摘要
以斑马鱼为研究对象,观察2,4,6-三氯酚(2,4,6-trichlorophenol)、4-氯-3-甲酚(4-chloro-3-methylphenol)和4-硝基苯酚(4-nitrophenol)3种酚类污染物对斑马鱼胚胎发育早期阶段形态学指标的影响,借助q-RT-PCR方法对斑马鱼胚胎分别暴露于0.1,0.5和2.5mg/L至120hpf幼鱼8个重要受体(雄激素受体(AR)、甲状腺激素受体(TR)、芳香烃受体(AhR)、雌激素受体(ER)、糖皮质激素受体(GR)、孕烷X受体(PXR)、盐皮质激素受体(MR)、过氧化物酶体增殖物激活受体(PPAR))相关基因的转录水平进行了研究;进一步采用分子对接和分子动力学模拟的方法研究污染物与筛选出的影响受体(ER、Ah R、PXR、GR以及MR)之间的相互作用过程.结果表明,在5和10mg/L的浓度下3种酚类污染物都显著影响斑马鱼的发育,造成脊柱弯曲、心包水肿等不良影响;基因结果显示2,4,6-三氯酚暴露导致ER的基因下调,雌激素受体α(er1)基因在2.5mg/L浓度下下调2.1倍.4-氯-3-甲酚显著影响ER、Ah R以及PXR的相关基因,表现为在0.5和2.5mg/L暴露er1基因显著下调1.8和2.2倍,ahr2和pxr基因在0.1和2.5mg/L的浓度下分别下调1.7和1.8倍和上调3.5和3.2倍.4-硝基苯酚暴露使GR、MR核心受体基因发生调控变化,糖皮质激素受体基因(gr)在低中浓度下分别显著上调2.0和2.1倍,最高浓度下下调1.9倍.盐皮质激素受体基因(mr)在中高浓度下分别下调1.9和2.1倍,在最低浓度下显著上调1.7倍;分子计算结果显示它们与相关受体通过疏水和氢键等相互作用而稳定结合,且均方根偏差(RMSD)在5ns后稳定.这3个典型酚类污染物主要通过斑马鱼ER、Ah R、PXR、GR、MR受体介导产生内分泌干扰效应并影响其早期生长发育.
2,4,6-trichlorophenol,4-chloro-3-methylphenol and 4-nitrophenol were the three most abundant phenols in various environmental media around chemical industry park in Nanjing,China.In this study,zebrafish embryos were exposedto 0.1,0.5,2.5,5 and 10 mg/L of three phenols to observe the effects of morphological indicators in the early stages of embryonic development,and then we investigated the transcriptional profiles on 61 genes associated with 8 important receptors(androgen receptor(AR),thyroid receptor(TR),aryl hydrocarbon receptor(AhR),estrogen receptor(ER),glucocorticoid receptor(GR),pregnane Xreceptor(PXR),mineralocorticoid receptor(MR),peroxisome proliferator activated receptor(PPAR))in zebrafish larvae by q-RT-PCR at 120 hpf.Moreover,the possible interaction processes between three phenols and their significantly affected nuclear receptors(including ER,AhR,PXR,GR and MR)were studied by in silico approaches(molecular docking and molecular dynamics simulation).Our results indicated that the three phenols at 5 mg/L and 10 mg/L concentrations all resulted in adverse effects on spinal curvature and edema in zebrafish embryos and larvae.The gene results of 2,4,6-trichlorophenol exposure could down-regulate gene of ER,the estrogen receptorα(er1)were down-regulated by 2.1-fold after exposure to 2.5 mg/L of 2,4,6-trichlorophenol.4-chloro-3-methylphenol significantly affected the genes of ER,AhR and PXR.er1 was significantly down-regulated by 1.8-fold and 2.2-fold respectively,at 0.5 and 2.5 mg/L,and aryl hydrocarbon receptor gene 2(ahr2)was down-regulated by 1.7-fold and 1.8-fold,and pregnane X receptor gene(pxr)was up-regulated by 3.5-fold and 3.2-fold at 0.1 and 2.5 mg/L.Exposure to 4-nitrophenol caused regulatory changes in the core receptor genes of GR and MR.The glucocorticoid receptor gene(gr)was significantly up-regulated by 2.0-fold and 2.1-fold at concentrations of 0.1 and 0.5 mg/L,and down-regulated by 1.9-fold at 2.5 mg/L.The glucocorticoid receptor gene(mr)was down-regulated by 1.9-fold and 2.1-fold at concentrations of 0.5 and 2.5 mg/L,and significantly up-regulated by 1.7-fold under the exposure concentration of 0.1 mg/L.In silico results showed that the three phenols were able to dock into the ligand-binding domain and stably bound to the related receptors through hydrophobic and hydrogen bonding interactions.Also,relative root mean square deviation(RMSD)fluctuations for the backbone atoms was stable after 5 ns.This study concluded that these three phenols could mediate endocrine disrupting effects and affect the early growth and development of zebrafish through ER,AhR,PXR,GR and MR receptors.
作者
宋静文
靳亚茹
刘红玲
SONG Jing-wen;JIN Ya-ru;LIU Hong-ling(State Key Laboratory of Pollution Control&Resource Reuse,School of the Environment,Nanjing University,Nanjing 210023,China)
出处
《中国环境科学》
EI
CAS
CSCD
北大核心
2020年第9期4065-4076,共12页
China Environmental Science
基金
国家自然科学基金资助项目(21677073)
国家重点研发计划(2018YFC1801505)
国家科技重大专项(2018ZX07208001,2017ZX07301002)。
关键词
2
4
6-三氯酚
4-氯-3-甲酚
4-硝基苯酚
早期发育毒性
核受体
分子动力学模拟
2,4,6-trichlorophenol
4-chloro-3-methylphenol
4-nitrophenol
embryonic development toxicity
nuclear receptor
molecular dynamics simulation
