Estrogenic Activity Of Water Extraction And Distribution Of PAEs In Donghu Lake

The environmental endocrine disruptors, a group of chemical substances which exist in the environment, are able to disturb the endocrine system of human and wild animals, and then result in disorders to their reproduction, growth and behavior. In 1995, the phthalic acid esters have been classified as an important category of endocrine disruptors by WHO, and they have already caused the environmental pollution globally. Here, we took the water of Donghu lake as the object of our study, chose DMP, DBP, DOP--as priority pollutants classified by both China and US, and used MCF-7 cell line which is sensitive to estrogen, to appraise the estrogen-like activity of DMP, DBP and DOP, as well as that of the water extraction of Donghu Lake. In order to know the existence level and the distributed situation of three kinds of PAEs in Donghu lake, we used solid phase extraction unify the HPLC-UV method to analysis water extraction of Donghu lake. The results are as follows:(1) Estrogen-sensitive human breast cancer cells MCF-7 are maintained in flask with phenolsulfonphthalein-free, high-glucose DMEM supplemented with 10% bovine calf serum--addressed with charcoal, and incubated in a humidified incubator. Cells are transferred into 96-well plates, then aspirate the medium, add an equal volume of fresh DMEM without calf serum containing test compounds and continue incubation for 2 days. E2 serially diluted from 10-8 M down to 10-12M, and 0.1% DMSO was used as positive and negative controls, respectively. Test compounds including DMP, DBP, DOP (diluted from 10-3M down to 10-8M, respectively) and Water extraction of Donghu Lake (extracted from 5~5,120ml, respectively). Five parallel wells are set for each concentration of the tested compounds. Estrogenic activity according to each test compound is preliminary evaluated by proliferation experiment results. The work demonstrated that after exposure to test compounds for 48h, comparing to negative controls, 10-7 M ~ 10-11M E2, 10-3 M ~10-5M DBP and Water extractions of Donghu Lake (80ml and 320ml respectively) can promote MCF-7 cell proliferation significantly(P<0.05 or P<0.01). The maximum effect of E2, DBP and water extraction respectively occur at concentration of 10-9 M, 10-4 M and 300ml water, with proliferation index values of 1.32, 1.17 and 1.19, accordingly. Response and potency of DBP relative to E2 is 88.64% and 1/10,000 respectively. Response of water extraction (300ml water) relative to E2 is 82.58%. No significant proliferation promoting effect was observed for DMP and DOP. Similar to nature estrogen E2, DBP and Donghu Lake water extraction can promote proliferation of MCF-7 cell line in a dose-effect manner when tested in a certain range of concentration, which indicates their estrogenic activity.(2) Six water samples were obtained from three different districts (Shuiguo Lake, Guozheng Lake, Tangling Lake) of Donghu Lake, and three other composite water samples were obtained from Donghu Lake, Yangtze River and Hanjiang River, respectively. After concentration, the water samples need to be analyzed by HPLC; the chromatograph condition is as follows: mobile phase: 95% methyl alcohol and 5% super-pure water; detector: ultraviolet detector and the wave length is 228nm; flow rate: 1.0 ml/min; column temperature: room temperature; injection volume: 20μL. The work demonstrated that all three PAEs were detected in main areas of Donghu Lake at concentration varied from 2.58×10-4 mg/L to12.47×10-4mg/L. DBP was detected in each sample at a significant higher concentration than the other two PAEs, which suggested that DBP is the predominant pollutant. Among different lake areas, the highest level of PAEs was found in Shuiguo Lake, less in Guozheng Lake and the lowest in Tanglin Lake. In Guozheng Lake and Tanglin Lake, there is an increasing tendency of PAEs level from north to south within the same lake area. PAEs were detected in water sample from Donghu Lake, Yangtze river and Hanjiang river, among which PAEs level was highest in Donghu Lake and lowest in Yangtze river. The results indicate that Donghu Lake has been polluted by PAEs to different extent.