Iso Coated V2 280 Cigarettes

Thousands of electronic cigarette refill fluids are commercially available. The concentrations of nicotine and the solvents, but not the flavor chemicals, are often disclosed on product labels.

The purpose of this study was to identify and quantify flavor chemicals in 39 commercial refill fluids that were previously evaluated for toxicity. Twelve flavor chemicals were identified with concentrations ≥1 mg/ml: cinnamaldehyde, menthol, benzyl alcohol, vanillin, eugenol, p-anisaldehyde, ethyl cinnamate, maltol, ethyl maltol, triacetin, benzaldehyde, and menthone. Transfer of these flavor chemicals into aerosols made at 3V and 5V was efficient (mean transfer = 98%). We produced lab-made refill fluids containing authentic standards of each flavor chemical and analyzed the toxicity of their aerosols produced at 3V and 5V using a tank Box Mod device.

Over 50% of the refill fluids in our sample contained high concentrations of flavor chemicals that transferred efficiently to aerosols at concentrations that produce cytotoxicity. When tested with two types of human lung cells, the aerosols made at 5V were generally more toxic than those made at 3V. These data will be valuable for consumers, physicians, public health officials, and regulatory agencies when discussing potential health concerns relating to flavor chemicals in electronic cigarette products. In 2014, more than 7,000 electronic cigarette (EC) refill fluid products with unique flavor names were commercially available online and from local shops, and the number of products continues to grow. In a study of 30 refill fluids, total flavor chemical concentrations ranged from 10–40 mg/ml.

Even though many of these flavor chemicals are generally regarded as safe (GRAS) for ingestion, the Flavors & Extract Manufacturers Association (FEMA) has cautioned that their GRAS designation does not extend to inhalation. It is important to gain a better understanding of which flavor chemicals are predominant ingredients in refill fluids, their concentrations, and the potential health effects that EC consumers may experience when inhaling high concentrations of flavor chemicals.As an initial step to identify refill fluids that may have adverse health effects, our laboratory evaluated the cytotoxicity of a library of refill fluids with a broad range of flavors which included buttery/creamy, minty, sweet/candy, fruit, tobacco and cinnamon/spiced. About a third of these products were highly cytotoxic to human pulmonary fibroblasts (hPF) and two types of stem cells. However, a cinnamon-flavored refill fluid was the most potent across all cell types. Cinnamaldehyde was subsequently identified as the dominant flavor chemical in a small library of commercial cinnamon-flavored refill fluids, and its concentration was directly correlated with its cytotoxicity in the MTT assay.The cytotoxicity of the refill fluids and aerosols made from the products in our original screen was then compared across brands. 74% of the refill fluid cytotoxicity data accurately predicted the toxicity of the corresponding aerosols.

The “creamy/buttery”-flavored refill fluids led to more cytotoxic aerosols than did fluids in other flavor classes, again suggesting that flavor chemicals are differentially important in determining cytotoxicity of EC products.The above studies have demonstrated a need for further identification and characterization of the potency of flavor chemicals in EC refill fluids. The current study evaluated the flavor chemicals and their cytotoxicity using the two refill fluid libraries from our previous studies, which included 39 commercial refill fluids and six duplicate bottles (45 products total). We specifically: (1) identified the flavor chemicals in each product, (2) produced “lab-made” refill fluids with authentic standards of those flavor chemicals present in concentrations ≥1 mg/ml, (3) determined how much of each flavor chemical is transferred from the lab-made refill fluids into aerosols made at high and low voltages, (4) examined the cytotoxicity of these aerosols using human lung cells, and (5) identified reaction products formed due to the aerosolization process. Identification of flavor chemicals in 39 refill fluids and selected duplicatesFifty-two of the 92 flavor chemicals on the GC/MS target analyte list used were identified and quantified in the combined library of 45 refill fluids (Fig. ). The total concentration of the flavor chemicals in these products ranged from 0.141 to 179 mg/ml. The chemicals in Fig.

Are arranged on the y-axis based on IC 50 (highest to lowest) from rat oral toxicity values found in online compilations. Products on the x-axis are identified by our laboratory inventory number (Supplemental Table ) and are arranged from highest (left) to lowest total flavor chemical concentration. Thirteen chemicals were identified as dominant flavor chemicals, being found at concentrations ≥1 mg/ml in at least one of the products and are identified in Fig.

With a red asterisk. The 13 th dominant flavor chemical, benzaldehyde PG (propylene glycol) acetal (13.8 mg/ml) was not available commercially for use as an authentic standard and was not examined in this work. Heat map of flavor chemicals identified and quantified in 45 commercial refill fluids. Chemicals ( y-axis) are classified by toxicity and ordered within each toxicity bracket from highest to lowest lethal dose based on rat oral toxicity data. Refill fluids ( x-axis) are represented by their inventory number and ranked with the left having the highest concentration of total flavor chemicals and the right having the lowest. The color gradient provides information on the concentration of each chemical.

Red asterisks represent the 12 flavor chemicals that were ≥1 mg/ml in at least one product. When arranged by toxicity classification according to rat oral data ( y-axis of Fig. ), eight of these 12 flavor chemicals were in the harmful bracket, three were in the irritant bracket, and one was not classified according to oral rat toxicity data (Fig. ). These dominant flavor chemicals include mainly minty, sweet, fruity, and creamy flavors (Table ). The highest concentrations for the 12 dominant flavor chemicals found in the refill fluids were: cinnamaldehyde (155 mg/ml), menthol (84 mg/ml), benzyl alcohol (39 mg/ml), vanillin (31 mg/ml), eugenol (12 mg/ml), p-anisaldehyde (9.0 mg/ml), ethyl cinnamate (8.5 mg/ml), maltol (4.9 mg/ml), ethyl maltol (4.2 mg/ml), triacetin (2.8 mg/ml), benzaldehyde (2.5 mg/ml), and menthone (1.4 mg/ml). Frequency of occurrence of the 12 dominant chemicalsThe frequency of occurrence and the concentration of each dominant flavor chemical from the GC/MS commercial refill fluid screen is shown in Figs. Duplicate bottles were not incorporated into the frequency calculation, but were included in the graphs to determine whether duplicate products were similar to each other. Six of the 12 dominant flavor chemicals were present less frequently (5–12 products) (Fig. ).

This included ethyl cinnamate in four products (10%), triacetin in five products (13%), eugenol in six products (15%), benzyl alcohol in seven products (18%), maltol in seven products (18%), and benzaldehyde in 12 products (31%). The other six dominant flavor chemicals were found more frequently (16–31 products) (Fig. ). Figure shows menthone in 16 products (41%), p-anisaldehyde in 17 products (44%), menthol in 17 products (44%), cinnamaldehyde in 20 products (51%), vanillin in 22 products (56%), and ethyl maltol in 31 products (80%).

Generally, for Figs and, the concentrations of flavor chemicals were similar in the duplicate bottles. Flavor chemicals present at ≥1 mg/ml that were in. Flavor chemicals present at ≥1 mg/ml that were in 35% of the 39 refill fluids. Duplicate bottles were not incorporated into the frequency calculation, but rather added to distinguish whether duplicate products were similar. ( a) Menthone was in 16 products (41%). ( b) p-Anisaldehyde was in 17 products (43.6%).

( c) Menthol was in 17 products (43.6%). ( d) Cinnamaldehyde was in 20 products (51.2%). ( e) Vanillin was in 22 products (56.4%). ( f) Ethyl maltol was in 31 products (79.5%). Dup = duplicate bottle purchased and screened.

Flavor chemical aerosolization transfer efficiencyAerosols that were captured in liquids and used for analytical chemistry or cytotoxicity experiments will be referred to as “aerosols”. GC/MS was used to identify and quantify flavor chemicals in aerosols produced at 3V (volts)/4.3 watts or 5V/11.9 watts from lab-made refill fluids that contained 80% propylene glycol and one of the 12 dominant flavor chemicals. For these analyses, the GC/MS target list included 178 flavor chemicals. Generally, for aerosols produced at 3V and 5V, the refill fluid to aerosol transfer efficiency was high (Fig. The 3V aerosolization process had a transfer efficiency of 80% or greater (except for ethyl maltol which was 62%) and an average transfer efficiency of 110% ± 8%, which was not significantly different than a theoretical mean of 100% (Table ). Aerosolization at 5V had a transfer efficiency of 70% or greater (with exception of maltol which was 58%) and an average transfer efficiency of 86% ± 4% which was significantly different than 100% (Table ). Identification of co-constituents and secondary reaction productsThe refill fluids and aerosols made from the three dominant chemicals with the highest concentrations (cinnamaldehyde, menthol, benzyl alcohol) contained several chemicals at low concentration that likely were co-constituents of the dominant flavor chemical (Fig. ).

To determine if new chemicals were produced upon aerosolization, refill fluids were compared to aerosols collected at 3V and 5V. No new chemicals from our target analyte list were found at significant concentrations for the 3V aerosol. Three new chemicals were present in aerosol made at 5V. Hydroxyacetone appeared in all 5V aerosols at concentrations ranging from 100 to 10,000 µg/mL (Fig. ).

Acetoin was present at 150 µg/mL in the 5V aerosol of triacetin (Fig. ), and 2,3-butanedione (diacetyl) was present in the 5V aerosol of cinnamaldehyde, benzyl alcohol, and triacetin at 20, 24 and 96 µg/mL, respectively. Cytotoxicity of hydroxyacetoneBecause hydroxyacetone appeared in all 5V aerosols, its cytotoxicity was evaluated using the MTT assay with hPF and the A549 CCL-185 lung epithelial cell line (A549) (Fig. ). Even at 1 mg/ml (1.35 × 10 −2 M), hydroxyacetone did not inhibit metabolic activity of hPF or A549 cells, and it was not deemed cytotoxic for either cell type using ISO 10993-5:2009(E) international standard, which categorizes cytotoxicity based on a treatment producing an absorbance in the MTT assay that is. Dose-response curves for hydroxyacetone and 80% propylene glycol blank. ( a) Hydroxyacetone diluted directly into culture medium for A549 cells and hPF. ( b) A549 cells and hPF treated with 80% propylene glycol aerosolized at 3V and 5V and captured in culture medium.

IC 50 represents the concentration that inhibited survival by 50% and is only indicated where applicable. Data are showing the means and their standard errors for three independent experiments. Red lines represent hPF dose-response curves. Blue lines represent A549 dose-response curves. Hash symbols (#) for 3V aerosols and asterisks (.) for 5V aerosols indicate the lowest concentration that is significantly different than the untreated controls. Cytotoxicity of aerosols made from propylene glycol and lab-made refill fluidsAn 80% propylene glycol/20% water blank aerosolized at 5V (but not at 3V) produced IC 50s for both cell types (Fig. ).

Using the cytotoxicity categorization from ISO 10993-5, 5V samples were cytotoxic and had a response that fell below 70% of control in the MTT assay, while 3V samples were not cytotoxic for either cell type (Table ), in agreement with our previous study.A549 cells and hPF were exposed to 3V and 5V aerosols from lab-made refill fluids that contained one dominant flavor chemical at its highest identified concentration (Table ) in 80% propylene glycol and 20% water (Fig. ). Generally, aerosols generated at 5V were more potent than those made at 3V (Fig. ). Exceptions to this were aerosols created from refill fluids containing cinnamaldehyde (at 155 mg/mL), vanillin (hPF) (at 31 mg/mL), and ethyl maltol (hPF) (at 4.2 mg/mL), for each of which the 3V and 5V aerosols produced similar results (Fig. ). When comparing sensitivity in the MTT assay using ISO 10993-5, 67% of the 3V aerosols and 92% of the 5V aerosols were cytotoxic to hPF, while 17% of the 3V aerosols and 100% 5V aerosol samples were cytotoxic to A549 (Table ).

The hPF were considerably more sensitive to cinnamaldehyde and menthol than the A549 cells. Dose-response curves for A549 cells and hPF treated with aerosols produced from lab-made refill fluids containing the dominant flavor chemicals.

The y-axis shows percent of survival in the MTT assay and is normalized to the untreated controls. The x-axis shows the mg of chemical per mL of culture medium. ( a–l) represents each of the 12 dominant chemicals, prepared in refill fluid form, then collected as an aerosol into culture medium. IC 50 represents the inhibitory concentration at 50% and is only indicated where applicable. Data are showing the means and their standard errors for three independent experiments. Red lines represent hPF dose-response curves. Blue lines represent A549 dose-response curves.

Hash symbols (#) for 3V aerosols and asterisks (.) for 5V aerosols indicate the lowest concentration that is significantly different from the untreated controls. Relatively little is known about the flavor chemicals used in EC refill fluids and their effects on human health. In this study, we identified and quantified flavor chemicals in 39 commercial refill fluids and evaluated their cytotoxicity. Because nicotine concentration and fluid color vary in duplicate bottles of refill fluids, we also compared six pairs of duplicate products, and in each case the duplicate bottle was generally similar to the original bottle’s flavor chemical profile. Total flavor chemical concentrations in these products ranged from 0.141 to 179 mg/ml with 29% of the products having concentrations 12 mg/ml, which is higher than the median nicotine concentration preferred by EC consumers. At least one of the 12 dominant flavor chemicals (i.e., those that were ≥1 mg/ml) was present in 56% of the original 39 products.

Some chemicals were used frequently. For example, menthone, p-anisaldehyde, menthol, cinnamaldehyde, vanillin, and ethyl maltol appeared in 41–80% of the refill fluids in our library.

Our data further showed that the flavor chemicals in lab-made refill fluids transferred efficiently into the aerosol. These data support the conclusion that flavor chemicals are a major component of EC refill fluids and that many commercial refill fluids would expose EC users to high concentrations of certain flavor chemicals.The cytotoxicity of the products screened by Bahl et al. Generally correlated with the concentration of a specific flavor chemical(s) or the total concentration of flavor chemicals. In Supplemental Table, the IC 50 data from the initial refill fluid screen on hPF, in which the highest dose tested was 1%, is compared to the total concentration of flavor chemicals and the individual dominant flavor chemicals that were in each product.

Of the nine highly cytotoxic refill fluids in the screen by Bahl et al.

ResultsUsing human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by the lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke.

Conversely, specific inhibitors of the JNK or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant NAC inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR. BACKGROUNDThe cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that plays a critical role in the lung by regulating airway fluid homeostasis allowing cilia to beat and clear pathogens. Absence of functional CFTR leads to cystic fibrosis a genetic disease associated with impaired mucus clearance, and chronic infection and inflammation. These past few years there has been a lot of interest in the negative regulation of CFTR by pollutants such as cigarette smoke, cadmium, and arsenic –.

We and others have shown that CFTR expression is reduced in the lung of patients who developed chronic obstructive pulmonary disease (COPD) after years of cigarette smoking –. These findings suggest that suppression of CFTR could contribute to the development of chronic bronchitis seen in COPD which is characterized by mucus secretion, infection and inflammation similarly to what is observed in the lungs of patients with Cystic Fibrosis.Suppression of CFTR can occur via degradation by two main pathways: the ubiquitin-proteasome pathway and the lysosomal pathway –.

Plasma membrane CFTR is rapidly endocytosed and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with more than 75% of endocytosed wild-type CFTR recycling back to the plasma membrane –. The plasma membrane stability of CFTR depends on its biosynthetic processing and post-maturational trafficking, which involves endocytic uptake followed by recycling to the plasma membrane or degradation in the lysosomes ,. The E3 ubiquitin ligase c-Cbl has been shown to facilitate CFTR endocytosis and ubiquitination with subsequent lysosomal degradation. The molecular mechanism by which cigarette smoke alters expression of the CFTR ion channel is still unknown. We have previously shown that using a heterologous expression system, cigarette smoke exposure causes rapid internalization of CFTR. During this internalization, CFTR does not co-localize with lysosomes but is instead internalized into an aggresome-like pathway in a calcium-dependent manner ,.Cigarette smoke activates several mitogen-activated protein kinase (MAPK) pathways including the MEK/Erk1/2 MAPK pathway. Activation of this latter MAPK pathway results in cell survival and proliferation.

It was recently shown that the MEK/Erk1/2 MAPK pathway can regulate the expression of the epithelial sodium channel ENaC by regulating its interaction with the E3 ubiquitin ligase Nedd4-2 leading to lysosomal degradation of ENaC ,. Whether the MEK/Erk1/2 MAPK pathway also regulates the expression of plasma membrane CFTR is unknown.Herein, we conducted this study to determine the underlying mechanisms by which cigarette smoke decreases CFTR abundance in human bronchial epithelial cells and determine the role of the MEK/Erk1/2 MAPK pathway in this process. We also evaluated whether the antioxidant N-acetyl-cysteine (NAC) could prevent the cigarette smoke-induced suppression of CFTR. Cell Culture and ReagentsThe human bronchial epithelial cell line 16HBE14o-, an immortalized human bronchial epithelial cell line, was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine, 10% FBS and penicillin (100 U/ml) and streptomycin (100 µg/ml). The tissue culture plates were coated using human fibronectin (1 mg/ml), collagen I bovine (3 mg/ml), and bovine serum albumin (1 mg/ml). All the cells used in the experiments were between passages 25 and 50, and were grown and maintained at 37 °C in a 5% CO 2 humidified incubator.

Primary human bronchial epithelial cells (HBECs) were isolated from excess donor tissue obtained at the time of lung transplantation under a protocol approved by UNC Medical School IRB. Primary HBECs were cultured as previously described and studied when fully differentiated ,. Lactacystin, UO126, UO124, SB203580, and SP600125 were from Calbiochem (La Jolla, CA). PD98059 was purchased from Cell Signaling Technology. The proteasome inhibitor MG132, and the lysosomal inhibitors, leupeptin and chloroquine, were purchased from Sigma-Aldrich (St. Cell Transfection16HBE14o- cells were transfected with Erk1 and Erk2 small interfering RNAs (siRNA), cbl siRNA (Ambion), or negative control #1 siRNA (Ambion) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

Forty eight hours after transfection, cells were treated with or without 10% cigarette smoke extract (CSE) prepared from Camel cigarettes (R.J. The cells were then lysed in PBS with 1% Triton X-100 containing a cocktail of protease inhibitors (Roche Diagnostics, IN) for protein analysis.

Cell Surface BiotinylationThe 16HBE14o- cells were rinsed with ice-cold phosphate-buffered saline (PBS) containing 0.1mM CaCl 2 and 1mM MgCl 2 to eliminate the proteins present in the media. Cell surface proteins were labeled with 1 mg/ml EZ-Link NHS-SS Biotin (Pierce) for 30 min at 4 °C.

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Biotinylation was stopped by removing the biotin solution and incubating the cells with PBS containing 1% bovine serum albumin for 10 min at 4 °C to quench any residual NHS-SS biotin. At the end of the experiment, the cells were lysed with PBS-1% Triton X-100 and protease inhibitors (Roche). Biotinylated proteins were incubated with streptavidin beads overnight at 4 °C. After extensive washings, bound proteins were subjected to Western blot analysis. Biotinylated CFTR was detected using a C-CFTR monoclonal antibody (24-1; R&D Systems). ImmunoblottingCells were lysed in PBS containing 1% Triton X-100 and a cocktail of protease inhibitors (Roche).

Western blotting was performed as previously described. In brief, 20 µg of the protein were separated with SDS-PAGE in 4–15% polyacrylamide gel and then transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% non-fat milk in PBS-Tween 20 and immunoblotted with primary antibodies against CFTR antibody (24-1, R&D Systems), phosphor-Erk1/2 (Cell Signaling), Erk1/2 (Cell Signaling), or β-actin (Santa Cruz Biotechnology) followed by treatment with appropriate HRP-conjugated secondary antibody (Pierce, Rockford, IL, USA). The signals were detected with enhanced chemiluminescence (Super Signal West Pico; Thermo Scientific) followed by exposure to X-ray films. The protein bands on the X-ray film were scanned, and band density was measured using ImageJ software (NIH). ASL height measurementsTo measure ASL height, PBS (20 µl) containing 2 mg/ml rhodamine-dextran (10 kDa; Invitrogen, USA) ± 10 µM MEK inhibitor was added to cultures at the start of the experiment for 10 mins. To measure ASL secretion, excess fluid was aspirated with a Pasteur pipette to bring ASL height down to 7 µm, as described by Tarran et al.

Before CS exposure, 10 µM MEK inhibitor was added basolateraly. In all cases, five predetermined points (one central, four 2 mm from the edge of the culture) were XZ scanned using a confocal microscope (Leica SP5; glycerol 63× immersion lens) as described. Cultures were returned to the incubator between time points. For all studies, PFC was added mucosally during imaging to prevent evaporation of the ASL. Cigarette smoke extract (CSE) preparation and whole Cigarette Smoke (CS) exposureCSE (100%) was prepared as previously described and used to treat 16HBE14o- cells. Primary HBECs were exposed to whole cigarette smoke (CS) after being placed in a specially built smoke exposure chamber that exposes apical but not basolateral surfaces ,.

CS was then generated according to the International Organization of Standardization (ISO) standards (35 ml draw over 2 s) using a LC1 smoke engine (Borgwaldt, Richmond, Virginia, USA) and applied to the cultures at a rate of 1 puff every 30 s until the cigarette is smoked ( 5 min; 12 puffs). For ASL experiments cells are treated with CS for about 5 min. This maneuver has previously been shown to drive removal of CFTR from the plasma membrane without inducing gross cellular toxicity ,. Confocal Microscopy16HBE14o- cells were fixed in ice-cold 100% methanol for 20 min at −20°C. The slides were then incubated in 1% bovine serum albumin (BSA)/PBS for 10 min, followed by incubation at 37 °C for 1 hr with primary antibody against CFTR (24-1; R&D Systems) and the lysosomal marker LAMP-1 (Cell Signaling Technology). After several washings, the slides were incubated at 37 °C for 45 min with appropriate Alexa Fluor® 488- and Alexa Fluor® 594-conjugated secondary antibody.

Coverslips were mounted onto slides with Vectashield mounting medium containing DAPI (Vector Laboratories) prior to being imaged on a Leica DMIRE2 inverted confocal microscope using a 63× objective lens. Effect of lysosomal and proteasome inhibitors on cigarette smoke extract (CSE)-induced decrease of CFTR protein in human airway epithelial cellsSeveral studies have recently shown that CSE decreases the expression of CFTR in human airway epithelial cells ,. Here we used the normal human bronchial epithelia cell line 16HBE14o- that endogenously expresses the ion channel CFTR. The main two pathways leading to CFTR degradation are the proteasomal and lysosomal pathways ,. In order to investigate whether the underlying pathway involves either lysosomes or the proteasome, 16HBE14o- cells were treated with CSE in presence of the lysosomal or proteasome inhibitors.

As expected, CSE reduced the expression of CFTR. It has to be noted that only mature CFTR (Band C) is seen on the blots. This result is in agreement with previous report showing that CFTR biogenesis is very efficient (close to 100%) in cells endogenously expressing CFTR such as 16HBE14o-. The lysosomal inhibitors chloroquine and leupeptin both significantly prevented the CSE-induced decrease of CFTR, but they both had no effect on steady state level of CFTR. As previously described the proteasomal inhibitor MG132 did not prevent CFTR diminution after CSE exposure. However MG132 alone decreased CFTR expression. We therefore used another proteasomal inhibitor lactacystin which had no effect on steady-state levels of CFTR.

Again, this inhibitor could not preclude the loss of CFTR induced by CSE exposure. Taken together our data show that cigarette smoke induces lysosomal degradation of CFTR. Effect of the lysosomal inhibitors leupeptin and chloroquine and the proteasomal inhibitor lactacystin on the expression of CFTR after exposure to cigarette smoke extract16HBE14o- cells were treated with 10% cigarette smoke extract (CSE) with or without the lysosomal inhibitor leupeptin (LP, 50 µg/ml) or chloroquine (CQ, 10 µM), or the proteasome inhibitor lactacystin (LC, 5 µM) for 48 hrs. CFTR protein was detected by immunoblotting as described in Methods. CTRL, Control. Role of MAPK pathways in CSE-induced suppression of CFTRCigarette smoke contains over 3,000 chemicals including reactive oxygen species (ROS) that can act on various pathways in the cell. Accordingly, CSE can stimulate multiple signaling pathways including mitogen-activated protein kinase (MAPK) pathways.

We therefore investigated whether the main classical MAPK pathways (i.e. P38, JNK, and MEK) contribute to the decrease in the expression of CFTR protein after CSE exposure.

As shown in, inhibition of the MEK/Erk1/2 MAPK pathway using two specific inhibitors, UO126 and PD98059, prevented the loss of CFTR induced by CSE. These results were further confirmed using UO124, the inactive form of UO126 which has no inhibitory property on MEK, and had no protective effect on CFTR after exposure to cigarette smoke. UO124 alone had no effect on the expression of CFTR ( p 0.05). Although UO126 alone has a trend to increase the expression of CFTR when compared with the control group, this increase failed to reach significance ( p = 0.063, ).

Conversely, inhibition of the p38 or JNK MAPK pathways had no effect on the suppression of CFTR after exposure of human bronchial epithelial cells 16HBE14o- to CSE. Role of MAPK inhibitors on CFTR expression after cigarette smoke exposure16HBE14o- cells were treated with 10% CSE with or without the MEK/Erk1/2 inhibitors UO126 (10 µM) or PD98059 (PD, 20 µM), the p38 inhibitor SB203580 (SB, 20 µM), the JNK inhibitor SP600125 (JNKi, 20 µM), or UO124 (10 µM) for 48 hrs.

CFTR protein was detected by immunoblotting. CTRL, Control.

Inhibition of the MEK/Erk1/2 MAPK pathway prevents the loss of CFTR from the plasma membrane of human airway epithelial cells after CSE exposureCFTR has to be present at the plasma membrane of bronchial epithelial cells to exert its role as a chloride channel and regulate the airway surface hydration. We therefore wanted to determine whether inhibition of the MEK/Erk1/2 MAPK pathway would have any protective effect on plasma membrane CFTR after exposure to CSE.

The human bronchial epithelial cells 16HBE14o- were incubated simultaneously with CSE and the MEK/Erk1/2 inhibitor UO126 and CFTR present at the plasma membrane was assessed using cell surface biotinylation. Not only inhibition of the MEK/Erk1/2 MAPK pathway prevented decrease of CFTR protein , it also prevented the loss of CFTR from the plasma membrane of airway epithelial cells. Inhibition of the MEK/Erk1/2 MAPK pathway prevents loss of plasma membrane CFTR after cigarette smoke exposure(A) and (B) 16HBE14o- cells were treated with 10% Camel cigarette smoke extract (CSE) with or without UO126 (10 µM) for 48 hrs. CFTR expression (total (A) or plasma membrane (B)) was detected as described in Methods.

CTRL, Control. (C) Primary human bronchial epithelial cells were pretreated with 10 µM UO126 and then exposed to air or cigarette smoke (CS) as described in Methods section. ASL was measured at the indicated time. N=6 from two normal donors.

Inhibition of the MEK MAPK pathway prevents cigarette smoke-induced decrease of airway surface liquid (ASL)CFTR is present at the plasma membrane of bronchial epithelial cells and regulates hydration of the airway surface liquid layer. As seen in cystic fibrosis, absence of functional CFTR results in impaired mucociliary clearance due to reduced ASL. We recently showed that cigarette smoke decreases expression of membrane CFTR in primary human bronchial epithelial cells resulting in impaired ASL. As shown above , inhibition of the MEK MAPK pathway prevented the decrease of plasma membrane CFTR. Accordingly, inhibition of the MEK pathway using the specific inhibitor UO126 prevented the reduction in the height of the ASL observed upon exposure to cigarette smoke. Role of the E3 ligase c-Cbl on CFTR expression after exposure to cigarette smokeUbiquitination of CFTR can lead to proteasomal or lysosomal degradation. The E3 ubiquitin ligase c-Cbl has been shown to be linked to lysosomal degradation of CFTR in airway epithelial cells ,.

To determine whether c-Cbl is involved in the CSE-induced degradation of CFTR, we used c-Cbl siRNA to decrease c-Cbl abundance. As shown in, transfection of c-Cbl siRNA reduced c-Cbl expression by 77%.

The CFTR expression was comparable between the control and c-Cbl siRNA groups in absence of CSE treatment. Conversely, addition of CSE significantly decreased the expression of CFTR in the control group, whereas suppression of c-Cbl expression using siRNA partly prevented the CSE-induced down-regulation of CFTR. These data indicate that the E3 ligase c-Cbl contributes to suppression of CFTR upon cigarette smoke exposure. Inhibition of the MEK/Erk1/2 pathway prevents lysosomal degradation of CFTRLysosome-associated membrane protein 1 (LAMP-1) is a marker of the lysosomes and was used to determine the intracellular localization of CFTR. As shown in and, using confocal microscopy the CFTR signal was reduced after exposure to CSE as expected.

No co-localization of CFTR with LAMP-1 was observed in 16HBE14o- cells in those conditions and could be due to CFTR degradation preventing its detection with the antibody used. However, inhibition of lysosmal degradation using chloroquine (CQ) allowed detection of CFTR in the lysosomes when the cells were exposed to CSE. The co-localization of CFTR with LAMP-1 was reduced in presence of the MEK/Erk1/2 inhibitor UO126. These results suggest that CSE induces lysosomal degradation of CFTR in human bronchial epithelial cells via activation of the MEK/Erk1/2 MAPK pathway. The antioxidant N-acetylcysteine (NAC) prevents the CSE-induced loss of CFTRCigarette smoke contains many chemical compounds as well as reactive oxygen species (ROS) that can trigger activation of signaling pathways such as MEK/Erk1/2. N-acetylcysteine (NAC) is an antioxidant that can inhibit ROS directly via the redox potential of its thiol or indirectly by increasing intracellular glutathione levels. We therefore investigated whether NAC could prevent the loss of CFTR after exposure to CSE.

Cells were treated with NAC and CSE simultaneously and CFTR expression was analyzed 24 hours later. As observed in, addition of 10 mM NAC prevented the loss of CFTR due to CSE exposure. NAC alone had no effect on CFTR expression. Since the data presented above show that CSE alters the expression of the CFTR protein via activation of the MEK/Erk1/2 MAPK pathway, we investigated whether NAC prevented the loss of CFTR after CSE exposure by blocking activation of Erk1/2 (phosphorylation). CSE induced activation of Erk1/2 as shown by detection of phosphorylated Erk1/2 which was inhibited in the presence of 10 mM NAC. A lower concentration of 2 mM NAC had little effect on CSE-induced decrease of CFTR or phosphorylation of Erk1/2. The antioxidant N-Acetylcysteine (NAC) prevents suppression of CFTR upon cigarette smoke exposure(A) 16HBE14o- cells were treated with 10% CSE with or without 0.5, 2, or 10 mM NAC.

CFTR protein was detected by immunoblotting. DISCUSSIONIn this study we investigated the molecular mechanism by which cigarette smoke suppresses expression of CFTR in human bronchial epithelial cells.

Our results revealed that cigarette smoke negatively regulates CFTR via activation of the MEK/Erk1/2 MAPK pathway. We found that cigarette smoke leads to internalization of the CFTR ion channel, and inhibition of the MEK/Erk1/2 MAPK pathway prevented the cigarette smoke-induced loss of CFTR as well as decreased of airway surface liquid (ASL). In addition we show that the antioxidant NAC prevented the loss of CFTR by inhibiting Erk1/2 phosphorylation.Dr. Welsh’s laboratory was the first to report that cigarette smoke inhibits chloride ion transport across tracheal epithelium. Several reports have shown that cigarette smoke inhibits the expression and function of the ion channels CFTR and ENaC and recently, a cigarette smoke-induced increase in intracellular calcium has been implicated in CFTR internalization. However, key questions remain regarding the molecular pathway leading to CFTR deregulation. In this study, we used normal bronchial epithelial cell line 16HBE14o- which endogenously expresses CFTR since many studies used cells derived from cancer or heterologous systems with cells overexpressing CFTR.

Consistent with a previous study where Bafilomycin A1 prevented CFTR inhibition, here we show that the lysosomal inhibitors, chloroquine and leupeptin, prevented the loss of CFTR, whereas inhibition of the proteasome had no effect. However, CFTR could not be detected in the lysosomes using immunohistochemistry in presence of CSE and could be due to CFTR degradation preventing its detection. Absence of co-localization of CFTR and LAMP-1 were previously reported even though lysosomes have been shown to contribute to CFTR degradation. Conversely, addition of the lysosmal inhibitor chloroquine, which prevents acidification of the lysosomes, allowed co-localization of CFTR with the lysosomal marker LAMP-1 (see ).

Bafilomycin A1 inhibits smoke-induced calcium release and also prevents CFTR diminution. Thus, sorting out effects caused by altered lysosomal calcium release versus inhibition of lysosomal degradation are hard to differentiate and additional studies will be required. Interestingly, the heavy metal arsenic has been shown to trigger lysosomal degradation of the CFTR ion channel in airway epithelial cells ,. Taken together, these results suggest that targeting the lysosomes would prevent CFTR degradation upon exposure to pollutants.Lysosmal degradation of membrane proteins is generally associated with monoubiquitination. C-Cbl is an E3 ligase previously reported to facilitate the lysosomal degradation of CD5, gp130, as well as CFTR ,.

However, decreasing the expression of c-Cbl using silencing RNA prevented the CSE-induced suppression of CFTR suggesting that c-Cbl contributes to regulation of CFTR in human bronchial epithelial cells.The epithelial sodium channel ENaC which interacts with CFTR in bronchial epithelial cells is degraded by the lysosomes after activation of the MEK/Erk1/2 MAPK pathway by interacting with the E3 ligase Nedd4-2 –. Here we show that pharmacological inhibition of MEK using UO126 or PD98059, or genetic inhibition of Erk1/2 using siRNAs prevented the cigarette smoke-induced suppression of CFTR.

Most importantly, inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of CFTR from the plasma membrane of the human bronchial epithelial cells 16HBE and most importantly prevented the cigarette smoke-induced decreased of ASL. This is an important finding since CFTR needs to be present at the apical membrane of airway epithelial cells to play its role as a chloride channel to maintain fluid homeostasis in the lung.

Activation of the Erk1/2 pathway by the pollutant cadmium was reported to increase CFTR activity in kidney cells. In this latter study, CFTR activity was measured 5 minutes after exposure to cadmium. It is therefore possible that activation of Erk1/2 has distinct effects depending of the type of cells studied (renal versus airway epithelial cells) and/or the time after MEK/Erk1/2 activation (short-term versus long-term). Since both CFTR and ENaC are downregulated following activation of the MEK/Erk1/2 MAPK pathway, it might be an unrecognized pathway to regulate plasma membrane ion channels.About 20% of smokers develop chronic obstructive pulmonary disease (COPD) but over 90–95% of patients with COPD were smokers. Some reports indicate that as many as 50% of smokers develop COPD if an advanced age is reached.

Cigarette smoke contains over 3,000 chemicals as well as reactive oxygen species that can lead to activation of several signaling pathways including the MEK/Erk1/2 MAPK pathway. For example our team has recently showed that cadmium, a toxic heavy metal present in cigarette smoke, induces secretion of the pro-inflammatory cytokine IL-8 via an Erk1/2-dependent pathway when added to human bronchial epithelial cells.

Interestingly, sustained activation of Erk1/2 has been found in mice and rats exposed to cigarette smoke ,. Most importantly, analysis of phospho-Erk1/2 revealed that patients with COPD (emphysema) have elevated Erk activation when compared to healthy control subjects ,. Based on our results we predict that sustained activation of Erk in the lung will contribute to suppression of CFTR expression.NAC has been used in patients with COPD with mixed success ,. Recently it was shown that higher doses might be required to obtain beneficial effects. Several studies reported that long-term high dose of NAC treatment may reduce the risk of exacerbations and improve lung function (FEV 1) ,. The doses used in our study are within the range of doses used in clinical practice (4–10 mM daily). We observed that 2 mM NAC had very little inhibitory effect on activation of Erk1/2 MAPK pathway, whereas 10 mM prevented activation of Erk1/2 and consequently loss of CFTR protein (see ).

It is important to note that the cells were not pre-treated with NAC so the protective effect of NAC is not due to increased levels of glutathione but rather by acting directly as an antioxidant. In addition, the inhibition of Erk1/2 activation was seen only after 5–10 minutes (see ). Interestingly, Varelogianni et al. Reported that NAC increases chloride efflux via activation of the CFTR chloride channel in human bronchial epithelial cells expressing the CFTR mutant deltaF508. This latter mutation is the most common mutation leading to cystic fibrosis (CF).

Since CF cells have higher Erk1/2 activation when compared to control non-CF cells it is possible that NAC could inhibit the MEK/Erk1/2 pathway in CF cells resulting in rescue of deltaF508-CFTR. Accordingly, a recent study identified kinase inhibitors, including inhibitors of the Ras/Raf/MEK/Erk1/2 pathway as potent correctors of deltaF508-CFTR.