The function of the oral mucosa is to protect underlying tissues and organs against physical, mechanical, chemical and microbial insults 1.
Exposure to cigarette smoke (CS) is one of the major risk factors for development of oral diseases. Besides its well-known effects increasing predisposition to oral cancer 2-4, CS can cause many benign mucosal conditions, including periodontal diseases, gingivitis, and other disorders 5-8.
Because CS enters the organism through the mouth via the oral cavity, the cytoarchitecture of the oral mucosa is essential in providing a barrier to counteract possible harmful consequences on the whole organism 9. Changes in distribution of cellular differentiation markers were detected as early as 3 h after CS exposure in basal and suprabasal layers of explanted oral epithelium in vitro, though no effects on intercellular adhesion and barrier functions were observed 9.
Smoking cessation is the primary way to avoid harmful effects of CS. However, switching to a modified risk tobacco product (MRTP), is a potential alternative to decrease risk of smoking-related diseases, including those of the oral cavity. Modified risk tobacco products, i.e. products with the potential to reduce individual risk and population harm in comparison with cigarettes, are developed. It is therefore important to assess the relative impact of exposure to these products compared with exposure to cigarettes.
Systems Toxicology strategies allow to adapt to new toxicity-assessment paradigms of environmental exposures proposed by the 21st Century Toxicology framework. This framework recommended that animal use should be minimized (see 3Rs below) and mechanistic data should be acquired using human cellular-based in vitro systems 10-13. In particular, a recent review outlines technological advances in in vitro assessment of tobacco products 13.
Applying the 3Rs principles (Refine, Reduce, Replace), aimed at reducing the use of laboratory animals14, the objective of the study was to assess the biological impact of an aerosol generated from a candidate MRTP, the Tobacco Heating System 2.2 (THS2.2), compared with smoke generated from 3R4F reference cigarettes, on a human organotypic buccal epithelial tissue culture model.
Organotypic cultures grown at the air liquid interface (ALI) retain the 3D structure and functional polarization typical of native epithelium, with an organized stratified epithelium (see figure below). Moreover, they show response characteristics similar to those of in vivo tissues15-18 and are therefore regarded as suitable, relevant models for investigating the biology, pathology and immunology of the oral mucosa, i.e. to study tissue innate responses to malignant transformation, pathogenic invasion, toxic stimuli and irritants under controlled conditions 17-22.
A Systems Toxicology approach summarized in the video below, was applied. Computational biology (i.e., a global analysis of mRNA and miRNA changes using a network-based approach) was used to complement well-established functional cellular assays (i.e., cytotoxicity assay, cytochrome P450 activity assay, measurement of secreted pro-inflammatory factors, and histological analysis) to uncover the cellular and molecular changes following exposure. For more details on the study design, results and methods applied, please read below.
To assess the biological impact of 3R4F smoke and THS2.2 aerosol, a series of four experimental repetitions was conducted. The repetition was performed to increase the robustness of the impact assessment.
Buccal organotypic tissue models were exposed to comparable doses of 3R4F smoke and THS2.2 aerosol. In addition, a high dose of THS2.2 was applied at a target nicotine concentration of 1.09 mg/L aerosol. Using the Vitrocell® Dilution/Distribution module, these specific nicotine concentrations were applied to the oral cultures by diluting 3R4F smoke or THS2.2 aerosol using the Vitrocell® exposure system.
Cytotoxicity of 3R4F smoke and THS2.2 aerosol exposure were assessed at various time points after exposure by measuring the activity of adenylate kinase (AK) released into the basolateral media of tissue cultures.
In parallel, a histological assessment was done to study the impact of the exposure on the morphology of the buccal tissue cultures. Note, to further evaluate the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted, in that the impact of a broader range of THS2.2 aerosol concentrations on the buccal epithelial histology and cytotoxicity were evaluated (up to 1.09 mg nicotine/L).
The impact of exposure on the activity of cytochrome P450 (CYP) 1A1 and 1B1, involved in metabolizing toxicants found in CS, such as polycyclic aromatic hydrocarbons (PAHs), nitrosamines, and arylamines, was assessed at different time points.
To determine the impact of 3R4F smoke and THS2.2 aerosol on the secretion of pro-inflammatory mediators, the concentrations of cytokines, chemokines, and growth factors were measured in the basolateral media of the buccal tissue cultures at various time points after exposure (as a cross sectional sampling).
Finally, the transcriptome (mRNAs and miRNAs) was analyzed at various time points after exposure.
A more detailed view of the study design is given in the figure below.
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 proinflammatory 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 in various 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.
With tableau reader you can filter, drill down, and view data details as prepared for review.
Data is plotted in a one or multiple different worksheet(s) for each endpoint, and you have the possibility to change the view by selecting the data you want to see, the treatment group(s) and time point(s).
Some tooltips will give some useful information as you mouse over the data.
At the start of the workbook, we have published a story (i.e., a sheet that contains a sequence of worksheets or dashboards that work together to convey information), which takes you through all endpoints available. You can already start interacting with the data on the story page, or by viewing individual pages.
For more information about Tableau and to get started, please refer to the tutorial on the Tableau tools page.