ABSTRACT
Transgenic embryonic stem cells were used to determine the embryotoxic effects of chemicals on the development of embryonic tissues. This investigation supports an ongoing validation study, aimed at reducing the time-consuming procedure currently in use, and at providing more-objective and more-detailed information on the embryotoxic potentials of chemicals. Green fluorescence protein (GFP) was used as a reporter gene and was linked to a human α-cardiac-specific promoter. The expression of GFP was switched on after specific activation of the human α-actin promoter. This permitted the easy quantification of cardiac cells by using a fluorescence-activated cell sorter (FACS). The percentage of cardiac precursor cells was calculated from the FACS-distribution pattern of cells which fluoresced versus the total number of cells. The percentage of cardiac precursor cells increased from 25% in embryoid bodies on day 3, to 86% on day 7. However, in 11-day-old embryoid bodies, the percentage decreased to 35%. Five chemicals with known embryotoxic potentials were compared with respect to the IC50 (concentration causing 50% inhibition of measured effect) values obtained by various in vitro endpoints (for example, cytotoxicity, morphology). The results showed a higher sensitivity of endpoints used for the analysis of specific effects on the selected target tissue. The data also showed the need to develop in vitro methods with specific endpoints which account for the complexity of embryotoxicology.
ABSTRACT
A project has been started using transgenic embryonic stem cells as a toxicological endpoint in order to register chemical effects on the development of embryonic tissues which are known to be sensitive during their differentiation. The green fluorescent protein (GFP) is used as a reporter gene and is linked to a cardiac specific promotor. This construct is integrated into the native DNA of undifferentiated embryonic stem cells. The expression of GFP was switched on after specific activation of the promotor (human-alpha-actin) which allows a quantification of cardiac cells using the fluorescence activated cell sorter. Kinetic analysis shows a differentiation of 25% on cells with activated human-alpha-actin promotor on day 3, increasing to 86% on day 7, and decreasing again to 35% on day 11. The known animal teratogens retinoic acid and 5-fluorouracil were chosen and the measurements were compared to the IC(50) values given by other in vitro endpoints in order to investigate the potential of this toxicological endpoint. The results show a higher sensitivity of endpoints which analysed specific effects on a selected target tissue. The exposure of embryonic stem cells to chemicals lead to the following IC(50) values: 1.149+/-0.170 ng/ml (cytotoxicity) versus 0.216+/-0.126 ng/ml (GFP expression) after treatment with retinoic acid and 54.2+/-5.2 ng/ml (cytotoxicity) versus 26.7+/-2 ng/ml (GFP expression) after treatment with 5-fluorouracil. The data shows the necessity to develop specific in vitro methods which take the complexity of embryotoxicology into account.
