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

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

workingsci4

Heightened 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 1 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 1 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 1 males also showed a reduced vitellogenic response when compared with F 0 males, indicating an acclimation to EE 2 exposure. Depuration studies found only a partial recovery in reproductive capacity after 5 months. Significantly, even though the F 1 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

Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish-2

<p><b>Copyright information:</b></p><p>Taken from "Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish"</p><p>Environmental Health Perspectives 2004;112(17):1725-1733.</p><p>Published online 4 Nov 2004</p><p>PMCID:PMC1253666.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish-4

<p><b>Copyright information:</b></p><p>Taken from "Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish"</p><p>Environmental Health Perspectives 2004;112(17):1725-1733.</p><p>Published online 4 Nov 2004</p><p>PMCID:PMC1253666.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish-0

<p><b>Copyright information:</b></p><p>Taken from "Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish"</p><p>Environmental Health Perspectives 2004;112(17):1725-1733.</p><p>Published online 4 Nov 2004</p><p>PMCID:PMC1253666.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish-5

<p><b>Copyright information:</b></p><p>Taken from "Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish"</p><p>Environmental Health Perspectives 2004;112(17):1725-1733.</p><p>Published online 4 Nov 2004</p><p>PMCID:PMC1253666.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish-1

<p><b>Copyright information:</b></p><p>Taken from "Long-Term Exposure to Environmental Concentrations of the Pharmaceutical Ethynylestradiol Causes Reproductive Failure in Fish"</p><p>Environmental Health Perspectives 2004;112(17):1725-1733.</p><p>Published online 4 Nov 2004</p><p>PMCID:PMC1253666.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p