Study On Combination Effect Of Environmental Estrogens By In Vivo And In Vitro Assays

So far the adverse effect of Environmental Estrogens (EEs) on human and wildlife had caused an increasing concern in the world, the research about them had become one of the important topics of environmental science this century. The study would optimize recombinant yeast estrogen system with which some kinds of environmental chemicals were to be detected and then evaluate the combination effect of EEs with both recombinant yeast estrogen system and uterus growth assay.Recombinant yeast estrogen system was sensitive, speedy, economical and simple. Due to its newly-built and nonstandard technique, Diethylstilbestrol (DES) and 4-nonyphenol (4-NP) were used as tested substance in order to compare the effects of vibrating and non-vibrating incubation, yeast multiplying time, exposure time and lysis methods on estrogen-like activity, to optimize the experiment conditions, and to detect some kinds of environmental chemicals to validate the reliability and practicability of the optimized recombinant yeast estrogen system.The results of the assay showed that vibrating incubation had no effect on estrogen-like activity, while yeast multiplying time had slender effect on it, and exposure time and lysis methods had huge effects on it. The optimal experimental conditions for the recombinant yeast estrogen system in environmental estrogens detection were that the yeast cells were multiplied for 24h under non-vibrating, the exposure time of tested substances was 30h, and yeast cells were broken by Y-PER. The optimized recombinant yeast estrogen system could detect and preliminary screen environmental chemicals. In alldetected chemicals, Genistein and Pueraria extracts had strong estrogenic effects, gasoline-fueled vehicle exhausts had weak estrogenic effects and others had no estrogenic effects in this experiment.The estrogenic combination effects of Diethylstilbestrol(DES), Bisphenol A(BPA), 4-nonyphenol(NP) and Estradiol Benzoate(E2B) were estimated with the optimized recombinant yeast estrogen system, which provided evidence for studying the adverse mechanism of EEs. The results indicated that when these chemicals were measured respectively, all of them exhibited estrogenic activity and had good dose-response relationship, so they all could combine and activate estrogen receptor; when these chemicals were combined by various ways, the three groups of DES+NP, BPA+NP and DES+BPA+E2B exhibited weak synergy effects while the combination effect of others were additive.The immature female SD rats (2Id) were administered BPA and DES as well as the mixture of the two chemicals in the uterus growth assay. The combination effect of EEs were evaluated by measuring uterine weight, uteri/body weight, thickness of the myometrium, height of luminal epithelium, the amount of uterine glands, the amount of mature follicul and vaginal opening rate . The results showed both of BPA and DES had estrogen-like activity, the estrogen-like activity of DES was 10000 times higher than that of BPA, and the combination effect of the mixture of BPA and DES was additive.Our study indicated it was efficient for the optimized recombinant yeast estrogen system to act as rapid screening methods in vitro on EEs, and it provided new content for the screening methods of EEs. In addition, it was the first time for us to detect the combination effect of EEs using in vivo and in vitro assay, which substantiated the possibility of synergy effect. The assay provided some evidences on study of EEs mechanism.