Abstract：
Zebrafish embryo assays are used by pharmaceutical and chemical companies as new approach methodologies (NAMs) in developmental toxicity screening. Despite an overall high concordance of zebrafish embryo assays with in vivo mammalian studies, false negative and false positive results have been reported. False negative results in risk assessment models are of particular concern for human safety, as developmental anomalies may be missed. Interestingly, for several chemicals and drugs that were reported to be false negative in zebrafish, skeletal findings were noted in the in vivo studies. As the number of skeletal endpoints assessed in zebrafish is very limited compared to the in vivo mammalian studies, the aim of this study was to investigate whether the sensitivity could be increased by including a skeletal staining method. Three staining methods were tested on zebrafish embryos that were exposed to four teratogens that caused skeletal anomalies in rats and/or rabbits and were false negative in zebrafish embryo assays. These methods included a fixed alizarin red-alcian blue staining, a calcein staining, and a live alizarin red staining. The results showed a high variability in staining intensity of larvae exposed to mammalian skeletal teratogens, as well as variability between control larvae originating from the same clutch of zebrafish. Hence, biological variability in (onset of) bone development in zebrafish hampers the detection of (subtle) treatment-related bone effects that are not picked-up by gross morphology. In conclusion, the used skeletal staining methods did not increase the sensitivity of zebrafish embryo developmental toxicity assays.
摘要：
制药和化学公司将斑马鱼胚胎测定法用作发育毒性筛选中的新方法方法（NAM）。尽管斑马鱼胚胎试验与体内哺乳动物研究总体高度一致，已经报道了假阴性和假阳性结果。风险评估模型中的假阴性结果对人类安全特别关注，因为发育异常可能会被遗漏。有趣的是,在斑马鱼中被报道为假阴性的几种化学物质和药物，在体内研究中注意到骨骼发现。由于与体内哺乳动物研究相比，斑马鱼评估的骨骼终点数量非常有限，这项研究的目的是研究是否可以通过包括骨骼染色方法来提高灵敏度。在斑马鱼胚胎上测试了三种染色方法，这些胚胎暴露于四种致畸剂，这些致畸剂导致大鼠和/或兔子的骨骼异常，并且在斑马鱼胚胎试验中呈假阴性。这些方法包括固定的茜素红-阿尔辛蓝染色，钙黄绿素染色，和鲜活的茜素红染色。结果表明，暴露于哺乳动物骨骼致畸剂的幼虫的染色强度具有很高的变异性，以及源自斑马鱼相同离合器的对照幼虫之间的可变性。因此，斑马鱼骨骼发育（开始）的生物学变异性阻碍了对（微妙的）与治疗相关的骨骼影响的检测，而总体形态学并未发现这些影响。总之,使用的骨骼染色方法没有增加斑马鱼胚胎发育毒性试验的敏感性。
