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Background: Recent studies show e-cigarette (EC) users have increased rates of chronic bronchitic symptoms that may be associated with depressed mucociliary clearance (MCC). Little is known about the acute or chronic effects of EC inhalation on in vivo MCC. Methods: In vivo  = 5 males, mean age = 21) after controlled inhalation of a radiolabeled (Tc99m sulfur colloid) aerosol. Whole-lung clearance of radiolabeled deposited particles was measured over a 90-minute period for baseline MCC and associated with controlled periodic vaping over the first 60 minutes of MCC measurements. The vaping challenge was administered from a fourth generation box mod EC containing unflavored e-liquid (65% propylene glycol/35% vegetable glycerin, 3 mg/mL freebase nicotine). The challenge was administered at the start of each 10-minute interval of MCC measurements and consisted of 1 puff every 30 seconds for 5 minutes (i.e., 10 puffs for each 10-minute period for a total of 60 puffs during the initial 60 minutes of MCC measurements). n  = 5 males, mean age = 21) after controlled inhalation of a radiolabeled (Tc99m sulfur colloid) aerosol. Whole-lung clearance of radiolabeled deposited particles was measured over a 90-minute period for baseline MCC and associated with controlled periodic vaping over the first 60 minutes of MCC measurements. The vaping challenge was administered from a fourth generation box mod EC containing unflavored e-liquid (65% propylene glycol/35% vegetable glycerin, 3 mg/mL freebase nicotine). The challenge was administered at the start of each 10-minute interval of MCC measurements and consisted of 1 puff every 30 seconds for 5 minutes (i.e., 10 puffs for each 10-minute period for a total of 60 puffs during the initial 60 minutes of MCC measurements). Results: Compared with baseline, peripheral lung average clearance (%) over the 90 minutes of MCC measures was enhanced, associated with EC challenge, 12 (±6) versus 24 (±6), respectively ( p  < 0.05 by Wilcoxon signed-rank test). Conclusions: MCC during EC challenge is contrary to recent studies showing nicotine-associated slowing of ciliary beat and mucus transport at higher nicotine levels than those used here. However, our findings are consistent with an acute increase in fluid volume and mucin secretion to the bronchial airway surface that is likely short lived. Research reported in this publication was supported by the National Institutes of Health R01HL139369 and registered with ClinicalTrials.gov (NCT03700892).in vivo MCC during EC challenge is contrary to recent studies showing nicotine-associated slowing of ciliary beat and mucus transport at higher nicotine levels than those used here. However, our findings are consistent with an acute increase in fluid volume and mucin secretion to the bronchial airway surface that is likely short lived. Research reported in this publication was supported by the National Institutes of Health R01HL139369 and registered with ClinicalTrials.gov (NCT03700892).

Nicotine is the primary psychoactive chemical in both traditional and electronic cigarettes (e-cigarettes). Nicotine levels in both traditional cigarettes and e-cigarettes are an important concern for public health. Nicotine exposure due to e-cigarette use is of importance primarily due to the addictive potential of nicotine, but there is also concern for nicotine poisoning in e-cigarette users. Nicotine concentrations in e-liquids vary widely. Additionally, there is significant genetic variability in the rate of metabolism of nicotine due to polymorphisms of CYP2A6, the enzyme responsible for the metabolism of approximately 80% of nicotine. Recent studies have shown CYP2A6 activity is also reduced by aromatic aldehydes such as those added to e-liquids as flavoring agents, which may increase nicotine serum concentrations. However, the impacts of flavored e-liquids on CYP2A6 activity are unknown. In this study, we investigated the impact of three flavored e-liquids on microsomal recombinant CYP2A6. Microsomal recombinant CYP2A6 was challenged at e-liquid concentrations ranging up to 0.125% (v/v) and monitored for metabolic activity using a probe molecule approach. Two e-liquids exhibited dose-dependent inhibition of CYP2A6 activity. Mass spectrometry was conducted to identify flavoring agents in flavored e-liquids that inhibited CYP2A6. Microsomal recombinant CYP2A6 was subsequently exposed to flavoring agents at concentrations ranging from 0.03 μM to 500 μM. Cinnamaldehyde and benzaldehyde were found to be the most potent inhibitors of microsomal CYP2A6 of the flavoring agents tested, with identified IC 50 values of 1.1 μM and 3.0 μM, respectively. These data indicate certain aromatic aldehyde flavoring agents are potent inhibitors of CYP2A6, which may reduce nicotine metabolism in vivo. These findings indicate an urgent need to evaluate the effects of flavoring agents in e-cigarette liquids on the pharmacokinetics of nicotine in vivo.

Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce ciliary beat frequency (CBF), leading to diminished mucociliary clearance (MCC). However, the effects of aldehyde e-cigarette flavorings on CBF are unknown. The purpose of this study was to investigate whether cinnamaldehyde, a flavoring agent commonly used in e-cigarettes, disrupts mitochondrial function and impairs CBF on well-differentiated human bronchial epithelial (hBE) cells. To this end, hBE cells were exposed to diluted cinnamon-flavored e-liquids and vaped aerosol and assessed for changes in CBF. hBE cells were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for effects on CBF. Changes in mitochondrial oxidative phosphorylation and glycolysis were evaluated by Seahorse Extracellular Flux Analyzer, and adenine nucleotide levels were quantified by HPLC. Both cinnamaldehyde-containing e-liquid and vaped aerosol rapidly yet transiently suppressed CBF, and exposure to cinnamaldehyde alone recapitulated this effect. Cinnamaldehyde impaired mitochondrial respiration and glycolysis in a dose-dependent manner, and intracellular ATP levels were significantly but temporarily reduced following exposure. Addition of nicotine had no effect on the cinnamaldehyde-induced suppression of CBF or mitochondrial function. These data indicate that cinnamaldehyde rapidly disrupts mitochondrial function, inhibits bioenergetic processes, and reduces ATP levels, which correlates with impaired CBF. Because normal ciliary motility and MCC are essential respiratory defenses, inhalation of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.