Formaldehyde and the Transient Receptor Potential Ankyrin-1 Contribute to Electronic Cigarette Aerosol-induced Endothelial Dysfunction in Mice.

Electronic Nicotine Delivery Systems (ENDS) aerosol exposures can induce endothelial dysfunction (ED) in healthy young humans and animals. Thermal degradation of ENDS solvents, propylene glycol and vegetable glycerin (PG: VG), generates abundant formaldehyde (FA) and other carbonyls. Because FA can activate the transient receptor potential ankyrin-1 (TRPA1) sensor, we hypothesized that FA in ENDS aerosols provokes TRPA1-mediated changes that include ED and 'respiratory braking' - biomarkers of harm. To test this, wild-type (WT) and TRPA1-null mice were exposed by inhalation to either filtered air, PG: VG-derived aerosol, or formaldehyde (FA, 5 ppm). Short-term exposures to PG: VG and FA induced ED in female WT but not in female TRPA1-null mice. Moreover, acute exposures to PG: VG and FA stimulated respiratory braking in WT but not in TRPA1-null female mice. Urinary metabolites of FA (ie, N  -1,3-thiazolidine-4-carboxylic acid, TCA; N  -1,3-thiazolidine-4-carbonyl glycine, TCG) and monoamines were measured by LC-MS/MS. PG: VG and FA exposures significantly increased urinary excretion of both TCA and TCG in both WT and TRPA1-null mice. To confirm that inhaled FA directly contributed to urinary TCA, mice were exposed to isotopic 13C-FA gas (1 ppm, 6 h).13C-FA exposure significantly increased the urine level of 13C-TCA in the early collection (0-3 h) supporting a direct relationship between inhaled FA and TCA. Collectively, these data suggest that ENDS use may increase CVD risk dependent on FA, TRPA1, and catecholamines, yet independently of either nicotine or flavorants. This study supports that levels of FA in ENDS-derived aerosols should be lowered to mitigate CVD risk in people who use ENDS.

Effects of electronic cigarette flavorants on human platelet aggregation ex vivo.

Because little is known about the effects of individual flavorants in electronic cigarette (e-cig) fluids on human platelet aggregation, we tested for the direct effects of 15 common e-cig flavorants on adenosine diphosphate (ADP)-induced human platelet aggregation ex vivo. To better understand a potential mechanism of action of flavorants, we quantified 2 phases of aggregation. Human platelet-rich plasma (PRP) was obtained from whole blood of healthy volunteers and used in a platelet aggregometry assay. PRP was incubated with 1 of 15 different flavorant compounds (e.g., benzyl alcohol, eugenol, citronellol, menthol, menthone, diacetyl, maltol, limonene, methylbutyric acid, isoamyl acetate, acetylpyridine, eucalyptol, 2,5-dimethylpyrazine, cinnamaldehyde, and vanillin) at 100 µM for 5 min at 37 °C prior to addition of ADP (10 µM). Subsequent ADP-induced platelet aggregation was tracked for 5 min using an aggregometer. Aggregation curves were analyzed for flavorant-induced effects on total (%) aggregation, Phase 1 and Phase 2 components, and compared with their ADP-only control via One-Way ANOVA. Notably, eugenol significantly inhibited total aggregation; an effect due solely to inhibition of Phase 2. No other flavor tested had any effect on total or phase-specific ADP-induced platelet aggregation. These results indicate that parent flavorant compounds commonly found in e-cig liquids neither activate nor inhibit ADP-induced human platelet aggregation. However, as flavorants are chemically altered during heating of e-cig, thermally-derived products of flavorants (e.g., flavor acetals) also will need to be tested for effects on platelet activation.

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/.

Tobacco Smoke and Endothelial Dysfunction: Role of Aldehydes?

PURPOSE OF REVIEW: Tobacco smoking is the most significant modifiable risk factor in the development of cardiovascular disease (CVD). Exposure to mainstream cigarette smoke (MCS) is associated with CVD through the development of endothelial dysfunction, a condition characterized by an imbalance of vasoactive factors in the vasculature. This dysfunction is thought to be induced in part by aldehydes generated at high levels in MCS. RECENT FINDINGS: Electronic cigarettes (e-cigs) may also pose CVD risk. Although the health effects of e-cigs are still largely unknown, the presence of aldehydes in e-cig aerosol suggests that e-cigs may induce adverse cardiovascular outcomes similar to those seen with MCS exposure. Herein, we review studies of traditional and emerging tobacco product use, shared harmful and potentially harmful constituents, and measures of biomarkers of harm (endothelial dysfunction) to examine a potential and distinct role of aldehydes in cardiovascular harm associated with cigarette and e-cig use.

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.