New models and molecular markers in evaluation of developmental toxicity

Mammalian and non-mammalian embryos and embryonic stem cells may be used as models in mechanistic studies and in testing embryotoxicity of compounds. In addition to conventional culture methods, genetic modifications and use of molecular markers offer significant advantages in mechanistic studies as well as in developing new test methods for embryotoxicity. Zebrafish model has been used for a long time and at present several applications are available. It is an easy vertebral non-mammalian model, whose genome is largely known and several genetic modifications are easily constructed to study gene expression or knocked down genes. Fluorescent marker proteins can be used also in zebrafish to indicate gene activation in transgenic models. Chemical genetics approach has been developed using zebrafish model. This is a new approach to screen small molecules that regulate signaling pathways. Embryonic stem cells have been used in mechanistic studies and mouse embryonic stem cell test has been validated to study embryotoxicity in vitro. This method has been improved using quantitative measurements of molecular endpoints by real-time RT-PCR or fluorescent activated cell sorting methods (FACS). Methods facilitating differentiation to several different cell types are available. We have studied preimplantation mouse embryos as a possible model for in vitro testing. In this method, superovulated and in vivo fertilized preimplantation embryos were collected at morula stage and cultured up to blastocysts. The mouse preimplantation culture test was improved by quantitative gene expression measurement using two-step real-time RT-PCR methods. New endpoints improve the tests of in vitro embryotoxicity because subjective assessments are replaced by objective measurements. In addition, automation is possible and less time is needed for analysis. Thus, high throughput screening will come possible to test large numbers of compounds.