Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish

International audienceHeightened concern over endocrine-disrupting chemicals is driven by the hypothesis that they could reduce reproductive success and affect wildlife populations, but there is little evidence for this expectation. The pharmaceutical ethynylestradiol (EE(2)) is a potent endocrine modulator and is present in the aquatic environment at biologically active concentrations. To investigate impacts on reproductive success and mechanisms of disruption, we exposed breeding populations (n = 12) of zebrafish (Danio rerio) over multiple generations to environmentally relevant concentrations of EE(2). Life-long exposure to 5 ng/L EE(2) in the F, generation caused a 56% reduction in fecundity and complete population failure with no fertilization. Conversely, the same level of exposure for up to 40 days in mature adults in the parental F(0) generation had no impact on reproductive success. Infertility in the F, generation after life-long exposure to 5 ng/L EE(2) was due to disturbed sexual differentiation, with males having no functional testes and either undifferentiated or intersex gonads. These F, males also showed a reduced vitellogenic response when compared with F(0) males, indicating an acclimation to EE(2) exposure. Deputation studies found only a partial recovery in reproductive capacity after 5 months. Significantly, even though the F(0) males lacked functional testes, they showed male-pattern reproductive behavior, inducing the spawning act and competing with healthy males to disrupt fertilization. Endocrine disruption is therefore likely to affect breeding dynamics and reproductive success in group-spawning fish. Our findings raise major concerns about the population-level impacts for wildlife of long-term exposure to low concentrations of estrogenic endocrine disruptors

Entwicklung eines Fischtests zur Erfassung von Stoffen mit endokrinen Wirkungen in Oberflaechengewaessern. Teilprojekt 3: Der neue Fischtest in der Gewaesserueberwachung durch eine Landesbehoerde Abschlussbericht

The aim of the presented study was to establish a method to detect estrogen-induced effects in the aquatic environment, basing on the determination of yolk-proteins in the blood of male or juvenile fish by gel electrophoresis (SDS-PAGE). The method is suitable for the use in routine testing of sewage treatment works (STW) effluents as well as monitoring of free living fish. Effects of 1 ng/L etylestradiol or 100 ng/L estradiol are reliably detectable. In surface waters no estrogen-induced effects could be found, whereas effluents of some industrial STWs showed a weak estrogenic effect. Histology of normal gonadal development of the test fish is compared to the situation after exposure to endocrine disrupters and to the gonadal development of roach caught in the wild. A method to detect effects on the gonadotropin mediated control of reproduction was tested within a monitoring programme. At certain locations a high degree of juvenile male perch showed gonadal hypertrophy (pubertas praecox). It could be shown by chemical analysis that the effects on perch were highly correlated with the tributyl tin (TBT) burden of the sediment from the respective location. Exposing small fish species to TBT in single compound studies confirmed the finding that TBT may act as an endocrine disrupter on vertebrates. (orig.)In der vorliegenden Studie wurde ein Verfahren zum Nachweis oestrogener Effekte in der aquatischen Umwelt etabliert. Die Methode basiert auf der Identifikation von Dotterproteinen im Blut maennlicher oder juveniler Fische mittels Gelelektrophorese und ist sowohl zum Einsatz in Monitoringprogrammen freilebender Fische als auch fuer die Routine der Abwasseruntersuchung nach DIN geeignet. Effekte von 1 ng/L Ethinyloestradiol und 100 ng/L Oestradiol sind sicher nachweisbar. Oestrogene Effekte von Oberflaechengewaessern wurden nicht gefunden, im Ablauf einzelner Industrieklaeranlagen war ein geringer oestrogener Effekt nachweisbar. Der Bericht enthaelt eine histologische Darstellung der Gonadenentwicklung der Versuchsfische insbesondere nach Exposition gegen endokrintoxische Stoffe. Die Gonadenhistologie von Rotaugen ist vergleichend dargestellt. Ein Verfahren zur Erfassung von Fremdstoffwirkungen auf die zentralnervoese Kontrolle der Fortpflanzung wurde im Monitoring erprobt. An Standorten mit einer erhoehten Belastung der Sedimente mit zinnorganischen Verbindungen trat bei maennlichen Barschen eine Pubertas praecox auf. Laborversuche mit Fischen belegen den Verdacht, dass sedimentgebundenes Tributylzinn diese Stoerung der Fortpflanzung bewirkt. (orig.)SIGLEAvailable from TIB Hannover: F01B1771+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing healthy stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology

Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells

AbstractIn vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.</jats:p

Entwicklung eines Fischtests zum Nachweis endokriner Wirkungen in Oberflaechengewaessern. T. 1: Entwicklung des Fischtests Schlussbericht

SIGLEAvailable from TIB Hannover: F01B198+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Umweltprobleme durch Arzneimitteln. Literaturstudie

The subject of the study was to examine whether the ecotoxicological hazard potential of pharmaceuticals or their metaboloties should be assessed. In a first step, publications on environmental problems known to be related to pharmaceuticlas were reviewed and evaluated. The data were discussed with concerned interest groups. In a second step, the data required by the 'Arzneimittelgesetz (AMG)' as part of the registration process were assessed under the aspect of suitability for ecotoxicological risk assessment. Additionally, it was evaluated which laboratory tests should be required to perform ecotoxicological risk assessments with pharmaceuticals. Preliminary risk assessments were conducted for different pharmaceutical groups if sufficient data were available. Taking into consideration the legal requirements for chemical notification within the EU, the necessity of further development of the AMG concerning ecotoxicological aspects was emphasized. (orig.)Available from TIB Hannover: RN 8422(1996,60) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Umwelt, Naturschutz und Reaktorsicherheit, Bonn (Germany)DEGerman