Effects Of Carbon Monoxide Releasing Molecules On The Development Of Zebrafish Embryos Irradiated By X-ray

Objective: To investigate the developmental effects and possible mechanisms of carbon monoxide release molecules(CORM-3) on zebrafish embryos irradiated by X-ray.Materials and Methods: The 4 hours post fertilization(4hpf) zebrafish embryos that developed normally were exposed to different concentrations of CORM-3 solution(0, 6.25, 12.5, 25, 50, 100, 200 and 400 μmol/l) and cultured to 144 hpf, the effects of CORM-3 on the development of zebrafish zembryos in terms of mortality, hatching rate, malformation, spontaneous movement, heart rate, body length and larval behavior were observed and recorded respectively at different time points.The normal zebrafish embryos of 4hpf were pre-treated by 10 μmol / L CORM-3 for 1h, and then irradiated by 4Gy X-ray, the mortality, hatching rate, malformation rate, spontaneous movement, and larval behavior were measured to evaluate the effect of CORM-3 on development of zebrafish irradiated by X-ray. The level of ROS in zebrafish embryos at 24 hpf was measured by DCFH-DA fluorescence probe. Apoptosis in vivo at 24 hpf were detected by acridine orange(AO) staining and flow cytometry analysis(FCM). The gene expression related to apoptosis were performed by Real-Time fluorescent quantitative analysis(RT-PCR) and the level of protein was decteced by Western blotting(WB).Results:(1) The effects of CORM-3 on the zebrafish embryonic development: Zebrafish embryos exposure to CORM-3 induced a concentration-dependant increase in mortality rate, caused a concentration-dependant decrease spontaneous movement, heart rate as well as body length of zebrafish larvae. The hatching rate was significantly inbibited start from the concentrations of 50 μmol/L CORM-3(P < 0.05). The concentration of CORM-3 higher than 100 μmol/L can lead to zebrafish larvae phenotypic abnormalities, mainly including yolk sac and pericardial edema. In the visible light, the larval behavior test of larvae showed that the total swimming distance of zebrafish exposed to CORM-3 was decreased in a dose-dependent manner, and larvae in 200 and 400 μmol/L group elicited remarkably hypoactivity(P < 0.05).(2) The effect of CORM-3 on the development of zebrafish embryos irradiated by X-ray: X-ray can induce a increase in the rate of mortality and malformation, and cause a drop in hatching rate, spontaneous movement as well as the total swimming distance of zebrafish embryos(P<0.05). CORM-3 pre-treatment can reduce the effect of X-ray on zebrafish embryonic development, showed a lower rate of mortality and malformation than that in the X-ray irradiation group, while the hatching rate, spontaneous movement and total swimming distance was significantly higher than that in the radiation group(P < 0.05).(3) The effect of CORM-3 on the level of reactive oxygen species and activity of SOD in zebrafish embryos induced by X-ray: At 24 hpf, the level of ROS was found to be significantly increased and the activity of SOD was decresed in embryos treated with X-ray irradiation(P < 0.001). CORM-3 pretreatment can promote the increase of SOD, reduce the level of ROS, and alleviate the accumulation of reactive oxygen species in the zebrafish embryos(P < 0.05).(4) Effect of CORM-3 on apoptosis and apoptosis related genes and protein level of zebrafish embryos after X-ray irradiation: Ao staining and Flow cytometry analysis showed that the early apoptotic cells zebrafish embryos in X-ray irradiated group were significantly higher than that of the control group, CORM-3 pretreatment can reduce the apoptosis induced by X-ray. RT-PCR shows that the expression of apoptosis gene Bax, Caspase9, Caspase3 in the X-ray group was significantly higher than that of control group(P < 0.05), wheras anti-apoptotic gene Bcl2 was lower than that of the control group. However, the situation was improved by CORM-3 pretreatment, the expression of Bax, caspase9, Caspase3 were down-regulated, and the expression of Bcl2 was up-regulated. Western Blot shows that CORM-3 pretreatment could alleviate the DNA damage in zebrafish embryos induced by X-ray, the protein level of γ-H2 AX and P53 were decreased, while the Bcl2 was increased.Conclusion:(1) The concentration of CORM-3 higher than 50 μmol/L is toxic on the development of zebrafish embryos; The 10 μmol/L CORM-3 has a potential protective effect on the development of zebrafish irradiated by X ray.(2) The 10 μmol/L CORM-3 can promot the activity of SOD, reduce the level of reactive oxygen species in the body and protect the X-ray injury of zebrafish embryos by anti-oxidative stress reaction.(3) The 10 μmol/L CORM-3 protect the X radiation damage in zebrafish embryos by regulating the expression of apoptosis related genes to produce anti apoptotic effects and promoting the repair of DNA damage.