Dynamics in zebrafish development define transcriptomic specificity after angiogenesis inhibitor exposure

Chemical exposure during embryonic development can pose significant risks potentially leading to birth defects. Testing of chemicals regarding their developmental toxicity potential requires huge efforts, animals, and costs. For reduction of those, the zebrafish embryo might be applied, however, suitability of the respective OECD test with regard to specificity needs to be confirmed. The OECD TG 236 Fish Embryo Acute Toxicity Test recommends testing within the 0-96 hours post-fertilization (hpf) window. Although this timeframe captures critical stages of organogenesis and vascular development, a more time resolved exposure scenario is needed to elucidate the dynamic influence of developmental toxicants. In this study, we investigated the effects on the zebrafish transcriptome after exposure to putative vascular disruptor compounds, the tyrosine kinase inhibitors, sorafenib and SU4312, and the putative vascular disruptor compound Rotenone. We used an early (0 hpf) and a late (24 hpf) exposure start to determine specificity and sensitivity of the developing zebrafish embryo model. In both exposure scenarios we measured changes on the zebrafish transcriptome at multiple time points and developed an analysis pipeline for the detection and evaluation of differentially expressed genes (DEGs) in this time series data based on generalized additive models (GAMs). We observed mainly unspecific developmental toxicity in the early exposure scenario, while a specific repression of vascular related genes was only partially observed. In contrast, differential expression of vascular-related genes could be identified clearly during with the late exposure scenario. Finally, we compared the toxicodynamic responses with the toxicokinetics of the compounds and show the influence of the internal dose kinetics on the gene expression patterns in zebrafish embryo.

胚胎发育阶段的化学物质暴露会带来显著风险，甚至可能引发出生缺陷。针对化学物质发育毒性潜力的检测工作需要耗费大量人力、动物实验资源与成本。为减少此类投入，可采用斑马鱼胚胎作为实验模型，但需验证对应经济合作与发展组织（Organisation for Economic Co-operation and Development，简称OECD）测试方法的特异性是否符合要求。经济合作与发展组织TG 236《鱼类胚胎急性毒性测试》建议在受精后0至96小时（hours post-fertilization，简称hpf）的窗口内开展测试。尽管该时间窗口覆盖了器官发生与血管发育的关键阶段，但仍需采用更高时间分辨率的暴露场景，以阐明发育性毒物的动态影响。本研究中，我们针对暴露于推定血管干扰物后的斑马鱼转录组变化展开了研究，所涉化合物包括酪氨酸激酶抑制剂索拉非尼（sorafenib）与SU4312，以及推定血管干扰物鱼藤酮（Rotenone）。我们分别设置早期（受精后0小时，即0 hpf）与晚期（受精后24小时，即24 hpf）两个暴露起始时间点，以评估发育中的斑马鱼胚胎模型的特异性与灵敏度。在两种暴露场景下，我们均在多个时间点检测了斑马鱼的转录组变化，并基于广义相加模型（generalized additive models，简称GAMs）开发了一套分析流程，用于该时间序列数据中差异表达基因（differentially expressed genes，简称DEGs）的检测与评估。在早期暴露场景中，我们主要观测到非特异性发育毒性，而血管相关基因的特异性抑制仅部分出现。与之相反，在晚期暴露场景中，我们可清晰检测到血管相关基因的差异表达情况。最后，我们将化合物的毒代动力学与毒效动力学响应进行了对比，并阐明了体内剂量动力学对斑马鱼胚胎基因表达模式的影响。