生物毒性测试法的研究与应用
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摘要
科学技术的发展促使人们合成越来越多的化学品,而环境作为工业废物的接纳点,受到的污染也越来越严重。传统的环境监测主要采用化学分析的手段,可以快速灵敏地测定出污染物的种类和浓度,但对为数众多的污染物进行一一测定需要耗费大量的时间和精力,也无法测定多种污染物共同暴露的综合效应。为弥补化学分析方法的不足,生物测试方法应运而生。其中,基于新的激素受体作用理论建立的重组受体基因／报道基因酵母测试系统由于更加接近哺乳动物的内分泌系统的作用机制,且具有简单、快速、灵敏的特点,适于批量筛选和检测,因而成为目前研究的热点。本论文采用重组基因酵母测试法研究了北京市污泥和一些取代酚的内分泌干扰效应,同时用离体的大白鼠肝癌细胞株(H4IIE)测试了北京市污泥的芳烃受体效应,通过两种方法的应用,进一步验证了重组基因酵母法的优越性及构建芳烃受体双杂交酵母的必要性。研究主要得到以下结论：
1.对北京市污水厂污泥的生物毒性测试结果表明所测污泥均不显示雄激素诱导效应和孕激素诱导效应,雌激素诱导效应处于较低水平；但雄激素抑制效应、孕激素抑制效应及芳烃受体效应较强。污泥经堆肥处理不能有效去除内分泌干扰物质和芳烃受体效应物质。
2.对酚的重组基因酵母测试结果表明酚类物质的类／抗雌激素效应与酚环上的取代基性质相关。烷基位于对位、烷基酚的吸电子基取代(氯原子)及分子结构的对称性均有利于表现酚的雌激素活性；而烷基位于邻位、硝基邻位取代、多羟基取代及氨基取代均会降低酚的雌激素活性。具有抗雌激素活性的酚类通常在单羟基酚环上有由至少三个非氢原子(不包括三个碳的烷基)组成的取代基。
3.采用基因克隆和酵母双杂交方法已成功克隆Ahr LBD基因片断并将其定向连接到酵母表达载体PGBKT7上构建PGBKT7-Ahr诱饵质粒,同时提取分离出PGAD424-GRIP1靶质粒并进行测序鉴定。
The development of science and technology has enabled people to synthesize more and more compounds, as a result, environment as the receiver of industrial and living wastes was sufferring from contamination greatly. The traditional method for environmental monitor was chemical analyse, by means of which the types and the concentration of pollutants could be detected quickly and sensitively. However, it wasted a lot of time and effort when we detected large pollutants one by one by this method; Besides, we couldn't get the comprehensive effect of many pollutants. In view of this, bioassay comed with the tide of fashion, especially the recombinant receptor gene/reporter gene yeast assay based on the new hormone-receptor communicated theory. The recombinant yeast assay is popular because of its simple, quickness, sensitiveness, and aptness to bulk screening and detection, what is more, the yeast test system is similar to the mechanism of mammal's endocrine system. In this paper, the endocrine disruptive effect of the sewage sludge in Beijing and some substituted phenols were tested using the recombinant gene yeast assay, meanwhile, the aromatic effect of the sewage sludge in Beijing was obtained using the vitro H4IIE assay. The application of the two assays validated the superiority of the recombinant gene yeast assay and the necessity of building two-hybrid yeast recombinating aromatic receptor gene. The main results were summarized as follows.
1 In the bioassay for the sewage sludge, results showed that both androgenic and progestogenic activity couldn't be found, estrogenic activity was at a low level; but there were the obviously inhibitory effect for androgen receptor and progestogen receptor, as well as the strong aromatic effect. The composting process played a little part in removing endocrine disruptors and aromatic compounds efficiently.
2 In the recombinant gene yeast assay for phenols, results revealed that the inducible/inhibitory effect for estrogenic receptor was related to the substituent structure on phenolic ring. All of the structure characters contribute to exhibit estrogenic activity, which included alkyl group in the para-position, the substituent impelling electron withdrawal on the alkylphenol ring and the structure symmetry; However, some features tended to decrease the estrogenic activity, which included alkyl group in the ortho-position、nitro-substitution in the ortho-position、multiple hydroxyl groups on a single ring and amidogen substituent on benzene ring. The key characteristic properties for inhibiting E2-inducing-β-Galactosidase activity are associated with the substitution of at least three nonhydrogen atoms (excluding three-carbon alkyl group) on a monohydroxy phenolic ring.
3 In this study Ahr LBD gene has been cloned successfully and joined to yeast expression vector PGBKT7 orientationally to build bait plasmid PGBKT7-Ahr using gene cloning and yeast two-hybrid assay; the prey plasmid PGAD424-GRIP1 was extracted and separated, with the following identification by sequencing.
1.对北京市污水厂污泥的生物毒性测试结果表明所测污泥均不显示雄激素诱导效应和孕激素诱导效应,雌激素诱导效应处于较低水平；但雄激素抑制效应、孕激素抑制效应及芳烃受体效应较强。污泥经堆肥处理不能有效去除内分泌干扰物质和芳烃受体效应物质。
2.对酚的重组基因酵母测试结果表明酚类物质的类／抗雌激素效应与酚环上的取代基性质相关。烷基位于对位、烷基酚的吸电子基取代(氯原子)及分子结构的对称性均有利于表现酚的雌激素活性；而烷基位于邻位、硝基邻位取代、多羟基取代及氨基取代均会降低酚的雌激素活性。具有抗雌激素活性的酚类通常在单羟基酚环上有由至少三个非氢原子(不包括三个碳的烷基)组成的取代基。
3.采用基因克隆和酵母双杂交方法已成功克隆Ahr LBD基因片断并将其定向连接到酵母表达载体PGBKT7上构建PGBKT7-Ahr诱饵质粒,同时提取分离出PGAD424-GRIP1靶质粒并进行测序鉴定。
The development of science and technology has enabled people to synthesize more and more compounds, as a result, environment as the receiver of industrial and living wastes was sufferring from contamination greatly. The traditional method for environmental monitor was chemical analyse, by means of which the types and the concentration of pollutants could be detected quickly and sensitively. However, it wasted a lot of time and effort when we detected large pollutants one by one by this method; Besides, we couldn't get the comprehensive effect of many pollutants. In view of this, bioassay comed with the tide of fashion, especially the recombinant receptor gene/reporter gene yeast assay based on the new hormone-receptor communicated theory. The recombinant yeast assay is popular because of its simple, quickness, sensitiveness, and aptness to bulk screening and detection, what is more, the yeast test system is similar to the mechanism of mammal's endocrine system. In this paper, the endocrine disruptive effect of the sewage sludge in Beijing and some substituted phenols were tested using the recombinant gene yeast assay, meanwhile, the aromatic effect of the sewage sludge in Beijing was obtained using the vitro H4IIE assay. The application of the two assays validated the superiority of the recombinant gene yeast assay and the necessity of building two-hybrid yeast recombinating aromatic receptor gene. The main results were summarized as follows.
1 In the bioassay for the sewage sludge, results showed that both androgenic and progestogenic activity couldn't be found, estrogenic activity was at a low level; but there were the obviously inhibitory effect for androgen receptor and progestogen receptor, as well as the strong aromatic effect. The composting process played a little part in removing endocrine disruptors and aromatic compounds efficiently.
2 In the recombinant gene yeast assay for phenols, results revealed that the inducible/inhibitory effect for estrogenic receptor was related to the substituent structure on phenolic ring. All of the structure characters contribute to exhibit estrogenic activity, which included alkyl group in the para-position, the substituent impelling electron withdrawal on the alkylphenol ring and the structure symmetry; However, some features tended to decrease the estrogenic activity, which included alkyl group in the ortho-position、nitro-substitution in the ortho-position、multiple hydroxyl groups on a single ring and amidogen substituent on benzene ring. The key characteristic properties for inhibiting E2-inducing-β-Galactosidase activity are associated with the substitution of at least three nonhydrogen atoms (excluding three-carbon alkyl group) on a monohydroxy phenolic ring.
3 In this study Ahr LBD gene has been cloned successfully and joined to yeast expression vector PGBKT7 orientationally to build bait plasmid PGBKT7-Ahr using gene cloning and yeast two-hybrid assay; the prey plasmid PGAD424-GRIP1 was extracted and separated, with the following identification by sequencing.
引文
[1]C.Waring, R.Stagg, K.Fretwell, et al.The impact of sewage sludge exposure on the reproduction of the sand goby, Pomatoschistus minutus[J]. Environmental Pollution,93(1):17-25
[2]Catriona Paul,Stewart M. Rhind, Carol E. Kyle,et al. Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge[J]. Environ Health Perspect,2005,113(11):1580-1587.
[3]Routledge, E.J., Sumpter, J.P. Structural Features of Alkylphenolic Chemicals Associated withEstrogenic Activity[J]. Biol Chem,272(6):3280-3288.
[4]Schultz,T.W., Kraut, D.H., Sayler, G.S., et al. Estrogenicity of selected biphenyls evaluated using A recombinant yeast assay[J]. Environ Toxicol Chem,17 (9): 1727-1729.
[5]Nishihara, T., Nishikawa, J., Kanayama, T., et al. Estrogenic Activities of 517 Chemicals by Yeast Two-Hybrid Assay[J]. Health Sci,46 (4):282-298.
[6]Shiau, A.K., Barstad, D., Loria, P.M., et al. The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen[J]. Cell,95:927-937.
[7]Brzozowski, A.M., Pike, A.C.W., Dauter, Z., et al. Molecular basis of agonism and antagonismin the oestrogen receptor[J]. Nature,389:753-758.
[8]王晓燕,尚伟.水体有毒有机污染物的危害及优先控制污染物[J].首都师范大学学报(自然科学版),2002,23(3)：73-78
[9]齐文启,孙宗光.痕量有机污染物的监测[M].北京：化学工业出版社.2001
[10]邓茂先,陈祥贵.环境内分泌干扰物研究进展[J].国外医学卫生学分册,2000,27(2)：65-68.
[11]农科.被怀疑为干扰内分泌作用的农药及其代谢物[J].农药科学与管理,2000,21(2)：10-12.
[12]宋宏宇,王捷.环境内分泌干扰物与农药[J].农药科学与管理,2001,22(2)：23-25.
[13]徐科峰,李忠,何莼等.持久性有机污染物(POPs)对人类健康的危害及其治理技术进展[J].四川环境,2003,22(4)：29-30.
[14]Kenaga, E.E.. Test organisms and methods useful for early assessment of acute toxicity of chemicals [J]. Environment Science and Techology,1978,12: 1322-1329
[15]Dohety, F.G.. Interspecies Correlations of Acute Aquatic Median Lethal Concentrations for four standard testing Species[J]. Environment Science and Techology,1983,17:661-665
[16]Mayer, F.L. and Ellersieck, M.R.. Manual for acute toxicity:interpretation and data base for 410 chemicals and 66 species of freshwater animals[M]. Washington D.C.:U.S. Dep. of the Interior, Fish, and Wildlife Service, Resource Publication,1986:505
[17]胡军,李杰.环境内分泌干扰物筛检方法研究进展[J].预防医学文献信息,2002,8(6)：706-708
[18]Payne J.F., Fancey A., Rahimtula D., et al. Review and perspective on thre use of mixd function oxygenase enzymes in biological monitoring[J]. Comp Biochem Physiol,1987,860:233-245
[19 ]许华夏,李培军,刘宛等.生物细胞色素P450的研究进展[J].农业环境保护,2002,21(2)：188-191
[20]霍传林,王菊英,韩庚辰等.鱼体内EROD活性对多氯联苯类的指示作用[J].海洋环境学,2002,21(1)：5-8
[21]Fujita S. P450 in wild animals as a biomarker of environmental impact[J]. Biomarkers,2001,6(1):19-25
[22]陈国胜,张甬元,徐盈等.鲫鱼肝脏中CYP1A1的诱导作为沉积物中二恶英的毒理学指标的研究[J].中国环境科学,1997,17(3)：264-267
[23 ]朱琳,钱芸,刘广良.细胞色素P450酶系及其在毒理学上的应用[J].上海环境科学,2001,202：88—91
[24]Marshall Adams S, Bevelluner Mark S, Mark Stephen Greeley, et al. Ecological risk assessment in a large river-reservoil:6. bioindicators of fish population health[J]. Environmental toxicology and chemistry,1999,18(4):628-640
[25]王咏,王春霞,王子健等.硝基芳烃对鲤鱼肝EROD活性影响的体外研究环境科学[J].环境科学,2001,22(4)：120-122
[26]Swanson H.I., Bradfield C.A.. The Ah-receptor:genetics, structure and function[J]. Pharmacogenetics,1993,3:213-230
[27]Nebert D.W., McKinnon R.A., Puga A.. Human drug-metabolizing enzyme polymorphism:effects on risk of toxicology and cancer[J]. DNA and Cell Biology, 1996,15(4):273-280
[28]Schriks M, Vrabie CM, Gutleb AC, 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.
[29]Hong H, Kohlit K, Trivedit A, et al. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors[J]. Proc. Natl. Acad. Sci,1996,93:4948-4952.
[30]何世华,梁增辉,战威等.环境雌激素重组酵母测评系统的建立[J].环境与健康杂志,2002,19(1)：57-59.
[31]Z Hong, E Ferrari, J Wright-Minogue, et al. Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system [J]. American Society for Microbiology, 1996,70(7):4261-4268
[32]D.Mangelsdorf, C.Thummel, M.Beato, et al. The nuclear receptor superfamily: The second decade[J]. Cell,83(6):835-839
[33]R. G. Jensen and J. A. Bassham. Photosynthesis by Isolated Chloroplasts. National Academy of Sciences[J].1966,56(4):1095-1101
[34]李季,吴为中.污泥处理处置技术及装置[M].北京：化学工业出版社,2003,7：1-11.
[35]金擂霖,刘永龄.污泥处置[M].北京：中国建筑工业出版社,1985,8：232-300
[36]李剑,崔青,马梅等.应用重组孕激素基因酵母测定饮用水中内分泌干扰物的方法[J].环境科学.2006,27(12)：2463-2466
[37]Mei Ma, Jian Li, Zijian Wang. Assessing the Detoxication Efficiencies of Wastewater Treatment Processes Using a Battery of Bioassays/Biomarkers[J]. Arch. Environ. Contam. Toxicol,2005,49:480-487
[38]黎雯,徐盈,吴文忠.利用离休大白鼠肝癌细胞的EROD诱导指示二恶英的复合毒性效应[J].动物学报,2001,47(1)：64-70
[39]BradfordM M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein2dye binding [J]. Anal Biochem,72:248—254
[40]张庆华,K.-W.Schramm,徐盈.利用96孔板和重组基因酵母筛选环境内分泌干扰物[J].环境科学学报,2003,23(5)：702-704
[41]Routledge E J, Sumpter J P. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen[J]. Environment Toxicology and Chemidtry,1996,15(3):241-248
[42]Gaido K W, Leonard L S, Lovell S,et al. Evaluation if chemicals with endocrine miodulating activity in a yeast-based steroid hormone receptor gene transcription assay[J]. Toxicol Appl Pharmacol,1997,143:205-212
[43]Jingxian Wang, Wenzhong Wu, Berhard Henkelmann, et al. Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay[J]. Atmospheric Environment,2003,37:3225-3235
[44]Thomas A. Ternes, Henrik Andersen, Daniel Gilberg, et al. Determination of Estrogens in Sludge and Sediments by Liquid Extraction and GC/MS/MS[J]. Anal. Chem.,2002,74 (14), pp 3498-3504
[45]Dray J, Dray F, Tiller F, et al. Hydrolysis of urine metabolites of different steroid hormones by-glucuronidase from Escherichia coli[J]. Ann Inst Pasteur, 123(6):853-857
[46]Desbrow C, Routledge E J, Brighty G C, et al. Identification of estrogenic chemicals in STW effluent.1. Chemical fractionation and in vitro biological screening[J]. Environ Sci Technol,32:1549-1558
[47]Tyler C R, Routledge E J. Oestrogenic effects in fish in English rivers with evidence of their causation[J]. Pure and Applied Chemistry,70:1795-1804
[48]Allen Y, Matthiesen P, Scott A P,et al. The extent of estrogenic contamination in the UK estuarine and marine environments—further survey of flounder[J]. The Science of the Total Environment,233(1-3):5-20.
[49]Ternes T A, Kreckel P, Mueller J. Behavior and occurrence of estrogens in municipal sewage treatment plants-II.Aer obic batch experiments with activated sludge[J]. Science of the Total Environment,225:91-99.
[50]Hamers T, Kamstra J H, Sonneveld E, Murk A J, Kester M H A,Andersson P L, Legler J, Brouwer A.2006. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants[J]. Toxicological Sciences,92:157-173.
[51]Miki M, Toyokazu K, Kei N, et al. The Assessment of the Estrogen and Androgen in the Compost Produced from the Sewage Sludge and Solid Waste of Livestock[J]. Journal of Environmental Chemistry,16(4):615-626
[52]Lange I G, Daxenberger A, Schiffer B, et al. Sex hormones originating from different livestock production systems:fate and potential disrupting activity in the environment[J]. Analytica Chimica Acta,473(1-2):27-37
[53]Labadie P, Budzinski H. Development of an analytical procedure for determination of selected estrogens and progestagens in water samples[J]. Analytical and bioanalytical chemistry,381:1199-1205
[54]Norpoth K, Nehrkorn A, Kirchner M, et al. Investigations on the problem of
solubility and stability of steroid ovulation inhibitors in water, waste water and activated sludge[J]. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Erste B Hyg Praev Med,156(6):500-511
[55]万小琼,吴文忠,贺纪正.利用草鱼原代肝细胞培养评价二恶英毒性效应[J].中国环境科学,2002,22(2)：114-117
[56]Lillemor KG, Nina K, Helena Olsman,et al. Fate of Ah Receptor Agonists during Biological Treatment of an Industrial Sludge Containing Explosives and Pharmaceutical Residues[J]. Environ Sci & Pollut Res,2004,11(6):379-387
[57]Magnus Engwall, Bjorn Brunstrom, Carina Naf, et al. Levels of dioxin-like compounds in sewage sludge determined with a bioassay based on erod induction in chicken embryo liver cultures[J]. Chemosphere,1999,38(10):2327-2343
[58]Routledge, E.J., Sumpter, J.P. Estrogenic activity of surfactants and some of Their degradation products assessed using a Recombinant yeast screen[J]. Environ Toxicol Chem,15(3):241-248.
[59]Schultz,T.W., Kraut, D.H., Sayler, G.S., et al. Estrogenicity of selected biphenyls evaluated using A recombinant yeast assay[J]. Environ Toxicol Chem,17 (9): 1727-1729.
[60]Austin, R.P., Davis, A.M., Manners, C.N. Partitioning of ionizing molecules between aqueous buffers and phospholipid vesicles[J].Pharmcol. Sci,84: 1180-1183.
[61]Schultz,T.W., Sinks, G.D., Cronin, M.T.D. Structure-activity relationships for gene activation oestrogenicity:Evaluation of a diverse set of aromatic compounds[J]. Environ. Toxicol,17:14-23.
[62]Shiau, A.K., Barstad, D., Loria, P.M., et al. The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen[J]. Cell,95:927-937.
[63]刘芸,李娜,马梅等.酚类化合物雌激素效应的比较研究[J].中国环境科学,2009,29(8)：873～878
[64]Ding XF, Anderson CM, Ma H. Nuclear Receptor-Binding Sites of Coactivators Glucocorticoid Receptor Interacting Protein 1 (GRIP1) and Steroid Receptor Coactivator 1 (SRC-1):Multiple Motifs with Different Binding Specificities[J]. Molecular Endocrinology,1998,12(2):302-313.
[65]王晓娟,金木梁,陈家宽.环境激素DBP对拟南芥试管形态发生的影响[J].西北植物学报,24(11)：1889-1893
[66]Norrgren L, Andersson T, Biork M. Liver morphology and cytochrome P450 activity in fry of rainbow trout after microinjection of lipid-soluble xenobiotics in the yolk-sac embryos[J]. Aquatic Toxicology,2:307-316
[67]Engwall M, Brunstroem B, Brewer A, et al. Cytochrome P450A induction by a coplanar PCB,a PAH mixture, and PCB-cotaminated sediment extracts following microinfection of rainbow trout sac-fry[J]. Aquatic Toxicology,30(4):311-324
[68]袁秀平,吴文忠,徐盈等.2,3,7,8-TCDD对鲍鲫幼体超微结构影响的观察[J].水生生物学报,23(2)：185-186
[69]张凤君,郑文彪,方展强等.PCBs对斑马鱼鳃及肝脏细胞超微结构的影响的初步研究[J].华南师范大学学报(自然科学版),(4)：112-114
[2]Catriona Paul,Stewart M. Rhind, Carol E. Kyle,et al. Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge[J]. Environ Health Perspect,2005,113(11):1580-1587.
[3]Routledge, E.J., Sumpter, J.P. Structural Features of Alkylphenolic Chemicals Associated withEstrogenic Activity[J]. Biol Chem,272(6):3280-3288.
[4]Schultz,T.W., Kraut, D.H., Sayler, G.S., et al. Estrogenicity of selected biphenyls evaluated using A recombinant yeast assay[J]. Environ Toxicol Chem,17 (9): 1727-1729.
[5]Nishihara, T., Nishikawa, J., Kanayama, T., et al. Estrogenic Activities of 517 Chemicals by Yeast Two-Hybrid Assay[J]. Health Sci,46 (4):282-298.
[6]Shiau, A.K., Barstad, D., Loria, P.M., et al. The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen[J]. Cell,95:927-937.
[7]Brzozowski, A.M., Pike, A.C.W., Dauter, Z., et al. Molecular basis of agonism and antagonismin the oestrogen receptor[J]. Nature,389:753-758.
[8]王晓燕,尚伟.水体有毒有机污染物的危害及优先控制污染物[J].首都师范大学学报(自然科学版),2002,23(3)：73-78
[9]齐文启,孙宗光.痕量有机污染物的监测[M].北京：化学工业出版社.2001
[10]邓茂先,陈祥贵.环境内分泌干扰物研究进展[J].国外医学卫生学分册,2000,27(2)：65-68.
[11]农科.被怀疑为干扰内分泌作用的农药及其代谢物[J].农药科学与管理,2000,21(2)：10-12.
[12]宋宏宇,王捷.环境内分泌干扰物与农药[J].农药科学与管理,2001,22(2)：23-25.
[13]徐科峰,李忠,何莼等.持久性有机污染物(POPs)对人类健康的危害及其治理技术进展[J].四川环境,2003,22(4)：29-30.
[14]Kenaga, E.E.. Test organisms and methods useful for early assessment of acute toxicity of chemicals [J]. Environment Science and Techology,1978,12: 1322-1329
[15]Dohety, F.G.. Interspecies Correlations of Acute Aquatic Median Lethal Concentrations for four standard testing Species[J]. Environment Science and Techology,1983,17:661-665
[16]Mayer, F.L. and Ellersieck, M.R.. Manual for acute toxicity:interpretation and data base for 410 chemicals and 66 species of freshwater animals[M]. Washington D.C.:U.S. Dep. of the Interior, Fish, and Wildlife Service, Resource Publication,1986:505
[17]胡军,李杰.环境内分泌干扰物筛检方法研究进展[J].预防医学文献信息,2002,8(6)：706-708
[18]Payne J.F., Fancey A., Rahimtula D., et al. Review and perspective on thre use of mixd function oxygenase enzymes in biological monitoring[J]. Comp Biochem Physiol,1987,860:233-245
[19 ]许华夏,李培军,刘宛等.生物细胞色素P450的研究进展[J].农业环境保护,2002,21(2)：188-191
[20]霍传林,王菊英,韩庚辰等.鱼体内EROD活性对多氯联苯类的指示作用[J].海洋环境学,2002,21(1)：5-8
[21]Fujita S. P450 in wild animals as a biomarker of environmental impact[J]. Biomarkers,2001,6(1):19-25
[22]陈国胜,张甬元,徐盈等.鲫鱼肝脏中CYP1A1的诱导作为沉积物中二恶英的毒理学指标的研究[J].中国环境科学,1997,17(3)：264-267
[23 ]朱琳,钱芸,刘广良.细胞色素P450酶系及其在毒理学上的应用[J].上海环境科学,2001,202：88—91
[24]Marshall Adams S, Bevelluner Mark S, Mark Stephen Greeley, et al. Ecological risk assessment in a large river-reservoil:6. bioindicators of fish population health[J]. Environmental toxicology and chemistry,1999,18(4):628-640
[25]王咏,王春霞,王子健等.硝基芳烃对鲤鱼肝EROD活性影响的体外研究环境科学[J].环境科学,2001,22(4)：120-122
[26]Swanson H.I., Bradfield C.A.. The Ah-receptor:genetics, structure and function[J]. Pharmacogenetics,1993,3:213-230
[27]Nebert D.W., McKinnon R.A., Puga A.. Human drug-metabolizing enzyme polymorphism:effects on risk of toxicology and cancer[J]. DNA and Cell Biology, 1996,15(4):273-280
[28]Schriks M, Vrabie CM, Gutleb AC, 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.
[29]Hong H, Kohlit K, Trivedit A, et al. GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors[J]. Proc. Natl. Acad. Sci,1996,93:4948-4952.
[30]何世华,梁增辉,战威等.环境雌激素重组酵母测评系统的建立[J].环境与健康杂志,2002,19(1)：57-59.
[31]Z Hong, E Ferrari, J Wright-Minogue, et al. Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells by using the herpes simplex virus amplicon system [J]. American Society for Microbiology, 1996,70(7):4261-4268
[32]D.Mangelsdorf, C.Thummel, M.Beato, et al. The nuclear receptor superfamily: The second decade[J]. Cell,83(6):835-839
[33]R. G. Jensen and J. A. Bassham. Photosynthesis by Isolated Chloroplasts. National Academy of Sciences[J].1966,56(4):1095-1101
[34]李季,吴为中.污泥处理处置技术及装置[M].北京：化学工业出版社,2003,7：1-11.
[35]金擂霖,刘永龄.污泥处置[M].北京：中国建筑工业出版社,1985,8：232-300
[36]李剑,崔青,马梅等.应用重组孕激素基因酵母测定饮用水中内分泌干扰物的方法[J].环境科学.2006,27(12)：2463-2466
[37]Mei Ma, Jian Li, Zijian Wang. Assessing the Detoxication Efficiencies of Wastewater Treatment Processes Using a Battery of Bioassays/Biomarkers[J]. Arch. Environ. Contam. Toxicol,2005,49:480-487
[38]黎雯,徐盈,吴文忠.利用离休大白鼠肝癌细胞的EROD诱导指示二恶英的复合毒性效应[J].动物学报,2001,47(1)：64-70
[39]BradfordM M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein2dye binding [J]. Anal Biochem,72:248—254
[40]张庆华,K.-W.Schramm,徐盈.利用96孔板和重组基因酵母筛选环境内分泌干扰物[J].环境科学学报,2003,23(5)：702-704
[41]Routledge E J, Sumpter J P. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen[J]. Environment Toxicology and Chemidtry,1996,15(3):241-248
[42]Gaido K W, Leonard L S, Lovell S,et al. Evaluation if chemicals with endocrine miodulating activity in a yeast-based steroid hormone receptor gene transcription assay[J]. Toxicol Appl Pharmacol,1997,143:205-212
[43]Jingxian Wang, Wenzhong Wu, Berhard Henkelmann, et al. Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay[J]. Atmospheric Environment,2003,37:3225-3235
[44]Thomas A. Ternes, Henrik Andersen, Daniel Gilberg, et al. Determination of Estrogens in Sludge and Sediments by Liquid Extraction and GC/MS/MS[J]. Anal. Chem.,2002,74 (14), pp 3498-3504
[45]Dray J, Dray F, Tiller F, et al. Hydrolysis of urine metabolites of different steroid hormones by-glucuronidase from Escherichia coli[J]. Ann Inst Pasteur, 123(6):853-857
[46]Desbrow C, Routledge E J, Brighty G C, et al. Identification of estrogenic chemicals in STW effluent.1. Chemical fractionation and in vitro biological screening[J]. Environ Sci Technol,32:1549-1558
[47]Tyler C R, Routledge E J. Oestrogenic effects in fish in English rivers with evidence of their causation[J]. Pure and Applied Chemistry,70:1795-1804
[48]Allen Y, Matthiesen P, Scott A P,et al. The extent of estrogenic contamination in the UK estuarine and marine environments—further survey of flounder[J]. The Science of the Total Environment,233(1-3):5-20.
[49]Ternes T A, Kreckel P, Mueller J. Behavior and occurrence of estrogens in municipal sewage treatment plants-II.Aer obic batch experiments with activated sludge[J]. Science of the Total Environment,225:91-99.
[50]Hamers T, Kamstra J H, Sonneveld E, Murk A J, Kester M H A,Andersson P L, Legler J, Brouwer A.2006. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants[J]. Toxicological Sciences,92:157-173.
[51]Miki M, Toyokazu K, Kei N, et al. The Assessment of the Estrogen and Androgen in the Compost Produced from the Sewage Sludge and Solid Waste of Livestock[J]. Journal of Environmental Chemistry,16(4):615-626
[52]Lange I G, Daxenberger A, Schiffer B, et al. Sex hormones originating from different livestock production systems:fate and potential disrupting activity in the environment[J]. Analytica Chimica Acta,473(1-2):27-37
[53]Labadie P, Budzinski H. Development of an analytical procedure for determination of selected estrogens and progestagens in water samples[J]. Analytical and bioanalytical chemistry,381:1199-1205
[54]Norpoth K, Nehrkorn A, Kirchner M, et al. Investigations on the problem of
solubility and stability of steroid ovulation inhibitors in water, waste water and activated sludge[J]. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Erste B Hyg Praev Med,156(6):500-511
[55]万小琼,吴文忠,贺纪正.利用草鱼原代肝细胞培养评价二恶英毒性效应[J].中国环境科学,2002,22(2)：114-117
[56]Lillemor KG, Nina K, Helena Olsman,et al. Fate of Ah Receptor Agonists during Biological Treatment of an Industrial Sludge Containing Explosives and Pharmaceutical Residues[J]. Environ Sci & Pollut Res,2004,11(6):379-387
[57]Magnus Engwall, Bjorn Brunstrom, Carina Naf, et al. Levels of dioxin-like compounds in sewage sludge determined with a bioassay based on erod induction in chicken embryo liver cultures[J]. Chemosphere,1999,38(10):2327-2343
[58]Routledge, E.J., Sumpter, J.P. Estrogenic activity of surfactants and some of Their degradation products assessed using a Recombinant yeast screen[J]. Environ Toxicol Chem,15(3):241-248.
[59]Schultz,T.W., Kraut, D.H., Sayler, G.S., et al. Estrogenicity of selected biphenyls evaluated using A recombinant yeast assay[J]. Environ Toxicol Chem,17 (9): 1727-1729.
[60]Austin, R.P., Davis, A.M., Manners, C.N. Partitioning of ionizing molecules between aqueous buffers and phospholipid vesicles[J].Pharmcol. Sci,84: 1180-1183.
[61]Schultz,T.W., Sinks, G.D., Cronin, M.T.D. Structure-activity relationships for gene activation oestrogenicity:Evaluation of a diverse set of aromatic compounds[J]. Environ. Toxicol,17:14-23.
[62]Shiau, A.K., Barstad, D., Loria, P.M., et al. The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen[J]. Cell,95:927-937.
[63]刘芸,李娜,马梅等.酚类化合物雌激素效应的比较研究[J].中国环境科学,2009,29(8)：873～878
[64]Ding XF, Anderson CM, Ma H. Nuclear Receptor-Binding Sites of Coactivators Glucocorticoid Receptor Interacting Protein 1 (GRIP1) and Steroid Receptor Coactivator 1 (SRC-1):Multiple Motifs with Different Binding Specificities[J]. Molecular Endocrinology,1998,12(2):302-313.
[65]王晓娟,金木梁,陈家宽.环境激素DBP对拟南芥试管形态发生的影响[J].西北植物学报,24(11)：1889-1893
[66]Norrgren L, Andersson T, Biork M. Liver morphology and cytochrome P450 activity in fry of rainbow trout after microinjection of lipid-soluble xenobiotics in the yolk-sac embryos[J]. Aquatic Toxicology,2:307-316
[67]Engwall M, Brunstroem B, Brewer A, et al. Cytochrome P450A induction by a coplanar PCB,a PAH mixture, and PCB-cotaminated sediment extracts following microinfection of rainbow trout sac-fry[J]. Aquatic Toxicology,30(4):311-324
[68]袁秀平,吴文忠,徐盈等.2,3,7,8-TCDD对鲍鲫幼体超微结构影响的观察[J].水生生物学报,23(2)：185-186
[69]张凤君,郑文彪,方展强等.PCBs对斑马鱼鳃及肝脏细胞超微结构的影响的初步研究[J].华南师范大学学报(自然科学版),(4)：112-114