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

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Why?

Growing concern over the toxicity of environmental pollutants and atmospheric aerosols has prompted research to investigate the mechanisms involved in lung injury and repair following exposure to toxicants1.

Although longitudinal clinical studies are valuable to assess potential human health risks associated with smoking, the adverse effects and disease manifestation often take decades to occur. Such long observation periods are difficult or even impossible to conduct. Invasive procedures inherent to tissue sampling further contribute to the impracticality in studying the human respiratory pathophysiology. Alternatively, animal models can be used to investigate adverse effects following exposure and their underlying mechanisms. However, inhalation studies in animals pose ethical, physiological, and practical challenges2. These challenges have prompted the search for alternative approaches. These approaches should be practical and cost-efficient, as well as provide more relevant insight to toxicity risk in humans3.

Studies have demonstrated the relevancy of in vitro organotypic human culture models to evaluate cellular and molecular changes at the tissue level. To study the impact of exposure via inhalation, e.g., of cigarette smoke (CS), organotypic human culture models reconstituted from primary bronchial epithelial cells have been used in numerous studies to investigate the impact of CS1,4-7.


How?

Applying the Three Rs principles, aimed at reducing the use of laboratory animals8, in a Systems Toxicology approach, the objective of the study was to assess the biological impact of an aerosol generated from a prototype tobacco heating system 2.2 (THS2.2) - a candidate MRTP - compared to smoke generated from conventional combustible 3R4F reference cigarettes, on a human organotypic bronchial epithelial tissue culture model.

Three dimensional (3D) organotypic bronchial 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 bronchial epithelium(see figure below). Similar to in vivo bronchial epithelium, in vitro bronchial cultures comprise basal cells, mucus-secreting (goblet) cells, and ciliated cells. These cells are held together by tight junctions, which control the permeability and maintain the polarity of the tissue. The polarized epithelium creates two distinct membrane-separated compartments: The apical (air-directed) and basolateral (medium-directed) side. The apical side of the culture mimics the characteristics of the in vivo mucociliary surface covered by a physiological mucus layer6,9,10.

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, 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 study11.

Study Design and Conduct

Study design


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

Bronchial 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 0.42 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).

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.

References

  1. BeruBe, K. et al. In vitro models of inhalation toxicity and disease. The report of a FRAME workshop. Alternatives to laboratory animals : ATLA 37, 89-141 (2009)
  2. Paur, H.-R. et al. In-vitro cell exposure studies for the assessment of nanoparticle toxicity in the lung--A dialog between aerosol science and biology. J Aerosol Sci 42, 668-692 (2011)
  3. Committee on Toxicity Testing and Assessment of Environmental Agents, National Research Council (ed National Research Council) (National Academies Press, Washington D.C., 2007)
  4. Mathis, C. et al. Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. American journal of physiology.Lung cellular and molecular physiology 304, L489-503 (2013)
  5. Maunders, H. et al. Human bronchial epithelial cell transcriptome: gene expression changes following acute exposure to whole cigarette smoke in vitro. American journal of physiology. Lung cellular and molecular physiology 292, L1248-1256 (2007)
  6. 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)
  7. Iskandar, A. R. et al. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts. Toxicological sciences : an official journal of the Society of Toxicology 147, 207-221 (2015)
  8. Balls, M. ATLA (Alternatives to Laboratory Animals): past, present and future. Alternatives to laboratory animals : ATLA 38, 437-441 (2010)
  9. 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)
  10. 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)
  11. 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 bronchial (THS2.2) Results

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Publications and Reports

Iskandar, A. R. et al. Toxicology in vitro 39: 29-51 (2016)

A systems toxicology approach for comparative assessment: Biological impact of an aerosol from a candidate modified-risk tobacco product and cigarette smoke on human organotypic bronchial epithelial cultures.

This study reports a comparative assessment of the biological impact of a heated tobacco aerosol from the tobacco heating system (THS) 2.2 and smoke from a combustible 3R4F cigarette. Human organotypic bronchial epithelial cultures were exposed to an aerosol from THS2.2 (a candidate modified-risk tobacco product) or 3R4F smoke at similar nicotine concentrations. A systems toxicology approach was applied to enable a comprehensive exposure impact assessment. Culture histology, cytotoxicity, secreted pro-inflammatory mediators, ciliary beating, and genome-wide mRNA/miRNA profiles were assessed at various time points post-exposure. Series of experimental repetitions were conducted to increase the robustness of the assessment. The results showed that at similar nicotine concentrations, THS2.2 aerosol elicited lower cytotoxicity compared with 3R4F smoke. At least four times of nicotine concentrations in the THS2.2 aerosol than that in 3R4F smoke was needed to elicit morphological changes. Lower levels of secreted mediators were detected in the basolateral media and fewer miRNA alterations were observed following THS2.2 than following 3R4F. A computational analysis of the transcriptomes showed less perturbed biological networks (in the context of Cell Fate, Cell Proliferation, Cell Stress, and Inflammatory Network Models) in cultures exposed to THS2.2 aerosol compared with 3R4F smoke exposure applied at similar nicotine concentrations.