Perturbation of Dopamine Metabolism by 3-Amino-2-(4Ј- halophenyl)propenes Leads to Increased Oxidative Stress and Apoptotic SH-SY5Y Cell Death

Abstract

ABSTRACT We have recently characterized a series of 3-amino-2-phenylpropene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-␤-monooxygenase (D␤M) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4Ј-position of the aromatic ring gradually increases VMAT inhibition potency from 4Ј-F to 4Ј-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4Ј-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4Ј-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants ␣-tocopherol, N-acetyl-L-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROSmediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4Ј-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death. Oxidative stress in the central and peripheral nervous systems plays a significant role in neurodegenerative disorders, aging, and the toxicity of a large number of neurotoxins Numerous studies indicate that efficient uptake, biosynthetic conversion, and storage of catecholamines in vesicles are mandatory for proper functioning of catecholaminergic neurons. The vital proteins responsible, including vesicular H ϩ -ATPase (V-H ϩ -ATPase), cytochrome b 561 (b 561 ), dopamine-␤-monooxygenase (D␤M), and vesicular monoamine transporter (VMAT), have been well characterized (Beer