Hydroxyurea Exposure Triggers Tissue-Specific Activation of p38 Mitogen-Activated Protein Kinase Signaling and the DNA Damage Response in Organogenesis-Stage Mouse Embryos

摘要

Hydroxyurea (HU) is commonly used to treat myeloproliferative diseases and sickle cell anemia. The administration of HU to gestation day 9 CD1 mice causes predominantly hindlimb, tail, and neural tube defects. HU induces oxidative stress and p38 mitogen-activated protein kinase (MAPK) signaling in embryos. HU also inactivates ribonucleotide reductase, leading to DNA replication stress and DNA damage response signaling. We hypothesize that HU exposure induces p38 MAPK activation and DNA damage response signaling during organogenesis preferentially in malformation-sensitive tissues. HU treatment (400 or 600mg/kg) induced the activation of MEK3/6, upstream MAP2K3 kinases, within 30min; phospho-MEK3/6 immunoreactivity was increased throughout the embryo. Activation of the downstream p38 MAPK peaked 3h post-HU treatment. At this time, phospho-p38 MAPK immunoreactivity was enhanced in the cytoplasm and nucleus of cells in the rostral and caudal neuroepithelium and neural tube; significant increases in p38 MAPK signaling were not observed in the somites or heart. Interestingly, the DNA damage response, as assessed by the formation of γH2AX foci, was increased at 3h in HU-exposed embryos in all tissues examined, including the somites and heart. Increases in pyknotic nuclei and cell fragmentation were observed in all tissues except the heart, an organ that is relatively resistant to HU-induced malformations. Thus, although HU induces a widespread DNA damage response, the activation of p38 MAPK is localized to the rostral and caudal neuroepithelium and neural tube, suggesting that p38 MAPK pathways may play a role in mediating the specific malformations observed after HU exposure.