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.

Cadmium induces a novel metallothionein and phytochelatin 2 in an aquatic fungus.

Cadmium stress response was measured at the thiol peptide level in an aquatic hyphomycete (Heliscus lugdunensis). In liquid culture, 0.1 mM cadmium increased the glutathione (GSH) content and induced the synthesis of additional thiol peptides. HPLC, electrospray ionization mass spectrometry, and Edman degradation confirmed that a novel small metallothionein as well as phytochelatin (PC2) were synthesized. The metallothionein has a high homology to family 8 metallothioneins (http://www.expasy.ch/cgi-bin/lists?metallo.txt). The bonding of at least two cadmium ions to the metallothionein was demonstrated by mass spectrometry (MALDI MS). This is the first time that simultaneous induction of metallothionein and phytochelatin accompanied by an increase in GSH level has been shown in a fungus under cadmium stress, indicating a potential function of these complexing agents for in vivo heavy metal detoxification. The method presented here should be applicable as biomarker tool.

Cadmium and zinc response of the fungi Heliscus lugdunensis and Verticillium cf. alboatrum isolated from highly polluted water.

The aquatic hyphomycete Heliscus lugdunensis and the terrestrial fungus Verticillium cf. alboatrum, both isolated from a highly polluted surface water, were investigated for their tolerance against Cd and Zn. Hl-H4 showed a 50% growth inhibition at 0.1 mM Cd, whereas at 0.7 mM Cd the growth of Va-H4 was only reduced by 30%. The fungi also showed a remarkable difference in their Zn-tolerance. The growth of Va-H4 was not inhibited at 1 mM Zn, whereas for Hl-H4 no growth occurred above 0.3 mM Zn. The biosorption and accumulation capacities for Cd or Zn of both fungi differed between the fungal species. In a 0.1 mM Cd-medium Hl-H4 biosorbed 15-fold and accumulated 39-fold more Cd than Va-H4. Exposure to 0.3 mM Zn resulted in a 13-fold higher biosorption and 11-fold higher accumulation for Hl-H4 than Va-H4. As glutathione (GSH) is known to be involved in the phytochelatin synthesis and other stress related processes we investigated its synthesis. Both fungi increased their synthesis of GSH in response to Cd. For Hl-H4 a concentration of 0.0125 mM Cd, corresponding to an intracellular Cd content of 2.1 nmol Cd mg(-1) dw, increased the GSH content, whereas Va-H4 only responded with a higher production of GSH at 1 mM Cd and a concomitant intracellular Cd content of 22.5 nmol Cd mg(-1) dw. An increased GSH synthesis under Zn-stress was only detectable for Va-H4 (20 mM).