TO INVESTIGATE THE LEVELS OF CYTOKINES BY THE EFFECT OF BETEL QUID/ARECA NUT ON ORAL EPITHELIUM

Abstract

Objective: The aims of this study were to investigate the in vitro effect of betel quid and its components on a stratified epithelium and to evaluate whether there was evidence of a site specific response to their effect

Methodology: The reconstituted human buccal epithelium model and human gingival epithelium model used in the study was prepared and supplied by Skin Ethic Laboratories, Nice, France. It is a three-dimensional tissue culture model obtained by culturing transformed oral keratinocytes (TR146) derived from a buccal carcinoma and oral keratinocytes derived from healthy gingiva. 50µl of freshly prepared  aqueous extract of paan, areca, lime, areca/lime mixture, tobacco and PBS(as control) was applied to the surface of the epithelium and the tissue incubated for upto 48 hours at 37ºC in 5 % CO_2  in a humidified atmosphere. The tissue was used to access the viability by MTT assay and the culture medium was also collected at 4 and 48 hours and used for the measurement of cytokines/chemokines release using ELISA technique.

Result: In this study the application of paan and its components caused up-regulation of cytokines and chemokines. On the buccal epithelium model after 4 hours of treatment lime caused increased release of IL-1α (26.9±14.3 pg/ml) compared to PBS Control (9.5±4.5 pg/ml), IL-6 (24.9±8.4 pg/ml) compared to PBS control (8.2±1.8 pg/ml), IL-8 (437.5±227.8 pg/ml) compared to PBS control (194±58.1pg/ml) and TGF-β after 24 hours (71.3±10.8 pg/ml) compared to PBS control (49.3±2.7pg/ml).

Areca caused increased release of IL-1 α (81.2±11.3 pg/ml) compared to PBS control (11.2±5.9 pg/ml) after 48 hours of treatment, whereas paan caused increased release of IL-6 (16.3±4.3 pg/ml) compared to PBS control (8.2± 1.7 pg/ml), IL-8 (2333.7±1252.3 pg/ml) compared to PBS control (294.8±126.5pg/ml) and TGF-β(35.8±0.9, 41.4± 1.4 pg/ml) compared to PBS control (62±4.2) after 48 hours of treatment.  In gingival epithelial model only paan caused increased IL-8 release (305.5±221.1 pg/ml) compared to PBS control (48.3±19.4) after 48 hours of treatment.  Areca inhibited the viability of buccal epithelial cells.

Conclusion: This study has confirmed that lime, areca and paan caused significant changes in cytokine release, viability and histology of the tissue. The release of proinflammatory cytokines may suggest that in the initial event of OSF these cytokines can act as a constant source of irritation to underlying tissue. The increase in TGF-β release suggests that it may act on the underlying fibroblasts and result in increased collagen synthesis, a feature of OSF.