trichostatin-a and Abnormalities--Drug-Induced

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

Topics: Abnormalities, Drug-Induced; Antigens, Differentiation; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cells, Cultured; Embryonic Stem Cells; Epigenesis, Genetic; Eye Proteins; Histone Deacetylase Inhibitors; Histones; Homeodomain Proteins; Humans; Hydroxamic Acids; Methylation; Nanog Homeobox Protein; Neural Plate; Neural Stem Cells; Neuroepithelial Cells; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Paired Box Transcription Factors; PAX6 Transcription Factor; Principal Component Analysis; Promoter Regions, Genetic; Protein Processing, Post-Translational; Repressor Proteins; Transcription, Genetic; Transcriptome; Valproic Acid

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.

Topics: Abnormalities, Drug-Induced; Animals; Butyrates; Cell Line; Embryonic Development; Embryonic Stem Cells; Fatty Acids, Unsaturated; Gene Expression Regulation, Developmental; Genetic Association Studies; Genetic Markers; Histone Deacetylase Inhibitors; Hydroxamic Acids; Mice; Microarray Analysis; Neural Tube Defects; RNA, Messenger; Teratogens; Toxicogenetics; Valproic Acid

Histone deacetylases (HDAC) control gene expression by changing histonic as well as non histonic protein conformation. HDAC inhibitors (HDACi) are considered to be among the most promising drugs for epigenetic treatment for cancer. Recently a strict relationship between histone hyperacetylation in specific tissues of mouse embryos exposed to two HDACi (valproic acid and trichostatin A) and specific axial skeleton malformations has been demonstrated. The aim of this study is to verify if boric acid (BA), that induces in rodents malformations similar to those valproic acid and trichostatin A-related, acts through similar mechanisms: HDAC inhibition and histone hyperacetylation. Pregnant mice were treated intraperitoneally with a teratogenic dose of BA (1000 mg/kg, day 8 of gestation). Western blot analysis and immunostaining were performed with anti hyperacetylated histone 4 (H4) antibody on embryos explanted 1, 3 or 4 h after treatment and revealed H4 hyperacetylation at the level of somites. HDAC enzyme assay was performed on embryonic nuclear extracts. A significant HDAC inhibition activity (compatible with a mixed type partial inhibition mechanism) was evident with BA. Kinetic analyses indicate that BA modifies substrate affinity by a factor alpha=0.51 and maximum velocity by a factor beta=0.70. This work provides the first evidence for HDAC inhibition by BA and suggests such a molecular mechanism for the induction of BA-related malformations.

Topics: Abnormalities, Drug-Induced; Animals; Blotting, Western; Bone and Bones; Boric Acids; Cell Nucleus; Enzyme Inhibitors; Female; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Immunohistochemistry; Kinetics; Male; Mice; Pregnancy; Teratogens; Valproic Acid

Chemically induced birth defects are an important public health and human problem. Here we use Xenopus and zebrafish as models to investigate the mechanism of action of a well-known teratogen, valproic acid (VPA). VPA is a drug used in treatment of epilepsy and bipolar disorder but causes spina bifida if taken during pregnancy. VPA has several biochemical activities, including inhibition of histone deacetylases (HDACs). To investigate the mechanism of action of VPA, we compared its effects in Xenopus and zebrafish embryos with those of known HDAC inhibitors and noninhibitory VPA analogs. We found that VPA and other HDAC inhibitors cause very similar and characteristic developmental defects whereas VPA analogs with poor inhibitory activity in vivo have little teratogenic effect. Unbiased microarray analysis revealed that the effects of VPA and trichostatin A (TSA), a structurally unrelated HDAC inhibitor, are strikingly concordant. The concordance is apparent both by en masse correlation of fold-changes and by detailed similarity of dose-response profiles of individual genes. Together, the results demonstrate that the teratogenic effects of VPA are very likely mediated specifically by inhibition of HDACs.

Topics: Abnormalities, Drug-Induced; Animals; Anticonvulsants; Enzyme Inhibitors; Female; Gene Expression Profiling; Histone Deacetylase Inhibitors; Hydroxamic Acids; Species Specificity; Spinal Dysraphism; Transcription, Genetic; Valproic Acid; Xenopus; Zebrafish

the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis.. Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination.. Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure.. In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.

Topics: Abnormalities, Drug-Induced; Acetylation; Animals; Bone and Bones; Cartilage; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Mice; Pregnancy; Valproic Acid