Adler Sarah, Paparella Martin, Pellizzer Cristian, Hartung Thomas, Bremer Susanne
ECVAM, Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra, Italy.
Altern Lab Anim. 2005 Apr;33(2):91-103. doi: 10.1177/026119290503300204.
The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.
对小鼠胚胎性畸胎瘤细胞系P19进行基因操作，以获取有关诱导分化化合物的初步信息。在没有化学诱导的情况下，P19细胞保持未分化状态，但可被诱导分化为特定细胞类型。例如，二甲基亚砜（DMSO）诱导心肌和骨骼肌分化，而视黄酸刺激神经元分化。用一个构建体转染P19细胞，该构建体包含一段鼠端粒酶逆转录酶（mTert）启动子序列与绿色荧光蛋白（GFP）基因，GFP基因作为报告基因。mTert表达是鼠端粒酶的逆转录酶成分，与端粒酶活性密切相关，在分化过程中下调。三种维甲酸和DMSO诱导了P19细胞的分化，这通过mTert_GFP表达的降低来确定，通过流式细胞术和共聚焦显微镜作为独立检测方法进行检测。一种受试物质乙醇和一种对照物质糖精未导致mTert_GFP表达降低。此外，可以证明mTert_GFP试验在非细胞毒性浓度下能检测到与发育相关的效应。mTert_GFP表达降低的ID50值低于用MTT试验检测到的IC50值，全反式维甲酸为21.4倍，9-顺式维甲酸为12.7倍，13-顺式维甲酸为29.6倍，DMSO为8.7倍。与P19细胞系的IC50值相比，乙醇在3T3细胞中获得了相似的IC50值，但DMSO增加了2倍。在所测试的浓度下，维甲酸对3T3细胞无细胞毒性。这种新开发的试验能够在4天内检测非细胞毒性浓度下诱导分化的化合物。它提供了一种检测具有特定毒理学机制的化学物质的方法，如维甲酸，通过使用不同的启动子，在胚胎毒性试验中可以提供额外信息。在此，我们报告使用这种新型试验系统成功分析了DMSO和三种具有不同体内致畸潜力的维甲酸。
