Matsumoto, H., Hayashi, S., Hatashita, M., Ohnishi, K., Shioura, H., Ohtsubo, T., Kitai, R., Ohnishi, T. and Kano, E. Induction of Radioresistance by a Nitric Oxide-Mediated Bystander Effect.

To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene. Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells. Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium. Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium. Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.

Sawant, S. G., Randers-Pehrson, G., Geard, C. R., Brenner, D. J. and Hall, E. J. The Bystander Effect in Radiation Oncogenesis: I. Transformation in C3H 10T½ Cells In Vitro can be Initiated in the Unirradiated Neighbors of Irradiated Cells.

It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of α particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by α particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of α particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight α particles through the nuclei of all or just 10% of C3H 10T½ cells. We show that (a) more cells can be inactivated than were actually traversed by α particles and (b) when 10% of the cells on a dish are exposed to α particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of α particles. These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells. This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.

Brenner, D. J., Little, J. B. and Sachs, R. K. The Bystander Effect in Radiation Oncogenesis: II. A Quantitative Model.

There is strong evidence that biological response to ionizing radiation has a contribution from unirradiated “bystander” cells that respond to signals emitted by irradiated cells. We discuss here an approach incorporating a radiobiological bystander response, superimposed on a direct response due to direct energy deposition in cell nuclei. A quantitative model based on this approach is described for α-particle-induced in vitro oncogenic transformation. The model postulates that the oncogenic bystander response is a binary “all or nothing” phenomenon in a small sensitive subpopulation of cells, and that cells from this sensitive subpopulation are also very sensitive to direct hits from α particles, generally resulting in a directly hit sensitive cell being inactivated. The model is applied to recent data on in vitro oncogenic transformation produced by broad-beam or microbeam α-particle irradiation. Two parameters are used in analyzing the data for transformation frequency. The analysis suggests that, at least for α-particle-induced oncogenic transformation, bystander effects are important only at small doses—here below about 0.2 Gy. At still lower doses, bystander effects may dominate the overall response, possibly leading to an underestimation of low-dose risks extrapolated from intermediate doses, where direct effects dominate.

Gottlöber, P., Steinert, M., Weiss, M., Bebeshko, V., Belyi, D., Nadejina, N., Stefani, F. H., Wagemaker, G., Fliedner, T. M. and Peter, R. U. The Outcome of Local Radiation Injuries: 14 Years of Follow-up after the Chernobyl Accident.

The Chernobyl nuclear power plant accident on April 26, 1986 was the largest in the history of the peaceful use of nuclear energy. Of the 237 individuals initially suspected to have been significantly exposed to radiation during or in the immediate aftermath of the accident, the diagnosis of acute radiation sickness (ARS) could be confirmed in 134 cases on the basis of clinical symptoms. Of these, 54 patients suffered from cutaneous radiation syndrome (CRS) to varying degrees. Among the 28 patients who died from the immediate consequences of accidental radiation exposure, acute hemopoietic syndrome due to bone marrow failure was the primary cause of death only in a minority. In 16 of these 28 deaths, the primary cause was attributed to CRS. This report describes the characteristic cutaneous sequelae as well as associated clinical symptoms and diseases of 15 survivors of the Chernobyl accident with severe localized exposure who were systematically followed up by our groups between 1991 and 2000. All patients presented with CRS of varying severity, showing xerosis, cutaneous telangiectasias and subungual splinter hemorrhages, hemangiomas and lymphangiomas, epidermal atrophy, disseminated keratoses, extensive dermal and subcutaneous fibrosis with partial ulcerations, and pigmentary changes including radiation lentigo. Surprisingly, no cutaneous malignancies have been detected so far in those areas that received large radiation exposures and that developed keratoses; however, two patients first presented in 1999 with basal cell carcinomas on the nape of the neck and the right lower eyelid, areas that received lower exposures. During the follow-up period, two patients were lost due to death from myelodysplastic syndrome in 1995 and acute myelogenous leukemia in 1998, respectively. Other radiation-induced diseases such as dry eye syndrome (3/15), radiation cataract (5/15), xerostomia (4/15) and increased FSH levels (7/15) indicating impaired fertility were also documented. This study, which analyzes 14 years in the clinical course of a cohort of patients with a unique exposure pattern, corroborates the requirement for long-term, if not life-long, follow-up not only in atomic bomb survivors, but also after predominantly local radiation exposure.

Cigarrán, S., Barquinero, J. F., Barrios, L., Ribas, M., Egozcue, J. and Caballín, M. R. Cytogenetic Analyses by Fluorescence In Situ Hybridization (FISH) in Hospital Workers Occupationally Exposed to Low Levels of Ionizing Radiation.

An occupationally exposed population has been studied to evaluate the suitability of FISH painting techniques to detect chronic exposures to very low doses of ionizing radiation by the analysis of translocations. Whole-chromosome painting probes for chromosomes 1, 4 and 11 in combination with a pancentromeric probe have been employed. For comparison, a matched control population has also been studied. The mean genomic frequencies per 100 cells of total translocations in the control and exposed populations were 0.90 ± 0.12 and 1.04 ± 0.11, respectively. In the occupationally exposed population, no correlation between the frequencies of translocations and the doses received was found. When the two populations were compared, no significant differences were observed for the frequencies of the different chromosomal abnormalities examined. The absence of differences between control and exposed populations could be attributed to the very low-dose exposures recorded in the occupationally exposed population and to the wide range of individual frequencies of translocations observed.

Ponsa, I., Barquinero, J. F., Miró, R., Egozcue, J. and Genescà, A. Non-disjunction and Chromosome Loss in Gamma-Irradiated Human Lymphocytes: A Fluorescence In Situ Hybridization Analysis Using Centromere-Specific Probes.

Centromere-specific DNA probes for chromosomes 4, 7 and 18 were used to simultaneously analyze chromosome loss, non-disjunction, breaks within the labeled region, and nucleoplasmic bridges induced by γ rays in binucleated human lymphocytes. The doses used were 0, 1, 2 and 4 Gy, and approximately 1000 cells were scored per dose. Micronucleus frequency increased in a linear-quadratic fashion. For chromosome loss, significant increases were observed at 2 and 4 Gy, whereas for non-disjunction significant increases were observed at 1 Gy; thus non-disjunction allowed us to detect the effects of radiation at a lower dose than chromosome loss. The use of centromere-specific probes allowed discrimination between the clastogenic and aneugenic effects of ionizing radiation. The analysis of chromosome loss, not taking fragmented signals into account, ensures the detection of an aneugenic effect, which was not possible using pancentromeric probes. The frequency of chromosome breakage within the labeled regions was higher in nuclei than in micronuclei, suggesting an increase in the engulfment of chromosomal material by nuclei as a consequence of the presence of cytochalasin B in the cultures. Chromatin filaments connecting main nuclei, the so-called nucleoplasmic bridges, were observed in irradiated samples, and are a manifestation of rearranged chromosomes producing anaphase bridges.

Suzuki, M., Piao, C., Hall, E. J. and Hei, T. K. Cell Killing and Chromatid Damage in Primary Human Bronchial Epithelial Cells Irradiated with Accelerated ⁵⁶Fe Ions

We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy ⁵⁶Fe ions. Cells were irradiated with graded doses of ⁵⁶Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for ⁵⁶Fe ions compared to ¹³⁷Cs γ rays was 1.99 for immediate plating and 2.73 for delayed plating at the D₁₀. The repair ratio (delayed plating/immediate plating) was 1.67 for ¹³⁷Cs γ rays and 1.22 for ⁵⁶Fe ions. The dose–response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for ¹³⁷Cs γ rays and ⁵⁶Fe ions. On the other hand, ∼85% of the fragments induced by ¹³⁷Cs γ rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for ⁵⁶Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by γ rays measured 24 h after irradiation was higher than that induced by ⁵⁶Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy ⁵⁶Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than ¹³⁷Cs γ rays in the target cells for radiation-induced lung cancers.

de Lara, C. M., Hill, M. A., Jenner, T. J., Papworth, D. and O'Neill, P. Dependence of the Yield of DNA Double-Strand Breaks in Chinese Hamster V79-4 Cells on the Photon Energy of Ultrasoft X Rays.

Induction of DNA DSBs by low-LET radiations reflects clustered damage produced predominantly by low-energy, secondary electron “track ends”. Cell inactivation and induction of DSBs and their rejoining, assayed using pulsed-field gel electrophoresis, were determined in Chinese hamster V79-4 cells irradiated as a monolayer with characteristic carbon K-shell (C_K) (0.28 keV), aluminum K-shell (Al_K) (1.49 keV), and titanium K-shell (Ti_K) (4.55 keV) ultrasoft X rays under aerobic and anaerobic conditions. Relative to ⁶⁰Co γ rays, the relative biological effectiveness (RBE) for cell inactivation at 10% survival and for induction of DSBs increases as the photon energy of the ultrasoft X rays decreases. The RBE values for cell inactivation and for induction of DSBs by C_K ultrasoft X rays are 2.8 ± 0.3 and 2.7 ± 0.3, respectively, and by Ti_K ultrasoft X rays are 1.5 ± 0.1 and 1.4 ± 0.1, respectively. Oxygen enhancement ratios (OERs) of ∼2 for cell inactivation and induction of DSBs by ultrasoft X rays are independent of the photon energy. The time scale for rejoining of DNA DSBs is similar for both ultrasoft X rays and ⁶⁰Co γ rays. From the size distribution of small DNA fragments down to 0.48 kbp, we concluded that DSBs are induced randomly by C_K and Al_K ultrasoft X rays. Therefore, ultrasoft X rays are more efficient per unit dose than γ radiation at inducing DNA DSBs, the yield of which increases with decreasing photon energy.

Akamatsu, K. and Yokoya, A. X-Ray Absorption near Edge Structures of DNA or its Components around the Oxygen K-shell Edge.

The initial process of radiation damage in DNA was investigated by measuring the X-ray absorption near edge structures (XANES) within the energy region around the oxygen K-shell absorption edge for DNA, cytosine and 2-deoxy-d-ribose. Irradiation and XANES experiments were performed with the BL23SU soft X-ray beamline, using synchrotron radiation from the 8 GeV electron storage ring at SPring-8. Samples were mounted on gold-coated plates in a vacuum chamber. The XANES spectra were obtained by measuring the photoelectron current of the samples. 2-Deoxy-d-ribose was exposed to X rays at the absorption peak corresponding to the oxygen (O) 1s→σ* transition energy (538 eV); the XANES spectra were obtained after each irradiation. DNA and cytosine, possessing characteristic XANES spectra, both had two major energy bands corresponding to the O 1s→π* and 1s→σ* transitions. Two new peaks appeared and gradually increased in the XANES spectra of 2-deoxy-d-ribose during irradiation. These results suggest that C–O bonds in 2-deoxy-d-ribose are transformed to C=O bonds by O 1s→σ* transition, suggesting that the molecules undergo chemical changes into carbonyl-containing compounds.

Trumbore, C. N., Ehrlich, R. S. and Myers, Y. N. Changes in DNA Conformation Induced by Gamma Irradiation in the Presence of Copper.

Gamma irradiation of DNA solutions containing copper causes changes in DNA conformation in oligonucleotides and in natural and synthetic DNAs. Diagnostic for these conformational changes is a ubiquitous 187-nm peak in the circular dichroism (CD) difference spectrum that has been predicted for a transformation from a right-handed to a left-handed helical DNA conformation. Changes in CD are correlated with changes in the UV spectrum. Reduction of DNA-bound Cu(II) to Cu(I) with ascorbic acid produces similar changes in CD spectra. These changes can be produced by the peroxy radical anion (O₂^· –) and the OH radical in the presence of copper. O₂^· – is approximately twice as efficient as ^·OH in initiating these changes in natural DNA. The changes in DNA conformation induced by ionizing radiation are remarkable in that they are dependent on the copper-ion concentration in a highly nonlinear manner at low copper concentrations and are not observed in the absence of copper ions. Possible implications of our results for radiobiological and oxidative damage in the cell nucleus are discussed.

O'Hara, J. A., Blumenthal, R. D., Grinberg, O. Y., Demidenko, E., Grinberg, S., Wilmot, C. M., Taylor, A. M., Goldenberg, D. M. and Swartz, H. M. Response to Radioimmunotherapy Correlates with Tumor pO₂ Measured by EPR Oximetry in Human Tumor Xenografts.

The efficacy of radiation treatment depends upon local oxygen concentration. We postulated that the variability in responsiveness of tumor xenografts to a fixed dose of radioimmunotherapy might be related to the tumor pO₂ at the time that radioimmunotherapy was administered. We evaluated the growth of xenografts of CALU-3 tumors, a non-small cell lung carcinoma, in response to an 8.9-MBq dose of ¹³¹I-RS-7-anti-EGP-1 and correlated tumor growth rate with initial tumor pO₂ measured by EPR oximetry. The greatest growth delay in response to radioimmunotherapy had the highest initial pO₂, and the fastest-growing tumors had the lowest initial pO₂. We then determined the dynamic effect of radioimmunotherapy on tumor pO₂ by serial measurements of pO₂ for 35 days after radioimmunotherapy. This information could be important for ascertaining the likelihood that a tumor will respond to additional doses as part of a multiple dose scheme. Serial tumor pO₂ measurements may help identify a window of opportunity when the surviving tumor regions will be responsive to a second round of radioimmunotherapy or a second therapeutic modality such as chemotherapy or an anti-vascular agent. After radioimmunotherapy, there was an increase in tumor pO₂ followed by a decrease below initial levels in most mice. Thus defined times may exist when a tumor is more or less radiosensitive after radioimmunotherapy.

Sharma, M., Sharma, R., McCarthy, E. T., Ge, X. L., Fish, B. L., Savin, V. J., Cohen, E. P. and Moulder, J. E. Early Detection of Radiation-Induced Glomerular Injury by Albumin Permeability Assay.

Renal irradiation leads predictably to glomerular vascular injury, cell lysis, matrix accumulation, sclerosis and loss of renal function. The immediate effects of renal irradiation that may be associated with glomerular pathology and proteinuria are not clear in the human disease or its rat model. We hypothesized that radiation-induced injury causes immediate and subtle alterations in glomerular physiology independent of the neurohumoral and hemodynamic regulatory mechanisms. We employed a sensitive in vitro functional assay of glomerular albumin permeability (P_alb) to demonstrate radiation-induced damage to the glomerular filtration barrier immediately after total-body irradiation of rats. In blinded experiments, control rats were sham-treated, and experimental rats received 9.5 Gy X rays. Rats were killed humanely at 1 h to 9 weeks after irradiation and glomeruli were isolated. In parallel experiments, glomeruli were isolated from normal rats and irradiated in vitro. The change in glomerular capillary permeability due to an experimental oncotic gradient was determined using videomicroscopy and P_alb was calculated. Results show that in vivo or in vitro irradiation of glomeruli caused an increased P_alb at 1 h. Increased P_alb was observed up to 3 weeks after irradiation. Glomeruli from mice irradiated with 9.5 or 19.0 Gy X rays did not show increased P_alb at 1 h postirradiation. We conclude that glomerular protein permeability of irradiated rats increases in a dose-dependent manner immediately after irradiation and that it appears to be independent of hemodynamic or systemic influences.

Robbins, M. E. C., O'Malley, Y., Zhao, W., Davis, C. S. and Bonsib, S. M. The Role of the Tubulointerstitium in Radiation-Induced Renal Fibrosis.

The functional and morphological response of the remaining hypertrophied kidney in unilaterally nephrectomized rats to single doses of 0–20 Gy X rays was investigated. Functional and histological end points were assessed serially 4–24 weeks postirradiation. Renal irradiation led to time- and dose-dependent reductions in renal function, seen in terms of a decreased glomerular filtration rate, increased blood urea nitrogen, and reduced hematocrit. These changes were accompanied by morphological changes in the glomerular, tubular and interstitial portions of the kidney. However, dose-dependent changes were observed only in terms of tubulointerstitial lesions. Significant increases in the degree of interstitial staining for collagen type III and fibronectin were observed 24 weeks postirradiation. These increases in extracellular matrix components were accompanied by a significant increase in interstitial α smooth muscle actin, suggesting activation of interstitial fibroblasts into myofibroblasts. There was no evidence of glomerular Tgfb after renal irradiation. A significant increase in tubular Tgfb staining was only seen 8 weeks postirradiation. In contrast, there was a shift of staining to the interstitium such that by 24 weeks postirradiation interstitial Tgfb staining was significantly greater than that seen in controls. These findings suggest that the tubule epithelial cell and the interstitial fibroblast are both active participants in the development and/or progression of radiation-induced renal fibrosis.

van Vliet-Vroegindeweij, C., Wheeler, F. J., Stecher-Rasmussen, F., Moss, R. and Huiskamp, R. Microdosimetry Model for Boron Neutron Capture Therapy: I. Determination of Microscopic Quantities of Heavy Particles on a Cellular Scale.

Due to the limitations of existing microdosimetry models, a new model called MICOR has been developed to analyze the spatial distribution of microscopic energy deposition for boron neutron capture therapy (BNCT). As in most existing models, the reactions independent of the incident neutron energy such as the boron and the nitrogen capture reactions can be considered. While other models do not include reactions that are dependent on the neutron energy such as the proton recoil reaction, the present model is designed so that the energy deposition resulting from these reactions is included. The model MICOR has been extended to enable the determination of the biological effects of BNCT, which cannot be done with the existing models. The present paper describes the determination of several microscopic quantities such as the number of hits, the energy deposition in the cell nucleus, and the distribution of lineal and specific energy deposition. The companion paper (Radiat. Res. 155, 000–000 2001) deals with the conversion of these microscopic quantities into biological effects. The model is used to analyze the results of a radiobiological experiment performed at the HB11 facility in the HFR in Petten. This analysis shows the value of the model in determining the dose depositions on a cellular scale and the importance of the extension to the energy deposition of the proton recoil.

van Vliet-Vroegindeweij, C., Wheeler, F. J., Stecher-Rasmussen, F. and Huiskamp, R. Microdosimetry Model for Boron Neutron Capture Therapy: II. Theoretical Estimation of the Effectiveness Function and Surviving Fractions.

A model has been developed to obtain a better understanding of the effects of boron neutron capture therapy (BNCT) on a cellular scale. This model, the microdosimetry model MICOR, has been developed to include all reactions important for BNCT. To make the model more powerful in the translation from energy deposition to biological effect, it has been designed to be capable of calculating the effectiveness function. Based on this function, the model can calculate surviving fractions, RBE values and boron concentration distributions. MICOR has been used to analyze an extensive set of biological experiments performed at the HB11 beam in Petten. For V79 Chinese hamster cells, the effectiveness function is determined and used to generate surviving fractions. These fractions are compared with measured surviving fractions, which results in a good agreement between the measured and calculated surviving fractions (within the uncertainties of the measurements).