Sutherland, B. M., Bennett, P. V., Sutherland, J. C. and Laval, J. Clustered DNA Damages Induced by X Rays in Human Cells. Radiat. Res. 157, 611–616 (2002).

Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages—two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns—are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.

Karger, C. P., Münter, M. W., Heiland, S., Peschke, P., Debus, J. and Hartmann, G. H. Dose–Response Curves and Tolerance Doses for Late Functional Changes in the Normal Rat Brain after Stereotactic Radiosurgery Evaluated by Magnetic Resonance Imaging: Influence of End Points and Follow-up Time. Radiat. Res. 157, 617–625 (2002).

Late reaction of normal tissue is still a limiting factor in radiotherapy and radiosurgery of patients with brain tumors. Few quantitative data in terms of dose–response curves are available. In the present study, 99 animals were irradiated stereotactically at the right frontal lobe using a linear accelerator and single doses between 26 and 50 Gy. The diameter of the spherical dose distribution was 4.7 mm (80% isodose). Dose–response curves for late changes in the normal brain at 20 months were measured using T1- and T2-weighted magnetic resonance imaging (MRI). The dependence of the dose–response curves on the follow-up time and the definition of the biological end point were determined. Tolerance doses were calculated at several effect probability levels and times after irradiation. The MRI changes were found to be dependent on dose and progressive in time. At 20 months, the tolerance doses at a 50% effect probability level were 39.6 ± 1.0 Gy and 42.4 ± 1.4 Gy for changes in T1- and T2-weighted images, respectively. These dose–response curves can be used for further quantitative investigations on the influence of various treatment parameters, such as the application of charged particles, radiopharmaceuticals or the variation of tissue oxygenation.

Rijken, P. F. J. W., Peters, J. P. W. and van der Kogel, A. J. Quantitative Analysis of Varying Profiles of Hypoxia in Relation to Functional Vessels in Different Human Glioma Xenograft Lines. Radiat. Res. 157, 626–632 (2002).

Tissue oxygenation influences the radiation response of tumors. To further investigate the underlying mechanisms of tumor hypoxia, the spatial distribution of hypoxic cells in relation to the vasculature was studied. In a panel of three human glioma xenograft lines (E2, E102, E106) with different growth characteristics, tumor line-specific patterns of hypoxia (pimonidazole) and (functional) vasculature (Hoechst 33342) were observed. Two of the three glioma lines showed a more homogeneous distribution of perfused vessels (E102 and E106) than the third glioma line (E2). Although all tumors showed hypoxia, the distance at which the steepest part of the gradient of the hypoxia marker was found varied significantly among the different glioma lines. The faster-growing E102 tumors had the longest distance (>300 μm). These results indicate that tumor line-specific factors, rather than vascular geometry alone, may determine the oxygenation status of a tumor. As a consequence, vascular density cannot be used as a surrogate parameter for tumor hypoxia when comparing different tumors. Additional hypoxia and perfusion markers will further improve our understanding of changes in tumor physiology at the microregional level explaining the relationship between the low oxygen levels and the response of tumors to treatment.

Kennel, S. J., Mirzadeh, S., Eckelman, W. C., Waldmann, T. A., Garmestani, K., Yordanov, A. T., Stabin, M. G. and Brechbiel, M. W. Vascular-Targeted Radioimmunotherapy with the Alpha-Particle Emitter ²¹¹At. Radiat. Res. 157, 633–641 (2002).

Astatine-211, an α-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[²¹¹At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered ²¹¹At to the lung (260–418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with ²¹¹At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25–40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of ²¹¹At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3–4 months after treatment, as did mice treated with 3700 kBq of the α-particle emitter ²¹³Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with ²¹¹At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted ²¹¹At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted ²¹¹At should be at least 10-fold more efficient for lung colony therapy than ²¹¹At bound to nontargeting controls. The data showed that ²¹¹At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of α-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.

Van der Meeren, A., Mouthon, M-A., Gaugler, M-H., Vandamme, M. and Gourmelon, P. Administration of Recombinant Human IL11 after Supralethal Radiation Exposure Promotes Survival in Mice: Interactive Effect with Thrombopoietin. Radiat. Res. 157, 642–649 (2002).

In the present study, we evaluated the therapeutic potential of recombinant human IL11 in lethally irradiated C57BL6/J mice exposed to γ rays. IL11 administered for 5 consecutive days beginning 2 h after total-body irradiation with 8 or 9 Gy ⁶⁰Co γ rays resulted in a significant increase in 30-day survival. When IL11 was administered, only a slight improvement in the hematopoietic status (both blood cell counts and progenitor cells) was observed after an 8-Gy exposure, and no improvement in hematopoietic reconstitution was observed after 9 Gy total-body irradiation. The enhancement of fibrinogen in the plasma of irradiated animals suggested the importance of infections in the death of animals. IL11 was able to limit the increase in fibrinogen levels. However, prevention of bacterial infections by antibiotic treatment, although it delayed death, was ineffective in promoting survival either in placebo-treated and IL11-treated mice. IL11 was administered along with thrombopoietin (TPO) or bone marrow transplantation to limit the hematopoietic syndrome, in addition to antibiotic treatment. When IL11 was combined with TPO, a potent stimulator of hematopoiesis, the survival of animals which had been irradiated with 10 Gy ¹³⁷Cs γ rays was increased significantly compared to those treated with IL11 or TPO alone. Furthermore, an interactive effect of TPO and IL11 on hematopoietic reconstitution was observed. Similarly, IL11 in combination with bone marrow transplantation enhanced survival after 15 Gy ¹³⁷Cs γ rays. These data suggest that the effect of IL11 on the hematopoietic system is only moderate when it is used alone in supralethally irradiated mice but that the effect is improved in the presence of a hematopoietic growth factor or bone marrow transplantation.

Kang, C-M., Park, K-P., Cho, C-K., Seo, J-S., Park, W-Y., Lee, S-J. and Lee, Y-S. Hspa4 (HSP70) is Involved in the Radioadaptive Response: Results from Mouse Splenocytes. Radiat. Res. 157, 650–655 (2002).

In a continuation of our earlier study on the involvement of HSP25 (now known as Hspb1) and HSP70 (now known as Hspa4) in the induction of an adaptive response, we examined the involvement of these proteins in the induction of the adaptive response using an animal model system. C57BL6 mice were irradiated with 5 cGy of γ radiation three times in 1 week (for a total of 15 cGy), and a high challenge dose (6 Gy) was given on the day after the last low-dose irradiation. The survival time of the low-dose preirradiated mice was increased to 30%. The induction of apoptosis induced by 6 Gy was also reduced by this low-dose preirradiation regimen. To elucidate any link existing between the HSPs and the induction of the adaptive response, reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis was performed using splenocytes. High-dose radiation up-regulated the expression of Hspb1 and especially Hspa4, while expression of other HSPs such as HSC70 (now know as Hspa8), Hsp90, and αB-crystalline (now known as Cryab) did not change. When splenocytes from Hspa4 transgenic mice were preirradiated with a low dose of radiation, a reduction in cell death after high-dose irradiation was observed. These results suggest that Hspa4 is a key molecule in the induction of the adaptive response.

Vujaskovic, Z., Feng, Q. F., Rabbani, Z. N., Anscher, M. S., Samulski, T. V. and Brizel, D. M. Radioprotection of Lungs by Amifostine is Associated with Reduction in Profibrogenic Cytokine Activity. Radiat. Res. 157, 656–660 (2002).

Radiation-induced pulmonary toxicity causes significant morbidity and mortality in patients irradiated for lung cancer, breast cancer, lymphoma or thymoma. Amifostine is an important drug in the emerging field of cytoprotection. Recent advances in our understanding of the mechanism of radiation-induced injury at the molecular and cellular levels have stimulated interest in the development of effective radioprotective strategies. Accumulation of macrophages with associated production of reactive oxygen species (ROS) and production and activation of cytokines is a key process involved in the pathophysiology of radiation injury in the lung. The purpose of this study was to determine whether the mechanism of radioprotection by amifostine includes reduction in both macrophage activity and the expression and activation of profibrogenic cytokines. Our results demonstrated a reduction in both functional and histological radiation-induced lung injury by amifostine. In addition, this study is the first to demonstrate that amifostine given prior to irradiation reduced both the accumulation of macrophages and the expression/activation of lung tissue Tgfb1 which was followed by the reduction of plasma Tgfb1 levels during the development of radiation-induced lung injury. Future studies are needed to determine whether administration of amifostine both during and after radiotherapy may further increase its radioprotective effect.

Shiraishi, K., Shimura, T., Taga, M., Uematsu, N., Gondo, Y., Ohtaki, M., Kominami, R. and Niwa, O. Persistent Induction of Somatic Reversions of the Pink-Eyed Unstable Mutation in F₁ Mice Born to Fathers Irradiated at the Spermatozoa Stage. Radiat. Res. 157, 661–667 (2002).

Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (p^un) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion p^um in F₁ mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F₁ mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F₁ mice. Somatic reversion occurred for the paternally derived p^un alleles. In addition, the reversion also occurred for the maternally derived, unirradiated p^un alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.

Carr, Z. A., Kleinerman, R. A., Stovall, M., Weinstock, R. M., Griem, M. L. and Land, C. E. Malignant Neoplasms after Radiation Therapy for Peptic Ulcer. Radiat. Res. 157, 668–677 (2002).

Most information on radiation-related cancer risk comes from the Life Span Study (LSS) of the Japanese atomic bomb survivors. Stomach cancer mortality rates are much higher in Japan than in the U.S., making the applicability of LSS findings to the U.S. population uncertain. A unique cohort of U.S. patients who were irradiated for peptic ulcer to control gastric secretion provides a different perspective on risk. Cancer mortality data were analyzed and relative risks estimated for 3719 subjects treated by radiotherapy (mean stomach dose 14.8 Gy) and/or by surgery and medication during the period 1936–1965 and followed through 1997 (average 25 years). Compared to the U.S. rates, stomach cancer mortality was significantly increased for irradiated and nonirradiated patients (observed/expected = 3.20 and 1.52, respectively). We observed strong evidence of exposure-related excess mortality from cancer of the stomach (RR 2.6, 95% CI 1.3, 5.1), pancreas (RR 2.7, 95% CI 1.5, 5.1), and lung (RR 1.5, 95% CI 1.1, 2.1), with commensurate radiation dose responses in analyses that included nonexposed patients. However, the dose responses for these cancers were not significant when restricted to exposed patients. Our excess relative risk per gray estimate of 0.20 at doses ≤10 Gy (95% CI 0, 0.73) is consistent with the estimate of 0.24 (95% CI 0.10, 0.40) obtained from the LSS study with the linear model.

Lee, T-K., Allison, R. R., O'Brien, K. F., Naves, J. L., Karlsson, U. L. and Wiley, A. L., Jr. Persistence of Micronuclei in Lymphocytes of Cancer Patients after Radiotherapy. Radiat. Res. 157, 678–684 (2002).

To verify the applicability of the micronucleus (MN) yield in peripheral blood lymphocytes (PBLs) as a quantitative biodosimeter for monitoring in vivo ionizing radiation damage, we applied the cytokinesis-blocked micronucleus assay in PBLs of cancer patients treated with partial-body radiotherapy. Dosimetric information on these 13 patients represented a wide range in the number of fractions, cumulative tumor dose, total integral dose, and equivalent total-body absorbed dose. We found in PBLs of these patients that (1) the MN yield increased linearly with the equivalent total-body absorbed dose (r = 0.8, P = 0.002), (2) the distributions of the MN yields deviated significantly from Poisson, and (3) there was a general decline in MN yields with increasing length of follow-up, but with considerable variation between individuals. The average rate of decline was found to be linear and was correlated with the equivalent total-body absorbed dose (r = 0.7, P = 0.007). Further, at 19–75 months of follow-up time, seven patients showed higher MN yields than their respective levels before radiotherapy, indicating the persistence of radiation-induced residual cytogenetic damage. Our findings suggest that the MN yield in human PBLs offers a reliable acute and perhaps chronic biodosimeter for in vivo radiation dose estimation. After the completion of radiotherapy, the persistence of elevated MN yield in PBLs is a reflection of the surviving population of radiation-induced genetically aberrant cells.

Marathe, D. L. and Mishra, K. P. Radiation-Induced Changes in Permeability in Unilamellar Phospholipid Liposomes. Radiat. Res. 157, 685–692 (2002).

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of α-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to γ radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.

Petitot, F., Morlier, J. P., Debroche, M., Pineau, J. F. and Chevillard, S. A New Method Specifically Designed to Expose Cells Isolated In Vitro to Radon and its Decay Products. Radiat. Res. 157, 693–699 (2002).

A system was set up to provide direct exposure of cells cultured in vitro to radon and its decay products. Radon gas emanating from a uranium source was introduced at a measured concentration in a closed 10-m³ exposure chamber. Cells were cultured on the microporous membrane of an insert that was floating over the culture medium in a six-well cluster plate. Plates with cells were placed in an open thermoregulated bath within the chamber. Under these conditions, cells were irradiated by direct deposition of radon and radon decay products. During exposure, all parameters, including radon gas concentrations, decay product activities, and potential α-particle energy concentrations, were determined by periodic air-grab samplings inside the chamber. The energy spectrum of deposited decay products was characterized. An estimation of α-particle flux density on the area containing cells was performed using CR-39 detector films that were exposed in cell-free wells during the cell exposure. The number of α-particle traversals per cell was deduced both from the mean number of CR-39 tracks per surface unit and from measurements of entire cells or nuclear surfaces. This paper describes the design of experiment, the dosimetry of radon and radon decay product, and the procedures for aerosol measurements. Our preliminary data show the usefulness of the in vitro cell culture approach to the study of the early cellular effects of radon and its decay products.

Chandra, S., Lorey, D. R., II and Smith, D. R. Quantitative Subcellular Secondary Ion Mass Spectrometry (SIMS) Imaging of Boron-10 and Boron-11 Isotopes in the Same Cell Delivered by Two Combined BNCT Drugs: In Vitro Studies on Human Glioblastoma T98G Cells. Radiat. Res. 157, 700–710 (2002).

Ion microscopy was used for subcellular quantitative imaging of the isotopes ¹⁰B and ¹¹B in the same cell to evaluate boron delivery using a mixture of two neutron capture therapy drugs, p-boronophenylalanine-fructose (BPA-F) and sodium borocaptate (BSH). The application of ¹⁰B-labeled BPA-F and ¹¹B-labeled BSH allowed independent imaging of both ¹⁰B and ¹¹B in the same cell using a CAMECA IMS-3f ion microscope. Mixed-drug treatments were compared to single-drug exposures given under identical conditions. ¹⁰BPA-F delivered ¹⁰B heterogeneously to T98G human glioblastoma cells, with a significantly reduced concentration in an organelle-rich perinuclear region. The intracellular distribution of ¹¹B from ¹¹BSH contrasted with that of the ¹⁰B from ¹⁰BPA-F, with ¹¹B distributed nearly homogeneously throughout cells. The subcellular distributions of ¹⁰B and ¹¹B were sustained in mixed-drug treatments and resembled their localizations after the single-drug treatments. In both single- and mixed-drug treatments, cellular levels of ¹⁰B from ¹⁰BPA-F nearly doubled between 1 h and 6 h, with a 3:1 intracellular to nutrient medium partitioning, while cellular levels of ¹¹BSH remained essentially unchanged. The net effect of the combined treatment with ¹⁰BPA-F and ¹¹BSH was an additive delivery of boron to cells. This study introduces a novel approach for checking potential synergistic, antagonistic or simple additive delivery of two mixed boronated compounds in cellular/subcellular compartments.

Vázquez-Gundín, F., Rivero, M. T., Gosálvez, J. and Fernández, J. L. Radiation-Induced DNA Breaks in Different Human Satellite DNA Sequence Areas, Analyzed by DNA Breakage Detection-Fluorescence In Situ Hybridization. Radiat. Res. 157, 711–720 (2002).

Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding–renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.

Gauter, B., Zlobinskaya, O. and Weber, K. J. Rejoining of Radiation-Induced DNA Double-Strand Breaks: Pulsed-Field Electrophoresis Analysis of Fragment Size Distributions after Incubation for Repair. Radiat. Res. 157, 721–733 (2002).

The repair of radiation-induced DNA double-strand breaks (DSBs) is frequently investigated by measuring the time-dependent decrease in the fraction of fragmented DNA that is able to enter electrophoresis gels. When transformed into equivalent doses without repair, such measurements are thought to reflect the removal of DSBs, and they typically exhibit a fast initial component and a decreasing rate at longer repair intervals. This formalism, however, assumes that the spatial distribution of unrejoined breakage resembles the pattern of induction of DSBs. While the size distributions for initial fragmentation, such as that resolved by conventional pulsed-field gel electrophoresis (PFGE) (between about 10⁵ and 10⁷ bp), are well known to agree with the prediction of random breakage, no data are available from studies explicitly testing this relationship for residual breakage. Therefore, Chinese hamster V79 cells and MeWo (human melanoma) cells were irradiated with different doses (10–100 Gy) or were incubated for repair for up to 4 h after a single dose of 100 Gy (V79) or 90 Gy (MeWo) before being subjected to PFGE. Fragment size distributions were calculated by convolution of the PFGE profiles with an appropriately generated size calibration function. The results clearly demonstrate an over-representation of smaller fragments (below about 2–3 Mbp) compared to the prediction of randomness for residual breakage. In consequence, the time-dependent decrease of dose-equivalent values calculated from data on the fraction released may not directly reflect DSB rejoining rates. The present findings are compatible with an earlier suggestion of slow rejoining of breaks which have been induced as multiple breaks (two or more) in large chromosomal loops, thus also predicting an increase of the slowly rejoining DSB fraction with increasing dose.

Hofland, I., Ramakers, B., Begg, A. C. and Vens, C. Rapid Fluorescence Ratio Assay for Detecting Changes in Radiosensitivity. Radiat. Res. 157, 734–739 (2002).

To test modifications in sensitization to radiation or drugs in preclinical studies of cancer therapy, the colony-forming assay is regarded as the gold standard. Because this assay is time consuming, somewhat laborious, and unsuitable for rapid screening, development of other assays is desirable. We describe here an assay based on the detection of enhanced green fluorescence protein (EGFP) with flow cytometry that is particularly suitable for genetic manipulation studies in which the gene of interest is introduced together with EGFP as reporter. It is easily adaptable to other reporters, however, whether naturally fluorescent or requiring immunochemical staining. Cells are irradiated as mixed populations of a known standard cell line (nonfluorescent) together with the genetically manipulated cell line expressing EGFP. Ratios of fluorescent and nonfluorescent cells are measured before treatment and several days after treatment. If the cell populations have equal radiosensitivities, the ratio remains unchanged. Changes in the ratio indicate changes in radiosensitivity. The assay was validated for two situations in which dominant negative peptides inhibiting DNA repair were expressed in A549 human lung cells and affected radiosensitivity.