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

Abstract

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/.

INTRODUCTION

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide (1, 2). In 2016, more than one-fourth of the almost 57 million global deaths were attributed to CVD deaths, and ischemic heart disease and stroke have been classified as the two leading causes of death for the last 20 years (2). Although a number of physical, behavioral, and socioeconomic factors have been linked to CVD (3), tobacco smoke has been characterized as the single most significant modifiable risk factor in CVD development (4, 5). Extensive evidence demonstrates that exposures to both mainstream (MCS) (6, 7) and secondhand (SHS) (6, 8) cigarette smoke increase the risk for CVD. Smoking increases the risk of heart attack, coronary artery disease, atherosclerosis, and stroke (9). Smoking is also linked with endothelium dysfunction (9), sine qua non in atherosclerosis. However, the constituents in tobacco smoke that promote CVD and the mechanisms by which these constituents enhance CVD risk remain enigmatic.

After more than a decade of electronic cigarette (E-cig) use in the United States, uncertainty persists regarding E-cig use and long-term CVD risk. Exposure to MCS or SHS significantly increases exposure to formaldehyde (FA) and acetaldehyde (AA) as these are major constituents (10, 11), and the Institute of Medicine ranks FA and AA as two of the most significant toxins in MCS, particularly in relationship to non-cancer disease risk, i.e., CVD (12). Similarly, E-cigs generate a variety of abundant aldehydes including saturated aldehydes (and to a much lesser quantity unsaturated aldehydes) dependent on propylene glycol (PG) and vegetable glycerin (VG) ratio (PG-VG), power of the device and user topography as key factors (13–17). Regardless of differences in E-cig device attributes, two saturated aldehydes, i.e., FA and AA, are generated abundantly (15, 16). Thus, we hypothesized that these aldehydes likely are potential mediators of PG-VG–induced harm.

FA and AA also are ubiquitous in a variety of food and drinks, and anthropogenic sources account for the majority of human exposures (18–20). FA is in consumer products, including carpets, manufactured wood products, paints, and preservatives, and both FA and AA are formed as products of combustion from industry, vehicle exhaust, and fires (18, 19, 21, 22). The negative cardiovascular effects of FA and AA exposure have been documented. FA causes hematotoxicity, decreasing both red blood cell (RBC) and white blood cell (WBC) and platelet counts (23–26) as well as the levels of myeloid progenitor cells (23, 26). FA exposure also decreases blood pressure via inhalation in rats (27) and ingestion in humans (28), consistent with FA-induced concentration-dependent vascular relaxation in vitro (29, 30). Occupational exposure to FA is also associated with increased incidence of heart disease (31–33), and a brief (90 min) exposure of young, healthy women to formalin led to acute onset of conduit blood vessel endothelial dysfunction (34). The cardiovascular effects of AA have largely been studied in relationship to aldehyde dehydrogenase (ALDH2), the enzyme responsible for AA metabolism, and specifically ALDH2 mutation that prevents AA metabolism, causing blood levels of AA to increase after ethanol consumption (35). Increased blood levels of AA are linked with AA-hemoglobin adducts (36, 37) and CVD risk (38–40).

Despite the significant exposures to FA and AA from MCS and E-cig–derived aerosols as well as other sources, few studies have addressed the effects of direct inhalation exposures to either FA or AA alone on cardiovascular biomarkers of harm such as endothelial dysfunction. To address this gap in knowledge regarding the potential cardiovascular toxicity of E-cig aerosols and two abundant saturated aldehydes (FA, AA), healthy adult male and female C57BL/6J mice were exposed acutely by inhalation to PG-VG, FA, or AA, and biomarkers of harm and exposure were measured.

MATERIALS AND METHODS

Materials

Reagent-grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Mice and Exposures

Mice.

Male and female C57BL/6J (wild-type, WT) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were treated according to the “Guiding Principles for the Care and Use of Animals in Research and Teaching” as adopted by the American Physiological Society, and all protocols were approved by the University of Louisville Institutional Animal Care and Use Committee. Before and during exposures, mice were housed under pathogen-free conditions, controlled temperatures, and a 12:12 h light-dark cycle. Mice were maintained on a standard chow diet (Rodent Diet 5010, 4.5% fat by weight, LabDiet, St. Louis, MO).

E-cigarette aerosol exposures.

A software-controlled (FlexiWare) cigarette-smoking robot (SCI-REQ, Montreal, Canada) system was used in the mechanical generation of aerosols from PG-VG mixtures. To control the generation of volatile organic compounds (VOCs) in E-cig aerosols, we used a defined E-cig platform. PG-VG mixture (50:50 or 30:70 ratio, vol/vol) was loaded into a refillable, clear tank atomizer with a fixed coil resistance (Mistic Bridge; ∼3.0 ohm; purchased online) coupled with a rechargeable bluPLUS+ (3.7 V) battery (power output of ∼8 W) (41). The atomizer tank was weighed before and after use to quantify solution consumption (g/puff). A 9-min session was composed of 18 puffs (4 s/puff, 91.1 mL/puff, 2 puffs/min). For respiratory parameters, each exposure had 3 PG-VG sessions evenly spaced over 1 h. For other exposures, 20 sessions were evenly spaced over a 6-h exposure per day for 4 consecutive days. Total suspended particulate (TSP) matter was monitored in real time with an inline infrared Microdust Pro 880 nm (Casella) positioned upstream of the exposure chamber (5 L, SCI-REQ). All whole body exposures were done between 7:00 AM and 2:00 PM in the absence of food or water.

Formaldehyde and acetaldehyde exposures.

For FA and AA exposures, naïve mice were exposed to either HEPA- and charcoal-filtered air, FA, or AA for either two 9-min sessions in 1 h (respiratory parameters) or for 4 consecutive days (5 ppm, 6 h/day) using an exposure system equipped with a certified permeation tube in a calibrated heating oven (Kin-Tek, LaMarque, TX) as described (42). Levels of AA were monitored real time with an inline photoionization detector (isobutylene calibration), whereas levels of FA were monitored real time with an inline electrochemical sensor (CO calibration) (MultiRAE Pro, RAE Systems, Burlington, VT). Both gases were monitored upstream of the exposure chamber (30 liter, flow 7 lpm). All whole body exposures were done between 7:00 AM and 2:00 PM in the absence of food or water.

Urine Collection and Metabolism

Urine collection.

Prior to exposures, mice were held and a small drop of d-glucose-saccharin solution (wt/wt 3.0%/0.125%; Sigma-Aldrich; St. Louis, MO) was touched to their lips and mouth. After 6 h of air and toxic exposures, mice were placed singly per metabolic cage (Harvard Apparatus, Cambridge, MA) without food but with glucose-saccharin solution in drinking water for urine collection (in graduated cylinders surrounded by 4°C water-jacketed organ baths). Urine was collected from 0 to 3 h post exposure, and in a second overnight collection (3–16 + h, overnight) during which mice were provided glucose-saccharin solution as well as food (43). Urine samples were centrifuged (1,800 g, 5 min; to pellet feces or food) before being decanted and stored at −80°C.

Urine metabolite analysis.

Urinary levels of formate and acetate, the primary metabolites of FA and AA, respectively, were measured by gas chromatography-mass spectrometry (GC-MS) as adapted and modified from previous reports (44, 45). Urine (20 µL) was mixed with sodium phosphate (20 µL; 0.5 M, pH 8.0) containing [¹³C]formate (2.3 mM) and [¹³C]acetate (0.23 mM) internal standards, and pentafluorobenzyl bromide (130 µL, 0.1 M). The mixture was vortexed for 1 min and then incubated at 60°C for 15 min, and the resulting reaction products were extracted using hexane (300 µL) before being transferred to glass tubes for GC-MS analysis. Analytes in urine samples were quantified using the peak area ratio based on seven-point standard curves that were run before and after the urine samples. MassHunter software (Agilent) was used for peak integration, calibration, and quantification. Measured formate and acetate sample concentrations were corrected for the natural abundance of ¹³C-isotopes, and normalized to urinary creatinine (41). Additionally, we estimated the total excreted formate and acetate by multiplying measured concentrations (ng/mL) and total volume (mL) of urine collected at each time point (0–3 h, overnight post exposure).

Systemic Outcomes

Pulmonary (irritant) reflexes.

To measure pulmonary irritant reflexes real time during exposures (i.e., respiratory rate, inspiratory and expiratory time, and amplitude), male mice were implanted with a pressure cannula tunneled into the esophageal serosa via the diaphragm for radiotelemetry recordings (PA-C10; DSI, St. Paul, MN). Mice recovered for 1-wk post surgery before any exposure. Signals were continuously acquired before (baseline), during, and after exposures (1 kHz). Respiratory rate (breaths per minute, bpm), amplitude (mmHg), and expiratory and inspiratory times (s) were calculated from pressure signal waveform (DSI) (46).

Euthanization.

Immediately following the final exposure, mice were euthanized with sodium pentobarbital (≈150 mg/kg ip), ventral thoracotomy, and exsanguination via right ventricle cardiac puncture for blood collection in EDTA-coated (0.2 M) syringes. Organs were removed, weighed, and snap frozen in liquid N₂ and kept at −80°C until further analysis.

Complete blood counts.

Complete blood counts (CBC) were measured (20 µL whole blood) with a hematology analyzer calibrated with multispecies hematological reference controls (Hemavet 950FS; Drew Scientific, Inc., Miami Lakes, FL) as described (43).

Plasma biomarkers.

Plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, albumin, total protein, alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), and creatinine were measured on a Cobas Mira Plus Clinical Chemistry Autoanalyzer (Roche Diagnostics, Indianapolis, IN) as previously described (43).

Flow Cytometry

Circulating angiogenic cells (CACs).

Blood CACs were identified by flow cytometry and quantified as events double positive for Flk-1 (endothelial marker; Vegfr2 homolog) and Sca-1 (hematopoietic stem cell marker) as previously described (47).

Platelet-leukocyte aggregates (PLAs).

PLAs were identified by flow cytometry and quantified as events double positive for CD41 (platelets) and CD45 (leukocytes) as previously described (43) with slight modifications. Briefly, aliquots of whole blood were diluted (1:4) with HEPES-Tyrode solution before fixation [paraformaldehyde, Fc 1.6%, room temperature (RT)]. Red blood cells were lysed (MilliQ water), and the sample was centrifuged (400 g, 5 min, RT). The sample pellet was incubated with 1% Fc Block (5 µL; 10 min) before staining for 30 min with FITC-labeled anti-CD41 and APC-labeled anti-CD45 or isotype-matched negative controls (FITC-IgG1; APC-IgG2bκ). Stained cells were washed with HEPES-Tyrode solution containing 1% BSA, centrifuged at 400 g for 5 min, and resuspended in HEPES-Tyrode solution (250 µL). A BD LSR Flow Cytometer (BD Biosciences, San Jose, CA) was used to analyze stained cells; a minimum of 20,000 events was collected for each sample.

Endothelium Dysfunction

Immediately after the final exposure (4th day), male and female mice were euthanized and the thoracic aorta was isolated and placed into cold (4°C) physiological salt solution (PSS). Thoracic aorta rings (3–4 mm) were carefully cleaned and prepared for isometric myography as described (42).

Physiological salt solutions (PSS).

PSS for aorta was (in mM) NaCl, 118; KCl, 4.7; CaCl₂, 2.5; KH₂PO₄, 1.2; MgSO₄, 1.2; NaHCO₃, 25; and, glucose, 5.5; pH 7.4. High K⁺ PSS (100 mM) was substituted with equimolar K⁺ for Na⁺ (29).

Aorta reactivity.

Organ baths contained PSS bubbled with 95% O₂-5% CO₂ at 37°C. Briefly, after 10 min without tension, aortic segments were equilibrated to ≈1 g of loading tension over 1 h. All segments were stimulated with high K⁺ to test for viability, washed three times with PSS over 30 min, and re-equilibrated to 1 g of resting tension. The segments were then stimulated again with high K⁺, followed by three bath changes and a re-equilibration to resting tension. To test whether exposures altered vascular reactivity, the following responses were measured: 1) contractions induced by high K⁺; 2) concentration-dependent contractions of phenylephrine (PE; 0.1 nM to 10 µM); 3) concentration-dependent relaxations of acetylcholine (ACh; 0.1 nM to 10 µM) in PE-precontracted segments; and 4) concentration-dependent relaxations of sodium nitroprusside [SNP; 0.01 nM to 10 µM; nitric oxide (NO) donor] in PE-precontracted segments after addition of N^G-nitro-l-arginine methyl ester (l-NAME) (100 µM) and addition of ACh (10 µM). Measures of efficacy (E_max: contractions normalized to aortic length and percentage relaxation of PE contraction) (48) and sensitivity (EC₅₀, effective concentration producing 50% response, i.e., cumulative concentration responses normalized to 100% with interpolation of EC₅₀) were calculated (49). To assess specific alteration in endothelial nitric oxide synthase (eNOS) function, the effect of l-NAME on PE-induced tension was calculated as a “PE contraction ratio”: PE tension_post-L-NAME/PE tension_pre-L-NAME.

Statistics

Data are presented as means ± SE. For two group comparisons, rank sum tests with Bonferroni’s post test or paired (or one-way repeated measures ANOVA) or unpaired t tests as appropriate were applied. For multiple group comparisons, one-way ANOVA with Bonferroni’s post test or when variation indicated Kruskal-Wallis ANOVA on ranks with Dunn’s post test were used (SigmaPlot, version 12.5; Systat Software, Inc., San Jose, CA). Statistical significance was set at P < 0.05.

RESULTS

Pulmonary Irritant Responses

Because PG-VG aerosols contain high levels of reactive aldehydes that can stimulate pulmonary irritant reflexes (e.g., respiratory braking, cough), we tested whether PG-VG aerosol was irritating to mice. We used radiotelemetry to monitor real-time changes in respiratory parameters before, during, and after exposures as previously performed during acrolein exposures in mice (46).

Propylene glycol-vegetable glycerin.

Within the first puff of PG-VG, respiratory parameters were significantly altered and remained altered until the end of the 9-min vaping session (18 puffs) after which parameters returned toward baseline (Fig. 1). Moreover, similar irritant responses (temporally, directionally, and magnitude) were observed in each successive vaping session. The ∼50% decrease in respiratory rate (Fig. 1A) with reciprocal increases in both expiratory (strongly) (Fig. 1B) and inspiratory (minimally) (Fig. 1C) times were hallmark changes of an “irritant” (or nocifensive) response. Amplitude (a measure of thoracic effort) also was lessened slightly by PG-VG aerosol perhaps reflecting shallower as well as slower breathing (Fig. 1D).

Figure 1.

Effects of PG-VG–derived aerosol on pulmonary function in mice. Male C57BL/6J mice instrumented with pressure transmitters to detect changes in pleural cavity pressure were exposed to filtered air or three 9-min sessions (18 puffs/session) of PG-VG–derived aerosol. Pressure waveforms were analyzed for respiratory rate (breaths per min, bpm; A), expiratory (B), and inspiratory (C) times (s), and, peak amplitude (mmHg, a measure of respiratory effort; D). Onset of exposure to PG-VG–derived aerosol provoked rapid and robust changes in respiratory rate (A), expiratory (B), and inspiratory (C) times, and, more modest changes in peak amplitude (D). Values are 1-min means ± SE (n = 4 mice per group) (*P < 0.05) vs. air control; 0.10 > #P > 0.05 vs. air control. Magenta line represents real-time total suspended particulate matter (TSP; g/m3) measured upstream of whole body exposure chamber (5-l) using a Microdust Pro 880 nm. ○, air; ●, PG-VG. PG, propylene glycol. VG, vegetable glycine.

Formaldehyde.

To test if the PG-VG–provoked response was potentially due to FA, an abundant aldehyde generated from PG-VG, mice were exposed to FA (5 ppm). Although initial exposure to 5 ppm FA had minimal effect on respiratory parameters, there was a rapid noticeable alteration in respiratory parameters during a second FA challenge when FA levels briefly increased to almost 10 ppm (Fig. 2). These changes included both robust depression of respiratory rate (Fig. 2A) and increased expiratory time (Fig. 2B) with less effect on inspiratory time (Fig. 2C) and amplitude (Fig. 2D). Thus, FA at levels between 5 and 10 ppm stimulated an irritant response that mimicked, in part, the robust response to PG-VG aerosol.

Figure 2.

Effects of formaldehyde (FA) on pulmonary function in mice. Male C57BL/6J mice instrumented with pressure transmitters to detect changes in pleural cavity pressure were exposed to filtered air or two 9-min sessions of FA (5-10 ppm). Pressure waveforms were analyzed for respiratory rate (breaths per min, bpm; A), expiratory (B), and inspiratory (C) times (s), and, peak amplitude (mmHg, a measure of respiratory effort; D). Onset of exposure to FA had modest effect at 5 ppm but when >5 ppm provoked rapid and robust changes in respiratory rate (A) and expiratory time (B). Less noticeable changes occurred in inspiratory (C) time and peak amplitude (D). Values are 1-min means ± SE (n = 2 mice per group); 0.10 > #P > 0.05 vs. air control. Blue line represents real time monitoring of FA level (ppm) measured upstream of whole body exposure chamber (30-L) with an inline MultiRAE Pro. ○, air; ●, FA.

Acetaldehyde.

As AA is also an abundant aldehyde generated in PG-VG aerosol, we tested for effects of AA. In contrast to “respiratory braking” effects observed with both PG-VG aerosol and FA, AA exposure (5–10 ppm) had no obvious effects on any respiratory parameter (Fig. 3). These data indicate AA did not trigger an irritant response at any level reached under these exposure conditions.

Figure 3.

Effects of acetaldehyde (AA) on pulmonary function in mice. Male C57BL/6J mice instrumented with pressure transmitters to detect changes in pleural cavity pressure were exposed to filtered air or two 9-min sessions of AA (5-10 ppm). Pressure waveforms were analyzed for respiratory rate (breaths per min, bpm; A), expiratory (B), and inspiratory (C) times (s), and, peak amplitude (mmHg, a measure of respiratory effort; D). Onset of exposure to AA had no noticeable effect at 5 or >5 ppm on respiratory rate (A), expiratory time (B), inspiratory time (C), and peak amplitude (D). Values are 1-min means ± SE (n = 3 mice per group). Blue line represents real time monitoring of AA level (ppm) measured upstream of whole body exposure chamber (30-L) with an inline MultiRAE Pro. ○, air; ●, AA.

Cardiovascular Biomarkers of Harm

Because brief exposures to PG-VG and FA provoked irritant responses, we hypothesized that PG-VG and FA exposures but not AA exposure also would share common changes in some cardiovascular biomarkers of harm.

Endothelial Dysfunction

Endothelial dysfunction is a common and sensitive biomarker of inhaled toxicant exposures including cigarette smoke. Male and female mice were exposed for 4 consecutive days, and then the aorta was isolated and vascular function was assessed by isometric myography.

Propylene glycol-vegetable glycerin.

Female mice “vaped” for 4 days had aortic endothelial dysfunction characterized as a decrease in ACh-dependent (endothelium) relaxation of a phenylephrine (PE)-induced contraction (Fig. 4A; Table 1). Notably, PG-VG aerosol exposure also dramatically reduced the sensitivity (×10) of female aorta to ACh (Fig. 4B; Table 1). Aortic relaxation to sodium nitroprusside (SNP), NO donor, was slightly enhanced in % relaxation (Fig. 4C) and in sensitivity (Fig. 4D) in vaped female mice versus air controls (Table 1). The PE contraction ratio (a measure of eNOS activity) was significantly depressed by PG-VG exposure (Fig. 4E; Table 1). By comparison, male mice similarly exposed to PG-VG aerosol over 4 days did not have endothelial dysfunction (as % relaxation to ACh), yet these mice did have a notable but not statistically significant decrease (>3 times) in aortic sensitivity to ACh (Table 1), which was in the same direction as this effect in female mice. No effect of PG-VG exposure on PE contraction ratio was observed in vaped male mice (Table 1).

Figure 4.

Vascular toxicity of acute PG-VG inhalation exposure in female C57BL/6 mice. Aortic function was measured ex vivo following 4 days of exposure of female C57BL/6J mice to either filtered air or PG-VG (30:70, 6 h/day, 4 days). Acetylcholine (ACh) was used to assess aortic endothelium-dependent relaxation efficacy (% relaxation; A) and sensitivity (EC₅₀; B) in phenylephrine (PE)-precontracted aortic rings by isometric myography. PG-VG exposure significantly diminished ACh efficacy (A) and shifted sensitivity (B). There were, however, modest changes in aortic efficacy (% relaxation; C) and sensitivity (EC₅₀; D) to the endothelium-independent vasorelaxant nitric oxide donor, sodium nitroprusside (SNP). The PE contraction ratio was significantly decreased after PG-VG exposure (E). Values are means ± SE (n = 9-10 mice per group) (*P < 0.05) vs. air control; 0.10 > #P > 0.05 vs. air control. ○, air; ●, PG-VG. PG, propylene glycol. VG, vegetable glycine.

Table 1.

Vascular function of isolated aorta from female and male C57BL/6 mice exposed 4 consecutive days (6 h/day) to either filtered air, PG-VG, FA, or AA

	Air	PG-VG (30:70)	FA (5 ppm)	AA (5 ppm)
Female mice
ACh, −% PE	−74.9 ± 3.7	−61.8 ± 4.2*	−56.3 ± 4.5*	−89.6 ± 4.6*
PE EC50, nM	136 ± 24	124 ± 26	252 ± 131	292 ± 88
ACh EC50, nM	157 ± 36	1,020 ± 435*	195 ± 105	98 ± 20
SNP EC50, nM	9.4 ± 1.0	8.8 ± 0.8	9.2 ± 1.0	5.2 ± 0.6
PE cont. ratio	1.50 ± 0.06	1.28 ± 0.03*	1.34 ± 0.09#	2.45 ± 0.36*
l-NAME+ACh, −% PE	−1.6 ± 1.4	−1.4 ± 0.7	0 ± 0	0 ± 0
Male mice
ACh, −% PE	−73.4 ± 3.5	−75.0 ± 6.2	−74.0 ± 6.3	ND
PE EC50, nM	133 ± 32	110 ± 43	206 ± 72	ND
ACh EC50, nM	142 ± 59	474 ± 323	92 ± 32	ND
SNP EC50, nM	12.4 ± 2.0	5.6 ± 0.3	46.8 ± 33.7	ND
PE cont. ratio	1.44 ± 0.05	1.47 ± 0.04	1.39 ± 0.05*	ND
l-NAME + ACh, −% PE	0 ± 0	0 ± 0	-0.4 ± 0.4	ND

Values are means ± SE; n = 5–18 mice per group. PG-VG, propylene glycol-vegetable glycerin; FA, formaldehyde; AA, acetaldehyde; ACh, acetylcholine; PE, phenylephrine; EC₅₀, effective concentration producing 50% of response; SNP, sodium nitroprusside; PE cont. ratio, PE contraction ratio; l-NAME, N^G-nitro arginine-l-methyl ester (100 µM); ND, not done. *P < 0.05 vs. air group. #P < 0.05 vs. AA group. “ACh −% PE” and “l-NAME + ACh (−% PE)” represent effect of maximal ACh at 10 µM.

Formaldehyde.

Because female mice were sensitive to PG-VG aerosol exposure, we tested whether female mice also were affected after FA exposure. Female mice exposed to FA for 4 days also had aortic endothelial dysfunction measured as a decreased % relaxation to ACh (Fig. 5A). However, the sensitivity of aorta to ACh was unaltered by FA exposure (Fig. 5B). The aortic relaxation response to SNP was unaffected in % relaxation (Fig. 5C) and in sensitivity (Fig. 5D). The aortic PE contraction ratio of FA-exposed female mice versus air controls was not statistically different but was trending in that direction (Fig. 5E). Although male mice exposed for 4 days to 5 ppm FA did not have aortic endothelial dysfunction (no change in % relaxation or sensitivity to ACh), the aortas of male mice had a significantly decreased PE contraction ratio compared with air controls (Table 1).

Figure 5.

Vascular toxicity of acute formaldehyde (FA) and acetaldehyde (AA) inhalation exposure in female C57BL/6 mice. Aortic function was measured ex vivo following 4 days of exposure of female C57BL/6 mice to either filtered air or FA or AA (5 ppm, 6 h/day, 4 days). Acetylcholine (ACh) was used to assess aortic endothelium-dependent relaxation efficacy (% relaxation; A) and sensitivity (EC₅₀; B) in phenylephrine (PE)-precontracted aortic rings by isometric myography. FA exposure significantly diminished while AA exposure increased ACh efficacy (A), but neither exposure affected ACh sensitivity (B). There was a modest loss of aortic efficacy to the endothelium-independent nitric oxide donor, sodium nitroprusside (SNP) after exposure to FA (% relaxation) but not after AA exposure (C). Neither FA nor AA exposure affected aortic sensitivity to SNP (EC₅₀; D). The PE contraction ratio was significantly increased after AA exposure but not FA exposure (E). Values are means ± SE (n = 5-12 mice per group) (*P < 0.05) vs. air control; #P < 0.05 vs. AA group. ○, air; ●, FA; ▾, AA.

Acetaldehyde.

Unlike the effects of PG-VG aerosol and FA in female mice, exposure of female mice to AA (5 ppm, 4d) enhanced aortic % relaxation to ACh (Fig. 5A). The sensitivity of aorta to ACh, however, was unaltered by AA exposure (Fig. 5B). Relaxation response to SNP was unaffected either in % relaxation (Fig. 5C) or in sensitivity (Fig. 5D) in aortas of AA-exposed female mice versus air controls.

Circulating angiogenic cells.

Decreased levels of circulating angiogenic cells (CAC) are associated with increased CVD risk, and it is thought that CAC levels serve as a predictor of overall cardiovascular health and future cardiovascular events (50). Altered CAC levels are noted in individuals exposed to SHS (51) and during smoking cessation (52). We previously show that acute (4 days) exposure of mice to the unsaturated aldehyde acrolein decreases levels of CACs by 50% (47). Female mice exposed to PG-VG (4 days) had a substantial (−45%) albeit highly variable and statistically insignificant decrease in the levels of CACs compared with air controls (Table 2). Similarly, female and male mice acutely exposed to FA or AA (4 days; 5 ppm) had no changes in blood levels of CACs (Tables 3, 4, 6 and 7).

Table 2.

Systemic and hematological parameters of female C57BL/6 mice acutely exposed to either air or PG-VG

	Exposure (4 days)
	Air control	PG-VG
Variable
Change in BWT, g	−1 ± 0	−2 ± 0*
Heart/BWT, mg/g	5.2 ± 0.2	4.8 ± 0.1
Lung/BWT, mg/g	6.4 ± 0.1	6.3 ± 0.2
Liver/BWT, mg/g	38.8 ± 1.3	38.0 ± 0.6
Kidney/BWT, mg/g	12.7 ± 0.3	11.8 ± 0.2
Plasma measurements
Cholesterol, mg/dL	44.74 ± 1.31	51.45 ± 1.77*
HDL, mg/dL	25.78 ± 1.05	30.36 ± 2.00
LDL, mg/dL	12.80 ± 0.85	16.14 ± 0.47*
Triglycerides, mg/dL	30.81 ± 5.26	26.28 ± 1.54
Albumin, g/dL	2.82 ± 0.07	2.98 ± 0.07
Total protein, g/dL	3.78 ± 0.11	3.94 ± 0.05
ALT, U/L	20.20 ± 0.56	16.18 ± 0.34*
AST, U/L	64.10 ± 4.40	57.11 ± 4.33
CK, U/L	164.16 ± 11.04	158.73 ± 24.29
LDH, U/L	125.46 ± 8.91	127.20 ± 10.71
Creatinine, mg/dL	0.21 ± 0.04	0.17 ± 0.01
Hematological measurements
White blood cell, K/µL	1.34 ± 0.16	0.70 ± 0.05*
Neutrophils, K/µL	0.30 ± 0.06	0.14 ± 0.01*
Lymphocytes, K/µL	1.00 ± 0.11	0.54 ± 0.04*
Monocytes, K/µL	0.04 ± 0.01	0.02 ± 0.01
Eosinophils, K/µL	0 ± 0	0 ± 0
Basophils, K/µL	0 ± 0	0 ± 0
Red blood cell, M/µL	8.73 ± 0.07	9.29 ± 0.07*
Hemoglobin, g/dL	12.1 ± 0.1	12.6 ± 0.1*
Hematocrit, %	44.9 ± 0.4	47.6 ± 0.5*
Mean corpuscular volume, fL	51.5 ± 0.5	51.2 ± 0.2
Mean corpuscular hemoglobin, pg	13.9 ± 0.2	13.6 ± 0.1#
Mean corpuscular hemoglobin concentration, g/dL	27.0 ± 0.3	26.5 ± 0.2
Red cell distribution width, %	17.0 ± 0.2	16.8 ± 0.1
Platelets, K/µL	656 ± 27	710 ± 36
Mean platelet volume, fL	4.4 ± 0	4.2 ± 0*
PLAs, % leukocytes	10.3 ± 1.2	12.6 ± 2.1
Circulating angiogenic cells, %air control	100 ± 28	55 ± 17
Circulating angiogenic cells, % leukocytes	0.02 ± 0	0.03 ± 0.02

Values are means ± SE; n = 5–10 mice per group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BWT, body weight; CK, creatine kinase; HDL, high-density lipoprotein; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; PG-VG, propylene glycol-vegetable glycerin; PLAs, platelet leukocyte aggregates. *P < 0.05 compared with air control; 0.05 ≤ #P ≤ 0.10 compared with air control.

Table 3.

Systemic parameters of female C57BL/6 mice acutely exposed to either air or FA

	Exposure (4 days)
	Air control	FA (5 ppm)
Variable
Final BWT, g	27 ± 1	26 ± 1
Plasma measurements
Cholesterol, mg/dL	48.15 ± 4.42	47.43 ± 3.62
Triglycerides, mg/dL	26.53 ± 2.48	26.83 ± 1.82
Albumin, g/dL	2.74 ± 0.08	2.60 ± 0.06
Total protein, g/dL	4.88 ± 0.10	4.47 ± 0.20
ALT, U/L	22.07 ± 2.76	16.77 ± 1.29
AST, U/L	73.04 ± 6.70	77.62 ± 15.65
CK, U/L	171.14 ± 9.48	229.07 ± 50.25
LDH, U/L	133.03 ± 12.85	139.31 ± 15.86
Creatinine, mg/dL	0.24 ± 0.01	0.25 ± 0.01
Hematological measurements
White blood cell, K/µL	2.13 ± 0.42	2.13 ± 0.34
Neutrophils, K/µL	0.57 ± 0.12	0.75 ± 0.20
Lymphocytes, K/µL	1.49 ± 0.33	1.34 ± 0.19
Monocytes, K/µL	0.07 ± 0.01	0.05 ± 0.01
Eosinophils, K/µL	0 ± 0	0 ± 0
Basophils, K/µL	0 ± 0	0 ± 0
Red blood cell, M/µL	8.23 ± 0.46	8.93 ± 0.20
Hemoglobin, g/dL	10.9 ± 0.6	12.1 ± 0.2
Hematocrit, %	34.3 ± 1.7	37.5 ± 0.7
Mean corpuscular volume, fL	41.7 ± 0.5	42.0 ± 0.4
Mean corpuscular hemoglobin, pg	13.3 ± 0.1	13.6 ± 0.2
Mean corpuscular hemoglobin concentration, g/dL	31.9 ± 0.3	32.3 ± 0.2
Red cell distribution width, %	17.5 ± 0.3	17.6 ± 0.2
Platelets, K/µL	814 ± 105	684 ± 49
Mean platelet volume, fL	5.0 ± 0.3	4.4 ± 0.1#
PLAs, % leukocytes	3.4 ± 1.3	5.5 ± 2.5
Circulating angiogenic cells, % air control	100 ± 37	90 ± 25
Circulating angiogenic cells, % leukocytes	0.28 ± 0.13	0.31 ± 0.05

Values are means ± SE; n = 6–8 mice per group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BWT, body weight; CK, creatine kinase; FA, formaldehyde; LDH, lactate dehydrogenase; PLAs, platelet leukocyte aggregates. 0.05 ≤ #P ≤ 0.10 compared with air control.

Table 4.

Systemic changes in female C57BL/6 mice acutely exposed to either air or AA

	Exposure (4 days)
	Air control	AA (5 ppm)
Variable
Final BWT, g	21 ± 0	23 ± 1
Heart/BWT, mg/g	5.0 ± 0.2	5.0 ± 0.2
Lung/BWT, mg/g	7.0 ± 0.2	6.8 ± 0.2
Liver/BWT, mg/g	44.8 ± 0.9	46.1 ± 1.8
Kidney/BWT, mg/g	12.6 ± 0.4	13.0 ± 0.3
Hematological measurements
White blood cell, K/µL	1.60 ± 0.34	1.15 ± 0.17
Neutrophils, K/µL	0.45 ± 0.14	0.28 ± 0.03
Lymphocytes, K/µL	1.10 ± 0.19	0.82 ± 0.14
Monocytes, K/µL	0.04 ± 0.01	0.04 ± 0.01
Eosinophils, K/µL	0 ± 0	0 ± 0
Basophils, K/µL	0 ± 0	0 ± 0
Red blood cell, M/µL	9.23 ± 0.05	9.32 ± 0.11
Hemoglobin, g/dL	11.9 ± 0.1	11.7 ± 0.2
Hematocrit, %	38.5 ± 0.3	38.1 ± 0.5
Mean corpuscular volume, fL	41.7 ± 0.3	40.9 ± 0.7
Mean corpuscular hemoglobin, pg	13.0 ± 0.1	12.5 ± 0.3
Mean corpuscular hemoglobin concentration, g/dL	31.0 ± 0.3	30.6 ± 0.4
Red cell distribution width, %	17.7 ± 0.2	17.6 ± 0.1
Platelets, K/µL	695 ± 36	680 ± 22
Mean platelet volume, fL	4.4 ± 0.1	4.4 ± 0.1
PLAs, % of leukocytes	6.7 ± 2.9	8.5 ± 2.7
Circulating angiogenic cells, % air control	100 ± 8	74 ± 15
Circulating angiogenic cells, %leukocytes	0.23 ± 0.02	0.17 ± 0.04

Values are means ± SE; n = 5–6 mice per group. AA, acetaldehyde; BWT, body weight; PLAs, platelet leukocyte aggregates.

Table 6.

Systemic parameters of male C57BL/6 mice acutely exposed to either air or FA

	Exposure (4 days)
	Air control	FA (5 ppm)
Variable
Final BWT, g	27 ± 0	28 ± 1
Heart/BWT, mg/g	5.0 ± 0.1	5.0 ± 0.1
Lung/BWT, mg/g	5.3 ± 0.1	5.4 ± 0.1
Liver/BWT, mg/g	43.9 ± 0.7	40.5 ± 0.8*
Kidney/BWT, mg/g	12.6 ± 0.3	12.1 ± 0.6
Plasma measurements
Cholesterol, mg/dL	95.53 ± 3.65	94.23 ± 2.32
HDL, mg/dL	71.91 ± 2.41	67.88 ± 2.90
Triglycerides, mg/dL	44.32 ± 2.12	43.64 ± 2.01
Albumin, g/dL	3.16 ± 0.04	3.21 ± 0.06
Total protein, g/dL	5.07 ± 0.08	5.18 ± 0.13
ALT, U/L	59.39 ± 17.39	38.44 ± 7.75
AST, U/L	116.32 ± 17.24	104.26 ± 14.34
CK, U/L	338.11 ± 65.73	314.13 ± 68.39
LDH, U/L	210.15 ± 19.99	188.06 ± 23.20
Creatinine, mg/dL	0.35 ± 0.01	0.37 ± 0.01
Hematological measurements
White blood cell, K/µL	2.21 ± 0.38	2.71 ± 0.42
Neutrophils, K/µL	0.55 ± 0.17	0.70 ± 0.14
Lymphocytes, K/µL	1.56 ± 0.24	1.94 ± 0.33
Monocytes, K/µL	0.06 ± 0.01	0.06 ± 0.01
Eosinophils, K/µL	0.01 ± 0	0.01 ± 0
Basophils, K/µL	0.01 ± 0	0.01 ± 0
Red blood cell, M/µL	8.82 ± 0.09	8.41 ± 0.16#
Hemoglobin, g/dL	12.2 ± 0.3	11.9 ± 0.4
Hematocrit, %	40.0 ± 1.5	36.8 ± 1.3#
Mean corpuscular volume, fL	45.3 ± 1.5	43.7 ± 1.5
Mean corpuscular hemoglobin, pg	13.9 ± 0.4	14.1 ± 0.4
Mean corpuscular hemoglobin concentration, g/dL	31.2 ± 1.8	32.7 ± 1.7
Red cell distribution width, %	17.0 ± 0.2	17.4 ± 0.3
Platelets, K/µL	801 ± 31	717 ± 26
Mean platelet Volume, fL	4.5 ± 0.1	4.5 ± 0
PLAs, % leukocytes	5.4 ± 0.9	7.4 ± 1.7
Circulating angiogenic cells, % air control	100 ± 11	103 ± 24
Circulating angiogenic cells, % leukocytes	0.44 ± 0.03	0.37 ± 0.05

Values are means ± SE; n = 6–13 mice per group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BWT, body weight; CK, creatine kinase; FR, formaldehyde; HDL, high-density lipoprotein; LDH, lactate dehydrogenase; PLAs, platelet leukocyte aggregates. *P < 0.05 compared with air control; 0.05 ≤ #P ≤ 0.10 compared with air control.

Table 7.

Systemic parameters of male C57BL/6 mice acutely exposed to either air or AA

	Exposure (4 days)
	Air control	AA (5 ppm)
Variable
Final BWT, g	30 ± 0	31 ± 1
Heart/BWT, mg/g	5.4 ± 0.4	5.3 ± 0.2
Lung/BWT, mg/g	5.3 ± 0.1	5.2 ± 0.1
Liver/BWT, mg/g	40.7 ± 3.2	38.4 ± 1.3
Kidney/BWT, mg/g	14.1 ± 0.5	13.2 ± 0.5
Plasma measurements
Cholesterol, mg/dL	69.69 ± 5.83	72.37 ± 4.30
HDL, mg/dL	41.68 ± 4.60	48.86 ± 4.24
LDL, mg/dL	5.49 ± 0.91	5.71 ± 1.07
Albumin, g/dL	2.87 ± 0.07	2.87 ± 0.04
Total protein, g/dL	4.89 ± 0.10	4.76 ± 0.06*
Hematological measurements
White blood cell, K/µL	1.74 ± 0.22	1.24 ± 0.19
Neutrophils, K/µL	0.52 ± 0.07	0.45 ± 0.19
Lymphocytes, K/µL	1.18 ± 0.17	0.73 ± 0.10
Monocytes, K/µL	0.05 ± 0.01	0.05 ± 0.01
Eosinophils, K/µL	0 ± 0	0 ± 0
Basophils, K/µL	0 ± 0	0 ± 0
Red blood cell, M/µL	8.33 ± 0.21	7.52 ± 0.53
Hemoglobin, g/dL	10.8 ± 0.3	10.5 ± 0.8
Hematocrit, %	35.1 ± 1.0	31.5 ± 2.2
Mean corpuscular volume, fL	42.2 ± 0.2	41.8 ± 0.2
Mean corpuscular hemoglobin, pg	13.0 ± 0.1	13.9 ± 0.2*
Mean corpuscular hemoglobin concentration, g/dL	30.8 ± 0.3	33.1 ± 0.4*
Red cell distribution width, %	18.8 ± 0.7	18.3 ± 0.4
Platelets, K/µL	829 ± 7	782 ± 81
Mean platelet volume, fL	4.7 ± 0	4.7 ± 0.1
Circulating angiogenic cells, % air control	100 ± 14	99 ± 23
Circulating angiogenic cells, % leukocytes	0.17 ± 0.03	0.25 ± 0.06

Values are means ± SE; n = 5 mice per group. AA, acetaldehyde; BWT, body weight; HDL, high-density lipoprotein; LDL, low-density lipoprotein. *P < 0.05 compared with air control.

Platelet-Leukocyte aggregates (PLAs).

A biomarker of thrombosis, PLAs reflect the balance of numerous forces that regulate platelet activation and aggregation. Previously, we show that multiday acrolein or MCS exposure in mice increases PLAs (53).

Propylene glycol-vegetable glycerin, formaldehyde, acetaldehyde.

Despite some effects of exposures on endothelial function in female mice (and to a lesser extent in male mice), exposures to PG-VG, FA, and AA had no effect on levels of PLAs in blood of either female or male mice (Tables 2, 3, 4, 5, 6 and 7).

Table 5.

Systemic and hematological parameters of male C57BL/6 mice acutely exposed to either air or PG-VG

	Exposure (4 days)
	Air control	PG-VG (30:70)
Variable
Change in BWT, g	−2.4 ± 0.2	−4.7 ± 0.3*
Heart/BWT, mg/g	5.0 ± 0.2	5.2 ± 0.1
Lung/BWT, mg/g	5.5 ± 0.2	5.7 ± 0.1
Liver/BWT, mg/g	39.5 ± 1.3	39.4 ± 1.2
Kidney/BWT, mg/g	13.4 ± 0.4	13.3 ± 0.3
Plasma measurements
Cholesterol, mg/dL	94.40 ± 3.04	86.20 ± 4.85
HDL, mg/dL	49.80 ± 2.71	47.80 ± 3.28
Triglycerides, mg/dL	44.20 ± 1.98	44.60 ± 2.16
Albumin, g/dL	2.56 ± 0.09	2.52 ± 0.06
Total protein, g/dL	4.28 ± 0.09	4.10 ± 0.08
ALT, U/L	12.40 ± 0.51	19.60 ± 2.98*
AST, U/L	41.80 ± 4.87	52.8 ± 7.34
CK, U/L	255.60 ± 129.13	196.00 ± 33.97
LDH, U/L	161.40 ± 20.68	167.00 ± 6.78
Creatinine, mg/dL	0.22 ± 0.04	0.23 ± 0.03
Hematological measurements
White blood cell, K/µL	1.99 ± 0.36	0.61 ± 0.02*
Neutrophils, K/µL	0.45 ± 0.06	0.13 ± 0.02*
Lymphocytes, K/µL	1.45 ± 0.29	0.42 ± 0.04*
Monocytes, K/µL	0.06 ± 0	0.04 ± 0.02
Eosinophils, K/µL	0.02 ± 0.01	0.02 ± 0.02
Basophils, K/µL	0 ± 0	0 ± 0
Red blood cell, M/µL	8.66 ± 0.21	8.50 ± 0.59
Hemoglobin, g/dL	12.2 ± 0.3	12.0 ± 0.7
Hematocrit, %	46.1 ± 1.2	44.9 ± 3.4
Mean corpuscular volume, fL	53.2 ± 0.2	52.7 ± 0.6
Mean corpuscular hemoglobin, pg	14.0 ± 0.1	14.2 ± 0.4
Mean corpuscular hemoglobin concentration, g/dL	26.4 ± 0.3	27.1 ± 0.9
Red cell distribution width, %	17.2 ± 0.4	16.5 ± 0.3
Platelets, K/µL	816 ± 41	968 ± 31*
Mean platelet volume, fL	4.8 ± 0	4.7 ± 0.1
PLAs, % leukocytes	8.2 ± 0.5	8.8 ± 0.2

Values are means ± SE; n = 5 mice per group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BWT, body weight; CK, creatine kinase; HDL, high-density lipoprotein; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; PG-VG, propylene glycol-vegetable glycerin; PLAs, platelet leukocyte aggregates. *P < 0.05 compared with air control.

Hematological and Systemic Biomarkers of Harm

CBCs.

As WBC, RBC, and platelets levels typically have a broad range of normal values, the CBC is considered a relatively crude indicator of health status.

Propylene glycol-vegetable glycerin.

Female mice acutely exposed (4 days) to PG-VG showed a greater change in body weight during the exposure period (−2 g versus −1 g; P = 0.01) but no changes in measured organ/body weight ratios (Table 2). Acute exposure had considerable effects on CBC measures. Exposed mice showed a significant decrease in WBCs (−47%; P = 0.003), driven by decreases in both neutrophils (−54%; P = 0.01) and lymphocytes (−46%; P = 0.003) (Table 2). Levels of RBCs were increased (+6%; P < 0.001) as were measures of hemoglobin (+4%; P = 0.002) and hematocrit (+6%; P = 0.002), whereas mean platelet volume (4%; P = 0.01) and, modestly, mean corpuscular hemoglobin (−2%; P = 0.06) were decreased (Table 2). In male mice, PG-VG aerosol exposure had similar toxic effects on body weight (decreased) and WBC (leukopenia) as in female mice but did not affect any RBC parameter while increasing platelet count (Table 5).

Formaldehyde.

Acute exposure to FA (4 days; 5 ppm) caused a modestly significant decreased mean platelet volume (−11%; P = 0.08) in female mice (Table 3). A similar exposure in male mice caused modest yet significant decreases in RBCs (−5%; P = 0.07) and hematocrit (−8%; P = 0.08) (Table 6).

Acetaldehyde.

Female mice acutely exposed to AA (4 days; 5 ppm) showed no significant changes (Table 4), whereas male mice had significant increases in mean corpuscular hemoglobin (+7%; P = 0.008) and mean corpuscular hemoglobin concentration (+8%; P = 0.008) after a similar exposure (Table 7).

Plasma Biomarkers of Harm

Propylene glycol-vegetable glycerin.

Measures of systemic biomarkers in PG-VG–exposed female mice (4 days) showed a significant increase in cholesterol (+15%; P = 0.03) driven by an increase in LDL (+26%; P = 0.03) (Table 2). Conversely, ALT was significantly decreased in exposed mice (−20%; P = 0.02) (Table 2). In male mice, PG-VG exposure modestly yet significantly increased plasma ALT (12.40 ± 0.51 versus 19.60 ± 2.98 U/L) (Table 5).

Formaldehyde.

Systemic toxicity outcomes of FA exposure (4 days; 5 ppm) were measured as before with acrolein (47). Female mice showed no significant changes (Table 3), whereas male mice showed significant changes in liver/body weight ratios (−8%; P = 0.004) (Table 6).

Acetaldehyde.

In male mice acutely exposed to AA (4 days; 5 ppm), plasma levels of total protein were significantly decreased (−3%; P = 0.008) (Table 7).

Urinary formaldehyde and acetaldehyde metabolites.

To better understand how PG-VG aerosol exposure related to FA and AA exposures, the primary metabolites of FA and AA, formate and acetate, respectively, were measured as urinary biomarkers of exposure using GC-MS (Fig. 6).

Figure 6.

Urinary excretion of absolute amounts of acetate and formate following 6 h exposure to air, acetaldehyde, and formaldehyde (FA) of PG-VG. Within the first 3 h post exposure, neither primary metabolite was increased by exposure of male mice to its parent aldehyde. Although acetate excretion (A) was increased overnight after PG-VG–derived aerosol exposure, acetate was not increased immediately (0-3 h) after exposure and formate excretion (B) was not increased at any point. Values are means ± SE (n = 4-30 male mice per group) (*P < 0.05) vs. air control. PG, propylene glycol. VG, vegetable glycine; AA, acetaldehyde.

Propylene glycol-vegetable glycerin.

Notably, PG-VG exposure (6 h) in male mice (but not in female mice; data not shown) enhanced only the absolute amount of urinary acetate excreted overnight post exposure (Fig. 6A), but did not increase urinary formate excreted in either period (Fig. 6B).

Formaldehyde and acetaldehyde.

Surprisingly, neither FA nor AA exposure (6 h, 5 ppm) in male mice had any effect on absolute urinary acetate or formate excretion, respectively, in the early (0–3 h) post-exposure urine collection (Fig. 6, A and B). However, FA exposure decreased the absolute urinary formate excretion in the overnight post exposure collection period (Fig. 6B). AA exposure had no effect on acetate excretion in the overnight post exposure collection period (Fig. 6A).

DISCUSSION

Our findings in this study add to the growing concern regarding cardiopulmonary disease risk in users of electronic nicotine delivery systems (ENDS). Although acute responses to ENDS aerosols including pulmonary inflammation and cough as well as endothelial dysfunction are reported in human and animal studies to date, our findings complement these studies by showing: 1) irritant responses and endothelial dysfunction occurred with exposure to both PG-VG aerosol and FA, indicating a potential link between FA formation and cardiopulmonary effects, 2) endothelial dysfunction was prominent in female (also in male) mice indicating potential sex-dependent sensitivity (e.g., basis for these differences is unclear but may be hormonal), and 3) despite the presence of abundant saturated aldehydes, FA and AA, in PG-VG–derived aerosols, currently used biomarkers of exposure, urinary formate and acetate, remain inadequate. This conclusion is re-enforced by equally poor utility of these urinary biomarkers even following controlled FA and AA exposures at 5 ppm in mice. Nonetheless, these findings provide data that implicate thermal degradation products of PG-VG in cardiopulmonary injury.

Pulmonary Irritation and Endothelial Dysfunction

Is there a connection between inhaled irritants and endothelial dysfunction? There certainly is a well-established connection between inhaled ambient fine particulate matter (PM_2.5) and cardiopulmonary disease risk (54). Additionally, the high irritant potential of inhaled volatile organic compounds (VOC) including aldehydes has been well-documented for over 5 decades especially in rodents (55). These reflex-mediated irritant responses including “respiratory braking” and coughing are triggered by irritants present in ENDS-derived aerosols (56–58). Our findings support the idea that irritants are generated in high amounts in each puff of PG-VG. Numerous studies have documented the formation of aldehydes including high levels (ppm) of FA and AA in aerosols of E-cigs (16, 59) (see Table 8), and, perhaps, many of these aerosol constituents contribute to rapid and reversible pulmonary irritant reflexes as observed in our study. We provide evidence that FA is one plausible constituent of PG-VG–derived aerosol that triggers an irritant reflex. Moreover, FA exposure recapitulates the effects of PG-VG–derived aerosol in mice by inducing: 1) “respiratory braking”, 2) endothelial dysfunction (decreased % relaxation to ACh), 3) decreased sensitivity of aorta to Ach, and, 4) decreased PE contraction ratio. We do not rule out a role of other constituents in PG-VG aerosols (e.g., particulates, other VOCs), although in our study, AA is “the exception that proves the rule.” At the same level as FA, AA exposure (5 ppm) alone did not induce an irritant reflex or endothelial dysfunction, and, in fact, AA exposure enhances the ACh-induced endothelial-dependent relaxation and the PE contraction ratio, effects opposite of both PG-VG and FA exposures. These results indicate that some constituents in PG-VG–derived aerosols may offset/cancel/balance out harmful effects of other constituents. It is an important and challenging problem to disentangle the contribution of each constituent in complex aerosols to disease etiology as counteracting effects may not be neatly added or subtracted. This quandary poses an important barrier for scientists to provide actionable data to regulatory agencies. In this regard, our study of FA exposure alone indicates a no-observable adverse-effect limit (NOAEL) is likely much lower than 5 ppm, a level of FA and AA likely reached in each puff of PG-VG, and theoretically these levels are exceeded greatly in aerosols of some E-cigarettes (mods) as well as cigarette smoke (16) (Table 8).

Table 8.

Levels of carbonyl compounds formed from e-cigarettes and e-liquids as converted to theoretical ppm

Product Code	AA µg/10 puffs	AA ppma	FA µg/10 puffs	FA ppma
bluClassic Tobaccob	0.57	0.35	0.55	0.49
bluMagnificent Mentholb	0.49	0.30	0.62	0.55
bluVivid Vanillab	0.52	0.32	0.43	0.38
bluCherry Crushb	0.15	0.09	0.18	0.16
eVo Black diamondb	63.1	38.37	26.8	23.90
Smooththolb	23.3	14.17	8.2	7.31
Perfected Vape/Clearwaterb	44.8	27.24	40.4	36.03
Halo Café Mochab	13.3	8.09	15.2	13.56
Halo Menthol Iceb	13.9	8.45	20.1	17.93
Halo Southern Classicb	15.2	9.24	21.8	19.44
PG-VG (25:75)c	0.88	0.54	0.88	0.78
PG-VG (50:50)c	1.33	0.81	1.75	1.56
PG-VG (75:25)c	2.63	1.60	2.18	1.94
Tobacco cigaretted	1,240.3	1,387.92	74.0	121.46

^aTheoretical ppm level calculated based on gas molar volume 24.4 L/mole and molecular weight according to the equation (under STP): [ppm = (mg/m³ × 24.4)/mol wt].

^bAerosol samples were generated at a battery power output of 4.6 W for the blu e-cigarettes and a battery power output of 9.1 W for e-liquids using a EVOD2 atomizer; puff volume of 91 mL, puff duration of 4 s, as described (9).

^cBased on data published in Conklin et al. (41).

^dData per cigarette based on Counts et al. (73).

Endothelial Dysfunction: Role of eNOS

To document the nature of vascular injury, we measured several indices. For example, we measured endothelial relaxation to ACh (efficacy and sensitivity), calculated a PE contraction ratio (ratio of PE-induced tension post-l-NAME:pre-l-NAME) as a real-time measure of eNOS activity that modulates PE-induced tension in the absence of Ach, measured sensitivity of aorta to contractile agonist PE, and measured sensitivity and efficacy of responses to an NO donor, SNP. In addition to endothelial dysfunction of PG-VG and FA, the PE contraction ratio is significantly lower in aortas of PG-VG–exposed female mice and trending in that direction with FA. The PE contraction ratio is a robust measure because the efficacy of l-NAME inhibition of eNOS is supported by near abolition of ACh-induced relaxation (>99% block) indicating the aorta remains completely dependent on eNOS and not on another EDRF/EDHF as in other blood vessels (60). Thus, these novel data support that the PE contraction ratio reflects not only a continuum of “endothelium functionality,” yet also provides data that are in agreement with the effects of PG-VG and FA exposures on ACh-induced relaxation. Although eNOS uncoupling due to oxidation of tetrahydrobiopterin (BH₄) is a known mechanism of endothelial dysfunction due to PM_2.5 exposure (and other conditions) in rodents (61), the mechanism by which PG-VG (or FA) exposure induces endothelial dysfunction is uncertain. However, our data are in agreement with the acute onset of endothelial dysfunction and specific loss of eNOS bioactivity as recorded in recent human and animal E-cig exposure studies (62–64). It remains unclear why female mice in our study appear more sensitive to the vascular effects of exposure to PG-VG aerosol than male mice, and, of course, more targeted mechanistic studies will be required to reveal underlying causes (e.g., hormonal). Regardless, a recent panel study of healthy, young adult women (n = 10) exposed acutely to FA (in cadaver laboratory) found that FA induced conduit blood vessel endothelial dysfunction (34). This study supports the biological plausibility of our findings in isolated aorta, and it indirectly suggests that natural variation in female biology (e.g., estrous cycle) may be inconsequential in conduit blood vessel function especially in this acute exposure setting (34).

Systemic and Hematological Biomarkers

Although the irritant responses and endothelial dysfunction are clearly highly sensitive biomarkers, we measured numerous other biomarkers of systemic and hematological injury to understand whether patterns of biomarkers change in concert or whether some biomarkers are more sensitive than others. Acute PG-VG exposure significantly decreased WBCs, which is contrary to what is seen in users of traditional cigarettes (65, 66), yet similarly seen in studies of individuals with secondhand smoke exposure (67). In contrast, PG-VG exposure significantly increases both RBCs and hemoglobin, similar changes are commonly seen in smokers (65, 66, 68, 69) and are believed to be a compensatory mechanism to account for lower blood oxygen levels. In our study, these changes may indicate that even acute exposure to PG-VG is perhaps sufficient to perturb gas levels in the blood, which may reflect repeated stimulation of reflex-driven bradypnea. In contrast, acute exposure to either FA or AA causes few, if any, significant systemic or hematological changes, which seems to demonstrate that additional changes present in PG-VG–exposed mice are due to constituents other than these 2 saturated aldehydes and/or are due to much higher levels of exposure to FA and AA than are tested herein.

Biomarkers of Exposure

In our previous study, we show that AA and FA are abundant in aerosols generated from different PG-VG ratios typical of e-liquids (75:25, 50:50, 25:75) (41). Despite showing that these aldehydes are formed abundantly, providing a biomarker of internal exposure is problematic (70). For example, we are unable to demonstrate any urinary peak of formate or acetate even though we exposed mice to 5 ppm level (continuous 6 h) and collected all urine post exposure (up to 18 h). We previously failed to show increases in either urinary acetate or formate excretion with blu E-cig exposures in mice (41). It is unlikely that 5 ppm FA or AA is too low as our estimates of PG-VG–derived levels of FA and AA are <3 ppm (Table 8). Our customized E-cig device (BluPlus battery coupled to a refillable Mistic bridge tank-style atomizer) has relatively low power output (<8 W) and is used under realistic vaping conditions (91.1-mL puff, 4-s puff, 2 puffs/min)(17). Nonetheless, and similar to that in vaping humans, our 91.1-mL puff is rapidly diluted (e.g., delivered to a 5-L whole body exposure chamber or to lungs), and thus, is diluted >50 times. Both urinary formate and acetate levels are further confounded because of dramatic increases overnight (with feeding) in rodents (71). A recent study, however, detects sulfur-containing metabolites of FA (thiazolidine carboxylic acid and thiazolidine carbonyl glycine) in urine following inhalation exposure to cigarette smoke spiked with [¹³C]PG and [¹³C]VG, indicating a new potential biomarker of FA inhalation exposure (72). Unfortunately, a similar marker of AA inhalation exposure is unavailable.

Conclusions

PG-VG–derived aerosols contain pulmonary irritants and constituents that can induce endothelial dysfunction thereby increasing concern that users of ENDS, independent of nicotine or flavorings, likely increase cardiopulmonary disease risk. FA, as an abundant thermal degradation product of PG-VG, is also an irritant and induces endothelial dysfunction, suggesting that FA may contribute to the deleterious effects of inhaled PG-VG–derived aerosols. As such, the U.S. Food and Drug Administration should consider the regulation of FA to levels that are below those that trigger acute cardiopulmonary injury.

GRANTS

This work was supported by National Institutes of Health Grants ES023716, GM127607, HL122676, HL149351, U54HL120163, and T32ES011564 and the Jewish Heritage Fund for Excellence.

DISCLOSURE

No conflicts of interest, financial or otherwise, are declared by the authors. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Food and Drug Administration or the American Heart Association.

AUTHOR CONTRIBUTIONS

S.S., D.J.C., and A.B. conceived and designed research; L.J., D.J.C., J.L., A.R., W.T., G.S., and A.H. performed experiments; L.J., D.J.C., J.L., A.R., P.L., and S.S. analyzed data; D.J.C., J.L., and A.R. interpreted results of experiments; L.J., D.J.C., and J.L. prepared figures; D.J.C. drafted manuscript; L.J., S.S., D.J.C., J.L., A.R., P.L., W.T., and G.S. edited and revised manuscript; L.J., S.S., D.J.C., J.L., A.R., P.L., S.S., W.T., G.S., A.H., and A.B. approved final version of the manuscript.