Isolation And Characterization Of The Estrogenic DBPs Based On The Affinity Chromatography With ER

Environmental estrogens are a class of nature or artificially synthesizedcompounds, which lead adverse effects on the reproduction and development of theexposed human and wildlife. Estrogens may react with the disinfection agents duringthe disinfection process in the water treatment plants, with the formation of somenovel estrogenic disinfection by-products (DBPs).Those estrogenic DBPs were firstlyisolated and identificated by some traditional chemical detective methods, such ashigh performance liquid chromatography (HPLC), mass spectrometry (MS) andnulear magnetic resonance (NMR), which was limited by the difficulties in thepurification of the DBPs. A more effective method is required for the purification anddetection of the estrogenic compounds. Based on the fact that most of the knownenvirontmtal estrogens impact the endocrine system by binding to the estrogenreceptor (ER), a new method that affinity chromatography with immobilizedrecombinant ER was desigen and manufactured in this study. Affinitychromatography combining with HPLC was used to isolate and characterize theestrogenic fractions from the extracts of the estrogens after chlorination. Theestrogenicity and generation laws of the estrogenic DBPs were then studied to acquiremore details for the strict control of the estrogens.Bacteria strain conservation method, culture conditions and the sonication timeused in the preparation of affinity column with ER would have an effect on therecovery efficiency of estrogens. The optimal method for affinity column preparationwas conducted as follow condition: using fresh strains, cultured in a LB mediumcontaining15%sucrose under a temperature of15degrees centigrade and released bya five-minute sonication.The recovery efficiency of the affinity chromatography was first tested withsome classic environmental estrogens, such as estradiol (E2) which is the naturalligand with the strongest biological activity. By detection with the HPLC, therecovery efficiency of the affinity chromatography for those estrogens ranged from60%to70%, due to the amount and the activity of the ER on the affinity column. It was found that the affinity chromatography showed a preferentially recoveries for theligands which had strong affinities with ER. Based on this phenomenon, aligand-competetive assay was designed with the affinity chromatography and HPLCfor the calculation of the relative binding abilities (RBA), which showed a positivecorrelationship with their estrogenic activities.By the use of ER affinity chromatography and HPLC, several estrogenic DBPswas isolated and characterizedsuch as the chloro-E1,chloro-E2andcchloro-BPAwhich had been reported already. And some novel estrogenic DBPs likedienestrol(DE), chloro-DES and chloro-DE were isolated from the extracts from DESafter chorination. The estrogenicities of the isolated novel DBPs were measured in atwo-hydrid yeast assay.The influences of reaction time and bromide ion on the DBPs formation andestrogenicities during DES chlorination were studied by adding dechlorination agentorsodiumbromide. It was found that long reaction time and bromide ion couldenhance the elimination of the DES and its estrogenic DBPs, along with a slowerdecrease of its estrogenicity.However new brominated DBPs would be formed as theproducts of HBrO generated by the bromide ion and HClO, which might exhibit ahigher toxicity on bodies.