Bremer S, van Dooren M, Paparella M, Kossolov E, Fleischmann B K, Hescheler J
ECVAM, JRC, Institute for Health and Consumer Protection, 21020 Ispra, Italy.
Toxicol In Vitro. 1999 Aug-Oct;13(4-5):645-50. doi: 10.1016/s0887-2333(99)00056-9.
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.
一个项目已经启动，该项目使用转基因胚胎干细胞作为毒理学终点，以记录化学物质对胚胎组织发育的影响，已知这些胚胎组织在分化过程中很敏感。绿色荧光蛋白（GFP）被用作报告基因，并与心脏特异性启动子相连。该构建体被整合到未分化胚胎干细胞的天然DNA中。在启动子（人α-肌动蛋白）特异性激活后，GFP的表达被开启，这使得可以使用荧光激活细胞分选仪对心脏细胞进行定量分析。动力学分析显示，在第3天，激活人α-肌动蛋白启动子的细胞中有25%发生分化，到第7天增加到86%，而在第11天又降至35%。选择了已知的动物致畸剂视黄酸和5-氟尿嘧啶，并将测量结果与其他体外终点给出的IC50值进行比较，以研究这个毒理学终点的潜力。结果表明，分析对选定靶组织的特定影响的终点具有更高的敏感性。胚胎干细胞暴露于化学物质后产生了以下IC50值：视黄酸处理后，细胞毒性为1.149±0.170 ng/ml，而GFP表达为0.216±0.126 ng/ml；5-氟尿嘧啶处理后，细胞毒性为54.2±5.2 ng/ml，而GFP表达为26.7±2 ng/ml。数据表明有必要开发考虑到胚胎毒理学复杂性的特定体外方法。
