title: Evaluation of novel high-throughput embryonic stem cell tests with new molecular markers for screening embryotoxic chemicals in vitro.
abstract: The embryonic stem cell test (EST) is a validated in vitro alternative test for prediction of embryotoxicity with inhibition of cardiomyocyte differentiation under the microscope as beating areas at day 10 as an endpoint. However, improvements are necessary for regulatory acceptance and application to high-throughput screening. We have previously reported that heart and neural crest derivatives expressed transcript 1 (Hand1), a transcription factor essential for mammalian heart development, and cardiomyopathy associated 1 (Cmya1), an intercalated disk protein implicated in cardiac morphogenesis, are quantitative and objective molecular endpoints for predicting embryotoxicity, detected at day 6 when mouse embryonic stem (ES) cells differentiate into cardiomyocytes. In established stable transgenic ES cells with Hand1 or Cmya1 promoters upstream of luciferase reporter gene, changes in each gene expression were found to be coincident with those in luciferase activities during cardiomyocyte differentiation, suggesting that monitoring might be possible by chemiluminescent determination. In our novel EST, differentiation toxicity and cytotoxicity of test chemicals were here analyzed using ES cells and 3T3 fibroblasts by this approach in 96-microwell plates. Extensive investigations were performed to explore predictive power and validity by comparing a set of 24 well-known test chemicals. The novel EST offers high predictability and accuracy with a reduced test duration and manpower compared with the original EST protocol, thus providing a new rapid and sensitive in vitro method for screening embryotoxicants.
authors.affiliation: Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd, Konohana-ku, Osaka 554-8558, Japan.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Suzuki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ando
authors.forename: Norihisa
authors.initials: N
authors.lastname: Yamashita
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Horie
authors.forename: Koichi
authors.initials: K
authors.lastname: Saito
mesh_terms: D016475:3T3 Cells; D000826:Animal Testing Alternatives; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013723:Teratogens / Q000633:toxicity*; D018675:Toxicity Tests / Q000379:methods*; D019076:Transgenes
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D008156:Luciferases; D009687:Nuclear Proteins; D013723:Teratogens; C572128:CMYA1 protein, mouse; C095572:Hand1 protein, mouse