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

Hurter, Etienne (2003-03)

Dissertation (PhD)--University of Stellenbosch, 2003.

Thesis

ENGLISH ABSTRACT: Concern has increased that widespread adverse effects are occurring in humans and wildlife populations as a result of exposure to environmental chemicals (mostly man-made) that disrupt the normal functioning of the endocrine system. Many pharmaceutical, agricultural and industrial chemicals, acting as endocrine modulating compounds (EDCs), have been detected 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 to evaluate biological activity. For this, animal models are used and relevant bioassays developed. These assays are based on biological markers, which are specific xenobiotically-induced physiological responses and are usually deviations in cellular or biochemical components, processes or structures. Vitellogenin is a large protein complex, produced in the liver under estrogen control and circulates in the plasma, destined for incorporation into the developing oocyte in female oviparous vertebrates. Since vitellogenin production is low or nonexistent in males, its presence may therefore be interpreted as evidence of exposure to estrogenic environmental contaminants. In this study the African Clawed Frog, Xenopus laevis was used as model to study the effects of estrogens on biochemical and physiological parameters associated with vitellogenesis. As a starting point the seasonal female reproductive cycle in a natural Xenopus laevis population in terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. It was shown that plasma vitellogenin and estrogen levels were seasonal and correlated to a seasonal ovarian cycle, which peaked during spring. However, although seasonality existed, there were reproductively active individuals at any time during the year. Increases in plasma estrogen levels predated increases in plasma vitellogenin levels and ovarian maturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride and phospholipid concentrations were determined and it was shown that estrogen affected these in such a way that these parameters could be used as additional markers in estrogen contamination studies. In order to develop an in vitro bioassay to screen for estrogenic activity, the use of hepatic tissue cultures was investigated. Optimal culture conditions were established and increased sensitivity in the estrogenic response was obtained by using liver slices from male frogs that were pre-treated with estrogen. Validation studies proved that this bioassay could be employed for routine screening of water and chemical samples. In order to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existing polyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies. Selected antibodies were characterised and ELISAs developed and validated. This study showed that the newly developed Xenopus laevis vitellogenin ELISA and liver slice bioassay have the potential to be employed in environmental monitoring programmes.

AFRIKAANSE OPSOMMING: Daar is toenemende besorgdheid dat afwykings in mens- en dierbevolkings voorkom as gevolg van blootstelling aan chemikalieë (hoofsaaklik mensgemaak) in die omgewing wat die normale werking van die endokrienstelsel versteur. Verskeie farmaseutiese, landbou- en industriële chemikalieë, wat as endokrienversteurders optree, is in die omgewing gevind. AI kan die vlakke van hierdie stowwe analities bepaal word, is dit belangrik om hulle biologiese aktiwiteit te evalueer. Hiervoor word diermodelle aangewend om toepaslike toetse daarvoor te ontwikkel. Hierdie toetse word baseer op biologiese merkers, spesifieke xenobioties-geïnduseerde fisiologiese reaksies, en is gewoonlik afwykings van sellulêre- of biochemiese komponente, -prosesse of - strukture. Vitellogeen ('n dooiervoorloper) is 'n lipoproteïenkompleks wat, onder estrogeenbeheer, in die lewer vervaardig word en in die plasma sirkuleer vir uiteindelike inkorporasie in ontwikkelende oësiete van vroulike, ovipare werweldiere. Aangesien daar min of geen vitellogeen in manlike diere geproduseer word, is die teenwoordigheid daarvan 'n aanduiding dat die dier aan estrogeniese omgewingsbesoedeling blootgestel is. In hierdie studie is die Platanna, Xenopus laevis, as model gebruik om die gevolge van estrogene op biochemiese en fisiologiese veranderlikes, wat met vitellogenese geassosieer word, te bestudeer. As vertrekpunt is die seisoenale voortplantingsiklus van die wyfie, in terme van vitellogeen en -estrogeen vlakke in die plasma en aktiwiteit van die ovaria bepaal. Daar is aangetoon dat die estrogeen- en vitellogeenvlakke in die plasma met die ovariumsiklus, wat gedurende die lente hoogtepunte bereik, korreleer. Alhoewel daar seisoenaliteit bestaan, was daar dwarsdeur die jaar wyfies wat ovarium dooierneerlegging getoon het. Verhoging in estrogeenvlakke het vitellogeenpieke en rypwording van die ovaria voorafgegaan. Lipoproteïenprofiele, sowel as die cholesterol- , trigliseried- en fosfolipiedkonsentrasies in die plasma is bepaal en daar is aangetoon dat estrogeen hierdie medeveranderlikes in só 'n mate affekteer dat hulle as bykomende biomerkers vir estrogeenblootstelling in besoedelingstudies gebruik kan word. In vitro Xenopus laevis lewersnitte in die weefselkultuur omgewing is ondersoek om 'n biotoets te onwikkel vir die gebruik in vinnige estrogenisiteits-toetsing van watermonsters en chemise stowwe. Die mees gunstige kultuurtoestande is bepaal en die sensitiwiteit van estrogeenreaksies is verhoog deur lewer van mannetjies, wat vooraf met estrogeen behandel is, te gebruik. Hierdie biotoets se geldigheid is gestaaf en kan in roetine eerstevlaktoetsing van watermonsters gebruik word. Die gebruik van poliklonale teenliggaampies in 'n bestaande enzyme-linked immunosorbent assay (ELISA) is vervang deur spesiaal-ontwikkelde monoklonale anti-Xenopus laevis vitellogeen teenliggaampies. Uitgesoekte teenliggaampies, spesifiek teen die estrogeengeïnduseerde proteïene, is gekarakteriseer en ELISAs saamgestel en die geldigheid gestaaf. Hierdie studie het aangetoon dat die nuut-onwikkelde Xenopus laevis vitellogeen-ELISA en lewerkultuurbiotoets die potensiaal het om In omgewingsmoniteringprogramme gebruik te word.

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