Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos

Abstract

The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies.

Keywords: bystander effect; ionizing radiation; nitric oxide; zebrafish embryos.

Figure 1

Comparison between the diaminofluorophore 4-amino-5-methylamino-2′-7′-difluorofluorescein diacetate (DAF-FM DA) fluorescence intensities (in arbitrary units; larger values corresponding to brighter fluorescence) in zebrafish embryos treated with 20 or 100 μM S-nitroso-N-acetylpenicillamine (SNAP) and their corresponding experimental controls treated with 0.02% or 0.1% dimethyl sulfoxide (DMSO), respectively. All experiments were carried out in triplicate on different days, and each set consisted of 15 examined zebrafish embryos. The data were shown as mean DAF-FM DA fluorescence intensities ± standard error of the mean (SEM). Cases with p < 0.05 are asterisked.

Figure 2

Representative images of acridine orange (AO)-stained embryos in different groups. (a) S20 group treated with 20 μM SNAP, and its experimental control D2 group; (b) S100 group treated with 100 μM SNAP, and its experimental control D10 group; (c) IS20 irradiated group treated with 20 μM SNAP, and its experimental control ID2 group; (d) IS100 irradiated group treated with 100 μM SNAP, and its experimental control ID10 group; (e) BS20 bystander group treated with 20 μM SNAP, and its experimental control BD2 group; (f) BS100 bystander group treated with 100 μM SNAP, and its experimental control BD10 group. (a–f) all corresponding control groups also shown. Images were captured using a florescent microscope with 40× magnification. Scale bar: 100 µm.

Figure 2

Representative images of acridine orange (AO)-stained embryos in different groups. (a) S20 group treated with 20 μM SNAP, and its experimental control D2 group; (b) S100 group treated with 100 μM SNAP, and its experimental control D10 group; (c) IS20 irradiated group treated with 20 μM SNAP, and its experimental control ID2 group; (d) IS100 irradiated group treated with 100 μM SNAP, and its experimental control ID10 group; (e) BS20 bystander group treated with 20 μM SNAP, and its experimental control BD2 group; (f) BS100 bystander group treated with 100 μM SNAP, and its experimental control BD10 group. (a–f) all corresponding control groups also shown. Images were captured using a florescent microscope with 40× magnification. Scale bar: 100 µm.

Figure 3

Comparison among different groups of embryos to test the cytotoxicity of SNAP.

Figure 4

Schematic diagram showing the protocols for studying the influence of SNAP on X-ray-induced targeted effects. Embryos were pretreated with 20 or 100 μM SNAP to form the irradiated groups IS20 or IS100, respectively, or pretreated with 0.02% or 0.1% DMSO for 2 h at 3 hpf to form the experimental control groups ID2 or ID10, respectively. After pretreatment, the irradiated groups and the experimental control groups were transferred to new E3 media and were then irradiated with 75 mGy of X-ray. The embryos were then incubated at 28.5 °C until 24 hpf for AO staining and analyses.

Figure 5

Schematic diagram showing the protocols for studying the influence of SNAP on X-ray-induced non-targeted bystander effects. Naïve embryos were pretreated with 20 or 100 μM SNAP to form the bystander groups BS20 or BS100, respectively, or pretreated with 0.02% or 0.1% DMSO for 2 h at 3 hpf to form the experimental control groups BD2 or BD10, respectively. After pretreatment, the bystander groups and the experimental control groups were transferred to new E3 media and were then partnered for 19 h with embryos already irradiated with 75 mGy of X-ray. The embryos were then incubated at 28.5 °C until 24 hpf for AO staining and analyses.