The Studies Of Using Vitellogenin Of Tanichthys Albonubes As A Biomarker To Monitor The Environmental Endocrine Disrupting Chemicals

Recently, the harm to wildlife and human beings by environmental endocrinedisrupting chemicals (EDCs) is the hot topic in environmental research. The findingsof both laboratory and field studies show that vitellogenin (Vtg) induction may beconsidered a useful tool in evaluating endocrine disruption in aquatic animals, andthus the potential risk associated with exposure to xenoestrogens. White Mountainminnow (Tanichthys albonubes), a special species in Guangzhou was considered asuitable fish used in EDCs studies. The aim of this study is to gained and purify theVtg from male T. albonubes induced by 17β-estradiol (E₂); to purify lipovitellin (Lv)from ovaries of mature female T. albonubes; to prove that if the Lv and Vtg areimmunogenic; to establish an indirect Enzyme-Linked Immunosorbent Assay (ELISA)for detecting Vtg content in the whole body homogenate of T. albonubes; and todetermine Vtg content in the whole body homogenate of DDTs treaded T. albonubes.Lv was purified from ovaries of mature female T. albonubes bynative-polyacrylamide gel electrophoresis (Native-PAGE) and sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in this study. Theresults indicated that the Lv appeared to exist as homotrimer of approximately 314kDin native-PAGE (4-10%). Polyclonal antiserum against Lv was raised in rat.Native-PAGE followed by Western blotting was performed to analyze the specificityof the antiserum and the immunological similarities between Lv and Vtg. The resultsshowed that only one protein band in the whole body homogenate from E₂ treatedmale T. albonubes and homogenate from ovaries respectively reacted with theantiserum. No protein bands from untreated males reacted with the anti-Lv serum andanti-high molecular weight (HMW) subunit of Lv serum. It was indicated that anti-HMW subunit of Lv could be used to develop immune analysis to measure Vtg.Vtg from the whole body homogenate of E₂ treaded T. albonubes was detected byNative-PAGE and purified by Mg²⁺-EDTA sedimentation and anion exchange chromatography. The results indicated that the Vtg having a molecular weight ofapproximately 440kDa.Anti-Lv serum was used to develop an indirect enzyme-linked immunosorbentassay (ELISA) to measure Vtg concentrations of whole body homogenate in T.albonubes. The intra-and ineerassay variabilities were 8.59% and 6.28%, respectively.The sensitivity was 8.1ng/mL.Adult male T. albonubes were exposured to a nominal water concentration of0.0275mg/mL, 0.0137mg/mL and 0.0067mg/mL of DDTs in a static exposure systemfor 20 days. Compared with the control group, Vtg concentration in exposured groupswere significant different. It is indicated that Vtg of the adult male T. albonubes couldbe as a valid biomarker for environmental estrogens.