Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures

July 12, 2016


Chem. Res. Toxicol.2016, 29, 1252−1269

Filippo  Zanetti, Alain  Sewer, Carole  Mathis, Anita  R.  Iskandar, Radina  Kostadinova, Walter K. Schlage, Patrice Leroy, Shoaib Majeed, Emmanuel Guedj, Keyur Trivedi, Florian Martin, Ashraf  Elamin, Celine  Merg, Nikolai  V.  Ivanov, Stefan  Frentzel, Manuel  C.  Peitsch, and  Julia  Hoeng*,

Philip Morris International Research and Development, Quai Jeanrenaud 5, 2000 Neuchatel, Switzerland
Biology Consultant, Max-Baermann-Str. 21, 51429 Bergisch Gladbach, Germany

The tobacco heating system (THS) 2.2 and the 3R4F reference cigarette smoke were exposed to human organotypic oral epithelial tissue cultures (EpiOral, MatTek)at the Vitrocell 24/48 exposure system. The results are shown by the evaluation of cytotoxicity and cytochrome P450 activity assays, mRNA and miRNA purification, measurements of secreted proin flammatory markers and histopathological analysis.


Cigarette smoke (CS) has been reported to increase predisposition to oral cancer and is also recognized as a risk factor for many conditions including periodontal diseases, gingivitis, and other benign mucosal disorders. Smoking cessation remains the most effective approach for minimizing the risk of smoking-related diseases. However, reduction of harmful constituents by heating rather than combusting tobacco, without modifying the amount of nicotine, is a promising new paradigm in harm reduction. In this study, we compared effects of exposure to aerosol derived from a candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, with those of CS generated from the 3R4F reference cigarette. Human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) were exposed for 28 min to 3R4F CS or THS2.2 aerosol, both diluted with air to comparable nicotine concentrations (0.32 or 0.51 mg nicotine/L aerosol/CS for 3R4F and 0.31 or 0.46 mg/L for THS2.2). We also tested one higher concentration (1.09 mg/L) of THS2.2. A systems toxicology approach was employed combining cellular assays (i.e., cytotoxicity and cytochrome P450 activity assays), comprehensive molecular investigations of the buccal epithelial transcriptome (mRNA and miRNA) by means of computational network biology, measurements of secreted proin flammatory markers, and histopathological analysis. We observed that the impact of 3R4F CS was greater than THS2.2 aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. Analysis of the transcriptomic changes in the exposed oral cultures revealed significant perturbations invarious network models such as apoptosis, necroptosis, senescence, xenobiotic metabolism, oxidative stress, and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) signaling. The stress responses following THS2.2 aerosol exposure were markedly decreased, and the exposed cultures recovered more completely compared with those exposed to 3R4F CS.

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