Dose-dependent changes after proton and photon irradiation in zebrafish model

Purpose/Objective: The laser-driven ionizing (LDI) beams have unique property of ultra-high dose rate, ultra-short pulses and carry the potential toward special clinical application. Our aim was to establish an in vivo zebrafish model for radiobiological research on later LDI radiation.
Material/methods: 24 hours post-fertilization (hpf) zebrafish (Danio rerio) embryos were irradiated at the University Proton Therapy Dresden with escalated doses (5, 10, 15, 20 and 30 Gy) at two positions along the proton depth-dose curve, at the plateau and at the middle of Spread Out Bragg Peak, and with reference 6 MV photon beams from a clinical linac (n=96 in each group). The experiment was 3 times repeated under the same conditions. On the 3th (96 hpf) and 4th (120 hpf) days after irradiation morphological malformations were documented (photo) and determined quantitatively. Two independent observers measured the length of the embryos, the degree of the yolk sac edema and the diameter of the eyes. Additionally, we have detected the DNA double-strand breaks immunohistochemically (gamma-H2AX foci) after 30 min of the irradiation at the two positions of the proton (mSOBP and plateau) and photon beams, at 5 Gy dose level.
Results: Dose-dependent organ developmental deteriorations could be detected morphologically at >10 Gy dose levels. The length of the embryo and the size of the eyes reduced, while the yolk sac edema increased significantly in dose dependent degree after 10 Gy, 15 Gy, 20 Gy and 30 Gy irradiation, at both developmental stages. At 5 Gy dose irradiation we have found significant elevation in the number of DNA double-strand breaks, as compared to the unirradiated control groups. Furthermore, data showed that after proton irradiation the degree of the DNA damage was higher, as compared to the photon irradiation.
Conclusion: We could establish a reliable quantitative morphological analysis of dose-dependent organ malformations using an in vivo vertebrate system. The zebrafish embryo model proved to be appropriate for complex evaluation of the irradiation-caused damages, molecular changes and for comparison of the biological effects of different radiation qualities. We could define the optimal parameters for future radiobiological experiments with the LDI beams.
Supported by: The ELI-ALPS project (GINOP-2.3.6-15-2015-00001) is supported by the European Union and co-financed by the European Regional Development Fund. The project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no 654148 Laserlab-Europe and by the German BMBF, grant no. 03Z1N511.