汞与镉雌激素样联合作用的实验研究
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摘要
近年来越来越多的研究表明，人类和野生动物的不良健康效应可能与环境中的某些化学物质有关。由于这些化学物质能干扰内分泌功能，故称为环境内分泌干扰物(environmental endocrine disruptors，EEDs)，其中环境雌激素(Environmental estrogens，EEs)是较为重要的一类环境内分泌干扰物，他能够引起肿瘤发病率增高、生殖功能损害、神经和免疫功能的损伤。关于环境雌激素样物质的研究，目前多集中在单一物质较大剂量对机体的影响，而环境中的化合物往往难以达到这个剂量；且绝大多数环境雌激素样物质的雌激素活性十分微弱，仅为E_2的1／1000至1／10000，如单独研究一种雌激素活性较弱的物质的雌激素样作用(特别是不具蓄积作用的环境雌激素样物质)，往往会发现他们对生物体几乎不产生任何损害作用。事实上，环境雌激素样物质常以混合物的形式存在于环境中，它们往往是以低剂量混合进入机体，对机体产生联合作用，因而其危害往往被低估了。随着对环境雌激素样物质的深入研究，环境雌激素样物质的联合作用已引起关注，目前关于环境雌激素样物质联合作用的研究，
郑州人学医学院2004届硕!论文
水与锡雌激素样联合作用的实验研究
主要集中在杀虫剂类(如:狄氏剂、硫丹、毒杀芬等)和某些工业化学物(如:
PCBs、双酚A等)等，重金属类雌激素样物质的联合作用尚未见报道。HgC12
和cdCI:是重要的工业化合物，他们进入人体后，一部分经代谢排出，但大部
分能够在体内蓄积，近年来己有报道认为汞和福是环境雌激素样物质。为此，
本研究用去卵巢大鼠子宫增重试验、大鼠子宫内膜增殖实验、MCF一7人乳腺癌
细胞增殖试验等，对低剂量HgC12与 CdCI:雌激素样联合作用进行初步探讨:
并用放射性配基结合分析法测定去卵巢大鼠子宫雌激素受体数量，进一步探讨
HgCI:与cdcl:是通过何种途径显示雌激素样作用。
方法
1.去卵巢大鼠子宫增重试验参照Diel介绍的方法，给大鼠行双侧去卵巢
手术，于术后第sd随机分11组，每组6只，分别为阴性对照组(蒸馏水)，
HgCI:低、中、高剂量组(o.04mg/kg、0.20mg/kg和1 .oomg/kg)，CdCI:低、中、
高剂量组(0.08mg/kg、0.40mg/kg和2.00mg/kg)，(HgCIZ+CdC12)低、中、高剂
量组[(0.04+0.08)mg/kg、( 0.04+0.40)mg/kg和(0.04+2.00)mg/kg]，阳性对
照组(E z0.08m留kg，以花生油为溶剂)，给药容积为2.oml/kg体重，每天腹腔注
射一次，连续3d。最后一次给药后24h，称重，颈椎脱臼处死大鼠，处死前2
h每只大鼠腹腔注射秋水仙素4mg/kg，使细胞分裂停留于有丝分裂中期。迅速
取出子宫，剔除周围结缔组织和脂肪，吸干子宫表面血渍和子宫内液，于万分
之一天平上称量。
2.去卵巢大鼠子宫内膜增殖实验于子宫中段垂直于子宫角长轴朝子宫体
方向切取约l。m子宫组织，经Bouin‘s液固定，在24h内，用体积分数为70%
乙醇洗涤直至苦味酸的黄色消失。常规石蜡包埋，一半切片进行常规HE染色;
另一半进行硝酸银染色;石蜡切片经二甲苯脱色后，逐级乙醇脱水，自来水和去
离子水冲洗，滴加新鲜配制的Ag一NORs银染工作液于切片上，室温(25℃)避光
0.5h，去离子水冲洗，逐级乙醇脱水后用二甲苯透明，中性树脂封片。
郑州大学医学院2004届硕十论文
汞与锡雌激素样联合作用的实验研究
3.MCF一7人乳腺癌细胞增殖实验按照Perez等的方法并做一些改良:细
胞采用开放式单层贴壁培养，培养液为RPMI一1 640(含体积分数为10ryo的小牛血
清，30U/IOOml胰岛素)，培养条件为37℃，5%C仇，100%相对饱和湿度。加受试物
前先将细胞用PBS洗涤，然后换为无酚红RPMI一1640培养液(含8W0经活性碳一
葡聚糖处理的胎牛血清)，继续培养2d，以耗尽细胞内储存的雌激素。生长至80%
融合时，用0.25%胰蛋白酶消化，以10000个细胞/ml接种于96孔板，每孔接种
200 pl，各剂量均设4个平行孔。贴壁培养24h后，弃孔内培养液，加入200
时含有不同剂量受试物、溶剂对照以及阳性物的不含酚红的RPMI一1 640培养
基。每3d换液1次，培养6d。在第6d细胞增殖的指数期检测细胞增长情况。
本实验采用MTT比色法检测总蛋白含量。培养6d后弃上清，每孔加入噬吟兰
(MTT)25曰，放置在培养箱中孵育4h，小心吸取干净孔内液体后，每孔加入
二甲基亚矾(DMSO)1 50 pl，振荡10min后，用酶联免疫检测仪于490nm波
长处测定吸光值。
4.去卵巢大鼠子宫雌激素受体测定实验用预冷生理盐水冲洗大鼠子宫增重试
验所得子宫，称重，计算所需组织抽提液的体积，剪碎，按1:10(mg/ml)加
入预冷的抽提缓冲液，在冰浴条件下用玻璃匀浆器匀浆，15000rpm，4℃离心
30min，上清液即为胞浆液。用考马斯亮蓝G一250/BSA法测定胞浆液中蛋白含
量，放射性配基结合分析法测定大鼠子宫雌激素受体含量。采用10nmol/L3H-
雌二醇作单点饱和分析，葡聚糖一活性炭(Dcc)法分离游离的和结合的3H一EZ
后，将上清液倒入sml的闪烁液中，用F小2，01G双道液体闪烁计数器计数。
结果
1.去卵巢大鼠子宫增重试验中，HgCI:和CdCI:各剂量组均能诱导去卵巢
大鼠子宫增殖，子宫脏器系数随着染毒剂量的增加而递增，且呈剂量一效应关
系，但与阴性对照组相比，仅高
Currently, more and more studies have reported that a few adverse health effects on human have been attributed to some chemicals existing in environment. Because these chemicals can affect the function of endocrine, they are called environmental endocrine disruptors(EEDs). Being one of the most important environmental endocrine disrupters , Environmental estrogens (EEs) have a few adverse health effects on human such as tumors dependent of hormone, abnormalities of immunity function, nerve system and reproductive and developmental function .Most of the studies about environmental estrogen focused effect of the single chemical with high dose on human,but it is difficult to up to this dose for the single chemical.The estrogenic activity of most
of EEs is only 1/ 1000 to 1/ 10000 of the activity of E2.If study the estrogenic effect of the chemical with weakly estrogenic activity ,you can find it hardly has harm to organism.Now many scientist had focused their studies on the joint effect of environmental estrogens. Most of studies on joint effect of environmental estrogens focused on insecticide and techchemical ,but there is not study on estrogenic joint effect of metal.some studies reported that Cadmium Chloride and Mercury Chloride have the estrogen-like effect ,so this study will evaluate estrogenic joint effect of Cadmium Chloride and Mercury Chloride with proliferation assay on MCF-7 human breast cancer cells, uterotrophic assay in ovariectomized SD rats and proliferation assay of mucous membrane. Methods
1. Uterotrophic assay : Female SD rats were ovariectomized,8 days later, the rats were divided into 11 groups at random, which is negative control group(distilled water), low Mercury Chloride group (0.04mg/kg) .middle dose group (0.20mg/kg), high dose group(1.00mg/kg), low dose Cadmium Chloride
group(0.08mg/kg),middle dose group(0.40mg/kg) , high dose group(2.00mg/kg), low dose (Mercury Chloride + Cadmium Chloride) group[(0.04+0.08)mg/kg] , middle dose group[(0.04+0.40)mg/kg], high dose group[(0.04+2.00)mg/kg] and positive control group(80
g/kg 17 3 -estradiol, solvent oil). The dose of 2.0ml/kg body weight was given to animals, and administration was given per day by intraperitonal injection for 3 days. All animals were injected Colchicine with the dose of 4mg/Kg. All animals' body weights were measured 24 hours after final injection, then all animals were sacrificed. Uterus were removed quickly, fat and connective tissue were peeled off , blood and liquid of uterus were absorbed with tissue, then uterus weights were measured.
2. Proliferation assay of cell of mucous membrane: Cut off a part of uterus plumbing horn of uterus at the middle part of uterus,and fox the uterus in the Bouin's ,then wash the uterus with 70% alcohol till destaining of yellow of the bitter acid. The uterus tissues was embeded with paraffin regularly, half of tissues are stained by HE,the half tissues are stained by silver nitrate .Put the slices in Dimethylbenzene,alcohol,tap water,distilled water in turn, then add the fresh prepared Ag-NoRs on the slices. After a half hour, put the slices in distilled water,alcohol,dimethybenzene in turn,then seal with neutral gum.
3. Proliferation assay of MCF-7 human breast cancer cells: each well seeded cells(1 104 cells/ml) in 96 wells plates in media of RPMI-1640 supplemented with 8% dextran/Charcoal-treated FBS and insulin(30u/mL). Cells were incubated at the environment of 37 C,5%CO2 for 24 hours after they being adhered to walls, then
removed the medium, and added 200 u 1 of RPMI-1640 medium with different doses of chemicals described above , blank media as negative control and 17 -estradiol as positive control. Each dose was repeated 4 times arranged at parallel wells. Media were changed once 3 days interval and were removed after 6 days , 25 1 of thiazolyl blue(MTT) was added to each well, then plates were put into incubator for 4 hours, then MTT was removed and 150 1 DMSO was added to each well.
郑州人学医学院2004届硕!论文
水与锡雌激素样联合作用的实验研究
主要集中在杀虫剂类(如:狄氏剂、硫丹、毒杀芬等)和某些工业化学物(如:
PCBs、双酚A等)等，重金属类雌激素样物质的联合作用尚未见报道。HgC12
和cdCI:是重要的工业化合物，他们进入人体后，一部分经代谢排出，但大部
分能够在体内蓄积，近年来己有报道认为汞和福是环境雌激素样物质。为此，
本研究用去卵巢大鼠子宫增重试验、大鼠子宫内膜增殖实验、MCF一7人乳腺癌
细胞增殖试验等，对低剂量HgC12与 CdCI:雌激素样联合作用进行初步探讨:
并用放射性配基结合分析法测定去卵巢大鼠子宫雌激素受体数量，进一步探讨
HgCI:与cdcl:是通过何种途径显示雌激素样作用。
方法
1.去卵巢大鼠子宫增重试验参照Diel介绍的方法，给大鼠行双侧去卵巢
手术，于术后第sd随机分11组，每组6只，分别为阴性对照组(蒸馏水)，
HgCI:低、中、高剂量组(o.04mg/kg、0.20mg/kg和1 .oomg/kg)，CdCI:低、中、
高剂量组(0.08mg/kg、0.40mg/kg和2.00mg/kg)，(HgCIZ+CdC12)低、中、高剂
量组[(0.04+0.08)mg/kg、( 0.04+0.40)mg/kg和(0.04+2.00)mg/kg]，阳性对
照组(E z0.08m留kg，以花生油为溶剂)，给药容积为2.oml/kg体重，每天腹腔注
射一次，连续3d。最后一次给药后24h，称重，颈椎脱臼处死大鼠，处死前2
h每只大鼠腹腔注射秋水仙素4mg/kg，使细胞分裂停留于有丝分裂中期。迅速
取出子宫，剔除周围结缔组织和脂肪，吸干子宫表面血渍和子宫内液，于万分
之一天平上称量。
2.去卵巢大鼠子宫内膜增殖实验于子宫中段垂直于子宫角长轴朝子宫体
方向切取约l。m子宫组织，经Bouin‘s液固定，在24h内，用体积分数为70%
乙醇洗涤直至苦味酸的黄色消失。常规石蜡包埋，一半切片进行常规HE染色;
另一半进行硝酸银染色;石蜡切片经二甲苯脱色后，逐级乙醇脱水，自来水和去
离子水冲洗，滴加新鲜配制的Ag一NORs银染工作液于切片上，室温(25℃)避光
0.5h，去离子水冲洗，逐级乙醇脱水后用二甲苯透明，中性树脂封片。
郑州大学医学院2004届硕十论文
汞与锡雌激素样联合作用的实验研究
3.MCF一7人乳腺癌细胞增殖实验按照Perez等的方法并做一些改良:细
胞采用开放式单层贴壁培养，培养液为RPMI一1 640(含体积分数为10ryo的小牛血
清，30U/IOOml胰岛素)，培养条件为37℃，5%C仇，100%相对饱和湿度。加受试物
前先将细胞用PBS洗涤，然后换为无酚红RPMI一1640培养液(含8W0经活性碳一
葡聚糖处理的胎牛血清)，继续培养2d，以耗尽细胞内储存的雌激素。生长至80%
融合时，用0.25%胰蛋白酶消化，以10000个细胞/ml接种于96孔板，每孔接种
200 pl，各剂量均设4个平行孔。贴壁培养24h后，弃孔内培养液，加入200
时含有不同剂量受试物、溶剂对照以及阳性物的不含酚红的RPMI一1 640培养
基。每3d换液1次，培养6d。在第6d细胞增殖的指数期检测细胞增长情况。
本实验采用MTT比色法检测总蛋白含量。培养6d后弃上清，每孔加入噬吟兰
(MTT)25曰，放置在培养箱中孵育4h，小心吸取干净孔内液体后，每孔加入
二甲基亚矾(DMSO)1 50 pl，振荡10min后，用酶联免疫检测仪于490nm波
长处测定吸光值。
4.去卵巢大鼠子宫雌激素受体测定实验用预冷生理盐水冲洗大鼠子宫增重试
验所得子宫，称重，计算所需组织抽提液的体积，剪碎，按1:10(mg/ml)加
入预冷的抽提缓冲液，在冰浴条件下用玻璃匀浆器匀浆，15000rpm，4℃离心
30min，上清液即为胞浆液。用考马斯亮蓝G一250/BSA法测定胞浆液中蛋白含
量，放射性配基结合分析法测定大鼠子宫雌激素受体含量。采用10nmol/L3H-
雌二醇作单点饱和分析，葡聚糖一活性炭(Dcc)法分离游离的和结合的3H一EZ
后，将上清液倒入sml的闪烁液中，用F小2，01G双道液体闪烁计数器计数。
结果
1.去卵巢大鼠子宫增重试验中，HgCI:和CdCI:各剂量组均能诱导去卵巢
大鼠子宫增殖，子宫脏器系数随着染毒剂量的增加而递增，且呈剂量一效应关
系，但与阴性对照组相比，仅高
Currently, more and more studies have reported that a few adverse health effects on human have been attributed to some chemicals existing in environment. Because these chemicals can affect the function of endocrine, they are called environmental endocrine disruptors(EEDs). Being one of the most important environmental endocrine disrupters , Environmental estrogens (EEs) have a few adverse health effects on human such as tumors dependent of hormone, abnormalities of immunity function, nerve system and reproductive and developmental function .Most of the studies about environmental estrogen focused effect of the single chemical with high dose on human,but it is difficult to up to this dose for the single chemical.The estrogenic activity of most
of EEs is only 1/ 1000 to 1/ 10000 of the activity of E2.If study the estrogenic effect of the chemical with weakly estrogenic activity ,you can find it hardly has harm to organism.Now many scientist had focused their studies on the joint effect of environmental estrogens. Most of studies on joint effect of environmental estrogens focused on insecticide and techchemical ,but there is not study on estrogenic joint effect of metal.some studies reported that Cadmium Chloride and Mercury Chloride have the estrogen-like effect ,so this study will evaluate estrogenic joint effect of Cadmium Chloride and Mercury Chloride with proliferation assay on MCF-7 human breast cancer cells, uterotrophic assay in ovariectomized SD rats and proliferation assay of mucous membrane. Methods
1. Uterotrophic assay : Female SD rats were ovariectomized,8 days later, the rats were divided into 11 groups at random, which is negative control group(distilled water), low Mercury Chloride group (0.04mg/kg) .middle dose group (0.20mg/kg), high dose group(1.00mg/kg), low dose Cadmium Chloride
group(0.08mg/kg),middle dose group(0.40mg/kg) , high dose group(2.00mg/kg), low dose (Mercury Chloride + Cadmium Chloride) group[(0.04+0.08)mg/kg] , middle dose group[(0.04+0.40)mg/kg], high dose group[(0.04+2.00)mg/kg] and positive control group(80
g/kg 17 3 -estradiol, solvent oil). The dose of 2.0ml/kg body weight was given to animals, and administration was given per day by intraperitonal injection for 3 days. All animals were injected Colchicine with the dose of 4mg/Kg. All animals' body weights were measured 24 hours after final injection, then all animals were sacrificed. Uterus were removed quickly, fat and connective tissue were peeled off , blood and liquid of uterus were absorbed with tissue, then uterus weights were measured.
2. Proliferation assay of cell of mucous membrane: Cut off a part of uterus plumbing horn of uterus at the middle part of uterus,and fox the uterus in the Bouin's ,then wash the uterus with 70% alcohol till destaining of yellow of the bitter acid. The uterus tissues was embeded with paraffin regularly, half of tissues are stained by HE,the half tissues are stained by silver nitrate .Put the slices in Dimethylbenzene,alcohol,tap water,distilled water in turn, then add the fresh prepared Ag-NoRs on the slices. After a half hour, put the slices in distilled water,alcohol,dimethybenzene in turn,then seal with neutral gum.
3. Proliferation assay of MCF-7 human breast cancer cells: each well seeded cells(1 104 cells/ml) in 96 wells plates in media of RPMI-1640 supplemented with 8% dextran/Charcoal-treated FBS and insulin(30u/mL). Cells were incubated at the environment of 37 C,5%CO2 for 24 hours after they being adhered to walls, then
removed the medium, and added 200 u 1 of RPMI-1640 medium with different doses of chemicals described above , blank media as negative control and 17 -estradiol as positive control. Each dose was repeated 4 times arranged at parallel wells. Media were changed once 3 days interval and were removed after 6 days , 25 1 of thiazolyl blue(MTT) was added to each well, then plates were put into incubator for 4 hours, then MTT was removed and 150 1 DMSO was added to each well.
引文
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14. Safe S.Doenvironmental estrogens play a role in development of breast ca ncer in wemen and male reproductive problems?Human Ecol Risk Assess, 1995,1:17-23.
15. Shekhar PVM,Werdell J,Basrur VS.Environmental estrogens stimulation of growth and estrogen rceptor function in preneoplastic and cancerous huma n breast cell lines.J Natl Cancer Inst,1997,89:1774-1782.
16. Reiter LW, Derosa C,Mac MJ,et al.The U.S.federal framework for research on endocrine disruptors and an analysis of research programs during fisc al year 1996.Environ Health Perspect,1998,106:105-113.
17. German Council of Environmental Advisors.Special Report 1999,Abbreviat ed Version,Section3.1,Item.39.
18. Arnold SF, Klotz DM,Collins BM,et al.Synergistic action of estrogen rece ptor with combination of environmental chemicals.Science,1996,2 72:1489-1492.
19. Ramamoorthy K,Wang F,Chen I,et al.Estrogenic activity of a dieldriin/toxa phene mix ture in the mouse uterus,MCF-7 human breast cancer ceils,and yeast-based estrogen receptor assays:no apparent synergism.Endocrinology, 1997,138:1520-1527.
20. Ashby J,Lefevre PA,Odum J,et al.Synthetic oestrogens?[Letter].Nature,1997, 385:494.
21. Mclachlan JA.Synergistic effect of environmental estrogens:report withdraw. Science, 1997,277:462-463.
22. Sumpter JP, Jobling S.Vitellogenesis as a biomarker for estrogenic contami nation of the aquatic environment.Environ Health Perspect,1995,103:173-17
8
23. Vonier PM,Crain DA,Arnold SF, et al.Interaction of environmental chemical s with the estrogen and progesterone receptors from the oviductof the A merican alligator.Environ Health Perspect, 1996,104:1318-1322.
24. Payne J, Scholze M,Kortenkamp A,et al. Mixtures of four organochlorines enchance human breast cancer cell proliferation.Environ Health Perspect, 2001, 109(4):391-397.
25. Shekhar P.V.M, Werdell J, Basrur V.S,et al. Environmental estrogen stimul ation of growth and estrogen receptor function in preneoplastic and cancer ous human breast cell lines. Journal of the National Cancer Institute, 199 7,89(23):1774-1780.
26. Steven F, Arnold, Diane M,et al. Synergistic activation of estrogen recept or with combinations of environmental chemicals. Science,1996,272:148 9—1491。
27. Rajapakse N, Ong D,Kortenkamp A,et al.Defining the impact of weakly e strogenicchemicals on the action of steroidal etrogens. Toxicol Sci ,2001, 60:296-304.
28. Rajapakse N,Silva E,Kortenkamp A,et al. Combining xenoestrogens at lev els below individual no-observed-effect concentrations dramatically enhan ces stero id hormone action. Environ Health Perspect,2002,110(9):917-921
29. Klaus Graumann,Astrid Breithofer, Alois Jungbauer, et al. Monitoring of es trogen imics by a recombinant yeast assay :synergy between natural and s ynthetic compounds? The Science of the total Environment,. 1999,225:69-79.
30. Andreas Stelzer, Hing Man Chan. The relative estrogenic activity of techn ical toxaphene mixture and two individual congeners. Toxicology, 1999,13 8:69-80.
31. Douglas B, tully, Virginia T, et al. Six high-priority organochlorine pestici des, either singly or in combination, are nonestrogenic in transfected HelL
a cells. Reproductive Toxicology, 2000,14:95-102.
32. Grantly D.Charles,Gennings C, Timothy R,et al. Assessment of interactio ns of diverse ternary mixtures in an estrogen receptor-α reporter assay. To xicology and Applied Pharmacology, 2002,180:11-21.
33. Wide M,Wide L.Estradiol receptor activity in uteri of pregnant mice given lead before implantation. Fertil Steril,1980,34(5):503-508
34. Garcia-Morales P, Saceda M,Kenney N,et al. Effect of cadmium on estroge n receptor and estrogen-induced responses human breast cancer cells. J Bi ol Chem. 1994,269(24): 16896-16901
35．夏世钧，吴中亮主编．分子毒理学基础．武汉：湖北科学技术出版社，2001
36．王亚东．汞、铬、锰雌激素样作用的实验研究(待发表)，2004．
37．申立军，周袁芬，金泰廙．镉雌激素样作用的实验研究．劳动医学，2001，18(2)：67-69．
38. Stoica A, Katzenellenbogen BS, Washington DC, et al. Activation of estro-gen receptor-alpha by the heavy metal cadium. Mol Endocrinol, 2000, 14(4): 545-553.
39. Guevel RL,Petit FG, Goff PL,et al.Inhibition of rainbow trout (Oncorhynch usmykiss) estrogen receptor activity by cadmium.2000,63(1):259-266.
40．镐英杰．一氧化氮供体防治去卵巢大鼠骨质疏松症的实验研究(硕士论文)．2004
41. Deil P, Schulz T, Smolnikar K,et al. Ability of xeno and phytoestrogens to modulate express of estrogen-sensitive genes in rat uterus:estrogenicity pro files and uterotrophic activity.[J].J Steroid Biochem Mol Biol,2000,73:1-10
42．严继承，朱心强，郑一凡等．硫丹对小鼠子宮内膜细胞的增殖作用．卫生毒理学杂志，2003，17(1)：14-17．
43. Wright NA,Appleton DR.The metaphase arrest technique critical review.Ce 11 Tissue Kinet,1980,13:634-663.
44. John crocker, David AR, Boldy, et al.How should we count AgNoRs?proposa Is for a standardized approach.Journal of pathology,1989,158:185-188.
45. Soto AM,Sonnenschein C,Chung KL. The E-screen assay as a tool to ide
ntify estrogens:An update on estrogenic environmental pollutants.[J].Enviro n Health Perspect, 1995,103(suppl.7): 113-122
46. Perez P,Pulgar R,Olea-Serrano F, et al.The estrogenicity of bisphenol A re lated diphenylalkanes with various substituents at the center carbon and th e hydroxy groups.Environ Health Perspect, 1998,106:167-173.
47．吕宝璋，卢建，安明榜主编．受体学．合肥：安徽科学技术出版社，2000．
48．吴景兰，丁一主编．实用非放射性分子生物学实验技术．郑州：河南医科大学出版社，1996．
49．朱国章，孙梅，张沅等．羟基磷灰石和葡聚糖活性炭法测定雌激素受体的比较．现代妇产科进展，1996，5：321-323
50. Bail RM,Fang H,Branham WS,et al. The estrogen receptor relative bindin g affinities of 188 natural and xenochemicals:structural diversity of ligand s. Toxicol Sci,2000,54:138-153
51. Anddreas Kortenkamp, Rolf Altenburger. Approaches to assessing combina tion effects of oestrogenic environmental pollutants. The Science of the T otal Environmental, 1999,233:131-140.
52．李寿祺主编．毒理学原理与方法．第二版．成都．四川大学出版社，2003．
53．王心如，陈海燕，肖杭等．有机磷与拟除虫菊酯农药联合作用的研究．中华劳动卫生职业病杂志，2001，19(4)：251-253．
54. Payne J, Rajapakse N ,Wilkins M,et al. Prediction and assessment of the effects of mixtures of four xenoestrogens. Environ Health Perspect, 2000,1 08(10):983-987.
55. Anddreas Kortenkamp, Rolf Altenburger. Synergisms with mixtures of xen oestrogens: A reevaluation using the method of isoboles. The Science of t he Total Environmental, 1998,221:59—73.
56. Soto AM,Chung KL,Sonnenshein C. The pesticides endosulfan toxaphene and dieldrin have estrogenic effects on human estrogen sensitive cell. Env iron Health Perspect,1994,102:380-384.
57. Dail P, Olff S,Schmidt S,et al. Effects of the environmental estrogens bisp henol A,o,p'-DDT, p-tert-octylphenol and coumestrol on apoptosis induction,
cell proliferation and the expression of estrogen sensitive molecular param eters in the human breast cancer cell line MCF-7.[J]. Steroid Biochem M ol Biol,2002,80(1):61—70.
58. Greenlee AR, Quail CA, Berg RL. The antiestrogen ICI182.780 abolishes developmental injury for murine embryos exposed in vitro to o,p'-DDT.[J] .Reprod Toxicol, 2000, 14(3):225-234.
59．陈海燕，王心如．环境雌激素的识别与评价．中国工业医学杂志，2001，14(6)：369—373．
60. Gray LE JR, Kelce WR,Wiese T, et,al. Endocrine screening methods worksh op report detection of estrogenic and androgenic hormonal and antihormo nal activity for chemicals that act via receptor or steroidogenic enzyme m echanisms.[J].Reprod Toxicol,1997, 11(5):719-750.
61. report detection of estrogenic and androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mecha nisms.[J].Reprod Toxicol,1997, 11(5):719-750.
62. Shelby MD. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect,1996, 104:1296-1300.
63. Soto AM,Chung KL,Sonnenschein C.The pesticides endosulfan,toxaphene,a nd diedrin have estrogenic effects on human estrogen-sensitive cells.Enviro n Health Perspect,1994,102:1296-1300.
64. Crocker J.Nucleolar organizer regions.Curr Top Pathol,1990,82:91-149.
65. Underwood JC,Ciri DD. Nucleolar organizer regions and diagnostic discri minants for malignancy.J Pathol,1988,155:95-95.
66. Kelce WR,Gray LE,Wilson EM.Antiandrogens as environmental endocrine disruotors. Reorod Fertil Dev., 1998,10(1):105-111.
67．龙鼎新．环境雌激素对生殖和发育毒性的分子机理．卫生研究，2002，31(2)：139-141．
68．刘兆平．环境雌激素活性物质的检测．国外医学卫生分册，2000，27(2)：96-100．
69．沈惠云，徐培渝，余文三等．对-壬基酚对MCF-7人类乳腺癌细胞雌激素受体表达的影响．四川大学学报(医学版)，2003，34(4)：641～644．
70. Predki PF, Sarkar, B.Metal replacement in "zinc finger" and its effect on D NA binding. Environ Health Perspect. 1994 Sep;102 Suppl 3:195-8.
71. Saceda M,Lippman ME,Chambon P, et al. Regulation of the estrogen recep tor in MCF-7 cells by estradiol. Mol Endocrinol. 1988, 2(12):1157-1162.
72. Saceda M,Lindsey RK, Solomon H,et al. Estradiol regulates estrogen recept or mRNA stability. J Steroid Biochem Mol Biol. 1998, 66(3): 113-20.
73. Ree AH,Landmark BF, Eskild w, et al. Autologous down-regulation of mess enger ribonucleic acid and protein levels for estrogen receptors in MCF-7 cells: an inverse correlation to progesterone receptor levels. Endocrinolog y. 1989, 124(5): 2577-83.
74. Jeng MH,Shupnik MA,Bender TP, et al. Estrogen receptor expression and f unction in long-term estrogen-deprived human breast cancer cells. Endocri nology. 1998, 139(10): 4164-74.
75. Ren L,Marquardt MA,Lech JJ. Estrogenic effects of nonylphenol on pS2, ER and MUCI gene expression in human breast cancer cells-MCF-7. Che m Biol Interact. 1997, 104(1):55-64.
2. Colborn T. Environmental estrogens: health implications for humans and wildlife. Environ Health Perspect,1995,103:135-136.
3．陈建锋，王心如．环境雌激素生物效应的受体途径研究．环境与职业医学，2002，19(6)：384—386．
4. Abraham EJ, Franuley LS ,et al. Octylphenol(op),an environmental estroge n ,stimulates prolactin(prl) gene expression.[J] life Sci, 1997,60(17):1457-1 465.
5. Matthew SJ , et al. [J] J Steroid Biachem Mol Bio, 2000,74(2):223-234.
6. Love Kamp-Swan T, Pavis BJ. Mechanism of phthalate ester toxicity in the female reproductive system. Environ Health Perspect,2003,111(2):139-145.
7. Bevan CL,Porter DM,Prasad A,et al. Environmental estrogens alter early de velopment in xenopus laevis. Environ Health Perspect,2003,111(4):448-496.
8. Kubo K,Arai O,Omura M,et al. Low dose effects of bisphenol A on sexua 1 differentiation of the brain and behavior in rats. Neurosci Res,2003,45(3) :345-356.
9. Althuis MD, Brogan DR, Coates BJ. Hormonal content and potency of ora 1 contraceptives and breast cancer risk among young women. Br J Cancer, 2003,88(1):50-57.
10. Sthoeger ZM,Zinger H,Mozes E. Beneficial effects of the anti-oestrogen ta moxifen on systemic lupus erythematosus of (NZBXNZW)F1 female mice are associated with specific reduction of IgGs autoantibodies. AnnRheum Dis,2003, 62(4):341-346.
11. Soto AM,Chung KL,Sonnenschein C.The pesticides endosulfan,toxaphene,a nd dieldrin have estrogenic effects on estrogen-sensitive cells.Environ Heal th Perspect,1994,102:380-383.
12. Arnold SF, Vonier PM,Collins BM,et al.In vitro synergistic interaction of al ligator and human estrogen receptors with combinations of environmental chemicals.Environ Health Perspect, 1997,105(3);615-618.
13. Soto AM,Chung KL, Sonnenschein C.The pesticides endosulfan,toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitive cells. En viron Health Perspect, 1994,102:180-183.
14. Safe S.Doenvironmental estrogens play a role in development of breast ca ncer in wemen and male reproductive problems?Human Ecol Risk Assess, 1995,1:17-23.
15. Shekhar PVM,Werdell J,Basrur VS.Environmental estrogens stimulation of growth and estrogen rceptor function in preneoplastic and cancerous huma n breast cell lines.J Natl Cancer Inst,1997,89:1774-1782.
16. Reiter LW, Derosa C,Mac MJ,et al.The U.S.federal framework for research on endocrine disruptors and an analysis of research programs during fisc al year 1996.Environ Health Perspect,1998,106:105-113.
17. German Council of Environmental Advisors.Special Report 1999,Abbreviat ed Version,Section3.1,Item.39.
18. Arnold SF, Klotz DM,Collins BM,et al.Synergistic action of estrogen rece ptor with combination of environmental chemicals.Science,1996,2 72:1489-1492.
19. Ramamoorthy K,Wang F,Chen I,et al.Estrogenic activity of a dieldriin/toxa phene mix ture in the mouse uterus,MCF-7 human breast cancer ceils,and yeast-based estrogen receptor assays:no apparent synergism.Endocrinology, 1997,138:1520-1527.
20. Ashby J,Lefevre PA,Odum J,et al.Synthetic oestrogens?[Letter].Nature,1997, 385:494.
21. Mclachlan JA.Synergistic effect of environmental estrogens:report withdraw. Science, 1997,277:462-463.
22. Sumpter JP, Jobling S.Vitellogenesis as a biomarker for estrogenic contami nation of the aquatic environment.Environ Health Perspect,1995,103:173-17
8
23. Vonier PM,Crain DA,Arnold SF, et al.Interaction of environmental chemical s with the estrogen and progesterone receptors from the oviductof the A merican alligator.Environ Health Perspect, 1996,104:1318-1322.
24. Payne J, Scholze M,Kortenkamp A,et al. Mixtures of four organochlorines enchance human breast cancer cell proliferation.Environ Health Perspect, 2001, 109(4):391-397.
25. Shekhar P.V.M, Werdell J, Basrur V.S,et al. Environmental estrogen stimul ation of growth and estrogen receptor function in preneoplastic and cancer ous human breast cell lines. Journal of the National Cancer Institute, 199 7,89(23):1774-1780.
26. Steven F, Arnold, Diane M,et al. Synergistic activation of estrogen recept or with combinations of environmental chemicals. Science,1996,272:148 9—1491。
27. Rajapakse N, Ong D,Kortenkamp A,et al.Defining the impact of weakly e strogenicchemicals on the action of steroidal etrogens. Toxicol Sci ,2001, 60:296-304.
28. Rajapakse N,Silva E,Kortenkamp A,et al. Combining xenoestrogens at lev els below individual no-observed-effect concentrations dramatically enhan ces stero id hormone action. Environ Health Perspect,2002,110(9):917-921
29. Klaus Graumann,Astrid Breithofer, Alois Jungbauer, et al. Monitoring of es trogen imics by a recombinant yeast assay :synergy between natural and s ynthetic compounds? The Science of the total Environment,. 1999,225:69-79.
30. Andreas Stelzer, Hing Man Chan. The relative estrogenic activity of techn ical toxaphene mixture and two individual congeners. Toxicology, 1999,13 8:69-80.
31. Douglas B, tully, Virginia T, et al. Six high-priority organochlorine pestici des, either singly or in combination, are nonestrogenic in transfected HelL
a cells. Reproductive Toxicology, 2000,14:95-102.
32. Grantly D.Charles,Gennings C, Timothy R,et al. Assessment of interactio ns of diverse ternary mixtures in an estrogen receptor-α reporter assay. To xicology and Applied Pharmacology, 2002,180:11-21.
33. Wide M,Wide L.Estradiol receptor activity in uteri of pregnant mice given lead before implantation. Fertil Steril,1980,34(5):503-508
34. Garcia-Morales P, Saceda M,Kenney N,et al. Effect of cadmium on estroge n receptor and estrogen-induced responses human breast cancer cells. J Bi ol Chem. 1994,269(24): 16896-16901
35．夏世钧，吴中亮主编．分子毒理学基础．武汉：湖北科学技术出版社，2001
36．王亚东．汞、铬、锰雌激素样作用的实验研究(待发表)，2004．
37．申立军，周袁芬，金泰廙．镉雌激素样作用的实验研究．劳动医学，2001，18(2)：67-69．
38. Stoica A, Katzenellenbogen BS, Washington DC, et al. Activation of estro-gen receptor-alpha by the heavy metal cadium. Mol Endocrinol, 2000, 14(4): 545-553.
39. Guevel RL,Petit FG, Goff PL,et al.Inhibition of rainbow trout (Oncorhynch usmykiss) estrogen receptor activity by cadmium.2000,63(1):259-266.
40．镐英杰．一氧化氮供体防治去卵巢大鼠骨质疏松症的实验研究(硕士论文)．2004
41. Deil P, Schulz T, Smolnikar K,et al. Ability of xeno and phytoestrogens to modulate express of estrogen-sensitive genes in rat uterus:estrogenicity pro files and uterotrophic activity.[J].J Steroid Biochem Mol Biol,2000,73:1-10
42．严继承，朱心强，郑一凡等．硫丹对小鼠子宮内膜细胞的增殖作用．卫生毒理学杂志，2003，17(1)：14-17．
43. Wright NA,Appleton DR.The metaphase arrest technique critical review.Ce 11 Tissue Kinet,1980,13:634-663.
44. John crocker, David AR, Boldy, et al.How should we count AgNoRs?proposa Is for a standardized approach.Journal of pathology,1989,158:185-188.
45. Soto AM,Sonnenschein C,Chung KL. The E-screen assay as a tool to ide
ntify estrogens:An update on estrogenic environmental pollutants.[J].Enviro n Health Perspect, 1995,103(suppl.7): 113-122
46. Perez P,Pulgar R,Olea-Serrano F, et al.The estrogenicity of bisphenol A re lated diphenylalkanes with various substituents at the center carbon and th e hydroxy groups.Environ Health Perspect, 1998,106:167-173.
47．吕宝璋，卢建，安明榜主编．受体学．合肥：安徽科学技术出版社，2000．
48．吴景兰，丁一主编．实用非放射性分子生物学实验技术．郑州：河南医科大学出版社，1996．
49．朱国章，孙梅，张沅等．羟基磷灰石和葡聚糖活性炭法测定雌激素受体的比较．现代妇产科进展，1996，5：321-323
50. Bail RM,Fang H,Branham WS,et al. The estrogen receptor relative bindin g affinities of 188 natural and xenochemicals:structural diversity of ligand s. Toxicol Sci,2000,54:138-153
51. Anddreas Kortenkamp, Rolf Altenburger. Approaches to assessing combina tion effects of oestrogenic environmental pollutants. The Science of the T otal Environmental, 1999,233:131-140.
52．李寿祺主编．毒理学原理与方法．第二版．成都．四川大学出版社，2003．
53．王心如，陈海燕，肖杭等．有机磷与拟除虫菊酯农药联合作用的研究．中华劳动卫生职业病杂志，2001，19(4)：251-253．
54. Payne J, Rajapakse N ,Wilkins M,et al. Prediction and assessment of the effects of mixtures of four xenoestrogens. Environ Health Perspect, 2000,1 08(10):983-987.
55. Anddreas Kortenkamp, Rolf Altenburger. Synergisms with mixtures of xen oestrogens: A reevaluation using the method of isoboles. The Science of t he Total Environmental, 1998,221:59—73.
56. Soto AM,Chung KL,Sonnenshein C. The pesticides endosulfan toxaphene and dieldrin have estrogenic effects on human estrogen sensitive cell. Env iron Health Perspect,1994,102:380-384.
57. Dail P, Olff S,Schmidt S,et al. Effects of the environmental estrogens bisp henol A,o,p'-DDT, p-tert-octylphenol and coumestrol on apoptosis induction,
cell proliferation and the expression of estrogen sensitive molecular param eters in the human breast cancer cell line MCF-7.[J]. Steroid Biochem M ol Biol,2002,80(1):61—70.
58. Greenlee AR, Quail CA, Berg RL. The antiestrogen ICI182.780 abolishes developmental injury for murine embryos exposed in vitro to o,p'-DDT.[J] .Reprod Toxicol, 2000, 14(3):225-234.
59．陈海燕，王心如．环境雌激素的识别与评价．中国工业医学杂志，2001，14(6)：369—373．
60. Gray LE JR, Kelce WR,Wiese T, et,al. Endocrine screening methods worksh op report detection of estrogenic and androgenic hormonal and antihormo nal activity for chemicals that act via receptor or steroidogenic enzyme m echanisms.[J].Reprod Toxicol,1997, 11(5):719-750.
61. report detection of estrogenic and androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mecha nisms.[J].Reprod Toxicol,1997, 11(5):719-750.
62. Shelby MD. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect,1996, 104:1296-1300.
63. Soto AM,Chung KL,Sonnenschein C.The pesticides endosulfan,toxaphene,a nd diedrin have estrogenic effects on human estrogen-sensitive cells.Enviro n Health Perspect,1994,102:1296-1300.
64. Crocker J.Nucleolar organizer regions.Curr Top Pathol,1990,82:91-149.
65. Underwood JC,Ciri DD. Nucleolar organizer regions and diagnostic discri minants for malignancy.J Pathol,1988,155:95-95.
66. Kelce WR,Gray LE,Wilson EM.Antiandrogens as environmental endocrine disruotors. Reorod Fertil Dev., 1998,10(1):105-111.
67．龙鼎新．环境雌激素对生殖和发育毒性的分子机理．卫生研究，2002，31(2)：139-141．
68．刘兆平．环境雌激素活性物质的检测．国外医学卫生分册，2000，27(2)：96-100．
69．沈惠云，徐培渝，余文三等．对-壬基酚对MCF-7人类乳腺癌细胞雌激素受体表达的影响．四川大学学报(医学版)，2003，34(4)：641～644．
70. Predki PF, Sarkar, B.Metal replacement in "zinc finger" and its effect on D NA binding. Environ Health Perspect. 1994 Sep;102 Suppl 3:195-8.
71. Saceda M,Lippman ME,Chambon P, et al. Regulation of the estrogen recep tor in MCF-7 cells by estradiol. Mol Endocrinol. 1988, 2(12):1157-1162.
72. Saceda M,Lindsey RK, Solomon H,et al. Estradiol regulates estrogen recept or mRNA stability. J Steroid Biochem Mol Biol. 1998, 66(3): 113-20.
73. Ree AH,Landmark BF, Eskild w, et al. Autologous down-regulation of mess enger ribonucleic acid and protein levels for estrogen receptors in MCF-7 cells: an inverse correlation to progesterone receptor levels. Endocrinolog y. 1989, 124(5): 2577-83.
74. Jeng MH,Shupnik MA,Bender TP, et al. Estrogen receptor expression and f unction in long-term estrogen-deprived human breast cancer cells. Endocri nology. 1998, 139(10): 4164-74.
75. Ren L,Marquardt MA,Lech JJ. Estrogenic effects of nonylphenol on pS2, ER and MUCI gene expression in human breast cancer cells-MCF-7. Che m Biol Interact. 1997, 104(1):55-64.
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