TNF-α protects embryos exposed to developmental toxicants

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) 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-alpha further, a comparative study was performed in TNF-alpha knockout and TNF-alpha 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-kappaB DNA-binding activity by electrophoretic mobility shift assay (EMSA) and the expression of RelA (an NF-kappaB subunit) and IkappaBalpha 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-alpha(-/-) females, than in TNF-alpha(+/+) mice. Excessive apoptosis and suppression of cell proliferation was found in the brain, and they were more prominent in TNF-alpha(-/-) than TNF-alpha(+ /+) embryos, when examined 24 hr after CP injection. Finally, CP-induced suppression of NF-kappaB DNA-binding activity was found to be enhanced in the brain of TNF-alpha(-/-) embryos, and the restoration of NF-kappaB DNA-binding activity was compromised. CONCLUSION: This work demonstrates for the first time that TNF-alpha may act as a protector of embryos exposed to teratogenic stress. One possible mechanism may be restoration of NF-kappaB activity in embryonic cells surviving the teratogenic insult.