No 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 wholeembryo 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-2Ј,7Ј-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.

Although exposure during pregnancy to many drugs and environmental chemicals is known to cause in utero death of the embryo of fetus, or initiate birth defects (teratogenesis) in the surviving offspring, surprisingly, little is known about the underlying biochemical and molecular mechanisms, or the determinants of teratological susceptibility, particularly in humans. In vitro and in vivo studies based primarily on rodent models suggest that many potential embryotoxic xenobiotics are actually proteratogens that must be bioactivated by enzymes such as the cytochromes P450 and peroxidases such as prostaglandin H synthase to teratogenic reactive intermediary metabolites. These reactive intermediates generally are electrophiles or free radicals that bind covalently (irreversibly) to, or directly of indirectly oxidize, embryonic cellular macromolecules such as DNA, protein, and lipid, irreversibly altering cellular function. Target oxidation, known as oxidase stress, often appears to be mediated by reactive oxygen species (ROS) such as hydroxyl radicals. The precise nature of the teratologically relevant molecular targets remains to be established, as do the relative conditions of the various types of macromolecular lesions. Teratological suseptibility appears to be determined in part by a balance among pathways of maternal xenobiotic elimination, embryonic xenobiotic bioactivation and detoxification of the xenobiotic reactive intermediate, direct and indirect pathways for the detoxification of ROS (cytoprotection), and repair of macromolecular lesions. Due largely to immature or otherwise compromised embryonic pathways for detoxification, Cytoprotection, and repair, the embryo is relatively susceptible to reactive intermediates, and teratogenesis via this mechanism can occur from exposure to therapeutic concentrations of drugs, or supposedly safe concentrations of environmental chemicals. Greater insight into the mechanisms involved in human reactive intermediate-mediated teratogenicity, and the determinants of individual teratological susceptibility, will be necessary to reduce the unwarranted embryonic attrition from xenobiotic exposure.

Valproic acid, a commonly used antiepileptic agent, is associated with a 1 to 2% incidence of neural tube defects when taken during pregnancy; however, the molecular mechanism by which this occurs has not been elucidated. Previous research suggests that valproic acid exposure leads to an increase in reactive oxygen species (ROS). DNA damage due to ROS can result in DNA double-strand breaks, which can be repaired through homologous recombination (HR), a process that is not error-free and can result in detrimental genetic changes. Because the developing embryo requires tight regulation of gene expression to develop properly, we propose that the loss or dysfunction of genes involved in embryonic development through aberrant HR may ultimately cause neural tube defects. To determine whether valproic acid induces HR, Chinese hamster ovary 3-6 cells, containing a neomycin direct repeat recombination substrate, were exposed to valproic acid for 4 or 24 h. A significant increase in HR after exposure to valproic acid (5 and 10 mM) for 24 h was observed, which seems to occur through a conservative HR mechanism. We also demonstrated that exposure to valproic acid (5 and 10 mM) significantly increased intracellular ROS levels, which were attenuated by preincubation with polyethylene glycol-conjugated (PEG)-catalase. A significant change in the ratio of 8-hydroxy-2Ј-deoxyguanosine/2Ј-de-oxyguanosine, a measure of DNA oxidation, was not observed after valproic acid exposure; however, preincubation with PEG-catalase significantly blocked the increase in HR. These data demonstrate that valproic acid increases HR frequency and provides a possible mechanism for valproic acid-induced neural tube defects.

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