| In China, organophosphate and pyrethroid pesticides has been extensively used in agriculture, ever since organochlorine pesticides was forbidden. According to the yearbook of agricultural statistics, annual production and consumption of agricultural chemicals are as tremendous as one million tons, and most of them actually enter into the aquatic environment. Up to the present, many fish populations have experienced serious difficulties in spawning and developing, leading to recruitment failure. These failures have been tentatively linked with exposure to pesticides that had been identified or suspected as environmental estrogens. Since consumption of contaminated fish may cause different kinds of developmental and neurological problems, the risk to human health by these pesticides should be seriously considered.Measurements of estrogen-regulated proteins are often used in bioassays of animal exposure to estrogenic estrogens. The yolk precursor protein, vitellogenin (Vtg), has been used most frequently for such assessments. In oviparous vertebrates, vitellogenin is synthesized by hepatocytes during oogenesis through stimulation by 17β-estradiol as female-specific protein. Males and immature fish normally do not produce significant amounts of vitellogenin, yet vitellogenesis can be induced by exposure to exogenous estrogens or xenoestrogens. Therefore, teleost vitellogenin has become an effective and sensitive biomarker for screening of commonly-used pesticides with estrogenic activities.Vitellogenin levels are often measured by means of immunoassays, such as Western blot and ELISA. Usually antibodies with high affinity and specificity are required for these methods. Considering that the primary structures of the vitellogenin molecules, however, generally differ between fish species, even between closely allied species, it's not surprising that heterologous antibodies may be less sensitive than their homologous counterparts, though there could be specific cross-reactivity to some extent. Consequently, vitellogenin from different fish species should first be purified from plasma or ascite for antibody production.The red drum, Sciaenops ocellatus, is an important commercial species in China. In this paper, we obtained highly purified vitellogenin from red drum, which was used to generate polyclonal antisera with Kunming mouse and New Zealand rabbit, respectively. Further the antisera was included in Western blot to evaluate the estrogenic activities of four extensively used pesticides, beta-cypermethrin, cypermethrin-malathion, dichlorvos, and omethoate. The results are as follows:Vitellogenesis was induced by intramascular injection of 17β-estradiol in red drum. After two weeks, blood was collected and plasma subjected to native-PAGE. Gels were stained with CBB R250, phospho-, lipo-, and glyco-protein staining methods, and three newly-emerged bands were identified as phospholipoglycoproteins, that were tentatively considered as red drum vitellogenin. Two purification procedures were employed to isolate vitellogenin form E2-treated plasma: gel filtration followed by anion exchange chromatography, or selective precipitation combined with anion exchange chromatography. Native-PAGE and SDS-PAGE analyses demonstrated that vitellogenin fractions from the latter procedure were highly purified and experienced little degradation. The molecular mass of intact vitellogenin was deteremined to be 560 kDa, while that of the monomer was 176 kDa under denaturing and reducing conditions.Polyclonal antisera were generated against Kunming mouse and New Zealand rabbit with purified vitellogenin, respectively. The titers both were measured to be 1:32 with double immunodiffusion test. Western blot showed that the antisera were highly specific and sensitive for red drum vitellogenin, the lower detection limit of the mouse antiserum was 0.90μg/ml, whereas that of the rabbit antiserum 0.45μg/ml.Using the polyclonal antisera against vitellogenin as the first antibodies, and goat anti-mouse/rabbit IgG-HRP as the second antibodies, the estrogenic activities of four commonly-used pesticides, beta-cypermethrin, cypermethrin-malathion, dichlorvos, and omethoate, were screened by Western blot. Positive reaction was detected between polyclonal antisera and plasma from all the exposed groups, though Vtg-176 was barely detectable in plasma exposed to omethoate of lower concentration, demonstrating the estrogenicity of beta-cypermethrin and cypermethrin-malathion were conspicuous, whereas dichlorvos and omethoate showed rather weak estrogenic activities.Aside from application to screening pesticides with estrogenicity, the antisera can be further used to detect vitellogenin from other species via cross-reaction. In this paper, we found Vtg-165 and Vtg-73 from stone flounder showed high affinity for the antisera, while Vtg-92 showed weak affinity. On the other hand, Vtg-106 derived from Vtg-520, was barely detectable by Western blot.In conclusion, it's effective for isolating vitellogenin from E2-treated plasma of red drum using selective precipitation followed by anion exchange chromatography, and the antisera were suitable for screening pesticides with estrogenic activities by Western blot. The results of this original research would be useful to improve the safety evaluation system and early-warning system of pesticides to some extent. |
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