In clinical use of heavy-ion beams, we cannot overlook skin damage appearing in the early time. We previously experimented effects of fractionated irradiation on the mouse skin with SOBP carbon-ion beams of 290 Mev / u . As an abrupt increase/decrease of normal tissue damage is caused by changing the number of fractions, we have concluded that, unlike photon therapy, skin damage should be carefully studied when the number of fractions is changed in new clinical trials. It is, however, still unclear whether any specific LETs cause such an abrupt change in skin reaction. Here we irradiated mice feet with mono-peak carbon ions, LET spectrum of which is much narrower than SOBP carbon ions, to obtain dose response of early skin reaction. Single doses and daily fractionation were used. We got the following results. With an increase of LET, repair of skin damage caused by radiation decreased. Analyzing isoeffect doses, α/β ratios of LET13.6 kev/μm , 28.4 kev/μm and the gamma rays were not different between each others. However, irradiation with LET 58 kev/μm carbon beams produced much stronger effect on skin so that Fe-plot, which was successfully brought α/β ratios for lower LET carbon beams, could not straightforwardly produce α/β ratios. Skin RBE was least affected by fractionation for LET lower than 28.4 kev/μm. In case of LET 58 kev/μm, however, RBE value of ~3 for single doses increased to 4~4.5 for fractionated irradiation. This result may account for clinical carbon therapy of lung tumors that requires larger doses than doses predicted by extrapolating from fractionation date.

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  Intestine is severely damaged by local and systemic exposure of high dose (above 10Gy) of ionizing radiation. To find the therapeutic methodology, we examined drugs which contribute to healing of the lethal intestinal damage following radiation using the experimental animal model.
  Since it was difficult to find the regions of lethal damage in the long intestine, survival rate was used as an indicator. Damaging of whole intestine was produced by abdominal exposure of 16.5 or 18.5 Gy of x-ray to anesthetized C3H/He mice. Parenteral nutrition and drugs were concomitantly injected to the mice from Day-1 to 10 after the irradiation. Even though the nutrition was introduced to the mice, the body-weight was decreased till Day-7. Although mice with increase in the weight at Day-8 were survived at least Day-28, others were died within 10 days.
  Co-administration of nutrition with sympatholytic drugs, parasympathomimetic agents or benzodiazepine derivatives decreased the survival rate. In contrast, co-injection with alpha-adrenergic receptor stimulators, parasympatholytic agents and antispasmodics shortened the period of the decrease in the body-weight and increased survival rate. This suggests that the repair of intestine following radiation is suppressed by the relaxation of the smooth muscle of intestine.

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Eleven molars donated by Nagasaki A-bomb survivors and collected by a Nagasaki University working group were subjected to ESR measurement to estimate radiation dose. The samples were selected to satisfy the conditions; molars, > 30mg of enamel is available, donor age > 10 years old at the time of exposure, and the donors are LSS subjects (highly likely to have estimated dose). The doses were estimated by the signal intensity of the samples using dose response curve obtained by irradiation of samples with known doses of 60Co gamma rays (calibration curve method).

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In most countries, radon is the largest natural source of exposure to ionizing radiation for the general population. Numerous case-control studies have been conducted using passive radon (Rn-222) detectors. Recently it has become aware that the reading of passive radon detectors that do not employ thoron (Rn-220) discrimination techniques are affected by thoron. Therefore, we conducted a simulation study to evaluate the possible effect of thoron on radon-related lung cancer risk. The results showed that thoron interference in radon measurement resulted in an approximately 90% downward bias in radon-related lung cancer risk. The standard error of the lung cancer risk estimates tended to be smaller when radon was measured without thoron discrimination than when radon was measured with thoron discrimination, indicating the error in the radon measurement due to thoron interference resulted in an apparent smaller uncertainty in risk estimates.

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A Chinese nuclear explosion disaster was evaluated without visiting radiologically risky local area. The dose prediction method RAPS was applied nuclear tests in Lop Nur. The results indicate dangerous nuclear radiation influences including fatal risk in the wide Uygur area with good agreement at boundary on a report from Kazakhstan. An iso-dose line due to radioactive fallout three megaton order nuclear explosions has been evaluated by RAPS method. The total area of the A-zone which becomes risky beyond acute semi lethality was estimated to be 24,000 square kilometers. The dead population is estimated to be 190,000 by using the average population density of 6.6-8.3 per a square kilometer in those years. The number of risky population who might become leukemia, other cancer and fetus influence in B and C-zones, is estimated at 1,290,000.

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It will not be exaggerating to say that modern people cannot spend a life independently of radiation. In addition to the fields of energy and medicine, radiation plays an important role in many fields such as inspection without break. However, probably because of the difficulty to sense the radiation without a special instrument, people tend to worry about their future health once they learned to have been exposed to radiation except the case of medical exposure with consent. Since mass media is almost a unique information source for general people, we reviewed the newspaper articles on health effects of radiation exposure by atomic bombs and the Chernobyl accident to evaluate how the mass media will influence people in their understanding radiation risk.

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Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine that has a wide variety of bioactivities. TNFα plays a central role in immune system, but over-production of TNFα leads to inflammatory disorders. Recent reports have shown that anti-TNFα agents prevent radiation-induced damage. We used wild-type (TNFα^+/+) and TNFα knockout (TNFα^-/-) BALB/c mice to clarify the role of TNFα in high-dose radiation exposure. We studied the survival rate of TNFα^+/+ and TNFα^-/- mice that were exposed to whole body irradiation. Thirty days after irradiation with 6 Gy, the survival rates were 100% and 54% in TNFα^+/+ and TNFα^-/- mice, respectively. Thus, the survival durations in TNFα^-/- mice were shorter than that in TNFα^+/+ mice. Administration of recombinant TNFα improved the survival rate on day 30 to 100% in irradiated TNFα^-/- mice. The numbers of RBC, Hb levels, and Ht values were significantly lower in TNFα^-/- mice than those in TNFα^+/+ mice on 15 days after irradiation. However, there was no significant difference in other hematological values between both groups. On the other hand, the level of serum iron in TNFα^-/- mice was approximately twofold higher compared with that in TNFα^+/+ mice, but there was no significant difference in other biochemical value such as liver function test between both groups. The histological analysis found no marked difference of injuries including hemorrhage between both mice. Our results suggest that endogenously produced TNFα may play important roles in erythropoiesis of irradiated mice. Further studies are in progress.

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Exposure to high dose radiation causes radiation injury. There are many reports that radiation activates the production of tumor necrosis factor α (TNFα) in various cells. TNFα is also thought to be a pro-inflammaroty cytokine and this factor plays a critical role in the initiation and continuation of inflammation and immunity. The excess production of TNFα leads to damage of organs. However, the roles of endogenously produced TNFα are not fully understood in radiation exposure. Using TNFα knock-out mice (TNFα^-/-), we investigated the role of TNFα in radiation-induced apoptosis of intestine of mice treated with lipopolysaccharide (LPS). Whole body irradiation with 10 Gy induced apoptosis in intestine of TNFα^+/+ and TNFα^-/-, but no difference of the magnitude in apoptosis was observed in both mice. The level of TNFα in serum was not detectable even in TNFα^-/- mice. TNFα and LPS are known as radioprotectors. Pre-treatment with either recombinant TNFα or LPS enhanced radiation-induced apoptosis in TNFα^-/-, although these factors inhibited apoptosis in TNFα^+/+ mice. The level of TNFα in serum was increased following to TNFα challenge, but there was no differnce of the levels between TNFα^+/+ and TNFα^-/- mice. On the other hand, administrations of LPS increased the serum levels of TNFα, IL-1α and IL-1β in TNFα^+/+, but not in TNFα^-/- mice. These results suggest that endogenously produced TNFα is not related to radiation-induced intestinal apoptosis, but may be essential for LPS-induced cytokine productions that can be associated with intestinal apoptosis.

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Risk of radiation and chemicals in humans is estimated by epidemiological studies, animal experiments and in vitro cell culture studies. We have succeeded to examine direct effects of radiation and chemicals on human organs and tissues maintained in improved SCID (severe combined immunodeficient) mice for long period (up to 3 years).
In this study, thyroid tissues from Graves' disease patients were transplanted s.c. to C57BL/6J-scid mice, and effects of nuclear radiations (UTR-KINKI, 0.2 Gy of neutron and 0.2 Gy of γ-rays/hour, weekly one hour exposure) on human thyroid tissues were examined in comparison with those of ¹³⁷Cs γ-rays (1.19 Gy/min, 0.23 mGy/min; biweekly one Gy exposure). This study is also aimed for the ground experiments of cosmic radiations.
Morphology and thyroid hormone secretion: Significant tissue damage and decrease of hormone secretion were observed by high dose rate γ-ray exposure (> 9 Gy) but not by low dose rate exposure. Similar changes were observed by neutron exposure (0.2 Gy x 4 times and more).
Gene mutation: High dose rate exposure to γ-rays (11-59 Gy, up to 2 years) induced significant increases of mutations in p53 and c-kit genes but not in K-ras, β-catenin, RET, bak and BRAF, although these mutations were not induced by low dose rate exposure and in unirradiated controls. Gene murations were not detected by neutron exposure (0.2 Gy x 6 times) during 5-13 months observation period).
Changes in gene expression: Changes in gene expression measured by GeneChip (Affymetrix) increased dose-dependently by 1-3 Gy of γ-ray exposure and by 2-4 times exposures to 0.2 Gy of neutron.
Thus, in human thyroid tissues, apparent dose rate effects were observed by γ-ray exposure, and neutron showed high RBE. (Supported by MEXT Japan and Japan Space Forum)

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We previously screened 506 progeny derived from X-irradiated mouse spermatogonia by two-dimensional DNA electrophoresis and identified 16 mutations in 20 mice (Asakawa et al., Rad. Res., 2004). The NotI fragments involved in the mutational events were cloned from a normal mouse DNA and each mutation was molecularly characterized. Southern blot analyses and quantitative PCR revealed that five of these mutations were deletions larger than 25 kb in length (one mutation detected among 190 mice in the control group: C1, four mutations detected among 306 mice in the exposed group: E1~E4). We have studied the size of each deletion mutation more accurately by using a microarray-CGH approach. A 4 x 44K custom array, purchased from Agilent Technologies, contained 44,000 probes spanning approximately 1-Mb region around the five regions of NotI sites involved in the deletions with mean probe spacing of 0.3 kb. The genomic DNA from the C1 mouse was used as the reference DNA in each experiment. The sizes of deletions were estimated as follows; C1: 61 kb, E1: >655 kb, E2: >690 kb, E3: 30 kb, and E4: 360 kb. The larger deletions, expanding more than several 100 kb, did not involved any genes. On the other hand, relatively small deletions located near the regions where many predictive genes are involved. This implies that a mouse lacking important genes can't be born, or can't grow up. The high-density microarray can screen the copy number changes throughout the genome with high resolution and our study demonstrated that this approach could detect relatively small deletions (several 10 kb).

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Leukemia was the first malignancy to be associated with radiation exposure in the atomic bomb survivors and has the highest relative risk of any type. The risk has decreased with time and the risk for those exposed early in life has decreased more rapidly than for those exposed at older ages. The issue of whether or not an increased risk of leukemia remains 50 or more years after exposure is of interest. The present report examined the temporal pattern of leukemia mortality risk during the period 1950-2003 among about 87,000 members with known DS02 doses in the Life Span Study (LSS) cohort followed by the Radiation Effects Research Foundation. There were 318 leukemia deaths during the follow-up period. The Excess Relative Risk (ERR) over the total period was 4.3 (95% CI; 3.1, 5.8) and the ERR in the period between 1996-2003 was 3.0 (95% CI; 1.1, 6.7) and was statistically significant (p<0.001). This indicates that leukemia risk has not disappeared even today. Most of the leukemia excess occurred in the early years after exposure. However, the excess tended to increase again in the recent period and this excess occurred among those exposed before age 20. Earlier reports observed that the risk in this group had decreased rapidly with time. The largest number of leukemia deaths is due to acute myeloid leukemia (AML), and the patterns seen here largely reflect AML deaths. Although the risk in the early period for those exposed when young was primarily due to acute lymphoid leukemia (ALL), there is a possibility that the recent risk for this group might derive from other types of leukemia, such as AML. We will continue the follow-up and will conduct detailed, type-specific analyses.

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Microcephaly is a malformation associated with in utero exposed atomic bomb survivors and can be induced in mice by fetal exposure to ionizing radiation (IR). The pathogenesis of IR-induced microcephaly, however, has not been fully understood. Our analyses of high-coverage expression profiling (HiCEP) demonstrated that the abnormal spindle-like microcephaly associated gene (ASPM) was down-regulated in irradiated human diploid fibroblasts. ASPM was recently reported as the causative gene for MCPH-5, the most common type of congenital microcephaly in humans. Here we show that the expression of the Aspm gene was significantly reduced by IR in various human and murine cells. Additionally, Aspm was found downregulated in the irradiated fetal mouse brain, particularly in the ventricular zones. A similar suppression was observed in the irradiated neurosphere cultures. This is the first report suggesting that the suppression of Aspm by IR could be the initial molecular target leading to the future microcephaly formation.

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The glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), is an antibiotic isolated from Streptomyces which has previously been described as an anti-tumor drug. The mechanism of teratogenesis and anti-tumor effects following DON exposure is not yet fully clarified. More specifically, compared to more commonly studied teratogenic subjects such as growth retardation, cleft palate, and limb defects, little has been reported on neurocristopathy and cardiovascular anomalies following maternal exposures to DON. In order to collect basic data for clinical application, prevention and safety, the biologically unique effects of DON and gamma-rays exposure need to be evaluated. In this study, Donryu rats in the experimental groups were given a single intraperitoneal DON injection and/or gamma-ray irradiation on day 10 of gestation. We reported on the relationships between full body maternal exposures to DON and subsequently observed embryonic lethality, external defects, and visceral anomalies, especially of cardiovascular nature. We observed a high frequency of craniofacial defects and cardiovascular anomalies, as well as embryonic lethality in the treated groups following day 10 of maternal exposure. These results indicate sensitivity to DON exposure of rat fetus leading to dysfunction and aberrant development of the neural crest, second heart field (SHF) in formation of neurocristopathy-induced cardiovascular anomalies, especially ventricular septal defects, tetralogy of Fallot, right aortic arch, vascular rings, double aortic arch and aortic arch anomalies. These types of cardiovascular anomalies are similar to those found in humans, termed DiGeorge syndrome, suggesting that similar chemicals and gamma-rays may play a role in dysfunction of neural crest, SHF and formation of human neurocristopathy as well as cardiovascular anomalies. It is crucial to consider their effects involving processes of DNA damage, mitochondrial damage, cell death and cell killing, dysfunction of neural crest, SHF and epithelial to mesenchymal transition (EMT) on the formation of these syndromes, as this animal model is expected to contribute in investigating the mechanism of such teratogenesis in human. The present data also suggests the need for further studies to specifically investigate the role played by the neural crest and SHF in the pathogenesis, prevention, clinical application of cardiovascular diseases and lethality.

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Microsatellites are composed of many tandemly repeated short-sequence units. They are known to have high spontaneous mutation rates in the number of repeat units in germ cells. Therefore, we chose microsatellite loci as targets for monitoring A-bomb radiation-induced mutation rates in germ cells. We measured mutation rates at 40 microsatellite loci among 129 offspring (62 and 4 are from radiation-exposed families having one and both exposed parents, respectively, and 63 from the unexposed controls). In the study we screened for mutations at a total of 2789 microsatellite alleles in 70 exposed germ cells (average dose 1.56 Gy) and at 7465 alleles in 188 unexposed germ cells.
For detecting length-modified mutant alleles via gain or loss of the repeat units, we amplified each microsatellite by PCR and analyzed products by capillary electrophoresis. We compared allele sizes in children with those of their parents. When allele sizes of the children were different from those of either of their parents, we scored the alleles as mutated. For the first screening, we analyzed DNA extracted from established lymphoblastoid cells. Because genetic instability at microsatellites in EBV-transformed cell lines was reported, we confirmed putative mutations detected in the first screening using DNA from uncultured cells.
We found 20 and 17 mutations in the children from the exposed and the control families, respectively. Although it is not yet possible to determine parental origins of four mutations, either from the exposed parents or the unexposed parents, the mean mutation rates of exposed and unexposed alleles are 0.39% (7+4/2789) and 0.35% (26/7465), respectively, even if we assume that all of the 4 mutations were derived from the exposed parents. In conclusion, the present results do not provide evidence for an increased mutation rate at microsatellite loci attributable to A-bomb radiation.

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[Objective] Recently, relationship between radiation dose and noncancer diseases such as circulatory disease have been suggested. Since metabolic syndrome (MetS) is an important risk factor for circulatory disease, we examined relationship between radiation dose and prevalence of MetS among Adult Health Study participants in Hiroshima. [Method] A total of 3166 Adult Health Study (AHS) participants, comprised of atomic-bomb survivors and their controls, who underwent health examinations between 1996 and 1997 (1046 men and 2120 women, mean age: 64.8 years and 68.5 years, respectively) were examined in this study. For diagnosis of MetS, both National Cholesterol Education Program (NCEP) criteria and Japanese criteria were used, although definition of abdominal obesity used the abdominal-girth criteria for Asians of more than 80cm for women and more than 90cm for men. [Result] Prevalence of NCEP-MetS was found to be 22.6% and 34.2% for men and women, respectively. In contrast, prevalence of Japanese-MetS was 10.0% and 21.5% for men and women, respectively. No significant relationship between radiation dose and prevalence of NCEP-MetS or Japanese-MetS was found in univariate and multivariate adjusted logistic regression analyses. [Conclusion] In the present study, no statistically significant relationship between radiation dose and prevalence of MetS was detected. However, further analysis examining such differences as risk by age at the time of the bombings will be needed in the future.

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At STS in Kazakhstan, more than 450 nuclear tests were performed between 1949 and 1989. Residents around STS were affected with chronic and repeated long-term exposure to low level mixed (external and internal) radiation. REA has been promoting "Study on Health Effects of Radiation in Residents in and around STS" since 2001, with cooperation of NNC, and CSPRE. Data were collected from Archives, Citizen Registration Office, etc. As of the end of July, 2008, personal data (date of birth, sex, race, etc.) were collected for 117,300 persons, including 46,400 of exposed population. Among this population, number of persons whose residential history between 1948 and 1963 was obtained and thereby whose dose can be calculated, was 18,200. Out of the latter, vital status became clear for 14,800 persons (alive, 7,000; dead, 7,800). Control population was not used in statistical analysis at present, because it has not been long after study of this population had been initiated. Among causes of death in exposed population, circulatory disorders were 42%, followed by neoplasms (21%), mainly of esophagus and stomach. Individual radiation doses were calculated with use of calculating formula advocated by Ministry of Health, Russian Federation. Relationship between radiation dose and ICD-10-coded cause of death was analyzed using data of exposed population. Positive trend between effective dose and mortality from malignant neoplasm and also circulatory disorders was suggested by the statistical analyses. Since period of this study is short, increase of study population and verification of various parameters, confounding factors and biases are needed hereafter. (This study is a part of projects supported by Special Accounts from MEXT, Japan.)

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Background and Purpose: We examined whether exposure to atomic bomb radiation altered cognitive function and/or incidence of dementia among the Adult Health Study cohort, consisting of atomic bomb survivors exposed at 13 years of age or older in the 1945 atomic bombings and their controls. Method: Study subjects were 2286 non-demented subjects, aged 60 years or older at 1992 baseline examination. A two-phase procedure, including cognitive function screening and neurological examination by a neurologist, was applied for identification of dementia cases. To estimate atomic radiation's effects on incidence rate of dementia, we applied Poisson regression analysis, with consideration paid to other potential risk factors. Information regarding history of radiotherapy among study subjects was obtained by hospital survey and questionnaire. Result: A-bomb radiation had no effect on cognitive function among survivors exposed at 13 years of age or older. A total of 206 subjects developed dementia based on DSM IV criteria during the average sex years of follow-up. Incidence per 1,000 person-years was 16.3 in the less than 5 mGy group, 17.0 in the 5-499 mGy group, and 15.2 in the 500 mGy or more group. AD was the predominant type of dementia. Poisson regression analysis showed that the incidence rates of all dementia, or its subtypes, were not affected by radiation exposure from the atomic bombings, even after adjustment was made for potential risk factors. While 68 subjects had histories of radiotherapy before this incidence study, only two dementia cases were diagnosed among them. Conclusion: No association was found between previous radiation exposure and cognitive impairment and/or development of dementia among atomic bomb survivors in this longitudinal study.

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[Objective] Dendritic cells (DCs) play a key role in the immune system. X-irradiated monocytes can differentiate into DCs, although some functions of DCs such as the matrix metalloproteinase-9 (MMP-9) activity are impaired in the DCs from X-irradiated monocytes (J. Radiat. Res., 49, 2008). In that study, we used TNF-α as maturation stimuli. The present study investigated whether the type of maturation stimulus influence DCs derived from X-irradiated monocytes using lipopolysaccharide (LPS) and a cytokine mixture (MIX). [Methods] Monocytes were separated from the buffy coat and exposed to X-rays at 0, 2, 5 and 10 Gy. To prepare immature DCs (iDCs), the irradiated monocytes were cultured in the presence of rhGM-CSF and rhIL-4. To prepare mature DCs, iDCs were stimulated with LPS or MIX (rhTNF-α, IL-1β, IL-6, PGE₂) for 48 hr. The phenotype of the DCs was analyzed by flow cytometry and the MMP-9 activity was assayed by zymography. [Results and Discussion] When LPS was used as the maturation stimuli, the expression level of the co-stimulatory molecule CD80 and DCs' maturation marker CD83 on the DCs from X-irradiated monocytes was lower than that of the DCs from non-irradiated monocytes. However, no decrease was observed in the DCs stimulated by MIX. The MMP-9 activity in the culture supernatants was impaired in the DCs from the X-irradiated monocytes matured with LPS, whereas there was no large difference in those matured with MIX. These results suggest that the influences of X-irradiation on monocytes regarding the maturation of DCs may depend on the type of maturation stimulus.

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²⁴¹Am in the marine environment originated mainly from the decay of ²⁴¹Pu. ²⁴¹Am and Pu isotopes are highly particle-reactive elements and those are therefore removed from the water column by scavenging onto settling particles. Compared with intensive studies of Pu isotopes, distributions and behavior of ²⁴¹Am in the ocean have been poorly investigated. The objectives of this study are to measure the activities of ²⁴¹Am, ^239+240Pu and ²¹⁰Pb in settling particles and surface sediments and to discuss the removal of reactive radionuclides by scavenging on the continental margin of the East China Sea. Settling particle samples were collected at four stations. Two types of sediment traps were used, cylindrical traps and conical time-series traps. Sediment samples were collected with a multiple core sampler. The ²⁴¹Am activities in settling particles ranged from 2.6 to 7.3 mBq/g, indicating that they increased gradually with increasing trap depth. The ²⁴¹Am/^239+240Pu activity ratios were 1.5 at 100-m depth and 2.1 at 600-m depth. The ²⁴¹Am fluxes ranged from 1.5 to 170 mBq/m²/day and increased with depth, with an especially large increase near-bottom. The activities and fluxes of ²⁴¹Am showed large seasonal variations. There was a tendency for ²⁴¹Am activities and ²⁴¹Am/^239+240Pu activity ratios in surface sediments (0-1 cm) to increase almost linearly with depth. This tendency was in good agreement with that of ²¹⁰Pb. All these results can be attributed to enhanced ²⁴¹Am scavenging by particles and removal near the front between the Kuroshio Current and the East China Sea shelf water. It appears that the same removal mechanism occur between ²⁴¹Am and ²¹⁰Pb.

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Excess relative risk of lung cancer among atomic-bomb survivors remains high even more than 60 years after the bombings. To assess effects of radiation on lung carcinogenesis, we first investigated mutations in the p53 (exons 5-8) and EGFR (exons 18, 19 and 21) genes examined association between the mutation status and radiation exposure among 20 atomic-radiation-exposed and 18 non-exposed patients with non-small cell lung cancer. Mutations of the p53 gene were found in 11 of the 20 exposed patients (55%) and in 6 of the 18 non-exposed patients (33%). Next, we separately analyzed p53 mutations in squamous cell carcinoma (Sq) and adenocarcinoma (Ad). Mutations of the p53 gene were found in 5 of 6 exposed Sq patients (83%) and in 2 of 5 non-exposed Sq patients (40%); in 5 of 12 exposed Ad patients (42%) and in 3 of 12 non-exposed Ad patients (25%). Furthermore, median radiation dose among lung cancer patients with p53 gene mutation of GC>TA transversion was higher than that of patients with other types of mutations or without mutation. On the other hand, among 12 radiation-exposed patients of lung adenocarcinoma, 2 had an EGFR mutation, a lower-than-expected frequency. These results suggest the possibility that frequencies of certain gene mutations (e.g. p53) and the types may be associated with radiation exposure in a histological dependent manner, although further analyses with increased number of cases are required.

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Alpha-crystallin, a major protein of mammalian lens, consists of two subunits, alpha A-crystallin and alpha B-crystallin. They interact to form an aggregate and play a prominent role in the maintenance of lens transparency. We evaluated the interaction between these subunits via surface plasmon resonance (SPR) using four combinations of immobilized protein and analyte: 1) AA: alpha A-crystallin was ligand immobilized onto the sensor and alpha A-crystallin was passed over the ligand, 2) AB: ligand - alpha A-crystallin, analyte - alpha B-crystallin, 3) BB: ligand - alpha B-crystallin, analyte- alpha B-crystallin, 4) BA: ligand - alpha B-crystallin, analyte - alpha A-crystallin. The order of rate of dissociation was AA ≈ BA > BB ≈ AB. We also examined the dissociation of gamma irradiated alpha A- and alpha B-crystallins. As radiation dose increased, so did the dissociation rate of all of the crystallins. The order of rate of dissociation of irradiated crystallins was BB > AB ≈ BA > AA. The results indicate that BB is the most susceptible to gamma-irradiation and that alpha B-crystallin forms a more stable aggregate than alpha A-crystallin under normal conditions. However, when alpha B is irradiated the aggregate becomes unstable.

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AIM: To investigate pathologically effects of internal radiation on human body, we determine radioactivity on samples of Nagasaki atomic bomb casualties. We have already investigated methodology of detecting residual radiation on formalin fixed tissue specimens and paraffin-embedded blocks of autopsy cases at acute stage of atomic bomb injury in Nagasaki and Thorotrast patients, who were internally exposed chronic low-dose-rate to alpha-radiation from thorium dioxide deposits following intravascular administration of the radiographic contrast agent. The liver specimen of Thorotrast patient were observed a gamma ray peak of Ac-228 by Whole body counter (gamma ray spectrum, NaI (T1) scintillator) and alpha tracks by autoradiography. However, we couldn't determine the radioactivity on the samples of Nagasaki atomic bomb casualties. In this study, we determined radioactivity on much more tissue specimens of Nagasaki atomic bomb causalities at acute stage injury, Thorotrast patient and non-exposed control subjects. MATERIAL and METHODS: 1)7 cases of Nagasaki atomic bomb casualties at acute stage radiation injury, 2)1 case of Thorotrast patient and 3)non-exposed control subjects; 4 cases from Nagasaki Medical Center and 3 cases from Kyusyu university were determined radioactivity in tissue specimen by autoradiography. RESULTS: A few number of alpha tracks on lung, kidney and bone of Nagasaki atomic bomb casualties at acute stage injury were observed by autoradiography, which had a tendency to be a little much more than in control subjects. It should identify the nuclide and determine residual radiation.

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The ground-based experiments using accelerated heavy ions are of critical importance for studying the biological effects from high-LET ionizing radiation concerning the human activities in space missions. We reported previously that heavy-ion irradiations on E15 generally induced markedly detrimental effects on prenatal gonads, postnatal testicular development and male breeding activity in Wistar rats. To explore the mechanisms underlying radiation-induced gonocyte apoptosis in fetal gonads, which played a vital role in the fate of postnatal testis development, carbon and neon-ion irradiations of cultured fetal rat testes were applied to investigations focused on cellular and molecular events with or without inhibitors of p53, gap junction, or pan-caspase, or nitric oxide specific scavenger. Results showed that, in addition to the clustered distribution, the time course and the percentage of apoptosis on E15 equivalent in vitro appeared similar to that in utero. Carbon-ion irradiations induced increased p53 expression and decreased expressions of p21 and Bcl-2 in a dose dependent manner. Pan-caspase inhibitor effectively inhibited apoptosis occurrence and reduced the extent of clustered distribution, while such effects were not observed with the presence of p53 or gap junction inhibitor, or nitric oxide specific scavenger. These findings indicated that irradiations of cultured fetal testes manifested pathologically similar gonocyte apoptosis to that in utero. The apoptosis was independent on p53, gap junction and nitric oxide. p53 expression was possibly responsible for the response to radiation damage rather than induction of apoptosis. The syncytial organization of gonocytes played a key role in formation of the clustered apoptosis.

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In a preveous study, we describe a novel method of rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain of medaka. Twenty to twenty four hours after the irradiation, living embryos were stained with a vital dye, acridine orange, for 2 h, and whole mount brains were examined under an epifluorescence microscope. The numbers of AO stained rosette shaped nuclear clusters and AO stained single nuclei increased in a dose dependent manner in the optic tectum.In this study, to examine the proliferation change, we carried out an immunohistochemical analysis using an antibody against a marker of proliferation, phospho histone H3.

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This study was designed to present evidence to clarify the relationships between learning ability, neuronal cell adhesion molecule L1 expression and hippocampal structural changes in the rat model received X-irradiation at an embryonic stage. Water maze task indicated that all of the irradiated rats failed to learn the task in the whole training procedure. Their latency to the platform and swimming distance were significant differences from those sham-treated controls. Histological studies showed that the hippocampal ectopias induced by X-rays in the CA1 were involved in the spatial learning impairment, in which they hampered normal processes in learning development and transmission of information. On the other hand, L1 expression in the hippocampus was examined with Western blot analysis. The results indicated a lower content of L1 in the irradiated rats. A decrease in L1 might be one of reasons to cause disorganization of the septohippocampal pathways. These findings suggest some mechanisms of spatial learning impairment can be attributed to the formation of the hippocampal ectopias and redaction of L1 following prenatal exposure to X-irradiation.

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The military use of depleted uranium has raised increasing concern about its toxicity on children. Renal toxicity is the hallmark effect of uranium exposure. In the present study, the dynamics of uranium in kidney and relationship between renal lesion and uranium distribution were examined in new born rats exposed to uranium acetate.
At 1 day after subcutaneous injection of uranium acetate at a dose of 2 mg as uranium/kg, the uranium content in kidney was 2.4% of the dose and the level was maintained up to 15 days. At 3 days after administration, uranium was distributed mainly in the proximal tubules of the outer stripe of the outer medulla, where apoptotic cells were observed. The kidney at 15 days had doubled weight of the value at 1 day. Uranium was still detected selectively in the renewed proximal tubules in the outer stripe of the outer medulla. These results were suggested that the renal damage induced by uranium was site-selective and could persist afterwards when newborn rats were exposed to uranium.

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A concern often occurs for an increase of infant congenital anomalies due to pollution sources such as nuclear power plant. Our purpose is to provide a reasonable interpretation for variation of infant mortality from congenital anomalies by calendar year and areas. Analysis of standard mortality ratio is mainly based on the 1972-2005 Japanese vital statistic data by 47 prefectures. General infant mortality in Japan continues to decrease but shows a strong positive relationship with that from congenital anomalies, especially after 1988-92. Relative high mortality areas for all combined congenital anomalies is not always same for specified one; circulatory system, others including chromosomal abnormalities. Proportion of circulatory system among the deaths of congenital anomalies became less than 50% after 1993-97. Decrease of observed deaths makes it difficult to understand temporal variation of mortality in small areas but potential risk near a pollution facility is very small in normal operation.

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It is well known from radiation surveys and sample measurements just after the bombing that the radioactive fallout from the Hiroshima atomic bomb contaminated the Koi-Takasu area located about 3 km west of the hypocenter. Meanwhile, "black rain" after the bombing was observed in the wider area, including villages 30 km away from the hypocenter, where radioactivity monitoring was not made just after the bombing. In 1970s measurements of ¹³⁷Cs and ⁹⁰Sr were carried out for soil samples taken from the black rain area. Unfortunately the contribution from the Hiroshima bomb could not be found because of large interference from the global fallout due to atmospheric nuclear tests. Recently, we succeeded to detect ²³⁶U in soil samples that was considered to be produced through the ²³⁵U(n,g)²³⁶U reaction in the Hiroshima bomb. We are now extending ²³⁶U measurements of soil samples from the black rain area and developing a method to evaluate the amount of fallout deposition based on the ²³⁶U data. The meteorological model is also investigated to evaluate the area of black rain as well as dosimetry models for external and internal irradiation.

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Early radiation surveys and sample measurements just after the bombing that the radioactive fallout from the Hiroshima atomic bomb has revealed that high radioactive contamination at the Koi-Takasu area located about 3 km west of the hypocenter. However, global fallout due to the nuclear test contaminated radioactivity originated to the atomic bomb. We have analyzed some small pieces obtained from plastered walls which has Black Rain streaks on it.The house located 3.7 km west to the hypocenter and the tainted part of the wall was cut and donated to Hiroshima Peace Museum in May, 2000.We applied gamma-ray spectrometry by a low-background Ge detector to detect Cs-137 and U-235 abundance by ICP-MS.It has shown that U-235/U-238 ratio in the black part of the wall was higher than natural ratio. The atom ratio Cs-137/U-235 was compared to the fission yield ratio. Some discussion of fractionation will be given.

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There was "black rain" after the Hiroshima aton bombing in the wide area that extended outside Hiroshima city more than 30 km to the north-west direction from the hypocentre. Previous attempts failed to detect fission products such as ¹³⁷Cs and ⁹⁰Sr from the Hiroshima bomb in the black rain area because of large contribution from global fallout of nuclear test in the atmosphere. According to unofficial reports, about 51 kg of ²³⁵U was loaded in Hiroshiba bomb, of which 912 g was consumed by the 16-kt explosion and some fraction was converted into ²³⁶U through the ²³⁵U(n,g)²³⁶U reaction. Although most of ²³⁶U were assumed to have been dispersed upto the high altitude in the sky some part could be deposited on the ground with the black rain. Thermal ionization mass spectrometry (TIMS) was applied to detect ²³⁶U from soil samples collected in 1976 from the black rain area around the Hiroshima city. So far, the ²³⁶U/²³⁸U atom ratios of (1.2-8.6)x10^-8 were obtained for 7 soil samples, while ²³⁶U was not detected in the sample taken from the control area. The ²³⁶U measurement using TIMS can provide valuable information about the local fallout contamination by the Hiroshima atom bomb.

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Supplementary UVB radiation inhibited growth, yield and grain development. One of principal causes of such UVB-induced damage is UVB-induced CPD, and increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition. How degree of growth inhibition was caused by UVB in natural sunlight? In this work, effects of solar UVB on growth and yield of rice under outdoor conditions at Miyagi pref. (2006-2008) and Kagoshima pref. (2008) were investigated using UV-resistant Koshihikari and the UV-sensitive chromosomal segment substitution line (SL-229) as experimental plant; chromosomal 10 region, on which CPD photolyase gene located, in SL-229 was homozygous for Kasalath allele, whereas all other chromosomal regions were homozygous for Koshihikari alleles. CPD photolyase activity of Kasalath was lower than that of Koshihikari. The grain size and weight of SL-229 under current outdoor conditions were reduced. These results mean that growth and yield of rice grown under current environmental conditions would be inhibited by UVB in natural sunlight.

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Cyclobutane pyrimidine dimer (CPD) is a major type of UV-induced DNA damage. CPD photolyase is a crucial factor for determining the sensitivity of plant to UVB. We previously reported that the native rice CPD photolyase, which was purified from rice leaves, was phosphorylated, whereas the E. coli-expressed rice CPD photolyase was not (Teranishi et al. Plant Physiol. 2008). Furthermore, the purified native rice CPD photolyase had significantly higher CPD photorepair activity than the E. coli-expressed CPD photolyase. These results suggested that the phosphorylation might affect the CPD photolyase activity. However, the purified native rice CPD photolyase possesses both pterin-like and FAD chromophores, whereas the E. coli-expressed rice CPD photolyase does not have the pterin-like chromophore. Thus, this result raises another possibility that the difference in the activity between the native rice CPD photolyase and E. coli-expressed rice CPD photolyase might be affected by the difference in the chromophore. To investigate the effect of phosphorylation on CPD photolyase activity, we separated phosphorylated and unphosphorylated CPD photolyase from the native rice CPD photolyase, and then we measured the activity of each CPD photolyase. As a result, there was no difference in the activity between phosphorylated and unphosphorylated CPD photolyase.

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We have reported that Tempo, a nitroxide that interacts with mitochondrial ETC complexes and produces ROS, sensitizes p53-defective human leukemic U937 cells to heat-induced cell death (Zhao et al, FRBM, 2006). Here we describe that in U937 cells, increasing hyperthermic intensity and Tempo concentration elicit two distinct modes of synergistic cell death: caspase-dependent apoptosis (CDA) and caspase-independent cell death (CICD).
We treated U937 cells with 5-10 mM Tempo and/or 10-30 min exposure to 44C, and analyzed the cell death mechanisms. The results were as follows. (1) CDA: The 5 mM Tempo/44C-10 min combination induced the synergistic CDA to the similar extent as did the 44C-30 min heating. The common CDA mechanisms involved (i) activation of the initiator caspase-8-tBid signaling, (ii) activation of the initiator caspase-2, and (iii) Bax-induced cytochrome c release, followed by activation of the initiator caspase-9 and effector caspase-3/7. Both treatments also resulted in mitochondrial dysfunction, such as ROS increase, pan-caspase inhibitor zVAD-suppressible membrane-potential loss due to increased mitochondrial Ca²⁺ levels, and decreased ATP synthesis. (2) CICD: Notably, a stronger combination of 10 mM Tempo/44C-30 min switched CDA to the particular CICD without obvious sign of autophagy, leading to irreversible cell proliferation. This CICD was characterized by multiple molecular marks, including (i) Bnip upregulation in mitochondria, related in part to (ii) irreversible mitochondrial dysfunction, (iii) failure in processing conformationally activated, full-length procaspases to active caspases, and (iv) inhibition of Bid cleavage to tBid, despite the heat-induced Bax activation and cytochrome c release.

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Eukaryotic cells have at least two major pathways to repair DNA double-strand break (DSB), i.e., non-homologous end joining (NHEJ) and homologous recombination (HR). Scid cells and hybrid cells were ideal to study the involvement of DSB repair in heat-induced cell killing, because their genetic backgrounds were identical except the pathway of NHEJ. Scid cells and hybrid cells were heated at 44'C for different periods. To clarify the relationship between thermotolerance and heat-induced DSB, the cells were conditioned by preheating at 44'C and then incubated at 37'C for various intervals. We analyzed the DNA double-strand breaks induced by heat treatment in cells using rH2AX foci formation assay. After a heat treatment, spots of phospholyrated histone 2AX (rH2AX), so-called foci, appear in the cell nucleus. H2AX surrounding the DSB point phosphorylated at the serine 139 residue within a few minute after the DSB-evoking stimuli. This assay is known to be quite sensitive and a specific indicator for DSB. Photograph of the cells were taken with a fluorescence microscope. Scid cells was sensitive to a heat treatment. After a heat treatment at 44'C, we observed a linear increase with time in the number of rH2AX foci. The number of rH2AX foci of scid cells was significantly high in comparison with hybrid scid cells. Th number of rH2AX foci was correlated with increasing thermosensitivity of the cells. Furthermore, we found that a preheat treatment led to heat-induced thermotoleranace. It was confirmed that the number of rH2AX foci was reduced in preheated cells later than treated with a challenge heat. These finding suggest that thermosensitivity and thermotolerance of cells might be associated with DNA double-strand break repair.

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The present study intends to calculate the organ doses and analyze the characteristics of the radiation field inside the bodies of a mouse and human. The voxel phantom of mouse had already been developed for an 8-week-old C3H/HeNrs mouse in the previous work. We upgraded this phantom to improve the resolution (voxel size: 0.1 x 0.1 x 0.1 mm³), and add the nine solid organs. The JM phantom is the voxel phantom of a Japanese adult male developed for the analysis of internal exposure from photons and electrons. We have converted the JM phantom to use in the dose calculation on external neutron exposure, and verified it through the calculation of the absorbed dose per unit neutron fluence at each organ for monoenergetic neutrons. The calculation results showed a good agreement with the reference values reported by ICRP. From the comparison between the organ doses of the mouse and human, it was found that the relativistic dose contribution of electron in the human body is greater than that in the mouse body. This is because the neutrons are moderated inside a large receptor such as the human body, and causes the thermal neutron capture reaction that generates gamma ray and consequently electron.

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Although it has been well established that radon exposure to high level radon in mining environment is a risk factor for lung cancer, it was not clear whether radon exposure in homes was a risk or not. Recently it has been revealed by large scale pool analyses of lung cancer case-controls studies that a level of 100 Bq/m³ in door radon does elevate a risk for lung cancer significantly. The levels of in door radon in Japan were estimated below the half level of world mean. However, there is a concern that the levels might elevate along with the increase of energy-saving houses with higher airtightness and lower ventilation rate. In the study, (1) we will measure radon at 3900 homes in Japan in order to obtain the population-weighted mean of in door radon in Japan, and (2) estimate the attributable fraction of radon for lung cancer among smokers and non-smokers in Japan.
A passive radon-thoron discriminative detector (RadoSys) will be set at either living or bed rooms for 6 months. Information will be obtained by questionnaire about possible confounding factors such as house structure, ventilation rate and location that will be used to grade the outdoor radon levels into three categories. In the present report, we will discuss about 820 and 900 measurements done from Oct. 2007 - Feb. 2008 and Mar. 2008 - Aug. 2008. At present, we have 820 measurements covering 28 metropolitan and prefectures. The distribution of indoor radon was compatible with log-normal distribution (Kolmogorov-Smirnov Test, p = 0.355), arithmetic mean and SD were 21.3 Bq/m³ and 21.0, geometric mean and geometric SD were 16.9 Bq/m³ and 1.95, lowest and highest values were 0.3 Bq/m³ and 437.9 Bq/m³, respectively. In door radon levels were statistically elevated if compared with those levels measured by RERF from 1993 to 1996.

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National Institute of Radiological Sciences (NIRS) has supported the assessment of cosmic radiation exposure of aircraft crew on request from domestic airline companies, according to the guideline settled on by the Radiation Council of the Japanese government. Radiation doses at aviation altitude are estimated by calculation using a particle transport model and are to be verified as to the accuracy of the estimated results by on-site measurements at high altitude. Thus, we have measured cosmic radiations at the summit of Mt. Fuji, the highest mountain in Japan (3,776m in altitude), and compared the results with calculations using a PHITS-based analytical model. Selected advanced neutron monitors coupled with exclusive data loggers were set in a building of the weather observatory for measurements from July 12 to August 25, 2008.

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Introduction Naturally occurring polyamines such as spermine and spermidine have been shown to protect DNA against radiation-induced single- and double-strand breaks. Several possible mechanisms for the radioprotective effects of polyamines have been proposed, including (1) direct scavenging of radiation-generated hydroxyl radical, and (2) the induction of DNA compaction or aggregation that would reduce susceptibility to radiation damage. However, most previous studies have been carried out without clearly distinguishing between the compaction of single DNA molecules and the aggregation of multiple DNA molecules. There has been no direct evidence that the conformational change of individual DNA molecules is a crucial factor in radioprotection. This may be due to the lack of a suitable experimental methodology. Further studies are needed to clarify which mechanism, i.e., the induction of compaction or radical scavenging, is the primary factor in the radioprotective effect of polyamines. Recently, we directly observed the conformational changes of single giant DNA molecules larger than 100 kbp in solution using fluorescence microscopy. We found that giant DNA molecules undergo a large discrete transition from an elongated state to a compact state upon the addition of various condensing agents including spermidine. In addition, it became clear that such a highly compacted DNA is unfolded by the addition of salt. Thus, in the present study we applied the above-mentioned experimental conditions to investigate the effect of g-ray irradiation on different DNA conformations, either an elongated coil state (treatment with spermidine in the presence of salt) or a folded compact state (treatment with spermidine in the absence of salt). Results and Discussion We performed single-DNA observation to measure double-strand breaks caused by g-ray irradiation. To analyze the efficiency of the breakage reaction in a semi-quantitative manner, we used T4 DNA, 166 kbp. The efficiency of g-ray-induced breakage for compact DNA in the presence of spermidine (3+) was ca 1/25 of that for elongated DNA. This finding suggests that the primary protective effect of polyamines is due to the compaction of DNA. References Y. Yoshiakwa, T. Mori, N. Magone, K. Hibino, and K. Yoshikawa, Chem. Phys. Lett., 456 (2008) 80-83.

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Although water is a main component of human cells and its interaction with ionizing radiations are of crucial importance, understanding of water radiolysis with heavy ions of energies comparable to those used in actual cancer treatments is not sufficient. Recently, such highly energetic heavy ions are available at the Heavy Ion Medical Accelerator in Chiba (HIMAC) of the National Institute of Radiological Sciences (NIRS), Chiba, Japan. We have carried out measurements of primary yields of main water decomposition products, hydrated electron (e^-_aq), hydroxyl radical (^•OH) and hydrogen peroxide (H₂O₂) with heavy ions from ⁴He²⁺ to ⁵⁶Fe²⁶⁺ of energies up to 28 GeV (corresponding LET varies from 2 to 700 keV/μm) provided from the HIMAC. Note that primary yields are defined as the yields at 100 ns after irradiation, which can be regarded as a time scale when diffusions and intra-track reactions of initial products are almost terminated. Then, such yields are important because they inherently involve information of initial track structure and dynamics of water decomposition products during track expansion. In the present study, not only such measurements but also discussions based on the measured data were conducted by using the two following simulations. One is stochastic simulation called Monte-Carlo method, and the other is deterministic one called Diffusion Kinetic model. From comparison between experimental and simulation results, validities of dose distribution models proposed in earlier works were also examined.

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Purpose: The efficiency of boron neutron capture therapy (BNCT) depends on the sufficient accumulation of boron-10 in cancer cells. Currently, prompt γ-ray analysis (PGA), inductively coupled plasma (ICP) and α-autoradiography facilities are widely used to measure the boron concentration. These methods are effective to determine the boron-10 average concentration in tumor tissues, but they can not demonstrate boron distribution in detail in tumor tissues and cancer cells. Here, we tried to clarify the boron-10 compound distribution using immunofluorescence staining.
Materials and methods: The human lung carcinoma cell line (A549) was cultured at subconfluent density. The boron-10 compound BPA (p-boronophenylalanine) was dissolved (¹⁰B: 1300 ppm) and prepared to study. The A549 cells were incubated with BPA at different ¹⁰B concentrations (1.625, 3.25, 6.5, 13, 26 and 52 ppm) for one hour. They were then processed for immunofluorescence staining with anti-BPA monoclonal antibody and Alexa Fluor labeling antibody. In addition, DAPI (4',6-diamino-2-phenylindole) staining was performed to observe the nucleus in cells. The immunofluorescence staining was observed by a fluorescence microscope and the intensity of BPA staining was measured by Adobe Photoshop Software.
Results: The double staining of BPA and DAPI showed that BPA was accumulated mostly in the surrounding of nucleus of A549 cells. The intensity of BPA staining increased with the increasing concentration of ¹⁰B. Up to 13 ppm ¹⁰B, the intensity of BPA staining increased in a linear dose-dependent manner. In addition, the peak of intensity histogram of 13 ppm ¹⁰B staining shifted to the higher intensity area compared with that of 3.25 ppm ¹⁰B staining.
Conclusions: In this study, the BPA accumulation in the surrounding of nucleus of A549 cells was investigated by an imaging method in vitro. In future, we will prefer to study the BPA distribution in tumor tissues with this method in vivo.

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In order to predict the radon emanation fraction of materials, model calculations have been studied. Most previous studies attempted to express mathematically the behavior of radon atoms and so cannot deal with a complicated model of grains such as soil. In this study, we developed three models of soil and then calculated the radon emanation fraction of soil grains by Monte Carlo simulation. In the three models, soil grains are packed in the simple cubic, body-centered cubic and faced-centered cubic structures. Some assumptions were introduced to simplify the calculation: for example, all grains are just composed of quartz (SiO₂) with the same diameter and the radon emanation results only from alpha recoil by the decay of radium atoms. The radon emanation fraction was calculated as a function of grain size, moisture content and radium distribution because these three factors are important in the radon emanation fraction. As a result, on the model of soil grains packed in the simple cubic structure, the radon emanation fraction greatly increased with increasing the grain size in the range of 10−100 μm and then was saturated when the moisture content was 0%. As the moisture content became higher, the radon emanation fraction increased and then was saturated at smaller grain size. Water in pore enhanced the radon emanation fraction because it plays an important role to stop radon atoms before embedded into adjacent grains. We are currently discussing the radon emanation fractions for the other models. On the other hand, the validity of the present model was evaluated in comparison with some experimental data. Since the model calculations were comparable with the experimental data, the present model would be more practical and available to estimate the radon emanation fraction of soil of the single grain structure.

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We have analyzed chemical compounds in environmental samples such as rock collected around radon hot springs and measured radium activities and radon emanation fractions of these samples. In previous studies, little attention was paid to some minerals of which the rock samples consist. In this study, the constituent minerals were first separated from the rock sampled at Badgastein in Austria and then measured the radium activity and the radon emanation fraction in order to clarify the radioactive characteristics of those minerals. The rock sample is fresh and composed of quartz (SiO₂) and muscovite (KAl₂(AlSi₃)O₁₀(OH)₂). The two minerals were separated from the crushed rock sample (250500 m) using a high-density agent, sodium polytungstate solution (SPT). The radium activities and the radon emanation fractions of the mineral samples were determined as follows. Each sample of several tens of grams was put in a U-8 container, sealed with an epoxy resin adhesive and gamma rays (C1) of ²¹⁴Pb from the sample were measured a high-purity germanium (HPGe) detector. After establishing the radioactive equilibrium among radium and its progeny (30 days), gamma rays (C2) of ²¹⁴Pb from the sample were measured using the HPGe detector and then the radium activity in the sample was determined. The radon emanation fraction is expressed as (C2-C1)/C1. As a result, the separated samples were confirmed by X-ray diffraction to verify the proper separation of the minerals. The radium activity in the rock was 7 Bq/g. And, the radium activity of muscovite (10 Bq/g) was about 10 times higher than that of quartz (1 Bq/g). We similarly intend to discuss the radon emanation fractions of those samples.

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Kyoto University Radiation Biology Center was founded in 1976 as a national cooperative research institute to promote the research of radiation biology. So far, many important researches were carried out as cooperative researches. The committee of cooperative researcher plays an important role for their promotion. The Japan Radiation Research Society contributed to establishment of Radiation Biology Center and has been supporting the management of this Center and the promotion of cooperative research. However, the focus of radiation biology research is recently changing in Japan and around the world. Therefore, we will present the current result of cooperative researches and discuss the role to promote the future research of radiation biology.

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Kyoto University Radiation Biology Center was founded in 1976 as a national cooperative research institute. Department of Radiation System Biology and Administration Division were established in this foundation. Department of Mutagenesis was established in 1978, Department of Late Effect Studies was established and Department of Genome Dynamics was established. So far, Radiaion Biology Center has been promoted the research of radiation biology with cooperative research. The committee of cooperative researcher plays an important role for their promotion.

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Kyoto University Radiation Biology Center possesses "Low Dose and Low Dose-rate Irradiation System", "Cs-137 Gammacell 40 Exactor", "High Dose-rate Orthovoltage X-ray Irradiator" and "Alpha-particle Irradiator". So far, cooperative researches has been carried our to use these machines. Currently, about forty cooperative researches were accepted in each year. Moreover, new kinds of cooperative researches, which use experimental techniques and knowledge of the staffs in Radiation Biology Center, are being carried out and many important results are expected in these researches.

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Radioadaptive response is a biological defense mechanism that is induced by low-dose ionizing irradiation for cellular resistance to the genotoxic effects of subsequent irradiation. We have reported the effect of pre-irradiation with X-rays of below 10 cGy prior to the challenging irradiation with 3 Gy on the induction of chromosome aberrations in quiescent mouse m5S fibroblasts. Similar effects have been reported by many other groups using various biological end-points, such as cell killing, mutation induction, the induction of micronuclei, and single-cell gel electrophoresis. These results suggest that low dose X-rays is effective for the induction of the radioadaptive response. However, the effect of dose-rate on the induction of the radioadaptive response has been still elusive. We examined the radioadaptive response induced by preirradiation with low dose-rate gamma-rays. When cells were irradiated with 0.8 mGy/min, we observed the adaptive effect with preirradiation of up to 16 cGy. These results suggest that dose-rate is an important factor for the induction of adaptive response. Moreover, we found that the radioadaptive response was suppressed by the down-regulation of protein kinase C alpha. Thus, it was suggested that continuous activation of PKC alpha is important for the induction of the radioadaptive response.

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RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, RECQ5/QE, which is Drosophila melanogaster RecQ5, is highly expressed in early embryos, suggesting an important role in DNA metabolism of early embryo. Embryos lacking maternally derived Recq5 contained irregular nuclei in early embryogenesis. The irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and located below surface monolayer of nuclei. By time-lapse microscopy, irregular nucleus did not divide, while all neighboring nuclei proceed through normal mitotic division with synchrony. These data suggested that the irregular nuclei exit from nuclear division cycle. The phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and is rescued by expression of RECQ5/QE. Thus, maternal supply of RECQ5/QE is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and X-ray induced chromosomal aberrations were increased in recq5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in recq5 mutants. Therefore, endogenous genomic damage may exist in Drosophila development, and RECQ5/QE would be involved in maintenance of genomic stability.

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It has been generally accepted that LDRE results from the SLD repair. To study the molecular mechanism of LDRE, we analyzed the knock-out mutants KU70-/-, RAD54-/-, and KU70-/-/RAD54-/- of the chicken B-cell line, DT40. Survival enhancement by LDR irradiation was observed in parent DT40 and RAD54-/- cells but not in NHEJ deficient KU70-/- and KU70-/-/RAD54-/- cells. Under continuous LDR irradiation, dividing NHEJ-deficient cells will be irradiated and killed in G1 phase. In the LDRE, NHEJ pathway was more important than HR pathway. We studied further the effect of low dose-rate irradiation using the deficient KU70-/- and KU70-/-/53BP1 -/- cells since 53BP1 is reported to play a role in new NHEJ repair. KU70-/- cells survived well under 0.5 Gy/day compared to 1.0 Gy/day dose rate irradiation. KU70-/-/53BP1 -/- cells were sensitive than KU70-/- cells under 0.5Gy/day dose rate irradiation. This suggests that 53BP1 dependent NHEJ is independent Ku/DNA-PK NHEJ and also that both NHEJ pathway play a role in the LDRE.

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Reverse genetics by gene disruption is an informative and powerful tool for investigating protein functions in the cells. The chicken B lymphocyte line DT40 is widely used because of the relative ease of gene manipulation including targeted gene disruption. Here we present our two resent research topics as follows: 1) Requirement of the non-homologous end-joining (NHEJ) DNA repair proteins DNA-PKcs and Ku70 required for inducing apoptosis following X-ray irradiation. Using altogether 11 mutant DT40 cell lines lacking various DNA repair related genes, we have examined apoptosis induction, judged by DNA ladder formation, after X-ray irradiation. Most of the cell lines showed similar apoptotic pattern as wild type cells, however, two cell lines, deficient in either DNA-PKcs or Ku70, failed to die by apoptosis. Neither DNA ladder formation nor mitochondrial membrane permeabilization could be detected in these cells. Loss of Artemis or DNA Ligase IV, downstream factors of the NHEJ pathway, did not affect X-ray induced apoptosis. These results suggested that DNA-PKcs and Ku70 themselves rather than the NHEJ pathway have essential roles in mediating apoptotic signals. 2) FancI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway A rare hereditary disorder Fanconi anemia (FA) is clinically characterized by progressive bone marrow failure, and developmental abnormalities. Altogether 13 genes have been implicated in FA, and their products constitute a common pathway in DNA damage signaling termed the "FA pathway". In response to DNA damage, the FA pathway is activated, leading to monoubiquitination and colocalizing foci formation of the key proteins FancD2 and FancI. In DT40 cell system, mutations in S/TQ motifs of FancI largely abrogate monoubiquitination as well as foci formation of both FancD2 and FancI, resulting in loss of DNA repair function. We propose that the multiple phosphorylation of FancI serves as a molecular switch in activation of the FA pathway.

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The tumor suppressor p53 is a transcriptional regulator that controls expression of its target genes involved in cell cycle arrest, apoptosis in response to various kinds of stress including DNA damage. The most famous p53 target gene is the cyclin-dependent kinase inhibitor p21, the principal mediator of p53-induced cell cycle arrest. A switch to an apoptotic responses lead an induction of p53-mediated apoptotic target genes like Bax and Puma. The spindle checkpoint is a guardian of genome instabilities. Mad2 is a key player in the spindle checkpoint mechanism. p31^comet serves as a silencer of Mad2-dependent spindle checkpoint. Here, we report an additional role of p31^comet in control of p53. p31^comet binds to p53 and p31^comet-depleted cells sensitized against DNA damage reagents and undergo p53-dependent apoptosis. Although p53 can induce apoptotic target genes under depletion of p31^comet, p53-mediated p21 induction is diminished. Upon depletion of p31^comet, p53 becomes highly acetylated at Lys120 within the DNA-binding domain of p53, which is catalyzed by Histone acetylase TIP60. Introduction of site-specific mutantion at Lys 120 residue can suppress apoptosis in p31^comet -depleted cells with DNA damage. p31^comet appears to determine the cell fate, death or survival, by controlling p53 through acetylation.

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