Skip to main content

In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts

Abstract

Objectives

Cigarette smoke aqueous aerosol extracts (AqE) have been used for assessing tobacco products, particularly with in vitro models such as oxidative stress and inflammation. These test articles can be generated easily, but there are no standardised methods for the generation and characterisation or stability. We investigated the effects of pro-oxidant smoke-derived chemicals by using 3R4F AqE generated under standardised conditioning and smoking regimes and assessed the stability over 31-week timeframe. Twenty batches generated from ten puffs per cigarette bubbled through 20 ml cell culture media were used fresh and thawed from frozen aliquots stored at – 80 ºC.

Results

Nicotine levels quantified by gas chromatography/mass spectrometry and optical density at 260 nm showed chemical and physical stability from week 0 (fresh sample) to weeks 1, 4, 8 and 31 (frozen samples). No significant change in H292 human bronchial epithelial cell viability or oxidative stress were observed between fresh AqE at week 0 and frozen AqE at 31 weeks. AqEs generated by our protocol were stable for up to 31 weeks for all tested end points, suggesting that it may not be necessary to use freshly generated AqE for each study, thus reducing batch-to-batch variability.

Introduction

Tobacco smoke is a mixture of more than 7000 constituents [1,2,3] partitioned in vapour and particulate phases of cigarette smoke aerosol. In vitro toxicological assessments of cigarette smoke have been conducted extensively in total particulate matter (TPM), whole aerosol, or aqueous aerosol extracts (AqE) and by a range of biological techniques [4,5,6,7]. TPM is well characterised and shows stability over 2 years [4]. Stability of whole aerosol approaches is less important for cigarettes because they are designed to generate and deliver the aerosol in seconds [5, 6]. Although the use of cigarette smoke AqE has been well documented, including the effective capturing of semi-volatiles, such as carbonyls [8,9,10,11], short-term and long-term stability of volatile and semi-volatile fractions remain uncharacterised. The generation of fresh extracts can be time consuming and subjected to a risk of batch-to-batch variability. Mitigation of variability might be possible if pooled batches are generated, stored and used throughout the lifecycle of a study.

AqE generation is further confounded by the lack of a standardised approach.

A cigarette smoke aqueous test matrix consists of one or more cigarettes smoked into a specified volume of solution and the resulting aqueous soluble fraction of the cigarette smoke (AqE) is diluted and applied to the cell surface. Studies have used multiple methods not fully described to allow replication [7, 10, 12, 13]. For instance, the puffing regime is not always specified, the capture solutions (e.g. cell culture medium) and the capture conditions (e.g. media temperature) differ, making comparisons of data difficult (Table 1).

Table 1 Different methods described for generation of AqE

Smoke-induced oxidative stress has been linked to chronic obstructive pulmonary disease [13, 17, 23] associated with impaired antioxidant defences and characterised by an accumulation of oxidised glutathione in the lungs of smokers. In this study, the effects of pro-oxidant smoke-derived chemicals in cigarette smoke (partitioned between the particulate and vapour phase) were investigated by use of AqE. Cigarette Smoke extracts containing both particulate and vapour phases have been shown to induce oxidative stress [24]. In addition, the stability of frozen compared with fresh AqE was assessed over 31 weeks.

Main text

Materials and methods

Study design

Cytotoxicity and oxidative stress responses were assessed by in vitro assays. Measurements of epithelial antioxidant responses to AqE exposure enabled observations of any reductions in AqE-contained oxidants, potentially due to loss of volatile or semi-volatile chemicals within the extract partitioned between the particulate and vapour phases. Viability of H292 cells following AqE exposure was quantified by cellular protease activity.

The physical and chemical stability of cigarette smoke AqE stored at – 80 °C was investigated by assessment of nicotine and tar concentrations in AqE used fresh and from frozen throughout the study. This served to assess if long-term storage of AqE resulted in significant changes in cytotoxicity and oxidative stress responses and if affected the ability to complete a full set of experiments in a study.

Materials and reagents

Unless otherwise stated all materials and reagents were purchased from Fisher Scientific (Loughborough, UK).

Test article

All cigarette smoke AqEs were obtained from Kentucky 3R4F reference cigarettes. The 3R4F is a US-blended king-sized product with a cellulose acetate filter and an International Organisation for Standardisation (ISO) tar yield of 9.4 mg per cigarette. The composition, construction and mainstream smoke chemistry yields from this product have been reported previously [25]. Prior to analysis, cigarettes were conditioned for at least 48 h at 22 ± °C in 60 ± 3% relative humidity and for no longer than 10 days, in accordance with ISO 3402:1999 [25].

Preparation of aqueous aerosol extracts

3R4F cigarettes were puffed on a Borgwaldt-KC RM20H rotary smoking machine (Borgwaldt, Richmond, VA, USA) using the Health Canada Intense T-115 regime (55 ml puff volume, 2 s puff duration every 30 s, and 100% ventilation blockage) [26]. Ten puffs from one 3R4F cigarette were bubbled through 20 ml cell culture media contained in an impinger per AqE. This produced a stock (100%) extract at a concentration of 0.5 puffs/ml; 20 AqE batches were produced on the same day by a single operator; all AqE batches were combined to provide a 400 ml pooled batch, which was stored at – 80 °C, except the fresh AqE aliquot used on the first day of the study. Frozen aliquots were thawed at room temperature overnight preceding the experiments and diluted with cell-type-specific culture media to the desired concentrations.

Dosimetry and quality control measurements

To confirm the capture of cigarette smoke constituents, assess batch-to-batch consistency of AqE, and test physical and chemical stability over time, three measurements were taken in the fresh AqE and after thawing of frozen AqE at 1, 4, 8, and 31-weeks. Nicotine concentration in the AqE was quantified via gas chromatography/mass spectrometry. An indirect measurement of nicotine concentration and the relative tar content of the extracts were also performed directly by optical density (OD) readings at 260 nm and 320 nm respectively, using a Spectramax M3 multimode spectrophotometer (Molecular Devices, San Jose, CA, USA) [7].

Human bronchial epithelial cell culture

Human bronchial epithelial cells (NCI-H292; American Type Culture Collection, Teddington, Middlesex, UK) were grown in cell culture flasks and maintained in RPMI 1640 medium containing sodium bicarbonate without l-glutamine, sterile-filtered and tested for endotoxins. The medium was supplemented with 10% foetal bovine serum (GE Healthcare Life Sciences, Hatfield, Hertfordshire, UK), 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. Experimental 96-well cell culture plates were seeded with 100 µl culture medium per well at a seeding density of 1 × 105 cells/ml cell suspension. Cultures were grown over 72 h at 37 °C in a humidified 5% carbon dioxide incubator prior to experimental exposures.

Measurement of epithelial viability

Cell viability was assessed by Apolive-Glo assay (Promega, Madison, WI, USA) in 96-well format. Viable cells were quantified through observations of live-cell protease activity. A cell permeable substrate (glycyl-phenylalanyl-amino fluorocoumarin; GF-AFC) that produces a fluorescent signal upon cleavage by viable cell proteases was co-incubated with the cells. Fluorescence signals, proportional to the viable cell number, were measured with a Spectramax M3 multimode plate reader. Exposure of cells to Triton X-100 (0.2%) allowed observations of complete cell death in the viability assay.

Quantification of epithelial oxidative stress

The ratio of reduced glutathione (GSH) to oxidised GSH (GSSG) was assessed in NCI-H292 cells and calculated after 4 h exposure to 100 µl AqE in a 96-well format assay (GSH/GSSG-Glo, Promega), as per the manufacturer’s protocol. Quantification of both GSH and GSSG was achieved through a GSH-dependent reaction, whereby a GSH probe, luciferin-NT (Promega), was converted to luciferin by a glutathione S-transferase enzyme that had been coupled to a firefly luciferase. The luminescent signal was proportional to the amount of GSH or GSSG present. Luminescent signals were measured with a Spectramax M3 multimode plate reader over a 1 s integration time. Cells were treated with potassium bromate (30 mM) to provide a pro-oxidant source required to lower the glutathione ratio, which was used as a positive control.

Statistics

All variables were measured at weeks 0, 1, 4, 8, and 31. Statistical analysis was performed by means of ANOVA, with one-sided Dunnett’s post hoc comparisons to assess differences in dosimetry, cell viability and GSH:GSSG between experiments performed at week 0 with fresh AqE and at week 31 with frozen AqE; p values < 0.05 were considered significant. AqE were assessed at an IC50 to compare shifts in responses. 50% viability was represented at 56% AqE dose (Clarified in Table 2). Nicotine and OD measurements were only conducted on one occasion per assessment after the first week of the study once the variability was established. This was to ensure that the test matrix was maximised for biological analysis.

Table 2 Summary of findings

Results

Dosimetry

Storage up to 31-weeks at – 80 °C was not associated with reductions in nicotine or tar concentrations compared with fresh AqE used at week 0 (Fig. 1).

Fig. 1
figure1

A1-3. QC measures. (A1) nicotine, (A2) OD260nm (A3) OD 320 nm. B1-3 Cell viability (B1) All data 0–31 weeks (B2) O vs. 31 weeks comparison (B3) Data at 56% AqE dilution. C1-3 Glutathione ratio (C1) All data 0–31 weeks (C2) O vs. 31 weeks comparison (C3) Data at 56% AqE dilution

Cellular viability

Little variation in the results for epithelial viability was observed between fresh and frozen AqE at any dilution (Fig. 1).

Glutathione ratio

The ratio of GSH to GSSG at different AqE dilutions was similar for all fresh and frozen aliquots (Fig. 1).

Summary of findings

Storage of 3R4F AqE at – 80 °C up to 31-weeks was not associated with any deterioration in nicotine and tar concentrations or any negative effects on cell biological activity (Table 2).

Discussion

Cigarette smoke AqE generation is complex and further confounded by differences in terminology, methodology and approaches, from study to study and between laboratories. This study addressed some of the misunderstandings and gaps in the current literature about the generation and stability of cigarette smoke AqE. The use of established methods and parameters based on published studies led to uniform generation of AqE and freezing and storing aliquots at – 80 ºC was a stable way of providing enough extract for long-term experimentation.

This methodology combined the use of an internationally recognised reference cigarette (3R4F), standard conditioning and smoking regimens [26, 27] with parameters that were the ‘average’ of those previously reported (Table 1), such as capture of ten puffs of smoke per cigarette in 20 ml cell culture media and dilution of aliquots with cell-type-specific media. This approach led to consistent generation of AqE that provided reproducibility, minimised batch-to-batch variation and allowed batches to be pooled for storage and consistent experimentation over 31-weeks.

Nicotine and tar concentrations were used as dosimetry and quality control markers for physical and chemical stability. Throughout the study, no values with frozen aliquots differed significantly from those achieved in the fresh samples. This finding indicates good stability in the storage conditions assessed. Nicotine has been reported as a stable analyte in culture media over 12 weeks at room temperature [28]. Our data supports this conclusion and suggest that nicotine concentration could be expected to remain stable for much longer than 31-weeks. OD measurements remained stable for the duration of the study.

The in vitro end points assessed determined whether biological activity would decrease after exposure to AqE stored at – 80 °C and thawed before use. The timepoints for assessment of frozen AqE (1, 4, 8 and 31 weeks) were selected to assess short-term and long-term effects of storage. Short-term effects are relevant, for example, for samples shipped to global locations, while longer-term effects account for the time that might be required to complete a full suite of experiments in a study. Both cytotoxicity and GSSG formation are thought to be driven by the semi-volatile and volatile vapour-phase fraction of cigarette smoke, which partition between the vapour and particulate phase. Analytically measuring these compounds is challenging, and reductions or shifts in analytical yields do not necessarily equate to a biological response. Therefore, cytotoxicity was investigated as a measure of AqE functionality and stability and the glutathione ratio as an indicator of oxidative stress. The results observed in fresh AqE are consistent with previously reported data [18]. After freezing, no significant differences from fresh AqE were observed up to 31-weeks.

Conclusions

Internationally recognised conditioning and smoking regimens and the use of a reference cigarette (3R4F) allowed consistent generation of AqE. The puff number and volume captured per batch were based on data collected from the literature and averaged to align protocols. The data from multiple end points suggest that the AqE are chemically and physically stable for weeks. This study has shown up to 31-weeks, that cigarette smoke AqE is stable in respect to cytotoxicity, glutathione ratio and nicotine. Cytotoxicity and glutathione ratio can be linked to the particulate and vapour phase of cigarette smoke, so observing little to no reduction in these biological end points is reassuring for the stability of the captured chemicals with the AqE matrix. Furthermore, pooling batches and creating single-use aliquots, ensuring consistency of AqE over time in a given cell line also provides an advantage to experimental design.

Limitations

More work is required to understand how extracts can be stored until biological activity decreases or is lost after 31-weeks. Future studies could investigate additional biological end points and more cell types, including primary cells, which might be more sensitive to subtle changes in AqE compared to immortalised cell lines. The use of more chemical markers to characterise AqE at various times would also be advantageous, especially in linking reduction in chemical stability to reduced biological responses. However, measuring these chemicals analytically at low levels and covering the required chemicals of interest could be challenging, which is why this study focused on reductions in biological effect rather than on chemical yields. Finally, this study focused on cigarette smoke extracts, however stability data are also required for tobacco heating products and electronic cigarette extracts, for which little to no information is available. This is extremely important, as the use of AqE to support testing of these novel products are increasing.

Availability of data and materials

Not applicable.

Abbreviations

AqE:

Aqueous extract

TPM:

Total particulate matter

3R4F:

Kentucky reference cigarette

GSH:

Glutathione

GSSG:

Oxidised glutathione

OD:

Optical density (at wavelengths 260 nm and 320 nm)

HCI:

Health Canada intense smoking regimen

ISO:

International standard

References

  1. 1.

    Rodgman A, Perfetti TA. The chemical components of tobacco and tobacco smoke. 2nd ed. Boca Raton: CRC Press; 2013.

    Google Scholar 

  2. 2.

    Borgerding M, Klus H. Analysis of complex mixtures—cigarette smoke. Exp Toxicol Pathol. 2005;57:43–73.

    CAS  Article  Google Scholar 

  3. 3.

    Fowles J, Dybing E. Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob Control. 2003;12:424–30.

    CAS  Article  Google Scholar 

  4. 4.

    Crooks I, Dillon DM, Scott JK, Ballantyne M, Meredith C. The effect of long-term storage on tobacco smoke particulate matter in in vitro genotoxicity and cytotoxicity assays. Regul Toxicol Pharmacol. 2013;65:196–200.

    CAS  Article  Google Scholar 

  5. 5.

    Thorne D, Adamson J. A review of in vitro cigarette smoke exposure systems. Exp Toxicol Pathol. 2013;65:1183–93.

    CAS  Article  Google Scholar 

  6. 6.

    Li X. In vitro toxicity testing of cigarette smoke based on the air-liquid interface exposure: a review. Toxicol In Vitro. 2016;36:105–13.

    CAS  Article  Google Scholar 

  7. 7.

    Taylor M, Carr T, Oluwatobiloba O, et al. E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke. Toxicol Mech Methods. 2016;26:465–76.

    CAS  Article  Google Scholar 

  8. 8.

    Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000;87:840–4.

    CAS  Article  Google Scholar 

  9. 9.

    Damico R, Simms T, Kim BS, et al. Inhibitory effects of macrophage migration inhibitor factor. Am J Respir Cell Mol Biol. 2011;44:323–32.

    CAS  Article  Google Scholar 

  10. 10.

    Munakata S, Ishimori K, Kitamura N, Ishikawa S, Takanami Y, Ito S. Oxidative stress responses in human bronchial epithelial cells exposed to cigarette smoke and vapor from tobacco- and nicotine-containing products. Regul Toxicol Pharmacol. 2018;99:122–8.

    CAS  Article  Google Scholar 

  11. 11.

    Donohue JF. Ageing, smoking and oxidative stress. Thorax. 2006;61:461–2.

    CAS  Article  Google Scholar 

  12. 12.

    Stabile AM, Marinucci L, Balloni S, et al. Long term effects of cigarette smoke extract or nicotine on nerve growth factor and its receptors in a bronchial epithelial cell line. Toxicol In Vitro. 2018;53:29–36.

    CAS  Article  Google Scholar 

  13. 13.

    Brunssen C, Giebe S, Hofmann A, Brux M, Morawietz H. Evaluation of cytotoxic, oxidative, and pro-inflammatory effects of aqueous cigarette smoke extract on human monocytes: a potential model system for assessment of next-generation tobacco and nicotine products. Appl In Vitro Toxicol. 2017;3:121–30.

    CAS  Article  Google Scholar 

  14. 14.

    Minghui J, Zhang Y, Li N, et al. Nicotine Component of cigarette smoke extract (CSE) decreases the cytotoxicity of CSE in BEAS-2B cells stably expressing human cytochrome P450 2A13. Int J Environ Res Public Health. 2017;14:1221.

    Article  Google Scholar 

  15. 15.

    Oke O, Azzopardi D, Corke S, et al. Assessment of acute in vitro human cellular responses to smoke extracts from a reduced toxicant prototype cigarette. Appl In Vitro Toxicol. 2017;3:182–92.

    CAS  Article  Google Scholar 

  16. 16.

    Gellner CA, Reynaga DD, Leslie FM. Cigarette smoke extract: a preclinical model of tobacco dependence. Curr Proto Neurosci. 2016;77:9541–95410.

    Article  Google Scholar 

  17. 17.

    Comer DM, Elborn JS, Ennis M. Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells. BMC Pulm Med. 2014;14:32.

    Article  Google Scholar 

  18. 18.

    Yoon CH, Park H-J, Cho Y-W, et al. Cigarette smoke extract-induced reduction in migration and contraction in normal human bronchial smooth muscle cells. Korean J Physiol Pharmacol. 2011;15:397–403.

    CAS  Article  Google Scholar 

  19. 19.

    Streck E, Jörres RA, Huber RM, Bergner A. Effects of cigarette smoke extract and nicotine on bronchial tone and acetylcholine-induced airway contraction in mouse lung slices. J Investig Allergol Clin Immunol. 2010;20:324–30.

    CAS  PubMed  Google Scholar 

  20. 20.

    Nana-Sinkam P, Lee JD, Sotto-Santiago S, et al. Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am J Respir Crit Care Med. 2007;175:676–85.

    CAS  Article  Google Scholar 

  21. 21.

    Hui JK, Liu X, Wng H, et al. Glutathione prevents inhibition of fibroblast-mediated collagen gel contraction by cigarette smoke. Am J Physiol Lung Cell Mol Physiol. 2002;283:L409–17.

    Article  Google Scholar 

  22. 22.

    Richter A, O’Donnell RA, Powell RM, et al. Autocrine ligands for the epidermal growth factor receptor mediate interleukin-8 release from bronchial epithelial cells in response to cigarette smoke. Am J Respir Cell Mol Biol. 2002;27:85–90.

    CAS  Article  Google Scholar 

  23. 23.

    Betsuyaku T. Oxidative stress in pathogenesis of COPD [article in Japanese]. Nihon Rinsho. 2007;65:633–66.

    PubMed  Google Scholar 

  24. 24.

    Ito S, Taylor M, Mori A, Thorne D, Nishino T, Breheny D, Gaca M, Yoshino K, Proctor C. An inter-laboratory in vitro assessment of cigarettes and next generation nicotine delivery products. Toxicol Lett. 2019;315:14–22.

    CAS  Article  Google Scholar 

  25. 25.

    Roemer E, Schamke H, Weiler H, et al. Mainstream smoke chemistry and in vitro and in vivo toxicity of the reference cigarettes 3R4F and 2R4F. Beiträge Tabakforschung Int. 2012;25:316–35.

    CAS  Google Scholar 

  26. 26.

    International Organisation for Standardisation. ISO 3402:1999. Tobacco and tobacco products – atmosphere for conditioning and testing. https://www.iso.org/standard/28324.html. Accessed 20 May 2020.

  27. 27.

    Health Canada. Health Canada Official Method T-115: determination of “tar”, nicotine and carbon monoxide in mainstream tobacco smoke. https://healthycanadians.gc.ca/en/open-information/tobacco/t100/nicotine. Accessed 20 May 2020.

  28. 28.

    Adamson J, Li X, Cui H, Thorne D, Xie F, Gaça M. Nicotine quantification in vitro: a consistent dosimetry marker for e-cigarette aerosol and cigarette smoke generation. Appl In Vitro Toxicol. 2017;3:14–27.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

All authors are (or were at the time of study conduct) employees of British American Tobacco. The work was funded by British American Tobacco.

Author information

Affiliations

Authors

Contributions

All authors designed the study and participated in its execution. DT and SS wrote the manuscript, supported by MT and NE. DT, SS and MT analysed the data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Simone Santopietro.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent to publish

The authors have all approved the final version. The data had not been published elsewhere, in part or its entirety.

Competing interests

All authors are (or were at the time of study conduct) employees of British American Tobacco.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Taylor, M., Santopietro, S., Baxter, A. et al. In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts. BMC Res Notes 13, 492 (2020). https://doi.org/10.1186/s13104-020-05337-2

Download citation

Keywords

  • In vitro
  • AqE
  • Aqueous extract
  • Cigarette smoke
  • Stability