Acute and Chronic Vascular Effects of Inhaled Crotonaldehyde in Mice: Role of TRPA1

Abstract

Although crotonaldehyde (CR) is an abundant α,β-unsaturated aldehyde in mainstream cigarette smoke (MCS), the cardiovascular toxicity of inhaled CR is largely unexplored. Thus, male C57BL/6J mice were exposed acutely (1h, 6h, 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/6J mice. Notably, CR exposure (4d, 1 ppm) had no effect on aortic function in female C57BL/6J 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.

INTRODUCTION

Smoking is the most significant modifiable risk factor in the development of cardiovascular disease (CVD) (Bhatnagar, 2006). Tobacco use is responsible for an estimated 7 million global deaths each year (World Health Organization, 2017), with many of these deaths due to CVD (Hitchman et al., 2012). Smoking has been shown to increase the risk of myocardial infarction, atherosclerosis, stroke, and thrombosis (Ambrose and Barua, 2004). Extensive evidence demonstrates that exposure to mainstream cigarette smoke (MCS) (U.S. Food & Drug Administration, 2012; U.S. Department of Health and Human Services, 2014) or secondhand tobacco smoke (U.S. Department of Health and Human Services, 2014) increases the risk of CVD. As MCS contains more than 7,000 compounds (U.S. Department of Health and Human Services, 2014), many of these even in limited quantities can contribute to disease development (Smith and Fischer, 2001; U.S. Food & Drug Administration, 2012; U.S. Department of Health and Human Services, 2014). Of these compounds, the unsaturated aldehydes (Smith and Fischer, 2001; U.S. Food & Drug Administration, 2012), including acrolein and crotonaldehyde (CR), are classified as the most significant toxins in tobacco smoke (Haussmann, 2012; Stabbert et al., 2017). More than 90% of the non-cancer health risk associated with smoking (i.e., cardiopulmonary disease risk) is attributable to aldehydes (Haussmann, 2012), and the majority of the in vitro cytotoxicity related to gas phase constituents of MCS is due to acrolein (88.5%) and CR (Stabbert et al., 2017).

As MCS is a known source of the unsaturated aldehydes—acrolein (18–98 μg/cigarette) and CR (10–43 μg/cigarette) (Roemer et al., 2012; Alwis et al., 2015)—the levels of the major urinary metabolites of acrolein (3-hydroxypropylmercapturic acid; 3HPMA) and CR (3-hydroxy-1-methylpropylmercapturic acid; HPMMA) are significantly increased in smokers versus non-smokers (Alwis et al., 2015; Bagchi et al., 2018). Baseline levels of 3HPMA and HPMMA in non-smokers are similar, and both are elevated nearly to the same median levels in smokers (approximately 3x), suggesting that there are common ambient and metabolic sources of acrolein and CR in non-smokers as well as generally equivalent levels of acrolein and CR in MCS.

Although much epidemiological and experimental evidence exists linking acrolein exposure with increased CVD risk, much less is known regarding CR-related cardiovascular toxicity in vivo despite its co-linearity with acrolein exposure in smokers. Both acute and chronic inhalation exposures to acrolein have been shown to suppress levels of circulating angiogenic cells (CACs) (Wheat et al., 2011; Conklin et al., 2017b), enhance thrombotic markers (Sithu et al., 2010), and initiate endothelium dysfunction in susceptible mice (Conklin et al., 2009), while acrolein gavage promotes atherosclerosis in mice (Srivastava et al., 2011). Moreover, urinary 3HPMA levels are associated with increased CVD risk, decreased levels of CACs, and increased platelet-leukocyte aggregates (PLAs) in humans (DeJarnett et al., 2014). By comparison, 1,3-butadiene, a parent source of CR, exposure is linked with an increased risk of atherosclerosis in African-American workers (Matanoski et al., 1990), and African-American men who worked in a 1,3-butadiene production plant for at least 6 months had increases in standardized mortality ratio for arteriosclerotic heart disease (Divine, 1990). Likewise, inhalation exposure to 1,3-butadiene, accelerates arteriosclerotic lesion development in cockerels (Penn and Snyder, 1996). Furthermore, although CR has been shown to induce oxidative stress in cells (Pei et al., 2014; Marescotti et al., 2016) and to cause cardiomyocyte dysfunction (Pei et al., 2014), CR inhalation studies with measures of cardiovascular injury have not been performed.

Notably, the transient receptor potential ankyrin 1 (TRPA1) channel is a pulmonary irritant receptor known to mediate responses to inhaled compounds such as MCS, diesel engine exhaust, or to individual MCS constituents such as acrolein and CR (Hazari et al., 2011; Conklin, 2016). Furthermore, the negative cardiovascular effects of inhaled acrolein (at levels found in cigarette smoke) are TRPA1-dependent (Kurhanewicz et al., 2018), yet the role of TRPA1 in the potential cardiovascular effects of inhaled CR is unknown. To address these gaps in knowledge regarding the potential cardiovascular toxicity of CR (and its mechanisms), healthy adult male and female C57BL/6J mice were exposed by inhalation to CR acutely or chronically (4 days or 12 weeks; 1 or 3 ppm), and biomarkers of cardiovascular harm were measured. To probe the role of TRPA1, we performed select CR exposures in TRPA1-null mice (Conklin et al., 2017b).

MATERIALS AND METHODS

Materials

Reagent-grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Mice and Crotonaldehyde Exposure

Mice

Male and female C57BL/6J (wild type, WT) mice were obtained from The Jackson Laboratories (Bar Harbor, ME). Male TRPA1-null mice (on a C57BL/6J background) were from a breeding colony at the University of Louisville (Conklin et al., 2017a). 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 12h:12h light:dark cycle. Mice were maintained on a standard chow diet (Rodent Diet 5010, 4.5% fat by weight, LabDiet; St. Louis, MO).

Crotonaldehyde and MCS exposures

To parallel our previous study of chronic (12 weeks, 1 ppm) acrolein exposure in mice (Conklin et al., 2017b), mice were exposed to CR at 1 ppm for 12 weeks. To demonstrate the presence of CR in MCS, naïve mice were exposed to MCS (50% of smoke of 12 KY Reference 3R4F cigarettes/6h) and urine collected as previously described (Conklin et al., 2018). For CR studies, naïve mice were exposed to either HEPA- and charcoal-filtered air or CR for 4 consecutive days (1 or 3 ppm, 6h/d) or 12 weeks (1 ppm; 6h/d, 5 d/week) using a custom exposure system and a certified permeation tube (Kin-Tek; LaMarque, TX) as previously described (Conklin et al., 2017b). Urine was collected during the 11^th week of CR exposure, and CR metabolites were quantified. Immediately following the final exposure, mice were euthanized by sodium pentobarbital (≈150 mg/kg, i.p.) and ventral thoracotomy and exsanguination with cardiac puncture for blood collection in EDTA-coated syringes. Organs were removed, weighed, and snap frozen in liquid N₂ and stored at −80°C until further analysis.

Urine Collection and Crotonaldehyde Metabolism

Urine collection

During the 11^th week of the chronic exposure, mice were weighed and briefly exposed to a D-glucose:saccharin solution (w/v; 3.0%/0.125%; Sigma-Aldrich; St. Louis, MO) immediately prior to 6h exposure. After CR and MCS exposures, mice were placed singly per metabolic cage (Harvard Apparatus; Cambridge, MA) with glucose:saccharin solution drinking water but without food for urine collection (in graduated cylinders surrounded by 4°C water-jacketed organ baths). Urine was collected in 1h increments up to 3h post-exposure followed by an overnight urine collection during which mice were provided glucose/saccharin solution as well as food (Conklin et al., 2017b). Collected urine samples were centrifuged (1,800xg, 5 min; to pellet any feces or food particles) before being decanted and stored at −80°C.

Crotonaldehyde metabolite analysis

The major metabolite of CR, HPMMA, was quantified in urine by ultrahigh performance tandem mass spectrometry (UPLC-MS/MS). Urine (25 μl) was diluted (40x) with 15 mM ammonium acetate (975 μl) containing HPMMA-d3 standard (667 ng) and filtered through a 0.2 mm PTFE membrane. Two microliters of the samples were applied on a UPLC-MS/MS instrument, and three multiple reaction monitoring (MRM) transitions were set up for metabolite quantification and measures of internal standard (Figs. 1Bi–1Biv). To account for urinary dilution, all values for urinary metabolites were normalized to urinary creatinine levels (mg/dl).

Fig. 1: Metabolism of crotonaldehyde (CR).

Proposed stepwise metabolism of CR with glutathione (GSH) conjugation by glutathione S-transferase (GST) followed by aldose reductase (AR)-mediated reduction and sequential renal processing to the main urinary excreted N-acetylated metabolite (3-hydroxy-1-methylpropylmercapturic acid, HPMMA) (A). Representative chromatogram of HPMMA standard showing transition (m/z 234 ➔ 105) (Bi). Multiple reaction monitoring (MRMs) of transition observed for air-exposed (Bii), 1 ppm CR-exposed (Biii) or HPMMA standard (Biv) specimens used for quantification. Two transition peaks represent HPMMA trans- and cis-isomers. Male mice were exposed (6h) to MCS (50% of smoke of 12 KY Reference 3R4F cigarettes) or CR (1 ppm; 11^th week of chronic exposure) after which urine was collected at 0–1, 1–2, 2–3, and 3–16h and HPMMA quantified; value are normalized to urinary creatinine (mg/dl) (C). Total excreted HPMMA (μg) between 0–3h and 3–16h post-air, CR or MCS exposure were calculated (D). Note: Baseline urinary HPMMA (t=0) was collected and measured after a matched air exposure (Air; 6h). Values are mean ± SE. Abbr.: AR, aldose reductase; CGT, cysteinylglycine transpeptidase; CR, crotonaldehyde; GGT, gamma-glutamyl transpeptidase; Glu, glutamate; Gly, glycine; GSH, glutathione, GST, glutathione s-transferase; HPMMA, 3-hydroxy-1-methylpropylmercapturic acid; MCS, mainstream cigarette smoke; NADPH, nicotinamide adenine dinucleotide phosphate; NAT, n-acetyltransferase. *, p<0.05 compared with HEPA, ^#, p<0.05 compared with CR.

Cardiovascular Outcomes

Non-invasive blood pressure

During the chronic exposure study, blood pressure was measured in male mice using the CODA tail-cuff blood pressure system (Kent Scientific; Torrington, CT) as described (Conklin et al., 2019a). Mice were acclimated for 5 consecutive days to the CODA system prior to collecting data. Blood pressure measurements were made immediately following an exposure once per week beginning in the second week of exposure and continuing for the remainder of the study. Briefly, mice (n=15 in both groups; 30 mice total) were put in a holding chamber on a warming platform, and 25 measurement cycles were performed on each mouse with a five second interval between each cycle (approx. 30 min).

Hemodynamics by telemetry

To measure real-time blood pressure and heart rate during CR exposure, mice were implanted with a pressure cannula in the aortic arch via the left carotid artery for radiotelemetry recordings (PA-C10; DSI, St. Paul, MN). Each implanted mouse was given one week post-surgery to recover before being placed in an exposure chamber for 15 min of baseline recording prior to onset of CR exposure; an additional 15–30 min baseline recording was taken at the end of the exposure. There were two CR exposure protocols: 1) 9 min session done twice in 1h; and, 2) 1 ppm for 3h, then increased to 3 ppm for 3h followed by a 15 min air exposure washout. Systolic blood pressure (mmHg), diastolic blood pressure (mmHg), mean blood pressure (mmHg), pulse pressure (mmHg), and heart rate (bpm) were continuously recorded (1 kHz).

Echocardiography

Echocardiography was performed in chronically-exposed male WT mice using a Vevo-770 echocardiography system (FUJIFILM Visual Sonics; Toronto, Canada). Body temperature was maintained (36.5–37.5°C) using a rectal thermometer interfaced with a servo-controlled heat lamp. Mice were anesthetized with 2% isoflurane and maintained under anesthesia with 1.5% isoflurane during the procedure. The 707-B (30MHz) scan head was used to obtain 2D images (100 fps) of the parasternal long axis. M-modes were taken from the same anatomical position. An apical four-chamber view of the heart was obtained, and pulse wave Doppler readings were taken of the mitral valve. Pulse wave Doppler readings were also taken in the ascending aorta, the innominate artery, and the left common carotid artery and in the descending aorta distal the left subclavian artery. Additional pulse wave Doppler readings were taken at the branches of the descending aorta and both the right and left renal arteries. Pulse wave velocity was calculated by dividing the distance (mm) between the left subclavian artery and the right and left renal arteries by the pulse wave travel time (ms).

Vascular Reactivity

Physiological buffers

Physiological salt solution (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 (10 mM) substituted equimolar K⁺ for Na⁺ (Jin et al., 2019b).

Aorta isolation and preparation

Exposed mice were anesthetized (sodium pentobarbital, ≈150 mg/kg, i.p.), and the aorta was removed via mid-ventral thoracotomy. Aortas were cleaned of perivascular adipose tissue and cut into rings. Thoracic aorta rings (3–4 mm) were hung on stainless steel hooks in 15-ml water-jacketed organ baths or in 5-ml heated organ baths of a MultiWire Myograph System 620M (DMT; Hinnerup, Denmark). The organ baths contained PSS bubbled with 95% O₂:5% CO₂ at 37°C. After 10 min without tension, aortic rings were equilibrated to ≈1 g (9.8 mN) loading tension over 1h. All rings were stimulated with High K⁺ to test for viability, washed three times with PSS over 30 min, and re-equilibrated to 1 g resting tension. The rings were then stimulated again with High K⁺ followed by three bath changes and a re-equilibration to resting tension. Transducer signals were recorded using LabChart software.

Post-CR exposure vascular evaluation

To test whether CR exposure altered vascular reactivity, the following responses were measured: 1) contractions induced by High K⁺; 2) concentration-dependent contractions of phenylephrine (PE; 0.1 nM-10 μM); 3) concentration-dependent relaxations of acetylcholine (ACh; 0.1 nM-10 μM) in PE-precontracted rings; and, 4) concentration-dependent relaxations of sodium nitroprusside (SNP; 0.01 nM-10 μM; nitric oxide (NO) donor) in PE-precontracted rings following addition of L-N^G-Nitro arginine methyl ester (L-NAME) (100 μM) and addition of ACh (10 μM). Measures of efficacy (Emax: contractions normalized to aortic length; percentage relaxation of PE contraction)(Jin et al., 2018) and sensitivity (EC₅₀, effective concentration producing 50% response, i.e., cumulative concentration responses normalized to 100% with interpolation of EC₅₀) were calculated as before (Conklin et al., 2009). The L-NAME effect on PE-induced tension was calculated as the ratio: [PE tension post-L-NAME+PE / PE tension pre-L-NAME].

Systemic Outcomes

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 (Conklin et al., 2017b).

Plasma Biomarkers

Plasma total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, albumin, total protein, aspartate transaminase (AST), alanine transaminase (ALT), 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 (Conklin et al., 2017b).

Glucose Tolerance Evaluation

In the 10^th week of the chronic exposure, a glucose tolerance test (GTT) was performed in male WT mice immediately after a 6h exposure (i.e., 6h fast) by injecting D-glucose (1 mg/g BWT, i.p., saline) and measuring blood glucose levels from tail blood using a handheld glucometer as previously described (Conklin et al., 2017b).

Flow Cytometry

Circulating Angiogenic Cells (CACs)

Blood CACs were identified by flow cytometry and quantified as events double positive for Flk-1 (endothelial; Vegfr2 homolog) and Sca-1 (hematopoietic stem cell) as previously described (Wheat et al., 2011).

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 (Conklin et al., 2017b) with slight modifications. Briefly, aliquots of whole blood were diluted with 400 μL of HEPES-Tyrode solution before fixation using 16% paraformaldehyde at room temperature. Red blood cells were lysed by dilution with MilliQ water, and subsequent collection of lysed cells was performed by centrifugation at 400xg for 5 min. The collected cells were incubated with 1% Fc Block (5 μL) for 10 min before staining with FITC-labeled anti-CD41 and APC-labeled antiCD11b or isotype matched negative controls (FITC IgG1 and APC IgG2b kappa) for 30 min. Stained cells were washed with HEPES-Tyrode solution containing 1% BSA, centrifuged at 400xg 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 the stained cells; a minimum of 20,000 events was collected for each sample.

Leukocyte Subpopulations

Circulating immune cell populations were analyzed as previously described (Conklin et al., 2017b). Monocytes double positive for Ly6C and CD62L were defined as a Ly6C^high subpopulation.

RNA Isolation & PCR Analysis

Real time quantitative PCR (RT-qPCR) analyses were performed on lung tissues of chronically-exposed male WT mice as described (Haberzettl et al., 2009). Expressions of a select set of inflammatory pathway gene mRNAs in the lungs of air- and CR-exposed mice were tracked based on previous chronic study with acrolein in mice (Conklin et al., 2017a). Primers for β−2 microglobulin (B2M) and chemokine ligand 15 (CXCL15) were purchased from Qiagen (Gaithersburg, MD), and primers for interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), glutathione S-transferase pi 1 (GSTP1), and ribosomal protein lateral stalk subunit P0 (RPLP0) were purchased from IDT Technologies (Coralville, IA). Relative mRNA amount was calculated by the ΔΔCT method using RPLP0 as control.

Statistics

Data are presented as mean ± standard error of mean (SE). Mann-Whitney U Rank sum tests with Bonferroni’s post-test were used for data comparison between two groups, and Kruskal-Wallis ANOVA on ranks with Dunn’s post-test was used when comparing more than two groups (SigmaPlot, ver. 12.5; Systat Software, Inc., San Jose, CA). Statistical significance was set at p<0.05.

RESULTS

Urinary Crotonaldehyde Metabolite in Cigarette Smoke- and Crotonaldehyde-Exposed Mice

The primary urinary metabolite of CR, HPMMA, is associated with combusted tobacco, and the number of cigarettes per day is a significant predictor of increased urinary HPMMA (Fig. 1A) (Bagchi et al., 2018). To better understand how CR-alone exposure relates to MCS exposure from a known number of cigarettes, we measured urinary HPMMA by UPLC-MS/MS (Figs. 1Bi–1Biv) as a biomarker of CR exposure (Conklin et al., 2018). For this, male mice were exposed to MCS, and urine was collected immediately after this single exposure session. Urinary HPMMA levels were highest within the first hour post-MCS exposure compared with undetectable urinary HPMMA after filtered air exposure (performed in same mice on the previous day) (Fig. 1C) (Conklin et al., 2018). Unlike humans, laboratory-housed, naïve mice have a low level (near detection limit) of HPMMA in urine, indicating minimal CR exposure via exogenous or endogenous sources (ambient air, food, water, intermediary metabolism). In the chronic CR (1 ppm) exposure study, the HPMMA level peaked within the first hour, and the peak level was about 2-times that measured following MCS exposure (Fig. 1C), indicating that MCS exposure was equivalent to about 0.5 ppm of continuous CR exposure. Although CR and MCS excretion profiles were qualitatively similar, after calculating absolute amount of urinary HPMMA excreted in the first 3h post-exposure and subsequently overnight (3–16h post-exposure), CR exposure led to a significantly greater amount of HPMMA excreted in the first 3h post-exposure compared with mice exposed to either HEPA-filtered air (p<0.001) or MCS (p<0.001) (Fig. 1D). However, total urinary HPMMA (μg) from overnight samples was significantly increased in MCS-exposed mice compared with mice exposed to either HEPA (p=0.002) or CR (p=0.006) (Fig. 1D) indicating differential excretion of HPMMA in MCS-exposed compared with CR only-exposed mice.

Crotonaldehyde and Hemodynamics

Acute

Because CR directly dilates blood vessels (Jin et al., 2020) and CR is metabolized and excreted rather quickly, the acute effects of CR inhalation exposure on hemodynamics were studied using invasive blood pressure monitoring via radiotelemetry. Representative tracings show that blood pressure (Fig. 2A) and heart rate (HR) (Fig. 2B) dropped rapidly upon CR exposure (1 ppm, 9 min), and both parameters reversed toward baseline upon cessation of CR exposure. Longer exposures to 1 ppm and then 3 ppm CR led to more sustained and level-dependent drops in blood pressures, especially diastolic pressure (Figs. 2C & 2E), while HR was less affected (Figs. 2D & 2F). These data show that acute CR exposure (1–3 ppm) can rapidly, yet reversibly, depress systemic arterial blood pressure.

Fig. 2: Hemodynamic effects of inhaled crotonaldehyde (CR) in mice.

Aortic arch blood pressure was measured via invasive radiotelemetry in a male C57BL/6J mouse during an acute CR exposure (9 min, 1 ppm). Diastolic blood pressure (DBP; A) and heart rate (HR; B) fell rapidly upon onset of CR exposure, and both parameters recovered quickly toward baseline levels within 10–12 min after ceasing CR exposure. Aortic arch blood pressure was measured via invasive radiotelemetry in male C57BL/6J mice during an acute CR exposure (3h, 1 ppm; 3h, 3 ppm) to simulate our acute and chronic exposure conditions. Although only modest drops in DBP (C) and HR (D) were observed upon beginning 1 ppm CR exposure, there were much more noticeable drops in DBP (C) and HR (D) when the CR level was increased to 3 ppm. As with brief CR exposure (9 min, 1 ppm), HR recovered quickly to baseline levels within 5 min of ceasing CR exposure. However, DBP was slow to recover and did not reach baseline levels after 15 min. Summary data of two mice exposed to CR (1 and 3 ppm, 3h each) showed that all blood pressures (systolic, mean, diastolic; E) were suppressed equally by CR exposure whereas mean HR was not (F). Blood pressure and heart rate were measured non-invasively by tail cuff method in C57BL/6J male mice weekly (within 1h after exposure) during the chronic air and CR exposures (1 ppm; 6h/d, 5 d/week, 12 weeks) (G, H). Blood pressure (mm Hg) was significantly lower in CR-exposed mice compared with air-exposed control mice over several weeks of the latter half of the exposure (G), but there was no consistent change in HR (bpm) throughout (H). Values = mean ± SE (n=9–10 mice per group, G, H; or, n=2, E, F); a, p<0.05 vs air control; b, p<0.05 vs baseline.

Chronic

To track chronic changes in hemodynamics, blood pressure and HR were measured each week immediately following a 6h exposure by non-invasive tail cuff approach in both air- and CR-exposed male mice (Figs. 2G & 2H). Compared with the air-exposed control group, CR-exposed mice had significantly lower systolic, mean, and diastolic blood pressures in the seventh, eighth, and tenth weeks of exposure. Diastolic and mean blood pressures also were significantly decreased in the CR-exposed mice in the fifth week compared with the baseline measurements made during the second week of exposure (Fig. 2G). Heart rates were not different between groups (Fig. 2H). Interestingly, at the end of the study, HR was significantly higher in mice exposed to CR than in air-exposed control mice measured by echocardiography under isoflurane anesthesia (Table 1). Isovolumic relaxation time (IVRT) also was significantly decreased in CR-exposed mice compared with air-exposed controls as detected by echocardiography (Table 1). There was no effect of chronic CR exposure on aortic stiffness as indexed by pulse wave velocity (PWV) and measured by ultrasound (Table 1).

Table 1.

Echocardiographic measures in male C57BL/6J mice chronically exposed to air or crotonaldehyde (CR; 1 ppm).

	Exposure
Parameter	Air Control	CR (1 ppm)
BWT (g)	29.1 ± 0.5	28.6 ± 0.5
HR (bpm)	476 ± 20	536 ± 13*
EDV (μL)	42 ± 7	41 ± 2
ESV (μL)	12 ± 2	11 ± 1
SV (μL)	30 ± 4	30 ± 2
EF (%)	72 ± 1	74 ± 2
CO (mL/min)	13.9 ± 1.4	16.2 ± 0.7
LVIDd (mm)	3.7 ± 0.1	3.4 ± 0.1
LVIDs (mm)	2.1 ± 0.1	1.9 ± 0.2
LVPWd (mm)	0.8 ± 0	1.0 ± 0.1
LVPWs (mm)	1.3 ± 0.1	1.4 ± 0.1
LVAWd (mm)	1.1 ± 0.1	1.1 ± 0
LVAWs (mm)	1.6 ± 0.1	1.6 ± 0
LVM (mg)	105.4 ± 6.9	103.6 ± 6.8
IVRT (ms)	13.3 ± 0.9	9.6 ± 1.2*
RWT (mm)	0.46 ± 0.03	0.56 ± 0.05
PWV (mm/s)	2.8 ± 0.1	2.9 ± 0.1

Values = mean ± SE (n=5 mice per group); Abbr.: BWT, body weight; CO, cardiac output; CR, crotonaldehyde; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; HR, heart rate; IVRT, isovolumic relaxation time; LCC, left coronary cusp; LVAWd, left ventricular anterior wall thickness at end-diastole; LVAWs, left ventricular anterior wall thickness at end-systole; LVIDd, left ventricular internal diameter diastole; LVIDs, left ventricular internal diameter systole; LVM, left ventricular mass; PWV, pulse wave velocity; RWT, relative wall thickness; SV, stroke volume

^*p<0.05 compared with air control based on Bonferroni’s post-test.

Chronic Exposure to Crotonaldehyde and Vascular Reactivity

As the aorta is an important site of systemic vascular pathology including endothelial dysfunction, aortic stiffness, aneurysm, and atherosclerosis, the aorta was isolated and examined by isometric myography for changes in vascular function using pharmacological agents: PE (contraction), ACh (endothelium-dependent relaxation), or SNP (endothelium-independent relaxation). There were no differences in aortic contractile efficacy and sensitivity to PE between groups. However, the ACh-induced relaxations were significantly stronger (% relaxation; Fig. 3A) and more potent (i.e., enhanced sensitivity; EC₅₀ rightward shift; Fig. 3B) in the chronic CR group versus the air control group (Tables 2 & 3). Importantly, the endothelium-independent relaxations of SNP (both efficacy and sensitivity) were not different between groups (Figs. 3C & 3D; Tables 2 & 3), indicating chronic CR exposure had a preferential and persistent effect on altering the endothelial cell response to ACh. Moreover, perhaps this ‘enhanced relaxation response’ may have contributed to the lower arterial blood pressures measured both in acute and in chronic CR-exposed mice compared with air-exposed control mice (Fig. 2). To test whether the enhanced aortic response to ACh was related to NO, aortic responses to PE, ACh, and SNP were measured in the presence of L-NAME, a nitric oxide synthase inhibitor. ACh-induced relaxations were ablated by L-NAME, indicating that CR did not alter canonical pathway of ACh stimulation of NO-dependent relaxation. Similarly, L-NAME did not alter aortic responses to PE or SNP, indicating chronic CR did not likely alter either basal NO production (affect PE response) or smooth muscle sensitivity to NO (affect SNP response) (Tables 2 & 3).

Fig. 3: Vascular toxicity of chronic crotonaldehyde (CR) inhalation exposure in mice.

Aortic function was measured ex vivo in male C57BL/6J mice following 12 weeks of exposure to either air or CR (1 ppm, 6h/d, 5 d/week, 12 weeks). 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. CR exposure significantly enhanced ACh sensitivity (B). There were, however, no changes in aortic efficacy (% relaxation; C) and sensitivity (EC₅₀; D) to the endothelium-independent vasorelaxant sodium nitroprusside (SNP). Values = mean ± SE (n=10 mice per group); *, p<0.05 vs air control.

Table 2.

Effects of either air or crotonaldehyde (CR; 1 or 3 ppm) exposure on efficacy responses of isolated thoracic aorta of C57BL/6J (wild type, WT) and TRPA1-null mice to pharmacological agents in the absence and presence of nitric oxide synthase inhibitor (L-NAME).

Exposure Conditions			PE, Emax [mN/mm]		ACh, Emax [%]
Strain (sex)	Level (ppm)	Duration	Air	CR	Air	CR
WT	1	4 days	4.1 ± 0.7	3.9 ± 0.6	−53.8 ± 4.4	−65.2 ± 5.2*
TRPA1-null	1	4 days	2.4 ± 0.5	2.1 ± 0.3	−64.6 ± 3.4	−59.8 ± 4.0
WT	3	4 days	4.0 ± 0.7	3.7 ± 0.7	−49.3 ± 4.6	−57.8 ± 4.4
WT	1	12 weeks	3.2 ± 0.6	3.6 ± 0.7	−51.2 ± 5.6	−62.6 ± 3.8
WT (f)	1	4 days	3.5 ± 0.6	4.0 ± 0.6	−76.4 ± 3.8	−77.4 ± 4.6
Exposure Conditions			PE Ratio, +L-NAME		ACh, [%], +L-NAME		SNP, [%], +L-NAME
Strain (sex)	Level (ppm)	Duration	Air	CR	Air	CR	Air	CR
WT	1	4 days	1.3 ± 0.03	1.5 ± 0.05*	−1.4 ± 0.5	−0.9 ± 0.5	−96.9 ± 0.7	−98.4 ± 0.6
TRPA1-null	1	4 days	1.8 ± 0.1	2.0 ± 0.2	−1.7 ± 1.1	0 ± 0	−99.7 ± 0.2	−99.6 ± 0.2
WT	3	4 days	1.4 ± 0.04	1.5 ± 0.04#	0 ± 0	0 ± 0	−98.6 ± 1.3	−99.1 ± 0.7
WT	1	12 weeks	1.5 ± 0.1	1.5 ± 0.1	−0.3 ± 0.2	−0.8 ± 0.5	−97.3 ± 1.3	−98.3 ± 0.6
WT (f)	1	4 days	1.5 ± 0.1	1.5 ± 0.05	−0.5 ± 0.4	−1.9 ± 1.5	−99.4 ± 0.5	−99.3 ± 0.3

Values = mean ± SE (n=9–11 mice per group); all values are of male mice except where (f) = female mice. Abbr.: ACh, acetylcholine; ACh, Emax and SNP, Emax equal percentage relaxation of PE-induced contraction; CR, crotonaldehyde; L-NAME, L-N^G-Nitro arginine methyl ester; +L-NAME, 100 μM added after PE but before addition of ACh (10 μM) and SNP (100 μM); PE, phenylephrine; PE, Emax, PE contractions normalized to aortic length; PE Ratio, PE+L-NAME tension/PE tension; SNP, sodium nitroprusside; TRPA1, transient receptor potential ankyrin 1

^*p<0.05 compared with air control based on Bonferroni’s or Dunn’s post-test

^#0.05≤p≤0.10 compared with air control based on Bonferroni’s or Dunn’s post-test.

Table 3.

Effects of either air or crotonaldehyde (CR; 1 or 3 ppm) exposure on sensitivity responses of isolated thoracic aorta of C57BL/6J (wild type, WT) and TRPA1-null mice to pharmacological agents.

Exposure Conditions			PE, EC50 [nM]		ACh, EC50 [nM]		SNP, EC50 [nM]1
Strain (sex)	Level (ppm)	Duration	Air	CR	Air	CR	Air	CR
WT	1	4 days	128 ± 30	201 ± 55	285 ± 79	135 ± 31#	11.5 ± 0.7	9.2 ± 0.9#
TRPA1-null	1	4 days	198 ± 34	252 ± 61	156 ± 30	296 ± 124	9.9 ± 0.4	9.9 ± 0.5
WT	3	4 days	139 ± 31	167 ± 26	236 ± 100	102 ± 9	18.8 ± 2.3	12.7 ± 2.8*
WT	1	12 weeks	108 ± 26	231 ± 109	238 ± 53	78 ± 8*	12.0 ± 2.0	8.6 ± 0.6
WT (f)	1	4 days	157 ± 45	234 ± 70	100 ± 15	68 ± 13	8.5 ± 1.9	7.5 ± 1.0

Values = mean ± SE (n=9–11 mice per group); all values are of male mice except where (f) = female mice.

¹SNP EC₅₀ measured in presence of L-NAME, 100 μM.

Abbr.: ACh, acetylcholine; CR, crotonaldehyde; EC₅₀, half maximal effective concentration; PE, phenylephrine; SNP, sodium nitroprusside; TRPA1, transient receptor potential ankyrin 1

^*p<0.05 compared with air control based on Bonferroni’s or Dunn’s post-test

^#0.05≤p≤0.10 compared with air control based on Bonferroni’s or Dunn’s post-test.

Acute Exposure to Crotonaldehyde and Vascular Reactivity

Because of the endothelium-dependent changes observed in the aortas of chronic CR-exposed WT male mice, vascular function was also measured in isolated aortas of WT male mice after acute (4-day) air and CR exposures (1 and 3 ppm). In the aortas of 4-day, CR-exposed (1 ppm) mice, there also was a significant increase in ACh % relaxation (efficacy) as well as a slightly enhanced sensitivity to ACh (Figs. 4A & 4B). Interestingly, there also was slightly increased sensitivity (0.05<p<0.10) in the aortic response to SNP of CR-exposed mice compared with air-exposed control mice, although no change in efficacy of SNP was observed (Figs. 4C & 4D). To test whether these changes were level-dependent, mice were exposed to air or 3 ppm CR for 4 days. Although aortic responses (both efficacy and sensitivity) to ACh and SNP were consistently enhanced, only sensitivity to SNP reached statistical significance (Suppl. Fig. 1; Table 3). In comparison, acute CR exposure (to 1 ppm) had no effect on female WT mice aortic efficacy or sensitivity (EC₅₀) to PE, ACh, and SNP (Tables 2 & 3), indicating vascular changes due to CR exposure at 1 ppm were sex-dependent. Because the changes in vascular reactivity after acute and chronic CR exposures appeared to involve NO, the effect of blocking NO formation with L-NAME was tested. L-NAME enhanced subsequent PE-induced tension development. The PE tension ratio (i.e., Tension_(PE+L-NAME) / Tension_(PE)) was enhanced significantly by 4-day, 1 ppm CR exposure (and slightly less so by 3 ppm) compared with air control group (Table 2). L-NAME effectively ablated ACh-induced relaxation in all groups (<2% ACh relaxation remained), demonstrating sole reliance of the aorta on endothelial-dependent NO release by ACh regardless of exposure (Table 2). Although L-NAME had no effect on the strength of SNP relaxations (Table 2), L-NAME revealed a significant increase in SNP sensitivity in aortas from 3 ppm CR-exposed mice (0.5<P<0.10 at 1 ppm) compared with air controls (Table 3). These data suggest that acute CR inhalation exposure rapidly enhanced the sensitivity of aortas to NO-dependent relaxation.

Fig. 4: Vascular toxicity of acute crotonaldehyde (CR) inhalation exposure in C57BL/6 mice.

Aortic function was measured ex vivo in male C57BL/6J mice following 4 days of exposure to either air or CR (1 ppm, 6h/d, 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. CR exposure significantly enhanced ACh efficacy (A) and slightly shifted sensitivity (B). There were, however, no changes in aortic efficacy (% relaxation; C) and sensitivity (EC₅₀; D) to the endothelium-independent vasorelaxant sodium nitroprusside (SNP). Values = mean ± SE (n=8–10 mice per group); *, p<0.05 vs air control. ^#, 0.05≤p≤0.10 vs air control.

Chronic Exposure to Crotonaldehyde and Circulating Angiogenic Cells (CACs)

Decreased levels of CACs have been associated with increased CVD risk, and it is thought that CAC levels serve as a predictor of overall cardiovascular health and future cardiovascular events (Hill et al., 2003). Altered CAC levels are noted in individuals exposed to secondhand smoke (Heiss et al., 2008) and during smoking cessation (Kondo et al., 2004). Previously, we showed that both acute (Wheat et al., 2011) and chronic (Conklin et al., 2017b) acrolein exposures in mice suppressed CACs by 50%. Despite the changes seen with acrolein, there was no significant change in the blood level of CACs (Flk-1⁺/Sca-1⁺ cells) after chronic CR exposure (1 ppm; Suppl. Fig. 2).

Acute Exposure to Crotonaldehyde and CACs

Acute exposure to 1 ppm CR increased the level of Sca-1⁺ cells (Fig. 5C). Sca-1⁺ cells are the hematopoietic stem cell pool from which CACs are derived. However, there was no change in the levels of CACs in WT male mice exposed to CR (Fig. 5D). Acute exposure to 3 ppm CR did not change the levels of Sca-1⁺ cells or CACs (Suppl. Fig. 3). Likewise, female WT mice had no change in the levels of CACs or Sca-1⁺ cells after acute CR (1 ppm) exposure (Suppl. Fig. 4).

Fig. 5: Effects of acute crotonaldehyde (CR) exposure on circulating stem cells in mice.

Hematopoietic stem cells (Sca-1⁺) and circulating angiogenic cells (CACs) were measured by flow cytometry in male C57BL/6J (wild type, WT) and TRPA1-null male mice exposed to either air or CR (1 ppm, 6h/d) for 4 days. The flow cytometry gating scheme for lymphocytes (A) and blood CAC (B) detection is shown for CR-exposed WT mice. After 4 days of exposure to CR, the level of circulating Sca-1⁺ cells was significantly increased in WT mice (C), however, no change in the level of CACs (Flk-1⁺/Sca-1⁺ cells) was detected relative to air-exposed control mice (D). In contrast, TRPA1-null mice had no change in blood levels of Sca-1⁺ cells (E) but had modestly increased levels of CACs (Flk-1⁺/Sca-1⁺ cells (F) after 4 days of exposure to CR compared with air-exposed control. Values = mean ± SE (n=6–10 mice per group); *, p<0.05 vs air control; ^#, 0.05≤p≤0.10 vs air control.

Chronic Exposure to Crotonaldehyde and Platelet Activation

Although both acute acrolein and acute MCS exposures have been shown to increase platelet activation in mice (Sithu et al., 2010), chronic acrolein did not affect total platelet count or the number of PLAs (Conklin et al., 2017b). Chronic exposure of mice to 1 ppm CR did not have a significant effect on the number of PLAs (as measured by flow cytometry) nor did CR exposure alter total platelet count in CBC (measured by Hemavet) (Table 4). These data suggest that, like acrolein, chronic exposure to 1 ppm CR did not induce persistent platelet activation.

Table 4.

Hematological measures in male C57BL/6J mice chronically exposed to air or crotonaldehyde (CR; 1 ppm).

	Exposure
Parameter	Air Control	CR (1 ppm)
White Blood Cell (K/μL)	1.96 ± 0.19	2.12 ± 0.18
Neutrophils (K/μL)	0.50 ± 0.08	0.60 ± 0.07
Lymphocytes (K/μL)	1.41 ± 0.12	1.47 ± 0.14
Monocytes (K/μL)	0.05 ± 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)	8.30 ± 0.15	8.32 ± 0.14
Hemoglobin (g/dL)	12.1 ± 0.1	12.0 ± 0.1
Hematocrit (%)	35.1 ± 0.7	34.8 ± 0.5
Mean Corpuscular Volume (fL)	42.3 ± 0.2	41.8 ± 0.3
Mean Corpuscular Hemoglobin (pg)	14.6 ± 0.2	14.4 ± 0.3
Mean Corpuscular Hemoglobin Concentration (g/dL)	34.6 ± 0.5	34.5 ± 0.5
Red Cell Distribution Width (%)	18.0 ± 0.3	18.0 ± 0.2
Platelets (K/μL)	728 ± 30	694 ± 31
PLAs (% of leukocytes)	7 ± 1	9 ± 1
Mean Platelet Volume (fL)	4.2 ± 0.0	4.3 ± 0.1
Blood Immune Cells
NK1.1+-Cells (count/μL)	8.22 ± 1.57	7.00 ± 1.11
CD19+ B-Cells (count/μL)	266.72 ± 31.36	291.24 ± 51.23
CD4+ T-Cells (count/μL)	31.43 ± 2.91	33.79 ± 3.10
CD8+ T-Cells (count/μL)	0.85 ± 0.17	1.04 ± 0.18
CD11b+ Monocytes (count/μL)	25.01 ± 1.95	27.06 ± 2.65
Ly6Chigh Cells (count/μL)	1.2 ± 0.2	1.9 ± 0.4
Ly6Clow Cells (count/μL)	23.8 ± 1.9	25.2 ± 2.5
Gr1+ Granulocytes (count/μL)	69.87 ± 5.21	82.52 ± 7.51

Values = mean ± SE (n=9–10 mice per group). Abbr.: CR, crotonaldehyde; PLAs, platelet-leukocyte aggregates.

Acute Exposure to Crotonaldehyde and Platelet Activation

Acute CR (1 or 3 ppm) had significant effects on PLAs in male WT mice (Table 5). Wherein CR exposure at 1 ppm decreased the level of PLAs (−52%; p=0.02), CR exposure at 3 ppm almost doubled the level of PLAs (+85%; p=0.03) (Table 5). However, acute exposure (4-day, 1 ppm) had no effect on level of PLAs in female WT mice (Table 1).

Table 5.

Systemic changes in male C57BL/6J (WT) and TRPA1-null mice acutely exposed to air or crotonaldehyde (CR; 1 or 3 ppm).

	Exposure Duration (4 days)
	Air Control		CR (1 ppm)		CR (3 ppm)
Variable	WT	TRPA1-null	WT	TRPA1-null	WT
Change in BWT (g)	−1 ± 0	−1 ± 1	−1 ± 0	−1 ± 0	−1 ± 0
Heart/BWT (mg/g)	4.9 ± 0.1	5.3 ± 0.2	5.2 ± 0.1	5.0 ± 0.2	4.9 ± 0.1
Lung/BWT (mg/g)	5.6 ± 0.1	5.7 ± 0.2	6.0 ± 0.1	5.4 ± 0.2	5.4 ± 0.1
Liver/BWT (mg/g)	44.6 ± 0.6	47.8 ± 2.0†	43.7 ± 1.5	47.8 ± 1.0†	43.6 ± 0.9
Kidney/BWT (mg/g)	12.5 ± 0.1	14.2 ± 0.4†	12.7 ± 0.3	12.9 ± 0.2*	12.4 ± 0.2
Blood Glucose (mg/dL)	177 ± 5	167 ± 8	172 ± 10	169 ± 8	175 ± 4
CBC Parameters
White Blood Cell (K/μL)	1.57 ± 0.13	1.70 ± 0.24	1.96 ± 0.16	1.57 ± 0.27	1.55 ± 0.14
Neutrophils (K/μL)	0.39 ± 0.06	0.59 ± 0.12	0.62 ± 0.10#	0.47 ± 0.09	0.29 ± 0.03
Lymphocytes (K/μL)	1.13 ± 0.09	1.06 ± 0.12	1.28 ± 0.10	1.06 ± 0.18	1.22 ± 0.11
Monocytes (K/μL)	0.04 ± 0	0.05 ± 0.01	0.06 ± 0.01	0.08 ± 0.04	0.04 ± 0
Eosinophils (K/μL)	0.01 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Basophils (K/μL)	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
Red Blood Cell (M/μL)	8.62 ± 0.10	8.40 ± 0.13	8.71 ± 0.10	8.72 ± 0.10	8.83 ± 0.11
Hemoglobin (g/dL)	11.7 ± 0.2	11.6 ± 0.2	11.7 ± 0.2	12.1 ± 0.1	11.9 ± 0.1
Hematocrit (%)	39.2 ± 0.8	37.8 ± 0.7	37.5 ± 0.7	39.5 ± 0.4	42.1 ± 0.5
Mean Corpuscular Volume (fL)	45.9 ± 0.5	45.0 ± 0.3	43.1 ± 0.5*	45.3 ± 0.3†	47.7 ± 0.3
Mean Corpuscular Hemoglobin (pg)	13.6 ± 0.2	13.9 ± 0.1	13.5 ± 0.3	13.8 ± 0.1	13.5 ± 0.1
Mean Corpuscular Hemoglobin Concentration (g/dL)	29.6 ± 0.6	30.8 ± 0.2	31.4 ± 1.1	30.6 ± 0.3	28.3 ± 0.1
Red Cell Distribution Width (%)	16.9 ± 0.1	18.3 ± 0.4†	17.9 ± 0.3*	17.7 ± 0.3	17.2 ± 0.3
Platelets (K/μL)	616 ± 16	869 ± 51†	670 ± 25	862 ± 56†	584 ± 15
PLAs (% of leukocytes)	6 ± 1	7 ± 3	3 ± 0*	6 ± 1	12 ± 2*
Mean Platelet Volume (fL)	4.3 ± 0	4.5 ± 0.1†	4.2 ± 0.1	4.6 ± 0.1†	4.3 ± 0

Values = mean ± SE (n=9–15 mice per group); Abbr.: BWT, body weight; CR, crotonaldehyde; PLAs, platelet-leukocyte aggregates; TRPA1, transient receptor potential ankyrin 1; WT, wild type

^*p<0.05 compared with air control based on Bonferroni’s or Dunn’s post-test

^#0.05≤p≤0.10 compared with air control based on Bonferroni’s or Dunn’s post-test

^†p<0.05 for WT vs TRPA1-null based on Dunn’s post-test.

Chronic Exposure to Crotonaldehyde and Hematology

Chronic exposures to acrolein (0.5 and 1 ppm) suppress a number of leukocyte subpopulations (Conklin et al., 2017b). Thus, hematological measures of circulating leukocytes and selective immune subpopulations were measured in mice chronically exposed to CR (1ppm, 12 weeks). There were no significant changes in major leukocyte classes or in levels of NK1.1⁺-cells, CD19⁺ B-cells, CD4⁺ or CD8⁺ T-cells, Gr1⁺ granulocytes, CD11b⁺ monocytes, or Ly6C^high or Ly6C^low monocyte sub-populations (Table 4).

Chronic Exposure to Crotonaldehyde and Cytokines

Smoking is known to induce systemic inflammation, and the risk of future cardiovascular events has been shown to be predicted by levels of pro-inflammatory cytokines (Conklin et al., 2019b). Thus, the expression levels of several key cytokine genes were measured in the lungs of mice chronically exposed to either air or CR (1 ppm). None of the mRNA levels were significantly different between exposure groups (Table 2). These data were similar to cytokine mRNA levels observed in male mice after chronic exposure to acrolein in that neither exposure induced significant changes in cytokine genes (Conklin et al., 2017b).

Systemic Toxicity of Chronic Exposure to Inhaled Crotonaldehyde

Systemic toxicity outcomes of chronic CR exposure (1 ppm) were measured as before with acrolein (1 ppm, 12 weeks) (Conklin et al., 2017b). Body weight and organ/body weight ratios and CBCs were not different between the air- and CR- exposed groups (Tables 4 & 6). Chronic CR also did not affect plasma levels of ALT and AST (liver), CK (striated muscle), and LDH (non-specific cell toxicity) enzymes, and creatinine, but did increase levels of plasma total cholesterol (+11%; p=0.04), HDL (+16%; p=0.01), triglycerides (+25%; p=0.01), albumin (+6%; p=0.01), and, modestly, total protein (+3%; p=0.08) compared with the air-exposed control group (Table 6).

Table 6.

Systemic parameters in male C57BL/6J mice exposed for 12 weeks to either air or crotonaldehyde (CR; 1 ppm).

	Exposure
Variable	Air Control	CR (1 ppm)
Change in BWT (g)	+5 ± 0	+4 ± 0
Heart/BWT (mg/g)	4.5 ± 0.1	4.6 ± 0.1
Lung/BWT (mg/g)	5.4 ± 0.2	5.4 ± 0.2
Liver/BWT (mg/g)	44.3 ± 1.6	41.8 ± 1.7
Kidney/BWT (mg/g)	12.3 ± 0.3	13.1 ± 0.5
Pancreas/BWT (mg/g)	3.9 ± 0.2	4.6 ± 0.4
Spleen/BWT (mg/g)	3.1 ± 0.2	3.0 ± 0.2
Epididymal Fat Pad/BWT (mg/g)	8.8 ± 0.5	9.0 ± 0.8
Blood Glucose (mg/dL)	160 ± 7	166 ± 12
GTT Area Under the Curve (AUC)	4753 ± 818	4333 ± 755
Plasma Measurements
Cholesterol (mg/dL)	58.83 ± 3.82	69.51 ± 2.10*
HDL (mg/dL)	41.93 ± 4.04	53.05 ± 0.94*
LDL (mg/dL)	4.94 ± 1.01	3.51 ± 0.33
Triglycerides (mg/dL)	37.12 ± 2.85	56.56 ± 7.31*
Albumin (g/dL)	2.81 ± 0.06	2.89 ± 0.07*
Total Protein (g/dL)	4.52 ± 0.08	4.67 ± 0.08#
ALT (U/I)	19.37 ± 3.76	15.95 ± 1.46
AST (U/I)	49.43 ± 13.16	46.74 ± 4.56
CK (U/I)	260.92 ± 39.83	149.57 ± 44.55
LDH (U/I)	83.03 ± 8.30	83.65 ± 4.80
Creatinine (mg/dL)	0.25 ± 0.02	0.25 ± 0.01

Values = mean ± SE (n=9–10 mice per group). Abbr.: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BWT, body weight; CK, creatine kinase; CR, crotonaldehyde; GTT, glucose tolerance test; HDL, high-density lipoprotein; LDH, lactate dehydrogenase; LDL, low-density lipoprotein

^*p<0.05 compared with air control based on Bonferroni’s post-test

^#0.05≤p≤0.10 compared with air control based on Bonferroni’s post-test.

Use of both cigarettes and moist snuff (150 g/week) increase the risk for type-2 diabetes (Persson et al., 2000), and a few studies have found associations between smoking status and systemic insulin resistance (Persson et al., 2000; Keith et al., 2016). Nonetheless, a 12-week exposure of WT mice on a normal chow diet to acrolein (1 ppm) induced no change in fasting blood glucose, glucose handling, or insulin levels (Conklin et al., 2017b). Similarly, chronic exposure of WT male mice to CR (1 ppm) had no effect on fasting blood glucose or glucose tolerance as measured by GTT Area Under the Curve (AUC) compared with air-exposed control mice (Table 6).

Systemic Toxicity of Acute Exposure to Inhaled Crotonaldehyde

Because chronic exposure to CR (1 ppm) modestly altered systemic toxicity (e.g., cholesterol, triglycerides), these factors were also measured after acute (4-day) exposure to better understand the temporal relationships between biomarkers (e.g., primary or secondary/compensatory response). Acute exposure of male mice to CR (1 or 3 ppm; 4 days) had modest to limited effects on organ and body weights (and ratios), CBC, and plasma markers. For example, male mice acutely exposed to 1 ppm CR had no change in overall body weight (Table 5), yet had an increased level of neutrophils (+60%; p=0.06), a decreased mean corpuscular volume (−6%; p<0.001), and an increased red blood cell distribution width (+6%; p=0.02) (Table 5). Plasma levels of ALT, ASK, CK, and LDH enzymes, albumin, and creatinine were unchanged with acute CR exposure in WT male mice, but levels of plasma cholesterol, LDL, triglycerides and total protein were decreased (−14%, p=0.002; −6%, p=0.02; −13%, p=0.02; −24%, p=0.002, respectively) (Table 7). Surprisingly, 4 days of CR (3 ppm) exposure in male mice had less of an effect overall on biomarkers of systemic toxicity. For example, there were no changes in levels of ALT, AST, CK, and LDH enzymes, albumin, creatinine or CBCs, with only modest effects on levels of cholesterol (+7%; p=0.09) and total protein (+5%; p=0.07) (Tables 5 & 7). Similarly, no changes in body weight, organ/body weight ratios, CBC, or plasma biomarkers were observed in female mice exposed for 4 days to 1 ppm CR (Table 1), indicating that male WT mice may be more sensitive to CR than female mice.

Table 7.

Plasma biomarkers in male C57BL/6J (WT) and TRPA1-null mice acutely exposed to air or crotonaldehyde (CR; 1 or 3 ppm).

	Exposure Duration (4 days)
	Air Control		CR (1 ppm)		CR (3 ppm)
Plasma Parameters	WT	TRPA1-null	WT	TRPA1-null	WT
Cholesterol (mg/dL)	69.97 ± 1.83	81.39 ± 3.13†	60.16 ±1.84*	67.27 ± 3.48*	74.72 ± 1.89#
HDL (mg/dL)	50.96 ± 1.21	49.95 ± 2.89	44.57 ± 2.31*	47.34 ± 2.80	53.10 ± 1.43
LDL (mg/dL)	4.49 ± 0.37	8.73 ± 0.96†	4.32 ± 0.62	4.49 ± 0.52*	5.52 ± 0.19
Triglycerides (mg/dL)	43.76 ± 1.39	40.77 ± 3.60	33.36 ± 2.39*	27.39 ± 1.66	41.31 ± 2.22
Albumin (g/dL)	2.81 ± 0.04	2.75 ± 0.10	2.65 ± 0.05	2.77 ± 0.07	2.85 ± 0.05
Total Protein (g/dL)	4.76 ± 0.07	5.11 ± 0.17†	4.45 ± 0.06*	4.83 ± 0.11†	5.01 ± 0.08#
ALT (U/I)	23.34 ± 1.25	28.91 ± 1.43	23.74 ± 2.92	33.71 ± 5.31	19.37 ± 0.63
AST (U/I)	61.99 ± 4.97	70.62 ± 4.13	56.39 ± 6.12	69.88 ± 8.82	52.86 ± 4.05
CK (U/I)	188.84 ± 21.28	222.70 ± 27.59	127.15 ± 13.94	233.81 ± 31.69	262.70 ± 33.38
LDH (U/I)	135.90 ± 7.72	138.03 ± 7.35	132.28 ± 11.21	160.28 ± 12.70	141.10 ± 10.36
Creatinine (mg/dL)	0.28 ± 0.01	0.25 ± 0.01	0.24 ± 0.01#	0.24 ± 0.01	0.31 ± 0.01

Values = mean ± SE (n=5–10 mice per group); Abbr.: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; CR, crotonaldehyde; HDL, high-density lipoprotein; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; TRPA1, transient receptor potential ankyrin 1; WT, wild type

^*p<0.05 compared with air control based on Bonferroni’s or Dunn’s post-test

^#0.05≤p≤0.10 compared with air control based on Bonferroni’s or Dunn’s post-test

^†p<0.05 for WT vs TRPA1-null based on Dunn’s post-test.

Acute Exposure to Crotonaldehyde and Vascular Responses: Role of TRPA1

Because CR is an agonist of TRPA1 in the vasculature (Jin et al., 2020), vascular function was measured in acutely CR- or air-exposed male TRPA1-null mice (1 ppm, 4-day). In contrast to significant effects observed in aorta of CR-exposed male WT mice, there were no significant changes in aortic function between CR- and air-exposed mice to any agonist (PE, ACh, SNP without or with L-NAME present) (Fig. 6; Tables 2 & 3). These data indicate that the acute effects of CR in male WT mice were mediated by TRPA1 activation.

Fig. 6: Vascular toxicity of acute crotonaldehyde (CR) inhalation exposure in TRPA1-null mice.

Aortic function was measured ex vivo in male TRPA1-null mice following 4 days of exposure to either air or CR (1 ppm, 6h/d, 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. CR exposure had no effect on efficacy (A) or sensitivity (B) of ACh-induced relaxations. Similarly, there were no CR-induced changes in aortic efficacy (% relaxation; C) or sensitivity (EC₅₀; D) in response to the endothelium-independent vasorelaxant sodium nitroprusside (SNP). Values = mean ± SE (n=10–11 mice per group).

Systemic Toxicity of Acute Exposure to Inhaled Crotonaldehyde: Role of TRPA1

In contrast to the effects observed in CR-exposed male WT mice, TRPA1-null mice had no systemic changes in CBC (Table 5), yet the mice did have a decreased kidney/body weight ratio (−9%; p<0.001) relative to air-exposed control mice (Table 5). Plasma levels of ALT, AST, CK, and LDH enzymes, albumin, and creatinine were unchanged between the air- and CR-exposed TRPA1-null mice (Table 7). Cholesterol was decreased by CR exposure in the TRPA1-null mice (−17%; p=0.003; a change also observed in WT mice at 1 ppm CR), a change due to decreased levels of LDL (−49%; p<0.001) rather than in HDL levels as observed in WT mice (Table 7). TRPA1-null mice acutely exposed to 1 ppm CR had no change in the levels of circulating Sca-1⁺ cells (Fig. 5E) but modestly increased the level of Flk-1⁺/Sca-1⁺ cells (0.05<p<0.10) (Fig. 5F). Interestingly, there was no change in the level of PLAs of TRPA1-null male mice as seen in WT male mice (Table 5).

DISCUSSION

Crotonaldehyde has been identified as a hazardous air pollutant of significant human concern, particularly as a component of MCS (International Agency for Research on Cancer, 1995), which can induce apoptosis and necrosis (Facchinetti et al., 2007; Liu et al., 2010), oxidative stress (Marescotti et al., 2016; Zhang et al., 2019), and inflammation (Zhang et al., 2019). CR is also a minor metabolite of 1,3-butadiene, a chemical associated with increased CVD risk and incidence (Divine, 1990; Matanoski et al., 1990; Penn and Snyder, 1996). Despite the well-recognized toxicity of CR, the chronic toxicity of inhaled CR and mechanisms underlying potential adverse effects are not well studied. To our knowledge, this is the first study to evaluate the cardiovascular effects of acute and chronic CR inhalation exposure in mice and to show that CR induces vascular alterations that are sensitive to TRPA1- and sex-dependent pathways.

We observe novel changes in aortic vascular function in male mice both acutely and chronically exposed to CR. In contrast to classically-defined endothelium dysfunction, i.e., impairment of endothelium-dependent dilation that necessarily precedes hypertension (Deanfield et al., 2007), CR induces functional “enhancement” of ACh-induced relaxations in isolated PE-precontracted aorta—characterized by increases in aortic sensitivity and efficacy to ACh. Although an atypical alteration, the overall change is robust (EC₅₀ shift was 3x; +15% relaxation), reproducible (both 1 ppm and 3 ppm), and persistent (both acute and chronic) as well as internally and externally consistent. For example, CR induces a ‘hypotensive phenotype’ both in real time (acutely) and post-exposure (chronically) with blood pressure being measured in two ways (invasive and non-invasively). Moreover, these findings are externally consistent with the direct CR-induced TRPA1- and NO-dependent vasorelaxation of isolated murine superior mesenteric artery (SMA) (Jin et al., 2020). A connection between inhaled CR and TRPA1 activation is supported in that aortic alterations are absent in TRPA1-null mice exposed to CR, indicating TRPA1-dependence and adding veracity to our primary observations. We also find that this enhanced aortic relaxation to ACh is, in part, due to an enhanced sensitivity of vascular smooth muscle to NO, indicating that perhaps NO bioavailability is compromised in vivo. Although speculative, perhaps, the increase in smooth muscle sensitivity to NO may be a compensatory mechanism. If so, then this phenomenon of “relaxation enhancement” due to inhaled CR should be classified as another form of ‘endothelium dysfunction.’ This remains to be discerned in future studies as does how this aortic change contributes to subsequent vascular pathophysiology, e.g., atherosclerosis, aneurysm.

Given that CR is a known agonist of TRPA1, the finding that inhaled CR triggers TRPA1-dependent changes is not necessarily surprising. TRPA1 is a non-selective, cation channel and a promiscuous receptor concentrated in sensory fibers that mediates pain reception/transmission as well as irritant-related pulmonary reflexes including cough, ‘respiratory braking’ (i.e., slowing rate and depth of breathing), and bronchoconstriction in response to CR and acrolein (Andre et al., 2008; Achanta and Jordt, 2017). Our previous study shows that the absence of TRPA1 both delays ‘respiratory braking’ response to high level acrolein (200 ppm) and promotes greater morbidity and mortality (Conklin et al., 2017a). Our present study uses much lower yet still potent levels of CR (1 and 3 ppm) that activate TRPA1 receptors (likely located in the upper airways) to induce systemic changes. As we expose mice in a whole-body setting, we cannot rule out a role of extra-pulmonary TRPA1 (e.g., neuronal or non-neuronal) in these outcomes. In fact, TRPA1 receptors are distributed in many non-neuronal sites, such as the heart, urothelium, and the vasculature including the endothelium (Earley et al., 2009; Sinharoy et al., 2017; Conklin et al., 2019a; Jin et al., 2019a). Activation of peripheral sensory TRPA1 by unsaturated aldehydes and TRPA1 agonists stimulates the release of vasoactive peptides including substance P and calcitonin gene-related protein (Trevisani et al., 2007; Russell et al., 2014); these peptides directly contribute to pain, locally-increased blood flow, increased vascular permeability, and increased leukocyte binding and extravasation—a collective process referred to as ‘neurogenic inflammation,’ which may contribute in a meaningful way to systemic changes we observe herein. Nonetheless, the precise anatomical location of TRPA1 activated by inhaled CR that is responsible for subsequent systemic effects in our study is unknown, and this question will need to be addressed using more specific models of TRPA1 deletion.

A potential site of TRPA1 that plausibly contributes to CR-induced hypotension is in the vasculature itself. As CR inhalation induces a relatively rapid-onset hypotension accompanied by decreased diastolic blood pressure, we infer this is due to dilation of resistance blood vessels. Because heart rate change is variable (and recovers), it is likely that persistent hypotension is independent of bradycardia and results from vascular dilation. Notably, and as mentioned above, CR potently relaxes isolated murine SMA in a TRPA1- and NO-dependent manner providing mechanistic support for CR-induced change in peripheral vascular resistance (Jin et al., 2020). Precedent for TRPA1-dependent hypotension comes from a study of infused anesthetic propofol (Sinha et al., 2015), and this effect is further supported by TRPA1 dependence of propofol-induced inhibition of U46,619-stimulated contractions in isolated murine coronary arteries (Sinharoy et al., 2017). Again, the precise location of TRPA1 responsible for this effect is unknown and will require a more specific model of TRPA1 deletion to test.

Although TRPA1 activation is known to trigger ‘neurogenic inflammation,’ we did not observe increases in measures of pulmonary inflammation in either acute or chronic CR exposure studies. We targeted known inflammatory markers in the lungs, and although IL-1β mRNA increases (approx. 2.41x), there is no widespread sign of chronic inflammation in the lungs and likewise no change in circulating leukocytes or specific immune cell differentials in either acute or chronic CR exposures. Of course, CR exposure could lead to the suppression of immune function (Yang et al., 2013; Wang et al., 2018) independent of leukocyte numbers as described for alveolar macrophages following acrolein exposure (Lambert et al., 2005). However, these other studies of CR or acrolein are hard to compare with our study due to the high level CR doses and concentrations given by intratracheal instillation that led to mortality (Wang et al., 2018) or to the use of in vitro testing (Lambert et al., 2005; Yang et al., 2013). For our present study, we infer that the immune system overall appears a less sensitive “target” of CR than is the vasculature.

In addition to alterations in vascular function, chronic CR exposure also increases other pro-atherosclerotic risk factors: cholesterol and triglycerides. High levels of cholesterol and triglycerides are known risk factors of ischemic (Jeppesen et al., 1998) and coronary heart disease (Sarwar et al., 2007). Triglycerides release pre-formed mediators of oxidative stress that influence endothelial cell function by stimulating intracellular production of reactive oxygen species (Wang et al., 2009), inducing endothelium dysfunction (Kajikawa et al., 2016; Lucero et al., 2016) and promoting atherosclerotic plaque formation (Rapp et al., 1994). Although levels of both cholesterol and triglycerides increase in our chronic study, neither increases after acute (4d) exposure in WT and TRPA1-null mice. In fact, both cholesterol and triglycerides significantly decrease in the CR-exposed WT group (cholesterol decreases/triglycerides increase in CR-exposed TRPA1-null mice), indicating that chronic increases in lipids occur at some point after vascular changes and not vice versa. Similarly, levels of PLAs, a marker of increased thrombosis, are altered inconsistently (unchanged chronically at 1 ppm; decreased acutely at 1 ppm; increased acutely at 3 ppm) unlike effects seen with acrolein (Sithu et al., 2011). These results reinforce the primacy of vascular dysfunction as a more sensitive and systemic biomarker of air pollution exposure per se, i.e., “ground zero” (Campen, 2009).

Over the past decade, we have found that exposure of mice and humans to air pollutants (PM_2.5, acrolein) leads to the suppression of CACs (aka endothelial progenitor cells, EPCs) (O’Toole et al., 2010; Wheat et al., 2011; DeJarnett et al., 2014), yet no change in CACs is observed in this CR study. We infer that 1 ppm CR is not as potent of a stimulus of CAC suppression as is either PM_2.5 or acrolein (1 ppm) yet it is a sufficient stimulus of TRPA1 as described above. We also conclude that changes in systemic blood pressure (hypotension) and the vasculature (enhanced sensitivity to NO) are more subtle measures of ‘vascular activation’ and independent of suppressed CAC mobilization. Thus, CR at levels found in MCS produces a complicated vascular phenotype that likely contributes to biological changes that ultimately enhance CVD risk in smokers and in those exposed to secondhand/environmental tobacco smoke. However, it is now recognized that certain tobacco smoke constituents, namely CO, may actually “mask” the effects of more injurious compounds by promoting vascular relaxation in healthy humans (Rezk-Hanna et al., 2019) similar to effects we observe after CR exposure in healthy mice. Perhaps, exposures with increasingly complex combinations of smoke constituents (e.g., acrolein and CR, CO and CR) may help us to begin to tease out the important interactions that surely manifest in vivo.

In conclusion, this study shows that chronic and acute exposures to CR lead to vascular changes that promote hypotension—an underappreciated cardiovascular risk factor for syncope, and, in regard to anesthesia-related hypotension, increased risk of post-operative mortality. Thus, CR induces an unconventional form of vascular/endothelium dysfunction that is both TRPA1- and sex-dependent. These novel effects of inhaled CR, although in parallel with conclusions reached for acrolein exposures in mice (Conklin et al., 2017a), require additional studies to identify the contribution of site-specific TRPA1 to these effects and to understand how CR exposure contributes to the cardiovascular pathology of real-world exposures to combustion-related aldehydes such as acrolein and CR. As smoking conventional tobacco cigarettes remains a global health threat, our study provides insights into relevant cardiovascular targets of CR while implicating chronic CR exposures as a risk factor for CVD.

Supplementary Material

2

Suppl. Fig1. 1: Vascular toxicity of acute crotonaldehyde (CR) inhalation exposure in C57BL/6 mice. Aortic function was measured ex vivo in male C57BL/6J mice following 4 days of exposure to either air or CR (3 ppm, 6h/d, 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. CR exposure significantly enhanced ACh efficacy (A) and slighted shifted sensitivity (B). There was no change in aortic efficacy (% relaxation; C) but there was an enhanced sensitivity (EC50; D) in response to the endothelium-independent vasorelaxant sodium nitroprusside (SNP). Values = mean ± SE (n=10 mice per group); *, p<0.05 vs air control. #, 0.05≤p≤0.10 vs air control.

Suppl. Fig1. 2: Effects of chronic crotonaldehyde (CR) inhalation exposure on circulating stem cells in mice. Hematopoietic stem cells (Sca-1+) and circulating angiogenic cells (CACs) were measured by flow cytometry gating (A) in male C57BL/6J (wild type, WT) mice exposed to either air or CR (1 ppm, 6h/d) for 12 weeks. Levels of circulating Sca-1+ cells (B) and CACs (Flk-1+/Sca-1+ cells; C) were unchanged in CR-exposed mice relative to air control mice. Values = mean ± SE (n=4-5 mice per group).

Suppl. Fig1. 3: Effects of acute crotonaldehyde (CR) inhalation exposure on circulating stem cells in mice. Hematopoietic stem cells (Sca-1+) and circulating angiogenic cells (CACs) were measured by flow cytometry gating (A) in male C57BL/6J (wild type, WT) mice exposed to either air or CR (3 ppm, 6h/d) for 4 days. Levels of circulating Sca-1+ cells (B) and CACs (Flk-1+/Sca-1+ cells; C) were unchanged in CR-exposed mice relative to air control mice. Values = mean ± SE (n=9-10 mice per group).

Suppl. Fig1. 4: Effects of acute crotonaldehyde (CR) inhalation exposure on circulating stem cells in mice. Hematopoietic stem cells (Sca-1+) and circulating angiogenic cells (CACs) were measured by flow cytometry gating (A) in female C57BL/6J (wild type, WT) mice exposed to either air or CR (1 ppm, 6h/d) for 4 days. Levels of circulating Sca-1+ cells (B) and CACs (Flk-1+/Sca-1+ cells; C) were unchanged in CR-exposed mice relative to air control mice. Values = mean ± SE (n=8-11 mice per group).

Highlights.

1. HPMMA is the primary urinary metabolite of crotonaldehyde exposure in mice.

2. Acute and chronic inhalation exposure of mice to crotonaldehyde (1 ppm) led to hypotension.

3. Acute and chronic inhalation exposure of mice to crotonaldehyde enhanced aortic relaxations.

4. Aortic dysfunction induced by crotonaldehyde exposure in mice was TRPA1-dependent.

Abbreviations

ACh,

acetylcholine

ALT,

alanine aminotransferase

AR,

aldose reductase

AST,

aspartate aminotransferase

AUC,

area under the curve

B2M,

β−2 microglobulin

BWT,

body weight

CAC,

circulating angiogenic cell

CBC,

complete blood count

CGT,

cysteinylglycine transpeptidase

CK,

creatine kinase

CO,

cardiac output

CR,

crotonaldehyde

CVD,

cardiovascular disease

CXCL15,

chemokine ligand 15

DBP;

diastolic blood pressure

E-cig,

electronic cigarette

EC₅₀,

half maximal effective concentration

EDV,

end-diastolic volume

EF,

ejection fraction

Emax,

efficacy of contractions or relaxations

EPC,

endothelial progenitor cell

ESV,

end-systolic volume

GGT,

gamma-glutamyl transpeptidase

Glu,

glutamate

Gly,

glycine

GSH,

glutathione

GST,

glutathione S-transferase

GSTP1,

glutathione S-transferase pi 1

GTT,

glucose tolerance test

HDL,

high-density lipoprotein

3HPMA,

3-hydroxypropylmercapturic acid

HPMMA,

3-hydroxy-1-methylpropylmercapturic acid

HR,

heart rate

IL-1β,

interleukin-1β

IL-6,

interleukin-6

IVRT,

isovolumic relaxation time

LCC,

left coronary cusp

LDH,

lactate dehydrogenase

LDL,

low-density lipoprotein

L-NAME,

L-N^G-Nitro arginine methyl ester

LVAWd,

left ventricular anterior wall thickness at end-diastole

LVAWs,

left ventricular anterior wall thickness at end-systole

LVIDd,

left ventricular internal diameter diastole

LVIDs,

left ventricular internal diameter systole

LVM,

left ventricular mass

MCS,

mainstream cigarette smoke

NADPH,

nicotinamide adenine dinucleotide phosphate

NAT,

n-acetyltransferase

NO,

nitric oxide

PE,

phenylephrine

PLA,

platelet-leukocyte aggregate

PM,

particulate matter

PSS,

physiological salt solution

PWV,

pulse wave velocity

RPLP0,

ribosomal protein lateral stalk subunit P0

RWT,

relative wall thickness

SMA,

superior mesenteric artery

SNP,

sodium nitroprusside

SV,

stroke volume

TNFα,

tumor necrosis factor alpha

TRPA1,

transient receptor potential ankyrin 1

WT,

wild type