Alpha-tocopherol monoglucoside (TMG) exhibits high antioxidant activity and radioprotective effect in mice irradiated at a dose of 6.6 Gy (Murase et.al, 1997, 1998, Shimanskaya et.al, 2001). We investigated TMG effect on irradiated mice hematopoiesis. CBA mice were whole-body exposed to 5.6 Gy of gamma rays (0,5 Gy per a minute) with using RUM-17 source (copper 0.5 mm + 1.0 mm aluminum, 200 kV, 5 mA). TMG was administered intraperitoneally at a dose of 600 mg/kg immediately after irradiation. Bone marrow and blood cell count were estimated. The largest reduction of myelokaryocytes amount was observed on day 8 after irradiation, and their level was twice higher in TMG treated animals as compared to control. The decrease of lymphocyte percentage in the bone marrow was also two-fold less pronounced in TMG treated mice versus control. TMG administration appeared to clearly intensify hematopoietic recovery starting on day 14 after irradiation as measured by the increase in bone marrow cellularity. The total level of blood leukocytes and their different components count was significantly higher in TMG treated mice in comparison with that of control mice for all days of observation. These data suggest, that TMG administration immediately post mice sublethal dose of gamma irradiation promotes hematopoiesis recovery.

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The effects of the heat shock protein 90 (Hsp90) inhibitor were examined on the radiosensitivity and signal transduction pathways in human cells. Cell lines were incubated for 16 hr in medium containing Hsp90 inhibitor geldanamycin The cells were then irradiated with X-rays and incubated with the drug for a further 8 hr. Geldanamycin sensitized human tumor cells, but not normal cells, to radiation. 17AAG, a geldanamycin derivative, also sensitized human squamous carcinoma cell lines. It was found that 17AAG reduced the expression of Akt and phosphorylated Akt. In addition, the ratio of apoptotic cells increased in tumor cells after a combination of 17AAG and X-rays. A similar results were obtained in tumor cells grown as multicellular spheroids, in vitro human tumor model. In conclution, targeting Hsp90 with 17AAG provides a promising strategy for radiosensitization of radioresistant carcinoma.

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Inhibition effects of ginseng on proliferation of the infectious Corynebacterium Kutcheri of the caecum in the whole body irradiation mice were studied. The mice (male ICR, 5weeks of age) were exposed to X rays (125Kv,217cGy/min) of 3 to 6Gy and were infected with Corynebacterium Kutc-heri peroral(p.o.). These mice were given 30% EtOH extract of ginseng p.o. at 40mg/0.2ml/mouse every other days for 5 times. The results obtained in the present study were as follows. 1. The bacteria count in the caecum and detection rate of the bacteria in the oral cavity, tracheal region of mice were increased in proportion to exposure dose. 2. The bacteria counts in the caecum and positive detection rate of the bacteria in the oral cavity, tracheal region of mice were decreased in the ginseng administration groups. 3. Life prolongation of the ginseng administration mice was increased, compared to the experiment control(irradiation + infection, no administration of ginseng. on the day 10 after irradiation).

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We have measured in vitro quenching of reactive oxygen species (ROS; superoxide anion radical and hydroxyl radical) and reactive nitrogen species (RNS; peroxynitrite) by tocophrol monoglucoside (TMG), a water-soluble vitamin E derivative, with ESR spin trapping and HPLC analysys, respectively. In vivo radioprotection by TMG was also evaluated with colony assay using L5178Y cells and 30 day survival of C3H mice after X-ray irradiation. TMG showed similar quenching activity against ROS and RNS with Trolox, another water-soluble vitamin E derivative. The quenching of superoxide radical by TMG or Trolox (IC₅₀=290 µM) was effective more than one order of magnitude than that of hydroxyl radical. In vivo radioprotection of C3H mice was observed by i.p. administration of TMG (about 650 mg/kg) either pre- or post-irradiation (whole body 7 Gy). In addition, the administration even at 30-60 min after irradiation was still effective for extending the survival.

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PCS syndrome is the human mitotic-spindle checkpoint disorder, characterized by mosaic variegated aneuploidy (MVA) and premature chromatid separation (PCS). The clinical manifestations include severe microcephaly, Dandy-Walker anomaly, and development of Wilms tumor. To determine the molecular basis of the disorder, we established immortalized skin fibroblast cell lines from Japanese patients. We found that the expression level of BubR1 protein was remarkably decreased in PCS cells, and only faint BubR1 signals were detected on kinetochores by immunofluorescence analysis. In PCS cells, p55cdc also failed to associate with the kinetochores. These abnormal features were normalized after introduction of BubR1 cDNA. Sequence analysis of the patients' cells detected no mutations in the BubR1 gene. These results indicated that the reduced expression of BubR1 protein might result in abolished kinetochore localization of p55cdc, and cause the mitotic checkpoint defects in PCS cells.

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UV sensitive syndrome (UV^sS) is a rare autosomal recessive disorder characterized by solar sensitivity and freckling, but no neurological abnormality and skin tumors. It is known that UV^sS does not belong to any complementation groups of xeroderma pigmentosum (XP) or Cockayne syndrome (CS), and that UV^sS lacks an ability of transcription-coupled repair (TCR). This fact suggests that UV^sS belongs to a new category of photosensitive disorder with defective TCR. To identify the gene responsible for UV^sS cells, UV^s1KO, we have performed microcell-mediated chromosome transfer based on functional complementation of its high-UV sensitivity. We found that UV^s1KO cells acquired the UV-resistance when chromosome 10 was transferred. Since the CSB gene is located on chromosome 10, we sequenced the CSB cDNA. Surprisingly, the homozygous nonsense mutation was detected on the CSB cDNA in the UV^s1KO cells. These results indicated that UV^sS is caused by the mutation on the CSB gene. To confirm these results, we transfected the wild type CSB cDNA into UV^s1KO cells. The transfectants exhibited the same UV-resistant character as wild type cells. These data indicated that UV^sS is caused by the mutation of CSB gene in spite of the absence of any growth and neurological failure observed in CS-B patients.

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The lymphoblast cells from a Japanese patient, characterized by severe combined immunodeficiency, growth retardation and congenital malformation, showed high incidence of both of spontaneous and radiation-induced chromosome aberrations. Here, we show that the cultured skin fibroblast cells from a patient are markedly sensitive to ionizing radiation. Western blot analysis revealed normal expression of NBS1, underlying gene of Nijmegen breakage syndrome, which shares the common features with the Japanese patient. The patient cells also showed normal expression of Mre11/Rad50, components of complex with NBS1, and formed their radiation-induced foci at the level similar to that of normal cells. Furthermore, the cells showed the normal phosphorylation of Chk2 kinase after irradiation. These results indicated that the syndrome reported here is unlikely ataxia-telangiectasia and Nijmegen breakage syndromes, suggesting a novel radiation sensitive disease.

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Ionizing radiation (IR) induces DNA doublestrand breaks(dsb), which are the major cause of the detrimental effects of radiation. DNA dsb activate checkpoint protein, that is ataxia telangiectasia mutated (ATM) kinase is triggered by its autophosphorylation at serine 1981, and phosphorylated ATM protein forms focus. And alsop53 binding-protein 1 (53BP1) was reported to form foci at DNA dsb, whose foci were colocalized with phosphorylated ATM foci. To investigate the dosedependency of induction of DNA dsb with low doses of X-ray, we examined the number and morphology of foci of phosphorylated ATM and 53BP1 induced by X-rays with doses between 1cGy and 100cGy using an immunofluorescence method. As expected, the number of foci of phosphorylated ATM and 53BP1 were found to be induced dose-dependently. In addition, the morphology of the foci were induced by both 1cGy and 100cGy were similar, indicating that DNA double strand breaks induced by low dose ionizing radiation are not qualitatively different from those by higher doses of radiation.

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Recently, we found that the accumulation of iNOS in wtp53 cells was induced by chronic irradiation with gamma rays followed by acute irradiation with X-rays, but not by each one, resulting in an increase in nitrite concentrations of medium. It is suggested that the accumulation of iNOS may be due to the depression of acute irradiation-induced p53 functions by pre-chronic irradiation. We found that chronic irradiation with gamma rays did not inhibit the accumulation of p53 after exposure to the conditioned medium from the irradiated mp53 cells. However, the decay of accumulated p53 was stimulated by chronic irradiation with gamma rays. At the same time, the accumulation of Hdm2 was observed; suggesting that chronic irradiation with gamma rays may stimulate the degradation of p53 accumulated by NO-mediated bystander effects. Furthermore, we found that the radiosensitivity of wtp53 cells against acute irradiation with X-rays was reduced after chronic irradiation with gamma rays. This reduction of radiosensitivity of wtp53cells was nearly completely suppressed by the addition of NO scavenger, carboxy-PTIO to the medium.
This reduction of radio-sensitivity of wtp53cells is just radiation-induced adaptive response, suggesting that NO-mediated bystander effect may considerably contribute to adaptive response induced by radiation.

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Purpose. The report is the study of the effect of nanosecond impulse irradiation of X-ray by SINUS-150 accelerator on tumor and normal cells proliferation. Materials and Methods. The SINUS-150 accelerator (peak electron energy 280 keV, peak beam current 4 kA, irradiation pulse rates is 6-25 Hz, pulse duration 4 ns) was developed by our Institute. The X-ray impulse irradiation dose rate is up to 0.05 mGy/pulse, the energy range of photon beam is 80-160 keV, the specific depth of penetration is about 7 cm. Total irradiation dose ranged between 36 and 144mGy. Cell proliferation was assayed by 3H-thymidine incorporation to DNA. Results. The 3H-thymidine incorporation of different tumor cells lines was almost completely inhibited by exposing to pulse irradiation with some different impulse frequencies. P-815 mastocytoma cells treatment with pulse irradiation resulted in a significant decrease of cell proliferation by 97%. On the contrary, no any inhibitory effect was detected after their exposition with the same doses of stationary irradiation. Inhibitory effect of pulse low dose X-ray on cell proliferation was depended on impulse frequency, but not dose of irradiation. Impulse X-ray had no effect on lymphocyte proliferation. Conclusion. The nanosecond impulse irradiation of low dose X-ray brought about high inhibition effect on tumor cells proliferation without damage of normal cells.

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Prenatal exposure to ionizing radiation of low doses in rodents induces deceleration of neuronal migration during cortical histogenesis. On the contrary, exposure to carbon beams during fetal period mainly affects cell survival. However, the molecular mechanisms underlying such difference in the effects between exposure to carbon beams and exposure to X-rays remain unknown. As the first step to shed light on the underlying mechanisms, we have attempted to elucidate whether the changes of gene expression after exposure to heavy ions differ from those after X-rays in fetal brains. Rab6A and its effector protein Rab-KIFL, involved in Golgi-to-endoplasmic reticulum retrograde transport, were found as the gene showing up-regulation in X-irradiated fetal brain. The changing pattern of expression of these genes was different between brains exposed to heavy-ion beams and those to X-rays. We report here the comparison of dose-dependent and temporal changes of mRNA expression of these genes in cell lines (Neuro2a, NIH/3T3) between exposure to carbon beams and exposure to X-rays.

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Since studies on the hemopoietic system have been recognized to be radio-sensitive, the mechanisms underlying cell proliferation and differentiationare well investigated.
We show here the effects of a continuous irradiation with low dose-rate ionizing radiation on the hemopoiesis of mice. SPF(specific pathogen free) female C3H/HeN mice were irradiated with various doses of 1-8 Gy ¹³⁷Cs γ-rays at the dose rate of 20 mGy/22h/day. After irradiation, the number of hemopoietic cells in the bone marrow was determined by the methods of CFU-S and CFU-GM assay, and the number of peripheral blood cells was also counted. It was shown that the day 12-CFU-S decreased as the dose increased. Decrease of 7days-CFU-S colonise and CFU-GM colonies. All of these colony-forming cells were reduced to about 50 % of the unirradiated controls at the dose of 8 Gy. Regardless of a decrease in the number of hemopoietic stem cells, no remarkable changes were observed in the number of peripheral blood cells, indicating the possibility of an enhanced differentiation of the precursor cells.

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A threshold was found in the dose-response relationship for Drosophila germ line mutation induced by X-ray irradiation. In the last annual meeting, we have reported that X-ray irradiation induced somatic mutation in Drosophila and that a threshold existed in the dose-response relationship. The threshold in a DNA repair defective strain was smaller than the one in the repair proficient wild type. Most of the results of the sex-linked recessive lethal assays performed in the early 20th century supported the linear non-threshold model. In these studies, however, mature sperms that lacked active DNA repair function were used. Our results suggest that a threshold can exist when the repair proficient immature sperms are used. Here, The sex-linked recessive lethal assay was performed using pre-meiotic young sperms. It was found that (1) mutation was induced in sperms irradiated with 10Gy (dose rate=0.5Gy/min) in a significantly higher frequency than in the non-irradiated control sperms (2) mutation frequency in the sperms irradiated with 10Gy (but dose rate=0.05Gy/min) was the same as control and (3) mutation frequency in the sperms irradiated with 0.2Gy (dose rate=0.05Gy/min) was lower than in control. A threshold was recognized in the dose-response relationship for mutation in immature sperms.

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In 1987, Planel et al. reported growth inhibition of Paramecium tetraurelia by shielding from background radiation. Here we examined this growth inhibition with an outperforming shield apparatus. We placed paramecium cultures in two identical chambers; the control chamber was a foam polystyrene box, and the shielded chamber was a foam polystyrene box but covered with aluminum film and surrounded by iron wall 15 cm thick and paraffin wall 10 cm thick. The temperature of both chambers was controlled by circulating water in constant temperature and the difference of the temperature between the control and shielded chambers was less than 0.1 degree C. Gamma ray dose rates, measured by a NaI scintillation counter, were 2.1 nGy/hr in the shielded chamber and 109 nGy/hr in the control chamber, and neutron, measured by a BF₃ detector, was diminished to 1/6 in the shielded chamber. We observed proliferation of paramecia for 10 days, however, we could not find any differences of their growth. Under our condition we could not admit the growth inhibition by shielding of background radiation.
Acknowledgment: Prof. Y. Takagi (Nara Women's University) kindly helped us to culture Paramecium tetraurelia.

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The reversed dose rate effects of mutation induction are often reported for fission neutron radiation. Our former investigation revealed that the reversed dose rate effects might be caused by hypersensitivity of G2/M cells for mutation induction by high LET radiation. To clarify whether this phenomenon could occur in any high LET radiations, a novel hyper-sensitive mutation detection system has been developed. The system uses hamster fibroblast cells carrying a normal human X-chromosome, and we found that the mutation frequencies obtained from our system is 100-fold higher than that from conventional system using an internal Hprt gene. This suggests that the system appears to be able to detect a wide spectrum of mutations, even mutations that affect expression of any important genes in neighbor of the Hprt. A lower limit of radiation dose for mutation induction with this system was found to be around 0.1 Gy. Using this system, we are investigating low dose-rate effects of heavy particle beam (carbon 290 MeV/u beam) with different LETs.

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In Caenorhabditis elegans (C. elegans), it is confirmed a radiation hormesis which extends life span by exposing to short term oxygen radicals. We have clarified that daf-16 gene which is a homolog of human fork head transcription factor is associated with hormesis. That is, daf-16(m26) animal has short lifespan and its lifespan extension by holmesis was not observed. The purpose of this presentation is to demonstrate an activated mechanism of the downstream genes which is directly related to the lifespan extension by hormesis.
Comparison of mRNA level using cDNA microarray between daf-16(m26) and age-1(hx546) animals showed decreased expression of metallothionein, protease and anti-oxidative genes in daf-16 animal. A mutation of age-1 gene functioned in the upstream within the signal transduction pathway of the DAF-16 leads to a remarkable lifespan extension by hormesis. In these genes, we measured the expression levels of genes with eight bases of the DAF-16 consensus binding element (DBE) in the 5'-UTR, and demonstrated that the expression of daf-16 mutant was less compared to age-1 mutant. Therefore, to express the life span extension effect by radiation hormesis, it is necessary for anti-oxidative and proteolysis systems to be activated. The possible action of daf-16 gene in the upstream for the activation is also suggested.

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Some epidemiology studies have reported that the relationship between radon and lung cancer was positive. Recently it was revealed that high thoron concentration exist at one of the reported area by our examination. Thoron is the isotope of radon, and its half-life is shorter than radon. On the other hands, the half-life of thoron decay products is longer than those for radon. Alpha energy of thoron decay products is also higher than radon. Dose conversion factor of thoron is also higher than radon. Thus thoron may affect the relationship between radon and lung cancer. To consider for effects of thoron, the biological experiment should be done with same experiment system of radon. The biological experiment for thoron is rare, so we tried to expose thoron with in vitro condition. The air-liquid interface culture that was constructed for radon experiments was used to culture system for thoron exposure. The preliminary data for thoron experiments will be reported.

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Linear non-threshold (LNT) model is a basic theory for radioprotection, but the adaptability of this hypothesis to biological responses at low doses or at low dose rates is not sufficiently investigated. In this study, some methods for quantitative risk assessment in dose response of biological reaction to low dose gamma radiation are proposed.
For defining statistically no risk level, we adapted some statistical models to quantitative data at low dose with statistically sufficient accuracy, using micronucleus formation of human osteosarcoma, and compared them with other statistical indexes corresponding to "threshold limit". The estimated values of statistically no risk levels remarkably increased with an increase of irradiation time. This indicates that the risk is reduced at low dose rates. Moreover, the values of statistically no risk levels and their confidence intervals depend on definitions of the value and on statistical models. These results suggest that we should statistically define the term "threshold" and include dose rate effects in the risk assessment model for estimating the low dose radiation risk and its uncertainty.

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LNT model is a basic theory for the regulation of radioprotection, it does not consider irradiation time or dose-rate. We have established Modified Exponential (MOE) model describing dose-rate effect of ionizing radiation. The model is not based on any physiological mechanism, but is derived from curve-fitting to data by an optimal function. Another model is designed based on a simple mechanistic hypothesis. The assumption is that the effects during one generation are restricted only in the generation, in other words that the effects in the previous generation are cancelled in the next generation. Cells then receive full dose from instantaneous dose, whereas the effects reduce with longer exposure. The model gives an iso-effect curve on the dose-dose-rate domain, converging to total dose as an asymptotic line in the high dose-rate region, and also to an asymptotic line with dose rate of total dose/generation time in the low dose-rate region. The data of the micronucleus formation with the rate 22% are compared with the model. The match was good in data up to 1200h, but the data shifted to higher dose-rate when exposure further extended. For the data of the inhibition of [³ H]thymidine uptake, the data for 50% inhibition showed fairly good agreement up to 170h, but the data shifted to low dose-rate in longer exposure.

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Cyclin kinase inhibitor p21 contributes to cellular responses to DNA damage, and its function decides the ultimate fate of damaged cells. Regulation of the expression of p21 in response to DNA damage has been extensively studied both at mRNA and protein levels. As for the degradation by proteasome, various characteristics of p21 have been reported; whereas p21 is ubiquitinated, p21 is degraded by proteasome without its ubiquitination, and the C-terminal 140-164 of p21 directly associates with proteasome 20S subunit. In this study, we present the region in p21 indespensable for the resistance to proteasome. We transfected eukaryotic cells with various tagged p21 deletion mutants and assessed the levels of del p21 expression with or without treatment with a proteasome inhibitor, lactacystin. High level of expression of full length (1-164), 1-157, or 1-147 was detected without lactacystin treatment, and lactacystin by itself did not affect the level. Theexpression of del1-20, del21-40 or del41-60 was very low without lactacystin, and lactacystin treatment significantly enhanced del p21 expression. The substantial level of expression of del61-80 was detected irrespective of the lactacystin treatment. To analyze the contribution of the structure of N-terminal region, we tested the deletion of α helix structure, 15-48 aa. The del26-35 was unstable without lactacystin, while del36-48 was stable. At least, 26-35 aa region may confer the stability of p21.

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Protein kinase C (PKC) plays an important role in radiation-induced apoptosis. However, each function of PKC subtypes remains unclear. In this study, signaling pathways of radiation-induced activation of PKCδ were assessed in radiation-sensitive mouse thymic lymphomas. We have already demonstrated that PKCδ is an important regulator in the radiation-induced apoptosis in 3SBH5 cells, one of radiation-sensitive mouse thymic lymphomas. Indeed, PKCδ signal detected by Western blot in cytosols of 3SBH5 cells was outstandingly decreased after irradiation. In contrast, in XR223, the resistant cells derived from 3SBH5, the PKCδ signal in the cytosols was decreased very slightly. Interestingly, the signal of endogenous PKCδ in cytosols in XR223 was stronger than that in 3SBH5 cells. On the other hand, mouse thymic lymphoma cells derived from Atm null(-/-) mouse were radioresistant than those from Atm wild-type mouse in terms of apoptosis. Besides, degradation of PKCδ by irradiation was observed in Atm(+/+) cells but not in Atm(-/-) cells. However, there was no difference in the distribution of endogenous PKCδ between the two cell lines. The regulation of PKCδ distribution, which is presumably distinct from the AT-mediated cascade, may determine radiation sensitivity in 3SB lines(3SBH5 and XR223).

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We have investigated the role of epidermal growth factor receptor (EGFR) and insulin-like growth factor I receptor (IGF-IR) in heat-induced activation of ERK and Akt. We utilized mouse embryo fibroblasts (MEFs) devoid of endogenous IGF-IR (R-) and MEFs overexpressing human IGF-IR (WT) and examined the activation kinetics of ERK and Akt following heat shock treatment. There were no differences in the kinetics or temperture dependence of activation of either ERK or Akt between the cell lines and the activation of IGF-IR or EGFR was not observed in MEFs. However, treatment of AG1478, which is a specific EGFR tyrosin kinase inhibitor, inhibited ERK activation following heat shock, but not Akt activation. Therefore, these results suggest that IGF-IR does not contribute to the heat shock-induced activation of ERK or Akt and that basal EGFR-TK activity, but not EGFR activation, is required for heat-induced ERK activation.

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It has been reported that ionizing radiation activates epidermal growth factor receptor (EGFR). Because over-expression of EGFR makes cells radio-resistant and inhibition of EGFR makes cells radio-sensitive, activation of EGFR is thought to be related to cellar radiation sensitivity. Activation of EGFR by ionizing radiation is not due to the binding of ligands to EGFR and further mechanisms remains to be elucidated. We are studying mechanisms of radiation-induced EGFR activation. The experiments were carried out using MDA-MB-468 and A431 cells. ERK was activated immediately after and 2-6 h after 2 Gy irradiation. These activations were inhibited by a EGFR inhibitor tyrphostin AG1478. SHP-2 was also activate immediately after and 2-6 h after 2 Gy irradiation. However phosphorylation of EGFR at Tyr1068 was decreased after 2 Gy irradiation. This contradicts with the results with tyrphostin AG1478. We further examined effects of 2 Gy irradiation of phosphatase activity using the pTPP assay system. Two Gy irradiation did not affected the phosphatase activity 30 minutes-6 h after irradiation. We are now investigating on the mechanisms of EGFR activation without auto-phosphorylation.

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Gene expression analysis is one of the most useful strategies to understand comprehensive biological responses. We applied a novel method (HiCEP) to normal human cells. The cells (HFLIII) were allowed to grow confluent before irradiation with X-rays (0.01, 0.1, 2.0 and 4.0 Gy). Total RNA was extracted 0, 2 and 4 hours after the irradiation and subjected to the expression profiling by the HiCEP. Approximately 30,000 peaks (presumably 70% of the total genes virtually expressed in a sample) were obtained from each sample. Comparison analyses were performed among the profiles from various challenges of dose and time courses. The expressions of less than 0.1% of the genes were found to be altered in a dose and time dependent manner. Those involved CDKN1A gene, which expression increased by 1.5 folds at 2 hours after X-irradiation (4 Gy). We are investigating other unexpected transcripts, some of which are located out of the predicted exons. Our studies shall provide useful clues to develop a research tool to accurately assess the risk of ionizing radiation on humans. (HiCEP is the registered trademark of MessengerScape Co.,Ltd for an expression profiling technology developed by Dr. M. Abe et al.)

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To re-evaluate the total amount of the Cs-137 global fallout, we analyzed worldwide historical data of Cs-137 observations in rain, seawater, soil and ice core. Another objective is to re-construct of temporal and spatial variation of Cs-137 global fallout as a source term for simulation of Cs-137 in the ocean using OGCM. A grid size is set 10 degree latitude by 10 degree longitude considering distribution of available data. 77 data for seawater column inventory, 179 data for deposition, 492 data for soil inventory and 3 data for ice core inventory were available for this study, respectively. 157 of 324 grid data obtained from the observational data in the Northern Hemisphere. For the grids in which no observational data was available, we fill them using the relationship between the fallout amount and precipitation amount at the neighboring grids. This study shows that total amount of Cs-137 fallout as of Jan. 1970 becomes ca. 770 PBq which largely exceeds previously reported value of 545 PBq in UNSCERA2000. The higher fallout regions are observed at the regions of warm current systems in the Pacific and Atlantic Oceans where higher precipitation amount occurs and the grater air mass exchange between stratosphere and troposphere also occurs. This study shows that UNSCEAR report on the part of total amount of Cs-137 fallout should be revised.

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ESR measurement using tooth is one of the valid dosimetry methods for residents near Semipalatinsk Nuclear Test Site in Kazakhstan. In this method, native people's teeth are brought to Japan by airplane at first. X-ray baggage scanning at airports may increase the exposure dose of the teeth. This study investigates the influence of X-ray baggage scanning on ESR dosimetry using tooth.
Two Japanese teeth, 'A' and 'B', were separated into six samples. Three samples were irradiated with X-ray from a baggage scanner at the Incheon airport in Seoul. The other samples were not irradiated as control. The doses of all the samples were measured and the results were compared between irradiated and un-irradiated groups.
The dose was evaluated to be 173.6 +/- 16.3 mGy for irradiated 'A'(inside) and 185.7 +/- 16.8 mGy for un-irradiated 'A' (inside). The results for the irradiated and un-irradiated samples are 195.4 +/- 19.0 mGy and 198.4 +/- 23.7 mGy for 'A'(outside), 119.1 +/- 11.9 mGy and 105.7 +/- 11.9 mGy for 'B', respectively. Differences are not observed between two groups. It seems unnecessary to consider the dose caused by X-ray baggage scanning in ESR dosimetry using tooth.

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Epidemiology investigation of the residents in Misasa, which is famous for radioactive hot spring, was carried out so far. Because dose from natural radiation would be relatively low, accuracy in the dose estimation should be important in the case of investigation for effects of exposure to natural radiation. It was found from the environmental investigations that there are many places where thoron concentration is very high as well as radon concentration. From these points of view, provisional study for measurements of the thoron concentrations in residence and the building for hot spring in Misasa were carried out. >From the result of the measurements, the thoron concentrations at the position of 10cm or 20cm from the surface of the wall ranged from about 0 to more than 1000 Bq/m³. It was found that there were locations of residence or hot spring where equilibrium equivalent thoron concentration (EETC) exceeds several Bq/m³. In the room where EETC are high, contribution of thoron to effective dose could be higher than that of radon. It was suggested that further investigation of measurements and dose estimation from thoron is necessary.

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At the Meteorological Research Institute (MRI, Tsukuba, Japan), we found that during the 1990s, the ¹³⁷Cs/⁹⁰Sr activity ratio resulting from atmospheric deposition and free from the direct influence of nuclear tests and accidents, was fairly low (average: 2.1). These values were not in accordance with those from Japanese surface soils (average: 4-7). This finding suggests that atmospheric deposition is currently a mixture of local and remote components. The remote component may be an aeolian dust which has been transported long distances from arid areas. The major component is likely to be Asian dust. It was further hypothesized from observations and model results that remote sources may exist other than Asian dust. To obtain more information, Saharan dust deposited in Monaco in 2002 and suspended dust collected in the Taklamakan area in 2001 were analyzed for ⁹⁰Sr and ¹³⁷Cs. The Taklamakan dust exhibited a ¹³⁷Cs/⁹⁰Sr ratio of about 4, within the range for atmospheric deposition recorded at the MRI, while the Saharan dust exhibited a higher ratio of about 13. Although the present Sahara datum was negative for our 'hyper-range transport' hypothesis, the seasonal change in the ¹³⁷Cs/⁹⁰Sr ratio in the atmospheric deposition implies an aeolian dust source other than Asian dust.

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Ingestion and organ content of stable isotopes of ¹³⁷ Cs, ⁹⁰ Sr and ¹³¹I, and ²³⁸U and ²³²Th, and K and Ca, chemically similar to Cs and Sr, were studied to obtain representative values for the adult in each participating country and in the Asian region. Diet samples from 9 countries and tissue samples from 4 were analyzed under the QC provided by IAEA and the regional central reference laboratory. Selected NIST reference materials, and Typical Japanese Diet newly developed for the project were characterized. The data were compiled and analyzed by CRL and IAEA. The obtained results are (1) ingestion intake, (2) organ content of these elements and nuclides, and (3) internal dose estimates for ²³⁸U and ²³²Th in the region. Out of 220 diet samples, 1160 determinations were obtained by use of RNAA, ICP-MS, ICP-AES, etc. Intakes of I, Th, U and Sr/Ca ratios showed characteristic tendencies when compared with ICRP Reference Man. The results support a use of ICRP metabolic models for the environmental dose assessment. An IAEA-TECDOC and scientific papers are being published.

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Scid mice have the spontaneous mutation on DNA-PKcs, exhibit the limited activities of DNA double strand break repair and are sensitive to ionizing radiation. Dose-response study of radiation induced thymic lymphoma by 0.05 to 3 Gy irradiation was done, and the quadratic equation was best fitted. The presence of the threshold was not statistically confirmed. In order to study the relationship between the defect of repair capacity for DNA double strand breaks and carcinogenesis in other target cells than thymocytes, the histochemical analysis was done using 1-3 Gy irradiated scid homozygotes, scid heterozygotes and C.B-17 mice. From the Kaplan-Meier analysis, significant difference in tumor free survaival between scid and wild type mice were observed in thymic lymphoma, but not in ovarian granulosa cell tumor, hepatocellular carcinoma, and lung adonocarcinoma. All of which were induced both in scid and wild type mice by 1 Gy gamma-irradiation. Dose-response of Notch1 abnormality in entire region of the gene was linear in scid mice. There are at least two pathways for induction of Notch1 deletions: illegitimate V(D)J recombination and microhomology-mediated NHEJ pathway. Microhomology-mediated NHEJ pathway and insertion of intracisternal A particle are likelly to be responsible for linear induction of Notch1 abnormalities, thereby contributing to thymic lymphoma induction.

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We have identified two pathways for the 5' deletion of Notch1 gene in radiation-induced thymic lymphomas; illegitimate V(D)J recombination in wild-type mice and microhomology-mediated nonhomologous end-joining (MNHEJ) in scid mice. We report that there are Rag2-dependent and Rag2-independet pathways for the induction of thymic lymphomas. The wild-type C.B-17 mice exhibited no induction of thymic lymphomas by 2 Gy gamma-irradiation, while Rag2^-/-, scid, and Rag2^-/- scid mice all displayed high frequencies of radiation-induced thymic lymphomas. This indicates that there is a Rag2-independent pathway for induction of thymic lymphomas. Notch1 abnormalities were induced by irradiation at high frequencies in these mice, suggesting that there is a Rag2-independent pathway for induction of Notch1 abnormality. In wild-type mice, the 5' deletions of Notch1 were formed via illegitimate V(D)J recombination and those in scid mice were formed via illegitimate V(D)J recombination and MNHEJ. In contrast, in Rag2^-/- or Rag2^-/- scid mice, the 5' deletions were formed via MNHEJ. These results indicate that in the presence of Rag2 gene, Rag2-dependent illegitimate V(D)J recombination functions as a main mechanism for the deletion formation, while in the absence of Rag2 gene, Rag2-independent MNHEJ works mainly in the deletion formation. The 5' deletions formed via Rag2-dependent illegitimate V(D)J recombination occur spontaneously in wild-type thymocytes.

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BALB/c mice are susceptible to radiation-induced lymphomagenesis, while STS mice are resistant. Previously we showed that more than one locus could be associated with the strain difference. By the use of a series of congenic lines with different portions of STS-derived chromosome 4 in the BALB/c background we also showed that lymphoma susceptibility was linked to the proximal half of chromosome 4. In the present study, we performed linkage analysis for susceptibility to lymphomagenesis using (BALB/c x STS)F₁ x BALB/c mice. Only females were used and subjected to 4 x 1.7 Gy of X-irradiation. Development of lymphomas was observed during 300 days after irradiation. Genotyping was carried out with PCR and PAGE using microsatellite markers. Linkage was analyzed by the Kaplan-Meier method. Significant linkage was detected both at D4Mit17 and in the D4Mit302-D4Mit255 segment more than 10 cM distal to D4Mit17 on chromosome 4. Analysis of congenic lines identified a susceptibility locus in the D4Mit302-D4Mit255 segment, but not in the D4Mit86-D4Mit302 segment between the two putative susceptibility loci. Further analysis of congenic lines is presently under way.

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We have proposed a hypothesis that excess risk of ALL following radiation exposure is not caused by induction of ALL-specific translocations but radiation affected on a small number of individuals who already had clonally expanded ALL-specific translocations spontaneously. In the present study, we show that the translocations probably occur in early fetal life. We used epidemiologic data of Oxford Survey of Childhood Cancer (OSCC) by Stewart et al, which showed that diagnostic X ray exposure (about 1 cGy) caused 50% increase of the background risk of childhood leukemia. The logic that we used is; if an early stage has not yet developed ALL-specific translocations, the excess risk should be substantially lower compared with the risk at later stages. The OSCC data showed that trimester 2 and 3 fetuses had the relative risks (RR) of 1.29 and 1.30, respectively, which means that there is no difference in the RR. In fetuses exposed at trimester 1, the RR was larger than 3 while the larger risk cannot be taken at face value because radiation dose per X-ray film was larger and the mean number of films taken was also larger in trimester 1 fetuses. Nonetheless, the trimester 1 fetuses do not seem less sensitive to radiation exposure. We conclude therefore that the ALL-specific translocations are most likely produced in the first trimester.

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C3H/He mice develop acute myeloid leukemia (AML) after whole-body irradiation, and typical chromosome 2 deletions are found in the leukemic cells. While the AML-type deletions are found in the bone marrow cells just after irradiation, the frequency of the individuals bearing the deletions rises after a year or so. These findings have suggested a hypothesis that the AML-type deletions in the primitive stem cells could result from delayed chromosomal aberrations. To test this hypothesis, we have cytogenetically examined primitive hematopoietic cells of irradiated animals. Ten-week male C3H/He mice were exposed to 3Gy x-rays and sacrificed after one or ninety days after irradiation. Bone marrow cells collected from each animal were divided into two batches. One batch was cultured in methylcellulose medium, and metaphase spreads were prepared from each growing colony. The other batch was sorted to obtain Lin⁺ and Lin^-Sca1⁺ cells, which were analyzed with FISH for the AML-type deletions. Non-clonal aberrations were found in colonies in methylcellulose medium, and its frequency was higher at 90 days after irradiation. However, no aberration has been observed in chromosome 2. FISH analysis of the sorted cells detected AML-type deletions in Lin^-Sca1⁺ subpopulation at one day after irradiation, and the deletions were retained in some individuals even after 90 days. These results do not necessarily support involvement of delayed chromosomal aberrations in radiation-induced AML in mice.

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To investigate effects of heterozygous deficient p53 (p53^+/-) and/or Atm (Atm^+/-) genes in radiation-induced mammary tumorigenesis, we examined the tumor induction by X-irradiation in p53^+/- and/or Atm^+/-- (BALB/cHeA x MSM/Ms) F₁ female mice. Both p53^+/-Atm^+/- and p53^+/-Atm^+/+ mice efficiently developed mammary adenocarcinomas (MC) by X-irradiation of 5 Gy at 5 weeks of age and the incidences of the MC were 58% and 24%, respectively. Average number of MC per mouse in p53^+/-Atm^+/- group was significantly larger than that in p53^+/-Atm^+/+ mice. X-irradiation considerably shortened latent period of the MC development. These MC were observed early in irradiated group (during 25 to 43 weeks of age) compared with non-irradiated group (during 45 to 60 weeks of age). However, almost all irradiated p53^+/+ mice did not develop the MC in spite of Atm gene status. To analyze putative tumor suppressor genes in mammary carcinogenesis, we examined loss of heterozygosity (LOH) in the MC. Frequent LOH was observed on chromosomes 5, 8, 10, 11 and 12. Precise allelotype analyses on chromosomes 8, 10 and 12 have been performed. Examination of RNA expression of some candidate genes are in progress.

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Though humans are continually exposed to various environmental carcinogens, the mechanism of combined carcinogenesis has been only deduced from oncogenic actions of individual agents. Here, we analyzed experimental mammary carcinogenesis caused by a combination of radiation and a chemical carcinogen, 1-methyl-1-nitrosourea (MNU). Seven-week-old female Sprague-Dawley rats were treated either with gamma-rays (2 Gy), MNU (40 mg/kg, i.p.), or a combination of gamma-rays and MNU (gamma/MNU). Rats were sacrificed by 50 weeks of age to collect tumors for histological and mutational analyses. The gamma/MNU treatment induced adenocarcinomas, but not fibroadenomas, more efficiently than gamma-rays or MNU alone. The H-ras mutation was not frequent in radiation-induced carcinomas, and was characteristic to MNU-induced carcinomas in individually treated groups. In the gamma/MNU group, H-ras-mutated, but not non-mutated, tumors were induced more efficiently than in the MNU-treated group. These results indicate that the combined exposure to radiation and a chemical carcinogen elicits an unexpected cooperativity in which pre-irradiation enhances chemical carcinogenesis predominantly through the mechanism involving H-ras mutation.

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Thorotrast is the trade name for 25% colloidal suspension of radioactive 232ThO2 that naturally emits alpha particles, and was used as a radiographic contrast agent in the second World War. After intravascular injection, more than 50% of the total amount of Thorotrast is located in the liver and is known to induce liver cancers several decades after injection. The aim of this study is to investigate the loss of heterozygosity (LOH) and microsatellite instability (MSI) in Thorotrast-induced liver angiosarcoma (AS). In addition, we performed the comparison between AS and intrahepatic cholangiocarcinoma (ICC) induced by Thorotrast. We found frequent LOH at 3p, 20p and 21q in Thorotrast-induced AS and non-Thorotrast AS. MSI did not significant different between Thorotrast and non-Thorotrast AS. The frequent LOH at 8q and 13q was observed in Thorotrast-induced AS and ICC. The results of our study suggested that the carcinogenic mechanism of Thorotrast-induced AS and ICC is partly shared.

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Cancer risk assessment was conducted on a population who received low dose-rate gamma-irradiation after occupying buildings containing ⁶⁰Co-contaminated steels for more than 10 years in Taiwan. More than 7,000 people were registered and the occurrence of various cancers ascertained till the end of 2002 via the National Cancer Registry Program in Taiwan. The cancer incidences were compared with the populations with the same temporal and geographic characteristics in Taiwan, by standardized incidence ratios (SIRs) after adjusting for age, gender, and latent periods for developing cancers and further association with cumulative radiation exposure via Cox regression. A total of 141 individuals with various cancers were observed, while the risks were significantly increased for acute lymphocytic leukemia, all leukemias, and hemato-lymphoid malignancies in men, and thyroid cancers in women. Women were also show with higher risks of all cancers combined and solid cancers than men. All cancers and solid cancers were further shown with exposure-dependent increased risks in those received initial exposure before 30 years old. The results indicated increased risks of developing various cancers in human population with protracted low dose-rate radiation exposure.

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The effects of I-131 on the thyroid have been examined using the rat model. (1) Kinetic analysis of I-131 accumulation into and excretion from the thyroid indicated that proportion of iodine in the thyroid was highest in 1-week-ols rats. Yet, this could be because of the amount of blood circulation proportional to the volume of whole body. (2) Carcinogenesis experiments indicated the higher effectiveness of external exposure to internal exposure irradiated at 1 week old. The mechanism how I-131 cause strand breaks of DNA of the thyroid epithelium was speculated. (1) I-131 could exist as a chemical component, HI surrounding the gonome. (2) I-131 could be incorporated into T3, form hormone-receptor complex, bind to specific sites on DNA (miri sec/transcription). Considering the topology of I-131 in the nuclei, the estimation of the frequency of DNA breaks were available for SSB/cell/Gy or for DSB/cell/Gy. The energy of electrons from I-131 (800keV) is similar to that of second electrons from Co-60 (1.25MeV), therefore, the biological effectiveness of the two radiations is thought to be similar. The dose rate should be contributed for the thyroid tumor igenesis.

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BRAF^T1796A transversion has been shown to be a prevalent event in human papillary thyroid carcinomas (PTCs). In this study the mutation prevalence was examined in radiation-associated PTCs. DNA from the post-Chernobyl (age at exposure below 18 years old) and sporadic PTCs was analyzed by direct sequencing and mutation allele-specific PCR. In the whole radiation-associated PTC group, BRAF^T1796A was found in 17/98 (17.3%) cases with no overall statistical difference from sporadic cases (14/53, 26.4%). However, when radiation-associated series was subdivided in the pediatric (age below 18 years old at diagnosis) and adult cases (age over 18 years old at diagnosis), it was found that in the younger patients the mutational frequency was significantly lower than that in the elder ones, 4/49 (8.2%) versus 13/49 (26.5%), p=0.03. Thus, the BRAF^T1796A mutational rate in PTCs does not associate with radiation history of patients but rather increases with individuals age.

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When mutation spectrum of haploid yeast cell is analyzed, various point mutations are detectable. On the other hand, when mutations of heterozygous locus from diploid cells are analyzed, allelic loss from functional counterpart is frequently observed. The event is referred to as loss of heterozygosity (LOH). LOH is caused by mitotic recombination between homologous chromosomes. Mitotic recombination functions as one of the DNA damage tolerant mechanism. The purpose of this study is to investigate the process of DNA damage repair by characterizing the mechanism of UV-induced LOH. To study the source of LOH events, we have constructed CAN1/can1 diploid yeast strains and examined canavanine-resistance (Can^R) mutations to can1/can1. The damage on the chromosome carrying CAN1 induced more Can^R mutants than the damage chromosome carrying can1, and a significant increase in Can^R mutants were observed when the damage were induced during DNA replication. These results indicate that mitotic recombination, that functions as DNA damage tolerant mechanism, occurs through a nonreciprocal strand transfer associated with DNA replication.

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DNA mismatch repair plays an important role in reducing errors created in DNA replication. Mlh1is one of the genes involved in the mismatch repair. Deficiency of the gene has been shown to result in high frequency of tumor incidence in mice, and also the elevated levels of mutation. Here we wondered when the elevation of mutation appears in the process of mouse development.
The mice used were Mlh1 knockout mice harboring LacZ gene made by crossing with Muta^TM mice. The level of mutation frequency at 12 day embryo was higher than those in Mlh1(+/+) or Mlh1(+/-) embryos. The high level was maintained at newborn, adult and middle-aged mice. The molecular characteristics of the mutants in Mlh1 (-/-) showed high frequency of deletion mutation which is unique to mismatch repair deficiency. In an attempt to investigate the consequence of high mutation level in Mlh1(-/-), we examined apoptosis in embryo and newborn mice. Preliminary results showed a low level of apoptosis. These indicate that the deficiency of mismatch repair results in an elevation of mutation at early stage of development and do not affect much in later stages. It is also suggested that the high level of mutation does not interfere with the developmental process in mice.

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We found previously, using lacZ-transgenic mice, that mutation induction in UVB-exposed skin epidermis was suppressed at a higher dose range and saturated to a leveled mutant frequency (Ikehata and Ono, Mutat. Res., 508:41-47, 2002). This epidermal phenomenon of mutation induction suppression (MIS) to UVB genotoxicity would be a biological defensive response unidentified before. We also observed the MIS response in sunlight-exposed epidermis, and the saturated mutant frequencies reached there were more than twice of that seen in the UVB-irradiated (Ikehata et al., Mutat. Res., in press). We will report a study on wavelength-dependency of the MIS response using a monochromatic UV source. We will also show some data on a relation of apoptosis to MIS.

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Mouse nuclear protein NP95 is colocalized with PCNA in early and mid-S phase cell nuclei and visualized as foci in proliferating cells. It apparently endows cells with the resistance against DNA damaging agents including X-rays and replication arrests incurred by hydroxyurea. As homozygously Np95-inactivated embryonic stem (ES) cells show an extremely high rate of spontaneous mutation, it is of interest if Np95-nulligous cells are also susceptible to X ray-induced mutagenesis. Whereas spontaneously-arising and 6 Gy (10 % survival)-induced TG-resistant mutation frequencies were 5 x 10e(-8) and 6.2 x 10e(-7), respectively, for wild-type E14 cells, unirradiated control and 3 Gy (10% survival) exposure brought about the Hprt mutations at 5.6 x 10e(-4) and 3.0 x 10e(-3), respectively, in Np95-nulligous 19.4 cells. Heterozygously Aprt-inactivated FM3A cells were transfected with anti-sense Np95 cDNA to see if LOH type mutation was affected by NP95 functions. The dose response of radiation-induced DAP-resistant mutations was higher in the parental Aprt(+/-) SR1 cells than in its Np95-inactivated derivative #15D, and the proportions of LOH mutant clones resulting from homologous recombination, those from deletion and those from point mutation were remarkably different between the parental cells and the Np95-inactivated subclones, implicating some critical roles for NP95 in LOH type mutagenesis induced by ionizing radiation.

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We have already reported that γ-irradiation with 3 Gy at 1.2 mGy/min was not mutagenic in TCR gene for p53(+/+) mice but mutagenic for p53(-/-) mice, and p53(-/-) mice kept the high frequencies of TCR mutation over the 2 months after irradiation. In order to elucidate the in vivo role of p53 gene in surveillance against mutation, we improved the method to observe the mutant frequency on TCR gene using a 3-color FACS analysis, which includes FITC-anti-CD3, PE-anti-CD4, and CyChrome-anti-CD44 monoclonal antibodies. We could detect the TCR mutant cells, which showed CD3^- and CD4⁺, both in CD44^- naive and CD44⁺ memory T cells after irradiation. The mutant frequencies of naive cells and memory cells at 6 weeks after irradiation were 11.4 and 17.9 (x 10^-4) in p53(+/+) mice, and 32.7 and 64.3 (x 10^-4) in p53(-/-) mice, respectively. These results indicate the high mutant frequency on TCR gene in p53(-/-) mice, especially in memory T cells. Previously we observed that the frequency of the cells dying by apoptosis after irradiation with 2 Gy greatly increased in p53(+/+) mice, but did not increase in p53(-/-) mice. Taken together, concerted DNA repair and p53-dependent apoptosis are likely to completely eliminate mutagenic damage at low doses or low dose-rates.

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Chromosomal double strand breaks (DSBs) occurring in human genome are generally repaired either of two pathways; End-joing (EJ) or homologous recombination (HR). We recently developed a system to trace the fate of DSBs occurring in human genome using meganuclease I-SceI site (Honma et al., EMM 42, 288-298, 2003). The lymphoblastoid cell lines TSCE5 and TSCE105 have single and double I-SceI site in the thymidine kinase gene (TK), respectively. Introducing highly efficient DSBs into the cells using Nucleofector^TM (Amaxa) made it possible to recover mutants by the DSBs without the phenotypic TK selection. The mutant frequencies of TSCE5 and TSCE105 were 3% and 30%, respectively. Most of mutations in TSCE5 were small deletions ranging from 1 to 30bp and others were over 30bp deletions and DNA rearrangements. On the other hand, TSCE105 mainly produced large deletions encompasing the two I-SceI sites. No mutants resulted by HR was observed in both cells. These results support an idea that majority of DSBs are repaired by EJ resulting small deletion, and HR rarely contributes for that. Error-free EJ mechanism may be possible, because single DSB in TSCE5 did not efficiently cause deletional mutations than expected.

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It has been shown that ionizing radiation generates oxidized nucleotids such as 8-oxo-dGTP or 8-oxo-dATP which are thought mutagenic substrate for DNA synthesis. Mammalian MTH1 hydrolyzes 8-oxo-dGTP or 8-oxo-dATP to 8-oxo-dGMP or 8-oxo-dAMP, respectively, thereby preventing occurrence of mutations. We have established Mth1-deficient mice, which were shown to have a higher susceptibility to spontaneous tumorigenesis in liver and lung. To examine in vivo mutation events due to the oxidized purines induced by ionizing irradiation, a reporter gene, rpsL of Escherichia coli, was introduced to Mth1-deficient mice. Using these mice, we examined the radiation induced mutagenesis. When analyzed DNA samples recovered from spleens of Mth1-deficient and wild-type mice exposed to 4 Gy of X-rays, we found a significant increase of A:T to G:C transition mutations in Mth1-deficient mice with X-ray irradiation, probably because 8-oxo-dATP formed in the nucleotide pools can not be sanitized in the absence of MTH1 protein. These results suggest that 8-oxo-dATP, generated by X-irradiation, can induce mutations and Mth1 prevents their occurrence by hydrolyzing 8-oxo-dATP.

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We have used Restriction Landmark Genome Scanning method to assess mutation induction rate at GC-rich sequences in mice after exposure of spermatogonial cells. In the present study, we used a restriction enzyme AflII to expand our knowledge to AT-rich sequences. To identify the parental origin of mutations, we used F1 mice prepared by crosses of irradiated BALB/c males (spermatogonia) with JF1 females and screened 1,120 selected spots (514 paternal and 606 maternal spots). We detected five mutations in the control group (184 animals, 92,655 paternal spots) and 11 mutations (including a cluster mutation of two that arose spontaneously) in the 5-Gy-exposed group (184 F1, 92,789 paternal spots). We also found 5 mutations (including a cluster mutation of three) in the un-irradiated maternal alleles (368 F1, 218,411 spots). Most of the DNA fragments mutated were found to comprise simple tandem repeats or satellite DNA, which hindered us from characterizing the mutations except for two mutations that appeared as due to base substitutions at recognition sites of the enzymes used. There was no indication that the mutation induction rates largely differed between GC- and AT-rich sequences. Our pooled estimate of mutation induction rate, including both GC- and AT-rich sequences, was ~1 x 10^-5/locus/Gy, which appeared to be substantially smaller than the mean induction rate at 7 loci of Russell (2~3 x 10^-5/locus/Gy).

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Although radiation-induced genomic instability has been demonstrated by the use of repeat sequence instabilities in most cases, little is known about its association with mutations at ordinary protein-coding genes. We introduced partial duplication within the HPRT gene (HPRT^dup) in human cells (HT1080), which can measure mutation rates at repeat and unique sequences in the same HPRT gene locus. Namely, the HPRT^dup cells are HPRT(-), hence 6TG resistant, while deletion of one of the duplicated sequences by recombination makes the cells HPRT(+), hence HAT resistant. Further, the revertants can be subjected to forward mutation assay using 6TG resistance as the marker. >From X-irradiated HPRT^dup cell pools, we have isolated a large number of clones that showed elevated reversion rates by 10 to 100-fold as a result of delayed mutation. Following measurements of forward mutation rate in each clone, we found that the two mutaion rates do not always correlate.

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When cells are maintained in a non-growing condition (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating, which is well known as repair of potentially lethal damage (RPLD). Although the factors affecting PLD and RPLD are well known, its mechanism remains still unknown. The effect of non-growing condition on recover (24-h incubation following irradiation) and chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) was compared with that of growing condition immediately after irradiation. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. In particular, complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. We conclude that misrepair under growing G1 can be the reason of PLD and decreased misrepair under G0 can be the mechanism of RPLD.

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