Testing for endocrine disruptors in South African waters : a comparative study employing in vitro and in vivo screening approaches

Truter, Johannes Christoff (2016-12)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Numerous natural and synthetic chemicals are known to interfere with the endocrine systems of wildlife and humans, i.e., endocrine disruptive chemicals (EDCs). Surface water bodies represent a major sink of EDCs and aquatic vertebrates including fish and amphibians are, therefore, at risk. Although research in the field of endocrine disruption has proliferated globally in the past two decades, only limited work describing the status of surface water has been performed in the South African context. The aims of this dissertation are: (1) to provide a detailed overview of the present literature on endocrine disruptive potential, i.e., EDC associated biological activity in South African surface waters; (2) address certain literature gaps by screening surface water, collected from different South African water bodies, using in vitro reporter gene assays (RGAs) and gene expression based biomarkers in Mozambique tilapia, Oreochromis mossambicus, exposed in vivo; (3) to compare the results of the in vitro hormone receptor RGAs and the in vivo gene expression based biomarkers to assess whether the risks predicted correspond; (4) evaluate the potential of O. mossambicus as environmental sentinel and source of biomarkers for potential disruption of the reproductive, thyroid and interrenal endocrine systems. The majority of research on endocrine disruption in South African surface waters has been focused on reproductive targets, whereas potential disruption of the thyroid, adrenal, pancreatic and other metabolism linked endocrine pathways are less well described. An inter-seasonal assessment of the Upper Olifants River (Mpumalanga) indicated alarmingly high concentrations of steroid estrogens and potent estrogenic activity in vitro in water collected from the close proximity of a waste water treatment plant (WWTP), indicating local fish may be adversely affected. Future studies evaluating the reproductive systems of wild captured fish inhabiting the Upper Olifants River will be of value. No association could be shown between altered endocrine signalling and the incidence of obesity and pansteatitis in the O. mossambicus population inhabiting Loskop Dam (Mpumalanga). Evidence of disrupted thyroid signalling was, however, observed in Loskop Dam fish relative to an alternative population. Gene expression biomarkers representing the reproductive, thyroid and interrenal systems in juvenile O. mossambicus suggest limited effects associated with exposure to surface water contaminated with neutralized acid mine drainage, containing high concentrations of Al, Mn, Ni, Co and Cu. In addition, evidence is given indicating the potential risk of crude oil contamination in fresh water to the reproductive and thyroid systems as well as lipid metabolism in wildlife. No significant changes in the expression of a selection of endocrine linked genes was, however, observed in Xenopus laevis tadpoles exposed for a short period to surface water collected from a freshwater pan into which crude oil contaminated water is periodically discharged. Further investigation, and in particular longer term exposures, as well as the evaluation of aquatic fauna collected from (environmental) fresh water bodies contaminated with crude oil are required. Surface water collected from river mouths and harbours in the eThekwini Metropolitan and City of Cape Town was shown to exhibit estrogenic and anti-androgenic activity in vitro, suggesting the reproduction of fish populations may be impaired. Future studies evaluating the endocrine systems of wild-captured fish from the aforementioned coastal systems are needed. The risks associated with exposure to surface water predicted by in vitro RGAs did not in all cases correspond to the potential biological activity indicated by in vivo gene expression based biomarkers. This study, therefore, provides evidence supporting the use of a combination of in vitro and in vivo techniques to evaluate surface water for endocrine disruptive activity/risk. Juvenile O. mossambicus exposed for a short period was not sensitive to estrogenic substances, but biomarkers relating to thyroid disruption are more promising.

AFRIKAANS OPSOMMING: Verskeie natuurlike en sintetiese chemikalieë of metale besit die vermoë om die endokriene sisteme van diere en mense te versteur, i.e., endokriene versteurende verbindings EVV’s). EVVs is geneig om te versamel in oppervlakwaterliggame, en hou dus ‘n risiko in vir akwatiese werweldiere insluitende visse en amfibieërs. Hoewel die hoeveelheid navorsing gefokus op endokriene versteuring wêreldwyd merkwaardig gegroei het in die afgelope twee dekades, is min bekend oor die toestand van oppervlakwater in Suid-Afrika in terme van potensiële endokriene versteuring. Die doelstellings van hierdie verhandeling is: (1) om 'n gedetailleerde oorsig van die huidige literatuur oor endokriene versteurende potensiaal, i.e., EVV-geassosieerde biologiese aktiwiteit in Suid-Afrikaanse oppervlakwaterliggame te verskaf; (2) om sekere gapings in die literatuur te vul deur oppervlakwater, wat versamel is uit verskillende Suid-Afrikaanse waterliggame, te toets vir endokriene versteurings potesiaal met die gebruik van in vitro verslaggewergeentoetse (VGT’s) en geenuitdrukking gebaseerde biomerkers in Mosambiekse kurpers, Oreochromis mossambicus, in vivo blootgestel; (3) om die resultate van die in vitro hormoonreseptor VGT’s en die in vivo geenuitdrukking gebaseerde biomerkers te vergelyk en sodoende te bepaal of die risiko’s wat voorspel word ooreenstem; (4) om die potensiaal van O. mossambicus as bron van biomerkers vir moontlike ontwrigting van die voortplanting, tiroïed en adrenale endokriene stelsels te evalueer. n Literatuuroorsig het aangetoon dat die meerderheid van navorsing rakende endokriene versteuring in Suid-Afrikaanse oppervlakwaterliggame tot hede gefokus was op die voortplantingsisteem, terwyl potensiële ontwrigting van die tiroïed, adrenale, pankreatiese en ander metabolisme gekoppelde endokrienebane minder goed beskryf is. 'n Inter-seisoenale studie op die Bo-Olifantsrivier (Mpumalanga) het onrusbarende hoë konsentrasies van steroïde estrogene en kragtige estrogeniese aktiwiteit in vitro aangetoon in water versamel in die nabyheid van 'n rioolsuiweringsaanleg, wat daarop dui dat die voortplanting van plaaslike vispopulasies nadelig beinvloed kan wees. Toekomstige studies waarin die hormoonsisteme van visse wat die Bo-Olifantsrivier bewoon bestuudeer word sal van waarde wees. Geen assosiasie kon getoon word tussen veranderde endokrieneseine en die voorkoms van vetsug en pansteatitis in die Loskopdam (Mpumalanga) O. mossambicus bevolking nie. Bewyse van ontwrigte tiroïedseine is egter waargeneem in Loskopdam kurpers relatief tot 'n ander kurper bevolking. Geenuitdrukking biomerkers wat die voortplanting-, tiroïed- en adrenale sisteme verteenwoordig in jong O. mossambicus, het aangetoon dat blootstelling aan oppervlakwater besmet met geneutraliseerde suurmynwater, met hoë konsentrasies van Al, Mn, Ni, Co en Cu, beperkte effekte op die hormoonsisteem het. Daarbenewens toon hierdie verhandeling dat ru-olie besoedeling in varswater die voortplantings- en tiroïed stelsels, asook lipiedmetabolisme in akwatiese werweldiere potensieel kan versteur. Geen beduidende veranderinge in die uitdrukking van 'n seleksie van endokriene-gekoppelde gene is egter waargeneem in platanna Xenopus laevis paddavisse blootgestel vir 'n kort tydperk aan water versamel vanuit 'n varswater pan waarin ru-olie besoedelde water van tyd tot tyd gestort word nie. Verdere ondersoek, en in die besonder langtermyn blootstellings, asook die evaluering van werweldiere versamel uit (omgewings) varswaterliggame besmet met ru-olie sal van waarde wees. Oppervlakwater versamel van riviermondings en hawens in die eThekwini Metropool en Stad-Kaapstad het estrogeniese en anti-androgeniese aktiwiteit in vitro gehad, wat daarop dui dat die voortplanting van visbevolkings versteur kan wees. Toekomstige studies waarin die endokriene stelsels van visse versamel uit die voorgenoemde kusstelsels ondersoek word is nodig. Die risiko’s wat verband hou met blootstelling aan oppervlakwater voorspel deur in vitro VGT’s het nie in alle gevalle ooreengestem met die potensiële biologiese aktiwiteit aangedui deur in vivo geenuitdrukking gebaseer biomerkers nie. Hierdie studie bied dus bewyse wat aantoon dat die gebruik van 'n kombinasie van in vitro en in vivo tegnieke nodig is om oppervlakwater te evalueer vir endokriene versteuring of die risiko daarvan. Jong O. mossambicus, blootgestel vir 'n kort tydperk, was nie sensitief vir estrogeniese stowwe nie, maar biomerkers wat verband hou met skildklier ontwrigting in die spesie blyk meer belowend vir toetsingsdoeleindes.

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