Purpose: Alpha-crystallin, a major protein of the ocular lens is found in large water soluble aggregates with an average molecular mass of 500-600 kDa. The aggregates consists of two types of subunits, alpha A- and alpha B crystallin comprising 173 and 175 amino acid residues, respectively. Alpha A and alpha B share 57% sequence homology, and have similar properties and can each act as chaperones. It is unclear why alpha-crystallin consists of heterosubunits of alpha A and alpha B. So we consider that alpha A and alpha B complement each other in protecting against various stresses. In this study we irradiated with gamma-rays to recombinant human alpha A and alpha B and analyzed changes of the structure and function of these proteins.
Methods: Human alpha A and alpha B proteins were expressed in E. coli. The purified those crystallins were subjected to gamma-irradiation (0-2.0 kGy). The irradiated proteins were measured size exclusion chromatography and chaperone activity.
Results: Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A and alpha B aggregates. The activity of irradiated alpha A was retained while that of irradiated alpha B was abolished after irradiation of >0.5 kGy. Our results indicate that alpha A is more stable to gamma irradiation than alpha B.

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Hematopoietic stem cells are defined by their ability to self-renew and to generate the multiple hematopoietic mature cells (multipotency) through a various types of hematopoietic progenitors. Normal hematopoiesis is regulated by a complex network of soluble physiological regulatory factors, stromal cells and the extracellular matrix. In the present study, we investigated whether X-irradiated hematopoietic stem cells can be generated to normal hematopoieis in vitro using mesenchymal stem cells (MSC) prepared from human placental/umbilical cord blood (CB) that have been demonstrated to be effective for supporting of hematopoiesis. The CD34⁺ cells were highly purified from CB, and the adherent MSC were expanded from CB mononuclear cells except CD34⁺ cells. The non-irradiated or 2 Gy irradiated CD34⁺ cells were cultured onto MSC supplemented with a combination of thrombopoietin + interleukine-3 + stem cell factor. An assay for the clonogenic potential of hematopoietic progenitor cells was evaluated using a methylcellulose culture for CFU-GM, BFU-E and CFU-Mix, and a plasma clot culture for CFU-Meg. After co-culture of non-irradiated or X-irradiated CD34⁺ cells with MSC, no significant differences in the total number of cells were observed in the cultures with, or without MSC. In the generated cells harvested from each culture, the significant increase of total CFU-GM numbers was observed in the co-culture of X-irradiated CD34⁺ cells with MSC in comparison to the control. These results suggest that MSC can induce nearly normal hematopoiesis from X-irradiated hematopoietic stem cells.

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A previous study demonstrated that treatment with a combination of recombinant human thrombopoietin (TPO) + interleukin-3 (IL-3) + stem cell factor (SCF) just slightly protected megakaryocytic progenitor cells (CD34⁺ colony-forming unit-megakaryocyte, [CFU-Meg]) from carbon-ion beams. In addition, the combination of TPO + IL-3 effectively stimulated the differentiation of the CD34⁺ CFU-Meg to megakaryocytes and platelets. However, each combination alone was insufficient to allow the CD34⁺ CFU-Meg to recover from heavy ion beam-induced damages. The effects of 2-step culture using the combinations of TPO + IL-3 + SCF and TPO + IL-3 on carbon-ion beam-irradiated CD34⁺ CFU-Meg isolated from the human placental/umbilical cord blood were evaluated to determine a more effective culture system for heavy ion beam-irradiated hematopoietic stem/progenitor cells. The CD34⁺ cells were exposed to carbon-ion beams (LET = 50 KeV/μm) at 2 Gy and then were cultured in IMDM-based medium supplemented with a combination of TPO + IL-3 + SCF for one day or 7 days. The medium was changed to a medium including the combination of TPO + IL-3 and these cultures were continued until analyses. The megakaryocytes and platelets numbers were assessed using a flow cytometer at 14 or 21 days after irradiation. However, the efficacy of each 2-step culture was equal to that of a 1-step culture using TPO + IL-3 + SCF combination alone and the 2-step culture could not protect hematopoietic stem/progenitor cells from high LET-radiation like carbon-ion beams. Therefore, additional cytokines and/or hematopoietic promoting compounds might be required to overcome damage to hematopoietic cells by heavy ion beams.

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We have previously reported that in mice irradiated in utero or soon after birth, the frequency of translocations did not persist when examined at the age of 20 weeks. One might suspect that this observation is due to longer interval between fetal or neonatal irradiation and chromosome test than that in adult exposures for elimination of aberrant cells. To test the possibility, fetal (day 15.5 p.c.) and adult mice were irradiated with 2 Gy of X-rays, and translocation yields were measured in bone marrow cells at 2-10 weeks after fetal irradiation and at 24-120 hours and 5-11weeks after adult irradiation. Thus, post irradiation time in fetuses is almost the same as in adults. Chromosomes 1 (yellow) and 3 (red) were painted with FISH for detection of translocations. Mean frequency of translocations involving the painted chromosomes was 0.7% at 2-10 weeks after fetal exposures. This value was almost the same as that observed in our previous study (0.5%) when mice were examined at 19-22 weeks after fetal exposures. On the other hand, the higher frequency was clearly observed in adults, i.e., about 3.4% at 24-120 hours and 5-11 weeks after irradiation. These results indicated that the lower frequency of translocations after fetal exposure was not affected by the longer post irradiation time, but a majority of aberrations disappeared from bone marrow in 2 weeks after irradiation of fetuses. We speculated that descendants from survived stem cells in fetuses, which were fortunately free from aberrations, diluted the aberration-bearing cells during the subsequent, normal growth period.

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It is well known that prenatal exposure of gamma-rays causes reduction of cell number and injury of neuronal migration in the developing cerebral cortex of mice. In order to provide information on the effects of neutrons on the fetal brain development, we previously determined the relative biological effectiveness (RBE) for 10 MeV neutrons in induction of apoptosis of the cerebral cortex neural cells of fetal mouse. RBE was about 10. Since RBE depends on neutron energy, we then aimed to examine the effect of 2MeV neutrons. Pregnant B6C3F1 mice were exposed to 2MeV neutrons (0.2Gy, 0.5Gy) or 137Cs gamma-rays (0.5Gy, 1.5Gy) on day 13.5 of gestation, and fetal brains were fixed at 1, 2, 4, 8, 12, 24, and 48hrs after exposure. The brains were sectioned at cerebral mid-plane and stained with HE or TUNEL, and the ventricular zone, intermediate zone and cortexanlage zone were analyzed quantitatively. It turned out that apoptotic cells was observed in the intermediate zone during 4 and 24 hrs after irradiation, and that the rate of apoptosis was highest at 12hrs after exposure in either neutron- or gamma-ray-exposure. It was of note that apoptosis was induced earlier by 2MeV neutrons than 10 MeV ones. Induction of p53 and caspase is under investigation.

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For developing human CNS, the period of 8-15 weeks post ovulations showed the most sensitve to radiation. This period, when neural cells proliferate rapidly, correspond to embryonic day 13-13.5 in the mouse and to the developmental stages 28-30 in the medaka embryos. Mammalian embryos develop in the mother`s uterus; hence, their developmental process cannot be examined directly. In contrast, fish embryos, such as those of zebrafish and medaka, develop outside the mother`s bodies and their chorions are transparent. Consequently, all gross abnormalities can be detected throughout the entire period of their development. In a previous study, transient radiation-induced apoptosis in the CNS is observed in all the living irradiated late medaka embryos (st.28-st.30) under a stereomicroscope, and dead cells are distributed mainly in the marginal proliferating regions of the optic tectum. Although radiation-induced brain cell death can be also visualized histologically by the TUNEL, we could succeed a versatile method of more rapidly visualizing and precisely observing radiation-induced apoptosis in the CNS using vital dye, acridine orange (AO), without preparing histological sections. Using this method, we could visualize the entire apoptotic processes from the beginning of nuclear condensation to the end of complete elimination in living irradiated medaka embryos.

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[Purpose] The association between breast cancer and radiation exposure is well known. The recent Life Span Study (LSS) report on solid cancer incidence also suggests that radiation exposure may be associated with uterine corpus cancer among those exposed to atomic bomb radiation when young. However, effects of risk factors other than age were not accounted for in these risk assessment. In this report, we assessed radiation risks by adjusting for factors possibly associated with gynecologic cancer incidence risk. We also examined an interaction between radiation dose and these factors. [Method] Study subjects were 36,116 females who responded to any of three mail surveys conducted for the LSS female cohort (1969, 1978, and 1991), and who had never been diagnosed with gynecologic cancer prior to their responses. Gynecologic cancer incidence data were obtained from the Hiroshima City/Prefecture and Nagasaki Prefecture tumor registries. Hazard ratios were obtained using Cox regression model. [Result] Breast cancer risk increased with increasing radiation dose even after adjusting for age at menarche and child-bearing experience. Interaction between radiation dose and age at the time of bombings (ATB) was observed; the group comprising those of young age ATB showed the tendency for greater risk increase with radiation dose. On the other hand, no significant interaction between radiation dose and other factors was observed. Among post-menopausal breast cancer cases, even with adjustment for age of menopause, significant association between radiation dose and breast cancer incidence risk remained. The association between uterine corpus cancer incidence and radiation dose was significant only for the group comprising those of young age ATB. This association was still significant even after adjustment for child-bearing experience.

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Japanese nuclear power plants (NPPs) located in four main islands from Hokkaido to Kyushu islands. Radiation dose to the public from NPP are too small in normal operation to be of concern for daily life. A relationship between geographical temporal pattern of 1993-97 cancer mortality and nine social-demographic factors including tobacco tax, physician number and financial index have been examined to provide a scientific interpretation of the observations in NPP areas by ecological study. Area variation analysis of standard mortality ratio (SMR) was mainly based on prefecture-level (47 units) in all Japan. Municipality-level analysis was limited to Ibaraki prefecture (85 units as the end of 1997) where the earliest NPP with the 1966 commissioning year was located. Baseline rate of SMR is thought to be weighted-average of each area-specific rate with the corresponding cancer death number. In prefecture-level the death number by area grouping with quintile of the above factors shows relatively equal for physician number and large unbalanced for financial index. Cancer mortality was high in the highest quintile of tobacco tax for lung and interestingly high in the lowest one for lymphoid-hemopoietic tissue although these SMR variations were small. In municipality-level the 5-years period observations limited to Ibaraki prefecture were not sufficient to recognize directly the relationship of the above social-demographic factors with cancer mortality. However, it is useful to demonstrate municipality frequency by these factors for leading to reasonable ecological interpretation.

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In May, 2006, The Radiation Council in Japan drew up a guideline for the control of cosmic radiation exposure of aircraft crew. They have requested domestic airline companies to construct a countermeasure against possible large solar flares. It is still difficult, however, to predict cosmic radiation doses precisely and shortly soon after the occurrence of a big flare. It is necessary to establish a monitoring system to predict exact aviation doses according to real-time observations obtained with sensitive monitoring instruments and to advice airlines what to do with how much priority. Thus, in the present study, we investigate the possibility of setting a new system at the summit of Mt. Fuji, at 3,770m in height above the sea, for the continuos monitoring of cosmic radiation enviroment at high altitude. Experiments were carried out for about 20 days from August to September, 2007. The data were compared to those obtained on ground and Mt. Norikura (about 2,700m).

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Arsenic is ubiquitous in the world as it is widely used as termiticide, wood preservatives and etc. Chronic ingestion of food and water contaminated with arsenic can cause several disorders including skin and lung cancer. To assess a chronic toxicity of low-level arsenic in the environment, we need to discover a sensitive biomarker for detecting a low concentration of arsenic. We applied a new novel sensitive comprehensive gene expression analysis method named HiCEP to normal human lung fibroblasts (HFLIII cells) and searched over 10,940 transcripts which responded to 1 microM sodium arsenite (NaAsO₂). We identified the transcript mostly up-regulated in response to 1 microM NaAsO₂ as heme oxygenase 1 (HMOX1). In order to determine the optimal incubation time required for the maximum expression of HMOX1 induced by NaAsO₂, we measured HMOX1 mRNA expression level in HFLIII cells exposed to 1-10 microM NaAsO₂ for 2, 4, 8, 12, or 24 hours by qPCR. It was acertained that HMOX1 expression reached to the maximum level after 4 hours' treatment with arsenite. As 1 microM NaAsO₂ hardly affect the cell viability, we examined the effect of less than 1 microM NaAsO₂ on the expression of HMOX1. HMOX1 expression reached to the maximum level after 2-4 hours' treatment with 0.3-1 microM NaAsO₂, and was significantly enhanced in HFLIII cells treated with the low concentration of arsenite compared with sham treated cells. These results indicate that HMOX1 is a good biomarker for the detection of a low level of arsenic in the environment. Next, we examined whether or not HMOX1 can be a biomarker for the assessment of the effect of ionizing radiation. X-ray irradiation didn't significantly affect the HMOX1 expression. Taken together, these results demonstrate HiCEP could be a useful method to find biomarkers for the assessment of the effect of toxicants including arsenic in the environment.

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Dolon village located 60 km from the border of the Semipalatinsk test site is known to be heavily contaminated by the radioactive plume originating from the first USSR atomic bomb test on August 29, 1949. External radiation of about 2 Sv was estimated in Dolon, while it was recently reevaluated to be about 0.5 Sv based on new TL measurements and other data. A possible explanation of this difference was that the previous estimates were based on the exposure rate measured just above the center-axis of the radioactive trace, but the location of Dolon village was several kilometers away from there. In October 2005, in order to confirm the position and width of the radioactive plume that contaminated Dolon village, an extensive soil sampling was carried out at 26 locations concentrating along the line perpendicular to the supposed center-axis of the radioactive plume. Radioactivities of ¹³⁷Cs and ^239,240Pu were measured and inventory values in soil (Bq/m²) were plotted as a function of the distance from the supposed center-axis. Peak-like shapes were observed in the spatial distribution both for ¹³⁷Cs and ^239,240Pu, making maxima near the supposed center-axis. By fitting Gaussian function to the obtained data, the position of plume centerline was estimated about 2 km north from Dolon, and the σ value of the plume width was 2 – 2.2 km. The width of the plume was very small considering the distance of 110 km between the ground-zero and Dolon. The peak ¹³⁷Cs value of 15 kBq/m² was evaluated at the centerline of the plume, while the average ¹³⁷Cs deposition of 6 kBq/m² was obtained in the area of Dolon village (1.8 – 2.6 km from the centerline). Meanwhile, 8.5 kBq/m² of ¹³⁷Cs deposition was previously obtained as the average of 49 soil samples taken around Dolon, which is not inconsistent with the current estimate based on the Gaussian spatial distribution.

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It is important to understand the distribution and fate of Pu in lakes due to their radioation threat and high chemical toxicity, since many lakes are the source of drinking-water.Moreover, studies indicated that Pu is useful for establishing deposition chronology of lakes, which has been commonly studied using 210Pb and 137Cs. However, differing from the analysis of 137Cs, samples must be destroyed during the analysis of Pu. Thus, a precise method with small sample size is always preferable for the determination of Pu in lake sediments. We report a simple and accurate Pu analytical method using ICP-SF-MS combined with chromatographic separation and purification for the studies of recent aquatic sedimentation. An anion-exchange resin (AG MP-1M) was used to purify Pu isotopes.The chemical yield was ca. 64 %. For the analysis of IAEA-368, both Pu activity of 31.6 mBq/g IAEA-368, both Pu activity of 31.6 mBq/g and 240Pu/239Pu atom ratio of 0.033 were comparable to other Pu analytical schemes. For SRM 4354, Pu activity of 3.90 mBq/g For SRM 4354, Pu activity of 3.90 mBq/g agrees well with the certified value. However, the mean 240Pu/239Pu atom ratio of the mean 240Pu/239Pu atom ratio of 0.144 +/-0.004 was lower than those reported by other labortories.Considering the fact that those reported values were different from each other, our results suggest thath this material may be isotopically inhomogeneous. The developed analytical method was applied to analyze Pu isotopes in sediment samples of Lake Poyang, East China. 2 sediment samples of Lake Poyang, East China. 240Pu/ samples of Lake Poyang, East China. 240Pu/239Pu atom ratios for these samples ramged from 0.185 to 0.192 with a mean value of 0.187, indicating no plutonium pollution originated from non-global fallout.

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Ukraine is one of the countries most seriously affected by the Chernobyl nuclear power plant accident. The UNSCEAR reported the foodstuffs, which were caused the major intake of radiocesium, are potatoes, meat, mushrooms, and milk, therefore, intake of radionuclides through critical foods were very interesting (or important) in the contaminated areas, if passing over for a long time after the accident. About 50 milk samples were collected from Ukraine and incinerated in a furnace. Radionuclides in milk and milk product samples were analyzed by gamma-spectrometer with Ge-detector. In all milk samples ¹³⁷Cs and ⁴⁰K were detected. The concentrations of 40K in milk in Ukraine were almost the same as reported values in Japanese. The concentrations of ¹³⁷Cs in milk were in the range of 2.0 - 530 Bq dm^-3 and geometric mean value is 64 Bq. The ¹³⁷Cs concentrations of milk in Ukraine were much higher than Japanese one (<0.29 Bq dm^-3). Cesium-134 was also found in some samples having higher ¹³⁷Cs contents. For Ukrainian, an annual effective dose (0.42mSv/y) calculated using the ¹³⁷Cs intake (64 Bq), was ca. 200 times higher than that of Japanese.

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The occurrence of epilation among the atomic bomb survivors in Hiroshima and Nagasaki has been discussed as a symptom for acute radiation syndrome. Epilation was also reported among "early entrance survivors" who were located far away from the hypocenter at the time of the atomic bomb explosion and entered the exposed area later. Considering that the early entrants were exposed to negligibly small dose by the initial neutron and γ rays due to fission reaction of the atomic bomb, the dose on such survivors was supposed to be mainly caused by radiation from neutron-activated materials around the ground. Therefore, in order to ascertain whether or not the epilation among the early entrants was caused by radiation, it is essential to evaluate absorbed dose to skin by the radionuclides produced in soil via activation by the atomic bomb neutrons.
Both &beta and γ rays can contribute to skin dose though only γ rays has been considered in the conventional atomic bomb dosimetry such as T65D, DS86 and DS02. In the case where radionuclides are deposited on skin surface, rather high dose could be delivered to skin tissue by β rays than by γ rays. The present study, therefore, evaluates the absorbed dose in two types of skin exposure, i.e., exposure from radionuclides in the ground soil and exposure from soil contamination on skin surface. Preliminary results are shown for early entrants in Hiroshima.

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241Am is present in the environment as a consequence of nuclear power plant accidents, authorized discharges from reprocessing plants, or atmospheric weapons tests. Compared with the intensive studies of plutonium in the environment, few investigations of 241Am have been performed. From a radiological health point of view, it is important to study the distribution of 241Am and its behavior in the environment because this radionuclide will be a major contributor to the dose to the public, considering the fact that the concentration of 241Am will continuously increase in the environment and to reach its maximum activity in the middle of 21st century. On the other hand, 241Am is useful tracer in understanding biogeochemical processes in the marine environment because of its high particle affinity. We report a rapid and simple SF-ICP-MS analytical method for 241Am in sediment samples. A selective CaF2 co-precipitation procedure followed by an extraction chromatography separation and purification using Eichrom TRU resin was employed to remove the major matrix and pre-concentrate 241Am. Because of the short-life of 241Am, effort was made to improve the sensitivity of SF-ICP-MS using a high efficiency sample introduction system. The achieved detection limit of 0.3 fg/g (0.04 mBq/g) is extremely low, which allowed the determination of 241Am for low-level environmental samples. The developed method was validated by the analysis of ocean sediment reference material (IAEA-368) with satisfactory result, and will be applied to the study of 241Am distribution in marine environment.

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Radioactive cesium in environment is derived from atmospheric deposition which is originated from weapon testing (1945~1960s) and Chernobyl accident(1986) fallouts. It is possible to estimate ¹³⁷Cs transportation, concentration and circulation in forest ecosystem as an atmospheric deposition indicator, because ¹³⁷Cs has long half-life period, 30 years, and is left for a long term in environment. It have been well known that ¹³⁷Cs is kept being accumulated in surface soil, even though it passed more than 20 years from Chernobyl accident.
The soil samples were collected at 4 sites at the southwest foot of Mt. Iwaki, Aomori, Japan, and analyzed for ¹³⁷Cs by gamma-ray multi-channel analyzer with a Ge-Li detector, 20 elements by X-ray fluorescence spectrometry, C and N contents by pyrolysis-gas chromatographic Analysis and loss on ignition of the soil samples. The maximum ¹³⁷Cs concentration in soils was 487Bq/kg. Soil ¹³⁷Cs was detected mostly within surface 10cm layer at most sites. Vertical soil ¹³⁷Cs profile indicates the logarithmic decrease with increase in depth, and is similar to that of loss on ignition and Pb content. No apparent peaks corresponding to both the testing and accident fallouts were found in ¹³⁷Cs soil profile in a beech forest of Mt. Iwaki, such as ones in swamp (Audry et al.,2004). Appling soil mixing model with a self-consistent least-squares method, it is suggested that most chemical concentrations of elements are explained by mixtures of four kinds of soil constituents with individual different chemical compositions. Thus, findings indicate that soil ¹³⁷Cs is contained in organic matter, 25%, and clay, 75%, respectively, and most of ¹³⁷Cs in surface soil is inorganically bounded to clay.

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The Dolon village is located 60 km from the border of the Semipalatinsk Nuclear Test Site (SNTS) in Kazakhstan where more than 450 nuclear tests were performed by the former USSR from 1949 to 1989. Dolon is known to be heavily contaminated by the radioactive plume from the first USSR nuclear test in 1949. To investigate thyroid dose, 131I contamination is important. So far no radioactive iodine measurement has been available around the SNTS because of the short half-life of ¹³¹I (8.02 d). It seemed to be possible, however, that other radioiodine, ¹²⁹I could be measured due to the long half-life of 129I (1.57 x 10⁷ y) by using accelerator mass spectrometry (AMS). Soil core samples around Dolon were taken in October 2005 by the joint university expedition team and 14 samples were measured at Tandem Accelerator of Mutsu Facility, Japan Atomic Energy Agency. The ¹²⁹I/¹²⁷I atom ratio was obtained to be from 3.3 x 10^-9 to 3.3 x 10^-7. These values are similar to the current background level in the environment (10^-9 - 10^-7) in Europe including contributions from global fallout of atmospheric nuclear tests and nuclear industries. The ¹²⁹I atom inventory measured in soil was ranged from 1.2 x 10¹³ to 1.5 x 10¹⁴ atoms m^-2, the average of which (7.3x10¹³) was slightly larger than the background level of (2 – 5) +/- 10¹³. Considering that the global fallout level of atmospheric nuclear tests and nuclear industries around the Semipalatinsk area is two to three times less than those in Europe and Russia, about 80 % of ¹²⁹I in soil samples from Dolon were deposited as local fall from the SNTS. These are the first results of our ¹²⁹I AMS measurement about the radioactive contamination around the SNTS.

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The main introduction routes of ¹³⁷Cs into the Pacific Ocean are worldwide global fallout from atmospheric nuclear weapons testing and close-in fallout from U. S. tests in the northern Marshall Islands. The ¹³⁷Cs activities and inventories in the water columns have been well investigated in the North Pacific Ocean, however, investigations on vertical profiles of ¹³⁷Cs activities and inventories have been limited in the South Pacific Ocean. The objectives of this study are to measure the ¹³⁷Cs activities in water columns collected in the western South Pacific Ocean and to discuss the processes controlling the ¹³⁷Cs inventory. The ¹³⁷Cs activities ranged from 1.4 to 2.3 Bq/m³ over the depth interval 0 - 250 m and decreased exponentially from the subsurface to 1000 m depth. The total ¹³⁷Cs inventories ranged from 850 Bq/m² in the Coral Sea Basin to 1270 Bq/m² in the South Fiji Basin. The ¹³⁷Cs inventories were 1.9 - 4.5 times higher than that of the expected deposition density of atmospheric global fallout at the same latitude. The possible sources of excess ¹³⁷Cs inventories might be attributable to both the inter-hemisphere dispersion of the atmospheric nuclear weapons testing ¹³⁷Cs from the northern stratosphere to the southern one and its subsequent deposition, and water-bearing transport of ¹³⁷Cs from the North Pacific Ocean to the South Pacific.

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Health effects for populations in depleted uranium-polluted areas and uranium mining areas have been reported and concerns of toxic effects on children have been increased. Uranium exhibits nephrotoxicity. The dynamics and distribution of uranium in kidney, however, have not been clearly understood. There is a little difficulty to find a suitable analytical methodology for the measurement of α-particle radionuclides e.g. uranium at trace levels in tissues. To overcome all problems, we have selected synchrotron radiation X-ray fluorescence analysis using high-energy incident X-rays and inductively coupled argon plasma-mass spectrometry for detecting a small amounts of uranium in renal samples. In the present study, time course studies of uranium distribution in kidney and renal damage were examined in new born and young (3-weeks old) rats exposed to uranium acetate.
After subcutaneous injection of uranium acetate at a dose of 2 mg as uranium/kg, uranium levels in kidney of the young rats were higher than those of the new born rats. While TUNEL-positive cells were observed in the proximal tubule of both the rats, the positive areas spread more in the young animals. These results suggest that there is an age-difference in renal damage induced by uranium.

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Risk assessment of technologies and social activities involves subjective judgment as one of its components, which depends on the risk perception of individuals. Medical radiation exposure is one of the main subjects of radiation protection and regulation. We undertook a survey of public perceptions of the social issues and risks related to radiation, particularly the use of medical radiation with children. The actual survey involved personal visit interviews carried out during October 2006, and resulted in 610 valid responses from men and 747 from women, aged 20 years and older. We also asked subjects for private information such as their sex, age, occupation and so on. We then compiled the results for each attribute. The majority identified global warming as highly risky among social issues related to technology, and smoking among health-damaging issues, but not radiation-related items such as natural radiation, artificial radiation, and X-ray/CT examinations. In general, a sexual distinction was observed regarding perceptions of food safety. Forty percent of the public inaccurately believed that the main source of daily exposure was nuclear facilities. For each of the attributes, the majority of the participants indicated that they were reluctant to expose children to radiation for cancer diagnosis or treatment. Mothers of children of elementary school age or younger demonstrated a higher acceptance of X-ray examinations for diagnoses of pneumonia or bone fractures, while women without children were less accepting of such risks. The results suggest that the public tends to rationally judge risk of medical exposure in the cases they are most likely to encounter. We also report the comparison of our results against those from earlier surveys and the analysis of the data from a socio-psychological perspective.

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Breast cancer, with a high average excess relative risk (ERR), is considered to be a highly radiogenic tumor-type among A-bomb survivors. Furthermore, the ERR of the early-onset cases (age ATB<20 and age at diagnosis<35) is estimated to be even higher. We assumed that the risk of early-onset breast cancer is high because those who inherited mutated breast cancer-related genes (heterozygotes) from their parent(s) may have lost the function of the wild-allele following radiation exposure. To test this possibility, we screened archived tissues for Japanese-specific founder mutations of breast cancer-related genes (two in the BRCA1 gene, one in the BRCA2 gene and one in the ATM gene) and of a founder mutation in the CHEK2 gene (European population) that has not been surveyed among Japanese, and investigated whether or not there was an accumulation of heterozygotes for these genes. Either PCR-RFLP or PCR-direct sequencing was used for analysis of founder mutations. Formalin-fixed and paraffin-embedded cancer tissues of the breast and ovary were used. Study subjects (about 550 cases) consisted of four groups (with almost the same number for each group): (I) Life span study (LSS) cohort, (II) non-LSS cohort, with ages at diagnosis 45 years old or younger, (III) LSS cohort, and (IV) non-LSS cohort, with ages at diagnosis between 55 and 69 years old. No heterozygote of the founder mutations was detected in the ATM and CHK2 genes. In the BRCA1 or BRCA2 gene, each founder mutation had one or two heterozygotes, and there were four heterozygotes in total (one in Group I, two in Group II, one in Group III, and none in Group IV). The results did not support the possibility that early-onset cases in A-bomb survivors (Group I) are attributable to founder mutations in the breast cancer-related genes (BRCA1, BRCA2, ATM or CHEK2 gene).

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As many as roughly 5% of men suffer from infertility; broadly defined as the inability to induce a pregnancy or the absence of offspring. Since mutations at a large number of genes (500 or more) can cause male infertility in Drosophila, it is possible that human male infertility is primarily caused by homozygosity for mutations at a fertility-related gene. In the present theoretical study, the effects of paternal exposure to 1 Gy of acute low LET radiation on infertility in the subsequent generation were estimated under the assumptions that (a) 100 to 4,000 genes are involved in human male fertility; (b) homozygosity for mutations at any one of these genes can cause infertility, and (c) the frequency of infertile males due to the homozygosity ranges from 1% to 20% in the population. The results indicate that, although the study endpoint is much more sensitive than that of monogenic hereditary disorders for the detection of mutagenic effects of radiation exposure, the predicted increase in the frequency of infertile men in the F1 generation would still be small. The maximum relative risk is estimated to be 1.013 under the conditions examined, and such a small increase would be undetectable. Overall, these results indicate that the detection of radiation effects in human germ cells would be an extremely difficult task, regardless of whether the investigators look at monogenic hereditary disorders or at diseases which involve multiple genes.

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Sintillation detectors have been widely for radiation detection. To achieve the high-sensitive, key elements for the scintillation detector performance are the photon production rate of the scintillator, the photon collection efficiency, the photo-electron efficiency and the energy resolution. To obtain the high-sensitive, a new detector was developed. The detector consists of plastic-scintillator plates(6*6*1cm3*3layer) and a NaI scintillator(6*6*1cm3*1layer). In this meeting, the performance of the new detector is discussed.

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The method of electron spin resonance (ESR) dosimetry was applied to human tooth enamel to obtain individual absorbed doses of residents of settlements in the vicinity of the Semipalatinsk Nuclear Test Site (SNTS), Kazakhstan. Most of settlements (Dolon, Mostik, Bodene, Cheremushki, etc.) are located near the central axis of radioactive fallout trace from the most contaminating surface nuclear test, which was conducted in 29, August 1949. The distances between investigated settlements and Ground Zero are in the range 70 - 200 km from SNTS. The other settlements located in the radioactive fallout trace as a result of surface nuclear tests in 24, August 1956 (Znamenka), in 12, August 1953 (Sarzhal) and in 7, August 1962 (Kurchatov). Semipalatinsk city was included to investigation as a biggest city, which located close to SNTS. Tooth samples were extracted according to medical indications in a course of ordinary dental treatment. (8 tooth samples were from control settlement Kokpekty, which were not subjected to any radioactive contamination and located 400 km to the Southeast from SNTS). Only molar teeth were used for dose determination, in which effects of solar ultraviolet on the radiation induced ESR signal in enamel are excluded. It was found that the excess doses obtained after subtraction of the contribution of natural background radiation ranged up to about 450 mGy for residents of Dolon, whose tooth enamel was formed before 1949, and do not exceed 100 mGy for younger residents. For residents of Mostik, excess doses do not exceed 100 mGy for all ages. For Bodene settlement, excess doses higher than 100 mGy be obtained for two samples from the residents having enamel formed before 1949. For residents of Sarzhal village, the maximum of excess dose were determined as 138.2 mGy and for Residents of Znamenka maximum excess dose was about 268 mGy and for residents of Kurchatov city excess dose was about 63 mGy.

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High energy radiation to DNA is suspected to proceed from the formation of transient charged radicals of cation and anion within the strand. In contrast to the mechanism in the oxidative process of cation radicals, much less is known about the process of excess electron transfer. It was suggested that pyrimidine radical anions C^•‒ and T^•‒ are most likely to serve as primary carriers for excess electron transfer. Protonation of C^•‒ and T^•‒ interfere with the electron hopping. However, there has been no previous report of direct observation of the protonation process of base in DNA. This report describes pulse radiolysis method with nanosecond time resolution to follow the protonation of radical anion of bases dA, dT, dG, dC and DNA. The anion radicals of deoxynucleotides were formed by the reaction of hydrated electron (e_aq^-). Subsequently, dT anion radical reacts with H+ to for neutral radical dT(H)• within 2.4 X 10¹⁰ M^-1s^-1. In dC, corresponding reaction of H⁺ was found to occur within 50 ns. The transient spectra of oligonucleotide AT and GC anion radicals were different from corresponding base anion radicals.

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 Standard procedures for transcriptome analysis usually requires more than one microgram of total RNA. For the HiCEP analysis, we first needed 1~2μg of polyA RNA (corresponded to 100μg of total RNA). However, actual transcriptome investigation often requires a new protocol which allows us to carry out analysis using less than 10,000 cells (corresponds to 0.1μg of total RNA). Here, therefore, we attempted to develop a new HiCEP way for a small number of cells.
 First we carried out our examination using diluted samples of high quality RNA fraction. We amplified HiCEP templates which are products of HiCEP reaction and have adaptors at either end, by PCR with primers designed in the adaptors. By this amplification, quantity of total RNA required for the analysis decreased to 0.1μg(corresponds to ~10,000cells), allowing us to conduct HiCEP analysis on wide range of applications, diagnosis with blood, small material obtained by biopsy and so on. Subsequently, we optimized the conditions in several reaction steps and succeeded in HiCEP analysis using 0.5 ng total RNA, which corresponds to 50 mammalian cells.
 Next, we used cells instead of the diluted RNA fractions as starting materials, namely we started HiCEP analysis with a small number of cells, which were collected under a microscope. Thus we obtained reproducible result of 10-cells analysis. Currently, we are focusing on single-cell analysis.

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 High Coverage gene expression profiling (HiCEP) is an AFLP-based method for transcriptome analysis, which doesn't require any sequence information beforehand and is thus applicable in all species. HiCEP observes more than 70% of the entire transcripts and its signals can be basically assigned to certain transcript with one-to-one relationship. Furthermore, it can discriminate 1.5-fold differences in gene expression.
 For spreading HiCEP technology through life science field, the analysis should be easy for everyone and high throughput system of it also should be established. For this purpose, we attempted to develop an automatic reaction robot (HiCEPer) which performs the main body of the HiCEP reaction, from 1st strand cDNA synthesis to adapter ligation. HiCEPer basically consists of three modules, for liquid handling, for incubation and for purification of nucleic acid with magnetic beads, and achieves 96reactions simultaneously with 1ng total RNA, which corresponds to 100 mammalian cells. Thus, the development of HiCEPer enabled us to perform massive and sensitive transcriptome analysis in a large number of applications.

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With the development of nuclear reactors and nuclear weapons during World War II the potential for both workers and members of the public to be exposed to ionizing radiation had increased significantly. At about the same time it was also becoming apparent that chronic exposures to low levels of ionizing radiations had the potential to cause carcinogenic and hereditary effects. In response to these developments the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) was formed by the General Assembly in 1955 to report on the sources and effects of ionizing radiation.

The detonation of 60 megaton of hydrogen bombs in the atmosphere from 1952 to 1954 significantly increased global radioactive fallout. The challenge for UNSCEAR was to develop techniques for the assessment of exposures from radioactive contamination and to assess the effects of these exposures on people. The Committee oversaw world wide programs for the assessment of contamination from fallout which was largely based around 90Sr measurements which was thought at the time to be one of the most important pathways for exposure to humans. In its 2000 report the Committee estimates that by 1963 world wide doses had increased five percent above normal background levels due to atmospheric weapons testing. The work of the Committee was instrumental in the adoption of the Partial Test Ban Treaty in 1963.

Over the last fifty years UNSCEAR has continued its work and has reviewed the sources of radiation exposure as well as the effects of ionizing radiations on human and non-human species. The Committee has assessed the radiological impact of the nuclear fuel cycle and other man-made sources, as well as medical radiation exposures and exposures from natural sources of ionizing radiation. Today it is apparent the doses from medical and natural sources are far greater than those from man-made sources.

In 2006 UNSCEAR finalised five annexes on the biological effects of radiation. • Sources-to-effects assessment for radon in homes and workplaces,
• Epidemiological studies of radiation and cancer,
• Epidemiological evaluation of cardiovascular disease and other non-cancer diseases following radiation exposure,
• Effects of ionizing radiation on the immune system and
• Non-targeted and delayed effects of exposure to ionizing radiation.

In summary the Committee expressed the view that the estimation of the health effects of radiation is based on epidemiological and experimental observations where there is a statistically significant dose-related increase in disease incidence. These direct observations of adverse health outcomes implicitly take account of mechanistic elements relating not only to the targeted (direct) effects of irradiation but also to the non-targeted and delayed effects. At low doses, an understanding of the range and nature of cellular and tissue responses is needed to provide insights into the mechanisms by which radiation exposure induces detrimental health effects. UNSCEAR continues to believe that the data on low dose effects in UNSCEAR 2000 provide a suitable foundation for judgements on mechanisms that affect risk estimation.

UNSCEAR is continuing its work and is currently preparing reviews on the consequences of the Chernobyl accident after 20 years, the effect on non-human biota of ionizing radiation and occupational public and medical exposures to ionizing radiation. At the 56th Session in 2008 the committee will approve a program of work for the next few years.

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The UNSCEAR documents are currently shifting more to the re-evaluation or overview, rather than database update, on radiation effects viewed from the rapidly growing biosciences and technology. Two topics presented here are not exceptions. Radiation effects on immune system were extensively discussed in 1977 report, but in terms of the crisis of the immune system. The present document was prepared from the entirely different view as a "tissue response". The document begins with the general outline of the functional differentiation of immune system and their interplay. The radiation effects are considered as a deregulation of stem cells and its recovery to the stable accommodation phase (stimulatory, sensitive, degradative phases inclusive therein). They are rate limiting process and hence constitute the reason for some controversies among experiments in the experimental animals. In contrast, changes seen after long-time stabilization (A-bomb cohort, and Techa river cohort) are relatively consistent and provide important information on the late effects of radiation. Untargeted and delayed effects have long been known; in bacterial system in 1970s and in sea urchin in 1940s, respectively. Recent development of the microbeam irradiation systems regained the interest on the issues in mammalian cells. They include untargeted mutagenesis, bystander effects, genetic instability and delayed mutagenesis. They are particularly important as they are beyond the scope of classical target theory of radiation action and hence have impact on the current estimate of radiation risk. Reviews in literature are consistent with the untargeted and delayed effects. However, some controversies among experimental systems and paucity of assessments in vivo remain the issues for further confirmation and more rigorous tests.

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Radiation and Cancer
Since the publication of the UNSCEAR 2000 Report, more information has become available from epidemiological studies of radiation-exposed populations.
There have been updates to the follow-up of RERF Life Span Study (LSS), both for solid cancer incidence and all cancer mortality. The analysis using the new DS02 indicate that cancer risk might decrease by about 8%, with no appreciable change in the shape of the dose response or in the age-time patterns of excess risks.
New findings have also been published from analyses of fractionated or chronic low-dose exposure, in particular the IARC 15-country nuclear worker study, and analyses of the Techa River and Semipalatinsk datasets. However, there are concerns about bias involved in all above three studies.
Cancer risks have been assessed for 24 different cancer sites in the report. There are cancers for which there is little evidence for an association with radiation (e.g. pancreatic cancer, melanoma of skin, prostate cancer, non-Hodgkin' s lymphoma, Hodgkin' s disease, multiple myeloma), and others where excess risks have only been seen following very high dose exposures (e.g. cancers of the small intestine, rectum, uterus, kidney).
The new results of LSS is again consistent with a linear dose-response for the risk of all solid cancers combined; therefore, as a first approximation, linear extrapolation can be used for estimating solid cancer risks at lower doses.

Radiation and Non-cancer Disease
There is an evidence of increased risk of cardiovascular disease (CVD) associated with high dose radiation to the heart delivered by radiotherapy. However to date, the evidence for an association between fatal CVD and radiation doses in the range of less than 1-2 Gy comes only from the analysis of LSS. Other studies have not provided clear or consistent evidence of a fatal CVD risk. It is the conclusion of the Committee that, given the inconsistent epidemiological data and the lack of a biologically plausible mechanism, the present scientific data are not sufficient to establish a causal relationship between ionizing radiation and CVD at doses less than 1-2 Gy.

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The new UNSCEAR report to be published in 2007 is composed of the main text and five annexes. In Annex E of "Sources-to-effects assessment for radon in workplaces and homes," the latest scientific findings on radon, the most dominant natural source to the public were reviewed and summarized. Findings from case-control studies of residential radon and lung cancer as well as those from cohort studies of underground miners were extensively reviewed in terms of health effects of radon.

Other annexes on "Medical radiation exposures", "Exposures of the public and workers from various sources of radiation", "Exposures from radiation accidents", "Effects of ionizing radiation on non-human biota", and "Health effects due to radiation from the Chernobyl accident", as well as one summary annex on "Sources of radiation and effects of exposure" are under preparation. The UNSCEAR has carried out a global survey to the member states to collect relevant data on radiation sources. The results of the survey are being analyzed and summarized in the annexes. In addition, the annexes on radiation effects on non-human biota and of Chernobyl accidents are deliberately being prepared. These annexes are expected to be completed in 2008 or later.

UNSCEAR reports could not appear without great contributions of the delegations (representatives and their advisors) of the UNSCEAR member states, the secretary of the UNSCEAR, and consultants. In Japan, systematic activity is in effect for reviewing the drafts of the UNSCEAR report, and for collecting and summarizing data on radiation sources. The expert panel of UNSCEAR, consisting of more than 100 experts is responsible for the activity.

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It has been more than 50 years since UNSCEAR was established in 1955 when the world was trembling with the fear of nuclear war. UNSCEAR set its task as assembling scientific data on the effect of radiation on human health and functioned well together with ICRP to evaluate radiation risk and to provide frameworks of radiation protection. However, UNSCEAR is now forced to face a transition from two sides; changes in politics and economy, and changes in the scientific framework. One such example of changes in the scientific framework can be the move to limit the overuse of the LNT based collective dose approach on risk assessment of a large population with trivial level of radiation doses. The collective dose when applied to the whole European population and trivial doses from the Chernobyl accident resulted in a number of causalities which has no meaning except a simple mathematical calculation. Another scientific issue surfaced out recently in UNSCEAR discussion is how to evaluate psychological effect of radiation accident. The most devastating effects were seen on the psychology of the inhabitants in the area contaminated by the Chernobyl accident, but the effects cannot be evaluated by the dose to the people. Thus, the issues handled in UNSCEAR may be expanding from natural science to sociological science. These changes will be discussed in relation to the future of UNSCEAR.

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UNSCEAR has proposed the radiation protection for human health to United Nations from the scientific results. For example, it is very hard to clarify the justice uncertain parts of radiation reports on human being of radiation accidents such as Chernobyl. In our JRRS, we has established special symposiums many times and published the special issue in JRR to seek accurate scientific results. In JRRS, we have studied about the radiation effects and risk estimation from the biological radiation effects of low dose and low dose-rate. On the other hand, we have no enough scientific results about risk estimation of radiation diagnosis. Therefore we have to study the field as soon as possible. On the other hand, our Japanese group has published many excellent papers about the biological effects of high LET radiations to normal tissue and tumors. These fields are focused from the word. In addition, recent progressed molecular biological techniques have contributed to the mechanisms of mutation induction and cancer events by study for cancer related genes and DNA repair. Especially, new approach such as mutagenic and knockout mice also has brought very important information of radiation effects. We are glad to accept the proposal about the projects which UNSCEAR requires. We have to challenge how we have to establish the proposed projects and prepare the grants for them in Japan. Then we have to produce high quality scientific products and arrange to summarize them. Finally I expect Japanese radiation scientists produce many important scientific papers which UNSCEAR cannot ignore.

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Translocations are an indicator of the effects of all kinds of clastogens such as chemicals and metabolic factors as well as radiation. We reported the frequencies of chromosome translocations in the peripheral lymphocytes of people in the normal living circumstance. Average genomic frequencies of translocations in 1,000 cells in 20 residents (61.2 year-old on average) in a large city and in 16 residents (64.4 year-old on average) in a remote village and in 8 children (12.3 year-old on average) in the remote village were 9.6, 8.4, and 3.2, respectively. Their standard deviations were 5.0, 3.1 and 2.0. As it is possible to calculate the dose with the frequency of chromosome aberrations, frequencies of translocations were converted to radiation dose according to the dose response formula of chromosome aberrations, assuming that all the translocations had been induced by radiation. Standard deviations of the calculated doses for non-smokers in a large city, non-smokers in a remote village and children in a remote village were 200 mSv, 124 mSv and 80 mSv, respectively in chronic exposures, and 153 mSv, 104 mSv and 72 mSv, respectively in acute exposures. Statistically it is not possible to distinguish the cohort if the difference is within the standard deviation of the control. Therefore, our findings suggest that it may not be possible to detect any effects of radiation to be caused in the human body at least up to124 mSv in adult and up to 80 mSv in child in chronic exposures, and up to 104 mSv in adult and up to 72 mSv in child in acute exposures.

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The sensitivity of the mouse gastrula embryonic stage for radiation induced mutations was studied using pUR288 transgenic reporter mice. 7.5 days old gastrula stage embryos were irradiated with 1Gy of X-rays followed by mutation analysis of liver, bone marrow, testis, kidney, brain and intestine 6 weeks later in surviving offspring. In all organs, increases in mutation frequencies were observed with in the irradiated animals a clear evidence for clonal expansion of mutation carrying cells. On a per Gy basis the gastrula stage showed with respect to the pUR288 system the highest mutation rate so far recorded in the literature suggesting a higher radiosensitivity of this phase of development compared to later embryonic and adult stages.

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It is well established that high dose exposure will result in severe lesions on cellular structure and physiological function, late carcinogenesis and even lethality, while low dose exposure might lead to adaptive response, bystander effect and hyper-radiosensitivity. In order to explore the molecular bases of these diversity effects induced by ionizing radiation, we have investigated the global changes of transcriptional profiles induced by different doses of gamma-rays. AHH-1 human lymphoblastoid cells were irradiated with 0, 0.05Gy, 0.2Gy 0.5Gy, 2Gy, 10Gy of gamma-ray respectively. After 4h of post-irradiation culture, the samples were subjected to microarray assay as compared with the sample from un-irradiated control cells. Our results demonstrate the following scenarios of genes expression alterations: 1) Dose specific, i.e. some genes’ expression change only occurred under a certain dose irradiation 2) Dose-dependent effect manner, the extent of down- or up-regulation in some genes increased along with the augment of doses, e.g. Connexin 43, IER5, XPC, tumor protein p53 inducible protein 3 gene, EI24, etc; 3) There were about 250 genes with expression changes in 10Gy irradiated cells, the largest amount among all the investigated doses, and most of these changed genes (95%) were depressed. Next is 0.5Gy irradiation with about 200 genes with changed expression (nearly half and half for the up- and down-regulation) and then 2Gy; 4) a few genes were found up-regulated by 0.05 Gy, e.g. GPR124, NOL6, LYK5, MAPK8IP2, ANXA13, connexin 43, GRIA3. These observations provide the further information for elucidating the molecular mechanisms of the diverse biological effects of ionizing radiation. * This work was supported by grants of Chinese National High Technology "863" Programs #2004AA221160 and Chinese National Natural Science Foundation #30270423.

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Radiation risk due to low dose-rate radiation has not been well established. It is generally considered that the animal study is a promising approach to this issue if it is combined with the study on mechanisms of radiation effects to extrapolate the animal data to human. Continuous low dose-rate irradiation of 4000 SPF mice for 400-days was carried out at IES, Japan, and it was reported that the life spans of mice irradiated at the dose-rate of 16 μGy/min were significantly shortened, but not at the dose-rate of 40 nGy/min. A significant life span-shortening in female mice irradiated at 800 nGy/min was observed. The observed life spans-shortening was due to early death from a variety of neoplasms and not from increased incidence of specific neoplasms. In order to investigate the molecular background for the life spans-shortening caused by low dose-rate irradiation, we examined the gene expression profile by using a cDNA microarray. By examining the de-regulated genes, it was found that activity of the mitochondrial respiration was elevated after irradiation at 650 nGy/min and 13 μGy/min in kidney. The resulting oxidative stress was thought to be one of the factors that cause early incidence of neoplasms. However it should be noted that alteration in the gene expression profile is different depending on the tissue. In testis, it was observed that genes related to the gene ontology categories of mitotic cell cycle, DNA replication, DNA repair, and response to DNA damage stimulus were down-regulated after 650 nGy/min and 13 μGy/min, and that genes related to heat-shock responses were up-regulated. It seemed as if the cells in testis were preparing for emergency by shutting down the general metabolisms as well as DNA repair functions. The molecular background for early incidence of neoplasms after low dose-rate irradiation may be different depending on target organs.

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In the present study, to investigate the role of mitochondria in the events of radiation induced bystander effects (RIBE), we used either mtDNA-depleted AL or normal AL cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments. Our results indicated that mtDNA-depleted cells or normal AL cells treated with the inhibitors for mitochondrial respiratory chain function had an attenuated gamma-H2AX induction in receptor cells. Moreover, it was found that treatment of normal AL donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (Ruthenium Red), gamma-H2AX induction in receptor cells was significantly decreased and that radiation could stimulate cellular NO and O2.- production in irradiated normal AL cells, but not in mtDNA-depleted AL cells. These observations, together with the findings that Ruthenium Red treatment significantly reduced the NO and O2.- levels in irradiated normal AL cells, suggest that mitochondria play a functional role in RIBE and calcium-dependent mitochondrial NOS might play an essential role in the process.

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Radiation induced bystander effect (RIBE) increases the probability of cellular response and therefore has important implications for cancer risk assessment following low dose irradiation and also for the likelihood of secondary cancers after radiotherapy. With a series of experiments of treating cells with NO inhibitor, TGF-beta1, or anti- TGF-beta1, the present study found that, when a fraction of glioma cells T98G were individually irradiated with a very low dose of helium ions, as a downstream product of irradiation-induced nitric oxide (NO), TGF-beta1 was released to the conditioned medium and caused further DNA damage in the bystander cells of either T98G or primary fibroblasts AG01522 co-cultured with irradiated T98G cells by inducing NO and ROS in the nonirradiated T98G and AG01522 cells, respectively. In addition, as an early response to bystander signal factors, calcium flux could be quickly triggered in the nonirradiated cells after receiving the conditioned medium from the glioma cells irradiated with a very low dose of helium ions. This calcium flux response could further induce micronucleus formation in the bystander cells through a pathway related to NO and ROS. In summary, NO, ROS, TGF-beta1 and calcium flux made up of a network of the bystander signaling factors.

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This report will be devoted to computational studies on DNA conductivity - the main message is that acoustic phonons can dramatically increase DNA conductivity. Such a theoretical result can explain Ohmic dependencies of electric currents through DNAs on applied voltage, just as it is measured in water solutions.

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Radiation-induced genomic instability (RIGI) encompasses a variety of effects such as delayed reproductive death, which occurs in the progeny of irradiated cells multiple generations after the initial insult. Whereas the biological effectiveness in irradiated cells has been shown to vary with the LET of the radiation, the LET dependence of the manifestation of RIGI is incompletely characterized. To address this, we have investigated the delayed effects arising in the progeny of normal human diploid fibroblasts surviving exposure to low-LET gamma-rays (0.2 keV/μm) or high-LET heavy ions (16.2-1610 keV/μm). First, we examined delayed loss of clonogenicity as an endpoint of delayed reproductive death, and found that carbon ions (18.3 MeV/amu, 108 keV/μm) were most effective at reducing the clonogenic survival both in primary and secondary colonies. Second, to gain insight into potential cellular mechanisms underpinning the dose- and LET-dependent delayed loss of clonogenicity, morphological changes induced in primary colonies were assessed. It was found that while the yield of differentiated cells having reduced capacity to further proliferate was increased in a dose- and LET-dependent fashion, the incidence of giant or multinucleated cells was much less frequent. Collectively, our results suggest that accelerated differentiation may be a major defensive response in the descendants of irradiated fibroblasts to minimize further expansion of aberrant cells, and may account for LET-dependent delayed loss of clonogenicity,

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Cellular senescence is generally defined as the physiological program of irreversible growth arrest, which can be caused either by telomere shortening (replicative senescence) or by telomere-independent signals (stress-induced premature senescence). In this study, we examined whether irradiation with carbon ion beam induces senescence of a human glioma-derived cell line, NP-2. The development of morphological phenotypes consistent with cellular senescence, that is, enlarged and flattened appearance was observed in irradiated NP-2 cells. This senescent nature was supported by positive staining for senescence-associated β-galactosidase (SA-β-Gal) activity, accumulation of lipofuscin, and increased lysosomal mass. Double labeling for 5-bromodeoxyuridine and SA-β-Gal indicated that most SA-β-Gal-positive NP-2 cells did not synthesize DNA. The mean telomere length of unirradiated NP-2 cells was approximately 4.5 kbp and its length did not significantly vary in SA-β-Gal-positive NP-2 cells. Since the status for p53 in NP-2 cells was determined as a mutant type, p53-independent growth arrest mechanism might be involved in induction of senescence-like phenotype in NP-2 cells. We conclude that carbon ion beam irradiation can induce a senescence-like phenotype associated with terminal growth arrest in human glioma-derived cells. Elucidation of gene(s) and regulatory mechanism(s) of cellular senescence in response to carbon ion beam irradiation should contribute for designing new therapeutic approaches to improve the efficacy for cancer radiation therapy.

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Muscular dystrophy is a heterogeneous group of hereditary diseases characterized by progressive muscle weakness. In many type of muscular dystrophy, microinjury of the plasma membrane is thought to result in muscle degeneration. However, no model system for inducing microinjury of the muscle plasma membrane and observing its resultant turnover has yet to be reported. To determine whether the muscle cell plasma membrane suffers microinjury and undergoes resultant turnover, we carried out target irradiation of isolated skeletal muscle fibers with heavy ion microbeams.
The effects of heavy ion microbeams on muscle fibers isolated from wild type mouse skeletal muscles were examined by electron microscopy. The plasma membranes of heavy ion beam-irradiated areas of muscle fibers showed irregular protrusions and invaginations. In the cytoplasm, an irregular distribution of microfilaments was found near the plasma membrane.Sarcoplasmic reticula in the irradiated regions showed a distended appearance with flocculent material within the lumen.Many autophagic vacuoles could be seen at 7 min after irradiation. At 22 min, the vacuoles became more prominent and showed more variety.We also examined the ultrastructural changes of the muscle fibers of SJL mouse, which is a model for human dysferlinopathy (limb-girdle muscular dystrophy type 2B and Miyoshi muscular dystrophy),after irradiation. In addition to the structural changes similar to the wild type, several regions which were thought to be formerly constructed by myofibril, were occupied by the autophagic vacuoles and unidentified membranes. These observations suggest that heavy ion beam irradiation causes disruption of the cellular architecture and the autophagy is involved in removal of this disruption.

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Purpose: Though the usefulness of carbon ion beam therapy has been reported recently, little has been known about effects of carbon ion beam on the normal brain tissue, especially at the developing stage. Hence, in this study, we investigated the effects of carbon ion beam on the early postnatal cerebellum using a modified organotypic slice culture system.
Methods: A cerebellum was dissected from 10-day-old Wistar rats. A vermis of the cerebellum was cut parasagittally into ∼600 μm-thick slices. The slices were treated with ¹²C ions (18.3 MeV/amu, 108 keV/µm, 0∼20 Gy) at JAEA, Takasaki. After irradiation, the slices were transferred into the incubation chamber and fixed at different times. Then, hematoxylin-eosin (HE) staining, in situ end labeling (TUNEL) methods, and immunostaining were performed.
Results: In the irradiated slices, the HE staining showed that the granule cells (GC) of the external granule layer (EGL) spread over the molecular layer and the zone of the EGL was extended. The GC in the irradiated slices at 5 days after irradiation still existed in the EGL, whereas almost all of the GC in the non-irradiated slices migrated toward internal granule layer. TUNEL methods showed that most of the GC in the EGL were densely stained at 24 h after irradiation. In addition, the processes of Bergmann glia retracted in the irradiated slices. These changes were similar to that occurred by x-irradiation.
Conclusions: The abnormality of EGL caused by carbon ion beam was as a result of the loss of migration ability of the GC.

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Purpose/Objective(s)
The radiosensitivity on neurons is believed to be difficult to be evaluated due to the complexity for culturing them alone. The growth cone collapse (GCC) assay has been reported as a useful means of quantifying the effects of various factors on cultured explants of nervous tissue. Therefore, we used the GCC assay to determine the radiosensitivity of neurons by estimating RBE of carbon-beams to X-ray on the cell neurons.
Materials/Methods
Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-16 (mature) and day-8 (immature) chick embryos and cultured for 20 h. Thereafter, neurons were exposed to graded doses of X-rays, or high-LET 12C ions (18.3 MeV/amu, 108 keV/μm). Morphological, time- and dose-dependence changes of the neurons were examined quantitatively by GCC assay. Apoptosis induction was examined using TUNEL assay.
Results
Carbon-beams induced GCC and neurite destruction in a time and dose–dependent manner. Day-8 neurons were more radiosensitive than day-16 neurons (p=0.01). At 12 h post-irradiation, 20 Gy carbon-beams and 30 Gy X-ray induced about 65% and 25% apoptosis, respectively. The simple regression analysis revealed that the carbon-beams RBE at day-8 DRG and SYMP using the GCC data were 4.6 and 4.2, respectively. Whilst, at day-16 DRG and SYMP were 3.4 and 3.3, respectively. However, the RBE at day-8 DRG and SYMP for apoptosis induction was 4.1 and 3.7, respectively, whereas that at day-16 DRG and SYMP was 4.2 and 3.7 respectively.
Conclusions
The carbon-beams were 3.3-4.6-fold more effective than X-rays for GCC and apoptosis induction in both day-16 and day-8 neurons. Growth cone collapse assay is potentially beneficial in assessing the effect of irradiation on neuron cells

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Purpose: To evaluate the relationship between ¹⁸F-FDG uptake and expression of glucose transporters (Gluts) and hexokinase II (HK II) in hepatic tumors.
Materials and Methods: This study included 31patients with HCC and 26 patients with CCC. All patients underwent to whole body ¹⁸F-FDG PET and CT examinations before treatment. PET acquisition was started at 60 minute after the injection of ¹⁸F-FDG (5-6 MBq/kg) Semiquantitative analysis using standardized uptake value (SUV) was measured for evaluation of tumor ¹⁸F-FDG uptake. Diagnosis were conformed by histological examination. Immunohistochemical staining of the section for glucose transporter 1, 2, 3, 4, 5 and HK II were performed using the streptoavidin-biotin methods.
Results: Immunohistochemistry activity were detected in Glut 1,Glut 2 and HK II in HCC where Glut 1, Glut 2 , Glut 3 and HKII in CCC. Gluts and HK II were often detected in moderately differentiated and poorly differentiated than well differentiated HCC. Significant association was revealed between the FDG uptake and expression of Gluts and HK II in HCC and CCC Conclusion: Present study showed significant association between ¹⁸F-FDG uptake and expression of Gluts and HK II in HCC and CCC even though ¹⁸F-FDG uptake was different, indicating that present finding may be valuable in management of both type of hepatic tumor.

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Treatment of cancer using heavy ion beams has shown excellent therapeutic results. It is expected that heavy ion irradiation will specifically affect properties of irradiated cells. For this, we analyzed effects of irradiation with heavy ion beams, X-ray or UV using virological methods as tools.
(1) Enhanced susceptibility of irradiated cells to HIV-1 Human cells irradiated with heavy ions but not X-ray or UV became more susceptible to HIV-1 than unirradiated cells. We examined whether the expression of genes that may be involved in HIV-1 infection steps was specifically affected by heavy ion irradiation: The expression of NF-kB was enhanced while that of Ku-80 or PARP was reduced.
(2) Effects of heavy ion irradiation on retrotransposition One of repeated genetic elements, LINE, is consisting 17% of the human genome and has a retrovirus-like genetic structure. We examined whether irradiation with heavy ions, X-ray, UV, etc. will enhance its transposition. Irradiation of cells by them lead to enhanced retrotransposition, but their rates were similar among different types of irradiation.
(3) Establishment of human cells expressing pprA and their susceptibilities to irradiation
D. radiodurans is a bacterium highly resistant to X-ray irradiation. pprA was identified as one of genes responsible for this trait. We have examined whether changes of cellular functions specific to heavy ion irradiation can be clarified using pprA gene. PprA protein was localized making peculiar patterns in cells. The cells became slightly more resistant to irradiation than the parental cells. We noticed that irradiation with heavy ions but not with X-ray or UV can evoke specific cellular responses which were detected upon analyses of genes possibly involved in HIV-1 infection

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In order to identify a new cellular factor required for human immunodeficiency virus type I (HIV-1) infection, we established a new indicator cell line to detect HIV-1 infection easily and real-time. Using irradiation with heavy ion beams, we tried to mutagenize human cells to isolate mutant cells that become resistant to HIV-1 infection. Human glioma-derived NP-2 cells transduced with HIV-1 receptor CD4 and coreceptor CCR5 were further transduced with the GFP gene that will be expressed under the control of HIV-1 long terminal repeat (LTR). We established N4R5/GFP cells that showed a low background of GFP expression before infection but became GFP-positive after HIV-1 infection. N4R5/GFP cells may be used as a model of cells latently infected with HIV-1. We examined the effects of irradiation with heavy ion, ultraviolet (UV-C), or X-ray, or treatment with TNF-α,DNA damaging agents or a histone deacetylase inhibitor (trichostatin A) on the appearance of GFP-positive N4R5/GFP cells.¹²C⁵⁺ and ⁴He²⁺ ions, UV-C, 4NQO, MNNG, cisplatin, and trichostatin A induced GFP expression in N4R5/GFP cells, although their induction efficiencies varied markedly. X-ray and 5-azaC showed hardly any effects. These results suggest that heavy ion beam may promote the reactivation of HIV-1. We next tried to isolate cells mutants in HIV-1 susceptibility. Cells irradiated with heavy ions were seeded to make their colonies and a dose (D10) that decreases the colony forming efficiency to 10% was determined. D10s for ⁴He²⁺ and ¹²C⁵⁺ irradiation were 2.6 and 0.9 Gy, respectively. We examined HIV-1 susceptibilities of cells derived from irradiated colonies. Although many colonial cell lines formed GFP-positive syncytia after HIV-1 infection, some lines formed GFP-negative syncytia, suggesting that heavy ion beams had affected transduced or cellular gene.

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[Background and Purpos]Despite recent improvements in surgical techniques and adjuvant therapies, the prognosis of patients with advanced esophageal cancer remains poor. Esophageal squamous cell carcinoma (ESCC) has high sensitivity for radiation and chemotherapy compared with other cancer of the digestive system. The purpose of this study was to elucidate the involvement of CDDP in ESCC using in air-micro PIXE (Particle Induced X-ray Emission).[Materials and method]Two human ESCC cell lines–TE-2 and TE-13–were examined for their response to CDDP. ESCC cells were treated various concentrations of CDDP, and we examined sensibility of cisplatin using MTT assay. We also performed real-time RT-PCR to evaluate the mRNA expression of multidrug resistant molecule in both cell lines. Dynamic state of element and intracellular metabolism were measured in CDDP treated cancer cells using air PIXE. Imaging data of individual cancer cell was captured in detail using this method. [Results]TE-2 cells were more highly sensitive to CDDP than TE-13 cells. The mRNA expressions of multidrug resistant molecules were differed between TE-2 and TE-13. Using air PIXE we observed distribution of P and K were localized at cytoplasm, and Br was localized at nuclear site. Air PIXE may be one of the new methods to measure CDDP combined with existing methods. [Discussion and Perspective]Intracellular concentration of Pt was measured using air PIXE, unfortunately we cannot confirm the difference of distribution of intranuclear Pt in two cells at this moment. Air PIXE may have role in application to predict CDDP sensitivity in cancer cells. Our results using air PIXE has limitation, since it only can be presented by the imaging semi quantitative data. Future study will be needed to establish a new method for precise quantitative data.

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Ashing and acid-digestion methods are necessary for analysis of microelements in biological sample. However, the analysis of two-dimensional distribution for the intracellular element was difficult. In-air micro-PIXE ( Particle Induced X-ray Emission) method is one of the few apparatus for measuring and visualizing the spatial distribution of elements in a single cell.
It is well known that cadmium (Cd) is toxic to various tissue, particularly the testis is highly sensitive to Cd toxicity. We have investigated the changes of micro element distribution in Cd-exposed testis.
1. Evaluation by in-air micro-PIXE method revealed that Cd and Fe distribution were increased in the interstitial tissues and seminiferous tubules. The histological findings indicated that the testicular tissue damage was advanced, which may have been caused by Fe flowing into seminiferous tubules followed by disintegration of the BTB. As a result, Fe was considered to enhance the tissue damage caused by Cd exposure.
2. We isolated Sertoli cells from rat testis and cultured them. Metallothionein(MT) levels, Heat shock protein 72(Hsp72) levels and Heme oxygenase-1(HO-1) levels were assayed in Sertoli cells, and these proteins were significantly increased by Cd exposure. Evaluation by in-air micro-PIXE method revealed that Cd and Fe distribution were increased in the Cd-exposed Sertoli cells, and Zn distribution was decreased.
These results suggested that Fe and Zn were closely associated with Heat shock proteins. And, HO-1 and MT is known as radical scavenger; therefore, these protein are suggested to protect Sertoli cells from Cd-toxicity.

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