Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression

Ionizing radiation from natural sources or anthropogenic activity has the potential to cause oxidative stress or genetic damage in living organisms, through the ionization and excitation of molecules and the subsequent production of free radicals and reactive oxygen species (ROS). The present work focuses on radiation-induced biological effects using the zebrafish (Danio rerio) vertebrate model. Changes in developmental traits and gene expression in zebrafish were assessed after continuous external gamma irradiation (0.4, 3.9, 15 and 38 mGy/h) with corresponding controls, starting at 2.5 hours post fertilization (hpf) and lasting through embryogenesis and the early larval stage. The lowest dose rate corresponded to recommended benchmarks at which adverse effects are not expected to occur in aquatic ecosystems (2–10 mGy/day). The survival observed at 96 hours post fertilization (hpf) in the 38 mGy/h group was significantly lower, while other groups showed no significant difference compared to controls. The total hatching was significantly lower from controls in the 15 mGy/h group and a delay in hatching onset in the 0.4 mGy/h group was observed. The deformity frequency was significantly increased by prolonged exposure duration at dose rates ≥ 0.4 mGy/h. Molecular responses analyzed by RNA-seq at gastrulation (5.5 hpf transcriptome) indicate that the radiation induced adverse effects occurred during the earliest stages of development. A dose-response relationship was found in the numbers of differentially regulated genes in exposure groups compared to controls at a total dose as low as 1.62 mGy. Ingenuity Pathway Analysis identified retinoic acid receptor activation, apoptosis, and glutathione mediated detoxification signaling as the most affected pathways in the lower dose rate (0.54 mGy/h), while eif2 and mTOR, i.e., involved in the modulation of angiogenesis, were most affected in higher dose rates (5.4 and 10.9 mGy/h). By comparing gene expression data, myc was found to be the most significant upstream regulator, followed by tp53, TNF, hnf4a, TGFb1 and cebpa, while crabp2b and vegfab were identified as most frequent downstream target genes. These genes are associated with various developmental processes. The present findings show that continuous gamma irradiation (≥ 0.54 mGy/h) during early gastrula causes gene expression changes that are linked to developmental defects in zebrafish embryos.

天然来源或人为活动产生的电离辐射，可通过对分子的电离与激发，以及后续自由基与活性氧物种（reactive oxygen species, ROS）的生成，对活生物体造成氧化应激或遗传损伤。本研究以脊椎动物斑马鱼（Danio rerio）为模型，聚焦辐射诱导的生物学效应。本实验于受精后2.5小时（hours post fertilization, hpf）开始，对斑马鱼进行持续体外γ射线辐照（辐照剂量率分别为0.4、3.9、15及38 mGy/h），并设置相应对照组，辐照持续至胚胎发生期与早期幼虫阶段，期间评估斑马鱼的发育性状与基因表达变化。该最低剂量率（0.4 mGy/h）对应水生生态系统未预期出现不良效应的推荐基准值（2–10 mGy/天）。受精后96小时（hpf）的观测结果显示，38 mGy/h组的存活率显著低于对照组，其余各组存活率与对照组无显著差异。15 mGy/h组的总孵化率显著低于对照组，而0.4 mGy/h组则出现孵化起始时间延迟的现象。当剂量率≥0.4 mGy/h时，持续辐照会显著提高畸形发生率。通过原肠胚形成期（5.5 hpf转录组）的RNA测序（RNA-seq）分析分子应答可知，辐射诱导的不良效应早在发育早期阶段就已发生。在总辐照剂量低至1.62 mGy的暴露组与对照组中，差异表达基因的数量呈现出显著的剂量-效应关系。Ingenuity通路分析（Ingenuity Pathway Analysis）显示，低剂量率（0.54 mGy/h）下受影响最显著的通路包括维甲酸受体激活、细胞凋亡以及谷胱甘肽介导的解毒信号通路；而高剂量率（5.4及10.9 mGy/h）下受影响最显著的通路则为涉及血管生成调控的eIF2与雷帕霉素靶蛋白（mTOR）通路。通过比对基因表达数据，本研究发现myc是最显著的上游调控因子，其次为tp53、TNF、hnf4a、TGFb1及cebpa；而crabp2b与vegfab则被鉴定为最常见的下游靶基因，这些基因与多种发育过程相关。本研究结果表明，原肠胚早期阶段的持续γ射线辐照（剂量率≥0.54 mGy/h）会引发与斑马鱼胚胎发育缺陷相关的基因表达变化。