title: Evaluation of the embryotoxic potency of compounds in a newly revised high throughput embryonic stem cell test.
abstract: The ability of murine-derived embryonic stem cells (D3) to differentiate into cardiomyocytes is the basis of the embryonic stem cell test (EST). With the EST, chemicals and pharmaceuticals can be assessed for their embryotoxic potency early on in the development process. In order to come to a higher throughput EST, a 96-well based method was developed based on low attachment well plates that allow for the formation of embryonic bodies from which the stem cells can differentiate. Twelve test compounds were selected based on their reported in vitro and in vivo embryotoxic potency. In the 96-well based EST, reportedly strong embryotoxic compounds 5-fluorouracil, 6-aminonicotinamide (6AN), methylmercury chloride, and hydroxyurea were correctly ranked with corresponding Relative Embryotoxic Potency values (REP, based on the EC(50) (microM) value of 6AN) of 2.6 +/- 2.9, 1, 2.0 +/- 3.1, and 0.07 +/- 0.05, respectively. Moderately embryotoxic compounds valproic acid, boric acid, methoxyacetic acid, and lithium chloride resulted in a correct ranking with REP values of 0.01 +/- 0.003, 0.001 +/- 0.001, 0.0007 +/- 0.001, and 0.0006 +/- 0.0004, respectively. The included nonembryotoxic compounds Penicillin G, acrylamide, and saccharin did not result in an inhibition of D3 cells to differentiate into cardiomyocytes, other than related to cytotoxicity (REP value of 0.00001). However, diphenhydramine resulted in an inhibitory effect similarly to the strong embryotoxic compound hydroxyurea, with a REP value of 0.40 +/- 0.36. However, further evaluation suggested this was due to direct inhibition of the contractile capacity of the D3 cardiomyocytes, rather than an embryotoxic mechanism. The 96-well based EST is a promising addition to the screening process of newly developed chemicals and pharmaceuticals.
authors.affiliation: Johnson & Johnson Pharmaceutical Research & Development (J&J-PRD), Department of Mechanistic Toxicology, Beerse, B-2340 Belgium. APeters5@prdbe.jnj.com
authors.forename: Annelieke K
authors.initials: AK
authors.lastname: Peters
authors.forename: Margino
authors.initials: M
authors.lastname: Steemans
authors.forename: Erik
authors.initials: E
authors.lastname: Hansen
authors.forename: Natalie
authors.initials: N
authors.lastname: Mesens
authors.forename: Geert R
authors.initials: GR
authors.lastname: Verheyen
authors.forename: Philippe
authors.initials: P
authors.lastname: Vanparys
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000473:pathology; D051379:Mice; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000473:pathology; D015203:Reproducibility of Results; D013723:Teratogens / Q000145:classification / Q000633:toxicity*; D018675:Toxicity Tests
chemical_list: D013723:Teratogens