Androgen controlled secondary sexual characters in the male African clawed frog, Xenopus laevis, as potential biomarkers for endocrine disruptor contaminants (with special reference to fungicides) in aquatic systems

Archer, Edward (2014-04)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Endocrine disrupting contaminants (EDCs) coming from households, industrial parks, wastewater (sewage) treatment and agricultural areas have been shown to pollute our freshwater systems. These contaminants may disrupt early development and reproductive systems in freshwater organisms (fish, frogs and crocodile species) as well as humans. Agricultural pesticides are shown as a large contributor to endocrine disruption activity in water catchment areas through spray drift, runoff, and/or groundwater leeching. Although South Africa is recognized as the largest consumer of agricultural pesticides in Africa, few studies have been undertaken to assess the prevalence and impact of endocrine disorders activities of pesticides in local freshwater systems. Recent studies have suggested that various agricultural pesticides, especially fungicides, might have adverse effects on the male endocrine system. There is therefore a need to test for a wider range of endocrine disrupting activities (mechanisms) in environmental waters other than conventional estrogenic (feminising) activities. Furthermore, there is a need to establish biomarkers in endemic species (bio-indicators) to show endocrine disruption in vertebrates (therefore also apply to humans). The specific objectives of the study were to: (1) describe and confirm the use of androgen-controlled breeding glands in male African clawed frogs (Xenopus laevis) as a biomarker for (anti)androgenic endocrine disruption activity (Chapter 2), (2) to investigate the premature development of breeding glands in X. laevis tadpoles (pre-metamorphic) and young froglets (post-metamorphic) (Chapter 2), (3) to investigate the disruption of male reproductive traits in adult X. laevis frogs by exposure to substances disrupting two different anti-androgenic endocrine disruption pathways (Chapter 3), (4) screen for (anti)androgenic activity of individual and binary mixtures of pesticides, which are regularly used in agricultural areas in the Western Cape Province of South Africa (Chapter 4), and (5) to test for (anti)androgenic and estrogenic endocrine disrupting activities by making use of in vitro assays as well as adult male X. laevis frogs collected from selected ponds surrounded by vineyards and fruit orchards in the Stellenbosch Winelands. The present study confirmed that male breeding glands can serve as biomarkers for (anti)androgenic endocrine disruption and that male reproductive and secondary sexual characteristics can be disrupted through two different biochemical control pathways. The study also confirmed that the expression of androgen-regulated breeding glands can be stimulated in pre-metamorphic tadpoles and immature, post-metamorphic frogs, and can thus be used for (anti)androgenic testing. The rapid testing and predictive value of an in vitro recombinant yeast screen for androgen receptor binding inhibition of selected individual or binary mixtures of pesticides was also confirmed. However, the current study showed that the predicted in vitro (anti)androgenic activity did not always correspond with in vivo (anti)androgenic biomarker outcomes. This It also confirmed that single-cell in vitro assays can be used as a first-level prediction for (anti)androgenic activities of individual or mixtures of agricultural pesticides. This study provides a better understanding for potential mixture interactions of commonly used agricultural pesticides, the hormonal control of secondary sexual characteristics in male frogs and the use of reproduction biomarkers to study long-term effects of endocrine disruptors in local water supplies.

AFRIKAANSE OPSOMMING: Endokriene versteurings-kontaminante (EVKe) wat vanaf huishoudings, industriële parke, afvalwater(riool)-behandeling en landbougebiede kom, besoedel ons varswaterstelsels. Hierdie kontaminante mag versteuring van vroeë ontwikkeling- en voorplantingstelsels in varswater-organismes (vis-, padda- en krokodil-spesies) sowel as die mens inhou. Landbou-plaagdoders word uitgesonder as ’n bydraer van endokriene versteuring-aktiwiteite in wateropvangs-gebiede deur spuitnewel, afloop-water en/of grondwater-deurvloei. Hoewel Suid-Afrika erken word as die grootste verbruiker van landbou-plaagdoders in Afrika, word min studies onderneem om die voorkoms en impak van endokriene versteurings-aktiwiteite van plaagdoders in plaaslike varswaterstelsels te ondersoek. Onlangse studies het voorgestel dat verskeie landbou-plaagdoders, veral swamdoders, nadelige uitwerkings kan hê op die manlike endokriene stelsel. Daar bestaan dus 'n behoefte om te toets vir 'n wyer verskeidenheid van endokriene versteurings-aktiwiteite (meganismes) in omgewingswater anders as konvensionele estrogeniese (vervroulikings) aktiwiteite. Verder bestaan daar ’n behoefte om biomerkers in endemiese spesies te gebruik as bio-indikators van endokriene versteuring in werweldiere (daarom ook van toepassing op die mens). Die spesifieke doelwitte van die studie het ingesluit om: (1) die gebruik van androgeen-beheerde parings- velkliere (“breeding glands”) in manlike platannas (Xenopus laevis) as 'n biomerker vir (anti)androgeniese endokriene versteuring-aktiwiteit te beskryf en bevestig (Hoofstuk 2); (2) ondersoek in te stel na die voortydige ontwikkeling van parings-kliere in X. laevis paddavisse (pre-metamorfose) asook jong paddas (post-metamorfose) as biomerkers van androgeniese (vermanlikheids) aktiwiteite (Hoofstuk 2); (3) ondersoek in te stel na die versteuring van manlike geslags-eienskappe in volwasse X. laevis paddas deur middel van blootstelling aan stowwe wat twee verskillende androgeniese endokrien reaksie-weë verteenwoordig (Hoofstuk 3); (4) toets vir (anti)androgeniese aktiwiteit van individuele en binêre mengsels van landbou-plaagdoders wat gereeld in die Westelike Provinsie van Suid Afrika gebruik word (Hoofstuk 4) en (5) te toets vir (anti)androgeniese en estrogeniese endokriene versteurings aktiwiteite deur gebruik te maak van in vitro toetse asook volwasse manlike X. laevis paddas wat uit geselekteerde damme (omring deur wingerde en vrugte boorde in die Stellenbosch wynland distrik) versamel was. Die huidige studie het bevestig dat die manlike parings-velkliere as biomerkers vir (anti)androgeniese versteuring kan dien en dat manlike voortplanting en sekondêre geslagskenmerke deur twee verskillende biochemiese beheer-weë ontwrig kan word. Die studie het verder bevestig dat die uitdrukking van androgeen-gereguleerde parings-velkliere voortydig gestimuleer kan word in pre-metamorfose paddavissies asook onvolwasse, post-metamorfose paddas. Die vinnige toetsing en voorspellingswaarde van 'n rekombinante in vitro gis toets om binding-inhibisie van die androgeen reseptor deur geselekteerde individuele of binêre mengsels van plaagdoders aan te toon is ook bevestig. Alhoewel, die huidige studie het getoon dat die voorspelde in vitro (anti)androgeniese aktiwiteit nie altyd ooreenstem met in vivo (anti)androgeniese biomerker uitkomstes nie. Hierdie studie bevestig dat enkel-sel in vitro toetse aangewend kan word as eerste vlak- en voorspelling-toetse vir (anti)androgeniese aktiwiteite van enkel of mengsels van landbou-plaagdoders. Sodoende is 'n beter begrip verkry vir potensiële mengsel-interaksies van algemeen-gebruikte landbou plaagdoders, die hormonale beheer van sekondêre geslagskenmerke in manlike paddas asook die aanwending van voortplantingsbiomerkers om langtermyn effekte van endokriene versteurders in plaaslike waterbronne te ondersoek.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/86508
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