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Journal Articles

The Role of nitric oxide in radiation-induced bystander cell-killing effect

Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Sakashita, Tetsuya; Suzuki, Michiyo; Kobayashi, Yasuhiko

JAEA-Review 2015-022, JAEA Takasaki Annual Report 2014, P. 67, 2016/02

The role of nitric oxide (NO) in bystander effect was investigated. Human fibroblasts were irradiated with $$gamma$$-rays (LET: 0.2 keV/$$mu$$m) or carbon-ion beam (108 keV/$$mu$$m), and then, co-cultured with the non-irradiated cells. After 24 h culture, the survival rates of non-irradiated cells and the concentrations of nitrate, an oxide of NO, in the medium were measured. The survival rates of non-irradiated cells decreased in dose-dependent and radiation quality-independent manners. Negative relationships between survival rates and nitrite concentrations existed, indicating the amounts of produced NO are an important determinant of bystander effects. Next, a reagent producing two molecules of NO in a half-life of 100 min was added in the culture medium. After incubation of 24 h the survival rates of treated cells did not decrease, suggesting NO produced intracellularly has an important role to lead the bystander effect but is not the signal molecule for intercellular communication.

Journal Articles

Characteristics of radiation-induced bystander effect; Participation of nitric oxide

Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Kobayashi, Yasuhiko

Isotope News, (741), p.21 - 25, 2016/01

Our article published on the International Journal of Radiation Biology (2015) was reviewed. We investigated the dependence of the bystander cell-killing effect on radiation dose and quality, and related molecular mechanisms. Human fibroblasts were irradiated with $$gamma$$-rays or carbon ions and co-cultured with non-irradiated cells. Survival rates of non-irradiated cells decreased and nitrite concentrations in co-culture medium increased with dose. Their dose responses were similar between $$gamma$$-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 depends on irradiation doses, but not on radiation quality. NO radical production appears to be an important determinant of bystander effects.

Journal Articles

Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

Tomita, Masanori*; Matsumoto, Hideki*; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke*; Maeda, Munetoshi*; Kobayashi, Yasuhiko

Life Sciences in Space Research, 6, p.36 - 43, 2015/07

A radiation-induced bystander response is generally known as a cellular response induced in unirradiated cell by receiving bystander signaling factors released from directly irradiated cells of a cell population. Bystander responses induced by high-LET heavy ions at low fluence are an important problem concerning the health of astronauts in the space environment. Here we set out NO-mediated bystander signal transductions induced by high-LET heavy-ion microbeam irradiation in normal human fibroblasts. Our findings suggest that Akt- and NF-$$kappa$$B-dependent signaling pathway involving COX-2 plays an important role in the NO-mediated high-LET heavy-ion-induced bystander responses. Additionally, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells, which are distinguish form directly irradiated cells.

Journal Articles

The Bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality

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:5 Percentile:34.97(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 $$gamma$$-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 $$gamma$$-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 $$gamma$$-ray- and carbon-ion-induced bystander effects.

Journal Articles

Responses of the salt chemotaxis learning in ${it C. elegans}$ mutants to microbeam irradiation

Sakashita, 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 ${it C. elegans}$ 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 ${it C. elegans}$. Microbeam irradiation is useful to analyze direct radiation effects at a cellular or tissue level. Thus, we applied the microbeam irradiation of the ${it C. elegans}$ nerve ring and examined the effect on the SCL.

Journal Articles

Effects of carbon-ion microbeam irradiation on locomotion and pharyngeal pumping motion in $textit{C. elegans}$

Suzuki, Michiyo; Hattori, Yuya; Sakashita, Tetsuya; Funayama, Tomoo; Yokota, Yuichiro; Ikeda, Hiroko; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 88, 2015/03

Journal Articles

Bystander effect mediated by nitric oxide depends on irradiation dose but not on radiation quality

Yokota, Yuichiro; Funayama, Tomoo; Ikeda, Hiroko; Sakashita, Tetsuya; Suzuki, Michiyo; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 75, 2015/03

We investigated the bystander effect induced by $$gamma$$-rays or carbon ions and analyzed the role of nitric oxide (NO) in the effect. Normal human fibroblasts were used. Cells inoculated on a porous membrane were irradiated with varying doses of $$gamma$$-rays or carbon ions. Irradiated cells were then non-contact co-cultured with non-irradiated cells for 24 h. After co-culture, the survival rates of non-irradiated bystander cells co-cultured with irradiated cells decreased with increasing dose and bottomed out at 0.5 Gy or higher doses. This indicates that the bystander effect is dependent on irradiation dose but independent of radiation quality. Next, a specific NO scavenger c-PTIO was added to the culture medium during irradiation and co-culture. This treatment prevented the reduction in survival rates of bystander cells, clearly indicating that NO has an important role in the bystander effect.

Journal Articles

Target irradiation of individual cells using focusing heavy-ion microbeam of JAEA-Takasaki, 5; Irradiation of individual cells with scanned heavy-ion microbeam

Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 73, 2015/03

Using a collimating heavy-ion microbeam system, we have explored various effects of heavy-ion hit on biological materials. However, there are limitations of the collimating system in the size of the microbeam spot and in the irradiation speed that cannot be overcome in principle. Thus, we started the development of a focusing microbeam system for target-irradiating individual cells more precisely. In this year, we established the protocol for irradiating "actual" cell sample with scanned beam. In the experiment, the HeLa cells were inoculated on a CR-39 film, then place on the sample stage. The microscopic image of cells was analyzed, and the cells were irradiated with scanned neon microbeam. After irradiation, we found the correspondence of the distribution pattern of the ion hit positions and the $$gamma$$-H2AX foci on cell nuclei, indicating rapid and accurate irradiation of individual cells with the focusing heavy-ion microbeam.

Journal Articles

Mechanisms for the induction of radioadaptive response by radiation-induced bystander response

Matsumoto, Hideki*; Tomita, Masanori*; Otsuka, Kensuke*; Hatashita, Masanori*; Maeda, Munetoshi*; Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Ikeda, Hiroko; et al.

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 76, 2015/03

The objective of this project is to elucidate molecular mechanisms for the induction of radioadaptive response through radiation-induced bystander responses induced by irradiation with heavy ion microbeams in JAEA. We found that the adaptive response was induced by Ar (520 MeV $$^{40}$$Ar$$^{14+}$$) microbeam-irradiation of a limited number of cells, followed by the broad beam-irradiation and that the adaptive response was almost completely suppressed by the addition of carboxy-PTIO, as a nitric oxide (NO) scavenger. In addition, we found several genes induced specifically and preferentially when radioadaptive response could be induced. We confirmed that ${it iNOS}$ expression was specifically induced only when radioadaptive response could be induced. Our findings strongly suggested that radioadaptive response can be induced by NO-mediated bystander responses evoked by irradiation with heavy ion microbeams.

Journal Articles

Analysis of bystander response in 3D cultured tissue induced by heavy-ion microbeam irradiation

Tomita, Masanori*; Matsumoto, Hideki*; Otsuka, Kensuke*; Funayama, Tomoo; Yokota, Yuichiro; Suzuki, Michiyo; Sakashita, Tetsuya; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 77, 2015/03

Radiation-induced bystander responses are defined as responses in cells that have not been directly targeted by radiation but are in the neighborhood of cells that have been directly exposed. In this study, we aim to clarify a role of bystander response to sustain the homeostasis of damaged tissue using heavy-ion microbeams. We established the heavy-ion microbeam irradiation method to a 3D cultured human epidermis. Using this method, a viable cell rate of the 3D cultured human epidermis irradiated with 260 MeV $$^{20}$$Ne-ion microbeams or broadbeams was analyzed by the MTT method.

Journal Articles

Ion-species dependent bystander mutagenic effect on ${it HPRT}$ locus in normal human fibroblasts induced by C-, Ne- and Ar-ion microbeams

Suzuki, 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 ${it HPRT}$ locus in normal human fibroblasts. The confluent culture were irradiated using a 256 (16$$times$$16)-cross-stripe method using C, Ne and Ar microbeam. Gene mutation on ${it HPRT}$ 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.

Journal Articles

Analysis of bystander effect induced by cell membrane response in glioma cells

Wada, Seiichi*; Ando, Tatsuhiko*; Watanabe, Aya*; Kakizaki, Takehiko*; Natsuhori, Masahiro*; Funayama, Tomoo; Sakashita, Tetsuya; Yokota, Yuichiro; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 79, 2015/03

So far, we clarified that X-ray irradiation induced cell killing by bystander effect mediated-secreted factor. This phenomenon was related with sphingomyelinase (SMase). In this study we analyzed mechanism of secreted SMase from irradiated cells after irradiation. SMase was detected in the culture medium after irradiation by SDS-PAGE. Then, SMase was detected in the exosome of culture medium, but not out of exosome after irradiation. This result indicates that SMase was secreted as exosome from the irradiated cells.

Journal Articles

Effect of heavy ion irradiation to the silkworm eggs at before fertilization and at nuclear cleavage stage

Ueda, Daisuke*; Shirai, Koji*; Funayama, Tomoo; Sakashita, Tetsuya; Yokota, Yuichiro; Kobayashi, Yasuhiko

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 84, 2015/03

In this study, we investigated the effects of irradiation to the silkworm eggs at various developmental stages. First, we tried the irradiation to the unfertilized eggs (at 1.5 hour after oviposition). At this stage, the female pronucleus and the sperm nucleus are observed in the eggs, but not fertilized. After irradiation, the irradiated eggs stopped the development after fertilization. About 2 hours after, the egg restarted the nuclear cleavage. This result indicates the DNA damage on pronuleus cannot prevent the fertilization. We also investigated the effects of irradiation to the egg at the nuclear cleavage stage (at 6 hour after oviposition). The egg also stopped the development after irradiation, but the duration time of the developmental arrest was almost two times longer (about 4 hours) than that of the egg irradiated at fertilization.

Journal Articles

Medaka blastoderm cells are capable of compensating the injured cells irradiated by carbon-ion micro-beam

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 $$mu$$m). We performed targeted irradiation by carbon-ion micro-beam (diameters of 120, 180 $$mu$$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 $$mu$$m diameter) could hatch normally in contrast to all embryos with 2 Gy of whole-body irradiation being lethal before hutching.

Journal Articles

Immunofluorescence observation of oxidative damage of DNA induced by heavy ions from TIARA

Kitabatake, Satomi*; Ushiroda, Tota*; Hirayama, Ryoichi*; Furusawa, Yoshiya*; Funayama, Tomoo; Yokota, Yuichiro; Okahata, Yoshio*; Ito, Atsushi*

JAEA-Review 2014-050, JAEA Takasaki Annual Report 2013, P. 86, 2015/03

Biological effects of high-LET radiation could be understood in terms of the ion track structure. Therefore the evaluation of the contribution of both core and penumbra regions to biological effects is an important issue for the study of high-LET effects. In the present study, we developed a protocol to make a uniform DNA sheet with insoluble nature in aqueous solution, and explored the applicability to the detection of 8-OHdG distributions after heavy-ion irradiation. Water-insoluble DNA sheet was irradiated with proton and neon ion beams at JAEA-Takasaki. After irradiation DNA samples were incubated with an 8-OHdG antibody followed by with a second antibody containing a fluorescence probe. The preliminary results indicated that upon ion irradiation randomly distributed dot-like fluorescence was observed, suggesting that these dots may be from incident ions.

JAEA Reports

Development of microbeam formation and single-ion hit technologies at the TIARA cyclotron

Yokota, Wataru; Sato, Takahiro; Kamiya, Tomihiro; Okumura, Susumu; Kurashima, Satoshi; Miyawaki, Nobumasa; Kashiwagi, Hirotsugu; Yoshida, Kenichi; Funayama, Tomoo; Sakashita, Tetsuya; et al.

JAEA-Technology 2014-018, 103 Pages, 2014/09

JAEA-Technology-2014-018.pdf:123.66MB

The world's first microbeam focusing technology for heavy ions of hundreds MeV accelerated by a cyclotron has been developed at the TIARA facility in the Takasaki Advanced Radiation Research Institute of the Japan Atomic Energy Agency. The technology enables us to form a microbeam of less than 1 $$mu$$m in diameter and to shoot a specified point on a target by one ion (single-ion hit) with spatial accuracy of microbeam size. In the course of the development, a cyclotron technology to accelerate a small energy-spread beam of hundres MeV, which is necessary for focusing to 1 $$mu$$m, has been developed as well as a beam focusing apparatus, beam size measurement and so forth based on the several-MeV microbeam/single-ion hit system of the TIARA electrostatic accelerators. Applicability of the technologies was examined by actual use in irradiation experiment and the result were fed back to them. This paper reports the process and the results of the development over ten years.

Journal Articles

Radiation-quality-dependent bystander effects induced by the microbeams with different radiation sources

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

Journal Articles

Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures; The Impact of radiation quality

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:39 Percentile:6.65(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.

Journal Articles

Effects of low- and high-LET radiation on the salt chemotaxis learning in ${it Caenorhabditis elegans}$

Sakashita, Tetsuya; Suzuki, Michiyo; Hamada, Nobuyuki*; Shimozawa, Yoko; Fukamoto, Kana*; Yokota, Yuichiro; Sora, Sakura*; Kakizaki, Takehiko*; Wada, Seiichi*; Funayama, Tomoo; et al.

Biological Sciences in Space, 26, p.21 - 25, 2012/10

High linear energy transfer (LET) radiation is important cosmic rays that has neurobiological effects: it is known to induce conditioned taste aversion, and suppress neurogenesis that may underlie cognitive impairment. However, the impact of high-LET radiation on other learning effects remains largely unknown. Here, we focus on kinetics of the radiation response for the salt chemotaxis learning (SCL) behavior in the nameatode, ${it Caenorhabditis elegans}$, because the SCL during the learning conditioning was modulated after low-LET $$gamma$$-irradiation. Firstly, the SCL ability was examined following high-LET irradiation ($$^{12}$$C, 18.3 MeV/u, LET = 113 keV/$$mu$$m), revealing its dose-dependent decrease after high- and low-LET exposure. Next, we demonstrate that the SCL at the early phase of the learning conditioning is greatly affected by high- and low-LET irradiation, and interestingly, the magnitude of these effects by high-LET radiation was smaller than that by low-LET one. Moreover, the analysis of ${it gpc-1}$ mutant showed that the G-protein $$gamma$$ subunit, GPC-1 is responsible for such early phase response. This study is the first to provide the evidence for the kinetics of changes in SCL after high-LET irradiation of C. ${it elegans}$.

Journal Articles

Behavioral resistance of ${it Caenorhabditis elegans}$ against high-LET radiation exposure

Sakashita, Tetsuya; Suzuki, Michiyo; Hamada, Nobuyuki*; Shimozawa, Yoko; Fukamoto, Kana*; Yokota, Yuichiro; Sora, Sakura*; Kakizaki, Takehiko*; Wada, Seiichi*; Funayama, Tomoo; et al.

Biological Sciences in Space, 26, p.7 - 11, 2012/07

Here, we investigated the resistance to high-LET radiation exposure for two behaviors of the nematode, ${it Caenorhabditis elegans}$, which is known as a model organism for the nervous system. Tested behaviors were locomotion and chemotaxis to NaCl. In addition, egg hatchability was examined as an indicator of high-LET radiation sensitivity. Relative biological effectiveness (RBE) of high-LET radiation ($$^{12}$$C, 18.3 MeV/u, LET = 113 keV/$$mu$$m) relative to low-LET radiation for hatchability was 4.5, whereas RBEs for locomotion and chemotaxis were 1.4 and 1.1, respectively. This study shows that the behavioral system for locomotion and chemotaxis of ${it C. elegans}$ is highly resistant to high-LET radiation exposure.

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