Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern C. Pellizzer, S. Adler, R. Corvi, T. Hartung, S. Bremer* 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 Received 21 July 2003; received in revised form 24 September 2003; accepted 24 September 2003 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 differ- entiation 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 (a-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, a-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 a-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. # 2003 Published by Elsevier Ltd. Keywords: Mouse embryonic stem cells; Teratogenicity; Gene expression; Retinoic acid; Lithium chloride; Alternative to animal experiments 1. Introduction Several in vitro methods for detecting the embry- otoxic hazard of chemicals have been developed (Brown et al., 1995). Currently, the most promising cell model is based on murine embryonic stem cells (ES). These cells arederivedfromtheinnercellmassofmouseblastocysts and can be maintained in an undifferentiated state in vitro. The cells are pluripotent and have the capacity to differentiate into all cell types of the three primary germ layers (Doetschman et al., 1985). This capability has been used to develop in vitro tests in order to detect embryotoxicity effects on pluripotent stem cells. The successful use of this test system has been demonstrated in several studies (Laschinski et al., 1991; Genschow et al., 1999). ECVAM’s (European Centre for the Validation of Alternative Methods) embryo- toxicity validation study has proven the reliability of an in vitro system using differentiating embryonic stem cells for the prediction of the embryotoxic hazard of chemicals (Scholz et al., 1999). However, in order to be considered for regulatory acceptance, this test needs to be improved. A panel of experts have reviewed the current status of the Embryonic Stem Cell Test (EST) within an ECVAM workshop in January 2003 and advised ECVAM to include additional endpoints that are, specifically, able to detect effects on the major target tissues such as the developing nervous system (Strubing et al., 1995) , the skeletal system (Zur Nieden et al., 2003) and the cardio- vascular system (Boheler et al., 2002). A project has been initiated that is identifying pre- dictive marker genes of the major target tissues during organogenesis. Effects on germ layer cells will most 0887-2333/$ - see front matter # 2003 Published by Elsevier Ltd. doi:10.1016/j.tiv.2003.09.007 Toxicology in Vitro 18 (2004) 325–335 www.elsevier.com/locate/toxinvit * Corresponding author. Tel.: +39-0332785914; fax: +39- 0332785336. E-mail address: susanne.bremer@jrc.it (S. Bremer).