Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma

Male Balb/C mice (18-22 g) were purchased from Charles River (Wilmington, Massachusetts) and housed in a conventional animal facility at McGill University. Animals were treated according to guidelines of the Canadian Council for Animal Care and protocols were approved by the Animal Care Committee of McGill University.

Mice were exposed to either room air (control) or Cl₂ gas diluted in room air for 5 minutes using a nose-only exposure chamber. An initial experiment was performed to assess an exposure level required to effect changes in airway responsiveness to methacholine (MCh) that was well tolerated by the animals. For this purpose we exposed mice to 100, 200 or 400 ppm Cl₂, and 24 hours later we performed MCh challenge and removed the lungs for histological analysis. Based on the results of this experiment we tested the effects of DMTU on animals exposed to 100 ppm Cl₂. The control mice were exposed to room air (Control; n = 6) and test mice were exposed to Cl₂ (Cl₂; 100 ppm; n = 6) with DMTU (100 mg/kg) treatment intraperitoneally either one hour before (DMTU/Cl₂; n = 6) or one hour after Cl₂ exposure (Cl₂/DMTU; n = 6). DMTU was prepared fresh prior to each exposure and a dose of 100 mg/kg in 500 μL of sterile phosphate buffered saline (PBS) was administered i.p. either one hour before or one hour following exposure to Cl₂. Control (air exposed) mice received 500 μL PBS i.p and Cl₂ exposed mice received 500 μL PBS i.p. either one hour before or one hour following exposure. We chose the dose of DMTU based on previous observations of efficacy against an oxidant pollutant in mice [11]. At 24 hours after Cl₂ exposure, lung function measurements including responsiveness to aerosolized MCh were performed and bronchoalveolar lavage fluid was obtained for assessment of inflammatory cell counts, total protein, nitrate/nitrite (nitric oxide) and glutathione levels. The lungs were removed for analysis of carbonylated proteins and 4-hydroxynonenal (4-HNE). Measurements of inflammatory cell counts and glutathione levels in BAL fluid were made also at 10 min and at 1 hr after Cl₂. Following exposure animals were returned to the animal facility and allowed food and water ad libitum.

Mice were restrained and exposed to Cl₂ gas for 5 minutes using a nose-only exposure device. Cl₂ gas was mixed with room air using a standardized calibrator (VICI Metronics, Dynacalibrator^®, model 230-28A). The Cl₂ delivery system has two main components, a gas generator, which includes a heated permeation chamber and air flow generator. Dynacal permeation tubes designed specifically for operation with the Dynacalibrator were used and contain the Cl₂. The permeation chamber and air flow generator control accuracy of the Cl₂ generated to within 1-3% of the desired concentration (manufacturer's specifications). Within the gas chamber, permeation tubes containing Cl₂ are housed for gas delivery. The Teflon permeation tubes contain Cl₂ in both gas and liquid phases. When the tube is heated the Cl₂ reaches a constant and increased vapor pressure and it permeates the tube at a constant rate. The desired concentration is delivered at an appropriate flow rate, as specified by the manufacturer. The device is attached to the exposure chamber and allowed to calibrate for 30 minutes until the optimum temperature of 30°C is reached and the Cl₂ flow is constant. Following removal of the animals from the exposure chamber, the chamber was continually flushed with the gas mix to ensure that the desired concentration of Cl₂ was maintained.

Mice were sedated with an intraperitoneal (i.p) injection of xylazine hydrochloride (8 mg/kg) and anaesthetized with i.p. injection of pentobarbital (30 mg/kg). Subsequently, the animal was tracheostomized using at 18 gauge cannula and connected to a small animal ventilator (FlexiVent, Scireq, Montreal, Canada). Muscle paralysis was induced with pancuronium bromide (0.2 mg/kg i.p.). The mice were ventilated in a quasi-sinusoidal fashion with the following settings: a tidal volume of 10 mL/kg, maximum inflation pressure of 30 cmH₂0, a positive end expiratory pressure (PEEP) of 3 cmH₂0 and a frequency of 150/min. Following an equilibration period of 3 minutes of tidal ventilation two lung inflations to a transrespiratory pressure of 25 cm H₂O were performed and baseline measurements were taken. The respiratory mechanics were estimated using a single compartment model and commercial software (Scireq). Baseline was established as the average of three perturbations. Following establishment of baseline, MCh was administered using an in-line nebulizer (Aeroneb Lab, standard mist model, Aerogen Ltd, Ireland) and progressively doubling concentrations ranging from 6.25 to 50 mg/ml were administered over 10 seconds synchronously with inspiration. Six perturbations were calculated at each dose of MCh to establish the peak response. The highest value was kept for analysis subject to a coefficient of determination above 0.85. Respiratory system resistance (Rrs) and respiratory system elastance (Edyn) were determined before challenge and after each dose of MCh.

Following euthanasia (60 mg/kg pentabarbital, i.p.), the lungs were lavaged with 600 μl of sterile saline, followed by four separate aliquots of 1 ml each as previously described [7]. The first 600 μlmL aliquot of BAL fluid was centrifuged at 1500 rpm for 5 minutes at 4°C and the supernatant was retained for measurements of nitric oxide, glutathione levels and protein levels using a Bradford Assay. The separate 1 mL aliquots were spun at 1500 rpm for 5 min at 4°C and the supernatant removed. The cell pellets were pooled for differential cell counts using 100 μl of the re-suspended cells. Cytospins were prepared, air-dried and stained (Diff-Quik^® method, Medical Diagnostics, Düdingen, Germany). A differential cell count was determined on a minimum of 300 cells.

Following harvesting, the lungs were perfused with saline until the effluent was clear. The right lung was inflated with 1 mL 10% buffered formalin, fixed overnight with formalin. Tissues were embedded in paraffin blocks, cut into 5 μm sections and stained with hematoxylin and eosin. Sections were evaluated for epithelial morphological changes. The absolute number of epithelial cells in the airways was determined by counting cells on hematoxylin and eosin stained slides at 200× magnification and data were expressed as the number of epithelial cells per mm of basement membrane perimeter (P_BM). Epithelial cell height was determined by measuring the distance between the basement membrane and the top of the epithelial cell in the four quadrants for each airway and averaged.

For the evaluation of nitric oxide, 0.6 N trichloroacetic acid was added to the supernatant of the BAL fluid to give a final concentration of 0.12 N to precipitate any protein. Samples were centrifuged for 10 minutes at 10,000 RPM followed by removal of the supernatant for analysis using previously described methods [7]. Total NO_x was measured in BAL as an index of NO production using the Griess reaction. Briefly, 80 μl of sample were pre-incubated with 20 μl of NO₃ reductase and 10 μl of its enzyme cofactor for 3 h at room temperature and then incubated with 100 μl of Griess reagent for 10 min. NO_x concentrations were determined using standard curves obtained from different concentrations of NaNO₂ or NaNO₃. Absorbance was measured at 540 nm with a plate reader (SLT 400 ATC; SLT Lab Instruments, Salzburg, Austria). No NO_x was detected in saline solutions using this assay.

An Oxyblot was performed on left lung tissue extracts taken 24 hours following Cl₂ challenge. Extracted proteins were denatured with 12% sodium dodecylsulfate (SDS) before derivatization with the addition of DNPH (2,4-dinitrophenylhydrazone-hydrazone). DNPH-derivatised proteins were separated on a 10% SDS-PAGE gel at 140 V for 2 h. Proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane with 11.6 mM Tris (Fisher), 95.9 mM glycine (Fisher) and 20% methanol (Fisher) at 25 V for 2 h. Membranes were then blocked with 1% bovine serum albumin-TTBS solution (0.02 M Tris base, 0.5 M NaCl, and 0.1% of Tween 20; Sigma) and were probed for 90 min with rabbit anti-DNP antibody (Intergen Company, Purchase, NY). The membranes were then rinsed in TTBS and incubated with HRP-conjugated goat anti-rabbit IgG (Intergen Company, Purchase, NY) for 1 h. A chemiluminescence detection system (ECL Plus; Amersham), Hyperfilm (Amersham), and Fluorochem 8000 software (Alpha Innotech Corporation, San Leandro, CA) were used for antibody detection and quantification by densitometry.

All reagents were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Frozen tissue, or a known amount of 4- HNE standard (Cayman Chemical, Ann Arbor, MI, USA), was placed in 2 ml of cold methanol (Thermo Fisher) containing 50 μg/ml butylated hydroxytoluene, with 10 ng d3-4-HNE (Cayman Chemical) internal standard added just before homogenization with the Ultra-Turrax T25 (Thermo Fisher). An EDTA solution (1 ml of 0.2 M, pH 7) was added. Derivatization was accomplished by the addition of 0.2 ml of 0.1 M HEPES containing 50 mM O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, pH 6.5. The mixture was then vortexed and held at room temperature. After 5 min, 1 ml of hexanes (Thermo Fisher) was added, and the samples were shaken vigorously. Brief centrifugation was performed to achieve phase separation and the O-pentafluorobenzyl-oxime derivatives were extracted from the upper hexane layer. The sample was dried under a stream of N2 gas and further derivatized into trimethylsilyl ethers by the addition of 15 μl each of pyridine and N, O bis(trimethylsilyl)trifluoroacetamide. The samples were vortexed and heated to 80°C for 5 min and then analyzed for 4-HNE content by GC/MS. GC/MS analysis was performed using a Focus GC coupled to a DSQ II mass spectrometer and an AS 3000 autosampler (Thermo Fisher).A15-m TR-5MS column (0.25-mm i.d., 0.25-μm film thickness; Thermo Fisher) was used with ultrahigh-purity helium as the carrier gas at a constant flow rate of 1.0 ml/min. Two microliters of sample was injected into the 270°C inlet using split mode with an injection ratio of 10 and a split flow of 10 ml/min. The initial oven temperature was 100°C and then ramped to 200°C at 15°C/min, followed by an increase in temperature to 300°C at 30°C/min, and held for 1 min. The MS transfer line temperature was held constant at 250°C and the quadrupole at 180°C. Analysis was done by negative-ion chemical ionization using 2.5 ml/min methane reagent gas. Ions were detected using SIM mode with a dwell time of 15.0 ms for each fragment of 4-HNE at m/z 152, 283, and 303, and d3-4-HNE at m/z 153, 286, and 306. Under these conditions, the larger, second peak of the two 4-HNE isomers was used for quantification and exhibited a retention time of 7.18 min, which was just preceded by the elution of d3-4-HNE at 7.17 min. Quantification was performed using a standard curve generated by graphing the area ratio of 4-HNE to d3-4-HNE versus concentration.

BAL fluid from control, chlorine exposed and DMTU pre-treated chlorine exposed mice was collected for glutathione evaluation by HPLC. Both glutathione (GSH) and glutathione disulfide (GSSG) were measured to determine if GSH had converted to GSSG. As GSH is found almost exclusively in its reduced form, a conversion to GSSG, which his inducible following oxidative stress, would indicate an increase in oxidative stress in the lung. BAL samples were collected at 10 minutes, one hour and 24 hours after Cl₂ challenge. Phosphoric acid (60 μL; 1 M) was added to BALF samples to prevent GSH degradation. BAL was centrifuged at 1500 RPM for 5 minutes, and the supernatant was removed for evaluation of extracellular GSH/GSSG and 150 μL of PBS and 15 μL 1 M phosphoric acid added was used to reconstitute the pellet for analysis of intracellular GSH and GSSG. CHAPPS (150 μL; 6 mM) was added to lyse the cells. GSH and GSSG were measured by RP-HPLC using a post-column derivatization procedure modified from the literature [14]. GSH and GSSG levels were determined in 50 μl aliquots by RP-HPLC using a gradient prepared from 0.05% trifluoroacetic acid (TFA) in water (solvent C) and 0.05% TFA in acetonitrile (solvent D) as follows: 0 min, 0% D; 10 min, 15% D. The flow rate was 1 ml/min and the stationery phase was a column (150 × 4.6 mm) of Ultracarb ODS (31% carbon loading; 5 μm particle size; 150 × 4.6 mm; Phenomenex, Torrance, CA). The eluate from the column was mixed with o-phthalaldehyde (370 μM) in 0.2 M tribasic sodium phosphate, pH 12, which was pumped into a T-fitting using an auxiliary pump (Waters Reagent Manager). The mixture then passed through a loop of PEEK tubing (6 m × 0.5 mm, i.d.; volume, 1.2 ml) that was placed in a water bath at 70°C. Under these conditions both GSH and GSSG are converted to a fluorescent isoindole adduct, which is measured using excitation and emission wavelengths of 336 and 420 nm, respectively. Prior to introduction into the fluorescence detector (Waters model 2475 Multi wavelength Fluorescence Detector), the mixture was cooled in a small ice-water bath and passed through a filter containing an OptiSolv 0.2 μm frit (Optimize Technologies). The amounts of GSH and GSSG were determined from a standard curve using the authentic compounds as external standards.

To establish a suitable submaximal concentration of Cl₂ for subsequent experiments animals were exposed to 100 ppm, 200 ppm or 400 ppm of Cl₂ for 5 minutes. The next day, the animals were challenged with doubling doses of MCh ranging from 6.25 to 50 mg/ml. Respiratory system resistance (Figure 1A) and elastance (Figure 1B) were evaluated. There was a dose-dependent increase in responsiveness to MCh reflected in both of the above parameters of lung function.

The effects of Cl₂ on airway architecture were assessed on hematoxylin and eosin stained lung sections obtained 24 hours after exposure (Figure 2). Lower concentrations of Cl₂ (100 ppm and 200 ppm) did not result in any detectable change under light microscopy to the airway epithelium (Figure 2A and 2B). There was an obvious thinning of the airway epithelium at a concentration of 400 ppm (Figure 2C). There were statistically significant differences observed in epithelial cell height caused by exposure to Cl₂ (Fig 2E). We also quantified the number of epithelial cells in the airway walls. While there was no significant difference in cell following exposure to Cl₂ at 100 ppm compared to control (Figure 2D), at 400 ppm, there were fewer epithelial cells compared to both control and 100 ppm (Fig 2D). Given the lack of gross histological change induced by 100 ppm of Cl₂ we chose to perform further studies using this concentration.

Airway responses to increasing doses of MCh (6.25-50 mg/ml) were elevated 24 h following Cl₂ challenge (Figure 3A). This effect was attenuated by administration of DMTU given both prior to and post Cl₂-exposure. Changes in respiratory system elastance in response to MCh paralleled those observed for resistance (Figure 3B). DMTU alone had no significant effect on MCh responsiveness.

To assess the effects of Cl₂ on airway inflammation and epithelial cell shedding bronchoalveolar lavage was performed at 10 minutes, one hour and at 24 hours after Cl₂ exposure. The fluid recovered by BAL averaged 75% of the volume instilled and did not differ significantly among the groups. Total cell counts were not significantly different at 10 minutes after exposure to Cl₂ (Figure 4A) but were significantly increased in Cl₂ treated groups by one and 24 hours compared to control (Figure 4B and 4C). At one hour, pre-treatment with DMTU reduced the total number of inflammatory cells present in the airways compared to Cl₂ only mice. At 24 hours, total cell counts were persistently elevated after Cl₂ and were attenuated only in mice post-treated with DMTU after Cl₂ exposure (Figure 4C). Cl₂ caused a significant increase in neutrophils and lymphocytes 24 hours following challenge, an effect attenuated by both pre- and post-treatment with DMTU (Figure 5C and 5D). There were no significant changes in any of the cell subsets at 10 mins (Figure 5A, B and 5E).

We measured the total protein level in BAL fluid harvested at 1 and 24 hours after Cl₂ exposure to assess the effects of Cl₂ on cell damage and protein levels. At both time points following Cl₂ exposure there was a significant increase in total protein in the BAL fluid as assessed by the Bradford assay. Treatment with DMTU, both before and after Cl₂ exposure reduced protein levels in BAL (Figure 6).

Nitric oxide concentrations were determined using the Griess reaction and no significant change was seen between any of groups 24 hours following Cl₂ challenge (Figure 7A). An OxyBlot was performed on lung extracts to detect proteins modified by oxygen metabolites 24 hours following Cl₂ exposure. Levels of carbonylation were quantified by densitometry and no substantial difference was seen among control, Cl₂ treated or DMTU treated animals (Figure 7B). Lungs were removed 24 hours following Cl₂ treatment for analysis of 4-HNE by GC-MS. Cl₂ induced a significant increase in 4-HNE levels (Figure 7C). DMTU given either pre- or post- Cl₂ treatment prevented any significant changes in 4-HNE levels (Figure 7C).

Cl₂ increased both intracellular (Figure 8A) and extracellular (Figure 8B) GSH levels in BAL after 10 min, but had no effect on GSH levels after 1 and 24 hours (Figure 8C and 8D). Treatment with DMTU prior to administration of Cl₂ blocked the increase in GSH in both compartments at 10 min (Figure 8A and 8B) but had no effect on GSH levels at the later time points (Figure 8C and 8D). Cl₂ induced a significant increase in GSSG levels in the intracellular and extracellular compartments at 10 min (Figure 9A and 9B). At 1 and 24 hours there was a decrease in GSSG levels in Cl₂ treated groups compared to control and DMTU treated groups that were restored by DMTU treatment (Figure 9C and 9D). The ratio of GSH/GSSG was significantly higher in the cell fraction of BAL in Cl₂ exposed mice than control and DMTU treated mice at 10 minutes (Figure 10A). There was a trend towards a decrease in GSH/GSSG ratio in the extracellular compartment of the BAL at the same time point, but this was not statistically significant. Additionally, at 24 hours, the GSH/GSSG ratio remained high in the Cl₂ treated mice but was attributable to a decline in GSSG at this time (Figure 10D). This effect was prevented by treatment with DMTU (Figure 10A and 10D).