| Summary: | Dissertation (PhD)--University of Stellenbosch, 2003. === 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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