Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test

Birth defects belong to the most serious side effects of pharmaceutical compounds or environmental chemicals. In vivo, teratogens most often affect the normal development of bones, causing growth retardation, limb defects or craniofacial malformations. The embryonic stem cell test (EST) is one of the most promising models that allow the in vitro prediction of embryotoxicity, with one of its endpoints being bone tissue development. The present study was designed to describe three novel inexpensive endpoints to assess developmental osteotoxicity using the model compounds penicillin G (non-teratogenic), 5-fluorouracil (strong teratogen) and all-trans retinoic acid (bone teratogen). These three endpoints were: quantification of matrix incorporated calcium by (1) morphometric analysis and (2) measurement of calcium levels as well as (3) activity of alkaline phosphatase, an enzyme involved in matrix calcification.To evaluate our data, we have compared the concentration curves and resulting ID50s of the new endpoints with mRNA expression for osteocalcin. Osteocalcin is an exclusive marker found only in mineralized tissues, is regulated upon compound treatment and reliably predicts the potential of a chemical entity acting as a bone teratogen. By comparing the new endpoints to quantitative expression of osteocalcin, which we previously identified as suitable to detect developmental osteotoxicity, we were ultimately able to illustrate IMAGE analysis and Ca2+ deposition assays as two reliable novel endpoints for the EST. This is of particular importance for routine industrial assessment of novel compounds as these two new endpoints may substitute previously used molecular read-out methods, which are often costly and time-consuming.