Protein Biomarkers for in Vitro Testing of Embryotoxicity

Abstract

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 β-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 β-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.

Food for thought ... considerations and guidelines for basic test method descriptions in toxicology

“…When dozens or hundreds of endpoints are used, such artificial correlations are likely to appear for at least one of them. typical examples are -omics studies suggesting a correlation between some metabolite or protein modification with toxicity (Groebe et al, 2010;West et al, 2010). For such studies, well-chosen statistics use measures to counteract the effect of multiple endpoints on apparent significances of effects.…”

Emodin induces liver injury by inhibiting the key enzymes of FADH/NADPH transport in rat liver

“…[12][13][14] Recently, mass spectrometry-based proteomics has been widely used to determine the modes of action and mechanisms involved in drug-or chemical-induced toxicity. [15][16][17][18][19][20][21][22] Candidate biomarkers for the earlier detection of geniposide-induced hepatotoxicity were identified using a label-free quantitative proteomics approach on a geniposide overdose-induced liver injury in a rat model. 23 In order to understand the antioxidant mechanism induced by dietary emodin in Megalobrama amblycephala liver, a comparative proteomic analysis was performed to investigate the proteome alteration under emodin administration; 24 a proteomic study using 2-D DIGE revealed that aloe-emodin affected multiple proteins associated with oxidative stress, cell cycle arrest, antimetastasis, and hepatitis C virus replication.…”

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis

“…If proteins are present in the media before culture, such information should necessarily be employed as a reference in the attempt to identify proteins secreted or metabolized by human embryos. Furthermore, embryo toxic factors have been identified that result in poor embryo quality and impaired developmental growth (Dokras et al, 1993;Groebe et al, 2010). The safety of culture media therefore makes it important to know the complete composition of the embryo culture media, and it would be advisable to test all factors for their positive and negative effects on embryo development and health effects for the offspring.…”