斑马鱼胚胎评价5种药物的发育毒性与模型验证

Model validation and evaluation of developmental toxicity of five drugs using zebrafish embryos

目的用斑马鱼胚胎探究环磷酰胺、乙酰水杨酸、盐酸四环素、乙酸地塞米松、阿扎胞苷5种已知对人类胚胎致畸药物的毒性和安全性。方法挑选4 hpf发育正常的受精卵,采用水浴染毒法,将药物添加到人工海水中,每种药物分别设置5个浓度组,另设空白对照组和溶剂对照组,观察给药120 hpf后斑马鱼的死亡情况,统计各实验组斑马鱼胚胎的死亡数、畸形数,并求出120 hpf时斑马鱼胚胎的死亡率、畸形率、半数致死浓度(LC50)、半数致畸浓度(EC50)、致畸指数(TI)。并利用公式:TI=LC50/EC50计算出阳性药物的致畸指数。根据已经测得的LC50,求出各药物的最大非致死浓度(MNLC),分别设置1/10 MNLC、1/3 MNLC,MNLC和LC104个浓度,以沙利度胺为阳性对照,维生素C为阴性对照,人工海水为空白对照,0.5%DMSO为溶剂对照,28.5℃下作用至120 h,每天观察胚胎的发育情况,统计胚胎死亡及畸形状态。结果 5种药物的LC50从大到小依次为:环磷酰胺>阿扎胞苷>盐酸四环素>乙酰水杨酸>乙酸地塞米松。EC50从大到小依次为:环磷酰胺>盐酸四环素>阿扎胞苷>乙酰水杨酸>乙酸地塞米松。环磷酰胺、乙酰水杨酸、盐酸四环素、乙酸地塞米松、阿扎胞苷TI值分别为1.92,1.11,1.05,1.44,2.99。结论斑马鱼胚胎模型可用于初步评价药物的发育毒性和安全性。

Aim To explore the toxicity and safety of five kinds of known positive drugs,cyclophosphamide,acetyl salicylic acid,tetracycline hydrochloride,dexamethasone acetate and azacitidine,using zebrafish embryos. Methods We selected normally developed 4hpf zygote,and used water bath infecting method to add the drug to the artificial seawater. Each drug had five concentrating groups,a separate control group and solvent control group. We observed the dead zebrafish embryos after 120 hpf drugs,counted the number of deaths and deformities of zebrafish embryos,and calculated mortality abnormal rate,the median lethal concentration( LC50),concentration for 50% of maximal effect( EC50),therapeutic index( TI) under 120 hpf condition. We also used the formula TI = LC50/ EC50 to calculate positive drug therapeutic index. Based on measured LC50 we calculated most nonlethal concentration( MNLC) of each drug setting,namely 1 /10 MNLC,1 /3 MNLC,MNLC,LC10 four concentration,thalidomide as a positive control,vitamin C as a negative control,artificial seawater as control,0. 5% DMSO as solvent control. Put in 28. 5 ℃ environment for 120hours,embryo development was observed daily for de velopmental state,mortality,deforming rate and abnor mal condition. Results The result of five drugs LC50 in descending order: cyclophosphamide > azacitidine> tetracycline hydrochloride > acetylsalicylic acid >dexamethasone acetate. EC50 in descending order: cy clophosphamide > tetracycline hydrochloride > azaciti dine > acetylsalicylic acid > dexamethasone acetate.The TI values of cyclophosphamide,acetyl salicylicacid,tetracycline hydrochloride,dexamethasone acetate, azacitidine were 1. 92, 1. 11, 1. 05, 1. 44,2. 99,respectively. Conclusion Zebrafish embryo model can be used in the preliminary evaluation of drugs,and the study of early developmental toxicity and safety.