SCID mice which have the defect of DNA-dependent protein kinase catalytic subunit, exhibit the limited activities of repair from DNA double strand breaks and are sensitive to ionizing radiation. In order to study the relationship between repair capacity for DNA double strand breaks and carcinogenesis, and the responsibitily at the low dose of gamma rays, the effects of ionizing radiation on tumor induction were studied using scid homozygote (scid/scid), and C.B-17(+/+)mice. Carcinogenesis experiments showed the significant increase of the incidence of thymic lymphomas at 25cGy to sublethal dose. At very low dose(5 and 10cGy), non-thymic B or T cell lymphomas were incuced in SCID mice, whereas there was no induction of these lymphomas in wild-type mice. No tumors other than lymphomas were observed, because of the life shortning effect by the induction of thymic lymphomas. Thus SCID mice is highly sensitive to the induction of non-thymic lymphomas at very low doses. [J Radiat Res 44:395 (2003)]

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Common polymorphism in the TP53 gene (encoding Arg72Pro in p53) was analyzed in radiation-associated post-Chernobyl and spontaneous series of adolescent and adult papillary thyroid cancer (PTC) in patients of Russia by real-time PCR allelic discrimination assay and direct sequencing. Reference data for sporadic PTC were available for German caucasians. The distributions of sequencing variants in both radiation-associated series, but not in spontaneous one differed significantly from the reference group. Radiation-associated groups harbored at least heterozygous 72Pro significantly more often, and homozygous 72Arg less often than the reference group. The radiation-associated and spontaneous adult groups statistically differed from each other for these indexes. Data imply the different distribution of allelic forms of the TP53 in PTCs of diverse ethiopathogenic groups. [J Radiat Res 44:395 (2003)]

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Genomic DNA of acute myeloid leukemia (AML) cells induced by x-ray in C3H/He mice is frequently accompanied with illegitimate insertion of the cDNA of the intracisternal A-particle (IAP) DNA element that is present in normal murine genome in two thousand copies per haploid and is closely related with provirus form of retrovirus in the structure. We established the PCR-based method to detect the characteristic integration site of IAP cDNA in tumors. From genomic DNAs of 11 lines of radiation-induced AML cells from C3H/He mice, 21 sites of the unique integration sites of IAP cDNA were amplified by this method. In contrast, occurrence of only 4 insertion of IAP cDNA were newly observed among 40 lines of thymic lymphoma cells from C3H/He mice. Since insertion of IAP element with H-type LTR was observed in all the characteristic site, suggesting that activation of the H-type LTR of IAP is common feature in hematopoietic tumors in C3H/He mice. [J Radiat Res 44:395 (2003)]

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Mouse genome possesses 2000 copies of the retrotransposon, intracisternal A-particle (IAP) DNA element, that closely resembles to provirus of retrovirus. We have previously classified long-terminal repeat (LTR) of IAP elements in genome by its nucleotide sequence variation to type-A to H. The integration of IAP cDNA with H-type LTR into genomic DNA to generate illegitimate structure is frequently observed in radiation-induced acute myeloid leukemia (AML) cells in C3H/He mice. Since production of the IAP RNA is the first step of retrotransposition mechanisms and is regulated by the 5'-LTR, we compared transcriptional activity of the LTRs of type-A, D and H by the reporter assay. Transcriptions driven by D-type LTR and H-type LTR were enhanced in Balb/c-based and C3H/He-based hematopoietic cell lines, respectively. Nucleotide sequences essential for mouse strain and tissue specific regulation site in U3-region of LTR were determined. [J Radiat Res 44:395-396 (2003)]

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Min(multiple intestinal neoplasia) mice carry a mutant allele of the murine Apc locus and are predisposed to adenoma formation in the intestinal tract. Tumorigenesis in Min mice is affected by various factors including genetic background and ionizing radiation. In this report, we addressed a possibility of genetic relevance between spontaneous and X-ray induced tumorigenesis in Min mice. MSM strain is highly resistant to intestinal tumorgenesis by Min mutation. QTL analysis indicated that a newly identified modifier locus Mom3 is a major determinant of the resistance, together with a resistant allele of Mom1 locus. A congenic strain of Mom 3 and a consomic strain of the chromosome in the B6-Min background resulted in more than five-fold decrease in tumor multiplicities in the intestine of Min mice. X-irradiation of the consomic mice increased their tumor incidence, almost reaching to a level in irradiated B6-Min mice. These data suggest a possibility of the crosstalk between induction by X-irradiation and suppression by Mom3 allele in the tumorigenesis in Min mice. [J Radiat Res 44:396 (2003)]

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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. We have examined the frequencies of the AML-type deletions in primitive hematopoietic cells that could be the targets of the leukemogenesis. Ten-week male C3H/He mice were exposed to 3Gy x-rays and sacrificed after certain periods of time. Bone marrow cells were collected from femora and a single-cell suspension from each animal was divided into two parts. One part was processed with MACS to obtain Lin⁺ and Lin⁻Sca1⁺ cells. Those cells were analyzed with FISH for the AML-type deletions. The other part of the cell suspension was cultured in methyl cellulose media, and metaphase spreads were prepared from each growing colony. Results obtained so far for 1 day post irradiation show that approximately 3% of the cells carried the AML-type deletions in both Lin⁺ and Lin⁻Sca1⁺ subpopulations while metaphases in GM colonies were mostly of normal karyotypes except for a small number of non-clonal aberrations. [J Radiat Res 44:396 (2003)]

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A high prevalence of aberrant beta-catenin has been reported in radiation-associated thyroid tumors (RATT). CyclinD1 is a target activated by Wnt/beta-catenin. While,Pin1 promotes cyclinD1 expression and p53 phosphorylation induced by irradiation. This study aimed to elucidate whether Pin1 was involved in cyclinD1 and beta-catenin expression in RATT. We examined follicular adenomas (FA) and papillary carcinomas (PC). All PC displayed cyclinD1 overexpression and a cytoplasmic beta-catenin. Overexpression of cyclinD1 mRNA was observed in 45.5% of FA and 54.5% of PC. Correlation was revealed between cyclinD1 and Pin1/cytoplasmic/membrane beta-catenin expressions, and between Pin1 and cytoplasmic/membrane beta-catenin expressions in RATT. Any mutations of beta-catenin gene could not be detected. Western blot analysis demonstrated a high level of cyclinD1 and beta-catenin as well as Pin1 expressions in a PC cell line. This study supported a significance of cyclinD1 overexpression and aberrant beta-catenin in RATT. Pin1 may be an important factor to regulate cyclinD1 and beta-catenin in RATT. [J Radiat Res 44:396 (2003)]

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We are living in the environment with numerous natural and man-made chemicals that are cancer-initiating and promoting potentials. Thus exposure to radiation could be a result of interaction with these agents. However, the quantitative assessment and mechanistic understanding of combined effects of radiation and chemical carcinogens are still insufficient. Mechanism-based cancer model is expected to greatly improve the risk assessment. We have set up an experiment for the combined effect of radiation and chemicals in order to determine the dose-response relationship including low doses. The animal models used in the present study are murine thymic lymphoma and rat mammary cancer. The mode of modification of radiation effect by chemicals in terms of interaction between X-rays and alkylating agents will be discussed from the viewpoint of molecular mechanisms of cancer development. [J Radiat Res 44:396-397 (2003)]

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A bacterial plasmid gene mucAB has an error-prone activity and is widely used for sensitive detection of mutagens as Ames test. The mucAB gene (1.7 Kbp) was cloned into a vector carrying the mammalian metallothionein promoter (pTE40), which enhanced transformation of cultured mouse cells in the previous study. In this study, mucAB was introduced into an embryo of C57Bl mouse to obtain mucAB transgenic mice. Among 93 mice from 439 injections, we found only one mice in which genome mucAB was integrated. Heterogenic mucAB mice from this founder were examined for tumor induction efficiency by subcutaneous injection of 0.02 mg methylchoranthrene. The mice developed fibrosarcomas with an elevated efficiency (24%) as compared with control mice without mucAB (8%) until the present moment. [J Radiat Res 44:397 (2003)]

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The risk of radiation carcinogenesis is influenced by various factors, including genetic and age factors. The human Apc gene was originally identified as a tumor suppressor gene in both sporadic and hereditary (familial adenomatous polyposis; FAP) colorectal cancers. Recently, missense mutations and promoter hypermethylations of Apc gene have been reported in human breast cancers, suggesting an involvement of Apc mutation in their development. Moreover, FAP model mice with germline Apc mutations have an increased susceptibility to mammary carcinogenesis. To investigate the effect of age at exposure on X ray-induced mammary carcinogenesis in the FAP model Apc^Min/+ mice, we examined the mammary tumor development after X irradiation (2 Gy) at various weeks of age. We found that, whereas no tumor was seen in the wild-type littermates, Apc^Min/+ mice developed mammary tumors, with significantly higher incidence in those irradiated at 7 and 10 weeks than those irradiated at 5 weeks. Histologically, all tumors were adenoacanthomas. We discuss the possibility that age dependency of the risk of radiation carcinogenesis is modulated by genetic factors. [J Radiat Res 44:397 (2003)]

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Mouse thymic lymphomas induced by gamma-irradiation exhibited homozygous deletions of Rit1/Bcl11b at a high frequency. Loss of one allele was due to internal chromosomal deletions of mostly exon 2 and exon 3 and the loss of the remaining allele due to allelic loss. To elucidate the mechanism of these internal deletions, we examined break and rejoining points by PCR-mapping and determined sequences in the vicinities. The mapping in lymphomas revealed clustering of recombination sites and subsequent sequencing showed the presence of cryptic sequences recognized by the RAG1/2 recombinase and the P and/or N nucleotides in the rejoining sites. This suggests that these deletions of Rit1 involve an illegitimate V(D)J recombinase activity in radiation-induced mouse thymic lymphomas. Interestingly, such aberrant recombination was detectable in the thymus of wild-type mice, but not of RAG2-deficient mice, and the recombination frequency was not increased by gamma-irradiation. Possible mechanisms for radiation lymphomagenesis will be discussed. [J Radiat Res 44:397 (2003)]

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Thorotrast is colloidal solution of natural alpha-emitter, thorium dioxide, which was used as a radiological contrast medium during World War II. Decades after injection, it caused hepatic tumors by local exposure to alpha-particles. Histological examination revealed that about 1/3 cases of Thorotrast-induced cancers consist of angiosarcoma (AS). In order to elucidate carcinogenic mechanisms of radiation-induced cancers, we performed genome wide scan of loss of heterozygosity (LOH) in Thorotrast-induced AS. As well as ICC, Thorotrast-induced AS showed more LOH frequency compared with non-Thorotrast cases. LOH frequency at loci common to liver cancer was low in AS. We found a Thorotrast-induced cancer specific locus on chromosome 8q. These indicate that LOH loci specific to the origin of cancer cells and Thorotrast-induced cancers exist, respectively. [J Radiat Res 44:397 (2003)]

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We have previously identified two pathways for the deletion formation of Notch1 gene in radiation-induced mouse thymic lymphomas: in wild-type mice, the 5' deletion of Notch1 was induced by illegitimate V(D)J recombination using recombination signal sequence-like elements present in the 5' region of Notch1, whereas in the absence of DNA-PKcs function in scid mice, microhomology-mediated end-joining pathway acts as a mechanism for deletion formation. While it was reported that there was no induction of thymic lymphoma by radiation in scid mice without Rag gene, we demonstrated that even in the absence of Rag2 gene, scid mice developed rediation-induced thymic lymphomas, which is consistent with the notion that there are two pathways in the deletion formation. To validate the presence of two pathways, the Notch1 abnormalities in radiation-induced thymic lymphomas developed in Rag2-KO, scid, and Rag2-KOscid mice were examined by Southern blot hybridization. The results showed that in the absence of Rag2 gene, the 5' deletions were formed, demonstrating that there is a Rag-independent deletion pathway. [J Radiat Res 44:398 (2003)]

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Ikaros is essential for the normal development of lymphoid cells. We previously showed that Ikaros was frequently inactivated in radiation-induced mouse T-cell lymphomas and that one of the inactivation mehanisms is abnormal expression in dominant-negative Ikaros isofoms. The pathway of leukemogenesis mediated by Ikaros inactivation has not determined yet. To clarify the mechanism of leukemogenesis caused by Ikaros inactivation, we attempted to identify genes that are altered in expression by induction of dominant-negative Ikaros isoform, IK6. We established cell lines that inducibly expressed IK6 under the control of a tetracycline-inducible promoter. Changes of mRNA expression profiles were monitored by DNA microarray after Tet-inducible expression of IK6. Then, we identified 60 genes that were up- or down-regulated. Expression of these genes was confirmed by quantitative real-time PCR analysis. As a result, we found that expressions of these genes were changed as a function of time after induction of IK6. Promoter activities of selected genes are currently examined using reporter plasmids. [J Radiat Res 44:398 (2003)]

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BALB/c-p53+/- (CP53+/-) mice develop a high incidence of mammary tumors within 36 weeks after 4 x 0.7 Gy of X-irradiation. In the present study, we examined development of mammary tumors induced by 4 x 0.35 Gy of X-irradiation in CP53+/- mice and F₁ hybrids (B6CP53) between CP53+/- and C57BL/6, a resistant strain for mammary tumors. Irradiated CP53+/- and B6CP53+/- mice started to develop lymphoma at 18 and 20 weeks, respectively, and mammary tumor at 24 and 34 weeks. Mortality of CP53+/- mice during 36 weeks was 18/23 (78%), resulted from 12 (52%) mammary tumors, 4 (17%) lymphomas and 2 (9%) unknown causes. Mortality of 23 B6CP53+/- mice during the same period was 6/23 (26%), caused by 1 (4%) mammary tumor, 4 (17%) lymphomas and 1 (4%) unknown cause. None of 14 B6CP53+/+ exposed to radiation died during the period. The lower mortality of B6CP53+/- mice compared to CP53+/- mice resulted from their resistance to mammary tumors. [J Radiat Res 44:398 (2003)]

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Radiation carcinogenesis in human is considered as a result of the combined effect of radiation and environment factor. The aims of current study are both to demonstrate the dose-response relationship for the combined treatment of radiation and chemical carcinogens (ENU) at low doses and to elucidate the underlying mechanism. The data in progress showed that the dose-response curve for radiation-induced TLs (0.2, 0.4, 0.8, 1.0, 1.2, 1.6, and 2.0Gy a week × 4 times) was sigmoidal with threshold dose at 0.8Gy, and that for ENU-induced TLs (50, 100, 200, and 400ppm, drinking for 4 weeks) was linear with threshold dose at 100ppm. Combined-treatment of both agents at the threshold dose increased the incidence of TLs synergistically. The treatment in the sequence of X-rays (1.0Gy) - ENU (100ppm) induced TLs at higher incidence than the reverse sequence of ENU - X-rays. The frequency of loss of heterozygousity was much reduced in the TLs induced by combined treatments compared with X-ray-induced one. [J Radiat Res 44:398-399 (2003)]

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To examine possible contributions of individual tumor suppressor genes to radiation mammary carcinogenesis, we have investigated the loss of heterozygosity (LOH) in the tumors from p53 and Atm heterozygous deficient mice. (BALB/cHeA-p53^+/KO x MSM/Ms-Atm^+/KO)F₁ hybrid female mice were exposed to single dose of 5Gy of X-rays at 5 weeks of age. Fifty-four mammary adenocarcinomas (MC) (25%) were observed in 219 irradiated female mice. The MC frequently developed between 211 and 240 days after birth. Twenty MC (37%) were observed in p53^+/KO-Atm^+/+ mice, and 34 MC (63%) developed in p53^+/KO-Atm^+/KO mice. Frequencies of LOH in MC were 30%, 52%, 20% 28%, 57% and 65% on chromosomes 5, 8, 9, 10, 11 and 12, respectively. The LOH pattern in the MC from p53^+/KO-Atm^+/+ mice was similar to that of the p53^+/KO-Atm^+/KO mice. Precise allelotype analysis on chromosome 8 is in progress. [J Radiat Res 44:399 (2003)]

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To analyze putative tumor suppressor genes in mammary carcinogenesis, we refined the regions with frequent loss of heterozygosity(LOH) on chromosomes 9,10 and 12 in the tumors developed in p53 and Atm heterozygous deficient mice. F₁ females produced by BALB/cHeA-p53^+/KO and MSM/Ms-Atm^+/KO were exposed to 5Gy of X-rays at five weeks of age. DNA was extracted from mammary adenocarcinomas and analyzed for LOH in detail. We found frequent LOH in 2 regions on chromosome 12. One region is located near centromere. The frequency of LOH reached 60%. Allele derived from BALB/c was preferentially lost, suggesting a contribution of this region to cancer susceptibility. The other region includes D12Mit132(52cM), and frequency of LOH was about 56%. Frequent allele losses were also found in this region of lymphomas. Notable LOH was also observed at D9Mit20(60cM)(20%) and D10Mit7(43cM)(36%). However, wild allele of Atm on chromosome 9 was retained. [J Radiat Res 44:399 (2003)]

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The total body irradiation of C3H/He mice with 1-3Gy produce myeloid leukemias as compared with non-irradiated controls that produce fewer than 1% of leukemia (Radiat. Res., 127: 146, 1991). The thymic lymphoma (TL) prone ATM-deficient 129 strain (Cell, 86: 159, 1996) was crossed back into C3H/He strain. Lethally irradiated wild type C3H/He mice to which Atm-deficient bone marrow cells were transplanted showed a considerably shortened life span either in total or in TL-free survivals. Posttransplantation irradiation of BMT mice with 3Gy of X-rays gave rise to a faster development of TL but no prevailing leukemia in contrast with the case of transfer of p53-/- or p53+/- bone marrow in which undifferentiated stem cell leukemia emerged (Leukemia Res., 26: 1085, 2002). Again, both median and mean survivals of mice transfused with Atm-/- bone marrow cells significantly diminished regardless of emergence of TL or leukemia. The significance of ATM function to support BMT recipient will be discussed. [J Radiat Res 44:399 (2003)]

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Radiation exposure increases the risk of both leukemia and solid tumors, including the cancer of mammary gland, which is one of the most radiosensitive organs in respect to carcinogenesis. The breast cancer risk is reported to be high in female flight attendants. It is therefore likely that female astronauts, also exposed to cosmic rays, and cancer patients treated with heavy ion therapy may have an increased risk of breast cancer. In order to obtain basic information on the heavy ion-induced mammary carcinogenesis, we irradiated rats of four strains (Sprague-Dawley, F344, Wistar and ACI/N) with 290 MeV carbon beam (LET 50 keV/µm) at doses of 0, 0.5, 1 and 2 Gy, and examined mammary tumor development up to 300 days. It was found that Sprague-Dawley and Wistar rats had high susceptibility, while F344 and ACI/N rats had low susceptibility. The incidence and multiplicity were dose dependent. Pathologically, tumors were mainly adenocarcinomas and fibroadenomas. These results suggest an existence of distinct strain- and dose-dependency in the heavy ion-induced mammary tumor development. [J Radiat Res 44:399-400 (2003)]

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DNA double strand break (DSB) is the most destructive damage caused by ionizing radiation and can lead to cell death and transformation if not repaired properly. The premature chromosome condensation (PCC) assay using cell fusion is a sensitive method to detect DSB at the chromosome level especially in G1 cells. However, the occurrence of repair during the incubation period of this method lowers its sensitivity. By using a DSB repair inhibitor, wortmannin (WM) during this critical period, we have improved the sensitivity of the PCC assay. Normal human fibroblasts were X-ray irradiated under cold condition and immediately fused with mitotic HeLa cells using Hemagglutinating virus of Japan envelop (HVJ-E). Wortmannin was added just before incubation of the PCC procedure. After 1 hour, cells were fixed and stained with Giemsa and was observed under light-microscope. We have observed almost twice as many chromosome breaks in WM treated cells as compared with cells without WM. We also were able to measure repair of chromosome breaks with X-ray doses less than 0.5 Gy. [J Radiat Res 44:400 (2003)]

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We developed a model to explain the frequency of clonal chromosome aberrations. We now show that application of our model to data from published reports gives good agreement between the calculated and observed numbers of clones. As our model, based on data of A-bomb survivors collected 50 years after irradiation, explains data for Chernobyl workers collected 5-6 years after the event, we conclude that clonal expansion is completed within the time needed for recovery from radiation-decreased lymphocyte counts. A recent study (Mori et al., PNAS 99; 8242, 2002) showed that not only ALL-specific translocations occurred in 1% of cord blood samples of ordinary newborns (100-fold more frequent than pediatric ALL frequency with the same translocation) but also that the translocation-bearing B (or pre-B) cells were clonally expanded to 1/1,000. We propose that ALL in A-bomb survivors was not due to induction of leukemia-specific translocations but that radiation affected a few individuals who, at the time of the bombing, had spontaneously-derived expanded levels of cells potentially leukemogenic, but not leukemia causing. [J Radiat Res 44:400 (2003)]

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Deletions of the chromosome 2 middle regions are associated with the radiation-induced mouse acute myeloid leukemias, while murine small eye mutants delete the chromosome 2 middle regions genetically. Expecting the predisposition to acute myeloid leukemia, the tumorigenicity of the two small eye mutants, Pax6Sey3H and Pax6Sey4H was examined. The commonly deleted region of the two small eye mutants was the segment of 3.2Mb between 106 and 109Mb, where the Wt1, Rcn, Pax6, Elp4 and other fourteen novel genes located. The two mutants produced glucagon in the islets, however, they impaired to elevate the blood glucose level up when loaded with insulin. Morphological anomaly of the Wirsung duct, pancreatitis or degeneration of the pancreas was observed with age. Both mutants developed intestinal tumors spontaneously (52.0% and 32.0%), which were not observed in the normal sibs. gamma-rays and MNU shortened the latency but did not increase the frequency of the hematopoetic tumors in the mutants. The hemizygous deletions of the 3.2Mb-segment of the chromosome 2 did not contribute for the development of hematopoietic tumors mainly. [J Radiat Res 44:400 (2003)]

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Cultured Human Normal Cells, GM 05389, were irradiated with heavy-ion beams and the chromosomal aberrations and its repairing kinetics were analysed by prematurely chromosome condencation method (PCC method) using calyculin A. The cells were irradiated with 290 MeV C, 490 MeV Si, 500 MeV Ar and 500 MeV Fe ions at HIMAC facility of the National Institute of Radiobiological Sciences in Chiba. Various LET values were selected at the irradiations of these ions. These were 13, 120, 136, 370 and 440 keV/mm. Chromatid-type breaks, isochromatid breaks and exchanges were scored for the samples from the cells keeping with various incubation time after irradiation. Some specific characteristics of repairing kinetecs were clarified for chormosomal aberrations induced by heavy-ion beams. [J Radiat Res 44:400-401 (2003)]

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We examined canavanine-resistance (Can^R) mutations in a CAN1/ CAN1D::LEU2 heterozygote S. cerevisiae formed by mating UV-irradiated and unirradiated haploids. All the Can^R diploid cells from unirradiated haploid cells were found to be due to chromosome rearrangement or chromosome loss and Can^R-frequency was 0.4×10⁻⁴/cell. To see the effect of UV for induction of LOH, UV irradiated haploid cells with functional CAN1 gene were mated with unirradiated haploid cells with CAN1-deficent and Can^R diploid cells were selected. The frequency was 1.4×10⁻³/cell. After irradiation to CAN1-deficient haploid cell, Can^R frequency was 0.4×10⁻³/cell. Consequently, irradiation of functional CAN1 cells causes higher induction of LOH than irradiation of CAN1-deficent cells. Therefore, an existence of DNA lesions on the chromosome with functional CAN1 yields effective induction of LOH. By analysis of chromosome structure of Can^R diploid mutant, we observed that LOH of the CAN1 gene was mostly associated with replacement of the CAN1 gene to telomere with can1D to telomere of homologous chromosome. We argue that LOH could have occurred during the repair of DNA lesions on a damaged chromosome due to nonreciprocal recombination. [J Radiat Res 44:401 (2003)]

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Ratio of reciprocal translocations vs. dicentrics was examined in 3 Gy X-irradiated lymphocytes. 123 reciprocal translocations and 105 dicentrics were detected in 948 metaphases by dual analyses of conventional Giemsa staining and chromosome painting (Nos. 2 and 4 were painted). The numbers of translocations and dicentrics originated from 2 chromosomes both of which consisted of a larger centric segment and a smaller acentric fragment were 89 and 86. They were statistically not different. Those originated from a pair consisting of a smaller centric segment and a larger acentric fragment were 4 and 3. They were too few for statistical analysis. Those originated from a heteromorphic pair (larger centric + smaller acentric and smaller centric + larger acentric) were 30 and 16. The yield of translocations was significantly higher than that of dicentrics. The larger (smaller) centric segments tended to join with the larger (smaller) acentric fragments, that resulted in more translocations in the irradiated lymphocytes. [J Radiat Res 44:401 (2003)]

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To evaluate the biological effects of neutrons with low energy spectrum, human Go-lymphocytes were irradiated with monoenergetic neutrons with 0.186 to 2.3MeV . We useed two biomarkers for radiation quality namely complex chromosome aberrations(CCA) as insertions and chromatid aberrations as a marker for genetic instability. Metaphase chromosomes were painted by FISH using chromosomes 1,2 and 4. Neutrons of 2.3, 1.0, 0.79, 0.57 0.37 and 0.186 MeV were generated and ganmma rays were used for comparison. The RBE was changed by energy spectrum and reached to 16.4 at 0.37MeV. Ratio of incidences of trans.and dic.chromosomes is influenced by source of radiation, dose rate and exposure dose.Number of chromosomes out of the 3 pairs of chromosomes associated with CCA were compared among neutrons with different energy levels at the exposure doses showing a same biological effects. 0.37MeV neutrons has highest chromatid aberration rate. These results suggest that formation of exchanged type of chromosome aberrations and genetic instability were influenced by neutron energy spctrume. [J Radiat Res 44:401 (2003)]

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Okadiac acid-induced prematurely condensed ring chromosomes (PCC rings) induced with X-irradiation were analyzed under a light microscope and then by an atomic force microscope (AFM) after Giemsa staining. PCC rings and small round-shaped chromosomes or chromatids without obvious holes that were difficult to recognize as PCC rings (PCC ring-like chromosome fragments or PCC ring-like chromatid fragments) by light microscopy were selected. Those Giemsa stained PCC rings and PCC ring-like fragments were visualized and analyzed by AFM. Section analysis by AFM revealed that some of the PCC ring-like chromatid fragments and PCC ring-like chromosome fragments were thicker than standard chromatids or chromosomes. These were considered as PCC rings turned sideways on slide. Atomic force microscopy made possible to identify these radiation-induced aberrations for improvement of the accuracy of dose estimation. [J Radiat Res 44:401-402 (2003)]

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We examine the effects of low dose X-irradiation on development in the fly. Following exposure of prepupal (day 5) flies to 0.5 Gy X-rays, the time to emergence was slightly shorter than in the sham controls. This tendency was increased when the X-ray exposure came during the pupal stage (day 7). In these flies, the time to eclosion decreased significantly, by an average of thirty hours sooner than sham controls. A further experiment examined whether such radiation effects could be observed in the unexposed F1 generation of exposed individuals. Greater radiation effects on early F1 emergence were seen when the time between exposure and mating was 3 days, indicating an effect on early spermatid development. Early F1 emergence was also observed after exposure of female flies to X-rays during late previtellogeny. Furthermore, rapid emergence could be induced in the F1 embryos of unexposed parents by transferring the polar cytoplasm from F1 embryos of exposed flies. [J Radiat Res 44:402 (2003)]

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Dose response relationship of the X-ray induced loss of heterozygosity (LOH) was examined in somatic cells of fruit fly Drosophila melanogaster. Using the wing spot test, the frequency of somatic mutation that induced LOH in two external marker genes (mwh and flr, both on the third chromosome) was measured in a post replication repair deficient mutant, mei-41 (a Drosophila homologue of the human ATM gene). In mei-41 heterozygotes in which the repair function is still active, the frequency of mutant spot in the high-dose groups (2 or 3Gy) was higher than that in the sham-exposed group, whereas that in low-dose groups (0.2 or 0.5Gy) was significantly lower than that in the sham-exposed group. There was a threshold at around 1Gy in the dose-response relationship in mei-41 heterozygotes, while in homozygous mei-41 siblings without repair function, the threshold was smaller, and the inclination of the dose-response curve was steeper than in heterozygotes. It is inferred that the DNA repair function is involved in the existence of the threshold. [J Radiat Res 44:402 (2003)]

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To evaluate quantitative dose-response relationship on the biological response to radiation, it is necessary to consider a model including cumulative dose, dose-rate and irradiation time. In this study, we measured micronucleus formation and [³H] thymidine uptake in human cells as indicators of biological response to gamma radiation and statistically analyzed the data. Effective dose (ED_x) was mathematically estimated by fitting a general logistic function to the dose-response relationship. It was obvious that ED_x increased with longer irradiation time and that the biological response depends on not only cumulative dose but also irradiation time. We represented the relationship among the variables with a three-dimensional curved surface using a general multiple logistic function. It was found that the biological response declined sharply when dose-rate was less than 0.01Gy/h. To further estimate the dose-rate effect, we proposed modified exponential (MOE) models. Our models indicated that log(ED_x) exponentially increased when dose-rate was less than 0.01Gy/h and that the risk approaches to 0 at infinitely low dose-rate. [J Radiat Res 44:402 (2003)]

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Effects of heavy ions on the proliferation and differentiation of embryonic mouse cells were investigated in this study. We selected melanocytes for this purpose, since melanin synthesized in melanocytes is easily observable in vivo. Pregnant females of mice crossed with males were whole body irradiated with a single acute dose of carbon ion to investigate its effects on embryonic melanoblasts. The effect was studied by scoring changes in the cutaneous coats of F₁ offspring three weeks after birth. White spots were found in the ventrum and tail tips of the offspring. The frequency of white ventral spots as well as white areas of tail spots were increased in a dose-dependent manner. In white areas of ventrum and tail-tips no melanoblasts and melanocytes were observed. These results suggest that carbon ion affects the proliferation and differentiation of melanocytes in the mouse skin. [J Radiat Res 44:402 (2003)]

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To elucidate the mechanism of low dose rate radiation-induced carcinogenesis, it is important to analyze spontaneous mutation frequencies at various tissues with aging and tissue specific mutation rates induced by radiation. Real time PCR method was used to observe four types deletion mutation of mitochondrial DNA from liver and brain in mice irradiated by γ-ray with 0.6Gy/min. Accumulations of four types of mtDNA deletions were observed with aging in all tissues. In contrast these deletion mutations in mitochondria were not induced by 7.5Gy of radiation. These findings suggest that the quantity of ROS (reactive oxygen species) in mitochondria produced by radiation may be within the limits which can remove with defense mechanism in the living cell. This work was supported by Aomori Prefecture, Japan. [J Radiat Res 44:403 (2003)]

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In order to elucidate the effects of ionizing radiation on a specific brain region individually without a mutual interaction with other brain regions, we have developed an experimental system for irradiating a small and restricted region of the rat brain, using accelerated heavy-ion beams to administer a large radiation dose in the vicinity of the endpoint in the beam range. Male Sprague-Dawley rats aged 10-14 weeks were used. The left cerebral hemispheres of the rat brains were irradiated at doses of 30, 50 or 100 Gy with charged carbon particles. The spread-out Bragg peak used here successfully and satisfactorily retained its high-dose localization in the defined region. Gross anatomy of the brain was examined using a clinical MRI system at different ages of the rats after accelerated heavy-ion irradiation. Behavioral and histological changes were observed with time course and these changes were compared with MRI examination of the rats of the same age. [J Radiat Res 44:403 (2003)]

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To assess effects of low-dose neutrons on fetuses, the relative biological effectiveness (RBE) should be known. In mice, prenatal exposure of gamma-rays caused several effects: reduction of cell proliferation and retention of neurocyte migration in cerebral cortex. However there had been few studies of neutron effects on fetuses. Therefore, we analyzed number of apoptotic neurocytes in mouse cerebral cortex as a marker of neutron effects on fetuses and estimated RBE values. B6C3F1 fetal mice were exposed to 10MeV fast neutrons (0.05 ~ 1.0Gy) or ¹³⁷Cs gamma-rays (0.4 ~ 2.0Gy) on day 13.5 of gestation and autopsied 24h later. Cross-sections at cerebral mid-plane were stained with HE or TUNEL method. Apoptosis of neurocytes were microscopically observed at cerebral cortex on HE stained sections and were quantitatively analyzed on TUNEL stained samples. Neurocyte death increased at manner of dose dependent for neutrons and also gamma-rays, and RBE was tentatively estimated to be about 3. [J Radiat Res 44:403 (2003)]

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To clarify the mechanism the heterogeneity in the response to ionizing irradiation arising from a genetic variation between individual humans, we here investigated the skin reaction of mice with fractionated irradiation. The fractionated irradiation was performed for 12 days with 10 fractions at a dose of 3, 4, 5, or 6 Gy. The order of radiosensitivity was obtained that C3H/HeMs was more resistant than A/J or C57BL/6J. This order was the same as the one after the experiments using a single dose. The inter-strain difference was not observed with dose-response analysis using the peak skin reaction score at each total dose group. The latent periods before the onset of skin reaction was various among the three strains or the dose of irradiation. As a result, inter-strain differences were observed in radiation susceptibility among three strains of mice using the functional endpoint of skin reaction after local fractionated irradiation. [J Radiat Res 44:403 (2003)]

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Three radiation sensitive mutant strains of medaka which showed significant increase of malformation by irradiation at organogenesis stage were established. Here, we examined the responses of early embryonic cells in the radiation sensitive mutant. After irradiation with 1Gy at morula stage, although no significant increase of embryonic abnormality was observed in wild-type (CAB strain) embryos, lower hatchability (about 21%) and higher malformation rate (about 21%) were shown in the mutant. Similarly, when embryos of the mutant strain were irradiated with 5Gy at late blastula stage, increase of embryonic abnormalities was shown in comparison with CAB strain. These results suggest that the mutant show high radiation sensitivity at both early embryonic stages and organogenesis stage. In addition, we have performed the TUNEL assay in flattened whole mounts to detected radiation-induced apoptotic cells and comet assay to examine DNA repair at early embryonic stages. [J Radiat Res 44:404 (2003)]

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H2AX is rapidly phosphorylated in response to ionizing radiation (IR)and is considered to be critical for recognition and repair of DNA doublestrand breaks (DSB). Moreover, the inhibition of DNA replication byhydroxyurea (HU) or UV-light has also been known to induce thephosphorylation and the foci formation of H2AX. We have recently shown that homozygouly Np95-inactivated embryonic stem(ES) cells are more sensitive to X-rays, UV-light, MNNG, and HU than ES wildtype or Np95+/-ES cells, suggesting that NP95 functions as a component inthe multiple DNA damage response pathways. In this study, we found that aconsiderable proportion of Np95-/- cells failed to stop at G2 phase andproceeded into mitosis after 8 Gy X-irradiation. We also observed thecolocalization of NP95 with H2AX following X-irradiation of m5S cells,suggesting that NP95 interacts with H2AX in the recognition and repair ofDSB. [J Radiat Res 44:404 (2003)]

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RNA interference (RNAi) is a process to knock down gene expression by injecting a small segment of double stranded RNA of the corresponding gene. Recent advances have allowed us to use this technique to silence a gene in the mammalian system. We applied RNAi to silence Ligase IV gene, a key molecule associated with nonhomologous end joining (NHEJ). HFL III normal human fibroblasts were transfected with Ligase IV siRNA using Hemagglutinating virus of Japan envelop (HVJ-E), and its protein expression and radiobiological characteristics were investigated. We have observed a reduction in Ligase IV protein expression associated with a decrease in X-ray survival in cells with Ligase IV si RNA when compared to cells treated with HVJ-E alone. These results indicate that the siRNA strategy can be applied in this repair gene. Further application and optimization of this technique are underway and will be reported. [J Radiat Res 44:404 (2003)]

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RNAi is a powerful tool for the analysis of gene function in vivo. The "Y-family" of DNA polymerases are characterized in terms of their low-fidelity synthesis on undamaged DNA and their ability to bypass DNA lesions in vitro which normally block replication by previously known polymerases.To analyze the function of these genes in vivo, we have tried to establish a transgenic line which can overexpress RNAi construct of Drosophila "Y-family" DNA polymerasegenes, Rad30A, Rad30B and Rev1. This trial, howevfer, did not showed satisfactory result, because of the low efficiency in the repression of the level of protein. Thus, we have established new RNAi transgenic lines using improved RNAi vector and in this paper we will report the results of their characterization. [J Radiat Res 44:404 (2003)]

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Although XPC-HR23B protein complex specifically binds to UV-induced (6-4) photoproduct, it also binds to non-damaged DNA non-specifically. Therefore, presence of a large excess of non-damaged DNA competitively inhibited the specific binding of XPC-HR23B to (6-4) photoproduct, thereby inhibiting nucleotide excision repair in vitro. This moderate damage specificity seems to be insufficient for XPC-HR23B to recognize a small number of lesions generated within the huge genomic DNA in cells. Since eukaryotic DNA is folded into nucleosomes, we examined effect of the nucleosome structure on the specific binding of XPC-HR23B to DNA damage. When non-damaged nucleosomal DNA was used as a competitor, the inhibitory effects on both the specific binding of XPC-HR23B to (6-4) photoproduct and the in vitro-nucleotide excision repair were suppressed. These results suggest that the presence of the nucleosome structure in non-damaged DNA regions may help specific targeting of XPC-HR23B to damaged sites by suppressing the non-specific DNA binding. [J Radiat Res 44:405 (2003)]

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In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients. CS causative mutations in such patients all produce truncated XPG proteins. To investigate the notion that the CS phenotype in XPG/CS patients results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (D811 residue to stop codon, XpgD811stop and deletion of exon 15, Xpgdeltaex15). In XpgD811stop and Xpgdeltaex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. The XpgD811stop homozygous mice exhibited growth retardation and short life span, but the Xpgdeltaex15 homozygous mice exhibited normal growth, indicating that the deletion of the last 360 amino acids causes CS phenotype but the deletion of the last 183 amino acids does not. Thus, the C-terminal truncation of the Xpg protein does not always cause CS phenotype. [J Radiat Res 44:405 (2003)]

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It has been suggested that Recql4 gene is the responsible gene for a subset of Rothmund-Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. It was reported that a knockout mice in which the Recql4 gene is disrupted at exons 5-8 exhibited embryonic lethality at embryonic day 3.5-6.5. We generated a helicase activity-inhibited mouse by replacing the exon 13 of Recql4 with a targeting vector, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The mutant mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. We compared the abnormalities in the Recql4-deficient mice with those in RTS patients. As a result, it was suggested that defects in the Recql4 gene may indeed be responsible for RTS. [J Radiat Res 44:405 (2003)]

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Radiation health risk estimation for radiation protection is based upon the epidemiological data of acute-exposure to relatively high-dose and high-dose rate radiation, and therefore, requires an extrapolation to low-dose and -dose rate to apply to the practical doses of radiation protection. The reduction factor is estimated by long-term animal experimental data and the numerical model analysis. Therefore, the storage of animal exposure experiment data and the reposition of derivative biomaterials of both passed and present studies are valuable and important. NIRS have started the archival and repository activities to collect, digitalize and storage the data/materials of long-term animal experiments. Activity includes the collection and digitalization of the primary data and information (detail exposure protocols, animal data and pathological diagnosis, etc.) and storage of the experimental materials (paraffin embedded tumor blocks, derivative slides). We here report the results of preliminary dose-response analysis of our archival data (C3H mouse) below 1Gy. [J Radiat Res 44:405 (2003)]

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Megakaryocytic progenitor cells with different radiosensitivity are detected in normal human peripheral blood (PB). CD34⁺ cells were purified by two and three isolation columns (i.e., CD34⁺(low) cells and CD34⁺(high) cells). Their purities were 74.8% and 88.8%, indicating a statistical significant difference. Thrombopoietin (TPO) supported 80.2 colonies per 1,000 CD34⁺(low) cells, but CD34⁺(high) cells were 42.2 colonies. X-irradiated CD34⁺(high) cells stimulated with TPO alone showed an exponential survival curve, however, no colonies were observed at dose over 4Gy. In the case of CD34⁺(low) cells, there were a few colonies also at 5Gy. TPO plus interleukin-3, which supports maximum radioprotection (Kashiwakura, Radiat Res 158, 202, 2002), showed that no significant differences in the survival curves between the both cells were observed. These findings suggest that different radiosensitivity observed in the both cells were depending on the differentiation pathway of CD34⁺ cells. Especially, there is a possibility that the expressions of Mpl receptor on the surface of CD34⁺ cells are different. [J Radiat Res 44:406 (2003)]

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When C3H mice were exposed to a sublethal dose (3Gy) of irradiation on day 7 after FLV infection, more than 90% of mice died within one month after irradiation. C3Hp53-/- mice exhibited no mortality. On the other hand, C3H Atm-/- mice manifested a significant enhancement of radiation-induced mortality and of radiation sensitivity of hematopoietic tissues. C3H SCID (DNA-PK) mice showed a significant but lesser degree of enhancement of radiation-induced mortality by FLV infection. We have so far failed to see a significant enhancement of radiation-induced hematopoietic injury to explain the enhancement of radiation-induced mortality in C3H SCID mice by FLV infection. The results suggest the significant role of p53 gene in the enhancement of radiation-induced mortality by FLV infection but no significant role of Atm gene. [J Radiat Res 44:406 (2003)]

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To measure the radiation damage, γ-H2AX foci in the irradiated human peripheral lymphocytes and mouse organ tissues were detected as fluorescent signals, since phosphorylation of H2AX at serine 139 (γ-H2AX) occurs following DNA double strand- but not single-strand break induction and is probably the earliest manifestation of the lesion. Human peripheral lymphocytes and mice were exposed to 0, 0.1, 0.2, 0.5, 1, 2 Gy (1 Gy/min) of γ-rays. The γ-H2AX foci were detected dose dependently as fluorescent signals in both cells at 15 minutes after irradiation. These results suggest that quantitation of DSBs by the γ-H2AX foci is an useful biodosimeter for detecting high and low dose radiation damage in human being and mice. It is also suggested that there is a threshold dose which can not be discriminated the additional radiation induced DSBs from spontaneous DSBs in a cell. [J Radiat Res 44:406 (2003)]

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For getting the biological data for the risk estimate of radon exposure, the biological effects for the tracheal epithelial cells were examined by in vitro exposure to radon. For radon exposure of cells, a ceramic radon source that was developed for radon experimental research was used. The radon gas was injected to the incubator through the filter. The exposed dose of radon was estimated by the method of passive dose measurement using CR39. The tracheal epithelial cells were isolated from Wister rat (WM/NIRS), and cultured by the air-liquid interface method. This culture method is expected to be especially useful for exposure to radon. The biological effects were examined by micronuleus assay as a function of exposed doses. When the exposed radon concentration was 1000 Bq/m³ , significant effects have not detected by micronuleus assay after one weeks exposure. The micronuclei rate was 2 times higher than control when the exposed radon concentration was 100,000 Bq/m³ . [J Radiat Res 44:406 (2003)]

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It is well known that radiation induces microcephaly or mental retardation when fetus is irradiated during brain development stage. Several researches have demonstrated that prenatal irradiation of low-LET radiation induced cerebral hypoplasia in rodents. However, little is known about neutrons. So, we examined the effects of prenatal exposure of neutrons on mice brains. B6C3F1 mice were irradiated with fast neutrons (0.1 to 1Gy) or gamma-rays (0.8, 1.5Gy) on embryonic day 13.5. At 8 weeks of age, offsprings of both sexes were necropsied. The brains were removed, weighed, and then were fixed in formalin for histopathology. Both types of radiation caused hypoplasia of cerebral cortex and brain absolute weight loss. These changes were remarkable at the highest dose groups. The brain weight loss was still noted at 0.1Gy of neutrons. Histopathologically, loss of cortical neurons were observed. The degrees of these changes were larger for neutrons, the RBE for brain weight loss was 2 to 3 for both sexes. [J Radiat Res 44:407 (2003)]

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It is well known that DNA-PKcs contributes to nonhomologous end-joining of DNA double-strand breaks, forming a complex with Ku70 and Ku80. We examined whether radio/heat sensitivity is enhanced by DNA-PKcs-targeted small interference (si)RNA which degrades DNA-PKcs mRNA. We used p53-null human cultured cells (H1299) transfected with wtp53 (H1299/wtp53) or mp53 gene (H1299/mp53) or SAS cells transfected with mp53 gene to study whether the effect of DNA-PKcs-targeted siRNA on radio/heat sensitivity of cells is p53-dependent or not. Colony formation assay showed X-ray sensitivity of those cells was remarkably enhanced by DNA-PKcs-targeted siRNA p53-independently but the heat sensitivity was not affected by the siRNA in the H1299 cells. In contrast, the heat sensitivity of SAS cells was slightly enhanced by the siRNA. In addition, heat-induced hsp70 accumulation was increased by the siRNA in the H1299 cells. Therefore, the increased hsp70 accumulation by the siRNA may introduce the no effectiveness about the heat sensitivity of the H1299 cells. These results suggest that DNA-PKcs-targeted siRNA is a strong candidate for p53-independent radiation sensitizer. [J Radiat Res 44:407 (2003)]

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