Abstract
Exposure to the anticonvulsant valproic acid (VPA) during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and, most frequently, neural tube defects (NTDs). The mechanisms by which VPA induces teratogenic effects are not fully understood, although previous studies support a role for oxidative stress. To investigate the effects of VPA on early development, a whole-embryo culture model was used to evaluate the protective effects of antioxidants, measure intracellular reactive oxygen species (ROS) levels, and assess markers of oxidative damage and apoptosis. Furthermore, in vivo teratological evaluations of antioxidant protection were also completed. VPA (0.60 mM in embryo culture, 400 mg/kg in vivo) induced significant decreases in embryonic growth and increases in NTDs. Of the antioxidants tested, catalase provided partial protection against VPA-mediated reductions in morphological and developmental growth parameters in both whole-embryo culture and in vivo systems. VPA exposure resulted in an increase in ROS staining in the head region, as assessed by whole-mount staining with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Markers of embryonic oxidative damage including 8-hydroxyguanosine, 4-hydroxynonenal adducts, and 3-nitrotyrosine were not affected by VPA treatment. Increased ROS levels were correlated with increased staining for apoptotic markers, as assessed by Western blotting and immunohistochemistry. Addition of catalase to the medium attenuated VPA-induced increases in ROS formation and apoptosis. These studies identify regions of the embryo susceptible to ROS and apoptosis induced by VPA, thus establishing a possible molecular pathway by which VPA exerts teratogenicity.
Footnotes
This work was supported by the Canadian Institutes of Health Research [Grant MOP 86593].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072314.
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ABBREVIATIONS:
- VPA
- valproic acid
- NTD
- neural tube defect
- ROS
- reactive oxygen species
- SOD
- superoxide dismutase
- GD
- gestational day
- PEG
- polyethylene glycol
- NAC
- N-acetylcysteine
- CM-H2DCFDA
- 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate
- TBST
- Tris-buffered saline with 0.1% Tween 20
- PARP
- poly(ADP-ribose) polymerase
- DAB
- 3,3′-diaminobenzidine
- HDAC
- histone deacetylase.
- Received March 19, 2011.
- Accepted August 25, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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