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Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko*; Ikeda, Hiroko; Kobayashi, Yasuhiko
International Journal of Radiation Biology, 91(5), p.383 - 388, 2015/05
Times Cited Count：13 Percentile：67.64(Biology)We investigated the dependence of the bystander cell-killing effect on radiation dose and quality, and related molecular mechanisms. Human fibroblasts were irradiated with 
-rays or carbon ions and co-cultured with non-irradiated cells. Survival rates of non-irradiated cells decreased and nitrite concentrations in culture medium increased with increasing doses. Their dose responses were similar between -rays and carbon ions. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of non-irradiated cells. Negative relationships were observed between survival rates and nitrite concentrations. From these results, it was concluded that the bystander cell-killing effect mediated by NO radicals in human fibroblasts depends on irradiation doses, but not on radiation quality. NO radical production appears to be an important determinant of -ray- and carbon-ion-induced bystander effects.
locus in normal human fibroblasts induced by C-, Ne- and Ar-ion microbeamsSuzuki, Masao*; Funayama, Tomoo; Yokota, Yuichiro; Muto, Yasuko*; Suzuki, Michiyo; Ikeda, Hiroko; Hattori, Yuya; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 78, 2015/03
We have been studying the radiation-quality dependent bystander cellular effects, such as cell killing, mutation induction and chromosomal damage, using heavy-ion microbeams with different ion species. This year we focused on the ion-species dependent bystander mutagenic effect on locus in normal human fibroblasts. The confluent culture were irradiated using a 256 (16
16)-cross-stripe method using C, Ne and Ar microbeam. Gene mutation on locus was detected with 6-thioguanine resistant clones. The mutation frequency in cells irradiated with C-ion microbeams was 6 times higher than that of non-irradiated control cells and of the sample treated with specific inhibitor of gap-junction cell-to-cell communication. On the other hand, no enhanced mutation frequencies were observed in cells irradiated with either Ne- or Ar-ion microbeams. There is clear evidence that the bystander mutagenic effect via gap-junction communication depends on radiation quality.
Yasuda, Takako*; Oda, Shoji*; Asaka, Tomomi*; Funayama, Tomoo; Yokota, Yuichiro; Muto, Yasuko*; Ikeda, Hiroko; Kobayashi, Yasuhiko; Mitani, Hiroshi*
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 85, 2015/03
In this present study, we examined the effects of heavy carbon-ions on development in pre-implantation period utilizing medaka blastula stage embryos (st. 11: blastderm diameter is about 500 
m). We performed targeted irradiation by carbon-ion micro-beam (diameters of 120, 180 m) to a central parts of blastoderm and observed the abnormalities during development compared with whole-body irradiated embryos. As a results, retardation and characteristic malformed eyes were observed during development when blastoderm cells were partially irradiated, However, more than half of 50 Gy-irradiated embryos (area size=120 m diameter) could hatch normally in contrast to all embryos with 2 Gy of whole-body irradiation being lethal before hutching.
mutants to microbeam irradiationSakashita, Tetsuya; Suzuki, Michiyo; Hattori, Yuya; Ikeda, Hiroko; Muto, Yasuko*; Yokota, Yuichiro; Funayama, Tomoo; Hamada, Nobuyuki*; Shirai, Kana*; Kobayashi, Yasuhiko
JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 74, 2015/03
An increasing body of data indicates that ionizing radiation affects the nervous system and alters its function. Recently, we reported that chemotaxis of during the salt chemotaxis learning (SCL), that is conditioned taste aversion to NaCl, was modulated by carbon ion irradiation, i.e. accelerated decrease in chemotaxis to NaCl during the SCL. However, we had no direct evidence for the interaction of ionizing radiation with the central neuronal tissue (nerve ring) in . Microbeam irradiation is useful to analyze direct radiation effects at a cellular or tissue level. Thus, we applied the microbeam irradiation of the nerve ring and examined the effect on the SCL.
Suzuki, Masao*; Autsavapromporn, N.*; Usami, Noriko*; Funayama, Tomoo; Plante, I.*; Yokota, Yuichiro; Muto, Yasuko*; Suzuki, Michiyo; Ikeda, Hiroko; Hattori, Yuya; et al.
Journal of Radiation Research, 55(Suppl.1), P. i54, 2014/03
Autsavapromporn, N.*; Suzuki, Masao*; Funayama, Tomoo; Usami, Noriko*; Plante, I.*; Yokota, Yuichiro; Muto, Yasuko*; Ikeda, Hiroko; Kobayashi, Katsumi*; Kobayashi, Yasuhiko; et al.
Radiation Research, 180(4), p.367 - 375, 2013/10
Times Cited Count：60 Percentile：87.87(Biology)We investigated the role of gapjunction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036-0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from X ray, carbon ion, neon ion or argon ion microbeams in the presence or absence of an inhibitor of GJIC. After 4 h incubation, the cells were assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. The results highlight the important role of radiation quality and dose in the observed effects.
Muto, Yasuko; Funayama, Tomoo; Yokota, Yuichiro; Kobayashi, Yasuhiko
International Journal of Radiation Biology, 88(3), p.258 - 266, 2012/03
Times Cited Count：13 Percentile：65.25(Biology)Hino, Mizuki*; Hamada, Nobuyuki*; Tajika, Yuki*; Funayama, Tomoo; Morimura, Yoshihiro*; Sakashita, Tetsuya; Yokota, Yuichiro; Fukamoto, Kana*; Muto, Yasuko; Kobayashi, Yasuhiko; et al.
Journal of Electron Microscopy, 59(6), p.495 - 501, 2010/12
Times Cited Count：18 Percentile：65.66(Microscopy)Muto, Yasuko; Tsukimoto, Mitsutoshi*; Homma, Takujiro*; Kojima, Shuji*
Journal of Health Science, 56(6), p.675 - 683, 2010/00
Times Cited Count：5 Percentile：12.79(Toxicology)Muto, Yasuko; Funayama, Tomoo; Yokota, Yuichiro; Kobayashi, Yasuhiko
no journal, ,
no abstracts in English
Ikeda, Hiroko; Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Kanai, Tatsuaki*; Kobayashi, Yasuhiko
no journal, ,
The purpose of this study is to detect bystander effect between normal fibroblasts and lung cancer cells with different p53 status and to elucidate its mechanisms. In the study, we used human lung normal fibroblasts WI-38 line and human lung cancer cells H1299/wtp53 line that can produce normal p53 proteins in their DNA damage response. We irradiated cells with carbon-ion broad beams (18.3 MeV/u, LET = 108 keV/m; Dose = 0.5 Gy) or -rays (Dose = 0.5 Gy), then calculated survival rates of bystander cells after 6 or 24 hours cocultute of irradiated and non-irradiated cells. When we cocultured irradiated H1299/wtp53 cells with non-irradiated WI-38 cells, it was found that survival rates of WI-38 cells were not decreased at all. Consequently, it was suggested that bystander factors were not released from irradiated H1299/wtp53 cells.
Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Ikeda, Hiroko; Kobayashi, Yasuhiko
no journal, ,
The purpose of this study is to clear a time dependency of carbon-ion-induced bystander cell-killing effect. Thus, we irradiated normal human fibroblasts with carbon ion microbeam (LET=103 keV/m), broad beam (108 keV/m) and -rays (0.2 keV/m). Survival rate of bystander cells was measured after 6-24 h co-culture with irradiated cells. The ratio of irradiated and bystander cells was <0.0005:1 in microbeam irradiation and 0.5:1 in broad beam and -ray irradiation, respectively. In microbeam-irradiated samples, the survival rate of bystander cells did not change at 6 h but decreased to about 85% of control at 24 h. In 0.13-Gy broad beam and 0.5-Gy -ray irradiated samples, the survival rate of bystander cells decreased to 80-90% of control at 6 h or later. From these results, it is found that bystander cell-killing effect is delayed when irradiated cells are extremely less than bystander cells.
Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Ikeda, Hiroko; Kobayashi, Yasuhiko
no journal, ,
In this study, we investigated a time dependency of bystander effect. In experiments, we irradiated normal human fibroblasts with carbon-ion microbeam (LET=103 keV/m, 10 particles/irradiated site), carbon-ion broad beam (0.13 Gy) and Co-60 -rays (0.2 keV/m, 0.5 Gy). In broad-beam and -ray irradiation, irradiated cells and non-irradiated cells were co-cultured with porous membrane. The ratio of irradiated cells and bystander cells was 1:20,000 in microbeam irradiation and 1:2 in broad-beam and -ray irradiation, respectively. In microbeam-irradiated samples, the survival rate of bystander cells did not change at 6 h but decreased to about 80% at 24 h. In broad-beam and -ray irradiated samples, the survival rate of bystander cells decreased to 80 to 90% at 6 h or later. From these results, it was found that bystander effect is dependent on time after irradiation and number of irradiated cells.
using heavy-ion microbeamSakashita, Tetsuya; Suzuki, Michiyo; Muto, Yasuko; Yokota, Yuichiro; Funayama, Tomoo; Hamada, Nobuyuki*; Fukamoto, Kana*; Kobayashi, Yasuhiko
no journal, ,
An increasing body of data indicates that ionizing radiation causes learning impairments. We recently found the modulatory effect of -rays on the salt chemotaxis learning that was manifested as a decrease in chemotaxis. However, we have no direct evidence for the interaction of ionizing radiation with the central neuronal tissue (nerve ring) of the nervous system in . Localized ionizing irradiation is useful to analyze radiation effects at a cellular or tissue level. Thus, to investigate the effects on the nerve ring, we used the heavy-ion microbeam system installed at the Takasaki Ion accelerators for Advanced Radiation Application of JAEA. To achieve during-learning irradiation, we have developed the live-targeting system for non-paralyzed using the micro-total analysis systems to restrict their motion, and this will be discussed.
Sakashita, Tetsuya; Suzuki, Michiyo; Muto, Yasuko; Yokota, Yuichiro; Funayama, Tomoo; Hamada, Nobuyuki*; Fukamoto, Kana*; Kobayashi, Yasuhiko
no journal, ,
To investigate the effects on the neuronal tissue (a nerve ring), we used the heavy-ion microbeam system. Well-fed adults of grown on the plate spread with the 
were used in all experiments. Anesthetized animals were irradiated locally with carbon-ion microbeams that were extracted into the air through an aperture. Immediately after microbeam irradiation, chemotaxis to NaCl of exposed animals was measured. Microbeam irradiation experiments in the both areas showed no significant effects on the ability of the salt chemotaxis learning. It indicates that the salt chemotaxis learning in is not affected by carbon-ion induced damage in Head or Tail area; possibly such as DNA strand breaks. Moreover, to challenge during-learning irradiation, we attempt the development of live-targeting system for non-paralyzed 
using the micro-device. We will discuss on the new approach.
Yokota, Yuichiro; Funayama, Tomoo; Muto, Yasuko; Sakashita, Tetsuya; Kikuchi, Masahiro; Kobayashi, Yasuhiko
no journal, ,
Heavy-ion-induced DNA double-strand breaks (DSBs) have been focused to reveal the underlying mechanism of the well-known findings that relative biological effectiveness is greater in high-LET heavy ions than in low-LET radiation. Since 1990s, DSBs have been quantified with pulsed-field gel electrophoresis (PFGE) technology. In conventional studies, DNA preparation from irradiated cells was performed in agarose plugs for avoiding excess DNA fragmentation during experimental procedures. However, we have found here that DNA fragments are partly lost from the agarose plug during DNA preparation. This makes difficult to reveal the total DSB yield in irradiated cells.
Yokota, Yuichiro; Funayama, Tomoo; Hamada, Nobuyuki*; Sakashita, Tetsuya; Suzuki, Michiyo; Muto, Yasuko; Kobayashi, Yasuhiko
no journal, ,
It is needed to accumulate the more evidence for combined therapy between heavy ions and chemical agents for overcoming refractory tumors. Here, we investigate a combination effect of carbon-ion irradiation and the telomerase inhibitor MST-312 treatment in human cervical cancer HeLa cells by means of a colony formation assay. Cells were cultured for 24 h with or without 1 M MST-312, and then, irradiated with carbon ions (LET: 110 keV/m) and -rays. MST-312 treatment additively decreased the survival of irradiated cells. Irradiation and MST-312 treatment might inactivate the clonogenic potential of tumor cells through independent mechanisms. The doses needed to reduce the survival of irradiated cells to 10% were decreased by MST-312 treatment from 1.2 Gy to 0.5 Gy in carbon-ion irradiation and from 5.4 Gy to 4.0 Gy in -ray irradiation, respectively. MST-312 pre-treatment may reduce the doses needed for a curative effect in heavy ion radiotherapy.
Suzuki, Masao*; Autsavapromporn, N.*; Funayama, Tomoo; Yokota, Yuichiro; Muto, Yasuko*; Ikeda, Hiroko; Suzuki, Michiyo; Hattori, Yuya; Sakashita, Tetsuya; Kobayashi, Yasuhiko; et al.
no journal, ,
To determine signal transduction factors that are expected to be secreted from microbeam-irradiated cells, the time course analysis of cell killing effect in microbeam irradiated cell population was carried out. To irradiate cell with heavy-ion microbeam, a collimating heavy-ion microbeam system of JAEA-Takasaki was used. After microbeam irradiation, the samples were incubated for 0.5, 3, 24 hours respectively, and the cell killing effect of the microbeam irradiation were measured by colony formation assay. The result suggested that some factors, which induces bystander cell killing, were secreted after argon-ion microbeam, and the factor can be inhibited by the addition of gap-junction inhibitor and DMSO, but not by the ascorbic acid.
-rays; Its dependencies on radiation quality and post-irradiation culture timeYokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Muto, Yasuko*; Suzuki, Michiyo; Sakashita, Tetsuya; Kobayashi, Yasuhiko
no journal, ,
We investigate radiation-induced bystander effect to elucidate its mechanism. Cells irradiated with up to 2 Gy of carbon ions (108 keV/m) and -rays (0.2 keV/m) were co-cultured with non-irradiated cells in the above and below of porous membrane. Non-irradiated bystander cells decreased their survival at 6 and 24 h in co-culture when 33% of cell population was irradiated. In addition, survival reduction of bystander cells was increased with dose and saturated at 0.5 Gy. The dose responses were similar between carbon ions and -rays, indicating that bystander effects were partly dependent on dose but independent of radiation quality. Next, 0.001% of cell population was irradiated with carbon ion and neon ion (380 keV/m) microbeams. Bystander cells decreased their survival at 24 h but not at 6 h in co-culture. From these, it was estimated that bystander effects were also dependent on the fraction of irradiated cells.
Oda, Shoji*; Yasuda, Takako*; Asaka, Tomomi*; Mitani, Hiroshi*; Ikeda, Hiroko; Muto, Yasuko*; Yokota, Yuichiro; Suzuki, Michiyo; Funayama, Tomoo; Kobayashi, Yasuhiko
no journal, ,
The method for targeting and irradiating the testis of fry and adult of Medaka fish using carbon-ion microbeam was established. To irradiate the testis of the fry, a transgenic Medaka that expresses GFP protein specifically in its testis was used. The position of the testis was observed under fluorescent microscopy and then the testis was targeted and irradiated with carbon ion microbeam. To irradiate the testis of the adult Medaka, a adult male of p53 knock out transgenic fish was used. The microbeam was irradiated on the estimated testis position; thereafter the success of irradiation was confirmed by the induction of testis-ova, which is known as induced by the radiation of testis in p53 knock out fish.
