Third-Hand Exposure to E-Cigarette Vapour Induces Pulmonary Effects in Mice

Abstract

In the last decade, e-cigarette usage has increased, with an estimated 82 million e-cigarette users globally. This is, in part, due to the common opinion that they are "healthier" than tobacco cigarettes or simply "water vapour". Third-hand e-vapour exposure is the chemical residue left behind from e-cigarette aerosols, which is of concern due to its invisible nature, especially among young children. However, there is limited information surrounding third-hand e-vapour exposure. This study aimed to investigate the pulmonary effects of sub-chronic third-hand e-vapour exposure in a murine model. BALB/c mice (4 weeks of age) were exposed to a towel containing nicotine free (0 mg) e-vapour, nicotine (18 mg) e-vapour, or no e-vapour (sham) and replaced daily for 4 weeks. At the endpoint, lung function was assessed, and bronchoalveolar lavage fluid and lungs were collected to measure inflammation and fibrosis. Mice exposed to third-hand e-vapour without nicotine had alveolar enlargement compared to sham exposed controls. Mice exposed to third-hand e-vapour with nicotine had reduced bronchial responsiveness to provocation, increased epithelial thickening in large airways, increased epithelial layers in small airways, alveolar enlargement, and increased small airway collagen deposition, compared to sham exposed controls. In conclusion, our study shows that third-hand e-vapour exposure, particularly in the presence of nicotine, negatively affects the lung health of mice and highlights the need for greater public awareness surrounding the dangers of third-hand exposure to e-cigarette vapour.

Keywords: e-cigarettes; e-vaping; fibrosis; lung function; nicotine; remodelling; vaping.

Figure 1

Exposure to third-hand e-vapour with nicotine reduces bronchial responsiveness to provocation, but has limited effects on airway inflammation in mice exposed to 4 weeks of third-hand e-vapour. Lung function in terms of (a) central airway resistance, (b) transpulmonary resistance, (c) tissue damping, and (d) tissue elastance, in response to increasing doses of nebulised methacholine. (e) Total leukocytes, (f) macrophages, (g) neutrophils, and (h) eosinophils in the bronchoalveolar lavage fluid. Data are expressed as the mean ± SEM and analysed by two-way ANOVA (a–d) or one-way ANOVA (e–h) with Tukey’s post hoc tests, n = 7–10/group. * p < 0.05, ** p < 0.01 versus sham at the same concentration of methacholine.

Figure 2

Exposure to third-hand e-vapour, with or without nicotine, has no effect on airway smooth muscle actin. Alpha smooth muscle actin staining and quantification in (a) small (20× magnification, a scale bar = 100 μm) and (b) large (10× magnification, a scale bar = 250 μm) airways of mice exposed to 4 weeks of third-hand e-vapour with (18 mg) or without (0 mg) nicotine. Data are expressed as the mean ± SEM and analysed by one-way ANOVA with Tukey’s post hoc tests, n = 6–9/group.

Figure 3

Exposure to third-hand e-vapour with nicotine induces remodelling of the airway epithelium and emphysema-like alveolar enlargement. Haematoxylin and Eosin staining and quantification in (a) small (20× magnification, a scale bar = 100 μm) and (b) large airways (10× magnification, a scale bar = 250 μm) airways of mice exposed to 4 weeks of third-hand e-vapour with (18 mg) or without (0 mg) nicotine. Representative images of parenchyma (40× magnification, a scale bar = 50 μm) and (c) mean linear intercept quantification Data are expressed as the mean ± SEM and analysed by one-way ANOVA with Tukey’s post hoc tests, n = 7–8/group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Exposure to third-hand e-vapour with nicotine increases small airway collagen deposition. Picrosirius red staining and quantification in (a) small (10× magnification, a scale bar = 250 μm) and (b) large (20× magnification, a scale bar = 100 μm) airways of mice exposed to 4 weeks of third-hand e-vapour with (18 mg) or without (0 mg) nicotine. Second-order harmonics merged representative images (25× magnification) and analyses of lung sections in small airways (c) and large airways (d). Gene expression of (e) collagen 1a1 and (f) collagen 4a1 expressed relative to B-actin. Data are expressed as the mean ± SEM and analysed by one-way ANOVA with Tukey’s post hoc tests, n = 3–8. * p < 0.05.