No Intercellular Regulation of the Cell Cycle among Human Cervical Carcinoma HeLa Cells Expressing Fluorescent Ubiquitination-Based Cell-Cycle Indicators in Modulated Radiation Fields.

The non-targeted effects of radiation have been known to induce significant alternations in cell survival. Although the effects might govern the progression of tumor sites following advanced radiotherapy, the impacts on the intercellular control of the cell cycle following radiation exposure with a modified field, remain to be determined. Recently, a fluorescent ubiquitination-based cell-cycle indicator (FUCCI), which can visualize the cell-cycle phases with fluorescence microscopy in real time, was developed for biological cell research. In this study, we investigated the non-targeted effects on the regulation of the cell cycle of human cervical carcinoma (HeLa) cells with imperfect p53 function that express the FUCCI (HeLa-FUCCI cells). The possible effects on the cell-cycle phases via soluble factors were analyzed following exposure to different field configurations, which were delivered using a 150 kVp X-ray irradiator. In addition, using synchrotron-generated, 5.35 keV monochromatic X-ray microbeams, high-precision 200 µm-slit microbeam irradiation was performed to investigate the possible impacts on the cell-cycle phases via cell-cell contacts. Collectively, we could not detect the intercellular regulation of the cell cycle in HeLa-FUCCI cells, which suggested that the unregulated cell growth was a malignant tumor. Our findings indicated that there was no significant intercellular control system of the cell cycle in malignant tumors during or after radiotherapy, highlighting the differences between normal tissue and tumor characteristics.

Health effects triggered by tritium: how do we get public understanding based on scientifically supported evidence?

The Commission for 'Corresponding to Radiation Disaster of the Japanese Radiation Research Society' formulated a description of potential health effects triggered by tritium. This was in response to the issue of discharging water containing tritium filtered by the Advanced Liquid Processing System (ALPS), generated and stored in Fukushima Daiichi Nuclear Power Station after the accident. In this review article, the contents of the description, originally provided in Japanese, which gives clear and detailed explanation about potential health effects triggered by tritium based on reliable scientific evidence in an understandable way for the public, were summarized. Then, additional information about biochemical or environmental behavior of organically bound tritium (OBT) were summarized in order to help scientists who communicate with general public.

Field size effects on DNA damage and proliferation in normal human cell populations irradiated with X-ray microbeams.

To clarify the health risks of internal radiation exposure, it is important to investigate the radiological effects of local exposure at cell levels from radioactive materials taken up by organs. Focusing on the response of cell populations post-irradiation, X-ray microbeams are very effective at reproducing the effects of local exposure within an internal exposure in vitro. The present study aims to clarify the effects of local exposure by investigating the response of normal human cell (MRC-5) populations irradiated with X-ray microbeams of different beam sizes to DNA damage. The populations of MRC-5 were locally irradiated with X-ray microbeams of 1 Gy at 0.02-1.89 mm2 field sizes, and analyzed whether the number of 53BP1 foci as DSB (DNA double strand break) per cell changed with the field size. We found that even at the same dose, the number of DSB per cell increased depending on the X-irradiated field size on the cell population. This result indicated that DNA damage repair of X-irradiated cells might be enhanced in small size fields surrounded by non-irradiated cells. This study suggests that X-irradiated cells received some signal (a rescue signal) from surrounding non-irradiated cells may be involved in the response of cell populations post-irradiation.

Spatially Fractionated Microbeam Analysis of Tissue-sparing Effect for Spermatogenesis.

Spatially fractionated radiation therapy (SFRT) has been based on the delivery of a single high-dose fraction to a large treatment area that has been divided into several smaller fields, reducing the overall toxicity and adverse effects. Complementary microbeam studies have also shown an effective tissue-sparing effect (TSE) in various tissue types and species after spatially fractionated irradiation at the microscale level; however, the underlying biological mechanism remains elusive. In the current study, using the combination of an ex vivo mouse spermatogenesis model and high-precision X-ray microbeams, we revealed the significant TSE for maintaining spermatogenesis after spatially fractionated microbeam irradiation. We used the following ratios of the irradiated to nonirradiated areas: 50:50, 150:50 and 350:50 µm-slit, where approximately 50, 75 and 87.5% of the sample was irradiated (using center-to-center distances of 100, 200 and 400 µm, respectively). We found that the 50 and 75% micro-slit irradiated testicular tissues showed an almost unadulterated TSE for spermatogenesis, whereas the 87.5% micro-slit irradiated tissues showed an incomplete TSE. This suggests that the TSE efficiency for spermatogenesis is dependent on the size of the nonirradiated spermatogonial stem cell pool in the irradiated testicular tissues. In addition, there would be a spatiotemporal limitation of stem cell migration/competition, resulting in the insufficient TSE for 87.5% micro-slit irradiated tissues. These stem cell characteristics are essential for the accurate prediction of tissue-level responses during or after SFRT, indicating the clinical potential for achieving better outcomes while preventing adverse effects.

Combination of agents modifying effects in hadrontherapy: modelization of the role of HO° free radicals.

Purpose: A study is presented of the irradiation of cancerous cervical cell line HeLa loaded with a platinum salt, betamethasone and deoxyglucose. The presence of the platinum increases the free-radical concentration and augments the cell death rate, whereas betamethasone or deoxyglucose induces radiosensitization by the alteration of metabolic pathways. Two by two combinations of these chemicals are made to investigate the possible benefit when two radiosensitizers are present. A model is proposed to understand the results of the presence of two modifying agents on the dose effects.Materials and methods: The cells were incubated for 6 h in the presence of the following molecules: dichloro terpyridine platinum, concentration C = 350 µM, betamethasone and deoxyglucose with concentrations of C = 0.2 µM and C = 6 mM, respectively. The cells were subsequently irradiated by carbon C6+ ion 290 MeV/amu up to a dose of 2.5 Gy, under atmospheric conditions.Results: The presence of the platinum salt or bethamethasone augments the cell death rate. The combination of betamethasone with the platinum salt also increases the cell death rate, but less than for the platinum salt alone. The explanation is that any radiosensitizer also behaves as a scavenger of free radicals. This dual behavior should be considered in any optimization of the design of radiosensitizers when different ionizing particles are used.

Enhancement of membrane lipid peroxidation in lung cancer cells irradiated with monoenergetic X-rays at the K-shell resonance absorption peak of phosphorus.

The aim of this study was to determine whether membrane lipid peroxidation in mammalian cells is enhanced by X-ray irradiation at the K-shell resonance absorption peak of phosphorus. A549 and wild-type p53-transfected H1299 (H1299/wtp53) cell lines derived from human lung carcinoma were irradiated with monoenergetic X-rays at 2.153 keV, the phosphorus K-shell resonance absorption peak, or those at 2.147 or 2.160 keV, which are off peaks. Immunofluorescence staining for 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, was used as marker for protein modification. In both cell lines, the HNE production was significantly enhanced after irradiation at 2.153 keV compared to sham-irradiation. The enhancement (E) was calculated as the ratio of the fluorescence intensity of irradiated cells to that of sham-irradiated cells. In both the cell lines, E2.153 was significantly larger than E2.147 and no significant difference between E2.147 and E2.160 was observed. The extra enhancement at 2.153 keV was possibly caused by energy transition within the phosphorus K-shell resonance absorption. Our results indicate that membrane lipid peroxidation in cells is enhanced by the Auger effect after irradiation at the K-shell resonance absorption peak of phosphorus rather than by the photoelectric effect of the constituent atoms in the membrane lipid at 2.147 keV.

High-precision microbeam radiotherapy reveals testicular tissue-sparing effects for male fertility preservation.

Microbeam radiotherapy (MRT) is based on a spatial fractionation of synchrotron X-ray microbeams at the microscale level. Although the tissue-sparing effect (TSE) in response to non-uniform radiation fields was recognized more than one century ago, the TSE of MRT in the testes and its clinical importance for preventing male fertility remain to be determined. In this study, using the combination of MRT techniques and a unique ex vivo testes organ culture, we show, for the first time, the MRT-mediated TSE for the preservation of spermatogenesis. Furthermore, our high-precision microbeam analysis revealed that the survival and potential migration steps of the non-irradiated germ stem cells in the irradiated testes tissue would be needed for the effective TSE for spermatogenesis. Our findings indicated the distribution of dose irradiated in the testes at the microscale level is of clinical importance for delivering high doses of radiation to the tumor, while still preserving male fertility.

Enhancement of DNA double-strand break induction and cell killing by K-shell absorption of phosphorus in human cell lines.

PURPOSE: To investigate an enhancement of DNA double-strand break (DSB) induction and cell killing effect by K-shell ionization of phosphorus atoms and Auger electrons on human cell lines. MATERIALS AND METHODS: Induction of DSB, DNA damage responses, cell cycle distributions, and cell killing effects were investigated after exposures of the cells with monochromatic synchrotron radiation soft X-rays of 2153 and 2147 eV, which were the resonance peak and off peak, respectively, of the K-shell photoabsorption of phosphorus. RESULTS: Higher biological effects in the cells irradiated with soft X-rays at 2153 eV than at 2147 eV were observed in (i) the efficiency of 53BP1/γ-H2AX co-localized foci formation per dose and residual number of foci, (ii) prolonged phosphorylation levels of DSB repair and/or cell cycle checkpoint related proteins and G2 arrest, (iii) the cell killing effects at the 10% survival level of normal human fibroblasts, HeLa cells, and human glioblastoma M059K cells (1.2-1.5 times higher) and that of human ataxia telangiectasia mutated (ATM)-defective cells and glioblastoma DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-defective cells (1.2 times). CONCLUSION: The yield of DSB and partly less-reparable complex DNA damage induction in human cells was enhanced by K-shell photoabsorption of phosphorus and low-energy Auger electrons.

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.

Gadolinium-based nanoparticles to improve the hadrontherapy performances.

Nanomedicine is proposed as a novel strategy to improve the performance of radiotherapy. High-Z nanoparticles are known to enhance the effects of ionizing radiation. Recently, multimodal nanoparticles such as gadolinium-based nanoagents were proposed to amplify the effects of x-rays and g-rays and to improve MRI diagnosis. For tumors sited in sensitive tissues, childhood cases and radioresistant cancers, hadrontherapy is considered superior to x-rays and g-rays. Hadrontherapy, based on fast ion radiation, has the advantage of avoiding damage to the tissues behind the tumor; however, the damage caused in front of the tumor is its major limitation. Here, we demonstrate that multimodal gadolinium-based nanoparticles amplify cell death with fast ions used as radiation. Molecular scale experiments give insights into the mechanisms underlying the amplification of radiation effects. This proof-of-concept opens up novel perspectives for multimodal nanomedicine in hadrontherapy, ultimately reducing negative radiation effects in healthy tissues in front of the tumor. FROM THE CLINICAL EDITOR: Gadolinium-chelating polysiloxane nanoparticles were previously reported to amplify the anti-tumor effects of x-rays and g-rays and to serve as MRI contrast agents. Fast ion radiation-based hadrontherapy avoids damage to the tissues behind the tumor, with a major limitation of tissue damage in front of the tumor. This study demonstrates a potential role for the above nanoagents in optimizing hadrontherapy with preventive effects in healthy tissue and amplified cell death in the tumor.

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.

X-ray-induced bystander responses reduce spontaneous mutations in V79 cells.

The potential for carcinogenic risks is increased by radiation-induced bystander responses; these responses are the biological effects in unirradiated cells that receive signals from the neighboring irradiated cells. Bystander responses have attracted attention in modern radiobiology because they are characterized by non-linear responses to low-dose radiation. We used a synchrotron X-ray microbeam irradiation system developed at the Photon Factory, High Energy Accelerator Research Organization, KEK, and showed that nitric oxide (NO)-mediated bystander cell death increased biphasically in a dose-dependent manner. Here, we irradiated five cell nuclei using 10 × 10 µm(2) 5.35 keV X-ray beams and then measured the mutation frequency at the hypoxanthine-guanosine phosphoribosyl transferase (HPRT) locus in bystander cells. The mutation frequency with the null radiation dose was 2.6 × 10(-)(5) (background level), and the frequency decreased to 5.3 × 10(-)(6) with a dose of approximately 1 Gy (absorbed dose in the nucleus of irradiated cells). At high doses, the mutation frequency returned to the background level. A similar biphasic dose-response effect was observed for bystander cell death. Furthermore, we found that incubation with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a specific scavenger of NO, suppressed not only the biphasic increase in bystander cell death but also the biphasic reduction in mutation frequency of bystander cells. These results indicate that the increase in bystander cell death involves mechanisms that suppress mutagenesis. This study has thus shown that radiation-induced bystander responses could affect processes that protect the cell against naturally occurring alterations such as mutations.

Comment on 'Therapeutic application of metallic nanoparticles combined with particle-induced x-ray emission effect'.

A recent paper (Kim et al 2010 Nanotechnology 21 425102) presented results on the combination of irradiation by atomic ions of cells loaded by particles made of heavy atoms. They propose that the projectile induced x-rays emission (PIXE) mechanism has an important contribution to the enhancement of the cell death rate. Experiments made in our group to study the effects of such a combination have shown that the Auger effect induced in the high-Z atoms and the following induction of surrounding water radiolysis has an important contribution to the enhancement of the cell death rate. In the light of our studies we propose an alternative interpretation of the results presented in the paper by Kim et al.

DNA damage and repair kinetics after microbeam radiation therapy emulation in living cells using monoenergetic synchrotron X-ray microbeams.

A novel synchrotron-based approach, known as microbeam radiation therapy (MRT), currently shows considerable promise in increased tumour control and reduced normal tissue damage compared with conventional radiotherapy. Different microbeam widths and separations were investigated using a controlled cell culture system and monoenergetic (5.35 keV) synchrotron X-rays in order to gain further insight into the underlying cellular response to MRT. DNA damage and repair was measured using fluorescent antibodies against phosphorylated histone H2AX, which also allowed us to verify the exact location of the microbeam path. Beam dimensions that reproduced promising MRT strategies were used to identify useful methods to study the underpinnings of MRT. These studies include the investigation of different spatial configurations on bystander effects. γH2AX foci number were robustly induced in directly hit cells and considerable DNA double-strand break repair occurred by 12 h post-10 Gy irradiation; however, many cells had some γH2AX foci at the 12 h time point. γH2AX foci at later time points did not directly correspond with the targeted regions suggesting cell movement or bystander effects as a potential mechanism for MRT effectiveness. Partial irradiation of single nuclei was also investigated and in most cases γH2AX foci were not observed outside the field of irradiation within 1 h after irradiation indicating very little chromatin movement in this time frame. These studies contribute to the understanding of the fundamental radiation biology relating to the MRT response, a potential new therapy for cancer patients.

Platinum nanoparticles: a promising material for future cancer therapy?

Recently, the use of gold nanoparticles as potential tumor selective radiosensitizers has been proposed as a breakthrough in radiotherapy. Experiments in living cells and in vivo have demonstrated the efficiency of the metal nanoparticles when combined with low energy x-ray radiations (below conventional 1 MeV Linac radiation). Further studies on DNA have been performed in order to better understand the fundamental processes of sensitization and to further improve the method. In this work, we propose a new strategy based on the combination of platinum nanoparticles with irradiation by fast ions effectively used in hadron therapy. It is observed in particular that nanoparticles enhance strongly lethal damage in DNA, with an efficiency factor close to 2 for double strand breaks. In order to disentangle the effect of the nano-design architecture, a comparison with the effects of dispersed metal atoms at the same concentration has been performed. It is thus shown that the sensitization in nanoparticles is enhanced due to auto-amplified electronic cascades inside the nanoparticles, which reinforces the energy deposition in the close vicinity of the metal. Finally, the combination of fast ion radiation (hadron therapy) with platinum nanoparticles should strongly improve cancer therapy protocols.

Preliminary study of laser welding for aortic dissection in a porcine model using a diode laser with indocyanine green.

BACKGROUND AND OBJECTIVES: The objective of this study was to determine whether a dissected aorta could be welded by a diode laser with a solder using an in vitro porcine aortic dissection model. STUDY DESIGN/MATERIALS AND METHODS: Porcine aortic strips were dissected into two flaps and the dissected faces were immersed in a solution of indocyanine green. The two flaps were pressed at 0.2 kg/cm2 with contact between the two immersed faces. The pressed flaps were irradiated with a diode laser (810 nm) at intensities of 170-425 W/cm2 for 8 seconds. The welded flaps were studied by light microscopy and the adhesive strengths were measured. RESULTS: The irradiated flaps were successfully welded. The breaking stress, the maximum stress recorded in a stress-strain curve, increased with increase in irradiation intensity up to 396 W/cm2 (2.7 x 10(2) mmHg) and decreased when the intensity reached 425 W/cm2. In the specimen irradiated at 396 W/cm2, the welded faces showed continuous fusion of elastin layers, while some voids were seen between the welded faces in the specimen irradiated at 425 W/cm2. CONCLUSIONS: The dissected porcine aortas were successfully welded using a laser with solder. The results suggest that the welded aorta can bear physiological blood pressure.