| Environmental estrogen is one of the most important kind of environmental endocrine disruptors. There are many different kinds of environmental estrogens, which generally exist in the real environment. Environmental estrogens can affect the normal functions of the endocrine and reproductive development system through low concentration for a long term exposure, then cause serious influence to human and animal health. Humans and ecosystems are continually exposed to a very complex mixture of environmental estrogen chemicals, which is the typical exposure. However, only individual compound is considered in the risk assessments of mixture effects, which may lead to some errors.In this study, MCF-7 hunman breast cancer cells were exposed to E2, BPA, NP, DBP, DEHP and TAM as well as the mixtures of them and the relative proliferation effect of MCF-7 cells and the expression levels of ERa and pS2 were detected on the basis of in vitro assays including E-screen assay and estrogen-responsive genes assay. Then based on the concentration-response curves of individual compounds, two models including concentration addition (CA) and independent action (IA) were employed to predict the joint toxicity of the mixtures. The concentration-response curves predicted by CA and IA were compared with the experimental data.The results show that data of single effect of E2, DBP, DEHP and TAM can be fitted by Weibull equation, while Hill function as the optimal fitting function of BPA and NP, and the correlation coefficient R2 are 0.8929,0.9598,0.9667,0.9666,0.9697 and 0.9878. The EC50 with 95% confidence interval which obtained by the equations were 4.184×10-5(6.18×10-6～3.535×10-4), 4.787(1.669～10.81),0.677 (0.271～1.525),10.09(8.547～1.173),1.139×10-3 (3.611×10-4-3.609×10-3) and 0.288 (0.180～0.398) μmol/L. The estrogen activity of E2 is strongest, BPA, NP, DBP, and DEHP is weaker relatively. TAM inhibites MCF-7 cell growth, but has weak estrogen activity on the low concentrations. The concentration-response relationships of mixtures are effectively fitted by Weibull, Hill, Logistic and ExpGrol equations, R2 are 0.969,0.889,0.967,0.996,0.986,0.966, 0.951,0.955,0.932,0.809 and 0.992. The joint effects of mixtures are relatively complex, binary mixture (E2+TAM) and quaternary mixture (BPA+NP+DBP+DEHP) show up as inhibition effect on cell proliferation, while the other mixtures have weaker estrogenic activity. Obviously, there are significant differences between combined effect and individual effect. Both individual environmental estrogen and mixtures can significantly change the genes expression levels of ERa and pS2 compared with the vehicle control. Environmental estrogens may promote and inhibit cell proliferation by up-regulating and down-regulating the expression levels of estrogen-responsive genes in MCF-7 cells. Within the certain concentration range, the mixture effects calculated by CA and IA models are more close to the experimental results which are significantly different from the results predicted by effect summation model in high concentration, and the index evaluation methods can not accurately represent the combined types of mixtures, due to the uncertainties.The tested compounds show estrogenicity or antiestrogenicity, and the mixture effects can be predicted based on the concentration-response data of individual compounds. Exogenous compounds in relatively low concentrations can produce significant mixture effect, and the joint action with endogenous estrogen is remarkable. CA and IA models compared with the index methods can quantitatively analyze the combation effects without depending on the concentration-response curve. From an overall perspective, results predicted by models were different from the experimental datas, but model curves is relatively close to the fitting curve of experiment results in low concentration, which can closely reflect the real exposure levels of environmental estrogens, so that CA and IA models can accurately predict the mixture effects. |
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