Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Short-time gene expression response to valproic acid and valproic acid analogs in mouse embryonic stem cells.

Prediction of developmental toxicity in vitro could be based on short-time toxicogenomic endpoints in embryo-derived cell lines. Microarray studies in P19 mouse embryocarcinoma cells and mouse embryos have indicated that valproic acid (VPA), an inducer of neural tube defects, deregulates the expression of many genes, including those critically involved in neural tube development. In this study, we exposed undifferentiated R1 mouse embryonic stem cells to VPA and VPA analogs for 6 h and used CodeLink whole-genome expression microarrays to define VPA-responsive genes correlating with teratogenicity. Compared with the nonteratogenic analog 2-ethyl-4-methylpentanoic acid, VPA and the teratogenic VPA analog (S)-2-pentyl-4-pentynoic acid deregulated a much larger number of genes. Five genes (of ∼2500 array probes correlating with the separation) were sufficient to effectively separate teratogens from nonteratogens. A large fraction of the target genes correlating with teratogenicity are functionally related to embryonic development and morphogenesis, including neural tube formation and closure. Similar responses in R1 were found for most genes previously identified as VPA responsive in P19 and embryos. A subset of target genes was evaluated as candidate markers predictive of potential teratogenicity against a range of known teratogens using TaqMan expression arrays. These marker genes showed a positive predictive value for the teratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoic acid are known histone deacetylase (HDAC) inhibitors but not for compounds that are likely to act by other mechanisms. This indicates that HDAC inhibition may be a major mechanism by which VPA induces gene deregulation and possibly teratogenicity.

Proliferative and molecular effects of the dual PPARalpha/gamma agonist tesaglitazar in rat adipose tissues: relevance for induction of fibrosarcoma.

The dual peroxisome-proliferator-activated receptor (PPAR) α/γ agonist tesaglitazar has been shown to produce fibrosarcomas in rats. Here, the authors studied morphology, proliferation, differentiation, and inflammation markers in adipose tissue from rats exposed to 1, 3, or 10 µmol/kg tesaglitazar for 2 or 12 weeks, including recovery groups (12 weeks treatment followed by 12 weeks recovery), and 3 or 10 µmol/kg tesaglitazar for 24 weeks. Subcutaneous white and brown fat revealed reversible dose-related histopathological alterations and after 12 and 24 weeks developed areas of thickened skin (fatty lumps). There was a dose-dependent increase in proliferation of interstitial cells in white and brown fat as shown by increased mitotic index in all dose groups after 2 weeks. This was limited to the high dose after 12 and 24 weeks in white fat. Gene expression analyses showed that while tesaglitazar induced differentiation of adipose tissue characterized with a switch in cyclin D1 and D3 mRNA by 12 weeks, longer exposure at high doses reversed this differentiation concurrent with a reappearance of early adipocyte and inflammatory markers. These data suggest that sustained increased turnover of mesenchymal cells in adipose tissues, concomitant with onset of inflammation and fibrosis, drives development of fibrosarcomas in rats treated with tesaglitazar.