The Experimental Study Of The Joint Effect Of Estrogens-like On Mercury And Chromium

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 mostof 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. Methods1. 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 Chloridegroup(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(80g/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, thenremoved 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.