Parallel Speaker Sessions

Abstract

From 1996-2000 ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, D) has co-ordinated the ECVAM (European Centre for the Validation of Alternative Methods, Ispra, I) prevalidation and validation study of three embryotoxicity tests: (a) a test employing embryonic stem cell lines (EST); (b) the micromass test (MM test); and (c) the post-implantation rat whole-embryo culture assay (WEC test). In the validation study for two of the in vitro tests, the EST and the WEC, the predicitvity (100%) for strong embryotoxic chemicals was excellent and the precision (81%) was good. The predictivity for non (72%) and weak (70%) embryotoxic compounds and the precision for non-embryotoxic compounds (70%) were sufficiently high (?65%). In contrast, the results for the MM test were in sufficient. Therefore, in 2001 the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the conclusions from the formal validation study of three in vitro embryotoxicity test that the embryonic stem cell test (EST) and post-implantation rat whole-embryo culture assay (WEC) are scientifically validated tests, which are ready to be considered for regulatory purposes while the MM test was not ready to be considered for regulatory purposes.Since the performance of the EST and of the WEC were identical in the ECVAM validation trial and since the EST does not require the use of pregnant animals and is less time consuming and expensive, from 2001-2003 we have improved the EST in collaboration with the three German drug companies Bayer AG, Boehringer Ingelheim and Schering AG. The EST is a reliable in vitro test system for the classification of compounds according to their teratogenic potential based on the morphological analysis of cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of beating areas and to adapt the EST to applications in high-throughput screening systems, we have improved and expanded the EST protocol by establishing molecular endpoints of differentiation.The quantitative expression of sarcomeric myosin heavy chain (MHC) and ?-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and ?-actinin and the differentiation into cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, ?-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data.Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to teratogenic potential using stem cell technology and technological advances in the field of gene expression analysis.