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A7626 - The E-Cigarette Flavoring Cinnamaldehyde Suppresses Mitochondrial Function and Transiently Impairs Cilia Beat Frequency in Human Bronchial Epithelial Cells
Author Block: P. Clapp1, K. Lavrich1, B. Reidel2, C. van Heusden3, M. Kesimer2, E. Lazarowski3, J. Carson4, I. Jaspers4; 1Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 2Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 3Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 4Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
Rationale:
Motile cilia on airway epithelial cells constitute an integral component of the mucociliary clearance (MCC) apparatus, which is essential for clearing inhaled particles and pathogens from the lungs. Ciliary beating is a highly coordinated process dependent upon normal mitochondria function and adequate ATP generation. Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce cilia beat frequency (CBF), leading to diminished MCC. However, the impact of aldehydic e-cigarette flavorings on cilia motility is completely unknown. Cinnamaldehyde, the α,β-unsaturated aldehyde that gives cinnamon its characteristic flavor, is contained in many flavored e-cigarettes and has structural similarities to toxic aldehydes in CS. We hypothesize that, like reactive aldehydes in CS, cinnamaldehyde is capable of disrupting normal mitochondria function and impairing motile cilia on primary human bronchial epithelial cells (HBECs).
Methods:
Well-differentiated HBEC cultures were exposed to diluted cinnamon e-liquids and e-liquid aerosol generated by a 3rd generation e-cigarette device. CBF was quantified over 120 minutes using a high speed digital camera and the Sisson Ammons Video Analysis (SAVA) system. The cinnamaldehyde content of each e-liquid was determined by gas chromatography-mass spectrometry (GC-MS) and HBECs were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for alterations of CBF. Changes in mitochondria oxidative phosphorylation were evaluated using a Seahorse Bioanalyzer and HBEC adenine nucleotide content (e.g. ATP, ADP, and AMP) was quantified by high-performance liquid chromatography (HPLC). Beas-2B cell cultures were exposed to cinnamaldehyde and cell lysates were screened for adducted proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomic analysis.
Results:
Both cinnamaldehyde-containing e-liquid and e-liquid aerosol rapidly suppressed CBF, which persisted for approximately 60 minutes. Quantitative GC-MS of the e-liquid revealed a cinnamaldehyde concentration of 1.131M±0.0199M and subsequent exposure to cinnamaldehyde alone recapitulated the transient ciliostasis observed with e-liquid exposure. Cinnamaldehyde rapidly impaired mitochondrial oxidative phosphorylation in a dose-dependent manner and intracellular ATP levels were significantly reduced following exposure. Proteomic analysis of cells exposed to cinnamaldehyde indicated cinnamaldehyde adducts with redox and energy metabolism proteins, including peroxiredoxin-6 and ATP synthase.
Conclusions:
Taken together, these data suggest that cinnamaldehyde, a ubiquitous flavoring agent commonly used in e-cigarettes, adducts to mitochondrial proteins, disrupts mitochondrial function, and significantly reduces intracellular ATP levels, which correlates with impaired CBF in airway epithelial cells. Considering that normal cilia motility and MCC are essential innate defenses in the lung, inhalational exposures of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.