溴敌隆对斑马鱼早期发育阶段的神经毒性作用

Neurodevelopmental toxicity of bromadiolone to early-staged zebrafish

[背景]溴敌隆是国内外广泛使用的第二代抗凝血类杀鼠剂,在生命早期暴露会导致神经发育毒性,但对于神经发育的毒性机制作用尚不明确。[目的]探究溴敌隆暴露导致斑马鱼胚胎神经发育毒性及可能的作用机制。[方法]将斑马鱼胚胎随机分为4组,分别为对照组(二甲基亚砜)以及0.39、0.78、1.18 mg·L^(−1)溴敌隆暴露组。暴露时间为受精后4~120 h。暴露至24 h记录每分钟内斑马鱼胚胎自主抽动次数,至120 h记录斑马鱼幼鱼120 min内的运动情况,检测幼鱼体内的乙酰胆碱酯酶(AChE)活性,通过荧光定量PCR方法检测神经发育相关基因(elavl3、gap43、mbp、syn2a)表达情况。[结果]与对照组相比,24 h时1.18 mg·L^(−1)溴敌隆暴露组的斑马鱼胚胎自主抽动次数下降40%(P<0.05)。120 h时,当溴敌隆暴露浓度达到0.78、1.18 mg·L^(−1)时,运动总距离相较于对照组分别下降60%、69%(P<0.05、P<0.01),运动总时间相较于对照组分别下降34%、65%(P<0.05、P<0.01)。与对照组相比,1.18 mg·L^(−1)暴露组AChE活性升高36%(P<0.05)。荧光定量PCR测试结果表明:与对照组相比,神经发育关键基因elavl3、syn2a和mbp的基因表达在1.18 mg·L^(−1)暴露组分别下调66%、69%、65%(P<0.01),gap43基因表达在溴敌隆浓度为0.78 mg·L^(−1)时上调56%(P<0.01),在浓度为1.18 mg·L^(−1)时,表达量下调34%(P<0.05)。[结论]溴敌隆暴露抑制斑马鱼胚胎自主抽动、运动行为能力,斑马鱼神经发育相关基因表达下调,阻碍神经传导递质释放,从而对斑马鱼产生神经发育毒性作用。

[Background]Bromadiolone is the second-generation anticoagulant rodenticide widely used all over the world.Exposure to bromadiolone in early life stage can lead to neurodevelopmental toxicity,but its toxic mechanism of neurodevelopment is not clear so far.[Objective]To investigate the developmental neurotoxicity and mechanism of bromadiolone to zebrafish embryos.[Methods]Zebrafish embryos were randomly divided into four groups:a solvent control group(dimethylsulphoxide)and three bromadiolone exposure groups(0.39,0.78,and 1.18 mg·L^(−1)).The exposure period was from 4 h to 120 h post-fertilization.The number of spontaneous movement per minute was recorded at 24 h post-treatment.The locomotor ability of zebrafish larvae and the activity of acetylcholinesterase(AChE)were tested at 120 h post-treatment.The relative expression levels of neurodevelopment-related genes(elavl3,gap43,mbp,and syn2a)were measured by fluorescence quantitative PCR.[Results]Compared with the control group,the number of spontaneous movement per minute at 24 h decreased significantly in the 1.18 mg·L^(−1)bromadiolone exposure group(P<0.05).Compared with the control group,the total distance travelled of the zebrafish larvae in the 0.78 and 1.18 mg·L^(−1)bromadiolone exposure groups decreased by 60%and 69%respectively(P<0.05,P<0.01),and the total movement time decreased by 34%and 65%respectively(P<0.05,P<0.01).The AChE activity in the 1.18 mg·L^(−1)bromadiolone exposure group increased by 36%when compared with the control group(P<0.05).The fluorescence quantitative PCR results showed that compared with the control group,the expression levels of neurodevelopment-related genes elavl3,syn2a,and mbp were significantly down-regulated by 66%,69%,and 65%in the 1.18 mg·L^(−1)bromadiolone exposure group respectively(P<0.01),the expression level of gap43 was up-regulated by 56%in the 0.78 mg·L^(−1)bromadiolone exposure group(P<0.01)and down-regulated by 34%in the 1.18 mg·L^(−1)bromadiolone exposure group(P<0.05).[Conclusion]Bromadiolone exposure could inhibit spontaneous movement and locomotive behavior,down-regulate the expression levels of neurodevelopment-related genes,hinder the release of neurotransmitters,and result in neurodevelopmental toxicity in the earlystaged zebrafish.