Administration of nitrite after chlorine gas exposure prevents lung injury: effect of administration modality

Abstract

Cl(2) gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl(2) exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1 mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30 min after Cl(2) exposure. Exposure of rats to Cl(2) gas (400 ppm, 30 min) significantly increased ALI and caused RAS 6-24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl(2)-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl(2)-exposed rats. Both ip and im nitrite improved airway histology compared to Cl(2) gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl(2) gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl(2) exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl(2)-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl(2) gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.

Figure 1. Effects of nitrite administration modality on protective mechanisms against Cl₂ induced lung accumulation of BAL protein and cells

Rats were exposed to Cl₂ gas (400ppm, 30min) and returned to room air. 30min after cessation of Cl₂ gas, saline or nitrite was administered by IP or IM injection, values in parentheses indicate nitrite dose in mg / Kg. 6h after Cl₂ gas administration, rats were sacrificed and BAL protein (panel A) and cells (panel B) measured as described in methods. Data show mean ± SEM (n= 4 – 19). *P < 0.01 relative to air alone, ^# P < 0.05 relative to respective Cl₂ + saline by t-test. No significant effect was observed for air + IP or IM nitrite vs. air alone.

Figure 2. Effects of IM nitrite on Cl₂ dependent increases in BAL levels of PMN

Rats were exposed to Cl₂ gas (400ppm, 30min) and returned to room air. 30min after cessation of Cl₂ gas, nitrite (1mg/Kg) was administered IM injection. 6h after Cl₂ gas administration, rats were sacrificed and BAL cells collected and differential analysis performed for PMN. Data show mean ± SEM (n= 5–13). *P < 0.05 relative to air, ^#P<0.05 relative to Cl₂ + saline as indicated by 1-way ANOVA with Tukey’s post –test.

Figure 3. Histopathological effects of chlorine on proximal airway epithelium 6 hours post-exposure

Representative light micrographs of proximal airway epithelium from rats exposed to 0 ppm chlorine (A), 400 ppm chlorine (B); 400 ppm chlorine plus post-exposure IM nitrite (C, E), or 400 ppm chlorine plus post-exposure IP nitrite (D). Tissues collected 6 hours post chlorine exposure, embedded in paraffin and stained with hematoxylin and eosin. Variability in the amount of intact airway epithelium with the IM nitrite treatment is shown in C and E. Thin arrows indicate squamous cells, CC = ciliated cells, NC = nonciliated cells, * indicates inflammatory cell. Magnification bar = 30 microns. Panels F and G show epithelial thickness and percent of images with attached epithelia as described in results. Data show mean ± SEM (n = 4–6 rats) (4–5 sections from each rat were analyzed). *P<0.001 by 1-way ANOVA with Bonferroni post test for panel F and **P<0.02 by t-test for panel G.

Figure 4. Effects of IP or IM nitrite on post Cl₂ exposure airway reactivity

Rats were exposed to air or Cl₂ (400ppm, 30min) and then returned to room air and IP or IM nitrite (both 1mg/Kg) administered 30min thereafter. 24h after initiating Cl₂ gas exposure, airway reactivity under basal and then increasing methacholine doses was determined by flexivent. Panels A, B show airway resistance and elastance respectively (□ = air, Δ = Cl₂ gas, ● = Cl₂ + IP nitrite, ▼ = Cl₂ + IM nitrite). Panels C, D show basal (pre methacholine) resistance and elastance respectively. Panels E, F show the methacholine dependence (between 0–5mg/ml) on increases in resistance and elastance respectively as calculated by linear regression fits of methacholine dependent changes between 0–5mg/ml. Data show mean ± SEM (n=3–6). P=<0.0001 for air vs Cl₂ and P<0.02 for Cl₂ vs. Cl₂ + IM nitrite by 2-way ANOVA for panel A and panel B. No significant differences between Cl₂ and Cl₂ + IP nitrite were observed. *P<0.05 for Cl₂ vs. air and **P<0.01 for Cl₂ vs Cl₂ + IM nitrite for panels C-F.

Figure 5. Effects of IP or IM nitrite on post Cl₂ exposure dependent changes in circulating leukocytes

Rats were exposed to air or Cl₂ (400ppm, 30min) and then returned to room air and IP or IM nitrite (both 1mg/Kg) administered 30min thereafter. 6h (Panel A) and 24h (Panel B) after initiating Cl₂ gas exposure, whole blood was collected and leukocyte counted. Data show mean ± SEM (n = 3–6). P = 0.05 by 1-way ANOVA with *P<0.05 relative to air by Tukeys post test for Panel A. P< 0.001 by 1-way ANOVA with *P<0.001 relative to air and **P<0.001 relative to Cl₂ by Tukeys post test for Panel B

Figure 6. Effects of allopurinol on IM nitrite dependent protection against PMN accumulation in the BAL

A) Rats were exposed to air or Cl₂ gas (400ppm, 30min) and then lung levels of XOR determined at 6h by western blotting. Data shown XOR normalized to β-actin and are mean ± SEM (n=4–5), * p<0.05 by t-test. Shown representative gel images were acquired from the same western blot. B) Rats were exposed to Cl₂ gas (400ppm, 30min) and then vehicle, allopurinol or IM nitrite (1mg/Kg) administered at 15 min (for allopurinol) and 30min (for nitrite) post Cl₂ gas exposure. Cells in the BAL were measured 6h thereafter. Data show mean ± SEM, n=5–6. *P< 0.03 relative to Cl₂ by t-test.

Figure 7. Nitrite and Nitrate Pharmacokinetics after IP or IM nitrite administration

Whole blood levels of nitrite (panel A) and nitrate (panel B) were measured after Cl₂ gas exposure and after IM (■) or IP (●) nitrite (1mg / Kg) administered 30min post cessation of Cl₂ gas. Data show mean ± SEM (n = 5–6) with time 0 indicating the time immediately prior to nitrite administration. *P < 0.05 by 2-way ANOVA with Bonferroni’s post test.