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Recently, the question of whether cancer risk is only accelerated but not increased by radiation exposure has been raised. To explore this matter, we analyzed whether the cumulative mortality of irradiated mice could be explained by x-axis (age) shifted cumulative mortality of nonirradiated mice. We reanalyzed publicly available data on observed cumulative mortality or prevalence in irradiated female B6C3F1 mice that lived their entire lifespan. The results showed that the irradiated curve was well matched to uniformly shifted nonirradiated curve for the cumulative mortality of all causes of death but not for the cumulative mortality of all solid tumors and prevalence of ovarian tumors as is. After adjusting lifetime mortalities, it was also well matched for all solid and ovarian tumors. The shifted days by irradiation were 71–116 days for all causes of death, 56–135 days for all solid tumors, and 41–140 days for ovarian tumors in the 1.9 Gy-irradiated group. The response was switched between irradiation at 35 and 105 days consistently for all the above indexes, supporting the hypothesis that radiation sensitivity differs between juvenile and adults. The shifted days of all causes of death showed a tendency of linear response to dose. This concept of shifting the age of death can be applied not only for all cause of death but also for mortality of all solid tumors after adjusting the magnitude. These findings contribute to the discussion on the application of the ‘shifting age of death’ concept to radiation protection.

Background: A comprehensive, traceable, and easy-to-understand radiation risk indicator is desired for radiological protection. The early-onset hypothesis could be used for this purpose.Materials and Methods: An indicator for early death (IED) was developed and calculated using the epidemiological dataset from the 14th Report of the Life Span Study (LSS) of Hiroshima and Nagasaki. By clarifying the calculation process, IED for all-cause mortality was estimated. In addition, the characteristics of IED for solid cancer mortality and cardiovascular mortality as well as those of men and women, and their dependence on age at exposure were investigated for detailed analysis.Results and Discussion: The IED for all-cause mortality was estimated to be approximately 4 years for an acute radiation exposure of 1 Gy regardless of the fitting dose range. The cumulative death rate for all solid cancers also indicated the early-death tendency (approximately 7–10 years at 1 Gy). Although, there is a slight difference in the characteristics of the risk obtained from the LSS study and this study, it is considered that the IED in a unit of years can also be used to show the overall picture of risk due to radiation exposure.Conclusion: We developed and calculated the indicator for early death, IED, for the cumulative mortality rate of all causes of death, all solid cancers, and circulatory diseases. The quantitative values of IED were estimated to be 4 years for all causes of death, 7–10 years for all solid cancers. IED has an advantage for intuitively understanding the meaning of radiation risk since it can be obtained by a simple and traceable method.

Background: For radiological protection and control, the International Commission on Radiological Protection (ICRP) provides the nominal risk coefficients related to radiation exposure, which can be extrapolated using the excess relative risk and excess absolute risk obtained from the Life Span Study of atomic bomb survivors in Hiroshima and Nagasaki with the dose and dose-rate effectiveness factor (DDREF). Materials and Methods: Since it is impossible to directly estimate the radiation risk at doses less than approximately 100 mSv only from epidemiological knowledge and data, support from radiation biology is absolutely imperative, and thus, several national and international bodies have advocated the importance of bridging knowledge between biology and epidemiology. Because of the accident at the Tokyo Electric Power Company (TEPCO)’s Fukushima Daiichi Nuclear Power Station in 2011, the exposure of the public to radiation has become a major concern and it was considered that the estimation of radiation risk should be more realistic to cope with the prevailing radiation exposure situation. Results and Discussion: To discuss the issues from wide aspects related to radiological protection, and to realize bridging knowledge between biology and epidemiology, we have established a research group to develop low-dose and low-dose-rate radiation risk estimation methodology, with the permission of the Japan Health Physics Society. Conclusion: The aim of the research group was to clarify the current situation and issues related to the risk estimation of low-dose and low-dose-rate radiation exposure from the viewpoints of different research fields, such as epidemiology, biology, modeling, and dosimetry, to identify a future strategy and roadmap to elucidate a more realistic estimation of risk against low-dose and low-dose-rate radiation exposure.

Background: For radiological protection and control, the International Commission on Ra- diological Protection (ICRP) provides the nominal risk coefficients related to radiation expo- sure, which can be extrapolated using the excess relative risk and excess absolute risk obtained from the Life Span Study of atomic bomb survivors in Hiroshima and Nagasaki with the dose and dose-rate effectiveness factor (DDREF). Materials and Methods: Since it is impossible to directly estimate the radiation risk at doses less than approximately 100 mSv only from epidemiological knowledge and data, support from radiation biology is absolutely imperative, and thus, several national and international bodies have advocated the importance of bridging knowledge between biology and epidemiology. Be- cause of the accident at the Tokyo Electric Power Company (TEPCO)’s Fukushima Daiichi Nu- clear Power Station in 2011, the exposure of the public to radiation has become a major concern and it was considered that the estimation of radiation risk should be more realistic to cope with the prevailing radiation exposure situation. Results and Discussion: To discuss the issues from wide aspects related to radiological protec- tion, and to realize bridging knowledge between biology and epidemiology, we have established a research group to develop low-dose and low-dose-rate radiation risk estimation methodology, with the permission of the Japan Health Physics Society. Conclusion: The aim of the research group was to clarify the current situation and issues relat- ed to the risk estimation of low-dose and low-dose-rate radiation exposure from the viewpoints of different research fields, such as epidemiology, biology, modeling, and dosimetry, to identify a future strategy and roadmap to elucidate a more realistic estimation of risk against low-dose and low-dose-rate radiation exposure. Keywords: Radiation Risk Estimation, Low Dose and Low Dose Rate, Epidemiology, Biology, Modeling, Dosimetry

Radiation response differs depending on the dose and dose rate in intestinal stem cells; however, the underlying mechanisms are not clear. To understand the effects of low-dose and low-dose-rate radiation, the authors established an organoid system that mimics the in vivo environment and sporadic low-dose-rate irradiation conditions in vitro. Organoid-forming potential and the number of stem cells in the organoids derived from 1 Gy-irradiated cells were lower than those from non-irradiated cells; however, the difference was not significant, although 1 Gy-irradiated stem cells exhibited significant growth disadvantage in the mixed-organoid with non-irradiated and irradiated stem cells. Furthermore, the authors irradiated a cell with X-ray microbeams and performed time-lapse observations and found that irradiated cells did not remain in the organoid. These results suggest that radiation-induced stem cell competition can occur in intestinal organoids and contribute to a low risk of cancers at low-dose-rate exposures.

Intestinal organoids are an in vitro cultured tissue model generated from intestinal stem cells, and they contain a mixture of epithelial cell types. We previously established an efficient ‘one cell/well’ sorting method, and defined organoid-forming potential (OFP) as a useful index to evaluate the stemness of individual cells. In this study, we assessed the response to radiation dose and dose-rate by measuring both OFP and the percentage of stem cells in the crypts. After high-dose-rate (HDR, 0.5 Gy/min) irradiation in vivo, the percentage of stem cells in the harvested crypt cells decreased, and the replenishment of cycling stem cells originating from dormant cells was enhanced, but OFP increased in cells irradiated with a total dose of >1 Gy. In contrast, at a total dose of 0.1 Gy the percentage of stem cells reduced slightly, but neither replenishment rate nor OFP changed. Furthermore, the response to 1 Gy of low-dose-rate (LDR) irradiation was similar to the response to 0.1 Gy HDR irradiation. These results suggest that 0.1 Gy HDR irradiation or 1 Gy LDR irradiation does not alter stemness. Additionally, the OFP increase in the colon in response to irradiation was smaller than that in the duodenum, similar to the percentage of stem cells. Understanding the differences in the response of stem cells between the colon and the duodenum to radiation is important to clarify the mechanisms underlying the development of radiation-associated intestinal cancers.

Stem cell competition could shed light on the tissue-based quality control mechanism that prevents carcinogenesis. To quantitatively evaluate stem cell competition in vitro, we developed a two-color intestinal organoid forming system. First, we improved a protocol of culturing organoids from intestinal leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)- enhanced green fluorescent protein (EGFP)high stem cells directly sorted on Matrigel without embedding. The organoid-forming potential (OFP) was 25% of Lgr5-EGFPhigh cells sorted at one cell per well. Using this culture protocol with lineage tracing, we established a two-color organoid culture system by mixing stem cells expressing different fluorescent colors. To analyze stem cell competition, two-color organoids were formed by mixing X-ray-irradiated and non-irradiated intestinal stem cells. In the two-color organoids, irradiated stem cells exhibited a growth disadvantage, although the OFP of irradiated cells alone did not decrease significantly from that of non-irradiated cells. These results suggest that stem cell competition can be evaluated quantitively in vitro using our new system.

For more than a century, ionizing radiation has been indispensable mainly in medicine and industry. Radiation research is a multidisciplinary field that investigates radiation effects. Radiation research was very active in the mid- to late 20th century, but has then faced challenges, during which time funding has fluctuated widely. Here we review historical changes in funding situations in the field of radiation research, particularly in Canada, European Union countries, Japan, South Korea, and the US. We also provide a brief overview of the current situations in education and training in this field. A better understanding of the biological consequences of radiation exposure is becoming more important with increasing public concerns on radiation risks and other radiation literacy. Continued funding for radiation research is needed, and education and training in this field are also important.

The effect of low-dose-rate exposure to ionizing radiation on cancer risk is a major issue associated with radiation protection. Tissue stem cells are regarded as one of the targets of radiation-induced carcinogenesis. However, it is hypothesized that the effect of radiation may be reduced if damaged stem cells are eliminated via stem cell competition between damaged and intact stem cells. This would be particularly effective under very low-dose-rate conditions, in which only a few stem cells in a stem cell pool may be affected by radiation. Following this hypothesis, we constructed a simple mathematical model to discuss the influence of stem cell competition attenuating the accumulation of damaged cells under very low-dose-rate conditions. In this model, a constant number of cells were introduced into a cell pool, and the numbers of intact and damaged cells were calculated via transition and turnover events. A transition event emulates radiation dose, whereby an intact cell is changed into a damaged cell with a given probability. On the other hand, a turnover event expresses cell competition, where reproduction and elimination of cells occur depending on the properties of cells. Under very low-dose-rate conditions, this model showed that radiation damage to the stem cell pool was strongly suppressed when the damaged cells were less reproductive and tended to be eliminated compared to the intact cells. Furthermore, the size of the stem cell pool was positively correlated with reduction in radiation damage.

We previously found that high-dose-rate radiation induced a replenishment of the colonic Lgr5+ stem cell pool, whereas low-dose-rate radiation did not. To identify key molecules that determine the dose-rate effects on this stem cell pool, we harvested colonic Lgr5+ stem cells by cell sorting at 2 weeks after exposure to 1 Gy of high-dose-rate (30 Gy/h) or low-dose-rate (0.003 Gy/h) radiation and analyzed their gene expression profiles using RNA-Seq. We found that pathways related to DNA damage response, cell growth, cell differentiation and cell death were upregulated in Lgr5+ stem cells irradiated with high dose rates, whereas pathways related to apical junctions and extracellular signaling were upregulated in low-dose-rate–irradiated colonic Lgr5+ stem cells. Interestingly, biological events involving apical junctions are known to play an important role in the exclusion of transformed cells that are surrounded by normal epithelial cells through ‘cell competition’. We speculated that cell competition, through apical junctions and extracellular ligands, might contribute to the dose-rate effect on Lgr5+ cell replenishment. To understand this mechanism, we focused on 69 genes that were significantly upregulated in low-dose-rate–irradiated cells, which we named DREDGE (Dose-Rate Effect Determining GEnes). Based on these findings, we propose a possible mechanism underlying the dose-rate effect observed in the colonic stem cell pool.

Radiation exposure such as A-bomb or radiation therapy is considered a major health-risk factor for cardiovascular disease. In order to understand the molecular mechanisms underlying the inflammatory reaction frequently encountered in the vascular system after exposure to ionizing radiation, we carried out a global scale microarray and computational gene expression analyses on human umbilical endothelial cells (HUVECs) exposed to X-ray (2.5Gy). The gene ontology analysis revealed that the down-regulated genes were associated with cell cycle regulation, whereas the up-regulated genes were associated with inflammatory responses, in particular, the type 1 interferon response. The computational analysis using ingenuity pathway analysis also identified a gene network containing the interferon response factor 7 (IRF7) and its transcriptional targets such as interferon-induced transcripts (IFITs) and Mx1, which have been known to be associated with inflammation in endothelial cells. The up-regulated genes and the gene network identified here may explain the inflammatory response induced by X-irradiation. These findings uncover part of the molecular basis of the mechanism(s) of the inflammatory disorder in response to X-irradiation in HUVECs. The dataset is publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE76484.

The biological effects on humans of low-dose and low-dose-rate exposures to ionizing radiation have always been of major interest. The most recent concept as suggested by the International Commission on Radiological Protection (ICRP) is to extrapolate existing epidemiological data at high doses and dose rates down to low doses and low dose rates relevant to radiological protection, using the so-called dose and dose-rate effectiveness factor (DDREF). The present paper summarizes what was presented and discussed by experts from ICRP and Japan at a dedicated workshop on this topic held in May 2015 in Kyoto, Japan. This paper describes the historical development of the DDREF concept in light of emerging scientific evidence on dose and dose-rate effects, summarizes the conclusions recently drawn by a number of international organizations (e.g., BEIR VII, ICRP, SSK, UNSCEAR, and WHO), mentions current scientific efforts to obtain more data on low-dose and low-dose-rate effects at molecular, cellular, animal and human levels, and discusses future options that could be useful to improve and optimize the DDREF concept for the purpose of radiological protection.

An understanding of the dynamics of intestinal Lgr5(+) stem cells is important for elucidating the mechanism of colonic cancer development. We previously established a method for evaluating Lgr5(+) stem cells by tamoxifen-dependent Lgr5-lineage tracing and showed that high-dose-rate radiation stimulated replenishment of colonic stem cells. In this study, we evaluated the effects of low-dose-rate radiation on stem cell maintenance. Tamoxifen (4OHT)-injected Lgr5-EGFP-IRES-Cre(ERT2) × ROSA-LSL-LacZ mice were used, LacZ-labeled colonic crypts were enumerated, and the loss of LacZ(+) crypts under low-dose-rate radiation was estimated. After 4OHT treatment, the number of LacZ-labeled Lgr5(+) stem cells was higher in the colon of infant mice than in adult mice. The percentage of LacZ-labeled crypts in infant mice rapidly decreased after 4OHT treatment. However, the percentage of labeled crypts plateaued at ∼2% at 4 weeks post-treatment and remained unchanged for up to 7 months. Thus, it will be advantageous to evaluate the long-term effects of low-dose-rate radiation. Next, we determined the percentages of LacZ-labeled crypts irradiated with 1 Gy administered at different dose rates. As reported in our previous study, mice exposed to high-dose-rate radiation (30 Gy/h) showed a marked replenishment (P = 0.04). However, mice exposed to low-dose-rate radiation (0.003 Gy/h) did not exhibit accelerated stem-cell replenishment (P = 0.47). These findings suggest the percentage of labeled crypts can serve as a useful indicator of the effects of dose rate on the stem cell pool.

In 2011, the International Commission on Radiological Protection issued a statement on tissue reactions (formerly termed non-stochastic or deterministic effects) to recommend lowering the threshold for cataracts and the occupational equivalent dose limit for the crystalline lens of the eye. Furthermore, this statement was the first to list circulatory disease (cardiovascular and cerebrovascular disease) as a health hazard of radiation exposure and to assign its threshold for the heart and brain. These changes have stimulated various discussions and may have impacts on some radiation workers, such as those in the medical sector. This paper considers emerging issues associated with cataracts and cardiovascular disease. For cataracts, topics dealt with herein include (i) the progressive nature, stochastic nature, target cells and trigger events of lens opacification, (ii) roles of lens protein denaturation, oxidative stress, calcium ions, tumor suppressors and DNA repair factors in cataractogenesis, (iii) dose rate effect, radiation weighting factor, and classification systems for cataracts, and (iv) estimation of the lens dose in clinical settings. Topics for cardiovascular disease include experimental animal models, relevant surrogate markers, latency period, target tissues, and roles of inflammation and cellular senescence. Future research needs are also discussed.

The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (?8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5+ stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at ?1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay., Journal of Radiation Research, 55(2), pp.381-390; 2014

This paper considers the dose–effect relationship for unstable chromosome aberration yields in human lymphocytes in very low-dose range. Data are presented for 60Co γ-ray doses of 0, 10, 20, 40 and 1000 mGy. More than 5,000 metaphases were scored for each data point at the very low doses, and each cell was double-checked using a semi-automated metaphase finding/relocation system. Aberration yields of dicentrics plus centric rings followed an excellent linear dose response down to zero dose; the yields were significantly above the control frequency from 20 mGy.

The aim of this study was to examine biological effects of nitric oxide (NO) on radiosensitivity and chromosome aberrations in different phases of the cell cycle in human cancer cells with a wild-type p53 (wtp53) genotype. H1299/wtp53 cells were pre-treated with isosorbide dinitrate (ISDN) at different concentrations or pre-irradiated with a low dose of X-rays, and then exposed to a high dose of X-rays. Cell synchronization was achieved with serum starvation. Cellular radiosensitivity, cell cycle distributions, and chromosome aberrations were assayed with colony-forming assays, flow cytometry and chromosome banding techniques, respectively. After treatment with ISDN at a low concentration or after an exposure to 0.02 Gy of X-rays, radioresistance and a reduction in the number of chromosome aberrations were observed mainly 17.5 h after plating mitotic cells. This radioadaptation effect was observed during a clearly shortened G(2)/M phase and a slightly prolonged S phase. In contrast, in the presence of a high concentration of ISDN, radiosensitization and the enhancement of chromosome aberrations were detected principally 17.5 h after plating mitotic cells, and this radiosensitization was observed during a significantly prolonged G(2)/M phase and a slightly shortened S phase. A range of concentrations of NO induced opposing effects on radiosensitivity and chromosome aberrations in human non-small cell lung cancer cells bearing wtp53 gene status, and these different effects produced by NO depended on the cell cycle phase.

Accepted: 2010-04-21, 資料番号: SA1002706000

To clarify the biological effects of space environment, especially space radiations, a proposal of "Rad Gene" was performed as the first life science experiment with two human lymphoblastoid cell lines bearing wild-type p53 gene (wtp53) and mutated p53 gene (mp53) in an International Space Station (ISS) for 133 days. We scheduled four projects: (1) DNA damage induced by space radiations including the high linear energy transfer (LET) particles was detected as a track of γH2AX foci in the nuclei of these frozen cells. (2) To examine the biological effects of microgravity and space radiations on gene and protein expression of p53-dependent regulated genes, these cells were grown under microgravity and 1 gravity in ISS, and on ground for 8 days and analyzed by DNA and protein arrays. (3) p53-Dependent regulated genes were analyzed in the cultured cells after spaceflight at frozen state exposed to space radiations. (4) To clarify the effects of space radiations on the radio-adaptive response, the space flown cells at frozen state were cultured, and then exposed to challenging X-ray-irradiation. All of the radio-adaptive responses of cell killing, apoptosis, chromosomal aberrations and mutations were found only in wtp53 cells, but not in the mp53 cells.

Female ICR mice, 6 weeks old, 35 in each group, were exposed to gamma-rays from a 137Cs source in the long-term low-dose rate irradiation facility at the Central Research Institute of Electric Power Industry (CRIEPI). The dose rate was 2.6 (A), 0.96 (B), or 0.30 mGy/h (C). Thirty-five days later, the mice were injected in the groin with 0.5 mg of methylcholanthrene (MC) dissolved in olive oil and irradiation was continued. Cumulative tumor incidences after 216 days following MC injection were 89% in group A, 76% in group B, and 94% in group C. The one in the non-irradiated control group was 94%. The difference in the tumor incidence between the control and position B was statistically significant, indicating the suppressive effect of the low-dose rate irradiation on the process of MC-induced carcinogenesis with an optimum dose rate around 1 mGy/h.

Tissue stem cells have self-renewal capability throughout their whole life, which is high enough to lead to the accumulation of DNA damage in a stem cell pool. Whether radiation-induced damage accumulates in tissue stem cells remains unknown, but could be investigated if the fate of tissue stem cells could be followed after irradiation. To realize this goal, we used an Lgr5-dependent lineage tracing system that allows the conditional in vivo labeling of Lgr5(+) intestinal stem cells and their progeny. We found that radiation induced loss of Lgr5(+) stem cells in the colon, but not in the duodenum. Interestingly, the loss of colonic Lgr5(+) cells was compensated by de novo production of Lgr5(+) cells, which increased after irradiation. These findings show that ionizing radiation effectively stimulates the turnover of colonic Lgr5(+) stem cells, implying that radiation-induced damage does not accumulate in the colonic Lgr5(+) stem cells by this mechanism.

A cDNA library enriched in clones for mRNA species (an equalized cDNA library) was constructed from human brain employing semi-solid self-hybridization system and cDNA amplification by in vitro transcription. Sequence analysis of the clones in the cDNA library indicated that (1) clones representing abundant species such as those of mitochondrial origin and expression sequence taq (EST) are drastically reduced in their proportion compared to the original library and (2) clones representing rare mRNA species or which do not have matched sequences in the database increased substantially in their proportion. cDNA libraries thus constructed, therefore, are a good source for cloning of cDNA for rare mRNA species from specific mammalian tissues and also for direct expression cloning since the inserts maintained their original molecular sizes after equalization.

Purpose The aim of this study was to clarify the effects of nitric oxide (NO) on radiation-induced cell killing and chromosome aberrations in two human lung cancer cell lines with a different p53 gene status. Methods and Materials We used wild-type (wt) p53 and mutated (m) p53 cell lines that were derived from the human lung cancer H1299 cell line, which is p53 null. The wt p53 and m p53 cell lines were generated by transfection of the appropriate p53 constructs into the parental cells. Cells were pretreated with different concentrations of isosorbide dinitrate (ISDN) (an NO donor) and/or 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (an NO scavenger) and then exposed to X-rays. Cell survival, apoptosis, and chromosome aberrations were scored by use of a colony-forming assay, Hoechst 33342 staining assay and TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP [deoxyuridine triphosphate] nick end labeling) assay, and chromosomal banding techniques, respectively. Results In wt p53 cells the induction of radioresistance and the inhibition of apoptosis and chromosome aberrations were observed in the presence of ISDN at low 2- to 10-μmol/L concentrations before X-irradiation. The addition of c-PTIO and ISDN into the culture medium 6 h before irradiation almost completely suppressed these effects. However, at high concentrations of ISDN (100–500 μmol/L), clear evidence of radiosensitization, enhancement of apoptosis, and chromosome aberrations was detected. However, these phenomena were not observed in m p53 cells at either concentration range with ISDN. Conclusions These results indicate that low and high concentrations of NO radicals can choreograph inverse radiosensitivity, apoptosis, and chromosome aberrations in human lung cancer cells and that NO radicals can affect the fate of wt p53 cells.

To examine the hypothesis that lymphocyte telomere length may be predictive of both breast cancer susceptibility and severity of acute reactions to radiotherapy.Peripheral blood lymphocyte cultures from breast cancer patients (with normal or severe skin reactions to radiotherapy) and normal individuals were assessed for in vitro radiosensitivity as measured by apoptosis, cell cycle delay and cytotoxicity. Telomere lengths were determined by a flow cytometric fluorescence in situ hybridization assay (FLOW-FISH).Female breast cancer cases (n = 24) had reduced lymphocyte telomere lengths by comparison with healthy controls (n = 20, p0.04). However, the average age of healthy controls was less (45.4) than cases (53). When the control group was modified to give a better age match (51.5, n = 13) the reduced telomere length in cases was not significantly different from controls. Lymphocytes from breast cancer cases also showed reduced cell cycle delay (p0.001) and increased apoptosis (p0.01) following irradiation in vitro at 3 and 5 Gy respectively, compared to healthy controls. Statistical significance was maintained with the improved age matching of groups. Comparison of lymphocytes from breast cancer patients with normal (n = 11) and severe (n = 13) skin reactions to radiotherapy failed to identify differences in telomere length or cellular radiosensitivity in this limited sample.This study adds to the evidence suggesting a correlation between altered cellular radiosensitivity and breast cancer. However, in the cases investigated, telomere length does not appear to be predictive of acute skin reactions to radiotherapy.

A photoluminescence glass dosimeter, GD-301, was applied to the measurement of low absorbed doses in mice exposed to low-dose rate 137Cs gamma-rays. The dosimeter system consists of small rod-shaped glass chip detectors capable of embedded in the body of a mouse and an automatic readout device equipped with a standard detector irradiated with 137Cs gamma-source. The measured absorbed doses were compared with the "exposure" estimated by an ionization chamber and with the doses measured by a BeO:Na thermoluminescence system. The results clearly demonstrate the superiority of the glass dosimetry regarding simplicity of operation, stability of long-term dose accumulation and good detector uniformity, which allow accurate tissue dosimetry.

Effects of low dose rate radiation on the process of carcinogenesis induced by a chemical carcinogen were examined. ICR female mice, 35 or 36 mice for each group, were kept and exposed to 137 Cs gamma-rays in the long- term low dose rate irradiation facility at the Central Research Institute of Electric Power Industry at a dose rate of 0.3, 0.96, or 2.5 mGy/h. Thirty-five days later, the mice were injected in the groin with 0.5 mg of 20-methylcholanthrene (MC) dissolved in olive oil, and irradiation was continued. Tumours started to appear 2 months after MC injection. Cumulative tumour incidences after 216 days following MC injection were 94% in the mice irradiated at 0.3 mGy/h, 76% at 0.95 mGy/h, 89% at 0.30 mGy/h, and 94% in non-irradiated control mice. The difference between the tumour incidence in the control mice and that in the mice irradiated at 0.95 mGy/h was statistically significant. These results indicate the suppressive effect of low dose rate irradiation on the process of tumour induction initiated by MC with an optimum dose rate of approximately 1 mGy/h.

The appearance of prostaglandins and thromboxane in mouse serum after X irradiation was observed by simultaneous quantitative analysis using gas chromatography/mass spectrometry/selected ion monitoring with stable isotope dilution methods. Mice of two strains (C57BL/CN Jcl and BALB/c) showed similar responses to X irradiation. In C57BL/6N Jcl mice, 0.2 Gy irradiation elicited a significant increase in generation of prostanoids: Immediately after irradiation, the 6-keto PGF1 alpha:TXB2 ratio and the level of PGE2 increased, after 20 min 6-keto PGF1 alpha and PGE2 increased, and after 4 h PGE1 and PGE2 increased. In BALB/c mice, generation of prostanoids was increased significantly immediately after irradiation (6-keto PGF1 alpha, 6-keto PGF1 alpha:TXB2 ratio, PGE2), and the increase was maintained from 20 min to 4 h (PGE1, PGE2) after 0.2 Gy irradiation. In C57BL/6N Jcl mice, a significant increase in production of 9alpha,11beta-PGF2 was observed at 20 min after irradiation. In BALB/c mice, a significant increase in 9alpha,11beta-PGF2 was seen immediately after irradiation and was maintained for 20 min. In C57BL/6N Jcl mice, the level of 8-epi PGF2 alpha was clearly increased 4 h after 4 Gy irradiation. A slight and slow increase was also seen after 0.2 Gy irradiation. In BALB/c mice, 8-epi PGF2 alpha was increased significantly at 20 min and 4 h after 4 Gy irradiation. These results show that 0.2 Gy irradiation stimulates production of prostanoids related to the inflammatory response in mice.

From a genomic library constructed from an EcoRI digest of Wistar rat brain DNA, we isolated a clone (BL-1) that gave a 0.6-kilobase restriction fragment only in brain (or lens) DNA upon Southern hybridization. The tissue-specific fragment was present in Wistar and other strains (Sprague-Dawley and Donryu) of rats regardless of their sex and age. Sequencing of the clone indicated that it is closely related to a part of the LINE 3 sequence, one of the highly repetitive sequences present throughout mammalian genomes. Polymerase chain reaction using primer sequences in the BL-1 clone indicated that it is derived from an amplified (rear-ranged) sequence, although other explanations are possible. These results suggest that there are tissue-specific changes in DNA primary structure during mammalian developmental processes.

Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3–5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.

By employing in gel competitive reassociation, which distinguishes two DNA preparations, to clone anonymous DNA fragments with altered primary structure, we isolated a clone (BL-1) from a rat DNA which gave an extra, apparently altered, DNA band in a specific tissue (brain) when we used it as a probe for Southern hybridization. The sequence of BL-1 was very similar to a portion of LINE, a highly repetitive sequence. Southern hybridization analysis with oligonucleotide probes representing a portion of the BL-1 sequence confirmed the presence of the brain-specific band. Another, different band specific to HindIII digests of heart DNA was also detected with the same oligonucleotide probes. Furthermore, we found that poly (A) +RNA hybridized with BL-1 was enriched in brain. These results suggest that there are tissue specific alteration of genomic DNA in rats which may be associated with transcriptional activation specific to the sequence. Alternative explanations of the results are also discussed.