Toxicity of Khat on Normal Human Oral Cells. In Vitro Studies Using Primary Human Oral Keratinocytes and Fibroblasts in Monolayer and Organotypic Cultures

Abstract

Khat is an evergreen shrub of the Celastraceae family grown in parts of the Middle East and Eastern Africa where its use is important for the social and economic wellbeing of the communities. Fresh leaves and shoots of the khat plant contain the chemical cathinone which has a psychoactive effect comparable to amphetamine. Habitual chewing of khat is widespread in Yemen and the horn of Africa, and its use as a stimulant is gradually spreading to other parts of the world especially in immigrant communities. Prolonged khat use has been reported to have adverse effects on the central nervous, cardiovascular and reproductive systems. In the oral cavity, khat chewing has been associated with histopathological changes like hyperkeratosis, epithelial hyperplasia and mild dysplasia. A higher incidence of head and neck cancer has been reported among khat chewers compared to non-chewers. However, studies on the toxicological potential and mechanisms of actions of khat remain scarce. The aim of this study was to investigate the toxic effects induced by an extract of khat on primary normal human oral keratinocytes and fibroblasts in monolayer and in vitro reconstructed human oral mucosa. Khat induced a concentration dependent inhibition of cell growth, with cells accumulating in the G1 phase of the cell cycle and showing an increased expression of cell cycle inhibitor proteins like p53, p21 and p16. The growth inhibition occurred earlier in fibroblasts when compared to keratinocytes. Unlike keratinocytes, fibroblasts also showed recovery of their proliferative potential on prolonged exposure. In reconstituted oral mucosa, khat induced a concentration dependent reduction in cell proliferation and a reduction in total epithelial thickness. An early and increased expression of p21, keratinocyte transglutaminase, involucrin and fillagrin, as well as decreased expression of cytokeratin 13 in tissues exposed to khat suggested premature differentiation and a switch from nonkeratinizing to keratinizing epithelium. These changes were accompanied by increase in p38 expression, and were reversed by inhibitors of p38. The results demonstrate the toxic potential of khat to oral tissues and identify p38 MAP kinase signalling as the mechanism involved in stress induced by khat. At higher concentrations, khat induced cell death that showed morphological and biochemical features consistent with apoptosis. Khat induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH). Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, fibroblasts were more sensitive to khat-induced cytotoxicity than keratinocytes. Cell death induced by khat was caspase-independent and showed a swift and sustained decrease in the mitochondrial membrane potential (ΔΨm) and release of mitochondrial apoptogenic proteins to the cytoplasm. The findings described in this study were observed at concentrations of khat comparable to those found in saliva among people chewing khat, and demonstrate the potential for khat to modulate key cellular functions such as proliferation, differentiation and cell death through specific signaling pathways. The results show that khat has toxic effects on human oral tissues and raises concerns about khat use and the development of various oral lesions.