Protein biomarkers for in vitro testing of embryotoxicity.

There are new challenges for hazard and risk assessment in the chemical industry with regard to REACH legislation in Europe and related activities in the U.S. and Japan, which require the development of novel in vitro models for the molecular characterization of drug- or chemical-related effects replacing conventional animal testing. In the frame of a European FP6 project on reproductive toxicology ( www.reprotect.eu ), we prepared protein samples from mouse embryonic stem cells differentiated into contracting cardiomyocytes according to the validated embryonic stem cell test (EST) protocol, which had been exposed to toxic substances selected by an expert committee from different in vivo categories of embryotoxicity. Lysates were used to carry out the following investigations: (i) identify optimal dose range conditions in the EST that are suitable for (ii) performing a differential quantitative proteomic study of underlying molecular pathways, (iii) define classes of substances with similar proteomic response patterns, (iv) relate these classes to the traditional in vivo categories of embryotoxicity with (v) the final goal to identify novel surrogate protein biomarker candidates for embryo toxicity. We found two distinct classes of toxic substances (Dinoseb, Ochratoxin-A, and Nitrofen vs ß-aminoproprionitril, Metoclopramide, Doxylamine succinate, and d-penicillamine) with clear pathway-related differences in their proteomic patterns. Most notably, different responses to cluster 1 and cluster 2 substances were observed for Heat shock protein ß-1, Ras-GTPase-activating protein SH3-domain binding protein, Ran binding protein 5, and Calreticulin, Dihydropyrimidinase-like 2 (Ulip2 protein). On the other hand, Heat shock protein 8 and Fscn1 protein were down-regulated by all compounds from both clusters.

Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin.

Novel molecular content for fast in vitro strategies in the context of safety tests concerning developmental toxicity has a potential to substantially reduce animal experiments according to the "3R" concept (Reduce/Refine/Replace). Here we present and discuss data from a differential proteomic profiling of samples generated using embryonic stem cell derived in vitro models treated with a set of model substances. Among substance-dependent proteomic changes, potential surrogate markers were some isoforms of heat shock proteins and a component of the Ras pathway, present in several redundant isoforms due to posttranslational modifications. Both proteins are implicated in cell migration, cell survival, growth and embryonic development. Using the examples of warfarin and lovastatin, two substances with entirely different primary targets, the surrogate marker signature nevertheless indicates a common embryotoxic mode of action. We discuss these findings observed in in vitro toxicity tests, in a context of clinical validation and evidence-based toxicology.