Noriyoshi Suya

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.

Bystander effect between zebrafish embryos in vivo induced by high-dose X-rays.

We employed embryos of the zebrafish, Danio rerio, for our studies on the in vivo bystander effect between embryos irradiated with high-dose X-rays and naive unirradiated embryos. The effects on the naive whole embryos were studied through quantification of apoptotic signals at 25 h post fertilization (hpf) through the terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay followed by counting the stained cells under a microscope. We report data showing that embryos at 5 hpf subjected to a 4-Gy X-ray irradiation could release a stress signal into the medium, which could induce a bystander effect in partnered naive embryos sharing the same medium. We further demonstrated that this bystander effect (induced through partnering) could be successfully suppressed through the addition of the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) into the medium but not through the addition of the CO liberator tricarbonylchloro(glycinato)ruthenium(II) (CORM-3). This shows that NO was involved in the bystander response between zebrafish embryos induced through X-ray irradiation. We also report data showing that the bystander effect could be successfully induced in naive embryos by introducing them into the irradiated embryo conditioned medium (IECM) alone, i.e., without partnering with the irradiated embryos. The IECM was harvested from the medium that had conditioned the zebrafish embryos irradiated at 5 hpf with 4-Gy X-ray until the irradiated embryos developed into 29 hpf. NO released from the irradiated embryos was unlikely to be involved in the bystander effect induced through the IECM because of the short life of NO. We further revealed that this bystander effect (induced through IECM) was rapidly abolished through diluting the IECM by a factor of 2× or greater, which agreed with the proposal that the bystander effect was an on/off response with a threshold.

Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity

Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high‐resolution quantitative in situ measurements by high‐energy synchrotron radiation X‐ray fluorescence analysis in renal sections from a rat model of uranium‐induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg–1 body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g–1, sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g–1). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50‐fold above mean renal concentration) in micro‐regions was found near the nuclei. These uranium levels were maintained up to 8 days post‐administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100‐fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site‐specific accumulation of uranium in micro‐regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney. Copyright

Non-induction of radioadaptive response in zebrafish embryos by neutrons

In vivo neutron-induced radioadaptive response (RAR) was studied using zebrafish (Danio rerio) embryos. The Neutron exposure Accelerator System for Biological Effect Experiments (NASBEE) facility at the National Institute of Radiological Sciences (NIRS), Japan, was employed to provide 2-MeV neutrons. Neutron doses of 0.6, 1, 25, 50 and 100 mGy were chosen as priming doses. An X-ray dose of 2 Gy was chosen as the challenging dose. Zebrafish embryos were dechorionated at 4 h post fertilization (hpf), irradiated with a chosen neutron dose at 5 hpf and the X-ray dose at 10 hpf. The responses of embryos were assessed at 25 hpf through the number of apoptotic signals. None of the neutron doses studied could induce RAR. Non-induction of RAR in embryos having received 0.6- and 1-mGy neutron doses was attributed to neutron-induced hormesis, which maintained the number of damaged cells at below the threshold for RAR induction. On the other hand, non-induction of RAR in embryos having received 25-, 50- and 100-mGy neutron doses was explained by gamma-ray hormesis, which mitigated neutron-induced damages through triggering high-fidelity DNA repair and removal of aberrant cells through apoptosis. Separate experimental results were obtained to verify that high-energy photons could disable RAR. Specifically, 5- or 10-mGy X-rays disabled the RAR induced by a priming dose of 0.88 mGy of alpha particles delivered to 5-hpf zebrafish embryos against a challenging dose of 2 Gy of X-rays delivered to the embryos at 10 hpf.

EVALUATION OF PRESSED POWDERS AND THIN SECTION STANDARDS FOR MULTI-ELEMENTAL ANALYSIS BY CONVENTIONAL AND MICRO-PIXE ANALYSIS

For muti-elemental analysis, various standards are used to quantify the elements consists of environmental and biological samples. In this paper two different configuration standards, pressed powders and thin section standards, were assessed for their purpose as standards by conventional and micro-PIXE analysis. Homogeneity of manganese, iron, zinc (Zn), copper and yttrium added to pressed powder standard materials were validated and the relative standard deviation (RSD) of the X-ray intensity of the standards was <10% within the range, 62.5–250 µg/g. We established linear relationships between the metal concentration and the specific X-ray intensity of standards containing up to 250 µg/g of these metals. A homogenous distribution of Zn added to thin section standard materials was also confirmed by 10-µm-step scanning of the standard within the range, 50–250 µg/g (RSD ~ 10%). The calibration line between the X-ray intensity obtained from a 10-µm2 area and the metal concentration was acceptable.

Micro-PIXE analysis system at NIRS-electrostatic accelerator facility for various applications

A micro-PIXE analysis system based on the ion beam analysis system by Oxford Microbeams Ltd. has been developed at the NIRS-electrostatic accelerator facility. The introduction of the CdTe X-ray detector dramatically improved the detection efficiencies for heavy elements that are important in the life sciences and environmental science. This system has been used for various projects and has provided several meaningful results, thus establishing the micro-PIXE system as an effective tool for the determination of elemental distribution with a high spatial resolution. In this paper, outline of the features of the micro-PIXE system at NIRS along with its recent application in research are introduced.

Research and development of focused proton microbeam irradiation system, SPICE for radio-biological studies.

There is continuing interest for the use of microbeam irradiation systems designed to deliver a defined number of charged particles on a single cell with a resolution of a few micrometers. Irradiation of an exact number of charged particles on a single cell means that the limitations of the Poisson distribution of the number of charged particles can be overcome. Moreover, microbeams are particularly useful for the field of radiation-induced non-targeted effects, so-called bystander effects that are considered to be one of the major effects in the low-dose region. Thus, microbeam technique is one of the powerful tools for investigating studies related to radiation effect and risk of low dose in space radiation for astronauts and cosmonauts. Our microbeam irradiation system, the Single-Particle Irradiation system to CEll (SPICE) provides a 3.4 MeV proton microbeam focused with a quadrupole magnetic lens on an upward vertical beam line. SPICE was severely damaged by the Tohoku-oki Earthquake on 11 March 2011, and was out of operation for about a year and a half. We have successfully reconstructed the facility, and it is now operational with system refinements. At present, SPICE is the only proton microbeam facility in Japan at which a single-ion single-cell irradiation can be performed on mammalian cells with stability and high throughput using an upward vertical beam of below 2-μm diameter, focused with a magnetic quadrupole triplet lens [ 1]. A variety of irradiation modes have been established for radiation-induced bystander effects, cytoplasm irradiation etc. An example of cells targeted with a multi-position targeting mode is shown in Fig. 1, which cells were targeted with five different positions in the nucleus with 50 protons per position. SPICE has been administrated as a ‘Joint-use facility for Collaborative Research’, and thus researchers outside NIRS can apply for beam time of SPICE after their research proposals are approved.Fig. 1. (A) Nuclei of HCT116 cells were targeted with five different positions (3-µm apart), and 50 protons were delivered to each position. (B) Cells were incubated for 1 h and then fixed for immunostaining against γ-H2AX.