BACKGROUND: Tumor necrosis factor α (ΤNF-α) has been implicated in mediating post-implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF-α further, a comparative study was performed in TNF-α knockout and TNF-α positive mice, exposed to a reference teratogen, cyclophosphamide (CP).
METHODS: Cyclophosphamide was injected on day 12 of pregnancy and 18-day fetuses were examined for external structural anomalies. Apoptosis and cell proliferation were measured by TdT-mediated biotin–dUTP nick-end labeling and 5′-bromo-2′-deoxyuridine incorporation, respectively, in the brain (an organ, sensitive to the teratogen) of embryos 24 hr after CP injection. NF-κB DNA-binding activity by electrophoretic mobility shift assay (EMSA) and the expression of RelA (an NF-κB subunit) and IκBα proteins by Western blot analysis were assessed in the brain of embryos tested 24 and 48 hr after CP treatment.
RESULTS: Surprisingly, the proportion of fetuses with craniofacial, trunk and severe limb reduction anomalies were significantly higher in TNF-α–/– females, than in TNF-α+/+ mice. Excessive apoptosis and suppression of cell proliferation was found in the brain, and they were more prominent in TNF-α–/– than TNF-α+/+ embryos, when examined 24 hr after CP injection. Finally, CP-induced suppression of NF-κB DNA-binding activity was found to be enhanced in the brain of TNF-α–/– embryos, and the restoration of NF-κB DNA-binding activity was compromised.
CONCLUSION: This work demonstrates for the first time that TNF-α may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-κB activity in embryonic cells surviving the teratogenic insult.