Tobacco-derived and tobacco-free nicotine cause differential inflammatory cell influx and MMP-9 in mouse lung

Abstract

Background: Electronic nicotine delivery systems (ENDS) or electronic cigarettes (e-cigarettes) aerosolize an e-liquid composed of propylene glycol (PG) and vegetable glycerin (VG) as humectants, flavoring chemicals, and nicotine. Nicotine naturally occurs in two isomers R- and S-nicotine, with tobacco-derived nicotine (TDN) composed of S-nicotine, and tobacco-free/synthetic nicotine (TFN) composed of a racemic mixture of R- and S-nicotine. Currently, there is limited knowledge of the potential differences in the toxicity of TFN versus TDN. We hypothesized that exposure of TFN and TDN salts to C57BL/6J mice would result in a differential response in lung inflammation and protease/ antiprotease imbalance.

Methods: Five-week-old male and female C57BL/6J mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), or PG/VG with TDN salts (TDN) by nose-only exposure. Lung inflammatory cell counts, cytokine/chemokine levels, and matrix metalloproteinase (MMP) protein abundance and activity levels were determined by flow cytometry, ELISA, immunoblotting, and gel zymography, respectively.

Results: Exposure to the humectants (PG/VG) alone increased cytokine levels- IL-6, KC, and MCP-1 in the BALF and KC levels in lung homogenate of exposed mice. While no change was observed in the cytokine levels in lung homogenate of TDN aerosol exposed mice, exposure to TFN aerosols resulted in an increase in KC levels in the lungs of these mice compared to air controls. Interestingly, exposure to TDN aerosols increased MMP-9 protein abundance in the lungs of female mice, while exposure to TFN aerosol showed no change. The metabolism of nicotine or the clearance of cotinine for TFN exposure may differ from that for TDN.

Conclusion: Exposure to humectants, PG/VG alone, induces an inflammatory response in C57BL/6J mice. TFN and TDN salts show distinct changes in inflammatory responses and lung proteases on acute exposures. These data suggest variable toxicological profiles of the two forms of nicotine in vivo. Future work is thus warranted to delineate the harmful effects of synthetic/natural nicotine with humectants to determine the potential toxicological risks for users.

Keywords: E-cigarettes; Flavors; MMPs; Natural nicotine (TDN); Synthetic nicotine (TFN).

Fig. 1

Differential effects of synthetic and tobacco-derived nicotine salts on infiltrating inflammatory cells in BALF. Mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), and PG/VG with TDN salts (TDN) for 5 days for 1 h per day. Mice were sacrificed 2 h after final exposure. A Total cell counts were measured in BALF by staining cells with AO/PI and counted with a cellometer. Flow cytometry was performed to determine the number of B Alveolar Macrophages (CD45⁺Siglec F⁺CD11b⁻), C Neutrophils (CD45⁺Siglec F⁻CD11b⁺Ly6G⁺), and D Eosinophils (CD45⁺CD11b⁺Ly6G⁻CD11c⁻Siglec F⁺) in BALF of control and experimental groups. Data represented as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparison with * p < 0.05, N = 6/group

Fig. 2

Differential effects of synthetic and tobacco-derived nicotine salts on pro-inflammatory cytokine production. Mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), and PG/VG with TDN salts (TDN) for five days for 1 h per day. Mice were sacrificed 2 h after final exposure. Pro-inflammatory cytokines, IL-6, KC, and MCP-1 were measured in (A) BALF and (B) lung homogenate. Data represented as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparison with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, N = 6/group

Fig. 3

Differential effects of synthetic and tobacco-derived nicotine salts on MMPs protein abundance in female mice. Mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), and PG/VG with TDN salts (TDN) for five days for 1 h per day. Mice were sacrificed 2 h after final exposure. The protein abundance of MMP-2, MMP-9, MMP-12, and TIMP-1 was measured in lung homogenate with GAPDH used as a loading control by western blot. A Representative images for MMP-2, MMP-9, MMP-12, TIMP-1, and GAPDH for exposed female mice. B Band intensity was measured by densitometry with relative fold change being measured compared to air exposed female mice. Data represented as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparison with * p < 0.05, N = 3/group. Images of full blots are shown in Additional file 1: Figs. S2A–S7A

Fig. 4

Differential effects of synthetic and tobacco-derived nicotine salts on MMPs protein abundance in male mice. Mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), and PG/VG with TDN salts (TDN) for five days for 1 h per day. Mice were sacrificed 2 h after final exposure. The protein abundance of MMP-2, MMP-9, MMP-12, and TIMP-1 was measured in lung homogenate with GAPDH used as a loading control by western blot. A Representative images for MMP-2, MMP-9, MMP-12, TIMP-1, and GAPDH for exposed male mice. B Band intensity was measured by densitometry with relative fold change being measured compared to air exposed male mice. Data represented as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparison with N = 3/group. Images of full blots are shown in Additional file 1: Figs. S2B–S7B

Fig. 5

Differential effects of synthetic and tobacco-derived nicotine salts on MMP-9 and MMP-2 activity level. Mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), and PG/VG with TDN salts (TDN) for five days for 1 h per day. Mice were sacrificed 2 h after final exposure. The activity of MMP-2 and MMP-9 was measured in lung homogenate by gelatin gel zymography. Representative images for MMP-2 and MMP-9 for exposed (A) female and (B) male mice. Band intensity was measured by densitometry with relative fold change being measured compared to air exposed (C) female and (D) male mice. Data represented as mean ± SEM and analyzed using one-way ANOVA with Tukey’s multiple comparison with * p < 0.05, N = 3/group. Images of full gelatin gels are shown in Additional file 1: Fig. S8