Differential teratogenic response of TNFalpha+/+ and TNFalpha-/- mice to cyclophosphamide: the possible role of NF-kappaB

We observed previously that tumor necrosis factor alpha (TNFalpha)-knockout embryos are more sensitive to a cyclophosphamide (CP)-induced teratogenic insult than their TNFalpha-positive counterparts, implicating molecules acting in TNFalpha-activated antiapoptotic pathways in the mechanisms underlying this phenomenon. The main goal of this study was to assess whether the transcription factor nuclear factor kappaB (NF-kappaB) may be 1 of those molecules. Such a choice is based by evidence demonstrating TNFalpha as a powerful activator of NF-kappaB and a key role of the transcription factor in the most effective TNFalpha-activated antiapoptotic cascade. Also, the expression pattern of active caspases 3, 8, and 9 was researched to assess the sensitivity of TNFalpha+/+ and TNFalpha-/- embryos to CP-induced apoptotic stimuli. TNFalpha-knockout mice were exposed to CP on day 12 of pregnancy, with or without an NF-kappaB 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-kappaB DNA-binding and activity of caspases 3, 8, and 9. PDTC potentiated the CP-induced teratogenic effect and augmented the CP-induced suppression of NF-kappaB DNA-binding. These effects were more prominent in TNFalpha-/- than TNFalpha+/+ embryos. CP-induced caspase activation was found to be similar in TNFalpha-/- and TNFalpha+/+ embryos at 24 hr after treatment. At 48 hr, TNFalpha-/- embryos exhibited higher levels of active caspases 8 and 9 than their TNFalpha-positive counterparts. The results of our study allow us to hypothesize that NF-kappaB may be a component of mechanisms underlying differential sensitivity of TNFalpha-/- and TNFalpha+/+ mice to CP-induced teratogenic insult.