Dysregulated Apoptosis in Teratogen-Induced Neural Tube Defects in Mice

Abstract

Dysregulation of apoptosis during development is a possible mechanism for teratogen-induced birth defects. Neural tube defects (NTDs) are the second most common fetal malformations. Non-specific stimulation of maternal immune system prevents birth defects. This study investigated the role of dysregulated apoptosis in formation of NTDs from two teratogens: valproic acid (VA) and an unknown teratogen found in tap water. Interferon- γ (IFN γ) was used to stimulate maternal immunity to evaluate the role of altered apoptosis in this protective mechanism.

Apoptosis was evaluated using flow cytometry, Terminal Transferase dUTP Nick End Labeling (TUNEL) assay and gene expression changes by RT2 Profiler PCR arrays. Additionally, changes in the expression of key signal transduction pathway genes that play a role in development were determined.

Increased apoptosis, suggesting involvement in VA teratogenicity, was observed along the neural tube in both normal and abnormal embryos from VA-exposed dams. Increased apoptosis in normal VA-exposed embryos suggests that VA may alter other cellular processes such as cell proliferation and differentiation in addition to apoptosis. Apoptotic percentages in embryos with NTDs from IFNγ+VA dams were similar to controls, which indicated resistance to teratogen-induced apoptosis. In IFNγ+VA-exposed embryos with NTDs, immune stimulation failed to prevent apoptosis. VA initiated both death and survival signaling in the embryos; however, upregulation of the apoptotic genes and down regulation of anti-apoptotic genes of p53 and Bcl2 family tended to shift the balance towards death signaling. This change in gene expression patterns could result in increased apoptosis and NTDs in VA-exposed embryos. Immune stimulation normalized changes in the expression of pro-apoptotic signaling molecules. These results suggest immune stimulation protects embryos from teratogenicity of VA by preventing VA-induced apoptosis.

VA altered the hedgehog, Wnt, retinoic acid and fibronectin signaling pathways in embryos with NTDs. These results suggest that VA also disrupted signaling pathways required for various morphogenic events during organogenesis. Immune stimulation normalized the expression of Fn1 and Hspb1 and thus may mediate protection through these signaling pathways.

In tap water exposed embryos, no change in apoptotic pattern was observed by flow cytometry, TUNEL assay and RT-PCR. Also, none of the signal transduction pathway genes tested were significantly altered in tap water-exposed embryos. This suggests that apoptosis is not a mechanism for teratogenicity resulting from exposure to the contaminant in tap water.