100 - The Role of N-Acetylcysteine and 3H-1,2-Dithiole-3-Thione in Maintaining Glutathione Status and Protection Against Advanced Glycation End Product Induced Neurite Degeneration in SH-SY5Y Cells

Abstract

Diabetes mellitus, a disease hallmarked by hyperglycemia, is one of the most common chronic diseases worldwide and new diagnoses are on an upward trajectory. Peripheral neuropathy (PN) affects at least 50% of diabetic patients. Unfortunately, medications available for patients with PN treat the symptoms but not the cellular damage, and come with severe side effects leading many to discontinue their use. The overall objective of this study was to gain a better understanding of the mechanism of action and potential impact of therapeutic substances, such as N-acetylcysteine (NAC), and preventive dietary components, such as the cruciferous vegetable constituent 3H-1,2-dithiole-3-thione (D3T), which is critically important for patient health. Previous studies in our lab have shown that when SH-SY5Y cells, a validated model for PN, are treated with advanced glycation end products (AGE), there is increased reactive oxygen species (ROS) generation, a significant decrease in glutathione (GSH) concentrations, and neurite loss. NAC was shown to protect against AGE-induced cell death, GSH depletion and neurite loss, indicating that maintaining GSH was important for neurite protection. Our lab has also shown that D3T, a potent inducer of nuclear factor (erythroid-derived 2)- like (Nrf2), can significantly increase SH-SY5Y cellular GSH concentrations and protect against H2O2 –induced cell death. Paradoxically, D3T conferred no protection against AGE-induced cell death or neurite degeneration. We report here follow up studies. The first aim of this study was to test whether NAC conferred protection through maintaining GSH, its intrinsic antioxidant properties, or both. Using buthionine sulfoximine (BSO) as a tool to evaluate the mechanistic role of NAC in neurite protection, we found that at high concentrations of NAC (2 mM) both intrinsic and GSH-maintaining properties were observed. At lower concentrations (1 mM), GSH-maintaining properties predominated. The second aim was to elucidate a mechanism for the paradoxical effect of D3T focusing on the role of NADPH-mediated ROS generation. NADPH is an electron donor for the oxidase enzyme that generates superoxide in response to the activation of the receptor for AGE (RAGE). D3T increased ROS generation and depleted GSH only in the presence of AGE, suggesting its role as a NADPH generating agent may promote oxidative stress rather than protect against it under these conditions. Inhibition of NADPH generation via glucose-6-phosphate dehydrogenase (G6PD), the protein product of a Nrf2-responsive gene, with dehydro-epiandrosterone (DHEA) was found to protect against AGE-induced loss of cell viability. These results suggest that the source of ROS is important to consider in evaluating the extent to which adaption to oxidative stress provides protection.