Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development.

High levels of homocysteine (Hcy) are related to an increased risk of the occurrence of congenital anomalies, including limb defects. However, few evaluations about how toxic levels of Hcy affect limb development have been reported. We investigated whether Hcy can affect the cell cycle proteins and proteins involved in mesenchymal cell differentiation during limb development, in a chicken embryo model. Embryos were treated with 20 µmol d-l Hcy/50 µl saline at embryonic day 2 and analyzed at embryonic day 6. Untreated control embryos received exclusively 50 µl saline solution. To identify cells in proliferation and cell cycle proteins, as well as Pax1/9 and Sox9 proteins, we performed immunolocalization and flow cytometry analyses using the antibodies anti-phosphohistone H3, anti-p53, anti-p21, anti-proliferating cell nuclear antigen, anti-Pax1, anti-Pax9 and anti-Sox9. No significant differences in cell proliferation were observed between Hcy-treated and untreated embryos. We observed a decrease of the proliferating cell nuclear antigen and p21 proteins, both involved in the G1 phase of cell cycle progression. On the other hand, in mesenchymal cells of the limbs, Hcy induces an increase of p53 protein, which can be activated by DNA damage. In cell differentiation, Hcy induced an increase mainly of Pax9 and Sox9 proteins. Our data indicate that the treatment with Hcy changes the mesenchymal cell dynamics during limb development, but does not change the morphology of the cartilage molds. These findings provide information to understand better the cellular basis of the toxicity of Hcy on chondrogenesis during limb development.