有机磷阻燃剂甲状腺干扰效应及其作用机制研究——以磷酸三(2-氯丙基)酯(TCPP)为例
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摘要
以典型有机磷阻燃剂磷酸三(2-氯丙基)酯(TCPP)为研究对象,应用GH3细胞增殖实验检测TCPP对甲状腺激素的干扰效应;应用重组甲状腺激素受体(TR)基因酵母实验和GH3(TRβ-)细胞增殖实验结合实时定量PCR技术初步探究TCPP甲状腺激素干扰作用机理.结果表明,TCPP在1×10~(-4)mol/L和2×10~(-4)mol/L浓度下对甲状腺激素T3诱导的GH3细胞增殖产生抑制效应;重组TR基因酵母实验和GH3(TRβ-)细胞增殖实验测试结果表明TCPP可能通过TR介导的基因组途径和非基因组途径诱导甲状腺激素干扰效应;实时定量PCR测试结果表明TCPP下调相关基因如:c-fos、TRβ、integrin-av和k-ras的m RNA表达,初步认为TCPP可能通过影响TRβ基因表达和激活αvβ3-ERK-1/2信号通路产生甲状腺激素干扰效应.
In the present study, typical organophosphorus flame retardants(OPFRs), tris(2-chloropropyl) phosphate(TCPP), was selected as the object of study. GH3 cell proliferation assay was used to test the thyroid disrupting activity of TCPP; a two-hybrid thyroid receptor(TR) yeast assay, GH3(TRβ-) cell proliferation assay and real time quantitative PCR were used to investigate the mechanism of action. The results revealed that TCPP decreased the GH3 cell proliferation at concentrations ranging from 1×10~(-4) mol/L to 2×10~(-4) mol/L. Furthermore, the results from two-hybrid TR yeast assay and GH3(TRβ-) cell proliferation assay suggested that TCPP could disrupt thyroid hormone activity through genomic and non-genomic pathway. The results of real time quantitative PCR supported the m RNA expression levels of c-fos, TRβ, integrin-av and k-ras were inhibited, suggesting that TCPP might induce thyroid hormone disruption by adjusting TRβ gene expression and activating αvβ3-ERK-1/2 signaling pathway. All of these results could provide the basic data and theoretical support for the environmental risk assessment of TCPP.
In the present study, typical organophosphorus flame retardants(OPFRs), tris(2-chloropropyl) phosphate(TCPP), was selected as the object of study. GH3 cell proliferation assay was used to test the thyroid disrupting activity of TCPP; a two-hybrid thyroid receptor(TR) yeast assay, GH3(TRβ-) cell proliferation assay and real time quantitative PCR were used to investigate the mechanism of action. The results revealed that TCPP decreased the GH3 cell proliferation at concentrations ranging from 1×10~(-4) mol/L to 2×10~(-4) mol/L. Furthermore, the results from two-hybrid TR yeast assay and GH3(TRβ-) cell proliferation assay suggested that TCPP could disrupt thyroid hormone activity through genomic and non-genomic pathway. The results of real time quantitative PCR supported the m RNA expression levels of c-fos, TRβ, integrin-av and k-ras were inhibited, suggesting that TCPP might induce thyroid hormone disruption by adjusting TRβ gene expression and activating αvβ3-ERK-1/2 signaling pathway. All of these results could provide the basic data and theoretical support for the environmental risk assessment of TCPP.
引文
[1]艾扬,孔东东,于畅,等.水环境中溶解态腐殖酸对锌抗甲状腺激素干扰效应的影响[J].环境科学,2017,38(1):195-200.
[2]Murk A.J,Rijntjes E,Blaauboer B J,et al.Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals[J].Toxicology in Vitro,2013,27(4):1320-1346.
[3]Shi W,Hu X X,Zhang F X,et al.Occurrence of thyroid hormone activities in drinking water from Eastern China:Contributions of phthalate esters[J].Environmental Science&Technology,2012,46(3):1811-1818.
[4]Boas M,Feldt-Rasmussen U,Main K M.Thyroid effects of endocrine disrupting chemicals[J].Molecular and Cellular Endocrinology,2012,355(2):240-248.
[5]穆希岩,李成龙,黄瑛,等.两种邻苯二甲酸酯类污染物对斑马鱼胚胎发育的影响[J].中国环境科学,2017,(9):3566-3575.
[6]王晓伟,刘景富,阴永光.有机磷酸酯阻燃剂污染现状与研究进展[J].化学进展,2010,22(10):1983-1992.
[7]Zhao F R,Chen M,Gao F M,et al.Organophosphorus flame retardants in pregnant women and their transfer to chorionic Villi[J].Environmental Science&Technology,2017,51(11):6489-6497.
[8]Khan M,Li,J,Zhang,G et al.First insight into the levels and distribution of flame retardants in potable water in Pakistan:An underestimated problem with an associated health risk diagnosis[J].Science of Total Environment,2016,565:346-359.
[9]Farhat A,Crump D,Chiu S,et al.In ovo effects of two organophosphate flame retardants-TCPP and TDCPP-on pipping success,development,m RNA expression,and thyroid hormone levels in chicken embryos[J].Toxicological Sciences,2013,134(1):92-102.
[10]van der Veen I,de Boer J.Phosphorus flame retardants:Properties,production,environmental occurrence,toxicity and analysis[J].Chemosphere,2012,88(10):1119-1153.
[11]皮天星,蔡磊明,蒋金花.新型阻燃剂TCPP对斑马鱼的毒性研究[J].生态毒理学报,2016,11(2):247-256.
[12]陆美娅.雌激素受体ER和甲状腺激素受体TR介导的典型环境内分泌干扰物效应研究[D].杭州:浙江工业大学,2015.
[13]Kojima H,Takeuchi S,Itoh T,et al.In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors[J].Toxicology,2013,314(1):76-83.
[14]Davis P J,Lin H Y,Mousa SA,et al.Overlapping nongenomic and genomic actions of thyroid hormone and steroids[J].Steroids,2011,76(9):829-833.
[15]Davis P J,Leonard J L,Davis F B.Mechanisms of nongenomic actions of thyroid hormone[J].Frontiers in Neuroendocrinology,2008,29(2):211-218.
[16]Liu C,Ha M,Cui Y,et al.JNK pathway decreases thyroid hormones via TRH receptor:A novel mechanism for disturbance of thyroid hormone homeostasis by PCB153[J].Toxicology,2012,302(1):68-76.
[17]周君.p,p'-DDE,PCB-153单独及联合作用通过ERK1/2信号转导通路对大鼠离体脑垂体细胞的类激素效应[D].武汉:华中科技大学,2013.
[18]Li J,Ma M,Wang Z J.A two-hybrid yeast assay to quantify the effects of xenobiotics on thyroid hormone-mediated gene expression[J].Environmental Toxicology and Chemistry,2008,27(1):159-167.
[19]刘芸,李剑,马梅,等.代谢活化作用对多溴代联苯类污染物类/抗甲状腺激素活性的影响[J].生态毒理学报,2008,3(1):27-33.
[20]Schriks M.,Vrabie C M,Gutleb A C,et al.T-screen to quantify functional potentiating,antagonistic and thyroid hormone-like activities of poly halogenated aromatic hydrocarbons(PHAHs)[J].Toxicology in Vitro,2006,20(4):490-498.
[21]Pruden A,Pei R T,Stortrboom H,et al.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado[J].Environmental Science and Technology,2006,40(23):7445-7450.
[22]李剑,任姝娟,马梅,等.改进型重组基因酵母TR-GRIP1检测化合物甲状腺激素干扰活性[J].环境科学研究,2011,24(10):1172-1177.
[23]文育,雷炳莉,康佳,等.双酚A类同系物的雌激素效应及对MCF-7细胞的毒性[J].上海大学学报(自然科学版),2015,21(4):515-524.
[24]Li J,Liu Y,Kong D,et al.T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium,mercury,and zinc[J].Environmental Science and Pollution Research,2016,23(10):9843-9851.
[25]Techer R,Houde M,Verreault J.Associations between organohalogen concentrations and transcription of thyroid-related genes in a highly contaminated gull population[J].Science of Total Environment,2016,545:289-298.
[26]Dingemans M M L,van den Berg M,Westerink RHS.Neurotoxicity of brominated flame retardants:(in)direct effects of parent and hydroxylated polybrominated diphenyl Ethers on the(Developing)Nervous System[J].Environmental Health Perspectives,2011,119(7):900-907.
[27]Sandler B,Webb P,Apriletti J W.Thyroxine-thyroid hormone receptor interactions[J].Journal of Biological Chemistry,2004,279(53):55801-55808.
[28]Arukwe A,Jenssen B M.Differential organ expression patterns of thyroid hormone receptor isoform genes in p.p’-DDE-treated adult male common frog Rana temporaria[J].Environmental Toxicology and Pharmacology,2005,20:485-492.
[29]Scarlett A,Parsons M P,Hanson P L,et al.Thyroid hormone stimulation of extracellular signal-regulated kinase and cell proliferation in human osteoblast-like cells is initiated at integrin V 3[J].Journal of Endocrinology,2008,196(3):509-517.
[30]Cohen K,Ellis M,Khoury S.Thyroid hormone is a MAPK dependent growth factor for human myeloma cells acting viaαvβ3integrin[J].Molecular Cancer Research,2011,9:1385-1394.
[31]Ashida R,Tominaga K,Sasaki E,et al.AP-1and colorectal cancer[J].Inflammopharmacology,2005,13:113-125.
[32]Augustine A,Bj?rn M J.Differential organ expression patterns of thyroid hormone receptor isoform genes in p,p’-DDE-treated adult male common frog,Rana temporaria[J].Environmental Toxicology and Pharmacology,2005,20:485–492.
[33]Shi X Y,Liu C S,Wu G Q,et al.Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae[J].Chemosphere,2009,77:1010-1018.
[34]Wu Y F,Koenig R J.Gene regulation by thyroid hormone[J].Trends in Endocrinology Metabolism,2000,11:207-211.
[35]邱志群.邻苯二甲酸二丁酯和苯并[a]芘对大鼠睾丸支持细胞的联合毒性研究[D].重庆:第三军医大学,2008.
[36]Cohen-Armon M.PARP-1activation in the ERK signaling pathway[J].Trends in pharmacological sciences,2007,28(11):556-560.
[2]Murk A.J,Rijntjes E,Blaauboer B J,et al.Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals[J].Toxicology in Vitro,2013,27(4):1320-1346.
[3]Shi W,Hu X X,Zhang F X,et al.Occurrence of thyroid hormone activities in drinking water from Eastern China:Contributions of phthalate esters[J].Environmental Science&Technology,2012,46(3):1811-1818.
[4]Boas M,Feldt-Rasmussen U,Main K M.Thyroid effects of endocrine disrupting chemicals[J].Molecular and Cellular Endocrinology,2012,355(2):240-248.
[5]穆希岩,李成龙,黄瑛,等.两种邻苯二甲酸酯类污染物对斑马鱼胚胎发育的影响[J].中国环境科学,2017,(9):3566-3575.
[6]王晓伟,刘景富,阴永光.有机磷酸酯阻燃剂污染现状与研究进展[J].化学进展,2010,22(10):1983-1992.
[7]Zhao F R,Chen M,Gao F M,et al.Organophosphorus flame retardants in pregnant women and their transfer to chorionic Villi[J].Environmental Science&Technology,2017,51(11):6489-6497.
[8]Khan M,Li,J,Zhang,G et al.First insight into the levels and distribution of flame retardants in potable water in Pakistan:An underestimated problem with an associated health risk diagnosis[J].Science of Total Environment,2016,565:346-359.
[9]Farhat A,Crump D,Chiu S,et al.In ovo effects of two organophosphate flame retardants-TCPP and TDCPP-on pipping success,development,m RNA expression,and thyroid hormone levels in chicken embryos[J].Toxicological Sciences,2013,134(1):92-102.
[10]van der Veen I,de Boer J.Phosphorus flame retardants:Properties,production,environmental occurrence,toxicity and analysis[J].Chemosphere,2012,88(10):1119-1153.
[11]皮天星,蔡磊明,蒋金花.新型阻燃剂TCPP对斑马鱼的毒性研究[J].生态毒理学报,2016,11(2):247-256.
[12]陆美娅.雌激素受体ER和甲状腺激素受体TR介导的典型环境内分泌干扰物效应研究[D].杭州:浙江工业大学,2015.
[13]Kojima H,Takeuchi S,Itoh T,et al.In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors[J].Toxicology,2013,314(1):76-83.
[14]Davis P J,Lin H Y,Mousa SA,et al.Overlapping nongenomic and genomic actions of thyroid hormone and steroids[J].Steroids,2011,76(9):829-833.
[15]Davis P J,Leonard J L,Davis F B.Mechanisms of nongenomic actions of thyroid hormone[J].Frontiers in Neuroendocrinology,2008,29(2):211-218.
[16]Liu C,Ha M,Cui Y,et al.JNK pathway decreases thyroid hormones via TRH receptor:A novel mechanism for disturbance of thyroid hormone homeostasis by PCB153[J].Toxicology,2012,302(1):68-76.
[17]周君.p,p'-DDE,PCB-153单独及联合作用通过ERK1/2信号转导通路对大鼠离体脑垂体细胞的类激素效应[D].武汉:华中科技大学,2013.
[18]Li J,Ma M,Wang Z J.A two-hybrid yeast assay to quantify the effects of xenobiotics on thyroid hormone-mediated gene expression[J].Environmental Toxicology and Chemistry,2008,27(1):159-167.
[19]刘芸,李剑,马梅,等.代谢活化作用对多溴代联苯类污染物类/抗甲状腺激素活性的影响[J].生态毒理学报,2008,3(1):27-33.
[20]Schriks M.,Vrabie C M,Gutleb A C,et al.T-screen to quantify functional potentiating,antagonistic and thyroid hormone-like activities of poly halogenated aromatic hydrocarbons(PHAHs)[J].Toxicology in Vitro,2006,20(4):490-498.
[21]Pruden A,Pei R T,Stortrboom H,et al.Antibiotic resistance genes as emerging contaminants:studies in northern Colorado[J].Environmental Science and Technology,2006,40(23):7445-7450.
[22]李剑,任姝娟,马梅,等.改进型重组基因酵母TR-GRIP1检测化合物甲状腺激素干扰活性[J].环境科学研究,2011,24(10):1172-1177.
[23]文育,雷炳莉,康佳,等.双酚A类同系物的雌激素效应及对MCF-7细胞的毒性[J].上海大学学报(自然科学版),2015,21(4):515-524.
[24]Li J,Liu Y,Kong D,et al.T-screen and yeast assay for the detection of the thyroid-disrupting activities of cadmium,mercury,and zinc[J].Environmental Science and Pollution Research,2016,23(10):9843-9851.
[25]Techer R,Houde M,Verreault J.Associations between organohalogen concentrations and transcription of thyroid-related genes in a highly contaminated gull population[J].Science of Total Environment,2016,545:289-298.
[26]Dingemans M M L,van den Berg M,Westerink RHS.Neurotoxicity of brominated flame retardants:(in)direct effects of parent and hydroxylated polybrominated diphenyl Ethers on the(Developing)Nervous System[J].Environmental Health Perspectives,2011,119(7):900-907.
[27]Sandler B,Webb P,Apriletti J W.Thyroxine-thyroid hormone receptor interactions[J].Journal of Biological Chemistry,2004,279(53):55801-55808.
[28]Arukwe A,Jenssen B M.Differential organ expression patterns of thyroid hormone receptor isoform genes in p.p’-DDE-treated adult male common frog Rana temporaria[J].Environmental Toxicology and Pharmacology,2005,20:485-492.
[29]Scarlett A,Parsons M P,Hanson P L,et al.Thyroid hormone stimulation of extracellular signal-regulated kinase and cell proliferation in human osteoblast-like cells is initiated at integrin V 3[J].Journal of Endocrinology,2008,196(3):509-517.
[30]Cohen K,Ellis M,Khoury S.Thyroid hormone is a MAPK dependent growth factor for human myeloma cells acting viaαvβ3integrin[J].Molecular Cancer Research,2011,9:1385-1394.
[31]Ashida R,Tominaga K,Sasaki E,et al.AP-1and colorectal cancer[J].Inflammopharmacology,2005,13:113-125.
[32]Augustine A,Bj?rn M J.Differential organ expression patterns of thyroid hormone receptor isoform genes in p,p’-DDE-treated adult male common frog,Rana temporaria[J].Environmental Toxicology and Pharmacology,2005,20:485–492.
[33]Shi X Y,Liu C S,Wu G Q,et al.Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae[J].Chemosphere,2009,77:1010-1018.
[34]Wu Y F,Koenig R J.Gene regulation by thyroid hormone[J].Trends in Endocrinology Metabolism,2000,11:207-211.
[35]邱志群.邻苯二甲酸二丁酯和苯并[a]芘对大鼠睾丸支持细胞的联合毒性研究[D].重庆:第三军医大学,2008.
[36]Cohen-Armon M.PARP-1activation in the ERK signaling pathway[J].Trends in pharmacological sciences,2007,28(11):556-560.