Catecholamine disturbance and SH-SY5Y cell toxicity of halogenated 3-amino-2 phenylpropenes

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects between 1% and 2% of the population over age 65. Despite decades of research and the development of many molecular models of PD, there is far from a consensus as to the etiology of this disease. Current molecular models, such as the role of the quaternary ammonium ion MPP+ and its effect on cell death, in the presence/absence of monoamine transporters as opposed to distinct intracellular activity are still disputed. The 3-amino-2-phenylpropene (APP) class of compounds has been previously characterized as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) as well as potent irreversible dopamine-β- monooxygenase (DβM) and monoamine oxidase (MAO) inhibitors. These effects result in perturbation of catecholamine uptake, storage and/or metabolism, leading to the potential for increased oxidative stress. Herein, we report that halogen substitution on the 4'-position of the aromatic ring gradually increases VMAT inhibition potency from 4'-F to 4'-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. In addition, these derivatives are highly toxic to human neuroblastoma SH-SY5Y cells, they are not toxic to several non-neuronal cell lines at similar concentrations. These compounds drastically perturb DA uptake and metabolism in SH-SY5Y cells under sub-lethal conditions, and are able to deplete both viii vesicular and cytosolic catecholamines similar to amphetamines. Additionally, (4'-iodo) 3-amino-2-phenylpropene (4'-IAPP) treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione reduced form (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants α- tocopherol, N-acetyl-L-cysteine (NAC) and glutathione (GSH). This suggests that ROS production is intricately involved with the mechanism of cell death. Although DNA fragmentation analysis supports that apoptosis occurs, the fact that a non-specific caspase inhibitor provided no significant protection suggests that that cell death is likely due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative stress leading to apoptotic cell death. These compounds, which induce catecholaminespecific neurotoxic effects following nonspecific cellular entry may be a unique resource in the modeling of PD both in vitro and in vivo.