Masakazu Oikawa

SPICE-NIRS microbeam: a focused vertical system for proton irradiation of a single cell for radiobiological research.

The Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) is a focused vertical microbeam system designed to irradiate the nuclei of adhesive mammalian cells with a defined number of 3.4 MeV protons. The approximately 2-μm diameter proton beam is focused with a magnetic quadrupole triplet lens and traverses the cells contained in dishes from bottom to top. All procedures for irradiation, such as cell image capturing, cell recognition and position calculation, are automated. The most distinctive characteristic of the system is its stability and high throughput; i.e. 3000 cells in a 5 mm × 5 mm area in a single dish can be routinely irradiated by the 2-μm beam within 15 min (the maximum irradiation speed is 400 cells/min). The number of protons can be set as low as one, at a precision measured by CR-39 detectors to be 99.0%. A variety of targeting modes such as fractional population targeting mode, multi-position targeting mode for nucleus irradiation and cytoplasm targeting mode are available. As an example of multi-position targeting irradiation of mammalian cells, five fluorescent spots in a cell nucleus were demonstrated using the γ-H2AX immune-staining technique. The SPICE performance modes described in this paper are in routine use. SPICE is a joint-use research facility of NIRS and its beam times are distributed for collaborative research.

Damaging and protective bystander cross-talk between human lung cancer and normal cells after proton microbeam irradiation

Most of the studies of radiation-induced bystander effects (RIBE) have been focused on understanding the radiobiological changes observed in bystander cells in response to the signals from irradiated cells in a normal cell population with implications to radiation risk assessment. However, reports on RIBE with relevance to cancer radiotherapy especially investigating the bidirectional and criss-cross bystander communications between cancer and normal cells are limited. Hence, in present study employing co-culture approach, we have investigated the bystander cross-talk between lung cancer (A549) and normal (WI38) cells after proton-microbeam irradiation using γ-H2AX foci fluorescence as a measure of DNA double-strand breaks (DSBs). We observed that in A549-A549 co-cultures, irradiated A549 cells exert damaging effects in bystander A549 cells, which were found to be mediated through gap junctional intercellular communication (GJIC). However, in A549-WI38 co-cultures, irradiated A549 did not affect bystander WI38 cells. Rather, bystander WI38 cells induced inverse protective signalling (rescue effect) in irradiated A549 cells, which was independent of GJIC. On the other hand, in response to irradiated WI38 cells neither of the bystander cells (A549 or WI38) showed significant increase in γ-H2AX foci. The observed bystander signalling between tumour and normal cells may have potential implications in therapeutic outcome of cancer radiotherapy.

The threshold number of protons to induce an adaptive response in zebrafish embryos

In this study, microbeam protons were used to provide the priming dose to induce an in vivo radioadaptive response (RAR) in the embryos of zebrafish, Danio rerio, against subsequent challenging doses provided by x-ray photons. The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed. The embryos were dechorionated at 4xa0h post fertilisation (hpf) and irradiated at 5xa0hpf by microbeam protons. For each embryo, one irradiation point was chosen, to which 5, 10, 20, 30, 40, 50, 100, 200, 300 and 500 protons each with an energy of 3.4xa0MeV were delivered. The embryos were returned to the incubator until 10 hpf to further receive the challenging exposure, which was achieved using 2xa0Gy of x-ray irradiation, and then again returned to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25xa0hpf were quantified through terminal dUTP transferase-mediated nick end-labelling (TUNEL) assay. The results revealed that at least 200 protons (with average radiation doses of about 300 and 650xa0mGy absorbed by an irradiated epithelial and deep cell, respectively) would be required to induce RAR in the zebrafish embryos in vivo. Our previous investigation showed that 5 protons delivered at 10 points on an embryo would already be sufficient to induce RAR in the zebrafish embryos. The difference was explained in terms of the radiation-induced bystander effect as well as the rescue effect.

Radioactive contamination mapping of northeastern and eastern Japan by a car-borne survey system, Radi-Probe

We constructed a new car-borne survey system called Radi-Probe with a portable germanium gamma-ray spectrometer onboard a cargo truck, to identify radionuclides and quantify surface contamination from the accident at Fukushima Dai-ichi Nuclear Power Station. The system can quickly survey a large area and obtain ambient dose equivalent rates and gamma-ray energy spectra with good energy resolution. We also developed a new calibration method for the system to deal with an actual nuclear disaster, and quantitative surface deposition densities of radionuclides, such as (134)Cs and (137)Cs, and kerma rates of each radionuclide can be calculated. We carried out car-borne survey over northeastern and eastern Japan (Tohoku and Kanto regions of Honshu) from 25 September through 7 October 2012. We discuss results of the distribution of ambient dose equivalent rate H(∗)(10), (134)Cs and (137)Cs surface deposition densities, spatial variation of (134)Cs/(137)Cs ratio, and the relationship between surface deposition densities of (134)Cs/(137)Cs and H(∗)(10). The ratio of (134)Cs/(137)Cs was nearly constant within our measurement precision, with average 1.06xa0±xa00.04 in northeastern and eastern Japan (decay-corrected to 11 March, 2011), although small variations from the average were observed.

Target irradiation induced bystander effects between stem-like and non stem-like cancer cells

Tumors are heterogeneous in nature and consist of multiple cell types. Among them, cancer stem-like cells (CSCs) are suggested to be the principal cause of tumor metastasis, resistance and recurrence. Therefore, understanding the behavior of CSCs in direct and indirect irradiations is crucial for clinical radiotherapy. Here, the CSCs and their counterpart non stem-like cancer cells (NSCCs) in human HT1080 fibrosarcoma cell line were sorted and labeled, then the two cell subtypes were mixed together and chosen separately to be irradiated via a proton microbeam. The radiation-induced bystander effect (RIBE) between the CSCs and NSCCs was measured by imaging 53BP1 foci, a widely used indicator for DNA double strand break (DSB). CSCs were found to be less active than NSCCs in both the generation and the response of bystander signals. Moreover, the nitric oxide (NO) scavenger c-PTIO can effectively alleviate the bystander effect in bystander NSCCs but not in bystander CSCs, indicating a difference of the two cell subtypes in NO signal response. To our knowledge, this is the first report shedding light on the RIBE between CSCs and NSCCs, which might contribute to a further understanding of the out-of-field effect in cancer radiotherapy.

Exogenous carbon monoxide suppresses adaptive response induced in zebrafish embryos in vivo by microbeam protons

Dechorionated embryos of the zebrafish, Danio rerio, irradiated at 5 h post-fertilization (hpf) with 30 protons delivered to 10 separate positions each with an energy of 3.4 MeV from the microbeam irradiation facility (Single-Particle Irradiation System to Cell, acronym as SPICE) at the National Institute of Radiological Sciences (NIRS), developed radioadaptive response (RAR) against a subsequent challenging exposure of 2 Gy of X-ray irradiation at 10 hpf, corroborated by reduced apoptotic signals at 25 hpf revealed through terminal dUTP transferase-mediated nick end-labeling assay. The effects of the CO liberator tricarbonylchloro(glycinato)ruthenium (II) (CORM-3) on the induction of RAR were examined by transferring the irradiated embryos to freshly prepared medium with the chemical at different time points after the application of the priming dose. Our results showed that transfer of irradiated embryos into media with CORM-3 at 0, 1, 2 and 3 h after application of priming exposure significantly suppressed RAR, while transfer at 5 h did not suppress RAR. This was attributed to the protection of bystander cells from the released CO, which caused less de novo synthesis of factors and thus less efficient induction of RAR. Once the factors were synthesized, RAR was induced, which would not be further affected by the application of CORM-3 introduced at 5 h after the application of the priming dose. Clinical Trial Registration number if required: None.

Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling

Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) targeted/irradiated A549-GFP cells; (2) neighboring/non-irradiated cells directly contacting the targeted cells; and (3) distant/non-irradiated cells, which were not in direct contact with the targeted cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi-directional signaling had extended a profound influence on cellular sensitivity to radiation as well as the sensitivity to RIBE.

Low 134Cs/137Cs ratio anomaly in the north-northwest direction from the Fukushima Dai-ichi Nuclear Power Station

A low 134Cs/137Cs ratio anomaly in the north-northwest (NNW) direction from the Fukushima Dai-ichi Nuclear Power Station (FDNPS) is identified by a new analysis of the 134Cs/137Cs ratio dataset which we had obtained in 2011-2015 by a series of car-borne surveys that employed a germanium gamma-ray spectrometer. We found that the 134Cs/137Cs ratio is slightly lower (0.95, decay-corrected to March 11, 2011) in an area with a length of about 15xa0km and a width of about 3xa0km in the NNW direction from the FDNPS than in other directions from the station. Furthermore, the area of this lower 134Cs/137Cs ratio anomaly corresponds to a narrow contamination band that runs NNW from the FDNPS and it is nearly parallel with the major and heaviest contamination band in the west-northwest. The plume trace with a low 134Cs/137Cs ratio previously found by other researchers within the 3-km radius of the FDNPS is in a part of the area with the lower 134Cs/137Cs ratio anomaly that we found. Our result suggests that this lower 134Cs/137Cs ratio anomaly is the area which was contaminated before March 13, 2011 (UTC) in association with the hydrogen explosion of Unit 1 on March 12, 2011xa0at 06:36 (UTC) and it was less influenced by later subsequent plumes.

Roles of nitric oxide in adaptive response induced in zebrafish embryos in vivo by microbeam protons.

Radioadaptive response (RAR) was successfully induced in dechorionated (5 h post-fertilization, hpf) embryos of the zebrafish, Danio rerio, by 3.4 MeV protons from the microbeam irradiation facility (Single-Particle Irradiation System to Cell, acronym as SPICE) [ 1] at the National Institute of Radiological Sciences (NIRS), against a challenging exposure of 2 Gy of X-ray irradiation at 10 hpf. The RAR induction was corroborated by reduced apoptotic signals at 25 hpf revealed through terminal dUTP transferase-mediated nick end-labeling assay. If de novo synthesis of factors was required for RAR induction, these should have already been synthesized at 5 h after the priming dose. Application of a nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) to the medium at 0, 1, 2, 3 or 5 h after application of priming exposure significantly suppressed RAR. The suppression of RAR with the application of cPTIO to the medium at 5 h after the priming dose irradiation, where de novo synthesis of factors should have been completed, suggested that NO scavenging impaired the repair machineries in the bystander cells. The suppression of RAR with the application of cPTIO to the medium at earlier than 5 h after the priming dose irradiation could be explained by the scavenging of bystander NO signals in the medium and thus deterring the de novo synthesis of factors.

Effects of experimental pastes containing surface pre-reacted glass ionomer fillers on inhibition of enamel demineralization

This study aimed to evaluate the inhibitory effect of experimental pastes containing surface pre-reacted glass ionomer (S-PRG) fillers on enamel demineralization. Bovine blocks were treated twice a day for 4 days by 7 groups; experimental pastes containing 0-30 wt% S-PRG filler (S00, S01, S05, S10, and S30), deionized water (DW) as negative control, and NaF paste (MP) as positive control. The surfaces were demineralized by acetic acid for 3 days. Mineral loss (ML) was calculated by micro-computed X-ray tomography. The treated surface was finally investigated with scanning electron microscope (SEM) and micro-focused particle induced X-ray emission (micro-PIXE). S05, S10 and S30 demonstrated significantly lower ML than S00, S01 and DW (p<0.05). S10 showed the greatest inhibitory effect, which was significantly greater than MP. The S-PRG filler containing experimental pastes demonstrated a potential to inhibit enamel demineralization. Sr ion incorporation was confirmed on the enamel surface with the experimental pastes.