Dynamics of Chromosome Aberrations and Cell Death in Zebrafish Embryos Exposed to 137Cs Total-Body Irradiation.

Ionizing radiation exposure induces significant DNA damage and cell death in aquatic species. Accurate sensing and quantification play pivotal roles in environmental monitoring and surveillance. Zebrafish (Danio rerio) is a well-suited animal model for research into this aspect, especially with recent development of cytogenetic and transgenic tools. In this study, we present time-course studies of chromosome aberrations and cell death in zebrafish embryos exposed to 2 Gy 137Cs total-body irradiation. Using a cytogenetic approach, we quantified chromosome and chromatid aberrations in irradiated embryos at 6, 14, 20, and 24 h postirradiation. Metaphases with aberrations showed rapid declining kinetics, accompanied by incomplete karyotypes and irregular chromatin contents. Using an apoptosis-reporting transgenic zebrafish, we found increasing cell death along these time points, with the embryonic eyes and brain contributing the majority of the cell death volumes. We provide evidence that self-proliferating progenitor cells form the underlying linkage between the two kinetics and their positions define radiosensitive niches in zebrafish embryos. Our results provide detailed chromosome aberration and cell death dynamics in 137Cs-irradiated zebrafish embryos and unveil the appropriate timeline and tissue positions for accurate sensing and quantification of radiation-induced damages in zebrafish embryos.

Imaging the radioprotective effect of amifostine in the developing brain using an apoptosis-reporting transgenic zebrafish.

PURPOSE: Normal tissue radioprotectants alleviate radiation-induced damages and preserve critical organ functions. Investigating their efficacy in vivo remains challenging, especially in enclosed organs like the brain. An animal model that enables direct visualization of radiation-induced apoptosis while possessing the structural complexity of a vertebrate brain facilitates these studies in a precise and effective manner. MATERIALS AND METHODS: We employed a secA5 transgenic zebrafish expressing secreted Annexin V fused with a yellow fluorescent protein to visualize radiation-induced apoptosis in vivo. We developed a semi-automated imaging method for standardized acquisition of apoptosis signals in batches of zebrafish larvae. Using these approaches, we studied the protective effect of amifostine (WR-2721) in the irradiated zebrafish larval brain. RESULTS: Upon 2 Gy total-body 137Cs irradiation, increased apoptosis could be visualized at high resolution in the secA5 brain at 2, 24, and 48 hour post irradiation (hpi). Amifostine treatment (4 mM) during irradiation reduced apoptosis significantly at 24 hpi and preserved Wnt active cells in the larval brain. When the 2 Gy irradiation was delivered in combination with cisplatin treatment (0.1 mM), the radioprotective effect of amifostine was also observed. CONCLUSIONS: Our study reveals the radioprotective effect of amifostine in the developing zebrafish larval brain, and highlights the utility of secA5 transgenic zebrafish as a novel system for investigating normal tissue radioprotectants in vivo.

In Vivo Imaging of Radiation-Induced Apoptosis at Single-Cell Resolution in Transgenic Zebrafish Embryos.

Among the various types of cell death induced by ionizing radiation, apoptosis is a highly regulated and well-characterized form. Investigating radiation-induced apoptosis in an intact organism offers advantages in capturing the dynamics of apoptosis under preserved physiology, although high resolution imaging remains challenging. Owing to their optical transparency and genetic amenability, zebrafish is an ideal animal model for research into this aspect. In this study, we present a secA5 transgenic zebrafish expressing genetically encoded secreted ANNEXIN V fused with mVenus, a yellow fluorescent protein that enables reporting of radiation-induced apoptosis. Using in vivo imaging approach, we show that after 2 Gy total-body irradiation, apoptosis could be visualized at single-cell resolution in different cell types throughout the embryo. Elevated apoptosis could be imaged and quantified in the neuroepithelium of the embryonic brain, as well as the proliferative zone and parenchyma of the larval brain. In addition, clearance of apoptotic cells by microglia, the professional phagocytes residing in the brain, could be imaged at single-cell resolution in irradiated larvae. These results establish transgenic secA5 zebrafish as a useful and versatile in vivo system for investigating the dynamic process of radiation-induced apoptosis.

Metaphase-Based Cytogenetic Approach Identifies Radiation-Induced Chromosome and Chromatid Aberrations in Zebrafish Embryos.

Metaphase-based cytogenetic methods based on scoring of chromosome aberrations for the estimation of the radiation dose received provide a powerful approach for evaluating the associated risk upon radiation exposure and form the bulk of our current knowledge of radiation-induced chromosome damages. They mainly rely on inducing quiescent peripheral lymphocytes into proliferation and blocking them at metaphases to quantify the damages at the chromosome level. However, human organs and tissues demonstrate various sensitivity towards radiation and within them, self-proliferating progenitor/stem cells are believed to be the most sensitive populations. The radiation-induced chromosome aberrations in these cells remain largely unknown, especially in the context of an intact living organism. Zebrafish is an ideal animal model for research into this aspect due to their small size and the large quantities of progenitor cells present during the embryonic stages. In this study, we employ a novel metaphase-based cytogenetic approach on zebrafish embryos and demonstrate that chromosome-type and chromatid-type aberrations could be identified in progenitor cells at different cell-cycle stages at the point of radiation exposure. Our work positions zebrafish at the forefront as a useful animal model for studying radiation-induced chromosome structural changes in vivo.

A Simple Method to Establish Metaphase Chromosomes from Individual Zebrafish Embryos.

Cytogenetic approach based on metaphase chromosomes established from dividing cells enables direct microscopic visualization of individual chromosomes, a powerful technique to investigate aneuploidy, chromosome aberrations, and genomic instability. In this study, we describe a simple method based on direct blocking of metaphases in individual zebrafish embryo and dropping slides with temperature changes, water vapor, and acetic acid treatment to increase the metaphase diameters. We demonstrate that well-separated metaphases could be established from single zebrafish embryos using this method. Our method could be further adapted for the analyses of DNA damage, chromosome aberrations, and genomic instability using zebrafish and other teleost models.