Evaluating the Inflammatory and Genotoxic Effects of Smokeless Tobacco using a Human Organotypic Model of Oral Epithelium

Objectives: In addition to the well-known effects of tobacco use on the causation of lung and cardiovascular disease, tobacco use is also implicated as a major cause of oral cavity disease that leads to thousands of deaths per year. Snus, a smokeless tobacco applied to the oral cavity, has been proposed as a less harmful alternative to smoking although its safety has not been adequately evaluated. The objective of this study was to evaluate the cytotoxic, genotoxic and inflammatory effects of snus using an in vitro model of human oral mucosa (EpiOral).

Methods: EpiOral tissues were treated topically with 5 or 25 milligrams of snus for 24-48 hours. Tissues were evaluated for cytotoxicity by MTT. Following treatment, an inflammation-specific cytokine panel was used to analyze markers of inflammation at 24 and 48 hours post treatment. As a measure of genotoxicity, the presence of γ-H2AX foci (specifically, phosphorlation at Serine 139) was evaluated in treated tissues. γ-H2AX is a phosphorylated derivative of the H2AX histone and is tightly bound to double strand DNA break sites, therefore serving as a biomarker of genotoxic insult.

Results: Tissues treated with 5 mg of snus had comparable viability to vehicle treated controls while those treated with 25 mg displayed approximately a 20% decrease in viability after 24 and 48 hours of exposure. Histological analysis revealed hyperchromic staining in tissues treated with 5 mg of snus at 24 hours post-treatment whereas tissues treated with 25 mg of snus displayed a significant amount of sloughing of the apical layers. Of the cytokines analyzed, significant increases (1.5-2 fold) in IP-10, GM-CSF and RANTES were observed at both 24 and 48 hours post treatment in tissues treated with 25 mg of snus. γ-H2AX foci were readily detected in the apical layer of tissues treated with 25 mg of snus at 24 and 48 hours post treatment.

Conclusions: These results demonstrate the utility of this organotypic oral tissue model to evaluate smokeless tobacco product safety.