Methionine Pretreatment Enhances the Effects of Valproate on Axial Development in a CD1 Mouse Model

Abstract

The present study was performed to evaluate the effect of methionine (Met) pretreatment on valproate (VPA) axial defects, induced in CD1 mice by a single intraperitoneal (i.p.) injection of 400 mg/kg VPA on E8 dams. This VPA dosage regimen has been in the past related to a specific pathogenic pathway cascade: (1) VPA in utero exposure, (2) H4 histone hyperacetylation (hAC) at the level of somites, (3) expression of pro-apoptotic factors in somite tissues, (4) apoptosis of somite cells, and (5) axial defects in embryos (abnormal or fused somites) and fetuses (fusions, duplications, respecifications of vertebrae, and/or ribs). On the basis of literature suggestion, E8 CD1 mice were i.p. injected with 70 mg/kg Met 30 min before the i.p. injection with 400 mg/kg VPA. Some females were sacrificed, 1 or 3 hr after the VPA injection, embryos explanted, and used to evaluate the H4 histone hAC. The remaining females were sacrificed at term (E18) and fetuses processed for external and skeletal examination. The pretreatment with Met worsened the axial skeletal malformative picture in fetuses (we observed a larger number of affected segment per fetus in respect to the groups treated with VPA alone). In embryos, Met pretreatment increased the H4 hAC index and shifted the timing of the H4 hAC peak. Our data suggest that Met pretreatment enhances the effects of VPA in deregulating the epigenetic control of gene expression in somites, and by consequence, induces more extended dysmorphogenic effects along the axial axis.