Qin, J. and Li, L. Molecular Anatomy of the DNA Damage and Replication Checkpoints. Radiat. Res. 159, 139–148 (2003).

Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption. The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained. DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively. Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type. These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage. Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge. We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses.

Malinen, E., Hult, E. A., Hole, E. O. and Sagstuen, E. Alanine Radicals, Part 4: Relative Amounts of Radical Species in Alanine Dosimeters after Exposure to 6–19 MeV Electrons and 10 kV–15 MV Photons. Radiat. Res. 159, 149–153 (2003).

The amino acid l-α-alanine can be used for high-precision dosimetry over a wide dose range, using EPR spectroscopy for monitoring radical concentrations. It is important, however, to understand the underlying composition of the observed EPR spectrum. In previous work, it was shown that the EPR signal from irradiated alanine consists of at least three different radical species, with the relative importance of each of these being almost independent of absorbed dose. However, it was not known whether the relative importance of each radical is independent of the radiation quality responsible for the EPR signal. In the present work, the relative contributions of the different radical species to the total EPR signal from alanine dosimeters irradiated with 6–19 MeV electrons and 10 kV–15 MV photons at a dose of 10 Gy were examined. By spectrum reconstruction using benchmark spectra generated from a simulation procedure, the relative amounts of the three different radical species were shown to be virtually independent of these radiation beam qualities.

Kinnison, J. D., Maurer, R. H., Roth, D. R. and Haight, R. C. High-Energy Neutron Spectroscopy with Thick Silicon Detectors. Radiat. Res. 159, 154–160 (2003).

The high-energy neutron component of the space radiation environment in thick structures such as the International Space Station contributes to the total radiation dose received by an astronaut. Detector design constraints such as size and mass have limited the energy range of neutron spectrum measurements in orbit to about 12 MeV in Space Shuttle studies. We present a new method for high-energy neutron spectroscopy using small silicon detectors that can extend these measurements to more than 500 MeV. The methodology is based on measurement of the detector response function for high-energy neutrons and inversion of this response function with measured deposition data to deduce neutron energy spectra. We also present the results of an initial shielding study performed with the thick silicon detector system for high-energy neutrons incident on polyethylene.

Gilbert, E. S., Stovall, M., Gospodarowicz, M., van Leeuwen, F. E., Andersson, M., Glimelius, B., Joensuu, T., Lynch, C. F., Curtis, R. E., Holowaty, E., Storm, H., Pukkala, E., van't Veer, M. B., Fraumeni, J. F., Jr., Boice, J. D., Jr., Clarke, E. A. and Travis, L. B. Lung Cancer after Treatment for Hodgkin's Disease: Focus on Radiation Effects. Radiat. Res. 159, 161–173 (2003).

Aspects of radiation-induced lung cancer were evaluated in an international study of Hodgkin's disease. The study population consisted of 227 patients with lung cancer and 455 matched controls. Unique features included dose determinations to the specific location in the lung where each cancer developed and quantitative data on both chemotherapy and tobacco use obtained from medical records. The estimated excess relative risk (ERR) per Gy was 0.15 (95% CI: 0.06–0.39), and there was little evidence of departure from linearity even though lung doses for the majority of Hodgkin's disease patients treated with radiotherapy exceeded 30 Gy. The interaction of radiation and chemotherapy that included alkylating agents was almost exactly additive, and a multiplicative relationship could be rejected (P = 0.017). Conversely, the interaction of radiation and smoking was consistent with a multiplicative relationship, but not with an additive relationship (P < 0.001). The ERR/Gy for males was about four times that for females, although the difference was not statistically significant. There was little evidence of modification of the ERR/Gy by time since exposure (after a 5-year minimum latent period), age at exposure, or attained age. Because of the very high radiation doses received by Hodgkin's disease patients and the immunodeficiency inherent to this lymphoma and that associated with chemotherapy, generalizing these findings to other populations receiving considerably lower doses of radiation should be done cautiously.

Kuin, A., Kruse, J. J. and Stewart, F. A. Proteinuria and Vascular Changes after Renal Irradiation: The Role of Reactive Oxygen Species (ROS) and Vascular Endothelial Growth Factor (Vegf). Radiat. Res. 159, 174–181 (2003).

Proteinuria occurs in all degrees of radiation nephropathy and can be present without other symptoms. In this study, radiation-induced proteinuria in C3H mice demonstrated a clear dose–response relationship and was apparent before the onset of significant structural vascular changes and decreases in renal function. This suggests that proteinuria is not a secondary event due to loss of the vascular structure. In an attempt to ameliorate radiation-induced proteinuria and progressive renal failure, two factors were studied. The influence of reactive oxygen species (ROS), which are generated by infiltrating neutrophils and mediate proteinuria in models of acute glomerular injury, was the first to be investigated. Short-term administration of the reactive oxygen scavengers superoxide dismutase (SOD) and catalase did not reverse an established radiation-induced proteinuria. Continuous administration of the antioxidant N-acetylcysteine (NAC) also failed to inhibit this proteinuria. However, since no direct assessment of the impact of these interventions on renal redox status was made, the putative role of ROS in radiation-induced proteinuria and nephropathy remains undefined. The second factor studied was vascular endothelial growth factor (Vegf), which is suggested to be involved in glomerular vessel permeability and the development of proteinuria in some models of renal disease. Northern blot analysis of mRNA from whole kidneys did not demonstrate any increased expression of Vegf after irradiation. There was also no change in the ratio of the different Vegf isoforms (PCR analysis), either in the whole kidney or in isolated glomeruli. No significant role for Vegf was identified for radiation-induced vascular changes or proteinuria, although post-transcriptional changes cannot be excluded.

Flynn, A. A., Pedley, R. B., Green, A. J., Dearling, J. L., El-Emir, E., Boxer, G. M., Boden, R. and Begent, R. H. J. The Nonuniformity of Antibody Distribution in the Kidney and its Influence on Dosimetry. Radiat. Res. 159, 182–189 (2003).

The therapeutic efficacy of radiolabeled antibody fragments can be limited by nephrotoxicity, particularly when the kidney is the major route of extraction from the circulation. Conventional dose estimates in kidney assume uniform dose deposition, but we have shown increased antibody localization in the cortex after glomerular filtration. The purpose of this study was to measure the radioactivity in cortex relative to medulla for a range of antibodies and to assess the validity of the assumption of uniformity of dose deposition in the whole kidney and in the cortex for these antibodies with a range of radionuclides. Storage phosphor plate technology (radioluminography) was used to acquire images of the distributions of a range of antibodies of various sizes, labeled with ¹²⁵I, in kidney sections. This allowed the calculation of the antibody concentration in the cortex relative to the medulla. Beta-particle point dose kernels were then used to generate the dose-rate distributions from ¹⁴C, ¹³¹I, ¹⁸⁶Re, ³²P and ⁹⁰Y. The correlation between the actual dose-rate distribution and the corresponding distribution calculated assuming uniform antibody distribution throughout the kidney was used to test the validity of estimating dose by assuming uniformity in the kidney and in the cortex. There was a strong inverse relationship between the ratio of the radioactivity in the cortex relative to that in the medulla and the antibody size. The nonuniformity of dose deposition was greatest with the smallest antibody fragments but became more uniform as the range of the emissions from the radionuclide increased. Furthermore, there was a strong correlation between the actual dose-rate distribution and the distribution when assuming a uniform source in the kidney for intact antibodies along with medium- to long-range radionuclides, but there was no correlation for small antibody fragments with any radioisotope or for short-range radionuclides with any antibody. However, when the cortex was separated from the whole kidney, the correlation between the actual dose-rate distribution and the assumed dose-rate distribution, if the source was uniform, increased significantly. During radioimmunotherapy, the extent of nonuniformity of dose deposition in the kidney depends on the properties of the antibody and radionuclide. For dosimetry estimates, the cortex should be taken as a separate source region when the radiopharmaceutical is small enough to be filtered by the glomerulus.

Schouwink, J. H., Oppelaar, H., Ruevekamp, M., Van der Valk, M., Hart, G., Baas, P. and Stewart, F. A. Oxygen Depletion during and after mTHPC-Mediated Photodynamic Therapy in RIF1 and H-MESO1 Tumors. Radiat. Res. 159, 190–198 (2003).

During photodynamic therapy (PDT), low oxygenation levels, induced both by oxygen consumption and by vascular occlusion, can lead to an inefficient photochemical reaction that may compromise the efficacy of PDT. In the present studies, tumor oxygenation was measured before, during and after meta-tetrahydroxyphenylchlorin (mTHPC)-mediated PDT of murine RIF1 tumors and human mesothelioma xenografts (H-MESO1). Tumor pO₂ was measured in real time with Eppendorf polarography, and the extent of relative hypoxia at specific times was measured by immunohistochemical staining. Significant decreases in median pO₂ values, as well as an increase in the number of values below 2.5 mmHg, were seen during and after PDT in RIF1 tumors, although there was a large intertumoral variation. Tumor pO₂ values did not change significantly in H-MESO1 tumors. Staining with antibodies against the hypoxia marker EF3 showed significant increases in relative hypoxia after PDT in both tumor types compared with separate groups of untreated controls. Our results are consistent with PDT-induced oxygen depletion (reduced pO₂) leading to an increase in relative hypoxia in RIF1 tumors. Extensive necrosis in the H-MESO1 tumors may have prevented the detection of PDT-induced hypoxia using the Eppendorf polarographic needle, whereas immunohistochemistry did reveal increases in relative hypoxia.

Benczik, J., Seppälä, T., Snellman, M., Joensuu, H., Morris, G. M. and Hopewell, J. W. Evaluation of the Relative Biological Effectiveness of a Clinical Epithermal Neutron Beam Using Dog Brain. Radiat. Res. 159, 199–209 (2003).

This investigation was designed to determine the relative biological effectiveness (RBE) of an epithermal neutron beam (FiR 1 beam) using the brains of dogs. The FiR 1 beam was developed for the treatment of patients with glioma using boron neutron capture therapy. Comparisons were made between the effects of whole-brain irradiation with epithermal neutrons and 6 MV photons. For irradiations with epithermal neutrons, three dose groups were used, 9.4 ± 0.1, 10.2 ± 0.1 and 11.5 ± 0.2 Gy. These physical doses were given as a single exposure and are quoted at the 90% isodose. Four groups of five dogs were irradiated with single doses of 10, 12, 14 or 16 Gy of 6 MV photons to the 100% isodose. Different reference isodoses were used to obtain the most comparable dose distribution in the brain for the two different irradiation modalities. Sequential magnetic resonance images (MRI) were taken for 77−115 weeks after irradiation to detect changes in the brain. Dose–effect relationships were established for changes in the brain as detected either by MRI or by subsequent gross morphology and histology. The doses that caused a specified response in 50% of the animals (ED₅₀) were calculated from these dose–effect curves for each end point, and these values were used to calculate the RBE values for the different end points. The RBE values for the FiR 1 beam, based on changes observed on MRI, were in the range 1.2−1.3. For microscopic and gross pathological lesions, the values were in the range 1.2−1.4. The corresponding RBE values for the MRI and pathological end points for the high-LET components (protons from nitrogen capture and recoil protons from fast neutrons) were in the ranges 3.5–4.0 and 3.4–4.4, respectively. This assumed a dose-rate reduction factor of 0.6 for the low-dose-rate γ-ray component of this beam. Finally, a comparison was made between experimentally derived photon doses, for a specified end point, with calculated photon equivalent doses, which were obtained using the weighting factors for clinical studies on the epithermal neutron beam on the Brookhaven Medical Research Reactor (BNL) in New York. This indicated that the radiation-induced lesions seen in the present study were, on average, detected at a 12% lower photon dose than predicted by the use of the BNL clinical weighting factors. This indicates the need for caution in the extrapolation of results from one reactor-based epithermal neutron beam to another.

Rijnkels, J. M., Moison, R. M. W., Podda, E. and Beijersbergen van Henegouwen, G. M. J. Photoprotection by Antioxidants against UVB-Radiation-Induced Damage in Pig Skin Organ Culture. Radiat. Res. 159, 210–217 (2003).

Topically applied antioxidants constitute an important group of protective agents against skin damage induced by ultraviolet radiation. The current study was performed to investigate whether a recently developed ex vivo pig skin model was suitable for short-term studies of the mechanism(s) of UVB-radiation-induced skin damage; the protective effect of topical application of α-tocopherol, l-ascorbic acid, α-lipoic acid, glutathione ethylester and N-acetylcysteine was tested. Increasing doses of the antioxidants were applied topically on ex vivo pig skin explants and allowed to penetrate for 60 min. Epidermal antioxidant bioavailability was measured before and 60 min after exposure to an ultraviolet B (UVB) radiation of 7.5 kJ/m². Cell viability (trypan blue dye exclusion) and apoptosis were measured 48 h later in isolated keratinocytes. UVB-radiation-induced epidermal lipid peroxidation was determined immediately after exposure of the skin to a UVB dose of 28 kJ/m². All antioxidants tested became bioavailable in pig skin epidermis, and none of them were depleted after UVB-radiation exposure. Increasing doses of the antioxidants tested decreased UVB-radiation-induced cell death and apoptosis. The highest doses of antioxidants prevented UVB-radiation-induced lipid peroxidation; α-lipoic acid only tended to decrease lipid peroxidation. In conclusion, a single topical dose of the above antioxidants on ex vivo pig skin can reduce UVB-radiation-induced oxidative stress and lipid peroxidation and thereby reduce apoptotic stimuli and cell death. Furthermore, the ex vivo pig skin model was a useful tool for testing compounds for their antioxidant activity.

Marfak, A., Trouillas, P., Allais, D. P., Calliste, C. A. and Duroux, J. L. Redox Reactions Obtained by γ Irradiation of Quercetin Methanol Solution are Similar to In Vivo Metabolism. Radiat. Res. 159, 218–227 (2003).

The flavonol quercetin is one of the most well-known antioxidant flavonoids. Its antioxidant potential has been studied extensively during the last 10 years, but little is known about the metabolites formed in vivo that lead to the formation of depside and small molecules such as benzoic acids. In this study, γ irradiation of a quercetin methanol solution was used as a model of certain oxidative reactions that occur in vivo. Qercetin at concentrations ranging from 5 × 10⁻⁵ M to 5 × 10⁻³ M, was irradiated with γ rays at doses of 2–14 kGy. Quercetin degradation was evaluated by HPLC analysis. The major radiolytic metabolite was identified as a depside by NMR and LC-MS. Formation of 3,4-dihydroxybenzoic acid was also observed. The presence of CH₃O^· formed during methanol radiolysis is invoked to explain depside formation. Transformation of the 8-methoxy substituted depside (Q1) to the 8-hydroxyl substituted depside (Q2) is discussed. The antioxidant properties of quercetin metabolites are evaluated according to their capacity to decrease the EPR DPPH signal and to inhibit superoxide radical formed by the enzymatic reaction (xanthine xanthine oxidase). For both assays, the IC50 of Q2 is twice as high as that of quercetin.

Iwasaki, T., Murata, M., Ohshima, S., Miyake, T., Kudo, S., Inoue, Y., Narita, M., Yoshimura, T., Akiba, S., Tango, T., Yoshimoto, Y., Shimizu, Y., Sobue, T., Kusumi, S., Yamagishi, C. and Matsudaira, H. Second Analysis of Mortality of Nuclear Industry Workers in Japan, 1986–1997. Radiat. Res. 159, 228–238 (2003).

A cohort study of nuclear industry workers was initiated in 1990 to determine the possible health effects of low-level radiation. A total of 5,527 deaths were ascertained among 176,000 male workers who had been retrospectively and/or prospectively followed for an average of 7.9 years during the observation period 1986–1997. Statistical analyses were made mainly on the prospective follow-up outcome of 120,000 workers followed for an average of 4.5 years. The standardized mortality ratio (and its 95% confidence interval) was 0.94 (0.90, 0.97) for 2,934 cases of all causes combined and 0.86 (0.82, 0.91) for 1,305 cases of non-malignant diseases combined, which suggested a healthy worker effect. For 1,191 cases of all cancers combined, it was 0.98 (0.93, 1.04), indicating no difference in mortality from that of the general population. In tests for trend of death rate with increasing radiation dose, no significant correlation was found for all cancers combined. For site-specific cancers, most cancers including leukemia showed no positive correlation with dose, except for cancers of the esophagus, stomach and rectum and multiple myeloma. External causes showed a significant correlation with dose. A separate questionnaire study indicated that these positive findings could be ascribed in part to lifestyle characteristics of the workers. For leukemia only, we attempted to estimate the excess relative risk per unit dose of radiation, which, with reservations because of its wide confidence interval, was within the range of variation of the risks reported in other radiation epidemiological studies. This population must be studied for a longer time and with a consideration of the possible effects of confounding factors.

Shagina, N. B., Tolstykh, E. I., Zalyapin, V. I., Degteva, M. O., Kozheurov, V. P., Tokareva, E. E., Anspaugh, L. R. and Napier, B. A. Evaluation of Age and Gender Dependences of the Rate of Strontium Elimination 25–45 Years after Intake: Analysis of Data from Residents Living along the Techa River. Radiat. Res. 159, 239–246 (2003).

The Mayak Production Association released large amounts of ⁹⁰Sr into the Techa River with peak amounts in 1950–1951. Residents near the Techa River ingested an average of ∼3,000 kBq of ⁹⁰Sr. The affected people have been followed by scientists at the Urals Research Center for Radiation Medicine. The whole-body content of ⁹⁰Sr of ∼15,000 individuals has been measured over a period of 24 years (1974–1997) using a special whole-body counter. This report evaluates the gender and age dependences of individual rates of strontium elimination. Data on persons who had been measured 12 or more times were selected for study. There were 108 men and 81 women older than 30 years who met this criterion. Individual measurement results were fitted to an exponential function and grouped mean averages of the rate of strontium elimination as a function of age for each sex were derived. For men, a significant increase (from 2.8% year^–1 to 3.2% year^–1) in the rate of strontium elimination after age 55 years is seen. For women, the increase in the rate of elimination was significant at age 45 and reached 5.8% year^–1 after the age of 60. The results may be used to develop a gender- and age-dependent model of strontium metabolism.

Bryant, P. E., Jones, C., Armstrong, G., Frankenberg-Schwager, M. and Frankenberg, D. Induction of Chromatid Breaks by Carbon K-Shell Ultrasoft X Rays. Radiat. Res. 159, 247–250 (2003).

Chromatid breaks have previously been shown to be induced in G₂-phase cells after exposure to ionizing radiation (X and γ rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for ⁶⁰Co γ rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.

Pastwa, E., Neumann, R. D., Mezhevaya, K. and Winters, T. A. Repair of Radiation-Induced DNA Double-Strand Breaks is Dependent upon Radiation Quality and the Structural Complexity of Double-Strand Breaks. Radiat. Res. 159, 251–261 (2003).

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, ⁶⁰Co γ rays, and the effects of ¹²⁵I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by γ rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by ¹²⁵I was near the lower limit of detection in our assay and was at least twofold lower than that of γ-ray-induced DSBs. In addition, DSB ends produced by ¹²⁵I were shown to be blocked by 3′-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Little, J. B., Nagasawa, H., Li, G. C. and Chen, D. J. Involvement of the Nonhomologous End Joining DNA Repair Pathway in the Bystander Effect for Chromosomal Aberrations. Radiat. Res. 159, 262–267 (2003).

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G₁ phase and exposed to very low fluences of α particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4–9% of wild-type cells and 36–55% of Xrcc5^−/− cells, whereas only 2–3% of the nuclei were traversed by an α particle and thus received any radiation exposure. G22p1^−/− cells responded similarly to Xrcc5^−/− cells, whereas Prkdc^−/− and Adprt^−/− cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5^−/− and G22p1^−/− cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc^−/− cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

Ferrieu, C., Ballester, B., Mathieu, J. and Drouet, E. Flow Cytometry Analysis of Gamma-Radiation-Induced Epstein-Barr Virus Reactivation in Lymphocytes. Radiat. Res. 159, 268–273 (2003).

Epstein-Barr virus (EBV), a member of the γ-herpesvirus family, is involved in the development of several diseases, and the infection is believed to persist for life in latent form. Ionizing radiation at clinically relevant doses may increase the amount of virus reactivation in B cells, and the combination of radiation with stress could amplify EBV reactivation. In vitro experiments were performed on several cell lines, including EBV-positive Burkitt lymphoma cells. The presence of the immediate-early protein ZEBRA, which is a hallmark of EBV reactivation, was evaluated using flow cytometry, which enabled us to measure the percentage of ZEBRA-positive cells. The process was studied previously in the EBV-positive Burkitt lymphoma cell line B95-8. Forty-eight hours after irradiation alone, 13.6 and 19.9% ZEBRA-positive cells were observed at 2 and 4 Gy, respectively, compared to the basal level of 1.85%. Thus irradiation induces EBV reactivation. The addition of a glucocorticoid (the final effector of the stress response) had no effect on EBV reactivation in our model. However, the combination of radiation and treatment with a glucocorticoid (dexamethasone) increased the expression of ZEBRA in B95-8 cells (15.8 and 28.75% of the cells was positive at 24 and 48 h after γ irradiation, respectively). Thus the combination of γ radiation and a glucocorticoid may play an important role in EBV reactivation.