TNF-alpha expression in embryos exposed to a teratogen

PROBLEM: The role of tumor necrosis factor (TNF)-alpha produced by embryoni c cells in normal and abnormal development is poorly understood. To assess to what extent TNF-alpha may be involved in the process of induced dysmorph ogenesis, the expression of TNF-alpha and TNF-alpha receptor (TNFRI) mRNA a s well as TNF-alpha protein was evaluated in embryos responding to a cyclop hosphamide (CP)-induced teratogenic insult. The effect of maternal immunost imulation increasing the embryo's tolerance to CP on TNF-alpha expression w as also investigated. METHOD OF STUDY: ICR female mice were treated intraperitoneally with 40 mg/ kg CP on day 12 of pregnancy. The immunostimulator, xenogeneic rat splenocy tes, was injected intrauterine 21 days before mating. Embryos were collecte d on days 13, 14, or 15 of pregnancy. TNF-alpha mRNA, TNFRI mRNA, and TNF-a protein expression were evaluated by in situ hybridization and immunostain ing techniques in control, teratogen-treated, and immunostimulated teratoge n-treated embryos. RESULTS: CP-treated embryos showed severe external brain and craniofacial a nomalies already visible on day 14 of pregnancy. TNF-alpha mRNA transcripts were detected in cells of the brain and the head of 13-day embryos, which preceded the occurrence of CP-induced external craniofacial anomalies. On d ay 15 of pregnancy, when severe craniofacial anomalies increased, a signifi cant increase in the intensity of TNF-alpha, TNFR1 mRNA transcripts, and TN F-alpha protein expression were observed in cells of the malformed regions of the head and the brain. In other nonmalformed organs of CP-treated embry os such as the liver (not macroscopically different from controls), neither TNF-alpha nor TNFR1 transcripts were detected. Immunostimulation substanti ally diminished the severity of CP-induced brain and craniofacial anomalies , decreased the resorption rate, and was associated with decreased intensit y of TNF-alpha mRNA transcripts detected on day 15 of pregnancy in the head and the brain of CP-treated embryos. CONCLUSIONS: TNF-alpha expressed in the embryo may be one of the molecules promoting the formation of CP-induced brain and craniofacial anomalies. The decrease of TNF-alpha expression in embryos of immunostimulated females ma y be one of the mechanisms responsible for the increased tolerance to the t eratogenic insult.