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ABSTRACT: Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, β-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.
Critical Reviews in Toxicology 05/2012; 42(5):443-64. · 5.16 Impact Factor
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Karlfried Groebe,
Katrin Hayess,
Martina Klemm-Manns,
Gerhard Schwall,
Woijciech Wozny,
Margino Steemans, Annelieke K Peters,
Chaturvedala Sastri,
Petra Jaeckel,
Werner Stegmann,
Helmut Zengerling,
Rainer Schopf,
Slobodan Poznanovic,
Tina C Stummann,
Andrea Seiler,
Horst Spielmann,
Andre Schrattenholz
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ABSTRACT: There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs β-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein β-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.
Journal of Proteome Research 10/2010; 9(11):5727-38. · 5.11 Impact Factor
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Karlfried Groebe,
Katrin Hayess,
Martina Klemm-Manns,
Gerhard Schwall,
Woijciech Wozny,
Margino Steemans, Annelieke K Peters,
Chaturvedala Sastri,
Petra Jaeckel,
Werner Stegmann,
Helmut Zengerling,
Rainer Schöpf,
Slobodan Poznanovic,
Tina C Stummann,
Andrea Seiler,
Horst Spielmann,
André Schrattenholz
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ABSTRACT: Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.
Reproductive Toxicology 08/2010; 30(1):121-30. · 3.23 Impact Factor
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ABSTRACT: In order to support drug research in the selection process for non-embryotoxic pharmaceutical compounds, a screening method for embryotoxicity is needed. The murine embryonic stem cell test (EST) is a validated in vitro test based on two permanent mouse cell lines and delivering results in 10-days. Implementation of this test within our laboratory, revealed variability in the differentiation potential of the embryonic stem cells and, as a consequence, a lot of assays needed to be rejected due the fact the acceptance criteria were not reached. In order to gain a better yield of contracting myocardial cells, we used (1) a stringent control of the cell growth during subcultivation and a standardised hanging drop culture method and (2) a non-enzymatic cell harvest instead of a trypsin/EDTA cell harvest. Implementing of these cell culture modifications resulted in a decreased variability in the size of embryonic bodies, an increase of the number of acceptable tests and a significant increase of the differentiation potential of embryonic cells into strong beating myocardium, which made scoring less time consuming. Testing of 6 reference compounds in the optimized EST showed that the cell culture modifications did not changed the in vitro classification.
Toxicology in Vitro 08/2008; 22(7):1789-96. · 2.78 Impact Factor
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ABSTRACT: The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals.
Toxicological Sciences 07/2008; 105(2):342-50. · 4.65 Impact Factor
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ABSTRACT: The embryonic stem cell test (EST) is an ECVAM-validated assay to detect embryotoxicity. The output of the assay is the effect of test compounds on the differentiation of murine-derived embryonic stem cells (D3 cells), recorded by visual analysis of contracting cardiomyocyte-like cells. Incorporation of a system to assess the contractility in an automated manner is proposed, to increase the throughput in the EST independent of observer bias. The automated system is based on image recording of each well, resulting in the area (pixels) and frequency of contractility (Hz). Four test compounds were assessed for their embryotoxic potency in the 96-well version of the EST, with both manual and automated analysis: 6-Aminonicotinamide, Valproic Acid, Boric Acid, and Penicillin G. There was no statistically significant difference in the outcome of both methods in the fraction of contractility (p < 0.05), resulting in the same rank-order of Relative Embryotoxic Potency (REP) values: 6-aminonicotinamide (1) > valproic acid (0.007–0.013) > Boric Acid (0.002–0.005) > Penicillin G (0.00001). The automated image recording of contractile cardiomyocyte-like cells in the EST allows for an unbiased high throughput method to assess the embryotoxic potency of test compounds, resulting in an outcome comparable to manual analysis.
Toxicology in Vitro.
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ABSTRACT: The in vitro embryonic stem cell test (EST) allows for categorisation of the embryotoxic potential of chemicals and drug candidates. For classification, a validated prediction model was developed based on the inhibition of differentiation of murine embryonic stem cells (D3 cells) into cardiomyocytes, and the cytotoxicity data of D3 cells and murine fibroblasts (3T3 cells). Alterations were made in order to simplify the experimental procedures of the EST; a low-cell-binding 96-well plate was used to obtain the embryonic bodies instead of the original hanging drop culture. Furthermore, we assessed the need to include the 3T3 cells and developed a new ranking system. D3 cells were exposed to test compounds either from day 0 or day 3 onwards and the compounds were ranked by their Relative Embryotoxic Potency (REP), relative to the positive control 6-Aminonicotinamide. This resulted in the following REP order; 6-Aminonicotinamide > Hydroxy urea > Valproic acid > Methoxyacetic acid > Penicillin G. A similar outcome was obtained when the validated prediction model was used. Exposure of cells from day 0 or day 3 onwards did not have any effect on the outcome as calculated with both methods. We propose a simplification of the in vitro EST procedure and REP values to rank compounds. Introduction The embryonic stem cell test (EST) was developed to assess the possible embryotoxic potential of chemicals and drug candidates in an in vitro system [1]. The application of the EST for chemical testing reduces time, testing costs and the amount of animal experimentation for embryotoxicity tests. For the test, murine embryonic stem cells (D3) are used. D3 cells are derived from the inner cell mass of pre-implantation embryos or blastocysts at day 3,5 of mouse development [2]. These pluripotent cells can differentiate into various tissue types of the three germ layers; endo-, meso-, and ectoderm. The pluripotent nature of D3 cells makes it an appropriate candidate for many in vitro embryotoxicity experiments. The cells spontaneously differentiate when cultured in the absence of murine leukaemia inhibitory factor (mLIF) and presence of foetal bovine serum. As a result, a culture of differentiated D3 cells will consist of a heterogeneous population with cells from various lineages (reviewed in [3]). The EST is based on the principles of formation of embryonic bodies (EB's) when D3 embryonic stem cells are placed in a 'hanging drop' culture. When subsequently seeded in tissue culture plates, the cardiomyocyte lineage is the predominant differentiation route, resulting in contractile areas in the EB [1,4]. These contractile areas can be quantified manually by microscopic analysis. A validated prediction model based on three representing endpoints: inhibition of proliferation of the embryonic D3 cells, as well as adult 3T3 cells (murine fibroblasts), and the inhibition of differentiation of ES cells into contracting EB's can be used to classify compounds as either non-embryotoxic, moderate embryotoxic, or strong embryotoxic [5-7]. Recent modifications to the 'classic EST' include addition of molecular markers as endpoints [8-11], assessment of extra cellular matrices [12], upscaling and alterations of the production of embryonic stem cell-derived cardiomyocytes [13,14].
