1-methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons - PubMed (original) (raw)

1-methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

Derek A Drechsel et al. Toxicol Appl Pharmacol. 2007.

Abstract

Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP(+)). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP(+) exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP(+) treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP(+).

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Figures

Figure 1

Figure 1

Effect of MPP+ on N27 cell viability after 24 (■) and 48 (▲) h incubations. Data presented as percentage of media LDH activity compared to total (media + cell lysate) LDH activity versus the log of MPP+ concentration (M). Data expressed as mean ± SE. *p < 0.05 versus time-matched controls, one-way ANOVA, n=6 per group.

Figure 2

Figure 2

Cellular GSH (A), GSSG (B) and GSH/GSSG (C) levels in N27 cells following MPP+ treatment for 24 h. Bars represent mean (% control) + SE. Bars with different letters were statistically different form one another (p < 0.05, one-way ANOVA, n=6 per group.

Figure 3

Figure 3

Cellular GSH levels in N27 cells following MPP+ treatment for 6, 12, or 24 h. Data points for control (■), 100 μM MPP+ (▲), and 1 mM MPP+ (▼) represent mean (% control) ± SE. *p < 0.05 versus controls, two-way ANOVA with Bonferroni post-test, n=6-7 per group.

Figure 4

Figure 4

Intracellular ROS levels measured by DCF fluorescence via flow cytometry in N27 cells following MPP+ treatment. (A) Representative histograms of DCF fluorescence in vehicle- and 1 mM MPP+-treated N27 cells at 6 h. Numbers represent the percentage of cells with DCF positive staining compared to unlabeled controls. (B) ROS levels in N27 cells following treatment with 100 μM or 1 mM MPP+ for 6, 12, and 24 h. Data expressed as percentage of cells staining positive for DCF compared to controls. Bars represent mean (% control) + SE. Bars with different letters were statistically different from one another (p < 0.05, one-way ANOVA, n=3 per group.

Figure 5

Figure 5

Glutathione reductase (GR, A) and glutathione peroxidase (GPx, B) enzyme activities in N27 cells following MPP+ treatment for 6,12, and 24 h. Bars represent mean (% control) + SE. Bars with different letters were statistically different from one another (p < 0.05, one-way ANOVA, n=3 per group.

Figure 6

Figure 6

Glutamate-cysteine ligase (GCL, A) and glutathione synthetase (GS, B) enzyme activities in N27 cells following MPP+ treatment for 6, 12, and 24 h. Bars represent mean (% control) + SE. Bars with different letters were statistically different from one another (p < 0.05, one-way ANOVA, n=3 per group.

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