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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

DNA double-strand breaks labeled with γ-H2A.X antibody analyzed by immunohistochemistry. γ-H2A.X-positive cells (arrows) were observed in MeHg-treated embryos. The graph displays the expression profile of γ-H2A.X in the spinal cord of control and MeHg-treated embryos. Integrated density of pixels of the fluorescence of the γ-H2A.X was determined, and data are expressed as mean ± SEM. ∗P < 0.05. Scale bar: 10 μm.
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fig5: DNA double-strand breaks labeled with γ-H2A.X antibody analyzed by immunohistochemistry. γ-H2A.X-positive cells (arrows) were observed in MeHg-treated embryos. The graph displays the expression profile of γ-H2A.X in the spinal cord of control and MeHg-treated embryos. Integrated density of pixels of the fluorescence of the γ-H2A.X was determined, and data are expressed as mean ± SEM. ∗P < 0.05. Scale bar: 10 μm.

Mentions: In order to verify if MeHg causes DNA damage, the expression of γ-H2A.X protein in response to DNA double-strand breaks was examined. After treatment, MeHg caused an increase in the expression of γ-H2A.X (53,740.89 ± 7,834.14 μm2) when compared to the control embryos (34,473.89 ± 670.97 μm2, P < 0.05). These anti-γ-H2A.X-positive cells were found mainly in the transition zone between the ependymal and mantle layers of the MeHg-treated embryos (Figure 5).


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)

DNA double-strand breaks labeled with γ-H2A.X antibody analyzed by immunohistochemistry. γ-H2A.X-positive cells (arrows) were observed in MeHg-treated embryos. The graph displays the expression profile of γ-H2A.X in the spinal cord of control and MeHg-treated embryos. Integrated density of pixels of the fluorescence of the γ-H2A.X was determined, and data are expressed as mean ± SEM. ∗P < 0.05. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: DNA double-strand breaks labeled with γ-H2A.X antibody analyzed by immunohistochemistry. γ-H2A.X-positive cells (arrows) were observed in MeHg-treated embryos. The graph displays the expression profile of γ-H2A.X in the spinal cord of control and MeHg-treated embryos. Integrated density of pixels of the fluorescence of the γ-H2A.X was determined, and data are expressed as mean ± SEM. ∗P < 0.05. Scale bar: 10 μm.
Mentions: In order to verify if MeHg causes DNA damage, the expression of γ-H2A.X protein in response to DNA double-strand breaks was examined. After treatment, MeHg caused an increase in the expression of γ-H2A.X (53,740.89 ± 7,834.14 μm2) when compared to the control embryos (34,473.89 ± 670.97 μm2, P < 0.05). These anti-γ-H2A.X-positive cells were found mainly in the transition zone between the ependymal and mantle layers of the MeHg-treated embryos (Figure 5).

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