Teruaki Konishi

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.

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.

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

Abstract 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.

Proliferation and differentiation of neural stem cells irradiated with X-rays in logarithmic growth phase.

Exposure of the fetal brain to ionizing radiation causes congenital brain abnormalities. Normal brain formation requires regionally and temporally appropriate proliferation and differentiation of neural stem cells (NSCs) into neurons and glia. Here, we investigated the effects of X-irradiation on proliferating homogenous NSCs prepared from mouse ES cells. Cells irradiated with X-rays at a dose of 1Gy maintained the capabilities for proliferation and differentiation but stopped proliferation temporarily. In contrast, the cells ceased proliferation following irradiation at a dose of >5Gy. These results suggest that irradiation of the fetal brain at relatively low doses may cause congenital brain abnormalities as with relatively high doses.

The differential role of human macrophage in triggering secondary bystander effects after either gamma-ray or carbon beam irradiation

The abscopal effect could be an underlying factor in evaluating prognosis of radiotherapy. This study established an in vitro system to examine whether tumor-generated bystander signals could be transmitted by macrophages to further trigger secondary cellular responses after different irradiations, where human lung cancer NCI-H446 cells were irradiated with either γ-rays or carbon ions and co-cultured with human macrophage U937 cells, then these U937 cells were used as a bystander signal transmitter and co-cultured with human bronchial epithelial cells BEAS-2B. Results showed that U937 cells were only activated by γ-irradiated NCI-H446 cells so that the secondary injuries in BEAS-2B cells under carbon ion irradiation were weaker than γ-rays. Both TNF-α and IL-1α were involved in the γ-irradiation induced secondary bystander effect but only TNF-α contributed to the carbon ion induced response. Further assay disclosed that IL-1α but not TNF-α was largely responsible for the activation of macrophages and the formation of micronucleus in BEAS-2B cells. These data suggest that macrophages could transfer secondary bystander signals and play a key role in the secondary bystander effect of photon irradiation, while carbon ion irradiation has conspicuous advantage due to its reduced secondary injury.

Single particle irradiation system to cell (SPICE) at NIRS

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 4 He 2þ ). For our primary goal, ‘‘irradiation of cell organelle with a single particle with a position resolution of 2 l mi n a reasonable irradiation time’’, special features are considered. Usage of a triplet Q-magnet for focussing the beam to micrometer levels is an outstanding 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 organelle (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.

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

Applicability of Polyimide Films as Etched-Track Detectors for Ultra-Heavy Cosmic Ray Components

The track registration property in polyimide Kapton has been examined for heavy ions, including 2.3 GeV Fe and 24 GeV Xe ions. Conventional track formation criteria fail to predict the thresholds of etch pit formation, while a chemical criterion stating that etchable tracks are formed when two adjacent diphenyl ethers are broken in the vicinity of the ions trajectory should be more appropriate. Discriminative detections of ultra-heavy components in cosmic rays, such as Bi, Th, and U ions, are possible by measuring the recorded track length.

Thresholds of Etchable Track Formation and Chemical Damage Parameters in Poly(ethylene terephthalate), Bisphenol A polycarbonate, and Poly(allyl diglycol carbonate) Films at the Stopping Powers Ranging from 10 to 12,000 keV/µm

The damage structure of latent tracks in poly(ethylene terephthalate) (PET) has been examined by Fourier transform infrared (FT-IR) measurements. Results are compared with those from previous studies on bisphenol A polycarbonate (PC) and poly(allyl diglycol carbonate) (PADC). These polymers are exposed to protons and heavy ions (He, C, Ne, Si, Ar, Fe, Kr, and Xe) in air with energies less than 6 MeV/n, as well as gamma rays from an intense Co-60 source. Chemical damage parameters, namely, damage density, which is the number of losses of considered functional groups per unit length of tracks, radial size of the track core, in which the considered chemical groups are lost, and radiation chemical yields (G values) for each group are evaluated as a function of the stopping power. It has been confirmed that latent tracks will be etchable when the radial track core size is larger than the distance between two adjacent breaking points of polymer chains. The predominant breaking points are the C–O bonds in ether, ester, and carbonate ester bonds.