathology. These changes may indicate fundamental changes in the structure and function of the hippocampus.

Moriconi, F., Christiansen, H., Raddatz, D., Dudas, J., Hermann, R. M., Rave-Fränk, M., Sheikh, N., Saile, B., Hess, C. F. and Ramadori, G. Effect of Radiation on Gene Expression of Rat Liver Chemokines: In Vivo and In Vitro Studies. Radiat. Res. 169, 162–169 (2008).

The aim of the study was to analyze the effect of a single irradiation on chemokine gene expression in the rat liver and in isolated rat hepatocytes. RNA extracted from livers and from hepatocytes within the first 48 h after irradiation was analyzed by real-time PCR and the Northern blot assay. The chemokine concentrations in the serum of irradiated rats were measured quantitatively by ELISA. A significant radiation-induced increase of CINC1, IP10, MCP1, MIP3α, MIP3β, MIG and ITAC gene expression could be detected at the RNA level in the liver. CINC1, MCP1 and IP10 serum levels were significantly increased. In rat hepatocytes in vitro, only MIP3α showed a radiation-induced increase in expression, while CINC1, IP10, MIP3β, MIG, MIP1α, ITAC and SDF1 RNA levels were significantly down-regulated. However, incubation of irradiated hepatocytes in vitro with either TNF-α, IL1β, or IL6 plus TNF-α led to up-regulation of MCP1, IP10 and MCP1 or CINC1 and MIP3β, respectively. Irradiation of the liver induces up-regulation of the genes of the main proinflammatory chemokines, probably through the action of locally synthesized proinflammatory cytokines. The reason for the lack of liver inflammation in this model has still to be clarified.

Rainaldi, G., Romano, R., Indovina, P., Ferrante, A., Motta, A., Indovina, P. L. and Santini, M. T. Metabolomics Using ¹H-NMR of Apoptosis and Necrosis in HL60 Leukemia Cells: Differences between the Two Types of Cell Death and Independence from the Stimulus of Apoptosis Used. Radiat. Res. 169, 170–180 (2008).

High-resolution proton nuclear magnetic resonance (¹H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The ¹H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH₂ and CH₃ mobile lipids, principally of CH₂, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of ¹H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH₂ mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in ¹H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.

Belloni, P., Meschini, R., Lewinska, D. and Palitti, F. Apoptosis Preferentially Eliminates Irradiated G₀ Human Lymphocytes Bearing Dicentric Chromosomes. Radiat. Res. 169, 181–187 (2008).

G₀ human peripheral blood lymphocytes were X-irradiated to determine whether there is a direct relationship between radiation-induced dicentric chromosomes and the triggering of apoptosis. Immediately after X-ray exposure, control and irradiated lymphocytes were analyzed for viability, apoptosis and chromosome damage using the premature chromosome condensation technique. A batch of lymphocytes was kept in liquid holding for 48 h and then loaded on Ficoll-Paque medium to separate apoptotic (high-density) and normal (normal-density) cells. Then the same end points were analyzed in high-density and normal-density fractions of control and irradiated lymphocytes. After 48 h of liquid holding, the majority of apoptotic cells contained dicentric chromosomes. These results demonstrate that in human lymphocytes, the type of chromosome damage influences the induction of programmed cell death and provide direct evidence that cells bearing dicentrics are eliminated by apoptosis. G₀ lymphocytes are the most common tissue used in biodosimetry studies, and the amount of chromosomal damage detected depends on the time between exposure and sampling. Since the radiation-induced apoptotic cells show the presence of dicentrics, radiation-induced damage can be underestimated. These results may have relevance in evaluations of the efficacy of radiotherapy based on the frequencies of chromosomal aberrations.

Ryan, L. A., Smith, R. W., Seymour, C. B. and Mothersill, C. E. Dilution of Irradiated Cell Conditioned Medium and the Bystander Effect. Radiat. Res. 169, 188–196 (2008).

While nontargeted and low-dose effects such as the bystander effect are now accepted, the mechanisms underlying the response have yet to be elucidated. It has been shown that the transfer of irradiated cell conditioned medium (ICCM) can kill cells that are not directly irradiated; however, to date the effect of ICCM concentration on cell killing has not been reported. The occurrence of a bystander effect was determined by measuring cell survival after exposure to various ICCM dilutions, using the colony-forming assay, in cells of six human cell lines with varied bystander responses and tumor/ p53 status. Autologous ICCM transfer for these cell lines induced a bystander effect as reported previously. ICCM from these cell lines was transferred to cells of a common reporter cell line (HPV-G) to investigate whether the lack of an induced bystander effect was due to their inability to generate or to respond to a bystander signal(s). ICCM from cells of four cell lines induced a bystander effect in HPV-G reporter cells, confirming that signal production is a critical factor. A saturation response was observed when ICCM was diluted. Survival was found to increase linearly until a plateau was reached and the bystander effect was abolished at 2× dilution. The effect of ICCM from the different cell lines reached a plateau at different dilutions, which were found to correlate with the cell line's radiosensitivity.

Zhang, Y., Zhou, J., Held, K. D., Redmond, R. W., Prise, K. M. and Liber, H. L. Deficiencies of Double-Strand Break Repair Factors and Effects on Mutagenesis in Directly γ-Irradiated and Medium-Mediated Bystander Human Lymphoblastoid Cells. Radiat. Res. 169, 197–206 (2008).

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H₂O₂ in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells.

Zölzer, F. and Streffer, C. Relative Biological Effectiveness of 6 MeV Neutrons with Respect to Cell Inactivation and Disturbances of the G₁ Phase. Radiat. Res. 169, 207–213 (2008).

The relative biological effectiveness (RBE) of neutrons and other types of densely ionizing radiation appears to be close to 1.0 for the induction of strand breaks, but considerably higher RBEs have been found for cellular end points such as colony-forming ability. This may be due to differences in the processing of strand breaks or to the involvement of other lesions whose yields are more dependent on radiation quality. Because cell cycle delays may be of great importance in the processing of DNA damage, we determined the RBE for disturbances of the G₁ phase in four different cell types (Be11 melanoma, 4197 squamous cell carcinoma, EA14 glioma, GM6419 fibroblasts) and compared them with the RBE for cell inactivation. The method we used to determine the progress from G₁ into S was as follows: Cells were serum-deprived for a number of days and then stimulated to grow with culture medium containing normal amounts of serum. Immediately before the change of medium, cells were exposed to graded doses of either 240 kV X rays or 6 MeV neutrons. At different times afterward, cells were labeled with BrdU and the numbers of active S-phase cells were assessed using two-parameter flow cytometry. For all four cell types, cells started to progress from G₁ into S after a few hours. Radiation suppressed this process in all cases, but there were some interesting differences. For Be11 and 4197 cells, the most obvious effect was a delay in G₁; the labeling index increased a few hours later in irradiated samples than in controls, and there was no significant effect on the maximum labeling index. For EA14 and GM6419 cells, although smaller doses were used because of greater radiosensitivity, a delay of the entry into S phase was again noticeable, but the most significant effect was a reduction in the maximum percentage of active S-phase cells after stimulation, indicating a permanent or long-term arrest in G₁. The RBE for the G₁ delay was the same for all four cell types, about 2.8, while the RBE for the G₁ arrest varied between 3.2 for the most resistant Be11 cells and 1.7 for the most sensitive GM6419 cells. This trend was similar to that observed for the RBE for cell inactivation. If, as described above, the same number of strand breaks per dose is induced by neutrons and by X rays, the signal transduction cascade translates them into a greater G₁ delay in the case of higher LET. This appears to be independent of repair capacity, because it is similar in all cell types we investigated. We therefore assume that a higher lesion density or the presence of other types of lesions is important for this relatively early effect. A G₁ arrest, however, is more closely related to the later events leading to cell inactivation, where strand break repair does play a major role, influencing X-ray sensitivity more strongly than sensitivity to neutrons because of a lower repairability of lesions induced by higher-LET radiation.

Cucinotta, F. A., Pluth, J. M., Anderson, J. A., Harper, J. V. and O'Neill, P. Biochemical Kinetics Model of DSB Repair and Induction of γ-H2AX Foci by Non-homologous End Joining. Radiat. Res. 169, 214–222 (2008).

We developed a biochemical kinetics approach to describe the repair of double-strand breaks (DSBs) produced by low-LET radiation by modeling molecular events associated with non-homologous end joining (NHEJ). A system of coupled nonlinear ordinary differential equations describes the induction of DSBs and activation pathways for major NHEJ components including Ku70/80, DNA-PKcs, and the ligase IV-XRCC4 heterodimer. The autophosphorylation of DNA-PKcs and subsequent induction of γ-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of regulation of repair by DNA-PKcs was developed with an initial step allowing access of other NHEJ components to breaks and a second step limiting access to ligase IV-XRCC4. Our model assumes that the transition from the first to the second step depends on DSB complexity, with a much slower rate for complex DSBs. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field gel electrophoresis (PFGE) at 10 min postirradiation or longer and quantification of the induction of γ-H2AX foci. A process that is independent of DNA-PKcs is required for the model to reproduce experimental data for rejoining before 10 min postirradiation. Predictions are made for the behaviors of NHEJ components at low doses and dose rates, and a steady state is found at dose rates of 0.1 Gy/h or lower.

Aydogan, B., Bolch, W. E., Swarts, S. G., Turner, J. E. and Marshall, D. T. Monte Carlo Simulations of Site-Specific Radical Attack to DNA Bases. Radiat. Res. 169, 223–231 (2008).

An atomistic biophysical model permitting the calculation of initial attacks to a 38-bp representation of B-DNA base moieties by water radicals is presented. This model is based on a previous radiation damage model developed by Aydogan et al. (Radiat. Res. 157, 38–44, 2002). Absolute efficiencies for radical attack to the 38-bp DNA molecule are calculated to be 41, 0.8 and 15% for hydroxyl radical (^·OH), hydrogen radical (H^·), and hydrated electron (e_aq)_, respectively. Among the nucleobases, guanine is found to have the highest percentage ^·OH attack probability at 36%. Adenine, cytosine and thymine moieties have initial attack probabilities of 24, 18 and 22%, respectively. A systematic study is performed to investigate ^·OH attack probabilities at each specified attack site in four molecular models including free bases, single nucleotides, single base pairs, and the central eight base pairs of the 38-bp DNA molecule. Cytosine is the free base moiety for which the closest agreement is observed between the model prediction and the experimental data. The initial ^·OH attack probabilities for cytosine as the free base are calculated to be 72 and 28%, while experimental data are reported at 87 and 13% for the C5 and C6 positions on the base, respectively. In this study, we incorporated atomic charges to scale the site-specific ^·OH reaction rates at the individual atomic positions on the pyrimidine and purine bases. Future updates to the RIDNA model will include the use of electron densities to scale the reaction rates. With respect to reactions of the aqueous electron with DNA, a comparison of the initial distribution of electron attack sites calculated in this study and experimental results suggests an extremely rapid and extensive redistribution of the e⁻_aq after their initial reactions with DNA.

Marrale, M., Brai, M., Gennaro, G., Bartolotta, A. and D'Oca, M. C. The Effect of Gadolinium on the ESR Response of Alanine and Ammonium Tartrate Exposed to Thermal Neutrons. Radiat. Res. 169, 232–239 (2008).

Many efforts have been made to develop neutron capture therapy (NCT) for cancer treatment. Among the challenges in using NCT is the characterization of the features of the mixed radiation field and of its components. In this study, we examined the enhancement of the ESR response of pellets of alanine and ammonium tartrate with gadolinium oxide exposed to a thermal neutron beam. In particular, the ESR response of these dosimeters as a function of the gadolinium content inside the dosimeter was analyzed. We found that the addition of gadolinium improves the sensitivity of both alanine and ammonium tartrate. However, the use of gadolinium reduces or abolishes tissue equivalence because of its high atomic number (Z_Gd = 64). Therefore, it is necessary to find the optimum compromise between the sensitivity to thermal neutrons and the reduction of tissue equivalence. Our analysis showed that a low concentration of gadolinium oxide (of the order of 5% of the total mass of the dosimeter) can enhance the thermal neutron sensitivity more than 13 times with an insignificant reduction of tissue equivalence.

Unoki, M., Bronner, C. and Mousli, M. A Concern Regarding the Current Confusion with the Human Homolog of Mouse Np95, ICBP90/UHRF1. Radiat. Res. 169, 240–244 (2008).

ICBP90/UHRF1, which is overexpressed in cancer cells and is down-regulated by p53, possesses a methylated CpG binding affinity and binds to the methylated promoters of tumor suppressor genes in cancer cells with HDAC1 and DNMT1, suggesting suppression of these genes and maintenance of methylation status which leads to carcinogenesis. Recently, it was reported that the human homolog of Np95 is different from ICBP90 but not from UHRF1. Because UHRF1 is the gene symbol of ICBP90, the claim is a little confusing; that is, UHRF1 and ICBP90 are identical. Because the previously published genomic structure of the ICBP90 gene needed to be revised and the registered ICBP90 sequence (AF129507) contains two rare polymorphisms or sequence errors, we think that confusion could occur. Here we show the revised ICBP90 gene structure and 366 polymorphisms in this gene. Our conclusion is that the human homolog of Np95 is ICBP90, whose gene symbol is UHRF1.