草甘膦暴露对斑马鱼胚胎发育的毒性效应及机制
Developmental Impact of Glyphosate Exposure on Zebrafish Embryos and Mechanism Involved
-
摘要: 为了探究环境中草甘膦的毒性效应及致毒机理。将斑马鱼(Danio rerio)胚胎暴露于草甘膦(1、10、100 μg·L-1)72 h,观察并记录胚胎孵化率、存活率、畸形率、心率,测定ATPase活性及胚胎发育相关基因(Tbx5、Nkx2.5、Tnnt2、Bmp2b、Ihh、Shh)表达水平。结果显示,高浓度组(10 μg·L-1和100 μg·L-1)中斑马鱼胚胎存活率和孵化率显著降低,心包水肿率、脊柱弯曲率和卵黄囊肿率显著升高;胚胎发育相关基因的转录水平发生了改变,在1 μg·L-1组中,Nkx2.5 mRNA水平无明显变化,但Tbx5、Tnnt2、Bmp2b、Shh水平明显升高,而Ihh水平明显降低;在10 μg·L-1和100 μg·L-1组中,Tbx5、Nkx2.5、Bmp2b、Ihh、Shh的mRNA表达显著降低;在10 μg·L-1和100 μg·L-1暴露组Na+/K+-ATP酶和Ca2+-ATP酶活性均受到显著抑制。较高浓度的草甘膦对于鱼类具有显著的发育毒性,毒性效应机制与胚胎发育相关基因转录水平变化及酶活性抑制有关。依据试验结果,草甘膦LOEC为10 μg·L-1,NOEC为1 μg·L-1,环境中的草甘膦残留量达到一定浓度(≥ 10 μg·L-1)时对鱼类构成潜在威胁,在实际应用中应全面考察环境草甘膦可能残留的浓度。Abstract: In order to explore the toxic effects of glyphosate and underlying mechanism, zebrafish (Danio rerio) embryos were exposed to glyphosate at 1, 10 and 100 μg·L-1 for 72 h, and embryonic hatching rate (48 h post-fertilization), survival rate, malformation rate, heart rate, ATPase activity and the transcription of development-related genes (Tbx5, Nkx2.5, Tnnt2, Bmp2b, Ihh, Shh) were examined. Both survival rate and hatching rate were significantly reduced in the high concentration groups (10 μg·L-1 and 100 μg·L-1), the rates of pericardial edema, spine curvature and yolk sac edema were all significantly increased. The transcriptional levels of development-related genes were altered, in the 1 μg·L-1 group, Nkx2.5 mRNA levels showed no significant change, the mRNA levels of Tbx5, Tnnt2, Bmp2b, Shh were significantly increased, while the transcription of Ihh were significantly decreased. Significantly decreased mRNA levels of Tbx5, Nkx2.5, Bmp2b, Ihh, Shh were observed in the 10 μg·L-1 and 100 μg·L-1 groups. The activities of Na+/K+-ATPase and Ca2+-ATPase were significantly inhibited in the 10 μg·L-1 and 100 μg·L-1 groups. Higher concentrations of glyphosate showed significant developmental toxicity for zebrafish, and the mechanisms underlying toxic effect might be related to the changes in the transcription level of genes related to embryonic development and the inhibition of the enzyme activities. Based on the results of this study, the LOEC of glyphosate is 10 μg·L-1 and NOEC is 1 μg·L-1. Thus, glyphosate residue in the environment at a certain concentration (≥ 10 μg·L-1) would pose a potential threat to fish. In practical application, the residual concentration of glyphosate in the environment should be fully investigated.
-
Key words:
- glyphosate /
- zebrafish /
- developmental toxicity /
- mechanism
-
-
孙克. 全球十大除草剂的市场与展望[J]. 农药, 2013, 52(5):317-322 Sun K. Market and outlook of the top ten herbicides in the world[J]. Agrochemicals, 2013, 52(5):317-322(in Chinese)
Williams A L, Watson R E, DeSesso J M. Developmental and reproductive outcomes in humans and animals after glyphosate exposure:A critical analysis[J]. Journal of Toxicology and Environmental Health Part B, Critical Reviews, 2012, 15(1):39-96 Bisson M, Hontela A. Cytotoxic and endocrine-disrupting potential of atrazine, diazinon, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro[J]. Toxicology and Applied Pharmacology, 2002, 180(2):110-117 Wang C, Lu G H, Cui J. Responses of AChE and GST activities to insecticide coexposure in Carassius auratus[J]. Environmental Toxicology, 2012, 27(1):50-57 宋秀杰, 陈博. 北京市农药化肥非点源污染防治的技术措施[J]. 环境保护, 2001, 29(9):30-32 Song X J, Chen B. Technical measures for prevention and control of non-point source pollution of pesticides and fertilizers in Beijing[J]. Environmental Protection, 2001, 29(9):30-32(in Chinese)
范瑾煜, 耿金菊, 王晓蓉. 离子色谱法测定太湖水体中草甘膦含量[C]//中国化学会环境化学专业委员会.第六届全国环境化学大会暨环境科学仪器与分析仪器展览会摘要集. 上海:中国化学会环境化学专业委员会, 2011:222-223 Contardo-Jara V, Klingelmann E, Wiegand C. Bioaccumulation of glyphosate and its formulation Roundup Ultra in Lumbriculus variegatus and its effects on biotransformation and antioxidant enzymes[J]. Environmental Pollution, 2009, 157(1):57-63 Stachowski-Haberkorn S, Becker B, Marie D, et al. Impact of Roundup on the marine microbial community, as shown by an in situ microcosm experiment[J]. Aquatic Toxicology, 2008, 89(4):232-241 Marchand J, Tanguy A, Charrier G, et al. Molecular identification and expression of differentially regulated genes of the European flounder, Platichthys flesus, submitted to pesticide exposure[J]. Marine Biotechnology, 2006, 8(3):275-294 Botta F, Lavison G, Couturier G, et al. Transfer of glyphosate and its degradate AMPA to surface waters through urban sewerage systems[J]. Chemosphere, 2009, 77(1):133-139 Vandenberg L N, Blumberg B, Antoniou M N, et al. Is it time to reassess current safety standards for glyphosate-based herbicides?[J]. Journal of Epidemiology and Community Health, 2017, 71(6):613-618 赵伟, 曹曦予, 吴艳萍, 等. 草甘膦致小鼠机体氧化损伤作用的研究[J]. 毒理学杂志, 2011, 25(5):364-366 Astiz M, de Alaniz M J T, Marra C A. Antioxidant defense system in rats simultaneously intoxicated with agrochemicals[J]. Environmental Toxicology and Pharmacology, 2009, 28(3):465-473 朱国念, 楼正云, 孙锦荷. 草甘膦对水生生物的毒性效应及环境安全性研[J]. 浙江大学学报(农业与生命科学版), 2000, 26(3):309-312 Zhu G N, Lou Z Y, Sun J H. Study on toxicity and environmental safety of glyphosate to aquatic organisms[J]. Journal of Zhejiang Agricultural University (Agric & Life Sci), 2000, 26(3):309-312(in Chinese) 范瑾煜, 耿金菊, 王晓蓉. 草甘膦及Roundup诱导鲫鱼肝脏自由基的产生及其氧化应激[C]//中国化学会. 中国化学会第28届学术年会第2分会场摘要集. 成都:中国化学会, 2012:78 南旭阳. 除草剂"草甘膦"对鲫鱼血细胞及血红蛋白影响的研究[J]. 甘肃科学学报, 2002, 14(4):66-70 Nan X Y. A study of hemoglobin and blood cells in Carassius auratus induced by the herbicide of glyphosate[J]. Journal of Gansu Sciences, 2002, 14(4):66-70(in Chinese)
南旭阳, 张艳丹, 黄小莲. 除草剂"草甘膦"对泥鳅白细胞的影响[J]. 温州师范学院学报(自然科学版), 2003, 24(2):72-74 Nan X Y, Zhang Y D, Huang X L. Effect of Misgurnus anguillicaudatus induced by glyphosate[J]. Journal of Wenzhou Teachers College, 2003, 24(2):72-74(in Chinese) Glusczak L, Miron D D S, Moraes B S, et al. Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)[J]. Comparative Biochemistry and Physiology Toxicology & Pharmacology, 2007, 146(4):519-524 Murussi C R, Costa M D, Leitemperger J W, et al. Exposure to different glyphosate formulations on the oxidative and histological status of Rhamdia quelen[J]. Fish Physiology and Biochemistry, 2016, 42(2):445-455 Çavaş T, Könen S. Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay[J]. Mutagenesis, 2007, 22(4):263-268 Lushchak O V, Kubrak O I, Storey J M, et al. Low toxic herbicide Roundup induces mild oxidative stress in goldfish tissues[J]. Chemosphere, 2009, 76(7):932-937 Folmar L C, Sanders H O, Julin A M. Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates[J]. Archives of Environmental Contamination and Toxicology, 1979, 8(3):269-278 Jiraungkoorskul W, Upatham E S, Kruatrachue M, et al. Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus)[J]. Environmental Toxicology, 2003, 18(4):260-267 Topal A, Atamanalp M, Uçar A, et al. Effects of glyphosate on juvenile rainbow trout (Oncorhynchus mykiss):Transcriptional and enzymatic analyses of antioxidant defence system, histopathological liver damage and swimming performance[J]. Ecotoxicology and Environmental Safety, 2015, 111:206-214 Armiliato N, Ammar D, Nezzi L, et al. Changes in ultrastructure and expression of steroidogenic factor-1 in ovaries of zebrafish Danio rerio exposed to glyphosate[J]. Journal of Toxicology and Environmental Health Part A, 2014, 77(7):405-414 傅建炜, 史梦竹, 李建宇, 等. 草甘膦对草鱼、鲢鱼和鲫鱼的毒性[J]. 生物安全学报, 2013, 22(2):119-122 Fu J W, Shi M Z, Li J Y, et al. Toxicity of glyphosate on grass carp (Ctenopharyngodon idellus), chub (Hypophthalmictuthys molitrix) and crucian (Carassius auratus)[J]. Journal of Biosafety, 2013, 22(2):119-122(in Chinese)
廖艺钰, 惠吕佳, 严吉祥, 等. 草甘膦农药对斑马鱼的急性毒性和慢性毒性研究[J]. 广州化工, 2020, 48(21):66-68 Liao Y Y, Hui L J, Yan J X, et al. Study on acute and chronic toxicity of glyphosate to zebrafish[J]. Guangzhou Chemical Industry, 2020, 48(21):66-68(in Chinese)
Clark M D, Hennig S, Herwig R, et al. An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library[J]. Genome Research, 2001, 11(9):1594-1602 Schulte C, Nagel R. Testing acute toxicity in the embryo of zebrafish, Brachydanio rerio, as an alternative to the acute fish test:Preliminary results[J]. Alternatives to Laboratory Animal, 1994, 22:12-19 Oberemm A. The use of a refined zebrafish embryo bioassay for the assessment of aquatic toxicity[J]. Lab Animal, 2000, 29(7):32-40 Organisation for Economic Co-operation and Development (OECD). Draft proposal for a new guideline fish two generation test guideline[R]. Paris:OECD, 2002 Hiroi Y, Kudoh S, Monzen K, et al. Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation[J]. Nature Genetics, 2001, 28(3):276-280 Mak K K, Kronenberg H M, Chuang P T, et al. Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy[J]. Development, 2008, 135(11):1947-1956 Kruithof B P, Xu J W, Fritz D T, et al. An in vivo map of bone morphogenetic protein 2 post-transcriptional repression in the heart[J]. Genesis, 2011, 49(11):841-850 Westerfield M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio)[M]. Eugene:University of Oregon Press, 2000:28-45 吴艳娣. 几种典型持久性有机污染物对斑马鱼胚胎发育的毒性效应研究[D]. 武汉:华中农业大学, 2008:17-18 Wu Y D. Study on the toxicity of several typical POPs exposed to zebrafish (Danio rerio) embryos[D]. Wuhan:Huazhong Agricultural University, 2008:17 -18(in Chinese)
Walker M K, Peterson R E. Aquatic Toxicity of Dioxins and Related Chemicals[M]//Schecter A. Dioxins and Health. Boston, MA:Springer, 1994:347-387 陈粉丽, 张松林, 陈红梅, 等. 氯代苯类化合物对斑马鱼胚胎的单一急性毒性[J]. 环境与职业医学, 2010, 27(8):472-475 Chen F L, Zhang S L, Chen H M, et al. Individual acute toxicity of chlorobenzenes on zebrafish embryo[J]. Journal of Environmental & Occupational Medicine, 2010, 27(8):472-475(in Chinese)
Goetz A K, Ren H Z, Schmid J E, et al. Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat[J]. Toxicological Sciences:An Official Journal of the Society of Toxicology, 2007, 95(1):227-239 Mu X Y, Pang S, Sun X Z, et al. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays[J]. Environmental Pollution, 2013, 175:147-157 Wu Y Q, Zuo Z H, Chen M, et al. The developmental effects of low-level procymidone towards zebrafish embryos and involved mechanism[J]. Chemosphere, 2018, 193:928-935 Wu Y Q, Yang Q H, Chen M, et al. Fenbuconazole exposure impacts the development of zebrafish embryos[J]. Ecotoxicology and Environmental Safety, 2018, 158:293-299 Organization for Economic Cooperation and Development (OECD). Guidelines for the testing of chemicals. Section 2:Effects on biotic systems test No.236:Fish embryo acute toxicity (FEAT) test[S]. Paris:OECD, 2013 Glickman N S, Yelon D. Cardiac development in zebrafish:Coordination of form and function[J]. Seminars in Cell & Developmental Biology, 2002, 13(6):507-513 Barrionuevo W R, Burggren W W. O2 consumption and heart rate in developing zebrafish (Danio rerio):Influence of temperature and ambient O2[J]. The American Journal of Physiology, 1999, 276(2):R505-R513 Kodde I F, van der Stok J, Smolenski R T, et al. Metabolic and genetic regulation of cardiac energy substrate preference[J]. Comparative Biochemistry and Physiology Part A, Molecular & Integrative Physiology, 2007, 146(1):26-39 Limas C J, Olivari M T, Goldenberg I F, et al. Calcium uptake by cardiac sarcoplasmic reticulum in human dilated cardiomyopathy[J]. Cardiovascular Research, 1987, 21(8):601-605 Yadwad V B, Kallapur V L, Basalingappa S. Inhibition of gill Na+ K+-ATPase activity in dragonfly larva, Pantala flavesens, by endosulfan[J]. Bulletin of Environmental Contamination and Toxicology, 1990, 44(4):585-589 王重刚, 余群, 郁昂, 等. 苯并(a)芘和芘暴露对梭鱼肝脏超氧化物歧化酶活性的影响[J]. 海洋环境科学, 2002, 21(4):10-13 Wang C G, Yu Q, Yu A, et al. Effect of benzo(a)pyrene and pyrene exposure on hepatic superoxide dismutase in Mugil so-iuy[J]. Marine Environmental Science, 2002, 21(4):10-13(in Chinese)
张玉, 张建明, 刘海涛, 等. 内蒙古不同水质湖泊瓦氏雅罗鱼Na+, K+-ATP酶活性的研究[J]. 内蒙古农业大学学报(自然科学版), 2013, 34(1):1-4 Zhang Y, Zhang J M, Liu H T, et al. Inner Mongolian different lake Valsalva jarraud fish study of blood physiological parameters[J]. Journal of Inner Mongolia Agricultural University (Natural Science Edition), 2013, 34(1):1-4(in Chinese) 夏勇, 傅剑云, 郑云燕, 等. 丁草胺对斑马鱼及其胚胎发育毒性影响的研究[J]. 浙江预防医学, 2011, 23(1):11-16 Xia Y, Fu J Y, Zheng Y Y, et al. Study on toxicity of butachlor on zebrafish and its embryo development[J]. Zhejiang Journal of Preventive Medicine, 2011, 23(1):11-16(in Chinese)
Garg V, Kathiriya I S, Barnes R, et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5[J]. Nature, 2003, 424(6947):443-447 Takeuchi J K, Bruneau B G. Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors[J]. Nature, 2009, 459(7247):708-711 Moskowitz I P, Pizard A, Patel V V, et al. The T-Box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system[J]. Development, 2004, 131(16):4107-4116 Chapman D L, Garvey N, Hancock S, et al. Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development[J]. Developmental Dynamics:An Official Publication of the American Association of Anatomists, 1996, 206(4):379-390 Bruneau B G, Nemer G, Schmitt J P, et al. A murine model of holt-oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease[J]. Cell, 2001, 106(6):709-721 Reiter J F, Verkade H, Stainier D Y R. Bmp2b and oep promote early myocardial differentiation through their regulation of gata5[J]. Developmental Biology, 2001, 234(2):330-338 Balci M M, Akdemir R. NKX2.5 mutations and congenital heart disease:Is it a marker of cardiac anomalies?[J]. International Journal of Cardiology, 2011, 147(3):e44-e45 Targoff K L, Colombo S, George V, et al. Nkx genes are essential for maintenance of ventricular identity[J]. Development, 2013, 140(20):4203-4213 Wu Y Q, Xu Z Y, Xu X B, et al. Exposure to metalaxyl disturbs the skeletal development of zebrafish embryos[J]. Bulletin of Environmental Contamination and Toxicology, 2020, 104(4):432-437 -
点击查看大图
计量
- 文章访问数: 1812
- HTML全文浏览数: 1812
- PDF下载数: 121
- 施引文献: 0
下载:
