Reactive Oxygen Species Mediate Khat Induced Cytotoxicity in Oral Cells

Objectives: To determine the role of reactive oxygen species (ROS) in khat induced cytotoxicity.

Methods: Normal oral keratinocytes and fibroblasts were isolated from samples taken from the buccal mucosa of adult volunteers undergoing surgical removal of wisdom teeth. The cells were cultured in their standard media. An organic extract of fresh Kenyan khat was supplemented in the media to test its effects on the cells. Generation of ROS and depletion of glutathione (GSH) were assessed using hydroethidium and 5-chloromethylfluorescein diacetate (CMF-DA) respectively, with or without ROS scavengers Tiron and N-acetyl-cysteine (NAC). Cell viability was evaluated by assessment of metabolic activity (WST-1 reagent).

Results: Khat concentrations above 100 µg/ml induced cell death which was consistently associated with depletion of glutathione, increased generation of ROS and loss of mitochondrial membrane potential. ROS scavengers (Tiron and NAC) offered significant protection against GSH depletion and loss of mitochondrial membrane potential, but only partial (insignificant) protection against cell death. On the other hand, low (less than 32 µg/ml) doses of khat strongly inhibited cell growth, with partial increase in ROS, depletion of GSH and no changes in mitochondrial membrane integrity and cell viability.

Conclusion: Intracellular ROS and GSH levels play an important role in khat induced cytotoxicity in oral cells, and this could occur in the oral cavity of khat users partly contributing to the reported oral lesions.

Funding: The study was supported partly by the L. Meltzer's College Fund, Gades Legat and the Norwegian government.