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Title: 1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

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{sup +}). 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{sup +} exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP{sup +} treatment was not associated with increasedmore » efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP{sup +}.« less

Authors:
 [1];  [1];  [2]
  1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, 4200 East Ninth Avenue, Box C238, Denver, CO 80262 (United States)
  2. Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, 4200 East Ninth Avenue, Box C238, Denver, CO 80262 (United States). E-mail: manisha.patel@uchsc.edu
Publication Date:
OSTI Identifier:
20976926
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 220; Journal Issue: 3; Other Information: DOI: 10.1016/j.taap.2007.02.002; PII: S0041-008X(07)00069-5; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; APOPTOSIS; BIOLOGICAL STRESS; CYSTEINE; GLUTATHIONE; LIGASES; NERVE CELLS; NERVOUS SYSTEM DISEASES; OXIDATION; OXIDIZERS; PEROXIDASES; RATS; STEADY-STATE CONDITIONS; TOXINS

Citation Formats

Drechsel, Derek A., Liang, L.-P., and Patel, Manisha. 1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons. United States: N. p., 2007. Web. doi:10.1016/j.taap.2007.02.002.
Drechsel, Derek A., Liang, L.-P., & Patel, Manisha. 1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons. United States. doi:10.1016/j.taap.2007.02.002.
Drechsel, Derek A., Liang, L.-P., and Patel, Manisha. Tue . "1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons". United States. doi:10.1016/j.taap.2007.02.002.
@article{osti_20976926,
title = {1-Methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons},
author = {Drechsel, Derek A. and Liang, L.-P. and Patel, Manisha},
abstractNote = {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{sup +}). 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{sup +} exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP{sup +} 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{sup +}.},
doi = {10.1016/j.taap.2007.02.002},
journal = {Toxicology and Applied Pharmacology},
number = 3,
volume = 220,
place = {United States},
year = {Tue May 01 00:00:00 EDT 2007},
month = {Tue May 01 00:00:00 EDT 2007}
}
  • Dopaminergic rat mesencephalic neurons in culture were exposed to a group of potential environmental neurotoxins. These cultures, which contained 0.5 to 1% dopaminergic neurons, were a suitable tool for determining nonselective and selective dopaminergic cytotoxicity. Selective toxicity was quantitated as the concentration which destroyed half of the population of dopaminergic neurons as visualized by tyrosine hydroxylase immunocytochemistry. Nonselective toxicity was defined as the concentration of test drug which destroyed half of the entire population of cultured cells as visualized by phase contrast microscopy. The compounds tested were selected to fulfill two molecular criteria underlying the toxic activity of 1-methyl-4-phenylpyridinium (MPP+),more » the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toward dopaminergic cells: 1) to be a substrate for the selective uptake system of the dopaminergic neurons and 2) to possess a delocalized positive charge related to their ability to inhibit mitochondrial electron transport. Of a total number of 29 compounds tested, MPP+ and its close derivatives, 2'-methyl-MPP+ and p-amino-MPP+, exhibited highly selective dopaminergic toxicity, hence the requirements for a selective dopaminergic neurotoxin are rather strict.« less
  • Mesencephalic cells in culture were exposed to various compounds which we hypothesized to be selective toxins for dopaminergic neurons. The culture system was previously shown suitable for assessing selective dopaminergic neurotoxicity, since 1-methyl-4-phenyl-pyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium, destroyed dopaminergic neurons without affecting other cells. Some compounds tested were selected to fulfill two criteria believed to underly the selective dopaminergic neurotoxicity of MPP+, i.e., to be a potential substrate for the uptake carrier for dopamine and to possess a strong delocalized positive charge to inhibit the mitochondrial respiratory system. Other compounds were chosen on the basis of clinical ormore » anecdotal evidence linking them to Parkinson's disease. Among the tested compounds two pyridinium analogs, 1-methyl-4-(4'-acetamidophenyl)pyridinium (MACPP+) and 1-methyl-4-cyclohexylpyridinium (MCP+) were found to be selectively toxic toward dopaminergic neurons. Incubation of cultures with both MACPP+ and MCP+ produced a dramatic reduction in the number of tyrosine hydroxylase-positive cells and the uptake of (3H)dopamine without reducing the number of cells visualized by phase-contrast microscopy or the uptake of (3H)aminobutyric acid. Besides MACPP+ and MCP+ none of the tested compounds exhibited any selective dopaminergic neurotoxicity. Together with earlier findings, these data suggest that the structural requirements are rather strict for a chemical to be a selective dopaminergic neurotoxin and make it unlikely that there is a wide spectrum of environmental dopaminergic toxins.« less
  • LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP{sup +}) toxicity at <= 5 muM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites.more » Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP{sup +} toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-L-tyrosine or 3-iodo-L-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP{sup +}-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP{sup +}-treated LUHMES.« less
  • Highlights: • DHA prevents PQ-induced dopaminergic neuronal loss via decreasing of excessive ROS. • DHA increases GR and GCLm derivate GSH pool by enhancement of Nrf2 expression. • Protective mechanism is removal of PQ-induced ROS via DHA-dependent GSH pool. • DHA may be a good preventive strategy for Parkinson’s disease (PD) therapy. - Abstract: Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinson’s disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinson’s disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuronmore » loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis.« less
  • Dissociated cell cultures from the pontine area of embryonic rat brain were used to study the sensitivity of serotonin (5-hydroxy-tryptamine (5-HT)) neurons to the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+). Treatment with MPTP (up to 100 microM) for 7 days did not cause degeneration of 5-HT neurons. A 50% inhibition of (3H)5-HT uptake caused by 100 microM MPTP was a direct effect on the 5-HT uptake carrier, reversed by washing for 7 days. Incubation of cultures with MPTP increased the intraneuronal levels of 5-HT and reduced the levels of 5-hydroxyindoleacetic acid, suggesting a reduction in 5-HT metabolism. MPTP reduced monoaminemore » oxidase activity in the cultures, which probably led to the reduction in 5-HT metabolism. Exposure to MPP+ (0.5-10 microM) for 4 to 7 days decreased (3H)5-HT uptake and induced loss of neurons stained with antibodies against 5-HT. Comparison between 5-HT and dopamine (DA) neurons indicated a differential sensitivity to MPP+ toxicity with DA neurons being more susceptible. Analysis of the competition of MPP+ with the natural substrates for uptake sites of 5-HT and DA neurons demonstrated higher affinity of MPP+ for DA compared to 5-HT neurons. The lower affinity of MPP+ for 5-HT neurons could be responsible for the accumulation of lower MPP+ levels observed in pontine cultures and explain the resistance of 5-HT neurons to this toxin.« less