Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx

Toxicol Rep. 2021 Apr 19:8:920-937. doi: 10.1016/j.toxrep.2021.04.007. eCollection 2021.

Abstract

The larynx is an essential organ in the respiratory tract and necessary for airway protection, respiration, and phonation. Cigarette smoking is a significant risk factor associated with benign and malignant laryngeal diseases. Despite this association, the underlying mechanisms by which cigarette smoke (CS) drives disease development are not well elucidated. In the current study, we developed a short-term murine whole body inhalation model to evaluate the first CS-induced cellular responses in the glottic [i.e. vocal fold (VF)] and subglottic regions of the larynx. Specifically, we investigated epithelial cell proliferation, cell death, surface topography, and mucus production, at various time points (1 day, 5 days, 10 days) after ∼ 2 h exposure to 3R4F cigarettes (Delivered dose: 5.6968 mg/kg per cigarette) and following cessation for 5 days after a 5 day CS exposure (CSE). CSE elevated levels of BrdU labeled proliferative cells and p63 labeled epithelial basal cells on day 1 in the VF. CSE increased proliferative cells in the subglottis at days 5, 10 and following cessation in the subglottis. Cleaved caspase-3 apoptotic activity was absent in VF at all time points and increased at day 1 in the subglottis. Evaluation of the VF surface by scanning electron microscopy (SEM) revealed significant epithelial microprojection damage at day 10 and early signs of necrosis at days 5 and 10 post-CSE. SEM visualizations additionally indicated the presence of deformed cilia at days 5 and 10 after CSE and post-cessation in the respiratory epithelium lined subglottis. In terms of mucin content, the impact of short-term CSE was observed only at day 10, with decreasing acidic mucin levels and increasing neutral mucin levels. Overall, these findings reveal regional differences in murine laryngeal cellular responses following short-term CSE and provide insight into potential mechanisms underlying CS-induced laryngeal disease development.

Keywords: AB/PAS, Alcian blue/Periodic acid Schiff; BLOQ, below limits of quantitation; BSA, bovine serum albumin; BrdU, 5-bromo-2′-deoxyuridine; CBF, ciliary beat frequency; CC3, cleaved caspase-3; CO, Carbon monoxide; CS, cigarette smoke; CSE, cigarette smoke exposure; Cell death; Cell proliferation; Cigarette smoke; DAB, 3,3′-diaminobenzidine; FTC/ISO, Federal Trade Commission/International Standard Organization; GSD, geometric standard deviation; H&E, Hematoxylin and Eosin; HIER, heat-induced antigen retrieval; HPF, high power field; MCC, mucociliary clearance; MMAD, Mass median aerodynamic diameter; Mucus production; Murine larynx; NMR, nicotine metabolite ratio; OECD, organization for economic co-operation and development; PAHs, polycyclic aromatic hydrocarbons; RE, respiratory epithelium; REV, reversibility; ROS, reactive oxygen species; SCIREQ, Scientific Respiratory Equipment Inc; SEM, scanning electron microscopy; SSE, stratified squamous epithelium; SWGTOX, Scientific Working Group for Forensic Toxicology; Surface topography; TBST, tris-buffered saline-tween 20; TPM, total particulate matter; TSNA, tobacco-specific nitrosamines; UPLC-MS/MS, ultra-performance liquid chromatography-tandem mass spectrometer; VF, vocal fold; VSC, veterinary service center.