National Radiation Emergency Medical Center(NREMC) was established in 2001, and authorized as the national medical center for radiation emergency in Republic of Korea in 2003. To establish the national network for radiation emergency medical preparedness, NREMC has designated 25 medical centers nationwide and offers training courses.The lecture will include :(1) Brief History (2) Functions- Medical Services- Radiation Emergency Preparedness- Education and Training- Research(3) Cases of Medical Response and Clinical Management to Radiation and Nuclear Accidents in Korea(4) International Cooperation - Japan - Russia - France

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In June 1980, the Nuclear Safety Commission came up with a guideline entitled "Off-site Emergency Planning and Preparedness for Nuclear Power Plants." This guideline nominated NIRS as a tertiary radiation emergency hospital that serves as the final stage hospital for receiving heavily exposed or contaminated victims due to nuclear or radiological accidents. Required aims as a tertiary emergency medicine hospital are as follows: (1) To accept exposed victims who require further expert diagnosis and treatment, (2) to dispatch a radiation emergency medical team to the local emergency site, (3) to facilitate exchange of information, research activities, and human resources, by constructing networks in cooperation with outside expert organizations, (4) to maintain and reinforce an efficient radiation emergency medicine system under normal conditions, and (5) to promote technical development and research on radiation emergency medicine. Strengthening its system to prepare for radiation emergencies three nation-wide network councils have been established, for medicine, chromosome analysis as bio-dosimetry, and physical dosimetry. The Radiation Emergency Medicine Network Council is a group of experts and medical organizations from which NIRS asks for their help at the time of a nuclear disaster or a radiological accident. The Chromosome Network Council and the Physical Dosimetry Network Council are nation-wide cooperation for the dose assessment. In addition to those network councils, educational training courses have been regularly conducted to medical professionals and disaster prevention personnel for the development of personnel for nuclear and radiological emergencies.

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WHO REMPAN (World Health Organization Radiation Emergency Medical Preparedness and Assistance Network) was established in 1988 as one of the consequences of Chernobyl accident occurred in April of 1986.
The main objectives of WHO REMPAN are 1) To promote the preparedness for radiation accidents among WHO member states; 2) To provide advice and assistance in the case of a radiation accident and radiological emergency; and 3) To assist in follow-up studies and rehabilitation.
As of March of 2004, there are 14 collaborating centers (CC) and 13 liaison institutions in the world and among which, Radiation Effects Research Foundation (RERF) is participating as CC named "WHO collaborating center for Radiation Effects Research on Humans at RERF ". This is one of the earliest CCs joined to this network and the main role is to help WHO REMPAN to achieve its 3^rd objectives.
WHO REMPAN has held biennial general meeting 10 times in the past and RERF participated in the 2^nd and subsequent meetings. RERF also held the 5^th meeting in Hiroshima in 1995. In addition to attending WHO REMAN meetings, RERF has been providing information on health effects of Atomic-bomb radiation based on long-term epidemiological follow-up studies of Atomic-bomb survivors.
RERF is now one of the members of the local network of radiation emergency medical preparedness which was established under the leadership of Hiroshima university. RERF should keep providing useful information to assist the operation of this local network as well.

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On the basis of the teachings of the critical radiation accident in Tokai village, the Nuclear Safety Commission of Japan completed a report of recommendations for our nation's medical system for radiation emergencies. The main point of this report is to prepare a radiation emergency medicine system that works according to the degree of each patient's injury, such as early stage (slight injury), secondary stage or tertiary stage (serious injury), in order to create a network in which effective medical treatment can be done with coordinated efforts during an emergency. The recommendation which was regarded as the most important in the plan was to prepare tertiary radiation emergency hospitals which have the ability to provide advanced medical care. Based on this report, the Ministry of Education, Culture, Sports, Science and Technology designated Hiroshima University and the National Institute of Radiological Sciences as a regional tertiary radiation emergency hospital for Western and Eastern Japan, respectively in 2004. Hiroshima University has the achievements of long years of atomic bomb medicine and a good staff of specialists in various fields of medical treatment of radiation-induced disorders. In addition, Hiroshima University's program titled "The Radiation Casualty Medical Research Center" was recently chosen as the 21st Century COE Program by the governments. The research and treatment of radiation-induced disorders has become a modern academic field. To promote such science and nurture younger doctors are two essential characteristics and role of Hiroshima University. From now on, we intend to play the role of a tertiary radiation emergency hospital and to contribute to society in this regard. We have established a Radiation Emergency Medicine Promotion Center and are currently proceeding with the preparation necessary for the development of our project.

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Although epidemiological studies have been the major source for the estimation of health risk from ionizing radiation, for low dose range, precise quantification is difficult based solely on epidemiology. Therefore, the epidemiological data should be supplemented with the knowledge of the biological mechanisms underlying the health effects of radiation. In Asia we have unique epidemiological data including that on those people living in high natural background radiation area in China and that on those living in apartments built with ⁶⁰Co-contaminated steel in Taiwan. In the high natural background area some biological study has been initiated to investigate radiation adaptive responses. On the other hand, in Korea and Japan animal experiments using low-dose rate irradiation facilities have been carried out. In this symposium, most recent information on these topics will be reviewed. And we would like to discuss our future perspective on the integration of biology and epidemiology will be discussed.

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The health effects of radiation from atomic explosion in Hiroshima and Nagasaki, Japan, is completely different to radiation from the Co-60 contaminated apartments in Taiwan. The acute radiation suddenly exposed to the Japanese in extremely high dose could kill them and the harmful health effects in lower doses could increases the cancer mortality, especially the leukemia of the survivors based on the LNT model. The residents received chronic radiation unknowingly in the Co-60 Contaminated apartments in Taiwan with doses even higher than the Japanese Survivors, yet no excess cancer deaths were observed based on LNT model, on contrary their spontaneous cancer deaths were reduced sharply to about only 2.5% of the general population, and hereditary defects to their offspring only 5-7% of the population. Therefore the residents in the Co-60 contamination apartments had coincidently accomplished an experiment on the health effects to human beings. The chronic radiation from the Co-60 contaminated houses is quite similar to the radiation exposure to the workers and public in the peaceful of the nuclear energy and medical radiation, and is also similar to the radiation in high natural background and radon concentration. Acute radiation from a nuclear accident could harm limited number of people, but chronic radiation might benefit tremendous people. People should fear no more of the chronic radiation and the traditional radiation protection policy and practices used in past 60 years should be earnestly revised based on the health effects observed in Taiwan.

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A cohort population of 7,271 people, who received low dose-rate γ-irradiation for about 10 years as a result of occupying buildings containing ⁶⁰Co-contaminated steel and with 103,225 person-years of follow-up, was assessed for their cancer risks via standardized incidence ratios (SIRs), as compared with those populations with the same temporal and geographic characteristics in Taiwan. The association of risks with excess cumulative exposure, 47.7 mSv in average, was further evaluated for hazard ratios (HR) by the Cox regression analyses.
The risks in SIRs were significantly higher for all leukemia combined and acute lymphocytic leukemia in men, and marginally significant for thyroid cancers in women. All cancers combined, all solid cancers, and a combination of leukemia and thyroid/breast cancers were further shown with exposure-dependent increased HRs in those received initial exposure before 30 years old.
Via the Cox proportional hazard models, significant higher Excess Relative Risks (ERR_1Sv) were observed in all cancers combined, breast cancers, and leukemia (except chronic lymphocytic leukemia), while marginally significant higher ERR_1Sv were shown in solid cancers (except thyroid cancers), all solid cancers combined, stomach cancers, and lung cancers.
Prolonged low dose-rate radiation exposure appeared to increase the risks of developing certain cancers in this population in Taiwan.

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The data from epidemiological studies are still insufficient to define implications of low-dose and low-dose-rate radiation for human health and especially for risk assessment for radiation field workers. In these studies, we have analyzed adaptive response in terms of the frequency of micronuclei in polychromatic erythrocytes (MN-PCE) and cranial abnormalities of mice to evaluate the modulation by split conditioning low-dose to challenging high-dose irradiated mice. Firstly, six-week-old ICR mice were exposed to 0.01 and 0.1Gy/day and irradiated with a challenging dose (2Gy) at 11 days. The split conditioning doses of 0.1Gy did not influence induction of an adaptive response. However, when a conditioning dose was given, then split with 0.01Gy, the subjects were able to modulate the response of the challenging dose of 2Gy. These results clearly showed the occurrence of adaptive response in hematopoietic microenvironment, the conditioning given in small fractions continuously seemed to be more effective. Secondly, pregnant ICR mice from 1.5 to 10.5 days post-coitus were exposed to 0.01 and 0.1Gy/day and irradiated with a challenging dose (2Gy) at 11 days. The animals were sacrificed on 4 months after delivered. These results showed that the occurrence of adaptive response in cranial abnormalities, the conditioning exposed in small fractions throughout the periods of pre-implantation seemed to be not effective. In this study, we excluded the effects of very low-dose-rate because further study is underway under Low Dose Irradiation Facility of RHRI, Korea.

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A cohort study on nuclear industry workers in Japan was initiated in 1990 to determine the health effects of low dose/ low dose-rate radiation with gamma- and X-rays. Among 176,000 male workers constituting the cohort of the second analysis with the observation period from 1991 to 1997, approximately 120,000 workers with prospective follow-up (average of 4.5 years) were subjected to statistical analyses. The standardized mortality ratio (and its 95% confidence interval) was 0.94 (0.90, 0.97) for 2,934 cases of all causes, 0.86 (0.82, 0.91) for 1,305 cases of non-neoplastic diseases and 0.98 (0.93, 1.04) for 1,191 cases of all cancers (External comparisons). In tests for trend of death rate with increasing radiation dose, no significant correlation was found for all cancers combined when the latest place of residence of workers as a potential confounder was adjusted for (Internal comparisons). For site-specific cancers, most cancers including leukemia showed no positive correlation with accumulated dose, except for cancers of the esophagus and some digestive tracts. A separate questionnaire study indicated that these positive results could be ascribed in part to lifestyle characteristics of workers including smoking. With the limited statistical power and the possible involvement of confounding factors, this cohort study has not yielded any definite evidence as to whether low-level radiation elevates cancer mortality.

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The Korean Electric Power Corporation (KEPCO) has operated the nuclear power plants since 1977. Among employees of KEPCO in 4 facilities - Kori, Ulchin, Wolsong, and Youngwang, the cohort was defined as the workers being employed on or from 1-1-1992, and worked for at least one year with complete monitoring the radiation exposure on duty. Personnel records of KEPCO provides information on the data of dose equivalent from external and internal exposure, as well as the information of beginning and end of employment, code of facility and main job title for each employment period. For obtaining some confounders – smoking habits, education levels etc., twice surveys were conducted through a self-administered structured questionnaire for all current workers in 1992 and 1999, respectively. For ascertaining vital status and cause of death, five databases – the National Death Certificate DB, the National Federacy of Medical Insurance DB, the Korean Central Cancer Registry, and 2 population-based regional cancer registries (Seoul, Busan). A total follow-up person-year was 133,304 in 19,912 subjects for 11 years(1992-2002). While 21.4% of subjects showed non-exposure of radiation, the median cumulative dose of exposure was 5.95 mSv. A total of 99 cancer incidents were ascertained so that the age-adjusted cancer incidence rate (ASR) was 60.18 per 100,000 person-year (95% CI: 36.39-84.07). The results in detail will be presented.

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In China, the first commercial nuclear power plant began opretion in 1991. As of July 2005, there are several thousands radiation workers working at nine nuclear power plants in three nuclear facilitiers. By 2020, about twenty more nuclear power plants are to be constructed. All the nuclear workers are monitored for radiation exposure at nuclear facilities and thier individual doses are recoreded. Mean annual dose received by nuclear workers is reported to be 1.76 mSv/a. Health surveillance of those workers are also under way. National Institute of Radiological Protection, Chinese CDC is now planning a nationwide cohort study of nuclear workers.

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Available data on cancer mortality in the employee populations of the five units of the Department of Atomic Energy (DAE) were analysed. The population groups consisted of employees from research reactors, power reactors, nuclear fuel fabrication facility and thorium processing industry. The group includes all the employees of these facilities, i.e. both radiation workers and others not directly associated with radiation work. Data available only in the serving employees, i.e. subjects in the age group 20-59 yrs and not on the retired employees are analysed. A total of 81 cancer deaths recorded in a database of 315519 Person-Years were analysed. Among these, 28 deaths were recorded among monitored radiation workers having a database 112363 Person-Years. Using background age-specific cancer death rates of the urban Indian populations as a tentative national baseline, the estimated Standardised Mortality Ratio (SMR) for combined DAE employees works out to be 111 (95% CI: 80-138). The SMR for radiation worker alone is 108 ((95% CI:72-156) as compared with the SMR of 113 (95% CI:84-149) for non-radiation workers. The relative risk of Radiation workers as compared to Non-radiation workers is 0.96(95% 0.64-1.39). The study shows that the radiation workers are not at any extra risk of cancer as compared to non-radiation workers. Also, when considered as a whole, comparison with the national baseline data does not indicate any extra cancer risk for atomic energy employees of India.

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Risk of cancer after low doses of ionising radiation: retrospective cohort study in 15 countriesE CardisIARC conducted pooled analysis of the cancer mortality data obtained from cohort studies nuclear workers in 15 countries in order to provide direct estimates of risk of cancer after protracted low doses of ionising radiation and to strengthen the scientific basis of radiation protection standards for environmental, occupational, and medical diagnostic exposures. The major finding of this study will be presented, and the significance of nuclear worker studies in Asia in future will be discussed.

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Evidence is accumulating that irradiated cells produce some signals which interact with non-exposed cells in the same population. Understanding this "Bystander effect" from ionizing radiation is important for understanding low dose effects on a cell population. In our COE program, we have been continuing a collaboration with the Gray Cancer Institute. The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander effects, because it permits the irradiation of only a single cell nucleus within a population. The results suggest that bystander signals eventually induce DNA double strand breaks in non-exposed cells. Also, radical species induced intracellularly by ionizing radiation are involved in producing bystander signals in exposed cells. In these experiments there was no cell-to-cell contact between exposed and non-exposed cells. Therefore, it is possible that bystander factors such as cytokines from exposed cells are secreted and this secretion is influenced by signal activation related to radical species produced outside of the cell nucleus of the targeted cells. As an advanced new ultrasoft-X-ray microbeam has been introduced from Gray Cancer Institute to Nagasaki University, this facility will be a powerful tool for radiation biology in Asia.

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Cellular exposure to DNA damaging agents like ionizing radiation can result in direct (targeted) damage to the genetic material and a number of non-targeted indirect effects that can manifest in the progeny of a damaged cell. Both targeted and non-targeted effects can result in DNA mutations, gene amplifications, chromosomal rearrangements, carcinogenesis, and even cell death. The paradigm for understanding how induced damage results in these cellular endpoints dictates that cellular responses to the induced damage, e.g., DNA repair, and cell cycle arrest fix the damage and thereby seal the fate of the irradiated cell. However, non-targeted effects cannot be accounted for by this paradigm. This presentation will focus on delayed genetic effects occurring in the progeny of cells after exposure to ionizing radiation. We will describe how the cellular micro-environment can perpetuate instability in clonally expanded populations of cells surviving irradiation. The emphasis will be on persistently elevated levels of reactive oxygen species that are found in many of the cell clones showing non-targeted effects of radiation exposure. These results will be discussed in terms of non-targeted bystander like effects where by cells that themselves were not irradiated exhibit many of the same detrimental effects as irradiated cells. In addition, other non-targeted effects associated with radiation exposure including clastogenic factors, the death inducing effect, hereditary effects, and abscopal effects of radiation and how these might impact on human disease will be discussed.

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Ionizing radiation produces various types of DNA damage, which have a potential to lead to specific gene mutations that initiate carcinogenesis. Chromosomal rearrangements are common after radiation exposure and most likely result from mis-rejoining of two radiogenic double-strand DNA breaks. Recent studies have shown that chromosomal rearrangements, such as those involving the RET and BRAF genes, are a dominant mechanism of carcinogenesis in radiation-induced thyroid cancer in humans, whereas point mutations of BRAF and RAS more frequently activate the same MAPK signaling pathway in sporadic tumors. RET/PTC rearrangements result from a paracentric inversion of chromosome 10q and are highly prevalent in radiation-associated thyroid tumors in human populations and can be induced by cell irradiation in vitro. Over the last decade, it has become evident that spatial proximity between potential recombinogenic partners is an important prerequisite for the generation of chromosomal exchanges. Interphase proximity of RET/PTC partners has been identified in normal human thyroid cells. This non-random positioning is most likely due to the large-scale chromosome folding in this region, which brings the recombinogenic-prone genes closer to each other in the interface nuclei of normal cells. Spatial proximity may facilitate the rearrangement by placing two free DNA ends close in space and time within the nuclear volume.

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Cellular mechanism for IR-induced perturbation of breast tissue microenvironmentZhi-Min Yuan, Harvard University School of Public Health, Boston, MA 02115Much of the research concerning IR-induced carcinogenesis has focused on its DNA damaging effects within the target cells. How IR affects the interactions of cells with their immediate surroundings and its role in IR-induced carcinogenesis are, however, less well understood. We show here that low dose IR can substantially perturb the tissue microenvironment by inducing premature senescence in human mammary fibroblasts (HMF), which is associated with marked morphological changes and upregulation of matrix metalloproteinases (MMPs) in these cells. By use of a 3-dimentional (3D) coculture system to model the critical interactions of different mammary cell types with their neighbors and with their environment, we have demonstrated that diminished pseudopods of senescent fibroblasts result in failure of mammary epithelial cells (MEC) to undergo glandular morphogenesis. MMP-mediated pericellular proteolysis loosens the growth constraint imposed by extracellular matrix (ECM), which enables breast cancer cells to fully express their malignant potential. .Our findings together support a model in which IR-induced senescence in stromal fibroblasts results in the perturbation of the tissue microenvironment, which leads to the dysregulation of cell-cell and cell-ECM interactions and promotion of tumor progression.

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Increase of leukemia among atomic bomb survivors in Hiroshima and Nagasaki started from 1947, reached its peak around 1952, and continued until 1980s. By contrast, increase of myelodysplastic syndrome (MDS) continued even 60 years after the bombing. Many researchers believe that early onset leukemia after atomic bomb were caused primarily by chimeric genes as a result of chromosomal translocations, although there is no way to confirm it. Point mutations likely contribute to late onset MDS, while chromosomal deletions are implicated in both of them.
In this lecture, I show two topics. One is AML1 point mutations in MDS. We found that AML1 mutations in approximately 20% of 'preleukemic' MDS and/or AML precedent with MDS. This gene anomaly is found especially at high frequencies (40-50%) in MDS patients among atomic bomb survivors in Hiroshima and in radiation-associated MDS/AML patients. Moreover, mutations in radiation-associated MDS were limited to the runt-homology domain, which mediates DNA binding, while those in sporadic MDS patients distributed throughout AML1 protein. Second, we isolated candidate genes for chromosomal deletion in the long arm of chromosome 7, which is frequently observed in MDS and AML, especially in radiation-associated MDS/AML. We developed a unique microarray CGH system to detect micro-deletions and isolated a region which is commonly deleted in the MDS/AML patients at a high frequency. These candidate genes likely encode novel DNA-repair proteins and we are now concentrated to elucidate their function and implications in leukemogenesis.

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We have generated cell lines and mice with deletions in the Rev1 and Rev3 translesion synthesis (TLS) polymerases. Cell lines with a deletion of the Brct domain of Rev1 are marginally hypersensitivity to genotoxic agents. UV light-induced mutagenesis was reduced in these cells, most significantly at TT dimers. UV induced many chromatid aberrations, but no increase in sister chromatid exchanges, in a subset of cells only. Remarkably, mice with the Rev1 Brct deletion display accelerated UV-induced tumorigenesis. Complete disruption of Rev1 confers sensitivity to all genotoxic agents tested, indicating a role for the protein in TLS beyond its dCTP transferase activity. Rev1-deficient mice are born in a sub-Mendelian ratio, are small, nearly infertile and are hypersensitive to UV. Rev3 deficiency results in embryonic lethality at mid-gestation accompanied with chromatid and chromosome aberrations. This phenotype could partially be rescued by introducing deficiency for DNA mismatch repair. Rev3-deficient primary fibroblasts display an S phase arrest and premature senescence; immortalized Rev3-deficient fibroblasts are extremely sensitive to all genotoxic agents tested. Together these data are consistent with a role of Rev1 and Rev3 in the mutagenic replication of endogenous and exogenous DNA damage, averting DNA damage-induced cell cycle arrests and cytogenetic aberrations. In addition, DNA mismatch repair appears an important player in eliciting these responses induced by endogenous DNA damage.

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The microbeam is expected internationally as a tool to advance the radiobiology research. There are two facilities prevent on the radiobiological studies outside Japan, i.e. proton and alpha-particle irradiation systems in Columbia University in USA, and alpha-particle and fluorescent X-ray systems in Gray Cancer Institute in UK. Some new overseas facilities in USA, Germany, France, Italy, and China are following them. In Japan, TIARA in JAERI Takasaki and PF in KEK has started some biological experiments, and SPICE in NIRS, Nagasaki University, Kyoto University, Wakasa-Wan Energy Research Center is now coming. We have different microbeam facilities having different quality of radiation, and it must be an advantage in Japan. We may possible to study new radiobiological researches with new biophysical points of view using those facilities by a combination of the different radiation qualities of the beams in Japan. In addition, to help and advance the studies using microbeams, the Research Association of Microbeam Biology in Japan also has established. Here, we summarize previous studies using microbeams in the world, and introduce all microbeam facilities in Japan together with in the other countries.

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Heavy-ion microbeam system of JAERI-Takasaki was designed for rapid, automatic irradiation with heavy-ion of the specific region of the bio-organisms. Using the system, we established the experimental system for target irradiation of individual cells, and found that heavy-ion hit on Chinese hamster ovary cell resulted in growth inhibition not only in ion-hit cells but also in non-hit bystander cells. For more detailed analysis of the bystander effect, we have upgraded the system. We installed two new off-line microscopes this year, which are used for generating positional database of target and observing radiation effect continuously after targeted irradiation. Installation of them improved the throughput of cell irradiation and observation of radiation effect at post-irradiation process. One microscope is based on inverted microscope with cooled CCD camera, with which we can continuously observe the change of localization of DNA repair proteins without killing the cells using GFP-fused recombinant protein. The other new off-line microscope is based on stereoscopic microscope, with which we can generate the positional database with certain height that makes it difficult to observe them by inverted microscope. Using this off-line microscope, irradiation of a sample with tallness like shoot of the plant and cultured organ sample can be achieved with high accuracy.

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Progress Report of the SPICE (Single Particle Irradiation system for CEll) in NIRSHitoshi Imaseki,National Institute of Radiological SciencesThe ion-microbeam technique adapted to cellular irradiation becomes currently interested in the field of radiation biological effects research. By using this method, it is prospected that important quantitive analysis in the field of radiobiological can be done.From 2000, we started to specify the Single Particle Irradiation system for CEll (SPICE), whose optical design bases the achievement of scanning microbeam PIXE system in NIRS, and installation was finished in 2003. From that time, we have been developing the system.The feature of this system is that it uses the triplet quadrupole lens for beam focusing and voice coil motor for the movement of the target stage, uses Proton (3.4MeV) and Alpha (5.1MeV), and is upward atmospheric radiation.The end goal of is that we put the both of the beam size and the targeting accuracy within 2um, and the irradiation speed above 2000 cells/hour.

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Synchrotron X-ray has a remarkable advantage in producing an X-ray microbeam due to its high directivity. We have developed an X-ray microbeam irradiation system, in which we can aim at cell nuclei with any rectangular microbeams larger than 5 micron square are available. We have already demonstrated its performance high enough to be able to obtain dose survival curves of mammalian cells. And this system is open to all the users of the Photon Factory, KEK. Characteristics of the system and perspective of the radiobiological work using it will be presented.

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The multi-purpose accelerator system of the Wakasa Wan Energy Research Center (W-MAST) is composed of two serially connected accelerators (a tandem accelerator and a synchrotron) and 7 beam lines.Our facility has two microbeam lines both connected to the tandem accelerator. One is magnetically focused microbeam, and the other is glass capillary-collimated microbeam.Magnetically focused microbeam line can focus proton beam to less than 1µm in diameter. It is mainly used for micro PIXE (particle induced X-ray emission) analysis.Collimated microbeam line is developed for biological use. It is also used for PIXE analysis in the air. A proton beam, being accelerated to 10 MeV by a tandem accelerator and vertically transported to the apparatus, is collimated by a glass capillary with an inner diameter of 10µm. The system is equipped with an irradiation stage with an optical microscope placed beneath the stage. Target cells are confirmed by the microscope and the location of the cells are recorded, then defined number of proton is automatically irradiated to the cells. The number and the energy of proton are detected by a silicon surface detector (SSD) put in the objective lens revolver. About 70% of irradiated proton is located within 20µm in diameter confined with CR39 track detector. This system is available to examine effects of proton beam on the individual cell attached to the culture matrix, however it is insufficient to study the effects of the beam on the cell organelles. We are improving the beam line for the irradiation to more restricted area.

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The charged-particle microbeam, which allows irradiation of cells individually with micron precision and with precise number of charge particles, provides a unique opportunity to address the effect of low levels of ionizing radiation. Heavy ions are particularly effective and different from low-LET radiation for charged particle therapy. Then the heavy-ion microbeam program for irradiation of cells is started. The ion beam was accelerated by an 8 MV tandem Van de Graaff accelerator. Using a pair of apertures with 5 &mum hole and 6 &mum hole as the final collimator, carbon microbeam of 42 MeV was extracted to the atmosphere with low background particles. The range of the beam is about 80 &mum in water and long enough. An 10 &mum transmission scintillator and a photomultiplier tube detected the extracted particles with good efficiency. New vertical beam line is installed in order to irradiate the biological targets supported horizontally.

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The use of microbeams for partial-cell irradiation, the selective exposure of microscopic fractions of living cells to ionizing radiation or UV light, has a long history. The early work used a microbeam as a tool for microsurgery to analyze their normal function of various cell parts. Later, as interest in the effects of ionizing radiation itself developed, these techniques were used as a radiobiological tool which yields information about the mechanisms of cellular radiation responses by determining which part of a cell was most sensitive to radiation damage. The earliest particle microbeam experiments were performed in 1953 using a 2 MV Van de Graaff accelerator and micro collimators to form a proton microbeam. In the next decade, an 11 MeV/amu proton and 22 MeV/amu deuteron microbeam was developed using a cyclotron. These earlier microbeam systems were helpful in studying radiation effects in living systems, especially to show that damage to the cytoplasm had a very limited effect on the survival of the cell. However, these systems were limited at relatively high doses. To investigate the effects at lower doses, it is necessary to establish single particle irradiation technique. After in 1990s, single-particle microbeams were developed in several facilities for targeting cells individually with a specific numbers of protons or heavy ions to elucidate the radiobiological processes in hit cells and bystander cells, in ways that cannot be achieved using conventional broad-field exposures.

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Heavy-ion micro-beams are extremely useful radio-surgical tools for studying bio-function and differentiation of various cells and tissues in insects. A spot irradiation to the cellular blastoderm stage eggs of the silkworm, Bombyx mori, brought about various abnormal embryos including deletion, duplication, and fusion. The location of the defects within the cuticle pattern induced on the resultant embryos correlated closely to the site of irradiation. Based on this correlation, a fate map of the Bombyx mori egg at the cellular blastoderm stage was established. Position-related cell fate and plasticity of the cell differentiation process were revealed with local irradiation of heavy ions. Locally targeted irradiation of heavy-ion beams of silkworm larva had no deleterious effects on survival, but induces marked functional disruption of the irradiated tissues or organs. For example, in instances where larval epidermis was locally irradiated with heavy-ions, scale development did not occur to various degrees in the irradiated area of the adult moths depending on the dose of irradiation. One of body-shape mutants of Bombyx mori, Knobbed, is characterized by the appearance of dermal protuberances (knobs) on the crescent and star-spot larval markings. To clarify the mechanism of Knob formation, we investigated the effects of heavy-ions on the corresponding epidermal cells of newly hatched larvae and found that the knob protuberances disappeared after the local irradiation.

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A classical paradigm of radiation biology asserts that all radiation effects on cells, tissues and organisms are due to the direct action of radiation. However, there has been a recent growth of interest in the indirect actions of radiation including the radiation-induced adaptive response, the bystander effect, low-dose hypersensitivity, and genomic instability, which are specific modes of stress exhibited in response to low-dose/low-dose rate radiation. Having become one of the triggers of this concern is development of the microbeam irradiation device that enables aim to be irradiated and advancement as for one cell. The research for the radiation-induced bystander effects using the microbeam irradiation device is actively done in Japan, USA and UK, and then many findings about the radiation-induced bystander effects have been accumulated.
Here, I would comprise recent research for radiation-induced bystander effects and the adaptive response, and discuss the contribution of the former to the latter.

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The observation of radiation-induced bystander responses, where cells respond to their neighbors being irradiated, indicates that the cancer risk of a low dose radiation may be greater than the predicted and it also indicates an opportunity to modulate the efficacy of radiotherapy. Growing evidence indicates that gap junctional intercellular communication (GJIC) plays an important role in the bystander responses. By irradiating a fraction of cells within a primary human fibroblasts population with either low doses of broad heavy ion beams or high-LET heavy ion microbeams, we found that the yields of radiation induced micronucleus (MN) and G1-phase arrest increased when the GJIC was enhanced by 8-Br-cAMP, but they were diminished when the cells were treated with DMSO and they were even eliminated when this GJIC was inhibited by PMA or lindane. Accordingly, both ROS and GJIC contribute to the bystander response in the confluent fibroblasts but the GJIC might play an essential role. With respect to tumor cells, however, the enhanced GJIC reduced radiation-induced cell responses of MN induction and G2-phase arrest, indicating that GJIC does not contribute to the bystander cellular damage. In fact, we found that some radiation-induced soluble factors including nitric oxide and their downstream products such as TGF-beta 1 were the bystander signaling molecules which could induce chromosome damage not only in the non-targeted tumor cells but also in their neighboring fibroblasts.

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Evidence is accumulating that irradiated cells produce some signals which interact with non-exposed cells in the same population. Understanding this Bystander effect from ionizing radiation is important for understanding radiation biology. Since 1990s, particle microbeam facilities have been available for biological study in Columbia University and Gray Cancer Institute. As it is possible to irradiate a specific number of cells on a cell population and a specific area of a cell in these facilities, it has been a powerful tool for bystander study. In this presentation, I would like to talk about some phenomena and mechanisms regarding bystander responses that have been elucidated by using these microbeam facilities.

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There are several methods of Retrospective Dosimetry (RD), which is being applied in order to support radiation epidemiological studies among population in the vicinity of Semipalatinsk Nuclear Test Site (SNTS). These methods are the following: Modeling Calculations (MC), which are being used with different radioecological models, based on archive data and on the results of contemporary measurements of radioactive contamination of the soils and foods; Luminescence Retrospective Dosimetry (LRD) with quartz inclusions in the bricks of the buildings; EPR Dosimetry with human tooth enamel; Biological Dosimetry (BD) methods (chromosome aberrations etc). Practically all of these methods were recently presented and discussed on 3^rd Dosimetry Workshop on the Semipalatinsk Nuclear Test Site Area (10^th Hiroshima International Symposium 9-11 of March 2005 RIRBM, Hiroshima University, Hiroshima, Japan). An overview of recent results of dose estimations by different Retrospective Dosimetry methods (Calculations and physical methods – LRD and EPR) is presented below. In order to provide the possibility to compare the dose estimations by different methods, the only results of dose reconstruction, which are related to the populations of the same village (the village of Dolon, Kazakhstan – the most radiation affected settlement), were considered.

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At International Symposium in Semipalatinsk (2002) a lot of differences were revealed in dose estimates, depending on the applied methods of retrospective dosimetry, for the areas affected by Semipalatinsk nuclear test site (SNTS) activity. It was suggested to perform international intercomparison of Luminescence Retrospective Dosimetry (LRD) method using brick samples from Dolon' village (Semipalatinsk region, Kazakhstan) affected by nuclear test of 29 August 1949. During field mission (autumn 2002), the team of specialists from Kazakhstan and Japan selected three buildings in the south-eastern part of Dolon' village as sampling locations. Four whole bricks were collected for international intercomparison of LDR method. Brick slices were distributed for measurements between laboratories in Finland, Germany, Japan, Russia, UK and USA. Data on sampling, locations, geographical coordinates, mean Cs-137 soil contamination density near sampling locations, mode of cut of samples for distribution, labeling, condition of transportation, order of samples distribution, as well as main results of the intercomparison were presented at 3rd Dosimetry Workshop on SNTS Area. There was good agreement in estimations of cumulative external absorbed dose by six laboratories. At the depth of 10 mm from external surface of the wall the overall mean value was 210±70 mGy. For given sampling conditions, the free-in-air dose in Dolon would be 420±140 mGy, which is in a good agreement with recent results (Imanaka et al., Simon et al., Stepanenko et al.) of external dose calculations.

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The method of electron paramagnetic resonance (EPR) spectroscopy of tooth enamel has been used for individual radiation doses determination at residents of settlements Dolon, Mostik and Bodene in vicinity of the Semipalatinsk Nuclear Test Site in Kazakhstan. These settlements are placed near the central axe of radioactive fallout trace of the most contaminating surface nuclear explosion, which was conducted in 1949. Samples were extracted for medical reasons during ordinary dental treatment. According to information obtained by questioning, no teeth donors were subjected to x-ray procedures of jaw. Only molar teeth were used for dose determination, therefore effects of solar ultraviolet on dosimetric EPR signal in enamel can be excluded. It was found that the excess doses obtained after subtraction of the contribution of natural background radiation are ranged up to about 450 mGy for residents of Dolon, whose tooth enamel was formed before 1949, and do not exceed 100 mGy for younger residents. For residents of Mostik, excess doses do not exceed 100 mGy for all ages excepting one resident, for whom extremely high dose of 1.25 Gy is registered. For Bodene settlement, doses are higher than 100 mGy were obtained for two samples from the residents having enamel formed before 1949. In general, the obtained results are in agreement with the pattern of radioactive contamination of territory after the nuclear test of 1949.

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Since 1994, environmental radioactive contamination levels in the vicinity of Semipalatinsk nuclear test site (SNTS) have been studied by our field mission. The village of Dolon is located about 100 km north-east of the hypocenter and is well known for receiving heavy exposure from radioactive fallout due to the 1st nuclear detonation on 29 August 1949. As one of the available methods external dose other than TL and/or ESR, there is a model calculation based on the long-lived radionuclide 137Cs (T1/2 = 30.17 y) in soil. Some investigators have reported the contamination levels of 90Sr, 137Cs and 239,240Pu in the area of Dolon settlement. But, the number of samples monitored was too scant to estimate the radiation dose around there. Furthermore, the assessment of plutonium isotopes (239,240Pu and 238Pu) is also important from the aspect of internal radiation. Such situation encouraged us to investigate in more detail the present situation of radioactive contamination of 137Cs and Pu isotope (239,240Pu and 238Pu). In this paper, we report the current radiological situation at Dolon and its adjoining three villages Mostik, Cheremshki and Budene. Emphasis was placed on (1) reevaluation of accumulated levels and (2) distributions of residual long-lived radionuclides 137Cs and Pu isotope in soil using long core samples up to a depth of 30 and 100 cm, and (3) characteristics of Pu in soil by using sequential leaching, particle size discrimination and a-track methods.

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Dolon village, located about 110 km ENE from the ground-zero point in the Semipalatinsk Nuclear Test Site, was heavily contaminated by the radioactive fallouts from the first USSR atomic bomb testing in 1949. The current level of Cs-137 soil contamination in Dolon was measured to be 2 – 10 kBq/m², showing a log-normal distribution with an average of 4.2 kBq/m². A 90 percentile of this log-normal distribution was chosen as a referential contamination in Dolon, which corresponded to 32 kBq/m² in 1949. A VB Excel program, FPCOMP.xls was developed to follow temporal change of FP composition after nuclear explosions, using JNDC FP Data Library containing 1,227 nuclides. From the results of the FPCOMP calculation for Pu-239 fission by fast neutrons, 29 gamma-emitting nuclides were selected as possible FPs that could contribute external radiation in Dolon village. Conversion coefficients from the unit deposition of each of 29 FPs to air-dose at 1 m above the ground were calculated using infinite plane model. Cumulative air-dose per unit Cs-137 deposition was calculated at arbitrary time after the fallout deposition. Air-dose of about 0.016 ± 0.004 Gy per 1 kBq/m² of Cs-137 deposition was obtained for the time period from 3 hr (assumed arrival time of radioactive clouds to Dolon village) to 1 yr after the explosion. Thus, combining this value with the Cs-137 deposition of 32 kBq/m², external radiation of 0.51 ± 0.13 Gy was evaluated as the referential air-dose in Dolon village.

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From 1949 to 1989, the former USSR conducted more than 450 nuclear explosion tests, including/ atomospheric and underground tests, at Semipalatinsk nuclear test site. The significant activity remaining includes long lived radioisotopes of Pu, Cs and Sr. Radioactive clouds moved on villages at the time of every nuclear test also emitted external radiation. Therefore, individuals lived near nuclear test site are considered to had been influenced by both internal and external exposures. In order to assess the prolonged radiation effects on individuals, chromosome analysis was performed in lymphocytes from 86 peoples living three villages, Dolon, Sarajar and Kaynar, where are from 80-150 km away from nuclear test site, and 25 control peoples in Kocpecti, being away from 700 km. Micronucleus assay was also done in 184 peoples in five different contaminated villages and one control village. Frequencies of dicentric and ring chromosomes in peoples at contaminated area(0.36-0.45 per 100 cells) were higher than those at non-contaminated area (0.12 per 100 cells). Peoples in the four villages had multiple complex chromosome aberrations(MCA) in 0.12-0.42%, with unknown reason. Micronucleus incidences in exposed group (9.36-12.3 per 1,000 lymphocytes) were also higher than non-exposed group(8.11 per 1,000 lymphocytes). Higher incidence of unstable type aberrations such as dicentric, ring chromosomes and micronucleus found in residents at contaminated areas seem to be mainly caused by internal exposure. Also, 1.2% of translocations were detected by FISH method.

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During last decade specialists from Semipalatinsk State Medical Academy, National Cancer Institute and Hiroshima University have been done a really significant contribution to thyroid studies around Semipalatinsk Nuclear Test Site. In 1998, during thyroid screening (Zh. Zhumadilov and C. Land) thyroid nodules were detected by ultrasound screening of 2998 persons who as children had lived around the Semipalatinsk nuclear test site in Kazakhstan. A total of 909 persons with thyroid nodules were identified . Nodule prevalence was positively and significantly (p < .001) associated with dose, with an estimated excess odds ratio per Gy (EOR) of 4.1, 95% confidence interval (CI) 1.6-7.8. In 1999-2002, Studies related to iodine deficiency in the Semipalatinsk region, thyroid dose reconstruction and rehabilitation program of the exposed population have been supported by Hiroshima University, Hiroshima-Semey Project and JICA, Japan (M. Hoshi). Those studies clarified external and internal radiation exposure doses, influences of iodine deficiency, exposure rates and rehabilitation program. Thyroid studies have suggested that genetic factors may play a role. A. Sigurdson and J. Struewing evaluated nodule prevalence, thyroid dose from fallout, and 18 single nucleotide polymorphisms (SNPs) in 10 genes relevant to DNA repair and thyroid tumorigenesis. Study results show that genetic factors are very important and more detailed research is needed in this field.

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Radiation Effects on thyroid were studied through the thyroid health examinations for the exposed people in Semipalatinsk and Hiroshima from 1999, using thyroid hormones (FT₃,FT₄,TSH) and thyroid autoimmune antibody (TgAb) in blood, iodine in urine and aspiration biopsy cytology of thyroid, and histopathology. In the nontumorous tissues of thyroid cancer cases, fibrosis thought be affected by expose seemed to be more pronounced in the exposed people in Semipalatinsk than that in Hiroshima. Urinary iodine level for Semipalatinsk people was lower than for Hiroshima people. Chronic thyroiditis was estimated not related to radiation expose. Rate of hypothyroidism was suspected higher in the most exposed village people in Semipalatinsk, and in the nontumorous thyroid lesions, nuclear abnormalities of micronucleus tended to be higher in those highly exposed villagers in Semipalatinsk. In Hiroshima, micronucleus was found in the nontumorous lesions of thyroid among the A-bomb exposed survivors in the cases of Hashimoto disease. Our urinary iodine level examined after one-week staying in Kazakhstan shoed apparent decrease. Thyroid stimulating hormone receptor (TSHR) sensitivity of noncancerous lesions of thyroid showed significant decrease with the increase of age (14-89 rears old). Amount of estrogen receptors in nontumorous tissues of thyroid cancer cases examined for female also decreased with age increasing. These data proved the truth of the hypothesis of oncology model of thyroid cancer development following irradiation, proposed by Takeichi in 1993.

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In lunar and Mars missions, astronauts fly outside the Earth magnetosphere and will be exposed usually to galactic cosmic rays (GCR) that are composed of high-energy protons and heavy ions (He-Fe). The effective dose rate is estimated to be about 1 mSv per day. Under this normal condition, their doses would not exceed 1Sv even for an interplanetary mission longer than two years. However, a greatest-scale solar particle event (SPE) would possibly bring a high-dose proton exposure for a short period (several hours to a cup of days), comparable to the total GCR dose. It is thus important to quantify the potential risks of SPE for lunar or Mars missions and to employ necessary countermeasures for reducing the risks. For establishing most effective countermeasures with the least increase of cost, we primarily need to construct a risk-evaluation method applicable to multiple exposures to GCR and SPE that are greatly different in regards to dose rate and its fluctuation. This task requires quantifying synergistic effects of peculiar conditions in space environment such as microgravity, in regard to carcinogenesis and other biological effects (cataracts, heart diseases, damage to central nervous system, etc.) that would be promoted by space radiation exposure.

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To explore synergistic effects of space radiation with other factors, we determined mutagenesis effect of 21 rice varieties with space flight (SP), simulated radiation (C 13.3 kev/µm Ne 31.0-80 kev/µm Fe450- 500 kev/µm in 2.0mGy) on ground comparing with their controls(CK). The results indicated that the cell damage effect is SP>Fe>Ne>C>CK. The japonica rice varieties were higher sensitivity to space environment than indica rice varieties. With active effect of physiology and cell level was in evidence in post-flight, the present study indicated that space radiation may be not only mutagenesis factors to rice seeds, but also other factors during space flight. The effect of mutagenesis on rice seeds of space environment is relative to genetic background of rice varieties.

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Space radiations consist of geomagnetic-trapped high LET particles from intensely energetic solar-particle events at a constant low dose and low dose rate. In particular, neutrons, a kind of high LET particles, have a reverse dose-rate effects with relationship on RBE, that is, the exposure at a low dose-rate shows severe effects compared with a high dose-rate. To protect human health, we need to measure the exact total doses of various space radiations over a long-term stay in space. Therefore, we need to know the maximum permissible doses of space radiations. Though physical monitoring of space radiations in recent space experiments indicated about 1 mSv per day, microgravity was reported increase the abnormal development induced by heavy particles in C. morosus and the mutation frequency induced space radiations in D. melanogaster. Our group reported the induction of stress protein (HSP72) in goldfish and a tumor suppressor gene product (p53) in rat. These findings may support that the biological effects of space radiations were enhanced by space environment, especially microgravity. It is quite important to assess the total permissible doses of space radiations from an aspect of space crew's health for long stay in space.

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Energetic heavy ions pose a great health risk to astronauts in extended ISS and future Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied chromosome aberrations in human lymphocytes and fibroblasts induced by both low- and high-LET radiation using FISH and multicolor fluorescence in situ hybridization (mFISH) techniques. In this study, we exposed human epithelial cells and lymphocytes in vitro to energetic particles of varying types and energies and dose rates at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or ¹³⁷Cs-gamma radiation source at the Baylor College, and analyzed chromosomal damages using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). The possible relationship between the frequency of inter- and intra-chromosomal exchanges and the track structure of radiation is discussed.

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The differential radiation environment encountered in space may cause deleterious effects in humans, and these effects are of special concern for prolonged space missions beyond the protective terrestrial magnetosphere. Furthermore, exposure to space radiation in a microgravity environment may induce a synergistic effect leading to compounded detrimental outcomes, and thus have a major impact on the risk assessment for space travel. Due to the fact that many cellular signaling pathways activated by physiological stresses and DNA damage overlap, interactions between these activated pathways could potentially alter the radiosensitivity of crewmembers on space missions. Ideally, investigations to elucidate the synergistic effects of space environmental factors and radiation require exposure of samples to a controlled radiation source in the microgravity environment. Over the last several decades, a number of flight experiments have been conducted to address the specific question of a possible synergism between radiation exposure and microgravity environment, but the issue remains unresolved due to the limitations of flight experiments and other factors. We will review the studies that addressed this specific question and discuss the limitations of flight experiments. Our data on a Space Shuttle astronaut will also be presented.

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Oxygen radicals are produced through normal cellular metabolism, and formation of such radicals is enhanced further by ionizing radiation and by various chemicals. The oxygen radicals attack DNA and its precursor nucleotides, and consequently induce a various oxidized form of bases in DNA within normally growing cells. Among such modified bases, 8-oxo-7, 8-dihydroguanine (8-oxoG) and 2-hydroxyadenine (2-OH-A) are highly mutagenic lesions, if not repaired, in DNA. MUTYH, a mammalian homolog of E. coli MutY, is a DNA glycosylase that excises adenine and 2-OH-A incorporated opposite guanine or 8-oxoG, thus expected to prevent G:C to T:A transversions in mammalian cells. To analyze mutagenesis in the mice, we constructed the Mutyh-deficient mice carrying the rpsL transgene as a mutation-reporter, and analyzed the mutation frequencies and spectra in Mutyh-deficient and wild type mice at the age of 24 weeks. Overall mutation frequencies observed in small intestines were not significantly different between the Mutyh-deficient and wild type mice. However, there are drastic differences between these two genotypes in class- and site-distributions of the rpsL^- mutations recovered from the mice; an increase in the frequency of G:C to T:A transversions was evident in Mutyh-deficient mice. These results suggest the contribution of the Mutyh protein in counteracting the mutagenic oxidative lesions in DNA, such as 8-oxoG, and consequently in suppressing spontaneous tumorigenesis in the intestine.

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To elucidate the molecular mechanism in radiation tumorigenesis in mice, we generated a mouse strain by substituting an entire chromosome18 in B6-Min mouse. The consomic-Min mouse strain is highly susceptible to radiation tumorigenesis; mice exposed to 2.0 Gy of X-rays at 2 weeks of age induced 7-fold small intestinal tumors and 29-fold colonic tumors over the unirradiated mice. The enhancement was dependent on the age at exposure; mice exposed at 7 weeks of age had lost the susceptibility to the radiation tumorigenesis. LOH analysis at the Apc locus revealed that radiation-induced colonic tumors showed significantly higher LOH frequency compared to the spontaneous tumors. Detailed LOH analysis using 24 SSLP markers on the chromosome 18 revealed that the vast majority of the radiation-induced intestinal tumors exhibited intra-chromosomal recombination, while spontaneous tumors showed entire loss of the chromosome 18 without any sign of the recombination. In all the cases mentioned above, normal allele of the Apc gene had been lost. Possible chromosome region where frequent recombination had occurred in the radiaton-induced tumors was suggested.

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BACKGROUNDS & AIM: X-ray is known to cause genetic damage and induce many types of carcinomas. Mouse model for human familial adenomatous polyposis, APC/min mouse, contains a truncating mutation in APC gene and spontaneously developes intestinal adenomas. To elucidate the role of X-ray in the intestinal tumor, we examined the promotion of carcinogenesis in APC/min mouse by X-ray irradiation.
METHODS: APC/min mice and wild-type were irradiated with a single whole body of 5 Gy X-rays at 8 weeks of age. After 20 weeks, all animals were sacrificed.
RESULTS: Total 51.9% cases of X-ray irradiated APC/min mice were highly observed invasive carcinoma in small intestine. 54.5% in male and 48.5% in female were showed adenocarcinoma with invasion to proprial muscle layer. Only 20.3% cases of non-irradiated APC/min mice were observed invasive carcinoma. 28.1% of male and 12.5% of female in non-irradiated APC/min mice were showed invasive carcinoma. There was statistical difference of the occurrence of invasive carcinoma between X-irradiated and non-irradiated APC/min male or female mice (p=0.019, 0.0017, respectively). X-irradiated APC/min mouse showed higher number of invasive carcinoma than non-irradiated mouse in male (p=0.041). The number of polyps did not show any difference between X-irradiated and non-irradiated Apc/min mice. Wild-type mice with or without X-irradiation were not occurred any invasive carcinoma.
CONCLUSIONS: Our results suggested that X-ray irradiation may promote the invasive activity of intestinal tumor in APC/min mouse.

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DNA mismatch repair (MMR) system is one of the important mechanisms to suppress cancer development. MMR deficiency is a cause of hereditary non-polyposis colorectal cancer (HNPCC) as well as mammary, gastric, pancreatic cancers and leukemia. Colon cancers in HNPCC patients and the lymphomas in Msh2-deficient mice have shown frequent frame shift mutations in TgfbrII. It has not yet examined whether MMR deficiency is involved in the susceptibility to radiation carcinogenesis. The aims of the current study are both to elucidate the susceptibility of Mlh1^-/- mice to radiation induced tumors and to determine the mutation of TgfbrII in the tumors. Eleven-day- and 10-week-old Mlh1^-/- mice were exposed to 2 Gy of X-rays. The mice were sacrificed at moribund and autopsied. Mutation of TgfbrII was analyzed in the DNA from pathological samples after microdissection or fresh samples. Thymic lymphomas (TL) after irradiation at 11-days and 10-weeks developed much earlier than spontaneous ones with the incidence of 87% (13/15) and 87%(13/15), respectively, which was significantly higher than that of spontaneous ones (61%, 11/18). Gastrointestinal tumors (GIT) also developed earlier when irradiated at 10-weeks, but not 11-days, old. The mutation of TgfbrII was not found in any tumors. We conclude that Mlh1 deficiency accelerates radiation-induced TL and GIT development. TgfbrII mutation, however, was not involved in cancer induction of Mlh1^-/- mice.

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The health effects of low-dose radiation should be evaluated in combination with chemicals as humans are continuously exposed to a variety of chemical agents. Carcinogenesis is one of the most important concerns regarding to the possible effects from low-dose radiation. Of various environmental hazardous compounds, the most prominent factor associated with the increase in cancer risk is cigarette smoke. Although cigarette smoke contains more than 40 human carcinogens, the most carcinogenic nitrosamino compound is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the present study, the effects of low dose-rate irradiation on the genotoxicity of NNK were examined. The female gpt delta mice (7 weeks of age) were either treated with gamma-irradiation at low dose rates (0, 0.5, 1.0 or 1.5 mGy/h x 22 h/day) for 31 days or combined with NNK (2 mg/mouse/day, i.p.) administration for 4 continuous days in the middle course of irradiation. The gpt and Spi^- mutations in the lung and liver of the mice were analyzed. NNK treatments enhanced the gpt mutation frequencies (MFs) 3-8 times and 12-19 times in the lung and in the liver, respectively. However, no modulating effects of low dose-rate radiation were observed in both organs. In the Spi^- MFs, large deletions with the size of more than 1 kb increased in the irradiated groups compared to those in the unirradiated groups. Analyses of the effects of low dose-rate irradiation on NNK-induced deletion mutations are in progress.

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To know the difference in sensitivity to ionizing radiation for mutation induction between somatic and male germ cells, we have carried out mutation assays using the transgenic mice, which is the gpt-delta mouse strain carrying the bacterial gpt gene as targets for mutagenesis. The spontaneous gpt gene mutation frequency in embryo cells was 1.04 x 10^-5. The mutation frequencies after exposure to 1, 2.5 or 5 Gy of X rays in embryo cells were 1.50, 2.43 or 3.46 x 10^-5, respectively. The induced mutation frequencies in embryo cells were increased linearly with increasing doses of X rays. The mutation frequencies (spontaneous or induced with 5 Gy of X rays) in spleen cells were 1.10 or 2.93 x 10^-5, respectively. The mutation frequencies for both somatic cells were nearly the same. To measure the induced mutation frequencies in male germ cells (spermatogonial cell stage), sperms were extracted at 65-83 days after irradiation of 1, 2.5 or 5 Gy of X rays, corresponding to the spermatogonial stem cell stage at the time of treatment. The spontaneous mutation frequency in male germ cells was 0.36 x 10^-5. The mutation frequencies in male germ cells irradiated with 1, 2.5 or 5 Gy of X rays at the spermatogonial stem cell stage were 0.39, 0.53 or 1.05 x 10^-5, respectively. The mutation frequencies (both spontaneous and induced) in spermatogonial cell stage were about three times lower than those in somatic cells. This difference would be due to the high base excision repair activity in male germ cells.

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Mutation in DNA is deleterious for maintenance of each individual. On the other hand, it is necessary for keeping variability of individuals. Thus, it is an interesting question to ask weather or not DNAs in somatic cells and germ-line cells are maintained in a same way. One of the approaches to answer the question is to examine mutations in these cells. Here, we compared spontaneous mutations in gonadal and somatic tissues in mouse embryo. We used lacZ-transgenic Muta(TM) mice. Mutants were selected as P-gal resistant lambda phage clones after retrieving lacZ-containing lambda DNA from mouse genome by a use of packaging procedure. To understand the molecular nature of mutations, the lacZ genes of mutants were sequenced and compared to the sequence of wild type gene. Testes and ovaries were separated under binocular from 14.5-day embryos. The remaining embryos were used as somatic tissues. The mutant frequencies in the male and female gonads were similar; about 3×10^-5. They were slightly lower than the level in the somatic tissue. The molecular nature of mutation as judged by spectrum of different kinds of mutation showed similarity among the tissues. The detailed analysis of deletion mutation, however, revealed a difference in the size of the deleted sequences between the gonads and the somatic tissue. It suggests that the genome maintenance system in germ-line cells is not the same as that working in somatic cells.

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