Detection of the Embryotoxic Potential of Cyclophosphamide by Using a Combined System of Metabolic Competent Cells and Embryonic Stem Cells
Susanne Bremer, Cristian Pellizzer, Sandra Coecke, Martin Paparella and Paolo Catalani
In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.
Development of a Testing Strategy for Detecting Embryotoxic Hazards of Chemicals In Vitro by using Embryonic Stem Cell Models
Susanne Bremer, Cristian Pellizzer, Sarah Adler, Martin Paparella and Jan de Lange
The importance of developing in vitro tests for embryotoxicity is discussed, and ECVAM's work with its collaborators is summarised. Studies are in progress to find new endpoints for use in the scientifically validated embryonic stem (ES) cell test, so that the potential for chemical effects on endodermal, mesodermal and/or ectodermal differentiation can be identified. This involves, inter alia, the use of genetically modified ES cells.
Validation of the Embryonic Stem Cell Test in the International ECVAM Validation Study on Three In Vitro Embryotoxicity Tests
Elke Genschow, Horst Spielmann, Gabriele Scholz, Ingeborg Pohl, Andrea Seiler, Nicole Clemann, Susanne Bremer and Klaus Becker
A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.