Toshiyasu Iwasaki

Suppression of carcinogenic processes in mice by chronic low dose rate gamma-irradiation

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.

Dose and dose-rate effects of ionizing radiation: a discussion in the light of radiological protection

Abstract 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.

Emerging issues in radiogenic cataracts and cardiovascular disease

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.

Ionizing Radiation Leads to the Replacement and de novo Production of Colonic Lgr5+ Stem Cells

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.

P53-DEPENDENT ADAPTIVE RESPONSES IN HUMAN CELLS EXPOSED TO SPACE RADIATIONS

PURPOSE It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. METHODS AND MATERIALS Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. RESULTS In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. CONCLUSION These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

The Dose Response of Chromosome Aberrations in Human Lymphocytes Induced In Vitro by Very Low-Dose γ Rays

Abstract 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.

Simultaneous quantitative analysis of prostaglandins and thromboxane after low-dose X irradiation

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.

A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation

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.

Biphasic Effects of Nitric Oxide Radicals on Radiation-Induced Lethality and Chromosome Aberrations in Human Lung Cancer Cells Carrying Different p53 Gene Status

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 wtp53 and mp53 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 wtp53 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-mumol/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 mumol/L), clear evidence of radiosensitization, enhancement of apoptosis, and chromosome aberrations was detected. However, these phenomena were not observed in mp53 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 wtp53 cells.

Lymphocyte telomere length correlates with in vitro radiosensitivity in breast cancer cases but is not predictive of acute normal tissue reactions to radiotherapy.

Purpose: To examine the hypothesis that lymphocyte telomere length may be predictive of both breast cancer susceptibility and severity of acute reactions to radiotherapy. Materials and methods: 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). Results: Female breast cancer cases (n = 24) had reduced lymphocyte telomere lengths by comparison with healthy controls (n = 20, p < 0.04). 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 (p < 0.001) and increased apoptosis (p < 0.01) 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. Conclusions: 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.