Biochemical and physiological changes associated with estrogenic activity in Xenopus laevis : a model for the detection of endocrine disruption

[摘要] ENGLISH ABSTRACT:Concern has increased that widespread adverse effects are occurring in humans and wildlifepopulations as a result of exposure to environmental chemicals (mostly man-made) thatdisrupt the normal functioning of the endocrine system. Many pharmaceutical, agriculturaland industrial chemicals, acting as endocrine modulating compounds (EDCs), have beendetected in an accumulated form in food, in drinking water and in the environment.Although the levels of these chemicals can be measured analytically, it is important toevaluate biological activity. For this, animal models are used and relevant bioassaysdeveloped. These assays are based on biological markers, which are specificxenobiotically-induced physiological responses and are usually deviations in cellular orbiochemical components, processes or structures. Vitellogenin is a large protein complex,produced in the liver under estrogen control and circulates in the plasma, destined forincorporation into the developing oocyte in female oviparous vertebrates. Sincevitellogenin production is low or nonexistent in males, its presence may therefore beinterpreted as evidence of exposure to estrogenic environmental contaminants. In this studythe African Clawed Frog, Xenopus laevis was used as model to study the effects ofestrogens on biochemical and physiological parameters associated with vitellogenesis. As astarting point the seasonal female reproductive cycle in a natural Xenopus laevis populationin terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. Itwas shown that plasma vitellogenin and estrogen levels were seasonal and correlated to aseasonal ovarian cycle, which peaked during spring. However, although seasonalityexisted, there were reproductively active individuals at any time during the year. Increasesin plasma estrogen levels predated increases in plasma vitellogenin levels and ovarianmaturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride andphospholipid concentrations were determined and it was shown that estrogen affected thesein such a way that these parameters could be used as additional markers in estrogencontamination studies. In order to develop an in vitro bioassay to screen for estrogenicactivity, the use of hepatic tissue cultures was investigated. Optimal culture conditions wereestablished and increased sensitivity in the estrogenic response was obtained by using liverslices from male frogs that were pre-treated with estrogen. Validation studies proved thatthis bioassay could be employed for routine screening of water and chemical samples. Inorder to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existingpolyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies.Selected antibodies were characterised and ELISAs developed and validated. This studyshowed that the newly developed Xenopus laevis vitellogenin ELISA and liver slicebioassay have the potential to be employed in environmental monitoring programmes.

[发布机构] Stellenbosch University