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 de velopment. We administered the cytidine analogue 5-aza-2'-deoxycytidin e (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death an d cell cycle. Groups of females were dosed intraperitoneally on gestat ion day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 h r later. Two embryos per litter were immediately stained with Nile blu e 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 accum ulation of cells in the S and G(2)/M phases was observed at 4 and 8 hr after maternal dosing. S-phase accumulation was the most sensitive in dicator of effect; a dose-related increase in the percentage of hindli mb 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 w as 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-assoc iated cytotoxicity, at doses of greater than or equal to 0.1 mg/kg. Ob servation of litters from additional darns killed at term revealed tha t at dosages of greater than or equal to 0.3 mg/kg, cleft palate and h indlimb 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 greater than or equal to 1 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 c ycle perturbations and cell death at dosages below those producing fra nk terata indicate some ability of the embryo to compensate for and/or repair dAZA-induced cellular damage. (C) 1994 Wiley-Liss, Inc.