Abstract E-cigarette (e-cig) aerosols are complex mixtures of various chemicals including humectants (propylene glycol (PG) and vegetable glycerin (VG)), nicotine, and various flavoring additives. Emerging research is beginning to challenge the “relatively safe” perception of e-cigarettes. Recent studies suggest e-cig aerosols provoke oxidative stress; however, details of the underlying molecular mechanisms remain unclear. Here we used a redox proteomics assay of thiol total oxidation to identify signatures of site-specific protein thiol modifications in Sprague-Dawley rat lungs following in vivo e-cig aerosol exposures. Histologic evaluation of rat lungs exposed acutely to e-cig aerosols revealed mild perturbations in lung structure. Bronchoalveolar lavage (BAL) fluid analysis demonstrated no significant change in cell count or differential. Conversely, total lung glutathione decreased significantly in rats exposed to e-cig aerosol compared to air controls. Redox proteomics quantified the levels of total oxidation for 6682 cysteine sites representing 2865 proteins. Protein thiol oxidation and alterations by e-cig exposure induced perturbations of protein quality control, inflammatory responses and redox homeostasis. Perturbations of protein quality control were confirmed with semi-quantification of total lung polyubiquitination and 20S proteasome activity. Our study highlights the importance of redox control in the pulmonary response to e-cig exposure and the utility of thiol-based redox proteomics as a tool for elucidating the molecular mechanisms underlying this response. Keywords: Electronic cigarette aerosol, Rat, Lung, Oxidative stress, Redox proteomics, Ubiquitin proteasome response (UPS) Graphical abstract Image 1 [41]Open in a new tab Highlights * • Acute exposure to electronic cigarettes (e-cig) aerosols decreased total lung glutathione in rats. * • E-cig exposures induced alterations in site-specific thiol oxidation and perturbated redox hemostasis. * • Acute e-cig exposures increased proteasome 20S activity in rat lungs following persistent e-cig oxidative stress. 1. Introduction Electronic cigarettes (e-cigs) are advertised as assistive devices for smoking cessation and a healthy substitute to tobacco with minimal-to-no harm. However, there is significant uncertainty as to the claimed a healthy outcome for users [[42][1], [43][2], [44][3], [45][4]]. E-cig aerosol is a complex mixture of e-liquid components including but not limited to propylene glycol (PG), vegetable glycerin (VG), nicotine, water, and flavoring additives. Several studies have assessed the in vivo toxicity of different mixtures of individual e-liquid components [[46][5], [47][6], [48][7], [49][8], [50][9]]. Werley et al. assessed 7-day and 28-day inhalation toxicity of 100% PG in Sprague-Dawley rats and observed relatively low toxicity with mild nasal irritation and laryngeal squamous metaplasia [[51]5]. Lechasseur et al. exposed BALB/c mice to 100% VG over an 8-week period and observed effects on the expression of circadian molecular clock genes in the exposed-lungs [[52]7]. This study also provided transcriptomic data but with no apparent sign of lung inflammation. In a more comprehensive analysis, Phillips et al. exposed Sprague-Dawley rats to varying PG/VG mixtures with or without nicotine over a 90-day exposure regime (sub-chronic inhalation) [[53]8]. Standard toxicological endpoints in the lung and liver complemented by molecular analyses using transcriptomics, proteomics, and lipidomics showed no signs of toxicity with limited molecular impacts. While the aforementioned studies reported e-cigs having mild or no adverse effect on lung tissue in animal models as observed by histopathological and -omics analyses, other studies have detected redox imbalance and increased inflammatory response in lung tissue of animal models elicited by e-cig exposures [[54][10], [55][11], [56][12]]. Lerner et al. reported increased expression of pro-inflammatory cytokines and diminished lung glutathione (GSH) levels following acute (3-day) e-cig exposure in C57BL/6J mice [[57]11]. Cirillo et al. exposed Sprague Dawley rats to e-cig aerosol for 28 days and assessed pulmonary inflammation, oxidative stress and tissue damage [[58]10]. Tissue morphometric analysis demonstrated alterations in lung structure with large areas of airflow collapse and tissue disruption. Modulation of antioxidant and phase II enzymes suggested a perturbation of the lung redox status. Glynos et al. reported negligible changes in proinflammatory cytokines and lung histology following acute (3 days) and subchronic (4 weeks) exposure of mice to e-cig aerosols [[59]12]. Further, three-day acute exposure, but not the 4-week subchronic exposure, significantly increased oxidative stress in lung tissue. Taken together, the prior studies described above support the notion that e-cig aerosol/vapor exposure induces oxidative stress and an inflammatory response in the lungs. However, details of the underlying molecular mechanisms remain unclear. Post-translational modifications (PTMs) of protein thiols in response to ROS/RNS provide important insights into the widespread consequences of inflammation and oxidative stress. Here we used a redox proteomics assay of thiol total oxidation to identify signatures of site-specific protein thiol modifications induced by e-cig exposures to provide new insights into e-cigarette-associated redox signaling in the lung. 2. Materials and methods 2.1. Animals The studies were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center (URMC). The investigators adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (1996), and the research was carried out in compliance with the Animal Welfare Act. Seven-week-old outbred male Charles River Sprague Dawley (SD) rats weighing 250 g–300 g (Charles River Laboratory, Wilmington, MA) were used in this study, maintained in an AAALAC-accredited animal care facility. All animals were provided commercially certified rat chow and water ad libitum. Rats were quarantined for 7 days following arrival to allow for acclimatization, housed two to three animals per cage, and their health status was monitored daily. 2.2. Chemicals & e-cigarette liquid Propylene glycol (PG), vegetable glycerin (VG), and (−)-nicotine free base (>99% GC) were purchased from Sigma-Aldrich (St. Louis, MO). E-liquid consisting of 50% PG, 50% VG and, in certain conditions, 25 mg/mL nicotine was prepared on the day of exposure. 2.3. Aerosol generation and characterization E-cigarette aerosol was generated using the InExpose tank system extension (E-cig mod; SciReq, Montreal, CA). The puff profile used for the exposure included a total volume of 70 mL, an exposure time of 3.3 s, with a sinusoidal wave, every 30 s (2 puffs per minute) with a bias flow of 2 L/min between puffs for 60 min. The e-cigarette atomizer consisted of a nickel-based, temperature-controlled coil with 0.15 Ohm resistance with stainless steel housing and cotton-based wick (sub-ohm coil, KangerTech, Shenzhen, China). The aerosol generated was characterized for total particulate mass (TPM), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD). Sampling from TPM, MMAD, and GSD was obtained from a nose-only port at a continuous flow of 250 mL/min for 10 min. A 25 mm Pallflex Emfab filter (Pall Corporation, Port Washington, NY) was weighed before and after sampling for TPM. MMAD and GSD were determined using a seven-stage cascade impactor (In-Tox Products, Moriarty, NM). 2.4. Animal exposures Prior to exposure, 1 mL of e-cig liquid was dispensed onto the atomizer ensuring saturation of the cotton wick and prevention of a dry burn. Rats were exposed to e-cigarette aerosol for 3 h per day for 3 consecutive days via a nose-only exposure system (IET 200B Chambers, IES 324 Inhalation Tower; Electro-Medical Measurement System (EMMS), Bordon, Hampshire, UK). After 60 min of exposure, animals were rested for 30 min prior to the next exposure (total of 3 h of exposure per day). Control animals were housed in identical nose-only chambers and were exposed to humidified (30–50%) filtered air only. 2.5. Euthanasia Animals were euthanized immediately following the last day of e-cigarette exposure. Euthanasia was performed via terminal sodium pentobarbital intraperitoneal (IP) injection (100 mg/kg; Abbott Laboratories, Abbott Park, IL) followed by diaphragmatic puncture and exsanguination. 2.6. Bronchoalveolar lavage fluid (BALF), tissue harvest and histopathology After terminal anesthesia and exsanguination, lung tissue was cleared of blood by flushing the right ventricle with 10 mL phosphate-buffered solution (PBS). The trachea was then cannulated, left bronchus clamped, and the right lung was lavaged with two 2.5 mL washes of normal saline (0.9%) solution. Right lung lobes were tied off and immediately snap frozen (−80 °C) following 10 mL PBS flush. The left lung was then unclamped and intratracheally fixed with 10% neutral buffered formalin at 20 cm H[2]O for 30 min prior to removal by gross dissection. Paraffin embedded tissues were sectioned at a thickness of 5 mm and stained for hematoxylin and eosin (H&E). BALF samples were pooled, centrifuged at 2000 g for 10 min at 4 °C, and supernatants frozen (−80 °C). Cell pellets from BALF were re-suspended using 1 mL PBS and cytospin slides (Thermo Shandon, Pittsburgh, PA) were prepared using 50,000 cells per slide. Differential cell counts (~400 cells/slide) were performed on cytospin-prepared slides stained with Diff-Quik (Siemens, DE). Total cell counts were expressed per microliter of BALF. 2.7. Cotinine assay Cotinine levels were measured in rat serum collected immediately after the third day of e-cigarette aerosol exposure by ELISA according to the manufacturer's instructions (Abnova, Taipei, TW). Briefly, three ml of fresh blood was collected from the descending aorta prior to exsanguination. The blood was allowed to clot, centrifuged at 10,000 g for 20 min at 4 °C, and the serum was collected for further analysis. Absorbance was measured at 450 nm using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA). Samples with cotinine concentrations greater than 100 ng/mL required a 1:2 dilution. 2.8. Total glutathione (GSH) assay Total glutathione (GSH) levels were measured by ELISA according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, Michigan, USA). All samples used for GSH measurement underwent deproteination in metaphophoric acid (MPA) reagent prior to performing the assay. Total lung glutathione (GSH), both oxidized and reduced forms, was determined from the assay; quantification of GSSG alone, the oxidized form of GSH and exclusive of total GSH, required further derivation with 2-vinylpyridine, which was not performed. Considering the sulfhydryl group of GSH reacts with 5, 5′-dithio-bis-2-(nitrobenzoic acid) (DTNB) producing 5-thio-2-nitrobenzoic acid (TNB), the concentration of total glutathione in the supernatant of lung homogenates was determined by measuring the absorbance of TNB at 405 nm. Results were expressed as the nmol of total glutathione (GSH) per mg total lung protein. All samples homogenates were prepared using RIPA buffer and underwent a single freeze thaw cycle prior to measuring glutathione levels. 2.9. Sample preparation for redox proteomics The thiol-based redox proteomics experiments were performed as previously described [[60][13], [61][14], [62][15], [63][16], [64][17]]. Briefly, frozen rat lung tissues were minced on dry ice quickly into small pieces. To quantify total thiol, a small portion of tissues from the control and e-cig samples (n = 4 for each) were pooled and incubated in homogenization buffer consisting of 250 mM MES, pH 6.0, 1% sodium dodecyl sulfate (SDS), and 1% Triton-X-100. Aliquots of the original 8 tissue samples were incubated in homogenization buffer with 100 mM N-ethyl-maleimide (NEM). Following incubation for 30 min on ice in the dark, all samples were then homogenized using a hand-held homogenizer. The resulting homogenate was transferred to a 2.0 mL centrifuge tube and then centrifuged at 14,000 rpm at 4 °C for 10 min to remove insoluble material. Alkylation reaction was carried out at 55 °C and 850 rpm in dark for 30 min, followed by protein precipitation with cold acetone (−20 °C) over night. Protein pellets were resuspended in 250 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), pH 7.0, 8 M urea and 0.1% SDS. Protein concentration was determined using the bicinchoninic acid assay (BCA). For each sample, 500 μg of proteins were subjected to buffer exchange via a 0.5 mL 10 K Amicon ultra filter with 8 M urea in 25 mM HEPES, pH 7.0. Samples were diluted to 500 μL using 25 mM HEPES buffer with 1 M urea in 25 mM HEPES, pH 7.6 and then reduced with 20 mM Dithiothreitol (DTT) in the presence of 0.2% SDS at 37 °C for 30 min. Excess DTT was removed by buffer exchange, and BCA was performed to determine the protein concentration prior to thiol enrichment. For each sample, 150 μg of proteins were incubated with 30 mg of thiopropyl Sepharose 6B resin in of 25 mM HEPES, pH 7.6 with 0.2% SDS at room temperature for 2 h, 850 rpm. The resin was washed sequentially with 1) 8 M urea, 2) 2 M NaCl, 3) 80% acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA), and 4) 25 mM HEPES, pH 7.0, each buffer 5 times. On-resin protein digestion, TMT labeling, and elution of peptides from the resin were essentially carried out as previously described [[65]14,[66]15]. All samples were combined, desalted by solid phase extraction and store in −80 °C before LC-MS/MS analysis. 2.10. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis Dried peptides were resuspended in H[2]O and diluted to 0.1 μg/μL. An aliquot of 5 μL was injected to a Waters nanoACQUITY UPLC system coupled with Q Exactive plus mass spectrometer (Thermo Fisher Scientific). Peptide separation was performed with an in-house packed C18 column (50 cm × 75 μm i.d., Phenomenex Jupiter, 3 μm particle size) using a binary mobile system (buffer A: 0.1% formic acid in H[2]O; buffer B: 0.1% formic acid in acetonitrile) with buffer B percentage increased linearly at the following points: 0.1% at 0 min, 8% at 4 min, 12% at 36 min, 30% at 135 min, 45% at 175 min, and 95% at 180 min. A data-dependent acquisition method was used to collect mass spectra. Each cycle consisted of a full MS scan and up to 12 MS/MS scans of the most abundant precursor ions. Full MS scan was performed in the Orbitrap mass analyzer at 400–2000 m/z with the following key parameters: resolution 70,000; automatic gain control (AGC): 1e6, and maximum injection time (IT): 20 ms. For MS/MS, precursor ions were selected by the quadrupole mass analyzer at an isolation window of 2 m/z, and then fragmented by higher-energy collisional dissociation (HCD) of normalized collision energy at 30. The resulting MS/MS spectra were analyzed in the Orbitrap with the following settings: resolution: 35,500; scan range 200–2000 m/z with the fixed first mass of 110 m/z; AGC: 1e5; and maximum IT of 100 ms. A 30 s dynamic exclusion was also applied. 2.11. Western blot for Lys48-linked ubiquitin and ubiquitin-C Rat lungs were homogenized in Radioimmunoprecipitation assay buffer (RIPA lysis buffer) (Abcam; Cambridge, MA) supplemented with a protease inhibitor cocktail (Roche; Indianapolis, IN). Following centrifugation at 12,000 rpm for 20 min at 4 °C, soluble supernatant fractions were collected for total protein and Western blot analysis. Total protein concentrations were determined by BCA assay kit (Thermo Scientific, Waltham, MA). 10 μg total protein were resolved in pre-casted 4–15% gradient Tris-Glycine ‘Stain-Free’ Mini-Protean gel (Bio-Rad, Hercules, CA) to assess for lysine (K)48-linked ubiquitin (1:1000, R&D Systems) and ubiquitin-C (1:1000, Invitrogen) expression. Gels were transferred to 0.1 μm nitrocellulose membrane (GE Healthcare). Horseradish peroxidase (HRP) and SuperSignal West Pico chemiluminescent substrates (Thermo Scientific) were used to detect protein signal intensity. Semi-quantification was performed using Image Lab software (Bio-Rad, Hercules, CA) and normalized relative to total protein imaged from the ‘Stain-Free’ gel. 2.12. 20S proteasome activity for rat lung homogenate Rat lung was lysed and collected in 50 mM HEPES (pH 7.5), 5 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl and 1% Triton X-100 supplemented with 2 mM ATP buffered solution. 20S proteasome activity was determined using the Chemicon 20S Proteasome Activity Assay (Millipore) according to the manufacturer's instructions. Luminescent signal was semi-quantified using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA). Enzymatic activity in e-cig-exposed samples was expressed as absolute activity (micrograms/milligram total rat lung) as well as relative activity to air controls. 2.13. Data analysis and statistics Raw MS data was searched using MS-GF + against an Uniprot Rattus norvegicus protein database (31,823 entries, downloaded October 2017) [[67]18]. Key search parameters were 20 ppm tolerance for precursor ion masses, 0.5 Da tolerance for fragment ions, dynamic oxidation of methionine (15.9949 Da), dynamic NEM modification of cysteine (125.0477 Da), and static 10-plex TMTs modification of lysine and peptide N-termini (229.1629 Da). The obtained peptide spectrum matches (PSMs) were filtered by 1) mass error within 10 ppm; and 2) PepQ value < 0.01, which controlled the false discovery rate (FDR) level below 1%. For quantitative analysis, MASIC was used to extract the reporter ion intensities from the raw data [[68]19]. An in-house R script was used to link the peptide identifications and quantifications as described before [[69]20]. Reporter ion intensities for PSMs corresponding to the same unique peptide were summed and log2 transformed. Peptides quantified in all samples were kept for further analysis. The site occupancy of total oxidation was calculated as the ratio of the average level of total oxidation (n = 4 e-cig or control samples) over total thiol (n = 1 for the pooled sample) in percentage. Student t-test was used to determine the statistical significance between the control and E-cig groups. Significant cysteine-containing peptides were defined as 1) p < 0.01 and 2) at least 20% in fold change (ie, log2FC < −0.26 or log2FC > 0.26). Gene ontology analysis of significant proteins were performed with DAVID ([70]https://david.ncifcrf.gov/summary.jsp). Functional and protein interaction network analysis was performed using Ingenuity pathway analysis ([71]www.ingenuity.com). 3. Results 3.1. Aerosol characterization E-cig aerosols were characterized for total particulate mass (TPM), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD). The average TPM for exposures was 0.968 ± 0.194 mg/mL. The average MMAD was 0.92 ± 0.04 μm, and the range GSD for all exposures was 1.4–1.6. This average TPM is similar to a medium concentration PG/VG exposure used for chronic studies in a prior rat e-cigarette exposure model [[72]8]. 3.2. E-cigarette exposures Seven-week-old Sprague-Dawley rats (average weight: 254.0 ± 10.2 g) were exposed to humidified room air (n = 15), 50% propylene glycol: 50% vegetable glycerin (PG/VG) (n = 14), or PG/VG + 25 (2.5%) mg/mL nicotine (n = 17) for 3 h per day for 3 consecutive days via nose-only exposure. At the end of exposures, rat serum was collected for cotinine levels. Both air controls and PG/VG + 0% nicotine rats had plasma cotinine levels less than 5 ng/mL (0.0 ± 0.0 ng/mL and 0.7 ± 1.2 ng/mL, respectively). Serum cotinine levels increased significantly in PG/VG + 2.5% nicotine as shown in [73]Fig. 1A (103.3 ± 10.5 ng/mL; ANOVA, ****p < 0.0001). Serum cotinine concentrations following acute exposure were similar in magnitude to that seen in newborn mice exposed to 1.8% nicotine with PG for ten days [[74]21]. Fig. 1. [75]Fig. 1 [76]Open in a new tab In vivo e-cigarette exposure and biological function. (A) Serum cotinine levels from air controls, PG/VG - propylene glycol/vegetable glycerin, and PG/VG+(25 mg/ml) Nicotine - propylene glycol/vegetable glycerin plus 25 mg/ml nicotine in rats (n = 7–10/group; ANOVA; ****p < 0.0001). (B) Hematoxylin and Eosin (H&E) stained lung sections from animals exposed to air, PG/VG and PG/VG + (25 mg/ml) Nicotine. (C) Bronchoalveolar lavage (BAL) fluid cell count in air controls (503 ± 66 cell/μL), PG/VG (454 ± 65 cell/μL), and PG/VG + (25 mg/ml) Nicotine (481 ± 137 cell/μL) (ANOVA; p = 0.56) and cell differentials with the average macrophage percent in air controls (92 ± 4%), PG/VG (91 ± 5%), and PG/VG + (25 mg/ml) Nicotine (91 ± 4%) (ANOVA; p = 0.77). 3.3. Histology and BALF analysis Left lungs of all exposed rats were fixed, embedded and sectioned for further histologic evaluation following e-cigarette exposure. H&E stained lung sections from animals exposed to air, PG/VG + 0% nicotine and PG/VG + 2.5% nicotine displayed preserved lung structure of the distal lung parenchyma and intrapulmonary bronchioles with rare foci of inflammatory infiltrates in the alveolar cell walls ([77]Fig. 1B). Consistent with histology, BAL total cell counts ([78]Fig. 1C) did not differ significantly between air controls (503 ± 66 cell/μL), PG/VG (454 ± 65 cell/μL), and PG/VG + 2.5% nicotine (481 ± 137 cell/μL) (ANOVA, p = 0.57). BAL cell differential ([79]Fig. 1C) also did not differ significant between groups with the average macrophage percent in air controls 92 ± 4%, PG/VG 91 ± 5%, and PG/VG + 2.5% nicotine 91 ± 4% (ANOVA, p = 0.77). 3.4. Decrease in total lung GSH GSH was assessed immediately following the end of e-cigarette exposures as a surrogate marker of oxidative stress. Normalized GSH was significantly reduced in rat lungs exposed to PG/VG (1.41 ± 0.52 nM/mg and PG/VG + 2.5% nicotine (1.63 ± 1.12 nM/mg) compared to lungs exposed to air control (5.28 ± 0.95 nM/mg; ANOVA, ****p < 0.0001) as shown in [80]Fig. 2. Normalized GSH did not differ between PG/VG and PG/VG + 2.5% nicotine exposed lungs (p = 0.78, ANOVA with Tukey's). Hence, acute e-cigarette exposure resulted in significant lung oxidative stress as demonstrated by reduced total lung glutathione and independent of nicotine concentration. Fig. 2. [81]Fig. 2 [82]Open in a new tab Total lung glutathione (GSH) assessment. GSH normal for total protein (nM/mg) in air control (5.28 ± 0.95 nM/mg; blue; n = 15), PG/VG (1.41 ± 0.52 nM/mg; red; n = 14) and PG/VG + (25 mg/ml) Nicotine (1.63 ± 1.12 nM/mg; dark red; n = 17) (ANOVA, ****p < 0.0001). (For interpretation of the references to colour in this figure legend, the