Craniofacial malformations induced by N-ethyl-N-nitrosourea in rat embryos in vivo and in vitro.

Teratogenic effects of N-ethyl-N-nitrosourea (ENU) on craniofacial development were investigated in rat embryos in vivo, and in vitro using a whole-embryo culture technique. A single intraperitoneal injection of ENU into the pregnant rat on day 9.5 of gestation induced several types of craniofacial malformations, e.g., cleft lip, cleft jaw, short muzzle and micrognathia in the offspring, but when the pregnant rats were treated with the drug on the later days (10.5 to 12.5), the incidence of the offspring exhibiting these malformations rapidly decreased. When the 9.5 day-old whole-embryos were cultured for 48 h with ENU, failure of neural tube closure, hypoplasia of frontal prominence, maxillary and mandibular swellings were evident. By light microscopy, the number of the neuroepithelial cells, migrating neural crest cells and mesenchymal cells in these swellings was significantly less than in the corresponding embryos cultured without ENU. By electron microscopy, almost all neuroepithelial cells, migrating neural crest cells, and many mesenchymal cells exhibited an electron-dense nucleus with faint nucleoli, and a dark cytoplasm containing various amount of phagosomes. These findings suggest that the craniofacial malformations induced by ENU are dependent on the day of the treatment with this drug, and also that the hypoplasia of the maxillary and mandibular swellings may be due to the degeneration of the neuroectodermal elements.