Clin Exp Otorhinolaryngol.  2021 Aug;14(3):303-311. 10.21053/ceo.2020.01907.

Changes in Mucin Production in Human Airway Epithelial Cells After Exposure to Electronic Cigarette Vapor With or Without Nicotine

Affiliations
  • 1Department of Otorhinolaryngology-Head and Neck Surgery, Yeungnam University College of Medicine, Daegu, Korea
  • 2Department of Medical Science, Yeungnam University College of Medicine, Daegu, Korea
  • 3Regional Center for Respiratory Diseases, Yeungnam University Medical Center, Daegu, Korea

Abstract


Objectives
. The emergence of electronic cigarettes (e-cigarettes) has created new perceptions of the tobacco market. Unlike traditional tobacco, the greatest advantage of e-cigarettes is that they have less smell and are convenient and inexpensive. Most e-cigarette smokers believe that e-cigarette smoking is less harmful than traditional smoking. Information on the effects of e-cigarettes on human health is limited, and the issue remains controversial.
Methods
. We studied the effects of e-cigarette vapor on mucin (MUC5AC and MUC5B) and the change of MUC5AC and MUC5B from e-cigarette liquid with or without nicotine in respiratory epithelial cells. The effects of e-cigarette vapor with or without nicotine on mucin, along with the involved signaling pathways, were investigated using reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, enzyme immunoassays, and immunoblot analysis with several specific inhibitors and small interfering RNA.
Results
. E-cigarette vapor with or without nicotine stimulated MUC5AC, but not MUC5B, expression in respiratory epithelial cells. In addition, we showed that e-cigarette vapor with and without nicotine induced MUC5AC expression via activation of the mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase [ERK] 1/2 and p38) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways in human airway epithelial cells.
Conclusion
. E-cigarette vapor with and with nicotine significantly increased MUC5AC expression in human airway epithelial cells.

Keyword

E-Cigarette Vapor; MUC5AC; Mitogen-Activated Protein Kinase; NF-Kappa B; Epithelial Cell

Figure

  • Fig. 1. Effects of e-cigarette vapor (with or without nicotine) on NCI-H292 cells and human nasal epithelial cells. (A) Result of cytotoxicity testing of NCI-H292 cells with water-soluble tetrazolium salt-1 (WST-1) treatment, showing that e-cigarette vapor did not display visible cell killing activity with up to 2 mL of e-liquid vapor when incubated for 24 hours. (B) Result of cytotoxicity testing of human nasal epithelial cells with WST-1 treatment, showing that e-cigarette vapor did not display visible cell killing activity with up to 2 mL of e-liquid vapor when incubated for 24 hours. e-cigarette, electronic cigarettes.

  • Fig. 2. Effects of e-cigarette vapor (with or without nicotine) on airway mucin expression in NCI-H292 cells and human nasal epithelial cells. (A) Real-time polymerase chain reaction (RT-PCR) results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) meaningfully stimulated MUC5AC, but not MUC5B, mRNA expression in NCI-H292 cells. (B) Enzyme-linked immunosorbent assay (ELISA) results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) significantly stimulated MUC5AC, but not MUC5B, protein levels in NCI-H292 cells. (C) RT-PCR results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) meaningfully stimulated MUC5AC, but not MUC5B, mRNA expression in human nasal epithelial cells. (D) ELISA results showing that e-cigarette vapor (with or without nicotine 24 mg/mL) significantly increased MUC5AC, but not MUC5B, protein levels in human nasal epithelial cells. Bars represent mean±standard deviation of three independent experiments performed in triplicate. PG, propylene glycol; VG, vegetable glycerin. a)P<0.05 vs. baseline.

  • Fig. 3. MUC5AC overexpression by e-cigarette vapor (with or without nicotine) in human airway NCI-H292 epithelial cells involves ERK1/2, p38, and NF-κB stimulation. (A, B) Western blot analysis showing that e-cigarette vapor with or without nicotine induced phosphorylation of ERK1/2, p38, and NF-κB. (C, D) Enzyme-linked immunosorbent assay (ELISA) results showing that U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), and BAY 11-7085 (an NF-κB inhibitor) treatment significantly attenuated the effect of e-cigarette vapor on MUC5AC protein levels. (E, F) ELISA results showing that knockdown of ERK1, ERK2, p38, and NF-κB by siRNA significantly blocked the effect of e-cigarette vapor on MUC5AC protein levels. Images are illustrative of three separate experiments performed in triplicate. Bars show mean±standard deviation of three independent experiments performed in triplicate. e-cigarette, electronic cigarettes; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; p-p38, phosphorylated p38; p-p65, phosphorylated p65; Con, control; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; siRNA, small interfering RNA. a)P<0.05 vs. baseline. b)P<0.05 vs. e-cigarette vapor alone.

  • Fig. 4. E-cigarette vapor (with or without nicotine)-induced MUC5AC expression in human nasal epithelial cells is associated with ERK1/2, p38, and NF-κB activation. (A) Immunofluorescence results showing that e-cigarette vapor without nicotine significantly induced MUC5AC expression, and this was significantly inhibited by pretreatment with U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or BAY 11-7085 (an NFκB inhibitor). E-cigarette vapor without nicotine also significantly increased MUC5AC expression in the cytoplasm. Each bar shows the mean± standard deviation of three independent experiments. (B) Immunofluorescence results showing that e-cigarette vapor with nicotine significantly increased MUC5AC expression, and this was significantly inhibited by pre-treatment with U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or BAY 11-7085 (an NF-κB inhibitor). Furthermore, e-cigarette vapor with nicotine significantly increased MUC5AC expression in the cytoplasm. Con, control; e-cigarette, electronic cigarettes; DAPI, 4ʹ,6-diamidino-2-phenylindole; ERK, extracellular signal-regulated kinase; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells.


Cited by  2 articles

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Min-Seok Rha, Chang-Hoon Kim
Clin Exp Otorhinolaryngol. 2021;14(3):249-250.    doi: 10.21053/ceo.2021.00906.

Ghrelin Downregulates Lipopolysaccharide/ Leptin-Induced MUC5AC Expression in Human Nasal Epithelial Cells
Yoon Seok Choi, Hyung Gyun Na, Chang Hoon Bae, Si-Youn Song, Yong-Dae Kim
Clin Exp Otorhinolaryngol. 2023;16(1):49-58.    doi: 10.21053/ceo.2022.00857.


Reference

1. Thornton SL, Oller L, Sawyer T. Fatal intravenous injection of electronic nicotine delivery system refilling solution. J Med Toxicol. 2014; Jun. 10(2):202–4.
Article
2. Chen EY, Sun A, Chen CS, Mintz AJ, Chin WC. Nicotine alters mucin rheological properties. Am J Physiol Lung Cell Mol Physiol. 2014; Jul. 307(2):L149–57.
Article
3. Kozlowski LT, Dreschel NA, Stellman SD, Wilkenfeld J, Weiss EB, Goldberg ME. An extremely compensatible cigarette by design: documentary evidence on industry awareness and reactions to the Barclay filter design cheating the tar testing system. Tob Control. 2005; Feb. 14(1):64–70.
Article
4. Farsalinos KE, Romagna G, Tsiapras D, Kyrzopoulos S, Voudris V. Evaluation of electronic cigarette use (vaping) topography and estimation of liquid consumption: implications for research protocol standards definition and for public health authorities’ regulation. Int J Environ Res Public Health. 2013; Jun. 10(6):2500–14.
Article
5. Wu Q, Jiang D, Minor M, Chu HW. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells. PLoS One. 2014; Sep. 9(9):e108342.
Article
6. Farsalinos KE, Romagna G, Allifranchini E, Ripamonti E, Bocchietto E, Todeschi S, et al. Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. Int J Environ Res Public Health. 2013; Oct. 10(10):5146–62.
7. Martey CA, Pollock SJ, Turner CK, O’Reilly KM, Baglole CJ, Phipps RP, et al. Cigarette smoke induces cyclooxygenase-2 and microsomal prostaglandin E2 synthase in human lung fibroblasts: implications for lung inflammation and cancer. Am J Physiol Lung Cell Mol Physiol. 2004; Nov. 287(5):L981–91.
Article
8. Farsalinos KE, Romagna G, Tsiapras D, Kyrzopoulos S, Voudris V. Evaluating nicotine levels selection and patterns of electronic cigarette use in a group of “vapers” who had achieved complete substitution of smoking. Subst Abuse. 2013; Sep. 7:139–46.
Article
9. Kwak S, Kim YD, Na HG, Bae CH, Song SY, Choi YS. Resistin upregulates MUC5AC/B mucin gene expression in human airway epithelial cells. Biochem Biophys Res Commun. 2018; May. 499(3):655–61.
Article
10. Reidel B, Radicioni G, Clapp PW, Ford AA, Abdelwahab S, Rebuli ME, et al. E-cigarette use causes a unique innate immune response in the lung, involving increased neutrophilic activation and altered mucin secretion. Am J Respir Crit Care Med. 2018; Feb. 197(4):492–501.
Article
11. Ghosh A, Coakley RC, Mascenik T, Rowell TR, Davis ES, Rogers K, et al. Chronic e-cigarette exposure alters the human bronchial epithelial proteome. Am J Respir Crit Care Med. 2018; Jul. 198(1):67–76.
Article
12. Alexander DJ, Collins CJ, Coombs DW, Gilkison IS, Hardy CJ, Healey G, et al. Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals. Inhal Toxicol. 2008; Oct. 20(13):1179–89.
Article
13. Renne RA, Wehner AP, Greenspan BJ, Deford HS, Ragan HA, Westerberg RB, et al. 2-Week and 13-week inhalation studies of aerosolized glycerol in rats. Inhal Toxicol. 1992; 4(2):95–111.
Article
14. Lechasseur A, Jubinville E, Routhier J, Berube JC, Hamel-Auger M, Talbot M, et al. Exposure to electronic cigarette vapors affects pulmonary and systemic expression of circadian molecular clock genes. Physiol Rep. 2017; Oct. 5(19):e13440.
Article
15. Lerner CA, Rutagarama P, Ahmad T, Sundar IK, Elder A, Rahman I. Electronic cigarette aerosols and copper nanoparticles induce mitochondrial stress and promote DNA fragmentation in lung fibroblasts. Biochem Biophys Res Commun. 2016; Sep. 477(4):620–5.
Article
16. Garcia-Arcos I, Geraghty P, Baumlin N, Campos M, Dabo AJ, Jundi B, et al. Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner. Thorax. 2016; Dec. 71(12):1119–29.
Article
17. Schweitzer KS, Chen SX, Law S, Van Demark M, Poirier C, Justice MJ, et al. Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures. Am J Physiol Lung Cell Mol Physiol. 2015; Jul. 309(2):L175–87.
Article
18. Margham J, McAdam K, Forster M, Liu C, Wright C, Mariner D, et al. Chemical composition of aerosol from an e-cigarette: a quantitative comparison with cigarette smoke. Chem Res Toxicol. 2016; Oct. 29(10):1662–78.
Article
19. Goniewicz ML, Knysak J, Gawron M, Kosmider L, Sobczak A, Kurek J, et al. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob Control. 2014; Mar. 23(2):133–9.
Article
20. Hiemstra PS, Bals R. Basic science of electronic cigarettes: assessment in cell culture and in vivo models. Respir Res. 2016; Oct. 17(1):127.
Article
21. Kaur G, Pinkston R, Mclemore B, Dorsey WC, Batra S. Immunological and toxicological risk assessment of e-cigarettes. Eur Respir Rev. 2018; Feb. 27(147):170119.
Article
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