Molecular multiple endpoint embryonic stem cell test--a possible approach to test for the teratogenic potential of compounds.
The embryonic stem cell test (EST) examines the cytotoxicity of chemical compounds on embryonic stem (ES) cells and 3T3.A31 fibroblasts. Additionally, the EST measures the ability of ES cells to differentiate into contracting cardiomyocytes following drug exposure. In this study, we introduce new endpoints to obtain a molecular multiple endpoint EST (mme-EST), enabling the identification of potential chemical effects on osteogenic, chondrogenic and neural differentiation in addition to the traditional endpoint of cardiomyocyte differentiation. Six compounds in three classes with known teratogenic in vivo potential were assayed with the mme-EST in a pilot study: penicillin G (non-teratogenic), 5-fluorouracil and retinoic acid (strongly teratogenic), diphenylhydantoin, valproic acid and thalidomide (moderately teratogenic). While the traditional EST measures a morphological endpoint, we included molecular markers of differentiation as endpoints. With the mme-EST, every compound could be classified correctly according to its known teratogenic potential in vivo. Penicillin G, 5-fluorouracil and diphenylhydantoin inhibited differentiation of all endpoints equally. Interestingly, valproic acid showed the strongest inhibition of neural differentiation, while thalidomide specifically inhibited osteogenic development. Retinoic acid, on the other hand, supported neural but inhibited chondrogenic and osteogenic differentiation concentration-dependently. Valproic acid and thalidomide, classified incorrectly with the established EST model, were classified correctly with the mme-EST according to their effects on specific endpoints. This pilot study indicates that the predictive value of the EST may be enhanced by including further differentiation endpoints.
zur Nieden
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NI
Research Toxicology, Bayer AG, Aprather Weg, D-42096 Wuppertal, Germany. nicole.zur.nieden@gmx.de
Kempka
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G
Ahr
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HJ
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D023381:Endpoint Determination; D005347:Fibroblasts / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D005819:Genetic Markers; D051379:Mice; D008807:Mice, Inbred BALB C; D032383:Myocytes, Cardiac / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D010006:Osteoblasts / Q000187:drug effects; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000187:drug effects*; D013723:Teratogens / Q000633:toxicity*
D005819:Genetic Markers; C034477:RNA primers; D013723:Teratogens; D012313:RNA