Balb/C mice were exposed to Cl₂ gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl₂, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl₂ exposure.

Mice exposed to Cl₂ had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl₂ exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl₂-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl₂ prevented lipid peroxidation in the lung. Following Cl₂ exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl₂ exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU.

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.