Nitrite therapy improves survival postexposure to chlorine gas

Abstract

Exposure to relatively high levels of chlorine (Cl₂) gas can occur in mass-casualty scenarios associated with accidental or intentional release. Recent studies have shown a significant postexposure injury phase to the airways, pulmonary, and systemic vasculatures mediated in part by oxidative stress, inflammation, and dysfunction in endogenous nitric oxide homeostasis pathways. However, there is a need for therapeutics that are amenable to rapid and easy administration in the field and that display efficacy toward toxicity after chlorine exposure. In this study, we tested whether nitric oxide repletion using nitrite, by intramuscular injection after Cl₂ exposure, could prevent Cl₂ gas toxicity. C57bl/6 male mice were exposed to 600 parts per million Cl₂ gas for 45 min, and 24-h survival was determined with or without postexposure intramuscular nitrite injection. A single injection of nitrite (10 mg/kg) administered either 30 or 60 min postexposure significantly improved 24-h survival (from ∼20% to 50%). Survival was associated with decreased neutrophil accumulation in the airways. Rendering mice neutropenic before Cl₂ exposure improved survival and resulted in loss of nitrite-dependent survival protection. Interestingly, female mice were more sensitive to Cl₂-induced toxicity compared with males and were also less responsive to postexposure nitrite therapy. These data provide evidence for efficacy and define therapeutic parameters for a single intramuscular injection of nitrite as a therapeutic after Cl₂ gas exposure that is amenable to administration in mass-casualty scenarios.

Keywords: acute lung injury; halogen; inflammation; nitric oxide.

Fig. 1.

Nitrite-dependent protection against chlorine-induced mortality in mice. A: male mice were exposed 2 at a time to Cl₂ 600 parts per million (ppm), 45 min and then brought back to room air. 30–60 min thereafter, mice received either saline or nitrite (Nit) at 0.1 mg/kg, 1.0 mg/kg, or 10.0 mg/kg at 30 min after Cl₂ exposure by intramuscular injection. Data show Kaplan-Meier survival curves. *P < 0.006 between 10 mg/kg nitrite 30 min and saline by the Log-rank test. No significant difference between 0.1 mg/kg nitrite (P = 0.33) or 1.0 mg/kg nitrite (P = 0.26) and saline was observed. B: male mice were exposed to Cl₂ at 600 ppm, 45 min and then brought back to room air, and nitrite was administered as indicated in figure by intramuscular (IM) injection. Data show Kaplan-Meier survival curves. P < 0.03 between saline and nitrite (10 mg/kg, 30 min) and P < 0.002 between saline and nitrite (10 mg/kg, 60 min). No significant difference was observed between saline and either multiple nitrite administration (P = 0.15) or nitrite (100 mg/kg, 60 min) (P = 0.75) groups.

Fig. 2.

Effect of nitrite therapy on indices of acute lung injury. Male mice were exposed to Cl₂ 600 ppm, 45 min and then brought back to room air and 30 min thereafter received saline or nitrite 10 mg/kg by IM injection. Mice were continually observed over the next 6 h, and, if they died during this time, bronchoalveolar lavage fluid (BALF) and blood were collected immediately. At 6 h, all mice were killed, and BALF and plasma were collected. A and B: BALF levels of protein and total cell accumulation. *P < 0.01 relative to air, #P < 0.05 relative to Cl₂ alone by 1-way ANOVA with Tukey's posttest. C: differential cell analysis. D: viability of BALF inflammatory cells. *P < 0.01 relative to respective + nitrite group by t-test. E: BALF and plasma macrophage inflammatory protein 2 (MIP-2) levels, *P < 0.01 relative to air and #P < 0.05 relative to Cl₂-alone group by 1-way ANOVA with Tukey's posttest. F: BALF and plasma keratinocyte chemokine (KC) levels, *P < 0.01 or #P < 0.05 relative to air, by 1-way ANOVA with Tukey's posttest. All data are means ± SE (n = 5–9).

Fig. 3.

Effects of neutropenia on Cl₂-induced mortality and nitrite therapy. Male mice were untreated (no pretreatment) or injected intraperitoneally with 200 μg of either anti Ly-6G or IgG2a isotype control antibody. 24 h later, all mice were exposed to Cl₂ gas (600 ppm, 45 min) and then treated with saline or nitrite (10 mg/kg) 30 min after Cl₂ exposure. Data show Kaplan-Meier survival curves. P < 0.01 between anti Ly-6G or IgG2a groups, P < 0.05 between IgG2a and IgG2a + nitrite groups, P = 0.6 between Ly-6G and Ly-6G + nitrite groups.

Fig. 4.

Nitrite and nitrate levels after nitrite therapy. Male mice were exposed to Cl₂ 600 ppm, 45 min and then brought back to room air and 30 min thereafter received saline or nitrite 10 mg/kg by IM injection. Plasma nitrite (A) and nitrate (B), lung nitrite (C) and nitrate (D), and BALF nitrite (E) and nitrate (F) were measured after 6 h. Data shown are means ± SE, n = 5–9. *P < 0.05 relative to air by for A and *P < 0.05 or **P < 0.01 relative to air and Cl₂ alone for E and F by 1-way ANOVA with Tukey's posttest.

Fig. 5.

Effect of sex on chlorine toxicity and nitrite therapy. Male or female mice were exposed to Cl₂ at 600 ppm, 45 min and then brought back to room air, and nitrite was administered as indicated in figure by IM injection 30 min postexposure. Data show Kaplan-Meier survival curves. P < 0.03 between male and female Cl₂-alone groups; P < 0.02 for nitrite therapy in males and P = 0.09 for nitrite therapy in females.