In the symposium, outline of Fukushima Neucear Power Station Accident will be reported and discussed, from the view points of engineering of plants, envionmental behavior, health effects and risk assessment.

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In this presentation, the outline of Fukushima Nuclear Power Plant Accident will be reported from the view point of engineering.

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Huge amount of radionuclides were released to the envionment at the Fukushima nuclear power plant accident. In this presentation, behavior in the environment and route of human exposure of the released radianuclide will be reported.

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Health Effects of Low-dose and Low-dose-rate radiation exposure are concerned after Fukushima nuclear power plant accident. In this symposium, health effect of low dose radiation effect are discussed with relation to the Fukushima accident from view points of radiation biology.

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Fukushima Nuclear Power Plant Accident caused severe environmental radioactive contamination. In this lecture, we assessed the responses of radiation specialists after the accident, and discussed what was, and will be demanded for us.

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Elucidation of the molecular mechanissm of cell death induced by ionizing radiation is a main topic in the field of radiation biology. Recently, many modes of cell death, such as apoptosis, autophagy, mitotic catastrophe, senescence like cell death etc. have been reported. These modes presented the main topics of the last year symposium which we organized under the title" Radiation induced cell death". In this year, we will organize a second symposium under the title "Reconsidering new roads for radiation-induced cell death" to focus on strategies in cancer therapy with a discussion on the mechanisms of cell killing and the implication in radiation treatment .

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The tumor suppressor p53 has been implicated in many important cellular processes, including regulation of apoptotic cell death, in the cellular response to DNA damage. When cells encounter genotoxic stress, certain sensors for DNA lesions stabilize and activate p53. Notably, the phosphorylation at serine-46 (Ser46) of p53 is essential for commitment to promote apoptosis. Recently, we have identified that DYRK2 is responsible for Ser46 phosphorylation in response to DNA damage. However, little is known about the pro-apoptotic genes induced by Ser46 phosphorylation. To address this issue, we have carried out the microarray screening and the ChIP-sequencing assay. Through the combination of these analyses and subsequent validation, we have identified several strong candidates that are specifically induced by Ser46 phosphorylation. I would like to discuss our progress based on these findings.

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Many biological findings including cell death have been reported in solid tumors after radiotherapy in a fractionated regimen. 4Rs were proposed as important kinetic factors occurring after fractionated radiotherapy in the 1960s, finally leading to the discoveries such as molecular mechanisms of DSB repair, cell cycle checkpoint, hypoxia response, and identification of cancer stem cells. As for apoptosis, at first, it was believed that apoptosis only occurs to lymphatic cells, but not to solid tumor cells after irradiation; however, development of sensitive methods identified apoptosis also in solid tumors albeit with a low frequency. Furthermore, many papers reported that the frequency is well correlated with clinical results of radiotherapy. Surprisingly, the activity of caspase-3 was recently reported to contribute to the accelerated repopulation after irradiation via secretion of prostaglandin E2. In this symposium, I will review the diversity of factors which significantly affect cell death occurring within solid tumors after irradiation.

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Ionizing radiation induces various damages on genomic DNA. Among those, DNA double strand breaks (DSBs) are the most critical damage and it might be a main cause of cell killing in cancer radiotherapy. There are at least two pathways for DSBs, one is non-homologous end joining (NHEJ) and the other is homologous recombination (HR). We report here that the partial inhibition of the HR pathways has significant potential for application for improvement of cancer radiotherapy through the slight but enough radio-sensitization. A specific, strong inhibitor of any HR protein should be an effective radio-sensitizer because it shut down the S/G2 specific DSB repair pathway. However, this efficient inhibition of HR results a strong cytotoxicity, in other words, the inhibitor cannot reach the bed side, due to its strong side effects. Our novel concept is that inhibiting the function of a specific domain of a DSB repair protein can partially suppress HR pathway and reduce cancer cell viability following irradiation with slight but enough efficiency. This weak radio-sensitization can be enhanced if radiation is given with a sprit dose because the recovery from sublethal damage depends on HR.

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Cell death induced by radiation therapy for patients in clinic is not as clear as in vitro study. p53-dependent apoptosis is frequently induced by radiation in radiosensitive tumor and normal cells in vivo; however, few apoptotic cell deaths are found in the majority of tumor and normal cells in vivo following irradiation. To investigate low LET X-ray- and high LET beam-induced cell death and gene expression profiles in human tumors with different p53 status, an ependymoblastoma with wild-type p53, a primitive neuroectodermal tumor with wild-type p53, and a glioblastoma with mutant-type p53, were transplanted into nude mice subcutaneously, and irradiated with 200kV X-rays or carbon ion beams (290MeV/u, 6 cm spread-out Bragg peak, NIRS). These tumors were excised 4, 6, or 24 hours (h) after 2Gy irradiation. Additionally, glioblastoma was examined 6 h after 8 or 16Gy irradiation. Total RNA was extracted for GeneChip expression microarray analysis. Hierarchical clustering, gene ontology analysis, and pathway analysis were also performed. Tumors with wild-type p53 showed significant changes in gene expression following 2Gy irradiation and most profiles by carbon ion and X-rays were similar. Glioblastoma with mutant-type p53 showed little change after 2Gy; however, significant changes in gene expression were induced by 8Gy or 16Gy, and the gene expression profiles induced by carbon ion beams were different from those by X-rays. p53, caspases, Fas, and TRAIL were not involved in the pathways, but NF-kB and IAP were suggested to be involved.

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Biological dose estimation in the radiological accident is considered to be very important as the first step in the medical treatment of persons exposed to radiation. The radiation dose is estimated by three methods; the clinical symptom, the count of the lymphocyte number and the analysis of chromosome aberrations peripheral blood lymphocytes (PBLs) of the exposed persons. Radiation induces many types (dicentric, translocation, ring etc.) of chromosome aberrations in the PBLs of the exposed individuals. Since dicentric chromosome is considered relatively radiation specific and the frequency of this aberration correlates with radiation dose , consequently, the dicentric chromosome is considered to be the most reliable, specific and sensitive biomarker for the dose estimation after acute and total radiation exposure. Therefore, this method is called as the "gold standard" for radiation biodosimetry. On the other hand, the analysis of chromosome translocation is used in the case of old exposure and chronic exposure because the lymphocytes with chromosome translocation remain for a long period after the radiation exposure. Furthermore, since the cells exposed to extremely high dose radiation will stop at G2 or earlier stage of the cell cycle and be dead due to apoptosis, the chromosomes are not identified in PBLs. However, in such cases, it is possible to analyze the chromosomes, in particular, ring chromosome, by using forcible condensation of DNA due to the treatment of chemical agents (PCC: premature chromosome condensation). This method is called the PCC-ring and used for the dose assessment in the case of higher radiation exposure. Also, in recent years, FISH techniques using DNA and PNA probe are applied to the dose estimation and will be discussed in this presentation.

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There is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person. We evaluated an immunocytofluorescence-based quantitation of gamma-H2AX foci as a biodosimeter of total-body radiation exposure (60Co gamma-rays) in a rhesus macaque (Macaca mulatta) model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation (TBI) doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual gamma-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs. Based on data obtained from the TBI study we introduce a partial-body irradiation (PBI) exposure analysis method. We established standard curves for PBI using Qgamma-H2AX (mean number of gamma-H2AX foci per damaged cell) and Fgamma-H2AX (fraction of damaged cells). Our findings indicate that quantitation of gamma-H2AX foci may make a robust biodosimeter for analyzing exposure to ionizing radiation in humans and could help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. This research was supported by the NIAID Radiation/Nuclear Countermeasures Program, the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH and the Armed Forces Radiobiology Research Institute under research work unit number BD-13 (RBB4AR).

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Chromosome aberrations have been used as a valuable marker for radiation dose assessment. However, as DNA double strand breaks are rapidly rejoined by DNA repair pathways, the initial number of DNA breaks is practically unable to measure by this method. Consequently, the numbers of dicentrics and translocations, which are caused by mis-rejoining of the initial DNA breaks, are counted for dose estimation. Thus, in order to increase the sensitivity of cytogenetic biodosimetry, some markers reflecting the initial number of breaks are required. Recently, it has been well established that DNA double strand breaks initiate activation of ATM, which phosphorylates various DNA damage checkpoint factors. For example, histone H2AX, a member of histone H2A, is phosphorylated at serine 139, and phosphorylation of histone H2AX results in a sequential accumulation of various DNA damage checkpoint factors at DNA damage sites. They form multiprotein complex, called ionizing radiation-induced foci, which are detectable under microscope. Recently, we found that the foci of phosphorylated H2AX and MDC1 persist into mitosis. Therefore, we established a method that visualizes phosphorylated H2AX foci on methaphase chromosome spreads. We confirmed that the foci were detectable on more than 95% of chromatid breaks. An application of this method for cytogenetic biodosimetry will be discussed.

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Both immediately after a large-scale radiological event, or in support of longer-term epidemiological studies after an event such as at Fukushima, there is a pressing need for ultra-high throughput biodosimetry. A logical approach to achieve this is complete automation of standard biodosimetric assays that are currently performed manually. We will discuss progress to date on the RABiT (Rapid Automated Biodosimetry Tool), designed to score micronuclei, gamma-H2AX fluorescence, and other related endpoints in lymphocytes derived from a single drop of blood from a fingerstick. The RABiT is designed to be completely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate, and to cover a wide dose range. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in situ analysis in filter-bottomed multi-well plates, implementation of robotic-plate and liquid handling, and new developments in high-speed imaging. The current RABiT potentially provides up to a 30,000 sample per day throughput. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. We will describe the ongoing development of the RABiT, current work on overall calibration and validation, as well as the infrastructure that would be required for its implementation. Garty G, Chen Y, Salerno A, Turner H, Zhang J, Lyulko O, Bertucci A, Xu Y, Wang H, Simaan N, Randers-Pehrson G, Yao YL, Amundson SA, Brenner DJ. The RABIT: a rapid automated biodosimetry tool for radiological triage. Health Phys. 98:209-217 (2010)

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ATM is required for the slow component of DNA double strand break (DSB) repair in mammalian cells. In previous studies, we have shown that this represents the repair of DSBs located within or close to regions of heterochromatin (HC). The damage response mediator proteins including H2AX, MDC1, the Mre11/Rad50/NBS1 complex, RNF8, RNF168 and 53BP1 are required for this component of DSB repair. We have shown previously that 53BP1 is required to tether ATM at DSBs and that this promotes dense phosphorylation of KAP1, generating pKAP-1, at HC-DSBs. Here we examine how pKAP1 enables the repair of HC-DSBs. Previous studies have shown that KAP1 is a sumo ligase and undergoes autosumoylation. Significantly, the sites undergoing sumoylation are close to the S824, the ATM-dependent phosphorylation site. Sumoylated KAP-1 interacts with the sumo-interacting motif (SIM) on the larger isoform of CHD3, a component of the NURD remodelling complex. Strikingly, we show that phosphorylation of KAP-1 at S824 does not impact upon sumoylation of KAP-1 but does impair the interaction between sumoylated KAP-1 and the SIM motif on CHD3. SUMO-SIM interactions are charge dependent and we suggest that the unstructured C-terminal region of KAP1, when phosphorylated, can out compete the SUMO-SIM interaction between KAP-1 and CHD3 via a charge-dependent reaction. Loss of CHD3 causes relaxation of the heterochromatic superstructure. siRNA of CHD3 overcomes the need for ATM for DSB repair, similar to the impact of siRNA KAP1. These findings represent a novel impact of phosphorylation and provide insight into the role of ATM in promoting the repair of DSBs. Interestingly, this mechanism for relaxing HC superstructure does not necessitate any changes to the epigenetic code.

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Metnase (also known as SETMAR) is a fusion gene expressed only in anthropoid or higher primates that contains a histone methyltranferase (SET) and a transposase domain derived from the Mariner transposase (MAR). Metnase improves the integration of foreign DNA, enhances DSB repair via the NHEJ pathway potentially through interaction with DNA Ligase IV, promotes replication-fork restart through interaction with PCNA, interacts with and stimulates TopoII alpha-dependent chromosome decatenation and suppresses chromosomal translocations Metnase is expressed higher levels in several cancer cell lines. To explore the role of Metnase in response to equitoxic doses of photon (2Gy 137Cs) or hadron particle (1Gy carbon and iron ion) radiation we constructed normal (BJ1 hTERT) and cancer (HT1080) human fibroblast cell lines bearing stably-integrated Metnase shRNA expression vectors. We examined replication recovery over time after irradiation by monitoring the incorporation of the nucleoside analog EdU in wild type and Metnase knockdown cells. We found that carbon and iron ion treatment suppressed replication recovery in both normal and cancer fibroblasts regardless of Metnase expression levels. Interestingly, Metnase knockdown promoted replication recovery in normal fibroblasts in response to gamma treatment suggesting that Metnase may have, as yet undetermined roles in cell cycle checkpoint in response to gamma radiation. These data also suggest that hadron-particle radiation evokes a stronger checkpoint response that may not be influenced by Metnase.

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When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase ATR and its partner ATRIP plays an important role in this response. Replication stress activates ATRIP-ATR via two distinct steps: first by local chromatin accumulation of the kinase through binding to the RPA-coated single stranded DNA; and second by direct association of ATRIP-ATR with the activation domain of TopBP1, leading to the increased kinase activity and Chk1 phosphorylation. Here we show evidence that the Fanconi anemia (FA) pathway is activated following genomic stress in a manner dependent on ATR. Furthermore, we have found that, following cellular exposure to DNA crosslinking damage, FA cells deficient in the core complex components have a defect in the first accumulation step in ATR signaling, and that the FA core complex but not FANCD2 enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. Our data now reveal a new mode of regulation of the ATR pathway via action of the multisubunit FA complex.

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Human cells are continuously threatened by endogenous and exogenous assaults on DNA. Following various DNA damages, PI3-kinase related protein kinases (PIKKs), including ATM, ATR and DNA-PKcs, take key roles in the DNA damage response (DDR). DNA double strand break (DSB) is a deleterious damage for cell survival and genome integrity, and major pathways for its repair are non-homologous end-joining (NHEJ) and homologous recombination (HR). ATM is critical for checkpoint signaling and DSB repair, especially HR, and DNA-PKcs is an essential component of NHEJ. ATR is activated by RPA-coated single-stranded DNA (ssDNA) that appears at a stalled replication fork (replication stress), and phosphorylates downstream factors in the S-phase checkpoint. However, recent studies have revealed that these PIKK family kinases function in conjunction with each other in DDR. For example, in response to DSB induced by ionizing radiation (IR), ATR is activated by ssDNA resulting from DNA end resection in ATM-dependent manner. We have shown that DNA-PKcs is phosphorylated by ATM and ATR in response to exposure to IR and ultraviolet light (UV), respectively. In addition, contrary to the rapid activation of the ATR pathway, delayed activation of ATM and DNA-PKcs kinases after UV-induced replication stress was found, suggesting that DNA double strand end (one-ended DSB) produced by replication fork collapse triggered their kinase activity. With a focus on our recent works, roles of PIKKs and their substrates in DSB response will be discussed.

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Biological effectiveness was compared among accelerated carbon, proton beam and gamma-rays. 290MeV/n carbon and 70MeV proton beam was generated by HIMAC and NIRS930 respectively. Biological effectiveness was calculated from cell survival obtained in special cell culture container, Opticells. Cell survival fraction was determined approximately each 2mm thickness till particle beam reaches its bragg peak. Dose was calculated from each data points and re-constitute the regular cell survival curves. CHO wild-type cells showed non-scattered straight line of survival curves as X-rays and gamma-rays. On the other hand, survival fraction after carbon ion exposure was not depending on dose of radiation. This result shows greater relative biological effectiveness for carbon beam but not for proton beam.

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In the Fukushima nuclear accident, radioactive materials released from the plant caused radiation exposure to general public. The exposure would be of long-term at low dose rate. The potential risk from internal exposure and effects on fetuses and children have drawn much attention. In this presentation information on these issues will be reviewed for discussion on future needs in radiation research.

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Fukushima

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A large amount of radioactive materials has been discharged to the atmosphere after the accident of the TEPCO's Fukushima Dai-ichi nuclear power stations (abbreviated to 1F). The total amount discharged from the reactors is estimated to be approximately 1.3 x 10¹⁷Bq of I-131 and 6.1x10¹⁵Bq of Cs-137. These radionuclides dispersed widely and some of them deposited on to the ground surface with rain water. The geographic pattern of contamination levels was much different from the shape of concentric circle; the most contaminated area is located along the line to the northwest from the 1F and the relatively high concentration is observed along the line from the east part of Fukushima city to a southwest region. The Ministry of Education, Culture, Sports, Science and Technology (MEXT) has measured ambient dose rates in the area beyond 20km from 1F from March 15, 2011 and observed relatively high dose rates in the northwest are such as 0.33 mSv/h on that day at about 20km from 1F and also 0.17 mSv/h on March 17 at about 30km from 1F. Regarding radiation exposure of residents who lived in the vicinity of 1F, MEXT has estimated the dose of the residents at about 30km in the northwest region from the noon on March 13 to May 30 to be 36mSv. In the south-by-sourthwest region, the integral dose is maintained at relatively low level though it is known that a highly radioactive plume passed there during the initial period of the accident. As to internal exposure, the local nuclear emergency response headquarter investigated thyroid exposure of 1,080 children in late March and found the radiation levels of all children who lived near the evacuation area were below the screening level. In addition, from the end of June to July, Fukushima prefecture performed whole-body-counter measurements for 122 residents in cooperation with the National Institute of Radiological Sciences and reported that the committed effective dose of radiocesium in all of them were below 1mSv.

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The 2011 off the Pacific Coast of Tohoku Earthquake with a magnitude of nine on the Richter scale happened at 14:46, 11 March, 2011. At the same time, several giant tsunami waves hit Iwate, Miyagi, Fukushima, Ibaraki and Chiba Prefectures. Furthermore, the Fukushima Daiichi nuclear power plant accident was caused by the earthquake and the tsunami. In the nuclear power plant, many nuclear fuel rods in the atomic reactor could not be cooled down by the loss of the entire electric source. Then, hydrogen explosions happened in the 1st, 3rd and 4th the nuclear power plant, the radioactive materials with 850,000 TBq were released into atmosphere, and foods, drinking waters, soils, ocean waters and so on were contaminated by them.
Since the movement involved in the Q&A support of JRRS has been started in mid-March, the members of the Q&A support of JRRS have been replying to too many questions about radioactivity and radiation from the public. At the same time, frequently-asked and important questions have been put on the website of JRRS. The contents have been brushed up according to an ever-changing condition. In addition, the Q&A support of JRRS is making on holding of the lecture meetings in Fukushima and surrounding Prefectures.
At the Workshop, we will introduce social impacts observed through the movement involved in the Q&A support of JRRS and discuss about the roles to play and the foresight of JRRS.

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Fancy and fact of low-dose effects: Social accountability and expectation to the Radiation Research Society Masao SASAKI Professor emeritus, Kyoto University Fukushima nuclear power plant accident smashed our contemporary understanding on the effects of low-dose radiation. It directly asked yet unsolved scientific problems of intensive debate lying behind the setting of radiation protection. Two things have been evoked; one is an urgent need for the quantification of low-dose effect, and another is the sound framework amenable to social need. These are mutually related and present key issues of the social accountability of the scientists. (1) The Life Span Study of A-bomb survivors have provided fundamental information on how radiation dose relates the health effects but have met ambiguity at low doses largely due to a limitation of statistical power. A newly devised statistical test system adds much new to the study, such as dose-restricted threshold, internal exposure, radiation quality, age-at-exposure effect, crosstalk with environmental mutagens, choice of DNA repair pathway. They pose a new paradigm for deep understanding of radiation effects particularly at low doses. (2) In Japan, the science of radiation effects was prompted in response to radioactive pollution from and exposures to Bikini test bombing. JRRS continued the effort in promoting the science of radiation effects through the Science Council of Japan. Currently, to promote the innovative science in today's economic growth rate, the MEXT proposed integrative research center, either individual or by network. The low-dose radiobiology is a sort of big science. Fortunately, we have such integrative research centers for environmental radioactivity measurement, radiation effects and medicine, international innovation center for health risk, radiation biology, and nuclear sciences, of international and nationwide. The science of radiation effects of the urgent need may be facilitated by tightly associated network corporation of those integrative research centers and COEs in the universities.

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Bystander effect is known as the signal transmission through secreted factors or GAP junction. This reaction is thought to be an important response against stress in cellular population. We observed bystander effects induced by X-irradiation and UV-irradiation. Severe damage are not induced so frequently in bystander response, but non-lethal change such as gene mutation are significantly occurring. On the other hand, vitamin C (ascorbic acid) is well known as anti-oxidant, and we want to know the possibilities of the biological usefulness. In our study, the results suggest that ascorbic acid is effective for non-lethal damage including bystander responses. The mitochondrial function is one of the key target in the reactions after irradiation, therefore we want to know the mechanisms to suppress the bystander response by the treatment of ascorbic acid.

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Chemical properties of vitamin C have been well studied by G. R. Buettner and his colleagues. Because pK_a of two OH group on the furan ring of L-ascorbic acid (AscH₂ ^-) are 4.1 and 11.8, ascorbate anions (AscH^-) are major chemical structure in biological system of weak alkaline. Although vitamin E (α-tocopherol) as antioxidants of oxidized lipid can reduce lipid radicals, vitamin E turn into α-tocopherol radicals those are in weakly oxidative. AscH^- can reduce α-tocopherol radicals by H atom (or H⁺ and e^-) transfer to be back in, α-tocopherol, and AscH^--- are oxidized to be Asc⋅^-. Because an unpaired electron in an Asc⋅^- are stabilized on π-conjugated system of furan ring with two ketons, Asc⋅^- are not reductive enough to produce super oxides by reduction of oxygen molecules. Two Asc⋅^- turned to an AscH^- and a DHA as dehydro-form by dismutation reaction, so that one Asc⋅^- can be recover as an AscH^-. DHA molecules can be reduced by GSH with enzymes to be AscH^- in biological system. We have been measured mutagenic long-lived radicals (LLRs) generated in irradiated hamster cells or non-irradiated recipient cells having irradiated medium with irradiated cells. LLRs in irradiated cells removed by the addition of vitamin C and/or N-acetylcysteine (NAC), however, NAC could not reduce LLRs in the recipient cells. Both vitamin C and NAC can be incorporated into cytsol of the cells, so that LLRs in irradiated cells may be distributed in the cytsol of the cells. On the other hand LLRs on the bystander effects could be produced in organelles having some proteins incorporating vitamin C such as GLUT-1 which can incorporate DHA from mitochondria intermembrane space to matrix. The difference in the reactivity of vitamin C and NAC with LLRs produced by bystander effects must be very important results for elucidating the mechanisms of inducing mutation from the LLRs.

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Background: X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation using ascorbic acid (vitamin C).
Method: Human embryonic fibroblasts (HE17) cells were used. HE17 cells were cultured and their confluent cultures were maintained for a week to synchronize their cell cycle at G1 phase, and then were irradiated by X-ray. To visualize the site of DNA DSBs, cells were fixed by formalin at designated periods (between 15 minutes and 24 hours after X-irradiation), and immunofluorescence staining on p53-binding protein-1 (53BP1) foci, which are known to accumulate on this site, was performed. Also, in order to visualize micronucleus formation, cells were fixed by formalin at 24 hours after X-irradiation and DAPI staining was performed. Moreover, we investigated the contents of micronuclei using immunofluorescence staining and fluorescence in situ hybridization (FISH). In this study, we examined the relationship between DNA DSBs and micronucleus formation using two different radical scavengers from the point of view of radioprotection ability. Dimethyl sulfoxide (DMSO) was used as an inhibitor for DNA DSBs, because DMSO can capture irradiation-derived reactive radicals and inhibit cell death. On the other hand, vitamin C was used as an un-inhibitor for DNA DSBs, because vitamin C cannot inhibit lethal effect by X-irradiation.
Results: First, we examined the effect of radical scavengers on radioprotection using colony formation assay. The result showed that treatment with vitamin C (5 mM) before X-irradiation cannot cause radioprotection effect, but treatment with DMSO (2%, 256 mM) can, by comparison of survival fraction with non-treatment controls. Moreover, in evaluation of DNA DSBs using 53BP1 foci, we found that treatment with vitamin C cannot inhibit the number of 53BP1 foci after X-irradiation at all, but treatment with DMSO can significantly do. These results showed that DMSO can inhibit the generation of DNA DSBs via inhibition of reactive radicals during X-irradiation and this inhibition can lead to decrease cell lethal effect, while vitamin C cannot do. Next, we examined the inhibitory effect of radical scavengers on X-ray induced formation of micronuclei. The result showed that treatment with DMSO before X-irradiation (between 0.5 Gy and 2 Gy) cannot inhibit X-ray induced formation of micronuclei, but treatment with vitamin C can do. If X-ray induced formation of micronuclei results from DNA DSBs, treatment with DMSO should inhibit it. Actually, vitamin C can significantly inhibit X-ray induced formation of micronuclei. In addition, we found micronuclei with centromeric signals. Overall, we suggest that abnormalities in the cell division mechanism may have something to do with micronucleus formation after X-irradiation.

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A kinetic study of the regeneration reactions of natural vitamin E by vitamin C and ubiquinol was carried out by means of double-mixing stopped-flow spectroscopy. A substantial deuterium kinetic-isotope effect was observed on the second-order rate constant and the activation energy for ubiquinol. In the regeneration reaction of α-tocopherol, deuteration of ubiquinol increased and decreased the activation energy and the second-order rate constant by 6.1 kJ/mol and a factor of 18.3, respectively. From this result, it is considered that proton tunneling plays an important role in the regeneration reaction of vitamin E by ubiquinol. In contrast, since such a substantial deuterium kinetic isotope effect was not found in the regeneration reaction of α-tocopherol by vitamin C, the tunneling effect may not play an important role under the present experimental conditions. However, for 5,7-diisopropyltocopherol a substantial deuterium kinetic isotope effect was observed. The conditions under which the tunneling effect becomes an important factor were discussed in conjunction of our experimental results. Vitamin E may inhibit the autooxidation of lipids in cellular membranes by taking advantage of proton tunneling. It is interesting that the microscopic quantum-mechanical tunneling effect could manifest itself in a macroscopic vital function. Although this is in conflict with our intuition at first glance, living cells might, in reality, know quantum mechanics very well and use it effectively.

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The application of localized radiation using heavy-ion microbeams eliminates the effect of non-uniform ion hits on cell population, since individual cells can be irradiated one by one with a defined number of energetic heavy ions. Another advantage associated with the use of heavy-ion microbeam irradiation concerns the precise detection of ion-hit position on micron-scale targets to obtain the information on the position of ion traversal and on cellular responses induced by ion hit simultaneously. Therefore microbeam is an operative means to elucidate initial cellular responses together with the relationship with ion track structure. The use of heavy-ion microbeams has not been restricted to the area of radiation biology. Targeted irradiation using heavy-ion microbeams has been applied to various biological studies, such as plant physiology or developmental biology, as a radio-microsurgical tool to inactivate specific tissue or cell populations in multicellular organisms and to investigate their function. The outlines of these studies, which were carried out using our collimated heavy-ion microbeam at JAEA-Takasaki, will be introduced.

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An increasing body of data indicates that ionizing radiation causes learning impairments. To understand the effects of ionizing radiation on the nervous system, we studied olfactory adaptation, salt chemotaxis learning and locomotion using Caenorhabditis elegans, a well-known model organism for the nervous system. We recently found the modulatory effect of gamma-rays on the salt chemotaxis learning that was manifested as a decrease in chemotaxis. In addition, our preliminary findings also show that high linear energy transfer carbon ions can decrease the chemotaxis during salt chemotaxis learning. However, we have no direct evidence for the interaction of ionizing radiation with the central neuronal tissue (nerve ring) of the nervous system in C. elegans. Localized ionizing irradiation is useful to analyze radiation effects at a cellular or tissue level. Thus, to investigate the effects on the nerve ring, we used the heavy-ion microbeam system installed at the Takasaki Ion accelerators for Advanced Radiation Application of JAEA. To achieve during-learning irradiation, we have developed the live-targeting system for non-paralyzed C. elegans using the micro-total analysis systems to restrict their motion, and this will be discussed.

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Microbeam irradiation systems have become significant tools in the field of radiation biology and have discovered much important evidence that have never been described in the study using conventional broad beam irradiation. Single Particle Irradiation System to Cell, SPICE at National Institute of Radiological Sciences (NIRS) is a proton microbeam irradiation system developed for low dose radiation effect studies, such as for the cellular response of targeted and non-targeted effects. SPICE provides 3.4 MeV proton microbeam with approximately, 2 μm in a diameter beam. Two thousand mammalian cells in 25 mm² area per dish can be irradiated at speed of 400 cells per minute which completes within 10 minutes including image capturing, cell recognition, and irradiation. With this SPICE, we made advancements in the targeting, image capturing system and sample dish to irradiate zebra fish embryos to expand radio-biological studies using microbeam technique to radio-adaptive response studies in in vivo. SPICE were used to provide the primimg dose and X-ray photons were employed to provide the challenging dose to investigate radio-adaptive response in developmental stage of zebra fish embryos. In detailed experimental setup and the first result of irradiation of zebra-fish embryos will be presented. Electrostatic accelerator facility of NIRS received a severe damage from the Tohoku-Kanto earthquake on March 11th, 2011, and currently, SPICE is under construction. Hopefully, SPICE will be renewed, and resume its operation by the beginning of April of 2012.

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Accumulating evidences have indicated that ionizing radiation can cause various effects in the cells that have not absorb radiation energy directly. These effects are now collectively called as non-targeted effects. Although the indirect lethal and mutagenic effects have been well described, its effect on differentiation remains to be determined. Cranial radiation causes dysfunctions of the normal central nervous system (CNS), including cognitive impairment, however, the mechanism is not fully understood. Since the CNS is composed of several types of differentiated cells from neural stem cells (NSCs), one possibility mechanism could be that ionizing radiation affects differentiation process of NSCs. Here, we investigated the indirect effects of local X-irradiation on NSCs differentiation using Nagasaki University X-ray microprobe. NSCs plated on coated-mylar membrane or coated glass slips were cultured at least 72 hours in proliferation-competent medium before irradiation. When NSCs were incubated in a differentiation-competent medium for 7 days, they predominantly differentiated into GFAP-positive astrocytes. Then, NSCs were locally-irradiated and incubated in a proliferation-competent medium. Cells in a targeted area were eliminated by apoptosis, however, the number of GFAP-positive cells was increased in the surrounding unirradiated areas. As NSCs differentiation is regulated by soluble factors, our observation might suggest a role of bystander factors, which are secreted by the targeted cells.

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It has been known that about two thirds of the biological effects of ionizing radiation (X-and gamma rays, IR) may be caused by an indirect action od free radicals (in which the recoil electrons interact with water molecules to produce hydroxyl radicals that difuse to target molecules). On the other hand, radiation-induced events which cannot be explained by the indirect effects, such as apoptosis, have been also reported. IR can induce reactive oxygen species (ROS) during irradiation and also irradiated cells can generate ROS on an extended time scale following exposure. Therefore, it is important to elucidate the time-and spatial distribution of intracellular ROS to evaluate their roles in these events. Here, we are focusing on the role of mitochondria in radiation-induced damage discussing on the significance of ROS in radiation biology.

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We previously demonstrated that manganese superoxide dismutase (MnSOD) stably transfected human hepatocellular carcinoma cell line, HLE (Motoori et al., Cancer Res. 61:5382-5388, 2001), was resistant to X-irradiation. Cell death induced by X-irradiation occurred through mitochondrial ROS induction and lipid peroxidation. Laser-microscopic quantitative examination of ROS using HPF (Setsukinai et al., J Biol Chem, 278:3170-3175, 2003; Indo et al., Mitochondrion 7:106-118, 2007) showed the development of intracellular ROS at mitochondrial region, and reached at maximum at two hours following x-irradiation. Further, the development of superoxide anions following x-irradiation, was proved by using EPR technique. To examine the main component of mitochondrial inner-membrane, cardiolipin was tested for the oxidation following x-irradiation. The results showed that cardiolipin extracted from irradiated cells showed lipid oxidation, while cardiolipin it-self did not show change in oxidation at two hours following x-irradiation. Thus, cardiolipin-oxidation of intra-mitochondrial membrane following x-irradiation was not originated by cardiolipin-itself oxidation, but by other mechanisms caused by in situ situation was suggested.

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Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In our study, we focused on superoxide dismutase (SOD2) and glutaredoxin (Grx2) proteins. It is well known that SOD eliminate superoxide anion, Grx reduces the oxidized macromolecules, and both SOD and Grx are conserved from bacteria to human. Therefore, SOD and Grx are essential enzymes in cellular homeostasis. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in both cells compared with those in the control cells. MitoSoxTM Red assay revealed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Flow cytometry with a fluorescent probe revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. Furthermore, we examined whether and how cellular sensitivity to ionizing radiation is modulated by the overexpression of mitochondrial Grx (Grx2) in the cultured human cells. As the results of overexpression of SOD2 and Grx2 in T-REx HeLa cells, morphological alterations of mitochondria and levels of mitochondrial superoxide, DNA double-stranded breaks, protein oxidation and OXR1 expression were suppressed. These results suggest that antioxidant enzymes in mitochondria play important roles in various cellular responses to ionizing radiation.

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While it has been well-known that ionizing radiation (IR) causes the formation of reactive oxygen species (ROS) through ionization and excitation of water, IR has been shown to stimulate intracellular ROS production several hours after irradiation. Recently, it is becoming clear that this secondarily-generated ROS after IR has a variety of biological roles, including apoptosis signaling, radiation-induced genetic instability, and bystander effect. However, the mechanism of IR-induced mitochondrial ROS production has not been fully elucidated. We have been investigated it by using tumor cell lines such as human lung carcinoma A549 cells. Our study revealed that IR increased mitochondrial ROS level, accompanied by the enhancement of mitochondria electron transport chain (ETC) activity. It was suggested that the increase of mitochondrial content in cells rather than the increase of ETC enzyme complex activities was involved in this IR-induced ETC activity. Irradiated cells undergo cell cycle arrest by checkpoint mechanisms. We examined the relationship between cell cycle and mitochondrial ROS level or mitochondrial content, and found that the cells at G2/M phase had higher mitochondrial ROS level and mitochondrial content than the cells at G1 or S phase. Therefore, it was suggested that IR-induced G2 arrest contributed to the increase of mitochondrial ROS level by accumulating cells at G2/M phase.
Recent evidences indicate that tumor cells have lower antioxidant capacity and are more susceptible to oxidative stress than normal cells. We thus hypothesized that drugs that increase IR-induced mitochondrial ROS production could enhance radiosensitivity of tumor cells. We have been testing several candidate compounds to verify this hypothesis. We would like to introduce our up-to-date data on it in this talk.

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It is widely thought that radiation carcinogenesis originated from DNA lesions. However, our results suggest presence of the route which does not assume DNA lesion origin besides the route which assumes DNA lesion origin, and it also suggest that this route is main in carcinogenesis. To examine the possibility, we studied it using primary cells derived from human, mouse and hamster. After irradiation, we examined expression of carcinogenic phenotypes and relationship of intracellular bioactivity changes. As a result, by radiation, disturbance of mitochondria function occurred temporarily, and electron leak from electron transfer chain. Electron elevated intra-cellular oxidation level. And oxidative radicals induced structural aberration of important molecules, such as telomere, sub-telomere and centrosome. Because aneuploid was observed in all cells being malignantly transformed, it is expected that chromosomal non-disjunction with centric structural aberration is main route of radiation carcinogenesis. We cannot distinguish this route from route of natural carcinogenesis. These results suggest that carcinogenesis by low dose radiation is bulk advance of natural carcinogenesis. *This work was supported by grants for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology-Japan (21310036) and for The Nuclear Safety Research Enhancement Actions from the Nuclear Safety Commission of Japan.

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Low dose or low fluences of ionizing radiation induces Poisson distribution of the exposure dose in a cell population (macro-scale). On the other hand, microbeam irradiation technique makes one-cell exposure with an exact quantity possible to do. However, there are many problems on understanding common or different points between macro and micro scale for radiation biology, because of a bystander response and an application limit of dose (J/kg). The present paper will discuss on these points.

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Each cell in a population is inhomogeneously irradiated with X- or γ-rays at low dose or low dose-rate, according to Poisson distribution. Thirty seven % of cells, which are not irradiated with X- or γ-rays at all, are contained within the cell population, however, it is much difficult that these unirradiated cells are distinguished. Om the other hand, since it is easy to analyze cellular response to radiation with distinguishing irradiated cells from unirradiated ones with microbeams of X-rays or heavy particles, many reports regarding the contribution of bystander response to classical cellular responses to radiation has been published now. In this Workshop, we must clarify the problems to establish a new global ‘concept of dose’ to compare easily certain phenomena after irradiation by broad beams with certain phenomena after irradiation by microbeams, and we must discuss strategy to solve these problems.
We would present several topics regarding bystander and adaptive responses induced after irradiation of heavy particles with different LET, in order to clarify the problems to establish a new global ‘concept of dose’ and to discuss strategy to solve these problems.

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We already reported our microbeam irradiation system using synchrotron monochromatic X-rays. Our system adopted a precise slit system to make X-ray microbeam, which enables us to change the beam size arbitrarily larger than 5 micron square. Using this advantage of our system, we have measured dose-survival relationships of V79 cell in two irradiation conditions with clonogenic assay method. One is to irradiate with 10-micron square beam aiming at nucleus only, the other with 50 micron square aiming at whole cell. This work revealed that hypersensitivity in low dose region is more enhanced in nucleus-irradiated cells than in whole-cell irradiated cells. We have observed also in bystander cells that bystander effect depends upon the energy-deposited area in the irradiated cells. These results suggest that intracellular communication between nucleus and cytoplasm plays an important role in determining the cell death in low dose region. For further investigation, we have developed a method to irradiate cytoplasm only without irradiating cell nucleus. In order to shield the nucleus in the uniform irradiation field, we made a gold mask, 15 micron in diameter and 20 micron thick, on a very thin (200 nm thick) SiN film. The thickness of the gold was determined to decrease the intensity of 5.35 keV X-rays to less than one-thousandth. It was mounted on a small X-Y stage and set in the system between the slit system and the sample stage. Using a scintillater dish, we adjusted the position of the mask and the size of the beam. Finally, we got a 50 micron by 50 micron beam with uniform intensity, at the center of which located was the gold mask of 15 micron diameter. When we want to irradiate only cytoplasm of the cells, center of the mask was recognized as the beam position and cytoplasm of cells, nuclei of which were stained with Hoechst dye and recognized by its fluorescence, were irradiated with X-rays, leaving nucleus unirradiated. When survival fraction of cytoplasm-irradiated cells was plotted against absorbed dose to cytoplasm (dose to nucleus is null), dose-survival relationship showed a single exponential behavior, which indicates that lethal damage in cytoplasm-irradiated cell may not be DNA damage. When we want to compare the lethal efficiencies of these three types of irradiation, we have to define a new metric or dose against which these survival fractions should be plotted.

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In the ICRU report 36 entitled "microdosimetry", the specific energy, z, is defined as the quotient of the energy imparted in microscopic site by its mass. The specific energy is a stochastic quantity, and its average value is equal to the macroscopic absorbed dose D. Introducing the concept of z, the difference between micro- and broad-beam experiments can be simply expressed by the difference of the probability densities of z in sub-cellular or cellular scales. As examples, cell survival fractions obtained using micro- and broad-beams were re-analyzed based on the probability densities of z in chromatin and cell-nucleus scales. The results of the analysis together with the discussion on the influence of the divergence of the probability density of z on the cell survival fractions will be presented at the meeting.

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Background: Late health effects of radiation exposure to the atomic bomb have been evaluated including the Life Span Study (LSS). Individual doses ranged widely from low- to high-dose level, which allows us to examine the effects at low-doses levels from many aspects.
Methods: Initial 120,321 people identified in 1950 were followed up until the end of 2003 using population registry system and death certificates. Among them, 86,611 subjects with known individual doses by the Dosimetry System 2002 (DS02) were analyzed for dose-response relationship for all solid cancer.
Results: Risk of all solid cancer was the best fit to the linear model in the whole dose range, but to the linear-quadratic model in the 0- to 2-Gy dose range. This finding suggested that the risk of radiation per unit exposure was lower at a low-dose level than at a high-dose level. However, this concave-upward curve was determined due to the relative lower estimates than the linear model at around 0.5 Gy level. The ERR/Gy was also higher, but not significantly, than the value that was estimated based on the whole dose range when the range was limited to 0.1 Gy or lower. The lowest range with significant excess relative risk of all solid cancer was around 0.2 Gy or larger.
Conclusions: Although no significant results were obtained at low-dose levels according to the RERF survivor cohort studies, including the latest study with the longest follow-up period, they are thought to support the linear no-threshold model. Analyses considering the confounding and interaction with radiation effects such as lifestyle, and medical radiation exposure are required to clarify the radiation effects at the low-dose levels.

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“Dose-rate effect” was known as a result of sub-lethal damage repair during chronic irradiation. However, there has not yet been well described the molecular mechanism of DNA double-strand break (DSB) repair under low dose-rate irradiation. In higher vertebrate cells, there are at least two major DSB repair pathways, namely non-homologous end-joining (NHEJ) and homologous recombination (HR). To elucidate the role of DSB repair mechanism in cellular dose-rate effect, the cellular sensitivity to low dose-rate radiation is investigating using DSB repair-defective cell lines. In chicken DT40 cells irradiated with γ-rays at a low dose-rate, NHEJ-related KU70-defective cells showed higher sensitivity than HR-related RAD54-defective cells and RAD54^-/-KU70^-/- cells. Next, we analyzed the causative cellular process of our obtained results at the Radiation Biology Center, Kyoto University. At 0.1 Gy/h, KU70^-/- cells showed significant G2 arrest in comparison with RAD54^-/- and RAD54^-/-KU70^-/- cells, following the induction of apoptosis.
We also analyzed the surviving fraction of human glioblastoma DNA-PKcs-defective cells after low-dose rate irradiation. The surviving fraction decreased gradually and leveled off after 3 weeks irradiation, suggesting cell turnover is one of the important factors under low dose-rate irradiation condition. When considering the effect of low dose-rate irradiation to the living tissue, the accumulation of DNA damage in tissue stem cells is one of the important issues. Nijnik et al. (Nature, 2007) reported that diminished DSB repair in the Lig4 mutated mouse strain causes a progressive loss of hematopoietic stem cells during ageing. The importance of DSB repair mechanisms in the tissues under low dose-rate irradiation condition will be discussed based on our obtained results.

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Radiation carcinogenesis is thought to be the results of a series of somatic mutations. Translesion synthesis is the major source of DNA damage-induced point mutations. Y family DNA polymerases which operate Translesion DNA synthesis TLS can bypass a various damage lesions with low fidelity. Rev1 has a regulatory role in TLS. However defect of Rev1 is sensitive to radiation, expression of Rev1 mutant, which is deleted its catalytic function in translesion synthesis, can recover this sensitivity. These demonstrate Rev1 has catalytic and non-catalytic function in radioresponse. However, little is known whether Rev1 protein has the precise role in radiation-induced tumorigenesis in vivo. We generated Rev1 transgenic mice that continuously express a Rev1transgene in various tissues. Both Rev1 Tg and wild-type (Wt) mice were subjected to gamma-irradiation of 1.6Gy four times at a weekly internal, starting at the age of 4 weeks. Our data demonstrates that overexpression of Rev1 may be associated with raditation-induced tumorigenesis.

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Previously, p53-dependent decrease of white blood cells in peripheral blood in mice by gamma ray irradiation at middle-dose-rate (MDR) [400 mGy/22h/day(18.2 mGy/h)] was reported. However, the effects of secreted molecules in serum from mice irradiated by low-dose-rate (LDR) or MDR gamma ray were not well investigated. In the present experiment, serum from mice irradiated by LDR or MDR gamma ray, were characterized. A cell-based-assay, which is a new method for measuring activity of substances in serum thorough gene expressions levels in mouse embryonic fibroblasts (MEFs), was performed. Expressing genes having two-fold higher gene-expressions levels in MDR irradiated mice than non-irradiated mice were analyzed for gene-expressions patterns. Moreover, the serum from LDR [(20 mGy/22h/day (0.91 mGy/h)] irradiated mice for 400 days were also used for the analysis. Numbers of identified genes in mice irradiated by MDR irradiation for 10 and 20 days with the total doses of 4 and 8 Gy, were 613 and 1,202, respectively. Caveolin signal pathway and insulin-receptor-signal pathway was found by pathway analysis in these genes. Interestingly, these genes-expressions patterns were quite similar to those of disease such as lipid metabolism or coronary artery disease. Moreover, the protein expression of Lipocalin2 in MEFs after adding of serum, which was reported to be a gene influenced by high-dose-rate gamma ray irradiation, was irreversibly decreased by MDR gamma ray irradiation for 8 Gy, while the change of Lipocalin2 in serum induced by LDR irradiation for 400 days for 8 Gy was not observed. Present results indicated the activity of secreted molecules in mouse serum was quantitatively and qualitatively changed by gamma ray irradiation at MDR, which might be influenced to metabolism. This study was performed under contract with the Aomori Prefectural Government, Japan.

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To understand health effects of low-dose and low-dose-rate radiation, many analyses on the changes at molecular levels have so far been conducted. The analyses of protein expression in connection with biological functions provided useful information for the evaluation of the health effects. We have reported changes of protein expression in the liver from mice irradiated at low-dose rates and for a long period. On the other hand, comparative studies between chronic and acute irradiation offer critical information for understanding of molecular mechanisms underlying a dose-rate effect. In the present work, changes in protein expression in the liver of mice irradiated with 4Gy (a sub-lethal dose) or 8Gy (a lethal dose) were investigated. In addition, the alteration of protein expression was investigated in mice irradiated with 8Gy delivered in a protracted period of 400 days. Since long-term exposure with a total dose of 8Gy is not a lethal dose for the mouse, the time-dependent change in protein expression after the end of irradiation was analyzed. Comparative analyses of the protein expression by the two-dimensional gel electrophoresis and the comprehensive analyses of changes in protein expression using antibody arrays were performed. We will discuss the responses of a living body to radiation and its defense systems with references to changes in protein expression based on the results of comparative analyses between sub-lethal and lethal doses as well as between acute and chronic irradiation.

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A large amount of tritium is required as the fuel source for the deuteron-tritium nuclear fusion reaction putting workers at a great risk for internal radiation exposure. As a result, one of the major issues arising in the development of the fusion reactor is the assessment of the biological effects of tritium released from nuclear fusion power plants during routine operation or accidents. In the present study, the mutagenic effect of tritiated water at low dose-rate was investigated using wold type and p53-deficient mice, then compared that of ¹³⁷Cs gamma rays. The p53 gene plays a key role in the cellular respomse to genotoxic stress. It is thought that p53 deficiency results in increased survival of cell with DNA damage, either by failure of DNA repair, or by failed deletion of mutation-bearing cells. The in vivo T-cell receptor (TCR) assay detects somatic mutation with high sensitivity.

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Genomic DNA is constantly damaged by a wide variety of endogenous and environmental agents. In addition to DNA strand breaks, radiations can induce diverse base lesions, especially through production of reactive oxygen species. Not only DNA strand break repair but also excision repair pathways must play important roles in suppression of radiation-induced carcinogenesis.
Nucleotide excision repair (NER) is a major DNA repair pathway, which can eliminate UV- and carcinogen-induced base lesions, as well as some oxidative lesions, such as 8,5'-cyclodeoxypurines. The xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition and initiation of NER throughout the genome. XPC functions as a versatile DNA damage sensor through interaction with oscillating unpaired bases, rather than damaged bases themselves. Thereafter, the basal transcription factor IIH (TFIIH) is recruited through interaction with XPC, and the presence of aberrant chemical modifications is finally checked through scanning of a DNA strand in 5'→3' direction by the XPD helicase.
We have reported that, even if XPC happens to bind to some helix-distorted sites devoid of damage, the NER machinery can search around and excise lesions at distal positions. This suggests the presence of a "patrolling" system, which may constantly survey the genome from XPC-anchored sites, including spontaneously opening sequences as well as endogenous DNA damage sites. Biological implications of such a mechanism will be discussed, also in light of the effects of low-dose radiations.

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Low levels of ionizing radiation and chemicals in the environment insult DNA continuously. Additionally metabolic intermediates also damage DNA. Although such DNA lesions usually inhibit DNA synthesis, cells can complete DNA replication within a normal S phase. Postreplication repair is a mechanism for damage tolerance; in which stalled primer ends are extended by specialized DNA polymerases beyond DNA lesions (translesion DNA synthesis, TLS) or by annealing with daughter strand as a template (template switching, TS). So far we have established in vitro reconstitution systems to analyze molecular mechanisms of postreplication repair. Here we report novel biochemical reactions underlying regulation of postreplication repair.

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Reactive oxygen species attack DNA and its precursor nucleotides, and consequently bases with a various modification are introduced in DNA within normally growing cells. One of such modified bases, 8-oxo-7, 8-dihydroguanine (8-oxoG) is highly mutagenic. Three enzymes, MTH1, OGG1, and MUTYH, play important roles in avoiding the 8-oxoG-related mutagenesis in mammalian cells. We have established an experimental system for oxidative DNA damage-induced mutagenesis and tumorigenesis in the gastrointestinal tracts of mice. Oral administration of KBrO₃ effectively induced G:C to T:A transversions and epithelial tumors in the small intestines of Mutyh-deficient mice, indicating the significance of Mutyh in the suppression of mutagenesis and tumorigenesis induced by KBrO₃. To elucidate the roles of other DNA repair genes in the tumor-suppression, we performed KBrO₃-induced tumorigenesis experiments using Ogg1-, Mth1-, Msh2- and Xpa-deficient mice. We observed an enhanced tumor-formation in the small intestines of Msh2-deficient, but not Xpa-deficient mice. Interestingly, the number of tumors marginally increased in either Ogg1- or Mth1-deficient mice, in contrast to Mutyh-deficient mice. Based on these findings, we will discuss the roles of these DNA repair-related factors in the suppression of oxidative DNA damage-induced mutagenesis and tumorigenesis in the gastrointestinal tracts.

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