The p53 Signaling Pathway is Activated in Response to Hydroxyurea Embryotoxicity During Organogenesis

Hydroxyurea (HU), an anticancer agent and potent teratogen, is used as a model drug to study the embryonic stress response during organogenesis. Previously, we demonstrated that HU activates a DNA damage response (DDR) pathway in the gestation day (GD) 9 mouse embryo (Toxicol Sci. 2013 133(2):298-308). The p53 tumor suppressor gene is a possible downstream effector of this pathway. P53 plays an important role in embryonic development, however its response to teratogens is debatable. We hypothesize that HU exposure at the organogenesis stage activates p53 which then mediates cell cycle arrest and cell death that result in the observed malformations. To test this hypothesis, CD-1 embryos at GD9 were exposed in vivo to saline (Control-CO) or two doses of HU (HU400= 400 mg/kg; HU600= 600 mg/kg); embryos were extracted after 3 hours and samples prepared to examine gene and protein expression. Microarray analysis of embryos in the HU400 group showed that the expression of 1346 genes significantly changed compared to control and that they were significantly associated with the p53 signaling pathway. The active form of p53 (phospho-p53) was significantly upregulated in HU-exposed embryos; Western blot and confocal microscopy showed increasing protein concentrations and nuclear translocation in major embryonic tissues. qRT-PCR showed that p53-regulated genes (Cdkn1A, Fas, p53inp1) were significantly upregulated and p53inp1 protein increased in a dose-dependent fashion with HU. Together, these data show that p53 signaling is the main pathway activated in response to HU during organogenesis, and leads to the upregulation of cell cycle arrest and cell death promoting factors. Gestational day 9 mouse embryos were treated with saline or 400 mg/kg hydroxyurea by intraperitoneal injection. Dams were euthanized after 3 hours and embryos explanted. 4 independent experiments were performed and analyzed for each sample.