Original paper
Does concentration influence viability of the bronchial epithelial cell line chronically exposed to antiasthmatic drugs?

Introduction

Asthma is the most common chronic respiratory disease in the pediatric population [1]. According to the current guidelines, chronic treatment of asthmatic patients is strongly recommended. However, the main problem in pediatric patients with asthma involves possible side effects that may appear after chronic treatment with anti-inflammatory agents (inhaled steroids) and bronchodilators (2-agonists and anticholinergic agents). Both groups of drugs are administered directly to the airways via inhalation of drug aerosols that provides the most efficient and safest method of drug delivery. Therefore, the epithelial cell layer, which is the first barrier that protects the underlying airways from the environmental influences, is directly exposed to the drugs.

It has been reported that airway epithelium plays an important role in the asthma pathogenesis. Bronchial biopsies performed in moderate and severe asthmatic patients show epithelial damage and metaplasia, thickening of the subepithelial basal lamina and an increased number of myofibroblasts [2, 3]. Those observations together with the other characteristics of airway remodeling were found not only in severe chronic asthma cases, but also in the airways of pediatric patients in relation to the disease onset [4-6]. Airway epithelium is chronically injured and unable to repair properly in asthmatic patients [7, 8] and the epithelial cell layer is more fragile due to the disruption of tight junctions and desmosomes [6, 9]. This results in the increased permeability, making airways more accessible to inhaled allergens, pollutants and other irritants (e.g. drug particles) [10].

The culture of epithelial cell layers to model the epithelia of the airway has undergone significant development for the last 10 years [11]. The 16HBE14o- cell line has been used in drug permeability and transport studies and it was reported that cells permeability correlates with the absorption coefficients observed in the isolated perfused rat lung [12]. The cells possess a columnar phenotype with apical microvilli and form functional tight junctions [13-16]. All these features support the use of this cell line in the analysis of changes in the morphology and physiology of airway epithelium chronically exposed to antiasthmatic drugs [1].

Commercially available anti-inflammatory and bronchodilatatory drugs are widely used in the treatment of asthma, however, their effects on viability and morphology of the airway epithelial cells have not been fully understood.

Aim

We aimed to investigate the effects of drugs routinely used in the asthma treatment on the bronchial epithelium in vitro under chronic exposure to different concentrations of drug solutions [17].

Material and methods

Chemicals and reagents



Fluticasone propionate and salbutamol were purchased from GlaxoSmithKline (Poland), budesonide from Astra Zeneca (Poland) and ipratropium bromide from Boehringer Ingelheim (Poland). Minimal Essential Medium (MEM), 10% Fetal Bovine Serum (FBS), L-glutamine, antibiotics (penicillin and streptomycin), Hanks’ balanced salt solution (HBSS), trypsin were purchased from Sigma (Poznan, Poland). LHC (Lechner and Laveck Medium) basal medium was purchased from Invitrogen; Bovine Serum Albumin (BSA), bovine collagen I and human fibronectin were purchased from BD Biosciences. Cell culture supports (flasks, Petri dishes, 6-well plates) were purchased from BD Falcon.



Cell culture



The simian virus (SV) 40-transformed bronchial epithelial cell line 16HBE14o- was purchased from Dieter C. Gruenert (California Pacific Medical Center, San Francisco, USA). Cell stocks were grown in monolayer in 25 and 75 cm² flasks in Minimal Essential Medium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS),

2 mmol/l L-glutamine and 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 95% air: 5% CO2 and subcultured weekly using 0.22% EDTA/0.25% trypsin. For the experiment, the cells were seeded at an initial density of 1.0 × 106 cells/8.55 cm² in total volume of 1 ml

(0.5 ml of MEM and 0.5 ml of drug solution) on 35 mm Petri dishes/6-well plates coated with the mixture containing LHC basal medium, 0.1% BSA (1 mg/ml), bovine collagen I (3 mg/ml) and human fibronectin (1 mg/ml).



Study design



We analyzed the influence of four drug solutions at different concentrations with the highest concentration being a half of the maximum dose recommended for the treatment of pediatric asthmatic patients according to GINA recommendations. The further concentrations were 2-fold serial dilutions of the highest concentration. The following drugs in four concentrations were analyzed:

• budesonide (Astra Zeneca): 0.25 mg/ml (5.8 × 10–5 M), 0.125 mg/ml (2.9 × 10–5 M), 0.0675 mg/ml (0.45 × 10–5 M), 0.03375 mg/ml (0.72 × 10–5 M);

• fluticasone propionate (GlaxoSmithKline): 0.5 mg/ml (1 × 10–6 M), 0.25 mg/ml (0.5 × 10–6 M), 0.125 mg/ml (0.25 × 10–6 M), 0.0675 mg/ml (0.125 × 10–6 M);

• salbutamol (GlaxoSmithKline): 1 mg/ml (4.2 × 10–3 M), 0.5 mg/ml (2.1 × 10–3 M), 0.25 mg/ml (1.05 × 10–3 M), 0.125 mg/ml (0.52 × 10–3 M);

• ipratropium bromide (Boehringer Ingelheim): 0.125 mg/ml (2.2 × 10–4 M), 0.0625 mg/ml (1.1 × 10–4 M), 0.03125 mg/ml (1.1 × 10–4 M), 0.015625 mg/ml (0.28 × 10–4 M).

The control cells were cultured in the same manner in MEM with PBS instead of drug solution.

Cells were continuously exposed to the drugs that were added every 24 h during medium change. Observations of cells were performed every 24 h for 4 days and the viability test was performed after 4 days of incubation with different drugs or with PBS in the control cells. All experiments were done in 8 replicates.



XTT viability test



The viability of cells was analyzed using the XTT colorimetric test, based on dynamics of reduction of XTT

pigment (tetrazoline-2,3-bis(2-methoxy-4-nitro-5-sulpho­phenyl)-2H-5-carboxyanilide) by the living cells and production of the colorful product. Intensity of the orange color formed in the reaction was measured at the wavelength of 450 nm. The percentage of growth inhibition was calculated according to the following equation:

100 – (OD of drug treated cells – OD of medium alone/OD of untreated cells-OD medium alone × 100), where OD is optical density.



Statistical analysis



Statistical analysis of the results was done with a non-parametric method (Kruskal-Wallis test) with post-hoc Dunn’s test using the Statistica ver.8 software (Statsoft, Krakow, Poland) and the demo version of GraphPad Instat3 software. Value of p < 0.05 was considered to represent the threshold of significance. After adjusting the significance level for multiple comparisons using Bonferroni’s correction, the corrected significance level was less than 0.008.



Fluorescence microscopy



Morphological evaluation and viability of epithelial cells 4 days after exposure to drug solutions was done with fluorescent dyes (Hoechst 33342, propidium iodide) and analyzed with fluorescent microscope (Eclipse TE200 Nikon). Apoptotic cells were identified as brightly fluorescing Hoechst 33342 stained cells (blue staining) displaying shrinkage with condensed, segmented nuclei. The necrotic cells were identified by brightly fluorescent Hoechst stained nuclei independent of chromatin condensation, coupled with PI fluorescence (red staining).



Ultrastructural analysis



After 72 h, cells allocated to ultrastructural analysis were fixed in situ directly in culture dishes with use of 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer pH 7.4. After that, cells were rinsed twice in phosphate buffer and the material was post-fixed for an hour in 1% of osmium tetroxide in phosphate buffer. After rinsing cells twice in phosphate buffer and distilled water, they were incubated for an hour with 0.5% uranyl acetate. After that, the material was washed once in distilled water and then dehydrated through a graded ethanol/HPMA series (Hydroxypropyl Methacrylate) and embedded in culture dishes with use of epon. Material prepared according to the above protocol was cut for ultrathin sections with use of Ultracut III microtome (Reichert) and stained with uranyl acetate and lead citrate. For ultrastructure examination, a transmission electron microscope JEM-100 S (Jeol, Japan) was used. All reagents and chemicals mentioned above were purchased from Sigma Aldrich.



Calcium measurement



Intracellular Ca2+ concentration in the cultured cells was performed with Fluo3 (Molecular Probes, USA) with use of LSM 510 confocal microscope (Zeiss, Germany).

Results

All the analyzed drugs in each concentration and its effect on epithelial cells viability was shown in Figure 1.

For the comparisons to the control cells, all drugs at each concentration were significantly different between the drug and the control cells. To test whether the differences between drug concentrations were significant we performed multiple comparisons between each drug concentration and the results are given in Table 1.

Budesonide at the highest concentration (0.25 mg/ml) led to the evident decrease in cells viability; however, for the drug concentration of 0.125 mg/ml and lower we did not observe such changes. The cells viability was inversely correlated to the drug concentration and those changes were significant in comparisons between all the concentrations except the highest and the lowest concentrations (Figure 1 and Table 1).

Changes in cells viability exposed to fluticasone propionate at different concentrations were significant (Table 1 and Figure 1) and after correction for multiple testing, the significance was observed for the following comparisons: 0.5 mg/ml vs. 0.125 mg/ml, 0.5 mg/ml vs. 0.067 mg/ml, 0.25 mg/ml vs. 0.067 mg/ml.

Incubation with salbutamol at different concentrations influenced cells viability at the concentrations of

1 mg/ml and 0.5 mg/ml (Table 1 and Figure 1) and the pattern of decreased viability with increasing concentration was similar to that observed for fluticasone propionate.

Cells exposed to ipratropium bromide showed an extremely high decrease in cells viability associated with necrosis and subsequent cells dissociation from the culture dish surface. The decreased cells viability was demonstrated for all concentrations with the highest concentration of 0.125 mg/ml being significantly more toxic in comparison to lower concentrations (Table 1 and Figure 1).

Ultrastructure of control cells and cells exposed to studied drug solutions did not differ significantly. However, in the case of cells exposed to salbutamol and fluticasone propionate, we observed alterations in cell junctions. In control cells, tight junctions with numerous desmosomes were observed, whereas cells exposed to drug solutions mentioned above lacked tight intercellular connections and we observed free spaces between the cells.

We also analyzed whether free calcium ions concentration in cultured cells may be altered due to exposure to different drug solutions. None of the analyzed drugs influenced free calcium ions concentration suggesting that this pathway may not be crucial for any of the drug action (data not shown).

Discussion

In our study we demonstrated that chronic exposure of bronchial epithelium to antiasthmatic drugs resulted in decreased cell viability of exposed cells as compared to the control cells. Moreover, decreasing drug concentration positively correlated with cell viability, with higher concentrations significantly more toxic in comparison to lower doses. Interestingly, the most cytotoxic drug was ipratropium bromide regardless of its concentration.

Chronic exposure to steroids at two highest concentrations resulted in a decrease in cell viability; however, this decrease was reversible after drugs were removed from the cell culture. Our observations for epithelial cells under chronic exposure to steroids are in concordance with the analysis performed after inhalation of a single dose of beclomethasone that resulted in mild damage of bronchial epithelium and moderate damage of tracheal epithelium [18]. Moreover, in our study we observed that budesonide was significantly more toxic than fluticasone propionate. Comparing our results to the other studies performed in vitro, it was reported by Stellato et al. [19] that glucocorticoids might be inhibitors of the epithelial cell function (in the range of concentrations from 10–13 mol/l to 10–6 mol/l) with fluticasone propionate being more potent than budesonide to inhibit VCAM-1 expression. In their study it was also reported that epithelial cells are the most sensitive cells to steroids in comparison to the other cells such as eosinophils and basophils. The different outcome for steroids cytotoxicity in our results and the study by Stellato may result from the different time of exposure (24 h in the study by Stellato as compared to 4 days in our experiment) as well as from different drug concentrations and readout (VCAM-1 expression).

In several studies it was reported that salbutamol at the concentration of 1 × 10–7 M added to the human bronchial epithelial cell culture for 48 h was as potent as 10% serum in stimulating cell growth [20, 21]. In contrast to the short-acting 2-agonist salbutamol, a consistent pro-apoptotic effect was observed with the long-acting 2-agonists, salmeterol and formoterol [22]. Our results demonstrated that chronic exposure (4 days) to salbutamol was associated with the decrease in cell viability for the two highest concentrations (1 mg/ml and 0.5 mg/ml). One of the possible explanations of this discrepancy is that chronic exposure to 2-adrenergic receptor agonists may affect cells proliferation via an increase in the cAMP level that may be responsible for G1 cell cycle arrest by inducing cyclin-dependent kinase inhibitor p27Kip1 [23]. Also, the cAMP increase contributes to downregulation of Bcl-2 expression and acceleration of cell apoptosis in asthmatics [24].

To our knowledge, there are no studies analyzing the influence of ipratropium bromide on decreased viability of epithelial cells, therefore we cannot verify or discuss our results on chronic exposure of ipratropium bromide on decrease in cells viability, regardless of the drug concentration.

The results regarding cells viability were obtained by two independent methods: fluorescent staining with Hoechst and propidium iodide and with use of colorimetric XTT test as a reference method. We also tried to apply flow cytometry in our analysis, however due to the presence of different cell subpopulations it was not possible to separate and count those cells.

As for research on respiratory epithelial permeability to drugs, a correlation between the in vitro model and in vivo conditions was observed [25]. However, the correlation between in vitro model and in vivo conditions in the cytotoxicity studies is significantly less obvious. Drug solutions at given concentrations in vitro have different concentrations and impact on epithelial cells in vivo where they are protected by several barriers, particularly the mucus layer rich in different protective substances and cells. Airway geometry, humidity, clearance mechanisms and presence of lung disease influence the drug deposition and therefore affect the therapeutic effectiveness of inhaled medications. Moreover, only 15-20% of the drug dose in the form of aerosol penetrates the lung, so the concentration of the drug targeting epithelial cells in vivo is lower than given in inhalation by both the bioavailability as well as limited inhalation efficiency. Therefore, our results may indicate only the need to undertake research on cytotoxicity of the inhaled drugs on respiratory epithelial cells, but for now it cannot be related directly to the clinical situation. Our study has been performed on the established cell line and requires verification in human bronchial primary epithelial cells before we can correlate our results to the in vivo situation.

Conclusions

In summary, despite different limitations of this study, our main observation that chronic exposure to lower drugs concentrations is generally less harmful for the epithelial cells, this finding can be considered as the treatment schedule is planned. However, these are preliminary results that need further confirmation before they can be used in clinical practice.

Acknowledgments

Dominika Oleś and Aleksandra Szczepankiewicz had an equal share in the preparation of this work. This study was supported by the Poznan University of Medical Sciences, grant no. 502-14-01105726-09185

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