Grantee Research Project Results
1999 Progress Report: Mechanism of Manganese-Induced Neurotoxicity
EPA Grant Number: R826248Title: Mechanism of Manganese-Induced Neurotoxicity
Investigators:
Institution: The State University of New York at Buffalo
EPA Project Officer: Chung, Serena
Project Period: October 1, 1997 through September 30, 2000
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $355,000
RFA: Ambient Air Quality (1997) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Air
Objective:
Atmospheric levels of manganese (Mn) have significantly increased in urban cities since its introduction as a fuel additive. Recent studies indicate that people with compromised liver function may be at considerably greater risk than the normal population to the toxic actions of Mn. Mn is a potent neurotoxin, capable of producing a variety of neurological symptoms characterized by severe extrapyramidal dysfunction resembling the dystonic movements associated with Parkinson's disease. With the realization of increased environmental exposure to Mn, it has become necessary to delineate the fundamental biochemical and molecular mechanisms responsible for its selective neurotoxic actions to prevent and identify individuals with Mn toxicity.Progress Summary:
Our laboratory chose to employ an in vitro cell culture system as a paradigm to study the selective biological processes affected by exposure to Mn. For these studies, we initially selected the rat pheochromocytoma cells (PC12) as our model system, because they possess much of the biochemical machinery associated with dopaminergic neuron. Rat PC12 cells have served as a model for studying the molecular mechanisms promoting neuronal differentiation induced by nerve growth factor (NGF) and other growth promoting agents, and these cells recently have been proposed to serve as an effective in vitro model for studying the mechanisms of apoptosis and neurotoxicity. We have observed that Mn can similarly induce neuronal differentiation in PC12 cells similar to that produced by NGF; however, in contrast to NGF treatment, exposure of these cells to Mn invariably leads to cell death in a time- and concentration-dependent manner. The mechanism by which Mn induces neurite outgrowth appears to be similar for that which promotes NGF-induced neuronal differentiation of PC12 cells (i.e., induction of the phosphorylation of the MAP kinases, ERK1 and 2 [p42 and p44]). Activation of both ERK1 and 2 is dependent on the interaction of the cell surface integrin receptors with that of the basement membrane proteins, fibronectin, and vitronectin. Inhibition of MAP kinase kinase (MEK) with the specific inhibitor, PD98059, blocked the phosphorylation of ERK1 and 2 and increased Mn2+ toxicity. These data support the hypothesis that integrin-mediated activation of the MAPK signal transduction pathway leading to the activation of ERK1 and 2 is required for Mn2+ induced PC12 differentiation and neurite outgrowth.
PC12 cell death induced by Mn is both time and concentration dependent with an inhibitory concentration (IC50) value of approximately 0.6 mM after 24 hours. Oxidative stress does not appear to contribute to Mn-induced cytotoxicity, because Mn did not increase lipid peroxidation; however, increases in the stress activated protein kinase, p38, and caspase-3 activity were observed in cells treated with Mn, suggesting that apoptosis may play a role in its cytotoxic actions. Inhibitors of p38, SKF86002, and SB203580 prevent Mn-induced PC12 cell death, but only at 24 hours of exposure, not at longer exposure times. These results suggest that both a p38-dependent and independent mechanism contribute to PC12 cell death. Selective and nonselective inhibitors of caspase-3 activity, DEVD-CHO and Z-VAD-FMK, respectively, failed to prevent Mn-induced cell death, demonstrating that activation of the caspases is not involved in PC12 cell death. Mn previously has been shown to affect mitochondrial function, and we have demonstrated that the production of ATP is inhibited in PC12 cells treated with Mn. In summary, these results demonstrate that the cytotoxic effects of Mn in PC12 cells are not brought about by oxidative stress, but most likely are generated by disruption of mitochondrial oxidative phosphorylation. We hypothesize that both apoptosis as well as necrosis contribute to Mn-induced PC12 cell death, although the necrotic events prevail even when the apoptitic signaling is inhibited.
Mn primarily is taken up into cells via the same transport protein responsible for iron uptake (i.e., the divalent metal transporter, DMT1). This membrane carrier has very broad substrate specificity and is responsible for the cellular transport of other divalent cations as well, including Cd+2, Zn+2, Co+2, Ni+2, Cu+2, and Pb+2. Two forms of DMT1 are present in mammalian cells, which result from post-transcriptional modification of a single gene product. The two forms differ primarily in their carboxy terminal end, such that only one of the two forms contains an iron response element (IRE) motif on the mRNA. The presence of the IRE within the message promotes the binding and subsequent iron-dependent activation of the iron response protein (IRP), causing stabilization of the message and increased expression of the protein. Accordingly, the form of DMT1 containing the +IRE is negatively regulated by iron status, such that if iron levels are low, DMT1 expression is elevated resulting in the increased transport of heavy metals. Thus, changes in iron levels have the potential to influence heavy metal cytotoxicity.
Based on RT-PCR and Western blot experiments, it has been shown that PC12 cells possess both forms of DMT1. Based on this, we currently are examining the interaction of iron and Mn in PC12 cells to determine whether iron can influence Mn toxicity. Preliminary studies demonstrate that the iron chelator, desferioxamine, enhances Mn toxicity and increases caspase-3 activity, suggesting that removal of iron from the growth media increases transport of Mn into PC12 cells.
Future Activities:
Recent studies have focused on the role of DMT1 in controlling Mn-induced PC12 cell death. It is known that iron can influence Mn levels in vivo and it is possible that DMT1 may play a major role in regulating the neurotoxic actions of this metal.Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 16 publications | 10 publications in selected types | All 10 journal articles |
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Larsen KE, Pacheco M, Roth J, Aletta JM. Increased MAP1B expression without increased phosphorylation in manganese-treated PC12Mn cells. Experimental Cell Research 1998;245(1):105-115. |
R826248 (1999) R826248 (2000) R826248 (Final) |
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Walowitz JL, Roth JA. Activation of ERK1 and ERK2 is required for manganese-induced neurite outgrowth in rat pheochromocytoma (PC12) cells. Journal of Neuroscience Research 1999;57(6):847-854. |
R826248 (1999) R826248 (2000) R826248 (Final) |
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Supplemental Keywords:
biology, heavy metals, manganese, urban exposure, human health, metabolism, neurotoxicology, apoptosis., Health, Scientific Discipline, PHYSICAL ASPECTS, Air, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Disease & Cumulative Effects, Physical Processes, Molecular Biology/Genetics, Biology, acute effects, ambient air quality, health effects, urban air toxics, exposure and effects, air pollutants, cellular metabolism, effects assessment, hazardous air pollutants, airway disease, ambient air, exposure, urban air pollutants, enzyme systems, neurotoxicity, human exposure, chronic effects, manganese, toxicity, inner city toxicants, public health, acute toxicity, hazardous air pollutants (HAPs), environmental toxicant, harmful environmental agents, toxicodynamics, human health, fuel additives, Parkinson's Disease, acute exposure, atmospheric chemistry, chronic toxicity, disease, heavy metals, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.