双酚AP和双酚AF对斑马鱼的早期神经发育毒性作用研究
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摘要
[目的]双酚AP(bisphenol AP,BPAP)和双酚AF(bisphenol AF,BPAF)大规模的生产和使用,对水生生态环境具有潜在威胁。关于BPAP和BPAF的神经毒性研究甚少,本文以斑马鱼作为模式动物,研究BPAP和BPAF对斑马鱼的早期神经发育毒性作用。[方法]本研究选取斑马鱼幼鱼作为实验对象,将斑马鱼胚胎随机分为5组,以BPAP(20μg/L和200μg/L)和BPAF(20μg/L和200μg/L)进行染毒,染毒时间为受精后4 h至受精后144 h。通过斑马鱼的运动行为学测试,原位杂交和荧光定量PCR方法来阐述BPAP和BPAF的早期神经发育毒性作用及其可能的作用机制。[结果]行为学测试结果显示,与对照组相比,染毒组的斑马鱼幼鱼的运动总距离和速度均明显下降,热图显示染毒组幼鱼有过多呆滞不动的现象,表明BPAP、BPAF均抑制了斑马鱼幼鱼的移动距离和速度。荧光定量PCR结果可见,与对照组相比,BPAP(20μg/L和200μg/L)和BPAF(20μg/L和200μg/L)都抑制了斑马鱼神经发育关键基因(mbp和syn2a)的表达(P <0.001),同时原位杂交结果也验证了基因的结果。[结论]初步认为BPAF和BPAP对斑马鱼具有潜在的神经发育毒性作用。
[Objective] Mass production and utilization of bisphenol AP(BPAP) and bisphenol AF(BPAF) has posed potential ecological risk to aquatic ecosystem. However, few studies have focused on the neurotoxicities of BPAP and BPAF. This study investigates the developmental neurotoxicities of BPAP and BPAF in the early life of zebrafish.[Methods] We selected zebrafish larvae as experimental subjects. Zebrafish embryos were randomly divided into five groups. BPAP(20 μg/L and 200 μg/L) and BPAF(20 μg/L and 200 μg/L) were administered with the designed drug exposure profile from 4 h to 144 h after fertilization. The early developmental neurotoxicities of BPAP and BPAF and their possible mechanisms were discussed by locomotion behavior test, in situ hybridization, and fluorescence quantitative PCR.[Results] Compared with the control group, the total moving distance and the speed of the zebrafish larvae in the exposed groups were significantly decreased, and the thermo gram showed that the larvae in the exposed groups were sluggish, indicating that both BPAP and BPAF inhibited the moving distance and speed of zebrafish larvae. The results of fluorescence quantitative PCR showed that compared with the control group, BPAP(20 μg/L and 200 μg/L) and BPAF(20 μg/L and 200 μg/L) significantly inhibited the expressions of key genes(mbp and syn2 a) involved in zebrafish neurodevelopment. The results of in situ hybridization also verified the results of the genes. [Conclusion] The findings tentatively suggest that BPAF and BPAP may have potential developmental neurotoxic effects on zebrafish.
[Objective] Mass production and utilization of bisphenol AP(BPAP) and bisphenol AF(BPAF) has posed potential ecological risk to aquatic ecosystem. However, few studies have focused on the neurotoxicities of BPAP and BPAF. This study investigates the developmental neurotoxicities of BPAP and BPAF in the early life of zebrafish.[Methods] We selected zebrafish larvae as experimental subjects. Zebrafish embryos were randomly divided into five groups. BPAP(20 μg/L and 200 μg/L) and BPAF(20 μg/L and 200 μg/L) were administered with the designed drug exposure profile from 4 h to 144 h after fertilization. The early developmental neurotoxicities of BPAP and BPAF and their possible mechanisms were discussed by locomotion behavior test, in situ hybridization, and fluorescence quantitative PCR.[Results] Compared with the control group, the total moving distance and the speed of the zebrafish larvae in the exposed groups were significantly decreased, and the thermo gram showed that the larvae in the exposed groups were sluggish, indicating that both BPAP and BPAF inhibited the moving distance and speed of zebrafish larvae. The results of fluorescence quantitative PCR showed that compared with the control group, BPAP(20 μg/L and 200 μg/L) and BPAF(20 μg/L and 200 μg/L) significantly inhibited the expressions of key genes(mbp and syn2 a) involved in zebrafish neurodevelopment. The results of in situ hybridization also verified the results of the genes. [Conclusion] The findings tentatively suggest that BPAF and BPAP may have potential developmental neurotoxic effects on zebrafish.
引文
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[12]杨洋,陈亚文,唐天乐,等.双酚AF暴露对胚胎期和幼鱼期斑马鱼的毒性效应[J].环境科学研究,2015,28(8):1219-1226.
[13]李靖.双酚AF(BPAF)对青春期前雄性大鼠睾丸毒性效应的研究[D].合肥:安徽大学,2015.
[14]CANO-NICOLAU J,VAILLANT C,PELLEGRINI E,et al.Estrogenic effects of several BPA analogs in the developing zebrafish brain[J].Front Neurosci,2016,10:112.
[15]徐静,刘济宁,汪贞,等.水体和沉积物加标法评估BPAP对摇蚊的毒性[J].生态毒理学报,2017,12(5):176-183.
[16]BR?SAMLE C,HALPERN M E.Characterization of myelination in the developing zebrafish[J].Glia,2002,39(1):47-57.
[17]MüLLER C,BAUER N M,SCHAFER I,et al.Making myelin basic protein-from mRNA transport to localized translation[J].Front Cell Neurosci,2013,7:169.
[18]KAO H T,PORTON B,CZERNIK A J,et al.A third member of the synapsin gene family[J].Proc Natl Acad Sci USA,1998,95(8):4667-4672.
[19]任文娟,汪贞,王蕾,等.双酚A及其类似物对斑马鱼胚胎及幼鱼的毒性效应[J].生态毒理学报,2017,12(1):184-192.
[20]朱小乔,唐天乐,彭翔,等.双酚AF暴露降低斑马鱼学习记忆能力并影响神经系统相关基因表达[J].生态毒理学报,2017,12(1):119-126.
[21]NERY L R,ELTZ N S,HACKMAN C,et al.Brain intraventricular injection of amyloid-βin zebrafish embryo impairs cognition and increases tau phosphorylation,effects reversed by lithium[J].PLoS One,2014,9(9):e105862.
[22]樊竑冶,彭忠禄,谢华平,等.斑马鱼整体原位杂交的技术改良[J].激光生物学报,2010,19(1):115-119.
[23]国先芬,王贻鑫,耿存珍,等.环境激素双酚A的毒理学研究进展[J].环境科学与技术,2013,36(2):86-92.
[24]LEE S,KIM Y K,SHIN T Y,et al.Neurotoxic effects of bisphenol AF on calcium-induced ROS and MAPKs[J].Neurotox Res,2013,23(3):249-259.
[25]COLEMAN K P,TOSCANO JR W A,WIESE T E.QSAR models of the in vitro estrogen activity of bisphenol a analogs[J].Mol Inform,2003,22(1):78-88.
[26]HE X,GAO J,DONG T,et al.Developmental neurotoxicity of methamidophos in the embryo-larval stages of zebrafish[J].Int J Environ Res Public Health,2017,14(1):E23.
[27]赵丹.急、慢性缺氧对斑马鱼幼鱼神经系统损伤机制的初步研究[D].广州:南方医科大学,2014.
[2]FISCHNALLER M,BAKRY R,BONN GK.A simple method for the enrichment of bisphenols using boron nitride[J].Food Chem,2016,194:149-155.
[3]CHEN D,KANNAN K,TAN H,et al.Bisphenol analogues other than BPA:environmental occurrence,human exposure,and toxicity-a review[J].Environ Sci Technol,2016,50(11):5438-5453.
[4]MATSUSHIMA A,LIU X,OKADA H,et al.Bisphenol AF is a full agonist for the estrogen receptor ERαbut a highly specific antagonist for ERβ[J].Environ Health Perspect,2010,118(9):1267-1272.
[5]VANDENBERG L N,COLBORN T,HAYES T B,et al.Hormones and endocrine-disrupting chemicals:low-dose effects and nonmonotonic dose responses[J].Endocr Rev,2012,33(3):378-455.
[6]JIN H,ZHU L.Occurrence and partitioning of bisphenol analogues in water and sediment from Liaohe River Basin and Taihu Lake,China[J].Water Res,2016,103:343-351.
[7]LIAO C,LIU F,GUO Y,et al.Occurrence of eight bisphenol analogues in indoor dust from the United States and several Asian countries:implications for human exposure[J].Environ Sci Technol,2012,46(16):9138-9145.
[8]LIAO C,LIU F,MOON H B,et al.Bisphenol analogues in sediments from industrialized areas in the United States,Japan,and Korea:spatial and temporal distributions[J].Environ Sci Technol,2012,46(21):11558-11565.
[9]SONG S,RUAN T,WANG T,et al.Distribution and preliminary exposure assessment of bisphenol AF(BPAF)in various environmental matrices around a manufacturing plant in China[J].Environ Sci Technol,2012,46(24):13136-13143.
[10]YANG Y,GUAN J,YIN J,et al.Urinary levels of bisphenol analogues in residents living near a manufacturing plant in south China[J].Chemosphere,2014,112:481-486.
[11]FERNáNDEZ M F,RIVAS A,OLEA-SERRANO F,et al.Assessment of total effective xenoestrogen burden in adipose tissue and identification of chemicals responsible for the combined estrogenic effect[J].Anal Bioanalyt Chem,2004,379(1):163-170.
[12]杨洋,陈亚文,唐天乐,等.双酚AF暴露对胚胎期和幼鱼期斑马鱼的毒性效应[J].环境科学研究,2015,28(8):1219-1226.
[13]李靖.双酚AF(BPAF)对青春期前雄性大鼠睾丸毒性效应的研究[D].合肥:安徽大学,2015.
[14]CANO-NICOLAU J,VAILLANT C,PELLEGRINI E,et al.Estrogenic effects of several BPA analogs in the developing zebrafish brain[J].Front Neurosci,2016,10:112.
[15]徐静,刘济宁,汪贞,等.水体和沉积物加标法评估BPAP对摇蚊的毒性[J].生态毒理学报,2017,12(5):176-183.
[16]BR?SAMLE C,HALPERN M E.Characterization of myelination in the developing zebrafish[J].Glia,2002,39(1):47-57.
[17]MüLLER C,BAUER N M,SCHAFER I,et al.Making myelin basic protein-from mRNA transport to localized translation[J].Front Cell Neurosci,2013,7:169.
[18]KAO H T,PORTON B,CZERNIK A J,et al.A third member of the synapsin gene family[J].Proc Natl Acad Sci USA,1998,95(8):4667-4672.
[19]任文娟,汪贞,王蕾,等.双酚A及其类似物对斑马鱼胚胎及幼鱼的毒性效应[J].生态毒理学报,2017,12(1):184-192.
[20]朱小乔,唐天乐,彭翔,等.双酚AF暴露降低斑马鱼学习记忆能力并影响神经系统相关基因表达[J].生态毒理学报,2017,12(1):119-126.
[21]NERY L R,ELTZ N S,HACKMAN C,et al.Brain intraventricular injection of amyloid-βin zebrafish embryo impairs cognition and increases tau phosphorylation,effects reversed by lithium[J].PLoS One,2014,9(9):e105862.
[22]樊竑冶,彭忠禄,谢华平,等.斑马鱼整体原位杂交的技术改良[J].激光生物学报,2010,19(1):115-119.
[23]国先芬,王贻鑫,耿存珍,等.环境激素双酚A的毒理学研究进展[J].环境科学与技术,2013,36(2):86-92.
[24]LEE S,KIM Y K,SHIN T Y,et al.Neurotoxic effects of bisphenol AF on calcium-induced ROS and MAPKs[J].Neurotox Res,2013,23(3):249-259.
[25]COLEMAN K P,TOSCANO JR W A,WIESE T E.QSAR models of the in vitro estrogen activity of bisphenol a analogs[J].Mol Inform,2003,22(1):78-88.
[26]HE X,GAO J,DONG T,et al.Developmental neurotoxicity of methamidophos in the embryo-larval stages of zebrafish[J].Int J Environ Res Public Health,2017,14(1):E23.
[27]赵丹.急、慢性缺氧对斑马鱼幼鱼神经系统损伤机制的初步研究[D].广州:南方医科大学,2014.