TNF-alpha 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 Re1lA (an NF-kappaB subunit) and I(kappa)B(alpha) 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.


CONCLUSIONS
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.
Torchinsky
Arkady
A
Department of Embryology and Teratology, Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel.
Shepshelovich
Jeanne
J
Orenstein
Hasida
H
Zaslavsky
Zeev
Z
Savion
Shoshana
S
Carp
Howard
H
Fain
Amos
A
Toder
Vladimir
V
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001921:Brain / Q000002:abnormalities / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D019465:Craniofacial Abnormalities / Q000139:chemically induced; D003520:Cyclophosphamide / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004622:Embryo, Mammalian / Q000002:abnormalities / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D020672:I-kappa B Proteins / Q000378:metabolism; D007107:Immune System / Q000002:abnormalities / Q000187:drug effects; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D011247:Pregnancy; D011485:Protein Binding; D013723:Teratogens / Q000633:toxicity*; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000502:physiology*
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D013723:Teratogens; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D003520:Cyclophosphamide