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Aldert H. Piersma - One of the best experts on this subject based on the ideXlab platform.
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Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis.
Toxicology and applied pharmacology, 2017Co-Authors: Myrto Dimopoulou, Jeroen L. A. Pennings, Aart Verhoef, Bennard Van Ravenzwaay, Ivonne M.c.m. Rietjens, Aldert H. PiersmaAbstract:Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the Embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their Embryotoxicity and pharmacological action. For evaluating Embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48h. The compounds tested showed Embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4h exposure at the ID10 (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed Embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds.
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Implementation of Transcriptomics in the Zebrafish Embryotoxicity Test
Toxicogenomics-Based Cellular Models, 2014Co-Authors: Sanne A.b. Hermsen, Aldert H. PiersmaAbstract:The zebrafish Embryotoxicity test (ZET) is considered a promising alternative model in predictive developmental toxicity testing. The basic principle of this test entails the exposure of zebrafish embryos to a test compound and the subsequent morphological assessment of developmental and teratogenic effects. To increase the sensitivity and predictability of the test by detecting subtle and more detailed responses, implementation of transcriptomics analyses may be of great value. Transcriptomics analysis provides an objective tool that enables the profound evaluation of biological processes and provides the possibility to gain insight into specific physiological, pathological, or toxicological mechanisms in a high-throughput manner. This chapter gives an overview of the current implementation of transcriptomic analysis in the zebrafish Embryotoxicity test for developmental toxicity testing and discusses future perspectives for this research.
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Results of the first phase of the ECVAM project "prevalidation and validation of three in vitro Embryotoxicity tests"
ALTEX, 1998Co-Authors: Gabriele Scholz, Aldert H. Piersma, Ingeborg Pohl, Andrea Seiler, Susanne Bremer, Nigel A. Brown, Hermann-georg Holzhütter, Horst SpielmannAbstract:The results of the experimental prevalidation of three in vitro Embryotoxicity tests in an ECVAM study are reported: the rat whole embryo culture assay (WEC), the micromass assay (MM) and the embryonic stem cell test (EST). Three test chemicals assigned to the three classes of Embryotoxicity, non-, moderate- and strong embryotoxic, and also a positive and a negative control chemical were repeatedly tested in each test in two laboratories. The data obtained were evaluated independently by a biostatistician. It could be shown that the standard protocols (SOPs) for the three in vitro Embryotoxicity tests could be transferred to a second laboratory and that reproducible results were obtained. The three methods were able to discriminate the test chemicals according to their embryotoxic potential. In addition, within the scope of this study, a test chemical database for in vitro Embryotoxicity testing was established that will be used in the following formal validation of the three in vitro tests.
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Interlaboratory evaluation of Embryotoxicity in the postimplantation rat embryo culture
Reproductive toxicology (Elmsford N.Y.), 1995Co-Authors: Aldert H. Piersma, Aart Verhoef, Patrick Attenon, Rudolf Bechter, Mirjam J.a.p. Govers, Nathalie Krafft, Beat P. Schmid, Jeanne Stadler, Christian VerseilAbstract:Abstract The Embryotoxicity of eight xenobiotic compounds in rat postimplantation whole embryo culture was blindly tested in four laboratories according to a standard protocol. The results show that the four nonteratogens amaranth, penicillin, isoniazid, and saccharin did not affect embryogenesis apart from general toxicity at very high concentrations in culture for amaranth and isoniazid. There was good concordance of results across the laboratories. The four teratogens (retinoic acid, 6-aminonicotmamide, acetylsalicylic acid, and vincristine) induced a variety of specific embryotoxic effects, which were in most cases similar in all laboratories. These results indicate that the definition for specific Embryotoxicity used, as well as the culture duration and embryonic age are crucial for concordant scoring. Other methodologic differences did not significantly influence scoring of Embryotoxicity. Therefore, within the limits of the end points and embryonic stage represented in the method, embryo culture appears as a useful method for Embryotoxicity screening, which can be reproducibly applied in different laboratories.
Carlo Cremisini - One of the best experts on this subject based on the ideXlab platform.
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Predictability of Copper, Irgarol, and Diuron Combined Effects on Sea Urchin Paracentrotus lividus
Archives of Environmental Contamination and Toxicology, 2008Co-Authors: S. Manzo, Silvia Buono, Carlo CremisiniAbstract:The aim of this work was to investigate the mixture toxicity of Irgarol (2-methylthio-4-t-butylamino-6-cyclopropylamino-s-triazine), Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and copper upon the sea urchin Paracentrotus lividus and to compare the observed data with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA). Copper spermiotoxicity was more sensitive (EC50 = 0.018 mg/L) than Embryotoxicity (EC50 = 0.046 mg/L). The offspring malformations were mainly P1 type (skeletal alterations) in both cases, probably because copper competes to fix Ca^2+. Irgarol and Diuron toxicity has been previously investigated. EC50 mixture Embryotoxicity showed an EC50 of 1.79 mg/L, whereas spermiotoxicity mixture effects were lower than 11%. Both CA and IA modeling approaches failed to predict accurately mixture toxicity. For Embryotoxicity, the IA model overestimated the mixture toxicity at effect levels of
Horst Spielmann - One of the best experts on this subject based on the ideXlab platform.
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The validated embryonic stem cell test to predict Embryotoxicity in vitro
Nature protocols, 2011Co-Authors: Andrea Seiler, Horst SpielmannAbstract:In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess Embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in Embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.
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the practical application of three validated in vitro Embryotoxicity tests the report and recommendations of an ecvam zebet workshop ecvam workshop 57
Atla-alternatives To Laboratory Animals, 2006Co-Authors: Horst Spielmann, Andrea Seiler, Susanne Bremer, Lars Hareng, Thomas Hartung, H J Ahr, Elaine M Faustman, Ulla Haas, Graeme J Moffat, Heinz NauAbstract:In line with the recommendations of an ECVAM workshop on reproductive toxicity, held in 1994 (1), ECVAM initiated and funded a validation study of three in vitro Embryotoxicity tests. In two of these assays, pregnant laboratory animals are used to obtain embryonic tissue — either primary embryonic cells in the mouse micromass (MM) test or embryos in the rat whole embryo culture (WEC) test. In contrast, in the embryonic stem cell test (EST) a permanent mouse embryonic stem cell line is used. The main objectives of the validation study were to assess the performance of the three in vitro tests in discriminating between non-embryotoxic, weak embryotoxic and strong embryotoxic compounds. All three in vitro Embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (2). The results obtained in the blind trial of the definitive phase of the ECVAM validation study were reproducible, both within and among laboratories, and the concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC test (with one prediction model [PM]), and sufficient for the MM test and the WEC test (with another PM), according to the performance criteria defined by the Management Team (MT) before the formal validation study (2). The ECVAM Scientific Advisory Committee (ESAC) concluded from the results that the three in vitro Embryotoxicity tests had been sufficiently validated and could be applied to the assessment of the embryotoxic potential of drugs and other chemicals (3). Furthermore, the ESAC recommended the setting up of a workshop with the aim of producing a guidance document on the applicability of the three scientifically valid test methods in the context of reproductive toxicity testing. ECVAM, therefore, held a second Embryotoxicity workshop on 28–30 January 2003, in Ispra, Italy, to further evaluate the practical applications of the three in vitro Embryotoxicity tests.
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use of murine embryonic stem cells in Embryotoxicity assays the embryonic stem cell test
Methods of Molecular Biology, 2006Co-Authors: Andrea Seiler, Roland Buesen, Anke Visan, Horst SpielmannAbstract:The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test Embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for Embryotoxicity testing based on the most important mechanisms in Embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro Embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process
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Results of the first phase of the ECVAM project "prevalidation and validation of three in vitro Embryotoxicity tests"
ALTEX, 1998Co-Authors: Gabriele Scholz, Aldert H. Piersma, Ingeborg Pohl, Andrea Seiler, Susanne Bremer, Nigel A. Brown, Hermann-georg Holzhütter, Horst SpielmannAbstract:The results of the experimental prevalidation of three in vitro Embryotoxicity tests in an ECVAM study are reported: the rat whole embryo culture assay (WEC), the micromass assay (MM) and the embryonic stem cell test (EST). Three test chemicals assigned to the three classes of Embryotoxicity, non-, moderate- and strong embryotoxic, and also a positive and a negative control chemical were repeatedly tested in each test in two laboratories. The data obtained were evaluated independently by a biostatistician. It could be shown that the standard protocols (SOPs) for the three in vitro Embryotoxicity tests could be transferred to a second laboratory and that reproducible results were obtained. The three methods were able to discriminate the test chemicals according to their embryotoxic potential. In addition, within the scope of this study, a test chemical database for in vitro Embryotoxicity testing was established that will be used in the following formal validation of the three in vitro tests.
Kurt J. Irgolic - One of the best experts on this subject based on the ideXlab platform.
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Arsenobetaine and arsenocholine: Two marine arsenic compounds without embryotoxity
Applied Organometallic Chemistry, 2004Co-Authors: T. Rick Irvin, Kurt J. IrgolicAbstract:The Embryotoxicity of carboxymethyl(trimethyl)arsonium bromide [arsenobetaine,(CH3)3As+CH2COO−] and of 2-hydroxyethyl(trimethyl)arsonium bromide [arsenobetaine, (CH3)3As+CH2CH2OHBr] was explored. Sprague-Dawley rat embryos with intact yolk sacs were removed on day 11 of gestation and grown in a culture medium for 24 h in the presence and absence of rat liver (S-9) homogenate. Solutions of arsenobetaine or arsenocholine in dimethyl sulfoxide [DMSO, (CH3)2SO] (0.03 cm3) were added to the media to achieve concentrations of 20 mg arsenic compound per cm3 of medium. After 24 h the circulation and heart beat were monitored (indicator of embryolethality); in addition the crown-to-rump lengths were measured and the neural structures (somites) and limb buds observed (indicator of Embryotoxicity). No evidence for Embryotoxicity or embryolethality was found in the absence or the presence of S-9. These results indicate that arsenobetaine, the most common arsenic compound found in seafood at concentrations from several micrograms to several hundred micrograms arsenic per gram, lacks subacute and acute prenatal toxicity.
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In‐vitro prenatal toxicity of trimethylarsine, trimethylarsine oxide and trimethylarsine sulfide
Applied Organometallic Chemistry, 1995Co-Authors: T. Rick Irvin, Kurt J. IrgolicAbstract:The embryolethality and the Embryotoxicity of trimethylarsine, trimethylarsine oxide and trimethylarsine sulfide were investigated employing Sprague–Dawley rat embryos with intact yolk sacs. The embryos were removed on day 11 of gestation and grown in a culture medium (Way-mouth's 725/1) spiked with the arsenic compounds to concentrations in the range 4–100 mM in the presence or absence of rat liver (S-9) homogenate. After 24 h the yolk-sac circulation and heart beat were monitored (indicator of embryolethality), the crown-to-rump lengths were measured, the neural structures (somites) counted, and the development of the limb buds evaluated (indicators of Embryotoxicity). At a trimethylarsine concentration of 18.7 mM 78% of the embryos were dead when no S-9 was present. In the presence of S-9 all embryos survived but were necrotic and malformed. Signs of Embryotoxicity were observed at concentrations of 18.7 and 9.3 mM. At the 4.7 mM concentration the embryos grew as well as the control embryos. Trimethylarsine oxide v as lethal at 100 mM and severely embryotoxic at 50 and 25 mM. At all but the lowest concentration (4.5 mM) the embryos looked sick, and were frequently necrotic, deformed and underdeveloped. Trimethylarsine sulfide exhibited severe Embryotoxicity at 50 mM concentration in the absence and in the presence of S-9. Signs of acute toxicity were observable at 9 mM concentrations of trimethylarsine and trimethylarsine oxide. Compared with other environmental toxicants that show effects at concentrations orders of magnitude smaller, these arsenic compounds cannot be classified as very toxic.
S. Manzo - One of the best experts on this subject based on the ideXlab platform.
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Predictability of Copper, Irgarol, and Diuron Combined Effects on Sea Urchin Paracentrotus lividus
Archives of Environmental Contamination and Toxicology, 2008Co-Authors: S. Manzo, Silvia Buono, Carlo CremisiniAbstract:The aim of this work was to investigate the mixture toxicity of Irgarol (2-methylthio-4-t-butylamino-6-cyclopropylamino-s-triazine), Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and copper upon the sea urchin Paracentrotus lividus and to compare the observed data with the predictions derived from approaches of Concentration Addition (CA) and Independent Action (IA). Copper spermiotoxicity was more sensitive (EC50 = 0.018 mg/L) than Embryotoxicity (EC50 = 0.046 mg/L). The offspring malformations were mainly P1 type (skeletal alterations) in both cases, probably because copper competes to fix Ca^2+. Irgarol and Diuron toxicity has been previously investigated. EC50 mixture Embryotoxicity showed an EC50 of 1.79 mg/L, whereas spermiotoxicity mixture effects were lower than 11%. Both CA and IA modeling approaches failed to predict accurately mixture toxicity. For Embryotoxicity, the IA model overestimated the mixture toxicity at effect levels of