Smokeless tobacco induces toxicity and apoptosis in neuronal cells: a mechanistic evaluation

Smokeless tobacco (SLT) or chewing tobacco has been a highly addictive practice in India across ages, posing major threat to the systemic health and possibly neurodegeneration. Earlier studies showed components of SLT could be harmful to neuronal health. However, mechanism of SLT in neurodegeneration remained unexplored. This study investigated the detrimental role of SLT on differentiated neuronal cell lines, PC12 and SH-SY5Y by using graded doses of water soluble lyophilised SLT. Reduced cell viability, compromised mitochondrial structure and functions were observed when neuronal cell lines were treated with SLT (6 mg/mL) for 24 h. There was reduction of oxidative phosphorylation and aerobic glycolysis as determined by diminution of ATP production (2.5X) and basal respiration (1.9X). Mitochondrial membrane potential was dropped by 3.5 times. Bid, a pro-apoptotic Bcl-2 family protein, has imperative role in regulating mitochondrial outer membrane permeabilization and subsequent cytochrome c release leading to apoptosis. This article for the first time indicated the involvement of Bid in SLT mediated neurotoxicity and possibly neurodegeneration. SLT treatment enhanced expression of cleaved-Bid in time dependent manner. The involvement of Bid was further confirmed by using Bid specific shRNA which reversed the effects of SLT and conferred significant protection from apoptosis up to 72 h. Thus, our results clearly indicated that SLT induced neuronal cell death occurred via production of ROS, alteration of mitochondrial morphology, membrane potential and oxidative phosphorylation, inactivation of survival pathway and activation of apoptotic markers mediated by Bid. Therefore, Bid could be a potential future therapeutic target for SLT induced neurodegeneration.