V. W. Y. Choi

Radioadaptive Response Induced by Alpha-Particle-Induced Stress Communicated in Vivo between Zebrafish Embryos

We report data demonstrating that zebrafish embryos irradiated by alpha particles can release a stress signal into the water, which can be communicated to the unirradiated zebrafish embryos sharing the same water medium and thereby inducing a radioadaptive response in these unirradiated zebrafish embryos. The effects of radiation on the whole embryos were studied through quantification of apoptotic signals at 24 h post fertilization through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. In these experiments, dechorionated embryos were irradiated and then partnered with two other groups of unirradiated embryos, namely the bystander group (no more further treatments) and adaptive group (subjected to a further challenging dose) of embryos. The adaptive group of embryos were then separately further irradiated with a challenging dose. The results show that the number of apoptotic signals for the adaptive group is smaller than that for the corresponding control group, while that for the bystander group is larger than that for the corresponding control group. These suggest that the stress communicated in vivo between the irradiated zebrafish embryos and those unirradiated embryos sharing the same medium will induce radioadaptive response in the unirradiated embryos.

Effects of alpha particles on zebrafish embryos

Dechorionated zebrafish embryos were irradiated at 1.5 h post fertilization (hpf) to low-doses of alpha particles, viz., 1.4, 2.8, 5.6, 11.2 mGy (determined using Monte Carlo simulations). At 24 hpf, these embryos were then examined for apoptotic cells through acridine orange staining. The mean number of apoptotic cells was found to decrease significantly from controls to 1.4-mGy irradiation, and then to increase almost linearly to 2.8, 5.6 and 11.2-mGy irradiation. This trend is a typical characteristic of a hormetic effect.

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.

Hormetic Effect Induced by Alpha-Particle-Induced Stress Communicated In Vivo between Zebrafish Embryos

We report data showing that embryos of the zebrafish, Danio rerio, at 1.5 h post fertilization (hpf) subjected to a low-dose alpha-particle irradiation can release a stress signal into the water, which can be communicated to unirradiated bystander zebrafish embryos sharing the same water medium to induce a hormetic effect in the bystander embryos. Hormetic responses are characterized as biphasic dose-response relationships exhibiting a low-dose stimulation and a high-dose inhibition. The effects on the whole embryos were studied through quantification of apoptotic signals at 24 hpf through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. The results show that, for low alpha-particle dose, the number of apoptotic signals decreases in the irradiated embryos and also in the unirradiated bystander embryos having partnered with the irradiated embryos. These suggested that alpha-particle-irradiated zebrafish embryos could release a stress signal into the water, which could be communicated to unirradiated bystander zebrafish embryos sharing the same water medium to induce a hormetic effect in the bystander embryos.

Adaptive response to ionising radiation induced by cadmium in zebrafish embryos.

An adaptive response is a biological response where the exposure of cells or animals to a low priming exposure induces mechanisms that protect the cells or animals against the detrimental effects of a subsequent larger challenging exposure. In realistic environmental situations, living organisms can be exposed to a mixture of stressors, and the resultant effects due to such exposures are referred to as multiple stressor effects. In the present work we demonstrated, via quantification of apoptosis in the embryos, that embryos of the zebrafish (Danio rerio) subjected to a priming exposure provided by one environmental stressor (cadmium in micromolar concentrations) could undergo an adaptive response against a subsequent challenging exposure provided by another environmental stressor (alpha particles). We concluded that zebrafish embryos treated with 1 to 10 μM Cd at 5 h postfertilisation (hpf) for both 1 and 5 h could undergo an adaptive response against subsequent ~4.4 mGy alpha-particle irradiation at 10 hpf, which could be interpreted as an antagonistic multiple stressor effect between Cd and ionising radiation. The zebrafish has become a popular vertebrate model for studying the in vivo response to ionising radiation. As such, our results suggested that multiple stressor effects should be carefully considered for human radiation risk assessment since the risk may be perturbed by another environmental stressor such as a heavy metal.

Some properties of the signals involved in unirradiated zebrafish embryos rescuing α-particle irradiated zebrafish embryos

Abstract Purpose: The in vivo radiation-induced bystander effect (RIBE) and radiation-induced rescue effect (RIRE) induced between embryos of the zebrafish (Danio rerio) by alpha-particle irradiation were studied through the number of apoptotic signals revealed at 24 h post fertilization (hpf) through vital dye acridine orange staining. Materials and methods: RIBE and RIRE were verified through the significant increase and decrease in apoptotic signals in the partnered bystander and irradiated embryos, respectively. Results: The medium transfer experiment where irradiated zebrafish embryos were rescued through immersion in the medium previously conditioned by a larger number of irradiated zebrafish embryos showed (a) the involvement of a released stress signal in the induction of RIRE, and (b) RIBE and RIRE signals had the same function. With the help of 500 μM of the specific nitric oxide (NO) scavenger cPTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide), NO was confirmed as an essential signaling molecule for inducing both the RIBE and RIRE. On the other hand, the treatment with 20 μM of the carbon monoxide (CO) releasing chemical CORM-3 (tricarbonylchloro(glycinato)ruthenium (II)) suppressed the manifestations of RIBE but did not suppress RIRE. Conclusions: In conclusion, unirradiated zebrafish embryos need NO but not NO-induced damages to rescue α-particle irradiated zebrafish embryos.

The multiple stressor effect in zebrafish embryos from simultaneous exposure to ionising radiation and cadmium

Living organisms are exposed to a mixture of environmental stressors, and the resultant effects are referred to as multiple stressor effects. In the present work, we studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to ionising radiation (alpha particles) and cadmium through quantification of apoptotic signals at 24 h postfertilisation (hpf) revealed by vital dye acridine orange staining. For each set of experiments, 32-40 dechorionated embryos were deployed, which were divided into four groups each having 8-10 embryos. The four groups of embryos were referred to as (1) the control group (C), which received no further treatments after dechorionation; (2) the Cd-dosed and irradiated group (CdIr), which was exposed to 100 μM Cd from 5 to 24 hpf, and also received about 4.4 mGy from alpha particles at 5 hpf; (3) the irradiated group (Ir), which received about 4.4 mGy from alpha particles at 5 hpf; and (4) the Cd-dosed group (Cd), which was exposed to 100 μM Cd from 5 to 24 hpf. In general, the CdIr, Ir and Cd groups had more apoptotic signals than the C group. Within the 12 sets of experimental results, two showed significant synergistic effects, one showed a weakly synergistic effect and nine showed additive effects. The multiple stressor effect of 100 μM Cd with ~4.4 mGy alpha-particle radiation resulted in an additive or synergistic effect, but no antagonistic effect. The failure to identify significant synergistic effects for some sets of data, and thus their subsequent classification as additive effects, might be a result of the relatively small magnitude of the synergistic effects. The results showed that the radiation risk could be perturbed by another environmental stressor such as a heavy metal, and as such a realistic human radiation risk assessment should in general take into account the multiple stressor effects.

Effects of exogenous carbon monoxide on radiation-induced bystander effect in zebrafish embryos in vivo

In the present work, the influence of a low concentration of exogenous carbon monoxide (CO) liberated from tricarbonylchloro(glycinato)ruthenium (II) (CORM-3) on the radiation induced bystander effect (RIBE) in vivo between embryos of the zebrafish was studied. RIBE was assessed through the number of apoptotic signals revealed on embryos at 25 h post fertilization (hpf). A significant attenuation of apoptosis on the bystander embryos induced by RIBE in a CO concentration dependent manner was observed.

A diamond nanocone array for improved osteoblastic differentiation

Efficient delivery of biomolecules to cells is of great importance in biology and medicine. To achieve this, we designed a novel type of densely packed diamond nanocone array to conveniently transport molecules to the cytoplasm of a great number of cells. The nanocone array was fabricated by depositing a thin layer of diamond film on a silicon substrate followed by bias-assisted reactive ion etching. The height of the diamond nanocones varied from 200 nm to 1 μm with tip radii of approximately 10 nm. Our fluorescein and propidium iodide staining results clearly demonstrated that dramatically enhanced delivery of fluorescein into cells was realized without leading to noticeable cell death with the aid of nanocone treatment. As a test case of the drug delivery application of the device, MC-3T3 cells in differentiation medium were applied to the nanocone array for enhanced intracellular delivery of the medium. This was confirmed by the fact that nanocone treated cells experienced much higher differentiation ability at an early stage in comparison with untreated cells. Overall, the results indicate that the diamond nanocone array provides a very simple but yet very effective approach to achieve delivery of molecules to a large number of cells.

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.