Abstract
BACKGROUND: We observed previously that tumor necrosis factor α (TNFα)–knockout embryos are more sensitive to a cyclophosphamide (CP)–induced teratogenic insult than their TNFα‐positive counterparts, implicating molecules acting in TNFα‐activated antiapoptotic pathways in the mechanisms underlying this phenomenon. The main goal of this study was to assess whether the transcription factor nuclear factor κB (NF‐κB) may be 1 of those molecules. Such a choice is based by evidence demonstrating TNFα as a powerful activator of NF‐κB and a key role of the transcription factor in the most effective TNFα‐activated antiapoptotic cascade. Also, the expression pattern of active caspases 3, 8, and 9 was researched to assess the sensitivity of TNFα^+/+^ and TNFα^−/−^ embryos to CP‐induced apoptotic stimuli. METHODS: TNFα‐knockout mice were exposed to CP on day 12 of pregnancy, with or without an NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC) and sacrificed on day 18 of pregnancy to evaluate the CP‐induced teratogenic effect. Embryos harvested 24 or 48 hr after the CP treatment were used to evaluate NF‐κB DNA‐binding and activity of caspases 3, 8, and 9. RESULTS: PDTC potentiated the CP‐induced teratogenic effect and augmented the CP‐induced suppression of NF‐κB DNA‐binding. These effects were more prominent in TNFα^−/−^ than TNFα^+/+^ embryos. CP‐induced caspase activation was found to be similar in TNFα^−/−^ and TNFα^+/+^ embryos at 24 hr after treatment. At 48 hr, TNFα^−/−^ embryos exhibited higher levels of active caspases 8 and 9 than their TNFα‐positive counterparts. CONCLUSIONS: The results of our study allow us to hypothesize that NF‐κB may be a component of mechanisms underlying differential sensitivity of TNFα^−/−^ and TNFα^+/+^ mice to CP‐induced teratogenic insult. Birth Defects Research (Part A) 76:437–444, 2006. © 2006 Wiley‐Liss, Inc.