| In the past years, more and more studies have reported that a few adverse health effects of human such as tumors dependent of hormone, abnormalities of immunity function, nerve system and reproductive and developmental function have been attributed to some chemicals in environment. Because these chemicals can disrupt the function of endocrine, they are called environmental endocrine disruptors(EEDs). While the mechanisms of EEDs are complicate, the estrogen-like effect is one of them. Many reports have showed some metals such as cadmium, lead and organic tin have endocrine disrupting effects. There are many other metals such as Mercury, Manganese and Chromium in working and living environment. They have adverse effects on human and animals. Now there is no exact proof whether this kind of effect is throughendocrine disrupting effect. This study will evaluate the estrogen-like effect of Mercury Chloride, Manganese Sulfate, Chromium Chloride and Chromium Trioxide with proliferation assay of MCF-7 human breast cancer cells, uterotrophic assay in ovariectomized SD rats, assay of peroxidase activity and estrogen receptor(ER) binding assay. The aim is to study endocrine disrupting effect of these compounds and provide experimental proof of their reproductive and developmental toxicity effects. Methods1. Proliferation assay of MCF-7 human breast cancer cells: cells(2-3 x104 cells/ml) were seeded in culture plates of 96 wells. Each well contained 100ul RPMI-1640 medium(phenol red-free) supplemented with 10% dextran/Charcoal-treated fetal bovine serum (FBS) and insulin(30u/mL). Cells were cultured 24 hours after they adhered to walls, then removed medium, and added 200 u 1 RPMI-1640 medium(phenol red-free) contained different doses of compounds, negative control group and 1 7 b -estradiol. Each dose contained 6 parallel wells. Medium was exchanged each 3 days and removed after being cultured 6 days , 25 u l thiazolyl blue(MTT) was added to each well, then plates were put into incubator. 4 hours later, liquid was removed and 150ul DMSO was added to each well. Plates were vibrated 10 min. At last, absorb light value was measured on enzyme link immunity detector with 490nm wave length.2. Uterotrophic assay : Female SD rats were ovariectomized,16 days later, the rats were divided into 5 groups at random, namely negative control group(distilled water), low dose Mercury Chloride group(0.04mg/kg), medium dose Mercury Chloride group(0.20mg/kg), high dose Mercury Chloride group(1.00mg/kg) and positive control group(100 u g/kg 17 3 -estradiol, solvent oil). Volume given to animals was 2.0ml/kg body, and administration was given per day by intraperitonal injection for 3 days. 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.3. Assay of peroxidase activity: Uterus were cut off with scissors, then 5% ice calcium chloride was added according to l:20(mg/ml). Uterus were homogenated with glass homogeniser in ice water. The homogenate was centrifuged at 12000rpm for 20 min at 4C, upper liquid was harvested and peroxidase activity was measured with assay of guaiacol colorimetry.4. Estrogen receptor of rat uterine binding assay: Mature female SD rats were sacrificed, uterus were peeled off and washed with ice Sodium Chloride, then weighted. The uterus were cut off and tissue buffer was added. The uterus were homogenated, the homogenate were centrifuged at 15000 rpm for 30 min at 4C. Upper liquid was harvested. The proteinin upper liquid was measured with Coomassic Brilliant Blue G-250/Bovine Serum Albumin(CBBG-250/BSA). Estrogen receptor of competing binding assay was done according to method of Zhu Guozhang: 3H -estradiol(3H-E2) of different concentration, 0.5nmol/L, lnmol/L, 2nmol/L, 4nmol/L, 8nmol/L were added t...
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