Nose-Only Exposure to Cherry- and Tobacco-Flavored E-Cigarettes Induced Lung Inflammation in Mice in a Sex-Dependent Manner

Abstract

Flavoring chemicals in electronic nicotine delivery systems have been shown to cause cellular inflammation; meanwhile, the effects of fruit and tobacco flavors on lung inflammation by nose-only exposures to mice are relatively unknown. We hypothesized that exposure to flavored e-cigarettes would cause lung inflammation in C57BL/6 J mice. The mice were exposed to air, propylene glycol/vegetable glycerin, and flavored e-liquids: Apple, Cherry, Strawberry, Wintergreen, and Smooth & Mild Tobacco, one hour per day for three days. Quantification of flavoring chemicals by proton nuclear magnetic resonance spectroscopy (¹H NMR), differential cell counts by flow cytometry, pro-inflammatory cytokines/chemokines by ELISA, and matrix metalloproteinase levels by western blot were performed. Exposure to PG/VG increased neutrophil cell count in lung bronchoalveolar lavage fluid (BALF). KC and IL6 levels were increased by PG/VG exposure and female mice exposed to Cherry flavored e-cigarettes, in lung homogenate. Mice exposed to PG/VG, Apple, Cherry, and Wintergreen increased MMP2 levels. Our results revealed flavor- and sex-based e-cigarette effects in female mice exposed to cherry-flavored e-liquids and male mice exposed to tobacco-flavored e-liquids, namely, increased lung inflammation.

Keywords: ENDS; e-cigarettes; flavors; inflammation; lung; mint; tobacco.

Figure 1

Sex-dependent effects of flavored e-cigarette exposure on inflammatory cell count in bronchoalveolar lavage fluid. Mice were exposed to air, PG/VG, and e-liquid flavors “Apple”, “Cherry”, “Strawberry”, “Wintergreen”, and “Smooth & Mild Tobacco” for 3 days for 1 h per day. Mice were sacrificed twenty-four hours after the final exposure. (A) Total cell counts were obtained by staining cells with AO/PI and counting with a cellometer. Differential cells were measured using flow cytometry: (B) F4/80+ macrophages, (C) Ly6 B.2+ neutrophils, (D) CD4+ T-cells, and (E) CD8+ T-cells. Data are shown as mean ± SEM with individual data points represented by the following symbols: Air (black circles), PG/VG (black squares), Apple (black triangles), Cherry (black diamonds), Strawberry (white circles), Wintergreen (white squares), Smooth & Mild Tobacco (white triangles), with * indicating p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. air controls. n = 6 for combined groups and n = 3 for male- and female-only groups.

Figure 2

Sex-dependent effects of flavored e-cigarette exposure on pro-inflammatory cytokines/chemokine release in bronchoalveolar lavage fluid. Mice were exposed to air, PG/VG, and e-liquid flavors “Apple”, “Cherry”, “Strawberry”, “Wintergreen”, and “Smooth & Mild Tobacco” for 3 days for 1 h per day. Mice were sacrificed twenty-four hours after the final exposure. Pro-inflammatory cytokines/chemokines were measured in BALF. (A) KC levels, (B) IL-6 levels, (C) MCP-1 levels. Data are shown as mean ± SEM with individual data points represented by the following symbols: Air (black circles), PG/VG (black squares), Apple (black triangles), Cherry (black diamonds), Strawberry (white circles), Wintergreen (white squares), Smooth & Mild Tobacco (white triangles), with ** p < 0.01 vs. air controls. n = 6 for combined groups and n = 3 for male- and female-only groups.

Figure 3

Sex-dependent effects of acute flavored e-cigarette exposure on pro-inflammatory cytokines/chemokine release in lung homogenate. Mice were exposed to air, PG/VG, and e-liquid flavors “Apple”, “Cherry”, “Strawberry”, “Wintergreen”, and “Smooth & Mild Tobacco” for 3 days for 1 h per day. Mice were sacrificed twenty-four hours after the final exposure. Pro-inflammatory cytokines/chemokines were measured in lung homogenate. (A) KC levels, (B) IL-6 levels, (C) MCP-1 levels. Data are shown as mean ± SEM with individual data points represented by the following symbols: Air (black circles), PG/VG (black squares), Apple (black triangles), Cherry (black diamonds), Strawberry (white circles), Wintergreen (white squares), Smooth & Mild Tobacco (white triangles), with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. air controls. n = 6 for combined groups and n = 3 for male- and female-only groups.

Figure 4

Effects of acute flavored e-cigarette exposure on matrix metalloprotease protein levels in lung homogenate. Mice were exposed to air, PG/VG, and e-liquid flavors “Apple”, “Cherry”, “Strawberry”, “Wintergreen”, and “Smooth & Mild Tobacco” for 3 days for 1 h per day. Mice were sacrificed twenty-four hours after the final exposure. Protein levels for matrix metalloproteinases were measured in lung homogenate using Western blot. (A) MMP2 and MMP9 protein abundance in mouse lung homogenate from male and female exposed mice. (B) Band intensity was measured using densitometry and data are shown as fold change compared to air control mice. Data are shown as mean ± SEM with individual data points represented by the following symbols: Air (black circles), PG/VG (black squares), Apple (black triangles), Cherry (black diamonds), Strawberry (white circles), Wintergreen (white squares), Smooth & Mild Tobacco (white triangles), with * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. air controls. n = 3 for male- and female-only groups.