药物神经发育毒性比较及筛查模型建立

Comparison of drug neurodevelopmental toxicity and establishment of screening model

目的:为建立药物神经发育毒性筛查模型选择可评价药物毒性的指标。方法:以受精后6 h(6 hpf)的斑马鱼胚胎为实验模型,E3培养液为溶剂对照组,不同药物不同浓度的暴露液为染毒组。计算受精后24,72,96,110 h的死亡率;受精后48,54,60,72,96 h的孵化率;观察受精后6 d(6 dpf)的幼鱼对强光刺激的惊恐逃避反射、黑暗状态下的运动行为及趋触性。结果:对6 hpf的斑马鱼胚胎染毒,氯丙嗪质量浓度>4.0mg·L^-1、丙戊酸钠质量浓度>2.0mg·L^-1可引起斑马鱼胚胎出现浓度依赖性死亡。氯丙嗪染毒时,与对照组、0.25 mg·L^-1染毒组相比,高浓度染毒组斑马鱼幼鱼总移动距离和平均速度均降低,且具有显著性差异(P<0.05);药物丙戊酸钠染毒时,与对照组相比,随浓度增加呈现移动距离和平均速度先增后降趋势,高浓度组2.0mg·L^-1移动距离和平均速度明显降低,且具有显著性差异(P<0.05)。氯丙嗪和丙戊酸钠染毒时,中间1 min光照期与前3 min钟黑暗期相比,运动平均速度均有所下降,具有显著性差异(P<0.05);后3 min黑暗期与中间1 min光照期相比,运动平均速度均有所上升,具有显著性差异(P<0.05),且丙戊酸钠染毒时各组后3 min黑暗期运动平均速度均大于前3 min黑暗期。结论:药物氯丙嗪和丙戊酸钠可能导致斑马鱼胚胎发育障碍以及幼鱼神经行为异常,且药物丙戊酸钠毒性大于氯丙嗪。初步认为模式动物斑马鱼作为一种高通量筛选模型,其死亡率、孵化率、半数致死浓度LC50、斑马鱼幼鱼总移动距离和平均速度可作为评价药物神经毒性的指标。

OBJECTIVE To establish a drug neurodevelopmental toxicity screening model and select indicators for evaluating drug toxicity.METHODS Zebrafish embryos at 6 hours after fertilization(6 hpf)were used as experimental models,E3medium as solvent control group,and exposure solution of different concentrations of drugs as exposure group.The mortality at 24,72,96 and 110 hours after fertilization,hatching rate at 48,54,60,72 and 96 hours after fertilization,frightening avoidance reflex to light stimulation,movement behavior and contact tendency in dark state of juvenile fish at 6 days after fertilization were observed.RESULTS The concentration of chlorpromazine>4.0mg·L^-1 and sodium valproate>2.0mg·L^-1 could cause the death of 6 hpf zebrafish embryos in a concentration-dependent manner.Compared with control group and 0.25 mg·L^-1 group,the total moving distance and average speed of juvenile zebrafish in high concentration exposure group decreased significantly(P<0.05),while that in sodium valproate group increased with the increase of concentration(P<0.05).At present,the moving distance and average speed increased first and then decreased.The moving distance and average speed of 2.0mg·L^-1 in high concentration group decreased significantly,and there was statistical difference(P<0.05).When chlorpromazine and sodium valproate were exposed,the average speed of exercise in the middle one-minute light period was lower than that in the first three-minute dark period(P<0.05),and the level of exercise in the last three-minute dark period was lower than that in the middle one-minute light period(P<0.05).The average velocity increased with statistical difference(P<0.05),and the average velocity in the dark period of the last three minutes was higher than that in the first three minutes when sodium valproate was exposed.CONCLUSION Drugs chlorpromazine and sodium valproate may cause embryonic dysplasia and neurobehavioral abnormalities in juvenile zebrafish,and the toxicity of sodium valproate is greater than that of chlorpromazine.It is preliminarily considered that zebrafish,a model animal,can be used as a high-throughput screening model,and its mortality rate,hatching rate,median lethal concentration LC50,total movement distance and average speed of zebrafish juveniles can be used as indicators to evaluate drug neurotoxicity.