E-cigarettes and their lone constituents induce cardiac arrhythmia and conduction defects in mice.

E-cigarette use has surged, but the long-term health effects remain unknown. E-cigarette aerosols containing nicotine and acrolein, a combustion and e-cigarette byproduct, may impair cardiac electrophysiology through autonomic imbalance. Here we show in mouse electrocardiograms that acute inhalation of e-cigarette aerosols disturbs cardiac conduction, in part through parasympathetic modulation. We demonstrate that, similar to acrolein or combustible cigarette smoke, aerosols from e-cigarette solvents (vegetable glycerin and propylene glycol) induce bradycardia, bradyarrhythmias, and elevations in heart rate variability during inhalation exposure, with inverse post-exposure effects. These effects are slighter with tobacco- or menthol-flavored aerosols containing nicotine, and in female mice. Yet, menthol-flavored and PG aerosols also increase ventricular arrhythmias and augment early ventricular repolarization (J amplitude), while menthol uniquely alters atrial and atrioventricular conduction. Exposure to e-cigarette aerosols from vegetable glycerin and its byproduct, acrolein, diminish heart rate and early repolarization. The pro-arrhythmic effects of solvent aerosols on ventricular repolarization and heart rate variability depend partly on parasympathetic modulation, whereas ventricular arrhythmias positively associate with early repolarization dependent on the presence of nicotine. Our study indicates that chemical constituents of e-cigarettes could contribute to cardiac risk by provoking pro-arrhythmic changes and stimulating autonomic reflexes.

Electronic Cigarette Solvents, JUUL E-Liquids, and Biomarkers of Exposure: In Vivo Evidence for Acrolein and Glycidol in E-Cig-Derived Aerosols.

Despite the increasing popularity of e-cigarettes, their long-term health effects remain unknown. In animal models, exposure to e-cigarette has been reported to result in pulmonary and cardiovascular injury, and in humans, the acute use of e-cigarettes increases heart rate and blood pressure and induces endothelial dysfunction. In both animal models and humans, cardiovascular dysfunction associated with e-cigarettes has been linked to reactive aldehydes such as formaldehyde and acrolein generated in e-cigarette aerosols. These aldehydes are known products of heating and degradation of vegetable glycerin (VG) present in e-liquids. Here, we report that in mice, acute exposure to a mixture of propylene glycol:vegetable glycerin (PG:VG) or to e-cigarette-derived aerosols significantly increased the urinary excretion of acrolein and glycidol metabolites─3-hydroxypropylmercapturic acid (3HPMA) and 2,3-dihydroxypropylmercapturic acid (23HPMA)─as measured by UPLC-MS/MS. In humans, the use of e-cigarettes led to an increase in the urinary levels of 23HPMA but not 3HPMA. Acute exposure of mice to aerosols derived from PG:13C3-VG significantly increased the 13C3 enrichment of both urinary metabolites 13C3-3HPMA and 13C3-23HPMA. Our stable isotope tracing experiments provide further evidence that thermal decomposition of vegetable glycerin in the e-cigarette solvent leads to generation of acrolein and glycidol. This suggests that the adverse health effects of e-cigarettes may be attributable in part to these reactive compounds formed through the process of aerosolizing nicotine. Our findings also support the notion that 23HPMA, but not 3HPMA, may be a relatively specific biomarker of e-cigarette use.

Electronic cigarette solvents, pulmonary irritation, and endothelial dysfunction: role of acetaldehyde and formaldehyde.

After more than a decade of electronic cigarette (E-cig) use in the United States, uncertainty persists regarding E-cig use and long-term cardiopulmonary disease risk. As all E-cigs use propylene glycol and vegetable glycerin (PG-VG) and generate abundant saturated aldehydes, mice were exposed by inhalation to PG-VG-derived aerosol, formaldehyde (FA), acetaldehyde (AA), or filtered air. Biomarkers of exposure and cardiopulmonary injury were monitored by mass spectrometry (urine metabolites), radiotelemetry (respiratory reflexes), isometric myography (aorta), and flow cytometry (blood markers). Acute PG-VG exposure significantly affected multiple biomarkers including pulmonary reflex (decreased respiratory rate, -50%), endothelium-dependent relaxation (-61.8 ± 4.2%), decreased WBC (-47 ± 7%), and, increased RBC (+6 ± 1%) and hemoglobin (+4 ± 1%) versus air control group. Notably, FA exposure recapitulated the prominent effects of PG-VG aerosol on pulmonary irritant reflex and endothelial dysfunction, whereas AA exposure did not. To attempt to link PG-VG exposure with FA or AA exposure, urinary formate and acetate levels were measured by GC-MS. Although neither FA nor AA exposure altered excretion of their primary metabolite, formate or acetate, respectively, compared with air-exposed controls, PG-VG aerosol exposure significantly increased post-exposure urinary acetate but not formate. These data suggest that E-cig use may increase cardiopulmonary disease risk independent of the presence of nicotine and/or flavorings. This study indicates that FA levels in tobacco product-derived aerosols should be regulated to levels that do not induce biomarkers of cardiopulmonary harm. There remains a need for reliable biomarkers of exposure to inhaled FA and AA.NEW & NOTEWORTHY Use of electronic cigarettes (E-cig) induces endothelial dysfunction (ED) in healthy humans, yet the specific constituents in E-cig aerosols that contribute to ED are unknown. Our study implicates formaldehyde that is formed in heating of E-cig solvents (propylene glycol, PG; vegetable glycerin, VG). Exposure to formaldehyde or PG-VG-derived aerosol alone stimulated ED in female mice. As ED was independent of nicotine and flavorants, these data reflect a "universal flaw" of E-cigs that use PG-VG.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/e-cigarettes-aldehydes-and-endothelial-dysfunction/.

Acute and chronic vascular effects of inhaled crotonaldehyde in mice: Role of TRPA1.

Although crotonaldehyde (CR) is an abundant α,ß-unsaturated aldehyde in mainstream cigarette smoke (MCS), the cardiovascular toxicity of inhaled CR is largely unexplored. Thus, male C57BL/6 J mice were exposed acutely (1 h, 6 h, and 4d) and chronically (12 weeks) to CR (at levels relevant to MCS; 1 and 3 ppm), and cardiovascular and systemic outcomes were measured in vivo and in vitro. Diastolic blood pressure was decreased (hypotension) by both acute and chronic CR exposure. Vascular toxicity of inhaled CR was quantified in isolated aorta in response to agonists of contraction (phenylephrine, PE) and relaxation (acetylcholine, ACh; sodium nitroprusside, SNP). Although no change in contractility was observed, ACh-induced relaxations were augmented after both acute and chronic CR exposures whereas SNP-induced relaxation was enhanced only following 3 ppm CR exposure. Because CR is a known agonist of the transient receptor potential ankyrin 1 (TRPA1) channel, male TRPA1-null mice were exposed to air or CR (4d, 1 ppm) and aortic function assessed in vitro. CR exposure had no effect on TRPA1-null aortic function indicating a role of TRPA1 in CR effects in C57BL/6 J mice. Notably, CR exposure (4d, 1 ppm) had no effect on aortic function in female C57BL/6 J mice. This study shows that CR inhalation exposure induces real-time and persistent vascular changes that promote hypotension-a known risk factor for stroke. Because of continued widespread exposures of humans to combustion-derived CR (environmental and tobacco products), CR may be an important cardiovascular disease risk factor.

Electronic cigarette-generated aldehydes: The contribution of e-liquid components to their formation and the use of urinary aldehyde metabolites as biomarkers of exposure.

Electronic cigarettes (e-cigarette) have emerged as a popular electronic nicotine delivery system (ENDS) in the last decade. Despite the absence of combustion products and toxins such as carbon monoxide (CO) and tobacco-specific nitrosamines (TSNA), carbonyls including short-chain, toxic aldehydes have been detected in e-cigarette-derived aerosols up to levels found in tobacco smoke. Given the health concerns regarding exposures to toxic aldehydes, understanding both aldehyde generation in e-cigarette and e-cigarette exposure is critical. Thus, we measured aldehydes generated in aerosols derived from propylene glycol (PG):vegetable glycerin (VG) mixtures and from commercial e-liquids with flavorants using a state-of-the-art carbonyl trap and mass spectrometry. To track e-cigarette exposure in mice, we measured urinary metabolites of 4 aldehydes using ULPC-MS/MS or GC-MS. Aldehyde levels, regardless of abundance (saturated: formaldehyde, acetaldehyde >> unsaturated: acrolein, crotonaldehyde), were dependent on the PG:VG ratio and the presence of flavorants. The metabolites of 3 aldehydes - formate, acetate and 3-hydroxypropyl mercapturic acid (3-HPMA; acrolein metabolite) -- were increased in urine after e-cigarette aerosol and mainstream cigarette smoke (MCS) exposures, but the crotonaldehyde metabolite (3-hydroxy-1-methylpropylmercapturic acid, HPMMA) was increased only after MCS exposure. Interestingly, exposure to menthol-flavored e-cigarette aerosol increased the levels of urinary 3-HPMA and sum of nicotine exposure (nicotine, cotinine, trans-3'-hydroxycotinine) relative to exposure to a Classic Tobacco-flavored e-cigarette aerosol. Comparing these findings with aerosols of other ENDS and by measuring aldehyde-derived metabolites in human urine following exposure to e-cigarette aerosols will further our understanding of the relationship between ENDS use, aldehyde exposure and health risk.

A novel method of nicotine quantification in electronic cigarette liquids and aerosols.

The electronic cigarette (e-cigarette) has emerged as popular electronic nicotine delivery devices (ENDs). However, the general safety and validity of e-cigarettes for nicotine delivery efficacy are still not well understood. This study developed a new method for efficient measurement of nicotine levels in both the liquids (e-liquids) used in e-cigarettes and the aerosols generated from the e-cigarettes. Protonation of the pyrrolidine nitrogen of nicotine molecules by addition of excess hydrochloric acid affords an aminium salt that is readily quantified by Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). The kinetics of nicotine protonation was studied using 1H NMR spectroscopy. Quantitative analyses of nicotine in commercial e-liquids and in the corresponding derived e-cigarette aerosols were carried out using direct infusion FT-ICR-MS. The 1H NMR study of nicotine protonation revealed a first order reaction and an activation energy of 30.05 kJ mol-1. The nicotine levels measured in the commercial e-liquids were within a wide and highly variable range of -2.94% to +25.20% around the manufacturer's stated values. The results indicated considerable differences between the measured levels and the advertised levels of nicotine in the e-liquids. The nicotine quantity measured in aerosols increased linearly both with nicotine level in e-liquids (same number of puffs) and with number of puffs (same e-liquids). These data show that quality control of e-liquids and use characteristics are major variables in efficacy of nicotine delivery.

Ozone inhalation modifies the rat liver proteome.

Ozone (O3) is a serious public health concern. Recent findings indicate that the damaging health effects of O3 extend to multiple systemic organ systems. Herein, we hypothesize that O3 inhalation will cause downstream alterations to the liver. To test this, male Sprague-Dawley rats were exposed to 0.5ppm O3 for 8h/day for 5 days. Plasma liver enzyme measurements showed that 5 day O3 exposure did not cause liver cell death. Proteomic and mass spectrometry analysis identified 10 proteins in the liver that were significantly altered in abundance following short-term O3 exposure and these included several stress responsive proteins. Glucose-regulated protein 78 and protein disulfide isomerase increased, whereas glutathione S-transferase M1 was significantly decreased by O3 inhalation. In contrast, no significant changes were detected for the stress response protein heme oxygenase-1 or cytochrome P450 2E1 and 2B in liver of O3 exposed rats compared to controls. In summary, these results show that an environmentally-relevant exposure to inhaled O3 can alter the expression of select proteins in the liver. We propose that O3 inhalation may represent an important unrecognized factor that can modulate hepatic metabolic functions.