In order to understand the role of BRCA2 protein in homologous recombination (HR) repair and radio-sensitization, we utilized RNA interference (RNAi) strategy in vitro and in vivo with tumor cells. HeLa cells transfected with BRCA2 siRNA (Qiagen) as well as negative-control siRNA for 48 hours were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock- or negative control siRNA transfected cells. DNA double strand break (DSB) repair as measured by constant field gel-electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno-staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the NHEJ proteins nor the phosphorylation of DNA-PKcs was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in vivo showed an efficient inhibition of tumor growth in xenograft model using HeLa cells. Our results demonstrate down-regulation of BRCA2 leads to radio-sensitization through inhibition of DSB repair, specifically the HR pathway, and a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise.

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Background.
Thyroid hormone receptors (THR) broadly regulate cell differentiation and metabolism acting as ligand-inducible transcription factors. Given a large variety of genes regulated by THRs and multiplicity of cellular processes potentially influenced by THRs, we addressed the role of THRΒ (thyroid hormone receptor beta) in cell radiosensitivity.
Material and Methods.
Wild-type and mutant THRΒ were overexpressed in several cell lines using an adenovirus-mediated gene delivery and their effects were examined after cell exposure to Γ-rays.
Results.
Wild-type THRΒ decreased clonogenic survival of the cell lines with low levels of endogenous THRΒ, retarded their growth and synergized with radiation in decreasing proliferative potential and promoting cellular senescence. These changes were accompanied by the accumulation of p21 and p16 inhibitors of cyclin-dependent kinases and by the decrease of Rb (retinoblastoma protein) phosphorylation. Mutant THRΒ produced a radioprotective effect, attenuated radiation-induced growth inhibition and cellular senescence.
Conclusions.
The results demonstrate that THRΒ is one of the factors that modulate cell radiosensitivity. This newly recognized property of THRΒ may be used for identification of the molecular mechanisms underlying cell radiosensitization and stress-induced senescence.

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Purpose:
Tumor microenvironment is dramatically altered after radiotherapy. Hypoxia-inducible Factor-1 (HIF-1), in response to the alteration, promotes tumor radioresistance by inducing various gene expressions. The purpose of this study is to analyze the postirradiation dynamism of intratumoral HIF-1 activity and the underlying molecular mechanisms.
Methods: Subcutaneous tumor xenografts with a novel HIF-1-dependent reporter gene were locally irradiated with 5 Gy of gamma-ray. The postirradiation change of intratumoral HIF-1 activity was optically imaged as the HIF-1-dependent bioluminescence.
Results:
Intratumoral HIF-1 activity was transiently decreased 6 h postirradiation in response to radiation-induced reoxygenation of hypoxic tumor cells. Later, the HIF-1 activity turned to increase, although the cells were still re-oxygenated. The former decrease of HIF-1 activity was suppressed in the von Hippel-Lindau (VHL)-deficient cell line. On the other hand, the later increase was suppressed by PI3K/Akt/mTOR pathway-inhibitors (LY294002 and rapamycin) or a non-metabolizable glucose analog (2-deoxy-D-glucose). In vitro studies confirmed that just after reoxygenation treatment, addition of glucose dramatically accelerated HIF-1alpha translation in PI3K/Akt/mTOR-dependent manner.
Conclusion:
After irradiation, intratumoral HIF-1 activity transiently decreases via VHL-dependent degradation of HIF-1alpha protein and subsequently increases via glucose-dependent up-regulation of HIF-1alpha translation. Therefore, we propose here that the that alteration of tumor microenvironments influences HIF-1 activity after irradiation; in postirradiation reoxygenation in early phase- and re-glucose (improvement of glucose distribution to hypoxic cells) in late phase conditions, respectively. To our knowledge, this is the first report emphasizing the importance of radiation-induced alteration of glucose- as well as hypoxic-microenvironment in the regulation of intratumoral HIF-1 activity.

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Yield of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in heavy-ion irradiated human HL-60 cells was measured over an LET range of 20 keV/μm to 440 keV/μm using carbon, neon, silicon and iron ions. The ion species dependence was also examined for carbon and neon ions at an LET of 80 keV/μm. After irradiation, DNA was extracted from the cells and digested into nucleosides. 8-OHdG was separated as a single peak on a chromatogram of ECD detector. 8-OHdG yield was decreased with increasing LET with a saturation tendency in the high LET region. The yield of 8-OHdG for neon ions were significantly higher than that for carbon ions at an LET of 80 keV/μm. This result could result from the larger low-LET penumbra area compared with the case of carbon ions. The LET dependence of the oxygen effect for 8-OHdG production was examined to evaluate reduced oxygen effect at the high LET region. The yield of 8-OHdG at the hypoxic condition had a tendency to be saturated above the LET value of about 100 keV/μm, in contrast with the decrease of the yield with increasing LET for the air-saturated condition. These results are compatible with the oxygen-in-the-track model to explain the absence of oxygen effect for high-LET radiations.

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Cell killing of X-rays is caused by direct action and indirect action. Two thirds of the effects are caused by indirect action under oxic condition. However, there are few reports on the effects under anoxic condition. We examined here contribution of indirect action under anoxic condition for cell killing. CHO cells were irradiated by X-rays (200kVp, 20mA) under oxic and anoxic conditions. Protective effect of DMSO that specifically caught OH radicals was estimated from the cell survival. Fractions of indirect action through OH radicals were calculated the contribution of the action using a method by Shinohara et al. (Acta Oncol., 35, 869-75. 1996). The portion in contributions of indirect action under oxic and condition was approximately 70%. However, it decreased to be 60% when cells were irradiated under hypoxic condition. G_(OH) value is independent with oxygen concentration (C. V. Sonntag, The Chemical Basis of Radiation Biology, 33. 1987). The difference of contribution of the action between oxic and anoxic conditions may be explained that oxygen participates in reaction with OH radicals and DNA.

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It has been long stated that cellular inactivation through neutron irradiation is mainly caused by energy deposition in DNA molecules from recoiled secondary charged particles. Complexities associated with neutrons, such as the generally broad energy spectrum and the inherently wide energy spectrum of the induced charged particles, not to mention that the dependence of cellular inactivation by charged particles on radiation quality is yet to be fully understood, make it difficult to check this statement. Recently a molecular model has been proposed that improves the quantitative explanation of the dependence of cellular inactivation by charged particles on radiation quality. An attempt was made to apply this model for analysis of neutron cellular inactivation. As a preliminary result it is suggested that neutron cellular inactivation is caused not only by secondary charged particles but also by an "atomic deletion" effect, generated by a stripped atom recoiling from a DNA molecule. This effect seems to be of significant importance, the inactivation cross section of this effect for fission neutrons is as much as 15% (aerobic conditions) or 55% (hypoxic) of the total, and the severity of one occurrence of atomic deletion by a single neutron is estimated as much as 3.1+/-1.1 times (aerobic) or 6.8+/-1.2 times (hypoxic) higher than the severity of one event by a single track of a charged particle interacting with DNA

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The LNT model was first proposed in a paper (Oliver, 1930) which described the results of the sex-linked recessive lethal test in Drosophila, using mature sperms. Today, however, it is known that mature sperms lack DNA repair function. We have previously reported that when immature spermatocytes with active DNA repair function were irradiated with low dose, low dose-rate X-rays, the mutation frequency became lower than in the non-irradiated control group and thus, there should be a threshold below which no increase in mutation frequency is observed. This phenomenon was not observed when a high dose rate irradiation was carried out, or when wild type flies were replaced by a mutant strain which is defective in DNA repair function. The background mutation frequency in this experiment was 10 to the –6th/gene which corresponds to that induced by a 5Gy irradiation. If a considerable fraction of the spontaneous damages are repaired without error, the background mutation frequency should be reduced, which compensates the increase in the induced mutation and thus would form a practical threshold. Here we present the results of further studies with various doses, which confirmed an U-shaped dose response relationship. The results suggests that the dose response in DNA repair defective cells is linear without thresholds, whereas in repair proficient cells, it is non-linear. It is possible that the LNT model is not suitable for estimation of human cancer risks.

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We have previously reported that irradiation of the Drosophila immature sperms using gamma rays of 500 µGy from the ⁶⁰Co in one and half minutes reduced the frequency of the sex-linked recessive lethal mutation up to 0.1% (P<0.01) compared with 0.3% in control. It is said that in this level irradiation rarely makes the DNA double strand break and the double strand break repair does not work. To elucidate this phenomenon, we examined the greatest dose that the mutation rate returned to the spontaneous mutation level. We found that the gamma ray irradiation of 40 µGy in one and half minutes raised the sex-linked recessive mutation to the 0.29%. This result was significantly higher than that at 500 µGy (P<0.01) and no difference to that in control significantly (P=0.86). Furthermore, we will report about the expression of the defense response genes and apoptosis genes which were changed the expression levels at the 500 µGy gamma ray irradiation.

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Because biological response to ionizing radiation is dependent on time or dose rate, irradiation with a same dose gives rise to different biological response at different dose rate or for different irradiation time. We have observed dose rate effect in micronucleus formation and growth inhibition of human cell lines, in which the effects are exponentially declined as the dose rate is decreased, and have proposed a new dose rate effect model named MOE model through the statistical analysis of the data. In this paper, we studied property of human cell lines exposed to low dose rate for long period more than five month. A human osteosarcoma, U2OS, and a human glioma, M059K, were continuously exposed to gamma-ray in an irradiation room bearing 50,000 Ci cobalt-60. Growth inhibition is equilibrated to depend on dose-rate after the several month continuous irradiation. Minimum inhibitory dose-rate for the growth inhibition was about 50 mGy/h. Cell cycle analysis demonstrated G1 arrest in the cells continuously exposed to low dose rate gamma-ray, while G2 arrest in the cells acutely exposed to high dose rate gamma-ray. Cells continuously exposed to low dose rate gamma ray exhibited delayed growth suppression even after one month culture under background environment, and kept high level expression of c-Jun and its phosphorylations at serine 63 and 73, whereas high level expression of p53 and its phosphorylations at serine 15 and 20 was returned to the normal levels. M059K cells continuously exposed to low dose rate radiation were resistant to apoptosis caused by staurosporine and chemotherapeutic agents. These results suggest that AP-1 activation is involved in delayed growth suppression induced by continuous low dose rate irradiation, and that optimization of irradiation schedule is crucial for radiation cancer therapy.

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Recently, we irradiated mice heterozygous at the Dlb-1 locus (Dlb-1^b/Dlb-1^a) with low dose-rate gamma-rays for 483 days and obtained experimental evidence for the accumulation of induced mutations in small intestinal stem cells. To determine whether the same is true after high dose-rate irradiation, heterozygous mice were irradiated repeatedly at an interval of two or three weeks with an X-ray dose of 40 cGy at a dose rate of 50 cGy/min, starting at an age of 10 weeks and ending at an age of 58 weeks. Small intestines were sampled from control and irradiated mice 2 weeks after doses of 0.4, 2, 4, 6 and 8 Gy and subjected to the Dlb-1 mutation assay in which evidence of mutations of Dlb-1^b gene in the stem cells was detected as mutant clones on the surface of villi. Frequency of mutant clones (F), i.e., the number of clones detected per 10000 villi, in the irradiated series increased approximately linearly as the number of treatments increased without significant dose- and age-dependent change in the size of mutant clones. F value in the control series also increased in a linear fashion. From dose-response data corrected for control F values, we estimated induced rate of mutant clones (10^-4 villi·Gy^-1) to be 4.35±0.33, which is greater by a factor of 4.8 than rate after long-term chronic gamma-irradiation. We thus may conclude that mutations induced by high dose-rate irradiation accumulate in somatic stem cells, as it was the case after chronic irradiation and that mutagenic effectiveness of long-term irradiation depends on the dose and dose-rate of radiation.

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The influence of p53 on the elevation of T-cell receptor (TCR) variant fractions and apoptosis induced by beta-rays from tritiated water (HTO) and gamma-rays from ¹³⁷Cs at a low-dose rate was examined in splenic T lymphocytes of wild-type p53^+/+, heterozygous p53^+/- and null p53^-/- mice on the 19 days after a single intraperitoneal injection of HTO and the start of simulation-irradiation of gamma-rays. The TCR variant fraction was defined as the number of events in the CD3^-CD4⁺ T-cell window divided by the total number of events in the CD3⁺CD4⁺ T-cell window. Frequency of the TCR variant fraction in p53^-/- mice was higher than that in p53^+/+ mice by both beta- and gamma-irradiation. Apoptosis was analyzed at 12 and 24 h after the start of irradiation. A number of apoptotic cells in spleens from p53^+/+ mice were greater than that from p53^+/- mice by both beta- and gamma-irradiation. The number of apoptotic cells in mice irradiated by &beta-rays were greater than that irradiated by gamma-rays in both p53^+/+ and p53^+/- mice. These results suggest that p53 is required to eliminate the damaged cells and RBE of beta-rays from tritium is larger than 1 at a low-dose rate in the case of TCR variant fractions assay and apoptosis assay.

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Time changes (kinetics ) of gene in murine tissues expression after γ-ray irradiation with low-dose-rate (< 0.1 mGy/min) or middle-dose-rate (0.1 – 99mGy/min) remains to be elucidated. Based on the results of our previous microarray analyses using murine NIH3T3/PG13Luc cells, 5 genes, including Trp53-dependent genes (CyclinG1/Ccng1 and p21/Cdkn1a), extra-cellular matrix-related genes (Tnc, Col1a2 and Fbln5), were selected for markers to evaluate gene expressions in spleens of mice after irradiation. Female C3H mice were continuously irradiated for 1- 20 days at 400 mGy/22 hr/day. Time changes of the gene expression levels were analyzed by the real-time PCR method. The Trp53-dependent genes (Ccng1, Cdkn1a) were up-regulated earlier after the initiation of irradiation at the doses higher than 0.8 Gy. On the other hand, extra-cellular matrix related-genes (Col1a2, Tnc and Fbln5) were up-regulated later after the initiation of irradiation at the doses higher than 4.0 Gy. Such kinetics for cells of spleen cells from irradiated mice were quite similar to those in murine cell lines irradiated at middle-dose rates. On the other hands, any time changes of those gene expressions were not observed in 20 mGy/22hr/day. Because of the up- regulation pathways for Trp53-independent genes in murine tissues were different from those of extra-cellular matrix related genes after middle-dose- rate γ-ray irradiation, the regulation of these genes between Trp53-dependent and independent might be different. This study was supported by a grant from Aomori Prefecture, Japan.

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We reported antioxidant capacities in various tissues of mice were enhanced by low dose-rate irradiation. We furthermore found Type II diabetes model mouse (DB mouse) consecutively for low dose-rate gamma ray (0.63 mGy/hr) irradiation changed biological effects, such as urine glucose level decreased to normal level, and lifespan prolonged. These phenomena might be thought increase antioxidative capacities by the irradiation was protected oxidative damages in pancreas of the mice, and insulin secretion function contributed. We used female DB mouse as elucidation of the action that low dose-rate irradiation gave to suppressive effect of pancreatic oxidation, and measured change of plasma glucose and insulin concentration, urinary glucose concentration and pancreatic antioxidant activities. Gamma ray source is 137-Cs (314 GBq). We started radiation from 10 wks old to 82 wks old with at a dose-rate of 0.30, 0.63 or 1.2 mGy/hr consecutively.
The irradiated groups were increased antioxidative capacities in the pancreas; in particular Mn-SOD activity and glutathione concentration of the 1.2 mGy/hr-irradiated group was clearly enhanced. Lipid peroxide concentration was significantly decreased in the 0.63 mGy/hr-irradiated group. In the irradiated groups, plasma glucose concentrations slightly decreased. On the other hand, plasma insulin concentrations in the irradiated groups were not significantly changed. Urinary glucose concentrations were not also different from with or without irradiation. There were not differences between the irradiated groups.
These results indicated that the antioxidative capacities in the pancreas protected from oxidative damage by continuous low dose-rate irradiation, and then to kept the insulin secretion function.

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To investigate the effects of pre-exposure of mouse testis to low-dose ¹²C⁶⁺ ions on cytogenetics of spermatogonia and spermatocytes induced by subsequent high-dose irradiation, the testes of outbred Kun-Ming strain mice were irradiated with 0.05 Gy of ¹²C⁶⁺ ions as the pre-exposure dose, and then irradiated with 2Gy as challenging dose at 4 h after per-exposure. Poly (ADP-ribose) polymerase (PARPs) activity and PARP-1 protein expression were respectively measured by using the enzymatic and Western blot assays at 4h after irradiation; chromosomal aberrations in spermatogonia and spermatocytes were analyzed by the air-drying method at 8 h after irradiation. The results showed that there was a significant increase in the frequency of chromosomal aberrations and significant reductions of PARP activity and PARP-1 expression level in the mouse testes irradiated with 2 Gy of ¹²C⁶⁺ ions. However, pre-exposure of mouse testes to a low dose of ¹²C⁶⁺ ions significantly increased PARPs activity and PARP-1 expression and alleviated the harmful effects induced by a subsequent high-dose irradiation. PARP activity inhibitor 3-aminobenzamide (3-AB) treatment blocked the effects of PARP-1 on cytogenetic adaptive response induced by low-dose ¹²C⁶⁺ ion irradiation. The data suggest that pre-exposure of testes to a low dose of heavy ions can induce cytogenetic adaptive response to subsequent high-dose irradiation. The increase of PARP-1 protein induced by the low-dose ionizing irradiation may be involved in the mechanism of these observations.

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Acquisition of radioresistance of cultured cells to high-dose irradiation induced by low-dose preirradiation is known to the adaptive response. It was found that not only cultured cells but also the survival rate of lethally irradiated mice was significantly improved by low-dose (0.45 Gy) preirradiation and that this phenomenon had a specific window of priming dose and interval until the acquisition of radioresistance. This type of whole-body adaptive response is sometimes referred to as the "Yonezawa effect". However, the mechanism of Yonezawa effect is still remained unknown. We focused on the regulation of hematopoietic cell differentiation after high-dose irradiation, and we found that myeloid cells (Gr-1⁺/Mac-1⁺) in femur were rapidly recovered after high-dose (6 Gy) irradiation in low-dose (0.5 Gy) preirradiated mice. According to those myeloid recovery, myeloid-stimulating cytokines in blood plasma also up-regulated after high-dose irradiation in low-dose preirradiated mice. Furthermore, the reduction of peripheral blood erythrocytes two weeks after high-dose irradiation was significantly suppressed, which is a possible mechanism that improved the survival rate of mice after high-dose irradiation. We would like to discuss the mechanism of Yonezawa effect regulated by those hematopoietic cell differentiation after high-dose irradiation in low-dose preirradiated mice.

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Radioadaptive response is a biodefensive response observed in a variety of mammalian cells and animals where exposure to low dose radiation induces resistance against the subsequent high dose radiation. Elucidation of its mechanisms is important for risk estimation of low dose radiation because the radioadaptive response implies that low dose radiation affects cells/individuals in a different manner from high dose radiation. In the present study, we explored molecular mechanisms of the radioadaptive response in human lymphoblastoid cells AHH-1 in terms of mutation at the HPRT gene locus. First we observed that preexposure to the priming dose in the range from 0.02 Gy to 0.2 Gy significantly reduced mutation frequency at HPRT gene locus after irradiation with 3 Gy of X rays. As no significant adaptive response was observed with the priming dose of 0.005 Gy, it was indicated that the lower limit of the priming dose to induce radioadaptive response may be between 0.005 Gy and 0.02 Gy. Second, we examined the effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose)polymerase1, which has been reported to inhibit the radioadaptive response in terms of chromosome aberration. However we could observe significant radioadaptive responses in terms of mutation in the presence of 3AB. These findings suggested that molecular mechanisms for the radioadaptive response in terms of mutation may be different from that for radioadaptive responses in terms of chromosomal aberration. Finally, by performing a comprehensive analysis of alterations in gene expression using HiCEP, we could identify 17 genes whose expressions were significantly altered 6h after irradiation with 0.02 Gy. We also found 17 and 20 genes, the expression of which was different with or without priming irradiation 3 and 18 hrs., respectively after challenging irradiation of 3 Gy. By analyzing the gene function, we indicated that expression of genes involved in intracellular signaling and redox-regulation is correlatively altered with radioadaptive responses.

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In a pile of documented investigations, the phenomenon of radioadaptive response (AR) is predominately classified into the category with beneficial effect. This is probably due mainly to that in many cell lines AR reduced gene mutations, chromosome aberrations, transformation, and cell death. However, little is known if the survivors are really normal, namely, being the same as their non-irradiated counterparts. In fact, the margin of limited applicable endpoints and the simplicity of cell lines makes it hardly possible to comprehensively justice AR using in vitro systems. In a series of studies on AR in fetal mice, different consequences were observed depending on the endpoints. Possibly as the good, which demonstrate the existence of AR in this model, the numbers of fetal mice rescued from prenatal death and malformations were significantly increased. Consequently as the bad, the survivors had a markedly high postnatal mortality, suffered severely from neurophysiological alteration and developmental retardation. The young adults were highly radiosensitive, not applicable to Yonezawa Effect. In the present study, life span shortening and life long low body weight will be reported. Taken together, these whole body studies indicate that AR is a simple phenomenon but with complicated late consequences. Concerns on such as ethics, well-being and quality of life must be taken into account before talking about possible AR application in humans in the future. This work was supported in part by the Budget for New Nuclear Crossover Research from MEXT, Japan.

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We reviewed immunization activity using a mouse to examine a hormesis effect of low dose radiation by this study. It reviewed mechanism of radio-hormesis such as a change with time, antioxidation of a blood cell count after irradiation, immunization activity in revelation environment with an Rn (Rn) seat to a mouse. The experimental group did it with four groups of control group, Rn sheet revelation group, 2Gy group, and Rn sheet revelation + 2Gy group in total. We assumed it white-cell count and lymphocyte counts, a granulocyte count, a monocyte count. We measured SOD activity in serum. In addition, we performed CD4 and analysis of CD8 and reviewed immunization activity. For white-cell count of 2Gy group, decrease of white-cell count by meaningful irradiation inhibited it in Rn sheet revelation + 2Gy group. A meaningful rise of SOD activity by Rn sheet revelation group was recognized when we measured SOD activity. CD4 and CD8 increased in Rn sheet revelation group, too. Cell stimulation by alpha-ray by Rn sheet revelation can expect an immunological enhancement effect as well as activity for SOD from these. Therefore, a cell stimulated by Rn sheet revelation can expect growth accelerated improvement of immune function, inhibition of sickness, extension of life.

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Recently, obesity, which is one of the main reasons for adult disease such as diabetes, hypertension, hyperlipemia and so on, has increased rapidly. It is thought that obesity is induced when precursor cells differentiate into adipocyte and the cells hypertrophied by accumulating fat. Therefore, many scientists are paying a special attention to mechanism of cell differentiation of preadipocyte. We discovered that marine microbe products inhibited cell differentiation of adipocyte recently. Surprisingly, some of products have dedifferentiation function of adipocyte.
It is well known that the cell-cycle arrest in G1/G0 phase triggers induction of the cell differentiation. In addition, there is a lot of evidence that radiation induce cell-cycle arrest. Low-dose radiation which is one of the environmental stress induces the cell cycle arrest. So, it is likely that that appropriate levels of radiation dose induce cell differentiation of adipocyte. Therefore, mechanistic study of stress response may contribute to obese prophylactic technical development. In this presentation I will present differentiation derivation by radiation and the depression mechanism.

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We have shown the environmental factors affecting the leaching rate of ²²²Rn from the natural radioactive minerals to water that exist at Misasa in Japan and at Badgastein in Austria, which are world-famous for the radon therapy, as part of the study to elucidate the mechanism of the radon therapy. However, no data for the radon emanation fraction from these minerals have been reported. In this study, we measured the ²²²Rn emanation fraction from those samples under some environmental conditions (temperature and grain size) in order to elucidate the factors affecting ²²²Rn emanation. The radioactive sludge (0.9±0.0 Bq/g) was obtained from the hot bathroom with a high concentration of radon at Misasa Medical Center of Okayama University Medical School and the radioactive rock (6.4±0.0 Bq/g) was collected at Badgastein in Austria. Moreover, the soil (10.8±0.3 Bq/g) sampled at Ningyo-toge, Okayama Prefecture was prepared. The ²²²Rn emanation fraction from the samples was measured by the γ-ray spectrometry as follows. The dried samples of grain size of less than 63, 63-250, 250-500, 500-1000 or 1000-2000 μm in sealed glass bottles stood for about 30 days and γ rays from the samples were measured by a high-purity germanium detector. After the first measurement, the bottles were placed under the condition of temperature of 5, 25, 40, 60 or 80°C for 2 days and then the stopples of them were opened to escape ²²²Rn under the same temperature. Subsequently, γ rays from the samples were measured again. The ²²²Rn emanation fraction was estimated by calculating γ-ray count ratio of ²¹⁴Pb or ²¹⁴Bi between two count measurements. As a result, the ²¹⁴Rn emanation fraction from the samples depended on the environmental temperature. In higher temperature, more amount of ²²²Rn exists in gas phase because higher temperature decreases adsorption of ²²²Rn on a solid surface. This may suggest that adsorption and desorption reactions of ²²²Rn dominate the emanation fraction in this experiment. We are currently discussing the ²²²Rn emanation fraction of those samples of the different grain sizes.

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The measurement of ²²²Rn emanation fraction from a natural radioactive mineral has been performed by counting α ray from ²²²Rn of gas phase in the sealed chamber where mineral is placed. Lower ²²²Rn emanation fraction of a sample can be accurately measured because emanated ²²²Rn itself is measured in the α-ray method. However, this method needs a dedicated detector for ²²²Rn which is correctly calibrated. On the other hand, the simple method of measuring ²²²Rn emanation fraction was developed, which is based on two count measurement of γ ray using an uncalibrated NaI (Tl) detector. However, ²²⁰Rn can significantly affect ²²²Rn emanation fraction measured by the γ-ray method if a sample contains high concentration of Th-series nucleus. In this study, the ²²²Rn emanation fraction was measured by γ-ray spectrometry to remove completely the influence of ²²⁰Rn. The radioactive sludge (0.9±0.0 Bq/g) was obtained from the hot bathroom with a high concentration of radon at Misasa Medical Center of Okayama University Medical School and the radioactive rock (6.4±0.0 Bq/g) was collected at Badgastein in Austria. The ²²²Rn emanation fraction from the samples was measured by the (1) γ-ray and (2) α-ray method as follows. (1) The dried samples in sealed glass bottles stood for about 30 days and γ rays from the samples were measured by a high-purity germanium detector. Subsequently, the stopples of the bottles were opened to escape ²²²Rn and γ rays from the samples were measured again. The ²²²Rn emanation fraction was estimated by calculating γ-ray count ratio of ²¹⁴Pb or ²¹⁴Bi between two count measurements. (2) The dried samples and a small ionization chamber were placed in a sealed camber. The ²²²Rn concentration in the chamber was measured for about 2 weeks and the ²²²Rn emanation fraction was estimated from the ²²²Rn growth curve. As a result, the ²²²Rn emanation fraction of the Misasa's sludge and Badgastein's rock measured by the α-ray method was 30 and 2%, respectively. We are currently discussing the ²²²Rn emanation fraction of those samples by the γ-ray method and the lowest ²²²Rn emanation fraction which we can measure by γ-ray method. Moreover, we discussed the detection limit of ²²²Rn emanation fraction in case of the γ-ray method.

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There are a lot of life style diseases that are relation to reactive oxygen species in indications of the radon therapy, and, the further clarification of mechanism is expected. Therefore, in this study, we investigated the activation of antioxidant function in some organs of mice by radon inhalation using the radon exposure device (the joint development with OZ PLAN (Okayama)). The exposure place was set that temperature: 25°C; humidity: 80%; radon concentration: 400Bq/m³ or 4000Bq/m³. The radon exposure was done for 8, 16, 24 or 48 hours for male BALB/c mice, 7-8 weeks of age. After exposure, we excised organs and made it to the sample. Results show that in the liver, in 400Bq/m³, both activities of superoxide dismutase (SOD) and catalase significantly increased for only 48 hours exposure. In 4000Bq/m³, both activities of SOD and catalase significantly increased for 8, 16, 24 or 48 hours exposure. The increase of antioxidant function in 4000Bq/m³ was significantly larger than that in 400Bq/m³. In the kidney, the result similar to the liver was shown. Moreover the activation of antioxidant function in liver was larger than that in kidney. In brain and lung, the increase in both activities of SOD and catalase in 400Bq/m³ were significantly larger than those in 4000Bq/m³. These findings suggested that the radon inhalation activates the antioxidant function in some organs of mice. And, for instance, the lipid solubility of radon is higher than that of the other gases, and tend to accumulate in the organs, such as liver, with a high content of fat. Therefore the activation in liver is significantly larger than that in kidney.

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We have reported that the radon and thermal therapy alleviated life style diseases, such as osteoarthritis. We examined the temporal changes in atrial natriuretic polypeptide (hANP) and arginine vasopressin (AVP) in human blood to elucidate the mechanism of alleviation of hypertension by the radon or the thermal treatment. Every 2 days, nasal inhalation of radon and mist using the radon and mist generator (the joint development with OZ PLAN (Okayama)) was performed for 30 min under a condition of high humidity (radon group;1496 Bq/m³, 30°C, thermal group; 33Bq/m³, 42°C). Blood samples were collected after each treatment on weeks 2 and 4 after first treatment. Results show that, in the radon group and in the thermal group, the maximum blood pressures and the minimum blood pressure in the hypertensive group (on weeks 2 and 4 after the first treatment) were significantly decreased compared with the control (before the first treatment). On weeks 4, in the hypertensive group and in the normal group, the hANP level in the radon group is larger than that in the thermal group. On weeks 4, in the hypertensive group, the AVP level in the radon group decreases to the normal level. These findings suggest that the radon treatment or the thermal treatment decreases blood pressure similar to normal level. Moreover the decrease of blood pressures is related to the increase of the hANP level and the decrease of the AVP level as the mechanism of alleviation of hypertension by the radon treatment. Moreover we will refer to the result of the synergistic radon and thermal effects.

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We have morphologically reported that low-dose irradiation inhibited ischemia-reperfusion injury. In this study, we histologically examined the changes of mouse paw after ischemia-reperfusion by prior low-dose X-irradiation. BALB/c mice were irradiated by sham or 0.5 Gy of X-ray. At 4 hrs after irradiation, the left hind leg was bound 10 times with a rubber ring at just above articulation for 0.5, 1, or 2 hrs. The left hind leg was excised, and divided into small blocks. These legs were stained conventionally with hematoxylin-eosin (HE). In addition, the interstitial fluid in interstitial edema is increased by ischemia-reperfusion and interstitial edema is increased. Therefore we calculated the percentage of interstitial edema (ration of cells spacing). Results show that in case of the ischemia for 0.5 or 1 hr, 0.5 Gy irradiation significantly inhibited the paw swelling thicknesses more than sham irradiation. In case of the ischemia for 0.5 or 1 hr, 0.5 Gy irradiation significantly decreased interstitial edema more than sham irradiation. In case of the ischemia for 1 hr, 0.5 Gy irradiation significantly decreased interstitial edema after 0.5, 2, or 24 hrs reperfusion more than sham irradiation. Moreover 0.5 Gy irradiation significantly increased superoxide dismutase (SOD) activity in serum after 1 hr reperfusion from 1 hr ischemia. These findings suggest that 0.5 Gy irradiation activated the antioxidant function in mouse paw, therefore low-dose irradiation alleviates ischemia-reperfusion injury.

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We have reported that low-dose irradiation enhanced antioxidant function and alleviated the ischemia-reperfusion injury in mouse. It was also reported that organ transplantation induced ischemia-reperfusion injury. In the present study, we examined the changes of the antioxidant substances in graft mouse liver after low-dose X-irradiation. BALB/c mouse liver was irradiated by sham, 0.25, 0.5, 1.0, or 5.0 Gy and stored for 4, 8, 24, or 48 hours in the preservative (Via Span) solution containing glutathione or saline solution. Results show that the activities of SOD and catalase were significantly increased for 24 hours soakage in saline solution after 0.25 or 0.5 Gy irradiation or in preservative solution after 1.0 Gy irradiation. The glutathione content was significantly decreased for 24 hours soakage in saline solution or preservative solution after every irradiation. On the other hand, after 0.5 Gy irradiation, the activities of SOD (4 or 24 hours) and catalase (8 hours) were significantly increased by soakage in saline solution. And the glutathione contents were significantly decreased for over 4 hours soakage in saline solution or preservative solution. Moreover the lipid peroxide level was significantly decreased for 8 hours soakage in saline solution. These findings suggested that low-dose irradiation significantly activates antioxidant function of mouse liver in saline solution or preservative solution. On the other hand, the preservative solution contains glutathione. And glutathione was made synthetically from ATP, which was decreased during ischemia. Therefore the glutathione content was significantly decreased in these conditions of irradiation and preservation.

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[Purpose] The thermo-enhancement effects of Parthenolide (PTL), a principal ingredient of herb which target on NF-kappaB, transcript factor, were investigated in human lung adenocarcinoma A549 cells with wtp53, show the low thermosensitivity. The expression of heat induced hsp70 proteins and p53 proteins, takes part in apoptosis induction, were also examined. The thermo- enhancement effects of PTL through the signaling pathway of NF-kappaB were analyzed. [Materials & Methods] A549 cells, human lung adenocarcinoma, were used in this study. Cytotoxicity or thermosensitivity to PTL, hyperthermia or the combination therapy in A549 cells were estimated by colony formation assay. The kinetics induction of hsp70 and p53 proteins after treatment with PTL, hyperthermia or the combination was analyzed by western blot assay. Induction of apoptosis was analyzed using fluorescent dye Hoechst33342. [Results] Combination therapy with PTL and hyperthermia in A549 cells showed thermo-enhancement effects. After treatment with hyperthermia alone, the accumulated amount of hsp70 increased markedly, but with PTL alone did not increase. Both hsp70 and p53 proteins were not induced significantly after combined treatment with PTL and hyperthermia. Apoptosis after treatment with PTL showed remarkable incidences, while apoptosis after combined treatment with PTL and hyperthermia showed significant incidences. [Summary] Thermosensitivity of human lung adenocarcinoma A549 cells was enhanced synergistically in combination therapy with PTL and hyperthermia. Heat-induced apoptosis after combined treatment with PTL and hyperthermia showed increase significantly. The thermo-enhancement effects of PTL were not inhibition of heat-induced hsp70, but due to induce apoptosis by blocking the activation of NF-kappaB.

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Exposure to high dose radiation causes to radiation injury. There are many reports that radiation activates the production of tumor necrosis factor α (TNFα) in various cells including monocytes/macrophages and granulocytes. TNF α is also considered to be a proinflammatory cytokine and this factor plays a critical role in the initiation and continuation of inflammation and immunity. The excess production of TNFα leads to damage of organs. However, the role(s) of TNFα is not fully understood in radiation exposure. In this study, we investigated the roles of TNFα in mice exposed to radiation. We compared the wild-type (TNFα^+/+) and the knockout (TNFα^-/-) BALB/c mice. Both mice were subjected to γ-ray radiation at a dose of 5-7 Gy. The survival durations in TNFα^-/- mice were shorter than in TNFα^+/+ mice. The survival rate in TNFα^+/+ mice exposed to 5 Gy was almost 95, 90 and 65% on days 5, 10, and 30 after radiation, respectively. On the other hand, those in TNFα^-/- mice were about 75, 35 and 15%. In order to investigate the cause of death, we compared weights of body and organs, the numbers of surviving intestinal crypts, apoptosis in crypts, liver function including plasma ALT, AST, or ALP level, and numbers of blood cells following radiation. We also performed histo-pathological study. However, there was no significant difference between TNFα^+/+ and TNFα^-/- mice. Our results from TNFα^-/- mice suggest that TNFα plays an important role in the radiation exposure, whereas the cause of death in TNFα^-/- mice exposed to radiation. Further studies are in progress.

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Differences of radiosensitivity between two mouse squamous cell carcinoma lines (SCCVII and NR-S1) measured by colony formation assay has been shown to reflect their in vivo radiation sensitivity differences measured by tumor growth delay assay in the body of transplanted animals. This study was carried out to characterize in vitro chronological behavior of the two cell lines after ionizing radiation exposure. Logarithmic growth phase culture of the two cell lines were exposed to 0 Gy (non-irradiation), 4 Gy and 8 Gy of X ray, respectively. Immediately after irradiation, the cells were harvested, counted and appropriate number of the cells was plated onto new culture dishes. They were incubated at 37 oC in a humidified 5% CO2 incubator for 24 hours, then transferred onto an incubation chamber equipped inside a timelapse microscopy. A phase-contrast image of individual cells was captured at every 5 min during 48 hours observation period, resulting in a total of 576 images. Chronological behavior of two cell lines on captured images was then compared. SCCVII showed mostly the non-synchronized proliferation among cells at non-irradiation condition. Cell fusions were rarely observed in this cell line. By 4 Gy and 8 Gy irradiations, most of cells showed remarkable growth retardation and enlargement. Even at this situation, cells proliferated individually and did not fuse with each other. On contrary, cells of NR-S1 showed the synchronized proliferation with neighboring cells at non-irradiation condition. The synchronized cells seemed to be fused with each other during mitosis then divided into multiple daughter cells. This feature was also observed in the cells exposed to 4 Gy and 8 Gy X ray. Growth retardation or enlargement of cells was not observed as in the cells of SCCVII. We are planning to investigate correlation of these behavioral features and radiation sensitivity.

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[Introduction]<BR/>  Microbeam radiation (MR) which irradiates arrays of parallel, microscopically thin planar slices of synchrotron-generated X-ray is expected as a new method of cancer therapy. We tried to make clear the feasibility of the therapy.<BR/> [Object]<BR/>  To examine tumor curative effect of MR and tolerable dose of nomal skin tissues.<BR/> [Materials and methods]<BR/>  Microbeam and broadbeam irradiation were performed at public beamline BL28B2 of the large synchrotron facility, SPring-8. White X-rays was transported by the beamline, passed through the multilayered slit collimator (25 μm width, 200 μm intervals) which was consisted of tungsten and Kapton polyimide film. The original beam, did not let a slit go through, was used for the broadbeam radiation (BR). NFSa cells were transplanted into the thighs of C3H male mice 8 days before irradiation. Hairs of C3H female mice on the right hind legs were removed at the same day. We observed tumor growth and skin reaction after irradiation. Tumor size was measured everyday to obtain tumor growth curves, and evalution of the skin damage was based on Skin Reaction Score table as previously reported.<BR/> [Result]<BR/>  MR sighnificantly prolonged the days for that a tumor volume was needed to reach the 1 cm³ compared to the control (0 Gy irradion) group. On the other hand, tumor growth was inhibited by BR than MR at the same absorbed dose. The skin reaction score resulted of MR was higher than that of BR at approximately 200 Gy at the 10th day after irradiation, but the skin reaction score after MR tended to lower than BR 14days after irradiation.<BR/> [Discussion]<BR/>  MR and BR have the equivalent inhibitory effects of cell growth at same dose, however the repair speed of the cell irradiated of the MR was faster than BR because of the faster recovery of the neighboring cells, which is suspected as actual irradiated area of the MR is 1/8 of the BR and the beam width are quite smaller than BR.

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Purpose: To clarify the radiobiological significance of the response of intratumor quiescent (Q) cells in vivo to accelerated carbon ion beams and reactor neutron beams.
Materials and Methods: SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating (P) cells. Then, they received accelerated carbon ion or gamma-ray irradiation with high dose-rate (HDR) or reduced dose-rate (RDR). Another tumor-bearing mice received reactor thermal or epithermal neutrons with RDR. Immediately after HDR and RDR irradiation or 12 hours after HDR irradiation, the response of Q cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The frequency in the total (= P+Q) tumor cells was determined from the BrdU non-treated tumors.
Results: The difference in radiosensitivity between total and Q cell populations under gamma-ray irradiation was markedly reduced with reactor neutron beams or accelerated carbon ion beams, especially with a higher linear energy transfer (LET) value. Clearer repair in Q cells than total cells through delayed assay or a decrease in dose-rate under gamma-ray irradiation was efficiently inhibited with carbon ion beams, especially with a higher LET. Under RDR irradiation, accelerated carbon ion beams with a higher LET showed almost similar radiobiological property to reactor thermal and epithermal neutron beams.
Conclusion: In terms of tumor cell-killing effect as a whole, including Q cells, accelerated carbon ion beams, especially with higher LET values, are very useful for suppressing the dependency on the heterogeneity within solid tumors as well as depositing radiation dose precisely.

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The effect of heavy-ion irradiation to brain on spontaneous motor activity is not reported. The present study was designed to determine whether irradiation (carbon particles 290 MeV/nucleon, Mono peak 60Gy, irradiation field 5-millimeter cube in hypothalamus) to rat's hypothalamus modifies the spontaneous motor activity of male rats. The irradiated field in rat's brain after carbon particles irradiation was identified by imaging plate method. The spontaneous motor activity was used by the wheel-running test. The activity was measured using an active increase effect of methamphetamine (MAP). The observation of wheel-running activity was conducted for 90 min after 2, 6, 14, 22, 26 or 30 weeks following irradiation. Results are as follows. The control group and irradiated group were highest motor activity in wheel-running test at first test (1st injection of MAP, 2 weeks after irradiation); however, motor activity of the control group decreased after from 2nd test (6 weeks) to 5th test (22 weeks). On the other hand, 60 Gy exposed rats were not decrement of wheel-running activity which depended on number of times in motor activity test (injection time of MAP). These results may be possible to describe that carbon irradiation to hypothalamus is caused edema at the irradiated field. Furthermore, it was supposed that an ambient blood flow increased to make up for disordered hemodynamics by the edema.

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The purpose of present study is to clarify experimentally significance of tumor heterogeneity. Two sarcomas of # 6107 and #9037 were transplanted into syngeneic C3H male mice. Single cell suspensions for each tumor were mixed together at various ratios just prior to transplantation. When tumors grew to reach 7.5-8.0 mm in diameter, leg tumors were locally irradiated with 290 MeV/n carbon ions at an LET of 74 keV/microm that were accelerated by HIMAC synchrotron in our institute. Tumor growth (TG) time was obtained by calculating days required for a tumor to reach 5 times initial volume, and Tumor Growth Delay time was used to obtain an isoeffect dose. RBE values were calculated by comparing isoeffect doses between carbon ions and reference Cs-137 gamma rays. Tumor control probabilities were calculated by counting number of mice free of tumors 150 days after irradiation. Tumors with different ratios of mixture (RM) showed various TG time such that TG times of 8.3, 3.8 and 6.1 days were for tumors with ratios of 9:1, 5:5 and 1:9 (#6107: #9073), respectively. Isoeffect doses for 5 tumors with different RM were ranging from 18 to 48 Gy for gamma rays, and from 8 to 18 Gy for carbon ions. A tumor with RM of 9:1 showed the largest isoeffect gamma-ray dose, and was more resistant than the either parental tumors of #6107 or #9073 tumors. However, the tumor with RM of 9:1 was modest radiosensitive to carbon ions. Conditional data of tumor control probabilities showed that a tumor with RM of 9:1 was more sensitive than tumors with RM of 1:9 after carbon-ion irradiation. Possible reasons for this interaction are either hypoxic c ell fraction or G1 cell cycle phase. Factor(s) and/or signaling responsible to the interaction are unknown. It is concluded that tumor heterogeneity strongly influences RBE of high LET radiation.

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1-(3-C-Ethynyl-β-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd) has previously shown to enhance the antitumor efficacy of X irradiation. However, the effect of TAS106 on hypoxic cells in tumors, which are associated with poor prognosis for radiotherapy, remains unknown. In this study, hypoxia (oxygen concentration < 20 mmHg) was achieved by passing 95% N₂ and 5% CO₂ gases continuously in a gas-exchangeable chamber. We showed that a low dose of TAS106 induced radiosensitization of apoptosis to human gastric adenocarcinoma MKN45 and MKN28 cells under hypoxia similarly to normoxia in vitro. The reduction of HIF-1α gene expression using the specific antisense oligodeoxynucleotides also enhances X-ray-induced cell death in hypoxic tumor cells dramatically. The accumulation of HIF-1α which was observed under hypoxia, was decreased to the level of normoxia in the presence of 0.1 μM TAS106. In vivo study, MKN45 cells were inoculated into the footpad in severe combined immunodeficient (SCID) mice. Histological analyses revealed a significant reduction of tumor hypoxic regions and apoptotic induction of hypoxic cells in mice treated with a low dose of X-rays (2 Gy) and TAS106 (0.5mg/kg), via probably the inhibition of HIF-1α expression by TAS106. These results suggested that TAS106 acted a strong radiosensitizer through the inhibition of HIF-1α pathway and a potent agent against radiotherapy-resistant hypoxic cells in solid tumors.

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High-LET heavy ions have been used clinically to cure malignant tumors not merely because of their excellent spatial dose distribution but because of their greater biological effectiveness compared to low-LET radiations. We have here investigated the potential impact of heavy-ion irradiation on the radioresistance of tumor cells overexpressing Bcl-2. While Bcl-2 overexpressing HeLa (HeLa/bcl-2) cells were more resistant to gamma-rays (0.2 keV/μm) and helium ions (16 keV/μm) than neomycin resistance gene expressing HeLa (HeLa/neo) cells, there was very little if any difference in susceptibility to heavy ions (76-1610 keV/μm) between HeLa/neo and HeLa/bcl-2 cells. This suggests that high-LET heavy ions overcome the radioresistance caused by Bcl-2 overexpression. Besides, after exposure to carbon ions (108 keV/μm) whose RBE was maximal among six different beams of heavy ions tested, the yield of apoptosis was lower but G2/M arrest was more prolonged in HeLa/bcl-2 than in Hela/neo cells. This result highlights that Bcl-2, in addition to its well-known anti-apoptotic function, may participate in G2/M checkpoint after heavy-ion irradiation. Clarification of the molecular underpinnings of Bcl-2 mediated G2/M checkpoint encourages further studies.

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It has been recognized the heavy-ion radiotherapy for cancer as effective. However, it is not clear the effect of heavy-ion exporsure on AT-heterozygote, whici is thought to occupy about 1% in population. In this study, we analyzed effects of carbon and iron-ion beam with various LET on AT-heterozygote cells. LETs of heavy-ion used were 24, 40, 50, 60 and 200 keV/mm. Survivals were determined by colony formation assay, and RBE was obtained referring the survival after X-ray radiation. In addition, cH2AX focus formation was analyzed. In a normal cell line, RBE was flat from 24-40 keV/mm but increased LET -dependently above 40 keV/mm. On the contrary, although RBEs of AT-heterozygote cell lines at 24 keV/mm were similar to that of normal cells, it increased LET-dependently above 24 keV/mm. On the other hand, in AT cell line, the RBE increased from 24-40 keV/mm. However, above 40 keV/mm, RBE showed plateau phase. In cH2AX analysis almost similar DSB-repair activities were observed in both normal and AT-heterozygote cells after various LET of carbon-ion beams. These results suggest ATM heterozygosity could influence to cellular survival after heavy-ion beams. We have to know the mechanisms how AT-heterozygotes show such radiosensitivity.

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Purpose: Tumor hypoxia and angiogenesis associated with malignant progression, radio- and chemotherapy resistance have been studied well. However, the relation between normal tissue injury and hypoxia is still unclear. In current investigation, we preferred to study the effect of hypoxia in radiation-induced late rectum injury in mice. Methods and Materials: The rectum of C57BL/6N mouse was irradiated locally with a single dose of 25 Gy. Radiation-induced fibrosis was observed at 90 days after irradiation by azan staining and the expression of transforming growth factor β1 (TGF-β1), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 were also assessed in accordance with fibrotic region using real-time PCR, immunohistochemistry and immunofluorescene. In addition, the effect of TGF-β1, VEGF and HIF-1α in radiation-induced injury was investigated by the administration of anti-TGF-β1, anti-VEGF or YC-1 respectively after irradiation. Results: The expression of TGF-β1, HIF-1α, VEGF and CD31 showed a significant increase with the formation of fibrosis induced by irradiation compared with unirradiated control. After treatment of anti-TGF-β1, anti-VEGF or YC-1, the development of irradiation-induced injury was attenuated. Conclusions: Our results suggest that radiation-induced hypoxia may play a role in late normal tissue injury. The paradigm may contribute to radiotherapy and understanding of radiation-induced late normal tissue injury.

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Potential advantage of greater biological effectiveness and excellent dose distribution over conventional low-LET radiations represents the rational for the clinical usage of high-LET heavy ions. Unavoidable, however, is exposure of normal cells that coexist with or surround tumors. Whereas fibrosis following radiotherapy is known to result from augmented differentiation of normal cells, the effect of heavy ions on differentiation is unknown. To address this, we have investigated the morphological differentiation of normal human fibroblasts irradiated with gamma-rays (0.2 keV/μm) or carbon ions (108 keV/μm). We found that carbon ions were fourfold more effective at inducing morphological differentiation at 5 days post-irradiation. Further experiments to examine the LET dependence of augmented differentiation and its underlying mechanism(s) are ongoing.

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We investigated the mechanisms for nitroxide Tempo to potentiate heat stress-induced apoptosis in human leukemic U937 cells. We treated the cells with either single or combined regiments of 5-10 mM Tempo, 44C/10 min or 44C/30 min hyperthermia and assessed apoptosis induction. The 5 mM Tempo-44C/10 min combination induced ∼80% apoptosis as did 44C/30 min heat stress, while 5 mM Tempo or 44C/10 min heating alone caused little apoptosis. The mechanistic basis for such a synergistic apoptotic enhancement involved (1) the activation of Bax and its translocation to mitochondria, leading to cytochrome c release and activation of the caspase-9 to -3 cascade, (2) activation of initiator caspases-8 and -2 as revealed by the biotin-zVAD-fmk pulldown experiment, and (3) zVAD-fmk-suppressible loss of mitochondrial membrane potential, which led to mitochondrial dysfunction. A stronger combination of 10 mM Tempo and 44C/30 min heating induced the caspase-3-independent cell death (CICD), involving zVAD-resistant loss of the potential (irreversible mitochondrial dysfunction) due partly to increases in ROS and mitochondria-associated Bnip3. Furthermore, transcriptional up regulation of inducible HSP70 and HSP27 by geldanamycin suppressed completely the 44C/30 min heat stress-induced apoptosis. The results together delineated the important mechanisms of hyperthermia-induced apoptosis and Tempo-mediated enhancement.

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Several nonsurgical approaches to the treatment of postradiation proctitis have been described, but no effective conservative treatment has yet been established. In order to establish whether extra-colonic cells contribute to the repair of colonic tissues, this study examined colons of 1) female rats that had received a male bone marrow transplant and 2) male rats that had received a Green Fluorescent Protein (GFP) transgenic bone marrow transplant. Colitis was induced by focal irradiation of 22.5 Gy X ray. By using in situ hybridization to detect Y-chromosomes and immunohistochemistry to detect GFP protein it could be demonstrated that circulating stem cells frequently engraft into the colon and differentiate into colonic epithelial cells. Female rat recipients of male bone marrow grafts showed localization of Y-chromosome. Y-chromozome-containing cells and GFP positive cells were observed within regenerative epithelium, with morphology and location appropriate for colonocytes. By using western blotting method GFP expression in the colon of irradiated area was higher than that in the colon of non-irradiated area. These data indicated that bone marrow cells contribute to regeneration of colonic epithelia after damage, and it is suggested that this could be exploited therapeutically.

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Malignant mesothelioma is a highly aggressive tumor arising from mesothelial cells of the pleura and peritoneum. Although this used to be a rare tumor, the incidence is expected to increase worldwide over the next several decades. Current treatment of choice for mesothelioma is surgical resection, which may be combined with chemotherapy and/or radiation. However, the prognosis of patients after this multimodality therapy remains poor and median survival of patients from diagnosis ranges from 8 to 18 months. Thus, it is urgently necessary to establish new and more effective treatments. To develop new treatments, non-invasive imaging for evaluating the efficacy of new treatments is essential. A positron emission tomography (PET) using F-18 fluorodeoxyglucose (FDG) is widely applied for the diagnosis of various cancers. However, since FDG also accumulates in inflammatory lesions, FDG-PET may become falsely positive in post-treatment tumors complicated with reactive inflammatory changes, which limits the usefulness of FDG-PET in evaluating treatment response. F-18 fluorothymidine (FLT) and C-11 thiothymidine (S-dThd), which are analogs of thymidine and tracers for measuring nucleic acid metabolism, are reported to accumulate less in inflammatory lesions than FDG. In this study, we established orthotopic implantation models of malignant pleural mesothelioma in mice and compared three PET tracers for imaging in these models.

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Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the human gastrointestinal (GI) tract. Overexpression of c-kit, encoding type III receptor tyrosine kinase, and its functional mutation is present in most GIST. [F-18]-fluoro-deoxyglucose (FDG) has been reported to highly accumulate in GIST, and FDG positron emission tomography (PET) is very useful for clinical tumor staging and also for the assessment of early response to GIST treatments using Imatinib (Gleevec). However, FDG-PET, in some cases, fails to detect relapse of GIST at early time point, and these cases showed c-kit positivity determined by biopsy. If we could develop a specific method to detect c-kit expression, it will lead to detect the early response and relapse of GISTs. In this study, we labeled anti-c-kit antibodies with I-125, and assessed its in vitro characteristics. We constructed an expressing vector of mutated c-kit and transfected it to HEK293 human embryonic kidney cells and isolated several stable clones. Immunofluorescence staining showed that c-kit is primarily localized to the cell membrane in c-kit stably expressing cells like the original GIST cell line. We radiolabeled anti-c-kit murine monoclonal antibodies with I-125 and performed cell binding, competition and internalization assays in vitro. These results showed that radiolabeled antibodies specifically bound to c-kit expressing cells and be internalized. Therefore, antibodies labeled with metal radionuclide such as In-111 seem suitable for in vivo application.

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Compared to popular teratogenesis subjects such as lethality, microcephaly and tumorigenesis, little has been reported on neurocristopathy, deficient epithelial-mesenchymal transition (EMT) and cardiovascular anomalies following maternal exposures to tritiated water (HTO) in mammals. In order to collect basic data on prevention and safety therapeutic applications in diseases regarding radiation, the biologically unique effects of HTO and gamma-rays exposure need to be evaluated. Here, pregnant rats in the experimental groups were given either a gamma-ray irradiation or a single intraperitoneal injection of HTO. We reported on the relationships between each full body maternal exposure and subsequently observed embryonic lethality, external malformations, and visceral malformations, especially of cardiovascular nature. We observed a high frequency of teratogenesis, especially cardiovascular and facial anomalies, in both treated groups. These results indicate sensitivity to gamma-rays exposure and HTO exposure of rat fetus leading to dysfunction of neural crest development in formation of neurocristopathy syndrome and neurocristopathy-induced HTO syndrome, respectively, and especially cardiovascular anomalies, including ventricular septal defects, tetralogy of Fallot, double outlet right ventricle, riding aorta, atrioventricular valve and septum defects, right aortic arch, coarctation of aorta and aortic arch anomalies. These types of cardiovascular anomalies are quite similar to those found in humans, termed DiGeorge syndrome and Aligellie syndrome, suggesting that gamma-rays and HTO may play a role in dysfunction of neural crest and formation of human neurocristopahty as well as cardiovascular anomalies. It is crucial to consider the their effects involving processes of cell killing and DNA damage, dysfunction of neural crest and epithelial to mesenchymal transition (EMT) on the formation of these syndromes. This animal model is expected to contribute in investigating the mechanism of such teratogenesis in human, as well as their relative biological effectiveness.

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The effects of heavy-ion-radiations on the differentiation of melanocytes as well as on the prenatal and postnatal development of mice were investigated by scoring changes in the ventral pigmentation and in the organogenesis. Pregnant females of C57BL/10J mice at 9 days of gestation were whole-body irradiated with a single acute dose of silicon- or argon-ion-radiations. The effect was studied by scoring changes in the pigmentation in cutaneous coats 22 days after birth in postnatal development of mice. The percentages of birth and survival to day 22 as well as the body weight at day 22 were decreased in irradiated mice. The effect of silicon- or argon-ion-radiations on the survival to day 22 was greater than that of gamma-rays (Hirobe, 1994). The frequency and size of white spots in the mid-ventrum were increased in irradiated mice. Silicon- and argon-ion-radiations were more effective than gamma-rays. As the prenatal effects, the frequencies of abnormalities in the fore and hind legs, tails and eyes as well as hemorrhage in 18-day-old embryos were increased from the dose of 0.1 Gy silicon-ion-radiations. The number of epidermal melanoblasts and melanocytes were decreased from the dose of 0.1 Gy silicon-ion-radiations. Similar results were obtained in the embryos exposed to argon-ion-radiations. These results suggest that silicon- and argon-ion-radiations have a greater effect on the melanocyte differentiation as well as on the prenatal and postnatal development of mice than gamma-rays.

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Objective: Colon and rectum are adjacent organs, and adenocarcinoma is a major histological type of cancer in both organs. However, a statistically significant radiation risk on developing cancer has been found only in colon in the Life Span Study (LSS) cohort. This study aims to examine the radiation risk by subsite of colon in the LSS.
Subjects and methods: Subjects were 105,427 LSS cohort members with individual dose estimates (DS02) who were not known to have had cancer prior to 1958, and were followed up through 1998. Cancers were ascertained by cancer registries in Hiroshima City/Prefecture and Nagasaki Prefecture. Mucosal tumors were excluded from the analyses. Colon was divided into two subsites: proximal colon (cecum, ascending colon and transverse colon) and distal colon (descending colon and sigmoid colon). Radiation-associated excess relative risks were calculated by Poisson regression models with adjustment for city, gender, attained age, birth year and location at the time of the bombing.
Results and Discussion: A total of 1,196 colon cancers (550 proximal and 646 distal) were ascertained. Excess relative risks (at age 70, per Gy) on proximal and distal colon were 0.35 and 0.64 in men and 0.39 and 0.24 in women, respectively. When proximal colon was further divided into two subsites, excess relative risks (at age 70, per Gy) on 1) cecum and ascending colon (394 cases) and 2) transverse colon (156 cases) were 0.00 and 0.87 in men and 0.01 and 1.21 in women, respectively. Although none of the excess relative risks shown above were statistically significant, it is indicated that radiation-associated relative risk of developing cancer is the lowest in the cecum and ascending colon.

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The molecular mechanism of carcinogenesis in atomic-bomb (A-bomb) survivors has not yet been fully understood. Recently, we found RET oncogene amplification (amp) in radiation-induced thyroid cancers, suggesting the involvement of genomic instability (GIN). A radiation etiology is also suggested in breast cancers among survivors. The aim of this study is to clarify the significance of oncogene amp in breast cancers from survivors. A total 593 cases of breast cancers were identified among survivors since 1961 through 1999 based on pathological reports. Total 67 cases of breast cancers from survivors were available in this study. Diagnosis was reviewed and determined histological grade according to a recent criteria. We analyzed the amps of HER2 and C-MYC by FISH and expressions of HER2 and hormone receptors by immunostaining on paraffin-embedded tissues. The incidence rate (IR) of breast cancer was 54.1 per 100,000 PY in survivors. The IR significantly increased as exposure distance decreased from the hypocenter. Incidences of both HER2 and C-MYC amps were significantly higher in proximal distance group (≤1.5km, n=35) than distal (>1.5km, n=32) or control group (others than survivors, n=30). Furthermore, a significant higher incidence of co-amp was found in proximal group as compared to distal or control group. In histological grading, both scores of nuclear size and mitotic counts were significantly higher in proximal group than distal or control group, and significantly correlated with gene amps. These results suggested a significance of A-bomb radiation exposure in HER2 and C-MYC amps in breast cancers from survivors. Gene amp is commonly associated with GIN in solid cancers. In breast cancers from the proximal distance group, the level of GIN might be high and resulted in inducing oncogene amps and tumor progression.

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The purpose of our research is to find ways of radiation protection for a nuclear terror. In this case study, using the dose level map for 1kt nuclear explosion in Tokyo1, we applied the lifetime table method2,3 to estimate the excess cancer mortality rate for the survivors. Data for the estimation were compiled by measuring the population and cancer mortality (according to National Vital Statistics in 2000) and the approximate daytime population density. The numbers of population affected by radioactive fallout with a wind velocity at 24 km/h in the elliptical spread area were estimated to be 50,000 in level-A (over 4Sv) , 110,000 in level-B (1-3Sv) and 690,000 in level-C (0.1-0.9Sv). The survival probability in a region of level A is presumed to be negligible. The excess risks of cancer mortality were estimated to be 3.4% for men and 2.5% for women in a region of level B, and 0.8% for men and 0.6% for women in a region of level C, respectively. The shortened lifespan of survivors were estimated to be 0.36 years for men and 0.29 years for women in a region of level-B, and 0.09 years for men and 0.07 years for women in a region of level-C, respectively. These figures for shortened lifespan are remarkably low for the survivors with high doses. The estimated figures from this study coincide with the lifespan for the survivors near the ground zero after the nuclear bombing in Hiroshima, 1945.
References [1] Takada J., Nuclear weapon terrorism in Tokyo, Kodansha (2004) (in Japanese). [2] Wum LM., et al., Estimating lifetime and age-conditional probabilities of developing cancer, Lifetime Data Anal. 4 (1998) 169-186. [3] Kamo.K. et al., Lifetime probability of developing cancer in Japan, Kouseino-Sihyou 52 (2005) 21-26 (in Japanese).

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We evaluated the accuracy of information on drinking and smoking habits in atomic bomb survivors by comparing the information obtained from two kind of surveys: one conducted mail survey through self-administrated questionnaires by Nagasaki City Government on 1 March 2003 and the other conducted interview survey at the time of receiving examination by Nagasaki Atomic Bomb Casualty Council Health Management Center between 1 April 2002 and 31 March 2004. 21,341 individuals (8,468 males and 12,873 females) who were in both surveys were selected. Average age of subjects was 69.4 years in males, 71.5 years in females. The frequency of respective answers for mail and interview survey about drinking in males was as follows: everyday drinking—35.9% vs. 44.8%; sometimes drinking—26.9% vs. 16.5%; never drinking—37.2% vs. 38.8%. In interview survey, the frequency of sometimes drinking was lower and everyday drinking was higher than those in mail survey. The consistency of answers for sometimes drinking was 43.8% in males and 35.3% in females. The frequency of respective answers for mail and interview survey about smoking in female was as follows: current smoking—4.9% vs. 4.5%; past smoking—4.4% vs. 0.7%; never smoking—90.6% vs. 94.9%. The consistency of answers for past smoking was 11.2 % in female. Importance of the procedure of interview about sometimes drinking and past smoking was indicated. No difference was observed in the frequency and consistency of drinking and smoking habits by distance from the hypocenter.

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Nagasaki prefecture designed the support program for elderly atomic bomb survivors. A total of 361 Nagasaki A-bomb survivors were interviewed in 2006 and were asked to totally support about mental health, problem on life and medical and welfare. First step was mail survey about visiting. Second step was interview about totally health. Their health condition assessed, for their mental health conditions using the 12-item version of General Health Questionnaire, for their QOL using WHO-QOL, for their atomic bomb impact scale using IES-R.
In the present study, we analyzed 171 Nagasaki A-bomb survivors who completely responded to all items. The subjects were classified into two groups (i.e. high-score and low-score groups) depending on whether their IES-R score exceeded 24. We used chi-square test and Wilcoxon test for the analysis.
Average of GHQ-score was 3.17 among high-score group and 1.42 among low-score group. Average of WHO-QOL score was 2.9 among high-score group and 3.13 among low-score group. Both score were significantly different among two groups.

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We used imaging plates (IP), sensitive to beta rays, in the present paper to obtain the images of beta ray emitters in the sample. Detection of radiation with IPs is based on optically stimulated luminescence which is accumulated in the lattice defect in the plate with (BaFBr:Eu²⁺) by radiation.
We examined mammal teeth taken in contaminated areas in the former Soviet Union. A part of the south Ural region, Russia, was contaminated by the chemical explosion of a radioactive waste facility in 1957. 467 nuclear tests were conducted from 1940 to 1949 at a Semipalatinsk test site, some of which contaminated nearby villages. The mammal tooth samples from these regions contain ⁹⁰Sr, detectable by IP. After slicing the samples to a thickness of 1mm, they were placed on an imaging plate together with standard samples for one week in a lead cave, protecting natural radiation from the outside. The plates were measured by Fuji BAS-1800-2, the obtained images processed with Multi Gauge v. 3.0. The average concentrations of ⁹⁰Sr in the samples were obtained by comparing the intensities of the luminescence to those of the standard samples.
A sample from Ozerskoe, Russia, with the highest contamination, showed 10.0 Bq/g of ⁹⁰Sr while one from Argayash, with lower contamination showed 0.03 Bq/g, which was correlated with the soil contamination level. A sample from a location close to the center of explosion at the Semipalatinsk test site showed 1.87 Bq/g, while one from the Maysk district showed 0.50 Bq/g where people are raising cows.

Key words
Imaging Plate, ⁹⁰Sr, radiation accident

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Long-lived radionuclides released from nuclear facilities, such as deep underground disposal facilities, could reach humans through several transfer pathways in the environment, in particular food consumption. Consequently, obtaining data on the root uptake of radionuclides to an edible part of a crop, namely the soil-to-plant transfer factor (TF), is important for long-term radiation dose assessment. Obtaining a precise TF under equilibrium is important in long-term dose assessment models to reasonably estimate radiation dose to humans. It would be better to obtain TFs under natural conditions since long-lived radionuclides remain in the environment for a long time. Moreover, safety assessment for people who live in Japan should consider rice and vegetables as critical foods. Unfortunately, many long-lived radionuclides generated in nuclear power plants do not exist in the natural environment and there is a general lack of knowledge on their environmental behavior over decades. To fill these gaps, measurement of TF values of naturally existing elements rather than radioactive nuclides can be a powerful tool to obtain TF values under equilibrium conditions. Thus, our purpose of this study is to obtain TFs of stable elements for rice.

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