Am J Physiol Lung Cell Mol Physiol 315: L662–L672, 2018.
First published August 9, 2018; doi:10.1152/ajplung.00389.2017.

RESEARCH ARTICLE

Electronic Cigarettes: Not All Good News?

Comparison of the effects of e-cigarette vapor with cigarette smoke on lung
function and inflammation in mice
Constantinos Glynos,1 Sofia-Iris Bibli,2,3 Paraskevi Katsaounou,1 Athanasia Pavlidou,1,2
Christina Magkou,4 Vassiliki Karavana,1 Stavros Topouzis,5 Ioannis Kalomenidis,1 Spyros Zakynthinos,1
and X Andreas Papapetropoulos1,2
1

George P. Livanos and Marianthi Simou Laboratories, Evangelismos Hospital, 1st Department of Pulmonary and Critical
Care, National, Kapodistrian University of Athens Medical School, Greece; 2Laboratory of Pharmacology, Faculty of
Pharmacy, National and Kapodistrian University of Athens, Athens, Greece; 3Institute for Vascular Signaling, Centre for
Molecular Medicine, Goethe University, Frankfurt am Main, Germany; 4Department of Histopathology, Evangelismos
Hospital, Athens, Greece; and 5Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras,
Patras, Greece
Submitted 29 August 2017; accepted in final form 7 August 2018

Glynos C, Bibli SI, Katsaounou P, Pavlidou A, Magkou C,
Karavana V, Topouzis S, Kalomenidis I, Zakynthinos S, Papapetropoulos A. Comparison of the effects of e-cigarette vapor with
cigarette smoke on lung function and inflammation in mice. Am J
Physiol Lung Cell Mol Physiol 315: L662–L672, 2018. First published August 9, 2018; doi:10.1152/ajplung.00389.2017.—Electronic
cigarettes (e-cigs) are advertised as a less harmful nicotine delivery
system or as a new smoking cessation tool. We aimed to assess the in
vivo effects of e-cig vapor in the lung and to compare them to those
of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days
or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene
glycol/vegetable glycerol (PG:VG-Sol; 1:1), 2) PG:VG with nicotine
(G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N⫹F) and
determined oxidative stress, inflammation, and pulmonary mechanics.
E-cig vapors, especially PG:VG-N⫹F, increased bronchoalveolar
lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF
and lung oxidative stress markers at least comparably and in many
cases more than CS. BALF protein content at both time points studied
was only elevated in the PG:VG-N⫹F group. After 3 days, PG:VGSol altered tissue elasticity, static compliance, and airway resistance,
whereas after 4 wk CS was the only treatment adversely affecting
these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N⫹F groups.
Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics.
In many cases, the added flavor in e-cigs exacerbated the detrimental
effects of e-cig vapor. We conclude that both e-cig vaping and
conventional cigarette smoking negatively impact lung biology.
cigarette smoking; electronic cigarettes; lung hyperresponsiveness;
lung inflammation; lung mechanics

INTRODUCTION

Smoking is the leading cause of morbidity and mortality in
men and women and for this reason, smoking prevention and
cessation strategies have tremendous potential for improving
overall public health (4). In recent years, electronic cigarettes
Address for reprint requests and other correspondence: A. Papapetropoulos,
Laboratory of Pharmacology, Faculty of Pharmacy, National and Kapodistrian
Univ. of Athens, Athens, Greece, 15771 (e-mail: apapapet@pharm.uoa.gr).
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(e-cigarettes or e-cigs) are increasingly advertised as a reduced-risk nicotine product and an attractive alternative smoking cessation tool (23, 24, 35). In fact, part of the medical
community believes that they can be used as a harm-reduction
strategy for smokers. E-cigs have become rapidly popular
worldwide (6, 35), although their effectiveness as a smoking
cessation tool has not been rigorously proven yet (29, 36).
Currently, their effects on human health have not been adequately addressed (6, 11, 36). Although some negative shortterm health effects have already been shown (6, 20, 36),
altogether there is still paucity of reliable data regarding
long-term exposure effects.
E-cigs are battery-powered devices, which do not contain or
burn natural tobacco. They consist of a rechargeable battery, a
heater, and a refillable cartridge with liquids, usually consisting
of propylene glycol, vegetable glycerol, nicotine, and flavorings (6, 11). When the battery-powered heater is activated, it
heats the solution to produce a vapor containing various heatproduced ingredients of variable concentration, which are inhaled by the user. Quality control of e-cigs among various
brands has been a matter of controversy, raising concerns about
their safety profile and their toxicity (12, 35, 46, 47). The
United States Food and Drug Administration has indicated that
e-cigs contain a number of toxins and carcinogens (i.e., nitrosamines, diethylene glycol) (8). Recently, there have been
attempts to regulate the market of the e-cig devices (6, 8). As
experiment-based scientific knowledge is still largely lacking
in the field, it is crucial to vigorously assess e-cig toxicity,
safety, and health effects.
The smoke of conventional cigarettes contains more than
4,000 chemicals with multiple immunomodulatory and other
effects on the lungs (9, 33). Compared with their effects, e-cig
vaping is advertised as less harmful. Based on the few published in vitro or in vivo studies, e-cig vapor seems to have
adverse effects on both cultured cells and on experimental
animals (reviewed in 6, 11). E-cig vapor induces inflammation, augments the development of allergic airway inflammation
in asthma models, suppresses the host defense, and triggers
effects associated with chronic obstructive pulmonary diseaselike tissue damage (26, 30, 42). In humans, clinical manifes-

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 E-CIGARETTE AND LUNG FUNCTION

tations such as acute eosinophilic pneumonia and lipoid pneumonia (31, 43) have been related to e-cig use. It has also been
shown recently that acute exposure to e-cig vapor increases
aortic stiffness, blood pressure (16, 23), and airway resistance
(45) whereas conversely, it decreases airway conductance (34)
in healthy subjects and changes the slope of phase III curve in
asthmatic smokers (34). It should be stressed that all the
above-mentioned studies focus on the acute pathophysiological
effects of the e-cig vapor with regard to the respiratory and
cardiovascular function. Thus, the potential longer-term effects
of e-cig vapor exposure in animals and humans still remain
unexplored and unknown.
The aim of the present study was to determine the effects of
both acute (3-day) and subchronic (4-wk) exposure of mice to
components of vaporized e-cig liquids on the respiratory functional parameters and inflammatory responses and to compare
them side-by side to those of air and classic cigarette smoke
(CS) exposure, using a well-established animal model (10).
MATERIAL AND METHODS

Animals. Sex disparity in response to chronic smoke exposure has
been observed in animal models (5). In the present study we used only
male mice for two reasons. First, it is estimated that men smoke nearly
five times as much as women worldwide (21). Second, limiting our
studies to male mice would be expected to reduce variability allowing
the use of a smaller number of animals that conforms with the 3Rs
principle (replacement, refinement, and reduction) in humane animal
research. Because of the abundance of information on C57BL6 and its
susceptibility to lung injury, this strain was chosen. Eight-to-twelveweek-old male C57BL/6 wild-type Pasteur Institute (Athens, Greece)
or Fleming Institute (Vari, Greece) mice, weighing 16 –24 g, were
exposed for 3 days or 4 wk to air (medical air grade), CS, or e-cig
vapor from 1) propylene glycol/vegetable glycerol (PG:VG-Sol; 1:1),
2) PG:VG with nicotine (PG:VG-N; 18 mg/ml), or 3) PG:VG-N⫹F
with flavor (PG:VG-N⫹F; tobacco blend) (Fig. 1E). A partial chemical characterization of the tobacco blend flavor (Nobacco American
Tobacco) has been previously published (14). It should be mentioned
that this product is not a tobacco extract. Mice were maintained in
standard conditions under a 12-h:12-h light-dark cycle and provided a
standard diet and chlorinated tap water ad libitum. All procedures
were in accordance to the European Union Directive for care and use
of laboratory animals and were approved by the competent Regional
Veterinary Service and the ethical committee of Evangelismos Hospital.
CS and e-cig vapor exposure. The apparatus used in our study is
shown in Fig. 1, A and B, it exposes the entire body of animals to the
treatment applied. The apparatus has been described before (10, 18)
and has been used by us in recent studies (10, 18). A pump connected
with five syringes was used to create positive/negative pressure cycles
to drive flow of the smoke or vapor to a chamber where the animals
were kept. The chamber volume was 7,500 cm3, and the flow of
medical air into the chamber was between 1.5 and 2 l/min. The puff
volume was 20 ml. In the chamber a smoke/air ratio of 1:6 was
obtained. All of these parameters were identical between the CS and
e-cig exposure. The e-cig exposure was performed using the same
system as for the CS exposure; the only change was the use of an
adaptor that held the e-cig in place because of its different diameter.
Three different experimental series of air, CS, and e-cig vapor exposures were performed for the acute study, and two experimental series
were performed for the subchronic study. Control mice were exposed
to medical air. For CS exposure, five reference cigarettes (3R4F,
University of Kentucky; Fig. 1C) were used, whereas for vaping five
eRoll devices (Joye Technology) were employed. The eRoll (Fig. 1D)
is a first generation e-cig device (39) and was chosen as it was among

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the most widely used in Greece when the study was initiated. The
smoke and vapor from all five cigarettes or e-cigs was directed toward
the chamber were the animals were. Mice were exposed to CS or e-cig
4 times a day with 30-min smoke-free intervals for 3 days or 4 wk. For
the CS treatment, 15 puffs were drawn per session, which sufficed to
burn the entire conventional cigarette. The eRoll cartridge holds 0.4
ml of liquid and contains a chromium coil. Eight puffs/min for two
minutes (i.e., a total of 16 puffs) were drawn during each session, with
4 sessions being used per day with 30-min intervals. The animal
whole-body exposure lasted 7 min in each session. Our protocol uses
less than half of the total amount of the 0.4 ml in the cartridge. The
cartridge was replaced after every vaping session, i.e., 4 times per day.
This procedure avoided overheating of the chromium coil. To eliminate the metal decay that could be relevant to the long-term treatment,
we changed the chromium coil every second week. Lack of overheating was empirically confirmed by regular e-cig users who vaped the
eRoll device at the same rate of 16 puffs over a 2-min period and
reported no heating or change in vapor taste.
Respiratory system mechanics. The function of the respiratory
system of mice after 3 days or 4 wk exposure to CS or e-cig vapor was
estimated with the use of the forced oscillation technique and by
performing static pressure volume curves, as previously described
(19). Following 3-day or 4-wk treatment, the animals were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and
xylazine (10 mg/kg). An anterior cervical incision was performed, and
the animals were tracheostomized below the level of banding with a
tracheal cannula (20-gauge). The animals were connected to a small
animal ventilator (Scireq, Montreal, Canada) and ventilated with a 7
ml/kg tidal volume, 150 breaths/min, and the end expiratory pressure
was set to 3 cmH2O. Following 3 min of ventilation, an intraperitoneal
injection of succinylcholine (8 mg/kg) was performed to cease spontaneous breathing, and after 1 min, three forced oscillation perturbations were performed with 1-min intervals, to estimate lung mechanics. A static pressure volume curve was also constructed following 1
min of ventilation after the last oscillation perturbation. Results from
repeated measures in every animal were averaged. Prior to measurements (30 s) the lung volume history was once standardized by one
inflation to total lung capacity, as estimated by airway opening
pressure at 30 cmH2O. During ventilation, the heart rate was monitored to ensure adequate depth of anesthesia.
Forced oscillation technique. The forced oscillation perturbation
consists of a pseudorandom waveform of low frequencies (0.5–19.75 Hz)
applied for 8 s with a peak-to-peak volume of 3 ml/kg. Pressure and
volume data are recorded, and the impedance of the respiratory system is
calculated using the Fast Fourier transformation. Impedance (Z) is then
fitted to constant phase model, where Zrs is the baseline measurement of
Z: Zrs(f) ⫽ Rn ⫹ i2␲fI ⫹ (G⫺iH)/(2␲f)␣, where Rn is the Newtonian
resistance of the airways, i is the imaginary unit, f is the frequency, I is
the inertance of the gas in the airways, G represents tissue viscance
(viscous dissipation of energy), H represents tissue elasticity, and ␣ can
be calculated through the equation ␣ ⫽ (2/␲)arctan(H/G). Data were
accepted only when the coefficient of determination (fit of the model) was
more than 0.9. For static pressure volume curve, static pressure volume
curves of the respiratory system were performed by gradually inflating
and deflating the lungs with a total volume of 40 ml/kg at 7 steps each.
The static compliance of the respiratory system was estimated by the
slope of the midlinear part in the expiratory limb of the pressure volume
curve. Hysteresis (area between inspiratory and expiratory limb) was
automatically calculated (FlexiVent software) (44).
Airway hyperresponsiveness. Twenty-four hours after the 3-day CS
exposure or e-cig vapor, mice were anesthetized, tracheostomized,
paralyzed, and ventilated with Flexivent (SCIREQ Scientific Respiratory Equipment, Inc., Montreal, Canada). After baseline Zrs, methacholine (2.5, 10, 20, 40, 60 mg/ml) or saline were delivered
(Aeroneb; SCIREQ) for 10 s. Afterwards, a 2-s forced oscillation
perturbation (1–20 Hz) was performed every 10 s for 3 min. Before
measurements and before every aerosol delivery, the volume history

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E-CIGARETTE AND LUNG FUNCTION

A

B

Modified cigarette smoking chamber Modified e-cigarette smoking chamber

C

cigarette smoking

D

e-cigarette vaping

3R4F cigarette
e-cigarette
Fig. 1. Equipment and treatment protocol. A: proprietary modified chamber used for both CS and
e-cigarette (e-cig) vapor exposure of mice. B: schematic representation of the protocol of exposure to
CS and e-cig vapor. BAL fluid, bronchoalveolar
lung fluid; CS, cigarette smoke.

E

3 days

X

3 days

X

cigarette smoking

4 weeks

e-cig. vaping

4 weeks

Lung Tissue
BAL fluid
• Cellularity
• Protein content
• Oxidaive stress
• Cytokines

Groups:
1.

Air

2.

Cigarette smoking (3R4F University of Kentucky, USA) (CS)

3.

Propylene glycol/ vegetable glycerol 1:1 (PV:VG)

4.

Propylene glycol/ vegetable glycerol with nicotine 18mg/mL (PV:VG-N)

5.

Propylene glycol/vegetable glycerol with nicotine 18mg/mL plus tobacco
blend flavour 4% (PV:VG-N+F)

of the lung was established with two 6-s deep inflations to a pressure
limit of 30 cmH2O. Measurements of Zrs were fit with the constant
phase model, where Rn is the Newtonian resistance of the airways, G
represents tissue resistance, and H represents tissue elasticity. After
each dose of methacholine, model parameters were expressed as %
ratio of the baseline (41, 44).
Bronchoalveolar lavage fluid. The animals were euthanized by
exsanguination (vena cava dissection) following anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg) intraperitoneally 24 h
after the last exposure to air, CS, or e-cig vapor (Fig. 1E). After
exsanguination, the trachea was cannulated with a 20-gauge plastic
catheter. Lungs were lavaged by infusing 1 ml warm saline, three
sequential times. The recovered bronchoalveolar lavage fluid (BALF)
was centrifuged; cells were collected and resuspended in PBS. Differential BALF cell counts were performed on Giemsa-stained cytospins, and percentages of eosinophils lymphocytes, neutrophils, and

macrophages were determined. Protein concentration was measured in
the BALF using the Lowry method, employing bovine serum albumin
as a standard.
Lung histology. Following bronchoalveolar lavage, the left lung
was harvested from mice and fixed using a 4% formaldehyde solution.
The tissue was embedded in paraffin wax, serially sectioned, and
stained with hematoxylin-eosin using standard methods. Two pathologists blinded for treatment evaluated the histopathological findings in
the lung. A scoring system to grade the degree of lung inflammation
has been used based on the following histological features: 1) capillary congestion, 2) intra-alveolar hemorrhage, 3) interstitial neutrophil
infiltration, 4) intra-alveolar neutrophil infiltration, and 5) focal thickening of alveolar membranes. A scale from 0 to 3 for each feature will
be used (0: absence, 1: mild, 2: moderate, 3: most severe) (32).
Immunohistochemistry. Mouse lung paraffin sections 0.5 ␮m thick
were applied to positive electrical charge-coated slides and left at

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 E-CIGARETTE AND LUNG FUNCTION

55°C to remove paraffin excess. Sections were deparaffinized by two
consecutive treatments (5 min each) with xylene. Rehydration was
performed with graded ethanols (90, 80, and 70%) for 4 min each.
Antigen retrieval was subsequently performed by boiling the sections
with 1⫻ Target Retrieval Solution pH 6.0 (Dako, Athens, Greece) in
a steamer for 30 min. Endogenous peroxidase was blocked with 3%
H2O2 in methanol for 15 min at room temperature. Primary antibody
mouse monoclonal Mucin 5AC (clone 45M1, PierceThermo Scientific) diluted 1:50 (vol/vol) in REAL Antibody Diluent (Dako) was
applied to sections and incubated for 30 min at room temperature.
Immunostaining reaction was developed using REAL EnVision Detection System Peroxidase/DAB⫹ Rabbit/Mouse (Dako) incubated for
30 min at room temperature. Washes were performed using Trisbuffered saline-tween 20 buffer for 10 min. Immunoreactivity was
detected using DAKO REAL DAB⫹ Chromogen reagent for 5 min.
Sections were counterstained with hematoxylin, dehydrated, mounted,
and examined. Sections from which the primary antibody was omitted
served as negative control. Immunohistochemistry slides were evaluated by light microscopy, and the immunosignal was scored using a
semiquantitative scoring system as previously described (1). An
intensity score was assigned representing the estimated average intensity of positive staining cells. The staining intensity was classified
into 4 scales scored as negative (0), weak (1⫹), moderate (2⫹), and
intensive (3⫹) (1).
Determination of oxidative stress in the lung and the BALF.
Biomarkers of oxidative stress were determined by measuring malondialdehyde (MDA) and protein carbonylation in the lung tissue. For
MDA measurement, lung tissue samples were pulverized and then

L665

minced in a small volume of ice-cold 20 mm Tris·HCl buffer (pH 7.4)
in a 1:10 wt/vol ratio and homogenized. After centrifugation at 3,000
g for 10 min at 4°C, the clear homogenate supernatant was used for
biochemical assay. For the determination of MDA, 0.65 ml of 10.3
mmol/l N-methyl-2-phenyl-indole in acetonitrile was added to 0.2 ml
of tissue sample. After vortexing for 3– 4 s, 0.15 ml of 15.4 mol/l
methanesulfonic acid was added, and samples were mixed well,
closed with a tight stopper, and incubated at 45°C for 40 min. The
samples were then cooled on ice, centrifuged, and the absorbance was
measured spectrophotometrically at 586 nm. A calibration curve,
made with standard MDA solutions (from 2 to 20 nmol/ml), was also
run for quantitation. Measurements were performed in triplicate.
MDA levels were expressed as ␮mol/mg protein (3). For protein
carbonylation, a modification of the technique of Levine et al. (27),
based on spectrophotometric measurement of 2,4-dinitrophenylhydrazine (DNPH) derivatives of protein carbonyls, was used to quantify
protein carbonyl content in the lung or the BALF of the mice. Briefly,
100 ␮l of the homogenized lung tissue or of the BALF was incubated
either with 500 ml DNPH or 2 mol/l HCl for 1 h at room temperature.
The samples were then reprecipated with 600 ␮l 20% trichloroacetic
acid, incubated for 5 min on ice, and subsequently extracted with
ethanol/ethyl acetate (1:1, vol/vol), 3 times at 11,000 g for 10 min at
4°C. The pellets were carefully drained and dissolved in 6 mol/l
guanidine solution in HO. The difference between the spectra of the
DNPH-treated sample and the HCl control was determined at 360 nm,
and the results are expressed as nmol protein carbonyl/mg protein
using a molar extinction coefficient of 22,000 mol/l. Protein concentration was determined using the Lowrey assay.

Fig. 2. Effects on BALF cellularity and protein content. Following an acute 3-day and a subchronic 4-wk exposure to CS
and e-cig vapor, BALF was obtained, and following centrifugation total cellularity was determined (A and D). Differential
counts, to assess cell type-specific contribution (B and E) were
performed on Giemsa-stained cytospins. Cell-free protein was
determined in the supernatant (C and F). Values are expressed
as mean ⫾ SD; n ⫽ 5–10 mice; *P ⬍ 0.05 vs. air, #P ⬍ 0.05
vs. CS. BALF, bronchoalveolar lung fluid; CS, cigarette smoke;
PG:VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N,
propylene glycol/vegetable glycerol with nicotine; PG:VGN⫹F, propylene glycol/vegetable glycerol with nicotine and
tobacco blend flavor.

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E-CIGARETTE AND LUNG FUNCTION

Determination of cytokine in the BALF. Cytokines levels (TNF-␣,
IL-1␤, IL-6) in the BALF were measured in 100 ␮l BALF using the
manufacturer’s protocol (HS Quantikine; R&D Systems, Minneapolis, MN). To determine the tissue cytokine levels, lungs were homogenized as for the MDA measurements, and TNF-␣, IL-1␤, and IL-6
were measured in the supernatant corresponding to similar amounts of
protein (2 mg protein).
Statistical analysis. Results are presented as means ⫾ SD of the
number of indicated observations. Statistical analysis was performed
with Sigma Stat software (SPSS 11.5, Chicago, IL) using nonparametric tests for continuous variables (Kruskall-Wallis, Mann-Whitney
U-test). Differences were considered significant when P ⬍ 0.05.
RESULTS

BALF cellularity and protein content. Total cell counts in
BALF of mice exposed to e-cig vapor for 3 days were in-

creased in all groups compared with air-breathing mice, mainly
because of macrophage influx. BALF cellularity in CS-exposed mice was also increased compared with air-breathing
mice, because of macrophage influx (90% of total cell count)
and to a lesser extent to neutrophils (7.1% of total cell count)
(Fig. 2, A and B). BALF cell-free protein content was increased
only in the PG:VG-N⫹F group (P ⫽ 0.001), compared with
air-breathing mice (Fig. 2C).
The results were different after 4 wk of exposure to CS or
e-cig vapor (Fig. 2, D–F). Total BALF cell count was elevated
only in the CS (P ⫽ 0.0001) and PG:VG-N⫹F (P ⫽ 0.0001)
groups, again mainly because of macrophage influx. These
results indicate that neither the PG:VG vehicle or nicotine
addition to e-cigs affected these parameters at 4 wk of vaping;
however, the addition of flavor to nicotine-containing e-cigs is

Fig. 3. Determination of markers of oxidative stress following
smoking and vaping. Following an acute 3-day and a subchronic 4-wk exposure to CS and e-cig vapor MDA (A, B, E, F)
and protein carbonyl (C, D, G, H) oxidative markers were
determined in the BALF (A, C, E, G) and lung homogenate (B,
D, F, H).Values are expressed as mean ⫾ SD; n ⫽ 5– 8 mice;
*P ⬍ 0.05 vs. air, #P ⬍ 0.05 vs. CS. BALF, bronchoalveolar
lung fluid; CS, cigarette smoke; MDA, malondialdehyde; PG:
VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N, propylene glycol/vegetable glycerol with nicotine; PG:VG-N⫹F,
propylene glycol/vegetable glycerol with nicotine and tobacco
blend flavor.

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 E-CIGARETTE AND LUNG FUNCTION

capable of significantly inducing macrophage influx into the
BALF. BALF cellularity of CS mice also increased because of
macrophage (87.5% of total cell count) and neutrophil (8.2% of
total cell count) influx (Fig. 2, D and E). BALF protein content
was elevated in the PG:VG-N (P ⫽ 0.014) and PG:VG-N⫹F
(P ⫽ 0.003) groups only, compared with air-breathing mice
(Fig. 2F), suggesting that exposure to nicotine through inhalation of e-cig vapor but not through CS exposure can increase
BALF protein content at this time point; in this case, addition
of flavor to the e-cig had no further effect.
Markers of oxidative stress in the BALF. A 3-day exposure
to CS or e-cig vapor resulted in 1) increased levels of MDA in
the PG:VG (P ⫽ 0.016) and PG:VG-N⫹F (P ⫽ 0.03) and 2)
increased protein carbonyls in all cigarette-/e-cig-exposed
groups compared with air-breathing mice (Figs. 3, A and C)
[CS mice (P ⫽ 0.013), PG:VG-Sol (P ⫽ 0.03), PG:VG-N
(P ⫽ 0.022), and PG:VG-N⫹F (P ⫽ 0.004)].
Four-week exposure to CS or e-cig vapor resulted in 1)
increased levels of MDA in CS (P ⫽ 0.011), PG:VG-N (P ⫽
0.002), and PG:VG-N⫹F (P ⫽ 0.003) groups, 2) increased
levels of protein carbonyls in BALF in CS (P ⫽ 0.009),
PG:VG-N (P ⫽ 0.019), and PG:VG-N⫹F (P ⫽ 0.004) groups
compared with air-breathing mice (Fig. 3, E and G). The only
group that did not show an increase in BALF protein oxidation
markers at 4 wk relative to air was the one exposed to PG:VG.
Markers of oxidative stress in lung tissue. Three-day exposure to CS or e-cig vapor resulted in 1) increased MDA levels
observed in the CS (P ⫽ 0.006), PG:VG-Sol (P ⫽ 0.004), and
PG:VG-N⫹F (P ⫽ 0.011) groups and 2) increased lung protein
carbonyls in the CS (P ⫽ 0.004) and PG:VG-N⫹F
(P ⫽ 0.004) compared with air-breathing mice in lung tissue
(Fig. 3, B and D). From these results, one can conclude that the
nicotine in the e-cig vapor seems to mitigate the effect of
PG:VG-Sol, whereas the additional inclusion of flavor reverses
this effect. This pattern is broadly reminiscent of the pattern in
oxidation markers seen in BALF (Fig. 3, A and C).
Four-week exposure to CS or e-cig vapor resulted in 1)
increased MDA levels only in the CS and PG:VG-N⫹F groups
and 2) similarly, increased protein carbonyls levels in CS (P ⫽
0.037) and in PG:VG-N⫹F (P ⫽ 0.005) groups (Fig. 3, F and
H). Again, as seen at 3 days of exposure, the addition of flavor
to the nicotine-containing e-cigs seemed to elevate the oxidation markers in the lung tissue to levels similar to CS.
Levels of proinflammatory cytokines in lung homogenates.
Exposure to CS or e-cig vapor for 3 days resulted in increased
levels of IL-1␤ and IL-6 in the PG:VG-N⫹F group only
(IL-1␤, P ⫽ 0.047 and IL-6, P ⫽ 0.047, Fig. 4, B and C)
without affecting TNF-␣ levels (Fig. 4A). Following 4 wk of
exposure to either CS or to e-cig vapor, in agreement with the
3-day exposure, TNF-␣ levels remained unchanged (Fig. 4D).
However, in contrast to the shorter exposure where IL-1␤ and
IL-6 were significantly increased only in the PG:VG-N⫹F
group, in this longer exposure levels of these two cytokines
were significantly elevated (IL-1␤, IL-6, P ⫽ 0.047) only in
the CS-exposed mice (Fig. 4, E and F).
Lung histopathology and Muc5a immunohistochemistry. Exposure of mice for 3 days to CS in comparison to air or e-cig
vapor resulted in a pronounced focal thickening and interstitial
inflammation. E-cig exposure failed to produce statistically
significant changes in the combined score (Fig. 5A). The
effects of inhaling CS was equally pronounced after 4 wk of

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Fig. 4. Determination of markers of inflammation following smoking and
vaping. Following an acute 3-day and a subchronic 4-wk exposure to CS and
e-cig vapor, cytokines were measured using commercial kits in lung homogenates. A and D: TNF-␣; B and E: IL-1␤; C and F: IL-6. Values are expressed
as mean ⫾ SD, n ⫽ 5 mice; *P ⬍ 0.05 vs. air. CS, cigarette smoking; IL,
interleukin; PG:VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N, propylene glycol/vegetable glycerol with nicotine; PG:VG-N⫹F, propylene glycol/vegetable glycerol with nicotine and tobacco blend flavor; TNF-␣: tumor
necrosis factor-␣.

exposure (Fig. 5B). Moreover, it seems that the increase in the
score is more prominent at 3 days versus 4 wk. Muc5ac protein
was homogenously detected in the apical surface of bronchial
epithelial cells. CS, PG:VG-Sol, and PG:VG-N⫹F groups
exhibited a pronounced Muc5ac production in the airways of
mice upon 3-day exposure compared with air-exposed mice
(Fig. 6).
Measurements of respiratory system mechanics following
CS or e-cig vapor inhalation. In addition, we assessed the
functional consequences of CS in mice, so we determined
airway resistance, tissue elasticity, and static compliance in the
various groups. Surprisingly, after 3 days of CS or e-cig vapor
exposure, all three parameters were significantly changed only
in the PG:VG-Sol group. This group presented increased airway resistance (P ⫽ 0.004) and tissue elasticity (P ⫽ 0.001)
and decreased static compliance (P ⫽ 0.001) compared with
either the air-exposed mice or to the CS-inhaling group (P ⫽
0.005 vs. CS for airway resistance; P ⫽ 0.001 for tissue
elasticity, and P ⫽ 0.001 for decreased compliance) (Fig. 7,
A–C). After a 4-wk exposure to CS the observed pattern was

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E-CIGARETTE AND LUNG FUNCTION

Fig. 5. Lung histological changes are evident
after smoking but not after vaping. Following
an acute 3-day (A) and a subchronic 4-wk (B)
exposure to CS and e-cig vapor, the left lung
was obtained and histopathology was performed on hematoxylin-eosin-fixed sections
according to Murao et al. (26).Values are expressed as mean ⫾ SD; n ⫽ 5– 8 mice; *P ⬍
0.05 vs. air, #P ⬍ 0.05 vs. CS. CS, cigarette
smoke; PG:VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N, propylene glycol/vegetable glycerol with nicotine; PG:VG-N⫹F,
propylene glycol/vegetable glycerol with nicotine and tobacco blend flavor.

different; only the CS-exposed mice presented significantly
altered functional parameters.
Determination of airway hyperresponsiveness. To test for
changes in pulmonary reactivity to a standard experimental

airway stimulus, we challenged mice that had previously
undergone a 3-day exposure to the same five inhaling
regimes to various doses of methacholine. Only two groups,
the CS group and the PG:VG-N⫹F vapor-exposed group,

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 E-CIGARETTE AND LUNG FUNCTION

L669

Fig. 6. Changes in lung Muc5ac following smoking
and vaping. After 3 days of exposure to CS or e-cig
vapor, mouse lung paraffin sections were stained
using an anti-Mucin 5ac antibody. IHC slides were
evaluated by light microscopy and the immune signal was scored using a semiquantitative scoring
system as previously described (1). Values are expressed as mean ⫾ SD; n ⫽ 5– 6 mice; *P ⬍ 0.05
vs. air, #P ⬍ 0.05 vs. CS. CS, cigarette smoke; IHC,
immunohistochemistry; PG:VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N, propylene glycol/
vegetable glycerol with nicotine; PG:VG-N⫹F, propylene glycol/vegetable glycerol with nicotine and
tobacco blend flavor.

exhibited increased airway hyperresponsiveness to increasing doses of methacholine compared with air-exposed mice
(Fig. 8, A and B).
DISCUSSION

The recent advent of e-cigs and their rapidly increasing use
among smokers puts pressure on the scientific community to
generate well-controlled preclinical and clinical studies of
pulmonary function to compare side-by-side the effects of
“classical” CS to the effects of vapors from e-cigs. Up to now,
there is only a limited number of in vitro studies on human and
murine pulmonary epithelial cell lines, fibroblasts, and stem
cells [(2, 15, 37) studies reviewed in (6)] that have addressed
the acute toxicological profile of refill liquid contents of e-cigs.
In a recent study of Scheffler et al. (38), direct exposure of
primary bronchial epithelial cells to e-cig vapor containing
glycerol/propylene glycol induced oxidative stress that was
less pronounced compared with the stress induced by conventional CS. In addition, the effect of chemicals found in the e-cig
flavors has also been shown to be responsible for toxic effects
on pulmonary fibroblasts (2, 7). Recent studies have addressed
the pulmonary effects of e-cigs in animal models in vivo (17,
22, 26, 42), and although they are very informative, they are
characterized by either use of a single time of exposure, by
focus on inflammatory and immune responses alone in the
absence of addressing functional lung mechanics, and lastly,
only occasionally do they address the effect of specific e-cig
components, such as the widely used vehicle PG:VG or added
flavors. To complete this gap in our in vivo study in mice, we
1) varied the length of exposure to either CS and e-cig vapor
and 2) we examined the effects of nicotine and flavorings
added to the main “vehicle” in e-cigs (PG:VG) on inflammatory markers and functional parameters of the exposed lungs.

Pulmonary irritation and other effects of propylene glycol (a
major constituent of e-cig fluid vehicle) have been noted in
humans (6, 20). In agreement with these observations, our
study showed that the propylene glycol-containing vehicle
(PG:VG-Sol) was able to increase BALF cellularity, induce
oxidative modifications, raise epithelial Muc5a production, and
negatively impact lung mechanics at 3 days of exposure. These
effects were mostly absent following exposure to PG:VG for 4
wk, suggesting a transient respiratory irritation by the vehicle
that subsides upon further exposure.
As far as nicotine content in e-cig liquids and its biological
effects are concerned, most previous studies have generally
reported a “nicotine effect,” usually by comparison to “air,”
without being able to distinguish whether the observed changes
are because of the e-cig liquid vehicle, the nicotine, or the
flavors, if present. In our experiments, nicotine addition was
found to exert variable effects either aggravating, causing no
change or ameliorating the detrimental effects of e-cig vapor
depending on the parameter and time point studied. E-cig
toxicity when tested in vitro with embryonic and adult cells
was not attributed to nicotine but correlated with the number
and concentration of chemicals used to flavor fluids (2). Only
one in vivo study, that of Garcia-Across et al. (17), has shown
clear nicotine-dependent effects on mouse airway hyperactivity, mucin production, distal airspace enlargement, cytokine
and protease expression, and impaired ciliary beat frequency.
Hundreds of flavors have been proven safe as food additives
but there are no data about their impact to the respiratory
system, and it is well known that the route of exposure plays a
significant role in the response to xenobiotics. In vitro as well
as human studies have revealed potential hazards of e-cig
vapor because of their flavoring components (17, 20, 26, 40).
In our study, tobacco-flavored, nicotine-containing e-cig vapor

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E-CIGARETTE AND LUNG FUNCTION

wk) exposure to e-cig vapor resulted in an increase in BALF
cellularity, BALF protein, and oxidative stress that were more
pronounced in the e-cig group or equal between e-cigs and CS.
In line with our observations, Lerner et al. (26) reported that
exposure of mice to e-cigs diminished lung glutathione levels.
Levels of inflammatory cytokines exhibited a different pattern
compared with BALF cellularity and oxidative stress. After 3
days of exposure, only PG:VG-N⫹F increased the levels of
IL-1␤ and IL-6 in the lung, whereas after 4 wk the effect of
PG:VG-N⫹F was absent and only CS elevated IL-1␤ and IL-6
in the lung. Our observations are in accordance with the study
of Lerner et al. (26) where acute (3 days) exposure to e-cig
containing both nicotine (16 mg) and classic tobacco flavor
elicited an increase in the levels of proinflammatory mediators,
such as IL-6, in the BALF. In a different study where mice
were exposed for 2 wk to e-cig vapor (42), although macrophage influx in the BALF was seen, IL-6 levels in the BALF
were lower than in mice that were exposed to air. This
discrepancy may be related to the different time of exposure
between the two studies and to the different compartment
analyzed (BALF in their work vs. lung homogenate herein). It
should be noted that in the work of Sussan et al. (42) it is not
entirely clear whether the exposure of these mice was to
flavored or unflavored e-cigs.
To examine further the potential harmful effect on the
airways and compare CS and e-cig effects, we measured mucin
production. After 3 days of exposure CS, PG:VG-Sol, and
PG:VG-N⫹F exposure showed higher levels of Muc5ac in the

Fig. 7. Changes in respiratory system mechanics following smoking and
vaping. Mouse lung function was measured using a forced oscillation before
the onset of cigarette exposure (baseline) and following exposure of mice for
either 3 days or 4 wk to CS or e-cig vapor. Parameters determined include R
(A and D), C (B and F), and H (C and E). Values are expressed as mean ⫾ SD;
n ⫽ 5–12 mice *P ⬍ 0.05 vs. air, #P ⬍ 0.05 vs. CS. C, static compliance; CS,
cigarette smoke; H, tissue elasticity; PG:VG-Sol, propylene glycol/vegetable
glycerol; PG:VG-N, propylene glycol/vegetable glycerol with nicotine; PG:
VG-N⫹F, propylene glycol/vegetable glycerol with nicotine and tobacco
blend flavor; R, airway resistance.

(PG:VG-N⫹F) showed significant effects in BALF cellularity
and protein content after both 3 days and 4 wk of exposure.
Notably, the determination of the two oxidative markers
(MDA, carbonyls) showed that the flavor contained in e-cig
vapor allowed for a marked effect in both the BALF and the
lung; in the latter case, the effect was not shown by nicotinealone e-cig exposure (PG:VG-N). Along the same lines, at the
3-day exposure, again among the various e-cig exposure regimes, only the PG:VG-N⫹F-exposed mice showed increases
in IL-6 and IL-1␤. Last, PG:VG-N⫹F exposure alone among
e-cig-inhalation regimes was able to exacerbate the methacholine response, as did CS. Thus, in many of the parameters
studied herein, the hazardous effects noted after exposure to
e-cig vapors were exacerbated or persisted longer when flavoring was added to the refill liquid. Given the thousands of
flavors available world-wide, meticulous examination of these
liquids and their components to prove which ones are safe
would be a daunting task.
Comparison of the effects of CS versus e-cig vapor yielded
interesting results. Both short-term (3-day) and long-term (4-

Fig. 8. Airway hyperresponsiveness in animals exposed to cigarette smoke or
e-cig vapor. Airway responsiveness mice was assessed before and after
methacholine challenge (2.5, 10, 20, 40, 60 mg/ml), 24 hours following a 3-day
exposure to room air, CS, or e-cig vapor. A: airway resistance (R) as percent
increase of baseline in response to methacholine. B: area under curve (AUC)
for R. Values are expressed as mean ⫾ SD; n ⫽ 5 mice; *P ⬍ 0.05 vs. air. CS,
cigarette smoke; PG:VG-Sol, propylene glycol/vegetable glycerol; PG:VG-N,
propylene glycol/vegetable glycerol with nicotine; PG:VG-N⫹F, propylene
glycol/vegetable glycerol with nicotine and tobacco blend flavor.

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 E-CIGARETTE AND LUNG FUNCTION

airways compared with air-exposed mice. Of note, among
mucins, Muc5ac is a predominant gel-forming mucin induced
in allergic murine lungs (13), suggesting a fundamental effector role in airway hyperreactivity. Our finding regarding
Muc5ac in mice exposed to CS and PG:VG-N⫹F correlates
with the observation that these groups of mice also exhibited
increased airway reactivity after methacholine challenge. In
line with this, Lim et al. (28) reported that e-cig vapor exacerbated the allergy-induced asthma symptoms in mice, although in this latter model the e-cig liquid was not vaporized
but was instilled intratracheally. Complementing our findings,
Garcia-Arcos et al. (17) also reported that long-term exposure
to nicotine-containing e-cig vapor increased airway hyperreactivity to methacholine.
Our results provide evidence for impairment of functional
lung parameters in mice after short-term exposure to e-cig
vapors. Interestingly, resistance, elastance, and compliance
were only affected in the PG:VG-Sol group. Larcombe et al.
(25) have also reported decrements in parenchymal lung function at both functional residual capacity and high transrespiratory pressures after exposure to e-cig vapors. The abovementioned preclinical data are in agreement with human studies; Vardavas et al. (45) have shown that short-term exposure
to e-cig vapor can increase the impedance and peripheral
airway flow resistance, suggesting that airway constriction
because of e-cig use is a result of the irritant effects of
propylene glycol. After more prolonged exposure to e-cig
vapors, the effect on pulmonary mechanics disappeared. At this
time point, pulmonary mechanics were adversely affected in
CS-exposed mice.
To study the effects of CS or e-cig vapors on lung structure
we used histology and determined inflammatory cell intraalveolar and interstitial recruitment, congestion, and induced
intra-alveolar and interstitial edema. Interestingly, we found a
high lung injury score only in CS-exposed mice both after 3
days or 4 wk of exposure, compared with e-cig vapor- or
air-exposed mice. Our data are aligned with the evidence of the
less toxic effect of e-cig vapor compared with tobacco smoke
(37), especially regarding the loss of lung integrity in mice
(22), albeit e-cig vapor exposure clearly promotes pulmonary
inflammatory effects (6).
In summary, we have shown that all ingredients of e-cig
refills, including the vehicle PG:VG, can induce lung inflammation and cause changes in respiratory mechanics. These
effects were exacerbated by the addition of flavoring to the
e-cig. The observed detrimental effects in the lung upon e-cig
vapor exposure in animal models highlight the need for further
investigation of safety and toxicity of these rapidly expanding
devices worldwide.
GRANTS
This study was funded in part by a joint grant from Nobacco and Alterego
(to A. Papapetropoulos).
DISCLOSURES
This study was funded in part by a grant by Nobacco and Alterego, vendors
of e-cigarettes, to A. Papapetropoulos.
AUTHOR CONTRIBUTIONS
A. Papapetropoulos conceived and designed research; C.G., S.-I.B., A.
Pavlidou, C.M., and V.K. performed experiments; C.G., S.-I.B., C.M., V.K.,
and A. Papapetropoulos analyzed data; C.G., P.K., S.T., I.K., S.Z., and A.

L671

Papapetropoulos interpreted results of experiments; C.G. and S.-I.B. prepared
figures; C.G., P.K., S.T., and A. Papapetropoulos drafted manuscript; I.K. and
S.Z. edited and revised manuscript; C.G., S.-I.B., P.K., A. Pavlidou, V.K.,
S.T., I.K., S.Z., and A. Papapetropoulos approved final version of manuscript.
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