纳米氧化铝对斑马鱼幼鱼的神经毒性及mTOR基因的作用
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摘要
[背景]纳米氧化铝(AlNPs)由于其独特的理化特性,在光电子学、颜料、催化剂和制陶业等领域有着广泛的应用。在生产过程中AINPs可能通过生产制造、加工和废料排放等途径直接或间接地进入环境中,然而现有的毒理学信息不足以评估AlNPs对水生生物、职业暴露人群及公众的潜在风险。[目的 ]本研究旨在探讨AlNPs对斑马鱼幼鱼的神经毒作用及特异性敲低mTOR基因对斑马鱼幼鱼的毒作用。[方法 ]斑马鱼胚胎在受精后6 h分别暴露于6.25、12.5、25.0、50.0、100 mg/L的AlNPs悬浊液,各组均为60颗受精卵。检测幼鱼相应的运动行为学指标,包括:在黑暗状态下的运动能力、趋触反应、光照惊恐反应和黑暗逃避反应;检测氧化应激指标,包括:超氧化物歧化酶活性、乳酸脱氢酶活性;检测幼鱼mTOR和Beclin1基因表达变化。另设置空白对照组、阴性对照组、AlNPs组、mTOR敲低组及mTOR敲低+AlNPs组,在斑马鱼受精卵受精后的20～60 min内,暴露于100 mg/L AlNPs悬浊液,并在显微镜下注射反义寡核苷酸特异性敲低m TOR基因,在受精后第6天检测幼鱼的运动行为学改变。[结果 ]随着AlNPs暴露剂量逐渐加大,斑马鱼在黑暗状态下的自发运动速率呈现逐渐减慢的趋势,在100 mg/L时运动速率低于对照组(P=0.003)。25、50、100 mg/L AlNPs组斑马鱼幼鱼趋触反应明显降低(P <0.05)。在50、100 mg/L时斑马鱼幼鱼光照惊恐反应能力降低(P <0.05)。在25、50、100 mg/L时斑马鱼幼鱼黑暗逃避反应能力降低(P <0.001)。各暴露组超氧化物歧化酶活性均降低(P <0.001);12.5 mg/L及以上组的乳酸脱氢酶活性均降低(P <0.05)。25、50、100 mg/L AlNPs组mTOR基因表达均降低(P <0.05),100 mg/L AlNPs组Beclin1基因表达升高(P=0.003)。与AlNPs组相比,mTOR敲低+AlNPs组斑马鱼光照惊恐反应和黑暗逃避反应能力降低(均P <0.05)。[结论 ]斑马鱼胚胎及幼鱼暴露AINPs可能导致幼鱼运动行为学改变,导致幼鱼产生氧化应激,其机制可能与mTOR基因的表达降低有关。特异性敲低mTOR基因表达使得AINPs对斑马鱼幼鱼毒性增强。
[Background] Al_2O_3 nanoparticles(AlNPs) are widely used in optoelectronics, pigments, catalysts, and ceramic industry because of its unique physical and chemical properties. AlNPs can be released into the environment directly or indirectly during processes such as product manufacturing, processing, and waste discharge. However, available toxicological information is insufficient to assess the potential risks of AlNPs to aquatic organisms, occupationally exposed populations, and the public. [Objective] We aims to explore the neurotoxicity of AlNPs on zebrafish larvae and the role of specifically knocked down mTOR gene in the relevant toxic effects.[Methods] Zebrafish embryos were exposed to 6.25, 12.5, 25.0, 50.0, and 100 mg/L AlNPs at 6 hours post-fertilization, with 60 fertilized eggs in each dose group. The testing indicators of larvae included motor behavioral indicators(locomotor activity in darkness, thigmotaxis, light-evoked startle response, and darkness-evoked escape response), oxidative stress indicators(superoxide dismutase activity and lactate dehydrogenase activity), and mTOR and Beclin1 mRNA expression changes. Another batch of embryos were divided into blank control group, negative control group, AINPs group, mTOR knockdown group, and mTOR knockdown+AINPs group. In 20-60 min after fertilization, the zebrafish eggs were exposed to 100 mg/L AlNPs and were injected with antisense oligonucleotides to specifically knock down mTOR gene under the microscope. The behavioral changes of larvae were detected on the 6 th day after fertilization.[Results] With increasing exposure dose of AINPs, the spontaneous movement velocity of zebrafish in darkness gradually decreased. The velocity in the 100 mg/L group was lower than that in the control group(P=0.003). Thigmotaxis was significantly lower in the 25, 50, and 100 mg/L AlNPs groups(P < 0.05). Light-evoked startle response was attenuated in the 50 and 100 mg/L groups(P < 0.05). Darkness-evoked escape response was attenuated in the 25, 50, and 100 mg/L groups(P < 0.001). The activity of superoxide dismutase was decreased in each exposed group(P < 0.001); the activity of lactate dehydrogenase was decreased in the groups exposed to AlNPs greater than or equal to 12.5 mg/L(P < 0.05). The expression of m TOR gene was down-regulated in the 25, 50, and 100 mg/L groups of AlNPs(P < 0.05), and the expression of Beclin1 gene was up-regulated in the 100 mg/L concentration group(P=0.003). The mTOR knockdown+AINPs group's zebrafish light-evoked startle response and darkness-evoked escape response were attenuated compared with the AINPs group(Ps < 0.05).[Conclusion] Exposure to AlNPs during embryo and larvae development may cause changes in motor behavior and oxidative stress in zebrafish larvae, which may be related to decreased expression of mTOR gene. Specific knockdown of mTOR gene could enhance the toxic effect of AlNPs on zebrafish larvae.
[Background] Al_2O_3 nanoparticles(AlNPs) are widely used in optoelectronics, pigments, catalysts, and ceramic industry because of its unique physical and chemical properties. AlNPs can be released into the environment directly or indirectly during processes such as product manufacturing, processing, and waste discharge. However, available toxicological information is insufficient to assess the potential risks of AlNPs to aquatic organisms, occupationally exposed populations, and the public. [Objective] We aims to explore the neurotoxicity of AlNPs on zebrafish larvae and the role of specifically knocked down mTOR gene in the relevant toxic effects.[Methods] Zebrafish embryos were exposed to 6.25, 12.5, 25.0, 50.0, and 100 mg/L AlNPs at 6 hours post-fertilization, with 60 fertilized eggs in each dose group. The testing indicators of larvae included motor behavioral indicators(locomotor activity in darkness, thigmotaxis, light-evoked startle response, and darkness-evoked escape response), oxidative stress indicators(superoxide dismutase activity and lactate dehydrogenase activity), and mTOR and Beclin1 mRNA expression changes. Another batch of embryos were divided into blank control group, negative control group, AINPs group, mTOR knockdown group, and mTOR knockdown+AINPs group. In 20-60 min after fertilization, the zebrafish eggs were exposed to 100 mg/L AlNPs and were injected with antisense oligonucleotides to specifically knock down mTOR gene under the microscope. The behavioral changes of larvae were detected on the 6 th day after fertilization.[Results] With increasing exposure dose of AINPs, the spontaneous movement velocity of zebrafish in darkness gradually decreased. The velocity in the 100 mg/L group was lower than that in the control group(P=0.003). Thigmotaxis was significantly lower in the 25, 50, and 100 mg/L AlNPs groups(P < 0.05). Light-evoked startle response was attenuated in the 50 and 100 mg/L groups(P < 0.05). Darkness-evoked escape response was attenuated in the 25, 50, and 100 mg/L groups(P < 0.001). The activity of superoxide dismutase was decreased in each exposed group(P < 0.001); the activity of lactate dehydrogenase was decreased in the groups exposed to AlNPs greater than or equal to 12.5 mg/L(P < 0.05). The expression of m TOR gene was down-regulated in the 25, 50, and 100 mg/L groups of AlNPs(P < 0.05), and the expression of Beclin1 gene was up-regulated in the 100 mg/L concentration group(P=0.003). The mTOR knockdown+AINPs group's zebrafish light-evoked startle response and darkness-evoked escape response were attenuated compared with the AINPs group(Ps < 0.05).[Conclusion] Exposure to AlNPs during embryo and larvae development may cause changes in motor behavior and oxidative stress in zebrafish larvae, which may be related to decreased expression of mTOR gene. Specific knockdown of mTOR gene could enhance the toxic effect of AlNPs on zebrafish larvae.
引文
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[15]XU J,HUAI Y,MENG N,et al.L-3-n-butylphthalide activates Akt/mTOR signaling,inhibits neuronal apoptosis and autophagy and improves cognitive impairment in mice with repeated cerebral ischemia-reperfusion injury[J].Neurochem Res,2017,42(10):2968-2981.
[16]贺凯宏.mTOR信号分子在纳米氧化铝致斑马鱼幼鱼早期神经行为损伤中的作用[D].太原:山西医科大学,2018.
[17]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2Method[J].Methods,2001,25(4):402-408.
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[19]SAMAEE S M,MANTEGHI N,YOKEL R A,et al.Morphometric characteristics and time to hatch as efficacious indicators for potential nanotoxicity assay in zebrafish[J].Environ Toxicol Chem,2018,37(12):3063-3076.
[20]SHIH Y J,SU C C,CHEN C W,et al.Adsorption characteristics of nano-TiO2 onto zebrafish embryos and its impacts on egg hatching[J].Chemosphere,2016,154:109-117.
[21]MCNEIL P L,BOYLE D,HENRY T B,et al.Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish(Danio rerio)[J].Aquat Toxicol,2014,152:318-323.
[22]SCHN?RR S J,STEENBERGEN P J,RICHARDSON M K,et al.Measuring thigmotaxis in larval zebrafish[J].Behav Brain Res,2012,228(2):367-374.
[23]MIAO W,ZHU B,XIAO X,et al.Effects of titanium dioxide nanoparticles on lead bioconcentration and toxicity on thyroid endocrine system and neuronal development in zebrafish larvae[J].Aquat Toxicol,2015,161:117-126.
[24]HU Q,GUO F,ZHAO F,et al.Effects of titanium dioxide nanoparticles exposure on parkinsonism in zebrafish larvae and PC12[J].Chemosphere,2017,173:373-379.
[2]YE N,WANG Z,WANG S,et al.Dissolved organic matter and aluminum oxide nanoparticles synergistically cause cellular responses in freshwater microalgae[J].J Environ Sci Health A Tox Hazard Subst Environ Eng,2018,53(7):651-658.
[3]MIRSHAFA A,NAZARI M,JAHANI D,et al.Size-dependent neurotoxicity of aluminum oxide particles:a comparison between nano-and micrometer size on the basis of mitochondrial oxidative damage[J].Biol Trace Elem Res,2018,183(2):261-269.
[4]MORSY G M,ABOU E K,ALI A A.Studies on fate and toxicity of nanoalumina in male albino rats:oxidative stress in the brain,liver and kidney[J].Toxicol Ind Health,2016,32(2):200-214.
[5]ZHANG X,XU Y,ZHOU L,et al.Sex-dependent depressionlike behavior induced by respiratory administration of aluminum oxide nanoparticles[J].Int J Environ Res Public Health,2015,12(12):15692-15705.
[6]LI X,YANG H,WU S,et al.Suppression of PTPN6exacerbates aluminum oxide nanoparticle-induced COPD-like lesions in mice through activation of STAT pathway[J].Part Fibre Toxicol,2017,14(1):53.
[7]LI X,ZHANG C,BIAN Q,et al.Integrative functional transcriptomic analyses implicate specific molecular pathways in pulmonary toxicity from exposure to aluminum oxide nanoparticles[J].Nanotoxicology,2016,10(7):957-969.
[8]SHAH S A,YOON G H,AHMAD A,et al.Nanoscale-alumina induces oxidative stress and accelerates amyloid beta(Aβ)production in ICR female mice[J].Nanoscale,2015,7(37):15225-15237.
[9]丁勇,杨川丽,陈佳慧,等.纳米氧化铝致雄性小鼠生殖系统损害[J].毒理学杂志,2015,29(3):177-180.
[10]贺凯宏,尚楠,陈建平,等.纳米氧化铝对斑马鱼幼鱼早期运动行为的影响[J].生态毒理学报,2018,13(3):165-171.
[11]ZHANG Q,WANG H,GE C,et al.Alumina at 50 and 13 nm nanoparticle sizes have potential genotoxicity[J].J Appl Toxicol,2017,37(9):1053-1064.
[12]丁勇.纳米氧化铝致小鼠神经发育毒性及其机制初探[D].太原:山西医科大学,2015.
[13]常丽俊,郭卫伟,葛翠翠,等.纳米氧化铝对新生Wistar乳大鼠皮质神经元线粒体自噬的影响[J].中国药理学与毒理学杂志,2014,28(5):737-742.
[14]LI X,ZHANG C,ZHANG X,et al.An acetyl-L-carnitine switch on mitochondrial dysfunction and rescue in the metabolomics study on aluminum oxide nanoparticles[J].Part Fibre Toxicol,2016,13:4.
[15]XU J,HUAI Y,MENG N,et al.L-3-n-butylphthalide activates Akt/mTOR signaling,inhibits neuronal apoptosis and autophagy and improves cognitive impairment in mice with repeated cerebral ischemia-reperfusion injury[J].Neurochem Res,2017,42(10):2968-2981.
[16]贺凯宏.mTOR信号分子在纳米氧化铝致斑马鱼幼鱼早期神经行为损伤中的作用[D].太原:山西医科大学,2018.
[17]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2Method[J].Methods,2001,25(4):402-408.
[18]JOHNSTON H J,VERDON R,GILLIES S,et al.Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials[J].Crit Rev Toxicol,2018,48(3):252-271.
[19]SAMAEE S M,MANTEGHI N,YOKEL R A,et al.Morphometric characteristics and time to hatch as efficacious indicators for potential nanotoxicity assay in zebrafish[J].Environ Toxicol Chem,2018,37(12):3063-3076.
[20]SHIH Y J,SU C C,CHEN C W,et al.Adsorption characteristics of nano-TiO2 onto zebrafish embryos and its impacts on egg hatching[J].Chemosphere,2016,154:109-117.
[21]MCNEIL P L,BOYLE D,HENRY T B,et al.Effects of metal nanoparticles on the lateral line system and behaviour in early life stages of zebrafish(Danio rerio)[J].Aquat Toxicol,2014,152:318-323.
[22]SCHN?RR S J,STEENBERGEN P J,RICHARDSON M K,et al.Measuring thigmotaxis in larval zebrafish[J].Behav Brain Res,2012,228(2):367-374.
[23]MIAO W,ZHU B,XIAO X,et al.Effects of titanium dioxide nanoparticles on lead bioconcentration and toxicity on thyroid endocrine system and neuronal development in zebrafish larvae[J].Aquat Toxicol,2015,161:117-126.
[24]HU Q,GUO F,ZHAO F,et al.Effects of titanium dioxide nanoparticles exposure on parkinsonism in zebrafish larvae and PC12[J].Chemosphere,2017,173:373-379.