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Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells
D C Jones1, P G Gunasekar, J L Borowitz
1Neurotoxicology Laboratory, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907- 1333, USA.
Abstract:
Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.
Insights
Dopamine oxidation and cyanide exposure increase neuronal cell death by generating reactive oxygen and nitric oxide species. This study shows cyanide worsens dopamine-induced neurotoxicity, highlighting oxidative stress mechanisms in neurological conditions.
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Dopamine (DA) oxidation and reactive oxygen species (ROS) contribute to dopaminergic neuron degeneration.
- Cyanide (KCN) mimics Parkinson's symptoms by inhibiting antioxidant systems and mitochondrial function.
Purpose of the Study:
- To investigate the mechanisms of DA-induced cytotoxicity, particularly in an impaired neuronal system mimicking cyanide exposure.
- To elucidate the roles of ROS, nitric oxide, and intracellular pathways in DA neurotoxicity.
Main Methods:
- Differentiated PC12 cells were exposed to DA and/or KCN.
- Cell death was assessed using lactate dehydrogenase (LDH) assays.
- Apoptosis was measured by Hoechst and TUNEL staining.
- Mechanisms were explored using DA reuptake inhibitors, antioxidants, glutathione synthesis inhibitors, nitric oxide synthase inhibitors, and peroxynitrite scavengers.
Main Results:
- Both DA and KCN induced concentration- and time-dependent cell death and apoptosis.
- The combination of DA and KCN significantly increased cytotoxicity and apoptosis.
- DA toxicity was partly mediated intracellularly, as indicated by protection with a DA reuptake inhibitor.
- Antioxidants attenuated DA- and KCN-induced apoptosis, confirming the role of oxidative species.
- Nitric oxide pathways were implicated through the effects of specific inhibitors and scavengers.
- Increased intracellular oxidant generation was observed.
Conclusions:
- DA neurotoxicity is exacerbated by cyanide-induced impairment.
- The study implicates both ROS and nitric oxide-mediated oxidative stress in initiating DA-induced apoptosis.
- Understanding these mechanisms is crucial for neurological conditions involving dopaminergic neuron degeneration.