We investigated whether chronic irradiation at a low dose-rate interferes with the p53-centered signal transduction pathway induced by radiation in human cultured cells and C57BL/6N mice. In in vitro experiments, we found that a challenge with X-ray irradiation immediately after chronic irradiation resulted in lower levels of p53 than those observed after the challenge alone in glioblastoma cells (A-172). In addition, the levels of p53-centered apoptosis and its related proteins after the challenge were strongly correlated with the above-mentioned phenomena in squamous cell carcinoma cells (SAS/neo). In in vivo experiments, the accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen at 12 h after a challenge with X-rays (3.0 Gy). However, we found significant suppression of p53 and Bax accumulation and the induction of apoptosis 12 h after challenge irradiation at 3.0 Gy with a high dose-rate following chronic pre-irradiation (1.5 Gy, 0.001 Gy/min). These findings suggest that chronic pre-irradiation suppressed the p53 function through radiation-induced signaling and/or p53 stability.

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In analyzing the response of quiescent (Q) cells in solid tumors, we have developed a combined method with a micronucleus (MN) assay and the identification of proliferating (P) cells by 5-bromo-2'-deoxyuridine (BrdU) and an anti-BrdU monoclonal antibody. Using this method, the responses of Q tumor cells as well as total tumor (P+Q) cells within murine solid tumors to various DNA-damaging treatments were evaluated. Based on this evaluation, combining with tirapazamine, a well-known bioreductive agent, and/or heat treatment at mild temperatures was thought to be a promising modality for cancer therapy in terms of conventional anticancer treatment-resistant Q cell control. Recently, our method for detecting the Q-cell response using P cell labeling with BrdU and the MN frequency assay was also shown to be applicable to an apoptosis detection assay. Meanwhile, our method for detecting the intratumor Q-cell response was also applicable toward high linear energy transfer radiation, including reactor neutrons. Thus, using our method, a new neutron capture compound that has the potential to be distributed in neutron capture therapy-resistant intratumor Q cell populations is now under development.

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In order to compare the radiotoxicity of alpha- and gamma-irradiations, primary cultures of tracheal epithelial cells from two rat strains, Sprague Dawley (SD) and Wistar Furth/Fisher F344 (WF/Fi) rats, were irradiated with ²⁴¹Am α-particles or ⁶⁰Co γ-rays. The survival ratio for each of the two rat strain cells appeared to be statistically different after high-LET irradiation. WF/Fi rat cells were 1.7-times more radiosensitive than SD rat cells, whereas no difference was observed following low-LET irradiation. A comparison of the cell survival yielded RBEs of 2.8 and 4.5 for SD and WF/Fi rat cells, respectively. As previously observed, with increasing LET of particles, the cell-survival curves approximate an exponential function of the dose. On the contrary, for low-LET, the survival curves showed a marked initial shoulder. This in vitro cellular model, using epithelial cells of the upper airway, provides a suitable system to estimate the mechanism involved in radiosensitivity after high-LET irradiation. The responses to radiation-induced lethal effects within a same type of cell were dependent on the irradiation parameters, but might be modulated by the individual sensitivity under genetic or epigenetic factor controls.

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In order to compare the cell transformation induced by α- and γ-irradiation, primary cultures of tracheal epithelial cells from two rat strains, Sprague Dawley (SD) and Wistar Furth / Fisher F344 (WF/Fi) rats, were irradiated with 241Am α-particles or ⁶⁰Co γ-rays. The relative transformation frequency (RTF) for WF/Fi primary cells was very close to the level of the spontaneous incidence and independent on the two irradiation types used. On the contrary, the RTF for the SD primary cells increased with a decrease of the LET radiation when the relative survival was higher than about 40%. Therefore, the RTF values reached 4-5 for α-particles and 10-12 for γ-rays. The RTF can be related to the intrinsic radiosensitivity of the rat epithelial cells. However, the difference in the radiation-induced RTF for SD or WF/Fi primary cells seems to be due to the development, under genetic control, of the initial lesion to the neoplastic state.

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The radiosensitizing effects of PR-350, a nitroimidazole derivative, were examined concerning the cell killing of human pancreatic cancer cell lines exposed to high doses of gamma-ray irradiation in vitro. The percentages of dead cells were analyzed with a multiwell plate reader to measure the fluorescence intensity of propidium iodide before and after a digitonin treatment. The sensitizing effect of PR-350 on cell killing by high-dose irradiation was confirmed by time-course, dose-dependency, and microscopic observations. In five of seven pancreatic cancer cell lines in which the number of dead cells was determined 5 days after 30 Gy irradiation in the presence of PR-350, the number was significantly increased under hypoxic conditions, but not under aerobic conditions. The selective radiosensitive effect of PR-350 on hypoxic cells was also confirmed by flow cytometry. The results indicate that PR-350 can enhance the killing of pancreatic cancer cells by high-dose irradiation under hypoxia, which supports its clinical radiosensitizing effects when administered during intraoperative irradiation to pancreatic cancer.

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The aim of the present study was to examine the excess relative risk for leukemia mortality and all cancers, except leukemia, among Hiroshima atomic-bomb survivors by applying ABS93D and ABS2000D. Particular attention was given to any difference in the neutron-dose estimates between the two dosimetry systems. The study subjects were 51,532 atomic-bomb survivors registered in a database of the Research Institute for Radiation Biology and Medicine of Hiroshima University (RIRBM). The results obtained by both dosimetry systems are similar: the excess relative risk per Sv for leukemia mortality and all cancers except leukemia is significantly higher compared to the control group. In addition, the difference in the excess relative risks between the two systems is not significant. Therefore, it is indicated that a modification of the neutron-dose estimates would not markedly change the conclusions about the cancer mortality risk.

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It is well known that the yield of DNA recovered form tissues preserved in formalin is inversely proportional to the stored duration. How is the quality? We tested the quality of DNA from archival tissues of atomic-bomb survivors stored in formalin for decades with the parameters of gene amplification efficiency by a polymerase chain reaction. All of the DNA extracted from the tissues preserved in formalin for 30 years amplified the 54-and 61-base pairs of the DNA fragments successfully. The direct sequencing of the PCR products confirmed the accurate amplification of the target sequence. A further trial to amplify the longer sequence of 111 base pairs succeeded in 20% of the samples tested. From these results, we propose a new utility of archival samples for the analysis of single nucleotide sequence polymorphism of genes, no matter how long the samples have been preserved in formalin.

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This study was performed to confirm the radiation-chemical properties of the 2-nitroimidazole derivative doranidazole, (±)-(2RS,3SR)-3-[(2-nitroimdazol-1-yl)-methoxy]butane-1,2,4-triol [CAS 137339-64-1], PR-350, which was synthesized as a hypoxic cell radiosensitizer with low toxicity. Radiation-chemical experiments using doranidazole showed that (1) unlike O₂, it had high reactivity toward not only hydrated electrons (e_aq^-), but also hydroxyl radicals (·OH), (2) the reduced intermediates of doranidasole had no ability to induce immediate strand breaks of colE1 plasmid DNA, (3) doranidazole enhanced radiation-induced DNA strand breaks of colE1 plasmid DNA in the aqueous state, whereas it did not enhance the base alteration, such as 8-oxo-deoxyguanosine, (4) it enhanced the radiation-induced formation of strand breaks with 3'-phosophate and 3'- phosphoglycolate termini, and (5) it was bound to DNA after irradiation. These facts revealed that the majority of radiation-chemical properties of doranidazole, except for the high reactivity toward ·OH, were similar to those of oxygen.

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The types and three-dimensional distribution of neocortical ectopias following prenatal exposure to X-irradiation were studied by a histological examination and computer reconstruction techniques. Pregnant ICR mice were subjected to X-irradiation at a dose of 1.5 Gy on embryonic day 13. The brains from 30-day-old mice were serially sectioned on the frontal plane at 15 μm, stained with HE and observed with a microscope. The image data for the sections were input to a computer, and then reconstructed to three-dimensional brain structures using the Magellan 3.6 program. Sectional images were then drawn on a computer display at 240 μm intervals, and the positions of the different types of neocortical ectopias were marked using color coding. Three types of neocortical ectopias were recognized in the irradiated brains. Neocortical Lay I ectopias were identified as small patches in the caudal occipital cortex, and were located more laterally in the neocortex in caudal sections than in the rostral sections. Periventricular ectopias were located more rostrally than Lay I ectopias, and were found from the most caudal extent of the presumed motor cortex to the most caudal extent of the lateral ventricle. Hippocampal ectopias appeared as continuous linear bands, and were frequently associated with the anterior parts of the periventricular ectopias.

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The reactions between tocopherol monoglucoside (TMG), a water-soluble vitamin-E derivative, with Br₂· ^-, N₃·, (SCN)₂·^-, NO₂·, OH· and various halogenated peroxyl radicals were examined using a pulse radiolysis technique. The results demonstrate that TMG forms a stable phenoxyl radical at pH > 6.8. The thus-formed phenoxyl radical shows pH-dependent decay kinetics and is disproportionated by 2nd order kinetics at pH 2.3. It was observed that the TMG reactivity towards a halogenated peroxyl radical increases with the number of halogen atoms at the carbon atom having a peroxyl group. The reaction between the TMG phenoxyl radical and ascorbic acid was also examined using a pulse radiolysis technique. The results indicate that the TMG phenoxyl radical is repaired by ascorbate. Kinetic studies indicate that TMG may act as an antioxidant to repair free-radical damage to some biologically important compounds. The one-electron reduction potential for TMG was found to be 0.522 V ± 0.06 vs. NHE.

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