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1、邻苯二甲酸酯类化合物的拟雌激素活性及对小鼠卵巢功能的影响1论文标题:邻苯二甲酸酯类化合物的拟雌激素活性及对小鼠卵巢功能的影响Estrogenic Activity of Phthalic Acid Esters and Its Effects on the Ovarian Functions of Mice论文作者 靳秋梅论文导师 孙增荣；吕严；吴丽娜；任大林,论文学位 硕士,论文专业 劳动卫生与环境卫生学 Endocrine discrupting chemicals; Di-(2-ethylhexyl) phthalate;Di-n-butyl phthalate; Estrogenic
2、activity; Human breast cancer MCF-7 cell line;Ovary目的:近年来,环境内分泌干扰物对人类和野生动物生殖发育的有害影响已成为国际性热 点问题，尤其受到欧美及日本等工业发达国家的高度重视。邻苯二甲酸酯类化合物(PAEs是世 界卫生组织(WHO)于1995年公布的、必须加强控制的、一类扰乱内分泌的化学物质,并且已 造成全球性的环境污染。其中邻苯二甲酸二(2-乙基己基)酯(di-(2-ethylhexyl)Phthalate,DEHP) 和邻苯二甲酸二丁酯(dibutyl phthalate,DBP)的应用最广泛、污染最严重,已被美国国家环保局 (EP
3、A)列为优先控制污染物。为评价PAEs类的拟雌激素活性及其对雌性小鼠性腺功能的影响, 探讨其对人类生殖健康的潜在危害,本课题选用 DBP 和 DEHP 为研究对象,通过体外实验和体 内实验,研究了二者的拟雌激素活性及对小鼠卵巢功能的影响。本研究弥补了当前 DBP 和 DEHP对雌性性腺卵巢毒性方面研究资料的不足，并为全面评价PAEs类化合物的毒理学作用 提供了科学依据。方法:(1) 体外实验:采用对雌激素敏感的人乳腺癌细胞系MCF-7细胞，在RPMI1640培养基中采 用开放式单层贴壁培养,开始实验前将细胞用磷酸盐缓冲液(PBS)洗涤后改为在无酚红 RPMI1640培养基中培养5d,实验设无水
4、乙醇(0.1%)溶剂对照组、雌二醇(E_2)(10(-9)mol/L雌 激素阳性对照组、DBP各剂量(10(-10)10(-3)mol/L)组和DEHP各剂量(10(-8) 10(-3)mol/L)组，采用MCF-7细胞增殖试验、细胞生长曲线、细胞核有丝分裂指数、细胞贴壁 率和细胞克隆形成试验,通过对MCF-7细胞的生长和增殖情况及细胞活力进行观察和分析，评 价其拟雌激素效应;(2) 体内实验:采用雌性性成熟昆明种小鼠,按体重随机分为高剂量、中剂量及低剂量三个 实验组和一个溶剂对照组，每组16只。受试物DBP和DEHP分别溶解于二甲基亚砜(DMSO) 中，采用腹腔注射染毒,DBP染毒剂量分别为
5、400、40、4mg/kg体重,DEHP染毒剂量分别为1250、Objective: Recently, environmental endocrine disrupting chemicals (EDCs) have been abundantly found in the environment. Their potential harms to the human health and wildlife have become the focus of world-wide researching, in particular, some developed countries such
6、 as America, Germany, and Japan have paid much more attention to their hazards. In 1995, World Health Organization (WHO) declared that phthalic acid esters (PAEs) could disrupt the functions of the endocrine system and their hazards must be controlled. Now, it has been suggested that PAEs isone of t
7、he global environmental pollutants. Some of them, such as di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) have been described as being among the most ubiquitous man-made environmental contaminations. DBP and DEHP have been classified as U.S. EPAs priority chemical pollutants for co
8、ntrolling. To evaluate the toxicity of PAEs on the reproduction-endocrine system and the potential hazards to the human health, DBP and DEHP1 摘自： were taken as the tested chemicals of this study, the estrogenic activities of DBP and DEHP and their effects on the ovarian functions of adult female mic
9、e were examined. This study made up the inadequation on toxic effect data of DBP and DEHP on the ovarian functions of adult female mice and provided scientific basis for overall evaluating the toxicological effects of PAEs.Methods: (1) In vitro assay: Human estrogen-dependent MCF-7 breast cancer cel
10、ls were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS). Five days before the addition of the tested compounds, the cells were washed by phosphate balanced solution (PBS), and the medium was substituted with a phenol red-free RPMI1640 medium containing 5% charcoal dextral-strippe
11、d FBS. The respective test compound was added into fresh medium and the negative control cells received only the vehicle (0.1% ethanol). In the treated groups, the cells were treated with 1010mol/L103mol/L DBP and 108mol/L103mol/L DEHP , respectively. In the postive control groups, the cells were re
12、ceived 109mol/L 17 A -estradiol (E2). The proliferation of MCF-7 cells was analyzed by the MTT assay, growth curves, mitotic index, plating efficiency and coloning efficiency. (2) In vivo assay: The healthy Kunming adult female mice with body weight from 22g to 30g were randomly divided into DBP and
13、 DEHP groups and control group, 16 female mice per group. There are three exposure groups and one reagent control group for each tested compound. In the DBP exposure groups, the mice were administrated by peritoneal injection with DBP at the doses of 400 40 and 4mg/kg body weight, respectively. In t
14、he DEHP exposure groups, the mice were administrated by the same way with DEHP at the doses of 1250A 125 and 12.5mg/kg body weight, respectively. The mice in control groups were administrated with the same amount of vehicle (dimethyl sulfoxide, DMSO). All the mice were administrated once a day, cont
15、inuously for 14 days. The estrous cycles were observed during the period of the whole experimental study in vivo. On the fifteenth and thirtieth days, the mice were sacrificed. The organ coefficients of uterus and ovary were determinded, and the histopathological changes of the left ovary were obser
16、ved.Results: (1) In vitro assay: Compared with the ethanol control cells, the proliferation of the tested cells treated with 1010103mol/L DBP and 10510 3mol/L DEHP, like E2, was markedly enhanced, and the activity of the cell proliferation reached the maximum at 105mol/L DBP and 103mol/L DEHP, respe
17、ctively. The relative proliferation effects of 105mol/L DBP and 103mol/L DEHP were 98.16% and 90.92% in comparison with 109mol/L E2, respectively. The relative proliferation potency of DBP and DEHP were 104 and 106 in comparsion with E2, respectively. During log phase, the mitotic index of the tested cells treated with DBP and DEHP, as well as E2, was significantly increased.
邻苯二甲酸酯类化合物的拟雌激素活性及对小鼠卵巢功能的影响
