High dose radiation significantly induces acute myeloid leukemia (AML) in C3H/He Nrs mice. The AML cells had the hemizygous deletion of chromosome 2. To detect the mutations of oncogenes related to radiation-induced AML, we analyzed the changes of whole-genomic DNA copy-numbers using array CGH, and the RNA expressions by RT-PCR and real-time PCR. The partial gain of chromosome 6 in 35 AMLs with the deletion of chromosome 2 (del(2)+ AML) and that of chromosome 15 in 4 AMLs without deletion of chromosome 2 (del(2)- AML) were observed, respectively. The point mutations in the remaining PU.1 allele were observed in 83% of del(2)+ AMLs, but not in del(2)- AMLs. AMLs induced in syngeneic C3H mice after transplantation of the primary AMLs, showed the similar chromosomal aberrations as well as the gain of chromosome X.
Analyses for the RNA expressions of 24 genes, including PU.1, AML1 and others within the altered chromosomal regions indicated that expressions of GATA1, M-CSFR and Wnt5b were lower in both del(2)+ AMLs and del(2)- AMLs than those in normal spleen cells. In contrast, expressions of PU.1 and C-myc were up-regulated more highly in del(2)+ AMLs than del(2)- AMLs. These results suggest a kind of regulation mechanism in which the expression of PU.1 on the deleted hemizygous allele can be complemented by up-regulation of the remaining allele. This work was supported by a grant from Aomori prefecture, Japan.

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It is well known that radiation-induced chromosomal translocation retains for a long time in hematopoietic cells in a dose-dependent manner. However, it is not yet clarified whether or not translocations retain for a long time after chronic low dose-rate radiation exposure. In the present study, we examined translocation rates in spleen cells from mice continuously exposed to low dose-rate gamma-rays. SPF C3H/HeN mice were continuously exposed from 8 weeks to 20 mGy/22 hr/day for a maximum of about 400 days. When the total accumulated doses reached 0.5, 1, 2, 4, and 8 Gy, spleen cells from mice were cultured for 48 hrs with LPS, Con A, and 2-ME for chromosome analysis. Chromosome aberrations of exchange type such as translocations were analyzed using multiplex-fluorescence in situ hybridization (M-FISH). Translocation rates did not increase until 456 days of age in non-exposed mice. In the irradiated mice, however, translocation rates increased depending on the total dose and were significantly higher than those of the age matched controls. These results showed that translocations induced by low dose-rate radiation can be retained in spleen cells, and could be accumulated in proportion to the total dose, implicating important findings to understand the in vivo mechanisms underlying induction of translocations after exposed to low dose-rate gamma-rays and also to assess the biological risk of exposed to low dose radiation. This work was supported by a grant from Aomori Prefecture, Japan.

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BALB/c mice show susceptibility to radiation-induction of lymphomas, while STS mice show resistance. Resistance to lymphomagenesis has been associated with the middle-proximal domain of STS-derived chromosome 4. Using DNA microarrays, gene expression profiling has been performed in different C.S congenic animals containing the STS alleles in this domain in comparison with the background strain BALB/c. The analysis revealed 7 and 12 spots with more than 2 of the median of ratios (Cy5/Cy3) in C.S7-86 and C.S302-9 animals, respectively. Among these spots 5 were common, and only 2 were on chromosome 4. Other spots are speculated to be downstream of the STS alleles in the congenics. To exclude false-positive signals possibly caused by different dye-labeling efficiency, comparison of the expression levels of these genes from the congenics to those from the BALB/c is presently under way using a real-time PCR analysis.

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We have studies combined effect of X-rays and ENU on the development of thymic lymphoma (TL) using B6C3F1 mice. Our previous results indicated that the induction of TL by ENU was suppressed by prior irradiation of low dose X-rays (0.2Gy or 0.4Gy for 4 consecutive weeks), while high dose irradiation showed synergistic effect. This result may imply that the mechanism of TL development after combined exposures was different between low and high dose irradiation. To provide a molecular clue of combined effect of X-rays and ENU, we examined mutation rate of thymic cells after combined exposure using gpt-delta transgenic mice with B6C3F1 background. [Material and method] . Four-weeks-old gpt delta mice were exposed weekly to whole body X irradiation at 0.2 or 1Gy for 4 consecutive weeks. After irradiation, mice were given ENU at concentration of 200ppm by free-choice drinking for 4 weeks. After 4 weeks of the end of ENU treatment, genome DNA was prepared from thymus of mice for gpt assay. Lambda EG 10 was recovered from the genome using in vitro packaging followed by transfection into E.coli. Mutated gpt genes were selected in the E.coli colonies recovered from 6-TG plates and were analyzed for DNA sequencing. [Results] .Mutant frequency of gpt gene in the thymic cells from ENU treated mice was significantly increased. Preliminary data indicated that the frequency was approximately 10^-4. Mutations were predominantly G:C to A:T and A:T to T:A. Further analysis in mutation of thymic cells from combined exposure is currently undertaken.

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Mortality in life-span studies of radiation carcinogenesis is a useful endpoint for risk assessment of radiation. Although various neoplasm have been reported in life-span studies, their biological profiles as well as nomenclature remain to be undetermined except some tumors which are induced by radiation. We investigated immunohistochemical profiles in the solid tumors (formalin-fixed and paraffin-embedded samples) observed in life-span studies using female B6C3F1 mice exposed to carbon ions and X-rays and control ones. We focused on reaction to proliferative cell nuclear antigen (PCNA) and p53 antibodies. The types of tumors were 10 well-differentiated adenocarcinomas in the lung, 14 granulosa cell tumors (GCTs), 4 ovarian tubular adenomas, 1 ovarian cancer, 3 endometrial adenocarcinomas, 6 fibrosarcomas, 5 angiosarcomas, 4 leiomyosarcomas, 2 malignant schwannomas, 1 rabdomyosarcoma and 1 histiocytic sarcoma. The lung, uterine tumors and GCTs were obtained from the animals exposed to the radiation. As a result, the ovarian, uterine and soft part tumors were strongly positive to PCNA, and some of them were often metastasized to the lung or other sites. Less PCNA positive cells were detected in most of the lung cancers. Interestingly, a half of GCTs showed positive reaction to p53, and negative to p21 and MDM2 antibodies. These results suggest the ovarian, uterine and soft tissue tumors have high proliferative cell activity, which might be related with metastasis, but growth of the lung tumors have low one. Expression of P53 might be a key process of GCT tumorigenesis.

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Oxygen is the essential material when we produce energy, but, it also induces adverse effects. Excessive oxidative stress is thought to be involved in the critical cause of life-style related disease, aging and cancer. These oxidative stresses are principally caused as by-products of energy metabolism, and radiation also induces them. As quantity of oxygen (quantity of energy metabolism) consumed throughout the life does not depend on a creature class. However, rodent cells spontaneously overcome replicative senescence (immortalization) whereas human cells rarely do so. This suggests that ability of oxygen metabolism control in human cells is better than that of in rodent cells. In addition, it is certain that oxygen stress contributes to in vitro carcinogenesis and/or genetic instability. Therefore this study was carried out to clarify mechanisms of intervention of oxygen in in vitro carcinogenesis process and the target of the oxygen in cells being cultured under low oxygen levels. We cultured rodent cells and human cells under atmospheric oxygen (20%) and hypoxic (2% and 0.5%) conditions, and measured cell growth and intracellular oxidation stress (DCFH examination) of each. As a result, by culture in 0.5% oxygen tension, human cells reduced their growth ability, but rodent cells increased it. In 2% oxygen tension, rodent cells showed the elevated growth ability. We will present the results of level of intracellular oxidation stress and chromosome damage of each cells, and discuss the relationship between cell transformation sensitivity and oxygen control ability.

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Radiation carcinogenesis in human is considered as a result of the combined effect of radiation and environment factors. We previously showed that the combined effect on the induction of thymic lymphoma (TL) after exposure to X-rays and N-ethyl-N-nitrosourea (ENU) was dependent upon dose of carcinogen and treatment order. Considering our real life, the simultaneous exposure of radiation and chemical carcinogen is the most common case. The aim of this study is to elucidate the mode and mechanism of the combined effect on TL development after simultaneous exposure of X-rays and ENU.
Four-weeks old female B6C3F1 mice were exposed to X-rays (0.2 to 1.0 Gy per week) for 4 consecutive weeks, and the same mice were simultaneously treated with ENU (0, 50, 100, 200 ppm) in drinking water. Combination of low dose of X-rays (0.2 and 0.4Gy x4) and ENU (50 ppm), the TL was not induced. However, in combination of higher dose of X-rays (0.8 and 1.0 Gy x4) and ENU (100 and 200 ppm), the TL development was synergistically enhanced. Molecular analysis demonstrated that the frequency of point mutations of Ikaros in the TL after simultaneous exposure was increased (46%) compared with that after single exposure of X-rays (27%) or ENU (24%). Mutation spectrum after simultaneous exposure was not only X-ray-induced or ENU-induced type but also additional new one. It is concluded that combined effect of simultaneous exposure is ascribed to the increased point mutation of Ikaros.

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Heterozygous germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary nonpolyposis colorectal cancer (HNPCC). In addition, homozygous germline mutations of MMR genes are also manifested by an early onset of childhood leukemias. We showed Mlh1-deficient mice spontaneously developed thymic lymphomas from 3 month after birth at the frequency of 61%. After X-ray-irradiation at 2-week- and 10-week-old mice, the frequency increased to 87% and 93%, respectively. Irradiation induced tumor more rapidly. The aim of this study was to elucidate the effect of radiation exposure on Ikaros mutation.
The thymic lymphomas developed in unirradiated, irradiated at 2-week- and 10-week-old Mlh1-deficient mice were analyzed by Western blot and sequencing.
The frequent lack of Ikaros protein was found in unirradiated, 2-week-old irradiated group at the frequency of 71%(5/7) and 85%(11/13), respectively, but that in 10-week-old irradiated group reduced to 46%(6/13). All lymphomas lacking Ikaros protein harbored the frameshift mutations in mononucleotide repeat sequence within Ikaros gene. On the other hand, lymphomas expressing Ikaros protein of 10-week-old irradiated group harbored the frequent point mutations (71%; 5/7) in DNA-binding domain. These results show the spectrum of Ikaros mutation is altered in an age-at-exposure dependent fashion in Mlh1-deficient mice.

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In radiation-induced murine myeloidleukemia (rML), a partial alleic loss of chromosome 2 (del2) occurs frequently. It has been believed, therefore, that there is a tumor suppressor gene in the common deletion region (cdr). PU.1, which locates within this cdr, is proposed as a causative gene of myeloid leukemia. We here examined mutation analysis of PU.1 in 41 gamma-ray-induced and 22 neutron(10MeV)-induced leukemia. Point mutations in DNA binding domain of PU.1 were observed in more than 70% of leukemia regardless of the radiation sources. These results suggest that PU.1 is a common target gene in radiation-induced myeloid leukemia, regardless of LET.

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Aberrant methylation of CpG islands, which are CpG dinucleotide rich areas located in the promoter region of many genes, serves as a mechanism for inactivation of tumor suppressor genes in many cancers. Recently, it is reported that lung tumors induced by plutonium and, to a lesser extent, X-rays showed higher frequency of estrogen receptor (ER) promoter than those induced by NNK, suggesting a close link between radiation exposure and promoter methylation. Recently we found 5' flanking region of membranous amino acid transporter gene, Slc7a10, was heavily methylated in MNU-induced rat mammary tumors (The 48th JRRS meeting, Hiroshima). Slc family is suspected as a new tumor suppressor gene in human colon and breast cancers. In the present study, we examined the status of expression and promoter methylation of Slc family genes in radiation (gamma-rays or carbon ions) -induced rat mammary tumors comparing with spontaneous and chemically (MNU or PhIP) induced ones. It was found that promoter of Slc7a10 was heavily methylated in chemically induced tumors, but less methylated in spontaneous and radiation-induced ones. It suggested that promoter methylation of Slc7a10 is dependent on the carcinogens in rat mammary tumors. Other Slc genes, which contain CpG islands in promoter, are now under investigation.

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We have analyzed the pathway for the development of thymic lymphomas and have identified two pathways, the illegitimate V(D)J recombination and the microhomology-mediated end joining, for the deletion formation of Notch1 gene, which is a major oncogene responsible for the development of thymic lymphomas. In the present study, we examined the induction of thymic lymphomas in Rag2Atm-double deficient mice to elucidate the effect of Atm deficiency on thymic lymphoma induction and to analyze the participation of Rag-mediated pathway for the development of thymic lymphomas in Atm-deficient mice. The Rag2Atm-double deficient mice exhibited the reduced frequencies of spontaneous and radiation-induced thymic lymphomas as compared to those in Atm-deficient mice. The Kaplan-Meier tumor-free survival curve revealed that the induction of thymic lymphomas in Rag2Atm-double deficient mice was significantly delayed as compared to that in Atm-deficient mice. The results indicate that thymic lymphomas are induced via both Rag-dependent and Rag-independent pathways in Atm-deficient mice. The frequencies of Notch1 abnormalities in thymic lymphomas were 30%, 56%, and 46% in Rag2, Atm, and Rag2Atm-double deficient mice, respectively, indicating that Notch1 gene is involved in the development of thymic lymphomas in these mice. It is speculated that the rearrangement of Notch1 gene via Rag-dependent and Rag-independent pathways might participate in the development of thymic lymphomas in Atm-deficient mice.

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We previously reported that life shortening induced by continuous gamma-ray irradiation at a low-dose rate in B6C3F1 mice appeared to be caused by death due to the early onset of malignant lymphomas. Life shortening and tumor development in C3H/He Nrs mice irradiated with ¹³⁷Cs-gamma-ray at a dose rate of 20 mGy/22 hr/day for about 400 days (total dose: 8 Gy) were examined to verify the strain differences and tumor types. Lifespan was significantly shortened for 141 and 39 days in irradiated male and female C3H/He Nrs mice, respectively, than that of non-irradiated mice. Significant life shortening was observed in mice died of malignant lymphomas, mammary tumors, ovarian tumors, liver tumors, acute myeloid leukemias (in female), and soft tissue tumors (in female). These results suggest that life shortening due to early onset of malignant lymphomas and the other various tumors occurs in irradiated C3H mice as well as B6C3F1 mice. This work was supported by a grant from Aomori prefecture, Japan.

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Dysregulation of cytokine receptor expression and downstream signal cascade plays important roles in lymphomagenesis. In this study, we compared activation of IL-9R-JAK-STAT cascade in radiation-induced thymic lymphoma (TL) between susceptible C57BL/6(B6) and resistant C3H mice. TL was induced by exposure to split-dose X-irradiation. Activation status of the pathway was investigated by western blotting and immunoprecipitation. The results obtained were as follows: (1) TL from B6 mice showed dramatically high expression of IL-9R protein as well as mRNA compared to normal thymocytes, and IL-9R was highly phosphorylated but less glycosylated, (2) high activation of JAK1, STAT3 and STAT5 were manifested in TL expressing high amount of IL-9R, and (3) in contrast to TL from B6, TL from C3H showed lower expression of IL-9R, and activation of JAK-STAT was also much infrequent. These findings suggest an association between the activation of JAK-STAT pathway possibly resulting from IL-9R over-expression and the TL-susceptibility in mice. Investigation of IL-9R transcription mechanism is in progress.

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We have proposed Long-Lived Radicals (LLRs) in proteins might be related to delayed radiation effects. Both heavy-ion or γ-ray radiation to mammalian cells with the same doses gives almost the same frequencies of the point mutation, but it is larger in complex mutation for the heavy-ion ray. In order to measure the ESR spectra of mammalian cells which have under an active metabolism, we have prepared instruments for cell culture. In this study, the cultured Syrian golden Hamster Embryo (SHE) cells were irradiated with C-ion beam at HIMAC (LET: 55.9 keV/μm) or γ-ray at Nagoya Univ. with the same dose and dose rate of 1 kGy and 0.82 kGy/h, respectively. Non-irradiated cultured cells showed a broadened ESR signal due to at least four radical species. Two species are assigned as a thiyl and a flavin anion radicals, and other two species might be some organic radicals. In addition to these species, a signal due to a hydrogen-added phenylalanine radical was observed in the ESR spectra of the γ-ray irradiated cells. Only a signal due to thiyl radical were detected in the cells irradiated by C-ion beam, and other radicals in non-irradiated or γ-irradiated cells were disappeared. The difference in existing radical species in the cells might be related to the stability of environments where the disappeared radicals exist against to γ-ray and C-ion irradiation. Further investigation is required for clarifying the issue of the difference, and we will investigate the delayed radiation effects through the measurements and identification of the LLRs.

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Mouse genome contains thousands of copies of retrotransposon, intracisternal A-particle (IAP) DNA element, that is constructed from gag-pol gene sandwiched with two LTR sequence. All the cells have the potential of IAP-mediated retrotransposition that includes IAP cDNA synthesis from the RNA and its integration to the mouse genome. We have previously found that genomic rearrangement by insertions of new copies of the IAP cDNA frequently occur in acute myeloid leukemia cells of C3H/He mouse. Since there are large amounts of IAP-related DNA/RNA molecules in the cells, it is difficult to analyze the IAP cDNA that has the potential to convert genomic information. To study the behavior of IAP cDNA, we constructed a series of reporter genes to detect unique reverse transcript and stably introduced into the mouse cells.
Reversely transcribed cDNAs from the RNAs of the exogenously introduced plasmids were confirmed by the determination of the nucleotide sequence of the amplified products by PCR for the unique structure of the retrotransposition. We succeeded in the establishmeht of the methodology to determine relative amounts of the unique cDNA per cell using real-time PCR by simultaneous amplification of a reference plasmid that possessed both fragments of the unique sequence and a single-copy gene.

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Parasitic organisms in cells are activated by damages in the host cells. In mammalian genomes, there are large numbers of retrotransposons that resemble to genomes of retrovirus. Although we found cDNA of retrotransposon, intracisternal A-particle (IAP), is frequently inserted into genomic DNA of radiation-induced myeloid leukemia cells in C3H/He mouse, the molecular process of IAP-mediated retrotransposition following to radiation in mouse cells has not yet been clarified. We studied radiation effect on IAP cDNA synthesis in RAW264.7 based on our original method to measure reverse transcript from expression vector by real-time PCR.
As reporter genes to measure reverse transcription, both entire and deletion types of IAP DNA element that possess unique nucleotides were constructed and stably introduced in RAW264.7 mouse macrophage cells. Transforming cell lines that continuously expressed IAP RNA were x-irradiated and the levels of RNA and cDNA from the transgene were quantified. We found that the level of cDNA from transgene IAP RNA increased 24 hours after radiation in the doses from 2 to 5 Gy, even though the RNA level was kept at a constant level. This suggests that reverse transcription is activated by radiation damage in mouse cells and that radiation stimulates obscure cellular mechanism including retroviral reverse transcription .

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C3H/He inbred mice frequently generate myeloid leukemia after sublethal doses of ionizing radiation. We previously found that the unique genomic insertion of cDNA of retrotransposon, intracisternal A-particle (IAP), is frequently detected in radiation-induced myeloid leukemia cells in the C3H/He mice. To investigate synthesis of IAP cDNA in C3H/He mouse, our original methodology to measure reverse transcription in cell lines was expanded to the generation of transgenic mouse in this study.
A transgene designed to express Idelta1-IAP RNA with 4 blocks of unique oligonucleotide as molecular markers was constructed. By the direct injection of the 12.5 kb DNA into embryos of C3H/He inbred mouse, we succeeded in generation of transgenic mice and bred the F1 and F2 offsprings. Quantitative analysis of nucleic acids from peripheral blood of each individual showed that copy numbers and RNA levels of the transgenic IAP element were stabilized in F1 generation and afterward. Using the molecular marker-specific PCR, we detected cDNA that was specifically synthesized from transgene IAP RNA, suggesting that retroviral reverse transcription was detectable in the transgenic mouse.

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We have analyzed delayed mutation as a part of cellular responses to genotoxic damage and found that this occurs at the pink-eyed unstable locus in the somatic cells of F₁ mice born to irradiated sperm. However, a whole body mouse system and tissue culture cells are too complex to elucidate the molecular mechanism of delayed mutation.
We have now examined delayed mutation in fission yeast. We constructed tester strains which carry reporter genes with tandem duplications. These reporters can revert to the wild type ura4 through homologous recombination. We mainly tested the strains carrying a pair of 200 bp repeats and found that X-irradiation increased recombination frequency of the reporter loci in a dose dependent manner before restarting from arrested cell cycle. In addition, the activation of recombination persisted for about 10 cell generations after irradiation within which damage repair was thought to have been completed. A Rad51 deficient strain did not exhibit both induction and continuation of high-frequent recombination.
These results suggest that untargeted and delayed activation of recombination system is induced not only in mice but in fission yeast by damage to the genome.

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It is important to understand non-targeted effects by ionizing radiation. In the present study, we examined a possibility of the induction of genomic instability by bystander effects. We have already reported that the yield of micronuclei in bystander xrs5 cells, that are defective in the function of non-homologous end joining pathway, is higher than that in normal CHO cells, therefore we examined the delayed effect in offspring of irradiated and bystander xrs5 cells. The result showed that the yields of micronuclei in progeny cells of irradiated xrs5 cells were similar to those in control cells. On the other hand, higher yields of micronuclei were observed in some progeny cells of bystander xrs5 cells, that were co-cultured with irradiated xrs5 cells, compare to those in control cells. These results suggest that genomic instability is induced by bystander signals from irradiated cells in non-targeted cells.

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Genetic instability has been suggested as one of the causes for the development of late effects following radiation exposure, whereas human data are limited. Our recent study on lymphocyte bearing clonal chromosome aberrations in A-bomb survivors did not indicate the presence of the instability in vivo. In the present study, we examined if the instability is observable during in vitro propagation of clonally-derived peripheral T lymphocytes from A-bomb survivors using the same indicator for the in vivo study. One survivor (estimated dose of 1.15Gy, case 1), and one control subject (0 Gy, matched by age and sex, case 2) were studied. Blood T cells were clonally propagated in vitro using standard methods, and metaphases were prepared for multi-color FISH (M-FISH) analysis. One hundred cells were examined for each clonal cell population. We obtained 22 clonal cell populations from case 1 (8 populations with normal karyotype, 8 with identical aberrations derived from clonal cells in vivo, and 6 with different aberrations), and 18 populations from case 2 (all normal karyotype). In case 1, 27 additional aberrations (translocation and derivative chromosome) were found in total among 2,200 cells (1.2%), and the corresponding value in case 2 was 0.3% (6/1,800). Thus, the frequency of additional, sporadic aberrations in the exposed case was several times higher than that in the control subject. While the number of cases studied is limited, present results suggest the presence of chromosome instability among the clonally expanded lymphocytes in vitro from A-bomb survivors.

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Regenerative medicine offers hope of cure for many kinds of disease. One of major fields of regenerative medicine is the application of stem cells in bone reconstruction. Mouse derived myoblast cell line C2C12 was differentiated to osteogenic lineage by treatment with bone morphogenic protein 2 (BMP-2), and radiation-induced suppression was reported when cells were induced to osteogenic lineage 6 h after or before irradiation. On the other hand, heparin maintained BMP-2 level in the culture medium and prolonged its activity. Taking our body into considerations, such effects of extracellular matrix could not be left out. To evaluate the effects of ionizing radiation on osteoblast differentiation including the effects of extracellular matrix, we direct C2C12 cells into osteogenic lineage in the presence of heparin just after or 6 h irradiation. We find that ionizing radiation decreased alkaline phosphatase activity and the expression of collagen type I mRNA in BMP-2 treated cells. These findings suggest that X ray irradiation might be suppressing the differentiation of osteoblasts.

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[Objective] Dendritic cells (DC) are one of antigen-presenting cells and play an essential role in the immune system. Recently, tumor immunotherapy using DC has been reported. However, whether immune response of DC is affected by radiotherapy remains to be eludicated. We therefore in an attempt to clarify the effect of radiation on the differentiation of DC, we focused on monocytes that could differentiate into myeloid DC, while also investigating whether X-irradiated monocytes could differentiate into DC. [Methods] Monocytes were separated from buffy coat, and then were exposed to X-ray of 5 Gy. For the preparation of immature DC (iDC), X-irradiated monocytes were cultured in RPMI 1640 supplemented with 2% human AB serum, rhGM-CSF and rhIL-4 for 5 days. To prepare mature DC (mDC), iDC were further incubated in the presence of rhTNF-alpha for 4 days. The phenotype of iDC and mDC and the phagocytic activity of iDC were analyzed by flow cytometry. [Results] The iDC and mDC generated from X-irradiated monocytes expressed each type of DC specific surface antigen. However, the expression of CD40 on iDC and CD80 on mDC derived from X-irradiated monocytes was higher than of the control, respectively. Furthermore, the degree of phagocytosis of iDC was also observed to be lower than of the control. [Discussion] Our results demonstrated that X-irradiated monocytes could differentiate into DC. In addition, since the expression of CD40 increases while phagocytosis decrease during maturation, these findings suggest the possibility that X irradiation may therefore enhance DC maturation.

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Radiation protection effect and an anti-tumor effect are recognized in AHCC educts by a precedence study, and, as for the action mechanism, a thing by immunization activity action and anti-oxidation action is made clear. In this study, we reviewed an anti-tumor effect with radiation exposure and presence of protection effect of immunization degradation by combined effect in anti-oxidation action and radiation with AHCC. We did oral of each AHCC 250mg/kg every other day for two weeks after having inoculated about 2*106 Scc-7 into ICR mice. In addition, we measured weight and tumor size later for cancer cell seeding two weeks. Furthermore, in AAPH method, we measured anti-oxidation action. As the days passed in the group that they inoculated only a cancer cell into, the tumor size increased, but decrease of tumor size to be clear in radiation independent exposure group and a AHCC and radiation combination group was recognized. It was the crowd whom they inoculated only a cancer cell into after exposure two weeks, and 2/3 lived in AHCC and radiation combination group for what died almost. In addition, the leukocyte, the lymphocyte count increased by the dosage of AHCC regardless of presence of radiation exposure. In addition, anti-oxidation action was clear, too. It think that we can contribute to an immunological enhancement effect in radiation cancer therapy and side effect prevention in addition to protective effect and a cure effect of cancer by taking in a AHCC.

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Our preceding studies have demonstrated that EF2001 extracts have some radiation-protection and anti-tumor effects, and, as for the mechanism of the action, the enhancement of immuno activity and the action of anti-oxidation have been elucidated. In this study, we examined anti-tumor effects, the protection of immuno disturbance and the anti-oxidation action, against radiation in EF2001. After an intra-abdominal inoculation of approximately 2 x 106 Scc-7, ICR mice were given an endoceliac dosage 100 mg/kg of EF2001 every other day for two weeks. 2 Gy of radiation was given three times and the numbers of leukocytes and lymphocyte were counted. Comparing with that the group inoculated only with cancer cells increased the tumor size with time, some decrease in tumor size was clearly demonstrated in the group of radiation exposure groups and administration of EF2001 group. Almost all the mice inoculated only with cancer cells died after two weeks of radiation, while two thirds of radiation and EF2001 groups lived. The leukocytes, the lymphocyte counts, increased when doses EF2001 were administered regardless of radiation exposure. Anti-oxidation action was also demonstrated in both groups of EF2001. These results may indicate that administration of EF2001 enhances the immunologic activity and prevent side effects during cancer radiotherapy and provides a supplemental tool for the cure of cancer.

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[Purpose] We have reported that human memory CD4 T cells in peripheral blood can be discriminated into three functionally different subsets (M1-3) by using HSCA-2 mAb against CD43 (J.Immunol,169:39,2002, J.Immunol,172: 7246,2004). The M1 subset consists of functionally maturated cells whose CD43 expression is relatively high; the M2 subset cells that express moderate levels of CD43 and respond weakly to TCR-mediated stimuli; and the M3 subset cells whose CD43 expression levels are relatively low and which are anergic to TCR-mediated stimuli and prone to spontaneous apoptosis. To evaluate how properly T-cell memory is maintained in A-bomb survivors, in this study we examined the relationship between the proportion of these memory CD4 T-cell subsets and aging or radiation exposure.
[Results and Discussion] The percentage of each memory T-cell subset in the CD4 T-cell population appeared to significantly increase with age but did not differ between males and females. It was also suggested that there were dose-dependent increases in the percentages of M2 (P = 0.036) and M3 (P = 0.059) subsets. Generally, individual ability to properly maintain T-cell memory is known to decline with aging. It is conceivable that the present study provided support to the hypothesis that such immunological aging has been accelerated in A-bomb survivors.

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In A-bomb survivors, there are significant radiation dose-dependent decreases in the percentages of naïve T cells, suggesting a reduction in the ability of thymus to produce T cells. In the present study, we made an attempt to evaluate individual thymic ability to produce T cells, by measuring the number of T cells bearing T-cell receptor-rearrangement excision circles (TRECs) as an endpoint for the number of naive T cells that have not undergone cell division after thymic emigration. The number of TRECs in each CD4 and CD8 T-cell fraction separated from peripheral blood lymphocytes using a cell sorter was measured by real-time PCR. In the 445 survivors so far been examined, multiple regression analysis indicated that the number of TRECs in the CD4 T-cell fraction was significantly higher in females than in males and decreased significantly with age in both males and females. This analysis also suggested a possible dose-dependent decrease in the number of TRECs in the CD4 T-cell fraction of the survivors who were less than 20 years of age at the time of bombing (P = 0.09). A similar statistically significant trend for gender difference or age was observed in the CD8 T-cell fraction of the survivors. However, there was no effect of radiation exposure on the number of TRECs in the CD8-T cell fraction. The results indicate the possibility that A-bomb radiation exposure may have induced a long-term impairment in thymic CD4 T-cell production. Further investigations in a larger study population are planned to test this hypothesis.

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Atopic dermatitis, an allergic disease of type 1, is a chronic disease that affects the skin. Recently, although a large number of studies have been made on effect of ultraviolet B (UVB) on immune system, little is known about the effect and mechanism of UVB irradiation on atopic dermatitis. In this study, we investigated whether UVB irradiation influences atopic dermatitis in an NC/Nga mouse model. Atopy score, ear thickness, and total IgE were measured at each time interval in all groups. On day 38, Cytokine production in the spleen lymphocytes (IL-4, IFN-γ, IL-10, IL-12) were measured with ELISA and subpopulations of spleen lymphocytes were performed by flow cytometer. The levels of TNF-α produced by peritoneal macrophage were measured by ELISA and NO production was analyzed by NOx analyzer system. UVB irradiation (0.1J/cm²) increased atopy score, ear thickness, and total IgE in comparison with the disease-control group. Levels of IL-4 and IL-10 were increased versus the disease-control group, resulting in a further decrease of the IFN-γ/IL-4 ratio compared with the disease-control group. In addition, the levels of TNF-α and NO production of peritoneal macrophage also were elevated. These results indicate that UVB irradiation may exacerbate atopic dermatitis. This may be because the UVB irradiation induces the changes of various cytokine in this atopic mouse model.

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Damage to gastrointestinal (GI) system is one of major problems in exposure to high dose radiation. However, treatment of the GI injuries and its mechanisms are poorly understood, since an in vitro experimental system of GI injury is not established. There are many cell lines derived from intestinal epithelial tissues, but most of these cell lines are transformed and acquired radioresistance. Using the primary culture system of GI epithelial cells from fetal rats (Fukamachi, H., 1992), we studied a mechanism of GI injury caused by radiation. Epithelial cells from forestomach (FS), glandular stomach (GS), and duodenum (D) were obtained from 17.5-day rat fetuses and cultured for three days. The numbers of these cells increased by 5-fold on Day 3. These cells were irradiated with 10 Gy of X-ray on Day 1 (24 hr after spread). Hoechst staining showed that irradiation induced apoptosis in all three kinds of epithelial cells within 24 hr postradiation. On the other hand, the apoptotic indexes varied among these cells; indexes were 5% and 15% in GS, and FS and D epithelial cells, respectively. When irradiated on Day 3, apoptosis was observed in FS and D epithelial cells but not in GS. When D epithelial cells were irradiated at various doses, the apoptotic index increased in a dose-dependent manner, and reached about 20% at 20 Gy. We conclude that this culture system is helpful for analysis of a mechanism for GI injury by irradiation. Further investigation on the different radiosensitivity among three epithelial cells is now in progress.

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The effects of iron ion beam (500 MeV/u, LET=200 KeV/micronm) 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 iron ions. The effect was studied by scoring changes in the pigmentation in cutaneous coats 22 days after birth and in the prenatal and postnatal development of mice. The percentage of birth, survival to day 22 and body weight at day 22 were decreased in irradiated mice. The effect of iron ions 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. Iron ions were more effective than gamma-rays. The frequency of abnormalities in the fore and hind legs, tails and eyes as well as of hemorrhage was increased as dose increased and the number of embryos as well as the body weight at 18 days of gestation was decreased. These results suggest that iron ions have a greater effect on the melanocyte differentiation as well as on prenatal and postnatal development of mice than gamma-rays.

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The effect of heavy-ion irradiation to brain on sexual behavior is not yet known. The present study was designed to determine whether irradiation (carbon particles 290 MeV/nucleon, Mono peak 15-120Gy, irradiation field 5-millimeter cube in hypothalamus) to rat's hypothalamus modifies the copulatory behavior of male rats. We planned to estimate the short-term effects of carbon-irradiation on the copulatory behavior using a relatively high doses, and observation of sexual behavior was conducted for 30 min after 1, 2 or 3 months following irradiation. Results are as follows. 1) At dosages of 15Gy, 30Gy, 45Gy and 60Gy these were no changes in copulatory behavior after one month following irradiation; however, the intromission and ejaculation was found to decrease after 3-month follow-up in rats exposed to 60Gy. 2) At higher doses such as 90Gy, 120Gy, the number of mounting, intromission (120Gy alone) and ejaculation (90Gy, 120Gy) were decreased. It may be possible to describe that carbon irradiation to hypothalamus does not inhibit the activity of copulatory behavior after short-term. Further experiments after long-term follow-up after irradiation are necessary to determine the chronic effects of heavy ions on the copulatory behavior.

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Formation of cerebral edema is acute response of brain tissue to a large radiation and may contribute to delayed pathology (vascular collapse and tissue necrosis) that often lead to permanent loss of function. Therefore, it is very important to reduce formation of cerebral edema for protect potential damage of normal brain tissue. Cyclooxygenase-2 (COX-2) is the inducible form of the 2 prostaglandin-synthesizing enzymes and is believed to be an important mediator of cell injury in inflammation. However, it is unclear what the role of COX2 activity in the brain irradiated with heavy-ion beams. In the present study, the left cerebral hemispheres of adult rats were irradiated at dose 50 Gy with heavy-ion beams, expression of COX2 protein and the effect of treatment with a COX2 inhibitor on cerebral edema were determined and compared at 2-24 hrs after irradiation. The results indicated that radiation-induced cerebral edema was dependent on COX2 activity.

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Public interest has been growing regarding various hazardous factors, which may affect the development of the central nervous system (CNS) of children. However, no simple method has been developed for identifying neuronal damages in developing mammalian embryos, because they develop in the mother's body and the developmental process cannot be examined directly. In contrast, medaka embryos develop outside the mother's body, and their chorions are transparent. Consequently, all gross abnormalities in these living embryos can be detected throughout the entire period of development using an ordinary stereomicroscope. In this study, early and late medaka embryos were subjected to a single acute dose of 10 Gy of X-ray irradiation, which is a lower dose than the LD₅₀ of the embryos.The effects of X-ray irradiation on the developing CNS were examined in the living embryos under a stereomicroscope for 10 days. After X-ray irradiation, all embryos survived; however, many clusters of dead cells were observed in the CNS of the embryos from 4h to 6h after irradiation. These dead cells disappeared thereafter, and the irradiated embryos continued to develop normally. At the hatching period, however, the irradiated embryos had smaller brains and exhibited abnormal histologies of the brain and retina. These results suggest that living medaka embryos are useful for screening the developmental neurotoxicity effects of various hazardous factors. Now, we are studying on the late medaka embryos, which were irradiated with a single acute dose of 5Gy of X-ray.

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Epidemiological studies on atomic bomb survivors exposed in utero have revealed that radiation causes abnormalities of brain development. Several studies of mice and rats have also shown that prenatal irradiation of low-LET radiation induces developmental anomaly of the brain. However, little is known about the effect of prenatal irradiation on the behavior pattern in the adulthood. Therefore, we investigated the effects of neutron exposure in utero on the postnatal behavior pattern of mice. B6C3F1 mice were exposed to cyclotron-derived fast neutrons with peak energy of 10 MeV (0.02-0.2 Gy) or Cs-137 gamma-rays (0.2-1.5 Gy) on gestation day 13.5. At 6.5-8 months of age, male offsprings were examined for their neurobehavior (RotaRod, locomotor activity, forced swimming). The brains were weighed, and examined histopathologically. Control group and neutron-irradiated groups were analyzed for accumulation of radiolabeled drugs to muscarinic acethylcholine receptor and serotonin receptor by tracer method. The results obtained were as follows; (1) the brain weights decreased in dose-dependent manner in both types of radiation, (2) locomotor activity during dark period and immobility time of forced swimming increased in 0.02Gy neutron-irradiated group. Further binding of drug to each receptor in vivo was increased in 0.02Gy neutron group. In conclusion, a certain 'low dose window' exists for radiation-induced changes in neurobehavior and binding to neurotransmitter receptors.

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Epidemiologic studies on A-bomb survivors indicate a significant increase of non-cancer risks (mainly life-style diseases). However, since no animal models are known, understanding of the biological mechanisms is slow. Here, I propose possible mechanisms that are biologically testable.
Low birth weight has been indicated as one of the risk factors for coronary heart disease, hypertension, and type II diabetes later in life, which raises a possibility that a part of life-style disease risk starts from fetal life. This observation is interpreted as that when nutrition level to fetuses was insufficient, babies are born small and acquire thrifty phenotype after birth as a result of adaptation. This phenotype is considered to be beneficial if the postnatal food conditions are suboptimal whereas if not it can lead to elevated risk of life-style diseases.
Two explanations are proposed; 1) Insufficient tissue recovery: it is possible that tissue recovery is not perfect. For example, insufficient cell number in pancreas can lead to suboptimal production of insulin years later. 2) Hypomethylation of DNA: A^vy(viable yellow) allele in the mouse is due to insertion of a retrotransposon (RT), which gives rise to a wide interindividual variation in methylation at RT sequences (dark brown = methylation, yellow = hypomethylation). When methyl-rich diet is given to pregnant mothers, the offspring becomes darker color and healthier. By contrast, radiation exposure and malnutrition may cause the opposite effect.

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DNA methylation in eukaryotes is the key event that governs epigenetic phenomena, including development and cell differentiation. Its collapse results in disorders such as cancer, aging and so on. Therefore it is essential to find a comprehensive method for detecting the change of methylation sites, for both basic and clinical research. Compared with gene expression profiling analysis, the analysis of methylation has several advantages. 1) It is rather easy to detect low-abundance transcripts. In gene expression profiling, detecting rarely-expressed genes is usually quite hard. 2) It is possible to identify unknown transcripts, including novel types of transcripts such as poly(A)-less ones. 3) It is possible to understand the epigenetic mechanisms. Recently we proposed a new highly sensitive, high-coverage method using a methylation-sensitive restriction enzyme in combination with the selective PCR of HiCEP. This enables us to analyze approximately 30,000 methylation sites simultaneously. In the present study, we examine whether this procedure accurately detects changes in methylation using bisulfate sequencing analysis, and whether the methylation closely correlates with the expression of adjacent genes. This is an ideal fragment analysis system since it is simple, has few peaks and few false signals. Peak simulation using sequence information in the public database seems to be realistic. We used this approach and evaluate its reliability. We also discuss a protocol for the analysis using several hundred cells.

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Thyroid hormone receptors (THRs) are transcription factors that regulate the expression of a variety of genes affecting cell growth and differentiation. Somatic mutations in the THR genes were found in human malignancies suggestive of their implication in carcinogenesis. In view of a broad range of intracellular processes dependent on the functional THRs, we set out to clarify the influence of THRB1 on cell radiosensitivity. Using an adenoviral vector, wild type (wt) and mutant (mut) THRB1 were overexpressed in TPC1, NPA, FRO, ARO , MCF-7 and COS-7 . Infected cells were irradiated with 0-7 Gy of gamma-rays and subjected to a colony formation assay. No significant changes in clonogenic survival were observed in TPC1, NPA and FRO cells (all display a high level of endogenous THRB1 protein). By contrast, in ARO, MCF-7 and COS-7 cells (with a low to absent endogenous THRB1), wtTHRB1 decreases and mutTHRB1 increased clonogenicity. Western blotting demonstrated that in the presence of wtTHRB1, ARO cells had elevated levels of cleaved PARP and markedly diminished levels of cyclin D1 as compared to mutTHRB1-infected and/or irradiated cells. In MCF-7, mutTHRB1 overexpression resulted in a strong upregulation of pospho-AKT. Thus, THRB1 can possibly mediate radiation effects by influencing apoptotic, cell cycle progression and survival programs. Dominance of each of signaling cascades appears to be cell type-specific. In conclusion, the net effect of wtTHRB1 is radiosensibilizing but that of mutTHRB1 – radioprotective.

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BackgroundInterleukin-11 (IL-11) is a well known anti-inflammatory cytokine that is associated with cell growth, and also participates in limiting X-ray irradiation induced intestinal mucosal injury.
AimThis study was performed to elucidate the effect of IL-11 in X-ray irradiated normal intestinal epithelial cells.
Materials and Methods Recombinant human IL-11 (rhIL-11) treated IEC-18 cells, normal rat ileum epithelial cells, were irradiated by X-ray and then examined for the cell viability. To evaluate the X-ray irradiation injury, we used trypan blue staining to detect the dead cells.
Results The viability of X-ray irradiated IEC-18 cells was up-regulated by rhIL-11 treatment and also resulted in the activation of p90 ribosomal S6 kinase (p90RSK) and S6 ribosomal protein (S6Rp). Wortmannin, a specific inhibitor of PI3K, suppressed the activation of S6Rp in rhIL-11 treated IEC-18 cells, and decreased the up-regulation of viability by rhIL-11 treatment in X-ray irradiated IEC-18 cells. We also performed TUNEL assay to estimate the rate of apoptosis in X-ray induced cell death. There was no difference in the result between in trypan blue staining and TUNEL assay. Further, rhIL-11 down-regulated the expression of cleaved caspase-3 in X-ray irradiated IEC-18 cells. ConclusionThese results suggest that rhIL-11 may play an important role to the protection of intesinal epithelial cell from X-ray irradiation injury.

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A body of clinical evidence has shown that expression levels of anti-apoptotic Bcl-2 proteins are increased in many types of malignant tumors, which are often resistant to low-LET ionizing radiation like X-rays and gamma-rays. However, little is actually known about influences of Bcl-2 expression levels on tumor radiosensitivity in vitro. To address this, we have therefore examined effects of Bcl-2 overexpression on vulnerability of cultured tumor cells to low-LET radiation. Colony formation assay revealed that 10% survival doses of ⁶⁰Co gamma-rays in HeLa cells overexpressing Bcl-2 (HeLa/bcl-2 cells, a kind gift from Prof. Masayuki Miura, Tokyo Univ., Japan) and in parent HeLa cells were 6.9 Gy and 4.8 Gy, respectively, indicating that increases in Bcl-2 expression lead to resistance to gamma-rays. Further experiments are under way, to examine killing of HeLa/bcl-2 cells by high-LET heavy charged particles, which have been applied to cancer therapy taking advantage of its greater biological effectiveness and more excellent selectivity of special dose distribution.

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Studies on the radioadaptive response (AR) in rodents retrospect to 1960 for the editio princeps by Maisin et al, stating a slight increased 30-day survival rate induced by preirradiation in rats. The first report on a significantly enhanced survival rate induced by preirradiation was by Yonezawa et al in 1990 in ICR mice. Later in 2001 Ohyama et al verified this so-called "Yonezawa Effect" and demonstrated that the AR could be induced in C57BL strain mice under the similar conditions. Namely, preirradiation of 6 weeks old mice with 0.5 Gy of X-rays before a second exposure 2 weeks later to a lethal dose of 6.5 Gy significantly enhanced the survival rate from 0 to about 77% in a 30-day survival study. On the other hand, Wang et al's work in 1998 and 2000 proved the existence of AR in fetal mice in both ICR and C57BL strains. However, the postnatal survivals rescued by the AR in utero were not healthy, being significantly suffered from neurophysiological alteration and developmental retardation. In this study, the responses of the postnatal survivals to the lethal irradiation and to the AR induction were investigated in C57BL mice using 30-day survival test. A significantly increased radiosensitivity to the killing effect was observed and the AR could not be induced in the AR postnatal survivals. For those only receiving the priming dose in utero, the response to lethal irradiation and AR induction was similar to the non-irradiated animals. These findings indicated that the postnatal survivals did not response to radiation the same as the control non-irradiated animals.

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We studied the localization of phosphorylated H2AX in cultured human fibroblasts (NB1RGB) and GFP-tagged rad51 in Chinese hamster CHO cells after irradiation with heavy ion beams. Asynchronous cells were irradiated with X-rays, carbon ion beam (LET is about 88 keV/um), Si ion beam (220 keV/um) and Fe ion beam (440 keV/um) at room temperature. Phosphorylation of H2AX in irradiated cells was measured from 0 to 24 h after irradiation monitored by using flow cytometry. Phosphorylated H2AX increased just after irradiation of each radiation and reached maximum around 30 min. Phosphorylated -H2AX was then decreased quickly for cells irradiated with X-rays but presented longer for Fe beams. Under the confocal microscopy, foci of gamma-H2AX on cell nucli was not visible just after X-irradiation, but obvious after Fe ion irradiation. Number of foci per nucleus was increased with increasing dose of each radiation at 30 min after irradiation. On the other hand, rad51 accumulation was observed about 2 h after irradiation. Foci of rad51 were visible about 10 h after irradiation.

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Radiotherapy treatment is expected to be applied to intraoral melanoma in a dog, since the surgical treatment accompanies massive degradation of the QOL and mental distress of an owner by a change of the exterior. Melanoma is resistant to X-ray, so it is expected to apply ion beams, which have Bragg peak and high biological effect, to the radiotherapy. But, the report about the radiosensitivity of canine spontaneous melanoma is not enough. We irradiated some of different radiation, carbon ion (LET=108 keV/ μm), proton (LET=2.7 keV/μm) and X-ray (LET=1.0 keV/μm) at TIARA for established cell line (CMM2) of canine spontaneous melanoma origin in this study. We evaluated the lethal effect in low dose from high dose range by clonogenic assay. After irradiation, the cells were plated at a moderate density. 8 days later, the cells were fixed and stained. We made survival curves from the results. While survival curves for the cell line after X-ray and proton irradiation had a shoulder in low dose, survival curve for the cell line after carbon ion irradiation didn't have a shoulder, and the surviving fraction in low dose was much lower than the extrapolated survival curve. When we compared the lethal effect at D10 to estimate the difference between radiation, lethal effect of cell was high in order of carbon ion, proton, X-ray, and it was observed that RBE based on X-ray was increasing with LET. This result indicates that the lethal effect of cell increase with LET, and it was thought that application of carbon ion to an intraoral melanoma was effective on the therapy.

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Production of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was examined in human leukemia HL-60 cells irradiated with carbon, neon and silicon ions. The LETs of carbon are 20, 50 and 80 keV/μm, those of neon ions 80 and 150 keV/μm, and those of silicon ions are 150 and 250 keV/μm. Irradiated cells under the air-saturated condition were subjected to DNA extraction using isopropanol, separation of DNA strands by heat treatment, digestion into nucleosides with nuclease P1 and alkaline phosphatase. 8-OHdG was separated as a single peak on a chromatogram of ECD detector. The linearity of the peak area with irradiation dose was confirmed using X-ray irradiated specimens. For all the ions tested, 8-OHdG yield was decreased with increasing LET with a saturation tendency in the high LET region. These results are in good agreement with those of irradiated dG solution which was previously reported by us. Ion species dependence in 8-OHdG yield was not so apparent through the comparison of 1) carbon and neon ions with an LET of 80 keV/μm and 2) neon and silicon ions with an LET of 150 keV/μm.

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(Purpose)
In order to quantitatively evaluate the generation of hydroxyl radicals in water and oxidative DNA damage caused by high LET radiation, we measured the ESR spectrum of DMPO radical adduct and 8-hydroxydeoxyguanosine (8-OHdG) in the salmon sperm DNA after irradiation.
(Materials and methods)
Pure water containing DMPO (200mM) and that containing salmon sperm DNA (1.0mg/ml) were irradiatied at a dose of 20Gy and 10Gy, respectively, with 290 MeV carbon-ion, 135 MeV carbon-ion and 490 MeV Silicon-ion beams at 4 and 37C. In addition, the sample was deoxygenized by argon to investigate the effect of oxygen on the OH generation. The DMPO solution was analyzed by ESR spectrometer 10 min fter irradiation. The 8-OHdG level in the ST-DNA were measured by HPLC with an electrochemical detector (ECD).
(Results)
Both levels of OH and 8-OHdG decreased as LET value increased. They were interfered with the temperature and the existence of oxygen during the irradiation.

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We previously reported tumor induction in mice legs after fractionated irradiation with carbon ions (J.Radiat.Res., 46,185-190, 2005), reporting that the RBE value of carbon ions was 2.9. What puzzles us is that dose responses of carbon ions as well as reference gamma rays are linear without decrease at large doses, which is contrary to other reports using whole body irradiation. We here studied and reported tumor induction in the same radiation conditions/endpoints after single doses. Right legs of female C3H mice were irradiated with either Cs-137 gamma rays or 290 MeV/u carbon ions of Spread-Out-Bragg peak (SOBP). Number of mice used was 50 for each dose, and a total of 1650 mice were irradiated. Irradiated legs were once a week observed and palpated to detect tumor induction up to 800 days. Tumor induction frequency for any radiation qualities increased with an increase of dose up to 40 Gy. The frequency saturated to increase at higher doses, and rather decreased at 80 Gy of gamma rays and at 60 Gy of 75 keV/micrometer carbon ions. RBE values of carbon ions were calculated by using a dose to induce 20 % tumor, and were 1.0, 1.2 and 1.9 for 15, 45 and 75 keV//micrometer, respectively. Maximum frequency of tumor induction by 15 keV/micrometer carbon ions was lower than that by gamma rays. Carcinogenic effects of carbon ions are quantitatively and qualitatively different from gamma rays. Low LET carbon ions could be less dangerous than gamma rays.

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Facilities for particle radiotherapy are increasing in number these days. Using mouse gut crypt survivals as an endpoint, we have investigated and here report the biological effectiveness of proton beams in domestic facilities. Also, we compared carbon-ion beams at Darmshtad (Germany) GSI and at Chiba HIMAC. Therapy facility and the irradiation condition: Wakasa wan energy research center 180MeV/u proton-beam 6cm-SOBP entrance plateau or middle position of SOBP (reference: Fukui medical university Linac4MeV/u). Shizuoka cancer center: 190MeV/u proton-beam 6cm-SOBP entrance plateau or middle position of SOBP (reference : Linac Shizuoka cancer center 6MeV/u). GSI: 290MeV/u carbon-beam 6cm-SOBP proximal, middle, distal HIMAC: 290MeV/u carbon-beam 6cm-SOBP proximal, middle, distal We obtained 3 isoeffect doses (D0, D30 and D10) from a crypt survival curve, and calculated a representative RBE value by averaging 3 RBE values obtained for the isoeffect doses. RBE of the proton beam in the Wakasa wan energy research center was 0.98 at the entrance plateau, and at the middle position of SOBP was 1.13. RBE of the proton beam in Shizuoka cancer center was 0.99 at the entrance plateau, and at the middle position of SOBP was 1.05. Commonly observed was that RBE at the middle position of SOBP was slightly larger than RBE in the entrance plateau. When we compared isoeffect doses for the carbon beam between GSI and HIMAC, biological effectiveness of HIMAC beams stronger than that of GSI by 5 %.

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Radiation impairs some functions of the central nervous system, which is one of the radiation-resistant tissues in the body, but the exact mechanisms are still unclear. We found that total body and head, but not abdominal, carbon irradiation induced a significant increase of the release of glutamate, the major excitatory neurotransmitter in the central nervous system, in the hypothalamus. We also found that the increase of glutamate is dependent on the Ca₂₊ ion, suggesting that the increased glutamate is derived from the release from neurons or glial cells. However, the underlying mechanisms of the increase of glutamate release are still unclear. In this study, we investigated that the effects of the glial selective metabolic inhibitor (L-aminoadipatic acid (L-AA), glutamine synthetase inhibitor (methionine sulfoximide (MSO)) and inhibitor of glutamate release from glial cell (carboxyphenylglycine (CPG)) on the increased glutamate measured by in vivo brain microdialysis. L-AA and MSO completely inhibited the heavy-ion-induced increase of glutamate, but CPG did not inhibit. Administration of glutamine recovered the increased glutamate level in the MSO-treated rats. These results suggested that neurons, but not glial cells, play an important role in the heavy-ion-induced increase of glutamate.

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Intestinal crypt stem cells have a high growth potential and radiosensitivity. It is dose-dependently reduced by heavy-ion irradiation and intestinal death occurs by arrest of epithelial cell supply in high dose area. Radiation to abdominal cancer, for example uterus and bladder, could give impairments not only on tumor but also on gut nearby target. Therefore, we believe that gut protective agents contributes to more effective and less harmful heavy-ion therapy. NMDA receptor is one of excitatory amino acid receptors and NMDA antagonist has been reported to prevent the radiation induced injury. We have hypothesized that peripheral NMDA receptors correlates with events induced by radiation and conducted in vivo experiments to test the hypothesis. The isotope-labeled [³H]MK-801 which is a noncompetitive NMDA antagonist was intravenously injected with C3H female mice and the biodistribution of MK-801 was investigated in time-dependent manner. Gut accumulation of MK-801 increased within 30 min after injection and reached maximum. MK-801 accumulation in gut after whole body irradiation of 9 Gy carbon-ion (290MeV/u, 20keV/µm) under unanesthesia were also examined. That increased until 24 hrs after irradiation and decreased afterward. These findings suggested that intestinal NMDA receptors are most activated at 24 hrs after carbon-ion irradiation and it is involved in gut impairments. Moreover, we compared the dose-dependent change of crypts after irradiation between treated with MK-801 and untreated mice in order to confirm a MK-801 protective effect on crypts.

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High-LET radiations are more effective for cell killing than low-LET ones, thereby having been applied to cancer therapy. Unavoidable, however, is exposure of normal cells which coexist with or surround tumors. Though many lines of evidence have shown that reproductive death occurs in the progeny of cells surviving radiations, its LET dependence remains elusive. We here investigated the cell-killing effectiveness of ⁶⁰Co gamma-rays (0.2 keV/μm) and six different types of heavy ions (16.2-1610 keV/μm) in normal human fibroblast AG01522 cells, being processed for primary and secondary colony formation. RBE values relative to iso-survival dose of gamma-rays peaked at around 100 keV/μm both in primary and secondary colonies, showing that delayed reproductive death observed in secondary colonies arose in a LET-dependent fashion. On the other hand, very little difference in LET was found in the RBE based on the secondary survival at the primary 10% survival doses, indicating that delayed reproductive death arising only during secondary colony formation depends upon initial damages which would have been fixed during primary colony formation. Further studies to clarify the LET dependence of heterogeneity in colonies derived from irradiated single cells and apoptosis induction are ongoing.

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We performed basal examination of ABM-FD (Freezing dryness ploysaccaharides, Agaricus Blazei Muril l; Himematsutake) about the safety and the protection effect of whole body X-ray irradiation in normal mice. In addition, combination effects with X-ray irradiation were investigated in Meth A tumor-bearing mice. The oral administration with safety for ABM-FD massive dose (7,200mg/kg/day x 4), there was not a change to mention specially in general findings after administration (body weight and organ findings in autopsy). However, increased 2.67 times (p<0.001) for control (distilled water administration) in ABM-FD administrated group was present in WBC count. Furthermore, the increase was found in an incorporation of 3H-thymidine of Con-A and LPS addition lymphocyte by the mitogen reaction of splenic lymphocytes in ABM-FD administrated group. The ABM-FD (680mg/kg/day x 15-18) administrated groups in which performed immediately after irradiation administrated group showed survival rate of 66%, but 6 days after irradiation administrated group and control group for survival rate 33%, it is a clear effect was found in protection from whole body exposure (8.0 Gy). The oral administration of ABM-FD (960mg/kg/day x 7-10) and X-ray irradiation (3 Gy/day x 4) moderately inhibited the growth of Meth A tumor cells implanted s.c. in mice. Development of implanted tumor was strongly inhibited by the combination of ABM-FD and irradiation. These result suggest that the radiation exposure protection effect of AMB-FD inhibits bone marrow death not action as radical scavenger.

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Oxidative modification of biomolecules has been implicated in the pathogenesis of many neuronal disorders including cognitive function. Antioxidant defense mechanisms in the brain might be insufficient to prevent increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. Present study, therefore, aimed to investigate the protective effect of α-lipoic acid against radiation-induced impairment in the water maze learning and spontaneous motor activity of mice. Acute intraperitoneal treatment of mice with α-lipoic acid (150 mg/kg body weight) prior to X-irradiation (6 Gy) significantly mitigated the radiation-induced decline in the spontaneous motor activities as well as water maze learning. Cyclic voltametric and deferential pulse voltametric evaluation indicated a profound protective potential of α-lipoic acid against radiation-induced decline in low molecular weight antioxidants. Biochemical estimation of protein carbonyls and melondieldehide in mice cerebellum indicated that radiation-induced augmented level of protein oxidation and lipid peroxidation products has been significantly (P<0.001) ameliorated in α-lipoic acid pre-treated mice cerebellum. Radiation-induced deficit of non protein sulfhydryl (NP-SH) content of cerebellum and plasma ferric reduced power (FRAP) was also normalized by α-lipoic acid pre-treatment. Results indicate the antioxidative as well as neuroprotective potential of α-lipoic acid against the radiation.

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