氯丙嗪在斑马鱼胚胎和早期幼鱼发育过程中的神经毒性作用

Developmental Neurotoxicity Effect of Chlorpromazine on Zebrafish Embryo and Larvae

目的采用模式动物斑马鱼作为研究对象,观察氯丙嗪(chlorpromazine,CPZ)暴露对胚胎和幼鱼早期神经发育的影响。方法在一般毒性评价的基础上,通过整体胚胎细胞凋亡检测和脑组织病理学检查,了解CPZ对神经发育的器质性改变;采用神经行为学方法,包括幼鱼触动逃避反应、自发运动以及惊恐逃避反射等,研究氯丙嗪暴露所致的神经发育功能性障碍。结果斑马鱼胚胎受精后6 h(6 hpf)～72 hpf暴露于CPZ(≥5 mg/L)可引起胚胎和幼鱼死亡、致畸和幼鱼孵化延迟,并呈浓度和时间依赖性;采用吖啶橙染色检测36 hpf整体胚胎凋亡细胞,发现凋亡细胞主要集中在胚胎中脑、后脑、丘脑以及中后脑连接区、脊索和尾部等处;脑组织病理学检测发现,7dpf幼鱼颅腔增大、脑体积减小、脑细胞缩小且细胞间隙增宽。6～72 hpf CPZ(≥0.0625 mg/L)暴露后,幼鱼神经行为学研究发现,CPZ(≥0.125 mg/L)可引起3dpf幼鱼触觉运动能力下降;CPZ(≥0.5 mg/L)可浓度依赖性地抑制幼鱼自发运动,并出现僵直不动、震颤或快速刻板式转圈运动等行为改变;光惊恐实验中,暗环境下各暴露组幼鱼对突发强光刺激均表现为惊跳逃避,并且暗-光交替期运动加速度变化与对照组无显著差异;在撤除光源后,1mg/L和2 mg/L暴露组幼鱼暗适应时程缩短,而0.125 mg/L和0.25 mg/L暴露组暗适应时程延长,提示CPZ对外界刺激引发的幼鱼活跃游动有抑制和促进双重毒性作用。结论 CPZ暴露对斑马鱼胚胎和幼鱼具有明显的神经发育毒性作用。模式动物斑马鱼作为一种高通量筛选模型在外源性化合物神经发育毒性评价中具有较好的应用前景。

Objective To realize the potentially developmental neurotoxieity of ehlorpromazine （CPZ） on zebrafish embryos and Larvae. Methods The effect of the general toxicity in zebrafish embryos and larvae were assessed after high doses of CPZ （≥5 mg/L） administered to zebrafish embryos on 6- 72 hours post fertilization （hpf）. Then, pathology change and cell apoptosis were investigated. Under teratogenie dosage of CPZ （ ≤ 2. 0 mg/L） administered to zebrafish embryos on 6 - 72 hpf, the escape times of 3 dpf larvae in the touched -evoke escape reaction was recorded. Moreover, spontaneous movement and light-evoked startle escape response of 6 dpf zebrafish were tracked and analyzed by using a video-tracking system. Results The lethal and teratogenic effects of CPZ was dose-dependent and stage- dependent. Histopathological examination revealed that brain volume and brain cells of 7 dpf larvae have reduced in size. The number of apoptotic cells of 36 hpf embryos has increased in midbrain, hindbrain, hypothalamus, midbrain hindbrain boundary, notochord as well as tail. The weaker tactile sensitivity was displayed when 3 dpf larvae were exposured to CPZ （ ≥0. 125 mg/L）. Hypoactivity was discovered when 6 dpf larvae were exposured to CPZ （ ≥0.5 mg/L）. Furthermore, CPZ also evokes tremor, freezing or stereotypic circling swimming in 6 dpf larvae. CPZ （1.0 mg/L or 2. 0 mg/L） can inhibit but CPZ （0. 125 mg/L or 0.25 rag/L） can promote hyperactivity by light-evoked startle reaction. Conclusion CPZ can cause developmental neurotoxicity on zebrafish embryos and larvae. In addition, zebrafish larvae have obvious advantages to make it a powerful alternative for the developmental neurotoxicity assessment of exogenous compounds by using high-throughput behavioral test methods.