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.