Dose-dependant increase of chromosome aberrations was found in the SPF C3H/HeN mice continuously irradiated with γ-rays at a low-dose-rate (LDR) of 20 mGy/22 h/day (0.91 mGy/h) from the age of 8 weeks up to a maximum of about 400 days. Translocations and dicentric chromosomes detected by M-FISH were increased almost in linear up to a total accumulated dose of 8000 mGy. Clones were appeared at 4000 mGy, and rapidly increased over 6000 mGy. Dose and dose-rate effectiveness factor (DDREF) was 2.6-4.1, varied for types of chromosome aberrations, by comparison of aberration rates at the same dose of 500 mGy between HDR (890 mGy/min) and continuous LDR (20 mGy/22 h/day) γ-irradiations.
Furthermore, mice were kept in SPF condition for 200 days or more, after continuous LDR irradiation for 200 days (4,000 mGy), it was examined whether or not chromosome aberration was reduced. Both frequencies of translocations and dicentric chromosomes were slowly decreased at 150 days after irradiation, but not down to those levels of age-matched non-irradiated control mice. A cause of slow reduction of dicentric chromosomes after irradiation can not only be explained by life span of splenic lymphocytes, but also by turn-over of splenic lymphocytes, in which more number of immature lymphocytes recruited into spleens from bone marrows or other tissues. These results suggest that mechanism for the effects of LDR irradiation on splenic lymphocytes could be different from that for HDR irradiation. The lymphocytes with dicentric chromosomes persisting for more than 150 days after irradiation without rapid reduction might be related to dicentric chromosomes increasing almost in linear depending on accumulated doses during the continuous LDR irradiation. These will be important information for radiation risk assessment of LDR radiation exposure. This study was performed under contact with Aomori Prefecture Government, Japan.

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Low-dose rate (DR) irradiation to mammalian cells elicits a unique cellular response even if the lethal impact could be negligible. In particular, the double strand break (DSB)-inducible endpoints, including somatic mutation, genetic recombination and chromosomal translocation, have been characterized by responsive curves that their frequency shows a limited decline or an increase (inverse effect) as DR is diminished. To search factors specific for such responses, the behavior of human DSB-interactive proteins as candidates was assessed by an in vitro assay system. We found that that continuous low-DR irradiation of γ-ray to lymphoblastoid cells introduces several changes into the aggregative profile of nucleoproteins on DNAs with ends, in contrast with the unchangeable pattern of DNA-dependent protein kinase components. Notably, this alteration appears to maintain over several decades of cell division without detectable changes of major proteins. A critical range of low-DR radiation may provide a state compatible with proliferation, in which the structure and the activity of aggregative proteins might be modified in an epigenetic manner.

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The biological effects of ionizing radiation are characterized by dose-rate effects (DREs). However the mechanisms of DREs have not been clarified. We hypothesize that the DREs might be induced quantitatively by the time-dependent accumulation of unrepaired DNA damages. It is necessary to examine the time course of radiation-induced DNA damage in detail to prove our hypothesis. First, we investigated the time course analysis of radiation –induced DNA damage based on phosphorylated ATM forms discrete foci in primary human fibroblasts. The result showed that the number of unrepaired DNA damages is 0.66 per cell at 30 min after 20 mGy. Next, we examined whether unrepaired DNA damages is accumulated by repetitive 20 mGy irradiation every 30 min. The number of unrepaired DNA damages was 0.65~0.7 per cell by 1~4 repetitive irradiation. This result indicated that unrepaired DNA damage is not accumulated by repetitive irradiation. Thus, we found that the mechanism of DREs is not able to explain from a model of DNA damage and repair.

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The hormetic effect, which extends lifespan by various stressors, has been confirmed in Caenorhabditis elegans (C. elegans). We have previously reported that oxidative stress resistance in a long-lived mutant age-1 is associated with the hormesis. In an age-1 allele that activates an insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway, the superoxide dismutase (SOD) and catalase activities increased during normal aging. We now demonstrate the changes of mitochondrial superoxide radical (^.O₂^-) levels in age-related strains under a hormetic condition. The ^.O₂^- levels in age-1 strain significantly decreased after intermittent hyperoxia exposure. On the other hand, this phenomenon was not observed in a daf-16 null mutant. This hormesis-dependent reduction of the ^.O₂^- levels was observed even if the mitochondrial Mn-SOD was experimentally reduced. Therefore, it is indicated that the hormesis is mediated by any event suppressing the mitochondrial ^.O₂^- production. Moreover, the expressions at the mRNA levels of some SOD gene in age-1 mutant under a hormetic condition were induced from the steady state. These data suggest that oxidative stress-inducible hormesis is associated with a reduction of mitochondrial ^.O₂^- production by activation of an antioxidant system via Ins/IGF-1 signaling pathway.

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We have examined the elevation of T-cell receptor (TCR) variant fractions in splenic T lymphocytes by β-rays from tritiated water (HTO) with 3 Gy by a single i.p. injection and ¹³⁷Cs γ-rays with 3 Gy at a low dose rate mimicking internal expose (simulation-irradiation). In p53^+/+ mice, although the TCR variant fraction did not increased at all by ¹³⁷Cs γ-rays, that by ³H β-rays increased 19 day after injection. In this study, We analyzed the p53 activity in T cells from mice after whole body exposure by ³H β-rays and ¹³⁷Cs γ-rays with 3 Gy.

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Therapy using radon or thoron gas is performed for various diseases such as diabetes mellitus and rheumatoid arthritis. The aim of this study is to analyze the effects of thoron and thermal treatment for life- style-diseases on humans. The subjects were 76 persons who divided into 3 groups: normal, diabetes mellitus and rheumatoid arthritis. The all subjects were inhaled thoron in a Hanamaki spa (Iwate, Japan) with a high concentration (about 4900 Bq/m³) for 2 weeks. The levels of free fatty acid, immunoreactive insulin and ketone bodies of diabetes mellitus group came to the normal levels by thoron and thermal treatment. The α-atrial natriuretic peptide level of rheumatoid arthritis group was increased and the blood pressure was significantly decreased. Superoxide dismutase activity of rheumatoid arthritis group was significantly increased by treatment. In addition, thoron and thermal treatment significantly enhanced concanavalin A-induce mitogen response and increased the level of CD4 positive cells; it decreased the level of CD8 positive cells. The results suggest that thoron and thermal treatment activates antioxidative function. Furthermore, these findings suggest the possibility that the thoron and thermal treatment prevents diabetic ketoacidosis, and, contributes to the prevention of life-style-disease, which relate to peroxidation reactions and immune suppression, by an enhancement of the immunity function. These indicate that may be a part of the mechanism for the alleviation of diabetes mellitus and rheumatoid arthritis brought about more by thoron and thermal therapy.

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We previously reported that low-dose irradiation activated antioxidant function and inhibited brain edema induced by cold injury in mouse. In the present study, we pathologically examined the development of tissue damage in the cerebral hemisphere after cold injury by prior low-dose X-irradiation. Mice were irradiated by sham or 0.5 Gy of X-ray. At 4 hr after irradiation, the right cerebral hemisphere was injured by placing a cooled metal probe on the skull surface. Brains were removed at 0 (control), 1, 4, 24, or 48 hr after the cold injury. Cells became atrophied after cold injury. The number of cells in lesion area of sham irradiated group significantly decreased at 4, 24, or 48 hr. Those of 0.5Gy irradiated group were significantly decreased at 24 or 48 hr. Non-structural areas in lesion tissue of sham and 0.5Gy irradiated groups were significantly increased at 4, 24, or 48 hr. At 4 hr, those of 0.5Gy irradiated group were lower than those of sham irradiated group. It was significantly decreased the number of neuron in lesion tissue after sham and 0.5 Gy irradiation. At 1 hr, the number of apoptotic cells of 0.5 Gy irradiated group were significantly lower than that of sham irradiated group. These findings suggested that low-dose irradiation alleviated tissue damage induced by cold injury. These results supported to the previous report on the inhibition of brain edema induced by cold injury using biochemical assay.

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We previously reported that radon inhalation or low-dose X-irradiation activated antioxidative functions and inhibited alcohol-induced or carbon tetrachloride (CCl₄)-induced oxidative damage in mouse liver. However, there are no reports on the best radon inhalation condition of these inhibitory effects. In the present study, we examined the inhibitory effects of radon inhalation on CCl₄-induced hepatopathy in mice and its best inhalation condition. BALB/c mice (age: eight weeks; body weight: approximately 25 g) were intraperitoneally injected CCl₄. Before or after CCl₄ injection, mice were inhaled approximately 20,000 Bq/m³ radon for 6 hours. We investigated the various biochemical parameters, such as the activities of transaminase and antioxidation in these hepatopathic mice. In result, the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in serum were significantly increased by CCl₄ injection, suggesting that CCl₄ injection induced hepatopathy. Histological examination also showed hepatopathy of these mice. Radon inhalation increased total glutathione content in liver, suggesting that radon inhalation activates antioxidative function. We will report the best radon inhalation condition of the inhibitory effects of hepatopathy in comparison with the other radon inhalation conditions.

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We previously reported that low-dose irradiation activated antioxidant functions and decreased lipid peroxide levels in some organs of mouse. Acetaldehyde, which is a metabolic product of alcohol, is one substance that reacts with glutathione (GSH). Alcohol administration leads to GSH depletion in liver; suggesting direct conjugation of GSH with acetaldehyde and reactive intermediates of alcohol oxidation. In this study, we examined the inhibitory effect of low-dose X-inhalation on chronic alcohol-induced damage of mouse liver. C57BL/6J mice (female, about 8 weeks old) were irradiated by sham or 0.5 Gy of X-ray, and fed for 2 weeks on a Lieber DeCarile liquid diet, which contains of 5 % alcohol. We investigated the various biochemical parameters, such as the activities of transaminase and antioxidative enzymes in these hepatopathic mice. In result, the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in serum were significantly increased by alcohol intake, suggesting that alcohol intake induced by hepatopathy. Histological examination also showed hepatopathy. We will more report the effects of low-dose X-irradiation on chronic alcohol-induced damage using the other biochemical parameters.

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The present study attempts to examine the radioactivity-modulated change of global gene expression patterns in the leaf of rice seedlings upon exposure to radioactive Chernobyl soil (CS), which contains high levels of cesium-137 and strontium-90 both fission products from the reactor core. An in vitro two-week-old rice seedling model system was used (Agrawal et al. 2003), and leaf segments from 3rd leaves were exposed to CS emitted radiation. The radioactivity in CS is measured to be 5.34 micro Gy/day; ca. 11-fold over the (shielded) control level. Leaves were sampled 96 h post-irradiation and total RNA was extracted and the quality checked before performing the Agilent 22K custom oligo DNA microarray experiment.
We detected radioactivity-modulated expression of 516 genes, among which 194 and 322 genes were either up- or down-regulated.
Rice genes were classified into up-/down-regulated 3 main functional categories of information storage and processing (2%/7%), cellular processes and signaling (15%/7%), and metabolism (13%/19%).
References
1.Kimura S, Shibato J, Agrawal GK, Kim YK, Nahm BH, Jwa NS, Iwahashi H, and Rakwal R.: Microarray analysis of rice leaf response to radioactivity from contaminated Chernobyl soil.: Rice Genetics Newsletter, 24: 52-54, 2008.
2.Rakwal R, Agrawal GK, Shibato J, Imanaka T, Fukutani S, Tamogami S, Endo S, Sahoo SK, Masuo Y, Kimura S.: Ultra Low-Dose Radiation: Stress Responses And Impacts Using Rice as a Grass Model : Int. J. Mol. Sci., 10: 1215-1225, 2009.

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Radiation-induced adaptive response (AR) was described in numerous biological systems. AR research is of great concern as investigations on the conditions essential for AR induction provide important scientific basis for risk estimates and offer significant insight into the novel biological defense mechanisms. However, at whole body level most of the investigations were performed using X-irradiations. In a series of study in progress, possible induction of AR by high LET irradiation of accelerated heavy ions is being tested both in vivo in young adult mice and in utero in fetal mice. Investigations are to verify 1) if priming low-dose X-rays could reduce the detrimental effects, i.e., growth retardation, death or malformation induced by the challenging high dose from heavy-ion irradiations, 2) if low dose of heavy-ion irradiation could induce any AR against the detrimental effects from high dose of X-rays, and 3) if low-dose heavy-ion irradiations could induce any AR against the detrimental effects from the high dose of heavy-ion irradiations. Accelerated heavy ion particles from mono beams of carbon, silicon and ion generated by HIMAC, with the LET values of about 15, 55, and 200 keV/micrometer respectively, are being examined. At the 51st annual meeting of JRRS, we reported that priming low-dose X-rays could modify the detrimental effects from challenging high dose of heavy-ion irradiations. In this work, we will present the new data obtained on AR induction by heavy-ion irradiations in vivo. This is for the first time, at the whole body level, that AR is demonstrated by priming low dose of high LET irradiations. Interestingly, the essential conditions for successful AR induction seem to be dependent on the particle and/or the LET value of the irradiation.

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Since radiation protection has been intended for humans, most studies on radiation biology have been made in mammals including humans. Recently, however, international organizations such as ICRP, UNSCEAR and IAEA began to make an effort for construction of a framework for radiation protection of non-human biota and environments. We have therefore started to study radiation effects on phytoplankton, which is important as a producer in aquatic ecosystems.
Transcriptome was analyzed in acutely gamma-irradiated green alga Pseudokirchneriella subcapitata, which is one of the species most commonly used in ecotoxicity tests of chemicals, but for which genomic sequence information is lacking. This transcriptome analysis was therefore carried out by HiCEP, which is based on AFLP and thus requires no sequence information¹⁾.
Approximately 7,000 expressed genes were detected. Expression levels of approximately 800-900 genes were affected at 100 to 300 Gy. Nucleotide sequences of 41 up-regulated genes were determined. The quantitative RT-PCR validated the up-regulation. Two genes had homology to genes involved in DNA repair. One resembled DEAD/DEAH box helicase genes, and the other resembled SNF2/RAD54 family and RAD26 genes²⁾. Further characterization of the affected genes will contribute to finding biomarkers for detection of radiation effects and elucidating molecular mechanisms of responses to radiation, which are expected to have some differences from those in mammals.
¹⁾ Fukumura et al., Nucleic Acids Research 31: e94, 2003.
²⁾ Fuma et al., Bulletin of Environmental Contamination and Toxicology 83, 301-306, 2009.

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The radioadaptive response is a defensive response where exposure to low dose radiation (priming dose) induces resistance against the subsequent high dose radiation (challenge dose). In order to identify the genes related to a radioadaptive response, we performed a DNA microarray analysis using the human lymphoblastoid-derived cell line AHH-1. The cells were unirradiated or irradiated with 0.02 Gy of priming dose, and the RNA was isolated 6 h after irradiation. In addition, cells unirradiated or irradiated with 0.02 Gy of priming dose were further irradiated with 3 Gy of challenge dose 6 h after priming irradiation, and the RNA was isolated 3 h after challenge irradiation. The gene expressions in cells irradiated with 0.02 Gy alone were compared with those in control cells using Student's t-test with the GeneSpring software, thereby 88 genes were selected as significantly modulated genes (p < 0.05). By a clustering analysis, it was revealed that the genes contained in a branch were significantly overlapped with a GO category 'Centrosome'. Similarly gene expressions in the cells irradiated with 3 Gy alone was compared with those in cells irradiated with 0.02 Gy plus 3 Gy. By a clustering analysis of 86 of significantly modulated genes, GO categories 'Metal ion binding', 'Zinc ion binding', 'Purine ribonucleoside salvage', 'Adenosine kinase activity', 'Ribonucleoside monophosphate biosynthetic process', 'Protein amino acid dephosphorylation' and 'Mitochondrion' were shown to be significantly overlapped with clusters of modulated genes. These GO categories are suggested to be the cellular functions related to the radioadaptive response of the AHH-1 cells.

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Radioadaptive response (AR) is a complex interplay between the irradiations and the organisms with the surrounding conditions involved. Genetic background is one of the crucial factors among the prerequisite conditions essential to the successful AR induction. As genetic diversity plays important roles in survival and adaptability of a species, laboratory mice provide good animal models for studying the differences among individuals in humans. On induction of AR, in vivo studies show clearly strain-specific differences in adult mice and in utero investigations demonstrate different efficient priming doses in fetal mice of different strains. For possible practical application of AR in tailor-made therapy, it is an approach to elucidate the similarity and dissimilarity of molecular mechanisms in different strains by comparing gene regulations under conditions efficient and inefficient in AR induction and correlating their functions with AR phenotype. As the first step, in this study AR induction in utero was tested in several strains of fetal mice using prenatal death, gross malformation and prenatal developmental retardation as the indexes. Two priming doses of X-irradiations efficient in C57BL strain were adopted and applied to the pure fetal mice of the C3H strain and BALB/c strain, and the hybrid fetal mice of mixed parentage from such as female C57BL and male C3H mice, female C3H and male C57BL mice and other combinations. Being different from the ICR and C57BL strains, AR was not observable in C3H and BALB/c strains. Interestingly, the hybrid fetal mice showed varied responses to these priming doses. These findings indicate the strain-specific differences in AR induction in fetal mice.

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Carcinogenic effect of radiation is known to depend on age of exposure. In mouse liver, irradiation at 0-7 days of age is reported to be most effective. In an attempt to understand the molecular mechanisms underlying the phenomena, we analyzed the alteration of gene expression at mRNA levels. Mice were exposed to 3.8 Gy whole body irradiation at 7 days of age or 10 weeks of age. Another group was sham-irradiated. Each group was divided further into two; fed normal-diet or caloric-restricted (CR) diet. At 10 months of age, we compared the gene expression levels in the liver of all 5 groups to that of control group (sham-irradiated, fed normal-diet). Microarray and quantitative RT-PCR analysis revealed that the expression of the 3 genes was altered in the liver irradiated at 7 days of age; two genes showed increased expression and one showed decreased expression. Expression levels of the 3 genes in the liver irradiated at 10 weeks of age were similar to those irradiated at 7 days of age. This suggested the change in expression levels of the 3 genes was not related to carcinogenic events. The CR effect on 3.8 Gy irradiated mice was complicated. The study could not find alteration of mRNA which would explain age-dependency of radiation induced liver cancer. But we found 3 genes whose expression levels were altered long time after irradiation in mouse liver.

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Relationship between X-ray doses and mRNA levels in hematopoietic cells was examined. Induction of p21 and mdm2 mRNAs and repression of c-myc mRNA were used as indicator of before growth-arrest, and enhancement of puma and bax mRNAs as apoptosis. These mRNA levels were quantified, based on a precise method of real-time RT-PCR with improvements as follows; 1) control of the amount and quality of template RNA; 2) optimization of reverse transcription reaction, and 3) simultaneous PCR using reference plasmid as standard.
A murine macrophage cell line, RAW264.7 was used as the first model. The cells were irradiated at various doses of X-rays, and were harvested at several time-points to examine the levels of mRNA, the rate of apoptosis by TUNEL-staining and the degree of proliferation by BrdU-staining. Before the increase in the rate of apoptotic cells, puma mRNA reached to the peak level. Attenuation of the growth determined by BrdU-staining was simultaneously occurred with decrease in c-myc mRNA level, followed by increase in mRNA levels of p21 and mdm2 to the peak. These peak levels of mRNA of p21, mdm2 and puma increased x-ray dose-dependently within the range from 0.1 to 1.0 Gy.
As the second model, peripheral blood and bone marrow cells from whole-body X-irradiated C3H/He mice were used. The levels of x-ray responsive induction of mRNAs were varied by the circadian rhythm of mice. Only the increase of puma and bax mRNA levels in marrow cells were dose-dependent manner between 0.1 to 1.0 Gy with less contribution of circadian rhythm. The results show that the precise quantification of suitable mRNAs can be used to estimate radiation damage.

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The effect of long-term continuous irradiation has relevance to study in radiation ecotoxicology. For investigation of biological responses to continuous irradiation in plants, we assessed gene expression in a model plant arabidopsis. Microarray analysis revealed 56 up-regulated genes in the plants exposed to gamma rays for 2 weeks at a dose rate of 20 Gy/day. The genes up-regulated by the irradiation could be classified into two types by the time-dependent expression patterns; early-responsive genes which responded within 1 day, and late-responsive genes which responded 3-7 days after the start of the continuous irradiation. The former type included some genes relating to DNA repair, whereas the latter other genes relating to protein and carbohydrate metabolism. Each type also included different genes of signal transduction and transcriptional control. These behaviors of gene expression probably reflect the responses of the plants to cope with progression of radiation damages. We compared the gene expression profile with those in previous studies not only about radiation effect but also other environmental stresses, to speculate biological responses relating to the damages on plant growth by continuous irradiation.

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[Objective] The biological effects of ionizing radiation are classified into direct action and indirect action, thus a low LET irradiation such as X-rays mainly induce an indirect action with the production of reactive oxygen species (ROS). Mitochondria act as both a source of ROS and a target organ of ROS in this process. Although hematopoietic stem cells (HSCs) are highly radio-sensitive cells, there is still little information regarding the intracellular ROS production and the involvement of the mitochondria. The present study examined these interactions in human HSCs.
[Methods] This study used highly purified CD34⁺ cells prepared from human placental/umbilical cord blood. The clonogenic potential was assayed by the methyl cellulose technique containing the optimal cytokine cocktails. The cells were treated with 2’,7’-dichlorofluoresceindiacetate and Mitotracker Green FM in order to estimate the intracellular ROS production and the fluorescence intensity of mitochondria, respectively. Each assay was analyzed by flow cytometry. X irradiation was performed by an X-ray generator under conditions of 150 kV, 20 mA, 0.5-mm Al plus 0.3-mm Cu filters at a dose of 90—100 cGy/min.
[Result and Discussion] No ROS were detected in the CD34⁺ cells immediately after irradiation, and little ROS production was observed in the cells cultured with cytokines at 3 to 6 h after irradiation. In a similar experiment, 10 to 20-fold increases in the ROS production were observed in human monocytic U937 cells and human normal fibroblasts WI-38 cells. However, the intensity of luminescence from mitochondria observed in CD34⁺cells was only half that seen in both cell lines. There was a positive correlation between the surviving fractions of 4 Gy irradiated-HSCs and the luminescence of mitochondria. Therefore, the present results suggest the possibility that the ROS production was either significantly reduced or eliminated by the mechanisms associated with mitochondria in HSCs.

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Purpose: Stem cells are defined as cells that self-renew indefinitely and also give rise to differentiated cells by suitable stimulation. There is increasing evidence that malignant tumors contain high-hierarchy cells that maintain the characteristics of tissue-specific stem cells. Recent findings support an idea that malignant gliomas can be generated from either a neural stem cells (NSCs) or glial lineage cells. The hypothesis can be viewed as cancer stem cells. The stem cells also are gaining attention of the application in regenerative medicine. However, radiosensitivity of stem cells is not fully determined due to difficulty of in vitro culture. The development of NSCs in vitro culture has been accomplished by neurosphere method. In this study, we demonstrated the induction of telomerase activity in mouse neural stem cells infected by retrovirus containing an hTERT. Additionally, we investigated radiation sensitivity of hTERT introduced neurosphere cells.
Methodology : NSCs were harvested from corpus striatum in E14.5d ICR mouse embryo. For 10 days culture, cells were grown as self-adherent complexes of cells, forming clusters known as neurosphere. An hTERT gene was introduced into neurosphere cells with retrovirus vector. The introduction of hTERT gene was checked by RT-PCR and telomerase activity of neurosphere cells was evaluated by ELISA. The radiosensitivity of neurosphere cells was determined by colony forming assay in soft agar.
Result : Telomerase activity of hTERT introduced neurosphere cells was elevated as compared with those of primary neurosphere cells and mouse embryo fibroblasts. Moreover, we defined that serum stimulation allowed NSCs to be differentiated to glial cells. We determined the radiosensitivity of primary, p53 and ATM deficient neurosphere cells, and hTERT introduced neurosphere cells. Finding radiation response in established NSCs contributes to the understanding of the role of stem cells in development of tumorigenesis, and to the estimation of the risk of stem cells in regenerative medicine.

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[in Japanese]

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Introduction:
X-irradiation induced the cell death by apoptosis in mucosal epithelial cells, and caused the impairment of physiological varrier against many antigens in gut lumen. Angiopoietin family is well known as a vasculogenic factor that signals through the Tie-2 receptor tyrosine kinase. Some groups reported that Angiopoietin (Ang)-1 prevented apoptosis induced by serum deprivation in endothelial cells of vasculature, through the regulation of signaling pathways.
Aim & Methods:
To elucidate the effect of Angiopoietin family in the normal intestinal epithelial cells, IEC-18 cell was treated with recombinant human Ang-1 (rhAng-1) or rhAng-2 followed by X-ray irradiation of 10Gy and then examined for the regulation of viability by the phosphatidylinositol 3-kinase (PI3K) and/or mitogen activated protein kinase (MAPK) pathway. Cell viability was counted by trypan blue staining and the TUNEL assay.
Results:
X-irradiation induced the cell death of IEC-18. When compared with non-rhAng-1 treated cells, the viability of the X-irradiated IEC-18 cells was up-regulated by rhAng-1 treatment and also resulted in the induction of phosphorylation of S6 ribosomal protein that represented the activity of PI3K pathway. However, p90 RSK and p38MAPK pathways were not up-regulated by rhAng-1 treatment. The activation of caspase-3 induced by X-irradiation was decreased with treatment of rhAng-1. However, the treatment of rhAng-2 did not show any protective effect on the cell death of IEC-18 with X-irradiation. Any activation of PI3K, p90RSK and p38MAPK pathways were not regulated by rhAng-2 in IEC-18 cell.
Conclusions:
These results suggested that Ang-1 has the possibility of protective effect in the intestinal disorder induced by X-irradiation.

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Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine that has a wide variety of bioactivities, and over-production of TNFα leads to damages of tissues. TNFα is known to be produced following irradiation in various tissues and anti-TNFα agents have been shown to prevent radiation-induced damage. To determine the role of TNFα in high-dose radiation exposure, we used wild-type (TNFα^+/+) and TNFα knockout (TNFα^-/-) BALB/c mice. We previously reported that the survival durations in TNFα^-/- mice were shorter than those in TNFα^+/+ mice after whole body irradiation with 6-6.5 Gy and that administration of recombinant TNFα improved the survival rate in irradiated TNFα^-/- mice. Furthermore, the numbers of red blood cells (RBC), the levels of hemoglobin (Hb), the hematocrit values (Ht) and the unsaturated iron binding capacity (UIBC) were significantly lower and the levels of serum iron (Fe) was higher in TNFα^-/- mice than TNFα^+/+ mice after irradiation. In the present study, we found that administration of recombinant TNFα before irradiation improved the numbers of RBC, Hb levels, Ht values, the Fe levels, and UIBC in irradiated TNFα^-/- mice. We also showed that erythroid burst-forming units (BFU-Es) and erythroid colony-forming units (CFU-Es) activities were significantly reduced in TNFα^-/- than in TNFα^+/+ mice following irradiation, whereas there were no difference in activities of BFU-E and CFU-E between control TNFα^+/+ and TNFα^-/- mice. Our results suggest that endogenously produced TNFα may play important roles in erythroid hematopoiesis following irradiation.

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[Purpose] Gliomblastoma is one of the most common tumor types resistant to radiation. Arsenite was reported to show synergistic radiosensitization effect in vitro and in vivo. But, the mechanism of radiosensitization is not well understood. Last year, we showed the difference of mechanism of SLGA between radiation and arsenite. This year, we have studied heterochomatin formation by treatment of arsenite associated with senescence-like growth arrest (SLGA).
[Results] We performed analysis using glioblastoma U87MG cells treated by arsenite. Formation of γH2AX foci observed after 6 hours. The results indicated DNA damaged by arsenite is indirectly. Double staining using antibody of histone H3 phospholylated or methylated with γH2AX foci shows the modification of histone H3 is on the cells with γH2AX. Thus, we thought the heterochromatin formation induced by DNA damage. Next, we performed about p53 dependency of heterochromatin formation. The results show γH2AX formation and phosphorylation of histone H3 observed, but methylation of histone H3 dose not observed at p53 deficient cells.

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Apoptosis is a well-known mode of cell death induced by ionizing radiation (IR). Our previous findings demonstrated that a major mode of IR-induced cell death in normal human fibroblasts was non-apoptotic type, that is, SLGA, which suggested a possibility of SLGA-induction in tumor cell exposed to IR. Here, we performed live-imaging analysis to examine a mode of cell death induced in individual breast carcinoma cells (MCF-7) exposed to 10Gy of X-irradiation.
We found that the majority of IR-induced cell death was a non-apoptotic mode. About 60 % of irradiated MCF-7 failed to enter into mitosis during 5 days incubation after IR, and they showed enlarged cytoplasm with mononuclear. Other 40 % of irradiated MCF-7 also failed to divide up to approximately 20 hrs after irradiation. Then, they started cell division and induced mitotic catastrophe. Interestingly, these cells underwent abnormal cell dividion, frequently fused again and swelled up cytoplasm with multi-nuclear. More than 70 % of cells were positive for senescence-associated ß-galactosidase, by which senescent cells were detected, subsequently, 20 % of all irradiated cells induced necrosis. These results suggest that SLGA-induction contributes to cell death by IR and to radiosensitivity in tumor cells.

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[Objective] Intracellular reactive oxygen species (ROS) generated by X-irradiation cause DNA damage. Transcriptional factor NF-E2-related factor 2 (Nrf2) regulates the expression of multiple antioxidant enzymes, including NAD(P)H-quinone oxidoreductase l (NQO1) and heme oxygenase l (HO-1). Although hematopoietic stem cells (HSCs) are radio-sensitive cells and their intracellular antioxidant system is very important, little is known about those mechanisms. The present study investigated the response of both HO-l and NQO1 in X-irradiated human HSCs. Furthermore, the relationship between the expression of both molecules and the radio-sensitivity of HSCs was also examined.
[Material and Methods] This study was approved by the Committee of Medica1 Ethics of Hirosaki University Graduate School of Medicine and informed consent was obtained from the mothers. Placental/umbilical cord blood was collected after full-term delivery. CD34⁺ cells (HSCs) were purified using a magnetic cell sorting kit. Total RNA was extracted from the cells. The expression of HO-l and NQO1 mRNA were assayed by quantitative RT-PCR. Moreover, the clonogenic potential was assayed by methylcellulose culture with a combination of cytokines (GM-CSF, G-CSF, EPO, IL-3, SCF). After 14 days of culture, the colonies containing more than 50 cells were counted as colony-forming cells (CFC).
[Results and Discussion] The CFC counts derived from 2 Gy-irradiated HSCs decreased to 80% of the non-irradiated control. At the same time, the expression of HO-l and NQO1 in X-irradiated HSCs showed a significant increase in comparison to the non-irradiated control. Therefore, the present results suggest the possibility that the radio-sensitivity of human HSCs is associated with the expressions of HO-l and NQO1 mRNA. Ongoing experiments are now investigating whether the expressions of HO-1, NQO1 and other response genes are associated with radio-sensitivity and individual differences in HSCs.

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Mesenchymal stem cells promote the proliferation of hematopoietic stem/progenitor cells (HSPCs) and maintain stem cells in a primitive state. A previous study demonstrated the beneficial effects of placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells on the generation of both non-irradiated and X-irradiated HSPCs^R). The present study examined the involvement of extracellular matrix components, which regulate the proliferation and differentiation of HSPCs, in the regeneration of X-irradiated HSPCs in the co-culture system. The co-culture experiments were performed as described in a previous report^R). The concentration of the extracellular matrix components, hyaluronic acid and sulfated glycosaminoglycans, contained in the conditioned media was assessed by an ELISA kit. The total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture supplemented with IL-3 plus SCF plus TPO. In addition, the number of CD34⁺ cells, CD34⁺/CD38^- cells, and immature HSPCs were also increased more than in the stroma-free culture. The hematopoiesis observed in the co-culture was equivalent to that when the cytokines were added 16 h later, although a dramatic decrease was seen in the stroma-free culture. The co-culture produced hyaluronic acid and sulfated glycosaminoglycan. In particular, X-irradiated cells alone produced levels of higher sulfated glycosaminoglycan than the non-irradiated control culture and this production was up-regulated by the stroma cells. These results suggest that the cell-cell interactions play a critical role in the co-culture system. Furthermore, the extracellular matrix components also act as a key factor in the regeneration of myeloid hematopoietic progenitor cells from X-irradiated HSCs in the co-culture system.
R) Life Sci. 84: 598 (2009).

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Purpose
Micronuclei induced in germinated onion seeds initially increase with radiation dose and next decrease with further amount of radiation dose. The main cause of the decrease seems not cell death but cell division delay because there is no conspicuous effect to the growth.
Method
Radiation sources used were heavy ions accelerated by HIMAC synchrotron at the National Institute of Radiological Sciences, Chiba, Japan and Cs-137 gamma-ray of irradiation system in Center for Frontier Life Sciences, Nagasaki University, Nagasaki, Japan.
Result and Conclusion
Peak time of micronuclei induction become later with increase of radiation dose and LET.

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The aim of this study was to clarify the effects of nitric oxide (NO) on radiation-induced cell killing and chromosomal aberrations in two human lung cancer cell lines with a different p53 gene status. Wild-type (wt) p53 and mutated (m) p53 cell lines were used which were derived from the human lung cancer H1299 cell line which is p53-null. The wtp53 and mp53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pre-treated with different concentrations of isosorbide dinitrate (ISDN, an NO donor) and/or 1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl) tetrahydroimidazole (c-PTIO, an NO scavenger), and then exposed to X-rays. Cellular sensitivity, apoptosis and chromosomal aberrations were scored using a colony-forming assay, Hoechst33342 staining and chromosomal banding techniques, respectively. In wtp53 cells, the induction of radioresistance and the inhibition of apoptosis and chromosomal aberrations were observed in the presence of ISDN at low 2-10 μM concentrations before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. At high concentrations of ISDN (100-500 μM), however, clear evidence for radiosensitization, enhancement of apoptosis and chromosomal aberrations was detected. On the other hand, these phenomena were not observed in mp53 cells at either concentration range with ISDN. These results indicate low and high concentrations of NO radicals can choreograph inverse radiosensitivity, apoptosis and chromosomal aberrations in human lung cancer cells, and NO radicals can affect the fate of cells through the regulation of p53 gene expression.

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All cells have elaborate mechanisms to maintain their genomes. DNA can be damaged by reactive metabolic byproducts and by environmental mutagens. Responding to and repairing DNA damage is crucial for cell viability and disease prevention. The DNA-damage response is a signal-transduction pathway that coordinates cell-cycle transitions, DNA replication, DNA repair and apoptosis. The major regulator of the DNA-damage response are two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). Both are large kinase with significant sequence homology and target an overlapping set of substrates that promote cell-cycle arrest and DNA repair. Both are involved in damage recognition step and thus play an important role on the initiation of cell cycle checkpoint. Another key player which mediates cell cycle checkpoints is tumor suppressor gene p53. p53 is activated and stabilized by ATM and serve as a transcription factor for the downstream cell cycle regulator. p53 pathway depends on the transcrption of its downstream gene and is therefore delayed after DNA damage and its role lies in the maintenance of cell cycle checkpoint. To study the role of these damage response pathways in vivo, we have isolated Medaka mutants for these three genes. In this paper we will show the phenotype of the obtained mutants.

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Radiation-induced G1 checkpoint activation plays a pivotal role in initiating irreversible G1 arrest in cells that have persistent chromatin damage. The ATM-p53-p21 pathway is critical for the initial activation of G1 checkpoint, however, its involvement in the maintenance of G1 arrest remains to be determined. In this study, we hypothesized a signal stabilization mechanism to maintain G1 arrest. To prove this, normal human diploid cells were synchronized in G0 phase, irradiated with 10 Gy of gamma rays, and released immediately following irradiation. Twelve hours after irradiation, the ATM kinase inhibitor, KU55933, was administrated. Cells were fixed at 24 hours, and the percentage of cells in S phase was determined by EdU incorporation. The percentages of EdU positive cells was decreased from 24.6% to 4.8 % by 10 Gy irradiation, while similar decrease was observed in KU55933 treated cells. Then, cells were incubated for up to 96 hours after irradiation with KU55933. We found that continuous inactivation of ATM gradually abandoned G1 arrest. These results indicate that ATM-dependent signal is involved in the maintenance of G1 arrest.

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53BP1 is rapidly recruited at sites of DNA double-strand breaks (DSBs) after treatment of cells with ionizing radiation or DNA-damaging agents, and is suggested to be involved in DNA damage checkpoint and DNA repair. Recently, the molecular mechanism of the recruitment of 53BP1 to DSB-sites has been revealed. In contrast, molecular bases for the functions of 53BP1, including regulation of DNA damage checkpoint and DNA repair, are still unclear. To investigate molecular mechanisms of 53BP1 function after the recruitment of 53BP1 to DSB-sites, we attempted to identify proteins which bind to the BRCT domain of 53BP1. The C-terminal region of 53BP1, including the nuclear localization signal and the BRCT domain, was fused downstream of the FLAG-HA tag and expressed in U2OS cells. The proteins co-immunoprecipitated with the FLAG-HA-BRCT protein were analyzed. We will show the results and discuss the molecular mechanism of 53BP1 in this meeting.

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Localized irradiation of specific regions within organisms using heavy-ion microbeam systems provides an attractive means of investigating the mechanism of heavy-ion radiation action. Therefore, we had developed the heavy-ion collimating microbeam system at TIARA of the Japan Atomic Energy Agency (JAEA), and utilized for analyzing heavy-ion induced biological effects. However, there is a difficulty in generating finer beam that is capable for carrying out precise subcellular irradiation in our current system, because of inevitable scattering of ions at the edge of micro collimator. Therefore, we installed new focusing microbeam system at another vertical beam line of AVF cyclotron of TIARA, JAEA. New system can focus heavy-ion beam to minimum 1 µm in vacuum using a quadruplet quadrupole lens system, and equipped with an X, Y beam scanner for fast hitting of single ion to micron scaled samples. To irradiate this finer microbeam to the specific region of individual cells, a cell targeting system, which equipped with a full-automatic inverted microscope and the set of automatic stages, was designed and installed under the beam extraction window. The position of focusing microbeam was detected under microscopic view using plastic scintillator film. The ions irradiated to the sample were detected by solid state ion detector installed on the objective revolver of the microscope, and counted by counter NIM module to control a fast beam shutter. Using the system, irradiation of HeLa cells which was stained with CellTracker Orange fluorescent dye and inoculated on ion track detector CR39 film of 100 µm thick, were carried out.

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[Purpose] Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome or basal cell nevus syndrome, is an autosomal dominant disorder that predisposes to both developmental defects and cancer. Fibroblast cells derived from (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. We recently reported that the induction of DNA synthesis after irradiation could be caused by the down-regulation of SUMO-2 gene expression in NBCCS cells. Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1 has been implicated in a wide variety of cellular stress responses. By contrast, the functions of SUMO-2 remain largely uncharacterized. At the last meeting, we reported that after X-ray irradiation, an increase in DNA synthesis activity was detected in HeLa cells with knockdown of SUMO-2. To investigate the molecules of DNA synthesis induction due to the down-regulation of SUMO-2, we searched for the proteins whose expression levels differ pre- and post-X-ray irradiation in HeLa cells with knockdown of SUMO-2.
[Methods] DNA synthesis activity was measured by a pulse-labeling method with 14C and 3H. When comparative proteomic analysis was performed between the siRNA for SUMO-2 and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, the NM23-H1 protein was identified as a candidate protein. Knockdowns of SUMO-2 and NM23-H1 were performed using their siRNAs. Immunoprecipitation assay was carried out to analyze of NM23-H1 modification by SUMO-2.
[Results and discussion] Eight hours after X-ray irradiation(2Gy), an increase in DNA synthesis activity was found in HeLa cells with knockdown of SUMO-2. Under these conditions, the expression levels of Nm23-H1, a metastatic suppressor gene, was altered. The decrease of NM23-H1 protein after X-ray irradiation was confirmed by western blot analysis. As expected, treatment of HeLa cells with the siRNA of NM23-H1 resulted in the enhancement of DNA synthesis. These findings suggest that the intimate relationship with the expression of SUMO-2 and the stability of NM23-H1 after X-ray irradiation, lead to the induction of DNA synthesis.
Nm23-H1 may be modified by SUMO-2 after X-ray irradiation. The reduction of Nm23-H1 seems to be casually related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis after X-ray irradiation.

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Bystander responses are phenomena in which cells adjacent to those that have been traversed by charged particles exhibit biological responses. Microbeam cell irradiation systems, which enable the observation of cellular responses of both non-irradiated and irradiated individual cells, are powerful tools for elucidating the mechanisms underlying these effects. We have been studying the differences in the lethal effects on V79 cells irradiated with microbeams of different sizes using synchrotron X-ray microbeam irradiation system developed at Photon Factory, KEK.
In a previous study, we observed a transient enhancement in bystander cell death in a dose-dependent manner when the nuclei alone were irradiated at low doses. On the other hand, the surviving fraction of bystander cells decreased monotonously when whole cells were irradiated. Moreover, addition of carboxy-PTIO, a specific scavenger of nitric oxide, NO, suppresses bystander cell death under both irradiation conditions. These results suggest that NO acts as a mediator in the induction of 2 types of bystander cell death, namely, the transient type and the monotonous type.
Recently, from the spatial analysis, we found that the transient type bystander cell death was principally induced in cells located within 1 mm from the irradiated cells, whereas majority of the monotonous type bystander cell death occurred in cells located beyond 1 mm from irradiated cells. Our findings clearly demonstrate that the induction of bystander cell death depend on the distance between irradiated cells and bystander cells and also on the energy-deposition sites in irradiated cells.

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ATM-dependent p53 activation is well recognized main pathway after gamma-irradiation. However, molecular mechanisms for ATM-independent cellular responses after irradiation are unknown. Comparative microarray analysis was done on gene expression profiles in cultures of ATM-deficient mouse embryonic fibroblasts (MEFs) and normal MEFs with 4.32 Gy of gamma-rays at middle-dose-rate (MDR; 60 mGy/h) or high-dose-rate (HDR; 0.9Gy/min), interferon-related genes such as Irf7 and Stat1 were up-regulated in ATM-deficient MEFs irradiated with MDR or HDR radiation. These gene expressions were also observed in cultures of normal MEFs irradiated with MDR or HDR radiation in the presence of ATM-specific inhibitor (KU55933). Up-regulation of Irf7 and Stat1 genes in normal MEFs were induced by HDR-irradiation alone or treatment of ATM-specific inhibitor alone, but not by MDR-irradiation alone. Furthermore, the addition of conditioned medium from HDR-irradiated normal MEFs augmented gene-expressions of Irf7 and Stat1 in non-irradiated normal MEFs. Analysis of the effect of conditioned medium from ATM-inactivated MEFs is under investigation. These results indicate that Irf7 and Stat1 genes were up-regulated by inactivation of ATM or HDR-irradiation, and the ATM-independent interferon-related gene expressions after irradiation might be due to bystander effect mediated by soluble factor(s). This study was performed under contract with the Aomori Prefectural Government, Japan.

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Purpose: Aim of the present study was to clarify the dependency of p53 status on migration/invasion potential of cancer cells irradiated with carbon ions. Materials and Methods: Human small cell lung cancer H1299 cells (neo: null of p53, mtp53: transfected with p53 mutant plasmid, wtp53: transfected with p53 wild type plasmid) and carcinoma A549 cells were used for experiments. Cells grown on T25 flasks in DMEM supplemented with 10% FBS were exposed to carbon ions (290MeV/n, 100keV/µm) at HIMAC. For migration/invasion assay at 24 hours after irradiation the cells were plated on culture insert membrane (8 micron pore size) coated with matrigel (1mg/ml). The number (Nm) of cells moved on lower side of membrane after 16 h were counted under microscope. Migration/invasion rate of cells was calculated as the ratio of Nm to total number of cells plated in the culture insert. Cell survivals were obtained from the colony formation assay immediately after irradiation. Results and Discussions: RBEs of H1299 and A549 cells irradiated with carbon-ions (100keV/µm) were 2.2~3.2. Migration/invasion rates of H1299neo, H1299wtp53 and H1299mtp53 irradiated with 0.5~4 Gy reduced as compared with that of uniradiated control: migration/invasion rate at 4Gy was about 60% of the control. After X-ray irradiation at 8 Gy the migration/invasion rate of H1299 and A549 cells reduced to 50~60% of the control. Present result suggests that carbon ion irradiation causes the reduction of migration/invasion potential of cells independently of p53 status of cells.

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We examined relationship between delayed production of ROS and delayed mitochondrial dysfunction in normal human cells exposed to γ-rays. Mitochondria are fused their membrane together and configurated tubular morphology in inter-phase cells. But they become fragmented at mitotic-phase or in pro-apoptotic cells. Particularly, in the latter case, mitochondrial fragmentation accelerates mitochondrial membrane permeablization, indicating that mitochondrial morphology directly correlated with its function. Therefore, we investigated here the mitochondrial morphological change after γ-irradiation.
Immortalized normal human diploid cells (BJ-hTERT) were used in this study. The amount of reactive oxygen species (ROS) was determined using aminophenyl fluorescein (APF) reagent. Levels of superoxide localized within mitochondria were measured by MitoSox Red reagent. Mitochondria were visualized by staining with MitoTracher reagent, and structural change was examined under fluorescence microscope.
While most of the unirradiated cells showed tubular mitochondria, mitochondrial fragmentation was observed in about 50 % cells 3 days after 6 Gy of γ-rays. We also observed increase in intracellular ROS level 2 to 3 days after irradiation, which was accompanied by the increased level of mitochondrial superoxide.
These results indicate that mitochondrial dysfunction caused by mitochondrial fragmentation induces delayed oxidative stress after γ-irradiation.

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Centrosomes are important cytoplasmic organelles which are involved in proper chromosome segregation. They are maintained through the activity of several factors, including DNA repair proteins such as BRCA1 and NBS1. It is shown here that ionizing radiation (IR) induces an excess number of centrosomes in a dose-dependent manner, and that the number of excess centrosomes significantly decreases after protracted exposures vs acute exposures; frequencies of the aberrant centrosomes, after an exposure to radiation at a dose rate of 0.5 mGy/min, were reduced to 70-80% of the extent observed after an acute exposure with a dose rate of 1 Gy/min. The excess number of centrosomes could be due to the overduplication of centrosomes, since the centrioles were also duplicated. Interestingly, when the IR-induced frequency of centrosome overduplication was plotted against radiation induced cell killing, similar almost coincident curves were seen for both types of exposures, in which the frequencies were significantly increased at low cell killing levels but reached a saturated level at higher cell killing levels. Moreover, these curves from Ku70- and DNA-PKcs-deficient cells overlapped with those of wild type cells, while cell killing was considerably enhanced by deficiencies in these proteins. These results suggest that IR-induced centrosome overduplication is caused by a common mechanism, which is involved in either WT cells or NHEJ-deficient cells, regardless of the radiation dose-rate. Conversely, the absence of either BRCA1 or NBS1 enhanced IR-induced overduplication frequencies by 2-4-fold and they were significantly increased even at higher cell killing levels.Thus, the present results indicate that protracted exposure to IR significantly alleviates the frequency of centrosome overduplication, although this type of centrosome aberration was pronounced in BRCA1- and NBS1-deficient cells even after irradiation at low dose-rates.

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Locomotory behavior (motility) is a vital importance in animals. We examined the effects of ionizing radiation (IR) on locomotory behavior using Caenorhabditis elegans, and reported an IR-induced reduction of locomotory rate in the absence of food [1], [2]. In the previous experiments, wild-type animals were irradiated with γ-rays in the whole body, and measured the motility using 'body bends' (the number of bends in the anterior body region at 20-s intervals) [3]. However, the IR-induced effects in the central and posterior body region were not evaluated by the body bends. Therefore, in the present study, to investigate the IR-induced effects in more detail, we propose a method evaluating the motility of whole body by using the video-based analysis. Animals were irradiated with graded doses of ⁶⁰Co γ-rays (< 1000 Gy) at a dose rate of 32 Gy/min. Immediately after irradiation, we video-recorded the animals, and analyzed the trajectories and bends at 17 points of the body. The proposed method provides some novel insight about the IR-induced changes on C. elegans's locomotion. In the presentation, we will show results of the analyses based on the conventional and novel evaluation standards, and would like to discuss about the importance of considering of an adequate standard to evaluate IR-induced effects. [1] Sakashita, T., et al. (2008) J. Radiat. Res. 49: 285-291. [2] Suzuki, M., et al. (2009) J. Radiat. Res. 50:119-125. [3] Sawin, E.R., et al. (2000) Neuron 26:619-631.

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Previously we reported irradiation-induced apoptosis in macrophages of C3H mice but not other strains of mice. The depletion of Mcl-1, an anti-apoptotic Bcl-2 family protein known to have a very short turnover time, through irradiation-induced arrest of global protein synthesis was identified as a major factor for the apoptosis. In the present study, the mechanism by which global protein synthesis was suppressed by irradiation was elucidated. Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) is a well documented mechanism of downregulating protein synthesis under a variety of stress conditions. Western blot analysis using phospho-eIF2alpha antibody to detect phosphorylation at serine 51 showed the increased phosphorylation of eIF2alpha in a dose dependent manner in irradiated macrophages of C3H mice, but not B6 mice. Four kinases have been identified as eIF2alpha kinases. PKR, PERK, GCN2 and HRI are activated by viral infection, endoplasmic reticulum stress, amino acid deprivation and hemin deficiency, respectively. The antibodies to detect the activation-specific phosphorylation site for PKR, PERK and GCN2 were available and used in Western blot analysis. Phospho-PKR and phospho-PERK antibody showed no change of phosphrylation at the specific site by irradiation. On the other hand, phosphorylation of GCN2 at Threonine 898 was markedly increased by irradiation in macrophages of C3H mice, but not B6 mice. Whether irradiation induces amino acid deprivation and increment of uncharged tRNA in C3H mouse macrophages is under investigation.

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We recently reported a novel suppressive effect of sodium orthovanadate (vanadate) on the DNA-binding activity of p53. In this study, we initially showed that vanadate had a more potent antiapoptotic activity than three other chemical p53 inhibitors, including pifithrin-α (PFTα), a well-known inhibitor of p53. Although the other agents inhibited the p53 transcriptional activity, they did not suppress p53-dependent apoptosis in irradiated MOLT-4 cells. Further investigations using cells with defective or impaired p53 function indicated vanadate’s specificity for p53 in suppressing DNA damage-induced apoptosis. To investigate the cause for the different effects of vanadate and the other inhibitors, we chose PFTα and PFTµ (an inhibitor of p53-mediated transcription-independent apoptotic pathway), as references, and determined the effect of them and vanadate on p53-mediated apoptosis, with a particular focus on the transcription-independent pathway. We found that vanadate suppressed the p53-associated apoptotic events at the mitochondria, such as the loss of the mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and its interaction with Bcl-2. Vanadate also suppressed the apoptosis-inducing activity of mitochondrially-targeted temperature-sensitive p53 in stable transfectants of the SaOS-2 cell line. Finally, we tested vanadate’s potential use as a radioprotector. Vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Our data demonstrate that vanadate can suppress both the transcription-dependent and the transcription-independent pathways, and suggest that both pathways must be inhibited to completely block p53-mediated apoptosis.

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[Purpose] Heat shock protein 27 (HSP27) is implicated in diverse biological functions. We have reported that HSP27 has a role in protecting human cells against UVC lethality. We identified annexin II as an HSP27-interacting protein. In this study, we sought to confirm the combined roles of HSP27 and annexin II in cell death after UVC irradiation.
[Methods] We used two human cell lines, UVC-sensitive RSa and its variant UVC-resistant AP^r-1. After UVC irradiation, the cellular amounts of annexin II protein were analyzed by Western blotting in those cells transfected with HSP27 cDNA and siRNA for HSP27. In those cells with up- or down-regulation of HSP27 or annexin II proteins, sensitivity to UVC irradiation was analyzed by colony formation assay. The capacity to remove UVC-damaged DNA was measured using antibody against the damage.
[Results and Conclusion] In RSa cells, expression of annexin II decreased after UVC irradiation, and over-production of annexin II in RSa cells resulted in increased resistance to UVC lethality. Transfection of RSa cells with HSP27 cDNA caused an increase in their resistance to UVC lethality and prevented the decline in annexin II expression. By contrast, AP^r-1 cells showed no decline in annexin II expression after UVC irradiation, while HSP27 siRNA-transfected AP^r-1 cells showed a decline and were sensitized to UVC lethality. The complex of HSP27 and annexin II was relocalized from the postnuclear to the nuclear fraction after UVC irradiation in AP^r-1 cells. Furthermore, annexin II siRNA-transfected AP^r-1 cells showed a decreased capacity to remove UVC-damage and were sensitized to UVC lethality. These results suggested that annexin II expression is involved in the repair of UVC-damaged DNA and the UVC susceptibility of human cells. As well, HSP27 appeared to regulate annexin II expression and/or localization after UVC irradiation as a molecular chaperone.

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INTRODUCTION
Magnetic resonance imaging (MRI) has been introduced to many hospitals and MRI systems with a much higher magnetic flux density have been under development. A main application of MRI systems with a much higher magnetic flux density is to analyze brain function. Therefore, it is becoming important to evaluate the biological effects of exposure to a strong SMF on the central nervous system. In this study, we evaluated the effects of exposure to strong SMFs on astrocyte differentiation in astrocyte progenitor cells.
MATERIALS AND METHODS
Astrocyte progenitor cells were were seeded on poly-L-lysin, fibronectin, laminin coated-well-plates, and exposed to SMF at a magnetic flux density of 6 T (41.7 T/m of magnetic field gradient) or 10 T (0 T/m), or sham-exposure for 30 minutes. Immediately after exposure, the culture medium was changed to transforming growth factor-β1 (TGF-β1) containing medium, and cultured for 2 days. The effects of exposure to SMFs were quantified by the expression of mRNA encoding cystatin C using a real-time PCR system with SYBR Green I as an intercalating dye.
RESULTS
Treatment with TGF-β1 markedly increased cystatin C mRNA expression. The exposure to SMFs did not affect cystatin C mRNA expression.

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[Purpose]
Reactive oxygen species (ROS) induced by ultraviolet rays (UVR) in human skin causes extra- and intracellular oxidative stresses. It is suggested that ROS induces photo-aging phenomenon such as activation of matrix metalloproteinases and melanogenesis, and photo-carcinogenesis such as melanoma. As an UVR response in the skin, melanogenesis in melanocyte is well known. This response needs the release of cytokines such as endothelin and alpha-MSH from keratinocyte. These cytokines stimulate melanocyte and promote melanogenesis. However, the relation between UV-induced ROS and cytokines is not clear. Aim of this study is to understand the relationship of ROS and cytokines in melanogenesis, using melanoma cell, melanocyte and keratinocyte.
[Methods]
Murine B16 melanoma cells were treated with H₂O₂ and UV. Then, we measured the melanin content and cellular tyrosinase activity.
[Results and discussion]
In B16 cells treated with 30 microM of H₂O₂, the cellular tyrosinase activity increased to 2.51 times higher than that of control 24 hours after treatment, and the melanin contents increased to 1.26 times. This shows that melanogenesis is induced by oxidative stress in murine B16 melanoma cell. By this report, we show influence of UV-induced ROS on melanogensis.

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Recently, it becomes practical that people are exposed to strong static magnetic fields (SMFs) up to several Tesla such as MRI. However, experimental evidences related to evaluation of biological effects of strong SMFs are not enough for health risk evaluation to date. Among the studies regarding biological effects of SMFs exposure, several reports suggested that exposure to SMFs have very weak mutagenicity by enhancement of oxidative stress or effects on life time of reactive oxygen species that is induced spontaneously or chemically within cells. In this study, we focused on effects of SMFs exposure on superoxide and the effects were investigated by evaluation of mutagenicity and co-mutagenicity of SMFs in SOD-deficient E. coli.
Superconducting magnet JMTD-10C13E-NC (JASTEC), which generates up to 13 T static vertical magnetic field, was used for SMFs exposure. Temperature in the bore was kept to be 37 deg C. In this study, E. coli QC774 (sodA sodB, Cm^r Km^r) and its parental strain GC4468 were used. These strains were exposed to 5, 10 or 13 T SMFs for 24 hr at 37 deg C in LB medium with or without 25 μM plumbagin. After the exposure, survival rate and mutation frequency from Thy⁺ to Thy^- was determined.
In the result, for both mutagenicity and co-mutagenicity, no statistically significant difference in Thy^- mutant frequency was observed between SMF-exposed cells and unexposed cells in all of magnetic flux densities for both E. coli QC774 and GC4468. It suggests that SMF up to 13T does not have mutagenicity nor affect mutagenicity of superoxide in SOD-deficient E. coli QC774.

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We showed that heat stress (44^oC/30 min) or SOD-mimic nitroxide Tempo (10 mM) induces apoptosis, while their combination swiches apoptosis to nonapoptotic cell death (Zhao et al., FBRM 40: 1131, 2006); however, this switch mechamism remains elusive. Here we identify that the above co-treatment induces autophagy in U937 cells. This high-dose of Tempo inhibited heat-induced apoptosis by specifically inhibiting the processing of activated procaspases-2, -8, -9 and -3 to active caspases downstream of tBid/Bax-mediated cytochrome c release, and instead induced the caspase-independent autophagy. Caspases were inactivated by a memchanism by which highly oxidative oxo-ammonium generated during Tempo's dismutation of superoxide oxidizes active-site-CysSH of all caspases. Autophagy was induced by the permeability transition pore opening-mediated mitochondrial dysfunction after the above co-treatment. The autophagic cells underwent propidium iodide-positive necrotic death in a delayed fashion, leading to complete inhibition of proliferation. Remarkably, ruthenium red and BAPTA (inhibitors of mitochondrial calcium transport) or cyclosporin A (a cyclophilin D inhibitor) facilitated autophagic cells to die of necrosis. In contrast, 5 mM Tempop-plus-44^oC/10 min or 44^oC/30 min induced more potently caspase-dependent apoptosis through tBid/Bax-mediated cytochrome c release than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.

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Recently it has been studied the effect of exposure to electromagnetic fields on human health in Japan. It begins to be also studied the effect of the exposure to the intermediate frequency (IF) magnetic field. Although a large number of scientific research in extremely low frequency (ELF) magnetic fields and radiofrequency (RF) fields has been done, there is few studies examining the biological effects of IF electromagnetic fields. Because the devices that are exposing IF electromagnetic fields have spread widely, it is important to evaluate the effect of human health effect.
We had already reported that the short exposure (2 hours) to an IF magnetic field at 532 μT_rms (85-fold of International Commission on Non-Ionizing Radiation Protection (ICNIRP) guideline) did not cause cellular genotoxic damage [1]. In this study, we evaluate the effect of exposure to 23 kHz an IF magnetic fields at more high density, 6 mT_rms (960-fold of ICNIRP guideline) on expression of heat shock proteins (HSPs). As a result, for the expression of Hsp27, p-HSP27, and HSP70, no significant changes were observed following exposure to the IF magnetic field using the method of Western blot and immunofluorescence. The expression of these HSPs was not affected by the IF magnetic field exposure. However, the possibility of effects on other cellular functions remains, and further studies on the cellular effects of IF magnetic fields are required.

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UV induces DNA lesions such as cyclobutane pyrimidine dimer (CPD), which can cause genetic mutations. Organisms have preventive measures for DNA lesions such as translesion DNA synthesis (TLS), which keeps DNA synthesis from being blocked by the lesions. Xeroderma pigmentosum (XP) variant group (XPV) is deficient in the Polh gene, which encodes one of the TLS-specific DNA polymerases, pol η. In this study, we attempted to clarify the roles of pol η in protecting the skin from harmful influences caused by UVB (280~320 nm), using XPV model mice. We prepared the XPV model mice (Polh-/-) harboring E. coli LacZ transgenes by mating lacZ-transgenic mice and Polh-knockout mice. After UVB irradiation (0~1 kJ/m²) to the mice, the exposed skin was removed, genomic DNA was extracted and the lacZ transgenes were recovered. Then we examined the mutant frequency of the transgene with a bacterial genetic assay. We also analyzed the mutation spectrum by DNA sequencing of the mutant lacZ genes recovered in the assay. The significance of the obtained results will be discussed.

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Premalignant lesions are strongly associated with the development of malignant neoplasia and must exist in γ-ray induced mouse atrophic thymus that persists until lymphoma development. Our previous studies of atrophic thymuses showed the presence of two different types of clonally expanding thymocytes. One is C type thymus that shows limited D-J rearrangement patterns at the TCRβ locus, indicative of clonal expansion in approximately 40% of thymuses at 40 days after. Those C type thymocytes mainly consist of CD4+CD8+ double positive (DP) cells, aberrant DP thymocytes bearing a given type of TCRβ chain on cell surface. The other is T type thymus that does not show limited D-J rearrangement patterns but shows allelic loss at Bcl11b. This suggested that loss of a Bcl11b allele probably contributed to clonal expansion and lymphoma development. Here we study the effect of Bcl11b heterozygous genotype on the development of C type thymocytes in Bcl11b ^KO/+ mice. Most of thymuses at 30 days after 3Gy of γ-ray at the age of 10 weeks showed decrease in cell number and approximately 50% of these thymuses consisted of C type thymocytes. Those thymuses were classified into three groups: (1) normal or T type thymus, (2) C type thymus consisting of CD4+CD8+DP cells, and (3) C type thymus consisting of immature cells before differentiating into DP cells, which was not found in irradiated wild type mice. These results suggest that Bcl11b heterozygous genotype promotes the development of C type thymocytes and differentiation arrest of C type thymocytes. In contrast, thymuses in mice irradiated at the age of 4 weeks rarely exhibited those changes, suggesting marked age-dependency in Bcl11b ^KO/+ mice.

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Recent studies have showed that microRNAs (miRNAs), a class of short noncoding RNAs, play important roles in tumorigenesis. The epidemiological study on atomic bomb survivors has suggested that the radiation-associated risk for breast cancer incidence is higher than that for most other sites.
In this study, to clarify the involvement of miRNA in radiation-induced mammary tumorigenesis, we analyzed the miRNA expression profiles in radiation-induced rat mammary carcinoma using miRNA microarrays (Agilent Technologies). Mammary carcinomas were collected from Sprague-Dawley rats irradiated with 2 Gy gamma-rays at 7 weeks of age or from nonirradiated rats that spontaneously developed tumors. The miRNA microarray analysis showed that 7 miRNAs were significantly up-regulated in radiation-induced mammary carcinomas compared with spontaneous mammary carcinomas. Of these 7 aberrantly expressed miRNAs, miR-31 and miR-135b showed an increased expression by more than 3-fold. Quantitative PCR analyses confirmed up-regulation of miR-135b. The biological function of miR-135b in vitro is currently under investigation and will be presented.

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