In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: An ESR spin-trapping investigation
In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a(N) =14.86 ± 0.03 G and a(H)(β) =2.48 ± 0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl(3) (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2(-)/(-)), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.
Murine models of acute and chronic lung infection
with P. aeruginosa have played a major role in the
search for the molecular mechanisms underlying the
pathogen virulence and host defence. In the last
decade, there has been accumulating evidence that
P. aeruginosa infection may result in acute lung injury
primarily by inducing the release of host-derived
mediators responsible for the inﬂ ux of phagocytes in
the lung; in their antibacterial action, the phagocytes
may release reactive oxygen species and reactive nitro-
gen species [4,5]. Several lines of evidence suggest
that animals challenged with P. aeruginosa undergo
increases in membrane lipid peroxidation, protein
oxidation and DNA damage that were positively
associated with indices of lung injury and neutrophil
In vivo evidence of free radical generation in the mouse lung
after exposure to Pseudomonas aeruginosa bacterium: An ESR
, JEAN CORBETT
, RONALD P. MASON
& MARIA B. KADIISKA
First Department of Biochemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Yoshino-Machi,
Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National
Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, North Carolina, USA
(Received date: 9 November 2011 ; Accepted date: 11 February 2012 )
In the Pseudomonas aeruginosa -induced rodent pneumonia model, it is thought that free radicals are signiﬁ cantly associated
with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo . Here
we used electron spin resonance (ESR) and in vivo spin trapping with α -(4-pyridyl-1-oxide)- N-tert -butylnitrone to investi-
gate free radical production in a murine model. We detected and identiﬁ ed generation of lipid-derived free radicals in vivo
⫽ 14.86 ⫾ 0.03 G and a
⫽ 2.48 ⫾ 0.09 G). To further investigate the mechanism of lipid radical production, we used
modulating agents and knockout mice. We found that with GdCl
(phagocytic toxicant), NADPH-oxidase knockout mice
), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased;
histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-
derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed
hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa .
Keywords: free radicals , mice , Pseudomonas aeruginosa pneumonia , NADPH-oxidase , xanthine-oxidase
Pseudomonas aeruginosa ( P. aeruginosa ) is a common
Gram-negative bacterium that is invasive and toxi-
genic, produces respiratory infections in patients with
abnormal host defences and is an essential nosoco-
mial pathogen . Frequently, lung infections caused
by this opportunistic pathogen can present as a spec-
trum of clinical symptoms from a rapidly fatal pneu-
monia in immuno-compromised patients with AIDS
to a life-threatening infection when the host has a low
level of immunity as in recipients of cancer chemo-
therapy, bone marrow or lung transplants. Even
patients with prolonged intubation of the airway
in the intensive care unit are often colonised with
P. aeruginosa [2 – 4].
Correspondence: Maria B. Kadiiska, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander
Drive, Research Triangle Park, NC 27709, US. Tel: ⫹ 919 - 541-0201. Fax: ⫹ 919 - 541 – 1043. E-mail: email@example.com
Free Radical Research, May 2012; 46(5): 645–655
ISSN 1071-5762 print/ISSN 1029-2470 online © 2012 Informa UK, Ltd.
DOI: 10.3109 /10715762.2012.667089
646 K. Sato et al.
inﬁ ltration [4,5]. The role of reactive oxygen species
in inducing injury to the lung and other tissues as a
result of the P. aeruginosa -induced inﬂ ammatory res-
ponse has been reported by other investigators but
direct evidence for their generation has been lacking.
Free radicals such as superoxide, nitric oxide and
peroxynitrite are thought to play important roles in
the pathogenesis of acute lung injury because SOD
(or its chemical mimics) , nitric oxide synthase
inhibitors  and N -acetylcysteine, all inhibit lypo-
polysaccharides(LPS)-induced damage [8,9]. There-
fore, lung damage is thought to be caused by these
reactive species either directly or indirectly.
P. aeruginosa also has LPS as one of its components.
Most Gram-negative endotoxins (LPS) are potent
immune stimulants through their interactions with
TLR4 . LPS isolated from P. aeruginosa has been
shown to possess immunogenic potential but is less
potent than LPS from other Gram-negative bacteria.
Recently, it has been proposed that production of
free radicals by various bacterial products such as pyo-
cyanin, LPS and exotoxin A and by overactive immune
responses of the host play vital roles in lung injury
induced by P. aeruginosa . Production of lipid-derived
free radicals in the acute respiratory distress syndrome
(ARDS) model induced by intratracheal instillation of
LPS was demonstrated in our previous studies, where
we also conﬁ rmed that activation of NADPH-oxidase
from inﬁ ltrated phagocytes is critical and plays an
important role in free radical generation by LPS .
Since LPS uses only one bacterial component, here
we have used P. aeruginosa as a source of bacterial
infection with the goal of ﬁ nding out where additional
treatments such as anti-oxidant therapy, anti-protease
therapy, etc. may be of use in supplementing antibi-
otic therapy. In this work, we have used the ESR
spin-trapping technique with α -(4-pyridyl-1-oxide)-
N-tert -butylnitrone (POBN) to show that lipid-
derived free radicals are generated during lung injury
in the P. aeruginosa -induced pneumonia model.
Materials and methods
2,2 ′ -dipyridyl (Abbott Laboratories, North Chicago,
IL), Desferal, pentobarbital, uric acid and allopurinol
(Sigma, St. Louis, MO) and modiﬁ ed Wright ’ s stain
kit (Fisher Chemicals, Pittsburgh, PA) were used
as received. The spin trap α -(4-pyridyl-1-oxide)-
N -tert-butylnitrone (POBN) was purchased from
Alexis, San Francisco, CA.
Animals and treatments
Adult male C57BL/6 mice weighing about 20 g (8
weeks) were used in this study. Mice were anesthe-
tised by pentobarbital (40 mg/kg), and pneumonia
was induced by intratracheal instillation of 2 ⫻ 10
cells of P. aeruginosa suspended in 0.05 ml saline.
Twenty-three hours after P. aeruginosa instillation,
the mice were anesthetised by pentobarbital (30 mg/kg)
and were injected with POBN intraperitoneally
(6 mmol/kg). One hour later, POBN-treated rats
were sacriﬁ ced, and lipid extracts of the lungs were
measured for radical adduct content. In all in vivo
and control experiments, sample preparations were
performed in situ with fresh lung tissue. Control
mice received 0.05 ml saline. Another group of
mice was pretreated with GdCl
(7 mg/kg, intrave-
nously, 24 hours before P. aeruginosa instillation),
allopurinol (2 mg/kg, intraperitoneally, 24 hours
and 2 hours before P. aeruginosa instillation) or
Desferal (50 mg/kg, intraperitoneally, 24 hours and
2 hours before P. aeruginosa instillation).
NADPH-oxidase knockout mice (Nox2
⫺ / ⫺
obtained from the Jackson Laboratory (Bar Harbor,
ME). Age-matched mice of the C57BL/6 strain that
possessed normal NADPH-oxidase activity served as
control animals for NADPH-oxidase deﬁ ciency
Both knockout mice and control mice were group-
housed in a temperature-controlled room at 23 – 25 ° C
with a 12/24 light/dark cycle and allowed free access
to food and acidic water. The studies adhered to the
National Institutes of Health guidelines for the care
and handling of experimental animals. All animal
studies were approved by the Institutional Review
In vivo ESR studies
Mice were treated with P. aeruginosa or saline as
shown in Animals and treatments . The lungs were
homogenised in 2.5 ml of 2:1 chloroform:methanol,
0.5 ml of 30 mM 2,2 ′ -dipyridyl, 2 ml of 1.2 mM
ultrapure phenol and 2 ml of deionized water using
a homogeniser (Fisher Scientiﬁ c Power Gen 125) in
an ice bath. The 2,2 ′ -dipyridyl was used to inhibit
ex vivo ferrous-dependent reactions. The phenol was
used as an antioxidant to protect from ex vivo oxida-
tion. In the case of pre-treatment with Desferal
(DFO), we used Desferal (100 mg/kg ip) instead of
2,2 ′ -dipyridyl (DP) to protect against artefactual pro-
duction of free radicals because using both DP and
DFO simultaneously sometimes increases free radical
production artefactually, presumably via electron
transfer between the DP-iron complex and the DFO-
To the homogenate obtained above, 16 ml of 2:1
chloroform:methanol was added and the resulting
sample was shaken, then centrifuged at 2000 rpm for
10 minutes (Beckman TJ-6) as described in [11,12].
The chloroform layer was isolated and dried by pass-
ing through a sodium sulphate column. The solvent
was evaporated to 0.5 ml of solution by bubbling with
In vivo free radical generation by Pseudomonas aeruginosa 647
. Sample handling lasted approximately 90 min-
utes for all experiments.
Immediately after solvent evaporation, ESR spectra
were recorded at room temperature using a quartz ﬂ at
cell in a Bruker EMX EPR spectrometer equipped
with a super high-Q cavity. Spectra were recorded on
an IBM-compatible computer interfaced with the
spectrometer with instrument settings of 9.79 GHz,
20.2-mW-microwave power, 100 kHz modulation
frequency, 1300 ms conversion time and 655 ms time
constant. The ESR spectra were simulated with a
computer optimisation procedure .
Control or treated lung tissue was removed 24 hours
after intratracheal instillation of P. aeruginosa and
ﬁ xed-inﬂ ated to 20 cm H
O pressure with 10% for-
malin. After ﬁ xation, all lobes of the lung were cut
sagittally through the centre of each lobe. Slices of
each pulmonary lobe, 2 – 3-mm thick, were embedded
in parafﬁ n. Tissue sections of 3 μ m thickness were
stained with hemotoxylin-eosin.
Broncho-alveolar lavage ﬂ uid and cell counts
As described previously , three injections and
aspirations with 1 ml of sterile ice-cold saline contain-
ing 1 mM EDTA were used to collect the broncho-
alveolar lavage ﬂ uid (BAL) ﬂ uid in the mice. The
lavage ﬂ uid was injected gently and then aspirated
three times by syringe. Cell counts from fresh BAL
ﬂ uid were determined by using a hemocytometer, and
differential cell counts were performed by cytospin
(Thermo Shandon) on 500 cells from BAL ﬂ uid with
a modiﬁ ed Wright ’ s stain.
Preparation of Pseudomonas aeruginosa
P. aeruginosa laboratory-type strain PAO1 was used
in our experiment. P. aeruginosa was cultured in tryp-
tic soy broth (Difco) at 37 ° C for 18 hours. The cells
were washed four times in sterile PBS and resus-
pended in PBS at a concentration of 2 ⫻ 10
forming units (CFU). A 50 μ l inoculum of a bacterial
suspension of the laboratory strain of P. aeruginosa
PAO1 was used for intratracheal instillation. A vol-
ume of 50 μ l of PBS was instilled into the trachea of
control mice. BAL ﬂ uid or homogenised tissue was
cultured in tryptic soy agar and viable CFU counted
24 hours later.
The statistical signiﬁ cance of the difference was deter-
mined by an unpaired Student ’ s t test. Data are
expressed as means ⫾ SD. Differences between groups
were considered statistically signiﬁ cant at the level of
p ⬍ 0.01.
Detection of free radicals in mouse lungs
instilled with P. aeruginosa
The technique of spin trapping involves the addition
of a primary free radical across the double bond of a
diamagnetic compound (spin trap) to form a radical
adduct more stable than the primary free radical. This
technique involves the indirect detection of primary
free radicals that cannot be directly observed by con-
ventional ESR due to low steady-state concentrations
and/or very short relaxation times, which lead to very
broad lines . It is known that the greatest limita-
tion of organic extraction is that only non-polar rad-
ical adducts that are soluble in chloroform, such as
the POBN, lipid-derived radicals, can be detected. In
addition, the radical adduct must be stable enough to
survive not only the biological environment in which
it was made but also homogenisation of the tissue
and the time required for solvent extraction and evap-
oration which, under our experimental conditions,
was approximately 90 minutes. We chose POBN as
our spin trap because it readily traps lipid radicals that
are stable in lipid extraction of tissue for the requisite
amount of time.
Twenty-four hours after administration of
P. aeru ginosa (2 ⫻ 10
cell count) and 90 minutes after
sample preparation, a stable six-line ESR spectrum
could be reproducibly detected in the lung extract of
POBN-injected mice (Figure 1A). The instillation of
saline instead of P. aeruginosa resulted in a much
weaker signal (Figure 1B). Without the spin trap, nei-
ther P. aeruginosa nor saline instillation yielded a detect-
able spectrum. The increase in signal intensity of the
POBN radical adduct in P. aeruginosa -treated lungs
compared to saline-treated lungs was statistically sig-
niﬁ cant ( P. aeruginosa infection, 20.7 ⫾ 8.7 mm, con-
trol, 6.5 ⫾ 1.4 mm, p ⬍ 0.01).
To evaluate the possibility of ex vivo free radical
generation, we performed a series of control experi-
ments. In the lung extract from a mouse that had
been treated with P. aeruginosa intratracheal instilla-
tion and then homogenised with POBN ex vivo , we
detected a much smaller signal than that formed in
vivo (Figure 1C). In the extract from a mouse treated
with POBN and then homogenised with P. aeruginosa
ex vivo , the signal was also quite weak (Figure 1D).
In a system where both POBN and P. aeruginosa were
added ex vivo to non-treated lungs and then homoge-
nised, there was no detectable ESR spectrum of any
radical adduct (Figure 1E). The ex vivo concentration
of POBN was chosen to be 5 mM on the basis of the
concentrations in blood, heart and liver reported
by Liu et al .  . The ex vivo concentration of
648 K. Sato et al.
P. aeruginosa (2 ⫻ 10
) was chosen to be high enough
to cause inﬂ ammation. In the system where both
POBN and P. aeruginosa (2 ⫻ 10
counts) were added
in vitro to a solution not containing lung tissue and
then homogenised, there was no detectable ESR
spectrum of any radical adduct (Figure 1F). These
experiments indicate that the radical adduct forma-
tion detected in lipid extracts of lung was not pro-
duced ex vivo.
In order to study the implications for pseudo -
monal endotoxin, we used antibiotic-inactivated or
boiled bacteria for intratracheal instillation (Figure 2).
Figure 2A shows a spectrum from lung lipid extract
of a mouse treated with P. aeruginosa and POBN for
ease of comparison. Inactivated bacteria decreased
the ESR signal by over 50% but did not eliminate it.
These results imply that pseudomonal endotoxin
produced nearly 50% of the lipid-derived free radical
Figure 1. ESR spectrum in mouse lung treated with intratracheal P. aeruginosa instillation. (A) Representative ESR spectrum of POBN
radical adduct(s) detected in lipid extracts of lung 24 hours after intratracheal instillation of P. aeruginosa and 1 hour after intraperitoneal
administration of POBN. (B) Same as in (A), but mice were not given P. aeruginosa . (C) ESR spectra of mouse lungs treated with
intratracheal P. aeruginosa instillation and ex vivo addition of POBN. (D) ESR spectra of non-treated mouse lungs 1 hour after intraperitoneal
administration of POBN and ex vivo addition of P. aeruginosa . (E) ESR spectra of non-treated mouse lungs with ex vivo addition of POBN
and P. aeruginosa . (F) ESR spectra of in vitro addition of POBN and P. aeruginosa without mouse lung.
(A) PA + POBN
(A) PA + POBN
treated PA + POBN
treated PA + POBN
(C) Boiled PA + POBN
(C) Boiled PA + POBN
Saline + POBN
Saline + POBN
Figure 2. LPS implication for free radical production in mouse lung treated with intratracheal Pseudomonas aeruginosa (PA) instillation.
(A) ESR spectrum of POBN radical adduct(s) detected in lipid extracts of lung 24 hours after intratracheal instillation of P. aeruginosa
and 1 h after intraperitoneal administration of POBN. (B) ESR spectrum of POBN radical adduct(s) from gentamycin-treated PA
(incubated with gentamycin for 2 hours at 37 ° C) was added instead of non-treated PA. (C) ESR spectrum of POBN radical adduct(s)
when boiled PA (PA was boiled for 1 hour at 100 ° C) was instilled instead of non-treated PA. (D) ESR spectrum of POBN radical adduct(s)
from saline control group.
In vivo free radical generation by Pseudomonas aeruginosa 649
in the absence of viable bacteria (Figures 2B and 2C).
The small POBN spin adduct signal on Figure 2D is
from a control mouse that had not been treated with
Computer simulation of the POBN radical adduct
spectrum and conﬁ rmation of in vivo generation of
When the ESR spectrum was simulated using a
computer program developed in this laboratory ,
the hyperﬁ ne coupling constants for the POBN
radical adducts were a
⫽ 14.86 ⫾ 0.03 G and
⫽ 2.48 ⫾ 0.09 G. To ascertain whether the
POBN radical adduct detected was derived from
lipid, we compared the hyperﬁ ne coupling constants
with literature values (Table I). There were only
minor variations in hyperﬁ ne coupling constants
between the P. aeruginosa -induced radical adducts
and other radical adducts of lipid-derived free
radicals identiﬁ ed as probably polyunsaturated fatty
acid-derived (Table I).
Based upon its hyperﬁ ne coupling constants, the
radical responsible for the 4-POBN radical adduct
is not hydroxyl or any other oxygen-centred radical
adduct. The radical adduct of the six-line spect -
rum in Figure 1A was derived from an endogenous
source (e.g. lipids). Although the coupling con-
stants of 4-POBN radical adducts are relatively
independent of the structure of the trapped radical
and, therefore, cannot be used to deﬁ nitively
identify the free radical intermediate, we have det-
ected, we provisionally assign it as a carbon-centred
PUFA-derived radical. We previously reported
the detection and identiﬁ cation of a chloroform-
soluble, 4-POBN radical adduct following LPS
instillation in the rat lung . The assignment of
these species was based on the hyperﬁ ne coupling
⫽ 14.94 G and a
⫽ 2.42 G) of
authentic ethyl and pentyl radical adducts of
4-POBN previously obtained in vitro [11,17].
The production of carbon-centred, lipid radical
adducts as a result of enhanced lipid peroxidation
in vivo is the most realistic assignment of the six-
line spectrum shown in Figure 1A as supported by
MS data [17,18].
The hydroxyl radical can initiate lipid peroxida-
tion by abstracting hydrogen from lipid molecules.
To investigate whether hydroxyl radical was pro-
duced in the lungs of P. aeruginosa -treated mice,
the hydroxyl radical scavenger dimethyl sulfoxide
(DMSO) was administered to mice with P. aerugi-
nosa because it is well known that a reaction between
DMSO and hydroxyl radical will yield ·CH
in the presence of O
, is converted to ·OCH
are then detected as POBN
adducts. The amount of POBN lipid radical did not
appear to be increased by the presence of DMSO,
and experiments with
C-labelled DMSO did not
change the appearance of the 6-line ESR signal
shown in Figure 1. Either this approach failed to
detect the hydroxyl radical, or another species is
responsible for the initiation of lipid peroxidation
Histopathological analysis of lung instilled
with P. aeruginosa
Murine models of acute and chronic lung infection
with P. aeruginosa have been used to study the molec-
ular mechanisms underlying the pathogen virulence
and host defence . Lung morphology and histo-
pathology by electron and light microscopy has
been reported for animal models and humans with
P. aeruginosa infection [5,19]. Our experimental pro-
tocol showed that 24 hours after intratracheal instil-
lation of P. aeruginosa , inﬂ ammatory responses were
conﬁ rmed by histological analysis of neutrophil inﬁ l-
tration and increasing cell counts of BAL ﬂ uid. In
the BAL ﬂ uid of mice treated intratracheally with
P. aeruginosa , neutrophil counts increased signiﬁ -
cantly ( p ⬍ 0.001) over those of the control group, but
alveolar macrophages did not increase (Figure 3).
These data suggest that, in this model, P. aeruginosa
caused severe lung inﬂ ammation in the form of
Table I. Hyperﬁ ne coupling constants of α -(4-pyridyl-1-oxide)- N-tert -butylnitrone (POBN) radical adducts.
Radical (system) aN a β H Solvent Reference
Pentyl ( in vitro , lung extract)
15.24 ⫾ 0.03 2.44 ⫾ 0.03
Ethyl ( in vitro , lung extract)
15.27 ⫾ 0.01 2.47 ⫾ 0.03
• C (arachidonic acid-derived)
15.12 ⫾ 0.01 2.35 ⫾ 0.01
• C (linoleic acid-derived)
15.04 ⫾ 0.01 2.31 ⫾ 0.01
• C (LPS, lung lipid extract)
14.94 ⫾ 0.07 2.42 ⫾ 0.06
• C (PA, lung lipid extract)
14.86 ⫾ 0.03 2.48 ⫾ 0.09
/MeOH this work
• C (PPIX, light, skin extract) 14.8 2.6 CHCl
• OOH (PPIX, light, skin extract) 13.8 1.8 CHCl
• OH ( in vitro ) 14.4 1.8 Benzene 55
• OH ( in vitro ) 14.9 1.6 Aqueous 55
LPS, liposaccharides; PA, Pseudomonas aeruginosa ; PP, protoporphyrin.
650 K. Sato et al.
Effect of GdCl
treatment to evaluate the role
is well known to decrease phagocyte activ-
ity , we evaluated its inhibitory effect on the pro-
duction of free radicals in the lungs of mice treated
intratracheally with P. aeruginosa . When GdCl
administered to mice 24 hours before P. aeruginosa
instillation, the production of free radicals in this
system decreased by 78.5% while the hyperﬁ ne
coupling constants ( a
⫽ 14.94 ⫾ 0.07 G and a
2.42 ⫾ 0.06 G) were unchanged (Figure 4). At the
same time, the levels of neutrophils and macrophages
in the BAL ﬂ uid also decreased signiﬁ cantly (data
not shown). Parallel to changes in the neutrophil
population, two lung-injury parameters, wet weight/
dry weight ratio ( p ⬍ 0.01) and the protein concentra-
tion in the BAL ﬂ uid ( p ⬍ 0.01), were considerably
decreased by GdCl
pre-treatment (Table II). In
the histopathological study, GdCl
resulted in a remarkable decrease in lung injury,
signiﬁ cantly inhibiting diffuse alveolar damage includ-
ing interstitial edema, inﬁ ltration with neutrophils
and monocytes, parenchymal hemorrhage, collapse of
air space and ﬁ brin exudation into alveolar space
(Figure 5). This inhibition applied not only to lung
injury parameters and histopathological ﬁ ndings but
also to free radical production in the lung. These
results conﬁ rm that free radical production in this
Control Control Control PA-IT
P < 0.001 P < 0.001
Cell counts (10
BALF cell counts
Figure 3. Cell counts in Broncho alveolar lavage ﬂ uid (BALF) of P. aeruginosa- treated and control mice. Bronchoalveolar lavage (BAL)
of mouse lung treated with or without intratracheal P. aeruginosa (PA) instillation was performed with saline containing 1 mM EDTA.
Cell counts from BAL were determined by using the hemocytometer method, and differential counts were performed on 400 cells from
BALF smears stained with a modiﬁ ed Wright ’ s stain. Results are expressed as means ⫾ SD of three independent experiments (n ⫽ 3).
% inhibition of free radical production
Figure 4. Inhibition of free radical generation in mouse lung treated
with intratracheal P . aeruginasa (PA) instillation using GdCl
NADPH oxidase KO mice, allopurinol (ALP), and Desferal
(DFO). ESR signal intensity of radical adducts detected in lipid
extract from lungs of GdCl
-pretreated mice (5 mg/kg iv), NADPH
knockout mice (Nox2
- / -
), allopurinol-pretreated mice (2 mg/kg ip),
and Desferal-treated mice (100 mg/kg ip) 24 hours after
intratracheal instillation of P. aeruginosa and 1 hour after
intraperitoneal administration of POBN. Results are expressed as
a percentage of the control mice and are the means ⫾ SD of four
independent experiments (n ⫽ 4).
Table II. GdCl
pre-treatment effect on the lung wet weight/dry
weight ratios and the lavage protein concentrations.
Lung wet weight/
dry weight ratio
4.69 ⫾ 0.28 0.26 ⫾ 0.01
5.87 ⫾ 0.06
2.17 ⫾ 0.22
⫹ PA-IT 4.95 ⫾ 0.07
0.96 ⫾ 0.11
Protein concentrations were measured by the bicinchoninic acid
p ⬍ 0.001 versus control group,
p ⬍ 0.01 versus
PA-intratracheally (IT) group,
p ⬍ 0.001 versus control group,
p ⬍ 0.01 versus PA-IT group. PA, Pseudomonas aeruginosa.
In vivo free radical generation by Pseudomonas aeruginosa 651
system depends on phagocytes, probably by inﬁ ltrat-
We evaluated the clearance of P. aeruginosa in this
model with or without the administration of GdCl
The counts of P. aeruginosa in the mouse lung
24 hours after bacterial inoculation are shown in
Table III. In this study, four different mice were used
for bacteria counts at each time point. Because we
observed an increase in bacterial yield with GdCl
treatment ( p ⬍ 0.001) at the same time that symptoms
decreased, these data suggest that lung injury in this
model results from phagocytic cell inﬁ ltration, not
from the bacteria itself (Table III).
Effect of modulating agents and knockout mice on
lipid-derived free radical production and
In order to analyse the mechanism of lipid-derived
free radical in detail in this system, we examined the
effects of a xanthine-oxidase inhibitor (allopurinol), a
metal chelater (Desferal) and NADPH-oxidase
knockout mice (Nox2
⫺ / ⫺
In the NADPH-oxidase knockout (Nox2
⫺ / ⫺
model, the production of lipid-derived free radical
was signiﬁ cantly decreased and lung injury was miti-
gated (Figures 4 and 5). Superoxide or superoxide-
derived free radicals are probably important in the
production of lipid-derived free radicals. All control
experiments were performed using a control strain
(age-matched C57BL/6) for NADPH-oxidase knock-
out mice (Nox2
⫺ / ⫺
). Free radical production, BAL
cell counts and histopathological ﬁ ndings in the con-
trol strain were similar to those in our previous exper-
iments (Figures 1,3 and 5).
We used Desferal to determine whether hydroxyl
radical and/or other iron-dependent species were
involved in the production of lipid-derived free rad-
icals in this model since Desferal readily chelates
iron to form the redox-inert Fe
prevents ·OH radical formation via the Fenton reac-
tion . When mice were pretreated with Desferal
2 hours and 24 hours before P. aeruginosa inocula-
tion, the production of lipid-derived free radicals was
decreased by 55%, and the histopathological ﬁ nd-
ings were noticeably improved (Figures 4 and 5).
We measured the bacterial counts of P. aeruginosa
with and without Desferal 24 hours after adminis-
tration of P. aeruginosa and found no difference
(data not shown).
Allopurinol, a competitive inhibitor of xanthine-
oxidase , also inhibited free radical production
and lung injury seen in histological examinations
(Figures 4 and 5). Xanthine-oxidase activity is a
new pathway in the production of lipid-derived free
radicals in P. aeruginosa -treated mice; the activity
was measured as described previously [18,19]. Its
activity in lung homogenate was signiﬁ cantly increased
after Pseudomonas infection (xanthine-oxidase activ-
ity: Control 0.002 ⫾ 0.001, Pseudomonas infection
0.082 ⫾ 0.005 nmol/min/ml). In the histopathological
NADPH OX KO Allo
Figure 5. Effect of modulating agents/KO mice on P. aeruginosa (PA)-infected lung in histological ﬁ ndings. Representative histopathological
ﬁ ndings from control lungs and lungs of PA-treated mice, NADPH knockout mice, allopurinol-pretreated mice, and Desferal-pretreated
mice 24 hours after intratracheal instillation of P. aeruginosa (n ⫽ 4). Hematoxilin-eosin stain was used (original magniﬁ cation, ⫻ 200).
Table III. GdCl
pre-treatment effect on bacteria counts by colony-
forming method (n ⫽ 4, mean ⫾ SD).
cfu/ml) Tissue (10
0.19 ⫾ 0.11 0.46 ⫾ 0.16
⫹ PA-IT 1.76 ⫾ 0.69
5.76 ⫾ 0.64
Bronchoalveolar lavage (BAL) and tissue homogenate were used
for counting by colony-forming methods.
p ⬍ 0.05 versus PA-IT
p ⬍ 0.001 versus PA-IT group.
652 K. Sato et al.
study, allopurinol was quite effective in limiting
Pseudomonas -mediated lung injury (Figure 5).
From these data, we have demonstrated that the
mechanism of lung injury from lipid-derived free
radical production involves two enzymatic pathways:
NADPH-oxidase and xanthine-oxidase.
We have provided ESR evidence that free radicals are
being generated in vivo in the lung and are dependent
on Pseudomonal endotoxin. Based on ESR spectral
simulations, we identiﬁ ed the radical adducts as
carbon-centred, lipid-derived. We suggest that the
carbon-centred radicals detected are probably an
intermediate of enhanced lipid peroxidation in the
lung caused by P. aeruginosa in vivo [5,23] and in our
previous in vivo studies [10,11]. In fact, we have con-
ﬁ rmed by various control experiments that the POBN-
radical adduct was formed in vivo and not during
sample collection or handling. Although the major
difﬁ culty of the spin-trapping technique in vivo is the
mere detection of a radical adduct, other factors must
be considered when spin traps are administered in
vivo . For example, it is not known whether the back-
ground ESR spectrum detected in the lipid extract of
the control animal after the injection of POBN was
formed during sample handling or in vivo prior to
sample collection. Based on our previous studies with
HPLC/MS, we propose that they are radical adducts
of ambient levels of endogenous radicals [17,24,25].
Since free radicals in biological systems are charac-
terised by their high reactivity, short lifetimes and low
concentrations, the type of spin trap used is an impor-
tant factor in determining how informative and sensi-
tive the spin-trapping technique may be for a given
free radical. A number of our previously reported
spin-trapping investigations have used the nitrone
spin trap α -(4-pyridyl-1-oxide)-N-tert-butylnitrone
(POBN) due to its relative hydrophilicity, low toxicity,
solubility and stability, leading to reproducible
results [10 – 12,37]. The detection of a free radical
generated in vivo is only possible if the spin trap and
free radical concentrations are high enough and the
rate of spin-trapping occurs rapidly. Due to the forma-
tion of relatively unreactive radical adducts, POBN
might be expected to protect the lung tissue from the
effects of free radicals. However, under the conditions
employed here, it is considered unlikely that POBN
will react with all but a very small fraction of radicals
generated (due to competing reactions of radicals with
biomolecules), and therefore, it is unlikely that the
small concentration of the spin-trapping agent (attrib-
utable to in vivo pharmacokinetics) will have a sig-
niﬁ cant effect on toxicity due to radical scavenging.
Histopathological analysis of the lung tissue instilled
with P. aeruginosa and the inﬂ ammatory response
through cell counts of BAL were followed as indices
of lung injury and were assessed to determine if rad-
ical generation preceded or was associated with
changes in the lungs. Although P. aeruginosa did not
signiﬁ cantly enhance alveolar macrophage numbers
in BAL, it signiﬁ cantly increased total cell and neu-
trophil counts. These data demonstrate that free
radical generation was associated with severe lung
inﬂ ammation as a result of neutrophil inﬁ ltration
caused by P. aeruginosa . This is consistent with other
studies, which showed increased oxidative stress in
patients or experimental animals with pneumonia [4,5].
It should be noted that the role of speciﬁ c virulence
factors, some lung enzyme activities, pulmonary
oxidant-antioxidant status, etc. in P. aeruginosa -
infected animals have been extensively studied [4,5]
and were not examined in the present study.
Lipid radicals can cause tissue injury via protein
damage [26,27] where protein is oxidised as a result
of the free radical chain reaction of lipid peroxidation.
When we examined the radical mechanism using
as a phagocytic toxicant  and NADPH-
oxidase knockout mice (Nox2
⫺ / ⫺
), we found a sig-
niﬁ cant decrease of the lipid-derived radical production
and improved histopathological parameters in the
lungs. With the use of GdCl
, we also demonstrated
that the participation of alveolar macrophages is
indispensable for free radical generation, neutrophilic
inﬂ ammation and lung injury. The inhibition of neu-
trophilic inﬁ ltration by GdCl
, which is probably the
result of enzyme blockage and factors related to neu-
trophil migration and adhesion that are released from
macrophages, has been reported previously in LPS-
and ozone-induced lung injury [10,29]. The produc-
tion of reactive oxygen species by the phagocyte
multicomponent NADPH-oxidase system is well
known as a critical component of antimicrobial host
defence [30,31]. Therefore, we concluded that
NADPH-oxidase activation from phagocytes plays an
essential role in the generation of free radicals in mice
with pneumonia caused by P. aeruginosa .
Recently, additional homologs of NADPH-oxidase
have been discovered and suggested to have speciﬁ c
involvement in respiratory and cardiovascular disease
[32,33]. In vitro studies have demonstrated that a
wide range of inﬂ ammatory factors upregulate the
expression of Nox2
⫺ / ⫺
, and superoxide auto-aug-
ments the formation of superoxide through an upreg-
ulation of NADPH-oxidase activity in pulmonary
artery endothelial cells . Other reports have
described the presence of NADPH-oxidases in differ-
ent types of cells with different functions . For
example, the oxidase in neutrophils releases large
amounts of superoxide in bursts, whereas the vascular
NADPH-oxidases continuously produce low levels of
superoxide . In this study, the data from geneti-
cally modiﬁ ed mice that lack an active catalytic sub-
unit of NADPH-oxidase, Nox2
⫺ / ⫺
, provide evidence
In vivo free radical generation by Pseudomonas aeruginosa 653
for the involvement of the Nox2
⫺ / ⫺
nent of the NADPH-oxidase system.
The xanthine/xanthine-oxidase system has been
shown to upregulate the expression of Nox2
⫺ / ⫺
although the xanthine-oxidase inhibitor allopurinol
had no effect [33,34]. However, the present study
does not exclude or imply the role of other cytosolic
NADPH-oxidases [30 – 32].
The contribution of xanthine-oxidase has been
reported by Wright et al. in cytokine-induced acute
lung injury in rats . Kahl et al . reported that LPS
increased xanthine-oxidase activity of plasma in an
LPS-induced sepsis model . LPS is also produ -
ced by Pseudomonas , a Gram-negative bacterium. In
P seudomonal pneumonia, LPS and other bacterial
extracts induced many kinds of cytokines, which have
been thought to increase the xanthine-oxidase activ-
ity. However, this increase has not been experimen-
tally conﬁ rmed.
We have recently reported the importance of
xanthine-oxidase activity in the pathogenesis of lung
injury caused by super-antigens . We have shown
that when 4-amino-6-hydroxypyrazolo(3,4-d)-pyrim-
idine (an allopurinol derivative) was used as an
antagonist of xanthine-oxidase, survival rate and his-
topathological ﬁ ndings of lung injury were signiﬁ -
cantly improved. In view of this, we have evaluated
here the efﬁ cacy of allopurinol in free radical produc-
tion and histological ﬁ ndings. We found an inhibitory
effect of allopurinol on radical production and a ben-
eﬁ cial effect of allopurinol for histopathological ﬁ nd-
ings. However, these experiments did not address the
potential role of xanthine oxido-reductase activity on
free radical generation in humans since much lower
activity has been reported in a variety of human tis-
sues relative to other species including rats and mice
[38 – 41]. Other studies have demonstrated that xan-
thine-oxidase and xanthine-dehydrogenase are inter-
convertible forms of the same enzyme, and some
cytokines or hypoxia upregulated their generation at
the translational and post-translational levels [42 – 44].
Tumour necrosis factor (TNF- α ), interleukin-1 and
IFN- γ were also shown to induce their activity in
epithelial cells . All these ﬁ ndings indicate that
xanthine-oxidase/dehydrogenase is regulated in a
cell-speciﬁ c manner and by inﬂ ammatory cytokines
and complex physiological and pathological events.
However, we do not have data to conﬁ rm or reject
these ﬁ ndings.
Desferal is a chelating agent for iron and other met-
als. Most stored iron is in the ferric state (Fe
release of which can create a pool of Fe which, in
turn, can participate in the Fenton reaction to form
hydroxyl radical. Desferal readily chelates iron to
form a redox-inert Fe
-complex and prevent
hydroxyl radical formation. Here Desferal had a
protective effect on lung injury most likely by Fe
chelation, thereby inhibiting lipid peroxidation and
free radical formation . In addition, iron is neces-
sary for bacterial growth. Although several papers
have shown that metal-chelating agents inhibit bacte-
rial growth in P. aeruginosa , there has been no
direct evidence that bacterial growth is inhibited by
Desferal. In our study , bacterial counts of P. aerugi-
nosa with and without Desferal 24 hours after admin-
istration of P. aeruginosa were not changed, thus
supporting the conclusion that Desferal inhibits free
radical production by inhibiting the Fenton reaction.
It has been reported that alterations in proinﬂ amma-
tory cytokines, adhesion molecules and chemotactic
gradients play an important role in the accumulation
of neutrophils during lung inﬂ ammation, and Des-
feral has been shown to interfere with the adhesion
functions of activated neutrophils. [47,48]. In addi-
tion, the effect of Desferal on the POBN radical
adduct detected by ESR as a result of P. aeruginosa
treatment may not reﬂ ect the role of free iron in lipid
peroxidation but rather the chain-breaking antioxi-
dant character of Desferal [49 – 51]. These data sug-
gest that there may be other mechanisms that account
for its protective effects in addition to the inhibition
of iron ’ s catalytic generation of hydroxyl radical. Lit-
erature data imply that products of P. aeruginosa may
also lead to oxidant-mediated tissue injury. For exam-
ple, pyocyanin produced by many strains of P. aerug-
inosa has been shown to undergo cellular redox
cycling, to interact synergistically with iron bound to
another P. aeruginosa secretory product, the sidero-
phore pyochelin, and to damage pulmonary endothe-
lial cells through production of • OH . ESR
spin-trapping evidence that pyocyanine and protease-
cleaved Fe-transferrin act synergistically to enhance
endothelial cell injury via formation of • OH has also
been demonstrated in vitro .
The experiments from our in vivo study do not
provide evidence of hydroxyl radical generation. The
hyperﬁ ne coupling constants of the radical adduct
that we did observe are not consistent with a POBN/
•OH adduct detected in either aqueous solution or
the non-polar solvent benzene [54,55] (Table I). Fur-
thermore, the POBN oxygen-centred radical detected
in a lipid extract of skin (assigned to •OOH) also had
coupling constants closer to those from POBN/•OH
than to carbon-centred adducts (Table I). Therefore,
we conclude that our radical adduct is a carbon-
centred adduct, as its coupling constants are very
similar to those of other POBN carbon-centred
adducts (Table I).
There are other potential mechanisms by which
free radical generation may be triggered in the lungs
of mice infected with P. aeruginosa in addition to
NADPH-oxidase and xanthine-oxidase involvement.
One potential common inﬂ ammatory mechanism is
participation of reactive nitrogen species such as nitric
oxide and peroxynitrite that lead to the nitration of
cellular lipids, proteins and nucleotides. Though we
654 K. Sato et al.
did not assess markers of reactive nitrogen species,
this is clearly an important focus for subsequent stud-
ies of the mechanisms of free radical generation in the
P. aeruginosa mouse model of pneumonia.
In conclusion, the present investigation has shown
(i) that lipid-derived radicals are generated in the
lungs of mice with pneumonia by P. aeruginosa ; (ii) that
NADPH- and xanthine-oxidase are required for the
generation of these radicals; and (iii) that metal-catal-
ysed, hydroxyl-like species play an essential role in lung
injury caused by P. aeruginosa . Taken together, our
results imply that NADPH-oxidase, xanthine-oxidase
and ferric iron work synergistically to generate free
radical metabolites in lung inﬂ ammation caused by
P. aeruginosa . In this respect, the primary role of mac-
rophage toxicants, xanthine-oxidase inhibitors and
iron chelators in inhibiting free radical-initiated per-
oxidative tissue injury is established to a great degree.
Thus, this report suggests that enzyme inhibitors might
be useful for the development of therapeutic agents in
supportive therapy not only for pseudomonal infection
but also for lung injury in general.
The authors wish to thank Dr. Ann Motten and
Mrs. Mary J. Mason for their excellent editorial help
in the preparation of the manuscript.
Declaration of interest
The authors report no conﬂ icts of interest. The authors
alone are responsible for the content and writing of
the paper. This research was supported by the Intra-
mural Research Program of the National Institutes of
Health and by the National Institute of Environmen-
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This paper was ﬁ rst published online on Early Online on
16 March 2012.