去水卫矛醇对斑马鱼神经发育毒性评价

Evaluation of Neurodevelopmental Toxicity of Dianhydrogalactitol in Zebrafish

关键词:  去水卫矛醇  斑马鱼  神经发育  多巴胺能神经元

Key words:  dianhydrogalactitol  zebrafish  neurodevelopment  dopaminergic neuron

摘要:

目的 探讨去水卫矛醇(dianhydrogalactitol，DAG)诱导斑马鱼胚胎及幼鱼神经发育毒性作用及其机制。方法 在一般毒性评价的基础上，对斑马鱼胚胎进行相应的分组暴露给药，采用幼鱼的自主运动反应、光照刺激反应等试验观察DAG对斑马鱼神经及行为的影响；通过脑部组织病理学检查、吖啶橙染色，观察DAG对斑马鱼脑部的组织影响；利用实时荧光定量PCR方法测定斑马鱼幼鱼体内多巴胺神经元相关基因(DAT、TH、GCH1)及神经抑/促凋亡相关基因(Bax、Bcl-2)的相对表达量。结果 DAG在20，40，75 mg·mL^-1下能明显抑制斑马鱼的自主运动，且自主运动抑制率呈现明显的浓度相关性；DAG在20，40，75 mg·mL^-1下对斑马鱼反应速度有较明显的抑制作用，反应能力下降率呈现浓度相关性；DAG各浓度组斑马鱼脑组织形态变小，但组织结构均未见显著异常；采用吖啶橙染色检测斑马鱼整体胚胎细胞凋亡情况，发现给药组头部绿色荧光比对照组明显，说明细胞凋亡增多，且细胞凋亡呈剂量依赖性增加，与表观一致；DAG在75，150，300，425，600 mg·L^-1下可导致斑马鱼幼鱼多巴胺能神经元相关基因DAT、TH、GCH1的mRNA相对表达量下调，Bax/Bcl-2的RNA相对表达量随着给药浓度增加而上升。结论 高浓度DAG对斑马鱼胚胎和幼鱼具有神经发育毒性作用，可能与多巴胺能神经元的抑制作用有关。

Abstract:

OBJECTIVE To explore dianhydrogalactitol(DAG)-induced neurodevelopmental toxicity and mechanisms in zebrafish embryos and larvae. METHODS On the basis of general toxicity evaluation, zebrafish embryos were exposed to DAG in groups, and the effects of DAG on zebrafish nerves and behaviors were observed by means of autonomic locomotor response and light stimulation response of juvenile fish; the effects of DAG on zebrafish brain tissues were observed by means of histopathological examination of brain and acridine orange staining. The relative expression of dopamine neuron-related genes(DAT, TH, GCH1) and neural inhibition/promotion of apoptosis-related genes(Bax, Bcl-2) was determined by real-time fluorescence quantitative PCR in zebrafish larvae. RESULTS DAG inhibited the voluntary movement of zebrafish under the 20, 40, 75 mg·L^-1, and the inhibition rate of voluntary movement showed obvious concentration correlation; DAG inhibited the reaction speed of zebrafish under the 20, 40, 75 mg·L^-1, and the rate of decrease of reaction ability showed concentration correlation; the brain tissue became smaller, but the tissue structure of zebrafish did not show any significant abnormality in the various concentration groups of DAG; acridine orange staining was used to detect the apoptosis of the overall embryonic cells of zebrafish, and it was found that the administration of DAG inhibited the apoptosis of zebrafish. Acridine orange staining was used to detect apoptosis in the whole embryonic cells of zebrafish, and it was found that the green fluorescence in the head of the administered group was more obvious than that in the control group, indicating that apoptosis increased, and apoptosis increased in a dose-dependent manner, which was in accordance with the apparent observation; DAG in 75, 150, 300, 425, 600 mg·L^-1 of the experimental condition could lead to a downward regulation of the relative expression of mRNA of the genes related to the zebrafish juvenile dopaminergic neuron, DAT, TH and GCH1. The Bax/Bcl-2 relative mRNA expression was up-regulated with increasing drug concentration. CONCLUSION The neurodevelopmental toxicity of DAG in zebrafish embryos and juveniles may be related to the inhibition of dopaminergic neurons.