Manganese-induced rat pheochromocytoma (PC12) cell death is independent of caspase activation
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1Department of Pharmacology and Toxicology, SUNY at Buffalo, Buffalo, NY 14214, USA. jaroth@buffalo.edu
Abstract:
Manganese (Mn) is an essential mineral that at high concentrations can produce an irreversible syndrome resembling Parkinson's disease. To examine the mechanism by which Mn elicits its toxic response, we have selected the rat pheochromocytoma cells (PC12) as our model system because it possesses much of the biochemical machinery associated with dopaminergic neurons. Mn-induced PC12 cell death is both time and concentration dependent with approximately 50% cell survival at 48 hr in the presence of 0.3 mM Mn. To determine whether oxidative stress contributed to cytotoxicity induced by Mn, lipid peroxidation was assessed in Mn-treated in PC12 cells. The highly sensitive HPLC assay that measures the lipid peroxide product, 9-HODE, was used and results of these experiments demonstrate there was no increase in the lipid peroxidation in cells exposed to 0.3 mM Mn for 24 hr. Mn was found to stimulate the activation of the apoptotic marker proteins, p38 and caspase-3 within the first 24 hr of treatment. The selective inhibitor of caspase-3, DEVD-CHO, and the nonselective caspase inhibitor, Z-VAD-FMK, however, fail to prevent Mn-induced PC12 cell death. Studies were performed to determine the role of mitochondria in initiating or supporting Mn cytotoxicity, because Mn has been reported to cause changes in membrane permeability. Mn caused a decrease in ATP levels in PC12 cells in both a time and concentration dependent manner. We hypothesize that both apoptosis and necrosis contribute to PC12 cell death although the necrotic events prevail even when the apoptotic signaling is inhibited.
Insights
High manganese (Mn) exposure causes Parkinsonism-like symptoms. This study shows Mn-induced cell death in PC12 cells involves apoptosis and necrosis, with necrosis prevailing even when apoptosis is blocked.
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Manganese (Mn) is essential but toxic at high concentrations, causing Parkinsonism-like symptoms.
- PC12 cells, a rat pheochromocytoma model, mimic dopaminergic neuron machinery for studying Mn toxicity.
- Mn-induced cytotoxicity is time and concentration-dependent, with 50% PC12 cell survival at 0.3 mM Mn after 48 hours.
Purpose of the Study:
- To investigate the mechanisms underlying manganese-induced cytotoxicity in PC12 cells.
- To determine the role of oxidative stress, apoptosis, and mitochondrial dysfunction in Mn toxicity.
Main Methods:
- PC12 cells were treated with varying concentrations of Mn.
- Lipid peroxidation was assessed using HPLC to measure 9-HODE.
- Apoptotic markers (p38, caspase-3) and ATP levels were measured.
- Inhibitors of caspase-3 (DEVD-CHO) and general caspases (Z-VAD-FMK) were used.
Main Results:
- Mn exposure did not increase lipid peroxidation, indicating oxidative stress is not the primary driver.
- Mn stimulated the activation of apoptotic markers p38 and caspase-3 within 24 hours.
- Caspase inhibitors failed to prevent Mn-induced PC12 cell death.
- Mn decreased ATP levels in a time- and concentration-dependent manner, suggesting mitochondrial involvement.
Conclusions:
- Mn-induced PC12 cell death involves both apoptotic and necrotic pathways.
- Necrotic cell death appears to be the predominant mechanism, even when apoptotic signaling is inhibited.
- Mitochondrial dysfunction, indicated by reduced ATP levels, plays a significant role in Mn toxicity.
