Exposure to homocysteine leads to cell cycle damage and reactive gliosis in the developing brain.

Studies that investigate the cellular effects of homocysteine (Hcy) on the differentiation of neural cells, and their involvement in establishment of cell layers in the developing brain are scarce. This study evaluated how Hcy affects the neural cell cycle and proteins involved in neuronal differentiation in the telencephalon and mesencephalon using the chicken embryo as a model. Embryos at embryonic day 2 (E2) received 20 µmol D-L Hcy/50 µl saline and analyzed at E6. The Hcy treatment induced an increase in the ventricular length of the telencephalon and also a reduction of the mantle layer thickness. We observed that Hcy induced impairments to the neural cell cycle and differentiation, which compromised the cell layers establishment in the developing brain. Hcy treatment also induced changes in gene and protein expression of astrocytes, characteristic of reactive gliosis. Our results point to new perspectives of evaluation of cellular targets of Hcy toxicity.

Impact of homocysteine on vasculogenic factors and bone formation in chicken embryos.

Developmental endochondral ossification requires constant blood supply, which is provided by the embryonic vascular network. High levels of homocysteine (Hcy) have vasculotoxic properties, but it remains unclear how Hcy disrupts blood vessel formation in endochondral ossification. Thus, we investigated the toxicity of Hcy on contents of vasculogenic factors (VEGF, VCAM-1, NOS3) and osteocalcin, using developing limbs as model. Chicken embryos were submitted to treatment with 20 µmol D-L Hcy at 12H&H and the analyses occur at 29H&H and 36H&H. We did not identify differences in the area of limb ossification in Hcy-treated (7.5 × 105 µm2 ± 3.9 × 104) and untreated embryos (7.6 × 105 µm2 ± 3.3 × 104) at 36H&H. In Hcy-treated embryos, we observed a significantly decrease of 46.8% at 29H&H and 26.0% at 36H&H in the number of VEGF-reactive cells. Also, treated embryos showed decrease of 98.7% in VCAM-1-reactive cells at 29H&H and 34.6% at 36H&H. The number of NOS3-reactive cells was reduced 54.0% at 29H&H and 91.5% at 36H&H, in the limbs of Hcy-treated embryos. Finally, in Hcy-treated embryos at 36H&H, we observed a reduction of 58.86% in the number of osteocalcin-reactive cells. Here, we demonstrated for the first time that the toxicity of Hcy is associated with a reduction in the contents of proteins involved in blood vessel formation and bone mineralization, which interferes with endochondral ossification of the limb during embryonic development. Graphical abstract.

Homocysteine-induced changes in cell proliferation and differentiation in the chick embryo spinal cord: implications for mechanisms of neural tube defects (NTD).

Maternal hyperhomocysteinemia during pregnancy is associated with increased risk of NTD in the offspring. Our study investigated the effects of homocysteine (Hcy) on proliferation and neuronal differentiation of the spinal cord cells in a chick embryo model. Embryos were treated with 20µmol D-L Hcy/50µL saline solution at embryonic day 2 (E2) and analyzed at embryonic days 4 (E4) and 6 (E6). Control embryos received exclusively 50µL saline solution. We performed immunolocalization and flow cytometry analyses using antibodies anti-phosphohistone H3 (pH3), anti-proliferating cell nuclear antigen (PCNA), anti-ß-tubulin III and anti-p53. Our results revealed that Hcy interferes in the proliferation of the neural cells, and that this effect is age-dependent and differed between Hcy-treated embryos with and without NTD. Also, Hcy induced a decrease of neuronal differentiation in the spinal cord at both embryonic ages. These findings contribute to clarifying the cellular bases of NTD genesis, under experimental hiperhomocysteinemia.

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.