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MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells.

Ferreira FF, Ammar D, Bourckhardt GF, Kobus-Bianchini K, Müller YM, Nazari EM - J Toxicol (2015)

Bottom Line: Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells.Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin.These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, UFSC, Campus Universitário, Trindade, 88040-900 Florianópolis, SC, Brazil; Instituto de Ciências Naturais Humanas e Sociais, UFMT, Avenida Alexandre Ferronato 1200, Setor Industrial, 78557287 Sinop, MT, Brazil.

ABSTRACT
The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus

βIII-tubulin protein analyzed by immunohistochemistry and flow cytometry. βIII-tubulin-positive cells (white arrows) observed mainly in the mantle layer. The graph displays the expression profile and relative frequency of βIII-tubulin in the spinal cord of the control and MeHg-treated embryos. Scale bar: 20 μm.
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fig7: βIII-tubulin protein analyzed by immunohistochemistry and flow cytometry. βIII-tubulin-positive cells (white arrows) observed mainly in the mantle layer. The graph displays the expression profile and relative frequency of βIII-tubulin in the spinal cord of the control and MeHg-treated embryos. Scale bar: 20 μm.

Mentions: We investigated whether MeHg compromises the neuronal differentiation in the developing spinal cord, considering that the major effects of MeHg were observed mainly in the mantle layer, where intense cell differentiation occurs. Immunofluorescence using anti-βIII-tubulin antibody revealed the expression of this protein in the mantle and marginal layers at E10. After treatment, no changes were observed in the expression of βIII-tubulin between the control (424.0 cells ± 40.91) and MeHg-treated embryos (517.33 cells ± 1.85, P > 0.05) (Figure 7). Additionally, when we analyze the expression of NeuN, recognized in postmitotic neurons and/or during neuronal differentiation, a significantly decrease in the NA of NeuN-positive cells was observed in the mantle layer of MeHg-treated embryos (14.32 cells/mm2  ± 4.1) when compared to controls (64.55 cells/mm2  ± 5.5, P < 0.0001) (Figure 8).


MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells.

Ferreira FF, Ammar D, Bourckhardt GF, Kobus-Bianchini K, Müller YM, Nazari EM - J Toxicol (2015)

βIII-tubulin protein analyzed by immunohistochemistry and flow cytometry. βIII-tubulin-positive cells (white arrows) observed mainly in the mantle layer. The graph displays the expression profile and relative frequency of βIII-tubulin in the spinal cord of the control and MeHg-treated embryos. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4697092&req=5

fig7: βIII-tubulin protein analyzed by immunohistochemistry and flow cytometry. βIII-tubulin-positive cells (white arrows) observed mainly in the mantle layer. The graph displays the expression profile and relative frequency of βIII-tubulin in the spinal cord of the control and MeHg-treated embryos. Scale bar: 20 μm.
Mentions: We investigated whether MeHg compromises the neuronal differentiation in the developing spinal cord, considering that the major effects of MeHg were observed mainly in the mantle layer, where intense cell differentiation occurs. Immunofluorescence using anti-βIII-tubulin antibody revealed the expression of this protein in the mantle and marginal layers at E10. After treatment, no changes were observed in the expression of βIII-tubulin between the control (424.0 cells ± 40.91) and MeHg-treated embryos (517.33 cells ± 1.85, P > 0.05) (Figure 7). Additionally, when we analyze the expression of NeuN, recognized in postmitotic neurons and/or during neuronal differentiation, a significantly decrease in the NA of NeuN-positive cells was observed in the mantle layer of MeHg-treated embryos (14.32 cells/mm2  ± 4.1) when compared to controls (64.55 cells/mm2  ± 5.5, P < 0.0001) (Figure 8).

Bottom Line: Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells.Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin.These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, UFSC, Campus Universitário, Trindade, 88040-900 Florianópolis, SC, Brazil; Instituto de Ciências Naturais Humanas e Sociais, UFMT, Avenida Alexandre Ferronato 1200, Setor Industrial, 78557287 Sinop, MT, Brazil.

ABSTRACT
The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus