Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos
Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos
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DOI：
10.3390/ijms20071712
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发表时间：
2019-04-01
影响因子：
5.6
通讯作者：
de Witte, Peter
中科院分区：
文献类型：
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作者：
Giusti, Arianna;Xuan-Bac Nguyen;de Witte, Peter
基于斑马鱼的平台最近成为毒性测试的有用工具，因为它们结合了体外和体内方法的优点。然而，据推测，斑马鱼胚胎代谢转化外源药物的能力很低。为了避免这一问题，我们开发了一种综合的方法，其中测试化合物（即对硫磷、马拉硫磷和氯霉素）首先在体外暴露于大鼠肝微粒体（RLM）中，在37℃下暴露1小时。加入甲醇后，对混合物进行超声波处理，在-20℃下放置2小时，离心，蒸发上清。将颗粒重悬于水中，采用超高效液相色谱-紫外-质谱（UHPLC-UV-MS）测定代谢转化和代谢物的存在。接下来，将受精后3天（dpf）的斑马鱼幼鳗胚胎暴露在丹尼氏培养基中稀释的代谢混合物中，在28℃下放置48小时，然后进行体视显微镜检查，看看是否有任何不良反应。该方法的新颖之处在于可以量化母体化合物的体外代谢率，并将3只dpf幼虫与稀释后的代谢混合物共孵育48小时，而不会产生主要的毒性作用。对硫磷的结果表明，该化合物的毒性潜力的预测能力有所提高。
Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 degrees C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at -20 degrees C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau's medium for 48 h at 28 degrees C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.