Mechanism of valproic acid induced dysmorphogenesis via oxidative stress and epigenetic regulation at the Hoxa2 gene promoter
Date
2015-10-20
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Degree Level
Doctoral
Abstract
Valproic acid (2-propylpentanoic acid, VPA) is a clinically used anti-epileptic drug and an
effective mood stabilizer. VPA is also a histone deacetylase inhibitor and can induce embryonic
malformations in both humans and mice. The mechanism(s) of VPA-induced teratogenicity are not
well characterized. The objectives of my study were three fold, to: (i) investigate the effect of VPA on
mouse embryonic development, (ii) characterize the putative mechanism(s) of VPA-induced
teratogenicity and, (iii) investigate VPA associated epigenetic regulation of Hoxa2 gene in cell lines
and in developing embryos. Whole mouse embryo cultures were treated with VPA at doses of 0, 50
(0.35 mM), 100 (0.70 mM), 200 (1.4 mM), and 400 µg/mL (2.8 mM), encompassing the therapeutic
range of 0.35 mM to 0.70 mM. Van Maele-Fabry’s morphologic scoring system was used to
quantitatively assess embryonic organ differentiation and development. Hoxa2 gene expression was
measured by quantitative real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). To
assess epigenetic changes on the Hoxa2 gene promoter, DNA methylation was determined by bisulfite
(BSP) sequencing and pyrosequencing. Histone “bivalent domains” H3K4me3 (histone 3 lysine 4
trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation) associated with gene activation
repression,
respectively,
analyzed
qChIP-PCR
(quantitative
chromatin
immunoprecipitation-PCR). Telomere length and telomerase activity were analyzed in mouse
embryos and in NIH3T3 cell line treated with VPA.
Results indicate significantly increased incidence of dysmorphogenesis in embryos (11.8%, 35.3%,
47.0% and 88.3%) exposed to increasing doses of VPA (0.35 mM, 0.70 mM, 1.4 mM and 2.8 mM
respectively). Van Maele-Fabry’s quantitative differentiation assessment of developing embryos
demonstrated a significantly lower score for the circulation system, central nervous system,
craniofacial development and limb development in VPA treated embryos (0.35 mM to 2.8 mM)
compared to the untreated control group. Glutathione homeostasis was altered as indicated by
decreased total glutathione content and increased GSSG/GSH ratio in all VPA treatment groups. In
addition, a dose-dependent inhibition of Hoxa2 gene expression was observed in embryos and in the
NIH3T3 cell line exposed to VPA. Pre-treatment with ascorbic acid [1000 µg/mL (5 mM)] restored
glutathione level and normalized Hoxa2 gene expression in embryos exposed to VPA. DNA
methylation status was characterized on the Hoxa2 gene promoter at the three CpG islands; CpG
island 1 (-277 to -620 bp), CpG island 2 (-919 to -1133 bp), and CpG island 3 (-1176 to -1301 bp) in
the two cells lines (NIH3T3 and EG7) and in developing embryos. CpG sites remained unmethylated
on the Hoxa2 gene promoter in the NIH3T3 cell line which expresses the Hoxa2 gene, whereas these
same CpG sites were methylated in EG7 cells that did not express Hoxa2. CpG island 1 is closest to
Hoxa2 transcription start site and its methylation status was most affected. In developing embryos,
CpG island 1 was found to be highly methylated at E6.5 when Hoxa2 is not expressed, whereas the
methylation status of CpG sites on the CpG island 1 declined between E8.5 and E10.5 when Hoxa2
expression is present. VPA induced methylation of several CpG sites on CpG island 1 in NIH3T3 cell
line and in E10.5 embryos when Hoxa2 expression was down regulated following VPA exposure. In
addition, embryos and the NIH3T3 cell line treated with VPA impacted the “bivalent domains”
resulting in increased H3K27me3 enrichment and decreased H3K4me3 enrichment on Hoxa2
promoter. Pre-treatment with ascorbic acid normalized Hoxa2 expression and histone bivalent domain
changes and prevented increased DNA methylation following VPA exposure. Moreover, the
telomerase activity and telomere length were both impacted by changes in glutathione redox potential
induced by VPA. Oxidative stress following VPA treatment reduced telomerase activity and
accelerated telomere shortening.
These results are the first to demonstrate: (i) a correlation between VPA dose and total
morphologic score in the developing mouse embryos. VPA impacted embryonic tissue differentiation
and neural system development in the dose range of 0.35 mM to 2.8 mM; (ii) VPA altered glutathione
homeostasis in cultured mouse embryos and inhibited Hoxa2 gene expression; (iii) Histone bivalent
domains of H3K27 and H3K4 trimethylation and DNA methylation status at the Hoxa2 gene
promoter region were altered following treatment with VPA. This appears to be the epigenetic event
in transcriptional silencing of Hoxa2 gene expression after VPA exposure; and (iv) Ascorbic acid
normalizes glutathione homeostasis, H3K27 and H3K4 trimethylation and DNA methylation status,
restoring Hoxa2 gene expression following VPA exposure. Taken together our results show VPA-
induced altered glutathione homeostasis, telomere shortening and telomerase dysfunction, and an
inhibition of Hoxa2 gene expression leads to developmental abnormalities. Exposure to ascorbic acid
had a protective effect on developing embryos exposed to VPA.
Description
Keywords
Valproic acid, dysmorphogenesis, oxidative stress, epigenetic regulation, Hoxa2 gene
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Pharmacy and Nutrition
Program
Pharmacy