Bystander Effect between Zebrafish Embryos in Vivo Induced by High-Dose X-rays

Abstract

ABSTRACT:We employed embryos of the zebrafish, Danio rerio, for our studies on the in vivo bystander effect between embryos irradiated with highdose 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 endlabeling (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.

Embryos of the zebrafish Danio rerio in studies of non-targeted effects of ionizing radiation

“…Choi et al [232] investigated the effect of carbon monoxide (CO) on the induction of the RIBE. More recently, the roles of NO and CO on the induction of the RIBE by high-dose X-ray irradiation in zebrafish embryos were also reported by Choi et al [52]. Considering the zebrafish embryo model as a good tumor model, the above results might be able to provide valuable information for Table 2 Key findings of non-targeted effects of ionizing radiation obtained using embryos of the zebrafish Danio rerio.…”

“…It would also be interesting and beneficial to study whether the CF can be transmitted at the inter-organismic level. Recently, there has been massive evidence of the RIBE occurring at the inter-organismic level, e.g., between mice [50,51], zebrafish [52] and medaka [53].…”

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

“…Interestingly, previous research suggested that scavenging NO with cPTIO could suppress RIBE in the bystander zebrafish embryos [12]. Taken together, both scavenging NO from bystander embryos and adding exogenous NO to bystander embryos were anti-apoptotic.…”

“…In a previous in vivo study [12], we examined the influence of NO on the bystander effect between embryos of the zebrafish, Danio rerio , irradiated with high-dose X-rays and naive unirradiated embryos. We demonstrated that the RIBE between partnered irradiated and naïve unirradiated zebrafish embryos was suppressed through the addition of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) into the medium [12].…”

“…We demonstrated that the RIBE between partnered irradiated and naïve unirradiated zebrafish embryos was suppressed through the addition of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) into the medium [12]. As RIBE was also induced in zebrafish naïve embryos introduced into the irradiated embryo conditioned medium (IECM) alone, i.e., without directly partnering with the irradiated embryos, and in view of the short life of NO in the IECM, NO should have been involved in the generation of bystander signals in irradiated embryos, or in generation of bystander response (in terms of apoptosis) in naïve unirradiated embryos upon receiving bystander signals, or both [12]. In relation to this, previous in vitro studies by other groups also demonstrated that pretreatment with c-PTIO could eliminate the RIBE [13,14,15].…”