Grantee Research Project Results
Final 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 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 increased significantly in urban cities since its introduction as a fuel additive. This particularly is relevant based on recent studies indicating 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 that is 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 becomes necessary to delineate the fundamental biochemical and molecular mechanisms responsible for its selective neurotoxic actions to prevent and identify individuals with Mn toxicity.
Summary/Accomplishments (Outputs/Outcomes):
The investigators 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, they initially selected the rat pheochromocytoma cells (PC12) as the model system because it possesses 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. It was observed that Mn can 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 mitogen-activated protein [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 the basement membrane proteins fibronectin and vitronectin. Function-blocking antisera specific for ß1 integrins block the neurite-promoting activity of Mn by 90 to 95 percent. Bioassays and biochemical studies with antisera specific for the v, α5, or α8 integrin subunit suggest that the αvß1 heterodimer is one of the principal ß1 integrins mediating the response of PC12 cells to Mn. This is corroborated by studies in which Mn failed to induce neurite outgrowth in a clone of PC12 cells that does not express αv at levels detectable by immunoprecipitation or immunocytochemistry. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) analysis of biotinylated surface proteins immunoprecipitated from Mn-responsive PC12 cells indicates that Mn increases the surface expression of av integrins. These data indicate that Mn induces neurite outgrowth in PC12 cells by upregulating av integrins, suggesting that Mn potentially represents an additional mechanism for regulating the rate and direction of neurite outgrowth during development and regeneration.
PC12 cell death induced by Mn is both time and concentration dependent with an IC50 value of approximately 0.6 µM 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 and not at longer exposure times. These results suggest that both a p38-dependent and independent mechanism contribute to PC12 cell death. Selective and non-selective inhibitors of caspase-3 activity, DEVD-CHO and Z-VAD-FMK, respectively, failed to prevent Mn-induced cell death, which demonstrates that activation of the caspases is not involved in PC12 cell death. Mn previously has been shown to effect mitochondrial function; in agreement with this, it was demonstrated that production of adenosine triphosphate (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, generated by disruption of mitochondrial oxidative phosphorylation. It was hypothesized that both apoptosis, as well as necrosis, contribute to Mn-induced PC12 cell death, although the necrotic events prevail even when the apoptotic 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 that result from post-transcriptional modification of a single gene product. The two forms differ primarily in their carboxy terminal end-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), which causes stabilization of the message and increased expression of the protein. Accordingly, the form of DMT1 containing the +IRE is negatively regulated by iron status; if iron levels are low, DMT1 expression is elevated and results in the increased transport of heavy metals. Thus, changes in iron levels have the potential to influence heavy metal cytotoxicity.
Recent studies have revealed that the subcellular distribution of the two isoforms of DMT1 is distinct and the IRE species accumulates in the nucleus of neuronal or neuronal-like cells. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis of PC12 cells reveal that these cells express both forms of DMT1. Immunofluorescence and immunoblotting studies, using immunospecific antibodies to the IRE forms of DMT1, demonstrate that this form of the transporter, in PC12 cells, predominantly is localized in the nucleus, cell membrane, and neurites, with only weak staining of the cell body. Studies using antibodies to the +IRE form indicate that this species of DMT1 is distributed within vesicles in the cell body and neurite projections, with minimal nuclear staining. Similar staining patterns are observed for the two forms of DMT1 in cultures of sympathetic ganglion neurons isolated from perinatal rat pups. To determine whether nuclear localization of the IRE form of DMT1 is constrained to neuronal or neuronal-like cells, immunocytochemical studies were performed with human HEK293T, HEP2G hepatoma and medulloblastoma, and rat Schwann cells. The IRE specific antibodies stained nuclei from medulloblastoma, whereas little nuclear staining was observed with HEK293T, hepatoma, or Schwann cells. The unexpected finding that the IRE species of DMT1 selectively accumulates in the nucleus of neuronal and neuronal-like cells leads us to postulate that the two proteins may have different functions in vivo.Iron inhibited Mn uptake onto PC12 cells with an IC50 value of approximately 20 µM. Cells were treated with the iron chelator. Accordingly, removal of iron from the growth media would be expected to result in increased Mn uptake into PC12 cells and augment its toxicity. Recent studies confirmed that the iron chelator, desferrioxamine (DfO; 10 µM), stimulated Mn-induced PC12 cell toxicity, but it inhibited the activity of the apoptotic marker, caspase 3. DfO also stimulated Mn-induced MAP kinase phosphorylation and neuronal differentiation. In addition, Western blots suggest that co-treatment of DfO with Mn stimulates the expression of both species of DMT1. Why both forms of DMT1 are increased is not known, but these results suggest that expression of this gene in PC12 cells may involve transcriptionally regulatory events. These results demonstrate that iron status is likely to have a direct effect on the uptake and biological actions of Mn and other heavy metals that are transported by DMT1.
Journal Articles on this Report : 10 Displayed | Download in RIS Format
Other project views: | All 16 publications | 10 publications in selected types | All 10 journal articles |
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Garrick MD, Dolan KG, Horbinski C, Ghio AJ, Higgins D, Porubcin M, Moore EG, Hainsworth LN, Umbreit JN, Conrad ME, Feng L, Lis A, Roth JA, Singleton S, Garrick LM. DMT1: a mammalian transporter for multiple metals. BioMetals 2003;16(1):41-54. |
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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. |
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Lein P, Gallagher PJ, Amodeo J, Howie H, Roth JA. Manganese induces neurite outgrowth in PC12 cells via upregulation of αv integrins. Brain Research 2000;885(2):220-230. |
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Roth JA, Kim BG, Lin WL, Cho MI. Melatonin promotes osteoblast differentiation and bone formation. Journal of Biological Chemistry 1999;274(31):22041-22047. |
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Roth JA, Feng L, Walowitz J, Browne RW. Manganese-induced rat pheochromocytoma (PC12) cell death is independent of caspase activation. Journal of Neuroscience Research 2000;61(2):162-171. |
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Roth JA, Horbinski C, Feng L, Dolan KG, Higgins D, Garrick MD. Differential localization of divalent metal transporter 1 with and without iron response element in rat PC12 and sympathetic neuronal cells. Journal of Neuroscience 2000;20(20):7595-7601. |
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Roth JA, Rosenblatt T, Lis A, Bucelli R. Melatonin-induced suppression of PC12 cell growth is mediated by its Gi coupled transmembrane receptors. Brain Research 2001;919(1):139-146. |
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Roth JA, Feng L, Dolan KG, Lis A, Garrick MD. Effect of the iron chelator desferrioxamine on manganese-induced toxicity of rat pheochromocytoma (PC12) cells. Journal of Neuroscience Research 2002;68(1):76-83. |
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Roth JA, Horbinski C, Higgins D, Lein P, Garrick MD. Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation. NeuroToxicology 2002;23(2):147-157. |
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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. |
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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, Disease & Cumulative Effects, Biochemistry, 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.