High energetic heavy ions give unique irradiation effects on target materials, and are promising tool for the applications in chemical and biological fields. These unique irradiation effects are resulted in the high density and non-homogeneous distributions of radicals and subsequent diffusions and reactions in track. Reactions in water under heavy ion irradiation are mainly induced by OH (hydroxyl) radicals. In the present study, we used aqueous solutions to estimate the primary yield of the OH radicals as the basic knowledge for the application of heavy ions to advanced cancer therapy, and so on. The primary yields of the OH radicals have been investigated by the product analysis. The aqueous phenol solutions were irradiated with He, C and Ne ions ranging from 2 to 18 MeV/n. The yields of irradiation products of phenol super-linearly increased with the incident energy of the ions. The yields of the OH radicals were estimated by analyzing the yields of the irradiation products of phenol. The yields of the OH radicals increased with the specific energy for each ion, but decreased with both the mass of each ion at the same specific energy and elapsed time after irradiation.

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Water radiolysis with heavy ions having energies up to 28 GeV has been investigated. Beams of ⁴He²⁺, ¹²C⁶⁺, ²⁰Ne¹⁰⁺, ²⁸Si¹⁴⁺, ⁴⁰Ar¹⁸⁺ and ⁵⁶Fe²⁶⁺ were taken for irradiations at HIMAC, NIRS. Corresponding LET values vary widely from 2 to 183 keV/μm. Aqueous solutions of MV (Methyl viologen) containing various concentrations of formate (0.01-2 M HCOO^-) were irradiated and the production G values of MV^+• were determined by absorption spectroscopy. Intra-track reactions were discussed from the MV^+• yields because they can approximately be regarded as the sum of the yields of e^-_aq, •OH and H•. With increasing LET and with increasing HCOO^- concentration, the MV^+• yield decreased, reflecting increase of track density and development of intra-track reactions, respectively.
In parallel to above measurement, a Monte-Carlo simulation has been conducted not only to reproduce experimental results but also to discuss track structures more deeply. After selection of additional reactions concerning scavengers, experimental results were reproduced well. Then, attention was paid to each intra-track reaction and there were two findings. The first was that reactions between the products in scavenging reactions as well as reactions between water decomposition radicals become significant in high LET track. The second was that one of such reactions, COO^-• + COO^-•, showed similar LET dependence with RBE value and this similarity could be an explanation of highly effective cell-killing induced by heavy ion irradiation. Such an explanation by using radiation chemical calculation has not been shown yet although further investigation is necessary.

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"How are the chemical structure,the yield,and the distribution of DNA damage (DNA damage spectrum)caused by the ionizing radiations different by the radiations quality and their energy?" The answer to this question becomes a vital information in clarifying the mutation and the carcinogenesis mechanism following to DNA damage. However, the unified view concerning this question has not been obtained because of a lot of uncertainties in the status of DNA, in the variety of DNA damage and in the distribution of radical scavengers and in the possibility of the directly and indirect effects, in the oxygen concentration, and furthermore in the artifact, etc., regardless of the long-term investigation for the past several ten years. Another reason why DNA damage research by radiations has not been advanced should be that there has not been enough analytical methods to detect the damage because irradiated DNA is extremely delicate against temperature and pH. Then, we have developed DNA damage spectrum analysis method that doesn't strictly stick to the chemical structure of individual damage and is not influenced easily by the weakness of irradiated DNA. This method makes us possible to quantify 3'termini with or without phosphate and the electron-withdrawing damaged bases and abasic sites piperidine-labile sites using phosphodiestertase I and alkaline phosphatase. We would like to introduce how DNA damage spectrum can be analyzed by this method, and discuss it by comparison with other information.

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We newly found novel slow-releasing long-lived radicals (SRLLRs) in 4 Gy gamma-ray irradiated cultured SHE cells by ESR spectroscopy without any spin-trapping agents. Levels of SRLLRs increased 18 and 36 % with an increase at intervals of 1 and 5 h AFTER irradiation, respectively, and were kept in the same until 12 h at least. Post-treatment of Vitamin C for 2 h suppressed the increase in the levels of SRLLRs. Addition of myxothiazol, as an inhibitor of electron transportation in mitochondria, suppressed the increase in the level of SRLLRs in irradiated cells, and direct addition of hydrogen peroxide to the cells increased the levels of them. These results indicate that SRLLRs are likely to be produced by hydrogen peroxide from dysfunctional mitochondria. Because SRLLRs have a long lifetime over 12 h and reduced by ascorbate, they might be responsible of late effects of radiation including point mutation.

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Deprotonation of the guanine radical cation in DNA is an important step in hole hopping and mutagenesis. The dynamics of one-electron oxidation of DNA have been investigated by pulse radiolysis. We proposed that the kinetics associated with the deprotonation process involves a shift of the N1 proton in G cation radical to its partner C, followed by final release of the proton into solution. In experiments using 5-substituted cytosine-modified oligonucleotides, changes of the cytosine C5 hydrogen to methyl or bromine groups resulted in an increase or decrease, respectively. Kinetic solvent isotope effects on the oxidation kinetics of dG and oligonucleotide duplexes were examined in H₂O and D₂O. The rate constant of formation of G radical in dG was larger by a factor of 1.7 than was the same rate constant in H₂O . In the oligonucleotide, on the other hand, a remarkable solvent isotope effect of 3.8 was observed. These results suggest that the slower kinetic phase is due to the deprotonation of oxidized hydrogen-bridged G-C base pair by a water molecule.

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Edaravone or RadiCut®, 3-methyl-1-phenyl-2-pyrazolin-5-one, is a newly developed free radical scavenger which has been approved in Japan as a neuroprotective drug since 2001. In the present work, the transient intermediates and the reactivity of edaravone towards ^·OH, N3^·, Br₂^·-, SO₄^·-, and CCl₃O₂^·- are investigated by pulse radiolysis techniques. The oxidation of edaravone by N3^·, Br₂^·-, SO₄^·-, and CCl₃O₂^·- results in an absorption band with λ_max = 345 nm (ε_345nm = 2600 M^-1cm^-1), which is assigned to the edaravone radical formed by H-abstraction or electron transfer. However, the transient species produced by the reaction of edaravone with ^·OH shows an absorption band with λ_max = 320 nm (ε_320nm = 4900 M^-1cm^-1). Accordingly, the main transient species by the reaction of edaravone with ^·OH in the absence of O₂ is attributed to OH-adducts. The rate constants of edaravone reacting with ^·OH, N3^·, SO₄^·-, and CCl₃O₂^·- are estimated to be 8.5×10⁹, 5.4×10⁹, 6×10⁸, and 5.1×10⁸ M^-1s^-1, respectively.

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It is inevitably important to estimate yield of hydroxyl radical (•OH) in radiolysis of neutral water with heavy ions because of the following two facts. One is that •OH is most responsible to indirect effect induced by radiation, and the other is that heavy ion irradiation shows highly effective cell-killing property. In the present work, it is aimed to apply fluorescence spectroscopy as a sensitive detection technique to such estimation.
It is known with ⁶⁰Co γ ray that Coumarin scavenges •OH and produces fluorescent species, 7-hydroxy-coumarin, which can be detected with resolution around 10 nM in concentration. The solubility of Coumarin is not high enough to investigate early events like intra-track reaction because the time scale of scavenging reaction is 10^-7 s at earliest. Therefore, a similar compound, Coumarin-3-carboxylic acid (CCA) was used in the present work because of its high solubility. Compared to Coumarin, there are little information about reactivity of CCA toward water decomposition radicals and that about reaction pathways from CCA to final, stable and fluorescent product, 7-hydroxy-CCA (7OH-CCA). Then, electron pulse radiolysis and product analysis with ⁶⁰Co γ ray irradiation were preliminary done to check the reactivity and to discuss the reaction pathways, respectively.
Subsequently, beams of ¹²C⁶⁺, ²⁸Si¹⁴⁺, ⁴⁰Ar¹⁸⁺ and ⁵⁶Fe²⁶⁺ having energies of several hundred MeV were taken for irradiation at HIMAC, NIRS. Irradiated samples were analyzed with reversed phase HPLC coupled with fluorescence spectrometer. Excitation and detection were done at 365 and 445 nm, respectively. It was found that the yield of the fluorescent product, 7-OH-CCA, decreases not only with increasing LET but also increasing scavenging capacity, reflecting the increase and the development of intra-track reactions, respectively. It is concluded that 7OH-CCA produced in CCA solution can be a probe of •OH although further investigation is necessary.

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Understanding of water radiolysis is necessary not only in basic science but also in application field such as radiation therapy of cancer because water is a main component of human body. However, water decomposition yields are not known well for highly energetic ions. In addition, there are few data obtained under neutral pH in spite of their crucial importance.
Then, heavy ions having energies up to 28 GeV were taken for irradiation at HIMAC, NIRS to determine the primary yields (primary g values) of main 3 species in water radiolysis, e^-_aq, •OH and H₂O₂. Because pulse irradiation was not available in the present experiment, steady-state irradiation was applied. The scavenger method was used to extract information of water decomposition products, which have high reactivity and diminish within short time after their production. 1-cm width cells were used for these irradiations to neglect LET changes in the samples. In addition, energy absorber made of PMMA was used to decrease beam energy, leading to the increase of LET. In this case, 2-mm width cells were used to minimize large LET change in the region close to Bragg peak. Thus, LET was varied widely from 2 to 700 keV/μm and measured yields were regarded as track segment yields as opposed to track averaged yields.
Above measurement showed that radical yields decrease with increasing LET while molecular yields increase. This is resulted from the increase of intra-track reactions. And lighter ion irradiation gave smaller radical yields and larger molecular yields than heavier ion irradiation having comparable LET, indicating that lighter ion irradiation generates smaller but denser track closer to its trajectory.
Further investigation with microscopic viewpoint is now being conducted by using simulation of intra-track reactions to clarify the mechanism of distinctive features seen in heavy-ion irradiation.

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The repair activities and mechanisms of both silybin (3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenil)-2-hydroxymethyl-1,4-bentreatment bone zodioxan-6-il]-chronan-4-one) and its analogues towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adduct were investigated with pulse radiolysis techniques. On pulse irradiation of nitrous oxide saturated 2.0 mM dGMP aqueous solution containing 0.01 mM silybin at neutral pH (2 mM phosphate buffer), the transient absorption spectrum of the dGMP hydroxyl radical adduct (dGMP-OH) decays following the formation of phenoxyl radical of silybin within tens of microseconds. This indicates that there is a repair reaction between dGMP-OH and silybin. The rate constant of the repair reaction was calculated to be 1.0×10⁹ M^-1s^-1 for silybin .The repair activity of hesperetin, naringin and naringenin towards dGMP-OH were also measured. A comparison of the rate constants of the four flavanones shows that silybin has highest reparation activities towards dGMP-OH adduct. These results imply that non-enzymatic fast repair might be a universal repair mechanism of phenolic antioxidants.

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X-ray microbeam irradiation system has been installed in Central Research Institute of Electric Power Industry (Tokyo, Japan) to elucidate biological effects of low dose and low dose-rate radiation including X-ray-induced bystander response. It has already been reported many results about cellular response to charged particle (i.e., alpha particle) microbeams. On the other hand, it has not yet fully recognized that to X-rays, though it has been speculated that the biological responses are different from high-LET charged particles. Our microbeam X-ray irradiation system was characterized by (1) desktop type (2) X-ray focusing system using Fresnel zone plate (FZP), and (3) on-line confocal laser microscope. Characteristic X-ray of aluminum (1.49 keV) was generated by focused electron bombardment of aluminum target. The bremsstrahlung radiation is removed by reflecting the radiation off an X-ray mirror. The FZP was used to focus aluminum K-shell X-ray. The size of X-ray beam is 2-3 μm in diameter detected by a scintillator. Confocal laser scanning microscope (FV300, Olympus) was equipped to obtain high resolution images from just irradiation. The details of the performance of our system and our visions will be present.

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  To elucidate the biological effects of low-dose radiation, it is important to study the responses in irradiated cells and in bystander cells. For this purpose, a microbeam irradiation system is a powerful tool, since we can observe individually the cellular responses of both irradiated cells and non-irradiated cells.
  We have been studying the differences in lethal effects of V79 cells irradiated with microbeam of different sizes using an X-ray microbeam irradiation system developed at the Photon Factory, KEK. This system can provide an arbitrarily sized X-ray microbeam larger than 5×5 μm².
  We reported last year that the low-dose hypersensitivity was more clearly observed in the nucleus-irradiated cell with 10×10 μm² beam than in the whole cell irradiation with 50×50 μm² beam. As the next step, we irradiated 5 cells in the dish with 10×10 μm² beam or with 50×50 μm² beam, and measured the survival of bystander cells. When nuclei only were irradiated, survival fraction of bystander cell decreased down to 90% around 1 Gy and increased back to 96% in higher dose region, whereas in the whole cell irradiation, it decreased monotonously to 92%. Dose-dependent enhancement of cell death was observed in the absence of energy deposition to cytoplasm in both irradiated and bystander cells. We speculate that the energy deposited in the cytoplasm might play an important role in cell killing in the low-dose region. The relationship between cell death (irradiated and bystander cells) and intracellular energy-deposited sites will be discussed.

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Synchrotron radiation is a useful tool to elucidate initial processes leading to various radiobiological effects, since we can induce selectively X-ray photoabsorption at desired elements in cells by tuning X-ray energy to the absorption edges of the element. At inner shell ionized or excited atoms, Auger effects occur and several low energy electrons are emitted around the photo-absorbing atom, producing complex and severe damages around the site.(element selective irradiation) On the other hand, we recently developed an X-ray microbeam irradiation system with monochromatic X-rays. Energy of X-rays is restricted to 5.35 keV, since the beam is deflected right angle upward using diffraction of Si(311), in order to irradiate the samples through the bottom of the dish. (site selective irradiation).
In order to realize combined irradiation of site-selective and element- selective, we have constructed an energy-tunable X-ray microbeam irradiation system. As a dish to irradiate cells we decided to employ a commercially available one with bottom made of thin glass, which was developed for use with confocal laser microscopes. Cells attached on the bottom are kept humid by covering with a thin Kapton film after the medium is suctioned out just before irradiation. Physiological condition of cells after treated in sham irradiation process was found good enough to obtain reliable and reproducible results. Several studies have started and some preliminary results will soon be reported.

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Heavy-ion microbeam system of JAEA-Takasaki is contributing for analysis of heavy-ion radiation effect and the bystander effects. Recently, there are increased interests in radiation effect of subcellular organelle such as mitochondria using fine subcellular microbeam irradiation. Nevertheless, it is difficult to generate finer beam that is capable for subcellular irradiation in our current system, because of technical difficulties in fabrication of finer micro collimator and inevitable scattering of ions at the edge of micro collimator. Thus, we installed new focusing microbeam system at another vertical beam line of AVF cyclotron of TIARA, JAEA. New system is equipped with a quadruplet quadrupole lens system for higher spatial resolution and with an X, Y beam scanner for fast hitting of single ion to micron scaled samples like a biological cell. In vacuum, a microbeam generated by the system had spatial resolution of less than 1 μm. The beam was extracted into the atmosphere, and its spatial distribution of ion was observed by irradiating ion track detector, CR39, at just beneath a vacuum window made by Kapton film of 8 μm thick. The spatial resolution of beam extracted in air was less than 5 μm, indicating that finer microbeam was generated by new focusing microbeam system. New cell targeting system for this microbeam system, which is designed to target and irradiate biological materials more precisely, is currently under development.

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The microbeam irradiation study of a cultured cell contributed to the development of the radiation-biology research, e.g. bystander effect ¹⁾. An advantage of a microbeam is localized irradiation, and the microbeam has been applied to the comparative study of radiation effects between a nucleus, cytoplasm and membrane. On the other hand, we have used Caenorhabditis elegans as a model multicellular organism which contained about 1000 cells, and push forward a heavy-ion microbeam irradiation individual-study. As for about 1.2 mm in a range of carbon ions in water, all cells and tissues of C. elegans are microbeam irradiation objects. At present, the target area is several dozens μm in a diameter, depending on the limits of beam-size of collimated microbeams and observation ability of the inverted microscope by the interference with the end part of the microbeam line. Using this system, Sugimoto et al. showed DNA-damage-induced cell cycle arrest and apoptosis in locally irradiated areas of C. elegans ²⁾. We focus the nervous system of C. elegans (occupied about 30% of all cells) and study the effects of localized irradiation on learning behavior (food-NaCl associative learning ³⁾). However, the anesthetic method for fixation of <I)C. elegans ²⁾ is not usable because the whole body irradiation of ⁶⁰Co gamma rays affected only transition stage of the conditioning for food-NaCl associative learning. Thus, now we are constructing the heavy-ion microbeam irradiation system for living target C. elegans.
References: 1) Shao et al., FASEB J 17, 1422-7 (2003), 2) Sugimoto et al., Int J Radiat Biol 82, 31-8 (2006), 3) Saeki et al., J Exp Biol 204, 1757-64 (2001).

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DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage remain elusive. We apply in situ analysis of DNA damage responses by using laser micro irradiation of living human cells. We found a human protein, PALF, with a FHA (forkhead-associated) domain and novel zinc-finger-like CYR (Cystein-Tyrosine-Arginine) motifs that are involved in responses to DNA damage. The CYR motif is widely distributed amongst DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, have AP-endonuclease and exonuclease activities. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with γH2AX foci. Furthermore, we report another approach to screen and identify damage accumulating proteins by laser-micro-irradiation as well.

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One of the goals of our study is to clarify the nature of clustered damage induced by densely ionizing radiation. Many studies on DNA damage to date have focused on yields of single-(SSB) and double-strand breaks (DSB) in closed-circular plasmid DNA. Oxidative base lesions and cluster damage composed of them have also been analyzed using base excision repair enzymes, which convert a base lesion to a detectable strand break. These studies, however, does have some limitations. For instance, previous simulation studies have shown that the proportion of SSB which are complex, i.e. one or more lesions close to SSB terminus, is significant. The complex SSBs as well as enzymatically induced SSBs, would be underestimated since these SSBs will not cause additional conformational changes if it is on the same strand as the prompt SSB or on the opposite strand but separated sufficiently (>6 bp) from the prompt SSB so as not to induce a DSB. In order to observe these secondary invisible SSBs, we developed a novel technique using DNA denaturation by which irradiated DNA is analyzed as single strand DNA. The prompt or enzymatically revealed additional SSBs which arise in both strands of pUC18 plasmid DNA, but do not induce a DSB, are measured as degradation of single strand (SS-DNA) using gel electrophoresis. To avoid induction of heat labile strand breaks by high temperature generally used for a denaturation treatment, we have determined much lower denaturation temperature (37 degrees centigrade) using formamide (50% v/v). Obtained preliminary results will be presented.

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Clustered DNA damage is a specific form of DNA damage induced by ionizing radiation, and it is thought to be mainly involved in radiation effect to any biological systems. We previously reported that it showed a clear inverse correlation between the yields of clustered DNA damage and LET of the ionizing radiation in DNA molecules irradiated in vitro. For demonstration of this correlation in cell, we analyzed yields of clustered DNA damage in cells irradiated with gamma-rays (0.2 keV/μm) and various accelerated ion particle beams including carbon (13 keV/μm), silicon (55 keV/μm), and iron ions (200 keV/μm), in the present study. Logarithmic growing chinese hamster ovary-AA8 cells were irradiated with respective radiations and embedded into agarose plug gels. After treatment with proteinase K, the plugs were subjected in static field gel electrophoresis for analysis of the clustered DNA damage. Surviving rate of the irradiated cells were also estimated by colony formation assay. Cell survivals decreased with an increase in LET. On the other hand, the yields of clustered DNA damage also decreased with an increase in LET, showing a correlation similar to the results observed in vitro, as our previous report. These results suggest that additional critical factors engage in severity of higher LET radiations, such as physiological processing of clustered DNA damage in vivo.

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Lewis lung carcinoma cells were exposed to 89.63MeV/u carbon ion and 6MV X-ray irradiations respectively, and cell reproductive death and DNA damage were examined. Lewis lung carcinoma cells were shown to be more sensitive to the carbon ion beam from the measured survival data using clonogenic assay. The relative biological effectiveness (RBE) value of the carbon ion beam at 10% survival level was up to 1.77, indicating that the damage induced by the carbon ion irradiation was more remarkable than that induced by the low linear energy transfer (LET) X-rays. The dose response curves of "Tail DNA (%)" (TD) and "Olive tail moment" (OTM) detected with comet assay for the carbon ion irradiation showed a saturated effect beyond about 8 Gy, while it was not found in the case of the X-ray irradiation. There was an inverse correlation that the high-LET carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment at 2 Gy (OTM2) than those for the X-ray irradiation. In conclusion, carbon ion beams having high LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cell in comparison with X-rays and comet assay could be a predictive assay solely applied or combining with clonogenic assay to assess the radiosensitivity of cancerous cells prior to clinical therapy.

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Mutagenesis is one of major consequences of irradiation of ionizing radiation. Oxidative DNA damage produced by ionizing radiation inhibits replicative DNA polymerases. Cells have a function to restore DNA replication called transleion DNA synthesis, in which specialized DNA polymerases could extend the primer termini by bypass synthesis of damage bases. It has been considered that the bypass synthesis is an error-prone process inducing mutations. To elucidate molecular mechanisms of polymerase switches, we reconstituted DNA replication in vitro using purified proteins including DNA polymerase δ (pol δ), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A (RPA). Here we describe dynamic properties of these proteins in the elongation step on single-stranded M13 template, providing evidence that pol δ has a distributive nature over the 7 kb of the template, repeating a frequent dissociation-association cycle at growing 3'-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder could slide out of the primer terminus or be unloaded once pol δ has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol δ has detached, or reload PCNA from solution to restart DNA synthesis. Based on these observations, we discuss the molecular mechanism of polymerase switches.

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[Purpose] Damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species induced by ionizing radiation. After damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, we examined whether a nucleotide excision repair enzyme, Eschericia coli UvrABC, could suppress mutations induced by oxidized deoxyribonucleotides in vivo.
[Methods] We introduced oxidized DNA precursors, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP (2-OH-dATP), into uvrA, uvrB, and uvrC E. coli strains, and analyzed mutations in the chromosomal rpoB gene, as a mutagenesis target. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with hydrogen peroxide (H₂O₂), and the rpoB mutant frequencies were calculated. We hypothesized that UvrABC incises the undamaged DNA strands, instead of the strands containing 8-hydroxyguanine (8-OH-Gua) and 2-hydroxyadenine (2-OH-Ade) formed by the incorporation of 8-OH-dGTP and 2-OH-dATP. Duplex oligonucleotides containing 8-OH-Gua and 2-OH-Ade were treated with the purified Thermus thermophilus HB8 UvrABC proteins.
[Results] Oxidized DNA precursors induced mutations much less efficiently in the uvrA and uvrB strains than in the wild type. In contrast, effect of the uvrC-deficiency was not observed. Frequency of the H₂O₂-induced mutations was increased in the all strains tested, and the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Although duplex oligonucleotides containing 8-OH-Gua and 2-OH-Ade were treated with UvrABC proteins, the expected activity was not observed.
[Conclusions] It is suggested that UvrA and UvrB are involved in the enhancement, but not in the suppression, of mutations induced by oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.

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It is believed that micronuclei (MN) originate from chromosome fragments or whole chromosomes that lag behind at anaphase during cell division. However, nobody looked its emergence in a living cell and nobody knows its consequence after cell division. Recently, a fluorescent microscope designed for live cell imaging enable to visualize cellular dynamics without cell killing by a direct detection of fluorescent-fused organelles and proteins. To understand the mechanism of MN formation derived by gamma-ray, we constructed dual-color fluorescent human lymphoblastoid TK6 cells in which histon H3 and tubulin-alpha were differentially expressed as fusion to mCherry (red) and EGFP (green) fluorescent proteins, respectively. Using a computer-controlled confocal microscope system, three-dimentional images of nuclei (chromosome) and microtubules were sequentially recorded by time-lapse during cell division (M phase) after exposure to gamma-ray. The moment of MN formation by gamma-ray was dynamically observed at anaphase. The MN emergence frequency in the live cell analysis was approximately consistent with that in conventional MN test. In addition, mitomycin C (MN-inducing agent) and vincristine (spindle poison agent) were also used in this study to compare with the style and phase of MN formation by gamma-ray. We will show some movies for the emergence of MN in the living cells exposed to gamma-ray, mitomycin C, and vincristine.

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We used fluorescence in situ hybridization with whole-chromosome probes for human chromosomes 1 and 3 to investigate the G0 and G1 phase chromosome aberrations after exposure to X-ray, silicon ions and iron ions . Non-dividing fibroblast cells (AG1522) were exposed to low- or high-LET radiations and chromosome aberrations in fusion premature chromosome condensation technique (PCC) after several hours repair (G0) or subcultured immediately after irradiation (G1) were scored. The repair process of chromosome fragments were determinted using the G0/G1 type premature chromosome condensation technique .We have found that chromosomal fragments were rejoined at a similar rate between G0 and G1 phases until 12hrs. However, around two times higher frequency of misrejoining was observed under growing G1 phase for X-rays, while similar frequency of misrejoining was observed after high-LET irradiations.

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The synaptonemal complex proteins, SCP1, SCP2, and SCP3, are components of the synaptonemal complex (SC), a meiosis-specific protein structure essential for synapsis of homologous chromosomes. Among these, at least SCP1 is ectopically expressed in some tumors, suggesting that SC proteins might belong to the cancer/testis antigens group. However, the role of SC proteins in tumorigenesis is still unknown. In this study, we investigated the tumorigenic function of another SC protein, SCP3. Several cancer cell lines deriving from various human tissues ectopically expressed SCP3. Forced expression of SCP3 in retinal pigmented epithelial cells resulted in reduction of growth rates. These cells frequently had one, three or four copies of chromosomes, suggesting that SCP3 induced aneuploidy. Moreover, these cells also showed hypersensitivity to ionizing radiation. Therefore, ectopic expression of an SC protein, SCP3, leads to genetic instability and could initiate tumorigenesis.

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Hypersensitivity to DNA damaging agents and chromosomal aberration including numerical chromosomal abnormality are found in cells with mutated homologous recombination (HR) proteins. Numerical chromosomal abnormalities are generally classified into aneuploidy and polyploidy, both of which are assumed to be critical condition, as they are observed frequently in cancer cells. Rad51 paralogs share sequence homology to Rad51, a central player of HR. They have been shown to play roles in HR in chicken DT40 and rodent CHO cells. We generated human HCT116 cell lines with mutated Rad51 paralogs by gene targeting; Rad51B^+/-/-/-, Rad51C^+/-/-/- and XRCC3^-/- cells. Human Rad51 paralogs are shown to play a role in HR in HCT116 cells, since the mutants showed hypersensitivity to MMC and decreases in sister chromatid exchange. Interestingly, numerical chromosomal abnormalities were increased in these cell lines. Tetraploidy was increased in XRCC3^-/-, while aneuploidy was increased in Rad51B^+/-/-/- and Rad51C^+/-/-/-. In addition, centrosome fragmentation and slow growth caused by cell cycle checkpoint activation were observed in Rad51B+^/-/-/- and Rad51C^+/-/-/-. We will discuss the mechanism of the development of polyploidy and aneuploidy in these cells.

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It has been believed that the serious biological effect observed after irradiation of heavy ions is due to formation of clustered-DNA-damage which may be difficult to be repaired. However, it is difficult to detect such kind of damages experimentally. The purpose of this study is to estimate the yields and the configuration of DNA damages as well as the spatial distribution of the damages along to the tracks of heavy ions using the Monte Carlo simulation. A new Monte Carlo track structure code for any kind of ions from hydrogen to uranium has been developed and applied for simulation of the induction process of DNA damage. Induction of strand breaks and base damages were simulated with direct and the radical generation and diffusion using Monte Carlo methods for atomistic model of DNA. The number and configuration of damages on the DNA segment of a few helical turns were estimated. In our presentation, the framework of the developed track code for ions, the calculated DNA damage spectrum for some selected types of ions for different energies will be shown and discussed by comparison with the related RBE data of double strand breaks and cell death.

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Polypeptide growth factors not only stimulate growth and regulate differentiation of various cell types, but some of them also exert cell-protecting activity through multiple signaling pathways. Utilization of such growth factors thus maybe helpful for preventing cellular damage induced by irradiation. Here we assessed the potential of fibroblast growth factor-1 (FGF1) and FGF7 (also known as keratinocyte growth factor: KGF) for preventing ultraviolet (UV) irradiation-induced apoptosis of HaCaT cells, human immortalized keratinocytes. HaCaT cells were exposed to UV-B (10 mJ/cm²) and 24 h later the resulting apoptotic effect was evaluated by the reactivity of the cell surface with annexin V using a flowcytometer. Compared to control HaCaT cells, the cells pretreated with FGF1 or FGF7 in the presence of heparin showed significant reduction in the number of the annexin V positive cells in the FGF-dose dependent manner. The anti-apoptotic activity of FGF1, that has wider receptor specificity than FGF7, was larger than that of FGF7. Similarly, whereas expression levels of apoptosis-related genes, c-jun and IL-6, kept increasing in the control HaCaT cells until 24 h after irradiation by UV-B, those in the cells pre-treated with FGF1 or FGF7 were suppressed; revealing FGF1 as more effective than FGF7. The results indicate that both FGF1 and FGF7 are capable of suppressing UV-induced apoptosis of HaCaT cells, and suggest that FGF1 protects various cell types from UV-induced apoptosis. The signaling mechanism underlying this activity is being investigated.

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Nuclear magnetic resonance imaging (MRI) has been introduced to many hospitals as a useful diagnostic tool because they do not cause any injury or fatigue. A static magnetic field (SMF) is one of the key components of MRI. Although most MRI systems are made up of a magnet generating SMF at a magnetic flux density of 1.5 T, systems with a magnetic flux density of 3 T have recently been used for diagnosis, and MRI systems with a much higher magnetic flux density have been developed and applied for potential medical diagnostic use. However, few studies have been performed to evaluate the effects of SMFs on biological systems. On the other hand, two hundred million people worldwide suffered from diabetes in 2003, and the incidence is rapidly increasing. In diabetes, the loss of pancreatic beta-cell mass and function are crucial. In this study, we evaluated the effects of SMFs on insulin-secreting cells. INS-1 cells were exposed to SMFs at a magnetic flux density of 3, 6 or 10 T for 0.5, 1, or 2 hours. After exposure, the effects were assessed by WST-1 cell proliferation assay, measurement of cellular insulin content, and secreted insulin levels in culture medium during the exposure. No effects were observed under each exposure condition assessed by WST-1 cell proliferation assay and measurement of cellular insulin content. Insulin secretion during exposure was slightly decreased. These findings suggested that the effects of SMFs on insulin-secreting cells were littele.

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Centrosome amplification can be detected in the tissues of p53–/– mice. In contrast, loss of p53 does not induce centrosome amplification in cultured human cells. However, examination of human cancer tissues and cultured cells has revealed a significant correlation between loss or mutational inactivation of p53 and occurrence of centrosome amplification, supporting the notion that p53 mutation alone is insufficient to induce centrosome amplification in human cells, and that additional regulatory mechanisms are involved. It has recently been shown that gamma irradiation of tumor cells induces centrosome amplification. However, the precise mechanism of radiation-induced centrosome amplification is not yet fully understood. In the present study, CCD32SK diploid normal human fibroblasts were transiently transfected with short interfering RNA (siRNA) specific for human p53 (CCD/p53i). There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (4.0%) without irradiation. In contrast, CCD/p53i cells after 5-Gy irradiation showed a marked increase in abnormal nuclear shapes and pronounced amplification of centrosomes (46.0%). At 12 h after irradiation, irradiated CCD/p53i cells were arrested in G2 phase. On laser scanning cytometry, abnormal mitosis with amplified centrosomes was frequently observed in the accumulating G2/M population at 48 h after irradiation. In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradiation, efficiently induces centrosome amplification and nuclear fragmentation, but these phenomena were not observed with either siRNA-mediated silencing of p53 or irradiation alone.

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We studied the localization of phosphorylated H2AX (γ-H2AX) in cultured human fibroblasts (NB1RGB) and GFP-tagged rad51 in Chinese hamster CHO cells after irradiation with heavy ion beams. Asynchronous cells were irradiated with X-rays, carbon ion beam (LET is about 30 or 88 keV/μm), Si ion beam (220 keV/μm), Ar (95 keV/μm) and Fe ion beam (440 keV/μm) at room temperature. Gamma-H2AX in irradiated cells was detecteded by immuno-staining from 0 to 24 h after irradiation. Gamma-H2AX monitored by using flow cytometry increased just after irradiation of each radiation and reached maximum around 30 min. Gamma-H2AX was then decreased quickly for cells irradiated with X-rays but presented longer for Si and Fe beams. Induction of γ-H2AX was similar between Si and Fe beams and bigger than X-rays. Under the confocal microscopy, foci of γ-H2AX on cell nucli was not visible just after X-irradiation, but obvious after Fe ion irradiation. In contrast to the data obtained by flowcytometry, induction of foci per nucleus was similar for all the radiations tested at 30 min after irradiation. On the other hand, rad51 accumulation was observed about 1 h after irradiation. Foci of rad51 were visible about 10 h after irradiation both for heavy ions and X-rays.

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To investigate the possible mechanisms of DNA damage repair in complex DNA aberrations produced by heavy charged particles, we demonstrated the experiments that various DNA repair deficient CHO cells were irradiated with X-ray and heavy charged particles (Carbon ions 290MeV/n LET13 or 70keV/micrometer, Iron ions 500MeV/n LET 200keV/micrometer) or treated with chemical agents, mitomycin C or cisplatin. DNA repair mutant CHO cell lines that we used in this study were V3 (DNA-PKcs deficient), xrs5 (Ku80 deficient), 51.D1 (rad51D mutant), and KO40 (fancg mutant). Asynchronous cell population was irradiated and immediately re-plated for colony formation assay for testing radio- or chemical sensitivities. Results will be presented in the meeting.

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Reactive oxygen species (ROS) are generated within cells by ionizing radiation via primary ionizing events as well as through secondary amplification systems including metabolic synthesis. Mediating oxidation of cell components, ROS not only cause dysfunction of the target molecules but also perturb intra- and inter-cellular signal transduction pathways. Our ultimate aim is to elucidate the contribution of ROS in cellular events, especially DNA damages, elicited by ionizing radiation. In the current study, we compared cellular responses to hydrogen peroxide (H₂O₂) and X-rays in DNA repair-deficient cells. XRCC4-deficient mutant cells exhibited lower survival rates than XRCC4-proficient parental cells after exposure to either H₂O₂ or X-irradiation. The LD37 values of the XRCC4^-/- and parental cells after a 1-hr treatment with H₂O₂ were 8.5 and 11.1 µM and those for X-irradiation were 0.5 and 1.7 Gy, respectively. XRCC4 is a component of the non-homologous end-joining, a predominant repair pathway for DNA double-strand breaks (DSBs). To date, no other functions of XRCC4 have not been reported yet. Thus, marked increases in death of the XRCC4^-/- cells exposed to H₂O₂ or X-irradiation must be attributed to DSBs. Consistently, γH2AX staining revealed a dose-dependent formation of DSBs in the X-irradiated cells (~50 DSBs/cell/Gy). However, the formation of DSBs was not so significant just after a 1-hr exposure to 0-50 µM H₂O₂ in both the mutant and parental cells. DSBs might be formed at later stages in the H₂O₂-treated cells through some long-term cellular response.

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Oxidative stress is believed to increase the risk of lifestyle-related diseases, such as cancer and heart disease. For the measurement of oxidative stress in vivo, 8-hydroxydeoxyguanosine (8-OH-dG) in DNA or urine is the most popular biomarker. However, there are some difficulties in the reproducible and accurate analysis of 8-OH-dG in DNA. In this study, we found that artifactual 8-OH-dG was elevated significantly with time in an analytical sample kept at 10 ˚C, but not for one kept at –80 ˚C. Furthermore, we developed a method for urinary 8-OH-dG analysis with high accuracy by an HPLC-ECD system, using anion-exchange- and reverse-phase-columns. This method can also be used for urinary and serum 8-hydroxyguanine (8-OH-Gua, free base) analyses, with a slight modification. By applying these improved methods, we confirmed the induction of oxidative stress with low dose (2 Gy) whole body X-ray irradiation of mice at a dose rate of 0.5 Gy / min. The 8-OH-dG levels in the mouse urine were increased about 4.2-fold by 2 Gy irradiation, in a dose-dependent manner. The 8-OH-Gua levels in the serum were also increased with 2 Gy of irradiation. These results suggest that our improved 8-OH-dG and 8-OH-Gua analysis methods are useful for measurements of oxidative stress in vivo.

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We have been prototyping a system which scans and detects dicentric chromosomes made by radiation from large amount of slide grasses, consists with an automated microscope and a computer for image recognition, for research of low-dose radiation and practicing of radiation accidents.
A Metaphase Finder is a part of the system, which automatically finds metaphases among the leukocytes of blood. It is known as a tool before radiation dosimetry of observing chromosomes by a microscope and counts the rate of dicentrics.
We have developed the Metaphase Finders and used at 7 domestic (increased 1 in this year) sites and 1 foreign site, but further development is continuing simultaneously. This year, we are reporting a new system whose camera for image processing is high-definition camera. It can capture the wide area at the same definition then its processing speed is become higher.
We are also to distribute the Training Software to show the normal and abnormal chromosomes from the database, which made the judgment of many viewers to be the same to the domestic sites.

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In this study, the effect of quantum ray irradiation product (bovine serum albumin which irradiated gamma-ray at 5 kGy) on DNA synthesis was examined. The mutation rate of DNA synthesis has determined for a single round of gap-filling reaction using M13mp18 DNA as a primer-template. The mutation rate was measured using the colored selection method. Then, the characteristics of mutation were examined using the sequence analysis.
The result shows that the mutation frequencies were 7.35×10^-5 for the control and 8.40×10^-4 in the presence of quantum ray irradiation product.
Mutation sites were determined by DNA sequencing. We sequenced 27 randomly chosen LacZ mutant plaques. The results show that the substitution mutations were located at the position 91 on the plus strand of LacZ gene, and that the mutation occurred next to homopolymeric runs.

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Ionizing radiation induces chromosome instability that is transmitted over many cell divisions after radiation exposure in the progeny of surviving cells. To know the transmissible trait of genomic instability induced by radiation, we transferred a human chromosome 6 or 8 exposed to 4 Gy of soft X-rays into unirradiated mouse recipient cells by microcell fusion and examined the numerical and structural changes of the transferred human chromosomes in the microcell hybrids detected by fluorescence in situ hybridization (FISH) using a fluorescent probe specific for the whole human chromosome 6 or 8. Transferred human chromosomes were structurally stable in all of eight microcell hybrids in which unirradiated human chromosomes 6 and 8 were introduced, suggesting that chromosome transfer itself does not destabilize the transferred chromosome. Interestingly, the copy number of transferred chromosome 8 but not 6 doubled in most of microcell hybrids in which both irradiated and unirradiated human chromosomes 8 were introduced, suggesting that human chromosome 8 is numerically unstable regardless of radiation effect. Multiple rearrangements involved in the transferred human chromosome emerged in four out of five microcell hybrids in which the irradiated human chromosome 6 was introduced, but in one out of four microcell hybrids in which the irradiated human chromosome 8 was introduced. Thus, the present study demonstrates that genomic instability is transmitted to the progeny of unirradiated cells by an irradiated chromosome and suggests that radiation effect on delayed chromosome instability is different between chromosomes 6 and 8.

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Recent studies have showed that the sharp of the LET-RBE curves of biological effects was different, when using different ion species even if similar LET values. We reported that the sharp of LET-RBE curves of cell killing and mutation induction were different, when using different ion species, such as carbon- neon- silicon- and iron-ion beams. Furthermore, we reported that deletion patterns of exons in mutants at the hprt locus were different when using different ion species. This study, we investigated LET and ion-species dependence in initially measured chromatid breaks and non-rejoining chromatid breaks induced by different ion beams. Normal human skin fibroblasts were irradiated with carbon-, neon-, silicon- and iron- ion beams generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Chromatid breaks were counted as excess fragments of prematurely condensed chromosomes using a technique of prematurely chromosome condensation (PCC).
The LET-RBE curves for initially measured chromatid breaks were not dependent on LETs and ion species. On the other hand, the LET-RBE curves of non-rejoining chromatid breaks for carbon-, neon- and silicon-ion beams showed a peaks around 85 keV/micrometer, 105 keV/micrometer, 133 keV/micrometer respectively, while it increased with increasing LETs for iron-ion beams.
These results suggest the LET and ion-species dependent biological effects could be affected by repair process of induced chromatid breaks.

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Accidental radiation exposure should be estimated as accurately as practicable. This aids physicians in deciding the appropriate medical procedures. Chromosomal aberrations in the peripheral lymphocytes are the most reliable indicators for biological dose estimation. The conventional method for estimating this dose uses score marker aberrations, such as dicentric and ring chromosomes in lymphocytes. However, because lymphocytes circulate in the peripheral blood, the dose estimated from these dicentric or ring values is the mean dose of whole-body cumulative radiation exposure. Therefore, in the case of partial body exposure, it is difficult to estimate the partial dose of radiation by this method. It is necessary to use the partial organ in order to estimate the dose of partial body exposure. The aim of this study is to establish an assay system to evaluate the dose of partial body exposure. For this purpose, we used the human hair root as the target organ and detected the indicators of dose estimation; further, we examined the method of culturing hair root cells for detecting chromosomal aberrations. The epilated hairs were dissected carefully, and single cells were isolated enzymatically. When the hair was irradiated with gamma radiation, DNA damage was detectable in the comet assay, and the cultured hair root cells were grown in vitro. These results suggest that dose estimation of partial body exposure is possible using hair root cells.

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Biological dosimetry is very important as the first step in the medical treatment of the irradiated persons. Since it has been indicated that the frequency of chromosome aberrations correlates with the radiation dose, it is considered to be the most reliable, specific and sensitive biomarker for dose estimation. In general, the peripheral lymphocytes are used for the analysis of the dicentric assay in the exposed persons. To obtain the accurate frequency of the dicentric chromosome in dose estimation, it may be significant to recognize clearly the primary constriction as the landmark of centromere on chromosome. It has also been suggested that the degree of chromosome condensation may have significant effects on the analysis of dicentric chromosome. In order to investigate the effects of Colcemid treatment on chromosome condensation, we compared the relative length of chromosome 2 in the lymphocytes which were treated with the concentrations of 0.01, 0.03, 0.05 and 0.1ug/ml for 2, 24 and 48hrs. It is very clear that the elongated chromosomes were obtained from the lymphocytes treated with 0.01ug/ml for 2 hrs. However, there was no significant differences in the relative length of chromosome 2 between the lymphocytes treated with 0.03, 0.05 and 0.1ug/ml for 24 and 48hrs.

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Biological dosimetry is considered to be very important as the first step in the medical treatment for the persons exposed to radiation. Since the frequency of dicentric chromosome correlates with the radiation dose, it is considered to be the most reliable, specific and sensitive biomarker for dose estimation. In the present study, a modified C-band technique was developed in order to analyze more accurately dicentric, tricentric and ring chromosomes in irradiated human peripheral lymphocytes. Instead of the original method due to the treatment with barium hydroxide Ba(OH)2, C-bands were obtained from the modification of heat treatment in formamide and following DAPI staining. This method was applied to the analysis of dicentric chromosome in irradiated human lymphocytes to examine its availability. The frequency of dicentric chromosome was almost equal between conventional Giemsa staining and the modified C-band technique. Although the analysis by Giemsa staining is comparatively difficult to recognize the centromere on the elongated chromosomes, the over-condensed chromosomes, a fragment and an acentric ring, our modified C-band method may be easier to identify the centromere on such chromosomes than Giemsa stain only. Thus, the modified C-band method may give more information about the location of centromere. Therefore, this method may be available and more useful for biological dose estimation due to the analysis of dicentric chromosome in human lymphocytes exposed to the radiation.

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The Long Evans Cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA double-strand break repair and is a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat completely covering the region associated with radiation susceptibility. We demonstrated that defective DNA repair in LEC is fully complemented by a 200-kb BAC, 65K18 in transient and stable transfected LEC cells. Further genetic analysis determined that the radiation susceptibility region is located in a 129-kb. We compared genome sequences of the region between Fischer 344 (F344) and LEC, and found that an intronless Rpl36a gene is inserted into the LEC genome, but not into the F344 genome. The gene expression of Rpl36a of LEC cells was lower than that of F344 cells, suggesting that the intronless Rpl36a in the region is a pseudogene like other intronless genes. We mapped four expressed sequence tags (ESTs) in the region. The expression of three ESTs was not different between F344 and LEC cells, but the remaining one (EST#4) was not expressed in LEC cells. To determine whether the EST#4 is associated with radiation susceptibility, we measured cell survival of EST#4 knockdown cells after irradiation. Survival of cells transfected with EST#4 siRNAs was not different from that of cells transfected with the negative control siRNA. From the present study, it is not clear whether EST#4 is associated with radiation susceptibility in the LEC rat. Further study will focus on cloning full-length of EST#4 and exploring more effective siRNAs.

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Radiotherapy is one of the three major treatment modalities used in eradicating malignant tumors. However, the existence of radioresistant cells remains a critical obstacle in radiotherapy and radiochemotherapy. In order to develop more efficient radiotherapy including suppression of the outbreak of radioresistant cells, we need to understand the features of radioresistant cells. Last year, we reported the establishment of radioresistant HepG2-8960-R cells those continue to grow even with daily exposure to 2 Gy of X-rays. Using neutral comet assay, we compared rejoining kinetics of DNA double strand breaks (dsbs) induced by X-rays between HepG2-8960-R cells and parental HepG2 cells. We also compared the induction rate of micronuclei and gamma-H2AX foci in both cells after administration of fractionated exposure to 2 Gy/day of X-rays for 5 consecutive days. The rejoining kinetics of dsbs in HepG2 cells was biphasic while that in HepG2-8960-R cells was monophasic, indicating that homologous recombination repair pathway is predominantly used in radioresistant cells. The numbers of micronuclei and gamma-H2AX foci were increased in HepG2, but not in HepG2-8960-R cells after fractionated exposure to X-rays. These results suggested that X-rays induced DNA dsbs were more accurately repaired in radioresistant cells than in HepG2 cells. Moreover, the number of micronuclei and gamma-H2AX foci after fractionated exposure would be the useful marker to predict cellular radioresistance.

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We have proposed that main route of radiation carcinogenesis is not targeted DNA molecules but induced from Long-Lived Radicals in intracellular proteins. Very recently we found Slow-Releasing Long-Lived Radicals (SRLLRs) in culture Syrian golden Hamster embryonic (SHE) cells those levels were gradually increased for 5h after gamma-ray irradiation of 4 Gy. Recent our experiments indicate that hydrogen peroxides derived from dysfunctional mitochondria by ionizing radiation, are responsible for producing SRLLRs. In order to confirm the effect of hydrogen peroxides on SRLLRs generation in the cells. DL-Buthionine-(S,R)-sulfoximine(BSO) or 3-Amino-1H-1,2,4-triazole(AT) for inhibiting glutathione synthesis or catalase, respectively, were treated to the cells for investigating relationships between the treatments and the levels of SRLLRs. Although increment of level of SRLLRs for 4 Gy irradiated cells at 5h after irradiation was 36%, that in BSO(0.1mM) treated cells was much increased as 86%. The treatment of AT and following gamma-ray irradiation of 4 Gy increased those levels 18 and 68%, respectively. These results indicate that glutathione or catalase ,as efficient scavengers of hydrogen peroxides in cells, are responsible for reducing levels of SRLLRs. Because addition of myxothiazole, as an inhibitor of electron transportation in mitochondria, suppressed the level of SRLLRs in irradiated cell, SRLLRs are likely to be produced by hydrogen peroxide from dysfunctional mitochondria by ionizing irradiation.

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In p53(+/+) mice, which were received a whole-body dose of 3 Gy X-rays at 8 weeks of age, T-cell receptor (TCR) mutation frequencies (MFs) increased to the maximum level 9 days after irradiation. The TCR MFs decreased to background levels at 16 weeks and 20 weeks of age, delayed TCR MF in irradiated mice increased from 60 weeks of age compared to that in non-irradiated mice. In p53(+/-) mice, delayed TCR MF in irradiated mice increased significantly compared to that in non-irradiated mice at 40 weeks of age. We suggested that the p53 gene plays a key role in the regulation of ionizing radiation-induced somatic mutation (Igari, K., et. al., Radiat Res, 166:55-60, 2006). We previously in the last meeting showed the reduction of apoptosis frequency and the expression of phospho-p53 at 72 weeks of age in p53(+/+) mice. We suggested that the rise in TCR MF in delayed mutation is caused by decreased Trp53-dependent apoptosis activity. Also, we showed the rise in translocation frequency in 11th chromosome. We speculated the p53 genome was mutated. We tried to detect p53 genomic mutations at 72 weeks of age in irradiated p53(+/+) mice, we extracted DNA from whole spleen, and then made PCR product in exon 4, 5-6, 7 and 8-9 of p53. However, we could not detect any mutation. In this study, we report how to select CD3-CD4+ cell from spleen cells and sequencing of the p53 genome in CD3-CD4+ cell, because CD3-CD4+ cell is the target cell in TCR MFs.

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We have recently reported a novel deletion in mitochondrial DNA (mtDNA). This deletion was induced in a cell line, HepG2 by radiation exposure. In addition, this novel deletion was detected by PCR only in the liver from patients administered with Thorotrast which emits alpha-particles. Therefore, this novel mtDNA deletion is considered to be a marker of radiation exposure. DNA base substitutions are frequently occurred in the D-loop region of mtDNA. In order to identify mutations specifically induced by radiation exposure, we analyzed the D-loop region. Whole genomic DNA separately extracted from tumor and non-tumor tissues of the liver from Thorotrast and non-Thorotrast patients was analyzed. Sequence analysis indicated that all of the mutations induced in the D-loop region associated with Thorortrast were transition type. Mitochondrial heteroplasmy of D-loop mutations was frequently observed in Thorotrast induced angiosarcoma (AS), Thorotrast induced intrahepatic cholangiocarcinoma (ICC) and non-Thorotrast-induced AS. Compared with non-tumor tissues the incidence of cases with mutations only in tumor part was higher in Thorotrast ICC than in non-Thorotrast ICC. No mutations including deletions specific to Thorotrast patients were found. These results suggest that mtDNA mutations are not induced by alpha-particles emitted from Thorotrast but are dependent on histological type of liver tumors.

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Exogenous genotoxic agents including irradiation randomly cause genetic damage on the genome and result mutations and chromosome alterations. To precisely understand the degree and character of the DNA damages by the agents, we must genome-widely analyze the genetic changes. Genomic DNA microarray technology enables to efficiently scan the whole genome and detect relatively small regions (<100kb) with deletions, amplification, and LOH. Using CGH-microarray (Agilent) and SNP-microarray, which are designed for detecting copy-number alterations and LOH, respectively, we characterize gamma-ray-inducing genetic alterations genome-widely. We irradiated human lymphoblastoid TK6 with 5Gy gamma-ray, and randomly isolated 25-survived clones (0.1% survival). Among 25 clones, 12 showed at least one chromosome alteration with >5cM in CGH- or SNP-microarray analyses. These chromosomes alterations were confirmed as deletion, amplification, translocation, or aneuploidy by Spectrum Karyotyping analysis. Deletions and amplifications were frequently observed as side-by-side on a chromosome, implying that Breakage-Fusion-Bridge cycle initiated by a DSB may contribute to generate the complicated changes. Four clones exhibited a same unreciprocal translocation with the breakpoint at chromosome 16q11.2. This breakpoint region may contain hot spot for ionizing irradiation.

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Mutation of Sfpi1 (PU.1) is crucial in the development of myeloid leukemia in mice exposed to radiation. The mutation is mostly missense point mutations at codon 235 in the DNA-binding Ets domain of PU.1. While they are unlikely to be a direct consequence of radiation-induced DNA damage, it is not evident when and how often this type of mutation arises in hematopoietic cells. To facilitate experimental approach to this issue, we have developed a technique that is able to detect a small number of mutated cells in the vast majority of non-mutated cells. The technique is based on the wild-type blocking PCR, in which LNA (locked nucleic acid) oligonucleotide blocks amplification of wild-type DNA during PCR while permitting amplification of mutants. A primer set to amplify a 443bp region within exon 5 of Sfpi1 was applied simultaneously with an 11mer LNA oligonucleotide that covers the mutation hot-spot in leukemic cells. Adjusting reaction condition has enabled two-step PCR to detect mutant DNA in 10³ times amount of wild-type DNA. DNA samples of exposed mice that are not affected with overt leukemia is being analyzed for the leukemia-specific Sfpi1 mutation with this technique.

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Chromosomal rearrangements are implicated in the etiology of T-cell lymphomagesesis and Rag-dependent and independent pathways participate in mouse thymic lymphomagenesis. However, Rag-dependent and -independent mechanisms for the rearrangements of cancer-related genes are not well characterized. We present the molecular mechanisms for Notch1 rearrangements by comparing susceptibility of Rag2^-/-, Atm^-/-, scid, and doubly mutated mice to thymic lymphomagenesis and by identifying sequence specificities of the junctions of Notch1 rearrangements in thymic lymphomas. Rag2^-/- mice spontaneously develop thymic lymphomas via Rag2-independent pathway. Scid mice develop radiation-induced thymic lymphomas mainly via Rag2-independent pathway. Atm^-/- mice develop them via both pathways. Total abnormalities in the Notch1 including rearrangements and mutations exceed 80% in thymic lymphomas, making Notch1 the most important oncogene for thymic lymphomagenesis. Analyses of Notch1 rearrangements show that Rag2-dependent mechanisms are the illegitimate V(D)J recombination and the Rag2-mediated pathway where Rag-cleaved DNA end and another DNA break are involved. Rag2-independent mechanisms include the microhomology-mediated end-joining, the nucleotide addition in deletion, the insertion of intracisternal A particle, inversion, duplication, and translocation. These mechanisms bring about the generation of Notch1 rearrangements, depending on the defects of mutant mice in repair and V(D)J recombination, indicating that errors in V(D)J recombination and DNA repair are involved in lymphomagenesis through the formation of rearrangements.

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Oxygen radicals are produced through normal cellular metabolism, and the formation of such radicals is further enhanced by exposure to either ionizing radiation or various chemicals. The oxygen radicals attack DNA and its precursor nucleotides, and consequently induce various oxidized forms 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. MUTYH is a DNA glycosylase that excises adenine or 2-OH-A incorporated opposite either 8-oxoG or guanine, respectively, thus considered to prevent G:C to T:A transversions in mammalian cells. The Mutyh-deficient mice showed a marked predisposition to spontaneous tumorigenesis in various tissues when examined at 18 months of age. The incidence of adenoma/carcinoma in the intestine significantly increased in Mutyh-deficient mice, as compared with wild-type mice. This high susceptibility of the mutant mice to intestinal tumor-development was well correlated with the condition observed in MAP (MUTYH-associated polyposis) patients. We performed mutation analysis of the tumor-associated genes amplified from the intestinal tumors developed in four mutant mice that had been treated with KBrO₃. Many tumors had G:C to T:A transversions in either Apc or Ctnnb1. No mutations were found in either k-ras (exon 2) or Trp53 (exon 5-8). Our findings indicate that the abnormality in the Wnt signaling pathway is causatively associated with oxidative stress-induced tumorigenesis in the small intestines of the Mutyh-deficient mice.

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Thoracic exposure to ionizing radiation is an established risk factor of breast cancer. Though there are numerous environmental carcinogens, their risk evaluation and regulation, including those for ionizing radiation, are carried out individually based on the assumption that their interactions are additive. We previously reported that rat mammary carcinoma was induced by a combination of ionizing radiation and methylnitrosourea (MNU) via an unexpectedly complex mechanism (Int J Cancer 115: 187-193). We herein analyzed its detailed mechanism by using Affymetrix expression microarrays. Female Sprague-Dawley rats were either gamma-irradiated (1 Gy), injected intraperitoneally with MNU (40 mg/kg) or subjected to their combination at 7 weeks of age, and observed until 50 weeks of age on a high corn oil diet with weekly palpations. Mammary tumors were examined histologically and only those diagnosed as carcinomas were used for H-rras mutation and microarray analyses. As a result, 0%, 54% and 78% of carcinomas harbored H-ras mutations in the radiation-alone, MNU-alone and combination-treated groups, respectively (P < 0.05 between the latter vs. either one of the formers), indicating that the combined exposure enhanced the development of carcinoma that carries H-ras mutation. We next compared the gene expression profiles between these groups and identified genes that were overexpressed in combination-induced carcinomas with H-ras mutation compared to MNU-induced counterparts. These results suggest an association between overexpression of these genes and the radiation enhancement of mammary carcinoma development with H-ras mutation.

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 Heterozygosity leading to haploinsufficiency for proteins involved in DNA damage signaling and repair pathways plays a role in tumorigenesis. Animals haploinsufficient for a specified protein have as low tumor development rates as their wild-type counterparts in normal conditions, however, they show much higher tumor incidence rates after DNA is damaged by exogenous stimulations. Mice heterozygous for DNA repair genes have a similar life span to the wild-types when not challenged with mutagens like ionizing radiation, nevertheless, individuals of different genotypes respond differently to the same stress conditions. Predisposition to cancer of individuals heterozygous for a specified gene is age-dependent and results in tumor development not in early stage but in aged one. Population carrying a certain heterozygous gene is close to tumor incidence while that carrying homozygous mutated gene is extremely lower. Apparently heterozygosity when one allele of a gene is inactivated contributes to tumorigenesis. Since one human cell has 20,000-25,000 protein-coding genes, it is quite possible that an individual carries more than one heterozygous gene. We suppose that heterozygosity for two or more genes critical for pathways controlling DNA damage signaling, repair, cell cycle checkpoints, apoptosis, and so on, may enhance tumor initiation.
 To verify this hypothesis, cells heterozygous for ATM, BRCA1, and both, were taken as experimental models since the both genes are key factors involved in DNA damage signaling and repair and related with each other. In particular, clinical statistics revealed that breast cancer in AT family is 1.5-14 times higher. In order to minimize the difference in genetic background except for the interested genes, mouse and mouse embryonic fibroblast (MEF) cells were isolated from littermates.
 The transformation of MEF cells exposed to 0.5Gy of 1GeV/n Iron was scored by morphological recognition. Transformation frequency of ATM/BRCA1 double heterozygotes was 7.2-, 3.5- and 2.0-fold higher than that of wild-type cells, ATM single heterozygotes and BRCA1 single heterozyogotes, respectively.
 Thymocytes are sensitive to radiation and they die in an apoptotic way. The main sub-population of thymocytes (CD4⁺CD8⁺ cells) was measured by using flow cytometer. The amount of wild-type cells, ATM single heterozygotes, and BRCA1 single heterozygotes decreased 86.5-, 44.0-, and 38.1- times, respectively, in 24 hours after exposed to 5Gy of γ-rays while that of ATM/BRCA1 double heterozygotes decreased only 9.8- times.
 γH2AX focus forming reflects DNA double strand breaks. Foci in wild-type cells exposed to 0.5Gy of γ-rays reached to the maximum as rapidly as 15min but it took ATM/BRCA1 double heterozygous cells 30min, which verified that double haploinsufficiency for ATM and BRCA1 had negative impact on DNA damage signaling.
 Four kinds of cells showed various kinetics of cell cycle progression although all of them were arrested in G2/M phase after exposed to the same dose of γ-rays. G2/M block was prolonged in ATM single heterozygotes but abrogated in BRCA1 single heterozygotes. Double heterozygotes for ATM/BRCA1 showed a compromised cell-cycle dynamics and the curve was similar to that of wild-type cells.
 In summary, haploinsufficiency resulted from heterozygosity for ATM and/or BRCA1 has negative impact on various pathways. It increases cell susceptibility to exogenous stress like ionizing radiation in transformation induction by disturbing cell cycle checkpoint, delaying DNA repair, and suppressing apoptosis. Consequently unrepaired lesions are transmitted to daughter cells, which leads to cell transformation and tumor initiation. Double heterozygosity multiplies the probability of tumor formation.

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