Fig 2. OX/ROS in ENDS vapor.

Aerosols or air-sham control drawn through DCFH OX/ROS

indicator solution.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

o An air flow pump was used to draw aerosols from Ecs through a fluorescein
dye (DCFH solution DCFH is an indicator for presence of reactive OX-ROS
in cell/cell-free systems. Activation of OX/ROS or ROS is indicated by DCF
which emits green fluorescence following excitation at 490nm. DCF is the
oxidated form of DCFH)
o Fig 2A) 2 different flavors of Blu ECs, one with nicotine, one without, (Classic
Tobacco 16 mg nicotine, Magnificent Menthol 0 mg nicotine) were used
for detection of OX/ROS in the vapor.
o Aerosols drawn through DCFH produced by both flavors resulted in increase
in H2O2 equivalents as compared to air-sham group.
o Comparison of both aerosols showed the one with nicotine resulted in
significantly reduced levels of H202 equivalents.
o Fig 2B) Ego Vision Spinner with 2.2 ohm wicked heating element and
clearomizer chamber (holds 4.5 mL e-liquid noticeably larger than Blu Ecs,
but produces aerosols in similar way)
o Many flavors of e-liquids available, but propylene glycol and glycerin are the
common humectants used for all of them.
o Results of this experiment show that OX/ROS are emanating from
Ecs/e-liquids and are associated with the aerosols drawn through the
DCFH indicator and nicotine was not likely a sole contributing factor in
increased OX/ROS

Table 2. DCF fluorescence values obtained for refillable ENDS aerosols or ambient air alone
drawn through DCFH in cell-free ROS assay.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Following the first set of experiments, it was unclear if the

OX/ROS detected in the study emanated from the ENDS
aerosols, or from the aerosols and the heating element.
To test this, the researchers drew air through the refillable
ENDS device by the pump, as the ENDS was activated.
There was no sign of aerosol production. There was OX/ROS
activity, despite the lack of aerosol. This suggests the heating
element is indeed a factor of OX/ROS activity.

Table 3. State of the refillable ENDS heating element and its influence over successive use to
generate OX/ROS in a cell-free ROS assay.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

 The researchers hypothesis supported that the OX/ROS activity is from the heating
element as well, and that the state of the element (new/used) is also a factor.
The researchers cleaned and refilled the removable clearomizer chambers with 2.0 mL
of either propylene glycol, glycerin, or a commercial refill e-liquid (flavor Vape Dudes
Classic tobacco, 0 mg nicotine.
They bought two new heating elements and also used 3 used heating elements (used
50+ times during previous experiments).
OX/ROS activity was seen with the pre-used heating element (methods were the same
as in the first experiment), compared to the air-sham group, which did not show an
appreciable level of reactivity.
Next, they ran the same humectant/e-liquids through the new
Results show the state of the heating element after activation affects the generation of
OX/ROS.
Next, they installed a new heating element and activated it without e-liquids for three
trials.
It was noticed that OX/ROS levels were high during the first use, but became similar to
air-sham group during second and third uses.
Next, they added a single drop of e-liquid onto the wick for absorption for a 4th trial. The
researchers hypothesized there would be a spike in OX/ROS due to the vaporization
process of the e-liquids. The 4th trial run was done in 5 minutes (half number of puffs).
They diluted the DCFH solution 1:10.
Results overall: There are at least 2 possible OX/ROS released from ENDS. 1)
heating element activation 2) vaporization of e-liquids process

ENDS dripping technique and

OX/ROS Generation (Table 3 &
Table 3 Experiment 3)
Usually, consumers use a refillable clearomizer chamber for e-liquids,
but another technique called dripping, similar to the method in trial #4
above, is becoming popular. This technique involves the consumer
dripping the e-liquid directly onto the heating element wick with a drip
tip.
-To test if the clearomizer technique produces more OX/ROS activity
than the dripping technique, a pre-used heating element was installed
into an ENDS for trial 1 & 2 (DCFH does not have to be diluted for this
method), and the aerosols were tested. They did show increased
OX/ROS.
-To test the dripping technique, the DCFH had to be diluted 1:10, and
then aerosols produced were measured after dripping small amounts of
e-liquid onto the wick. This method showed higher OX/ROS than in
trial 1 & 2

Fig 3. E-liquid reactivity with DCFH exhibits differences between nicotine content and flavor
additives.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Table 4. DCF fluorescence of refillable e-liquids with different flavors and nicotine
concentrations after addition of DCFH solution analyzed by a cell-free ROS assay.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Reactivity of Commercial e-fluids with DCFH

- Variety of e-liquids of differing flavor and nicotine content were
reacted directly with the DCFH solution.
- Water and humectants (propylene glycol and glycerin) were also
tested, but showed no reaction with the DCFH solution.
- Table 4 shows the differing reactions of various flavors.
-It was noticed that the e-liquids without nicotine exhibited significantly
more DCFH reactivity than those with nicotine. (regardless of flavor or
brand)
-E-liquids with added flavoring (such as the dessert and sweet flavors)
other than regular tobacco flavor, revealed higher reactivity than
tobacco flavor.
-This experiment suggests that the fun non-tobacco flavors are more
OX/ROS reactive and therefore more injurious.

Fig 4. Addition of e-liquids to cell culture media induces morphological changes in human lung
fibroblasts.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Human Lung Fibroblasts Exhibited Stress and Morphological Change in Response

to E-liquids/Humectants
-The researchers wanted to test the OX/ROS activity in cell conditions as well.
-Human lung cells were used to test for morphological changes, which are a sign of stress.
-There were various alterations in the fibroblasts (treated after 24 hours; near confluence)
-The fibroblasts were treated with 1% or 5% propylene glycol, glycerin, or tobacco flavored
e-liquid.
-Fig 4B) reduction in number of cells per count area in fibroblasts cultured with e-liquid and
CSE.
-Many were enlarged and vacuolarized greater in CSE treated fibroblasts than 5% e-liquid
-E-liquid in 1% concentration (no nicotine) showed similar morphological alterations to 1%
propylene glycol.
-1% e-liquid treated cells with nicotine were more similar to cells treated with 1% CSE
-Cell overlap and mixed directional orientation throughout image field for cells treated with
1% e-liquid with nicotine.
-Control shows a fusiform structure, which was completely lost in the cells treated with 1%
CSE.
-CSE and e-liquid tx shows prevalence of larger adhered circular cells halo effect
-Overall results: e-liquids applied to directly to lung fibroblasts = signs of cell stress and
other phenotypic abnormalities
-Nicotine seems to make the stress worse.

Table 5. Effect of e-liquids on HFL-1 cell viability in small 24-well culture area after 24 hours.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Normal human lung fibroblasts were cultured in 35 mm dishes and grown to 90%
confluence to assess cell viability after exposure to e-liquids/humectants v. CSE.
-Cells were added to mediums consisting of the various substances tested, and
measured after 24 hours.
-CSE 1%: high density maintained normal lung fibroblasts for over 24 hours
without sig cell viability
-2.5% propylene glycol, glycerin, commercial e-liquids) not much diff than the
control
-2.5% CSE: signif cell death, leading to less than 20% viability after 24 hours.
-Lung fibroblasts = more sensitive to CSE than e-liquids/humectants which did
not effect cell viability when treated with a greater concentration than the CSE.
-Smaller growth areas for the cultures showed differing results, which shows that
the susceptibility to loss of cell viability by direct addition of e-liquids to culture
media is dependent on size of the cell population.

Fig 5. Inflammatory mediators secreted by human lung fibroblasts (HFL-1) treated with eliquids/humectants and human epithelial airway cells (H292) treated by air-liquid interface with
e-cigarette aerosols.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Air-liquid interface culture system used to expose human lung

H292 epithelial cells directly to tobacco flavor Blu e-cig
Aerosols.
-IL-8 and IL-6 secretion measured at 16 hours: showed higher
secretion than air groups
-Release of IL-6 into culture media occurred dosedependently to aeros exposures
-10-min exposures to e-cig aerosols were much higher than 5min
-IL-8 levels were increased compared to air group, but were
not dose dependent

Fig 6. Air-liquid interface deposition of fluorescent substance on human bronchial airway

epithelial cells.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

C57BL/6J mice were acutely exposed to

classic tobacco flavor e-cigs with nicotine
- Macrophage

counts were higher in e-cig exposed mice, but not

signif compared to control
-Pulmonary inflammation was seen in BAL fluid collected 24 hours
after 3-day exposure
-IL-1alpha and Ili-13 were higher in e-cigs aerosol exposed mice
than in control
-IL-1alpha, IL-1beta, and IL-13 were slightly increased in e-cig
aerosol exposed mice but not signif.
-Overall: acute side-stream exposure in mouse lung is sufficient to
elicit an inflame response due to
Increased levels of proinflammatory mediators

Fig 7. Acute e-cigarette aerosol exposure causes lung inflammation and pro-inflammatory
response in mouse lungs.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Mice sacrificed after acute blu e-cig exposure (3 rd

day; 5 hours expos)
-Blood was collected and used to measure cotinine
levels which is a nicotine metabolite
-Plasma showed 10.78 +- 7.80 ng/ml for e-cig
exposed mice, but plasma collected from mice who
were sacrificed
24 hours after exposure (instead of immediately) did
not show detectable cotinine levels

Fig 8. Intracellular glutathione levels in mouse lung following acute e-cigarette aerosol
exposure.

Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, et al. (2015) Vapors Produced by Electronic Cigarettes and E-Juices with
Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response in Lung Epithelial Cells and in Mouse Lung. PLoS ONE
10(2): e0116732. doi:10.1371/journal.pone.0116732
http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0116732

Total and oxidized glutathione assessed.

-Lung lysates of mice exposed to side stream
aerosols showed depleted glutathione levels
-Oxidized levels were also lower.
-Ratios of ttl Glutathione to oxidized Glutathione
revealed that ttl glutathione levels are reduced
by e-cig aerosols
And the redox balance b/t both forms is affected
by side-stream e-cig aerosol inhalation