title: Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern.
abstract: The development of in vitro methods for regulatory embryotoxicity testing is challenging since the understanding of chemical effects on the mammalian development is still poor. The aim of the project is to identify marker genes during in vitro cell differentiation of murine embryonic stem cells, in order to predict chemical effects on cell differentiation of specific target tissues. The present study is focusing on the expression pattern by using semi-quantitative reverse transcriptase (RT)-PCR of key genes involved in cardiomyocytes development; i.e. Oct-4, Brachyury, Nkx2.5 and alpha myosin heavy chain (alpha-MHC). Two reference chemicals with well-known in vivo data have been analysed by using this approach: retinoic acid and lithium chloride. Retinoic acid has been selected as a teratogen affecting several target tissues, whereas lithium chloride has been described to affect the development of the cardiovascular system. We demonstrate that retinoic acid already affects in the early stage of germ layer formation, which was demonstrated by a change of Oct-4 and Brachyury gene expression. As we expected, the expression of cardiac specific genes (Nkx2.5, alpha-MHC) has been also modified. In contrary, the Oct-4 and Brachyury expression was not changed by lithium treatment. In this case, we observed a modification in the normal gene expression pattern, for alpha-MHC and Nkx2.5, demonstrating that lithium chloride affects the later stage of heart development. These data suggest that the inclusion of selective target organ genes in an established embryotoxicity test allows to predict effects of chemicals and drugs to the heart development.
authors.affiliation: ECVAM (European Centre for the Validation of Alternative Methods) IHCP (Institute for Health and Consumer Protection), J.R.C (Joint Research Centre), Via E. Fermi n.1 21020 Ispra (VA), Italy.
authors.forename: C
authors.initials: C
authors.lastname: Pellizzer
authors.forename: S
authors.initials: S
authors.lastname: Adler
authors.forename: R
authors.initials: R
authors.lastname: Corvi
authors.forename: T
authors.initials: T
authors.lastname: Hartung
authors.forename: S
authors.initials: S
authors.lastname: Bremer
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D046149:Embryo Culture Techniques; D005326:Fetal Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D006321:Heart / Q000187:drug effects* / Q000196:embryology; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics; D018021:Lithium Chloride / Q000633:toxicity*; D051379:Mice; D018995:Myosin Heavy Chains / Q000235:genetics; D050814:Octamer Transcription Factor-3; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects*; D020825:T-Box Domain Proteins / Q000235:genetics; D018675:Toxicity Tests / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics; D014212:Tretinoin / Q000633:toxicity*; D000098908:Brachyury Protein
chemical_list: D004268:DNA-Binding Proteins; D005326:Fetal Proteins; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D018021:Lithium Chloride; D018995:Myosin Heavy Chains; D050814:Octamer Transcription Factor-3; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D014212:Tretinoin; D000098908:Brachyury Protein; C484469:Nkx2-5 protein, mouse; C494174:Pou5f1 protein, mouse