J Vet Sci.  2014 Mar;15(1):61-71. 10.4142/jvs.2014.15.1.61.

Toxic effects of methylmercury, arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells

Affiliations
  • 1Toxicology and Residue Chemistry Division, Animal and Plant Quarantine Agency, Anyang 430-824, Korea. kanghg67@korea.kr
  • 2GLP Research Center, College of Natural Sciences, Hoseo University, Industrial-academy Corporation Center I, Asan 336-795, Korea.

Abstract

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA(A)-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 microM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.

Keyword

arsanilic acid; danofloxacin; embryonic stem cell test; methylmercury; neural cell

MeSH Terms

Acetylcholinesterase/metabolism
Animals
Arsanilic Acid/*toxicity
Cell Differentiation/*drug effects
Embryonic Stem Cells/cytology/*drug effects
Environmental Pollutants/*toxicity
Fluorescent Antibody Technique
Fluoroquinolones/*toxicity
Gene Expression Regulation/drug effects
Methylmercury Compounds/*toxicity
Mice
Nerve Tissue Proteins/metabolism
Neurons/cytology/*drug effects
Tetrazolium Salts/metabolism
Thiazoles/metabolism
Acetylcholinesterase
Arsanilic Acid
Environmental Pollutants
Fluoroquinolones
Methylmercury Compounds
Nerve Tissue Proteins
Tetrazolium Salts
Thiazoles

Figure

  • Fig. 1 Quantitative changes of marker protein expression during the differentiation of ESCs into neural cells. Mouse ESCs were transformed into neural cells by culturing the cells under specific conditions for 14 days. The expressions of POU5F1 (■), GABAA-R (●), GFAP (▲), Nestin (□), Tuj1 (○), and MAP2 (△) were measured by immunocytochemical staining (A). Changes of their expression levels were measured as fluorescent intensities expressed as a percentage of the control (B). The expression of POU5F1 as a marker of undifferentiated cells significantly decreased after 6 days of differentiation. Meanwhile, expression of the neural lineage marker proteins GABAA-R, GFAP, Nestin, Tuj1, and MAP2 gradually increased until Day 8. After this time, no further significant changes were observed. Therefore, the effects of MM, AA, and DF observed before and after differentiation were evaluated separately (C): incubation with the differentiating neural cells for 8 days (differentiating stage) and incubation with the fully differentiated neural cells for 6 days following 8 days of differentiation (differentiated stage). Values are presented as the percentage of the cells relative to the controls and expressed as the mean ± SE (n = 3).

  • Fig. 2 Cytotoxicity of MM, AA, and DF in mouse ESCs. Effects of the three chemicals were measured with an MTT was assay after 16 h of treatment. IC15 values for MM, AA, and DF were 1,000 nM, 4 mM and 80 µM, respectively (A~C). Data are presented as the mean ± SE (n = 3). Statistically significant differences (*p < 0.05 or **p < 0.01) were identified by comparison to the control.

  • Fig. 3 The expression of marker proteins during the differentiating and differentiated stages after treatment with MM. (A) POU5F1, (B) GABAA-R, (C) GFAP, (D) Nestin, (E) Tuj1, and (F) MAP2. The levels of protein expression were normalized to that of the untreated control. Data are presented as the mean ± SE (n = 3). Statistically significant differences (*p < 0.05 and **p < 0.01) were identified by comparison to the control.

  • Fig. 4 Relative quantification of marker protein expression during the differentiating and differentiated stages after treatment with AA. (A) POU5F1, (B) GABAA-R, (C) GFAP, (D) Nestin, (E) Tuj1, and (F) MAP2. The levels of marker expression were normalized to that of the untreated control. Data are presented as the mean ± SE (n = 3). Statistically significant differences (*p < 0.05 and **p < 0.01) were identified by comparison to the control.

  • Fig. 5 Quantitative changes of marker protein levels during the differentiating and differentiated stages after treatment with DF. (A) POU5F1, (B) GABAA-R, (C) GFAP, (D) Nestin, (E) Tuj1, and (F) MAP2. The levels of protein expression were normalized to that of the untreated control. Data are presented as the mean ± SE (n = 3). Statistically significant differences (*p < 0.05 and **p < 0.01) were identified by comparison to the control.

  • Fig. 6 The changes of AChE activity induced by exposure to MM, AA, and DF. The activity levels of AChE were estimated for the control during the differentiating and differentiated stages (A). Data are presented as the mean AChE activity (µM/mg/min). T-test results indicated a statistically significant difference between the groups (p = 0.0026). Neurotoxicities of MM, AA, and DF as indicated by AChE activity inhibition during the differentiating (B~D) and differentiated (E~G) stages relative to the stage-specific control. Data are presented as the mean ± SE (n = 3). Statistically significant differences (*p < 0.05 and **p < 0.01) were identified by comparison to the control.


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