Purpose Solid tumors are highly heterogeneous in oxygenation and contain hypoxic regions resulting from imbalance between cell proliferation and angiogenesis. These hypoxic regions result in the higher possibility to survive after treatments than normoxic regions due to resistance to radiation and anticancer drug treatments. Under these circumstances, we found that tempol, one of nitoxides, strongly induced the expression of hypoxia inducible-factor (HIF)-1alpha with a combination of hypoxic conditions. This induction mechanism seems to enable us to enhance the hypoxic cell killing applying the vector bearing the suicide gene downstream of HIF-1 binding site, hypoxia responsive element (HRE). In order to realize this idea, we constructed the plasmids that regulate the expression of suicide gene with tempol and then evaluated the enhancement of the cell killing effect at in vitro level.
Materials and Methods The properties of enhancement of HIF-1alpha expression by the addition of tempol were determined by luciferase (Luc) assay using the plasmid inserted four copies of a HRE in promoter sequence and oxygen-dependent degradation domain upstream and downstream of the Luc gene, respectively. We then performed a colony assay to evaluate the enhancement of cell killing effect controlled by tempol after transfection of the plasmid replaced Luc gene with FcyFur fusion gene whose product converts the prodrug, 5-FC to the anticancer drug, 5-FU into MCF7 cells.
Results and Conclusion The Luc activity of cells with tempol addition at 1%O₂ increased up to about 217-fold induction, compared to that without tempol addition at 20%O₂ (about 10-fold induction compared to that without tempol addition at 1%O₂). In addition, the transfected cells with the plasmid encoding FcyFur fusion gene indicated the highest cell killing effect after treatment of 5-FC, tempol and hypoxic mimic CoCl₂. These results indicate the expression of the suicide gene can be enhanced specifically in hypoxic regions by the addition of tempol. Therefore, this strategy might become one of the candidates to eradicate hypoxic cancer cell.

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Our previous investigation showed that 5-Fluorouracil (5FU) was a electron-affinic compound, which could show oxygen-mimic effects in DNA, as the result of molecular orbital calculation. The present study aimed to clarify the radiosensitization of hypoxic human lung cancer cells (H1299), which were transfected with a wild-type p53 expression vector (H1299wtp53) and mutant vector (H1299mtp53). Exponential growing cells were suspended in media with or without 5FU and then irradiated with X-rays (150 kV) in the glass tube, into which air or nitrogen gas was flowed. Cell survival was assessed with the colony formation assay. 5FU (0.1 mM) was a effective radiosensitizer toward killing of hypoxic cells irradiated with X-rays: ER(enhancement ratio) of 1.26 on H1299wtp53, 1.14 on H1299mtp53. No potentiation of aerated cells to X-irradiation killing was observed. The fluorescence intensity of foci of gammaH2AX produced in hypoxic cells after X-irradiation was enhanced in the presence of 5FU. It was suggested that hypoxic cell killing radiosensitization by 5FU correlated with the enhancement of DNA double strand breaks.

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Total radiation dose was limited by the tolerance dose of normal tissue in Boron Neutron Capture Therapy (BNCT). The radio-sensitivity of normal tissue, that is to say individual radiation sensitivity is the important element of success of BNCT. The RBEs of BNCR were estimated using radiation sensitive cell, Xrs5 (Ku80 deficient CHO mutant cell) and SCID mice (are well-known for the DNA double strand breaks (DSBs) repair deficiency due to DNA-PKs defect). The RBE values were estimated by the study of the cell killing effect, oral radiation death. In the radiation sensitive cell study, the RBE of xrs5 cell was 1.4-1.8. In the radiation sensitive mice study, the RBE of SCID mice was 1.6. CHO-K1 and the wild type mice, C3H. The RBE of CHO-K1 cell was 2.0-2.1. The RBE of C3H mice was 2.0. Our result suggested that the RBEs of radiation sensitive cells and mice was smaller than the wild type cells, that is to say, the hyper radiation sensitivity does not have a disadvantage in BNCT.

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[Purpose] Boron neutron capture therapy (BNCT) is a system, involving the taking up of boron-10 (¹⁰B) compounds by tumour cells and irradiation with thermal neutrons. ¹⁰B absorbs a thermal neutron and yields an α(⁴He) particle and a lithium (⁷Li) nucleus, which have high linear energy transfer (LET) and path lengths. We investigated the role of non-homologous end joining (NHEJ) repair gene, Lig4 for DNA damage induced by BNCT. [Methods] We used the cultured mouse embryonic fibroblasts deficient in Lig4 and their parental cells. We examined the cell survival after neutron or γ ray irradiation by colony forming assay. Our neutron beam facility includes γ ray up to 50%. To exclude the effect of the γ ray, the pure contribution of Lig4 for repair of DNA damage by neutron-α particle reaction was examined using relative survival with various concentration of ¹⁰B at 1.5Gy. [Results] Lig4-/- cells were extremely more sensitive to neutron beam with or without ¹⁰B (10ppm) than the parental cells. In addition, relative survival showed no difference between Lig4-/- and the parental cells. [Conclusion] It is suggested that BNCT caused irreparable DNA damage by Lig4.

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To evaluate if D-methionine, the dextro-isomer of the common amino acid L-methionine, can prevent oral mucositis resulting from heavy ion irradiation during treatment for head and neck cancers. For the first study, RBE of carbon-ion beams for epithelial thickness on mouse tongues was evaluated. The head of female C3H mice, 8 weeks old, were irradiated by five fractionated doses of carbon ion beams (290MeV/u) or 150kV X-rays to induce oral mucositis (ulcers on tongue). Six days after the last irradiation, mice were sacrificed. Excised tongues were evaluated macroscopically and micro- scopically. The thickness of the lingual epithelium was measured. The RBE of 6-cm SOBP (290MeV/u) for epithelium on mouse tongues after fractionated doses is 1.82. It was almost the equivalent value as that for gut crypt survivals reported by enormous studies.

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To investigate whether a carbon ion beam has beneficial effects compared to X-rays by targeting putative cancer stem cells, human liver cancer stem-like cells sorted from HepG2 and Huh7 cells were treated with or without carbon ion or X-ray irradiation and then colony formation assay, spheroid formation assay, FACS analysis, as well as in vivo tumorigenesis analyses were performed. FACS analysis showed that the proportion of CD133+/CD90+, CD44+/ESA+ cells were 6.4% and 1.5 % in Huh7, whereas only 0.6% and 0.2% in HepG2 cells. The proportion of CD133+/CD90+, CD44+/ESA+ was more significantly enriched by X-rays compared to carbon-ion beam. CD133+/CD90+ and CD44+/ESA+ cells significantly have higher number of colony and spheroid compared to CD133-/CD90- and CD44-/ESA- cells. In addition, CD133+/CD90+ and CD44+/ESA+ cells have shown more potential to form tumors in SCID mice than CD133-/CD90- and CD44-/ESA- cells. Colony assay showed that CD133+/CD90+ and CD44+/ESA+ cells appeared to be radioresistant to both X-ray and carbon ion beam, but carbon beam was more effective at killing those of cancer stem-like cells. In conclusion, CD133+/CD90+ and CD44+/ESA+ cells are putative liver cancer stem cells, and carbon ion beam potentially benefit for targeting liver cancer stem-like cells compared to X-rays.

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Radiotherapy is one of the established treatments for solid tumors. Investigation of tumor radioresistance is important for future tumor radiotherapy. Biological bases on the resistance have not been fully understood. Recent studies have demonstrated that there is a small fraction in solid tumor which is highly resistant to ionizing radiation and the radioresistant tumor cells often expressed some stem cell markers. Resistance of tumors to both radiation and chemotherapeutic agents can be attributed to the features of the cancer stem cells. In the present study, we show that a human glioblastoma cell line A172 transiently becomes to cancer stem cell-like when cultured with non-serum media which is supplemented with growth factors. The treated cell was found significantly resistant to X-rays and heavy ion particles (carbon) compared with the A172 cells cultured with the normal media. Phosphorylation of histon H2Ax was induced in both forms, however, recovered earlier in the stem cell-like population than in the parental cells. Our result is consistent with the hypothesis that cancer stemness causes radioresistance through the efficient activity of DNA double-strand break repair.

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[Background and Purpose] The fractionated irradiation is standard protocol for radiotherapy including carbon-ion therapy. There are big differences between single and fractionation, for example on DNA damage repair, reoxygenation of hypoxic region, etc. It is necessary to get the results using fractionated irradiation to know the effects in clinical. The purpose of this study is to examine the effects of fractionated irradiation for metastasis using C-ions and X-rays. [Materials and Methods] A mouse osteosarcoma cells, LM8 were used. All irradiation were performed with C-ions at the center of 6cm-SOBP and X-rays as reference beam. in vitro: The cytotoxic effects were examined using colony formation assay, and the inhibit effects of migration and invasion were examined using Boyden chamber assay and Matrigel invasion assay, respectively. in vivo: LM8 tumors inoculated into right hind leg of mice were irradiated. Radiosensitivity for whole tumors was obtained by tumor growth curves and for individual cell in a tumor was obtained by in vivo-in vitro assay. The metastatic effects were analyzed with the spontaneous lung metastasis model. [Results] in vitro: After fractionated irradiation, cell survival, migration and invasion activity were enhanced with the increment of fraction number. The degree of enhancement was more significant after X-rays than C-ions. in vivo: Anti-tumor effects were decreased and the number of lung metastatic nodules were increased depend on the fraction number. These effects were also more remarkable by X-rays than C-ions. [Conclusion] It is confirmed that the anti-metastatic effects after fractionated irradiation were decreased compared with single irradiation, however the degree was more smaller for C-ions with high LETs than X-rays with low LETs. It was suggested that carbon ion therapy was superior to photon therapy to suppress the metastasis even for clinical fractionated irradiation.

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Cancer therapy with carbon ions at the HIMAC has started in 1994, more than 5,000 lesions have been treated by 2010, and fruitful results have been demonstrated. Following those results, few heavy-ion treatment facilities are working, some are under construction and many new facilities are planning all over the world now. To start treatments with HIMAC clinical experiences such as treatment planning, the beam quality at those new facilities must be very similar to the HIMAC in physics and biology. Biological results are easy affected by the biological conditions, techniques or so on. We tried to establish a standard biological protocol for this purpose.
Both C3H/He mice bred at NIRS (N) and Charles River Co. (C) were employed, and the crypt cell survival assay 3.5 days after the irradiation was performed with whole body single irradiation of carbon 290 MeV/u beam having 6 cm SOBP at HIMAC. Crypt survival data was taken from (N) and (C) mice. An expert and a new face measured crypt of the prepared slide.
The radio-sensitivity of the mice crypt was different by the breeders. The Dq values were similar between them (C and N), but the D_o for (N) was higher than that for (C). Recognition level of the existing crypts at counting was different by the observers, but this technical difference was conquerable by simple training. Change in the recognition level with elapsed time could not found for an observer within 6 years.
Use of mice supplied by worldwide breeder with good quality control may be required for intercomparison of RBE. Short training could unify technique of new observer.

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Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5-8 weeks. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy. Therefore, we clarified the radiosensitivity of heavy-ion beams in X-ray resistant cells.
We used clinically relevant X-ray-resistant SAS-R cells from human tongue cancer SAS cells. The SAS and SAS-R cells were exposed to X-rays (TITAN-225S, Shimadzu) or carbon-ion beams (290 MeV/u, 6 cm-SOBP, Gunma University Heavy Ion Medical Center). We examined the cell survival after irradiation by modified high-density survival assay by scoring the total number of surviving cells using cell counter (TC10, BIO RAD).
Our results show that 20% survival dose of X-rays were 7.5 and 12 Gy in SAS cells and SAS-R cells, respectively. The 20% survival dose of carbon-ion beams were 3.0 and 3.2 Gy in SAS cells and SAS-R cells, respectively. Relative biological effectiveness (RBE) at 20% survival in SAS cells and SAS-R cells was 3.8 and 2.5, respectively. It is suggested that heavy-ion beams are useful in cancer therapy.

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We have measured radioactive nucleides contained in soils and some materials near Fukushima Daiichi Nuclear Power Station with germanium semiconductor detectors. Te-129, Te-129m, I-131, Te-132, I-132, Cs-134, Cs-136, Cs-137, Ba-140, La-140, Tc-99m, Nb-95 and Ag-110m were detected. Proportion of Te-129, Te-129m, Cs-134, Cs-137 was almost same irrespective of sampling point. Proportion of I-131 among the radionucleides was a little dependent on the sampling point, and proportions of Nb-95 and Ag-110m were respectively largely different by the sampling point. Radioacitivity concentrations of moss and pine leaves were larger than the neighbor soil, strongly correlated with those of the soil and the proportion of the radioactive nucleides was almost same as the soil.

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Following the Fukushima Daiichi nuclear power plant accident on March 11th 2011, the Village of Iitate located 25 - 45 km northwest from the plant is found with radioactive contamination and suspected as a Hot Spot, where the contamination is greater than other parts of Fukushima Prefecture. Given the urgency of the situation of residents living in the region and in order to grasp the extent and spread of Hot Spot, radiation survey activities have been carried out with the cooperation of Iitate Village and related authorities on March 28th and 29th. With radiation detectors brought into a van provided by Iitate Village office, the radiation dose rate inside the vehicle was measured as it travelled within the village. The detectors used were ALOKA Co. pocket survey meter PDR-101(CsI detector) and ALOKA Co. Ionization Chamber Survey Meter ICS-313. The air dose rates at 130 locations have been measured. The survey was conducted in and outside the vehicle at several locations, and this confirmed the shielding effect of the vehicle. In addition, soil samples were obtained at 5 locations for the propose of the radionuclide analysis, and later with Ge-detector, gamma ray radionuclide analysis was carried out at Hiroshima University. Radionuclides of 132Te, 131I, 129Te, 129mTe, 137Cs, 134Cs, 136Cs and 140La were identified. The time variation of air dose rate was calculated by using the contamination density. The estimated accumulative exposure to radiation over 90 days beginning from the March 15 low is 74mSv for Nagadoro-Magata and 22mSv for the Iitate Village Hal. A cumulative exposure map in Iitate village are has been evaluated from the above mentioned data. The cumulative exposure map is compared with the cumulative exposure map by MEXT. In conclusion, the both cumulative exposure maps show a similar trend. Our map shows the detailed distribution cumulative exposure.

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Dosimetry system; so-called DS86 and DS02 are used for dose estimation of the Hiroshima and Nagasaki Atomic bomb survivors. For the early entrance survivors, radiation doses came from activated materials induced by the atomic bomb neutrons. For estimation of such doses, element composition in soil is necessary. Especially Sc density is important for estimating radiation doses during the period after several ten days from the explosion. Scandium-46 which has the half-life of 84 days-, is induced by neutrons from stable 46Sc(abundance 100%). However, few data of Sc density in soil are available both of Hiroshima and Nagasaki (Hashizume 1967, DS86) cities. Purpose of this study is evaluation of Sc density in soil and its variation using neutron activation analysis. Soil samples were taken from 11 locations within 4 km from the hypocenter of Hiroshima city. The soil samples were dried up in a condition of 120o for over night and grained in a mortar. The grained samples were activated by neutrons in Kyoto University Reactor (KUR). The activated soil samples are measured by Ge-detectors. From gamma-ray spectrum, 23 element compositions including Al, Mn, Na and Sc are obtained. The obtained element compositions are roughly the same as the DS86-reported values. Sc density in Hiroshima soil was estimated to be 5.12(59) (ppm). It was found SD value of Sc density in soils for 11 location of Hiroshima city is about 12%. Using element compositions obtained from the activation analysis, time variation of radiation exposure rate by activated soil were estimated. It was found that exposure rate in the few minutes was time dominated by 28Al, in the several days by 24Na, and then by 46Sc. This estimated result was compared with measured dose rate values after three months after the explosion by Pace and Smith (1959). The estimated dose rate was quite similar to the measured one.

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Radioactivity in air have been surveyed at Hiroshima after the reactor accident of Fukushima Daiichi Nuclear Power Plant on March 11, 2011. Sampling was started on March 20 with a high volume air sampler (SHIBATA HVA-500N) with 500 l/min for 8 hours in the daytime. A glass fiber filter (TOYO GB100R) was used for air sampling. Gamma-ray measurements were performed with a low-background Ge detector (EG&G ORTEC GWL-120230-S) To reduce the background gamma-rays from radon progeny nuclei (214Pb, 214Bi), the measurement was started after 4 h from the end of sampling. Radionuclide 131I was first observed on March 30, thereafter 134Cs, 137Cs and 132Te were observed. The concentration of radionuclides were reached at maximum on April 7 and April 17 again, then gradually decreased. Radioactivity was not observed at the end of April any more. The maximum concentrations were 0.0066 Bq/m3 for 131I, 0.0078 Bq/m3 for 137Cs, 0.0068 Bq/m3 for 134Cs and 0.00011 Bq/m3 for 132Te. 131I was also observed in rain water on April 8 with concentration 0.044 Bq/l. Comparison of observed radionuclide and those of Chernobyl reactor accident will be given.

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The system is composed of a NaI(Tl) scintillator of 3 – 1 inch diameter with lightweit portable MCA. Measurements were carried out in a car between Fukushima and iwaki, kitakata and namie, in Fukushima Prefecture.

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The Fukushima nuclear power plant disaster which occurred by the East Japan great earthquake caused the extensive radioactive contamination. It is very difficult to know how long this radioactive contamination continues. However, when taking into consideration the future decontamination work, the refuge duration, etc., it is considered one of the important factors. I tried a speculation of the ecological half-life of ¹³⁷Cs in the animal which inhabits the Fukushima nuclear power plant disaster contaminated zone based on the several data. The following data were referred to for a speculation of ecological half-life. 1) The half-life of the ¹³⁷Cs whole body count originating radioactive fallout to Japan by nuclear test in the atmosphere (1964-1969) and the Chernobyl nuclear plant accident of 1986 (Uchiyama,M. et al. 1996), 2) The ecological half-life of the ¹³⁷Cs in fish of lake in Norway (Jonsson,B. et al. 1999) and UK (Smith,JT. et al. 2000) , and 3) The ¹³⁷Cs pace of decrease in the Chernobyl contaminated zone animal body in 1997 to 2005 (our report of previous JRR meeting: Nakajima,H. et al. 2006). It was speculated that the ecological half-life of ¹³⁷Cs of the beginning in Fukushima is about 1.5 years.

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The Fukushima Daiichi accident on March 11, 2011 became the most serious accident that can be compared only with the Chernobyl accident on April 26, 1986. The radioactivity composition released by these two accidents was different each other, depending the reactor type and the accident process. Chernobyl was a power surge accident at one reactor, while Fukushima was loss-of-coolant accidents at three reactors. In the case of Chernobyl, the radioactivity composition of the initial release caused by an explosion due to the power surge was similar to that contained in the reactor core, and then the graphite fire of the moderator continued about ten days. According to the USSR report presented to IAEA in August 1986, the total release of ¹³¹I and ¹³⁷Cs was estimated to be 2.710¹⁷ and 3.710¹⁶ Bq, respectively. Meanwhile, in the case of Fukushima, the large amount of volatile radionuclides was released into the environment when the integrity of containment, the last protecting shield was destroyed during the course of pressure increases inside containments as well as hydrogen explosions after the core melt-downs. According to the press release from the Nuclear Safety Commission on April 12, the released amount of ¹³¹I and ¹³⁷Cs was estimated to be 1.510¹⁷ and 1.210¹⁶ Bq, respectively. From these figures, the similar level of radioactivity release occurred for volatile radionuclides although the Chernobyl case was a little larger than the Fukushima. Regarding refractory radionuclides such as ⁹⁰Sr and ⁹⁵Zr, the released estimates of 8.110¹⁵ and 1.410¹⁷ Bq were given for ⁹⁰Sr and ⁹⁵Zr for Chernobyl, respectively, while, according to the Japanese government report to IAEA in June, they were 1.410¹⁴ and 1.710¹³ Bq for Fukushima, respectively. The amount of ⁹⁰Sr and ⁹⁵Zr released by the Fukushima accident was estimated 1/60 and 1/8000 of the Chernobyl accident, respectively. In our presentation discussion will be given for uncertainties of the above values as well as a comparison of radiation situation in the contaminated areas between Fukushima and Chernobyl.

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The eastern Japan where has been affected by the nuclear event formed in Fukushima 1st nuclear power plant after March 12th in 2011 was studied by a scientist in a wide range of 800km on nuclear hazards and affected population a month later of earthquake disaster and 3 months later. The annual external dose of the eastern Japan except for Fukushima prefecture is less than 1mSv in 2011. That one is evaluated to be generally less than 10 mSv for population in Fukushima considering the long period of the indoor sojourn time. Radioactivity of iodine 131 in thyroid of 76 people has been measured and then the dose was evaluated to be less than 8 mGy. Portable whole body counter on Fukushima 33 people was carried out and showed annual internal doses to be less than 0.1 mSv. These low doses indicate us no health effects in Fukushima incident.

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Since Nagasaki city is hilly, some people exposed to the atomic bomb over mountains might be shielded. The people exposed in the shielded areas should have received less radiation dose and will be probable lower in cancer mortality than those exposed in the unshielded areas at the same distance from the hypocenter.
We first defined shielded and unshielded areas located 2-5 km from the hypocenter into 5 groups using GIS, i.e., shielded and unshielded areas located at 2-3 km from the hypocenter (denoted by S2-3 and U2-3, respectively), and unshielded areas located at 3-4 km (U3-4) and 4-5 km (U4-5). We compared the cancer mortality among atomic bomb survivors exposed in the respective areas on the basis of the Cox proportional hazard model after adjusting for sex, age at the bombing, and attained age.
Of 37,357 survivors who were exposed at the age of less 30 years and were living in Nagasaki city on 1 January 1970, those exposed in areas of U2-3, S2-3, U3-4 and U4-5 were 1663, 2341, 5062 and 3313, respectively. From 1 January 1970 through 31 December 2009, the number (percent) of cancer deaths observed among survivors exposed in areas of U2-3, S2-3, U3-4 and U4-5 were 198 (11.9%), 202 (8.6%), 435 (8.6%) and 267 (8.1%), respectively.
The hazard ratio of cancer mortality in S2-3 was 0.76-fold of that in U2-3 (95% confidence interval: 0.63-0.93), that is 24% reduction. Similarly, the hazards of cancer mortality in U3-4 and U4-5 were 0.75-fold (0.64-0.89) and 0.80-fold (0.67-0.97) of that in U2-3, respectively. No significant difference was observed in hazards of cancer was observed among in S2-3, U3-4 and U4-5.
The present study demonstrates the terrain shielding effects and suggests that such effects are similar to those by remoteness from the hypocenter for the distance of 3 km or over.

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We examined the effects of lifestyle on the mortality in Nagasaki A-bomb survivors on the basis of the mail-survey results collected from 35,035 Nagasaki A-bomb survivors in 2003. Items in the questionnaire included smoking, drinking, exercise, health checkup and mental health conditions. Mental health conditions were assessed by 12-item version of General Health Questionnaire. In 15,864 survivors finally remained for the analysis, 3,142 died from 1 April 2003 through 31 March 2010; 1,088 died from cancer, 821 from circulatory system diseases and 1,233 from other diseases. We analyzed the effects of smoking, drinking, exercise, health checkup and mental health conditions on mortality separately using Cox proportional model after adjusting for sex and age. In the comparison of survivors without and with exercise, the hazard of death from cancer in the former group was 1.34-fold of that in the latter group, the hazard of death from circulatory system in the former group was 1.52-fold of that in the latter group, and the hazard of death from other causes in the former group was 1.42-fold of that in the latter group. Similarly, in the comparison of survivors without and with health checkup, the hazard of death from cancer in the former group was 1.39-fold of that in the latter group, the hazard of death from circulatory system in the former group was 1.50-fold of that in the latter group, and the hazard of death from other causes in the former group was 1.36-fold of that in the latter group. Effects of drinking, smoking and mental health conditions showed a similar tendency, respectively.

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After the accident at Chernobyl Nuclear Power Plant (CNPP) on 26 April 1986, the incidence of thyroid cancer was increased in children. Recently, strong relationship between the incidence of solid thyroid nodules and of thyroid cancer was shown in atomic bomb survivors. In this study, we perform the epidemiological study aimed for inhabitants who were pointed out thyroid nodules by ultrasonography, to assess the long-term risk of thyroid nodules for future thyroid cancer around CNPP. We investigated 160 inhabitants who have been pointed out thyroid nodules (Case) and age and sex matched 160 inhabitants who have not been pointed out thyroid nodules (Control) by ultrasonography during 1991 to 2001. All participants were aged 0-10 years old at the time of accident. All participants with thyroid solid nodules underwent FNAB under ultrasound guidance. Furthermore, blood samples were collected for measurements of fT4, TSH, thyroglobulin (Tg), antithyroglobulin antibodies (TgAb) and thyroid peroxidase antibodies (TPOAb). Obtained data was statistically analyzed to compare the incidence of thyroid abnormalities, including cancer. There were no significant differences of age and sex ratio between Case and Control groups. There was no difference in FT4, TSH, positive ratios of TgAb and TPOAb between both groups. On the other hand, Tg was significantly higher in Case group than Control group (p<0.001). In Case group, number and size of nodules were significantly increased during the second screening compared with the first screening. No nodule was observed in Control group, whereas three participants were diagnosed as malignant by follow-up FNAB in Case group. The ratio of malignancy in Case group diagnosed by FNAB was relatively, but not significantly higher than in Control group (p=0.082). Since 9 of Case group were considered as indeterminate by FNAB, the ratio of Case group with indeterminate and malignant diagnosed by FNAB was significantly higher than that of Control group (p<0.001). These results suggest that thyroid nodule during childhood is a potent risk for the development of malignant tumor in irradiated individuals around CNPP.

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The pollution caused by the military use of depleted uranium and the nuclear accident have raised increasing concern about its toxicity. We reported previously that uranium accumulated selectively into the renal proximal tubules and caused renal lesions in adult rats. In the present study, uranium localization in the proximal tubules was examined in rats exposed to uranium acetate by the combination of high energy synchrotron radiation X-ray fluorescence analysis using microprobe with immunohistochemical staining of the down stream of the renal proximal tubules, toxic target sites of uranium. At 1 day after administration, uranium was found in the inner areas of the cortex, where uranium was highly concentrated more than 50-fold of the mean uranium concentration in kidney. Damaged tubules were observed at 8 days. At 15days, regenerated tubules were found and the uranium concentrated areas decreased, but still contained uranium at almost equal levels at 1 day.

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It is important to examine the neurocytotoxic effects of radiation on the developing central nervous system (CNS) because of its high sensitiveness to radiation. Medaka is an ideal model for studying the effects of developing CNS of vertebrates, because the transparency of their eggs and embryos makes it possible to detect morphological abnormalities in the CNS easily using a conventional stereomicroscope. Wild-type and p53(-/-) medaka embryos at developmental stage 28, when neural cells proliferate rapidly especially in the optic tectum (OT), were irradiated with 10 Gy gamma-rays which does not affect to hathability. Time-lapse changes of radiation-induced apoptosis were examined in whole-mount specimens using an acridine orange (AO) assay. By AO assays, apoptotic cells in irradiated wild-type OT appeared as AO-positive small single nuclei at 3h after the irradiation. At 8-10 h after the irradiation, we found two types of AO-stained cells, namely,a single single nuclei and rosette-shaped clusters. At 15 h after the irradiation, the AO-positive clusters became bigger and more obvious, and the number of them gradually decreased at 35 h after the irradiation. On the other hand, in p53(-/-) irradiated OT, AO-positive single nuclei started to appear 3-5 h later than that of wild-type embryos, and the size of AO-positive rosette-shaped clusters was apparently smaller than that of wild-type. In order to prevent the diffusion of damaging degradation products into the surrounding tissue, apoptotic neuronal cells are quickly phagocytosed by the microglia in the brain. Here, we investigated how microglia act to phagocytose rosette-shaped clusters of apoptotic cells in wild-type and p53(-/-) irradiated brain at 24 h after the irradiation. The gene expressions of apolipoprotein-E (Apo-E) protein, which is specifically expressed in micriglia, determined by whole-mount in situ hybridization revealed a high-level of transcripts in the retinal ganglion cell layer and in the OT, where rosette-shaped clusters of apoptosis were observed on histological sections in both type of irradiated OT. Almost no differences were observed in expression patterns of ApoE in both type of irradiated embryos.

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It is important to examine the neurocytotoxic effects of radiation on the developing central nervous system (CNS) because of its high sensitiveness to radiation. Mammalian embryos develop in the uteri, hence, their developmental process cannot be examined directly. In contrast, medaka is an ideal model for studying the effects of developing CNS of vertebrates, because the transparency of their eggs and embryos makes it possible to detect morphological abnormalities in the CNS easily using a conventional stereomicroscope. Moreover, the smaller size of their embryos compared with that of mammalian embryos provides the advantage of easy examination of whole-mount specimens. Wild-type Hd-rR medaka embryos at developmental stage 28, when neural cells proliferate rapidly especially in the optic tectum (OT), were irradiated with 10Gy gamma-rays, which does not affect to the hatchability. Radiation-induced apoptosis were examined in whole-mount specimens using an acridine orange (AO) assay. At 8-10 h after the irradiation, we found distinct two types of AO-stained cells, namely, a single condensed nuclei and rosette-shaped clusters of them. This characteristic staining pattern was continually observed up to 35 h after the irradiation. Electron microscopic observation of irradiated OT at 24 h after the irradiation revealed that rosette-shaped clusters were aggregates of 10-15 apoptotic cells and these aggregated apoptotic cells were phagocytosed inside a single large cell. In order to prevent the diffusion of damaging degradation products into the surrounding tissue, apoptotic neuronal cells are quickly phagocytosed by a resident immune cell, known as the microglia in the brain. The phagocytic motility of microglia for radiation-induced apoptosis in irradiated developing brain was not well studied. Here, we investigated how microglia act to phagocytose rosette-shaped clusters of apoptotic cells in Hd-rR irradiated brain at 24 h after the irradiation. The gene expressions of apolipoprotein-E (Apo-E) protein, which is specifically expressed in micriglia, determined by whole-mount in situ hybridization revealed higher levels of transcripts in the irradiated embryos especially in the eyes and OT than that of non-irradiated controls. Imaging in 3D of the microglial motility was based on the serial sections of irradiated in situ stained brain by utilizing 3D application tools for computational method.

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One of the principal risks which associated with nuclear power arise from health effects of radiation. Since radiation event victims may be exposed to various dose of radiation, early biomarkers of radiation exposure would be useful for the evaluation and medical management of victims. In order to identify new biomarkers for the triage, we analyzed the urine peptides and proteins in irradiated mice by proteomics approach.
The urine samples were collected from mice which were exposed to whole body γ-irradiation (0.25 ~ 6.0 Gy). The urine was separated by HPLC and analyzed by MALDI-TOF MS. It was found that the peak at m/z 2821 specifically increased in mice which were irradiated by γ-ray. According to MS/MS analysis, the peak was identified as hepcidin 2. Hepcidin 2 levels in the urine were increased in time and dose dependent manner. The mRNA expression of hepcidin 2 in the liver also showed the similar expression pattern in urine hepcidin 2. These results indicated that urinary hepcidin peptides might be a potential biomarker for acute ionizing radiation exposure.
We also identified another potential biomarker for radiation exposure. The dose response and time course of these biomarkers will be shown in this speech.

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In analyses of radiation epidemiology, the regression method is applied to investigate the dose-response curve. Errors in the outcome variable is assumed in the usual regression model, and this may resulted in wider confidence intervals, not in biased estimates. Although errors in covariates are not included in the models, and this may be resulted in biased estimates. To decrease the bias, the measurement error models are proposed, which adjust the bias by quantifying the magnitude of covariate errors and using the information of these. In radiation epidemiology, two kinds of covariate errors (Berkson and classical) are known to exist, and several models which assume these errors and adjust them. We have continued the evaluation of these methods since last year.

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The background doses for the teeth of residents of Fukushima Prefecture, Japan, before the nuclear power plant accidents in Mar. 2011, were obtained by electron spin resonance (ESR) method. The doses obtained from 77 of 92 samples are less than 100 mGy while the doses of the other samples are high up to 250 mGy. The doses for buccal part of the teeth are higher than lingual part possibly due to contributions from dental X ray examination. A positive correlation was found between the ages of the donors and the obtained doses. The averaged annual ESR dose was calculated to be 0.87 mGy/y, being higher than the natural dose rate of 0.33mGy/y in this region. These doses have to be considered in actual retrospective dosimetry studies for the Fukushima nuclear power plant accidents. The statistically significant critical level for Japanese residents would be about 200 mGy for individual doses and about 100 mGy for averaged group doses for α=5 % for those with ages older than 50.

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Ultraviolet-B (UVB) radiation induces photodamage in DNA, including the formation of cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts]. Two major repair mechanisms for such DNA damage are excision repair and photoreactivation. In photoreactivation, the enzyme photolyase mediates the repair of damaged DNA by binding to CPDs or (6-4) photoproducts in a lesion-specific manner. In plants, the CPD photolyase is a crucial factor for determining UVB sensitivity. In rice, however, the relative contribution of (6-4) photolyase to UVB resistance is unclear. We measured the activity of (6-4) photolyase and the UVB sensitivity of three rice cultivars. There was no correlation between (6-4) photolyase activity and UVB sensitivity. Furthermore, we investigated the UVB sensitivity of (6-4) photolyase-deficient mutant lines induced by rice retrotransposon Tos17. No difference was found between wild-type rice and the mutant. The Arabidopsis mutant defective in (6-4) photolyase was more sensitive to UVB than the wild-type. These results suggested that the activity of (6-4) photolyase was not significantly affected the UVB sensitivity of rice.

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The risk of exposure to environmental UV such as sunlight has been one of the public greatest concerns because of its potent genotoxic hazard for the skin. The risk for human skin is currently estimated mainly by an empirical index of erythema, which is specifically detectable on UV-exposed skin. The erythema-based assessment has been useful for protection against UV, but cannot provide any quantitative prediction of the future effects of UV exposure because erythema induction is a threshold event that does not correlate linearly with the UV dose. To solve the problem, quantifiable indices which correlate more directly with the UV genotoxicity should have been introduced. For such indices, we adopted mutation, which has a direct correlation with the UV genotoxicity. Importantly, we utilized it from two aspects based on the analysis of mutation induction kinetics in the UV-exposed skin. The two indices of mutation are mutagenicity (the initial increment of mutant frequency) and mutation induction suppression (MIS), a skin-specific protective response to UV found in our previous study. With these indices, we determined a set of action spectra of UV genotoxicity and developed a UV risk estimation system for the skin. Based on the system, we actually estimated mutant frequencies (MFs) induced in the skin after one-hour exposure to the sunlight at noon on a clear summer day in Japan, using data on the spectral solar UV intensities, which are measured regularly by Japan Meteorological Agency and available online (http://www.woudc.org). The predicted MFs were found to become more reliable and accurate by the introduction of the MIS index and to show a good agreement with the observed values we directly measured before. (This study was carried out under the NIBB Cooperative Research Programs for the Okazaki Large Spectrograph, 10-501.)

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  We have previously demonstrated that the excessive mitochondrial O₂^•- production caused by mitochondrial complex II SDHC mutations results in premature death in C. elegans and Drosophila and cancer in mouse embryonic fibroblast cells (M. Tsuda, et al. BBRC 2007, T. Ishii, et al. Cancer Res. 2005, N. Ishii, et al. Nature 1998). In humans, it has been reported that mutations in SDHB, SDHC or SDHD often result in inherited head and neck paragangliomas (PGLs).
  Recently, we established Tet-mev-1 conditional transgenic mice using our uniquely developed Tet-On/Off system, which can induce the mutated SDHC gene to be equally and competitively expressed compared to the endogenous wild-type SDHC gene (T. Ishii, et al. Mitochondrion 2011). These mice experienced mitochondrial respiratory chain dysfunction that resulted in O₂^•- overproduction. The oxidative stress caused excessive apoptosis leading to low birth weight and growth retardation in the neonatal developmental phase in Tet-mev-1 mice. Moreover Tet-mev-1 mice have low pregnancy and fertility rates, and occasionally result in maternal death. In an in vitro fertilization assay, the ovulation rate and sperm activity were slightly reduced, but early embryogenesis was not changed.
  In this study, the results suggest that SDHC V69E mutation might significantly affect some roles at the time of implantation or placental functions. We hypothesize that the mutation causes not only excessive O₂^•- production by mitochondrial respiratory chain abnormality but also mimic-hypoxic condition by HIF-induced signal transductions leading to infertility, habitual abortion and maternal death.

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Calculations of radon-exposure doses have intensively made due to its importance from the standpoint of radiation protection. To determine radiation doses from radon inhalation, we should take into account the following exposure patterns: (1) Deposition of inhaled radon progeny; (2) Retention of radon gas in lung airways; (3) Biokinetics of inhaled radon gas. Almost all studies reported so far paid attention to the pattern (1), which was the greatest contributor to total radiation doses. We have conducted radon exposure experiments using mice to observe their biological changes. Since organs other than lungs were also analyzed, absorbed doses of many organs and tissues must be assessed. Taking into account the above three patterns, we computed the doses for small laboratory animals with dose calculation models that have been established for humans. These assessments will be important for considering the radiation protection of environment. The result of mice is shown here. Exposure pattern (1): the absorbed doses were 51.6, 4.6 and 35.9 nGy/(Bq/m3)/h for the tracheobronchial and alveolar regions, and whole lung, respectively. Exposure pattern (2): 0.036, 0.015 and 0.029 nGy/(Bq/m3)/h for the tracheobronchial and alveolar regions, and whole lung, respectively. Exposure pattern (3): 0.056, 0.029 and 0.002 nGy/(Bq/m3)/h for adipose tissue, red bone marrow and the rest (average), respectively. It was confirmed that, in descending order, the pattern (1), (2) and (3) give the high doses. This trend was seen for rats, which is the same as humans. In our presentation, we will talk about the dose calculation models used, the comparison of doses between humans and small animals, and future issues.

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In charged particle therapy with passive method, Spread-Out Bragg Peaks (SOBP) with a ridge or wheel filter are used to achieve uniform biological responses in the irradiated tumor. Therefore, there are primary and fragment particles with variable energies from the filter in the SOBP. Currently, the dosimetry in SOBP beams using the ionization chambers has been performed with the beam quality correction factors for monoenergetic beams due to unknown physical quantities within the SOBP. Calorimetry is a most direct method to measure the absolute dose. We determined precisely the absorbed dose in the SOBP using the graphite calorimeter at the center of SOBP, 60 mm for irradiation fields of diameter of 10 cm and 5 cm in 290 MeV/n carbon ion beams. Under the same geometry and measurement position, the dosimetry was performed using the parallel and cylindrical ionization chambers. Next, the ionization chamber dosimetry was done in a water phantom at the center of SOBP. We compared and verified the dose using the ionization chambers with that from the calorimetry. At the center of SOBP in 290 MeV/n carbon ion beams, the comparisons to our calorimeter measurements revealed that the absorbed dose using the ionization chambers and beam quality correction factors from IAEA TRS 398 comes out low by about 3 %. For irradiation fields of diameter of 10 cm and 5 cm in 290 MeV/n carbon ion beams, the detailed information on dosimetry using the calorimeter and beam quality correction factors are reported.

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The external and internal radiation doses were estimated for 15 residents who lived at around 37 km north-west from the Fukushima Daiichi nuclear power plant which released radioactive plume due to the accident on March 11, 2011. An interview has been performed to ask where they stayed and what they ate after the accident. To estimate the external radiation dose for each person, the air dose rate at the person's yard was measured and cumulative dose up to the 54th day from the release has been calculated. With regard to the internal radiation dose, urinary bioassay method has been performed on the 54th day and the 78-85th day after the deposition, by using a low-background Ge spectrometer. As a result, the average external dose was 10.5mSv among the adults, and 6.3mSv among the children. The average committed effective dose from <134>Cs and <137>Cs was 0.055mSv among the adults and 0.034mSv among the children. <131>I was detected in five persons; four adults and one child. The average committed effective dose from <131>I was 2.5mSv among the adults and 2.2mSv of the child. The ratio of external dose to internal dose from <134>Cs and <137>Cs ranged from 87:1 to 566:1 among them.

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The accident at Fukushima Daiichi Nuclear Power Plant occurred after the Great East Japan Earthquake Disaster resulted in the release of radionuclides to atmosphere. Although data on ambient dose rates measured at monitoring posts have been released since the accident, there is no comparison of ambient dose rate with external radiation dose evaluated by personal dosimeters. In this study,we evaluated differences between ambient dose rates measured at Fukushima city office and individual external radiation dose evaluated by personal dosimeters. Doctors, nurses and radiological technologists who were dispatched from Nagasaki University Hospital as members of disaster relief team during March 15 and July 27 participated in this study(n=7). All members worked mainly at Fukushima Medical College Hospital, Fukushima city. The maximum ambient dose rate of the Fukushima city was 12.34μSv/h on March 18, and the minimum was 0.94μSv/h on July 23. On the other hand, maximum individual external radiation dose was 1.63μSv/h and minimum was 0.08μSv/h. All personal external radiation doses were below ambient dose rate at the corresponding dates. Our results suggested that individual external radiation exposure dose is much lower than ambient dose rate in Fukushima. Further evaluation is needed to clarify the individual external radiation doses in general population of Fukushima.

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To evaluate the environmental contamination and contributory external exposure immediately after the accident at the Fukushima Nuclear Power Plant (FNPP) and to evaluate the current environmental contamination and contributory external exposure at the Chernobyl Nuclear Power Plant (CNPP), the concentrations of artificial radionuclides in soil samples from each area were analyzed by gamma spectrometry. Six artificial radionuclides (iodine-131, cesium-134, cesium-137, tellurium-129m, niobium-95, and cesium-136) were detected in soil samples around FNPP, whereas cesium-137 was only detected in soil samples around CNPP and in Nagasaki. Calculated external effective doses from artificial radionuclide contamination in soil samples around FNPP were 3.7-5.7 micro-Sv/h in Fukushima city as of March 22, 2011, 0.11 micro-Sv/h in Iwaki city, and 0.18 micro-Sv/h in Ono town as of April 7, 2011. On the other hand, calculated external effective doses in soil samples around CNPP were 0.00023 micro-Sv/h in Gomel city and 0.00041 micro-Sv/h in Klincy city in January 2011. Moreover, the calculated external effective dose in soil was 0.00010 micro-Sv/h in Nagasaki city in August 2010. The present study revealed that artificial radionuclides derived from the accident were detected in soil samples around FNPP even though current levels are decreasing gradually. Also, the dose rates from external exposure around FNPP immediately after the accident, especially in Fukushima city, were higher than those around CNPP and in Nagasaki. Long-term environmental monitoring around FNPP, as well as evaluation of health effects in the population residing around these areas, contribute to radiation safety with a reduction in unnecessary exposure to the residents.

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Recently, it was recognized the importance to estimation impacts of radiation about non-human biota. ICRP reported estimation methods for non- human biota dose in ICRP Publication 108. On the other hand, ambient dose was broadly measured and reported after the nuclear accident because the measurement of ambient dose is easier than the measurement of radioactivity about each radionuclides. Some studies were estimated the radiation effects of non-human biota using ambient dose, however, ambient dose could not directly apply to the estimation of non-human biota. In this study, the ambient dose to biota absorbed dose conversion factors for ICRP reference terrestrial organisms were calculated and we assessed environmental effect for the case in Fukushima Daiichi power plant accident.

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Double-strand breaks (DSBs) in the DNA are a major threat for all of the living cells on the earth. Because of the biological significance, there have appeared many studies to try to measure DSBs. However, it is known that, by use of usual biochemical methodologies, quantitative evaluation is applicable only for high dose conditions. We have currently been developing the experimental method of single DNA observation on giant DNA molecules with fluorescence measurement. By applying single DNA observation, it has become possible to evaluate the probability of DSBs down to the low probability on the order of one breakage per several hundreds kbp. Actually, we have demonstrated that the DSBs induced by active oxygen species generated by photon are highly suppressed by the addition of polyamine, where the effect of polyamine is not to diminish the active oxygen species but to collapse the giant DNA into a compact state.
In the present paper, as the extension of this methodology we report the result of quantitative evaluation of DSBs induced by gamma-ray. It is demonstrated that the probability of DSBs per base pair is proportional to gamma-ray radiation dose (less than 100Gy) and that 1.7 DSBs are induced by 1Gy radiation dose per 10Mbp, which corresponds to one DSB per single human cell by 1mGy dose. Next challenge is the evaluation on the DSBs for the condensed human genomic DNA molecules caused by gamma-ray.

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One common feature of radioresistant bacteria is that they contain red carotenoid pigments. Their colorless mutants are more sensitive than wild types to ionizing radiation. Therefore, these red carotenoid pigments are thought to contribute to defense mechanisms of radioresistant bacteria against ionizing radiation. Carotenoid pigments are localized within the lipids of the cell surfaces in prokaryotes. From these facts, we consider that red carotenoid pigments in radioresistant bacteria must defense the lipids of the cell surfaces of these bacteria against ionizing radiation. In this study, the effects of beta-carotene and astaxanthin, which are typical carotenoids, on the degradation and peroxidation of a biological lipid (linolenic acid) induced by gamma irradiation were analyzed. Linolenic acid was dissolved in benzene at a final concentration of 0.5 M, and the carotenoids were added at final concentrations of 5.0 x 10^-8 to 8.5 x 10^-3 M. The prepared solutions were irradiated with ⁶⁰Co gamma-rays at a dose of 30 kGy. The effects of the carotenoids on the degradation of linolenic acid induced by gamma irraidation were analyzed by TBA reaction and measurement of the absorbance at 532 nm of the reaction solutions. The effects of the carotenoids on the peroxidation of linolenic acid induced by gamma irraidation were analyzed by measurement of the absorbance at 230-236 nm of the n-hexane solutions of the irradiated samples. In these experimental conditions, the relatively high concentrations of beta-carotene and astaxanthin inhibited the degradation of linolenic acid induced by gamma irradiation, while the relatively low concentrations of the carotenoids promoted the degradation of linolenic acid. The carotenoids did not affect the peroxidation of linolenic acid induced by gamma irraidation. These results suggest that carotenoid pigments are active in the prevention of damage to biological molecules after the formation of lipid peroxide induced by ionizing irradiation in radioresistant bacteria and that concentrations of the carotenoid pigments in the cells are strictly regulated.

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It is well known that neutron has more damaging effects with high RBE or a weighing factor depending on neutron energy, compared with low-LET ionizing radiations. In the present study, we aimed to evaluate the contribution of indirect action induced by radicals for the purpose of studying mechanisms of fast neutron effects or mechanisms of boron neutron capture therapy (BNCT) using relatively lower energy neutrons. Plasmid pBR322 DNA was irradiated with fast neutrons (1-10MeV) in the "Yayoi" fast neutron source reactor core installed at the University of Tokyo, and thermalized neutrons passing through a phantom made of acrylic resin to simulate human body. The single strand breaks (ssb) and double strand breaks (dsb) of the plasmid were detected by agarose gel electrophoresis. The amount of native DNA with a closed circular form decreased exponentially with the absorbed dose of fast neutron. The decrease was inhibited by DMSO in a concentration dependent manner. Contributions of the indirect action to strand breaks by fast neutrons and thermalized neutrons were calculated as 50 to 65 %, which was slightly lower than the value for X-rays. In the case of thermalized neutrons, ssbs and dsbs were increased by the addition of boric acid. Moreover, the increase of the concentration of 10B was found to increase the ratio of dsb/ssb.

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DNA-damaging agents from endogenous and environmental sources induce DNA-protein crosslink (DPC) damage, where proteins are irreversibly trapped on DNA through covalent bonds. For the elucidation of the biological effect of DPCs, it is crucial to monitor the induction and intracellular dynamics of DPCs. Several methods have been developed for the detection of DPCs. These include the alkaline elution, nitrocellulose filter binding, sodium dodecyl sulfate-potassium ion precipitation, and single cell gel electrophoresis (comet) methods. Although these methods provided valuable information about the induction and elimination of genomic DPCs induced by various chemical and physical agents, they are all indirect detection methods of DPCs based on the measurement of DNA and not crosslinked proteins, hence offering only semiquantitative information of DPCs. Thus, it would be useful to develop a direct detection method for quantitative analysis of DPCs. In the present study, we have developed a novel direct DPC detection method, where crosslinked proteins were specifically labeled with FITC and assayed for fluorescence. Cells were treated with various aldehydes, typical DPC-inducing agents, and genomic DNA was isolated. The induction and removal of DPCs in cells as well as their in vitro stability were monitored by the FITC-labeling method. These data will be presented in the meeting.

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The object of the study is to reveal whether the induction process of an abasic site (AP site) and the clustered DNA damage which contain AP sites (AP cluster) depends on ionizing density of radiation. Many studies using synthetic AP clusters incorporated into oligonucleotides reported that AP clusters retard base excision repair processes. Sutherland et al. (2002) reported that AP clusters are induced efficiently in human cells by low LET X-irradiation with the similar yields with those for the clusters which contain pyrimidine or purine base lesions. There, however, has been very little knowledge of AP sites and AP clusters induced by heavy ion beam irradiation. In order to clarify the relation between track structure of C ions (290MeV/nucleon, LET 13, 60keV/um) or X-rays and the induction processes of an AP or AP clusters, we measure the yield of AP sites visualized by the treatment of irradiated pUC18 plasmid DNA with the AP endonuclease (Nfo), which converts an AP site to detectable single strand break. Several scavenging capacities of the samples are tested to estimate the effect of indirect action of diffusible OH radicals in the induction of AP site. These experimental data will be discussed with theoretical radiation track structure in the respect of repair susceptibility of the AP cluster.

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Reactive oxygen species (ROS) are generated within cells by ionizing radiation via primary ionizing events as well as through activating metabolic cascades. ROS not only cause dysfunction of the target molecules but also perturb intra- and inter-cellular signal transduction pathways. XRCC4-deficient (XRCC4^-/-) cells generated in our programs from a human colon adenocarcinoma cell line HCT116 by a gene targeting exhibit a lower survival rate and a higher frequency of chromosomal aberration than XRCC4-proficient parental cells after exposure to either X-rays or hydrogen peroxide (H₂O₂). Since XRCC4 is a key component of the non-homologous end-joining, a predominant repair pathway for DNA double-strand breaks (DSBs), the increase in susceptibility of XRCC4^-/- cells to these insults might be attributed to the induction of DSBs. Consistently, formation of γH2AX foci, a marker of DSBs, is evidently observed in both cell lines exposed to H₂O₂ as well as X-rays. In the current study, further kinetic analyses of nuclear foci formation were carried out to elucidate role(s) of H₂O₂ on DNA damage induction. γH2AX foci induced in H₂O₂-treated cells were different in several features from those induced in X-irradiated cells. First, H₂O₂-induced γH2AX foci showed obscure contours in contrast to distinct round shapes of X-ray-induced ones. Second, the cells treated with H₂O₂ were not uniform in the number of γH2AX foci. Third, most of the H₂O₂-induced γH2AX foci did not colocalize with ATM[pS1981] foci, while the colocalization of γH2AX with ATM[pS1981] foci was apparently observed in X-irradiated cells. Fourth, γH2AX foci mostly disappeared by 8-h after H₂O₂ treatment, and then reappeared at 24-h after the treatment. These results suggest the presence of different processes for DSB-induction by H₂O₂ and X-rays. An association between cell-cycle phases and formation of γH2AX foci after H₂O₂ treatment is currently under investigation.

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Ionizing radiations induce a variety of oxidative DNA damage including isolated and clustered DNA damage. The latter damage is a specific one with ionizing radiation, which might be involved in expression of radiation effect. On the other hand, the former one is same as common oxidative damage with other oxidative stresses. Since the yield of isolated DNA damage is tens of times corresponding clustered ones, both isolated and clustered DNA damage are thought to be similarly important in expression of molecular mechanism of radiation effect. In the present study, we estimated both damage in cultured cells irradiated with heavy particle beams. Chinese hamster ovary AA8 cells were irradiated with gamma-rays, and carbon ions (C), silicon ions (Si) and argon ions (Ar) beams. Their linear energy transfer (LET) were 0.2, 13, 55 and 90keV/µm, respectively. For isolated DNA damage, chromosomal DNA was extracted from the irradiated cells by NaI, and it was analyzed with aldehyde reactive probe quantified by the chemiluminescence. Oxidized pyrimidine and purine lesions were exposed by enzymatic treatment used with endonuclease III (Endo III) and human OGG1. The total yields of intracellular isolated DNA damage decreased with increasing LET [Gamma > C > Si > Ar]. On the other hand, yield of intracellular clustered DNA damage were analyzed by static field gel electrophoresis with similar enzymatic treatment used with Endo III and Fpg. The total yields of intracellular clustered DNA damage showed similar tendency [Gamma > C > Si > Ar]. These results conform to our previous results in test tube. The irradiated cells showed decrease of survival frequencies with increasing LET. Our present and previous results indicate that isolated and clustered DNA damage impact not only quantitatively but also qualitatively radiation effects of heavy particle beams.

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It is known that DNA lesions induced by ionizing radiation and chemicals can cause mutation and carcinogenesis. In particular, "clustered damage" site, that is a DNA region with multiple lesions within a few helical turns, is believed to hardly be repaired. This type of damage is considered to be induced around high-LET radiation tracks and at track-end of secondary electron. However, details of the clustered damage sites are not known. In order to get experimental information about the hypothetical damage, we have developed an analytical method for measuring the degree of dispersion of distances between DNA lesions using Förster resonance energy transfer (FRET). Experimentally-obtained FRET efficiencies of random distributed AP-sites on heat-treated DNA almost correspond to theoretical ones calculated on the basis of exponential distribution. Now we have tried to apply the FRET methodology to plasmid DNA irradiated with some radiation qualities. The results and the perspective will be discussed.

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We have investigated the site selective damage induction in plasmid DNA by tuning soft X-ray energy using synchrotron radiation as a light source. We reported that the relative yields of base lesions to single strand breaks are strongly depend on the ionization of nitrogen or oxygen atoms in DNA (Fujii et al., 2009). The yield of base lesions at oxygen K-edge is highly more greater than those at nitrogen K-edge. The specific lesions are thought to be induced by direct ionization of DNA. In this study, we aim to reveal the role of direct ionization at the energy region of oxygen K-edge in the selective DNA damage. We measure the yields of single strand breaks (SSBs), base lesions, and AP sites produced in thin film of pUC18 plasmid DNA by irradiation of several soft X-rays which excite a1s electron to anti-bonding energy states or a vacuum level. The yields of base lesions and AP sites are determined by post-irradiation-treatment of the DNA with enzymatic probes, which excise (Nth and Fpg for pyrimidine and purine base lesion, respectively, and Nfo for AP site) and convert the lesions into detectable SSBs. The obtained data will be compared with our previous data obtained by soft X-ray irradiation around carbon and nitrogen K-edge region.

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In order to clarify the mechanism of DNA damage induced by K-shell photoabsorption of nitrogen and oxygen atoms, we have developed an X-band JES-TE300 electron spin resonance (ESR) spectrometer (JEOL, Japan) system at a synchrotron soft X-ray beamline BL23SU in SPring-8 (Japan), and examined the ESR 'in situ' signal during the soft X-ray irradiation. Pyrimidine nucleobases and Calf thymus DNA were used as sample without further purification. Samples were introduced into a high vacuum ESR chamber (~10^-7Pa) for soft x-ray irradiation, ESR measurement and X-ray absorption measurement. Monochromatic soft X-ray photons were provided using a grazing-incidence monochromatic equipped with variable-line-spacing-plane-gratings. Observed ESR spectra were numerically double integrated to obtain the relative spin concentrations. The g-factor of 2.000 of the unpaired electron arising only during irradiation in calf thymus DNA film is obtained not only around nitrogen but also around oxygen K-edge by comparison with standard ESR sample of Mn²⁺/MgO, and it is significantly lower than that of a free electron (2.0023). The photon energy dependence of the ESR intensity around the K-edge regions of nitrogen and oxygen is not simply proportional to X-ray absorption near edge structure (XANES), indicating that the production of unpaired electron species in DNA enhanced by the nitrogen and oxygen K-shell photoabsorption. It is well known that the hydrated water which strongly bond to the DNA exists even in vacuum. Such hydrated water may be plays an important role in the induction of unpaired electron around the oxygen K-edge. On the other hand, an enhancement of the yield of the unpaired electron species around the both nitrogen and oxygen K-edge is observed for evaporated cytosine film presumably due to electron capturing by cytosine. The enhanced unpaired electron yields will be discussed in respect of stable DNA damage formation and the electron capturing of the DNA molecules will be discussed.

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In order to clarify the characteristics of DNA damage and dose-rate effect induced by neutron irradiation, the induction of DNA double strand breaks (DNA-DSBs) was investigated in CHO-K1 cells and xrs5 cells (Ku-80 deficient mutant cells). The cells were incubated according to the ordinary procedures, and then exposed to a mixed field of neutron and gamma-ray (total doses: approximately 2Gy/50min and 2Gy/10min) in the D₂O neutron irradiation facility of Kyoto University Reactor. The cells were also irradiated with ⁶⁰Co gamma-ray (approximately 2Gy/50min) as a control. At one hour after irradiation, the cells were fixed with 4% formalin and stained immunochemically with gamma-H2AX or 53BP1 antibody. The number of DNA-DSBs was determined by counting the gamma-H2AX or 53BP1 foci. The numbers of gamma-H2AX or 53BP1 foci induced by gamma-ray irradiation increased with the dose up to 2Gy. The number of 53BP1 foci was larger in the xrs5 cells than in the CHO-K1 cells, in both of neutron and gamma-ray irradiations. This may suggest that the repair of DNA-DSBs induced by neutron was impaired in xrs5 cells, as well known in gamma-ray irradiation.

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We have estimated the DNA damage spectrum by track structure simulation method. The simulation result shows that the increase of the complexity of initial DNA damage and cell lethality show partially similar trend in their LET dependence. The goal of our study is to elucidate and modeling the relationship between initial DNA damage and radiation-induced cellular response by using the calculated DNA damage spectrum. For this purpose, in addition to the detail of the clustered DNA damage in nanometer scale, the spatial distribution of the damage in larger scale should be considered. In this presentation, The DNA damage spectrum calculated for gamma-rays, X-rays and heavy ions will be shown. The calculation was performed using the simulation system which had been improved in DNA model and damage spectrum analysis method. Next, the spatial distribution of each type of DNA damage around the radiation track will be shown. The simulated DNA damage spectrum and the spatial distribution such as the distance between two clustered DNA damages in chromosome domain in cell nucleus model was discussed in comparison with the yield of lethal damage estimated from the experimentally obtained survival curves.

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We have reported that selective induction of DNA strand breaks or base lesions can be partially realized using synchrotron monochromatic soft X-rays tuned in to particular energy of innershell ionization of DNA constituent atoms (Fujii et al., 2009). These evidences suggest that the monochromatic soft X-irradiation could be a powerful tool to reveal the process of the induction of complex DNA damage involved in biological effects such as cell killing or mutation induction. The ionized DNA molecule produced through innershell electron excitation and successive Auger relaxation is thought to be a precursor of DNA damage. When the energy of soft X-rays is close to the threshold of the innershell ionization, a produced photoelectron might be recaptured by the atom because the slow photoelectron could be overtaken by an Auger electron during the relaxation. This event causes a sharp decline of the Coulomb potential of the atom, and accordingly the photoelectron could be trapped by the atom again. In this study we semiclassically analyze the probability of the photoelectron recapture by DNA constituent atoms. Obtained results are discussed by comparison with our previous data of the unpaired electron showing significant change of the yield in energy regions of particular K-shell ionizations.

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Chromatin remodeling is important for the localization and function of many proteins involved in transcription, DNA replication and repair. Regulation of such activity within the chromatin context requires posttranslational modifications (PTM) of histones, such as ubiquitination, acetylation, methylation, sumoylation and phosphorylation that counterbalance the repressive nature of chromatin. In mammals, the FACT complex is essential for transcription initiation, relaxing chromatin condensation through displacement of the nucleosome. Being crucial in chromatin remodeling during transcription, and considering its general function as a chromatin remodeling facilitator, here we investigate the potential role of FACT during DNA repair mediated by homologous recombination (HR). In accordance with other previous works, our results also show that during transcription, FACT and RNF20, an E3 ubiquitin ligase recent found by our lab and others to be involved in DNA repair, interact with themselves. Moreover, this interaction seems to take place also during DNA damage response, which appears not to be related to transcription. Furthermore, depletion of FACT led to compromised DNA end resection through RAD51 and BRCA1 recruitment, whereas H2AX phosphorylation showed no major change. Additionally, RNF20 recruitment to the damage site and subsequent H2B ubiquitination, an important marker of chromatin relaxation, were also suppressed. Therefore, our data suggest that FACT might play a deciding function in chromatin assessment of other DNA repair proteins following DNA damage by loading of RNF20 to the damage sites. Together, they might play a critical role in HR independently of H2AX, revealing an alternative pathway of the chromatin remodeling during repair.

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