Schmid, E., Krumrey, M., Ulm, G., Roos, H. and Regulla, D. The Maximum Low-Dose RBE of 17.4 and 40 keV Monochromatic X Rays for the Induction of Dicentric Chromosomes in Human Peripheral Lymphocytes. Radiat. Res. 160, 499–504 (2003).

Schmid et al. (Radiat. Res. 158, 771–777, 2002) recently reported on the maximum low-dose RBE for mammography X rays (29 kV) for the induction of dicentrics in human lymphocytes. To obtain additional information on the RBE for this radiation quality, experiments with monochromatized synchrotron radiation were performed. Monochromatic 17.4 keV X rays were chosen for comparison with the diagnostic mammography X-ray spectrum to evaluate the spectral influence, while monochromatic 40 keV X rays represent a higher-energy reference radiation, within the experiment. The induction of dicentric chromosomes in human lymphocytes from one blood donor irradiated in vitro with 17.4 keV and 40 keV monochromatic X rays resulted in α coefficients of (3.44 ± 0.87) × 10^–2 Gy^–1 and (2.37 ± 0.93) × 10^–2 Gy^–1, respectively. These biological effects are only about half of the α coefficients reported earlier for exposure of blood from the same donor with the broad energy spectra of 29 kV X rays (mean energy of 17.4 keV) and 60 kV X rays (mean energy of 48 keV). A similar behavior is evident in terms of RBE_M. Relative to weakly filtered 220 kV X rays, the RBE_M for 17.4 and 40 keV monochromatic X rays is 0.86 ± 0.23 and 0.59 ± 0.24, respectively, which is in contrast to the RBE_M of 1.64 ± 0.27 for 29 kV X rays and 1.10 ± 0.19 for 60 kV X rays. It is evident that the monochromatic radiations are less effective in inducing dicentric chromosomes than broad-spectrum X rays with the corresponding mean energy value. Therefore, it can be assumed that, for these X-ray qualities with broad energy spectra, a large fraction of the effects should be attributed predominantly to photons with energies well below the mean energy.

Schettino, G., Folkard, M., Prise, K. M., Vojnovic, B., Held, K. D. and Michael, B. D. Low-Dose Studies of Bystander Cell Killing with Targeted Soft X Rays. Radiat. Res. 160, 505–511 (2003).

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic C_K X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 μm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a C_K X-ray beam was measured in the 0–2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (∼25 mm²). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Zhou, H., Randers-Pehrson, G., Geard, C. R., Brenner, D. J., Hall, E. J. and Hei, T. K. Interaction between Radiation-Induced Adaptive Response and Bystander Mutagenesis in Mammalian Cells. Radiat. Res. 160, 512–516 (2003).

Two conflicting phenomena, the bystander effect and the adaptive response, are important in determining biological responses at low doses of radiation and have the potential to have an impact on the shape of the dose–response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive A_L cell mutagenic assay, we reported previously that nonirradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been traversed with either a single or 20 α particles each. Here we show that pretreatment of cells with a low dose of X rays 4 h before α-particle irradiation significantly decreased this bystander mutagenic response. Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X rays. Results from the present study address some of the pressing issues regarding both the actual target size and the radiation dose response and can improve on our current understanding of radiation risk assessment.

Miller, S. C., Lloyd, R. D., Bruenger, F. W., Krahenbuhl, M. P. and Romanov, S. A. Comparisons of the Skeletal Locations of Putative Plutonium-Induced Osteosarcomas in Humans with those in Beagle Dogs and with Naturally Occurring Tumors in both Species. Radiat. Res. 160, 517–523 (2003).

Osteosarcomas occur from exposures to bone-seeking, α-particle-emitting isotopes, particularly plutonium. The skeletal distribution of putative ²³⁹Pu-induced osteosarcomas reported in Mayak Metallurgical and Radiochemical Plutonium Plant workers is compared with those observed in canine studies, and these are compared with distributions of naturally occurring osteosarcomas in both species. In the Mayak workers, 29% and 71% of the osteosarcomas were in the peripheral and central skeleton, respectively, with the spine having the most tumors (36%). An almost identical distribution of plutonium-induced osteosarcomas was reported for dogs injected with ²³⁹Pu as young adults. This distribution of osteosarcomas is quite different from the distributions of naturally occurring osteosarcomas for both species. In the Cooperative Osteosarcoma Study Group in humans (1,736 osteosarcomas from all ages), over 91% of the tumors occurred in the peripheral skeleton. In the Mayo Clinic group of older individuals (>40 years old), over 60% of the osteosarcomas appeared in the peripheral skeleton. The distribution of naturally occurring osteosarcomas in the canine is similar to that in the adult human. The similarities of the distributions of plutonium-associated osteosarcomas in the Mayak workers with those found in experimental studies suggest that many of the reported osteosarcomas may have been associated with plutonium exposures. These results also support the experimental paradigm that plutonium osteosarcomas have a preference for well vascularized cancellous bone sites. These sites have a greater initial deposition of plutonium, but also greater turnover due to elevated bone remodeling rates.

Wang, J., Zheng, H., Hollenberg, M. D., Wijesuriya, S. J., Ou, X. and Hauer-Jensen, M. Up-regulation and Activation of Proteinase-Activated Receptor 2 in Early and Delayed Radiation Injury in the Rat Intestine: Influence of Biological Activators of Proteinase-Activated Receptor 2. Radiat. Res. 160, 524–535 (2003).

Proteinase-activated receptor 2 (Par2, F2rl1, also designated PAR-2 or PAR₂) is prominently expressed in the intestine and has been suggested as a mediator of inflammatory, mitogenic and fibrogenic responses to injury. Mast cell proteinases and pancreatic trypsin, both of which have been shown to affect the intestinal radiation response, are the major biological activators of Par2. Conventional Sprague-Dawley rats, mast cell-deficient rats, and rats in which pancreatic exocrine secretion was blocked pharmacologically by octreotide underwent localized irradiation of a 4-cm loop of small bowel. Radiation injury was assessed 2 weeks after irradiation (early, inflammatory phase) and 26 weeks after irradiation (chronic, fibrotic phase). Par2 expression and activation were assessed by in situ hybridization and immunohistochemistry, using antibodies that distinguished between total (preactivated and activated) Par2 and preactivated Par2. Compared to unirradiated intestine, irradiated intestine exhibited increased Par2 expression, particularly in areas of myofibroblast proliferation and collagen accumulation, after both single-dose and fractionated irradiation. The majority of Par2 expressed in fibrotic areas was activated. Postirradiation Par2 overexpression was greatly attenuated in both mast cell-deficient and octreotide-treated rats. The severity of acute mucosal injury did not affect postirradiation Par2 expression. Mast cells and pancreatic proteinases may exert their fibro-proliferative effects partly through activation of Par2. Par2 may be a potential target for modulating the intestinal radiation response, particularly delayed intestinal wall fibrosis.

Debus, J., Scholz, M., Haberer, T., Peschke, Jäkel, O., Karger, C. P. and Wannenmacher, M. Radiation Tolerance of the Rat Spinal Cord after Single and Split Doses of Photons and Carbon Ions. Radiat. Res. 160, 536–542 (2003).

The sensitivity of the rat spinal cord to single and split doses of radiation and the resulting relative biological effectiveness (RBE) were determined for carbon-ion irradiations (¹²C) in the plateau and Bragg-peak regions. The cranial part of the cervical and thoracic spinal cords of 180 rats were irradiated with one or two fractions of ¹²C ions or photons, respectively. Dose–response curves for the end point symptomatic myelopathy were established, and the resulting values for the ED₅₀ (dose for 50% complication probability) were used to determine the RBEs. A median latency for myelopathy of 167 days (range, 121–288 days) was found. The ED₅₀ values were 17.1 ± 0.8 Gy, 24.9 ± 0.7 Gy (one and two fractions, ¹²C plateau) and 13.9 ± 0.8, 15.8 ± 0.7 Gy (one and two fractions, ¹²C Bragg peak), respectively. For photons we obtained ED₅₀ values of 24.5 ± 0.8 Gy for single doses and 34.2 ± 0.7 Gy when two fractions were applied. The corresponding RBEs were 1.43 ± 0.08, 1.37 ± 0.12 (one and two fractions, ¹²C plateau) and 1.76 ± 0.05, 2.16 ± 0.11 (one and two fractions, ¹²C Bragg peak), respectively. Hematoxylin and eosin staining revealed necrosis of the white matter in the spinal cord in all symptomatic animals. In summary, from one- and two-fraction photon, ¹²C plateau and Bragg-peak irradiation of the rat spinal cord, we have established RBEs as well as the individual ED₅₀'s. From the latter there is a clear indication of repair processes for fractionated photons and ¹²C plateau ions which are significantly reduced by using Bragg-peak ions. Additional studies are being carried with 6 and 18 fractions to further refine and define the RBE and ED₅₀ values and estimate the α/β ratios.

Magae, J., Hoshi, Y., Furukawa, C., Kawakami, Y. and Ogata, H. Quantitative Analysis of Biological Responses to Ionizing Radiation, Including Dose, Irradiation Time, and Dose Rate. Radiat. Res. 160, 543–548 (2003).

Because biological responses to radiation are complex processes that depend on both irradiation time and total dose, consideration of both dose and dose rate is necessary to predict the risk from long-term irradiations at low dose rates. Here we mathematically and statistically analyzed the quantitative relationships between dose, dose rate and irradiation time using micronucleus formation and inhibition of proliferation of human osteosarcoma cells as indicators of biological response. While the dose–response curves did not change with exposure times of less than 20 h, at a given dose, both biological responses clearly were reduced as exposure time increased to more than 8 days. These responses became dependent on dose rate rather than on total dose when cells were irradiated for 20 to 27 days. Mathematical analysis demonstrates that the relationship between effective dose and dose rate is well described by an exponential function when the logarithm of effective dose is plotted as a function of the logarithm of dose rate. These results suggest that our model, the modified exponential (ME) model, can be applied to predict the risk from exposure to low-dose/low-dose-rate radiation.

Furuno-Fukushi, I., Masumura, K., Furuse, T., Noda, Y., Takahagi, M., Saito, T., Hoki, Y., Suzuki, H., Wynshaw-Boris, A., Nohmi, T. and Tatsumi, K. Effect of Atm Disruption on Spontaneously Arising and Radiation-Induced Deletion Mutations in Mouse Liver. Radiat. Res. 160, 549–558 (2003).

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse “gpt-delta” system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi⁻ (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm ^/ genotype showed a dose-dependent increase in the Spi⁻ mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8 × 10⁻⁶. The livers from Atm^−/− mice yielded a virtually identical dose–response curve for X rays with a background fraction of 2.4 × 10⁻⁶. Structural analyses revealed no significant difference in the proportion of −1 frameshifts and larger deletions between Atm ^/ and Atm^−/− mice, although larger deletions prevailed in X-ray-induced Spi⁻ mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Hahn, P. J., Lai, Z-W., Nevaldine, B., Schiff, N., Fiore, N. C. and Silverstone, A. E. Bcl2-Independent Chromatin Cleavage is a Very Early Event during Induction of Apoptosis in Mouse Thymocytes after Treatment with Either Dexamethasone or Ionizing Radiation. Radiat. Res. 160, 559–567 (2003).

We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 μM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4–6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.

Epperly, M. W., Gretton, J. E., Sikora, C. A., Jefferson, M., Bernarding, M., Nie, S. and Greenberger, J. S. Mitochondrial Localization of Superoxide Dismutase is Required for Decreasing Radiation-Induced Cellular Damage. Radiat. Res. 160, 568–578 (2003).

We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D₀ = 0.89 ± 0.01 and 1.08 ± 0.02 Gy, respectively) compared to parent line 32D cl 3 (D₀ = 1.15 ± 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D₀ = 2.1 ± 0.1 and 1.97 ± 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.

Lenarczyk, M., Ueno, A., Vannais, D. B., Kraemer, S., Kronenberg, A., Roberts, J. C., Tatsumi, K., Hei, T. K. and Waldren, C. A. The “Pro-drug” RibCys Decreases the Mutagenicity of High-LET Radiation in Cultured Mammalian Cells. Radiat. Res. 160, 579–583 (2003).

We are carrying out studies aimed at reducing the mutagenic effects of high-LET ⁵⁶Fe ions and ¹²C ions (⁵⁶Fe ions, 143 keV/μm; ¹²C ions, 100 keV/μm) with certain drugs, including RibCys [2-(R,S)-d-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)-thiazolidine-4(R)-carboxylic acid]. RibCys, formed by condensation of l-cysteine with d-ribose, is designed so that the sulfhydryl amino acid l-cysteine is released intracellularly through nonenzymatic ring opening and hydrolysis leading to increased levels of glutathione (GSH). RibCys (4 or 10 mM), which was present during irradiation and for a few hours after, significantly decreased the yield of CD59⁻ mutants induced by radiation in A_L human–hamster hybrid cells. RibCys did not affect the clonogenic survival of irradiated cells, nor was it mutagenic itself. These results, together with the minimal side effects reported in mice and pigs, indicate that RibCys may be useful, perhaps even when used prophylactically, in reducing the mutation load created by high-LET radiation in astronauts or other exposed individuals.

Jagetia, G. C., Baliga, M. S., Venkatesh, P. and Ulloor, J. N. Influence of Ginger Rhizome (Zingiber officinale Rosc) on Survival, Glutathione and Lipid Peroxidation in Mice after Whole-Body Exposure to Gamma Radiation. Radiat. Res. 160, 584–592 (2003).

The radioprotective effect of the hydroalcoholic extract of ginger rhizome, Zingiber officinale (ZOE), was studied. Mice were given 10 mg/kg ZOE intraperitoneally once daily for five consecutive days before exposure to 6–12 Gy of γ radiation and were monitored daily up to 30 days postirradiation for the development of symptoms of radiation sickness and mortality. Pretreatment of mice with ZOE reduced the severity of radiation sickness and the mortality at all doses. The ZOE treatment protected mice from GI syndrome as well as bone marrow syndrome. The dose reduction factor for ZOE was found to be 1.15. The optimum protective dose of 10 mg/kg ZOE was ¹₅₀ of the LD₅₀ (500 mg/kg). Irradiation of the animals resulted in a dose-dependent elevation in the lipid peroxidation and depletion of GSH on day 31 postirradiation; both effects were lessened by pretreatment with ZOE. ZOE also had a dose-dependent antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Candida albicans.

Mouthon, M-A., Vereycken-Holler, V., Van der Meeren, A. and Gaugler, M-H. Irradiation Increases the Interactions of Platelets with the Endothelium In Vivo: Analysis by Intravital Microscopy. Radiat. Res. 160, 593–599 (2003).

Adhesion of platelets to the endothelium is believed to be a major factor contributing to thrombosis and vascular occlusion after radiotherapy or endovascular irradiation. In the present study, platelet–endothelium interactions were analyzed in vivo by intravital microscopy in mesenteric venules of mice according to three parameters: (1) platelet rolling, (2) platelet adhesion, and (3) the presence of platelet clusters. A 10-Gy total-body irradiation of mice resulted in an increase in the frequency of appearance of these three types of platelet–endothelium interactions in postcapillary venules 6 and 24 h after exposure, whereas only minor alterations were seen in large venules. In addition, the duration of platelet adhesion was increased 24 h after irradiation in both postcapillary and large venules. However, P-selectin was not up-regulated on the platelet membrane and platelet-leukocytes were not seen rolling together, suggesting that changes in platelet–endothelial cell interaction result from endothelial cell activation rather than platelet activation. Our data suggest that irradiation transforms resting endothelial cells to a pro-adhesive surface for platelets, which could ultimately lead to thrombosis.

Gatta, L., Pinto, R., Ubaldi, V., Pace, L., Galloni, P., Lovisolo, G. A., Marino, C. and Pioli, C. Effects of In Vivo Exposure to GSM-Modulated 900 MHz Radiation on Mouse Peripheral Lymphocytes. Radiat. Res. 160, 600–605 (2003).

The aim of this study was to evaluate whether daily whole-body exposure to 900 MHz GSM-modulated radiation could affect spleen lymphocytes. C57BL/6 mice were exposed 2 h/day for 1, 2 or 4 weeks in a TEM cell to an SAR of 1 or 2 W/kg. Untreated and sham-exposed groups were also examined. At the end of the exposure, mice were killed humanely and spleen cells were collected. The number of spleen cells, the percentages of B and T cells, and the distribution of T-cell subpopulations (CD4 and CD8) were not altered by the exposure. T and B cells were also stimulated ex vivo using specific monoclonal antibodies or LPS to induce cell proliferation, cytokine production and expression of activation markers. The results did not show relevant differences in either T or B lymphocytes from mice exposed to an SAR of 1 or 2 W/kg and sham-exposed mice with few exceptions. After 1 week of exposure to 1 or 2 W/kg, an increase in IFN-γ (Ifng) production was observed that was not evident when the exposure was prolonged to 2 or 4 weeks. This suggests that the immune system might have adapted to RF radiation as it does with other stressing agents. All together, our in vivo data indicate that the T- and B-cell compartments were not substantially affected by exposure to RF radiation and that a clinically relevant effect of RF radiation on the immune system is unlikely to occur.

Laakso, J., Ruokonen, I., Lapatto, R. and Kallio, M. Inborn Errors in Metabolism and 4-Boronophenylalanine–Fructose-Based Boron Neutron Capture Therapy. Radiat. Res. 160, 606–609 (2003)

Infusions of boronophenylalanine–fructose complex (BPA-F), at doses up to 900 mg/kg of BPA and 860 mg/kg of fructose, have been used to deliver boron to cancer tissue for boron neutron capture therapy (BNCT). In patients with phenylketonuria (PKU), phenylalanine accumulates, which is harmful in the long run. PKU has been an exclusion criterion for BPA-F-mediated BNCT. Fructose is harmful to individuals with hereditary fructose intolerance (HFI) in amounts currently used in BNCT. The harmful effects are mediated through induction of hypoglycemia and acidosis, which may lead to irreversible organ damage or even death. Consequently, HFI should be added as an exclusion criterion for BNCT if fructose-containing solutions are used in boron carriers. Non-HFI subjects may also develop symptoms, such as gastrointestinal pain, if the fructose infusion rate is high. We therefore recommend monitoring of glucose levels and correcting possible hypoglycemia promptly. Except for some populations with extremely low PKU prevalence, HFI and PKU prevalences are similar, approximately 1 or 2 per 20,000.