As a part of studies on physiological factors that affect radiosensitivity, we examined the in vitro effect of estradiol (E2) on the yield of radiation-induced chromosome aberrations in human peripheral lymphocytes. Lymphocytes were cultured for 3 days in the medium containing E2 at 0-100000 ng/ml. On the second day, they were irradiated by X-rays at 3 Gy, and then 2% phytohemagglutinin and 0.05 μ g/ml colcemid were added to the medium. After further 48 h, mitotic indices and the yields of chromosome aberrations were examined at various E2 concentrations. E2 treatment at concentrations above 1000 ng/ml resulted in dose-related inhibition of mitosis. Repeated experiments showed that the yield of dicentrics plus centric rings in the culture containing E2 at 100 ng/ml was significantly higher than the yields at 0 ng/ml. Similarly, the yield of total chromosome breaks in the culture containing E2 at 100 ng/ml was significantly higher than that at 1 ng/ml. This study provides the direct evidence in human that radiosensitivity may vary in relation to hormonal conditions.

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Radioprotective effects of sodium tungstate (ST) on ⁶⁰Co γ -ray induced decrease in hematocrit value and in survival rate in Wistar strain male rats were examined. A long term administration of ST (less than 150 mg/kg body weight/day) for 60-300 days had no significant effects on body and organs weights and survival days. The LD_50/60 in 20 weeks old rats was 220 mg/kg body weight/day. Daily administration of 38, 75 or 150 mg from 7 days before and after irradiation to 60 days significantly mitigated the decrease in hematocrit values, especially at 23 days after irradiation (P< 0.05). The highest mitigation rate of the decrease in hematocrit value was observed in rats administered at a dose of 38mg ST/day. Simultaneously, a dose of 38 mg ST/day inhibited lethal effect of ⁶⁰Co γ -rays significantly. The dose-reduction factor for survival of 38 mg ST administered rats was 1.14.

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Bacillus subtilis spores were exposed to three types of photons, monochromatic soft X-rays with the energy corresponding to the absorption peak of phosphorus K-shell electron (2,153eV) and with the slightly lower energy (2,147eV), and ⁶⁰Co γ -rays. From the irradiated spores, 233 mutants exhibiting nalidixic acid resistance were isolated, and together with 94 spontaneous mutants, the sequence changes in the 5′ -terminal region of the gyrA gene coding for DNA gyrase subunit A were determined. Among eighteen alleles of the gyrA mutations, eight were single-base substitutions, nine were tandem double-base substitutions, and one was a double substitution skipping a middle base pair. About 6% of the radiation-induced mutations were tandem double-base substitutions, whereas none was observed among the spontaneous ones. Among spontaneous mutations, A: T and G: C pairs were equally subjected to mutations, whereas the substitutions from G: C pairs and those to A: T pairs predominated among those induced with soft X-rays. The peak-energy X-rays were more effective in killing and causing mutations than the low-energy X-rays, however, there seemed no base-change events uniquely attributable to phosphorus K-shell absorption.

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Prediction of radiosensitivity would be valuable for heavy-ion radiotherapy. Premature chromosome condensation (PCC) technique has been a potential predictive assay in photon radiotherapy, but has not been investigated for hepatomas receiving heavy ions. Two human hepatoma cell lines, i.e., HLE and HLF, were irradiated with either 290MeV/u carbon ions or 200kVp X rays. Cell lethality was assayed by colony formation and compared with the unrejoined fraction of chromatin breaks as measured by PCC technique. Carbon ions at linear energy transfer (LET) of 76keV/μ m produced cell death more effectively than those of 13keV/μ m and X rays. For the cell killing, the relative biological effectiveness (RBE) of 13 and 76 keV/μ m carbon ions compared with X rays was 1.10-1.24 and 2.57-2.59, respectively. Mean number of chromosomes in HLE and HLF cells was similar to each other, i.e., 60.48 and 60.28.RBEs for chromatin breaks of 13 and 76keV/μ m carbon ions were 1.30-1.31 and 2.64-2.79, respectively. A strong correlation between unrejoined chromatin breaks and cell killing for human hepatoma cells was observed irrespective of radiation quality. We conclude that PCC provides a potential predictor for the radiosensitivity of individual hepatoma that are treated with photon as well as heavy ion irradiation.

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Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that γ -irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following γ -irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation.

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We measured strand breaks of pBR322 plasmid DNA irradiated with ultrasoft X-rays using monochromatic synchrotron radiation as a light source. Three photon energies, 388, 435 and 573 eV, a value below and above the nitrogen K-edge and above the oxygen K-edge, respectively, were chosen for the irradiation experiments as they have an equivalent photon transmittance of the sample. Irradiated DNA was analyzed by agarose gel electrophoresis and the numbers of single- and double-strand breaks (ssb and dsb) were determined by measuring the band intensity on the gel after ethidium bromide staining. The action cross-sections for the ssb and dsb slightly increased with the photon energy. The ratio between 388 and 573 eV was about 1.5 for both forms of strand breaks. The absorbed energy required for a strand break was about 60 eV for ssb and 1 keV for dsb, less than one fifth of the values obtained previously in the 2 keV region. On the other hand, the absorbed energies per strand break, as well as the ratio of the action cross-section for the ssb to that for the dsb, were constant regardless of the photon energy used. The K-shell photoabsorption on carbon, nitrogen and oxygen atoms in the DNA molecule, followed by an Auger cascade, induced DNA strand breaks with a constant efficiency in terms of the absorbed energy. These results indicate that the strand breaks of the DNA molecule in the solid state are mainly caused by the photo- and Auger-electrons and the efficiency of the strand breaks little depends on the atoms ejecting these secondary electrons.

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To clarify the mechanism of leukemogenesis in atomic bomb survivors, leukemic cells were investigated using fluorescence in situ hybridization (FISH) analysis on the basis of conventional G-banding in patients with a history of radiation exposure and also in de novo patients. Conventional G-banding showed higher incidences (p< 0.005) of structural and numerical abnormalities without any specific types of chromosome aberrations in the group exposed to a dose of more than one Gy, compared to the non-exposed group. FISH analysis revealed significantly higher incidences (P< 0.05) of subclones with monosomy 7 and deletion of the 20q13.2 region, which were not found in conventional cytogenetic analysis in the exposed group (more than one Gy) compared to the non-exposed controls. Furthermore, segmental jumping translocation (SJT) of the c-MYC gene region was observed only in the exposed group. These chromosomal instability suggested that the leukemic cells from the heavily exposed patients contained persistent cellular genetic instability which may strongly influence the development of leukemia in people exposed to radiation.

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The depth profile of ¹⁵²Eu activity induced in a large granite stone pillar by Hiroshima atomic bomb neutrons was calculated by a Monte Carlo N-Particle Transport Code (MCNP). The pillar was on the Motoyasu Bridge, located at a distance of 132m (WSW) from the hypocenter. It was a square column with a horizontal sectional size of 82.5cm× 82.5cm and height of 179cm. Twenty-one cells from the north to south surface at the central height of the column were specified for the calculation and ¹⁵²Eu activities for each cell were calculated. The incident neutron spectrum was assumed to be the angular fluence data of the Dosimetry System 1986 (DS86). The angular dependence of the spectrum was taken into account by dividing the whole solid angle into twenty-six directions. The calculated depth profile of specific activity did not agree with the measured profile. A discrepancy was found in the absolute values at each depth with a mean multiplication factor of 0.58 and also in the shape of the relative profile. The results indicated that a reassessment of the neutron energy spectrum in DS86 is required for correct dose estimation.

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DNA-dependent protein kinase (DNA-PK) is considered a critical enzyme in the repair and/or signal transduction of DNA double-strand breaks. DNA-PK activity has been mostly measured through “ DNA-plus-minus” or “ DNA-pull-down” procedures using synthetic peptide as substrate followed by filter-binding analysis, i.e. liquid scintillation counting of acid-insoluble radioactivity bound to phosphocellulose filter. Considering that non-specific phosphorylation of other cellular proteins in filter-bound acid-insoluble count could interfere with the detection of specific phosphorylation of peptide substrate, we examined the specificity and characteristics of these assay procedures by SDS gel-electrophoresis of the reaction mixture. The electrophoretic pattern showed phosphorylation in wide range of non-specific protein bands other than the specific substrate. The very low DNA-PK activity shown by murine L5178Y or FSA1233 cells was unambiguously detectable as the count in substrate band. Even following DNA-pull-down procedure, which would separate DNA-PK from most of other protein kinases, substantial amount of phosphorylation of other cellular proteins were still contaminated. Thus by selectively counting the particular bands, small amount of specific phosphorylation of peptide substrate was reliably quantified. These results indicated that the DNA-PK activity through filter-binding analysis was, as suspected, contaminated by non-specific phosphorylation of other cellular proteins and also that the gel-electrophoretic analysis would improve detectability of specific phosphorylation by DNA-PK of synthetic peptide substrate and, therefore, would improve the kinase assay in both sensitivity and specificity.

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Wistar rats inhaled ²³⁹PuO₂ particles prepared by the calcination of ²³⁹Pu hydroxide at 1150 and 400° C. Lung retention, fecal and urinary excretion, and translocation of ²³⁹Pu were compared between the two calcination temperatures. The clearance of ²³⁹Pu from the lungs was significantly faster in the rats exposed to ²³⁹PuO₂ calcined at 400° C (low-temperature group) than those exposed to ²³⁹PuO₂ calcined at 1150° C (high-temperature group). Both the fecal excretion of ²³⁹Pu and the ratio of fecal excretion to urinary excretion was greater in the low-temperature group than in high-temperature group. The amounts of ²³⁹Pu translocated from the lungs to the other organs were very small. Even in the liver, which accumulated the largest amount of ²³⁹Pu except for the lungs, only 0.13-0.20% of the initial lung burden was retained 1 year after inhalation. The amount of ²³⁹Pu deposited in the liver was greater in the high-temperature group than in the low-temperature group both at 1 month and 1 year after the inhalation. These findings clearly suggest that the lung retention of ²³⁹Pu in rats is significantly affected by the calcination temperature of²³⁹PuO₂.

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