E-cigarettes and flavorings induce inflammatory and pro-senescence responses in oral epithelial cells and periodontal fibroblasts

Abstract

Electronic-cigarettes (e-cigs) represent a significant and increasing proportion of tobacco product consumption, which may pose an oral health concern. Oxidative/carbonyl stress via protein carbonylation is an important factor in causing inflammation and DNA damage. This results in stress-induced premature senescence (a state of irreversible growth arrest which re-enforces chronic inflammation) in gingival epithelium, which may contribute to the pathogenesis of oral diseases. We show that e-cigs with flavorings cause increased oxidative/carbonyl stress and inflammatory cytokine release in human periodontal ligament fibroblasts, Human Gingival Epithelium Progenitors pooled (HGEPp), and epigingival 3D epithelium. We further show increased levels of prostaglandin-E2 and cycloxygenase-2 are associated with upregulation of the receptor for advanced glycation end products (RAGE) by e-cig exposure-mediated carbonyl stress in gingival epithelium/tissue. Further, e-cigs cause increased oxidative/carbonyl and inflammatory responses, and DNA damage along with histone deacetylase 2 (HDAC2) reduction via RAGE-dependent mechanisms in gingival epithelium. A greater response is elicited by flavored e-cigs. Increased oxidative stress, pro-inflammatory and pro-senescence responses (DNA damage and HDAC2 reduction) can result in dysregulated repair due to proinflammatory and pro-senescence responses in periodontal cells. These data highlight the pathologic role of e-cig aerosol and its flavoring to cells and tissues of the oral cavity in compromised oral health.

Full text

Oncotarget1
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www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2016
E-cigarettes and avorings induce inammatory and pro-
senescence responses in oral epithelial cells and periodontal
broblasts
Isaac K. Sundar1, Fawad Javed2, Georgios E. Romanos3, Irfan Rahman1
1Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY
2Department of General Dentistry, Eastman Institute for Oral Health University of Rochester, Rochester, NY
3Department of Periodontology, School of Dental Medicine, Stony Brook University, Stony Brook, NY
Correspondence to: Irfan Rahman, email: irfan_rahman@urmc.rochester.edu
Keywords: e-cigarettes, e-juices, RAGE, COX2, PGE2
Received: August 02, 2016 Accepted: October 14, 2016 Published: October 24, 2016
ABSTRACT
Electronic-cigarettes (e-cigs) represent a signicant and increasing proportion
of tobacco product consumption, which may pose an oral health concern. Oxidative/
carbonyl stress via protein carbonylation is an important factor in causing inammation
and DNA damage. This results in stress-induced premature senescence (a state of
irreversible growth arrest which re-enforces chronic inammation) in gingival
epithelium, which may contribute to the pathogenesis of oral diseases. We show that
e-cigs with avorings cause increased oxidative/carbonyl stress and inammatory
cytokine release in human periodontal ligament broblasts, Human Gingival Epithelium
Progenitors pooled (HGEPp), and epigingival 3D epithelium. We further show increased
levels of prostaglandin-E2 and cycloxygenase-2 are associated with upregulation of
the receptor for advanced glycation end products (RAGE) by e-cig exposure-mediated
carbonyl stress in gingival epithelium/tissue. Further, e-cigs cause increased oxidative/
carbonyl and inammatory responses, and DNA damage along with histone deacetylase
2 (HDAC2) reduction via RAGE-dependent mechanisms in gingival epithelium. A greater
response is elicited by avored e-cigs. Increased oxidative stress, pro-inammatory
and pro-senescence responses (DNA damage and HDAC2 reduction) can result
in dysregulated repair due to proinammatory and pro-senescence responses in
periodontal cells. These data highlight the pathologic role of e-cig aerosol and its
avoring to cells and tissues of the oral cavity in compromised oral health.
INTRODUCTION
The use of electronic-cigarettes (e-cigs) is increasing
in the United States, which may pose oral health concerns
[1]. E-cigs are battery operated devices, which consist
of a metal heating element in a stainless steel shell, a
cartridge, an atomizer and a battery. The heating element
vaporizes a solution containing a mixture of chemicals
including nicotine and other additives/humectants, such as
base/carrying agents propylene glycol, glycerin/glycerol,
and avoring agents including fruit and candy avors.
Apart from inhaled nicotine, variable levels of aldehydes
and carbonyls are detected in e-cig aerosols during
vaporizations [2, 3]. Aldehyde causes carbonyl/oxidative
stress, DNA adducts/damage, as well as stress-induced
cellular senescence (a state of irreversible growth arrest
which re-enforces chronic inammation) [4, 5] leading to
oral health problems [6–8].
Periodontal disease is characterized by chronic
inammation of the supporting tissues of the teeth.
Periodontal ligament and gingival broblasts as well as
epithelial cells are the most abundant structural cells in
periodontal tissue. Upon stimulation or stress, these cells
are able to incite and maintain inammatory responses
[9]. There is an association between smoking and tooth
loss, periodontal attachment level, deeper periodontal
pockets, and more extensive alveolar bone loss along with
the destruction of connective tissue and matrix, leading

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to increased risk of periodontitis [10]. Clinical studies
[11, 12] have also shown that habitual tobacco smokers
exhibit a greater number of sites with plaque accumulation,
clinical attachment loss and probing depth (≥ 4 mm) as
compared to individuals who had never used tobacco in
any form. It is important to mention that bleeding upon
probing (a classical marker of periodontal disease activity)
is masked in tobacco smokers than non-smokers [11, 12].
This most likely occurs as a result of the vasoconstrictive
effect of nicotine (nicotine is the main component in
e-cigs) on gingival blood vessels. Therefore, tobacco
smokers may remain unaware of ongoing periodontal
destruction until the inammatory process reaches a stage
where tooth mobility becomes evident.
We have recently shown the comparable oxidants/
reactive oxygen species (ROS) reactivity in e-cig
aerosols compared to conventional cigarette smoke
[13, 14]. Smoking tobacco contributes to the progression
of periodontal disease [10]. However, there is no
information available regarding the e-cig aerosols vaping
on periodontal/gingival oral health effects, especially in
response to e-cig avoring agents and nicotine. Periodontal/
gingival cells in the oral cavity are the rst targets by
aerosols of e-cigs. Furthermore, the effects of e-cig aerosols
on carbonyl stress, inammation, and pro-senescence have
not been studied on oral health. Here, we have determined
the mechanism of gingival epithelial inammation and pro-
senescence by e-cig aerosols with avorings in human oral
epithelial cells and periodontal ligament broblasts.
RESULTS
E-cigarette exposure in human periodontal
ligament broblasts (HPdLFs) and human
gingival epithelium progenitors, pooled
(HGEPp) increases protein carbonylation and
pro-inammatory responses
It is possible that ROS produced by e-cig vapors
in cultured cells can augment protein carbonylation. The
oxyblot assay was performed for immunodetection of the
carbonyl groups that were introduced into protein side
chains by e-cig-induced oxidative stress. We found that
e-cig avoring (BLU® Classic Tobacco, 16 mg nicotine
and Magnicent Menthol, zero nicotine) had different
levels of protein oxidation as conrmed by OxyBlot.
E-cigarette vapors from BLU® Classic Tobacco showed
signicant increase in protein carbonylation compared
to air exposed controls. E-cigarette vapors from BLU®
Magnicent Menthol avor also showed a trend towards
increased protein carbonylation but not signicant
compared to the control (Figure 1A–1B).
HPdLFs and HGEPp were exposed to BLU®
e-cig vapors (Classic Tobacco, 16 mg nicotine; and
Magnicent Menthol, zero nicotine) using ALI system
for 15 min. Conditioned medium was collected from
both air exposed (control) and e-cig vapor exposed cells
24 hrs post ALI exposure. IL-8 secretion in conditioned
medium 24 hrs after ALI exposure was signicantly
increased in both BLU® e-cig vapors. PGE2 secretion
showed a trend of incremental increase, but was not
signicant compared to air exposed controls (Figure
1C). IL-8 release was signicantly higher in both BLU
®
Classic Tobacco (16 mg nicotine) and Magnicent
Menthol (zero nicotine) e-cig vapor exposed HPdLF cells
compared to the controls. PGE
2
release was signicantly
higher in BLU® Magnicent Menthol (zero nicotine)
e-cig vapor exposed cells compared to the controls
(Figure 1C). Similarly, HGEPp cells also showed an
increased trend in IL-8 release in BLU® Classic Tobacco
(16 mg nicotine) e-cig vapor exposed cells compared to
the controls. We did not see a signicant increase in IL-8
release in HGEPp cells exposed to BLU® Magnicent
Menthol (zero nicotine) e-cig vapor compared to control
(Figure 1D). Overall, e-cig vapors with avoring induce
protein carbonylation and increase in pro-inammatory
cytokines release which are indicative of oxidative stress
and inammatory response cause by e-cig vapors in
HPdLF and HGEPp.
E-cigarette exposure in human periodontal
ligament broblasts (HPdLFs) increases
inammation and DNA damage markers
HPdLFs exposed to e-cig vapor from BLU® e-cig
(Classic Tobacco, 16 mg nicotine; and Magnicent
Menthol, zero nicotine) for 15 min using ALI system and
incubated for 24 h. Then, we measured several markers of
inammation such as PGE2-mediated COX-2 induction,
HDAC2, S100A8, RAGE and phosphorylated γH2A.X
(Ser139) in whole cell lysates. We found E-cig avoring
BLU® Classic Tobacco (16 mg nicotine) and Magnicent
Menthol (zero nicotine) showed a differential effect on
levels of COX-2, HDAC2, S100A8, RAGE and γH2A.X
in HPdLFs in vitro (Figure 2A). HPdLFs exposed to
BLU® e-cig vapors (Magnicent Menthol, zero nicotine)
showed a signicant increase in all the inammatory
markers (COX-2, S100A8, RAGE), a trend towards
reduced HDAC2 and increased phosphorylated γH2A.X
(Ser139), a DNA damage marker compared to control.
HPdLFs exposed to BLU® e-cig (Classic Tobacco, 16 mg
nicotine) also showed a signicant increase in S100A8 and
γH2A.X, and an increased trend in COX-2 compared to
the controls. We did not observe any signicant increase in
RAGE or a signicant decrease in HDAC2 levels in BLU
®
E-cig vapor (Classic Tobacco, 16 mg nicotine) exposed
HPdLFs compared to the controls (Figure 2A). Likewise,
the HPdLFs exposed to e-cig vapor from BLU® e-cig
(Classic Tobacco, 16 mg nicotine) showed a signicant
increase in DNA damage as measured by the percentage of
DNA in tail using the Comet assay (Figure 2B). Overall,
we showed that e-cig vapors with avoring differentially

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affects inammatory response and DNA damage markers
as a result of oxidative stress and inammatory response
caused by e-cig vapors in HPdLFs.
E-cigarette exposure in human gingival
epithelium progenitors, pooled (HGEPp)
increases inammation and DNA damage
markers
We found E-cig avoring BLU® Classic Tobacco
(16 mg nicotine) and Magnicent Menthol (zero
nicotine) showed differential effects on levels of COX-
2, S100A8, RAGE and γH2A.X in HGEPp cells in
vitro (Figure 2A). HGEPp cells exposed to BLU® e-cig
vapors (Classic Tobacco, 16 mg nicotine and Magnicent
Menthol, zero nicotine) showed a signicant increase
in the inammatory markers (COX-2 and RAGE), and
DNA damage marker (phosphorylated γH2A.X Ser139)
compared to the controls. The effect of BLU
®
e-cig vapors
(Magnicent Menthol, zero nicotine) on HGEPp cells
was signicantly higher compared to BLU
®
e-cig vapors
Classic Tobacco (16 mg nicotine) and the controls. We
did not observe any signicant increase in COX-2 and
S100A8 levels in BLU
®
e-cig vapor from Classic Tobacco
(16 mg nicotine) as well as S100A8 levels in Magnicent
Menthol (zero nicotine) exposed HGEPp cells compared
to the controls (Figure 3). Overall, we conrmed that
e-cig vapors with avoring affects inammatory response
and DNA damage markers as a result of oxidative stress
and inammatory response caused by e-cig vapors in
HGEPp cells.
E-cigarette exposure in human 3D in vitro model
of EpiGingival tissue increases inammation and
DNA damage markers
Human 3D model of EpiGingival tissues were
exposed to e-cig vapor from BLU
®
(Classic Tobacco, 16 mg
nicotine; and Magnicent Menthol, zero nicotine) for 15 min
using the modied ALI system without culture media. The
human 3D EpiGingival tissue insert was contained within
35 mm culture dishes along with 900 μl culture medium
during e-cig vapor exposure, and incubated for 24 h. Then,
we measured pro-inammatory cytokines IL-8 and PGE2
Figure 1: E-cig aerosol resulted in increased protein carbonylation and inammatory responses in human periodontal
ligament broblasts (HPdLFs) and human gingival epithelium progenitors, pooled (HGEPp). HPdLFs (A–C) and HGEPp
(D) were exposed to aerosols from BLU® e-cig (Classic Tobacco, 16 mg nicotine; and Magnicent Menthol, ‘zero’ nicotine) (2 puffs/min;
4–5 sec/puff every 25 sec) using air-liquid interface system for 15 min, and then incubated at 37°C and 5% CO2 for 24 h. (A, B) Protein
carbonylation in cell lysates was determined by the oxyblots. Gel pictures shown are representative of at least 3 separate experiments for
protein carbonylation. (C, D) Levels of IL-8 and PGE2 in supernatants were determined by ELISA. Data are means ± SE (n = 3–6/group)
and signicance determined using 1-way ANOVA. *P < 0.05, vs. air. MWM: Molecular weight markers; Negative: without derivitizing
reagent 2,4-dinitrophenylhydrazine (DNPH).

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in conditioned medium collected 24 h post last exposure.
Markers of inammation and DNA damage, such as RAGE,
PGE2-mediated COX-2 induction and γH2A.X were
measured using whole tissue lysates from 3D EpiGingival
cultures. We found e-cig avoring BLU® Classic Tobacco
(16 mg nicotine) and Magnicent Menthol (zero nicotine)
showed an increasing trend in the levels of IL-8 release
and a signicant increase in the levels of PGE2 release
compared to the controls (Figure 4A). Additionally, only
e-cig avoring BLU
®
Magnicent Menthol (zero nicotine)
showed an increasing trend in RAGE, COX-2, and γH2A.X
(immunoblot analysis and immunohistochemistry) in 3D
EpiGingival tissues in vitro compared to the controls (Figure
4B–4C). The effect of BLU® e-cig vapors (Magnicent
Menthol, zero nicotine) on 3D EpiGingival tissues was
signicantly higher compared to BLU
®
e-cig vapors Classic
Tobacco (16 mg nicotine) and the controls. Overall, we
reproduced the above ndings in 3D models that e-cig
vapors with avoring differentially affects inammatory
response and DNA damage markers as a result of oxidative
stress and inammatory response caused by e-cig vapors in
3D in vitro model of EpiGingival tissues.
DISCUSSION
In this study, we show that e-cigs with avorings
cause increased oxidative/carbonyl stress and inammatory
cytokine release in human periodontal ligament broblasts,
Human Gingival Epithelium Progenitors pooled (HGEPp),
and EpiGingival 3D epithelium. We further show increased
levels of prostaglandin-E2 and cycloxygenase-2 were
associated with upregulation of the receptor for advanced
glycation end products (RAGE) by e-cig exposure-
mediated carbonyl stress in gingival epithelium/tissue.
Further, e-cigs cause increased oxidative/carbonyl stress
and inammatory responses, and DNA damage along with
histone deacetylase 2 (HDAC2) reduction via RAGE-
dependent mechanisms in gingival epithelium, with
greater response by avored e-cigs. Increased oxidative
stress, pro-inammatory and pro-senescence responses
(DNA damage and HDAC2 reduction) can result in
dysregulated repair due to proinammatory and pro-
senescence responses in periodontal cells. Our data also
implicate that e-cig affects the regenerative potential of
human progenitor cells due to increased inammatory and
DNA damage responses. It is well known that the mTOR
pathway is activated by most oncogenes that induce
cellular senescence (i.e., by Ras, Raf, MEK, and Akt).
Further, the mechanistic target of rapamycin (mTOR) is
generally activated in proliferating cells. During acute
DNA damage, mTOR induces DNA damage response
(DDR) and cell cycle arrest (induction of p21 and 16) [15].
Such arrested cells where mTOR is active play an
important role in geroconversion (converts quiescence into
senescence) to their pro-senescent phenotype [15, 16]. In
case of oncogene-induced senescence, DDR causes cell
Figure 2: E-cig vapor exposure caused inammatory responses and DNA damage in human periodontal ligament
broblasts (HPdLFs). (A) HPdLFs were exposed to aerosols from BLU® e-cig (Classic Tobacco, 16 mg nicotine; and Magnicent
Menthol, ‘zero’ nicotine) for 15 min (2 puffs/min; 4–5 sec/puff every 25 sec) using air-liquid interface system, and then incubated at 37°C
and 5% CO2 for 24 h. (A) Levels of COX-2, S100A8, RAGE, γH2AX, and HDAC2 in cell lysates were measured by Western blotting. (B)
DNA damage, indicated by an increase in uorescent tail length, was measured by the Comet assay. Dash lines indicate DNA in tails. Data
are means ± SE (n = 3–6/group) and signicance determined using 1-way ANOVA or 2-tail t-test. *P < 0.05, **P < 0.01, ***P < 0.001
vs. air.

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cycle arrest, leading to cell senescence [16]. This may
be one of the mechanisms of e-cigarette induce DDR
response via mTOR activation.
Conventional cigarette smoke has been shown to
cause deleterious effects on oral and periodontal health
[10]. However, the role of e-cig vaping and its association
with carbonyl stress, inammation, and DNA damage-
triggered senescence on oral/periodontal epithelium
remains unknown. Periodontal ligament and gingival
broblasts as well as epithelial cells are the most abundant
structural cells in periodontal tissue, and are the direct
targets of e-cigs upon vaping. Upon stimulation or stress,
these cells are able to incite and maintain inammatory
responses [9]. There is an association between smoking
and tooth loss, periodontal attachment loss, deeper
periodontal pockets, and more extensive alveolar bone
loss along with the destruction of connective tissue and
matrix , leading to an increased risk of periodontitis [10].
However, no studies are available to document the effects
of e-cig vaping especially in response to e-cig avoring
agents on periodontal health in terms of oxidative/
carbonyl stress and inammation in human gingival
epithelial cells in vitro.
We have recently shown oxidants/ROS reactivity
from e-cig aerosols is comparable to conventional
cigarette smoke [13, 14]. We show that e-cig avoring
caused protein oxidation as reected in increased protein
carbonylation. This was associated with increased IL-8
and PGE2 secretion from HPdLFs and HGEPp cells
upon exposure to e-cig aerosols. Direct exposure to
e-liquids (this is not the case when users vape e-cigs i.e.
users do not consume e-liquids) has also been shown
to produce harmful effects in periodontal ligament
and gingival broblasts in culture [17, 18]. While the
contribution of smoking tobacco to the progression
of periodontal disease and other adverse oral health
outcomes is well described [10, 19], no information is
available regarding the impact of e-cig aerosols vaping
on periodontal/gingival oral health effects, especially in
response to e-cig avoring agents. We determined the
effect of avoring on oxidative and pro-inammatory
responses, and upon exposure of periodontal ligament
broblasts, Human Gingival Epithelium Progenitors
pooled (HGEPp), and epigingival 3D epithelium to
menthol avoring agent resulted in increased oxidative/
carbonyl stress, and IL-8 release. Menthol acts on
transient receptor potential ankyrin 1 (TRPA1) receptors
to activate inammatory responses [20, 21]. It may be
conceived that activation of TRPA receptors by e-cig
aerosols will drive COX-PGE2 mediated responses
in periodontal tissues, leading to augmentation of
inammatory and pro-brotic and pro-carcinogenic
responses. However, further studies are required to
understand the augmented response by BLU® menthol
avoring than BLU® classic tobacco.
Protein carbonylation leads to autoantibody
production which may lead to destruction of matrix
and bone loss during periodontitis [6, 7]. Further, it is
possible that carbonyls/aldehydes play an important
role in e-cig aerosol-induced oral toxicity. Conventional
tobacco smoke is known to cause oxidative burden
leading to DNA damage and inammatory responses
[22]. The RAGE is a pattern-recognition receptor
implicated in immune and inammatory diseases
including dental pulp inammation and periodontitis
[23–27]. RAGE is involved in smoking-related disorders
and known to cause cellular senescence via oxidant stress
[28, 29]. However, the mechanism of RAGE-mediated
Figure 3: E-cig vapor exposure caused inammatory responses and DNA damage in human gingival epithelium
progenitors, pooled cells (HGEPp). (A) HGEPp cells were exposed to aerosols from BLU® e-cig (Classic Tobacco, and Magnicent
Menthol) (2 puffs/min; 4–5 sec/puff every 25 sec) using air-liquid interface system for 15 min, and then incubated at 37°C and 5% CO2 for
24 h. Levels of COX-2, S100A8, RAGE, and γH2AX in cell lysates were measured by Western blotting. Data are means ± SE (n = 3–6/
group) and signicance determined using 1-way ANOVA. *P < 0.05, ***P < 0.001, vs. air.

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Figure 4: E-cig vapor caused inammatory responses and DNA damage in normal human 3D in vitro model of
EpiGingival tissues. Normal human 3D in vitro model of EpiGingival tissue (Cat#: GIN-100, MatTek) were exposed to aerosols from
BLU® e-cig (Classic Tobacco, and Magnicent Menthol) using air-liquid interface system for 15 min, and then incubated at 37°C and 5%
CO2 for 24 h. (A) Levels of IL-8 and PGE2 in culture media were determined by ELISA. (B) Levels of RAGE, COX-2, and γH2AX in tissue
lysates were measured by Western blotting. (C) Representative images of EpiGingival tissues used for ALI exposures stained with H&E
(showing histological features) and γH2AX staining 24 h post exposure to control (air) and avored e-cig aerosols. Immunohistochemistry
revealed a distinct staining in both the avored BLU e-cig exposed EpiGingival tissues for γH2AX. Data are means ± SE (n = 4–6/group)
and signicance determined using 1-way ANOVA. ***P < 0.001,vs. air.

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signaling especially via its ligand S100A8 as a
susceptible factor in inducing gingival epithelial
inammation and senescence by e-cigs is not known.
E-cig vapor exhibited signicant inammatory response
(COX2, RAGE S100A8), DNA damage as determined
by the Comet assay and γ-H2AX levels (a marker of
DNA damage) in gingival epithelium/tissue. Our data
showing inammatory and pro-DNA damage responses
are unique in light of primary cells and 3D tissue culture
models which mimic closely with users vaping of e-cigs.
Our results further attest the pro-oxidant, DNA damaging
and pro-inammatory effects of e-cig vapor exposure.
The increased levels of pro-inammatory mediators IL-8
and PGE2 would cause remodeling of the ECM during
periodontitis by e-cig due to cellular senescence, where
these cells have secretory phenotype to perpetuate the
inammatory responses.
It has been reported that outcomes of periodontal
therapy are compromised in smokers compared with
non-smokers [30, 31]. A variety of mechanisms have
been proposed in this regard. For example, increased
expression of RAGE occurs in gingival epithelial cells
of smokers as compared to non-smokers. Furthermore,
it has been reported that nornicotine (a metabolite of
nicotine), upregulates RAGE expression in the gingivae
of smokers and elicits a proinammatory response by
stimulating the secretion of cytokines and ROS which
are involved in destruction of the periodontal tissue
[32]. The vasoconstrictive effects of nicotine increase
platelet adhesiveness, increases the risk of microvascular
occlusion and causes tissue ischemia [32]. Furthermore,
tobacco smoking is also associated with catecholamines
release resulting in vasoconstriction and decreased
tissue perfusion [33]. Therefore, it is hypothesized that
the outcomes of periodontal surgery are compromised
in e-cig users compared with non-smokers/non-users
through the mechanisms comparable to those stated above.
However, further studies are needed to test this contention
in a clinical cohort of users and non-users of e-cigs vs
conventional smokers.
In conclusion, our data showed that e-cig aerosol
cause increased oxidative/carbonyl stress and inammatory
responses, and cellular senescence associated with
persistent DNA damage via RAGE-HDAC2-dependent
mechanisms in gingival epithelium, with greater response
by avored e-cigs. Further understanding of the chronic
effect of vaping could lead to molecular mechanisms for
susceptibility (inammatory, DNA damage and senescence
responses) to the development of periodontitis, and
therapeutic targets or biomarkers in determining vaping-
avoring mediated oral complications in cells and tissues
of the oral cavity. Our data also implicate that e-cig affects
the regenerative potential of human progenitor cells due
to increased inammatory and DNA damage responses.
Overall, our data suggest the pathogenic role of e-cig
aerosol to cells and tissues of the oral cavity, leading to
compromised periodontal health.
MATERIALS AND METHODS
E-cigarettes
BLU® rechargeable e-cigs (Lorillard Technologies,
Inc.) containing two different disposable cartomizers
[Flavors: classic tobacco (16 mg nicotine), and magnicent
menthol (zero nicotine or 13–16 mg nicotine)] with pre-
loaded e-liquid were used. The BLU® e-cigarette device
and disposable cartomizer cartridges were purchased from
local retailers.
Cell culture and air-liquid interface (ALI)
culture/exposures
Clonetics
TM
Human periodontal ligament broblast
(HPdLF, CC-7049; Lonza) were grown at 37ºC in 5% CO
2
incubator to 80–90% conuence in stromal cell medium
(SCGM
TM
BulletKit, Lonza; CC-3205) supplemented with
hFGF-B (0.5 ml), insulin (0.5 ml), FBS (25 ml) and GA-
1000 (0.5 ml) according to manufacturer recommendations.
Human gingival epithelium progenitors, (HGEPp; gingival
epithelial cells) were grown in CnT-Prime medium (CnT-
PR), epithelial culture medium as recommended by
CELLnTEC Advanced Cell Systems. Transwell cultures
were then placed into air-liquid interface (ALI) exposure
chamber [14, 34]. BLU® e-cigarette vapor (Classic Tobacco
containing 16 mg nicotine or Magnicent Menthol avor
containing zero or 13–16 mg nicotine) was drawn into the
ALI exposure chamber (2 puffs every 1 min), 4–5 second
puff followed by 25 second pause for different time
durations 5, 10, and 15 minutes respectively.
Human EpiGingival tissue model
Human gingival tissues (EpiGingival, GIN-100)
were obtained from MatTek Corporation (Ashland,
MA). The 3D tissue model is a reconstructed oral
epithelial tissue that are derived from human primary oral
keratinocytes. This model and allowed to differentiate to
a structure characteristic to that of in vivo. EpiGingival
tissues were exposed to air (control) or BLU
®
e-cigarette
vapors (Classic Tobacco and Magnicent Menthol). After
15 min exposure, the conditioned medium was collected to
measure pro-inammatory cytokines and tissues harvested
for Western blotting and immunohistochemistry according
to the recommendations of the manufacturer. The protein
levels were measured using a BCA kit (Pierce, IL, USA).
Comet assay
Comet assay was performed as per the instructions
of the manufacturer (Trevigen, Gaithersburg, MD) [34].
Comet images were captured using a Nikon ECLIPSE Ni
uorescent microscope. The images were analyzed using
OpenComet software. The extent of DNA damage was
expressed as a measure of percentage of DNA in tail.

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www.impactjournals.com/oncotarget
Protein carbonylation/oxyblot
Protein oxidation was determined using the
OxyBlot protein oxidation detection kit following the
manufacturer’s instruction (Millipore, S7150). Equal
amount of protein was loaded for oxyblot analysis, and
the results were quantied by densitometry using Image J.
Pro-inammatory cytokine analysis
Following 24 hrs after BLU® e-cigarette vapor
exposure using air-liquid interface approach, conditioned
media was collected and stored at −80ºC for measuring pro-
inammatory mediators. IL-8 (Life Technologies, Carlsbad,
CA) and PGE
2
(Cayman Chemical, Ann Arbor, MI) levels
were measured by enzyme-linked immunosorbent assay
(ELISA) according to manufacturer’s instructions.
Western blotting
For Western blots, 25 μg protein samples were
separated on a 4–15% gradient and 7.5% SDS-PAGE gels.
Then probed with specic primary antibodies (1:1000
dilution in 5% milk in PBS containing 0.1% Tween
20 (v/v), such as anti-COX2 (Cayman chemical; #160112),
HDAC2 (ab32117), S100A8 (ab92331), RAGE (ab37647)
and γH2A.X phospho S139 (ab2893) from Abcam at 4°C
overnight. The bound complexes were detected using
ECL method with the ChemiDocTM MP Imaging System
(Bio-Rad). Equal loading of the samples was determined
by quantitation of proteins and by stripping and re-
probing membranes for β-actin or GAPDH (Santa Cruz
Biotechnology, sc-1616 and sc-365062) and the results
were quantied by densitometry using Image J.
Statistical analysis
Statistical analysis of signicance was calculated
using unpaired Student’s t-test for comparison between
two groups control vs. e-cigarette. Probability of
signicance compared to control for more than
two treatment groups (different e-cigarette avors)
was analyzed by 1-way ANOVA (Tukey’s multiple
comparisons test) using GraphPad Prism 6 as indicated in
gure legends. The results are shown as the mean ± SEM
unless otherwise indicated. P < 0.05 is considered as
statistically signicant.
ACKNOWLEDGMENTS
We thank Ms. Janice Gerloff for technical assistance.
CONFLICTS OF INTEREST
The authors have declared that no conicts of
interests exists.
GRANT SUPPORT
This study was supported by the
NIH 2R01HL085613 and 3R01HL085613-07S2 (to I.R.).
Authors’ contributions
IKS, IR: Conceived and designed the experiments;
IKS: Performed the experiments; IKS: Analyzed the data;
FJ, GER: Provided inputs and thoughtful discussion as
well as edited the manuscript; IKS, IR: Wrote and edited
the manuscript.
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