Am J Physiol Lung Cell Mol Physiol 307: L888–L894, 2014.
First published October 17, 2014; doi:10.1152/ajplung.00079.2014.
Nitrite therapy improves survival postexposure to chlorine gas
Jaideep Honavar,1 Stephen Doran,2 Joo-Yeun Oh,1 Chad Steele,3 Sadis Matalon,2,4 and Rakesh P. Patel1,4
1
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama; 2Department of Anesthesiology
University of Alabama at Birmingham, Birmingham, Alabama; 3Department of Medicine University of Alabama at
Birmingham, Birmingham, Alabama; 4Center for Free Radical Biology and Lung Injury and Repair Center, University
of Alabama at Birmingham, Birmingham, Alabama
Submitted 25 March 2014; accepted in final form 13 October 2014
halogen; acute lung injury; nitric oxide; inflammation
(Cl2) and resultant toxicity are concerns
in civilian and military settings. Several examples pertaining to
the use of Cl2 as a chemical weapon and large-scale accidental
exposures attributable to the derailment of trains transporting Cl2
through populated areas have been documented (3, 8, 21, 31, 34).
The potential for future incidents remains because of the demand
for Cl2 in multiple industrial processes (8). Several studies have
provided insight into the pathogenesis of Cl2 toxicity, which is
characterized by injury to the airways during the initial halogen
insult. This is followed by a postexposure injury phase occurring
over a time span ranging from days to months, which affects the
airways and pulmonary and systemic vasculature (9, 11, 20, 25,
26, 30, 37). Clinically, this presents as reactive airway syndrome,
hypoxemia, noncardiogenic edema leading to acute lung injury,
and development of acute respiratory distress syndrome, which
chronically leads to reactive airway disease, increased sensitivity
to pulmonary infections, fibrosis, and bronchiolitis obliterans, as
EXPOSURE TO CHLORINE GAS
Address for reprint requests and other correspondence: R. Patel, Dept. of
Pathology, Univ. of Alabama at Birmingham, 901 19th St. South, BMR-2, Rm.
532, Birmingham, AL 35294 (e-mail: [email protected]).
L888
well as dermal injury (9, 10, 12, 17, 24, 26, 28, 30). Exciting
recent studies are providing insights into the postexposure mechanisms that include increased oxidative stress, neurogenic and
airway inflammation, fibrosis, loss of nitric oxide (NO) signaling
homeostasis, and loss of endogenous airway repair mechanisms
(2, 9 –11, 15, 20, 22, 25, 26, 36). This information has led to the
testing and demonstration of efficacy for antioxidant therapy (9,
16, 22, 23, 37), anti-inflammatory (using corticosteroids) treatment (5), ␤2-agonists (29), phosphodiesterase inhibitors (4), transient receptor potential channel inhibitors (1, 2), and strategies that
improve NO bioavailability (28, 36).
An important consideration for Cl2 toxicity is that potential
therapies are effective when administered postexposure. They
must also be amenable to stockpiling with a rapid and easy
method of administration. These are all key features for masscasualty scenarios. In this context, intramuscular (IM) injection is
a preferred modality. Given the multitude of mechanisms involved in postexposure toxicity, it is unlikely that one therapeutic
will prevent all aspects of pathogenesis of Cl2 toxicity. Moreover,
in some cases, therapeutics targeted to specific aspects of Cl2
toxicity may not be amenable to rapid administration in a masscasualty scenario. For example, inhaled therapies, although being
the most efficient means of delivering large amount of therapeutic
agents to lung epithelia, are logistically more challenging as a
first-line therapeutic in a mass-casualty scenario but more feasible
if given in a hospital setting. Thus therapeutics that improve
immediate/short-term survival after Cl2 exposure are also critical
to develop, the goal being that this will allow for administration of
other targeted therapies that can be administered in a more
controlled and clinical environment.
NO is a critical mediator of homeostasis in all organ systems. In addition to its generation by one of three nitric oxide
synthases, multiple studies over the last decade have shown
that enzymatic production of NO is complemented by nonenzymatic mechanisms for NO generation from nitrite (18). A
functional role for the latter pathway has been demonstrated in
both physiological and pathophysiological contexts, and perhaps the most evidence is in therapeutics where the goal is to
improve or replete NO bioactivity in ischemic and inflammatory diseases (14, 19, 33). Consistent with this, our recent
studies showed that nitrite administered by intraperitoneal (IP)
or IM injections, after Cl2 exposure prevented acute lung injury
and development of reactive airways in rats (28, 36). Importantly, nitrite also meets the therapeutic criteria outlined above
for mass-casualty scenarios and has an established safety
profile for use in humans in the context of cyanide antidotes,
which is further supported by more recent phase 1/2 studies. In
this study, using a model of lethal Cl2 exposure, we demonstrate the therapeutic potential for nitrite administered postex-
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Honavar J, Doran S, Oh J-Y, Steele C, Matalon S, Patel RP.
Nitrite therapy improves survival postexposure to chlorine gas. Am J
Physiol Lung Cell Mol Physiol 307: L888 –L894, 2014. First published October 17, 2014; doi:10.1152/ajplung.00079.2014.—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.
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NITRITE AND CHLORINE GAS TOXICITY
posure to improve survival and provide insights into underlying mechanisms of protection.
MATERIALS AND METHODS
min, 180 min, and 300 min postexposure. The numbers of replicates
are indicated in the figure legends. All analyses were performed using
GraphPad Prism (La Jolla, CA). Significance was set at P ⬍ 0.05.
RESULTS
Nitrite administration after chlorine exposure improves 24-h
survival. The goals of this study were to test whether nitrite
administered by IM injection postexposure is an effective
postexposure therapy in a lethal model of Cl2 exposure. To
A
Paired Cl 2 600ppm n=25
Paired Cl2 600ppm+10mg/kg Nit n=17
Paired Cl2 600ppm+1mg/kg Nit n=18
Paired Cl2 600ppm+0.1mg/kg Nit n=7
Percent survival
100
80
vs.
P<0.006
60
40
20
0
0
4
8
12
16
20
24
Hours post Cl2 exposure
B
Paired Cl 2 600ppm n=25
Paired Cl2 600ppm+10mg/kg Nit n=11
Cl2 600ppm+10mg/kg Nit (60MIN) n=32
Cl2 600ppm+10mg/kg Nit(1,3,5h) n= 14
Cl2600ppm+100mg/kg Nit (60MiN) n=11
Percent survival
100
80
vs.
P<0.03
vs.
P<0.002
60
40
20
0
0
4
8
12
16
20
24
Hours post Cl2 exposure
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.
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Materials. Unless stated otherwise all reagents were purchased
from Sigma (St. Louis, MO). Quantitative chemokine (CXCL2 and
CXCL1) ELISA kits were purchased from R&D Systems (Minneapolis, MN). Bio-Rad Protein Assay Reagent Kits were purchased from
Bio-Rad (Hercules, CA). Male (25–27 g, 8 –11 wk of age) and female
(18 –20 g, 8 –11 wk of age) C57bl/6 mice were purchased from Harlan
(Indianapolis, IN) and kept on 12-h:12-h light/dark cycles with access
to standard chow and water ad libitum before and after chlorine gas
exposure.
Mouse exposure to chlorine gas. Whole body exposures of male
and female mice to Cl2 gas were performed as previously described
(16, 37). Exposures were performed with either two or five mice in the
same chamber at any one time, and all exposures were performed
between 8:00 AM and 12:00 PM. Exposure conditions were either 400
parts per million (ppm) Cl2 in air for 30 min or 600 ppm for 45 min
using 400-ppm and 600-ppm Cl2 tanks, respectively. Tanks were
replaced when the pressure in the tanks reached 500 psi. In each case,
immediately following exposure, mice were returned to room air. The
mice were monitored hourly for 12 h and every 6 h thereafter for 24 h. All
experiments involving animals were conducted according to protocols
approved by the UAB IACUC.
IM nitrite administration. Mice received a single injection of
sodium nitrite in PBS (0.1–100 mg/kg final concentration) in the
gluteus maximus region at either 30 min or 60 min after cessation of
Cl2 exposure. In addition, a multiple-dose nitrite therapy protocol (10
mg/kg at 60 min, 180 min, and 300 min) was also tested. Nitrite stocks
were prepared daily in sterile PBS with injection volumes of 25 ␮l.
Acute lung injury assessment. Mice were euthanized with intraperitoneal ketamine and xylazine (100 and 10 mg/kg body wt, respectively). An incision was made at the neck to expose the trachea, and
a 3-mm endotracheal cannula inserted. Lungs were lavaged with 3 ⫻
1 ml of PBS; ⬃1.8 –1.9 ml was recovered in all groups. Mice were
then exsanguinated by cardiac puncture for collection of blood.
Recovered aliquots of lavage fluid were kept on ice and centrifuged
immediately at 300 g for 10 min to pellet cells. Supernatants were
removed and stored on ice for protein analysis using the Bio-Rad
Protein Assay Reagent Kit compared with BSA standards. Cells were
resuspended in 100 ␮l PBS and counted using a Neubauer hemocytometer. Cells were then placed on slides using a cellspin (Tharmac,
Drosselweg, Germany) and stained using a two-stain set consisting of
eosin Y and a solution of thiazine dyes (Quik-Stain; Siemens, Washington, DC). Differential counts (specifically monocytes, neutrophils,
and lymphocytes) were then performed on slides via light microscopy.
Chemokine measurement. Mouse CXCL1 (keratinocyte-derived
chemokine, KC) and CXCL2 (macrophage inflammatory protein 2,
MIP-2) were measured in the plasma and bronchoalveolar lavage fluid
(BALF) by ELISA per manufacturer protocols.
Inducing neutropenia. C57Bl/6 mice were rendered neutropenic as
described previously (35) by IP injection with 200 ␮g of either anti
Ly-6G (clone 1A8) (cat. no. BE0075-1; Bxcel, London, UK) or IgG2a
Isotype control (cat. no. BE0089, Bxcel) 24 h before Cl2 gas exposure.
Mice were exposed to Cl2 gas and received nitrite (10 mg/kg, 30 min
after Cl2 exposure) therapy as described above.
Survival analysis. Survival was assessed using the Log rank (Mantel-Cox) test. For each exposure, mice were preassigned to either be
exposed to Cl2 only or Cl2 followed by nitrite therapy. In some cases,
mice assigned to a nitrite therapy group died before receiving nitrite,
i.e., during or within 30 min or 60 min postexposure. These animals
were excluded from the study because they did not receive therapy
and constituted n ⫽ 2 for 1 mg/kg nitrite, n ⫽ 5 for 10 mg/kg nitrite
at 30 min postexposure; n ⫽ 4 for 10 mg/kg nitrite, n ⫽ 1 for 100
mg/kg nitrite at 60 min postexposure; n ⫽ 1 for 10 mg/kg nitrite at 60
L890
NITRITE AND CHLORINE GAS TOXICITY
BALF Protein (mg/mL)
3
*
*
2
1
*
300
200
#
100
0
0
Air
Cl2
Cl2
+ nitrite
Air
Cl2
+ nitrite
Cl2
Macrophage
D
Neutrophil
100
Cells in BALF (% of total)
Cells in BALF (% of total)
C
100
*
80
60
40
*
20
Live
Dead
*
80
60
40
*
20
0
0
Air
Cl2
Cl2
+ nitrite
E
Air
#
BALF
160
Cl2
Cl2
+ nitrite
F
#
BALF
600
Plasma
Plasma
120
*
80
KC (pg/mL)
MIP-2 (pg/ml)
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).
B
Cells in BALF*10^4 / ml
A
*
40
0
400
*
200
0
Air
Cl2
Cl2 Air
+ nitrite
Cl2
Cl2
+ nitrite
Air
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00079.2014 • www.ajplung.org
Cl2
Cl2 Air
+ nitrite
Cl2
Cl2
+ nitrite
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no different compared with nitrite administered at 30 min. The
protective effect of nitrite injection at 60 min after Cl2 exposure was lost, however, if a higher dose (100 mg/kg) was used,
consistent with previous studies showing that nitrite has an
optimum therapeutic concentration (7, 28). Because the circulating half-life of nitrite is ⬃20 – 40 min, we also tested a
multiple-dose regimen to establish whether a single injection of
nitrite was sufficient to afford protection or whether multiple
doses would provide better protection. Nitrite (10 mg/kg)
administered at 1 h, 3 h, and 5 h after Cl2 exposure did not
improve survival relative to Cl2 alone although a trend toward
protection was noted (P ⫽ 0.15, Figure 1B). We also noted
that, when combined, data from Fig. 1, A and B, indicated that
the greatest degree of survival benefit was observed over the
first 12–16 h postexposure.
Role of polymorphonuclear leukocytes in nitrite-dependent
protection. To evaluate the potential mechanisms by which IM
nitrite afforded survival benefit, mice were exposed to Cl2 gas
date, the beneficial effects of nitrite therapy on sublethal Cl2
injury have been established (28, 36). Using male mice, initial
studies determined exposure conditions leading to ⬎75% mortality over 24 h. When five mice at a time were exposed to 600
ppm of Cl2 for 45 min, ⬍10% mortality was observed over 24 h;
however, mortality significantly increased (⬎85% by 24 h) if
only two mice at a time were exposed (not shown). Consistent
with our previous studies, 400 ppm Cl2 for 30 min did not
cause any mortality despite causing significant lung injury (29,
36, 37). We therefore chose exposure conditions of 600 ppm,
45 min with two mice being exposed at any given time.
Figure 1A shows the effects of nitrite (0.1, 1.0, and 10.0
mg/kg) administered by IM injection 30 min after Cl2 exposure. Neither 0.1 mg/kg nor 1.0 mg/kg nitrite affected mortality. However, a single administration of 10.0 mg/kg at 30 min
postexposure significantly improved 24-h survival by ⬃50%.
Figure 1B shows that nitrite (10 mg/kg) also afforded protection when administered 60 min after Cl2 exposure, which was
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NITRITE AND CHLORINE GAS TOXICITY
observed in Fig. 1. However, nitrite did not statistically improve survival in females although a trend (P ⫽ 0.09) was
noted.
DISCUSSION
In this study, we provide evidence that nitrite therapy improves 24-h survival in mice exposed to Cl2. Importantly,
nitrite was efficacious when administered at 30 min or 60 min
after Cl2 exposure, providing the first example, to our knowledge, of a postexposure IM injectable therapeutic that was
effective in improving mortality in a model of Cl2 toxicity.
Together with the potential ability of nitrite to be stockpiled,
these data underscore the potential for nitrite therapy as a front
line therapeutic for inhaled toxicants in mass-casualty situations.
The last decade has seen many studies document the potential for nitrite to replete NO signaling in acute and chronic
diseases afflicting all major organ systems, which are characterized by loss of endogenous NO function (14, 18, 33). We
chose to test nitrite therapy on the basis that endothelial NO
synthase-dependent signaling is inhibited, and nitrite levels are
decreased during the initial stages (hours) after Cl2 exposure
(Fig. 4) (11). Also, nitrite afforded protection against acute
lung injury in a model of sublethal Cl2 exposure, and similar
results have been shown in a model of mechanical ventilatorinduced injury (27, 28, 36). This suggests that the therapeutic
mechanisms of action of nitrite may target aspects of lung
injury common to distinct initiators of acute lung injury.
Further studies testing nitrite therapy for lung injury induced
by other infectious and noninfectious/mechanical stimuli are
warranted.
Less clear is how nitrite improves survival after Cl2 exposure. We have not characterized precisely how mice were
No pre-treatment (n=6)
Anti Ly-6g 1A8 (n=12)
Ig2a Isotype control (n=12)
Anti Ly-6g 1A8 + nitrite (n=12)
Ig2a Isotype control + nitrite (n=11)
Percent survival
100
80
vs.
P<0.01
vs.
P<0.05
60
40
20
0
0
3
6
9
12
15
Hours post Cl2 exposure
Fig. 3. Effects of neutropenia on Cl2-induced mortality and nitrite therapy.
Male mice were untreated (no pretreatment) or injected intraperitoneally with
200 ␮g 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.
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600 ppm, 45 min and administered saline or IM nitrite (10
mg/kg) 30 min postexposure. Mice were continually observed
over the next 6 h, and, if they died during this time, BALF and
blood were collected immediately for assessment of MIP-2 or
KC and accumulation of protein and inflammatory cells. At
6 h, all mice were killed, and similar measurements of acute
lung injury were made (5/8 mice in the Cl2 gas-only group and
9/9 mice in the nitrite-treatment group survived to 6 h). Figure
2A shows that BAL protein increased to similar extents in both
Cl2 and Cl2 ⫹ nitrite groups. However, BAL levels of inflammatory cells were markedly lower in nitrite-treated mice compared with Cl2-alone-exposed mice (Fig. 2B); this effect was
specific for polymorphonuclear leukocytes (PMN) (Fig. 2C).
Figure 2D shows that, in addition to decreasing PMN infiltration, nitrite therapy also improved viability of cells collected
from the BALF. To gain insights into how nitrite prevented
PMN trafficking into the lungs, BAL and plasma levels of the
proneutrophilic chemokines MIP-2 (CXCL2) and KC
(CXCL1) were measured (Fig. 2, E and F, respectively).
Surprisingly, highest chemokine levels were observed in the
nitrite group relative to air, with trends toward increased levels
relative to Cl2 also noted.
Effects of neutropenia on Cl2 toxicity and nitrite therapy.
Figure 2 suggests an association between preventing PMN
accumulation in the BAL and short-term survival benefit after
Cl2 gas exposure. To directly test whether PMN are involved,
mice were rendered neutropenic first, then exposed to Cl2 gas,
and mortality followed over 12 h. Neutropenia was confirmed
by no change in blood macrophage counts and ⬎90% depletion
of blood PMN 24 h after treatment with anti-Ly6 antibody
compared with isotype control (not shown). Figure 3 shows
that mortality was no different between mice exposed to Cl2
only vs. mice exposed to Cl2 after treatment with isotype
control antibody. However, mortality was significantly lower
in neutropenic mice. Moreover, nitrite therapy still afforded
protection in mice treated with isotype control antibody but had
no effect in neutropenic mice.
Effects of nitrite therapy on nitrite and nitrate concentrations.
Similar to our previous studies (11), circulating nitrite levels
decreased in Cl2-exposed mice (Fig. 4A) with no significant
changes in nitrate (Fig. 4B). We have also shown previously
that IM injection of nitrite maximally increased circulating
levels ⬃5 min after administration and with a half-life of ⬃20
min. (28). Consistent with this short plasma half-life, nitrite
therapy had no effect on circulating nitrite at 6 h postadministration. Although not significant, trends toward increased
plasma nitrate formation were evident, however, consistent
with nitrite oxidation. More interestingly, trends (P between
0.08 and 0.1) toward increased lung tissue levels of nitrite and
nitrate were observed in Cl2-exposed animals. Most striking,
however, were the significant increases in nitrite and nitrate in
the BALF in nitrite-therapy groups (Fig. 4, E and F).
Effects of sex on nitrite-dependent protection against Cl2
toxicity. Recent studies have shown potential differences in the
anti-platelet effects of therapeutic nitrate in males vs. females,
nitrite efficacy being lost in females (32). To test the general
applicability of nitrite therapy and assess whether sex is a
modifying factor for Cl2-induced toxicity, matched male and
female mice were compared. Figure 5 shows that female mice
are more sensitive to Cl2-induced injury compared with males.
Nitrite therapy afforded protection in males similar to that
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NITRITE AND CHLORINE GAS TOXICITY
B
0.6
0.4
*
0.2
50
Plasma Nitrate (µM)
Plasma Nitrite (µM)
A 0.8
*
0.0
20
10
1
0
Cl2
0.2
0.1
0.0
Cl2
+ nitrite
Air
Cl2
Cl2
+ nitrite
2000
1500
1000
500
0
Cl2
+ nitrite
*
0.3
Cl2
D
Lung nitrate (nmol/ g tissue)
2
Air
F
100
**
80
60
40
20
0
Air
Cl2
dying in these studies with hypoxemic stress and critical-end
organ dysfunction likely key. Notably, nitrite was able to
improve 24-h survival even when administered 1 h after Cl2
exposure, a time over which much damage to the lungs has
already occurred and induction of inflammation likely. Our
previous study using a sublethal Cl2 exposure protocol showed
that nitrite protected against both increased permeability and
inflammation components of Cl2 injury, which was associated
with increased airway reepithelialization (28, 36). In the current study, we demonstrate a more prominent role for PMN,
which may be attributable to a different model being used here
(mice and lethal exposure protocol), testing of only one nitrite
dose, and assessment of acute lung injury at one time point.
Neutropenia alone improved survival and resulted in a loss of
nitrite-dependent protection, suggesting that prevention of
PMN-dependent injury is one mechanism by which nitrite
elicits protective effects. A recent study also noted attenuated
nitrogen mustard-induced skin injury in mice lacking the predominantly neutrophilic enzyme myeloperoxidase (13), suggesting a central role for PMN in mediating postexposure
toxicities to diverse chemical threat agents. The anti-PMN
Cl2
+ nitrite
Air
Cl2
Cl2
+ nitrite
effects of nitrite were not mediated by altering MIP-2 or KC,
two principal chemokines responsible for PMN homing to the
lungs in acute lung injury. In fact, nitrite therapy increased
levels of these chemokines in the BALF and plasma. At first
glance, such increases in MIP-2 and KC are expected to
further stimulate PMN egress into the lungs; however, it is
also possible that nitrite increased plasma chemokines more
so than BALF levels, which would dissipate the chemokine
gradient into the lung, lowering the driving force for PMN
infiltration from the circulation into the pulmonary compartment. Other potential mechanisms by which nitrite could
affect PMN infiltration include modulation of endothelial
adhesion molecules and PMN activation, and future studies
need to evaluate whether nitrite modulates PMN-dependent
clearance of pathogens, which we have shown are inhibited
after Cl2 exposure (10).
Other potential protective mechanisms of nitrite include
improving blood flow, protecting against cell death, and allowing endogenous repair mechanisms to function. In the latter
context, macrophage viability in the BAL was also improved
by nitrite therapy. The dose of nitrite that was effective in the
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Lung nitrite (nmol / g tissue)
3
Air
BALF Nitrite (µM)
Cl2
+ nitrite
BALF Nitrate (µM)
Cl2
C
E
30
0
Air
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.
40
NITRITE AND CHLORINE GAS TOXICITY
Male Cl2 (n=14)
Male Cl2 + nitrite (10mg/kg, 30min (n=12))
Female Cl2 (n=14)
Female Cl2 + nitrite (10mg/kg, 30min (n=12))
Percent survival
100
80
vs.
P<0.03
vs.
P<0.02
60
40
20
0
4
8
12
16
20
24
Hours post Cl2 exposure
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.
current study (10 mg/kg) was 10-fold higher compared with
our previous studies that employed a sublethal Cl2-exposure
protocol (28, 36). Thus the precise dose of nitrite required
appears to depend in part on the exposure and the end point
being assessed. In the current study, use of 10-fold higher or
lower dose of nitrite resulted in a loss of protection. Similar
“U”-shaped dependence for nitrite therapy has been observed
previously for ischemia-reperfusion injury (7, 27). That said,
even at the highest dose tested here, nitrite did not increase Cl2
toxicity (see Fig. 1), suggesting that a relatively large therapeutic dose and time window exist.
Interestingly, female mice were more sensitive to Cl2-induced mortality compared with males. Whether this is a consequence of estrogen remains to be tested. We used agematched males and females, and therefore females were lighter
compared with males; whether this is an effector for different
responses also requires further investigation. Our primary
goals were to test whether nitrite therapy was effective in
female mice also. Recent studies have shown that nitrite-based
anticoagulation therapy (via nitrate supplementation) may be
less effective in female human volunteers (32). Similarly, our
data show that nitrite was less effective at preventing Cl2induced mortality in female mice compared with male mice.
This could be a consequence for greater injury in females vs.
males and/or less sensitivity for nitrite-based protection in
females. That said, a trend toward significance was still noted,
however, suggesting the general utility for nitrite therapy for
males and females, although we note that this requires much
further study.
We also observed that the degree of Cl2-induced toxicity can
vary significantly depending on the exposure protocol. We
utilized whole body exposure protocols, in contrast to noseonly exposure. The pros and cons of these approaches have
been compared previously (6). Cl2 toxicity was significantly
lower if five mice were exposed at the same time compared
with two. We speculate that “huddling of mice” may have
limited exposure of Cl2 to individual mice in the former setting
as well as potentially introducing uncontrolled variables. For
these reasons, we opted for a protocol with two animals per
exposure. Moreover, variances in Cl2 toxicity responses were
observed between different experiments. For example, for all
data shown in this study, the time for 50% mortality in Cl2
gas-only groups varied from 3–12 h. The precise mechanisms
for such variance is not clear but underscores the importance of
ensuring that exposures are performed with a pairwise manner
when comparing different experimental/therapeutic groups.
The purpose of this study was to test whether IM injection of
nitrite may be an effective postexposure therapeutic in a lethal
model of Cl2 exposure. The extent of Cl2-induced injury
depends on the dose and exposure time. Although these variables can be controlled experimentally, testing of therapeutics
across all potential Cl2 exposure scenarios that likely occur in
an accident or military setting is not feasible. We therefore
opted to use 12–24-h mortality as the end point, with the
rationale being that a postexposure therapeutic that can be
administered within 60 min of exposure, to model first-responder times, and which also improves short-term survival
will be key in allowing victims to reach a primary care facility
where targeted therapeutics can be initiated. A limitation of
this study is the lack of testing of this concept and assessing
mortality effects beyond 24 h. Future studies evaluating longterm outcomes after postexposure nitrite therapy are required.
GRANTS
This research was supported by the CounterACT Program, National
Institutes of Health, Office of the Director, and the National Institute of
Environmental Health Sciences, Grant Number 1U01ES023759 and
1R21ES024027-01
DISCLOSURES
R. Patel is a coinventor on a patent for use of nitrite salts for the treatment
of cardiovascular conditions. No other conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
J.H., S.F.D., C.S., S.M., and R.P.P. conception and design of research; J.H.,
S.F.D., and J.-Y.O. performed experiments; J.H., J.-Y.O., and R.P.P. analyzed
data; J.H., S.F.D., J.-Y.O., S.M., and R.P.P. edited and revised manuscript;
J.H., S.F.D., J.-Y.O., C.S., S.M., and R.P.P. approved final version of manuscript; S.M. and R.P.P. interpreted results of experiments; R.P.P. prepared
figures; R.P.P. drafted manuscript.
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