Toxicological endpoints to assess developmental bone toxicity in vitro

Alemán. To predict the toxic potential of industrial chemicals and pharmaceutical products on human bone development, rodent and non-rodent (e.g. rabbits) in vivo models are commonly used.The assessment of osteogenic toxicity in the embryo is integrated into the OECD testing guideline #414, which covers prenatal developmental toxicity. In experiments using rodents, onehalf of the litter (approximately 450 animals per test substance) is sacrificed for the examination of skeletal damage. In addition to the high number of laboratory animals required, these invivo studies are costly and time-consuming. Until today, there is no validated in vitro test to assess developmental bone toxicity.Therefore, current work at ZEBET in the frame of a joint project, funded by the German Ministry for Education and Research (BMBF), focuses on the development of a robust assay with the capacity to predict osteotoxicity in the embryo.Embryonic stem cells are pluripotent cells which can differentiate into a multitude of diverse cell types. Their capacity for unlimited self-renewal together with their ability to faithfullyrecapitulate early developmental programmes in the embryo in vitro makes them an extremely attractive model for embryonic toxicity studies.Crucial stages during osteogenesis involve the sequential expression of a tightly regulated set of molecular markers and the mineralisation of the extracellular matrix. We are currentlyassessing whether any of these markers can serve as a predictive endpoint to assess bone toxicity using a diverse range of molecular biological methods, e.g. real-time PCR, Western blot,flow cytometry, Fourier transform infrared spectroscopy (FTIR) and cytochemical staining. Mouse embryonic stem cells (line D3) are currently employed as a model system to study osteogenesis.As a next step this approach will be expanded to other cellular systems including stem cells from rhesus monkey, human iPS cells as well as human mesenchymal stem cells. The applicability of the different molecular markers and themorphological approach depending on the method is discussed regarding their potential as toxicological endpoints