Oral Lesions Induced by Chronic Khat Use Consist Essentially of Thickened Hyperkeratinized Epithelium

Objectives. The habit of khat chewing is prevalent in many Middle Eastern and African cultures and has been associated with various adverse conditions in humans. This study aimed to describe histological changes induced by chronic khat chewing on the buccal mucosa. Methods. Biopsies of the buccal mucosa from 14 chronic khat chewers, 20 chronic khat chewers who also smoked tobacco, and 8 nonchewers were compared for epithelial thickness, degree and type of keratinization, and connective tissue changes. Results. Tissues from khat chewers depicted abnormal keratinization of the superficial cell layer and showed increased epithelial thickness affecting all layers. Epithelial thickness in control samples was 205 ± 26 μm whereas thickness in khat chewers and khat chewers who smoked tobacco was significantly higher measuring 330 ± 35 μm and 335 ± 19 μm, respectively. Tissues from khat chewers also showed increased intracellular edema, increased melanin pigment deposits, and increased number of rete pegs most of which were thin and deep. Conclusions. These results show that oral lesions induced by chronic chewing of khat in the buccal mucosa present with white and brown discoloration due to increased epithelial thickness, increased keratinization, and melanin deposition

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s).publishedVersio

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

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

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast-derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D-organotypic (3D-OT) cultures were constructed. Various growth factors were added at a range of 0.1-100 ng/ml. 3D-OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D-OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage-colony stimulating factor (GM-CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM-CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1-integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL-positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM-CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast-derived diffusible factor(s)