邻苯二甲酸二(2-乙基己基)酯对罗非鱼肝脏转录组影响研究
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摘要
邻苯二甲酸二(2-乙基己基)酯(DEHP)作为生产量和使用量最多的一种邻苯二甲酸酯类化合物,其对水生生物具有多种毒性.本研究选用中国南方地区最重要的经济鱼类尼罗罗非鱼(Oreochromis niloticus)作为实验对象,研究DEHP(50mg/kg bw)作用下罗非鱼肝脏组织内转录组的响应特征.利用IlluminaTM HiSeq 4000测序平台对DEHP处理组和对照组尼罗罗非鱼肝脏RNA样品分别进行转录组测序.对照组和DEHP暴露组分别获得30.10Mb和30.16Mb待分析数据(clean reads),对转录组数据进行组装并去冗余后得到58,585个Unigene.将DEHP处理组和对照组的转录组数据进行比较一共获得5,008个差异表达基因,其中上调表达基因和下调表达基因分别为3,217个和1,791个.通过GO和KEGG功能分析后发现这些差异表达基因主要为机体免疫、生殖内分泌以及脂类代谢相关基因,这表明DEHP对尼罗罗非鱼肝脏毒性主要表现为免疫毒性、生殖毒性和脂类代谢紊乱.另外,实时荧光定量PCR验证结果发现转录组分析数据基本可靠.上述结果为在转录组水平筛选DEHP生物标志物,解析DEHP对罗非鱼毒性作用的分子机制提供了科学参考.
Di-(2-ethylhexyl) phthalate(DEHP) is currently the most frequently detected phthalic acid esters(PAEs) and can induce diverse toxicities on aquatic organisms. To understand the transcriptomic responses of fish exposed to DEHP, we performed transcriptomic profiles in liver of tilapia(Oreochromis niloticus), which is the most important commercial fishes in Guangdong province. Transcriptome sequencing in liver of Nile tilapia exposed to olive oil(control group) and 50 mg kg-1body weight of DEHP for 7days was performed respectively using Illumina HiSeq 4000 platform. A total of 30.10 Mb and 30.16 Mb clean reads were retrieved from the control and DEHP treated libraries, respectively. De-novo assembly of all the clean reads obtained 58,585 unigenes. After comparing the two libraries, 3,217 and 1,791 genes were identified as significantly increased and depressed, respectively. Gene ontology(GO) classification system and Kyoto Encyclopedia of Genes and Genomes(KEGG) database analysis demonstrated that DEHP significantly disturbed the expression level of genes associated with immunity, endocrine and reproductive system, lipid metabolism and so on. Quantitative real-time PCR was performed to validate the results of RNA-sequencing(RNA-seq) analysis. The resulting data provide new insights for exploring the molecular basis of tilapia response to DEHP exposure.
Di-(2-ethylhexyl) phthalate(DEHP) is currently the most frequently detected phthalic acid esters(PAEs) and can induce diverse toxicities on aquatic organisms. To understand the transcriptomic responses of fish exposed to DEHP, we performed transcriptomic profiles in liver of tilapia(Oreochromis niloticus), which is the most important commercial fishes in Guangdong province. Transcriptome sequencing in liver of Nile tilapia exposed to olive oil(control group) and 50 mg kg-1body weight of DEHP for 7days was performed respectively using Illumina HiSeq 4000 platform. A total of 30.10 Mb and 30.16 Mb clean reads were retrieved from the control and DEHP treated libraries, respectively. De-novo assembly of all the clean reads obtained 58,585 unigenes. After comparing the two libraries, 3,217 and 1,791 genes were identified as significantly increased and depressed, respectively. Gene ontology(GO) classification system and Kyoto Encyclopedia of Genes and Genomes(KEGG) database analysis demonstrated that DEHP significantly disturbed the expression level of genes associated with immunity, endocrine and reproductive system, lipid metabolism and so on. Quantitative real-time PCR was performed to validate the results of RNA-sequencing(RNA-seq) analysis. The resulting data provide new insights for exploring the molecular basis of tilapia response to DEHP exposure.
引文
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[14]Xiang N,Zhao C,Diao X,et al.Dynamic responses of antioxidant enzymes in pearl oyster Pinctada martensii exposed to di(2-ethylhexyl)phthalate(DEHP)[J].Environ Toxicol Pharmacol,2017,54:184-190.
[15]Ye T,Kang M,Huang Q,et al.Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka(Oryzias melastigma)[J].Aquatic Toxicology,2014,146(146C):115-126.
[16]Yuan L,Li M,Meng F,et al.Growth,blood health,antioxidant status,immune response and resistance to Aeromonas hydrophila of juvenile yellow catfish exposed to di-2-ethylhexyl phthalate(DEHP)[J].Comparative Biochemistry Physiology,2017,202C:79-84.
[17]Li C,Chen X,Zhang P,et al.Di-(2-ethylhexyl)phthalate mediates glycolysis and the TCA cycle in clam Venerupis philippinarum[J].Invertebrate Survival Journal,2014,11:174-181.
[18]周远扬,王玉梅,曹俊明,等.2015年广东罗非鱼产业发展形势与对策建议[J].广东农业科学,2016,43(6):23-27.Zhou Y Y,Wang Y M,Cao J M,et al.Development situation and countermeasures of Guangdong tilapia industry in 2015[J].Guangdong Agricultural Sciences,2016,43(6):23-27.
[19]余嘉欣,吴芳,孙成飞,等.尼罗罗非鱼γ-干扰素基因的克隆、结构分析及组织表达[J].南方水产科学,2017,(13)1:85-93.Yu J X,Wu F,Sun C F,et al.Cloning,structure analysis and expression of interferon gamma(IFN-γ)gene in Oreochromis niloticus[J].South China Fisheries Science,2017,(13)1:85-93.
[20]Grabherr M G,Haas B J,Yassour M,et al.Trinity:reconstructing a full-length transcriptome without a genome from RNA-Seq data[J].Nature Biotechnology,2011,29(7):644-652.
[21]Pertea G,Huang X,Liang F,et al.TIGR Gene Indices clustering tools(TGICL):a software system for fast clustering of large EST datasets[J].Bioinformatics,2003,19(5):651-652.
[22]Li B,Dewey CN.RSEM:accurate transcript quantification from RNA-Seq data with or without a reference genome[J].BMCBioinformatics,2011,12(1):323.
[23]Benjamini Y,Yekutieli D.The control of the false discovery rate in multiple testing under dependency[J].Annals of Statistics,2001,29(4):1165-1188.
[24]Griffitt R J,Lavelle C M,Kane A S,et al.Chronic nanoparticulate silver exposure results in tissue accumulation and transcriptomic changes in zebrafish[J].Aquatic Toxicology,2013,130(2):192-200.
[25]Jiang X,Qiu L,Zhao H,et al.Transcriptomic responses of Perna viridis embryo to benzo(a)pyrene exposure elucidated by RNAsequencing[J].Chemosphere,2016,163:125-132.
[26]Moreira R,Balseiro P,Planas J V,et al.Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing[J].Plos One,2012,7(4):e35009.
[27]金伯泉.细胞和分子免疫学(第二版)[M].北京:科学出版社,2001.Jin B Q.Cellular and molecular immunology[M].2nd ed.Beijing:Science Press,2001.
[28]Volanakis J E,Frank M.The human complement system in health and disease[D].New York:Marcel Deiierk Inc,1998.
[29]Ghai R,Waters P,Roumenina L T,et al.C1q and its growing family[J].Immunobiology,2007,212(4/5):253-266.
[30]Bennett J E,Dolin R,Blaser M J.Principles and Practice of Infectious Diseases[D].Elsevier Health Sciences,2014.
[31]Mix M C.Shellfish diseases in relation to toxic chemicals[J].Aquatic.Toxicology,1988,11(1):29-42.
[32]Martins K,Applegate B,Hagedorn B,et al.Di(2-ethylhexyl)phthalate inhibits B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues of the rainbow trout[J].Fish Shellfish Immunology,2015,44(1):332-341.
[33]Lu Y,Zhang P,Li C,et al.Characterisation of immune-related gene expression in clam(Venerupis philippinarum)under exposure to di(2-ethylhexyl)phthalate[J].Fish&Shellfish Immunology,2013,34(1):142-146.
[34]Wang Y X,Zeng Q,Sun Y,et al.Phthalate exposure in association with serum hormone levels,sperm DNA damage and spermatozoa apoptosis:A cross-sectional study in China[J].Environmental Research,2016,150:557-565.
[35]Zarean M,Keikha M,Poursafa P,et al.A systematic review on the adverse health effects of di-2-ethylhexyl phthalate[J].Environmental Science and Pollution Research,2016,23(24):24642-24693.
[36]Chen T T,Reid P C,Beneden R V,et al.Effect of Aroclor 1254and mirex on estradiol-lnduced vitellogenin production in juvenile rainbow trout(Salmo gairdneri)[J].Journal Canadien Des Sciences Halieutiques Et Aquatiques,1986,43(1):169-173.
[37]Kim E J,Kim J W,Lee S K.Inhibition of oocyte development in Japanese medaka(Oryzias latipes)exposed to di-2-ethylhexyl phthalate[J].Environment International,2003,28(5):359-365.
[38]Urenwebster T M,Lewis C,Filby A L,et al.Mechanisms of toxicity of di(2-ethylhexyl)phthalate on the reproductive health of male zebrafish[J].Aquatic Toxicology,2010,99(3):360-369.
[39]Wang X,Yang Y,Zhang L,et al.Endocrine disruption by di-(2-ethylhexyl)-phthalate in Chinese rare minnow(Gobiocypris rarus)[J].Environmental Toxicology Chemistry,2013,32(8):1846-1854.
[40]Nanton D A,Vegusdal A,Amb R,et al.Muscle lipid storage pattern,composition,and adipocyte distribution in different parts of Atlantic salmon(Salmo salar)fed fish oil and vegetableoil[J].Aquaculture,2007,265(1):230-243.
[41]Wilbanks M S,Gust K A,Atwa S,et al.Validation of a genomicsbased hypothetical adverse outcome pathway:2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance[J].Toxicological Sciences,2014,141(1):44-58.
[42]Jia Y,Wu C,Kim J,et al.Astaxanthin reduces hepatic lipid accumulations in high-fat-fed C57BL/6J mice via activation of peroxisome proliferator-activated receptor(PPAR)alpha and inhibition of PPAR gamma and Akt[J].Journal of Nutritional Biochemistry,2016,28:9-18.
[43]Chmurzyńska A.The multigene family of fatty acid-binding proteins(FABPs):Function,structure and polymorphism[J].Journal of Applied Genetics,2006,47(1):39-48.
[44]Nakamura M T,Nara T Y.Structure,junction,and dietary regulation of Delta 6,Delta 5,and Delta 9desaturases[J].Annual Review of Nutrition,2004,24:345-376.
[45]Huang C,Chen Q L,Luo Z,et al.Time-dependent effects of waterborne copper exposure influencing hepatic lipid deposition and metabolism in javelin goby Synechogobius hasta and their mechanism[J].Aquatic Toxicology,2014,155(4):291-300.
[46]Xu Y,Agrawal S,Cook T J,et al.Maternal di-(2-ethylhexyl)-phthalate exposure influences essential fatty acid homeostasis in rat placenta[J].Placenta,2008,29(11):962-969.
[2]Ma T,Zhou W,Chen L,et al.Toxicity effects of di-(2-ethylhexyl)phthalate to Eisenia fetida at enzyme,cellular and genetic levels[J].PLoS ONE,2017,12(3):e0173957.
[3]李潇,聂湘平,潘德博,等.养殖鱼体邻苯二甲酸酯含量与分布特征[J].环境与健康杂志,2008,25(3):202-205.Li X,Nie X P,Pan D B,et al.Analysis of PAEs in muscle tissue of freshwater fish from fishponds in Pearl River Delta[J].Journal of Environmental Health,2008,25(3):202-205.
[4]Cheng Z,Nie X P,Wang H S,et al.Risk assessments of human exposure to bioaccessible phthalate esters through market fish consumption[J].Environment International,2013,57-58:75-80.
[5]林钦,石凤琼,柯常亮,等.水产品中邻苯二甲酸酯残留与健康风险评价研究进展[J].南方水产科学,2014,(10)1:92-99.Lin Q,Shi F Q,Ke C L,et al.Residues of phthalic acid esters in aquatic products and their health risk assessment[J].South China Fisheries Science,2014,(10)1:92-99.
[6]Li C,Zhao Y,Li L,et al.Exposure assessment of phthalates in non-occupational populations in China[J].Science of the Total Environment,2012,427-428(12):60-69.
[7]Kong S,Ji Y,Liu L,et al.Diversities of phthalate esters in suburban agricultural soils and wasteland soil appeared with urbanization in China[J].Environmental Pollution,2012,170(8):161-168.
[8]Liu N,Wang Y,Yang Q,et al.Probabilistic assessment of risks of diethylhexyl phthalate(DEHP)in surface waters of China on reproduction of fish[J].Environmental Pollution,2016,213(213):482-488.
[9]Zhan Y,Sun J,Luo Y,et al.Estimating emissions and environmental fate of di-(2-ethylhexyl)phthalate in Yangtze River Delta,China:Application of inverse modeling[J].Environmental Science&Technology,2016,50(5):2450-2458.
[10]Zhang L,Liu J,Liu H,et al.The occurrence and ecological risk assessment of phthalate esters(PAEs)in urban aquatic environments of China[J].Ecotoxicology,2016,24(5):1-18.
[11]Huang Q,Chen Y,Chi Y,et al.Immunotoxic effects of perfluorooctane sulfonate and di(2-ethylhexyl)phthalate on the marine fish Oryzias melastigma[J].Fish&Shellfish Immunology,2015,44(1):302-306.
[12]Ma T,Zhou W,Chen L,et al.Toxicity effects of di-(2-ethylhexyl)phthalate to Eisenia fetida at enzyme,cellular and genetic levels[J].Plos One,2017,12(3):e0173957.
[13]Meng F X,Li M,Song M Z,et al.Di-2-ethylhexyl phthalate(DEHP)exposure disturbs lipid metabolism in juvenile yellow catfish Tachysurus fulvidraco[J].Journal of Fish Biology,2017,92:85-93.
[14]Xiang N,Zhao C,Diao X,et al.Dynamic responses of antioxidant enzymes in pearl oyster Pinctada martensii exposed to di(2-ethylhexyl)phthalate(DEHP)[J].Environ Toxicol Pharmacol,2017,54:184-190.
[15]Ye T,Kang M,Huang Q,et al.Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka(Oryzias melastigma)[J].Aquatic Toxicology,2014,146(146C):115-126.
[16]Yuan L,Li M,Meng F,et al.Growth,blood health,antioxidant status,immune response and resistance to Aeromonas hydrophila of juvenile yellow catfish exposed to di-2-ethylhexyl phthalate(DEHP)[J].Comparative Biochemistry Physiology,2017,202C:79-84.
[17]Li C,Chen X,Zhang P,et al.Di-(2-ethylhexyl)phthalate mediates glycolysis and the TCA cycle in clam Venerupis philippinarum[J].Invertebrate Survival Journal,2014,11:174-181.
[18]周远扬,王玉梅,曹俊明,等.2015年广东罗非鱼产业发展形势与对策建议[J].广东农业科学,2016,43(6):23-27.Zhou Y Y,Wang Y M,Cao J M,et al.Development situation and countermeasures of Guangdong tilapia industry in 2015[J].Guangdong Agricultural Sciences,2016,43(6):23-27.
[19]余嘉欣,吴芳,孙成飞,等.尼罗罗非鱼γ-干扰素基因的克隆、结构分析及组织表达[J].南方水产科学,2017,(13)1:85-93.Yu J X,Wu F,Sun C F,et al.Cloning,structure analysis and expression of interferon gamma(IFN-γ)gene in Oreochromis niloticus[J].South China Fisheries Science,2017,(13)1:85-93.
[20]Grabherr M G,Haas B J,Yassour M,et al.Trinity:reconstructing a full-length transcriptome without a genome from RNA-Seq data[J].Nature Biotechnology,2011,29(7):644-652.
[21]Pertea G,Huang X,Liang F,et al.TIGR Gene Indices clustering tools(TGICL):a software system for fast clustering of large EST datasets[J].Bioinformatics,2003,19(5):651-652.
[22]Li B,Dewey CN.RSEM:accurate transcript quantification from RNA-Seq data with or without a reference genome[J].BMCBioinformatics,2011,12(1):323.
[23]Benjamini Y,Yekutieli D.The control of the false discovery rate in multiple testing under dependency[J].Annals of Statistics,2001,29(4):1165-1188.
[24]Griffitt R J,Lavelle C M,Kane A S,et al.Chronic nanoparticulate silver exposure results in tissue accumulation and transcriptomic changes in zebrafish[J].Aquatic Toxicology,2013,130(2):192-200.
[25]Jiang X,Qiu L,Zhao H,et al.Transcriptomic responses of Perna viridis embryo to benzo(a)pyrene exposure elucidated by RNAsequencing[J].Chemosphere,2016,163:125-132.
[26]Moreira R,Balseiro P,Planas J V,et al.Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing[J].Plos One,2012,7(4):e35009.
[27]金伯泉.细胞和分子免疫学(第二版)[M].北京:科学出版社,2001.Jin B Q.Cellular and molecular immunology[M].2nd ed.Beijing:Science Press,2001.
[28]Volanakis J E,Frank M.The human complement system in health and disease[D].New York:Marcel Deiierk Inc,1998.
[29]Ghai R,Waters P,Roumenina L T,et al.C1q and its growing family[J].Immunobiology,2007,212(4/5):253-266.
[30]Bennett J E,Dolin R,Blaser M J.Principles and Practice of Infectious Diseases[D].Elsevier Health Sciences,2014.
[31]Mix M C.Shellfish diseases in relation to toxic chemicals[J].Aquatic.Toxicology,1988,11(1):29-42.
[32]Martins K,Applegate B,Hagedorn B,et al.Di(2-ethylhexyl)phthalate inhibits B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues of the rainbow trout[J].Fish Shellfish Immunology,2015,44(1):332-341.
[33]Lu Y,Zhang P,Li C,et al.Characterisation of immune-related gene expression in clam(Venerupis philippinarum)under exposure to di(2-ethylhexyl)phthalate[J].Fish&Shellfish Immunology,2013,34(1):142-146.
[34]Wang Y X,Zeng Q,Sun Y,et al.Phthalate exposure in association with serum hormone levels,sperm DNA damage and spermatozoa apoptosis:A cross-sectional study in China[J].Environmental Research,2016,150:557-565.
[35]Zarean M,Keikha M,Poursafa P,et al.A systematic review on the adverse health effects of di-2-ethylhexyl phthalate[J].Environmental Science and Pollution Research,2016,23(24):24642-24693.
[36]Chen T T,Reid P C,Beneden R V,et al.Effect of Aroclor 1254and mirex on estradiol-lnduced vitellogenin production in juvenile rainbow trout(Salmo gairdneri)[J].Journal Canadien Des Sciences Halieutiques Et Aquatiques,1986,43(1):169-173.
[37]Kim E J,Kim J W,Lee S K.Inhibition of oocyte development in Japanese medaka(Oryzias latipes)exposed to di-2-ethylhexyl phthalate[J].Environment International,2003,28(5):359-365.
[38]Urenwebster T M,Lewis C,Filby A L,et al.Mechanisms of toxicity of di(2-ethylhexyl)phthalate on the reproductive health of male zebrafish[J].Aquatic Toxicology,2010,99(3):360-369.
[39]Wang X,Yang Y,Zhang L,et al.Endocrine disruption by di-(2-ethylhexyl)-phthalate in Chinese rare minnow(Gobiocypris rarus)[J].Environmental Toxicology Chemistry,2013,32(8):1846-1854.
[40]Nanton D A,Vegusdal A,Amb R,et al.Muscle lipid storage pattern,composition,and adipocyte distribution in different parts of Atlantic salmon(Salmo salar)fed fish oil and vegetableoil[J].Aquaculture,2007,265(1):230-243.
[41]Wilbanks M S,Gust K A,Atwa S,et al.Validation of a genomicsbased hypothetical adverse outcome pathway:2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance[J].Toxicological Sciences,2014,141(1):44-58.
[42]Jia Y,Wu C,Kim J,et al.Astaxanthin reduces hepatic lipid accumulations in high-fat-fed C57BL/6J mice via activation of peroxisome proliferator-activated receptor(PPAR)alpha and inhibition of PPAR gamma and Akt[J].Journal of Nutritional Biochemistry,2016,28:9-18.
[43]Chmurzyńska A.The multigene family of fatty acid-binding proteins(FABPs):Function,structure and polymorphism[J].Journal of Applied Genetics,2006,47(1):39-48.
[44]Nakamura M T,Nara T Y.Structure,junction,and dietary regulation of Delta 6,Delta 5,and Delta 9desaturases[J].Annual Review of Nutrition,2004,24:345-376.
[45]Huang C,Chen Q L,Luo Z,et al.Time-dependent effects of waterborne copper exposure influencing hepatic lipid deposition and metabolism in javelin goby Synechogobius hasta and their mechanism[J].Aquatic Toxicology,2014,155(4):291-300.
[46]Xu Y,Agrawal S,Cook T J,et al.Maternal di-(2-ethylhexyl)-phthalate exposure influences essential fatty acid homeostasis in rat placenta[J].Placenta,2008,29(11):962-969.