Dopamine‐Induced Apoptosis Is Mediated by Oxidative Stress and Is Enhanced by Cyanide in Differentiated PC12 Cells

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 M) and KCN (100 -500 M) 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 M) and/or KCN (100 M) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60 -70% of the cells from DAand 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.

Urate as a Marker of Risk and Progression of Neurodegenerative Disease

“…In vivo, genetic manipulation of urate oxidase and resulting increased concentrations of urate in the CNS led to improved phenotype and histopathologic findings in PD mouse models [35,49]. These data substantiate earlier findings in PC12 cells, in which urate blocked cell death and oxidative damage induced by either dopamine [50] or 6-hydroxydopamine [51]. Similarly, in a rotenone toxicity model, urate prevented death of dopaminergic cells [52].…”

Oxidative Stress and Neurotoxicity

“…Indeed, dopamine is toxic to PC12 cells via oxidative stress, leading to apoptosis (225). It must be realized that metabolic deamination of dopamine and its O-methyl metabolite by MAO results in H 2 O 2 as a byproduct.…”

Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs

“…Furthermore, O 2 U − reacts with nitric oxide, forming the highly toxic peroxinitrite (Cadet and Brannock, 1998). Thus, dopamine is both a neurotransmitter and a neurotoxin, and changes in dopamine metabolism may induce oxidative stress and cell death in dopaminergic or surrounding cells (Jones et al, 2000) if the antioxidant systems are not able to deal with the increase in the levels of ROS. An increase in ROS levels may lead to cell death due to the oxidation of important cellular macromolecules such as amino acids, phospholipids and nucleic acids (Cadet and Brannock, 1998).…”