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Purpose: Considerable evidence exists regarding ionizing radiation (IR) induced bystander effect has important implication in clinical radiotherapy [1]. Their persistence in the progeny of human cells may contribute to risk of long-term radiation-related human health effect, including cancer [2]. The objective of this study is to elucidate the involvement of radiation quality and gap junction intercellular communication (GJIC) in the propagation of delayed stressful effects in the progeny of bystander normal human cells. Materials and method: Confluent human skin fibroblasts (NB1RGB) were exposed to various types of microbeam with a different linear energy transfer (LET) at mean absorbed doses 0.4 Gy, wherein 0.036-0.4% of the cells were targeted by IR. Following 20 populations post-irradiation, the cells were harvested and assayed for micronucleus formation, mutation assay and protein oxidation. Results: Our results showed that the delayed stressful effects in the progeny of bystander cells are dependent on radiation quality or LET. The progeny of bystander cells exposed to X rays (LET ~6 keV/µm) and protons (LET ~11 keV/µm) showed the persistence of oxidative stress, and correlate with the increased micronucleus formation and mutant fraction. However, such effects were not observed after irradiation by carbon ions (LET~103 keV/µm). Interestingly, inhibition of GJIC mitigated the damaging effects in the progeny of bystander cells exposed to protons and carbon ions but not X rays. Conclusions: These data show, for the first time, carbon ions can reduce cancer risk after microbeam irradiation compared with X rays or protons, and GJIC may be a critical mediator in the observed effect., The 12th International Workshop on Microbeam Probes of Cellular Radiation Response

Purpose: Treatment plans of carbon-ion radiotherapy have been made on the assumption that the beams are delivered instantaneously irrespective to the dose delivery time as well as the interruption time. The single fractionated irradiation against non-small cell lung cancer (NSCLC) requires more time to deliver a fractional dose than the conventional multi-fractionated ones, since four beams at different directions are delivered in one fraction with a patient re-setup among them. The purpose of this study is to investigate the effects of the beam interruption time for the patient re-setup on tumor control probability (TCP) of NSCLC for single fractionated carbon-ion radiotherapy. \nMethods: The rate equations defined in the microdosimetric kinetic model (MKM) for primary lesions caused in DNA were applied to the single fractionated irradiation of NSCLC. The rate constant characterizing the time of the primary non-lethal lesions to repair was experimentally determined for human salivary gland tumor cells. Treatment plans were made for a NSCLC case for various fraction sizes ranging from 25 to 50 Gy (RBE), on the assumption that the beams are delivered instantaneously. These plans were recalculated by varying the interruption time ranging from 3 to 60 min for continuous irradiations of 3 min per beam based on the MKM with the determined repair rate constant. The curative dose which gives TCP of 95% was investigated for respective interruption times. \nResults: The rate constant for non-lethal lesion to repair is 2.19±0.40 h-1. The biological effectiveness in the target decreases with the interruption time τ compared to the planned one due to the repair of non-lethal lesions during the interruption. The curative dose for planned instantaneous irradiation was 33.8 Gy (RBE), while it increased to 35.8, 37.9, 39.5 and 41.1 Gy (RBE) for τ=3, 15, 30 and 60 min. \nConclusions: We investigated the effects of beam interruption time on TCP of NSCLC for single fractionated carbon-ion radiotherapy. The biological effectiveness of therapeutic carbon-ion beam decreases with the interruption time. The curative dose can increase by 20% or more as compared to the planned dose if the interruption time prolongs to 30 min or longer. These effects can be considered in designing the carbon-ion treatment planning., The 15th International Congress of Radiation Research (ICRR 2015)

Purpose: Ionizing Radiation (IR)-induced bystander effects and genomic instability have important implication in radiotherapy and radioprotection. Their persistence in the progeny of normal cells may contribute to risk of long-term radiation-related health effect in human, including cancer. Hence, this study investigates the role of gap junction intercellular communication (GJIC) and the quality of radiation in the propagation of stressful effects in the unirradiated bystander cells and their progeny. Material and methods: Human skin fibroblasts in the confluent state were exposed to microbeam irradiations with different linear energy transfer (LET) at mean absorbed dose of 0.4 Gy, in the presence or absence of GJIC inhibitor (AGA) by which 0.036-0.4% of cells were directly targeted by IR. After 4 h irradiation or following 20 population doublings, the cells were harvested and assayed for micronucleus (MN) formation, gene mutation and protein oxidation. Results: Our results showed that high-LET carbon microbeams (LET ~103 keV/μm) and high-LET proton microbeams (LET~11 keV/μm) were more effective than low-LET X ray microbeam (LET ~6 keV/μm) in the induction of DNA damage (MN formation) in bystander cells. Interestingly, significant attenuation of MN formation occurred in bystander cells in the presence of AGA after proton and carbon microbeams. In contrast, incubation of the cells with AGA did not significantly affect the induction of MN formation in bystander cells during confluent holding after X irradiation. Further, the progeny of bystander cells exposed to X rays or protons showed persistent oxidative stress which correlated MN formation and mutation frequency. Such effects were not observed after irradiation by carbon ions. Importantly, the progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultured were exposed to X rays. Taken together, the overall results show the expression of stressful effects in the bystander cells and their progeny are dependent on the radiation quality or LET. Conclusions: Our findings suggest that the involvement of GJIC-dependent of radiation quality in the propagation of radiation induces stressful effects to bystander cells and their progeny. In addition, this work provides a strong support to the fact that carbon ions can significantly reduce the risk of cancer and have potential implications in the therapeutic outcome of radiotherapy compared to X rays or protons., American Association for Cancer Research Annual Meeting 2015

第15回宇宙科学シンポジウム (2015年1月6日-7日. 宇宙航空研究開発機構宇宙科学研究所(JAXA)(ISAS)相模原キャンパス), 相模原市, 神奈川県, 15th Space Science Symposium (January 6-7, 2015. Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency(JAXA)(ISAS)Sagamihara Campus), Sagamihara, Kanagawa Japan, 資料番号: SA6000034244, レポート番号: P-245

The controversial use of a linear, no threshold extrapolation model for low dose risk assessment is based on the accepted dogma that the deleterious effects of ionizing radiation such as mutagenesis and carcinogenesis are attributable mainly to direct damage to DNA. However, this extropolation was challenged by the recent reports on the bystander phenomenon. The bystander effect contributes to this debate by implying that the biological effects of low doses, where not all cells are traversed by a charged particle, are amplified by the transfer of factors to un-irradiated neighbors. This interested phenomenon implies that a linear extrapolation of risks from high to low doses may underestimate rather than over estimate low dose risks. Together with some radiation-induced phenomena such as adaptive response and genomic instability, the radiobiological response at low doses is likely to be a complex interplay among many factors., Acta medica Nagasakiensia. 2005, 50(supl.1), p.73-77

Radiation-induced damage to living cells results from either a direct hit to cellular DNA, or from indirect action which leads to DNA damage from radiation produced radicals. However, in recent years there is evidence that biological effects such as cell killing, mutation induction, chromosomal damage and modification of gene expression can occur in a cell population exposed to low doses of alpha particles. In fact these doses are so low that not all cells in the population will be hit directly by the radiation. Using a precision alpha-particle microbeam, it has been recently demonstrated that irradiated target cells can induce a bystander mutagenic response in neighboring "bystander" cells which were not directly hit by alpha particles. Furthermore, these results suggest that gap-junction mediated cell-to-cell communication plays a critical role in this bystander phenomenon. The purpose of this section is to describe recent studies on bystander biological effects. The recent work described here utilized heavy charged particles for irradiation, and investigated the role of gap-junction mediated cell-cell communication in this phenomenon.

Selective irradiation by an ionizing particle of a targeted cell organelle may disclose such mechanisms as signal transaction among cell organelles and cell-to-cell communication in the processes toward an endpoint observed. Bystander effect, existence of which has been clearly evidenced by application of the particle microbeam to biological experiments, suggests potential deviation from the conventional risk estimation at low particle fluence rates, such as in an environment of space radiation in International Space Station.To promote these studies we started the construction of a microbeam facility (named as SPICE) by using our HVEE Tandem accelerator (3.4 MeV proton and 5.1 MeV 4He2+). For our primary goal,"irradiation of cell organelle with a single praticle with a position resolution of 2 mum in a reasonable irradiation time", special features are considered. Usage of a triplet Q-magnet for focussing the beam to micrometer levels is an out standing feature compared to facilities of other institutes. Other features are almost similar to those of other institutes. Those are precise position control of a cell dish holder, design of the cell dish, data acquisition of microscopic image of a cell oraganelle (cell nucleus), data processing, reliable particle detection, soft and hard wares to integrate all these related data and system to control and irradiate a targeted spot with exactly determined number of particles.

At a time when manned space exploration is more a reality with the planned the International Space Station (ISS) well underway, the biological effects for chronically low-dose (rate) irradiation in the field of low-flux galactic cosmic rays (GCR) have become one of the major concerns of space science. It is, therefore, important to estimate radiation risk in human body of high-energy & charged (HZE) particles, especially secondary scattered radiation field produced by the interaction between heavy ions and structural materials of a space station etc.In this study we designed an experimental field for chronically low-dose irradiation of secondary radiation scattered with heavy-ion radiation field and examined cellular responses in the field.

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It is well established that the RBE (relative biological effectiveness) for cell killing depends on LET (linear energy transfer), and that a maximum RBE is observed at ~150 keV/mm. However, the therapeutic gain depends on the ratio of the RBEs for the effects on the cancer cell population and the effects on normal tissues. The RBE of a given radiation quality depends on LET but also on dose, biological system and effect, and irradiation conditions. There is no data available to answer the question: which LET is suitable to improve the biological therapeutic gain of carbon ions? Here, we selected 3 different LET values of 290 MeV/u carbon ions, and compared the relative biological effectiveness between tumor-growth retardation and skin damage using a murine transplantable tumor. Larger RBE values for tumors after than the skin type were obtained when carbon-ions of intermediate LET were daily delivered for 2 through 5 fractions. The biological therapeutic gain would be high for the carbon-ion SOBP if the number of fractions were correctly selected in clinical trials.

The goal of this study is to investigate the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of delayed stressful effects in the progeny of bystander human skin fibroblasts cultures (NB1RGB). Briefly, confluent NB1RGB cells in the presence and absence of gap junction inhibitor (AGA) were exposed to ionizing radiation (IR) with a different linear energy transfer (LET) either 5.35 keV X rays (LET 6 keV/m) or 18.3 MeV/u carbon (LET 103 keV/m) microbeam radiations. Following 20 populations post-irradiation, the progeny of bystander NB1RGB cells were harvested and assayed for several of biological endpoints. Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to low-LET X rays showed the persistence of oxidative stress and it was correlated with the increased mutant fraction. Such effect were not observed after high-LET carbon ions. Interestingly, inhibition of GJIC mitigated the toxic effects in the progeny of bystander cells. Together, the results contribute to the understanding of the fundamental radiation biology relating to the high-LET carbon ions to mitigate cancer risk after radiotherapy. Furthermore, GJIC be considered as a critical mediator in the bystander mutagenic effect.

A woman started to feel intractable pain on her lower legs when she was 76. At the age of 78, she was diagnosed as having Parkinson’s disease (PD). The leg pain was suspected to be a symptom of PD after eliminating other causes. The patient also suffered from nonmotor symptoms, depression, anxiety, hot flashes, and paroxysmal sweating. Though the patient had received pharmacotherapy including levodopa for 5 years, she still suffered from the nonmotor symptoms and was referred to our department. We treated her with acupuncture based on the Chinese traditional medicine and electroacupuncture five times per week. After the 2-week treatment, the assessment for the symptoms was as follows; visual analogue scale (VAS) score of the leg pain was 16 mm (70 mm, before), Hamilton’s rating scales for depression (HAM-D) score was 9 (18, before), timed 3 m Up and Go took 20 steps in 30 sec (24 steps in 38 sec, before), and the Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part 1 score was 13 (21, before). Autonomic symptoms, hot flashes and paroxysmal sweating, were also alleviated. Acupuncture may be a good treatment modality for nonmotor symptoms in PD.

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炭素イオン及びＸ線マイクロビームを低フルエンスでヒト正常細胞に照射した時に誘導される細胞致死と突然変異誘発に関する細胞応答（バイスタンダー効果）の線質依存性を調べた。マイクロビームを全細胞数の0.20%の細胞に限定的に照射した時、炭素イオンでは直接照射された細胞数を遙かに超えた細胞致死と突然変異誘発が観察された一方で、Ｘ線ではその様な現象は観察されなかった。

We have begun to investigate cellular bystander responses in normal human fibroblasts induced by microbeams of different radiation types. This year, we used two different microbeams, such as carbon ions as high-LET (Linear Energy Transfer) radiations and X rays as low-LET radiations. Carbon-ion microbeams ((12)C(5+), 220 MeV) were generated with the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) at Japan Atomic Energy Agency. Mono-energetic X-ray (5.35 keV) microbeams were produced with the Photon Factory (PF) at High Energy Accelerator Research Organization. We designed that only 0.2 percent of total cells was irradiated with each microbeam. The percent cell survival in microbeam-irradiated dishes was ranging from 83 percent to 94 percent for carbon ions, while around 100 percent for X rays. Mutation frequency for carbon-ion-microbeam irradiated cells was about 6 times higher than that for non-irradiated control cells, but there was no difference in mutation frequency between X-ray-microbeam irradiated and control cells. There is clear evidence that bystander cellular effects induced by low-dose radiations depends on a radiation quality., 資料番号: AA0063706092

We have studied biological effects in normal human fibroblasts induced by low-fluence high-LET (Linear Energy Transfer) ions to examine what kinds of radiation species induce genomic instability. Cells were pre-treated with low-fluence ions (approximately 1 milli-Gy/7 to 8 h) of either carbon or helium before following irradiation with X-ray challenging dose (1.5 Gy). There observed no difference in cell-killing effect in pre-treatment with either carbon or helium, when comparing to the pre-treatment with low-dose Cs-137 gamma rays. In mutation induction there was no difference in induced mutation frequency at 1.5 Gy of X-ray challenging dose between un-pretreated and gamma-ray pre-treated cells. On the other hand, it was around 4.0 times higher in carbon-ion pre-treated cells and 1.9 times higher in helium-ion pre-treated cells than in un-pretreated cells. There is evidence that genomic instability in mutation induction induced by the pre-treatment of low-fluence ions depends upon both LET and ion species., 資料番号: AA0063349086

We studied the effect of repeated irradiation with a low dose rate (about 0.0012 cGy per mm) of ^ Co γ-rays on the in vitro life-span of human embryo (HE) cells. HE cells were cultured in an incubator that was set in a ^Co γ-ray-irradiation room, and the irradiation was repeated throughout the life-span of the HE cells (for about 150 to 160 days) on every day base. During this period, the cells accumulated 106 to 123 cGy. The life-span of the irradiated cells prolonged 1.14 to 1.35 times when compared to that of non-irradiated cells. The incidence of cells with chromosome bridge and micronuclei significantly increased in the irradiated cells. Although the number of chromosomes gradually changed with repetition of culture in both non-irradiated and irradiated cells, the frequencies of aneuploid cells in irradiated cells were about two times higher than that in non-irradiated cells. These results indicate that repeated irradiation with a low dose of γ-rays produces chromosome instability, and it may be a cause of numerical chromosome abnormalities and life-span extension of irradiated cells., Journal of radiation research. 1998, 39(3), p.203-213

Treatment plans of c-ion radiotherapy have been made on the assumption that the beams in one fraction are delivered instantaneously by neglecting the dose delivery time as well as the interruption time. However, in practical treatments, it takes 1-10 minutes or longer to deliver a fractional dose to a patient depending on the prescribed dose level, the dose delivery technique and the performance of the delivery system. Especially, the advanced therapeutic techniques such as a hypo-fractionation and a respiratory gating usually require more time to deliver a fractional dose than the convetional one. In addition, the dose delivery can be interrupted due to some reasons such as the necessity of a patient re-setup between fields in a treatment fraction and machine troubles. Cell killing tends to decrease with the fraction time because of sublethal damage repair (SLDR). Thus, it is important to investigate the quantity of SLDR during the fraction time and to evaluate the effects of dose-delivery time structure on the biological effectiveness of therapeutic c-ion beams. In this study, the Microdosimetric Kinetic Model (MKM) was adopted to address this issue., 4D Treatment planning workshop 2013

We investigated the role of intercellular communication and oxidative metabolism in determining the biological responses of human cells to heavy ion radiotherapy. We focused on the communication of signaling events between carbon-irradiated cancer cells and bystander normal cells. Here, human glioblastoma cells (T98G) were irradiated with a single dose of 6 Gy or doses divided into 3 fractions given at consecutive days (3 x 2 Gy) of high-LET carbon ions (LET -76 keV/um). Then were co-cultured with normal human skin fibroblasts (NB1RGB), in the presence or absence of gap-junction inhibitor (18-alpha-glycyrrhetinic: AGA) or antioxidant (Superoxide Dismutase: SOD) for 4 hrs. The layered tissue culture system is used to allow co-culturing the irradiated-T98G cells growing in wells with bystander NB1RGB cells growing in inserts, and also medium transfer strategy. Bystander NB1RGB cells were then harvested and assayed for clonogenic survival, micronucleus formation (MN) or allowed to grow for 10 weeks and assayed for MN. As expected, relative to control, both single dose irradiation and fractionation of the dose into three fractions show the bystander NB1RGB cells exhibited reduced cloning efficiency and consistence with the increase of MN formation. In contrast, treatment with AGA or SOD shows a significant increase of survival and decrease of MN formation in bystander cells. Interestingly, the condition medium harvested from irradiated T98 cells receiving fractionated irradiation presented lower cloning efficiency and a higher MN formation than a single-dose irradiation. These results suggested that fractionated irradiation removed the effect of dose sparing that was observed after conventional fractionated regime. Furthermore, the progeny of bystander cells that were co-cultured with irradiated T98G cells for 10 weeks showed that there was induction of MN. Interestingly, the level of MN is reduced if there is the inhibition of GJIC. In addition, the findings also indicated that intercellular communication and oxidative metabolism amplifies the stressful effects of high-LET carbon ions in bystander cells and their progeny. Our results provide new evidence that high-LET carbon ions-induced the bystander effect and genomic instability in both single dose irradiation and fractionated irradiation. They were observed in bystander normal skin fibroblasts and their progeny indicating that irradiated cancer cells can induce damage in normal human cells. Characterizing the nature of the communicated molecules(s) would have translational implication in radiotherapy and radioprotection. Supported by the Japan Society for the Promotion of Sciences and the National Institute of Radiological Sciences, 59th Radiation Research Society Annual Meeting