GN_honors thesis_finalversion.docx

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of the Effects of Electronic Cigarettes

Grace Nipp

Honors Thesis ENHS

Department of Environmental Sciences and Engineering Gillings School of Global Public Health

The University of North Carolina at Chapel Hill April, 2019

Approved

:

________________________________ Dr. Ilona Jaspers (Advisor) UNC Department of Pediatrics

________________________________ Dr. Jason Surratt (Advisor/Reader)

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Acknowledgements

This research is funded by R01 HL139369-01 in addition to T32 ES007126. Research reported

in this thesis was in part supported by NIH and the FDA Center for Tobacco Products. The

content is solely the responsibility of the author and does not necessarily represent the official

views of the National Institutes of Health or the Food and Drug Administration.

Additional funding was provided by William W. and Ida W. Taylor Undergraduate Research

Fellowship.

In Vitro exposures and analysis were completed with the aid of Yael Escobar, and chemical

analysis was completed with the help of Tianqu Cui, both of whom deserve much thanks and

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1. Abstract

Use of electronic cigarettes (e-cigs) has proliferated in recent years. However, there

remains little consensus on e-cigs’ health effects. With the plethora of devices, e-liquids, and

flavorings available, a controlled yet flexible exposure system is needed, allowing for

comparisons between e-cig devices at varying settings. This study developed and optimized a

versatile e-cig exposure system and performed initial exposures to determine biological effects

associated with specific e-cig chemical constituents. Mock exposures using a fourth-generation

e-cig device were performed to determine exposure chamber parameters, which yield

homogeneous aerosol deposition. Initial exposures of human bronchial epithelial cells to vaped

propylene glycol (PG) and glycerin (GLY) were conducted at PG:GLY ratios of 100:0, 55:45,

and 0:100. IL-6 and IL-8 transcript and protein levels were analyzed following acute exposure.

Results indicate that this exposure system will allow for a controlled, replicable exposure to

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2. List of Tables

Table 1. Chemical characterization of collected condensate from e-cig aerosol created at 85 W and 40 W.

Characterization completed using HILIC/ESI(–)-HR-QTOFMS_________________________________________25

Table 2. Solvent Volume and Mass Collected for 40W Samples_________________________________________32

Table 3. Solvent Volume and Mass Collected for 85W Samples_________________________________________32

3. List of Figures

Figure 1. Depiction of the aerosol exposure chamber_________________________________________________11

Figure 2. Procedure for collecting and measuring aerosol condensate_____________________________________12

Figure 3. Schema for labeling transwells and tissue culture plate placement_______________________________12

Figure 4. Mass deposited onto each coverslip (Area = 0.503 cm2_{) after exposure to vaporized E-liquid with different}

chamber permutations. n=3, mean ± SD.____________________________________________________________20

Figure 5. Mass deposited onto each coverslip (Area = 0.503 cm2_{) after 40W exposure to vaporized (A) PG, (B)}

GLY and (C) 55:45 PG/GL. n=3, mean ± SD._______________________________________________________20

Figure 6. Mass deposited onto each coverslip (Area = 0.503 cm2) after 80W exposure to vaporized (A) PG, (B) GLY and (C) 55:45 PG/GLY, n=3, mean ±SD.______________________________________________________21

Figure 7. IL-6 transcript levels 2 hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001_______________________________________________________________________________22

Figure 8. IL-8 transcript levels in 2 hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001__________________________________________________________________________23

Figure 9. IL-6 protein levels (pg/mL) 24 hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001__________________________________________________________________________23

Figure 10. IL-8 protein levels (pg/mL) 24 hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001__________________________________________________________________________24

Figure 11. Chemical characterization of collected condensate from e-cig aerosol created at 40W and 85W.

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4. Introduction

4.1 Background

E-cigs were introduced to U.S. markets in 2007 as nicotine-delivery products and were

marketed as a safe alternative to traditional tobacco use (Besaratinia et al, 2014). However, there

is no scientific consensus that e-cigs are indeed safe (CDC, 2019, Besaratinia et al, 2014). E-cig

popularity has nevertheless skyrocketed, despite decades of decline in tobacco use (CDC, 2018,

Besartinia et al., 2014). E-cigs are becoming particularly popular among U.S. youth. Between

2011 and 2015, e-cig use grew by 900% among U.S. youth (Murphy 2016). Between 2017 and

2018, the CDC reported that the number of U.S. youth using e-cigs increased by 1.5 million

(CDC, 2019). In 2018, while conventional cigarette smoking dropped to less than 8.1% among

U.S. youth, 20.8% of U.S. high school students and 4.9% of middle school students reported

having used e-cig devices in the last thirty days (Cullen et al, 2018). Given the prevalence and

proliferation of e-cig use among youth, research is urgently needed to assess potential health

consequences of their usage.

4.2 Navigating the E-Cig Market

E-cigs operate by rapidly heating a mixture of propylene glycol (PG), glycerol (GLY),

nicotine, and flavoring up an atomizing coil. The mixture (e-liquid) is vaporized under intense

heat, and then cooled quickly into the aerosol particle phase. A third generation of e-cigs called

“Advanced Personal Vaporizers” (APVs) was released to the U.S. market in 2014. These APVs

allow the user to manually control the wattage, voltage, and air flow of the e-cig device, and

have much higher wattage potential than prior generations, allowing wicking materials, metal

heating coils, and e-liquids to be heated to much higher temperatures. For example, Sigelei’s

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at much lower wattages of less that 10 W (Protano et al. 2018). Those who are experienced e-cig

users frequently choose APVs as they offer greater control over device settings and deliver larger

“vapor” plumes, while also providing a stronger “throat hit” (McQueen et al. 2011).

4.3 Existing Methods Regarding E-cig Aerosol Exposures

Aerosol chemistry and physics vary with particle size, ambient humidity, and

temperature. As such, aerosol production can be affected by the composition of the e-liquid and

temperature of the atomizing coil. There is concern that, with the greater heating capacity seen in

APVs, thermal decomposition of e-liquid components will occur, forming more toxic thermal

decomposition products. The Surgeon General reported in 2016 that “the detection and level of

carcinogens [in e-cig aerosol] depend on several factors, including the concentration of e-liquid

and the strength of the heating device” (Murphy 2016). While previous studies have shown that

e-cig aerosol particles may contain carbonyls (e.g., formaldehyde, acetylide, and acrolein),

gas-phase metals from the coil, and other uncharacterized, potentially toxic organic compounds, few

studies have been published which assess particle phase constituents as a factor of wattage under

standardized exposure conditions (Sleiman et al. 2016, Goniecwiz et al. 2014, Kosmider et al.

2014).

Despite the increasingly urgent need for conclusive research regarding both the chemical

composition of e-cig aerosol and its health effects, there remains a gap in the literature regarding

a replicable exposure chamber allowing for both in vitro exposure and chemical characterization

while not compromising or influencing the natural behavior of the aerosol. One method of

exposure is to “trap” liquid aerosol and to then expose cells to the condensate via conditioned

media (Bengalli et al. 2017). This method, however, completely separates the e-cig aerosol from

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aerosol are an important facet of e-cig exposure. Another established exposure method is the

Vitrocell® exposure system, which was designed for traditional cigarette exposures and has been

retrofitted for e-cig exposure experiments. It is a method preferred by many because of its

ability to expose in vitro cells to aerosol at an air-liquid interface without separating or removing

the gas-phase chemical components (Lucci et al. 2018). When using systems such as the

Vitrocell® or others like it (Cultex®, XposeALI®, P.R.I.T® ExpoCube®, or Gillings Sampler)

for e-cig exposures, however, the e-cig aerosol is greatly diluted such that the cells are only

exposed to a fraction of the original aerosol, yielding a low deposition efficiency of around

15-30% (Lucci et al. 2018, Zavala et al. 2014, Thorne and Adamson 2013). The dilution built into

these systems was necessary to mimic traditional cigarette smoking profiles, but may disrupt the

chemistry of e-cig aerosol, thus not producing accurate exposures. Additionally, these systems

all feature high amounts of tubing through which the aerosol must travel. This is not ideal, as it

enhances wall losses and is not easily cleaned (Liu et al. 1985).

4.4 Chromatographic Techniques for Analyzing Aerosol

There has been extensive research into the chemical character of the e-liquid sold on the

open market. Many researchers have found that the concentrations of PG and GLY in these

mixtures match the advertised percentages (Beauval et al. 2017). Sleiman et al. (2016) analyzed

the composition of an e-liquid in addition to the volatile phase of phase of the vaped e-liquid

using gas chromatography coupled to mass spectrometry (GC/MS), and were able to aldehydes

and carbonyls such as acrolein, glycidol, and formaldehyde, which are known carcinogens also

found in cigarette smoke. Sleiman et al.’s (2016) results are additionally supported by other

findings (Goniewicz et al. 2013, Uchiyama et al. 2013). Furthermore, Sleiman et al. found that

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Despite these chemical studies, there is a gap in the literature in regards to potentially harmful

oligomers formed as a byproduct of e-cig operation. Whereas most e-cig studies make use of

GC/MS, the GC/MS operates at too high a temperature to study oligomers without concern of

fragmenting these potentially large compounds (Sleiman et al. 2016, Cui et al. 2018). Liquid

chromatography coupled to mass spectrometry (LC/MS) is thus favored, and utilized in these

studies as a method to analyze potential oligomers formed from propylene glycol and glycerin

during vaping because it operates at a lower temperature (Cui et al. 2018).

4.5 Using in vitro Exposure to Understand E-Cig Toxicity

Airway epithelial cells represent the first natural line of defense when humans are

exposed to airborne toxicants, like e-cigs. In response to challenges, epithelial cells produce

cytokines ultimately resulting in inflammatory and immune cells, which comprise the initial

innate immune response (Boris et al. 2017). E-cig use in particular has been shown to induce an

inflammatory innate immune response in humans (Reidel et al. 2018, Lerner et al. 2015).

Chronic inflammation induced by continual exposure to airborne toxicants may cause

irreversible scarring and damage lung fibroblasts and the lung cells (Gerloff et al. 2017,

Rubenstein et al. 2015). While there has been previous research assessing human lung epithelial

cell inflammation in response to flavorings (Gerloff et al. 2017, Begnalli et al. 2017), there is not

an abundance of research characterizing airway responses to only PG, GLY, or a mixture

thereof. As such, an exposure system is needed which allows for airway epithelial cell exposure,

more accurately mimicking e-cigarette use profiles, and permitting the analysis of inflammatory

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4.6 Objectives

Ultimately, we aim to describe our newly developed unique system, which allows for

easy exposure to e-cig aerosol, minimizes loss or disruption of aerosol and its gas-phase

chemical constituents, and produces homogenous aerosol deposition for in vitro exposures and

chemical analysis. Our interdisciplinary approach of analyzing the aerosol particles produced by

e-cigs produces a comprehensive analysis of aerosol-phase components of the vaped e-liquid,

which, in addition to in vitro inflammatory responses, allow us to demonstrate the merits and

versatility of our proposed exposure system.

5. Methods

5.1 Chamber and Basic Exposure Parameters

As shown in Figure 1, a 7.6 cm by 7.0 cm by 4.0 cm Plexiglas chamber was used for

e-cig exposures. The chamber was sealed using vacuum grease (Dow Corning®). At the front-right

corner of the chamber, an inlet port was constructed into which the e-cig device is inserted and

can be operated to mimic vaping. The inlet is sealed with a rubber stopper when the e-cig is not

being operated. Within the chamber, in front of the inlet, hangs a YaKoo® DC Brushless fan

(Model DC12V), used to circulate the incoming e-cig aerosol. The fan can be turned on and off

from outside the chamber. At the back-left corner of the chamber, and outlet was constructed, to

which a vacuum line was attached.

To conduct an exposure, cells were placed in the chamber, the e-cig was inserted into the

inlet and the vacuum line was attached. The e-cig device used for all exposures was a 3rd

generation DNA 200 Lava Box with a SMOK TFV4 mini tank with a TF-S6 sextuple (Kanthal)

coil. It was operated at 85W and 40W, which are representative of the high and low wattage

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second inter-puff period during which the chamber was sealed. Each exposure lasted 10 minutes,

with a total of 20 puffs per exposure. The vacuum flow rate was set to 2.5 liters per minute using

a flowmeter (Dwyer, Inc.). A HEPA capsule filter (Product No. 12144, Gelman Sciences) and

water trap were implemented upstream of the flow meter to keep outgoing e-cig aerosol from

clogging the flow meter.

Figure 1. Depiction of the aerosol exposure chamber featuring the fan, vacuum, filters, and flow meter.

5.2 Measuring Aerosol Deposition

In order to confirm even and sufficient aerosol deposition, 12-mm glass cover slips were

first weighed using a Precision Balance (Sartorius), then inserted into a 12-well transwell cell

culture plate (Costar, Corning, NY). The cover slips and plate were then placed in the chamber

and an exposure was completed using a 3rd generation DNA 200 Lava Box with a SMOK TFV4

six coil mini tank filled with GLY and set to 85W. The puff and exposure parameters described

in 5.1 were utilized. Following the exposures, the cover slips were removed from the cell culture

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subtracting the pre-exposure coverslip weight from the post-exposure coverslip weight (Figure

2). The 85W exposures were repeated, placing the tissue culture plate in each of the 4 corners of

the chamber (Figure 3). At each corner, the following permutations were tested: (1) fan and

vacuum on for entire 10-minute exposure; (2) Fan off, vacuum always on during full 10-minute

exposure; and (3) Fan and vacuum on only during the 4 second puff and off during each 26

second inter-puff interval, of the 10-minute exposure.

Further deposition experiments were conducted to ascertain aerosol mass deposition

using a more sensitive microbalance (Mettler Toledo) and utilizing 8-mm glass coverslips. These

exposures were performed similar to above, but utilized only permutation (3) and used all three

PG:GLY mixtures (100:0, 0:100, and 55:45) at both 85 and 40W.

Figure 2. 12-mm glass cover slips (A), were weighed pre-exposure on a microbalance (B), they were then inserted into a 12-mm insert transwell cell culture plate (C), the culture plate and coverslips were then placed in our exposure chamber (D), we ran the exposure and the cover slips were removed from the chamber and culture plate and weight post-exposure on the microbalance (E). Exposure condensate was then calculated by subtracting the pre-exposure coverslip weight from the post-exposure coverslip weight.

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Inlet

Back

Left

Back

Right

Front

Left

Front

Right

Vacuum

1 2 3 4

A

B

C

Figure 3. Schema for labeling transwells and tissue culture plate placement within the exposure chamber.

5.3 Cell Culture

16HBE14o (16HBE) cells, an SV-40-transformed human male bronchial epithelial cell

line and gift from Dr. D. C. Gruenert (University of California at San Francisco), were used in

these experiments. They were grown in minimal essential media (MEM) (Gibco) with 10% Fetal

Bovine Serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine (Life Technologies, Inc.)

and subcultured in T-75 flasks with care being taken that they did not become overly confluent.

Subcultured 16HBE cells were plated onto 12-mm insert transwells (Costar, Corning, NY).

Flasks and transwells were coated with a Fibronectin solution (Medium (Life Technologies,

Inc.), 0.01% BSA (Sigma), 1% Vitrogen (Advanced Bio Matrix, San Diego), and 1% human

fibronectin (BD Biosciences)) to encourage adherence of the cells. Two days prior to exposure,

the apical medium covering the cells grown on Transwells was removed to establish air-liquid

interface (ALI). One day prior to exposure, the cells were serum starved using MEM media with

2% FBS. Immediately prior to exposures, the apical side of the cells was washed with DPBS and

the basolateral media changed using 2% FBS media. Further details regarding cell culture

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5.4 In Vitro Exposure

Cultured cells were exposed to PG:GLY mixtures universal to all e-cig devices: 100:0,

0:100, and 55:45. For in vitro exposures, samples were collected at four time points following

cell exposure: 10 minutes, 2 hours, 8 hours, and 24 hours. At each time point, the apical wash,

basolateral supernatant, and cell lysate were collected. For each time point, there were three

technical replicates, and for each endpoint, there were three biological replicates. As such, we

repeated our 85W exposures and 40W exposures on three separate days each.

5.5 Gene Transcript Analysis

Quantitative Polymerase Chain Reaction (qPCR) was used to determine gene expression

of Interleukin-6 (IL-6) and Interleukin-8 (IL-8) relative to -actin. Total RNA was extracted

using 1% BME in Ambion Pure-LinkTM_{Lysis Buffer according to the manufacturer’s protocol}

(Life Technologies, Inc.). cDNA was prepared using the Ambion Pure-LinkTM_kit

(Thermo-Fischer). Real-time polymerase chain reaction (RT-PCR) using quantitative fluorogenic

amplification of first strand cDNAs was performed using the ABI Prism 7700 Sequence Detector

System (PE Biosystems, Foster City, CA), TaqMan Universal PCR Master Mix (PE

Biosystems), and primers and fluorophore-labeled probes.

Real-time measurements of fluorescence were taken and used to determine the threshold

intensity (Ct) for each amplification by calculating the number of cycles required to reach a

fluorescence intensity of 10 standard deviations greater than baseline fluorescence intensity

(Jaspers, 2001). Threshold intensity of the gene of interest was compared to the threshold

intensity of a constitutively expressed gene transcript (-actin) generated from the same sample.

This comparison determines Ct. The Ct from the sample was then compared to the Ct of a

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2-Ct _{and averaged in three biological replicates, each with three technical replicates. This}

process was used to analyze relative gene expression for both IL-6 and IL-8.

5.6 Protein Analysis

Enzyme-linked immunosorbent assays (ELISAs) were used to assess relative abundance

of proinflammatory cytokines IL-6 and IL-8 in basolateral supernatant collected from ALI

cultures post-exposure. Protein isolation and antibody detection were performed according to

manufacturer’s protocol (BD OptEIATM_{). The capture antibody was an Anti-Human IL-8}

monoclonal antibody, the detection antibody was a Biotinylated Anti-Human IL-8 monoclonal

antibody, and the enzyme reagent was Streptavidin-horseradish peroxidase conjugate

(SAv-HRP). Standards were created using lyophilized recombinant human IL-8 and IL-6. Total protein

concentration in samples is reported in pg/mL.

5.7 Statistical Analysis

Data are presented as mean  standard deviation (SD). Triplicate technical replicates

(n=3) were averaged and then compared to their corresponding biological replicates (n=3).

Statistical analyses comparing the biological replicates were completed using One-Way

ANOVAs against air control samples using Dunnett’s Post-Hoc tests. Significance is indicated

as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

5.8 Chemical Analysis

5.8.1 Sterilization and Sample Collection

12-mm insert transwells (Costar, Corning, NY) and 8-mm diameter glass coverslips were

sonicated in 50:50 Methanol and water for 10 minutes and then dried using house air. Coverslips

were then weighed as described previously (Section 5.3) and returned to the transwells for

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ten-minute exposure, the 8-mm glass coverslips were carefully removed from the transwell and

weighed in the microbalance. Care was taken to not push the glass coverslip around in the

transwell and the coverslip was transferred and weighed exposure-side-up. This was to limit

unintended mass gain or loss. Following weighing, the glass coverslip was transferred to an

empty, clean 20 mL borosilicate vial and stored until extraction.

5.8.2 Sample Extraction and Preparation

Two high performance liquid chromatographic methods coupled to electrospray

ionization high-resolution quadrupole time-of-flight mass spectrometry (ESI-HR-QTOFMS)

were used for the chemical analysis of e-cig aerosol collected: (1) reverse-phase liquid

chromatography (RPLC) operated in the positive (+) ion mode of ESI

(RPLC/ESI(+)-HR-QTOFMS) and hydrophilic interaction liquid chromatography operated in the negative (–) ion

mode of ESI (HILIC/ESI(–)-HR-QTOFMS). For e-cig aerosol samples analyzed via

RPLC/ESI(+)-HR-QTOFMS, methanol (LC-MS Chromasolv®, 99.9%, Fisher) and Milli-q

water (18.2 M) in a 50:50 (v/v) solution were added to the samples. For the samples analyzed

via HILIC/ESI(–)-HR-QTOFMS, a solution of 95:5 (v/v) of acetonitrile (Sigma Aldrich) and

Milli-q water (18.2 M) was added to the samples. Following addition of the solvent, the

samples were sonicated for 10 minutes to extract the collected aerosol mass into solution. Three

samples each were prepared for analysis by both RPLC/ESI(+)-HR-QTOFMS and

HILIC/ESI(–)-HR-QTOFMS. The volume of solvent varied by mass collected. For 40W, 5mL

and 2 mL of solvent was used and for 85W, 10mL of solvent was used. This was done to

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For analysis of the 40W samples by RPLC/ESI(+)-HR-QTOFMS, the samples were

additionally diluted such that there would be a mass concentration of only 50 ppm in the

solution, which help to avoid saturating the detector and allowing for better quantification of PG

and GLY.

5.8.3 Standards Preparation

RPLC/ESI(+)-HR-QTOFMS:

For analysis of the 85W samples in RPLC/ESI(+)-HR-QTOFMS, one set of standards

containing masses of PG and GLY from the same stock of PG and GLY used in the e-cig device.

The standards were prepared at 500, 100, 10 and 1 ppm of GLY and 615, 123, 12.3 and 1.26

ppm PG (R2 _{= 0.9999 for GLY and R}2_{= 0.9984 for PG). For analysis of the 40W samples in}

RPLC/ESI(+)-HR-QTOFMS, standards were diluted to 50 ppm from the measured collected

mass. Separate standards were created for PG and GLY. Both sets of standards were 25, 10, 2.5,

1 and 0.25 ppm PG and GLY, respectively (R2 _{= 0.9983 for GLY and R}2_{= 0.9991 for PG).}

HILIC/ESI(–)-HR-QTOFMS:

For analysis of the 85W samples using the HILIC/ESI(–)-HR-QTOFMS method, we

created one set of standards containing PG and GLY at a concentrations of 7400, 740, 74, and

7.4 ppm and 1000, 100, 10, and 1 ppm, respectively. R2_{= 1.0 for the GLY standards, but there}

was no response for PG using the HILIC method. For the 40W standards, we created one set of

standards containing only GLY at concentrations of 1, 2.5, 10, 25, 100 and 500 ppm (R2₌

0.9991).

5.8.4 LC/MS Operation

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The chromatographic separations were carried out using a Waters ACQUITY UPLC HSS

(high-strength silica) column (2.1×100 mm, 1.8 µm particle size) at 45°C. The mobile phases

consisted of eluent (A) 0.1% acetic acid in water (LC-MS ChromaSolv-Grade, Sigma-Aldrich)

and eluent (B) 0.1% acetic acid in methanol (LC-MS ChromaSolvGrade, Sigma-Aldrich). The

applied 15 min gradient elution program was as follows: the concentration of eluent B was 100%

for the first 2 min, decreased to 10% from 2 to 10 min, held constant at 10% between 10-11 min,

then increased to 100% from 11 to 15 min, and held at 100% during a 5-min post run. The flow

rate and sample injection volume were 0.3 mL min−1_{and 25-50 µL, respectively. Data were}

acquired and analyzed by Mass Hunter Version B.03.01 Build 3.1.346.0 software. At the

beginning of each analysis period, the Q-TOFMS instrument was calibrated using a

commercially available ESI-L low concentration tuning mixture (Agilent Technologies), which

was composed of a 95:5 (v/v) solvent mixture of acetonitrile and water. This external calibration

was done in the low mass-to-charge ratio (m/z) range. Six specific ions were used from the

commercial tuning mixture during calibration, and include 68.9958, 112.9856, 301.9981,

601.9790, 1033.9881, 1333.9689, and 1633.9498 m/z. During the chromatographic runs, the

Q-TOFMS was continually calibrated by the constant injection of the following reference

compounds in the ESI source: purine, leucine enkephalin, and HP-0921 acetate adduct (Agilent

Technologies) (Cui et al. 2018).

HILIC/ESI(–)-HR-QTOFMS

An Agilent 6520 Series Accurate Mass Q-TOFMS instrument interfaced to an Agilent

6500 Series UPLC system, equipped with an ESI source operated in the negative (−) ion mode,

was used to chemically characterize PG and GLY standards, as well as the generated samples.

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out using a Waters ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm particle size,

Waters) at 35 °C. The mobile phases consisted of eluent (A) 0.1% ammonium acetate in water,

and eluent (B) 0.1% ammonium acetate in a 95 : 5 (v/v) ACN (HPLC Grade, 99.9%, Fisher

Scientific)/Milli-Q water. Both eluents were adjusted to a pH of ∼9.0 with NH4OH (Hettiyadura

et al. 2015). The gradient elution program was eluent A, 0% for 4 min, increasing to 15% from 4

to 20 min, constant at 15% between 20 and 25 min, decreasing to 0% from 25 to 26 min, and

constant at 0% from 26 to 30 min and during a 5-minute post-run. The flow rate and sample

injection volume were 0.3 mL min−1_{and 5 μL, respectively. Data were acquired and analyzed by}

Mass Hunter Version B.06.00 Build 6.0.633.0 software (Agilent Technologies). At the beginning

of each analysis period, the mass spectrometer was calibrated using a commercially available

ESI-L low-mass concentration tuning mixture (Agilent Technologies) in a 95:5 (v/v)

ACN/Milli-Q water. Instrument mass axis calibration was conducted in the low-mass range (m/z 50–1700).

Seven masses were used for calibration: m/z 68.9958, 112.9856, 301.9981, 601.9790, 1033.9881,

1333.9689, and 1633.9498. The adduct of hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazene

+ acetate, purine, and leucine enkephalin were continuously infused for real-time mass axis

correction. The mass resolution of the ESI-HR-QTOFMS was approximately 8000–12300 from

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6. Results and Discussion

6.1 Uniform and Substantial Aerosol Deposition

Figure 4. Mass deposited onto each coverslip (Area = 1.131 cm2_{) (Figure 3) after exposure to}

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Figure 5. Mass deposited onto each coverslip (Area = 0.503 cm2_{) after 40W exposure to}

vaporized (A) PG, (B) GLY and (C) 55:45 PG/GLY. n=3, Mean ± SD.

Figure 6. Mass deposited onto each coverslip (Area = 0.503 cm2_{) after 85W exposure to}

vaporized (A) PG, (B) GLY and (C) 55:45 PG/GLY, n=3, Mean ±SD.

Our initial deposition experiment, permutation 1 (Figure 4A) at 85W, yielded even

deposition averaging 0.43mg (SD  0.098) (Figure 4A). However, constitutively running the fan

introduces a potential disruption to the normal aerosol nucleation and partitioning and also

increases potential wall loss. In order to address these concerns, we next ran deposition

experiments at the back right, back left, and front left corners of our chambers without running

the fan, while still operating the vacuum for the entire exposure (Permutation 2). Yet, the

deposition was clearly uneven and variable across the tissue culture plate at every corner of the

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As a compromise, we decided to run the fan and the vacuum only for the four-second

puffs during our exposure (Permutation 3). Deposition experiments were conducted at both 85W

and 40W using this permutation, and the deposited condensate was measured using a

microbalance, allowing for a more precise understanding of the deposition in our different

exposures (Figures 5 and 6, respectively). There were no statistical differences in condensate

between any of the wells of the plates, indicating homogenous aerosol deposition (Figures 5 and

6). Furthermore, deposition was found to be on the scale mg/cm2_{, which is approximately 3}

orders of magnitude greater than mass deposition seen in other systems such as the Vitrocel®

and Gillings Sampler, which saw deposition on the scale of g/cm2_{(Lucci et al. 2018, Zavala et}

al. 2014, Delaval et al. 2019). Such a large magnitude of deposition allows for assessment of

more acute exposures to the e-cig aerosol, which may allow for detection of in vitro responses

that might only be seen at these higher exposure levels. Additionally, the e-cig aerosol generated

using this exposure system travels through zero tubing prior to its entry into the chamber. This

allows for a direct exposure of cells to the e-cig aerosol and minimizes loss of aerosol to the

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6.2 Increased Pro-Inflammatory Transcript and Protein Levels in response to PG

Figure 7. IL-6 transcript levels 2-hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett’s post hoc test *p < 0.05, **p < 0.01

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Figure 9. IL-6 protein levels (pg/mL) 24-hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett’s post hoc test *p < 0.05, **p < 0.01, ***p < 0.001

Figure 10. IL-8 protein levels (pg/mL) 24-hours post-exposure in 16HBE cells exposed to e-cig aerosol produced at (A) 40W and (B) 85W. n=3. Mean ± SD. One-Way ANOVA, Dunnett’s post hoc test.

Proinflammatory cytokines IL-6 and IL-8 were assessed as a measure of inflammatory

innate immune response. Transcript levels of IL-6 (Figure 7) and IL-8 (Figure 8) in the cells

exposed to PG, GLY, and PG/GLY at 40 and 85W were compared to their respective air controls

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transcript levels. However, only IL-6 protein levels were significantly increased at 40W 24-hours

after 100% PG exposure. At 85W, cells exposed to 100:0 PG and 100:0 GLY had significantly

increased IL-6 transcript levels, but no increase in IL-8 transcripts. The protein data for IL-6 and

IL-8 followed these trends (Figures 9 and 10, respectively), with a significant increase in IL-6

protein levels for the PG and GLY exposures 24-hours post-exposure.

These results provide evidence for a proinflammatory response due to exposure to vaped

humectants, particularly PG at both 40 and 85W exposures.

6.3 Propylene Glycol and Glycerin as Major Components of Collected Aerosol

PG GLY PG/GLY 0% 25% 50% 75% 100% 77% 29% 59% 36%

(A) 40W

M as s Fr ac ti o n

PG GLY PG/GLY 0% 25% 50% 75% 100% 24% 5% 90% 70%

(B) 85W

Unknown GLY PG

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Table 1. Chemical characterization of collected condensate from e-cig aerosol created at 85 W and 40 W. Characterization completed using HILIC/ESI(–)-HR-QTOFMS

Wattage E-cig liquid Extraction Volume

GLY Collected mass GLY/mass

mL ug/mL mg 0%

1 0.0 0.786 0%

10 0.0 0.805 0%

10 0.0 0.785 0%

1 426.4 0.672 63%

10 36.3 0.629 58%

10 47.8 0.824 58%

1 785.4 0.962 82%

10 82.4 0.952 87%

10 89.7 0.939 96%

1 0.0 0.5 0%

5 0.0 0.5 0%

5 0.0 0.451 0%

1 188.73 0.383 49%

5 33.90 0.339 50%

5 33.95 0.363 47%

1 189.36 0.229 83%

2 94.35 0.213 89%

2 93.66 0.266 70%

HILIC (-) 40W PG pure PG/GLY (55:45) GLY (100:0) 85W PG (100:0) PG/GLY (55:45) GLY (100:0)

Offline Liquid Chromatography coupled to Mass Spectrometry was utilized in order to

chemically characterize the aerosol condensate collected in the wells of the tissue culture plate.

The RPLC/ESI(+)-HR-QTOFMS method was used in order to isolate water soluble compounds,

while the HILIC/ESI(–)-HR-QTOFMS was used in order to isolate extremely water-soluble

compounds. As compared to the RPLC/ESI(+)-HR-QTOFMS method, the

HILIC/ESI(–)-HR-QTOFMS method provides more sensitive measurements and quantifications. However, the

HILIC/ESI(–)-HR-QTOFMS method could only resolve GLY, leaving PG undetectable. This

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Because of this, RPLC/ESI(+)-HR-QTOFMS was useful for comparing relative amounts of PG

and GLY, while HILIC/ESI(–)-HR-QTOFMS may be more useful for indicating precise

measurements of GLY and also allowing the identification of other, less abundant products in the

samples.

Chromatographs isolated from RPLC/ESI(+)-HR-QTOFMS corresponding to PG (m/z

99.0417, RT = 2.2 min) and GLY (m/z 115.0366, RT=2.1 min) indicate that PG and GLY

account for a large fraction of collected aerosol condensate. While we hypothesized that at

higher wattages there would be a corresponding increase in thermal degradation of the vaped

humectants, this does not appear to hold true for both PG and GLY. Interestingly, PG appears to

undergo greater degradation at higher wattages than GLY does: PG was 77% of the mass

fraction at 40W, while only 24% of the mass fraction at 85W in the PG samples (Figure 11).

However, there is an abundance of unknown mass collected in our RPLC/ESI(+)-HR-QTOFMS

samples, and further analysis is needed in order to identify this mass.

At 85W we were able to isolate responses from PG and GLY using a single set of

standards containing both PG and GLY. However, at 40W, we discovered that it was necessary

to create two separate standards for PG and GLY. It is also likely that at 40W there was a greater

relative abundance of PG and GLY, such that the detector was saturated and the differential

ability of PG and GLY to cluster sodium ions became more apparent. It was thus additionally

necessary to dilute the 40W samples down to 50 ppm and also increase the concentration of

sodium in the eluent. More analyses need to be completed using the latter procedure to better

control for the competition effect between PG and GLY during LC/ESI-MS. Encouragingly, the

HILIC/ESI(–)-HR-QTOFMS quantification of GLY largely corresponds to the percentage of

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HILIC/ESI(–)-HR-QTOFMS to be a useful check on quantification via

RPLC/ESI(+)-HR-QTOFMS.

The aerosol concentration inside the chamber, following operation of the e-cig, far

exceeded the capacity of the differential mobility analyzer (DMA, BMI) coupled to a mixing

condensation particle counter (MCPC, Model 1710, BMI). As such, the size distribution of the

e-cig aerosol particles in the exposure system remain unknown.

7. Conclusions and Future Directions

The described exposure system provided substantial and significant deposition across the

tissue culture plate while also minimizing necessary tubing, demonstrating that it is suitable for

in vitro exposures and chemical analysis. Subsequent in vitro exposures demonstrated a robust

proinflammatory response through elevated IL-6 transcript and protein levels due to exposure to

vaped 100:0 PG. Chemical analysis shows that PG and GLY remain a substantial fraction of the

e-cig condensate at both high and low wattages, though there remains an uncharacterized mass

which though which may contain unique oligomers derived from these humectants.

Further chemical analysis will identify and quantify potential oligomers in the unknown

fraction of aerosol condensate. Additionally, gas-only in vitro exposures will be conducted in

order to assess whether the cellular proinflammatory effects are gas-phase or particle-phase

dependent. This will be accomplished by filtering out the e-cig aerosols’ particles prior to their

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9. Appendix

Table 2. Solvent Volume and Mass Collected for 40W Samples

Method

Exposure Solvent Volume Solvent

Mass of Condensate (mg)

PG 95:5 ACN 1 mL 0.500

HILIC _PG _{95:5 ACN} _{5 mL} _0.550

PG 95:5 ACN 5 mL 0.451

RPLC PG 50:50 MeOH 5 mL 0.507

PG 50:50 MeOH 5 mL 0.516

PG/GLY 95:5 ACN 1 mL 0.383

HILIC _PG/GLY _{95:5 ACN} _{5 mL} _0.339

PG/GLY 95:5 ACN 5 mL 0.363

RPLC PG/GLY 50:50 MeOH 5 mL 0.414

PG/GLY 50:50 MeOH 5 mL 0.394

GLY 95:5 ACN 1 mL 0.229

HILIC _GLY _{95:5 ACN} _{2 mL} _0.213

GLY 95:5 ACN 2 mL 0.266

RPLC GLY 50:50 MeOH 2 mL 0.232

GLY 50:50 MeOH 2 mL 0.197

Table 3. Solvent Volume and Mass Collected for 85W Samples

Method

Exposure Solvent Volume Solvent Condensate (mg)Mass of

PG 95:5 ACN 1 mL 0.786

HILIC _PG _{95:5 ACN} _{10 mL} _0.805

PG 95:5 ACN 10 mL 0.785

RPLC PG 50:50 MeOH 10 mL 0.93

PG 50:50 MeOH 10 mL 0.818

PG/GLY 95:5 ACN 1 mL 0.672

HILIC _PG/GLY _{95:5 ACN} _{10 mL} _0.629

PG/GLY 95:5 ACN 10 mL 0.824

RPLC PG/GLY 50:50 MeOH 10 mL 0.691

PG/GLY 50:50 MeOH 10 mL 0.646

GLY 95:5 ACN 1 mL 0.962

HILIC _GLY _{95:5 ACN} _{10 mL} _0.952

GLY 95:5 ACN 10 mL 0.939

RPLC GLY 50:50 MeOH 10 mL 1.096

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GN_honors thesis_finalversion.docx

of the Effects of Electronic Cigarettes

Grace Nipp

Approved

Acknowledgements

Table of Contents

1. Abstract

2. List of Tables

3. List of Figures

4. Introduction

5. Methods

Back

Left

Back

Right

Front

Left

Front

Right

1 2 3 4

A

B

C

6. Results and Discussion

(A) 40W

(B) 85W

7. Conclusions and Future Directions

8. References

9. Appendix