AEOL10150: a novel therapeutic for rescue treatment after toxic gas lung injury.
ABSTRACT New therapeutics designed as rescue treatments after toxic gas injury such as from chlorine (Cl(2)) are an emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities, on Cl(2)-induced airway injury. Balb/C mice received 100ppm Cl(2) gas for 5 min. Four groups were studied: Cl(2) only, Cl(2) followed by AEOL10150 1 and 9 h after exposure, AEOL10150 only, and control. Twenty-four hours after Cl(2) gas exposure airway responsiveness to aerosolized methacholine (6.25-50mg/ml) was measured using a small-animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at 72 h after Cl(2) injury to evaluate epithelial cell proliferation. Mice exposed to Cl(2) showed a significantly higher airway resistance compared to control, Cl(2)/AEOL10150, or AEOL10150-only treated animals in response to methacholine challenge. Eosinophils, neutrophils, and macrophages were elevated in BAL of Cl(2)-exposed mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl(2)-induced increase in BAL fluid protein. Chlorine induced an increase in the number of proliferating airway epithelial cells, an effect AEOL10150 attenuated. 4-Hydroxynonenal levels in the lung were increased after Cl(2) and this effect was prevented with AEOL10150. AEOL10150 is an effective rescue treatment for Cl(2)-induced airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration, and oxidative stress.
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ABSTRACT: Exposure to chlorine (Cl2) gas during industrial accidents or chemical warfare leads to significant airway and distal lung epithelial injury that continues post exposure. While lung epithelial injury is prevalent, relatively little is known about whether Cl2 gas also promotes injury to the pulmonary vasculature. To determine this, rats were subjected to a sub-lethal Cl2 gas exposure (400ppm, 30min) and then brought back to room air. Pulmonary arteries (PA) were isolated from rats at various times post-exposure and contractile (phenylephrine) and nitric oxide (NO)-dependent vasodilation (acetylcholine and mahmanonoate) responses measured ex-vivo. PA contractility did not change, however significant inhibition of NO-dependent vasodilation was observed that was maximal at 24-48hours post exposure. Superoxide dismutase restored NO-dependent vasodilation suggesting a role for increased superoxide formation. This was supported by ∼2-fold increase in superoxide formation (measured using 2-hydroethidine oxidation to 2-OH-E(+)) from PA isolated from Cl2 exposed rats. We next measured PA pressures in anesthetized rats. Surprisingly, PA pressures were significantly (∼4mmHg) lower in rats that had been exposed to Cl2 gas 24hours earlier suggesting that deficit in NO-signaling observed in isolated PA experiments did not manifest as increased PA pressures in vivo. Administration of the iNOS selective inhibitor 1400W, restored PA pressures to normal in Cl2 exposed, but not control rats suggesting that any deficit in NO-signaling due to increased superoxide formation in the PA, is offset by increased NO-formation from iNOS. These data indicate that disruption of endogenous NO-signaling mechanisms that maintain PA tone is an important aspect of post-Cl2 gas exposure toxicity.Toxicology 04/2014; · 3.75 Impact Factor
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ABSTRACT: Our previous studies and other published reports with the chemical warfare agent sulfur mustard (SM) and its analog 2-chloroethyl ethyl sulfide (CEES) have indicated a role of oxidative stress in skin injuries caused by these vesicating agents. We examined the effects of the catalytic antioxidant AEOL 10150 in attenuation of CEES-induced toxicity in our established skin injury models (skin epidermal cells and SKH-1 hairless mice) to validate the role of oxidative stress in the pathophysiology of mustard vesicating agents. Treatment of mouse epidermal JB6 and human HaCaT cells with AEOL 10150 (50μM) 1h post CEES exposure resulted in significant (p<0.05) reversal of CEES-induced decreases in both cell viability and DNA synthesis. Similarly, AEOL 10150 treatment 1h after CEES exposure attenuated CEES-induced DNA damage in these cells. Similar AEOL 10150 treatments also caused significant (p<0.05) reversal of CEES-induced decreases in cell viability in normal human epidermal keratinocytes. Cytoplasmic and mitochondrial reactive oxygen species measurements showed that AEOL 10150 treatment drastically ameliorated the CEES-induced oxidative stress in both JB6 and HaCaT cells. Based on AEOL 10150 pharmacokinetic studies in SKH-1 mouse skin, mice were treated with topical formulation plus subcutaneous (injection; 5mg/kg) AEOL 10150, 1h after CEES (4mg/mouse) exposure and every 4h thereafter for 12h. This AEOL 10150 treatment regimen resulted in over 50% (p<0.05) reversal in CEES-induced skin bi-fold and epidermal thickness, myeloperoxidase activity, and DNA oxidation in mouse skin. Results from this study demonstrate potential therapeutic efficacy of AEOL 10150 against CEES-mediated cutaneous lesions supporting AEOL 10150 as a medical countermeasure against SM-induced skin injuries.Free Radical Biology and Medicine 05/2014; · 5.27 Impact Factor
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ABSTRACT: Significance SOD enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2.-; no wonder thus that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored. Recent advances Structure-activity relationship (E1/2 vs log kcat), originally reported for Mn porphyrins(MnP), is valid for any other class of SOD mimics, as it is dominated by the superoxide reduction and oxidation potential. The biocompatible E1/2 of ~+300 mV vs NHE allows powerful SOD mimics to be both mild oxidants and antioxidants (alike O2.-) and therefore readily traffic electrons among reactive species and signaling proteins, serving as fine mediators of redox-based signaling pathways. Based on similar thermodynamics both SOD enzymes and their mimics undergo similar reactions, yet, due to vastly different sterics, with different rate constants. Critical Issues Whereas log kcat(O2.-) is a good measure of therapeutic potential of SOD mimics, discussions of their in vivo mechanisms of actions remain mostly of speculative character. Most recently, the therapeutic and mechanistic relevance of glutathionylation and oxidation of protein thiols and ascorbate by MnP-based SOD mimics in oxidation and subsequent inactivation of NF-B has been substantiated in rescuing normal and killing cancer cell. Interaction of MnPs with thiols seems to be at least in part involved in upregulation of endogenous antioxidative defenses leading to the healing of diseased cell. Future directions Mechanistic explorations of single and combined therapeutic strategies, along with studies of bioavailability and translational aspects, will comprise future work in optimizing redox-active drugs.Antioxidants & Redox Signaling 07/2013; · 8.20 Impact Factor
AEOL10150: A novel therapeutic for rescue treatment after toxic gas lung injury
Toby McGoverna, Brian J. Dayb, Carl W. Whiteb, William S. Powella, James G. Martina,⁎
aMeakins Christie Laboratories, Department of Medicine, McGill University, Montreal, QC H2X 2P2, Canada
bDepartment of Pediatrics, National Jewish Health, Denver, CO 80206, USA
a b s t r a c ta r t i c l ei n f o
Received 4 August 2010
Revised 22 November 2010
Accepted 1 December 2010
Available online 13 December 2010
Acute airway injury
New therapeutics designed as rescue treatments after toxic gas injury such as from chlorine (Cl2) are an
emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a
compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities,
on Cl2-induced airway injury. Balb/C mice received 100 ppm Cl2gas for 5 min. Four groups were studied: Cl2
only, Cl2followed by AEOL10150 1 and 9 h after exposure, AEOL10150 only, and control. Twenty-four hours
after Cl2gas exposure airway responsiveness to aerosolized methacholine (6.25–50 mg/ml) was measured
using a small-animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation
and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at
72 h after Cl2injury to evaluate epithelial cell proliferation. Mice exposed to Cl2showed a significantly higher
airway resistance compared to control, Cl2/AEOL10150, or AEOL10150-only treated animals in response to
methacholine challenge. Eosinophils, neutrophils, and macrophages were elevated in BAL of Cl2-exposed
mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl2-
induced increase in BAL fluid protein. Chlorine induced an increase in the number of proliferating airway
epithelial cells, an effect AEOL10150 attenuated. 4-Hydroxynonenal levels in the lung were increased after Cl2
and this effect was prevented with AEOL10150. AEOL10150 is an effective rescue treatment for Cl2-induced
airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration, and
© 2010 Elsevier Inc. All rights reserved.
Chlorine (Cl2) is a highly reactive oxidant gas that is used in the
bleaching of paper, in the production of hydrocarbon solvents, in the
year cumulative data between 1988 and 1992 from the American
reported 27,788 exposures to Cl2 in the United States . Acute
human exposures have occurred as a result of industrial accidents or
during wartime that have led to long-term respiratory dysfunction
and even death . Residual effects after acute Cl2damage can persist
for years and include decreased vital capacity, reduced diffusing
capacity, and lowered total lung capacity with a trend toward higher
airway resistance [4,5]. There are no effective pharmacological rescue
treatments currently available.
There have been several experimental and case studies performed
in both animal and human models characterizing the effects of Cl2gas
is generally characterized by an influx of inflammatory cells into the
airways, specifically neutrophils, lymphocytes, eosinophils, and
hyperresponsiveness can occur [7,8]. Epithelial cell damage has been
observed in rodents exposed to Cl2gas, including denudation of the
epithelium, followed by repopulation of the epithelial cell layers .
The molecular properties of Cl2are such that it has an extremely
high propensity to oxidize. It has been shown to have greater toxicity
than nitrogen dioxide (NO2), oxygen (O2) or ozone (O3), a property
that may be related, in part, to its high water solubility . The
hydrationof Cl2leads to the productionof hydrochloric acid (HCl) and
hypochlorous acid (HOCl). It is therefore likely that oxidative injury is
also involved in the damage and repair processes [10,11]. Consistent
with this idea, Cl2gas is about 30-fold more potent than hydrochloric
acid, further emphasizing its oxidant, rather than acidic, properties as
being the predominant mechanism responsible for its actions [3,12].
When administered into the airways hydrochloric acid causes airway
hyperresponsiveness in mice by mechanisms that have been
suggested to relate to epithelial barrier function . Epithelial cells
are particularlysusceptibletoCl2damage and have beenimplicatedas
key targets in the damage and repair process. They are among the first
cells to encounter Cl2in the airway and may be affected by the direct
toxicity of Cl2or indirectly through its by-products HOCl and HCl.
Additionally, epithelial cells are capable of storing, producing, and
releasinglarge quantitiesof the antioxidantglutathionein responseto
oxidative stress .
The aim of this study was to assess the efficacy of a novel catalytic
antioxidant in ameliorating airway damage when administered after
Free Radical Biology & Medicine 50 (2011) 602–608
⁎ Corresponding author.
E-mail address: firstname.lastname@example.org (J.G. Martin).
0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Free Radical Biology & Medicine
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an acute exposure to inhaled Cl2gas. For this purpose, we utilized a
catalytic metalloporphyrin that is a member of a novel class of low-
molecular-weight antioxidants. The compound, Mn(III) tetrakis-(N,N′
-diethylimidizolium-2-yl) porphyrin, AEOL10150 (AEOL), efficiently
scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and
catalase-like activities . Recent studies using the sulfur mustard
analog 2-chloroethylethyl sulfide (CEES), a compound that also can
induce oxidative stress in the lung, have demonstrated that AEOL is
effective in reducing cytotoxicity and mitochondrial dysfunction
when given 1 h after CEES exposure. In vivo, AEOL has shown
promising therapeutic properties in protecting the lungs of rats
exposed to CEES against inflammatory cell infiltration, reactive
oxygen species, and DNA damage . In this study, we used a
mouse model to further characterize the effects of Cl2gas as well as
the ability of AEOL to rescue the lungs from damage caused by Cl2
inhalation by examining airway function, airway inflammation,
bronchial epithelial cell proliferation, and markers of oxidative stress.
Animals and protocol
Male Balb/C mice (18–22 g) were purchased from Charles River
(Wilmington, MA, USA) and housed in a conventional animal facility
at McGill University. Animals were treated according to the guidelines
of the Canadian Council for Animal Care and protocols were approved
by the Animal Care Committee of McGill University. Animals were
provided with water and food ad libitum throughout the experiment.
Four groups were studied: Cl2only (n=10), Cl2followed by AEOL
(n=10), AEOL only (n=10), and control (n=10). Mice in groups
treated with AEOL weregiven 5 mg/kg intraperitoneally (ip) 1 and 9 h
after Cl2exposure. Mice in control or Cl2-only groups were given 1 ml
of phosphate-buffered saline (PBS; pH 7.4) ip 1 and 9 h after air or Cl2
exposure. Mice were studied at 24 h after initial Cl2exposure. In
separate groups (n=6/group) mice were studied at 72 h after initial
Cl2exposureto evaluateepithelial cell proliferationin thelungs.There
was no significant weight loss or behavioral signs of distress at any
time point after Cl2.
AEOL10150 pharmacokinetics and safety profile
Male C57Bl/6 mice were given a single bolus dose of AEOL
intravenously by tail-vein injection and blood samples were drawn
upon sacrifice by cardiac puncture (two mice/time point). Blood
samples were obtained at 5, 10, 15, and 30 min and 1, 2, 4, 8, 12, and
24 h after dosing. Plasma AEOL concentrations were measured using a
previously described HPLC analytical method . Pharmacokinetic
data fitting was done using a WinNonlin noncompartmental model
(Pharsight, Cary, NC, USA) (Table 1).
AEOL10150 safety assessments were performed in mice, rats,
nonhuman primates, and humans as described in FDA IND No. 67,741.
In mice, AEOL's no observable adverse effect level was 40 mg/kg/day
and the maximum tolerated dose was 160 mg/kg/day (data not
shown). The structure of AEOL is shown in Fig. 1.
Exposure to Cl2
Mice were restrained and exposed to Cl2gas for 5 min using a
nose-only exposure device. Cl2gas 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 an air-
flow generator. Dynacal permeation tubes designed specifically for
operation with the Dynacalibrator were used and contained the Cl2.
The permeation chamber and air-flow generator control the accuracy
of the Cl2generated to within 1–3% of the desired concentration
according to the manufacturer's specifications. Teflon permeation
tubes containing Cl2in both gas and liquid phases are contained
within the permeation chamber. When the tube is heated the Cl2
reaches a constant vapor pressure such that 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 min until the
optimum temperature of 30 °C is reached and the Cl2flow is constant.
After removal of the animal from the exposure chamber, the chamber
was continually flushed with the gas mix to ensure that the desired
concentration of Cl2was maintained between mouse exposures.
Mice were sedated with xylazine hydrochloride (8 mg/kg, ip) and
anesthetized with pentobarbital (30 mg/kg, ip). Subsequently, the
animals were given tracheotomies using an 18-gauge cannula and
connected to a small-animal ventilator (FlexiVent; Scireq, Montreal,
QC, Canada). Muscle paralysis was induced with pancuronium
bromide (0.2 mg/kg ip). 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 cm H2O, a positive end
expiratory pressure of 3 cm H2O, and a frequency of 150/min. After an
equilibration period of 3 min of tidal ventilation,two lung inflationsto
a transrespiratory pressure of 25 cm H2O were performed and
baseline measurements were taken. The template used was Snap-
shot-150 version 5.2 and mechanics were calculated using the single-
compartment model. Baseline was established as the average of three
perturbations. After establishment of baseline, methacholine (MCh)
was administered using an inline nebulizer (Aeroneb Lab, standard
mist model; Aerogen Ltd., Galway, Ireland), and progressively
doubling concentrations ranging from 6.25 to 50 mg/ml were
administered over 10 s synchronous with inspiration. Six measure-
ments were made 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 and respi-
ratory system elastance were determined before challenge and after
each dose of MCh.
Mouse pharmacokinetics of AEOL10150
(2 mg/kg, iv)
Values were calculated from curve fit data from two mice per time point.
Fig. 1. Structure of AEOL10150. The skeletal formula for compound Mn(III) tetrakis-(N,
N′-diethylimidizolium-2-yl) porphyrin, AEOL10150, is shown.
T. McGovern et al. / Free Radical Biology & Medicine 50 (2011) 602–608
Twenty-four hours after Cl2exposure the mice were euthanized
with an overdose of sodium pentobarbital (30 mg/kg, ip). The mice
weregiventracheotomies and0.5 mlof sterile salinewasinstilled into
the lungs and the fluid recovered was placed in 1.5-ml Eppendorf
tubes and kept on ice. Fluid recovered from the first wash was
centrifuged at 1500 rpm for 5 min at 4 °C and the supernatant was
retained for extracellular glutathione analysis. Three subsequent
washes were done with 1-ml aliquots of sterile saline to recover
lung inflammatory cells and placed in a 15-ml tube for centrifugation.
The bronchoalveolar lavage fluid (BAL) was centrifuged at 1500 rpm
for 5 min at 4 °C and total live and dead cells were counted using
trypan blue exclusion. Cytospin slides were prepared using a
cytocentrifuge (Shandon, Pittsburgh, PA, USA) and stained with Diff
Quick (Jorgensen Labs, Loveland, CO, USA). Differential cell counts
were determined based on a count of 300 cells/slide. Total protein in
the BAL supernatant was quantified using a Bradford colorimetric
assay (Bio-Rad, Hercules, CA, USA) and determined by spectropho-
tometry at 620 nm against a standard curve of bovine serum albumin.
Tissue preparation for assessment of epithelial regeneration
Mice were allowed to recover for 72 h in separate groups after Cl2
exposure before they were euthanized using an overdose of sodium
pentobarbital (30 mg/kg, ip). The pulmonary circulation was flushed
with sterile saline via the right ventricle until the effluent was clear.
After removal the lungs were fixed by intratracheal perfusion with
10% buffered formalin at a constantpressure of 25 cm H2O for a period
of 24 h. Immunohistochemistry was done after cutting of 5-μm
paraffin-embedded sections, which were stained with Ki-67 nuclear
staining to determine epithelial cell proliferation.
Cells undergoing proliferation were detected in tissue sections by
immunostaining for Ki-67. Immunohistochemical staining for Ki-67
was performed with the Vectastatin avidin–biotin peroxidase com-
plex kit (Vector Laboratories, Burlingame, CA, USA). After deparaffi-
nation of sections, the slides were immersed in antigen unmasking
solution (Vector Laboratories) for 8 min. Sections were washed for
5 min twice with Tris-buffered saline (TBS; 0.5 M Tris–HCl, 1.5 M
NaCl, pH 7.6). Lung sections were permeabilized using 0.2% Triton
X-100 for 20 min. After being washed, nonspecific binding sites were
saturated with Universal Blocking Solution (Vector Laboratories) for
20 min.PrimaryantibodiesagainstgoatKi-67(CaymanChemical, Ann
Arbor, MI, USA) and control normal goat serum (Vector Laboratories)
used at a dilution of 1:250 were applied to tissue sections for
incubation in a humidified chamber at room temperature for 1 h. The
sections were then washed with TBS twice for 5 min. Biotinylated
rabbit anti-goat IgG (Vector Laboratories) was applied to the tissue
sections at a concentration of 1:50 and incubated at room temper-
ature for 45 min. Sections were washed twice with TBS for 5 min.
Then the slides were incubated with avidin–biotin complex alkaline
phosphatase (Vector Laboratories) for 45 min. Last, Vector red
alkaline phosphatase (Vector Laboratories) was used to develop the
sections for 15 min. Sections were dehydrated by moving slides
through three baths of xylene and mounted with Vectamount
mounting medium (Vector Laboratories). Lung sections were visual-
ized for positive Ki-67 staining by light microscopy.
Quantitative morphology on airway sections
Numbers of Ki-67-positive cells were quantified after staining.
staining for Ki-67. Only airways with a major/minor (long axis/short
axis) diameter ratio of b2.5 in cross section were selected for analysis.
positive cells in the epithelium wasquantified under a lightmicroscope
using a 40× objective. The airway basement membrane length was
measured by superimposing the image of the airway onto a calibrated
digitizing tablet (Jandel Scientific, Chicago, IL, USA), with a microscope
equipped with a camera lucida projection system (Leica Microsystems,
Richmond Hill, ON, Canada). The numbers of proliferating cells
corrected for airway size were expressed as Ki-67+cells/mm of
basement membrane perimeter (PBM).
The BAL fluid from control, Cl2-exposed, and AEOL-treated mice
was collected for glutathione evaluation by HPLC. BAL samples were
collected 24 h after Cl2challenge. Phosphoric acid (60 μl; 1 M) was
added to BAL fluid samples to prevent glutathione (GSH) degradation.
BAL was centrifuged at 1500 rpm for 5 min, and the supernatant was
removed for evaluation of extracellular GSH/GSSG. The pellet was
reconstituted with 150 μl of PBS, 150 μl of 12 mM Chaps, and 15 μl of
1 M phosphoric acid for analysis of intracellular GSH and GSSG. GSH
and GSSG were measured in 50-μl aliquots by RP-HPLC using
postcolumn derivatization as described previously . The mobile
phase was a gradient between 0 and 15% acetonitrile containing 0.05%
trifluoroacetic acid over 10 min at a flow rate of 1 ml/min. The
GSSG in the column eluate were converted to a fluorescent isoindole
derivative by continuously mixing the column eluate with 'o-
phthalaldehyde (370 μM) in 0.2 M tribasic sodium phosphate, pH 12,
at 70 °C. Fluorescence was monitored using excitation and emission
GSSG were determined from a standard curve using the authentic
compounds as external standards.
Lung 4-hydroxynonenal (4-HNE) assay
4-HNE assay was performed as described previously . 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,
Waltham, MA, USA) containing 50 μg/ml butylated hydroxytoluene,
with10 ngd3-4-HNE(CaymanChemical)internalstandard addedjust
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 incubation with 0.2 ml of 0.1 M Hepes containing
50 mM O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochlo-
ride, pH 6.5, for 5 min at room temperature. The O-pentafluoroben-
zyl–oxime derivatives were then extracted with hexane, converted to
trimethylsilyl ethers by the addition of 15 μl each of pyridine and N,O-
bis(trimethylsilyl)trifluoroacetamide, and analyzed by gas chroma-
tography/mass spectrometry using a Focus gas chromatograph
coupled to a DSQ II mass spectrometer (Thermo Fisher). The
stationary phase was an A15-m TR-5MS column (0.25-mm i.d.,
0.25-μm film thickness; Thermo Fisher) and the carrier gas was
helium (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 temper-
ature 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,
T. McGovern et al. / Free Radical Biology & Medicine 50 (2011) 602–608
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.
Differences in responsiveness to methacholine were analyzed by
repeated-measures ANOVA and a post hoc Bonferroni test. One-way
analysis of variance was used to determine the drug treatment effect
on other variables and the post hoc comparisons were performed
using a Dunnett multiple comparison test for differential cell counts.
Pb0.05 was considered significant.
Effect of AEOL on MCh responsiveness after Cl2challenge
Airway responsiveness to inhaled aerosolized MCh in increasing
concentrations (6.25–50 mg/ml) was elevated 24 h after Cl2exposure.
Cl2significantly increased respiratory system resistance over control
and AEOL-treated groups at the two highest doses of MCh (Fig. 2A).
Respiratory system elastance was increased in the Cl2-only exposed
group compared with control at the two highest doses of MCh
(Fig.2B).AEOLdid notsignificantlyattenuatetheincreasein elastance
compared to Cl2-only treated mice (Fig. 2B). However, there was also
no significant difference between control groups and Cl2/AEOL-
treated mice (Fig. 2B).
Changes in bronchoalveolar lavage cells after Cl2gas exposure
To assess the airwayinflammationinduced by Cl2, bronchoalveolar
lavage was performed at 24 h after Cl2 exposure. The BAL fluid
recovered averaged75% of the initial volume instilled anddid not vary
significantly between groups. Total cell counts were significantly
increased in the Cl2-exposed group compared to control groups as
well as Cl2/AEOL (Fig. 3). Although AEOL was able to reduce total
inflammatory cell influx after Cl2exposure compared with Cl2-only
exposed mice, there was still a significant difference between Cl2/
AEOL-treated groups and control groups. AEOL alone had no effect on
total inflammatory cell numbers.
Differential cell counts were performed to examine the pattern of
inflammatory cell recruitment to the airways after Cl2exposure. Cl2
exposure significantly increased total numbers of macrophages,
neutrophils, and eosinophils (Figs. 4A–C) above control groups but
did not significantly increase lymphocytes or epithelial cells (Figs. 4D
and E). Treatment with AEOL attenuated the increase in macrophage
and neutrophil numbers recruited to the airway after Cl2exposure but
did not significantly affect eosinophil, lymphocyte, or epithelial cell
Changes in protein level after Cl2exposure
Total protein levels were measured in BAL fluid using a Bradford
assay 24 h after Cl2exposure to assess cell damage and microvascular
leak. Cl2caused a twofold increase in the total protein present in the
BAL compared to control groups. Treatment with AEOL after Cl2
exposure significantly reduced the amount of protein in the BAL fluid
Epithelial cell proliferation
We have previously reported that epithelial proliferation occurs as
early as 48 h after high-level Cl2exposure . We measured the level
of cellular hyperplasia as an indirect method of assessment of
epithelial damage. We chose to quantify the level of proliferation of
epithelial cells at 72 h after exposure. Ki-67 nuclear staining was
performed to determine numbers of proliferating epithelial cells. The
total numbers of positive cells were quantified in each airway and
corrected for airway size by dividing by the perimeter of the basement
membrane of the airway. A minimum ofsix animals totalingatleast 24
airways were studied in each group. Cl2induced an increase in the total
number of proliferating epithelial cells in the airways (Fig. 6A).
Treatment with AEOL significantly reduced the number of proliferating
Fig. 2. AEOL reduces airway responsiveness to methacholine after Cl2challenge. Airway
responsiveness was determined by FlexiVent 24 h after Cl2 exposure. (A) Total
respiratory system resistance after MCh in control, Cl2-only, Cl2/AEOL, and AEOL-only
treated mice. A two-way ANOVA showed a significant decrease between mice
posttreated with AEOL compared to animals receiving Cl2only at the two highest
doses of MCh. There was no difference between AEOL-treated mice or control groups.
(B) The effects of Cl2 exposure and AEOL treatment on total respiratory system
elastance. There were differences only in control vs Cl2-only mice at the two highest
doses of MCh. AEOL-treated groups were not significantly different from control or Cl2-
only treated animals (n=10 per group; **Pb0.01, ***Pb0.001).
Fig. 3. Effects of Cl2exposure on the numbers of cells in BAL fluid. Mice were sacrificed
24 h after Cl2exposure. Cl2led to a significant increase in total leukocytes compared to
controls and AEOL-treated groups. AEOL only was not different from control; however,
Cl2/AEOL-treated mice had more total cells in their BAL compared to AEOL-only mice
(n=10 per group; *Pb0.05, **Pb0.01, ***Pb0.001).
T. McGovern et al. / Free Radical Biology & Medicine 50 (2011) 602–608
epithelial cells compared to Cl2-only treatment. Figs. 6B–D show
representative pictures of control (Fig. 6B), Cl2-only (Fig. 6C), and Cl2/
AEOL (Fig. 6D) cells. Darker nuclei indicate Ki-67-positive cells.
BAL GSH and GSSG levels at 24 h are unaffected by Cl2
At 24 h after Cl2and AEOL treatments, extracellular GSH levels
were measured in BAL fluid (Fig. 7A). There were no significant
differences in the levels of either GSH (Fig. 7A) or GSSG (Fig. 7B)
among the four groups.
AEOL protects against Cl2-induced increases in 4-NHE
Twenty-four hours after Cl2exposure, lungs were harvested and
lung homogenates analyzed for 4-HNE. Cl2 induced a significant
increase in 4-HNE levels compared to control and AEOL-treated
groups (Fig. 8). AEOL alone had no effect on 4-HNE levels, but
completely blocked the response to Cl2.
In this study we wished to explore whether rescue treatment with
the synthetic metalloporphyrin AEOL could prevent the deleterious
effects of Cl2on the lungs, including airway hyperresponsiveness to
MCh, inflammatory cell influx into the airways, epithelial cell
proliferation,andoxidative damage. Ourresultsshowedthatalthough
AEOL itself had no effect on the lungs, it was effective in attenuating
the effects of Cl2on all of the above parameters. BAL glutathione levels
were not perturbed at 24 h after chlorine. However, at the same time,
there was clear evidence of oxidative lung damage measured by 4-
HNE that was prevented by AEOL. The data show that post hoc
treatment is effective and support the concept that oxidative damage
Fig. 4. Cellular composition of BAL fluid after Cl2exposure. Differential cell counts were done 24 h after Cl2exposure. (A–C) Macrophages, neutrophils, and eosinophils were
significantly elevated in Cl2groups compared to control. (A and B) AEOL attenuated this increase in macrophages and neutrophils. (A–E) There was no difference between control
and AEOL-treated groups. (D and E) There were no differences in any of the other cell types assessed (n=10 per group; *Pb0.05, **Pb0.01, ***Pb0.001).
T. McGovern et al. / Free Radical Biology & Medicine 50 (2011) 602–608
continues beyond the immediate effects of acid and oxidant damage
caused by Cl2.
Previous studies have shown that exposure to Cl2causes increased
airway responsiveness (AHR) in human, rat, and mouse models
[3,8,15,16]. The mechanism of AHR after Cl2may be similar to that of
the tissues . In our study, mice received a single dose of Cl2and
increase in respiratory system resistance and elastance in response to
inhaled MCh. We noted a marked increase in numbers of proliferating
epithelial cells in Cl2-exposed mice compared to control and AEOL-
treated groups, an indication of the intensity of the epithelial
regeneration necessitated by prior damage by Cl2 exposure. AEOL
attenuation of this effect indicates a protective effect on the airways,
of AEOL's action on the airways is that it inhibits epithelial cell repair.
However, this is improbable considering AEOL's favorable effects on
other study outcomes. We did not, however, see high numbers of shed
epithelial cells in the BAL fluid after Cl2, which suggests that shed cells
may already have been removed by phagocytosis or that we may not
have sampled BAL fluid early enough to detect the signal. Whether
airway hyperresponsiveness is caused by loss of epithelial-derived
bronchorelaxant factors or through disruption of barrier function
remains to be determined.
Consistent with previous studies, we noted an increase in
macrophage, neutrophil, and eosinophil populations after Cl2expo-
sure . In addressing potential mechanisms associated with chlorine
damage, these cells, especially neutrophils, produce reactive oxygen
species from activation of endogenous enzymes such as myeloperox-
idase and NADPH oxidase. Activated neutrophils exacerbate airway
damage, causing an increase in microvascular permeability with the
escape of proteinaceous fluid into the airways. AEOL was effective at
Fig. 5. Effects of Cl2exposure and AEOL treatment on BAL fluid protein. Protein levels in
BAL fluid were assessed by Bradford assay. There was a twofold increase in total protein
at 24 h after Cl2exposure compared to control groups. Treatment with AEOL after Cl2
significantly limited the increase in protein content in the BAL fluid (n=10 per group;
Fig. 6. Epithelial cell proliferation after Cl2exposure. Actively proliferating cells were
labeled using Ki-67 staining 72 h after Cl2exposure. (A) Cl2caused a significant increase
in numbers of proliferating epithelial cells in the airways compared to control. AEOL
treatment attenuated this effect; however, Cl2/AEOL groups were significantly
increased compared to controls. (B–D) Representative examples of airway sections
stained with Ki-67 at 250× original magnification. Dark nuclei indicate a positive cell
against the methyl green counterstain (n=6 per group; *Pb0.05, ***Pb0.001).
Fig. 7. Effects of Cl2exposure and AEOL treatment on glutathione levels in BAL fluid.
(A) 24 h after Cl2exposure, GSH levels in the BAL cell fraction show no increase in any
of the treatment groups. (B) GSSG levels show no change in any group 24 h after
Cl2exposure (n=10 per group).
Fig. 8. 4-HNE levels in total lung homogenates. Lungs were collected 24 h after Cl2
exposure and snap-frozen. Total lung homogenates were assessed for 4-HNE levels. Cl2
induced a significant increase in 4-HNE production compared to controls and AEOL-
treatment groups. There were no differences between any of the control and the AEOL-
treatment groups (n=6 per group; *Pb0.05).
T. McGovern et al. / Free Radical Biology & Medicine 50 (2011) 602–608
reducing BAL fluid protein, probably reflecting favorable effects on
cellular necrosis and microvascular permeability after Cl2exposure
[18,19]. Production of reactive oxygen species may contribute to Cl2-
induced AHR. Previous studies report that exposure to ozone induces
several genes such as thyroid hormone-β receptor, nitric oxide
synthase, and glutathione reductase . Nitric oxide synthase has
been implicated as a potential mechanism leading to airway hyperre-
(1400 W) abrogated the Cl2-induced changes in responsiveness .
The increase in eosinophils is a result consistent with previous reports
showing oxidative stress induces eosinophilia . Eosinophilia in an
oxidative injury model may be of consequence, as oxidative stress
induces production of 5-oxo-6,8,11,14-eicosatetraenoic acid, a potent
chemoattractant for granulocytes that function through G-protein-
stress may increase inflammatory cell influx .
Because the effects of Cl2exposure could be at least partially
mediated by oxidative stress we measured two markers: glutathione
and 4-HNE. The glutathione redox system responds rapidly to
oxidative stress, resulting in the oxidation of GSH to GSSG. This can
lead to an initial reduction in the ratio of GSH to GSSG, but this is often
temporary, as there are compensatory changes in the enzymes
involved in glutathione biosynthesis and metabolism. Although
there are studies reporting an increase in glutathione 24 h after Cl2
inhalation, these studies used high concentrations of Cl2for longer
periods of time [7,8,16,23]. In accordance with our findings, a recent
study using similar doses of Cl2but for longer periods of exposure
found that GSH/GSSG levels were reduced at 1 h but had recovered by
24 h after Cl2exposure . Additionally, our previous work has
shown an increase in BAL fluid GSH levels 10 min after chlorine, but
not at 1 h, indicating a rapid response to oxidative stress . The
present study was designed to investigate the longer term (≥24 h)
protective effects of AEOL and it is likely that these times were too late
to detect changes in GSH and GSSG.
4-HNE is a biologically active aldehyde and a stable end product of
lipid peroxidation. The evaluation of lipid peroxidation through the
assessment of 4-HNE is a highly sensitive way of measuring oxidative
stress. Increases in 4-HNE levels have previously been reported to be
associated with endothelial barrier dysfunction and increases in
microvascular leak . Importantly, in this study mouse lung levels
of 4-HNE were reduced to baseline by post-Cl2treatment with AEOL,
suggesting that lipid peroxidation is an important mechanism for Cl2-
induced lung damage.
AEOL10150 is a member of a class of catalytic metalloporphyrins
characterized as small-molecular-weight antioxidants . AEOL
effectively scavenges peroxynitrite and lipid peroxides and has high
SOD and catalase-like activities . Additionally, AEOL is also
effective as a scavenger of lipid hydroperoxides. It is unique as a
potential therapeutic as it reacts with and detoxifies multiple reactive
oxygen and nitrogen species in a catalytic rather than a stoichiometric
fashion. As a highly effective superoxide scavenger AEOL may have
prevented the neutrophil-induced damage to airway epithelial cells
by reducing the toxicity of superoxide by-products released from
In summary, we have shown that AEOL is an effective rescue
hyperresponsiveness, airway inflammation, oxidative stress, and epi-
thelial proliferation. O'Neill et al. have shown that AEOL is also effective
in preventing the effects of other chemical toxins such as half mustard
This work was supported by the CounterACT Program, National
Institutes of Health Office of the Director, and the National Institute of
Environmental Health Sciences,Grant U54 ES015678 (B.J.D., C.W.W., J.
G.M., and T.M.). Dr. Day is a consultant for and holds equity in Aeolus
Pharmaceuticals, which is developing metalloporphyrin catalytic
antioxidants as potential therapeutic agents.
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