Plutonium in monthly wet and dry deposition samples collected in Daejeon (KINS), Korea was determined during the period from January 2000 to August 2000. Monthly ^239,240Pu depositions in Korea show a typical seasonal variation with a maximum in spring season (March to May), which corresponds to seasonal cycle of soil dusts originating from the East Asian arid area. This seasonal change was similar to that in Nagasaki. The cumulative amount of the ^239,240Pu deposition observed in Korea in the period of Jan. 2000 to Aug. 2000 was 16.7 mBq m⁻², which was about three times greater than the annual sum of ^239,240Pu deposition in Japan. The trajectory analysis suggests that ^239,240Pu deposition in Korea in spring is originating from plutonium-bearing surface soil particles from the East Asian arid areas. A significant part of the ^239,240Pu deposition in spring season is attributable to dry deposition. [J Radiat Res 44:432 (2003)]

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Iodine is an essential element and plays an important role in thyroid function. It is known that transfer of radioiodine into thyroid gland is influenced by the coexisting stable iodine. Therefore, it is important to study the iodine levels in diet. In order to estimate the dietary intake of iodine by Japanese we have determined iodine concentrations in 30 total diet samples. The samples were collected within the framework of an IAEA/RCA project. From the analytical results we estimated the daily intake of iodine through foodstuffs. The range of iodine intake was 0.036 - 8.4 mg/day. The high value might be caused by the consumption of seaweeds. The mean and median values are 0.93 mg/d and 0.29 mg/d, respectively. These values are higher than the value recommended by ICRP (0.2 mg/day). However, about 30% cases were lower than the ICRP value. [J Radiat Res 44:432 (2003)]

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Among the chemical forms of tritium released in the environment, tritiated water (HTO) has a very high contribution to the dose. But, the dose due to organically bound tritium (OBT) is 2.3 times higher than that due to HTO. Heavy water (D₂O) vapor release experiments were carried out in a greenhouse using D₂O as a substitute for HTO and uptake and loss kinetics of D₂O in leaves and formation, translocation and retention of organically bound deuterium (OBD) in rice were investigated using different growth process of rice. Rate constants of D₂O uptake in leaves of rice plant in the daytime and nighttime release, rate constants and the half time of D₂O loss in leaves after release were determined. After D₂O release, OBD concentration in unhulled rice increased gradually until 4 days after the exposure and then decreased with time due to metabolic consumption in rice grain. [J Radiat Res 44:432 (2003)]

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Heavy water (D₂O) release experiments were carried out in a greenhouse to determine uptake and loss kinetics of D2O in leaves of Japanese mandarin orange (Citrus unshiu Marc.) and formation, translocation and retention of organically bound deuterium (OBD) in orange fruit in daytime and nighttime release experiments held at different growth process. Potted oranges were exposed D₂O vapor in the greenhouse for 8 h in the daytime or nighttime. The same kinds of parameters were obtained in D₂O supply experiments in soil of orange pots to estimate the contribution of contaminated soil. Although translocation rates of soil water D₂O to leaves were less than one-third those of atmospheric D₂O to leaves, those of leave D₂O derived from soil D₂O to OBD in orange fruit at harvest were more than those of leave D₂O derived from atmospheric D₂O to OBD in orange fruit at harvest. [J Radiat Res 44:432 (2003)]

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Technetium was rare nuclide in the natural environment but now the nuclides are increasing in the environment by nuclear weapon tests, nuclear accidents, nuclear fuel fabrication and reprocessing. Especially ⁹⁹Tc attracts public concern because of its long physical half-times as 2.1 × 10⁵ years. To get information for accumulation of the nuclide by aquatic organisms, radioisotopes tracer experiments using ^95mTc(chemical form : TcO₄⁻) was made in laboratory conditions. Among the mollusca seaweed-eating gastropods(abalone and turbo) showed higher accumulation of ^95mTc from water than other carnivorous mollusca. The abalone Haliotis discusgave the highest concentration factor as 400 for whole body in the mollusca. The mollusca absorbed well the nuclide from labelled food regardless of feeding habit. The organisms retained more than 50% of radioactivity in food 3 to 5 days after feeding. Fishes except a shark(elasmobranch fish) showed lower accumulation ability than the mollusca for the nuclide from water and also from food. The shark Scyliorhinus torazame indicated a whole-body concentration factor of 20 in the uptake experiment from water and retained about 60% of radioactivity in food 3 to 5 days after feeding. [J Radiat Res 44:433 (2003)]

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For nuclear safety assessment, it is required to know the behavior of radioiodine in the environment because of its affinity to the thyroid gland. We have already estimated the soil-to-plant transfer factors (TFs) of radioiodine using Andosol, the most abundant soil types in Japan. TF showed the concentration ratio of the nuclide between crop and soil at harvest. It is, however, necessary to obtain the plant/soil ratio at different growth stages in order to understand the uptake pattern during the cultivation. Therefore, we have carried out radiotracer experiments on the transfer of radioiodine from Andosol to radish. The mean values of the concentration ratio (on a wet weight basis) of radioiodine for roots (tubers) of radish at 17, 24, 31 and 38 days after sowing were 0.010, 0.0057, 0.0040 and 0.0028, respectively, suggesting the values decreased with time. Contrary to these, the concentration ratios observed for leaves of radish increased with time, i. e. the ratios at 10, 17, 24, 31 and 38 days after sowing were 0.007, 0.008, 0.013, 0.011 and 0.011, respectively. [J Radiat Res 44:433 (2003)]

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The production of thymine decomposition products and their radical precursors by monochromatic ultrasoft X(USX)- or ⁶⁰Co γ-irradiation were studied using HPLC and EPR spectroscopy. By HPLC analysis of irradiated thymine at the photon energy of 395, 407, 538 eV using a soft X-ray beamline in SPring-8, some well-known products such as dihydrothymine were assigned and several hydrophobic products were also found. Although the kind of the product by the USX-irradiation was almost similar to those by the γ-irradiation, a quantitative difference was shown: the relative strength of each hydrophobic product to dihydrothymine in the USX-irradiated samples was larger than that of the γ-irradiation. EPR experiment of thymine was performed using an EPR device installed in the beamline. The signal intensity of the EPR spectrum at 407 eV preferred to be plateaued than that at 538 eV. These HPLC and EPR results suggest that the possibility of K-shell excitation/ionization and the difference in the secondary electron energy spectra should influence in the relationships between absorption energy and mass of the decomposition product. [J Radiat Res 44:433 (2003)]

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Oxidized forms of nucleotides within DNA or precursor pools were generated by oxygen radicals, and are thought to play an important role in mutagenesis as well as carcinogenesis. In particular, 8-oxo-dGTP is a highly mutagenic substrate for DNA synthesis. Mammalian MTH1 hydrolyzes 8-oxo-dGTP to monophosphate, thereby preventing occurrence of mutations caused by oxygen radicals. Since ionizing radiation induces oxygen radicals in mammalian cells, Mth1 deficient mice might show a hypermutability in response to ionizing radiation. In the present study, we examine this possibility by analyzing X-ray induced mutagenesis in Mth1 deficient mice carrying E. coli rpsL transgene as a reporter.When analysed DNA samples recovered from spleens of Mth1 deficient and of wild-type mice, we found a slight increase in mutation frequency in spleens of Mth1 deficient mice as compared with findings in wild-type mice after exposure to 4Gy of X-rays. Mutation spectrum analysis revealed an increase in the frequency of A:T to G:C transition in Mth1 deficient mice irradiated by X-ray. [J Radiat Res 44:433 (2003)]

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Oxygen radicals are though to play an important role in mutagenesis and tumorgenesis.Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine(8-oxoguanine) is most important because of its abundance and mutagenicity. In mammals, MutY homolog(MUTYH) have been detected and in vitro analysis suggested that MUTYH protein excises adenine paired with 8-oxoguanine in DNA. To investigate MUTYH function in vivo,we analyzed spontaneous tumorgenesis and mutagenesis inMutyh^−/- mice.When examined 18 months after birth, a greater number of tumors were formed in different tissues,including small intestines,ofMutyh^−/- mice, as compared with wild-type mice. Using the rpsL transgene as a reporter, DNA samples recovered from spleen and small intestine of these mice were analyzed for spontaneous mutation. The net frequency of rpsL forward mutants showed no apparent increase in Mutyh^−/- mice as compared to Mutyh^+/+mice,except for a significant increase in frequency of G:C to T:A transversion.These result suggest that MUTYH prevent mutagenesis as well as tumorgenesis caused by oxygen-induced DNA damages. [J Radiat Res 44:434 (2003)]

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The LEC rat, a mutant strain, spontaneously develops heritable hepatitis due to copper accumulation caused by mutation of copper transporting ATPase gene (Atp7b). Iimmunodeficiency and radio-sensitivity had also been reported. We, firstly, analyzed linkage between radio-sensitivity and the mutation responsible for LEC hepatitis in hybrid animals of cross with F344 rats. Results demonstrated the absence of any significant association. Radio-sensitive rats were twice as numerous as their radio-resistant counterparts, suggesting possibility of control by two or more recessive genes. In order to select radio-sensitivity gene of the LEC rat, we wished to develop congenic line of F344 rats using a phenotype-driven breeding protocol: rats of each backcross generation were mated with LEC rats and their offspring were irradiated with 4.5 Gy X-rays, which dose cause death in LEC rats but did not in F344 rats. Lately we also combined using genomic-wide genotyping in each backcross generation and their progeny to select heterogeneous rat. Backcross exceeded 16 generations. This radio-sensitivity gene locates on chromosome 4. [J Radiat Res 44:434 (2003)]

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Germ-line mutation is detected in mice but not in humans. To estimate genetic risk of humans, a new approach to detect radiation-induced mutations in man is expected. We have developed a new method to detect germ-line mutations by directly comparing DNA sequences of parents and children. The nucleotide sequences of STS markers containing SNPs of parents and children DNA were determined by a direct sequencing. At each radiation dose, 5 Mb DNA sequences were determined. We found 7 deletions and 1 point mutation in 3 Gy irradiated mice but not in 1 Gy and control mice. The mutation frequency was about 5.3 × 10-7/bp/Gy or 2.2 × 10-4/locus/Gy, and suggested the non-linear increase of mutation rate with dose. [J Radiat Res 44:434 (2003)]

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3-Nitrobenzanthrone (NBA) is a powerfully mutagenic nitrated aromatic hydrocarbon found in diesel exhaust. NBA forms an unusual DNA adduct that has a C-C bond between the C-8 position of guanine and the C-2 position of NBA. Here, we analyzed translesion DNA synthesis (TLS) over a single adduct in lacZ' gene in a plasmid in uvrA, uvrApolB, uvrAdinB, uvrAumuDC Escherichia coli. The result showed that the adduct blocked DNA replication and an observed TLS frequency was about 5.2% in all non-SOS-induced E. coli. All progenies after the TLS had no mutation. On the other hand, in SOS-induced E. coli, TLS increased to about 10.7% in uvrA, uvrApolB, uvrAdinB cells, but no increase was observed in uvrAumuDC cells. After the TLS, 4.8% of the uvrA cells had mutations with mostly G to T. These results suggest that the umuDC polymerase takes part in the TLS and partially in the mutagenesis of this unusual NBA adduct. [J Radiat Res 44:434-435 (2003)]

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SLD repair after high-LET radiations were determined with low-LET X-ray beam. Chinese Hamster V79 cells were exposed to priming ion-beams having different LETs. The cells were repaired in room temperature and exposed to testing X-ray. Resulting from kinetics study, surviving fractions of the cell quickly increased with the repair time, and reached to a plateau in 3-4 hours, when cells has received 440 keV/um iron ion priming dose, as well as X-ray. The shoulder of survival curves for testing X-ray after all priming high-LET ion-beams and 3 hours repair were the same to that for acute X-ray dose. These results suggest that SLD after high-LET radiation is well repaired as low-LET radiations. Ion beam may produce lethal damages in the cell when cell was hit by core of the track. When a cell was hit by the penumbra, SLDs may be produced in the cell, because the LETs in the penumbra are low. [J Radiat Res 44:435 (2003)]

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In tobacco protoplasts from BY-2 cell line and CHO-K1 cells irradiated with ⁶⁰Co gamma rays, DNA double-strand breaks (DSB) were measured by pulsed-field gel electrophoresis. Linear relationship between dose and number of DSB was found, and number of DSB per Gy were 27.8 +/- 1.4 in tobacco cells and 39.6 +/- 1.2 in CHO-K1 cells. On the other hand, mean lethal doses (D₀) of gamma rays based on the colony formation assay were 10.7 Gy in tobacco cells and 1.4 Gy in CHO-K1 cells. Consequently, the average numbers of DSB needed to induce cell killing were calculated to be 297.5 +/- 14.9 in tobacco cells and 55.4 +/- 1.7 in CHO-K1 cells. From these, it is suggested that the efficiencies of induction and repair of DSB are different between tobacco cells and CHO-K1 cells. [J Radiat Res 44:435 (2003)]

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Production of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was examined in 2'-deoxyguanosine (dG) solution irradiated with X-rays, carbon ions, neon ions or silicon ions. The range of LET was 2-300keV/micrometer. The dG solution was saturated with air or with the mixture of 95% nitrogen and 5% carbon dioxide for a hypoxic condition during irradiation. 8-OHdG was detected by an HPLC-ECD system. In both conditions, the yield of 8-OHdG decreased with increasing LET, which can be explained by the lower yield of OH radicals in the high LET region. However, the yield showed constant or rather increasing tendency above 100 keV/micrometer under the hypoxic condition. Consequently the ratio of 8-OHdG yields between the air-saturated and the hypoxic condition became small. These results are consistent with oxygen-in-the-track hypothesis that proposes the oxygen production along ion tracks at the high LET region. [J Radiat Res 44:435 (2003)]

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Nucleotide excision repair (NER) comprises of two pathways, namely global genome repair (GGR) and transcription-coupled repair (TCR). GGR recognizes all lesions present in the genome whereas TCR is confine to transcribed regions of the genome. In the mechanism of NER, it is thought that TCR is very similar to GGR expect that RNA polymerase II (RNAPII) replaces the role of XPC-HR23B complex as the damage sensor and initial NER. These subpathways contribute in removal of ultraviolet light (UV)-induced photolesions, such as cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP). To investigate how RNA polymerase II stalls these UV-induced lesions, purified RNAPII and oligo(dC)-tailed template containing a single lesion on the transcribed strand were used for transcription elongation assays. Results showed that mammalian RNAPII comes to a halt at CPDs and (6-4) PPs at similar rates. This suggest that TCR subpathway can eliminate different lesions at similar rates. In cells, (6-4) PPs are eliminated from the global genome much faster than CPDs. Thus, theoretically, GGR is thought to be dominant in living cells. [J Radiat Res 44:435-436 (2003)]

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We have investigated whether each ion traversal in a cell nucleus induced a columnar distribution of the DSB cluster. Cells on the surface of CR-39 were irradiated by Ar ions and then the DSBs were visualized by fluorescence immunostaining using gamma-H2AX, which is an indicator of the DSBs. Bright and well-defined fluorescent circular foci were observed in the cell nuclei. The locations of ion traversals were determined by etch-pits produced in the surface of the CR-39. The locations of the gamma-H2AX foci coincided with the location of etch-pits, demonstrating experimentally that the DSBs were produced in a local columnar distribution along the ion traversals. Similar studies were performed using NBS1 foci. NBS1 focus was observed at each location of ion traversal. [J Radiat Res 44:436 (2003)]

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Nitrosative deamination of cytosine and adenine exclusively results in uracil (U) and hypoxanthine (Hx), respectively, whereas that of guanine lead to formation of xanthine (Xan) and oxanine (Oxa). The resulting U, Hx and Xan are excised from DNA by specific DNA glycosylases that hydrolyze the N-glycosidic bond. However, DNA glycosylases tested so far exhibit no or only marginal activity for Oxa, indicating that Oxa is a long persisting lesion. We have recently shown that Oxa forms covalent adducts with polyamines and DNA binding protein such as histone and DNA glycosylases. In the present study, we assessed the repair mechanism of a crosslink product formed between of Oxa and a cellular polyamine (spermine). A duplex oligonucleotide substrate containing an Oxa-spermine crosslink at the defined position was prepared and tested for purified repair enzymes and E. coli cell free extracts. The results will be presented in the meeting. [J Radiat Res 44:436 (2003)]

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Ionizing radiation generates various types of DNA lesions such as base damage and strand breaks. The spectra of DNA damage induced by high LET radiation has been relatively less clarified than that by low LET radiation such as gamma or X rays. In the present study, we have quantitated isolated and clustered oxidized pyrimidine and purine lesions and double strand breaks generated high LET particles. Lambda phage DNA or pDEL plasmid DNA was irradiated with C or Fe ions in phosphate or Tris buffer using the HIMAC accelerator at NIRS in Chiba. After irradiation, the DNA treated without or with endonuclease III or hOGG1 was subjected to pulse field gel electrophoresis to quantitate radiation-induced double strand breaks and clustered oxidized base lesions. The DNA was also assayed for oxidized base lesions using the aldehyde reactive probe (ARP) method. [J Radiat Res 44:436 (2003)]

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Detection of DNA strand breaks by radiation was carried out using real-time PCR amplification. The method is able to target to given sequences. Gamma irradiation to pBR322 plasmid solutions was done by 137Cs, gammacell 40, as dose from 0 to 250Gy. 1000bp, 750bp, 500bp and 250bp fragments were amplified by combined primer sets to irradiated pBR322 plasmid DNA solution including 0~2mM Tris. The results were compared with simulation calculation and then the condition of p=0.2 was more fit to actual data for linear plasmid. The data from DNA solution containing closed circular (CC)plasmids was not fit between data from PCR and simulation.When this closed circular shape might be very difficult to be proceeded by PCR reaction, predicted calculation was fit to actual data of PCR. Therefore, we might estimate contents of CC DNA in the given plasmid solution. [J Radiat Res 44:436 (2003)]

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We have studied oxidative DNA damage using 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) and thymine glycol (TG) as biomarkers for occupational exposure to diesel exhaust. DNA was extracted from lymphocyte of 19 exposed bus garage and waste collection workers, and 18 controls. The measurement of 8-oxodG was carried out by HPLC with an EC detector. High individual variations between 8-oxodG levels were observed, however, the mean 8-oxodG obtained from the exposed workers was not statistically different from the control persons. Thymine glycol (TG) in urine of exposed and control persons was analyzed with TG-d4 as an internal standard. Five ml of urine was treated with the affinity column and C18 cartridges, then HPLC separations were processed. The fractions containing endogenous TG and the deuterated internal standard were derivatized with BSTFA. The GC/MS analysis was performed by JMS-700 with the high resolution SIM mode. The TG levels in human urine were low and the purification procedure of urine samples needs to be further developed. [J Radiat Res 44:437 (2003)]

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Apurinic/apyrimidinic (AP) sites are produced as the intermediates in the course of the base excision repair (BER) via several specific DNA glycosylases. We have recently developed a method for detection and quantitation of AP sites in DNA via Aldehyde Reactive Probe (ARP) which specifically reacts with aldehyde group of AP sites. In this study, we have directly analyzed intracellular AP sites using Fluorescein Aldehyde Reactive Probe-1 (FARP-1, DOJINDO Laboratories). After incubating HeLa RC355 cells with 0, 2.5 and 5.0 mM methylmethanesulfonate (MMS) for 1 hour to produce AP sites, cells were harvested, treated in Triton X-100 and RNase A/PBS(-) solution, and then stained with FARP-1 for 2 hours. They were analyzed by a flowcytometer. The signal intensities were constant at the concentrations more than 0.1 mM. A linear relationship between MMS concentrations and the signal intensities was observed. By establishing this new method, it will be possible to investigate in situ repair process of DNA damages in the cell. [J Radiat Res 44:437 (2003)]

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Nijmegen breakage syndrome (NBS) cells exhibit hyper-radiosensitivity, abnormal S phase checkpoint and chromosomal instability. NBS1 forms a complex with Mre11/Rad50 to play critical roles in DNA double strands break (DSB) repair. DSB repair is accomplished by accurate homologous recombination (HR) repair or by error-prone non-homologous end joining (NHEJ). Recent reports suggest that the Mre11 complex is essential for HR repair pathway and that NBS1 regulates the complex by controlling their subnuclear localization and enzymatic activities. To analyze functional domains of NBS1 in HR repair pathway, a SCneo reporter was introduced into NBS fibroblast cells. Full length or mutant NBS1 was expressed in the cells, and then DSB was induced by transient expression of I-SceI endonuclease. When full length NBS1 was expressed in NBS cells, HR frequency increased up to 100 times higher than that in parental cell line. Further analysis is in progress. [J Radiat Res 44:437 (2003)]

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Non-homologous end-joining (NHEJ) is crucial for repair of DNA double-strand breaks. This pathway is processed through the catalytic joining of DNA ends by the complex of DNA ligase IV and XRCC4. In addition, this process is regulated by DNA-dependent serine/threonine protein kinase (DNA-PK) components, including a large catalytic subunit (DNA-PKcs) and a DNA-binding complex of Ku70 and Ku86 subunits known as the Ku autoantigen. The function of the DNA-PK complex remains to be fully defined in the context of the end-joining reaction. To obtain biochemical evidence about this issue, we attempted to separate and characterize of a DNA-PK mediated DNA end-joining activity from human placenta, which has a high amount of DNA-PK components and a moderate level of the end-joining activity. Here we demonstrated the valid scheme for separation of the end-joining activity that is regulated by DNA-PK activity, whose inhibitor, wortmannin, simultaneously suppresses the end-joining activity. [J Radiat Res 44:437 (2003)]

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Mammalian genes, NEIL1, NEIL2, and NEIL3, have been discovered as E.coli Nei/Fpg glycosylase family. Although repair activities of NEIL1 and NEIL2 for oxidative damage have been characterized, enzymatic activity of NEIL3 remains unclear. Here, we present a structural model for NEIL3 and examine the recombinant protein on its possible DNA repair activity. We find no clear glycosylase activity of NEIL3 as well as its DNA glycosylase domain to several types of DNA substrate containing a modified base. Nevertheless, NEIL3 retains DNA binding activity and AP-lyase activity. Moreover, NEIL3 can partially rescue E.coli nth nei mutant from hydrogen peroxide sensitivity. These results suggest that NEIL3 can work as a DNA glycosylase to repair detrimental base lesions generated by oxidative stress in vivo. [J Radiat Res 44:438 (2003)]

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Our laboratory previously reported a concordant increase of relative mitochondrial DNA (mtDNA) content and the number of large-scale mtDNA deletions in radiation-associated post-Chernobyl papillary thyroid carcinoma. In this study, we investigated the same indices in several kinds of tissues of irradiated rats, to determine whether mtDNA might be used as a bioindicator of a genotoxic exposure. Five-week old rats were acutely irradiated with single doses of 0.5 and 1Gy of X-rays, and sacrificed at the age of 6 months. All solid organs show that specific number of large-scale deletions in mtDNA does not change significantly in any tissue, in irradiated or control animals, as normalized for the relative mtDNA content. Our data suggest that the number of large-scale deletions in mtDNA and mtDNA levels in a given tissue does not appear to be associated with exposure. [J Radiat Res 44:438 (2003)]

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High LET radiation such as heavy ions, are predicted to induce densely clustered DNA lesions unlike low LET radiations. We are interested in the possibility of the difference of the mutational spectra due to the LET difference. Bacillus subtilis strain HA101 spores were irradiated with Ar and Fe ions near the Bragg peak. From irradiated spores, 41 mutants exhibiting rifampicin resistance were isolated, and together with 25 spontaneous mutants, sequence changes in the rpoB gene were determined. Among the spontaneous ones, 24 single-base substitutions (SBS) and 1 three-base insertion were found. Among the irradiated ones, 37 SBS, 2 tandem-double substitutions, 1 three-base insertion and 1 double substitution skipping 6 bases were found. Among 37 SBS, 20 were an identical substitution from G:C to A:T forming a hot spot. So far, no difference in base substitution spectra between Ar and Fe ions was detected. Further investigation with low LET radiations are in progress. [J Radiat Res 44:438 (2003)]

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We report that RibCys (2(R,S)-D-ribo-1',2',3',4'-tetrahydroxybutly-thiazolidene-4(R)-cariboxylic acid), previously shown to protect against radiation induced pathology in rats and pigs, scavenges long-lived radicals (LLR), mainly in proteins assigned as sulfinyl radicals (R-CH₂SO). LLR levels were directly measured by electron spin resonance (ESR) spectroscopy at 77 K in Syrian Golden Hamster embryo (SHE) cells un-irradiated or gamma-ray irradiated (5 kGy) at room temperature. RibCys (480 mM) was added 2 h after irradiation. The rate constant between LLRs and RibCys was estimated as 3.2 x 10⁻⁴ M⁻¹s⁻¹, about 20 times slower than for vitamin C. In mutation experiments, RibCys (4 mM) added after radiation with carbon ions (3 Gy) decreased levels of CD59⁻ mutants in Chinese hamster ovary hybrid A_L cells by about 50%, compared with 70% with Vitamin C. These results provide further evidence that LLR are mutagenic and may trigger genomic instability. [J Radiat Res 44:438 (2003)]

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Radon exposure contributes to nearly half of the environmental radiation, however, its molecular nature of the biological effects has not been elucidated. We, therefore, have started the development of the experimental procedure for detecting the biological effects, especially genetic one, caused by radon. In the control experiments, mouse FM3A cells were grown on soft-agar plates, and irradiated by up to 10Gy of X-ray. DNA sample was extracted from the cells, and analyzed by Southern blot hybridization technique using hypervariable repeat Pc-1 DNA as a probe. Dynamic mutation at Pc-1 locus was not observed in the cells exposed by up to 10Gy of X-ray. Also the cells growing on soft-agar plates were exposed to 100K-1MBq/cubic-meter of Radon, and the Pc-1 locus was analyzed. The experiments are still on the half way, and we will describe the results and the reliability of this new experimental procedure. [J Radiat Res 44:439 (2003)]

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We have developed a novel automated system for the quantitative evaluation of DNA damage based on an atomic force microscopy (AFM). By visualizing DNA molecules and measuring their lengths directly and automatically, we succeeded to detect the increase in DNA damage caused by several hundred Gy-irradiation with the statistical significance (p < 0.05). The result demonstrates the improvement of sensitivity by one order of magnitude compared to previously reported AFM-based systems. We found AFM more suitable for the quantitative detection of DNA break than electron microscopy, because it is easy to operate, and can be used in high throughput analysis. These results suggest that our automatic analyzer is powerful tool for the quantitative evaluation of DNA break. [J Radiat Res 44:439 (2003)]

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Werner syndrome (WS) is autosomal recessive disorder exhibits features of premature aging.The precise role of werner protein (WRN) remain to be determined. In order to reveal the causes of abnormalities, We introduced human normal chromosome 8 codes WRN gene into the ws cell immortalized by htert.Ws cell has previously been shown to have hypersensitivity to 4-nitroquinoline1-oxide (4NQO), and cis-platinum (II) diamine dichloride (CDDP), hydroxyurea (HU) and have increased chromosomal instability after X-ray irradiated. So we tested these agents and treatment. the results show the hyper sensitivity to 4nqo and cddp, chromosomal instability were complemented. And hu was partially complemented. These findings suggest WRN protein has functions concerning recombinational DNA repair. Further more, these complementation have not seen in the ws cell has human normal chromosome 8 immortalized by SV40. This may be the result of occurring further mutation caused by deficient of WRN protein. [J Radiat Res 44:439 (2003)]

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We analyzed the nucleosome positioning in the promoter of the human c-FOS gene, using ionizing radiation and 4,5',8-trimethylpsoralen as in vivo nucleosome footprinting agents. The distribution of the lesions induced by these agents was visualized by ligation-mediated PCR or terminal transferase-dependent PCR. We firstly studied the influence of the transcriptional activation of the c-FOS gene on a nucleosome positioned in the promoter of this gene. When human fibroblasts were serum-induced, we could not detect any change in the position of this nucleosome, although concurrent chemical modifications of nucleosomes in the c-FOS region upon induction have been reported. Then we studied cell cycle-dependent changes in the positioning of this nucleosome. In mitotic HeLa cells, the positioning of the nucleosome appeared to be lost. This result suggests random distribution of nucleosomes in this promoter in mitotic chromosomes. [J Radiat Res 44:439 (2003)]

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Monochromatic X-rays at the energy (2.153 keV) of K-shell absorption peak of phosphorus are strongly absorbed by phosphorus K-shell and followed by Auger processes. We irradiated V79 (Chinese hamster) cells by X-rays of 2.153 keV (peak), and 2.147 keV (low), which is 6 eV lower off the peak. Survival curves revealed that the doses at the survival 10% were 0.83 times less at the peak than at the low; namely, peak X-rays inactivated the cells 1.20 times efficiently. Induction and repair of DSBs were measured with Pulse Field Gel Electrophoresis (PFGE). The percentage of DNA fragments size of below 4.6 Mbp was adopted as measure of DSB amount: the 2.153 keV induced DSBs 1.40 times higher, although the rejoining of induced DSBs were 0.85 times less compare to the 2.147 keV. [J Radiat Res 44:440 (2003)]

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X-irradiation induces permanent cell cycle arrest, named senescence-like growth arrest (SLGA), in cells which have unreparable DNA damage. In this study, we compared localization of phosphorylated H2AX and phosphorylated p53 at Ser15 to determine whether ATM-dependent phosphorylation is involved in SLGA. Phosphorylated H2AX formed several speckle foci in all irradiated cells 30 min after 4 Gy irradiation. Phosphorylated p53 was observed at 30 min and discrete foci were formed 2-4 hrs after irradiation. The number of phosphorylated H2AX foci and phosphorylated p53 foci gradually decreased, but large foci were still remained 24hrs after irradiation. Those foci were detected at 5 days post-irradiation, when cells began showing SLGA. Both foci co-localized in more than 80% of cells, although not all the foci were co-localized. These results indicate that ATM-dependent phosphorylation is activated in cells inducing SLGA, however, because not all the phosphorylated p53 co-localized with phosphorylated H2AX foci, other factor(s) may play a role in the induction of SLGA. [J Radiat Res 44:440 (2003)]

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It's well-known that low LET ionizing radiation, such as X-rays and gamma-rays, produces a unique form of DNA damage called "clustered damages", which is two or more lesions induced within the one or two helical turns of the DNA. E. coli mutM nth nei triple mutants were less sensitive to gamma-rays and X-rays than wild-type strain. The triple mutant with plasmid bearing mutM gene was more sensitive than wild-type. On the other hand, the triple mutants showed higher sensitivity to H₂O₂ than wild-type. Clustered damages formed by ionizing radiation might be converted to lethal DSB during attempted base excision repair. Recently we found that overexpression of hOGG1 also enhanced the sensitivity to gamma-rays in E. coli. In this report we showed HeLaS3 cells transfected by hOGG1 type1a plasmid was more sensitive to gamma-rays than HeLaS3 cells without plasmid. We are now studying the biological effects of clustered damages in human cells. [J Radiat Res 44:440 (2003)]

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To develop an in situ detection system of 2-acetylaminofluorene(AAF)-DNA adducts, we tried to establish monoclonal antibodies against them. Mice were immunized with AAF-ssDNA coupled with protein, and then the spleen cells were fused with myeloma cells. Out of 359 hybridoma cells, 5 produced antibodies showing preferential binding to AAF-DNA than to DNA. After clonings, 5 types of monoclonal antibodies were obtained. The antibodies showed the high binding to AAF-DNA, but undetectable or minimal binding to undamaged DNA or UV-irradiated DNA. The competitive inhibition experiments revealed that the epitope is dG-C8-AAF in DNA, but deacetylated dG-C8-AF is also recognized with less efficiency. Using the most promising antibody AAF-1, we could measure the formation of AAF-DNA adducts in DNA of xeroderma pigmentosum group A (XP-A) cells following exposure of N-acetoxy-2-AAF (25-150 uM). Moreover, we could detect AAF-DNA adducts in XP-A cells in situ. Thus, we succeeded in establishing, for the first time, the monoclonal antibodies applicable to in situ detection of AAF-DNA adducts. [J Radiat Res 44:440 (2003)]

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Genomic instability is induced in the progeny of radiation irradiated surviving cells. Which is manifested by the expression of various delayed phenotypes. In the present study, we examined the molecular mechanism of delayed mutagenesis in CHO-LacZ cells, which harbor the reporter plasmid of LacZ gene. As this CHO-LacZeo cells produced beta-galactosidase, they formed blue-stained colonies (LacZ⁺) in the presence of X-gal as substrate, on the other hand, these cells formed white colonies (LacZ⁻) when this gene mutate. After X-irradiation, frequency of LacZ⁻ colonies increased depending to dose, which indicated that X-irradiation caused the LacZ gene mutation in CHO-LacZ cells. Delayed mutagenesis was examined 15 PDN after irradiation, and the frequency of LacZ⁻ colonies in X-ray-surviving cells was higher than that in control cells. Next, we studied mutation spectrum of LacZ⁻ clones. Using PCR, the LacZ gene was absent in approximately 75% in delayed LacZ⁻ clones. This result was like to mutation spectrum in the spontaneous LacZ⁻ clones (72%). These results indicate that delayed mutagenesis rise spontaneous mutation level. [J Radiat Res 44:441 (2003)]

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We previously demonstrated that scid mouse cells were hypersensitive to genetic instability induced by radiation. To confirm this result, we established scid mouse cells expressing human DNA-PKcs by the introduction of a human chromosome 8. The human DNA-PKcs complemented the hypersensitivity to the induction of delayed reproductive death in scid mouse cells, indicating that NHEJ play a role to suppress the induction of genetic instability. To know a role of double strand break repair in the induction of genetic instability further, we investigated delayed reproductive death in chicken Rad54^−/- DT40 cells. The result indicated no difference in delayed reproductive death between wild-type and Rad54^−/- DT40 cells, suggesting that homologous recombination might play little role in the induction of delayed reproductive death. [J Radiat Res 44:441 (2003)]

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The gene responsible for ataxia telangiectasia (AT) encodes ATM protein that plays a major role in the network of a signal transduction initiated by DNA-double strand breaks. To elucidate how radiation-induced genomic instability is modulated by dysfunction of ATM protein, we examined delayed chromosomal instability in individual cell lines established from wild type Atm^+/+ , heterozygote Atm^+/- and knock out Atm^−/- mouse embryos. The Atm^−/- mouse cells show elevated chromosomal and telomeric instability compared with Atm^+/+ mouse cells. The telomeric instability was characterized by the abnormal telomere FISH signals including loss and gain of the signals in the end of chromosome. This suggests that Atm deficiency makes telomeres vulnerable to breakage. Thus, the present study shows that ATM protein plays an essential role to maintain telomere integrity and prevent chromosomes from end-to-end fusions. [J Radiat Res 44:441 (2003)]

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Ionizing radiation induces chromosomal instability that is transmitted over many generations after irradiation in the progeny of surviving cells. It is suggested that delayed chromosomal instability plays a role in the development of cancers. However, the trigger for inducing the instability remains unknown. To know the trigger, we constructed two types of microcell hybrids by microcell-mediated chromosome transfer where a human chromosome 11 was transferred into a mouse cell line. In one type of microcell hybrids, 4 Gy-irradiated human chromosome 11 was introduced into unirradiated mouse cells, and in the other type of microcell hybrids, unirradiated human chromosome 11 was introduced into 4 Gy-irradiated mouse cells. Chromosome aberrations were analyzed by fluorescence in situ hybridization. The results indicated that not only the irradiated chromosome per se but also the irradiated recipient cells contribute to the trigger for the induction of delayed chromosomal instability by radiation. [J Radiat Res 44:441 (2003)]

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Radiation exposure causes genetic instability in mammalian cells while the underlying mechanisms are not well understood. Although most of the animal studies used repeat sequences as the target for detection of the instability (e.g., minisatellites and pink eye-unstable locus), no pertinent assay systems are available in cultured cells. We therefore thought it useful to create cultured cells bearing partial, tandem duplication of a gene. We have chosen HPRT gene as a model since the duplication makes the cells HPRT(-) and hence selectable in the presence of 6TG, and HPRT(+) revertants due to intragenic recombination of the duplicated sequences in HAT medium. We constructed a Neo-tagged vector carrying 8.4kb genomic DNA that covers HPRT exons 2-3. Transfection of human fibrosarcoma cells (HT1080) with the vector yielded Neo-resistant and 6TG-resistant clones. These cells reverted spontaneously from HPRT(-) to HPRT(+) with relatively high frequencies as expected [i.e., 10(-5) to 10(-4)]. Preliminary results will be presented on the relation between the reversion and forward mutation frequencies at the HPRT gene of some clones. [J Radiat Res 44:442 (2003)]

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Radiation generates reactive oxygen species (ROS) that may contribute to the induction of genetic instability. We examined the suppressive effect of a radical scavenger, ascorbic acid phosphate magnesium salt (APM), on the induction of delayed reproductive cell death by radiation. The delayed cell death was determined by two successive colony formation periods. The treatment with APM was applied either primary and secondary colony formation. The result indicated that the APM treatment during primary colony formation, but not secondary colony formation, suppressed the delayed reproductive cell death. We also demonstrated that the rapid increase and then decrease of the amount of hydrogen peroxide (ROS) in X-ray-irradiated cells by 5 hr postirradiation and then the level of hydrogen peroxide gradually decrease to a base line within two weeks. The APM treatment kept the hydrogen peroxide production in a lower level than an untreated control. These results suggest that the cause of genetic instability might be fixed by partly ROS during 2 weeks postirradiation. [J Radiat Res 44:442 (2003)]

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In this study, we examined whether X-ray-induced gene deletion at the HPRT locus induces delayed instability in X-chromosome. SV40-immortalized normal human fibroblasts, GM638, were irradiated with 3 Gy of X-rays, and the HPRT mutants were isolated in the medium containing 60 µM of 6-thioguanine. The molecular structure of the HPRT mutations was determined by multiplex polymerase chain reaction of the eight exons of the HPRT gene. The size of deletions in total HPRT deletion mutants were determined by PCR amplification using sequence tagged site (STS) primers. Delayed chromosomal instability in X-chromosome was analyzed by whole human X-chromosome paint FISH. GM638 and spontaneous mutants, which showed no detectable change in the HPRT gene, did not induce X-chromosomal rearrangements, however, 3 Gy-induced mutants with large deletion showed delayed instability involving X-chromosome. These results suggest that ionizing radiation-induced large deletion is involved in initiation and perpetuation of radiation-induced genomic instability. [J Radiat Res 44:442 (2003)]

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We previously reported that the genomic instability was observed on F1 mice born with irradiated sperm. In this report, we examined whether this phenomenon had the dependency of irradiation stage or not. It was only observed as spermatozoa stage irradiation, but not spermatid, -cyte and -gonia stage. Moreover, in two-next generation (F2) mice, the mutation frequency does not increase. These findings suggest that the genomic instability which we estimated especially occurs at spermatozoa stage. [J Radiat Res 44:442 (2003)]

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Mutations in the Recql helicase domain cause several genetic disorders in humans, including Werner syndrome, Rothmund-Thomson syndrome and Bloom syndrome. Common phenotypes of these diseases include premature aging and shortened lifespan. Genomic instability is also observed in the cells of these patients. In order to understand the role of RECQL4, which is the product of the gene responsible for the Rothmund-Thomson syndrome, we prepared two independent +/- ES cell lines and established two -/- cell lines from one and one -/- cell line from the other. Using these, we measured sensitivity to several genotoxical reagents, including hydroxyurea, mitomycin C, UV and IR. We discuss the role of RECQL4 using the results of our assay. [J Radiat Res 44:442-443 (2003)]

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In an attempt to elucidate a possible involvement of Xpc gene in the maintenance of genomic DNA in adult organs, we examined spontaneous mutation in brain, liver and spleen of lacZ-transgemic mice at different ages. The three organs showed similar levels of mutant frequency at 2 months of age. The levels were comparable to those of wild type mice. At old age, however, liver and spleen showed increases in mutant frequency and they were higher than those found in wild type mice. Preliminary studies of DNA sequences of the mutants showed that the spectrum of mutation might be different between young and old tissues. The results suggest that the Xpc gene is involved in suppression of mutation in liver and spleen of adult mice. Old brain revealed a slight increase but no difference between Xpc(-/-) and Xpc(+/+). [J Radiat Res 44:443 (2003)]

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A single cell irradiation system has been developed at JAERI-Takasaki. Individual mammalian cultured cells are irradiated in the atmosphere on the cell dish, the bottom of which is made of ion-track-detector CR-39, with a single or precise numbers of 13.0 MeV/u ²⁰Ne and 11.5 MeV/u ⁴⁰Ar ions. Positional data of the target cells are obtained at the off-line microscope before irradiation. Targeting and irradiation of the cells are performed automatically at the on-line microscope of the microbeam apparatus according to the obtained data. About 50-100 cells are irradiated in several minutes. The number of ions traversed the cells attached on the ion track detector CR-39 were counted with a plastic scintillator-photomultiplier tube assembly and a constant fraction discriminator. Immediately after the irradiation, the bottom of the cell dish was etched at 37 C to check the spatial distribution of irradiated ions. The one batch of cell sample is treated in about 1 hr including the phases of cell finding, irradiation, and etching/observing of the ion track detector CR-39. [J Radiat Res 44:443 (2003)]

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To analyze the effect of heavy ion radiation on mammalian cultured cells, we developed the cell irradiation system under the high-energy collimated heavy ion microbeam apparatus in JAERI Takasaki, and obtained some preliminary results of inhibitory effect on cellular growth by heavy ion hit. The apparatus itself and the experimental procedures of the cell irradiation were modified for improving cell irradiation throughput for obtaining detailed data. The improvement of cell irradiation throughput of the system enabled us to confirm our preliminary results: 1. The irradiation of ⁴⁰Ar¹³⁺ (11.5 MeV/u, LET=1260 keV/µm) ion on cell nucleus resulted to the complete inhibition of the cell growth. 2. The bystander cells in the irradiated dish showed reduced cell growth rate. [J Radiat Res 44:443 (2003)]

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In order to evaluate the risk of low dose radiation, a microbeam irradiation system has been developed, by which we can irradiate individually identified cells with monochromatic (5.35 keV) synchrotron X-ray microbeam. The system is composed of a focussing mirror system or an adjustable 4-D slit system to obtain a microbeam, a fluorescence microscopy (Olympus BX51WIS) equipped with a precise motor-controlled sample stage (Prior H101, stroke 100mm × 75 mm, reproducibility of positioning 1 micron), and a cell image capture system (Hamamatsu ORCA-ER-1394). Beam size routinely available is 5 micron square or larger, depending upon the request. Beam intensity is about 6 Gy per min, independent of the beam size. Also we have developed a system software to control all the process with Visual Basic calling Image Pro plus as a subroutine. Presently, we can automatically scan and identify the target in desired area of the dish and also irradiate them one by one automatically. [J Radiat Res 44:443-444 (2003)]

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X-ray bystander effect was demonstrated by the observation of X-ray induced p53 expression area extended from the irradiated point of X-ray microbeam. X-ray microbeam was prepared from micro-capillary equipped in the apparatus for X-ray fluorescence analysis at local areas with operation voltage of 15 kV (XGT-2700, HORIBA, Ltd.). Confluent cells of NB1RBG (human normal skin fibroblast) and A549 (human lung carcinoma) were irradiated with X-ray microbeam emitted from a 100 micrometer capillary for 1 min. The expression area of p53 was determined by the fluorescent antibody technique after 6hr incubation from irradiation. Beam diameter and irradiation dose were estimated by the density on X-ray film. Beam size was found to be enlarged to the diameter of 165 micrometer. The dose rate was approximately 0.3 Gy/min. The results showed that the p53 area was about the same as the size of a beam diameter for A549 cells, while for NB1RGB cells the area exceeded the size of an X-ray microbeam. The present observation can be explained by the presence of the gap junctional function. [J Radiat Res 44:444 (2003)]

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