Assessment of acute THS2.2 aerosol exposure in in vitro Human nasal epithelial cultures

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The nasal mucosa helps to guard the more sensitive lower airway tissues against harmful exposures, including air pollutants, airborne particulate matter, and other agents in the environment. Toxicant-induced injury in the nasal tissue can lead to increased risk of infection and susceptibility for respiratory tract diseases 1.

Exposure to cigarette smoke (CS), in particular, has been associated with nasal irritation leading to local inflammatory and physiological processes, such as the release of cytokines and transport of small molecules 2-4. Moreover, alterations of gene expression in nasal epithelial cells attributed to smoking, has been reported to closely resemble that in the lower airway epithelial cells 5, despite some differences 4.

Collectively, studies have supported that the nasal epithelium can be a surrogate tissue and is a relevant tissue to evaluate many aspects of the pathophysiological impact of CS exposure on the lower airway.

Modified risk tobacco products (MRTPs), i.e. products with the potential to reduce individual risk and population harm in comparison with smoking cigarettes, are developed. Switching from cigarette smoking to potentially-reduced harm/risk nicotine products is one possible approach to reduce the prevalence of smoking-related diseases, including those of the nasal cavity. It is therefore important to assess the relative impact of exposure to these MRTPs compared with cigarettes.

Systems Toxicology strategies are developed 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 and mechanistic data should be acquired using human cellular-based in vitro systems 6-9.


Applying the 3Rs principles, aimed at reducing the use of laboratory animals 10, in a Systems Toxicology approach, 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 nasal epithelial tissue culture model.

The recently established three dimensional (3D) organotypic nasal epithelial culture models are grown on top of an artificial porous membrane at the air-liquid interface (ALI), allowing them to develop a morphology that closely resembles the characteristics of the in vivo nasal epithelium—a pseudostratified epithelium comprising basal cells, goblet cells, and ciliated cells forming a columnar epithelium 11,14.

A Systems Toxicology approach, summarized in the video below, was applied. Briefly, 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, histological analysis, and cilia beating analysis) to uncover the cellular and molecular changes following exposure.

A video and accompanying manuscript have been published to explain the concepts and methods used in a similar study 15.

For more details on the study design, results and methods applied, please read below.

Study Design and Conduct

Study design

To assess the biological impact of 3R4F smoke and THS2.2 aerosol, a series of five experimental repetitions was conducted. The repetition was performed to increase the robustness of the impact assessment.

Nasal epithelial cultures 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 0.44 mg/L aerosol). Using the Vitrocell® Dilution/Distribution module, these specific nicotine concentrations were applied to the nasal cultures by diluting 3R4F smoke or THS2.2 aerosol during using the Vitrocell® exposure system.

A systems toxicology study

Cytotoxicity of 3R4F smoke and THS2.2 aerosol exposure was assessed at various time-points after exposure by measuring the activity of adenylate kinase (AK) released into the basolateral media of tissue cultures post-exposure.

In parallel, a histological assessment was done to observe the impact of the exposure on the morphology of the nasal 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 nasal epithelial histology and cytotoxicity was evaluated (up to 1.79 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 nasal 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.


  1. Harkema, J. R. et al. The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicologic pathology 34, 252-269 (2006)
  2. Lee, H. S. & Kim, J. Cigarette smoke inhibits nasal airway epithelial cell growth and survival. International forum of allergy & rhinology (Wiley Online Library) 3:3, 188-192 (2013)
  3. Lan, M.-Y. et al. Cigarette smoke extract induces cytotoxicity on human nasal epithelial cells. American journal of rhinology 21, 218-223 (2007)
  4. Huvenne, W. et al. Different regulation of cigarette smoke induced inflammation in upper versus lower airways. Respiratory research 11, 100 (2010)
  5. Sridhar, S. et al. Smoking-induced gene expression changes in the bronchial airway are reflected in nasal and buccal epithelium. BMC genomics 9, 259 (2008)
  6. Sheldon, L. S. & Cohen Hubal, E. A. Exposure as part of a systems approach for assessing risk. Environ Health Perspect 117, 119-1194 (2009)
  7. Berg, N. et al. Toxicology in the 21st century--working our way towards a visionary reality. Toxicology in vitro : an international journal published in association with BIBRA 25, 874-881 (2011)
  8. Rovida, C. et al. Toxicity testing in the 21st century beyond environmental chemicals. Altex 32, 171-181 (2015)
  9. Manuppello, J. R. & Sullivan, K. M. Toxicity assessment of tobacco products in vitro. Alternatives to laboratory animals : ATLA 43, 39-67 (2015)
  10. Balls, M. ATLA (Alternatives to Laboratory Animals): past, present and future. Alternatives to laboratory animals : ATLA 38, 437-441 (2010)
  11. Dimova, S. et al.The use of human nasal in vitro cell systems during drug discovery and development. Toxicology in vitro 19, 107-122 (2005)
  12. Werner, U. & Kissel, T. In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies. Pharmaceutical research 13, 978-988 (1996)
  13. Constant, S. et al. The Use of in vitro 3D Cell Models of Human Airway Epithelia (MucilAir™) in Inhalation Toxicity. In Cellular In Vitro Testing: Methods and Protocols (Haycock, J., Whluwalia, A., and Wilkinson, M., Eds.), pp. 15-33. CRC Press, London, UK., (CRC Press, 2014)
  14. Talikka, M. et al. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure. International journal of toxicology 33, 506-517 (2014)
  15. Kuehn, D. et al. Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke. Journal of visualized experiments : JoVE 96 (2015)

Organotypic nasal (THS2.2) Results

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Publications and Reports

Iskandar, A. R. et al. Altex (2016)

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product.

In vitro toxicology approaches have evolved, from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS)2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understand the cellular/molecular changes that occur following exposure. In agreement with the Vision and Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was substantially greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles.

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