The anticonvulsant drug VPA has been directly implicated as a potent neural tube teratogen, producing a 1–2% spina bifida response frequency in exposed human fetuses (65). This represents a 10- to 20-fold increase in prevalence over the spina bifida rates observed in the general population (66). Since spina bifida develops in only a small percentage of VPA-exposed fetuses, the data suggest that the affected fetuses have a genetically determined predisposition for VPA-induced NTDs. In utero VPA exposure in humans also has been associated with craniofacial, cardiovascular, and skeletal defects (62,67,68), although the developing nervous system appears to be particularly sensitive to disruption after exposure to this drug.
Humans are not unique in their response to VPA, as this drug has been shown to induce exencephaly and spina bifida in rodents and other laboratory animals (69,70). In the various animal species exposed in utero to VPA, abnormalities of the skeletal system were the most commonly reported developmental defect. The principal malformation associated with VPA exposure in utero in experimental animals has been NTDs, including exencephaly and spina bifida. Nau (71) produced exencephaly in mouse embryos from dams exposed to sufficiently high dosages of VPA to produce maternal plasma concentrations in excess of 230 µg/ml, irrespective of the route of administration. This concentration represents a two- to fivefold increase over the recommended human therapeutic level (72). Probit analysis indicated that a single subcutaneous injection of the drug administered on gestational day 8 must produce maternal plasma concentrations of 445 µg/ml to produce a 10% increase in the rate of exencephaly over that observed in the controls (71). A multiple-injection treatment regimen will produce the same increased response frequency for NTDs at maternal plasma concentrations of only 225 µg/ml, whereas osmotic minipumps can be used to induce this same NTD response frequency while delivering a steady state plasma VPA concentration of 248 µg/ml (71).
With respect to posterior NTDs, Ehlers and colleagues (73,74) demonstrated that multiple doses of VPA (200 mg/kg, i.p.) administered 6 h apart beginning on gestational day 9.0 can produce a 10% response frequency of spina bifida occulta, which increases to 95% with a VPA dosage increase to 500 mg/kg body weight. A significant degree of malformation of the ribs and vertebrae was apparent when the exposed fetuses were examined after alcian blue–alizarin red skeletal staining (73,74). A low frequency (4–6%) of spina bifida aperta was also induced by the same VPA treatment regimen in the Han:NMRI mouse strain, resulting in a highly disorganized and necrotic spinal cord within the vertebral canal in the lumbosacral region of the developing fetus. The absence of neuronal tissue indicated an almost complete localized ablation of the neural tube in the VPA-exposed fetuses (74).
Murine model systems have been exploited in an effort to learn more about the genetic basis of susceptibility to VPA-induced NTDs. Such studies have revealed a strain-dependent hierarchy of NTD susceptibility after single maternal intraperitoneal injections of 600 mg/kg VPA on gestational day 8.5 (70). In these studies, SWV/Fnn mice demonstrated high susceptibility to exencephaly, LM/Bc/Fnn embryos demonstrated a more modest NTD response, and C57BL/6J and DBA/2J mice were completely resistant (70). There are several possible theories to explain a genetically regulated mechanism for susceptibility to VPA-induced NTDs, one of which involves the documented inhibition of folate metabolism by VPA (75,76). Interference with selected steps in the folate pathway could potentially result in a decreased rate of methylation of essential, developmentally regulated genes during critical periods of embryogenesis. This would significantly enhance the sensitivity of the embryos to specific malformations. Such a difference in methylation patterns between embryos of several inbred strains might explain their different sensitivity to VPA-induced NTDs. However, definite interactions between folate metabolism, VPA therapy, and gene regulation have yet to be documented.
The pathogenesis of VPA-induced NTDs may also arise from alterations in neuroepithelial mitotic rates that drive the normal timing of neurulation. Thus, at discrete time points VPA exposure may perturb mitosis, leading to insufficient neuroepithelial cellular proliferation that culminates in a failure of neural fold elevation and fusion. VPA exposure has been shown to inhibit the proliferation of neuronal cells in culture. At concentrations that had previously been reported to be teratogenic in both humans and mice, VPA caused a 50% reduction in the proliferation rate of C6 glioma cells by impeding the cell cycle during the G1 phase (77,78). If exposure of C6 glioma cells to VPA occurred after this specific cell cycle restriction point, the proliferation of these cells was not affected (77). Furthermore, agents that inhibited cell proliferation in the C6 glial cell line within twice their therapeutic dose were consistently associated with major NTDs (78). Collectively these data illustrate the necessity for stable cellular proliferation within the developing neuroepithelia in order for NTC to occur, and provide compelling evidence of a potential mechanism for VPA teratogenicity.
As previously outlined, the process of neurulation is complex and requires elevation, apposition, and fusion of the neural folds to form the neural tube. This process involves many genes and their corresponding proteins. To ascertain whether changes in the expression of genes might play a role in determining the susceptibility to VPA exposure, the transcriptional activity of genes known to encode proteins involved in NTC was analyzed in embryos exposed to VPA. With the advent of newer molecular biological approaches, such as in situ transcription and antisense RNA amplification, it was possible to examine gene expression directly in the neural tubes of developing embryos (79). The experimental protocols for the gene expression studies have been previously reviewed in some detail (80,81). Briefly, NTC stage embryos from control- or VPA-treated dams were harvested at selected timepoints, generally gestational days 8.5, 9.0, and 9.5. The gene expression patterns in the embryos were analyzed by univariate and multivariate statistical approaches. In general, it appeared that teratogenic concentrations of VPA elicited strain-dependent effects on the expression of several genes that are important to normal embryonic development. These genes included cell cycle and apoptosis genes such as bcl-2 and p53. Strain-dependent changes were also observed in a number of growth factor genes, including brain-derived neurotrophic factor (bdnf), nerve growth factor (ngf), and its receptor (ngf-R). Folate pathway genes, including the folbp-1 and folbp-2 genes, as well as the MTHFR gene, were examined (79). The gene expression data collected to date suggest that subtle collective changes in several molecules, each of which by itself may be developmentally harmless, together produce the adverse phenotypic changes that may result in the observed NTDs. Clearly, cell cycle and growth factor genes are involved, and these changes may well be folate responsive.
The literature concerning the teratogenic potential of the front-line AED CBZ is much more limited than that which exists for either PHT or VPA (for review see ref. 82). Nonetheless, it has become increasingly clear in recent years that the risk for NTDs in infants exposed in utero to CBZ rivals that of VPA. Hernandez-Diaz and colleagues (83) observed a sevenfold increased risk for NTDs among women who used CBZ during pregnancy between 1976 and 1998. This supports the earlier work of Rosa (84), who first reported the association between CBZ and the risk for NTDs. In a meta-analysis of the literature on CBZ teratogenicity, Matalon and colleagues (85) reviewed the outcomes of 1,255 prospectively ascertained gestations compromised by the drug. They reported a significantly increased rate of congenital malformations, primarily NTDs, among the CBZ-exposed infants. Evaluating the effects of polytherapy, the same authors failed to identify any increased rate of malformations when CBZ was administered with one other AED (p > 0.05); however, when CBZ exposure occurred in the presence of two or more other AEDs, the teratogenic risk to the exposed infant was significantly elevated (85). In summary, the teratologic evidence collected in the past 25 years suggests that in utero exposure to CBZ, whether the drug is used as monotherapy or polytherapy by a pregnant woman with epilepsy, poses a significant risk for NTDs. The evidence will become more clear with the maturing collection and analysis of data from the AED pregnancy registries that exist in both the United States and Europe (EURAP).