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
Authors: Stochelski Mateusz Andrzej , Wilmanski Tomasz , Burgess John R 
Published: November 2023

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 the study was to test the hypothesis that NAC confers protection in this PN model via its role in stimulating GSH synthesis rather than through exerting intrinsic antioxidant properties. After differentiation, cells were treated with 100 μM buthionine sulfoximine (BSO) for three days to inhibit glutamate‐cysteine ligase (GCL) and deplete GSH. Following GSH depletion, cells were treated with AGE or BSA alone or in combination with NAC (2 mM). Three day BSO treatment resulted in near complete loss of GSH and partial neurite loss. In control cells (BSA), 2 mM NAC treatment increased GSH concentrations slightly and conferred partial protection against BSO‐induced neurite loss, but NAC‐treatment had little effect on AGE‐treated cells suggesting that both mechanisms might be functioning. The second aim of this study was to elucidate a mechanism for the paradoxical effect of D3T in this PN model initially 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). Inhibition of NADPH generation via glucose‐6‐phosphate dehydrogenase (G6PD), the protein product of a Nrf2‐responsive gene, with dehydroepiandrosterone (DHEA) was found to protect against AGE‐induced loss of cell viability. However, co‐treatment of SH‐SY5Y cells with both AGE and D3T further depleted cellular GSH concentrations in comparison to AGE alone. Conversely, co‐treatment with DHEA and D3T restores AGE‐treated cell viability. Further studies are underway to determine the extent to which D3T upregulates G6PD expression and mediates the observed paradoxical effect.