Histological study of cell death in digital malformations induced by 5-azacytidine: suppressive effect of caffeine.

The present study investigated microscopically the process of 5-azacytidine (5-AC)-induced digital teratogenesis and caffeine's suppressive effect on this process. Three distinct zones of programmed cell death were observed in control and caffeine-treated embryos 3 hours after 5-AC injection: the preaxial and postaxial ectodermal regions and the central part of the mesodermal regions. 5-AC temporarily suppressed programmed cell death in the ectoderm and mesoderm 3 hours after it was injected. However, caffeine promoted programmed cell death; normal programmed cell death was observed in the limb buds of embryos whose dams were treated with 5-AC and caffeine. The percentage of total cell death in hindlimb buds of embryos treated with 5-AC and caffeine was higher than that from embryos treated with 5-AC, whereas 5-AC-induced digital malformations were reduced by post-treatment with caffeine. Cell death reached a maximum 12 hours after the injection in limb buds from 5-AC and caffeine-treated embryos and at 24 hours in the 5-AC treated embryos. Furthermore, in the 5-AC and caffeine-treated embryos, the frequency of cell deaths at 12 hours increased almost linearly with the doses of caffeine in parallel with the reduction of 5-AC-induced malformation frequency by caffeine. These results suggest that although induced cell death may be one of the factors leading to digital malformations produced by 5-AC, it is not essential, and the existence of other factors affecting the pattern formation of the limb bud is proposed.