Ascorbate and deferoxamine administration after chlorine exposure decrease mortality and lung injury in mice

Abstract

Chlorine (Cl(2)) gas exposure poses an environmental and occupational hazard that frequently results in acute lung injury. There is no effective treatment. We assessed the efficacy of antioxidants, administered after exposure, in decreasing mortality and lung injury in C57BL/6 mice exposed to 600 ppm of Cl(2) for 45 minutes and returned to room air. Ascorbate and deferoxamine were administered intramuscularly every 12 hours and by nose-only inhalation every 24 hours for 3 days starting after 1 hour after exposure. Control mice were exposed to Cl(2) and treated with vehicle (saline or water). Mortality was reduced fourfold in the treatment group compared with the control group (22 versus 78%; P = 0.007). Surviving animals in the treatment group had significantly lower protein concentrations, cell counts, and epithelial cells in their bronchoalveolar lavage (BAL). Lung tissue ascorbate correlated inversely with BAL protein as well as with the number of neutrophils and epithelial cells. In addition, lipid peroxidation was reduced threefold in the BAL of mice treated with ascorbate and deferoxamine when compared with the control group. Administration of ascorbate and deferoxamine reduces mortality and decreases lung injury through reduction of alveolar-capillary permeability, inflammation, and epithelial sloughing and lipid peroxidation.

Figure 1.

Continuous recordings of chlorine (Cl₂) concentrations in the exposure chamber. Six mice were placed in the glass chamber and breathed air for approximately 10 minutes. At time zero, one of the mass flow controllers was connected to a Cl₂ cylinder (1,000 ppm in air), while the other one remained connected to room air. The relative flow rates were adjusted to achieve a nominal concentration of 600 ppm. Cl₂ concentrations were monitored continuously with an Interscan Corporation (model RM34-1000 m) Cl₂ detector, connected to a data logger for data storage. After 45 minutes, the Cl₂ cylinder was switched off and the compressed air flow rate was increased to 5 L/min. Differences in Cl₂ profiles in the presence and absence of mice are probably due to adsorption and reaction of Cl₂ with animal fur. Shown are typical measurements that were repeated six times with identical results.

Figure 2.

Particle size distribution of aerosolized antioxidants. Cascade impactor samples were collected from the inhalation exposure plenum and analyzed using SigmaPlot software (see the online supplement for additional information). Particle size distribution is presented as mass median aerodynamic diameter (MMAD) and geometric SD (GSD) (both in microns). d ln d_ae, the change of the natural log of the aerodynamic equivalent diameter of the particles collected; d M/M_o, the change of mass fraction of particles collected on each stage of the impactor; M_o, total particle mass collected. Shown are results of a typical experiment that was repeated nine times with identical results.

Figure 3.

After-exposure antioxidants increase survival of Cl₂-exposed mice returned to room air. Mice were exposed to 600 ppm of Cl₂ for 45 minutes and returned to room air. Each data point shows the number of mice that were alive at the indicated time after Cl₂ exposure. The experimental group (gray line, solid circles; n = 18) received intramuscular injections of ascorbate (2 mg) and deferoxamine (0.3 mg) in saline starting at 1 hour after exposure and every 12 hours thereafter up to 60 hours after exposure. They also received aerosols of ascorbate (150 mg/ml) and deferoxamine (0.3577 mg/ml) at 1.5, 24, and 48 hours after exposure in sterile water, as described in the online supplement. The control group (black line, solid squares; n = 18) received vehicle (saline for intramuscular injections; sterile water for aerosols) instead of antioxidants using identical protocols. A total of 14 mice were alive at 72 hours after exposure in the antioxidant group, and 4 in the saline group, respectively. Data points were fitted with Kaplan-Meier survival curves and compared with the log-rank test (P = 0.0007).

Figure 4.

After-exposure antioxidants decreased total bronchoalveolar lavage (BAL) protein in mice exposed to Cl₂. Mice were exposed to 600 ppm of Cl₂ for 45 minutes and returned to room air and treated with either ascorbate and deferoxamine (Cl₂ Asc/Def) or vehicle (Cl₂ Vehicle), as described in Materials and Methods. Surviving mice were killed at 72 hours after Cl₂ exposure and used for these measurements. A third group of mice was exposed to air (Air Control) and did not receive antioxidants. Protein concentrations in cell-free BAL were measured by the BCA Protein Assay. Values are means (±SE). *P < 0.001 compared with the Air Control group; ^#P < 0.05 compared with the Cl₂ vehicle. Statistical analysis was performed after square root transformation of the data to meet assumption of normal distribution, followed by one-way ANOVA and Tukey multiple comparisons post test. The number of animals in each group was: air control, 18; Cl₂ Asc/Def, 9; Cl₂ vehicle, 4.

Figure 5.

After-exposure antioxidants decrease BAL albumin and IgM in mice exposed to Cl₂. Mice were exposed to 600 ppm of Cl₂ for 45 minutes and returned to room air and treated with either ascorbate and deferoxamine (Cl₂ Asc/Def) or vehicle (Cl₂ Vehicle), as described in Materials and Methods. Surviving mice were killed 72 hours after Cl₂ exposure and used for these measurements. A third group of mice was exposed to air (Air Control) and did not receive antioxidants. Equal volumes (40 μl) of BAL were separated by 10% SDS-PAGE and transferred polyvinylidene difluoride (PVDF) membranes and blotted for (A) albumin and (B) IgM. Bands were digitized and mean values are shown in C and D. Values of densitometry units are expressed as means (±SE). Shown are results of a typical experiment.

Figure 6.

After-exposure antioxidants decrease number of cells in BAL in mice exposed to Cl₂. Mice were exposed to 600 ppm of Cl₂ for 45 minutes, returned to room air, and treated with either ascorbate and deferoxamine (Cl₂ Asc/Def) or vehicle (Cl₂ Vehicle), as described in Materials and Methods. Surviving mice were killed 72 hours after Cl₂ exposure and used for these measurements. A third group of mice was not exposed to Cl₂, and did not receive any treatment serving as baseline. After being killed, the lungs of mice were lavaged and BAL samples were spun at 300 × g for 10 minutes at 4°C to pellet cells. (A) Total number of cells in BAL; (B) the indicated types of inflammatory and epithelial cells. Values are means (±SEM); number of animals in each group: (A) Air Control, 18; Cl₂ Asc/Def, 9; Cl₂ Vehicle, 4; (B) Air Control, 7; Cl₂ Asc/Def, 9; Cl₂ Vehicle, 4. Statistical analysis was performed after square root transformation of the data to meet assumption of normal distribution, followed by one-way ANOVA and Tukey multiple comparisons post test (A), and two-way ANOVA and Bonferroni post test. Values are expressed as means (±SE). *P < 0.001 compared with air control; ^#P < 0.001 compared with Cl₂ vehicle.

Figure 7.

BAL indices of inflammation and lung injury depend on ascorbate. Mice were exposed to 600 ppm of Cl₂ for 45 minutes and returned to room air and treated with either ascorbate and deferoxamine (Cl₂ Asc/Def) or vehicle (Cl₂ Vehicle) as described in Materials and Methods. Surviving mice were killed 72 hours after Cl₂ exposure and used for these measurements. Ascorbate levels in BAL and lung tissues were measured by HPLC as described previously (5). No statistical significant differences were found in either BAL or lung tissue ascorbate between these two groups of mice because of large variations. Therefore data were grouped and the presence of significant correlations among total cells (top left), neutrophils (top right), epithelial cell (bottom left), and total protein (bottom right) in the BAL and BAL ascorbate were examined using Spearman's rank correlation coefficient (ρ), derived from the actual data. Values of ρ and its P values are shown in each panel.

Figure 8.

Ascorbate and deferoxamine treatment decreased BAL lipid peroxidation in Cl₂-exposed mice. Mice were exposed to 600 ppm of Cl₂ for 45 minutes, returned to room air, and treated with either ascorbate and deferoxamine (Cl₂ Asc/Def) or vehicle (Cl₂ Vehicle), as described in Materials and Methods. Surviving mice were killed 72 hours after Cl₂ exposure and were used for these measurements. A third group of mice was exposed to air and did not receive any treatment. Equal amounts of proteins (5 μg) from cell-free BAL were separated by 10% SDS-PAGE and transferred to PVDF membranes. Lipid peroxidation was detected using a primary rabbit anti-malondialdehyde (MDA) antibody. Values of densitometry units are expressed as means (±SE). ^#P < 0.05 versus Cl₂ saline. Statistical analysis was performed after square root transformation of the data to meet assumption of normal distribution, followed by one-way ANOVA and Tukey multiple comparisons post test. Number of animals in each group was: Air Control, 2; Cl₂ Asc/Def, 4; Cl₂ Vehicle, 4.