A. Tachibana

H. Tauchi

J. Kobayashi

J. Magae

M. Sasatani-Toyoshima

T. Ono

T. Shimura

T. Uda

T. Umata

Y. Masuda

National Institute for Fusion Science (NIFS-Repository)

 Because of the technical difficulties, we do not have enough experimental data for assessing the biological effects of low dose (less than 0.2 Gy) or low dose rate radiation. Concerning to the biological effects of tritium, the situation is the same. Therefore, to prepare the data for biological effects of low dose tritium, the present study focused on, i) analysis of biological effects of tritium by using genetically engineered animals, ii) analysis of the mechanism of DNA damage response, and iii) analysis of the biological effects of tritium at the cellular level.  Followings are summary of the results. i) Analysis of biological effects of tritium by using genetically engineered animals We have established a novel experimental system that can examine the biological effects of low dose (rate) tritium radiation at animal level. We used two strains of transgenic mice, the gpt-delta mice1) and Rev1 mice2). The gpt-delta mice carry a mutation reporter gene, and Rev1 mice are over-expressing Rev1. Rev1 is a gene for translesion synthesis (TLS), an error prone repair pathway, which acts on DNA damage such as DNA base modification induced by UV or ionizing radiation. We found that Rev1 transgenic mouse may be a useful model system for the study of risk estimation of tritium-induced cancers. We also used the p53 (a tumor suppressor gene) knockout mice3), in order to clarify the importance of the regulation of DNA damage checkpoint in prevention of teratogenic effects by low dose tritium exposure. The gpt-delta mouse is a novel system that can be successfully applied to experiments assessing the biological effects of low dose radiation. Tritiated water (HTO) was administrated to the gpt-delta mice at low dose rate, and the mutation frequency at several tissues are analyzed. With the total dose of 3 Gy, the mutant frequency was elevated approximately 2-fold above control level, and it remained at a similar level up to 6 Gy. These results indicate that gpt delta mouse could be a good model animal to study genotoxicity (induction of mutation) of tritium. ii) Analysis of the mechanism of DNA damage response Understanding the molecular mechanism of cellular DNA damage responses is another important point of view to understand the biological risk of low dose (rate) radiation. If the mechanisms are fully clarified, we believe that one can simulate the biological responses to low dose tritium radiation.  Activation of the DNA-dependent protein kinase (DNA-PK) and AKT signaling pathway was found in cellular response to X-ray exposure. Therefore, we tested whether the pathway is also activated by HTO exposure. As well as X-rays, exposure of HepG2 and HeLa cells to HTO activated AKT, which was shown by phosphorylated-AKT at Serine 473 residue. The activation of DNA-PK was also observed in these cells by X-ray irradiation but not by exposure to HTO4) These suggest the existence of difference in cellular response between X-rays and HTO.  We also investigated molecular function of DNA damage repair-related proteins such as histone H2AX, WRN, and Rad18. WRN protein rapidly accumulated at DNA damage sites and formed discrete nuclear foci only during S phase, but not in G1 phase. WRN-defective Werner Syndrome (WS) cells showed the spontaneous accumulation of gamma-H2AX (DSB marker), suggesting that WRN could function to repair the S phase-dependent DNA damage. WS cells also exhibit an increase in spontaneous focus formation of Rad18, suggesting that WRN could work for the regulation of TLS pathway5).  iii) Analysis of the biological effects of tritium at the cellular level We previously found that the human-X-carrying hamster cell system appeared to be able to detect a wide range of mutation spectrum, even if those mutations affect the expression of important human genes for cell survival. The system showed about 100-fold sensitivity compared to the conventional system that uses endogenous Hprt gene. By using the cell system, we are testing the mutation induction by HTO exposure at low dose rate. Our results suggested that the reverse dose rate effect does not apply in the case of mutation induction by HTO and that exposure to low level of HTO (0.1 Gy with 12mGy/day) could merely enhance the spontaneous mutagenesis6). Radio-adaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. We have demonstrated that the recognition of primary-dose and adaptive response could be mediated by a feedback signal pathway which involves protein kinase C (PKC), p38 mitogen activated protein kinase (p38/MAPK), and phospholipase C (PLC). We are doing experiments to clarify the effect of PKC knockdown by siRNA on radio-adaptive response. By the experiments, we may verify the importance of PKC pathway for expression of radio-adaptive responses. In addition, we found that an adaptive response was induced by pre-treatment of the cells with low concentration of tritium compound such as 3H-thymidine for a few days7).  1) Ono, T., et al. Fusion Sci. Tech. 60, 1183-1185, 2011. 2) Toyoshima, M., et al. Fusion Sci. Tech. 60, 1204-1207, 2011. 3) Umata, T. et al. Fusion Sci. Tech. 60, 1193-1196, 2011. 4) Shimura T, et al. Fusion Sci. Tech. 60, 1190-1192, 2011. 5) Kobayashi, J. et al. Fusion Sci. Tech. 60, 1186-1189, 2011. 6) Tauchi, H. et al. Fusion Sci. Tech. 60, 1173-1178, 2011. 7) Tachibana, A. et al. Fusion Sci. Tech. 60, 1197-1199, 2011.  222§23. Study on Biological Effects of Tritium at Animal LevelUmata, T. (Univ. Occupational & Environ. Health),  Tauchi, H., Tachibana, A. (Fac. Sci., Ibaraki Univ.),  Kobayashi, J. (Rad. Biol. Cntr, Kyoto Univ.),  Sasatani-Toyoshima, M., Masuda, Y. (RIRBM, Hiroshima Univ.),  Ono, T., Shimura, T. (Tohoku Univ. Grad. School Med),  Magae, J. (Central Res. Inst, Electric Power Industry),  Uda, T.

Study on Biological Effects of Tritium at Animal Level

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Study on Biological Effects of Tritium at Animal Level

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