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DNA hypomethylating drug induces oxidative stress in the mammalian embryo and placenta during gestation

A large body of evidence from our previous experimental work has shown that the DNA hypomethylating drug 5-azacytidine (5azaC) is a teratogen that causes intrauterine growth restriction (IUGR), malformations in the embryos of treated dams, and disruption of placental development. Such effects were ameliorated by the antioxidant acetylsalicylic acid, recently proposed as a prophylactic agent for the adverse perinatal outcome in humans. To investigate in more detail the possible impact of 5azaC on the induction of oxidative stress markers, we also used a free-radical scavenger N-tert-butyl-a- phenylnitron (PBN) as a pretreatment. On 12-13 GD Fisher rat dams were pretreated by PBN (40 mg/ kg, i.v.) and one hour later by 5azaC (5 mg/kg, i.p.) or only with 5azaC or PBN. Embryonic, fetal (Sobocan et al. Stem Cells Dev 28, 717–733), and placental samples were evaluated compared to controls on 15 and 20 GD by classical histology, stereological quantification by numerical density (Nv) of proliferating cell nuclear antigen and oxidative markers 8-oxoDG and nitrotyrosine. Apoptotic index was calculated, and global DNA- methylation was assessed by pyrosequencing. 5azaC significantly lowered the global DNA methylation that was partly rescued by PBN pretreatment in limb buds. The impact of 5azaC on the PCNA level was tissue-dependent (from lower to higher compared to controls). PBN-pretreatment was able to significantly ameliorate survival and growth of embryos and placentas, diminish the level of severe malformations, markers of oxidative/nitrosative processes, and apoptosis in embryos and placentas of treated dams. We may conclude that a DNA hypomethylating agent caused the oxidative stress during mammalian development that was partly prevented by a free radical scavenger’s prophylactic impact. This supplements hypothesis that ROS is the primary cause of global DNA hypomethylation as based on cancer and aging research.

oxidative stress ; placenta ; embryo ; methylation

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