Tobacco-Derived and Tobacco-Free Nicotine cause differential inflammatory cell influx and MMP9 in mouse lung

Electronic nicotine delivery systems (ENDS) or electronic cigarettes (e-cigarettes) have propylene glycol (PG) and vegetable glycerin (VG) as humectants, flavoring chemicals, and nicotine. Nicotine naturally occurs in two isomers R- and S-nicotine, with both tobacco-derived nicotine (TDN) composed of S-nicotine and synthetic nicotine (TFN) composed of a racemic mixture of R- and S-nicotine. Currently there is limited knowledge of the potential differences in the toxicity of TFN vs TDN. We hypothesized that exposure of TFN salts to C57BL/6J mice will result in a differential response in inflammation and lung protease and antiprotease imbalance compared to TDN salts exposed mice. We studied the toxicological impact of these isomers by exposing mice to air, PG/VG, PG/VG with TFN salts, or PG/VG with TDN salts by nose-only exposure and measured the cytokine levels in BALF and lung homogenate along with MMP protein abundance in the lungs of exposed mice. Exposure to the humectants, PG/VG, used in e-cigarettes alone was able to increase cytokine levels-IL-6, KC, and MCP-1 in BALF and KC levels in lung homogenate. Further, it showed differential responses on exposure to PG/VG with TDN salts and PG/VG with TFN salts since PG/VG with TDN salts did not alter the cytokine levels in lung homogenate while PG/VG with TFN salts resulted in an increase in KC levels. PG/VG with TDN salts increased the levels of MMP9 protein abundance in female exposed mice, while PG/VG with TFN salts did not alter MMP9 levels in female mice. The metabolism of nicotine or the clearance of cotinine from TFN may differ from the metabolism of nicotine or the clearance of cotinine from TDN. Thus exposure of humectants alone to induce an inflammatory response while PG/VG with TFN salts and PG/VG with TDN salts may differentially alter inflammatory responses and lung proteases in acute exposures. These data suggest the harmful effects of synthetic/natural nicotine and PG/VG and potential toxicological risk for users.


Introduction
Ever since the February 2020 ban by the US Food and Drug Administration (FDA) on the avored cartridge-based electronic cigarettes (e-cigs), the national sales of disposable e-cigarette device have increased drastically [1].Shortly afterwards, avored products claiming to contain 'tobacco free nicotine' (TFN) entered the markets.These products are being advertised as; 'cleaner', 'purer', 'tastier' and 'having higher quality', as compared to traditionally available 'tobacco-derived nicotine' (TDN) products.While these strategies have bene tted the sale of avored e-cig products, they have also created confusion as many users believe them to contain 'no tobacco'.Prior to April 2022, there was a regulatory gap that meant the FDA could not regulate TFN since it is not made or derived from tobacco which allowed tobacco companies to keep products on the market without needing to go through the pre-market approval process [1,2].In April 2022, this regulatory gap was closed after the new legislation allowed the FDA to regulate TFN products [1].Yet, there is little to no knowledge with regards to the toxic effects of the use of TFN-containing products.
Nicotine is a naturally occurring alkaloid extracted from tobacco leaves that exists in two isomers: (R)-(+)nicotine and (S)-(-)-nicotine [3,4].Traditionally tobacco products have utilized nicotine extracted from tobacco leaves which contains predominantly S-nicotine [5,6].TFN, on the other hand, is a chemically synthesized nicotine containing a racemic (50:50) mixture of R-and S-forms.[5] Besides these two isomers, nicotine can also be found in two forms-a protonated and a free base form [8]. The protonated form of nicotine is formed from the addition of acid to freebase nicotine to form nicotine salts.The two most commonly used acids for this purpose being lactic and benzoic acid [9].
Nicotine salts in e-liquids are found to have a lower pH level than freebase nicotine even with higher levels of nicotine present [10].This reduction in pH is believed to reduce the throat irritation and harshness due to high levels of nicotine [11].In clinical studies, protonated nicotine has been found to result in a higher nicotine absorption than free-base nicotine, although, contrary evidence exists though in vivo and in vitro studies, thus warranting further investigation [8,12].
Nicotine is known to have adverse effects on the respiratory system [13].Previous work within our lab using freebase nicotine, has shown that acute exposure (3 days; 2 hr/day) of C57BL/6J mice to PG with nicotine resulted in an increase in pro-in ammatory cytokines and MMP2 levels in exposed mice as compared to the air controls [14].Furthermore, sub-chronic (3 weeks; 3 h/d; 5 d/wk) exposures of C57BL/6J mice to PG/VG with freebase nicotine has also demonstrated sex-speci c alterations in lipogenic and myogenic gene expression and levels of matrix metalloproteinase 9 (MMP9) [15].Our lab has also previously conducted research on e-cigarettes with nicotine salts, and has found pods containing nicotine salts result in an increase in pro-in ammatory cytokines, oxidative stress, epithelial barrier dysfunction and DNA damage in lung epithelial cells [16].These results provide evidence for the adverse toxicological effects of both free-form and protonated form of nicotine.However, there is limited knowledge about the pharmacological effects of R-nicotine.Current data has indicated that R-nicotine is a less potent agonist of nicotinic acetylcholine receptors andcan bind and inhibit acetylcholinesterase.
Further, R-nicotine and R-cotinine has been found to have a faster clearance than S-nicotine or S-cotinine [5,17,18].
We thus hypothesized that exposure of C57BL/6J mice to TFN salts will result in a differential response in in ammation and lung protease/antiprotease imbalance compared to TDN salts exposed mice.To test this hypothesis, we exposed mice to air, PG/VG, PG/VG with TFN salts, or PG/VG with TDN salts, and measured the cytokine levels in BALF and lung homogenate along with MMP protein abundance in the lungs of exposed mice.

Ethics Statement
Experiments were conducted using the standards established by the United States Animal Welfare Act.Animal experimental protocols conducted at the University of Rochester were approved by the University Committee on Animal Resources.All laboratory studies were approved by the Institutional Biosafety Committee of the University of Rochester Medical Center.

Mouse Exposures
An equal number of male (12) and female (12) C57BL/6J mice at ve weeks old were ordered from Jackson Laboratory.Mice were housed for one week at the University of Rochester Vivarium prior to being moved to the inhalation suite to begin nose-only tower training.In order to acclimatize the mice to the mesh restraints of the nose-only tower, one week prior to beginning the e-cig exposure mice were placed in the mesh restraints and held in the tower.Mice were trained for ve days following the methodology described in Lamb, et al. [19].The rst day mice were held in the restraints for fteen minutes, the second day mice were held in the restraints for thirty minutes, the third day mice were held in the restraints for forty-ve minutes, and the nal two days mice were held in the restraints for one hour.

E-cigarette Device and E-liquid
A Joyetech eVic-VTC mini and cubis pro atomizer (SCIREQ, Montreal, Canada), with a BF SS316 1.0 Ω coil (Joyetech, Shenzhen, China) and the Scireq nose-only tower (SCIREQ, Montreal, Canada) were utilized for all e-cigarette exposures.PG and VG from "EC Blend" were purchased through local vendors/online vendors.A 1:1 mixture of PG/VG was used for PG/VG exposures and a 1:1 mixture of PG/VG mixed with a 1:1 mixture of lactic acid with R-nicotine ((±)-Nicotine, Sigma-Aldrich, Cat# N0267) or S-nicotine ((-)-Nicotine, Sigma-Aldrich, Cat#N3876) at a concentration of 50 mg/mL was used for PG/VG with TFN salts and PG/VG with TDN salts exposure, respectively.Nicotine e-liquids utilized for exposures were analyzed by proton nuclear magnetic resonance ( 1 H NMR) to con rm nicotine concentration and R/S ratio.In brief 1 H NMR following the same basic methodology as described in Lamb, et al. was used to determine nicotine concentrations for PG/VG with TFN salts at 52.09 mg/mL and for PG/VG with TDN salts at 48.35 mg/mL [19].The ratio of R-nicotine to S-nicotine was determined by 1 H NMR following a similar methodology as described in Duell, et al. and determined the R/S ratio of nicotine in PG/VG with TFN salts to be 56/44 and in PG/VG with TDN salts to be 0/100 [20].

E-cigarette Exposure
Nose-only e-cigarette exposure was conducted utilizing the Scireq InExpose system with the Scireq exiware software controlling the Joyetech eVic-VTC mini device.Mice were exposed to a pu ng pro le of two puffs per minute with a puff volume of 51 mL, puff duration of three seconds, and an inter puff interval of twenty seven seconds with a 2 L/min bias ow between puffs [21].Mice were split into four groups-(i) air, (ii) PG/VG, (iii) PG/VG with 50 mg/mL TDN salts (labelled as TDN), and (iv) PG/VG with 50 mg/mL TFN salts (labelled as TFN), of equal number of male (3) and female (3) mice.Mice were exposed to the described pu ng pro le for one hour per day (120 puffs) for a total of ve days, with air mice being exposed to room air [22].Temperature, humidity, and CO levels were measured at the starting, mid-, and end point of the exposure utilizing Q-Trak Indoor Air Quality Monitor (TSI, SKU#7575).Total particulate matter (TPM) measurements were taken at the exhaust tubing at the half-way point of the exposure and at the inlet tubing connected to the top of the nose-only tower immediately after the end point of the exposure.TPM was measured by weighing a glass ber lter pad (Pall Corporation, P/N#61630) before and after collecting aerosol over the course of ve minutes.Cotinine levels in the blood serum of exposed mice were measured using an ELISA based assay (Calbiotech, Cat#C0096D) following manufacturer's protocol.

Mouse Sacri ce
Mice were sacri ced two hours after the nal e-cigarette exposure and were anesthetized with a mixture of ketamine and xylazine.Blood was drawn from the inferior vena cava, and allowed to sit for roughly thirty minutes before being centrifuged at 2000 rpm for fteen minutes.After being centrifuged, the serum was collected and stored at -80°C.Manual lung perfusion was performed by taking 3 mL of 1x PBS in a 3 mL syringe and slowly injecting the 1x PBS into the heart of the mouse until the lung lobes turned white.Mice were lavaged via catherization three separate times with 0.6 ml of 0.05% FBS in 0.9% NaCl.The combined lavage uids were centrifuged at 3000 rpm for 10 minutes at 4°C.The supernatant was recovered and stored at -80°C until further experimentation, while the cell pellet was re-suspended in 1 ml of 1x PBS for determination of immune cell population.Mouse lung lobes were harvested from exposed mice and washed with 1x PBS, one lobe was left in 1 mL 1x PBS while the rest were blotted dry using a lter pad, and then ash frozen by dry ice before being stored at -80°C.

Lung Digest, Cell Count, and Flow Cytometry
Lung lobes to be used for lung digestion were minced nely, and placed into a 50 mL conical tube with a liberase enzymatic cocktail (0.5 mL of 5 mg/mL liberase with 2 mL DMEM and 3 µL of 100 mg/mL DNAse I).Tissue samples were dissociated using gentleMACS Dissociator (Miltenyi, Biotec, Gaithersburg, MD), placed on a rocker and incubated at 37°C for thirty minutes.After incubation, cell suspensions were strained through a 70 micron cell strainer into a new 50 mL conical tube and then remaining cells were collected by adding 5 mL DMEM (10% FBS) through the cell strainer.Cells were spun at 300 g for ve minutes at 4°C, afterwards, the supernatant was removed and 1 mL of RBC lysis buffer was added to the cell pellet and incubated on ice for one minute.After incubation 5 mL of DMEM (10% FBS) was added to the cell suspension and then centrifuged at 300 g for ve minutes at 4°C.Supernatant was removed and the cell pellet was re-suspended in 2 mL DMEM (10% FBS).Total cell counts for BALF and lung digest were measured by staining cells with AO/PI and counted using the Nexcelom Cellometer Auto 2000 cell viability counter.Differential cell counts were determined by ow cytometry using the BD LSRFortessa cell analyzer.Cells from both lung digest and BALF were stained with CD16/32 (Cat#70-0161-u500, Tonbo Biosciences, 1:10 dilution) to block nonspeci c binding and then stained with a master mix of

Protein Extraction
Roughly 20-30 mg of ash frozen lung tissue was added to 350 µL of RIPA buffer containing protease inhibitor (Cat#87785, Thermo Fisher Scienti c) and EDTA (Cat#R1021, Thermo Fisher Scienti c) and mechanically homogenized while on ice.After homogenization, samples remained on ice for forty-ve minutes and then spun at 14000 rpm for thirty minutes at 4°C.The supernatant was collected and 50 µL aliquots were stored at -80°C.Total protein concentration for each sample was determined using the Pierce BCA Protein Assay kit (Cat#23225, Thermo Fisher Scienti c) with BSA being utilized as the protein standard.
Band intensity was determined using densitometry analysis using image lab software and normalized to the levels of GAPDH.Fold change in protein abundance were relative to the protein abundance of air exposed mice.

Gelatin Gel Zymography
Total MMP activity levels were measured by following the gelatin zymography protocol from Abcam with slight modi cations.The night prior to the start of the assay, the gelatin gel was prepared with a 15-well comb utilizing the Mini-PROTEAN Tetra Cell Casting Module (Cat#1658022 BioRad).After the gels had solidi ed, the gels were left at 4°C in 1x running buffer.An equal concentration of protein (50 µg) from each lung homogenate samples were loaded per well of the gelatin gel, with 10 µl of Precision Kaleidoscope Prestained Protein Standards (Cat# #1610375, BioRad) added to the rst well.Proteins were separated based on size through the stacking gel and separating gel.Once the protein are separated, the gels were washed with washing buffer two times for thirty minutes, rinsed twice with incubation buffer for 10 minutes per rinse while rocking, and then fresh incubation buffer was added to cover the gels which were then incubated for twenty-four hours at 37°C.After incubations, the gels were stained with staining solution for one hour with rocking at room temperature.After staining, the gels were rinsed with ddH 2 O and then destaining solution was added and incubated until bands could be visualized.Images of membranes were collected utilizing the Bio-Rad ChemiDoc MP Imaging system (Bio-Rad Laboratories).After imaging, band intensity was determined using densitometry analysis using image lab software and fold change in activity levels were relative to the activity levels of air exposed mice.

Statistical Analysis
Analysis was performed using GraphPad Prisma utilizing One-Way ANOVA with Tukey's multiple comparisons test with data shown as mean ± SEM.

Results
PG/VG with synthetic nicotine salts exposure alters in ammatory cells in ltration in exposed mice.
In order to determine the effects of TFN and TDN salts on in ammatory cell in ux in vivo, C57BL/6J mice were exposed to nose-only exposure to aerosols from PG/VG, TFN and TDN salts as described earlier.
Average TPM measurements at the inlet for for all the exposures was comparable with the values being: 2755.33 mg/m 3 , 3300.67 mg/m 3 for TFN, and 3367.00 mg/m 3 for PG/VG, TFN and TDN exposures respectively.We further performed Cotinine assay to ensure exposure to tobacco in our samples and found the levels of cotinine in the blood serum to be signi cantly varied between TFN (53.69 ± 9.63 ng/mL) and TDN (104.41 ± 28.09 ng/mL) exposed mice (Supplementary Fig. 1A).
Lung in ammation following acute exposures to TFN and TDN salts was determined using ow cytometry.Interestingly, we observed a signi cant increase in the total cell counts in BALF from TFN exposed mice as compared to the mouse exposed to TDN salts pointing towards elicitation of varied immune responses in C57Bl/6J mice on exposure to TFN salts versus TDN (Fig. 1A).Mice exposed to PG/VG, TFN salts, and TDN salts did not alter the differential cell counts of alveolar macrophage, eosinophils or neutrophils in the BALF compared to air exposed mice (Fig. 1B-D).We did not observe any change in the differential cell counts of alveolar macrophages, neutrophils and eosinophils in the lung homogenates from TFN and TDN exposed mouse lungs as compared to air controls.It is pertinent to mention, though, that the neutrophilic responses in the lung tissues of TFN exposed mice (73487 ± 55121.28) was higher than those exposed to TDN (28523 ± 5202.428) (Supplementary Fig. 1B-D).

PG/VG exposure alters in ammatory cytokines in BALF
In order to determine the potential of TFN and TDN salts exposure to induce an in ammatory response, pro-in ammatory cytokines were measured in BALF and lung homogenate.Mice exposed to PG/VG resulted in a signi cant increase in IL-6, KC, and MCP-1 levels in BALF compared to air, TFN, and TDN salts exposed mice (Fig. 2A).Contrarily, mice exposed to PG/VG, TFN, or TDN salts did not signi cantly alter IL-6 and MCP-1 levels in lung homogenate compared to air exposed mice (Fig. 2B).Mice exposed to PG/VG and TFN salts signi cantly increased KC levels compared to air exposed mice (Fig. 2B).
PG/VG with tobacco-derived nicotine salts alters MMP9 protein abundance In order to determine the effect of PG/VG with TFN and PG/VG with TDN salts to alter lung protease levels, protein abundance of MMPs was measured.In PG/VG exposure, both male and female mice had no change in MMP2, MMP9, MMP12, or TIMP-1 protein abundance compared to air exposed mice (Figs. 3, 4).In TFN salt exposed mice, both female and male mice had no change in MMP2, MMP9, MMP12, or TIMP-1 protein abundance compared to air exposed mice (Figs. 3, 4).In TDN salt exposed mice, both male and female mice had no change in MMP2, MMP12, or TIMP-1 protein abundance compared to control mice.In female mice exposed to PG/VG with TDN salts a signi cant increase in MMP9 protein abundance was observed compared to air exposed mice while no change in the MMP9 protein abundance was observed in male mice exposed to TDN salts compared to air controls (Fig. 3, 4).

PG/VG with tobacco-derived nicotine salts alters MMP9 and MMP2 activity levels
In order to determine the effects of PG/VG with TFN and PG/VG with TDN salts to alter lung protease activity, total activity levels of MMP2 and MMP9 were determined using gel zymography.In PG/VG exposure, both male and female mice did not result in a signi cant change in MMP2 or MMP9 activity levels compared to air exposed mice (Fig. 5).In TFN salt exposed mice, both male and female mice did not result in a signi cant change in MMP2 or MMP9 activity levels compared to air exposed mice (Fig. 5).
In TDN salt exposed mice, MMP9 activity levels were signi cantly increased in female mice, whereas no change was observed in the males as compared to mice exposed to PG/VG only (Fig. 5).Furthermore, TDN salts exposure resulted in a signi cant increase in MMP2 activity levels compared to TFN salts exposed female mice; while no change was observed for male mice (Fig. 5).

Discussion
Although TFN has only recently begun to be used in tobacco products, TFN has been around for many years.However, unlike TDN, the health effects of TFN salts are relatively unknown [5,7,17,18].This study attempted to understand the potential toxicological differences between tobacco-derived nicotine (TDN) and synthetic nicotine (TFN).PG/VG with TFN salts and PG/VG with TDN salts were found to be at concentrations of roughly 50 mg/mL in e-liquids.Despite the similar exposures between the two exposure groups, there is a signi cantly increased level of serum cotinine in PG/VG with TDN salts exposed mice.This difference in serum cotinine levels indicates the possibility that TFN may be metabolized in mice differently than TDN.While there is limited knowledge about the metabolism and clearance of R-and Scotinine in humans, a 1988 study studied the disposition kinetics of nicotine and cotinine enantiomers in rabbits and beagle dogs.This study showed that while the clearance of R-and S-nicotine differed in beagle dogs; the clearance of R-cotinine in rabbits was twice that of S-cotinine [18].This substantiates our claim that biotransformation of the two enantiomers could be different for the TFN versus TDN and requires further research.Besides the differences in cotinine levels between TFN salts and TDN salts, serum cotinine levels in the air and PG/VG only exposure groups indicates that alteration in in ammatory cytokines and protease levels are due to exposure to the humectants alone.
Our results found that PG/VG with TFN salts increased total cell counts in the BALF but did not alter macrophage, neutrophil or eosinophil cell counts in BALF.While we looked into the myeloid cell population of immune cells in this study, there is a possibility that the exposures to TFN salts results in an increase in the lymphocytic (B-cell and T-cell) populations in these mice that could explain the signi cant increase in the total cell counts in mice exposed to TFN salts compared to controls.Future work in this area might be able to shed more light into this mechanism.Though not signi cant, the exposure to TFN salts resulted in a 1.6 fold increase in the neutrophil cell count in the lung homogenate as compared to the TDN salt exposed mouse lungs.This proves that the immunological responses on exposure to both TFN and TDN salts are distinct.Although our study found no change in eosinophil levels in either nicotine exposures, we observed a decrease in eosinophil count in lung digest of PG/VG exposed mice.This is contrary to previous observations.Chapman, et al., found that exposure of Balb/c mice to avored e-liquids with nicotine resulted in a signi cant decrease in the eosinophil cell counts after treatment with house dust mites [26].While Ahmad, et al., found that exposure of nicotine aerosol to male Sprague-Dawley rats led to elevated levels of eosinophil counts in the blood of exposed rats [27].Although these exposures found contrary results, this may be due to different pu ng topography, a higher temperature set for the heating of e-liquids, or the use of a nicotine solution not within PG or VG, indicating that the alterations in exposure methodologies can result in differential responses in rodents.Furthermore, it is also important to note that each of these studies used different mouse strains which may not show comparable results as each strain has a distinct innate immune response [28].
Our results found that exposure to TDN salts and TFN salts had a signi cant decrease in IL-6, KC, and MCP-1 levels compared to exposure PG/VG alone exposed mice.Similar to these results, other investigations that utilized male Balb/c mice treated with lipopolysaccharide (LPS) by intratracheal administration and treated with nicotine by intraperitoneal administration, resulted in a decrease in IL-1, IL-6, and TNF-α cytokine levels in BALF compared to LPS treated alone mice [29].Glynos, et al., exposed male C57BL/6J mice to PV/VG with nicotine similarly resulted in no alteration in lung homogenate cytokine levels of IL-1β, TNF-α, and IL-6 at an acute and sub-chronic exposure time points [30].Although our study and previous work have shown anti-in ammatory effects of nicotine, contradictory evidences also exist.Ahmad, et al., found that male Sprague-Dawley rats exposed to nicotine aerosols results in an increase in the mRNA expression of IL-1α and CXCL1 along with increases in IL-1α protein levels [27].
Garcia-Arcos, et al. demonstrated an increase in the mRNA expression of IL-1β, MCP-1, and IL-6 in A/J mice exposed to PG/VG with nicotine as compared to PG/VG only [31].These variations from our results and between previous studies could be a result of variations in the pu ng topography and exposure duration.Further, these studies utilize free-base TDN while our study used both TDN and TFN salts for exposure which could explain the obtained results.Nevertheless, our results indicate that nicotine salts may have altered effects compared to free-base nicotine exposures.
PG/VG alone exposure resulted in an increase in cytokine levels of KC, IL-6, and MCP-1 in BALF.Previous studies have also show in a signi cant increase in the IL6 and IL8 production in lung epithelial cells (16-HBE), exposed separately to propylene glycol or vegetable glycerin only [32], thus indicating that base humectants alone may pose a risk to e-cigarette users.
Our results showed that exposure to PG/VG and PG/VG with TFN salts did not alter the protein abundance of MMPs or TIMP-1 levels while PG/VG with TDN salts did increase MMP9 protein abundance in female mice.A previous study has reported an increase in the mRNA expression of MMP9 and MMP12, along with other lung proteases in Cathepsin K and Cathepsin L1 in PG/VG with nicotine exposed study A/J mice as compare to only PG/VG exposures [31].Similarly another study, demonstrated an increase in MMP9 and MMP12 levels in BALF of C57BL/6J mice exposed to PG/VG with nicotine [33].Similarly, Wang, et al. determined sex-speci c alterations in the expressions of MMPs and TIMP-1 in pups born from pregnant dams exposed to PG/VG with nicotine [15].Though the changes observed in this work were contrary to our results, but it still substantiates the fact that responses towards exposure to TFN and TDN salts varies based on sex which should be an important consideration in future work.Previous work has also shown cell-speci c changes in MMP production on e-cig aerosol exposure.In vitro exposure to avored JUUL pods with 5% nicotine salts in lung epithelial cells (Beas2b) resulted in an upregulation of MMP12 but downregulation of MMP9 gene expression while in murine macrophages( Raw264.7)resulted in an upregulation in MMP12 and no change in MMP9 gene expression [34].These alterations suggest the potential for lung extracellular matrix remodeling and the development of respiratory diseases caused by alterations in MMPs [35,36].
It is pertinent to mention that there were few limitations in determining the source of nicotine used TDN salts exposure in this study.Current analysis could de nitively determine the presence of R-and Snicotine in the e-liquids prepared using TFN salts but could only determine the presence of S-nicotine in the e-liquids prepared using TDN salts.Without conducting radiocarbon analysis to determine the C 14 content of the nicotine, the e-liquids prepared using TDN salts cannot be determined to be Tobaccoderived [5].Furthermore, the duration of exposure for this study was too short to observe noticeable phenotypic changes within the lung.Future work with longer exposure durations might be able to shed more light on the toxicity and immune-modulators on TFN exposures in vivo.Despite these shortcomings, this study is one of the rst studies to determine the effects of exposure to TFN salts in mice and also compares the differences in the inhalation of S-nicotine and TFN.Besides being one of the rst in vivo studies on TFN, this study utilized a nose-only exposure system and a pu ng pro le that mimics current e-cigarette user pu ng topography, allowing this study to best mimic an exposure relevant to human users.

Conclusions
Overall, this study was able to show that exposure to the humectants, PG/VG, used in e-cigarettes alone was able to increase cytokine levels, IL-6, KC, and MCP-1 in BALF and KC levels in lung homogenate of exposed C57Bl/6J mice.It also demonstrated that despite few changes in the differential immune cell counts in the BALF and lung homogenates, the level of KC, IL6 and MCP1 remained comparable in both TFN and TDN-exposed groups.We further provide evidence of sex-speci c changes in th the expression and activity of MMP9 in TFN salt exposed mouse lungs.This study also suggests that the metabolism of nicotine or the clearance of cotinine from TFN may differ from the metabolism of nicotine or the clearance of cotinine from TDN.These ndings indicate that exposure of humectants alone can induce an in ammatory response while exposure to TFN or TDN salts may differentially alter in ammatory responses and lung proteases production.

Figures Figure 1
Figures

Figure 4 Differential
Figure 4 -In ammatory Cytokines/Chemokines Levels -15% Criterion Precast Gel (Cat#5671085, BioRad) with 10 µl of Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards (Cat# #1610375, BioRad) added to the rst well.Protein were separated based on size using gel electrophoresis before being transferred to a nitrocellulose membrane.Membranes were blocked for one hour with 5% BSA in 1x TBST or with 5% non-fat milk in 1x