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  • 1
    Language: English
    In: Mut.Res.-Genetic Toxicology and Environmental Mutagenesis, 2011, Vol.723(2), pp.152-157
    Description: Over the last years, extensive research has documented endocrine-disrupting activities for a significant number of substances including, among others, hormones, pharmaceuticals, pesticides and surfactants. Nonetheless, for most endocrine disruptors, toxicological profiles are still incomplete or even lacking. A systematic review has shown that a number of endocrine disruptors with steroid-modulating effects may also exert mutagenic and carcinogenic activities. For trenbolone, an androgenic compound, there is controversy about its genotoxic properties in the literature, apparently with a strong dependence on the choice of the test system. Since fish and other aquatic animals are at risk of exposure to run-offs from cattle feedlots or sewage-discharge sites containing trenbolone, potential consequences to aquatic ecosystems need to be assessed. To this end, the potential genotoxic hazard of trenbolone was tested in the permanent rainbow trout-liver cell-line RTL-W1, as well as in primary cell cultures derived from zebrafish ( ) embryos after exposure. In either test system, a potential genotoxic hazard characterized by biphasic dose–response curves could be documented even at exposure concentrations of 30 μg/L. These results thus confirm the conclusion that the steroid trenbolone may act as a genotoxic substance.
    Keywords: Genotoxicity ; Micronucleus Assay ; Comet Assay ; In Vivo ; In Vitro ; Trenbolone ; Biology ; Public Health
    ISSN: 1383-5718
    E-ISSN: 1879-3592
    E-ISSN: 1873135X
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  • 2
    Language: English
    In: Science of the Total Environment, 2011, Vol.409(11), pp.2114-2119
    Description: The metal platinum is used for industrial and medical purposes. Due to its application in automobile catalytic converters and as an anti-cancer drug, Pt enters the aquatic environment road runoff and hospital sewage and raises concerns about its environmental impact and toxicity to organisms. Therefore, the genotoxicity of Pt at 0, 0.1, 1, 10, 50, 100 and 200 μg/l PtCl was tested on two freshwater organisms, zebrafish ( ) and ramshorn snail ( ) using the single cell gel electrophoresis, also called comet assay. PtCl did not show any genotoxicity for at the tested concentrations, whereas significantly elevated DNA damage was observed in at 1 μg/l PtCl and beyond. The results of the study suggest a high sensitivity of concerning the genotoxic impact of PtCl . ► We tested the genotoxicity of the heavy metal platinum using the comet assay. ► Model organisms were the freshwater species Danio rerio and Marisa cornuarietis. ► Genotoxicity of the tested concentrations was only found for M. cornuarietis.
    Keywords: Aquatic Organisms ; Ptcl 2 ; DNA Damage ; Comet Assay ; Environmental Sciences ; Biology ; Public Health
    ISSN: 0048-9697
    E-ISSN: 1879-1026
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  • 3
    Language: English
    In: PLoS ONE, 2014, Vol. 9(9)
    Description: Purpose: Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity.Materials and Methods: Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances.Results and Discussion: The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects asconfirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals.Conclusions: The apparent difference between the exposure scenarios is an important aspect that needs to be addressed when conducting analyses of alterations in the expression level of mRNA.
    Keywords: Natural Sciences ; Earth And Related Environmental Sciences ; Environmental Sciences ; Naturvetenskap ; Geovetenskap Och Miljövetenskap ; Miljövetenskap ; Enviromental Science ; Miljövetenskap
    ISSN: 1932-6203
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  • 4
    Language: English
    In: Science of the Total Environment, 15 February 2018, Vol.615, pp.330-347
    Description: The visualization of specific activation of the aryl hydrocarbon receptor (AhR) directly in the zebrafish embryo ( ) live-imaging is a reliable tool to investigate the presence of dioxin-like substances in environmental samples. The co-existence of inducers and inhibitors of cytochrome P450-dependent monooxygenases (CYP1A) is typical of complex environmental mixtures and requires modifications of the EROD assay: For this end, zebrafish embryos were used to evaluate the EROD-modifying potentials of common single-compound exposures as well as binary mixtures with the PAH-type Ah-receptor agonist β-naphthoflavone. For chemical testing, chlorpyrifos and Aroclor 1254 were selected; β-naphthoflavone served as maximum EROD induction control. Chlorpyrifos (≤ EC ) could be documented to be a strong CYP1A inhibitor causing characteristic edema-related toxicity. Aroclor 1254 resulted in inhibition of CYP1A catalytic activity in a concentration- and specific time-dependent manner. Next to a fast CYP1A induction, CYP1A inhibition could also be detected after 3 h short-term exposure of zebrafish embryos to chlorpyrifos. This communication also describes techniques for the quantification of fluorescence signals densitometry as a basis for subsequent statistical assessment. The co-exposure approach with zebrafish embryos accounts for the nature of potential interaction between CYP1A inducers and inhibitors and thus pays tribute to the complexity of environmental mixtures. The co-exposure EROD live-imaging assay thus facilitates a better understanding of mixture effects and allows a better assessment and interpretation of (embryo) toxic potentials.
    Keywords: Erod Induction ; Cyp1a Inhibition ; Live-Imaging ; Zebrafish Embryo ; Densitometry ; Environmental Sciences ; Biology ; Public Health
    ISSN: 0048-9697
    E-ISSN: 1879-1026
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  • 5
    Language: English
    In: Science of the Total Environment, 15 April 2018, Vol.621, pp.827-838
    Description: The measurement of EROD (ethoxyresorufin- -deethylase) activity to determine the induction of CYP1A after exposure to dioxin-like substances is a well-established biomarker in fish. For reasons of animal welfare and implementations of new chemicals regulations (REACh), methods using zebrafish ( ) and medaka ( ) embryos have recently been developed to quantify CYP1A induction, which is visualized as mean intensity of the autofluorescent resorufin formed in living anaesthetized embryos. In the present study, concentration ranges of three PAHs (benzo[ ]pyrene, β-naphthoflavone, benzo[ ]fluoranthene) as examples of known CYP1A inducers as well as extracts of two well-characterized sediment samples of the lower Neckar river (Southern Germany) were used to determine the suitability of the fathead minnow ( ) embryo for the EROD assay. Data for zebrafish embryos were generated for comparison. Fathead minnow embryos were principally suitable to show EROD induction live-imaging. Since in fathead minnow embryos both signal area and fluorescence intensities are lower than in zebrafish embryos, the induction potentials of the three model PAHs and the environmental samples proved to be species-dependent. Among the three PAHs tested, benzo[ ]fluoranthene lead to the strongest EROD signal followed by β-naphthoflavone and benzo[ ]pyrene in comparison to the positive control. Whereas benzo[ ]fluoranthene and β-naphthoflavone showed a dose-response relationship for the EROD induction, benzo[ ]pyrene failed to induce a significant signal in fathead minnow embryos. If compared to the model PAHs, the extracts of both sediments from the lower Neckar River induced stronger EROD signals in both fathead minnow and zebrafish embryos. Observations thus documented fathead minnow embryos to be as suitable for biomonitoring purposes as are zebrafish embryos.
    Keywords: Fathead Minnow ; Erod Assay ; Pahs ; Sediments ; Cytochrome P4501a ; Environmental Sciences ; Biology ; Public Health
    ISSN: 0048-9697
    E-ISSN: 1879-1026
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  • 6
    Language: English
    In: Science of the Total Environment, 15 July 2017, Vol.590-591, pp.269-280
    Description: The present study compares two alternative approaches for the measurement of ethoxyresorufin- -deethylase (EROD) activity in zebrafish ( ) following exposure to acetonic model sediment extracts: (1) the live-imaging EROD assay for the direct detection of EROD induction in individual livers epifluorescence, and (2) the fish embryo EROD assay in subcellular fractions derived from entire zebrafish embryos after exposure. For toxicity assessment, each sediment extract was tested with the standard fish embryo test (FET). Upon completion of a functioning liver after 72 h, the embryos gave a distinct fluorescent signal in the liver, and a corresponding EROD activity could be detected in the fish embryo EROD assay. The exposure time in the live-imaging EROD assay was reduced to 3 h, which resulted in a stronger, less variable and more sensitive EROD response. Overall, the live-imaging and the fish embryo EROD assays showed the same tendencies and gave comparable results, a concentration-dependent increase in EROD activity at concentrations one order of magnitude below concentrations producing macroscopically visible abnormalities. At higher concentrations, however, a decrease of EROD activity was observed in either test. Both tests ranked the three model sediment extracts in the same order. Results indicate that both test systems complement each other and together provide a rapid and reliable tool to investigate the presence of dioxin-like substances in environmental samples.
    Keywords: Erod Induction ; Live-Imaging ; Cyp 1a ; In Vivo ; Zebrafish ; Embryo ; Sediment ; Environmental Sciences ; Biology ; Public Health
    ISSN: 0048-9697
    E-ISSN: 1879-1026
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  • 7
    Language: English
    In: Environmental Science and Pollution Research, 2015, Vol.22(21), pp.16247-16261
    Description: Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish ( Danio rerio ) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If—for ethical reasons—not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has—at least for the substances tested so far—compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight 〉3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.
    Keywords: Fish embryo test ; FET ; Validation ; Alternative test method ; OECD guideline ; Acute toxicity ; Teratogenicity ; Genotoxicity ; Endocrine disruption ; Neurotoxicity ; Biotransformation ; Cytochrome P450
    ISSN: 0944-1344
    E-ISSN: 1614-7499
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  • 8
    Language: English
    In: Environmental Pollution, April 2018, Vol.235, pp.918-930
    Description: Since only a few studies have investigated effects of microplastics (MPs) by routes other than ingestion, this study was designed to analyze the accumulation patterns and transfer of toxic substances associated with microplastic exposure by simple attachment to (1) adult zebrafish ( ) gills and (2) zebrafish embryos. Two sizes of fluorescently labelled polymers (1–5 and 10–20 μm) loaded with the model polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) were used to analyze fate, accumulation and transfer of microplastic-associated persistent organic pollutants (POPs) on gills and embryos. Results indicate that microplastics did not permanently accumulate at high amounts in adult zebrafish gills after 6 nor 24 h of incubation: Most particles only superficially adhered to the mucus layer on the filaments, which is constantly being excreted. In contrast, the smaller and heavier MPs (1–5 μm) accumulated in high numbers on the surface of zebrafish egg chorions. In both exposure scenarios, transfer of BaP could be visualized with fluorescence microscopy: A prominent BaP signal was visible both in gill filaments and arches after 6 and 24 h incubation and in zebrafish embryos after exposure to BaP-spiked microplastics. Furthermore, the gill EROD (Ethoxyresorufin-O-deethylase) assay showed a clear trend to CYP 1A (Cytochrom P450 1 A) induction exposure to BaP-spiked microplastics. However, BaP from spiked microplastics did not reach sufficiently high concentrations to be able to induce morphological effects in the fish embryo toxicity test (FET). In contrast, control exposure to waterborne BaP did induce effects in the FET. As a conclusion, microplastics can also transfer POPs not only ingestion, but also by simple attachment to epithelia or the water column. However, further studies are needed to clarify if these interactions are of environmental concern relative to waterborne exposure to toxic substances.
    Keywords: Microplastics ; Zebrafish Gills ; Zebrafish Embryos ; Pop Transfer ; Benzo[a]Pyrene ; Engineering ; Environmental Sciences ; Anatomy & Physiology
    ISSN: 0269-7491
    E-ISSN: 1873-6424
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  • 9
    Language: English
    In: Chemosphere, October 2014, Vol.112, pp.77-84
    Description: As part of the risk assessment process within REACh, prior to manufacturing and distribution of chemical substances their (eco)toxicological impacts have to be investigated. The fish embryo toxicity test (FET) with the zebrafish has gained a high significance as an alternative to animal testing in (eco)toxicology. However, for hydrophobic organic chemicals it remains a technical challenge to ensure constant freely dissolved concentration at the maximum exposure level during such biotests. Passive dosing with PDMS silicone was thus applied to control the freely dissolved concentration of ten PAHs at their saturation level in the FET. The experiments gave repeatable results, with the toxicity of the PAHs generally increasing with the maximum chemical activities of the PAHs. HPLC analysis confirmed constant exposure at the saturation level. In additional experiments, fish embryos without direct contact to the silicone surface showed similar mortalities as those exposed with direct contact to the silicone. Silicone oil overlaying the water phase as a novel passive dosing phase had no observable effects on the development of the fish embryos until hatching. This study provides further data to support the close relationship between the chemical activity and the toxicity of hydrophobic organic compounds. Passive dosing from PDMS silicone enabled reliable toxicity testing of (highly) hydrophobic substances at aqueous solubility, providing a practical way to control toxicity exactly at the maximum exposure level. This approach is therefore expected to be useful as a cost-effective initial screening of hydrophobic chemicals for potential adverse effects to freshwater vertebrates.
    Keywords: Passive Dosing ; Pdms Silicone ; Fish Embryo Toxicity ; Danio Rerio ; Pahs ; Chemical Activity ; Chemistry ; Ecology
    ISSN: 0045-6535
    E-ISSN: 1879-1298
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  • 10
    Language: English
    In: PLoS ONE, Sept 4, 2014, Vol.9(9)
    Description: Purpose Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio) embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity. Materials and Methods Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances. Results and Discussion The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects as confirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract). This effect might be related to different bioavailability of chemicals. Conclusions The apparent difference between the exposure scenarios is an important aspect that needs to be addressed when conducting analyses of alterations in the expression level of mRNA.
    Keywords: Dioxins – Analysis ; Dioxins – Investigations ; Embryonic Development – Analysis ; Embryonic Development – Investigations ; RNA – Analysis ; RNA – Investigations ; Gene Expression – Analysis ; Gene Expression – Investigations ; Sediments (Geology) – Analysis ; Sediments (Geology) – Investigations
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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