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Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity - PubMed

  • ️Sun Jan 01 2017

Comparative Study

Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

Dina Popova et al. BMC Pharmacol Toxicol. 2017.

Abstract

Background: Exposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide.

Methods: Retinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein βIII-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue™ cell viability assay and the TMRE mitochondrial membrane potential assay.

Results: Differentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 μM in the P19 neurons and 1 μM in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (ΔΨm) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells.

Conclusions: P19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Keywords: Acrylamide; Glutathione; In vitro cytotoxicity; Methylmercury; Nerve growth factor-treated PC12 cells; Neuronal cell cultures; Neurotoxicity; Okadaic acid; Retinoic acid-treated P19 cells; Retinoic acid-treated SH-SY5Y cells.

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Figures

Fig. 1
Fig. 1

Development of neurons derived from RA-treated P19 and SH-SY5Y cells, and NGF-stimulated PC12 cells up to 10 days in culture. The cells were plated at a density of 500 cells/mm2 and immunostained against the neuron-specific protein βIII-tubulin. a Representative fluorescence microscopy images of neurons (20 × magnification). b Fluorescence of anti-βIII-tubulin antibodies measured in a microplate reader and expressed as relative fluorescence units (RFU). Data are means ± SEM of 3–4 independent experiments

Fig. 2
Fig. 2

Concentration-dependent effects of MeHg, okadaic acid and acrylamide on cell viability and expression of the neuron-specific protein βIII-tubulin in neuronally differentiated P19, PC12 and SH-SY5Y cells. The cells were plated at a density of 500 cells/mm2 and cultured for 6 days in the differentiation media, followed by exposure to the test compounds for 48 h. Effects of MeHg (a, b), okadaic acid (c, d) and acrylamide (e, f) on the cell viability were assessed by using the calcein-AM assay (a, c, e), and the immunofluorescence of βIII-tubulin (b, d, f). The data are means ± SEM of n = 6 independent experiments (n = 3 for okadaic acid in the βIII-tubulin assay; panel d). The results are expressed as percentage of non-treated cells or cells treated with 0.1% DMSO (used as vehicle). Wells treated with 2% Triton X-100 for 30 min served as controls for maximal cell death. Statistical analysis was performed using repeated measures one-way ANOVA with post hoc Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) compared to corresponding controls

Fig. 3
Fig. 3

Representative fluorescence microscopy images of neuronally differentiated P19, PC12 and SH-SY5Y cells exposed to 1 μM methylmercury, 10 nM okadaic acid and 1 mM acrylamide. The cells were plated at a density of 500 cells/mm2 and cultured for 6 days in the differentiation media, followed by exposure to the test compounds for 48 h. The cells were immunolabeled against the neuron-specific protein βIII-tubulin and the fluorescence microscopy images were obtained at 20 × magnification

Fig. 4
Fig. 4

Effects of MeHg, BSO and GSH on the viability of RA-treated P19 cells (a) and SH-SY5Y cells (b). Cells cultured for 6 days in the differentiation media were pre-treated with 100 μM BSO for 17 h or 1 mM GSH for 1 h followed by exposure to 1 μM MeHg for 24 h. Cell viability was assessed with the PrestoBlue assay that measures cellular metabolic reduction, and extracellular LDH activity assay. Data are means ± SEM of n = 6 independent experiments. For the PrestoBlue assay, data are expressed as percentage of non-treated or 0.1% DMSO vehicle-treated cells. For the LDH assay, the data are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was undertaken using one-way ANOVA with post hoc Bonferroni’s multiple comparisons test: *p < 0.05, **p < 0.01, and ****p < 0.0001 (the effect of the treatment compared to the corresponding vehicle control, or the effect of the combination of MeHg + GSH or MeHg + BSO compared to MeHg per se). ND = not determined

Fig. 5
Fig. 5

The effects of MeHg, GSH and BSO on TMRE fluorescence in RA-treated P19 (a) and SH-SY5Y cells (b). Cells cultured for 6 days in the differentiation media were pre-treated with 100 μM BSO for 17 h or with 1 mM GSH for 1 h followed by exposure to 1 μM MeHg for 24 h. Alterations in mitochondrial membrane potential were measured with the TMRE assay. Data are means ± SEM of n = 6 independent experiments, and expressed as percentage of non-treated or 0.1% DMSO vehicle-treated cells. Statistical analysis was undertaken using one-way ANOVA with post hoc Bonferroni’s multiple comparisons test: *p <0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (the effect of the treatment compared to the corresponding vehicle control, or the effect of the combination of MeHg + GSH or MeHg + BSO compared to MeHg per se)

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