Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5‐Aza‐2′‐deoxycytidine

DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5‐aza‐2′‐deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05‐3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose‐related accumulation of cells in the S and G₂/M phases was observed at 4 and 8 hr after maternal dosing. S‐phase accumulation was the most sensitive indicator of effect; a dose‐related increase in the percentage of hindlimb bud cells in S‐phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication‐associated cytotoxicity, at doses of ≥0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of ≥0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased. Embryonic mortality did not increase significantly at dosages of < 2 mg/kg but reached 100% at 3 mg/kg. Vertebral and pelvic girdle malformations were seen at doses of 2l mg/kg. These results indicate that the demethylating agent dAZA perturbs embryonal DNA synthesis and results in the death of rapidly proliferating cells. The observation of cell cycle perturbations and cell death at dosages below those producing frank terata indicate some ability of the embryo to compensate for and/or repair dAZA‐induced cellular damage. © 1994 Wiley‐Liss, Inc.