Flavorings in Tobacco Products Induce Endothelial Cell Dysfunction

Abstract

Objective: Use of alternative tobacco products including electronic cigarettes is rapidly rising. The wide variety of flavored tobacco products available is of great appeal to smokers and youth. The flavorings added to tobacco products have been deemed safe for ingestion, but the cardiovascular health effects are unknown. The purpose of this study was to examine the effect of 9 flavors on vascular endothelial cell function.

Approach and results: Freshly isolated endothelial cells from participants who use nonmenthol- or menthol-flavored tobacco cigarettes showed impaired A23187-stimulated nitric oxide production compared with endothelial cells from nonsmoking participants. Treatment of endothelial cells isolated from nonsmoking participants with either menthol (0.01 mmol/L) or eugenol (0.01 mmol/L) decreased A23187-stimulated nitric oxide production. To further evaluate the effects of flavoring compounds on endothelial cell phenotype, commercially available human aortic endothelial cells were incubated with vanillin, menthol, cinnamaldehyde, eugenol, dimethylpyrazine, diacetyl, isoamyl acetate, eucalyptol, and acetylpyrazine (0.1-100 mmol/L) for 90 minutes. Cell death, reactive oxygen species production, expression of the proinflammatory marker IL-6 (interleukin-6), and nitric oxide production were measured. Cell death and reactive oxygen species production were induced only at high concentrations unlikely to be achieved in vivo. Lower concentrations of selected flavors (vanillin, menthol, cinnamaldehyde, eugenol, and acetylpyridine) induced both inflammation and impaired A23187-stimulated nitric oxide production consistent with endothelial dysfunction.

Conclusions: Our data suggest that short-term exposure of endothelial cells to flavoring compounds used in tobacco products have adverse effects on endothelial cell phenotype that may have relevance to cardiovascular toxicity.

Keywords: endothelial cells; eugenol; inflammation; nitric oxide; tobacco.

Figure 1.

Menthol and eugenol impair nitric oxide production in freshly isolated endothelial cells from human participants. Endothelial cells from menthol (n=6) and nonmenthol cigarette smokers (n=6) had a lower change in nitric oxide measured by 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence in response to A23187 stimulation compared with endothelial cells from nonsmokers (n=9, ‡P<0.01, †P<0.05; A). Treatment of endothelial cells freshly isolated from healthy participants with 0.01 mmol/L menthol (B) or eugenol (C) decreased DAF-2DA fluorescence in response to A23187 stimulation (n=5, ‡P<0.01 for menthol; n=5, ‡P<0.01 for eugenol). Data are expressed as mean±SEM.

Figure 2.

Tobacco flavoring compounds induce cell death. The percentage of cells staining positive for DNA strand breaks (TUNEL positive [terminal deoxynucleotidyl transferase dUTP nick-end labeling]) after a 90 min incubation with varying concentrations of flavor compounds were detected using immunofluorescence (n=3–4, *P<0.001, ‡P<0.01, †P<0.05 compared with vehicle control). Data are expressed as mean±SEM.

Figure 3.

Tobacco flavoring compounds increase oxidative stress. Oxidative stress was measured by quantifying the fluorescent dye dihydroethidium after exposure of human aortic endothelial cells to flavoring compounds at varying concentrations (n=3, *P<0.001). Antimycin A (AA), a stimulus for mitochondrial oxidant generation, served as a positive control. Data are expressed as mean±SEM.

Figure 4.

Tobacco flavoring compounds increase endothelial cell inflammation. IL-6 (interleukin-6) expression was quantified using reverse transcription-quantitative polymerase chain reaction 3 h after initial flavoring exposure in human aortic endothelial cells. All data are expressed as the relative quantification compared with vehicle alone treated cells (n=3–6, ‡P<0.01, †P<0.05). Data are expressed as mean±SEM.

Figure 5.

Tobacco flavoring compounds impair nitric oxide production in human aortic endothelial cells (HAECs). Nitric oxide production (4,5-diaminofluorescein diacetate [DAF-2DA] fluorescence) in response to A23187 stimulation was decreased in HAECs treated with flavoring compounds (n=3, *P<0.001, ‡P<0.01, †P<0.05 compared with untreated control). Data are presented as percent change in DAF-2DA fluorescence in response to A23187 stimulation. Data are expressed as mean±SEM.

Figure 6.

Differential effects of aerosolizing tobacco flavoring compounds on A23187-stimulated nitric oxide production in human aortic endothelial cells (HAECs). Treatment of HAECs with vanillin aerosolized at 200°C impaired A23187-stimulated nitric oxide production (4,5-diaminofluorescein diacetate [DAF-2DA] fluorescence), but this impairment was not observed with vanillin aerosolized at 700°C (n=3, †P<0.05 compared with untreated vehicle control, A). HAECs treated with menthol aerosolized at 200°C or 700°C had no effect on A23187-stimulated nitric oxide production (n=3, B). Treatment of HAECs with eugenol aerosolized at 200°C and 700°C impaired nitric oxide production in response to A23187 stimulation (n=3, *P<0.001, ‡P<0.01, compared with vehicle control, C). Data are presented as percent change in DAF-2DA fluorescence in response to A23187 stimulation. Data are expressed as mean±SEM.