Dose-rate-effects on chromosome aberrations at low dose rate region has no been evaluated. Incidences of chromosome aberrations were analyzed in splenic lymphocytes from female SPF C3H mice continuously irradiated with low- or medium-dose-rate (LDR, MDR) of Cs 134 gamma-rays from 56 days of age to evaluate the dose-rate effects. Dose response relationship of dicentrics chromosome aberration frequencies was obtained at each dose rate (400 mGy/22h/day, 200 mGy/22h/day, 20 mGy/22h/day and 1 mGy/22h/day) using age-adjusted multiple liner regression analysis and a test for difference between radiation group and non-irradiated group on the assumption that the relationship is shown by a linear or a linear quadratic model. Values of the linear term, which are shown as slope, were decreased with reduction of dose rates from 400 mGy/day(18.2 mGy/h) to 1 mGy/day(0.045 mGy/h), indicating positive dose-rate-effects. Incidences of dicentric chromosomes at LDR (20 mGy/day) were compared with those in HDR(890 mGy/min) irradiation at each total dose to obtain dose and dose-rate effectiveness factor (DDREF). Incidence of translocation detected by M-FISH method in irradiated mice at 20 mGy/day was also used for obtaining DDREF. Dose and dose-rate effectiveness factor (DDREF) were obtained to be 4.5 for dicentrics and 2.3 for translocations using chromosome aberration rate. These results will be a useful for risk estimation of LDR radiation exposure. This study was performed under contract with the Aomori Prefectural Government, Japan.

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Study on dose and dose-rate-effect of chromosome aberrations in chronic low-dose-rate (LDR) exposure in human is very difficult, because they are usually exposed to from extremely low dose radiation and they are influenced by the effect of confounding factors, such as smoking. Therefore, we examined dose and dose-rate-effect of chromosome aberrations using mice chronologically irradiated by radiation different LDRs. In this study, C3H/HeN female mice were long-term exposed to ¹³⁷Cs gamma-rays at 20 mGy/22h/day(910 uGy/h), 1 mGy/22h/day(45.5 uGy/h) and 0.05 mGy/22h/day(2.27 uGy/h) from the age of 8 weeks up to a maximum of about 700 days in SPF condition. In present analysis, we observed that both incidences of translocations and dicentric chromosomes in spleen lymphocytes detected by M-FISH were increased almost in linear up to a total accumulated dose of 8000 mGy, at a LDR of 20 mGy/22h/day. We observed dose and dose-rate-effect of both chromosome aberrations at a LDR of 1 mGy/22h/day. In addition, the chromosome aberration rates were compared between two LDRs (20 mGy/22h/day and 1 mGy/22h/day).This study was performed under contact with Aomori Prefecture Government, Japan.

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It hasn't been clear known how an individual biological response affects the risk of radiation at a dose of low dose/low dose-rate radiation. We think the response would be elucidated, because the effects of the low level radiation on tissues and whole body might be important rolls. We would use model mice about the carcinogenesis regarded as the main factor of the radiation risk. We would evaluate dose/dose-rate dependence of the onset diseases in the intestine, and here shall present its preliminary results.

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Pyroelectric crystal emits high energy electrons in a vacuum by heating or cooling. X rays will be produced by bombarding a metal target with the high energy electrons. This makes it possible to make a compact X-ray source without a high voltage power supply. The compact X-ray source will be powerful tool in the low dose radiation research because the source can irradiate local region with a high space resolution. However, there are few studies of fundamentals for the X-ray sources. In the present work, the energy of produced X ray were measured at low pressures and compared with the calculated value. In the experiment, an LiTaO₃ single crystal (z-cut, 10 mm X 10 mm X 0.5 mmt) was used. The +z surface of crystal faced to a stainless electrode (30 mm diam., 2 mm thickness) at intervals of about 4.5 mm. A central hole of 1 mm diam. of the electrode was covered with a copper foil of 5 um thickness. A CdZnTe or CdTe detector was positioned at the intervals of about 3 mm from the copper foil. The crystal was heated form the room temperature to 120 ^oC. X rays were detected with an average rate of temperature change of 1.5 K/s and the pressure range of 10-25 Pa. In the same pressure, the measured results did not show the same results. However, X-ray energy and intensity increased with the decrease of the pressure. The maximum voltage obtained in the present work was about 22 kV. The voltage calculated from the geometry of the pyroelectric crystal was estimated to be about 21 kV. The estimated value agrees with the measured value. This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science (22659221).

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A hyper-radiosensitivity (HRS) has been reported in several mammalian cells irradiated with doses lower than 0.3 Gy. However, a mechanism for the HRS remains unknown. In the present study, we investigated the HRS in normal human fibroblast cells to elucidate the mechanism for this phenomenon. To determine the radiosensitivity, we irradiated normal human fibroblast cells with a soft X-ray machine at graded doses of 0.1-1.0 Gy at a dose rate of 0.596 Gy/min and allowed the cells to form colonies for two weeks. The result revealed that the sensitivity was composed of a biphasic survival curve; the slope in a range of 0-0.3 Gy (D₀=0.42 Gy) is steeper than that in a range of 0.4-1.0 Gy (D₀=0.57 Gy). This indicates that the radiosensitivity of normal human fibroblast cells in doses lower than 0.3 Gy is higher than that in doses of 0.4-1.0 Gy. Then, we examined the radiosensitivity in the cells enriched with a G1 phase in 70% and obtained a similar biphasic survival curve as that shown in the cells of random culture (G1 phase, 50%). On the other hand, to examine the possibility that the cells in the G2 phase are involved in the HRS, we determined mitotic index after 1 Gy irradiation with combined treatment with aphidicolin. The result indicated that G2 arrest was seen until 6 h and released 8 h post-irradiation. Therefore, we assume that the cells that go through the G2 checkpoint might be responsible for the induction of the HRS.

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To assess the biological effects of long-term low-dose-rate gamma irradiation, we have analyzed the induced transcriptional changes by DNA microarray using Affymetrix Mouse Genome 430 2.0 GeneChips. Eight week-old male C57BL/6J mice were exposed to whole body irradiation at a dose-rate of 20 mGy/22h/d for 400 days. The expression levels in 6 irradiated mice were individually compared with two pooled samples from control animals. Our results showed that the expression levels of at least 20 genes were modulated to more than 1.5-fold in liver of all 6 mice immediately after the irradiation (Radiat. Res. (2010) 174, p 611-7). In the present study, we have investigated the modulation kinetics of gene expression after long-term low-dose-rate irradiation. While the expression levels of most of the 20 genes were kept modulated for several hours, a few genes were modulated for up to 24 hours, after which, the modulation of expression was attenuated and was not detectable at 12 days after the irradiation. As some effects of low-dose-rate irradiation have been reported to emerge late after the end of irradiation, we also performed the microarray analysis of the liver 3 months after the irradiation. Our results suggest that there are a few genes wherein the expression was modulated at 3 months after the low-dose-rate irradiation.

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Assessment of biological effects of low dose and/or low dose-rate radiation is an important subject for radiation safety. To clarify the dose rate effects for low dose exposure, we established a hyper-sensitive mutation detection system. The hyper-sensitive mutation detection system is based on hprt deficient hamster cell line carrying a human X-chromosome. Even if a large deletion occurs on the human X chromosome and several essential genes in neighbor of the hprt are lost, those cells are viable and detected as mutants. We confirmed the system is 50-100-fold sensitive compared to conventional system. The cells were cultured in the medium containing HTO to irradiate with low dose rate. Then, mutation frequencies were calculated from the number of 6-thioguanine-resistant colonies. Our results suggest that induced mutation frequency could not be affected at dose rates over 0.09cGy/h.

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The radioadaptive response is the induction of cellular resistance to moderate or high doses of radiation by previous exposure to low doses. We have shown that cellular signaling pathways involving protein kinase C and p38 MAPK are involved in the induction of the radioadaptive response. It has been shown that, by using relatively high dose-rate X-rays, the priming doses of 1-10 cGy were effective for the induction of the adaptive response, whereas the pre-irradiation with higher doses (>10 cGy) was not effective for, but rather inhibitory to, the adaptive response in mouse m5S cells. Since in most situations, cells or organisms might be exposed with radiation at very low dose-rate in nature, it would be important to clarify if radioadaptive response could be induced by low dose-rate irradiation, and to examine the dose range for the induction of the adaptive response by low dose-rat radiation. Mouse m5S cells were primed with -rays (dose rate: about 1 mGy/min) by using Low Dose and Low Dose-Rate Irradiation System at Radiation Biology Center, Kyoto University, which is equipped with 137Cs as a -rays source. Cells were incubated for 5 hours after pre-irradiation, and then irradiated with 5 Gy -rays, followed by micronuclei assay to examine the adaptive response. The results showed that the micronuclei frequencies after 5 Gy irradiation were reduced in cells primed with 1 mGy/min irradiation comparing to in unprimed cells, indicating that the adaptive response was induced by pre-irradiation with low dose-rate -irradiation. Adaptive response was observed in cells treated with priming dose of 30-350 mGy, suggesting that the total dose effective for the induction of radioadaptive response with the low dose-rate irradiation might be much higher than that with high dose-rate irradiation.

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[Purpose] Frequency of micronuclei observed in germinating onion seed root tip cells 18 hours after irradiation increased with increase of absorbed dose in the low absorbed dose region and decreased with further amount of the dose. We firstly supposed that the disappearance was caused by cell death. However growth of the root was not stopped with as high as 160 Gy of γ-ray. No remarkable effects were observed about the growth of the seedlings with the high dose. Therefore, we turned the idea that the main cause of the disappearance was not cell death but temporal division delay after the irradiation. To verify the idea, we studied relationship between delay of cell division and micronuclei occurrence frequency in germinating onion seed root tip cells irradiated with gamma ray. [Method] Cs-137 γ-ray was irradiated to germinating onion seed at the irradiator at Division of Radiation Biology and Protection, Center for Frontier Life Sciences, Nagasaki University. Time dependence of micronucleus frequency after irradiation was observed. [Results] As increase of absorbed dose, peak of micronuclei incidence became late. Occurrence frequency of micronuclei increased with increasing absorbed dose until 4 Gy and decreased with further amount of the dose. As increase of absorbed dose, mitotic index peak of cell became late too.

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The extracellular effect of fibroblast growth factor-12 (FGF12) remains unknown because FGF12 cannot activate any fibroblast growth factor receptors (FGFRs). In contrast, we found that recombinant FGF12 was able to be internalized into the cytoplasm of the cells and identified two cell-penetrating peptide domains (CPP-M, CPP-C), which played a role in penetrating through plasma membranes. We also reported that FGF12 played an intracellular role in the inhibition of radiation-induced apoptosis and intraperitoneally added FGF12 inhibited radiation-induced apoptosis in the intestinal epithelial cells of BALB/c mice. This study evaluated the protective activity of FGF12 against radiation-induced intestinal injuries. FGF12 was administered intraperitoneally to BALB/c mice 24 h before or after total body irradiation (TBI) and the numbers of surviving crypts were determined 3.5 days after TBI with gamma-rays at 10 Gy. As a result, FGF12 significantly increased crypt survival even in the absence of heparin and FGF12-treatment significantly increased BrdU incorporation into the crypts, the depth of the crypts and the epithelial differentiation. In addition, we examined thirteen synthesized, 30-amino-acid partial peptides of the FGF12B polypeptide for their radioprotective activity using the above assays. We identified two peptides with radioprotective activity. One contained CPP-C sequence, and the other contained CPP-M sequence. These finding indicate that extracellular FGF12 strongly protects the jejunum against radiation-induced injury and suggests that cell-penetrating peptide domains are involved in this activity of FGF12.

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  Mucosa of small intestine is radiation-sensitive and damaged severely by abdominal expose of high dose radiation caused by nuclear accident or tumor therapy. To find the therapeutic methodology for the intestinal radiation damage, we constructed an experimental mouse model to estimate the effects of drugs. Damage of the mucosa of small intestine was induced by abdominal exposure of LD50 dose of 15.7 Gy of x-ray to anesthetized C3H/He mice. Regeneration of damaged mucosa was initiated 4 days after radiation and the levels reflected BrdU-incorporating microcolony rate and c-myb mRNA level in mucosa. Various drugs were injected with nutrient for 1 to 10 days after radiation and the effects of the drugs were compared. Drugs such as histamine, antithyroid drug and antidiuretic drug accelerated the regeneration rate and increased the survival rate. On the contrary, thyroid hormone and antihistamine drugs decreased the survival. Similarly to our previous report of the effects of anabolic steroids (Radiat. Res. 175, 367), conventional pharmaceutical drugs can be used to ameliorate intestinal mucosal damage by radiation.

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Radiation damage to DNA originates from DNA radicals directly induced by radiation or through chemical reactions with OH radicals produced in water radiolysis. Generally lifetimes of some DNA radicals are a few seconds (Hildebrand, 1997), which is significantly longer than that of OH radical in biological systems (a few nanoseconds (Roots, 1972)). Thus some reducing agents such as antioxidants may react with DNA radicals to chemically repair them from the radical forms, even under the low concentrations of the chemicals. O'Neill et al. (1983) showed this process by measuring reaction of dGMP radicals with antioxidants. The chemical repair mechanism mediated by antioxidants could be an important radioprotection process in a living system.
Edaravone has clinically been used as an antioxidant for treatment of brain infarct. Recently a radioprotection effect of edaravone in terms of reactive oxidative species (ROS) scavenging was also demonstrated through an in vivo experiment (Anzai, 2004). We infer that such effects can be attributed to its chemical repair property as well as its high reactivity with OH radical. In this study we verify the chemical repair property of edaravone.
At first, edaravone radicals produced in the reaction of edaravone with dGMP radical were observed by pulse radiolysis method. In order to verify the chemical repair of radicals in DNA macromolecules by edaravone we irradiated aqueous solutions of plasmid DNA with gamma-rays. In addition to strand breaks we quantified the chemical yields of AP sites and base lesions using base excision repair enzymes. Productions of AP sites and base lesions were more significantly inhibited than that of strand breaks by addition of edaravone. This result indicates that edaravone can chemically repair DNA radicals resulting in AP sites or base lesions on DNA.

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Purpose: In this study, we reviewed a radiation protection effect and immunological enhancement effect for Shunkokan which was quality of natural product.
Methods: Six week old male ICR mice were purchased from Japan SLC Inc. and kept under standard conditions (room temperature 22+/-3°, humidity 60%), and consistent feeding and drinking water . Mice were used for experiments after at least two weeks of Shunkokan administration and Shunkokan administration was continued until the end of the experiment . Measurement of peripheral blood cell counts in mice. We divided an experimental group into control group, Shunkokan treated group and 2Gy irradiation group and Shunkokan + 2Gy irradiation group in total four groups. We used an SOD activity detection kit as an antioxidation experiment and tested it. Statistical analysis was performed by a parametric ANOVA test among the groups to determine significant difference in blood cell counts for each group.
Result: The measurement of number of leukocytes revealed increase of leukocytes of a Shunkokan treated group in comparison with control group. In addition, a radiation protection effect was seen in Shunkokan+2Gy irradiation group after irradiation with lymphocyte counts. In the SOD-like active measurement, a rise was seen in a Shunkokan treated group.
Conclusions: The radioprotection effect can be expected of Shunkokan treated group. Mouse peripheral blood antioxidant activity was increased by Shunkokan , and a relationship between the suppressive effect on radiation-induced leukopenia and the antioxidant effect was suggested.

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Objectives: To evaluate if D-methionine, the dextro-isomer of the common amino acid L-methionine, can prevent oral mucositis resulting from irradiation (IR) during treatment for head and neck cancers. Methods: Female C3H mice, between 8-10 weeks of age, received IR without the use of anesthetics. Lead shields assured that only the head of the immobilized animal was irradiated. Either a single dose of 22.5 Gy or five fractionated doses of 8 Gy were delivered to the head of the mouse at a dose rate of 1.9 Gy/min. Thirty minutes before each IR, D-methionine, L-methionine (each at 150 mg/kg) or saline was administered by gavage. Animals were sacrificed on day 7 for single dose studies, or day 10 for fractionated dosing. Excised tongues were stained in 1% toluidine blue in 10% acetic acid for histologic evaluation. Macroscopic and microscopic images pictures were taken and were examined using NIH Image J software. The area of all ulcers, stained in dark blue, and the thickness of the lingual epithelium were measured. Results: A single dose of 22.5 Gy induced severe ulcers on the dorsum of tongues. Similarly, five fractionated doses of 8 Gy caused ulcers on the tongues, though not as severe as the single dose IR. In fractionated dose IR experiments, the size of ulcers and the thickness of keratinized epithelium demonstrated that administration of D-methionine reduced ulcer size compared to saline- or L-methionine-administration. D-methionine, however, did not prevent severe ulcers induced by a single 22.5 Gy dose. Conclusions: D-methionine can prevent IR-induced oral mucositis in mice and may be useful clinically to prevent such oral mucositis in head and neck cancer patients.

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 Fibroblast growth factors (FGFs) are a structurally related family of proteins and play important roles in numerous biological events, and are expected to be used for protecting various organs against radiation-induced injuries. Recently radioprotective agents that modify biological responses are eagerly desired. Here we summarize our study on FGFs as radioprotective agents and discuss their perspectives.

 We have evaluated the effect of FGF1 on damage of intestinal crypts and bone marrow cells caused by gamma-ray (or X-ray) irradiation. We found that when administered to animals prior to exposure to a high dose of radiation, FGF1 reduced the intestinal damage with the highest effectiveness among several wild-type FGFs examined. However, structural instability of wild-type FGF1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. We have developed an FGF1:FGF2 chimera (FGFC) that demonstrates structural stability and independence from exogenous heparin for its optimal activity. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. FGFC was superior to FGF1 in its protective activity determined by several protocols, including post-radiation administration. FGFC also reduced the radiation-induced damage to bone marrow cells.

 These results indicate that FGFs, especially FGFC, are effective radioprotective agents, and prompts their use in clinical settings.

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[Introduction]
The cancer therapy with ascorbic acid (AsA) is expected as a therapy with little effects, thus its use alone or combined with chemotherapy. However, little information has so far been reported regarding the combination of AsA with radiation. It is known that AsA shows cytotoxic effects to tumor cells which less content of intracellular catalase neutralizing H ₂O₂, therefore, the generation of reactive oxygen species (ROS) derived from H ₂O₂ is thought to be involved. While, most of cell death induced by X-irradiation depend on the intracellular ROS production. In the present study, the action of AsA combined with radiation and the role of ROS was studied by using human promyelocytic leukemia HL60 cells.
[Material and Methods]
HL60 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum. A final concentration 0.01 mM - 2.5 mM AsA was added to the culture and the number of a viable cell was counted after 24 h. Catalase (1,300 U/mL ) was added to the culture, then the cells were treated with AsA, 2 Gy X-irradiation and a combination of them. The viable cells were counted after 24 h. The intracellular ROS production was measured by a flow cytometer using ROS-sensitive fluorescent probe CM-H ₂DCFDA.
[Result and Discussion]
AsA showed cytotoxic effects on the growth of HL60 cells with a dose-dependent manner. When X-irradiation was performed after AsA treatment, the additive cytotoxic effects were observed. When catalase was added to the culture with AsA alone, the cytotoxic effects of AsA were disappeared. In addition, the additive cytotoxic effects were decreased to the same level obtained by X-irradiation alone. The intracellular ROS production reached to the peak at 12 h after X-irradiation, but AsA resulted in the decrease of ROS. These results suggest that the action pathway of hydroxyl radical derived from H ₂O₂ was different from between AsA and X-irradiation.

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We have found specific base positions in the DNA cleavage by the irradiation of X-rays after association of a cisplatin with DNA. In cancer therapy it is well known that application of X-rays radiation with cisplatin dosage enhances the anti-cancer effect. However, the mechanism of enhancement in the anti-cancer has not been elucidated in the level of DNA base. We have developed a new approach for the determination of the DNA cleavage position using the Sanger method. Here we have specified the positions of base cleaved by X-ray radiation with cisplatin association with DNA. The cleavage patterns of the DNA irradiated by X-ray show that the DNA was cleaved by 100 Gy irradiation, and the four kinds of bases were cleaved. Ciplatin associated DNA without uthe irradiation was not cleaved. On the other hand, the ciplatin associated DNA specifically enhanced the cleavage at the bases of "guanine" and "adenine". When the irradiation increased to 150 Gy, the specific cleavage at the "guanine" and "adenine" were still detected. These results indicate that supra-additive effect by cisplatin association and X-ray irradiation on the DNA damage was caused by the specific cleavage at the base positions of "guanine" and "adenine"

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NKG2D ligand express in raft area on the cellular membrane. Tumor cells express much NKG2D ligand by stress, such as radiation. But tumor cells do not exclude in the immune system, because of secretion of NKG2D ligand by mechanism of tumor cells. This mechanism is not clear. In this study, we elucidated the mechanism of X-ray induced-expression and secretion of NKG2D ligand on the tumor cell surface. NKG2D ligand was retained on the membrane raft and the vicinity of raft after irradiation for 1hr, this retention was suppressed by SMase inhibitor. When MMP14 in the exosome observed after irradiation, active MMP14 was observed in the exosome, and MMP14 was not observed in the exosome by SMase inhibitor. These results indicate that degradation of NKG2D ligand was inhibited by the secretion of MMP14 that mediated SMase activation by irradiation.

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To evaluate the effects of radiation exposure or radiation treatment, it is important to know the biological effects of radiation. Depending on the LET or radiation type, the level of those effects vary most probably due to the difference of the radiation damage and its repair processes. In this study, effects of growth conditions were examined, using X-rays and high-LET He ions (LET = 89 keV⁄μm).
The E. coli strain CSH100 (wild-type) and recA deficient strains were used. After irradiation, the survival rate and the lacI mutation frequency were measured. Rich medium and minimal medium were used to look at the effect of the culture condition on radiation sensitivity.
As a result, when rich medium was used for pre- and post-irradiation culture of X-irradiation, the survival rate was higher than when minimal medium was used. When rich medium was used for either pre- or post-irradiation culture only, the enhancement was not observed. On the other hand, the medium-dependent radiation sensitivity was not observed in CSH100recA. Therefore, homologous recombination or SOS response is implicated to play a role in the medium-dependent radiation sensitivity.
In the case of He ions, the medium-dependent radiation sensitivity was not observed. The mutation frequency after He-irradiation decreased slightly compared with that of X-ray irradiation. Previous studies reported that He-irradiation causes SOS response to cell. These results suggests that different types of DNA damages are produced after X- and He-irradiation, and this leads to the difference in medium-dependent radiation sensitivity and mutation frequency.

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According to the depth of the cancer in a body, three different accelerated energy of carbon beams; 290, 350, 400 MeV/u are used for therapy at HIMAC. The difference in biological effectiveness by the difference in the accelerated energy is however not well known. We measured biological effectiveness for different energy of carbon beams with the same SOBP (Spread Out Bragg Peak) to evaluate the difference. We have measured the physical characteristics of the beams, and perform biological experiment. Here, we used human salivary grand tumor originated HSG cells that have been used to measure biological effectiveness for particle beams. We used 290 and 400 MeV/u carbon beams at proximal (-25 mm), middle (0), distal (+25 mm) and distal end (+28 mm) in the 60 mm SOBP beam. Cells were exposed to the beam at those position and applied conventional cell survival assay after 14 days after irradiation. The survival curves were fitted to Linear-Quadratic model, and D10 dose and RBEs to 200 kV X-rays were obtained. Physical depth-dose distribution to the depth was steeper in the SOBP at 290 MeV/u compared to that at 400 MeV/u. In the other hand, depth-RBE distribution was increases tenderly to 290 MeV/u than that to 400 MeV/u beam. Finally the uniformity of the biological effectiveness (Dose x RBE) becomes flat as well as pre-clinical study at start of HIMAC clinical trial. Both biological uniformity and absolute biological efficiency to the 290 and 400 MeV/u beams were similar each other.

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The aim of this study was to evaluate the contribution of cellular alternations in the hippocampus of rats after prenatal x-ray irradiation using manganese-enhanced MRI (MEMRI). All radiation-exposed rat brains showed a prominent dilatation of the cerebral ventricle. The enhanced areas by MEMRI decreased between the DG (dentate gyrus) and CA3 (cornu ammonis region 3) areas in radiation-exposed rats compared to normal rats. Compared to non-exposed rats, whole-brain volumes of radiation-exposed rats significantly decreased. Moreover, no histopathological abnormalities were observed except for a decrease in cell density in the hippocampal region. In conclusion, MEMRI can detect developmental abnormalities of the brain in the hippocampus in a radiation-exposed rat model.

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AIM: To investigate pathological effects of internal radiation on human body, we have determined radioactivity on samples of Nagasaki acute atomic bomb casualties. Alpha particle forms in the paraffin-embedded specimen were detected by a classical method of autoradiography. We have already reported that the presence of a genomic instability, 53BP1-focus formation, in the atomic bomb survivors as a predisposition to cancer. 53BP1 rapidly forms nuclear foci at the sites of DNA double strand breaks. The much 53BP1-focus expression was observed in the cells surrounding Thorotrast granule of liver. In this study, we determine 53BP1-focus formation on tissue specimens. The much expression of 53BP1-focus was also observed on the liver of Nagasaki atomic bomb acute casualties, who was outdoor 0.5km from the bomb hypocenter. Hyposensitivity to radiation induced apoptosis was observed in liver, kidney and lung. In this study we investigated radiation induced DNA damage response and autophage. Autophage involves the sequestration of cytosolic proteins and organelles within double-membrane structures termen autophagosomes and their subsequent degradation via lysosomal hydrases. MATERIAL and METHODS: 1)Rats were exposured 8Gy by X ray. The liver, kidney and lung specimen were prepared after 3,6 and 24 hours and determined 53BP1 and LC3 by immunohistochemistry, and autophage by electron microscope. 2) The liver specimen from Thorotrast patient were studied. RESULTS: 1) 53BP1-focus expression, LC3 expression and autophage by electron microscope were observed in the rat liver, . 2) The much 53BP1-focus expression and LC3 expression were observed in the cells surrounding Thorotrast granule of liver. These suggested internal radiation exposure triggers DNA double strand brake and forms 53BP1-focus. The much expression of 53BP1-focus was also observed on the spleen of Nagasaki atomic bomb acute casualties, who was outdoor 0.5km from the bomb hypocenter and died at 68th day. and spleen. These suggested internal radiation exposure triggers damage response and autophage.

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Medaka is a teleost fish native to the Asian countries. It has long been the study object of biology in various fields including radiation biology, especially in Japan. Documented data on radiation effects in the medaka system are accumulating. Recently, the establishment of a transgenic medaka, cab-Tg(rag1-egfp), makes it possible to directly visualize the thymus in vivo in a real time manner (Li J. et al., Journal of Immunology, 2007). In our previous investigation, decrease in the volume of thymus was confirmed in the cab-Tg(rag1-egfp) medaka after exposure to a dose from 5 to 10 Gy of X-rays. After 10 Gy of X-ray irradiation, the volume of medaka thymus was decreased on the next day, and started to recover from 5 day. After exposure to a dose at 30Gy of X-ray, the thymus was disappeared after 2 day and recovered at 22 day. To comparatively study the effects between low and high LET irradiations on medaka thymus, in the present work, irradiations from accelerated Fe ions was verified. A total of 36 medaka fish were used in the control and the experimental groups exposed to a dose from 0.5 to 10 Gy. The preliminary data obtained so far showed that decrease in the volume of thymus in few medaka could be detectable in the 0.5 Gy-irradiated group, while this effect was observable in all the medaka receiving a dose at 2 Gy or more. The RBE of Fe-ion was estimated around 3. Further investigations are needed. These findings suggest that the cab-Tg(rag1-egfp) would be a good tool for detecting the radiation effects by monitoring the volume decrease in thymus at live.

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[Introduction] In cases of high-dose radiation exposure, one of the first priorities is to reduce and recover from radiation damage to high-reproducibility tissue, such as the hematopoietic system, intestinal mucosa, and skin. Medications can be used for primary care because hematopoietic stem cell transplant is not appropriate for use in the case of mass casualties with respect to timely treatment. However, almost all the drugs/growth factors shown to be effective in this regard in the past are not prescription drugs in Japan. There are problems with respect to standing and emergency in prompt response; therefore, appropriate countermeasures based on prescription drugs are required. In this study, appropriate therapeutic strategies for high-dose radiation casualties, especially with respect to damage to the hematopoietic system and alimentary canal, were studied using a combination of prescription drugs in a mouse model.
[Materials and Methods] Eight-week-old female C57BL/6J jcl mice were exposed to 7 Gy total body irradiation by using a ¹³⁷Cs gamma source, and test drugs were given after irradiation. Recombinant human EPO, G-CSF, Nplate and nandrolone decanoate (ND) were used as test drugs. On day 30 after irradiation, survival mice of body weight, peripheral blood cells, bone marrow cells, hematopoietic progenitors, and cell surface antigens were analyzed.
[Results and Discussion] Approximately 10% of irradiated mice survived for 27 days, but all the mice died at 30 days. Of the mice that were administered EPO+G-CSF+ND or EPO+G-CSF+ND+Nplate for 3 or 5 days, 40-50% survived for 30 days. However, no significant differences were observed between the value of the parameters evaluated for irradiated mice and control. These results show that an appropriate combination of prescription drugs may help treat individuals exposed to high-dose radiation.

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An increased thyroid cancer risk is most concerned in Fukushima multiple commercial nuclear reactors accident with total discharged amount of ¹³¹I 130∼160 PBq officially estimated. A summary was provided mainly from the related information opened in Web for the public. The evacuation started 6 hours after the March 11 earthquake while the discharge of radioactive materials began the next day. Sometimes the facility location and wind direction made the radioactive plume to arrive in the Pacific Ocean. In the areas, deposited large amount of radioactive materials probably on March 15 and 16, the averaged daily air dose rate basically decreased after March 17. Within Fukushima pref., Ouu Mountains made it difficult for the plume to arrive in Aizu area compared with Nakadori and Hamadori areas. Air dose rate data were mainly behind a fear of increased thyroid cancer risk in the northwest area from the NPP. On the other hand limited monitoring data were provided in Miyagi pref. and ¹³¹I atmospheric concentration was not systematically monitored even in Fukushima pref. specifically before March 17. The earliest available concentration within the NPP facility was 5940 Bq/m³ in March 19 and in the following two days some areas could be over 1000 Bq/m³ at one time in south as well as in northwest. About 20% was less than 20 years old among two million in Fukushima or among 146000 individuals of high risk areas involved in the accident. Most of thyroid cancer cases are nonfatal and effectiveness of protective measures taken such as evacuation and contaminated food control will be confirmed only through non-biased risk assessment based on such as cancer registry.

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The present study described the results of the surveys of public perceptions of the social issues and risks related to radiation in 2006-2007. One survey involved personal visit interviews carried out, 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. And nuclear power-related items such as nuclear weapons, nuclear wastes and nuclear facilities were regarded as the terrible items. However, majority did not bring up any image from the terms of radiation and health effects of radiation. In the second survey, we asked Japanese adults to rank 30 items related to various types of technologies and human activities according to their subjective judgments on the order of perceived magnitude of risk using Web-based questionnaires. As a result, the risk perceptions of the Japanese, irrespective of gender, age and occupation, have been uniform during the last 25 years, and the majority regarded hand guns, nuclear power and smoking as the high-risk items. The third questionnaire survey was conducted on radiation risk, targeted at nurses (170 females). Most of them tended to assess exposed parts, doses and damage potentially suffered based on their professional experiences and knowledge in order to distinguish acceptable risks rationally from unacceptable ones. Then, nurses with children perceived food exposure as more risky and infertility as less concerned compared with those without. The risk communication in consideration of these risk perception is the problem we should solve immediately.

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Melanogenesis in melanocytes is considered one of the dermal reactions against ultraviolet (UV) rays. The melanogenesis in melanocytes is induced by cytokines, which are discharged from keratinocytes that exist in the intercellular space around the melanocyte, as a result of UV irradiation. However, the mechanism of self-induced melanogenesis in a single melanocyte, or co-induced melanogenesis between multiple melanocytes, has not been fully explained. We conducted a verification study evaluating the bystander effect, in an attempt to elucidate the mechanism of the co-induced melanogenesis by multiple melanocytes.
We successfully identified the bystander effect indicated by the melanogenesis, as a result of medium transfer of B16 melanoma cells, which were cultured for 24 hours after being exposed to UVA irradiation, to non-irradiated cells (bystander cells). Through our confirmation study regarding the functional mechanism of bystander cells, we found reduced level of mitochondrial membrane potential after 1-3 hours from the medium transfer, increased level of intracellular oxidation after 12 hours, and the generation of melanin radicals, as well as delayed long-lived radicals after 24 hours from the medium transfer. Further study on bystander factors revealed that the administration of EGTA treatment at the time of medium transfer leads to inhibition of melanogenesis, and neutralization of mitochondrial membrane potential level, as well as the intracellular oxidation level to the control level. The results showed that the UVA irradiation bystander effect in the B16 mouse melanoma cells, indicated by melanogenesis, was induced by the increase of intracellular oxidation level, as a result of calcium ions, one of the bystander factors, acting on mitochondria.

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Biologically uncommon D-beta-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. In this study, in order to clarify the relationship between the formation of D-beta-Asp and CML, immunohistochemical analysis using anti-D-beta-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice skin. Immunohistochemical analyses and the western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that the epidermal proteins were immunoreactive towards antibodies for both D-beta-Asp containing protein and CML, while the dermal proteins were immnoreactive for an anti-D-beta-Asp containing peptide antibody but not for an anti-CML antibodies. The epidermal and the dermal proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10 and keratin-14 , and the collagen type III, respectively.

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Parthenolide (PTL), a nuclear factor-kappaB (NF-kappaB) inhibitor, has significant thermo-enhancement effects. It is known that step-up hyperthermia (SUH), induce thermotolerance in cells by primary heating and these cells show thermo-resistance to second heating. Modification of thermosensitivity by treatment with PTL prior to hyperthermia was investigated in the human prostate cancer androgen-independent cell lines PC3 and DU145. In addition, we analyzed the mechanisms related to induction of apoptosis or G2/M cell-cycle arrest via the effects of ERK1/2, p38 and SAPK/JNK signaling on mitogen-activated protein kinase (MAPK). Lethal damage caused by mild hyperthermia at 41.0ºC or 42.0ºC in both cell lines resulted in a low level of thermosensitivity, while sequential combination with PTL showed significant thermosensitization. PTL pretreated prior to SUH (42ºC for 30 minutes, 43.0ºC or 43.5ºC for various periods) prevented thermal tolerance of the cells to second heating and resulted in significant thermo-sensitization. Induction of apoptosis by combination of PTL and hyperthermia at 44.0ºC was determined by the ratio of sub-G1 division phase by flow cytometry, which increased significantly in comparison with single treatment, and was more effective with PC3 than DU145 cells. The behavior of ERK1/2, p38, and SAPK/JNK signaling in the MAPK cascade by combined treatment with PTL and hyperthermia were examined by western blotting. As for PC3 cells, ras-downstream p-ERK1/2 was activated and p-p38 slightly activated by combined treatment with PTL and hyperthermia in comparison with each alone. As for DU145 cells, ERK1/2 was not changed, while p38 and SAPK/JNK were slightly activated by combination treatment. Those results were related to increases in induction of apoptosis, G2/M cell-cycle arrest, and lethal damage of cells via the MAPK cascade. Together, our findings demonstrate that PTL is an effective thermo-sensitizing agent for multidisciplinary therapy for human prostate cancer.

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We have shown that the combination of heat stress and superoxide dismutase-mimic nitroxide, TEMPO, causes apoptotic and autophagic cell death, in a dose- and time-dependent manner (Zhao et al., Apoptosis 15: 1270, 2010). In this study, we used the mitochondria-targeting derivative MitoTEMPO. Here, we identified that (1) 5 mM TEMPO-44^oC/30 min combination induced apoptosis, while 5 mM TEMPO-44^oC/60 min combination induced delayed autophagic cell death in HeLa cells. This co-treatment inhibited the processing of heat-activated procaspases-3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy; (2) the combination of 0.1-0.5 mM MitoTEMPO and 44^oC/60 min enhanced the two modes HeLa cell death in a dose-dependent manner. Together, upon combination with heat stress, MitoTEMPO induced approximately 10-fold higher apoptotic and autophagic cell death than TEMPO. Thus, the MitoTEMPO is a unique thermosensitizer which synergistically induce more apoptotic and autophagic cell death than TEMPO.

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Effects of chronic gamma-irradiation were investigated in the experimental ecosystem model, i.e., microcosm, consisting of flagellate algae Euglena gracilis as producers, ciliate protozoa Tetrahymena thermophila as consumers and bacteria Escherichia coli as decomposers. At 1.1 Gy/day, no effects were observed. At 5.1 Gy/day, cell densities of E. coli showed a tendency to be lower than those of controls. At 9.7 and 24.7 Gy/day, population decrease was observed in E. coli. E. gracilis and T. thermophila died out after temporal population decrease and subsequent population increase in T. thermophila. It is likely that this temporal population increase was an indirect effect due to interspecies interactions. A dose rate-effect relationship was determined using the effect index for microcosm (EIM), which holistically represents differences in populations of all the constituent species between irradiated and control microcosms. A 10 % effect dose rate, at which EIM was 10 %, was calculated to be 3.4 Gy/day. Similarly, 10 % effect concentrations for some heavy metals were calculated from data which the authors had previously obtained in this microcosm. Comparison of these community-level effects data with environmental exposure data suggests that ionizing radiation, gadolinium and dysprosium have low risks to affect aquatic microbial communities while manganese, nickel and copper have considerable risks.

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The study on the effects of high LET radiation and chronic exposure on animals and plants is important in environmental radiation protection, because radiation effects in environment could be attributed to the chronic exposure to alpha emitters as well as gamma and beta emitters. Therefore, we studied the effects of heavy ions at NIRS-HIMAC with various high LET and of chronic exposure to gamma radiation on the growth of Enchytraeus japonensis (E. japonensis), a species of terrestrial earthworm, kindly donated by Dr. M. Myohara, (National Institute of Agrobiological Sciences, Tsukuba, Japan). The earthworm is known to grow very rapidly and increase more than 10 fold in population every half a month. The earthworm was exposed to gamma radiation or C, Ne, Si, Ar, Fe ion with the energy of 290, 400, 490, 500 and 500 MeV/u, respectively. The earthworm was then reared on plain agar medium in Petri dishes by occasional supply with powdered rolled oats and autoclaved distilled water, and the number of worms was enumerated 30 days after irradiation. Chronic exposure to gamma radiation was performed during 30 days rearing period. Heavy ions clearly showed stronger effects than gamma ray with respect to the growth inhibition of the earthworm. As LET was increased, heavy ions appeared to inhibit more effectively the growth, however the effects of Si, Ar and Fe ion were not significantly different. The dose or dose rate required to suppress 50% of growth was approximately 5Gy/day, total dose of 150Gy in chronic exposure compared with 22Gy in acute exposure.

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Native plants are continuously exposed to ionizing radiation in radiologically contaminated areas. For detection of biological responses of the plants to the continuous irradiation in the natural environment, radiation-induced gene expression was examined in a Japanese native conifer, Japanese cedar (Cryptomeria japonica). An initial screening of the transcripts from the plants by the HICEP (High Coverage Expression Profiling) analysis selected 53 transcripts that exhibited 3 times higher or lower expression levels after gamma ray exposure for 25 days at a dose rate of 100 mGy/day. Many of the transcripts showed homology to known coniferous ESTs, 17 % (17 out of 53) matched the EST sequences of Japanese cedar and 60 % (32 out of 53) were homologues to those of any coniferous plant species. Additionally, 58 % (31 out of 53) of the transcripts were homologous to the genes of a model plant arabidopsis, which enabled functional estimation of the transcripts. The transcripts were further analyzed for irradiation dependent expression patterns by the real time PCR method to be identified as the radiation responsive transcripts. Use of the transcripts as biomarkers for the radiation effects in Japanese cedar will be discussed.

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The method of human tooth enamel electron spin resonance (ESR) dosimetry was used to obtain individual absorbed doses of residents of settlements in the vicinity of the uranium mine and uranium processing plants in Stepnogorsk city, Akmola oblast, Kazakhstan. Measured teeth samples were extracted according to medical indications. In total, 33 tooth enamel samples were analyzed from the residents of Stepnogorsk city (180 km from Astana city, Kazakhstan). Some results of dose estimation of residents staying in Stepnogorsk city and workers of uranium enterprises were included. About 10 tooth samples have been collected from the workers of uranium plant. Results of tooth enamel dose estimation shows us small influence of working conditions to workers, the maximum excess dose is less then 100 mGy. This is pilot study of ESR dose estimation, for a final conclusion additional sample is required. Some results of excess estimation by ESR dosimetry method from Ust-Kamenogorsk region have been included to this presentation. This region is well known place of uranium processing industry.

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   Radiation therapy, demand of which is recently increasing, requires irradiation of a tumor with reducing dose onto normal tissues surrounding the tumor in order to reduce risk of side effect of the irradiation such as secondary carcinogenesis. Such requirement led to development of advanced methods such as IMRT and ion-beam therapy, which enable us to plan complicated dose distributions. However, most typically used dosimeters such as ionization chambers and film dosimeters allows us only to measure dose at a certain point or two-dimensional dose distribution on the film plane.
   Polymer gel dosimeters are proposed as a promising tool to check three-dimensional dose distributions, however, widespread polymer gel dosimeters contain toxic components such as acrylamide and methacrylic acid, leading to difficulties in handling. It was tried to solve this problem by employing naturally-derived polymer hydroxypropylcellulose (HPC) as a base matrix. Solutes dissolved into hydrogel matrix of HPC were also selected from the viewpoint of low toxicity, and hydroxyethyl 2-methacrylic acid and poly-ethylene glycol ester were selected.
   Such environment-friendly polymer gel dosimeter was irradiated with ⁶⁰Co γ-rays in a practically important dose range of 1-10 Gy, and an increase of white turbidity was observed. This turbidity would be attributed to macromolecule products in radiation-induced polymerization reactions. UV-vis absorption analysis was also done for more brief but quantitative estimation of the turbidity increase. It was found that absorbance dependences on dose are different for different wavelengths and that absorbance at shorter wavelength (300-500 nm) tends to saturate at relatively low dose of about 4 Gy while that at longer wavelength (500-800 nm) is not so much wavelength dependent. In addition, effects of solutes composition on these behaviors will also be discussed.

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The purpose of this study was to investigate possible linear energy transfer (LET) dependence for a polymer gel dosimeter using proton beam absorbed dose measurements. Polymer gel containing the antioxidant tetrakis-hydroxymethyl-phosphonium chloride was irradiated with 210 MeV protons, and the gel absorbed dose response was evaluated using MRI. The LET distribution for proton beams was calculated as a function of depth using the Monte Carlo code PHITS. PHITS simulations of a proton beam-line under a range of beam conditions have been compared with MR analysis of irradiated polymer gel. The result calculated by using a part of LET corresponded with the gel's one.

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