A Method to Locally Irradiate Specific Organ in Model Organisms Using a Focused Heavy-Ion Microbeam.

The functions of organisms are performed by various tissues composed of different cell types. Localized irradiation with heavy-ion microbeams, which inactivate only a portion of the constituent cells without destroying the physical intercellular connections of the tissue, is a practical approach for elucidating tissue functions. However, conventional collimated microbeams are limited in the shape of the area that can be irradiated. Therefore, using a focused heavy-ion microbeam that generates a highly precise beam spot, we developed a technology to uniformly irradiate specific tissues of an organism with a defined dose, which conventional methods cannot achieve. The performance of the developed paint irradiation technology was evaluated. By irradiating the CR-39 ion track detector, we confirmed that the new method, in which each ion hit position is placed uniformly in the irradiated area, makes it possible to uniformly paint the area at a specified dose. The targeted irradiation of the pharynx and gonads of living Caenorhabditis elegans demonstrated that the irradiated ions were distributed in the same shape as the targeted tissue observed under a microscope. This technology will elucidate biological mechanisms that are difficult to analyze with conventional collimated microbeam irradiation.

Responses of hematopoietic cells after ionizing-irradiation in anemic adult medaka (Oryzias latipes).

PURPOSE: Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Oryzias latipes), which has been a model animal and a large body of knowledge has been accumulated in radiation biology. METHODS: Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated. RESULTS: When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction. CONCLUSIONS: The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

Repair Kinetics of DNA Double Strand Breaks Induced by Simulated Space Radiation.

Radiation is unavoidable in space. Energetic particles in space radiation are reported to induce cluster DNA damage that is difficult to repair. In this study, normal human fibroblasts were irradiated with components of space radiation such as proton, helium, or carbon ion beams. Immunostaining for γ-H2AX and 53BP1 was performed over time to evaluate the kinetics of DNA damage repair. Our data clearly show that the repair kinetics of DNA double strand breaks (DSBs) induced by carbon ion irradiation, which has a high linear energy transfer (LET), are significantly slower than those of proton and helium ion irradiation. Mixed irradiation with carbon ions, followed by helium ions, did not have an additive effect on the DSB repair kinetics. Interestingly, the mean γ-H2AX focus size was shown to increase with LET, suggesting that the delay in repair kinetics was due to damage that is more complex. Further, the 53BP1 focus size also increased in an LET-dependent manner. Repair of DSBs, characterized by large 53BP1 foci, was a slow process within the biphasic kinetics of DSB repair, suggesting non-homologous end joining with error-prone end resection. Our data suggest that the biological effects of space radiation may be significantly influenced by the dose as well as the type of radiation exposure.

Space Radiation Biology for "Living in Space".

Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.

Distinct modes of death in human neural stem and glioblastoma cells irradiated with carbon-ion radiation and gamma-rays.

Purpose: Accumulated damage in neural stem cells (NSCs) during brain tumor radiotherapy causes cognitive dysfunction to the patients. Carbon-ion radiotherapy can reduce undesired irradiation of normal tissues more efficiently than conventional photon radiotherapy. This study elucidates the responses of NSCs to carbon-ion radiation.Methods: Human NSCs and glioblastoma A-172 cells were irradiated with carbon-ion radiation and γ-rays, which have different linear-energy-transfer (LET) values of 108 and 0.2 keV/µm, respectively. After irradiation, growth rates were measured, apoptotic cells were detected by flow cytometry, and DNA synthesizing cells were immunocytochemically visualized.Results: Growth rates of NSCs and A-172 cells were decreased after irradiation. The percentages of apoptotic cells were remarkably increased in NSCs but not in A-172 cells. In contrast, the fractions of DNA synthesizing A-172 cells were decreased in a dose-dependent manner. These results indicate that apoptosis induction and DNA synthesis inhibition contribute to the growth inhibition of NSCs and glioblastoma cells, respectively. In addition, high-LET carbon ions induced more profound effects than low-LET γ-rays.Conclusions: Apoptosis is an important clinical target to protect NSCs during brain tumor radiotherapy using carbon-ion radiation as well as conventional X-rays.

Histone Deacetylase Inhibitors Sensitize Murine B16F10 Melanoma Cells to Carbon Ion Irradiation by Inducing G1 Phase Arrest.

Epigenetic processes, in addition to genetic abnormalities, play a critical role in refractory malignant diseases and cause the unresponsiveness to various chemotherapeutic regimens and radiotherapy. Herein we demonstrate that histone deacetylase inhibitors (HDACis) can be used to sensitize malignant melanoma B16F10 cells to carbon ion irradiation. The cells were first treated with HDACis (romidepsin [FK228, depsipeptide], trichostatin A [TSA], valproic acid [VPA], and suberanilohydroxamic acid [SAHA, vorinostat]) and were then exposed to two types of radiation (carbon ions and gamma-rays). We found that HDACis enhanced the radiation-induced apoptosis and suppression of clonogenicity that was induced by irradiation, having a greater effect with carbon ion irradiation than with gamma-rays. Carbon ion irradiation and the HDACi treatment induced G2/M and G0/G1 cell cycle arrest, respectively. Thus, it is considered that HDACi treatment enhanced the killing effects of carbon ion irradiation against melanoma cells by inducing the arrest of G1 phase cells, which are sensitive to radiation due to a lack of DNA homologous recombination repair. Based on these findings, we propose that pretreatment with HDACis as radiosensitizers to induce G1 arrest combined with carbon ion irradiation may have clinical efficacy against refractory cancer.

In vivo 3D analysis of systemic effects after local heavy-ion beam irradiation in an animal model.

Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body, and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

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

In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time after irradiation.

Dependence of the bystander effect for micronucleus formation on dose of heavy-ion radiation in normal human fibroblasts.

Ionising radiation-induced bystander effects are well recognised, but its dependence on dose or linear energy transfer (LET) is still a matter of debate. To test this, 49 sites in confluent cultures of AG01522D normal human fibroblasts were targeted with microbeams of carbon (103 keV µm(-1)), neon (375 keV µm(-1)) and argon ions (1260 keV µm(-1)) and evaluated for the bystander-induced formation of micronucleus that is a kind of a chromosome aberration. Targeted exposure to neon and argon ions significantly increased the micronucleus frequency in bystander cells to the similar extent irrespective of the particle numbers per site of 1-6. In contrast, the bystander micronucleus frequency increased with increasing the number of carbon-ion particles in a range between 1 and 3 particles per site and was similar in a range between 3 and 8 particles per site. These results suggest that the bystander effect of heavy ions for micronucleus formation depends on dose.

A mathematical model of radiation-induced responses in a cellular population including cell-to-cell communications.

Cell-to-cell communication is an important factor for understanding the mechanisms of radiation-induced responses such as bystander effects. In this study, a new mathematical model of intercellular signalling between individual cells in a cellular population is proposed. The authors considered two types of transmission of signals: via culture medium and via gap junction. They focus on the effects that radiation and intercellular signalling have on cell-cycle modification. The cell cycle is represented as a virtual clock that includes several checkpoint pathways within a cyclic process. They also develop a grid model and set up diffusion equations to model the propagation of signals to and from spatially located cells. The authors have also considered the role that DNA damage plays in the cycle of cells which can progress through the cell cycle or stop at the G1, S, G2 or M-phase checkpoints. Results of testing show that the proposed model can simulate intercellular signalling and cell-cycle progression in individual cells during and after irradiation.

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

PURPOSE: To investigate the dependence of the bystander cell-killing effect on radiation dose and quality, and to elucidate related molecular mechanisms. MATERIALS AND METHODS: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method. RESULTS: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ≤ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations. CONCLUSION: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.

Genetic changes in progeny of bystander human fibroblasts after microbeam irradiation with X-rays, protons or carbon ions: the relevance to cancer risk.

PURPOSE: Radiation-induced bystander effects have important implications in radiotherapy. Their persistence in normal cells may contribute to risk of health hazards, including cancer. This study investigates the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of harmful effects in progeny of bystander cells. MATERIALS AND METHODS: Confluent human skin fibroblasts were exposed to microbeam radiations with different linear energy transfer (LET) at mean absorbed doses of 0.4 Gy by which 0.036-0.4% of the cells were directly targeted by radiation. Following 20 population doublings, the cells were harvested and assayed for micronucleus formation, gene mutation and protein oxidation. RESULTS: 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 X-rays (LET ∼6 keV/µm) or protons (LET ∼11 keV/µm) showed persistent oxidative stress, which correlated with increased micronucleus formation and mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus. Such effects were not observed after irradiation by carbon ions (LET ∼103 keV/µm). Interestingly, 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 cultures were exposed to X-rays. CONCLUSIONS: These findings suggest that cellular exposure to proton and heavy charged particle with LET properties similar to those used here can reduce the risk of lesions associated with cancer. The ability of cells to communicate via gap junctions at the time of irradiation appears to impact residual damage in progeny of bystander cells.

Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation.

DNA double-strand breaks (DSBs) induced by ionizing radiation pose a major threat to cell survival. The cell can respond to the presence of DSBs through two major repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Higher levels of cell death are induced by high-linear energy transfer (LET) radiation when compared to low-LET radiation, even at the same physical doses, due to less effective and efficient DNA repair. To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene (NHEJ-related Lig4 and/or HR-related Rad54) knockouts (KO) were used and their responses were compared to wild-type cells. The cells were exposed to X rays, spread-out Bragg peak (SOBP) carbon ion beams as well as with carbon, iron, neon and argon ions. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells > Lig4-KO cells > Rad54-KO cells > wild-type cells. Although Rad54-KO cells had an almost constant SER value, Lig4-KO cells showed a high-SER value when compared to Rad54-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair.

Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells.

This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.

Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures: the impact of radiation quality.

Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction 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 monochromatic 5.35 keV X ray (LET ~6 keV/µm), 18.3 MeV/u carbon ion (LET ~103 keV/µm), 13 MeV/u neon ion (LET ~380 keV/µm) or 11.5 MeV/u argon ion (LET ~1,260 keV/µm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and 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, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that 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. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects.

Synergistic effect of heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin and X-rays, but not carbon-ion beams, on lethality in human oral squamous cell carcinoma cells.

The purpose of this study is to clarify the effect of a heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in combination with X-rays or carbon-ion beams on cell killing in human oral squamous cell carcinoma LMF4 cells. Cell survival was measured by colony formation assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of DNA repair-related proteins was investigated by western blotting. The results showed 17-AAG to have synergistic effects on cell lethality with X-rays, but not with carbon-ion beams. The 17-AAG decreased G(2)/M arrest induced by X-rays, but not by carbon-ion beams. Both X-ray and carbon-ion irradiation up-regulated expression of non-homologous end-joining-associated proteins, Ku70 and Ku80, but 17-AAG inhibited only X-ray-induced up-regulation of these proteins. These results show that 17-AAG with X-rays releases G(2)/M phase arrest; cells carrying misrepaired DNA damage then move on to the G(1) phase. We demonstrate, for the first time, that the radiosensitization effect of 17-AAG is not seen with carbon-ion beams because 17-AAG does not affect these changes.

Ionising irradiation alters the dynamics of human long interspersed nuclear elements 1 (LINE1) retrotransposon.

It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (>5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

Involvement of bystander effect in suppression of the cytokine production induced by heavy-ion broad beams.

PURPOSE: Immune cells accumulate in and around cancers and cooperate with each other using specific cytokines to attack the cancer cells. The heavy-ion beams for cancer therapy may stimulate immune cells and affect on the immune system. However, it is still poorly understood how the immune cells are stimulated by ion-beams. Here, we irradiated immune cells using heavy-ion beams and analyzed changes in production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) that are important cytokine for the cancer treatment. MATERIALS AND METHODS: The human THP-1 monocytes were differentiated into macrophages and then irradiated using carbon-ion broad-beams (108 keV µm(-1)). To examine the bystander response after heavy-ion irradiation, a very small fraction (approx. 0.45%) of the cell population was irradiated using heavy-ion microbeams. After irradiation, we examined the cytokine productions. RESULTS: When cells were irradiated with 5 Gy, cytokine levels were reduced after both microbeam irradiation and broad-beam irradiation. TNF-α production of macrophages with the nitric oxide (NO) inhibitor-treatment increased after carbon-ion broad-beam. NO was involved in the radiation-induced suppression of TNF-α production. CONCLUSIONS: The suppression of cytokine production arose after irradiation with heavy-ions, and may also be induced in the surrounding non-irradiated cells via the bystander effect.

Effectiveness of carbon-ion beams for apoptosis induction in rat primary immature hippocampal neurons.

The direct biological effects of radiation, particularly accelerated heavy particle ions, on neurons are not fully known. Hence, the direct effect of carbon-ion beams on immature neurons was investigated by comparing to the effect of X-rays in vitro using primary hippocampal neurons. Primary neurons were prepared from hippocampi of fetal rats at embryonic day 18 from timed pregnant Wistar rats and cultured with Banker's methods. At 7 Days In Vitro (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/µm). The cells were fixed with 4% paraformaldehyde at 12 hours after irradiation. Then, the cells were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI staining for measuring the percentage of apoptosis (apoptotic index: AI). AI in sham-irradiated hippocampal neurons was 18%. The value of AI (AIs) of the cells irradiated with X-rays at 10 or 30 Gy were 15% or 23%, respectively. AI in cells irradiated with carbon-ion beams at 1 Gy, 3 Gy, 5 Gy and 10 Gy were 22%, 23%, 24% and 33%, respectively. AI was significantly increased by carbon-ion beams at 10 Gy (p < 0.001). The apoptosis of hippocampal neurons increased in a dose-dependent manner following both X-ray and carbon-ion beams irradiation. Carbon-ion beams were about 10-fold more effective than X-rays for apoptosis induction in immature hippocampal neurons.

Enhanced micronucleus formation in the descendants of gamma-ray-irradiated tobacco cells: evidence for radiation-induced genomic instability in plant cells.

Ionizing radiation-induced genomic instability has been documented in various end points such as chromosomal aberrations and mutations, which arises in the descendants of irradiated mammalian or yeast cells many generations after the initial insult. This study aimed at addressing radiation-induced genomic instability in higher plant tobacco cells. We thus investigated micronucleus (MN) formation and cell proliferation in tobacco cells irradiated with gamma-rays and their descendants. In gamma-irradiated cells, cell cycle was arrested at G2/M phase at around 24 h post-irradiation but released afterward. In contrast, MN frequency peaked at 48 h post-irradiation. Almost half of 40 Gy-irradiated cells had MN at 48 h post-irradiation, but proliferated as actively as sham-irradiated cells up to 120 h post-irradiation. Moreover, the descendants that have undergone at least 22 generations after irradiation still showed a two-fold MN frequency compared to sham-irradiated cells. This is the direct evidence for radiation-induced genomic instability in tobacco cells.