典型酚类污染物内分泌干扰效应研究——对斑马鱼发育及核受体介导基因调控的分子影响

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (5709 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

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、AhR、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、AhR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、AhR、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.

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	宋静文
	靳亚茹
	刘红玲

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

Abstract：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 10mg/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 5mg/L and 10mg/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 receptor α (er1) were down-regulated by 2.1-fold after exposure to 2.5mg/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 2.5mg/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 2.5mg/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 0.5mg/L, and down-regulated by 1.9-fold at 2.5mg/L. The glucocorticoid receptor gene (mr) was down-regulated by 1.9-fold and 2.1-fold at concentrations of 0.5 and 2.5mg/L, and significantly up-regulated by 1.7-fold under the exposure concentration of 0.1mg/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 5ns. 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.

Key words： 2,4,6-trichlorophenol 4-chloro-3-methylphenol 4-nitrophenol embryonic development toxicity nuclear receptor molecular dynamics simulation

收稿日期: 2020-02-04

PACS:

X503.2

基金资助: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）

通讯作者: 刘红玲,副教授,hlliu@nju.edu.cn E-mail: hlliu@nju.edu.cn

作者简介: 宋静文(1995-),男,安徽池州人,南京大学硕士研究生,研究方向为环境毒理学.

引用本文:

宋静文, 靳亚茹, 刘红玲. 典型酚类污染物内分泌干扰效应研究——对斑马鱼发育及核受体介导基因调控的分子影响[J]. 中国环境科学, 2020, 40(9): 4065-4076. SONG Jing-wen, JIN Ya-ru, LIU Hong-ling. Research on the endocrine disruption effect of typical phenolic pollutants: The embryonic development effects and molecule effects of gene regulation mediated by nuclear receptor on Zebrafish. CHINA ENVIRONMENTAL SCIENCECE, 2020, 40(9): 4065-4076.

链接本文:

http://www.zghjkx.com.cn/CN/ 或 http://www.zghjkx.com.cn/CN/Y2020/V40/I9/4065

[1]	罗茜,查金苗,雷炳莉,等.三种氯代酚的水生态毒理和水质基准[J]. 环境科学学报, 2009,29(11):2241-9. Luo Q, Cha J M, Lei B L, et al. Review of the aquatic ecotoxicology and water quality criteria of three chlorophenols[J]. Acta Scientiae Circumstantiae, 2009,29(11):2241-9.
[2]	陈洁霞,韦恩泽,鲜啟鸣.长江沿岸某化工园区土壤、底泥中酚类化合物的污染现状[J]. 色谱, 2014,32(8):843-848. Chen J X, Wei E Z, Xian Q M. Pollution status of phenolic compounds in the soil and sedimentfrom a chemical industrial park along the Yangtze River[J]. Chinese Journal of Chromatography, 2014,32(8):843-848.
[3]	纪靓靓,李法云,罗义,等.2,4,6-三氯苯酚诱导鲫鱼肝脏自由基的产生及其氧化应激[J]. 应用生态学报, 2007,18(1):129-132. Ji L L, Li F Y, Luo Y, et al. Free radicals in Carassius auratus liver:Their generation andoxidative stress induced by 2, 4, 6-trichlorophenol[J]. Chinese Journal of Applied Ecology, 2007,18(1):129-132.
[4]	Yin D, Zhu H, Hu P, et al. Genotoxic effect of 2,4,6-trichlorophenol on P53 gene in zebrafish liver[J]. Environmental Toxicology & Chemistry, 2008,28(3):603-608.
[5]	王力超,邢立群,戴建军.2, 4-二氯酚和2, 4, 6-三氯酚对斑马鱼胚胎发育的影响[J]. 环境科学与技术, 2015,38(5):25-29. Wang L C, Xing L Q, Dai J J. Effect of 2,4-Dichlorophenol and 2,4,6-Trichlorophenol to embryonic development of Zebrafish (Brachydanio rerio)[J]. Environmental Science & Technology, 2015, 38(5):25-29.
[6]	Zhang Y, Piao Y, Li Y, et al. 4-Nitrophenol induces Leydig cells hyperplasia, which may contribute to the differential modulation of the androgen receptor and estrogen receptor-α and -β expression in male rat testes[J]. Toxicology Letters, 2013,223(2):228-235.
[7]	Furuta, C. Estrogenic activities of nitrophenols in diesel exhaust particles[J]. Biology of Reproduction, 2004,70(5):1527-33.
[8]	Li C M, Taneda S, Suzuki A K, et al. Estrogenic and anti-androgenic activities of 4-nitrophenol in diesel exhaust particles[J]. Toxicology and applied pharmacology, 2006,217(1):1-6.
[9]	Lam S H, Ung C Y, Hlaing M M, et al. Molecular insights into 4-nitrophenol-induced hepatotoxicity in zebrafish:transcriptomic, histological and targeted gene expression analyses[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2013,1830(10):4778-89.
[10]	Ramamoorthy S, Ramamoorthy S. Chlorinated organic compounds in the environment:regulatory and monitoring assessment[M]. CRC press, 1997.
[11]	Vinggaard A M, Körner W, Lund K H, et al. Identification and quantification of estrogenic compounds in recycled and virgin paper for household use as determined by an in vitro yeast estrogen screen and chemical analysis[J]. Chemical research in toxicology, 2000, 13(12):1214-22.
[12]	Zhang L, Jin Y, Han Z, et al. Integrated in silico and in vivo approaches to investigate effects of BDE-99mediated by the nuclear receptors on developing zebrafish[J]. Environmental Toxicology & Chemistry, 2018,37(3):780-7.
[13]	Liu C, Yan W, Zhou B, et al. Characterization of a bystander effect induced by the endocrine-disrupting chemical 6-propyl-2-thiouracil in zebrafish embryos[J]. Aquatic toxicology, 2012,118:108-115.
[14]	Gasteiger J, Marsili M. Iterative partial equalization of orbital electronegativity-a rapid access to atomic charges[J]. Tetrahedron, 1980,36(22):3219-3228.
[15]	Clark M, Cramer R D, Opdenbosch N V, et al. Validation of the general purpose TRIPOS 5.2force field[J]. Journal of Computational Chemistry, 1989,10(8):982-1012.
[16]	Florian K, Konstantin A, Michael K, et al. The SWISS-MODEL Repository and associated resources[J]. Nucleic acids research, 2009,37(suppl_1):D387-D392.
[17]	Liu H, Tang S, Zheng X, et al. Bioaccumulation, biotransformation, and toxicity of BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47in early life-stages of zebrafish (Danio rerio)[J]. Environmental Science & Technology, 2015,49(3):1823-33.
[18]	Saito R, Smoot M E, Ono K, et al. A travel guide to Cytoscape plugins[J]. Nature methods, 2012,9(11):1069.
[19]	Wu B, Zhang Y, Kong J, et al. In silico predication of nuclear hormone receptors for organic pollutants by homology modeling and molecular docking[J]. Toxicology letters, 2009,191(1):69-73.
[20]	Chen Q, Wang X, Shi W, et al. Identification of thyroid hormone disruptors among HO-PBDEs:in vitro investigations and coregulator involved simulations[J]. Environmental Science &Technology, 2016,50(22):12429-38.
[21]	Braunbeck T, Lammer E. Fish embryo toxicity assays[J]. German Federal Environment Agency, 2006:298.
[22]	Hen Chow E S, Cheng S H. Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis[J]. Toxicological Sciences, 2003,73(1):149-59.
[23]	Zhang Y, Liu M, Liu J, et al. Combined toxicity of triclosan, 2,4-dichlorophenol and 2,4,6-trichlorophenol to zebrafish (Danio rerio)[J]. Environmental Toxicology & Pharmacology, 2018,57:9-18.
[24]	Ceylan Z, Şişman T, Yazıcı Z, et al. Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio)[J]. Toxicology and industrial health, 2016,32(8):1414-1422.
[25]	Ma Z, Liu H, Yu H. Triclosan Affects Ca2+ Regulatory Module and Musculature Development in Skeletal Myocyte during Early Life Stages of Zebrafish (Danio rerio)[J]. Environmental Science & Technology, 2019,53(20):11988-98.
[26]	Aardema M J, Macgregor J T. Toxicology and genetic toxicology in the new era of "toxicogenomics":impact of "-omics" technologies[M]. Toxicogenomics. Springer, 2003:171-93.
[27]	Zheng X, Zhu Y, Liu C, et al. Accumulation and biotransformation of BDE-47 by zebrafish larvae and teratogenicity and expression of genes along the hypothalamus-pituitary-thyroid axis[J]. Environmental Science &Technology, 2012,46(23):12943-51.
[28]	Liu C, Wang Q, Liang K, et al. Effects of tris (1, 3-dichloro-2-propyl) phosphate and triphenyl phosphate on receptor-associated mRNA expression in zebrafish embryos/larvae[J]. Aquatic toxicology, 2013, 128:147-157.
[29]	Ma Z, Yu Y, Tang S, et al. Differential modulation of expression of nuclear receptor mediated genes by tris (2-butoxyethyl) phosphate (TBOEP) on early life stages of zebrafish (Danio rerio)[J]. Aquatic Toxicology, 2015,169:196-203.
[30]	Xie X, Jin Y, Ma Z, et al. Underlying mechanisms of reproductive toxicity caused by multigenerational exposure of 2, bromo-4, 6-dinitroaniline (BDNA) to Zebrafish (Danio rerio) at environmental relevant levels[J]. Aquatic Toxicology, 2019,216:105285.
[31]	Ma Y, Han J, Guo Y, et al. Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2, 4-dichlorophenol[J]. Aquatic toxicology, 2012,106:173-81.
[32]	Mattingly C J, Toscano W A. Posttranscriptional silencing of cytochrome P4501A1(CYP1A1) during zebrafish (Danio rerio) development[J]. Developmental dynamics:an official publication of the American Association of Anatomists, 2001,222(4):645-54.
[33]	Swedenborg E, Pongratz I. AhR and ARNT modulate ER signaling[J]. Toxicology, 2010,268(3):132-138.
[34]	Körner W, Hanf V, Schuller W, et al. Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals[J]. Chemosphere, 1998,37(9-12):2395-407.
[35]	Krüger T, Long M, Bonefeld-Jørgensen E C. Plastic components affect the activation of the aryl hydrocarbon and the androgen receptor[J]. Toxicology, 2008,246(2/3):112-23.
[36]	Takahashi H, Sakamoto T. The role of ‘mineralocorticoids’ in teleost fish:relative importance of glucocorticoid signaling in the osmoregulation and ‘central’ actions of mineralocorticoid receptor[J]. General and comparative endocrinology, 2013,181:223-228.
[37]	Berger S, Bleich M, Schmid W, et al. Mineralocorticoid receptor knockout mice:pathophysiology of Na+ metabolism[J]. Proceedings of the National Academy of Sciences, 1998,95(16):9424-9.
[38]	Barnes P J. How corticosteroids control inflammation:quintiles prize lecture 2005[J]. British journal of pharmacology, 2006,148(3):245-54.
[39]	De Kloet E R, Joëls M, Holsboer F. Stress and the brain:from adaptation to disease[J]. Nature reviews neuroscience, 2005,6(6):463-475.
[40]	Wang M. The role of glucocorticoid action in the pathophysiology of the metabolic syndrome[J]. Nutrition & metabolism, 2005,2(1):3.
[41]	Migliaccio S, Brama M, Fornari R, et al. Glucocorticoid-induced osteoporosis:an osteoblastic disease[J]. Aging clinical and experimental research, 2007,19(3Suppl):5-10.
[42]	Chen J, Song M, Li Y, et al. The effect of phytosterol protects rats against 4-nitrophenol-induced liver damage[J]. Environmental toxicology and pharmacology, 2016,41:266-271.
[43]	Stöcklin E, Wissler M, Gouilleux F, et al. Functional interactions between Stat5 and the glucocorticoid receptor[J]. Nature, 1996,383(6602):726-8.
[44]	Vijayan M M, Raptis S, Sathiyaa R. Cortisol treatment affects glucocorticoid receptor and glucocorticoid-responsive genes in the liver of rainbow trout[J]. General and comparative endocrinology, 2003,132(2):256-63.
[45]	Hansch C, Kiehs K, Lawrence G L. The role of substituents in the hydrophobic bonding of phenols by serum and mitochondrial proteins[J]. Journal of the American Chemical Society, 1965,87(24):5770-3.