J Rhinol.  2020 May;27(1):34-40. 10.18787/jr.2019.00301.

Effect of Tobacco-specific Nitrosamines on MUC5AC Expression in Human Airway Epithelial Cells

• Kwak S
• Choi YS
• Na HG
• Bae CH
• Song SY
• Kim YD

• KMID: 2502792
• DOI: http://doi.org/10.18787/jr.2019.00301

Abstract

Background and Objectives
Nicotine is oxidized into tobacco-specific nitrosamines (TSNAs; NAB, NAT, NNN, NNAL, NNK) at high temperature and high pressure. TSNAs are associated with airway diseases characterized by mucus hypersecretion as a major pathophysiologic phenomenon. The aim of study is to investigate the effect of TSNAs on mucin overexpression and its molecular mechanism in human airway epithelial cells. Materials and Method: The cytotoxicity of TSNAs was evaluated using EX-Cytox and inverted microscopy. The mRNA and protein levels of MUC5AC and MUC5B were measured using real-time PCR and ELISA.
Results
NAB, NNN, NNAL, and NNK did not affect cell viability. NAT did not affect cell viability up to a concentration of 100 μM in human airway epithelial cells. NAT, NNN, NNAL, and NNK significantly induced MUC5AC expression, but not MUC5B expression. NAB did not affect the expression of MUC5AC and MUC5B. Propranolol (a β-adrenergic receptor antagonist) inhibited NAT, NNN, NNAL, and NNK-induced MUC5AC expression, whereas α-bungarotoxin (an α7-nicotinic acetylcholine receptor antagonist) only inhibited NNN- and NNK-induced MUC5AC expression.
Conclusion
These results suggested that NAT, NNN, NNAL, and NNK induce MUC5AC expression through β-adrenergic receptor and/or α7-nicotinic acetylcholine receptor in human airway epithelial cells, which may be involved in mucus hypersecretion in inflammatory airway diseases.

Keyword

Tobacco-specific nitrosamines; MUC5AC; Human airway epithelial cell; 담배특이니트로사민; 점액유전자; 인간호흡기상피세포

Figure

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  Fig. 1. Effects of NAB, NAT, NNN, NNAL, and NNK on cell viability in NCI-H292 cells. WST-1 assays were performed in human airway epithelial NCI-H292 cells treated with NAB, NAT, NNN, NNAL, and NNK for 24 h. NAB, NNN, NNAL, and NNK did not affect cell viability up to 100 μM. However, NAT reduced cell viability at 100 μM. Bars indicate the mean±S.D. of three independent experiments performed in triplicate. *: p<0.05 compared with zero value. TSNAs: Tobacco-specific nitrosamines, WST-1: water soluble tetrazolium salt-1.

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  Fig. 2. Effects of NAB, NAT, NNN, NNAL, and NNK on MUC5AC expression in NCI-H292 cells. NCI-H292 cells were treated with various concentration of NAB, NAT, NNN, NNAL, and NNK for 8 h. A, B: Real time-PCR and ELISA showed that NAT, NNN, NNAL, and NNK significantly induced MUC5AC gene and protein expression. However NAB did not induced MUC5AC expression. C: NAT, NNN, NNAL, and NNK-induced MUC5AC mRNA expression was peaked at 8 h after treatment with NAT, NNN, NNAL, and NNK. Bars indicate the mean±S.D. of three independent experiments performed in triplicate. *: p<0.05 compared with zero value. TSNAs: Tobacco-specific nitrosamines, Real time-PCR: real time-polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay.

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  Fig. 3. Effects of NAB, NAT, NNN, NNAL, and NNK on MUC5B expression in NCI-H292 cells. NCI-H292 cells were treated with various concentration of NAB, NAT, NNN, NNAL, and NNK for 8 h. A, B: Real time-PCR and ELISA showed that NAB, NAT, NNN, NNAL, and NNK did not induced MUC5B gene and protein expression. Bars indicate the mean±S.D. of three independent experiments performed in triplicate. TSNAs: Tobacco-specific nitrosamines, Real time-PCR: real time-polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay.

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  Fig. 4. Effects of NAT, NNN, NNAL, and NNK on related-receptor mediated expression on MUC5AC expression in NCI-H292 cells. NCI-H292 cells were pretreated with propranolol (β-adrenergic receptor inhibitor, 20 μM) or α-bungarotoxin (α-BTX, a α7-nicotinic acetylcholine receptor inhibitor, 100 nM) for 2 h. A, B: Real-time PCR and ELISA showed that NAT and NNAL-induced MUC5AC expression were inhibited by propranolol and α-BTX in NCI-H292 cells. NNN and NNK-induced MUC5AC were inhibited by propranolol but not α-BTX. Bars indicate the mean±S.D. of three independent experiments performed in triplicate. *: p<0.05 compared with zero value. †: p<0.05 versus NAT, NNN, NNAL, and NNK (10 μM), respectively. TSNAs: Tobacco-specific nitrosamines, Real time-PCR: real time-polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay.

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  Fig. 5. The schematic signaling pathway of TSNAs-induced MUC5AC expression. TSNAs-induced MUC5AC expression through β-adrenergic receptor and/or α7-nicotinic acetylcholine receptor in human airway epithelial cells. TSNAs: Tobacco-specific nitrosamines, α7-nAChRs: α7-nicotinic acetylcholine receptor, β-AdrRs: β-adrenergic receptor, α-BTX: α-bungarotoxin.

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