Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
Reprod Toxicol. 2010 Aug;30(1):113-20. doi: 10.1016/j.reprotox.2010.01.009. Epub 2010 Feb 2.
The murine embryonic stem cell test (EST) represents a validated alternative method for in vivo embryotoxicity testing. In the present study, primary hepatocytes were combined with the EST by a preincubation approach to improve its predictivity on bioactivation caused teratogenicity. As substances the well-known proteratogens cyclophosphamide (CPA) and valpromide (VPD) were used. The embryotoxic potential of CPA was detected by a strong decrease of the resulting ID(50)-concentration (50% inhibition of ES cell differentiation) after incubation with murine hepatocytes. Interspecies variation in metabolism was detected by testing VPD. After incubation of VPD with murine hepatocytes no inhibition of ES cell differentiation was observed, since hardly any teratogenic VPD metabolites were formed. In contrast, with human hepatocytes a significant conversion of VPD into the teratogen valproic acid (VPA) was observed. In summary we developed a co-culture approach for embryotoxicity testing, whereby the test compounds were incubated with hepatocytes and the supernatant was added to the ES cell culture to obtain a dose dependency of the preincubated test substances.
鼠胚胎干细胞试验（EST）代表了一种经过验证的替代体内胚胎毒性测试的方法。在本研究中，通过预孵育方法将原代肝细胞与 EST 结合，以提高其对生物活化引起致畸性的预测能力。使用了两种众所周知的前致畸剂环磷酰胺（CPA）和丙戊酸（VPD）作为物质。用小鼠肝细胞孵育后，由于形成的 ID（50）浓度（ES 细胞分化的 50%抑制）明显降低，检测到 CPA 的胚胎毒性潜力。通过测试 VPD 检测到种间代谢的差异。用小鼠肝细胞孵育 VPD 后，未观察到 ES 细胞分化的抑制，因为几乎没有形成致畸性的 VPD 代谢物。相比之下，用人肝细胞观察到 VPD 显著转化为致畸剂丙戊酸（VPA）。总之，我们开发了一种用于胚胎毒性测试的共培养方法，其中将测试化合物与肝细胞孵育，并将上清液添加到 ES 细胞培养物中，以获得预孵育测试物质的剂量依赖性。
