Assessment of Cytotoxicity of Smokeless Tobacco (Shammah) In Hepg2 and WRL68 Cells Line

Shammah is a traditional form of chewing tobacco [Smokeless tobacco, (ST)] that is commonly used in the Middle east specially Saudi Arabia (KSA), Yemen and Sudan. The cytotoxicity of Sudanese and Yemenis ST hexane and methanol extracts was evaluated using MTT assay. Annexin-V assay has been used to detect the induction of apoptosis. Luminescence based assay also been conducted to check the level of caspases enzyme. The involvement of cell cycle check point arrest has been performed using flow cytometry analysis. The current study found that ST has the capacity to induce cell toxicity in human liver cells. The inhibitory capacity of ST in HepG2 and WRL 68 has been found to be 151 ± 2.5 and 305 ± 11.5 μg/ml for 24 h. An early apoptosis induction in HepG2 cells was observed by annexin V assay, which clearly exhibited significantly increased early and late apoptosis phases both at 24 and 48 h. Both the caspases-8 and-9 level was found to be increased by the introduction of ST to HepG2 cells significantly (p<0.05). Moreover the ST extract was able to arrest the cell cycle check point at G2/M phase. A significantly increasing pattern of hypodiploid phases of cells also been observed, which confirm the apoptosis induction again. Collectively, results presented in this study demonstrated that the ST, which is used as a euphoritic substance of abuse also, has significant level of toxicity in human cells. Moreover the mode of cell death was found to be though programmed cell death which is closely associated with cell cycle arrest.

Key words: Hepatotoxicity, In vitro models, Saudi Arabia, Smokeless tobacco, Substance Absue Research Centre, Shammah.