摘要:
为探讨重金属Cd对斑马鱼胚胎发育的毒性效应,将受精1 h后(1 hpf)的斑马鱼胚胎暴露于不同浓度的CdCl2溶液中,观察CdCl2处理对胚胎死亡、孵化及幼鱼畸形的影响。采用吖啶橙(AO)染色,定性观察胚胎细胞凋亡情况;以活性氧(ROS)荧光探针DCFH-DA染色法检测胚胎ROS水平,TBA比色法测定胚胎脂质过氧化水平,DTNB比色法测定还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)水平。结果表明,10.0~30.0 mg·L-1 CdCl2浓度依赖性地诱导斑马鱼胚胎死亡和幼鱼畸形,胚胎孵化率亦降低。CdCl2处理引起斑马鱼胚胎心脏水肿,尾部弯曲和胚胎发育阻滞。胚胎半数致死浓度(LC50)为18.9 mg· L-1,R2=0.973,幼鱼半数致畸浓度(EC50)为13.7 mg·L-1,R2=0.967。20.0 mg·L-1 CdCl2处理组ROS水平、MDA含量明显升高,GSH/GSSG比值明显降低(P < 0.01)。20 mg·L-1 CdCl2处理后,胚胎头部和尾部可见大量细胞凋亡。10 mg·L-1 N-乙酰半胱氨酸(NAC)与20 mg·L-1 CdCl2共同处理组斑马鱼胚胎的死亡率和畸形率明显降低,孵化率明显升高,ROS水平、MDA含量以及GSH/GSSG比值趋于正常。以上结果说明,CdCl2暴露对斑马鱼胚胎发育的毒性效应可能与CdCl2诱导的氧化应激相关。
Abstract:
To investigate the developmental toxicology of CdCl2 to the embryonic stage of zebrafish. Zebrafish embryos (1 hour post fertilization, 1 hpf) were exposed to different concentrations of CdCl2. The survival and hatching of embryos and juvenile malformations were examined. Cell apoptosis in embryos was observed by the staining of acridine orange (AO). Embryonic reactive oxygen species (ROS) level was measured by fluorescent probe of DCFH-DA. Lipid peroxidation degree in embryos was measured by the TBA colorimetric method, as well as levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were assayed by DTNB colorimetric method. Results showed that exposure to CdCl2 for 96 hours could cause severe lethality of embryo, malformation of juvenile and the failure of hatching in a concentration-dependent manner. CdCl2 treatment caused malformation of heart, tail and growth retardation in embryos. The half lethal concentration (LC50) of CdCl2 on zebrafish embryos was 18.9 mg·L-1(R2=0.973), and the half effective concentration (EC50) with respect to malformation was 13.7 mg·L-1 (R2=0.967). ROS and MDA levels in zebrafish embryos increased significantly at exposure concentration of 20 mg·L-1 or above (P < 0.01) with decreased ratio of GSH to GSSG. Cell apoptosis in the head and tail could be observed obviously after exposure. When an anti-oxidative reagent N -acetyl cysteine (NAC, 10 mg·L-1) and CdCl2 (20 mg·L-1) were co-existed, the lethal and teratogenic effects ameliorated significantly, and levels of ROS, MDA and the ratio of GSH to GSSG due to exposure of CdCl2 resumed to normal. Therefore, it can be concluded that exposure of cadmium chloride could cause lesions in the developmental stage of zebrafish embryos, which probably resulted from the oxidative stress.
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