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Development and Application Ofin Vitroandin Vivoreporter Gene Assays Development and application ofin vitro and in vivoreporte r gene assays for the assessment of (xeno-)estrogenic compounds in the aquatic environment Juliette Legler Promotor: Prof. Dr.J.H .Koema n Hoogleraar ind e Toxicologie Wageningen Universiteit Co-promotores: Dr.A .J .Mur k Leerstoelgroep Toxicologie Wageningen Universiteit Dr. B.va nde rBur g Nederlands Instituut voor Ontwikkelingsbiologie Promotiecommissie: Prof. Dr.W.B .va nMuiswinke l (Wageningen Universiteit) Dr.J .Kome n(Wage.iinge n Universiteit) Prof. Dr.S.E .Wendelaa r Bonga (Katholieke Universiteit Nijmegen) Prof.Dr .J.P . Sumpter (Brunei University,U.K. ) Prof.Dr .W .Seine n (Universiteit Utrecht) Development and application of* « vitro and /n vivoreporte r gene assaysfo r the assessment of(xeno-)estrogeni c compounds in the aquatic environment Juliette Legler Proefschrift ter verkrijging van de graadva n doctor op gezagva n de rector magnificus van Wageningen Universiteit Prof.Dr . Ir. L. Speelman, inhe topenbaa r te verdedigen op dinsdag 27 februari 2001 desnamiddag st evie r uur in de Aula. The research described in this thesis was carried out at the Department of Toxicology, Wageningen University, and the Netherlands Institute for Developmental Biology (Hubrecht Laboratory), Utrecht. Title Development and application of in vitro and in vivo reporter gene assays for the assessment of (xeno-)estrogenic compounds in the aquatic environment Author J. Legler Thesis Wageningen University - with ref. - With summary in Dutch ISBN 90-5808-362-4 This research was funded by The Ministry of Transport, Public Works and Water Management, National Institute of Coastal and Marine Management (Rijkswaterstaat- RWS/RIKZ). Financial support was also provided by the National Sciences and Engineering Research Council of Canada. for myparents Stellingen 1. Met behulp van de nieuwe invitro e n in vivoreporte r gen assays, die kunnen worden ingezet voor het snel determineren van de aanwezigheid van biologisch-actieve oestrogene stoffen, is aangetoond dat op enkele plaatsen in het Nederlandse aquatische milieu al niveaus van (xeno) oestrogenen voorkomen, die zeer waarschijnlijk ongewenste effecten op de voortplanting van vissen zouden kunnen veroorzaken. (ditproefsckrift) 2. Terwijl persistente stoffen invivo hoger e oestrogene activiteit vertonen dan voorspeld wordt met in vitro bioassays, kan de oestrogene potentie van snel metaboliseerbare stoffen mogelijk overschat worden door invitro bioassays . (ditproefschrift) 3. De synergistische effecten van gecombineerde blootstelling aan de pesticiden paraquat en maneb op het dopamine systeem ind ehersene n van de rat kan betekenen, dat blootstelling aan mengselsva n pesticiden een bijdrage zou kunnen leveren aan het ontstaan van deziekt e van Parkinson. (Thiruchelvam, M. etal. (2000)J. Neuroscience, 20, 9207-9214) 4. Subtiele ecologische-relevante effecten, zoals verlengde metamorfose bij kikkers door PCB-blootstelling, worden niet aangetoond met klassieke toxicologische toetsten. (now:Gutleb, A.C. etal.(1999) Environ. Toxicol. Pharmacol. 8, 1-14) 5. De aanwezigheid van lage niveaus van farmaceutica in het milieu is zorgwekkend, omdat deze stoffen continu terechtkomen inoppervlaktewate r via rioolwaterzuivering effluenten en bovendien zeer weinig bekend is over de aquatische toxiciteit van deze stoffen. (Daughton, C.G. andTerms, T.A. (1999) Environ. HealthPerspect, 107, 907-938) 6. Met haar plannen om de fok van zogenaamde "agressieve" hondensoorten te verbieden legt de Nederlandse overheid de verantwoordelijkheid voor deze problematiek bij dehon d inplaat sva n bij de eigenaar. 7. De werkelijke morele beproeving van de mens, de meest essentiele (zo diep opgeborgen dat die zich aan onze blik onttrekt),berus t op zijn verhouding tot wie aan hem zijn overgeleverd: de dieren. (Kundera, M. (1983) "De ondraaglijke lichtheidvan het bestaan") 8. Buitenlanders zouden het beste in Nederland kunnen integreren door drie dingen te leren: de Nederlandse taal, fietsen en vergaderen. (naar: Van Vree, W.(1994) "Nederlandals vergaderland") Stellingen behorendbi j hetproefschrif t "Developmentand application ofin vitro andin vivo reporter geneassays for the assessment of (xeno-)estrogenic compounds in the aquatic environment" door JulietteLegler ,Wageningen ,2 7februar i 2001. Propositions 1. Application of the newly developed in vitro and in vivo reporter gene assays, which can be used to rapidly determine the presence of biologically-active estrogenic compounds, has shown that insom e localised areas in the Dutch aquatic environment, fish may be exposed to (xeno-)estrogens at levels that may impact their reproductive success, (this dissertation) 2. While persistent compounds may be more estrogenic in vivo than predicted by in vitro assays, the estrogenic potency of rapidly metabolised compounds may be overestimated by invitro assays , (thisdissertation) 3. Thesynergisti c effects of combined exposure toth e pesticides paraquat and manebo n the brain dopaminergic system in the rat suggests that exposure to mixtures of pesticides may play a role in the development of Parkinson's disease. (Thiruchelvam, M. etal. (2000)J. Neuroscience, 20, 9207-9214) 4. Subtle, ecologically relevant effects, such as prolonged metamorphosis in PCB- exposed frogs, are not detected with classical toxicological tests, (based on: Gutleb, A.C. etal.(1999) Environ. Toxicol. Pharmacol. 8, 1-14) 5. The presence of low levels of pharmaceuticals in the environment is alarming, as these compounds are continually introduced to surface waters via sewage treatment effluent and very little is known of their aquatic toxicity. (Daughton, C.G. and Terms, T.A. (1999) Environ. HealthPerspect, 107, 907-938) 6. By outlawing the breeding of so-called "aggressive" dog-breeds, the Dutch government is placing the burden of responsibility for this issue upon the dog instead ofth e owner. 7. Mankind's true moral test, its fundamental test (which lies deeply buried from view), consists of its attitude towards those who are at its mercy: animals. (Kundera, M. (1983) "Theunbearable lightness ofbeing") 8. Foreigners would integrate best in The Netherlands by learning three things: the Dutch language, bicycling, and "meeting" skills, (based on: Van Vree, W. (1994) "The Netherlandsas a meeting-place ") Propositions belonging toth e PhD dissertation "Development andapplication ofin vitro andin vivo reporter gene assaysfor the assessment of(xeno-)estrogenic compounds inthe aquatic environment" byJuliett eLegler ,Wageningen ,Februar y27,2001 . Contents Chapter 1 General Introduction Chapter 2 Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line 17 Chapter 3 Detection of estrogenic activity in sediment-associated compounds usingi nvitr oreporte r gene assays 39 Chapter 4 Measurement of estrogenic activity in human urine and fish bile usingth e invitro ER-CALUXreporte r gene assay 57 Chapter 5 A novel in vivo bioassay for (xeno-)estrogens using transgenic zebrafish 71 Chapter 6 Value of in vivo transgenic zebrafish and in vitro reporter gene assays for determination of estrogenic activity in environmental matrices 87 Chapter 7 General Discussion (summary andconcludin g remarks) 103 References 111 Samenvatting 123 Curriculum vitae 128 List of publications 129 Dankwoord 131 Chapter 1 General Introduction The research presented inthi sthesi s describes the development of new bioassays to determine the exposure to and early effects of substances with an estrogenic mode of action. An invitro reporter gene assay using a human breast cancer cell line, and an in vivo reporter gene assay using stably transformed (transgenic) zebrafish, were developed. Special emphasis was placed on application of the assays to environmental mixtures to monitor levels of biologically active estrogenic substances inth e aquatic ecosystem. Chapter 1 Endocrinedisruption: a "stolenfuture"for humansand wildlife? In 1992, Carlsen, Skakkebaek and colleagues published a controversial study reporting that a significant, worldwide decrease in sperm quality has occurred over the past 50 years. In addition to decreased sperm counts, increased incidence of testicular cancer and cryptorchidism were reported (Giwercman et al., 1993). Evidence in wildlife species that deleterious effects on male reproductive health were occurring also mounted. In Florida panthers, males were found with low sperm counts and cryptorchidism (Facemire et al., 1995). Male alligators in Lake Apopka in Florida demonstrated an array of reproductive abnormalities, including reduced penis size, abnormal testis morphology and abnormal testicular steroidogenesis (Guillette et al., 1994). In seagulls, altered sex ratio and feminization of sexual behaviour in males was observed (Fry and Toone, 1981). Wild male fish captured in waters receiving sewage effluents showed feminized gonads and production of a yolk protein, which normally is produced in females (see below). These alarming reports lead to the hypothesis that declines in sperm counts and related disorders of the male reproductive system in both humans and wildlife could have arisen because of exposure of the developing fetus or during neonatal life to estrogens (Sharpe and Skakkebebaek, 1993; Colborn etal., 1993).On e potential source of increased estrogen exposure was via chemicals in the environment that can mimic natural estrogens, the so-called xeno-estrogens. The term "endocrine disruption" was born. Endocrine disruption has been officially
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